Temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal communities with ontogenetic and phenological development in Prasophyllum (Orchidaceae).

BACKGROUND AND AIMS: Plant-fungus symbioses may experience temporal turnover during the host's ontogenetic or phenological development, which can influence the host plant's ecological requirements. This study investigates temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal (OMF) communities in Prasophyllum (Orchidaceae), asking if OMF communities are subject to temporal change due to orchid phenology or ontogeny. METHODS: Roots of adult Prasophyllum frenchii, P. lindleyanum and P. sp. aff. validum from Australia were sampled between autumn and spring. Seed was sown in situ as 'baits' to explore the mycorrhizal associations of germinating protocorms, which were compared to OMF in roots of co-occurring adult plants. Culture dependent and independent sequencing methods were used to amplify the internal transcribed spacer and mitochondrial large subunit loci, with sequences assigned to Operational Taxonomic Units (OTUs) in phylogenetic analyses. Germination trials were used to determine if fungal OTUs were mycorrhizal. KEY RESULTS: A persistent core of OMF associated with Prasophyllum, with Ceratobasidiaceae OMF dominant in all three species. Phenological turnover occurred in P. lindleyanum and P. sp. aff. validum, but not in P. frenchii, which displayed specificity to a single OTU. Ontogenetic turnover occurred in all species. However, phenological and ontogenetic turnover was typically driven by the presence or absence of infrequently detected OTUs in populations that otherwise displayed specificity to one or two dominant OTUs. Ex situ germination trials showed 13 of 14 tested OTUs supported seed germination in their host orchid, including eight OTUs that were not found in protocorms in situ. CONCLUSIONS: An understanding of OMF turnover can have practical importance for the conservation of threatened orchids and their mycorrhizal partners. However, frameworks for classifying OMF turnover should focus on OTUs important to the life cycle of the host plant, which we suggest are likely to be those that are frequently detected or functionally significant.

Culture age of Tulasnella affects symbiotic germination of the critically endangered Wyong sun orchid Thelymitra adorata (Orchidaceae).

Orchids (Orchidaceae) are dependent on mycorrhizal fungi for germination and to a varying extent as adult plants. We isolated fungi from wild plants of the critically endangered terrestrial orchid Thelymitra adorata and identified them using a multi-region barcoding approach as two undescribed Tulasnella species, one in each of phylogenetic group II and III (OTU1) of the Tulasnellaceae. Using symbiotic propagation methods, we investigated the role of Tulasnella identity (species and isolate) and age post isolation, on the fungus's ability and efficacy in germinating T. adorata. The group II isolate did not support germination. Seed germination experiments were conducted using either (i) three different isolates of OTU1, (ii) 4- and 12-week-old fungal cultures (post isolation) of a single isolate of OTU1, and (iii) T. subasymmetrica which is widespread and known to associate with other species of Thelymitra. Culture age and fungal species significantly (P < 0.05) affected the time to germination and percentage of seed germination, with greater and faster germination with 4-week-old cultures. Tulasnella subasymmetrica was able to germinate T. adorata to leaf stage, although at slightly lower germination percentages than OTU1. The ability of T. adorata to germinate with T. subasymmetrica may allow for translocation sites to be considered outside of its native range. Our findings on the age of Tulasnella culture affecting germination may have applications for improving the symbiotic germination success of other orchids. Furthermore, storage of Tulasnella may need to take account of the culture age post-isolation, with storage at - 80 °C as soon as possible recommended, post isolation.

Predominant Clonal Reproduction with Infrequent Genetic Recombination of Phaeoacremonium minimum in Western Cape Vineyards.

Phaeoacremonium minimum is an important esca and Petri disease pathogen that causes dieback of grapevines in South Africa. Little is known regarding the reproductive strategy of the pathogen. Sexual reproduction could lead to a better adaptation of the pathogen to disease management strategies by combining alleles through recombination. The study aimed to investigate the genetic diversity and recombination potential of eight populations in the Western Cape, from six commercial vineyards and two nursery rootstock mother blocks. This was achieved by developing and applying nine polymorphic microsatellites and mating-type-specific markers. Thirty-seven genotypes were identified from 295 isolates. Populations were characterised by the same dominant genotype (MLG20 occurring 65.43%), low genotypic diversity (H) and high numbers of clones (81.36% of dataset). However, genotypes from the same sampling sites were not closely related based on a minimum spanning network and had high molecular variation within populations (94%), suggesting that multiple introductions of different genotypes occurred over time. Significant linkage disequilibrium among loci (r̅d) further indicated a dominant asexual cycle, even though perithecia have been observed in these four populations. The two rootstock mother blocks had unique genotypes and genotypes shared with the vineyard populations. Propagation material obtained from infected rootstock mother blocks could lead to the spread of more genotypes to newly established vineyards. Based on our results, it is important to determine the health status of rootstock mother blocks. Management strategies must focus on reducing aerial inoculum to prevent repeated infections and further spread of P. minimum genotypes.

Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids.

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host-switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well-resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1-6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event-based method also revealed significant congruence in Drakaeinae-Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host-switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae-Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages.

Cryptostylis species (Orchidaceae) from a broad geographic and habitat range associate with a phylogenetically narrow lineage of Tulasnellaceae fungi.

While many Australian terrestrial orchids have highly specialized mycorrhizal associations, we tested the hypothesis that the geographically widespread orchid genus Cryptostylis associates with a diversity of fungal species. Using fungal isolation and molecular approaches, we investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites). Like related orchid genera, Tulasnellaceae formed the main fungal associations of the Cryptostylis species we sampled, although some ectomycorrhizal, ericoid and saprotrophic fungi were detected infrequently. Each species of Australian Cryptostylis associated with three to seven Tulasnella Operational Taxonomic Units (OTUs), except for C. hunteriana where only one Tulasnella OTU was detected. In total, eleven Tulasnella OTUs associated with Australian Cryptostylis. The Asiatic Cryptostylis associated with four different Tulasnella OTUs belonging to the same lineage as the Australian species. While five Tulasnella OTUs (T. australiensis, T. prima, T. warcupii, T. densa, and T. punctata) were used by multiple species of Australian Cryptostylis, the most commonly used OTU differed between orchid species. The association with different Tulasnella fungi by Cryptostylis species co-occurring at the same site suggests that in any given environmental condition, Cryptostylis species may intrinsically favour different fungal OTUs.

New species of Tulasnella associated with Australian terrestrial orchids in the subtribes Megastylidinae and Thelymitrinae.

Tulasnella (Tulasnellaceae) is a genus of fungus that can form mycorrhizal associations with orchids (Orchidaceae). Here we used molecular phylogenetic analyses and morphological characteristics of pure cultures across four different media to support the description of five new Tulasnella species associated with commonly occurring and endangered Australian orchids. Tulasnella nerrigaensis associates with Calochilus; T. subasymmetrica and T. kiataensis with Thelymitra; and T. korungensis and T. multinucleata with Pyrorchis and Rimacola respectively. The newly described species were primarily delimited by analyses of five loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), C14436 (adenosine triphosphate [ATP] synthase), C4102 (glutamate synthase), C3304 (ATP helicase), and mt large subunit 16S rDNA (mtLSU). Tulasnella subasymmetrica is introduced for some isolates previously identified as T. asymmetrica, and this latter species is characterized from multilocus sequencing of a new isolate that matches ITS sequences from the ex-type culture. Morphological differences between the new species are slight. Tulasnella multinucleata has 6-12 nuclei per hyphal compartment which is the first instance of multinucleate rather than binucleate or trinucleate hyphal compartments in Tulasnella. The formal description of these species of Tulasnella will aid in future evolutionary and ecological studies of orchid-fungal interactions.

Seven new Serendipita species associated with Australian terrestrial orchids.

Serendipita is one of the main fungal genera that form mutualistic associations with species of orchids (Orchidaceae). Here, seven new Serendipita species associated with various Australian orchid genera are described. These Serendipita species were originally characterized by multilocus DNA sequence species delimitation analyses (three mtDNA and four nuclear genes) and confirmed as distinct with addition of further isolates and reanalysis of nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and nuc 28S rDNA (28S). Culture morphology and microscopic features are presented for each species, three of which are binucleate and four multinucleate. For the ITS region, the seven species have within-species sequence divergence between 1.07% and 4.31%, and all but one of the species pairs is separated by interspecific divergence of at least 4.35%. The newly described Serendipita species, S. australiana, S. communis, S. occidentalis, S. rarihospitum, S. secunda, S. talbotii, and S. warcupii, are shown to be separate species from S. vermifera on the basis of comparison against a sequence from the type. Isolates originally identified by Warcup as Sebacina "vermifera" from Caladenia orchids are revised and shown to belong to three of the species newly described here. Some non-Caladenia isolates identified by Warcup as S. "vermifera" are also shown to be non-conspecific with the type of S. vermifera. On the basis of ITS sequences, 346 isolates from 26 other studies, previously identified under provisional designations, are accommodated under the novel species. The species of Serendipta described here associate with the Australian orchid genera Caladenia, Cyanicula, Elythranthera, Ericksonella, Eriochilus, Glossodia, and Pheladenia. Most of the novel Serendipita species occur widely across Australia, often with widely distributed hosts, but one species, Serendipita rarihospitum, associates with narrowly distributed orchid species.

Continental-scale distribution and diversity of Ceratobasidium orchid mycorrhizal fungi in Australia.

BACKGROUND AND AIMS: Mycorrhizal fungi are a critical component of the ecological niche of most plants and can potentially constrain their geographical range. Unlike other types of mycorrhizal fungi, the distributions of orchid mycorrhizal fungi (OMF) at large spatial scales are not well understood. Here, we investigate the distribution and diversity of Ceratobasidium OMF in orchids and soils across the Australian continent. METHODS: We sampled 217 Ceratobasidium isolates from 111 orchid species across southern Australia and combined these with 311 Ceratobasidium sequences from GenBank. To estimate the taxonomic diversity of Ceratobasidium associating with orchids, phylogenetic analysis of the ITS sequence locus was undertaken. Sequence data from the continent-wide Australian Microbiome Initiative were used to determine the geographical range of operational taxonomic units (OTUs) detected in orchids, with the distribution and climatic correlates of the two most frequently detected OTUs modelled using MaxEnt. KEY RESULTS: We identified 23 Ceratobasidium OTUs associating with Australian orchids, primarily from the orchid genera Pterostylis, Prasophyllum, Rhizanthella and Sarcochilus. OTUs isolated from orchids were closely related to, but distinct from, known pathogenic fungi. Data from soils and orchids revealed that ten of these OTUs occur on both east and west sides of the continent, while 13 OTUs were recorded at three locations or fewer. MaxEnt models suggested that the distributions of two widespread OTUs are correlated with temperature and soil moisture of the wettest quarter and far exceeded the distributions of their host orchid species. CONCLUSIONS: Ceratobasidium OMF with cross-continental distributions are common in Australian soils and frequently have geographical ranges that exceed that of their host orchid species, suggesting these fungi are not limiting the distributions of their host orchids at large spatial scales. Most OTUs were distributed within southern Australia, although several OTUs had distributions extending into central and northern parts of the continent, illustrating their tolerance of an extraordinarily wide range of environmental conditions.

A multitiered sequence capture strategy spanning broad evolutionary scales: Application for phylogenetic and phylogeographic studies of orchids.

With over 25,000 species, the drivers of diversity in the Orchidaceae remain to be fully understood. Here, we outline a multitiered sequence capture strategy aimed at capturing hundreds of loci to enable phylogenetic resolution from subtribe to subspecific levels in orchids of the tribe Diurideae. For the probe design, we mined subsets of 18 transcriptomes, to give five target sequence sets aimed at the tribe (Sets 1 & 2), subtribe (Set 3), and within subtribe levels (Sets 4 & 5). Analysis included alternative de novo and reference-guided assembly, before target sequence extraction, annotation and alignment, and application of a homology-aware k-mer block phylogenomic approach, prior to maximum likelihood and coalescence-based phylogenetic inference. Our evaluation considered 87 taxa in two test data sets: 67 samples spanning the tribe, and 72 samples involving 24 closely related Caladenia species. The tiered design achieved high target loci recovery (>89%), with the median number of recovered loci in Sets 1-5 as follows: 212, 219, 816, 1024, and 1009, respectively. Interestingly, as a first test of the homologous k-mer approach for targeted sequence capture data, our study revealed its potential for enabling robust phylogenetic species tree inferences. Specifically, we found matching, and in one case improved phylogenetic resolution within species complexes, compared to conventional phylogenetic analysis involving target gene extraction. Our findings indicate that a customized multitiered sequence capture strategy, in combination with promising yet underutilized phylogenomic approaches, will be effective for groups where interspecific divergence is recent, but information on deeper phylogenetic relationships is also required.

New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae.

Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4-6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and » potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.

Spatial and Temporal Genetic Analyses of Phyllosticta citricarpa in Two Lemon Orchards in South Africa Reveal a Role of Asexual Reproduction Within Sexually Reproducing Populations.

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is a disease that affects citrus worldwide. In different regions of the world where both mating types occur, reports differ as to whether asexually produced pycnidiospores play an important role in the epidemiology of CBS and fruit infections. Therefore, we investigated the potential role of pycnidiospores in two lemon orchards in South Africa by using microsatellite-based analysis of fruit populations over time (two seasons) and space (distance). The two orchards were situated in the semiarid North West province (NW) and subtropical Mpumalanga province (MP). Each population contained both mating types in 1:1 ratios, and linkage disequilibrium analysis indicated a random mating population. A total of 109 and 94 multilocus genotypes (MLGs) were detected across the two seasons in the NW and MP orchards, respectively. Psex analyses indicated that most MLGs probably resulted from sexual reproduction, but there were six predominant MLGs in each orchard that were probably replicated via asexual reproduction. Each of the predominant MLGs was monomorphic for mating type. In the NW, five predominant and widespread MLGs caused 46 and 44% of the fruit infections in the two seasons, whereas in MP, three MLGs caused 34 and 48% of the infections. Asexual reproduction in both orchards was supported by low MLG evenness values in all populations. In both orchards, distance was not a reliable predictor of population genetic substructuring or season. Populations of P. citricarpa in the MP and NW orchards were significantly genetically differentiated from each other.

Specific mycorrhizal associations involving the same fungal taxa in common and threatened Caladenia (Orchidaceae): implications for conservation.

BACKGROUND AND AIMS: In orchid conservation, quantifying the specificity of mycorrhizal associations, and establishing which orchid species use the same fungal taxa, is important for sourcing suitable fungi for symbiotic propagation and selecting sites for conservation translocation. For Caladenia subgenus Calonema (Orchidaceae), which contains 58 threatened species, we ask the following questions. (1) How many taxa of Serendipita mycorrhizal fungi do threatened species of Caladenia associate with? (2) Do threatened Caladenia share orchid mycorrhizal fungi with common Caladenia? (3) How geographically widespread are mycorrhizal fungi associated with Caladenia? METHODS: Fungi were isolated from 127 Caladenia species followed by DNA sequencing of the internal transcibed spacer (ITS) sequence locus. We used a 4.1-6 % sequence divergence cut-off range to delimit Serendipita operational taxonomic units (OTUs). We conducted trials testing the ability of fungal isolates to support germination and plant growth. A total of 597 Serendipita isolates from Caladenia, collected from across the Australian continent, were used to estimate the geographic range of OTUs. KEY RESULTS: Across the genus, Caladenia associated with ten OTUs of Serendipita (Serendipitaceae) mycorrhizal fungi. Specificity was high, with 19 of the 23 threatened Caladenia species sampled in detail associating solely with OTU A, which supported plants from germination to adulthood. The majority of populations of Caladenia associated with one OTU per site. Fungal sharing was extensive, with 62 of the 79 Caladenia sampled in subgenus Calonema associating with OTU A. Most Serendipita OTUs were geographically widespread. CONCLUSIONS: Mycorrhizal fungi can be isolated from related common species to propagate threatened Caladenia. Because of high specificity of most Caladenia species, only small numbers of OTUs typically need to be considered for conservation translocation. When selecting translocation sites, the geographic range of the fungi is not a limiting factor, and using related Caladenia species to infer the presence of suitable fungal OTUs may be feasible.

Host specialisation and disparate evolution of Pyrenophora teres f. teres on barley and barley grass.

BACKGROUND: Pathogens evolve in an arms race, frequently evolving virulence that defeats resistance genes in their hosts. Infection of multiple hosts may accelerate this virulence evolution. Theory predicts that host diversity affects pathogen diversity, with more diverse hosts expected to harbour more diverse pathogens that reproduce sexually. We tested this hypothesis by comparing the microsatellite (SSR) genetic diversity of the barley leaf pathogen Pyrenophora teres f. teres (Ptt) from barley (monoculture) and barley grass (outbreeding). We also aim to investigate host specificity and attempt to track virulence on two barley cultivars, Maritime and Keel. RESULTS: Genetic diversity in barley Ptt populations was higher than in populations from barley grass. Barley Ptt populations also had higher linkage disequilibrium levels, indicating less frequent sexual reproduction, consistent with the Red Queen hypothesis theory that genetically diverse hosts should select for higher levels of sexual reproduction of the pathogen. SSR analyses indicate that host-associated Ptt populations do not share genotypes and have independent evolutionary histories. Pathogenicity studies showed host specificity as host-associated Ptt isolates could not cross-infect hosts. Minimum spanning network analyses indicated two major clusters of barley Ptt. One cluster represents Maritime virulent and isolates from Western Australia (WA). Low PhiPt population differentiation between WA populations and those from Maritime and Keel, indicated a WA origin of the Maritime and Keel virulences. The main minimum spanning network cluster is represented by a panmictic population structure, represented by isolates from all over Australia. CONCLUSIONS: Although barley Ptt populations are more diverse than barley grass Ptt populations, this may be a result of the size and number of founder Ptt populations to Australia, with larger and more barley Ptt populations introduced. More frequent sexual reproduction of Ptt on barley grass support the Red Queen Hypothesis and suggest evolutionary potential of pathogens on diverse hosts are high. Extensive gene flow of Ptt between regions in Australia is suggested to maintain a panmictic population structure, with human-mediated dispersal aiding in virulence evolution of Ptt on barley.

Matching symbiotic associations of an endangered orchid to habitat to improve conservation outcomes.

Background and Aims: An understanding of mycorrhizal variation, orchid seed germination temperature and the effect of co-occurring plant species could be critical for optimizing conservation translocations of endangered plants with specialized mycorrhizal associations. Methods: Focusing on the orchid Thelymitra epipactoides, we isolated mycorrhizal fungi from ten plants within each of three sites; Shallow Sands Woodland (SSW), Damp Heathland (DH) and Coastal Heathland Scrub (CHS). Twenty-seven fungal isolates were tested for symbiotic germination under three 24 h temperature cycles: 12 °C for 16 h-16 °C for 8 h, 16 °C for 16 h-24 °C for 8 h or 27 °C constant. Fungi were sequenced using the internal transcribed spacer (ITS), nuclear large subunit 1 (nLSU1), nLSU2 and mitochondrial large rRNA gene (mtLSU). Orchids were grown to maturity and co-planted with each of ten associated plant species in a glasshouse experiment with tuber width measured at 12 months after co-planting. Key Results: Two Tulasnella fungal lineages were isolated and identified by phylogenetic analyses, operational taxonomic unit 1 (OTU1) and 'T. asymmetrica'. Fungal lineages were specific to sites and did not co-occur. OTU1 (from the SSW site) germinated seed predominantly at 12-16 °C (typical of autumn-winter temperature) whereas 'T. asymmetrica' (from the DH and CHS sites) germinated seed across all three temperature ranges. There was no difference in the growth of adult orchids germinated with different OTUs. There was a significant reduction in tuber size of T. epipactoides when co-planted with six of the commonly co-occurring plant species. Conclusions: We found that orchid fungal lineages and their germination temperature can change with habitat, and established that translocation sites can be optimized with knowledge of co-occurring plant interactions. For conservation translocations, particularly under a changing climate, we recommend that plants should be grown with mycorrhizal fungi tailored to the recipient site.

New species of Tulasnella associated with terrestrial orchids in Australia.

Recent studies using sequence data from eight sequence loci and coalescent-based species delimitation methods have revealed several species-level lineages of Tulasnella associated with the orchid genera Arthrochilus, Caleana, Chiloglottis, and Drakaea in Australia. Here we formally describe three of those species, Tulasnella prima, T. secunda, and T. warcupii spp. nov., as well as an additional Tulasnella species associated with Chiloglottis growing in Sphagnum, T. sphagneti sp. nov. Species were identified by phylogenetic analyses of the ITS with up to 1.3 % sequence divergence within taxa and a minimum of 7.6 % intraspecific divergence. These new Tulasnella (Tulasnellaceae, Cantharellales) species are currently only known from orchid hosts, with each fungal species showing a strong relationship with an orchid genus. In this study, T. prima and T. sphagneti associate with Chiloglottis, while T. secunda associates with Drakaea and Caleana, and T. warcupii associates with Arthrochilus oreophilus.

Evidence and Consequence of a Highly Adapted Clonal Haplotype within the Australian Ascochyta rabiei Population.

The Australian Ascochyta rabiei (Pass.) Labr. (syn. Phoma rabiei) population has low genotypic diversity with only one mating type detected to date, potentially precluding substantial evolution through recombination. However, a large diversity in aggressiveness exists. In an effort to better understand the risk from selective adaptation to currently used resistance sources and chemical control strategies, the population was examined in detail. For this, a total of 598 isolates were quasi-hierarchically sampled between 2013 and 2015 across all major Australian chickpea growing regions and commonly grown host genotypes. Although a large number of haplotypes were identified (66) through short sequence repeat (SSR) genotyping, overall low gene diversity (Hexp = 0.066) and genotypic diversity (D = 0.57) was detected. Almost 70% of the isolates assessed were of a single dominant haplotype (ARH01). Disease screening on a differential host set, including three commonly deployed resistance sources, revealed distinct aggressiveness among the isolates, with 17% of all isolates identified as highly aggressive. Almost 75% of these were of the ARH01 haplotype. A similar pattern was observed at the host level, with 46% of all isolates collected from the commonly grown host genotype Genesis090 (classified as "resistant" during the term of collection) identified as highly aggressive. Of these, 63% belonged to the ARH01 haplotype. In conclusion, the ARH01 haplotype represents a significant risk to the Australian chickpea industry, being not only widely adapted to the diverse agro-geographical environments of the Australian chickpea growing regions, but also containing a disproportionately large number of aggressive isolates, indicating fitness to survive and replicate on the best resistance sources in the Australian germplasm.

Weeds, as ancillary hosts, pose disproportionate risk for virulent pathogen transfer to crops.

BACKGROUND: The outcome of the arms race between hosts and pathogens depends heavily on the interactions between their genetic diversity, population size and transmission ability. Theory predicts that genetically diverse hosts will select for higher virulence and more diverse pathogens than hosts with low genetic diversity. Cultivated hosts typically have lower genetic diversity and thus small effective population sizes, but can potentially harbour large pathogen population sizes. On the other hand, hosts, such as weeds, which are genetically more diverse and thus have larger effective population sizes, usually harbour smaller pathogen population sizes. Large pathogen population sizes may lead to more opportunities for mutation and hence more diverse pathogens. Here we test the predictions that pathogen neutral genetic diversity will increase with large pathogen population sizes and host diversity, whereas diversity under selection will increase with host diversity. We assessed and compared the diversity of a fungal pathogen, Rhynchosporium commune, on weedy barley grass (which have a large effective population size) and cultivated barley (low genetic diversity) using microsatellites, effector locus nip1 diversity and pathogen aggressiveness in order to assess the importance of weeds in the evolution of the neutral and selected diversity of pathogens. RESULTS: The findings indicated that the large barley acreage and low host diversity maintains higher pathogen neutral genetic diversity and lower linkage disequilibrium, while the weed maintains more pathotypes and higher virulence diversity at nip1. Strong evidence for more pathogen migration from barley grass to barley suggests transmission of virulence from barley grass to barley is common. CONCLUSIONS: Pathogen census population size is a better predictor for neutral genetic diversity than host diversity. Despite maintaining a smaller pathogen census population size, barley grass acts as an important ancillary host to R. commune, harbouring highly virulent pathogen types capable of transmission to barley. Management of disease on crops must therefore include management of weedy ancillary hosts, which may harbour disproportionate supplies of virulent pathogen strains.

Defence gene expression profiling to Ascochyta rabiei aggressiveness in chickpea.

KEY MESSAGE: Significant differences in defence pathway-related gene expression were observed among chickpea cultivars following A. rabiei infection. Differential gene expression is indicative of diverse resistances, a theoretical tool for selective breeding. A high number of Ascochyta rabiei pathotypes infecting chickpea in Australia has severely hampered efforts towards breeding for sustained quantitative resistance in chickpea. Breeding for sustained resistance will be aided by detailed knowledge of defence responses to isolates with different aggressiveness. As an initial step, the conserved and differential expressions of a suit of previously characterised genes known to be involved in fungal defence mechanisms were assessed among resistant and susceptible host genotypes following inoculation with high or low aggressive A. rabiei isolates. Using quantitative Real-Time PCR (qRT-PCR), 15 defence-related genes, normalised with two reference genes, were temporally differentially expressed (P < 0.005) as early as 2 h post inoculation of Genesis090 (resistant) or Kaniva (susceptible). The highly aggressive isolate, 09KAL09, induced vastly different expression profiles of eight key defence-related genes among resistant and susceptible genotypes. Six of these same genes were differentially expressed among ten host genotypes, inclusive of the best resistance sources within the Australian chickpea breeding program, indicating potential use for discrimination and selection of resistance "type" in future breeding pursuits.

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids.

BACKGROUND AND AIMS: Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale. METHODS: Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia. KEY RESULTS: With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent. CONCLUSIONS: The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids.

Development of phylogenetic markers for Sebacina (Sebacinaceae) mycorrhizal fungi associated with Australian orchids.

PREMISE OF THE STUDY: To investigate fungal species identity and diversity in mycorrhizal fungi of order Sebacinales, we developed phylogenetic markers. These new markers will enable future studies investigating species delineation and phylogenetic relationships of the fungal symbionts and facilitate investigations into evolutionary interactions among Sebacina species and their orchid hosts. • METHODS AND RESULTS: We generated partial genome sequences for a Sebacina symbiont originating from Caladenia huegelii with 454 genome sequencing and from three symbionts from Eriochilus dilatatus and one from E. pulchellus using Illumina sequencing. Six nuclear and two mitochondrial loci showed high variability (10-31% parsimony informative sites) for Sebacinales mycorrhizal fungi across four genera of Australian orchids (Caladenia, Eriochilus, Elythranthera, and Glossodia). • CONCLUSIONS: We obtained highly informative DNA markers that will allow investigation of mycorrhizal diversity of Sebacinaceae fungi associated with terrestrial orchids in Australia and worldwide.