Morpho-physiological and Biochemical Variability in Greengram [Vigna radiata (L.) Wilczek] Varieties for Mungbean Yellow Mosaic Virus (MYMV) Resistance under Natural Field Conditions

Greengram is one of the most important pulse crop grown in India after pigeonpea, chickpea and blackgram. Greengram is popularised by the name moong and mungbean. It is an important legume food crop because of its high protein, dietary fibres, vitamins and mineral contents. It is highly adaptable to a wide range of environmental conditions which favours it to grow under varied climatic conditions.
       
The wider adaptability of greengram is affected by both biotic and abiotic stresses. The biotic stresses include the complex viral diseases that are being transmitted by their insect vectors. In India, the major viral diseases that affect the mungbean production are mungbean yellow mosaic virus disease (MYMV) transmitted by whitefly, Urdbean Leaf crinkle virus (ULCV) transmitted by both aphids and whitefly and Leaf curl virus disease (LCV) transmitted by thrips. Among the three diseases MYMV is of highly important causing significant yield losses of 22.3 – 61.7% and might cause 100% yield losses when the disease appear in the early stages (Yadav and Brar, 2010).
       
MYMV disease is caused by a Begomovirus that belongs to the family Geminiviridae and order Geplafuvirales which has become a major threat to the pulse crop production in India. The major symptom of this disease is the yellow colour mottling on the leaves and later turn the whole leaf to yellow colour under severe conditions the total foliage appears yellow causing the major photosynthetic losses. During the early stages of crop growth this disease leads to malformations in the growing tips and younger leaves affecting growth of the plant.
               
Several approaches were being adopted for the control of MYMV disease with in the field conditions such as use of insecticide sprays for the control of vectors,  but it is near impossible to achieve full control of the disease. Development of resistant varieties is the most economical and effective solution for the control of disease, that is why there is a need to develop greengram varieties of high resistance to MYMV. The present field experiment was conducted to study the physiological and biochemical basis of resistance to MYMV disease in greengram varieties including both resistant and susceptible varieties.

A field experiment was conducted during rabi 2017-18 and 2018-19 at RARS Lam Guntur in a randomized block design with eight greengram genotypes and replicated thrice under receding soil moisture condition. Experiment was conducted using eight genotypes of greengram (WGG 42, Pusa Vishal, LGG 607, LGG 630, LGG 601, LGG 610, LGG 460 and LGG 450) obtained from RARS Lam Guntur. The plot size was 4 rows with 4 meter length and sown with a spacing of 30x10 cm. The estimation of sugars, phenols, tannins, trichome density and SCMR was done at vegetative and reproductive stages of crop growth. The SCMR value was measured using Minolta SPAD-502 Chlorophyll meter. The seed yield was recorded at maturity. The experimental data was statistically analyzed using OPSTAT.
 
Trichome density
 
The trifoliate leaves from ten randomly selected plants of a plot were brought from the field and were cut into leaf discs of 1 cm diameter. These discs were observed under a stereo microscope to count the number of hairs present on the leaf disc and the average value of the trichome density was calculated as cm² leaf area.
 
SPAD Chlorophyll meter reading (SCMR)
 
The SPAD meter reading was recorded from ten randomly selected plants within the plot. The most exposed leaf was preferred to record the SCMR value.
 
Total sugars (mg g^-1 FW)
 
Total sugar content was estimated using anthrone reagent method (Hedge and Hofreiter, 1962). Fresh leaf sample of 0.25 g was ground in 5 mL of 70% hot ethanol and centrifuged at 5000 rpm for 15 min. Then the supernatant was discarded and the pellet was air dried. After air drying 5 ml of anthrone reagent (2 g of anthrone in 1 L 10:4 v/v Conc. H₂SO₄) was added to the pellet and the tubes were kept in ice bath at 4°C for 10 min and immediately the tubes were transferred to water bath at 80°C for 30 min. Later, the tubes were cooled to room temperature and the absorbance was read at 620 nm. A series of standards were prepared using fructose (1 mg per 10 ml) stock solution.
   
Phenols (mg g^-1 FW)
 
Leaf Phenol content was estimated (Malik and Singh, 1980) using folin’s reagent in which 0.25 g of fresh leaf was ground in 5 ml of 80% ethanol and kept it for 1 hour. 1 ml of aliquot was collected and 2 ml of 1 M Sodium Carbonate (10.6 g in 100 ml distilled water) was added. Later, 1 ml of folin reagent (1:9 v/v with water) was added and the tubes were kept in water bath for 20 min at 70°C. The absorbance was measured at 765 nm. Standard stock was prepared by using 0.5 g gallic acid in 10 mL ethanol and 90 mL of water and series of different concentrations as standards were made. 
 
Tannins (mg g^-1 DW)
 
A powdered dry leaf sample of 0.5 g was taken in test tube and 7.5 ml of water was added to it. The test tubes were kept in boiling water bath of 100°C for 30 min. The contents were centrifuged at 10,000 rpm for 20 min and the supernatant was collected. The volume was made to 10 ml with water. The extract obtained was used for assay with Folin-Denis’ reagent by adding 0.5 ml of the reagent to the 0.1 ml of plant extract and also added 1 ml of 35% sodium carbonate solution and shaken well kept at room temperature for 30 min to take the readings at 700nm. A set of standard solutions were made of tannic acid (10, 20, 30, 40, 50 μg ml^-1) and the tannins content of the samples was expressed in mg g^-1 dry weight of the sample (Sadasivam and Manickam,1992).
 
Disease score
 
It is natural disease appearing during crop season. The plot wise symptoms on the plants were observed and per cent incidence of the disease was calculated by dividing the number of infected plants by total plant population of the plot and multiplied by hundred. Disease scoring was given based on formula given by Alice and Nadarajan, (2007).

Sugars
 
The sugar content of the leaves of greengram varieties was estimated by anthrone method and the values are given in Table 1. A significant difference was observed for total sugars among the varieties at vegetative and reproductive stages during both rabi 2017-18 and 2018-19. At vegetative stage during rabi 2017-18, higher total sugar content was recorded in the variety LGG 450 (45.27 mg g^-1 FW) followed by LGG 460 (43.65 mg g^-1FW) and LGG 601 (41.21 mgg^-1FW) which are at par with each other and the lowest sugar content was recorded in the variety LGG 630 (35.40 mg g^-1 FW) preceded by WGG 42 (36.13 mg g^-1 FW). At reproductive stage during rabi 2017-18, highest sugar content was recorded in variety LGG 450 (34.42 mg g^-1 FW) followed by LGG 460 (33.53 mg g^-1 FW) which are significantly higher than the other varieties and are at par with each other followed by LGG 601 (30.58 mg g^-1 FW) and are at par with each other and the lowest sugar content was recorded in the variety LGG 630 (25.55 mg g^-1 FW) preceded by WGG 42 (26.25 mg g^-1 FW).


 

       
At vegetative stage during rabi 2018-19 higher sugar content was recorded in the varieties LGG 450 (47.32 mg g^-1 FW) followed by LGG 460 (45.70 mg g^-1 FW) and are at par with one another and the lowest sugar content was recorded in the variety LGG 630 (37.45 mg g^-1 FW) preceded by WGG 42 (38.18 mg g^-1FW). At reproductive stage the leaf sugar content of the greengram varieties followed the trend that was observed earlier with higher sugar content in the variety LGG 450 (36.47 mg g^-1FW) and the lower sugar content was record in the variety LGG 630 (27.60 mg g^-1 FW).
       
The leaf total sugar content decreased from vegetative stage to reproductive stage in all the greengram varieties tested. The total sugar content of the plants contributes as a nutritious diet to whitefly, insect vector that transmits the MYMV disease. From the correlation studies it is evident that the total sugar content of the plants has a significant positive association with the whitefly population (Table 8). The increased total sugar content after MYMV infection in the greengram varieties increased the whiteflies populations especially on susceptible varieties. The increased whitefly populations increase the disease incidence on the susceptible varieties. The MYMV disease infection has reduced the total sugar content in all the varieties and the reduction was higher in susceptible varieties (Ghuge et al., 2018). Similar results were previously reported by Narasimhulu et al., (2014) in greengram varieties.
 
Phenolic content
 
A significant variation was observed among the varieties tested for leaf phenolic content at vegetative and reproductive stages during rabi 2017-18 and 2018-19 (Table 2). The leaf phenolic content at reproductive stage during rabi 2017-18 was observed to be higher in the variety LGG 630 (25.78 mg g^-1 FW) followed by WGG 42 (25.18 mg g^-1FW) and PusaVishal (23.32 mg g^-1 FW) which are in line with one another. The lowest phenolic content was recorded in the variety LGG 450 (15.37 mg g^-1 FW). At vegetative stage during rabi 2017-18 highest phenolic content was recorded in the variety LGG 630 (21.40 mg g^-1 FW) followed by WGG42 (20.45 mg g^-1 FW) and LGG 607 (18.40 mg g^-1 FW).

At reproductive stage during rabi 2018-19, higher phenolic content was recorded in the variety LGG 630 (26.83 mg g^-1 FW) followed by WGG 42 (26.23 mg g^-1 FW) both are on a par with each other and lowest phenolic content was recorded in the variety LGG 450 (16.43 mg g^-1 FW). At vegetative stage during rabi 2018-19 the highest phenolic content was recorded in the variety WGG 42 (21.50 mg g^-1 FW) followed by LGG 630 (20.44 mg g^-1 FW) and the lowest was recorded in the variety LGG 460 (13.14 mg g^-1 FW).
 

       
A significantly higher phenolic content was observed in the leaves of varieties that showed lower disease incidence under field conditions indicating the importance of phenolic content for MYMV resistance. The phenolic compounds have attributed to the resistance of host plant towards the whitefly as the toxic nature of phenol causes damage to the insect. Further, phenolic compounds are capable of increasing the anti-oxidant enzyme activities such as phenyl alanine lyases and trigger the antioxidant activities of catalase and peroxidase. Similar results were obtained in greengram varieties by Mantesh et al., (2020) and Basavaraj et al., (2019) in soybean by Singh et al., (2019).
 
Tannins
 
The tannin content of the dried leaf samples was measured at vegetative and reproductive stages during both rabi 2017-18 and 2018-19 and the mean values were tabulated in Table 3 in mg g^-1 DW and a significant variation was observed among the varieties tested. At reproductive stage during rabi 2017-18 the higher tannin content was recorded in the variety LGG 630 (0.055 mg g^-1 DW) followed by WGG 42 (0.053 mg g^-1 DW) and the lowest tannin content was recorded in the variety LGG 450 (0.035 mg g^-1DW). At 30 DAS stage during 2017-18, highest tannin content was recorded in the variety LGG 630 (0.040 mg g^-1 DW) followed by WGG 42 (0.038 mg g^-1 DW) and the lowest was recorded in the variety LGG 450 (0.022 mg g^-1 DW).
 

       
During rabi 2018-19 at reproductive stage the higher tannins content was recorded in the variety LGG 630 (0.053 mg g^-1 DW) of the leaf followed by WGG 42 (0.051 mg g^-1 DW) and the lower tannins content was recorded in the variety LGG 450 (0.033 mg g^-1 DW). At vegetative stage during rabi 2018-19 the higher tannins content was recorded in the variety LGG 630 (0.038 mg g^-1 DW) followed by WGG 42 (0.036 mg g^-1 DW) and the lower tannins content was recorded in the variety LGG 450 (0.020 mg g^-1 DW).
       
The tannins are a group of phenolic compounds which are known for their astringent nature and protect the plant from insect pests through antibiosis. The higher tannins content values are recorded in the varieties with lower disease score. The tannins content has significantly affected the vector population which has reduced the per cent disease incidence. The tannins production along with the pigmentation has influence on host plant resistance (Davies, 2004). A significant higher tannins and flavonoids content were observed in the MYMV resistant varieties of blackgram (Taggar et al., 2014).
 
Trichome density
 
The trichome density of the ten randomly selected most exposed leaves from each plot were taken and the average number of trichomes was calculated per cm² and the values were enumerated in Table 4. The trichome density values indicate the average number of trichomes per cm² of the leaf lamina. The pubescence of the leaf plays an important role in repelling the pests.
 

       
At vegetative stage during rabi 2017-18 the higher trichome density was recorded in the variety LGG 630 (162/cm² of leaf area) (Plate 1A) followed by WGG 42 (149/cm² of leaf area) and the lower trichome density was observed in the variety LGG 450 (95/cm² of leaf area). At reproductive stage during rabi 2017-18 the highest trichome density was recorded in the variety LGG 630 (153/cm² of leaf area) followed by WGG 42 (142/cm² leaf area) and the lowest was recorded in the variety LGG 450 (77/cm² of leaf area).


 
At vegetative stage during rabi 2018-19 the highest trichome density was recorded in the variety LGG 630 (160/cm² of leaf area) followed by WGG 42 (147/cm² of leaf area) and the lowest trichome density was observed in the variety LGG 460 (93/cm² of leaf area) (Plate 1B). At reproductive stage during rabi 2018-19 the highest trichome density was recorded in the variety LGG 630 (151/cm² of leaf area) followed by WGG 42 (140/cm² leaf area) and the lowest was recorded in the variety LGG 460 (75/cm² of leaf area).
       
The trichome density showed a significant effect on whitefly population in greengram varieties. Higher trichome density reduced the feeding preference of the whitefly on plants and reduced the per cent disease incidence and score. The resistant varieties showed higher values of trichome density. Mantesh et al., (2020) observed similar results in greengram varieties against whitefly infestation. A reduced whitefly population was observed in the blackgram varieties with higher trichome density (Taggar and Gill, 2012).
 
SCMR (SPAD Chlorophyll meter reading)
 
At vegetative stage during rabi 2017-18 the higher SCMR value (Table 5) was recorded in the variety LGG 630 (52.29) followed by WGG 42 (50.50) and the lower SCMR was observed in the variety LGG 450 (38.12). At reproductive stage during rabi 2017-18 the higher SCMR value was recorded in the variety LGG 630 (56.67) followed by WGG 42 (53.54) and the lower value was recorded in the variety LGG 450 (43.07). At vegetative stage during rabi 2018-19 the higher SCMR value was recorded in the variety LGG 630 (54.29) followed by WGG 42 (52.50) and the lower value was observed in the variety LGG 460 (35.12). At reproductive stage during rabi 2018-19 the higher SCMR value was recorded in the variety LGG 630 (58.67) followed by WGG 42 (55.54) and the lower value was recorded in the variety LGG 450 (36.07).
 

       
The MYMV disease infection reduced the chlorophyll contents of the plants. The varieties with higher SCMR values were observed having darker foliage which in turn reduces the whitefly population in those varieties. The reduction in chlorophyll was higher in the susceptible varieties which has reduced the SCMR value significantly. The total chlorophyll content was higher in resistant varieties of greengram (Mantesh et al., 2020).
 
Disease score
 
The disease score values recorded at reproductive stage in all the varieties showed observable variation among the varieties (Table 6). The varieties WGG 42 and LGG 630 showed a score of ‘2’ during both rabi 2017-18 and 2018-19 which indicates highly resistant type. The varieties PusaVishal, LGG 607 and LGG 610 showed a score of ‘3’ during both rabi 2017-18 and 2018-19 indicating resistant type. The variety LGG 601 recorded a score of ‘5’ during both rabi 2017-18 and 2018-19 indicating moderately susceptible type. The variety LGG 460 recorded a score of ‘6’ during rabi 2017-18 and 2018-19 which showed susceptible nature of the variety. The variety LGG 450 showed a score of ‘8’ during both rabi 2017-18 and 2018-19 showing highly susceptible nature. The score given by Alice and Natarajan, (2007) was previously used by Raj et al., (2019) in screening greengram varieties and Gopi et al., (2018) in the screening of blackgram varieties.
 

 
Yield
 
During rabi 2017-18 the highest seed yield (Table 7) was recorded in the variety LGG 630 (1076 kg ha^-1) followed by WGG 42 (980 kg ha^-1) and the lowest seed yield was recorded in the variety LGG 450 (650 kg ha^-1). During rabi 2018-19 the highest seed yield was recorded in the variety LGG 630 (1122 kg ha^-1) followed by WGG 42 (1030 kg ha^-1) and the lower seed yield was recorded in the variety LGG 460 (655 kg ha^-1). The disease incidence has significantly reduced the yield of the susceptible varieties and the yields of resistant varieties stayed unaffected.
 

 
Correlations
 
The correlation studies indicated that the disease score showed a highly significant negative correlation with phenols, tannins content, trichome density, SCMR and yield and a highly significant positive correlation with total sugars content (Table 8). Mantesh et al., (2020) observed significant negative correlations between trichome density, phenols and chlorophyll contents among the greengram varieties.

From these results it can be inferred that higher phenols, tannins content and trichome density and less sugars content traits contributed MYMV resistance in LGG 630 and WGG 42 of greengram. The genotypes WGG 42 and LGG 630 were grouped under highly resistant category with higher phenol and tannin content and higher trichome density. The genotypes Pusa Vishal, LGG 607 and LGG 610 were categorised under resistant group. The varieties LGG 460 and LGG 450 were grouped under susceptible and highly susceptible categories respectively with lower phenol and tannin content and lower trichome density.