​Induction of Systemic Resistance against Phytophthora Blight in Pigeonpea Through the Interaction of Plant Growth Promoting Rhizobacteria: In vivo and in vitro Study

Current soil management strategies are mainly dependent on inorganic chemical-based fertilizers, which cause a serious threat to human health as well as the environment. The use of PGPR in agriculture is steadily increasing and it offers an appealing alternative to artificial fertilizers and pesticides. The beneficial microbes in biofertilizer have transformed agriculture because it encourages sustainable crop production.
       
Pigeonpea [Cajanus cajan (L.) Millsp.] is a versatile and profitable legume crop with several applications as food, fodder and fuel. With 20 to 22% protein, it has been identified as a rich source of protein (Saxena et al., 2002) particularly in underdeveloped nations where a large population relies on vegetarian diet. Phytophthora blight of pigeonpea caused by Phytophthora drechsleri Tucker f. sp. cajani is a soil-borne disease that is difficult to manage. Phytophthora  drechsleri causes seedlings to die suddenly as in damping-off disease. Its outbreak in the Deccan Plateau region of India was reported with 11.0-31.5% disease incidence (Naik et al., 2020). In contrast with chemical fertilizers, plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria which have capabilities to act as biocontrol agents against soil-borne pathogens. Therefore, co-inoculation with antagonistic efficient strains of Rhizobium with plant growth promoting rhizobacteria (PGPR) shows effective plant growth promotion and resistance to disease. The antagonistic potential of PGPR is due to antifungal plant growth-promoting traits i.e., siderophores having the efficiency to chelate iron from soil-borne pathogens in soil, synthesis of volatile antifungal metabolites such as ammonia, aldehydes and ketones (Attia et al., 2020).
               
The present study was undertaken to isolate rhizobacteria from pigeonpea rhizosphere with potential antagonistic activity to control Phytophthora blight disease and the effect of symbiotic efficiency of Rhizobium with isolated PGP traits under greenhouse pot and field conditions.

Isolation and characterization of rhizobacteria
 
Phytophthora drechsleri was isolated from the infected main stem of pigeonpea as per the method given by (Sosa et al., 2015) and pure culture was maintained on V8 juice agar slants. All the isolates were isolated from 12 different locations of Punjab by the standard isolation methods given by (Kumar et al., 2010).
       
Morphological, physiological and biochemical characteristics of isolated rhizobacteria were determined as per the standard techniques given by (Cappuccino and Sherman, 1992; Holt et al., 1994).
 
In vitro antagonistic activity of rhizoisolates and effect of culture filtrates against Phytophthora drechsleri 
 
Antagonistic properties of rhizobacterial isolates were tested against Phytophthora drechsleri on V8 juice agar plates using the dual culture technique (Sharma et al., 2006). Radial growth and percentage growth inhibition were calculated using the formulae: 
 
Where
r = Radius of the fungal colony opposite the bacterial colony.  R = Maximum radius of the fungal colony in the absence of the bacterial colony.
       
Growth inhibition of Phytophthora drechsleri by culture filtrates was recorded as per the method given by (Kumar et al., 2010).
 
Characterization of antagonistic rhizobacteria for Multifarious PGP traits 
 
Siderophore production of isolates was done both qualitatively and quantitatively as per the method described by (Schwyn and Neilands, 1987) and (Arnow, 1937) respectively. Hydroxamate siderphores were evaluated by the method of (Csaky, 1948). Characterization of isolates for the production of IAA was determined as per the method given by (Bakker and Schippers, 1987). Qualitative and quantitative phosphate solubilizing activity was measured according to the method described by (Jackson, 1973) and (Nautiyal, 1999) (incubated at 28±2°C for 15 days). Zinc solubilization efficiency using Zinc Oxide (ZnO) supplemented in Tris-minimal medium was assayed (incubated in dark at 28±2°C for 7 days) according to (Batool et al., 2021). The selected antagonistic bacterial isolates were also characterized for indirect multifunctional PGP traits, production of volatile antifungal metabolite Hydrogen Cyanide (HCN) production (Bakker and Schippers, 1987), production of ammonia in peptone water (Cappuccino and Sherman, 1992). Bacterial strains were assayed for the production of hydrolytic enzymes such as chitinase (El-Katatany et al., 2003). cellulase (β-1-4-gluconase) (Ariffin et al., 2006) and protease (Chaiharn et al., 2008) were tested for all the isolates.
 
Scanning electron microscopy (SEM) of the interaction between rhizobacteria and test fungus
 
SEM mycelia fixation preparation for topography visualization was given by Kang et al., (2010). Two random fields of view as per sample were photographed. 
 
In vitro biocompatibility interaction of potential antagonistic rhizoisolates
 
The compatibility of antagonistic rhizoisolates was tested on the soybean digest agar disc plate as per the method given by (Subramanian et al., 2015).
 
Evaluation of bioantagonistic potential under greenhouse conditions and field study 
 
To study the disease suppression capability of the 4 selected antagonists i.e., S-2, S-4, S-18 and S-30 pot assay was conducted as per the method given by (Ghassemi-Golezani et al., 2008). Seedling vigor index (SVI) was calculated by shoot and root lengths of dried seedlings using the formulae
         
Field experiments were conducted during the Kharif season of 2017 on pigeonpea at the research farms of Punjab Agricultural University, Ludhiana, Punjab, India (30°54'5"N 75°47'53"E)(Table 1). This experiment was performed as per the recommended practices (Daspute et al., 2014).  Growth parameters 
 
Different growth parameters such as nodule number, nodule dry weight, the number of pods per plant, the number of seeds per pod and plant height were determined given by methods (Singh et al., 2011). Chlorophyll content was estimated by the method of Witham et al., (1971) and leghemoglobin content by the method of (Wilson et al., 1963). Grain yield from each plot (g/plot) was recorded at the final harvest and was expressed in kg/ha. Nitrogen content in the grains was determined by using the method given by (Nwokolo, 1987). The grain protein content was calculated by multiplying grain N content with 6.25 and expressed in %. Harvest index calculating by (Thanki et al., 2010).

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).
 
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).
 
 
 
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. 
 

 
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. 
 
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.
 
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 mm) 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.
 
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).
 
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.
 

 
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 spectrophoto metrically 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 600 nm) 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 9^th 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.
 
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.
 
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.

The results of this study strongly suggest that potential rhizobacteria (S-2 and S-18) can be investigated as potent inoculants for pigeonpea biofertilization, in addition to the recommended Rhizobium, PAU (LAR-06), in the future. Moreover, it promoted systemic resistance to soil pathogens under low-input technology for increasing pulses production in sustainable agriculture.

The authors would like to thank the Department of Plant breeding and genetics and Microbiology, Punjab Agricultural University, Ludhiana for providing laboratory facilities.

None.