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The family Sclerotiniaceae includes important phytopathogens, such as Botrytis cinerea and Sclerotinia sclerotiorum, that activate plant immune responses to facilitate infection propagation. The mechanisms of plant resistance to these necrotrophic pathogens are still poorly understood. To discover mechanisms of resistance, we used the Ciborinia camelliae (Sclerotiniaceae)–Camellia spp. pathosystem. This fungus induces rapid infection of the blooms of susceptible cultivar Nicky Crisp (Camellia japonica × Camellia pitardii var. pitardii), while Camellia lutchuensis is highly resistant. Genome-wide analysis of gene expression in resistant plants revealed fast modulation of host transcriptional activity 6 h after ascospore inoculation. Ascospores induced the same defense pathways in the susceptible Camellia cultivar but much delayed and coinciding with disease development. We next tested the hypothesis that differences in defense timing influences disease outcome. We induced early defense in the susceptible cultivar using methyl jasmonate and this strongly reduced disease development. Conversely, delaying the response in the resistant species, by infecting it with actively growing fungal mycelium, increased susceptibility. The same plant defense pathways, therefore, contribute to both resistance and susceptibility, suggesting that defense timing is a critical factor in plant health, and resistance against necrotrophic pathogens may occur during the initial biotrophy-like stages.

Fungal effector proteins facilitate host‐plant colonization and have generally been characterized as small secreted proteins (SSPs). We classified and functionally tested SSPs from the secretomes of three closely related necrotrophic phytopathogens: Ciborinia camelliae, Botrytis cinerea, and Sclerotinia sclerotiorum. Alignment of predicted SSPs identified a large protein family that share greater than 41% amino acid identity and that have key characteristics of previously described microbe‐associated molecular patterns (MAMPs). Strikingly, 73 of the 75 SSP family members were predicted within the secretome of the host‐specialist C. camelliae with single‐copy homologs identified in the secretomes of the host generalists S. sclerotiorum and B. cinerea. To explore the potential function of this family of SSPs, 10 of the 73 C. camelliae proteins, together with the single‐copy homologs from S. sclerotiorum (SsSSP3) and B. cinerea (BcSSP2), were cloned and expressed as recombinant proteins. Infiltration of SsSSP3 and BcSSP2 into host tissue induced rapid necrosis. In contrast, only one of the 10 tested C. camelliae SSPs was able to induce a limited amount of necrosis. Analysis of chimeric proteins consisting of domains from both a necrosis‐inducing and a non‐necrosis‐inducing SSP demonstrated that the C‐terminus of the S. sclerotiorum SSP is essential for necrosis‐inducing function. Deletion of the BcSSP2 homolog from B. cinerea did not affect growth or pathogenesis. Thus, this research uncovered a family of highly conserved SSPs present in diverse ascomycetes that exhibit contrasting necrosis‐inducing functions., The C‐terminus of previously uncharacterized small, secreted, cysteine‐rich proteins from three fungal pathogens of the Sclerotiniaceae is responsible for necrosis‐inducing function in plant tissues.

Forests are under threat from pests, pathogens, and changing climate. One of the major forest pathogens worldwide isDothistroma septosporum, which causes dothistroma needle blight (DNB) of pines.D. septosporumis a hemibiotrophic fungus related to well-studied Dothideomycete pathogens, such asCladosporium fulvum. These pathogens use small secreted proteins, termed effectors, to facilitate the infection of their hosts. The same effectors, however, can be recognised by plants carrying corresponding immune receptors, resulting in resistance responses. Hence, effectors are increasingly being exploited to identify and select disease resistance in crop species. In gymnosperms, however, such research is scarce. We predicted and investigated apoplasticD. septosporumcandidate effectors (DsCEs) using bioinformatics and plant-based experiments. We discovered secreted proteins that trigger cell death in the angiospermNicotianaspp., suggesting their recognition by immune receptors in non-host plants. In a first for foliar forest pathogens, we also developed a novel protein infiltration method to show that tissue-cultured pine shoots can respond with a cell death response to one of our DsCEs, as well as to a reference cell death-inducing protein. These results contribute to our understanding of forest pathogens and may ultimately provide clues to disease immunity in both commercial and natural forests.

Summary Dothistroma needle blight is one of the most devastating pine tree diseases worldwide. New and emerging epidemics have been frequent over the last 25 years, particularly in the Northern Hemisphere, where they are in part associated with changing weather patterns. One of the main Dothistroma needle blight pathogens, Dothistroma septosporum, has a global distribution but most molecular plant pathology research has been confined to Southern Hemisphere populations that have limited genetic diversity. Extensive genomic and transcriptomic data are available for a D. septosporum reference strain from New Zealand, where an introduced clonal population of the pathogen predominates. Due to the global importance of this pathogen, we determined whether the genome of this reference strain is representative of the species worldwide by sequencing the genomes of 18 strains sampled globally from different pine hosts. Genomic polymorphism shows substantial variation within the species, clustered into two distinct groups of strains with centres of diversity in Central and South America. A reciprocal chromosome translocation uniquely identifies the New Zealand strains. Globally, strains differ in their production of the virulence factor dothistromin, with extremely high production levels in strain ALP3 from Germany. Comparisons with the New Zealand reference revealed that several strains are aneuploids; for example, ALP3 has duplications of three chromosomes. Increased gene copy numbers therefore appear to contribute to increased production of dothistromin, emphasizing that studies of population structure are a necessary adjunct to functional analyses of genetic polymorphisms to identify the molecular basis of virulence in this important forest pathogen.

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

BackgroundScab, or black spot, caused by the filamentous fungal pathogenVenturia inaequalis, is the most economically important disease of apple (Malusxdomestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity ofV. inaequalisis required. A major bottleneck for the genetic characterization ofV. inaequalisis the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions inV. inaequalishave yet been published. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool inV. inaequalis.ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway genetrihydroxynaphthalene reductase(THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) targeted to theTHNgene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an~16.7% gene inactivation efficiency. Notably, of these sixTHNmutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants ofV. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening.ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing inV. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing‐based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant–pathogen interactions in gymnosperm forest trees, including kauri., RXLR effectors of the kauri dieback pathogen Phytophthora agathidicida triggered or suppressed immunity in Nicotiana, showing that RXLRs from a gymnosperm pathogen can interact with an angiosperm immune system.

Phytophthora pluvialis is the causal agent of red needle cast on Pinus radiata in New Zealand. It was first isolated in 2008 but had previously been recovered from tanoak (Notholithocarpus densiflorus) and Douglas‐fir (Pseudotsuga menziesii) trees in Oregon, USA in 2002. Phytophthora pluvialis was subsequently described as a new species in 2013 and classified as a clade III Phytophthora species. The aim of this study was to gain a better understanding of the genetic diversity, population structure and origin of this pathogen. A total of 360 P. pluvialis isolates were collected from the USA and New Zealand. The genome sequences of two P. pluvialis isolates were used to identify 27 single nucleotide polymorphism (SNP) markers that were then used to genotype the two populations. The genotypic data showed that the USA population of P. pluvialis had twice the genetic diversity and a greater number of multilocus genotypes (MLGs) compared to the New Zealand population, with 126 and 24 MLGs, respectively. The majority of the subpopulations within the USA and New Zealand showed linkage disequilibrium. All subpopulations had a negative fixation index, indicating that clonal reproduction is prevalent in both countries. A minimum spanning network (MSN) showed two unique clusters of isolates in the New Zealand population, suggesting two potential introductions of P. pluvialis into New Zealand from the USA. There was no significant structure within the New Zealand or USA populations. This study provides novel insight into the genetic structure of P. pluvialis in New Zealand and the USA.

Genes required for fungal secondary metabolite production are usually clustered, co-regulated and expressed in stationary growth phase. Chromatin modification has an important role in co-regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP-qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin-level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.

The detrimental effect of fungal pathogens on forest trees is an increasingly important problem that has implications for the health of our planet. Despite this, the study of molecular plant-microbe interactions in forest trees is in its infancy, and very little is known about the roles of effector molecules from forest pathogens. Dothistroma septosporum causes a devastating needle blight disease of pines, and intriguingly, is closely related to Cladosporium fulvum, a tomato pathogen in which pioneering effector biology studies have been carried out. Here, we studied D. septosporum effectors that are shared with C. fulvum, by comparing gene sequences from global isolates of D. septosporum and assessing effector function in both host and non-host plants. Many of the effectors were predicted to be non-functional in D. septosporum due to their pseudogenization or low expression in planta, suggesting adaptation to lifestyle and host. Effector sequences were polymorphic among a global collection of D. septosporum isolates, but there was no evidence for positive selection. The DsEcp2-1 effector elicited cell death in the non-host plant Nicotiana tabacum, whilst D. septosporum DsEcp2-1 mutants showed increased colonization of pine needles. Together these results suggest that DsEcp2-1 might be recognized by an immune receptor in both angiosperm and gymnosperm plants. This work may lead to the identification of plant targets for DsEcp2-1 that will provide much needed information on the molecular basis of gymnosperm-pathogen interactions in forests, and may also lead to novel methods of disease control.

Fungal secondary metabolites have important functions for the fungi that produce them, such as roles in virulence and competition. The hemibiotrophic pine needle pathogen Dothistroma septosporum has one of the lowest complements of secondary metabolite (SM) backbone genes of plant pathogenic fungi, indicating that this fungus produces a limited range of SMs. Amongst these SMs is dothistromin, a well-characterised polyketide toxin and virulence factor that is required for expansion of disease lesions in Dothistroma needle blight disease. Dothistromin genes are dispersed across six loci on one chromosome, rather than being clustered as for most SM genes. We explored other D. septosporum SM genes to determine if they are associated with gene clusters, and to predict what their likely products and functions might be. Of nine functional SM backbone genes in the D. septosporum genome, only four were expressed under a range of in planta and in culture conditions, one of which was the dothistromin PKS backbone gene. Of the other three expressed genes, gene knockout studies suggested that DsPks1 and DsPks2 are not required for virulence and attempts to determine a functional squalestatin-like SM product for DsPks2 were not successful. However preliminary evidence suggested that DsNps3, the only SM backbone gene to be most highly expressed in the early stage of disease, appears to be a virulence factor. Thus, despite the small number of SM backbone genes in D. septosporum, most of them appear to be poorly expressed or dispensable for virulence in planta. This work contributes to a growing body of evidence that many fungal secondary metabolite gene clusters might be non-functional and may be evolutionary relics.

Summary We present genome‐wide gene expression patterns as a time series through the infection cycle of the fungal pine needle blight pathogen, Dothistroma septosporum, as it invades its gymnosperm host, Pinus radiata. We determined the molecular changes at three stages of the disease cycle: epiphytic/biotrophic (early), initial necrosis (mid) and mature sporulating lesion (late). Over 1.7 billion combined plant and fungal reads were sequenced to obtain 3.2 million fungal‐specific reads, which comprised as little as 0.1% of the sample reads early in infection. This enriched dataset shows that the initial biotrophic stage is characterized by the up‐regulation of genes encoding fungal cell wall‐modifying enzymes and signalling proteins. Later necrotrophic stages show the up‐regulation of genes for secondary metabolism, putative effectors, oxidoreductases, transporters and starch degradation. This in‐depth through‐time transcriptomic study provides our first snapshot of the gene expression dynamics that characterize infection by this fungal pathogen in its gymnosperm host.

Biosynthesis by Aspergillus parasiticus of aflatoxin, one of the most potent known naturally occurring carcinogens, requires the activity of two regulatory proteins, AflR and AflJ, which are encoded by divergently transcribed genes within the aflatoxin gene cluster. Although the Zn2Cys6 transcription factor, AflR, has been well-studied, the role of AflJ as a transcription regulatory factor is not well understood. An AflJ-like gene (DsAflJ) is also present in the genome of the pine needle pathogen Dothistroma septosporum and is similarly divergently transcribed from an AflR orthologue (DsAflR). These genes are involved in biosynthesis of dothistromin, a toxic virulence factor related to aflatoxin. DsAflJ mutants produced low levels of dothistromin (

CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.

Tomato leaf mould disease is caused by the biotrophic fungusCladosporium fulvum. During infection,C. fulvumproduces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed byCfimmune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent.C. fulvumstrains capable of overcoming one or more of all clonedCfgenes have now emerged. To combat these strains, newCfgenes are required. An effectoromics approach was employed to identify wild tomato accessions carrying newCfgenes. Proteomics and transcriptome sequencing were first used to identify 70 apoplasticin planta-inducedC. fulvumSSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe−microbe interactionsin planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs using thePotato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry newCfgenes available for incorporation into cultivated tomato.

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.

Summary Dothistroma needle blight is a forest disease of increasing international importance due to its ability to kill as well as to retard the growth of pines. It is caused by fungi in the genus Dothistroma that produce dothistromin, a non-host selective toxin and virulence factor that is involved in necrosis of pine tissue. Recent studies of the genome of one of the main pathogenic species, Dothistroma septosporum, showed that it contains many similarities to that of the biotroph Cladosporium fulvum, including the presence of candidate biotrophic effector genes, which supported the hypothesis that D. septosporum has a hemi-biotrophic lifestyle. Using Pinus radiata as a host, we used a combination of microscopy, histological and molecular tools to further test this hypothesis and to determine the stage of the disease cycle in which dothistromin toxin is produced. The results showed a biotrophic-type phase in which the fungus grew over the needle surface, penetrated through stomatal pores and colonized epistomatal chambers. The subsequent necrotrophic phase was characterized by colonization of the mesophyll and production of dothistromin, with a >100-fold increase in dothistromin levels from early necrotic lesion to sporulating lesion stages. This is consistent with the role of dothistromin as a virulence factor that is involved in lesion expansion.

Dothistromin is a broad-spectrum mycotoxin produced by the Dothideomycete pine needle pathogen Dothistroma septosporum. It accumulates in lesions, causing characteristic red bands on needles infected with this fungus. Dothistromin is similar in structure to the aflatoxin precursor versicolorin B and the biosynthetic pathways of these toxins share many common gene products. Although dothistromin is not essential for pathogenicity in dothistroma needle blight, its presence in infected needles suggests it might have a role in the disease process. The hypothesis that dothistromin is a virulence factor was tested by studying Pinus radiata infected with dothistromin-deficient mutants of D. septosporum. The mutants were able to infect pine needles and complete their life cycle as previously shown, and were unaffected in spore germination, epiphytic growth or needle penetration. However, colonization of the mesophyll by the mutants was restricted compared to the wild type. Correspondingly, lesions produced by the mutants were smaller and produced significantly fewer spores than lesions produced by wildtype strains. Interestingly, ‘green islands’, in which chlorophyll was maintained at a higher level than in adjacent chlorotic and necrotic regions, surrounded early-appearing lesions caused by both wildtype and mutant strains. At a later stage of disease development green islands were still present in the mutant but appeared to be masked by the extended dothistromin-containing lesions in the wild type. Overall the results support a role for dothistromin in virulence in dothistroma needle blight.

Pathogen incursions are a major impediment for global forest health. How pathogens and forest trees coexist over time, without pathogens simply killing their long-lived hosts, is a critical but unanswered question. The Dothistroma Needle Blight pathogen Dothistroma septosporum was introduced into New Zealand in the 1960s and remains a low-diversity, asexual population, providing a unique opportunity to analyze the evolution of a forest pathogen. Isolates of D. septosporum collected from commercial pine forests over 50 years were compared at whole-genome and phenotype levels. Limited genome diversity and increased diversification among recent isolates support the premise of a single introduction event. Isolates from the 1960s show significantly elevated virulence against Pinus radiata seedlings and produce higher levels of the virulence factor dothistromin compared to isolates collected in the 1990s and 2000s. However, later isolates have no increased tolerance to copper, used in fungicide treatments of infested forests and traditionally assumed to be a strong selection pressure. The isolated New Zealand population of this forest pathogen therefore appears to have become less virulent over time, likely in part to maintain the viability of its long-lived host. This finding has broad implications for forest health and highlights the benefits of long-term pathogen surveys.

Lecanosticta acicola is an ascomycete that causes brown spot needle blight of pine species in many regions of the world. This pathogen is responsible for a major disease of Pinus palustris in the USA and is a quarantine organism in Europe. In order to study the genetic diversity and patterns of spread of L. acicola, eleven microsatellite markers and two mating type markers were developed. An enrichment protocol was used to isolate microsatellite-rich DNA regions, and 18 primer pairs were designed to flank these regions, of which eleven were polymorphic. A total of 93 alleles were obtained across all loci from forty isolates of L. acicola from the USA with an allelic diversity range of 0.095 to 0.931 per locus. Cross-species amplification with some of the markers was obtained with L. gloeospora, L. guatemalensis and Dothistroma septosporum, but not with D. pini. Mating type (MAT) markers amplifying both idiomorphs were also developed to determine mating type distribution in populations. These markers were designed based on alignments of both idiomorphs of nine closely related plant pathogens, and a protocol for multiplex PCR amplification of the MAT loci was optimised. The MAT markers are not species specific and also amplify the MAT loci in Dothistroma septosporum, D. pini, L. gloeospora and L. guatemalensis. Both types of genetic markers developed in this study will be valuable for future investigations of the population structure, genetic diversity and invasion history of L. acicola on a global scale.

Dothistroma septosporum causes Dothistroma needle blight (DNB) on pines throughout the world. An increase in incidence and severity of DNB, associated with climatic factors, raises an urgent need to screen for resistance in plant hosts and to detect changes in virulence of the pathogen. The availability of the genome sequence for this fungal pathogen also opens up possibilities for biochemical and molecular studies leading to new methods of disease control. All these activities require a reliable and reproducible DNB pathogenicity assay, but this has been notoriously difficult to achieve in controlled glasshouse conditions. We therefore aimed to improve DNB assays by investigating the effects of various pathogen, environment and host factors, using Pinus radiata. The production of spores in sufficient quantity and quality for infection was optimised by using pine needle minimal medium with glucose (PMMG). The critical environmental parameter for achieving high levels of DNB (>80 % of needles with disease symptoms) was leaf wetness; use of individual covers for 4–7 days post inoculation, followed by incubation in covered chambers containing water foggers for the remainder of the infection cycle, enabled reliably high rates of disease incidence. Using this assay, it was possible to distinguish between trees from families with different levels of field resistance, using clones taken as cuttings from young trees (

In filamentous fungi both pathway-specific and global regulators regulate genes involved in the biosynthesis of secondary metabolites. LaeA is a global regulator that was named for its mutant phenotype, loss of aflR expression, due to its effect on the aflatoxin-pathway regulator AflR in Aspergillus spp. The pine needle pathogen Dothistroma septosporum produces a polyketide virulence factor, dothistromin, that is chemically related to aflatoxin and whose pathway genes are also regulated by an ortholog of AflR. However, dothistromin biosynthesis is distinctive because it is switched on during early (rather than late) exponential growth phase and the genes are dispersed in six loci across one chromosome instead of being clustered. It was therefore of interest to determine whether the function of the global regulator LaeA is conserved in D. septosporum. To address this question, a LaeA ortholog (DsLaeA) was identified and its function analyzed in D. septosporum. In contrast to aflatoxin production in Aspergillus spp., deletion of DsLaeA resulted in enhanced dothistromin production and increased expression of the pathway regulatory gene DsAflR. Although expression of other putative secondary metabolite genes in D. septosporum showed a range of different responses to loss of DsLaeA function, thin layer chromatography revealed increased levels of a previously unknown metabolite in DsLaeA mutants. In addition, these mutants exhibited reduced asexual sporulation, germination and hydrophobicity. Our data suggest that although the developmental regulatory role of DsLaeA is conserved, its role in the regulation of secondary metabolism differs from that of LaeA in A. nidulans and appears to be species specific. This study provides a step towards understanding fundamental differences in regulation of clustered and fragmented groups of secondary metabolite genes that may shed light on understanding functional adaptation in secondary metabolism.

Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.

Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.

Summary Dothistroma septosporum and D. pini are the causal agents of Dothistroma needle blight (DNB) of Pinus spp. in natural forests and plantations. The main aim of this study was to develop molecular diagnostic procedures to distinguish between isolates within D. septosporum, for use in biosecurity and forest health surveillance programmes. This is of particular interest for New Zealand where the population is clonal and introduction of a new isolate of the opposite mating type could have serious consequences. Areas of diversity in the dothistromin toxin gene clusters were identified in D. septosporum (51 isolates) and D. pini (6 isolates) and used as the basis of two types of diagnostic tests. PCR-restriction fragment length polymorphism (RFLP) of part of the dothistromin polyketide synthase gene (pksA) enabled distinction between two groups of D. septosporum isolates (A and B) as well as distinguishing D. septosporum and D. pini. The intergenic region between the epoA and avfA genes allowed further resolution between some of the A group isolates in RFLP assays. These regions were analysed further to develop a rapid real-time PCR method for diagnosis by high-resolution melting (HRM) curve analysis. The pksA gene enabled rapid discrimination between D. septosporum and D. pini, whilst the epoA–avfA region distinguished the New Zealand isolate from most other isolates in the collection, including some isolates from DNB epidemics in Canada and Europe. Although this study is focused on differences between the New Zealand isolate and other global isolates, this type of diagnostic system could be used more generally for high-throughput screening of D. septosporum isolates.

The peanut leaf spot pathogen Passalora arachidicola (Mycosphaerella arachidis) is known to produce dothistromin, a mycotoxin related to aflatoxin. This is a feature shared with the pine needle pathogen Dothistroma septosporum (Mycosphaerella pini). Dothistromin biosynthesis in D. septosporum commences at an unusually early stage of growth in culture compared to most other fungal secondary metabolites, and the biosynthetic genes are arranged in fragmented groups, in contrast to aflatoxin gene clusters. Dothistromin biosynthetic genes were identified and studied in P. arachidicola to determine if the attributes described in D. septosporum are shared by another dothistromin-producing species within the Class Dothideomycetes. It was shown that dothistromin biosynthesis is very similar in the two species with regard to gene sequence and gene synteny. Functional complementation of D. septosporum mutants with P. arachidicola dothistromin genes was also possible. These similarities support a vertical mode of dothistromin gene transmission. P. arachidicola also produced dothistromin at an early growth stage in culture, suggesting that this type of regulation pattern may be relevant to the biological role of dothistromin.

Dothistroma needle blight is one of the most devastating fungal pine diseases worldwide. The disease is characterized by accumulation in pine needles of a red toxin, dothistromin, that is chemically related to aflatoxin (AF) and sterigmatocystin (ST). This review updates current knowledge of the genetics of dothistromin biosynthesis by the Dothistroma septosporum pathogen and highlights differences in gene organization and regulation that have been discovered between the dothistromin and AF/ST systems. Some previously reported genes are promoted or demoted as 'dothistromin genes' based on recent research. A new dothistromin gene, norB, is reported, and evidence of dothistromin gene homologs in other Dothideomycete fungi is presented. A hypothesis for the biological role of dothistromin is outlined. Finally, the impact that the availability of the D. septosporum genome sequence will have on dothistromin research is discussed.

Summary The most abundant metabolite of the eucalyptus leaf spot pathogen Mycosphaerella cryptica was extracted from agar cultures. The structure of the compound was elucidated by detailed studies of NMR and MS data and by comparison with derivatives. The compound is a previously undescribed diphenylether structurally related to pannaric acid found in lichens. Culture extracts from another species of Mycosphaerella isolated from the same environment yielded 5-hydroxymethylfuran3-carboxylic acid, a furan acidic compound previously isolated from a basidiomycete fungus. Assays for bioactivity of these metabolites revealed no evidence for antimicrobial activity. Some phytotoxicity was seen on newly emerged leaves of Eucalyptus globulus, but not on juvenile or adult leaves, when treated with either metabolite.

The forest pathogen Dothistroma septosporum produces the polyketide dothistromin, a mycotoxin very similar in structure to versicolorin B, a precursor of aflatoxin (AF). Dothistromin is a broad-range toxin and possibly involved in red-band needle blight disease. As the role of dothistromin in the disease is unknown the expression of dothistromin genes was studied to reveal clues to its function. Although the genes of AF and dothistromin biosynthesis are very similar, this study revealed remarkable differences in the timing of their expression. Secondary metabolites, like AF, are usually produced during late exponential phase. Previously identified dothistromin genes, as well as a newly reported versicolorin B synthase gene, vbsA, showed high levels of expression during the onset of exponential growth. This unusual early expression was also seen in transformants containing a green fluorescent protein (GFP) gene regulated by a dothistromin gene promoter, where the highest GFP expression occurred in young mycelium. Two hypotheses for the biological role of dothistromin are proposed based on these results. The study of dothistromin genes will improve current knowledge about secondary metabolite genes, their putative biological roles, and their regulation.

Summary Dothistroma needle blight (DNB) is one of the most important diseases of pine. Although its notoriety stems from Southern Hemisphere epidemics in Pinus radiata plantations, the disease has increased in prevalence and severity in areas of the Northern Hemisphere, including Europe, during the last two decades. This increase has largely been attributed to expanded planting of susceptible hosts, anthropogenic dispersal of the causative pathogens and changes in climate conducive to disease development. The last comprehensive review of DNB was published in 2004, with updates on geographic distribution and host species in 2009. Importantly, the recognition that two species, Dothistroma septosporum and D. pini, cause DNB emerged only relatively recently in 2004. These two species are morphologically very similar, and DNA-based techniques are needed to distinguish between them. Consequently, many records of host species affected or geographic location of DNB prior to 2004 are inconclusive or even misleading. The objectives of this review were (i) to provide a new database in which detailed records of DNB from 62 countries are collated; (ii) to chart the current global distribution of D. septosporum and D. pini; (iii) to list all known host species and to consider their susceptibility globally; (iv) to collate the published results of provenance trials; and (v) to consider the effects of site factors on disease incidence and severity. The review shows that DNB occurs in 76 countries, with D. septosporum confirmed to occur in 44 and D. pini in 13. There are now 109 documented Pinaceae host taxa for Dothistroma species, spanning six genera (Abies, Cedrus, Larix, Picea, Pinus and Pseudotsuga), with Pinus being the dominant host genus, accounting for 95 host taxa. The relative susceptibilities of these hosts to Dothistroma species are reported, providing a resource to inform species choice in forest planting. Country records show that most DNB outbreaks in Europe occur on Pinus nigra and its subspecies. It is anticipated that the collaborative work described in this review will both underpin a broader global research strategy to manage DNB in the future and provide a model for the study of other forest pathogens.

Advances in protoplast technology underpinned many crucial developments in our understanding of the molecular biology of filamentous fungi. This review follows one of these developments, namely the discovery and analysis of difuran toxin gene clusters. Our understanding of the biosynthetic pathway of the agriculturally important toxin, aflatoxin, has been dramatically enhanced by the use of protoplasts and protoplast-based gene transformation methods. Since the identification of the first pathway genes by complementation of mutants with transforming DNA, transformation has continued to play a critical role in the elucidation of gene function and regulation. But despite the wealth of knowledge accumulated so far some fundamental questions remain to be answered. How did these gene clusters evolve? What is the biological role of aflatoxin? The discovery of homologues of aflatoxin genes in other fungal species such as the pine needle pathogen Dothistroma septosporum may help to shed some light on these questions.

Fungi have now well and truly entered the genomic age. We currently know the complete DNA sequence for 18 fungal species and many more fungal genome sequencing projects are in progress. Whilst yeasts dominated the early genomic years, recently there has been a dramatic increase in filamentous fungal genome projects. The implications of this wealth of genetic information for mycologists worldwide is immense. In this review we summarise the background to fungal genome projects with an emphasis on the filamentous fungi. We discuss efforts to determine gene function and to compare genomes from different species. Since this is such a fast-moving field, useful web sites are listed that will enable the reader to keep up to date with developments.

La maladie des bandes rouges causee par Dothistroma est un probleme dans les plantations de pins exotiques de l'hemisphere sud depuis de nombreuses annees. La prevalence de cette maladie est en augmentation dans l'hemisphere nord et affecte maintenant les pins dans leurs regions d'origine. Le champignon pathogene Dothistroma pini, responsable de la maladie, produit une toxine, la dothistromine, proche de l'aflatoxine qui est un puissant carcinogene. Ceci pose donc la question des effets possibles sur la sante des travailleurs forestiers. Cette revue repose sur une large couverture de la litterature concernant aussi bien la maladie que la toxine. Le champignon a une taxonomie complexe avec de nombreux synonymes, et seul l'anamorphe se rencontre dans de nombreux pays. C'est un champignon necrotrophe qui tue les tissus de l'aiguille et realise son cycle biologique dans la lesion ainsi formee. La dissemination de la maladie s'effectue principalement par eclaboussures de pluie contenant les conidies mais une dissemination a longue distance a ete mise en evidence par transport de materiel contamine ou par dissemination des spores par le vent ou les nuages dans les courants aeriens. La severite de la maladie est affectee par l'humidite, la temperature et la lumiere. Il existe des differences de sensibilite entre especes de Pinus, et certaines presentent une resistance accrue avec l'âge. La methode actuelle de lutte dans les forets de plantations de l'hemisphere sud consiste a pulveriser des fongicides a base de cuivre ; dans le cas de Pinus radiata, une augmentation de la resistance a ete obtenue grâce a un programme d'amelioration genetique. La toxine dothistromine est une difuroanthraquinone, similaire en structure a la versicolorine B, precurseur de l'aflatoxine. Une partie d'une batterie de genes comprenant des genes de biosynthese de la dothistromine a ete clonee, confirmant les analogies entre les voies de biosynthese de la dothistromine et de l'aflatoxine. La dothistromine produit des radicaux oxygene nocifs par activation de la reduction de l'oxygene plutot que par photosensibilisation, mais ses effets toxiques s'exercent aussi probablement sur des sites cellulaires specifiques. Des etudes montrent que la dothistromine est un mutagene et clastogene faible, et donc potentiellement carcinogene. Bien que les risques pour les ouvriers forestiers soient consideres comme tres faibles, il est prudent d'eviter dans la mesure du possible de s'exposer dans les periodes ou les niveaux de dothistromine sont supposes eleves.

A microsatellite-based DNA profiling system was developed that can be used to distinguish genetically diverse isolates of a forest pathogen, Dothistroma pini. All isolates of this pathogen identified in New Zealand so far appear to be isogenic and the disease is kept under control by aerial applications of copper fungicide. Although New Zealand has strict importation controls in place, the possibility of genetically diverse isolates of D. pini being introduced from other countries poses a severe biosecurity threat to forest health. Therefore a DNA-based monitoring system was developed. Two informative microsatellite loci were found serendipitously in D. pini sequence data available in our laboratory. Further microsatellite loci were obtained using a rapid 5’-anchored PCR amplification technique. For each informative locus identified, specific primers were designed to flank the repeated sequence and subsequently used to generate DNA profiles for the D. pini strains. The profiles obtained from five microsatellite loci were sufficient to distinguish most isolates tested. The anchored primer technique provides an efficient tool for the identification of polymorphic loci that can be used to screen for genetic differences between fungi.

The forest pathogen Dothistroma pini (Scirrhia pini) infects the needles of many pine species, causing needle loss and consequently retarded wood growth. Only one strain of Dothistroma pini is present in New Zealand. Because over 90% of commercial forests in New Zealand are planted with the susceptible species Pinus radiata, a study of the global diversity of D. pini strains was initiated to assess the threat of further unwanted introductions of the pathogen. A collection of D. pini strains from eight countries was studied in the UK. The production of dothistromin toxin by the strains, and DNA sequence analysis of the ribosomal ITS region, confirmed their identification as D. pini, although strains from the central USA contained two nucleotide substitutions in the ITS region. Colony morphologies and growth rates were diverse, but all strains which sporulated showed a similar wide range of spore size. The morphological features examined did not support separation of the strains into the two groups shown by ITS sequences. Most striking was the production, in axenic culture, of extremely high levels of dothistromin toxin by strains from Germany and, to a lesser extent, some from the USA (> 500 times and > 40 times as much as the New Zealand strain, respectively). The high level of production of dothistromin toxin by some strains is a concern for forest health as well as for forest workers and needs to be evaluated further.

Extensive adaptability and diversity in fungi, even among closely related species, enable them to occupy various ecological niches. Of particular importance for niche adaptation is the production of fungal secondary metabolites (SM) because they can offer a distinct selective advantage in specific environments. This chapter explores two examples of SM clusters: aflatoxin-like gene clusters in fungi such as Aspergillus flavus and ACE1 gene clusters that were originally described in Magnaporthe oryzae. Both clusters occur in different classes of fungi, and a heterogeneous collection of gene clusters in related and more distant fungal taxa can help explain the evolutionary processes that led to their formation, maintenance, and inactivation/loss. Together, they illustrate the interplay of recombination and horizontal gene transfer (HGT), as well as other genomic modifications that can impact SM biosynthetic pathways in fungi. This edition first published 2014

The forest pathogen Dothistroma pini, which produces the polyketide toxin, dothistromin, was transformed with the pAN7–1 plasmid containing the Escherichia coli hygromycin B phosphotransferase gene (hph). The frequency of hygromycin resistant transformants was 9–48 μg-1 DNA. Southern blot analysis indicated that a single copy of the vector DNA had integrated into the genome in at least six of the eight transformants tested and that the site of integration was different in each case. All transformants analysed were mitotically stable through several subcultures without selective pressure.

Perennial ryegrass ( Lolium perenne ) is a widely used pasture grass, which is frequently infected by Neotyphodium lolii endophytes that enhance grass performance but can produce alkaloids inducing toxicosis in livestock. Several selected endophyte strains with reduced livestock toxicity, but that confer insect resistance, are now in common use. Little is known regarding the survival and persistence of these endophytes when in competition with common toxic endophytes. This is mainly because there are currently no assays available to easily and reliably quantify different endophytes in pastures or in batches of seeds infected with multiple strains. We developed real time PCR assays, based on secondary metabolite genes known to differ between N. lolii endophyte strains, to quantify two selected endophytes, AR1 and AR37, and a common toxic ecotype used in New Zealand. A duplex PCR allowed assessment of endophyte:grass DNA ratios with high sensitivity, specificity and precision. Endophyte specific primers/probes could detect contamination of AR37 seeds with other endophytes down to a level of 3–25%. We demonstrated that it is possible to quantify different endophyte strains simultaneously using multiplex PCR. This method has potential applications in management of endophytes in pastures and in fundamental research into this important plant-microbe symbiosis.

Ciborinia camelliae is the causal agent of Camellia flower blight. This fungal pathogen is a significant pest of the Camellia floriculture industry because it specifically infects the floral tissue of ornamental camellia cultivars leading to the rapid development of necrotic lesions and blight. This study aims to characterize natural resistance to Ciborinia camelliae within a selection of Camellia spp. Based on macroscopic lesion development, Camellia ‘Nicky Crisp’ and Camellia lutchuensis were chosen as compatible and incompatible hosts, respectively. Microscopic analyses of the incompatible Camellia lutchuensis–Ciborinia camelliae interaction revealed several hallmarks of induced plant resistance, including papillae formation, H2O2 accumulation, and localized cell death. The compatible Camellia Nicky Crisp–Ciborinia camelliae interaction failed to trigger a similar resistance response. Ciborinia camelliae growth in compatible tissue demonstrated a switch from biotrophy to necrotrophy, evident from the simultaneous development of secondary hyphae and necrotic lesions. Extension of resistance analyses to 39 additional Camellia spp. identified variable levels of resistance within the Camellia genus. The evidence presented supports a resistance breeding strategy for controlling Ciborinia camelliae on ornamental Camellia hybrids.

Summary Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary timescale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D. septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D. septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.

In fungi, genes involved in the production of secondary metabolites are generally clustered at one location. There are some exceptions, such as genes required for synthesis of dothistromin, a toxin that is a chemical analog of the aflatoxin precursor versicolorin A and made by the pine needle pathogen Dothistroma septosporum. The availability of the D. septosporum genome sequence enabled identification of putative dothistromin genes, including an ortholog of the aflatoxin regulatory gene AflR, and revealed that most of the genes are spread over six separate regions (loci) on chromosome 12 (1.3 Mb). Here we show that levels of expression of the widely dispersed genes in D. septosporum are not correlated with gene location with respect to their distance from a telomere, but that AflR regulates them. The production of dothistromin by D. septosporum in which the AflR gene was knocked out (ΔDsAflR) was drastically reduced, but still detectable. This is in contrast to orthologous ΔAflR mutants in Aspergillus species that lack any aflatoxin production. Expression patterns in ΔDsAflR mutants helped to predict the complete set of genes involved in dothistromin production. This included a short-chain aryl alcohol dehydrogenase (NorB), which is located on chromosome 11 rather than chromosome 12, but was 24-fold down regulated in ΔDsAflR. An orthologous set of dothistromin genes, organized in a similar fragmented cluster arrangement to that seen in D. septosporum, was found in the closely related tomato pathogen Cladosporium fulvum even though this species does not produce dothistromin. In C. fulvum, pseudogenization of key biosynthetic genes explains the lack of dothistromin production. The fragmented arrangement of dothistromin genes provides an example of coordinated control of a dispersed set of secondary metabolite genes; it also provides an example where loss of dothistromin production might have allowed adaptation to a new pathogenic lifestyle.

Biological control agents (BCA) are beneficial organisms that are applied to protect plants from pests. Many fungi of the genusTrichodermaare successful BCAs but the underlying mechanisms are not yet fully understood.Trichoderma cf. atroviridestrain LU132 is a remarkably effective BCA compared toT. cf. atroviridestrain LU140 but these strains were found to be highly similar at the DNA sequence level. This unusual combination of phenotypic variability and high DNA sequence similarity between separately isolated strains prompted us to undertake a genome comparison study in order to identify DNA polymorphisms. We further investigated if the polymorphisms had functional effects on the phenotypes. The two strains were clearly identified as individuals, exhibiting different growth rates, conidiation and metabolism. Superior pathogen control demonstrated by LU132 depended on its faster growth, which is a prerequisite for successful distribution and competition. Genome sequencing identified only one non-synonymous single nucleotide polymorphism (SNP) between the strains. Based on this SNP, we successfully designed and validated an RFLP protocol that can be used to differentiate LU132 from LU140 and otherTrichodermastrains. This SNP changed the amino acid sequence of SERF, encoded by the previously undescribed single copy gene “small EDRK-rich factor” (serf). A deletion ofserfin the two strains did not lead to identical phenotypes, suggesting that, in addition to the single functional SNP between the nearly clonalTrichoderma cf. atroviridestrains, other non-genomic factors contribute to their phenotypic variation. This finding is significant as it shows that genomics is an extremely useful but not exhaustive tool for the study of biocontrol complexity and for strain typing.

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress., Author Summary Dothideomycetes is the largest and most ecologically diverse class of fungi that includes many plant pathogens with high economic impact. Currently 18 genome sequences of Dothideomycetes are available, 14 of which are newly described in this paper and in several companion papers, allowing unprecedented resolution in comparative analyses. These 18 organisms have diverse lifestyles and strategies of plant pathogenesis. Three feed on dead organic matter only, six are necrotrophs (killing the host plant cells), one is a biotroph (forming an association with and thus feeding on the living cells of the host plant cells) and 8 are hemibiotrophs (having an initial biotrophic stage, and killing the host plant at a later stage). These various lifestyles are also reflected in the gene sets present in each group. For example, sets of genes involved in carbohydrate degradation and secondary metabolism are expanded in necrotrophs. Many genes involved in pathogenesis are located near repetitive sequences, which are believed to speed up their evolution. Blocks of genes with conserved gene order were identified. In addition to this we deduce that the mechanism for mesosynteny, a type of genome evolution particular to Dothideomycetes, is by intra-chromosomal inversions.