Gavin Ash

Low level of genetic diversity in cultivated pigeonpea compared to its wild relatives is revealed by diversity arrays technology

Understanding the distribution of genetic diversity among individuals, populations and gene pools is crucial for the efficient management of germplasm collections and breeding programs. Diversity analysis is routinely carried out using sequencing of selected gene(s) or molecular marker technologies. Here we report on the development of Diversity Arrays Technology (DArT) for pigeonpea (Cajanus cajan) and its wild relatives. DArT tests thousands of genomic loci for polymorphism and provides the binary scores for hundreds of markers in a single hybridization-based assay. We tested eight complexity reduction methods using various combinations of restriction enzymes and selected PstI/HaeIII genomic representation with the largest frequency of polymorphic clones (19.8%) to produce genotyping arrays. The performance of the PstI/HaeIII array was evaluated by typing 96 accessions representing nearly 20 species of Cajanus. A total of nearly 700 markers were identified with the average call rate of 96.0% and the scoring reproducibility of 99.7%. DArT markers revealed genetic relationships among the accessions consistent with the available information and systematic classification. Most of the diversity was among the wild relatives of pigeonpea or between the wild species and the cultivated C. cajan. Only 64 markers were polymorphic among the cultivated accessions. Such narrow genetic base is likely to represent a serious impediment to breeding progress in pigeonpea. Our study shows that DArT can be effectively applied in molecular systematics and biodiversity studies.

AN EXPERT SYSTEM FOR THE MANAGEMENT OF BOTRYTIS CINEREA IN AUSTRALIAN VINEYARDS. I. DEVELOPMENT

Abstract An expert system was developed with the aim of improving decision-making by grape growers in their management of Botrytis cinerea . Knowledge was obtained from the refereed literature on B. cinerea and also from the more general literature on plant pathology and viticulture. The decision to apply a fungicide was based on estimated disease risk, economic threshold, and duration of protective cover provided by fungicides. Up to six factors were used to estimate disease risk. These were ‘Growth Stage’, ‘Conidial Infection’, ‘Mycelial Infection’, ‘Injury’, ‘Symptoms’ and ‘Cultivar Susceptibility’. The economic threshold depended on whether grapes were destined for premium or bulk wine, and length of protective cover of a fungicide was estimated from use patterns advocated on product labels. The knowledge was structured into a dependency network of objects, with the object determining the need to apply a fungicide at the apex of the network. The knowledge was then represented in the knowledge base of the expert system in a series of IF-THEN rules.

Genetic characterization of a novel Phomopsis sp., a putative biocontrol agent for Carthamus lanatus

A large number of isolates of Phomopsis sp. have been collected from the weed Carthamus lanatus (saffron thistle) in Australia, and their potential as biological control agents for weeds of the Asteraceae has been demonstrated. An analysis of their genetic diversity and a multigene phylogenetic analysis were undertaken to ascertain whether these isolates were distinct from other species of Phomopsis that commonly attack crop species in Australia. Minimal variation was found between the Phomopsis spp. isolated from saffron thistle, except two isolates that appeared to share identity with Diaporthe helianthii and P. viticola. Analysis of the selected isolates from saffron thistle with the nucleotide sequence of the partial ITS and tef1-α regions demonstrated that the sequences were distinct from all other species of Phomopsis so far described from crops in Australia. These findings provide strong support for the recognition of these isolates as a separate species of Phomopsis. The implications of these findings are discussed in relation to biological control of saffron thistle.

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species

BackgroundMetarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).ResultsWhole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.ConclusionsThe annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Clarifying the Nomenclature in Microbial Weed Control

Correct terminology is essential to promote the concepts of biological control. In the current literature there are many terms used interchangeably and often inappropriately. The purpose of this paper is to examine the overlapping and sometimes contradictory terms used in biological weed control and to suggest a standardized usage to clarify existing terms. This is achieved through a hierarchical classification of existing terms (bioherbicide, mycoherbicide) and the introduction of new terms such as biopestistat (an inundatively applied, living organism which reduces the competitive ability of the target weed to below a desired threshold) and microbially derived phytotoxin (secondary metabolite used in weed management practices as an analogue of a chemical herbicide).

Gulypyrones A and B and Phomentrioloxins B and C Produced by Diaporthe gulyae, a Potential Mycoherbicide for Saffron Thistle (Carthamus lanatus).

A virulent strain of Diaporthe gulyae, isolated from stem cankers of sunflower and known to be pathogenic to saffron thistle, has been shown to produce both known and previously undescribed metabolites when grown in either static liquid culture or a bioreactor. Together with phomentrioloxin, a phytotoxic geranylcyclohexenetriol recently isolated from a strain of Phomopsis sp., two new phytotoxic trisubstituted α-pyrones, named gulypyrones A and B (1 and 2), and two new 1,O- and 2,O-dehydro derivatives of phomentrioloxin, named phomentrioloxins B and C (3 and 4), were isolated from the liquid culture filtrates of D. gulyae. These four metabolites were characterized as 6-[(2S)2-hydroxy-1-methylpropyl]-4-methoxy-5-methylpyran-2-one (1), 6-[(1E)-3-hydroxy-1-methylpropenyl]-4-methoxy-3-methylpyran-2-one (2), 4,6-dihydroxy-5-methoxy-2-(7-methyl-3-methyleneoct-6-en-1-ynyl)cyclohex-2-enone (3), and 2,5-dihydroxy-6-methoxy-3-(7-methyl-3-methyleneoct-6-en-1-ynyl)cyclohex-3-enone (4) using spectroscopic and chemical methods. The absolute configuration of the hydroxylated secondary carbon of the 2-hydroxy-1-methylpropyl side chain at C-6 of gulypyrone A was determined as S by applying a modified Moshers method. Other well-known metabolites were also isolated including 3-nitropropionic, succinic, and p-hydroxy- and p-methylbenzoic acids, p-hydroxybenzaldehyde, and nectriapyrone. When assayed using a 5 mM concentration on punctured leaf disks of weedy and crop plants, apart from 3-nitropropionic acid (the main metabolite responsible for the strong phytotoxicity of the culture filtrate), phomentrioloxin B caused small, but clear, necrotic spots on a number of plant species, whereas gulypyrone A caused leaf necrosis on Helianthus annuus plantlets. All other compounds were weakly active or inactive.

Effects of temperature on the response of some Australian wheat cultivars to Puccinia striiformis f. sp. tritici

The response of 83 Australian wheat cultivars to Puccinia striiformis f. sp. tritici was tested at the seedling stage under controlled conditions of low (18/5 °C) and high (24/15 °C) post-inoculation temperature. Twenty cultivars differed in their response to P. striiformis f. sp. tritici at high and low post-inoculation temperatures, and in all of these cases a shift toward resistance at higher temperatures was recorded. Detailed studies made on a selection of 13 cultivars confirmed these results and further indicated that the high temperature treatment used did not adversely affect pathogen development in susceptible cultivars. The resistance observed in the wheat cultivars Cook, Timgalen, and Suneca at higher post-inoculation temperatures was not affected by pre-inoculation temperature.

CLIMEX and DYMEX simulations of the potential occurrence of rice blast disease in south-eastern Australia

Rice blast, caused by Magnaporthe grisea does not occur in Australia. The potential for infection and sporulation events by M. grisea under Australian conditions was investigated using two software programs, DYMEX and CLIMEX. The climate of Deniliquin, NSW, representative of the southern Australian rice-growing areas, was projected to the rest of the world and compared, using CLIMEX, with foreign regions where rice blast occurs. Most of the locations whose climates matched that of Deniliquin were within the distribution range of rice blast and the potential for establishment of the disease therefore appears high. A model was also developed and run under DYMEX to predict the potential number of infection and sporulation events of the pathogen. The model was run for the period 1988–1999 with the meteorological data of four representative locations in the Australian ricegrowing region. Out of a possible 11 rice-growing seasons, the number of years favourable for M. grisea ranged from two at Griffith to nine at Yanco. The rice blast model confirmed the CLIMEX results and highlighted the potential threat of rice blast to the Australian rice industry. This paper reports the first disease model developed using DYMEX.

Survey of Botryosphaeriaceae associated with grapevine decline in the Hunter Valley and Mudgee grape growing regions of New South Wales

Species belonging to the fungal family Botryosphaeriaceae are important pathogens of grapevines in Australia. A survey of declining grapevines in the Hunter Valley and Mudgee grape growing regions of New South Wales revealed 36% were infected with species belonging to the Botryosphaeriaceae. The incidence of Diplodia seriata was greatest, followed by Neofusicoccum parvum, Botryosphaeria dothidea and Lasiodiplodia theobromae. These identifications were made using a combination of molecular and morphological characters. Although D. seriata was the most common species found, its role as a primary pathogen of grapevines in Australia is yet to be ascertained. The accuracy of estimating the incidence of species was increased by surveying up to 25 grapevines per vineyard and by sampling both the trunks and cordons. Other pathogens capable of causing trunk diseases were also isolated in this survey, demonstrating that diagnosis based on symptoms alone is not sufficient and isolations on artificial media and sometimes DNA sequencing are required for a definitive diagnosis of the causal organism of decline.

Prevalence and survival, with emphasis on stubble burning, of Rhizoctonia spp., causal agents of sheath diseases of rice in Australia

Aggregate sheath spot and sheath spot of rice were found in Australia in 2001. A disease survey revealed that both diseases are already well distributed within the Australian rice growing areas and that disease severity can be relatively important in some crops. Epidemiological studies showed that under Australian conditions, both Rhizoctonia oryzae and R. oryzae-sativae could overwinter as mycelium on straw debris, regardless of whether the straw is left on the ground or buried. Mycelium of R. oryzae-sativae present on rice straw was also found to be able to produce sclerotia, as a saprophyte, during the overwintering period. Results also strongly suggest that overwintered hyphal fragments present in the debris supplement the sclerotia as a primary source of inoculum, and also highlight the importance of straw management to reduce the inoculum of both pathogens in rice paddocks. The effect of burning stubble on the survival of laboratory-produced sclerotia of R. oryzae-sativae was investigated and it was shown that the vast majority of the sclerotia present on the soil surface survived stubble burning regardless of whether it was a ‘cold burn’ or a ‘hot burn’. A threshold temperature for sclerotial mortality was found to be between 93 and 12 PC.