Survey for the incidence of natural entomopathogens in S. frugiperda
Surveys were conducted to explore the natural occurrence of entomopathogens on S. frugiperdaat major maize growing districts of Tamil Nadu during 2019-2022 in farmers’ field. A sum of 24 bacterial cadavers and 55 fungal cadavers of S. frugiperda were collected from various farmers field of Perambalur, Dindigul, Tiruchirappalli and Madurai districts (Table 1). The bacterial cadavers collected, ranged from 2.0 to 12.0 per cent and that of fungal cadaver ranged from 4.0 to 32.0 per cent (Table 2).
Collection and isolation of B.thuringiensis from diseased fall armyworm cadavers
Out of 24 dead cadavers of S. frugiperda collected from four different districts of Tamil Nadu (Perambalur, Dindigul, Tiruchirapalli and Madurai), 15 screened for the presence ofB.thuringiensis. The 15 isolates yielded and named as TRY 1, TRY2, TRY3, TRY 4, TRY 5, TRY 6, TRY 7, TRY 8, TRY 9, TRY 10, TRY 11, TRY 12, TRY 13, TRY 14 and TRY 15. All the isolates were cultured on B.t selective medium (T3 media) for the cultural characterization and confirmation of B. thuringiensis. On post overnight incubation, all the isolates produced milky white colonies. The shape of each colonies were circular or irregular with raised appearances having serrate or undulated margins.Surveys were conducted to explore the natural occurrence of entomopathogens on fall armyworm at four districts of Tamil Nadu during 2019-20 in farmers’ field. Totally 24 bacterial dead cadavers and 55 fungal dead cadavers of S. frugiperda were collected from Perambalur, Dindigul, Tiruchirappalli and Madurai Districts (Fig. 2). Theunis et al. (1998) collected about 801 samples from rice grain dust, soil, rice field insects (dead and live) for isolation of Bacillus thuringiensisin Philippines and yielded about 3950 isolates of B.tin total. Asokan (2007) isolated B. thuringiensisfrom soil, leaf, seed dust and dead insect cadavers in Karnataka and yielded about 33 isolates of B.t. Anandhi et al. (2013) isolated and identified native B. thuringiensisfrom diverse habitats like soil, warehouse grains and insect cadavers in Allahabad. Similarly, Saravanan and Gujar (2006) isolated B. thuringiensisfrom warehouse samples and insect cadavers. Jaber et al. (2016) have isolated and identified 42 fungal strains from 17 different arthropod cadavers. Sun and Liu (2008) have isolated 6 species of insect pathogenic fungi from the forest soil samples. Wakil et al. (2014) evaluated and isolated over 195 isolates of 24 different fungal species from 25,720 nos. of stored pests in Punjab and Pakistan. Strasser (2001) had reported that Beauveriabassiana occurs naturally in more than 700 species of arthropod hosts.
Morphological characterization of B.t isolates
A total of 345 colonies were isolated based on the physical appearances like colony colour, colony shape, colony margin, colony elevation and colony texture. Colonies of all isolates produced creamy milky white to yellowish white colonies with irregular or regular shapes, entire or serrate or undulated margins, raised or flat elevation and rough or smooth texture. Isolates such as TRY 1, TRY 3, TRY 6, TRY 8, TRY 10, TRY 11, TRY 14 and TRY 15 produced creamy white colonies while TRY 2, TRY 5, TRY 9 and TRY 12 produced milky white colonies. Yellowish white colonies were observed in TRY 4, TRY 7 and TRY 13. Irregular shape was observed in TRY 1, TRY 2, TRY 3, TRY 5, TRY 7, TRY 8, TRY 10, TRY 13 and TRY 14 whereas regular shape was observed in TRY 4, TRY 6, TRY 9, TRY 11, TRY 12 and TRY 15. Entire type of margin was present in TRY 1, TRY 3, TRY 11 and TRY 13; while serrate type was observed in TRY 2, TRY 5, TRY 6, TRY 9, TRY 10, TRY 12 and TRY 15 whereas undulated type was present in TRY 4, TRY 7, TRY 8 and TRY 14. Raised elevation was observed in TRY 1, TRY 3, TRY 4, TRY 6, TRY 7, TRY 8, TRY 9, TRY 11, TRY 12, TRY 13 and TRY 14; whereas flat surface was present in TRY 2, TRY 5, TRY 10 and TRY 15. Isolates TRY 1, TRY 3, TRY 4, TRY 6, TRY 8, TRY 10, TRY 11 and TRY 14 have smooth texture while isolates TRY 2, TRY 5, TRY 7, TRY 9, TRY 12, TRY 13 and TRY 15 showed rough texture (Table 3).Out of 24 dead cadavers collected from four different districts of Tamil Nadu (Perambalur, Dindigul, Tiruchirapalli and Madurai), 15 isolates were screened for the presence of Bacillus thuringiensis. On post overnight incubation, all the isolates produced milky white colonies. The shape of each colonies was circular or irregular with raised appearances having serrate or undulated margins (Plate 6). In the present investigation, out of 345 colonies, 205 colonies were morphologically similar to Bacillus and 66 colonies produced crystal proteins (Plate 7). Distribution frequency ofB. thuringiensis in dead cadavers of S.frugiperda was 0.17- 0.43 per cent. Occurrence of B. thuringiensis was highest in isolate TRY 1 (0.43 %) followed by isolate TRY 6 (0.40 %) and lowest frequency was observed in isolates TRY 3 and TRY 14 (0.17 %) (Fig. 9). The mean B.t index was 0.32 per cent.The mean of B.t index in the present study was relatively lower than other research findings which was reported by Martin and Travers (1989) and Obeidat et al. (2004). The concentration of protein of different isolates were TRY 6 (17.49 mg), TRY 3 (14.61 mg), TRY 11 (13.26 mg), TRY 1 (10.72 mg), TRY 8 (9.16 mg) and TRY 14 (7.57 mg) (Fig. 10).Alves and Lemos (2000)has estimated the crystal protein content of Bacillus thuringiensis var. israelensisand obtained 5.7 to 34 per cent of protein in different samples analyzed.Correspondingly Barreto et al. (1999) estimated the protein content of
B. thuringiensis strains and analyzed their mortality against S. frugiperda.
Colonies of all the isolates were creamy white/milky white/yellowish white in color with regular/irregular shape, entire/serrate/undulated margin, raised/flat elevation and smooth/rough in texture. The findings of the present study was similar to the findings of Rampersad and Ammons (2005) and Renganathan et al. (2011), as they observed similar colony morphologies like creamy white to off white color with smooth edges and flat to slightly raised elevation in their studies. The results are in line with the findings of Chatterjee et al. (2007) who reported white, flat, and undulate colonies were observed in his studies. Similar colonial morphology results were also announced by Mukhija and Khanna (2018)
Biochemical characterization of B.t isolates
Bacillus sp. is a gram positive bacteria, which produces rod shaped violet colored cells. All the 15 isolates were gram positive. Isolates TRY 2, TRY 5, TRY 7, TRY 12 and TRY 13 were non-motile while the other isolates were motile. Isolates such as TRY 2, TRY 5, TRY 10 and TRY 15 showed negative result to methyl red (MR) test whereas the other isolates showed positive results. For VogesProskauer (VP) test, except isolate such as TRY 2, TRY 5, TRY 7, TRY 10, TRY 13 and TRY 15 showed positive results, while remaining isolates showed negative results for VP test. For catalase test, TRY 2, TRY 4, TRY 7, TRY 10 and TRY 12 isolates showed negative, while the other isolates showed positive results. Isolates viz., TRY 4, TRY 9, TRY 13 and TRY 15 showed negative result for starch hydrolysis test, whereas the other isolates showed positive results (Table 4).All the 15 isolates produced rod shaped, violet color cells which specified the presence of gram positive Bacillusspecies and similar result was reported by Das et al. (2015).Biochemical tests were done for 15 strains and one reference strain B.t-HD1. Isolates TRY 2, TRY 5, TRY 7, TRY 12 and TRY 13 were non-motile while the other isolates were motile. Isolates such as TRY 2, TRY 5, TRY 10 and TRY 15 showed negative result to methyl red (MR) test whereas the other isolates showed positive results. For VogesProskauer (VP) test, except isolate such as TRY 2, TRY 5, TRY 7, TRY 10, TRY 13 and TRY 15 showed positive results, while remaining isolates showed negative results for VP test. For catalase test, TRY 2, TRY 4, TRY 7, TRY 10 and TRY 12 isolates showed negative, while the other isolates showed positive results (Plate 8). Isolates viz., TRY 4, TRY 9, TRY 13 and TRY 15 showed negative result for starch hydrolysis test whereas the other isolates showed positive results.Similar results was observed by Gorashi et al. (2014); Das et al. (2015) and Rajashekhar et al. (2017) who reported that all strains showed positive reaction for catalase and showed negative results for VogesProskauer test. El-Kersh et al. (2016) reported that isolated B. thuringiensis strains showed 90% motile activities.
Crystal Protein Profiles of Bacillus thuringiensis Isolates
From the selected isolates only 6 isolates produced spore crystals viz., TRY 1, TRY 3, TRY 6, TRY 8, TRY 11 and TRY 14, producing a total of 66 colonies out of 345 colonies. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to compare protein profiles of B. thuringiensis isolates. The results of the protein profiling analysis of B.tisolates revealed that the 6 isolates such as TRY 1, TRY 3, TRY 6, TRY 8, TRY 11 and TRY 14 showedprotein bands around 60-75 kDawhile HD-1 showed protein bands around 130kDa. These proteins belonged to Cry2 protoxin and their activation products. The isolate kept as standard, B. thuringiensissubsp. kurstaki, (HD-1) had a protein profile of 130 kDa which settled their specificity for Lepidoptera. Molecular weight of standard Cry1 class proteins was approximately 130-140 kDa, whereas standard Cry2 and Cry3 protein groups, which are act2wive against Lepidoptera and Coleoptera, will have a weight of 65-70 kDa.
Estimation of B.t index and Protein
Distribution frequency of B. thuringiensis in dead cadavers of S.frugiperdaranged from 0.17 to 0.43 per cent. Occurrence of B. thuringiensis was highest on isolate TRY 1 (0.43 %) followed by isolate TRY 6 (0.40 %) while lowest frequency was observed in isolates TRY 3 and TRY 14 (0.17 %). The mean Bt index was 0.32 per cent. Six isolates and reference strain HD1 were subjected to protein estimation. Protein concentration was estimated by Lowry’s method. The concentration of protein of different isolates were TRY 6 (17.49 mg), TRY 3 (14.61 mg), TRY 11 (13.26 mg), TRY 1 (10.72 mg), TRY 8 (9.16 mg) and TRY 14 (7.57 mg). Reference strain Bt-HD1 had 16.63 mg protein concentration (Table 5).
Molecular characterization and identification of Bacillus sp. isolates
Six different isolates of B.t producing crystalliferous proteins were taken for molecular characterization and identification. All the six samples viz., TRY 1, TRY 3, TRY 6, TRY 8, TRY 11 and TRY 14 got amplified using forward primer 27F
(5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3')and sequenced. Results of PCR amplification of 16S rRNA from the selected samples are provided below (Plate1). The sequences of the 16S rRNA from six samples such as: TRY 1, 3, 6,8,11,14.The results obtained are similar to the findings of Rajashekhar et al. (2017), who used 16S rRNA gene sequencing for identification of Bacillus thuringiensisobtained from the cadavers of Spodopteralitura and Aphis craccivora. Similarly,Gorashi et al. (2014) has identified 12 Bacillus thuringiensisfrom the various samples like infested grains, soil samples and air of Sudan using the 16S rRNA genome sequencing method.
In-silicoAnalysis: Species confirmation was done from the sequence information of 16S rRNA of bacterial culture through BLAST of NCBI. Phylogenetic tree was constructed for the six B. thuringiensisstrains and three sequences were retrieved from GenBank using Maximum likelihood with 1000 iterations. The sequences formed two clusters. Strains from TRY 1 and TRY 8 ofPerambalur and TRY 3 and TRY 14 of Dindigul formed a single clade with the retrieved sequences NR114581 and NR112780. The TRY 1 ofPerambalur and TRY 11 of Dindigul were similar to NR043403.
Pathogenicity of B.t isolates against S. frugiperda
The pathogenicity potential of crystal proteins of different B.t isolates against S. frugiperda as assessed by a preliminary bioassay. The results revealed that the collected B.tisolates differed greatly in their toxicity to the second instar larvae of S. frugiperdaand the mortality ranged from 20 to 47.5 per cent as compared to 10 per cent in control. The mortality of S. frugiperdagradually increased in all the isolates. Among the B.t isolates, TRY 3 caused maximum mortality of 47.5±4.79 per cent on the seventh day, followed by TRY 11 and TRY 6 which produced 45±6.45 and 45±2.89 per cent mortality on the seventh day. In contrast, the least mortality was caused by TRY 1 as 20±4.08 per cent on the seventh day. The reference strain, HD-1 showed the highest mortality of 80±4.08 per cent on the seventh day. The per cent reduction in weight of the surviving larva over the control after the seventh day was observed as minimum in the HD-1 reference strain (27.32 %), followed by the isolate TRY 11 (46.38 %) and the maximum was observed in the isolate TRY 6 (64.89 %) (Table 6).Bohorova et al. (1997) had reported that cry1D and cry1F gene of B.t produced highest mortality against S. frugiperdalarvae. Jara et al. (2006) reported thatB.t isolates obtained from phylloplane and soil of maizeproduced mortality against S. frugiperda. The susceptibility of S. frugiperdadiffers with the different strains of B.t. The most toxic strain against S. frugiperdawereCIBCM-166, S811, IB412, and LBIT27 and 147-5501(Monnerat et al., 2006). dos Santos et al. (2009)reported that out of 100 strains, seven B.tstrains were screened which showed above 70 per cent mortality against S. eridania, S. cosmioides and S. frugiperda. BR9, BR37, BR45 S608 and S1905 strains were toxic against S. frugiperda larvae. del Valle Loto et al. (2019) studied the efficacy of native strains B.t RT and B.tk HD1 on the third instar larvae of S. frugiperda. The native B.t RT strain showed higher toxicity of 71 per cent compared to the HD 1 strain which produced 52 per cent mortality.