Functional genomics identifies a small secreted protein that plays a role during the biotrophic to necrotrophic shift in the root rot pathogen Phytophthora medicaginis.

Introduction: Hemibiotrophic Phytophthora are a group of agriculturally and ecologically important pathogenic oomycetes causing severe decline in plant growth and fitness. The lifestyle of these pathogens consists of an initial biotrophic phase followed by a switch to a necrotrophic phase in the latter stages of infection. Between these two phases is the biotrophic to necrotrophic switch (BNS) phase, the timing and controls of which are not well understood particularly in Phytophthora spp. where host resistance has a purely quantitative genetic basis. Methods: To investigate this we sequenced and annotated the genome of Phytophthora medicaginis, causal agent of root rot and substantial yield losses to Fabaceae hosts. We analyzed the transcriptome of P. medicaginis across three phases of colonization of a susceptible chickpea host (Cicer arietinum) and performed co-regulatory analysis to identify putative small secreted protein (SSP) effectors that influence timing of the BNS in a quantitative pathosystem. Results: The genome of P. medicaginis is ~78 Mb, comparable to P. fragariae and P. rubi which also cause root rot. Despite this, it encodes the second smallest number of RxLR (arginine-any amino acid-leucine-arginine) containing proteins of currently sequenced Phytophthora species. Only quantitative resistance is known in chickpea to P. medicaginis, however, we found that many RxLR, Crinkler (CRN), and Nep1-like protein (NLP) proteins and carbohydrate active enzymes (CAZymes) were regulated during infection. Characterization of one of these, Phytmed_10271, which encodes an RxLR effector demonstrates that it plays a role in the timing of the BNS phase and root cell death. Discussion: These findings provide an important framework and resource for understanding the role of pathogenicity factors in purely quantitative Phytophthora pathosystems and their implications to the timing of the BNS phase.

Chickpea Roots Undergoing Colonisation by Phytophthora medicaginis Exhibit Opposing Jasmonic Acid and Salicylic Acid Accumulation and Signalling Profiles to Leaf Hemibiotrophic Models.

Hemibiotrophic pathogens cause significant losses within agriculture, threatening the sustainability of food systems globally. These microbes colonise plant tissues in three phases: a biotrophic phase followed by a biotrophic-to-necrotrophic switch phase and ending with necrotrophy. Each of these phases is characterized by both common and discrete host transcriptional responses. Plant hormones play an important role in these phases, with foliar models showing that salicylic acid accumulates during the biotrophic phase and jasmonic acid/ethylene responses occur during the necrotrophic phase. The appropriateness of this model to plant roots has been challenged in recent years. The need to understand root responses to hemibiotrophic pathogens of agronomic importance necessitates further research. In this study, using the root hemibiotroph Phytophthora medicaginis, we define the duration of each phase of pathogenesis in Cicer arietinum (chickpea) roots. Using transcriptional profiling, we demonstrate that susceptible chickpea roots display some similarities in response to disease progression as previously documented in leaf plant-pathogen hemibiotrophic interactions. However, our transcriptomic results also show that chickpea roots do not conform to the phytohormone responses typically found in leaf colonisation by hemibiotrophs. We found that quantified levels of salicylic acid concentrations in root tissues decreased significantly during biotrophy while jasmonic acid concentrations were significantly induced. This study demonstrated that a wider spectrum of plant species should be investigated in the future to understand the physiological changes in plants during colonisation by soil-borne hemibiotrophic pathogens before we can better manage these economically important microbes.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

Background: The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. Methods: A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Results: Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Conclusions: Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

A Near-Complete Haplotype-Phased Genome of the Dikaryotic Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici Reveals High Interhaplotype Diversity.

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism's evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.

Speed breeding is a powerful tool to accelerate crop research and breeding.

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand 1 . This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.

Draft Genome Sequence of the Fungus Lecanicillium psalliotae Strain HWLR35, Isolated from a Wheat Leaf Infected with Leaf Rust (Caused by Puccinia triticina).

Lecanicillium psalliotae is an entomopathogenic, mycoparasitical, and nematophagous fungus known to produce antibiotic and antifungal compounds. Here, we report the first 36-Mb draft genome sequence of L. psalliotae strain HWLR35. The draft genome contains 197 scaffolds and is predicted to have 11,009 protein-coding genes.

Research investment implications of shifts in the global geography of wheat stripe rust.

Breeding new crop varieties with resistance to the biotic stresses that undermine crop yields is tantamount to increasing the amount and quality of biological capital in agriculture. However, the success of genes that confer resistance to pests induces a co-evolutionary response that depreciates the biological capital embodied in the crop, as pests evolve the capacity to overcome the crop's new defences. Thus, simply maintaining this biological capital, and the beneficial production and economic outcomes it bestows, requires continual reinvestment in new crop defences. Here we use observed and modelled data on stripe rust occurrence to gauge changes in the geographic spread of the disease over recent decades. We document a significant increase in the spread of stripe rust since 1960, with 88% of the world's wheat production now susceptible to infection. Using a probabilistic Monte Carlo simulation model we estimate that 5.47 million tonnes of wheat are lost to the pathogen each year, equivalent to a loss of US$979 million per year. Comparing the cost of developing stripe-rust-resistant varieties of wheat with the cost of stripe-rust-induced yield losses, we estimate that a sustained annual research investment of at least US$32 million into stripe rust resistance is economically justified.

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils.

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths > 10 cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.

Host selection of symbiotic cyanobacteria in 31 species of the Australian cycad genus: Macrozamia (Zamiaceae).

The nitrogen-fixing cyanobacterium Nostoc is a commonly occurring terrestrial and aquatic cyanobacterium often found in symbiosis with a wide range of plant, algal, and fungal species. We investigated the diversity of cyanobacterial species occurring within the coralloid roots of different Macrozamia cycad species at diverse locations throughout Australia. In all, 74 coralloid root samples were processed and 56 endosymbiotic cyanobacteria were cultured. DNA was isolated from unialgal cultures and a segment of the 16S rRNA gene was amplified and sequenced. Microscopic analysis was performed on representative isolates. Twenty-two cyanobacterial species were identified, comprising mostly Nostoc spp. and a Calothrix sp. No correlation was observed between a cycad species and its resident cyanobiont species. The predominant cyanobacterium isolated from 18 root samples occurred over a diverse range of environmental conditions and within 14 different Macrozamia spp. Phylogenetic analysis indicated that endosymbionts were not restricted to previously described terrestrial species. An isolate clustering with Nostoc PCC7120, an aquatic strain, was identified. This is the first comprehensive study to identify the endosymbionts within a cycad genus using samples obtained from their natural habitats. These results indicate that there is negligible host specialization of cyanobacterial endosymbionts within the cycad genus Macrozamia in the wild.