Alternaria Alternata Apple Pathotype Research Articles

Molecular Mechanism of Resistance to Alternaria alternata Apple Pathotype in Apple by Alternative Splicing of Transcription Factor MdMYB6-like.

As a fruit tree with great economic value, apple is widely cultivated in China. However, apple leaf spot disease causes significant damage to apple quality and economic value. In our study, we found that MdMYB6-like is a transcription factor without auto-activation activity and with three alternative spliced variants. Among them, MdMYB6-like-β responded positively to the pathogen infection. Overexpression of MdMYB6-like-β increased the lignin content of leaves and improved the pathogenic resistance of apple flesh callus. In addition, all three alternative spliced variants of MdMYB6-like could bind to the promoter of MdBGLU H. Therefore, we believe that MdMYB6-like plays an important role in the infection process of the pathogen and lays a solid foundation for breeding disease-resistant cultivars of apple in the future.

Comparative transcriptome analysis reveals key genes and pathways in response to Alternaria alternata apple pathotype infection

Apple leaf spot, caused by the Alternaria alternata apple pathotype (AAAP), is an important fungal disease of apple. To understand the molecular basis of resistance and pathogenesis in apple leaf spot, the transcriptomes of two apple cultivars ‘Hanfu’ (HF) (resistant) and ‘Golden Delicious’(GD) (susceptible) were analyzed at 0, 6, 18, 24 and 48 h after AAAP inoculation by RNA-Seq. At each time point, a large number of significantly differentially expressed genes (DEGs) were screened between AAAP-inoculated and uninoculated apple leaves. Analysis of the common DEGs at four time points revealed significant differences in the resistance of ‘HF’ and ‘GD’ apple to AAAP infection. RLP, RNL, and JA signal-related genes were upregulated in both cultivars to restrict AAAP development. However, genes encoding CNLs, TNLs, WRKYs, and AP2s were only activated in ‘HF’ as part of the resistance response, of which, some play major roles in the regulation of ET and SA signal transduction. Further analysis showed that many DEGs with opposite expression trends in the two hosts may play important regulatory roles in response to AAAP infection. Transient expression of one such gene MdERF110 in ‘GD’ apple leaves improved AAAP resistance. Collectively, this study highlights the reasons for differential resistance to AAAP infection between ‘HF’ and ‘GD’ apples which can theoretically assist the molecular breeding of disease-resistant apple crops.

Insertion of a TRIM-like sequence in MdFLS2-1 promoter is associated with its allele-specific expression in response to Alternaria alternata in apple.

Alternaria blotch disease, caused by Alternaria alternata apple pathotype (AAAP), is one of the major fungal diseases in apple. Early field observations revealed, the anther-derived homozygote Hanfu line (HFTH1) was highly susceptible to AAAP, whereas Hanfu (HF) exhibited resistance to AAAP. To understand the molecular mechanisms underlying the difference in sensitivity of HF and HFTH1 to AAAP, we performed allele-specific expression (ASE) analysis and comparative transcriptomic analysis before and after AAAP inoculation. We reported an important immune gene, namely, MdFLS2, which displayed strong ASE in HF with much lower expression levels of HFTH1-derived alleles. Transient overexpression of the dominant allele of MdFLS2-1 from HF in GL-3 apple leaves could enhance resistance to AAAP and induce expression of genes related to salicylic acid pathway. In addition, MdFLS2-1 was identified with an insertion of an 85-bp terminal-repeat retrotransposon in miniature (TRIM) element-like sequence in the upstream region of the nonreference allele. In contrast, only one terminal direct repeat (TDR) from TRIM-like sequence was present in the upstream region of the HFTH1-derived allele MdFLS2-2. Furthermore, the results of luciferase and β-glucuronidase reporter assays demonstrated that the intact TRIM-like sequence has enhancer activity. This suggested that insertion of the TRIM-like sequence regulates the expression level of the allele of MdFLS2, in turn, affecting the sensitivity of HF and HFTH1 to AAAP.

Comparative Analysis of Alternative Splicing in Two Contrasting Apple Cultivars Defense against Alternaria alternata Apple Pathotype Infection.

Alternaria blotch disease, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of the most serious fungal diseases in apples. Alternative splicing (AS), one of the pivotal post-transcriptional regulatory mechanisms, plays essential roles in various disease resistance responses. Here, we performed RNA-Seq for two apple cultivars (resistant cultivar 'Jonathan' (J) and susceptible cultivar 'Starking Delicious' (SD)) infected by A. alternata AP to further investigate their AS divergence. In total, 1454, 1780, 1367 and 1698 specifically regulated differential alternative splicing (DAS) events were detected in J36, J72, SD36 and SD72 groups, respectively. Retained intron (RI) was the dominant AS pattern. Conformably, 642, 764, 585 and 742 uniquely regulated differentially spliced genes (DSGs) were found during A. alternata AP infection. Comparative analysis of AS genes in differential splicing and expression levels suggested that only a small proportion of DSGs overlapped with differentially expressed genes (DEGs). Gene ontology (GO) enrichment analysis demonstrated that the DSGs were significantly enriched at multiple levels of gene expression regulation. Briefly, the specific AS was triggered in apple defense against A. alternata AP. Therefore, this study facilitates our understanding on the roles of AS regulation in response to A. alternata AP infection in apples.

Comparative transcriptomics analysis reveals MdGRAS53 contributes to disease resistance against Alternaria blotch of apple

Alternaria blotch disease, caused by Alternaria alternata apple pathotype (AAAP), is one of the most prevalent diseases in apple production. To identify AAAP resistance-related genes and provide a theoretical basis for Alternaria blotch disease resistance breeding, we used two apple cultivars, ‘Jonathan’, a variety resistant to AAAP infection, and ‘Starking Delicious’, a variety susceptible to AAAP infection, as materials to perform transcriptome sequencing of apple leaves 72 h after AAAP infection. A Venn diagram showed that a total of 5229 DEGs of ‘Jonathan’ and 4326 DEGs of ‘Starking Delicious’ were identified. GO analysis showed that these DEGs were clustered into 25 GO terms, primarily “metabolic process” and “catalytic activity.” Functional classification analyses of the DEGs indicated that “MAPK signaling pathway-plant pathway” is the most significant metabolic pathway among the top 15 KEGG pathways, followed by the “plant hormone signal transduction” pathway. There are more DEGs in ‘Jonathan’ that are significantly classified GO terms and KEGG pathways than in ‘Starking Delicious’. Specifically, 13 DEGs were identified as involved in the GA-GID1-DELLA module, and the expression of MdGRAS53, a homologous gene of DELLA, was significantly upregulated in ‘Jonathan’ compared with ‘Starking Delicious’. Phenotype analysis revealed that exogenous hormone GA3 suppressed apple resistance to AAAP infection and reduced the expression of MdGRAS53. The opposite result was observed for exogenous spraying of paclobutrazol (PAC), an inhibitor of gibberellin synthesis. Overexpression of MdGRAS53 in apple leaves by transient transformation decreased lesion area and the number of spores in leaves infected with AAAP, while silencing MdGRAS53 showed the opposite result. Meanwhile, SA/JA signaling pathway-related genes were upregulated significantly in MdGRAS53-overexpressed leaves and downregulated significantly in MdGRAS53-silenced leaves. The findings suggest that the GA-GID1-DELLA module is involved in apple resistance to AAAP, and MdGRAS53, a DELLA homologous gene, may play a positive role in this resistance by modulating cooperative JA- and SA-dependent pathways.

MdWRKY75e enhances resistance to Alternaria alternata in Malus domestica

The Alternaria alternata apple pathotype adversely affects apple (Malus domestica Borkh.) cultivation. However, the molecular mechanisms underlying enhanced resistance to this pathogen in apple remain poorly understood. We have previously reported that MdWRKY75 expression is upregulated by A. alternata infection in ‘Sushuai’ apples. In this study, we discovered that overexpression of MdWRKY75e increased the resistance of transgenic apple lines to A. alternata infection, whereas silencing this gene enhanced susceptibility to A. alternata infection. Furthermore, we found that MdWRKY75e directly binds to the MdLAC7 promoter to regulate the biosynthesis of laccase and increase the biosynthesis of lignin during A. alternata infection. Moreover, the thickening of the cell wall enhanced the mechanical defense capabilities of apple. In addition, we found that jasmonic acid remarkably induced MdWRKY75e expression, and its levels in transgenic apple lines were elevated. These results indicate that MdWRKY75e confers resistance to the A. alternata apple pathotype mainly via the jasmonic acid pathway and that pathogenesis-related genes and antioxidant-related enzyme activity are involved in the disease resistance of MdWRKY75e transgenic plants. In conclusion, our findings provide insights into the importance of MdWRKY75e for resistance to A. alternata infection in apples.

Identification of microRNA transcriptome in apple response to Alternaria alternata infection and evidence that miR390 is negative regulator of defense response

MicroRNAs (miRNAs) play an important regulatory role in plant growth and development, as well as in response to multiple stresses. In recent years, multiple studies have shown that miRNAs participate in regulating diverse aspects of the plant-pathogen interactions, but little is known about the potential existence and functional roles of pathogen-responsive miRNAs in apple (Malus × domestica Borkh.). Here, we identified pathogen-responsive miRNAs in apple leaves challenged by Alternaria alternata apple pathotype (AAAP). These AAAP-responsive miRNAs were classified into 41 families and predicted to target 673 genes, including multiple R genes and transcription factors, such as LRR, RPK2, LACCASE, and MYB, among others. A follow-up analysis revealed that some of the AAAP-responsive miRNAs and their predicted targets exhibited differential expression patterns in the disease-susceptible vs. disease-resistant apple cultivars, i.e. ‘Starking Delicious’ vs. ‘Jonathon’, indicating that these miRNAs are involved in the regulation of apple resistance to AAAP infection. Then, we focused specifically on a potential negative regulatory impact of mdm-miR390a on disease resistance and predicted the impact may result from targeting and inhibiting the expression of the MdRPK2 and MdLRR8 genes, which was confirmed experimentally in both Agrobacterium co-infiltration assays in tobacco (Nicotiana benthamiana) and apple (Malus × domestica ‘Gala’). The results showed that overexpression of mdm-miR390a enhanced the sensitivity to AAAP infection, and overexpression of MdLRR8 or MdRPK2 improved the resistance to AAAP while co-expression with mdm-miR390a counteracted the resistance. Above all, beyond presenting a large data resource for studies of miRNA species and miRNA-based regulation mechanisms in tree fruit, our study uncovered a fascinating case wherein a fungus-induced plant-genome-encoded miRNA apparently increases the susceptibility of the plant host to fungal infection via a post-transcriptional R-gene-knockdown mechanism.

Cloning, sequencing, and expression analysis of 32 NAC transcription factors (MdNAC) in apple.

BackgroundNAC transcription factors play important roles in the regulation of plant growth, development, abiotic and biotic stress responses. The transcriptional level of MdNACs in different tissues and under various biotic and abiotic stress treatments was determined to provide a solid foundation for studying the function of MdNACs.MethodsThirty-two full-length cDNA sequences of Md NACs were isolated by homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. The prediction of subcellular locations of MdNAC proteins was performed using CELLO v.2.5, PSORT, and SoftBerry ProtComp 9.0. Expression levels of MdNACs were detected in 16 different tissues using an array. Expression patterns of MdNACs were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq, and the expression of MdNACs was analyzed under NaCl and mannitol treatments using RT-qPCR.ResultsThe sequencing results produced 32 cDNAs (designated as MdNAC24-39, MdNAC54-65, and MdNAC67-70 with GenBank accession No. MG099861–MG099876, MG099891–MG099902, and MG099904–MG099907, respectively). Phylogenetic analysis revealed that MdNAC34 belonged to the ATAF group, MdNAC63 belonged to the AtNAC3 group, MdNAC24, MdNAC26-30, MdNAC32-33, MdNAC35, MdNAC37-39, MdNAC56-57, MdNAC59-62, MdNAC64-65, and MdNAC67-70 belonged to the NAM group, and MdNAC25, MdNAC36, MdNAC54-55, and MdNAC58 belonged to the VND group. Predictions of subcellular localization showed that MdNAC24-27, MdNAC29-30, MdNAC33-37, MdNAC39, MdNAC54-65, and MdNAC67-70 proteins were located in the nucleus, MdNAC28 proteins were located in the cytoplasm, MdNAC31-32 proteins were located in the nucleus and cytoplasm, and MdNAC38 proteins were located in the nucleus and plasma membrane. Array results indicated that 32 MdNACs were expressed in all examined tissues at various expression levels. RNA-seq results showed that expression levels of MdNAC26-28, MdNAC33-34, MdNAC60, MdNAC62-65, and MdNAC68 were induced, but MdNAC24, MdNAC32, and MdNAC58 were down-regulated in response to AAAP infection. Under salt treatment, MdNAC24, MdNAC27, MdNAC29, MdNAC34, MdNAC37, MdNAC39, MdNAC54, MdNAC59, and MdNAC63 transcription levels were induced. Under mannitol treatment, MdNAC32 and MdNAC54 transcription levels were induced, but MdNAC24, MdNAC28, MdNAC30, MdNAC33, MdNAC35, MdNAC37, MdNAC55, MdNAC56, MdNAC58, and MdNAC59 were down-regulated. Taken together, the results indicated that the cloned MdNAC genes were expressed constitutively in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol, which suggested they may be involved in the regulation of growth, development, and stress response in apple.

Major Latex Protein MdMLP423 Negatively Regulates Defense against Fungal Infections in Apple.

Major latex proteins (MLPs) play critical roles in plants defense and stress responses. However, the roles of MLPs from apple (Malus × domestica) have not been clearly identified. In this study, we focused on the biological role of MdMLP423, which had been previously characterized as a potential pathogenesis-related gene. Phylogenetic analysis and conserved domain analysis indicated that MdMLP423 is a protein with a ‘Gly-rich loop’ (GXGGXG) domain belonging to the Bet v_1 subfamily. Gene expression profiles showed that MdMLP423 is mainly expressed in flowers. In addition, the expression of MdMLP423 was significantly inhibited by Botryosphaeria berengeriana f. sp. piricola (BB) and Alternaria alternata apple pathotype (AAAP) infections. Apple calli overexpressing MdMLP423 had lower expression of resistance-related genes, and were more sensitive to infection with BB and AAAP compared with non-transgenic calli. RNA-seq analysis of MdMLP423-overexpressing calli and non-transgenic calli indicated that MdMLP423 regulated the expression of a number of differentially expressed genes (DEGs) and transcription factors, including genes involved in phytohormone signaling pathways, cell wall reinforcement, and genes encoding the defense-related proteins, AP2-EREBP, WRKY, MYB, NAC, Zinc finger protein, and ABI3. Taken together, our results demonstrate that MdMLP423 negatively regulates apple resistance to BB and AAAP infections by inhibiting the expression of defense- and stress-related genes and transcription factors.

Isolation, sequencing, and expression analysis of 30 AP2/ERF transcription factors in apple.

BackgroundAP2/ERF transcription factors are involved in the regulation of plant growth, development, and stress responses. Our research objective was to characterize novel apple (Malus × domestica Borkh.) genes encoding AP2/ERF transcription factors involved in regulation of plant growth, development, and stress response. The transcriptional level of apple AP2/ERF genes in different tissues and under various biotic and abiotic stress was determined to provide valuable insights into the function of AP2/ERF transcription factors in apple.MethodsThirty full-length cDNA sequences of apple AP2/ERF genes were isolated from ‘Zihong Fuji’ apple (Malus × domestica cv. Zihong Fuji) via homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. Expression levels of apple AP2/ERF genes were detected in 16 different tissues using a known array. Expression patterns of apple AP2/ERF genes were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq with existing data, and the expression of apple AP2/ERF genes was analyzed under NaCl and mannitol treatments using qRT-PCR.ResultsThe sequencing results produced 30 cDNAs (designated as MdERF3-8, MdERF11, MdERF16-19, MdERF22-28, MdERF31-35, MdERF39, MdAP2D60, MdAP2D62-65, and MdRAV2). Phylogenetic analysis revealed that MdERF11/16, MdERF33/35, MdERF34/39, and MdERF18/23 belonged to groups A-2, A-4, A-5, and A-6 of the DREB subfamily, respectively; MdERF31, MdERF19, MdERF4/25/28/32, MdERF24, MdERF5/6/27, and MdERF3/7/8/17/22/26 belonged to groups B-1, B-2, B-3, B-4, B-5, and B-6 of the ERF subfamily, respectively; MdAP2D60 and MdAP2D62/63/64/65 belonged to the AP2 subfamily; and MdRAV2 belonged to the RAV subfamily. Array results indicated that 30 apple AP2/ERF genes were expressed in all examined tissues to different degrees. RNA-seq results using previously reported data showed that many members of the apple ERF and DREB subfamilies were induced by Alternaria alternate apple pathotype (AAAP) infection. Under salt treatment, many members in the apple ERF and DREB subfamilies were transcriptionally up or down-regulated. Under mannitol treatment, many members of the apple ERF, DREB, and AP2 subfamilies were induced at the transcriptional level. Taken together, the results indicated that the cloned apple AP2/ERF genes were expressed in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol treatment, which suggested they may be involved in regulating growth, development, and stress response in apple.

Genome-wide annotation and expression responses to biotic stresses of the WALL-ASSOCIATED KINASE - RECEPTOR-LIKE KINASE (WAK-RLK) gene family in Apple (Malus domestica)

The WALL ASSOCIATED-KINASE - RECETOR-LIKE KINASE (WAK-RLK) gene family has been reported to act as a sensor for disease. Apple (Malus domestica) can be affected by multiple biotic stresses, such as fungal diseases from Valsa mali (Vm), Alternariaalternata Apple Pathotype (AaAP), and Pythium ultimum (Pu). However, there has been no report of WAK-RLK genes involved in apple biotic stress response. In this paper, we performed a comprehensive study including genome-wide annotation, characterization and gene expression analysis of WAK-RLKs in apple (MdWAK-RLKs). We found 44 members based on structural domain identification. The number of amino acids, molecular weight, and theoretical pI of these identified members ranged from 302 to 998, 33.63 to 110.35 kD, and 5.1 to 9.26, respectively. Members of the family were anchored to 16 out of 17 chromosomes and were classified into six phylogenetic groups. We found two phylogenetic groups specific to the apple genome. Synteny analysis revealed that 11 gene pairs arose from segmental duplications and 7 gene clusters resulted from tandem duplications. Cis-elements in the promoter region of MdWAK-RLKs were found mainly in response to circadian rhythm, hormones, and multiple stresses. The large number of members that showed high expression in multiple tissues and differential expressed in response to stress revealed that the different functional roles of MdWAK-RLKs under physiological or pathological conditions. Several genes, such as MDP0000278283, MDP0000153539, MDP0000170906, and MDP0000251865, were significantly influenced by multiple diseases. This study provides new insights into the potential function of WAK-RLKs in Malus and in Rosaceae and its contribution to disease resistance.

Comparative iTRAQ proteomic profiling of susceptible and resistant apple cultivars infected by Alternaria alternata apple pathotype

Alternaria blotch, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of serious pathogen of apples. In order to better understand the molecular mechanisms that underlie the defense responses of apple resistance to Alternaria blotch disease, a comparative proteomic approach was applied to analyze of susceptible and resistant apple cultivars response to A. alternata AP infection using iTRAQ (isobaric tags for relative and absolute quantitation) technique. A total of 4225 proteins were identified, and 1226 proteins were quantified. Of the quantified proteins, 280 and 34 expressed differentially (fold change >1.5) at 72 h post-infection (HPI) in the susceptible (“Starking Delicious”) and the resistant (“Jonathan”) apple cultivars, respectively, compared with mock-inoculated controls. Most of the differentially expressed proteins (DEPs) were associated with host plant resistance to pathogens, including signal transduction, stress and defense, and photosynthesis metabolism. Among these proteins, beta-1,3-glucanase(PR2), thaumatin-like protein (PR5), and lipoxygenase were found in both susceptible and resistant hosts. However, endochitinase and (+)-neomenthol dehydrogenase were only detected in the resistant cultivar and increased in abundance in response to the pathogen attack. To study the role of pathogenesis-related (PR) proteins in the early infection process, their expressions at 6, 18, 36, and 72 HPI were analyzed by western blot. It showed that PR5 were accumulated to a high level at 6 HPI in “Jonathan,” while cannot be detected in “Starking Delicious” until 18 HPI. The above results suggested that endochitinase and (+)-neomenthol dehydrogenase, as well as PR5 which exerts function at early stage, play important roles in apple plant against A. alternata AP infestation.

Resistance to Marssonina coronaria and Alternaria alternata Apple Pathotype in the Major Apple Cultivars and Rootstocks Used in China

A comparison of field tolerance of 25 apple (Malus ×domestica) cultivars and 39 apple rootstocks to the pathogens Marssonina coronaria and Alternaria alternata apple pathotype was performed. Although most apple cultivars grown in China were susceptible or highly susceptible to both pathogens causing two diseases, considerable variation in the level of resistance to both pathogens was observed. Only three cultivars, Qinguan, Jiguan, and Xiangyanghong, exhibited resistance to both pathogens. Among the rootstocks tested, 30 of 39 (77%) were susceptible or highly susceptible to M. coronaria and 32 (82%) were susceptible to the A. alternata apple pathotype. Four rootstocks, ‘P.22’, ‘SH12’, ‘Za’ai 76’, and ‘Qingdao598’, were resistant to both pathogens. The correlation between resistance to both pathogens was highly significant in the 74 apple cultivars and rootstocks tested suggesting common genetic resistance factors to these two pathogens. Data represented horizontal resistance in the germplasm. The resistant local germplasm should be used in disease resistance breeding programs.

A Simple Sequence Repeat Marker Linked to the Susceptibility of Apple to Alternaria Blotch Caused by Alternaria alternata Apple Pathotype

Alternaria alternata apple pathotype (previously A. mali) causes alternaria blotch disease of apple (Malus ×domestica), which may result in leaf spots and up to 70% premature leaf drop in serious cases. This disease is of worldwide importance but is most serious in eastern Asia (Japan, Korea, and China) and in parts of the United States. The excessive use of fungicides not only adds cost to apple growers, but also pollutes the environment. In this study, we characterized a 5-year F1 population from a cross of a resistant cultivar (Huacui) and a susceptible cultivar (Golden Delicious) consisting of 110 individuals along with 14-year-old parent trees (10 each). A field evaluation of disease severity was conducted in 2008 and 2009 under the natural conditions in Liaoning, China (lat. 40°37′ N, long. 120°44′ E). Based on the field data, 110 F1 plants were divided into five groups. Artificial inoculation was carried out both on the living trees and on the detached leaves in 2009 to ensure that A. alternata apple pathotype was the causative agent. Eighty primer pairs of simple sequence repeat (SSR) were screened against the four genomic DNA pools, respectively, from six highly susceptible F1 plants, six most resistant F1 plants, one tree of the seed parent, and the one tree of the pollen parent. One pair of primers (CH05g07) was shown to be linked to the DNA pools of susceptible F1 and the parent tree, but not to the DNA pools of resistant F1 and parent trees. This primer pair was then used to screen all individual 110 F1 progenies and two parent trees. The differentiation of 103 individuals (97.3%) with the marker matched the field disease resistance rating. This marker was further screened with 20 cultivars with known susceptibility or resistance to A. alternata apple pathotype and its linkage to susceptibility was validated. These results suggest that this marker can be used in marker-assisted selection for resistance/susceptibility to alternaria blotch disease in apple.

Structure-activity relationship study of host-specific phytotoxins (AM-toxin analogs) using a new assay method with leaves from apple meristem culture.

AM-toxins are host-specific phytotoxins of the Alternaria alternata apple pathotype, which induce necrosis on apple leaves. In this study, we developed a new assay to measure the necrotic activity of AM-toxin analogs using cultured leaves from meristem cells. This method was not only more sensitive to AM-toxin I, but also more reliable than the previous one that used tree leaves due to the homogeneous nature of cultured leaves and to the method of application of toxins. Using this assay method we investigated a structure-activity relationship of AM-toxin analogs synthesized in this study. Most residues and the macrocyclic ring structure were strictly recognized by AM-toxin putative receptor, whereas the L-Ala binding subsite of the receptor allowed for side chain structures with various stereoelectronic properties. These findings are important for designing ligands for further experimental probing of the nature of the receptor.

Spontaneous loss of a conditionally dispensable chromosome from the Alternaria alternata apple pathotype leads to loss of toxin production and pathogenicity.

The Alternaria alternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide, host-specific toxin, AM-toxin. We recently cloned a cyclic peptide synthetase gene, AMT, whose product catalyzes the production of AM-toxin and showed that it resides on chromosomes of 1.8 Mb or less, depending on the A. alternata apple pathotype strain. Reverse transcriptase (RT)-PCR, using primers specific to AMT, on laboratory sub-cultured strains previously shown to produce AM-toxin, identified one isolate that did not express the gene. A leaf necrosis bioassay confirmed an AM-toxin-minus phenotype. However, an original isolate of this strain which had not undergone sub-culture gave a positive result by both RTPCR and bioassay. Contour-clamped homogeneous electric field electrophoresis and Southern hybridization demonstrated the loss of a 1.1-Mb chromosome in the non-toxin-producing isolate. Since this chromosome can be entirely lost without affecting growth, but is necessary for pathogenicity, we propose it is a conditionally dispensable chromosome.

A Polymerase Chain Reaction-Based Method to Specifically Detect Alternaria alternata Apple Pathotype (A. mali), the Causal Agent of Alternaria Blotch of Apple

ABSTRACT Alternaria alternata apple pathotype (previously A. mali) causes Alternaria blotch on susceptible apple cultivars through the production of a host-specific toxin, AM-toxin. Identification of some Alternaria species, especially those that produce host-specific toxins, has been extremely difficult due to a high level of variability which extends even to nonpathogenic isolates. We have recently cloned and characterized a gene (AMT) that plays a crucial role in AM-toxin biosynthesis and demonstrated that it is only present in isolates of A. alternata apple pathotype. Using primers designed for the AMT gene, we developed a polymerase chainreaction-based method to specifically detect AM-toxin producing isolates of A. alternata apple pathotype.

Synergistic Mechanism of Polyoxin B and Iminoctadine Triacetate in Polybelin<sup>®</sup> on Polyoxin-Resistant <i>Alternaria alternata</i> Apple Pathotype

ポリベリン (ポリオキシンBとイミノクタジン酢酸塩を3:1の割合で含む複合殺菌剤) はポリオキシン耐性リンゴ斑点落葉病菌の胞子発芽管を膨潤化した. ポリベリンはポリオキシン耐性菌の菌糸生育を50%阻害する33.3μg/mlにおいて電解質漏出を誘起し, キチン生合成系阻害活性を示した. ポリベリンの成分の一つであるポリオキシンBは25μg/mlでポリオキシン耐性菌に対して電解質漏出およびキチン生合成系阻害活性を示さず, またもう一つの成分であるイミノクタジン酢酸塩は8.3μg/mlでポリベリン8.3μg/mlとほぼ同等の電解質漏出活性を示したものの, キチン生合成系を阻害しなかった. これらの結果は, ポリオキシン耐性菌に対するポリベリンの共力作用機構がイミノクタジン酢酸塩の細胞膜機能阻害による菌体内へのポリオキシン取込み量の増加に起因することを示唆するものである.

Leakage Sites of Electrolytes from Susceptible Apple Leaf Cells Treated with AM-toxin I of Alternaria alternata Apple Pathotype.

Leakage Sites of Electrolytes from Susceptible Apple Leaf Cells Treated with AM-toxin I of Alternaria alternata Apple Pathotype.

Effects of Light and SH-reagent on Ultrastructural Changes in Leaf Cells Induced by AM-toxin from Alternaria alternata Apple Pathotype.

[in Japanese]