First Report of the β-tubulin E198A Allele for Fungicide Resistance in Monilinia fructicola from South Carolina

Resistance to methyl benzimidazole carbamates (MBCs) in Monilinia fructicola, the causal agent of brown rot of stone fruits, is known to be present in South Carolina peach orchards, but the molecular mechanism of resistance has not been investigated. Nine isolates were collected from peach in five counties in South Carolina and examined in petri dish assays on potato dextrose agar (PDA) for resistance to the MBC fungicide thiophanate-methyl (Topsin-M 70WP; Ceraxagri, King of Prussia, PA) at the discriminatory dose of 50 μg/ml. Isolates that grew on the fungicide-amended medium were considered highly resistant (HR). The β-tubulin gene from four sensitive (S) and five HR M. fructicola isolates was PCR-amplified with primer pair TubA and TubR1 as described previously (1). Sequence analysis revealed several silent mutations in introns and exons in S and HR isolates and the presence of the previously described E198A allele in HR but not S isolates (1). Nucleotide sequences of the β-tubulin gene from three S (BS, S2, MfEgpc1) and two HR isolates (MfPdt6 and BR2) were submitted to GenBank under accession numbers HM051379, HM051380, HM051381, HM051382, and HM051383, respectively. To our knowledge, this is the first report of the E198A in M. fructicola isolates from South Carolina and the East Coast. This allele is responsible for high levels of MBC resistance in M. fructicola (1). A previously reported PCR-based method using primers HRF+HRR designed to detect the E198A mutation in M. fructicola HR isolates (1) was improved by adding primer TR739 (5′-TCA CGA CGA ACA ACA TCA AGA-3′) to the PCR cocktail. This additional internal primer amplified a 222-bp fragment from all S and HR isolates and therefore provided a useful, additional control. The confirmation of the E198A allele in M. fructicola isolates provides another useful tool to detect MBC resistance in commercial peach orchards in South Carolina. Reference: (1) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003.

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First Report of the β-Tubulin E198A Mutation Conferring Resistance to Methyl Benzimidazole Carbamates in European Isolates of Monilinia fructicola

Plant Disease ◽

10.1094/pdis-12-10-0922 ◽

2011 ◽

Vol 95 (4) ◽

pp. 497-497 ◽

Cited By ~ 6

Author(s):

J. Weger ◽

M. Schanze ◽

M. Hilber-Bodmer ◽

T. H. M. Smits ◽

A. Patocchi

Keyword(s):

South Carolina ◽

Molecular Mechanisms ◽

Brown Rot ◽

The United States ◽

Monilinia Fructicola ◽

Tubulin Gene ◽

First Report ◽

Benzimidazole Carbamates ◽

High Level ◽

Β Tubulin

The causal agent of brown rot on stone and pome fruits, Monilinia fructicola (G. Wint.), is a quarantine pathogen in Europe. It has been detected in Austria (later eradicated), Spain, the Czech Republic, Italy, Germany, and Switzerland (1). In the United States and other countries, M. fructicola isolates were reported to show resistance to different classes of fungicides, including methyl benzimidazole carbamates (MBC) (2). Lichou et al. (2) reported the presence of isolates resistant to the MBC carbendazim in France, but the mechanisms inducing MBC resistance in these isolates were not studied. Ma et al. (3) in California, and more recently, Zhu et al. (4) in South Carolina, demonstrated that the molecular mechanisms accounting for low and high levels of resistance to MBC fungicides in M. fructicola isolates were the mutations H6Y and E198A, respectively, in the β-tubulin gene. Four M. fructicola isolates each from Italy, France, Spain, and Switzerland (16 isolates total), all having an unknown level of MBC resistance, were selected. In each isolate, the section of the β-tubulin gene containing the two potentially mutant codons was PCR-amplified with the primers TubA and TubR1 (3) and the amplicons were sequenced directly. Sequence analysis revealed the amino acid histidine (H) at codon 6 in all the isolates, which would not predict MBC resistance, while alanine (A) at codon 198 (the mutation predictive of a high level of MBC resistance) was found in all isolates from Spain and Switzerland and in three isolates each from France and Italy. A representative sequence of the four identical partial β-tubulin gene sequences from the Swiss isolates was submitted to GenBank under the Accession No. HQ709265. All isolates were tested in a potato dextrose agar (PDA) petri dish assay for resistance to the MBC fungicide thiophanate-methyl (Nippon Soda Co., Ltd., Tokyo, Japan) at the discriminatory dose of 50 μg/ml (4). All isolates with the E198A mutation were able to grow on the media, while the two isolates without the E198A mutation were not able to grow. The result indicated that most isolates had a high level of resistance to the MBC fungicide. To our knowledge, this is the first report of the presence of the E198A mutation conferring resistance to MBC fungicides in European isolates of M. fructicola. As the mutation appears to be widely distributed, we anticipate that MBC fungicides may be ineffective at controlling brown rot in countries with occurrence of M. fructicola. References: (1) M. Hilber-Bodmer et al. Plant Dis. 94:643, 2010. (2) J. Lichou et al. Phytoma 547:22, 2002. (3) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003. (4) F. X. Zhu et al. Plant Dis. 94:1511, 2010.

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First Report of Brown Rot Caused by Monilinia fructicola Affecting Peach Orchards in Slovenia

Plant Disease ◽

10.1094/pdis-94-9-1166a ◽

2010 ◽

Vol 94 (9) ◽

pp. 1166-1166 ◽

Cited By ~ 15

Author(s):

A. Munda ◽

M. Viršček Marn

Keyword(s):

Prunus Persica ◽

Brown Rot ◽

Fruit Rot ◽

Morphological Identification ◽

Monilinia Fructicola ◽

Conidial Suspension ◽

Stone Fruits ◽

The European Union ◽

First Report ◽

Representative Isolate

Monilinia fructicola, the causal agent of brown rot, is a destructive fungal pathogen that affects mainly stone fruits (Prunoideae). It causes fruit rot, blossom wilt, twig blight, and canker formation and is common in North and South America, Australia, and New Zealand. M. fructicola is listed as a quarantine pathogen in the European Union and was absent from this region until 2001 when it was detected in France. In August 2009, mature peaches (Prunus persica cv. Royal Glory) with brown rot were found in a 5-year-old orchard in Goriška, western Slovenia. Symptoms included fruit lesions and mummified fruits. Lesions were brown, round, rapidly extending, and covered with abundant gray-to-buff conidial tufts. The pathogen was isolated in pure culture and identified based on morphological and molecular characters. Colonies on potato dextrose agar (PDA) incubated at 25°C in darkness had an average daily growth rate of 7.7 mm. They were initially colorless and later they were light gray with black stromatal plates and dense, hazel sporogenous mycelium. Colony margins were even. Sporulation was abundant and usually developed in distinct concentric zones. Limoniform conidia, produced in branched chains, measured 10.1 to 17.7 μm (mean = 12.1 μm) × 6.2 to 8.6 μm (mean = 7.3 μm) on PDA. Germinating conidia produced single germ tubes whose mean length ranged from 251 to 415 μm. Microconidia were abundant, globose, and 3 μm in diameter. Morphological characters resembled those described for M. fructicola (1). Morphological identification was confirmed by amplifying genomic DNA of isolates with M. fructicola species-specific primers (2–4). Sequence of the internal transcribed spacer (ITS) region (spanning ITS1 and ITS 2 plus 5.8 rDNA) of a representative isolate was generated using primers ITS1 and ITS4 and deposited in GenBank (Accession No. GU967379). BLAST analysis of the 516-bp PCR product revealed 100% identity with several sequences deposited for M. fructicola in NCBI GenBank. Pathogenicity was tested by inoculating five mature surface-sterilized peaches with 10 μl of a conidial suspension (104 conidia ml–1) obtained from one representative isolate. Sterile distilled water was used as a control. Peaches were wounded prior to inoculation. After 5 days of incubation at room temperature and 100% relative humidity, typical brown rot symptoms developed around the inoculation point, while controls showed no symptoms. M. fructicola was reisolated from lesion margins. Peach and nectarine orchards in a 5-km radius from the outbreak site were surveyed in September 2009 and M. fructicola was confirmed on mummified fruits from seven orchards. The pathogen was not detected in orchards from other regions of the country, where only the two endemic species M. laxa and M. fructigena were present. To our knowledge, this is the first report of M. fructicola associated with brown rot of stone fruits in Slovenia. References: (1) L. R. Batra. Page 106 in: World Species of Monilinia (Fungi): Their Ecology, Biosystematics and Control. J. Cramer, Berlin, 1991. (2) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (3) K. J. D. Hughes et al. EPPO Bull. 30:507, 2000. (4) R. Ioos and P. Frey. Eur. J. Plant Pathol. 106:373, 2000.

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Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7145-7152.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7145-7152 ◽

Cited By ~ 127

Author(s):

Zhonghua Ma ◽

Michael A. Yoshimura ◽

Themis J. Michailides

Keyword(s):

Dna Sequences ◽

Point Mutations ◽

Brown Rot ◽

Stone Fruit ◽

Monilinia Fructicola ◽

Tubulin Gene ◽

Benzimidazole Fungicides ◽

Specific Pcr ◽

Allele Specific ◽

Fruit Orchards

ABSTRACT Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of theβ -tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

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First Report of Fusarium Maize Ear Rot Caused by Fusarium kyushuense in China

Plant Disease ◽

10.1094/pdis-05-13-0558-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 279-279 ◽

Cited By ~ 2

Author(s):

J.-H. Wang ◽

H.-P. Li ◽

J.-B. Zhang ◽

B.-T. Wang ◽

Y.-C. Liao

Keyword(s):

Fusarium Species ◽

Morphological Characteristics ◽

Distilled Water ◽

Ear Rot ◽

Tubulin Gene ◽

Average Growth ◽

First Report ◽

Maize Ear Rot ◽

Maize Ear ◽

Β Tubulin

From September 2009 to October 2012, surveys to determine population structure of Fusarium species on maize were conducted in 22 provinces in China, where the disease incidence ranged from 5 to 20% in individual fields. Maize ears with clear symptoms of Fusarium ear rot (with a white to pink- or salmon-colored mold at the ear tip) were collected from fields. Symptomatic kernels were surface-sterilized (1 min in 0.1% HgCl2, and 30 s in 70% ethanol, followed by three rinses with sterile distilled water), dried, and placed on PDA. After incubation for 3 to 5 days at 28°C in the dark, fungal colonies displaying morphological characteristics of Fusarium spp. (2) were purified by transferring single spores and identified to species level by morphological characteristics (2), and DNA sequence analysis of translation elongation factor-1α (TEF) and β-tubulin genes. A large number of Fusarium species (mainly F. graminearum species complex, F. verticillioides, and F. proliferatum) were identified. These Fusarium species are the main causal agents of maize ear rot (2). Morphological characteristics of six strains from Anhui, Hubei, and Yunnan provinces were found to be identical to those of F. kyushuense (1), which was mixed with other Fusarium species in the natural infection in the field. Colonies grew fast on PDA with reddish-white and floccose mycelia. The average growth rate was 7 to 9 mm per day at 25°C in the dark. Reverse pigmentation was deep red. Microconidia were obovate, ellipsoidal to clavate, and 5.4 to 13.6 (average 8.8) μm in length. Macroconidia were straight or slightly curved, 3- to 5-septate, with a curved and acute apical cell, and 26.0 to 50.3 (average 38.7) μm in length. No chlamydospores were observed. Identity of the fungus was further investigated by sequence comparison of the partial TEF gene (primers EF1/2) and β-tubulin gene (primers T1/22) of one isolate (3). BLASTn analysis of the TEF amplicon (KC964133) and β-tubulin gene (KC964152) obtained with cognate sequences available in GenBank database revealed 99.3 and 99.8% sequence identity, respectively, to F. kyushuense. Pathogenicity tests were conducted twice by injecting 2 ml of a prepared spore suspension (5 × 105 spores/ml) into maize ears (10 per isolate of cv. Zhengdan958) through silk channel 4 days post-silk emergence under field conditions in Wuhan, China. Control plants were inoculated with sterile distilled water. The ears were harvested and evaluated 30 days post-inoculation. Reddish-white mold was observed on inoculated ears and the infected kernels were brown. No symptoms were observed on water controls. Koch's postulates were fulfilled by re-isolating the pathogen from infected kernels. F. kyushuense, first described on wheat in Japan (1), has also been isolated from rice seeds in China (4). It was reported to produce both Type A and Type B trichothecene mycotoxins (1), which cause toxicosis in animals. To our knowledge, this is the first report of F. kyushuense causing maize ear rot in China and this disease could represent a serious risk of yield losses and mycotoxin contamination in maize and other crops. The disease must be considered in existing disease management practices. References: (1) T. Aoki and K. O'Donnell. Mycoscience 39:1, 1998. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) F. Van Hove et al. Mycologia 103:570, 2011. (4) Z. H. Zhao and G. Z. Lu. Mycotaxon 102:119, 2007.

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Reduced Sensitivity in Monilinia fructicola to Propiconazole Following Prolonged Exposure in Peach Orchards

Plant Disease ◽

10.1094/pdis.1999.83.10.913 ◽

1999 ◽

Vol 83 (10) ◽

pp. 913-916 ◽

Cited By ~ 54

Author(s):

Eldon I. Zehr ◽

Lynn A. Luszcz ◽

William C. Olien ◽

W. C. Newall ◽

Joe E. Toler

Keyword(s):

South Carolina ◽

Radial Growth ◽

Prolonged Exposure ◽

Agar Medium ◽

Brown Rot ◽

Initial Population ◽

Monilinia Fructicola ◽

Baseline Sensitivity ◽

Dmi Fungicides ◽

Peach Orchard

The baseline sensitivity of Monilinia fructicola in a peach orchard not previously exposed to demethylation-inhibiting (DMI) fungicides was determined for propiconazole, using the concentration in an agar medium required to suppress radial growth of mycelium by 50% (EC50) The baseline sensitivity was found to be approximately 0.03 μg/ml. Prolonged, regular exposure of the natural population of M. fructicola to propiconazole in the test orchard over a 3-year period (29 total applications) resulted in a wider range of sensitivity (EC50 of 0.02 to 2.16μg/ml) among isolates than was observed in the initial population (EC50 of 0.02 to 0.15 μg/ml). Comparisons with isolates from commercial orchards where DMI fungicides were used regularly showed that sensitivities were comparable to, or less than, those of isolates from the population in the test orchard that had been exposed to propiconazole for the 3-year period. M. fructicola in South Carolina peach orchards might now be less sensitive to DMI fungicides than when those fungicides were first introduced for brown rot control, although effective disease control in the field has been maintained.

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First Report of Eutypella spp. Associated with Branch Canker of Citrus in California

Plant Disease ◽

10.1094/pdis-01-11-0047 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1187-1187 ◽

Cited By ~ 1

Author(s):

A. O. Adesemoye ◽

A. Eskalen

Keyword(s):

San Diego ◽

Its Sequences ◽

Control Treatment ◽

Molecular Characteristics ◽

Its2 Region ◽

Petroleum Jelly ◽

Tubulin Gene ◽

First Report ◽

Β Tubulin ◽

Branch Canker

Eutypella is one of the few genera in the Diatrypaceae considered plant pathogens (1). In California, E. vitis and other members of the Diatrypaceae cause branch and trunk canker on grapevine (3,4). Eutypella spp. have not previously been documented as pathogens of citrus. In a 2010 survey on citrus branch canker and dieback in six citrus-growing counties of California, four isolates of Eutypella species were detected in Riverside and San Diego counties. Canker symptoms included dieback and bark cracking, and cuts made through symptomatic trees showed that the cankers were expanding through the center of the tree. Branch samples were collected from 10 trees per orchard and 5 to 10 orchards per county (102 trees for two counties). Pieces of symptomatic tissue (1 to 2 mm2) were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated at 25°C for 4 days. All isolates were identified by morphological and molecular characteristics. PCR of isolates was performed in a thermal cycler using two primer pairs, ITS4/5 and Bt2a/2b for amplifying the internal transcribed spacer (ITS1), 5.8S, and ITS2 region and the β-tubulin gene, respectively (2,3). PCR products were sequenced at the University of California, Riverside Genomics Core and the sequences compared in a BLAST search. Four isolates identified as Eutypella spp. included two (UCR1088 and UCR1101) from San Diego County and two (UCR1148 and UCR1149) from the Riverside County samples. The sequences were deposited in GenBank (HQ880579, JF758610, HQ880581, and HQ880582 and HQ880583, JF758611, HQ880585, and HQ880586 for the ITS regions and β-tubulin gene, respectively. ITS sequences for UCR1088 and UCR1101 had 98 and 100% match, respectively, to Eutypella spp. ITS sequences in GenBank (GQ293959 to GQ293961), while UCR1148 and UCR1149 matched 99% (GQ293956 to GQ293958). On the basis of morphological characteristics, UCR1088 and UCR1101 were similar to Eutypella spp. group 1, while UCR1148 and UCR1149 were similar to Eutypella spp. group 3 (4). Pathogenicity tests were conducted with all four isolates on detached shoots from healthy citrus trees of the same cultivar/rootstock from which each isolate was obtained. One wound per shoot was made on 1-year-old, green, detached shoots using a 3-mm-diameter cork borer and the wounded surfaces were inoculated with 3-mm-diameter mycelial plugs of 5-day-old cultures of each isolate growing on PDA-tet. Inoculated wounds and shoot ends were covered with petroleum jelly and wrapped with Parafilm (3). Control shoots were inoculated with sterile agar plugs. There were 10 inoculated shoots per isolate and noninoculated control treatment. Shoots were incubated at 25°C in moist chambers for 6 weeks. Lesions similar to those on the original infected shoots were observed on all inoculated shoots except the control treatment. Reisolation and identification of fungi from inoculated and control shoots were done using methods described above. Inoculated isolates were recovered from 100% of inoculated shoots but none was recovered from noninoculated shoots, indicating association of Eutypella spp. with citrus branch canker. To our knowledge, this is the first report of Eutypella spp. associated with cankers on citrus in California. References: (1) B. Piskur et al. Plant Dis. 91:1579, 2007. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) F. P. Trouillas and W. D. Gubler. Plant Dis. 94:867, 2010. (4) F. P. Trouillas et al. Mycologia 102:319, 2010.

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First Report of Blueberry Leaf Spot Caused by Cylindrocladium colhounii in China

Plant Disease ◽

10.1094/pd-90-1553a ◽

2006 ◽

Vol 90 (12) ◽

pp. 1553-1553 ◽

Cited By ~ 2

Author(s):

Y. S. Luan ◽

L. Feng ◽

L. J. An

Keyword(s):

Leaf Spot ◽

Morphological Traits ◽

Internal Transcribed Spacers ◽

Fluorescent Light ◽

Adaxial Side ◽

Single Spore ◽

Tubulin Gene ◽

First Report ◽

Plastic Bags ◽

Β Tubulin

During late July and early August of 2005, leaf spot symptoms were observed in a blueberry nursery at a plantation in Dalian, which to our knowledge, lies within the largest blueberry-production area in China. Symptoms were observed primarily on lowbush species, for example Blomidon, as well as half-highbush cultivars. A slow-growing, white mycelium from the margin of necrotic leaf spots was recovered on potato dextrose agar (PDA). The following morphological traits were observed: erect conidiophores that branch twice and were terminated in a stiped, clavate phialide; hyaline, cylindrical, four-celled conidia; and globose, reddish brown, aggregated chlamydospores. Conidiophores (including stipes and terminal phialides) were 305 to 420 × 5 to 9 μm; primary branches were 9 to 45 × 5 to 6.3 μm; secondary branches were 9 to 17.3 × 3.1 to 4.5 μm; phialides were 7.8 to 17.5 × 2.5 to 6 μm; stipes (from the highest branch area to vesicle) were 150 to 270 μm long; and vesicles were 13 to 30 × 2 to 4.5 μm. Conidia were 50 to 72 × 4 to 5.5 μm. Chlamydospores were 15 to 20 μm in diameter. Koch's postulates were fulfilled by spray inoculating two healthy cultivars with conidiophores homogenized in axenic water. As a control, two healthy plants were sprayed with axenic water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and 20°C in darkness for 10 h. After 2 days, the plastic bags were removed and plants were maintained under the same conditions. After 4 days, small-to-medium brown spots with purplish margins were observed on the adaxial side of leaves from inoculated plants, but not from control plants. Fungi isolated from these lesions had the same morphological traits as the ones isolated previously from field plants. The morphological descriptions and measurements were similar to Cylindorocladium colhounii (2). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and the β-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 primers and T1/Bt2b primers, respectively, and sequenced (1). The ITS and β-tubulin gene sequences were similar to C. colhounii STE-U 1237 (99%; GenBank Accession No. AF231953) and C. colhounii STE-U 705 (99%; GenBank Accession No. AF231954), respectively. The morphology, secondary conidiation, and sequences of ITS and β-tubulin gene identify the causal fungus as C. colhounii. To our knowledge, this is the first report of C. colhounii on blueberry in China or in the world. References: (1) P. W. Crous et al. Can. J. Bot. 77:1813, 1999. (2) T. Watanabe. Page 222 in: Dictorial Atlas of Soil and Seed Fungi. CRC Press, Inc., Boca Raton, Fl, 1994.

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First Report of Brown Rot on Peach, Nectarine, Cherry, and Plum Fruits Caused by Monilinia fructicola in Bulgaria

Plant Disease ◽

10.1094/pdis-10-19-2094-pdn ◽

2020 ◽

Vol 104 (5) ◽

pp. 1561-1561

Author(s):

S. G. Bobev ◽

L. T. Angelov ◽

K. Van Poucke ◽

M. Maes

Keyword(s):

Brown Rot ◽

Monilinia Fructicola ◽

First Report

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First Report of the Peach Brown Rot Fungus Monilinia fructicola Resistant to Demethylation Inhibitor Fungicides in New Jersey

Plant Disease ◽

10.1094/pdis-94-1-0126a ◽

2010 ◽

Vol 94 (1) ◽

pp. 126-126 ◽

Cited By ~ 16

Author(s):

A. Burnett ◽

N. Lalancette ◽

K. McFarland

Keyword(s):

South Carolina ◽

New Jersey ◽

Cold Storage ◽

Prunus Persica ◽

Crop Protection ◽

Brown Rot ◽

Polymorphism Analysis ◽

Monilinia Fructicola ◽

Resistant Strains

Reduced sensitivity and resistance of Monilinia fructicola to demethylation inhibitors (DMIs; fungicide group 3) have been previously found in stone fruit orchards in Georgia, South Carolina, Ohio, and New York (2). Resistance development is a major concern because of the importance of DMIs for brown rot management. Eleven single-spore isolates, originally collected during 2006 from separate commercial peach (Prunus persica) orchards in southern New Jersey, were removed from cold storage (5°C) in early 2008 and examined in vitro for resistance to the DMI propiconazole (Orbit 3.6EC; Syngenta Crop Protection, Inc., Greensboro, NC). After 19 months at 5°C, isolate 7 was inhibited 53.4% in growth on potato dextrose agar (PDA) amended at the discretionary dose of 0.3 μg/ml propiconazole; inhibition of the remaining isolates ranged from 81.4 to 100%. Inhibition values were based on two replications of eight colonies per isolate performed after incubation at 25°C for 4 days. Because of the previously reported relationship between duration of cold storage and propiconazole sensitivity, isolate 7 was tentatively deemed resistant (1). To confirm the in vitro results, isolates were grown at 25°C for 7 days on cellophane over PDA. Genomic DNA was isolated from mycelium with the DNeasy Plant Mini Kit (Qiagen, Inc., Valencia, CA). PCR with primers INS65-F and INS65-R was conducted on a GeneAmp thermal cycler (Applied Biosystems, Inc., Foster City, CA) as described previously to amplify a 65-bp region named ‘Mona’ associated with DMI resistance (2). PCR products were separated via electrophoresis on 0.8% agarose gel. The primers amplified a 376-bp fragment from isolate 7 and a 311-bp fragment from all other isolates, thus indicating the presence of Mona in isolate 7. Restriction fragment length polymorphism analysis using the BsrBI enzyme, specific to a single restriction site within Mona, was conducted on the amplified fragments from all isolates. Electrophoresis results showed digestion of the 376-bp fragment from isolate 7 into 140-bp and 236-bp fragments, thereby confirming the presence of Mona; none of the 311-bp fragments from the remaining isolates were cut by BsrBI. Although economic loss from brown rot has not been reported in New Jersey, these results show that propiconazole-resistant strains have been detected since 2006 and it is most likely that resistant strains of the pathogen are still present in commercial peach orchards. To combat this risk, current brown rot control recommendations are incorporating quinone outside inhibitors (QoIs; fungicide group 11) and carboxamides (fungicide group 7) into control programs as a resistance management strategy. More extensive sampling is planned to ascertain the prevalence and location of resistant strains. References: (1) K. D. Cox et al. Phytopathology 97:448, 2007. (2) C.-X. Luo et al. Plant Dis. 92:1099, 2008.

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First Report of a Pestalotiopsis sp. Causing Leaf Spot of Blueberry in China

Plant Disease ◽

10.1094/pdis-92-1-0171a ◽

2008 ◽

Vol 92 (1) ◽

pp. 171-171 ◽

Cited By ~ 10

Author(s):

Y. S. Luan ◽

Z. T. Shang ◽

Q. Su ◽

L. Feng ◽

L. J. An

Keyword(s):

Leaf Spot ◽

Apical Cell ◽

Vaccinium Corymbosum ◽

Internal Transcribed Spacers ◽

Fluorescent Light ◽

Tubulin Gene ◽

First Report ◽

Plastic Bags ◽

Plant Nursery ◽

Β Tubulin

In August 2006, leaf spots were observed on half-high blueberry (Vaccinium corymbosum) in a plant nursery in Dalian, China. The symptomatic potted 1-year-old blueberry plants were located in parts of a plant nursery with poor ventilation. The primary symptom was a leaf spot, 0.4 to 0.8 cm in diameter, with brown margins that enlarged and coalesced. Mycelium grew from symptomatic and green leaf tissue removed from the margin of a necrotic leaf spot. Plant tissues were surface disinfested with 0.1% mercuric chloride for 3 min and 70% ethyl alcohol for 30 s before plating onto potato dextrose agar. The resulting colonies were white with a regular margin and a rough surface. The cultures were covered with black and globular acervuli with a diameter of 100 to 200 μm. The base of each conidiophore was swollen and globose with phialides growing from the apical end. Mature conidia were straight to fusiform, measuring 19.0 to 27.5 × 6.3 to 9.2 μm, and five-celled with the three middle cells brown and darker than the end cells. The apical cell was triangular and hyaline with three simple setulae that were 17.2 to 29.7 μm long. The base cell terminated in a point 4.0 to 8.6 μm long. Koch's postulates were fulfilled for the fungus by spray inoculating two healthy young plants with 2 × 105 conidia per ml of sterile distilled water. As a control, two similar plants were sprayed with sterile water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and at 20°C in darkness for 10 h. After 3 days, the plastic bags were removed and plants were maintained under the same conditions. More than 20 days after inoculation, symptoms on inoculated plants were similar to those previously described in the nursery. Control plants did not show any symptoms. Cultures isolated from the lesions were similar to those isolated previously from plants in the nursery. The morphological descriptions and measurements were similar to Pestalotiopsis clavispora (1). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and partial β-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 and T1/Bt2b primers (2) respectively, and sequenced (GenBank Accession Nos. EF119336 and EF152585). The ITS sequences were most similar to the ITS regions of P. clavispora TA-8 (98%; GenBank Accession No. AY924264), P. clavispora TA-6 (98%; GenBank Accession No. AY924263), and P. clavispora PSHI 2002 Endo 389 (96%; GenBank Accession No. AY682929). The partial β-tubulin gene sequence was identical to Pestalotiopsis sp. isolate PSHI 2004 Endo 86 (100%; GenBank Accession No. DQ657901). The morphology and sequence data support the identity of the causal fungus as P. clavispora. To our knowledge, this is the first report on the presence of a Pestalotiopsis sp. causing a disease of blueberry in China. References: (1) E. F. Guba. Monograph of Monochaetia and Pestalotia. Harvard University Press, Cambridge, MA, 1961. (2) W. Tao et al. Mol. Cell Biol. 27:689, 2007.

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