A conditionally dispensable chromosome controls host-specific pathogenicity in the fungal plant pathogen Alternaria alternata

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce host-specific toxins and cause diseases on different plants. Previously, the gene cluster involved in host-specific AK-toxin biosynthesis of the Japanese pear pathotype was isolated, and four genes, named AKT genes, were identified. The AKT homologs were also found in the strawberry and tangerine pathotypes, which produce AF-toxin and ACT-toxin, respectively. This result is consistent with the fact that the toxins of these pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid structural moiety. In this study, three of the AKT homologs (AFT1-1, AFTR-1, and AFT3-1) were isolated on a single cosmid clone from strain NAF8 of the strawberry pathotype. In NAF8, all of the AKT homologs were present in multiple copies on a 1.05-Mb chromosome. Transformation-mediated targeting of AFT1-1 and AFT3-1 in NAF8 produced AF-toxin-minus, nonpathogenic mutants. All of the mutants lacked the 1.05-Mb chromosome encoding the AFT genes. This chromosome was not essential for saprophytic growth of this pathogen. Thus, we propose that a conditionally dispensable chromosome controls host-specific pathogenicity of this pathogen.     

Protective Effect of Homologues of Host-Specific AF-Toxin I Produced by Alternaria alternata Strawberry Pathotype on Strawberry Cells

The protective effect of homologues of host-specific AF-toxin I from Alternaria alternata strawberry pathotype on susceptible strawberry cells was quantitatively examined by using the cultured cells. Among three AF-toxin molecules, AF-toxin II did not exhibit toxicity to strawberry cells, although AF-toxin I and III were highly toxic to the cells. AF-toxin II protected strawberry cells from AF-toxin I action. The protection was remarkable when the cultured cells were exposed to excess amounts of AF-toxin II both prior to and simultaneously with AF-toxin I addition. The simultaneous treatment, however, was most effective at preventing AF-toxin I action: it gave complete protection at a 50 : 1 (toxin II : toxin I) ratio on a molar basis. The epoxy-decatrienoic acid moiety of the AF-toxins also prevented the cell death caused by AF-toxin I, but was less effective than AF-toxin II. Protection was apparent when the cultured cells were exposed to AF-toxin II for 3 h, rinsed to remove free AF-toxin II and then exposed to AF-toxin I. These results suggest that the toxoids of AF-toxin I, such as AF-toxin II and the decatrienoic acid, act as competitive inhibitors of AF-toxin I.     

Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata.

The Japanese pear pathotype of Alternaria alternata causes black spot of Japanese pear by producing a host-specific toxin known as AK-toxin. Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for toxin biosynthesis. Protoplasts of a wild-type strain were treated with a linearized plasmid along with the restriction enzyme used to linearize the plasmid. Of 984 REMI transformants recovered, three produced no detectable AK-toxin and lost pathogenicity on pear leaves. Genomic DNA flanking the integrated plasmid was recovered from one of the mutants. With the recovered DNA used as a probe, a cosmid clone of the wild-type strain was isolated. Structural and functional analyses of an 8.0-kb region corresponding to the tagged site indicated the presence of two genes. One, designated AKT1, encodes a member of the class of carboxyl-activating enzymes. The other, AKT2, encodes a protein of unknown function. The essential roles of these two genes in both AK-toxin production and pathogenicity were confirmed by transformation-mediated gene disruption experiments. DNA gel blot analysis detected AKT1 and AKT2 homologues not only in the Japanese pear pathotype strains but also in strains from the tangerine and strawberry pathotypes. The host-specific toxins of these two pathotypes are similar in structure to AK-toxin. Homologues were not detected in other pathotypes or in non-pathogenic strains of A. alternata, suggesting acquisition of AKT1 and AKT2 by horizontal transfer.     

Morphological and molecular characterization of the strawberry black leaf spot pathogen referred to as the strawberry pathotype of Alternaria alternata

The remaining unclarified taxon among the seven known pathotypes of host-selective toxin (HST)-producing Alternaria alternata, namely, the strawberry pathotype (the strawberry black leaf spot pathogen), is taxonomically revised and re-described herein. According to our morphological observations, reference isolates of strawberry and Japanese pear pathotypes, which are toxic to leaves of Japanese pear ‘Nijisseiki’, have conidia that are formed in chains of 3–13, usually without lateral branches, after 7?d incubation on potato-carrot agar. The mean size of the conidia is 27–31?×?11–13?μm. Morphological characteristics of the examined isolates are identical to those of A. gaisen rather than A. alternata. A phylogenetic tree obtained by analysis of a combined dataset of ITS, gapdh, rpb2, tef1, Alt a 1, and endoPG sequences also strongly supports both pathotypes as one species, A. gaisen. We re-describe the fungus as A. gaisen Nagano ex Bokura and propose two formae speciales of the species, A. gaisen f. sp. fragariae producing AF-toxin and f. sp. pyri producing AK-toxin. The epitype specimen and ex-epitype culture of A. gaisen are newly designated.     

Utilization of Bacterial Xanthine Oxidase for Inorganic Phosphorus Determination

Two host-specific phytotoxic metabolites, AK-toxin I and II, were isolated from a culture broth of Alternaria alternata Japanese pear pathotype, the fungus causing black spot disease of susceptible Japanese pear cultivars. From chemical, spectral and X-ray crystallographic data, AK-toxin I was characterized as 8-(2′S, 3′S)-2′-acetylamino-3′-methyl-3′-phenyl-propionyloxy]-(8R,9S)-9,10-epoxy-9-methyl-deca-(2E,4Z,6E)-trienoic acid. The structure of AK-toxin II was also assigned to be 3′-demethyl derivative of AK-toxin I by comparing the spectral data with those of AK-toxin I.     

Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range

Plant pathogenic fungi adapt quickly to changing environments including overcoming plant disease resistance genes. This is usually achieved by mutations in single effector genes of the pathogens, enabling them to avoid recognition by the host plant. In addition, horizontal gene transfer (HGT) and horizontal chromosome transfer (HCT) provide a means for pathogens to broaden their host range. Recently, several reports have appeared in the literature on HGT, HCT and hybridization between plant pathogenic fungi that affect their host range, including species of Stagonospora/Pyrenophora, Fusarium and Alternaria. Evidence is given that HGT of the ToxA gene from Stagonospora nodorum to Pyrenophora tritici-repentis enabled the latter fungus to cause a serious disease in wheat. A nonpathogenic Fusarium species can become pathogenic on tomato by HCT of a pathogenicity chromosome from Fusarium oxysporum f.sp lycopersici, a well-known pathogen of tomato. Similarly, Alternaria species can broaden their host range by HCT of a single chromosome carrying a cluster of genes encoding host-specific toxins that enabled them to become pathogenic on new hosts such as apple, Japanese pear, strawberry and tomato, respectively. The mechanisms HGT and HCT and their impact on potential emergence of fungal plant pathogens adapted to new host plants will be discussed.     

Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria

Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD⁺ in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon.     

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific,toxin-producing fungus Alternaria alternata Japanese pear pathotype

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant–pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.     

Cloning and characterization of a cyclic peptide synthetase gene from Alternaria alternata apple pathotype whose product is involved in AM-toxin synthesis and pathogenicity

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.     

ACTG-Toxins Produced by a Strain of Alternaria altemata That Does Not Originate From Citrus Species

AGTG-toxins designated as host-specific toxins were isolated at first from a pathotype of Alternaria alternata attacking Citrus species. A strain of Alternaria alternata isolated from Brassica sinensis in Central Germany and improved to a high tentoxin content, also produces two ACTG-toxins. It is doubtful, therefore, if both substances can be denoted as host-specific toxins.     

Plant pathotoxins from Alternaria citri: The minor ACRL toxins

Five host-specific pathotoxins, ACRL toxins II, III, III′, IV and IV′, were isolated from the culture broth of Alternaria citri, the fungus causing brown spot disease of rough lemon. These toxins are related structurally to the major ACRL toxin, toxin I, and to its derivative compound A. Chemical and spectral studies indicated that the ACRL minor toxins were a group of analogous compounds of different chain lengths all of which have a α-pyrone group, in contrast to the dihydro-α-pyrone group in toxin I. Toxin II showed a very low biological activity (ED₅₀ greater than 10 μg/ml) whereas the other minor toxins had slightly higher activities ranging from 1 to 10 μg/ml. The dihydropyrone group in ACRL toxin I was correlated with high biological activity (ED₅₀ = 18–30 ng/ml).     

Durable and broad-spectrum disease protection measure against airborne phytopathogenic fungi by using the detachment action of gelatinolytic bacteria

We had previously obtained collagenolytic/gelatinolytic bacteria, which degrade the fungal extracellular matrix, to establish a novel biological control measure that inhibits germling adhesion of airborne phytopathogenic fungi on the host plant surface. By using barley-Magnaporthe oryzae pathosystem, Chryseobacterium sp. was most effective biocontrol agents as tested. The selected bacteria were evaluated for durable disease protection against M. oryzae on barley leaves by using chloramphenicol-resistant mutants. Chryseobacterium sp. from the soil was less likely to settle on leaf surfaces. Therefore, we tried to manipulate Chryseobacterium sp. to inhabit the leaf’s surface. The gelatin supplementation dramatically improved the settlement of gelatinolytic bacteria Chryseobacterium sp. from the soil, and the disease protection effect lasted for more than 2 weeks on barley. Moreover, exploitation of Chryseobacterium sp. for disease protection was extended against other airborne pathogens, Alternaria alternata Japanese pear pathotype on Japanese pear and Colletotrichum orbiculare on cucumber.     

A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.     

Production of a Host-Specific Toxin by Germinating Spores of Alternaria tenuissima Causing Leaf Spot of Pigeon Pea

Production of a host-specific toxin by Alternaria tenuissima , the cause of pigeon pea leaf spot, was investigated in spore-germination fluids (SGF). The SGF selectively induced necrosis on pigeon pea leaves in a deteched leaf assay. Necrotic lesions were observed when a toxin from SGF was applied onto detached young leaves of the pigeon pea cultivar Bahar at concentration as low as 5 ng/ml. The resistant line Tanzania and nonhosts tolerated at least 20,000 times higher concentration of the toxin. The differential activity of the toxin on hosts and nonhosts of the fungus, as well as on susceptible and resistant cultivars or lines, suggested host-specific property of the toxin. At a concentration of 10 ng/ml, the toxin induced susceptibility of pigeon pea leaves to a non-pathogenic isolate of Alternaria alternata. The toxin possibly plays a role as a disease determinant of A. tenuissima , because the toxin was released from germinating spores as early as 3 h of incubation andthe, amount detected within 9 h was about 6 times of the concentration required for necrotic toxicity.     

Plant pathotoxins from Alternaria citri: The major toxin specific for rough lemon plants

A pathotype of the fungus Alternaria citri that attacks rough lemon plants produces several toxins in culture which specifically damage rough lemon and Rangpur lime plants. The major toxin produced, Toxin I, was by far the most potent compound (ED₅₀ = 30 ng/ml). Five other minor toxins were active at ED₅₀ levels greater than 1 μg/ml. On the basis of mass, ¹H and ¹³C NMR spectra and decoupling studies of Toxin I and derivative, Toxin I is a 19 carbon polyalcohol with an α-dihydropyrone ring. The γ-dihydropyrone tautomer was less predominant. Culture filtrates of A. citri also contained a biologically inactive, partially analogous, component possessing a tetrahydropyran ring. It probably arises from decarboxylation of Toxin I. Toxin I was highly specific and did not affect nonhost plants at 10 000 times the concentrations affecting rough lemon.     

Genetic mapping of genes for susceptibility to black spot disease in Japanese pears.

Black spot disease, which is caused by the Japanese pear pathotype of Alternaria alternata (Fr.) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We identified the exact positions and linkage groups (LGs) of the genes for susceptibility to black spot in the Japanese pear (Pyrus pyrifolia Nakai) cultivars 'Osa Nijisseiki' (gene Ani) and 'Nansui' (gene Ana). Segregation of susceptibility and resistance fitted the expected ratio of 1:1 in progeny of 'Nansui' but showed a slight distortion in progeny of 'Osa Nijisseiki'. We mapped the genes for susceptibility to black spot in both populations using a genome scanning approach. The simple sequence repeat (SSR) markers CH04h02 and CH03d02 showed tight linkage to Ani and Ana. Although Ani and Ana are derived from different sources, both genes are located at the top region of LG 11. Information about the positions of the susceptibility genes and the molecular markers linked to them will be useful for marker-assisted selection in pear breeding programs.     

Binding affinity of the methyl ester of AK-toxin I to membrane fractions from Japanese pear leaves

The binding site of AK-toxin, a host-specific toxin against Japanese pear, was searched for in the membrane fractions of the pear leaves, using 3H-labeled AK-toxin I methyl ester. Binding activity, which was displaceable by the unlabeled ligand, was observed for microsomal fraction from a toxin-susceptible cultivar, Nijisseiki. However, the binding was also observed for those from toxin-resistant cultivars, Kosui and Hosui. Detection of the specific binding failed for the plasma membrane fraction which was prepared from microsomal fraction of the toxin-susceptible cultivar by aqueous two-phase separation, and the hitherto presumed model of the AK-toxin receptor in the plasma membrane could not be verified.     

Circular DNA Plasmid in the Phytopathogenic Fungus Alternaria Alternata: Its Temperature-Dependent Curing and Association with Pathogenicity

We found the presence of plasmid DNA in strain T88-56 of the Japanese pear pathotype of Alternaria alternata, which causes black spot of certain cultivars of Japanese pear by producing host-specific AK-toxin. The plasmid, designated pAAT56, was identified to be an ~5.4-kilobase (kb) circular molecule by electron microscopic observation and restriction endonuclease mapping. Southern blot analysis showed that pAAT56 DNA had no homology with either nuclear or mitochondrial DNA. Cultures of strain T88-56 grown at 26° showed markedly reduced plasmid levels relative to those grown at lower temperatures. The strain was completely cured of pAAT56 during growth at 29°. Temperature-dependent curing of pAAT56 was confirmed by using single-protoplast isolates from mycelia grown at 23°, most of which maintained the plasmid, and from mycelia grown at 29°, most of which had lost the plasmid. Northern blot analysis detected the presence of three RNA species (~1.7, 2.7 and 5.4 kb) transcribed from pAAT56. The biological function of pAAT56 was observed using single-protoplast isolates from mycelia that either contained or had been cured of pAAT56. The plasmid-containing isolates tended to be reduced in AK-toxin production and pathogenicity compared with the plasmid-cured isolates.