Endophytic Establishment of Beauveria Bassiana in Grapevine Plants As A Sustainable Pest Management Strategy

Endophytic establishment of Beauveria bassiana
in grapevine plants as a sustainable pest

management strategy
Von der Naturwissenschaftlichen Fakultät
der Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades
Doktorin der Gartenbauwissenschaften
(Dr. rer. hort.)
genehmigte Dissertation
von
Yvonne Rondot, M. Sc.
2019
Referent: Prof. Dr. rer. nat. Dr. rer. hort. habil. Hans-Michael Poehling
Korreferentin: Prof. Dr. sc. agr. Annette Reineke
Tag der Promotion: 30. August 2019

Abstract
Fungal entomopathogens like Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) are
known as antagonists of insects with multiple functional and ecological roles and have
attracted increased attention as biocontrol agents in integrated pest management programs.
Besides its entomopathogenic habit, evidence has accumulated that the fungus can also
establish as an endophyte in a wide array of plant species. However, only limited information
is currently available on the endophytic colonization of grapevine, Vitis vinifera (L.), plants
with B. bassiana. In addition, the functional role of the fungus in planta, and/or the plant’s
response to colonization by B. bassiana as well as the mechanisms underlying these responses
and putative protection effects, still require elucidation. Therefore, the objectives of this thesis
were to investigate whether the fungus B. bassiana is able to colonize grapevine plants, still
maintains its entomopathogenic potential against insect pests, and can provide additional
protection against plant fungal pathogens or limit their damaging effects. The investigation
focused on the interaction between B. bassiana, grapevine plants, and potential target insect
pests as well as fungal pathogens to gain more knowledge on this particular tritrophic
interaction with regard to potential biological control strategies.
In the present thesis, greenhouse and field experiments were conducted to optimize
endophytic establishment of the entomopathogenic fungus B. bassiana in potted and mature
grapevine plants. Two different commercialized B. bassiana strains (ATCC 74040/product
®
Naturalis and GHA) were applied on the leaf surfaces of grapevine plants. To determine if
endophytic colonization of grapevine leaves by B. bassiana was successful, a culture
dependent approach was used and the assessment was verified by the amplification of strain-
specific microsatellite markers. Endophytic survival of B. bassiana inside leaf tissues was
evident for at least 21 days after inoculation in potted grapevine plants and up to five weeks
after the last application in mature grapevine plants in the vineyard. The antagonistic activity
of endophytic B. bassiana against putative target pest insects like the vine mealybug
Planococcus ficus was assessed in a bioassay using surface sterilized leaves. Infestation rate
and growth of P. ficus were significantly reduced. Possible effects of endophytic B. bassiana on
the host choice preference of adult black vine weevil Otiorhynchus sulcatus choosing between
control and B. bassiana inoculated plants were examined through choice assays. Adult O.
sulcatus chose significantly more often the control plants as a host plant compared to
®
grapevine plants treated with Naturalis , where B. bassiana putatively had established as an
endophyte. These results suggest that adult black vine weevils are able to detect and
subsequently avoid plants treated with B. bassiana and indicate a new mode of action of plant-
associated entomopathogenic fungi. Furthermore, the protective potential of endophytic B.
bassiana against grapevine downy mildew Plasmopara viticola was investigated in greenhouse
experiments. A significant effect on the disease severity and disease incidence of downy
mildew on grapevine leaves was observed if plants were treated with B. bassiana 3 and 7 days
before inoculation with P. viticola. To work out fundamental aspects of genes involved in the
interaction between grapevine and the endophytic fungus B. bassiana, a microarray and an
RT-qPCR analysis were performed. The results indicate an up-regulation of diverse defense-
related genes in grapevine as a response to the endophytic establishment of B. bassiana.
In conclusion, the results of this thesis indicate that endophytic establishment of an
entomopathogenic fungus such as B. bassiana in grapevine plants can represent an alternative
and sustainable plant protection strategy, with the potential for reducing pesticide
applications in viticulture.
Keywords: Beauveria bassiana, grapevine, endophyte, biological control
III
Zusammenfassung
Im integrierten und ökologischen Pflanzenschutz stellen entomopathogene Pilze bei der
Bekämpfung verschiedener Arthropoden eine gute Alternative zu chemischen
Pflanzenschutzmitteln dar. Dieses Potential wird allerdings bislang noch unzureichend
ausgeschöpft. Insbesondere ist über die Fähigkeit dieser Pilze, sich endophytisch in Pflanzen
zu etablieren nur wenig bekannt. Durch eine endophytische Etablierung könnten
entomopathogene Pilze wie Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) zum
einen eine Infektionsquelle für Schädlinge darstellen oder zum anderen über Mechanismen
der induzierten Resistenz Abwehrrektionen gegen Schaderreger in der Pflanze aktivieren. Ein
verbessertes Wissen über diese Interaktionen könnte eine vermehrte und effizientere Nutzung
entomopathogener Pilze in biologischen Pflanzenschutzstrategien unterstützen.
Im Rahmen der vorliegenden Dissertation wurde ein Verfahren für die endophytische
Etablierung des entomopathogenen Pilzes B. bassiana in Reben Vitis vinifera (L.) entwickelt.
®
Dazu wurden zwei Stämme des Pilzes (ATCC 74040/Präparat Naturalis und GHA) verwendet.
Die Behandlung von Topfreben im Gewächshaus und von ausgewachsenen Reben im
Weinberg erfolgte mittels Sprühapplikation auf die Blattober- sowie Blattunterseiten. Der
Nachweis einer endophytischen Besiedelung der Blätter wurde durch Blattscheibentests auf
Selektivmedium mit anschließender Verifizierung durch Amplifikation mittels stamm-
spezifischer Mikrosatelliten erbracht. Es konnte gezeigt werden, dass sich B. bassiana in
Topfreben über einen Zeitraum von mindestens drei Wochen endophytisch etablieren konnte
und auch in Weinbergsreben fünf Wochen nach der letzten Applikation nachweisbar war. Das
antagonistische Potential von endophytisch etabliertem B. bassiana gegenüber Schmierläusen
(Planococccus ficus) wurde unter Verwendung von oberflächensterilisierten Blättern von
behandelten Topfreben in einem Bioassay bewertet. Der endophytische B. bassiana hatte
einen signifikanten Einfluss auf die Mortalität und das Wachstum von P. ficus in der ersten
Woche nach der anfänglichen Festsetzungsphase. Mögliche Auswirkungen des
endophytischen B. bassiana auf die Wirtspflanzenwahl von adulten Rüsselkäfern Otiorhynchus
sulcatus wurden durch Olfaktometer-Tests mit Kontrollpflanzen und mit B. bassiana
inokulierten Pflanzen untersucht. Adulte O. sulcatus wählten signifikant häufiger
®
Kontrollpflanzen als Wirtspflanze verglichen mit Naturalis behandelte Reben, bei denen sich
B. bassiana mutmaßlich als Endophyt etabliert hatte. Diese Ergebnisse legen nahe, dass der
Gefurchte Dickmaulrüssler in der Lage ist, mit B. bassiana behandelte Pflanzen zu erkennen
und aufgrund dessen zu meiden und deuten auf einen neuen Wirkmechanismus
pflanzenassoziierter entomopathogener Pilze hin. Zusätzlich wurde das protektive Potential
von B. bassiana gegenüber dem Erreger des Falschen Rebenmehltaus Plasmopara viticola an
Topfreben untersucht. Bei einer protektiven Behandlung von Reben mit B. bassiana 3 und 7
Tage vor einer Inokulation mit P. viticola konnte eine signifikante Reduktion der Befallsstärke
und -häufigkeit von Falschem Mehltau an Topfreben beobachtet werden. Um grundlegende
Aspekte der Wechselwirkung zwischen Weinrebe und endophytischem B. bassiana auf
Genebene aufzudecken, wurden ein Microarray und eine RT-qPCR-Analyse durchgeführt. Die
Ergebnisse zeigen nach der Behandlung mit B. bassiana eine erhöhte Expression verschiedener
Gene der Weinrebe, welche in Zusammenhang mit der Abwehrreaktion von Pflanzen stehen.
Zusammenfassend lässt sich festhalten, dass die endophytische Etablierung eines
entomopathogenen Pilzes wie B. bassiana in Weinreben eine alternative und nachhaltige
Pflanzenschutzstrategie darstellen kann, mit dem Potenzial, den synthetisch-chemischen
Wirkstoffeinsatz im Weinbau zu reduzieren.
Schlüsselwörter: Beauveria bassiana, Weinrebe, Endophyt, biologischer Pflanzenschutz
IV
Contents
Abstract ............................................................................................................................................................ III
Zusammenfassung .........................................................................................................................................IV
List of Abbreviations ................................................................................................................................... VII
List of Figures .............................................................................................................................................. VIII
List of Tables ...................................................................................................................................................IX
1 General Introduction ................................................................................................................................. 1
1.1 Plant protection in viticulture .................................................................................................... 1
1.2 Biological control of pests and plant diseases ........................................................................ 2
1.2.1 Microbial control agents ......................................................................................................... 2
1.2.2 Entomopathogens ..................................................................................................................... 4
1.3 Beauveria bassiana ......................................................................................................................... 7
1.3.1 Taxonomy and morphology ................................................................................................... 7
1.3.2 Beauveria bassiana as an entomopathogen......................................................................... 9
1.3.3 Plant colonization by Beauveria bassiana ........................................................................... 9
®
1.3.4 The product Naturalis ..........................................................................................................12
2 Objectives...................................................................................................................................................18
3 Endophytic Beauveria bassiana in grapevine Vitis vinifera (L.) reduces infestation with
piercing-sucking insects .........................................................................................................................20
3.1 Introduction ..................................................................................................................................22
3.2 Materials and methods ...............................................................................................................23
3.2.1 Fungal material ........................................................................................................................23
3.2.2 Endophytic establishment in potted grapevine plants ..................................................24
3.2.3 Re-isolation of B. bassiana ....................................................................................................24
3.2.4 Strain-specific detection of B. bassiana .............................................................................25
3.2.5 Mealybug bioassay..................................................................................................................26
3.2.6 Field trial ...................................................................................................................................27
3.3 Results ............................................................................................................................................28
3.3.1 Endophytic colonization of potted grapevine plants .....................................................28
3.3.2 Strain-specific detection of endophytic B. bassiana .......................................................29
3.3.3 Mealybug bioassay..................................................................................................................29
3.3.4 Field trial ...................................................................................................................................31
3.4 Discussion .....................................................................................................................................32
4 Association of Beauveria bassiana with grapevine plants deters adult black vine weevils,
V
Otiorhynchus sulcatus ..............................................................................................................................36
4.1 Introduction ..................................................................................................................................37
4.2.1 Source of fungus, insects, and plants .................................................................................39
4.2.2 Design and validation of the two-choice olfactometer..................................................40
4.2.3 Experimental design ...............................................................................................................41
4.3 Results ............................................................................................................................................42
4.4 Discussion .....................................................................................................................................44
5 Endophytic Beauveria bassiana activates expression of defense genes in grapevine and
prevents infections by grapevine downy mildew Plasmopara viticola .......................................46
5.1 Introduction ..................................................................................................................................47
5.2.1 Plant and fungal material ......................................................................................................49
5.2.2 Treatment of plants with B. bassiana ................................................................................. 50
5.2.3 Inoculation of plants with P. viticola .................................................................................. 50
5.2.4 Disease assessment .................................................................................................................51
5.2.5 Assessment of endophytic colonization ............................................................................52
5.2.6 RNA isolation ...........................................................................................................................53
5.2.7 Microarray analysis ................................................................................................................53
5.2.8 RT-qPCR ....................................................................................................................................54
5.3 Results ............................................................................................................................................56
5.3.1 Endophytic colonization .......................................................................................................56
5.3.2 Preventive activity against Plasmopara viticola ..............................................................56
5.3.3 Changes in expression patterns after treatment with B. bassiana ..............................60
5.4 Discussion .....................................................................................................................................65
6 General discussion ...................................................................................................................................69
7 References .................................................................................................................................................. 78
A Supplementary Material .........................................................................................................................94
A1 Figures ............................................................................................................................................94
A2 Tables..............................................................................................................................................96
A3 Manuscripts...................................................................................................................................99
B Danksagung/Acknowledgements ......................................................................................................117
C Curriculum vitae ................................................................................................................................... 119
D List of Publications ................................................................................................................................121
VI
List of Abbreviations
ANOVA analysis of variance

ATCC American Type Culture Collection
BSM Beauveria selective medium
CI confidence intervall
CNRQs calibrated normalized relative quantities
cv cultivar
DAI days after inoculation
DBU Deutsche Bundesstiftung Umwelt
DNA deoxyribonucleic acid
EC European Commission
EPF entomopathogenic fungi
ET ethylene
EtOH ethanol
EU European Union
GEP good experimental practice
GLRaV grapevine leafroll associated virus
hpt hours post treatment
HR hypersensitive response
ISR induced systemic resistance
JA jasmonic acid
MAMPs microbe-associated molecular patterns
NaOCl sodium hypochlorite or active chlorine
OD oil dispersion
OIV International Organisation of Vine and Wine
P bzw p propability
PDA potato dextrose agar
PGPR plant growth promoting rhizobacteria
PR pathogenesis-related
RH relative humidity
(RT-q)PCR (quantitative reverse transcription) polymerase chain reaction
SA salicylic acid
SAR systemic acquired resistance
SD/SE standard deviation/error
SSR simple sequence repeats
UV ultra violet
VOCs volatile organic compounds
VII
List of Figures
Figure 1: Diagram illustrating how entomopathogenic fungi infect arthropod hosts by spores,
proliferate, and disperse.. ....................................................................................................................... 6
Figure 2: Left: Conidiophores with conidiogenic cells of Beauveria bassiana, Right: Picture of the
conidiospores of B. bassiana by electron microscopy...................................................................... 8
Figure 3: Characteristic white, woolly colonies of B. bassiana on a solid culture medium ...................... 8
Figure 4: Left: B. bassiana re-emerges from the cadaver and produces the characteristic white
covering called “white muscardine”. Right: Endophytic B. bassiana re-emerges from leaves
discs of grapevine plants ........................................................................................................................ 9
Figure 5: Mean (±SE) percentage colonization of Vitis vinifera leaf discs 7, 14 and 21 days after
inoculation (DAI) with B. bassiana. ................................................................................................... 28
Figure 6: Mean (± 95% CI) size of vine mealybug larvae (P. ficus) after feeding for three weeks on
detached grapevine leaves of control plants and plants with endophytic B. bassiana in two
replicates.................................................................................................................................................. 30
Figure 7: Mean (± 95% CI) infestation rate of vine mealybug larvae (P. ficus) after feeding for three
weeks on detached grapevine leaves of control plants and plants with endophytic
B. bassiana in two replicates................................................................................................................ 31
Figure 8: Design of the two-choice still-air olfactometer used in the experiments. ................................ 41
Figure 9: Percentage of adult black vine weevils O. sulcatus recovered when released in an
olfactometer containing (i) control plants and no plant, (ii) control plants and plants treated
with Naturalis®, (iii) control plants and plants treated with the formulation, and (iv) plants
treated with Naturalis® and plants treated with the formulation. ............................................. 43
Figure 10: Time schedule of experiments and assignment of leaf levels of potted grapevine plants for
the disease severity assessment.......................................................................................................... 52
Figure 11: Mean percentage of downy mildew disease severity (+/-SE) of ten leaves of grapevine
plants treated with B. bassiana 3 und 7 days before the inoculation with P. viticola.
Experimental replicate of July 2013. .................................................................................................. 57
Experimental replicate of autumn 2013. ........................................................................................... 58
Experimental replicate of July 2014. .................................................................................................. 58
Figure 14: Boxplots of percentage disease incidence of grapevine plants inoculated with P. viticola 3
und 7 days after a treatment with endophytic B. bassiana. .......................................................... 59
Figure 15: Main groups of GO slim classes concerning the biological processes affected in grapevine
plants after treatment with B. bassiana.. .......................................................................................... 62
Figure 16: Graphical abstract of manuscript I .................................................................................................... 94
Figure 17: Assessment of disease severity of grapevine leaves with P. viticola .......................................... 95
VIII
List of Tables
Table 1: Studies reporting natural and artificial establishment of endophytic entomopathogenic B.
bassiana and effects on herbivorous insect or plant pathogens .................................................. 14
Table 2: Amplification of B. bassiana strain GHA or ATCC 74040 specific SSR markers in a subset of
eight obtained mycelia samples from leaf discs of the different treatments 14 and 21 days
after inoculation (DAI) with B. bassiana. ......................................................................................... 29
Table 3: Number of leaf discs assessed (n) and showing B. bassiana outgrowth collected from
Naturalis® treated and control plots of a grapevine field trial in 2014. .................................... 32
Table 4: Mean number (±SE) of grape leafhopper E. vitis larvae in four control and four Naturalis®
treated plots assessed at five observation dates in a grapevine field trial in 2014 ................. 32
Table 5: Primer sequences and PCR characteristics of two grapevine reference genes (Actin,
GADPH) and three defense-related genes used in RT-qPCR experiments. .............................. 55
Table 6: Percentage of colonized leaves per plant 3, 7 and 14 dat with a B. bassiana in three different
experimental replicates (July 2013, autumn 2013 and July 2014). ............................................... 56
Table 7: Mean efficiency according to Abbott against downy mildew on grapevine leaves by a
treatment with B. bassiana 3 und 7 days before the inoculation with P. viticola in three
different experimental replicates (July 2013, autumn 2013 and July 2014). .............................. 60
Table 8: Mean [95% CI] expression levels of three genes 24, 72 and 168 hpt of grapevine with B.
bassiana analyzed with RT-qPCR....................................................................................................... 64
Table 9: Induced and repressed genes in grapevine plants 24 hpt with B. bassiana. ............................. 96
Table 10: Induced and repressed genes in grapevine plants 168 hpt with B. bassiana. ........................... 98
IX
1 General Introduction
1.1 Plant protection in viticulture

Grapevine (Vitis vinifera L.) is an economically important fruit crop, which is mostly
cultivated in the temperate climatic belt (between 40°N and 50°N and between 30°S and 40°S)
with the area dedicated to viticulture exceeding 7.5 million ha worldwide (OIV 2017).
However, most cultivars of V. vinifera commonly used are highly susceptible to a considerable
number of pests and pathogens, which has a significant effect on both yield and quality of the
must and wine (Flaherty 1992). The application of chemical control agents is still the most
effective and predominantly used method to control these pests and pathogens. Accordingly,
viticulture is considered to be very input intense, both in terms of frequency and intensity of
pesticide (herbicides, fungicides, and insecticides) applications during the growing season
(Roßberg 2007). Indeed, a report on the use of plant protection products in the European
Union over the period 1992–2003 (EUROSTAT EC 2007) indicated that on average 71% of all
fungicides applied to crops in the EU were applied to grapevines in European vineyards while
viticulture only accounted for 4.6% of the cultivated area in that period. Since fungal and
oomycete infections are one of the primary reasons for losses in grape quality and yield, most
pesticides applied in viticulture are fungicides, with an average of 12-15, in some years up to
25-30 applications in the most problematic conditions (Pertot et al. 2017). They are
predominantly used to control downy mildew (causal agent: Plasmopara viticola (Berk. and
Curt) Berl. and de Toni), powdery mildew (causal agent: Erysiphe necator Schw., formerly
Uncinula necator (Schw.) Burr.) and grey mould (causal agent: Botrytis cinerea Pers.: Fr.
(teleomorph Botryotinia fuckeliana (de Bary) Whetzel)). However, the inadequate use of
pesticides in viticulture can cause increased concentrations of their residues in vineyard soils,
other environmental compartments and the wine (Romić et al. 2014; Hildebrandt et al. 2008;
Cabras and Angioni 2000), raising public concerns (Jacobson et al. 2005). Furthermore,
especially in organic viticulture, copper is the most widely used fungicide, because of its
natural origin and wide-spectrum activity (Dagostin et al. 2011; Gessler et al. 2011). The long-
term use of copper-containing fungicides in vineyards resulted in their persistence and
accumulation in the soil, with putative detrimental effects on soil microorganisms or
microbial activity (Komárek et al. 2010; Jacobson et al. 2005). Therefore, one of the major goals
of sustainable viticulture is the reduction of pesticide and copper input in vineyards.
Although grapevines are hosts of various arthropod pests, pesticide use against them is
usually low to moderate with one to four insecticide applications on average per year (Pertot
et al. 2017). Damage by insect pests occurs at different parts of the plant such as roots, buds,
1
berries or leaves and is caused either directly due to feeding activities or indirect via the
transmission of pathogens such as bacteria or viruses. Pests that threaten grapevine include
phytophagous mites, leafhoppers, piercing-sucking insects and leaf-eating or cluster- feeding
Lepidoptera.
Current predictions on the possible effects of climate change on disease and pest pressure
suggest that even more pesticide applications will be necessary in the future (Salinari et al.
2006; Caffarra et al. 2012; Reineke and Thiéry 2016). Recent findings suggest that the impact of
some pest insects will increase with increasing temperatures, but the implications of climate
change on plant diseases and arthropod development in global viticulture seem to be more
complex than expected (Caffarra et al. 2012; Reineke and Thiéry 2016; Gregory et al. 2009).
They are either affected directly through impacts on their life history and epidemiology or
indirectly by changes of grapevine physiology and phenology. Even if precise predictions are
not yet possible, imaginable changes include a) an increase of incidence of pests and diseases
in viticulture; b) a shift in species causing problematic situations; c) a change in pests and
diseases biological cycles that will make their control more difficult; d) and increased
difficulty in forecasting due to extreme variation in climatic conditions and, consequently, in
the vine growth and in pests and diseases development.
Although viticulture has been pioneering in terms of the adoption of several alternatives to
synthetic chemical pesticides, it is still regularly depending on multiple applications of
synthetic pesticides for pest and disease management. Therefore, grape growers face
increasing pressure by politicians, retailers, and consumers to reduce their reliance on
conventional chemical pesticides (Jacobson et al. 2005; Komárek et al. 2010). Hence, there is an
increasing interest to identify alternative treatments and more sustainable methods of pest
management (Dagostin et al. 2011; Gessler et al. 2011).
1.2 Biological control of pests and plant diseases
1.2.1 Microbial control agents
The use of microorganisms for biological control of plant pests and diseases is a promising
alternative to the use of chemical pesticides. Microbial biological control agents consist of
bacteria, fungi, or viruses (and sometimes include the metabolites that bacteria or fungi
produce as well) and are used as active substances to control different kinds of crop pests
(Montessinos and Bonaterra 2009). Microbial pesticides are often considered to have a low risk
to the environment and generate little or no toxic residues when compared to chemical
pesticides. They can also have a high level of selectivity as well as lower production costs
compared to conventional pesticides. Due to these positive characteristics, biological control is
2
currently receiving a lot of attention and support by politicians, policy makers, retailers,
consumers, growers, and grower organizations (van Lenteren et al. 2018). With its Sustainable
Use of Pesticides Directive, the European Union (EU) has been recommending the use of
biological control since 2009 (EC 2009). Worldwide, biological control is currently applied on
more than 30 million ha. However, the global market of biological control agents represents
merely less than 2% of the pesticide market (van Lenteren et al. 2018). Although various
studies showed promising results about the use of microbial agents, only rather few
antagonistic microorganisms were registered as biological control agents (Fravel 2005). The
most limiting factor is their inconsistent efficiency, which was particularly observed in studies
done under field conditions (Alabouvette et al. 2006; Fravel 2005; Butt and Copping 2000;
Lacey et al. 2015). Biotic and abiotic factors in the environment can greatly influence and alter
the growth, survival, and pathogenicity of microorganism and thus, bring variability and
uncertainty in their activity and efficiency as biological control agents.
Microbial biological control agents can be characterized by their modes of action or the
mechanisms underlying their protection. Potential modes of action, which might be involved
in the control of plant diseases, range from antibiosis, mycoparasitism, and competition to
induced resistance (Alabouvette et al. 2006; Jaber and Ownley 2018; Pal and McSpadden
Gardener 2006).
Antibiosis is a form of direct interaction resulting from the production of secondary

metabolites by one microorganism, which inhibits other microorganisms. Secondary
metabolites involved in the mechanism are reported to be antibiotics, bioactive volatile
organic compounds (VOCs), or enzymes (Ownley et al. 2010). Antibiosis is a well-described
phenomenon responsible for the activity of a range of biological control agents (Alabouvette
et al. 2006). Mycoparasitism represents another mechanism of direct antagonism. It involves
specific recognition between the antagonist and its target pathogen. Due to the production of
lytic enzymes, which break down cell wall components, the parasite can penetrate the cell
wall and enter the hyphae of the pathogen. Enzymes involved in mycoparasitism of plant
pathogens are distinctly different from those involved in antibiosis. A prerequisite for the
third direct interaction, competition for space and nutrition between an antagonist and a
pathogen, is that both share the same ecological niche while the resources are limited. A more
rapid colonization of plant tissues by the antagonist will reduce the amount of available
nutrients as well as the available space for the pathogen, resulting in reduced spore
germination and reduced growth of the pathogen. Therefore, successful competition is often a
matter of timing.
An indirect mechanism of biological control is the activation of the plant’s inherent defense
system, known as induced resistance, whereby the biocontrol agent and the phytopathogen do
3
not have direct physical contact with one another. Induced resistance can be either local
(hypersensitive response, HR) or systemic throughout the plant and is defined as the process
of active resistance dependent on the host plant’s physical or chemical barriers, activated by
biotic or abiotic agents (Kloepper et al. 1992). It can be activated by microbial pathogens or
insect herbivores, but also by beneficial microbes, abiotic stresses or chemical applications.
Forms of induced resistance that have so far been described are systemic acquired resistance
(SAR), induced systemic resistance (ISR) and herbivore-induced direct defense and indirect
defense (Van Loon et al. 1998; Pieterse and Dicke 2007).
SAR is a form of induced resistance that is activated throughout a plant typically following
infection by a pathogen that causes localized necrotic lesions induced by a pathogenic disease
or as a result of a hypersensitive response (HR) (Ryals et al. 1996). This causes a local
accumulation of salicylic acid (SA) that stimulates a signal to the rest of the plant, and the
plant becomes resistant to pathogens in areas distant from the original infection. Therefore,
SAR often involves the signal molecule SA and is accompanied by the accumulation of genes
encoding pathogenesis-related (PR) proteins and their protein products (Durrant and Dong
2004). These PR proteins include, amongst others, the antifungal chitinases, β-(1,3)-glucanases,
peroxidases, as well as PR-1 and PR-5 proteins that have anti-oomycete activity (Verberne et
al. 2000; Van Loon et al. 2006). The development of SAR takes several days, but it is persistent
for weeks to months and protects the plant against secondary infections by a broad spectrum
of microorganisms including bacteria, true fungi, oomycetes, and viruses.
ISR develops in response to the interaction with certain plant growth promoting rhizobacteria
(PGPR) that do not induce a necrotic response or cause visible damage. The term was coined
by Kloepper et al. (1992) to distinguish resistance induced by PGPR from SAR, which has
different underlying mechanisms. Unlike SAR, ISR does not result in the systemic expression
of PR genes, but its induction is dependent on signaling pathways of the plant hormones
ethylene (ET) and jasmonic acid (JA) (Van Loon et al. 1998). Although SAR and ISR work
through different pathways, they can act antagonistically, complementary, or synergistically
depending on the intensity and duration of the signals provided to the host plant (Mur et al.
2006).
Many microbial biological control agents do not exclusively feature one single mode of action.
Strong evidence suggests that a combination of these mechanisms is involved in biological
control of pests and pathogens (Ownley et al. 2010; Elad 2000; Hubbard et al. 2014).
1.2.2 Entomopathogens
The term “entomopathogenic fungi” (EPF) refers to a polyphyletic group of fungi, which are
natural pathogens of a wide variety of insects and other arthropods (Hegedus and
4
Khachatourians 1995). Since EPF are found ubiquitously in the soil throughout the world and
are effective against a wide variety of insect pests, they have attracted increased attention as
environmentally friendly biological control agents (Hajek 1994). According to Keller (2008),
with approximately 800 fungal taxa, only a fraction of the existing entomopathogenic fungi
are described so far. The best-known among them were assigned to the anamorphic fungi
(Deuteromycota), the Clavicipetales (Ascomycetes), or the Entomophthorales (Zygomycota)
by the long-term valid taxonomic nomenclature (Keller 2008).
The infection of the host by EPF takes place mainly via the cuticle, unlike to most other
insect-pathogenic organisms. Accordingly, for a successful infection, only the contact of the
pathogen with the insect is necessary and therefore, consumption by feeding is not obligatory.
The basic steps of the infection process, illustrated in Figure 1, are summarized in four steps
by Schmutterer und Huber (2005):
- adhesion of the fungal spores and subsequent germination on the insect’s cuticle
- mechanical and enzymatical penetration of the cuticle layers
- interaction with the host's immune system, colonization of the hemolymph and
destruction of the host due to several factors (release of fungal toxins, invasion of
organs, water and nutrients depletion, and physical obstruction)
- saprophytic re-emergence of the fungus from the host with the characteristic
outgrowth of fungal mycelia on the cadaver followed by sporulation
A more detailed version is given by Hegedus and Khachatourians (1995) and a comprehensive
description of the infection steps can be found in Mora et al. (2017). Hence, entomopathogenic
fungi also have a saprophytic phase in addition to the pathogenic one. Both phases are
influenced by different abiotic and biotic factors such as temperature, relative humidity, or
UV-portion of the solar radiation as well as other microorganisms present in the soil or inside
or outside of the host insect (Schmutterer and Huber 2005; Wraight et al. 2007; Vega 2018).
They are also dependent on the pathogenicity and virulence of the fungal isolate. In this
context, the pathogenicity describes the basic ability of the pathogen to infect a host and cause
disease symptoms, whereas the virulence defines the magnitude of this ability. Both
parameters are in turn influenced by the genetic constitution of the fungus as well as the
physiology and the developmental stage of the host.
5
Figure 1: Illustration of how entomopathogenic fungi (on the example of B. bassiana) infect
arthropod hosts by spores, proliferate, and disperse. A conidium adheres to the insect host to induce
germination (step 1). This is followed by germination and production of a germ tube (step 2).
Mechanical pressure and secretion of enzymes are employed to breach the cuticle (step 3). The fungus
colonizes the host hemocoel through hyphal growth or blastospores, where it feeds on sugars in the
hemolymph (step 4). The secretion of toxins facilitates the death of the host. After the host has died,
the fungus breaches the cuticle again from the inside and sporulates on the cadaver (step 5).
A prerequisite for the use of EPF as a biological insecticide (mycoinsecticide) is that highly
virulent strains of the fungus are available through sufficient selection. Also, mass-production
methods and application techniques must have been developed and reviewed for product
registration. Schmutterer and Huber (2005) explained that resistance of host insects to EPF is
improbable to develop due to the multifactorial virulence, but it cannot be excluded with
absolute certainty (Shelton et al. 2007). It is also possible to combine EPF with chemical
insecticides and, in some cases, this can even lead to increased efficacy of entomopathogenic
fungi (Butt and Ansari 2011; Lacey et al. 2015). The combination with chemical fungicides,
however, can lead to a reduction in efficacy. Shah and Pell (2003) explained that
6
entomopathogenic fungi are best used when total elimination of a pest is not required, but
instead, insect populations should stay below an economic threshold.
In recent years, various biological insecticides have been developed, which are mainly based
on various species of the entomopathogenic fungi Beauveria, Metarhizium, Lecanicillium, and
Isaria. Despite the commercial availability of approximately 150 mycoinsecticides (Jaronski
2010) and the recent growth of biological control programs (van Lenteren et al. 2018), their
potential application as biological control of insect pests is, according to Zimmermann (2007),
still not fully exploited. Reasons for this limited use are costs, product quality, efficacy, and
the handling of the products as well as aspects of regulatory restrictions on the environmental
impact such as effects on non-target organisms (Jackson et al. 2010; Jaronski 2010). However,
Roy (2010) emphasizes that limited success is also caused by a lack of some basic
understanding of the ecology and evolution of entomopathogens. Within the terrestrial
ecosystem, EPF have diverse functions, but their different roles have rarely been studied (Vega
et al. 2009). However, studying their ecology is a prerequisite for developing efficient plant
protection strategies basing on them (Vega et al. 2009).
1.3 Beauveria bassiana
1.3.1 Taxonomy and morphology
Beauveria bassiana (Balsamo-Crivelli), Vuillemin (Ascomycota: Hypocreales) is a well-known

and worldwide distributed entomopathogenic fungus, which normally occurs in the soil. The
fungus was first described in 1835 by the Italian scientist Agostino Bassi as a cause of the
"White Muscardine" in silkworms and received different taxonomic names before Vuillemin
(1912) named the genus with Beauveria as an independent genus.
Taxonomically, the fungus was assigned to the Deuteromycota (Fungi imperfecti) due to its
anamorphic developmental cycle. Deuteromycetes classified fungi, which have only asexual
reproduction or whose sexual propagation stages are not known so far. According to Rehner
(2005), this classification is no longer common, and most Deuteromycetes can be classified
between their sexual relatives with the help of new molecular biology methods. B. bassiana is
presently assigned to the Cordycipitaceae family in the order of Hypocreales and therefore
belongs to the Ascomycota. The telomeric form of B. bassiana, Cordyceps bassiana, has only
been discovered and proved in Asia (Li et al. 2001).
The Ascomycetes are dominated by asexual propagation, which is mainly responsible for the
rapid spreading of the fungus (Raven et al. 2006). The dissemination structures used are
mononuclear conidiospores, called conidia, which are usually formed by special conidiogenic
cells (Figure 2). These usually sit at the tip of specialized hyphae, the conidiophores. The dense
7
clusters of short-globose to flask-shaped conidiogenic cells result in the characteristic white,

woolly colonies of B. bassiana (Figure 3). The hyaline (colorless) conidia themselves are single
cellular, haploid, and hydrophobic and sit on a zigzag-shaped rhachis (Rehner 2005). Although
the conidiophores tend to become finer and less dense after longer periods of artificial culture,
the fungus is morphologically relatively easy to determine. Rehner (2005) explains that a
morphological differentiation of the individual species within the genus Beauveria, however,
is only possible based on the exact shape and size of the conidia. Since these characteristics
vary according to the culture conditions, a routine determination of the species is problematic
and questionable. Therefore, molecular methods to identify the species are essential. With
microsatellite markers, even a strain-specific determination (Rehner and Buckley 2003) and
detection (Reineke et al. 2014) of B. bassiana in different habitats are possible.
Figure 2: Left: Conidiophores with conidiogenic cells of B. bassiana, Right: Picture of the conidio-
spores of B. bassiana by electron microscopy (x 4000) (pictures taken from Domsch et al. (1980))
Figure 3: Characteristic white, woolly colonies of B. bassiana on a solid culture medium (photo
courtesy of Winfried Schönbach)
8
1.3.2 Beauveria bassiana as an entomopathogen
Beauveria bassiana has a broad host range and infects Coleoptera, Lepidoptera, Diptera, and
other insect orders in both temperate and tropical climates (Domsch et al. 1980). As an
entomopathogenic fungus, it is capable of entering the insect directly through the cuticle
instead of depending on being eaten by the insect or on the opportunity to enter through a
natural opening like other entomopathogens (e.g., nematodes or viruses). The spores can be
transported by wind or just be picked up by the insect as it moves through its environment.
B. bassiana has a dimorphic mode of growth and passes through an asexual vegetative life
cycle in the absence of an insect host. On contact with a susceptible host, Beauveria switches
to the pathogenic life cycle. After germination the hyphal tube uses mechanical pressure by
specialized physical structures, such as appressoria, to penetrate the insect host cuticle
directly. Toxic metabolites are secreted by the fungus, which may assist in the infection
process. When having entered the hemocoel, the fungus alters its growth morphology to a
yeast-like phase with the production of blastospores and/or hyphal bodies, circulating in the
hemolymph and multiplying by budding. The death of the host is caused by the proliferation
of the fungus and due to dehydration or depletion of nutrients (Ladurner et al. 2008). During
the infection process, toxic secondary metabolites can be secreted by the fungus, that assist in
parasitism of insects but are not required (Griffin 2007). Following the death of the host, B.
bassiana re-emerges from the cadaver and produces new conidia in the form of the
characteristic white covering called “white muscardine” (Figure 4).
Figure 4: Left: B. bassiana re-emerges from the cadaver and produces the characteristic white
covering called “white muscardine”. Right: Endophytic B. bassiana re-emerges from leaves discs of
grapevine plants (photos courtesy of Winfried Schönbach)
1.3.3 Plant colonization by Beauveria bassiana
Besides being important natural enemies of many insects, recent studies reported various
additional roles of EPF in nature, including endophytism and rhizosphere colonization as well
as plant disease antagonism or plant growth promotion (Vega et al. 2009). The importance and
9
complexity of these ecological roles are not yet fully understood but can provide opportunities
for the use as alternative pest management strategies (Jaber and Enkerli 2017). In the present
thesis, the role as endophyte will be subject of closer examination.
The term endophyte was first introduced by the German scientist Anton de Bary (1884). Many
other definitions have been used ever since, changing in accordance with the increased
understanding of endophytic lifestyle (Wilson 1995). While initially, the term refers to all
microorganisms living inside plants, it was subsequently restricted to organisms living
asymptomatic within the plant by Carroll (1986). As further studies revealed that the same
organism could switch between different lifestyles, the definition was expanded to the
following and still most commonly used one:
“[Endophytes] include all organisms inhabiting plant organs that at some time in their life, can
colonize internal plant tissues without causing apparent harm to their host.”
(Petrini 1991)
So defined, endophytes cover a diverse polyphyletic group of microorganisms that can exhibit
more than one type of life stages, including true symbionts as well as latent pathogens
(Arnold and Lewis 2005).
Fungal endophytes are ubiquitous amongst terrestrial and agricultural plants and are reported
to protect host plants against pathogens, plant parasitic nematodes or herbivores (Arnold and
Lewis 2005; Vidal and Jaber 2015; West et al. 1988; Schulz and Boyle 2005). Vega et al. (2008)
provided an overview of the diversity of fungi traditionally known as insect pathogens, which
have been isolated as endophytes. Both, naturally occurring and artificially introduced
entomopathogenic fungi are mentioned in the literature, including publications on endophytic
colonization by Beauveria bassiana.
The first observation of endophytically growing B. bassiana was made by Lewis and
Cossentine (1986) and Bing and Lewis (1992a) in corn plants Zea mays L. (Poaceae). These
authors not only proved the endophytic colonization of corn plant tissues by this fungus but
also found antagonistic potential against the European corn borer (Ostrinia nublialis Hbn.)
(Lepidoptera: Pyralidae). Besides higher mortality when feeding on the plants endophytically
colonized by this fungus (Bing and Lewis 1993), a season-long suppression of larvae –
measured as reduced tunneling by the corn borer – was reported (Lewis and Cossentine 1986).
Subsequent work by Lewis and colleagues examined the in planta growth of the fungus and is
reviewed in Arnold and Lewis (2005). Movement of B. bassiana has been detected, but the
10
mechanism of movement is poorly understood. Wagner and Lewis (2000) observed in their
studies on maize plants using light and electron microscopy that B. bassiana can colonize the
plant systemically. In addition to the observation of hyphal growth between the parenchyma
cells in the apoplast, hyphae could also be detected in the xylem vessels of the plants, where
they may move passively. Quesada-Moraga et al. (2006) confirmed these observations, as they,
too, could not detect any intracellular colonization by B. bassiana on opium poppy, but the
fungus did indeed spread with hyphae in the xylem.
In addition to corn plants, a wide variety of plants have also been shown to host B. bassiana as
an endophyte. Table 1 summarizes the literature about colonized plant species, the reported
materials including used strain, inoculated and analyzed tissue type as well as (if available)
investigations concerning the antagonistic potential against pests and pathogens. In most of
the studies published so far on endophytic B. bassiana or other entomopathogenic fungi,
mycosis of insects has either not been tested or was not observed (see Table 3 in Vidal and
Jaber (2015)). The traditional mode of infection by fungal entomopathogens takes place via
direct contact with the cuticula. Therefore, Arnold and Lewis (2005) do not regard the hyphal
state of endophytes in planta and the consumption of infected plant tissues as a significant
source of entomopathogenic infections. In addition, the conidia of B. bassiana, which are
usually the infective propagule, have not yet been observed inside plant tissues or the vascular
system (Vega 2008). However, some investigations suggest that infections of chewing or
sucking pest insects by endophytic B. bassiana can occur (Gurulingappa et al. 2010; Quesada-
Moraga et al. 2009; Reddy et al. 2009). The described lack of mycosis and the lack of conidia
inside the plants suggest other modes of action against insects than direct fungal infections. It
is speculated that the protective effects are mediated by secondary metabolites, produced by
the fungus and causing feeding deterrence or antibiosis (Cherry et al. 2004; Akello et al. 2008b;
Vega 2008; Gurulingappa et al. 2010). Despite accumulating evidence on the potential of
endophytic B. bassiana, the mechanisms underpinning the protective effects remain little
understood. The colonization with B. bassiana showed to induce proteins related to plant
defense and stress response (Gómez-Vidal et al. 2009) suggesting that endophytic colonization
by entomopathogenic fungi induces plant defense responses, probably by activating the plant
immune system.
In addition to its biocontrol activity against insect pests, there is substantial evidence that
endophytic B. bassiana may also demonstrate antagonistic activity against plant pathogens
and therefore effectively suppresses plant diseases (Goettel et al. 2008; Ownley et al. 2008).
Research on the control for plant pathogens by B. bassiana as has been mostly limited to in
vitro studies with an array of soilborne and foliar plant pathogens and were summarized in
Table 1 in Ownley et al. (2010). Only a few studies investigated the antagonistic potential by
11
using soil-borne pathogens and seed treatments in greenhouse trials. Seed treatment with B.
bassiana strain 11–98 resulted in suppression of damping-off caused by the soil-borne
pathogens Rhizoctonia solani Kuhn (Basidiomycota: Cantharellales) and Pythium myriotylum
Drechsler (Oomycota: Pythiales) in tomato (Ownley et al. 2004; Clark et al. 2006) and cotton
seedlings (Griffin 2007; Ownley et al. 2008). The treatment of cotton seedlings with the same
B. bassiana strain has also been reported to reduce the severity of bacterial blight caused by
Xanthomonas axonopodis pv. malvacearum (Xam) (Griffin et al. 2006; Ownley et al. 2008).
Following foliar inoculation of plants with B. bassiana, a reduced incidence and severity of
Zucchini yellow mosaic virus in squash (Jaber and Salem 2014) and downy mildew in
grapevines (Jaber 2015) was recently reported. These findings provide promising potential for
the multiple uses of fungal entomopathogens as biopesticides against both insects and pests in
integrated plant protection strategies when used as endophytes. The precise mechanisms
underlying such protection mediated by (endophytic) B. bassiana remain at an early stage. It is
assumed that the mechanisms of plant disease antagonism involve competition for space,
induced systemic resistance, and the production of various secondary metabolites (Ownley et
al. 2008; Griffin et al. 2006; Ownley et al. 2010; Vega et al. 2009).
1.3.4 The product Naturalis®
Beauveria bassiana is one of the most frequently commercialized fungal mycoinsecticides

(Faria and Wraight 2007). In the present thesis, B. bassiana strain ATCC 74040 was also used
®
in the form of the commercial product Naturalis (CBC (Europe) S.r.l. – BIOGARD Division).
® 7
Naturalis is formulated as oil dispersion (OD) and contains approximately 2.3 x 10 colony
forming units/ml of B. bassiana strain ATCC 74040 as active ingredient. The strain ATCC
74040 has been isolated from the cotton boll weevil Anthonomus grandis (Boheman), in the
Lower Rio Grande Valley, Texas, USA, and does not produce any toxins (Copping 2004). In
2005, Intrachem Bio International S.A. (Geneva, Switzerland), now CBC Europe S.r.l. (Italien),
®
acquired production and marketing rights for Naturalis from Troy Biosciences Inc. (Phoenix,
USA). Manufacturing takes now place under the control of CBC Europe.
st
Initially, in Germany, the product was only allowed to be used from the 1 of April 2008 for a
period of 120 days in order to combat wireworms on potatoes (including Limonius spp. and
Agriotes spp.) as part of a temporary approval according to former § 11 (2) now Art. 53
PflSchG ("Gefahr im Verzug"). Since January 2009, the strains ATCC 74040 and GHA of the
fungus B. bassiana have been listed in Annex 2 (positive list for active substances) of EC
Regulation No. 1107/2009 (formerly Annex I of EU Directive 91/414) (European Commission
2011). On this basis, national approvals already existed in several EU member states, including
Spain, France, Italy, Sweden, and the Netherlands. Furthermore, products containing the same
strain are also registered in the USA. Since 2015 the product is registered in Germany, and
12
other EU member states, e.g. for control of whiteflies in tomato, herbs, and ornamentals in
greenhouses.
The identification of an appropriate fungal pathogen or strain for development as

mycoinsecticide can be complex and expensive till market launch (Jackson et al. 2010).
Therefore the present thesis focused on the already registered and formulated strain ATCC
®
74040 and the product Naturalis in order to speed up the implementation in and the
development of alternative plant protection strategies in viticulture.
13
14
Table 1: Studies reporting natural and artificial establishment of endophytic entomopathogenic B. bassiana and effects on herbivorous insect or plant pathogens
Reference Plant species Strain Plants parts treated – sampled Insect/pathogen

observed
Akello et al. (2007), (2009) Banana Musa spp. G41 Roots, rhizomes –pseudostems
Akello et al. (2008a), (2008b) Banana Musa spp. G41 Roots, rhizomes –pseudostems Cosmopolites sordidus
Akello and Sikora (2012) Bean Vicia faba G1LU3; S4SU1 Seeds – roots Acyrthosiphon pisum,
Aphis fabae
Akutse et al. (2014) Bean V. faba G1LU3, S4SU1 and Seeds – leaves, stems, roots Phaedrotoma cabriventris,
ICIPE 279, Diglyphus isaea
Akutse et al. (2013) Bean V. faba and Phaseolus Three different isolates Seeds – leaves, stems, roots Liriomyza huidobrensis
vulgaris
Amin et al. (2014) Cocoa Theobroma cacoa Pods – pods Conopomorpha cramerella
Barta (2018) Horse-chestnut trees (Aesculus AM_EF0111, AM_EP0715 Leaves – leaves Cameraria ohridella
hippocastanum L.)
Behie et al. (2015) Bean P. vulgaris ARSEF 252 Roots – stems, leaves, hypocotyl,
roots
Bing and Lewis (1991), (1992a), Corn Zea mays ARSEF 3113 Leaves – stems, whorl stage Ostrinia nubilalis
(1992b), (1993) parts, pith
Lewis and Bing 1991, Lewis et
al. (1996, 2001, 2002)
Bills and Polishook (1991) Ironwood Carpinus caroliniana Isolation from natural habitat –
Bark
Biswas et al. (2012) Jute Corchorus olitorius ITTC 4796 Seeds – roots, leaf, stem, capsule
Biswas et al. (2013) White jute Corchorus capsularis Nine different strains Seeds, roots – leaves and Apion corchori
nonspecified segments
Brownbridge et al. (2012) Pine Pinus radiata F647 (Genbank GU237004), Seeds, roots – roots, needles
F668 (Genbank GU237005)
Canassa et al. (2019) Bean P. vulgaris ESALQ 3375 Seeds – leaves, stems, roots Tetranychus urticae,
Phytoseiulus persimilis
Castillo Lopez et al. (2014) Cotton Gossypium sp. Strain from Botanigard® Seeds – leaves, stems, roots Aphis gossypii
Table 1: continued
Castillo Lopez and Sword (2015) Cotton Gossypium sp. Strain from Botanigard® Seeds Helicoverpa zea
Cherry et al. (1999) Corn Z. mays Five different isolates Injection – stems Sesamia calamistis
Cherry et al. (2004) Corn Z. mays Six different isolates Seeds, leaves, stems – stems Sesamia calamistis
Clifton et al. (2018) Soybean Glycine max L. GHA Seeds – stems, leaves Aphis glycines
Dara et al. (2013) Strawberry Fragaria X Roots – roots, foliage
ananassa
Dash et al. (2018) Bean P. vulgaris B12, B13, B16 Seeds – root, stem, leaves Tetranychus urticae
El-Deeb et al. (2012) Tomato Solanum lycopersicum Injection – non specified Tomato leaf curl virus,
Bemisia tabaci
Evans et al. (2003) Cocoa Theobroma gileri
Ganley and Newcombe (2006) Western white pine Pinus Natural habitant – Seeds, needles
monticola
Gomez-Vidal et al. (2006), Date palm Phoenix dactylifera Petioles – petioles
(2009)
Greenfield et al. (2016) Cassava Manihot esculenta Five isolates Soil – stems, leaves, roots
Guesmi-Jouini et al. (2014) Artichoke Cynara scolymus L. Leaves – leaves
Gurulingappa et al. (2010), Cotton Gossypium sp., GenBank AN GU953211, AN Leaves – leaves Aphis gossypii; Chortoicetes
(2011) wheat Triticum GU953212 terminifera
aestivum, bean P. vulgaris,
corn Z. mays, tomato
Lycopersicum esculentum,
pumpkin Cucurbita maxima
Jaber (2015) Grapevine Vitis vinifera ATP01, ATP05, EABb04/01-Tip Leaves – leaves Plasmopara viticola
and ATCC 74040
Jaber und Araj (2018) Sweet pepper Capsicum annum ATCC 74040 Soil – roots, stems, leaves Myzus persicae, Aphidius
colemani
Jaber et al. (2018) Brassica oleracea ATCC 74040 Leaves - leaves, stems, roots Bremisia tabaci
Jaber (2018) Wheat Triticum aestivum L. ATCC 74040 Seeds – shoots, roots Fusarium culmorum
Jaber and Alananbeh (2018) Sweet pepper C. annuum L. ATCC 74040 Roots – roots, stems, leaves Fusarium spp.
Jaber and Enkerli (2016) Broad bean V. faba ATCC 74040 Seed – root, leaf, and stem
Jaber and Salem (2014) Squash Cucurbita pepo ATCC 74040 Leaves – leaves Zucchini yellow mosaic
virus
15
16
Table 1: continued
Jia et al. (2013) Rice Oryza sp. Bb0062 (Bb-4 and Bb-7) Leaves – leaves, stems, roots,
seeds
Klieber and Reineke (2016) Tomato S. lycopersicon ATTC 74040 Leaves – leaves Tuta absoluta
Landa et al. (2013) Opium poppy Papaver EABb 04/01-Tip Leaves – leaves
somniferum
Lewis and Bing (1991), Lewis et Corn Zea mays ARSEF 3113 Leaves – stems, whorl stage Ostrinia nubilalis
al. (1996), (2001) parts, pith
Mantzoukas et al. (2015) Sweet sorghum Sorghum IGE3 Leaves – leaves, stems Sesamia nonagrioides
bicolor
McKinnon et al. (2018) Corn Z. mays BG11, FRh2 and J18) Roots – roots
Moloinyane and Nchu (2019) Grapevine V. vinifera SM3 Roots – leaves Planococcus ficus
Ownley et al. (2008) Tomato S. lycopersicon; cotton 11-98 Seeds – seedlings Rhizoctonia solani,
Gossypium sp. Pythium myriotylum
Parsa et al. (2013) Bean P. vulgaris GHA Leaves, soil – leaves, stems, roots
Parsa et al. (2018) Bean P. vulgaris 11 isolates Seeds – leaves, stems, roots
Pelizza et al. (2017) Corn Z. mays LPSc 1067 (accession number Leaves – leaves Dichroplus maculipennis
KF500409)
Posada et al. (2007) Coffee Coffea arabica ARSEF 5486, ARSEF 2687, Leaves, stems, roots – leaves,
ARSEF 1480 stems, roots
Posada and Vega (2005) Cocoa T. cacoa IC-5486, CS16-1 Topical – leaves, stems, roots
Posada and Vega (2006) Coffee C. arabica IC-5486, CS16-1 Roots – leaves, stems, roots
Posada et al. (2010) Cocoa T. cacao Bb04005 Flowers – pods
Powell et al. (2009) Tomato S. lycopersicum 11 - 98 Seeds – leaves, stems, roots
Qayyum et al. (2015) Tomato S. lycopersicum WG-40, WG-14, WG-1 Roots, stems, leaves – leaves Helicoverpa armigera
Quesada-Moraga et al. (2006) Opium poppy P. somniferum EABb 04/01-Tip Leaves – leaves
Quesada-Moraga et al. (2009) Opium poppy P. somniferum EABb 04/01-Tip Seeds, soil, leaves – leaves Iraella luteipes
Quesada-Moraga et al. (2014) Opium poppy P. somniferum EABb 04/01-Tip Seeds – leaves
Razinger et al. (2014) Cauliflower Brassica oleracea Isolate 1174, ATCC 74040 Soil – roots, stems
Reay et al. (2010) New Zealand pine P. radiata Isolation from natural habitat
Reddy et al. (2009) Sorghum Sorghum bicolor ITCC 4688 Leaves – stems Chilo partellus
Renuka et al. (2016) Corn Z. mays NBAII-Bb-5a, 7, 14, 19, 23, 45 Leaves – leaves, stems
Table 1: continued
Resquín-Romero et al. (2016) Alfalfa Medicago sativa, tomato EABb 01/33-Su Leaves – roots, stems, leaves Spodoptera littoralis
L. esculentum, melon
Cucumis melo
Rondot and Reineke (2017) Grapevine V. vinifera ATTC 74040 Leaves Otiorhynchus sulcatus
Rondot and Reineke (2018) Grapevine V. vinifera ATCC 74040, GHA Leaves – leaves Planococcus ficus,
Empoasca vitis
Russo et al. (2015) Tobacco Nicotiana tabacum; LPSC 1067 Seeds, roots, leaves – leaves
corn Z. mays, wheat T.
aestivum; soybean Glycine
max
Russo et al. (2019) Corn Z. mays, LPSc 1098 (GenBank KT16325) Leaves – Leaves, seeds Rachiplusia nu
Sánchez-Rodríguez et al (2018) Bread wheat Triticum aestivum, EABb 04/01-Tip Soil, seed, leaf - grains Spodoptera littoralis
durum wheat T. durum
Tefera and Vidal (2009) Sorghum S. bicolor Bb-04 Roots – leaves, stems, roots
Vidal and Jaber (2015) Bean V. faba; Oilseed rape Eight different isolates Leaves – leaves Helicoverpa armigera
Brassica napus
Wagner and Lewis (2000) Corn Z. mays ARSEF 3113 Leaves – leaves
17
2 Objectives
Fungal entomopathogens are important antagonists of arthropod pests and have attracted
attention as biocontrol agents. In addition to colonizing arthropods, evidence has accumulated
that some entomopathogenic fungi like Beauveria bassiana can endophytically colonize a wide
array of plant species. However, only limited information is currently available on the
endophytic colonization of grapevine plants with B. bassiana. In addition, the functional role
of the fungus in planta, and/or the plant response to colonization by B. bassiana and the
mechanisms underlying these responses, still require elucidation. The objectives of the present
study were to investigate whether the fungus B. bassiana is able to colonize grapevine plants,
still maintains its entomopathogenic potential against insect pests, and has additional
antagonistic potential against other fungal pathogens. The investigation focused on the
interaction between B. bassiana, grapevine plants, and potential target insect pests as well as
fungal pathogens to gain more knowledge of this particular tritrophic interaction with regard
to potential biological control strategies in viticulture.
The specific objectives were:
• to optimize the endophytic establishment of B. bassiana in grapevine plants via

artificial application in the greenhouse and under field conditions (chapter 3)
• to characterize the entomopathogenic potential of endophytic B. bassiana against

selected insects pests attacking grapevine (chapter 3 and 4)
• to evaluate the antagonistic potential of endophytic B. bassiana against a key-fungal

pathogen in viticulture, the causal agent of downy mildew (chapter 5)
• to study the effects of the endophytic establishment of B. bassiana in grapevine plants

on gene level (chapter 5)
This thesis is based on three (peer-reviewed) manuscripts included in the following chapters
as follows:
I. Yvonne Rondot and Annette Reineke (2018): Endophytic Beauveria bassiana

in grapevine Vitis vinifera (L.) reduces infestation with piercing-sucking
insects. Biological Control, 116, 82-89
II. Yvonne Rondot and Annette Reineke (2017): Endophytic Beauveria bassiana
in grapevine plants influences host plant selection of adult black vine weevils,
Otiorhynchus sulcatus. Biocontrol Science and Technology, 27(7), 811-820
18
2 Objectives
III. Yvonne Rondot and Annette Reineke: Endophytic Beauveria bassiana

activates expression of defence genes in grapevine and prevents infections by
grapevine downy mildew Plasmopara viticola. Chapter 5 under revision
The contribution of Yvonne Rondot to the manuscripts listed above was as follows:
I. Planned the experiments together with the co-author. Performed most of the
experimental work in the greenhouse and the laboratory as well as some of the
experimental work in the field. Supervised a student in performing some parts of the
experimental work in the laboratory. Evaluated and statistically analyzed all data.
Prepared all figures and tables. Wrote the manuscript together with the co-author.
II. Planned the experiments together with the co-author. Performed most of the
experimental work and supervised a student in performing some parts of the
experimental work. Evaluated and statistically analyzed all data. Prepared all result
figures and tables. Wrote the manuscript together with the co-author.
III. Planned the experiments together with the co-author. Performed all of the
experimental work in the greenhouse as well as parts of the experimental work in the
laboratory. Evaluated and statistically analyzed the data. Prepared all figures and
tables. Wrote the manuscript together with the co-author.
19
3 Endophytic Beauveria bassiana in grapevine Vitis
vinifera (L.) reduces infestation with piercing-sucking
insects
Yvonne Rondot and Annette Reineke
Geisenheim University, Department of Crop Protection, Von-Lade-Str. 1, D-65366 Geisenheim,

Germany, e-mail: yvonne.rondot@hs-gm.de, fax: 0049-(0)6722-502 410
Type of authorship: First author
Type of article: Research article
Contribution to the article: Planned and performed most of the experiments

Evaluated and statistically analyzed all data
Prepared all figures and tables
Wrote the manuscript
Contribution of other authors: Annette Reineke contributed to experimental design and

writing the paper
Journal: Biological Control, 116, 82-89
Impact Factor: 2.112
Date of manuscript submission: 27. April 2016
DOI: https://doi.org/10.1016/j.biocontrol.2016.10.006
This publication included a graphical abstract (see Figure 16, supplementary material)
20
3 Manuscript I
Highlights
• Successful endophytic establishment of the entomopathogen B. bassiana in grapevine
plants.
• In potted plants, endophytic survival of B. bassiana was evident for at least 21 days
after inoculation.
• Endophytic B. bassiana reduces infestation rate and growth of vine mealybugs.
• In the vineyard, B. bassiana was detected as an endophyte up to five weeks after last
application.
• B. bassiana reduces infestation with grape leafhopper in the vineyard.
Abstract
Fungal entomopathogens are important antagonists of arthropod pests and have attracted
increased attention as biocontrol agents. In addition to colonizing arthropods, evidence has
accumulated that some entomopathogenic fungi like Beauveria bassiana can endophytically
colonize a wide array of plant species. However, only limited information is currently
available on the endophytic colonization of grapevines with B. bassiana and whether the
fungus still maintains its antagonistic potential against insect pests.
Greenhouse and field experiments were conducted to optimize endophytic establishment of

the entomopathogenic fungus B. bassiana in potted and mature grapevine plants. We used two
different commercialized B. bassiana strains and applied them as conidial suspensions or as
®
the formulated product Naturalis on grapevine leaves. The antagonistic activity of
endophytic B. bassiana against putative target pest insects like the vine mealybug Planococcus
ficus was assessed in a bioassay using surface sterilized leaves. Endophytic survival of B.
bassiana inside leaf tissues of potted plants was evident for at least 21 days after inoculation,
irrespective of the inoculum used. Endophytic B. bassiana reduces infestation rate and growth
of P. ficus. In the vineyard, B. bassiana was detected as an endophyte in mature grapevine
plants up to five weeks after last application with a significant impact on infestation with
grape leafhopper, Empoasca vitis.
Keywords:
Beauveria bassiana; endophyte; entomopathogenic fungi; Vitis vinifera; Planococcus ficus;
interactions
21
3.1 Introduction
3.1 Introduction
The entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) is
a well-known microbial antagonist of a diverse range of arthropod species. Hence, this species
has attracted increased attention as a potential microbial biocontrol agent for integrated pest
management of arthropod pests with a couple of B. bassiana based commercial products being
available on the market (Zimmermann 2007; Jackson et al. 2010). Generally, for control of
target species, preparations of blastospores or aerial conidia formulated in oil or other
adjuvants are sprayed onto the plant’s phylloplane. Besides its entomopathogenic habit of life
style, this fungus has also been shown to be able to thrive saprophytically in the soil, to
colonize the rhizosphere of plants, to have antagonistic activities against plant pathogens, as
well as to grow endophytically inside plants (Vega et al. 2009). As far as the latter is
concerned, a few studies have shown that B. bassiana is occurring as part of the natural
endophytic community of certain plant species (Ormond et al. 2010; Reay et al. 2010; Vega et
al. 2008). Moreover, endophytic establishment of B. bassiana has been achieved via an artificial
application of this fungus on the plant’s tissue following a subsequent colonization of the
entire host plant. Using such an approach, successful endophytic establishment of B. bassiana
has been proved for a variety of crop plant species including cocoa (Posada and Vega 2005)
and pine seedlings (Brownbridge et al. 2012), corn (Wagner and Lewis 2000), coffee (Posada et
al. 2007), sorghum (Reddy et al. 2009; Tefera and Vidal 2009), tomato(Klieber and Reineke
2016), banana (Akello et al. 2009), and jute (Biswas et al. 2012; Biswas et al. 2013). So far, no
negative effects of the presence of endophytic B. bassiana on performance of the colonized
host plant were evident in a range of studies (Akello et al. 2009; Tefera and Vidal 2009;
Wagner and Lewis 2000; Klieber and Reineke 2016). Endophytic B. bassiana has been reported
to provide systemic protection against several insect pests or to inhibit insect development
and establishment (Quesada-Moraga et al. 2009; Reddy et al. 2009; Gurulingappa et al. 2010;
Biswas et al. 2013). At the same time, presence of endophytic B. bassiana has been shown to
reduce disease symptoms caused by a variety of fungal pathogens (Griffin et al. 2005; Ownley
et al. 2010; Ownley et al. 2008; Jaber 2015) Therefore, defining means of ensuring an
endophytic establishment of B. bassiana strains in target crop plants is currently receiving
increased attention, as this would represent a dual biocontrol strategy both against insect
pests and plant pathogens.
Grapevine (Vitis vinifera L.) is an important global commodity crop which is planted
throughout temperate regions worldwide. A substantial number of different insect pests and
pathogens are associated with grapevine and are significant factors influencing both the
quantity of the yield as well as the quality of must and wine (Flaherty 1992). Accordingly,
grapevine cultivation is regarded as being quite input intensive, in particular regarding the
22
3 Manuscript I
frequency and intensity of fungicide and insecticide applications throughout the year
(Roßberg 2007). Insects with a piercing-sucking mode of feeding are frequently attacking
grapevines and cause damage either by extracting sap fluids or feeding in mesophyll cells or
by transmitting grapevine pathogens. The grape leafhopper Empoasca vitis (Goethe)
(Homoptera: Cicadellidae, Typhlocybinae) feeds on mesophyll cells or on phloem sap and is
recognized as a major insect pest in many European grapevine growing areas (Olivier et al.
2012). Moreover, the vine mealybug Planococcus ficus (Signoret) (Homoptera: Pseudococcidae)
is regarded as a key pest in many countries around the world with grapevine cultivation
(Daane et al. 2012). Planoccocus ficus causes direct damage to grapevine due to phloem-feeding
on leaves and fruit and excretion of honeydew. Additionally, P. ficus acts as a vector for
grapevine leafroll associated virus (GLRaV), one of the most economically destructive
grapevine viruses that occur in all the major grape-growing regions of the world (Almeida et
al. 2013). Accordingly, a combination of methods including insecticide applications, biological
control via antagonists or mating disruption is usually applied by growers to control P. ficus
(Almeida et al. 2013). The system grapevine (as an input intensive crop) - P. ficus and E. vitis
(as phloem-feeding pest insects) - B. bassiana (as a commercially available biopesticide) is thus
ideal for studying tritrophic interactions between plants, insects and entomopathogenic
endophytic fungi. Endophytic establishment of an entomopathogenic fungus like B. bassiana
still having antagonistic activity against insect pests and fungal pathogens would therefore
represent a novel and sustainable plant protection strategy in viticulture, with the potential to
reduce frequency of pesticide applications.
Here we demonstrate for the first time, that endophytic establishment of commercially
available B. bassiana strains in grapevine displays antagonistic activity against insects with a
piercing-sucking mode of feeding. Moreover, we proved that an endophytic colonization of B.
bassiana is possible, both in greenhouse potted grapevine plants as well as in mature and
lignified plants grown in the field.
3.2 Materials and methods
3.2.1 Fungal material
Beauveria bassiana strains ATCC 74040 and GHA were isolated from the commercial products
® ®
Naturalis (CBC (Europe) S.r.l., Italy) and Botanigard 22WP (BioWorks, Inc., USA),
® 7
respectively. Naturalis is formulated as an oily fluid and contains approximately 2.3 x 10
colony forming units/ml of B. bassiana strain ATCC 74040 as active ingredient. The isolates
were maintained on a Beauveria medium at 24 °C in the dark. The medium consisted of 10 g
soy peptone (AppliChem, Germany), 20 g glucose (Sigma-Aldrich, Germany) and 18 g
23
Bacto™Agar (BD Difco, USA) dissolved in 1000 ml ultrapure water and was subsequently
autoclaved for 20 min at 120 °C.
To obtain spore suspensions, conidia were harvested by gently scraping the surface of Petri
dishes containing 8-day-old B. bassiana cultures and suspending them in 20 ml sterile 1/8
concentrated Ringer’s solution containing 0.02% Tween 80. The conidia concentration was
7
determined using a Thoma haemocytometer and adjusted to 2 × 10 conidia/ml for strain GHA
7 7
and to 1 × 10 and 2 × 10 conidia/ml for strain ATCC 74040. Both, the freshly collected
®
conidia suspensions and the formulated product Naturalis (at concentrations of 3% and 5%),
were used in the experiments. Aliquots of 50 µl of spore suspensions were plated on Beauveria
medium using the Spiralplater WASP 2 (Meintrup DWS Laborgeräte GmbH, Germany).
Concentrations of viable conidial spores were calculated using the colony forming unit’s
®
method. Germination rate was 100% for conidial spores present in Naturalis and around 70%
for the spore suspensions of isolates ATCC 74040 and GHA. Accordingly, concentrations of
7 6
viable conidia applied onto plants were 1.4 × 10 conidia/ml for strain GHA and 7 × 10 (conc.
7
1) and 1.4 × 10 (conc. 2) conidia/ml for strain ATCC 74040.
3.2.2 Endophytic establishment in potted grapevine plants
Grapevine plants, Vitis vinifera (L.) cv. 'Riesling', were obtained from hardwood cuttings. After
root development, the plants were potted and grown in a greenhouse chamber at 22-25 °C.
Seven-week-old grapevine plants with 4-7 fully expanded leaves were used for inoculation
®
with either B. bassiana conidial suspensions or the commercial product Naturalis (3% and
5%). For each treatment, 10 replicate plants were inoculated by spraying the adaxial and the
abaxial surfaces of all fully expanded leaves until run-off using a 1 l one-hand pressure
sprayer. The control plants were sprayed with sterile 1/8 concentrated Ringer’s solution
containing 0.02% Tween 80. Position of the last fully expanded leaf used for inoculation was
labeled using a tapener (Max tapener HT-B, Max Staple, Japan). Inoculated and non-inoculated
plants were kept in a greenhouse chamber for three weeks (daily mean temperature 23-25° C,
daily mean relative humidity 50-70%) and were watered regularly.
3.2.3 Re-isolation of B. bassiana
Endophytic colonization of plants by B. bassiana was assessed 7, 14, and 21 days after
inoculation (DAI) by re-isolation following surface sterilization. No newly developed leaves
were included in the present study. At each sampling period one leaf from each of the 10
replicate plants was excised and transported to the laboratory on ice. The leaves were
individually surface sterilized under sterile conditions by dipping them in 0.5% NaOCl (active
chlorine) containing 0.05% Tween 80 for 2 min, followed by 70% EtOH for 2 min and rinsed
twice with sterile distilled water according to Akello et al. (2009). The success of this
24
3 Manuscript I
disinfection process was assessed by plating three replicates of 200 µl of the residual rinse
water on PDA (potato dextrose agar). No fungal growth was recorded in any of the rinse
water samples after 21 days of incubation. After surface sterilization, six leaf discs (d = 1.2 cm)
were obtained with a sterile cork borer from each leaf. Leaf discs were placed on Beauveria
selective medium (BSM), the same medium as indicated above (2.1) but supplemented with 0.1
g/l streptomycin (Sigma-Aldrich, Germany), 0.05 g/l tetracycline (Sigma-Aldrich, Germany),
®
0.1 g/l dodine (as aliquot of the product Syllit , Spiess-Urania Chemicals, Germany) and 0.05
g/l cyclohexamide (Sigma-Aldrich, Germany). This medium is based on a medium initially
described by Strasser et al. (1996) for the isolation of Beauveria brongniartii and adapted by
Meyling and Eilenberg (2006) for isolation of B. bassiana. Plates were incubated at room
-1 -1
temperature with a 12:12 h light:dark photoperiod (mean light intensity of 11.2 µmol m s ).
After 7 and 14 days leaf discs were examined visually for the presence of any fungal growth.
Fungal tissue was characterized as endophytic B. bassiana if characteristic white dense
mycelia, becoming creamy at the edge (Humber 1997) grew from internal plant tissues of
surface sterilized leaf discs. Final assessment of the presence of endophytic B. bassiana was
recorded after 14 days and was expressed as percentage colonization by dividing the number
of leaf discs exhibiting B. bassiana outgrowth by the number of total leaf discs and multiplying
the obtained value with 100. If one of the six leaf discs obtained from a single plant showed
fungal outgrowth the total leaf was classified as being endophytically colonized. Differences
in percentage colonization of plant tissues at the different sampling dates were analyzed for
statistical significance with a Kruskal-Wallis-ANOVA using Dell Statistica data analysis
software system (Dell Inc., version 13, software.dell.com).
3.2.4 Strain-specific detection of B. bassiana
To ensure that fungal tissue present at the edges of grapevine leaf discs originated from the
respective inoculated B. bassiana strain (ATCC 74040 or GHA), now internally colonizing
plant tissues as an endophyte, a subset of mycelia samples was further analyzed with
molecular techniques. DNA was extracted from fungal tissues using the MasterPure™ DNA
Purification Kit (Epicentre Biotechnologies, USA) according to the manufacturer’s instructions
with an additional step for 30 min on ice after the recovering step with isopropanol.
Accordingly, extracted fungal DNAs were subjected to strain-specific PCR analysis using
three B. bassiana microsatellite (simple sequence repeats, SSR) primers, namely Ba01, Ba12
and Ba13 (Rehner and Buckley 2003). In previous studies, these primers have proved to allow
a confident discrimination among different B. bassiana isolates (Reineke et al. 2014).
For fluorescent labelling of the generated PCR products, a M13(-21) tail was placed at the 5’-
end of each forward primer and a CY5 labelled universal primer M13(-21) was added to the
25
PCRs according to the method described by Schuelke (2000). PCR amplifications were set up
in a total volume of 15 µl consisting of 90 ng DNA, 10x reaction buffer, 5 pmol of forward
primer, 10 pmol of reverse primer, 2.25 mM MgCl2, 3 mM dNTPs and 0.5 U of Dream Taq
Polymerase (Fermentas, St. Leon-Rot, Germany). PCRs were performed under the following
conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 60.2
°C for 45 s and 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. An aliquot of
each PCR product was checked for successful amplification on a 1% agarose gel. PCR products
were analyzed for SSR sizes via capillary electrophoresis on a Beckman GenomeLab GeXP
DNA Genetic Analysis System (Beckman Coulter, Inc., CA, USA). As different fluorescent
primers were used for labeling the obtained PCR products (DY-751 for Ba01, BMN5 for Ba12
and DY-681 for Ba13) reactions were loaded as a multiplex analysis with 1 µl of each PCR
product, mixed with 36.7 µl sample loading solution (Beckman Coulter, Inc., CA, USA) and 0.3
µl of a 400 bp size standard. Allele sizes were determined using GenomeLab GeXP Version
10.2 (Beckman Coulter, Inc., CA, USA).
3.2.5 Mealybug bioassay
The antagonistic activity of endophytic B. bassiana against piercing-sucking insects was tested
with a detached leaf assay and vine mealybugs, P. ficus. 60 potted grapevine plants cv.
®
'Riesling' were inoculated with Naturalis (3%) or water as control as described above. The
whole experiment was repeated twice. Two weeks after inoculation two leaves per plant were
obtained, with one leaf used for the bioassay and the other leaf to verify endophytic
establishment by re-isolation as described above. To ensure that mealybugs were only
influenced by endophytic and not by epiphytic fungal propagules, all grapevine leaves were
surface disinfected before the bioassay according to the procedure described above. With a
pretest (data not shown) we verified that any leftovers of NaOCl still present on the leaves did
not harm the mealybugs.
Vine mealybugs were grown on potato sprouts in a growth room with 23 ± 1 °C, 60–65% RH
and 16:8 h light:dark period. In all experiments, first instar P. ficus individuals were used,
which were removed from potato tubers by irritation with a paintbrush until their stylets
were withdrawn. Ten P. ficus larvae each were carefully transferred with a paintbrush to the
surface sterilized leaves. Leaves with mealybugs were maintained in enclosed transparent
plastic containers (height 10 cm, diameter 13.5 cm) with water provided for the leaf and were
placed in a growth chamber under the conditions mentioned above.
After two days infestation rate was calculated as the number of remaining larvae on the leaf
in relation to the initially used ten individuals. This procedure was repeated once a week over
a period of three weeks (7, 14, and 21 days after initial settlement) and was supplemented by
26
3 Manuscript I
determination of the size of all individual mealybug larvae with a binocular microscope and
measurement software (Leica Microsystems, Application Suite, Switzerland). A total of 300
mealybugs werw assessed for each, the endophytic and the control leaves. Size and infestation
rate were analyzed for statistical significance between endophytic and control leaves with a
Mann-Whitney-U-Test (α = 0.05) using Dell Statistica data analysis software system (Dell Inc.,
version 13, software.dell.com).
3.2.6 Field trial
In addition to greenhouse and laboratory experiments we conducted a field trial as proof of

principle to get preliminary evidence of efficacy of endophytic establishment of B. bassiana
and its antagonistic potential against insect pests in the field. The field trial was realized in the
framework of GEP (good experimental practice) certified efficacy tests of plant protection
products. The experimental vineyard was located in the Rheingau region, Germany (49°58'N,
7°57'E) and included 0.3 ha of grapevine plants, Vitis vinifera (L.) cv. 'Riesling' planted in 1999.
The experiment was conducted in a completely randomized block design with 4 plots
(replicates) of 114 m2 size and 14 vines per plot.
® ®
Naturalis (1%) was applied in the vertical canopy by a tunnel sprayer with 8 Teejet flat
spray nozzles and driving speed of 0.7 m/s. Control plots were treated with water. Application
was carried out in step with other plant protection measures (fungicide applications against
® ® ®
powdery mildew, Erysiphe necator, using the products Vivando , Talius , Luna Experience
®
and Topas in rotation). Interval between applications was approximately 10 days depending
on weather and disease pressure of other grapevine pathogens with a first application on 15
®
May 2014. In two of the four Naturalis treated plots treatment included nine applications
during the season. The other two plots were treated twice in the beginning of the season to
determine how long the fungus can be detected endophytically (15 and 26 May 2014).
Endophytic establishment in grapevine leaves was evaluated at 4 dates (22 May, 12 June, 2
July, and 23 July 2014) in 10 leaves per plot according to the method described above. In
addition we assessed the infestation with grape leafhopper, Empoasca vitis, at 5 dates (15 July,
23 July, 31 July, 07 Aug and 12 Aug 2014) in treated plots and control plots by counting E. vitis
larvae on 25 leaves per plot. Infestation data was analyzed for statistically significant
differences with nonparametric Wilcoxon signed-rank test (McDonald 2014).
27
3.3 Results
3.3 Results
3.3.1 Endophytic colonization of potted grapevine plants
During the assessment period of 7, 14, and 21 DAI B. bassiana was successfully re-isolated
from 46%, 40%, and 46% of all inoculated grapevine plants, respectively. None of the leaf discs
obtained from control plants showed signs of fungal outgrowth, thus none of the control
plants were colonized by the fungus. Not all leaf discs from colonized plants showed fungal
outgrowth, causing a high variance in percentage colonization in all treatments (Figure 5). In
some instances contaminating fungi and bacteria were occasionally found growing from leaf
discs of both inoculated and control plants (data not shown).
Figure 5: Mean (±SE) percentage colonization of Vitis vinifera leaf discs 7, 14 and 21 days after
inoculation (DAI) with a conidial suspension of Beauveria bassiana strains GHA (1.4 × 107 conidia/ml) or
ATCC 74040 (conc. 1: 7 × 106 conidia/ml; conc. 2: 1.4 × 107 conidia/ml) or with the formulated product
Naturalis® (3% and 5%). Differences between treatments were not statistically significant (p<0.05). In
control leaves (treated with Ringer’s solution) no B. bassiana was present (not shown).
If applied as a conidial suspension on foliage of grapevine plants, both B. bassiana strains

(GHA and ATCC 74040) were able to establish as an endophyte, with no significant
differences in percentage colonization being evident between the different spore
concentrations and the strains applied (Figure 5). The same was obvious if B. bassiana strain
®
ATCC 74040 was applied as the formulated product Naturalis , with colonization rates being
not significantly different for both concentrations (3% and 5%) applied (Figure 5). During the
assessment period, no significant decline or increase in percentage colonization by endophytic
B. bassiana was observed 7, 14, and 21 DAI.
28
3 Manuscript I
3.3.2 Strain-specific detection of endophytic B. bassiana
In capillary electrophoresis, DNA from all analyzed mycelia samples obtained from
endophytic fungal tissues showed the respective strain-specific peaks after amplification with
three B. bassiana microsatellite primers (Table 2). Amplicons of primer pairs Ba01, Ba12 and
Ba13 showed peaks at 117 bp, 231 bp and 216 bp for strain ATCC 74040 and 117 bp, 222 bp and
168 bp for strain GHA, respectively. These results indicate that endophytic B. bassiana re-
isolated from inoculated leaf discs originated from the previously applied strains.
Table 2: Amplification of B. bassiana strain GHA or ATCC 74040 specific SSR markers in a subset of
eight obtained mycelia samples from leaf discs of the different treatments 14 and 21 days after
inoculation (DAI) with B. bassiana.
Treatment DAI No. of leaf discs used and screened positive with
three SSR markers
n total Ba01 Ba12 Ba13
ATCC 74040 (1.4 x 107 conidia/ml) 14 2 2 2 2
® 14 2 2 2 2
Naturalis 3%
7 21 1 1 1 1
GHA (1.4 × 10 conidia/ml)
6 21 2 2 2 2
ATCC 74040 (7 x 10 conidia/ml)
7
ATCC 74040 (1.4 x 10 conidia/ml) 21 1 1 1 1
3.3.3 Mealybug bioassay
Antagonistic potential of endophytic B. bassiana against vine mealybug larvae was assessed
®
on detached and surface sterilized grapevine leaves of Naturalis treated and control
grapevine plants. Endophytic establishment in the repective grapevine plants was 30% in the
first and 60% in the second experimental replicate (data not shown). Because of this difference
in endophytic establishment results of the two replicates were not combined but were
analyzed separately.
®
Mealybug larvae were smaller when feeding for a period of 3 weeks on Naturalis treated
leaves compared to those feeding on control leaves (Figure 6). In the first experimental
replicate this difference was not statistically significant (Mann-Whitney-U-Test: 1 week p =
0.057; 2 weeks p = 0.159; 3 weeks p = 0.286), while in the second replicate the mean size of the
mealybugs on treated leaves was significant smaller over the whole assessment period of
three weeks (Mann-Whitney-U-Test: 1 week p<0.001; 2 weeks p<0.005; 3 weeks p<0.05).
In the first experimental replicate significantly less mealybug larvae stayed alive on leaves
®
with endophytic B. bassiana (Naturalis ) compared to control leaves (p<0.05) over the period
of the bioassay (Figure 7). In the second replicate this effect was only observed at the
beginning of the assessment period (1 week of feeding).
29
3.3 Results
Figure 6: Mean (± 95% CI) size of vine mealybug larvae (P. ficus) after feeding for three weeks on
detached grapevine leaves of control plants and plants with endophytic B. bassiana (Naturalis®) in two
replicates (A and B). Statistical significant differences between treatments were analyzed with a Mann-
Whitney-U-Test (α=0.05).
30
3 Manuscript I
Figure 7: Mean (± 95% CI) infestation rate of vine mealybug larvae (P. ficus) after feeding for three
weeks on detached grapevine leaves of control plants and plants with endophytic B. bassiana
(Naturalis®) in two replicates (A and B). Statistical significant differences between treatments were
analyzed with a Mann-Whitney-U-Test (α=0.05).
3.3.4 Field trial
Re-isolation of B. bassiana after application in the field showed that the fungus was able to
establish as an endophyte in perennial and lignified grapevine plants in the vineyard (Table 3).
®
In the plots treated several times with Naturalis the fungus could be detected at all sampling
dates (22 May, 12 June, 02 July and 23 July 2014). Detection rate declined over the season. In
plots treated only at the beginning of the season B. bassiana was successfully re-isolated up to
®
five weeks after the last application of Naturalis . In control plots no B. bassiana was re-
isolated from the leaves.
31
3.3 Results
Table 3: Number of leaf discs assessed (n) and showing B. bassiana outgrowth collected from
Naturalis® treated and control plots of a grapevine field trial in 2014.
No. of leaf discs with endophytic

Treatment n
B. bassiana
22 May 12 June 02 July 23 July
Control 80 0 0 0 0
Naturalis® (2 applications) 160 16 8 8 0
Naturalis® (9 applications) 160 47 19 10 13
Infestation rate with grape leafhopper E. vitis in the vineyard was overall low in the year 2014.
®
At all five monitoring dates the mean number of larvae was lower in Naturalis treated plots
than in control plots (Table 4). Over the whole assessment period the total number of E. vitis
®
larvae was higher in control than in Naturalis treated plots (246 vs. 183 individuals).
Wilcoxon signed-rank test showed that the median difference between the mean number of
®
grape leafhopper larvae per monitoring date in control plots vs. Naturalis treated plots was
significantly greater than zero (W=0, P<0.001)
Table 4: Mean number (±SE) of grape leafhopper E. vitis larvae in four control and four Naturalis®
treated plots (25 leaves/plot) assessed at five observation dates in a grapevine field trial in 2014. Mean
number of grape leafhopper larvae per monitoring date in control plots vs. Naturalis® treated plots
were statistically different (Wilcoxon signed-rank test; W=0, P<0.001).
Date 2014 Mean number (± SD) of E. vitis larvae
Control Naturalis®
15 July 15,50 (± 5,07) 12,75 (± 2,22)
23 July 18,75 (± 9,71) 12,75 (± 6,60)
31 July 14,00 (± 5,48) 13,50 (± 5,20)
07 Aug 6,00 (± 2,94) 4,50 (± 3,11)
12 Aug 4,75 (± 2,06) 2,25 (± 0,96)
3.4 Discussion
Successful endophytic colonization of both potted grapevine plants in the greenhouse as well
as mature plants in the field with two different commercially available B. bassiana strains was
achieved via artificial spray inoculation. Analysis of fungal mycelia obtained after re-isolation
with strain-specific molecular markers confirmed our initial assessment based on morphology
of endophytic fungal mycelia obtained from colonized grapevine plants. In greenhouse
experiments, no significant difference in percentage colonization by endophytic B. bassiana
was observed during the assessment period of 21 DAI. This suggests that endophytic
colonization of grapevine by B. bassiana was evident as early as 7 DAI and did not decline
32
3 Manuscript I
during the period of screening for presence of endophytic B. bassiana of 21 DAI. Moreover,
percentage colonization of grapevine plants did not vary significantly among the different
strains or inoculum doses used. This may be a consequence of the relatively small number of
positive samples identified and the apparent variability in isolation success.
In the present study, mean colonization rates of potted grapevine plants by B. bassiana were
between 5% and 23% and were thus rather low compared to colonization rates of leaves of
other plant species like corn (Wagner and Lewis 2000), tomato(Klieber and Reineke 2016),
sorghum (Tefera and Vidal 2009), and jute (Biswas et al. 2013). In contrast to these plants
grapevines are deciduous, woody perennial plants, and plants used for our greenhouse trials
were cultivated from hardwood cuttings. Seed treatment as an alternative inoculation method
as it has been successfully shown for tomato, cotton (Ownley et al. 2008), opium poppy
(Quesada-Moraga et al. 2009) and sorghum (Tefera and Vidal 2009) is therefore not possible.
Previous grapevine inoculation trials via root dipping or soil inoculation resulted in no
colonization at all (data not shown). Root dipping or soil inoculation has been used for
endophytic establishment of B. bassiana in banana (Akello et al. 2007), sorghum (Tefera and
Vidal 2009) and pine seedlings (Brownbridge et al. 2012). Therefore, inoculation via spray
application is apparently the only option for endophytic inoculation of grapevine plants and
we have shown here that such an application is compatible with viticultural practice.
Jaber (2015) reported slightly higher colonization rates of up to 50% of grapevine plants by B.
bassiana after artificial spray inoculation. Endophytic establishment of entomopathogenic
fungi is known to be dependent on plant cultivar, fungal strain and many other environmental
conditions (Vidal and Jaber 2015). However, to the best of our knowledge it was not yet
possible to prove systemic establishment of B. bassiana in grapevine plants.
Here, we used molecular SSR markers to prove that the re-isolated B. bassiana strain was the
one previously applied. Direct detection with PCR-based techniques of endophytic B. bassiana
after spray application is difficult because of the likelihood of contamination with epiphytic
propagules and is thus only applicable for systemic establishment. In addition, surface
sterilization is regarded as an insufficient technique for subsequent molecular assessment of
endophytic establishment (McKinnon et al. 2014). Evidence has accumulated that for culture-
based techniques surface sterilization can result in underestimated colonization rates, due to
diffusion of the chemicals used for sterilization into the leaves (Lohse et al. 2015; Ownley et al.
2008). In consequence and in line with other reports only a combination of different detection
methods will result in sound qualitative and quantitative data about endophytic colonization
of plants (Lohse et al. 2015). In this context methods must be adapted for every plant species
and different plant material.
33
3.4 Discussion
Despite the comparatively low endophytic colonization rates of B. bassiana in grapevine we

observed significant antagonistic effects of endophytic B. bassiana on infestation and size of
vine mealybug larvae in bioassays. Moreover, grape leafhopper E. vitis larvae were
significantly more abundant on control than on endophytic B. bassiana grapevine plants in the
field. Usually, fungal entomopathogens infect their insect hosts via cuticular penetration by
germinating propagules (Arnold and Lewis 2005). Infection by endophytic entomopathogens
via consumption of infected plant tissue or ingestion of hyphae or spores seems to be unlikely
and rare (Vidal and Jaber 2015). Existing reports about mycosis due to endophytic
entomopathogens are so far restricted to insects living inside plant tissues like stem-borers or
leafmining larvae (Akello et al. 2009; Klieber and Reineke 2016), where a direct contact of
insects feeding inside plant tissues and endophytic fungal propagules can be envisaged. In
contrast to stem-borers or leaf-miners mealybug larvae live on the plant surface and have a
piercing-sucking feeding habit with the consequence that a direct mode of action due to direct
contact is not likely to occur. On surface disinfected leaves previously treated with B.
bassiana, mealybug larvae were smaller and mortality rates were higher than on control
leaves, but none of the dead larvae exhibited symptoms of mycosis. These results suggest a
mode of action involving feeding deterrence, antibiosis or changes in metabolism of the host
plant and thus host plant quality rather than a direct fungal infection of the insects.
Colonization of grapevine plants was different in the two replicates of the bioassay. At a
higher B. bassiana colonization rate, size of vine mealybug larvae was significantly smaller
after feeding on endophytic leaves compared to control leaves. Vice versa, at a lower
colonization rate, we detected significant differences in vine mealybug infestation rates.
Accordingly, two different modes of action of endophytic entomopathogens might account for
these observations, depending on rate of tissue colonization by B. bassiana. In any case, we
have shown that the presence of entomopathogens as endophytes negatively influences insect
performance, yet further investigations are required to determine the mechanisms underlying
these effects. Results presented here point to the importance to also study sublethal effects of
endophytic entomopathogens on insects in order to understand tritrophic interactions
between plants, endophytes, and insects.
In the present study we have shown for the first time that an endophytic establishment of B.
bassiana in mature grapevine plants under field conditions is possible. Our results also
indicate the potential for a long term establishment of the fungus in grapevine plants and that
endophytic establishment does apparently not interfere with common viticultural
management practices. In this regard, a couple of studies have shown that B. bassiana is
sensitive against various pesticides (Todorova et al. 1998; Sapieha-Waszkiewicz et al. 2004;
Kos and Celar 2013). However, even though synthetic fungicides were simultaneously applied
in our experimental vineyard, an endophytic establishment of B. bassiana in the mature plats
34
3 Manuscript I
was successful. Moreover, B. bassiana conidia are known to be extremely sensitive to

ultraviolet radiation and consequently persistence as well as germination of conidial
suspensions applied on the foliage is limited. Lohse et al. (2015) emphasized the importance of
an adequate formulation for endophytic establishment of entomopathogenic fungi. Here, we
®
used a commercially available fungal-based product (Naturalis , active ingredient B. bassiana
isolate ATCC 74040) formulated as an oily dispersion, which may provide a benefit for the
colonization process of B. bassiana on grapevine plants. This product is registered in some EU
member states i.e. for control of whiteflies in tomato thus having the perspective of a rapid
registration for other applications.
Overall, grapevine plants seemed not to be negatively affected by the presence of endophytic
B. bassiana, as growth and performance of the respective inoculated plants was visually
similar to control plants during the period of observation (data not shown). This is in
accordance with previous studies on plant performance after endophytic establishment of
entomopathogenic fungi (Akello et al. 2009; Tefera and Vidal 2009; Wagner and Lewis 2000;
Klieber and Reineke 2016). However, whether presence of endophytic B. bassiana in grapevine
plants has an effect on quality and sensory attributes of must and wine still remains to be
tested with fruit-bearing grapevine plants.
Endophytic establishment of an entomopathogenic fungus such as B. bassiana in grapevine

plants represents a new and sustainable plant protection strategy. The implementation of the
indirect effects (endophyte) in combination with direct effects (epiphyte) of entomopathogens
on both plant and insect herbivores will show their full potential value in insect pest
management. Further research should also include an in-depth study on the mode of action of
endophytic entomopathogens against insects as well as identifying possible effects on induced
resistance mechanisms against both grapevine pathogens and insect pests.
Acknowledgements
This work was supported by Deutsche Bundesstiftung Umwelt (DBU). We thank Edith
®
Ladurna from CBC (Europe) S.r.l., Italy, for providing the product Naturalis and Ottmar Baus
for support during the field trial. Gordan Behaderovic is kindly acknowledged for help in
mealybug bioassays.
35
4 Association of Beauveria bassiana with grapevine
plants deters adult black vine weevils, Otiorhynchus
sulcatus

Germany, email: yvonne.rondot@hs-gm.de, fax: 0049-(0)6722-502 410
Contribution to the article: Planned all experiments

Performed most of the experimental work
Evaluated and statistically analyzed all data
Contribution of other authors: Annette Reineke contributed to experimental design and

writing the paper
Journal: Biocontrol Science and Technology
Date of manuscript submission: 22. Feb 2017
DOI: https://doi.org/10.1080/09583157.2017.1347604
36
4 Manuscript II
Abstract
Fungal entomopathogens are known as microbial pathogens of insects, colonizing multiple
habitats and ecosystems. Besides being an entomopathogen, the fungus Beauveria bassiana
can also establish as an endophyte in plants. Limited knowledge is so far available on the
ability of plant-associated B. bassiana to influence plant-feeding insects. Here, we assessed the
capability of adult black vine weevils Otiorhynchus sulcatus to select grapevine as a host plant
in the presence of plant-associated B. bassiana after foliar application of a commercially
®
available mycoinsecticide (product Naturalis ) on young potted grapevine plants. Three
pairwise comparisons of weevil behaviour were conducted when weevils were released in a
two-choice olfactometer and were given the choice between (i) control plants and plants
®
treated with Naturalis , (ii) control plants and plants treated with the formulation of
® ®
Naturalis without fungal propagules, and (iii) plants treated with Naturalis and plants
treated with the formulation. Adult O. sulcatus were significantly deterred by plants treated
®
with Naturalis or the formulation in comparison to control plants. In a direct comparison
®
between plants treated either with Naturalis or the formulation weevils significantly
®
preferred plants treated with the formulation and avoided Naturalis treated plants, where B.
bassiana putatively had established as an endophyte. These results suggest that adult black
vine weevils are able to detect and subsequently avoid plants treated with B. bassiana and
indicate a new mode of action of plant-associated entomopathogenic fungi when integrated in
pest management programmes.
Keywords
Beauveria bassiana, endophyte, entomopathogen, Otiorhynchus sulcatus, choice assay,
olfactometer
4.1 Introduction
Endophytes, a term first defined by De Bary (1884), are fungi or bacteria occurring within
plant tissues without causing visible disease symptoms in the colonized plant. Even though
their presence does not seem to negatively influence the plant, some endophytes have
profound impacts on plant communities or have the ability to influence interactions between
plants and their natural enemies. For example, certain endophytes can enhance overall plant
fitness (Rodriguez et al. 2009) or increase resistance of plants against herbivores or pathogens
as well as limit their spread and damage (Arnold and Lewis 2005; Ownley et al. 2010; Backman
and Sikora 2008; Vega 2008). Although endophytes are present in most, if not all, plants in
natural as well as in agricultural ecosystems, their function in shaping plant-insect
interactions is yet not fully understood and their potentially beneficial role in sustainable
37
4.1 Introduction
plant production is not exploited so far. However, the ability of endophytes to colonize
internal host tissues could be used to improve crop performance or pest management
strategies. Reduced herbivory on endophyte hosting plants can be a direct result from
decreased survival rates of herbivorous insects, which is often attributed to the production of
defensive compounds or toxins (Clay 1993). In addition, alterations in the plant’s nutritional
quality as well as changes in plant volatile profiles or secondary plant metabolites of
endophyte-associated plants may influence developmental time, fecundity, host location, or
oviposition behaviour of herbivorous insects (Jallow et al. 2008; Vega 2008).
Host plant selection by herbivorous insects includes a series of behavioural and decision
events. The ability to detect the presence of natural enemies or pathogens in the respective
host plant’s environment and to react accordingly would be advantageous for any insect
during foraging or oviposition site selection. Entomopathogenic fungi, such as Beauveria
bassiana (Bals.) Vuill. (Ascomycota: Hypocreales), are important mortality factors of insect
pests. Some potential pest insects are able to detect entomopathogenic fungi and to avoid
contact with them. The common flower bug Anthocoris nemorum for example can recognise
its natural enemy B. bassiana and has been shown to avoid fungus infected leaves (Meyling
and Pell 2006). Another example of such a prevention strategy in the presence of the
entomopathogenic fungus B. bassiana has been proved for the seven-spot ladybird (Coccinella
septempunctata), which is able to avoid lethal densities of B. bassiana conidia in soil or on
leaves (Ormond et al. 2011). Other insects, such as the termite Coptotermes lacteus, are capable
of recognising the presence of the entomopathogenic fungus Metarhizium anisopliae and were
shown to avoid direct contact with this fungus (Staples and Milner 2000). In contrast, Kepler
and Bruck (2006) described a significant attraction of black vine weevil Otiorhynchus sulcatus
larvae to pots containing plants and the fungus M. anisopliae, which is likely due to changes in
volatile profiles when roots are colonized by this entomopathogenic fungus. Pivotal for these
reactions are active detection mechanisms by the insects. However, the exact processes are
not yet fully understood. In this context, Elliot et al. (2000) extended the herbivory-bodyguard
hypothesis describing tritrophic interactions among plants, herbivores and their predators or
parasitoids also to entomopathogens and their plant association.
The black vine weevil, O. sulcatus F. (Coleoptera: Curculionidae), is a serious insect pest of
economic importance in nursery, ornamental and soft fruit production worldwide (Moorhouse
et al. 1992). While adult weevils are nocturnal and cause mostly cosmetic damage by feeding
on the leaves, larvae are ground dwelling and feed on root systems, which may result in high
levels of plant damage and subsequently kill the plant (Shah et al. 2007). Because O. sulcatus
has a parthenogenetic mode of reproduction, a single weevil left uncontrolled can lay up to
38
4 Manuscript II
900 eggs, resulting in the infestation of an entire nursery (Bruck 2007). Keeping the insect out
of nurseries is one main issue in its control.
Infestation by larval stages of O. sulcatus can be limited by the incorporation of synthetic

insecticides into the potting media (Kepler and Bruck 2006). An alternative biological control
strategy is the application of entomopathogenic nematodes. However, practical use of this
group of biological control agents is limited due to insufficient efficacy at low temperatures, a
short shelf life, and high application costs (Johnson and Rasmann 2015; Lu et al. 2016).
Moreover, the management of adult O. sulcatus includes foliar applications of pesticides,
however, adult weevils are active at night, which necessitates and complicates an application
at the right site and right time. Entomopathogenic fungi showed considerable potential as
biological control agents against adults and larvae of the black vine weevil (Bruck 2007; Shah
et al. 2007; Ansari et al. 2008; Hirsch and Reineke 2014). Accordingly, the simultaneous use of
endophytic entomopathogens as plant bodyguards as defined by Elliot et al. (2000) in addition
to the already proved direct effect of entomopathogenic fungi against O. sulcatus would
represent a dual mode of action of entomopathogens against this pest insect. For example, the
presence of endophytic entomopathogenic fungi might influence host choice behaviour of
adult weevils, resulting in an avoidance of the colonized plant.
In the present study we assessed the behaviour of adult black vine weevils when given the
choice between grapevine plants treated several weeks before with B. bassiana containing
mycosinsecticide and control plants. We hypothesised that weevils are able to detect and
avoid B. bassiana when actively searching for a host plant. The results presented here will
provide information on the potential of endophytic fungi to influence herbivore host choice
behaviour, and promote the development of improved management strategies for insect pests.
4.2.1 Source of fungus, insects, and plants
Beauveria bassiana strain ATCC 74040 was used in the form of the commercial product
® ®
Naturalis (CBC (Europe) S.r.l. – BIOGARD Division). Naturalis is formulated as an oil
7
dispersion (OD) and contains approximately 2.3 x 10 colony forming units/ml of B. bassiana
strain ATCC 74040 as active ingredient. The product is registered in some EU member states,
e.g. for the control of whiteflies in tomato. In addition, the pure formulation of this product
without conidia of B. bassiana was used as a control in our experiments (CBC (Europe) S.r.l. –
BIOGARD Division).
A population of black vine weevil, O. sulcatus, was kept at 22° C and fed with grapevine
leaves. Egg and larval development was completed in boxes (h = 9 cm, w = 22 cm, l = 34 cm)
39
filled with 8 cm soil and using Impatiens walleriana plants and carrots as food source. In all
assays, we used adult weevils that had emerged from pupae in the soil boxes at least 4weeks
but not more than 12 weeks before the start of experiments. At this age, weevils are in the
period of maturation feeding; accordingly foliar feeding on the respective host plant is
required for egg production. Prior infection experiments carried out with the same O. sulcatus
population and the B. bassiana strain via direct inoculation resulted in 48 to 65% mortality of
adult weevils within 28 days (Hirsch and Reineke 2014).
Grapevine plants, Vitis vinifera (L.) cv. 'Riesling', were propagated from hardwood cuttings.
After root development, the plants were potted and grown in a greenhouse chamber at
22-25 °C. Seven-week-old grapevine plants with four to six fully expanded leaves were used
® ®
for treatment either with the commercial product Naturalis (3%), the Naturalis formulation
without conidia (3%) or water. For each treatment, 40 plant replicates were inoculated
separately by spraying the adaxial and abaxial surfaces of all fully expanded leaves using a 2-l
handheld pressure sprayer. Approximately 10 ml were applied at each plant. Prior to the
experiments, treated plants were retained in a greenhouse chamber for 1-3 weeks (mean
temperature 23-25° C, mean relative humidity 50-70%) and were watered as required. The rate
of endophytic establishment of B. bassiana in grapevine plants was tested under the same
conditions and using plants of the same origin and age in parallel experiments. As reported by
Rondot and Reineke (2018), between 30-60% of plants could be detected as having B. bassiana
as an endophyte. In this study, endophytic colonization of 7-week-old potted grapevine plants
by B. bassiana was assessed 7, 14, and 21 days after inoculation (DAI) by re-isolation from leaf
tissues after surface sterilization. For the experiments described here, we excluded additional
re-isolations of B. bassiana or other leaf sampling analysis as described by Rondot and Reineke
(2018), in order to prevent activation of plant defense reactions by mechanical damage of
leaves. Since treatment and cultivation conditions were similar in both experiments, we
expect identical colonization rates.
4.2.2 Design and validation of the two-choice olfactometer
In order to assess host choice behaviour of adult black vine weevils we constructed a two-
choice still-air olfactometer (Figure 8). Transparent plastic cylinders (h = 30 cm, d = 13.5 cm)
were modified by drilling a hole (d = 2.5 cm) into the side of each cylinder (6 cm from the
bottom) and fitting a horizontal connection tube (l = 6.5 cm, d = 2.3 cm) into the hole. Two of
these tubes were connected by a T-shaped piece of PVC pipe (d = 2.5 cm). The middle section
of the T-shaped piece was plugged with a small petri dish lid which served as a release point
for the weevils. Each cylinder was placed on the soil surface of a potted grapevine plant and
was sealed with gauze to allow sufficient air flow within the olfactometer and prevent
excessive moisture. Prior to the experiments, the newly designed olfactometer was validated
40
4 Manuscript II
by releasing weevils in the T-shaped middle section and giving them the choice between a
control plant placed inside a cylinder and an empty cylinder in order to observe if black vine
weevils were generally able to choose the host plant in this test system. The design of the
olfactometer permitted the weevils to change sides after an initial selection.
Figure 8: Design of the two-choice still-air olfactometer used in the experiments. Adult black vine
weevils were placed inside the lid of the T-shaped piece connecting the two cylinders and were
allowed 1 h to choose between plants in the cylinders.
4.2.3 Experimental design
With the olfactometer described earlier, three different pairwise comparisons of weevil host
choice behaviour were performed. Adult black vine weevils were allowed to choose between
®
(i) control plants and grapevine plants treated 7-21 days before with Naturalis , (ii) control
®
plants and plants treated 7-21 days before with the Naturalis formulation, and (iii) plants
® ®
treated with Naturalis and plants treated with the Naturalis formulation. Plants were not
surface sterilized before use in the olfactometer trials.
Because O. sulcatus has a nocturnal lifestyle and trials should be performed in the active
period of adults, the natural daily rhythm of adult O. sulcatus was switched by 12 h with the
help of artificial lighting. Additionally, weevils were deprived of food for 24-36 h prior to
testing. Each test lasted 1 h, starting when weevils were in the active period for food
searching (2-4 h after “sunset”). Pretests indicated that most of the weevils had made their
decision within 1 h with no significant changes occurring compared to their initial selection
even if they were given more time (data not shown).
Moreover, we decided to assess host choice behaviour of single adult O. sulcatus instead of
releasing several weevils at the same time to avoid aggregation behavioural effects. All trials
41
were performed in a dark room (24 ± 2 °C, 55 ± 8 % RH). Nine olfactometers were used
simultaneously, three for each pairwise comparison. Orientation of the olfactometer in the
room was changed for every replicate. Adult O. sulcatus were used only once and plants were
replaced every second day or when feedings sites were visible on the leaves. Cylinders and
connecting tubes were thoroughly washed before each experimental day. Trials were repeated
3 times a day for 12 days, so that 108 decisions were realised and documented for every
pairwise comparison. The whole experimental set-up was repeated twice in two subsequent
years (2013 and 2014).
The preference of adult O. sulcatus was compared relative to each other. Weevils that
remained in the connecting T-tube were categorised as unresponsive. Each pairwise
comparison as well as the validation experiment were analysed separately. Number of
decisions for each side was counted and the proportion out of the total number of responsive
adult O. sulcatus in the trial was analysed with an exact binominal test (McDonald 2014). The
number of responsive weevils and the number of unresponsive weevils in all comparisons
throughout the study were compared using a two sample t-test (McDonald 2014).
4.3 Results
The usefulness of the designed olfactometer was validated by the black vine weevils’ ability to
select a cylinder with a grapevine plant over an empty cylinder. In both experimental
replicates (years 2013 and 2014), the majority of adult O. sulcatus was recovered from
cylinders containing a plant (2013: p = .03; 2014: p < .001; Figure 2). Throughout all
comparisons, the percentage of responsive weevils was generally high (mean = 82%) and was
significantly greater than the percentage of unresponsive weevils (p < .0001). The weevils in
the first experimental replicate in 2013 were less decisive (70% responsive) than in the second
experimental replicate in 2014 (94% responsive).
When black vine weevils were allowed to choose between control grapevine plants and plants
®
treated 7-21 days before with Naturalis , significantly more weevils decided for the cylinders
with a control plant (Figure 9). In the first replicate (2013), we recovered 67 weevils from the
cylinders with a control plant, 9 weevils from the cylinders with plants treated with
®
Naturalis (p < .0001), and 32 weevils were categorised as unresponsive. In the second
replicate (2014), the proportion was 62 versus 41 recovered weevils, respectively (p = .048),
and 5 weevils were categorised as unresponsive. In both replicates, the distribution of weevils
significantly differed from random.
When weevils were given a choice between control plants and plants treated 7-21 days before
®
with the Naturalis formulation, significantly more weevils decided for the cylinders with a
control plant (Figure 9). In the first replicate (2013), we recovered 64 weevils from the
42
4 Manuscript II
cylinders with a control plant, 14 weevils from cylinders with plants treated with formulation
(p < .0001), and 30 weevils were categorised as unresponsive. In the second replicate (2014),
the proportion was 69 versus 35 recovered weevils, respectively (p = .001), and 4 weevils were
categorised as unresponsive. In both replicates, the distribution of weevils significantly
differed from random.
When weevils were allowed a choice between plants treated 7 to 21 days before with the
® ®
Naturalis formulation without B. bassiana conidia or plants treated with Naturalis ,
significantly more weevils decided for the cylinders with plants treated with the formulation
(Figure 9). In the first replicate (2013), we recovered 64 weevils from the cylinders with a
®
formulation-treated plant and 3 weevils from cylinders with plants treated with Naturalis (p
< .0001), and 41 weevils were categorised as unresponsive. In the second replicate (2014), the
proportion was 62 versus 38 recovered weevils, respectively (p = .02), and 8 weevils were
categorised as unresponsive. In both replicates, the distribution of weevils significantly
differed from random.
Figure 9: Percentage of adult black vine weevils O. sulcatus recovered when released in an
olfactometer containing (i) control plants and no plant (validation assay), (ii) control plants and plants
treated with Naturalis®, (iii) control plants and plants treated with the formulation, and (iv) plants
treated with Naturalis® and plants treated with the formulation. Results from two independent
replicates (2013 and 2014) are shown. Asterisks indicate significant differences from even distribution
with p ≤ .05 (*), p ≤ .01 (**) or p ≤ .001 (***). Right side of the graph depicts percentage of responsive
weevils in the respective experiments.
43
4.4 Discussion
4.4 Discussion
In this study, we proved that adult black vine weevils are able to identify grapevine plants that
have been treated with a B. bassiana containing mycoinsecticide when actively searching for a
host plant. In choice tests carried out in our newly developed plant olfactometer, weevils
avoided grapevine plants treated 1-3 weeks earlier with the B. bassiana containing product
®
Naturalis as well as plants treated with the formulation of the same product. Since in a direct
comparison, weevils significantly preferred plants treated with the formulation over
®
Naturalis -treated plants, we suppose that the presence of plant-associated B. bassiana is the
chief factor influencing host choice behaviour. In parallel experiments using the same
conditions as reported here, we have shown that the endophytic establishment of B. bassiana
®
in grapevine plants can be achieved via spray inoculation of the product Naturalis , with 30 –
60% of the plants being colonized between 7 and 21 DAI (Rondot and Reineke 2018). However,
fungal inoculum or other foliar residues still present on the leaf surface could be another
factor contributing to adult O. sulcatus host plant choice behaviour, because the plants were
®
not cleansed of any residual Naturalis or formulation carrier before being used. Taken
together, these results suggest that adult black vine weevils are able to discriminate between
plants previously treated and not treated with B. bassiana and subsequently avoid treated
plants, where B. bassiana is present or has established as an endophyte. Although black vine
weevils are polyphagous herbivores, known to feed and reproduce on over 140 different plant
species (Bruck 2007), it has been previously shown that adults are able to discriminate
between different plant species and are attracted to the odour of some but not all host plants
(Van Tol et al. 2002). Moreover, in the same study weevils were attracted to volatiles of
weevil-damaged foliage of certain host plants (Van Tol et al. 2002). Visual as well as chemical
cues (volatiles, aggregation pheromones, or leaf surface chemicals) are involved in the
attraction of insect herbivores towards feeding or oviposition sites (Bernays and Chapman
1994). In the context of fungal endophytes, the biochemical cues thereto can be altered directly
by the growth of the fungus or indirectly mediated by the response of the plant to the fungal
infection. Plants can detect the mere presence of microbes on their cuticle via microbe-
associated molecular patterns (MAMPs) and respond with a number of biochemical changes
(Newman et al. 2013). We ascribe the mechanism underlying this tritrophic interaction
between the grapevine plant, B. bassiana and the insect O. sulcatus to a complex process,
mediated, e.g. through the combination of metabolic and hormonal changes in the colonized
plant.
The mechanisms involved in the detection of endophytic B. bassiana by adult O. sulcatus were
not examined in this study. Preliminary studies have, however, indicated that the volatile
profile of endophytic B. bassiana grapevine plants is different compared to non-endophytic
44
4 Manuscript II
plants (Peiter 2013). A quantitative or qualitative change in plant volatile profiles may thus
play a key role for O. sulcatus to discriminate between endophytically colonized and
endophyte-free plants. In this regard, Jallow et al. (2008) have detected significant quantitative
differences in certain volatiles of tomato plants when roots were colonized by the endophytic
fungus Acremonium strictum, which accordingly influenced host selection by adult Helicoverpa
armigera moths. In a similar way, the colonization of perennial ryegrass plants (Lolium
perenne) by an endophytic fungus altered the composition of volatile compounds, which
significantly influenced attraction of plants to adult African black beetles (Heteronychus
arator) (Qawasmeh et al. 2015). Yet in our study, it is also possible that the establishment of
endophytic B. bassiana altered visual, contact chemoreception and mechanoreception cues, or
changed the leaf surface itself. Since weevils were able to freely move around in the plant
olfactometer and behavioural assays were carried out in the dark without observing weevils
during the 1 h period of the choice assays, it might as well be possible that weevils decided to
leave a plant after initial contact.
Assessing putative behavioural responses of insect pests including recognition and avoidance
of fungal entomopathogens present as an epiphyte or endophyte on or inside the respective
host plant is pivotal for designing successful biological control strategies. The observed effects
on the behaviour of O. sulcatus in the presence of the entomopathogen B. bassiana are
contributing to our increased understanding of the function of entomopathogens as
bodyguards of plants. Endophytic establishment of an entomopathogenic fungus such as B.
bassiana in grapevine plants might thus represent a new and sustainable plant protection
strategy. Moreover, the combination of indirect effects (endophyte) and direct effects
(epiphyte) of entomopathogens on insect herbivores represents a dual-control strategy of
entomopathogenic fungi when integrated in pest management programmes. In this regard,
future experiments should also simulate field conditions, where usually all plants are treated
in the same way and O. sulcatus would have no choice between B. bassiana associated and
non-associated plants. Under these conditions, we speculate an overall reduction in feeding
rates and/or an increase in unresponsive weevils. Further research should also focus on the
mode of action of endophytic entomopathogens as plant bodyguards against insect pests as
well as on the identification of possible effects on induced resistance mechanisms in the host
plant itself targeting both pathogens and insect pests.
Acknowledgements
This work was supported by Deutsche Bundesstiftung Umwelt (DBU). We thank Edith
®
Ladurner from CBC (Europe) S.r.l. – BIOGARD Division for providing the product Naturalis
and the formulation without conidia. Roman Peiter is kindly acknowledged for help in choice
assays and Sandra Linck for graphical illustration of the olfactometer.
45
5 Endophytic Beauveria bassiana activates expression of
defense genes in grapevine and prevents infections
by grapevine downy mildew Plasmopara viticola

Germany, email: yvonne.rondot@hs-gm.de, fax: 0049-(0)6722-502 410
Contribution to the article: Planned all experiments

Performed most of the experimental work
Evaluated and statistically analyzed the data
Contribution of other authors: Annette Reineke contributed to experimental design,

data analysis and writing the paper
Journal: Plant Pathology
Date of manuscript submission: 9. June 2019
DOI: https://doi.org/10.1111/ppa.13089
46
5 Manuscript III
Abstract
Fungal entomopathogens like Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) are
known as antagonist of insects with multiple functional and ecological roles and have
attracted increased attention as biocontrol agents in integrated pest management programs.
For some crop plants, it has been proven that endophytic B. bassiana, besides its
entomopathogenic habit, can provide protection against plant pathogens or limit their
damaging effects. For grapevine, limited knowledge is however available on the influence of
endophytic B. bassiana on fungal pathogens and about the mechanisms underlying putative
protection effects.
Here, we assessed the protective potential of endophytic B. bassiana against grapevine downy
mildew Plasmopara viticola in greenhouse experiments. Three and seven days after a
B. bassiana treatment, respectively, potted grapevine plants were inoculated with P. viticola
and the evolving disease severity was assessed. Disease severity was significantly reduced in
B. bassiana-treated plants compared to control plants depending on the age of leaves.
Furthermore, a microarray and an RT-qPCR analysis were performed to work out
fundamental aspects of genes involved in the interaction between grapevine and the
endophytic fungus B. bassiana. The results indicate an up-regulation of diverse defense-
related genes in grapevine as a response to endophytic establishment of B. bassiana. Thus,
endophytic establishment of an entomopathogenic fungus such as B. bassiana in grapevine
plants would represent an alternative and sustainable plant protection strategy, with the
potential for reducing pesticide applications in viticulture.
Keywords
Beauveria bassiana, endophyte, Plasmopara viticola, gene expression, microarray analysis,
biological control, Vitis vinifera
5.1 Introduction
Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, the causal agent of grapevine downy
mildew, is one of the most destructive fungal diseases of European grapevine (Vitis vinifera L.)
plants. As an obligate biotrophic oomycete, it attacks all green parts of the vine, negatively
influencing both the quantity of the yield as well as the quality of must and wine. In
consequence, the repeated application of fungicides each vegetation period is practically
inevitable to limit the pathogen infections. Under optimal weather conditions for the fungus
(moist and moderately warm) and high disease pressure, an average of 12-15 fungicide
applications may be necessary for cool climate viticulture to keep the infection level under
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5.1 Introduction
control (Pertot et al. 2017). Current predictions on the effects of climate change on downy
mildew disease pressure suggest that even more fungicide applications will be necessary in
the future (Salinari et al. 2006). Concerns about environmental safety, the appearance of
resistant pathogen strains, and the economic costs associated with these applications require
alternative strategies for disease management. In organic viticulture, even shorter application
intervals are necessary due to the lower persistence of copper-containing products (Dagostin
et al. 2011). Given the poor soil-ecological properties of copper (persistence and accumulation
in the soil) and the limited availability of other organic fungicides, the exploration and
provision of alternative treatment strategies are thus becoming increasingly important.
Biological control agents suitable for use in organic as well as integrated viticulture originate
from many different sources (e.g., plant, microbial or mineral) and exhibit different modes of
action (e.g., antibiosis, competition or hyperparasitism). Besides the production of
preinfectional defense substances (e.g., stilbenes, saponins), plants have evolved inherent
effective defense mechanisms against phytopathogenic fungi, herbivorous insects or abiotic
stressors (Kaplan et al. 2008; Pieterse and Dicke 2007). In order to successfully prevent
infection or infestation, these mechanisms must be already activated or the defense reactions
initiated prior to infection. In the first case, a so-called acquired resistance can occur in the
plant against a later infection due to an initial infection by apathogenic microorganisms. In
the second case, the plant may be put into a state by which it can react more rapidly and
intensively to an attack by treatment with certain microorganisms or substances (Conrath et
al. 2006). This phenomenon is called "priming".
Fungal entomopathogens are traditionally known as microbial pathogens of insects but have
recently shown to play additional roles in nature and colonize multiple habitats and
ecosystems. These newly emerging ecological roles, including endophytism, plant disease
antagonism, plant growth promotion, and rhizosphere colonization, provide opportunities for
the multiple uses of fungal entomopathogens in integrated pest management (IPM) strategies
(Vega et al. 2009; Lacey et al. 2015). Among the group of entomopathogenic fungi, Beauveria
bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) is the most widely researched as an
endophyte (Parsa et al. 2013) and is commercialized in the form of mycopesticides (Faria and
Wraight 2007). Several studies have demonstrated that endophytic B. bassiana can protect its
host plant against plant pathogens (Griffin et al. 2005; Ownley et al. 2010; Ownley et al. 2008).
However, so far, only limited knowledge is available about the mechanisms underlying such
protection. Jaber and Ownley (2018) suggest that a combination of mechanisms might be used
by endophytic fungal entomopathogens against plant pathogens, rather than a single
mechanism. The colonization of date palm with B. bassiana showed to induce proteins related
to plant defense and stress response (Gómez-Vidal et al. 2009). This suggests that endophytic
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colonization by entomopathogenic fungi induces plant defense responses, probably by

activating the plant immune system.
The objective of the present study was to examine the biocontrol potential of endophytic
B. bassiana against P. viticola by pre-infection application of B. bassiana inoculum on
grapevine leaves. In addition, we investigated the effects of an inoculation with endophytic B.
bassiana on the innate plants’ defense reactions of grapevine by gene-based analyses
(microarray and RT-qPCR) of gene expression levels. Analyses of expression of respective
defense-related genes can expand our understanding of interactions between an endophytic
fungus and host plant and may provide a future basis for novel pest management approaches
with beneficial microorganisms.
5.2.1 Plant and fungal material
Two-eye hardwood cuttings of Vitis vinifera cv. Riesling were obtained from mature shoots
from a vineyard in the Rheingau region, Germany (49.58°N, 7.57°E) after the first frost. After
®
disinfection (0.5% Chinoplant solution for 12 h) they were stored at 4°C and 95% rel. humidity
until use. For rooting the lower eye was removed, and the cuttings were put in boxes filled
with a mixture of 50% perlite and 50% standard substrate. Thereafter, plants were potted in 2 l
containers with standard substrate (ED 73) and cultivated in a greenhouse chamber at an
average temperature of 24:22°C day:night with a photoperiod of 12 hours. Seven-week-old
grapevine plants with four to seven fully expanded leaves were used in all experiments.
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Beauveria bassiana strain ATCC 74040 was isolated from the commercial product Naturalis
®
(CBC (Europe) S.r.l. – BIOGARD Division, Italy). Naturalis is formulated as an oily dispersion
and contains 69.1 g/l of B. bassiana strain ATCC 74040 as active ingredient with a
7
concentration of at least 2.3 x 10 /ml viable spores. The isolate was maintained on a solid
medium at 24°C in the dark. The medium consisted of 10 g soy peptone (AppliChem,
Germany), 20 g glucose (Sigma-Aldrich, Germany) and 18 g Bacto™Agar (BD Difco, USA)
dissolved in 1000 ml ultrapure water and subsequently autoclaved for 20 min at 120°C.
To obtain spore suspensions, the conidia were harvested by gently scraping the surface of
Petri dishes containing 8-day-old B. bassiana cultures and suspending them in 20 ml sterile 1/8
concentrated Ringer’s solution containing 0.02% Tween 80. The conidia concentration was
7
determined using a Thoma haemocytometer and was adjusted to 2 × 10 conidia/ml. Both, the
®
freshly collected conidia suspensions and the formulated product Naturalis (1%) were used in
the experiments. Aliquots of 50 µl of spore suspensions were plated on Beauveria medium
using the Spiralplater WASP 2 (Meintrup DWS Laborgeräte GmbH). Concentrations of viable
49
conidial spores were calculated using the colony forming unit’s method. Germination rate was
®
100% for conidial spores present in Naturalis and around 70% for the spore suspensions of
isolate ATCC 74040. Accordingly, the concentration of viable conidia applied onto plants was
7
1.4 × 10 conidia/ml.
Plasmopara viticola was maintained on potted grapevine plants (in vivo) and infected leaves
with visible sporangia on the abaxial side were collected and stored at -20°C. For inoculation
of grapevine plants, these leaves were used to prepare a suspension containing approximately
5
1 × 10 sporangia/ml. One week before inoculation of the plants used for the experiments, one
infection cycle was carried out on living plants to get fresh sporangial material.
5.2.2 Treatment of plants with B. bassiana
Seven-week-old grapevine plants with four to seven fully expanded leaves were used for
®
treatment with either B. bassiana conidial suspensions or the commercial product Naturalis
(1%). For each treatment, 40 replicate plants were sprayed at the adaxial and the abaxial
surfaces of all fully expanded leaves using a 2 l one-hand pressure sprayer. Control plants
were sprayed with tap water. Position of the last fully expanded leaf was labeled using a
tapener (Max tapener HT-B, Max Staple, Japan). Treated plants were kept in a greenhouse
chamber (daily mean temperature 23-25 °C, daily mean relative humidity 50-70%) and were
watered regularly. This procedure has been shown to allow the successful endophytic
establishment of B. bassiana in grapevine plants (Rondot and Reineke 2018).
For analyzing effects of endophytic B. bassiana on grapevine gene expression levels, 30 potted
grapevine plants were treated with a B. bassiana conidia suspension or sterile 1/8 concentrated
Ringer’s solution containing 0.02% Tween 80 as a control as described above. 24, 72 and 168
hours post treatment (hpt) one leaf of each grapevine plant was carefully cut at its base from
the plant and was immediately shock-frozen in liquid nitrogen before storage at -80°C prior to
RNA extraction.
5.2.3 Inoculation of plants with P. viticola
For assessment of the preventive activity of endophytic B. bassiana against downy mildew
grapevine plants with endophytic B. bassiana were inoculated with P. viticola three and seven
days after treatment (dat) with B. bassiana, respectively. To obtain fresh P. viticola sporangia
containing zoospores infected leaves were carefully washed by spraying tap water at the
5 6
abaxial side. The concentration of the sporangial solution was adjusted to 10 -10 sporangia
-1
ml using a Thoma haemocytometer.
Ten plants per treatment were inoculated in the first replicate experiment. For the second and
third replicate experiment the number of plants was increased to 15 per treatment.
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5 Manuscript III
Sporangia suspension was sprayed, using a handheld sprayer, on the abaxial leaf surface.
After inoculation, grapevine plants were immediately covered with a dark plastic wrap,
previously moistened with tap water, for 24 h to create an ideal microclimate for the infection
process and disease development. After 24 h the plastic wrap was removed. In order to induce
sporulation, plants were wrapped again for twelve hours overnight at the end of the
incubation period on day seven after inoculation. The whole experimental setup was
replicated individually in July 2013, autumn 2013, and July 2014.
5.2.4 Disease assessment
Disease severity (percentage of leaf surface covered by sporulation) was visually estimated on
ten leaves of each plant using the disease severity scheme from guideline EPPO/OEPP PP 1/31
(3) by the European Plant Protection Organization (EPPO 2001). Example leaves of each
disease severity group can be found in Figure 17 (supplementary material). Leaves were
selected according to the labeling conducted before treatment and as indicated in Figure 10
and assigned to one of the twelve grades of disease severity (Figure 17, supplementary
material). Based on the disease severity found in the treatment and in the control, the efficacy
of the treatments with endophytic B. bassiana was determined according to Abbott’s formula
(Abbott 1925). Disease incidence was calculated as the number of leaves with visible
sporulation divided by the total number of leaves and was expressed as percentage.
Differences between treatments in mean disease severity of each leaf level (Figure 10) were
analyzed with Kruskal-Wallis test using Dell Statistica data analysis software system (Dell
Inc., version 13, software.dell.com). Disease incidences were compared using a Kruskal-Wallis
rank sum test followed by multiple comparisons by Dunn (1964) with p-values adjusted by the
Benjamini-Hochberg method. These analyses were calculated using the R-programming
language (R Core Team 2019) and graphs were produced with the R-Package ggplot2
(Wickham 2016).
51
Figure 10: Time schedule of experiments and assignment of leaf levels of potted grapevine plants
for the disease severity assessment. As an example, assessment of disease severity 14 days after
treatment with B. bassiana and 7 days after inoculation with P. viticola is shown. Disease severity
assessment was conducted based on a scheme with twelve grades of disease severity, according to
EPPO (2001) and Figure 17.
5.2.5 Assessment of endophytic colonization
Ten plants of each treatment were used for confirmation of endophytic colonization of B.
bassiana by re-isolation of the fungus following surface sterilization of the leaves. At each day
of inoculation and at the end of the experiment (3, 7 and 14 dat) one leaf from each of the 10
replicate plants per treatment was excised and individually surface sterilized under sterile
conditions by dipping in 0.5% NaOCl (active chlorine) containing 0.05% Tween 80 for 2 min,
followed by 70% EtOH for 2 min. Finally, the leaves were dipped twice in sterile water each
for 1 min and additionally rinsed with sterile distilled water. The success of this disinfection
process was assessed by plating three replicates of 200 µl of the residual rinse water on PDA
(potato dextrose agar). No fungal growth was recorded in any of the rinse water samples after
21 days of incubation. After surface sterilization, eight leaf discs (d = 0.8 cm) were obtained
with a sterile cork borer from each leaf. The leaf discs were placed on Beauveria selective
medium (BSM), the same solid medium as indicated above but supplemented with 0.1 g/l
streptomycin (Sigma-Aldrich, Germany), 0.05 g/l tetracycline (Sigma-Aldrich, Germany), 0.1
g/l dodine (as aliquot of the product Syllit, Spiess-Urania Chemicals, Germany) and 0.05 g/l
cycloheximide (Sigma-Aldrich, Germany). This medium is based on a medium initially
described by Strasser et al. (1996) for the isolation of B. brongniartii and adapted by Meyling
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5 Manuscript III
and Eilenberg (2006) for isolation of B. bassiana. The plates were incubated at 24° C in the
dark.
After 7 and 14 days of incubation the leaf discs were examined visually for the presence of any
fungal growth. Fungal tissue was characterized as endophytic B. bassiana, if characteristic
white dense mycelia, becoming creamy at the edge (Humber 1997) grew from internal plant
tissues of surface sterilized leaf discs. A final assessment of the presence of endophytic
B. bassiana was recorded after 14 days and was expressed as percentage colonization by
dividing the number of leaf discs exhibiting B. bassiana outgrowth by the number of total leaf
discs and multiplying the obtained value with 100. If one of the eight leaf discs obtained from
a single plant showed fungal outgrowth the total leaf was classified as being endophytically
colonized with B. bassiana.
5.2.6 RNA isolation
RNA was extracted from individual leaves using the Spectrum™ Plant Total RNA Kit (Sigma-
Aldrich, Steinheim, Germany) following the manufacturer’s protocol. Leaves were crushed
using liquid nitrogen and a total of up to 100 mg leaf tissue was used for RNA extraction.
Contaminating DNA was removed by digestion with 0.8 U DNase (Ambion, Heidelberg,
Germany) followed by lithium chloride precipitation. RNA purity and quantity were assessed
based on the absorbance ratio of 1.8 to 2.0 at 260/280 nm using a Nanodrop ND-1000
spectrophotometer (NanoDrop Technologies, Wilmington, USA).
5.2.7 Microarray analysis
For microarray analysis, twelve independent pools of RNA samples were constructed: For
both time points (24 h and 168 h) and B. bassiana treated or control plants, respectively, three
RNA pools each were generated. Each RNA pool contained an individual leaf from 9 biological
®
replicates. In this study, the Affymetrix GeneChip Vitis vinifera Genome Array was used.
Sample preparation for microarray hybridization was carried out as described in the
Affymetrix GeneChip 3’ IVT Express Kit User Manual (Affymetrix, Inc., Santa Clara, CA,
USA). In brief, 250 ng of total RNA were reverse transcribed into double-stranded copy DNA
(cDNA) followed by an in vitro transcription generating biotin-labeled amplified RNA (aRNA).
The length of the purified aRNA products was assessed using an Agilent 2100 bioanalyzer
(Agilent Technologies, Palo Alto, USA). Following fragmentation, 12 µg aRNA were
hybridized to Affymetrix GeneChip Vitis vinifera Genome Arrays for 16 h at 45° C and 60 rpm
in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an
Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an
Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by the
Affymetrix GeneChip Command Console v4.1.3 software. Sample processing and Affymetrix
53
microarray hybridization were performed at an Affymetrix Service Provider and Core Facility
(KFB - Center of Excellence for Fluorescent Bioanalytics; KFB, University of Regensburg,
Germany; www.kfb-regensburg.de).
Summarized probe set signals in log2 scale were background-adjusted, quantile normalized
and log-transformed by using the robust multi-chip average (RMA) algorithm (Irizarry et al.
2003) with the Affymetrix GeneChip Expression Console v1.4 Software. After exporting into
Microsoft Excel, average signal values, comparison fold changes, and significance p-values
(student’s t test) were calculated. Genes were regarded as being significantly up- or down-
regulated when the log ratio of the change in expression between a B. bassiana treated and a
control sample was >= 1 or <= -1 and the adjusted p-value was <= 0.05, with a log ratio of 1
representing a two-fold change in expression. Affymetrix probesets were annotated using the
NetAffx Annotation Files. Sequence information included public content from GenBank and
dbEST and was used to retrieve Gene Ontology (GO) annotations. To group similar classes
into wider groups, GO categories were associated to related biological processes using the
owltool map2slim (https://github.com/owlcollab/owltools/wiki/Map2Slim) on the basis of GO-
BASIC.obo und the PLANT-subset (http://geneontology.org/docs/download-ontology/).
Additionally, some terms were manually associated to related biological processes (TAIR). GO
terms were reconstructed using the R-programming language (R Core Team 2019) and the
GO.db-package (Carlson 2018). The complete microarray data set has been deposited in
NCBI's Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) (Edgar et al. 2002) and are
accessible through GEO Series accession number GSE132311.
5.2.8 RT-qPCR
Gene expression levels of three genes known to be associated with defense responses to
herbivore or pathogen attack (Table 5) were additionally assessed using RT-qPCR in eight B.
bassiana treated or control grapevine plants 24, 72 and 168 hpt, respectively. Gene-specific
®
primers were designed using the software Geneious 6.1.7 (Biomatters, New Zealand). For
primer design, a stringent set of criteria was used, which included a predicted melting
o
temperature of 60-65 C, primer lengths of 18-24 nucleotides, GC contents of 40-60 % and PCR
amplicon lengths of 80-200 nucleotides. Grapevine genes coding for actin and GAPDH were
used as a stable set of reference genes for endogenous quantification controls of gene
expression data. Primer sequences for reference genes were used as designed by Timm and
Reineke (2014). Melt-curve analysis was performed to check the specificity of each primer
pair. Furthermore, the efficiency and amplification performance of each primer pair was
evaluated using a tenfold-dilution series of a known template, analyzed with a minimum of
three independent technical replicates.
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5 Manuscript III
Individual RNAs from single leaves of each treated and control plants for each of the three
time points (24, 72 and 168 hpt) were used for RT-qPCR. The single RNA samples were diluted
to 100 ng/µl before cDNA synthesis. First strand cDNA was synthesized using 1 µg RNA with
the DyNAmo M-MuLV reverse transcription system (Finnzymes) with an oligo (dT)15 primer.
Quantitative real-time PCR reactions were performed on an iQ5 Multicolor iCycler (Bio-Rad)
using the DyNAmo™ ColorFlash SYBR® Green Kit (Finnzymes) according to the
manufacturer’s instructions. The single cDNA samples were diluted 1:40 before qPCR
analysis. Amplifications were performed in a total volume of 25 µl using 2 µl cDNA as
template, 10 pmol of each primer and 12.5 µl DyNAmo master mix. As control reactions,
nuclease-free water replaced the cDNA template. For standard template reactions, a two-step
cycling program was used consisting of 95°C for 3 min followed by 40 cycles of 95°C for 10 s
and 60°C for 30 s. A minimum of three independent technical replicates was performed for
each cDNA template with each primer pair.
Table 5: Primer sequences and PCR characteristics of two grapevine reference genes (Actin,
GADPH) and three defense-related genes used in RT-qPCR experiments.
Amp. PCR
Gene Identification Primer Sequence 5'-3'
Length (bp) efficiency (%)
Actin AY847627 for GCCTGATGGGCAAGTCAT 244 92.3
rev TGGGAGCAAGAGCAGTG
GAPDH EF192466 for TCAAGGTCAAGGACTCTAACACC 226 97.0
rev CCAACAACGAACATAGGAGCA
ATPase LOC100251261 for TTTCGCCCATCAGGTACAGC 146 95.1
rev TGAAACGCCTTGAGCTGGAA
PR-1-like LOC100256515 for GTCACAAACAACCCGAGCAC 168 94.3
rev AACGGCGATACATGGACTCC
beta-1,3- LOC100233076 for GACAGGACGCCACTCTTGAA 148 124.4
Glucanase rev TTGTTCTCCCTGCCATGCAA
Quantification cycle (Cq) values were calculated using the iQ5 version 2 software (BioRad).
Reference genes were evaluated based on expression stability (M values) and coefficients of
variation (CV) using qBasePlus software (Biogazelle, Zulte, Belgium). Target sample
expression levels were normalized based on three independent technical replicates with
relation to mean Cq values of the two reference genes. Quantification of gene expression was
calculated using the method implemented in qBase software (Hellemans et al., 2007), which
allows the inclusion of multiple reference genes for normalization and corrects for different
amplification efficiencies. Statistical differences between expression levels of treated and
control leaves at the three time points were calculated on the basis of the calibrated
normalized relative quantities (CNRQs) using the Mann-Whitney-U test, with a p-value of <
0.05 considered to be significant.
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5.3 Results
5.3 Results
5.3.1 Endophytic colonization
Plant colonization with B. bassiana was determined 3, 7, and 14 days after treatment with a
®
conidia suspension or the product Naturalis by culture-based re-isolation of the fungus.
Depending on sampling date and experimental replicate between 10 and 100% of the ten tested
leaves per plant were categorized as being endophytically colonized (Table 6). Throughout the
experiments, a higher re-isolation rate was achieved after treatment with a conidia suspension
®
in comparison with a Naturalis treatment. The highest colonization percentage was recorded
7 dat in plants inoculated with conidia suspension in July 2014 (100%). We could not detect
any decline in colonization percentage over time but recorded a high variability between the
three experimental replicates. No fungal colonization was observed in any of the control
plants.
Table 6: Percentage of colonized leaves per plant 3, 7 and 14 dat with a B. bassiana conidia
suspension or the product Naturalis® in three different experimental replicates (July 2013, autumn
2013 and July 2014). None of the control plants showed fungal outgrowth (not shown).
Colonized leaves per plant (%)

3 dat 7 dat 14 dat
July 2013 30% 20% 30%
Conidia
susp.
Autum 2013 80% 80% 60%

July 2014 90% 100% 80%
Naturalis®
July 2013 10% 10% 20%

Autum 2013 10% 10% 10%
July 2014 40% 80% 60%
5.3.2 Preventive activity against Plasmopara viticola
A treatment of potted grapevine plants with B. bassiana as conidia suspension or the product
®
Naturalis three and seven days before inoculation with P. viticola resulted in a reduction in
disease severity (percentage leaf area infected) of downy mildew in all experimental
replicates. The results of the experimental replicates in July 2013 (Figure 11), autumn 2013
(Figure 12) and July 2014 (Figure 13) are presented as mean disease severity for the ten
assessed leaves per plant and treatment. The varying disease severity on the ten leaves of the
control plants reflects the different susceptibility of the leaves depending on their leaf level (1-
10) and thus age. Basically, the susceptibility of grapevine leaves to downy mildew decreases
with increasing leaf age. Despite of this, the uppermost leaves (leaf level 1-3) usually showed a
lower disease severity level, since they were not yet unfolded or still very small at the time of
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5 Manuscript III
inoculation. This effect was less pronounced with a longer period (7 dat) between the
treatment with B. bassiana and the inoculation with P. viticola. Both in July 2013 and in July
2014, therefore, a significant reduction (p<0.05) in disease severity was only evident starting at
leaf level 3 and subjacent. Although the disease pressure of P. viticola was lower in 2014, the
antagonistic effect of an application of B. bassiana conidia suspension or the product
®
Naturalis was visible. A significant reduction (p<0.05) of mean disease severity of both
treatments was examined on all leaf levels except leaf level 1 and 2 with a P. viticola
inoculation 3 dat with B. bassiana.
plants treated with B. bassiana (conidia suspension or Naturalis®) A) 3 und B) 7 days before the
inoculation with P. viticola. Leaves were examined from the upper (1) to the lower (10) leaf level of the
plants (see Figure 10). Asterisks indicate significant differences between the treatments with p ≤ 0.05
(*). Experimental replicate of July 2013.
57
5.3 Results
(*). Experimental replicate of autumn 2013.
(*). Experimental replicate of July 2014.
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5 Manuscript III
Figure 14: Boxplots of percentage disease incidence of grapevine plants inoculated with P. viticola
3 und 7 days after a treatment with endophytic B. bassiana (conidia suspension or Naturalis®). Control
plants were only inoculated with P. viticola. Different letters indicate significant differences between
the treatments at p<0.05 (Dunn’s multiple comparison after Kruskal-Wallis rank sum test).
Experimental replicates of A) July 2013, B) autumn 2013, and C) July 2014.
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5.3 Results
Incidence of downy mildew (percentage of leaves with visible sporulation) was significantly
reduced following a treatment with endophytic B. bassiana. In experimental replicate of July
®
2014 (Figure 14 C) disease incidence of grapevine plants after treatments with Naturalis or
conidia suspension differed significantly from control plants for both inoculation time points
-05 -06 -06 -04
(3 dat: p = 6.58x10 and p = 6.57 x 10 , 7 dat: p = 1.78 x 10 and p = 1.31 x 10 ,
respectively). Also in experimental replicate of autumn 2013 (Figure 14 B) disease incidence of
®
grapevine plants after treatments with Naturalis or conidia suspension differed significantly
from control plants for both inoculation time points (3 dat: p = 0.00781 and p = 0.00368, 7 dat:
p = 0.0214 and p = 0.000840, respectively). In experimental replicate of July 2013 (Figure 14 A)
disease incidence of grapevine plants only differed significantly from control plants after
®
treatment with Naturalis for inoculation time point 3 dat (p = 0.0484).
Table 7: Mean efficiency according to Abbott against downy mildew on grapevine leaves by a
treatment with B. bassiana (conidia suspension or Naturalis®) 3 und 7 days before the inoculation with
P. viticola in three different experimental replicates (July 2013, autumn 2013 and July 2014).
Mean efficiency according to Abbott

3 dat 7 dat
conidia conidia
Naturalis® Naturalis®
susp. susp.
July 2013 3.45 32.13 43.15 61.82
autumn 2013 64.63 45.68 28.31 26.43
July 2014 89.03 72.06 78.41 67.89
The calculated mean efficiencies according to Abbott (Table 7) confirm the observations of a
reduction in downy mildew disease severity and disease incidence due to treatment with
endophytic B. bassiana. For data obtained in the last experimental run in July 2014, the
efficiencies differed between 67 and almost 90%, despite a low disease pressure of P. viticola. In
the previous runs carried out in 2013, the efficiencies were significantly lower, usually below
50%. The efficiencies were averaged over all leaves, thus including also the upper leaves which
were probably not colonized with B. bassiana.
5.3.3 Changes in expression patterns after treatment with B. bassiana

®
The Affymetrix GeneChip V. vinifera genome array represents comprehensive parts of the
30,344 genes predicted in V. vinifera. It consists of 16,436 probesets: 14,496 derived from V.
vinifera transcripts and 1940 derived from other Vitis species or hybrids transcripts. The
®
design of the Vitis GeneChip is based on sequences selected from GenBank, dbEST, and NCBI
Reference Sequences (RefSeq).
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5 Manuscript III
With microarray analysis changes in global gene expression of B. bassiana treated grapevine
plants were found both 24 and 168 h after treatment. Differences in gene expression between
B. bassiana treated and control leaves allowed the identification of differentially expressed
genes which were up-regulated or down-regulated as a response to endophytic B. bassiana.
Overall, the transcriptional response of grapevine plants due to treatment with B. bassiana
was higher 24 hpt and declined 168 hpt. At 24 hpt 65 transcripts of the 16,436 analyzed
transcripts were significantly up-regulated (fold change in gene expression levels >2), and 25
transcripts were significantly down-regulated (fold change <-2) compared to the control
plants. Whereas 168 hpt with B. bassiana only 14 genes were significantly induced (fold
change >2) and 14 genes were repressed (fold change <-2) in response to endophytic
B. bassiana. The strongest transcriptional response of grapevine plants was evident at an early
stage of the colonization process (24 hpt) with B. bassiana with 16 transcripts being
significantly up-regulated with a fold change greater than 3 and two transcripts being
significantly down-regulated with a fold change lower than -3 compared to the control plants.
One transcript with unknown gene ontology was up-regulated more than six-fold as a
response to B. bassiana treatment. 168 hpt, no transcript showed higher changes than three in
the expression level due to B. bassiana treatment. The significantly regulated genes (factor > 2
and < -2 against the control) 24 and 168 hpt with B. bassiana are reported in Table 9 and Table
10 (supplementary material).
Most of the genes associated to biological processes like defense response or response to biotic
stimulus had similar expression patterns as a result of treatment with B. bassiana 24 and 168
hpt. However, some genes like the pathogenesis-related protein 10.3 (RefSeq Protein ID
XP_002274483), major allergen Pru ar 1-like (RefSeq Protein ID XP_002274785) and disease
resistance response protein 206-like (RefSeq Protein ID XP_002266825) were induced 24 hpt
with endophytic B. bassiana. In addition we found that the expression level of genes encoding
pathogenesis-related proteins PR-2 (b-1,3-glucanases; RefSeq Protein ID XP_002277446), PR-3
(chitinases; e.g. RefSeq Protein ID XP_002266583), and PR-5 (thaumatin-like proteins; e.g.
RefSeq Protein ID XP_002274443) increased upon treatment with B. bassiana within 24 h.
However, expression of protein PR-1-like (RefSeq Protein ID XP_002276867) was repressed in
grapevine plants 24 hpt with endophytic B. bassiana. While expression of all four genes was
not significantly affected after 168 hpt with B. bassiana, pathogenesis-related protein PR-1
(RefSeq Protein ID XP_002273788) was significantly down-regulated in grapevine plants as a
response to treatment with endophytic B. bassiana. Also, various genes involved in stilbene
synthesis and related genes (e.g., RefSeq Protein ID XP_002269293 or XP_003634066), which
are predominantly categorized to the GO response to stress, were up-regulated 24 hpt with
endophytic B. bassiana.
61
5.3 Results
Gene Ontology slim tools were used to identify major groups of biological processes affected
by treatment with endophytic B. bassiana. Main groups of GO slim classes associated to
significantly regulated genes are represented as a bar chart in Figure 15. 168 hpt only nine (4
induced, 7 repressed) GO slim categories were influenced by treatment with B. bassiana,
whereas 24 hpt all 14 categories were influenced (9 induced, 11 repressed). Categories
involved in anatomical structure morphogenesis, cell death, flower development, and signal
transduction were only represented by inhibited genes 24 hpt. As highlighted in Figure 15,
some GO slim categories were mostly represented by induced genes 24 hpt with B. bassiana.
In particular, more genes categorized to metabolic process (GO:0008152), response to stimulus
(GO:0050896) and response to stress (GO:0006950) were up-regulated than down-regulated.
These were also the three processes, which were most strongly affected by treatment with
endophytic B. bassiana, with each process representing around 20% of all significantly
regulated and assigned transcripts.
Figure 15: Main groups of GO slim classes (y-axis labels, ordered regarding their impact level)
concerning the biological processes affected in grapevine plants after treatment with B. bassiana. The
figure shows the number of transcripts assigned to terms of Gene Ontology biological processes
induced or repressed by B. bassiana treatment 24 hpt and 168 hpt. GO slim classes with N=1 are not
shown.
62
5 Manuscript III
In a second experimental approach, differences in expression levels of three selected defense-

related genes were assessed by RT-qPCR in grapevine plants after treatment with B. bassiana
(24, 72 and 168 hpt). The three designed primer pairs showed adequate performance in
amplification, in melt curve analysis and in investigation of the efficiency by means of
standard curves. A combination of the two grapevine housekeeping genes (GADPH and actin),
was found to be suitable as reference for normalization of gene expression (M = 0.850,
CV = 0.299 and M = 0.850, CV = 0.293, respectively). After normalization and correction of the
differences between the individual reaction plates, only the PR1-like gene showed a significant
difference in the expression levels between B. bassiana treated and control plants 24 and 168
hpt (Table 8). These findings confirm the result of the microarray analysis, in which the
transcript of this gene was also down-regulated by a factor of three 24 hpt.
63
5.3 Results 64
Table 8: Mean [95% CI] expression levels of three genes 24, 72 and 168 hpt of grapevine with B. bassiana analyzed with RT-qPCR. Asterisks indicate a significant
difference between treated and control plants with p ≤ 0.05 (*)
24 hpt 72 hpt 168 hpt
conidia susp. control p-value conidia susp. control p-value conidia susp. control p-value
0.151 0.54 0.01119* 0.531 1.555 0.3618 9.021 2.590 0.04545*

PR-1-like
[0.102, 0.225] [0.263, 1.109] [0.114, 2.477] [1.007, 2.402] [6.283,12.95] [0.915, 7.335]
beta-1,3- 1.589 0.667 0.06014 0.924 1.015 0.9551 1.341 0.963 0.2695
Glucanase [1.09, 2.317] [0.364, 1.258] [0.409, 2.087] [0.562, 1.835] [1.047, 1.719] [0.545, 1.700]
0.851 1.259 0.152 0.937 0.896 0.9551 1.431 0.985 0.3939

ATPase
[0.573, 1.263] [0.867, 1.829] [0.578, 1.52] [0.505, 1.591] [0.711, 2.879] [0.407, 2.384]
5 Manuscript III
5.4 Discussion
Here, we showed that endophytic B. bassiana is able to reduce downy mildew disease severity
and incidence on grapevine plants and that its colonization triggers the plant's inherent
defense system. The results of this study add to the increasing evidence of supplementary
positive effects of entomopathogenic fungi when present as an endophyte in crop plants as
already described by Vega et al. (2009) and Vidal (2011). In addition to the recently proven
antagonistic effect of endophytic B. bassiana against grapevine insect pests (Rondot and
Reineke 2017, 2018), a protective effect of endophytic B. bassiana against the causal agent of
grapevine downy mildew, P. viticola, was evident. In line with results reported by Jaber (2015),
both the disease incidence and severity on grapevine plants were reduced following treatment
with B. bassiana. Grapevine plants were treated with B. bassiana before the infection with
P. viticola. Therefore, our experimental set-up only allows an assessment of the protective
potential of B. bassiana. As explained by Gessler et al. (2011), it is hard to control P. viticola
with biocontrol antagonists after infection because the fungus quickly penetrates and
develops inside host tissues.
The observed positive effect of a treatment with B. bassiana against downy mildew was
particularly noticeable in older leaves and after a longer establishing period of seven
compared to three days. A period of seven days between the treatment with B. bassiana and
the inoculation with P. viticola also showed substantial reductions in disease severity in the
study by Jaber (2015). These observations, therefore, indicate the necessary time period the
fungus B. bassiana needs for endophytic establishment. Accordingly, the state of plant
development and leaf growth must be taken into account, when considering the protective
effects of a treatment with B. bassiana against any fungal pathogen. To our knowledge, leave
susceptibility and timing of the treatment were not yet considered in previous experiments
regarding the tritrophic interaction between grapevine plants, endophytes, and
phytopathogens. However, on leaves developed after the treatment with B. bassiana, downy
mildew disease severity was not significantly reduced, supporting previous studies, where
systemic colonization of grapevine plants by B. bassiana could not be detected (Rondot and
Reineke 2018).
With a reduction of 3 to 89 % of P. viticola disease severity by colonization with endophytic

B. bassiana, efficiencies are lower than those reported and expected for a treatment with
synthetic fungicides. Our findings showed a higher variation in downy mildew disease
reduction of B. bassiana strain ATCC 74040 than observed by Jaber (2015) but are in line with
research results of other potential microorganisms against P. viticola reviewed by Gessler et al.
(2011). Despite an overall good activity of the respective microorganisms, they were not
65
5.4 Discussion
capable of completely controlling downy mildew disease. As already mentioned above, one
reason for this can be found in the nature of P. viticola, which penetrates the leaves very
rapidly through the stomata. Moreover, most of the microorganisms tested so far are
insufficient to control this disease, because they have a low persistence with only short
periods of activity after application or are easily washed off by precipitation (Pertot et al.
2017). The endophytic lifestyle of B. bassiana in grapevine plants may accordingly lead to
longer persistence and thus longer periods of activity. However, this assumption warrants
further studies.
In the present constellation of an endophytic entomopathogenic fungus, grapevine and downy

mildew, the mechanisms underlying the protective potential of the endophyte against the
pathogen are not yet known. In general, the following mechanisms are possible: antibiosis,
competition for space and nutrients, parasitism, and induction of plant defense. Since it has
been shown that the fungus colonizes the plants, competition for space or resources might be
involved in the protection mechanism. Successful competition depends on both timing and
magnitude of colonization as resources and nutrients are supposed to go to the initial and best
plant colonizer. In the present study, plant colonization with B. bassiana was confirmed by the
time of plant inoculation with P. viticola (3 dat and 7 dat) as well as the latest date of disease
assessment (14 dat). Although percent colonization of plants differed among the replicates and
the tested treatments, B. bassiana was able to provide protection against downy mildew in all
experiments. A direct linkage between colonization rate and disease severity was not possible
due to methodical aspects. However, this supports the hypothesis of Vega et al. (2009) and
Ownley et al. (2010) that multiple mechanisms of biocontrol might be operating in B. bassiana-
colonized plants.
Activation of plant-mediated systemic resistance could be another possible mechanism of

suppression of P. viticola in B. bassiana-colonized grapevines. Griffin et al. (2006) and Ownley
et al. (2008) suspect this mechanism to operate against Xanthomonas spp. in cotton seedlings
after treatment with endophytic B. bassiana. A similar ability to induce systemic resistance in
grapevine plants against grapevine downy mildew, the disease investigated here, has already
been demonstrated for other non-pathogenic fungi such as Trichoderma harzianum, which
caused a direct modulation of defense-related genes and the activation of priming (Perazzolli
et al. 2008). However, in contrast to our findings, where we could not detect a systemic effect
of B. bassiana against downy mildew, in the aforementioned study, homogeneous disease
resistance was observed, independent of leaf position.
In the present study, we observed that a set of genes in grapevine leaves were up- or down-
regulated following the treatment with endophytic B. bassiana. Genes associated to biological
processes like defense response or response to biotic stimulus are of particular interest for
66
5 Manuscript III
understanding the tritrophic interaction between grapevine and endophytic B. bassiana and its
antagonistic potential against insects or pathogens. We observed that the expression level of
genes encoding PR-2 (b-1,3-glucanases), PR-3 (chitinases), PR-5 (thaumatin-like proteins), and
the PR-protein 10.3 were increased upon the treatment with B. bassiana within 24 h after
treatment. These results confirm previous reports that these genes are involved in the defense
response of vines to infestation with pathogens (Enoki and Suzuki 2016; Fung et al. 2008;
Albertazzi et al. 2009; Jacobs et al. 1999; Ferreira et al. 2004; Kortekamp 2006; Adrian et al.
2012) and are also related in response to colonization by biotrophic fungi (Perazzolli et al.
2008). In addition, other defense-related genes like genes associated to the stilbene synthesis
and related genes were found to be regulated. Stilbenes are also known to be involved in the
plant-pathogen interaction of grapevine (Schnee et al. 2008; Olivier et al. 2018; Adrian et al.
2012), therefore providing indications of the interaction between endophytic fungus and
grapevine plant. Induced expression of key defense genes strongly suggested that a defense
response was activated in grapevine plants due to treatment with endophytic B. bassiana.
However, most of the genes were only regulated 24 hpt and expression of defense-related
genes, including PR genes and genes associated to the stilbene synthesis, declined within 168
hpt. Similar results are reported for the grapevine reaction to powdery mildew by Fung et al.
(2008), who observed the expression of defense-related genes and secondary metabolite
biosynthesis genes to reach a maximum level at 12 hours post inoculation and then declined.
Such a decline in expression of defense-related genes suggests that the plant and B. bassiana
may establish a symbiotic relationship. Perazzolli (2008) emphasizes the importance of
repeated applications of T. harzianum T39 to significantly induce plant resistance against
downy mildew in grapevine plants. A similar approach with B. bassiana remains to be
investigated. We already found significant reductions of downy mildew with a single
protective treatment of endophytic B. bassiana. Yet, we did not analyze the expression level of
defense-related genes after a post treatment inoculation with P. viticola. So we can only
hypothesize if grapevine enters a ‘‘primed state’’ that results in broad-spectrum resistance to
pathogens, insects, or abiotic stress as described by Conrath et al. (2006).
Successful plant protection strategies against P. viticola based on microorganisms will need to
target P. viticola at multiple sites and multiple stages of its life cycle (Vecchione et al. 2007).
B. bassiana, with its diverse roles and multiple modes of action, could represent one
component in such a strategy with the potential of reducing frequencies of chemical pesticide
applications. Its ability to act both as an epiphyte and endophyte with effects against both
insect pests and pathogens might be the answer to overcome the poor persistence and efficacy
of other microorganisms used as a biocontrol control agents, which are applied on the leaf
surface. Nevertheless, for optimal exploitation of B. bassiana as part of a new and sustainable
67
5.4 Discussion
plant protection strategy in viticulture, an identification of the relevant operating mechanisms

is required.
Acknowledgments
This work was financially supported by Deutsche Bundesstiftung Umwelt (DBU). We thank
®
Edith Ladurna from CBC Europe S.r.l. (Italy) for providing the product Naturalis and Dustin
Kulanek for help in the performance of the RT-qPCRs. We kindly acknowledged Dominik
Schmidt for support with the GO slim tools and Matthias Olberz for assistance in statistical
analyses in ‘R’.
68
6 General discussion
The increased awareness of additional roles that entomopathogenic Hypocreales may play in
the ecosystem led to numerous research projects during the last years. Particularly, the
endophytic lifestyle is viewed as a promising function with enormous potential in the
development of novel integrated crop protection tools and as a component of environmentally
friendly pest management strategies. However, despite the newly recognized importance of
this additional role much remains unknown about the ecology and environmental interaction
of endophytic entomopathogens. For instance, only limited information is available on the
tritrophic interaction between entomopathogenic endophytes, grapevine plants, and potential
target insect pests as well as fungal pathogens. Therefore, the objective of the present thesis
was to investigate whether the fungus B. bassiana is able to colonize grapevine plants, still
maintains its entomopathogenic habit against insect pests, and has additional antagonistic
potential against fungal pathogens. Additionally, this thesis focused on the plant response to
endophytic colonization on gene level. The investigations were carried out with regard to the
development of potential biological control strategies.
The results of the three conducted studies were already discussed in the respective
manuscripts. Here, the main findings and conclusions will be summarized again by referring
back to the objectives (see chapter 2). Furthermore, additional aspects will be emphasized
based on limitations as well as applications and implications of the studies to associate the
experiments to the context of current research. Therefore, this supplementary discussion
focuses on three aspects: (1) alternative strategies for and restrictions in the endophytic
establishment of B. bassiana in grapevine plants; (2) challenges in characterization of the
antagonistic potential of endophytic B. bassiana; (3) potential applications of the experimental
results in viticultural practice. Finally, future prospects for research objectives are given.
Alternative strategies for and restrictions in the endophytic establishment of B.

bassiana in grapevine plants
The aim of this thesis was to optimize the endophytic establishment of B. bassiana in
grapevine plants via artificial application. Amongst others, the endophytic establishment of
entomopathogenic fungi is known to be dependent on plant cultivar and fungal strain (Vidal
and Jaber 2015). For both experimental sites, the greenhouse and the field, we used Vitis
vinifera cv. ‘Riesling’ as these plants are most frequently planted in the German Rheingau
region where the experiments were carried out. Since the identification of strains suitable for
69
endophytic establishment is time-consuming, we decided to focus on the already registered

® ®
and formulated strains ATCC 74040 (product Naturalis ) and GHA (product Botanigard ).
Strain ATCC 74040 was also reported to be the strain with the highest colonization rates by
Jaber (2015), who compared the endophytic establishment in grapevine plants of four different
B. bassiana strains. Another possibility to achieve a successful endophytic establishment is to
select a suitable inoculation method. Since plants used for our greenhouse trials were
cultivated from hardwood cuttings according to common practice for cultivation and grafting
of grapevine plants in nurseries, seed treatment as an alternative inoculation method as it has
been successfully shown for tomato, cotton (Ownley et al. 2008), opium poppy (Quesada-
Moraga et al. 2009) and sorghum (Tefera and Vidal 2009) was not possible. Inoculation trials
via root dipping or soil inoculation by drenching or mixing conidia containing material in the
planting substrate resulted in no colonization of the leaves (own unpublished results).
Therefore, inoculation via spray application is apparently the only option for endophytic
inoculation of grapevine plants/leaves and in addition simple to implement in viticultural
practice due to already existing spray equipment for other pesticides.
Greenhouse and field experiments showed that endophytic establishment by the

entomopathogenic fungus B. bassiana in potted and mature grapevine plants after artificial
spay inoculation is possible. Although B. bassiana is reported to colonize some plants, i.e.
opium poppy and corn, systemically (Quesada-Moraga et al. 2006; Quesada-Moraga et al. 2009;
Landa et al. 2013; Wagner and Lewis 2000), in all of our studies we could not detect systemic
colonization by B. bassiana in the grapevine plants. In contrast to the plants referred to in the
aforementioned studies, grapevines are deciduous, woody perennial plants, which might be
the reason for the different behavior of B. bassiana inside this crop plant. As already pointed
out by several authors, many endophytes of leaves or other plant tissues are host, host genus
or host family specific (Arnold 2007; Hyde and Soytong 2008). Further tests regarding the
optimization of endophytic establishment of B. bassiana in grapevine, therefore, should
consider other grape varieties as well as different fungal strains. Although, the literature
indicates a frequent association of B. bassiana with multiple plant species, with reports of
isolation from different plant organs (see Table 1), the genotypic basis – of the fungus as well
as of the host – for endophytism and the biological mechanism involved in proliferation
within a host plant have not been elucidated. As soon as the plant recognizes the presence of a
potential fungal invader, defense pathways are activated, leading to the suppression or death
of the fungus (Dangl and Jones 2001). Therefore, fungal endophytes must somehow be able to
indicate that they are not pathogens (Redman et al. 2001; Rodriguez et al. 2009). The
endophyte may be restricted in terms of distribution and metabolic activity within plant
tissues; remain localized in a nearly dormant phase or proliferate systemically throughout
multiple tissues of the host (Rodriguez et al. 2009). Studies reporting the exact location of the
70
fungi within plant tissues are extremely rare. So far, colonization by endophytic B. bassiana is
reported to occur through intercellular spaces and vascular xylem elements directly after the
fungus has penetrated the plant epidermis (Wagner and Lewis 2000; Quesada-Moraga et al.
2006; Landa et al. 2013). The attempt to visualize the endophytic colonization of B. bassiana in
Z. mays by light and electron microscopy demonstrated fungal colonization of inoculated
plant tissue (Wagner and Lewis 2000; Gómez-Vidal et al. 2006). However, potential other
resident fungal endophytes were neither identified nor differentiated from the inoculum.
More recently, B. bassiana isolate EABb-04/01-Tip was transformed with a green fluorescent
protein (GFP) to visualize association with opium poppy using a confocal microscope (Landa
et al. 2013). During a stay at Rusty Rodriguez’ company Adaptive Symbiotic Technologies,
Seattle (USA) within a short term scientific mission (STSM) of the Cost Action Fa1103
‘Endophytes in Biotechnology and Agriculture’ we also tried a GFP-transformation of
B. bassiana strain ATCC 74040 according to a method described by Maciá-Vicente et al. (2009)
to visualize the fungus in planta (own unpublished results). The transformation by
electroporation of the Agrobacterium tumefaciens AGL-1 strain was successful. However, it
was not possible to select the transformed colonies from untransformed ones with the method
of choice because of an existing tolerance of the used B. bassiana strain against hygromycin –
the agent chosen for the selection. Due to time restriction, an adaption of the method could
not be realized during the stay. Although, Quesada-Moraga et al. (2006) and Landa et al. (2013)
predominantly observed hyphal growth on or near epidermal cells and not into stomata, Jaber
(2015) hypothesizes the survival of B. bassiana in substomatal chambers of the leaves as was
described for a strain of Burkholderia sp when colonizing grapevines (Compant et al. 2005;
Compant et al. 2008).
Summarized over all conducted experiments, we found plant colonization by endophytic

B. bassiana to be highly inconsistent even with high inoculum loads. According to Wagner
and Lewis (2000), who observed the mode of penetration of B. bassiana conidia into the leaves
of maize, approximately 3% of the applied conidia germinate and less than 1% succeeded to
penetrate the leaf surface. Also, Quesada-Moraga et al. (2006) rarely observed germinating and
penetrating conidia on the leaves of opium poppy, Papaver somniferum. With a Color 3D Laser
Scanning Microscope, we observed conidia on the abaxial and axial surface of grapevine
leaves after application, hypothesizing that they belong to B. bassiana (own unpublished
results). In addition some hyphae, but no germinating conidia were detected. However, it was
not possible to definitively differentiate between other fungal residues and the inoculum
fungus. The endophytic growth and occurrence of B. bassiana in the plant that was detected in
our studies as well as in many other reports appears to be opportunistic (random) irrespective
of the used inoculation method (Wagner and Lewis 2000; Quesada-Moraga et al. 2006; Behie et
al. 2015; Landa et al. 2013; McKinnon et al. 2017). Despite the comparatively low endophytic
71
colonization rates of B. bassiana in grapevine, we observed antagonistic effects of endophytic

B. bassiana on vine mealybug larvae, black vine weevil, and downy mildew. As will be further
addressed in the next section of the discussion, not all of these effects can be assigned
exclusively to the endophytic lifestyle of B. bassiana.
Challenges in characterizing the antagonistic potential of endophytic B. bassiana
The study of endophytes is generally regarded as method-dependent, thus problematic and

accompanied by some flaws (Hyde and Soytong 2008). The main challenge of endophyte
research is to differentiate endophytes from epiphytes and to assign observed effects to each
lifestyle. The widely used surface sterilization is shown to be inadequate or ineffectively to
remove epiphytic DNA or inoculum depending on the type of experiment (McKinnon et al.
2017). Therefore it is advised by McKinnon et al. (2017) to consider the fungus present on the
plant surface as an epiphyte as an additional contribution to observed effects on plants, plant-
associated insects or the plant’s microbiome. In the experiments with P. viticola, the
microarray experiments as well as in the choice assays with O. sulcatus we used whole potted
grapevine plants, accordingly it was not possible to surface disinfect these plants before usage.
Therefore, it is stated that not all effects after spay application of B. bassiana reported in this
thesis could be assigned to the endophytic lifestyle of the fungus. The possibility that residual
B. bassiana surviving on the leaf surface as an epiphyte could also have contributed to the
observed effects against insects and pathogens cannot be ruled out completely (Rondot and
Reineke 2017). However, the persistence of B. bassiana conidia in the phyllosphere is very
poor due to the high sensitivity of the fungus to ultraviolet radiation (Inglis et al. 1993). A
decrease in persistence and viability up to a complete loss of B. bassiana conidia in the
phyllosphere within few days after application is reported by several authors (Daoust and
Pereira 1986; Gardner et al. 1977; Inglis et al. 1993, 1995). Vega (2018) pointed out that
amongst others, the susceptibility to ultraviolet (UV) light might have been an impulse to use
fungal entomopathogens as endophytes to overcome this characteristic. However, most of our
experiments were carried out in a greenhouse roofed with float glass, which absorbs most of
the detrimental UV-B waves. Therefore, we cannot assume that residues on the phyllosphere
of grapevine plants were limited due to UV-susceptibility, as was hypothesized by Jaber
(2015), and shelf life of B. bassiana conidia on grapevine stays to be tested under different
conditions. As a conclusion, residues on the plant surfaces may provide a greater contribution
to observed effects in plants such as growth promotion, indirect effects to insect herbivores or
the induction of plants defense system than realized so far. Nevertheless, the combination of
an epiphytic and (temporary) endophytic lifestyle, when using B. bassiana as an antagonist to
pests and pathogens, may optimize overall effectiveness and its full potential value may be
72
retrieved. We conclude that the present thesis gives hints that B. bassiana can be endophytic
in grapevine plants and its presence – endophytic as well as epiphytic – negatively influences
insect pests and pathogens.
The common infection pathway of the insects by fungal entomopathogens is via cuticular
penetration by germinating propagules (Arnold and Lewis 2005). Most of the reports about
mycosis due to endophytic entomopathogens are so far restricted to insects living inside plant
tissues (Akello et al. 2008b), where direct contact between insects and endophytic fungal
propagules are imaginable (Klieber and Reineke 2016). As explained by Vega (2018), there are
no studies elucidating why endophyte sporulation should be inhibited inside plants. However,
reports about conidia of B. bassiana and other entomopathogenic fungi inside plants lack of
additional information to give sufficient evidence on infecting propagules and therefore, it
remains elusive what mechanism led to reported mycosis in association with endophytes, if
no infecting propagules were present (Vega 2018). McKinnon (2017) emphasized that clear
verifications are missing in most of the studies, that the endophytic form of the
entomopathogens and no residual plant surface inocula caused the infection. This again points
to the already mentioned challenge of assigning observed effects to the endophytic lifestyle.
Consumption of infected plant material or ingestion of hyphae of endophytic
entomopathogens seems to be unlikely to cause an infection (Vidal and Jaber 2015). However,
negative effects on insect herbivores performance due to these incidences might be possible,
but not well investigated (Vega 2018). Additional effects of endophytic entomopathogenic
fungi on insects and possible operating mechanisms are reviewed and summarized amongst
others by Vega (2008, 2018) and McKinnon et al. (2017). Whereas Jaber and Ownley (2018) and
Ownley et al. (2010) also discuss mechanisms of plant disease suppression. Summarizing,
fungal metabolites, produced in planta and causing feeding deterrence or antibiosis, are
suggested to cause negative effects against herbivorous insect. In addition induced systemic
plant resistance is also considered as the mode of action. However, potential effects against
plant pathogens are attributed to mycoparasitism, competition and antibiosis directly caused
by the endophytic entomopathogenic fungi or those mechanisms mediated through the host
plant, like induction of systemic plant resistance or stimulation of plant secondary
metabolites. Only recently, a meta-analysis about entomopathogenic endophytes was
performed with the aim to identify reasons within the analyzed studies (e.g. experimental
conditions, used methods) for the inconsistency of reported effects against herbivores insects
(Gange et al. 2019).
The results presented in this thesis suggest that different modes of action accounted for the
diverse observed effects on insect performance and behavior as well as on plant pathogens
including feeding deterrence, antibiosis or changes in metabolism of the host plant and thus
73
host plant quality or induction of the plant defense system. Indeed, the results of the
microarray analysis indicated that also the host plant transcriptome reacts to the inoculation
with endophytic B. bassiana, for instance, by the up-regulation of various genes involved in
plant defense signaling pathways such as genes encoding for several PR-proteins (b-1,3-
glucanases, chitinases, thaumatin-like proteins and PR-protein 10.3) and genes associated to
the stilbene synthesis (Chapter 5.3.3). However, the response of the plant to a subsequent
inoculation with downy mildew with the aim to detect possible priming mechanism was not
analyzed.
Potential applications and practical implementations of the experimental results
The inundative application of EPF in the field still suffers from inconsistency and provides
only limited disease control (Vega 2018), presumably because of a lack of understanding their
ecology and biology (Roy et al. 2010). In addition, Jackson et al. (2010) pointed out the
importance to link the new insight to ecology and biology to production and formulation
aspects as well as to consider environmental conditions. Also Lohse et al. (2015) emphasized
the importance of an adequate formulation for endophytic establishment of entomopathogenic
fungi. To provide a benefit for the colonization process of B. bassiana on grapevine plants, we
®
used a commercially available fungal-based product (Naturalis , active ingredient B. bassiana
isolate ATCC 74040) formulated as oil dispersion.
However, already more than twenty years ago Waage (1998) explained, why the main
mistakes regarding biological control agents are to apply the “chemical model”, to create false
expectations of chemical-like efficacy and to under evaluate their properties. Therefore, we
suggest that entomopathogenic fungi should be considered as an additional option within
integrated crop management strategies rather than to directly replace synthetic pesticides. In
this regard, a couple of studies have shown that B. bassiana is sensitive against various
pesticides (Todorova et al. 1998; Sapieha-Waszkiewicz et al. 2004; Kos and Celar 2013), but
combinable/compatible with a range of insecticides (Faraji et al. 2016; Alizadeh et al. 2007) and
acaricides (Oliveira and Neves 2004). Due to the high frequencies in the application of
fungicides, compatibility with fungicides seems to be most important in viticulture, but also
most challenging and depending on the spectrum (range of controlled pathogens) of the used
fungicide. Differences in compatibility between broad-spectrum multisite, site-specific
systemic and specific action based fungicides depending on the timing of the application
would be expected. Application of contact fungicides after the endophytic establishment of B.
bassiana certainly appears to be conceivable. In our experimental vineyard, an endophytic
establishment of B. bassiana in the mature plants under field conditions was successful, even
though synthetic fungicides against powdery mildew (Uncinula necator) were simultaneously
74
applied (Rondot and Reineke 2018). Possible limitations or synergies of a combination between
B. bassiana and a diverse range of other plant protection products used in viticulture under
laboratory as well as under field conditions remain to be tested. Our results also indicate the
potential for a long term establishment of the fungus in grapevine plants and that endophytic
establishment does apparently not interfere with common viticultural management (Rondot
and Reineke 2018), as inoculation via spray application is simple to implement in viticultural
practice due to already existing spray equipment for other pesticides.
A temporal colonization of grapevine plants, activation of the plants defense system or a long
term establishment of (endophytic) B. bassiana in the ecosystems of the vineyards could
improve the overall effect of the treatment as biological control agent and overcome some of
their limitations addressed by several authors (Butt and Copping 2000; Copping and Menn
2000; Lacey et al. 2015). Our results showed a limited, but significant effect of endophytic
B. bassiana on the performance of vine mealybug P. ficus and the leafhopper E. vitis as well as
an impact on host choice behavior of O. sulcatus (Rondot and Reineke 2017, 2018). In addition,
we observed a reduction in disease incidence and severity of downy mildew P. viticola due to a
protective treatment with B. bassiana. (see chapter 5) However, in a preliminary field trial
with repeated curative applications, we could not confirm the suppressive effect on P. viticola
(own unpublished results) and point to the still existing potential for optimization under field
conditions. Even though results from our studies allow tantalizing glimpses on the potential of
B. bassiana in viticulture with various effects on different trophic levels, the long term impact
on grapevine, the surrounding agroecosystem and associated (micro)organisms remains
elusive. However, due to its multi-layer effects, the utilization of B. bassiana in vineyards as
well as vine nurseries is imaginable with the restriction of conducting further investigations.
Finally, whether the presence of endophytic B. bassiana in grapevine plants has an effect on
the quality and sensory attributes of must and wine still remains to be investigated with fruit-
bearing grapevine plants (Rondot and Reineke 2018). The need to consider these and further
“unusual impacts” was also addressed by Vega (2018) in his review. To our knowledge,
sensory attributes and valuable/secondary compounds as important parameters of the inner
plant quality are issues not yet attributed in connection with B. bassiana as an endophyte in
plants giving first contents for future endophyte research in addition to those prospects
addressed in the next section.
Future prospects of endophyte research
In this thesis, different dimensions of the multitrophic interaction between endophyte, plant,
and pest/pathogen were addressed, however much remains elusive. Some research gaps,
75
particularly those regarding the endophytic establishment of B. bassiana in grapevine plants

and its consequences, were already indicated in the previous sections and should be integrated
into future studies. Furthermore, additional, more general prospects in endophyte research
remain to be elucidated and are described hereafter. As already pointed out previously,
endophyte research is challenging, complex, and method-depending. Supporting evidence for
method dependency of reported results and the influence of experimental design comes from
the meta-analysis conducted by Gange et al. (2019). Therefore, first of all, methods and
protocols for determining endophytism, the ecology of endophytes and their effects must be
validated and adapted to prevent ambiguity of reported results. The need for stringent
protocols is also emphasized by McKinnon et al. (2017), who reviewed current methods and
elucidated associated pitfalls.
Although many studies on the endophytic establishment of B. bassiana were conducted in the
past years (see Table 1, chapter 1), further studies are certainly warranted to explore the
impact of endophytic entomopathogens on multitrophic levels. Previous studies primarily
aimed at introducing those fungi into a wider array of plants or focused on their potential
activity against insects and plant pathogens when in planta. However, evidence has
accumulated that there is an increasing relevance to understand their ecology and complete
life history in association with plants (Vega et al. 2009; Roy et al. 2010; Lacey et al. 2015). Thus
the understanding and the optimization of conditions and mechanisms underlying fungal
endophytism as well as the response of the plant, herbivorous insects and plant pathogens
need to be the focus of future research efforts (Vega 2018). Summarizing, the following issues,
amongst others, within the different multitrophic levels should be further addressed:
• Understanding the conditions that facilitate, as well as those that impede endophytic
colonization by fungal entomopathogens. This includes monitoring fungal habits and
the extent and persistence of endophytic fungal colonization within the plant.
Exploring the reasons for variation in plant colonization ability of fungal
entomopathogens could contribute to reproducible introductions into crops and to
precise predictions of their outcome.
• Characterization of the range of host plant responses to the endophytic

establishment, including the production of secondary metabolites, shifts in volatile
profile, and induction of transcriptional changes. To provide more detailed data on
gene expression by the plant, studies using next-generation sequencing technology
(RNAseq) could be conducted.
• Modifications in the fungal biology by changes in gene expression of the fungus

during colonization should also be taken into account. However, these investigations
require a more and improved annotation of the fungal genomes. First, but still quite
76
limited information is available on B. bassiana genome sequences and transcriptional

responses of the fungus to insect cuticles, insect hemocoel, and plant root exudates
(Xiao et al. 2012).
• Hypothesis about the mechanisms underlying the antagonistic effects on insect pests
and plant pathogens must be verified.
• Analysis of the interactions between entomopathogenic fungi and other plant-

associated microorganisms (internal and external) apart from pathogens. Thus,
increased attention must be paid to the impact on or by the functional microbiome
due to changes in microbial community diversity, density, and activity. Here, too,
next-generation sequencing technology could contribute to more detailed data on, e.g.
the microbial diversity.
• Compatibility with other (biological and chemical) control measures must be

evaluated to improve the incorporation of fungal entomopathogen-based biological
control agents within IPM programs.
• In this context, higher trophic levels like parasitoids and predators must be
considered and shifts in communities of (non-target) insects monitored.
• Finally, more evaluation of entomopathogenic endophytes under field conditions is

required to ascertain long term effects and impacts on natural habitats.
All issues mentioned above are individual pieces, but contributing to the complete picture, and
not supposed to be addressed merely separated. The need for a holistic approach is also
espoused by Vega (2018), who stated: “The more we hunker down and focus on simple things,
(…) the more likely we are to miss the big picture”. Hence, collaboration among insect
pathologists, plant biologists, endophyte specialists, chemists, system biologists, and scientists
of other disciplines, as also advised by McKinnon at al. (2017) and Vega (2018), is needed to
shed light on the whole system of endophytes and understand the complexity of the
multitrophic interaction. Therefore, combining methods from different disciplines like
entomology, mycology, and botany will be necessary and utilization of new technologies such
as transcriptomics and proteomics helpful to elucidate the genuine biocontrol potential of
entomopathogenic endophytes like B. bassiana and make them work as a pest management
strategy. Although limited, the results of the studies conducted in this thesis contributed to
our understanding of the ecology of B. bassiana. To conclude, endophyte research, in
particular the exploration of entomopathogenic endophytes, is challenging and exhibits a
number of knowledge gaps. Though, the knowledge about the negative impacts of chemical
control and the intention to further promote biological control is worth the extensive efforts.
77
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A Supplementary Material
A1 Figures
Figure 16: Graphical abstract of manuscript I
94
0% 5% 10 %
20 % 30 % 40 %
50 % 60 % 70 %
80 % 90 % 100 %
Figure 17: Assessment of disease severity of grapevine leaves with P. viticola
95
A2 Tables
Table 9: Induced and repressed genes in grapevine plants 24 hpt with B. bassiana. The List includes
genes that are significantly regulated more than twofold in response to B. bassiana treatment in
comparison to control plants (p < 0.05).
Probe Set Fold Representative UniGene

ID Change p-value Public ID ID Gene Title
1608304_at 6.32 0.0253 CD011601 Vvi.3800 ---
1610704_at 5.92 0.0002 CA809376 Vvi.24460 major allergen Pru ar 1-like
1618568_s_at 5.66 0.0002 CF205010.1 --- major allergen Pru ar 1-like
1617963_at 4.97 0.0149 CF074697 Vvi.8718 ---
1610850_at 4.64 0.0041 S63225.1 Vvi.27488 stilbene synthase 1-like
1609696_x_at 4.55 0.0034 S63225.1 Vvi.27488 stilbene synthase 1-like
1615118_at 4.38 0.0000 CF373171 Vvi.15301 3-ketoacyl-CoA synthase 12-like
1618663_s_at 4.16 0.0007 BM436446 Vvi.9447 uncharacterized LOC100263839
1610824_s_at 3.66 0.0002 AY059639.1 Vvi.25322 stilbene synthase 2-like
1609697_at 3.62 0.0002 CF207058.1 Vvi.27363 Stilbene synthase 4
1612804_at 3.58 0.0001 X76892.1 Vvi.25322 stilbene synthase 2-like
1620964_s_at 3.57 0.0014 S63225.1 Vvi.27488 resveratrol synthase
1622638_x_at 3.45 0.0004 X76892.1 Vvi.25322 stilbene synthase 2-like
1608009_s_at 3.33 0.0001 S63221.1 Vvi.25322 stilbene synthase 2-like
1611190_s_at 3.24 0.0010 AF274281.1 Vvi.27488 stilbene synthase
1619034_at 3.17 0.0006 CK136955.1 --- cytochrome P450 87A3-like
1620792_at 2.83 0.0005 CF605390 --- uncharacterized LOC100242276
1621102_at 2.75 0.0370 CB971771 Vvi.5498 acidic endochitinase-like
1614436_at 2.72 0.0011 CF415505 Vvi.13239 uncharacterized LOC100855161
1618260_s_at 2.67 0.0018 CD799434 Vvi.9106 myb-related protein Myb4-like
1613999_x_at 2.63 0.0014 CF202364.1 --- chitinase
1610800_at 2.59 0.0122 CF204250.1 --- ---
1619517_at 2.58 0.0071 CF202125.1 Vvi.25952 resveratrol O-methyltransferase
1616788_at 2.58 0.0002 CF202817.1 Vvi.20128 high affinity nitrate transporter 2.5-like
1619986_s_at 2.51 0.0099 CD800813 Vvi.8580 anthocyanidin 5,3-O-
glucosyltransferase-like
1611117_at 2.43 0.0012 CF201368.1 --- ---
1615967_at 2.42 0.0010 CF211449 Vvi.14598 peroxidase 73-like
1618373_at 2.40 0.0026 Z68123.1 Vvi.18 acidic chitinase
1622455_at 2.39 0.0006 CF207039.1 Vvi.674 peptide transporter PTR3-A-like
1622633_at 2.39 0.0014 CF201563.1 --- ---
1614487_at 2.37 0.0000 CF403809 Vvi.15496 3-hydroxy-3-methylglutaryl-coenzyme A
reductase 1-like
1607619_s_at 2.36 0.0003 CA814423 Vvi.5009 uncharacterized LOC100243642
1613871_at 2.35 0.0010 CF207387 Vvi.8893 endochitinase PR4-like
1608864_s_at 2.35 0.0026 CF202364.1 Vvi.18 acidic chitinase
1613006_at 2.34 0.0022 CF204981.1 --- ---
1614769_at 2.33 0.0001 CF207048.1 --- ---
1610011_s_at 2.33 0.0027 CF200913.1 --- ---
1614404_x_at 2.32 0.0011 CF201563.1 --- ---
1622745_at 2.32 0.0001 BQ796736 --- flavoprotein wrbA-like
1620245_at 2.26 0.0018 CF202722.1 --- cytochrome P450 71A1-like
1608538_at 2.26 0.0021 CA813555 Vvi.5021 uncharacterized LOC100855082
1613141_at 2.25 0.0061 CF518362 Vvi.11367 NAC domain-containing protein 42-like
1616575_at 2.22 0.0010 AF418567.1 --- ---
96
1621553_at 2.21 0.0188 CB002757 Vvi.9919 ---
1610914_at 2.19 0.0089 CF203251.1 Vvi.25984 copper transporter
1613344_at 2.16 0.0002 CF202628.1 --- ---
1613811_a_at 2.15 0.0103 CB920849 Vvi.9248 VVTL1
1614803_at 2.14 0.0029 AY046416.1 Vvi.161 proline-rich protein 1
1621371_at 2.14 0.0006 CF202171.1 Vvi.19808 disease resistance response protein 206-
like
1616413_at 2.13 0.0066 AF003007.1 Vvi.9248 VVTL1
1615401_at 2.11 0.0008 CB342555 Vvi.1393 putative UDP-glucose flavonoid
3-O-glucosyltransferase 3-like
1620063_at 2.10 0.0042 CB921343 Vvi.644 beta-1,3-glucanase
1607193_at 2.10 0.0000 BQ796845 Vvi.683 alternative oxidase 3, mitochondrial-like
1615458_at 2.10 0.0020 CB969727 Vvi.5161 uncharacterized LOC100255664
1621970_at 2.09 0.0007 CD713131 Vvi.5009 uncharacterized LOC100243642
1616822_at 2.08 0.0042 AF220196.1 Vvi.161 proline-rich protein 1
1620390_s_at 2.07 0.0318 AF532965.1 Vvi.8525 thaumatin-like protein
1606453_x_at 2.07 0.0216 CF203408.1 Vvi.24387 pathogenesis-related protein 10.3
1609653_at 2.06 0.0005 BQ797078 --- ---
1609156_at 2.05 0.0130 CF211313 --- valencene synthase-like
1615789_at 2.04 0.0161 BQ795769 Vvi.553 extensin-3-like
1622369_at 2.02 0.0009 CB342790 Vvi.7017 germin-like protein subfamily T member
2-like
1610243_at 2.02 0.0006 BM437744 Vvi.9617 probable glutathione S-transferase-like
1622550_at 2.02 0.0006 AY427148.1 --- ---
1618589_s_at -2.01 0.0051 CF206361.1 Vvi.27529 uncharacterized LOC100260620
1610488_at -2.02 0.0054 CK138238.1 --- ---
1609901_at -2.03 0.0172 CF212785 Vvi.14816 monothiol glutaredoxin-S1-like
1612562_at -2.04 0.0024 CA808714 Vvi.7197 uncharacterized LOC100264675
1609749_at -2.08 0.0071 CD716155 Vvi.5707 gibberellin 3-beta-dioxygenase 4-like
1611996_at -2.10 0.0161 CF373384 Vvi.15425 ---
1607561_at -2.14 0.0086 CF209184 Vvi.5178 ABC transporter G family member 5-like
1613022_s_at -2.15 0.0032 CF569215.1 Vvi.7621 non-specific lipid-transfer protein P5-
like
1613301_at -2.20 0.0141 CF372159 Vvi.27809 stem-specific protein TSJT1-like
1617940_at -2.33 0.0035 CA809342 Vvi.2349 nuclease S1-like
1610299_at -2.40 0.0114 CF373165 Vvi.5632 MLP-like protein 423
1618921_at -2.43 0.0209 CK138176.1 --- ---
1613442_at -2.43 0.0085 CF415231 Vvi.21002 glutaredoxin-C1-like
1617400_at -2.45 0.0078 BQ798101 Vvi.6741 sulfate transporter 3.1-like
1607541_at -2.46 0.0049 CF208308 --- uncharacterized LOC100257913
1622416_at -2.60 0.0051 CF518913 Vvi.7621 non-specific lipid-transfer protein P5-
like
1615985_at -2.63 0.0035 CF516133 Vvi.13054 auxin-induced protein 22D-like
1608268_at -2.67 0.0028 CB970701 Vvi.5372 thebaine 6-O-demethylase-like
1615445_at -2.70 0.0296 BQ794327 Vvi.4693 metallothionein-like protein type 2-like
1608175_at -2.73 0.0168 CF404148 Vvi.15636 non-specific lipid-transfer protein-like
1617786_at -2.80 0.0081 CB972580 Vvi.5653 uncharacterized LOC100263887
1619751_at -2.85 0.0076 CB341549 Vvi.13054 auxin-induced protein 22D-like
1613471_at -2.92 0.0327 CF215857 Vvi.14794 pathogenesis-related protein PR-1-like
1615971_a_at -3.89 0.0046 CB980630 --- uncharacterized LOC100262468
1615109_at -3.90 0.0293 CK138176.1 --- ---
97
Table 10: Induced and repressed genes in grapevine plants 168 hpt with B. bassiana. The List includes
genes that are significantly regulated more than twofold in response to B. bassiana treatment in
comparison to control plants (p < 0.05).

1622767_at 2.17 0.0013 CB982859 Vvi.7837 hypothetical protein
1621587_at 2.11 0.0115 CF214129 Vvi.5464 adenosine 5'-phosphosulfate reductase
1617074_s_at 2.19 0.0210 CF204027.1 --- ---
1622010_at 2.14 0.0213 CF207538 Vvi.9839 ---
1621593_s_at 2.07 0.0032 AF347624.1 --- ---
1613911_s_at 2.53 0.0310 CF201679.1 --- ---
1613371_s_at 2.09 0.0088 CB340927 --- ---
1609916_s_at 2.24 0.0085 CD005933 Vvi.3813 ---
1609373_at 2.23 0.0052 CD800734 Vvi.11248 30S ribosomal protein S7, chloroplastic-
like
1616528_s_at 2.94 0.0353 CD801342 --- uncharacterized LOC100242887
1607341_at 3.09 0.0441 CB970018 --- uncharacterized LOC100242887
1613853_s_at 2.03 0.0057 CF203997.1 --- ---
1615527_at 2.01 0.0326 CF372317 Vvi.10595 uncharacterized LOC100246222
1619204_at 2.14 0.0409 CF373337 Vvi.15313 dnaJ homolog subfamily C member 1-like
1622374_at -2.03 0.0254 CB920589 Vvi.11581 osmotin-like protein
1610638_at -2.11 0.0035 CD719790 Vvi.10998 uncharacterized LOC100855409
1607353_at -2.08 0.0032 CF206245.1 --- uncharacterized LOC100249186
1608852_at -2.36 0.0418 CF516023 Vvi.3488 probable sulfate transporter 3.4-like
1615169_at -2.10 0.0353 BQ792970 Vvi.1874 uncharacterized LOC100258290
1608692_s_at -2.14 0.0213 CF074673 Vvi.8923 pathogenesis-related protein
1609391_s_at -2.05 0.0101 CF404650 Vvi.13978 blue copper protein-like
1617047_at -2.14 0.0299 BQ792281 Vvi.7462 peptidyl-prolyl cis-trans isomerase-like
1619479_a_at -2.14 0.0014 CB980068 Vvi.7570 lanC-like protein 2
1608229_s_at -2.03 0.0086 CD003870 Vvi.9783 glycogenin-2-like
1620065_at -2.23 0.0415 CD798903 Vvi.888 probable sulfate transporter 3.5-like
1621688_at -2.01 0.0393 CF415096 Vvi.3163 glycerophosphodiester phosphodiesterase
GDE1-like
1612443_at -2.65 0.0026 CF211151 Vvi.2313 CBL-interacting protein kinase 16
1621592_s_at -2.70 0.0424 BQ792954 Vvi.4682 dehydrin
98
A3 Manuscripts Biological Control 116 (2018) 82–89
Contents lists available at ScienceDirect
Biological Control
journal homepage: www.elsevier.com/locate/ybcon
Endophytic Beauveria bassiana in grapevine Vitis vinifera (L.) reduces

infestation with piercing-sucking insects
Yvonne Rondot ⇑, Annette Reineke
Geisenheim University, Department of Phytomedicine, Von-Lade-Str. 1, D-65366 Geisenheim, Germany
h i g h l i g h t s g r a p h i c a l a b s t r a c t
Successful endophytic establishment

of the entomopathogen B. bassiana in
grapevine plants.
In potted plants endophytic survival
of B. bassiana was evident for at least
21 days after inoculation.
Endophytic B. bassiana reduces
infestation rate and growth of vine
mealybugs.
In the vineyard B. bassiana was
detected as an endophyte up to five
weeks after last application.
B. bassiana reduces infestation with
grape leafhopper in the vineyard.
a r t i c l e i n f o a b s t r a c t
Article history: Fungi are important natural pathogens of arthropod pests and are successfully used as biocontrol agents
Received 27 April 2016 in various crops. In addition to colonizing arthropods, evidence has accumulated that some ento-
Revised 11 October 2016 mopathogenic fungi like Beauveria bassiana can endophytically colonize a wide array of plant species.
Accepted 21 October 2016
However, only limited information is currently available on the endophytic colonization of grapevines
Available online 22 October 2016
with B. bassiana and whether the fungus still maintains its pathogenic habit against insect pests.
Greenhouse and field experiments were conducted to optimize endophytic establishment of the ento-
Keywords:
mopathogenic fungus B. bassiana in younger, potted plants and mature grapevine plants in the vineyard.
Beauveria bassiana
Endophyte
We used two different commercialized B. bassiana strains, applied either as conidial suspensions (ATCC
Entomopathogenic fungi 74040 and GHA) or as a formulated product (NaturalisÒ, strain ATCC 74040) on grapevine leaves. The
Vitis vinifera potential of endophytic B. bassiana to provide protection against putative target pest insects like the vine
Planococcus ficus mealybug Planococcus ficus was assessed in a bioassay using surface sterilized leaves. Endophytic survival
Empoasca vitis of B. bassiana inside leaf tissues of seven-week-old potted plants was evident for at least 21 days after
Interactions inoculation, irrespective of the inoculum used. Endophytic B. bassiana reduces infestation rate and
growth of P. ficus. In the vineyard B. bassiana was detected as an endophyte in mature grapevine plants
up to five weeks after last application with significant reduction of infestation with grape leafhopper,
Empoasca vitis.
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction mopathogen of a diverse range of arthropod species. Hence, this

species is successfully used as a microbial biocontrol agent for inte-
The hypocrealean fungus Beauveria bassiana (Bals.) Vuill. grated pest management of arthropod pests with many B. bassiana
(Ascomycota: Hypocreales) is a well-known microbial ento- based commercial products being available on the market (Jackson
et al., 2010; Zimmermann, 2007). Generally, for control of target
⇑ Corresponding author. species, preparations of blastospores or aerial conidia formulated
E-mail address: yvonne.rondot@hs-gm.de (Y. Rondot). in oil or other adjuvants are sprayed onto the plant’s phylloplane.
http://dx.doi.org/10.1016/j.biocontrol.2016.10.006
1049-9644/Ó 2016 Elsevier Inc. All rights reserved.
99
Y. Rondot, A. Reineke / Biological Control 116 (2018) 82–89 83
Besides its entomopathogenic habit of life style, this fungus has plants, insects and entomopathogenic endophytic fungi. Endo-
also been shown to be able to thrive saprophytically in the soil, phytic establishment of an entomopathogenic fungus like B. bassi-
to colonize the rhizosphere of plants, to have antagonistic activities ana still having antagonistic activity against insect pests and fungal
against plant pathogens, as well as to grow endophytically inside pathogens would therefore represent a novel and sustainable plant
plants (Vega et al., 2009). As far as the latter is concerned, a few protection strategy in viticulture, with the potential to reduce fre-
studies have shown that B. bassiana is occurring as part of the nat- quency of pesticide applications.
ural endophytic community of certain plant species (Ormond et al., In the present study we have addressed the following main
2010; Reay et al., 2010; Vega et al., 2008). Moreover, endophytic questions (1) Is an endophytic establishment of commercially
establishment of B. bassiana has been achieved via an artificial available B. bassiana strains possible both in young potted green-
application of this fungus on the plant’s tissue following a subse- house grapevine plants as well as in mature and lignified plants
quent colonization of specific parts of the plant or the entire host grown in the field? (2) Does an endophytic B. bassiana strain pre-
plant. Using such an approach, successful endophytic establish- sent in grapevine have negative impacts on insects with a
ment of B. bassiana has been proved for a variety of crop plant spe- piercing-sucking mode of feeding?
cies including cocoa (Posada and Vega, 2005) and pine seedlings
(Brownbridge et al., 2012), corn (Wagner and Lewis, 2000), coffee
2. Materials and methods
(Posada et al., 2007), sorghum (Reddy et al., 2009; Tefera and
Vidal, 2009), tomato (Klieber and Reineke, 2016), banana (Akello
2.1. Fungal material
et al., 2009), and jute (Biswas et al., 2012, 2013). So far, no negative
effects of the presence of endophytic B. bassiana on performance of
Beauveria bassiana strains ATCC 74040 and GHA were isolated
the colonized host plant have been reported in a range of studies
from the commercial products NaturalisÒ (CBC (Europe) S.r.l. –
(Akello et al., 2009; Klieber and Reineke, 2016; Tefera and Vidal,
BIOGARD Division, Italy) and BotanigardÒ 22WP (BioWorks, Inc.,
2009; Wagner and Lewis, 2000). Endophytic B. bassiana has been
USA), respectively. NaturalisÒ is formulated as an oily fluid and
reported to provide systemic protection against several insect
contains approximately 2.3 107 colony forming units/ml of B.
pests or to inhibit insect development and establishment (Biswas
bassiana strain ATCC 74040 as active ingredient. The isolates were
et al., 2013; Gurulingappa et al., 2010; Quesada-Moraga et al.,
maintained on a solid medium at 24 °C in the dark. The medium
2009; Reddy et al., 2009). At the same time, presence of endophytic
consisted of 10 g soy peptone (AppliChem, Germany), 20 g glucose
B. bassiana has been shown to reduce disease symptoms caused by
(Sigma-Aldrich, Germany) and 18 g BactoTMAgar (BD Difco, USA)
a variety of fungal pathogens (Griffin et al., 2005; Jaber, 2015;
dissolved in 1000 ml ultrapure water and was subsequently auto-
Ownley et al., 2010, 2008) Therefore, defining means of ensuring
claved for 20 min at 120 °C.
an endophytic establishment of B. bassiana strains in target crop
To obtain spore suspensions, conidia were harvested by gently
plants is currently the focus of several studies, as this would repre-
scraping the surface of Petri dishes containing 8-day-old B. bassi-
sent a dual biocontrol strategy both against insect pests and plant
ana cultures and suspending them in 20 ml sterile 1/8 concen-
pathogens. Thus, the use of endophytes for the purpose of pest and
trated Ringer’s solution containing 0.02% Tween 80. The conidia
disease control is of particular interest for perennial crops like
concentration was determined using a Thoma haemocytometer
grapevine, which regularly require frequent and intensive applica-
and adjusted to 2 107 conidia/ml for strain GHA and to 1 107
tions of pesticides.
and 2 107 conidia/ml for strain ATCC 74040. Both, the freshly col-
Grapevine (Vitis vinifera L.) is an important global commodity
lected conidia suspensions and the formulated product NaturalisÒ
crop which is planted throughout temperate regions worldwide.
(at concentrations of 3% and 5%), were used in the experiments.
A substantial number of different insect pests and pathogens are
Aliquots of 50 ll of serially diluted spore suspensions were plated
associated with grapevine and are significant factors influencing
on solid medium mentioned above using the Spiralplater WASP 2
both the quantity of the yield as well as the quality of must and
(Meintrup DWS Laborgeräte GmbH, Germany). Germination rates
vine (Flaherty, 1992). As a result, grapevine cultivation is regarded
were thereafter assessed by plate counts of viable conidial spores
as being input intensive, in particular regarding the frequency and
and were calculated using the colony forming unit’s (CFU) method
intensity of fungicide and insecticide applications throughout the
(Goldman and Green, 2008). Germination rate was 100% for coni-
year (Roßberg, 2007). Insects with a piercing-sucking mode of
dial spores present in NaturalisÒ and around 70% for the spore sus-
feeding frequently attack grapevines and cause damage either by
pensions of isolates ATCC 74040 and GHA. Accordingly,
extracting sap fluids or feeding in mesophyll cells or by transmit-
concentrations of viable conidia applied onto plants were
ting grapevine pathogens. The grape leafhopper Empoasca vitis
1.4 107 conidia/ml for strain GHA and 7 106 (conc. 1) or
(Goethe) (Homoptera: Cicadellidae, Typhlocybinae) feeds on mes-
1.4 107 (conc. 2) conidia/ml for strain ATCC 74040.
ophyll cells or on phloem sap and is recognized as a major insect
pest in many European grapevine growing areas (Olivier et al.,
2012). Moreover, the vine mealybug Planococcus ficus (Signoret) 2.2. Endophtic establishment in potted grapevine plants
(Homoptera: Pseudococcidae) is regarded as a key pest in many
countries around the world (Daane et al., 2012). Planococcus ficus Grapevine plants, Vitis vinifera (L.) cv. ‘Riesling’, were obtained
causes direct damage to grapevine due to phloem-feeding on from hardwood cuttings. After root development the plants were
leaves and fruit and excretion of honeydew. Additionally, P. ficus potted in a clay/white peat substrate ED73 (Patzer, Sinntal,
acts as a vector for grapevine leafroll associated virus (GLRaV), Germany) and grown in a greenhouse chamber at 22–25 °C.
one of the most economically destructive grapevine viruses that Seven-week-old grapevine plants with 4–7 fully expanded leaves
occur in all the major grape-growing regions of the world were used for inoculation with either B. bassiana conidial suspen-
(Almeida et al., 2013). Accordingly, a combination of methods sions or the commercial product NaturalisÒ (3% and 5%). For each
including insecticide applications, biological control via predators treatment, 10 replicate plants were inoculated by spraying the
and parasitoids or mating disruption is usually applied by growers adaxial and the abaxial surfaces of all fully expanded leaves using
to control P. ficus (Almeida et al., 2013). The system grapevine (as a 1 L one-hand pressure sprayer. During application, pots with
an input intensive crop) - P. ficus and E. vitis (as phloem-feeding plants were held in an almost horizontal position so that any
pest insects) - B. bassiana (as a commercially available biopesti- run-off was not contaminating the soil. Control plants were
cide) is thus ideal for studying tritrophic interactions between sprayed with sterile 1/8 concentrated Ringer’s solution containing
100
84 Y. Rondot, A. Reineke / Biological Control 116 (2018) 82–89
0.02% Tween 80. Position of the last fully expanded leaf used for ies, these primers have proved to allow a confident discrimination
inoculation was labeled using a tapener (Max tapener HT-B, Max among different B. bassiana isolates (Reineke et al., 2014).
Staple, Japan). Inoculated and non-inoculated plants were random- For fluorescent labeling of the generated PCR products, a M13
ized in blocks and were kept in a greenhouse chamber for three (21) tail was placed at the 50 -end of each forward primer and a
weeks (daily mean temperature 23–25° C, daily mean relative CY5 labeled universal primer M13(21) was added to the PCRs
humidity 50–70%). Plants were watered as needed. according to the method described by Schuelke (2000). PCR ampli-
fications were set up in a total volume of 15 ll consisting of 90 ng
2.3. Re-isolation of B. bassiana DNA, 10x reaction buffer, 5 pmol of forward primer, 10 pmol of
reverse primer, 2.25 mM MgCl2, 3 mM dNTPs and 0. U of Dream
Endophytic colonization of plants by B. bassiana was assessed 7, Taq Polymerase (Fermentas, St. Leon-Rot, Germany). PCRs were
14, and 21 days after inoculation (DAI) by re-isolation following performed under the following conditions: initial denaturation at
surface sterilization. No newly developed leaves were included in 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 60.2 °C
the present study. At each sampling period one leaf from each of for 45 s and 72 °C for 45 s, followed by a final extension at 72 °C
the 10 replicate plants was excised and transported to the labora- for 10 min. An aliquot of each PCR product was checked for suc-
tory on ice. The leaves were individually surface sterilized under cessful amplification on a 1% agarose gel. PCR products were ana-
sterile conditions by dipping them in 0.5% NaOCl (active chlorine) lyzed for SSR sizes via capillary electrophoresis on a Beckman
containing 0.05% Tween 80 for 2 min, followed by 70% EtOH for GenomeLab GeXP DNA Genetic Analysis System (Beckman Coulter,
2 min and rinsed twice with sterile distilled water according to Inc., CA, USA). As different fluorescent primers were used for label-
Akello et al. (2009). The success of this disinfection process was ing the obtained PCR products (DY-751 for Ba01, BMN5 for Ba12
assessed by plating three replicates of 200 ll of the residual rinse and DY-681 for Ba13) reactions were loaded as a multiplex analysis
water on PDA (potato dextrose agar). No fungal growth was with 1 ll of each PCR product, mixed with 36.7 ll sample loading
recorded in any of the rinse water samples after 21 days of incuba- solution (Beckman Coulter, Inc., CA, USA) and 0.3 ll of a 400 bp size
tion. After surface sterilization, six leaf discs (d = 1.2 cm) were standard. Allele sizes were determined using GenomeLab GeXP
obtained with a sterile cork borer from each leaf. Leaf discs were Version 10.2 (Beckman Coulter, Inc., CA, USA).
placed on Beauveria selective medium, the same solid medium as
indicated above (2.1) but supplemented with 0.1 g/l streptomycin
(Sigma-Aldrich, Germany), 0.05 g/l tetracycline (Sigma-Aldrich, 2.5. Mealybug bioassay
Germany), 0.1 g/l dodine (as aliquot of the product SyllitÒ,
Spiess-Urania Chemicals, Germany) and 0.05 g/l cycloheximide The potential of endophytic B. bassiana to provide protection
(Sigma-Aldrich, Germany). This medium is based on a medium ini- against piercing-sucking insects was tested with a detached leaf
tially described by Strasser et al. (1996) for the isolation of B. assay and vine mealybugs, P. ficus. Sixty-seven weeks old potted
brongniartii and adapted by Meyling and Eilenberg (2006) for isola- grapevine plants cv. ’Riesling’ were inoculated with NaturalisÒ
tion of B. bassiana. Plates were incubated at room temperature with (3%) or water as control as described above. The experiment was
a 12:12 h light:dark photoperiod (mean light intensity of repeated twice. Two weeks after inoculation two leaves per plant
11.2 lmol m1 s1). were obtained, with one leaf used for the bioassay and the other
After 7 and 14 days leaf discs were examined visually for the leaf to verify endophytic establishment by re-isolation as described
presence of any fungal growth. Fungal tissue was characterized above. To ensure that mealybugs were only influenced by endo-
as endophytic B. bassiana if characteristic white dense mycelia, phytic and not by epiphytic fungal propagules, all grapevine leaves
becoming creamy at the edge (Humber, 1997) grew from internal were surface disinfected before the bioassay according to the pro-
plant tissues of surface sterilized leaf discs. Final assessment of cedure described above. With a pretest (data not shown) we veri-
the presence of endophytic B. bassiana was recorded after 14 days fied that any leftovers of NaOCl still present on the leaves did not
and was expressed as percentage colonization by dividing the harm the mealybugs.
number of leaf discs exhibiting B. bassiana outgrowth by the num- Vine mealybugs were grown on potato sprouts in a growth
ber of total leaf discs and multiplying the obtained value with 100. room with 23 ± 1 °C, 60–65% RH and 16:8 h light:dark period. In
If one of the six leaf discs obtained from a single plant showed fun- all experiments, first instar P. ficus individuals were used, which
gal outgrowth the total leaf was classified as being endophytically were removed from potato tubers by irritation with a paintbrush
colonized. Differences in percentage colonization of plant tissues at until their stylets were withdrawn. Ten P. ficus larvae each were
the different sampling dates were analyzed for statistical signifi- carefully transferred with a paintbrush to the surface sterilized
cance with a Kruskal-Wallis-ANOVA using Dell Statistica data anal- leaves. Leaves with mealybugs were maintained in enclosed trans-
ysis software system (Dell Inc., version 13, software.dell.com). parent plastic containers (height 10 cm, diameter 13.5 cm) with
water provided for the leaf and were placed in a growth chamber
2.4. Strain-specific detection of B. bassiana under the conditions mentioned above.
After two days infestation rate was calculated as the number of
To ensure that fungal tissue present at the edges of grapevine remaining larvae on the leaf in relation to the initially used ten
leaf discs originated from the respective inoculated B. bassiana individuals. This procedure was repeated once a week over a per-
strain (ATCC 74040 or GHA), now internally colonizing plant tis- iod of three weeks (7, 14, and 21 days after initial settlement)
sues as an endophyte, a subset of mycelia samples was further ana- and was supplemented by determination of the size of all individ-
lyzed with molecular techniques. DNA was extracted from fungal ual mealybug larvae with a binocular microscope and measure-
tissues using the MasterPureTM DNA Purification Kit (Epicentre ment software (Leica Microsystems, Application Suite,
Biotechnologies, USA) according to the manufacturer’s instructions Switzerland). A total of 300 mealybugs were assessed for each,
with an additional step for 30 min on ice after the recovering step the endophytic and control leaves. Size and infestation rate were
with isopropanol. Accordingly, extracted fungal DNAs were sub- analyzed for statistical significance between endophytic and con-
jected to strain-specific PCR analysis using three B. bassiana trol leaves with a Mann-Whitney-U-Test (a = 0.05) using Dell Sta-
microsatellite (simple sequence repeats, SSR) primers, namely tistica data analysis software system (Dell Inc., version 13,
Ba01, Ba12 and Ba13 (Rehner and Buckley, 2003). In previous stud- software.dell.com).
101
2.6. Field trial variance in percentage colonization in all treatments (Fig. 1). In
some instances contaminating fungi and bacteria were occasion-
In addition to greenhouse and laboratory experiments we con- ally found growing from leaf discs of both inoculated and control
ducted a field trial as proof of principle to get preliminary evidence plants (data not shown).
of efficacy of endophytic establishment of B. bassiana and its poten- If applied as a conidial suspension on foliage of grapevine
tial to control insect pests in the field. The field trial was realized in plants, both B. bassiana strains (GHA and ATCC 74040) were able
the framework of GEP (good experimental practice) certified effi- to establish as an endophyte, with no significant differences in per-
cacy tests of plant protection products (EPPO, 2012). The experi- centage colonization being evident between the different spore
mental vineyard was located in the Rheingau region, Germany concentrations and the strains applied (Fig. 1). The same was obvi-
(49°580 N, 7°570 E, 95 masl) and included 0.3 ha of grapevine ous if B. bassiana strain ATCC 74040 was applied as the formulated
plantscv. ’Riesling’ planted in 1999. The experiment was conducted product NaturalisÒ, with colonization rates being not significantly
in a completely randomized block design with 4 plots (replicates) different for both concentrations (3% and 5%) applied (Fig. 1). Dur-
of 114 m2 size and 14 vines per plot. ing the assessment period, no significant decline or increase in per-
NaturalisÒ (1%) was applied in the vertical canopy by a tunnel centage colonization by endophytic B. bassiana was observed 7, 14,
sprayer with 8 TeejetÒ flat spray nozzles and driving speed of and 21 DAI.
0.7 m/s. Control plots were treated with water. Applications were
carried out at the same time with other plant protection measures
3.2. Strain-specific detection of endophytic B. bassiana
(fungicide applications against powdery mildew, Erysiphe necator,
using the products VivandoÒ, TaliusÒ, LunaÒ Experience and
In capillary electrophoresis, DNA from all analyzed mycelia
TopasÒ in rotation). Interval between applications was approxi-
samples obtained from endophytic fungal tissues showed the
mately 10 days depending on weather and disease pressure of
respective strain-specific peaks after amplification with three B.
other grapevine pathogens with a first application on 15 May
bassiana microsatellite primers (Table 1). Amplicons of primer
2014. In two of the four NaturalisÒ treated plots, treatment
pairs Ba01, Ba12 and Ba13 showed peaks at 117 bp, 231 bp and
included nine applications during the season. The other two plots
216 bp for strain ATCC 74040 and 117 bp, 222 bp and 168 bp for
were treated twice in the beginning of the season to determine
strain GHA, respectively. These results indicate that endophytic B.
how long the fungus can be detected endophytically (15 and 26
bassiana re-isolated from inoculated leaf discs originated from
May 2014).
the previously applied strains.
Endophytic establishment in grapevine leaves was evaluated at
four dates (22 May, 12 June, 2 July, and 23 July 2014) in 10 leaves
per plot according to the method described above. In addition we 3.3. Mealybug bioassay
assessed the infestation with grape leafhopper, Empoasca vitis, at
five dates (15 July, 23 July, 31 July, 07 Aug and 12 Aug 2014) in Negative potential of endophytic B. bassiana against vine mealy-
treated plots and control plots by counting E. vitis larvae on 25 bug larvae was assessed on detached and surface sterilized grape-
leaves per plot. Infestation data was analyzed for statistically sig- vine leaves of NaturalisÒ treated and control grapevine plants.
nificant differences with nonparametric Wilcoxon signed-rank test Table 1
(McDonald, 2014). Amplification of B. bassiana strain GHA or ATCC 74040 specific SSR markers in a
subset of eight obtained mycelia samples from leaf discs of the different treatments
3. Results 14 and 21 days after inoculation (DAI) with B. bassiana.
Treatment DAI No. of leaf discs used and

3.1. Endophytic colonization of potted grapevine plants screened positive with three
SSR markers
During the assessment period of 7, 14, and 21 DAI B. bassiana n total Ba01 Ba12 Ba13
was successfully re-isolated from 46%, 40%, and 46% of all inocu- ATCC 74040 (1.4 107 conidia/ml) 14 2 2 2 2
lated grapevine plants, respectively. None of the leaf discs obtained NaturalisÒ 3% 14 2 2 2 2
from control plants showed signs of fungal outgrowth, thus none of GHA (1.4 107 conidia/ml) 21 1 1 1 1
ATCC 74040 (7 106 conidia/ml) 21 2 2 2 2
the control plants were colonized by the fungus. Not all leaf discs
ATCC 74040 (1.4 107 conidia/ml) 21 1 1 1 1
from colonized plants showed fungal outgrowth, causing a high
50
GHA
45 ATCC 74040 conc. 1
40 ATCC 74040 conc. 2
Naturalis® 3 %
35 Naturalis® 5 %
Colonization [%]
30
25
20
15
10
0
7 DAI 14 DAI 21 DAI
Fig. 1. Mean (±SE) percentage colonization of Vitis vinifera leaf discs 7, 14 and 21 days after inoculation (DAI) with a conidial suspension of Beauveria bassiana strains GHA
(1.4 107 conidia/ml) or ATCC 74040 (conc. 1: 7 106 conidia/ml; conc. 2: 1.4 107 conidia/ml) or with the formulated product NaturalisÒ (3% and 5%). Differences between
treatments were not statistically significant (p < 0.05). In control leaves (treated with Ringer’s solution) no B. bassiana was present (not shown).
102
A Table 2
Number of leaf discs assessed (n) and showing B. bassiana outgrowth collected from
2400 Control
2200 NaturalisÒ treated and control plots of a grapevine field trial in 2014.
Naturalis®
2000 p = 0.286
1800
Treatment n No. of leaf discs with endophytic B.
1600 p = 0.159 bassiana
Size [µm]
1400
22 May 12 June 02 July 23 July
1200
p = 0.057
1000 Control 80 0 0 0 0
800 NaturalisÒ (2 applications) 160 16 8 8 0
600
NaturalisÒ (9 applications) 160 47 19 10 13
400
200
0
1 week 2 weeks 3 weeks
B assessed on a separate leaf obtained from the same plant as the
p = 0.019
2400 one used in the mealybug bioassay.
2200
2000
Mealybug larvae were smaller when feeding for a period of
1800 3 weeks on NaturalisÒ treated leaves compared to those feeding
p = 0.004
1600 on control leaves (Fig. 2). In the first experimental replicate this
Size [µm]
1400
1200
p = 0.000 difference was not statistically significant (Mann-Whitney-U-
1000 Test: 1 week p = 0.057; 2 weeks p = 0.159; 3 weeks p = 0.286),
800 while in the second replicate the mean size of the mealybugs on
600
400 treated leaves was significant smaller over the whole assessment
200 period of three weeks (Mann-Whitney-U-Test: 1 week p < 0.001;
0
1 week 2 weeks 3 weeks
2 weeks p < 0.005; 3 weeks p < 0.05).
Assessment period In the first experimental replicate significantly less mealybug
larvae stayed alive on leaves with endophytic B. bassiana (Natu-
Fig. 2. Mean (±95% CI) size of vine mealybug larvae (P. ficus) after feeding for three ralisÒ) compared to control leaves (p < 0.05) over the period of
weeks on detached grapevine leaves of control plants and plants with endophytic
the bioassay. In the second replicate this effect was only observed
B. bassiana (NaturalisÒ) in two replicates (A and B). Statistical significant differences
between treatments were analyzed with a Mann-Whitney-U-Test (a = 0.05).
at the beginning of the assessment period (1 week of feeding) (see
fig. 3).
Endophytic establishment in the respective grapevine plants was

30% in the first and 60% in the second experimental replicate (data 3.4. Field trial
not shown). Because of this difference in endophytic establishment
results of the two replicates were not combined but were analyzed re-isolation of B. bassiana after application in the field showed
separately. Data on success of endophytic establishment were that the fungus was able to establish as an endophyte in perennial
and lignified grapevine plants in the vineyard (Table 2). In the plots
treated several times with NaturalisÒ the fungus could be detected
at all sampling dates (22 May, 12 June, 02 July and 23 July 2014).
A Detection rate declined over the season. In plots treated only at
100
p = 0.014 Control the beginning of the season B. bassiana was successfully re-
90
p = 0.000
80 Naturalis®
isolated up to five weeks after the last application of NaturalisÒ.
Infestation rate [%]
70 p = 0.042
p = 0.006
In control plots no B. bassiana was re-isolated from the leaves.
60
50
Infestation rate with grape leafhopper E. vitis in the vineyard
40 was overall low in the year 2014. At all five monitoring dates the
30 mean number of larvae was lower in NaturalisÒ treated plots than
20 in control plots (Table 3). Over the whole assessment period the
10
total number of E. vitis larvae was higher in control than in Natu-
0
2 days 1 week 2 weeks 3 weeks ralisÒ treated plots (236 vs. 183 individuals). Wilcoxon signed-
B
rank test showed that the median difference between the mean
100 p = 0.956
number of grape leafhopper larvae per monitoring date in control
90
p = 0.004
plots vs. NaturalisÒ treated plots was significantly greater than
80 zero (W = 0, P < 0.001)
Infestation rate [%]
p = 0.618
70
p = 0.757
60 Table 3
50 Mean number of grape leafhopper E. vitis larvae in four control and four NaturalisÒ
40 treated plots (25 leaves/plot) assessed at five observation dates in a grapevine field
30 trial in 2014. Mean number of grape leafhopper larvae per monitoring date in control
20 plots vs. NaturalisÒ treated plots were statistically different (Wilcoxon signed-rank
10 test; W = 0, P < 0.001).
0
2 days 1 week 2 weeks 3 weeks Date 2014 Mean number (± SD) of E. vitis larvae
Assessment period Control NaturalisÒ

15 July 15.50 (±5.07) 12.75 (±2.22)
Fig. 3. Mean (±95% CI) infestation rate of vine mealybug larvae (P. ficus) after
23 July 18.75 (±9.71) 12.75 (±6.60)
feeding for three weeks on detached grapevine leaves of control plants and plants
31 July 14.00 (±5.48) 13.50 (±5.20)
with endophytic B. bassiana (NaturalisÒ) in two replicates (A and B). Statistical
07 Aug 6.00 (±2.94) 4.50 (±3.11)
significant differences between treatments were analyzed with a Mann-Whitney-
12 Aug 4.75 (±2.06) 2.25 (±0.96)
U-Test (a = 0.05).
103
4. Discussion endophytic B. bassiana on infestation and size of vine mealybug lar-

vae in bioassays. Moreover, grape leafhopper E. vitis larvae were
Successful endophytic colonization of both young potted grape- significantly more abundant on control than on endophytic B.
vine plants in the greenhouse as well as mature plants in the field bassiana grapevine plants in the field. Usually, fungal ento-
with two different commercially available B. bassiana strains was mopathogens infect their insect hosts via cuticular penetration
achieved via artificial spray inoculation. Analysis of fungal mycelia by germinating propagules (Boomsma et al., 2014) (Arnold and
obtained after re-isolation with strain-specific molecular markers Lewis, 2005). Infection by endophytic entomopathogens via con-
confirmed our initial assessment based on morphology of endo- sumption of infected plant tissue or ingestion of hyphae or spores
phytic fungal mycelia obtained from colonized grapevine plants. seems to be unlikely and rare (Vidal and Jaber, 2015). Existing
In greenhouse experiments, no significant difference in percentage reports about mycosis due to endophytic entomopathogens are
colonization by endophytic B. bassiana was observed during the so far restricted to insects living inside plant tissues like stem-
assessment period of 21 DAI. This suggests that endophytic colo- borers or leafmining larvae (Akello et al., 2009; Klieber and
nization of grapevine by B. bassiana was evident as early as 7 DAI Reineke, 2016), where a direct contact of insects feeding inside
and did not decline during the period of screening for presence plant tissues and endophytic fungal propagules can be envisaged.
of endophytic B. bassiana of 21 DAI. Moreover, percentage coloniza- In contrast to stem-borers or leaf-miners mealybug larvae live on
tion of grapevine plants did not vary significantly among the differ- the plant surface and have a piercing-sucking feeding habit with
ent strains or inoculum doses used. This may be a consequence of the consequence that a direct mode of action due to direct contact
the relatively small number of positive samples identified and the is not likely to occur. On surface disinfected leaves previously trea-
apparent variability in isolation success. ted with B. bassiana, mealybug larvae were smaller and mortality
Mean colonization rates of potted grapevine plants by B. bassi- rates were higher than on control leaves, but none of the dead lar-
ana were between 5% and 23% and were thus rather low compared vae exhibited symptoms of mycosis. These results suggest a mode
to colonization rates of leaves of other plant species like corn of action involving feeding deterrence, antibiosis or changes in
(Wagner and Lewis, 2000), tomato (Klieber and Reineke, 2016), metabolism of the host plant and thus host plant quality rather
sorghum (Tefera and Vidal, 2009), and jute (Biswas et al., 2013). than a direct fungal infection of the insects (Vega, 2008; Vega
In contrast to these plants, grapevines are deciduous woody peren- et al., 2008). Colonization of grapevine plants was different in the
nial plants and plants used for our greenhouse trials were culti- two replicates of the bioassay. At a higher B. bassiana colonization
vated from hardwood cuttings. Seed treatment as an alternative rate, size of vine mealybug larvae was significantly smaller after
inoculation method as it has been successfully shown for tomato, feeding on endophytic leaves compared to control leaves. Vice
cotton (Ownley et al., 2008), opium poppy (Quesada-Moraga versa, at a lower colonization rate, we detected significant differ-
et al., 2009) and sorghum (Tefera and Vidal, 2009) is therefore ences in vine mealybug infestation rates. Accordingly, two differ-
not possible. Previous grapevine inoculation trials via root dipping ent modes of action of endophytic entomopathogens might
or soil inoculation resulted in no colonization at all (data not account for these observations, depending on rate of tissue colo-
shown). Root dipping or soil inoculation has been used for endo- nization by B. bassiana. In any case, we have shown that the pres-
phytic establishment of B. bassiana in banana (Akello et al., ence of entomopathogens as endophytes negatively influences
2007), sorghum (Tefera and Vidal, 2009), pine seedlings insect performance, yet further investigations are required to
(Brownbridge et al., 2012) and for cassava (Greenfield et al., determine the mechanisms underlying these effects. Results pre-
2016). Therefore, inoculation via spray application is apparently sented here point to the importance to also study sublethal effects
the only option for endophytic inoculation of grapevine plants. of endophytic entomopathogens on insects in order to understand
Moreover we have shown here that such an application is also tritrophic interactions between plants, endophytes, and insects.
compatible with viticultural practice in the field. We have shown for the first time that an endophytic establish-
Jaber (2015) reported slightly higher colonization rates of up to ment of B. bassiana in mature grapevine plants under field condi-
50% of young grapevine plants by B. bassiana after artificial spray tions is possible. Our results also indicate the potential for a long
inoculation. In these experiments as well as in the present study term establishment of the fungus in grapevine plants and that
no systemic establishment of B. bassiana in grapevine plants was endophytic establishment does apparently not interfere with com-
proved. Endophytic establishment of entomopathogenic fungi is mon viticultural management practices. In this regard, a couple of
known to be dependent on plant cultivar, fungal strain and many studies have shown that B. bassiana is sensitive against various
other environmental conditions (Vidal and Jaber, 2015). pesticides (Kos and Celar, 2013; Sapieha-Waszkiewicz et al.,
Here, we used molecular SSR markers to prove that the re- 2004; Todorova et al., 1998). However, even though synthetic
isolated B. bassiana strain was the one previously applied. In fungicides were simultaneously applied in our experimental vine-
general, direct detection of endophytic B. bassiana after spray yard, an endophytic establishment of B. bassiana in the mature
application using PCR-based techniques is difficult because of the plats was successful. Moreover, B. bassiana conidia are known to
likelihood of contamination with epiphytic propagules. In addition, be extremely sensitive to ultraviolet radiation and consequently
surface sterilization is regarded as an insufficient technique for persistence as well as germination of conidial suspensions applied
subsequent molecular assessment of endophytic establishment on the foliage is limited. Lohse et al. (2015) emphasized the impor-
(McKinnon et al., 2014). Evidence has accumulated that for tance of an adequate formulation for endophytic establishment of
culture-based techniques surface sterilization can result in under- entomopathogenic fungi. Here, we used a commercially available
estimated colonization rates, due to diffusion of the chemicals used fungal-based product (NaturalisÒ, active ingredient B. bassiana iso-
for sterilization into the leaves (Lohse et al., 2015; Ownley et al., late ATCC 74040) formulated as an oily dispersion, which may pro-
2008). In consequence and in line with other reports only a combi- vide a benefit for the colonization process of B. bassiana on
nation of different detection methods will result in sound qualita- grapevine plants. This product is registered in some EU member
tive and quantitative data about endophytic colonization of plants states i.e. for control of whiteflies in tomato thus having the per-
(Lohse et al., 2015). In this context methods must be adapted for spective of a rapid registration for other applications.
every plant species and different plant material. Overall, grapevine plants seemed not to be negatively affected
Despite the comparatively low endophytic colonization rates of by the presence of endophytic B. bassiana, as growth and perfor-
B. bassiana in grapevine we observed significant negative effects of mance of the respective inoculated plants was visually similar to
104
control plants during the period of observation (data not shown). Humber, R.A., 1997. Chapter V-1 – fungi: identification. In: Lawrence, A.L. (Ed.),
Manual of Techniques in Insect Pathology. Academic Press, London, pp. 153–
This is in accordance with previous studies on plant performance
185.
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B Danksagung/Acknowledgements
An dieser Stelle möchte ich allen herzlich danken, die mich während der (langen) Phase der
Promotion begleitet und durch ihre fachliche oder persönliche Unterstützung zum Gelingen
dieser Arbeit beigetragen haben.
Liebe Annette, dir möchte ich ganz herzlich danken für die Unterstützung bei der Bewerbung
um das Stipendium sowie für die Offenheit, die du der Umsetzung meiner persönlichen Pläne
entgegengebracht hast und dass du dennoch stets an den Abschluss der Arbeit geglaubt hast.
Während der Promotion hast du mir die Möglichkeit gegeben, Erfahrungen auf
internationalen Tagungen zu sammeln, mich zur Selbständigkeit im wissenschaftlichen
Arbeiten ermutigt und trotzdem immer eine offene Tür für mich gehabt. Danke dafür!
Herr Poehling, der diese Arbeit ebenfalls mitbetreut hat und mir immer sein vollstes
Vertrauen entgegengebracht hat, Ihnen möchte ich danken für die bereitwillige Übernahme
der Betreuung seitens der Leibniz Universität, Ihre Ideen und die umgehenden Antworten auf
meine Nachrichten unabhängig von Ihrem Aufenthaltsort.
Frau Winkelmann und Herrn Maiß danke ich für die spontane Bereitschaft zur Mitwirkung an
der Promotionskommission.
Der Deutschen Bundesstiftung Umwelt (DBU) für die finanzielle Unterstützung und die
Möglichkeit, auf Seminaren viele tolle Menschen kennenzulernen und den Ansporn immer
wieder über den Tellerrand zu blicken. Frau Dr. Schlegel-Starmann für die Betreuung von
Seiten der DBU und die besonderen Einblicke in die Stiftungsarbeit durch mein Amt als
Sprecherin.
Many thanks to Rusty Rodriguez and his team of Adaptive Symbiotic Technologies for being
my host during the short term scientific mission, for providing insights in their work and for
the unforgettable stay in rainy Seattle.
Ein weiterer Dank geht an das gesamte Institut für Phytomedizin der Hochschule Geisenheim
„Allen Phytos“, die mich herzlich in ihr Team aufgenommen und mich mit Rat und Tat
unterstützt haben. Danke für die stete Hilfsbereitschaft und die hervorragende
Arbeitsatmosphäre, die zum Gelingen dieser Arbeit beigetragen haben. Dabei bedanke ich
mich besonders bei:
Winfried für die tollen Fotos von allen Arbeitsbereichen, die unermüdliche Bereitstellung von
Topfreben sowie sein offenes Ohr und kreativen Lösungsvorschlägen bei Fragen zur
Versuchsumsetzung; Mirjam und Dustin für die Unterstützung im Gewächshaus und im
Labor; Ottmar für die Umsetzung und Unterstützung bei dem Freilandversuch; Helga für die
Weitergabe ihrer Erfahrung mit Pero.
117
Justine, Elizabeth und Jacqui, ihr habt dafür gesorgt, dass mir das Ankommen in Geisenheim,
der Phyto und der Promotion leicht fiel und viel Spaß gemacht hat. Ich wollte nicht mehr weg
und bin deswegen geblieben. Danke für die unvergessliche gemeinsame Zeit! Auch bei Holger,
Deniz und Ginger möchte ich mich für die unendlich vielen tollen und lustigen Stunden im
Büro und im Labor bedanken. Holger, danke für die Aufmunterung und den Ansporn bis zu
Letzt „Do it!“. Tina für die vielen lustigen Stunden in der WG und das Abschalten nach dem
Arbeitstag. Sandra für die Anfertigung wundervoller Graphiken zum besseren Verständnis
meiner Arbeit.
Besonders möchte ich meiner Familie danken, die immer hinter mir steht und mich bei
meinem Lebensweg unterstützt. Ein großer Dank geht an meine Eltern, die uns in den letzten
Jahren immer wieder in ihr Haus aufgenommen haben, um mir das Schreiben am Stück zu
ermöglichen und meinen beiden Kindern gleichzeitig eine unvergessliche Zeit geschenkt
haben. Danke für die viele Mühe, die uneingeschränkte Rückendeckung, das Vertrauen.
Lieber Michael, lieber Johann, lieber Anton, danke, dass es euch gibt!
118
Curriculum vitae
Persönliche Daten
ame
N Yvonne Rondot
Anschrift Adalbert-Stifter-Str. 26
65375 Oestrich-Winkel
Mobil 0179/38 92 942
E-Mail yvonne@rondot.de
Geburtsdatum 04. November 1982
Geburtsort Bonn-Bad Godesberg
Nationalität deutsch
Akademischer Werdegang
seit 03/2017 Wissenschaftliche Mitarbeiterin

am Institut für Gemüsebau der Hochschule Geisenheim
Projektkoordination des EIP-AGRI Projektes zur Ursachenfindung und Bekämpfung
der Gelben Welke an Feldsalat (GeWeKom)
Elternzeit: April 2018 - September 208
01/2012 – 09/2016 Promotionstipendium der Deutschen Bundestiftung Umwelt

Thema der Dissertation: „Endophytic establishment of Beauveria
bassiana in grapevine plants as sustainable pest management strategy”
Naturwissenschaftlichen Fakultät der Leibniz Universität Hannover
Durchführung am Institut für Phytomedizin der Hochschule Geisenheim
Elternzeit: September 2014 - August 2015

3/2010 – 07/2011 Studium M. Sc. Gartenbauwissenschaft Hochschule RheinMain
Abschluss: Master of Science (Gesamtnote: 1,0)
bschlussarbeit: „Endophytische und edaphische Etablierung des entomopathoge-
A
nen Pilzes Beauveria bassiana Stamm ATCC 74040 sowie Untersuchungen zu dessen
Virulenz“ (Note: 1,0)
09/2005 – 08/2009 Studium Gartenbau Fachhochschule Osnabrück

Abschluss: Diplom (Gesamtnote: 1,48)
bschlussarbeit: „Einfluss von Wassergehalt und Lagerungsdichte auf die Sauerstoff-
A
diffusion in gärtnerischen Substraten“ (Note: 1,3)
Schul- und Berufsausbildung
08/2002 – 07/2005 Ausbildung zur Bauzeichnerin

Architekturbüro Schubert+Thillmann, Koblenz
1993 – 2002 Martinus-Gymnasium Linz am Rhein
Abschluss: Allgemeine Hochschulreife (1,8)
119
weitere Praxiserfahrungen
05/2010 – 12/2011 Wissenschaftliche Hilfskraft am Fachgebiet Phytomedizin,

Forschungsanstalt Geisenheim
Mitarbeit in ein einem Forschungsprojekt zum Einsatz von mikrobiologischen
Präparaten zur Regulierung von Krankheiten an Erdbeeren
01/2010 – 02/2010 Volontärin am Julius-Kühn-Institut, Institut für Züchtungsforschung

an gartenbaulichen Kulturen und Obst, Quedlinburg
Mitarbeit bei der Etablierung biotechnologischer und molekularer Methoden zur
züchterischen Nutzung von Resistenzmerkmalen an Pelargonien
10/2009 – 12/2009 Gastwissenschaftlerin am Forschungszentrum Jülich, ICG-3,

Institut für Chemie und Geosphäre – Phytosphäre
Mitarbeit im Projekt PlantPower - Lebende Pflanzen in mikrobiellen
Brennstoffzellen für saubere, erneuerbare, nachhaltige, effiziente, in-situ
Erzeugung von Bioenergie
07/2008 – 12/2008 Praxissemester beim Pflanzenschutzdienst,

Landwirtschaftskammer NRW, Bonn
Arbeitsschwerpunkte:
Pflanzenschutzmittelprüfungen, Betreuung von Insektenzuchten
Förderungen und Auszeichnungen
hort-Term-Scientific-Mission im Rahmen der COST action FA1103

S
“Endophytes in Biotechnology and Agriculture” bei der Firma Adaptive
Symbiotic Technologies, Seattle, USA (03/2014 - 04/2014)
Arbeitsschwerpunkte: Untersuchung der Interaktion von Pflanzen mit endophyti-
schen Pilzen und deren Effekte auf die Stresstoleranz von Pflanzen; Transformation
des Pilzes Beauveria bassiana
1. Platz für eine hervorragende Posterpräsentation während der 58.

Deutschen Pflanzenschutztagung in Braunschweig, Titel des Posters: “The
entomopathogen Beauveria bassiana as an endophyte in Vitis vinifera”
Auszeichnung der Rudolf-Hermanns-Stiftung für eine herausragende
Masterarbeit im Studiengang Gartenbauwissenschaften
120
List of Publications
Peer-reviewed
Rondot Y. & A. Reineke (2019): Endophytic Beauveria bassiana activates expression of defence genes in
grapevine and prevents infections by grapevine downy mildew Plasmopara viticola. Plant Pathology
68, 1719-31.
Rondot, Y. & A. Reineke (2018): Endophytic Beauveria bassiana in grapevine Vitis vinifera (L.) reduces in-
festation with piercing-sucking insects. Biological Control. 116, 82-89.
Rondot, Y., & A. Reineke (2017). Association of Beauveria bassiana with grapevine plants deters adult black
vine weevils, Otiorhynchus sulcatus. Biocontrol Science and Technology, 27(7), 811-820.
Reineke, A., Bischoff-Schaefer, M., Rondot, Y., Galidevara, S., Hirsch, J., Uma Devi, K., (2014): Microsatellite
markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in
different environments: Technical validation and first applications. Biological Control 70, 1-8.
Oral Presentations
Rondot, Y. & A. Reineke (2014): Potential of endophytic Beauveria bassiana in grapevine against insects.
47th Annual Meeting of the Society for Invertebrate Pathology and International Congress on Inverte-
brate Pathology. Berichte aus dem Julius-Kühn-Institut 174:68
Rondot, Y. & A. Reineke (2014): Interaktionen des endophytisch etablierten entomopathogenen Pilzes
Beauveria bassiana mit Reben (Vitis vinifera) und deren Schaderregern. Julius-Kühn-Archiv 447: 390-
391
Rondot, Y. & A. Reineke (2014): Endophytic establishment of the entomopathogenic fungus Beauveria bas-
siana in grapevine Vitis vinifera. Journal of Plant Diseases and Protection 121: 97
Rondot, Y. & A. Reineke (2013): Potential of the entomopathogenic fungus Beauveria bassiana as an end-
ophyte in grapevine Vitis vinifera plants. 14th Meeting of the IOBC/wprs Working Group “Integrated
Protection and Production in Viticulture”, 13-17 October 2013, Ascona.
Rondot, Y. & A. Reineke (2013): The entomopathogenic fungus Beauveria bassiana as an endophyte in
garpevine Vitis vinifera (L.) plants. Proceedings of the International Symposium on Plant Protection
and Plant Health in Europe, „Endophytes for plant protection: the state of the art“. Berlin, Germany
26-29 May 2013
Poster Presentations
Rondot, Y. & A. Reineke (2013): Endophytic establishment of the entomopathogen Beauveria bassiana in
Vitis vinifera plants. 14th Meeting of the IOBC/wprs Working Group “Insect pathogens and entomopa-
rasitic nematodes”, 16-20 June 2013, Zagreb. IOBC-WPRS Bulletin Vol. 90, 2013 p. 129
Rondot, Y. & A. Reineke (2013): The entomopathogen Beauveria bassiana as an endophyte in Vitis vinifera.
COST Action FA1103: Endophytes in biotechnologie and agriculture, Working Group 1-4 Meeting, 14-
16 November 2012, Fondazione Edmund Mach, Italy p.45
Rondot, Y. & A. Reineke (2012): Endophytische Etablierung des entomopathogenen Pilzes Beauveria
bassiana in Reben (Vitis vinifera). Abstracts of the 58th German Plant Protection Conference, Braun-
schweig, Germany, 11-14 September 2012.
121

Endophytic Establishment of Beauveria Bassiana in Grapevine Plants As A Sustainable Pest Management Strategy

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Endophytic establishment of Beauveria bassiana

in grapevine plants as a sustainable pest

Von der Naturwissenschaftlichen Fakultät

der Gottfried Wilhelm Leibniz Universität Hannover

zur Erlangung des Grades

Doktorin der Gartenbauwissenschaften

(Dr. rer. hort.)

Yvonne Rondot, M. Sc.

Korreferentin: Prof. Dr. sc. agr. Annette Reineke

Tag der Promotion: 30. August 2019

ANOVA analysis of variance

Figure 16: Graphical abstract of manuscript I .................................................................................................... 94

1.1 Plant protection in viticulture

1.2 Biological control of pests and plant diseases

1.2.1 Microbial control agents

Antibiosis is a form of direct interaction resulting from the production of secondary

- mechanical and enzymatical penetration of the cuticle layers

1.3 Beauveria bassiana

1.3.1 Taxonomy and morphology

Beauveria bassiana (Balsamo-Crivelli), Vuillemin (Ascomycota: Hypocreales) is a well-known

clusters of short-globose to flask-shaped conidiogenic cells result in the characteristic white,

1.3.2 Beauveria bassiana as an entomopathogen

1.3.3 Plant colonization by Beauveria bassiana

1.3.4 The product Naturalis®

Beauveria bassiana is one of the most frequently commercialized fungal mycoinsecticides

The identification of an appropriate fungal pathogen or strain for development as

Reference Plant species Strain Plants parts treated – sampled Insect/pathogen

The specific objectives were:

• to optimize the endophytic establishment of B. bassiana in grapevine plants via

• to characterize the entomopathogenic potential of endophytic B. bassiana against

• to evaluate the antagonistic potential of endophytic B. bassiana against a key-fungal

• to study the effects of the endophytic establishment of B. bassiana in grapevine plants

I. Yvonne Rondot and Annette Reineke (2018): Endophytic Beauveria bassiana

III. Yvonne Rondot and Annette Reineke: Endophytic Beauveria bassiana

Yvonne Rondot and Annette Reineke

Geisenheim University, Department of Crop Protection, Von-Lade-Str. 1, D-65366 Geisenheim,

Type of authorship: First author

Type of article: Research article

Contribution to the article: Planned and performed most of the experiments

Contribution of other authors: Annette Reineke contributed to experimental design and

Journal: Biological Control, 116, 82-89

Impact Factor: 2.112

Date of manuscript submission: 27. April 2016

• Endophytic B. bassiana reduces infestation rate and growth of vine mealybugs.

• B. bassiana reduces infestation with grape leafhopper in the vineyard.

Greenhouse and field experiments were conducted to optimize endophytic establishment of

3.2 Materials and methods

3.2.1 Fungal material

3.2.2 Endophytic establishment in potted grapevine plants

3.2.3 Re-isolation of B. bassiana

3.2.4 Strain-specific detection of B. bassiana

3.2.5 Mealybug bioassay

3.2.6 Field trial

In addition to greenhouse and laboratory experiments we conducted a field trial as proof of

3.3.1 Endophytic colonization of potted grapevine plants

If applied as a conidial suspension on foliage of grapevine plants, both B. bassiana strains

3.3.2 Strain-specific detection of endophytic B. bassiana

3.3.3 Mealybug bioassay

3.3.4 Field trial