Gerhard Pietersen

Plant virus disease problems in the developing world.

Publisher Summary This chapter focuses on the plant virus disease problem in the developing world. The viruses of greatest importance are those affecting the crops that feed the greatest number of people rather than those destined for export. This chapter focuses on major disease problems, especially those affecting the major food crops for subsistence agriculture and important emerging diseases in the developing world. Although in many instances virus infections are suspected, these infections are never satisfactorily identified, and in many other cases it is not known whether crops are infected, let alone what effect the infection has on yield. It discusses recent information and/or information on less-well characterized viruses. Virus diseases of rice, wheat, maize, sweet potato, cassava, and banana are described in the chapter. Other important considerations in discussing the most important viruses affecting a developing countrys agriculture are agricultural practices in the country and the depth/coverage of the reporting of plant virus diseases.

Ecology and management of grapevine leafroll disease

Grapevine leafroll disease (GLD) is caused by a complex of vector-borne virus species in the family Closteroviridae. GLD is present in all grape-growing regions of the world, primarily affecting wine grape varieties. The disease has emerged in the last two decades as one of the major factors affecting grape fruit quality, leading to research efforts aimed at reducing its economic impact. Most research has focused on the pathogens themselves, such as improved detection protocols, with limited work directed toward disease ecology and the development of management practices. Here we discuss the ecology and management of GLD, focusing primarily on Grapevine leafroll-associated virus 3, the most important virus species within the complex. We contextualize research done on this system within an ecological framework that forms the backbone of the discussion regarding current and potential GLD management strategies. To reach this goal, we introduce various aspects of GLD biology and ecology, followed by disease management case studies from four different countries and continents (South Africa, New Zealand, California-USA, and France). We review ongoing regional efforts that serve as models for improved strategies to control this economically important and worldwide disease, highlighting scientific gaps that must be filled for the development of knowledge-based sustainable GLD management practices.

Molecular characterization of a subgroup I geminivirus from a legume in South Africa

A South African geminivirus for which we propose the name bean yellow dwarf virus (BeYDV) has been isolated from French bean (Phaseolus vulgaris cv. Bonus) showing stunting, chlorosis and leaf curl symptoms. A full-length cloned copy of the viral genome produced characteristic symptoms of the disease when reintroduced into French bean by agroinoculation, and was systemically infectious in Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum, Datura stramonium and Arabidopsis thaliana. BeYDV resembles subgroup I geminiviruses which infect monocotyledonous plants in having a single DNA component, two non-overlapping virion-sense (V1 and V2) and two overlapping complementary-sense (C1 and C2) coding regions, and an intron within the complementary-sense coding regions that is excised to produce a C1C2 fusion protein. It is most closely related to tobacco yellow dwarf virus from Australia, the only subgroup I geminivirus previously known to infect dicotyledonous plants, although it is sufficiently dissimilar (65% nucleotide sequence identity) to be considered a distinct virus.

A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae)

Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5′UTR

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.

Diversity of Dicotyledenous-Infecting Geminiviruses and Their Associated DNA Molecules in Southern Africa, Including the South-West Indian Ocean Islands

The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-βs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world.

Genotype composition of populations of grapefruit-cross- protecting citrus tristeza virus strain GFMS12 in different host plants and aphid-transmitted sub-isolates

Citrus tristeza virus (CTV) causes severe losses in grapefruit production in South Africa and requires mild-strain cross-protection to maintain production. Unfortunately, cross-protection breakdown of the pre-immunizing CTV grapefruit mild source GFMS12 is prevalent in grapefruit in South Africa. The CTV genotype composition of the GFMS12 population inoculated onto different hosts was determined by sequencing part of ORF1a and the p23 gene of multiple clones from each plant. Analysis of the GFMS12 population in Mexican lime and Marsh and Star Ruby grapefruit varieties revealed that at least four genotypes occur in the GFMS12 population and that genotype compositions differed amongst the populations in different host plants. Single-aphid-transmitted sub-isolates derived from the GFMS12 mother population on Mexican lime appeared to contain three populations of a mixture of VT-like and recombinant B165/VT-like genotypes; a mixture of recombinant RB/VT- and B165/VT-like genotypes; and a single recombinant B165/VT-like genotype. This study underlines the importance of determining the genotype composition of a potential CTV pre-immunizing source on a range of inoculated host species before utilization.

Transmission of Activated-Episomal Banana streak OL (badna)virus (BSOLV) to cv. Williams Banana (Musa sp.) by Three Mealybug Species

Four different mealybug species (Dysmicoccus brevipes, Planococcus citri, P. ficus, and Pseudococcus longispinus) were evaluated for their ability to transmit putative activated-episomal Banana streak OL (badna)virus (BSOLV) to banana cv. Williams (Cavendish subgroup, AAA). Expressible endogenous sequences of banana streak viruses (BSVs) have been reported to be present in the DNA of various Musa hybrids, including FHIA-21 (AAAB). To obtain activated episomal BSOLV for this experimental transmission study, intentional stress by tissue culture propagation was applied to indexed FHIA-21 which, while free of other viruses, can contain activated episomal BSOLV. Immunocapture polymerase chain reaction and triple-antibody sandwich enzyme-linked immunosorbent assay results revealed that 13.4% of the derived progeny of the mother plants were infected with episomal BSOLV. Four of these BSOLV-infected progeny were used as sources of episomal virus for transmission studies. D. brevipes, Planococcus citri, and P. ficus mealybugs were able to transmit the putative activated episomal BSOLV. Control plants for the transmission experiments included FHIA-21 corms with no background history of tissue culture, as well as virus-free Williams plants. Episomal Banana streak GF (badna)virus (BSGFV) was transmitted from asymptomatic corm-derived FHIA-21 plants by P. citri and P. ficus. This is the first report of P. ficus as a vector of BSVs.

Distribution of grapevine leafroll associated virus-3 variants in South African vineyards

Three genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) were identified from vineyards of the Western Cape, South Africa. In a previous study, three full genome sequences of isolates representing each of the variant groups were determined. The three variant groups were represented by accessions 621, 623 and PL-20, of variant groups I, II and III respectively. A specific single strand conformation polymorphism (SSCP) profile was assigned to each variant which was used as a quick, reliable detection and differentiation method. In this study we analysed the occurrence of these three GLRaV-3 variants in mother blocks in different cultivars and from different vine growing regions using SSCP. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis of ORF5 amplified PCR products. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows.

Control of Grapevine Leafroll Disease Spread at a Commercial Wine Estate in South Africa: A Case Study

Grapevine leafroll disease (LR) is a serious disease of grapevine worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent virus associated with this disease in South Africa and, despite a successful virus-elimination strategy within a certification scheme, spreads rapidly in local commercial vineyards. Since 2002 an integrated control strategy was used at a commercial wine estate to control LR and serve as a case study for the local and international wine industries to show that control in a commercial setting is possible. The strategy included planting of certified material tested free of detectable viruses, use of herbicide and subsequent removal of infected vine material, fallow periods during which time volunteer hosts were removed, and use of systemic and contact insecticides, sanitation, and horticultural practices to minimize spread of viruliferous mealybugs. Leafroll was reduced from a 100% infection in 2002 on 41.26 ha (111,431 vines) planted mainly from 1989 to 1992, to only 58 LR infected vines detected in 2012 on 77.84 ha (209,626 vines), an incidence of 0.027%. This reduction was achieved by replacing the fully infected vineyards and roguing 3105 infected vines within young and replaced new vineyards. The control strategies were successful in curtailing the spread of LR disease and have resulted in the removal of the disease from the majority of individual vineyards. Leafroll currently occurs at sufficiently low levels in the remaining vineyards that local eradication may be possible in these, in contrast to the general situation in the South African industry where the majority of producers do not apply LR control strategies and leafroll is widespread.