Sandra C. Lamprecht

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.

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.

Fusarium andiyazi sp. nov., a new species from sorghum

We describe a new Fusarium species in sec- tion Liseola from sorghum in Africa and the United States. This species is distinguished morphologically by the production of unique pseudochlamydospores in carnation leaf pieces on carnation leaf agar, and appears to be most closely related to F thapsinum. The new species also can be distinguished molecu- larly by means of Amplified Fragment Length Poly- morphisms, AFLPs. Strains with both MAT-1 and MAT-2 mating types were identified, but no sexual stage was generated in crosses made under labora- tory conditions.

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.

Pathogenicity of three Fusarium species associated with asparagus decline in South Africa

Isolations were made from crown, root, and stem lesions of symptomatic U.C. 157 F2 asparagus plants and from soil debris sampled in a declining asparagus field. Fusarium oxysporum, F. proliferatum, and to a lesser extent F. solani were the three dominant fungi isolated. Isolates of each species were evaluated for pathogenicity by inoculating U.C. 157 F2 plants in a greenhouse assay and an in vitro agar-test-tube assay. All three Fusarium spp. were pathogenic to asparagus, although they differed significantly in their disease-causing ability. In an in vitro assay, F. proliferatum isolates had a mean disease rating class of 4 compared with a class 3 for F. oxysporum and 2 for F. solani, on a scale of 1-5. Significant differences between F. proliferatum isolates were also detected. The high frequency of isolation and virulence of both F. oxysporum and F. proliferatum indicate that both species are important pathogens associated with asparagus decline in South Africa

Effect of crop rotation on crown rot and the incidence of Fusarium pseudograminearum in wheat in the Western Cape, South Africa

The effect of different crop rotation treatments and within-treatment crop sequences on crown rot and the incidence of Fusarium pseudograminearum in crowns of wheat from a long-term crop rotation experiment at Langgewens Experimental Farm, Moorreesburg, South Africa, were studied in 2000, 2001 and 2002. Crop rotation treatments included the following crops: canola (C), lupin (L), medic (M), medic-clover mixture (Mc) and wheat (W). Rotation treatments (RT) and within-treatment crop sequences were: RT 1=W-W-W-W, RT 2=C-W-W-W, W-C-W-W,W-W-C-W, RT 3=C-W-L-W, L-W-C-W, RT 4=W-L-C-W, L-C-W-W, RT 5=M-W-M-W, RT 6=Mc-W-Mc-W and RT 7=M-C-M-W. Crop rotation significantly affected the incidence of F. pseudograminearum, crown rot incidence and severity, dry mass and grain yield. The incidence of F. Pseudograminearum was significantly higher on wheat for the rotation treatment that included 3 years of wheat and 1 year of canola compared with the other treatments, and the highest severities and incidences of crown rot and the lowest yields were also recorded for this treatment and the monoculture wheat. The lowest incidences of the fungus, as well as crown rot incidences and severities, and the highest yields were recorded for the rotation treatment that alternated wheat with a medic-clover mixture and the treatment that included wheat after 3 years of rotation with broadleaf crops. Crop rotation should, therefore, be an important component of management strategies against crown rot of wheat.

Characterisation and pathogenicity of Rhizoctonia isolates associated with black root rot of strawberries in the Western Cape Province, South Africa

Black root rot is an important disease of strawberry caused by a complex of fungi including species of Rhizoctonia. In this study, the Rhizoctonia species and anastomosis groups isolated from diseased strawberries in the Western Cape Province of South Africa were determined and their pathogenicity and relative virulence assessed. Both binucleate and multinucleate types were recovered from diseased roots and identified as R. fragariae and R. solani, respectively. Anastomosis grouping of the isolates was carried out on a sub-sample using the conventional method of hyphal fusion, and molecular techniques were employed to confirm results of the former. RFLP analysis of the 28S RNA gene was used to further characterise relationships among the isolates of Rhizoctonia. The molecular results correlated with those obtained from the conventional methods. In the sub-sample tested, all isolates of R. solani were members of Anastomosis Group 6, whereas three AG types were identified among isolates of R. fragariae, viz. AG-A, AG-G and AG-I at a relative occurrence of 69%, 25% and 6%, respectively. Pathogenicity trials were conducted on 8-week-old cv. Tiobelle plants. All Rhizoctonia isolates tested were pathogenic to strawberry, but R. solani (AG 6) was the most virulent causing severe stunting of plants. R. fragariae AG-A and AG-G were not as virulent as R. solani but also caused stunting. R. fragariae AG-I was the least virulent, and did not cause stunting of the plants; however, it incited small, pale, spreading lesions on infected roots. This is the first species confirmation and AG type identification of Rhizoctonia taxa causing root rot of strawberries in South Africa.

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.

Nodulation of rooibos (Aspalathus linearis Burm. f.), an indigenous South African legume, by members of both the α-Proteobacteria and β-Proteobacteria

Rooibos (Aspalathus linearis) has been reported to be nodulated by rhizobia belonging to members of the genus Bradyrhizobium but based solely on slow growth rate on growth media in vitro. Because there is very little information about the rhizobia that nodulate and fix nitrogen in rooibos, the characterization of rhizobial strains and their ability to nodulate A. linearis was investigated in this study. Soils intially collected from the rhizosphere of different Aspalathus populations were used in a baiting experiment to trap rhizobia by rooibos roots. The rhizobia trapped in the nodules were re-isolated and used in Koch’s postulate experiment using the Leonard jar assembly in the glasshouse. The strains that formed on the average between five and 12 indeterminate pink nodules per plant resulted in statistically significant (P = 0.05) increase in shoot and root dry weights. Phylogenetic analysis of the 16S ribosomal RNA sequence of the isolates from the root nodules revealed for the first time that A. linearis is nodulated by different groups of rhizobia belonging to members of both the α-Proteobacteria and the β-Proteobacteria. It was also found that only 2% of the total rhizobia isolated from the root nodules of rooibos were represented by the genus Bradyrhizobium. The finding that rooibos is nodulated by different groups of α-rhizobia and β-rhizobia provides valuable information both in the study of the microbial ecology of rooibos and in the selection of highly efficient nitrogen fixing strains for the commercial cultivation of this indigenous legume.

Pathogenicity of Pythium irregulare, P. sylvaticum and P. ultimum var. ultimum to lucerne (Medicago sativa)

The ability of Pythium irregulare, P. sylvaticum and P. ultimum var. ultimum to induce damping-off of lucerne (Medicago sativa) seedlings cv. SA Standard was tested in artificially infested planting medium. Three levels of sand-bran inoculum were used: 5, 25 and 50 g inoculum per kg planting medium. All the Pythium species caused more than 80% damping-off at each inoculum level and are therefore pathogenic to lucerne. There were differences in virulence among the species and isolates tested. Pythium sylvaticum was the most virulent while P. irregulare was the least virulent species tested. The highest survival rates in inoculated soil were recorded for seedlings growing in soil containing the lowest levels of inoculum for each species tested. At the high inoculum levels (25 and 50 gkg) in some cases (P, sylvaticum and P. ultimum var. ultimum) survival was so low that intra-species differences in virulence could no longer be detected. Since there were differences in virulence among the species and isolates tested, it is important to test a number of isolates of a species before drawing conclusions about the pathogenicity of the species as a whole to a particular host, and to select appropriate levels of inoculum to elucidate these differences. As far as could be determined this is the first valid report of P. sylvaticum as a pathogen of lucerne seedlings.