The ilv2 gene, encoding acetolactate synthase for branched chain amino acid biosynthesis, is required for plant pathogenicity by Leptosphaeria maculans.

BACKGROUND: Control of blackleg disease of canola caused by the fungus Leptosphaeria maculans relies on strategies such as the inhibition of growth with fungicides. However, other chemicals are used during canola cultivation, including fertilizers and herbicides. There is widespread use of herbicides that target the acetolactate synthase (ALS) enzyme involved in branched chain amino acid synthesis and low levels of these amino acids within leaves of Brassica species. In L. maculans the ilv2 gene encodes ALS and thus ALS-inhibiting herbicides may inadvertently impact the fungus. METHODS AND RESULTS: Here, the impact of a commercial herbicide targeting ALS and mutation of the homologous ilv2 gene in L. maculans was explored. Exposure to herbicide had limited impact on growth in vitro but reduced lesion sizes in plant disease experiments. Furthermore, the mutation of the ilv2 gene via CRISPR-Cas9 gene editing rendered the fungus non-pathogenic. CONCLUSION: Herbicide applications can influence disease outcome, but likely to a minor extent.

Genetic Transformation of Cryptococcus Species with Agrobacterium Transfer DNA.

Transformation of foreign DNA into Cryptococcus species is a powerful tool for exploring gene functions in these human pathogens. Agrobacterium tumefaciens-mediated transformation (AtMT) has been used for the stable introduction of exogenous DNA into Cryptococcus for over two decades, being particularly impactful for insertional mutagenesis screens to discover new genes involved in fungal biology. A detailed protocol to conduct this transformation method is provided in the chapter. Scope for modifications and the benefits and disadvantages of using AtMT in Cryptococcus species are also presented.

Potential for Use of Species in the Subfamily Erynioideae for Biological Control and Biotechnology.

The fungal order Entomophthorales in the Zoopagomycota includes many fungal pathogens of arthropods. This review explores six genera in the subfamily Erynioideae within the family Entomophthoraceae, namely, Erynia, Furia, Orthomyces, Pandora, Strongwellsea, and Zoophthora. This is the largest subfamily in the Entomophthorales, including 126 described species. The species diversity, global distribution, and host range of this subfamily are summarized. Relatively few taxa are geographically widespread, and few have broad host ranges, which contrasts with many species with single reports from one location and one host species. The insect orders infected by the greatest numbers of species are the Diptera and Hemiptera. Across the subfamily, relatively few species have been cultivated in vitro, and those that have require more specialized media than many other fungi. Given their potential to attack arthropods and their position in the fungal evolutionary tree, we discuss which species might be adopted for biological control purposes or biotechnological innovations. Current challenges in the implementation of these species in biotechnology include the limited ability or difficulty in culturing many in vitro, a correlated paucity of genomic resources, and considerations regarding the host ranges of different species.

Hormonal and proteomic analyses of southern blight disease caused by Athelia rolfsii and root chitosan priming on Cannabis sativa in an in vitro hydroponic system.

Southern blight disease, caused by the fungal pathogen Athelia rolfsii, suppresses plant growth and reduces product yield in Cannabis sativa agriculture. Mechanisms of pathology of this soil-borne disease remain poorly understood, with disease management strategies reliant upon broad-spectrum antifungal use. Exposure to chitosan, a natural elicitor, has been proposed as an alternative method to control diverse fungal diseases in an eco-friendly manner. In this study, C. sativa plants were grown in the Root-TRAPR system, a transparent hydroponic growth device, where plant roots were primed with .2% colloidal chitosan prior to A. rolfsii inoculation. Both chitosan-primed and unprimed inoculated plants displayed classical symptoms of wilting and yellowish leaves, indicating successful infection. Non-primed infected plants showed increased shoot defense responses with doubling of peroxidase and chitinase activities. The levels of growth and defense hormones including auxin, cytokinin, and jasmonic acid were increased 2-5-fold. In chitosan-primed infected plants, shoot peroxidase activity and phytohormone levels were decreased 1.5-4-fold relative to the unprimed infected plants. When compared with shoots, roots were less impacted by A. rolfsii infection, but the pathogen secreted cell wall-degrading enzymes into the root-growth solution. Chitosan priming inhibited root growth, with root lengths of chitosan-primed plants approximately 65% shorter than the control, but activated root defense responses, with root peroxidase activity increased 2.7-fold along with increased secretion of defense proteins. The results suggest that chitosan could be an alternative platform to manage southern blight disease in C. sativa cultivation; however, further optimization is required to maximize effectiveness of chitosan.

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades-primarily plant-associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans.

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

Effects of chitin and chitosan on root growth, biochemical defense response and exudate proteome of Cannabis sativa.

Fungal pathogens pose a major threat to Cannabis sativa production, requiring safe and effective management procedures to control disease. Chitin and chitosan are natural molecules that elicit plant defense responses. Investigation of their effects on C. sativa will advance understanding of plant responses towards elicitors and provide a potential pathway to enhance plant resistance against diseases. Plants were grown in the in vitro Root-TRAPR system and treated with colloidal chitin and chitosan. Plant morphology was monitored, then plant tissues and exudates were collected for enzymatic activity assays, phytohormone quantification, qPCR analysis and proteomics profiling. Chitosan treatments showed increased total chitinase activity and expression of pathogenesis-related (PR) genes by 3-5 times in the root tissues. In the exudates, total peroxidase and chitinase activities and levels of defense proteins such as PR protein 1 and endochitinase 2 were increased. Shoot development was unaffected, but root development was inhibited after chitosan exposure. In contrast, chitin treatments had no significant impact on any defense parameters, including enzymatic activities, hormone quantities, gene expression levels and root secreted proteins. These results indicate that colloidal chitosan, significantly enhancing defense responses in C. sativa root system, could be used as a potential elicitor, particularly in hydroponic scenarios to manage crop diseases.

Isolation of strains and their genome sequencing to analyze the mating system of Ophiocordyceps robertsii.

The fungal genus Ophiocordyceps contains a number of insect pathogens. One of the best known of these is Ophiocordyceps sinensis, which is used in Chinese medicine and its overharvesting threatens sustainability; hence, alternative species are being sought. Ophiocordyceps robertsii, found in Australia and New Zealand, has been proposed to be a close relative to O. sinensis, but little is known about this species despite being also of historical significance. Here, O. robertsii strains were isolated into culture and high coverage draft genome sequences obtained and analyzed. This species has a large genome expansion, as also occurred in O. sinensis. The mating type locus was characterized, indicating a heterothallic arrangement whereby each strain has an idiomorphic region of two (MAT1-2-1, MAT1-2-2) or three (MAT1-1-1, MAT1-1-2, MAT1-1-3) genes flanked by the conserved APN2 and SLA2 genes. These resources provide a new opportunity for understanding the evolution of the expanded genome in the homothallic species O. sinensis, as well as capabilities to explore the pharmaceutical potential in a species endemic to Australia and New Zealand.

Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases.

Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.

Globisporangium and Pythium Species Associated with Yield Decline of Pyrethrum (Tanacetum cinerariifolium) in Australia.

Pyrethrum (Tanacetum cinerariifolium) cultivation in Australia, which accounts for the majority of global production of natural insecticidal pyrethrins, is affected by a persistent yield decline which in part is caused by a complex of pathogens. Globisporangium and Pythium species were isolated from crown and roots of pyrethrum plants showing stunting and brown discoloration of crown tissue, and from soil adjacent to diseased plants from yield-decline-affected sites in Tasmania and Victoria, Australia. Ten known Globisporangium species (Globisporangium attrantheridium, G. erinaceum, G. intermedium, G. irregulare, G. macrosporum, G. recalcitrans, G. rostratifingens, G. sylvaticum, G. terrestris and G. ultimum var. ultimum), two new Globisporangium species (Globisporangium capense sp. nov. and Globisporangium commune sp. nov.) and three Pythium species (Pythium diclinum/lutarium, P. tracheiphilum and P. vanterpoolii) were identified through morphological studies and multigene phylogenetic analyses using ITS and Cox1 sequences. Globisporangium ultimum var. ultimum, G. sylvaticum, G. commune sp. nov. and G. irregulare were most abundant. Globisporangium attrantheridium, G. macrosporum and G. terrestris were reported for the first time in Australia. Seven Globisporangium species were pathogenic on both pyrethrum seeds (in vitro assays) and seedlings (glasshouse bioassays), while two Globisporangium species and three Pythium species only caused significant symptoms on pyrethrum seeds. Globisporangium irregulare and G. ultimum var. ultimum were the most aggressive species, causing pyrethrum seed rot, seedling damping-off and significant plant biomass reduction. This is the first report of Globisporangium and Pythium species causing disease in pyrethrum globally and suggests that oomycete species in the family Pythiaceae may have an important role in the yield decline of pyrethrum in Australia.

A Polyphasic Approach including Whole Genome Sequencing Reveals Paecilomyces paravariotii sp. nov. as a Cryptic Sister Species to P. variotii.

Whole genome sequencing is rapidly increasing phylogenetic resolution across many groups of fungi. To improve sequencing coverage in the genus Paecilomyces (Eurotiales), we report nine new Paecilomyces genomes representing five different species. Phylogenetic comparison between these genomes and those reported previously showed that Paecilomyces paravariotii is a distinct species from its close relative P. variotii. The independence of P. paravariotii is supported by analysis of overall gene identify (via BLAST), differences in secondary metabolism and an inability to form ascomata when paired with a fertile P. variotii strain of opposite mating type. Furthermore, whole genome sequencing resolves the P. formosus clade into three separate species, one of which lacked a valid name that is now provided.

Isolation of a fungal calcineurin A mutant suggests that amoebae can counter-select virulence attributes of microbes.

Evolutionary selection pressures that resulted in microbes found within environmental reservoirs that can cause diseases in animals are unknown. One hypothesis is that predatory organisms select microbes able to counteract animal immune cells. Here, a non-pathogenic yeast, Sporobolomyces primogenomicus, was exposed to predation by Acanthamoeba castellanii. Strains emerged that were resistant to being killed by this amoeba. All these strains had altered morphology, growing as pseudohyphae. The mutation in one strain was identified: CNA1 encodes the calcineurin A subunit that is highly conserved in fungi and where it is essential for their virulence in hosts including mammals, insects, and plants.

One hypothesis why some microbes cause disease in humans is that they have been exposed to selection pressures in the environment, like predation by amoebae. This study selected yeast strains resistant to amoeba. One is due to the loss of calcineurin, a protein required for disease.

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins.

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.

Sexual reproduction is the null hypothesis for life cycles of rust fungi.

Sexual reproduction, mutation, and reassortment of nuclei increase genotypic diversity in rust fungi. Sexual reproduction is inherent to rust fungi, coupled with their coevolved plant hosts in native pathosystems. Rust fungi are hypothesised to exchange nuclei by somatic hybridisation with an outcome of increased genotypic diversity, independent of sexual reproduction. We provide criteria to demonstrate whether somatic exchange has occurred, including knowledge of parental haplotypes and rejection of fertilisation in normal rust life cycles.

Root-TRAPR: a modular plant growth device to visualize root development and monitor growth parameters, as applied to an elicitor response of Cannabis sativa.

BACKGROUND: Plant growth devices, for example, rhizoponics, rhizoboxes, and ecosystem fabrication (EcoFAB), have been developed to facilitate studies of plant root morphology and plant-microbe interactions in controlled laboratory settings. However, several of these designs are suitable only for studying small model plants such as Arabidopsis thaliana and Brachypodium distachyon and therefore require modification to be extended to larger plant species like crop plants. In addition, specific tools and technical skills needed for fabricating these devices may not be available to researchers. Hence, this study aimed to establish an alternative protocol to generate a larger, modular and reusable plant growth device based on different available resources. RESULTS: Root-TRAPR (Root-Transparent, Reusable, Affordable three-dimensional Printed Rhizo-hydroponic) system was successfully developed. It consists of two main parts, an internal root growth chamber and an external structural frame. The internal root growth chamber comprises a polydimethylsiloxane (PDMS) gasket, microscope slide and acrylic sheet, while the external frame is printed from a three-dimensional (3D) printer and secured with nylon screws. To test the efficiency and applicability of the system, industrial hemp (Cannabis sativa) was grown with or without exposure to chitosan, a well-known plant elicitor used for stimulating plant defense. Plant root morphology was detected in the system, and plant tissues were easily collected and processed to examine plant biological responses. Upon chitosan treatment, chitinase and peroxidase activities increased in root tissues (1.7- and 2.3-fold, respectively) and exudates (7.2- and 21.6-fold, respectively). In addition, root to shoot ratio of phytohormone contents were increased in response to chitosan. Within 2 weeks of observation, hemp plants exhibited dwarf growth in the Root-TRAPR system, easing plant handling and allowing increased replication under limited growing space. CONCLUSION: The Root-TRAPR system facilitates the exploration of root morphology and root exudate of C. sativa under controlled conditions and at a smaller scale. The device is easy to fabricate and applicable for investigating plant responses toward elicitor challenge. In addition, this fabrication protocol is adaptable to study other plants and can be applied to investigate plant physiology in different biological contexts, such as plant responses against biotic and abiotic stresses.

Independent breakdown events of the Brassica napus Rlm7 resistance gene including via the off-target impact of a dual-specificity avirulence interaction.

Protection of many crops is achieved through the use of genetic resistance. Leptosphaeria maculans, the causal agent of blackleg disease of Brassica napus, has emerged as a model for understanding gene-for-gene interactions that occur between plants and pathogens. Whilst many of the characterized avirulence effector genes interact with a single resistance gene in the host, the AvrLm4-7 avirulence gene is recognized by two resistance genes, Rlm4 and Rlm7. Here, we report the "breakdown" of the Rlm7 resistance gene in Australia, under two different field conditions. The first, and more typical, breakdown probably resulted from widescale use of Rlm7-containing cultivars whereby selection has led to an increase of individuals in the L. maculans population that have undergone repeat-induced point (RIP) mutations at the AvrLm4-7 locus. This has rendered the AvrLm4-7 gene ineffective and therefore these isolates have become virulent towards both Rlm4 and Rlm7. The second, more atypical, situation was the widescale use of Rlm4 cultivars. Whilst a single-nucleotide polymorphism is the more common mechanism of virulence towards Rlm4, in this field situation, RIP mutations have been selected leading to the breakdown of resistance for both Rlm4 and Rlm7. This is an example of a resistance gene being rendered ineffective without having grown cultivars with the corresponding resistance gene due to the dual specificity of the avirulence gene. These findings highlight the value of pathogen surveillance in the context of expanded knowledge about potential complexities for Avr-R interactions for the deployment of appropriate resistance gene strategies.

A large transposable element mediates metal resistance in the fungus Paecilomyces variotii.

The horizontal transfer of large gene clusters by mobile elements is a key driver of prokaryotic adaptation in response to environmental stresses. Eukaryotic microbes face similar stresses; however, a parallel role for mobile elements has not been established. A stress faced by many microorganisms is toxic metal ions in their environment. In fungi, identified mechanisms for protection against metals generally rely on genes that are dispersed within an organism's genome. Here, we discover a large (∼85 kb) region that confers tolerance to five metal/metalloid ions (arsenate, cadmium, copper, lead, and zinc) in the genomes of some, but not all, strains of a fungus, Paecilomyces variotii. We name this region HEPHAESTUS (Hφ) and present evidence that it is mobile within the P. variotii genome with features characteristic of a transposable element. HEPHAESTUS contains the greatest complement of host-beneficial genes carried by a transposable element in eukaryotes, suggesting that eukaryotic transposable elements might play a role analogous to bacteria in the horizontal transfer of large regions of host-beneficial DNA. Genes within HEPHAESTUS responsible for individual metal tolerances include those encoding a P-type ATPase transporter-PcaA-required for cadmium and lead tolerance, a transporter-ZrcA-providing tolerance to zinc, and a multicopper oxidase-McoA-conferring tolerance to copper. In addition, a subregion of Hφ confers tolerance to arsenate. The genome sequences of other fungi in the Eurotiales contain further examples of HEPHAESTUS, suggesting that it is responsible for independently assembling tolerance to a diverse array of ions, including chromium, mercury, and sodium.

Identification of the ergC gene involved in polyene drug sensitivity in the Mucorales species Phycomyces blakesleeanus.

BACKGROUND: A strain of Phycomyces blakesleeanus (Mucorales, Mucoromycota) that was previously isolated after ultraviolet mutagenesis has altered responses to polyene antifungal drugs, sterol profiles, and phototropism of its sporangia. In this study, the genetic basis for these changes was sought. METHODS AND RESULTS: Two base pair substitutions were identified in the mutant within a P. blakelesleeanus gene that is homologous to others characterized from fungi, such as the Saccharomyces cerevisiae ERG3 gene, encoding sterol Δ5,6-desaturase. The polyene resistance and growth reduction phenotypes co-segregated with mutations in the gene in genetic crosses. The P. blakelesleeanus wild type ergC gene complemented a S. cerevisiae deletion strain of ERG3. CONCLUSIONS: This gene discovery may contribute towards better antifungal use in treating mucormycoses diseases caused by related species in the order Mucorales.

The "Bipartite" Structure of the First Genome of Ampelomyces quisqualis, a Common Hyperparasite and Biocontrol Agent of Powdery Mildews, May Point to Its Evolutionary Origin from Plant Pathogenic Fungi.

Powdery mildews are among the most important plant pathogens worldwide, which are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. The taxonomy of the genus Ampelomyces is unresolved, but well-supported molecular operational taxonomic units were repeatedly defined suggesting that the genus may include at least four to seven species. Some Ampelomyces strains were commercialized as biocontrol agents of crop pathogenic powdery mildews. However, the genomic mechanisms underlying their mycoparasitism are still poorly understood. To date, the draft genome of a single Ampelomyces strain, designated as HMLAC 05119, has been released. We report a high-quality, annotated hybrid draft genome assembly of A. quisqualis strain BRIP 72107, which, based on phylogenetic analyses, is not conspecific with HMLAC 05119. The constructed genome is 40.38 Mb in size, consisting of 24 scaffolds with an N50 of 2.99 Mb and 96.2% completeness. Our analyses revealed "bipartite" structure of Ampelomyces genomes, where GC-balanced genomic regions are interspersed by longer or shorter stretches of AT-rich regions. This is also a hallmark of many plant pathogenic fungi and provides further evidence for evolutionary affinity of Ampelomyces species to plant pathogenic fungi. The high-quality genome and annotation produced here provide an important resource for future genomic studies of mycoparasitisim to decipher molecular mechanisms underlying biocontrol processes and natural tritrophic interactions.