Factors Affecting the Infection of Sweet Cherry (Prunus avium) Fruit by Podosphaera cerasi.

Powdery mildew caused by Podosphaera cerasi is the most important fungal disease of sweet cherries in the Pacific Northwest of the United States. In this study, several factors related to disease epidemiology were evaluated. The experiments were conducted to investigate flower susceptibility to P. cerasi infection by in planta and in vitro inoculation. The susceptibility of fruit at various developmental stages was investigated using defined concentrations of P. cerasi conidia. Furthermore, the threshold of conidial concentration required for fruit infection was determined. The pathogen activity during full bloom was limited and not related to fruit disease incidence and severity at harvest. Foliar infections always preceded fruit infections by an average of 42 days during the 3 years of the study. The onset of fruit infection followed, on average, 66 days after full bloom and appeared simultaneously on all susceptible cherry cultivars in the research orchard. Disease symptoms were only observed on fruit in Biologische Bundesanstalt, Bundessortenamt, and Chemical Industry scale 8 (maturity) in all cultivars examined. During this stage, a concentration of 500 conidia/ml was sufficient to cause fruit infection at harvest. Interaction between the inoculation dates and conidial concentration revealed a dependency of disease development on the host stage at the time of inoculation; the younger the fruit, the more conidia are needed to cause disease at harvest. Molecular studies showed a rapid increase in conidia viability at the transition from asymptomatic to the symptomatic disease of fruit. No evidence of ontogenic resistance of fruit to powdery mildew infection was observed.

Biocontrol potential of native yeast strains against Aspergillus flavus and aflatoxin production in pistachio.

ASPERGILLUS FLAVUS: is the main aflatoxin producer in food and feed and has wide ecological niches. Contamination of food products such as pistachio nuts and aflatoxin secretion directly affects food safety and international food product trades. Abilities of 13 yeast strains isolated from 200 soil and pistachio nut samples collected in Iranian orchards to reduce the growth of A. flavus as well as aflatoxin production were assessed in dual culture, volatile and non-volatile compounds tests. The growth of A. flavus was reduced by 32-60%, 13-31% and 40-61% in dual culture, volatile and non-volatile compounds, respectively, while aflatoxin B1 production was diminished by 90.6-98.3%. Based on these assays, five yeast strains were selected for co-inoculation experiments using soil, pistachio hulls and leaf. A significant reduction in colony-forming units (CFU) ranging from 23% to 110% (p < .05) was observed. Molecular, physiological and morphological identification revealed these were strains of Pichia kudriavzevii and Lachansea thermotolerans. Aflatoxin biocontrol with yeast strains possesses many advantages including the ease of commercial production and organic application which is an environmental approach. More investigation is required to understand the efficiency of selective strains to inhibit A. flavus and aflatoxin production as well as withstand predominant abiotic stress in pistachio orchards and mass production in field application.

DNA barcoding for the identification of mold species in bakery plants and products.

Mold identification at the species level in environmental samples is a major challenge. Molecular techniques have been widely used for fungal classification, but as most primers are genus-specific, it is laborious to identify unknown samples. In this study, a PCR-based method for the identification of mold at the species level was developed. Therefore, common sequencing primers and combinations of them, targeting specific DNA regions, were tested. Here we present a combination of eight primer pairs to identify mold within a single PCR run. The approach correctly identified mold of unknown species from samples taken at a local bakery, including Penicillium chrysogenum, Penicillium citrinum, Cladosporium sphaerospermum, Paecilomyces formosus, Rhizopus oryzae and Aspergillus niger. Results obtained from the PCR method were successfully validated by chromatographic mycotoxin and microscopy analysis. Findings highlight DNA barcoding as an appropriate tool for mold identification; however, its efficacy is essentially dependent on DNA quality and primer selection.

Risk Factors for Bud Perennation of Podosphaera macularis on Hop.

The hop powdery mildew fungus Podosphaera macularis persists from season to season in the Pacific Northwestern United States through infection of crown buds because only one of the mating types needed to produce the ascigerous stage is presently found in this region. Bud infection and successful overwintering of the fungus leads to the emergence of heavily infected shoots in early spring (termed flag shoots). Historical data of flag shoot occurrence and incidence in Oregon and Washington State during 2000 to 2017 were analyzed to identify their association with the incidence of powdery mildew, growers' use of fungicides, autumn and winter temperature, and other production factors. During this period, flag shoots were found on 0.05% of plants evaluated in Oregon and 0.57% in Washington. In Oregon, the incidence of powdery mildew on leaves was most severe and the number of fungicide applications made by growers greatest in yards where flag shoots were found in spring. Similarly, the incidence of plants with powdery mildew in Washington was significantly associated with the number of flag shoots present in early spring, although the number of fungicide applications made was independent of flag shoot occurrence. The occurrence of flag shoots was associated with prior occurrence of flag shoots in a yard, the incidence of foliar powdery mildew in the previous year, grower pruning method, and, in Washington, winter temperature. A census of hop yards in the eastern extent of the Oregon production region during 2014 to 2017 found flag shoots in 27 of 489 yards evaluated. In yards without flag shoots, 338 yards (73.2%) were chemically pruning or not pruned, whereas the remaining 124 (26.8%) were mechanically pruned. Of the 27 yards with flag shoots, 22 were either chemically pruned or not pruned and 4 were mechanically pruned in mid-April, well after the initial emergence of flag shoots. The prevalence of yards with flag shoots also was related to thoroughness of pruning in spring (8.1% of yards with incomplete pruning versus 1.9% of yards with thorough pruning). A Bayesian logistic regression model was fit to the data from the intensively assessed yards in Oregon, with binary risk factors for occurrence of a flag shoot in the previous year, occurrence of foliar mildew in the previous year, and thoroughness of pruning in spring. The model indicated that the median and 95% highest posterior density interval of the probability of flag shoot occurrence was 0.0008 (0.0000 to 0.0053) when a yard had no risk factors but risk increased to 0.0065 (0.0000 to 0.0283) to 0.43 (0.175 to 0.709) when one to all three of the risk factors were present. The entirety of this research indicates that P. macularis appears to persist in a subset of chronically affected hop yards, particularly yards where spring pruning is conducted poorly. Targeted management of the disease in a subset of fields most at risk for producing flag shoots could potentially influence powdery mildew development regionwide.

Distribution and Characterization of Podosphaera macularis Virulent on Hop Cultivars Possessing R6-Based Resistance to Powdery Mildew.

Host resistance, both quantitative and qualitative, is the preferred long-term approach for disease management in many pathosystems, including powdery mildew of hop (Podosphaera macularis). In 2012, an epidemic of powdery mildew occurred in Washington and Idaho on previously resistant cultivars whose resistance was putatively based on the gene designated R6. In 2013, isolates capable of causing severe disease on cultivars with R6-based resistance were confirmed in Oregon and became widespread during 2014. Surveys of commercial hop yards during 2012 to 2014 documented that powdery mildew is now widespread on cultivars possessing R6 resistance in Washington and Oregon, and the incidence of disease is progressively increasing. Pathogenic fitness, race, and mating type of R6-virulent isolates were compared with isolates of P. macularis lacking R6 virulence. All isolates were positive for the mating type idiomorph MAT1-1 and were able to overcome resistance genes Rb, R3, and R5 but not R1 or R2. In addition, R6-virulent isolates were shown to infect differential cultivars reported to possess the R6 gene and also the R4 gene, although R4 has not yet been broadly deployed in the United States. R6-virulent isolates were not detected from the eastern United States during 2012 to 2015. In growth chamber studies, R6-virulent isolates of P. macularis had a significantly longer latent period and produced fewer lesions on plants with R6 as compared with plants lacking R6, indicating a fitness cost to the fungus. R6-virulent isolates also produced fewer conidia when compared with isolates lacking R6 virulence, independent of whether the isolates were grown on a plant with or without R6. Thus, it is possible that the fitness cost of R6 virulence occurs regardless of host genotype. In field studies, powdery mildew was suppressed by at least 50% on plants possessing R6 as compared with those without R6 when coinoculated with R6-virulent and avirulent isolates. R6 virulence in P. macularis appears to be race specific and, at this time, imposes a measurable fitness penalty on the fungus. Resistance genes R1 and R2 appear to remain effective against R6-virulent isolates of P. macularis in the U.S. Pacific Northwest.

Interaction of Basal Foliage Removal and Late-Season Fungicide Applications in Management of Hop Powdery Mildew.

Canopy management is an important aspect of control of powdery mildew diseases and may influence the intensity of fungicide applications required to suppress disease. In hop, powdery mildew (caused by Podosphaera macularis) is most damaging to cones when infection occurs during bloom and the juvenile stages of cone development. Experiments were conducted over 3 years to evaluate whether fungicide applications could be ceased after the most susceptible stages of cone development (late July) without unduly affecting crop yield and quality when disease pressure was moderated with varying levels of basal foliage removal. In experimental plots of 'Galena' hop, the incidence of leaves with powdery mildew was similar whether fungicides were ceased in late July or made in late August. Disease levels on leaves were unaffected by the intensity of basal foliage removal, whereas the intensity of basal foliage removal interacted with the duration of fungicide applications to affect disease levels on cones. Similar experiments conducted in large plots of 'Tomahawk' hop in a commercial hop yard similarly found no significant impact on disease levels on leaves from either the duration of fungicide applications or intensity of basal foliage removal. In contrast, on cones, application of fungicides into August had a modest, suppressive effect on powdery mildew. There was also some evidence that the level of powdery mildew on cones associated with fungicide treatment was influenced by the intensity of basal foliage removal. When fungicide applications ceased in late July, there was a progressive decrease in the incidence of cones with powdery mildew with increasing intensity of basal foliage removal. Removing basal foliage two to three times allowed fungicide applications to be terminated in late July rather than late August without diminishing disease control on cones, yield, or cone quality factors. Thus, this study further establishes that fungicide applications made during the early stages of hop cone development have the strongest effect on suppression of powdery mildew on cones. The additive effect of fungicide applications targeted to the periods of greatest cone susceptibility and canopy management to reduce disease favorability may obviate the need for fungicide applications later in the season. This appears to be a viable strategy in mature hop yards of certain cultivars when disease pressure is not excessively high.

Hop Powdery Mildew Control Through Alteration of Spring Pruning Practices.

Podosphaera macularis, the causal agent of hop powdery mildew, is a recurrent threat to hops in the Pacific Northwest because of the potential to reduce cone yield and quality. Early-season pruning is a common practice in hop production for horticultural reasons. Studies were conducted over a 3-year period in a commercial hop yard to quantify the effect of pruning method and timing on disease development, yield, and cone quality factors. A 4-week delay in pruning reduced the incidence of leaves with powdery mildew from 46 to 10% and cones from 9 to 1%, with the specific effect being season dependent. Pruning using chemical desiccants rather than by mechanical means had similar effects on disease levels on leaves. On cones, though, chemical pruning had a small but significant reduction in the incidence of powdery mildew compared with mechanical pruning. Cone yield, levels of bittering-acids, and color were not negatively affected in any individual year or cumulatively over three seasons when pruning treatments were applied repeatedly to the same plots during the study period. Delayed pruning may offer a low-cost means of reducing both the incidence of powdery mildew and early-season fungicide inputs in certain cultivars.

Aspergillus flavus diversity on crops and in the environment can be exploited to reduce aflatoxin exposure and improve health.

Humans and animals are exposed to aflatoxins, toxic carcinogenic fungal metabolites, through consumption of contaminated food and feed. Aspergillus flavus, the primary causal agent of crop aflatoxin contamination, is composed of phenotypically and genotypically diverse vegetative compatibility groups (VCGs). Molecular data suggest that VCGs largely behave as clones with certain VCGs exhibiting niche preference. VCGs vary in aflatoxin-producing ability, ranging from highly aflatoxigenic to atoxigenic. The prevalence of individual VCGs is dictated by competition during growth and reproduction under variable biotic and abiotic conditions. Agronomic practices influence structures and average aflatoxin-producing potentials of A. flavus populations and, as a result, incidences and severities of crop contamination. Application of atoxigenic strains has successfully reduced crop aflatoxin contamination across large areas in the United States. This strategy uses components of the endemic diversity to alter structures of A. flavus populations and improve safety of food, feed, and the overall environment.

Relationships between in vivo and in vitro aflatoxin production: reliable prediction of fungal ability to contaminate maize with aflatoxins.

Aflatoxins are highly carcinogenic mycotoxins frequently produced by Aspergillus flavus. Contamination of maize with aflatoxins imposes both economic and health burdens in many regions. Identification of the most important etiologic agents of contamination is complicated by mixed infections and varying aflatoxin-producing potential of fungal species and individuals. In order to know the potential importance of an isolate to cause a contamination event, the ability of the isolate to produce aflatoxins on the living host must be determined. Aflatoxin production in vitro (synthetic and natural media) was contrasted with in vivo (viable maize kernels) in order to determine ability of in vitro techniques to predict the relative importance of causal agents to maize contamination events. Several media types and fermentation techniques (aerated, non-aerated, fermentation volume) were compared. There was no correlation between aflatoxin production in viable maize and production in any of the tested liquid fermentation media using any of the fermentation techniques. Isolates that produced aflatoxins on viable maize frequently failed to produce detectable (limit of detection=1ppb) aflatoxin concentrations in synthetic media. Aflatoxin production on autoclaved maize kernels was highly correlated with production on viable maize kernels. The results have important implications for researchers seeking to either identify causal agents of contamination events or characterize atoxigenic isolates for biological control.

Outbreak of an acute aflatoxicosis in Kenya in 2004: identification of the causal agent.

Maize contaminated with aflatoxins has been implicated in deadly epidemics in Kenya three times since 1981, but the fungi contaminating the maize with aflatoxins have not been characterized. Here we associate the S strain of Aspergillus flavus with lethal aflatoxicoses that took more than 125 lives in 2004.