Strawberry Pathotype Research Articles

Genomics Evolutionary History and Diagnostics of the Alternaria alternata Species Group Including Apple and Asian Pear Pathotypes.

The Alternaria section alternaria (Alternaria alternata species group) represents a diverse group of saprotroph, human allergens, and plant pathogens. Alternaria taxonomy has benefited from recent phylogenetic revision but the basis of differentiation between major phylogenetic clades within the group is not yet understood. Furthermore, genomic resources have been limited for the study of host-specific pathotypes. We report near complete genomes of the apple and Asian pear pathotypes as well as draft assemblies for a further 10 isolates representing Alternaria tenuissima and Alternaria arborescens lineages. These assemblies provide the first insights into differentiation of these taxa as well as allowing the description of effector and non-effector profiles of apple and pear conditionally dispensable chromosomes (CDCs). We define the phylogenetic relationship between the isolates sequenced in this study and a further 23 Alternaria spp. based on available genomes. We determine which of these genomes represent MAT1-1-1 or MAT1-2-1 idiomorphs and designate host-specific pathotypes. We show for the first time that the apple pathotype is polyphyletic, present in both the A. arborescens and A. tenuissima lineages. Furthermore, we profile a wider set of 89 isolates for both mating type idiomorphs and toxin gene markers. Mating-type distribution indicated that gene flow has occurred since the formation of A. tenuissima and A. arborescens lineages. We also developed primers designed to AMT14, a gene from the apple pathotype toxin gene cluster with homologs in all tested pathotypes. These primers allow identification and differentiation of apple, pear, and strawberry pathotypes, providing new tools for pathogen diagnostics.

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

Abstract 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.

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.

Complex regulation of secondary metabolism controlling pathogenicity in the phytopathogenic fungus Alternaria alternata

The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes), which produce different host-selective toxins and cause disease on different plants. The Japanese pear, strawberry and tangerine pathotypes produce AK-toxin, AF-toxin and ACT-toxin, respectively, which have a common structural moiety, 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid (EDA). Here, we identified a new gene, AKT7 (AK-toxin biosynthetic gene 7), from the Japanese pear pathotype, which encodes a cytochrome P450 monooxygenase and functions to limit AK-toxin production. AKT7 homologs were found in the strawberry pathotype, but not the tangerine pathotype. However, the strawberry pathotype homolog appeared to include a premature stop codon. Although the Japanese pear pathotype strain has multiple copies of AKT7, a single-copy disruption resulted in mutants with increased production of AK-toxin and EDA. AKT7 overexpression in the three pathotypes caused marked reductions of toxin and EDA production, suggesting that Akt7 catalyzes a side reaction of EDA or its precursor. AKT7 overexpression caused reduced virulence in these pathotypes. We also found that AKT7 transcripts predominantly include misspliced mRNAs, which have premature stop codons. Our observations suggest that the AK-toxin production required for full virulence is regulated in a complex way by the copy number and intron information content of AKT7.

Function of Genes Encoding Acyl-CoA Synthetase and Enoyl-CoA Hydratase for Host-Selective ACT-Toxin Biosynthesis in the Tangerine Pathotype of Alternaria alternata

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease. Sequence analysis of a genomic cosmid clone identified a part of the ACTT gene cluster and implicated two genes, ACTT5 encoding an acyl-CoA synthetase and ACTT6 encoding an enoyl-CoA hydratase, in the biosynthesis of ACT-toxin. Genomic Southern blots demonstrated that both genes were present in tangerine pathotype isolates producing ACT-toxin and also in Japanese pear pathotype isolates producing AK-toxin and strawberry pathotype isolates producing AF-toxin. ACT-, AK-, and AF-toxins from these three pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Targeted gene disruption of two copies of ACTT5 significantly reduced ACT-toxin production and virulence. Targeted gene disruption of two copies of ACTT6 led to complete loss of ACT-toxin production and pathogenicity and a putative decatrienoic acid intermediate in ACT-toxin biosynthesis accumulated in mycelial mats. These results indicate that ACTT5 and ACTT6 are essential genes in ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and both are required for full virulence of this fungus.

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of Alternaria alternata

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity.

Multiple copies of AMT2 are prerequisite for the apple pathotype of Alternaria alternata to produce enough AM-toxin for expressing pathogenicity

The apple pathotype of Alternaria alternata produces the cyclic depsipeptide AM-toxin and causes Alternaria blotch of apple. Previously, we cloned AMT2 from the apple pathotype as an orthologue of AFTS1, which is required for biosynthesis of the decatrienoic acid ester AF-toxin I of the strawberry pathotype. These genes were predicted to encode aldo-keto reductases involved in biosynthesis of a common precursor, 2-hydroxy-isovaleric acid, of AF-toxin I and AM-toxin. In this study, we analyzed the function of AMT2 in AM-toxin biosynthesis in the apple pathotype. DNA gel blot analysis of the apple pathotype strain IFO8984 with five restriction enzymes suggested that this strain has a single copy of AMT2 in the genome. However, gene disruption experiments showed that IFO8984 probably has three copies of AMT2. We made mutants having one or two copies of AMT2 disrupted. The single-copy mutants produced less AM-toxin than did the wild type and were still as pathogenic as the wild type. The two-copy mutants produced trace or undetectable amounts of AM-toxin and were markedly reduced in pathogenicity. Thus, AMT2 was verified to be required for AM-toxin biosynthesis and hence pathogenicity. The fact that the two-copy mutants have a remaining copy of AMT2 suggests that multiple copies of AMT2 are prerequisite for the pathogen to produce enough AM-toxin for full pathogenicity.

DNA transposon fossils present on the conditionally dispensable chromosome controlling AF-toxin biosynthesis and pathogenicity of Alternaria alternata

The strawberry pathotype of Alternaria alternata produces host-specific AF-toxin and causes Alternaria black spot of strawberry. Previously, we isolated cosmid clones pcAFT-1 and pcAFT-2 from strain NAF8 of the strawberry pathotype that contain AF-toxin biosynthetic genes, named AFT genes. In a molecular characterization here of pcAFT-1 and pcAFT-2, 11 AFT genes and five transposon-like sequences, named TLS-S1 to TLS-S5, were detected. The nucleotide sequences of TLS-S1 and TLS-S4 share high homology, and their putative products have similarity to transposases of the hAT family transposons. Thus, TLS-S1 and TLS-S4 were renamed TLS-S1-1 and TLS-S1-2, respectively. Amino acid sequences deduced from TLS-S2, TLS-S3, and TLS-S5 have similarity to transposases of the Fot1/Pogo family transposons, but they are significantly different. All five sequences have incomplete open reading frames (ORFs) for transposases owing to deletions, termination codons, and/or frameshifts, indicating that they are inactivated elements. Analysis of genomic distribution of these sequences revealed that they are specifically distributed on a 1.05-Mb chromosome of NAF8, which has been identified as a conditionally dispensable (CD) chromosome encoding AFT genes. The presence of three, four, and three copies of TLS-S1, TLS-S2, and TLS-S3, respectively, and a single copy of TLS-S5 on the CD chromosome were estimated by DNA gel blot analysis. The remaining copy of TLS-S1 and the three copies of TLS-S2 were isolated and identified to also encode incomplete ORFs. Thus, it appears that all copies of the transposon-like sequences identified are inactivated elements (fossils) unique to the CD chromosome in the genome of the strawberry pathotype.

Structural analysis of cosmid clone pcAFT-2 carrying AFT10-1 encoding an acyl-CoA dehydrogenase involved in AF-toxin production in the strawberry pathotype of Alternaria alternata

The strawberry pathotype of Alternaria alternata produces the host-specific AF-toxin and causes black spot of strawberry. The genes in the toxin gene cluster are currently being identified and characterized. In this study, a genomic cosmid clone, pcAFT-2, was structurally characterized. This cosmid contains AFT homologs, which were found to be involved in AF-toxin biosynthesis. These homologs were designated AFTR-2 and AFT3-2. Four new open reading frames (ORFs) (AFT9-1, AFT10-1, AFT11-1, AFT12-1) and two transposon-like sequences (TLS-S4, TLS-S5) were also identified. These ORFs were shown to encode for polyketide synthase, acyl-CoA dehydrogenase, P450 monooxygenase, and an oxidoreductase, respectively. Transcripts of all the ORFs were detected. DNA gel blot analysis detected homologs of these four ORFs only in the tangerine, strawberry, and Japanese pear pathotypes, which share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety in their toxin structure. Targeting of AFT10-1, which encodes an acyl-CoA dehydrogenase, produced single- and double-copy mutants with highly reduced numbers of lesions on host leaves concomitant with reduced toxin production, confirming its role in pathogenicity. Thus, AFT10-1 exists in multiple copies in the genome of Alternaria alternata; and based on the presence of homologs in the tangerine and Japanese pear pathotypes, it is involved in the formation of the 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety of the toxin molecule.

Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism.

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce different host-specific toxins and cause diseases on different plants. The strawberry pathotype produces host-specific AF-toxin and causes Alternaria black spot of strawberry. This pathotype is also pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype that produces AK-toxin. The strawberry pathotype produces two related molecular species, AF-toxins I and II: toxin I is toxic to both strawberry and pear, and toxin II is toxic only to pear. Previously, we isolated a cosmid clone pcAFT-1 from the strawberry pathotype that contains three genes involved in AF-toxin biosynthesis. Here, we have identified a new gene, designated AFTS1, from pcAFT-1. AFTS1 encodes a protein with similarity to enzymes of the aldo-ketoreductase superfamily. Targeted mutation of AFTS1 diminished the host range of the strawberry pathotype: Delta aftS1 mutants were pathogenic to pear, but not to strawberry, as is the Japanese pear pathotype. These mutants were found to produce AF-toxin II, but not AF-toxin I. These data represent a novel example of how the host range of a plant pathogenic fungus can be restricted by modification of secondary metabolism.

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.

Distribution and Characterization of AKT Homologs in the Tangerine Pathotype of Alternaria alternata

ABSTRACT The tangerine pathotype of Alternaria alternata produces a host-selective toxin (HST), known as ACT-toxin, and causes Alternaria brown spot disease of citrus. The structure of ACT-toxin is closely related to AK- and AF-toxins, which are HSTs produced by the Japanese pear and strawberry pathotypes of A. alternata, respectively. AC-, AK-, and AF-toxins are chemically similar and share a 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Two genes controlling AK-toxin biosynthesis (AKT1 and AKT2) were recently cloned from the Japanese pear pathotype of A. alternata. Portions of these genes were used as heterologous probes in Southern blots, that detected homologs in 13 isolates of A. alternata tangerine pathotype from Minneola tangelo in Florida. Partial sequencing of the homologs in one of these isolates demonstrated high sequence similarity to AKT1 (89.8%) and to AKT2 (90.7%). AKT homologs were not detected in nine isolates of A. alternata from rough lemon, six isolates of nonpathogenic A. alternata, and one isolate of A. citri that causes citrus black rot. The presence of homologs in the Minneola isolates and not in the rough lemon isolates, nonpathogens or black rot isolates, correlates perfectly to pathogenicity on Iyo tangerine and ACT-toxin production. Functionality of the homologs was demonstrated by detection of transcripts using reverse transcription-polymerase chain reaction (RT-PCR) in total RNA of the tangerine pathotype of A. alternata. The high sequence similarity of AKT and AKT homologs in the tangerine patho-type, combined with the structural similarity of AK-toxin and ACT-toxin, may indicate that these homologs are involved in the biosynthesis of the decatrienoic acid moiety of ACT-toxin.

(+)-Catechin Acts as an Infection-Inhibiting Factor in Strawberry Leaf

ABSTRACT An infection-inhibiting factor (IIF) was isolated from strawberry leaves and identified as (+)-catechin. This compound inhibited the formation of infection hyphae from appressoria of Alternaria alternata, but allowed both spore germination and appressorial formation. It is a normal component of strawberry leaves, but further accumulates as the major IIF in response to inoculation with nonpathogenic spores of A. alternata. The accumulation of (+)-catechin on a susceptible host was not induced, however, by inoculation with pathogenic spores of the strawberry pathotype or by inoculation with nonpathogenic spores supplemented with host-specific toxin (AF-toxin I). These results imply that (+)-catechin acts as a protective agent during induced resistance and that AF-toxin I acts as a fungal suppressor of induced resistance.

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.

Temporary Acceleration of Exocytosis of Polysaccharides in Susceptible Strawberry Leaves by AF-toxin I from Alternaria alternata Strawberry Pathotype.

Host responses of AF-toxin I-treated susceptible strawberry leaves associated with accumulation of polysaccharides outside plasmodesmatal plasma membranes were examined using morphometrical analysis. The volume of chloroplasts, microbodies, nuclei, and vacuoles in the toxin-treated susceptible cells and the water-treated ones did not change. On the contrary, the number of exocytotic Golgi vesicles and the volume of cristae, Golgi regions, nucleoli, and rough endoplasmic reticulum in susceptible cells increased within 3hr after toxin exposure, suggesting that the biosynthetic and secretory functions for polysaccharides were activated in susceptible cells at an early stage of toxin exposure. However, both the volume of organelles and suborganelles, and the number of Golgi vesicles then decreased dramatically at 6 or 10hr after toxin exposure. These results suggested that the acceleration of both biosynthesis and secretion of the materials associated with exocytotic phenomena was temporary before necrosis.

Characteristics of Alternaria alternata Strawberry Pathotype Isolated in New Zealand and the Resistance of the 'Akita Berry' Strawberry to the Fungus

Cultural characteristics, morphology, and pathogenicities of ICMP-2487, a New Zea-land isolate of Alternaria alternata strawberry pathotype were compared with 5 Japanese isolates (OH-5, 90-12, MO-1, SN-3, and MA-2). Concurrently, the resistance of a new strawberry cultivar designated as 'Akita Berry', a somaclonal variant to a New Zealand isolate (ICMP-2487) was examined. The following results were obtained.1. No differences between the New Zealand and Japanese isolates were found in the growth and colors of colonies, shapes, sizes, and septa of conidia.2. Pathogenicities of ICMP-2487 to 58 strawberry cultivars and strains, 4 pear culti-vars, 3 cabbage cultivars, 3 tomato cultivars, 3 Welsh onion cultivars, 2 carrot cultivars, and 2 sunflower cultivars were similar to those of Japanese isolates.3. No lesions on 'Akita Berry' were formed by inoculation of ICMP-2487 and Japanese isolates. This cultivar is, therefore, resistant to the isolates of Alternaria alternata straw-berry pathotype occuring in Japan and in New Zealand.

Occurrence of the Strawberry Pathotype ofAlternaria alternatain Italy

Recently in Italy, Alternaria black spot-like symptoms were observed on leaves and petioles of the strawberry cultivars Cesena, Dana, and Miss. The pathogen was identified as Alternaria alternata based on conidial morphology. Isolates of the fungus from Italy were pathogenic to susceptible cultivars of Japanese pear as well as strawberry among differential plants used to determine susceptibility to host-specific Alternaria toxins. Bioassay and high-performance liquid chromatography analysis showed that the fungus released host-specific AF-toxin I during spore germination. These data strongly suggest that the outbreak of Alternaria black spot of strawberry in Italy is caused by the strawberry pathotype of A. alternata.

東北・北海道におけるイチゴ黒斑病菌の分離菌株に対するイチゴ‘盛岡１６号’の体細胞変異株‘Ｍ１６‐ＡＲ１，２および３’の抵抗性

In order to confirm the resistance of 'M16-AR 1, 2 and 3' to isolates of Alternaria alternata strawberry pathotype occurring in Tohoku and Hokkaido, 'M16-AR' lines were inoculated with 8 isolates of the pathogen collected from 8 municipalities in both districts.Conidia of these 8 isolates were morphologically undifferentiable. However, the isolates differed slightly in their cultural characters.As a result of inoculating each of 8 isolates into leaf blades of various test plants, black spot, a typical symptom of this disease was formed only on strawberry cvs. Morioka-16 and Robinson and the Japanese pear cvs. Nijisseiki and Shinsui. Thus, it was confirmed Ithat the host range of the isolates was not different from that of Alternaria alternata strawberry pathotype reported previously in Japan.'M16-AR' lines inoculated with the 8 isolates were found to be resistant. Furthermore, these lines cultured for three years (1989 to 1991) under field conditions were not affected by Alternaria black spot of strawberry. Consequently, it seems that selections of 'M16-AR' lines may be the preferred cultivars to plant instead of 'Morioka-16' for Tohoku and Hokkaido in Japan.

Stimulation of phospholipase A 2 activity in strawberry cells treated with AF-toxin I produced by Alternaria alternata strawberry pathotype

The effect of AF-toxin I from Alternaria alternata strawberry pathotype on phospholipid metabolism in susceptible host cells was examined by using strawberry cultured cells and their protoplasts. When the susceptible and resistant cells were treated with AF-toxin I, phospholipase A 2 (PLA 2) activity in the microsomes of the susceptible cells increased at relatively low concentrations of AF-toxin I, but not in microsomes of resistant cells. Quinacrine, a known inhibitor of phospholipases, significantly suppressed the PLA 2 activity elevated by toxin treatment in a dose-dependent manner. In vitro toxin treatment of the microsomes prepared from susceptible cells also enhanced the PLA 2 activity. In strawberry protoplasts treated with AF-toxin I, the total amount of phospholipids in the microsomes decreased in the susceptible protoplasts but not in the resistant ones. In vitro treatment of isolated microsomes with the toxin also caused a significant decrease in the amount of phospholipids in the fraction from susceptible cells, but not in the resistant ones. These results indicate that PLA 2 activation and the degradation of phospholipids in the microsomes of susceptible host cells may be involved in the specific toxic action of AF-toxin.

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.