Molecular Characterization of Cassava Brown Streak Viruses and Identification of their Alternative Hosts in Mozambique

Abstract

Full genome sequences of CBSD-associated virus isolates contribute to the understanding of genetic diversity and the development of new diagnostic primers that can be used for early detection of the viruses, a key aspect for disease management and control. In the first objective, cassava fields were assessed 15 Km regularly, samples in the field were collected randomly (Z format). A total of 100 cassava leaf samples were collected in Nampula, Zambezia, Niassa and C. Delgado provinces. Thirty (30) samples were first screened for U/CBSV by PCR, and 10 were selected for deep sequencing (NGS). The study determined seven new whole CBSV genomes from total RNA isolated from cassava leaves in Mozambique. Phylogenetic analyses of the new genomes with published CBSV and UCBSV sequences in GenBank grouped the CBSV isolates from Mozambique into two distinct clades together with CBSV isolates from Tanzania. Clade 1 and 2 isolates shared lower nucleotide (79.1–80.4%) and amino acid (86.5–88.2%) sequence identity. Further, comparisons within the seven new CBSV isolates, and between them and the single published complete CBSV sequence (CBSV_MO_83_FN434436) from Mozambique, revealed nucleotide sequence identities of 79.3–100% and 79.3–98%, respectively, and amino acid identities of 86.7–100% and 86.7–98.8%. Using several comprehensive evolutionary models and statistical programs, it was confirmed that CBSV and UCBSV are distinct virus species, with an additional probable new species (clade 2). In objective two, full genome sequences of CBSD associated viruses were retrieved from Genebank, Intra and inter specific genetic diversity and differentiation ( number of mutation, synonymous and nonsynonymous substatitions, neutrality index , nucleotide diversity, genetic distance between species and population from Mozambique Kenya and Tanzania) was determined using whole genome and individual genes. Data indicate the highest nucleotide diversity (0.09251) of CBSV clade 2 across the three closest species and clade 1 is the least diverse amongst the species associated with CBSD. MacDonald Kreitman (MK) Test across the 10 genomic regions, mean values of Neutrality index (NI) observed in two genes (P1 and P3), indicates an excess of nonsilent divergence which suggest that polymorphic change observed in these genes is due of strong positive diversifying, in contrast to 6K1, Nlb, CP and HAM1 which the overall change is exclusive to the polymorphism. The level of genetic diversity/ polymorphism varied according geographical origin. Amongst the three population, the highest diversity was observed in CBSV population from Tanzania indicating that some genetic exchange has occurred, in contrast to CBSV population from Mozambique which showed lowest genetic diversity across individual sequences. CBSV population from Tanzania and Kenya shows frequent gene flow, it appears that there is higher connectivity between CBSV from Tanzania and Kenya than between Mozambique and Tanzania/Kenya, which suggests that some genetic exchange has occurred between CBSV isolates from both geographical locations (Tanzania and Kenya) due to exchange of cassava cuttings. These findings reinforce that exchange of cassava cuttings between regions must be anticipated by the CBSV screening test. In the third objective, a total of 120 samples (leaf, cuttings and seeds) were collected from 15 plant species showing virus-like disease symptoms. Total RNA was extracted from the leaf samples and using RT-PCR to amplify partial coat protein nucleotide sequences, CBSV was detected for the first time occurring in two non-cassava perennial wild plant species: Zanha africana (Radlk.) Exell.and Trichodesma zeylanicum (Burm.f.) R.Br., that occur widely within and near cassava fields in Nampula, Zambezia, Niassa, and Cabo Delgado provinces. In addition, CBSV and UCBSV were also detected in Manihot carthaginensis subsp. Glaziovii (Muell-Arg.) Allem., a wild cassava relative. These findings were verified in biological assays through mechanical inoculation of CBSV to T. zeylanicum, albeit at low rates of infection. Phylogenetic analysis clustered the CBSV isolates from the non-cassava plant species with those from cultivated cassava, with high sequence homology among CBSV (91.0–99.6%) and with UCBSV (84–92%) isolates. These results provide definitive evidence of a wider host range for CBSV and UCBSV in Mozambique, indicating that these viruses are not restricted to cultivated cassava. The findings are key to understanding the epidemiology of CBSD and will aid in the development of management strategies for the disease. This study concluded that there is a high diversity of CBSD associated viruses in Mozambique. CBSV is not single species as it appear, there are two closest species within CBSV. There is a high genetic differentiation across CBSD associated viruses, information which can take into consideration by breeders during the development of new cassava varieties. CBSV population from three countries has a different level of polymorphism indicate and suggesting that geographical origins could partially explain the difference of level of genetic polymorphism. CBSV population from Mozambique is the least diverse and Tanzania the most diverse. Cassava brown streak viruses are not restricted to cassava crops or related cassava plants, the viruses associated with CBSD are also found in wild plants.