3.1 Morphological pathogen identification
On V8 tomato juice agar, the fungus forms the circular, slightly petaloid colonies with compact hyphae. The structure of sporangium varied in different isolates from broadly ovoid, obpyriform to elongate, and nonpiliated. The identity of morphological characters of the pathogen was done as described in the manual on phytophthora (Gallegly and Hong, 2008).
3.2 Isolation and biochemical characterization of rhizobacterial isolates
Isolated seven (S-2, S-4, S-18, S-28, S-30, S-32, and S-34) antagonistic rhizobacteria on different respective media were assessed. The morphologically and biochemical characteristics of rhizobacteria given at (Table 2). Based on these tests, the isolates were tentatively placed into three genera: Bacillus, Pseudomonas, and Rhizobium. The outcomes were coinciding with previous investigations documented by new (Kumar et al. 2010).
3.3.1 In vitro screening for antagonistic rhizobacteria against Phytophthora dreschsleri
The percent growth inhibition was found to range between 17.8-39.6% in dual culture against P. drechsleri as compared to control. Out of 7 antagonists S-18 (39.6%) isolate showed the highest mycelial growth inhibition against the test fungus as compared to others (Table 3). Similar studies were conducted by (Anjum et al. 2019) reported the bioactivity of biocontrol agents against Phytophthora drechsleri infection was studied in vitro. T. asperellum (47.3%) showed the highest growth rate of Phytophthora drechsleri in potato dextrose.
3.3.2 Quantitative evaluation of antagonism
In vitro, broth-based dual cultures offer a better method for the evaluation of antagonistic efficiency of the biocontrol agents. The maximum percent biomass inhibition on a dry weight basis was recorded after 5 days of incubation by S-2 (79.3%) followed by isolates S-18 (71.4%) and were highest as compared to others (Table 3). Mathur and Mathur, (2021) reported similar results of inhibition fungus biomass in broth-based dual culture was revealed by 11 antagonists in chickpea.
3.3.3 Antagonism of siderophore producing bacteria against P. drechsleri
All seven isolates showed a distinct orange halo on CAS plates indicating siderophore production. The highest amount of catechol and hydroxamate type siderophore was produced by S-18 (78.2 μg/ml-1 and 70.8 μg/ml-1) respectively as compared to other isolates after 6 days of incubation (Table 3). Siderophore production from rhizobacteria has been reported by several researchers, Gupta et al. (2020) found that under the iron-deficient condition all the isolated rhizobacteria inhibited the vegetative growth of P. drechsleri and also benefited the growth of heterologous microbes in the soil.
3.3.4 Characterization of antagonistic rhizobacteria for Multifarious PGP Traits
All the seven antagonistic rhizobacteria solubilized inorganic phosphate on pikovskya’s agar, after 24 hrs. of incubation. The maximum solubilization index was showed by S-18 (21.5) as compared to others (Table 3). IAA as evidenced by the development of pink color without the addition of tryptophan into the culture media. Rhizobacteria S-2 (10.8) produced maximum IAA without the addition of tryptophan as compared to other rhizoisolates (Table 3). A similar pattern of IAA production and phosphate solubilization has been reported by (Tewari et al. 2021). All seven strains of rhizobacteria produced ammonia and HCN which is indicated by the change of filter paper color from yellow to brown and reddish-brown. Aggarwal et al. (2010) reported the role of HCN and ammonia in inhibiting the growth of P. drechsleri. A marked variation in the ability to produce ammonia was observed amongst the isolates indicated by the intensity of color developed.
3.3.5 Cell wall degrading enzymes produced by rhizobacteria
All the antagonistic isolates produced cellulase and protease on CMC and skim milk agar media, respectively (Table 3). Clear halo on skim milk agar medium with a diameter ranging from 1.96 to 5.02 cm for protease enzyme production. It has been demonstrated that cellulase and protease synthesized by rhizobacteria digest and lyse the mycelium of P. dreschsleri (Panth et al. 2020).
3.4 Scanning electron microscopic (SEM) observation of post-interaction abnormalities between rhizoisolates and test fungal mycelia
Scanning electron micrographs depicted the morphological abnormalities in the hyphae of P. dreschsleri obtained from the zone of interaction during dual culture. Loss of structural integrity of conidia of P. dreschsleri, hyphal perforations, and swelling were clearly observed (Fig. 1). The SEM results complied with Kumar et al. (2010) reported that allelochemicals (volatile and non-volatile) HCN, antibiotics, and enzymes produced by antagonistic rhizobacteria resulting in the lysis of mycelial structure and hence curbing the growth of Phytophthora dreschsleri.
3.5 Biocompatibility of potential microbial consortium in pigeonpea
Four potential antagonistic rhizoisolates (S-2, S-4, S-18, and S-30) were found compatible without producing any zone of inhibition on tryptone soy agar plate assay. These isolates with positive compatibility were assessed spectrophotometrically for mutual interaction in Luria broth. Dual inoculation enhanced the growth as compared to monoculture treatment. The highest growth in terms of optical density (OD at 600nm) was recorded with LAR06+ S-2 treatment (1.31) followed by LAR06+ S-18 (1.05), LAR06+S-4 (0.95), and S-18 (0.91) as compared to respective single inoculants S-2, S-18, S-4 and S-30 (0.82 and 0.81, 0.74 and 0.64 respectively) at 9th day of incubation. These combinations showed a sustained population of bacterial growth at different incubation periods. Subramanian et al. (2015) reported the positive interaction of rhizobacteria done on soybean digest agar disc plate with pre-seeded rhizobacteria in in-vitro conditions. This protocooperation is due to the release of non-reactive metabolites during co-inoculation of rhizobacteria
3.6.1 In vitro effect of antagonistic rhizobacteria on the incidence of Phytophthora blight under pathogen stress conditions
Antagonists S-2, S-4, S-18 and S-30 further authenticated in vitro tests, providing a strong confirmation efficiency of these isolates in suppressing Phytophthora blight in pigeonpea. Maximum Seed vigor Index (SVI) shown by dual inoculation of Rhizobium with S-2 (8678.67) followed by S-18 (8518.51), S-4 (8057.17), S-30 (8262.68) as compared to recommended Rhizobium alone under pathogen stress conditions (Fig. 2). Treatment with bio antagonist S-18 alone, showed the highest SVI (7907.72) as compared to others and recommended Rhizobium (7250.4). In the case of negative control, SVI (4770.44) was recorded.
Similar findings have been reported by Anjum et al. (2019) revealed that reduction in Phytophthora blight incidence was due to their synergistic nitrogen fixation and antagonistic effect treatment with PGPR strains and improved the germination rate of seeds as compared to others.
3.6.2 In vivo effects of compatible rhizobacteria on incidence of blight and symbiotic growth parameters
Based on strong in vitro antagonistic PGP activities, strains S-2, S-4, S-18 and S-30 were selected for in vivo experimentation. Percentage of incidence of blight was found to range between (27.7-41.6%) as compared to others. Co-inoculation of seeds with recommended rhizobium enhanced various plant growth parameters (plant height, number of nodules, nodule dry weight, number of pods, and number of seeds compared to control (Table 4). After 30 DAS, Chlorophyll content was enhanced in dual inoculation of S-2 (2.73 mg/g) and Leghemoglobin in dual inoculation S-18 (1.65 mg/g). Similar trend was observed in both chlorophyll leghemoglobin content after 60 DAS (Table 3). These results were coinciding with Bhowmik and Das, (2018) reported that co-inoculation of rhizobacteria with a recommended dose of biofertilizer significantly improved plant growth parameters as compared to un-inoculated control treatment in pigeonpea.
Post-harvest analyses indicated maximum grain yield was found in the combination treatment of rhizobium and S-18 (1091 kg/ha), followed by the other combination S-2 (1086 kg/ha) as compared to control (1037 kg/ha) and recommended rhizobium (LAR-06) ((1062 kg/ha). The protein content of seeds in pigeonpea (Table 4) showed that co-inoculation of S-2 + Rhizobium exhibited maximum protein content (4.5%) compared to control (3.6%) and Rhizobium alone (3.9%). A similar trend was observed in other treatments too. Earlier also, Tewari et al. (2020) reported that combined inoculation of biofertilizers increased protein content and seed yield by 1.2- and 2.2-fold increments respectively in comparison with control treatment in pigeonpea.