Genetic variation in Pythium myriotylum based on SNP typing and development of a PCR-RFLP detection of isolates recovered from Pythium soft rot ginger.

Pythium myriotylum is responsible for severe losses in both capsicum and ginger crops in Australia under different regimes. Intraspecific genomic variation within the pathogen might explain the differences in aggressiveness and pathogenicity on diverse hosts. In this study, whole genome data of four P. myriotylum isolates recovered from three hosts and one Pythium zingiberis isolate were derived and analysed for sequence diversity based on single nucleotide polymorphisms (SNPs). A higher number of true and unique SNPs occurred in P. myriotylum isolates obtained from ginger with symptoms of Pythium soft rot (PSR) in Australia compared to other P. myriotylum isolates. Overall, SNPs were discovered more in the mitochondrial genome than those in the nuclear genome. Among the SNPs, a single substitution from the cytosine (C) to the thymine (T) in the partially sequenced CoxII gene of 14 representatives of PSR P. myriotylum isolates was within a restriction site of HinP1I enzyme which was used in the PCR-RFLP for detection and identification of the isolates without sequencing. The PCR-RFLP was also sensitive to detect PSR P. myriotylum strains from artificially infected ginger without the need for isolation for pure cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of intraspecific variants of Pythium myriotylum isolates recovered from different hosts and origins based on single nucleotide polymorphism (SNP) genotyping of multiple genes. The SNPs discovered provide valuable makers for detection and identification of P. myriotylum strains initially isolated from Pythium soft rot (PSR) ginger by using PCR-RFLP of the CoxII locus. The PCR-RFLP was also sensitive to detect P. myriotylum directly from PSR ginger sampled from pot trials without the need of isolation for pure cultures.

Green and brown bridges between weeds and crops reveal novel Diaporthe species in Australia.

Diaporthe (syn. Phomopsis) species are well-known saprobes, endophytes or pathogens on a range of plants. Several species have wide host ranges and multiple species may sometimes colonise the same host species. This study describes eight novel Diaporthe species isolated from live and/or dead tissue from the broad acre crops lupin, maize, mungbean, soybean and sunflower, and associated weed species in Queensland and New South Wales, as well as the environmental weed bitou bush (Chrysanthemoides monilifera subsp. rotundata) in eastern Australia. The new taxa are differentiated on the basis of morphology and DNA sequence analyses based on the nuclear ribosomal internal transcribed spacer region, and part of the translation elongation factor-1α and ß-tubulin genes. The possible agricultural significance of live weeds and crop residues ('green bridges') as well as dead weeds and crop residues ('brown bridges') in aiding survival of the newly described Diaporthe species is discussed.

Stem cankers on sunflower (Helianthus annuus) in Australia reveal a complex of pathogenic Diaporthe (Phomopsis) species.

The identification of Diaporthe (anamorph Phomopsis) species associated with stem canker of sunflower (Helianthus annuus) in Australia was studied using morphology, DNA sequence analysis and pathology. Phylogenetic analysis revealed three clades that did not correspond with known taxa, and these are believed to represent novel species. Diaporthe gulyae sp. nov. is described for isolates that caused a severe stem canker, specifically pale brown to dark brown, irregularly shaped lesions centred at the stem nodes with pith deterioration and mid-stem lodging. This pathogenicity of D. gulyae was confirmed by satisfying Koch's Postulates. These symptoms are almost identical to those of sunflower stem canker caused by D. helianthi that can cause yield reductions of up to 40 % in Europe and the USA, although it has not been found in Australia. We show that there has been broad misapplication of the name D. helianthi to many isolates of Diaporthe (Phomopsis) found causing, or associated with, stem cankers on sunflower. In GenBank, a number of isolates had been identified as D. helianthi, which were accommodated in several clades by molecular phylogenetic analysis. Two less damaging species, D. kochmanii sp. nov. and D. kongii sp. nov., are also described from cankers on sunflower in Australia.

QTL analysis of ergot resistance in sorghum.

Sorghum ergot, caused predominantly by Claviceps africana Frederickson, Mantle, de Milliano, is a significant threat to the sorghum industry worldwide. The objectives of this study were firstly, to identify molecular markers linked to ergot resistance and to two pollen traits, pollen quantity (PQ) and pollen viability (PV), and secondly, to assess the relationship between the two pollen traits and ergot resistance in sorghum. A genetic linkage map of sorghum RIL population R931945-2-2 x IS 8525 (resistance source) was constructed using 303 markers including 36 SSR, 117 AFLP , 148 DArT and two morphological trait loci. Composite interval mapping identified nine, five, and four QTL linked to molecular markers for percentage ergot infection (PCERGOT), PQ and PV, respectively, at a LOD >2.0. Co-location/linkage of QTL were identified on four chromosomes while other QTL for the three traits mapped independently, indicating that both pollen and non pollen-based mechanisms of ergot resistance were operating in this sorghum population. Of the nine QTL identified for PCERGOT, five were identified using the overall data set while four were specific to the group data sets defined by temperature and humidity. QTL identified on SBI-02 and SBI-06 were further validated in additional populations. This is the first report of QTL associated with ergot resistance in sorghum. The markers reported herein could be used for marker-assisted selection for this important disease of sorghum.

First Report of Pythium splendens Associated with Severe Wilt of Muskmelon (Cucumis melo) in Oman.

Muskmelon (Cucumis melo L.) is one of the most important vegetable crops in Oman. In the fall of 2004, sudden wilt was observed in muskmelon grown in a field at Sultan Qaboos University, Muscat. The disease was characterized by rapid collapse of vines and muskmelon plants at the fruit production to maturation stage, associated with brown-to-dark brown rotted primary and secondary roots. The disease resulted in death of more than 85% of muskmelon plants in that field. On potato dextrose agar (PDA), with published methods (1), Pythium spp. were consistently isolated from crowns and roots of plants showing wilt symptoms. Further identification of five isolates of Pythium with sequences of the internal transcribed spacer (ITS) of the ribosomal DNA (1) using ITS1 and ITS4 primers produced a nucleotide sequence 806 bp long, which was identical among all isolates. Comparison with sequences deposited at the National Center for Biotechnology Information revealed 100% nucleotide similarity to a previously published sequence (Accession No. DQ381808) of isolate P091 of P. splendens from cucumber from Oman, for which identification has also been confirmed by morphological characteristics. The sequence of one isolate of P. splendens (P222) was assigned GenBank Accession No. EF546436 and deposited at CBS under Accession No. CBS121855. In pathogenicity tests conducted in a greenhouse, P. splendens induced damping-off symptoms on 7-day-old muskmelon seedlings and also reproduced the same wilt symptoms observed in the field when 2-month-old muskmelon plants were inoculated with 3-day-old P. splendens grown in PDA. To our knowledge, this is the first report of association of P. splendens with wilt of muskmelon in Oman. Reference: (1) A. M. Al-Sa'di et al. Plant Pathol. 56:140, 2007.

First Report of Glomerella cingulata (Colletotrichum gloeosporioides) Causing Anthracnose and Tip Dieback of Lygodium microphyllum and L. japonicum in Australia.

The Old World climbing fern, Lygodium microphyllum (Cav.) R. Br., and Japanese climbing fern, L. japonicum (Thunb.) Sw., are invasive noxious weeds in Florida (1). Exploratory surveys for classical biological control agents of L. microphyllum in the fern's native range of Australia and Asia have focused on aboveground herbivores (1). From February to August 2006, fungi were isolated from symptomatic foliage, including lesions associated with leaf curls caused by the mite Flocarus perrepae Knihinicki & Boczek., obtained from L. microphyllum at sites across southeast Queensland, Australia and from both fern species grown at the CSIRO Long Pocket Laboratories in Brisbane, Australia. Anthracnose symptoms with chlorotic margins, initiating at the tip or base of the individual pinnules, were observed on fronds. Dieback symptoms affected growing tips, with sunken lesions and a gradual necrotic wilt as far as the next growth junction of pinnae. Sections from diseased margins were surface sterilized, placed onto water agar, and incubated at 23°C with a 16-h photoperiod. Variable colonies of white-to-gray mycelia, felted or tufted with complete margins, grew well on oatmeal agar and potato dextrose agar. Conidia were hyaline to light salmon, aseptate, straight, and cylindrical (10.4 to 18.2 × 2.6 to 5.2 µm), borne in salmon-to-bright orange masses at 25°C, and consistent with previous descriptions of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (3), anamorph of Glomerella cingulata (2). Asci that formed after 3 to 4 weeks in culture were eight-spored, clavate to cylindrical (46.8 to 62.4 × 9.1 to 11.7 µm), and thickened at the apex, and ascospores were cylindrical (11.7 to 18.2 × 3.9 to 5.2 µm), slightly curved, unicellular and hyaline, which is consistent with descriptions of G. cingulata (2). No fruiting bodies were observed in planta; acervuli, setae, and perethecia were not observed. Identification was further confirmed by molecular analysis using the primer pair ITS1/ITS4 (4) (GenBank Accession No. EU697014), indicating 100% similarity to isolates of G. cingulata. To confirm pathogenicity, Koch's postulates were performed on three plants of L. japonicum and 12 plants of L. microphyllum, with an equal number of controls. Conidial suspensions were made to 1.7 × 106 conidia ml-1. During the experiments in the glasshouse, temperatures ranged from 12.6 to 40°C and relative humidity from 39 to 85%. Tips and fronds were collected after 2 to 8 weeks and isolation and identification performed. G. cingulata was consistently reisolated from diseased tissue. No symptoms appeared on controls and isolations did not yield the pathogen. To our knowledge, this is the first report of G. cingulata infecting L. microphyllum and L. japonicum in Australia. Its potential as a biological control agent in the ferns' introduced range remains to be tested. References: (1) J. A. Goolsby et al. Biol. Control. 28:33, 2003. (2) J. E. M. Mordue. Glomerella cingulata. No. 315 in: CMI Descriptions of Pathogenic Fungi and Bacteria. CAB, Kew, UK, 1971. (3) B. C. Sutton. The Genus Glomerella and its Anamorph Colletotrichum. In: Colletotrichum: Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International, Wallingford, UK, 1992. (4) T. M. White et al. Amplification and Direct Sequencing of Fungal Ribosomal RNA for Phylogenetics. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

The Genetic Structure of Australian Populations of Mycosphaerella musicola Suggests Restricted Gene Flow at the Continental Scale.

ABSTRACT Mycosphaerella musicola causes Sigatoka disease of banana and is endemic to Australia. The population genetic structure of M. musicola in Australia was examined by applying single-copy restriction fragment length polymorphism probes to hierarchically sampled populations collected along the Australian east coast. The 363 isolates studied were from 16 plantations at 12 sites in four different regions, and comprised 11 populations. These populations displayed moderate levels of gene diversity (H = 0.142 to 0.369) and similar levels of genotypic richness and evenness. Populations were dominated by unique genotypes, but isolates sharing the same genotype (putative clones) were detected. Genotype distribution was highly localized within each population, and the majority of putative clones were detected for isolates sampled from different sporodochia in the same lesion or different lesions on a plant. Multilocus gametic disequilibrium tests provided further evidence of a degree of clonality within the populations at the plant scale. A complex pattern of population differentiation was detected for M. musicola in Australia. Populations sampled from plantations outside the two major production areas were genetically very different to all other populations. Differentiation was much lower between populations of the two major production areas, despite their geographic separation of over 1,000 km. These results suggest low gene flow at the continental scale due to limited spore dispersal and the movement of infected plant material.

Identification of a major locus conferring resistance to powdery mildew (Erysiphe polygoni DC) in mungbean (Vigna radiata L. Wilczek) by QTL analysis.

A major locus conferring resistance to the causal organism of powdery mildew, Erysiphe polygoni DC, in mungbean (Vigna radiata L. Wilczek) was identified using QTL analysis with a population of 147 recombinant inbred individuals. The population was derived from a cross between 'Berken', a highly susceptible variety, and ATF 3640, a highly resistant line. To test for response to powdery mildew, F7 and F8 lines were inoculated by dispersing decaying mungbean leaves with residual conidia of E. polygoni amongst the young plants to create an artificial epidemic and assayed in a glasshouse facility. To generate a linkage map, 322 RFLP clones were tested against the two parents and 51 of these were selected to screen the mapping population. The 51 probes generated 52 mapped loci, which were used to construct a linkage map spanning 350 cM of the mungbean genome over 10 linkage groups. Using these markers, a single locus was identified that explained up to a maximum of 86% of the total variation in the resistance response to the pathogen.