Cassava Frogskin Disease: Current Knowledge on a Re-Emerging Disease in the Americas.

Cassava frogskin disease (CFSD) is a graft-transmissible disease of cassava reported for the first time in the 1970s, in Colombia. The disease is characterized by the formation of longitudinal lip-like fissures on the peel of the cassava storage roots and a progressive reduction in fresh weight and starch content. Since its first report, different pathogens have been identified in CFSD-affected plants and improved sequencing technologies have unraveled complex mixed infections building up in plants with severe root symptoms. The re-emergence of the disease in Colombia during 2019-2020 is again threatening the food security of low-income farmers and the growing local cassava starch industry. Here, we review some results obtained over several years of CFSD pathology research at CIAT, and provide insights on the biology of the disease coming from works on symptoms' characterization, associated pathogens, means of transmission, carbohydrate accumulation, and management. We expect this work will contribute to a better understanding of the disease, which will reflect on lowering its impact in the Americas and minimize the risk of its spread elsewhere.

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(including, but not limited to, Rolling Circle Amplification and Illumina and Nanopore sequencing), but is currently unpublished.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.•DNA purity estimations reveal OD260/280 ratios above 2.0 and OD260/230 ratios above 1.7, even for samples stored in silica gel for several months.•The high quality of the extracts is suitable for detection of DNA and RNA viruses, with high efficiency.•We suggest this method could be used as part of a gold standard kit for virus detection in cassava.

First report of Sri Lankan cassava mosaic virus and Cassava Mosaic Disease in Laos.

Cassava (Manihot esculenta Crantz) has been traditionally grown as a subsistence crop in Laos, but in recent years cassava cultivation in this country has expanded and is becoming a 'cash crop' for farmers (Malik et al., 2020). This also means that cassava vegetative seed (stakes) is rapidly multiplied and distributed. One of the most important diseases affecting cassava in the world is the Cassava Mosaic Disease (CMD), caused by several species of begomoviruses and disseminated by infected stakes or vectored by the whitefly Bemisia tabaci (Legg et al., 2014). Sri Lankan cassava mosaic virus (SLCMV), a bipartite begomovirus, is the virus species causing CMD in Southeast Asia (SEA) and is widespread in Cambodia, Vietnam, Thailand and south China (Siriwan et al., 2020). During field surveys on July 12 to 14, 2020, the team in south Laos, surveyed 8 fields along the border with Cambodia, in the southern provinces of Attapeu and Champassack and identified CMD symptoms (Supplementary Figure 1A) in only one of the fields, located at Kong District of the Champassack province (GPS coordinates 13.94325, 105.99102). From these 8 fields, samples were collected from every third plant in an X pattern. Photographs from each sampled plant were taken and uploaded into CIAT's PestDisPlace platform (https://pestdisplace.org), for CMD symptom confirmation (Supplementary Figure 1B). Leaf samples were sent to the laboratory for PCR using primers SLCMV-F 5'-ATGTCGAAGCGACCAGCAGATATAAT-3' and SLCMV-R 5'-TTAATTGCTGACCGAATCGTAGAAG-3' targeting the AV1 gene (Dutt et al., 2005), following the protocol described in Siriwan et al. (2020) and primers SLCMV-B-F1 5'-ACCGGATGGCCGCGCCCCCCTCT-3' and SLCMV-B-606R 5'-CACCTACCCTGTTATCGCTAAG-3' targeting part of the BV1 gene. Out of 60 samples collected for the field in Kong district, eleven (18.3%) resulted PCR positive to SLCMV (to DNA-A and DNA-B) but only four plants (6.7%) showed symptoms of CMD (see Supplementary Figure 1B and 1C). None of the samples in the other seven fields had CMD symptoms nor was SLCMV detected in any of these plants. Furthermore, the presence of CMD symptoms in the old leaves of the plants in the affected field suggests that the virus was introduced with contaminated stakes. The complete bipartite genome of one isolate (Champ1), was amplified by Rolling Circle Amplification and sequenced with the nanopore MinION technology as described by Leiva et al. (2020). The sequences were submitted to GenBank under accession nos MT946533 (DNA-A) and MT946534 (DNA-B). A phylogenetic tree for SLCMV and a link to the open SLCMV Nextstrain map (Hadfield et al., 2018) is included in Supplementary Figure 2. The sequences of the DNA-A and DNA-B components of the Champ1 isolate were nearly identical to those of anisolate of SLCMV from Ratanakiri, Cambodia (99.72% for DNA-A and 99.82 for DNA-B; Wang et al., 2016). Phylogenetic analysis (Supplementary Figure 2), grouped isolate Champ1 with those that form the cluster of SEA isolates that contain the shorter version of the rep gene (Siriwan et al., 2020). This short version of rep present a deletion of 7 amino acids at the C-terminus, which is involved in host responses to SLCMV (Wang et al., 2020). The confirmation of CMD and SLCMV in the border between Laos and Cambodia should be followed by disease containment and management strategies, particularly given that the majority cassava varieties grown in Laos are from neighbor countries, most of which have already reported the presence of CMD. Acknowledgements We thank all staff from the CIAT's Cassava Program and the Plant Protection Center of Laos in Vientiane. We acknowledge financial support from the Australian Centre for International Agricultural Research (ACIAR) and the CGIAR Research Program on Roots, Tubers and Bananas (RTB) (https://www.cgiar.org/funders/).