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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.

Blue mold, caused by

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.

Sequencing herbarium specimens can be instrumental in answering ecological, evolutionary, and taxonomic inquiries. We developed a protocol for sequencing herbarium specimens of rust fungi (Pucciniales) and proceeded to sequence specimens ranging from 4 to 211 yr old from five different genera. We then obtained sequences from an economically important biological control agent, Puccinia suaveolens, to highlight the potential of sequencing herbarium specimens in an ecological sense and to evaluate the following hypotheses: (1) The population structure of a plant pathogen changes over time, and (2) introduced pathogens are more diverse in their native range. Our efforts resulted in sequences from 87 herbarium specimens that revealed a high level of diversity with a population structure that exhibited spatial-temporal patterns. The specimens sequenced from Europe showed more diversity than the ones from North America, uncovering an invasion pattern likely related to its European native host in North America. Additionally, to the best of our knowledge, the specimen from France collected in c. 1811 is the oldest herbarium specimen sequenced from kingdom Fungi. In conclusion, sequencing old herbarium specimens is an important tool that can be extrapolated to better understand plant-microbe evolution and to evaluate old type specimens to solidify the taxonomy of plant pathogenic fungi.

Fungi secrete a variety of compounds that have wide ranging effects on society and govern the outcome of host–pathogen interactions. The secreted products range from powerful toxins and carcinogens, to beneficial compounds such as ethanol used in common commercial practices, and the ‘wonder drug’ penicillin. Much research in the past 50 y has focused on identifying the genes and their functions relating to the fungal secretome. Recent advances into the mechanisms by which phytopathogenic fungal secretion systems function and modulate virulence have broad implications for the agricultural and biotechnological industries. In this review, we focus on secretion mechanisms in phytopathogenic fungi with examples from key plant–pathogen systems. Current progress and knowledge gaps regarding secretion pathways and their regulation are discussed. We highlight possible approaches to using novel molecular techniques to generate alternative control methods to synthetic pesticides.

This perspective presents a synopsis of the topics contained in the Phytopathology Pathogen Spotlight on Botrytis spp. causing gray mold, including pathogen biology and systematics, genomic characterization of new species, perspectives on genome editing, and fungicide resistance. A timely breakthrough to engineer host plant resistance against the gray mold fungus has been demonstrated in planta and may augment chemical controls in the near future. While B. cinerea has garnered much of the research attention, other economically important Botrytis spp. have been identified and characterized via morphological and genome-based approaches. Gray mold control is achieved primarily through fungicide applications but resistance to various chemical classes is a major concern that threatens global plant health and food security. In this issue, new information on molecular mechanism(s) of fungicide resistance and ways to manage control failures are presented. Finally, a significant leap in fundamental pathogen biology has been achieved via development of CRISPR/Cas9 to assess gene function in the fungus which likely will spawn new control mechanisms and facilitate gene discovery studies.

BACKGROUND: The new approach of using UV-C irradiation followed by a specific dark period to control plant diseases has the potential to become a mainstream treatment in the production of strawberries and other fruits and vegetables. Thus, it is imperative to study the effects of this treatment on fruit quality. METHODS: In this study, short-day ‘Chandler’ strawberry plants grown in growth chamber from bloom to harvest were irradiated twice a week with nighttime UV-C light (253 nm peak value 12.36 J m–2 and the total dose of 50 J m–2 in the range of 240 –260 nm). The effects on the content of key phenolic compounds in mature fruit were studied with targeted analysis and a non-targeted metabolomic approach based on ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS). Analysis of variance-principal component analysis (ANOVA-PCA) was used to associate variance with different experimental factors. RESULTS: Results indicate that the content of anthocyanins, glucosides and glucuronides of quercetin and kaempferol, catechin, pelargonidin rutinoside, and ellagic acid was not affected by UV-C treatment. ANOVA-PCA analysis of the metabolomic dataset showed no significant differences in composition and content of strawberry metabolites between UV-C and control groups; however, differences were observed between harvest times. CONCLUSIONS: Our findings strongly indicate that UV-C treatment of 50 J/m2 twice per week did not affect nutritional values of strawberry fruit. The metabolomic approach combined with ANOVA-PCA used to evaluate strawberry fruit quality after UV-C irradiation proved to be a very powerful tool in providing an overall insight into fruit quality and accurately determines the influence of each experimental factor.

Blue mould, caused primarily by Penicillium expansum, is a major threat to the global pome fruit industry, causing multimillion‐dollar losses annually. The blue mould fungus negatively affects fruit quality, thereby reducing fresh fruit consumption, and significantly contributes to food loss. P. expansum also produces an array of mycotoxins that are detrimental to human health. Management options are limited and the emergence of fungicide‐resistant Penicillium spp. makes disease management difficult, therefore new approaches and tools are needed to combat blue mould in storage. This species profile comprises a comprehensive literature review of this aggressive pathogen associated with pomes (apple, pear, quince), focusing on biology, mechanisms of disease, control, genomics, and the newest developments in disease management. Taxonomy Penicillium expansum Link 1809. Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Subphylum Pezizomycotina, Class Eurotiomycetes, Subclass: Eurotiomycetidae, Order Eurotiales; Family Trichocomaceae, Genus Penicillium, Species expansum. Biology A wide host range necrotrophic postharvest pathogen that requires a wound (e.g., stem pull, punctures, bruises, shoulder cracks) or natural openings (e.g., lenticel, stem end, calyx sinus) to gain ingress and infect. Toxins Patulin, citrinin, chaetoglobosins, communesins, roquefortine C, expansolides A and B, ochratoxin A, penitrem A, rubratoxin B, and penicillic acid. Host range Primarily apples, European pear, Asian pear, medlar, and quince. Blue mould has also been reported on stone fruits (cherry, plum, peach), small fruits (grape, strawberry, kiwi), and hazel nut. Disease symptoms Blue mould initially appears as light tan to dark brown circular lesions with a defined margin between the decayed and healthy tissues. The decayed tissue is soft and watery, and blue‐green spore masses appear on the decayed area, starting at the infection site and radiating outward as the decayed area ages. Disease control Preharvest fungicides with postharvest activity and postharvest fungicides are primarily used to control decay. Orchard and packinghouse sanitation methods are also critical components of an integrated pest management strategy. Useful websites Penn State Tree Fruit Production Guide (https://extension.psu.edu/forage‐and‐food‐crops/fruit), Washington State Comprehensive Tree Fruit (http://treefruit.wsu.edu/crop‐protection/disease‐management/blue‐mold/), The Apple Rot Doctor (https://waynejurick.wixsite.com/applerotdr), penicillium expansum genome sequences and resources (https://www.ncbi.nlm.nih.gov/genome/browse/#!/eukaryotes/11336/)., This article is a synthesis and compilation of the latest information on the mycotoxingenic blue mould fungus from multiple perspectives that entail omics, biology, and tools for decay control.

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.

Lambertella corni-maris was isolated from a dark brown lesion on apple fruit cv. Golden Delicious collected from an orchard, and from stored apple fruit cv. Idared previously colonized with Monilia polystroma. The contrasting origin of the isolates led us to hypothesize that this fungus behaves differently in various contexts, which triggered the investigation of L. corni-maris as a pathogen and antagonist. Following identification based on cultural and molecular characteristics, isolates of different origin were characterized and their cultural, ecological, pathogenic, and antagonistic features were compared. This study has shown that L. corni-maris, originally isolated as an apple fruit pathogen and one as an antagonist of M. polystroma, have similar features in vitro and in vivo. The isolates vary widely in growth rate, cultural morphology, crystal production, intensity of dark zone and co-antagonism with two Monilinia species but can also function as apple fruit pathogens and inhibit and replace different Monilinia spp. on apple fruit. These findings reinforce their dual nature in different host-pathogen interactions. The data from this study are biologically intriguing and practically relevant, and have translative potential to exploit L. corni-maris as a model system for developing strategies to block brown rot of apple and to pinpoint virulence factors in the fungus for yellow rot management.

Blue mold, caused by Penicillium spp., is one of the most economically important postharvest diseases of pome fruits, globally. Pome fruits, in particular apple, is the most widely grown pome fruit in Serbia, and the distribution of Penicillium spp. responsible for postharvest decay is unknown. A two-year survey was conducted in 2014 and 2015, where four pome fruits (apple, pear, quince, and medlar) with blue mold symptoms were collected from 20 storage locations throughout Serbia. Detailed morphological characterization, analysis of virulence in three apple cultivars, and multilocus phylogeny revealed three main Penicillium spp. in order of abundance: P. expansum, P. crustosum, and P. solitum. Interestingly, P. expansum split into two distinct clades with strong statistical support that coincided with several morphological observations. Findings from this study are significant and showed previously undocumented diversity in blue mold fungi responsible for postharvest decay including the first finding of P. crustosum, and P. solitum as postharvest pathogens of quince and P. crustosum of medlar fruit in the world, and P. expansum of quince in Serbia. Data from this study provide timely information regarding phenotypic, morphological and genotypic plasticity in P. expansum that will impact the design of species-specific detection tools and guide the development of blue mold management strategies.

Postharvest fungal pathogens of stored fruits, nuts, and vegetables cause food spoilage and some produce mycotoxins that harm human health. These fungi can develop resistance to the chemicals used for their control despite judicious use, rotating different chemistries, and routine resistance monitoring. Once antimicrobial resistance develops, these fungi are difficult to control and persist in the field, packing, and storage environments. Therefore, new tools and approaches for control with reduced emphasis on chemicals and movement toward durable, innovative approaches (e.g. double-stranded RNA, translational metagenomics, and host-induced gene silencing) are warranted. The focus of this review is on formative breakthroughs to combat postharvest pathogens and the mycotoxins they produce via translation of fundamental science using biotechnology tools.

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.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. NO fumigation must be conducted under ultralow oxygen conditions because NO reacts with O2 to produce nitrogen dioxide (NO2). In this study, NO fumigations under different O2 concentrations were conducted on Aspergillus flavus spores to determine effectiveness of NO and NO2 in inactivating spores. Spores on gridded cellulose filter discs in Petri dishes were subjected to six fumigation treatments including a control with varying levels of NO under different O2 conditions for 3 h at 15 °C. The discs with spores were then cultured on Aspergillus Differentiation medium plates after fumigation for four days at 25 °C to count A. flavus colonies. Untreated control discs each had over 50 A. flavus colonies. Three fumigation treatments with 0.1% NO2 or 1.0% NO caused complete inactivation of A. flavus spores. The study demonstrated that NO fumigation with certain levels of NO2 can effectively inactivate A. flavus spores. The results suggest that NO fumigation has potential to be an alternative treatment to control both pests and microbes on stored products.

The 2014–2015 U.S. nationwide outbreak of listeriosis linked to apples used in commercially produced, prepackaged caramel apples was the first implication of whole apples in outbreaks of foodborne illnesses. Two case patients of this outbreak didn’t consume caramel apples but did eat whole apples, suggesting that contaminated whole apple may serve as a vehicle for foodborne listeriosis. The current study evaluated the effect of conventional fruit coating with wax and that of apple cultivar on the survival of outbreak-associated and non-outbreak Listeria monocytogenes strains on Red Delicious, Granny Smith and Fuji apples during 160 days under simulated commercial storage. L. monocytogenes survived in calyxes and stem ends of apples of all 3 cultivars through the duration of the experiment. After 2 months of storage, significantly (p L. monocytogenes populations were recovered from apples coated with wax than those un-waxed, regardless of the cultivar. No differences in survival amongst L. monocytogenes strains (serotypes 1/2a and 4b) from clinical, food, and environmental sources were observed. The observation that coating with wax facilitates prolonged survival of L. monocytogenes on whole apples is novel and reveals gaps in understanding of microbiological risks associated with postharvest practices of tree fruit production.

Inhibition of spore germination offers an attractive and effective target for controlling fungal species involved in food spoilage. Mushroom alcohol (1-octen-3-ol) functions as a natural self-inhibitor of spore germination for many fungi and, therefore, provides a useful tool for probing the molecular events controlling the early stages of fungal growth. In Penicillium spp., the R and S enantiomers of 1-octen-3-ol delayed spore germination and sporulation in four species of Penicillium involved in soils of fruit and grains, but to different degrees. Because of its well-annotated genome, we used Penicillium chrysogenum to perform a comprehensive comparative transcriptomic analysis of cultures treated with the two enantiomers. Altogether, about 80% of the high-quality reads could be mapped to 11,396 genes in the reference genome. The top three active pathways were metabolic (978 transcripts), biosynthesis of secondary metabolites (420 transcripts), and microbial metabolism in diverse environments (318 transcripts). When compared to the control, treatment with (R)-(-)-1-octen-3-ol affected the transcription levels of 91 genes, while (S)-(+)-1-octen-3-ol affected only 41 genes. Most of the affected transcripts were annotated and predicted to be involved in transport, establishment of localization, and transmembrane transport. Alternative splicing and SNPs’ analyses indicated that, compared to the control, the R enantiomer had greater effects on the gene expression pattern of Penicillium chrysogenum than the S enantiomer. A qRT-PCR analysis of 28 randomly selected differentially expressed genes confirmed the transcriptome data. The transcriptomic data have been deposited in NCBI SRA under the accession number SRX1065226.

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.

Fungicides are the primary tools to control a wide range of postharvest fungal pathogens. Fungicide resistance is a widespread problem that has reduced the efficacy of fungicides. Resistance to FRAC-1 (Fungicide Resistance Action Committee-1) chemistries is associated with mutations in amino acid position 198 in the β-tubulin gene. In our study, we conducted a meta-analysis of β-tubulin sequences to infer temporal, spatial, plant host, and pathogen genus patterns of fungicide resistance in postharvest fungal pathogens. In total, data were acquired from 2,647 specimens from 12 genera of fungal phytopathogens residing in 53 countries on >200 hosts collected between 1926 and 2020. The specimens containing a position 198 mutation were globally distributed in a variety of pathosystems. Analyses showed that there are associations among the mutation and the year an isolate was collected, the pathogen genus, the pathogen host, and the collection region. Interestingly, fungicide-resistant β-tubulin genotypes have been in a decline since their peak between 2005 and 2009. FRAC-1 fungicide usage data followed a similar pattern in that applications have been in a decline since their peak between 1997 and 2003. The data show that, with the reduction of selection pressure, FRAC-1 fungicide resistance in fungal populations will decline within 5 to 10 years. Based on this line of evidence, we contend that a β-tubulin position 198 mutation has uncharacterized fitness cost(s) on fungi in nature. The compiled dataset can inform end users on the regions and hosts that are most prone to contain resistant pathogens and assist decisions concerning fungicide resistance management strategies.

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.

Several wild apple accessions (Malus sieversii) from Kazakhstan and two (Malus × soulardii, Malus sylvestris) from other parts of the world are highly resistant to blue mold decay caused by Penicillium expansum. Previous studies on the wound responses of these apples to infection by this fungus suggest multiple mechanisms of resistance including innate immunity. In this study, the phenolic composition of extracts from mature wild apples resistant (GMAL 4317.f, PI 589391, and PI 369855) and susceptible (GMAL 3623.i) to blue mold, as well as the susceptible cultivar ‘Golden Delicious’ (Malus × domestica) were investigated using ultra-high-performance liquid chromatography coupled to diode array detection and high resolution multiple stage mass spectrometry (UHPLC/DAD/HRMS). The metabolomic and quantitative results of this study support the hypothesis of the possible relationship between the phenolic content of wild apples and their resistance to P. expansum. Apple accessions resistant to P. expansum had higher concentrations of procyanidins, dihydrochalocone, flavonols, and hydroxycinnamic acids. Findings from this study may lead to development of the physiological markers of resistance that could be used by breeders in evaluating crosses for resistance to blue mold and may be helpful to further define apple defense mechanism(s) against P. expansum.

The apple scab pathogen

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.

Background Hydroxycinnamoyl-spermine conjugates (HCSpm) are a class of hydroxycinnamic acid amides (HCAAs), which not only are instrumental in plant development and stress response, but also benefit human health. However, HCSpm are not commonly produced in plants, and the mechanism of their biosynthesis remains unclear. In previous investigations of phenolics in Solanum fruits related to eggplant (Solanum melongena L.), we discovered that Solanum richardii, an African wild relative of eggplant, was rich in HCSpms in fruits. Results The putative spermine hydroxycinnamoyl transferase (HT) SpmHT was isolated from S. richardii and eggplant. SrSpmHT expression was high in flowers and fruit, and was associated with HCSpm accumulation in S. richardii; however, SpmHT was hardly detected in eggplant cultivars and other wild relatives. Recombinant SpmHT exclusively selected spermine as the acyl acceptor substrate, while showing donor substrate preference in the following order: caffeoyl-CoA, feruloyl-CoA, and p-coumaroyl-CoA. Molecular docking revealed that substrate binding pockets of SpmHT could properly accommodate spermine but not the shorter, more common spermidine. Conclusion SrSpmHT is a novel spermine hydroxycinnamoyl transferase that uses Spm exclusively as the acyl acceptor substrate to produce HCSpms. Our findings shed light on the HCSpm biosynthetic pathway that may allow an increase of health beneficial metabolites in Solanum crops via methods such as introgression or engineering HCAA metabolism. Electronic supplementary material The online version of this article (10.1186/s12870-019-1846-3) contains supplementary material, which is available to authorized users.

Soybean rust (SBR) survival and host availability (kudzu, Pueraria spp.) were assessed from November 2006 through April 2007 at six sites from the panhandle to southwest Florida. Micro loggers recorded both temperature and relative humidity hourly at each location. Periods of drought and cumulative hours below 0°C correlated with kudzu defoliation. Inoculum potential from detached kudzu leaves was evaluated in vitro under various temperature and relative humidity levels. Kudzu leaves with SBR kept at 4°C produced viable urediniospores with the highest germination at all moisture levels over time. Freezing temperatures (–4 and –20°C) drastically reduced spore germination. However, when leaves were incubated at low (

Blue mold decay occurs during long term storage of apples and is predominantly caused by Penicillium expansum Link. Apples harvested in 2010 were stored in a controlled atmosphere at a commercial Pennsylvania apple packing and storage facility, and were examined for occurrence of decay in May 2011. Several decayed apples from different cultivars, exhibiting blue mold symptoms with a sporulating fungus were collected. One isolate recovered from a decayed ‘Golden Delicious’ apple fruit was identified as P. carneum Frisvad. Genomic DNA was isolated, 800 bp of the 3′ end of the β-tubulin locus was amplified using gene specific primers and sequenced (4). The recovered nucleotide sequence (GenBank Accession No. JX127312) indicated 99% sequence identity with P. carneum strain IBT 3472 (GenBank Accession No. JF302650) (3). The P. carneum colonies strongly sporulated and had a blue green color on potato dextrose agar (PDA), Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) media at 25°C after 7 days. The colonies also had a beige color on plate reverse on CYA and YES media. The species tested positive for the production of alkaloids, as indicated by a violet reaction for the Ehrlich test, and grew on CYA at 30°C and on Czapek with 1,000 ppm propionic acid agar at 25°C; all of which are diagnostic characters of this species (2). The conidiophores were hyaline and tetraverticillate with a finely rough stipe. Conida were produced in long columns, blue green, globose, and averaged 2.9 μm in diameter. To prove pathogenicity, Koch's postulates were conducted using 20 ‘Golden Delicious’ apple fruits. Fruits were washed, surface sterilized with 70% ethanol, and placed onto fruit trays. Using a nail, 3-mm wounds were created and inoculated with 50 μl of a 106/ml conidial suspension or water only as a negative control. The fruit trays were placed into boxes and were stored in the laboratory at 20°C for 7 days. The inoculated fruit developed soft watery lesions, with hard defined edges 37 ± 4 mm in diameter. The sporulating fungus was reisolated from infected tissue of all conidia inoculated apples and confirmed to be P. carneum by polymerase chain reaction (PCR) using the β-tubulin locus as described. Water inoculated control apples were symptomless. Originally grouped with P. roqueforti, P. carneum was reclassified in 1996 as a separate species (1). P. carneum is typically associated with meat products, beverages, and bread spoilage and produces patulin, which is not produced by P. roqueforti (1,2). Our isolate of P. carneum was susceptible to the thiabendazole (TBZ) fungicide at 250 ppm, which is below the recommended labeled application rate of 600 ppm. The susceptibility to TBZ suggests that this P. carneum isolate has been recently introduced because resistance to TBZ has evolved rapidly in P. expansum (4). To the best of our knowledge, P. carneum has not previously been described on apple, and this is the first report of P. carneum causing postharvest decay on apple fruits obtained from storage in Pennsylvania. References: (1) M. Boyson et al. Microbiology 142:541, 1996. (2) J. C. Frisvad and R. A. Samson. Stud. Mycol. 49:1, 2004. (3) B. G. Hansen et al. BMC Microbiology 11:202, 2011. (4) P. L. Sholberg et al. Postharvest Biol. Technol. 36:41, 2005.

Apples in the United States are stored in low-temperature controlled atmospheres for 9 to 12 months and are highly susceptible to blue mold decay. Penicillium spp. cause significant economic losses worldwide and produce mycotoxins that contaminate processed apple products. Blue mold is managed by a combination of cultural practices and the application of fungicides. In 2004, a new postharvest fungicide, pyrimethanil (Penbotec 400 SC, Janseen PMP, Beerse, Belgium) was registered for use in the United States to control blue mold on pome fruits (1). In this study, 10 blue mold symptomatic ‘Red Delicious’ apples were collected in May 2011 from wooden bins at a commercial facility located in Pennsylvania. These fruit had been treated with Penbotec prior to controlled atmosphere storage. Ten single-spore Penicillium spp. isolates were analyzed for growth using 96-well microtiter plates containing Richards minimal medium amended with a range of technical grade pyrimethanil from 0 to 500 μg/ml. Conidial suspensions adjusted to 1 × 105 conidia/ml were added to three 96-well plates for each experiment; all experiments were repeated three times. Nine resistant isolates had prolific mycelial growth at 500 μg/ml, which is 1,000 times the discriminatory dose that inhibited baseline sensitive P. expansum isolates from Washington State (1). However, one isolate (R13) had limited conidial germination and no mycelial proliferation at 0.5 μg/ml and was categorized as sensitive. One resistant (R22) and one sensitive (R13) isolate were selected on the basis of their different sensitivities to pyrimethanil. Both isolates were identified as P. expansum via conventional PCR using β-tubulin gene-specific primers according to Sholberg et al. (2). Analysis of the 2X consensus amplicon sequences from R13 and R22 matched perfectly (100% identity and 0.0 E value) with other P. expansum accessions in GenBank including JN872743.1, which was isolated from decayed apple fruit from Washington State. To determine if pyrimethanil applied at the labeled rate of 500 μg/ml would control R13 or R22 in vivo, organic ‘Gala’ apple fruit were wounded, inoculated with 50 μl of a conidial suspension (1 × 104 conidia/ml) of either isolate, dipped in Penbotec fungicide or sterile water, and stored at 25°C for 7 days. Twenty fruit composed a replicate within a treatment and the experiment was performed twice. Non-inoculated water-only controls were symptomless, while water-dipped inoculated fruit had 100% decay with mean lesion diameters of 36.8 ± 2.68 mm for R22 and 38.5 ± 2.61 mm for R13. The R22 isolate caused 30% decay with 21.6 ± 5.44 mm lesions when inoculated onto Penbotec-treated apples, while the R13 isolate had 7.5% decay incidence with mean lesion diameters of 23.1 ± 3.41 mm. The results from this study demonstrate that P. expansum pyrimethanil-resistant strains are virulent on Penbotec-treated apple fruit and have the potential to manifest in decay during storage. To the best of our knowledge, this is the first report of pyrimethanil resistance in P. expansum from Pennsylvania, a major apple growing region for the United States. Moreover, these results illuminate the need to develop additional chemical, cultural, and biological methods to control this fungus. References: (1) H. X. Li and C. L. Xiao. Phytopathology 98:427, 2008. (2) P. L. Sholberg et al. Postharvest Biol. Technol. 36:41, 2005.

Mucor piriformis E. Fischer causes Mucor rot of pome and stone fruits during storage and has been reported in Australia, Canada, Germany, Northern Ireland, South Africa, and portions of the United States (1,2). Currently, there is no fungicide in the United States labeled to control this wound pathogen on apple. Cultural practices of orchard sanitation, placing dry fruit in storage, and chlorine treatment of dump tanks and flumes are critical for decay management (3,4). Cultivars like ‘Gala’ that are prone to cracking are particularly vulnerable as the openings provide ingress for the fungus. Mucor rot was observed in February 2013 at a commercial packing facility in Pennsylvania. Decay incidence was ~15% on ‘Gala’ apples from bins removed directly from controlled atmosphere storage. Rot was evident mainly at the stem end and was light brown, watery, soft, and covered with fuzzy mycelia. Salt-and-pepper colored sporangiophores bearing terminal sporangiospores protruded through the skin. Five infected apple fruit were collected, placed in an 80-count apple box on trays, and temporarily stored at 4°C. Isolates were obtained aseptically from decayed tissue, placed on potato dextrose agar (PDA) petri plates, and incubated at 25°C with natural light. Five single sporangiospore isolates were identified as Mucor piriformis based on cultural characteristics according to Michailides and Spotts (1). The isolates produced columellate sporangia attached terminally on short and tall, branched and unbranched sporangiophores. Sporangiospores were ellipsoidal, subspherical, and smooth. Chlamydospore-like resting structures (gemmae), isogametangia, and zygospores were not evident in culture. Mycelial growth was examined on PDA, apple agar (AA), and V8 agar (V8) at 25°C with natural light. Isolates grew best on PDA at rates that ranged from 38.4 ± 5.3 to 34.5 ± 2.41 mm/day, followed by AA from 30.5 ± 1.22 to 28.5 ± 2.51 mm/day, and V8 from 29.2 ± 3.0 to 26.7 ± 2.17 mm/day. Species-level identification was conducted by isolating genomic DNA, amplifying a portion of the 28S rDNA gene, and directly sequencing the products. MegaBLAST analysis of the 2X consensus sequences revealed that all five isolates were 99% identical to M. piriformis (GenBank Accession No. JN2064761) with E values of 0.0, which confirms the morphological identification. Koch's postulates were conducted using organic ‘Gala’ apples that were surface sanitized with soap and water, then sprayed with 70% ethanol and allowed to air dry. Wounds 3 mm deep were created using the point of a finishing nail and then inoculated with 50 μl of a sporangiospore suspension (1 × 105 sporangiospores/ml) for each isolate. Ten fruit were inoculated with each isolate, and the experiment was repeated. The fruit were stored at 25°C in 80-count boxes on paper trays for 14 days. Decay observed on inoculated ‘Gala’ fruit was similar to symptoms originally observed on ‘Gala’ apples from storage and the pathogen was re-isolated from inoculated fruit. This is the first report of M. piriformis causing postharvest decay on stored apples in Pennsylvania and reinforces the need for the development of additional tools to manage this economically important pathogen. References: (1) T. J. Michailides, and R. A. Spotts. Plant Dis. 74:537, 1990. (2) P. L. Sholberg and T. J. Michailides. Plant Dis. 81:550, 1997. (3) W. L. Smith et al. Phytopathology 69:865, 1979. (4) R. A. Spotts. Compendium of Apple and Pear Diseases and Pests: Second Edition. APS Press, St. Paul, MN, 2014.

Bitter rot of apple is caused by Colletotrichum acutatum and C. gleosporioides and is an economically important disease in the mid-Atlantic and southern regions of the United States (1). However, other Colletotrichum spp. have been found to infect apple and pear fruit in Croatia that include C. fioriniae and C. clavatum (3). The disease is favorable under wet, humid conditions and can occur in the field or during storage causing postharvest decay (2). In February 2013, ‘Nittany’ apples with round, brown, dry, firm lesions having acervuli in concentric rings were observed at a commercial cold storage facility in Pennsylvania. Samples were placed on a paper tray in an 80-count apple box and immediately transported to the lab. Fruit were rinsed with sterile water, and lesions were sprayed with 70% ethanol until runoff. The skin was aseptically removed with a scalpel, and tissue under the lesion was placed onto potato dextrose agar (PDA) petri dishes. Dishes were incubated at 25°C with constant light, and a single-spore isolate was propagated on PDA. Permanent cultures were maintained as PDA slants stored at 4°C in darkness. The isolate was identified as a Colletotrichum sp. based on culture morphology, having light gray mycelium with a pinkish reverse and abundant pin-shaped melanized acervuli oozing pink conidia on PDA. Conidia were fusiform, pointed at one or both ends, one-celled, thin-walled, aseptate, hyaline, and averaged 10.5 μm (7.5 to 20 μm) long and 5.1 μm (5 to 10 μm) wide (n = 50). Genomic DNA was extracted from mycelia and amplified using conventional PCR and gene specific primers for 313 bp of the Histone 3 gene and with ITS4/5 primers for the internal transcribed spacer (ITS) rDNA region. MegaBLAST analysis of both gene sequences showed that our isolate was identical to other Colletotrichum fioriniae sequences in GenBank and was 100% identical to culture-collection C. fioriniae isolate CBS:128517, thus confirming the morphological identification. To prove pathogenicity, Koch's postulates were conducted using organic ‘Gala’ apple fruit that were washed with soap and water, sprayed with 70% ethanol, and wiped dry. The fruit were wounded with a sterile nail to a 3-mm depth, inoculated with 50 μl of a conidial suspension (1 × 104 conidia/ml), and stored at 25°C in 80-count boxes on paper trays for 14 days. Lesion diameter was measured from 10 replicate fruit with a digital micrometer and averaged 31.2 mm (±2.5 mm) over two experiments (n = 20). Water-only controls were symptomless. Artificially inoculated ‘Gala’ apples had identical external and internal symptoms (v-shaped decay pattern when the fruit were cut in half) to those observed on ‘Nittany’ apples that were originally obtained from cold storage. Bitter rot caused by C. fioriniae may become an emerging problem for the pome fruit growing industry in the near future, and may require investigation of new disease management practices to control this fungus. This is the first report of postharvest decay caused by C. fioriniae on apple fruit from cold storage in the United States. References: (1) H. W. Anderson. Diseases of Fruit Crops. McGraw-Hill, New York, 1956. (2) A. R. Biggs et al. Plant Dis. 85:657, 2001. (3) D. Ivic et al. J. Phytopathol. 161:284, 2013.

Alternaria rot, caused by Alternaria alternata (Fr.) Keissl., occurs on apple fruit (Malus × domestica Borkh) worldwide and is not controlled with postharvest fungicides currently registered for apple in the United States (1). Initial infections can occur in the orchard prior to harvest, or during cold storage, and appear as small red dots located around lenticels (1). The symptoms appear on fruits within a 2 month period after placement into cold storage (3). In February 2013, ‘Nittany’ apple fruit with round, dark, dry, spongy lesions were collected from bins at commercial storage facility located in Pennsylvania. Symptomatic apples (n = 2 fruits) were placed on paper trays in an 80 count apple box and immediately transported to the laboratory. Fruit were rinsed with sterile water, and the lesions were superficially disinfected with 70% ethanol. The skin was removed with a sterile scalpel, and tissues underneath the lesion were cultured on potato dextrose agar (PDA) and incubated at 25°C with constant light. Two single-spore isolates were propagated on PDA, and permanent cultures were maintained on PDA slants and stored at 4°C in darkness. Colonies varied from light gray to olive green in color, produced abundant aerial hyphae, and had fluffy mycelial growth on PDA after 14 days. Both isolates were tentatively identified as Alternaria based on multicelled conidial morphology resembling “fragmentation grenades” that were medium brown in color, and obclavate to obpyriform catentulate with longitudinal and transverse septa attached in chains on simple conidiophores (2). Conidia ranged from 15 to 60 μm (mean 25.5 μm) long and 10 to 25 μm (mean 13.6 μm) wide (n = 50) with 1 to 6 transverse and 0 to 1 longitudinal septa per spore. To identify both isolates to the species level, genomic DNA was extracted from mycelial plugs and gene specific primers (ALT-HIS3F/R) were used via conventional PCR to amplify a portion of the histone gene (357 bp) (Jurick II, unpublished). Amplicons were sequenced using the Sanger method, and the forward and reverse sequences of each amplicon were assembled into a consensus representing 2× coverage. A megaBLAST analysis revealed that the isolates were 99% identical to Alternaria alternata sequences in GenBank (Accession No. AF404617), which was previously identified to cause decay on stored apple fruit in South Africa. To prove pathogenicity, Koch's postulates were conducted using organic ‘Gala’ apples. The fruit were surface disinfested with soap and water and sprayed with 70% ethanol to runoff. Wounds, 3 mm deep, were done using a sterile nail and 50 μl of a conidial suspension (1 × 104 conidia/ml) was introduced into each wound per fruit. Fruit were then stored at 25°C in 80 count boxes on paper trays for 21 days. Water only was used as a control. Ten fruit were inoculated with each isolate or water only (control) and the experiment was repeated once. Symptoms of decay observed on inoculated were ‘Gala’ apple fruit were identical to the symptoms initially observed on ‘Nittany’ apples obtained from cold storage after 21 days. No symptoms developed on fruit in the controls. A. alternata was re-isolated 100% from apple inoculated with the fungus, completing Koch's postulates. A. alternata has been documented as a pre- and postharvest pathogen on Malus spp. (3). To our knowledge, this is the first report of postharvest decay caused by A. alternata on apple fruit during cold storage in Pennsylvania. References: (1) A. L. Biggs et al. Plant Dis. 77:976, 1993. (2) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Center, Utrecht, the Netherlands, 2007. (3) R. S. Spotts. Pages 56-57 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinkle, eds. American Phytopathological Society, St. Paul, MN, 1990.