Characterization of Tomato curly stunt virus: a new tomato‐infecting begomovirus from South Africa

The biological and molecular characterization of a virus recognized as a distinct begomovirus species, Tomato curly stunt virus (ToCSV), first observed in South Africa in 1997, is reported here. Whitefly‐transmission and host‐range studies were carried out using a Bemisia tabaci colony identified as the B‐biotype. The experimental host range of ToCSV spanned primarily species in the Solanaceae and Fabaceae. The complete ToCSV genome (2·766 kb) was amplified by PCR, cloned, and the DNA sequence determined. Phylogenetic analysis revealed that ToCSV was most closely related to Tobacco leaf curl Zimbabwe virus (TbLCZV), at 84% nucleotide identity, indicating that ToCSV is a new species in the genus Begomovirus that is probably endemic to southern Africa. The ToCSV genome sequence contained all of the hallmark coding and non‐coding features characteristic of other previously recognized monopartite begomoviruses. ToCSV is only the second begomovirus described from southern Africa that infects solanaceous species. Neither a begomoviral DNA‐B component nor a satellite‐like DNA molecule was detected by PCR in extracts of ToCSV‐infected plants.     

Wide genetic diversity of Ralstonia solanacearum strains affecting tomato in Trinidad,West Indies

The genetic diversity of Ralstonia solanacearum causing bacterial wilt of tomato in Trinidad was assessed using the hierarchical phylotyping scheme and rep‐PCR DNA fingerprinting. Seventy‐one isolates were collected in 2003 on infected tomato crops in the four main vegetable cropping areas of Trinidad (North, Central, South‐East and South). Two phylotypes were present, with phylotype II being much more prevalent (66%) than phylotype I (34%). Phylotype II strains consisted mainly of sequevar 7 in Central and South‐East, and sequevar 35 in North, South‐East and South. This is the first report of sequevar 7 outside south‐eastern USA. In contrast, no ‘brown rot’ (phylotype IIB/1, race 3 biovar 2) or emerging strains of phylotype IIB/4NPB were identified. Rep‐PCR data were used to assess population genetic structure. No significant clustering by geographical distance was found, suggesting regular gene flow among cropping areas (via waterways, plant or soil). However, the population from Central was significantly differentiated from the others, containing only phylotype II/seq 7 strains, with a high degree of clonality, suggesting a possible recent introduction from abroad. The South population was less aggressive and more genetically diverse, suggesting horizontal gene transfers within the population, even among isolates of different phylotypes. Phylotype I and phylotype II populations differed slightly in clonality levels, with indications of more frequent recombination events within phylotype I populations. Possible factors influencing genetic diversity and distribution within the island are discussed.     

Analyses of seven new whole genome sequences of cassava brown streak viruses in Mozambique reveals two distinct clades: evidence for new species

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) is a major constraint to cassava production in Mozambique. Full genome sequences of CBSD-associated virus isolates contribute to the understanding of genetic diversity and the development of new diagnostic primers that can be used for early detection of the viruses for sustainable disease management. This study determined seven new whole CBSV genomes from total RNA isolated from cassava leaves with CBSD symptoms collected from Nampula and Zambezia in Mozambique. Phylogenetic analyses of the new genomes with published CBSV and UCBSV sequences in GenBank grouped the CBSV isolates from Mozambique into two distinct clades together with CBSV isolates from Tanzania. Clade 1 and 2 isolates shared low nucleotide (79.1–80.4%) and amino acid (86.5–88.2%) sequence identity. Further, comparisons within the seven new CBSV isolates, and between them and the single published complete CBSV sequence (CBSV_MO_83_FN434436) from Mozambique, revealed nucleotide sequence identities of 79.3–100% and 79.3–98%, respectively, and amino acid identities of 86.7–100% and 86.7–98.8%. In addition, using RDP4, a recombination analysis comprising all CBSV and UCBSV genome sequences from GenBank detect 11 recombination events. Using several comprehensive evolutionary models and statistical programs, it was confirmed that CBSV and UCBSV are distinct virus species, with an additional probable new species (clade 2).     

Molecular identification of a begomovirus causing yellow vein disease on Calendula officinalis in India

A whitefly transmitted begomovirus was detected by PCR using begomovirus-specific primers from naturally infected Calendula officinalis plants showing yellow vein disease symptoms. An approximately 800 bp PCR amplicon was cloned and sequenced to identify the species of the virus isolate. Analysis of nucleotide sequence data resulted in its identification as the complete coat protein gene open reading frame (CP ORF) of 771 bp, which encoded 256 amino acid residues. The coat protein of the virus isolate shared maximum identities of 96–97% with four strains of Tobacco curly shoot virus (ToCSV) and an Ageratum enation virus (AgEV) during BLAST analysis of sequence data. Nucleotide- and amino acid-based phylogenetic analysis revealed the close relationship of the isolate with ToCSV strains, therefore it has been identified as an isolate of ToCSV and C. officinalis is considered to be a new host of ToCSV begomovirus. Association of a DNA-β molecule with the virus isolate was also detected by PCR and Southern hybridization tests using DNA-β specific primers and probe.     

Phakopsora myrtacearum sp. nov., a newly described rust (Pucciniales) on eucalypts in eastern and southern Africa

Outbreaks of a rust disease in eucalypt forestry plantations and nurseries in Kenya, Mozambique and South Africa occurred between 2009 and 2014. The pathogen was identified using morphology and molecular phylogenetic analyses as an undescribed species in the Phakopsoraceae. A systematic study, based on nuclear ribosomal DNA, showed that it is a species of Phakopsora, herein named Phakopsora myrtacearum sp. nov. This new species of rust is the second validly described species on Eucalyptus, along with Puccinia psidii. Phakopsora myrtacearum is distinguished from P. psidii by leaf symptoms, morphology of the urediniospores and distinct phylogenetic placement. Phakopsora myrtacearum has been found on three species of Eucalyptus in Kenya, Mozambique and South Africa, and it may have future negative implications for commercial forestry in these areas.     

Multilocus sequence analysis reveals a novel phylogroup of Xanthomonas euvesicatoria pv. perforans causing bacterial spot of tomato in Iran

A multilocus sequence analysis (MLSA) was performed on five housekeeping genes (fusA, gapA, gltA, lacF and lepA) of 22 Xanthomonas euvesicatoria strains recently isolated from alfalfa, pepper and tomato plants in Iran. In addition, 161 strains isolated worldwide from pepper, poinsettia, rose and tomato plants were included in the analysis. All X. euvesicatoria pv. perforans isolates from tomato plants in Iran clustered in a monophyletic group, although five MLSA haplotypes were detected among them. The Iranian tomato strains presented 10 nucleotide differences in the lepA gene sequences compared to the known worldwide population of X. euvesicatoria pv. perforans. Statistical analyses revealed a recombination event that had occurred in the lepA gene of the strains isolated from tomato in Iran. BOX‐PCR analysis confirmed the inclusion of Iranian tomato strains within X. euvesicatoria pv. perforans. Furthermore, X. euvesicatoria pv. euvesicatoria strains isolated from pepper in Iran differed in one nucleotide in the lepA gene sequence from the known worldwide population of the pathovar, and clustered in a group containing strains isolated in Nigeria. The strains isolated from alfalfa in Iran clustered with the type strain of X. euvesicatoria pv. alfalfae. Altogether, the results reveal the existence of a phylogenetically novel population of X. euvesicatoria pv. perforans in Iran which needs further in‐depth analysis to pinpoint the epidemiological impact of these strains.     

Diversity of stem borer parasitoids and their associated wild host plants in South Africa and Mozambique

The diversity of lepidopterous stem borers, their parasitoids and their associated wild host plants was studied in South Africa between 2006 and 2009 and in Mozambique between 2005 and 2010. In South Africa, 20 species of parasitoids were recovered from 17 stem borer species collected on 16 wild host plant species. From Mozambique, 14 parasitoid species were recorded from 16 stem borer species collected on 14 wild host plant species. The highest diversity of parasitoids was recorded on stem borers that attacked the host plants Phragmites australis (7 spp.) and Panicum maximum (6 spp.), in South Africa and Mozambique, respectively. Bracon sp. (Hymenoptera: Braconidae) and Procerochasmias nigromaculatus (Hymenoptera: Ichneumonidae) were the most recorded parasitoid species in South Africa while Cotesia sesamiae (Hymenoptera: Braconidae) parasitized most stem borer species in Mozambique. The most common tachinid parasitoid recorded in this study was Sturmiopsis parasitica (Diptera: Tachinidae). Parasitism of stem borers during the off season was previously thought to occur mainly in natural habitats but this study shows that although natural habitats provided refuges for some parasitoid species, stem borer parasitism was generally low in wild host plants, irrespective of whether collections were done during the cropping or off-season. Parasitoid beta diversity did not depend on habitat or host plant species, but was determined by stem borer diversity.     

Diversity analysis of Tomato leaf curl New Delhi virus-[potato], causing apical leaf curl disease of potato in India

The complete coat protein (cp) gene sequence of eighty Tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) isolates collected from eleven states were determined. Phylogenetic analysis based on cp gene grouped the isolates into two major clades (I & II) and they shared 95.9–100.0% identity. The DNA A and DNA B of eight representative isolates (six from clade I and two from clade II) were 2739–2740 and 2692–2694 nts long and shared 94.6–99.4% and 97.2–99.5% homology within the isolates, respectively. Among the eight isolates, the DNA A of two isolates (Clade II), GWA-5 and FAI-19 had 94.6–95.3% sequence identity to other six isolates and formed a sub-clade within the ToLCNDV-[potato] isolates. Similar grouping was also revealed with AC1 and AC4 genes of these eight isolates. The DNA A components shared more than 90.0% identity with the DNA A of ToLCNDV isolates from cucurbitaceous crops, tomato, bhendi, 89.0–90.0% with ToLCNDV-papaya isolates and 70.4–74.0% with other tomato leaf curl viruses. Hence, the begomovirus infecting potatoes are the ToLCNDV isolates, designated as ToLCNDV-[potato]. Whereas, the DNA B components shared 86.6–91.7% identity with ToLCNDV isolates from cucurbits, tomato and bhendi. Evidence for intra-species recombination was detected only in DNA A with a maximum of three events in GWA-5 and FAI-19 isolates. Analysis of cp gene, DNA A, iterons and recombination events clearly indicate that two groups of ToLCNDV-[potato] infects potato in India.     

Melanaspis corticosa: a new insect pest of olive trees in Europe

The presence of the South African Obscure Scale, Melanaspis corticosa (Brain) (Hemiptera, Diaspididae), was detected infesting olive trees, in Portugal. The identity of the scale insect was confirmed based on both morphological and molecular studies. Until now, this species was only known in a few African countries, including Guinea, Mozambique, South Africa and Zimbabwe. This is the first record of this species in Europe and in the Palearctic region. The scale was observed in 15 different locations, in the Algarve, since its first detection at the end of 2016. Samples were collected between 21 December 2016 and 10 March 2022, covering all seasonal periods. Most of the sampling sites resulted from private requests from farmers and proprietaries received by the Plant Protection Division of the Regional Directorate of Agriculture. Although it is considered a polyphagous species, it was not observed in other plant species, besides olive trees. The actual dispersion in the region suggests that M. corticosa became established and has been expanded its distribution since its arrival. This scale insect is a potential injurious pest of olive trees and needs to be studied to clarify its pest status and develop effective pest management strategies.

     

Genetic Relationships Between Cercospora kikuchii Populations from South America and Japan

ABSTRACT A collection 160 isolates of Cercospora kikuchii was made from South America and 245 from Japan. DNA fingerprint patterns were analyzed based on amplified fragment length polymorphism among the sample isolates, dividing the isolates into seven lineages (I to VII). Partial nucleotide sequence analyses of the beta-tubulin gene supported this division into seven lineages. Lineages I and III commonly existed in South America and Japan. In all, 136 of the 160 isolates from South America and 223 of the 245 isolates from Japan belonged to lineage I, indicating that lineage I was the major lineage in each area; 5 isolates from South America and 8 isolates from Japan belonged to lineage III. Lineages II (12 isolates) and IV (2 isolates) were specific to Japan and lineages V (3 isolates), VI (1 isolate), and VII (15 isolates) specifically existed in South America. These results suggest that the population genetic structure of C. kikuchii was different between South America and Japan, but the dominance of lineage I was common between the two areas.     

Grapevine virus A variants of group II associated with Shiraz disease in South Africa are present in plants affected by Australian Shiraz disease,and have also been detected in the USA

Primers were designed for RT‐nested PCR amplification of the highly variable 293‐nt fragment from the 5′ terminal part of the Grapevine virus A (GVA) replicase gene, specific to South African variants of molecular groups I and II. This new technique, along with RT‐PCR for simultaneous amplification of variants of groups I, II and III, as well as cloning of amplicons, single‐strand conformation polymorphism (SSCP) analysis of clones and sequencing, were used to investigate the populations of variants infecting 16 local Shiraz grapevines with different Shiraz disease (SD) status. The techniques were also used to study variants in GVA‐infected grapevines from Australia and the USA. The Australian grapevines included seven plants of cvs Shiraz and Merlot affected by Australian Shiraz disease (AuSD), and one plant of cv. Crimson Seedless with unknown AuSD status. Grapevines from the USA included plants of cvs Chardonnay, Thompson Seedless and an unknown cultivar. The results confirmed the association of certain genetic variants of group II with SD and showed the common presence of these variants in AuSD‐affected grapevines from Australia. Interestingly, a variant of this group was also detected in grapevine cv. Chardonnay from the USA, although the disease has not yet been reported from that country. The study also supports an earlier observation that members of group II, closely related to variant GTR1‐2, are not associated with the disease. The variants were found only in SD‐free grapevines. Results show that variants of the most divergent group III, which are common in South Africa, are also present in Australia and the USA. These variants are not associated with SD, but frequently occur in mixed infections with members of group II in plants affected by this disease in South Africa.     

Sequence Analysis and Detection of Ralstonia solanacearum by Multiplex PCR Amplification of 16S–23S Ribosomal Intergenic Spacer Region with Internal Positive Control

Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II) of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.     

Genetic variation of the invasive Campuloclinium macrocephalum,Asteraceae in South Africa,inferred from molecular markers

Campuloclinium macrocephalum is native to Central and South America, but is a highly invasive weed in South Africa, where it is commonly known as the ‘pompom weed’. It is targeted for biological control, the success of which will depend on host specificity and biotype compatibility to its full genetic diversity in South Africa. We investigated the genetic diversity and phylogeography of 52 specimens from across South Africa, 14 from Argentina and three from Brazil using nuclear ribosomal ITS regions. We further explored the AFLP marker diversity in 54 South African, 25 Argentine and three Brazilian specimens. Maximum parsimony analysis of the ITS sequence data produced an unresolved phylogeny. However, three haplotypes were recognised via network analysis. All South African, one Brazilian and all bar one of the Argentine individuals shared a single haplotype. AFLP analyses generated two genetic clusters with a low net nucleotide distance of 0.115 and further revealed that most plants were a mixture of alleles from these two genetic clusters. Although there was a significant genetic variation among the populations, genetic differentiation and mean heterozygosity were low, suggesting that clonal reproduction may be occurring. The current South African populations may therefore be clonal products with different proportions of genetic admixture introduced more than once. The original point of entry appears to be Gauteng, South Africa. Long‐distance dispersal appears to have played a major role in its spread across South Africa. Candidate Argentine biological control agents should therefore be effective on C. macrocephalum in South Africa.     

A new aggressive strain of Verticillium albo-atrum in Verticillium resistant cultivars of tomato in the Netherlands

In 1991 serious losses caused byVerticillium wilt were found on two holdings in the Westland glasshouse district in the Netherlands in which theVerticillium resistant tomato cultivars Calypso and Criterium were grown in soilless systems. Isolates from diseased plants were identified asVerticillium albo-atrum.In inoculation experimentsVerticillium resistant tomato cultivars were seriously affected by the new isolates but not by a control isolate. Moneydor, a susceptible cultivar without the Ve gene, was the most seriously diseased by all isolates. The isolates from theVerticillium resistant tomato cultivars were less virulent on the susceptible cultivar than the control isolate.     

Diversity and differentiation in two populations of Gibberella circinata in South Africa

Gibberella circinata [anamorph Fusarium circinatum (=  F. subglutinans f.sp. pini )] causes pitch canker and is an important pathogen in South African pine nurseries. The initial outbreak of the pitch canker fungus was limited to a single nursery at Ngodwana in Mpumalanga Province. Subsequently, several other pine nurseries in South Africa became infected. Most of these outbreaks were relatively small except for the outbreak in the Klipkraal nursery (Mpumalanga Province). The genetic diversity, population differentiation and relative frequencies of the sexual and asexual cycles among two South African subpopulations were determined to establish whether immigration, mutation and/or recombination contributed towards population structure. The allelic diversity of the initial population (Ngodwana) was observed to be lower (0·16) than that of the more recent Klipkraal population (0·25). Approximately 4% ( G _ST = 0·04) of total gene diversity could be attributed to differences among the subpopulations. Furthermore, six new vegetative compatibility groups (VCGs) have been identified since the initial outbreak of G. circinata in South Africa 10 years ago. The relatively low allelic diversity and low level of genetic differentiation suggest restricted gene flow among subpopulations, and indicate that the pathogen has been introduced recently. However, the amount of allelic and VCG diversity suggests that multiple genotypes have been introduced into South Africa. The increases in effective population number, allelic diversity and new VCGs over the past 10 years suggest that sexual reproduction might be occurring.     

Comparison of populations of the wilt pathogen Ceratocystis albifundus in South Africa and Uganda

Ceratocystis albifundus is an important fungal pathogen of Acacia mearnsii trees in South Africa. In a previous study, a high level of gene diversity was demonstrated in a South African population of C. albifundus . This, together with the occurrence of the pathogen on native Protea species and its exclusive occurrence in South Africa, led to the hypothesis that C. albifundus is probably native to that country. More recently, C. albifundus has been reported from A. mearnsii in south-western Uganda. The aim of this study was to compare the populations of C. albifundus from Uganda and South Africa based on genetic diversity, population structure and possible gene flow. This was achieved using codominant microsatellite markers developed for the closely related species Ceratocystis fimbriata . Available isolates for comparison were from six different areas of South Africa and six jungle stands in Uganda. Eight of the 11 available markers amplified loci in C. albifundus . Gene diversity was higher in the Ugandan population, but genotypic diversity was greater for the South African isolates. There were no common genotypes between the two populations and they shared only 22% of the total alleles. The populations were genetically isolated from each other and highly substructured within. There was no association between isolates collected from the same geographic locations, and gene flow between the two populations was low. Results suggest that C. albifundus was probably not introduced into Uganda from South Africa but rather that an ancestral population, yet to be discovered, is the source of both populations.     

番茄斑萎病毒核衣壳蛋白基因的克隆与分析

从云南表现褪绿黄化症状的番茄上分离到番茄斑萎病毒(Tomato spotted wilt virus)YN-1株系,利用RT-PCR方法克隆了该株系的核衣壳蛋白基因,并测定了该基因的序列,全长为777 bp,编码258个氨基酸。对该基因的氨基酸序列分析结果表明,YN-1 N基因与韩国3个株系的氨基酸序列相似性均高达97%。