Would You Like Wood or Plastic? Bin Material, Sanitation Treatments, and Bin Inoculum Levels Impact Blue Mold Decay of Stored Apple Fruit.

Blue mold, caused primarily by Penicillium expansum, is a significant postharvest disease of apples. It not only causes economic losses but also produces mycotoxins that contaminate processed fruit products, which contributes to food waste and loss. Previous research has shown that packing and storage bins harbor Penicillium spores and that steam and hot water efficiently reduce spore inoculum levels. However, studies using wooden and plastic bins regarding their ability to harbor spores, the effect of chemical sanitation treatments on spore levels, and the impact of rinsate from treated bins on apple fruit decay have not been investigated for the Mid-Atlantic area (Okull et al. 2006; Rosenberger 2009). We evaluated different sanitation treatments (chemical and physical) to reduce P. expansum inoculum levels on wooden and plastic bins. We determined that wooden bins bound P. expansum spores four orders of magnitude higher than plastic. When both bin types were treated with steam (wooden) or sterile hot water (plastic), Thyme Guard, or Academy, all treatments resulted in significantly (P < 0.05) lower spore levels compared to untreated controls. Although, plastic bins retained lower numbers of spores after inoculation with contaminated spore rinsate and required much higher concentrations of P. expansum spores in rinsate to retain spores at levels that would lead to decay on apple fruit. Overall, we demonstrated that plastic bins retain fewer spores than wooden bins and that both can be sanitized by various physical or chemical treatments. We envision that our findings will be applicable in the future as the techniques implemented in this study were used to investigate industry-relevant questions. Our goal is that the research techniques and findings become feasible with advancements in technology and/or accompany other shifts in existing processes in commercial pome fruit packing and storage facilities.

More than a Virulence Factor: Patulin Is a Non-Host-Specific Toxin that Inhibits Postharvest Phytopathogens and Requires Efflux for Penicillium Tolerance.

Mycotoxin contamination is a leading cause of food spoilage and waste on a global scale. Patulin, a mycotoxin produced by Penicillium spp. during postharvest pome fruit decay, causes acute and chronic effects in humans, withstands pasteurization, and is not eliminated by fermentation. While much is known about the impact of patulin on human health, there are significant knowledge gaps concerning the effect of patulin during postharvest fruit-pathogen interactions. Application of patulin on six apple cultivars reproduced some blue mold symptoms that were cultivar-independent and dose-dependent. Identical symptoms were also observed in pear and mandarin orange. Six Penicillium isolates exposed to exogenous patulin exhibited delayed germination after 24 h, yet all produced viable colonies in 7 days. However, four common postharvest phytopathogenic fungi were completely inhibited by patulin during conidial germination and growth, suggesting the toxin is important for Penicillium to dominate the postharvest niche. Using clorgyline, a broad-spectrum efflux pump inhibitor, we demonstrated that efflux plays a role in Penicillium auto-resistance to patulin during conidial germination. The work presented here contributes new knowledge of patulin auto-resistance, its mode of action, and inhibitory role in fungal-fungal interactions. Our findings provide a solid foundation to develop toxin and decay mitigation approaches.

Blistering1 Modulates Penicillium expansum Virulence Via Vesicle-mediated Protein Secretion.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

Global transcriptomic responses orchestrate difenoconazole resistance in Penicillium spp. causing blue mold of stored apple fruit.

BACKGROUND: Blue mold is a globally important and economically impactful postharvest disease of apples caused by multiple Penicillium spp. There are currently four postharvest fungicides registered for blue mold control, and some isolates have developed resistance manifesting in decay on fungicide-treated fruit during storage. To date, mechanisms of fungicide resistance have not been explored in this fungus using a transcriptomic approach. RESULTS: We have conducted a comparative transcriptomic study by exposing naturally-occurring difenoconazole (DIF) resistant (G10) and sensitive (P11) blue mold isolates to technical grade difenoconazole, an azole fungicide in the commercial postharvest product Academy (Syngenta Crop Protection, LLC). Dynamic changes in gene expression patterns were observed encompassing candidates involved in active efflux and transcriptional regulators between the resistant and sensitive isolates. Unlike other systems, 3 isoforms of cytochrome P450 monoxygenase (CYP51A-C) were discovered and expressed in both sensitive and resistant strains upon difenoconazole treatment. Active efflux pumps were coordinately regulated in the resistant isolate and were shown to mediate the global resistance response as their inhibition reversed the difenoconazole-resistant phenotype in vitro. CONCLUSIONS: Our data support the observation that global transcriptional changes modulate difenoconazole resistance in Penicillium spp. While the dogma of CYP51 overexpression is supported in the resistant isolate, our studies shed light on additional new mechanisms of difenoconazole resistance on a global scale in Penicillium spp. These new findings broaden our fundamental understanding of azole fungicide resistance in fungi, which has identified multiple genetic targets, that can be used for the detection, management, and abatement of difenoconazole-resistant blue mold isolates during long-term storage of apples.

A Genome Resource for Several North American Venturia inaequalis Isolates with Multiple Fungicide Resistance Phenotypes.

The apple scab pathogen, Venturia inaequalis, is among the most economically important fungal pathogens that affects apples. Fungicide applications are an essential part of disease management. Implementation of cultural practices and genetic sources of resistance in the host are vital components of scab management. This is the first presentation of multiple, high quality, well-annotated genomes of four North American V. inaequalis isolates having both sensitive and multiple fungicide resistance phenotypes. We envision that these isolates will enable investigations into fungicide resistance mechanisms, exploring fungal virulence factors and delineating phylogenomic relationships among apple scab isolates from around the world.

Baseline Sensitivity of Penicillium spp. to Difenoconazole.

Penicillium spp. cause blue mold of stored pome fruit. These fungi reduce fruit quality and produce mycotoxins that are regulated for processed fruit products. Control of blue mold is achieved by fungicide application, and in 2015 Academy (active ingredients fludioxonil and difenoconazole) was released for use on pome fruit to manage postharvest blue mold. Baseline sensitivity for fludioxonil but not difenoconazole has been determined for P. expansum. To establish the distribution of sensitivity to difenoconazole before commercial use of Academy, 97 unexposed single-spore isolates from the United States and abroad were tested in vitro. Baseline EC50 values ranged from 0.038 to 0.827 µg/ml of difenoconazole with an average of 0.16 µg/ml. Complete inhibition of mycelial growth for all but three isolates occurred at 5 µg/ml of difenoconazole, whereas 10 µg/ml did not support growth for any of the isolates examined. Hence, 5 µg/ml of difenoconazole is recommended for phenotyping Penicillium spp. isolates with reduced sensitivity. Isolates with resistance to pyrimethanil and to both thiabendazole and pyrimethanil were observed among the isolates from the baseline collection. Academy applied at the labeled rate had both curative and protectant activities and controlled four representative Penicillium spp. from the baseline population. This information can be used to monitor future shifts in sensitivity to this new postharvest fungicide in Penicillium spp. populations.

Characterization of Postharvest Fungicide-Resistant Botrytis cinerea Isolates From Commercially Stored Apple Fruit.

Botrytis cinerea causes gray mold and is an economically important postharvest pathogen of fruit, vegetables, and ornamentals. Fludioxonil-sensitive B. cinerea isolates were collected in 2011 and 2013 from commercial storage in Pennsylvania. Eight isolates had values for effective concentrations for inhibiting 50% of mycelial growth of 0.0004 to 0.0038 µg/ml for fludioxonil and were dual resistant to pyrimethanil and thiabendazole. Resistance was generated in vitro, following exposure to a sublethal dose of fludioxonil, in seven of eight dual-resistant B. cinerea isolates. Three vigorously growing B. cinerea isolates with multiresistance to postharvest fungicides were further characterized and found to be osmosensitive and retained resistance in the absence of selection pressure. A representative multiresistant B. cinerea strain caused decay on apple fruit treated with postharvest fungicides, which confirmed the in vitro results. The R632I mutation in the Mrr1 gene, associated with fludioxonil resistance in B. cinerea, was not detected in multipostharvest fungicide-resistant B. cinerea isolates, suggesting that the fungus may be using additional mechanisms to mediate resistance. Results from this study show for the first time that B. cinerea with dual resistance to pyrimethanil and thiabendazole can also rapidly develop resistance to fludioxonil, which may pose control challenges in the packinghouse environment and during long-term storage.

Mining the Penicillium expansum Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit.

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434KD) were generated for three loci. Data show that the ∆t-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

Avirulent Isolates of Penicillium chrysogenum to Control the Blue Mold of Apple Caused by P. expansum.

Blue mold is an economically significant postharvest disease of pome fruit that is primarily caused by Penicillium expansum. To manage this disease and sustain product quality, novel decay intervention strategies are needed that also maintain long-term efficacy. Biocontrol organisms and natural products are promising tools for managing postharvest diseases. Here, two Penicillium chrysogenum isolates, 404 and 413, were investigated as potential biocontrol agents against P. expansum in apple. Notably, 404 and 413 were non-pathogenic in apple, yet they grew vigorously in vitro when compared to the highly aggressive P. expansum R19 and Pe21 isolates. Whole-genome sequencing and species-specific barcoding identified both strains as P. chrysogenum. Each P. chrysogenum strain was inoculated in apple with the subsequent co-inoculation of R19 or Pe21 simultaneously, 3, or 7 days after prior inoculation with 404 or 413. The co-inoculation of these isolates showed reduced decay incidence and severity, with the most significant reduction from the longer establishment of P. chrysogenum. In vitro growth showed no antagonism between species, further suggesting competitive niche colonization as the mode of action for decay reduction. Both P. chrysogenum isolates had incomplete patulin gene clusters but tolerated patulin treatment. Finally, hygromycin resistance was observed for both P. chrysogenum isolates, yet they are not multiresistant to apple postharvest fungicides. Overall, we demonstrate the translative potential of P. chrysogenum to serve as an effective biocontrol agent against blue mold decay in apples, pending practical optimization and formulation.

Penicillium solitum produces a polygalacturonase isozyme in decayed Anjou pear fruit capable of macerating host tissue in vitro.

A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5-6, with an optimum at pH 4.5. PG activity was detectable 0-70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca(2+), Mg(2+), Mn(2+) and Fe(2+) and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple.

Comparative Penicillium spp. Transcriptomics: Conserved Pathways and Processes Revealed in Ungerminated Conidia and during Postharvest Apple Fruit Decay.

Blue mold, caused by Penicillium spp., is an impactful postharvest disease resulting in significant economic losses due to reduced pome fruit quality and mycotoxin contamination. Using two Penicillium species with different levels of aggressiveness, transcriptomics were implemented in order to identify genes expressed during apple fruit decay and loci expressed in ungerminated conidia. Total RNA was isolated from ungerminated conidia and decayed apple fruit infected with P. expansum R19 or P. polonicum RS1. There were 2442 differentially expressed genes (DEGs) between the R19 and RS1 in apple. Comparisons within species between apple and conidia revealed 4404 DEGs for R19 and 2935 for RS1, respectively. Gene ontology (GO) analysis revealed differential regulation in fungal transport and metabolism genes during decay, suggesting a flux in nutrient acquisition and detoxification strategies. In R19, the oxidoreductase GO category comprised 20% of all DEG groups in apple verses conidia. Ungerminated conidia from both species showed DEGs encoding the glyoxylate shunt and beta-oxidation, specifying the earliest metabolic requirements for germination. This is the first study to identify pre-loaded transcripts in conidia from blue mold fungi, reveal unique genes between species expressed during apple decay, and show the expression dynamics of known fungal virulence factors. These findings will enable development of targeted approaches for blue mold abatement strategies.

Purification and biochemical characterization of polygalacturonase produced by penicillium expansum during postharvest decay of 'Anjou' pear.

A polygalacturonase (PG) was extracted and purified from decayed tissue of 'Anjou' pear fruit inoculated with Penicillium expansum. Ammonium sulfate precipitation, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. PG enzyme activity from healthy and wounded pear tissue was undetectable, which supports the claim that the purified PG is of fungal origin. The purified enzyme had a molecular mass of 41 kDa and a pI of 7.8. Activity of the PG was not associated with a glycosylated protein. The enzyme was active over a broad pH range from 3 to 6, with optimal activity at 4.5 in sodium citrate and sodium acetate buffers. The optimal temperature for activity was 37 degrees C but the enzyme was also active at 0, 5, 10, 20, and 50 degrees C. Thin-layer chromatographic analysis of PG hydrolysis products showed that the enzyme exhibits endo- and exo-activity. The purified enzyme macerated tissue in vitro causing approximately 30% reduction in mass of pear plugs compared with approximately 17% reduction for apple. Additionally, it produced 1.5-fold more soluble polyuronides on pear than apple tissue. This work shows for the first time the production of a PG by P. expansum during postharvest decay of pear fruit is different from the previously described PG produced in decayed apple fruit by the same pathogen.

Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay.

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other's closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit-fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.

Genome Sequence of Penicillium solitum RS1, Which Causes Postharvest Apple Decay.

Penicillium species cause postharvest decay, commonly known as blue mold, in pome fruits, such as apples and pears. To devise novel strategies to prevent and reduce economic losses during storage, the genome sequence of Penicillium solitum RS1 is reported here for the first time.

Draft Genome Sequence of Penicillium expansum Strain R19, Which Causes Postharvest Decay of Apple Fruit.

Among the species that cause blue mold, isolates of Penicillium expansum are the most prevalent and virulent species, causing more than 50 percent of postharvest decay. We report the draft genome sequence of P. expansum R19 in order to identify fungal virulence factors and to understand the mechanism of infection.