RESUMEN
Avocado sunblotch viroid (ASBVd) is a subcellular pathogen of avocado that reduces yield from a tree, diminishes the appearance of the fruit by causing unsightly scarring and impedes trade because of quarantine conditions that are imposed to prevent spread of the pathogen via seed-borne inoculum. For countries where ASBVd is officially reported, permission to export fruit to another country may only be granted if an orchard can be demonstrated to be a pest free production site. The survey requirements to demonstrate pest freedom are usually defined in export protocols that have been mutually agreed upon by the trading partners. In this paper, we introduce a flexible statistical protocol for use in optimizing sampling strategies to establish pest free status from ASBVd in avocado orchards. The protocol, which is supported by an interactive app, integrates statistical considerations of multistage sampling of trees in orchards with a RT-qPCR assay allowing for detection of infection in pooled samples of leaves taken from multiple trees. While this study was motivated by a need to design a survey protocol for ASBVd, the theoretical framework and the accompanying app have broader applicability to a range of plant pathogens in which hierarchical sampling of a target population is coupled with pooling of material prior to diagnosis.
Asunto(s)
Persea , Virus de Plantas , Viroides , Viroides/genética , ARN Viral , Virus de Plantas/genéticaRESUMEN
Banana bunchy top disease is the most devastating viral disease of bananas worldwide and is caused by banana bunchy top virus (BBTV). The disease is spread by the banana aphid Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) and through infected propagation material. In 2016, the virus was detected for the first time in an isolated area in the South Coast region of KwaZulu-Natal Province (KZN), South Africa. The aim of this study was to conduct surveys across all banana-producing regions in South Africa, viz. KwaZulu-Natal, Mpumalanga, and Limpopo provinces. Over 1700 plant and aphid samples were collected from commercial farms and rural households in the three provinces, and more-intense sampling was done in the affected KZN region. A BBTV-specific PCR targeting DNA-R (encoding the master replication initiation protein, M-Rep) was used to detect virus-infected samples, and amplicons of the expected size were sequenced. Comparative phylogenetic analysis showed that the South African BBTV isolates clustered within the Pacific Indian Oceans genomic group, which includes isolates from India and other regions in Africa, with a bootstrap value of 94%. To date, the virus has been identified only in the South Coast region of KwaZulu-Natal Province. Intense management strategies, including scouting, removal of infected plants, and control of aphids, have been implemented in areas where positive samples were identified to minimize the spread of the virus.
Asunto(s)
Áfidos , Babuvirus , Musa , Animales , Babuvirus/genética , ADN Viral/genética , Filogenia , Sudáfrica/epidemiologíaRESUMEN
Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5' ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5'UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.
Asunto(s)
Regiones no Traducidas 5' , Closteroviridae/genética , Closteroviridae/aislamiento & purificación , Polimorfismo Genético , ARN Viral/genética , Vitis/virología , Closteroviridae/clasificación , Análisis por Conglomerados , Dermatoglifia del ADN , Genotipo , Datos de Secuencia Molecular , Filogenia , Polimorfismo Conformacional Retorcido-Simple , Alineación de Secuencia , Análisis de Secuencia de ADN , SudáfricaRESUMEN
RT-PCRs, designed for the specific detection of molecular variants of Grapevine virus A (GVA), were applied to analysis of the virus from various grapevines and isolates recovered from these grapevines in Nicotiana benthamiana. Results of SSCP, cloning and sequencing revealed that the combination of these RT-PCR techniques permits reliable and rapid identification of grapevines mixed-infected with divergent variants of GVA. The results suggest that such grapevines are very common among GVA-infected grapevines in vineyards in South Africa.