First Report of Penicillium crustosum Causing Blue Mold on Stored Apple Fruit in Serbia.

Penicillium crustosum Thom (1930) causes blue mold on pome fruits and is also regularly found on cheese, nuts, and soil (1,3). The fungus produces a wide range of mycotoxins such as penitrem A, roquefortine C, terrestric acid, and cyclopenol, which impact human health (1). In January and February 2013, 20 decayed apples, 'Golden Delicious' and 'Jonagold' (Malus × domestica Borkh.) with blue mold symptoms were collected from cold storages in Svilajnac and Bela Crkva, Serbia. Decayed areas were light to medium brown with blue green sporulation on the surface of the lesion. Decayed tissue was soft and watery with a sharp margin between the diseased and healthy areas. One isolate from each cultivar was designated JP2 ('Golden Delicious') and JBC7 ('Jonagold') and further characterized. Conidiophores of both isolates were terverticillate, stipes were septate with rough walls, and phialides were ampulliform. Conidia were smooth, borne in columns, and were spherical to subglobose. Conidial dimensions for JP2 were 3.2 to 4.56 (3.73) × 2.64 to 4.3 (3.32) µm and for JBC7 were 3.1 to 4.46 (3.65) × 2.81 to 4.27 (3.31) µm (n = 50). The isolates were cultured on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) media and incubated at 25°C for 7 days. Mycelia were white with heavy sporulation yielding grayish green colonies on all media. Colonies were radially sulcate and velutinous, with clear exudate, and produced a yellow to orange reverse on CYA and YES. On MEA, colonies were plane, low, and mycelia subsurface with conidia having a dry powdery appearance. Crusts of conidial masses formed after 10 or more days. No growth was observed on CYA when these isolates were incubated at 37°C. Both isolates were identified as P. crustosum Thom using morphological characters according to (2) and (1). Species level identification was confirmed by isolating genomic DNA followed by amplification of the ß-tubulin locus using gene specific primers via conventional PCR (4). MegaBLAST analysis of the 2X consensus nucleotide sequences revealed that JP2 and JBC7 (GenBank KJ433984 and 85) were 99% identical to P. crustosum culture collection isolate IBT 21518 (JN112030.1). Koch's postulates were examined using two apple cvs. Idared and Kolacara. Ten fruit per cultivar per isolate were inoculated on two sides of each fruit; 20 fruit were used as water-only inoculated controls. Fruit were washed with soap and water, surface sanitized with 70% ethanol, and placed into polyethylene boxes. Using a finishing nail, 4-mm wounds were created and inoculated with 50 µl of a 3 × 105/ml conidial suspension or Tween-treated sterile distilled water. Boxes with inoculated and control fruit were stored at 25°C for 10 days. The inoculated fruit developed small, soft, watery lesions, which enlarged into decayed areas with defined edges and abundant sporulation on the surface. Symptoms were identical to the original ones, while the control fruit remained symptomless. The fungus was re-isolated from infected tissue and showed the same morphological characteristics as the original isolates, thus completing Koch's postulates. Blue mold occurs during long term storage of apples and is predominantly caused by P. expansum. This is the first report of P. crustosum causing postharvest blue mold decay on apple fruit obtained from storage in Serbia and indicates that P. crustosum is an emerging pathogen for the Serbian pome fruit growing and packing industry. References: (1) J. C. Frisvad and R. A. Samson. Stud. Mycol. 49:1, 2004. (2) J. I. Pitt and A. D. Hocking. Fungi and Food Spoilage, 239. Springer, 2009. (3) P. G. Sanderson and R. A. Spotts. Phytopathology 85:103. 1995. (4) P. L. Sholberg et al. Postharvest Biol. Technol. 36:41, 2005.

First Report of Fusarium avenaceum Causing Postharvest Decay of 'Gala' Apple Fruit in the United States.

Apples are kept in controlled atmosphere cold storage for 9 to 12 months and are highly susceptible to postharvest decay caused by various fungi. Fusarium avenaceum is a wound pathogen that has been shown to account for the majority of Fusarium rot on apple fruit in Croatia (1). F. avenaceum produces an array of mycotoxins including moniliformin, acuminatopyrone, and chrysogine, which are of primary concern for the apple processing industry (2). In February 2013, 'Gala' apple fruits with soft, circular, brown, watery lesions with characteristic abundant whitish mycelium covering the surface of the colonized fruit were obtained from bins from a commercial storage facility located in Pennsylvania. Several samples were collected and prepared for pathogen isolation. Apples were rinsed with sterile water, and the lesions were sprayed with 70% ethanol until runoff. The apple skin was aseptically removed with a scalpel, and asymptomatic tissue was placed onto full strength potato dextrose agar (PDA) petri plates without antibiotics and incubated at 25°C under natural light. Two single-spore isolates were propagated on PDA and permanent cultures were maintained as slants and stored in a cold room at 4°C in the dark. Fungal colonies initially formed abundant fluffy white mycelium and produced a golden orange pigment on PDA at 25°C. Isolates were identified as Fusarium based on cultural and conidial morphology as macroconidia were slightly falcate, thin-walled, usually 3 to 5 septate, with a tapering apical cell that was on average 23.6 µm long × 5.0 µm wide (n = 50). Microconidia were produced on PDA plates while chlamydospores were not evident. Identity of the isolates was confirmed through DNA extraction followed by amplification and sequencing of the translation elongation factor (EF-1α, 350 bp) gene region. The amplicons were sequenced using the forward and reverse primers and assembled into a consensus representing 2X coverage. MegaBLAST analysis revealed that both isolates were 100% identical with many other culture collection F. avenaceum sequences in Genbank (Accessions JQ949291.1, JQ949305.1, and JQ949283.1), which confirms their identification in conjunction with the morphological observations. Koch's postulates were conducted to determine pathogenicity using organic 'Gala' apple fruit that were surface sanitized with soap and water, sprayed with 70% ethanol, and wiped dry. The fruit were wounded with a finishing nail to 3 mm depth, inoculated with 50 µl of a conidial suspension (1 × 104 conidia/ml) using a hemocytometer, and stored at 25°C in 80-count boxes on paper trays for 21 days. Water-only controls were symptomless. Ten fruit composed a replicate for each isolate, and the experiment was repeated. Symptoms observed on artificially inoculated 'Gala' apple fruit were identical to the decay observed on 'Gala' apples that were obtained from cold storage. Decay caused by F. avenaceum may represent an emerging problem for the apple storage and processing industry. Therefore, it is important to monitor for this pathogen to prevent future losses and mycotoxin contamination of processed fruit products caused by this fungus. To the best of our knowledge, this is the first report of Fusarium rot caused by F. avenaceum on apple fruit from cold storage in the United States. References: (1) Z. Sever et al. Arch. Ind. Hygiene Toxicol. 63:463, 2012. (2) J. L. Sorenson. J. Agric. Food Chem. 57:1632, 2009.

First Report of Alternaria tenuissima Causing Postharvest Decay on Apple Fruit from Cold Storage in the United States.

Apples are grown and stored for 9 to 12 months under controlled atmosphere conditions in the United States. During storage, apples are susceptible to various fungal pathogens, including several Alternaria species (2). Alternaria tenuissima (Nees) Wiltshire causes dry core rot (DCR) on apples during storage and has recently occurred in South Africa (1). Losses range widely, but typically occur at 6 to 8% annually due to this disease (2). In February 2013, 'Nittany' apples with round, dark-colored, dry, spongy lesions were obtained from wooden bins in a commercial cold storage facility located in Pennsylvania. Symptomatic fruits were transported to the lab, rinsed with sterile water, and the lesions were sprayed with 70% ethanol until runoff and wiped dry. The skin was aseptically removed with a scalpel, and asymptomatic tissue was placed onto potato dextrose agar (PDA) and incubated at 25°C. Two single-spore isolates were propagated on PDA and permanent cultures were maintained as slants and stored at 4°C. The fungus produced a cottony white mycelium that turned olive-green to brown with abundant aerial hyphae and had a dark brown to black reverse on PDA. Isolates were identified as Alternaria based on conidial morphology as the spores were slightly melanized and obclavate to obpyriform catentulate with longitudinal and transverse septa attached in unbranched chains on simple short conidiophores. Conidia ranged from 10 to 70 µm long (mean 27.7 µm) and 5 to 15 µm wide (mean 5.25 µm) (n = 50) with 1 to 6 transverse and 0 to 2 longitudinal septa. Conidial beaks, when present, were short (5 µm or less) and tapered. Mycelial genomic DNA was extracted, and a portion of the histone gene (357 bp) was amplified via gene specific primers (Alt-His3-F/R) using conventional PCR (Jurick II, unpublished). The forward and reverse sequences were assembled into a consensus representing 2× coverage and MegaBLAST analysis showed that both isolates were 100% identical to Alternaria tenuissima isolates including CR27 (GenBank Accession No. AF404622.1) that caused DCR on apple fruit during storage in South Africa. Koch's postulates were conducted using 10 organic 'Gala' apple fruit that were surface sterilized with soap and water, sprayed with 70% ethanol, and wiped dry. The fruit were aseptically wounded with a nail to a 3 mm depth, inoculated with 50 µl of a conidial suspension (1 × 104 conidia/ml), and stored at 25°C in 80 count boxes on paper trays for 21 days. Mean lesion diameters on inoculated 'Gala' apple fruit were 19.1 mm (±7.4), water only controls (n = 10 fruit) were symptomless, and the experiment was repeated. Symptoms observed on artificially inoculated 'Gala' apple fruit were similar to the decay observed on 'Nittany' apples from cold storage. Based on our findings, it is possible that A. tenuissima can cause decay that originates from wounded tissue in addition to dry core rot, which has been reported (1). Since A. tenuissima produces potent mycotoxins, even low levels of the pathogen could pose a health problem for contaminated fruit destined for processing and may impact export to other countries. To the best of our knowledge, this is the first report of alternaria rot caused by A. tenuissima on apple fruit from cold storage in the United States. References: (1) J. C. Combrink et al. Decid. Fruit Grow. 34:88, 1984. (2) M. Serdani et al. Mycol. Res. 106:562, 2002. (3) E. E. Stinson et al. J. Agric. Food Chem. 28:960, 1980.

First Report of Alternaria alternata Causing Postharvest Decay on Apple Fruit During Cold Storage in Pennsylvania.

Alternaria rot, caused by Alternaria alternata (Fr.) Keissl., occurs on apple fruit (Malus × domestica Borkh) worldwide and is not controlled with postharvest fungicides currently registered for apple in the United States (1). Initial infections can occur in the orchard prior to harvest, or during cold storage, and appear as small red dots located around lenticels (1). The symptoms appear on fruits within a 2 month period after placement into cold storage (3). In February 2013, 'Nittany' apple fruit with round, dark, dry, spongy lesions were collected from bins at commercial storage facility located in Pennsylvania. Symptomatic apples (n = 2 fruits) were placed on paper trays in an 80 count apple box and immediately transported to the laboratory. Fruit were rinsed with sterile water, and the lesions were superficially disinfected with 70% ethanol. The skin was removed with a sterile scalpel, and tissues underneath the lesion were cultured on potato dextrose agar (PDA) and incubated at 25°C with constant light. Two single-spore isolates were propagated on PDA, and permanent cultures were maintained on PDA slants and stored at 4°C in darkness. Colonies varied from light gray to olive green in color, produced abundant aerial hyphae, and had fluffy mycelial growth on PDA after 14 days. Both isolates were tentatively identified as Alternaria based on multicelled conidial morphology resembling "fragmentation grenades" that were medium brown in color, and obclavate to obpyriform catentulate with longitudinal and transverse septa attached in chains on simple conidiophores (2). Conidia ranged from 15 to 60 µm (mean 25.5 µm) long and 10 to 25 µm (mean 13.6 µm) wide (n = 50) with 1 to 6 transverse and 0 to 1 longitudinal septa per spore. To identify both isolates to the species level, genomic DNA was extracted from mycelial plugs and gene specific primers (ALT-HIS3F/R) were used via conventional PCR to amplify a portion of the histone gene (357 bp) (Jurick II, unpublished). Amplicons were sequenced using the Sanger method, and the forward and reverse sequences of each amplicon were assembled into a consensus representing 2× coverage. A megaBLAST analysis revealed that the isolates were 99% identical to Alternaria alternata sequences in GenBank (Accession No. AF404617), which was previously identified to cause decay on stored apple fruit in South Africa. To prove pathogenicity, Koch's postulates were conducted using organic 'Gala' apples. The fruit were surface disinfested with soap and water and sprayed with 70% ethanol to runoff. Wounds, 3 mm deep, were done using a sterile nail and 50 µl of a conidial suspension (1 × 104 conidia/ml) was introduced into each wound per fruit. Fruit were then stored at 25°C in 80 count boxes on paper trays for 21 days. Water only was used as a control. Ten fruit were inoculated with each isolate or water only (control) and the experiment was repeated once. Symptoms of decay observed on inoculated were 'Gala' apple fruit were identical to the symptoms initially observed on 'Nittany' apples obtained from cold storage after 21 days. No symptoms developed on fruit in the controls. A. alternata was re-isolated 100% from apple inoculated with the fungus, completing Koch's postulates. A. alternata has been documented as a pre- and postharvest pathogen on Malus spp. (3). To our knowledge, this is the first report of postharvest decay caused by A. alternata on apple fruit during cold storage in Pennsylvania. References: (1) A. L. Biggs et al. Plant Dis. 77:976, 1993. (2) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Center, Utrecht, the Netherlands, 2007. (3) R. S. Spotts. Pages 56-57 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinkle, eds. American Phytopathological Society, St. Paul, MN, 1990.

First Report of Colletotrichum fioriniae Causing Postharvest Decay on 'Nittany' Apple Fruit in the United States.

Bitter rot of apple is caused by Colletotrichum acutatum and C. gleosporioides and is an economically important disease in the mid-Atlantic and southern regions of the United States (1). However, other Colletotrichum spp. have been found to infect apple and pear fruit in Croatia that include C. fioriniae and C. clavatum (3). The disease is favorable under wet, humid conditions and can occur in the field or during storage causing postharvest decay (2). In February 2013, 'Nittany' apples with round, brown, dry, firm lesions having acervuli in concentric rings were observed at a commercial cold storage facility in Pennsylvania. Samples were placed on a paper tray in an 80-count apple box and immediately transported to the lab. Fruit were rinsed with sterile water, and lesions were sprayed with 70% ethanol until runoff. The skin was aseptically removed with a scalpel, and tissue under the lesion was placed onto potato dextrose agar (PDA) petri dishes. Dishes were incubated at 25°C with constant light, and a single-spore isolate was propagated on PDA. Permanent cultures were maintained as PDA slants stored at 4°C in darkness. The isolate was identified as a Colletotrichum sp. based on culture morphology, having light gray mycelium with a pinkish reverse and abundant pin-shaped melanized acervuli oozing pink conidia on PDA. Conidia were fusiform, pointed at one or both ends, one-celled, thin-walled, aseptate, hyaline, and averaged 10.5 µm (7.5 to 20 µm) long and 5.1 µm (5 to 10 µm) wide (n = 50). Genomic DNA was extracted from mycelia and amplified using conventional PCR and gene specific primers for 313 bp of the Histone 3 gene and with ITS4/5 primers for the internal transcribed spacer (ITS) rDNA region. MegaBLAST analysis of both gene sequences showed that our isolate was identical to other Colletotrichum fioriniae sequences in GenBank and was 100% identical to culture-collection C. fioriniae isolate CBS:128517, thus confirming the morphological identification. To prove pathogenicity, Koch's postulates were conducted using organic 'Gala' apple fruit that were washed with soap and water, sprayed with 70% ethanol, and wiped dry. The fruit were wounded with a sterile nail to a 3-mm depth, inoculated with 50 µl of a conidial suspension (1 × 104 conidia/ml), and stored at 25°C in 80-count boxes on paper trays for 14 days. Lesion diameter was measured from 10 replicate fruit with a digital micrometer and averaged 31.2 mm (±2.5 mm) over two experiments (n = 20). Water-only controls were symptomless. Artificially inoculated 'Gala' apples had identical external and internal symptoms (v-shaped decay pattern when the fruit were cut in half) to those observed on 'Nittany' apples that were originally obtained from cold storage. Bitter rot caused by C. fioriniae may become an emerging problem for the pome fruit growing industry in the near future, and may require investigation of new disease management practices to control this fungus. This is the first report of postharvest decay caused by C. fioriniae on apple fruit from cold storage in the United States. References: (1) H. W. Anderson. Diseases of Fruit Crops. McGraw-Hill, New York, 1956. (2) A. R. Biggs et al. Plant Dis. 85:657, 2001. (3) D. Ivic et al. J. Phytopathol. 161:284, 2013.

First Report of Pyrimethanil Resistance in Botrytis cinerea from Stored Apples in Pennsylvania.

Botrytis cinerea Pers.: Fr. (teleomorph Botryotinia fuckeliana [de Bary] Whetzel) causes gray mold on apple fruit. A survey of commercial packinghouses in Washington State revealed that it accounted for 28% of the decay in storage (1). Fungicide application coupled with cultural practices are the primary method of control as all commercial apple cultivars are susceptible to gray mold. In February 2013, gray mold was observed at ~5% incidence for commercially packed 'Gala' apple fruit that had been treated with Penbotec (active ingredient: pyrimethanil, Shield-Bright, Pace International) prior to controlled atmosphere storage in Pennsylvania. Eight infected apple fruit were collected, placed in 80 count boxes on cardboard trays, and stored at 4°C. One isolate was obtained from each decayed apple, placed on potato dextrose agar (PDA) petri plates, and incubated at 20°C with natural light. Eight single-spore isolates were identified as B. cinerea based on cultural characteristics. Species level identification was executed by obtaining mycelial genomic DNA, amplifying the ITS rDNA, and sequencing the ~550-bp amplicon directly (2). MegaBLAST analysis of the 2X consensus for the 8 isolates revealed 100% identity to B. cinerea ITS sequences in GenBank (KF156296.1 and JX867227.1) with E values of 0.0, thus confirming the morphological identification. Minimum inhibitory concentration (MIC) was determined using conidial suspensions obtained from ~14-day-old plates (104 spores/ml) and a range (0 to 500 µg/ml) of technical grade pyrimethanil on three replicated 96-well microtiter plates containing a defined medium for each experiment. Conidial proliferation was inhibited at 250 µg/ml for all eight isolates and the experiment was conducted four times. To further define the resistance levels between the isolates, mycelial growth analysis using a plug of actively growing mycelium from the margin of ~3-day-old plates was conducted with a defined medium three times with technical grade pyrimethanil with three plates per experiment. Five isolates grew at 250 µg/ml (highly resistant), while three did not (moderately resistant). To assess resistance in vivo, organic 'Gala' apples were rinsed with soap and water, sprayed with 70% ethanol, placed on trays, and allowed to air dry. Apples were wounded with a sterile finishing nail, inoculated with 20 µl of a conidial suspension (104 spores/ml) of either a moderately or a highly resistant isolate, and dipped in the labeled application rate of Penbotec at 500 µg/ml or sterile water for 30 s. Fruit were stored in 100 count boxes at 22°C for 5 days and decay incidence and severity were recorded. Ten fruit composed a replicate per treatment and the experiment was repeated. Water inoculated controls were symptomless and water-dipped inoculated fruit had 100% decay. Penbotec-treated fruit had 100% decay incidence and mean lesion diameters of 37.6 (±13.1 mm) for the highly, and 35.7 (±9.0 mm) for the moderately resistant isolate. This is the first report of pyrimethanil resistance in B. cinerea from decayed apples collected from a commercial packinghouse in Pennsylvania. The results indicate that pyrimethanil resistance has developed in B. cinerea, which can result in control failures on Penbotec-treated fruit during storage. Furthermore, it emphasizes the need for additional tools to manage gray mold on apple fruit and may pose issues for export concerning the spread of fungicide-resistant inoculum. References: (1) Y.-K. Kim and C. L. Xiao. Plant Dis. 92:940, 2008. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Penicillium expansum Isolates Resistant to Pyrimethanil from Stored Apple Fruit in Pennsylvania.

Apples in the United States are stored in low-temperature controlled atmospheres for 9 to 12 months and are highly susceptible to blue mold decay. Penicillium spp. cause significant economic losses worldwide and produce mycotoxins that contaminate processed apple products. Blue mold is managed by a combination of cultural practices and the application of fungicides. In 2004, a new postharvest fungicide, pyrimethanil (Penbotec 400 SC, Janseen PMP, Beerse, Belgium) was registered for use in the United States to control blue mold on pome fruits (1). In this study, 10 blue mold symptomatic 'Red Delicious' apples were collected in May 2011 from wooden bins at a commercial facility located in Pennsylvania. These fruit had been treated with Penbotec prior to controlled atmosphere storage. Ten single-spore Penicillium spp. isolates were analyzed for growth using 96-well microtiter plates containing Richards minimal medium amended with a range of technical grade pyrimethanil from 0 to 500 µg/ml. Conidial suspensions adjusted to 1 × 105 conidia/ml were added to three 96-well plates for each experiment; all experiments were repeated three times. Nine resistant isolates had prolific mycelial growth at 500 µg/ml, which is 1,000 times the discriminatory dose that inhibited baseline sensitive P. expansum isolates from Washington State (1). However, one isolate (R13) had limited conidial germination and no mycelial proliferation at 0.5 µg/ml and was categorized as sensitive. One resistant (R22) and one sensitive (R13) isolate were selected on the basis of their different sensitivities to pyrimethanil. Both isolates were identified as P. expansum via conventional PCR using ß-tubulin gene-specific primers according to Sholberg et al. (2). Analysis of the 2X consensus amplicon sequences from R13 and R22 matched perfectly (100% identity and 0.0 E value) with other P. expansum accessions in GenBank including JN872743.1, which was isolated from decayed apple fruit from Washington State. To determine if pyrimethanil applied at the labeled rate of 500 µg/ml would control R13 or R22 in vivo, organic 'Gala' apple fruit were wounded, inoculated with 50 µl of a conidial suspension (1 × 104 conidia/ml) of either isolate, dipped in Penbotec fungicide or sterile water, and stored at 25°C for 7 days. Twenty fruit composed a replicate within a treatment and the experiment was performed twice. Non-inoculated water-only controls were symptomless, while water-dipped inoculated fruit had 100% decay with mean lesion diameters of 36.8 ± 2.68 mm for R22 and 38.5 ± 2.61 mm for R13. The R22 isolate caused 30% decay with 21.6 ± 5.44 mm lesions when inoculated onto Penbotec-treated apples, while the R13 isolate had 7.5% decay incidence with mean lesion diameters of 23.1 ± 3.41 mm. The results from this study demonstrate that P. expansum pyrimethanil-resistant strains are virulent on Penbotec-treated apple fruit and have the potential to manifest in decay during storage. To the best of our knowledge, this is the first report of pyrimethanil resistance in P. expansum from Pennsylvania, a major apple growing region for the United States. Moreover, these results illuminate the need to develop additional chemical, cultural, and biological methods to control this fungus. References: (1) H. X. Li and C. L. Xiao. Phytopathology 98:427, 2008. (2) P. L. Sholberg et al. Postharvest Biol. Technol. 36:41, 2005.

First Report of Botryosphaeria dothidea Causing White Rot on Apple Fruit in Maryland.

Botryosphaeria dothidea (Moug.:Fr.) Ces. De Not. causes perennial cankers on apple trees and causes white rot on apple fruit in the field and during storage (1). Prolonged periods of warm wet weather favor rapid disease outbreaks that result in severe losses, which range from 25 to 50% for the southeastern United States (3). A B. dothidea isolate was obtained from decayed 'Fuji' apple fruit exhibiting white rot symptoms from a local farm market in Beltsville, MD, in May 2010. The fruit had characteristic large dark brown lesions with irregular margins and decay expanded unevenly toward the core and the tissue was soft. The pathogen was isolated from symptomatic tissue by spraying the lesion surface with 70% ethanol. The skin with aseptically removed with a scalpel and small pieces of tissue were placed on potato dextrose agar (PDA) and incubated at 20°C. Once fungal growth was evident, the cultures were hyphal-tip transferred to individual PDA plates and incubated at 20°C. The B. dothidea isolate produced black aerial mycelium with a white margin on PDA and had a black reverse. Conidiomata were evident after 10 to 14 days at 20°C only on oatmeal agar. Conidia were hyaline, smooth and straight, fusiform with an subobtuse apex and a truncate base 20 to 26 (24.33) × 4 to 7 (5) µm (n = 50). Genomic DNA was isolated from the fungus and amplified with gene specific primers (ITS 4 and 5) for the ribosomal DNA internal transcribed spacer region ITSI-5.8S-ITS2 as described by White et al. (4). Both forward and reverse strands of the 542-bp amplicon were sequenced and assembled into a contig. The nucleotide sequence (GenBank Accession No. KC473852) indicated 99% identity to B. dothidea isolate CMM3938 (JX513645.1) and to voucher specimens CMW 25686, 25696, and 25222 (FM955381.1, FM955379.1, and FM955377,1). Koch's postulates were conducted using three 'Golden Delicious' apple fruit that were wound-inoculated with 50 µl of a mycelial suspension of the fungus, obtained from aseptically scraping a 7-day-old PDA culture, and was also repeated using 'Fuji' apple fruit. Large, brown, slightly sunken, soft lesions with undefined edges developed 5 days after inoculation at 20°C and water-only inoculated fruit were symptomless. The fungus was reisolated from infected tissue and was morphologically identical to the original isolate from decayed apple fruit. To determine if the B. dothidea isolate was resistant to postharvest fungicides, the minimum inhibitory concentration (MIC) was conducted using the 96 well plate method with a mycelial suspension of the fungus as described by Pianzzola et al. (2). The MIC for the isolate was >1 ppm for Mertect and Scholar and 50 ppm for Penbotec, which are well below the labeled rates for these postharvest fungicides and the experiment was repeated. To our knowledge, this is the first report of B. dothidea causing white rot on apple fruit in Maryland. References: (1) A. R. Biggs and S. S. Miller. HortScience 38:400, 2003. (2) M. J. Pianzzola et al. Plant Dis. 88:23, 2004. (3) T. B. Sutton. White rot and black rot. Pages 16-20 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinckle, eds. The American Phytopathological Society, St Paul, MN, 1991. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Application. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Neofusicoccum ribis Causing Postharvest Decay of Apple Fruit from Cold Storage in Pennsylvania.

Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.), previously known as Botryosphaeria ribis (Grossenb. & Duggar), is an aggressive fungal plant pathogen that is part of the N. ribis/N. parvum species complex that causes stem cankers on a variety of woody plant species (2). An isolate of N. ribis was obtained from decayed 'Honeycrisp' apple fruit from a commercial cold storage facility located in Pennsylvania in October of 2011. The decayed apple fruit sample had a brownish lesion that was soft, dry, and leathery on the surface while sporulation was not evident. To conduct Koch's postulates, three 'Golden Delicious' apple fruits were wound-inoculated with a 50-µl mycelial suspension, obtained from aseptically scraping a 7-day-old potato dextrose agar (PDA) culture of the fungus, and was repeated using 'Fuji' apple fruit. The inoculated fruit developed lesions, while water-inoculated fruit were symptomless after 5 days at 20°C. N. ribis was reisolated from infected tissue and was morphologically identical to the original isolate. Genomic DNA was isolated, a portion of the ß-tubulin gene was amplified with the gene specific primers, and the amplicon was sequenced and analyzed using BLAST (1). The nucleotide sequence (GenBank Accession No. KC47853) had 99% identity with N. ribis SEGA8 isolate (JN607146.1). The N. ribis isolate produced a grayish-white mycelium with abundant aerial hyphae on PDA and had an olive-colored reverse. Microscopic investigation revealed septate mycelia with right angle branching and conidiomata were not evident on PDA, V8, oatmeal agar (OMA), or water agar (WA). Growth on WA was sparse and transparent, and aerial mycelial growth was not produced. Growth rate analyses were conducted on PDA, V8, and OMA and were 10.1 (±1.39), 20.4 (±1.15), and 17.6 (±0.70) mm/day at 20°C and the experiment was repeated. The minimum inhibitory concentrations (MIC) for the N. ribis isolate was carried out for three postharvest fungicides as described by Pianzzola et al. (3). Briefly, 96 well plates were filled with PDA alone (0 ppm) and PDA amended with 10 fungicide concentrations ranging from 1 to 1,200 ppm for thiabendazole (Mertect), and 1 to 1,000 ppm for fludioxonil (Scholar) and pyrimethanil (Penbotec). A mycelial suspension of the fungus was obtained from pure culture, 50 µl of the mycelial suspension was pipetted into each well, and allowed to grow for 72 h at 25°C. The experiment was conducted twice. The N. ribis isolate displayed MIC values of >1 ppm thiabendazole (Mertect), >1 ppm fludioxonil (Scholar), and 50 ppm pyrimethanil (Penbotec), which are all well below the labeled application rates for these postharvest fungicides. To our knowledge, this is the first report of N. ribis causing postharvest decay on apple fruit obtained from a commercial storage facility in Pennsylvania. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) D. Pavlic et al. Mycologia 101:636, 2009. (3) M. J. Pianzzola et al. Plant Dis. 88:23, 2004.

First Report of Penicillium carneum Causing Blue Mold on Stored Apples in Pennsylvania.

Blue mold decay occurs during long term storage of apples and is predominantly caused by Penicillium expansum Link. Apples harvested in 2010 were stored in a controlled atmosphere at a commercial Pennsylvania apple packing and storage facility, and were examined for occurrence of decay in May 2011. Several decayed apples from different cultivars, exhibiting blue mold symptoms with a sporulating fungus were collected. One isolate recovered from a decayed 'Golden Delicious' apple fruit was identified as P. carneum Frisvad. Genomic DNA was isolated, 800 bp of the 3' end of the ß-tubulin locus was amplified using gene specific primers and sequenced (4). The recovered nucleotide sequence (GenBank Accession No. JX127312) indicated 99% sequence identity with P. carneum strain IBT 3472 (GenBank Accession No. JF302650) (3). The P. carneum colonies strongly sporulated and had a blue green color on potato dextrose agar (PDA), Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) media at 25°C after 7 days. The colonies also had a beige color on plate reverse on CYA and YES media. The species tested positive for the production of alkaloids, as indicated by a violet reaction for the Ehrlich test, and grew on CYA at 30°C and on Czapek with 1,000 ppm propionic acid agar at 25°C; all of which are diagnostic characters of this species (2). The conidiophores were hyaline and tetraverticillate with a finely rough stipe. Conida were produced in long columns, blue green, globose, and averaged 2.9 µm in diameter. To prove pathogenicity, Koch's postulates were conducted using 20 'Golden Delicious' apple fruits. Fruits were washed, surface sterilized with 70% ethanol, and placed onto fruit trays. Using a nail, 3-mm wounds were created and inoculated with 50 µl of a 106/ml conidial suspension or water only as a negative control. The fruit trays were placed into boxes and were stored in the laboratory at 20°C for 7 days. The inoculated fruit developed soft watery lesions, with hard defined edges 37 ± 4 mm in diameter. The sporulating fungus was reisolated from infected tissue of all conidia inoculated apples and confirmed to be P. carneum by polymerase chain reaction (PCR) using the ß-tubulin locus as described. Water inoculated control apples were symptomless. Originally grouped with P. roqueforti, P. carneum was reclassified in 1996 as a separate species (1). P. carneum is typically associated with meat products, beverages, and bread spoilage and produces patulin, which is not produced by P. roqueforti (1,2). Our isolate of P. carneum was susceptible to the thiabendazole (TBZ) fungicide at 250 ppm, which is below the recommended labeled application rate of 600 ppm. The susceptibility to TBZ suggests that this P. carneum isolate has been recently introduced because resistance to TBZ has evolved rapidly in P. expansum (4). To the best of our knowledge, P. carneum has not previously been described on apple, and this is the first report of P. carneum causing postharvest decay on apple fruits obtained from storage in Pennsylvania. References: (1) M. Boyson et al. Microbiology 142:541, 1996. (2) J. C. Frisvad and R. A. Samson. Stud. Mycol. 49:1, 2004. (3) B. G. Hansen et al. BMC Microbiology 11:202, 2011. (4) P. L. Sholberg et al. Postharvest Biol. Technol. 36:41, 2005.