Yared T. Tewoldemedhin

Cylindrocarpon species associated with apple tree roots in South Africa and their quantification using real-time PCR

Cylindrocarpon species are known to be a component of the pathogen/pest complex that incites apple replant disease. In South Africa, no information is available on apple associated Cylindrocarpon species and their pathogenicity. Therefore, these aspects were investigated. Among the isolates recovered from apple roots in South Africa, four species (C. destructans, C. liriodendri, C. macrodidymum and C. pauciseptatum) were identified using β-tubulin gene sequencing and phylogenetic analysis. This is the first report of C. liriodendri, C. macrodidymum and C. pauciseptatum on apple trees. Cylindrocarpon macrodidymum was the most prevalent. Isolates within each of the four species were pathogenic towards apple seedlings, but varied in their virulence. With a single exception, all isolates were able to induce lesion development on seedling roots. Only 57% of the isolates, which represented all four species, were able to cause a significant reduction in seedling weight and/or height. The greatest seedling growth reductions were caused by two isolates of C. destructans, and one isolate each of C. liriodendri and C. macrodidymum. A quantitative real-time polymerase chain reaction (qPCR) method was developed for simultaneous detection of all four Cylindrocarpon species. qPCR analyses of Cylindrocarpon from the roots of inoculated seedlings showed that the amount of Cylindrocarpon DNA in roots was not correlated to seedling growth reductions (weight and height) or root rot. The qPCR method is, however, very useful for the rapid identification of apple associated Cylindrocarpon species in roots. The technique may also hold potential for being indicative of Cylindrocarpon disease potential if rhizosphere soil rather than roots are used.

Morphological and phylogenetic analyses of Pythium species in South Africa

The genus Pythium is important in agriculture, since it contains many plant pathogenic species, as well as species that can promote plant growth and some that have biocontrol potential. In South Africa, very little is known about the diversity of Pythium species within agricultural soil, irrigation and hydroponic systems. Therefore, the aim of the study was to characterise a selection of 85 Pythium isolates collected in South Africa from 1991 through to 2007. The isolates were characterised morphologically as well as through sequence and phylogenetic analyses of the internal transcribed spacer regions (ITS) and the 5.8S gene of the nuclear ribosomal DNA. Phylogenetic analyses showed that the isolates represented ten of the 11 published Pythium clades [Lévesque & De Cock, 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycological Research 108: 1363-1383]. Characterisation of isolates in clade D and J suggested that the phylogenetic concept of Pythium acanthicum and Pythium perplexum respectively, needs further investigation in order to enable reliable species identification within these clades. Our phylogenetic analyses of Pythium species in clade B also showed that species with globose sporangia group basal within this clade, and are not dispersed within the clade as previously reported. The 85 South African isolates represented 34 known species, of which 20 species have not been reported previously in South Africa. Additionally, three isolates (PPRI 8428, 8300 and 8418) were identified that may each represent putative new species, Pythium sp. WJB-1 to WJB-3.

Characterization of Rhizoctonia spp. Recovered from Crop Plants Used in Rotational Cropping Systems in the Western Cape Province of South Africa

Isolates of Rhizoctonia spp. associated with barley, canola, clover, lucerne, lupin, annual Medicago spp. (medic), and wheat were recovered during the conduct of a 4-year (2000 to 2003) crop rotation trial in the Western Cape province of South Africa. These isolates were characterized by determining their anastomosis group (AG), in vitro optimum growth temperature, and pathogenicity toward emerging and 14-day-old seedlings of all the aforementioned crops. During the 4-year rotational trial, 428 Rhizoctonia isolates, in all, were obtained. The most abundant multinucleate AG was AG-4 HG-II (69%), followed by AG-2-1 (19%), AG-3 (8%), AG-2-2 (2%), and AG-11 (2%). The population of binucleate Rhizoctonia spp. comprised AG-K (53%), AG-A (10%), AG-I (5%), and unidentified AGs (32%). The optimal time for isolating Rhizoctonia spp. was found to be at the flowering or seedpod stage (20 to 22 weeks after planting). Temperature studies showed that isolates belonging to AG-2-2, AG-4 HG-II, and AG-K had significantly higher optimum growth temperatures than those from other AGs. In pathogenicity assays conducted on emerging as well as 14-day-old seedlings, isolates of AG-2-2 and AG-4 HG-II were the most virulent on all crops. Rhizoctonia solani AG-2-1 was highly virulent on canola, moderately virulent on medic and lupin, weakly virulent on lucerne and barley, and nonpathogenic on wheat. AG-11 isolates were moderate to weakly virulent on all crops, with the exception of barley and wheat. AG-3 was weakly virulent on canola, lupin, and medic. AG-K was the only binucleate Rhizoctonia sp. capable of inciting disease in our assays. This is the first comprehensive study to elucidate the identity and potential importance of Rhizoctonia spp. as a yield limiting factor in crop production systems in the Western Cape province of 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.

Evaluation of strategies for the control of canola and lupin seedling diseases caused by Rhizoctonia anastomosis groups

Several methods with potential for the management of Rhizoctonia diseases of canola and lupin including plant resistance, fungicide seed treatment and biological control using binucleate Rhizoctonia anastomosis groups (AGs) were evaluated under glasshouse conditions. Screening included the examination of resistance of eight canola and eight lupin cultivars/selections to damping-off and hypocotyl/root rot caused by the multinucleate Rhizoctonia solani AG-2-1, 2–2, 4 and 11. All canola cultivars were highly susceptible to AG-2-1, but Rocket, Spectrum and 44C11 were more resistant than the other cultivars. Spectrum and 44C73 were also more resistant to AG-4 than the other canola cultivars. On lupin, R. solani AG-2-2 and 4 were most virulent, and the cultivar Cedara 6150 and selection E16 were most resistant to AG-2-2; Cedara 6150, E16, Mandelup and Quilinock were more resistant to AG-4 than the other cultivars/selections. The Lupinus luteus selections, E80.1.1.2 and E82.1.1 were most susceptible to AG-2-2, 4 and 11. Seed treatment with the fungicides Cruiser OSR (a.i. difenconazole, fludioxonil, metalaxyl-M, thiamethoxam) and SA-combination (a.i. iprodione, metalaxyl, thiram) significantly increased survival of canola and lupin seedlings, decreased hypocotyl/root rot and improved the percentage of healthy seedlings, with the SA-combination being significantly more effective than Cruiser OSR. Application of the binucleate Rhizoctonia AGs (A, Bo, K and I) significantly increased the survival of lupin seedlings inoculated with R. solani AG-2-2 and 4, and AG-I and K significantly improved survival of canola in the presence of AG-4. This is the first report of the potential of binucleate AGs to protect canola and lupin seedlings against infection by multinucleate AGs.

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. Kochs 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.

Rhizoctonia Anastomosis Groups Associated with Diseased Rooibos Seedlings and the Potential of Compost as Soil Amendment for Disease Suppression

Rhizoctonia spp. associated with rooibos in the Western Cape province of South Africa were recovered during the 2008 season by planting seedlings in rhizosphere soils collected from 14 rooibos nurseries. In all, 75 Rhizoctonia isolates were obtained, of which 67 were multinucleate and 8 were binucleate Rhizoctonia spp. The identity of these isolates to anastomosis group (AG) was determined through sequence analysis of the ribosomal DNA internal transcribed spacer region. The collection of multinucleate isolates included representatives of AG-2-2 (67%), AG-4 HGI (14%), AG-11 (5%), and R. zeae (3%). Binucleate AGs included AG-Bo (4%) and AG-K (4%) and an unidentified binucleate Rhizoctonia (3%). Rhizoctonia solani AG-2-2 was the most widely distributed species of Rhizoctonia detected among the 11 nurseries sampled. All AGs recovered from rooibos have been previously reported on crop plants in South Africa, with the exception of R. zeae. However, this is the first study to classify the Rhizoctonia AGs recovered from rooibos. In glasshouse bioassays, the most virulent Rhizoctonia AGs on rooibos and lupin were AG-2-2, AG-4 HGI, and AG-11. Although plant damage was less than that observed for lupin and rooibos, oat was significantly affected by AG-2-2 and AG-4 HGI. Two composts sourced from independent suppliers were evaluated for disease suppression under glasshouse conditions. Compost amendment suppressed damping-off by most R. solani AGs, except for AG-4 HGI. Furthermore, within AG-2-2, suppression by compost was isolate specific.

Effect of cropping system on composition of the Rhizoctonia populations recovered from canola and lupin in a winter rainfall region of South Africa

Rhizoctonia spp. anastomosis groups (AGs) associated with canola and lupin in the southern and western production areas of the Western Cape province of South Africa were recovered during the 2006 and 2007 growing seasons and identified using sequence analyses of the rDNA internal transcribed spacer regions. The effect of crop rotation systems and tillage practices on the recovery of Rhizoctonia spp. was evaluated at Tygerhoek (southern Cape, Riviersonderend) and Langgewens (western Cape, Moorreesburg) experimental farms. Isolations were conducted from canola planted after barley, medic/clover mixture and wheat, and lupin planted after barley and wheat, with sampling at the seedling, mid-season and seedpod growth stages. In the 2006 study, 93.5% of the Rhizoctonia isolates recovered were binucleate and 6.5% multinucleate; in 2007, 72.8% were binucleate and 27.2% were multinucleate. The most abundant AGs within the population recovered included A, Bo, I and K, among binucleate isolates and 2-1, 2-2 and 11 among multinucleate isolates. Crop rotation sequence, tillage and plant growth stage at sampling all affected the incidence of recovery of Rhizoctonia, but certain effects were site-specific. The binucleate group was more frequently isolated from lupin and the multinucleate group from canola. AG-2-1 was only isolated from canola and AG-11 only from lupin. This study showed that important Rhizoctonia AGs such as AG-2-1, 2-2 and 11 occur in both the southern and the western production areas of the Western Cape province and that crop rotation consistently influences the incidence and composition of the Rhizoctonia community recovered from the cropping system.