Entomopathogenic Fungi

Some species of the fungal genus Ophiocordyceps that parasitize ants have evolved some degree of behavioral manipulation to increase their own transmission. Carpenter ants (Camponotus spp.) infected by Ophiocordyceps unilateralis species climb vegetation and bite leaves or other plant parts before death, while other ants infected by other Ophiocordyceps species die buried within or on the leaf-litter, attached to stems and tree trunks. The microenvironment of the host's death location is important for fungal development and dispersal to new hosts, being an interesting example of an extended phenotype. This study investigated the effect of humidity variation in space (15 km 2) and time (14 months) on the occurrence and expression of the extended Ophiocordyceps phenotype parasitizing ants in Central Amazonia. We found O. unilateralis s.l., O. kniphofioides s.l. and O. australis s.l. parasitizing 30 ant species, but O. unilateralis s.l. was by far the most abundant fungus. Parasitized Camponotus atriceps and C. bispinosus were more abundant and died in higher places in periods with greater air humidity. Otherwise, the abundance and height of C. senex cadavers were higher in drier plots. These results provide insights on how the spatial and temporal variation in air humidity may shape the occurrence and behavioral manipulation of ants infected by entomopathogenic fungi at larger scales.

Fungi share an ancient historical bond with humans which is completely fascinating.
The Strange History of Life
Humans have always been fascinated with life in all its forms, but thousands of years ago, we only classified life on Earth into two categories – plants and animals. We can thank Aristotle for that, and he further divided animals into those with and without blood, as well as those in the land, sea, and air. That rudimentary system remained in place until the 1600s. In the 18th century, Carolus Linnaeus divided life into the kingdoms of animals and plants, and then further began dividing those into different genera and species, which is why we have the two-part naming system in science (Homo sapiens, for example; Homo is the genus and sapiens is the species.

It wasn’t until the middle of the 19th century that single-celled organisms were finally given their due as a separate kingdom of life (Protista). Seventy years later, single-celled organisms were divided into eukaryotes and prokaryotes, so bacteria became the 4th kingdom of life. Although fungi had been recognized as a unique part of the animal kingdom, it was not separated into its own kingdom until 1969. This five-kingdom system remains the most widely accepted format for classifying life on Earth.
All classifications of life until recently, including the expansion from two kingdoms to five, were based on physical observations of how things looked, even under a microscope. This is how the closeness and relationships between species, genera, classes, orders and kingdoms were decided. Given that fact, it comes as no surprise that most people classified fungi as plants for so long. The similarity in appearance is pretty clear, like little red and white trees…

However, thanks to modern technology, the analysis of genetic relationships between species and organisms is now possible, and it shows something very interesting. By examining the genes of different species, both animal and fungi, the mutational changes can be observed, and genealogical relationships can be determined that go back millions of years.

As it turns out, animals and fungi share a common ancestor and branched away from plants at some point about 1.1 billion years ago. It was only later that animals and fungi separated on the genealogical tree of life, making mushrooms more closely related to humans than plants. Most likely, this common ancestor was a single-celled organism that had sperm-like characteristics (like an animal) and then a later developmental stage with a stronger cell wall (fungi).

Are mushrooms vegetables?
Simple answer? No, it’s not a vegetable. Mushrooms are fruiting bodies of macroscopic filamentous fungi. Earlier when mycology (the study of fungi) arose, it was a part of botany. This happened as fungi were considered to be primitive plants. The major difference between a plant (vegetable) and mushroom is how they acquire their food. In the case of plants, they possess chlorophyll and make their food via photosynthesis. Fungi, on the other hand, exist on decaying material in nature. Also the obvious structural differences, such as lack of leaves, roots, and seeds.So fungi now have their own kingdom on the basis of cellular organization.

However, this is the science side of things. Now let us take a look at the other side, the side of food! In terms of food and nutrition, mushrooms are often put in the category of vegetables. The reason for this lies in the nutritional value. Mushrooms and plants have similar nutritional attributes. The nutrients provided by mushroom are spread across various core food groups such as meat and grains. All this has made mushroom a valuable asset in the kitchen both for taste and nutrition.

The Connection Between Mushrooms and Men
Haven’t you ever noticed that eating a perfectly cooked portobello mushroom is closer to eating meat than a salad? Well, that isn’t exactly a scientific explanation of the connection, but this new revelation of our link to fungi does shed some light on the trouble we have with fungal infections in the body. It is notoriously difficult to treat fungal infections, rather than viral and bacterial pathogens, and this close genetic link may explain why.

Fungi and animals are both eukaryotes, with nuclei and other complex structures inside their cell membranes, whereas bacteria are much simpler – composed of just genetic material and a cell wall. Therefore, when we try to fight a bacterial infection, the body is able to easily differentiate and neutralize the threat. Unfortunately, with fungal infections, the body sometimes thinks that the infection is…. us. Drugs that are synthesized to fight the cells of fungal infections may also attack healthy human cells, as there is very little structural difference.

This study was aimed at developing a physical extraction method of M. ansiopliae and B. bassiana conidia from precooked rice culture media by agitation with inerts. Evaluation was made of the abrasive effect of the following inerts: Caulin SAZ, Caulin ML, Filito Cremogeo, Argila Lopes, Diatomita and Leucita Calcinada, at different times of the agitation. It was verified that Diatomita was the most  efficient for M. anisopliae conidia extraction and the sieve for B. bassiana.

The objective of this work was to evaluate the pathogenicity of Beauveria bassiana to Diaphorina citri (Hemiptera: Psyllidae) nymphs, and to check the compatibility of the fungus with phytosanitary products, and its persistence in citrus plants. Nymphs of D. citri were sprayed with B. bassiana in the concentrations 5x106, 1x107, 5x107, 1x108, 5x108 and 1x109 conidia mL-1 for lethal concentration determination. In order to evaluate the compatibility of the phytosanitary products with the fungus, neem extract and five insecticides of four different chemical groups were individually added to PDA culture medium, in which the fungus was cultivated. Vegetative growth, sporulation and viability of the entomopathogen were evaluated. In the greenhouse, citrus plants were first treated with the phytosanitary products and, then, with the pathogen. Exposure times of 24 hours, 7 and 14 days were evaluated. Fungus pathogenicity to D. citri nymphs  was confirmed; LC50 of 0.4 x107 and LC90 of 6.7 x107 conidia mL-1 were determined at the tenth day of evaluation. In the laboratory, the phytosanitary products reduced fungus growth. In greenhouse, the products did not affect fungus survival on citrus plants.

The susceptibility of the adult and larval stage of housefly, Musca domestica L. (Diptera: Muscidae), to two entomopathogenic fungi, Metarhizium anisopliae (Metsch.) Sor. and Beauveria bassiana (Bals.) Vuill., was evaluated under laboratory and simulated field bioassays. Bioassays on adult houseflies were carried out at different conidial concentrations ranging from 103 to 109 conidia/ml in petri plate and minichamber assays. Absolute mortality was observed within 4–5 days at all the concentrations tested. M. anisopliae was found to be more effective with LC50 of 6.75 × 107 conidia/ml compared with 1.21 × 108 conidia/ml of B. bassiana in petri plate bioassay. Similar trend was observed in minichamber bioassay. Larvicidal activity evaluated through petri plate bioassay also indicated that M. anisopliae was more effective larvicide with LC50 of 4.1 × 108 conidia/ml as against 3.31 × 109 conidia/ml of B. bassiana. Larvicidal activity was further evaluated in simulated field condition of decaying waste matrix using dry conidial formulations (108 conidia/g) of both the fungi. Larval mortality obtained in this assay was 43% (B. bassiana) and 63% (M. anisopliae). Remarkably better performance of M. anisopliae as an adulticidal and larvicidal agent over B. bassiana in laboratory bioassays as well as simulated field conditions suggests that it may have good potential to become part of an integrated housefly control program.

Isaria poprawskii is described as a new entomopathogenic species similar to Isaria javanica (=Paecilomyces javanicus). It was discovered on the sweetpotato whitefly, Bemisia tabaci biotype B
in the Lower Rio Grande Valley of Texas (LRGV), USA. Morphological and DNA examinations indicated the distinctness of I. poprawskii from the ex-type isolate of I. javanica.  I. poprawskii
produced light yellow young colonies to darker yellow with a grayish-violet center to a taupe or a brownish-gray mature conidial mass; conidia hyaline, one-celled, 3.9 (2.9-4.6) mm long x 1.6
(1.4-2.1) mm wide; colored synnemata, but I. javanica ex-type produced white colony, hyaline conidia and no synnemata. A phylogenetic position of I. poprawskii was inferred by a nucleotide
sequence analysis of b-tubulin along with standard b-tubulin sequences from GenBank. Fifteen unsequenced isolates, including eight from the LRGV, were investigated.The analysis confirmed
that I. poprawskii could be recovered from LRGV fields, and that both I. javanica and I. poprawskii are present in the LRGV in sympatry. I. poprawskii was shown to be closely related to I. javanica; however, it formed its own unique clade, thus confirming its status as a new fungal species.

Entomopathogenic fungi are rich sources of bioactive secondary metabolites that possess insecticidal properties. The present study reported a novel approach for the identification of insecticidal compounds produced by Lecanicillium lecanii 09 and to assess their toxicity against the diamondback moth Plutella xylostella L. The cyclic peptides groups of toxic substances were separated from L. lecanii 09 through submerged liquid state fermentation. The most abundant toxic metabolite, Bassianolide was purified by high-performance liquid chromatography (HPLC) and its molecular weight and purity were determined by Liquid chromatography – mass spectroscopy (LC-MS), Fourier transformed infrared spectroscopy (FT-IR), and H 1 nuclear magnetic resonance (NMR) respectively. Subsequently, the toxicity of bassianolide was tested against third instar larvae of P. xylostella at three different concentrations (0.01, 0.1, 0.5 mg/ml). The results showed that higher concentration of 0.5 mg/ml had significant maximum mortality at 120 hour post inoculation. Furthermore, we investigated the ligand-target interaction of secondary metabolite binding with target insect immune receptor proteins and predicted the role of toxicity against insect host. This is the first study to report the infection process and the interaction of fungal mediated cyclicdepsipeptide compound (bassianolide) from L. lecanii 09 against the insect host P. xylostella. This novel approach provides a potential impact on biological control using natural toxic compound which acts as good inhibitor on pest insect and prevents toxicity hazards, pollution as well as ecocidal effects killing several beneficial insects.

Tkaczuk C., Krzyżanowski R., Leszczyński B., Krzyczkowski T., Papierok B., 2010. The effect of selected plant volatiles on conidial germination of aphid-pathogenic fungi. 43th Annual Meeting of the Society for Invertebrate Pathology (SIP), 11-15 July, Trabzon (Turkey).

Global agriculture is currently challenged to provide increasing supplies of food for a growing population due to the changing trends of insect attacks and climatic conditions. There is overwhelming evidence that the use of chemical pesticides have created many other serious problems like ecological backlashes in pest species, environmental pollution and degradation, threat to biodiversity conservation, loss of beneficial fauna (predators, parasites, pollinators etc) and human beings in particular. Keeping in view, the new generation of pesticides have attracted attention in pest management in recent years, Biorational pesticides as “third-generation pesticides” are derived from some natural source and impose minimum or no adversarial threats on the environment or beneficial organisms. Some examples of biorational pesticides are the microbial pesticide Bacillus thuringiensis (Kurstaki), neonicotinoids, avermectins, phenlpyrazoles, spinosyns, pyrroles, oxadiazines and various groups of insect growth regulators. With a narrower target range of pests, they also tend to have a more specific mode of action. This new class of pesticides are often designed to control a pest population to a manageable level rather than completely eradicate a target pest. These products have great potential for replacing the persistent conventional insecticides, confirming effective cost-benefit ratio, tackling ecological backlashes and ensuring food security with safe environment. The field of biorational pesticides is deep; consequently they are a source of both optimism and concern. However, these bio-products are not only winning the reliability of the market and end user; but also demonstrating their worth and potential in sustainable integrated pest management (IPM) program.

“Studies on Management of Insect Pests of Chickpea vis-a-vis Organic
Farming” was conducted at Research Farm, College of Agriculture, Bikaner during
rabi, 2015-16.
The moth catches in traps of H. armigera started in 48 standard
meteorological week and reached peak in 11th standard meteorological week during
rabi, 2015-16. A significant positive correlation was computed between male moth
catches in the traps where septa were chanced after every 15 days and 30 days
interval with maximum and minimum temperature, rainfall and sunshine hours while
maximum and minimum relative humidity had no affection H. armigera moth catches.
Out of ten bio-agents/insecticides treatments evaluted for termite control in
chickpea, seed treatment with Imidacloprid 70 WS @ 2 g per kg seed followed by soil
application of imidacloprid 17.8 SL @ 300 ml/ha and seed treatment with
Imidacloprid 70 WS @ 2 g per kg seed were found most effective against termite,
However, seed treatment with M. anisopliae and B. bassiana @ 5 and 10 g per kg
seed followed by soil application @ kg per ha of respective bio-agent also proved
better for the control of termite. The minimum infestation at harvest was obtained in
seed treatment with Imidacloprid 70 WS @ 2 g per kg seed followed by soil
application of imidacloprid 17.8 SL @ 300 ml/ha (9.17%) whereas it was maximum in
seed treatment with M. anisopliae @ 5 g per kg seed (15.67%).The maximum seed
yield was obtained in seed treatment with Imidacloprid 70 WS @ 2 g per kg seed
followed by soil application of imidacloprid 17.8 SL @ 300 ml/ha (13.67 q ha-1
)
whereas it was minimum in seed treatment with B. bassiana @ 5 g per kg seed(8.00
q ha-1
).
Out of six insecticide/bio-pesticide evaluated for H. armigera control, two
spray of each treatment viz., indoxacarb 14.5 SC (1.0 ml/l) and NSKE (5.0 ml/l) was
found most effective in crop protection. Azadirachtin 0.3 EC (5.0 ml/l), HaNPV (250
LE/ha) and B. thuringiensis (1000 ml/ha) were found moderately effective in reducing
pod borer population, while garlic extract (10 ml/lit.) was found least effective. The
minimum pod damage (4.69%) was recorded in treatment of indoxacarb followed by
NSKE (10.41%) and azadirachtin (10.84%). The maximum pod damage (14.27%)
was found in garlic extract treatment. While it was 20.95% in control. The maximum
seed yield was obtained in the treatment of indoxacarb 14.5 SC (14.83 q ha-1
)
whereas it was minimum in garlic extract (10.00 q ha-1
). Maximum B:C ratio was
obtained in the treatment NSKE (13.62), while minimum in B. thuringiensis (2.29).

Nowadays, the use of entomopathogenic nematodes as a biological control agent is a key component in IPM system. Entomopathogenic nematodes of the genera Steinernema and Heterorhabditis (Nematoda: Rhabditida) are extraordinarily lethal to many important insect pests, yet are safe for plants and animals. They are the only insect-parasitic nematodes possessing an optimal balance of biological control attributes. The effect of temperature on the virulence of three species of entomopathogenic nematodes, Steinernema thesami, Heterorhabditis bacteriophora and Steinernema feltiae was investigated. Last instar of Tenebrio molitor larvae were choosing for experiment. In the laboratory, all three nematode species successfully reproduced inside T. molitor larvae. H. bacteriophora produced the highest number of infective juveniles per larva at 30°C than S. feltiae and S. thesami. S. feltiae caused the highest mortality of larvae at 20°C, whereas S. thesami infected T. molitor larvae at the widest temperature range and killed insects between 8-35°C. Based on the present study, we indicate that entomopathogenic nematodes have well-defined thermal breadths for their development and reproduction.

The objectives of this research were: to evaluate, in laboratory, the pathogenicity of fungi isolates and nematodes in the control of fruit fly prepupae and adults in the laboratory and the application of these entomopathogens in the greenhouse. Different Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorok. concentrations were tested on Ceratitis capitata (Wied.) prepupae in sterilized and natural soil under laboratory conditions. The efficiency of six B. bassiana and M. anisopliae isolates was evaluated on C. capitata prepupae with application this selected isolates at a concentration of 5×108 conidia/mL in soil pots containing citrus seedlings in the greenhouse. In addition, the pathogenicity of the entomopathogenic nematode Heterorhabditis sp. (isolate IBCBn 05) was evaluated by applying 200 infective juveniles (JI)/ prepupae with the same methodology used for the fungi. Transmission of the above fungi in C. capitata adults was also studied under laboratory conditions. It was verified that the fungi B. bassiana and M. anisopliae were pathogenic to C. capitata prepupae, with the isolates IBCB 66 and IBCB 425, respectively, were the most virulent. Heterorhabditis sp. was also pathogenic at the concentration applied. In the greenhouse, the B. bassiana fungus reached a prepupal control efficiency of 66.6%. The fungi were transmitted among the C. capitata adult population even when only 10% of the population was initially contaminated.

The virulence of ten isolates of Beauvena bassiana (Balsamo) Vuillemin to the maize weevil, Sitophilus zeamais Motsch., was tested in the laboratory. All isolates tested were capable of infecting S. zeamais but their virulence, determined by adult mortalities and median lethal time, varied. A total of five (I89-481, I90-520, I89-477, I90-533, and I94-907) highly virulent, three (I92-736, I93-906 and I92-761A) intermediate and two (I93-868 and I93-870) weakly virulent isolates were identified for S. zeamais. The growth and sporulation rates and persistency of water formulated conidia of the most virulent isolates were also studied.I90-520 was selected for further work and dose-mortality relationships and the infectivity of dry conidia against Sitophilus zeamais were determined. The lowest dose (104 conidia ml−1) caused about 88% mortality within eight days. To assess the infectivity of a dry formulation of the isolate, conidial doses of 0, 0.1, 0.5 and 1 g of B. bassiana per 25 g of popping maize seeds were compared with pirimiphos-methyl at 10 ppm. This demonstrated the potential of dry conidia of the isolate to infect and cause mycosis to S. zeamais. Adult mortality of S. zeamais and damage to maize seeds treated with different levels of dry conidia were not statistically significantly different from pirimiphos-methyl-treated maize after 14 days of storage period, but the chemical pesticide killed all target insects within 24 h, whereas mortality began at day 3 for the best B. bassiana treatment.

The pathogenicity of a naturally occurring entomopathogenic fungus, Isaria sp., found during natural epizootics on whiteflies in the Lower Rio Grande Valley of Texas, against the sweet potato whitefly,
Bemisia tabaci (Gennadius) biotype B, was tested under laboratory conditions (27 C, 70% RH and a photoperiod of 14:10 h light:dark). Exposure of second-, third- and fourth-instar nymphs to 20, 200, and 1000 spores/mm2, on sweet potato leaves resulted in insect mortality. Median lethal concentrations for second-instar nymphs (72–118 spores/mm2) were similar to those for third-instar nymphs (101–170 spores/mm2), which were significantly more susceptible than fourth-instar nymphs (166–295 spores/mm2). The mean time to death was less for second instars (3 days) than for third instars (4 days) when exposed to 1000 spores/mm2. Mycosis in adult whiteflies became evident after delayed infections of sweet potato whitefly caused by this fungus. These results indicate that Isaria sp. is pathogenic to B. tabaci nymphs, and to adults through delayed infections caused by this fungus. Its low lethal concentrations and high virulence against B. tabaci make this fungus a promising biocontrol agent to control whitefly.