Evaluation of potential bio-control agents on root-knot nematode Meloidogyne incognita and wilt causing fungus Fusarium oxysporum f.sp. conglutinans in vitro

Indigenous strains of Trichoderma viride (ITCC No. 6889), Pseudomonas fluorescens (ITCC No. B0034) and Purpureocillium lilacinum (ITCC No.6887) were isolated from undisturbed forest eco-system of Southern India. These three bio-mediators were evaluated for their antagonism towards root knot nematode, Meloidogyne incognita and Fusarium oxysporum f.sp. conglutinans in vitro. Cell free culture filtrate of these strains significantly inhibited the egg hatching and caused juvenile (J2) mortality of M. incognita at 25, 50, 75 and 100% concentrations. Maximum inhibition in egg hatching and juvenile mortality were recorded in P. lilacinum as 94.21 and 91.28%, respectively after 120 h. It was followed by T. viride and P. fluorescens which recorded 92.72 and 91.46% and 89.12 and 90.14% inhibition in egg hatching and juvenile mortality, respectively after 120 h. Antagonism of T. viride on F. oxysporum was recorded maximum on the 5th day as 45.82%. Similarly, the antagonism on the 5th day for both the bioagents of P. lilacinum and P. fluorescens were recorded as 45.26 and 44.19%, respectively.


INTRODUCTION
Root-Knot nematodes are causing a notable damage to a wide range of vegetable crops causing significant yield loss in tropical and sub-tropical agriculture (Sikora and Fernandez, 2005).The symptoms of nematode disease are manifested by the formation of root galls accompanied by stunted growth, chlorosis and loss of viability of the plant (Babu et al., 1999).Fusarium wilt is soil borne fungal pathogen which can sustain many years in the soil without a host (Ignjatov et al., 2012).F. oxysporum has a worldwide distribution and causes severe root rot or vascular wilt in ample range of plant families (Enya et al., 2008;Lievens et al., 2008;Michielse and Rep, 2009).This fungal pathogen infects the seed and early stages of seedling growth, causing seed decay and damping-off (Punja et al., 2004).
Trichoderma viride is an effective bio-control agent against numerous soil borne plant pathogens and can easily colonize plant rhizosphere and helps in the plant growth promotion (Verma et al., 2007;Savazzini et al., 2009;John et al., 2010).T. viride proved to be effective against root knot nematode, Meloidogyne species and reduced its damage on several crops (Meyer et al., 2001;Abd et al., 2007).Various reports on nematophagous fungus Purpureocillium lilacinum prove its efficacy as effective bio-control agent on Meloidogyne spp. on various crops (Jatala, 1986;Rao and Reddy, 1994;Rao et al., 1999;Khan and Williams, 1998;Mohd et al., 2009;Brand et al., 2010).It has more rate of occurrence in tropical and subtropical areas (Morgan-Jones et al., 1984;Chen et al., 1996).
Process of DNA extraction from fungal cultures eliminates many unknown interfering substances which allow to identify species specific organisms using ribosomal DNA by PCR (Don et al., 2000;Bryan et al., 1995).Currently, many methods are available for the isolation of fungal genomic DNA (Plaza et al., 2004;Melo et al., 2006).Pseudomonas species were isolated from soil eco-system that was naturally suppressive to many plant diseases like Fusarium wilt and black rot of tobacco (Thomashow et al., 1990;Raaijmakers and Weller, 1998;De Boer et al., 1999;William et al., 1991).
Culture filtrates of bio-control agents are reported to be antagonistic to a wide range of plant parasitic nematodes in in vitro (Reibinger, 1995;Hallaman and Sikora, 1994;Meyer et al., 2004;Vu, 2005;Abd et al., 2007).In the current investigation, efforts were made to isolate the bioagents and evaluate the effect of three bio-agents, namely, T. viride, P. fluorescens and P. lilacinum against M. incognita and Fusarium oxysporum f.sp.conglutinans in in vitro.

Isolation of bio-agents, collection of M. incognita egg mass and F. oxysporum
Cultures of T. viride (ITCC No.6889), P. fluorescens (ITCC No. B0034) and P. lilacinum (ITCC No. 6887) were maintained on nutrient agar (NA) (Himedia chemicals, India) for bacterial cultures and Potato dextrose agar (PDA) (HIMEDIA chemicals, India) for fungi by cryopreservation method (Sudheer, 2010).The isolates were sub-cultured and used for further study.Root knot nematode culture was obtained from infected cauliflower plants grown in farmers field (Doddaballapur, Bengaluru rural district, Karnataka, India).Identification of M. incognita was confirmed by perineal cuticular pattern (PCP) under stereo microscope and used for further studies.F. oxysporum f. sp.conglutinans was isolated from fusarium infected cauliflower plants.Preparation of inoculum was made by culturing the F. oxysporum isolate in PDA amended with streptomycin sulphate for 7 days at 25 ± 2°C.
DNA isolation from T. viride, P. lilacinum and P. fluorescens and PCR Genomic DNA was isolated from cultures of T. viride and P. lilacinus as per the protocol of Raeder and Broda (1985).Bacterial genomic DNA of P. fluorescens was isolated using Nucleospin tissue extraction kit (Macherey-Nagal, Germany).Polymerization Chain Reaction (PCR) of 25 µl PCR mixture was prepared for molecular identification of T. viride and P. lilacinum with the following protocol.Final PCR volume contained 19.7 μl of PCR grade water, 0.5 μl of each forward and reverse primers (0.2 pmol; Bio-serve Pvt.Ltd, India), 0.5 μl of dNTPs (10 mM; Fermentas Inc, Canada), 2.5 μl (10X) of Taq buffer (Bangalore Genei Pvt.Ltd, India), and 0.3 μl of Taq DNA polymerase (Bangalore Genei Pvt.Ltd, India) and 1 μl of template DNA (25 ng/μl).PCR amplifications were carried out in Eppendorf master cycler gradient (vapo.protect,Germany).Amplification reactions were performed in master cycler with heated lid.The primer pairs ITS F (TCCGTAGGTGAACCTGCGG) and ITSR (TCCTCCGCTTA-TTGATATGC) were used for the amplification of region including the ITS 1, 5.8 S and ITS 2 (Hurtado et al., 2008).The initial denaturation for 5 min at 94°C was followed by 35 cycles of 45 s at 94°C, 35 s at 43°C, 40 s at 72°C and a final extension of 10 min at 72°C.
16s rDNA amplification of P. fluorescens was carried out using universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) (Weisburg et al., 1991) in thermo cycler (Eppendorf vapo.protect,Germany) using the aforementioned components which followed for final PCR mixture of 25 µl except 16s primers and DNA template (25 ng/μl).The PCR cycle of initial denaturation was done at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 45 s, primer annealing at 51°C for 35 s and elongation at 72°C for 40 s.Final extended elongation was done for 10 min at 72°C.The obtained PCR products were gel electrophoresis on 1.5% agarose gel (Figures 1 and 2).
The PCR products were sequenced at Bio-serve, Hyderabad, Telangana state, India.All the sequenced PCR products were confirmed using NCBI mega blast for its species identity of ITS region and 16s region.The molecular identified strains were submitted at NCBI.

Effect of culture filtrate of P. fluorescens on egg hatching of M. incognita
A single colony from pure culture of P. fluorescens taken from 24 h old culture plates was inoculated into 50 ml of sterilized King's B broth (HIMEDIA chemicals, India) in 100 ml Erlenmeyer flasks.These flasks were incubated in a shaker incubator at 150 rpm speed and 37°C for 24 h.The bacterial growth after 24 h was tested for their luminosity under transilluminator at 250 to 260 nm.P. fluorescens culture filtrate was obtained by centrifugation (Eppendorf refrigerated centrifuge 5415) at 10,000 rpm for 15 min at 4°C.The supernatant culture filtrate was collected and passed through syringe filter of 0.22 µm (Millipore PVDF Durapore 13 mm diameter).Consequently, collected culture filtrate was tested for the absence of any viable cell and used to study the effect on egg hatching and juvenile mortality of M. incognita.P. fluorescens culture filtrate was made into four concentrations of 100, 75, 50 and 25% by adding sterile distilled water.Three millilters of each concentrations of culture filtrate was transferred to sterile Petri-dishes of 5 cm diameter.M. incognita egg masses  were collected and surface sterilized using 0.1% sodium hypochlorite for 30 s and rinse the treated egg masses with sterile distilled water.Each petri dish was placed with five egg masses containing culture filtrate of each concentration and incubated at room temperature.M. incognita egg masses placed in King' B and distilled water served as control.The number of juveniles (J2) hatched in all the four concentrations were recorded at 24, 48, 72, 96 and 120 h of exposure.
The percentage suppression in hatching of juveniles (J2) was calculated using the following formula: Where, Ht is the number of juveniles hatched in treatment and Hc is the number of juveniles hatched in control.

Effect of culture filtrate of P. fluorescens on mortality of M. incognita juveniles
Culture filtrate was made into four concentrations of 100, 75, 50 and 25% by adding sterile distilled water.Three millilters culture filtrate of each concentration was transferred to sterile Petri-dishes of 5 cm diameter.Freshly hatched 100 M. incognita juveniles (J2) were placed in each Petri dish and incubated at room temperature (25 to 30°C).Petri dishes containing sterile water and autoclaved King's B broth placed with juveniles (J2) served as control and treatment.. Total number of dead nematodes were counted after 24, 48, 72, 96 and 120 h of exposure and percentage mortality of juveniles was calculated.

Effect of culture filtrate of T. viride and P. lilacinum on egg hatching of M. incognita
Freshly sub-cultured T. viride and P. lilacinum of 5 mm disc were inoculated in 100 ml sterilized potato dextrose broth -PDB (HIMEDIA chemicals, India) in 250 ml Erlenmeyer flask.The flasks were incubated at 27 ± 1°C for 8 days.From each culture, 50 ml of broth containing 2.8 × 10 6 spores (CFU/ml) was centrifuged at 13000 rpm at 4°C for 20 min (Eppendorf refrigerated centrifuge 5415).The obtained pellet was discarded and supernatant was collected which passed through 0.45 µm syringe filter (Millipore PVDF Durapore 13 mm diameter).The culture filtrate thus obtained was tested for the absence of any fungal spores by plating it on PDA.T. viride and P. lilacinum culture filtrate was made into four concentrations of 100, 75, 50 and 25% by adding sterile distilled water.They were tested for hatching and J2 mortality as per the aforementioned procedure for P. fluorescens.

Effect of T. viride and P. lilacinum antagonists against F. oxysporum f.sp. conglutinans in in vitro
Five day old cultures of T. viride (TV-15), P. lilacinum (PL-29) and F. oxysporum discs (5 mm diameter) were grown on PDA and punched in the periphery in 90 mm petri plates.Each culture was inoculated separately on PDA plates as control.Cultures of T. viride with F. oxysporum and P. lilacinum with F. oxysporum were inoculated separately on PDA plates at 20 mm distance from periphery of the petriplate as per dual culture method.Each treatment was replicated thrice and incubated at 27±2°C for antagonistic study.Observations were recorded on growth of each fungal culture for 5 days.The percentage of inhibition of F. oxysporum was calculated (Vincent, 1927).

PI =C-T/C×100
Where, PI is the percentage inhibition over control, C is the control of F. oxysporum without T. viride (mm), and T is the growth of F. oxysporum with T. viride (mm).

Effect of P. fluorescens antagonists against F. oxysporum f.sp. conglutinans in in vitro
Both the cultures of 1 day old P. fluorescens and 5 days old F. oxysporum were inoculated on Nutrient Agar (NA) and PDA plates (90 mm), respectively.As per dual culture method, P. fluorescens was inoculated by spread plate method on PDA plate.F. oxysporum disc (5 mm) was inoculated in the centre of PDA plate.Half streak of P. fluorescens and 5 mm F. oxysporum disc inoculated 20 mm distance from periphery of the plate in the same PDA plates were also maintained for antagonistic study.The same procedure for T. viride and P. lilacinum antagonistic study was followed.

Molecular identification of bio-agents using gene specific primers
Ribosomal DNA internal transcribed spacers region was amplified using fungal genus specific ITS-1 and ITS-4 primers (Figure 1).Bacterial strain was amplified using 16s primers (Figure 2).Molecular identification of these PCR amplified strains were confirmed using NCBI mega blast.T. viride and P. lilacinum matching to National Centre for Biotechnology Information (NCBI) were 94 and 96%, respectively and P. fluorescens was 97%.These strains were submitted at NCBI with the following accession numbers T. viride -KP271026, P. lilacinum -KP271028 and P. fluorescens -KP27102.

Effect of culture filtrates of three bio-agents on M. incognita
Results of studies on culture filtrates showed that the efficacy of the isolates of T. viride, P. lilacinum and P. fluorescens on hatching of M. incognita eggs and J 2 mortality increased with the increase in concentration of the culture filtrates.These investigations clearly indicated that as the duration of exposure to culture filtrate of the bacterium and fungal cultures increased, suppression in hatching and mortality of juveniles (J 2 ) also increased.The maximum suppression in egg hatching was recorded in P. lilacinum as 94.21% and juvenile mortality as 91.28% after 120 h at 100% concentration (Table 1).It was followed by T. viride which recorded the inhibition in egg hatching as 92.72%, and juvenile mortality as 89.12% after 120 h (Table 2).In P. fluorescens, suppression in egg hatching and juvenile mortality was recorded as 91.46 and 90.14%, respectively after 120 h (Table 3).

Effect of bio-agents antagonism on F. oxysporum
T. viride showed antagonistic activity on F. oxysporum on the 5th day which was recorded as 45.82%.It was followed by P. lilacinum and P. fluorescens, for which the antagonism was recorded on the 5th day as 45.26 and 44.19% (Figure 3).

DISCUSSION
During the experimental investigations, native strains of T. viride, P. lilacinum and P. fluorescens were isolated from different regions of South India.The identity of these isolates were confirmed through molecular techniques and evaluated for their antinemic and antifungal activity in vitro.The culture filtrate studies revealed that all the biocontrol agents were effective in suppressing the egg hatching of M. incognita and causing J 2 mortality which increased with increase in time of exposure of eggs to cell free culture filtrate as well with increase in concentration.Deformation of juveniles was observed in most of the eggs in the present study.The results    et al. (1999) who reported that mortality of M. incognita increased with increase in exposure time as well as the concentration of culture filtrate.Bin et al. (2005) showed that culture filtrates of rhizobacterium are heat stable and resistant to extreme pH values, which suggested that the antibiotic rather than protein might be responsible for the nematicidal activity.
Antagonism was observed in 3 bio-control agents, namely, T. viride, P. lilacinum and P. fluorescens against F. oxysporum in vitro.Trichoderma species are widely used as biocontrol agents to reduce the disease incidence caused by plant pathogenic fungi and many soil borne pathogens (Papavizas, 1985;Sivan and Chet, 1986).Pau et al. (2012) investigated the effect of antagonism on P. lilacinum in in vitro.The culture filtrates and their antagonism proved the biocontrol efficiency of these microbes.Results on antagonism clearly indicated the effect of bio-agents in in vitro on F. oxysporum.More antagonism was recorded with T. viride followed by P. lilacinum and P. fluorescens.Hence, it can be exploited further in development of formulations and evaluation under field conditions.

Conclusion
The findings on the effect of culture filtrates of three bioagents, namely, T. viride, P. fluorescens and P. lilacinum showed on root-knot nematode M. incognita in in vitro.The experiments also proved the antagonism of these bio-control agents on suppression of F. oxysporum f.sp.conglutinans.For the resultant data, the effect on M. incognita and on F. oxysporum was an apparent indication to control the nematode induced disease complex in in vivo in cauliflower (Rajinikanth et al., 2013) by application of eco-friendly bio-control agents of bionematicide (P.lilacinum), bio-fungicide (T.viride) and biobactericide (P.fluorescens).

Figure 3 .
Figure 3.Effect of T. viride, P. fluorescens and P. lilacinum antagonism on growth inhibition of F. oxysporum in in vitro.

Table 1 .
Effects of the cell-free culture filtrate of T. viride on egg hatching and mortality of M. incognita in in vitro.
Figures in parentheses arearc sine transformed values.

Table 2 .
Effects of cell-free culture filtrate of P. lilacinum on egg hatching and mortality of M. incognita in in vitro.

Table 3 .
Effects of the cell-free culture filtrate of P. fluorescens on egg hatching and mortality of M. incognita in in vitro.
indicated the production of nematicidal compounds in the culture filtrates.These nematicidal compounds produced by T. viride, P. fluorescens and P. lilacinum seemed to play an important role in causing nematode mortality.Many soil borne nematode trapping fungi, endoparasitic fungi, parasites of nematode eggs and cysts were reported to produce toxic metabolites against nematodes(Li et al., 2007).In earlier reports,