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.

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.

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.

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.

Apples (Malus domestica, Rosaceae) are one of the most widely grown and economically valuable fruits worldwide. In Hood River County, Oregon in 1991 decayed apples exhibiting blue mold signs and symptoms were collected and spores from the causal agent of the disease were isolated. The decayed area of the infected apples was brown colored with soft, decayed tissue, which had bluish-green colored spores on the fruit surface. The whole genome of this isolate was sequenced (GenBank number: JYNM00000000) and it was originally identified as Penicillium solitum strain RS1 (Yu et al. 2016). Subsequent genome-wide species-level investigations showed higher homology to P. polonicum. Therefore, we taxonomically and phylogenetically reevaluated the fungus in question. Colonies were analyzed growing on potato dextrose agar (PDA), Czapek yeast autolysate agar (CYA) and malt extract agar (MEA) at 25°C. Colonies on PDA were blue-green and growth was moderately deep and raised at the center with low margins. Colonies on CYA were blue-green. The range of the colony diameter after 7 days at 25ºC was 24-27 mm on CYA and 20-25 mm on MEA. Colony reverse color on CYA was yellow-brown and on MEA was cream. Conidiophores were terverticillate. Stipes were septate with smooth walls and measured 62-250 × 3-5 µm, x̄ = 111.1 × 3.8 µm with 1-4 branches per stipe. Branches measured 8-25 × 2-6 µm, x̄ = 4.9 × 16.3 µm with 2-4 metulae per branch. Metulae measured 7-14 × 2-5 µm, x̄ = 10.1 × 3.6 µm with 1-3 phialides per metulae. Phialides were flask shaped and measured 5-11 × 2-5 µm, x̄ = 7.5 × 3.4 µm. Conidia were globose to subglobose, borne in columns measuring 2.2-5.4 × 2.1-5.3 µm, x̄ = 3.6 × 3.4 µm. Morphologically, the fungal strain RS1 matched the description of Penicillium polonicum K. Zaleski from Bashir et al. (2017), Duduk et al. (2014) and Frisvad and Sampson (2004) with some minor differences. The ITS, TUB and RpB2 sequences of the strain RS1 were extracted from GenBank accession number JYNM00000000. The sequences were then submitted for nucleotide BLAST (NCBI) analysis in GenBank and evaluated. The ITS sequence aligned 100% with the type specimen (CBS 222.28) of P. polonicum (GenBank number: NR_103687). The TUB sequence aligned over 99% with P. polonicum (GenBank numbers: MK450898, MK450935, MK450899). The RpB2 sequences aligned 99.9% or higher with multiple P. polonicum specimens deposited in CBS and CMV (GenBank numbers: MK450847, MK450846, JN985414 and JN985415). Koch's postulates were conducted. Ten apples were wounded with the point of a 16-penny nail, and 10ul of a conidial suspension adjusted to 106 conidia-distilled water/tween solution was added to the wound. Ten separate apples served as a control that were wounded and 10ul of sterile Tween treated water was used to simulate inoculation. None of the control apples developed signs or symptoms of the disease. The inoculated apples all developed typical blue mold symptoms. The fungus was reisolated from the fruit and deemed to be morphologically identical to those of the original RS1, P. polonicum isolate. To the best of our knowledge this is the first report of blue mold caused by P. polonicum in the USA on apples (Farr and Rossman 2021). This information is important for the apple industry for which blue mold is a major problem.

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.