DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine.

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.

Genome-wide gene expression dynamics of the fungal pathogen Dothistroma septosporum throughout its infection cycle of the gymnosperm host Pinus radiata.

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.

A genetically homogenous population of Fusarium circinatum causes pitch canker of Pinus radiata in the Basque Country, Spain.

Pitch canker, caused by Fusarium circinatum, is a destructive disease of Pinus species and has recently been shown to represent a substantial threat to natural and commercial forests in northern Spain. The genetic diversity of F. circinatum in the Basque Country of Spain was assessed by characterising 96 isolates based on vegetative compatibility groups (VCGs), mating type assays, polymorphic DNA-markers and amplified fragment length polymorphism (AFLP) analyses. For this purpose, F. circinatum isolates were collected from diseased Pinus radiata as well as from insects associated with this host. Overall, a low level of diversity was detected in the population. The isolates represented only two VCGs and they were all of the same mating type. AFLP analyses revealed three genotypes and polymorphic DNA-markers specific for F. circinatum showed nine genotypes. The most common genotypes represented 97% of all isolates for AFLP analysis and 68% of isolates for the polymorphic DNA-marker sets. Over all, this indicates that pitch canker in the Basque Country of Spain is caused by a clonally propagating population of F. circinatum, typical of a recently introduced pathogen.

Fungal endophytes in seeds and needles of Pinus monticola.

Using a sequence-based approach, we investigated the transmission of diverse fungal endophytes in seed and needles of Pinus monticola, western white pine. We isolated 2003 fungal endophytes from 750 surface-sterilized needles. In contrast, only 16 endophytic isolates were obtained from 800 surface-sterilized seeds. The ITS region was sequenced from a representative selection of these endophytes. Isolates were then assigned to the most closely related taxa in GenBank. Although 95% of the endophytes in needles from mature trees belonged to the Rhytismataceae, 82 unique ITS sequences were obtained from at least 21 genera and 10 different orders of fungi. Significantly, none of the endophytes in seed were rhytismataceous (chi(2) = 180; P < 0.001). Similarly, needles of greenhouse seedlings yielded only non-rhytismataceous isolates, whereas seedlings of the same age that had naturally regenerated near older white pines in roadless areas were colonized by rhytismataceous endophytes almost to the same extent as in mature trees. Only one of 17 rhytismataceous isolates were able to grow on a medium containing only 0.17% nitrogen, whereas 25 of 31 non-rhytismataceous endophytes grew. Rhytismataceous endophytes are dominant in needles of P. monticola, but they appear to be absent in seed, and unlikely colonists of nitrogen-limiting host tissues such as the apoplast.

A community of unknown, endophytic fungi in western white pine.

The endophytic fungi of woody plants may be diverse as often claimed, and likewise, they may be functionally novel as demonstrated in a few studies. However, the endophyte taxa that are most frequently reported tend to belong to fungal groups composed of morphologically similar endophytes and parasites. Thus, it is plausible that endophytes are known (i.e., described) parasites in a latent phase within the host. If this null hypothesis were true, endophytes would represent neither additional fungal diversity distinct from parasite diversity nor a symbiont community likely to be novel ecologically. To be synonymous with parasites of the host, endophytes should at least be most closely related to those same parasites. Here we report that seven distinct parasites of Pinus monticola do not occur as endophytes. The majority of endophytes of P. monticola (90% of 2,019 cultures) belonged to one fungal family, the Rhytismataceae. However, not a single rhytismataceous endophyte was found to be most closely related by sequence homology to the three known rhytismataceous parasites of P. monticola. Similarly, neither endophytic Mycosphaerella nor endophytic Rhizosphaera isolates were most closely related to known parasites of P. monticola. Morphologically, the endophytes of P. monticola can be confounded with the parasites of the same host. However, they are actually most closely related to, but distinct from, parasites of other species of Pinus. If endophytes are generally unknown species, then estimates of 1 million endophytes (i.e., approximately 1 in 14 of all species of life) seem reasonable.

Dothistroma pini, a forest pathogen, contains homologs of aflatoxin biosynthetic pathway genes.

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.