Four Tulasnella taxa associated with populations of the Australian evergreen terrestrial orchid Cryptostylis ovata.

Of the more than 400 indigenous orchid species in Western Australia, Cryptostylis ovata is the only species that retains its leaves all year round. It exists as a terrestrial herb and occasionally as an epiphyte in forested areas. Like all terrestrial orchids, C. ovata plants associate with mycorrhizal fungi, but their identities have not previously been investigated. Fungi were isolated from pelotons in rhizomes collected from three southern and two northern populations of C. ovata on six occasions over two years. Phylogenetic analysis of ITS sequences temporally and spatially revealed that all the fungal isolates were of Tulasnella species of four distinct groups. One Tulasnella group was present only in the three southern orchid populations, and it closely resembled T. prima isolates previously described from Chiloglottis sp. orchids from eastern Australia. Isolates collected from plants in the two northern populations were of three undescribed Tulasnella groups. Analysis of intra-group diversity using inter-simple sequence repeat markers revealed that plants were usually colonised by a single genotype of Tulasnella at each sampling period, and this genotype usually, but not always, persisted with the host plant over both years tested.

Infection processes and involvement of defense-related genes in the expression of resistance in cultivars of subterranean clover (Trifolium subterraneum) to Phytophthora clandestina.

Studies on infection processes and gene expression were done to determine differential responses of cultivars of Trifolium subterraneum resistant and susceptible to infection by races of Phytophthora clandestina. In the infection process study, one race was inoculated onto the roots of T. subterraneum cvs. Woogenellup and Junee (compatible or incompatible interactions, respectively). There were no differences in relation to the processes of cyst attachment, germination, and hyphal penetration. There were, however, major differences in infection progression observed post-penetration between compatible and incompatible interactions. In susceptible cv. Woogenellup, hyphae grew into the vascular bundles and produced intercellular antheridia and oogonia in the cortex and stele by 4 days postinoculation (dpi), oospores in the cortex and stele by 8 dpi, when sporangia were evident on the surface of the root. Infected taproots were discolored. Early destruction of taproots prevented emergence of lateral roots. Roots of resistant cv. Junee showed no oospores or sporangia and no disease at 8 dpi. In the gene expression studies, two races of P. clandestina were inoculated onto three cultivars of T. subterraneum. Results showed that three genes known to be associated with plant defense against plant pathogens were differentially expressed in the roots during compatible and incompatible interactions. Phenylalanine ammonia lyase and chalcone synthase genes were activated 4 h postinoculation (hpi) and cytochrome P450 trans-cinnamic acid 4-monooxygenase gene was activated 8 hpi in the incompatible interactions in cvs. Denmark and Junee following inoculation with Race 177. In contrast, in compatible interactions in cv. Woogenellup, there were no significant changes in the activation of these three genes following inoculation, indicating that these three genes were associated with the expression of resistance to Race 177 of the pathogen by the host. To confirm this result, in the second test, cv. Woogenellup was challenged by Race 000 of P. clandestina. In this incompatible interaction, cv. Woogenellup was resistant and expressed highly all three genes in the manner similar to the incompatible interactions observed in the first test.

Secondary metabolites produced by a root-inhabiting sterile fungus antagonistic towards pathogenic fungi.

AIMS: A sterile red fungus (SRF) isolated from cortices of roots of both wheat (Triticum aestivum cv. Gamenya) and ryegrass (Lolium rigidum cv. Wimmera) was found to protect the hosts from phytopathogens and promote plant growth. In this work, the major secondary metabolites produced by this SRF were analysed, and their antibiotic and plant-growth-promoting activities investigated. METHODS AND RESULTS: Two main compounds, veratryl alcohol (VA) and 4-(hydroxymethyl)-quinoline, were isolated from the culture filtrate of the fungus. In antifungal assays, VA inhibited the growth of Sclerotinia sclerotiorum and Pythium irregulare even at low amounts, while high concentrations (>100 microg per plug) of 4-(hydroxymethyl)-quinoline were needed. Both metabolites revealed weak inhibition of Rhizoctonia solani. Furthermore, both compounds showed a growth promotion activity on canola (Brassica napus) seedlings used as bioassays. CONCLUSIONS: Isolation and characterization of the main secondary metabolites from culture filtrates of a root-inhabiting sterile fungus are reported. The biological assays indicate that these secondary metabolites may have a role in both plant growth regulation and antifungal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a better understanding of the metabolism of a cortical fungus that may have a useful role in the biological suppression of root-infecting soil-borne plant pathogens.

Molecular Genetic Characterization of Olpidium virulentus Isolates Associated with Big-Vein Diseased Lettuce Plants.

Lettuce plants showing symptoms of lettuce big-vein disease were collected from fields in the Perth Metropolitan region of southwest Australia. When root extracts from each plant were tested by polymerase chain reaction (PCR) using primers specific to the rDNA internal transcribed spacer (ITS) region of Olpidium brassicae and O. virulentus, only O. virulentus was detected in each of them. The nucleotide sequences of the complete rDNA ITS regions of isolates from five of the root samples and 10 isolates of O. virulentus from Europe and Japan showed 97.9 to 100% identities. However, with the six O. brassicae isolates, their identities were only 76.9 to 79.4%. On phylogenetic analysis of the complete rDNA-ITS region sequences of the five Australian isolates and 10 others, the Australian isolates fitted within two clades of O. virulentus (I and II), and within clade I into two of its four subclades (Ia and Id). Japanese isolates had greatest sequence diversity fitting into both clades and into all of clade I subclades except Ib, while European isolates were restricted to subclades Ib and Id. When the partial rDNA-ITS region sequences of two additional southwest Australian isolates, four from Europe, and four from the Americas were included in the analyses, the Australian isolates were within O. virulentus subclades Ia and Id, the European isolates within subclade Ic, and the American isolates within subclades Ia and Ib. These findings suggest that there may have been at least three separate introductions of O. virulentus into the isolated Australian continent since plant cultivation was introduced following its colonization by Europeans. They also have implications regarding numbers of different introductions to other isolated regions. Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus were both detected when symptomatic lettuce leaf tissue samples corresponding to the root samples from southwest Australia were tested using virus-specific primers in reverse transcription-PCR, so presence of both viruses was associated with O. virulentus occurrence.

Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens.

AIMS: Strains of Trichoderma spp. produce numerous bioactive secondary metabolites. The in vitro production and antibiotic activities of the major compounds synthesized by Trichoderma harzianum strains T22 and T39 against Leptosphaeria maculans, Phytophthora cinnamomi and Botrytis cinerea were evaluated. Moreover, the eliciting effect of viable or nonviable biomasses of Rhizoctonia solani, Pythium ultimum or B. cinerea on the in vitro production of these metabolites was also investigated. METHODS AND RESULTS: T22azaphilone, 1-hydroxy-3-methyl-anthraquinone, 1,8-dihydroxy-3-methyl-anthraquinone, T39butenolide, harzianolide, harzianopyridone were purified, characterized and used as standards. In antifungal assays, T22azaphilone and harzianopyridone inhibited the growth of the pathogens tested even at low doses (1-10 microg per plug), while high concentrations of T39butenolide and harzianolide were needed (>100 microg per plug) for inhibition. The in vitro accumulation of these metabolites was quantified by LC/MS. T22azaphilone production was not enhanced by the presence of the tested pathogens, despite its antibiotic activity. On the other hand, the anthraquinones, which showed no pathogen inhibition, were stimulated by the presence of P. ultimum. The production of T39butenolide was significantly enhanced by co-cultivation with R. solani or B. cinerea. Similarly, viable and nonviable biomasses of R. solani or B. cinerea increased the accumulation of harzianopyridone. Finally, harzianolide was not detected in any of the interactions examined. CONCLUSIONS: The secondary metabolites analysed in this study showed different levels of antibiotic activity. Their production in vitro varied in relation to: (i) the specific compound; (ii) the phytopathogen used for the elicitation; (iii) the viability of the elicitor; and (iv) the balance between elicited biosynthesis and biotransformation rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of cultures of phytopathogens to enhance yields of Trichoderma metabolites could improve the production and application of novel biopesticides and biofertilizers based on the active compounds instead of the living microbe. This could have a significant beneficial impact on the management of diseases in crop plants.

Plant growth promotion and biological control of Pythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes.

AIMS: To evaluate the potential of Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis endophytic in cucumber roots, to promote plant growth and to protect seedlings and mature plants of cucumber from diseases caused by Pythium aphanidermatum, under greenhouse conditions. METHODS AND RESULTS: Three endophytic isolates, out of 29, were selected through tests aimed at understanding their mechanisms of action as biocontrol agents and plant growth promoters. When applied individually or in combination, they significantly promoted plant growth and reduced damping-off and crown and root rot of cucumber. The combination of the three isolates resulted in significantly better suppression of diseases and plant growth promotion, than where the plants were exposed to individual strains. CONCLUSIONS: The three selected actinomycete isolates colonized cucumber roots endophytically for 8 weeks, promoted plant growth and suppressed pathogenic activities of P. aphanidermatum on seedling and mature cucumber plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The results clearly show that the endophytic, glucanase-producing actinomycetes used, especially as a combined treatment, could replace metalaxyl, which is the currently recommended fungicide for Pythium diseases in the United Arab Emirates. These endophytic isolates also have the potential to perform as plant growth promoters, which is a useful attribute for crop production in nutrient impoverished soils.

Occurrence of a new subclade of Leptosphaeria biglobosa in Western Australia.

Stem canker of crucifers is caused by an ascomycete species complex comprising of two main species, Leptosphaeria maculans and L. biglobosa. These are composed of at least seven distinct subclades based on biochemical data or on sequences of internal transcribed spacer (ITS), the mating type MAT1-2 or fragments of actin or beta-tubulin genes. In the course of a wide-scale characterization of the race structure of L. maculans from Western Australia, a few isolates from two locations failed to amplify specific sequences of L. maculans, i.e., the mating-type or minisatellite alleles. Based on both pathogenicity tests and ITS size, these isolates were classified as belonging to the L. biglobosa species. Parsimony and distance analyses performed on ITS, actin and beta-tubulin sequences revealed that these isolates formed a new L. biglobosa subclade, more related to the Canadian L. biglobosa 'canadensis' subclade than to the L. biglobosa 'australensis' isolates previously described in Australia (Victoria). They are termed here as L. biglobosa 'occiaustralensis'. These isolates were mainly recovered from resistant oilseed rape cultivars that included the Brassica rapa sp. sylvestris-derived resistance source, but not from the susceptible cv. Westar. The pathogenicity of L. biglobosa 'occiaustralensis' to cotyledons of most oilseed rape genotypes was higher than that of L. biglobosa 'canadensis' or L. biglobosa 'australensis' isolates.

Stabilization of Resistance to Leptosphaeria maculans in Brassica napus-B. juncea Recombinant Lines and Its Introgression into Spring-Type Brassica napus.

The value of Katanning Early Maturing (KEM) breeding lines from Western Australia, derived from Brassica napus × B. juncea crosses, was assessed as a source of germplasm for resistance to blackleg disease (caused by Leptosphaeria maculans) in spring-type oilseed rape cultivars. The stability of blackleg resistance in these KEM lines was related to key cytological characteristics to determine why there are poor levels of introgression of this resistance into progeny. Promising recombinant KEM lines were crossed with the spring-type B. napus cv. Dunkeld, which has useful polygenic resistance to blackleg, and screened for resistance. The lines were analyzed cytologically for pairing of bivalents in each generation to aid in the selection of stable recombinant lines. KEM recombinant lines showing regular meiotic behavior and a high level of blackleg resistance were obtained for the first time. We also showed that the stable introgression of the B. juncea resistance from the KEM lines into a 'Dunkeld' background was possible. Inoculation of selfing and backcross populations with isolates of L. maculans having different AvrLm genes indicated that the B. juncea resistance gene, Rlm6, had been introgressed into a B. napus spring-type cultivar carrying polygenic resistance. The combination of both resistances would enhance the overall effectiveness of resistance against L. maculans. This is clearly needed in Australia and France where cultivars relying upon single dominant gene-based resistance for their effectiveness have proved not durable.

First Report of Powdery Mildew Caused by Erysiphe cruciferarum on Brassica juncea in Australia.

In Australia, Brassica juncea (L.) Czern & Coss (Indian mustard) has the potential as a more drought-tolerant oilseed crop than the B. napus L., with the first canola-quality B. juncea varieties released in Australia in 2006 and first sown for commercial production in 2007. Increased production of B. juncea is expected to result in the appearance of diseases previously unreported in Australia. In the spring of 2007 at the University of Western Australia field plots at Crawley (31.99°S, 115.82°E), Western Australia, plants of B. juncea genotypes from Australia and China had extensive stem colonization by powdery mildew at the end of the flowering period, with whitish patches ranging in size from 3 mm to 3 cm long. These patches coalesced to form a dense, white, powdery layer as they expanded. Pathogenicity was demonstrated by gently pressing infected stems containing abundant sporulation onto leaves of potted B. juncea seedlings of variety JM-18, incubating the plants in a moist chamber for 48 h, and then maintaining the plants in a controlled-environment room at 18/13°C for day/night. Signs of powdery mildew appeared at 7 days after inoculation, and by 10 days, it was well developed. Uninoculated control plants did not have powdery mildew. When symptomatic plants were examined, abundant conidia were typical of Erysiphe cruciferarum Opiz ex Junell, with cylindrical conidia borne singly or in short chains as described previously (2). Mycelia were amphigenous, in patches, and often spreading to become effused. Conidiophores were straight, foot cells were cylindrical, and conidia were mostly produced singly and measured 21.2 to 35.4 (mean 26.7 µm) × 8.8 to 15.9 µm (mean 11.9 µm) from measurements of 100 conidia. The spore size that we measured approximated what was found for E. cruciferarum (2) (30 to 40 × 12 to 16 µm), since we found 35 and 50% of spores falling within this range in terms of length and width, respectively. Conidia were, however, generally smaller in size than that reported on broccoli raab in California (1) (35 to 50 × 12 to 21 µm). We confirmed a length-to-width ratio greater than 2 as was found previously (1,2). Infected leaves showed signs of early senescence. While powdery mildew caused by E. cruciferarum is an important disease of B. juncea in India where yield losses as much as 17% have been reported (4), its potential impact in Australia is yet to be determined. To our knowledge, this is the first record of E. cruciferarum on B. juncea in Australia. In Western Australia, E. cruciferarum has been recorded on B. napus (oilseed rape) since 1986 and on B. napus L. var. napobrassica (L.) Reichenb. (swede) since 1971 (3). In other regions of Australia, it has been recorded on B. rapa in Queensland since 1913 and on B. napus (oilseed rape) in South Australia since 1973. References: (1) S. T. Koike and G. S. Saenz. Plant Dis. 81:1093, 1997. (2) T. J. Purnell and A. Sivanesan. No 251 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1970. (3) R. G. Shivas. J. R. Soc. West. Aust. 72:1, 1989. (4) A. K. Shukla et al. Manual on Management of Rapeseed-Mustard Diseases. National Research Centre on Rapeseed-Mustard, Bharatpur, India, 2003.

Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions.

The diversity of mycorrhizal fungi associated with an introduced weed-like South African orchid (Disa bracteata) and a disturbance-intolerant, widespread, native West Australian orchid (Pyrorchis nigricans) were compared by molecular identification of the fungi isolated from single pelotons. Molecular identification revealed both orchids were associated with fungi from diverse groups in the Rhizoctonia complex with worldwide distribution. Symbiotic germination assays confirmed the majority of fungi isolated from pelotons were mycorrhizal and a factorial experiment uncovered complex webs of compatibility between six terrestrial orchids and 12 fungi from Australia and South Africa. Two weed-like (disturbance-tolerant rapidly spreading) orchids - D. bracteata and the indigenous Australian Microtis media, had the broadest webs of mycorrhizal fungi. In contrast, other native orchids had relatively small webs of fungi (Diuris magnifica and Thelymitra crinita), or germinated exclusively with their own fungus (Caladenia falcata and Pterostylis sanguinea). Orchids, such as D. bracteata and M. media, which form relationships with diverse webs of fungi, had apparent specificity that decreased with time, as some fungi had brief encounters with orchids that supported protocorm formation but not subsequent seedling growth. The interactions between orchid mycorrhizal fungi and their hosts are discussed.

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens.

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.

Annual Medicago: from a model crop challenged by a spectrum of necrotrophic pathogens to a model plant to explore the nature of disease resistance.

BACKGROUND: Annual Medicago spp., including M. truncatula, play an important agronomic role in dryland farming regions of the world where they are often an integral component of cropping systems, particularly in regions with a Mediterranean or Mediterranean-type climate where they grow as winter annuals that provide both nitrogen and disease breaks for rotational crops. Necrotrophic foliar and soil-borne pathogens dominate these regions and challenge the productivity of annual Medicago and crop legume species. SCOPE: This review outlines some of the major and/or widespread diseases these necrotrophic pathogens cause on Medicago spp. It then explores the potential for using the spectrum of necrotrophic pathogen-host interactions, with annual Medicago as the host plant, to better understand and model pathosystems within the diseases caused by nectrotrophic pathogens across forage and grain legume crops. CONCLUSIONS: Host resistance clearly offers the best strategy for cost-effective, long-term control of necrotrophic foliar and soil-borne pathogens, particularly as useful resistance to a number of these diseases has been identified. Recently and initially, the annual M. truncatula has emerged as a more appropriate and agronomically relevant substitute to Arabidopsis thaliana as a model plant for legumes, and is proving an excellent model to understand the mechanisms of resistance both to individual pathogens and more generally to most forage and grain legume necrotrophic pathogens.

Analysis of Leptosphaeria maculans Race Structure in a Worldwide Collection of Isolates.

ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape, develops gene-for-gene interactions with its hosts. To date, eight L. maculans avirulence (Avr) genes, AvrLm1 to AvrLm8, have been genetically characterized. An additional Avr gene, AvrLm9, that interacts with the resistance gene Rlm9, was genetically characterized here following in vitro crosses of the pathogen. A worldwide collection of 63 isolates, including the International Blackleg of Crucifers Network collection, was genotyped at these nine Avr loci. In a first step, isolates were classified into pathogenicity groups (PGs) using two published differential sets. This analysis revealed geographical disparities as regards the proportion of each PG. Genotyping of isolates at all Avr loci confirmed the disparities between continents, in terms of Avr allele frequencies, particularly for AvrLm2, AvrLm3, AvrLm7, AvrLm8, and AvrLm9, or in terms of race structure, diversity, and complexity. Twenty-six distinct races were identified in the collection. A larger number of races (n = 18) was found in Australia than in Europe (n = 8). Mean number of virulence alleles per isolate was also higher in Australia (5.11 virulence alleles) than in Europe (4.33) and Canada (3.46). Due to the diversity of populations of L. maculans evidenced here at the race level, a new, open terminology is proposed for L. maculans race designation, indicating all Avr loci for which the isolate is avirulent.

Absence of Oospores of Downy Mildew of Grape Caused by Plasmopara viticola as the Source of Primary Inoculum in Most Western Australian Vineyards.

Grapevine downy mildew, caused by the obligate, oomycete pathogen, Plasmopara viticola, was first recorded in Western Australia (W.A.) in 1998 (2) and has subsequently been observed in most viticultural regions of the state. Heterothallism in P. viticola was established by Wong et al. (3), whereby more than one mating type of the pathogen is required for sexual reproduction to occur. Oospores are considered to be the source of primary inoculum for this disease with further, secondary infection being advanced by asexual inoculum. However, recent research in European vineyards suggests that the majority of infection throughout the growing season arises via sexually derived (oosporic) inoculum (1). Since downy mildew is relatively new to W.A., few surveys have been conducted to study populations of the pathogen within the state. It is also noteworthy that the incidence of oospores in Australian vineyards has not been reported. The objective of this research was to assess the occurrence and type of inoculum of P. viticola in W.A. vineyards. A total of 1,266 P. viticola-infected leaf discs (LD) from eight wine-grape (775 LD), five table-grape (450 LD), and seven unknown (41 LD) cultivars grown in 16 vineyards in 10 geographically separate regions of W.A. were collected in the growing seasons of 2001-2003. These regions range from Chittering in the north to Albany in the south and received 700 to 1,200 mm annual rainfall, mostly in winter. Each LD was cleared in 1 M KOH at 60°C for 12 to 24 h and then was assessed for the presence of oospores with light microscopy. Leaves showing "mosaic"-type lesions (older infection) late in the season were collected where possible to ensure colony maturity and an increased likelihood of oospore formation. All LD from all regions were negative for the presence of oospores except for samples from a single vineyard (approximately 1,200 mm annual rainfall), where all 140 LD from six wine-grape cultivars contained oospores. The discovery that oospores were present in only one of 16 sampled vineyards provides a rare opportunity to study gene flow in field populations of the pathogen with time and to determine sources of primary inoculum where overwintering of P. viticola may not involve oospores. References: (1) S. McKirdy et al. Plant Dis. 83:301, 1999. (2) A. Rumbou et al. Eur. J. Plant Pathol. 110:379, 2004. (3) F. P. Wong et al. Plant Pathol. 50:427, 2001.

First Report of an Alternaria Leaf Spot Caused by Alternaria brassicae on Crambe abyssinicia in Australia.

Crambe abyssinicia Hochst. is grown sporadically worldwide for its value as a source of high erucic acid industrial oils and secondary commercial products. While there is increasing interest in cropping C. abyssinicia in Australia, for these potentials and also as a source of oil for biodiesel production, currently, there have been no commercial crops of this species. In September 2004, inspection of a small experimental field crop in Beverley, Western Australia indicated the presence of significant leaf spotting just prior to commencement of flowering. The symptoms of this disease included as many as 10 to 15 spot lesions per leaf that were generally rounded and varied between 0.5 to 11 mm in diameter. Clusters of these lesions were often associated with chlorosis of the region of leaves where they occurred. More than 95% of plants inspected showed these symptoms. When affected leaves were incubated in moist chambers, typical conidia of Alternaria brassicae (Berk.) Sacc. were observed. The description of these conidia matched that of the Commonwealth Mycological Institute for this pathogen (1) showing obclavate conidia 105 to 210 µm long and 20 to 30 µm thick, with 11 to 15 transverse septa and 0 to 3 longitudinal or oblique septa, predominantly with a pronounced beak 5 to 8 µm thick extending 0.3 to 0.5 µm of the length of the conidium. Single-spore isolations were made onto potato dextrose agar. Subcultures of these isolates were identified using a polymerase chain reaction (PCR)- based assay (2). This assay involved the use of two sets of A. brassicae-specific primers selected for conventional and real-time PCR. The colonies were confirmed to belong to A. brassicae. In a pathogenicity test to confirm Koch's postulates, single-spore isolates were inoculated onto cotyledons and leaves of 10-day-old C. abyssinicia seedlings. Symptoms on inoculated plants appeared within a period of 14 days of inoculation, matching those found on the affected plants in the field, and A brassicae was reisolated. A. brassicae causes an important worldwide disease of crucifers, for example, it can be a devastating disease of rapeseed and the other cruciferous crops in the United States and Canada. Since A. brassicae has already been reported on other species of crucifers Australia-wide, it may pose a threat to any potential Crambe spp. industry in this country. References: (1) M. B. Ellis No. 162 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, England, 1966. (2) T. Guillemette et al. Plant Dis. 88:490, 2004.

Production of leucinostatins and nematicidal activity of Australian isolates of Paecilomyces lilacinus (Thom) Samson.

AIMS: To evaluate the relationship between leucinostatin production by Paecilomyces lilacinus isolates and their biological activities. METHODS AND RESULTS: The nematicidal, parasitic and enzymatic activity of Australian P. lilacinus isolates were investigated. Nematicidal activities of culture filtrates were measured by mortality and inhibition of reproduction of Caenorhabditis elegans, whereas egg-parasitic activity was measured by colonization on Meloidogyne javanica. Enzymatic activities (protease and chitinase) were assayed on solid media. The results suggest that leucinostatins in P. lilacinus are indicators of nematicidal activity, whereas chitinase activity might be related to parasitism. CONCLUSIONS: Nematicidal activity of culture filtrates of Paecilomyces lilacinus strains related to their ability to produce leucinostatins. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing the leucinostatins as nematicides.

Endodontic sequelae of dental erosion.

BACKGROUND: The incidence of pulp involvement in patients with excessive wear has not been extensively documented. METHODS: Clinical records of 448 patients with excessive tooth wear were reviewed and 52 cases (11.6 per cent) with near or frank pulp exposures or root canal treatments were found and their numbers and sites were tabulated. Light microscopy of study models was used to determine aetiology at each site of exposure as attrition, erosion or abrasion, scanning electron microscopy (SEM) was performed on some individual teeth. RESULTS: Forty sites of near exposure and 57 sites of frank exposures or root canal treatments were found, some cases had both types of exposure. The commonest sites exposed by erosion were the palatal surfaces of maxillary, and the incisal surfaces of mandibular anterior teeth. Posterior teeth were not commonly affected. Toothbrush abrasion had exacerbated some lesions as shown by SEM. CONCLUSIONS: Endodontic sequelae were found in 11 per cent of tooth wear patients as late stages of dental erosion. Near and frank exposures of the pulp thus constitute a small but significant, problem for the Australian dental profession's concern in the management of the tooth wear cases.

Breakdown of a Brassica rapa subsp. sylvestris Single Dominant Blackleg Resistance Gene in B. napus Rapeseed by Leptosphaeria maculans Field Isolates in Australia.

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus) grown in Canada, Europe, and Australia. Cv. Surpass 400 was released in Australia in 2000 as the most resistant cultivar to L. maculans. It carries a single dominant resistance gene from B. rapa subsp. sylvestris. This cultivar usually shows a hypersensitive response to L. maculans characterized by small, dark brown lesions that are necrotic, localized, and without pycnidia on cotyledons, leaves, and stems. However, in 2001 on a Western Australian experimental farm, a small proportion of the lesions on the lower stem and crown region of cv. Surpass 400 were typical of those observed in susceptible cultivars, which were brown, necrotic lesions with a darker margin, but they contained fewer pycnidia. Forty seedlings of cv. Surpass 400 and susceptible cv. Westar were inoculated with pycnidiospore suspensions (106/ml) of each of 18 isolates taken from lesions on cv. Surpass 400. All 18 isolates caused collapse of cotyledons of susceptible cv. Westar. Four of these isolates caused large cotyledon lesions with some pycnidia on cv. Surpass 400. Three of these four isolates were subsequently inoculated onto 60 seedlings per isolate, at each of the four cotyledon lobes of each seedling of the two cultivars. Inoculated plants were assessed for disease severity on cotyledons and transplanted to the field 14 days after inoculation. The cotyledons of inoculated cv. Surpass 400 showed characteristic large, necrotic lesions with pycnidia, while the cotyledons of cv. Westar had collapsed and contained a mass of pycnidia. Blackleg disease severity in the crown region of the stem was assessed at 2 weeks before harvest. Fifty-four percent of the cv. Surpass 400 transplanted inoculated plants subsequently developed susceptible symptoms of crown cankers on stems. These symptoms were deep, girdling, brown lesions on the plant crowns with some pycnidia. One hundred percent of cv. Westar plants were infected and dead at this stage. This confirmed the ability of these field isolates to overcome the single dominant resistance gene present in cv. Surpass 400. To our knowledge, this is the first report of breakdown of a single dominant B. rapa subsp. sylvestris gene based resistance to blackleg in oilseed rape in the field.

Pathogenesis of Streptoverticillium albireticuli on Caenorhabditis elegans and its antagonism to soil-borne fungal pathogens.

AIMS: To examine the biological activity of Streptoverticillium albireticuli. METHODS: Isolation of S. albireticuli was carried out using the dry-heat technique. Nematicidal and pathogenic activity on Caenorhabditis elegans was measured by mortality in metabolites and colonization rate on fishmeal extract agar. Antifungal and enzymatic activities of S. albireticuli were measured by the agar plate method and the semidefined solid media method, respectively. RESULTS: S. albireticuli showed strong nematicidal activity against C. elegans. Pathogenic activity was also evident with the colonized nematode by the isolate on fishmeal extract agar. It also showed antifungal activity against certain fungal pathogens such as Rhizoctonia solani, Phytophthora cinnamomi and Fusarium oxysporum. SIGNIFICANCE AND IMPACT OF THE STUDY: The discovery of an actinomycete showing pathogenic activity against the nematode may indicate the potential for it to be used as a biocontrol agent of parasitic nematodes, in addition to its ability to suppress fungal pathogens.

Dental erosion in asthma: a case-control study from south east Queensland.

BACKGROUND: Asthma medication places patients at risk of dental erosion by reducing salivary protection against extrinsic or intrinsic acids. But patterns of lesions in asthmatics may differ from patterns in non-asthmatics, because gastro-oesophageal reflux (GOR) is found in 60 per cent of asthmatics. METHODS: The lesions in 44 asthma cases were compared to those of age and sex match controls with no history of asthma or medications drawn from the dental records of 423 patients referred concerning excessive tooth wear. The subjects were 70 males age range 15 to 55 years and 18 females age range 18 to 45. Anamnestic clinical data were compared between the two groups. Models of all 88 subjects were examined by light microscopy, and wear patterns were recorded on permanent central incisor, canine, premolar and first molar teeth. RESULTS: Clinical differences were a higher incidence of tooth hypersensitivity, xerostomia, salivary gland abnormalities, gastric complaints, and self induced vomiting in the cases. No differences were found between the cases and controls on citrus fruit and acid soft drink consumption. More occlusal erosion sites were found in cases, whereas more attrition sites were found in the controls. There were no significant differences in palatal erosion on maxillary anterior teeth found between cases and controls. Lingual erosion of the mandibular incisors, found only in GOR patients, was not observed. CONCLUSIONS: A higher incidence of erosion was found in asthmatics. Gastro-oesophageal reflux symptoms were not associated with the sign of lingual mandibular incisor erosion. The clinical significance is that asthmatics are at risk of dental erosion from extrinsic acid, but GOR does not appear to contribute in a site-specific manner.