Nematicidal activity of native Bacillus thuringiensis against the root knot nematode, Meloidogyne incognita (Kofoid and White)

In this study, potential of the native Bacillus thuringiensis Berliner was investigated against the root knot nematode, Meloidogyne incognita (Kofoid and White) under laboratory and greenhouse conditions. Fourteen out of 50 Bt isolates obtained from root zone of vegetable crops of north western zone of Tamil Nadu, India, were found to be encoded with nematicidal cry genes, confirmed by gene-specific PCR. Nine different Cry protein profiles were obtained from native Bt. Spore-crystal mixtures of 6 isolates out of 14 (at 0.5%) showed 100% inhibition to J2 juveniles emergence from egg masses of M. incognita within 72 h of treatment. Analysis by SEM revealed that fluffy egg masses were observed in untreated samples due to hatching, whereas egg masses were covered by gelatinous matrix, when treated with spore-crystal mixture of nematicidal Bt. Two (BC and BD) of the 6 isolates were selected based on their highest nematicidal activity against J2 of M. incognita with LC50 values of 0.12 and 0.23 μg/ml of protein. The spore-crystal mixtures of isolates BC and BD when applied to tomato plants under greenhouse conditions exhibited the enhanced biocontrol potential by suppressing number of egg masses, reduction of female population and decreased root gall index, when compared to control and chemical treatments.


Background
Meloidogyne spp. are the most devastating plant-parasitic nematodes (PPNs) causing serious economic damage to agricultural and horticultural crops. The genus has 98 species among which M. incognita causes root galls in almost all vascular plants (Jones et al. 2013). Meloidogyne spp. are very difficult to control owing to their quick generation time, high reproduction rate, entophytic and sedentary nature (Engelbrecht et al. 2018).
The uses of chemical nematicides are under scrutiny now because of growing awareness of their harmful effects on soil, water, environment and non-targeted organisms. Besides, Meloidogyne spp. have developed resistance to many of these nematicides. Hence, there is a lot of emphasis on finding a sustainable alternative to chemical nematicides for the control of Meloidogyne spp.
Biological control as an environmental friendly alternative in integrated pest management strategy is gaining momentum recently (Pocurull et al. 2020;Radwan et al. 2012;Siddiqui and Futai 2009). Bacillus thuringiensis (Bt), a gram positive soil bacterium possessing nematicidal crystal proteins, is being used widely to control PPNs. For instance, BtCR371 was evaluated and found effective against a model nematode, Caenorhabditis elegans under in vitro and in vivo conditions (Zuckerman and Dicklow, 1993). Mohammed et al. (2008) studied the biocontrol efficacy of Bt against M. incognita and observed a reduction of egg masses and number of eggs over untreated samples. BtDB27 produced 2 novel protoxins namely, Cry21Fa1 and Cry21Ha1, which exhibited synergistic action towards Caenorhabditis elegans (Iatsenko et al. 2014). Scientists also explained the mechanism of nematode control by Cry proteins. The crystal proteins of different molecular weights are known to enter via stylet of nematodes, cause pores in the esophagus and thereby kill them. Naturally isolated Bt often acts as an effective location-specific control agent. The reason might be the bacterium and the nematode might coevolved in that particular ecosystem. The toxicity of location-specific Bt is always found to be effective and more novel proteins can be expected out of them.
North western zone of Tamil Nadu, India, one of the highly vegetable growing regions of the nation, with severe nematode infestation, was selected for the present study where the crop yield losses due to M. incognita were more than 60%.
In this study, native Bt was isolated to control M. incognita. The efficacy of spore-crystal mixture (SCM) of the native Bt isolates was evaluated under lab and greenhouse conditions.

Isolation, screening and identification of Bacillus thuringiensis
The Bt was isolated from 5 soil samples collected from each of the 15 different blocks of the north western zone of Tamil Nadu state, India, and stained with Coomassie Brilliant Blue (CBB) (0.133% CBB in acetic acid 50%) for identification of crystal morphology as described by (Ramalakshmi and Udayasuriyan 2010). Twenty-four hours old broth culture of crystal positive isolates were prepared as glycerol stocks and stored at − 70°C for further studies. Molecular identification, using 16SrRNA gene amplification by PCR was performed to identify the crystal positive isolates. The phylogenetic relationships of isolates are given in Fig. (S3).

Preparation of spore-crystal mixtures (SCM) and SDS-PAGE
The spore-crystal mixtures were prepared from Bt isolates as described earlier (Ramalakshmi and Udayasuriyan 2010). Five microlitres of SCM were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970).

Screening for nematicidal cry genes by PCR
The total genomic DNA from Bt isolates was extracted, using appropriate methods (Sambrook and Russell 2001). The nematode-specific cry gene primers were used to identify the cry5, cry6, cry14 and cry21 gene families in Bt isolates (Porcar and Juárez-Pérez 2003).
The 25 μl PCR reaction mixture consisted of 1X Taq buffer, 2.5 mM MgCl2, 0.5 μM forward and reverse primers, 0.25 mM dNTP mixtures and 3 U of Taq polymerase (all from Fermentas, USA). PCR condition includes 95°C for 5 min (initial denaturation), 35 cycles of 95°C for 1 min (denaturation), 55°C for 1 min (annealing), 72°C for 1 min (primer extension), followed by 72°C for 10 min (final extension). Amplification was performed in Biorad T100 PCR. Four reference strains of Bt, 4 M1, 4Q1, 4E1 and 4BG1 obtained from Bacillus Genetic Stock Centre (BGSC), the Ohio State University, Columbus, Ohio 43210, were used as a positive control for screening of native Bt isolates by PCR.

Root knot nematode and maintenance of egg masses
The egg masses of root knot nematode (RKN), M. incognita were obtained from the Department of Nematology, TNAU, Coimbatore, and maintained in pots having tomato plants.

In vitro inhibition of M. incognita egg masses by SCM of Bacillus thuringiensis
The Bt isolates were grown in T 3 broth till more than 95% cell lysis. After lysis, the different crystal protein suspensions 0.1, 0.2, 0.3, 0.4 and 0.5% with sterile water were prepared. The egg masses were collected from the pot culture experiment and washed with sterile distilled water before exposure to the crystal protein suspension. Three egg masses were released into each concentration of suspension in 6 mm dia petriplates. The sterile water with egg masses was used as control. The number of hatched J 2 was recorded every 24 h up to 3 days under binocular light microscope (model Olympus); the images were recorded in Cannon camera 20 MP colour CMOS attached to the Olympus microscope. All the treatments were carried out with three replications.

Scanning electron microscope analysis
The virulence of Bt isolates against M. incognita egg masses was confirmed by SEM analysis. The unhatched egg masses of M. incognita were observed under SEM after 3 days incubation with different concentrations of SCM of Bt isolates, reference strains and control. Sample preparation was made by the modified method (Orion et al. 1994;Wergin and Orion 1981). Briefly, the egg samples were fixed at 4°C in closed tubes containing 1.25% glutaraldehyde and 1.25% paraformaldehyde in 0.05 M cacodylate buffer (pH 7.2) for 4 h. The samples were treated serially for 15 min with 50, 70, 90 and 100% ethanol, and butanol. Then the samples are vacuum freeze-dried for 24 h. The samples were coated by carbon grid with platinum coating observed under a SEMFEI-Quanta 250.

In vitro inhibition of J 2 juveniles by SCM of Bacillus thuringiensis
The spore-crystal mixtures from nematicidal positive Bt isolates were further tested against M. incognita (J2) stage. Known concentration of 5 μg/ml of SCM was transferred into glass plates containing 100 nos of surface sterilised infective juveniles (J2). Four replications of each treatment were made to assess the juvenile mortality. The dead juveniles were counted from each treatment up to 48 h. Juveniles not showed any movement upon addition of water were considered as dead ones. Juveniles released in sterile water served as control. The experiments were performed at 30 ± 0.5°C.

Greenhouse experiment
A pot culture experiment was conducted under greenhouse conditions for testing the biocontrol efficacy of Bt against M. incognita. Sterilised cocopeat mixture obtained from Orchard, TNAU, Coimbatore, was prepared and filled in 96 well portrays. Tomato seeds (PKM 2 variety obtained from HC and RI, TNAU, Coimbatore) were planted in portrays and maintained for better establishment. Tomato seedlings after 25 days were transferred to earthen pots of 10 kg capacity which were filled by steam sterilised pot mixture. M. incognita was inoculated at 1 J2/g of soil around the roots of tomato plants by making 3 holes around the plant and covered with sterilised soil. Ten millilitres of spore-crystal mixture of isolates BC and BD along with reference strain (4 M1) were applied near root zone. As a standard check Carbofuran 3G at 1 kg a.i/ha and an untreated control were included. Regular watering was done with tap water passed through 325 mesh sieve. The experiment consisted of 4 treatments with 3 replications in a completely randomised block design. Pots were placed at room temperature of 35 ± 2°C under controlled conditions in the greenhouse.
Observations on shoot and root length, shoot and root weight were recorded after 60 days of planting. Plants were carefully uprooted and observation on gall index, number of females, number of egg masses and number of eggs per egg mass were observed. Population of nematode in soil was assessed by (KOPPENHõFER et al. 1998;Schindler 1961).

Statistical analysis
Data were pooled and analysed in the SPSS 16 software. Duncan's multiple range tests were performed for treatment with spore-crystal mixture. The P value < 0.005 was kept as cutoff value for significance. The LC 50 values were determined by probit analysis using SAS 8.0.

Results and discussion
Screening of nematicidal Bt against M. incognita Fifty native isolates were identified as Bt based on the presence of crystalline inclusions. The types of crystalline inclusion present in 50 isolates are presented in fig. (S1). All the 50 isolates were subjected to PCR for confirmation of nematicidal cry genes, using specific primers and simultaneously the spore-crystal mixtures of all the 50 isolates were run on SDS-PAGE to identify the molecular weight of the cry protein. Crystal protein profiles of 50 isolates were depicted in Fig. (S2). Out of 50 isolates, 14 harboured nematicidal genes and showed amplification of expected size of cry5, cry6, cry14 and cry21 genes either in combination or alone ( Fig. 1 and Table 1). All the 14 isolates showed variation in their molecular weight of the protein (Table 1 and Fig. 2).
Biological control of insect's pests is known to be very old practice (Laznik et al. 2012). Several studies were attempted on Bacillus sp. based nematode control. However, Bt possessing nematicidal cry genes are more efficient in controlling specific nematodes when compared to Bacillus sp. Moreover, native isolates of Bt might harbour novel cry genes for nematode management. Furthermore, studies pertaining to Bt-based nematode control are limited. Iatsenko et al. 2014 extracted 2 novel protoxins viz., Cry21Fa1 and Cry21Ha1, from native Bt against C. elegans. Similarly, the native Bt BRC-XQ12 harbouring CryEa11 was active against pine wood needle nematode, Bursaphelenchus xylophilus (Huang et al. 2018). Guo and co-workers (2008) reported that most of the nematicidal Cry proteins are large proteins (90 to 140 kDa), except Cry6 (Guo et al. 2008). In the present study, 43-135 kDa proteins in native Bt isolates were reported. The two Bt isolates shared the same cry5 amplification profile with~130 kDa protein and had the highest nematicidal activity against pinewood nematode (Wang et al. 2012). The same result was noticed in the present study also. Fourteen isolates harbouring nematicidal cry genes were screened by bioassay against egg masses and 2nd stage juveniles (J2) of M. incognita for identification of potential isolate. The result revealed that 6 isolates were effective in inhibiting the emergence of J2 juveniles from egg masses of M. Incognita (Figs. 3 and 4). All the 6 isolates at 0.5% of SCM inhibited the emergence of J2 juveniles by 100%. Inhibition percentage of 80-90% of J2 juvenile emergence was observed at 0.3-0.4% SCM of all isolates (Fig. 4). The lowest inhibition was noticed in the concentration of 0.1-0.2% among the isolates. Microscopic images confirmed the inhibition of emergence of J2 by spore-crystal mixture of native isolates of Bt when compared to control (Fig. 3.) Similarly, mortality of J2 was observed under microscope and the results are presented in Fig. 5. It was  evident from the figure that the spore-crystal mixturetreated juveniles were dead observed as straight ones, and no movement were noticed even after disturbance with the needle, whereas untreated J2 were found live by their curved and comma-shaped movement. (Fig. 5).

SEM analysis of changes in egg structure
The SEM analysis of egg mass treated with SCM of native isolates of Bt showed that the eggs were embedded in a gelatinous matrix with smooth egg shells, whereas the egg shells were fluffy owing to hatching in untreated samples (Fig. 5). From the SEM results, it is evident that the proteins inhibited the emergence of J2 from egg masses.

LC 50 value
The spore-crystal mixture of 6 isolates were further analysed on J2 of M. incognita and LC 50 values were found out. The result revealed that lethal concentration varied from 0.12 to 0.23 μg of protein (Fig. 6). The isolate BC recorded lowest LC 50 value of 0.12 μg of protein/millilitre, followed by the isolate BD (0.13 μg of protein/millilitre). The highest LC 50 value of 0.23 mg of protein/millilitre was recorded in isolate CD2 (Fig. 6). The 2 isolates with low LC 50 values, namely BD and BC, were selected for greenhouse experiment using host plants. The LC 50 value obtained in this study was much lower than already reported Cry5Ba protein whose LC 50 value was146.05 μg of protein/millilitre (Peng et al.,  2011) (Fig. 7). Similarly, the LC 50 value of this study could be compared to Huang et al. (2018) where in purified Cry1Ea11 had LC 50 of 32.53 and 23.23 μg of protein/millilitre after 24 and 48 h, respectively.

Greenhouse experiments
Biocontrol potential of the 2 native Bt isolates (BC and BD) was evaluated for their effective control of root knot nematode under greenhouse conditions. The results were compared by chemical nematicide, carbofuran and with reference strain 4MI harbouring cry5 nematicidal gene. Parameters such as number of egg masses/5 g root, number of eggs/egg mass, number of adult females/5 g root, final nematode population/kilogram soil and root gall index were evaluated. As shown in Table 2, the 2 native Bt isolates exhibited superior biocontrol potential of root knot nematode over the reference strain and chemical control. The highest percent decreased of the number of egg masses per 5 g of root was observed for the isolate BC to a tune of 79.35, followed by BD (76.09%). The percent decrease of reference strain 4MI was 66.66 and the lowest percent decrease of egg masses was noticed in the treatment with carbofuran with a value of 31.50. Similarly, the results on number of female population per 5 g of root also followed the same trend. The highest percent reduction of number of female population per 5 g of root was observed for isolate BC (68.14), followed by the isolate BD with a percent reduction of 64.53. The percent reduction of reference strain 4MI was 52.72%. Reduction percent of 25.45 was observed for treatment with chemicals. Final nematode population per kilogram of soil was estimated and the isolate BD had the lowest number nematode population with 73.15% decreased over control, followed by isolate BC, which had percent decrease of 71.96%.The lowest values of percent reduction (40.92) in final nematode population were recorded at the treatment with carbofuran. Root gall index was found to be low (1.66) in the native isolates of Bt and high of 3.33 was noticed in reference strain 4MI and in treatment with carbofuran. Bio efficacy of cry6 proteins on nematode M. hapla in pot culture experiments with tomato host plant was evaluated by Yu et al. (2015). The results indicated that egg masses were reduced to 62.3

Conclusion
The present study demonstrated biocontrol efficiency of 2 native isolates of Bt for efficient control of root knot nematode. The study also investigated the changes in egg mass treated with spore-crystal mixture of native isolates of Bt by SEM analysis. The LC 50 values of the 2 native isolates were much lower than other isolates. Molecular weight of 135 kDa and nematicidal cry5 gene(s) were noticed in these isolates. Pot culture experiment showed that the 2 isolates were better than reference strain and chemical in controlling egg mass, nematode population and root gall index of tomato plants. The crystal proteins of native isolates of Bt, BC and BD, could be potential candidates for a biocontrol agents in controlling root knot nematode.
Additional file 1: Fig. S1. Types of crystalline inclusions in native isolates of B. thuringiensis. Fig. S2. Crystal protein profiles of native isolates of B. thuringiensis. Fig. S3. Phylogenetic relationship of native isolates of Bacillus thuringiensis