Effect of some Bacterial Bioagents against Root- Knot Nematode (Meloidogyne incognita race2)

The effect of culture filtrate of different bacterial isolates on egg hatching and juvenile mortality of root-knot nematodes (Meloidogyne incognita race 2) was studied in vitro and the effective strains were selected. The per cent mortality and hatching inhibition was proportional to the concentration of culture filtrate and the duration of exposure period. Culture filtrate of all the isolates of bacteria significantly induced mortality and inhibition of egg hatching of M. incognita juveniles. The highest percentage of inhibition of egg hatching was recorded for Bacillus thuringiensis followed by Bacillus sp. and Pseudomonas fluorescens whereas the highest percentage of mortality of juvenile was recorded for Bacillus thuringiensis followed by Pseudomonas fluorescens and Pseudomonas sp.


INTRODUCTION
Root-knot nematode Meloidogyne incognita race 2 is one of the major constraints in crop production in Assam. Present strategies for nematode management largely depend on cultural pract ices such as crop rotations, use of resistant varieties and use of nematicides which has several disadvantages. Utilization of biocontrol agent is of advantage over the above mentioned nematode management strategies. Many species of Pseudomonas and Bacillus have been reported as plant growth pro moting rhizobacteria (PGPR) wh ich produces iron-chelating siderophores, antibiotics or hydrogen cyanide, and these compounds have been implicated in the reduction of deleterious and pathogenic rhizosphere microorganisms, creating an environment more favourable for root growth [1] .It has been demonstrated that bio-agents produce different metabolites and antibiotics which directly or indirectly stimulate plant growth [2] . A number of bacterial species has been used as biocontrol agents against Meloidogyne spp. [3][4][5][6][7][8][9][10][11][12]. However, few biocontrol products are currently commercially available and it is necessary to find different strains of antagonistic bacteria for controlling plantparasitic nematodes more efficiently. Therefore, a laboratory bioassay was carried out to test some of the isolated strains of Bacillus and Pseudomonas for their nematicidal activity.

II. MATERIAL AND METHODS Isolation of bacteria
A survey was conducted during Feb 2018 to May 2018 in different localit ies of the district Jorhat, Assam co mprising an area appro ximately 500 ha, in order to identify the nematodes infection and to isolate the bacterial antagonists fro m crops rhizosphere viz. cucurbits, tomato, brin jal, okra, cabbage, banana, citrus, and tea. A total of 50 soil (500 g) sample were collected fro m the rhizosphere of each crop. Serial d ilution technique was used for the isolation of bacterial strain. One g of rhizosphere soil was dispensed in 9 ml sterile water, fro m the 10 -6 dilution, 50 μl were inoculated over Petri plates containing Nutrient Agar and King's B agar media (autoclaved at 121°C for 15 min) separately. The plates were incubated at room temperature 28±2°C for 48-72h.The colonies obtained on plates were picked and streaked on more plates and purified by restreaking. The isolates were initially categorized into two broad groups based on Gram staining by Hucker's modified method [13]. The isolated strains were identified by up to generic level by Bergey's manual and using standard protocol [14,15].Two isolates namely Pseudomonas sp. and Bacillus sp. were co llected fro m soil rh izosphere of cowpea and banana respectively. Pseudomonas fluorescens, Bacillus thuringiensis, B.brevis were procured fro m the Depart ment of Plant Pathology, AAU, Jorhat, Assam. All the strains were evaluated for nematicidal properties against nematodes.

Nematode inoculum and mass culturing
The inoculu m o f root-knot nematode M. incognita race 2 was collected fro m naturally infested tomato crop in field and single egg mass was used to raise pure culture. Mass culturing of nematodes was done on tomato variety Sel 7, in order to get regular supply of the inoculums for the experiments. One month old tomato seedlings were inoculated with small volu me of egg suspension approximately consisting of 2000 eggs of M.incognita race 2. These pots were watered and kept in glasshouse at temperature 28-35°C. Preparation of culture filtrate and test for nematoxicity of bacterial culture filtrate The bacterial isolates were inoculated in the respective med iu m (40 ml). Pseudomonas on King's B broth and Bacillus on Nutrient broth were separately incubated at 30°C on a shaker for 48 hrs. The liquid culture was filtered through Whatman No.1 filter paper and passed through bacterial filter [16] .Filtrates were centrifuged at 6000 rp m for 15 min. The supernatant was taken and the suspended residue was discarded. Juvenile mortality test One ml of S/ 2, S/8, S/32 dilutions of each cell-free culture filtrates was mixed with 1 ml nematode suspension (containing ca. 100 M.incognita J2) in cav ity block to obtain S/4, S/16 and S/64 dilutions and tested for per cent mortality of nematodes. There were 3 replications for each treatment. Observations were taken after 24 h, 48 h and 72 h. The immob ile J2 were confirmed for mortality by randomly disturbing with a needle. Sterile water and med iu m alone served as control. The per cent juvenile mortality was calculated by the following formula.

Percent mortality =
Total number of dead juveniles Total number of juveniles 100

Egg hatching test
To determine the effect of culture filtrate on the hatching of eggs of J2 of M. incognita eggmasses of M.incognita were dissolved in NaOCl to release individual eggs [17]. Sterilized Petri d ishes of 5 cm dia were separately pipette 1 ml of culture filtrate with dilution S/2, S/8 and S/ 32. Hundred eggs of M.incognita in 1 ml water suspension were transferred to each dish. Mediu m alone and water alone served as control. All Petri dishes were kept at 28±2°C, rep licated thrice. Observations were recorded on after 5, 10 and 15 days. The per cent egg hatch was calculated by the following formula.

Statistical analysis
Per cent egg hatch and per cent mortality data was subjected to statistical analysis using the three factorial co mp letely randomized design statistical package. The critical differences in main effects i.e. isolates, concentration, and time of exposure as well as in their interactions were tested at P=0.05.

III. RESULTS
Two nu mbers of isolate of bacteria were isolated fro m the root rhizosphere of M.incognita race 2 infected plants. Bacillus sp. and Pseudomonas sp. were isolated fro m rhizospheric soil of banana and brinjal, respectively. The results presented revealed significant differences in juvenile mortality of M.incognita among isolates (biocontrol agent) (T), concentration of culture filtrate(C) and exposure period (t). The data showed in the Table 1 revealed that all the culture filtrates of bacterial isolates were having nematicidal effect of varying degree on M.incognita race 2. Per cent mortality of nematodes was directly proportional to the concentration of culture filtrate and the period of exposure. Irrespective of concentration of culture filtrate(C) and duration of exposure (t), six isolates namely Bacillus sp., B.brevis B.thuringiensis, Pseudomonas sp., P.florescence and P.aeruginosa were exhibited nematicidal effects on M. incognita J2. Irrespective of concentration of culture filtrate and exposure period the mortality rates of M. incognita J2 caused by B.thuringiensis reached 75.77% at which is at par with P.florescence (75.44%).All the new isolate namely Bacillus sp, and Pseudomonas sp displayed more than 40% juvenile mortality during 24 h exposure time at S/16 concentration of culture filtrate. On the other hand, P.aeruginosa showed the lowest toxicity, caused only 48% juvenile mortality S/64 at concentration of cu lture filtrate during 48 h exposure time. Irrespective of isolate and period of exposure the mo rtality rate of M. incognita J2, at lowest concentration (S/64) is 43.62% whereas at highest concentration (S/4) mortality rate is 51.66%.Similarly irrespective of isolate and culture filt rate concentration the mortality rate of M. incognita J2 is highest (63.07%) at 72 h exposure period and lowest (34.85%) at 24h exposure period. B.thuringiensis and P.florescence showed highest toxicity (100% J2 mo rtality) at S/16 concentration of culture filtrate during 72 h exposure period. There was least effect of media on juvenile mortality of M. incognita. The results presented in Table 2, revealed significant differences among isolates (T), concentration of culture filtrate(C) and exposure period (t) in egg hatching of M. incognita. Irrespective of concentration of culture filtrate (C) and time of exposure period (t), the culture filtrate of

IV.
DISCUSSION Culture filt rates of several soil borne microbes are known to exhibit nematicidal action besides inhibiting larval emergence of p lant parasitic nematodes. Antibiotics, extracellular en zy mes and other to xic co mpounds present in metabolites of rhizobateria are probably involved in the increase of J2 mortality and the inhib ition of egg hatching [18]. The lethal effect of the cultural filt rates of these bacteria may be attributed to the production of nematicidal metabolites i.e. lyt ic enzy mes (gelat inase, protease and chitinase) and volatile co mpounds in the cultural med ia. Similar results were reported by Ali et al.
[19] who found that cultural filtrates of Pseudomonas sp. caused juvenile mortality of M. javanica. The nematicidal volatile products that were produced by Bacillus and were characterized to include mainly the benzene acetaldehyde, 2-nonanone, decanal, 2-undecanone and dimethyl disulphide, wh ich were active against M. incognita juveniles and eggs at the concentration of 0.5 mmo l [20]. Ahl et al., [21] proposed the mechanis m of reduction of nematode population which was due to premature egg hatching and reduction in viability and mortality of juveniles induced by secondary metabolites such as 2,4 Diacetylphloroglucinol and lytic enzy mes [22], antibiot ics and hydrogen cyanide produced by Pseudomonas spp. and non cellular ext ract and to xic metabolites like bacillopeptidase, subtillin E and a lactamase fro m Bacillus spp. Regina et al. [23] and Hanna et al. [24] have reported that mortality of M. incognita increased with increase in exposure time as well as the concentration. The effects of rhizobacterial to xins include the suppression of nematode reproduction, egg hatching and juvenile survival, as well as direct killing of nematodes by causing paralysis and convulsive movements [4]. Dhawan et al. [25] evaluated four strains of Bacillus thuringiensis, and found that the mobility of M.incognita juveniles completely ceased after 24h exposure in S and S/10 d ilutions. Ho wever, all dilutions above S/25 were ineffective. Production of surfactin by Bacillus is a characteristic that supports their persistence under extreme conditions [26,27]. El-Hamshary et al. [28] found that Pseudomonas fluorescens and Pseudomonas aeruginosa affected M. incognita juvenile survival in vitro study, and the mortality percentages of the nematode were dependent on the bacterial concentration and exposure time. Terefe et al. [29] found that an aqueous suspension of Bacillus firmus at 2.5 and 3% concentration caused 100% inhibit ion of mobility M. incognita, 24 hrs after treat ment. The mortality percentage of Bacillus circulans (KSB2) at dilutions of 1/100 and 1/ 1000 were the h ighest inhibitors comparing with the other tested bacteria being 97.8 and 40.3%, respectively [30]. Xia et al. [31] reported that the culture supernatant of B. subtilis strains caused a higher mortality of M. incognita J2. Nagesh et al. [32] observed that, cell-free cu lture filt rates of B. cereus reduced egg hatching (90%) and caused 100% mo rtality of juveniles. Bin et al. [33] ment ioned that culture filtrates of rhizobacteriu m are heat stable and resistant to extreme pH values, which suggested that the antibiotic action might be responsible for the nematicidal activity. The exposure of M. incognita to various concentrations (5-100%) of cultural filtrate of Paenibacillus polymyxa in vitro conditions significantly reduced egg hatching and caused substantial mortality of its juveniles [34]. These results are similar with the findings of Ying et al. [35] who reported Bacillus spp. culture could significantly inhibit ing the hatching of eggs and increases the mortality of second stage juveniles and reduce infection of the nematode through production of nematicidal volat iles. Antibiotic production viz., 2, 4 DAPG, phenazine and pyocyanine were also well pronounced in the Pseudomonas strain, Pf 128 through thin layer chro matography [36] .The failure to regain its activity of M. incognita after separating from culture filtrate of Pseudomonas spp. placed over sterile water demonstrates the numbers of metabolites that had longer systemic activity [37,38].

V.
CONCLUSION It is clear fro m this work that, there are many bacteria which have potentialities for controlling root-knot nematode in plant rh izosphere region. A mong six bacterial isolates Bacillus thuringiensis and Pseudomonas fluorescens were exhibited the highest production of nematicidal activit ies against root-knot nematode M. incognita race 2.

ACKNOWLEDGEMENT
The authors are thankful to the Head of the Department, Depart ment of Nematology fo r provid ing necessary facilities for conducting the experiment.