Cassava torrado-like virus encodes a gene that facilitates the mechanical transmission to Nicotiana benthamiana of Cassava virus X

Cassava-torrado-like virus (CsTLV) is a bipartite single-stranded RNA virus belonging to the family Secoviridae. The virus has been reported in Brazil and Colombia, and is usually found in mixed infections, and in plants affected by Cassava Frogskin Disease (CFSD), an endemic cassava disease in the Americas. Genome analysis of CsTLV identifies a gene in RNA2 implicated in pathogen movement in other torradoviruses. This gene (RNA2-ORF1) and another one no related to virus movement (Maf/Ham1) were amplified by PCR and cloned into constructs under the 35S promoter of cauliflower mosaic virus (CaMV), which then were transfered to agrobacterium binary vectors. When agro-infiltrated in Nicotiana benthamiana plants, only RNA2-ORF1 had a positive effect on the mechanical inoculation of cassava virus X (CsVX), a potexvirus that has a low rate of mechanical infection in N. benthamiana. Efficiency of CsVX mechanical transmission was measured by the number of infected plants, presence of symptoms, and titers of CsVX as measured by ELISA, two weeks after infection. On average, CsVX could infect 2.3 times more plants when these were previously agro-infiltrated with A. tumefaciens binary vector encoding CsTLV RNA2-ORF1. We conclude that the novel secovirid CsTLV associated with leaf spot symptoms in cassava, encodes a gene that could enhance other viral infections in N. benthamiana. Further studies are required to elucidate this effect and its role in mixed infections, often observed in cassava plants affected by CFSD

An optimized nucleic acid isolation protocol for virus diagnostics in cassava (Manihot esculenta Crantz.)

Our group works on the detection and characterization of cassava viruses, supporting projects that involve large scale pathogen surveillance activities and resistance screening assays in multiple and remote locations. In order to comply with these applications, nucleic acid isolation protocols need to be cost effective, adjusted for samples that will stand long distance transport and harsh storage conditions, while maximizing the yield and quality of the nucleic acid extracts obtained. The method we describe here has been widely used and validated using different downstream tests (including, but not limited to, Rolling Circle Amplification and Illumina and Nanopore sequencing), but is currently unpublished. The protocol begins with milligram amounts of dry leaf samples stored in silica gel, does not require liquid Nitrogen nor phenol extraction and produces an average of 2.11 µg of nucleic acids per mg of dry tissue

Susceptibility of cassava varieties to disease caused by Sri Lankan cassava mosaic virus and impacts on yield by use of asymptomatic and virus-free planting material

Cassava (Manihot esculenta Crantz) is a rainfed, smallholder-produced crop in mainland Southeast Asia, and is currently facing a serious challenge posed by the introduction of cassava mosaic disease (CMD). This study assessed the susceptibility of popular Asian varieties to CMD, yield penalties associated with the disease, and the efficacy of selecting clean or asymptomatic plants as seed for the following season. Field experiments evaluated agronomic management practices (ie, fertilizer application, use of symptomatic and asymptomatic seed stakes) in Cambodia with six to nine popular varieties over three seasons under natural disease pressure. Popular cassava varieties KU50 and Huaybong60 showed superior CMD tolerance, with consistently fewer symptomatic plants, lower disease progress measures, and higher yields. Plants demonstrating symptoms at early stages of development, ie, 60 days after planting, yielded significantly less than those developing symptoms later (ie, 270 DAP) or not at all. Plants grown from clean stems yielded on average 20% to 2.7-fold higher than those grown from symptomatic planting material. A yield decline of~ 50% was recorded with symptomatic planting materials of susceptible varieties (eg, SC8,~ 25 t ha− 1) over successive years. The findings emphasize that farmers could use positive selection by choosing asymptomatic plants to significantly reduce yield losses

Complete genome sequence of a novel secovirid infecting cassava in the Americas

We report the complete genome sequence of a field isolate of a novel bipartite secovirid infecting cassava in Colombia, provisionally named "cassava torrado-like virus" (CsTLV). The genome sequence was obtained using Oxford Nanopore Technology, and the 5’ ends were confirmed by RACE. The RNA1 is 7252 nucleotides (nt) long, encoding a polyprotein of 2336 amino acids (aa) containing the typical “replication block”, conserved torradovirus motifs, and a Maf/Ham1 domain, which is not commonly found in viral genomes. The RNA2 is 4469 nt long and contains two overlapping ORFs encoding proteins of 226 and 1179 aa, showing the characteristic genome arrangement of members of the genus Torradovirus

Diagnostic and diversity of a novel secovirid affecting cassava in the Americas

We have developed a protocol for the detection, and characterization of pathogens in cassava (Manihot esculenta Crantz, Fam. Euphorbiaceae) widely cultivated in the tropics of Africa, Asia and Latin America. These processes guarantee the extraction of nucleic acids of high quality and yield to ensure their use in the detection of DNA and RNA viruses and the sequencing of viral genomes. An average of 2.11 μg of nucleic acids per mg of dry tissue can be obtained using silica gel as a desiccant avoiding the use of liquid nitrogen and phenol. Diverse families of viruses infect cassava, causing significant economic losses and affecting food security. In the Americas, Cassava torrado-like virus (CsTLV) belongs to the Family Secoviridae, Genus Torradovirus have been found in mixed virus infections in Colombia and Brazil, associated with severe disease symptoms in leaves and roots of cassava. In single infections CsTLV can induce leaf chlorotic spots symptoms. Using Oxford Nanopore Technology we complete the genome sequence of a field isolate of CsTLV, showing the characteristic genome arrangement of members of the genus Torradovirus. interestingly CsTLV encodes an atypical Maf / HAM1 domain that functions in eukaryotes to protect cells against mutagenesis only present in heterologous viruses of the Potyviridae family. Using this protocol, we analyzed field surveys in Colombia and a cassava country-wide collection from Peru, detecting the diverse isolates of CsTLV and the occurrence of the virus in the Americas

A protocol for measuring disease resistance to Cassava Bacterial Blight, using machine-learning image analysis

A protocol for measuring disease resistance to Cassava Bacterial Blight, using machine-learning image analysis Cassava (Manihot esculenta Crantz), a starchy root crop, is the fourth most important source of dietary carbohydrate in developing countries and important source of industrial raw material in tropical regions especially in southeast Asia (SEA). Xanthomonas phaseoli pv. manihotis formerly known as Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of cassava bacterial blight (CBB) disease. CBB is the major bacterial disease of cassava worldwide. To support breeding for resistance programs, we standardized a screen house protocol for the reliable, simple, space- and time-efficient and high throughput tool based on machine learning image analysis using the PhenotyperCV software. Bacterial strains from three edaphoclimatic zones in Colombia (isolates CIO 156, CIO 59, CIO 1238, CIO 482, CIAT 1075), were sequenced at the genome level using Nanopore technology and their pathogenicity profile was evaluated by inoculation in cassava leaves of 3 month old plants, under greenhouse conditions. Inoculations were carried out by infiltration using a needleless syringe of bacterial suspensions at OD600=0.05. Cassava leaves were detached from the plants and imaged at 0, 4, 6, 9, and 12-days post inoculation (DPI), and water-soaked spots were measured to calculate the area under the disease progress curve (AUDPC). This protocol allows us to identify distinct pathogenic strains and evaluate the characteristic CBB water soaked symptoms at different DPI by performing spots measurements in an automated way generating AUDPC curves over time that can be used for pathogenic characterization of isolates and subsequent varietal resistance screenings, a susceptible material will have a significantly higher AUDPC compared to a less susceptible or resistant material. Key words: Cassava bacterial blight , screen house protocol, machine learning, pathogenicity, AUDPC

SLCMV and CMD-field survey dataset using a standard BSP for Cambodia, Laos and Vietnam

Standard surveillance and diagnostic protocols need to be evaluated and implemented at regional level, for early warning and coordination of rapid responses among neighbor countries. This dataset provides a description of 2020 surveillance data for cassava mosaic disease (CMD) in Laos, East Cambodia and Northern Vietnam, indicating location of the surveyed fields, and presence/absence of visual symptoms. The image data was accessed and validated through the PestDisPlace platform by experts working with CMD. All data was collected using standard protocols (e.g. 60 observations per Hectare). Detailed information is available upon request