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Phacidiopycnis washingtonensis is the cause of speck rot of apple and leaf blight of Pacific madrone in Washington State. In total, 314 isolates were collected from apple production areas in eastern Washington and Pacific madrone in western Washington. Using eight microsatellite markers designed in this study, 58 unique multilocus haplotypes were identified. Only one of the haplotypes was shared between the apple and Pacific madrone populations. Analysis of molecular variance showed no genetic differentiation between the apple and Pacific madrone populations. Genetic variation was present within each subpopulation of apple from different geographic locations. The apple population possessed higher genotypic diversity than the Pacific madrone population, suggesting that isolates from apple may represent an older population and could have been introduced into the native habitat of Pacific madrone. P. washingtonensis likely reproduces asexually because populations examined in this study were not in linkage equilibrium. In pathogenicity tests, representative isolates from apple and Pacific madrone all incited leaf blight on Pacific madrone and speck rot on apple fruit regardless of their host of origin. Overall, our findings indicate that the P. washingtonensis population in Washington State is largely asexual, with high genotypic flow and that apple, crabapple, and Pacific madrone could serve as sources of P. washingtonensis inoculum for these hosts.

Members of the genera Neofabraea and Phlyctema have been reported to incite canker diseases of apple trees and a postharvest decay of apple fruit referred to as "bull's-eye rot." Neofabraea kienholzii was recently identified as participating in the bull's-eye rot disease complex of apple and other pome fruit. In this study, apple twigs inoculated with N. kienholzii were shown to develop symptoms of a canker disease closely resembling perennial canker of pome fruit trees caused by N. perennans. Cankers resulting from infection by either Neofabraea spp. were more likely to be induced when twig inoculations occurred in October, and to a lesser degree in April, compared with all other inoculation dates evaluated in this study. Although N. kienholzii tended to induce cankers that were smaller in size compared with N. perennans, both pathogens shared similar seasonal trends in the initiation and expansion of tree cankers. N. perennans and N. kienholzii were recovered from inoculated twigs 6 months postinoculation regardless of when inoculations were conducted, indicating that both pathogens can survive on diseased twigs year-round. In addition, acervuli were observed more often on twigs inoculated in September and April compared with those inoculated in other months. Data from this work should help further our understanding of the epidemiology of N. kienholzii. This information also highlights the importance of proper branch pruning, canker removal, and aphid control. Such management activities should be conducted in a manner that helps minimize further spread of the pathogen.

Brown rot is a severe disease affecting stone and pome fruit. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, M. laxa, M. fructigena, Monilia polystroma, M. mumecola, and M. yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase-2 genes. Additionally, based on sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limits of 10 to 100 fg of DNA, which is equivalent to one to seven conidia. The assays were also able to detect the target pathogens in a mixed DNA sample comprising all six Monilinia spp. and related species. The real-time PCR assays accurately detected target fungi from infected apple fruit. Furthermore, the identification results were consistent with those of traditional morphological methods.

Bull's-eye rot is a major postharvest disease of apple caused by several fungi belonging to the Neofabraea and Phlyctema genera. Chemical control of these fungi is a crucial component of disease management for apples that are conventionally grown. The efficacy of several preharvest and postharvest applied fungicides were evaluated to identify effective chemistries that can control bull's-eye rot incited by Neofabraea perennans and N. kienholzii on apples. In general, the preharvest fungicide thiophanate-methyl was found to be effective at reducing disease caused by N. perennans and N. kienholzii. Two postharvest fungicides, thiabendazole and pyrimethanil, also provided disease control that was far superior to other chemical compounds evaluated in this study. The efficacy of thiabendazole and pyrimethanil was unaffected by application method (fungicide dip compared with thermofog). Despite providing satisfactory control of bull's-eye rot, integration of these three chemicals into disease management programs should proceed judiciously with consideration of their impact on the development of fungicide resistance and influence on diversity in populations of apple postharvest pathogens.

Aim: To identify media and environmental conditions suitable for rapid mycelial growth and sporulation of Diplocarpon mali., Methods and Results: Liquid shake cultures were used to evaluate effects of media and environmental conditions on mycelial growth and conidial production of D. mali. Carrot sucrose broth (CSB), potato and carrot dextrose broth (PCDB) and potato and carrot sucrose broth (PCSB) were most favourable for rapid mycelial growth. PCDB, PCSB, PCB (potato and carrot broth) and carrot dextrose broth (CDB) were favourable for conidial production. All carbon sources tested and peptone favoured for mycelial growth. Carbon and nitrogen sources tested did not significantly stimulate conidial production. The optimum temperature for mycelial growth and conidial production was 25 degrees C. No mycelial growth occurred at 5 or 30 degrees C, but D. mali survived at these temperatures. Active mycelial growth occurred at pH 5-7, and pH 5-8 was favourable for sporulation., Conclusions: PCDB and PCSB incubated at 25 degrees C for 14 day are recommended for mycelial growth and conidial production of D. mali., Significance and Impact of the Study: The information generated in this study will facilitate mycological and pathological research on D. mali and Marssonina leaf blotch of apple caused by D. mali.

The objectives of this study were i) to give a taxonomic description of a fungus phylogenetically related to Neofabraea and assign the name Cryptosporiopsis kienholzii to this fungus, ii) to expand previous Neofabraea species profiles from infected apple and pear fruit collected from major pome fruit production districts in Oregon and Washington, and iii) to determine the sensitivity of Neofabraea alba, Neofabraea malicorticis, Neofabraea perennans, and C. kienholzii to a range of fungicides. A name is given herein to the anamorph of the fungus previously called 'Neofabraea sp. nov.' in the literature, and the fungus is designated as C. kienholzii. The teleomorph of this fungus has never been observed in vivo or in vitro. N. alba, N. perennans, and C. kienholzii accounted for 61.3, 35.6, and 3.1 %, respectively, of 608 isolates obtained from pear fruit and 6.0, 81.3, and 12.7 % of 150 isolates from apple fruit. N. alba was the most common species in Oregon and N. perennans was most common in Washington. N. malicorticis was not found in any of the districts and may be limited to the wet climates west of the Cascade mountain range. C. kienholzii occurs in pome fruit orchards from southern Oregon to North Central Washington, and this is the first report of C. kienholzii in the state of Washington. The four most effective fungicides for control of bull's-eye rot caused by all species were thiabendazole, thiophanate-methyl, pyrimethanil, and pyraclostrobin+boscalid. Other fungicides gave acceptable control of some species of Neofabraea but not others. There was good agreement of the microbioassay with fungicide evaluations using wound-inoculated pear fruit for five of seven fungicides, but the microbioassay was not consistent enough for determination of the sensitivity of Neofabraea species to new fungicides.

Potebniamyces pyri is the cause of Phacidiopycnis rot of d'Anjou pear, which is grown primarily in Washington and Oregon. To estimate the population structure of P. pyri, 146 single-spore isolates were sampled from five major pear-production areas and scored for variation at eight sequence-characterized amplified region (SCAR) loci. Significant genetic differentiation was detected among five subpopulations and a total of 54 multilocus genotypes were identified, with significant genotypic diversity in each subpopulation. No genotype was shared among more than three subpopulations. To estimate the relationship between phenotype and multilocus SCAR genotype, four to five representative isolates of each dominant SCAR genotype in each subpopulation were assayed for growth rate on oatmeal agar and virulence on d'Anjou pear fruit. Significant differences in daily growth rates and virulence were detected among genotypes; however, genotype was not predictive of virulence. To assess the mating system of the pathogen, 10 ascospores were sampled from each of 20 apothecia from a commercial orchard and scored for five SCAR markers. Segregation of alleles at one or more SCAR loci was detected among 18 of 20 ascospore progeny sets, indicating that P. pyri is likely a heterothallic fungus with a predominantly outcrossing mating system.

A new species of Phacidiopycnis associated with pome fruits is described. The fungus causes fruit rot on apples during storage and is associated with a twig dieback and canker disease of crabapple trees and dead twigs of pear trees. To characterize the biology of the fungus and compare it with Ph. piri, the type species of the genus, effects of nine media and light on mycelial growth and pycnidial production, mycelial growth in response to temperature and mode of conidial germination in response to nutrient were determined. Apple-juice agar, pear-juice agar, prune-juice agar, potato-dextrose agar (PDA) and malt-extract agar, Czapek-Dox agar and oatmeal agar (OMA) favored mycelial growth. Cornmeal agar (CMA) did not favor mycelial growth. Light effect on pycnidial formation was medium dependent. Abundant pycnidia with mature conidia formed in 14 d old PDA and OMA cultures at 20 C, regardless of light, whereas none or very few pycnidia formed on other media in the dark. Fluorescent light stimulated formation of pycnidia except on CMA. The fungus grew at -3-25 C, with optimum growth at 15-20 C. Conidia germinated either by forming germ tubes or less often by budding. Budding of conidia occurred in 1 and 10% pear-juice solutions but not in 100% pear-juice solution. Six isolates of Ph. washingtonensis from different species of pome fruits had identical ITS sequences. The sizes of the ITS region were the same for both Ph. washingtonensis and Ph. piri, and four polymorphic nucleotide sites were found in the ITS region between Ph. washingtonensis and Ph. piri. The similarity in ITS sequences between these two taxa is confirmatory evidence for the erection of the new species of Phacidiopycnis associated with pome fruits we describe here.

Sphaeropsis pyriputrescens, the causal agent of Sphaeropsis rot of pears and apples, is a recently described species. In this study the effects of culture media, temperature, water potential, pH and light on mycelial growth and pycnidial production of S. pyriputrescens were evaluated. Apple juice agar and pear juice agar were most suitable for mycelial growth of all six isolates tested. Cornmeal agar was not suitable for either mycelial growth or pycnidial production. The fungus grew from -3 to 25 C, with optimum growth at 20 C and no growth at 30 C. The fungus grew at water potential as low as -5.6 MPa on potassium chloride-amended potato-dextrose agar (PDA). Hyphal extension was not observed at -7.3 MPa after 10 d incubation, but growth resumed when the inoculum plugs were placed on PDA. The fungus grew at pH 3.3-6.3 and optimum growth was at pH 3.3-4.2. No mycelial growth was observed at pH above 7.2 after 10 d incubation, but growth resumed when the inoculum plugs were transferred onto PDA. Regardless of medium tested, few pycnidia formed at 20 C in the dark. Pycnidial production was enhanced significantly by fluorescent light, but continuous light appeared to reduce pycnidial production, depending on the medium. Oatmeal agar (OMA) was most suitable for production of pycnidia and conidia. Pycnidia that formed on 3 wk old OMA cultures at 20 C under 12 h light/12 h dark produced abundant conidia, and the technique is recommended for inoculum production.

Potebniamyces pyri (anamorph Phacidiopycnis piri) is the causal agent of Phacidiopycnis rot of apples and pears. The disease has recently been recognized in pears in the USA. Little information on the basic biology of the fungus is available. In this paper, we report the effects of culture media, temperature, water potential and pH on mycelial growth, and the effects of media and light on pycnidia production. Prune juice agar was the best for rapid mycelial growth. Pear juice agar, apple juice agar, potato dextrose agar, and oatmeal agar (OMA) also favoured mycelial growth. Czapek-Dox agar was not suitable for mycelial growth. The fungus was able to grow at temperatures from -3 to 25 degrees C. Optimal mycelial growth occurred between 15 and 20 degrees. The average radial growth rate on OMA was 3.9 mm d(-1) at 15 degrees and 4.4 mm d(-1) at 20 degrees. Mycelial growth was not observed after 10 d at 30 degrees, but growth resumed at 20 degrees. The fungus failed to resume growth at 20 degrees after being incubated for 10 d at 35 degrees. The fungus was able to grow at water potentials as low as -4 MPa but no growth took place at -7.3 MPa. Active mycelial growth was observed on OMA at pH between 3.2 and 6.1. Optimal growth was observed at pH around 4 and no growth was observed at pH 7.1. No pycnidia or very few formed on the nine media at 20 degrees in the dark. Fluorescent light significantly stimulated formation of pycnidia. OMA was the best medium for production of pycnidia and macroconidia. Pycnidia that formed on 8-wk-old OMA cultures incubated at 20 degrees under 12 h dark/12 h light produced abundant macroconidia and the technique is recommended for inoculum production of conidia for research.