Nodule Occupancy Behaviour of Bacteriocinogenic Rhizobium spp. in Mungbean (Vigna radiata)

Legume-Rhizobium symbiosis is the most promising plant bacterium association for increase in grain yield through biological nitrogen fixation. Rhizobium spp. have ability to colonize root nodules of leguminous plants and are routinely applied as biofertilizers. The efficiency of this association depends upon the establishment of chosen strains, which are able to compete with native rhizobia. However, sometimes inoculated Rhizobium strains often fail to compete with indigenous rhizobia and do not increase nodulation. Bacteriocins are one of the major factors affecting competition among rhizobia (Maan and Garcha, 2018). Bacteriocin production plays important role in inter-specific and intra-specific competition. Bacteriocin-producing rhizobial strains have shown competitive advantage in mixed culture (Schwinghamer and Brockwell, 1978). Mixed cultures help to improve legume inoculants potency, competitivity and survival after inoculation (Warda et al., 2014).
        
Bacteriocin producing Rhizobium strains can be applied in three different approaches (1) using a dual culture consisting of a bacteriocin producer and bacteriocin resistant nitrogen fixer strain (2) using bacteriocin producer which also fixes nitrogen (3) and transfer of the genetic characters for bacteriocin production to efficient nitrogen fixer (Maan et al., 2019).The present work was carried out to investigate the advantage of dual inoculation in terms of plant growth, yield and nodule occupancy behavior of bacteriocinogenic Rhizobium isolates.

Pot experiment was carried out in the experimental fields of Department of Microbiology, Punjab Agricultural University,  Ludhiana, during Kharif 2016 under glasshouse. Four bacteriocin producing Rhizobium strains namely N8, S1, S6 and S13 were isolated from nodule and soil samples drawn from the fields of mungbean (Vigna radiata) in Punjab. Bacteriocin production was demonstrated in vitro quantified and characterized (Maan et al., 2019). The bacteriocinogenic Rhizobium (N8, S1, S6 and S13) and indicator strain (Rhizobium spp. procured from the Department of Microbiology) inoculums were prepared by inoculating them in yeast extract mannitol (YEM) broth followed by incubation at 28°C for 24 h at 150  rpm. Medium black clayey soil  from local  mungbean  field was  collected and autoclaved 3x (1h 121°C) at 12 hours interval. 3 kg of sterilized soil was filled per pot. Seeds of Mungbean variety SML 668 were procured from the Department of Seed Technology Centre. Mungbean SML 668 seeds were  surface sterilized by immersing in 1 percent sodium hypochlorite  solution for 10 min and then washed three times with distilled water. Previously prepared  5 ml samples of each inoculant (bacteriocinogenic Rhizobium strain and indicator strain) were uniformly applied on mungbean seeds as  single and co-inoculation and ten seeds were be sown in each pot. Before inoculation, an equal volume of different strains (1:1) was mixed and allowed to stand for 30 minutes at room temperature. Inoculated seeds were dried at room temperature under shade before sowing.

The crop was sown on 3^rd May 2016 and harvested on 7^th July 2016. The experiment was conducted with ten treatments (Un-inoculated control, Indicator strain, bacteriocinogenic N8 strain, bacteriocinogenic S1 strain, bacteriocinogenic S6 strain, bacteriocinogenic S13 strain, Indicator+N8, Indicator+S1, Indicator+S6 and Indicator+ S13) having three replications each. Three approaches were used in the investigation (un-inoculated control, single culture inoculation and dual culture inoculation). The crop was raised as per recommendations outlined Package of Practices printed by Punjab Agricultural University, Ludhiana (Anonymous, 2016). Observations for number and dry  weight  of  nodules, Fresh and dry weight of shoot and  root,  nodule index (number of nodules per plant/root length) and Plant height were recorded at both 35 DAS and harvesting stage. Three randomly selected plants were  carefully uprooted from each pot at 35 DAS and harvesting, with root system intact. The roots were washed in running tap water and nodules were detached carefully with forceps and the total number of nodules was counted. The number of nodules per plant  was  recorded by taking an average. The detached  nodules  were  dried in oven at 60°C for 2 days and their dry weight per plant was recorded in milligram. Three randomly selected plants  were uprooted from each pot and fresh weight of shoot and root per plant was measured in gram. These were then dried in an oven at 60°C for 2 days and dry weight  per  plant  was  measured in gram. Chlorophyll content (Witham et al., 1971) and leghaemoglobin content (Wilson and Reisenauer, 1963) were recorded at 35 DAS. Cultures of indicator strain and bacteriocinogenic Rhizobium were challenged against a broad spectrum of antibiotics to determine their intrinsic antibiotic resistance characteristics (Maan et al., 2019). Nodule suspensions were plated on YEMA medium supplemented with antibiotics (gentamicin, streptomycin and penicillin) for analyzing total viable cell count. Nodule occupancy behavior exhibited by inoculants (Indicator strain was resistant and bacteriocingenic strain were sensitive to Gentamicin 30 mcg/discs) was recorded at 35 DAS (Josey et al., 1979). Observations like primary branches, secondary branches, leaflet length, leaflet breadth, no. of pods, seeds, seed set per cent (number of seeds per inflorescence/ number of florets per inflorescence), nitrate reductase assay (Jawarski,1971), Nitrogen-content in grains and straw (McKenzie and Wallace, 1954), grain yield was recorded after harvesting.  
 
Statistical analysis
 
The collected data were analyzed statistically using the CPCS 1 software developed by Department of Statistics, Punjab Agricultural University, Ludhiana and Punjab, India. Differences among treatment and varieties were determined using comparion method at 5% level of significance.

Nodulation
 
Nodule number, nodule dry weight, fresh weight and nodule index
 
Dual inoculation of bacteriocinogenic Rhizobium spp. with indicator strain (I+N8) registered significantly higher number of nodules, fresh weight and nodule index (24.3 nn/plant, 25.2 mg/plant and 1.36, respectively) followed by (I+S6) treatment (21.3 nn/plant, 24.1 mg/plant and 1.34) as compared to indicator alone (18.3nn/plant, 14.2 mg/plant and 1.22) (Table 1). The data was supported by Rao (1983) that dual inoculation of bacteriocin producing Rhizobium sp. Rt1B and Rt1103M (72.0 nn/plant) produced more number of nodules as compared to Rhizobium Rt1B (68.7nn/plant) and Rt1103M (51.7 nn/plant) alone. Maximum enhancement of nodule dry weight was recorded with co-inoculation of bacteriocinogenic Rhizobium N8 and indicator strain (I+N8) (23.4 mg/plant) as compared to co-inoculation of bacteriocingenic S6 and indicator (I+S6) (23.0 mg/plant) (Table 1). Sharma and Khurana (2007) reported that the indicator Rhizobium favored bacteriocinogenic Rhizobium inoculation to form more nodules either by favoring its survival in the rhizosphere or the synthesis of plant growth regulators which result in more root hair development and leading to more infection. Pramar and Dadarwal (1999) also reported rhizobacteria enhancing the production of flavonoid like compound or phytoalexins in roots of several crop plants enhancing nodulation.
 

 
Plant growth parameters
 
Dry, fresh weight of root and shoot
 
Dual inoculation of bacteriocinogenic Rhizobium N8+ indicator strains has maximum fresh weight of shoot and root significantly in comparison to un-inoculated control at both flowering and harvesting stages (Table 2). Significant increase in fresh weight of shoot and root were recorded in bacteriocinogenic Rhizobium N8 + indicator strain (13.2 g/plant and 4.02 g/plant, respectively), over indicator strain alone (7.91 g/plant and 2.57 g/plant, respectively). A significant  difference  for  dry  weight  of  shoot  was  observed  for  dual inoculation over mono-inoculation and un-inoculated control. Similar trend was followed for dry weight of root. Co-inoculation of bacteriocinogenic Rhizobium N8 with indicator strain (I+N8) gave maximum shoot dry weight (0.79 g/plant) followed by I+S6 (0.75 g/plant) and I+S13 (0.71 g/plant) whereas I+N8 (1.65 g/plant) and I+S6 (1.62 g/plant) depicted maximum dry weight of root at harvesting stage. Dry weight of shoot is considered as best indirect measurement of nitrogen supplied by rhizobial strain to host cultivar. Similarly, higher shoot dry weight in plants inoculated with rhizobial strains might be ascribed to more N supply to crop through N fixation by bacteria (Meghvansi et al., 2010). Improved root dry weight in indicator Rhizobium inoculation with bacteriocinogenic Rhizobium could be due to the ability of rhizobia to conserve carbohydrates that further increases the total plant biomass (Rahmani and Rstin, 2001).
 

 
Number of branches, leaflet length, breadth and plant height
 
The results summarized in Table 3 depicted that bateriocinogenic Rhizobium N8 spp. with indicator strain (I+N8) has maximum primary branches (7.33), secondary branches (22.3), leaflet length (8.16) and leaflet breadth (5.83) as compared to other dual inoculation. Application of indicator Rhizobium with bacteriocinogenic Rhizobium to seeds significantly increases plant height, number of branches, number of leaves as reported by Adesemooye et al., (2009). The increased leaf area produced greater amount of chlorophyll, which resulted into more efficient use of absorption of sunlight and higher rate of photosynthesis.
 

 
On the basis of data collected at harvesting stage, maximum height was recorded with dual inoculation of bacteriocinogenic Rhizobium and indicator strain of I+N8 (96cm) followed by I+S6 (89cm) and I+S13(85.3cm) (Table 3). This investigation has been found coherent between the results of Waseem et al., (2014) who reported the significant increase in plant height of dual inoculation of Rhizobium + PGPR in mungbean (22.7%) over control and 10.8% over Rhizobium inoculation alone. Kaur et al., (2015) described improvement in plant height with PGPR or Mesorhizobium sp alone or dual over control plants in both varieties of chickpea could be attributed to presence of phytohormone (IAA) which functions as signal molecule in the regulation of plant development.
 
Number of pods/plant, number of seeds/pod and seed set percent
 
Dual inoculation with indicator + N8 showed maximum increase in number of pods, seeds and seed set per cent by 9.4%, 39%, 17% over that obtained with use of bacteriocinogenic N8 alone (Table 4). Increase in number of pods/plant, number of seeds/pods and seed set percent was exhibited by dual inoculation of bacteriocinogenic Rhizobium N8 with indicator strain (15.9, 13.8 and 86.79%, respectively) as compared to un-inoculated control (12.6, 7.5 and 59.52%, respectively). These results were coherent with Raza et al., (2004), who reported that dual inoculation of Rhizobium and PGPR strain Q14 significantly increased number of pods/plant by 87 and 66% when compared with un-inoculated control and Rhizobium inoculation alone, respectively. Similar significant increase in the number of pods bearing branches was also reported by Brar and Lal (1991).
 

 
Plant pigments
 
Dual inoculation of bacteriocinogenic Rhizobium spp. with Indicator strain showed significant differences for chlorophyll content as compared to indicator strain alone (Table 4). Significant higher chlorophyll content was observed in co-inoculation of bacteriocinogenic Rhizobium with indicator strain I+N8 (1.34 mg/g leaf tissue) followed by I+S6 (1.31mg/g leaf tissue). Ladha et al., (1998) observed the improvement in chlorophyll content may be due to increased N uptake by a larger root surface areas associated with additional root hairs and lateral root development and/or to BNF, either directly by the inoculant strains or indirectly by stimulating BNF activity of the associated rhizosphere community. Mishra et al., (2012) also reported that Rhizobium leguminosarum-PR1 inoculation showed significant increases in Chl a, Chl b and total Chl contents as compared to non-inoculated control plants in field pea.

Maximum content of leghaemoglobin was observed in dual inoculation of bacteriocinogenic Rhizobium N8 with indicator strain (I+N8) (3.43 mg/g fresh weight of nodules) over indicator spp. alone (1.05 mg/g fresh weight of nodules) (Table 4). Kaur et al., (2015) reported that co-inoculation of Rhizobium + PGPR (J-17) (1.07 and 2.10 mg/g) showed significant increase in leghaemoglobin content as compared to Rhizobium (0.649 and 1.64 mg/g), respectively at 60 and 90 DAS in lentil. Our data also supported with studies of Mahmoud and Abd-Alla (2001) where microbial siderophores may be involved in biosynthesis of leghaemoglobin by facilitating the uptake of iron, a constituent of key proteins such as nitrogenase and leghaemoglobin content from environment and helps in its enhancement on dual inoculation of bacteriocinogenic Rhizobium with indicator Rhizobium in mungbean.
 
Biochemical parameters
 
Nitrogen content is an important growth parameter, which has a direct bearing with biological nitrogen fixation. Dual inoculation of bacteriocinogenic Rhizobium N8 with indicator strain (I+N8), showed highest nitrate reductase activity (0.984) as compared to indicator strain alone (0.856) (Table 5). N content ranged from 3.30% to 4.02% in grains and from 0.82% to 2.32% in straw. Co-inoculation of bacteriocingenic Rhizobium N8 with indicator Rhizobium (I+N8) showed an increase in N content in grains (4.02%) followed by I+S6 (3.73%) as compared to use of indicator strain (3.45%) alone. Similarly, in straw samples, indicator strain + bacteriocinogenic N8 and indicator + bacteriocinogenic S6 (2.32% and 1.39%, respectively) were significantly more than use of indicator alone (1.04%) (Table 5).
 

 
Co-inoculation of Rhizobium spp. with LK-786 and LK-884 showed increase in N uptake by grains than individual inoculation of Rhizobium spp. LK-786 and LK-884 as reported by Kumar and Chandra (2008). Khan et al., (2006) also reported that co-inoculation resulted in more N2 fixation and P solubilization due to release of protons by Rhizobium during biological nitrogen fixation results into lowering of soil pH and by producing organic acids. These results are in close agreement with the findings of Barea et al., (2005) who demonstrated that the interactive effect of rhizobia and rhizobacteria mediated the number of soil processes and thus enhanced the availability of nutrients.
 
Grain yield
 
The grain yield was recorded at harvesting stage  to  analyse  best cultures that can  be  further  used  as  biofertilizers  in  mungbean (Table 5). Inoculation of bacteriocinogenic Rhizobium and indicator Rhizobium considerably enhanced the grain yield, while the effect was more pronounced when they were applied in combination as compared to uninoculated ones. The highest grain yield was recorded by co-inoculation of bacteriocinogenic Rhizobium N8 and indicator Rhizobium (I+N8) (1.69 g/plant) followed by indicator alone (1.43 g/plant) and un-inoculated control (0.85 g/plant). Dual inoculation has been reported to enhance grain yield of lentil by about of 13.6% (Rhizobium + Pseudomonas-1), 14.6% (Rhizobium + Klebsiella-133) and 14.9% (Rhizobium + Bacillus-40) over the un-inoculated control (Saini and Khanna, 2012). Inoculation with free living diazotrophs increased the signal exchange between host legumes and resulting in more N2 fixing sites and ultimately higher nutrient concentration and yield of legume as reported by Parmar and Dadarwal, (1999) and Qureshi et al., (2009).
 
Nodule occupancy exhibited by inoculants
 
Inoculated strains have been monitored through antibiotic resistance sensitivity pattern presented in Table 6. Assessment of nodule competitiveness of rhizobial strain were identified using antibiotic resistance method. Total viable cell count of nodule suspension was analysed for nodule occupancy in Table 7. Results of nodule occupancy indicated that the bacteriocinogenic Rhizobium isolates namely N8 dominated the nodule microbiota when used in combination with the indicator strain. Their number was generally higher than the indicator strain by a factor of 100. It is able to occupy all root nodules and fully exert its Nitrogen fixation ability. Co-inoculant has greater impact over use of single bacteriocinogenic isolates. Kumar and Chandra (2008) also reported the influence of PGPR and PSB on Rhizobium leguminosarum bv. viciae strain competition and symbiotic performance in lentil. In the present study, co-inoculation of bacteriocinogenic Rhizobium N8 with indicator presented more viable cell count as compared to other co-inoculant strain. Co-inoculation of I+N8 revealed better nodule occupancy as compared to indicator strain alone which might be due to bacteriocin production and release of growth hormone (IAA) by rhizobacterial strain. These results are in close agreement with Parmar and Dadarwal (1999) that also correlated improvement in the nodule occupancy due to siderophores production and release of growth hormones of rhizobia in rhizosphere of chickpea. This enhancement in nodule occupancy might be due to well adaption of bacteriocinogenic rhizobacteria in soil with bacteriocin production and plant growth promotional activities.
 

 

Rhizobium are diazotrophic bacteria that fix nitrogen and are readily used as  an effective and cheap biofertilizer. They contribute towards sustainability of agriculture. Dual inoculation strategy of using an indicator strain (Rhizobium spp - mungbean biofertilizer) in this study and bacteriocin producing Rhizobium isolates gave significantly better results for nodulation, plant growth and yield attributes. Bacteriocin plays a role in inter-specific and intra-specific competition. Strains have the ability to produce bacteriocin are known to dominate the native microflora as demonstrated in this investigation. Bacteriocinogenic Rhizobium isolates have also demonstrated nitrogen fixing ability in the preliminary tests carried out in this investigation. Dual inoculation of bacteriocinogenic Rhizobium sp. with Indicator (Rhizobium spp.) is effective as bio-fertilizer to enhance plant growth, symbiotic efficiency, nutrient uptake and yield in mungbean.