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

2010 ◽  
Vol 100 (2) ◽  
pp. 120-126 ◽  
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.

scholarly journals ACTTS3 Encoding a Polyketide Synthase Is Essential for the Biosynthesis of ACT-Toxin and Pathogenicity in the Tangerine Pathotype of Alternaria alternata

2010 ◽  
Vol 23 (4) ◽  
pp. 406-414 ◽  
Author(s):  
Y. Miyamoto ◽  
A. Masunaka ◽  
T. Tsuge ◽  
M. Yamamoto ◽  
K. Ohtani ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Polyketide Synthase ◽  
Essential Gene ◽  
Attachment Site ◽  
Toxin Production ◽  
Open Reading Frames ◽  
Brown Spot ◽  
Bac Clone ◽  
Chain Reactions ◽  
Highly Correlated

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerine and tangerine hybrids. Sequence analysis of a genomic BAC clone identified part of the ACT-toxin TOX (ACTT) gene cluster, and knockout experiments have implicated several open reading frames (ORF) contained within the cluster in the biosynthesis of ACT-toxin. One of the ORF, designated ACTTS3, encoding a putative polyketide synthase, was isolated by rapid amplification of cDNA ends and genomic/reverse transcription-polymerase chain reactions using the specific primers designed from the BAC sequences. The 7,374-bp ORF encodes a polyketide synthase with putative β-ketoacyl synthase, acyltransferase, methyltransferase, β-ketoacyl reductase, and phosphopantetheine attachment site domains. Genomic Southern blots demonstrated that ACTTS3 is present on the smallest chromosome in the tangerine pathotype of A. alternata, and the presence of ACTTS3 is highly correlated with ACT-toxin production and pathogenicity. Targeted gene disruption of two copies of ACTTS3 led to a complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS3 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.

scholarly journals Role of the Pathotype-Specific ACRTS1 Gene Encoding a Hydroxylase Involved in the Biosynthesis of Host-Selective ACR-Toxin in the Rough Lemon Pathotype of Alternaria alternata

2012 ◽  
Vol 102 (8) ◽  
pp. 741-748 ◽  
Author(s):  
Yuriko Izumi ◽  
Eri Kamei ◽  
Yoko Miyamoto ◽  
Kouhei Ohtani ◽  
Akira Masunaka ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Essential Gene ◽  
Toxin Production ◽  
Open Reading Frames ◽  
Leaf Spot Disease ◽  
Bac Clone ◽  
Biosynthesis Gene Cluster ◽  
Rough Lemon ◽  
Reading Frames

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of the rootstock species rough lemon (Citrus jambhiri) and Rangpur lime (C. limonia). Genes controlling toxin production were localized to a 1.5-Mb chromosome carrying the ACR-toxin biosynthesis gene cluster (ACRT) in the genome of the rough lemon pathotype. A genomic BAC clone containing a portion of the ACRT cluster was sequenced which allowed identification of three open reading frames present only in the genomes of ACR-toxin producing isolates. We studied the functional role of one of these open reading frames, ACRTS1 encoding a putative hydroxylase, in ACR-toxin production by homologous recombination-mediated gene disruption. There are at least three copies of ACRTS1 gene in the genome and disruption of two copies of this gene significantly reduced ACR-toxin production as well as pathogenicity; however, transcription of ACRTS1 and production of ACR-toxin were not completely eliminated due to remaining functional copies of the gene. RNA-silencing was used to knock down the remaining ACRTS1 transcripts to levels undetectable by reverse transcription-polymerase chain reaction. The silenced transformants did not produce detectable ACR-toxin and were not pathogenic. These results indicate that ACRTS1 is an essential gene in ACR-toxin biosynthesis in the rough lemon pathotype of A. alternata and is required for full virulence of this fungus.

scholarly journals Function of Genes Encoding Acyl-CoA Synthetase and Enoyl-CoA Hydratase for Host-Selective ACT-Toxin Biosynthesis in the Tangerine Pathotype of Alternaria alternata

2009 ◽  
Vol 99 (4) ◽  
pp. 369-377 ◽  
Author(s):  
Y. Miyamoto ◽  
Y. Ishii ◽  
A. Honda ◽  
A. Masunaka ◽  
T. Tsuge ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Gene Disruption ◽  
Toxin Production ◽  
Brown Spot ◽  
Acid Moiety ◽  
Targeted Gene Disruption ◽  
Brown Spot Disease ◽  
Alternaria Brown Spot ◽  
Genes Encoding ◽  
Enoyl Coa Hydratase

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease. Sequence analysis of a genomic cosmid clone identified a part of the ACTT gene cluster and implicated two genes, ACTT5 encoding an acyl-CoA synthetase and ACTT6 encoding an enoyl-CoA hydratase, in the biosynthesis of ACT-toxin. Genomic Southern blots demonstrated that both genes were present in tangerine pathotype isolates producing ACT-toxin and also in Japanese pear pathotype isolates producing AK-toxin and strawberry pathotype isolates producing AF-toxin. ACT-, AK-, and AF-toxins from these three pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Targeted gene disruption of two copies of ACTT5 significantly reduced ACT-toxin production and virulence. Targeted gene disruption of two copies of ACTT6 led to complete loss of ACT-toxin production and pathogenicity and a putative decatrienoic acid intermediate in ACT-toxin biosynthesis accumulated in mycelial mats. These results indicate that ACTT5 and ACTT6 are essential genes in ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and both are required for full virulence of this fungus.

scholarly journals Functional Analysis of a Multicopy Host-Selective ACT-Toxin Biosynthesis Gene in the Tangerine Pathotype of Alternaria alternata Using RNA Silencing

2008 ◽  
Vol 21 (12) ◽  
pp. 1591-1599 ◽  
Author(s):  
Y. Miyamoto ◽  
A. Masunaka ◽  
T. Tsuge ◽  
M. Yamamoto ◽  
K. Ohtani ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Rna Silencing ◽  
Toxin Production ◽  
Brown Spot ◽  
Plasmid Construct ◽  
Dispensable Chromosome ◽  
Alternaria Brown Spot ◽  
Multiple Copies ◽  
Commercially Important ◽  
Serious Disease

Alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is a serious disease of commercially important tangerines and their hybrids. The pathogen produces host-selective ACT toxin, and several genes (named ACTT) responsible for ACT-toxin biosynthesis have been identified. These genes have many paralogs, which are clustered on a small, conditionally dispensable chromosome, making it difficult to disrupt entire functional copies of ACTT genes using homologous recombination-mediated gene disruption. To overcome this problem, we attempted to use RNA silencing, which has never been employed in Alternaria spp., to knock down the functional copies of one ACTT gene with a single silencing event. ACTT2, which encodes a putative hydrolase and is present in multiple copies in the genome, was silenced by transforming the fungus with a plasmid construct expressing hairpin ACTT2 RNAs. The ACTT2 RNA-silenced transformant (S-7-24-2) completely lost ACTT2 transcripts and ACT-toxin production as well as pathogenicity. These results indicated that RNA silencing may be a useful technique for studying the role of ACTT genes responsible for host-selective toxin biosynthesis in A. alternata. Further, this technique may be broadly applicable to the analysis of many genes present in multiple copies in fungal genomes that are difficult to analyze using recombination-mediated knockdowns.

scholarly journals Distribution and Characterization of AKT Homologs in the Tangerine Pathotype of Alternaria alternata

2000 ◽  
Vol 90 (7) ◽  
pp. 762-768 ◽  
Author(s):  
A. Masunaka ◽  
A. Tanaka ◽  
T. Tsuge ◽  
T. L. Peever ◽  
L. W. Timmer ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
Toxin Production ◽  
Brown Spot ◽  
Japanese Pear ◽  
Black Rot ◽  
Acid Moiety ◽  
High Sequence Similarity ◽  
Rough Lemon

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.

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

Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 231-239 ◽  
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.

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

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.

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

Plant Disease ◽  
2010 ◽  
Vol 94 (3) ◽  
pp. 375-375 ◽  
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.

scholarly journals Evaluation of the Alter-Rater Model for Timing of Fungicide Applications for Control of Alternaria Brown Spot of Citrus

Plant Disease ◽  
2003 ◽  
Vol 87 (9) ◽  
pp. 1089-1093 ◽  
Author(s):  
Alka Bhatia ◽  
P. D. Roberts ◽  
L. W. Timmer
Keyword(s):  
Disease Control ◽  
Fruit Quality ◽  
Alternaria Alternata ◽  
The Other ◽  
Brown Spot ◽  
Leaf Wetness ◽  
Yield Losses ◽  
Copper Fungicides ◽  
Alternaria Brown Spot

Alternaria brown spot, caused by Alternaria alternata, results in serious yield losses of tangerines and their hybrids in Florida. The Alter-Rater model predicts the need for fungicide applications based on daily cumulative points that are assigned on the basis of rainfall, leaf wetness, and temperature. Previously, Alter-Rater threshold or trigger values of 50, 75, 100, and 150 points for application of copper fungicides were suggested for groves with different cultivars and disease histories. In this study, we evaluated thresholds of 50, 100, and 150 points in four Minneola tangelo and Murcott tangor groves in 2000 and 2001. For comparison, copper fungicides were applied according to the DISC Copper Model in 2000 and according to calendar sprays in 2001. Use of the Alter-Rater model resulted in fewer sprays in three of the four groves in 2000 and better fruit quality in the other grove than the Copper Model. Compared to a calendar spray schedule in 2001, use of the Alter-Rater model resulted in fewer sprays in two of the four groves but more sprays in one grove. The results confirmed that the Alter-Rater is a valuable tool for timing fungicide applications and that its use results in better disease control or fewer sprays.

scholarly journals Positive relationship between citrus leaf miner and alternaria brown spot

2015 ◽  
Vol 45 (7) ◽  
pp. 1160-1163 ◽  
Author(s):  
Fernando Alves de Azevedo ◽  
Ivan Bortolato Martelli ◽  
Denis Augusto Polydoro ◽  
Camilla de Andrade Pacheco ◽  
Evandro Henrique Schinor ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Brown Spot ◽  
Leaf Miner ◽  
Lesion Area ◽  
Citrus Leaf ◽  
The World ◽  
Alternaria Brown Spot ◽  
Detached Leaves ◽  
The Relationship ◽  
Diagrammatic Scale

Alternaria brown spot, or ABS (Alternaria alternata), is the most prevalent fungal disease of tangerines in the world. Field observations have revealed ABS lesions on leaves wounded by the citrus leaf miner (CLM). Thus, the objective of this study was to assess the relationship between CLM and ABS. Leaves from young Murcott tangor plants and detached leaves from 16 different varieties of tangerines with and without lesions caused by CLM were inoculated with A. alternata. The symptoms of the plants were subsequently quantified by counting the number of lesions, and the lesion area was estimated using a diagrammatic scale. The presence of CLM damage aggravates the severity of Alternaria alternata fungus infections in susceptible tangerine varieties.

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