The characterization of chitinolytic soil bacterial isolates for their antagonistic activity against root-knot nematode Meloidogyne incognita: An effort towards developing 'green' nematicidal agents.

Abstract

Root-knot nematodes (Meloidogyne spp.) are one of the most infective pests for a wide range of cultivated plants. The study was aimed to isolate chitinolytic soil bacteria, characterizing their properties, and to determine their in vitro antagonism against root-knot nematode eggs and juveniles. Out of the Ninety-five isolated strains, twenty-five gram-positive and non-hemolytic strains were selected and screened for production of extra-cellular enzymes, such as chitinase, protease, lipase, gelatinase, etc., as well as for biofilm formation, anti-fungal activity, and in vitro antagonism against root-knot nematodes. Eleven strains demonstrated broad anti-fungal activity against common plant pathogenic fungi, like Fusarium oxysporum and Alternaria alternata. Among the twenty-five strains, ten strains exhibited statistically significant (P˂0.05) second stage juvenile (J2s) percent mortality (>60 %) and inhibited egg hatching. The in-vitro activity of these ten strains was found to be correlated with their biofilm formation, enzyme production, and anti-fungal activity. Further, out of these ten strains, nine strains were assigned to the Bacilli group (99-100% similarity), and one was identified as Paenibacillus xylanilyticus (96 % similarity) after sequencing the gyrB gene. Overall, the present in vitro study suggested that the chitinolytic bacterial strains showing biofilm formation, enzyme production, and anti-fungal activity demonstrate an important trait to exhibit antagonism against root-knot nematodes.

1. Introduction

Soil is one of the most complex ecosystems present in the environment, which harbors diverse organisms that exhibit a highly complex network of interactions. Root-knot nematodes (RKN) belonging to Meloidogyne spp., are one of the most infective soil pests that affect a wide range of crop plants and account for a considerable loss in agricultural production. Root-knot nematodes are microscopic and sedentary endoparasitic nematodes found in the roots of infected plants. Among the RKNs, M. incognita, M. javanica, M. hapla, and M. arenaria are the most common species that encounter infections. Additionally, other RKN species like M. enterolobii, M. paranaensis, M. exigua (from tropical and subtropical regions) and, M. fallax, M. chitwoodi (from temperate regions) were previously considered minor agricultural pests, but now they are presenting a significant agricultural threat (Escobar et al., 2015; Poornima et al., 2016).

Root-knot nematodes cause substantial agricultural economic losses. It was observed that RKN caused approximately 12.6% of annual global agricultural losses during 2010-13, accounting for more than $100 billion (Askary & Martinelli, 2015). Considering the severity of agricultural losses contributed by the RKNs, there is an immense need to design management strategies for curbing them. Chemical nematicides are effective, easy to apply, and show rapid effects; however, they lead to several adverse effects on human health and the environment. Thus, research pertaining to biocontrol agents that are antagonistic to RKNs, stable, and are environmentally benign has become pertinent.

Plant growth-promoting rhizobacteria (PGPR) are known to enhance plant growth by colonizing over plants' rhizospheric region. Some PGPRs have also been reported to demonstrate nematicidal activity against plant-parasitic nematodes. In recent years, several investigations have proven the effect of rhizobacteria, such as Bacillus, Pasteuria, Pseudomonas, etc., on RKNs as they represent a dominant genera of nematophagous soil bacteria (Mhatre et al., 2019; Sharma & Sharma; 2017.). Further, chitinolytic bacteria was assessed for its potential biological control against RKNs. Gupta et al.,(2017) observed that the chitinolytic microbes inhibit the M.incognita multiplication and significantly reduce the infestation. Additionally, bacterial antagonism results from the actions of metabolites and hydrolytic enzymes like chitinases, proteases, lipases, gelatinases, etc. (Lee & Kim, 2015; Lee et al., 2015). Many rhizobacteria produce extra-cellular polysaccharides and form biofilms in the rhizosphere region (Nayak et al., 2020), inhibiting infective juvenile nematodes' penetration (J2) as they have been reported to protect the pathogen infection sites. In addition to enzymes, bacteria produce metabolites that can kill nematode larvae and inhibit egg hatching in Meloidogyne spp. (Zheng et al., 2018). Endospore-forming Bacillus strains produce non-ribosomal lipopeptides like surfactins, iturins, fengycins, plipastatins, etc., which act as broad-spectrum biocontrol agents against plant pathogenic fungi and nematodes for agricultural applications (Ongena & Jacques, 2008; Tapi et al., 2010). Thus, in this investigation, we have aimed to explore the nematicidal activity of gram-positive, spore-forming bacterial soil isolates as 'green' nematicidal agents and it was found that strains showing biofilm formation, extra-cellular enzyme production, and anti-fungal activity demonstrate an important trait to exhibit better antagonism against root-knot nematodes.

2. Material And Methods

2.1 Isolation of chitinolytic bacterial strains

The bacteria used in this study (Table 1) were isolated from various soil samples (rhizospheric fertile soil, rhizospheric nematode infected soil, rhizospheric nematode non-infected soil, rhizospheric saline soil, crab shell rich soil, etc.) from diverse locations across the states of Maharashtra and Gujarat in India on colloidal chitin agar (CCA) media. Colloidal chitin was prepared as per the protocol by Hsu and Lockwood (1975), using chitin flakes (Himedia-GRM1356, India). A CCA medium was prepared as described by Shahbaz and Yu (2020) and used to isolate chitinase-positive strains using a serial dilution scheme. Plates were incubated at 30 ± 2 ⁰C for seven days. Colonies surrounded by a clear zone on the CCA plate are considered as chitinolytic bacteria. These strains were maintained as glycerol (SDFCL-38454 L05, India) stocks and stored at -80˚C till further studies.

2.2 Characterization of the selected bacterial isolates

The pure colony of each bacterial strain was inoculated separately in a 500ml Erlenmeyer flask containing 50ml nutrient broth (Himedia- M002, India) and incubated at 37 °C, at 200 rpm, for 18h to determine the Gram nature by the Gram's staining method (Coico, 2005). The sporulation capacity of the isolates was evaluated by inoculating pure colonies in nutrient broth (Himedia- M002, India) and incubating for 48 h at 37 °C, 200 rpm by spore staining technique (Schaeffer and Fulton, 1933). A hemolysis test was conducted by spot inoculating sheep blood agar plates (Himedia-MP1301, India) with the bacterial cultures cultivated overnight and further incubating the plates for 24–72 hrs at 30 ± 2 ⁰C. The zones of clearance around the colonies were considered as an indicator of the presence of hemolysis (Nayak and Mukherjee, 2011). Further, a tube staining assay was employed to qualitatively assess the exopolysaccharide (EPS) production as an indication for biofilm formation capacity. (Jain et al., 2013).

2.3 Qualitative extra-cellular enzyme analysis

Cultures were assessed for their ability to produce extra-cellular hydrolytic enzymes like chitinase, protease, lipase, and gelatinase. A total of twenty-five Gram-positive, sporulating, and non-hemolytic bacterial isolates were selected for enzyme analysis. Enzyme analysis was qualitatively conducted using an agar (Himedia-RM026, India) medium, with a suitable substrate for each enzyme. A protease was determined on skim milk powder (Himedia- GRM1254, India) agar media as suggested by Vazquez & Mac Cormack,(2002), lipase activity was determined on tributyrin agar (Himedia- M157, FD081, India) media as described by Veerapagu et al., (2013), gelatinase activity was determined on gelatinase agar (Merck-M9512jh70, Germany) as described by Sharma et al., (2015) and colloidal chitin agar was used to determine the chitinase activity as suggested by Jha et al., (2016). The cultures' enzyme activity was reported as mentioned by (Tennalli, 2012).

2.4 Anti-fungal activity of bacterial isolates

Anti-fungal activity of the selected twenty-five strains was analyzed against common plant pathogenic fungi, Fusarium oxysporum NFCCI 651, and Alternaria alternata NFCCI 261. These fungal strains were procured from the National fungal culture collection of India (NFCCI) and preserved on potato dextrose agar (Himedia-M096, India) slants. The activity was determined by the agar well-diffusion method suggested by El Barnossi et al. (2020), with modifications as required. Briefly, an aliquot consisting of 1 mL of the inoculum, containing approximately 10⁴ spores of the pathogenic fungal strain, was added to 20 mL of melted potato dextrose agar (Himedia- M096) medium, maintained at 45 °C. This was then poured into 90 mm diameter size sterile petri plates and allowed to solidify at room temperature for an hour. Small wells were created in the agar plates using a cork borer (5 mm). A 25µl of overnight grown bacterial isolates, exhibiting an optical density of 1.0 (pre-adjusted with sterile distilled water) at 600 nm, was loaded into the wells. The petri plates were maintained at 4 °C for 2 hrs to allow uniform diffusion of culture inoculum into the agar. Later, plates were incubated aerobically at 30 ± 2 ⁰C for 48 h, and the diameters of the clearance zones were recorded, as published earlier. (Oyedele & Ogunbanwo, 2014).

2.5 In vitro activity

The in vitro nematicidal activity was evaluated by the egg hatching inhibition assay and by studying the mortality of second-stage juveniles (J2) of the M. incognita.

2.5.1 Preparation of nematode inoculum

The RKN-infected roots from tomato plants were used for extracting the nematode eggs. Briefly, the infected roots were chopped into pieces having a length of 2-3 cm and were treated for 3 min with 0.05% w/v of sodium hypochlorite (SDFCL-33040 L05, India) under stirring. The released eggs were harvested by passing through a series of sieve (1mm, 0.250mm, 0.045mm and 0.025mm), followed by (36%, w/v) sucrose (Himedia-MB025, India) gradient centrifugation at 3000 RPM. The supernatant was poured over a sieve of the size 500 mesh (0.025mm), washed with sterile distilled water several times, and collected as a suspension. The contents of this suspension were examined under a stereo zoom microscope (Olympus- SZ61, Japan) to identify the presence and density of eggs. Further, J2s were obtained by pouring the egg suspension over 4-ply tissue paper mounted on a piece of wire mesh, as described by Kumar et al. (2018). This assembly was fixed in a petri plate, to which fresh water was added and maintained at 24 ± 1°C till the eggs hatched. The freshly hatched J2s were used during the experiments.

2.5.2 Identification of RKNs

The galls from tomato roots were dissected to collect the female nematodes. The perineal pattern of these female nematodes was studied under a compound microscope (Olympus-CX43 RF, Japan, 40X resolution) as described by (Taylor & Netscher, 1974).

2.5.3 Effect on egg hatching

The effect of twenty-five bacterial isolates on egg hatching was evaluated by the method reported by Kumar et al. (2018), with necessary modifications. The test was conducted in pre-sterilized 6-well tissue culture (Tarson. Cat. No.980010, India) plates, with each well having a capacity of 12 ml volume. In brief, 1.0 mL of egg suspension containing approximately 100 eggs of M. incognita was mixed with 2.5 ml of sterile distilled water and 0.5 ml of bacterial suspension, having an optical density of 1.0 (pre-adjusted with sterile distilled water) at 600 nm, and grown overnight in a nutrient broth (Himedia-M002, India). All the analyses were performed with three replications, and the plates were incubated at 28°C for five days. Nutrient broth and water were maintained as controls. The effect of bacterial isolate on egg hatching was observed microscopically (Olympus- SZ61, Japan) on 3^rd, 4^th and 5^th day of incubation and calculated by the formula (Sikandar et al., 2020) stated in equation 1.

2.5.4 Effect on mortality of juveniles

Juvenile mortality was evaluated after slight modifications in the method stated by Kumar et al. (2018). An aliquot of 0.5 mL of the respective overnight grown in a nutrient broth (Himedia-M002, India) bacterial suspensions (at 1.0 OD_600nm) was mixed with 2.5 ml sterile distilled water and 1.0 mL of nematode suspension containing approximately 100 M. incognita J2's, in 6-well sterile tissue-culture plates, to test the percent mortality of nematodes. Sterile water and nutrient broth medium served as controls. The mobility of the J2's was confirmed microscopically by probing the nematodes with a needle, while the immobile ones were considered dead. All the analyses were performed with three replications. The percentage mortality was recorded after 2-4 days and calculated with the formula (Sikandar et al., 2020) stated in equation 2.  

2.6 Electron microscopic observations of bacteria-treated eggs

The virulence of the isolates against M. incognita eggs was confirmed by environmental scanning electron microscopy (E-SEM) analyses. For this, 1mL of suspension containing unhatched eggs of M. incognita was treated with 0.5 mL of actively growing ZB-HT4 bacterial isolate (OD_600nm-1.0) and maintained for three days at room temperature. The treated and non-treated eggs specimen were prepared for analysis as described by  Sousa et al. (2020) and observed under the scanning electron microscope. The e-SEM analysis was carried out at 20kV under a low vacuum. FEI Quanta 200 (Netherlands) was used for the SEM analysis at 1000X, 2000X, and 10000X magnification.

2.7 Molecular identification of selected strains.

The ten most effective strains were selected based on their properties and antagonism against RKN eggs and juveniles for molecular identification using the Illumina MiSeq platform. Genomic DNA was isolated using the QIAampPowerFecal Pro DNA kit, Cat: 51804 (Qiagen, Germany), as per the manufacturer's instructions. A genomic library for each strain was constructed in alignment with the recommendations for whole genome sequencing using Illumina Nextera^TM DNA flex library preparation kit, cat: 20060060 (Illumina Inc., USA) (Prjibelski et al., 2020) A gyrB gene was used as a marker for phylogenetic identification (Wang et al.,2007). The phylogenetic tree was constructed based on subjecting the gyrB sequence of the selected isolates to BLASTN search against the NCBI nr/nt database, using default parameters (Tatusova et al., 2016). The sequences from the top 10 hits of alignments were downloaded and aligned against the query using clustalW. The multi-aligned sequences were imported into MEGA X and Tamura Nei model, in association with gamma distribution, to construct a phylogenetic tree using the bootstrap value of 3000. The phylogenetic tree was downloaded in Newick format and visualized in iTOL.

2.8 Statistical Analysis

The data obtained from the in vitro studies were analyzed statistically using analysis of variance. Tukey's test was applied for the one-way analysis of variance (ANOVA) using SPSS 20.0 (IBM, SPSS statistics 20) with a statistical significance level of 0.05. 

3. Results

3.1 Isolation and strain characterization

The bacterial strains were isolated from diverse soil samples. The colonies that exhibited a clear halo around themselves on chitin agar were considered chitinase positive and were selected to evaluate their hemolytic nature, gram nature, and sporulation capability. A total of Ninety-five different chitinase-positive bacterial cultures were obtained. Among them, twenty-five isolates were selected based on their Gram-positive nature and non-hemolytic property for biofilm-formation capacity. Sixteen strains were capable of producing EPS responsible for biofilm formation. Figure 1 depicts the representative image for ring formation as an indication for biofilm formation. The majority of these selected twenty-five isolates possess extra-cellular enzyme production ability. Protease, lipase, chitinase, and gelatinase were qualitatively tested to utilize their respective substrates as nutritional sources. The enzyme production capacity of all twenty-five strains has been stated in Table 1. A total of twelve strains were able to produce all four extra-cellular enzymes. 

3.2 Anti-fungal activity

All twenty-five strains were tested for their anti-fungal activity against Fusarium oxysporum and Alternaria alternata. Thirteen strains exhibited anti-fungal activity against Fusarium oxysporum, while twenty strains exhibited antagonistic action against Alternaria alternata. Eleven strains, namely ZB-AC4, ZB-BD4, ZB-BT5, ZB-CD12, ZB-CL7, ZB-DA10, ZB-DC5, ZB-EB1, ZB-EY1, ZB-IM5 and ZB-JE1 exhibited antagonistic action against both the fungal phytopathogens. Remarkably, strain ZB-CD12 exhibited maximum antagonistic activity against both fungal phytopathogens, followed by ZB-DC5, ZB-EB1, ZB-BT5, and ZB-CL7. The results of all twenty-five strains have been stated in table 1. 

3.3 In vitro trials

The perineal patterns of female nematodes exhibited an angularly oval structure with a high dorsal arch (Fig. 2), and the presence of stylet on J2 confirmed that the RKN belonged to the species Meloidogyne incognita. The bacterial isolates were evaluated for their antagonistic effect on juvenile mortality and egg hatching at the same embryonic stage for each treatment. The results were statistically significant (F = 41.77, df = 26, P ˂ 0.05) for percent J2 mortality after 96 h and for percent egg hatching after 120 h (F = 112.11, df = 26, P ˂ 0.05). The medium control and non-treated plates demonstrated statistically similar hatching percentages (70% and 73% respectively) and J2 mortality (9.5% and 9.9% respectively), indicating that the media components did not contribute towards the antagonism as exhibited by the bacterial isolates. J2 mortality after 96 h of incubation ranged between 26-67%, while inhibition of egg hatching at the end of 120 h was found to be between 17-67 % tested at a similar concentration of cultured broth for each strain. An increase in incubation time increased the percent mortality and inhibited egg hatching (Table 2). Ten strains, namely ZB-BT5, ZB-CD12, ZB-CL7, ZB-CP9, ZB-DA10, ZB-DC5, ZB-EB1, ZB-HT4, ZB-IM5, and ZB-S3 demonstrated broad spectrum of antagonism. They were found to be statistically significant regarding mortality and egg hatching inhibition, which confirmed their nematicidal potential. Strain ZB-DA10 was observed to be highly antagonistic with regard to the mortality of J2s (67.3%) and while strain ZB-EJ5 resulted in the lowest egg hatching (17.9%)

Further, microscopic analysis supported the antagonistic properties of bacterial isolates on nematode eggs. Eggs of nematodes were affected by treatment with bacteria, including morphological changes, such as deformation, vacuolation, loss of internal content, and subsequent death. Bacteria-treated eggs did not undergo hatching even after six days of incubation. The morphological changes observed in the treated and non-treated eggs have been depicted in fig. 3.

3.4 Identification of the bacterial strains

Molecular identification of the ten selected antagonistic strains was conducted upto the species level by sequencing the gyrB gene and comparing it with the NCBI nucleotide database. The taxonomy check module of PGAP used average nucleotide identity (ANI) to compare the input draft genome. An ANI threshold of 96% identity and a minimum coverage threshold of 80% of both the query and the type assembly was used to declare the identification. The bacterial isolates were identified as Bacillus spp. and Paenibacillus spp. (Table 3). Among these, ZB-IM5 and ZB-HT4 were identified as Bacillus licheniformis, ZB-CL7 and ZB-S3 as Bacillus paralicheniformis, ZB-BT5 and ZB-DC5 as Bacillus velezensis, ZB-DA10, ZB-CP9 and ZB-CD12 as Bacillus cereus and ZB-EB1 as Paenibacillus xylanilyticus. A Phylogenetic tree construct prepared for all ten strains presented in fig.4.

4. Discussion

RKNs belonging to the Meloidogyne spp. are a group of well-known plant pathogens. Infestation of fruits and vegetable crops with Meloidogyne spp. can significantly impact their growth and quality of produce and eventually affect the agricultural yield. The use of biological control is recommended as chemical nematicides are harmful to the soil and can adversely affect human health. Identifying effective biocontrol agents against RKNs is an important area of research. In this study, we have designed a screening strategy to select the potent anti-nematicidal bacterial isolates. We investigated the chitinase producing ability of soil bacteria and screened them for antagonism against RKNs. The antagonistic strains were chosen for studies based on their safety aspects, including Gram's nature, sporulation ability, non-hemolytic nature, etc. The chitinolytic strains were screened for biofilm formation capability, hydrolytic enzyme production ability, and anti-fungal properties. The antagonism of selected strains was confirmed via in vitro studies, such as the effect on egg hatching and J2s mortality. From this exercise, we could isolate Ninety-five bacterial strains and further selected ten isolates with potent anti-nematicidal activity confirmed by in-vitro juvenile mortality and inhibition of egg hatching.

The activity of biocontrol agents against nematodes is a complex process that includes a series of events such as host recognition, adherence, penetration and digestion of the nematode body (Zhang et al., 2020). The success of an effective biocontrol agent against nematodes is likely to depend on all these properties. At the time of infection of nematodes, the nematophagous bacteria must first contact and penetrate the host cuticle (Gan et al., 2007). It has become more apparent that chitinases are involved in penetration through the cuticle and cellular digestion of nematodes. (Soares et al., 2015). The eggshell cuticle of RKNs is a vital infection site for chitinolytic antagonistic microorganisms. In our study, Ninety-five chitinolytic bacterial strains were efficiently isolated from soil samples collected using a colloidal chitin agar medium. Colonies surrounded by a clear zone on the CCA medium confers the hydrolysis of colloidal chitin and are considered as an indication of chitinase-producing bacteria. The gram-negative and hemolytic strains were excluded from further investigations, considering their pathogenicity. The twenty-five chitinolytic strains were studied for their EPS production capability. The EPS production contributes to the biofilm-forming nature of bacteria. The study conducted by Ghahremani et al. (2020) revealed that Bacillus firmus I-1582 could form biofilms on nematode eggs, which helped in the degradation of nematode eggshells. EPSs are also essential in maintaining higher moisture content and growth of plants under severe drought conditions by forming a rhizo sheath around the roots, consequently protecting the plant roots from desiccation (Khan & Bano, 2019). In our study, we observed that fourteen isolates were able to produce EPS. Most of the EPS positive isolates such as, ZB-BT5, ZB-DW5, ZB-DC5, ZB-IM5, ZB-S3, etc. exhibited better antagonism during the in- vitro studies.

Extra-cellular enzymes produced by many bacteria are important virulence factors due to their ability to result in membrane damage and mediate tissue destruction in nematodes to provide nutrients for the toxin-producing bacteria. A study by Lee et al. (2015) on Lysobacter capsici YS1215 revealed that chitinases and gelatinase contributed to RKN antagonism. Basyony and Abo-Zaid (2018) have described the ability of Bacillus spp. to produce lytic enzymes such as chitinase, glucanase and protease, which affect the nematode cuticles. We studied several bacterial isolates qualitatively for their ability to produce lytic enzymes such as chitinase, protease, lipase and gelatinase. The enzyme production data presented in table 1 indicates that most isolates exhibited a broad enzyme production ability. The selected twenty-five strains were tested for their anti-fungal activity against Fusarium oxysporum and Alternaria alternata. Wilt fungus such as Fusarium spp. causes wilting of nematode-infected plants, leading to plant death. Infection of roots by RKNs makes them vulnerable to infection by root-infecting fungi, resulting in the development of disease complexes (Son et al., 2009). It was mentioned by Adam et al. (2014) that bacteria that possess anti-fungal properties also suppressed M. incognita population in tomato plants under greenhouse conditions. Therefore, the anti-fungal activity of nematicidal strains was anticipated to control the disease complexes. Our study observed that the bacterial strains that produced protease and chitinase enzymes exhibited higher anti-fungal activity, viz., ZB-BT5, ZB-IM5, ZB-DC5, ZB-CD12, and ZB-EB1. All twenty-five strains were evaluated via in vitro studies against the M. incognita J2s and hatching eggs in RKNs. Among the twenty-five strains, ten strains viz. ZB-BT5, ZB-EB1, ZB-HT4, ZB-IM5, ZB-CP9, ZB-S3, ZB-DC5, ZB-DA10, ZB-CD12, and ZB-CL7 displayed statistically significant (P˂0.05) juvenile mortality and inhibited the egg hatching in RKNs. The in-vitro activity of these strains is co-related with their EPS production, enzyme activity, and anti-fungal activity. These strains were belonged to the Bacillus group, as confirmed via sequencing of gyrB gene with the exception of ZB-EB1 as Paenibacillus xylanilyticus. We observed higher J2 mortality in strains that produced chitinase, protease, and lipase enzymes. The chitinase enzyme produced by bacteria initiates antagonism against nematodes by hydrolyzing the insoluble peptide bonds present in chitin to their oligo N-acetyl-D-glucosamine components (Shahbaz & Yu, 2020). RKNs eggshell is rich with chitin. The higher chitinase-producing isolates evidently inhibit the egg hatching during the in-vitro studies, viz., ZB-CP9, ZB-DC5, ZB-CD12, ZB-DA10, ZB-CLO26, ZB-GE4a etc. We have Light microscopic and scanning electron microscopic studies that revealed the deformation of nematode eggs. This deformation was attributed to be caused by hydrolytic enzymes like chitinase and proteases secreted by the bacteria. The colonization of bacteria disrupted the embryonic development in nematode eggs. On the other side, the strains which does not produce EPS and had low enzyme and anti-fungal activity, did not show significant antagonism at in-vitro studies e.g. ZB-AB4, ZB-BD4, ZB-GB2, ZB-SI8 etc.

Hence, our results indicate that strains showing biofilm formation, extra-cellular enzyme production, and anti-fungal activity are essential trait to exhibit better antagonism against root-knot nematodes and provides a screening platform for the research community.

5. Conclusion And Way Forward

In conclusion, we have identified ten Bacillus strains having good nematicidal activity. The strains were studied for their biofilm formation, enzyme production, and anti-fungal activity. These strains exhibited anti-fungal activity, an additional advantage to preventing the formation of the disease complex produced by fungi in nematode-infected plants. They demonstrated significant in-vitro activity against both, J2s and eggs of M. incognita. Therefore, it was concluded that strains ZB-BT5, ZB-CD12, ZB-CL7, ZB-CP9, ZB-DA10, ZB-DC, ZB-EB1, ZB-HT4, ZB-IM5, and ZB-S3 may be further evaluated in pot studies and subsequent field trials as a biocontrol strategy for RKN infected plants. The whole genome sequence data obtained will be further studied to characterize strains for the presence of putative biocontrol genes. Strains exhibiting maximum activity in field trials will be used for formulation preparation, stability study, and efficiency as a biocontrol formulation.

Declarations

DISCLOSURE STATEMENT

No potential conflict of interest was reported by the authors.

ACKNOWLEDGMENT

The study was financially supported by Zytex Biotech Private Limited, Mumbai, as an in-house Ph.D. project on bionematicide. The research was carried out in coordination with the Institute of Chemical Technology, Mumbai. The authors would like to express their gratitude towards the Director, Department of Nematology, Tamil Nadu Agricultural University, for approving the professional training at Tamil Nadu Agricultural University, Coimbatore, India.

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Tables

Table 1 Enzyme activity and anti-fungal activity of bacterial isolates

Table 1: Biofilm production and enzyme production capability of the isolated strains. Note: Enzyme activity "+", "++" ,"+++" and "++++"represent the average ratio of zone of clearance diameter to zone of colony diameter as 1-1.5 mm, 1.5 to 2.0 mm, 2.0-2.5 mm and 2.5 mm & above, respectively. Antifungal activity  "+", "++" ,"+++", "++++" "++++"represent the average inhibition radius are less than 3 mm, between 3 mm and 5 mm, between 5 mm and 8 mm, and more than 8 mm, respectively 

Table 2 In-vitro activity of bacterial isolates on J2s mortality and egg hatching of M. incognita.

Table 2 In-vitro effect of culture broth of bacterial isolates on mortality and egg hatching of M. incognita. Data presents the mean value of each treatment followed by standard deviation and identical letter according to Tukey's test P<0.05 level.

Table 3 Analysis of variance of the In-vitro antagonism of bacterial isolates against M. incognita

Table 3 Analysis of variance of the effect of bacteria isolates on J2 mortality (%) and egg hatching (%). Whereas:

S.S (Sum of square); M.S (Mean square); df (Degree of freedom); F (F-value), and P (significant value)

Table 4:  BLAST analysis outcomes for gyrB sequences of the selected strains