Indian Phytopathology

https://doi.org/10.1007/s42360-019-00153-6

RESEARCH ARTICLE

Occurrence and molecular identification of nematodes associated

with brinjal (Solanum melongena L.) var. Mattu Gulla
Annamalai Muthusamy1 · Rashmika Singh1 · Bobby Paul2 · Mattu Radhakrishna Rao1 · Thokur Sreepathy Murali3

Received: 22 November 2018 / Revised: 17 May 2019 / Accepted: 11 July 2019

© Indian Phytopathological Society 2019

Abstract
The nematodes which belong to the family Longidoridae are obligatory plant parasites and are considered as economically
significant pests since they cause severe crop losses around the world. Conventional identification of nematodes using mor-
phological features is a time-consuming and challenging task. To overcome the challenges in conventional identification
methods, rapid and reliable identification methods need to be established which will not only enable proper identification
but also provide effective management program for nematodes which devastate the root system. In the present study, we
have observed the nematode infection in a unique brinjal variety (Solanum melongena L. var. Mattu Gulla) grown in Mattu
Village, Udupi District of Karnataka, India and identified the nematodes on the basis of molecular characterization using D3
expansion region of 28S rRNA gene sequence. Cephalobus cubaensis was found from the roots of Mattu Gulla for the first
time. Our study will facilitate further screening and characterization of the root-knot and root-lesion nematode infections in
both nursery as well as post-transplantation seedlings of Mattu Gulla in the field conditions.

Keywords Brinjal · Nematode infection · 28S rRNA sequencing · Identification · Cephalobus cubaensis

Introduction further sub-categorised into ectoparasites, semi-endopara-

sites and migratory and sedentary endoparasites (Bernhard
Nematodes constitute 80–90% of all multicellular animals 1992; Ruehle 1973). Currently, more than 4100 species of
in numbers and occupy various environments such as soil, plant-parasitic nematodes have been studied and described
marine or freshwater as free-living opportunistic animals (Decraemer and Hunt 2006).
with different nutrition sources (Jones et al. 2013; Khan Collectively, the nematodes represent a major threat to
2015). Nematodes which are abundant in soil are con- global food security because of the damage they cause to
sidered to be reliable ecological indicators of varied soil agricultural crops. It is estimated that nematode infection
environments and are classified into five different trophic can lead up to 10.7% yield loss in 20 major food crops while
groups such as plant-parasitic, fungivorous, bacterivorous, in other economically important crops, this figure can be as
omnivorous and predatory nematodes (Ferris et al. 2001; high as 14% in developing countries including India (Gan-
Neher 2001; Yeates 2003). The parasitic nematodes are guly and Dutta 2012). Besides their direct impact on plant
fitness, nematodes also act as wounding agents, vectors, host
modifiers, rhizosphere modifiers and resistance breakers and
* Annamalai Muthusamy play a significant role in establishment of disease complexes
amsamy.slsmu@gmail.com in association with other pathogens in plants (Brussaard
1
Department of Plant Sciences, Manipal School of Life et al. 2001; Desaeger et al. 2004). There are 12 different
Sciences, Manipal Academy of Higher Education (MAHE), species of nematodes reported from India (Khan et al. 2010)
Planetarium Complex, Manipal, Karnataka 576104, India while 97 species of root lesion nematode have been reported
2
Department of Bioinformatics, Manipal School of Life from cool, temperate and tropical environments (Handop
Sciences, Manipal Academy of Higher Education (MAHE), et al. 2008). Among the environmental factors, the host crop
Manipal, Karnataka 576104, India has been shown to have the foremost influence on the build-
3
Department of Biotechnology, Manipal School of Life up of nematode population subsequently leading to reduc-
Sciences, Manipal Academy of Higher Education (MAHE), tion in crop yield.
Manipal, Karnataka 576104, India

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 Indian Phytopathology

Identification of nematodes based on the morphological packed separately, sealed and brought to laboratory. The
features is challenging and a time-consuming process. The infected plants were preserved at − 80 °C for future use
diagnostic morphological features of nematodes are cir- and the soil samples were stored at 20–25 °C (Gazaway
cumscribed, indication of phenotypic plasticity and overlay et al. 1995). The nematodes were isolated from plant roots
among the nematode species. Rapid and consistent iden- using Jar incubation method while the nematodes from soil
tification of nematode is imperative for effective manage- were isolated following a modified protocol of Baermann
ment and to establish active integrated pest control programs (Barrière and Félix 2006). The isolated nematodes from
(Luc 1987; Madani et al. 2005; Sato et al. 2007). Therefore, infected roots and soil samples were cultured separately
molecular techniques such as DNA based diagnostic tools on nematode growth medium (NGM) (Stiernagle 1999).
along with phylogenetic analysis is used extensively to chal- The NGM was seeded with E. coli OP 50 liquid culture and
lenge the difficulties associated with conventional identifica- incubated at 37 °C for 8 h. 200 µL of nematode suspension
tion methods (Munera et al. 2009). DNA based molecular from soil and root samples were added on NGM with E.
approach have been used since three decades for the iden- coli and incubated at 20–22 °C for nematode multiplication
tification and discrimination of nematode species (Waey- and growth. The temporary mounts of nematodes from soil
enberge et al. 2000; Al-Banna et al. 2004; Inserra et al. and root samples as well as cultured plates were prepared
2007; Subbotin et al. 2008). For instance, 28S rDNA and and observed.
18S rDNA genes have been routinely used as biomarkers for The genomic DNA was isolated from nematodes
identification and characterization of plant parasitic and soil obtained from soil, infected root and culture plates (Keiko
nematodes (Bhadury et al. 2005; Madani et al. 2005; Jones and Mitani 2000) and used for polymerase chain reaction
et al. 2006; Munera et al. 2009) and species-specific primers (PCR) amplification (Al-Banna et al. 2004) and sequenc-
have been designed to differentiate the species among the ing. The D3 expansion region of 28S rDNA fragment was
major nematode genera (Uehara et al. 1998; Waeyenberge amplified with the following primers: D3A 5′-GAC​CCG​
et al. 2000). TCT​T GA​A AC​ACG​GA-3′, and D3B 5′-TCG​GAA​G GA​
Mattu Gulla (MG), a unique, round and green variety of ACC​AGC​TAC​TA-3′ (BioServe, Hyderabad, India). The
brinjal is being cultivated since 500 years in Mattu village PCR amplification was carried out with 1X PCR buffer,
of Udupi District in Karnataka, India (Bhat and Madhyastha 0.4 mM of dNTPs, 1% BSA, 0.5 mM of ­MgCl2, 10–30 ng
2007). Geographical Indication Registry, Chennai, India of template DNA, and 100 ng of primer, each in a total
has granted Geographic Indication status to MG consider- volume of 10 µL and 1U of Taq DNA polymerase (Invit-
ing the uniqueness of the variety by Geographical Indica- rogen). The PCR was performed in a Mastercycler Nexus
tions Journal (2011). MG is highly susceptible to diseases Thermal Cycler (Eppendorf, USA) and initially DNA was
by pathogens, insect pests and nematode infections, which denatured at 95 °C for 3 min and PCR was carried out with
affect the regular growth and development, and leads to 35 cycles of denaturation, annealing and extension at 95 °C
reduction in agronomic characters. Therefore, the present for 3 min, 51 °C for 1 min and extension at 72 °C for 1 min
investigation was aimed to identify the genus and species of and kept for a final extension at 72 °C for 7 min.
nematodes associated with Mattu Gulla plant using PCR- Amplified fragments were electrophoresed on 1%
based methods. agarose gel, gel-eluted for purification and sequenced in
ABI3130 sequencer (Applied Biosystem, USA) using
BigDye™ dye Terminator v3.1 cycle sequencing kit
Materials and methods (ABI, USA) according to manufacturer’s instructions. The
obtained sequences were manually edited and aligned with
The field at Mattu Village (N = 13º 16′ 58.0974″; E = 74º already available sequences using BLASTN program (Alts-
44′ 15.8274″), Udupi District, Karnataka, India was chul et al. 1990).
observed carefully from brinjal seedling transplantation
stage and the infected part of the field was noted. The plant
and soil samples were collected from healthy and infested Results and discussion
spots from five different locations of the field. The brinjal
plants from infected region were uprooted gradually for The plants and its wide range of diversity offers energy
isolation of nematodes and around 500 g of soil (around and nutrient source to the soil microbial communities of
the infected plants) was also collected in separate labelled which plant parasitic nematodes are major organisms (Soh-
polythene bags. The collected plants and soil samples were lenius 1980). Being an invisible enemy of crop plants, it

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Indian Phytopathology

has been extensively documented as the most overwhelm- nematodes are not constantly observable. Currently the
ing pest among various groups of pathogenic organisms, DNA sequence signature-based methods are extensively
which depends on crop and other plants for their persis- used as a tool for nematode community analysis (Holter-
tence (Sasser and Freckman 1987). Majority of the plant man et al. 2008). In the present study, we have isolated
parasitic nematodes can feed and reproduce on a wide nematodes successfully from soil, infected roots of MG
range of plant species whereas some nematode species plant and cultured plates, and confirmed the identity of
feed and reproduce in large numbers only in association the nematodes using D3 expansion region of 28S rDNA
with specific plant species as food source (Cook and Yeates fragment by PCR and BLAST.
1993). Several control measures have been developed and The brinjal field was carefully observed from develop-
are still further needed to improve the existing methods of ment of nursery, transplantation of seedlings and further
identification, controlling the infection and multiplication growth of the plantlets. The healthy plants in the field
of nematodes. The conventional identification of nematode showed normal growth without any symptoms (Fig. 1a)
species in soil and plant communities using light micros- whereas the stunted, unthrifty and patchy growth was
copy is time consuming and difficult for relatively small noted under infected areas (Fig. 1b). The symptoms fol-
samples. Further, juvenile morphological characters of lowing infection included leaf yellowing and reduction

Fig. 1  a Normal Mattu Gulla

field. b Arrow showing infected
area of the field. c Normal
Mattu gulla plant. d Infected
plant showing symptoms of
toppling. e Highly infected plant
showing yellowing of fruit and
death of leaves. f Infected plant
undergoing the process of death

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 Indian Phytopathology

Fig. 2  a, b Nematode infected

seedling in different places of
Mattu village

of foliage (Fig. 1c), day time wilting, reduced foliage, Many economically important crops are infected by
chlorosis, necrosis and formation of white tips (Fig. 1d). reniform nematodes; cowpea tomato, okra, potato, onion,
The progressive decline in leaves and stem (Fig. 1e) due sweet potato, beans, and brinjal are the vegetables infested
to infection led to permanent wilting and death of plants with nematode species (Reddy et al. 1979; Jain 1992). Our
(Fig. 1f). Besides the above ground abnormalities, the results are in conformity with earlier reports by Korno-
root abnormalities such as thick roots with less number bis et al. (2015) for morphometrics and molecular char-
and growth of primary as well as secondary roots was acterization of Paralongidorus rex using 18S, ITS1 and
also observed (Fig. 2). The nematodes were isolated from D2-D3 of 28S rRNA. Further, PCR based discrimination
infected roots and soil (Fig. 3) as well as from cultured of Pratylenchus have been reported earlier (Waeyenberge
plates (Fig. 4). et al. 2000; Al-Banna et al. 2004; Carrasco-Ballesteros
The D3 region was amplified from both isolated and et al. 2007; Subbotin et al. 1999, 2008). Similarly, Munera
cultured nematodes from soil and infected roots. The et al. (2009) reported the morphological and molecular
345 bp PCR fragment was observed from soil, root and characterization of root-lesion nematode, Pratylenchus
cultured nematodes (Fig. 5). The PCR products were araucensis. The PCR-based diagnostic method has wide
eluted, sequenced using ABI3130 sequencer, analyzed range of application and is extensively utilized for iden-
with NCBI BLAST and compared to earlier reported tification as well as to distinguish the major species of
sequences from NCBI GenBank database. The nematode nematodes, which suppress the plant growth and develop-
sequence showed maximum pairwise alignment score of ment eventually leading to drastic reduction in agronomic
593 and 98% identity with 28S rRNA gene sequence from characters. The rapid identification and characterization of
Cephalobus cubaensis. The sequence alignment of large nematodes will be very useful for the control of nematode
ribosomal subunit (28S-D3 region) from the nematode infection as well as in integrated pest management system.
isolated from Mattu Gulla fields were identical with that Kerrigan and Rogers (2013) have reported the widespread
of Cephalobus cubaensis (GenBank accession number occurrence of Cephalobus cubaensis in soils and plant
EU253570). roots.

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Indian Phytopathology

Fig. 3  a Modified Baer-

mann–Petri plate set up. b–d
Microphotographs of nematode
(10X). e Microphotographs of
Nematode (40X)

Currently, the PCR based methods and marker-assisted Mattu Village, Udupi District of Karnataka, India and iden-
selection are used extensively for prompt identification of tified the nematode based on molecular characterization
nematodes and facilitate the novel approaches for nematode using D3 expansion region of 28S rRNA gene sequence
management program (Banu et al. 2017). Based on present as Cephalobus cubaensis. Our study will facilitate fur-
findings, it can be concluded that the nematode infection ther screening and characterization of the root-knot and
in brinjal was observed along with other diseases, which root-lesion nematode infections in both nursery as well as
devastates the crop. Our study has shown the presence of post-transplantation seedlings of Mattu Gulla in the field
nematode infection in S. melongena L. var. Mattu Gulla at conditions.

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 Indian Phytopathology

Fig. 4  a Nematode eggs

scattered on the plate (4X). b
Nematodes feeding and interact-
ing with other nematodes (4X).
c Nematodes at × 20 magnifica-
tion under inverted microscope

Acknowledgements We are grateful to Manipal Academy of Higher

Education (MAHE), Manipal, Karnataka, India and TIFAC-CORE
and FIST, DST, New Delhi and K-FIST, VGST, Govt. of Karnataka
for the facilities. We are indebted to Prof. K. Satyamoorthy, Director,
Manipal School of Life Sciences, MAHE for encouragement, support
and critical comments. We would like to thank Mrs. Shashikala Tantry
for her experimental assistance and Mr. Kiran K.R. for the format of
article; Shri Mohan Rao and Lakshmana Rao, Members of Mattu Gulla
Growers Association, Mattu Village, Udupi District, Karnataka, India
for their help in collection of plant samples.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of

interest.

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