Sources and Availability of Inoculum and Seasonal Survival of Sphaeropsis pyriputrescens in Apple Orchards.

Sphaeropsis pyriputrescens is the cause of Sphaeropsis rot, a recently reported postharvest fruit rot disease of apple. Infection of apple fruit by the fungus is believed to occur in the orchard, and symptoms develop during storage or in the market. S. pyriputrescens also is the cause of a twig dieback and canker disease of apple and crabapple trees. To determine sources of pathogen inoculum in the orchard, twigs with dieback and canker symptoms, dead fruit spurs, dead bark, and fruit mummies on the trees were collected and examined for the presence of pycnidia of S. pyriputrescens. To monitor inoculum availability during the growing season from early May to early November, dead fruit spurs or twigs from Fuji trees, and twigs with dieback from crabapple trees (as a source of pollen for apple production) in a Fuji orchard as well as dead fruit spurs and dead bark from Red Delicious trees in a Red Delicious orchard were sampled periodically and examined for the presence and viability of pycnidia of S. pyriputrescens. To determine seasonal survival and production of pycnidia of the fungus on twigs, apple twigs were inoculated in early December, sampled periodically for up to 12 months after inoculation, examined for the presence of pycnidia, and subjected to isolation of the fungus from diseased tissues to determine its survival. Pycnidia of S. pyriputrescens were observed on diseased twigs, dead fruit spurs and bark, and mummified fruit on both apple and crabapple trees, suggesting that these tissues were the sources of inoculum for fruit infection in the orchard. With the combined observations from two orchards during three growing seasons, viable pycnidia of the fungus were present throughout the year and observed in 50 to 100% of the Fuji trees, >90% of crabapple trees, and 0 to 50% of the Red Delicious trees. S. pyriputrescens was recovered from diseased tissues of inoculated twigs at all sampling times up to 12 months after inoculation. The results suggest that S. pyriputrescens can survive as mycelium in diseased twigs in north-central Washington State and that availability of viable S. pyriputrescens pycnidia is unlikely a limiting factor for infection of apple fruit in the orchard leading to Sphaeropsis rot during storage.

Control of Sphaeropsis Rot in Stored Apple Fruit Caused by Sphaeropsis pyriputrescens with Postharvest Fungicides.

Sphaeropsis rot caused by Sphaeropsis pyriputrescens is a recently reported postharvest fruit rot disease of apple grown in Washington State. The objective of this study was to develop chemical-based mitigation measures for Sphaeropsis rot in stored apple fruit. To determine in vitro sensitivity of S. pyriputrescens to the three registered postharvest fungicides thiabendazole, fludioxonil, and pyrimethanil, 30 isolates of S. pyriputrescens obtained from various sources were tested for mycelial growth and conidial germination on fungicide-amended media. Golden Delicious apple fruit were inoculated with the pathogen in the orchard at 2 or 5 weeks before harvest. After harvest, fruit were either nontreated or dipped in thiabendazole, fludioxonil, or pyrimethanil solutions, stored at 0°C, and monitored for decay development for up to 9 months after harvest. The mean effective concentration of a fungicide that inhibits mycelial growth or spore germination by 50% relative to the nonamended control (EC50) values of thiabendazole, fludioxonil, and pyrimethanil on mycelial growth were 0.791, 0.0005, and 2.829 µg/ml, respectively. Fludioxonil and pyrimethanil also were effective in inhibiting conidial germination of the fungus with EC50 values of 0.02 µg/ml for fludioxonil and 5.626 µg/ml for pyrimethanil. All three postharvest fungicides applied at label rates immediately after harvest were equally effective in controlling Sphaeropsis rot in stored apple fruit, reducing disease incidence by 92 to 100% compared with the nontreated control. The results indicated that Sphaeropsis rot may be effectively controlled by the currently registered postharvest fungicides thiabendazole, fludioxonil, and pyrimethanil.

First Report of Occurrence of Pyrimethanil Resistance in Penicillium expansum from Stored Apples in Washington State.

Blue mold caused by Penicillium expansum is a major postharvest fruit rot disease of apples (Malus domestica) worldwide. Pyrimethanil was registered in late 2004 in the United States for postharvest use on apples. Since then, pyrimethanil has been increasingly used in Washington State as a postharvest drench treatment for control of blue mold and other postharvest diseases in apples. Baseline sensitivity to pyrimethanil in P. expansum populations from apples in Washington State has been established and all isolates in the baseline population were sensitive to pyrimethanil (1). To monitor resistance to pyrimethanil in P. expansum populations, blue mold-like decayed apple fruit were sampled from May to August 2009 from the fruit that had been drenched with pyrimethanil prior to storage from fruit packinghouses. Isolation of Penicillium species from decayed fruit was attempted. Isolates of Penicillium species were identified to species according to the descriptions by Pitt (2). In total, 186 P. expansum isolates were collected and tested for resistance to pyrimethanil in a conidial germination assay on an agar medium amended with pyrimethanil at the discriminatory concentration of 0.5 µg ml-1 (1). Isolates that were able to germinate were considered resistant to pyrimethanil. Of the 186 isolates tested, one was resistant to pyrimethanil. EC50 (the effective concentration that inhibits fungal growth by 50% relative to the control) of pyrimethanil for the resistant isolate was determined according to a method described previously (1) and the test was done twice. EC50 values of pyrimethanil on mycelial growth and conidial germination for the resistant isolate were 9.9 and 3.1 µg/ml, respectively, which were 7.4-fold and 16.5-fold higher than the means of the baseline population (1). To evaluate whether pyrimethanil at label rate is still able to control this resistant isolate, 'Fuji' apples were wounded, inoculated with conidial suspensions (1 × 104 conidia ml-1) of either the resistant isolate or a pyrimethanil-sensitive isolate, treated with either pyrimethanil or sterile water as controls, and stored at 20°C for 10 days following a method described previously (1). There were four 20-fruit replicates for each treatment. The experiment was performed twice. All inoculated fruit in the nontreated controls were decayed. Pyrimethanil applied at label rate completely controlled blue mold incited by a pyrimethanil-sensitive isolate, but 75% of the fruit that were inoculated with the resistant isolate and treated with pyrimethanil developed blue mold. To our knowledge, this is the first report of pyrimethanil resistance in P. expansum from decayed apple fruit collected from commercial packing houses. The pyrimethanil-resistant isolate was obtained from a packing house in which pyrimethanil had been used as a postharvest drench treatment in each of four consecutive years, suggesting that pyrimethanil-resistant individuals are emerging in P. expansum populations in Washington State after repeated use of pyrimethanil. Our results also indicate that pyrimethanil resistance in P. expansum reported in this study can result in failure of blue mold control in apples with pyrimethanil. References: (1) H. X. Li and C. L. Xiao. Postharvest Biol. Technol. 47:239, 2008. (2) J. I. Pitt. A Laboratory Guide to Common Penicillium species. Food Science Australia, North Ryde NSW, Australia, 2002.

Residual Activity of Fludioxonil and Pyrimethanil Against Penicillium expansum on Apple Fruit.

Blue mold caused by Penicillium expansum is a major postharvest disease of apples (Malus × domestica). Residual activity of fludioxonil and pyrimethanil in apple fruit against P. expansum was investigated during 2005 to 2008. Fruit of the cultivar Delicious harvested from commercial orchards where fungicides were not used were either not treated or drenched with fludioxonil, pyrimethanil, or thiabendazole prior to storage and then stored in controlled atmosphere at 0°C for 5 or 7 months, after which time the fruit were removed from storage and subjected to washing and brushing, practices that are done at the time of packing. Fruit were then wounded and inoculated with conidial suspensions of P. expansum. Inoculated fruit were treated with either sterile water or fungicides. Fruit were stored at 0°C for 8 weeks and at room temperature for one additional week after cold storage. To determine distribution of fungicide residues in the fruit flesh, fruit were cut horizontally at the equator, sprayed with the conidial suspension of P. expansum, incubated at room temperature, and examined for inhibition of blue mold on the cut fruit 4 days after inoculation. Fungicide residues on/in the fruit were analyzed using a gas chromatograph. Zero to 26% blue mold incidence was observed on fludioxonil-drenched fruit that were inoculated and not treated with fungicides at packing. No decay or <4% blue mold incidence was observed on pyrimethanil-drenched fruit that were inoculated and not treated with fungicides at packing, whereas 65 to 99% blue mold incidence was observed on thiabendazole-drenched fruit that were not treated with fungicides at packing. An average of >32 mm inhibition zone and approximately 5 mm inhibition zone measured from the fruit peel toward the fruit core were observed on pyrimethanil-drenched and fludioxonil-drenched fruit, respectively. Washing and brushing at the time of packing 5 and 7 months after harvest did not remove or only partially removed residues of fludioxonil and pyrimethanil from apple fruit. The results suggest that residues of fludioxonil and pyrimethanil on/in apple fruit are persistent and that residual protection of apple fruit by the two fungicides can last for at least 7 months under apple-storage conditions.

Preharvest Application of a Boscalid and Pyraclostrobin Mixture to Control Postharvest Gray Mold and Blue Mold in Apples.

After harvest, apples (Malus × domestica) may be kept in cold storage for up to 12 months prior to packing. Gray mold caused by Botrytis cinerea and blue mold caused by Penicillium expansum are common postharvest fruit rot diseases affecting apples and are controlled commonly by applications of fungicides after harvest. To search for an alternative strategy, Pristine (a premixed formulation of boscalid and pyraclostrobin) as a preharvest treatment was evaluated for control of postharvest gray mold and blue mold in cultivars Fuji and Red Delicious apples during 2004 to 2006. Pristine (0.36 g per liter of water) was applied 1, 7, or 14 days before harvest. For comparison, thiram (2.04 g per liter of water) was applied 7 days before harvest and ziram (2.4 g per liter of water) was applied 14 days before harvest, to Fuji and Red Delicious, respectively. Fruit were harvested at commercial maturity, wounded with a finishing nail head, inoculated with conidial suspensions of either B. cinerea or P. expansum, stored in air at 0°C, and evaluated for decay after 8 or 12 weeks. In 2004 and 2005, Pristine was equally effective when applied to Fuji 1 or 7 days before harvest, reducing gray mold incidence by 93 to 99% and blue mold incidence by 78 to 94% compared with the nontreated control. Thiram reduced gray mold incidence by 38 to 85%. Thiram reduced blue mold incidence by 22% in 2004 but not in 2005. On Red Delicious, Pristine was equally effective when applied 7 or 14 days before harvest and reduced gray mold incidence by 69 to 85% and blue mold incidence by 41 to 70%. Ziram applied 2 weeks before harvest reduced gray mold incidence by 97 and 94% in 2005 and 2006, respectively, but it did not reduce blue mold incidence. The results indicate that Pristine applied within 2 weeks before harvest may be an effective alternative to postharvest fungicides for control of postharvest gray mold and blue mold in Fuji and Red Delicious apples.

Distribution of Potebniamyces pyri in the U.S. Pacific Northwest and Its Association with a Canker and Twig Dieback Disease of Pear Trees.

Winter pears (Pyrus communis) in the United States are produced primarily in the Pacific Northwest. Potebniamyces pyri (anamorph Phacidiopycnis piri) is the causal agent of Phacidiopycnis rot, a recently reported postharvest disease on pears in the United States. Infection of fruit by P. pyri occurs in the orchard, and symptoms develop during storage. P. pyri was observed to be associated with cankers, dead bark, and twig dieback of pear trees. P. pyri was isolated from 40 to 50% of the twig samples exhibiting dieback symptoms from three commercial d'Anjou pear orchards, and 35% of dying bark samples from one orchard. However, little information is available regarding pathogenicity of P. pyri on pear trees. To determine the distribution of P. pyri, dying and dead bark samples were collected from pear orchards in various pear-producing areas in Oregon and Washington, and examined for presence of fruiting bodies (pycnidia or apothecia) of P. pyri. In the orchard, 2-year-old twigs were wounded using a sterile cork borer with or without spraying with a commercial aerosol tissue-freezing product at the wound sites. Wounds were then inoculated with either mycelial plugs from an agar medium or conidial suspensions of P. pyri. In a separate experiment, freshly made pruning wounds were inoculated with conidial suspensions of P. pyri. Canker development was monitored approximately monthly for up to 6 months after inoculation, at which time reisolation of P. pyri was attempted. P. pyri was found to be widespread in the Pacific Northwest. Incidence of trees infected by P. pyri based on presence of viable pycnidia in pear orchards ranged from 0 to 100%. Monthly tree inoculations in the orchard indicated that P. pyri in general did not cause cankers on non-cold-injured, wound-inoculated twigs, but apparently became established on cold-injured, wound-inoculated twigs and caused small cankers. Minor dieback developed on twigs inoculated at pruning wounds. At 6 months after inoculation, P. pyri was recovered from the majority of inoculated twigs. Thus, P. pyri appears to be a weak canker-causing pathogen on pear trees.

A New Canker and Twig Dieback Disease of Apple and Crabapple Trees Caused by Sphaeropsis pyriputrescens in Washington State.

Crabapple (Malus sylvestris) is commonly used as a source of pollen in apple production. During September and October 2003, a canker and twig dieback disease of 'Manchurian' crabapple trees was observed in some commercial apple (Malus × domestica Borkh.) orchards (7- to 10-years-old) in north-central Washington State. A fungus was consistently isolated from 40 to 77% of sampled crabapple trees. During May 2004, the same symptoms and fungal association were observed in an 8-year-old 'Fuji' apple orchard in which all crabapple pollenizers and 43% of the Fuji trees were diseased. Canker and dieback appeared to originate from infection of dying or dead fruit spurs or pruning wounds. Cankered areas were slightly sunken, brown and the margin of diseased area often developed cracks in the cortical tissue. Pycnidia were often present in older areas of the lesion. Pycnidia were black, 0.3 to 0.6 mm in diameter, separate to aggregated in small numbers, and partially immersed to nearly superficial in the diseased tissue. To isolate the fungus, outer bark tissues of diseased twigs were scraped and small tissue segments were cut from the canker margin. Tissue segments were surface disinfested for 5 min in 0.5% sodium hypochlorite solution, rinsed three times with sterile water, cut into small pieces, and placed on acidified potato-dextrose agar (APDA, 4.0 ml of a 25% solution of lactic acid per liter of medium). Isolation plates were incubated at 20°C in the dark. Colonies of the fungus first appeared as dense colorless mycelium that later turned light yellow to yellow. The fungus was identified as Sphaeropsis pyriputrescens Xiao & J. D. Rogers (1). To complete Koch's postulates, two isolates (one each from apple and crabapple) were used in pathogenicity tests on 'Fuji' apple and 'Manchurian' crabapple trees. In the orchard, selected 2-year-old twigs were sprayed with 70% ethanol and allowed to dry. Twigs were wounded to a depth of 1 to 2 mm with a sterile 5-mm-diameter cork borer; a 5-mm mycelial plug from 4-day-old PDA cultures of S. pyriputrescens was placed into each wound. Twigs wounded and treated with sterile APDA plugs were used as controls. Inoculation sites were covered with moist cheesecloth and sealed with Parafilm that was removed 3 weeks after inoculation. Four twigs per isolate on each of four trees were inoculated. The experiment was conducted twice (April and November 2004 for apple; two different locations in March 2004 for crabapple). At 2 and 6 months after inoculation, two apple twigs per treatment were removed from each tree. All crabapple twigs were removed 2 months after inoculation. Canker sizes were measured and reisolation of the fungus was attempted as described above. Both isolates caused cankers on apple and crabapple twigs. Mean canker sizes at 6 months after inoculation were 11 and 32 mm on apple twigs inoculated in April and November 2004, respectively and 7 to 8 mm on crabapple twigs at 2 months after inoculation. No cankers developed on control twigs. S. pyriputrescens was reisolated from all inoculated twigs and was not recovered from noninoculated controls. S. pyriputrescens is the cause of Sphaeropsis rot, a recently reported postharvest fruit rot disease of apple and pear (1,2). To our knowledge, this is the first report of this fungus causing cankers and twig dieback on apple and crabapple trees. Reference: (1) C. L. Xiao and J. D. Rogers. Plant Dis. 88:114, 2004. (2) C. L. Xiao et al. Plant Dis. 88:223, 2004.

First Report of a New Postharvest Fruit Rot on Apple Caused by Sphaeropsis pyriputrescens.

During March to July 2003, a postharvest fruit rot was observed on 'Golden Delicious', 'Granny Smith', and 'Red Delicious' apples (Malus × domestica Borkh.) sampled from commercial packinghouses in Washington State. Losses as high as 24% in storage bins were observed in July on 'Red Delicious'. The disease started at the stem bowl area or the calyx end of the fruit. Decayed fruit was apparently not wounded. Decayed areas were brown and firm. Internal decayed flesh appeared yellowish brown. On 'Red Delicious' apples, decayed fruit was apparently discolored from red to brown. As the disease advanced, pycnidia of a fungus might form on the stem, sepals, or the surface of decayed fruit. Pycnidia were 0.3 to 0.7 mm in diameter, black, and partially immersed in decayed tissues. To isolate the causal agent, decayed fruit was lightly sprayed with 70% ethanol and air dried. Fragments of diseased tissue were removed from the margin of diseased and healthy tissue and plated on acidified potato dextrose agar (PDA). A fungus was consistently isolated from decayed fruit with the symptoms described above. On PDA, the colonies of the fungus first appeared with dense hyaline mycelium and later turned light yellow to yellow. Black pycnidia of the fungus formed on 2- to 3-week-old oatmeal agar cultures at 20°C under 12-h alternating cycles of fluorescent light and dark. The fungus was identified as Sphaeropsis pyriputrescens Xiao & J. D. Rogers, based on the description of the fungus (1). Voucher specimens were deposited at the WSU Mycological Herbarium. Two isolates of the fungus recovered from decayed apples were tested for pathogenicity on apple. Fruit of 'Golden Delicious' and 'Gala' were surface-disinfested for 5 min in 0.5% NaOCl, rinsed, and air dried. Fruit was wounded with a sterile 4-mm-diameter nail head. A 4-mm-diameter plug from the leading edge of a 3-day-old PDA culture or plain PDA (control) was placed in the wound of each of 10 replicate fruit for each isolate or control. Fruit was tray packed with polyethylene liners and stored in cardboard boxes in air at 3°C, and decay was evaluated 2 weeks after inoculation. Five decayed fruits from each treatment were selected for reisolation of the causal agent. The experiment was conducted twice. In a separate pathogenicity test, two isolates (one each from apple and pear) were included in the test. Fruit of 'Red Delicious' apple was prepared and inoculated as the same manner described above, but fruit was stored in air at 0°C. The experiment was conducted twice. All fruit that were inoculated with the fungus developed decay symptoms. No decay developed on fruit in the controls. The same fungus was reisolated from decayed fruit. This indicates that isolates from apple and pear were pathogenic to apple. S. pyriputrescens is the causal agent of a newly reported postharvest disease on 'd'Anjou' pears (1). To our knowledge, this is the first report of this fungus causing postharvest fruit rot on apple. We propose 'Sphaeropsis rot' as the name of this new disease on apple and pear. Preliminary evidence suggests that infection of fruit by this fungus occurred in the orchard prior to storage. Reference: (1) C. L. Xiao and J. D. Rogers. Plant Dis. 88:114, 2004.

Prevalence and Incidence of Phacidiopycnis Rot in d'Anjou Pears in Washington State.

Phacidiopycnis rot, caused by Phacidiopycnis piri, is a newly recognized postharvest disease in pear fruit (Pyrus communis cv. d'Anjou) in the United States. To determine the prevalence and incidence of this disease, decayed fruit were sampled during packing and repacking operations from four packinghouses in 2001 and 2002. During March to May (repacking) in 2001, Phacidiopycnis rot was found in packed fruit that were stored in cardboard boxes from 22 of 26 grower lots (orchards), and accounted for 5 to 71% of the total decay. Phacidiopycnis rot, gray mold caused by Botrytis cinerea, and blue mold caused by Penicillium spp. accounted for an average of 34.1, 10.3, and 33.6% of decayed fruit from conventional orchards, respectively; and 22.8, 35.7, and 23.5% of decayed fruit from organic orchards, respectively. During November 2001 to January 2002 (packing), Phacidiopycnis rot was observed in fruit that were stored in field bins before packing from 30 of 33 grower lots, accounting for 18.4% of decayed fruit sampled. During March to May in 2002, Phacidiopycnis rot was responsible for 2 to 68% of decayed fruit sampled from 36 of 39 grower lots. Phacidiopycnis rot, gray mold, and blue mold accounted for an average of 19.6, 26.8, and 37.4% of decayed fruit from conventional orchards, respectively; and 42.2, 25.7, and 8.2% of decayed fruit from organic orchards, respectively. Most Phacidiopycnis rot that occurred in field bins before packing appeared to originate from wound infections; whereas after packing, approximately 60 and 30% of Phacidiopycnis rot originated from stem and calyx infections, respectively. This study indicates that Phacidiopycnis rot should be considered one of the targets for control of postharvest diseases in d'Anjou pears in the region.

Occurrence and Identification of Phytophthora spp. Pathogenic to Pear Fruit in Irrigation Water in the Wenatchee River Valley of Washington State.

ABSTRACT Seven hundred forty-nine isolates of Phytophthora spp. were obtained from irrigation canals in eastern Washington State during the 1992 to 1995 and 1999 growing seasons. Isolates were retrieved using pear baiting techniques. All isolates were pathogenic to pear and were present in irrigation water beginning early in fruit development. Over the course of the 5 year study, 10 and 5% of isolates were identified as P. cactorum and P. citricola, respectively, using morphological criteria. The remaining isolates could not be identified using morphological criteria. Colony morphology of these isolates was characterized during all years of the study. In 1999, more detailed studies utilizing polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of entire internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) of ribosomal DNA for 180 isolates, and sequence analysis of ITS2 for 50 isolates, were used to investigate genetic variation and phylogenetic relationships among isolates. Isolates were divided into 12 groups based on their growth type on corn meal agar. Restriction digestion of the entire ITS region with three enzymes revealed 11 restriction digestion patterns among 180 isolates. PCR-RFLP and sequence data were obtained for 12 reference Phytophthora spp. (two species in each of Waterhouse's six morphological groups). Phylogenetic analysis of ITS2 regions revealed nine clades, each with strong bootstrap support. Molecular analyses revealed 23 isolates that were in the P. gonapodyides clade, 9 in the P. parasitica clade, 1 in the P. cactorum clade, 7 in the P. citricola/capsici clade, and 4 in the P. cambivora/pseudotsugae clade. The three isolates comprising clade 5 were significantly distinct from all other Phytophthora spp. in the databases and may represent a new Phytophthora sp. Colony morphology was not consistently correlated to PCR-RFLP pattern or ITS2 phylogeny, suggesting that the former criterion is insufficient for species identification. The results of this study indicate that at least nine phylogenetically distinct taxa of Phytophthora pathogenic to pear are present in irrigation water in North Central Washington.