Brown spot of Indian mulberry (Morinda citrifolia L.) caused by Alternaria alternata.

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

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Worldwide Phylogeography of the Citrus Brown Spot Pathogen, Alternaria alternata

Phytopathology ◽

10.1094/phyto.2002.92.7.794 ◽

2002 ◽

Vol 92 (7) ◽

pp. 794-802 ◽

Cited By ~ 57

Author(s):

T. L. Peever ◽

A. Ibañez ◽

K. Akimitsu ◽

L. W. Timmer

Keyword(s):

United States ◽

South Africa ◽

Alternaria Alternata ◽

Sequence Data ◽

Random Amplified Polymorphic Dna ◽

Disease Incidence ◽

The United States ◽

Brown Spot ◽

Bootstrap Support ◽

Nucleotide Substitutions

Sixty-five isolates of Alternaria alternata were sampled from brown spot lesions on tangerines and mandarins (Citrus reticulata) and tangerine × grapefruit (C. reticulata × 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.

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Role of the Host-Selective ACT-Toxin Synthesis Gene ACTTS2 Encoding an Enoyl-Reductase in Pathogenicity of the Tangerine Pathotype of Alternaria alternata

Phytopathology ◽

10.1094/phyto-100-2-0120 ◽

2010 ◽

Vol 100 (2) ◽

pp. 120-126 ◽

Cited By ~ 33

Author(s):

Naoya Ajiro ◽

Yoko Miyamoto ◽

Akira Masunaka ◽

Takashi Tsuge ◽

Mikihiro Yamamoto ◽

...

Keyword(s):

Alternaria Alternata ◽

Essential Gene ◽

Toxin Production ◽

Brown Spot ◽

Gene Encoding ◽

Bac Clone ◽

Biosynthesis Gene Cluster ◽

Enoyl Reductase ◽

Alternaria Brown Spot ◽

Rapid Amplification

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerines and tangerine hybrids. Sequence analysis of a genomic BAC clone identified a previously uncharacterized portion of the ACT-toxin biosynthesis gene cluster (ACTT). A 1,034-bp gene encoding a putative enoyl-reductase was identified by using rapid amplification of cDNA ends and polymerase chain reaction and designated ACTTS2. Genomic Southern blots demonstrated that ACTTS2 is present only in ACT-toxin producers and is carried on a 1.9 Mb conditionally dispensable chromosome by the tangerine pathotype. Targeted gene disruption of ACTTS2 led to a reduction in ACT-toxin production and pathogenicity, and transcriptional knockdown of ACTTS2 using RNA silencing resulted in complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS2 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.

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Sensitivity of Alternaria alternata from Citrus to Boscalid and Polymorphism in Iron-Sulfur and in Anchored Membrane Subunits of Succinate Dehydrogenase

Plant Disease ◽

10.1094/pdis-04-14-0374-re ◽

2015 ◽

Vol 99 (2) ◽

pp. 231-239 ◽

Cited By ~ 5

Author(s):

Byron Vega ◽

Megan M. Dewdney

Keyword(s):

Succinate Dehydrogenase ◽

Alternaria Alternata ◽

Mycelial Growth ◽

Conidial Germination ◽

Brown Spot ◽

Sequence Comparisons ◽

Sensitivity Tests ◽

Alternaria Brown Spot ◽

Succinate Dehydrogenase Inhibitor

Boscalid, a succinate dehydrogenase inhibitor (SDHI), was registered in 2011 to control Alternaria brown spot (ABS) of citrus, caused by Alternaria alternata. In this study, the effect of boscalid on mycelial growth, conidial germination, and resazurin reduction was established in a subset of 16 sensitive isolates using three different media. Conidial germination and mycelial growth inhibition were not suppressed even at higher concentrations of boscalid, although effective concentration to inhibit 50% growth (EC50) values were established with each method. Resazurin reduction produced the lowest EC50 values and was selected for further sensitivity tests. In total, 419 isolates, never exposed to boscalid and collected from Florida tangerine orchards between 1996 to 2012, were tested for boscalid sensitivity. The sensitivity distribution was a unimodal curve with a mean EC50 value of 0.60 μg/ml and a range of 0.07 to 5.84 μg/ml. The molecular characterization of the succinate dehydrogenase (SDH) genes were also determined in a subset of 15 isolates, exhibiting great variability in boscalid sensitivity, by cloning and sequencing the sdhB, sdhC, and sdhD genes. Sequence comparisons of the SDH complex revealed the presence of mutations in 14 of 15 isolates. In total, 21 mutations were identified. Double and multiple mutations were observed in SDHC and SDHD, respectively. In SDHB, 4 mutations were observed while, in SDHC and SDHD, 5 and 12 mutations were detected, respectively. No mutations were found in the highly conserved histidine residues at positions 277 in SDHB, 134 in SDHC, and 133 in SDHD, typically observed in SDHI-resistant isolates. Our findings suggest that A. alternata populations from Florida are sensitive to boscalid and it could be used in ABS spray programs. Boscalid resistance is currently not a problem, although further monitoring for resistance is advisable.

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The YAP1 Homolog–Mediated Oxidative Stress Tolerance Is Crucial for Pathogenicity of the Necrotrophic Fungus Alternaria alternata in Citrus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-8-0942 ◽

2009 ◽

Vol 22 (8) ◽

pp. 942-952 ◽

Cited By ~ 85

Author(s):

Ching-Hsuan Lin ◽

Siwy Ling Yang ◽

Kuang-Ren Chung

Keyword(s):

Oxidative Stress ◽

Alternaria Alternata ◽

Critical Role ◽

Disease Process ◽

Brown Spot ◽

Null Mutant ◽

Wild Type ◽

Protein Fusion ◽

Fungal Pathogenicity

Citrus brown spot disease is caused by the necrotrophic fungus Alternaria alternata. Its pathogenic capability has been thought to depend exclusively on the production of host-selective ACT toxin. However, circumvention of plant defenses is also likely to be important for the disease process. To investigate the fungal response to host-generated reactive oxygen species (ROS), we cloned and characterized the AaAP1 gene of A. alternata, which encodes a polypeptide resembling yeast YAP1-like transcriptional activators implicated in cellular responses to stress. Expression of the AaAP1 gene in a wild-type strain was primarily induced by H2O2 or ROS-generating oxidants. Using a loss-of-function mutation in the AaAP1 gene, we demonstrated an essential requirement for oxidative tolerance during the host invasion step. Mutants lacking AaAP1 showed increased sensitivity to H2O2 and loss of fungal pathogenicity. The ΔAaAP1 null mutant did not cause any visible necrotic lesions on wounded or unwounded leaves of citrus cv. Minneola. Compared with the wild type, the null mutant displayed lower catalase, peroxidase, and superoxide dismutase activities. All mutant phenotypes were restored to the wild type in fungal strains expressing a functional copy of AaAP1. Upon exposure to H2O2, the AaAP1::sGFP (synthetic green fluorescent protein) fusion protein became localized in the nucleus. Inoculation of the mutant with NADPH oxidase inhibitors partially restored fungal pathogenicity. Our results highlight the global regulatory role of a YAP1 homolog in response to oxidative stress in A. alternata and provide insights into the critical role of ROS detoxification in the pathogenicity of A. alternata.

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Chromosome-scale genome sequence of Alternaria alternata causing Alternaria brown spot of citrus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-20-0278-sc ◽

2021 ◽

Author(s):

Yunpeng Gai ◽

Haijie Ma ◽

Yanan Chen ◽

Lei Li ◽

Yingze Cao ◽

...

Keyword(s):

Genome Sequence ◽

Genome Assembly ◽

Alternaria Alternata ◽

Draft Genome ◽

Gc Content ◽

Brown Spot ◽

Toxin Gene ◽

Alternaria Brown Spot ◽

Alternaria Species ◽

Chromosome Level

Alternaria brown spot (ABS) caused by Alternaria alternata is an economically important fungal disease of citrus worldwide. The ABS pathogen A. alternata tangerine pathotype can produce a host-specific ACT toxin, which is regulated by ACT toxin gene cluster located in the conditionally dispensable chromosome (CDC). Previously, we have assembled a draft genome of A. alternata tangerine pathotype strain Z7, which comprises 165 contigs. In this study, we report a chromosome-level genome assembly of A. alternata Z7 through the combination of Oxford nanopore sequencing and Illumina sequencing technologies. The assembly of A. alternata Z7 had a total size of 34.28 Mb, with a GC content of 51.01% and contig N50 of Mb. The genome is encompassed 12067 protein-coding genes, 34 rRNAs, and 107 tRNAs. Interestingly, A. alternata Z7 is composed of 10 essential chromosomes (ECs) and 2 conditionally dispensable chromosomes (CDCs), which is consistent with the experimental evidences of pulsed-field gel electrophoresis (PFGE). To our best knowledge, this is the first chromosome-level genome assembly of A. alternata. In addition, a database for citrus-related Alternaria genomes has been established to provide public resources for the sequences, annotation and comparative genomics data of Alternaria species. The improved genome sequence and annotation at the chromosome level is a significant step toward a better understanding of the pathogenicity of A. alternata. The database will be updated regularly whenever the genomes of newly isolated Alternaria species are available. The citrus-related Alternaria genomes database is open accessible through http://www.zjudata.com/alternaria/blast.php.

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Genomic features and evolution of the conditionally dispensable chromosome in the tangerine pathotype of Alternaria alternata

10.1101/207746 ◽

2017 ◽

Author(s):

Mingshuang Wang ◽

Huilan Fu ◽

Xing-Xing Shen ◽

Ruoxin Ruan ◽

Nicholas Pun ◽

...

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Gene Cluster ◽

Alternaria Alternata ◽

Deletion Mutant ◽

Metabolic Process ◽

Brown Spot ◽

P Value ◽

Dispensable Chromosome ◽

Cdc Genes

AbstractThe tangerine pathotype of the ascomycete fungus Alternaria alternata is the causal agent of citrus brown spot, which can result in significant losses of both yield and marketability for tangerines and tangerine hybrids worldwide. A conditionally dispensable chromosome (CDC), which harbors the host-selective ACT toxin gene cluster, is required for tangerine pathogenicity of A. alternata. To understand the genetic makeup and evolution of the tangerine pathotype CDC, we analyzed the function and evolution of the CDC genes present in the A. alternata Z7 strain. The 1.84Mb long CDC contains 512 predicted protein-coding genes, which are enriched in functional categories associated with ‘metabolic process’ (132 genes, p-value = 0.00192) including ‘oxidation-reduction process’ (48 genes, p-value = 0.00021) and ‘lipid metabolic process’ (11 genes, p-value = 0.04591). Relatively few of the CDC genes can be classified as CAZymes (13), kinases (3) and transporters (20). Differential transcriptome analysis of H2O2 treatment and control conditions revealed that 29 CDC genes were significantly up-regulated and 14 were significantly down-regulated, suggesting that CDC genes may play a role in coping with oxidative stress. Evolutionary analysis of the 512 CDC proteins showed that their evolutionary conservation tends to be restricted within the genus Alternaria and that the CDC genes evolve faster than genes in the essential chromosomes. Interestingly, phylogenetic analysis suggested that the genes of 13 enzymes and one sugar transporter residing in the CDC were likely horizontally transferred from distantly related species. Among these, one carboxylesterase gene was transferred from bacteria but functionally knocking out this gene revealed no obvious biological role. Another 4 genes might have been transferred from Colletotrichum (Sordariomycetes) and 5 were likely transferred as a physically linked cluster of genes from Cryptococcus (Basidiomycota) or Penicillium (Eurotiomycetes). Functionally knocking out the 5-gene cluster resulted in an 80% decrease in asexual spore production in the deletion mutant. These results provide new insights into the function and evolution of CDC genes in Alternaria.Author SummaryMany fungal phytopathogens harbor conditionally dispensable chromosomes (CDCs). CDCs are variable in size, contain many genes involved in virulence, but their evolution remains obscure. In this study, we investigated the origin of the CDC present in the tangerine pathotype of Alternaria alternata Z7 strain. We found that most of the Z7 CDC proteins are highly conserved within the genus Alternaria but poorly conserved outside the genus. We also discovered that a small number of genes originated via horizontal gene transfer (HGT) from distantly related fungi and bacteria. These horizontally transferred genes include a carboxylesterase gene that was likely acquired from bacteria, a cluster of 4 physically linked genes likely transferred from Colletotrichum, and a cluster of 5 physically linked genes likely transferred from Cryptococcus (Basidiomycota) or Penicillium (Eurotiomycetes). To gain insight into the functions of these transferred genes, we knocked out the bacterial carboxylesterase and the 5-gene cluster. Whereas the carboxylesterase deletion mutant showed no obvious phenotype, the 5-gene cluster mutant showed a dramatically reduced production of asexual spores (conidia). The results of our study suggest that Alternaria CDCs are largely comprised from rapidly evolving native genes; although only a few genes were acquired via horizontal gene transfer, some of them appear to be involved in functions critical to the phytopathogenic lifestyle.

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First Report of Alternaria Brown Spot of Citrus Caused by Alternaria alternata in Yunnan Province, China

Plant Disease ◽

10.1094/pdis-94-3-0375c ◽

2010 ◽

Vol 94 (3) ◽

pp. 375-375 ◽

Cited By ~ 7

Author(s):

X. F. Wang ◽

Z. A. Li ◽

K. Z. Tang ◽

C. Y. Zhou ◽

L. Yi

Keyword(s):

Alternaria Alternata ◽

Yunnan Province ◽

Morphological Characteristics ◽

Control Measures ◽

Brown Spot ◽

Control Treatment ◽

Infected Leaf ◽

Conidial Suspension ◽

First Report ◽

Alternaria Brown Spot

Brown spot of citrus is considered a major problem on the fruit of many citrus cultivars grown for fresh markets including tangerines (Citrus reticulata) and their hybrids. It causes lesions on leaves, stems, and fruit and reduces yield and fruit quality (2). In 2003 in southern Wenshan Municipality, Yunnan Province in China, sporadic occurrence of Alternaria brown spot was observed on Tangfang mandarin, a local citrus cultivar identified preliminarily as a kind of mandarin hybrid. From 2006 to 2008, nearly 80% of local orchards were infected with the disease. Fruit symptoms typical of Alternaria brown spot ranging from light brown, slightly depressed spots to circular and dark brown areas were observed. Leaves showed small, brown, circular spots and irregular blighted areas with characteristic yellow halos. Tissues from the margin of fruit spots or infected leaf parts of eight different trees were surface sterilized in 1.5% sodium hypochlorite for 1 min, plated on potato dextrose agar (PDA), and then incubated at 27°C in the dark for 1 week. Dark brown mycelia and pigmented septate conidia with lengths of 10 to 35 μm and widths of 5 to 13 μm were produced. On the basis of conidial morphological characteristics, the pathogen was identified as Alternaria alternata (Fr.:Fr.) Keissl (1). Detached young healthy leaves of ‘Minneola’ tangelo (C. reticulata × C. paradisi) were sprayed with a conidial suspension of 105 conidia per ml and incubated in a moist chamber at 27°C. A control treatment with an equal number of leaves was sprayed with distilled water only. After 48 h, seven of these isolates caused necrotic lesions on detached leaves, characteristic of the disease, whereas there were no symptoms on leaves of the water control. Pure cultures were recovered on PDA from symptomatic tissues and the morphological characteristics of the conidia closely fit the description of A. alternata, confirming Koch's postulates. Currently, the distribution of Alternaria brown spot of citrus is confined to southern Wenshan Municipality in Yunnan Province where it is a serious disease problem on the most important commercial cultivar in this region. The identification of the pathogen now allows for appropriate field management and control measures. To our knowledge, this is the first report of Alternaria brown spot of citrus in China. References: (1) Z. Solel. Plant Pathol. 40:145, 1991. (2) J. O. Whiteside. Plant Dis. Rep. 60:326, 1976.

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