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Legume Research, volume 45 issue 3 (march 2022) : 363-370

Survey for Yellow Mosaic Disease Occurring on Blackgram [Vigna mungo (L.) Hepper] and Weed Hosts in Four Major Blackgram Growing Districts of Andhra Pradesh and Characterization of Associated Viruses using Coat Protein Gene

Vallabhaneni Tilak Chowdary1, V. Manoj Kumar1, P. Kishore Varma2, B. Sreekanth3, V. Srinivasa Rao4
1Department of Plant Pathology, Agriculture College, Acharya NG Ranga Agricultural University, Bapatla-522 101, Guntur, Andhra Pradesh, India.
2Department of Plant Pathology, RARS, Acharya N.G. Ranga Agricultural University, Anakapalle-531 001, Guntur, Andhra Pradesh, India.
3Department of Crop Physiology, AICRP on Cotton, RARS, Lam, Acharya N.G. Ranga Agricultural University, Guntur-522 034, Andhra Pradesh, India.
4Department of Statistics and Computer Applications, Agricultural College, Acharya N.G. Ranga Agricultural University, Bapatla-522 101, Guntur, Andhra Pradesh, India.

  • Submitted17-07-2021|

  • Accepted25-10-2021|

  • First Online 27-11-2021|

  • doi 10.18805/LR-4738

Cite article:- Chowdary Tilak Vallabhaneni, Kumar Manoj V., Varma Kishore P., Sreekanth B., Rao Srinivasa V. (2022). Survey for Yellow Mosaic Disease Occurring on Blackgram [Vigna mungo (L.) Hepper] and Weed Hosts in Four Major Blackgram Growing Districts of Andhra Pradesh and Characterization of Associated Viruses using Coat Protein Gene . Legume Research. 45(3): 363-370. doi: 10.18805/LR-4738.

ABSTRACT

Background: Yellow mosaic disease (YMD) caused by Yellow mosaic virus is one of the major constraints in the pulse production in Andhra Pradesh (A.P.) due to fast evolution of strains, like Mungbean yellow mosaic India virus (MYMIV). Keeping this in view, a survey was undertaken in the major blackgram growing districts of A.P. to know the YMD incidence in blackgram and weed hosts and were characterized based on genetic features by comparing with other YMV isolates from different hosts and locations across the world.

Methods: Roving survey was conducted during rabi 2019-20 in major blackgram growing districts of A.P. viz., Krishna, Guntur, West Godavari and Prakasam districts for YMD incidence. Blackgram plants showing characteristic symptoms were collected as representative samples from each mandal along with the suspected weed plants and were subjected to amplification using coat protein (CP) specific primers followed by molecular characterization. Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny.

Result: Out of the four districts surveyed, the highest disease incidence was recorded at Machavaram village of Prakasam district (43.22%), whereas least disease incidence was recorded at Chinaganjam village of Praksam district (2.4%). Six weeds viz., Ageratum conyzoides, Amaranthus viridis, Parthenium hysterophporus, Vigna trilobata, Abelmoscus moschatus, Desmodium laxiflorum have showed positive result in PCR amplification with MYMIV specific coat protein primers. Four isolates from blackgram samples and two from weed plants shared 94.85 to 99.58% nucleotide identity among themselves. 

INTRODUCTION

Blackgram is one of the important pulse crops grown throughout India which is consumed in the form of ‘dal’ (whole or split, husked and un-husked) or perched, its high value of lysine makes it an excellent complement to rice in terms of balanced human nutrition. India is one of the major producers of blackgram cultivated in 349.91 lakh ha with a production of 232.41 lakh tonnes in 2017 (Directorate of Economics and Statistics, M/A, GoI.). In India, during kharif 2019-20, area covered under blackgram is 37.52 lakh ha. with the major contributions from Madhya Pradesh (16.50 lakh ha), Uttar Pradesh (7.01 lakh ha), Rajasthan (4.56 lakh ha), Maharashtra (2.87 lakh ha), Karnataka (0.687 lakh ha) and Andhra Pradesh (0.11 lakh ha) (Blackgram Outlook Agricultural Market Intelligence Centre, PJTSAU, 2019). In Andhra Pradesh, the majority of the area is under Krishna, Guntur, West Godavari and Prakasam districts covering an area of 2.03 lakh ha. out of 3.18 lakh ha with a production of 2.35 lakh tonnes in which major area is covered in Krishna, Guntur, West Godavari and Prakasam districts. The area under blackgram cultivation is being reduced from 4.55 lakh hectares in 2015-16 to 3.18 lakh ha in 2018-19 (Statistical Abstract Andhra Pradesh 2019). The decline in the production was mainly due to biotic (viral and fungal diseases) and abiotic factors.
       
Among the viral diseases, YMD is one of the major constraints in the pulse production. It was reported that YMD was caused by four viruses - MYMV (Mungbean yellow mosaic virus), MYMIV (Mungbean yellow mosaic india virus), HgYMV (Horsegram yellow mosaic virus) and DoYMV (Dolichus yellow mosaic virus) across southern Asia which are collectively known as Yellow mosaic viruses (YMVs) (Qazi et al., 2007). In general, MYMV was the major isolate infecting pulse crops in Western and Southern India, Thailand and Indonesia whereas MYMIV isolate in Central, Eastern, Northern India, Pakistan, Bangladesh, Nepal and Vietnam (Malathi and John, 2009). However, first report of MYMIV from Andhra Pradesh was reported by Reddy et al., (2015).
       
Survey was undertaken in major blackgram growing districts of Andhra Pradesh to study the incidence of YMD in blackgram and weeds. The collected samples were subjected to molecular characterization of coat protein (CP) in blackgram and suspected weed isolates and compared with other YMV’s isolates occurring on different hosts and locations.

MATERIALS AND METHODS

Field and molecular lab studies were conducted in Department of Plant Pathology, Agricultural College, Bapatla and a part of molecular work was conducted at Plant Virology Lab, National Research Centre for Banana (NRCB), Tiruchirapalli.
 
Survey
 
The survey was carried out to have first hand information on the occurrence of YMD in blackgram and weed hosts during rabi 2019-2020 in major blackgram growing districts of Andhra Pradesh viz., Krishna, Guntur, West Godavari and Prakasam districts. In each district, three mandals and in each mandal three villages and three fields from each village were surveyed. Five locations, i.e., at four corners of the field and one at the centre and in each location of one sq. m, incidence of YMD was recorded based on the symptoms. Disease incidence was calculated by using the formula given below. Samples of infected blackgram plants and suspected weed hosts were collected in plastic bags and brought to the laboratory for further analysis.
       
Data pertaining to the variety sown, predominant weed species, preceding/succeeding crop, surrounding crop was also recorded during the survey (Table1).
                                      
                             
 
 

Table 1: Incidence of yellow mosaic disease in major blackgram growing districts of Andhra Pradesh.


 
Molecular characterization of CP gene
 
DNA extraction
 
The total genomic DNA was isolated from the representative sample from each mandal (12 samples) and weed hosts (15 samples) by using a protocol given by Rouhibakhsh et al., (2008) with slight modification.
 
PCR
 
The genomic DNA was subjected to PCR with coat protein specific primers (Table 2). The PCR amplifications were carried out in thermal cycler (Eppendorf master cycler gradient). The components of the reaction mixture were 1μl of genomic DNA (50 ng), 0.5 μl each of 10 pm forward and reverse primers, 2.5 μl of 10X PCR buffer, 1.5 μl of 25 mM MgCl2, 0.5 μl of 10 mM dNTPs, 0.5 μl of Taq DNA polymerase (5U/μl) in nuclease free water to make up final volume up to 25 μl. (All the above reaction components were procured from Thermo scientific company). PCR profiles for different primers used are mentioned in the (Table 3). Subsequently, PCR product was run on one per cent agarose gel for an hour at 80 volts.
 

Table 2: Primers used in the present study.


 

Table 3: PCR profiles for YMV’s amplification.


       
Each representative YMV isolate showing the highest disease incidence in that corresponding district were selected. YMV-KR (Krishna), YMV-GN (Guntur), YMV-WG (West Godavari) and YMV-PR (Prakasam) and two isolates from weed hosts YMV-ABEL (Abelmoschus moschatus), YMV-DES (Desmodium laxiflorum) were used for molecular characterization. The total genomic DNA isolated was amplified with MYMIV specific primers (~1065 bp) and the amplified products were outsourced to sequencing facility at Bangalore (Eurofins Genomics Bangalore India). Low quality nucleotide sequences at both the ends were trimmed by Chromas (version 2.6.6) and the trimmed sequences were assembled using Bioedit (version 7.0.5.3). Sequence identities among the isolates were obtained using Species Demarcation Tool (SDTv1.2) (Muhire et al., 2014). Assembled sequence of partial CP gene was evaluated using BLASTN (www.ncbi.nlm.nih.gov) to know the identity and homology with other reported YMV isolates. For further analysis, reference sequences of CP gene sequences of YMV available in database were downloaded from NCBI website. Partial CP gene sequences of YMV were compared with seventeen reported YMV isolates from different parts of the world infecting various pulses and ToLCNdV is taken as an out group member. All the sequences were aligned using MUSCLE algorithm of MEGAX (version 10.1.1). The phylogenetic tree for CP gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny of MEGAX (Sudhir et al., 2018).

RESULTS AND DISCUSSION

Roving survey
 
Survey was undertaken in four major blackgram growing districts of Andhra Pradesh (12 mandals and 36 villages). Blackgram crop was in the mid-vegetative stage to pod formation stage across the surveyed fields. Plants showing the symptoms of YMD like general yellowing, mosaic pattern, stunted growth of the plant and curved pods were recorded. To confirm the presence of YMV, representative sample from each surveyed mandal was subjected to PCR using MYMV and MYMIV specific primers, where in, all samples were tested positive for MYMIV with an amplicon length of ~1065 bp (Fig 1).
 

Fig 1: PCR amplification of Coat protein (CP) gene of MYMIV in blackgram isolates collected from different locations in Andhra Pradesh.


       
The survey results indicate that there was a significant difference in the per cent disease incidence of YMD during rabi 2019-20 in different districts of Andhra Pradesh ranging from 2.43% (Chinaganjam village of Ongole mandal in Prakasam district) to 43.22% (Machavaram village of Naguluppalapadu mandal in Prakasam district) (Table 1). High disease incidence recorded in some villages of Prakasam district might be attributed to the cultivation of blackgram as preceding crop that might have resulted in higher whitefly population. Further, large scale cultivation of susceptible local varieties could also be ascribed to high disease incidence (Archith et al., 2017). Prevalence of YMV susceptible weed hosts in Prakasam district might have helped in the perpetuation of the virus during offseason. Earlier studies in Koppal district of Karnataka emphasized the role of favourable weather conditions for biological development of whitefly for horizontal spread of virus during summer months and the role of weed hosts in perpetuation of YMD (Meghashree et al., 2017).
       
Mean disease incidences of major blackgram growing districts of Andhra Pradesh viz., Krishna, Guntur, West Godavari and Prakasam districts were 20.51, 14.58, 18.73 and 23.03% respectively and the mean incidence among all the surveyed districts was 19.21% with the lowest incidence in Guntur district (14.58%) followed by West Godavari (18.73%), Krishna (20.15%) and Prakasam (23.03%). The lowest disease incidence in Guntur, West Godavari and Krishna district might be ascribed to the rice fallow pulse cropping system with concomitant sowing dates and consequently less whitefly population.
       
Panduranga et al., (2012) surveyed Warangal and Khammam districts of Telangana and reported that the variation in YMD incidence was due to variation in sowing time of the crop and prevalence of different weeds which have contributed to the population dynamics of whitefly and subsequent incidence of YMD.
       
Similar studies done in different agro climatic zones of Karnataka (Jayappa et al., 2017) reported the highest incidence of YMD on sole crop of mungbean compared to intercrop and established a positive correlation between YMD incidence and vector population. Manjunatha et al., (2013) reported the prevalence of B Biotype whitefly (Bemesia tabaci) and large-scale cultivation of susceptible varieties led to the increase in the incidence of YMD in Southern Karnataka. Salam et al., (2011) surveyed Dharwad, Gadag, Haveri, Gulbarga and Bidar districts of Karnataka and reported the highest disease incidence of YMD in mungbean in Bidar district and the variation in disease incidence over locations was attributed to the varied population dynamics of whitefly as influenced by temperature and relative humidity.
 
Weed hosts
 
During survey, suspected weeds were identified, varied symptoms were observed (Table 4) and samples were collected for conformational studies using specific primers. Out of 15 weed species, six weeds viz., Ageratum conyzoides, Amaranthus viridis, Parthenium hysterophporus, Vigna trilobata, Abelmoscus moschatus, Desmodium laxiflorum were tested positive for MYMIV showing an amplicon length of 4 1065 bp (Fig 2). None of the weed species was tested positive for MYMV. Begomovirus species cause numerous diseases in cultivated crops and weeds of the families Cucurbitaceae, Solanaceae, Fabaceae and Malvaceae which pose a threat to cultivation in different parts of India (Varma and Malathi, 2003).
 

Table 4: Weed hosts collected in the present study.


 

Fig 2: PCR amplification of Coat protein (CP) gene of MYMIV in different weed species collected from different locations in Andhra Pradesh.


       
Naimuddin et al., (2014) reported Ageratum conyzoides, a common weed growing throughout the year, as a new host for MYMIV and concluded that prevalence of this weed act as an important source of primary inoculum and is responsible for recurrence of yellow mosaic disease in grain legumes in Northern India. Bhanu et al., (2015) reported different weeds viz., Ageratum conyzoides, Amaranthus viridis, Parthenium hysterophporus and Vigna trilobata as alternate hosts by detecting them using MYMV specific primers whereas, in the present study MYMIV was noticed in the same weeds which emphasize the distribution of new species of YMD in Andhra Pradesh. Marabi et al., (2017) suspected ten weeds as potential weed hosts for YMV based on the visual symptoms but only four viz., Ageratum conizoides, Vigna trilobata, Corchorus olitorius and Alternanthera sessilis were found to be true alternate hosts of MYMIV when tested using MYMIV specific markers. Cross inoculation studies conducted by Deepa et al., (2017) using the insect vector whitefly (Bemisia tabaci) could not transmit the YMD to Parthenium hysterophorus concluding the weed as non host for MYMIV. In the present study, 15 weed hosts were suspected positive for YMD based on visual symptoms, of which six were found positive for MYMIV. Hence, molecular confirmation of the YMD suspected samples with MYMIV specific molecular markers could be a reliable technique to detect alternative hosts of MYMIV.
 
Molecular characterization of CP gene of Isolates
 
The representative YMD isolates, were sequenced and submitted to NCBI with the following accession numbers, YMV-KR (MZ475993), YMV-GN (MZ475994), YMV-WG (MZ475996), YMV-PR (MZ475995), YMV-ABEL (MZ475997) and YMV-DES (MZ475998).
       
A phylogenetic dendrogram based on an alignment of coat protein gene sequences of six isolates under the study with sequences available in NCBI database (Fig 3) revealed that all the test isolates clustered into MYMIV group and formed a separate clad with other species of the begomoviruses i.e., MYMV, HgYMV, DoYMV. All the test isolates showed a unique feature of close clustering with MYMIV isolates originated from South India and distantly related with the foreign isolates (Indonesia and Bangladesh). The isolates YMV-GN, YMV-PR, YMV-DES, YMV-ABEL formed a closed clustering with Andhra Pradesh isolates from Guntur (JN181003), Tirupati (JX110618) and Krishna (MT270285) whereas, the isolates YMV-KR and YMV-WG formed a close clustering with the isolates from Andhra Pradesh (KJ747961, JN181004), Tamil Nadu (KJ747962) and Karnataka (MN698280). The CP gene is known to be highly conserved gene in the family Geminiviridae which effectively predicts discrete strains, species and taxonomic lineage of begomoviruses that has been accepted by ICTV as desirable marker for virus identity when a full-length genomic sequence is not available (Rybicki 1998). Coat protein genes have traditionally proven useful for plant virus identification and classification (Mayo and Pringle 1997). According to these guidelines, the present isolates from crop and weed hosts i.e., YMV-KR, YMV-GN, YMV-PR, YMV-WG, YMV-ABEL, YMV-DES belongs to MYMIV. It is clear from the above results that six isolates causing YMD of blackgram in Andhra Pradesh are closely related to MYMIV (old world geminiviruses) than MYMV (New world geminiviruses).
 

Fig 3: Phylogenetic comparison based on nucleotide sequences of coat protein (CP) gene of MYMIV isolates of present study with previously reported YMV isolates from India and other countries.


       
The sequence analysis of six isolates using SDTv1.2 revealed that YMV-GN and YMV-DES shared 99.58% homology which is the highest among the tested isolates whereas least homology of 94.85% was found between YMV-PR and YMV-WG. Whereas the two weed species YMV-DES and YMV-ABEL shared an identity of 99.30% at nucleotide level. The isolates collected from crop species i.e., YMV-KR, YMV-GN, YMV-WG, YMV-PR shared a nucleotide similarity of 94.85 - 97.77%. Reddy et al., (2015) reported two variants of MYMIV infecting blackgram in Andhra Pradesh based on CP gene homology. Naimuddin et al., (2011) elucidated the causal agent associated with YMD of V. mungo var. silvestris, as MYMIV based on CP gene homology of more than 97% at nucleotide level and 99% at amino acid level. Usharani et al., (2004) studied MYMIV infecting soybean in Tamil Nadu and found that CP region has more than 96% homology with MYMIV isolates and less than 80% with MYMV isolates. The earlier studies in Southern part of India on different isolates of YMV infecting blackgram, greengram and cowpea revealed no variation among MYMV isolates. However, YMV infecting horsegram was found similar to HgYMV which could be deciphered using CP gene. The study disclosed the efficiency of CP gene in early detection of YMV infection of pulses by MYMV and HYMV (Maheshwari et al., 2014).
       
Chaitanya et al., (2020) reported the presence of both the species of begomoviruses i.e., MYMIV and MYMV in Andhra Pradesh where MYMIV was found predominant compared to MYMV which are contradictory to the present results wherein, only MYMIV is reported in all the districts surveyed. These results suggests that MYMIV has replaced MYMV due to evolutionary changes.

CONCLUSION

Previous survey reports for YMD in Andhra Pradesh revealed the prevalence of MYMV and MYMIV, but in the present study causal organism for the YMD was reported as MYMIV in all four surveyed districts. The molecular characterization of different isolates revealed that MYMIV population in Indian subcontinent is highly conserved irrespective of hosts and locations based on CP gene sequences. The variation in the incidence level might be due to stage of the crop at which infection occurred, variety grown, cropping system, cropping pattern and the management practices followed by the farmers. Further research on YMD management should be based on the development of resistant varieties using the genetic information of MYMIV blackgram isolates from various districts to develop coat protein mediated resistance due to its highly conserved nature over the years.

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