Deployment of yellow mosaic disease resistance in mungbean [Vigna radiata (L.) Wilczek] through interspecific hybridization

 
YMD infected mungbean leaf samples from kharif and summer season were collected during 2013-14. Total DNA was extracted from YMD infected leaf samples and amplified with Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) species specific primers (Naimuddin et al. 2011). PCR reaction mixture was prepared with 40 ng of total genomic DNA along with 1.5 mM magnesium chloride, 1.0X of PCR buffer, 0.15 of dNTP mix, 10 p mol of each forward and reverse primers and 1 U of Taq DNA polymerase as final concentration. Visualization of viral components was done on 1% agarose gel and all the samples were eluted, purified by gel purification kit (Favor Prep GEL/PCR Purification mini kit) and sequenced (Eurofins Genomics India Pvt. Ltd.).  Retrieved sequences were further analyzed using BLASTn programme.
 
Screening of interspecific derivatives lines of mungbean for YMD resistance
 
Short duration interspecific derivative lines along with three resistant checks viz; ML 818, ML 2037 and ML 2056 were evaluated for YMD resistance by modified double infector row technique in kharif season during 2014-16. The observations on whitefly incidence were made twice in the season at 40 and 55 days after sowing. Whitefly population was recorded using Split cage for counting adult whiteflies (Kooner and Cheema 2007). The promising lines were further evaluated by whitefly mediated artificial inoculation technique for confirmation of resistance during 2017. Pedigree of promising interspecific derivative lines of mungbean is given in Table 1.
 

 
Modified double infector row technique
 
The test entries along with checks were sown in kharif season for three consecutive years (2014-2016). Two rows (three meter length) of each test entry were sown between two infector rows of susceptible check SML 1082. Each entry was sown with three replications. All recommended package and practices were followed for raising experimental material. YMD score was recorded on 1-9 scale (Singh and Bhan 1998) at weekly intervals after first appearance of disease on susceptible check till 12^th week (Table 2). Data was also recorded on per cent disease incidence (number of infected plants) and disease severity (number of infected leaves per plant). The formulas used for calculation of per cent disease incident and disease severity were as follows:  


Based on the disease severity, symptom severity grades, designated with numerical values of 0-4 and a scale of response value (0-1) corresponding to such grades was given to each entry. Coefficient of infection was calculated by multiplying the percent disease incidence to the response value. Disease reaction categories were assigned to each test entry on the basis of coefficient of infection as per scale given by Singh and Singh (2000), described in Table 3. The data was analysed using CPCS software.
 

 
Whitefly mediated inoculations under controlled conditions
 
Pure culture of whitefly (Bemisia tabaci) was maintained on cotton plants in pots and kept in cages made up of muslin cloth and black chart to provide dark condition for whitefly to establish on cotton plants. YMD infected plants from field were transplanted in pots and kept in insect proof cages. Non-viruliferous whiteflies from cotton were allowed to feed on YMD infected plant for 24 hours. After 24 hours acquisition access period, these viruliferous whiteflies were released on healthy mungbean plants of susceptible variety SML 1082 for 48 hours kept in insect proof cages separately. The inoculated plants expressed YMD symptoms after 10 days and were further used as inoculum for screening. The viruliferous whiteflies were released on test entries for 48 hours of inoculation access period (IAP) at true leaf stage of seedlings. The YMD inoculum maintained on SML 1082 was also kept among test entries to enhance disease development. After IAP, whiteflies were removed and test entries were transferred to insect proof glasshouse. YMD score was recorded on 1-9 scale (Singh and Bhan 1998) after 2 days of inoculations and at every 2 days interval till 24 days.
 
Confirmation of interspecific derivative lines with SSR marker
 
Genomic DNA was extracted from interspecific derivative line SML 1827 along with parents i.e. ML 881 and RBL 35. A set of 121 Vigna specific SSR markers were used to amplify the genomic DNA of parents. Out of 121, three markers viz; VR 0293, CedG-048 and MBM-050 were polymorphic between parents. These three markers were used to amplify the genomic DNA of SML 1827. The sequence of these SSR markers is given in Table 4. PCR reaction mixture was prepared with 40 ng of total genomic DNA along with 1.5 mM magnesium chloride, 1.0X of PCR buffer, 0.15 of dNTP mix, 5 p mol of each forward and reverse primers and 1 U of Taq DNA polymerase as final concentration. PCR products were visualized under 6 per cent PAGE (Poly Acrylamide Gel Electrophoresis).
 

 
DNA A component of begomovirus amplified as 1000 bp size amplicon and showed 94-99% sequence homology with Mungbean yellow mosaic virus [Urdbean: New Delhi: 2011] Accession no JQ398669.1 in all summer as well as kharif samples. Recently, in an extensive study, MYMV- Urdbean strain was found prevalent in North India (Nair et al. 2017) whereas, both MYMV and MYMIV species were detected at New Delhi (Bag et al. 2014). Since similar strain was observed in both the seasons, the summer mungbean lines were evaluated in kharif seasons, which was highly favourable for YMD epidemics. 
 
Interspecific derivative lines of mungbean with stable YMD resistance
 
In preliminary evaluation by modified double infector row technique for three years (2014-2016), YMD symptoms were observed on fourth week after sowing and disease continued to progress up to 8^th week of sowing. The mean YMD score, PDI and disease severity of three years is given in Table 5. The YMD score, per cent disease incidence and disease severity in all lines differed significantly. However, the influence of seasonal variation was non-significant on disease severity. Least per cent disease incidence (PDI) was observed in case of SML 1825 whereas least disease severity was observed in case of SML1827. On the basis of PDI and disease severity, fourteen lines namely; SML: 1808, 1809, 1810, 1811, 1815, 1822, 1824, 1825, 1826, 1827, 1829, 1829, 1841 and 1843 showed resistant reaction with coefficient of infection less than 9 whereas four lines viz; SML 1828, 1829, 1837 and 1844 were classified as moderately resistant with coefficient of infection ranging from 10-19. The interspecific lines, showing resistant reaction were derived from either mungbean × urdbean or mungbean × rice bean crosses, whereas highly susceptible line was derived from mungbean × mungbean cross. YMD score of these lines were also recorded for comparison with parental data, which was available in form of YMD score only (Table 6). It was observed that the interspecific derivative lines derived from R × R cross between mungbean and ricebean, showed transgressive segregation for YMD resistance. Similarly, in all R × R crosses between mungbean and urdbean, the derivative lines showed YMD score lower than parents, indicating transgressive segregants. Further, in case of all interspecific R × S crosses, the resistance in derivative lines clearly showed introgression from ricebean and urdbean.  Infector row technique has also been used in earlier studies to explore durable resistance in mungbean at hot spot locations over the years. In one study, multi environment testing of mungbean genotypes at 28 hot spot locations was resulted in identification of three genotypes viz; ML 818, ML 1349 and IPM 2-14 with stable resistant reaction across the test environments. GGE biplot analysis revealed that out of six, Pantnagar, Vamban and Ludhiana were the best test environment for screening YMD resistance (Parihar et al. 2017). YMD resistant interspecific lines with good yield performance were selected and subjected to whitefly mediated artificial inoculations for confirmation of resistance. The disease symptoms were appeared after the 10 days of inoculations and the symptoms were appeared first on susceptible check (SML 1082). All the interspecific derivatives lines along with three resistant checks showed no symptoms except SML 1829 (Table 7). The interspecific lines showed similar disease reaction as in case of infector row technique; however, level of disease severity differed in both the cases. The low level of disease severity, observed in case of artificial screening technique due to less inoculum pressure as compared to field conditions. It suggests that double infector technique is more reliable and efficient technique for screening large number of germplasm at hotspot location. Whitefly mediated inoculation technique can be used for confirmation of resistance in elite lines. The yield performance of promising line is given in Table 8. SML 1827 was found to be most promising with 1427 kg/ha yield followed by SML 1829.
 

 

 

 


Identification of donor segments in interspecific derivative line SML 1827 with SSR markers
 
Since SML 1827 was most promising and released as first interspecific mungbean variety for cultivation in Punjab, it was critical to identify donor segments in SML 1827 introgressed from ricebean. Out of 121 markers, three markers, viz; VR-0293, CedG-048 and MBM-050 were found to be polymorphic between ML 881 and RBL 35. The rice bean parent (RBL 35) specific segment of ~200 bp and ~250 bp were amplified in promising interspecific line SML 1827 with markers VR- 0293 and MBM – 050 respectively (Fig 1).
 

Despite of several efforts of extensive resistance breeding, stable resistance against YMD could not be achieved due to highly variable nature of the virus. However, in rice bean and few germplasm lines of urdbean, high level of resistance has been observed. There is an urgent need for transferring this resistance into high yielding mungbean lines. Modern short duration varieties of mungbean offer a great scope for diversification as summer/spring crop between rice and wheat/potato. The landmark summer mungbean variety SML 668 became popular in Punjab and adjoining states as catch cum cash crop. Summer mungbean crop encounters low incidence of YMD in North India due to high temperature and low humidity. However, last several seasons have witnessed severe YMD epidemics in few pockets of Punjab. Upsurgence of whitefly during last few years further aggravated the situation and revisiting the population structure of begomovirus species associated with YMD became urgent. It has implications for breeding YMD resistant short duration lines suitable for summer season-the largest pulse cultivation segment in Punjab. In present study, it was found that Mungbean yellow mosaic virus [Urdbean: New Delhi: 2011] Accession no. JQ398669.1 was prevalent in both summer and kharif season of mungbean. Therefore, strategy was devised to screen all summer germplasm during kharif season which was favourable for YMD epidemic caused by similar viral strain.