Meloidogyne graminicola infestation in selected Sri Lankan rice varieties, Oryza sativa L. and nemato-toxic effect of Trichoderma viride to reduce infectivity

Meloidogyne graminicola is a nematode parasite of rice, Oryza sativa L., in some rice growing areas in Sri Lanka. M. graminicola infestation levels in eight rice varieties namely Bg 407, Bg 366, Bg 403, Bg 251, Bg 369, Bg 380, Bg 310, and Bg 745 and nematotoxic effect of Trichoderma viride against the nematode infectivity in a susceptible rice variety were evaluated in this study. Results revealed that varieties Bg 366 and Bg 251 were susceptible; varieties Bg 407, Bg 369, Bg 380, Bg 310, and Bg 745 were moderately susceptible while Bg 403 was moderately resistant to M. graminicola. There was a significant reduction of root galls (p = 0.000) and weight of the fresh roots (p = 0.001) of T. viride treated Bg 366 susceptible rice variety compared to the untreated tillers of the same rice variety. This study concludes that none of the rice varieties tested were totally resistant to M. graminicola infestation, hence, T. viride treatment can be integrated into nematode management practices to reduce the nematode population in susceptible rice varieties.


INTRODUCTION
The root-knot nematode, Meloidogyne graminicola (Golden and Birchfield), is a major threat to Sri Lankan rice cultivation (Ravindran, et al., 2017). It was first reported to infest rice (Oryza sativa L.) fields in Sri Lanka in early 1990s and it has now dispersed in to major rice growing areas of the country (Ekanayake et al., 2001, Nugaliyedda et al., 2001. The infestation level varies from mild through moderate to severe (Nugaliyedde et al., 2001).
The infestation of M. graminicola was severe in rice fields in some parts of the dry zone of the country. Dispersion of this nematode occurs mainly through irrigated water and soil.
Mature female nematodes produce a large number of eggs at once leading to a sudden increase of the pathogen population causing severe outbreaks in a very short period of time.
It has been found that there is a decline of rice yield when more than 75% of the roots of an infested plant is affected by nematodes (Nugaliyedde et al., 2001). Experiments have proven that infestation level varies based on the rice variety (Amarasinghe, 2011;Amarasinghe et al., 2007).
Adult female and juvenile nematodes cause the damage by altering physical changes in the host plant. The juveniles continuously feed on cells close to the vascular system of the root enlargement, hence, vascular disruption is occurred. If the meristematic cells near the root tip are damaged by the juveniles, the root elongation will be interrupted (Norton & Niblack, 1991). Gradually, nutrient and water absorption of the root system is blocked and upward translocation is interrupted. This situation indirectly affects photosynthesis that leads to the condition known as 'chlorosis' (Bridge et al., 1994). M. graminicola affected rice plants show stunting due to the characteristic terminal swellings/galls on the root tips which ultimately result in severe reduction in growth and yield (Jaina et al., 2011). Wilting of seedling occurs, when the population density of M. graminicola is high (Ou, 1985).
Elimination of the favorable environmental conditions that is required for completing the life cycle of M. graminicola is an efficient way to control the nematode. Use of resistant rice varieties can also be implemented to keep the nematode population under control. Bg 352, Bg 300 and Bg 357 are some of the existing rice varieties identified as resistance to the nematode infestation in Sri Lanka (Amarasinghe et al., 2011). Several indices have been developed to identify the level of resistance of different crop varieties. Some indices were based on the ratio between final nematode population and the initial nematode population in an infested rice plant and some were based on the galling index, the number of galls produced in an infested rice plant by the nematode (Bridge et al., 1994). Apart from varying susceptibility levels, several authors have described the successful use of biological control agents against M. graminicola. Fungal species such as Paecilomyces lilacinus, Trichoderma harzianum and other Trichoderma sp. (Huong et al. 2009) the bacterium, Bacillus subtilis (Narasimhamurthy et al., 2017)

Rising up the inoculum of Meloidogyne graminicola
According to the approval and guidance of the officers at the Regional Office of the Department of Agriculture at Dehiaththakandiya, Sri Lanka, a minimum number of infested rice plants (n = 6) were collected from infested rice fields. The infested plants were kept inside polythene bags (30 cm×15 cm) to avoid the exposure of the plants to the outside environment and brought to the laboratory. Dry soil from apparently uninfested, tilled paddy fields in Kurunegala district was collected into transparent polythene bags. Soil bags were exposed to direct sunlight for five hours continuously each day for two weeks to defaunation.
De-faunated soil was filled into plastic basins (25 dia. × 22hei. cm) with adequate water and germinated seed paddy were sown in these containers. Infested rice plants were transplanted in them. The plants were kept under in-situ conditions. NPK fertilizer was added according to the recommended rate where necessary and watered regularly. Prior to the experiments, required amount of J2 juveniles were collected from root-galls. The roots were washed and root galls were separated into a watch glass. The galls were crushed using a glass rod to expose the nematodes. The suspension of right stage of the nematode (J2 juveniles) was prepared in a 100 mL beaker.

Preparation of the inoculum of Trichoderma viride
Ten number of Potato Dextrose Agar (PDA) plates (9 cm × 1.5 cm) were prepared.
A loop of fungal hyphae from the laboratory maintained Trichoderma viride cultures originally prepared by (Amarasinghe & Madurusinghe 2012) were placed on the agar plates under sterilized conditions. The pure cultures were identified as T. viride as described by (Amarasinghe & Madurusinghe 2012) and maintained in the incubator at 10 ℃ until they were used for the experiment.

Levels of susceptibility of rice varieties to Meloidogyne graminicola
About 50 seeds each of rice varieties of Bg 407, Bg 366, Bg 403, Bg 251, Bg 369, Bg 380, Bg 310, Bg 745 were soaked in glass beakers (100 mL) each containing 50 mL of water for 24 hours. They were transferred to a moistened filter paper and were covered with a transparent polythene sheet until seed germination. Square shaped plastic pots (7.5 L × 7.5 W × 11.5 H cm) (n = 96) were filled with 250 g of de-faunated soil and arranged in 12 rows.
Twelve seedlings each from each rice variety (V1-V8) were planted in randomly selected pots. Six of the randomly selected plants of each variety were assigned as control pots (C1-C6) and the other six were assigned for nematode inoculum (T1-T6). After two weeks, 1 mL suspension containing five J2 juveniles was injected to the rhizosphere of each test plant using a pasture pipette. The plants that were maintained as controls (C1-C6) were treated with distilled water. Experiment was arranged in fully randomized design. This experiment was continued for two month. All plants were watered daily. The water level was maintained about 2 mm above the soil layer in the pots for three weeks. Thereafter, adequate amount of water was added only to retain the moisture of the soil throughout the remaining period of the experiment. A known amount of NPK fertilizer was added once in two weeks. The plant growth was monitored daily. After eight weeks, number of yellow leaves, root galls per plant and wet weight of the root system were recorded by destructive sampling. The gall index (percent gall bearing roots/total roots) for each variety was calculated. The final nematode population (Pf) in each plant was counted. Reproduction factor (Pf/Pi) was calculated (Pi = initial population).

Nemato-toxic effect of Trichoderma viride on Meloidogyne graminicola
A 100 mL of water containing pre-determined concentration of the Trichoderma viride, 1×10 4 spores/mL (Amarasinghe & Madurusinghe 2012) was prepared from a stock suspension (4.05×10 5 spores/mL). Bg 366 variety rice plants were raised individually in sixty plastic pots (7.5 L × 7.5 W × 11.5 H cm) filled with un-infested soil (250 g). The tillers were allowed to grow for two weeks. Following three treatments (T1 to T3) were applied randomly to 20 pots each as follows; T1 -distilled water (10 mL) spread around the rice tillers, T2 -10 mL of 1 ×10 4 spores/mL Trichoderma viride applied to the soil around the tiller and T3-10 mL of 1×10 4 spores/mL T.viride injected at the root level. Each tiller was inoculated with J2 juveniles at the rate of five individuals per rice tiller as described above.
The plants were allowed to grow for eight weeks in a screen house. During the experimental period, the plants were monitored daily. NPK fertilizers were added once in two weeks. Posttreatment assessment was done after uprooting the plants. The number of yellow leaves was recorded. Root system was carefully washed and the number of root galls per plant and wet weight of the roots were recorded.

Statistical analysis
Two sample t-test was carried out to determine whether there were significant differences among control and test plants. One-way ANOVA was performed to ascertain the effects among the different treatments. Tukey's test was carried out to compare the means (Minitab Version 14)

Symptoms of M. graminicola infestation on rice varieties:
Chlorosis: The percentage of yellow leaves increased in nematode infested rice tillers compared to uninfested control rice plants in all the varieties (Table 1) (Table 2).

Susceptibility level of rice varieties:
The levels of susceptibility in tested rice varieties were determined by using the index prepared by Ravindra et al., (2015) according to the number of galls per root system i.e. zero galls -Immune; 1-2 gallsresistant; 3-10 gallsmoderately resistant ; 11-30 gallsmoderately susceptible; 31-100 gallssusceptible; >100 gallshighly susceptible. respectively) ( Table 2). The highest reproduction factor was also observed in Bg 366 rice variety. The lowest reproduction factor was observed in Bg 403 rice variety that had the lowest number of galls.

Nemato-toxic effect of Trichodermaviride on Meloidogyne graminicola
There was a reduction in mean percentage of yellow leaves in T2 and T3 compared to T1 untreated control (Table 3). The percentage of yellow leaves in treatment 02 was lower than that of treatment 03. However, they were not significantly different (p= 0.731, F=0.32, DF = 2). The mean weight of the fresh root of tillers in treatment 1 was significantly higher than that of treatment 2 and treatment 3 (p = 0.001,F = 12.89, DF = 2). The mean fresh root weight of the T2 tillers dipped in T. viride (1×10 4 spores/mL) suspension was lower than that of T3, but it was not significant. The number of root galls were significantly higher in untreated plants (p = 0.000, F =68.92 DF = 2) compared to treated plants. However, there was no significant difference between T2 and T3. Though they were not significant, the number of galls was lower in treatment 2 than that of treatment 3. shown by Endo (1975) this study reveals that the intensity of root galling is directly proportional to the increment of root weight. The number of galls per root system increased with the increment of M. graminicola population and the number of feeder roots. Kumar et al., (2012) reported that T.viride reduces gall formation and production of egg masses of Meloidogyne incognita, which infects okra. In the present study, the application of T. viride against M. graminicola, leads to a similar outcome. The present study also revealed that T. viride activity is not specific either to the crop or Meloidogyne spp. Jegathambigai, et al., (2011) stated that Trichoderma spp parasitize and prevent hatching of the eggs and the growth of the second stage juveniles of M. incognita . According to this author, fungal hyphae penetrate the parasite eggs and larval cuticle by dissolving the chitin layer through enzymatic activity. They proliferate within the organism and produce toxic metabolites leading to the reduction of the nematode population, which explains the reduced number of galls.
According to the results obtained, susceptible and moderately susceptible varieties can be recommended for the integration of T.viride to reduce nematode infection. Goswami