Microbial preparation
Trichoderma and Bacillus isolates were obtained from the rice rhizosphere of different regions of Telangana State, using standard serial dilution method. Trichoderma specific medium (TSM) and peptone yeast extract medium (PYEM) were used as selective medium for isolation and purification of fungal and bacterial antagonists, respectively (Cavaglieri et al. 2004). Key morphological and microscopic characters were used for identification of Trichoderma isolates (Gams and Bissett 1998), and Bacillus isolates (Sneath 1986). Scanning electron microscopy (SEM) was done as described by Bozzola and Russell (1999). Samples were fixed in 2.5% glutaraldehyde for 24 h at 4 °C, followed by 2% aqueous osmium tetroxide for 4 h. After dehydration in series of graded alcohols, the samples were mounted and observed in SEM (JEOL JSM-5600).
Pathogens
R. solani, collected from Plant Pathology Laboratory, Hyderabad (Yugander et al. 2015) and Xoo (Accession number: MZ158566), were used for the experimental study. Their pathogenicity was proved according to Koch’s postulates on TN1 cultivar.
Genomic DNA from bacterial and fungal antagonists
Bacillus isolates, viz., B2, B3 and B4, and Trichoderma isolate T7 were cultured in Luria Bertani (LB) and potato dextrose broth (PDB), respectively. Bacillus cells were harvested by centrifuging at 8000 × g for 10–15 min and fresh mycelial mats of Trichoderma were used for the isolation and purification of genomic DNA. NucleoSpin® genomic DNA isolation and purification kit (Machery-Nagel) were used as per the manufacturer’s instructions. The isolated DNA was quantified using both nanodrop spectrophotometer (Thermofischer) and 0.8% agarose gel pre-stained with ethidium bromide.
PCR amplification
Bacillus isolates were identified using amplification of 16S rRNA (~ 1500 bp) by universal primer pairs 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′). The polymerase chain reaction (PCR) mixture consists of 30–50 ng DNA template, 2.5 mM dNTP mix, 0.5 μM primers, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 5U Taq polymerase. Amplification conditions were as follows: denaturation for 2 min. at 94 °C; 30 cycles of amplification; 40 s. denaturation at 94 °C; 45 s. annealing at 54 °C; 1 min. extension at 72 °C followed by final extension 10 min. at 72 °C. In case of Trichoderma, 18S rRNA gene (partial sequence), internal transcribed spacer 1, 5.8S rRNA gene internal transcribed spacer 2 (complete sequence) and 28S rRNA gene (partial sequence) were amplified by using the primer combinations ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and LR3R (5′-GGTCCGTGTTTCAAGAC-3′) with fragment size of ~ 1200 bp; using the following conditions, viz., 1 min initial denaturation at 94 °C; 30 cycles of 1 min. denaturation at 94 °C; 1 min primer annealing at 50 °C; 90 s. extension at 74 °C and a final extension period of 7 min. at 74 °C. Size of the PCR amplicons were analysed on 1% agarose gel.
Purification of PCR product and sequencing
PCR amplicon fragments were purified using Promega Wizard® SV Gel and PCR Clean-Up System kit, and the purified products of about concentration 50–100 ng/ul were sequenced using Sangers sequencing method. The nucleotide sequences were submitted to NCBI GenBank database and NCBI accession numbers were obtained. These sequences were further aligned and compared with the sequences of Bacillus and Trichoderma species available with NCBI. NCBI BLAST homology searches of the respective gene sequences which performed to assess homologous sequences available in NCBI. Computational analysis of DNA sequence data, sequence editing, multisequence alignment and molecular phylogeny were performed using EMBL-EBI.
In vitro inhibition of Xoo by Trichoderma and Bacillus sp.
Inhibitory efficiency of Trichoderma on Xoo was analysed using dual culture competition-suppression assay (Sinclair and Dingra 2017). A loop of individual isolates Trichoderma mycelia along with spore and Xoo was placed exactly opposite to each other on a Petri plate maintaining equidistance from centre, containing modified Wakimoto media (MWM). Control plate was maintained by inoculating Xoo alone. Radial growth of Xoo was measured in treated as well as control plate and percent inhibition was calculated by the formula (Gangwar and Sinha 2010)
$${\text{Per}}\,{\text{cent}}\,{\text{inhibition}}\,\left( \% \right) = \frac{C - T}{C} \times 100$$
where C = colony growth in centimetre in control plate, T = colony growth in centimetre in treated plate.
Efficacy of f Bacillus on Xoo was established by slightly modifying the agar well diffusion technique (Sinclair and Dingra 2017). Four diffusion wells opposite sides to each other on PYEM plate containing one day old colony of Xoo were added with the broth of Bacillus. The suppression was observed as overgrowth of Bacillus isolates over Xoo. Quantitative estimation of Bacillus efficiency against Xoo was obtained by measuring the optical density (OD600nm) of broth containing Bacillus and Xoo. Bacillus isolates were grown on LB broth at 37 °C in rotatory shaker at 160 rpm for 48 h. Culture filtrates were obtained by centrifugation of broth containing culture @16,000 rpm for 10 min and filtered sterilized twice through 0.22 µm filter. Different concentrations (10, 25, 50, 75 and 100%) of culture filtrate were tested to finalize the lethal concentration for pathogen. In 20 ml of broth containing different concentrations of Bacillus filtrates, a 100 µl of Xoo broth culture was added and kept for incubation at 28 ± 2° for 48 h. Negative control was maintained with Xoo alone in the LB broth (Elshakh et al. 2016). OD600nm was measured after 72 h of incubation.
In vitro inhibition of R. solani by Trichoderma and Bacillus sp.
Antagonistic potential of both the antagonists against R. solani was studied using dual plate competition assay (Marzano et al. 2013). Five mm mycelial discs of both Trichoderma isolates and R. solani were placed opposite to each other equidistance from centre of a Petri plate containing PDA. The radial growth of R. solani with Trichoderma isolates was recorded along with control. The efficiency of Bacillus isolates on R. solani was estimated in the similar method as done for Trichoderma sp. with slight modification to make up for the differences in the rate of growth of the Bacillus when compared to R. solani (Huang et al. 2012). R. solani was grown for 24 h at a corner of a plate with NA, followed by streaking a loop of individual Bacillus at the opposite edge. Plates with pathogen alone served as control. The plates were incubated at room temperature for 2 days, and thereafter, the radial growth of R. solani was measured and recorded.
Percentage inhibition was calculated for both the protocol using above-mentioned formula (Gangwar and Sinha 2010).
Compatibility of consortia mixtures
Isolates of Trichoderma and Bacillus were individually tested against each other for their compatibility (Siddiqui and Shaukat, 2003). Different isolates of Bacillus grown separately on PYEM plates were streaked perpendicular to each other on a fresh plate containing 20 ml PYEM. Similarly, 5-mm disc from 7-day-old cultures of the isolates of Trichoderma grown separately was placed in a fresh plate containing PDA, maintaining equal distance with each other. Both the consortia were incubated at 27 ± 2 °C (Fig. 1). Zone of inhibition, if any formed was measured as the incompatibility against the two antagonists. The test was replicated multiple times.
Mass multiplication of antagonists for seed and soil application
Antagonists slurry made from 4-day-old cultures was suspended in sterile water to make a uniform suspension and used for seed treatments. For soil application, the antagonists were cultured in their respective broths. About 100 ml of broth (× 106 and × 104 CFU/ml, respectively, for Bacillus and Trichoderma) was mixed with 1 kg of powdered rice bran and carboxymethyl cellulose (CMC) @2 g/Kg. This mixture was used @100 g per pot as per the treatment schedule. Consortia of compatible microbes were formulated as above by mixing both the antagonists in their respective seed and soil formulations in equal proportions. In all the above formulations, the inoculum density of 2.14 × 107/ml (OD600nm ~ 1.0) for Trichoderma and 1.08 × 108/ml (OD600nm ~ 0.4) for Bacillus were maintained in the liquid suspension (Beal et al. 2020).
In vivo screening of isolated antagonists against Xoo and R. solani in net-house
The experiments were conducted under controlled conditions in net-house. The seeds of susceptible rice cultivar TN were soaked in sterile water for 24 h and after draining the excess water, were mixed with the antagonist suspension (@10 ml/Kg seeds) and incubated for 12 h. Treated seeds were then kept on blotting paper to test the germination percentage. About 25 seeds from the initial lot were then placed in nursery trays and monitored for 10 days to calculate morphological parameters like seedling length, seedling dry weight, vigour index-1 (germination % X seedling length) and vigour index-2 (germination % X seedling dry weight). About 25-day-old seedlings from the nursery were transplanted in pots of size 30 × 25 cm with about 5–7 kg of soil. Thirty days after transplanting (DAT), antagonists were applied to soil @ 10 g/Kg of soil. The pathogens were inoculated at 40 DAT. Xoo was inoculated by leaf clipping method by diluting with 10 mM MgCl2 and maintaining the 0.5 OD600 (Ke et al. 2017). Inoculation of R. solani was done by placing about 0.5 mg of sclerotia in rice sheath and covering it by moist cotton swab (Singh et al. 2002). The experiments were repeated during 2 seasons with 3 replications under controlled conditions in net-house and the values were averaged.
Statistical analysis
The experiments were conducted in completely randomized design (CRD) and data were subjected for one-way analysis of variance (ANOVA), using post hoc test with Duncan’s multiple range test (DMRT) at 5% (P ≤ 0.05) significance level in SPSS 20.0.1 version. Correlation analysis and graphs were made using Microsoft excel 2019. Three replications were maintained during each experiment.
