Molecular detection and characterisation of begomovirus causing bean yellowing disease in Sri Lanka

Bean (Phaseolus vulgaris L.) is one of the major vegetable crops cultivated in tropical, sub-tropical and temperate regions in the world. Bean yellowing disease still a serious viral threat to bean cultivation causing severe yield reduction. In this study, polymerase chain reaction (PCR) assays using degenerate (universal) primers were conducted with the aim of developing molecular techniques to detect the virus. Two degenerate primer pairs, namely, Deng 540/541 association of a begomovirus with the BYD by giving the desired core coat protein amplicons of 520 bp and 550 bp, respectively. The resulting amplicons were subjected to DNA sequencing and the sequence data were analysed to determine the phylogenetic and molecular evolutionary relationships with other related begomovirus sequences obtained from the GenBank. The analysis revealed that the virus associated with BYD (BYVD-GN-SL-Partial) is closely related to Horsegram [ isolate (HgYMV-LK: 09-Bean) reported in Sri Lanka. Further, the DNA sequence of BYVD-GN-SL-Partial was distinctively clustered with the Indian HgYMV sequences and positioned in between the Mungbean yellow mosaic virus (MYMV) sequences.


INTRODUCTION
Bean (Phaseolus vulgaris L.) is one of the major vegetable crops cultivated in Sri Lanka up to an extent of 8000 ha and has an annual production of 40,000 mt.This accounts for 23 % of the total extent of up country vegetables (Anon, 2010).Among the biotic constraints of bean threat in Sri Lanka due to the spread of bean yellowing disease (BYD) in epidemic proportions.BYD is caused by the Horsegram yellow mosaic virus (HgYMV), which et al., 2010) and continues to be the major threat for bean cultivation in Sri Lanka.Hence, accurate detection and characterisation of the virus is a crucial prerequisite when formulating management strategies.Early diagnosis of causal agents is immensely important in managing such outbreaks (Ozalan et al., 2006).Using symptomatology alone to detect viral diseases is not always accurate due to similar symptoms being caused by other pathogens.Polymerase chain reaction (PCR) technique using method widely adopted in virus detection.Therefore in this study, two degenerate (universal) primer pairs, namely, Deng 540/541 and AV494/AC1048 were used to detect begomovirus associated with beans in Sri Lanka.Certain weeds act as alternate hosts of begomovirus, hence management of the viral diseases becomes more challenging.The present study also focused on using molecular methods to identify local weed species capable of harbouring BYD causing viruses.Begomoviruses have been categorised as major quarantine pests in the world (Hamilton, 2000).Comparing viral genome sequence information is vital in predicting possible evolutionary pathways and transboundary movements, which is ultimately important in executing appropriate reveal important molecular level information useful for effective management of the BYD in Sri Lanka.

September 2016
Journal of the National Science Foundation of Sri Lanka 44(3)

Bean leaf samples for PCR
Tender bean leaf samples of various cultivars showing typical BYD symptoms were collected from farmer namely, Kandy (e.g.Kadugannawa and Yatinuwara), Matale (e.g.Naula), Nuwara Eliya (e.g.Seetaeliya) and Badulla (e.g.Bandarawela).Apparently healthy leaf samples of the same variety were also collected from the same locations as control samples.All the leaf samples were collected when the plants were at the early same growing season.water to make up the required volume.

Seeds of
The reaction mixture was subjected to one cycle of initial denaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 ° extension at 72 °C for 10 min for Deng 540 and Deng 541 primers.

Optimising PCR conditions for AV 494 and AC 1048 primers
The gradient PCR reactions were performed in the range °C under two dilutions of genomic DNA template [1/10 (200 -250 ng) and 1/25 (80to determine the optimum annealing temperature for AV 494 and AC 1048 and optimum dilution of DNA, respectively.

Analysis of PCR products
The PCR products were analysed on 1.0 % agarose gel at 60 V for 1 hr in 1× TBE buffer [100 mM Tris (pH 8), size was estimated using a 100 bp DNA size marker (Promega, WI, USA).

DNA sequencing of partial genome of BYD
A fragment of the core coat protein gene present on the genome A component of the causal virus of BYD was for DNA sequencing to Genetech Pvt. Ltd., Sri Lanka.The sequence information obtained was subjected to homology search using the basic local alignment tool (BLAST) (Altschul et al., 1990) available at www.ncbi.nlm.nih.gov/BLAST(GenBank).

Phylogenetic and molecular evolutionary analysis
Thirty accessions showing higher homology with the DNA sequence obtained from the present study (designated as BYVD-GN-SL-Partial) were selected from the DNA databases available at www.ncbi.nlm.nih.gov/BLAST (GenBank).Using MEGA 4.0 software package, phylogenetic and molecular evolutionary relationships of the DNA sequences were determined by neighbour-joining method at 1000 bootstrap value (Tamura et al., 2007).

Appearance of symptoms on weed plants inoculated
Virus-like symptoms appeared on the leaves of H. corymbosa after two weeks of inoculation using different to the typical yellowing symptoms seen on beans and showed crinkling of both adaxial and abaxial surfaces of the leaves (Figure 1).In contrast, Ageratum spp.did not show any virus-like symptom (data not shown).

PCR by degenerate primers
obtained from DNA extracted from all bean leaf samples, which showed characteristic yellowing symptoms, and also in the two virus-inoculated weed species (i.e.Ageratum spp.and H. corymbosa) with Deng 540/541 gradient PCR using AV 494/AC 1048 primers showed faint bands of ~550 bp at the annealing temperature of 56 °C for 2 min (data not shown) and was found to be non-consistent over PCR assays performed within the ° DNA extracted from apparently healthy leaves of bean plants and weeds with any of the above primer pairs.

Phylogenetic and molecular evolutionary analysis
Closely related sequences to the DNA sequence of BYVD-GN-SL-Partial obtained through DNA homology search are given in Table 2. Phylogenetic and molecular evolutionary relationships obtained after analysing the 30 sequences using MEGA 4.0 software at 1000 bootstrap value are illustrated in Figure 3.The present study provides important information on rapid and precise detection of the BYD, its genetic characteristics and evolutionary relationships, and potential weed hosts.This information can be utilised to formulate future management strategies useful for effective management of the virus through crop management and quarantine practices.

Figure 2 :Figure 3 :
Figure 2: inoculated weeds using Deng 540/541 primers.(Lane M: 100 bp marker; Lanes 1, 2, 3, and 4: BYD infected beans collected from Kandy, Matale, Nuwara Eliya and Badulla Districts respectively; Lane 5: Ageratum spp.; Lane 6: H. corymbosa) (2010) from beans in Sri Lanka, indicating the genetic similarity of BYVD-GN-SL-Partial with HgYMV-LK:09-Bean.Furthermore, both sequences (BYVD-GN-SL and HgYMV-LK:09-Bean) formed a distinct cluster with other HgYMV isolates, namely, HgYMV-CP-Ban-IN, HgYMV-A-Hg-Ban1-IN, HgYMV-A-Lb-Ban2-IN and HgYMV-A-Hg-Coi-IN reported in India suggesting the close genetic relationship of the begomovirus associated with bean yellowing disease in Sri Lanka with the HgYMV isolates in India.Thus phylogenetic analysis provides information that the begomovirus associated with BYD can be a pseudorecombinant of begomoviruses associated with the Horsegram yellow mosaic disease caused by HgYMV and mungbean yellow mosaic disease caused by mungbean yellow mosaic virus (MYMV).Similarly, Qazi et al. (2007) have suggested pseudorecombination as a possible reason for clustering of MYMV-(IN:Mad:Sb) along with HgYMV.Further, higher degree of genetic similarity of the mungbean yellow mosaic virus with HgYMV-(IN:Coi) has been reported by Girish and Usha (2005).

Transmission of the virus into weed plants using
Hedyotis corymbosa (Rubiaceae) and Ageratum spp.(Compositae) were sown in pots containing sterilised soil maintained in insect-proof cages.The seeds of Ageratum spp.were obtained from stock cultures maintained at the Division of Plant Pathology,

Table 1 :
(3)uences of selected degenerate primersJournal of the National Science Foundation of Sri Lanka 44(3)September 2016for 5 min at 12,000 rpm.The pellet was then washed with 70 % ethanol and dried at room temperature for 20 min.Finally the pellet was dissolved in 100 µL of 1× TE buffer and stored at -20 °C.
Monger et al. (2010)has been isolated from beans in Sri Lanka byMonger et al. (2010).BYVD-GN-SL-Partial also showed isolates reported from India in Horsegram, Lima bean and French bean (Table2).The DNA sequence of the present study formed a sub cluster with HgYMV-LK:09-Bean isolated byMonger et al.