Identification and characterization of viruses infecting soybean (glycine max. L) in KwaZulu-Natal, South Africa.

Abstract

Soybean (Glycine max L.) is the world's most important seed legume, primarily used as an oil crop and protein source. Plant viruses are a major limiting factor to soybean production worldwide. Many destructive plant viruses have been discovered to infect soybean on a global scale. In South Africa, few viruses have been detected infecting soybean crops. The studies of viruses on soybean were undertaken several years ago and it is possible that the virus population structure may have evolved over time. The aim of the study was to identify and characterise viruses infecting some soybean cultivars grown in KwaZulu-Natal. Additionally, to determine the incidence of seed transmitted viruses. The first part of the study was undertaken to detect and identify viruses presently infecting soybean grown in the province of KwaZulu-Natal, South Africa and determine the incidence of any seed transmitted viruses. Fifty-four soybean leaf samples exhibiting virus-like symptoms were collected from breeding lines growing in a Plant Pathology disease garden and greenhouses at the University of KwaZulu-Natal during the 2018 - 2019 and 2019 - 2020 growing seasons. Mechanical inoculation using inoculum prepared from the soybean field samples was done on Nicotiana tabacum L. to propagate the viruses in the collected samples. Symptom development was monitored on inoculated N. tabacum plants for 2-3 weeks after inoculation. The field samples were also subjected to Reverse Transcription Polymerase Chain Reaction (RT-PCR) and PCR to detect viruses known to infect soybean worldwide. Generic and specific primers were used to target specific coding regions of the viruses tested. Antibodies specific to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) were used to test for virus presence in the field samples using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The study also focused on determining the incidence of seed transmitted viruses by planting different soybean varieties/lines and testing for virus presence after the plants had germinated. Virus presence was based on symptoms exhibited by the germinated plants and by DAS-ELISA. Results of this study showed that the inoculated N. tabacum plants developed virus-like symptoms. Soybean mosaic virus (SMV), TMV, CMV, and hibiscus chlorotic ringspot virus (HCRSV) were identified in the field samples based on PCR results. Seed transmission assays did not demonstrate the presence of viruses based on symptomatology and DAS-ELISA tests. The second part of the study was undertaken by using Next Generation Sequencing (NGS) to analyse the complete genome sequence of HCRSV infecting soybean in the province of KwaZulu-Natal, South frica Total RNA extracted from soybean samples exhibiting virus-like symptoms was combined into one sample and used as template for NGS analysis. The sequence data generated was analysed using Genome Detective Virus Tool version 1.133.

The HCRSV complete genome sequence obtained was compared with other HCRSV sequences from GenBank database using BLASTN. Pairwise and Multiple sequence alignments of the sequences were done using ClustalW tool available in MEGA X. Phylogenetic analysis was done using nine closely related HCRSV sequences including turnip crinkle virus (TCV) which was used as an outgroup. The open reading frames (ORFs) for the HCRSV genome were determined using ORF finder and protein sizes were measured using Protein Molecular Weight software. Recombination events were analysed using RDP4 software. NGS data analysis revealed that HCRSV, CMV and TMV were present in the infected soybean samples. Results from the phylogenetic analysis showed that the NdlovuNS_HCRSV-SA isolate from this study (Accession number: OK636421) was closely related to isolate XM from China with a bootstrap value of 99%. Genome organisation analysis of the NdlovuNS_HCRSV_SA isolate compared with other HCRSV isolates suggested high levels of similarity. The BLAST analysis correlated with the results from the genome organisation data, with the HCRSV isolates sharing 87.87% - 97.10% nucleotide identity. Recombination analyses showed a single event confirming that the NdlovuNS_HCRSV-SA isolate is a recombinant strain. Accurate detection and identification of viruses plays an important role in virus disease management. Undetected viruses many occur and cause severe losses in soybean production. In this study, molecular detection techniques were used to accurately detectand identify the viruses infecting soybean field samples. It is important to emphasize that accurate and early detection of viruses is crucial for application of proper and effective control measures. The findings of this study will contribute to the body of knowledge on viruses infecting soybean in South Africa and will help in developing effective control measures.