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Methods of Screening of Jute (corchorus Sp.) Germplasms for Resistance Sources Against Nematodes

 

M. R. Khan

Nematologist, AICRP (Nematode), Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya, P.O. Kalyani, Nadia, PIN-741235, West Bengal, India

Email: mrkhanbckv@rediffmail.com

Exploitation of resistance in crops is one of the most effective and ecofriendly components of integrated pest management and inclusion of this property ensures increased crop yield in the presence of nematode. The nematodes resistance in host plant is manifested by reduced rates of nematode reproduction and consequently low nematode population densities in the crop rhizosphere than that of a susceptible one. The crops cultivars showing high degree of resistance with all acceptable agronomic features are commonly recommended for nematode infested fields either as a routine crop or in a rotational sequence of the crops. Therefore, the growing of nematode resistant cultivars against the target nematode species demands correct identity of nematodes with the prevailing race(s) existing in the area.

 Screening of germplasms/varieties against plant parasitic nematodes require preparation of pure inoculum of identified nematode species and races, sterile soil, inoculation technique and assessment or rating scale for resistance response to nematodes. For preliminary screening, a simple technique of assaying is most essential for handling large number of genetic materials. The primary objective of screening assays is exposing the plant population to the nematode parasite for differentiating resistant genotypes from the susceptible ones. Often for assaying against nematodes, final nematode population (Pf) is compared with the initial population inoculated (Pi) for calculating reproduction factor (RF=Pf/Pi). Generally resistance in host is characterized by the ability of the nematodes to reproduce on the host’s genotype and/or their compatibility reactions.

Root knot nematodes induce a most common and conspicuous symptoms of root galls and egg masses on the highly susceptible hosts. The size of galls vary from the small (1-2mm in diameter) to large gall (>1cm in diameter) depending upon the hosts. The gall appears on the root system either discretely or numerous galls merged together forming a multiple gall. The larger galls may contain more than one female while small galls contain only one female. The gall formation on the root is not necessarily meant there should have a Meloidogyne female because it’s a symptom of nematode attack. Therefore, clinical diagnosis on the root system is essential through dissection of roots under microscope for confirmation of nematode involvement with the symptoms(Taylor & Sasser, 978).

For routine screening process of crop germplasms, the most commonly used term’ ‘resistant’ and ‘susceptible’ with various intermediate categories are used to express the degree of resistant response on the test genotypes. To evaluate the resistant response against nematode, host suitability/compatibility for nematode is determined with their reproduction ability on the host. Usually, in the highly resistant plant, reproduction of nematode is less as compared to moderate and slightly resistant plants. Thus susceptibility is primarily measured by the reproduction of the nematode on the host and not on the basis of root galling alone.

The plant parasitic nematodes are obligate parasite need nutritional requirement and favourable environment from the host for its survival but any sort of inhibition or resistance response exhibited by the host to establish the feeding site results in poor parasitic development. After entering into the host by the second stage juveniles, the reproduction of the nematode on the host much depend on induction of giant cell. If the host and nematode are not suitable smaller giant cells are formed and a few juveniles (J2) enter into adult stage.

The formation of root galls by the Meloidogyne species can also be observed on the immune and the highly resistant plants. The presence of J2 inside the test plant may be found but further development into adult and reproduction (egg mass production) is inhibited. There could be several fate of juveniles (J2) entering into the resistant plants: i) they may develop to maturity as female but no production of egg mass or defective eggs, ii) develop in males, iii) further development may be arrested, iv)  be killed by an immune response (cell may die around the nematode head or release of toxic substances, and v) the juvenile may egress out from the root penetrated and freshly enter into a new site/host to induce gall(Taylor & Sasser,1978).

 Thus, resistance property of host may influence the nematode at any stage of nematode development. The occurrence of nematode resistance genes into the host plants makes the root less attractive for attacking nematodes. There may be possibility of interception of signal transduction in host recognition process of the nematode. Resistance and susceptibility to plant parasitic nematodes reflect the effect of the plant on the nematode.

 Steps for screening of jute germplasms:

Preparation of sterile soil:

Soil preferably sandy soils is mixed with river sand at 3:1 ratio (v/v) and sterilized for 30 minutes at 15 psi for two-three consecutive days. The steam sterilized soils is then exposed to open sunlight by spreading it over a polythene sheet for aeration. The soil can also be sterilized with formalin in a heap and subsequent covering with polythene sheet at least for 10-15 days.

Pot filling:

The usual size of 10 to 15cm diameter earthen pot (preferably new/fresh) is filled up with the sterile soil leaving the space on the top for watering the plants. The provision for proper drainage in the each pot below is essential to prevent water logging or stagnation of water.

Sowing of seeds:

Seeds of jute germplasms are sown in each pot along with a susceptible check (susceptible variety) for comparison. Usually after 15 days of seed emergence is thinned to one healthy seedling in each pot.

Preparation and maintenance of pure inoculum of Meloidogyne species:

A single egg mass of Meloidogyne species hand picked with the help of a forceps from an infected susceptible plant root and placed in a watch glass containing water. This is then transferred to a small coarse sieve lined with tissue paper to cover the bottom of the sieve that was placed over a Petri plate containing water. The Petri plate is then incubated at room temperature ((27±5) °C) for 2-4 days. Seedlings of susceptible plants, be tomato or brinjal are grown in sterile soil were inoculated with J2 and the process is repeated to get the pure progeny of the single egg mass for inoculating the experiment.

 Collection and preparation of nematode inoculum:

The pure nematode inoculum of identified species and race of Meloidogyne species is prepared by gently uprooting the infested plants from the culture pots, washed gently in tap water and egg masses are picked up with a forcep under the natural light or using lamp. Egg masses are usually kept in water before using them as inoculum. Generally second stage juveniles (J2) of Meloidogyne species are inoculated for resistance screening. For inoculation with juveniles, egg masses are placed over a double layered tissue paper fitted with wire gauge placed over a Petri plate containing water. Within 2-4 days, most of the J2 will hatch out and wriggle down to plain water in the Petri plate. The number of J2 per ml of water is counted and its exact density per ml suspension is estimated to determine the volume of nematode suspension to be inoculated. For inoculation with eggs, the infected roots are chopped into small pieces and treated with 1.0% NaOCl solution for 3-5 minutes to dissolve the egg masses and release the eggs from the gelatinous matrix. The eggs can be collected through passing 200 mesh (for removing debris) and 500 mesh (25um) sieves. The egg mass can also be directly used in the pot for the screening studies.

Inoculation with nematode:

Each seedling of at least 15 day-old or two leaf stage in the pot may be inoculated with eggs, juveniles or egg masses. Usually, one second stage juvenile per g of soil is inoculated around the seedling with a pipette. At least three holes of 3-5cm are made around base of each seedling and nematode suspension of known number is poured into the holes and plugged with soil immediately. Uninoculated pot for each entry serves as controls for that particular entry. Regular watering of the pots is essential and the plants become ready for taking observation after 40-45 days of inoculation.

Methods of assay:

For root knot nematode, root galling of root system is considered as main criterion for screening resistance in test plant. But galling/knotting alone is not sufficient to evaluate the resistance response against the nematode. The reproduction of nematode on the test plant is best measured either by egg mass index (EMI) or determining reproduction factor (RF). The determination of gall index is much easier and convenient than measuring of reproduction of nematode. Thus, it would be better to select resistant genotypes based on root-knot index for preliminary evaluations, and nematode reproduction could be measured for advanced evaluation.

The most commonly used methodology on a 1-5 scale for measuring resistance is given below: 1= No galls/egg masses(highly resistant), 2=1-10 galls/egg masses(resistant), 3=11-30 galls/egg masses(moderately resistant), 4=31-100 galls/egg masses(susceptible), 5= above 100 galls/egg masses(highly susceptible)[AICRP, Nematode]. Visual galling on root system:  0%= Highly resistant, 1-20%=resistant, 21-50%=moderately resistant,51-80%= susceptible and 81-100%=highly susceptible is most convenient for preliminary asssessment.

It should be noted that a variety/germplasm exhibiting susceptibility even in one replication may be considered susceptible (Gaur et al, 2001). However, the plants in one or more pots not showing root galls may be useful for identifying the sources of resistance. Therefore, care must be taken while discarding the plants free from galls in the screening process.

Experimentation either in net house or glass house:

The pots used for assaying the jute germplasms should be placed on the concrete floor to prevent any short of contamination. All the pots should be kept in randomized block design and each entry should have at least five replications. There should be proper sanitations in using equipments and hands of the workers.

 References for further reading:

 ABD-ELGAWAD, M.M. 1991. A new rating scale for screening plant genotypes against root knot and reniform nematodes. Anz.scadlingskde., Pflanzenschutz, Umweltschutz 64(37): 37-39.

BHATTI, D.S. AND JAIN, R.K.1994.  Crop cultivars resitant to nematodes. Pp. 217-227. In: Nematode pest management in crops. (D.S.Bhatti and R.K.Walia eds.). CBS Publishers & Distributors, New Delhi, India, 350pp.

BRIDGE, J. AND PAGE, S.L.J., 1980. Estimation of root-knot nematodes infestation levels on roots using a rating chart. Tropical Pest Management  26 (3), 296--298.

CANTO-SAENZ, M., 1983. The nature of resistance to Meloidogyne incognita (Kofoid & White, 1919) Chitwood 1949. Proc. Third Res. & Plan. Conf. March 22--26, 1982, ed. C.C. Carter. Int. Meloidogyne project, Lima, Peru, 233 pp.,160--165.

COOK, R. AND EVANS,K. 1987. Resistance and tolerance, pp.180-231. In: Principles and practices of nematode control in crops (R.H.Brown and B.R.Kerry, eds.) Acadmic Press, Australia. 467pp.

GAUR, H.S.; R.V. SINGH, KUMAR, S.; KUMAR, V. AND SINGH, J.V. 2001. Search for nematode resistance in crops. 84pp.

HADISOEGA-DA, W.W. AND SASSER, J.N., 1982. Resistance of tomato, bean, southern pea, and garden pea cultivars to root-knot nematodes based on host suitability. Pl. Dis. 66, 145--150.

KHAN, M.R.AND MUKHOPADHYAY, A.K. 2004. Screening of crop germplasms for resistance against root-knot nematode, Meloidogyne incognita race 2. Envir. & Ecol., 22 (Spl-3): 445-448.

SASSER, J.N.; CARTER, C.C. AND HA.RTMAN, M.M., 1984. Standardization of host suitability studies and reporting of resistance to root-knot nematodes. Coop. Pub. Dep. Plant Path., North Carolina State Univ. and U.S. Agency Int. Dev.Raleigh, N.C. 7pp.

TAYLOR, A.L.1971. FAO guide on plant parasitic nematodes. pp.14-15.

TAYLOR, A.L. AND SASSER, J.N., 1978. Biology, identification and control of root-knot nematodes (Meloidogyne spp.) Coop. Pub. Dep. Plant Pathol., North Carolina State Univ. and U.S. Agency Int. Dev. Raleigh, N.C. 111 pp.

VAN GUNDY, S. D.; THOMASON, I.J. AND RACKMNN, R. L., 1959. The reaction of three citrus species to three Meloidogyne species. PI. Dis. Reptr. 43: 970-971.

ZECK, W.M., 1971. A rating scheme for field evaluation of root-knot nematode infestation. Pflanzenschutz-Naehr. Bayer 24, 141--144.

 

About the Author

Dr. Khan is a Nematologist and Reader of Nematology in the Department of Agricultural Entomology, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalya(BCKV), Mohanpur, Nadia-741235, West Bengal, India. He has published more than 80 research papers in National and International Journals. He was the recepient of Youmg Scientist Award in 2005. His main interest on working with nematode biosystematics, nematode problems of flower, rice, jute, tea and fruit crops. Recently he along with his team eatblished a Plant Health Clinic laboratory at Directorate of Research, Kalyani, Nadia, West Bengal for extending Pest advisory services to the growers of West Bengal.