Management of Root Knot Nematode Meloidogyne incognita in Banana (Cv. Robusta) through Biocontrol Agents and Neem Cake

Isolation and formulation of Bioagents
 
Native soil sample were collected from rhizosphere of banana plants to isolate biocontrol agents, Pseudomonas fluorescens and Trichoderma viride by serial dilution plate technique. 0.1 ml of each 10-5 and 10-6 dilution was pipetted out into serial Petri plates containing King’s B medium (King et al., 1954) for P. fluorescens, Potato Dextrose Agar (PDA) and Malt extract agar (Gams and Bisset, 1998) for T. viride. The confirmation of P. fluorescens using various morphological and biochemical key’s given at Beargy’s manual of Bacteriology. The PDA and Malt extract agar containing plates were incubated at 25°C for 7 days. T. viride colonies were identified according to the identification key based on branching of conidiophores, shape of phialides, emergence of phialides and spore characters (Gams and Bisset, 1998).
 
The confirmative bioagents were formulated in purified talc powder (sterilized at 105°C for 12h) with calcium carbonate 15g and carboxyl methyl cellulose (CMC) 10g/kg. (Vidhyasekaran and Muthamilan, 1995). At the time of application, the population of P. fluorescens in talc formulation was 2×10⁸ cfu/g and T. viride 2×10⁸ cfu/g. These were tested separately and in combination with and without neem cake against root-knot nematode M. incognita infesting banana under shade net condition.
 
Pure culture of Meloidogyne incognita
 
Egg masses of root-knot nematode, M. incognita were collected from pure culture maintained on coleus plants. These egg masses were transferred into a beaker containing tap water. The top of the beaker was closed with a piece of polythene sheet with small holes for aeration. This was incubated at room temperature for 3-4 days. The water at the top was removed and then fresh water was added and aerated every day. After three days, freshly hatched juveniles (J2) of M. incognita were collected and used as a source of inoculum. To estimate the number of J2 used for treatments, J2 individuals obtained per ml were counted in triplicates and later averaged. Distilled water was used to adjust to 2000 J2 per treatment. The collection and preparation of J2 was described by Sundararaju and Kiruthika, (2009).
 
Experimental details          
     
 The pot culture experiment was conducted at Post Graduate and Research Department of Zoology, Government Arts College (Autonomous), Karur-639 005, TamilNadu, India, during 2016-2017. Uniform size banana suckers (cv.Robusta) were planted in pots (size: 30 cm width and 60 cm height) containing sterilized soil mixture (Red soil: sand: FYM) in the ratio of 2:1:1. After one month of planting freshly hatched J2 of M.incognita were inoculated at the rate of 2000 J₂/plant except uninoculated control. After 1 week of inoculation the bioagent and neem cake were applied in the treatments given below.
 
T1. Neem cake @ 100 gm/plant
T2. Pseudomonas fluorescens @ 10 gm/plant
T3. Trichoderma viride @ 10 gm/plant
T4. Pseudomonas fluorescens @ 10 gm/plant + Neem cake @ 100 gm/plant
T5. Trichoderma viride @ 10 gm/plant +Neem cake @ 100 gm/plant
T6. Pseudomonas fluorescens @ 10 gm/plant + Trichoderma viride @ 10 gm/plant + Neem cake @ 100 gm/plant
T7. Nematode alone
T8. Untreated Control (Check)
 
Each treatment was replicated thrice and arranged in randomized block design (RBD). All the treated plants were watered periodically. The experiment was terminated after 120 days after planting. At the time of termination, the plants were carefully uprooted with intact root system. The soil and root samples were collected for estimating final nematode population. Observations were recorded on plant height (cm), number of leaves, Pseudostem girth (cm), root length (cm), root weight (g), number of infected roots, number of healthy roots and root-knot index. The root galling was assessed by examining the roots on the outside by visual observation of the whole root system based on the percentage of galled roots. The percentage was converted to a scale of 0-5 as suggested by Sundararaju and Kiruthika, (2009). After this, roots were cut into small pieces, mixed thoroughly and samples of 5gm each were stained in boiling acid fuschin-lacto phenol. These were blended and nematode populations were assessed. Soil samples (250cc) were also collected from each pot for estimating the nematode population. Nematode population from soil was estimated by using Cobb’s sieving and decanting method (Cobb’s 1918) followed by modified Baermann’s funnel method (Christie and Perry, 1951).
 
The data were subjected to statistical scrutiny by analysis of variance, standard error of deviation and co-efficient of variance using AgRes statistical software (1994, Pascal International Software Solutions). Critical difference at P=0.05% were calculated by the statistical method developed by Panse and Sukhatme (1978).

Significant increases in plant growth and root indices were observed in all the treatments over nematode alone inoculated treatment and untreated control. The combined application of neem cake along with bio-agents P.fluorescens, T.viride against M. incognita resulted that significant increase in plant height (38.8 cm), number of leaves (7) and pseudostem girth (15.0 cm), root length (25.6 cm), root weight (37.8 gm), total number of roots (20.3) number of healthy roots (17.0) number of infected roots (3.3), minimum root gall index (1) and nematode population of 55.0 from soils and 90.0 from roots was recorded over nematode alone treated control (Table 1 and 2). The present results were confirmative with earlier workers Pandey et al. (2005) who reported an eco-friendly management of root-knot nematode and Fusarium wilt disease infecting chickpea by integrating fungal bio-agents and oil cakes. The application of organic amendments and use of biocontrol agents on plant parasitic nematodes were gaining much importance (Kalaiarasan et al. 2006). Konsam et al., (2013) have reported that application of jatropha cake, neem cake and castor cake could enhance growth and yield of cucumber by reducing nematode population.
 

 

 
The significant   increase in plant height (35.0 cm), number of leaves (6) and pseudostem girth (13.6 cm), root length (23.0), root weight (36.2 gm), total number of roots (18.0) number of healthy roots (14.0) and number of infected roots (4.0), medium root gall index (3) and nematode population of 175.3 from soils and 302.0 from roots were recorded from plants treated with neem cake and P. fluorescens over nematode alone treated control (Table 1 and 2). The plant growth promoting rhizobacteria such as P.fluorescens would effective against root-knot nematode, Meloidogyne incognita in banana (Jonathan et al.  2006). Nama and Sharma (2017) further reported that P. fluorescens could increase the plant growth by reducing nematode population. Kar et al., (2018) also reported that the application of P.fluorescens @ 20 g m² + neem cake @ 100 g m² increased the cowpea production by reducing nematode population.

Increase in plant height (34.3 cm), number of leaves (5) and pseudostem girth (13.0 cm), root length (22.1 cm), root weight (34.8 gm), total number of roots (17.3), number of healthy roots (13.0), number of infected roots (4.3), minimum root gall index (2) and nematode population of 230.0 from soils and 419.0 were recorded from plants treated with neem cake and bio-agent T.viride over nematode alone treated control. The rest of other treatments were superior over nematode alone and untreated control treatment plots (Table 1 and 2). The combined application of T. viride and neem cake reduced the multiplication rate of nematodes (Farooq Azam et al., 2007). The neem cake and T.viride applications supported the growth in addition to their adverse effects on M. incognita which ultimately led to improved plant growth characteristics (Sunil Kumar and Anju Khanna, 2006). The above results were confirmed by the workers of Shanthi and Rajendran, (2006) who reported that the application of P. fluorescens at 20 g/plant reduced the populations of Radopholus similis, Pratylenchus coffeae and Helicotylenchus multicinctus by 48.7, 46.3 and 44.3%, respectively in banana. P. fluorescens at 10⁹. CFU/ml decreased Meloidogyne javanica infections, compared to the control. Also, it was able to cause destruction of nematode egg mass matrix and significantly decreased nematode egg hatching level (Norabadi et al., 2014). Combined applications of bioagents were more effective in reducing the disease incidence than the individual treatments. T. harzianum + P. fluorescens + Bacillus subtilis minimized the nematode population and increased plant growth parameters in Ginger (Dohroo and Meenu Gupta, 2014).

From the results of the present study it is inferred that though the individual application of P. fluorescens and neem cake is effective in the control of root-knot nematode M. incognita, the combined application of P. fluorescens @10 g/plant or T. viride @10g/plant with neem cake @ 100 g/plant is more effective in controlling the root-knot nematode but also increasing the growth of the plant.

We gratefully acknowledge to the Principal, Government Arts College (Autonomous), Karur-639 005, Tamil Nadu and Professor and Head, PG and Research Department of Zoology, Government Arts College (Autonomous), Karur-639 005, Tamil Nadu for supporting us to carry out this work in various ways.