Role of aminopeptidase N-like in the acquisition of begomoviruses by Bemisia tabaci, the whitefly vector.

Sri Lankan cassava mosaic virus (SLCMV) is a prominent causative agent of cassava mosaic disease in Asia and relies on the whitefly Bemisia tabaci cryptic complex for its transmission. However, the molecular mechanisms involved in SLCMV transmission by B. tabaci have yet to be understood. In this study, we identified an aminopeptidase N-like protein (BtAPN) in B. tabaci Asia II 1, an efficient vector of SLCMV, which is involved in the SLCMV transmission process. Through the use of glutathione S-transferase pull-down assay and LC-MS/MS analysis, we demonstrated the interaction between BtAPN and the coat protein (CP) of SLCMV. This interaction was further confirmed in vitro, and we observed an induction of BtAPN gene expression following SLCMV infection. By interfering with the function of BtAPN, the quantities of SLCMV were significantly reduced in various parts of B. tabaci Asia II 1, including the whole body, midgut, hemolymph, and primary salivary gland. Furthermore, we discovered that BtAPN is conserved in B. tabaci Middle East-Asia Minor 1 (MEAM1) and interacts with the CP of tomato yellow leaf curl virus (TYLCV), a begomovirus known to cause severe damage to tomato production. Blocking BtAPN with antibody led to a significant reduction in the quantities of TYLCV in whitefly whole body and organs/tissues. These results demonstrate that BtAPN plays a generic role in interacting with the CP of begomoviruses and positively regulates their acquisition by the whitefly.

Ceratobasidium sp. is associated with cassava witches' broom disease, a re-emerging threat to cassava cultivation in Southeast Asia.

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.

New Technologies Provide Innovative Opportunities to Enhance Understanding of Major Virus Diseases Threatening Global Food Security.

Plant viruses pose a continuous and serious threat to crop production worldwide, and globalization and climate change are exacerbating the establishment and rapid spread of new viruses. Simultaneously, developments in genome sequencing technology, nucleic acid amplification methods, and epidemiological modeling are providing plant health specialists with unprecedented opportunities to confront these major threats to the food security and livelihoods of millions of resource-constrained smallholders. In this perspective, we have used recent examples of integrated application of these technologies to enhance understanding of the emergence of plant viral diseases of key food security crops in low- and middle-income countries. We highlight how international funding and collaboration have enabled high-throughput sequencing-based surveillance approaches, targeted field and lab-based diagnostic tools, and modeling approaches that can be effectively used to support surveillance and preparedness against existing and emerging plant viral threats. The importance of national and international collaboration and the future role of CGIAR in further supporting these efforts, including building capabilities to make optimal use of these technologies in low- and middle-income countries, are discussed. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Cassava Witches' Broom Disease in Southeast Asia: A Review of Its Distribution and Associated Symptoms.

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.

Translating virome analyses to support biosecurity, on-farm management, and crop breeding.

Virome analysis via high-throughput sequencing (HTS) allows rapid and massive virus identification and diagnoses, expanding our focus from individual samples to the ecological distribution of viruses in agroecological landscapes. Decreases in sequencing costs combined with technological advances, such as automation and robotics, allow for efficient processing and analysis of numerous samples in plant disease clinics, tissue culture laboratories, and breeding programs. There are many opportunities for translating virome analysis to support plant health. For example, virome analysis can be employed in the development of biosecurity strategies and policies, including the implementation of virome risk assessments to support regulation and reduce the movement of infected plant material. A challenge is to identify which new viruses discovered through HTS require regulation and which can be allowed to move in germplasm and trade. On-farm management strategies can incorporate information from high-throughput surveillance, monitoring for new and known viruses across scales, to rapidly identify important agricultural viruses and understand their abundance and spread. Virome indexing programs can be used to generate clean germplasm and seed, crucial for the maintenance of seed system production and health, particularly in vegetatively propagated crops such as roots, tubers, and bananas. Virome analysis in breeding programs can provide insight into virus expression levels by generating relative abundance data, aiding in breeding cultivars resistant, or at least tolerant, to viruses. The integration of network analysis and machine learning techniques can facilitate designing and implementing management strategies, using novel forms of information to provide a scalable, replicable, and practical approach to developing management strategies for viromes. In the long run, these management strategies will be designed by generating sequence databases and building on the foundation of pre-existing knowledge about virus taxonomy, distribution, and host range. In conclusion, virome analysis will support the early adoption and implementation of integrated control strategies, impacting global markets, reducing the risk of introducing novel viruses, and limiting virus spread. The effective translation of virome analysis depends on capacity building to make benefits available globally.

Mitochondrial Genetic Diversity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Associated with Cassava in Lao PDR.

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.

Draft Genome Sequence of Fusarium oxysporum f. sp. cubense Tropical Race 4 from Peru, Obtained by Nanopore and Illumina Hybrid Assembly.

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.

Genome Analysis and Pathobiology of Cassava-Infecting Torradoviruses Containing a Putative Maf/HAM1 Pyrophosphatase Domain.

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.

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.

Gut-Expressed Vitellogenin Facilitates the Movement of a Plant Virus across the Midgut Wall in Its Insect Vector.

Many viral pathogens of global importance to plant and animal health are persistently transmitted by insect vectors. Midgut of insects forms the first major barrier that these viruses encounter during their entry into the vectors. However, the vector ligand(s) involved in the movement of plant viruses across the midgut barrier remains largely uncharacterized. Begomoviruses, many of which are disease agents of some major crops worldwide, are persistently transmitted by whiteflies (Bemisia tabaci). Here, in order to identify whitefly midgut proteins that interact with a devastating begomovirus, tomato yellow leaf curl virus (TYLCV), we performed midgut-specific TYLCV coat protein (CP) immunoprecipitation followed by high-throughput mass spectrometry proteomic analysis. We find that vitellogenin (Vg), a critical insect reproductive protein that has been considered to be synthesized by the fat body, is also synthesized by and interacts with TYLCV CP in the whitefly midgut. TYLCV appears to be internalized into midgut epithelial cells as a complex with Vg through endocytosis. Virus-containing vesicles then deliver the virus-Vg complexes to early endosomes for intracellular transport. Systematic silencing of Vg or midgut-specific immune blocking of Vg inhibited virus movement across the midgut wall and decreased viral acquisition and transmission by whitefly. Our findings show that a functional Vg protein is synthesized in the midgut of an insect and suggest a novel Vg mechanism that facilitates virus movement across the midgut barrier of its insect vector. IMPORTANCE An essential step in the life cycle of many viruses is transmission to a new host by insect vectors, and one critical step in the transmission of persistently transmitted viruses is overcoming the midgut barrier to enter vectors and complete their cycle. Most viruses enter vector midgut epithelial cells via specific interaction between viral structural proteins and vector cell surface receptor complexes. Tomato yellow leaf curl virus (TYLCV) is persistently transmitted by the whitefly Bemisia tabaci between host plants. Here, we find that TYLCV coat protein interacts with vitellogenin (Vg) in the whitefly midgut. This interaction is required for the movement of the virus crossing the midgut wall and thus facilitates viral acquisition and transmission by whitefly. This study reveals a novel mechanism of virus overcoming the insect midgut barrier and provides new insights into the function of Vg beyond serving as nutrition for developing embryos in insects.

A simple PCR-based method for the rapid and accurate identification of spider mites (Tetranychidae) on cassava.

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.

Global Cropland Connectivity: A Risk Factor for Invasion and Saturation by Emerging Pathogens and Pests.

The geographic pattern of cropland is an important risk factor for invasion and saturation by crop-specific pathogens and arthropods. Understanding cropland networks supports smart pest sampling and mitigation strategies. We evaluate global networks of cropland connectivity for key vegetatively propagated crops (banana and plantain, cassava, potato, sweet potato, and yam) important for food security in the tropics. For each crop, potential movement between geographic location pairs was evaluated using a gravity model, with associated uncertainty quantification. The highly linked hub and bridge locations in cropland connectivity risk maps are likely priorities for surveillance and management, and for tracing intraregion movement of pathogens and pests. Important locations are identified beyond those locations that simply have high crop density. Cropland connectivity risk maps provide a new risk component for integration with other factors-such as climatic suitability, genetic resistance, and global trade routes-to inform pest risk assessment and mitigation.

Genome Sequence of SARS-CoV-2 Isolate Cali-01, from Colombia, Obtained Using Oxford Nanopore MinION Sequencing.

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.

Surveillance and diagnostics of the emergent Sri Lankan cassava mosaic virus (Fam. Geminiviridae) in Southeast Asia.

Emergent agricultural pathogens cause severe damage worldwide and their invasive potential is significantly increased by global trade, crop intensification and climate change. Standard surveillance and diagnostic protocols need to be evaluated and implemented, particularly with diseases caused by a wide range of pathogens that induce similar symptoms. Such is the case with Cassava Mosaic Disease (CMD) present in Africa and Asia, and associated with mixed virus infections and recombinant and re-assorted virus strains. CMD has been recently reported in Southeast Asia (SEA) and is already widely spread throughout this region. This communication offers an update on protocols and tools used to track the distribution of CMD and to characterize the pathogen associated with it in SEA.

Complete Genome Sequence of the Plant Pathogen Ralstonia solanacearum Strain CIAT-078, Isolated in Colombia, Obtained Using Oxford Nanopore Technology.

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.

Badnaviruses of Sweet Potato: Symptomless Coinhabitants on a Global Scale.

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.

Characterization of distinct strains of an aphid-transmitted ilarvirus (Fam. Bromoviridae) infecting different hosts from South America.

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.

Nanopore-Based Complete Genome Sequence of a Sri Lankan Cassava Mosaic Virus (Geminivirus) Strain from Thailand.

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.

Complete Genome Sequence of Rice hoja blanca tenuivirus Isolated from a Susceptible Rice Cultivar in Colombia.

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.

Resolution of cassava-infecting alphaflexiviruses: Molecular and biological characterization of a novel group of potexviruses lacking the TGB3 gene.

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.