​Study of Morpho-physiological and Biochemical Traits for Resistance to Yellow Mosaic Virus (YMV) in Blackgram [Vigna mungo (L.) Hepper] Varieties

Blackgram is an important tropical pulse crop widely distributed in the South Asian countries. The higher protein and antioxidant concentration mainly anthocyanin which is the reason for its dark hue makes blackgram nutritionally more important. It is sometimes described as “poor man’s meat” because of its balanced protein and carbohydrate diet when combined with cereal food grains. Blackgram is a rich source of dietary micronutrient Iron (Fe). The blackgram production in India accounted for more than 25% of the world with 25.23 Million tonnes (Mt) during 2017-18 over a cultivated area of greater than 29 Million Hectares (Mha) with an average productivity of 841 kg ha^-1. Andhra Pradesh has an area of 3.81 Lakh ha under blackgram cultivation with contribution of 7.57% (Pulses Revolution from Food to Nutritional Security, 2018).

Blackgram is a short duration pulse crop grows on the marginal lands with low inputs due to which it is affected by many abiotic and biotic stresses. Insect pests, viral diseases and fungal diseases are different biotic stresses and among them whitefly is the most important insect pest which transmits the viral disease yellow mosaic virus (YMV) which results in severe yield losses and often total failure of the crop, if the incidence occurs during early stages of crop growth. The incidence of YMV disease results in yellow mottled appearance of the leaf with chlorotic spots and patches on older leaves and the younger leaves show complete yellowing in case of susceptible genotypes; yellowing and chlorosis leads to reduction in the photosynthetic efficiency of the plant, thereby finally resulting in the reduction of 25 to 78% yield in Mothbean (Vir, 1984).

To overcome the problem of YMV, host plant resistance studies plays a prominent role in resisting the insect pest attack on the crop throughout its growth period. The morphological, physiological and biochemical traits such as sugar, phenols, tannins, trichome density and SCMR values play a pivotal role in host plant resistance. In view of the above, an experiment has been conducted to study the host plant and insect interactions through the variability in the morpho-physiological and biochemical traits that influence the YMV disease resistance in blackgram varieties.

A field experiment was conducted during rabi 2017-18 and 2018-19 at RARS Lam Guntur in a randomized block design with eight blackgram varieties and replicated thrice under receding soil moisture condition. Experiment was conducted using eight varieties of blackgram (LBG 787, GBG 1, TBG 104, LBG 884, LBG 806, LBG 808, LBG 623 and LBG 752) 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 (30 DAS) and reproductive stages (50 DAS) 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 “Statistical Software Package for Agricultural Research Workers” (Sheoran et al., 1998).
 
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 Natarajan, (2007).

Sugars
 
The sugar content of the leaves of blackgram 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. The leaf total sugar content decreased  from  vegetative stage to flowering stage in all genotypes in both the  years. At vegetative stage during rabi  2017-18, higher total sugar content was recorded in the variety LBG 623 (47.78 mg g^-1 FW) followed by LBG 787 (46.31 mg g^-1 FW) and LBG 752 (45.16 mg g^-1 FW) and are at par with each other and the lowest sugar content was recorded in the variety LBG 884 (35.50 mg g^-1 FW) preceded by GBG 1 ( 40.77 mg g^-1 FW). At reproductive stage during rabi 2017-18, highest sugar content was recorded in variety LBG 623 (39.36 mg g^-1 FW) followed by LBG 787 (37.20 mg g^-1 FW) and LBG 752 (36.34 mg g^-1FW ) which are significantly higher than the other varieties and are at par with each other and the lowest sugar content was recorded in the variety LBG 884 (25.16 mgg-1FW) preceded by GBG 1  (30.42 mg g^-1 FW).



At vegetative stage during rabi 2018-19 higher sugar content was recorded in the varieties LBG 623  (49.82 mg g^-1 FW) followed by LBG 787 (48.36 mg g^-1 FW) and LBG 752 (47.21 mg g^-1 FW) and are at par with one another and the lowest sugar content was recorded in the variety LBG 884 (38.55 mg g^-1 FW) preceded by GBG 1 (42.83 mg g^-1 FW). At reproductive stage the leaf sugar content of the blackgram varieties followed the trend that was observed earlier with higher sugar content in the variety LBG 623 (41.41 mg g^-1 FW) and the lower sugar content was record in the variety LBG 884  (27.21 mg g^-1 FW).

The leaf total sugar content decreased from vegetative stage to reproductive stage in all the blackgram varieties tested and the variety with higher total sugar content showed high YMV incidence with reduced yield, whereas, the variety with lower total sugar content recorded higher yields with lower YMV disease incidence. The increase in the total sugar content of the leaves was observed to increase the susceptibility of the host plant to YMV. The total sugar content of the plants contributes as a nutritious diet to whitefly, insect vector that transmits the YMV 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 YMV infection in the blackgram varieties increased the whiteflies populations especially on susceptible varieties. The increased whitefly populations increase the disease incidence on the susceptible varieties. The sugar contents, especially non reducing sugar acts as a good nutrient source to the whitefly population on blackgram, thereby increasing the whitefly population significantly to spread of YMV disease which shows the higher disease incidence percentage in the genotypes with higher leaf total sugar content (Gurumurthy et al., 2019). The rapid accumulation of total sugar in the susceptible genotypes increase the susceptibility further and also increase the rate of transmission of YMV. The accumulation of total sugar was observed to be lower in the resistant genotypes which reduce the whitefly preference and less disease incidence was observed in such genotypes. These results are in line with the findings of Ashfaq et al., (2014); Rajitha et al. (2018) and Vannirajan et al. (2019) in blackgram
 
Phenolic content
 
A significant variation was observed among the varieties tested for leaf total phenolic content at vegetative and reproductive stages during rabi 2017-18 and 2018-19 (Table 2). The leaf total phenolic content  increased from  vegetative stage to flowering stage in all genotypes in both  the years  The leaf total phenols content is indispensable for the host plant resistance towards pest which acts as an anti-feedant that makes the pest to not show any propinquity towards the genotypes with higher total phenols contents. The leaf phenolic content at vegetative  stage during rabi 2017-18 was observed to be higher in the variety LBG 884 (19.38 mg g^-1 FW) followed by GBG 1 (17.66 mg g^-1 FW) and TBG 104 (17.08 mg g^-1 FW) which are in line with one another. The lowest phenolic content was recorded in the variety LBG 623 (10.26 mg g^-1 FW). At reproductive stage during rabi 2017-18 highest phenolic content was recorded in the variety LBG 884 (22.50 mg g^-1 FW) followed by GBG 1 (20.70 mg g^-1 FW) and TBG 104  (20.20 mg g^-1 FW).



At vegetative stage during rabi 2018-19, higher phenolic content was recorded in the variety LBG 884 (20.42 mg g^-1 FW) followed by GBG 1(18.70 mg g^-1 FW) and TBG 104 (18.13 g g-1 FW) and  which are  on  par with each other and lowest phenolic content was recorded in the variety LBG 623 (11.31 mg g^-1 FW) followed by LBG 787 (12.31 mg g^-1 FW). At reproductive stage during rabi 2018-19 the highest phenolic content was recorded in the variety LBG 884 (23.55 mg g^-1 FW) followed by TBG 104 (22.25 mg g^-1 FW) and GBG 1( 21.75 mg g^-1 FW). The lowest phenolic content was recorded in the variety LBG 623 (14.18 mg g^-1 FW) followed by LBG 787(15.19 mg g^-1 FW) and LBG 752(15.55 mg g^-1 FW).

The rapid accumulation of phenolic compounds immediately after the whitefly infestation in resistant genotypes is the most common adaptive response of resistant genotypes. In blackgram genotypes phytic acid accumulation was also observed along with the phenolic compound accumulation. Accumulation of phenols acts as the precursors for the formation of highly toxic quinines and other substances that determine the whitefly population in those genotypes. The total phenols content not only imparts the antibiosis resistant to whitefly but also acts as a trigger to the anti-oxidative response of plant to the virus infection. 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 YMV 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. These compounds also directly act as activators of protective enzymes like polyphenol oxidases. In the current study, it was observed that the genotypes with high phenols content showed less incidence of YMV and the above results were corroborated with the studies of Tamilzharasi et al., (2020); Rajitha et al., (2018) and Vannirajan et al. (2019) in blackgram.
 
Tannin
 
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. The tannins  content  increased from  vegetative stage to flowering stage in all genotypes in both the  years.  The tannins  content at vegetative stage during rabi 2017-18 was observed to be higher in the variety LBG 884 (0.047 mg g^-1 DW) followed by GBG 1 (0.040 mg g^-1DW) and TBG 104 (0.035 mg g^-1 DW) which are on par with each another. The lowest tannins content was recorded in the variety LBG 623 (0.016 mg g^-1 DW) followed by LBG 787(0.020 mg g^-1 DW). At reproductive stage during rabi 2017-18 highest tannins content was recorded in the variety LBG 884 (0.052 mg g-1 DW) followed by TBG 104 (0.041  mg g^-1DW) and GBG1 (0.040 mg g^-1 DW). The lowest tannins content was recorded in the variety LBG 623 (0.027 mg g^-1 DW) followed by LBG 752 (0.032 mg g^-1 DW).

At vegetative stage during rabi 2018-19, higher tannins content was recorded in the variety LBG 884 (0.045 mg g^-1 DW) followed by GBG 1(0.038 mg g-1DW) and TBG 104 (0.033 g g^-1DW) and are on  par with each other and lowest tannins content was recorded in the variety LBG 623 (0.014 mg g^-1 DW) followed by LBG 787 (0.018 mg g-1 DW). At reproductive stage during rabi 2018-19 the highest tannins content was recorded in the variety LBG 884 (0.050 mg g^-1 DW) followed by TBG 104 (0.039  mg g^-1 DW) and GBG 1 (0.038 mg g^-1 DW). The lowest tannins content was recorded in the variety LBG 623 (0.025 mg g^-1DW) followed by LBG 752(0.030 mg g^-1 FW).

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 tannins are of two types present in the plants those are of condensed tannins and hydrolysable tannins. Condensed tannins are the type of tannins that are involved in imparting resistance against aphids and whitefly. The astringent nature of the condensed tannins imparts anti-feedant character to the leaves of resistant genotypes. 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 YMV resistant varieties of blackgram (Taggar et al., 2014). In this study, tannins content in the dried leaves of blackgram genotypes showed that higher the tannin content lesser the whitefly incidence and the similar results were reported by Taggar et al. (2014) in blackgram.

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 LBG 884 (74.59/cm² of leaf area) (Plate 1A) followed by GBG 1 (72.52/cm² of leaf area) and TBG 104 (68.40/cm² of leaf area)  and the lower trichome density was observed in the variety LBG 623 (20.43/cm² of leaf area) followed by LBG 787 (48.06/cm² of leaf area). At reproductive stage during rabi 2017-18 the highest trichome density was recorded in the variety LBG 884(67.82/cm² of leaf area) followed by GBG 1 (65.00/cm² leaf area) amd TBG 104(61.51/cm² leaf area)  and the lowest trichome density was recorded in the variety LBG 623 (14.86/cm² of leaf area) followed by LBG 787( 41.40/cm² of leaf area).

At vegetative stage during rabi 2018-19 the highest trichome density was recorded in the variety LBG 884 (72.64/cm² of leaf area) followed by GBG 1 (70.57/cm2 of leaf area) and TBG 104 (66.45/cm² of leaf area). The lowest trichome density was observed in the variety LBG 623 (18.48/cm² of leaf area) (Plate 1B) followed by LBG 787(46.10 /cm2 of leaf area). At reproductive stage during rabi 2018-19 the highest trichome density was recorded in the variety LBG 884 (65.87/cm² of leaf area) followed by GBG 1 (63.05/cm² leaf area) and TBG 104 (59.56/cm² leaf area) the lowest was recorded in the variety LBG 623 (12.91/cm² of leaf area) followed by LBG 787(39.45 /cm² of leaf area).

Variations in trichome density of LBG 884 and LBG 623 are depicted in Plate 1. The trichome density showed a significant effect on whitefly population in blackgram varieties. This trichome density plays an important role as a morphological anti-feedant which reduces the pest attack and subsequently lowers the disease incidence and the plants with high trichome density showed less YMV symptoms. The higher trichome density hinders the free movement of white fly nymphs and adults making the genotypes with higher trichome density less preferable.The resistant varieties showed higher values of trichome density The study was emphasized on trichome density which showed that the genotypes with high trichome density was less effected by YMV in blackgram (Devi et al., 2019; Taggar and Gill, 2012 and Suman et al., 2018).


 
SCMR (SPAD Chlorophyll meter reading)
 
The SPAD Chlorophyll meter reading (SCMR) is the actual value of a factor representing the number of chlorophyll molecules present per unit leaf area (Table 5). In the present study, SCMR values varied significantly among the genotypes at different growth stages during both the seasons of rabi 2017-18 and 2018-19. All the genotypes showed an increase in the SCMR values from 30 DAS to 50 DAS.At vegetative stage during rabi 2017-18 the higher SCMR value (Table 5) was recorded in the variety LBG 884 (45.12) followed by GBG 1 (44.70) and TBG 104 (43.10) .The  lower SCMR was observed in the variety LBG 623  (32.21) followed by LBG 787(40.65). At reproductive stage during rabi 2017-18 the higher SCMR value was recorded in the variety LBG 884  (53.17) followed by GBG 1 (51.27) and TBG 104(50.27).The lower SCMR  value was recorded in the variety LBG 623 (37.13) followed by LBG 787 (45.25).



At vegetative stage during rabi 2018-19 the higher SCMR value was recorded in the variety LBG 884 (47.12) followed by TBG 104 (45.10) and GBG 1(44.70) which are on par with each other. The lower SCMR value was observed in the variety LBG 623 (24.21) followed by LBG 787 (42.65). At reproductive stage during rabi 2018-19 the higher SCMR value was recorded in the variety LBG 884 (55.17) followed by GBG 1(53.27) and TBG 104 ( 52.27) which are at par with each other. The lower SCMR value was recorded in the variety LBG 623 ( 25.13) followed by LBG 787 (47.25).

The genotypes with darker leaves were less preferred by whiteflies. The SCMR value is a parameter that can be recorded easily with in the field and all the remaining morpho-physiological and biochemical parameters that impart resistance to YMV disease showed a highly significant correlation with SCMR. The above characteristic features of SCMR made it a dependable and easy screening technique for YMV resistant genotypes. SCMR gives the absorbance of chlorophyll molecules in the leaf tissue of specific area measured at Infrared and far infrared wavelengths which give the arbitrary value of chlorophyll content of that area. The chlorophyll content also affects the whitefly population and YMV disease, decreased chlorophyll values were recorded in susceptible genotypes. The genotypes with higher chlorophyll values showed tolerance to YMV disease; similar results were found by Rajitha et al., (2018). in blackgram
 
Disease score
 
The disease score values recorded at reproductive stage in all the genotypes showed observable variation among the varieties (Table 6). The varieties LBG 884, TBG 104 and GBG 1 showed a score of ‘2’ during both rabi 2017-18 and 2018-19 which indicates highly resistant type. The varieties LBG 808 and LBG 806 showed a score of ‘3’ during rabi 2018-19 and 2017-18 indicating resistant category. The variety LBG 752 showed a score of ‘4’ during rabi 2017-18 and ‘4’ during rabi 2018-19 indicating that, the variety LBG 752 was moderately resistant to YMV disease under field condition. The variety LBG 787 recorded scores of 5 and 5 during rabi 2017-18 and 2018-19 respectively falling under the category of moderately susceptible genotype. The variety LBG 623 showed a score of ‘9’ during both rabi 2017-18 and 2018-19 showing highly susceptible nature. These results are in confirmation with the studies of Raj et al., (2019) in screening greengram varieties and Gopi et al., (2018), Devi et al., (2019) and Vannirajan et al. (2019) in screening of blackgram genotypes.


 
Yield
 
Higher seed yield was noticed in the variety LBG 884 (1191 and 1240 kg ha^-1) in both rabi 2017-18 and 2018-19 due to least YMV disease incidence followed by GBG 1 (1070 and 1120 kg ha^-1) and TBG 104 (1010 and 1060 kg ha^-1) in both the seasons (Table 7). The lower seed yield was observed in genotype LBG 623 (700 and 751 kg ha^-1) in both the seasons. The susceptible genotypes showed high YMV disease incidence with lower yields, whereas, the resistant genotypes recorded higher yields with less YMV incidence (Devi et al., 2019 and Subedi et al., 2016).


 
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). Devi et al., (2019); Taggar and Gill (2012); Taggar et al., (2014) and Vannirajan et al. (2019) similar results were reported  in blackgram.

From these results it can be inferred that the higher phenols, tannins content and trichome density and less sugar content traits contributed YMV resistance in LBG 884, GBG 1 and TBG 104 of blackgram. The varieties  LBG 884, GBG 1 and TBG 104 were grouped under highly resistant category with higher phenols, tannins content and trichome density. The varieties LBG 806 and LBG 808 were categorized under resistant group. The variety  LBG 752 was grouped under moderately resistant group. The variety LBG 787 and LBG 623 were grouped under moderately susceptible and highly susceptible category respectively with lower phenol and tannins and trichome density and higher sugar content.