Reconstructing the early evolution of Fungi using a six-gene phylogeny.

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

Bacterial Canker of Sweet Cherry in Oregon-Infection of Horticultural and Natural Wounds, and Resistance of Cultivar and Rootstock Combinations.

This study was done to (i) compare seven types of natural or horticultural injuries and wounds for incidence, severity, and mortality of infection of sweet cherry (Prunus avium) by Pseudomonas syringae pv. syringae; (ii) determine the relative resistance to bacterial canker of 14 cultivar-rootstock combinations; (iii) determine if P. syringae pv. syringae is transmitted by contaminated pruning tools; and (iv) determine if summer and winter pruning cuts become resistant to infection. Infection occurred at all of the seven types of injury and wound sites on both cvs. Sunset Bing and Golden Heart. Infection of inoculated wounds made in spring and summer (heading cuts when trees were planted, scoring cuts, and summer pruning) resulted in the greatest canker incidence and severity. Inoculation of heading cuts resulted in the highest tree mortality (86%). 'Bing' and 'Sweetheart' were the most susceptible cultivars while 'Regina' and 'Rainier' appeared to be more resistant. Bing trees had the highest mortality of any cultivar with 70% dead at the end of the 3-year study. Canker severity of the three rootstocks varied considerably but mortality was greatest for trees on Gisela 6 (77%). Bacterial canker was not transmitted in summer or winter by cutting through active cankers, then immediately using the same pruning tool to make heading cuts on healthy trees. Heading cuts became resistant to infection after about 1 week in summer and 3 weeks in winter. Results are discussed as part of an integrated management program for bacterial canker of sweet cherry.

Penicillium Species Associated with Preharvest Wet Core Rot in South Africa and Their Pathogenicity on Apple.

Symptoms associated with the core region of apple fruits (Malus domestica) can be classified as moldy core (MC), wet core rot (WCR), and dry core rot (DCR). Infections leading to WCR are thought to occur primarily postharvest, although in South Africa preharvest symptoms also have been reported. The first aim of this study was to investigate the causative agent(s) of preharvest WCR by isolating fungi from eight internal positions in asymptomatic, MC, WCR, and DCR fruits. Secondly, the pathogenicity and virulence of all Penicillium isolates were investigated using three apple fruit inoculation methods: surface wounding, deep wounding, and nonwounding. Isolation of fungi from WCR fruits showed that Penicillium was the predominant fungal genus from most isolation positions including the lesion area. Penicillium ramulosum was the predominant species isolated from all fruits. However, in WCR fruits, the incidence (58%) of P. ramulosum was much higher than in MC (6%), DCR (7%), or asymptomatic (7%) fruits. Less frequently isolated Penicillium species included P. expansum and a few other species. Pathogenicity testing using the nonwounding method was best at discriminating highly virulent isolates. P. expansum was the most virulent species, followed by a putative new Penicillium species with closest sequence similarity to P. dendriticum. P. ramulosum isolates, although showing varying degrees of virulence, all had low virulence, causing only small lesions in wounded apple fruits.

Description of Cryptosporiopsis kienholzii and species profiles of Neofabraea in major pome fruit growing districts in the Pacific Northwest USA.

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.

Effects of Environmental Factors and Cultural Practices on Bull's Eye Rot of Pear.

Bull's eye rot of pome fruits caused by Neofabraea spp. is characterized by infection occurring in the orchard throughout the growing season whereas rot lesions develop during long-term storage after harvest. Bull's eye rot was observed on pear fruit exposed to natural infection for any of six to nine sequential 1-to-2-week exposure periods during two growing seasons. Highest infection levels were associated with exposure closest to harvest. Over-tree irrigation and late harvest resulted in higher bull's eye rot incidence than under-tree irrigation and early or mid-season harvest. Fruit were inoculated prior to harvest with Neofabraea perennans to determine the effect of environmental factors on the development of bull's eye rot. The effect of temperature was inconsistent; disease was greatest at 10°C in one year of study but greatest at 30°C in the second year. Bull's eye rot developed independently of wetness durations longer than 0.5 h.

Methodology for Determining Relationships Between Inoculum Concentration of Botrytis cinerea and Penicillium expansum and Stem End Decay of Pear Fruit.

The objective of this research was to determine quantitative relationships between incidence of stem end decay of pear fruit and inoculum concentration of Botrytis cinerea and Penicillium expansum using dry conidia applied to pear fruit in a settling tower. Five concentrations of conidia were applied to pear fruit, fruit were stored at -1°C for 8 months, and stem end decay was evaluated. In addition, conidia were washed from the surface of inoculated fruit, and DNA was extracted and quantified with real-time polymerase chain reaction (PCR). The linear regression relationships between percent stem end gray mold and B. cinerea conidia per liter of air or per square centimeter of fruit surface were significant (P = 0.01). At the highest inoculum dose introduced into the settling tower, conidia per liter of air, conidia per square centimeter, and percent stem end gray mold at 8 months after inoculation were 12, 31, and 39, respectively for 2000 and 6, 33, and 67, respectively for 2001. Similarly, the linear regression relationships between percent stem end blue mold and P. expansum conidia per liter of air or per square centimeter of fruit surface were significant (P = 0.01 and 0.05, respectively). At the highest inoculum dose introduced into the settling tower, conidia per square centimeter and percent stem end blue mold at 8 months after inoculation were 39 and 26, respectively for 2000 and 66 and 23, respectively for 2003. Real-time PCR provided a rapid, quantitative measure of B. cinerea and P. expansum DNA on pear fruit surfaces. Because of possible year-to-year shifts in susceptibility of fruit to decay, disease incidence:inoculum dose relationships may be of most value compared within years rather than across years. This would facilitate comparison of decay risk among orchards in order to determine which fruit is most suitable for long-term storage.

Inoculum Sources of Botrytis cinerea Important to Pear Orchards in Oregon.

Botrytis cinerea was capable of colonizing and persisting in tissue pieces of six common weed species found in pear orchards in Oregon from midsummer until pear harvest in early September. Sporulation occurred on blackberry mummies in orchard perimeters and ranged from 5,700 to 55,000 conidia per mummy at pear harvest. Conidia of B. cinerea also were observed on fresh blackberry fruit in 2 of 3 years. Sclerotia were present on only a few of the naturally infected pear fruit on the orchard floor through November; however, sclerotia production increased greatly between mid-November and mid-December. Over 90% of sclerotia from naturally infected, overwintered fruit germinated in the laboratory when collected 6 to 8 months after infection. Sclerotia formed on pear fruit inoculated with 28 different paired combinations of 22 isolates in the laboratory. Viability of sclerotia in inoculated pear fruit that overwintered on the orchard floor was 44 to 59% in March and April, and then declined steadily to about 25% at pear harvest. Germination of sclerotia was both sporogenic and myceliogenic. No apothecia were observed in the field or laboratory. An integrated approach to control is needed to deal with these multiple sources of potential inoculum.

Induction of Cankers on Pear Tree Branches by Neofabraea alba and N. perennans, and Fungicide Effects on Conidial Production on Cankers.

Cankers formed on pear branches after inoculations with mycelia of Neofabraea alba or N. perennans, causal agents of bull's eye rot of pear fruit. The highest proportions of successful infections followed inoculations made in fall and winter. Cankers induced by N. perennans were larger than those induced by N. alba. Small, superficial cankers were obtained after inoculations with conidia of N. perennans on wounded branches of pear trees. Sporulation of both pathogens on mycelial-induced cankers occurred throughout the year, with the largest amount of conidia produced at the end of summer and during fall. Sporulation on cankers induced by N. perennans spanned at least 2 years. Copper sulfate reduced sporulation on cankers induced by N. alba, while copper sulfate, trifloxystrobin, and ziram applied to sporulating cankers reduced germination of conidia of N. perennans.

Control of Postharvest Decay in Pear by Four Laboratory-Grown Yeasts and Two Registered Biocontrol Products.

Control of blue mold decay in Bosc pears was studied with the laboratory-grown yeasts Rhodotorula glutinis, Cryptococcus infirmo-miniatus, and two strains of Cryptococcus laurentii, as well as registered biocontrol products Aspire, containing the yeast Candida oleophila, and Bio-Save 11 (now Bio-Save 110), containing the bacterium Pseudomonas syringae. Both thiabendazole (TBZ)-sensitive and TBZ-resistant strains of Penicillium expansum were used. Aspire treatment reduced the average lesion diameter by approximately 65 and 45%, and reduced decay incidence by 27 and 9% with TBZ-resistant and TBZ-sensitive P. expansum, respectively, in the first year of the study, but did not result in significant decay control in the second year. Bio-Save 11 reduced decay lesion diameter by 32 to 72% and incidence by 21 to 40% over the 2 years. In both years, TBZ-sensitive P. expansum was completely controlled by the combination of either C. laurentii (both strains), R. glutinis, or C. infirmo-miniatus with 100 ppm TBZ. With TBZ-resistant P. expansum, control of wound infection with these yeasts alone or with 100 ppm TBZ ranged from 62.9 to 100%. In a packinghouse trial, control by Bio-Save 110 + 100 ppm TBZ and Aspire + 100 ppm TBZ was not different than control by TBZ at 569 ppm, the maximum label rate. The amount of decay following Aspire + 100 ppm TBZ treatment was significantly less than the amount of decay following Bio-Save 110 + 100 ppm TBZ treatment.

Sensitivity of Populations of Botrytis cinerea from Pear-Related Sources to Benzimidazole and Dicarboximide Fungicides.

Botrytis cinerea is responsible for a major portion of postharvest decay in winter pears in the Pacific Northwest. The baseline sensitivity levels (mean EC50 values) of a wild-type B. cinerea population to thiabendazole and iprodione were 6.66 and 0.56 mg/liter, respectively. B. cinerea from commercial orchards not treated with a benzimidazole had significantly lower incidence of resistance (0.59%) to a discriminatory concentration of thiabendazole at 10 mg/liter than did isolates from orchards in which benomyl had been applied for experimental purposes (16.0%), unsprayed control trees in benomyl-sprayed orchards (5.34%), and isolates from packinghouses where thiabendazole was applied as a prestorage drench or packingline spray (3.23%). The mean EC50 value of isolates in the wild-type population was lower than those of resistant isolates from all other sources. High-level thiabendazole resistance (EC50 > 100 mg/liter) was found in 0.20% of isolates from unsprayed commercial orchards, 9.33% of isolates from benomyl-sprayed orchards, and 2.67% of isolates from unsprayed control trees in these benomyl-sprayed orchards. In isolates from packinghouses where a thiabendazole line spray was applied, 1.52% had high-level thiabendazole resistance. All isolates from all pear-related sources tested were sensitive to iprodione at 10 mg/liter. This study provides evidence supporting current recommendations of a single postharvest application of a benzimidazole to control decay caused by B. cinerea, and no application of benzimidazole fungicides in the orchard.

Disease Incidence-Inoculum Dose Relationships for Botrytis cinerea and Penicillium expansum and Decay of Pear Fruit Using Dry, Airborne Conidia.

The objective of this research was to determine quantitative relationships between incidence of pear fruit decay and inoculum dose of Botrytis cinerea and Penicillium expansum using dry conidia applied to wet or dry pears in a settling tower. On wet fruit, incidence of gray mold fruit rot increased from 0.1 to 83.1% as the airborne concentration of B. cinerea conidia increased from 0 to 8.6 spores per liter of air. Significantly less decay occurred in fruit inoculated dry compared to wet, particularly in fruit wounded after inoculation. Incidence of blue mold increased from 1 to 100% as the airborne concentration of P. expansum conidia increased from 0.1 to 803.5 spores per liter of air. Blue mold incidence was not affected by fruit wetness or time of wounding relative to inoculation. All regressions of decay incidence versus airborne and surface conidial concentrations were highly significant (P = 0.01).

Etiology of Bull's Eye Rot of Pear Caused by Neofabraea spp. in Oregon, Washington, and California.

A collection of Neofabraea isolates from pear fruit grown in Oregon, Washington, and California was screened with species-specific primers in a multiplex polymerase chain reaction (PCR). Neofabraea alba was identified most frequently in samples from Oregon and California, whereas N. perennans was found most frequently in samples from Washington. N. alba also was identified from tissue of small cankers and pruning stubs on pear trees using PCR. Bull's eye rot pathogens were isolated from fruit of nine different European pear cultivars, Asian pear, and quince. Overall, N. alba was the most prevalent species in 2001 whereas N. perennans was more prevalent in 2002. An undescribed species of Neofabraea was identified in samples from Medford, OR that corresponds to a species previously found by others using molecular methods. This information increases the known geographic distribution of this undescribed species.

Incidence of Postharvest Decay of 'd'Anjou' Pear and Control with a Thiabendazole Drench.

Postharvest decay causes losses to commercial pear industries. The incidence of decay in air and controlled-atmosphere (CA)-stored 'd'Anjou' pear fruit was investigated, and the effect of a prestorage thiabendazole drench on decay in CA-stored fruit was determined. In air storage, bull's-eye rot (31.37%) was most prevalent in 1996, whereas incidence of gray and blue mold (1996 and 1997) and bull's-eye rot (1997) were similar. Mucor, Alternaria, and Coprinus rot levels were low. Incidence of stem-end gray mold (2.58%) was significantly higher than calyx-end (0.73%) and puncture gray mold (0.61%). Incidence of gray mold (2.26%) was higher than all other decay in nondrenched CA-storage, and incidence of other decay types were similar. Incidence of puncture gray mold (1.13%) was higher than stem-end gray mold (0.84%), which in turn was higher than calyx-end gray mold (0.36%) in nondrenched CA-storage. Incidence of gray mold (1.04%) in CA-stored fruit was reduced by a prestorage thiabendazole drench. Drenching reduced stem-end (0.34%) and puncture gray mold (0.40%) but had no effect on all other decay or the total decay incidence. These results support the current recommendations of a single postharvest application of thiabendazole to control gray mold in 'd'Anjou' pear fruit.

Pear Scab: Components of Potential Ascospore Dose and Validation of an Ascospore Maturity Model.

Variables necessary to calculate potential ascospore dose (PAD) for Venturia pirina, the causal agent of pear scab, were measured: area of individual leaves averaged 20.0 cm2, number of lesions per infected leaf ranged from 1.8 to 2.2, 13.5 to 41.7% of lesions were fertile (contained pseudothecia), number of pseudothecia per fertile lesion ranged from 15.2 to 24.4, and number of asci per pseudothecium averaged 131.5. The proportions of orchard floor covered with leaves (leaf litter density) were 0.34 and 0.67 for mowed and nonmowed orchards, respectively. Incidence of infected leaves ranged from 0.1 to 32.3%. Calculated PAD ranged from 1,114 to 708,320 ascospores per m2 in a commercial and an unsprayed orchard, respectively. For validation of a previously published model relating ascospore maturity to degree-days with a 0°C base, a linear regression equation was developed using 11 data sets collected between 1981 and 1998. The slope of the line was not different (P = 0.951) from that of the original model.

Populations of Botrytis cinerea and Penicillium spp. on Pear Fruit, and in Orchards and Packinghouses, and Their Relationship to Postharvest Decay.

The aims of this study were to examine the sources and population sizes of Botrytis cinerea and Penicillium spp. in 'd'Anjou' pear orchards, packinghouses, and storage, and to determine the relationship between population sizes and postharvest decay. Densities of B. cinerea ranged from nondetectable to 4.0 CFU/cm2 on fruit, nondetectable to 3.1 CFU/liter in orchard air, and nondetectable to 1,167 CFU/g in orchard litter. The majority of packinghouse air and orchard soil samples collected yielded no B. cinerea inoculum. Densities of Penicillium spp. ranged from nondetectable to 2.7 CFU/cm2 on pear fruit, nondetectable to 3.13 CFU/liter in orchard air, nondetectable to 11.8 CFU/liter in packingline air, nondetectable to 3.9 CFU/liter in cold-storage air, 38 to 431 CFU/g in orchard soil, and 131 to 1,128 CFU/g in orchard litter. The mean incidence of gray mold in stored d'Anjou pear fruit ranged from 0.7 to 10.7%. Incidence of blue mold ranged from 0 to 16.5%. Significant positive correlations were observed between decay and fruit surface populations of B. cinerea and Penicillium spp. In conclusion, inoculum levels of these important postharvest pathogens in orchard and packinghouse air, and orchard soil and litter, cannot be used as indicators of postharvest decay; whereas the inoculum level on fruit surfaces may be a useful predictor of decay.