RESUMO

In Southeast Asia (SEA) fastidious fungi of the Ceratobasidium genus are associated with proliferation of sprouts and vascular necrosis in cacao and cassava, crops that were introduced from the tropical Americas to this region. Here, we report the isolation and in vitro culture of a Ceratobasidium sp. isolated from cassava with symptoms of witches' broom disease (CWBD), a devastating disease of this crop in SEA. The genome characterization using a hybrid assembly strategy identifies the fungus as an isolate of the species C. theobromae, the causal agent of vascular streak dieback of cacao in SEA. Both fungi have a genome size > 31 Mb (G+C content 49%), share > 98% nucleotide identity of the Internal Transcribed Spacer (ITS) and > 94% in genes used for species-level identification. Using RNAscope® we traced the pathogen and confirmed its irregular distribution in the xylem and epidermis along the cassava stem, which explains the obtention of healthy planting material from symptom-free parts of a diseased plant. These results are essential for understanding the epidemiology of CWBD, as a basis for disease management including measures to prevent further spread and minimize the risk of introducing C. theobromae via long-distance movement of cassava materials to Africa and the Americas.

Assuntos

RESUMO

Cassava witches' broom disease (CWBD) is a devastating disease of cassava in Southeast Asia (SEA), of unknown etiology. Affected plants show reduced internodal length, proliferation of leaves and weakening of stems. This results in poor germination of infected stem cuttings (i.e., planting material) and significant reductions in fresh root yields and starch content, causing economic losses for farmers and processors. Using a metagenomic approach, we identified a fungus belonging to the Ceratobasidium genus, sharing more than 98.3-99.7% nucleotide identity at the Internal Transcribed Spacer (ITS), with Ceratobasidium theobromae a pathogen causing similar symptoms in cacao. Microscopy analysis confirmed the identity of the fungus and specific designed PCR tests readily showed (1) Ceratobasidium sp. of cassava is strongly associated with CWBD symptoms, (2) the fungus is present in diseased samples collected since the first recorded CWBD outbreaks in SEA and (3) the fungus is transmissible by grafting. No phytoplasma sequences were detected in diseased plants. Current disease management efforts include adjustment of quarantine protocols and guarantee the production and distribution of Ceratobasidium-free planting material. Implications of related Ceratobasidium fungi, infecting cassava, and cacao in SEA and in other potential risk areas are discussed.

Assuntos

RESUMO

Cassava witches' broom disease (CWBD) is one of the main diseases of cassava in Southeast Asia (SEA). Affected cassava plants show reduced internodal length and proliferation of leaves (phyllody) in the middle and top part of the plant, which results in reduced root yields of 50% or more. It is thought to be caused by phytoplasma; however, despite its widespread distribution in SEA still little is known about CWBD pathology. The overarching goal of this study was to review and corroborate published information on CWBD biology and epidemiology considering recent field observations. We report the following: (1) CWBD symptoms are conserved and persistent in SEA and are distinct from what has been reported as witches' broom in Argentina and Brazil. (2) In comparison with cassava mosaic disease, another major disease of cassava in SEA, symptoms of CWBD develop later. (3) Phytoplasma detected in CWBD-affected plants belong to different ribosomal groups and there is no association study available indicating phytoplasma as the causing agent of CWBD. These findings are essential clues for designing surveillance and management strategies and for future studies to better understand the biology, tissue localization and spatial spread of CWBD in SEA and other potential risk areas.

RESUMO

Cassava Mosaic Disease (CMD) caused by Sri Lankan cassava mosaic virus (SLCMV), has rapidly spread in Southeast Asia (SEA) since 2016. Recently it has been documented in Lao PDR. Previous reports have identified whitefly species of B. tabaci as potential vectors of CMD in SEA, but their occurrence and distribution in cassava fields is not well known. We conducted a countrywide survey in Lao PDR for adult whiteflies in cassava fields, and determined the abundance and genetic diversity of the B. tabaci species complex using mitochondrial cytochrome oxidase I (mtCOI) sequencing. In order to expedite the process, PCR amplifications were performed directly on whitefly adults without DNA extraction, and mtCOI sequences obtained using nanopore portable-sequencing technology. Low whitefly abundances and two cryptic species of the B. tabaci complex, Asia II 1 and Asia II 6, were identified. This is the first work on abundance and genetic identification of whiteflies associated with cassava in Lao PDR. This study indicates currently only a secondary role for Asia II in spreading CMD or as a pest. Routine monitoring and transmission studies on Asia II 6 should be carried out to establish its potential role as a vector of SLCMV in this region.

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Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is the causal agent of Fusarium wilt, a major threat to the banana industry worldwide. Here, we report the genome of a Foc TR4 strain from Peru, sequenced using a combination of Illumina and Oxford Nanopore Technologies.

RESUMO

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.

RESUMO

Next generation sequencing has been used to identify and characterize the full genome sequence of a cassava-infecting torradovirus, revealing the presence of a Maf/HAM1 domain downstream of the RNA-dependent RNA-polymerase (RdRp) domain in RNA1 in all isolates sequenced. A similar domain is also found in unrelated potyvirids infecting Euphorbiaceae hosts in the Americas and cassava in Africa. Even though cassava torrado-like virus (CsTLV) could not be mechanically transmitted to a series of herbaceous hosts, it can be efficiently transmitted by bud graft-inoculation to different cassava landraces. Our bioassays show that CsTLV has a narrow host range. Crystal-like structures of isometric virus-like particles were observed in cells of plants with single infection by CsTLV, and consistently induced chlorotic leaf spots and affected root yields significantly. Moreover, CsTLV infection induces changes in the accumulation of total sugars in storage roots. Field surveys indicated the presence of CsTLV in the main cassava growing regions of Colombia, and the occurrence of two different cassava-infecting torradovirus species. Profiles of small RNAs of 21 to 24 nucleotides in length, derived from CsTLV RNAs targeted by cassava RNA silencing defense mechanisms, are also reported.

Assuntos

RESUMO

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.

Assuntos

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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.

RESUMO

The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism-polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.

Assuntos

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We report the genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate obtained from a patient with symptoms of coronavirus disease 2019 (COVID-19) who was infected in Cali, Colombia. The patient had no recent travel record and did not require hospitalization. The virus genome was obtained using Oxford Nanopore MinION sequencing.

RESUMO

Moko is one of the main diseases affecting banana and plantain in Colombia. Here, we report the genome sequence of the causal agent, the bacterium Ralstonia solanacearum (Smith) strain CIAT-078, collected in 2004 from affected plantains in central-west Colombia. The assembled genome was obtained using Oxford Nanopore Technology.

RESUMO

Sweet potato is among the most important root crops worldwide, particularly in developing countries, and its production is affected severely by a variety of virus diseases. During the last decade, a number of new viruses have been discovered in sweet potatoes through next-generation sequencing studies. Among them are viruses belonging to the genus Badnavirus and collectively assigned to the species sweet potato pakakuy virus (SPPV). We determined the complete genome sequence of two SPPV isolates and show the ubiquitous presence of similar viruses in germplasm and field material from around the globe. We show that SPPV is not integrated into the sweet potato genome, occurs only at extremely low titers, and is efficiently transmitted through seeds and cuttings. They are unaffected by virus elimination therapy and do not induce discernible symptoms in sweet potatoes or indicator host plants. They show considerable variation in their nucleotide sequences and correspond to several genetic lineages. Studies of their interaction with the two most important sweet potato viruses showed only limited synergistic increase in the titers of one of two SPPV isolates. We contend that these viruses may pose little threat to sweet potato production and more likely represent a new type of persistent virus in sweet potato.

RESUMO

Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range and RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA3 sequences from potato and partial sequences of RNA1, 2 and 3 of isolates infecting potato and yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently described isolate from Solanum muricatum (pepino dulce), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus.

Assuntos

RESUMO

Sri Lankan cassava mosaic virus is an emerging pathogen in Southeast Asia. Here, we report the complete genome of a Thai isolate obtained using Nanopore technology. The isolate was collected in 2019 from the northeastern province of Surin, soon after disease eradication was reported in the country.

RESUMO

We describe here the complete genome of Rice hoja blanca tenuivirus The sequenced isolate was obtained by insect vector transmission from a symptomatic rice sample grown in Colombia. Sequence data from the four RNA components were obtained by deep sequencing (Illumina), and infections were confirmed by enzyme-linked immunosorbent assay and Sanger sequencing.

RESUMO

Several potexviruses (Family Alphaflexiviridae) have been reported infecting cassava (Manihot esculenta Crantz) in the Americas. They were isolated from severely diseased plants during the last 30-40 years and include: Cassava common mosaic virus (CsCMV), Cassava Caribbean mosaic virus (CsCaMV), Cassava Colombian symptomless virus (CsCSV) and Cassava virus X (CsVX). However, their definitive classification as distinct species remains unresolved for several reasons, including the lack of sequence data and unavailability of samples from original isolates. This complicates disease diagnostics, cassava germplasm exchange certification, evaluation of virus cleaning protocols and epidemiological studies. Furthermore, a recently detected novel alphaflexivirus, indicates that cassava-infecting potexviruses may be more diverse. To solve the identity of these viruses, we started indexing samples from different parts of Colombia using different sets of PCR primers, antisera available and inoculation to indicator plants. Results show that there are three major phylogenetic groups of potexviruses infecting cassava, and they correspond to CsCMV, CsVX and the newly identified Cassava new alphaflexivirus (CsNAV). Bioassays and sequence analysis established that isolates of CsNAV and CsVX cause latent infections in different cassava landraces, they are not efficiently transmitted to the indicator plant Nicotiana benthamiana and they lack the gene 3 of the conserved potexviral 'triple gene block' (TGB). In contrast, all isolates of CsCMV (which have a characteristic potexvirus genome arrangement) caused Cassava Common Mosaic Disease (CCMD) in single infections and were efficiently transmitted to N. benthamiana. Although phylogenetic analysis of the replicase sequence placed CsNAV and CsVX as members of the Potexvirus genus, their distinct genome arrangement and biological characteristics suggest they can be considered as members of a separate taxonomic group.

Assuntos

RESUMO

A copy of the complete genome of a novel bipartite begomovirus infecting common bean (Phaseolus vulgaris L.) in Colombia was obtained by rolling-circle amplification (RCA), cloned, and sequenced. The virus is associated with leaf crumple symptoms and significant yield losses in Andean and Mesoamerican beans. Such symptoms have been reported increasingly in Colombia since at least 2002, and we detected the virus in leaf material collected since 2008. Sequence analysis showed that the virus is a member of a distinct species, sharing 81% and 76% nucleotide (nt) sequence identity (in DNA-A and DNA-B, respectively) to other begomoviruses infecting common bean in the Americas. The data obtained support the taxonomic status of this virus (putatively named 'bean leaf crumple virus', BLCrV) as a member of a novel species in the genus Begomovirus.

Assuntos

RESUMO

The complete bipartite genome (RNA1 and RNA2) of a new nepovirus infecting potato was obtained using small RNA sequencing and assembly complemented by Sanger sequencing. Each RNA encodes a single polyprotein, flanked by 5' and 3' untranslate regions (UTR) and followed by a poly (A) tail. The putative polyproteins encoded by RNA1 and RNA2 had sets of motifs which are characteristic of viruses in the genus Nepovirus. Sequence comparisons using the Pro-Pol region and the coat protein, including phylogenetic analysis of these regions, showed closest relationships with nepoviruses. The data obtained support the taxonomical status of this new virus (putative named Potato virus B, PVB) as a member of the genus Nepovirus, subgroup B.

Assuntos

RESUMO

An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB.

Assuntos