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Almond production is a significant horticulture crop for Australia. Serious yield losses can be caused by the fungal disease anthracnose, caused by Colletotrichum species that infect the fruit and leaves. Genomic sequence data for gene extraction, along with multigene phylogenetic analyses and whole genome average nucleotide identity (ANI) analysis were used to determine the phylogeny of Colletotrichum isolates collected from almonds across Australia. Multigene phylogenetic analyses of six gene regions (ITS, tub2, gapdh, chs-1, act and his3) identified C. acutatum sensu stricto, C. fioriniae and C. simmondsii as pathogens of almond. Similar topology was observed using ANI, which provided increased resolution within C. acutatum, with isolates separating largely according to geographic origin. The ANI analysis also supported three separate species with isolates within each species sharing >98.7% ANI (species cut off = 95%), while between species there was

Verticillium wilt, caused by the soilborne fungi Verticillium dahliae and Verticillium albo-atrum, is a serious disease of potato as well as many other crops. Potato seed tuber surveys (2010 to 2012) from Victoria and Tasmania, Australia identified V. dahliae, V. albo-atrum and V. tricorpus infecting the stem end vascular tissue of seed tubers. The species were identified by traditional morphology and phylogenetic analysis of the ITS region. Isolation of V. dahliae within a seed lot varied greatly and ranged from 0 to 55%. Verticillium spp. were isolated from the stem-end vascular tissue of tubers from seed lots from Victoria and Tasmania with an overall percent infection of 27.7 (V. dahliae), 8.4 (V. albo-atrum) and 4.8 (V. tricorpus). Verticillium dahliae was isolated from 11% of tubers with discoloured stem-end vascular tissue and 3.3% of tubers without stem-end vascular decolourisation suggesting that tuber stem end vascular discolouration symptoms were not a reliable indication of Verticillium wilt infection. In glasshouse trials, V. dahliae isolates from different geographical locations varied in pathogenicity during infection of susceptible potato cv Shepody, moderately resistant cv Ranger Russet and eggplant cv Black Beauty. The majority of V. dahliae isolates were highly aggressive in potato and eggplant, especially the Tasmanian V. dahliae isolates. Infected plants of cv Shepody inoculated with V. albo-atrum and V. tricorpus showed typical wilt symptoms however, the severity of infection caused by V. tricorpus was substantially lower compared to highly aggressive isolates of V. dahliae. In eggplant, V. dahliae isolates also varied in pathogenicity in terms of disease severity but all isolates significantly (p ≤ 0.05) reduced eggplant growth.

Yield decline in pyrethrum fields of northern Tasmania was thought to be due to an interaction between soil-borne pathogens and abiotic stresses. Glasshouse trials were conducted to assess the influence of a 4-day waterlogging period on growth of pyrethrum plants already infected with the crown and root rot pathogens, Fusarium oxysporum, F. avenaceum and Paraphoma vinacea. In plants grown at optimum soil water capacity, F. oxysporum and P. vinacea significantly reduced the below-ground and total biomass of plants before waterlogging (0 bw = 2 months after inoculation, 2 mai), at 2 months after waterlogging (2 maw = 4 months after inoculation, 4 mai) and 6 months after waterlogging (6 maw = 8 months after inoculation, 8 mai) but had little effect on above-ground biomass. Although F. avenaceum was pathogenic it only had a significant effect on below-ground biomass at 4 and 8 mai. At 7 days after waterlogging treatment, plants infected with P. vinacea had more severe wilting, necrosis and chlorosis of the basal leaves and petioles than plants infected with the other two pathogens or non-infected plants. Significant interaction between pathogen treatments and waterlogging occurred at 2 maw, whereas at 6 maw plants had recovered and no significant interaction was observed between the pathogen treatments and waterlogging. The effect of waterlogging on below-ground dry weight of the plants infected with F. oxysporum and P. vinacea at 2 maw was more severe than those infected with F. avenaceum. There was no significant interaction between waterlogging and pathogens on photosynthesis 2 maw however, plants infected by each of the pathogens had the lowest photosynthesis rate in both waterlogged and the non-waterlogged treatments. At 6 maw the number of flowers, flower stems, petioles and leaves were significantly reduced by waterlogging however, there was no significant effect by pathogens nor an interaction between pathogens and waterlogging on these growth parameters. Overall, waterlogging exacerbated the effect of F. oxysporum, F. avenaceum and P. vinacea on below-ground dry weight and total biomass of the root-dip inoculated pyrethrum plants 2 maw and affected the flower and flower stem production.

Perithecia at the base of dead flower stems of pyrethrum (Tanacetum cinerariifolium) plants in yield-decline affected fields of northern Tasmania were identified as Alternaria infectoria and Stemphylium herbarum. Identification was based on morphological description of cultures established from single ascospores; and conidiospores; ascospore shape and septation; and multigene phylogenetic analyses using the internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1), polymerase II second largest subunit (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes. Stemphylium herbarum produced necrotic leaf lesions on both sides of the spray inoculated pyrethrum leaves which coalesced to encompass the entire leaf in in vivo and in vitro experiments. Alternaria infectoria produced small necrotic leaf lesions on both sides of the leaves two weeks after inoculation which did not expand and hence, was considered as a minor pathogen of pyrethrum. This is the first report of A. infectoria and S. herbarum as pathogens of pyrethrum in Australia. The role of A. infectoria and S. herbarum in pyrethrum yield-decline in northern Tasmania needs to be evaluated.

Pyrethrum (Tanacetum cinerariifolium) is commercially cultivated in Australia for the production of pyrethrins. Ray blight caused by Stagonosporopsis tanaceti is considered a major limiting factor for pyrethrum production. Histopathology of infected pyrethrum plants identified the importance of crown infection where the infection hyphae were confined to the parenchyma cells of the epidermis, hypodermis and cortex without infecting the vascular tissues. Throughout the growth of pyrethrum plants, no vascular infection occurred in the petioles, flower stems and crowns. In infected leaf petioles and flower stems, pycnidia and extra-cellular anthocyanin material developed within 1–2 weeks of infection. The symptoms of infection included a dark brown discolouration or necrosis of infected cells caused by intra- or intercellular colonisation of hyphae. Secondary infection of leaves, flower stems and flower buds most likely occurred through wind, rain or water splash of pycnidiospores released from pycnidia in the leaves and petioles. Roots of the infected plants remained uninfected throughout the growth of the plants. Detailed understanding of the disease cycle of ray blight disease in pyrethrum plants will enable the development of more efficient, targeted control measures.

Ray blight is a destructive disease of Asteraceae affecting chrysanthemum and pyrethrum industries worldwide. Three morphologically similar but phylogenetically distinct species of the family Didymellaceae; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti are associated with the disease. Despite their close evolutionary relationship and cross host pathogenicity, these species have marked differences in their biology and epidemiology. Stagonosporopsis chrysanthemi and S. inoxydabilis are both homothallic with a MAT locus that carries both mating type genes. Ascomata play a major role in survival and dispersal of S. chrysanthemi, contributing to the onset of ray blight epidemics on chrysanthemum. However, S. tanaceti is either asexual or heterothallic due to the presence of only MAT1-2 idiomorph in its genome. Morphological similarity of the species causing ray blight on various Asteraceae, multiple changes in their taxonomy and historical confusion with other Phoma-like species have resulted in lack of clarity in their taxonomy, host range and global distribution. Host specificity studies and reports on global distribution of S. chrysanthemi published before its separation from S. inoxydabilis should be treated with caution. A recently developed species-specific multiplex PCR assay provides a rapid and robust tool to study the global distribution of these important quarantine pathogens. When global populations from cultivated and/or wild Asteraceae are available, population genetic analyses may aid in understanding the origin, evolutionary history and global migration patterns of the Stagonosporopsis spp. associated with ray blight of Asteraceae.

Colonization of stems, symptom development, yield, progeny tuber infection, quantification of soil inoculum and contribution of soil inoculum on subsequent disease development by Verticillium dahliae, causal agent of Verticillium wilt of potato was evaluated in the glasshouse using naturally infected and non-infected seed tubers of seven cultivars in two trials. Internal seed tuber infection of seven potato cultivars did not contribute to severe disease development although the pathogen was isolated from symptomless plants. Susceptible cultivars Atlantic, Trent and Russet Burbank had the highest incidence of petiole infection while the moderate resistant cv Ranger Russet had the lowest incidence at 65 days after planting. Progeny tuber infection in various cultivars after harvest ranged from 0 (cv Ranger Russet) to 13 % (cv Atlantic). Planting infected tubers contributed to V. dahliae inoculum in the potting soil. Mean levels were highest from cv Russet Burbank (70 pg DNA/g of soil; range from 6 to 149) while the least was recorded in cv Ranger Russet planted soil (11 pg DNA/g of soil; range from 3 to 31). Soil inoculum originating from naturally infected tubers did not contribute to subsequent foliar disease development in Russet Burbank and Shepody cultivars that were grown in this infected soil. However, V. dahliae was isolated from the petiole of some symptomless plants. Progeny tubers were completely free from V. dahliae. Tubers of cv Nicola, infected with Verticillium tricorpus, produced very few Verticillium wilt symptoms but the fungus was re-isolated from the petiole of symptomless plants. Only 4 % of progeny tubers were infected by the pathogen and the level of V. tricorpus inoculum in the soil at maturity was only 1 cfu/g of dry soil. There was no significant difference in the yield of tubers between plants originating from infected and non-infected tubers.

The disease cycle of the chili (Capsicum annuum) anthracnose fungus Colletotrichum truncatum (formerly C. capsici) was elucidated from a study of infection and colonization of seed, leaves and fruit. Microscopic observations of detached leaves and fruit inoculated with a virulent pathotype (F83B), revealed direct cuticle penetration and, intramural, endophytic and necrotrophic phases of colonization. Seedling and fruit ripening stages were very susceptible to infection with the pathogen causing pre- and post-emergence damage and postharvest fruit rot. Furthermore, a quiescent stage, following leaf infection during the vegetative phase of plant growth served as a potential primary inoculum source for fruit infection. Leaf epidermal cells of the resistant C. chinense PBC932 expressed a strong hypersensitive response 48 h after infection (HAI) to both highly virulent (F83B) and less virulent (BRIP 26,974) pathotypes. Infected cells had thickened cell walls, cytoplasm aggregation, and high levels of reactive oxygen species produced 12 HAI. In contrast, the infected epidermal cells of the susceptible C. annuum cultivar Bangchang showed necrosis and rapid cell death after infection by either pathotype. Knowledge of the disease cycle of C. truncatum will be helpful in understanding the behaviour of the pathogen in chili fields which will lead to more efficient application of control measures.

Twenty four Ascochyta rabiei isolates, collected from the 2009 eastern Australian chickpea growing regions, were assessed for their ability to infect 12 previously characterised chickpea genotypes. Comparison of means analysis and principal components analysis of area under the disease progress curve measures resulted in continua rather than discrete isolate groupings, suggesting that a broad diversity of pathogenicity exists in the current A. rabiei population. Breeders can now choose isolates that represent the variation found in the field to screen their germplasm, providing greater knowledge on the likely longevity of cultivars before they are commercially released.

Screening and identification of disease resistance to Botrytis grey mould (BGM, Botrytis cinerea) of chickpea is an important component to the development of integrated disease management strategies. The disease reaction to BGM with C. arietinum genotypes and C. bijugum accessions were assessed in growth chamber bioassays. The infection typically progressed with more severe necrosis and wilting from the shoot tip that could spread at least 2–4 internodes with the most susceptible reaction resulting in plant collapse and death. Fungal mycelium was commonly observed growing externally on shoot tips and sporulation occurred within 7–14 days. Initial screening to evaluate the disease progression from C. arietinum genotypes and C. bijugum accessions showed that disease symptoms were indicative of severity and increased with time. Moderate resistance was detected in four C. arietinum genotypes 99314–1003, FLIP97-529C, FLIP94-089C, FLIP94-508C and all six C. bijugum accessions. A detached leaf assay was effective in discriminating resistance in C. bijugum ILWC240 and susceptibility in FLIP97-90C and Lasseter genotypes, based on lesion number per leaflet. The detached leaf assay could be used for screening susceptible genotypes in less time than growth chamber bioassays. Resistance in C. bijugum indicated the potential use of wild Cicer species for novel sources of BGM resistance. Microscopic examination of the initial infection process with whole leaflet sections showed spore germination, appressoria formation as hyphal tip swelling and penetration directly through the cuticle, stomata and glandular stalks on the leaf surface. However, no remarkable differences between resistant and susceptible genotypes were shown within 72 h after inoculation (hai). This indicated that defence responses were likely to have been effective after 72 hai during the post-penetration or colonisation phase.

Ascochyta blight, caused by Ascochyta lentis Vassilievsky, is an important fungal disease of lentil (Lens culinaris subsp. culinaris). Manifestation of disease in plants is due to differential expression of genes in the both host and the pathogen. Identification of genes that are differentially expressed in varieties with resistance to A. lentis will lead to the accurate selection and development of crop varieties with increased resistance. To elucidate the complex network of genes underlying A. lentis resistance in lentil, a targeted genomics approach was utilised. The present study reports for the first time the use of microarray technology to study gene expression in lentil, specifically in response to A. lentis inoculation in a highly resistant (ILL7537) and highly susceptible (ILL6002) lentil variety. Ninety genes were differentially expressed in ILL7537 and 95 genes were differentially expressed in ILL6002. The expression profiles of the two varieties showed substantial difference in type and time of genes that were expressed in response to A. lentis. The resistant variety showed early upregulation of PR4 and 10 proteins and other defence-related genes. The susceptible genotype showed early downregulation of defence-related genes. Real-time RT-PCR was used to verify microarray expression ratios. The resistant and susceptible lentil varieties differ not only in the type of genes expressed but also in the time and level of expression in response to A. lentis inoculation. Different components of the defence mechanism and key putative defence genes were identified by comparing the transcriptional profiles of the susceptible and resistant lentil genotypes. Following further functional characterisation, these candidate ascochyta blight resistance genes may be used in future strategic A. lentis resistance breeding programs.

This paper reports on the strategy being used to study chickpea (Cicer arietinum) quantitative trait loci (QTL) conferring resistance to the necrotrophic fungal pathogen Ascochyta rabiei. The three chickpea linkage groups (LG), harbouring six QTL conditioning resistance against A. rabiei, were compared with the most comprehensive chickpea map (W-Ca-LG) and to the orthologous pseudochromosomes of the legume model plant Medicago truncatula. QTL1 was located in the subcentromere region of the chickpeaW-Ca-LG3 (chromosome C) and on the short arm of the M. truncatula pseudochromosome 7. QTL2 and QTL3 were located on the long arm of the W-Ca-LG8 (chromosome H) and on the short arm of the M. truncatula pseudochromosome 5. QTL4, QTL5 and QTL6 were located in the subcentromere region of the W-Ca-LG4 (chromosome B) and in a similar location of the M. truncatula pseudochromosome 1. To further delimit the genomic regions where the QTL were located, 25 legume expressed sequence tags (ESTs), which were shown to be upregulated in resistant chickpea accessions challenged with A. rabiei, were selected and BLASTed against M. truncatula genome sequences using a chromosome visualisation tool. Eleven of the 25 ESTs were mapped in silico on the nine M. truncatula pseudochromosomes with 1–4 copies. The association of mapped ESTs with chickpea resistance to A. rabiei requires further characterisation.

Colletotrichum truncatum is an important exotic pathogen threat to the Australian lentil industry. Canadian isolates of C. truncatum from lentil were assessed for morphological and molecular differences against Australian and international isolates of C. truncatum from hosts other than lentil and other Colletotrichum species. Although similar in dimension, conidiospores from lentil C. truncatum were ellipsoidal and those from C. truncatum originating from Australian Glycine max, Xanthium occidentale and Arachis hypogaea were falcate in shape. Conidiospores of Australian C. trifolii, C. gloeosporioides and C. destructivum were cylindrical. Molecular analysis, using both RAPD markers and 18–25S rDNA sequences, demonstrated the genetic relatedness of C. truncatum isolates from the same host species and discriminated among isolates from lentil and other host species. The Canadian isolates of C. truncatum from lentil formed a single distinct cluster, separate from that of the C. truncatum isolates originating from other legume host species and other Colletotrichum species from within and outside Australia. Conserved nucleotide sequence was used to develop a sensitive and highly specific diagnostic marker for the Canadian lentil isolates. The marker detected the pathogen in as little as 0.1 ng of DNA from infected plant tissue and represents a major step towards preventing or delaying the introduction and establishment of lentil anthracnose in Australia.

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

Ascochyta blight resistance in Pisum sativum (field pea), P. fulvum and Lathyrus species was examined in glasshouse experiments using an isolate of the fungal pathogen Mycosphaerella pinodes that had been isolated from field pea. In the genus Pisum there was significant variation in stem infection among the primitive field pea lines, the field pea cultivars and the P. fulvum lines. Two P. fulvum lines and one primitive field pea line exhibited significantly less stem infection than the two field pea cultivars. Leaf infection of the primitive field pea lines was not significantly different from that of the field pea cultivars. P. fulvum accession PS1115 had the least stem infection and the least leaf infection among the Pisum germplasm. Examination of stem infection in Lathyrus showed that L. sativus, L. ochrus and L. clymenum accessions were significantly more resistant to stem infection than the field pea cultivars. Six of the eight accessions of Lathyrus were also significantly more resistant to leaf infection than the field pea cultivars. Among ten accessions of L. sativus, there was significant variation in severity of stem infection but not leaf infection. This is the first report comparing ascochyta blight resistance between Lathyrus and Pisum species and among Lathyrus accessions and the results show that Lathyrus species may be a source of resistance alleles that could be exploited to develop ascochyta blight resistance in field pea.

Ascochyta blight, caused by Ascochyta rabiei, is a major constraint to chickpea production in Australia. A diagnostic test based on restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified DNA was developed to detect A. rabiei in infected leaves and seeds of chickpea. The test was developed by using primers designed to conserved sequences of the 18–25S ribosomal genes to amplify the internal transcribed spacer (ITS) regions of A. rabiei and other closely related Ascochyta species commonly found in pulses (A. lentis, A. pinodes and A. fabae) and identifying restriction enzymes that specifically cut the A. rabiei ITS region. The test was so sensitive that it detected DNA of A. rabiei at a quantity of 0.1 pg. The test also detected DNA extracted from infected leaf or seed in dilutions to 1×10−4 and 1 × 10−5, respectively, thus indicating that fungal genomic DNA can be specifically amplified by PCR from a plant genomic DNA background. This study also showed that the best method to detect chickpea seeds infected with A. rabiei was based on incubating surfacesterilised chickpea seeds in a liquid, fungal-growth medium (Czapek-Dox) for 18 h prior to the PCR and RFLP methods. This test was sensitive enough to detect a 1% infection.

A collection of 114 Cicer accessions including 95 wild Cicer accessions from four different species (C. bijugum K. H. Rech., C. echinospermum P. H. Davis, C. pinnatifidum Jaub. & Sp. and C. reticulatum Ladiz.) was screened in glasshouse trials for resistance to ascochyta blight (caused by Ascochyta rabiei (Pass.) Lab.). Resistance was identified in accessions from all four wild Cicer species. There was variation for resistance within accessions of C. echinospermum, C. pinnatifidum and C. reticulatum, with only C. bijugum containing accessions in which all plants were completely resistant. Resistant accessions from C. echinospermum and C. reticulatum may offer accessible sources of resistance because fully fertile hybrids with cultivated chickpea can be readily obtained.

Bacteria serologically related to Leifsonia xyli ssp. xyli, the causal bacterium of ratoon stunting disease (RSD) in sugarcane, were detected using the fluorescent antibody direct count on filter (FADCF) technique in grasses in eastern Australia. In a survey of 191 grass, sedge and bullrush samples comprising 53 plant species, 90 (47%) of the samples tested harboured bacteria which reacted positively with L. xyli ssp. xyli polyclonal antiserum. A total of 18 grass species was found to be naturally colonised with bacteria serologically related to and morphologically similar to L. xyli ssp. xyli. Grasses colonised by these L. xyli-like bacteria were present in areas both adjacent to, and removed from, sugarcane crops. When L. xyli-like bacteria from Rhodes grass (Chloris gayana) were inoculated into sugarcane, they multiplied at a lower rate than L. xyli ssp. xyli. L. xyli-like bacteria in Rhodes grass were isolated in axenic culture and exhibited growth rates, colony size and pigmentation similar to those of L. xyli ssp. cynodontis, a bacterial pathogen of Cynodon dactylon (couch grass). Further, using a polymerase chain reaction (PCR) test that could differentiate L. xyli spp. xyli from L. xyli spp. cynodontis, the L. xyli-like bacteria infecting Rhodes grass, couch grass and panic grass (Panicum maximum) generated a product of the same size as L. xyli spp. cynodontis. We conclude that L. xyli ssp. cynodontis or closely related bacteria are common endophytes of grasses in eastern Australia.

The genetic variation of Ascochyta lentis in Australia was investigated using random amplified polymorphic DNA (RAPD) analysis of 23 isolates. Of the 87 loci assessed, 86 were polymorphic. The most closely related isolates were from Victoria and South Australia, and they shared identical banding profiles. The most genetically distant isolates were from South Australiaand Western Australia (83% dissimilar). Analysis of molecular variance showed low genetic variation between groups of isolates originating from either within or outside Australia (4%). Large variation within each group (96%) indicated similar pathogen variability within and outside Australia. Partitioning of variation within Australian states was shown. Isolates collected within South Australia were least variable (1%) and isolates collected within Western Australia were most variable (61%). Increased variation in Western Australia is thought to have occurred through multiple introductions andlor the frequent presence of both mating types. Furthermore, two RAPD markers used in unison were able to explaino ver 50% of the variation in isolate pathogenicity. No RAPD markers were found that discriminated mating type.