Co-existence of Mungbean yellow mosaic India virus with Cowpea mild mottle virus on soybean (Glycine max.) in North-East India

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Co-existence of Mungbean yellow mosaic India virus with Cowpea mild mottle virus on soybean (Glycine max.) in North-East India

https://doi.org/10.21203/rs.3.rs-4358766/v1

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Soybean (Glycine max. L.) plants with virus-like systemic mosaic and mottling symptoms were observed in both experimental and commercial fields in Assam State, India. Begomo and Carlaviruses are the most destructive viruses infecting legume crops in India. A diagnostic survey was carried out to assess the prevalence of major viruses in soybean growing tracts of Assam and North-East India. Infected plants showing severe mosaic, mottling, and crinkling of leaves were collected. Molecular indexing was performed using reverse transcription polymerase chain reaction (RT-PCR) with a coat protein gene-specific primer of Begomovirus (Mungbean yellow mosaic virus) and a Carlavirus degenerate primer corresponding to the coat protein gene. Apparently, amplifications were seen for both Begomovirus and Carlavirus infections alone and in combination. According to the sequence analysis, the combination infections in soybean are caused by the Mungbean yellow mosaic virus (MYMIV) and the Cowpea mild mottle virus (CPMMV). This is the first molecular report of the co-existence of the Mungbean yellow mosaic India virus strain and the Cowpea mild mottle Carlavirus on soybean in N.E. India and India.

Soybean (Glycine max. L.), is the most important source of animal protein feed and vegetable oil in the world. It is considered an economically significant leguminous crop containing significant quantities of vital amino acids, oil, minerals, vitamins, and nutrient storage. It is also the world's most important legume, used for a variety of purposes such as human nutrition such as soy milk, oil, animal feed, and soil improvement. Soybean has a higher protein level than any other major vegetable or animal food source, containing an average of 40% protein. On a dry matter basis, soybean seeds contain around 20% oil, which is 85% unsaturated and cholesterol-free. It is highly industrialized in wealthy nations, supplying over one-fourth of the world's food. According to 2023 USDA data, the major global soybean producers are Brazil, the United States, Argentina, China, India, Paraguay, Canada, Russia, and Ukraine. In India, soybean is cultivated on an acreage of 12.7 million ha, with a production of around 12 million MT and a productivity of 0.9 T/ha (USDA 2023). Soybean is susceptible to infection by a number of viruses that significantly affect yield and quality. Typically, virus infections in soybean result in yield losses of 10–30%, while losses of 50–100% have been documented during severe outbreaks. Mungbean yellow mosaic India virus (MYMV) is one of the most significant viruses that infect soybeans globally. However, many other viruses, such as soybean mosaic virus (SMV), cucumber mosaic virus (CMV), cowpea mild mottle virus (CPMMV), and alfalfa mosaic virus, have also been reported (Gupta et al., 2022, Hill and Whitham, 2014).

Mungbean yellow mosaic India Virus (MYMIV), a type member of the genus Begomovirus in the family Geminiviridae, is considered a major constraint to the productivity of legume crops across the Indian subcontinent. The majority of legume crops are infected by MYMIV, including soybean (Glycine max), mungbean (Vigna radiata), black gram (V. mungo), pigeon pea (Cajanus cajan), moth bean (Vigna aconitifolia), and common bean (Phaseolus vulgaris) (Usharani et al., 2004; Karthikeyan et al., 2004). MYMIV infection causes an average yield loss of 10–100%, depending on crop genotype and infection stage (Bashir et al., 2006). MYMIV is persistently transmitted through the whitefly, Bemisia tabaci, and is not transmitted through soil or seed (Malathi and John, 2008). Virus infected plants exhibit severe yellow mosaic, chlorosis, and mottling of the leaf, which finally results in complete yellowing, drying, and withering of the leaf (Varma and Malathi, 2003). The genome of MYMIV is found to be isometric and geminate, containing two DNA molecules, DNA and DNA B, of 2726 and 2775 nucleotides, respectively (Hull 2004). DNA A encodes two open reading frames (ORFs) in the viral sense, (ORF AV1—coat protein gene; ORF AV2—movement or pre-coat protein gene), and the viral complementary sense strand encodes five proteins (ORF AC1—replication-associated protein, Rep; ORF AC2—transcriptor activator protein, TrAP; ORF AC3—replication enhancer protein, Ren; AC4—symptom expression; and AC5—pathogenicity determinant) (Li et al., 2015). The DNA B harbors two genes, viz., BV1 and BC1, encoding nuclear shuttle protein and movement protein, respectively (Cavalvizhi et al., 2015).

Cowpea mild mottle virus (CPMMV) is considered another important virus affecting legume crops around the world and was first reported from cowpea in Ghana (Zanardo and Carvalho, 2017). CPMMV, the causal agent for stem necrosis in soybeans, caused huge havoc in soybean production and was considered a major economic problem for Brazilian soybean production in the early 2000s (Almeida et al., 2005). CPMMV is a single-stranded positive-sense RNA virus that belongs to the genus Carlavirus of the family Betaflexiviridae and consists of 8200 nucleotides, a cap structure [m7GpppG] linked at the 5’ terminus, and a poly A tail at the 3’ end of the genome (Adams et al., 2012). The genome is organized into 6 open reading frames (ORFs) that encode a specific function (ORF 1: RdRp replicase protein activity; ORFs 2, 3, and 4: triple gene block proteins TGB1, TGB2, and TGB3 essential for virus movement; ORF 5: coat protein activity; and ORF 6: nucleic acid-binding activity) (Lukhovitskaya et al., 2009). Symptoms associated with CPMMV in soybean may vary from mild (mosaic, yellowing, crinkled and blistered leaves, and vein clearing) to severe (necrosis, dwarfing, and bud blight) (Zonardo et al., 2014). Generally, the carlavirus group is transmitted by aphids in a non-persistent manner (Fauquett et al., 2005); however, CPMMV is transmitted by whiteflies (Bemisia tabaci) in a non-persistent manner (Zanarado and Carvalho, 2017). Recently, CPMMV was found infecting legume crops in central and southern India (Baranwal et al., 2015), but the distribution, importance, and biological characteristics of CPMMV carlavirus in the soybean of North-East India are largely unknown. The characterization of plant virus is essential as it will be helpful in disease management using modern technologies like RNAi, CRISPR and many gene editing technologies (Routhu et al., 2020).

In this study, we collected soybean plants with virus-like symptoms from different regions of North-East India to investigate the presence of viruses. A survey was conducted in soybean-growing districts of North-East India during 2021-2022 to investigate the natural occurrence of mosaic disease in soybeans, and the disease's incidence was found to be high. Infected plants showed severe yellow mosaic, chlorosis, and mottling at the later stage of infection. The prevalence of whiteflies in infected soybean fields prompted us to investigate the possibility of a begomovirus infection. To the best of our knowledge, this is the first report of co-existence of MYMIV and CPMMV infecting the soybean of India.

Virus Isolates

Leaves of virus infected soybean plants expressing typical symptoms were collected from soybean growing districts of Assam and North-East India during 2021-22. Survey was conducted in four districts namely Jorhat comprising of Upper Brahmaputra Valley Zone, Lakhimpur and Dhemaji comprising of North Bank Plain Zone and Nagaon of Central Brahmaputra Valley Zone which comprise the major soybean growing areas of N.E.India (Fig. 1). In each field, pools of leaves from three to five symptomatic plants were collected in plastic bags and frozen with dry ice. The virus sap extracts were mechanically sap inoculated on to susceptible soybean variety. Sap extracts were prepared from 1 g of virus infected leaves homogenized with 15ml of 0.01M phosphate buffered saline, pH 7.0.Mechanical inoculation onto host plants was done by rubbing with celite powder on newly emerged leaves and the inoculums finally rinsed with tap water. The inoculated plants were maintained at 25+2˚C under green house with insect proof cage.

Total RNA/DNA isolation

The presence of mixed symptoms and whiteflies suspected the existence of multiple viruses in soybean and thereby it was suggested to extract total RNA and DNA from the soybean leaves. Total DNA was isolated from the soybean leaf samples following a modified CTAB extraction method (Murray and Thompson, 1980). In other case, total RNA was isolated from each leaf sample by the TRIzol Reagent (Invitrogen, United States). Approximately 100 ng of extracted RNA were used for cDNA synthesis using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, U.S.A.) following standard protocols.

Polymerase chain reaction

For the detection of MYMIV, specific primers were designed from the conserved region of coat protein region from other isolates (FJ663015.1, MK409378.1, MK409377.1, DQ389146.1, MN698273.1, JQ004982.1). Firstly, multiple sequence alignment was performed by CLUSTAL W program (Clustal Omega) and the primers were designed within a conserved region in Primer 3 selection tool (http://primer3.ut.ee). For the detection of CPMMV Carlavirus group in soybean leaves, a set of degenerate primers were used which were used in the study conducted by Gaspar et al., in 2008. The primers that were used in the present study along with their product length and annealing temperatures were listed in Table 1. Polymerase chain reaction (PCR) was performed in of reaction mixture using 1X PCR reaction buffer, 2.5mM of MgCl2, 10mM of each dNTPs, 10 pmols of each primer, 2.5 U/μl of Taq DNA polymerase (Thermo Scientific^TM) and 100 ng of DNA template and final volume was adjusted to 25μl by adding nuclease free water. The reaction mixtures were amplified by using a thermal cycler (Applied Biosystems, 96 well veriti^TM thermal cycler). The PCR amplification program consists of initial denaturation at 94°C for 2 minutes, followed by 35 cycles of denaturation (94°C for 30 seconds), annealing (56/55°C for 1 min), extension (72°C for 1 min), and a final extension step of 72°C for 5 minutes. After the PCR program, the products were separated on a 1.2% (w/v) agarose gel electrophoresis stained with ethidium bromide in 1 X TAE buffer for 2 hours at 70V and then visualized in a gel documentation system (BioRad, USA).

Table 1: Primeres used for the detection of CPMMV carlavirus and MYMIV in soybean of N.E. Inida

Primer Name	Nucleotide sequence	Target molecule	Product size	Annealing temp.
CPMMV Carlavirus	CPMMV F- GGTTTYYYCGTGCCCACTYA	Coat protein	524 bp	56˚C
	CPMMV R- TTTTNNNGTTATTTTCCTGGTN
MYMIV	MYMIV CP F- GGTTGTGAYYYACCATGTANG	Coat protein	940 bp	55˚C
	MYMIV CP R- TACACNGGATTTGAYYCANGAG

Cloning, Sequencing and Phylogenetic analysis

The amplified PCR products were eluted and purified using Nucleospin (Machery-Nagel) accoerding to the manufactures instructions. The purified PCR product was ligated into pTZ57R/T using T4 DNA ligase (New England BioLabs) and finally transformed into DH5α strain of E.coli cells following standard transformation protocols. Recombinant clones were selected through colony PCR and restriction digestion. The selected recombinant clones were sequenced by BigDye Terminator kit and characterized through Di-deoxy chain terminator method on a Applied Biosystem 373A sequencer (Applied Biosystems crop., U.S.A.).

The genome sequences obtained in the study and some reported isolates retrieved from NCBI database were aligned by CLUSTAL W method in MEGA X software (Routhu et al., 2022). Phylogenetic analysis was performed using Neighbour Joining method using the best-fitting model: Jukes-Cantor model. For reliability of the phylogenetic trees, the bootstrap test with 1,000 replications was used. For the construction of CPMMV phylogenetic tree, Indian citrus ringspot virus (Mandavirus, Alphaflexiviridae) was used as an outgroup. Tomato Leaf curl Virus (Begomovirus, Geminiviridae) was used as the outgroup for MYMIV phylogenetic tree construct.

Occurrence of CPMMV and MYMIV in N.E. India

Soybean plants expressing typical mosaic, mottling and crinkling symptoms on their leaves were collected from major soybean growing districts of N.E. Inida (Figure 2a-2c). Through visual observation it was found that the incidence of viral disease is higher and it ranges from 60-80% of disease incidence. The mechanically inoculated plants exhibited mosaic symptoms on leaf as observed in field conditions after 7-8 days of post inoculation (Fig 2d).

Polymerase chain reaction (PCR)

From the isolated RNA/DNA, polymerase chain reaction was performed using specific primers. Through PCR, MYMIV was detected using CP gene specific primers by yielding an expected amplicons size of 524bp in gel documentation system (Figure 3). CPMMV virus was also successfully detected by generating an amplicon size of 940bp in agarose gel electrophoresis (Figure 4). The amplified PCR products were successfully cloned into pTZ57R/T and finally confirmed through colony PCR and restriction digestion analysis.

Sequencing and Phylogenetic analysis

The positive clones were subjected to Sanger di-deoxy sequencing, the obtained sequence were deposited in NCBI database with accession numbers (MYMIV-OQ309176; CPMMV-OQ309178). Sequence analysis revealed that isolated MYMIV (OQ309176) coat protein gene shares 97-95% similarity with other MYMIV isolates around the world and maximum similarity (97.14%) with Indian isolate (GQ387501). By Neighbor joining method in MEGA X software, it was observed that isolated MYMIV shares maximum homology with Indian isolates (Figure 5). On the other hand, isolated CPMMV sequence (OQ309178) shares 99-95% similarity with other CPMMV isolates around the world, where maximum similarity (99%) is found with CPMMV (EF635061), an isolate from Brazil. The isolated CPMMV sequence consists of 510 nucleotides of coat protein gene, 332 nucleotides of 11K gene and 88 nucleotides of short untranslated sequence before the poly (A) tail. Phylogenetic analysis based on neighbor joining method revealed that OQ309178 shares maximum homology with Brazil isolates therby forming a common clade (Figure 6). Presently, mosaic disease caused by MYMIV and CPMMV is the major viral disease in soybean crops of N.E. India.

The North-Eastern part of India is considered a hotspot of biodiversity, comprising 7 states: Assam, Arunachal Pradesh, Meghalaya, Nagaland, Tripura, Manipur, and Mizoram. In recent years, due to the increase in demand for edible oils in India, soybean is considered an alternative to meet the needs of edible oils in India as well as in North-East India. Besides its oil content, soybean is a rich source of protein and a decent source of carbohydrates and fats. Due to its position as an important crop, more research into disease management can contribute to the better production of the crop. Proper identification of the pathogen is necessary to adopt proper control measures for the disease.

A large number of viruses infect soybean, due to the vector being active and present in appreciable numbers. However, to date, no studies have been undertaken to identify the viruses infecting soybean in North-East India. In our study, we surveyed mosaic disease incidence in soybean during 2021-22 in N.E. states and observed severe yellow mosaic disease symptoms (Kumar et al., 2016). The incidence of typical mosaic and mottling symptoms on soybean leaf and prevalence of whiteflies in soybean growing areas led us to investigate the presence of multiple viruses in an association with begomoviruses in soybean.

Yellow mosaic disease (YMD) is considered as the major constraint to the productivity of grain legumes across the Indian subcontinent (Varma et al. 1992). Mungbean yellow mosaic India virus (MYMIV), one of the four legumoviruses in begomovirus group initially restricted to northern India, is now reported in North East India mainly infecting Soybean, blackgram, greengram and various other pulses. MYMIV is now increasing its host range by infecting various weed plants and perpetuating during offseason. The CP gene is the most conserved gene within the Geminiviridae family. This sequence, which accurately predicts the strains, species, and taxonomic lineage of begomoviruses, has been acknowledged by ICTV as a suitable identifier for virus identity in the absence of a full-length genomic sequence (Rybicki 1998). In the present study, MYMIV infecting soybean leaves was successfully detected using CP gene specific primers by generating a band size of 52bp in agarose gel. The sequence analysis of present isolate (OQ309176) with other isolates in NCBI database shows that jorhat isolate has 97% homology with MYMIV and less than 79% homology with MYMV. In phylogenetic analysis, the present isolate shares common homology with Indian isolates and form a common clade in the phylogenetic tree. The viruses involved with cowpea golden mosaic disease in Kanpur were identified as MYMIV based on CP gene homology greater than 95% (Naimuddin & Akram 2010). MYMIV infecting soybeans in Tamil Nadu shares a CP area with more than 96% similarity with MYMIV isolates and fewer than 80% similarity with MYMV isolates. On the basis of phylogenetic analysis, it was determined that YMV infecting soybean in Tamil Nadu formed a distinct cluster with MYMIV (Usharani et al., 2004). According to the most recent Begomovirus classification rules, if the nucleotide identity at the coat protein or full DNA-A sequence is 85–93%, they will be deemed strains of the same virus, 94–100% will be considered variants, and 85% will be considered different species (Fauquet et al. 2008). According to these guidelines, the present isolate belong to MYMIV having 97% homology with other Indian isolates of MYMIV at nucleotide level.

CPMMV was first reported on infecting cowpea in Ghana during 1973 (Brunt and Kenten, 1973). In the 1980s, it was found to be infecting soybeans in Thailand and the Ivory Coast (Thouvenel et al., 1982). CPMMV led to disease outbreaks and considered as a major problem for soybean production in Brazil (Zanardo et al., 2014). In India, CPMMV induces systemic mottling, mosaic and leaf deformation on legume crops of mungbean and urdbean causing considerable loss in production and productivity (Baranwal et al., 2015). Polymerase chain reaction (PCR) can easily amplify specific DNA sequences and has various advantages over other traditional diagnostic procedures. In the present study, a set of degenerate primers were used for the detection of Carlavirus group of RNA virus. These degenerate primers can detect three different viruses simultaneously viz., Potato virus S, Cowpea mild mottle virus and Cole latent virus (Gaspar et al., 2008). In our study, we have successfully detected the presence of cowpea mild mottle virus infection in soybean by generating an amplicons size of 940 bp. Through sequence analysis it was found that isolated CPMMV sequence (OQ309178) from Jorhat shares maximum homology with isolates from Brazil and there by forming a common clade in phylogenetic analysis. Phylogenetic study based on comparison of coat protein sequence of twelve CPMMV isolates with other isolates revealed a unique feature of clustering with CPMMV isolates originated from Brazil and South-East Asia. The isolated CPMMV sequence from Jorhat consists of partial CP gene, 11K gene and short untranslated sequence before poly A tail. The presence of CPMMV in soybean in North India was detected using carlavirus specific primers by amplifying an expected size of 940bp (Yadav et al., 2013).

To our knowledge, this is the first report for combined infection of Begomovirus and Carlavirus in soybean of N.EIndia as well as in Inida. This preliminary study aimed to aid future research on Begomovirus and Carlavirus co-infections with other plant viruses. Nevertheless, the mechanism behind the interaction of viruses from various groups on a single host during mixed infections has yet to be investigated in detail.

Acknowledgement: The Authors are grateful to Department of Plant Pathology, Assam Agricultural University, Jorhat and All India Cooerdinated Research Project on Soybean (AICRP-Soybean) for providing lab facilities to carry on the research work.

Conflicts of interest/Competing interests: The authors declare no conflict of interest.

Consent for publication (include appropriate statements): The authors declare that there is no objection for publication of this article.

Availability of data and material (data transparency): All the data and material are available to the corresponding author.

Code availability (software application or custom code): Not Applicable

Authors' contributions: All authors have equal contribution.

Conflict of Interest: The authors declare that they have no conflict of interest.

Research involving human participants and/ or animals: This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent: Informed consent was obtained from all individual participants included in the study.

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Co-existence of Mungbean yellow mosaic India virus with Cowpea mild mottle virus on soybean (Glycine max.) in North-East India

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