Salicylic Acid Induced Resistance against Mungbean Yellow Mosaic Virus (MYMV) and Enhanced Seed Yield in Resistant and Susceptible Urdbean [Vigna mungo (L.) Heper] Genotypes

Urdbean [also known as black gram, Vigna mungo (L.) Heper] is an important pulse crop in South East Asia including India, Bangladesh, Pakistan, etc. (Kumari et al., 2020). The production and productivity of urdbean oscillates upon various environmental stresses. Among them, mungbean yellow mosaic virus (MYMV) causing yellow mosaic disease (YMD) is a most destructive disease of urdbean (Kumari et al., 2020). A plethora of reports estimates yield losses due to MYMV ranging from 10-100% depending upon susceptibility of urdbean genotypes, stage of crop infection and population of whitefly (Bemisia tabaci) (Nene 1972; Kumari et al., 2020). The MYMV infection also impairs the grain size and quality of urdbean (Kumari et al., 2020). Controlling this disease largely depends upon planting resistant genotypes and restricting whitefly population, a well-known natural transmitter of MYMV (Abubakar et al., 2018). Although several tolerant urdbean genotypes against MYMV have been identified (Saha et al., 2017; Kumari et al., 2020), but most of the tolerant genotypes are lacking yield potential under diverse environmental conditions. Further, breakdown of MYMV resistance is a problem associated with breeding for resistance in urdbean against MYMV due to the rapid formation of new pathotypes (Sehrawat et al., 2016; Saha et al., 2017).
        
Induction of Systemic acquired resistance (SAR) by exogenous application of salicylic acid (SA) is well established in plants (Umar et al., 2019). The role of SA in conferring MYMV resistance in mungbean (Umar et al., 2019) and urdbean were reported earlier (Kundu et al., 2011). Interestingly, application of SA was also linked with increase in seed yield in urdbean (Rawat et al., 2019). However, no systemic effort has been made to see the effect of SA in management of MYMV and seed yield in urdbean under field conditions. We chose to address these questions by analyzing the effect of SA on induction of defense related enzymes and further comparing its effect on MYMV resistance and grain yield in 39 urdbean genotypes. Overall, a net positive impact on MYMV tolerance and enhanced seed yield was achieved by exogenous application of SA in urdbean genotypes. These findings also provide a strong base for developing SA-based management programs for urdbean improvement.

All the field experiments were conducted for two consecutive seasons during Kharif, 2018 and 2019 at Tirhut College of Agriculture (TCA), Dholi; Dr. Rajendra Prasad Central Agricultural University, Pusa, Bihar, India.
 
Activation of defense related enzymes
 
Three week-old healthy urdbean plants grown in earthen pot (inner dimension 20 × 27 sq. cm) were sprayed with 3 different concentrations of SA (50, 100 and 150 µM) along with a mock (sprayed with distilled water only). The induction of POD, SOD and CAT activities were observed at 24, 48 and 72 h after SA spraying. The experiments were laid out in a randomized block design (RBD) with six replications per treatment.
        
Furthermore, a pot experiment in RBD with six replications per treatment was performed to evaluate the effect of SA on initial induction of antioxidant enzymes maintained over a longer period of time. Three week-old healthy urdbean plants grown in earthen pots were sprayed with 100 µM SA, along with a mock treatment and transferred to an infected urdbean field with high MYMV disease pressure. After the first, second and third weeks of post-inoculation, the enzymatic activities of POD, SOD and CAT were determined in LBG 623. The enzyme activities of all the 39 urdbean genotypes were also estimated after two week of post-inoculation.
 
Antioxidant enzyme assay
 
POD enzyme activity was analyzed spectrophotometrically using guaiacol as substrate (Singhai et al., 2011). CAT activity was estimated spectrophotometrically by measuring the rate of H₂O₂ disappearance at 240 nm (Miyagawa et al., 2000). SOD activity was determined spectrophotometrically by measuring the inhibition of nitro-blue tetrazolium (Beauchamp et al., 1971). Total soluble protein was calculated using the method of Bradford (Bradford 1976). Enzyme specific activity was expressed as the number of enzyme units per milligram of protein.
 
Effect of SA on MYMV resistance in urdbean
 
The urdbean genotypes were tested for MYMV resistance under natural field conditions by planting two sets of experiments. In 1^st set, foliar application of SA (100 µM) were applied on three week-old urdbean genotypes, whereas in 2^nd set, plants were sprayed with distilled water only (mock). Both the experiments were laid out in RBD with two replications. Two rows of spreader (highly susceptible urdbean genotype, LBG 623) were planted all around the experiment in order to attract white fly and enhance infection of MYMV. All the cultural practices were adopted except for application of insecticide in order to encourage the population of whitefly for natural disease spreading.
 
MYMV incidence analysis
 
The urdbean genotypes were tested for MYMV resistance by visually assessing symptoms in the field and categorizing them from resistant to highly susceptible on a scale of 1 to 9 (Kumari et al., 2020). The per cent disease index (PDI) was calculated using Wheeler’s formula at weekly intervals (1969).
   
Seed yield
 
Ripened pods from each genotype were hand-picked, allowed to dry for 10 days, then separated and weighed to determine yield. The mean data for seed yield in both years and their pooled data were subjected to analysis of variance for RBD.
 
Data analysis
 
All the experiments were repeated twice and pooled data were analysed with SPSS and ANOVA. The least significant difference (LSD) was used to compare treatment means using a multiple mean-comparison test (Steel and Torrie, 1980).

The wide host range of the MYMV and whitefly vectors makes managing this notorious disease very challenging (Varma et al., 1998). The use of SA, a known inducer of SAR in plant, has recently been employed in controlling MYMV via induction of antioxidant enzymes (Umar et al., 2019). However, the effect of SA in controlling MYMV and seed yield in urdbean under field conditions are less explored. The need for improved management strategy against MYMV in urdbean encouraged us to investigate the role of SA in the induction of antioxidant enzymes viz. POD, SOD and CAT leading to enhanc disease resistance and higher seed yield in urdbean.
 
Activation of defense related enzymes
 
To examine the effect of SA on the induction of defense-related enzymes, three week-old healthy urdbean plants were sprayed with three different concentrations of SA (50, 100 and 150 M), as well as a mock treatment. The gradual increase in the activities of all the three enzymes were observed as the concentration of SA treatment was increased in all times point (24, 48 and 72 h after treatment) studied (Fig 1). Although the maximum enzymatic activities were observed at 150 µM SA treatment but the difference in enzymatic activities observed at 100 and 150 µM SA treatments were not significant (Fig 1). However, significant increase in enzymatic activities was recorded either 100 µM or 150 µM over 50 µM of SA treatment (Fig 1). The exogenous application of SA induced the activity of antioxidant enzymes viz. POD, SOD, CAT and PAL in mungbean supports our study (Umar et al., 2019; Ali and Mahmoud, 2013).
 

        
Three week-old pot grown urdbean plants (LBG 623), sprayed with 100 M SA along with the mock, were transferred to MYMV infected urdbean field with high MYMV disease pressure to evaluate the effect of SA on initial induction of defence related enzymes maintained over a longer period of time. There was a ~3-fold increase in the production of antioxidant enzymes in SA treated urdbean plants challenged with MYMV compared to mock treated plants (Fig 2). However, no significant differences in the enzymatic activities of POD, CAT, and SOD were observed at 1^st, 2^nd and 3^rd week post treatment (Fig 2). Further, enzymatic activities were estimated in all the 39 urdbean genotypes (SA and mock treated) at 2 week post-inoculation of MYMV. The POD activities in SA treatment followed by MYMV infection showed more than 4-fold rise in disease free or highly resistant genotypes, while ~3-fold increase in susceptible urdbean genotypes were observed (Fig 3). The resistant or moderately resistant genotypes showed up to 4 fold increase in POD activities (Fig 3). SA treatment followed by MYMV infection increase CAT activities by ~4-fold, ~3.5, and ~3-fold in disease free or highly resistant, moderately resistant and susceptible urdbean genotypes, respectively (Fig 4). Similarly, the SOD showed more than 3.5-fold increase in disease free or highly resistant genotypes, while ~2-fold rise in susceptible urdbean genotypes were observed (Fig 5). The resistant or moderately resistant genotypes showed up to 3.5-fold increase in SOD activities (Fig 5). Taken together, greater induction in POD, CAT and SOD activities were observed in genotypes belonging to resistant group.
 

 

 

 

        
The induction of isoforms of SOD and POD in urdbean leaves upon SA treatment supports our results (Kundu et al., 2011). The high level of antioxidant enzymes produced due to the application of SA before the pathogen challenge led the plant to induce defence responses comfortably and effectively (Umar et al., 2019). The maintenance of a high level of antioxidant enzymes for several weeks upon SA treatment was also supported by previous study on mungbean (Umar et al., 2019).
 
Effect of SA on MYMV resistance in urdbean
 
We have evaluated the effect of SA on MYMV resistance in urdbean genotypes at field conditions. At pod formation stage disease severity in 39 urdbean genotypes were analysed and grouped into different reactions group (Table 1). In control set, only six genotypes were found to be either disease free or highly resistance (HR), whereas 20 genotypes in SA treated set were observed to be either disease free or HR in both the year tested (Table 1). In control set, 6 genotypes showed highly susceptible (HS) reaction in both the year tested, whereas none of the genotypes showed HS reaction against MYMV (Table 1). These results clearly demonstrate the profound effect of SA treatment on urdbean genotypes against MYMV. The induced resistance was observed in all the genotypes irrespective of tolerant or susceptible genotypes. Therefore the protection observed might be due to the induction of SAR. Our findings were duly supported by previous studies on the effect of SA in treatment on different mosaic viruses (Kundu et al., 2011; Farooq et al., 2018). The mechanisms by which SA promotes tolerance against MYMV may consist of higher basal defense preparedness, as indicated by the early induction of antioxidant enzymes. Previous studies showed that SA facilitates production of antioxidant enzymes, which induced SAR mediated disease resistance against several viral diseases (Elbadry et al., 2006; Siddique et al., 2014) including MYMV in mungbean and urdbean (Umar et al., 2019; Kundu et al., 2011). Taken together, exogenous application of SA leads to the activation of antioxidant enzymes, which persist for a long period and provide effective protection against MYMV in urdbean. The enhanced resistance observed in this study could be due to synergistic effect of SA mediated induction of SAR and antioxidant enzymes.
 

 
Effect of SA on seed yield of urdbean
 
SA is known for its pleiotropic effect on plants including crop yield (Liu et al., 2015; Ali and Mahmoud 2013). Therefore, we tempted to analyse the increase in seed yield of urdbean genotypes on SA treatment under field conditions. The yield analysis showed that there were increase in the yield of all SA treated urdbean genotypes (Table 2). The increase in yield was ranging from 23.98 to 31.97% (Table 2). The maximum increase in yield was observed in DKU 90 followed by IPU 12-5 and AKU 1608. A plethora of reports indicate that foliar application of SA had a beneficial effect on growth and photosynthesis in crop plants, leading to an increase in seed yield in normal as well as under stress conditions (Ali and Mahmoud 2013; Rawat et al., 2019).
 

        
Based on the results obtained in this study, a model incorporating MYMV resistance couple with increased seed yield mediated directly by SA or via induction of antioxidant enzymes in urdbean are depicted in Fig 6.
 

In this study, we found that exogenous application of SA causes an initial induction of antioxidant enzymes that is sustained over time, which aids in the management of MYMV disease. The field experiment revealed that exogenous SA application significantly reduced MYMV incidence and increased seed yield in urdbean genotypes.

The authors acknowledge AICRP on MULLaRP, India for providing the seed materials for conducting this study.

The authors declare that they have no conflict of interest.