A Rapid Resazurin-Based Microtiter Assay to Evaluate QoI Sensitivity for Alternaria alternata Isolates and Their Molecular Characterization

Chemical management of Alternaria brown spot of citrus is based upon the timely application of site-specific fungicides, many of which are vulnerable to the development of fungicide resistance. A rapid microtiter bioassay based on the colorimetric changes of resazurin (RZ) dye was developed to evaluate the sensitivity of Alternaria alternata to quinone outside inhibitor (QoI) fungicides. Four liquid media (complete medium, minimal medium, potato dextrose broth, and yeast peptone dextrose broth), five conidia concentrations (from 101 to 105 conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 μM) were evaluated. Complete medium at 105 conidia/ml and 40 μM RZ were identified as optimal for measuring RZ reduction. The effective concentration of two QoI fungicides (azoxystrobin and pyraclostrobin) needed to reduce RZ by 50% (EC50) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC50 values were observed (R2 = 0.923; P < 0.0001) from both methods. Resistant phenotypes were further characterized by the partial sequencing of the cytochrome b gene. Genetic variability associated with the presence or absence of two introns was observed among isolates. The identified resistant isolates had the amino acid substitution G143A, typical of QoI resistance in other fungi.

Download Full-text

Distribution of QoI Resistance in Populations of Tangerine-Infecting Alternaria alternata in Florida

Plant Disease ◽

10.1094/pdis-04-13-0449-re ◽

2014 ◽

Vol 98 (1) ◽

pp. 67-76 ◽

Cited By ~ 15

Author(s):

Byron Vega ◽

Megan M. Dewdney

Keyword(s):

Alternaria Alternata ◽

Brown Spot ◽

Cross Resistance ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Citrus Orchards ◽

Application Frequency ◽

Polymerase Chain ◽

Highly Correlated ◽

Qoi Resistance

Chemical control, based on copper and quinone outside inhibitor (QoI) fungicides, has been essential for the management of brown spot of citrus, caused by Alternaria alternata. However, QoI control failures were detected recently in Florida. From 2008 to 2012, 817 monoconidial isolates of A. alternata from 46 citrus orchards were examined for sensitivity to azoxystrobin (AZ) and pyraclostrobin (PYR). Of the isolates, 57.6% were resistant to both fungicides, with effective concentration to inhibit 50% growth (EC50) values greater than 5 μg/ml for AZ and 1 μg/ml for PYR. The mean EC50 values for sensitive isolates were 0.139 and 0.020 μg/ml for AZ and PYR, respectively. The EC50 values of both fungicides were highly correlated (P < 0.0001), indicating cross resistance. The proportion of resistant isolates differed significantly (P < 0.0001) among cultivars and with QoI application frequency (P < 0.0001). However, resistance was not significantly related (P = 0.364) to disease severity in the field (low, moderate, and high) or isolate virulence (P = 0.397). The molecular basis for QoI resistance was determined for a subset of 235 isolates using polymerase chain reaction restriction fragment length polymorphism of the cytochrome b gene. All resistant isolates showed the point mutation G143A. Based on the presence of one or two introns, isolates were classified as profile I and profile II, respectively. The resistance frequency was significantly higher (P < 0.0001) in isolate profile II, suggesting a higher selection pressure for resistant population profile II.

Download Full-text

First Report on Quinone Outside Inhibitor Resistance of Alternaria alternata Causing Alternaria Brown Spot in Tangerines in São Paulo, Brazil

Plant Health Progress ◽

10.1094/php-12-18-0079-br ◽

2019 ◽

Vol 20 (2) ◽

pp. 94-94 ◽

Cited By ~ 1

Author(s):

Gabriel M. Chitolina ◽

Geraldo J. Silva-Junior ◽

Eduardo Feichtenberger ◽

Rosana G. Pereira ◽

Lilian Amorim

Keyword(s):

Alternaria Alternata ◽

Sao Paulo ◽

Field Resistance ◽

Brown Spot ◽

São Paulo ◽

Qoi Fungicides ◽

First Report ◽

Quinone Outside Inhibitor ◽

Alternaria Brown Spot ◽

Inhibitor Resistance

Field resistance to quinone outside inhibitor (QoI) fungicides in Alternaria alternata causing Alternaria brown spot of mandarins was reported by growers in Brazil in 2017. Conidial germination tests were performed, and isolates showed effective concentration to inhibit 50% of conidia germination (EC50) to be over 100 ppm. This is the first report of QoI resistance in A. alternata causing Alternaria brown spot in the state of São Paulo, Brazil.

Download Full-text

Distribution and Stability of Quinone Outside Inhibitor Fungicide Resistance in Populations of Potato Pathogenic Alternaria spp. in Wisconsin

Plant Disease ◽

10.1094/pdis-11-18-1978-re ◽

2019 ◽

Vol 103 (8) ◽

pp. 2033-2040 ◽

Cited By ~ 1

Author(s):

Shunping Ding ◽

Dennis A. Halterman ◽

Kiana Meinholz ◽

Amanda J. Gevens

Keyword(s):

Cytochrome B ◽

Point Mutations ◽

Alternaria Solani ◽

Cytochrome B Gene ◽

Brown Spot ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Before And After ◽

Pathogen Populations ◽

Qoi Resistance

Quinone outside inhibitor (QoI) fungicides have been an important class in managing potato early blight caused by Alternaria solani and brown spot caused by A. alternata. Because of the single-site mode of action character of QoI fungicides, which are relied on for management of diseases in Wisconsin, and the abundant asexual conidia production of the Alternaria species, pathogen isolates with QoI resistance have been detected after just a few years of QoI fungicide usage in commercial production fields. Resistance to QoIs has been attributed to amino acid substitutions F129L and G143A in cytochrome b of A. solani and A. alternata, respectively, as a result of point mutations. The aim of this study was to assess Alternaria populations in Wisconsin for QoI resistance before and after fungicide applications in order to evaluate resistance stability. A TaqMan single nucleotide polymorphism genotyping assay was designed based on the sequences of the cytochrome b gene from Alternaria isolates collected in Wisconsin to profile QoI resistance in Alternaria populations as well as to explore factors that may influence frequency of QoI resistance in the pathogen populations. This assay successfully identified the mutations conferring QoI resistance in isolates collected from four locations each year from 2015 to 2017. During the course of this study, the frequency of A. solani isolates with the F129L mutation was consistently high and showed primarily the TTA mutation type. The frequency of A. alternata isolates with the G143A mutation started relatively low and increased at the end of the production season in each year (P = 0.0109, P = 0.2083, and P = 0.0159). A potato field managed without use of QoI fungicides showed a significantly lower (P < 0.05) frequency of A. alternata isolates carrying G143A than conventionally managed potato fields. The overall frequency of A. alternata isolates carrying G143A in the four locations was similar over the 3 years (P = 0.2971). The QoI resistance characteristics of the isolates were stable even when QoI selection pressure was removed for at least five subculture transfers, and the mutation types of codons 129 and 143 in the cytochrome b gene in A. solani and A. alternata, respectively, remained the same. This indicated that the application of QoIs in the field is not the sole factor responsible for the variation of the frequency of QoI resistance in the pathogen populations.

Download Full-text

Sensitivity of Bipolaris oryzae Isolates Pathogenic on Cultivated Wild Rice to the Quinone Outside Inhibitor Azoxystrobin

Plant Disease ◽

10.1094/pdis-12-18-2267-re ◽

2019 ◽

Vol 103 (8) ◽

pp. 1910-1917 ◽

Cited By ~ 2

Author(s):

Claudia V. Castell-Miller ◽

Deborah A. Samac

Keyword(s):

Cytochrome B ◽

Fungicide Resistance ◽

Wild Rice ◽

Brown Spot ◽

Bipolaris Oryzae ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Group I ◽

Mitochondrial Cytochrome B

The occurrence of fungal brown spot, caused by Bipolaris oryzae, has increased in cultivated wild rice (Zizania palustris) paddies in spite of the use of azoxystrobin-based fungicides. The active ingredient blocks electron transfer at the quinone outside inhibitor (QoI) site in the mitochondrial cytochrome b within the bc1 complex, thus obstructing respiration. The in vitro averaged EC50 of baseline isolates collected in 2007 before widespread fungicide use was estimated to be 0.394 µg/ml with PROBIT and 0.427 µg/ml with linear regression analyses. Isolates collected during 2008, 2015, and 2016 had a range of sensitivity as measured by relative spore germination (RG) at a discriminatory dose of 0.4 µg/ml azoxystrobin. Isolates with a higher (≥80%) and lower RG (≤40%) had the wild type nucleotides at amino acid positions F129, G137, and G143 of cytochrome b, sites known to be associated with QoI fungicide resistance. Two Group I introns were found in the QoI target area. The splicing site for the second intron was found immediately after the codon for G143. A mutation for fungicide resistance at this location would hinder splicing and severely reduce fitness. B. oryzae expresses an alternative oxidase in vitro, which allows the fungus to survive inhibition of respiration by azoxystrobin. This research indicates that B. oryzae has not developed resistance to QoI fungicides, although monitoring for changes in sensitivity should be continued. Judicious use of QoI fungicides within an integrated disease management system will promote an effective and environmentally sound control of the pathogen in wild rice paddies.

Download Full-text

Widespread QoI Fungicide Resistance Revealed Among Corynespora cassiicola Tomato Isolates in Florida

Plant Disease ◽

10.1094/pdis-03-19-0460-re ◽

2020 ◽

Vol 104 (3) ◽

pp. 893-903 ◽

Cited By ~ 2

Author(s):

Keevan J. MacKenzie ◽

Katia V. Xavier ◽

Aimin Wen ◽

Sujan Timilsina ◽

Heather M. Adkison ◽

...

Keyword(s):

Amino Acid Position ◽

Rapid Screening ◽

Silent Mutation ◽

Screening Methods ◽

Corynespora Cassiicola ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Target Spot ◽

Moderately Resistant ◽

Qoi Resistance

Target spot of tomato caused by Corynespora cassiicola is one of the most economically destructive diseases of tomato in Florida. A collection of 123 isolates from eight counties in Florida were evaluated for sensitivity to azoxystrobin and fenamidone based on mycelial growth inhibition (MGI), spore germination (SG), detached leaflet assays (DLAs), and sequence-based analysis of the cytochrome b gene (cytb). Cleavage of cytb by restriction enzyme (Fnu4HI) revealed the presence of a mutation conferring a glycine (G) to alanine (A) mutation at amino acid position 143 (G143A) in approximately 90% of the population, correlating with quinone outside inhibitor (QoI) resistance based on MGI (<40% at 5 μg/ml), SG (<50% at 1 and 10 μg/ml), and DLA (<10% severity reduction). The mutation conferring a phenylalanine (F) to leucine (L) substitution at position 129 (F129L) was confirmed in moderately resistant isolates (#9, #19, and #74) based on MGI (40 to 50% at 5 μg/ml), SG (<50% at 1 μg/ml and >50% at 10 μg/ml), and DLA (>10% and <43% severity reduction) for both QoI fungicides, whereas sensitive isolates (#1, #4, #7, #28, #29, #46, #61, #74, #75, #76, #91, #95, and #118) based on MGI (>50% at 5 μg/ml), SG (>50% at 1 μg/ml and 10 μg/ml), and DLA (>50% severity reduction) correlated to non-mutation-containing isolates or those with a silent mutation. This study indicates that QoI resistance among C. cassiicola isolates from tomato is widespread in Florida and validates rapid screening methods using MGI or molecular assays to identify resistant isolates in future studies.

Download Full-text

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.

Download Full-text

Distribution of Alternaria alternata isolates with resistance to quinone outside inhibitor (QoI) fungicides in Brazilian orchards of tangerines and their hybrids

Crop Protection ◽

10.1016/j.cropro.2020.105493 ◽

2021 ◽

Vol 141 ◽

pp. 105493

Author(s):

Gabriel Moraes Chitolina ◽

Geraldo José Silva-Junior ◽

Eduardo Feichtenberger ◽

Rosana G. Pereira ◽

Lilian Amorim

Keyword(s):

Alternaria Alternata ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text