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.     

Citrus Black Rot is Caused by Phylogenetically Distinct Lineages of Alternaria alternata

ABSTRACT Phylogenetic analysis revealed that isolates of Alternaria alternata causing black rot of citrus were associated with six well-supported evolutionary lineages. Isolates recovered from brown spot lesions on Minneola tangelo, leaf spot lesions on rough lemon, and healthy citrus tissue and noncitrus hosts were related closely to isolates from black-rotted fruit. Phylogenies estimated independently from DNA sequence data from an endopolygalacturonase gene (endoPG) and two anonymous regions of the genome (OPA1-3 and OPA2-1) had similar topologies, and phylogenetic analysis was performed on the combined data set. In the combined phylogeny, isolates from diverse ecological niches on citrus and noncitrus hosts were distributed in eight clades. Isolates from all lineages, regardless of ecological or host association, caused black rot in fruit inoculation assays, demonstrating that small-spored Alternaria isolates associated with different ecological niches on citrus and other plant hosts are potential black rot pathogens. These data also indicated that the fungi associated with black-rotted fruit do not form a natural evolutionary group distinct from other Alternaria pathogens and saprophytes associated with citrus. The use of the name A. citri to describe fungi associated with citrus black rot is not justified and it is proposed that citrus black rot fungi be referred to as A. alternata.     

An Isolate of Alternaria alternata That Is Pathogenic to Both Tangerines and Rough Lemon and Produces Two Host-Selective Toxins, ACT- and ACR-Toxins

ABSTRACT Two different pathotypes of Alternaria alternata cause Alternaria brown spot of tangerines and Alternaria leaf spot of rough lemon. The former produces the host-selective ACT-toxin and the latter produces ACR-toxin. Both pathogens induce similar symptoms on leaves or young fruits of their respective hosts, but the host ranges of these pathogens are distinct and one pathogen can be easily distinguished from another by comparing host ranges. We isolated strain BC3-5-1-OS2A from a leaf spot on rough lemon in Florida, and this isolate is pathogenic on both cv. Iyokan tangor and rough lemon and also produces both ACT-toxin and ACR-toxin. Isolate BC3-5-1-OS2A carries both genomic regions, one of which was known only to be present in ACT-toxin producers and the other was known to exist only in ACR-toxin producers. Each of the genomic regions is present on distinct small chromosomes, one of 1.05 Mb and the other of 2.0 Mb. Alternaria species have no known sexual or parasexual cycle in nature and populations of A. alternata on citrus are clonal. Therefore, the ability to produce both toxins was not likely acquired through meiotic or mitotic recombination. We hypothesize that a dispensable chromosome carrying the gene cluster controlling biosynthesis of one of the host-selective toxins was transferred horizontally and rearranged by duplication or translocation in another isolate of the fungus carrying genes for biosynthesis of the other host-selective toxin.     

中国柑橘黑腐病和褐斑病病原菌的系统发育分析

 近些年链格孢属真菌的分类有了长足的进展,但柑橘褐斑病和黑腐病的病原却还是存在着一些争议。本研究从中国柑橘果实上收集了49株黑腐病菌菌株,并选取了8株从柑橘叶片上分离的具代表性的褐斑病菌菌株,基于Alta1 、endoPG、LSU、OPA10-2、OPAl-3和OPA2-1 等6个基因位点串联序列构建系统发育树。结果表明,柑橘黑腐病和褐斑病都可由不止一种链格孢菌引起,均以Alternaria alternata为主。两种病害的病原菌之间不能通过该系统发育树区分,但在致病性上存在差异,且能通过扩增ACT毒素合成基因进行区分。为了使两种病害的病原更加方便阐述,作者建议以它们的主要类群对其进行命名,柑橘褐斑病病原学名还是遵循前人的称呼,为the tangerine pathotype of A. alternata,即链格孢菌橘致病型,而柑橘黑腐病病原应为A. alternata,即链格孢菌。     

中国柑橘黑腐病和褐斑病病原菌的系统发育分析

 近些年链格孢属真菌的分类有了长足的进展,但柑橘褐斑病和黑腐病的病原却还是存在着一些争议。本研究从中国柑橘果实上收集了49株黑腐病菌菌株,并选取了8株从柑橘叶片上分离的具代表性的褐斑病菌菌株,基于Alta1 、endoPG、LSU、OPA10-2、OPAl-3和OPA2-1 等6个基因位点串联序列构建系统发育树。结果表明,柑橘黑腐病和褐斑病都可由不止一种链格孢菌引起,均以Alternaria alternata为主。两种病害的病原菌之间不能通过该系统发育树区分,但在致病性上存在差异,且能通过扩增ACT毒素合成基因进行区分。为了使两种病害的病原更加方便阐述,作者建议以它们的主要类群对其进行命名,柑橘褐斑病病原学名还是遵循前人的称呼,为the tangerine pathotype of A. alternata,即链格孢菌橘致病型,而柑橘黑腐病病原应为A. alternata,即链格孢菌。     

基于刃天青微平板法测定交链格孢菌橘致病型对戊唑醇和苯醚甲环唑的敏感性

由交链格孢菌橘致病型(Alternaria alternata tangerine pathotype)引起的柑橘链格孢褐斑病(Alternaria brown spot,ABS)是柑橘上的主要病害,目前主要采用杀菌剂防治。本研究以采自中国各柑橘链格孢褐斑病发生区的54个交链格孢菌橘致病型菌株为供试群体,采用刃天青微平板法测定了病原菌孢子萌发和菌丝生长对戊唑醇和苯醚甲环唑的敏感性。结果表明:刃天青微平板法最佳体系中的培养基为完全培养基(CM),刃天青终浓度为40 μmol/L,孢子浓度为105个/mL。戊唑醇和苯醚甲环唑的有效抑制中浓度(EC50)范围分别为0.304~0.885 μg/mL和0.367~0.873 μg/mL,平均值分别为0.562和0.609 μg/mL。正态分布检测结果显示:这2种药剂抑制交链格孢菌橘致病型群体的EC50值均符合正态分布,因此认为所得EC50值可作为病原菌对戊唑醇和苯醚甲环唑的敏感性基线。本研究结果可为田间防治褐斑病和监测交链格孢菌对戊唑醇和苯醚甲环唑的抗药性奠定基础。     

Alternaria brown spot on Minneola tangelos in Israel

Brown spots were observed on the rind of fruit and foliage of Minneola tangelo (Citrus reticulata C. paradisi) for the first time in Israel. The causal organism was isolated in culture and identified as Alternaria alternata, citrus pathotype. Typical disease symptoms were produced on inoculated leaves and fruit.     

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.     

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.     

Detection of fungi producing infection-inhibiting metabolites against <Emphasis Type="Italic">Alternaria alternata</Emphasis> Japanese pear pathotype from fungi inhabiting internal tissues of Japanese pear shoots

Fungi inhabiting Japanese pear were isolated from internal tissues of cv. Nijisseiki, and culture filtrates (CFs) of 100 isolates were evaluated for their inhibitory activity against infection by Alternaria alternata Japanese pear pathotype. CFs of 11 isolates inhibited lesion formation on the pear by the pathogen. Among these isolates, CFs of five isolates inhibited spore germination. CFs of the six other isolates inhibited appressorial formation, infection hypha formation, AK-toxin production, or a combination of these actions. Analysis of sequence homology in the rDNA ITS1 regions of these isolates showed that most isolates had high homology with some fungal endophytes.     

Genetic Differentiation and Host Specificity Among Populations of Alternaria spp. Causing Brown Spot of Grapefruit and Tangerine x Grapefruit Hybrids in Florida

ABSTRACT Alternaria spp. were sampled from brown spot lesions in several geographically separated citrus groves and different grapefruit and tangerine x grapefruit hybrid cultivars in Florida and screened for variation at 16 putative random amplified polymorphic DNA loci. Populations of the pathogen on two hybrids, Minneola and Orlando, in five locations throughout Florida were moderately differentiated (Nei's coefficient of gene differentiation [G(ST)] = 0.12) among locations. The hypothesis that host-specialized forms of Alternaria spp. cause brown spot on different Citrus spp. and cultivars was tested by estimating genetic differentiation among isolates sampled from different hosts and by pathogenicity assays. Isolates sampled from grapefruit and the hybrid cv. Nova were genetically distinct from isolates sampled from other hybrid cultivars including Robinson, Sunburst, Minneola, Orlando, and Murcott. No differentiation could be detected among isolates sampled from this latter group of hybrids. Quantitative pathogenicity assays on leaves using spray inoculation revealed that 'Nova' isolates were not significantly more pathogenic on 'Nova' compared with isolates from 'Minneola' and 'Orlando'. Similarly, grapefruit isolates were not significantly more pathogenic on grapefruit compared with isolates from 'Minneola'. Isolates from all hosts had similar disease rankings on each inoculated cultivar, with 'Minneola' the most susceptible, followed in decreasing order of susceptibility by 'Orlando', 'Sunburst', 'Nova', and 'Duncan' grapefruit. Rough lemon was generally immune to all isolates tested; however, occasional brown spot lesions were observed on leaves of this host with isolates from grapefruit. No evidence was found to support the hypothesis that unique genotypes of the pathogen, which are more virulent on 'Sunburst' or grapefruit, have been introduced to Florida. Populations of Alternaria spp. causing brown spot of citrus on grapefruit and 'Nova' in Florida are genetically distinct from isolates on other cultivars, and we speculate that these populations are in the early stages of adaptation to and possible speciation on these hosts.     

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.     

Identification of putative defense-related genes in Japanese pear against <Emphasis Type="Italic">Alternaria alternata</Emphasis> infection using suppression subtractive hybridization and expression analysis

The Japanese pear pathotype of Alternaria alternata, a toxin-dependent necrotrophic pathogen, causes black spot of Japanese pear by producing the host-specific AK-toxin. Pre-inoculation with nonpathogenic A. alternata or pretreatment with an elicitor prepared from A. alternata reduced disease symptoms caused by the pathogen. Salicylic acid- and jasmonic acid-dependent signaling pathways are not involved in the induced resistance to infection by the pathogen. The expression of multiple defense-related genes in Japanese pear leaves inoculated with nonpathogenic A. alternata was examined using suppression subtractive hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database as accessions DC993229–DC993535.     

Worldwide Phylogeography of the Citrus Brown Spot Pathogen, Alternaria alternata

ABSTRACT Sixty-five isolates of Alternaria alternata were sampled from brown spot lesions on tangerines and mandarins (Citrus reticulata) and tangerine x grapefruit (C. reticulata x C. paradisi) hybrids in the United States, Colombia, Australia, Turkey, South Africa, and Israel to investigate the worldwide phylogeography of the fungus. Genetic variation was scored at 15 putative random amplified polymorphic DNA (RAPD) loci and 465 bp of an endo-polygalacturonase (endo-PG) gene was sequenced for each isolate. Cluster analysis of RAPD genotypes revealed significant differentiation between United State and Colombia isolates and Turkey, South Africa, Israel, and Australia isolates. Sequencing of endo-PG revealed 21 variable sites when the outgroup A. gaisen (AK-toxin-producing pathogen of Japanese pear) was included and 13 variable sites among the sampled isolates. Nucleotide substitutions at 10 of 13 variable sites represented silent mutations when endo-PG was translated in frame. Eight distinct endo-PG haplotypes were found among the sampled isolates and estimation of a phylogeny with endo-PG sequence data revealed three clades, each with strong bootstrap support. The most basal clade (clade 1) was inferred based on its similarity to the outgroup A. gaisen and consisted exclusively of pathogenic isolates from the United States and Colombia. Clade 2 consisted of pathogenic and nonpathogenic isolates from the United States, Australia, South Africa, and Israel and clade 3 contained pathogenic and nonpathogenic isolates from Australia, South Africa, Israel, and Turkey. Quantitative estimates of virulence (disease incidence) were obtained for isolates from the United States, Colombia, South Africa, Israel, and Turkey by spray inoculating detached citrus leaves and counting the number of lesions 24 h after inoculation. Large differences in virulence were detected among isolates within each location and isolates from the United States were significantly more virulent than isolates from other locations. Several isolates from Colombia, South Africa, Israel, and Turkey had low virulence and 8% of all isolates were nonpathogenic. All but one of the nonpathogenic isolates were found in clade 2 of the endo-PG phylogeny, which also included the most highly virulent isolates sampled.     

A Virulence-Reducing Mutation in the Postharvest Citrus Pathogen Alternaria citri

ABSTRACT Alternaria citri causes Alternaria black rot, a postharvest fruit disease, on a broad range of citrus cultivars. We previously described that an endopolygalacturonase minus mutant of A. citri caused significantly less black rot in citrus fruit. To search for other essential factors causing symptoms in addition to endopolygalacturonase, a random mutation analysis of pathogenicity was performed using restriction enzyme-mediated integration. Three isolates among 1,694 transformants of A. citri had a loss in pathogenicity in a citrus peel assay, and one of these three mutants was a histidine auxotroph. Gene AcIGPD that encodes imidazole glycerol phosphate dehydratase, the sixth enzyme in the histidine biosynthetic pathway, was cloned, and the mutant containing the disrupted target gene, AcIGPD, caused less black rot.