Yasunori Akagi

Horizontal chromosome transfer, a mechanism for the evolution and differentiation of a plant-pathogenic fungus.

ABSTRACT The tomato pathotype of Alternaria alternata produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene, ALT1, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of ALT1 and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five A. alternata tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the A. alternata strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among A. alternata strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.

Taxonomic study of Favolus and Neofavolus gen. nov. segregated from Polyporus (Basidiomycota, Polyporales)

We present a taxonomic study of ‘group Favolus’ and related species in Polyporus. Phylogenetic analyses of nurLSU and ITS regions revealed that the infrageneric ‘group Favolus’ is divided into two main clades. Fungi within the group share laterally stipitate basidiocarps, with non-crustose stipe surfaces, and are distinguishable by the morphology of the pileus surface. One clade is characterized by species with hyaline to brown cutis, composed of hyaline to brown agglutinated generative hyphae. The other clade accommodates species with radially striate pileus, and lacks any distinct cutis of agglutinated hyphae. We propose Neofavolus gen. nov., typified by N. alveolaris, for the former clade, and revise the genus Favolus, typified by F. brasiliensis, for the latter clade. Neofavolus includes N. mikawai and N. cremeoalbidus sp. nov., known only from temperate eastern Asia, in addition to N. alveolaris. Favolus includes members of the Polyporus grammocephalus complex, the P. tenuiculus complex, and P. pseudobetulinus. We reveal that the polypore known as ‘P. grammocephalus’ in Asia includes F. acervatus and F. emerici (= P. grammocephalus), whereas ‘P. tenuiculus’ includes three distinct species; F. brasiliensis from tropical America, and F. spathulatus and F. roseus from tropical Asia. Detailed descriptions and illustrations are provided for the accepted species in Favolus and Neofavolus.

Functional analysis of the melanin biosynthesis genes ALM1 and BRM2-1 in the tomato pathotype of Alternaria alternata

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.

Detection of fungi producing infection-inhibiting metabolites against Alternaria alternata Japanese pear pathotype from fungi inhabiting internal tissues of Japanese pear shoots

Fungi inhabiting Japanese pear were isolated from internal tissues of cv. Nijisseiki, and culture filtrates (CFs) of 100 isolates were evaluated for their inhibitory activity against infection by Alternaria alternata Japanese pear pathotype. CFs of 11 isolates inhibited lesion formation on the pear by the pathogen. Among these isolates, CFs of five isolates inhibited spore germination. CFs of the six other isolates inhibited appressorial formation, infection hypha formation, AK-toxin production, or a combination of these actions. Analysis of sequence homology in the rDNA ITS1 regions of these isolates showed that most isolates had high homology with some fungal endophytes.

Characterization of Trichoderma species isolated in Ecuador and their antagonistic activities against phytopathogenic fungi from Ecuador and Japan

Native Trichoderma spp. were isolated from agricultural fields in several regions of Ecuador. These isolates were characterized via morphological observation as well as molecular phylogenetic analysis based on DNA sequences of the rDNA internal transcribed spacer region, elongation factor-1α gene and RNA polymerase subunit II gene. Fifteen native Trichoderma spp. were identified as T. harzianum, T. asperellum, T. virens and T. reesei. Some of these strains showed strong antagonistic activities against several important pathogens in Ecuador, such as Fusarium oxysporum f. sp. cubense (Panama disease) and Mycosphaerella fijiensis (black Sigatoka) on banana, as well as Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease) on cacao. The isolates also showed inhibitory effects on in vitro colony growth tests against Japanese isolates of Fusarium oxysporum f. sp. lycopersici, Alternaria alternata and Rosellinia necatrix. The native Trichoderma strains characterized here are potential biocontrol agents against important pathogens of banana and cacao in Ecuador.

The global regulator LaeA controls biosynthesis of host-specific toxins, pathogenicity and development of Alternaria alternata pathotypes

The global regulator LaeA is required for the expression of biosynthetic genes for secondary metabolites in filamentous fungi such as Aspergillus nidulans. To learn more about the regulation of these genes in Alternaria alternata, which has at least seven pathogenic variants (pathotypes) that produce host-specific toxins (HSTs) and cause severe diseases in their host plants, we identified LaeA homologs encoding methyltransferase in tomato, strawberry and apple pathotypes of A. alternata, designating them AtLAE1, AsLAE1 and AaLAE1, respectively. In the AtLAE1-deleted mutant of the tomato pathotype (∆AtLAE1), expression of the AAL-toxin biosynthetic gene ALT1 was lower than that in the wild type. Correspondingly, AAL-toxin production and virulence of the mutant were significantly lower as were spore production and hyphal growth. Similarly, the AsLAE1-deleted mutant of the strawberry pathotype (∆AsLAE1) and the AaLAE1-deleted mutant of the apple pathotype (∆AaLAE1) produced less of their host-specific toxin (AF- and AM-toxin, respectively), had reduced virulence on the host plant, and hyphal growth and sporulation were defective. Thus, the global regulator gene LaeA positively regulates HST biosynthesis, pathogenicity, growth and differentiation in A. alternata pathotypes.

Functional Analysis of the Ceramide Synthase Gene ALT7, A Homologof the Disease Resistance Gene Asc1, in the Plant Pathogen Alternariaalternata

The tomato pathotype of Alternaria alternata produces a host-specific AAL-toxin and causes Alternaria stem canker on susceptible tomato cultivars. AAL-toxin is a sphinganine-analog mycotoxin which induces apoptotic cell death in tomato cells and mammalian cells by inhibiting ceramide biosynthesis. Insensitivity to the AAL-toxin in resistant tomatoes and other plants is conferred by the Asc1 gene, a homolog of the yeast ceramide synthase gene Lag1 . The ALT7 gene, a putative acyl-CoA-dependent ceramide synthase, was found to be located in the AAL-toxin biosynthetic ( ALT ) gene cluster of the tomato pathotype of A. alternata . ALT7 and Asc1 have the TLC (TRAM/Lag1/ CLN8) domain characteristic of proteins involved in ceramide biosynthesis and are members of the LASS/Lag family. To test the hypothesis that ALT7 and Asc1 , both of which are Lag1 ceramide synthase gene homologs, might share a common biological function as toxin tolerance genes, we have cloned and characterized ALT7. ALT7 -deleted mutants were generated to investigate the effects of the deletion on vegetative growth, sporulation, toxin-sensitivity, toxin-production and pathogenicity. The deletion of ALT7 has no deleterious effect on the toxin-producing pathogen, indicating that the gene does not act as a resistance/self-tolerance factor against the toxin in the toxin biosynthetic gene cluster.

Functional characterization of putative G protein-coupled receptors in the tomato pathotype of Alternaria alternata

G protein-coupled receptors (GPCRs) are a large transmembrane receptor superfamily that is involved in many cellular signaling pathways. In the present study, GPCR-family genes from the toxigenic and necrotrophic plant pathogen Alternaria alternata were cloned and characterized. Three GPCR-encoding genes, AaGPR1, AaGPR2, and AaGPR3 were identified in the draft genomic data of the A. alternata tomato pathotype, which produces the host-specific AAL-toxin. AaGPR1, AaGPR2, and AaGPR3 each encodes a protein containing a seven transmembrane domain that is characteristic of GPCRs. Targeted deletion of AaGPR1, AaGPR2, or AaGPR3 in the A. alternata tomato pathotype was conducted to understand the influence of G-protein signaling mechanisms on developmental processes and virulence of this pathogen. No changes in colony morphology or AAL-toxin production were observed for the deletion strain ΔAaGPR1, 2, and 3, compared with the wild-type strain. However, one deletion strain, ΔAaGPR3, exhibited aberrant conidial morphology including decreased conidial length and beak formation. The ability to induce the formation of necrotic lesions on susceptible leaves also significantly decreased in ΔAaGPR3, indicating a reduction in virulence. These defects are similar to the phenotypes found for the Gα gene (AGA1) mutant of A. alternata. These results indicate that the G-protein signal transduction pathway appears to be involved in conidial development and virulence of A. alternata.

Involvement of ThSNF1 in the development and virulence of biocontrol agent Trichoderma harzianum

Trichoderma harzianum, a biocontrol agent for various plant pathogens, is known to degrade fungal cell walls; this mycoparasitism is believed to require secretion of cell-wall-degrading enzymes against host pathogens. In this study, we identified a homologue of yeast SNF1 (sucrose nonfermenting 1) encoding protein kinase in T. harzianum (ThSNF1) by draft genome sequencing of strain T36. Targeted gene disruption of ThSNF1 was performed using the PEG method with fusion PCR products. Growth of mutant ΔThSNF1 was markedly less than for the wild-type strain on minimal medium with chitin as a carbon source. The mutant exhibited reduced expression of the genes encoding chitinase and polygalacturonase and markedly reduced spore production. Mycoparasitism against plant pathogens such as Fusarium oxysporum f. sp. cubense (Panama disease) and Fusarium graminearum (Fusarium head blight) was clearly impaired in the mutant. The results suggest that ThSNF1 is critical for asexual development, utilization of certain carbon sources and virulence on fungi, and is therefore important for the biocontrol ability of T. harzianum.

4 Fungal Toxins of Agricultural Importance

Fungi produce a diverse array of toxins that contribute both positively and negatively to agriculture. We describe phytotoxins which have defined roles in plant disease, as well as mycotoxins which generally have no direct role in disease but have significant impacts on animals that feed on infected host plants. We have included examples of host-specific phytotoxins which act upon a very narrow range of susceptible host genotypes, and non-specific phytotoxins which can affect a wide range of plants. We also describe fungal secondary metabolites that underpin certain mutualistic symbioses. Toxins are described based on their core biosynthesis, and include agriculturally important examples of non-ribosomally synthesised peptides, polyketide-based peptides, proteinaceous phytotoxins, sesquiterpenoid-containing compounds and miscellaneaous toxins of herbivores. The role of each toxin in the plant–fungus interaction, as well as the biochemistry and molecular basis of toxin biosynthesis, will be described.