Chief EditorJ. S. Sandhu
Print ISSN 0250-5371
Online ISSN 0976-0571
NAAS Rating 6.67
Impact Factor 0.8 (2023)
Identification and Confirmation of Resistance in Mungbean [Vigna radiata (L.) Wilczek] Derivatives to Mungbean Yellow Mosaic Virus (MYMV)
- Email email@example.com
First Online 21-12-2020|
Methods: Four interspecific derivatives (VGGRU 1, VGGRU 2, VGGRU 3 and VGGRU 4) generated by making crosses between mungbean VRM (Gg) 1 and rice bean (TNAU RED) along with the susceptible check VRM (Gg) 1 were agroinoculated with the MYMV infectious clone VA 239 (KA30 DNA A + KA27 DNA) and are further substantiated through whitefly transmission studies from the artificially reared whiteflies.
Result: The agroinoculation results revealed that among the four interspecific derivatives, VGGRU 1 was found to be completely resistant to MYMV. The substantiation of the obtained result through whitefly transmission also revealed that 24 h Acquisition Access Period (AAP) and 24 h Inoculation Access Period (IAP) with Bemisia tabaci able to cause 65% infectivity in susceptible plant VRM (Gg) 1 and zero infectivity in VGGRU 1 and the results were PCR confirmed for the presence of viral DNA.
MYMV is found to be one of the prime viral diseases of pulses in Tamil Nadu state. Development of resistant mungbean cultivars against MYMV has long been a major objective in disease resistance breeding programmes. Owing to the fact that MYMV is transmitted by whiteflies, the confirmation of resistance through field screening always remains a hassle. The MYMV symptoms may not always appear in the fields due to some factors such as environmental changes, whitefly genotypes, host factors etc., which makes failure in the development of infections in field and also it makes intricate to identify the true resistant lines.
In this perspective and with a view towards developing a reliable laboratory screening protocol for assessing resistance/susceptibility of mungbean accessions against MYMV, Rogers et al., (1986) developed a new technique called “Agroinfection” using the Ti plasmid of Agrobacterium for viral infection and demonstrated on tomato golden mosaic virus. In this technique, the infectious viral clones are introduced into plants using A. tumefacians and this A. tumefaciens subsequently deliver the infectious viral DNA from the T-DNA into a plant cell and initiates the infection. Thus, this technique produces a uniform disease outbreak among the test genotypes rather than the natural infestation and acts as a finer tool for the confirmation of resistance. Practicability of using an in vitro agroinoculation technique in MYMV screening was demonstrated by various researchers on urdbean, mungbean and soybean (Jacob et al., 2003; Usharani et al., 2005; Haq et al., 2010; Karthikeyan et al., 2011; Sudha et al., 2013). With the above leads, the study was carried out for (i) confirmation of resistance in mungbean and rice bean derivatives to MYMV through agroinoculation (ii) assessing the MYMV disease intensity at different day intervals on the infected plants (iii) further validation of the resistance was done with artificial whitefly transmission.
MATERIALS AND METHODS
A total of four interspecific derivatives (VGGRU 1, VGGRU 2, VGGRU 3 and VGGRU 4) generated by making crosses between mungbean VRM (Gg)1 and ricebean (TNAU RED), obtained from a previous breeding programme made by Dr. M. Pandiyan, Agricultural College and Research Institute, Echankottai, Thanjavur and one susceptible mungbean variety VRM (Gg)1 as check were used in this study.
MYMV construct for agroinoculation
Balaji et al., (2004) formerly constructed an infectious clone named VA 239 (KA30 DNA A + KA27 DNA B) which was isolated from the YMV infected black gram leaves collected from Vamban village of Pudukkottai District, Tamil Nadu and mobilized into the A. tumefaciens strain C 58. The above mentioned construct was obtained and utilized for agroinoculation in the present study.
The experiment on agroinoculation was conducted in the Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore. Agroinoculation was done on surface sterilized overnight sprouted seeds of four interspecific derivatives (VGGRU 1, VGGRU 2, VGGRU 3 and VGGRU 4) and VRM (Gg) 1. Agrobacterium tumefaciens strains harbouring the partial tandem repeat clone VA 239 were grown to 1 Optical Density at 600 nm in 2 mL AB minimal medium (pH 7.0) containing the antibiotics like streptomycin (150 mg L-1), spectinomycin (50 mg L-1) and tetracycline (5 mg L-1) at 28°C at 220 rpm. From this, 1 mL of the culture was taken and inoculated into another 50 mL of AB minimal medium (pH -7.0) containing the above mentioned antibiotics and grown to 1 OD at 600 nm at 28°C at 220 rpm. The culture was spinned at 4000 rpm for 10 min at 25°C. The obtained cells were re-suspended in 50 mL of AB minimal medium (pH -5.6) with 100 μL acetosyringone (100 μm). Seed coat of the pre-soaked sprouted seeds was removed by using forceps and pricked around the hypocotyl region and were immediately immersed in the culture of A. tumefaciens which carries MYMV construct VA239. After the overnight incubation, seeds were washed with distilled water and sown in pots containing autoclaved sand and vermiculite in the ratio of 1:1. Agroinoculated plants were maintained in a growth chamber at 25°C with 60-70% relative humidity and a photoperiod of 16/18 h, for the proper growth of the plants twice in a week hoagland’s solution was applied and the plants were transferred to green house after 15 days for symptom observation. The development of yellow mosaic symptoms on the plant in a given time is considered as susceptible plant and the absence of yellow mosaic symptoms on the plant is scored as resistance against the disease. The percentage of infectivity after agroinoculation was calculated based on the number of infected plants to the portion of the number of plants inoculated.
DNA extraction and PCR analysis
The leaf samples collected from both agroinoculated control and infected plants after symptom expression were subjected to DNA isolation using CTAB method as described by Sudha et al., (2009). The DNA was quantified using a Nano-Drop spectrophotometer (ND-1000 Spectro photo meter, NanoDrop Technologies, USA) and quality was checked in 0.8% agarose gel. DNA concentration was normalized to 25 ng/µl for PCR reaction after the necessary quality and quantity checks. The amplification was done using the MYMV coat protein (CP) gene-specific primers FP1 5¢GCGGAATTACGATACCGCC3' and RP1 5'GATGCAT GAGTACATGCC3' (Richa Maheswari et al., 2014) for both control and agroinoculated plants in MyCycler (BioRAD, USA). A standard volume of 10 μl reaction consists of 6 μl PCR mix 2X SMART master mix (readymade mix of taq polymerase, dNTPs and PCR buffer), 1.0 µL of 10 µM primer (forward and reverse each) (First Base, Singapore), 2.0 µL of 25 ng µL-1 DNA. The temperature cycles were as follows: 5 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 56°C and 1 min at 72°C. The final elongation step was extended to 10 min at 72°C and finally maintained at 4°C. The amplified products were separated on a 1.2% ethidium bromide pre-stained agarose gel and are visualized on a digital gel documentation and image analysis system (Alpha Imager 1200, Alpha Innotech Corp., USA).
Confirmation of viral DNA in plants
DNA was extracted in ten days interval from both the control and infected plants of susceptible VRM (Gg)1 and resistant VGGRU 1 after the expression of symptoms i.e. 25, 35, 45 and 55 days after sowing (DAS) and the infectivity was confirmed by PCR (35 cycles) assay of virus using coat protein primers.
Collection and maintenance of whiteflies
To initiate transmission studies, adults of Bemisia tabaci were collected from different fields of cotton and brinjal in Tamil Nadu Agricultural University, Coimbatore and mass cultured in insect-proof glass house using different host plants (cotton, brinjal and bhendi). The whitefly culture was maintained by regular transfer of fresh batch of plants after every six weeks. The new adults after three generations were collected using aspirator and used for transmission studies.
DNA extraction and mt goi gene amplification for white fly species confirmation
The total DNA was isolated from the maintained whiteflies through lysis method (Zeidan and Czosnek, 1991) and subjected to amplification using LCOI 1490 forward primer 5'GGTCAACAAATCATAAAGATATTGG 3' and HCOI 2198 reverse primer 5' TAAACTTCAGGGTGACCAAAAAATCA 3' (Folmer et al., 1994) for white fly species confirmation. The PCR was performed with initial denaturation at 94oC for 3 minutes, followed by 40 cycles each consists of 30 secs at 94°C, 40 secs at 53°C, 1 min at 72°C followed by final extension for 20 minutes at 72°C. The PCR products were gel purified and sequenced in Bioserve Biotechnologies (India) Private Limited, Hyderabad.
White fly transmission studies
Ten to fifteen B. tabaci adults were collected in nylon clip cages from the maintained culture with the help of an aspirator and are allowed for starvation. After starvation, the clip cage containing whiteflies were clipped on to the MYMV agro-infected mungbean plants and allowed to feed for an acquisition period of 24h. After 24 h acquisition access period (AAP), B. tabaci adults were removed from MYMV agro-infected mungbean plants and transferred into a separate insect free chamber containing healthy VGGRU 1 and VRM (Gg) 1 plants for inoculation access period (IAP) of 24 h. After 24 h of IAP, the B. tabaci adults were removed and the plants were sprayed with an insecticide (Dimethoate 30 EC at 1 ml/L) and evaluated for MYMV symptom development 10-20 days later.
Further to substantiate the agroinoculation results, attempt was also made for whitefly screening. Artificial rearing of whitefly was done for obtaining pure MYMV inoculum and also to avoid the mixed infections that are commonly seen during field level.
PCR assay for virus in viruliferous and non-viruliferous whiteflies
Total DNA was extracted from the viruliferous and non-viruliferous whiteflies through lysis method as mentioned above and observed for the presence of MYMV CP gene using polymerase chain reaction (PCR). The PCR reaction mixture includes the MYMV coat protein (CP) gene-specific primers with the same temperature conditions and reaction volume as mentioned above for MYMV infected leaf samples.
RESULTS AND DISCUSSION
On screening for MYMV resistance through agroinoculation, among the four interspecific derivatives of mungbean (namely VGGRU 1, VGGRU 2, VGGRU 3, VGGRU 4 that are generated by making crosses between mungbean VRM (Gg) 1 and ricebean (TNAU RED), the line VGGRU 1 was found to be highly resistant, while the others along with the susceptible check [VRM (Gg)1] developed yellow mosaic symptoms in their trifoliate leaves from 15 to 25th days after agroinoculation and there was no symptom development in the control (Fig 1).
In the field screening, three lines namely VGGRU 2, VGGRU 3 and VGGRU 4 were found to be resistant based on the rating scale (Table 1) suggested by Singh et al., (1988) but developed MYMV symptoms on agroinoculation (Table 2). It is clear from the result that the resistance at field conditions may be due to some factors that restrict the insect transmission. To confirm the results, the agroinoculation experiment was repeated twice and comparable results were obtained and by this technique 50-100% infectivity could be observed. Similar to the above results, Jacob et al., (2003) followed a single strain strategy of agroinfection by co-delivery of MYMV DNA A and DNA B from one Agrobacterium strain and yielded 100% agroinfection. Usharani et al., (2005) conducted infectivity analysis of MYMV through agroinoculation in soybean isolate and obtained infectivity of about 70-95 per cent. Similarly, Sudha et al., (2013) reported that the MYMV infectivity on agroinoculated plants ranged between 0-100 per cent even for the field-resistant genotypes. Based on the above result, it is clearly understood that the resistance can be easily identified through reliable agroinoculation technique and the results were further confirmed through PCR to verify the presence of viral DNA in the host genome using oligonucleotide primers that are specific to the MYMV coat protein gene of DNA A. The expected amplicon size of 703 bp was obtained in all the infected samples which showed yellow mosaic symptoms (Fig 2). These results are in accordance with the reports of Usharani et al., (2005) and Sudha et al., (2013), signifying the presence of viral DNA in agroinoculated symptomatic plants and their absence in asymptomatic plants.
Interestingly, VGGRU1 was found to exhibit higher level of resistance and did not develop any mosaic symptoms even after 2 months from inoculation. Further confirmation by PCR with CP gene specific primer using DNA samples taken at ten days interval i.e. 25 35, 45 and 55 DAS from both resistant (VGGRU1) and susceptible (VRM (Gg)1 showed a visible increase in the intensity of the amplified bands of VRM (Gg)1 from 25 to 55 DAS but the intensity of the bands in VGGRU1 resistant line was comparatively less and reduced to half its intensity at 35 DAS and there was no amplification as the days get older (Fig 3). The presence of viral load was found high in the susceptible plants and the viral load for resistance gradually decreases as plants get matured due to activation of defense genes. Usharani et al., (2005) and Kayalvizhi et al., (2015) also reported the same results which indicated that viral intensity on the resistant plant gets decreased when the plants get matured (reproductive stage) and it may be due to the expression of defense genes in resistant plants.
Whitefly transmission experiment was attempted to prove the Koch’s postulates for MYMV under controlled conditions during the course of present investigation. The genotype of non-viruliferous whiteflies collected from different fields of cotton and brinjal was identified and confirmed as Asia I type after partial gene sequencing (accession no: MK333460). This genotype results correlated with the findings of Prasanna et al., (2015) and Elango et al., (2015). The PCR results using MYMV CP primers for both viruliferous and non-viruliferous whiteflies after 24 h of AAP revealed the presence of MYMV amplicons only on the viruliferous whiteflies which contains MYMV in it and there was no amplification in non-viruliferous whiteflies which had no AAP treatment along with the water blank (Fig 4). The transmission experiment was done after whitefly genotype confirmation and the results revealed that 24 hr AAP and 24 hr IAP with B. tabaci were able to cause 65% infectivity in susceptible plant VRM (Gg) 1 and zero infectivity in VGGRU 1 (Table 3). The results are in confirmation with the report of Govindan et al., (2013), where MYMV DNA fragment of 703 bp was observed only in viruliferous adults of B. tabaci. Similarly, Usharani et al., (2005) defined the host range of MYMV using both whitefly transmission and agroinoculation. It was found that the vector was able to transmit the virus from agroinoculated plants to the healthy plants.
- Anonymous, (2018). Annual Report 2016-17, Ministry of Agriculture and Farmers Welfare (Department of Agriculture, Cooperation and Farmers Welfare), Directorate of Pulses Development, Vindhyachal Bhavan, New Delhi.
- Balaji, V., Vanitharani, R., Karthikeyan, A.S., Anbalagan, S., Veluthambi, K. (2004). Infectivity analysis of two variable DNA B components of Mungbean yellow mosaic virus-Vigna in Vigna mungo and Vigna radiata. Journal of Biosciences. 29: 297-308.
- Biswas, K.K. and Varma, A. (2000). Identification of variants of mungbean yellow mosaic Gemini virus by host reaction and nucleic acid spot hybridization. 53(2): 134-141.
- Cayalvizhi, B., Nagarajan, P., Raveeendran, M., Rabindran, R., Selvam, N.J., Bapu, J.K., Senthil, N. (2015). Unraveling the responses of mungbean (Vigna radiata) to mungbean yellow mosaic virus throug 2D-protein expression. Physiological and Molecular Plant Pathology. 90: 65-77.
- Dhakar, K., Gupta, V.K., Rathore, M.S., Gaur, R.K. (2010). Virus resistance and gene silencing in plants infected with begomovirus. Journal of Applied Sciences. 10: 1787-1791.
- Ellango, R., Singh, S.T., Raina, H., Chaubey, R., Ramamurthy, V.V., Rana, V.S., Naveen, N.C., Asokan, R., Gayatri Priya, N., Riaz Mahmood., Rajagopal, R. (2015). Distribution of Genetic Groups in India. Environmental Entomology. 44(3): 1-7.
- Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. (1994). DNA primers for amplifcation of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular marine biology and biotechnology. 3(5): 294.
- Govindan, K., Nagarajan, P., Angappan, K. (2014). Molecular studies on transmission of mung bean yellow mosaic virus (MYMV) by Bemisia tabaci Genn. in Mungbean. African Journal of Agricultural Research. 9(38): 2874-2879.
- Habib, S., Shad, N., Javaid, A., Iqbal, U. (2007). Screening of mungbean germplasm for resistance/tolerance against yellow mosaic disease. Mycopathology. 5: 89-94.
- Haq, Q.M.I., Rouhibakhsh, A., Ali, A., Malathi, V.G. (2011). Infectivity analysis of a black gram isolate of Mungbean yellow mosaic virus and genetic assortment with MYMIV in selective hosts. Virus Genes. 42: 429-439.
- Jacob, S.S., Vanitharani, R., Karthikeyan, A.S, Chinchore, Y., Thillaichidambaram, P., Veluthambi, K. (2003). Mungbean yellow mosaic virus-Vi agroinfection by co delivery of DNA A and DNA B from one Agrobacterium strain. Plant Disease. 87: 247-251.
- Karthikeyan, A., Sudha, M., Pandiyan, M., Senthil, N., Shobhana, V.G., Nagarajan, P. (2011). Screening of MYMV resistant mungbean (Vigna radiata (L) Wilczek) progenies through agroinoculation. International Journal of Plant Pathology. 2: 115-125.
- Nariani, T.K. (1960). Yellow mosaic of mung (Phaseolus aureus L.). Indian Phytopathology. 13: 24-29.
- Paul, P.C., Biswas, M.K., Mandal, D., Pal, P. (2013). Studies on host resistance of mungbean against Mungbean Yellow Mosaic Virus of lateritic zone of West Bengal. The Bioscan. 8: 583-587.
- Prasanna, H.C., Kanakala, S., Archana, K., Jyothsna, P., Varma, R.K., Malathi, V.G. (2015). Cryptic species composition and genetic diversity within Bemisia tabaci complex in soybean in India revealed by mtCOI DNA sequence. Journal of Integrative Agriculture. 14(9): 1786-1795.
- Richa Maheshwari, Panigrahi, G., Angappan, K. (2014). Molecular characterization of distinct YMV (Yellow mosaic virus) isolates affecting pulses in India with the aid of coat protein gene as a marker for identification. Molecular Biology Reports. 41: 2635-2644.
- Richa Maheswari. (2008). DNA finger printing of Mungbean yellow mosaic virus (MYMV) affecting pulses. M.Sc Thesis, Tamil Nadu Agricultural University, Coimbatore, India pp. 48.
- Rogers, S.G., Bisaro, D.M., Horsch, R.B., Fraley, R.T., Hoffmann, N.L., Brand, L., Elmer, J.S., Lloyd, A.M. (1986). Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cell. 45(4): 593-600.
- Sidhu, J.S., Mann, R.S., Butter, N.S. (2009). Deleterious effects of cotton leaf curl virus on longevity and fecundity of whitefly, Bemisia tabaci (Gennadius). Journal of Entomology. 6: 62-66.
- Singh, G., Sharma, Y., Kaur, L. (1992). Methods of rating yellow mosaic virus of mungbean and urdbean. Plant Disease Research. 7:1-6.
- Sudha Manickam. (2009). DNA isolation protocol for Vigna radiata with free of phenolics. Protocol Exchange. Doi: 10.5281/zenodo.15918.
- Sudha, M ., Karthikeyan, A., Nagarajan, P., Raveendran, M., Senthil, N., Pandiyan, M., Angappan, K., Ramalingam, J., Bharathi, M., Rabindran, R., Veluthambi, K., Balasubramanian, P. (2013). Screening of mungbean (Vigna radiata) germplasm for resistance to Mungbean yellow mosaic virus using agroinoculation. Canadian Journal of Plant Pathology. 35 (3): 424-430.
- Usharani, K.S., Sundernath, B., Haq, Q.M.R., Malathi, V.G. (2004). Yellow Mosaic Virus infecting soybean in northern India is distinct from species infecting soybean in southern and western India. Current Science. 86: 845-850.
- Van Regenmortel, M.H., Fauquet, C.M., Bishop, D.H., Carstens, E.B., Estes, M.K., Lemon, S.M., Wickner, R.B. (2000). Virus taxonomy: Classification and nomenclature of viruses. Seventh report of the International Committee on Taxonomy of Viruses. Academic Press. New York.
- Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 3(5): 294.
- Zeidan, M. and Czosnek, H. (1991). Acquisition of tomato yellow leaf curl virus by the whitefly Bemisia tabaci. Journal of General Virology. 72: 2607-2614.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.