Status of cassava mosaic disease and molecular characterization of associated geminiviruses in Malawi

Abstract

A study was conducted to evaluate cassava mosaic disease (CMD) incidence, severity, distribution and genetic diversity of responsible viruses across the major cassava production areas of Malawi. A field survey to determine CMD incidence, severity and distribution was conducted in April 2013 in 120 cassava fields across three administrative regions of Malawi. Separate surveys were conducted in August 2013 and June 2016 to determine the genetic diversity of the responsible viruses. A total of 200 symptomatic and asymptomatic cassava leaf samples were collected from 100 fields, surveyed in 10 districts in the August 2013 survey. DNA was extracted using Dellaporta method. Universal and specific primers were used to detect cassava mosaic begomoviruses (CMBs). An aliquot of the extracted DNA was subjected to circle amplification (RCA) and subsequent virus detection using specific primers. Illumina library construction and sequencing was performed on the RCA products. In the June, 2016 survey, 116 symptomatic cassava leaf samples were collected from 112 fields, surveyed in 11 districts of the country. Total RNA was extracted using CTAB method and samples were shipped to Switzerland for small RNA library construction and next generation sequencing (NGS). Results from the April 2013 survey showed a country-wide occurrence of CMD with incidence ranging from 4 to 52% (mean 24%). CMD symptom severity scores ranged between 2.2 and 3.8 (mean 3.0). The southern and central regions of the country registered the highest and lowest disease incidence at 41% and 10%, respectively. In contrast, the southern region recorded the lowest average disease severity score (2.7) while the northern region registered the highest (3.6). Mchinji and Chitipa districts had significantly lower disease incidence (4% and 5%, respectively) yet the latter recorded the highest severity score alongside Nkhatabay (3.8). Adult whitefly abundances were generally very low across the country with an average of 0.376 whiteflies per plant. Using virus specific primers, all known species reported in Africa were detected with the exception of ACMV. On the other hand, analysis of RCA and sRNA-based NGS data revealed that three CMB species (EACMV, EACMMV and EACMZV) are associated with CMD in Malawi and that some CMB isolates from Malawi are recombinants of EACMV/EACMMV and SACMV. RCA-based NGS data further suggested the presence of a putative species - Cassava mosaic virus [MW-KK3S-2013] isolated from the central region. In addition to CMB species, Cassava virus C was also recovered from samples from Chitipa district using sRNA-derived NGS data. Findings of this study unveils an increasing threat to cassava production in Malawi from not only well-known but also recombinant CMBs and other previously unreported viruses.