The distribution and type B trichothecene chemotype of Fusarium species associated with head blight of wheat in South Africa during 2008 and 2009.

Fusarium head blight (FHB) of wheat occurs commonly in irrigation regions of South Africa and less frequently in dryland regions. Previous surveys of Fusarium species causing FHB identified isolates using morphological characters only. This study reports on a comprehensive characterisation of FHB pathogens conducted in 2008 and 2009. Symptomatic wheat heads were collected from the Northern Cape, KwaZulu-Natal (KZN), Bushveld and eastern Free State (irrigation regions), and from one field in the Western Cape (dryland region). Fusarium isolates were identified with species-specific primers or analysis of partial EF-1α sequences. A representative subset of isolates was characterized morphologically. In total, 1047 Fusarium isolates were collected, comprising 24 species from seven broad species complexes. The F. sambucinum (FSAMSC) and F. incarnatum-equiseti species complexes (FIESC) were most common (83.5% and 13.3% of isolates, respectively). The F. chlamydosporum (FCSC), F. fujikuroi (FFSC), F. oxysporum (FOSC), F. solani (FSSC), and F. tricinctum species complexes (FTSC) were also observed. Within the FSAMSC, 90.7% of isolates belonged to the F. graminearum species complex (FGSC), accounting for 75.7% of isolates. The FGSC was the dominant Fusaria in all four irrigation regions. F. pseudograminearum dominated at the dryland field in the Western Cape. The Northern Cape had the highest species diversity (16 Fusarium species from all seven species complexes). The type B trichothecene chemotype of FGSC and related species was inferred with PCR. Chemotype diversity was limited (15-ADON = 90.1%) and highly structured in relation to species differences. These results expand the known species diversity associated with FHB in South Africa and include first reports of F. acuminatum, F. armeniacum, F. avenaceum, F. temperatum, and F. pseudograminearum from wheat heads in South Africa, and of F. brachygibbosum, F. lunulosporum and F. transvaalense from wheat globally. Potentially novel species were identified within the FCSC, FFSC, FOSC, FSAMSC, FIESC and FTSC.

Emergence of ß-rhizobia as new root nodulating bacteria in legumes and current status of the legume-rhizobium host specificity dogma.

Recent developments in the legume rhizobium symbiotic interaction particularly those related to the emergence of novel strains of bacteria that nodulate and fix nitrogen in legumes is gaining momentum. These novel strains of bacteria were mostly isolated from the root nodules of indigenous and invasive legumes belonging to the sub families Papilionoideae and Mimosoideae in South Africa, South America and South East China. These rhizobia are phylogenetically and taxonomically different from the traditional 'alpha rhizobia' and are termed 'ß-rhizobia' as they belong to the ß-sub class of Proteobacteria. There are also new reports of novel species of root nodulating bacteria from the α-Proteobacteria, not known for several decades since the discovery of rhizobia. However, in this review focus is given to the emerging ß-rhizobia isolated from the indigenous Papilionoid legumes in the Cape Floristic regions in South Africa and the indigenous and invasive Mimosoid legumes in South America and South East Asia respectively. The nodulation of the indigenous South African Papilionoid legumes including that of Aspalathus linearis (rooibos) is discussed in a bit detail. Previous reports indicated that A. linearis is very specific in its rhizobium requirement and was reported to be nodulated by the slow growing Bradyrhizobium spp. This review however summarizes that the bacteria associated with the root nodules of A. linearis belong to members of both the alpha (α) Proteobacteria that include Mesorhizobium, Rhizobium and Bradyrhizobium spp. and the beta (ß) Proteobacteria represented by the genus Burkholderia (now reclassified as Paraburkholderia). In addition, the occurrence of Paraburkholderia as the newly emerging root nodule symbionts of various other legumes has been discussed. In doing so, the review highlights that nodulation is no longer restricted to the traditional 'rhizobia' group following the emergence of the new beta rhizobia as potential nodulators of various indigenous legumes. It thus provides some insights on the status of the legume-rhizobium host specificity concept and the loss of this specificity in several symbiotic associations that puts the long held dogma of host specificity of the legume rhizobium symbiosis in a dilemma.

Draft Genome Sequence of Mesorhizobium sp. Strain SARCC-RB16n, an Effective Nodulating and Nitrogen-Fixing Symbiont of Rooibos [Aspalathus linearis (Burm. f.)] in South Africa.

The draft genome sequence of Mesorhizobium sp. strain SARCC-RB16n reveals the presence of major symbiotic (nod and nif) and additional plant growth-promoting (PGPR) genes associated with enhanced growth of Aspalathus linearis (Burm. f.) in South Africa. The genome sequence provides vital information for the development of a commercial inoculant for rooibos cultivation.

Soybean SDS in South Africa is Caused by Fusarium brasiliense and a Novel Undescribed Fusarium sp.

Soybean sudden death syndrome (SDS) was detected in South Africa for the first time during pathogen surveys conducted in 2013 to 2014. The primary objective of this study was to characterize the 16 slow-growing Fusarium strains that were isolated from the roots of symptomatic plants. Molecular phylogenetic analyses of a portion of translation elongation factor 1-α (TEF1) and the nuclear ribosomal intergenic spacer region (IGS rDNA) indicated that the etiological agents were Fusarium brasiliense and a novel, undescribed Fusarium sp. This is the first report of F. brasiliense outside of Brazil and Argentina and the novel Fusarium sp. causing soybean SDS. Koch's postulates were completed for both fusaria on seven soybean cultivars that are commercially available in South Africa. Results of the pathogenicity experiment revealed that the strains of F. brasiliense and Fusarium sp. differed in aggressiveness to soybean, as reflected in differences in foliar symptoms, root rot, and reduction in shoot length. Cell-free culture filtrates of the two soybean SDS pathogens from South Africa and two positive control strains of F. virguliforme from the United States induced typical SDS symptoms on susceptible soybean cultivars in a whole-seedling assay, indicating that they contained phytotoxins.

Pythium spp. Associated with Rooibos Seedlings, and Their Pathogenicity Toward Rooibos, Lupin, and Oat.

Rooibos (Aspalathus linearis) is an important indigenous crop in South Africa. Oomycetes are a common problem in rooibos nurseries, causing serious losses, but limited information is available on the species involved. Molecular and morphological analyses of 117 oomycete isolates from 19 rooibos nurseries and 33 isolates from 11 native rooibos sites revealed the presence of several Pythium spp., including Pythium acanthicum, P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, P. cederbergense, and Pythium RB II, and Phytophthora cinnamomi (native site). Most of the species were identified in nurseries and native rooibos, with Pythium irregulare being the most common species occurring in all nurseries and 46% of the native sites. Phylogenetic analyses of the internal transcribed spacer region of the P. irregulare isolates showed that isolates within this species complex fit into three subclades, of which only two have previously been reported. On rooibos, all species except P. acanthicum and the previously characterized P. cederbergense and Pythium RB II were pathogenic and highly virulent. On lupin and oat, rotation crops in nurseries, the three aforementioned species were also nonpathogenic. All the other oomycete species were pathogenic on lupin but less so than on rooibos. On oat, only P. irregulare, P. myriotylum, and P. pyrilobum were pathogenic. This is the first report of P. mamillatum, P. pyrilobum, and P. myriotylum as pathogens of lupin, and P. irregulare and P. pyrilobum as pathogens of oat. The three nonpathogenic Pythium spp. were able to significantly reduce disease caused by pathogenic species in the less susceptible lupin and oat but not on rooibos. On lupin, the nonpathogenic species enhanced the virulence of Phytophthora cinnamomi.

Pythium cederbergense sp. nov. and related taxa from Pythium clade G associated with the South African indigenous plant Aspalathus linearis (rooibos).

The genus Pythium consists of more than 120 species and is subdivided into 11 phylogenetic clades (A-K) based on internal transcribed spacer (ITS) region sequence data. Pythium clade G contains only seven known species, with most not being well described. Our study characterized 12 Pythium isolates from Aspalathus linearis (rooibos) that fit into clade G. Phylogenetic analyses of the ITS region and a combined phylogeny of four gene regions (ITS, ß-tubulin, COX1 and COX2 [cytochrome c oxidase subunits I, II]) identified five clade G subclades. The rooibos isolates formed two groups, Pythium Rooibos I (RB I) and II (RB II), that clustered into two separate clades within subclade 1. The nine Pythium RB I isolates formed a distinct clade from P. iwayamai and is described here as a new species, Pythium cederbergense sp. nov. The three Pythium RB II isolates had P. canariense and P. violae as their closest relatives and were genetically diverse, suggesting the presence of several new species or a species complex that cannot be resolved with the current data, thus precluding a species description of this group. Morphological analyses showed that P. cederbergense and Pythium RB II were indistinguishable from each other but distinct from known clade G species. Clade G studies are being hampered by imprecise morphological descriptions of P. violae, P. canariense and P. iwayamai and each species being represented by only one isolate. The P. cederbergense and Pythium RB II isolates all were nonpathogenic toward rooibos, lupin and oats seedlings. One oligonucleotide was developed for each of P. cederbergense and Pythium RB II, which was able to differentiate the isolates with DNA macro-array analyses.

Suppression of Pythium and Phytophthora Damping-Off of Rooibos by Compost and a Combination of Compost and Nonpathogenic Pythium Taxa.

Pathogenic oomycetes, including Phytophthora cinnamomi and several Pythium spp. (Pythium irregulare, P. mamillatum, P. myriotylum, and P. pyrilobum), cause serious damping-off problems in rooibos (Aspalathus linearis) nurseries. The management of these pathogens in organic nurseries is problematic, because phenylamide fungicides may not be used. Compost, or compost in combination with Pythium taxa that are nonpathogenic to rooibos (P. acanthicum, P. cederbergense, and Pythium RB II), were investigated as alternative management options. Compost was able to suppress damping-off caused by several oomycete isolates but there was within- and between-species variation among the 30 evaluated isolates. This phenomenon was observed using two compost batches (A and B) sourced from independent suppliers. Compost B significantly reduced damping-off caused by 60% of the isolates, whereas compost A controlled only 37% of the isolates. The pathogens that were more readily controlled by both composts included P. mamillatum and P. pyrilobum, whereas the composts were ineffective at suppressing damping-off caused by >62% of P. irregulare and >50% of P. myriotylum isolates. Based on the evaluation of one Phytophthora cinnamomi isolate, this pathogen may also be controlled by compost. Neither of the composts as a stand-alone treatment could suppress damping-off caused by a combination of pathogenic species (P. cinnamomi, Pythium irregulare, P. mamillatum, P. myriotylum, and P. pyrilobum). However, damping-off was significantly reduced when nonpathogenic Pythium taxa (P. acanthicum, P. cederbergense, and Pythium RB II) were combined with the composts. Similarly, damping-off caused by a P. irregulare isolate that was not suppressed by either of the composts alone was significantly suppressed when the two composts were inoculated with the nonpathogenic Pythium taxa.

Analysis of the Fusarium graminearum species complex from wheat, barley and maize in South Africa provides evidence of species-specific differences in host preference.

Species identity and trichothecene toxin potential of 560 members of the Fusarium graminearum species complex (FGSC) collected from diseased wheat, barley and maize in South Africa was determined using a microsphere-based multilocus genotyping assay. Although three trichothecene types (3-ADON, 15-ADON and NIV) were represented among these isolates, strains with the 15-ADON type predominated on all three hosts. A significant difference, however, was identified in the composition of FGSC pathogens associated with Gibberella ear rot (GER) of maize as compared to Fusarium head blight (FHB) of wheat or barley (P<0.001). F. graminearum accounted for more than 85% of the FGSC isolates associated with FHB of wheat and barley (N=425), and was also the dominant species among isolates from maize roots (N=35). However, with the exception of a single isolate identified as an interspecific hybrid between Fusariumboothii and F. graminearum, GER of maize (N=100) was exclusively associated with F. boothii. The predominance of F. graminearum among FHB isolates, and the near exclusivity of F. boothii among GER isolates, was observed across all cultivars, collection dates, and provinces sampled. Because these results suggest a difference in host preference among species of the FGSC, we hypothesize that F. graminearum may be less well adapted to infect maize ears than other members of the FGSC.

Diaporthaceae associated with root and crown rot of maize.

Several isolates of coelomycetous fungi with pigmented conidia were consistently isolated from diseased roots of Zea mays in irrigated plots monitored in the KwaZulu-Natal Province of South Africa. Based on their morphology, these isolates could be identified as representative of Stenocarpella macrospora, S. maydis, and Phaeocytostroma ambiguum. Although species of Stenocarpella are well-known as causal agents of cob and stalk rot and leaf blight of maize in South Africa, the occurrence and importance of P. ambiguum is less well documented and understood. To determine the role of P. ambiguum as a root pathogen of maize, pathogenicity tests were conducted under glasshouse conditions at 18 °C night and 28 °C day temperatures using a pasteurised soil, river sand and perlite medium and a 0.5 % sand-bran inoculum. Based on these results, P. ambiguum was shown to be a primary pathogen of maize, but to be less virulent than the positive control, S. maydis. Furthermore, to clarify the higher-level phylogeny of these fungal genera, isolates were subjected to DNA sequencing of the nuclear ribosomal DNA (ITS & LSU). Partial gene sequences of the translation elongation factor 1-alpha gene were added to confirm the species monophyly. To resolve the generic placement of Phaeocytostroma, additional species such as P. sacchari, P. plurivorum and P. megalosporum were also added to the analysis. Based on these results, Stenocarpella and Phaeocytostroma were shown to be two well defined genera, belonging to Diaporthales, Diaporthaceae, being closely allied to Phomopsis (Diaporthe). All three genera were also observed to form alpha as well as beta conidia, and although this phenomenon is well documented for Phomopsis and Phaeocytostroma, it is a new observation for Stenocarpella. In spite of the differences in conidial pigmentation, no support could be obtained for polyphyly in Diaporthaceae, suggesting that as observed in Botryosphaeriaceae (Botryosphaeriales), conidial pigmentation is not informative at the family level in Diaporthales.

Taxonomy and phylogeny of the Fusarium dimerum species group.

The morphospecies Fusarium dimerum, known only from its anamorph, comprises at least 12 phylogenetically distinct species. Analyses of the large subunit ribosomal DNA (LSU rDNA) show they are taxa of the Nectriaceae (Hypocreales), related to F. domesticum and form a phylogenetically distinct clade within Fusarium. Fusarium dimerum, for which no herbarium material could be located, is characterized by macroconidia with a single, median septum, according to the original description and illustration. Fusarium lunatum (= F. dimerum var. violaceum) forms similar but longer macroconidia and purple, catenate or clustered chlamydospores. Fusarium delphinoides sp. nov., F. biseptatum sp. nov., F. penzigii sp. nov., F. nectrioides comb. nov. (= F. dimerum var. nectrioides) and two unnamed Fusarium spp. produce macroconidia with mostly two or rarely three septa. The name F. dimerum, which originally was applied to a fungus from a citron, is used for a taxon including isolates causing infections in immunocompetent and immunocompromised patients. Fusarium nectrioides, F. delphinoides, F. penzigii and F. biseptatum are known from soil and dead plant substrata or rarely as agents of trauma-related eye infections of humans. Fusarium lunatum is an inhabitant of the cladodes of species within the cactus genera Opuntia and Gymnocalycium. Its unnamed closest sister taxon, which also forms 1-septate macroconidia and purple, clustered chlamydospores, was isolated from a human sinus. Fusarium delphinoides is a pathogen of the cactus-like African species Hoodia gordonii (Apocynaceae). Phylogenetic analyses based on combined sequences of the internal transcribed spacer region, LSU rDNA and partial sequences of the elongation factor 1-alpha and beta-tubulin genes identified a clade of several species producing predominately 2-septate macroconidia as the reciprocally monophyletic sister of F. dimerum. The basal sister group of the two aforementioned clades includes Fusarium lunatum and two undescribed species, all of which form 1-septate macroconidia.

Characterisation of Phomopsis spp. associated with die-back of rooibos (Aspalathus linearis) in South Africa.

Die-back of rooibos (Aspalathus linearis) causes substantial losses in commercial Aspalathus plantations in South Africa. In the past, the disease has been attributed to Phomopsis phaseoli (teleomorph: Diaporthe phaseolorum). Isolates obtained from diseased plants, however, were highly variable with regard to morphology and pathogenicity. The aim of the present study was thus to identify the Phomopsis species associated with die-back of rooibos. Isolates were subjected to DNA sequence comparisons of the internal transcribed spacer region (ITS1, 5.8S, ITS2) and partial sequences of the translation elongation factor-1 alpha gene. Furthermore, isolates were also compared in glasshouse inoculation trials on 8-mo-old potted plants to evaluate their pathogenicity. Five species were identified, of which D. aspalathi (formerly identified as D. phaseolorum or D. phaseolorum var. meridionalis) proved to be the most virulent, followed by D. ambigua, Phomopsis theicola, one species of Libertella and Phomopsis, respectively, and a newly described species, P. cuppatea. A description is also provided for D. ambigua based on a newly designated epitype specimen.

Toxicity, pathogenicity, and genetic differentiation of five species of fusarium from sorghum and millet.

ABSTRACT Fusarium isolates recovered from sorghum and millet are commonly identified as F. moniliforme, but with the recognition of new species in this group, the strains given this name are being re-evaluated. We analyzed five strains each from five Fusarium species (F. andiyazi, F. nygamai, F. pseudonygamai, F. thapsinum, and F. verticillioides) often associated with sorghum and millet for their ability to produce fumonisin and moniliformin, their toxicity to ducklings, and their ability to cause disease on sorghum seedlings in vitro. These species can be distinguished with isozymes (fumarase, NADP-dependent isocitrate dehydrogenase, and malate dehydrogenase) and with banding patterns resulting from amplified fragment length polymorphisms. Two species, F. pseudonygamai and F. thapsinum, produced high levels of moniliformin, but little or no fumonisins, and were consistently highly toxigenic in the duckling tests. Two species, F. verticillioides and F. nygamai, produced high levels of fumonisins, but little or no moniliformin, and also were toxigenic in the duckling tests. F. andiyazi produced little or no toxin and was the least toxigenic in the duckling tests. In sorghum seedling pathogenicity tests, F. thapsinum was the most virulent followed by F. andiyazi, then F. verticillioides, and finally F. nygamai and F. pseudonygamai, which were similar to each other. Thus, these five species, which would once have all been called F. moniliforme, differ sufficiently in terms of plant pathogenicity and toxin production profile, that their previous misidentification could explain inconsistencies in the literature and differences observed by researchers who thought they were all working with the same fungal species.

Characterization of Colletotrichum species associated with diseases of Proteaceae.

Colletotrichum spp. are known to occur on and cause diseases of Proteaceae, but their identities are confused and poorly understood. The aim of the present study thus was to identify accurately the Colletotrichum spp. associated with diseases of cultivated Proteaceae. Colletotrichum spp. associated with proteaceous hosts growing in various parts of the world were identified based on morphology, sequence data of the internal transcribed spacer region (ITS-1, ITS-2), the 5.8S gene, and partial sequences of the ß-tubulin gene. Four species of Colletotrichum were found to be associated with Proteaceae. Colletotrichum gloeosporioides, a cosmopolitan species known to occur on numerous hosts, was isolated from Protea cynaroides cultivated in South Africa and Zimbabwe, and from a Leucospermum sp. in Portugal. A recently described species, C. boninense was associated with Zimbabwean and Australian Proteaceae but also occurred on a Eucalyptus sp. in South Africa. This represents a major geographical and host extension for the species and a description of the African strains is provided. Colletotrichum crassipes was represented by a single isolate obtained from a Dryandra plant in Madeira. Colletotrichum acutatum was isolated from Protea and Leucadendron in South Africa as well as from other hosts occurring elsewhere. A pathologically distinct population of this species was found to occur on Hakea in South Africa. This population is described as C. acutatum f. sp. hakeae, and its relationship with other strains of C. acutatum is discussed. Contrary to earlier literature reports linking C. gloeosporioides to anthracnose of Proteaceae, the present study has shown that several distinct species of Colletotrichum are associated with different diseases of this crop, which has serious implications for quarantine and disease control practices.