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.     

Interactions of Apple and the Alternaria alternata Apple Pathotype

Apple is one of the most cultivated tree fruits worldwide, and is susceptible to many diseases. Understanding the interactions between the host and pathogen is critical in implementing disease management strategies and developing resistant cultivars. This review provides an update on the interactions of apple with Alternaria alternata apple pathotype, which causes Alternaria blotch, with a brief history about the discovery of the disease and pathogen and its damage and epidemiology. The focus of the review is placed on the physiological and genetic response of the host to pathogen infection, including resistance and susceptibility, and the molecular markers associated with them. Of the response of the pathogen to the host, the emphasis is placed on the role of the selective toxins on pathogenicity and their genetic controls and regulations. The review ends with a perspective on future directions in the research on the apple-A. alternata pathosystem in the era of genomics and post genomics, particularly on how to identify candidate genes from both host and pathogen for potential genetic engineering for disease resistant cultivars.     

Comparative iTRAQ proteomic profiling of susceptible and resistant apple cultivars infected by <Emphasis Type="Italic">Alternaria alternata</Emphasis> 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.     

Expression profiles of genes encoded by the supernumerary chromosome controlling AM-toxin biosynthesis and pathogenicity in the apple pathotype of Alternaria alternata

The apple pathotype of Alternaria alternata produces host-specific AM-toxin and causes Alternaria blotch of apple. Previously, we cloned two genes, AMT1 and AMT2, required for AM-toxin biosynthesis and found that these genes are encoded by small, supernumerary chromosomes of <1.8 Mb in the apple pathotype strains. Here, we performed expressed sequence tag analysis of the 1.4-Mb chromosome encoding AMT genes in strain IFO8984. A cDNA library was constructed using RNA from AM-toxin-producing cultures. A total of 40,980 clones were screened with the 1.4-Mb chromosome probe, and 196 clones encoded by the chromosome were isolated. Sequence analyses of these clones identified 80 unigenes, including AMT1 and AMT2, and revealed that the functions of 43 (54%) genes are unknown. The expression levels of the 80 genes in AM-toxin-producing and nonproducing cultures were analyzed by real-time quantitative polymerase chain reaction (PCR). Most of the genes were found to be expressed in both cultures at markedly lower levels than the translation elongation factor 1-alpha gene used as an internal control. Comparison of the expression levels of these genes between two cultures showed that 21 genes, including AMT1 and AMT2, were upregulated (>10-fold) in AM-toxin-producing cultures. Two of the upregulated genes were newly identified to be involved in AM-toxin biosynthesis by the gene disruption experiments and were named AMT3 and AMT4. Thus, the genes upregulated in AM-toxin-producing cultures contain ideal candidates for novel AM-toxin biosynthetic genes.     

Pathogenicity of Alternaria alternata and Fusarium moniliforme and Phytotoxicity of AAL-toxin and Fumonisin B1 on Tomato Cultivars

Phytotoxicity of AAL-toxin and fumonisin B₁ to six cultivars of tomato was compared with the pathogenicity of their fungal sources, Alternaria alternata and Fusarium moniliforme, respectively. These include two AAL-toxin susceptible cultivars with genotypes(asc/asc), three resistant cultivars (Asc/Asc), and a heterozygous cultivar (Asc/asc.) A. alternata spores were pathogenic to the susceptible but not to the resistant cultivars F. moniliforme was not pathogenic to any of the tomatoes. Filtrates of both fungi grown on rice containing their respective toxins caused necrosis within 48 h and eventually mortality on susceptible cultivars but not on the resistant lines. The heterozygous cultivar Asc/asc showed minimal damage and no mortality after 14 days exposure to both filtrates and both toxins. The spores of both fungi had no effect on heterozygous intact plants. Tomato leaf disc bioassays with AAL-toxin and fumonisin B₁ at 1μM caused cellular leakage and reduced chlorophyll content in susceptible cultivars and minimal effects on the heterozygous and resistant varieties.     

Growth inhibition effect of pyroligneous acid on pathogenic fungus,<Emphasis Type="Italic">Alternaria mali</Emphasis>, the agent of Alternaria blotch of apple

We investigated the growth inhibition effect of pyroligneous acid on the pathogenic fungus,Alternaria mali, which is known to be the agent of Alternaria blotch of apple plants. Chemical control ofA. mali could be achieved through the use of agrochemical fungicides, while the substitute for agrochemical control is gradually increasing. It was observed that pyroligneous acid exhibited antifungal activity against some plant pathogenic fungi. More specifically, the growth ofA. mali was completely inhibited in pyroligneous acid at a dilution of 1∶32. When its antifungal activity was compared to that of polyoxin B, which is used for the chemical control of Alternaria blotch of apple, it was observed that the antifungal activity of pyroligneous acid diluted at 1∶32 corresponded to 2.0 mg/mL of polyoxin B. Consequently, it is concluded that the diluted pyroligneous acid can substitute for polyoxin B, thereby reducing the use of the agrochemical for the control of Alternaria blotch of apple.     

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.     

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M₂ mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.     

Effect of storage conditions on virulence of Fusarium avenaceum and Alternaria alternata on apple fruits

To overcome difficulty in phytopathogenic fungi control during storage of apple fruits, the effect of different storage conditions on the occurrence and development of Fusarium avenaceum and Alternaria alternata infections on apple cultivar “Cripps Pink” was investigated during and after storage. Inhibitory effects of wild oregano essential oil on apple fruit rots caused by F. avenaceum and A. alternata were also tested as possible rot control measure. Artificially inoculated apple fruits were kept in cold storage with normal (NA) and controlled (CA) atmosphere for 95 days and at room temperature only. The obtained results indicated that different storage conditions significantly affect necrosis development on apple fruits caused by F. avenaceum and A. alternata after storage, as well as during shelf life.     

Virulence and the Production of Endo-1,4-β-glucanase by Isolates of Alternaria alternata Involved in the Moldy-core Disease of Apples

Alternaria alternata, is the predominant fungal pathogen responsible for moldy‐core in apple cultivars of the Red Delicious group. Here we report on the association between virulence of natural isolates of A. alternata, their production of endo‐1,4‐β‐glucanase (EG) and moldy‐core development in apple fruits. Based on decay development following wound inoculations of mature fruits, three of 150 isolates, collected in three orchards in northern Israel and representing low, moderate and high virulence, were selected and designated Rm44, Er30 and Sh42, respectively. All three isolates secreted EG when grown on enzyme‐inducing medium (EIM) containing commercial cellulose or apple cell walls and this production was related to their degree of virulence. Polyacrylamide gel electrophoresis (PAGE) revealed quantitative differences between the three isolates, relative to their virulence. When fungal extracts were run in native gels, a single band with a molecular mass of 23 kDa showing EG activity was produced by the high‐ (Sh42) and the medium‐virulence (Er30) isolate but not by the low‐virulence (Rm44) isolate. A commercial cellulase preparation (containing endo‐ and exo‐1,4‐β‐glucanase) placed on pricked fruit led to the formation of symptoms similar to those developing on A. alternata‐inoculated fruits within 2–4 days. Inoculation of bloom clusters at full bloom with the highly virulent isolate (Sh42) of A. alternata resulted in a significantly higher infection in fruits (58%) than in those inoculated with the low‐virulence isolate (Rm44) (30%). Our results suggest that the moldy‐core symptoms caused by A. alternata in apple, could be related to the ability of the fungus to produce EG in developing lesions.     

A cAMP‐Dependent Protein Kinase Gene,aapk1, Is Required for Mycelia Growth,Toxicity and Pathogenicity of Alternaria alternata on Tobacco

The fungus Alternaria alternata is a common spot‐producing plant pathogen. During the past decade, tobacco brown spot disease caused by this fungus has became prevalent in China and lead to significant losses. To better understand the molecular pathogenesis of this fungus, the aapk1 gene encoding a cAMP‐dependent protein kinase catalytic subunit was cloned, sequenced and characterized. The aapk1 deletion mutants were identified from hygromycin‐resistant transformants by PCR strategy and confirmed by Southern blot analysis and RT‐PCR. The aapk1 deletion mutant exhibited reduced vegetative growth and was less toxic than the wild‐type strain sd1. Deletion of aapk1 also delayed disease development on detached tobacco leaves. Thus, we propose that the cAMP signalling pathway is involved in mycelia growth and pathogenic phenotype of Alternaria alternata.     

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.     

First report of Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler on Sonchus oleraceus L. and Convolvulus arvensis L. in Algeria

Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler was found on sow thistle (Sonchus oleraceus L.) and field bindweed (Convolvulus arvensis L.) in the experimental station of ENSA (Ecole Nationale Supérieure d'Agronomie) in Algiers, Algeria, during the winter of 2016. Necrotic spots in the form of concentric circles were observed on the leaves of both weeds with disease incidence of approximately 70% and disease severity ranging from 50% to 70%. Fungi were isolated from the infected leaves and identified as A. alternata, based on morphological and molecular analyses (using genetic marker internal transcribed spacer, ITS of rDNA). Pathogenicity tests confirmed that A. alternata is the causing agent of leaf spot disease of sow thistle and field bindweed in accordance with the original symptoms. To the best of our knowledge, this is the first report of sow thistle and field bindweed naturally infected by A. alternata in Algeria.     

CCR-NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica)

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CC_R-NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.     

Nuclear magnetic resonance study of ring conformations of cyclic tetradepsipeptide AM-toxins and related peptides

Cyclic tetradepsipeptides, AM-toxin I and II, are the host-specific phytotoxins of Alternaria mali. In order to elucidate conformation-toxicity relationships, we analyzed the 270-MHz proton nmr spectra of AM-toxins and hydrogenated analogs, (D -Ala²)AM-toxin I (toxic) and (L -Ala²)AM-toxin I (not toxic), in (C²H₃)₂SO. These cyclic tetradepsipeptides do not contain N-substituted amino acid residues, and all the peptide and ester groups have been found to be transoid. Two conformers with very unequal populations have been found for AM-toxin I and II; the C^β?C^α? C?O conformations of the Dha² residues are nonplanar S-trans in the major conformer and nonplanar S-cis in the minor conformer. Only one ring conformation has been found for each of (L -Ala²) and (D -Ala²)AM-toxin I. (L -Ala²)AM-toxin I takes a C₄-type ring conformation; all the C?O groups and C^α-H bonds are oriented to the same side of the ring. (D -Ala²)AM-toxin I takes a new ring conformation; the side chain and C?O group of the L -Amp¹ residue are oriented to the same side of the ring. This new conformation is also found for the major conformers of AM-toxin I and II and thus appears to be required for the toxicity. The ring conformations of Tyr(OCH₃)¹-bearing analog tetradepsipeptides have been found to be much the same as those of Amp¹-bearing depsipeptides. Furthermore, on the basis of the two distinct conformations of (D -Ala²) and (L -Ala²)AM-toxin I, an empirical rule is proposed for the stable ring conformations of cyclic tetra-D ,L -peptides, not containing N-substituted amino acid residues.     

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.     

Oxidative Protective Mechanisms and Resistance to the Dicarboximide Fungicide, Iprodione, in Alternaria alternata

The free radical scavengers α-tocopherol and butylated hydroxytoluene, but not ascorbate, diminished the growth-inhibiting effects of the dicarboximide fungicide, iprodione in Alternaria alternata. Growth of A. alternata in the presence of iprodione increased the activities of superoxide dismutase and glutathione reductase while catalase was unaffected. Four iprodione sensitive and four iprodione resistant isolates of A. alternata were compared for activity of free radical enzymes. The isolates of A. alternata resistant to iprodione had more catalase activity than those which were sensitive, but did not differ in superoxide dismutase of glutathione reductase, activities. 3-Amino-1.24.-triazole, a specific inhibitor of catalas, reduced the ability of DAR 69775, a dicarboximide resistant isolate of A. alternata. to grow in the presence of iprodione. In A. alternata dicarboximide resistance appeats to be at least partially mediated by enhanced activitiesof, catalase.     

In vitro Analysis of Defence Mechanism in the System Solanum tuberosum-Alternaria alternata

In a preliminary study on the interaction between an Alternaria alternata pathotype and Solanum tuberosum a series of parameters, indicative of active and passive defence processes, was investigated in vitro using tolerant (Chiquita) and susceptible (Superior) cultivars. Ion leakage experiments along with growth of suspension cultures and plating efficiencies in the presence of fungal culture filtrates suggest that toxin tolerance is a valuable character in distinguishing between resistant and susceptible genotypes. As far as active defence was concerned, although TLC patterns suggested the synthesis of different fluorescent compounds, probably phenolic acid, when fungal elicitors were applied to Chiquita or susceptible tissue,cultures, no consistent pattern suggesting a relationship with defence could be observed. On the other hand, peroxidase isozyme analysis after different dual culture periods showed the activation of ionically and covalently bound peroxidases in the prese,nce of the pathogen only in cv. Chiquita.     

Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

Molecular characterization of Alternaria alternata population isolated from Upper Egyptian tomato fruits

Alternaria alternata is the most common fungal pathogen of tomatoes in Upper Egypt. Morphological identification of this fungus is challenging; therefore, this study searched for new classification tools based on molecular techniques. Using a dilution plating method, 67 strains of A. alternata were isolated from 34 samples of rotten tomato fruits representing the Giza 80 and Edkawy cultivars. The collected strains were identified using the amplification products of the internal transcribed spacer (ITS) region, glyceraldehyde 3‐phosphate dehydrogenase (Gpd) and Alt a1, which is a gene involved in the production of most of the allergens produced by A. alternata. The screening revealed that A. alternata constituted more than half of the total fungi recovered from rotten tomatoes in this study. According to the phylogenetic analysis using these three loci, the collected strains clustered in accordance with the host cultivar type from which they had been isolated. Specific gene random primer polymerase chain reaction (SGRP‐PCR) techniques indicated that the A. alternata population in the tested region has a high genetic diversity. The pathogenicity test showed that most of the A. alternata isolates (67.2%) were highly pathogenic, and no correlation was found between the phylogenetic analysis and pathogenicity. In addition, the influence of the fungicide Disan 80% on the collected strains showed significant differences that were attributed to the source of isolation.