Construction and Genetic Analysis of Hybrid Strains between Apple and Tomato Pathotypes of Alternaria alternata by Protoplast Fusion

The genetic controls of host-specific toxin (HST) biosynthesis and the pathogenicity of A. alternata pathogens have been limited by the asexual nature of the life cycle of these fungi. We used a protoplast fusion system for A. alternata to analyze the genetics of HST production and its relation to the specific pathogenicity of these pathogens. Drug-resistant transformants were isolated by genetic transformation, using vectors conferring resistance to hygromycin B and geneticin, for the A. alternata apple pathotype (AM-toxin producer) and A. alternata tomato pathotype (AAL-toxin producer), respectively. Protoplasts of the respective transformants were fused by electrofusion. The majority of resultant stable fusants produced both AM- and AAL-toxins and were pathogenic to susceptible cultivars of both apple and tomato. Pulsed-field gel electrophoresis analysis demonstrated that these fusants (or hybrids) carried small 1.7-and 1.1-Mb chromosomes, characteristic of the parental strains of the apple and tomato pathotypes, respectively. Detection of the AMT gene, involved in AM-toxin biosynthesis, by polymerase chain reaction revealed that all fusants pathogenic to apple maintained this gene. Microfluorimetry analysis using propidium iodide staining suggested that the fusants might be diploid. Received 14 November 2000/ Accepted in revised form 11 December 2000     

AAL-Toxin-Deficient Mutants of Alternaria alternata Tomato Pathotype by Restriction Enzyme-Mediated Integration

ABSTRACT Host-specific toxins are produced by three pathotypes of Alternaria alternata: AM-toxin, which affects apple; AK-toxin, which affects Japanese pear; and AAL-toxin, which affects tomato. Each toxin has a role in pathogenesis. To facilitate molecular genetic analysis of toxin production, isolation of toxin-deficient mutants utilizing ectopic integration of plasmid DNA has been attempted. However, the transformation frequency was low, and integration events in most transformants were complicated. Addition of a restriction enzyme during transformation has been reported to increase transformation frequencies significantly and results in simple plasmid integration events. We have, therefore, optimized this technique, known as restriction enzyme-mediated integration (REMI), for A. alternata pathotypes. Plasmid pAN7-1, conferring resistance to hygromycin B, with no detectable homology to the fungal genome was used as the transforming DNA. Among the three restriction enzymes examined, HindIII was most effective, as it increased transformation frequency two-to 10-fold depending on the pathotype, facilitating generation of several hundred transformants with a 1-day protocol. BamHI and XbaI had no significant effect on transformation frequencies in A. alternata pathotypes. Furthermore, the transforming plasmid tended to integrate as a single copy at single sites in the genome, compared with trials without addition of enzyme. Libraries of plasmid-tagged transformants obtained with and without addition of restriction enzyme were constructed for the tomato pathotype of A. alternata and were screened for toxin production. Three AAL-toxin-deficient mutants were isolated from a library of transformants obtained with addition of enzyme. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity of the fungus. Characterization of the plasmid integration sites and rescue of flanking sequences are in progress.     

Evolution of pathogenicity controlled by small,dispensable chromosomes in Alternaria alternata pathogens

Alternaria alternata includes seven pathogenic variants, called pathotypes, which produce host-selective toxins (HSTs) as determinant factors for pathogenicity. The gene clusters for HST biosynthesis were identified from six pathotypes (Japanese pear, strawberry, tangerine, apple, tomato and rough lemon) and were found to reside on small chromosomes of <2.0 Mb in most strains tested. We isolated mutants lacking the small chromosomes from the strawberry, apple and tomato pathotypes and showed that the small chromosomes are dispensable for growth. In this review, we summarize our current understanding of the evolution of pathogenicity controlled by small, dispensable chromosomes in Alternaria alternata pathogens.     

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

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. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers AB469331to AB469354.     

Pathological evaluation of host-specific AAL-toxins and fumonisin mycotoxins produced by Alternaria and Fusarium species

Host-specific AAL-toxins and mycotoxin fumonisins are structurally related and were originally isolated from the tomato pathotype of Alternaria alternata and from Fusarium verticillioides, respectively. Previous reports on the production of fumonisin derivatives by the tomato pathotype suggested a possible involvement in the pathogenicity of the pathogen. Here, we have evaluated the role of fumonisin in A. alternata–tomato interactions. The results indicate that highly pathogenic isolates of A. alternata tomato pathotype produce AAL-toxin as the sole toxin, strongly implicating it as a pathogenicity factor. The related compound, fumonisin, is also toxigenic and has infection-inducing activity on susceptible tomato plants.     

Alternaria host-selective toxins: determinant factors of plant disease

Seven diseases caused by pathotypes of Alternaria alternata, which produced host-selective toxins (HSTs), a diverse group of low-molecular-weight secondary metabolites, are known, and each HST has an essential role as a determinant of pathogenicity in all interactions between the plant host and A. alternata. Although these HST-producing pathotypes are morphologically indistinguishable, each has a distinct host range and can be distinguished by its specificity on the respective host plant, hence their designation as pathotypes of A. alternata. In 1933, Tanaka made the first discovery of a HST; fungus-free culture filtrates of A. kikuchiana (now called A. alternata Japanese pear pathotype) were toxic to susceptible cultivar Nijisseiki, but not to resistant cultivars. Over the 80 years since then, the structure of HST molecules, target sites and mode of actions of HSTs, and the molecular genetics of HST production regulating by supernumerary chromosomes encoding HST gene clusters have been studied extensively. We focus this review on studies of low-molecular-weight HSTs produced by A. alternata and give an overview of various types of HST studies.     

Citrus as a molecular contact point for co-evolution of Alternaria pathogens

Alternaria black rot, Alternaria leaf spot of rough lemon, and Alternaria brown spot of tangerines are three major citrus Alternaria pathogens. Citrus could be considered as a molecular contact point for host-selective toxin (HST)-mediated co-evolution of these Alternaria pathogens and susceptibility in the field. ACR-toxin is an HST produced by the rough lemon pathotype, and the target site of the toxin was identified as rough lemon mitochondria. The biosynthetic gene cluster for ACR-toxin production is on the 1.5 Mb-chromosome of the rough lemon pathotype. Another gene cluster for ACT-toxin production is located on the 1.9 Mb-chromosome of the tangerine pathotype. These TOX genes shown to have a role in ACR- or ACT-toxin biosynthesis by using gene disruption and silencing.     

Induced resistance in tomato plants to the toxin-dependent necrotrophic pathogen Alternaria alternata

A necrotrophic pathogen, the tomato pathotype of Alternaria alternata (Aa) causes Alternaria stem canker on tomato. Its pathogenicity depends on the production of host-specific AAL-toxin. Pre-inoculation with nonpathogenic Aa or pretreatment an elicitor prepared from Aa reduced disease symptoms by the pathogen. Salicylic acid (SA)- and jasmonic acid (JA)-dependent defense responses in tomato are not involved in the resistance to the pathogen induced by nonpathogenic Aa. The results suggest that an alternative and unknown signaling pathway independent of SA- and JA-signaling might modulate the induced resistance by activating the expression of the multiple defense genes.     

Detoxification of α-tomatine by tomato pathogens Alternaria alternata tomato pathotype and Corynespora cassiicola and its role in infection

In tomato plants, α-tomatine, a steroidal glycoalkaloid saponin, inhibits fungal growth. Tomato pathogens that produce host-specific toxins, Alternaria alternata tomato pathotype causing Alternaria stem canker and Corynespora cassiicola causing Corynespora target spot, were investigated for sensitivity to α-tomatine. Although spore germination of A. alternata pathogenic and nonpathogenic to tomato and of C. cassiicola pathogenic to tomato was not affected by 0.1 mM α-tomatine, spore germination of C. cassiicola nonpathogenic to tomato was significantly inhibited. This result showed that A. alternata, regardless of its pathogenicity, and only the C. cassiicola pathogenic to tomato are resistant to α-tomatine. Germinating spores of A. alternata and C. cassiicola resistant to α-tomatine detoxified α-tomatine by degrading it to a less polar product. After inoculation of tomato leaves, spores of A. alternata and C. cassiicola nonpathogenic to tomato germinated and formed appressoria, but did not form infection hyphae in host tissues. When a host-specific toxin (CCT-toxin) produced by C. cassiicola pathogenic to tomato was added to nonpathogenic spores, colonization within leaves was observed in A. alternata, but not in C. cassiicola. On the other hand, when spores of C. cassiicola nonpathogenic to tomato were suspended in spore germination fluid of nonpathogenic A. alternata with α-tomatine detoxification activity, the fungus could be induced to colonize leaves in the presence of CCT-toxin. These results indicate that A. alternata tomato pathotype and C. cassiicola pathogenic to tomato detoxify α-tomatine during infection and that this detoxification is essential for host colonization by pathogens that produce host-specific toxins.     

Specific Detection of Tomato Pathotype of Verticillium dahliae by PCR Assays

Verticillium dahliae Klebahn is the causal agent of tomato wilt disease. Isolates of V. dahliae can be classified based on pathogenicity to tomato, but the pathotypes are indistinguishable in morphology. We designed PCR primers for specific detection of isolates pathogenic to tomato (tomato pathotype) from the sequences of a pathotype-specific gene, vdt1. With the primer pair Tg5/Tc3, a PCR product (approximately 3.2 kb) specific to tomato pathotype was amplified from the genomic DNA of isolates. Using the primer pair, a tomato pathotype isolate was specifically detected from hypocotyls of inoculated tomato and eggplant. On the other hand, no amplification was observed from non-tomato pathotype isolates of V. dahliae, some other wilt pathogens of tomato and a healthy host plant. Therefore, the primer pair can be useful for pathotype-specific detection of V. dahliae as well as for diagnosis of wilt disease of tomato plant. Received 7 September 2001/ Accepted in revised form 3 December 2001     

Functional analysis of the melanin biosynthesis genes <Emphasis Type="Italic">ALM1</Emphasis> and <Emphasis Type="Italic">BRM2</Emphasis>-<Emphasis Type="Italic">1</Emphasis> in the tomato pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

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

Multiple copies of <Emphasis Type="Italic">AMT2</Emphasis> are prerequisite for the apple pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis> 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.     

梨黑斑病菌遗传操作体系的建立与RFP标记转化子的致病性分析

由链格孢(Alternaria alternata)引起的梨黑斑病是我国梨生产上的主要病害之一,导致梨叶与果实产生黑斑症状及早期落叶,对我国梨产业的健康发展构成严重威胁。本实验在前期获得强致病性梨黑斑病菌菌株HN-5基础上,建立该病菌的遗传转化体系。通过优化原生质体制备过程以及转化体系发现梨黑斑病菌HN-5原生质体制备的最优条件为:菌丝在M R液体培养基中培养36 h;以0.7 M NaCl+0.8 M Mannitol为稳渗剂,配置复合细胞壁裂解酶(1%崩溃酶+1%裂解酶+1%纤维素酶+1%蜗牛酶),酶解菌丝3 h,原生质体的产量可达0.5×10~7个·mL~(-1)。通过PEG介导的原生质体遗传转化体系,将含有RFP基因的质粒p KD7转入链格孢菌HN-5中,转化效率为6个·μg~(-1)DNA。PCR检测和转化子荧光观察均表明RFP基因整合到了HN-5基因组中并成功表达。致病性分析发现RFP (Red Fluorescent Protein)标记转化子致病性未发生变化。本研究成功建立了PEG (Polyethylene glycol)介导的梨黑斑病菌遗传转化体系,构建了RFP标记菌株,为研究该病菌的致病机理奠定基础。     

辣椒胶孢炭疽菌遗传转化体系的建立

由辣椒胶孢炭疽菌Colletotrichum gloeosporioides导致的辣椒炭疽病是辣椒生产上最为严重的真菌病害之一。本文以辣椒胶孢炭疽菌CSLL11为供试菌株,采用PEG-CaCl_2介导的原生质体转化法,将含有潮霉素B抗性基因和eGFP表达基因的DNA片段成功转入辣椒胶孢炭疽菌的原生质体中,获得了稳定表达绿色荧光的转化子,从而成功建立了辣椒胶孢炭疽菌的遗传转化体系。试验结果表明,可有效筛选阳性转化子的潮霉素B浓度为500 mg/L;PCR及Southern blot结果显示,eGFP表达基因已单拷贝整合至辣椒胶孢炭疽菌转化子的基因组中;使用荧光显微镜观察第一代及继代培养后的转化子,菌丝与分生孢子均表现出强烈的绿色荧光信号,说明GFP能在转化子中稳定遗传;将转化子与野生型菌株相比,菌落形态、生长速率及致病力水平无明显差异。本研究建立了辣椒胶孢炭疽菌遗传转化体系并获得了稳定表达绿色荧光蛋白的转化子,对辣椒炭疽菌与寄主互作的研究及病害防治具有重要意义。     

Differentiation ofVerticillium dahliae populations on the basis of vegetative compatibility and pathogenicity on cotton

Complementary auxotrophic nitrate-nonutilizing (nit) mutants were used to investigate vegetative compatibility within 27 strains ofVerticillium dahliae isolated from several hosts originating from Africa, Asia, Europe and the United States. Using about 500nit mutants generated from these strains, three vegetative compatibility groups, 1, 2 and 4, were identified. Simultaneously, virulence of each strain was assessed on cultivars ofGossypium hirsutum, G. barbadense andG. arboreum, based upon Foliar Alteration Index (FAI) and Browning Index (BI) estimation. The strains in VCG1 were of both the cotton-defoliating pathotype and race 3 (on cotton) but were non pathogenic on tomato; those in VCG2 and VCG4 were of the nondefoliating pathotype and belonged to different races on cotton and on tomato. Hyaline mutants deriving from parental wild-type strain showed differences in pathogenicity but were always assigned to the parental VCG. A relationship was established between VCGs and the taxonomic position of host plants. Data fromnit pairings indicated that the sub-populations ofV. dahliae (VCGs) may not be completely isolated genetically.     

向日葵黄萎病菌突变体库的建立及其阳性转化子的鉴定

为揭示向日葵大丽轮枝菌Verticillium dahliae Kleb.的致病机理,利用农杆菌介导法将带有潮霉素抗性标记和绿色荧光蛋白报告基因的双元载体转入大丽轮枝菌的分生孢子中并获得阳性转化子,以野生型菌株为对照,对随机挑取的阳性转化子的菌落形态、菌丝生长速率、产孢量、粗毒素分泌量和致病力进行了研究。结果表明,共获得800株阳性转化子,随机选取的40株阳性转化子中有2株的菌落只产生白色气生菌丝,不能形成黑色微菌核。与对照相比,40株转化子的生长速率均有不同程度降低,其中转化子A1生长速度降低最显著,菌落直径仅为3.28 cm,比对照下降了38.92%。40株转化子中有3株的产孢量高于对照,其中转化子A9的产孢量最高,为3.50×10~7个/mL,比对照提高1.10倍;转化子A1的产孢量最低,仅为1.35×10~7个/mL,比对照下降了19.16%。40株转化子中有4株的粗毒素分泌量较对照显著升高,占测定菌株的10%,有24株较对照显著降低,占60%,其余12株与对照无显著差异。40株转化子中有3株的致病力较对照显著增强,占测定菌株的7.5%;有7株的致病力较对照显著降低,占17.5%;其余30株与对照无显著差异。     

Alternaria Leaf Spot on Three Species of Pelargonium Caused by Alternaria alternata in Japan

A new disease of pelargonium (Pelargonium domesticum Bailey), ivy geranium (P. peltatum (L.) L'Hér. ex Ait.) and scented geranium (P. graveolens L'Hér.), primarily causing brown spots on leaves, was found in Kawasaki-shi in Kanagawa Prefecture and Tachikawa-shi in Tokyo. An Alternaria sp. was consistently isolated from these diseased leaves, and the isolates were pathogenic to their host leaves. Based on morphological characteristics, the causal fungus in all three cases was identified as Alternaria alternata (Fr.) Keissler. Because Alternaria leaf spot of geranium by A. alternata has already been reported, the pathogenicity of isolates from four groups of genus Pelargonium was investigated. The isolates from scented geranium were pathogenic only to their original host, but the isolates from pelargonium, ivy geranium and geranium were pathogenic to all groups of pelargonium. This is the first report of this disease on pelargonium, ivy geranium and scented geranium caused by A. alternata in Japan. We propose the names for these diseases as Alternaria leaf spot of pelargonium (kappan-byo), Alternaria leaf spot of ivy geranium (kappan-byo) and Alternaria leaf spot of scented geranium (kappan-byo). Received 11 December 2000/ Accepted in revised form 19 July 2001     

山茱萸褐斑病病原鉴定

 山茱萸(Cornus officinalis)为山茱萸科(Cornaceae)山茱萸属植物, 其成熟干燥果肉(俗称枣皮)为我国常用名贵中药材。河南省南阳市(西峡、内乡和南召等县)为我国山茱萸主产区之一, 仅西峡县截止1990年已发展到500万株, 年产枣皮315万kg, 产值达4 200万元, 居全国首位。近年, 作者对西峡县山茱萸产区进行病害调查时发现, 由链格孢菌(Alternaria sp.)引起的山茱萸褐斑病发生普遍, 该病严重发生时可致植株95%以上叶片枯死, 落果率高达87%, 造成山茱萸大幅减产, 给当地果农带来严重经济损失。迄今, 未见链格孢菌引起的山茱萸病害报道。本研究对该病的病原菌进行分离鉴定, 以期为病害的防控提供依据。