First Report of Fludioxonil Resistance in Botrytis cinerea from a Blackberry Field in Georgia

Botrytis cinerea Pers. is an important plant-pathogenic fungi responsible for gray mold on more than 230 plant species worldwide, including blackberry (Rubus). One of the main strategies to control the disease involves the application of different classes of fungicides. The phenylpyrrole fludioxonil is currently marketed in combination with the anilinopyrimidine cyprodinil as Switch 62.5WG (Syngenta Crop Protection Inc., Greensboro, NC) for gray mold control. In August 2013, blackberries affected with symptoms resembling gray mold were collected from a field located in Berrien County (Georgia), where Switch 62.5WG had been used extensively over the last 5 years. Three single-spore isolates, each from a different fruit, were obtained and identified as B. cinerea on the basis of morphology and confirmed by a 238-bp PCR amplification product obtained with primer set G3PDH-F1 (5′-GGACCCGAGCTAATTTATGTCACGT-3′), G3PDH-F2 (5′-GGGTGTCAACAACGAGACCTACACT-3′), and G3PDH-R (5′-ACCGGTGCTCGATGGGATGAT-3′). In vitro sensitivity to fludioxonil (Scholar SC, Syngenta) was determined on 1% malt extract agar (MEA) using a conidial germination assay as previously described (4). One isolate was moderately resistant due to growth on medium amended with the discriminatory dose of 0.1 μg/ml fludioxonil and residual growth at 10 μg/ml (4). To assess performance of fludioxonil in detached fruit assays, commercially grown strawberries (24 in total for each isolate and treatment) were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water (control fruit) or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Scholar SC was used because fludioxonil was the sole active ingredient in this product and strawberries were used because latent infections in fresh blackberry fruit interfered with inoculation experiments. This dose reflects the rate recommended for postharvest gray mold control according to the Scholar label. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-μl droplet of conidia suspension (106 spores/ml) of the two sensitive or the resistant isolate. After inoculation, the fruit were kept at 22°C for 4 days. The sensitive isolates developed gray mold on non-treated (2.7 cm lesion diameter) but not on Scholar SC-treated fruit (0.0 cm lesion diameter). The resistant isolate developed gray mold disease on the water-treated control fruit (2.5 cm lesion diameter) and the fungicide-treated fruit (1.8 cm lesion diameter). EC50 values were determined in microtiter assays as described previously (3) using the concentrations of 0.01, 0.04, 0.12, 0.37, 1.1, 3.3, and 10 μg/ml fludioxonil. Values were 0.02 and 0.05 μg/ml for the two sensitive isolates and 3.15 μg/ml for the resistant isolate. All experiments were performed twice. This is the first report of fludioxonil resistance in B. cinerea from blackberry in Georgia. Prior to this study, resistance to fludioxonil in B. cinerea was reported in France, Germany, and only a few states in the United States including Maryland, South Carolina, Virginia, and Washington (1,2). The emergence of resistance to fludioxonil emphasizes the importance of resistance management strategies. References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 98:692, 2013. (3) M. Kretschmer et al. PLOS Pathog. 5:e1000696, 2009. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

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First Report of Fludioxonil Resistance in Botrytis cinerea, the Causal Agent of Gray Mold, from Strawberry Fields in Maryland and South Carolina

Plant Disease ◽

10.1094/pdis-08-13-0875-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 692-692 ◽

Cited By ~ 8

Author(s):

D. Fernández-Ortuño ◽

A. Grabke ◽

P. K. Bryson ◽

R. J. Rouse ◽

P. Rollins ◽

...

Keyword(s):

United States ◽

South Carolina ◽

Botrytis Cinerea ◽

Crop Protection ◽

The United States ◽

Gray Mold ◽

Causal Agent ◽

First Report ◽

Lesion Diameter

Botrytis cinerea Pers. is the causal agent of gray mold and one of the most economically important plant-pathogenic fungi affecting strawberry (Fragaria × ananassa). Control of gray mold mainly depends on the use of site-specific fungicides, including the phenylpyrrole fludioxonil. This fungicide is currently registered in combination with cyprodinil in form of Switch 62.5WG (Syngenta Crop Protection, Greensboro, NC) for gray mold control of small fruits in the United States. In June 2013, strawberries affected with symptoms resembling gray mold were observed despite the application of Switch in one field located in Federalsburg, MD, and one located near Chesnee, SC. Ten single-spore isolates, each from a different fruit, were obtained from each location and confirmed to be B. cinerea using cultural and molecular tools as described previously (3). In vitro sensitivity to fludioxonil (Scholar SC, 20.4% [v/v] active ingredient, Syngenta Crop Protection, Greensboro, NC) was determined using a conidial germination assay as previously described (4). Eight of the 20 isolates (six from Maryland and two from South Carolina) were moderately resistant to fludioxonil, i.e., they grew on medium amended with 0.1 μg/ml fludixonil and showed residual growth at 10 μg/ml (4). The in vitro assay was repeated obtaining the same results. To assess in vivo sensitivity on fungicide-treated fruit, commercially grown strawberries were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-μl droplet of conidia suspension (106 spores/ml) of either two sensitive or four resistant isolates (two isolates from Maryland and two isolates from South Carolina). Each isolate/treatment combination consisted of 24 mature but still firm strawberry fruit with three 8-fruit replicates. The fruit were kept at 22°C and lesion diameters were measured after 4 days of inoculation. The sensitive isolates developed gray mold symptoms on nontreated (2.5 cm lesion diameter) but not on Scholar SC-treated fruit. The resistant isolates developed gray mold on both, the water-treated control (2.3 cm lesion diameter), and the fungicide-treated fruit (1.8 cm lesion diameter). The experiment was performed twice. To our knowledge this is the first report of fludioxonil resistance in B. cinerea from strawberry fields in Maryland and South Carolina. Resistance to fludioxonil is still rare in the United States and has only been reported in B. cinerea isolates from a Virginia strawberry field (1). The increase in occurrence of resistance to fludioxonil may be a result of increased use of Switch following reports of resistance to other chemical classes in this pathogen in southern strawberry fields (2). References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 96:1198, 2012. (3) D. Fernández-Ortuño et al. Plant Dis. 95:1482, 2011. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

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First Report of Fludioxonil Resistance in Botrytis cinerea from a Strawberry Field in Virginia

Plant Disease ◽

10.1094/pdis-01-13-0012-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 848-848 ◽

Cited By ~ 12

Author(s):

D. Fernández-Ortuño ◽

P. K. Bryson ◽

A. Grabke ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Botrytis Cinerea ◽

Disease Incidence ◽

Crop Protection ◽

Gray Mold ◽

Resistant Isolate ◽

Single Spore ◽

First Report ◽

Single Spore Isolate

Gray mold caused by Botrytis cinerea Pers.:Fr. is one of the most economically important diseases of cultivated strawberry (Fragaria × ananassa) worldwide. Control of gray mold mainly depends on fungicides, including the phenylpyrrole fludioxonil, which is currently marketed in combination with cyprodinil as Switch 62.5WG (Syngenta Crop Protection, Research Triangle Park, Raleigh, NC). In 2012, 790 strains of B. cinerea were collected from 76 strawberry fields in eight states, including Arkansas, Florida, Georgia, Kansas, Maryland, North Carolina, South Carolina, and Virginia. Strains were collected from sporulating flowers and fruit and sensitivity to fludioxonil was determined using a conidial germination assay as previously described (2). Only one isolate from a farm located in Westmoreland County, Virginia, grew on medium amended with the discriminatory dose of 0.1 μg/ml fludioxonil and was therefore considered low resistant. The isolate did not grow on 10 μg/ml. All other 789 isolates did not grow at either of the two doses. This assay was repeated twice with a single-spore culture of the same strain. In both cases, residual growth was observed on the fludioxonil-amended medium of 0.1 μg/ml. The single spore isolate was confirmed to be B. cinerea Pers. using cultural and molecular tools as described previously (1). To assess resistance in vivo, commercially grown ripe strawberry fruit were rinsed with sterile water, dried, placed into plastic boxes (eight strawberries per box for each of the three replicates per treatment), and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of fludioxonil (Scholar SC, Syngenta) to runoff using a hand mister. This dose reflects the rate recommended for gray mold control according to the Scholar label. Each fruit was stabbed at three equidistant points, each about 1 cm apart and 1 cm deep using a syringe tip. Wounds were injected with a 30-μl droplet of conidia suspension (106 spores/ml) of either 5 sensitive or the resistant isolate. Control fruit were inoculated with water. After inoculation, the fruit were kept at 22°C for 4 days. In two independent experiments, sensitive and low resistant isolates were indistinguishable in pathogenicity on detached, unsprayed fruit. The low resistant isolate developed gray mold disease on all treated and untreated fruit (100% disease incidence) as determined by the absence or presence of gray mold symptoms. The sensitive isolates only developed disease on untreated fruit. The EC50 values, determined in microtiter assays with concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μg/ml fludioxonil, were 0.01 μg/ml for the sensitive isolates and 0.26 μg/ml for the resistant isolate. To our knowledge, this is the first report of fludioxonil resistance in B. cinerea from strawberry in North America. Our monitoring results indicate that resistance is emerging 10 years after the introduction of fludioxonil and stress the importance of chemical rotation for gray mold control. References: (1) X. P. Li et al. Plant Dis. 96:1634, 2012. (2) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

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Characterization of Botrytis cinerea Isolates from Strawberry with Reduced Sensitivity to Polyoxin D Zinc Salt

Plant Disease ◽

10.1094/pdis-02-16-0166-re ◽

2016 ◽

Vol 100 (10) ◽

pp. 2057-2061 ◽

Cited By ~ 5

Author(s):

Madeline E. Dowling ◽

Meng-Jun Hu ◽

Linus T. Schmitz ◽

Jennifer R. Wilson ◽

Guido Schnabel

Keyword(s):

United States ◽

South Carolina ◽

Botrytis Cinerea ◽

Tomato Fruit ◽

Resistance Management ◽

The United States ◽

Gray Mold ◽

Zinc Salt ◽

Malt Extract ◽

Action Committee

Polyoxin D is a Fungicide Resistance Action Committee (FRAC) code 19 fungicide that was recently registered for gray mold control of strawberry in the United States. In this study, we determined the sensitivity to polyoxin D zinc salt (hereafter, polyoxin D) of Botrytis cinerea isolates from 41 commercial strawberry farms in South Carolina, North Carolina, Maryland, Virginia, and Ohio and investigated the fitness of sensitive (S) and reduced sensitive (RS) isolates. Relative mycelial growth ranged between 0 and over 100% on malt extract agar amended with a discriminatory dose of polyoxin D at 5 μg/ml. Isolates that grew more than 70% at that dose were designated RS and were found in three of the five states. The 50% effective dose (EC50) values of three S and three RS isolates ranged from 0.59 to 2.27 and 4.6 to 5.8 μg/ml, respectively. The three RS isolates grew faster on detached tomato fruit treated with Ph-D WDG at recommended label dosage than S isolates (P < 0.008). In all, 25 randomly selected RS isolates exhibited reduced sporulation ability (P < 0.0001) and growth rate (P < 0.0001) but increased production of sclerotia (P < 0.0386) compared with 25 S isolates. Of 10 isolates tested per phenotype, the number of RS isolates producing sporulating lesions on apple, tomato, and strawberry was significantly lower compared with S isolates (P < 0.0001 for each fruit type). The results of this study indicate that resistance management is necessary for fungicides containing polyoxin D. To our knowledge, this is the first study demonstrating reduced sensitivity to FRAC 19 fungicides in B. cinerea isolates from the United States.

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First Report of Iprodione Resistance in Botrytis cinerea on Blackberry from South Carolina

Plant Disease ◽

10.1094/pdis-07-11-0543 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1481-1481

Author(s):

F. P. Chen ◽

X. L. Liu ◽

X. P. Li ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Botrytis Cinerea ◽

Disease Incidence ◽

Gray Mold ◽

Resistant Isolate ◽

Cross Resistance ◽

Broad Host Range ◽

Paper Strip ◽

Single Spore ◽

First Report

Botrytis cinerea Pers.:Fr., is a necrotrophic fungus with a broad host range that causes gray mold on hundreds of plant species (2). Control of gray mold mainly depends on fungicides, including the dicarboxamide iprodione. Thirty-nine diseased blackberry fruit were collected from four orchards in South Carolina and the sensitivity of single-spore isolates to iprodione was examined by Spiral Plater assays (1) on potato dextrose agar (PDA). Briefly, a 5.3 cm long paper strip containing mycelia was placed along the concentration gradient of the PDA and 50% inhibition (EC50 value) was calculated after 2 days of incubation with the Spiral Gradient Endpoint (SGE) software (Spiral Biotech, Norwood, MA). Each isolate was tested in duplicates. Sensitivity ranged from 0.043 to 2.596 μg/ml, with a maximum resistance factor of 60.4. Isolates with EC50 values greater than 2 μg/ml were found in two orchards. Those isolates represented 40 and 7.1% of the total isolates from each orchard. Two isolates with high (EC50 value of 2.596 μg/ml) and low (EC50 value of 0.062 μg/ml) values were chosen to determine the efficacy of iprodione formulated product Rovral 4 Fl (Bayer CropSciences, Research Triangle Park, NC) on detached apple fruit. Fifteen apples were used for each isolate and experiment. Each fruit was wounded on the surface in three locations with a sterile syringe and inoculated with 15 μl of a spore suspension (106 conidia/ml) at the wounded sites. Rovral was applied at the recommended label rate either 24 h before (protective treatment) or 48 h after inoculation (curative treatment). The experiment was conducted three times. Blackberry fruit were not found suitable for this assay because of persistent contamination problems likely from latent infections of a symptomatic fruit. Disease incidence and lesion diameter were recorded 7 days after incubation. Disease incidence following inoculation of the sensitive and resistant isolates on non-fungicide-treated fruit was 100 and 86.7%, respectively. Disease incidence on fungicide-treated apples was 4.4% for the sensitive isolate and 75.6% for the resistant isolate with corresponding mean lesion areas of 0.36 mm and 9.37 mm, respectively. Both isolates were controlled effectively in protective treatments, however, indicating low levels of resistance. To our knowledge, this is the first report of iprodione resistance in B. cinerea from blackberry or any other field-grown crop in South Carolina. This finding adds to a study from 1999 (3) documenting resistance to the dicarboxamide fungicide vinclozolin in B. cinerea collected from ornamentals in South Carolinian greenhouses and suggests that resistance to iprodione needs to be considered in the design of gray mold control strategies in commercial blackberry orchards. No cross resistance between the phenylpyrrole fludioxonil and iprodione was found. References: (1) H. Forster et al. Phytopathology 94:163, 2004. (2) B. Williamson et al. Mol. Plant Pathol. 8:561. 2007. (3) L. F. Yourman and S. N. Jeffers. Plant Dis. 83:569, 1999.

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First Report of Gray Mold of Blackberry Caused by Botrytis cinerea in South Carolina

Plant Disease ◽

10.1094/pdis-06-11-0530 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1592-1592 ◽

Cited By ~ 1

Author(s):

X. Li ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Botrytis Cinerea ◽

Signs And Symptoms ◽

Gray Mold ◽

Fluorescent Light ◽

Its2 Region ◽

Conidial Suspension ◽

Heat Shock Protein 60 ◽

First Report ◽

Control Fruit

Botrytis cinerea Pers.: Fr. is a causal agent of gray mold of blackberry but may also affect grapevine, tomato, bulb flowers, and ornamental crops (2). In August 2010, blackberries (Rubus fruticosus and other species) showing gray mold symptoms were found in Longcreek, Six Mile, and Cheddar, SC. Symptomatic blackberry fruit exhibited patterns of brown-to-gray mycelia and conidiophores. Upon isolation, the mycelium grew at a rate of 12.3 mm per day at 22°C on potato dextrose agar, forming pale white-to-gray colonies with concentric rings and conidiophores (less than 12 h of fluorescent light per day). Some isolates formed dark brown sclerotia in the dark after 18 days. The lemon-shaped spores averaged 12 × 9 μm and were consistent with descriptions of B. cinerea. (1) The ribosomal internal transcribed spacer (ITS) ITS1-5.8S-ITS2 region was amplified via PCR from genomic DNA obtained from mycelia using primers ITS1 and ITS4. A BLAST search in GenBank revealed highest similarity (99 to 100%) to sequences from various Botrytis spp. collected in China, Canada, and Spain (GenBank Accession Nos. FJ169666.1, GU934505.1, and EF207414.1). The ITS sequence amplified from the blackberry isolate was submitted to GenBank (Accession No. JN164269). The pathogen was further identified to the species level as B. cinerea using glyceraldehyde-3-phosphate dehydrogenase, heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) gene sequences (2) (GenBank Accession Nos. JN164270, JN164271, JN164272). Pathogenicity was confirmed by inoculating three surface-sterilized (soaked in 5% bleach for 15 min), mature blackberry fruit (R. fruticosus) with a conidial suspension (105 spores/ml) of the blackberry isolate. A 20-μl droplet was placed on the fruit; control fruit received sterile water without conidia. After 5 days of incubation at room temperature in an air-tight Magenta box, the inoculated fruit developed typical signs and symptoms of gray mold. The developing spores on inoculated fruit were confirmed to be B. cinerea. All control fruit remained healthy. To our knowledge, this is the first report of B. cinerea on blackberry in South Carolina. The disease must be managed with fungicides to obtain high quality fruit with market-requested shelf life. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , June 17, 2011. (2) M. Staats et al. Mol. Biol. Evol. 22:333, 2005.

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First Report of Laurel Wilt Caused by Raffaelea lauricola on Sassafras in Mississippi

Plant Disease ◽

10.1094/pdis-05-11-0446 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1479-1479 ◽

Cited By ~ 10

Author(s):

J. J. Riggins ◽

S. W. Fraedrich ◽

T. C. Harrington

Keyword(s):

United States ◽

South Carolina ◽

The United States ◽

Ambrosia Beetle ◽

Malt Extract ◽

Laurel Wilt ◽

First Report ◽

Raffaelea Lauricola ◽

Redbay Ambrosia Beetle ◽

Soil Interface

Laurel wilt is caused by the fungus Raffaelea lauricola T.C. Harrin., Aghayeva & Fraedrich and is lethal to redbay (Persea borbonia (L.) Spreng.), sassafras (Sassafras albidum (Nutt.) Nees), and other species in the Lauraceae (1). The fungus is carried by the redbay ambrosia beetle (Xyleborus glabratus Eichh.), which is native to Asia. After being discovered in Georgia in 2002 (1), X. glabratus and R. lauricola have spread rapidly, causing extensive redbay mortality in South Carolina, Georgia, Florida, and Mississippi (1,4). The disease has also been confirmed on sassafras in Florida, South Carolina (1), and Georgia. Questions remain as to whether laurel wilt will continue to spread on sassafras, which often occurs as scattered trees in the eastern United States. In June 2010, a homeowner reported that a sassafras tree north of Van Cleave, MS (30.668°N, 88.686°W) had begun wilting in late May. This landscape tree had three 10-m high stems (~20 cm in diameter at breast height). Dark staining in the xylem was observed around the entire circumference of all three stems and nearly all leaves were bronze colored and wilted. No ambrosia beetle tunnels were observed in the stems. No other symptomatic Lauraceae were encountered in the wooded area within 300 m. The nearest known location with laurel wilt on redbay was ~15 km away (4). A Lindgren funnel trap baited with manuka oil (2) was placed at the site in June and monitored biweekly until November, but no X. glabratus adults were captured. Chips from discolored xylem of the sassafras were surface sterilized, plated on cycloheximide-streptomycin malt agar, and R. lauricola was readily isolated (1). Identity of the fungus (isolate C2792 in collection of T. Harrington) was confirmed by using partial sequences of the 28S rDNA (3). The sassafras sequence was identical to that of all known sequences of R. lauricola in the United States, including GenBank No. EU123076 (the holotype isolate from redbay). To confirm pathogenicity, isolate C2792 was grown on malt extract agar and three redbay (average: 141 cm high and 12 mm in diameter at soil interface) and three sassafras (average: 170 cm high and 17 mm in diameter at soil interface) potted plants were wound inoculated with 0.2 ml of a spore suspension (4.9 × 106 conidia/ml) (1). Three control plants of each species were inoculated with sterile deionized water. After 8 weeks in a growth chamber at 26°C, all inoculated redbay and sassafras plants exhibited xylem discoloration above and below the inoculation point, two of the redbay and two of the sassafras had died, and the other plant of each species exhibited partial wilt (the main terminal or one or more branches). All control plants were asymptomatic. R. lauricola was reisolated from all inoculated symptomatic plants but not from controls. To our knowledge, this is the first report of laurel wilt on sassafras in Mississippi. Both redbay (4) and sassafras appear to be highly susceptible to the disease as it moves westward. Sassafras is less attractive than redbay to X. glabratus and it was thought that this might contribute to slowing the spread of laurel wilt once outside the range of redbay (2). Nonetheless, our observations confirm that sassafras can be infected where laurel wilt on redbay is not in the immediate vicinity. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. L. Hanula et al. J. Econ. Entomol. 101:1276, 2008. (3) T. C. Harrington et al. Mycotaxon 111:337, 2010. (4) J. J. Riggins et al. Plant Dis. 94:634, 2010.

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First Report on Resistance to Pyraclostrobin, Thiophanate-methyl, Fenhexamid and Boscalid in Botrytis cinerea from Eucalyptus Seedlings in Florida Greenhouses

Plant Disease ◽

10.1094/pdis-10-13-1107-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 851-851 ◽

Cited By ~ 6

Author(s):

A. Amiri ◽

A. I. Zuniga ◽

J. Mertely ◽

N. A. Peres

Keyword(s):

Botrytis Cinerea ◽

Germ Tube ◽

Crop Protection ◽

Malt Extract ◽

Thiophanate Methyl ◽

First Report ◽

Research Triangle ◽

Extract Agar ◽

Bayer Cropscience ◽

Syngenta Crop Protection

Botryotinia fuckeliana de Bary (anamorph Botrytis cinerea Pers.) is an ubiquitous plant pathogen causing gray mold disease on more than 200 crops grown in the field or in greenhouses. Eucalyptus seedlings originating from three different greenhouses showing stem lesions were submitted to the Gulf Coast Research and Education Center Disease Clinic in June 2012. Ten single spore isolates of B. cinerea were obtained and tested for sensitivity using spore germination and germ tube elongation assays described previously (4). Fungicides tested were pyraclostrobin at 100 μg/ml (Cabrio, BASF, Research Triangle Park, NC), thiophanate-methyl at 100 μg/ml (Topsin-M, UPI, King of Prussia, PA), fenhexamid at 1 and 50 μg/ml (Elevate, Arysta Life Sciences, Cary, NC), fludioxonil at 0.1 and 10 μg/ml (Medallion, Syngenta Crop Protection, Research Triangle Park, NC), and iprodione at 5 and 50 μg/ml (Rovral, Bayer CropScience, Greensboro, NC) on 1% malt extract agar (MEA, 10 g malt extract and 15 g agar), and to cyprodinil at 1 and 25 μg/ml (Vanguard, Syngenta Crop Protection) on 0.5% sucrose agar (4). Sensitivity to the succinate dehydrogenase inhibitors (SDHIs) boscalid at 5 μg/ml (Endura, BASF), penthiopyrad at 1 and 3 μg/ml (Fontelis, DuPont Crop Protection, Willington, DE), and fluopyram at 3 μg/ml (Luna Privilege, Bayer CropScience) was evaluated on yeast bacto acetate agar (YBA) (3). The discriminatory dose for boscalid was adapted from (2) whereas those used for penthiopyrad and fluopyram were developed in this study. Isolates were grown on malt yeast extract agar for 7 to 10 days and spore suspensions were prepared in sterile distilled water and diluted to 106 conidia/ml. Respective media in 9-cm petri dishes were seeded with 7-μl droplets from each isolate allowing testing for all isolates on one plate. Two plates were used for each fungicide and sensitivity tests were repeated twice. Germination and germ tube growth were assessed microscopically after 16 to 24 h incubation at 22°C. The frequency of isolates resistant to two, three, and four fungicides was 90, 60, and 10%, respectively. Nine isolates (90%) were resistant to thiophanate-methyl and pyraclostrobin, simultaneously, whereas six (60%) and two isolates (20%) were resistant to boscalid and fenhexamid, respectively. All boscalid-resistant isolates were also resistant to pyraclostrobin and thiophanate-methyl, but one fenhexamid-resistant isolate was sensitive to the other three fungicides. Eight isolates that germinated at 5 μg/ml iprodione but not at 50 μg/ml were considered sensitive. All isolates were sensitive to the SDHIs penthiopyrad and fluopyram as well as to cyprodinil and fludioxonil. To our knowledge, this is the first report of resistance to pyraclostrobin, thiophanate-methyl, fenhexamid, and boscalid in B. cinerea from eucalyptus seedlings in Florida. The absence of resistance to fludioxonil and iprodione is likely because these fungicides are not registered in nurseries as well as fluopyram and penthiopyrad which were developed only recently. Management practices should be developed to limit the selection and spread of additional resistant populations in eucalyptus nurseries as has occurred in Florida strawberries where multi-fungicide resistance is widespread (1). References: (1) A. Amiri et al. Plant Dis. 97:393, 2013. (2) M. Leroch et al. Appl. Environ. Microbiol. 79:159, 2013. (3) G. Stammler and J. Speakman. J. Phytopathol. 154:508, 2006. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

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First Report of Resistance to Mefenoxam in Phytophthora cactorum in the United States and Elsewhere

Plant Disease ◽

10.1094/pdis.2004.88.5.576a ◽

2004 ◽

Vol 88 (5) ◽

pp. 576-576 ◽

Cited By ~ 18

Author(s):

S. N. Jeffers ◽

G. Schnabel ◽

J. P. Smith

Keyword(s):

United States ◽

South Carolina ◽

Tap Water ◽

Southeastern United States ◽

Crop Protection ◽

The United States ◽

Crown Rot ◽

Phytophthora Cactorum ◽

Mycelium Growth ◽

First Report

Phytophthora cactorum causes crown rot of strawberry (Fragaria × ananassa) (2), a disease that has been particularly severe during the last 5 years in the southeastern United States. In the fall of 2001, strawberry plants (cv. Camarosa) in a field in Lexington County, South Carolina exhibited typical crown rot symptoms (2) 1 to 2 weeks after transplanting, even though plants had been drenched with mefenoxam (Ridomil Gold; Syngenta Crop Protection, Greensboro, NC) immediately after transplanting. Initially, we observed leaves that had marginal necrosis, were smaller than normal, and were discolored. Soon after, diseased plants appeared stunted and unthrifty compared with other plants in the field, and some of these plants eventually wilted and died. Severely affected plants had necrotic roots and decayed crowns. Ten symptomatic plants were collected for isolation. In the laboratory, root and crown tissues were rinsed in running tap water and blotted dry, small pieces of necrotic tissue were placed aseptically on PAR-V8 selective medium (1), and isolation plates were placed at 20°C in the dark for up to 7 days. P. cactorum was recovered from six plants. Isolates produced characteristic asexual and sexual structures directly on the isolation plates (i.e., papillate sporangia on sympodial sporangiophores and oospores with paragynous antheridia) (2). A single hypha of an isolate from each plant was transferred to fresh PAR-V8, and pure cultures were stored on cornmeal agar in glass vials at 15°C in the dark. All six isolates from the Lexington County field and nine other isolates of P. cactorum from strawberry (three from South Carolina, three from North Carolina, and three from Florida) were tested for sensitivity to mefenoxam on fungicide-amended medium. Mefenoxam was added to 10% clarified V8 juice agar (cV8A) after autoclaving so the concentration in the medium was 100 ppm. Agar plugs from active colonies were transferred to mefenoxam-amended and nonamended cV8A (three replicates per treatment), plates were placed at 25°C in the dark for 3 days, and linear mycelium growth was measured. All six isolates from Lexington County were highly resistant to mefenoxam with mycelium growth relatively unrestricted on mefenoxam-amended medium (73 to 89% of that on nonamended medium). In comparison, the other nine isolates were sensitive to mefenoxam with mycelium growth severely restricted by 100 ppm of mefenoxam (0 to 7% of that on nonamended medium). To our knowledge, this is the first report of mefenoxam resistance in P. cactorum on strawberry or any other crop in the United States and elsewhere. Because mefenoxam is the primary fungicide used to manage Phytophthora crown rot in the southeastern United States, resistance may limit use of this fungicide in strawberry production. References: (1) A. J. Ferguson and S. N. Jeffers. Plant Dis. 83:1129, 1999. (2) E. Seemüller. Crown rot. Pages 50–51 in: Compendium of Strawberry Diseases, 2nd ed. J. L. Maas, ed. The American Phytopathological Society, St. Paul, MN, 1998.

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First Report of Gray Mold of Strawberry Caused by Botrytis cinerea in South Carolina

Plant Disease ◽

10.1094/pdis-06-11-0529 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1482-1482 ◽

Cited By ~ 4

Author(s):

D. Fernández-Ortuño ◽

X. Li ◽

W. Chai ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Botrytis Cinerea ◽

Species Identification ◽

Morphological Characteristics ◽

Pcr Amplification ◽

Gray Mold ◽

Tween 20 ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Control Fruit

Gray mold caused by Botrytis spp. is one of the most economically important diseases of cultivated strawberry (Fragaria × ananassa) worldwide. From April to June 2011, strawberries with symptoms resembling gray mold disease were collected from different locations (Chesnee, Florence, Lexington, McBee, Monetta, and North Augusta) in South Carolina. Fruit infections began as small, firm, light brown lesions that enlarged quickly, becoming covered with a gray, fuzzy mass of spores followed by a soft rot. To isolate the causal agent, spores from symptomatic fruit were suspended in 1% Tween 20, streaked onto the surface of potato dextrose agar plates, and incubated at 22°C. Fungal colonies from single spores were at first colorless and later became gray to brown when the conidiphores and conidia developed. Conidia were identified by their morphological characteristics: an average size of 14 × 9 μm, ellipsoid to rounded without internal structure, and with a scar on the point of union to the conidiophore (1). Sclerotia produced in culture were hard, dark, irregular shaped, and formed after 2 weeks. The pathogen was identified as Botrytis cinerea Pers.: on the basis of morphology and confirmed by a restriction digest with ApoI of the 413-kb PCR amplification product obtained with BA2f/BA1r primers (2). Koch's postulates were conducted by inoculating 10 surface-sterilized strawberries with a conidial suspension (105 spores/ml) of a randomly chosen B. cinerea isolate previously characterized; 10 control fruit received sterile water without conidia. The inoculated fruit were incubated for 3 days at room temperature in air-tight plastic bags. Inoculated fruit developed typical gray mold symptoms with gray sporulating lesions. The developing spores on inoculated fruit were confirmed to be B. cinerea. All control fruit remained healthy. For many Botrytis spp., the internal transcribed spacer region does not reveal nucleotide variations and thus is useless for species identification. We used additional, more appropriate genetic markers for molecular-based species identification and verified that strawberries in South Carolina are affected by gray mold disease caused by B. cinerea. To our knowledge, this is the first scientific report of B. cinerea causing gray mold of strawberry in South Carolina. References: (1) W. R. Jarvis. Botryotinia and Botrytis Species: Taxonomy, Physiology and Pathogenicity. A Guide to the Literature. Monograph no. 15. Canada Department of Agriculture, Research Branch, Ottawa, 1977. (2) K. Nielsen et al. Plant Dis. 86:682, 2002.

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First Report of Gray Mold in Tomato Caused by Botrytis cinerea in Baja California, Mexico

Plant Disease ◽

10.1094/pd-89-0528b ◽

2005 ◽

Vol 89 (5) ◽

pp. 528-528 ◽

Cited By ~ 1

Author(s):

R. J. Holguín-Peña ◽

F. G. Arcos

Keyword(s):

Botrytis Cinerea ◽

Baja California ◽

Polymerase Chain Reaction Amplification ◽

Random Amplified Polymorphic Dna ◽

Gray Mold ◽

Malt Extract ◽

Conidial Suspension ◽

Sterile Water ◽

Tomato Production ◽

First Report

San Quintin Valley, a 60-mile-long coastal plain (30°30′N, 116°W) in the Baja California Peninsula, is one of the major fresh tomato (Lycopersicon esculentum Mill.) production areas in Mexico with more than 8,000 ha. During the last 10 years, the valley's tomato production has declined because of gray mold and stem canker diseases. Flower rot, reddish brown margins on the leaves and stems, and fruit with a gray mold were observed on field-grown tomato plants (Roma type cv. Tequila) in the autumn of 2003. Severity ranging from 55 to 60% was observed at harvest. Infected tissues were sampled and disinfested by immersion in 1% NaOCl for 1 min, rinsed in sterile water, and placed on malt extract agar at 22°C. Fungal conidia were then transferred to 2% potato dextrose agar (PDA). The resulting fungal colonies were definitively identified as Botrytis cinerea Pers.:Fr. The colonies of B. cinerea were first hyaline and white and became dark gray after 96 h. Mycelia were septate with dark branched conidiophores. Conidia were unicellular, ellipsoid, and ranged from 5 to 8 × 8 to 14 μm. Profuse black sclerotia developed in 7-day-old cultures. Infection site analyses in diseased flowers at different stages during the bloom were done with scanning electron microscopy. Fungal hyphae were located predominantly on the receptacle areas, whereas conidia were located in the ovaries as described previously (3). The identity of B. cinerea was confirmed by a restriction digest with ApoI of the 413-kb polymerase chain reaction amplification product obtained with BA2f/BA1r primers (1) and random amplified polymorphic DNA banding patterns (2). Pathogenicity tests were done by spray inoculation of 1-ml aqueous conidial suspension (106 CFU/ml) on 20 healthy plants during the blossom stage. An equal number of plants sprayed with sterile water was used as the control. Plants were incubated at 20 ± 2°C for 5 days. The fungus was reisolated from diseased flowers and peduncles after surface disinfestation (2.5% NaOCl) and plating on PDA. No symptoms were observed in the noninoculated controls. To our knowledge, this is the first report of B. cinerea causing gray mold disease on tomato in Baja California. References: (1) K. Nielsen et al. Plant Dis. 86:682, 2002. (2) S. Rigotti et al. FEMS Microbiol. Lett. 209:169, 2002. (3) O. Viret et al. Phytopathology 94:850, 2004.

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