First Report of Brown Rot Caused by Monilia polystroma on Apple in Serbia

Monilia polystroma van Leeuwen is a new Japanese species, similar to M. fructigena but distinguishable based on morphological and molecular characteristics (3). After its first discovery on apple in Japan, occurance of M. polystroma in Europe has been reported in Hungary, the Czech Republic, and Switzerland (2,3,4). In October 2011, during a survey for apple fungal pathogens in the Bela Crkva district, 15 apple fruit (Malus domestica Borkh.) cv. Golden Delicious were collected. Two isolates of Monilinia polystroma were obtained from apple fruit showing brown rot, covered with small yellowish sporodohia. The pathogen was identified as M. polystroma based on morphological and molecular features (1,3). Upon isolation, colonies cultivated on PDA were white to grayish and the mycelium grew 8.85 mm per day at 22 ± 1°C in 12-h light/12-h dark regime. After 6 to 8 days of incubation, black stromatal plates were observed on the reverse sides of the inoculated petri dishes. Conidia were one-celled, limoniform, hyaline, 14.7 to 21.88 μm (16.2 mean) × 7.85 to 12.92 μm (10.8 mean), and were produced in branched monilioid chains on inoculated apple fruit. Morphological identification was confirmed by PCR (1) using genomic DNA extracted from the mycelium of pure cultures, and amplified products of 425 bp in length, specific for M. polystroma were amplified as expected with primers MO368-5 and MO368-8R. For one isolate, the ribosomal ITS1-5.8S-ITS2 region was obtained, using primers ITS1 and ITS4, and deposited in GenBank (Accession No JX315717). The sequence was 498 bp in length and showed 100% identity with sequences deposited for M. polystroma in NCBI GenBank (JN128835, AM937114, GU067539). Pathogenicity was confirmed by wound-inoculating five surface-sterilized, mature apple fruit with mycelium plugs (5 mm in diameter) of both isolates grown on PDA. Control fruit were inoculated with sterile PDA plugs. After 3 days of incubation in plastic containers, under high humidity (RH 90 to 95%) at 22 ± 1°C, typical symptoms of brown rot developed on inoculated fruit, while control fruit remained symptomless. Isolates recovered from symptomatic fruit showed the same morphological and molecular characteristics as original isolates. To the best of our knowledge, this is the first report of M. polystroma in Serbia. Further studies are necessary to estimate the economic importance and geographic distribution of this organism in Serbia. References: (1) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (2) M. Hilber-Bodmer et al. Plant Dis. 96: 146, 2012. (3) G. C. M. van Leeuwen et al. Mycol. Res. 106: 444, 2002. (4) OEPP/EPPO Reporting Service. Retrieved from http://archives.eppo.int/EPPOReporting/2011/Rse-1106.pdf

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First Report of Brown Rot Caused by Monilinia fructicola on Stored Apple in Serbia

Plant Disease ◽

10.1094/pdis-06-11-0531 ◽

2012 ◽

Vol 96 (3) ◽

pp. 456-456 ◽

Cited By ~ 10

Author(s):

M. Vasić ◽

N. Duduk ◽

M. M. Ivanović ◽

A. Obradović ◽

M. S. Ivanović

Keyword(s):

Online Publication ◽

Slovak Republic ◽

Brown Rot ◽

Pathogenic Fungi ◽

Its2 Region ◽

Morphological Identification ◽

Monilinia Fructicola ◽

First Report ◽

Molecular Features ◽

Quarantine Organism

Monilinia fructicola (G. Winter) Honey is a causal agent of brown rot of stone fruits, occasionally affecting pome fruits as well. The pathogen is commonly present in North and South America, Oceania, and Asia, but listed as a quarantine organism in Europe (4). After its first discovery in France in 2001, its occurrence has been reported in Germany, Hungary, Italy, Poland, Romania, Slovenia, Spain, Switzerland, Austria, and the Slovak Republic (1). In February 2011, during a survey for fungal postharvest pathogens in cold storage conditions, apple fruits (Malus domestica Borkh.) grown and stored in the Grocka Region, Serbia, were collected. All pathogens from symptomatic fruits were isolated on potato dextrose agar (PDA). One isolate from apple fruit cv. Golden Delicious with brown rot symptoms was identified as M. fructicola based on morphological and molecular characters. Colonies cultivated on PDA at 22°C in darkness were colorless, but later became grayish, developing mass of spores in concentric rings. Colony margins were even. Conidia were one-celled, limoniform, hyaline, measured 12.19 to 17.37 (mean 13.8) × 8.62 to 11.43 μm (mean 9.9), and were produced in branched monilioid chains (3). Morphological identification was confirmed by PCR (2) using genomic DNA extracted from the mycelium of pure culture, and an amplified product of 535 bp, specific for the species M. fructicola, was obtained. Sequence of the ribosomal (internal transcribed spacer) ITS1-5.8S-ITS2 region was obtained using primers ITS1 and ITS4 and deposited in GenBank (Accession No. JN176564). Control fruits were inoculated with sterile PDA plugs. After 3 days of incubation in plastic containers with high humidity at room temperature, typical symptoms of brown rot developed on inoculated fruits, while control fruits remained symptomless. The isolate recovered from symptomatic fruits showed the same morphological and molecular features of the original isolate. To our knowledge, this is the first report of M. fructicola in Serbia. Further studies are necessary for estimation of economic importance and geographic distribution of this quarantine organism in Serbia. References: (1) R. Baker et al. European Food Safety Authority. Online publication. www.efsa.europa.eu/efsajournal . EFSA J. 9(4):2119, 2011. (2) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (3) J. E. M. Mordue. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 616, 1979. (4) OEPP/EPPO. EPPO A2 List of Pests Recommended for Regulation as Quarantine Pests. Online publication. Version 2010-09. Retrieved from http://www.eppo.org/QUARANTINE/listA2.htm , June 27, 2011.

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First Report of Brown Rot Caused by Monilinia fructicola on Imported Peach in Hungary

Plant Disease ◽

10.1094/pd-90-0375b ◽

2006 ◽

Vol 90 (3) ◽

pp. 375-375 ◽

Cited By ~ 21

Author(s):

M. Petróczy ◽

L. Palkovics

Keyword(s):

Molecular Genetic ◽

Brown Rot ◽

Glass Container ◽

Brown Spot ◽

Genetic Identification ◽

Molecular Characteristics ◽

Fungal Mycelium ◽

Morphological Identification ◽

Monilinia Fructicola ◽

First Report

Brown rot caused by Monilinia fructicola is one of the most important diseases of peach. The pathogen is included in the EPPO A2 list of quarantine organisms for Europe (2). M. laxa and M. fructigena are common in Hungary, but M. fructicola has never been reported in orchards, in trade, or in markets. In early October 2005, brown rot was observed on imported peaches from Italy and Spain at a vegetable market and some supermarkets in Budapest. The variety of peach was identified as ‘Michellini’ by colleagues in the Department of Pomology of Corvinus University. The pathogen was identified as M. fructicola on the basis of morphological and molecular characteristics. Symptoms began with a small, circular brown spot, and the rot spread rapidly. At the same time, numerous small, grayish stromata developed. Finally, the whole surface of the fruit was covered with conidial tufts. The conidia were one-celled, lemon-shaped, hyaline, 15.7 × 10.3 μm, and produced in branched monilioid chains. Conidia from infected fruit were transferred to potato dextrose agar. Fungal mycelium grew at a linear rate of 10.7 mm per 24 h at 22°C in the dark. The color of the colony was grayish, and the sporulation showing concentric rings was abundant (sporulation is sparse in M. laxa or M. fructigena). The colony was not rosetted and the margin was not lobed, in contrast with M. laxa. Pathogenicity was tested by inoculating surface-sterilized, mature peach fruits with conidia. Inoculated and control fruits were placed in a sterilized glass container at room temperature. After 5 days of incubation, typical brown rot symptoms developed on inoculated fruits while control fruits remained healthy. M. fructicola was reisolated from the inoculated fruits. PCR was used to identify the fungus (1). Species-specific internal transcribed spacer (ITS) primers for M. fructicola, M. laxa, and M. fructigena were used to amplify the DNA of isolates. Three type-cultures were used as the positive control. Following the removal of the mycelia from the agar, total DNA was extracted using a cetyltrimethylammoniumbromide extraction. The nucleic acid-containing pellet was resuspended in RNase containing Tris-EDTA buffer. DNA quality was assessed by gel electrophoresis on 1% agarose gel stained with ethidium bromide. The molecular genetic identification method confirmed the results of morphological identification. To our knowledge, this is the first report of M. fructicola on peaches in Hungary or in eastern Europe. References: (1) R. Ioos and P. Frey. Eur. J. Plant Pathol. 106:373, 2000. (2) OEPP/EPPO. List of A2pests regulated as quarantine pests in the EPPO region. Version 2005-09. Online publication, 2005.

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First Report of Brown Rot Caused by Monilinia fructicola on Various Stone and Pome Fruits in the Czech Republic

Plant Disease ◽

10.1094/pdis-91-7-0907b ◽

2007 ◽

Vol 91 (7) ◽

pp. 907-907 ◽

Cited By ~ 23

Author(s):

J. Duchoslavová ◽

I. Širučková ◽

E. Zapletalová ◽

M. Navrátil ◽

D. Šafářová

Keyword(s):

Czech Republic ◽

Malus Domestica ◽

Prunus Persica ◽

Brown Rot ◽

Prunus Armeniaca ◽

Molecular Characteristics ◽

Morphological Identification ◽

Monilinia Fructicola ◽

The Czech Republic ◽

First Report

Monilinia fructicola, a causal agent of brown rot, is one of the most important fungal pathogens of stone fruits. The disease causes major crop losses in peach, plum, prune, nectarine, and apricot. M. fructicola is commonly present in Asia, North and South America, and Australia. This is a quarantined pathogen in Europe; restricted occurrence has been observed in Austria and France. Recently, it was detected in Hungary and Switzerland on peach and nectarine fruits imported from Italy and Spain (1,4). During a survey in the summer of 2006, 56 samples were tested for the presence of Monilinia spp. M. fructicola was detected in 15 samples from 11 locations in the western area (Bohemia) of the Czech Republic, mainly on peaches (Prunus persica), apples (Malus × domestica), and sweet and sour cherries (Cerasus avium and C. vulgaris) and rarely on flowering plum (Prunus triloba) and Malus × moerlandsii cv. Liset. On the other hand, the pathogen was not detected on fruits of apricot (Prunus armeniaca) or pear (Pyrus communis). In all cases, M. fructicola was detected on fruits except for a single occurrence of the pathogen on a shoot of the Malus × domestica. The pathogen was always detected in mixed infections with M. fructigena and/or M. laxa. On both fruits and the shoot, symptoms appeared as brown, sunken lesions covered with grayish pustules. Many infected fruits became dry and mummified because rot progressed through the fruit surface. The infected shoot died back (3). M. fructicola was identified by means of colony and conidial morphology and molecular characteristics. The colonies cultivated on potato dextrose agar were entire and the colony surface was even. The color of the colony was gray, and sporulating colonies showed concentric rings that changed to a hazel color. Conidia were ellipsoid, hyaline, and 13.5 to 17.7 × 8.3 to 10.5 μm. Preliminary morphological identification was confirmed by PCR (2) on DNA isolated directly from mycelium on the examined fruits. A product that was 280 bp long was obtained in all cases. The BLAST analysis of our PCR product sequences showed 100% homology to sequences of M. fructicola (GenBank Accession Nos. DQ491506, AY2891185, Z73778, and AB125615). One sequence from our study was deposited in GenBank (Accession No. EF378628). To our knowledge, this is the first report of the quarantined fungus M. fructicola in the Czech Republic. References: (1) E. Bosshard et al. Plant Dis. 90:1554, 2006. (2) K. J. D. Hughes et al. EPPO Bull. 30:507, 2000. (3) J. M. Ogawa et al., eds. Compendium of Stone Fruit Diseases. The American Phytopathological Society, St. Paul, MN, 1995. (4) M. Petróczy and L. Palkovics. Plant Dis. 90:375, 2006.

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First Report of Brown Rot Caused by Monilinia fructicola on Apple in Italy

Plant Disease ◽

10.1094/pdis-09-12-0869-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 689-689 ◽

Cited By ~ 6

Author(s):

C. Martini ◽

A. Spadoni ◽

M. Mari

Keyword(s):

Fungal Pathogens ◽

Brown Rot ◽

Its2 Region ◽

Monilinia Fructicola ◽

Malt Extract ◽

Conidial Suspension ◽

First Report ◽

Molecular Features ◽

Sterile Needle ◽

Emilia Romagna Region

Monilinia fructicola (G. Wint.) Honey, the causal agent of brown rot, is one of the most important fungal pathogens of stone fruit but may also affect pome fruits. The pathogen is common in North America, Oceania, South America, and Asia. It is a quarantined pathogen in Europe (3), but was recently detected in apple from the Czech Republic, Germany, and Serbia (1,2,4). In January 2012, during a survey for fungal postharvest pathogens, stored apple (Malus domestica Borkh.) belonging to the cultivars Gala and Pink Lady showing brown rot symptoms were observed in the Emilia Romagna region, Italy. Typical decay spots were circular and brown, tending toward black. Decayed tissues remained firm, and numerous grayish pustules containing spores appeared on rotted areas. The pathogen was isolated on V8 juice agar and culture plates were incubated at 25°C in darkness for 5 days. A conidial suspension was spread on malt extract agar and single spores were selected. The colonies were morphologically identified as M. fructigena. Two colonies developing a gray mass of spores in concentric rings with the reverse side black were further studied by molecular tools. The colony margins were even and the conidia were one-celled, limoniform, hyaline, and 12.1 to 17.4 × 8.4 to 11.2 μm. The ribosomal ITS1-5.8S-ITS2 region was PCR-amplified from genomic DNA obtained from mycelium using primers ITS1 and ITS4. A BLAST search in GenBank revealed the highest similarity (99%) to M. fructicola sequences (GenBank Accession Nos. HQ893748.1 and FJ515894.1). Pathogenicity was confirmed using surface-sterilized mature ‘Gala’ apples, wounded with a sterile needle, and inoculated with an isolate conidial suspension (103 spores/ml). A 20 μl droplet was placed in the wound; control fruits received sterile water without conidia. After 5 days of incubation at 20°C in plastic containers with high humidity, typical symptoms of brown rot developed on inoculated fruits, while control fruits remained symptomless. The fungus isolated from inoculated fruit exhibited the same morphological and molecular features shown by the original isolates. To our knowledge, this is the first report of the fungus M. fructicola on apple in Italy. Further studies are necessary to determine geographic distribution, prevalence and economic importance of this quarantine organism in Italy. References: (1) J. Duchoslavovà et al. Plant Dis.91:907, 2007. (2) A. Grabke et al. Plant Dis. 95:772, 2011. (3) OEPP/EPPO. EPPO A2 list of pests recommended for regulation as quarantine pests. Version 2010-09. Retrieved from http://www.eppo.int/QUARANTINE/listA2.htm , 2010. (4) M. Vasic et al. Plant Dis. 96:456, 2012.

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First Report of Botryosphaeria dothidea Causing White Rot of Apple Fruit in Serbia

Plant Disease ◽

10.1094/pdis-05-13-0493-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1659-1659 ◽

Cited By ~ 4

Author(s):

M. Vasić ◽

N. Duduk ◽

I. Vico ◽

M. S. Ivanović

Keyword(s):

Morphological Characteristics ◽

Its Region ◽

Apple Fruit ◽

White Rot ◽

Morphological Identification ◽

Botryosphaeria Dothidea ◽

Apple Trees ◽

First Report ◽

Surface Sterilization ◽

Control Fruit

Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not has a worldwide distribution infecting species from over 80 genera of plants (1). Apart from being an important pathogen of apple trees in many countries, B. dothidea can cause pre- and postharvest decay on apple fruit (2). It has been known to cause canker and dieback of forest trees in Serbia (3), but has not been recorded either on apple trees or apple fruit. In December 2010, apple fruit cv. Idared (Malus × domestica Borkh.) with symptoms of white rot were collected from one storage in the area of Svilajnac in Serbia. The incidence of the disease was low but the symptoms were severe. Affected fruit were brown, soft, and almost completely decayed, while the internal decayed tissue appeared watery and brown. A fungus was isolated from symptomatic tissue of one fruit after surface sterilization with 70% ethanol (without rinsing) and aseptic removal of the skin. Small fragments of decayed tissue were placed on potato dextrose agar (PDA) and incubated in a chamber at 22°C under alternating light and dark conditions (12/12 h). Fungal colonies were initially whitish, but started turning dark gray to black after 5 to 6 days. Pycnidia were produced after 20 to 25 days of incubation at 22°C and contained one-celled, elliptical, hyaline conidia. Conidia were 17.19 to 23.74 μm (mean 18.93) × 3.72 to 4.93 μm (mean 4.45) (n = 50). These morphological characteristics are in accordance with those described for the fungus B. dothidea (4). Genomic DNA was isolated from the fungus and internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The nucleotide sequence has been assigned to GenBank Accession No. KC994640. BLAST analysis of the 528-bp segment showed a 100% similarity with several sequences of B. dothidea deposited in NCBI GenBank, which confirmed morphological identification. Pathogenicity was tested by wound inoculation of five surface-sterilized, mature apple fruit cv. Idared with mycelium plugs (5 mm in diameter) of the isolate grown on PDA. Five control fruit were inoculated with sterile PDA plugs. After 5 days of incubation in plastic containers, under high humidity (RH 90 to 95%) at 22°C, typical symptoms of white rot developed on inoculated fruit, while wounded, uninoculated, control fruit remained symptomless. The isolate recovered from symptomatic fruit showed the same morphological features as original isolate. To the best of our knowledge, this is the first report of B. dothidea on apple fruit in Serbia. Apple is widely grown in Serbia and it is important to further investigate the presence of this pathogen in apple storage, as well as in orchards since B. dothidea may cause rapid disease outbreaks that result in severe losses. References: (1) G. H. Hapting Agriculture Handbook 386, USDA, Forest Service, 1971. (2) A. L. Jones and H. S. Aldwinckle Compendium of Apple and Pear Diseases. APS Press, St. Paul, MN, 1990. (3) D. Karadžic et al. Glasnik Šumarskog Fakulteta 83:87, 2000. (4) B. Slippers et al. Mycologia 96:83, 2004.

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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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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 Sphaeropsis Rot of Apple Caused by Sphaeropsis pyriputrescens in New York

Plant Disease ◽

10.1094/pdis-03-13-0320-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1257-1257 ◽

Cited By ~ 4

Author(s):

Y. K. Kim ◽

R. Caiazzo ◽

P. Sikdar ◽

C. L. Xiao

Keyword(s):

New York ◽

New York State ◽

Apple Fruit ◽

Fruit Rot ◽

Economic Losses ◽

Sodium Hypochlorite Solution ◽

First Report ◽

Control Fruit ◽

Representative Isolate ◽

Sphaeropsis Rot

In March 2012, decayed ‘Empire’ apple fruit (Malus × domestica Borkh.) were sampled from apples grown in Albion (Orleans County) in New York State and stored in bins for 6 months under controlled atmosphere at a commercial packinghouse. At the packinghouse following storage prior to be packed, the fruit were completely rotten, spongy to firm, and light brown without pycnidia. All fruit rots originated from either stem-end or calyx-end infections but no wound infections were observed. The incidence of fruit with these symptoms in the total decay was relatively low (0.1%). To isolate the causal agent, small fragments of fruit flesh from 12 decayed fruit were cut and placed on potato dextrose agar (PDA) acidified with 0.1% lactic acid. The plates were incubated at 20°C for 4 days and sub-cultured on PDA to obtain a pure culture. The colonies initially appeared with dense hyaline mycelium and later turned light yellow to yellow, and black pycnidia formed after about 2 weeks of incubation under a 24-h fluorescent light at 20°C. Conidia were light brown to brown, clavate to subglobose to irregular, and 15 × 8 μm on average. The fungus was identified as Sphaeropsis pyriputrescens Xiao & J.D. Rogers based on the morphology of the fungus (3). The identity of a representative isolate was further confirmed by analysis of nucleotide sequences of the internal transcribed spacer (ITS) regions amplified using the primers ITS1/ITS4. A BLAST search in GenBank showed that the sequence had 99% homology to an S. pyriputrescens sequence (Accession No. GQ374241). One representative isolate was tested for pathogenicity on apple fruit. Organic ‘Red Delicious’ apple fruit were surface-disinfected in 0.6% sodium hypochlorite solution for 5 min, rinsed twice with deionized water, and air-dried. Each fruit was wounded with a sterilized finish-nail head (3 mm in depth and 4 mm in diameter) and inoculated by placing a 4-mm-diameter plug from the leading edge of a 4-day-old PDA culture on the wound. Control fruit were treated with sterile PDA plugs. The inoculation site was covered with two layers of moist cheesecloth to avoid dehydration. There were four 10-fruit replicates for each treatment, and fruit were placed in plastic crispers and stored at 4°C for 4 weeks. The experiments were conducted twice. Sphaeropsis rot developed on all inoculated fruit, while no decays appeared on the control fruit. Koch's postulates were fulfilled by reisolating the fungus from the decayed fruit. Sphaeropsis rot is a recently reported postharvest fruit rot disease of apple and pear (1,3). The disease was first observed on ‘d'Anjou’ pears, and later more serious economic losses were observed in apples in Washington State (1). The disease has also since been reported in British Columbia, Canada (2). To the best of our knowledge, this is the first report of the occurrence of Sphaeropsis rot caused by S. pyriputrescens on apple in New York or in any region outside of the Pacific Northwest in North America. References: (1) Y. K. Kim and C. L. Xiao. Plant Dis. 92:940, 2008. (2) P. L. Sholberg et al. Plant Dis. 93:843, 2009. (3) C. L. Xiao et al. Plant Dis. 88:223, 2004.

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First Report of Penicillium griseofulvum Causing Blue Mold on Stored Apples in Italy (Piedmont)

Plant Disease ◽

10.1094/pdis-08-10-0568 ◽

2011 ◽

Vol 95 (1) ◽

pp. 76-76 ◽

Cited By ~ 3

Author(s):

D. Spadaro ◽

A. Lorè ◽

M. T. Amatulli ◽

A. Garibaldi ◽

M. L. Gullino

Keyword(s):

High Performance ◽

The United States ◽

Apple Fruit ◽

Morphological Identification ◽

Blue Mold ◽

Sequence Coverage ◽

First Report ◽

Golden Delicious ◽

Penicillium Griseofulvum ◽

Piedmont Region

In northern Italy, blue mold can occur generally on apples after 3 months of storage under controlled atmospheres. The mold can be caused by Penicillium griseofulvum Dierckx (synonym P. urticae Bainier). During 2008, several postharvest fruit rots were observed on apples (cv. Golden Delicious) after 180 to 240 days of storage at 1°C. Approximately 8% of the fruits showed blue mold. Apples had been cultivated in Aosta (Aosta Valley Region) and Lagnasco (Piedmont Region). Infected fruits showed soft, watery, brown spots enlarging rapidly at 20°C. There was a distinct margin between soft rotted flesh and firm healthy tissues. Under high humidity, masses of blue-green spores formed on the surface of the lesion. Apple fruit excisions from the margin between the healthy and diseased tissues were plated on potato dextrose agar (PDA), pH 5.6. The recovered fungus produced abundant mycelium and conidia, with the colonies attaining a diameter of 2.0 to 2.4 cm after 7 days at 20 ± 2°C on PDA. Colonies were mostly yellow-green, with a yellowish-to-orange brown underside. Conidiophores were mononematous or loosely synnematous, hyaline, with branches strongly divergent. Phialides were cylindrical with a very short neck. Conidia were ellipsoidal, sometimes subglobose, 2.5 to 3.5 × 2.2 to 2.5 μm, hyaline to greenish. Preliminary morphological identification of the fungus (2) was confirmed by PCR using genomic DNA extracted from mycelia of pure cultures. Two sequences, obtained through the amplification of ribosomal region ITS1-5.8S-ITS2 (1), were blast searched in GenBank and showed 99% sequence coverage and 99% similarity to ribosomal sequences of P. griseofulvum. Two sequences were deposited in GenBank with Accession Nos. HQ012498 (a strain from Aosta Valley) and HQ012499 (a strain from the Piedmont Region). Pathogenicity was tested on 20 ripe fruits each of four apple cultivars (Golden Delicious, Red Chief, Granny Smith, and Royal Gala). Fruits were surface sterilized with 1% sodium hypochlorite. Conidial suspensions (30 μl of 105 conidia/ml) of the fungus were placed on artificial wounds generated on the apple surface. Control fruits were treated with sterile water. Seven days after inoculation, the symptoms were reproduced on the four cultivars and P. griseofulvum was reisolated on PDA from the inoculated fruits of all four cultivars. Control fruits were symptomless. An analysis using high-performance liquid chromatography with diode array of the rotting tissues associated with inoculated fruits of all four cultivars (4) confirmed, as in the case of other strains of P. griseofulvum, the production of the mycotoxin patulin (12.1 to 44.4 mg kg–1). Previously, P. griseofulvum was reported on apple in other countries such as the United States (3), Japan, Egypt, and Brazil. To our knowledge, this is the first report of P. griseofulvum on apples during storage in Italy. References: (1) R. Nilsson et al. FEMS Microbiol. Lett. 296:97, 2009. (2) R. A. Samson and J. L. Pitt. Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification. Harwood Academic Publishers, Singapore, 2001. (3) P. G. Sanderson and R. A. Spotts. Phytopathology 85:103, 1995. (4) D. Spadaro et al. Food Addit. Contam. B 1:134, 2008.

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First Report of Peach Brown Rot Caused by Monilinia fructicola in Central and Western China

Plant Disease ◽

10.1094/pdis-03-13-0310-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1255-1255 ◽

Cited By ~ 2

Author(s):

L. F. Yin ◽

S. N. Chen ◽

N. N. Yuan ◽

L. X. Zhai ◽

G. Q. Li ◽

...

Keyword(s):

Prunus Persica ◽

Direct Sequencing ◽

Eastern China ◽

Morphological Characteristics ◽

Brown Rot ◽

Its Sequences ◽

Western China ◽

Its2 Region ◽

Monilinia Fructicola ◽

First Report

Brown rot of peach (Prunus persica) in China has been reported to be caused by at least three Monilinia species (1). In the present study, peaches with symptoms resembling brown rot caused by Monilinia species were collected from commercial orchards in the northwestern province of Gansu in August 2010, the southwestern province of Yunnan in July 2011, and in the central province of Hubei in July 2012. Affected fruit showed the typical symptoms of brown rot with zones of sporulation. Fungal isolates were single-spored and cultured on potato dextrose agar (PDA). Colonies showed grayness with concentric rings of sporulation after incubation at 25°C in the dark. Mean mycelial growth of isolates YHC11-1a and YHC11-2a from Yunnan, GTC10-1a and GTC10-2a from Gansu, and HWC12-14a and HWC12-23a from Hubei, was 4.6 ± 0.4 and 7.5 ± 0.7 cm after 3 and 5 days incubation, respectively. Conidia were lemon shaped and formed in branched monilioid chains, and the mean size was 9.3 (6.7 to 11.5) × 12.5 (7.9 to 17.8) μm, which was consistent with the characteristics of Monilinia fructicola (1,2). The species identification was confirmed by sequencing of the ribosomal ITS sequences. The ribosomal ITS1-5.8S-ITS2 region was amplified from each of the six isolates using primers ITS1 and ITS4 (3). Results indicated that the ITS sequences of these isolates were identical and showed the highest similarity (100%) with M. fructicola ITS sequences from isolates collected in China (GenBank Accession Nos. HQ893748, FJ515894, and AM887528), Slovenia (GU967379), Italy (FJ411109), and Spain (EF207423). The pathogen was also confirmed to be M. fructicola based on the detection of an M. fructicola- specific band (534 bp) using a PCR-based molecular tool developed for distinguishing Chinese Monilinia species affecting peach (1). Pathogenicity was tested on surface-sterilized, mature peaches (Shui Mi Tao) with representative isolates. Fruits were holed at three equidistant positions to a depth of 5 mm using a sterile cork borer. Mycelial plugs (5 mm in diameter) from the periphery of a 4-day-old colony of each isolate were placed upside down into each hole, control fruits received water agar. After 3 days of incubation at 22°C in a moist chamber, inoculated fruits developed typical brown rot symptoms while control fruits remained healthy. Pathogens from the inoculated fruit were confirmed to be M. fructicola based on morphological characteristics. To our knowledge, this is the first report of occurrence of M. fructicola in Gansu, Yunnan, and Hubei provinces, thousands of kilometers away from eastern China where occurrence of peach brown rot caused by M. fructicola has been confirmed (2,4). The results indicated the further geographical spread of the M. fructicola in China. References: (1) M. J. Hu et al. Plos One 6(9):e24990, 2011. (2) M. J. Hu et al. Plant Dis. 95:225, 2011. (3) T. J. White et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, San Diego, 1990. (4) X. Q. Zhu et al. Plant Pathol. 54:575, 2005.

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