Integrated Management of Meloidogyne incognita Infecting Vigna radiata L. using Biocontrol Agent Purpureocillium lilacinum

MATERIALS AND METHODS

Host plant and pathogen: Mung bean (Vigna radiata L.) cv. ‘PDM-139’ (Family-Fabaceae) was selected as host crop. The root-knot nematode, Meloidogyne incognita was selected as test pathogen.

Preparation of fungal biocontrol agents: The pure culture of the fungal biocontrol agent, Purpureocillium lilacinum (ITCC No. 6064) was purchased from the Indian Agriculture Research Institute (IARI), New Delhi, India. Fungal culture was maintained on Potato Dextrose Agar (PDA) medium¹⁰.

Nematode management by individual, sequential and concomitant application of Purpureocillium lilacinum with Meloidogyne incognita on mung bean: The glasshouse experiments were conducted for evaluating the nematicidal efficacy of biocontrol agent, P. lilacinum against the root-knot development caused by the root-knot nematode, M. incognita and their potential in enhancing the plant growth parameters of Mung bean cv. ‘PDM-139’. Clay pots (15 cm in diameter) filled with 1 kg autoclaved soil mixed with farmyard manure in the ratio of 4:1 were treated with biocontrol agent, P. lilacinum at 1.5 g /pot. The experiment was designed as follows:

Observations
Chlorophyll estimation: The chlorophyll content in the fresh leaves was estimated following the method of Mackinney¹¹. One gram of finely cut fresh leaves was grounded to a fine pulp using a mortar and pestle after pouring 20 mL of 80% acetone. The mixture was centrifuged at 5000 rpm for 5 min. The supernatant was collected in 100 mL volumetric flask. The residue was washed three times, using 80% acetone. Each washing was collected in the same volumetric flask and the volume was made up to the mark using 80% acetone. The absorbance was read at the wavelength of 645 and 663 nm against blank (80% acetone) on spectrophotometer (UV 1700, Shimadzu, Japan). The chlorophyll content present in the extract (mg g^–1 tissue) was calculated by using the following equation:

Nitrate reductase activity (NRA): The nitrate reductase activity in fresh leaves was estimated by the following method of Jaworski¹². The leaves were cut into small pieces (1-2 cm). About 200 mg of these chopped leaves were weighed and transferred to plastic vials. To each vial, 2.5 mL of phosphate buffer pH 7.5 and 0.5 mL of potassium nitrate solution was added followed by the addition of 2.5 mL of 5% of isopropanol. These vials were incubated in BOD incubator for 2 h at 30±2°C in dark. Incubated mixture of 0.4 mL was taken in a test tube to which 0.3 mL each of sulphanilamide solution and NED-HCl were added. The test tubes were left for 20 min for maximum colour development. The mixture was diluted to 5 mL Double Distilled Water (DDW). The absorbance (O.D.) was read at 540 nm using spectrophotometer. A blank was run simultaneously with each sample. Standard curve was plotted by using known graded concentration of NaNO₂ (Sodium nitrite) solution. The absorbance (O.D) of each sample was compared with that of calibration curve and nitrate reductase activity expressed at nm (μm h^–1 g^–1).

Pathological parameters
Eggmasses per plant: The eggmasses were counted by following the procedure of Daykin and Hussey¹³. The roots were diped in Phloxine B solution (0.015%) for 20 min and were then washed with running tap water to remove the residual Phloxine B. The eggmasses stained a pink red colour where as the roots remain colorless or stain lightly.

Root-knot index: The degree of root-knot nematode infection was recorded according to rating degree given by Taylor and Sasser¹⁴ as shown in Table 1.

Table 1:	Degree of root-Knot nematode infection

Statistical analysis: The data of the experiments were analyzed statistically by using the Statistical Package for the Social Sciences SPSS 12.00 Software (SPSS Inc., Chicago, IL, USA) for analysis of variances (ANOVA). All the values were presented as the mean which were compared according to Least Significant Differences/Critical Differences (C.D) at p = 0.05 and p = 0.01 level. Duncan's Multiple Range Test was employed to test for significant difference between the treatments.

RESULTS

The present experiment was conducted under glasshouse conditions to evaluate the effect of individual, concomitant and sequential inoculation of Purpureocillium lilacinum with Meloidogyne incognita against the root-knot development and on growth and physiological parameters of mung bean cv. ‘PDM-139’. The data presented in Table 2 and 3 revealed that inoculation of mung bean plant with root-knot nematode, M. incognita caused significant reduction in growth and physiological parameters and enhancement in pathological parameters as compared to untreated uninoculated control.

There was significant enhancement in plant growth parameters viz., plant length (cm), fresh weight (g) and dry weight (g) were found when P. lilacinum applied alone. Concomitant and sequential inoculation of P. lilacinum with M. incognita also improve the plant growth parameters. Highest increase in growth parameters of mung bean was observed when P. lilacinum was applied 15 days prior the inoculation of M. incognita followed by simultaneously inoculation of P. lilacinum and M. incognita and sequential inoculation of P. lilacinum 15 days after the inoculation of M. incognita as compare to untreated inoculated control (Table 2).

The highest number of nodules, flowers and pods were found when P. lilacinum applied alone. Significant increase in number of nodules, flowers and pods were also observed in sequential inoculation of P. lilacinum 15 days prior the M. incognita inoculation followed by simultaneously inoculation of M. incognita with P. lilacinum and sequential inoculation of P. lilacinum 15 days after the inoculation of M. incognita as compare to untreated inoculated control (Table 3).

When the biocontrol agent P. lilacinum applied 15 days prior to M. incognita inoculation was found highly effective in enhancing the growth parameters viz., plant length, fresh and dry weight of plant, number of flowers, pods and nodules and least improvement was observed in those pots treated with P. lilacinum applied after 15 day to M. incognita inoculation as shown in Table 2 and 3.

Table 2:	Effect of individual, sequential and concomitant inoculation of Meloidogyne incognita and Purpureocillium lilacinum on the plant growth of mung bean cv. ‘PDM-139’ in pots
Values are mean of five replicates, Means in each column followed by the same letter are not significantly different according to duncan multiple range test (p<0.05), Mi→Pl15: Meloidogyne incognita inoculated 15 days before treatment, Pl→Mi15: Purpureocillium lilacinum treatment given 15 days prior nematode inoculation, UIC: Untreated inoculated control, UUC: Untreated uninoculated control

Table 3:	Effect of individual, sequential and concomitant inoculation of Meloidogyne incognita and Purpureocillium lilacinum on pathologicaland physiological parameters of mung bean cv. ‘PDM-139’ in pots
Values are mean of four replicates, Means in each column followed by the same letter are not significantly different according to duncan multiple range test (p<0.05), Mi→Pl15: Meloidogyne incognita inoculated 15 days before treatment, Pl→Mi15: Purpureocillium lilacinum treatment given 15 days prior nematode inoculation, NRA: Nitrate reductase activity, UIC: Untreated inoculated control, UUC: Untreated uninoculated control

Highest increase in biochemical parameters viz., chlorophyll content (mg g^–1) and nitrate reductase activity (μm h^–1g^–1) was found when P. lilacinum applied alone. Significant improvement in chlorophyll content and nitrate reductase activity were also observed in sequential inoculation of P. lilacinum 15 days prior M. incognita inoculation followed by simultaneously inoculation of M. incognita and P. lilacinum and sequential inoculation of P. lilacinum 15 days after the inoculation of M. incognita as compare to untreated inoculated control (Table 3).

The data presented in Table 2 revealed that highest multiplication of root-knot nematode M. incognita was found in case of M. incognita alone inoculated pots. The highest reduction in number of eggmasses was observed in sequential application of P. lilacinum 15 days prior M. incognita inoculation followed by simultaneously inoculation of M. incognita with P. lilacinum and sequential application of P. lilacinum 15 days after M. incognita inoculation as compare to untreated inoculated control (Table 3). The least root-knot index was found when pots were treated with the application of P. lilacinum 15 days prior M. incognita inoculation followed by simultaneously inoculation of M. incognita with P. lilacinum and sequential application of P. lilacinum 15 days after M. incognita inoculation as compare to untreated inoculated control (Table 3).

The data present in Table 3 predicted that highest enhancement in physiological parameters viz., chlorophyll content and nitrate reductase activity and highest reduction in pathological parameters like number of egg mass per plant and root knot index was found in those pots when the biocontrol agent P. lilacinum applied 15 days prior to M. incognita inoculation and least was observed in those pots treated with P. lilacinum applied after 15 day to M. incognita inoculation.

From the above results, it can be say that biocontrol agent P. lilacinum reduced the root knot infestation cause by root knot nematode, M. incognita on mung bean in eco-friendly manner.

DISCUSSION

These results indicated that P. lilacinum has significant potential as biocontrol agent against the root-knot nematode, M. incognita under glasshouse conditions. Our results are in conformity with several scientists^15-17. Inoculation of chickpea with P. lilacinum can significantly reduce the population of root-knot nematode and disease severity caused by root-knot nematode. It may be found that biocontrol agent significantly brought down the population of root-knot nematode and some compounds released from bio-agent reduced the nematode infestation. Biocontrol agent, P. lilacinus contained protease and chitinase, play an important role in the degradation of the egg shell³. Ganaie and Khan¹⁸ reported significant reduction in number of galls, eggmasses, eggs/eggmass and final nematode population of M. javanica on sequential inoculation of P. lilacinus 10 days prior to M. javanica treatment. Esfahani and Pour¹⁹ observed that P. lilacinus, a biocontrol agent was most effective when the fungus and root-knot nematode, M. javanica were inoculated simultaneously or the fungus proceeded the nematode in sequential inoculation on tomato. Paecilomyces lilacinus by direct hyphal penetration colonizes the root surface and parasitizes eggs, eggmasses, second stage juveniles and females of Meloidogyne spp.²⁰. Paecilomyces lilacinus reported as a good bicontrol agent of root-knot nematodes and other plant-parasitic nematodes^21,22. Therefore, it was concluded that the severe infection caused by root-knot nematode could be lowered by the use of P. lilacinum as bioagent in view of eco-friendly environment. This has an advantage against expensive and hazardous chemical nematicides.

CONCLUSION

From the above study it may be concluded that biocontrol agent, P. lilacinum showed nematicidal potential against root-knot nematode, M. incognita and showed improvement in growth and physiological parameters of mung bean cv. ‘PDM-139’ under glasshouse conditions. It may be due to the presence of various secondary metabolites released from biocontrol agent which showed nematicidal effect against root-knot nematode. This method of root-knot nematode control may contribute to minimize toxic effect of chemical nematicides on flora and fauna. Hence, the outcome of the results predict that the use of biocontrol agent P. lilacinum may provide safe and environmentally reliable approach for the root-knot nematode management programme for sustainable environment.

SIGNIFICANCE STATEMENT

The root-knot nematode, Meloidogyne incognita is a sedentary endoparasite attacking a wide range of crops including pulses, vegetables other ornamental crops worldwide and caused dramatic yield loss. So, that management strategy is urgently needed, chemical nematicides is best way for the management of root knot nematodes, but on the other hand, these chemical nematicides were harmful to flora and fauna and very costly so, farmers does not applied these chemical nematicides in our field. So, biocontrol agent, Purpureocillium lilacinum is better option for the management of M. incognita because this method of nematode management is eco-friendly and does not leave any toxic impact on environment.