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.

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ∼7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.

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.

Apples are kept in controlled atmosphere cold storage for 9 to 12 months and are highly susceptible to postharvest decay caused by various fungi. Fusarium avenaceum is a wound pathogen that has been shown to account for the majority of Fusarium rot on apple fruit in Croatia (1). F. avenaceum produces an array of mycotoxins including moniliformin, acuminatopyrone, and chrysogine, which are of primary concern for the apple processing industry (2). In February 2013, ‘Gala’ apple fruits with soft, circular, brown, watery lesions with characteristic abundant whitish mycelium covering the surface of the colonized fruit were obtained from bins from a commercial storage facility located in Pennsylvania. Several samples were collected and prepared for pathogen isolation. Apples were rinsed with sterile water, and the lesions were sprayed with 70% ethanol until runoff. The apple skin was aseptically removed with a scalpel, and asymptomatic tissue was placed onto full strength potato dextrose agar (PDA) petri plates without antibiotics and incubated at 25°C under natural light. Two single-spore isolates were propagated on PDA and permanent cultures were maintained as slants and stored in a cold room at 4°C in the dark. Fungal colonies initially formed abundant fluffy white mycelium and produced a golden orange pigment on PDA at 25°C. Isolates were identified as Fusarium based on cultural and conidial morphology as macroconidia were slightly falcate, thin-walled, usually 3 to 5 septate, with a tapering apical cell that was on average 23.6 μm long × 5.0 μm wide (n = 50). Microconidia were produced on PDA plates while chlamydospores were not evident. Identity of the isolates was confirmed through DNA extraction followed by amplification and sequencing of the translation elongation factor (EF-1α, 350 bp) gene region. The amplicons were sequenced using the forward and reverse primers and assembled into a consensus representing 2X coverage. MegaBLAST analysis revealed that both isolates were 100% identical with many other culture collection F. avenaceum sequences in Genbank (Accessions JQ949291.1, JQ949305.1, and JQ949283.1), which confirms their identification in conjunction with the morphological observations. Koch's postulates were conducted to determine pathogenicity using organic ‘Gala’ apple fruit that were surface sanitized with soap and water, sprayed with 70% ethanol, and wiped dry. The fruit were wounded with a finishing nail to 3 mm depth, inoculated with 50 μl of a conidial suspension (1 × 10⁴ conidia/ml) using a hemocytometer, and stored at 25°C in 80-count boxes on paper trays for 21 days. Water-only controls were symptomless. Ten fruit composed a replicate for each isolate, and the experiment was repeated. Symptoms observed on artificially inoculated ‘Gala’ apple fruit were identical to the decay observed on ‘Gala’ apples that were obtained from cold storage. Decay caused by F. avenaceum may represent an emerging problem for the apple storage and processing industry. Therefore, it is important to monitor for this pathogen to prevent future losses and mycotoxin contamination of processed fruit products caused by this fungus. To the best of our knowledge, this is the first report of Fusarium rot caused by F. avenaceum on apple fruit from cold storage in the United States. References: (1) Z. Sever et al. Arch. Ind. Hygiene Toxicol. 63:463, 2012. (2) J. L. Sorenson. J. Agric. Food Chem. 57:1632, 2009.

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.