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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.

Blue mold caused primarily by Penicillium expansum and to a lesser extent other Penicillium spp. is the most destructive disease of stored apples in the US and worldwide. It was recently shown that resistance to blue mold exists in wild apple germplasms, Malus sieversii, from Kazakhstan and in other species from different regions maintained as a collection in Geneva, NY. We initiated studies to determine the durability and the mechanism(s) of resistance to P. expansum in select wild apple accessions. Wound responses (up to 96 h in 24 h intervals), affecting P. expansum infection, and related cytological changes were determined in accessions with varying levels of resistance. In general, the more resistant the accession, the quicker the wound response that prevented the fungus from infecting tissue and causing decay. No decay developed on immune apple accessions, even when inoculated immediately after wounding at the inoculum concentration of 105 conidia/mL. On a moderately resistant accession, a 24 h interval between wounding and inoculation was sufficient to avert decay. Reactive oxygen species (ROS) were detected at high levels immediately after wounding in the immune as well as susceptible accessions. Callose and lignin/suberin appears to play a minor role in resistance responses. Our results indicate the presence of a high level of durable resistance/immunity in the wild apples which is governed by several mechanism(s). This presents a new challenge for explaining the observed resistance and at the same time creates an opportunity for exploiting these resistant mechanisms in breeding programs to incorporate resistance to fruit decays into commercial cultivars.

A growing number of bacteria and fungi have been found to promote plant growth through mutualistic interactions involving elements such as volatile organic compounds (VOCs). Here, we report the identification of an environmentally isolated strain of Cladosporium sphaerospermum (herein named TC09), that substantially enhances plant growth after exposure in vitro beyond what has previously been reported. When cultured on Murashige and Skoog (MS) medium under in vitro conditions, tobacco seedlings (Nicotiana tabacum) exposed to TC09 cultures for 20 days increased stem height and whole plant biomass up to 25- and 15-fold, respectively, over controls without exposure. TC09-mediated growth promotion required >5 g/L sucrose in the plant culture medium and was influenced by the duration of exposure ranging from one to 10 days, beyond which no differences were detected. When transplanted to soil under greenhouse conditions, TC09-exposed tobacco plants retained higher rates of growth. Comparative transcriptome analyses using tobacco seedlings exposed to TC09 for 10 days uncovered differentially expressed genes (DEGs) associated with diverse biological processes including cell expansion and cell cycle, photosynthesis, phytohormone homeostasis and defense responses. To test the potential efficacy of TC09-mediated growth promotion on agricultural productivity, pepper plants (Capsicum annuum L.) of two different varieties, Cayenne and Minisweet, were pre-exposed to TC09 and planted in the greenhouse to monitor growth, flowering, and fruit production. Results showed that treated pepper plants flowered 20 days earlier and yielded up to 213% more fruit than untreated controls. Altogether the data suggest that exposure of young plants to C. sphaerospermum produced VOCs may provide a useful tool to improve crop productivity.

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.

Species in the genus

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae. Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Brown rot, caused by Monilinia spp., is an economically important pre- and postharvest disease of pome and stone fruits worldwide. In Serbia, apple is the most widely grown pome fruit, and the distribution of economically important Monilinia spp. responsible for apple brown rot is unknown. Hence, we conducted a three year survey, from 2010 to 2012, where 349 isolates were obtained from six orchards and four storage facilities from five different apple cultivars with brown rot symptoms. Morphological characterization of the isolates, multiplex PCR, and phylogenetic analysis revealed four species: M. fructigena, M. laxa, M. fructicola, and Monilia polystroma. All species were found in the orchard and in storage, with M. fructigena predominating, followed by M. polystroma. Representative isolates were analyzed in vitro and in vivo where differences in growth rate, sporulation, and virulence on apple fruit were observed. Findings from this investigation demonstrate diversity in the species responsible for pre- and postharvest apple brown rot, which has significant implications for pathogen detection and for developing disease-specific management strategies.

Maintaining proper storage temperature is critical for ensuring the quality and safety of fresh-cut products. The US Food and Drug Administration Food Code recommend that packaged fresh-cut leafy green vegetables be kept no warmer than 5 °C at all times to ensure food safety. Substantial temperature variations, however, within the widely used open refrigerated display cases used in retail stores are known to present the technical challenge of complying with this federal guidance for industry. This study determined the extent of the spatial and temporal temperature variations within two commercial open-refrigerated display cases under different operating conditions, and their impact on the quality and microbial growth of packaged baby spinach products. The packaged products were received within 2 d of commercial processing and temperature data loggers were placed inside-and-outside of each bag. All bags were immediately loaded in the display cases and the overall visual quality, tissue electrolyte leakage, total aerobic mesophilic bacteria and psychrotrophic bacteria were evaluated for each bag. Results from this study showed that the temperature variation in the cases was dependent on spatial location, thermostat setting, and defrost cycle interval and duration of defrost. The largest temperature differentials were found for samples located in the front and back rows of the display cases. Samples located in the front rows had the highest temperature due to heat penetration from the surrounding ambient environment, while those in the back were damaged as temperatures fell below freezing. These products received low quality scores and had higher tissue electrolyte leakage. In order to reduce the large temperature variations in the display cases, insulating foam boards were installed which significantly (P

Penicillium is a large genus of common molds with over 400 described species; however, identification of individual species is difficult, including for those species that cause postharvest rots. In this study, blue rot fungi from stored apples and pears were isolated from a variety of hosts, locations, and years. Based on morphological and cultural characteristics and partial amplification of the β-tubulin locus, the isolates were provisionally identified as several different species of Penicillium. These isolates were investigated further using a suite of molecular DNA markers and compared to sequences of the ex-type for cognate species in GenBank, and were identified as P. expansum (3 isolates), P. solitum (3 isolates), P. carneum (1 isolate), and P. paneum (1 isolate). Three of the markers we used (ITS, internal transcribed spacer rDNA sequence; benA, β-tubulin; CaM, calmodulin) were suitable for distinguishing most of our isolates from one another at the species level. In contrast, we were unable to amplify RPB2 sequences from four of the isolates. Comparison of our sequences with cognate sequences in GenBank from isolates with the same species names did not always give coherent data, reinforcing earlier studies that have shown large intraspecific variability in many Penicillium species, as well as possible errors in some sequence data deposited in GenBank.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean and other major food crops. Here we sequenced the genome of the sugar beet pathogen C. beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae. Although cercosporin biosynthesis has been thought to-date to rely on an eight gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein (CFP) previously shown to be involved with cercosporin auto-resistance, and four additional genes required for cercosporin biosynthesis including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Finally, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide new insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.Significance StatementSpecies in the fungal genus Cercospora cause diseases in many important crops worldwide. Their success as pathogens is largely due to the secretion of cercosporin during infection. We report that the cercosporin toxin biosynthesis (CTB) cluster is ancient and was horizontally transferred to diverse fungal pathogens on an unprecedented scale. Since these analyses revealed genes adjacent to the established CTB cluster, we evaluated their role in C. beticola to show that four are necessary for cercosporin biosynthesis. Finally, we confirmed that the apple pathogen Colletotrichum fioriniae produces cercosporin, the first case outside the family Mycosphaerellaceae. Other Colletotrichum plant pathogens also harbor the CTB cluster, which points to a wider concern that this toxin may play in virulence and human health.

Penicillium spp. has been genetically manipulated and gene function studies have utilized single gene deletion strains for phenotypic analysis. Fungal transformation experiments have relied on hygromycin and hygromycin phosphotransferase (hph) as the main dominant selectable marker (DSM) system in Penicillium spp. This poses a limitation on the number of loci that can be analyzed and complemented in reverse genetic studies. Additionally, many economically important Penicillium spp. have not been evaluated to determine the utility of additional chemicals that can serve as DSMs. Therefore, six compounds were examined for 15 blue mold strains and their Minimum Inhibitory Concentrations (MICs) determined. Phleomycin, neomycin and G418 were deemed ineffective, as Penicillium spp. growth was observed on media amended with 1000 μg/ml of each compound. The efficacy of bialophos to inhibit fungal growth was intermediate, with MICs ranging from 250 to 1000 μg/ml and was species-dependent. However, chlorimuron ethyl and benlate had the lowest MIC values and minimal variation in efficacy within and between species. Therefore, benlate and chlorimuron ethyl are good candidates for use as since corresponding fungal resistance genes have been cloned, characterized and are available from a variety of public and academic sources.

A non-destructive method to analyze the freshness of raw milk was developed using a FT-NIR spectrometer and a fiber optic probe. Diffuse transmittance spectra were acquired in the spectral range 833 ~ 2,500 nm from raw milk samples collected from Northwest AF University Animal Husbandry Station. After each spectral acquisition, quality parameters such as acidity, pH, and lactose content were measured by traditional detection methods. For all milk samples, PLS (partial least square regression), MLR (multiple linear regression), and ANN (artificial neural networks) analyses were carried out in order to develop models to predict parameters that were indicative of freshness. Predictive models showed R(2) values up to 0.9647, 0.9876 and 0.8772 for acidity, pH, and lactose content, respectively (validation set validations). The similarity analysis and classification between raw milk freshness during storage was also conducted by means of hierarchical cluster analysis. Over an 8 day storage period, the highest heterogeneity was evident between days 1 and 2.

Calmodulin, a ubiquitous calcium sensor, plays an important role in decoding stress-triggered intracellular calcium changes and regulates the functions of numerous target proteins involved in various plant physiological responses. To determine the functions of calmodulin in fleshy fruit, expression studies were performed on a family of six calmodulin genes (SlCaMs) in mature-green stage tomato fruit in response to mechanical injury and Botrytis cinerea infection. Both wounding and pathogen inoculation triggered expression of all those genes, with SlCaM2 being the most responsive one to both treatments. Furthermore, all calmodulin genes were upregulated by salicylic acid and methyl jasmonate, two signaling molecules involved in plant immunity. In addition to SlCaM2, SlCaM1 was highly responsive to salicylic acid and methyl jasmonate. However, SlCaM2 exhibited a more rapid and stronger response than SlCaM1. Overexpression of SlCaM2 in tomato fruit enhanced resistance to Botrytis-induced decay, whereas reducing its expression resulted in increased lesion development. These results indicate that calmodulin is a positive regulator of plant defense in fruit by activating defense pathways including salicylate- and jasmonate-signaling pathways, and SlCaM2 is the major calmodulin gene responsible for this event.

Antibiotics are routinely added to milk products and pose potential harm to public health. The objective of this study was to use an innovative and nondestructive application of an electronic nose instrument for rapid detection of penicillin G in goat milk. The PEN3 electronic nose system was utilized to detect volatile substances in goat milk after the addition of penicillin G sodium salt at concentrations of 0, 50, 100 and 200 μg/L. The data were extracted at 60 s to carry out a linear discriminant analysis. Additional statistical analysis was conducted using neural networks to predict the penicillin G concentration in goat milk samples. Accuracy rates for the two methods were 98.0 and 96.7% for training samples, and 97.0 and 94.9% for testing samples, respectively. The results from this study show that the electronic nose system can be utilized to predict the penicillin G concentrations in goat milk samples. Practical Applications Antibiotics are routinely added to animal-derived food products and have been reported to cause potential harm to human health. However, traditional analytical chemical methods (i.e., gas, liquid and high-performance liquid chromatography) can detect trace amounts of additives and have some drawbacks, such as complicated operation, high costs of implementation and lengthy analysis time. This research has examined the feasibility, accuracy and effectiveness of a metal oxide semiconductor gas sensor type electronic nose device (the PEN3 e-nose) to detect and discriminate among different concentrations of penicillin G in goat milk. Data from this study show that the electronic nose system can be used to predict the penicillin G concentration in goat milk samples.

Temperature abuse of fresh-cut products occurs routinely during transport and retail store display. However, the stage of product shelf life during temperature abuse and its impact on sensory attributes have not been studied. This study evaluated the effect of temperature abuse occurring immediately after processing and late in shelf life through measurements of sensory attributes, and membrane integrity of commercially packaged ready-to-eat baby spinach. The packaged products were received within 2 days of processing. Samples subject to early temperature abuse were immediately placed at 1, 4, 8, 12, 16 and 20 °C storage upon arrival, and those subject to late temperature abuse were stored at 1 °C for six days, and then transferred to 4, 8, 12, 16 and 20 °C storage. Package headspace gas composition, in-package visual appeal, purchase intent, product color, off-odor, decay, texture, overall quality, and tissue electrolyte leakage were evaluated every 1–2 day up to 16 day total. Results indicate that when the product temperature is maintained at 1–4 °C, the quality of commercially packaged baby spinach can be retained for up to 18 days post-processing. However, storage temperature of 8 °C or above, significantly ( P

Improved postharvest quality is an important goal for fresh-market raspberry breeding programs. To determine if warm or cold storage following harvest would better facilitate the breeding selection process for the assessment of postharvest decay and juice leakage, pesticide-free fruit from cultivars and breeding selections of red, yellow, purple, and black raspberries were stored at two temperatures. Following storage fruits were examined for decay and juice leakage rate at room-temperature (25 °C) and at a cooler temperature (5 °C). The rate of decay was much faster in room-temperature storage than in cooler storage; however, classification of genotypes as parents or discards was not always in agreement between these two temperatures. This suggests that a breeder should determine whether room-temperature storage or cooler storage more closely resembles the postharvest environment for the targeted growers. For many leakage rate comparisons, there was no advantage from either storage temperature. However, when an advantage was evident, cold storage evaluation identified a greater number of classes comparing black raspberry and purple raspberry genotypes, but warm storage evaluation identified a greater number of classes comparing red and yellow raspberry genotypes. There was complete agreement on genotype breeding disposition, indicating that a breeder could evaluate genotypes for leakage in the same storage temperature chosen to evaluate decay. Selection decisions made from evaluating floricane fruit were not always in agreement with decisions made from evaluating primocane fruit, indicating that genotypes should be evaluated in both fruiting seasons.

Bacterial and yeast antagonists isolated from fruit surfaces have been effective in controlling various post-harvest diseases, and several microbial antagonists have been developed into commercial products. Our knowledge of the fruit microbial community, with the exception of grapes, apples and some citrus fruit, is rudimentary and the potential of the resident yeasts for biocontrol remains largely unknown. We determined the occurrence of yeasts on plum surfaces during fruit development from the pre-hardening stage until harvest for 2 years. A total of 16 species from 13 genera were isolated. Species from three genera, basidiomycetes Rhodotorula (29.5%) and Sporidiobolus (24.7%) and the dimorphic ascomycete genus Aureobasidium (24.7%), constituted 78.7% of all isolations and were recovered throughout fruit development, while Cryptococcus spp. constituted only 6.2% of the total plum isolates. The yeast community in the final sampling was significantly different from the first three samplings, reflecting a rapidly changing fruit habitat during the maturation of fruit. For example, Hanseniaspora, Pichia, Zygosaccharomyces and Wickerhamomyces occurred only on the most mature fruit. Screening of the yeasts for antagonistic activity against Monilinia fructicola, a fungus that causes brown rot, revealed a range of biocontrol activities. Several isolates provided complete control of the decay on plums, challenged with a pathogen suspension of 10(3) conidia/ml and > 90% of control on fruit inoculated with the pathogen at a concentration 10 times higher. Some of the best antagonists included A. pullulans and R. phylloplana. Populations of both of these antagonists increased rapidly by several orders of magnitude in wounds of plums incubated at 24oC and 4oC. Our results indicate that plum surfaces harbour several yeast species, with excellent potential for use in biological control of brown rot of stone fruits.

Raspberries are a delicate, high-value crop with an extremely short shelf life exacerbated by postharvest decay caused by Botrytis cinerea Pers. European red raspberry (Rubus idaeus L.) is the most widely grown variety. Yellow (R. idaeus L.), black (R. occidentalis L.), and purple raspberries (R. ×neglectus Peck. or R. occidentalis ×idaeus hybrids) are available mainly at local markets and U-pick farms. To compare the postharvest quality of the raspberry color groups, pesticide-free fruit from cultivars and breeding selections of red, yellow, purple, and black raspberries were examined for oxygen radical absorbance capacity (ORAC), phenolics, anthocyanins, soluble solids, titratable acids, pH, color, firmness, decay and juice leakage rates, ethylene evolution, and respiration. There were significant correlations between decay rate and physiochemical properties. Both decay and leakage rates were correlated with weather conditions before harvest, but each color group responded differently to different weather factors. There were no correlations among changes in color, firmness, decay, or juice leakage rates. All the other color groups were less acidic than the familiar red raspberry. Yellow raspberries had the worst decay rates but the best leakage rates. Black and purple raspberries, with the highest phenolics and anthocyanins and the lowest ethylene evolution rates, resisted decay the longest but bled soonest.

Monilinia fructicola, the causal agent of brown rot, was recovered from decayed stone fruits (peach, plum and nectarine) in 11 West Virginia orchards. There was significant variation among these isolates with respect to colony morphology, growth rate, sporulation level, sensitivity to fenbuconazole, vegetative compatibility and virulence. Species identification was confirmed using ITS sequences from the nuclear ribosomal RNA gene. The cultural phenotypes on potato dextrose agar (PDA) ranged from white to dark, melanized colonies. The growth rate of the isolates on PDA ranged from 0.3 to 3.2 mm day−1 at 4 °C, from 2.9 to 7.6 mm day−1 at 10 °C, and from 4.8 to 19 mm day−1 at 24 °C. There was a statistically significant relationship between the growth of the isolates on PDA and their aggressiveness on nectarines at 24 °C, especially at higher inoculum concentrations. Sporulation of 3-day-old cultures on peach agar at 24 °C varied from profuse to no sporulation, with some isolates sporulating only sp...

Calcium has been shown to enhance stress tolerance, maintain firmness and reduce decay in fruits. Previously we reported that seven tomato SlSRs encode calcium/calmodulin-regulated proteins, and that their expressions are developmentally regulated during fruit development and ripening, and are also responsive to ethylene. To study their expressions in response to stresses encountered during postharvest handling, tomato fruit at the mature-green stage was subjected to chilling and wounding injuries, infected with Botrytis cinerea and treated with salicylic acid or methyl jasmonate. Gene expression studies revealed that the seven SlSRs differentially respond to different stress signals. SlSR2 was the only gene upregulated by all the treatments. SlSR4 acted as a late pathogen-induced gene; it was upregulated by salicylic acid and methyl jasmonate, but downregulated by cold treatment. SlSR3L was cold- and wound-responsive and was also induced by salicylic acid. SlSR1 and SlSR1L were repressed by cold, wounding and pathogen infection, but were upregulated by salicylic acid and methyl jasmonate. Overall, results of these expression studies indicate that individual SlSRs have distinct roles in responses to the specific stress signals, and SlSRs may act as a coordinator(s) connecting calcium-mediated signaling with other stress signal transduction pathways during fruit ripening and storage.

Fruit microflora have been the richest source of antagonists against fruit decays and the active ingredient in all currently available commercial biocontrol products. A comprehensive evaluation of plum bacteria for biocontrol activity against Monilinia fructicola , which causes brown rot of stone fruit, is important for determining their biocontrol potential. Resident culturable bacterial microflora of plums from early fruit development until maturity were characterized. The most dominant genera were Curtobacterium (19.88%), Pseudomonas (15.06%), Microbacterium (13.86%), and Clavibacter (12.65%). These genera occurred at all four isolation times and accounted for 61.45% of all isolates. Microbacterium and Curtobacterium dominated at the early stage of fruit development while Pseudomonas and Clavibacter were dominant at the end of the season. Less prevalent genera were Enterobacter (5.42%), Chrysomonas (4.82%), and Pantoea (4.22%). Most frequently isolated species were Microbacterium lacticum , Clavibacter michiganensis , Curtobacterium flaccumfaciens , Enterobacter intermedius , and Chrysomonas luteola. The seasonal succession of genera was observed in both MANOVA and frequency analysis. Primary and secondary screening of plum-inhabiting bacteria for control of brown rot on wounded fruit resulted in selection of several antagonists among which Pantonea agglomerans and Citrobacter freundii were the most effective. These antagonists grew well in plum wounds and increased by four log units during first three days at 24 °C, and two log units after seven days at 4 °C. Results indicate that plum microflora are an excellent source of antagonists against brown rot decay originating from wounds after harvest.

Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that have an important role in plant development and adaptation to environmental changes. In this study, we identified and characterized a spermidine hydroxycinnamoyl transferase (SHT) from eggplant (Solanum melongena) and its wild relative S. richardii, designated as SmSHT and SrSHT, respectively. SmSHT was abundant in flowers and fruits, whereas the level of SrSHT was remarkably low in all tissues. Heat-shock/drought treatment stimulated the expression of SmSHT in both leaves and fruits, indicating its involvement in plant stress response. Both SHT polypeptides had extremely high identity with just five amino-acid substitutions. Recombinant SmSHT catalyzed the synthesis of mono-, bi- and tri- acylated polyamines. Using caffeoyl-CoA as the acyl donor, SmSHT preferred spermidine as the acyl acceptor. When spermidine was the acyl acceptor, the donor preference order for SmSHT was caffeoyl-CoA>feruloyl-CoA>ρ-coumaroyl-CoA. SrSHT exhibited the same substrate specificity as SmSHT, yet exhibited significantly higher catalytic activity than SmSHT. For example, under caffeoyl-CoA and spermidine, Kcat of SrSHT was 37.3% higher than SmSHT. Molecular modeling suggests that five amino-acid substitutions in SrSHT result in four alterations in their predicted 3D structures. In particular, the conserved Lys402 adjacent to the DFGWG motif, and Cys200 in the crossover loop in SmSHT were replaced by Glu and Ser in SrSHT. These substitutions may contribute to the enhanced activity in SrSHT. Our study provides a platform to generate HCAA rich fruits for eggplant and other solanaceous crops.

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.

Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that have an important role in plant development and adaptation to environmental changes. In this study, we identified and characterized a spermidine hydroxycinnamoyl transferase (SHT) from eggplant (Solanum melongena) and its wild relative S. richardii, designated as SmSHT and SrSHT, respectively. SmSHT was abundant in flowers and fruits, whereas the level of SrSHT was remarkably low in all tissues. Heat-shock/drought treatment stimulated the expression of SmSHT in both leaves and fruits, indicating its involvement in plant stress response. Both SHT polypeptides had extremely high identity with just five amino-acid substitutions. Recombinant SmSHT catalyzed the synthesis of mono-, bi- and tri- acylated polyamines. Using caffeoyl-CoA as the acyl donor, SmSHT preferred spermidine as the acyl acceptor. When spermidine was the acyl acceptor, the donor preference order for SmSHT was caffeoyl-CoA>feruloyl-CoA>ρ-coumaroyl-CoA. SrSHT exhibited the same substrate specificity as SmSHT, yet exhibited significantly higher catalytic activity than SmSHT. For example, under caffeoyl-CoA and spermidine, Kcat of SrSHT was 37.3% higher than SmSHT. Molecular modeling suggests that five amino-acid substitutions in SrSHT result in four alterations in their predicted 3D structures. In particular, the conserved Lys402 adjacent to the DFGWG motif, and Cys200 in the crossover loop in SmSHT were replaced by Glu and Ser in SrSHT. These substitutions may contribute to the enhanced activity in SrSHT. Our study provides a platform to generate HCAA rich fruits for eggplant and other solanaceous crops.

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.

Strawberry powdery mildew, caused by Podosphaera aphanis, significantly reduces fruit yield and quality and predisposes fruit to other diseases. Fungicides have been routinely used to control this disease; however, their limitations necessitate the development of alternative approaches, especially for protective culture and organic production. A disease-control strategy was developed that combines treating strawberry plants with a low dose of UV-C (low dose/short duration) followed by a specific period of darkness, which greatly increases the lethality of UV-C. A leaf disc assay was developed to evaluate the effectiveness of the UV-C treatment in controlling powdery mildew on strawberry. Irradiation for only 15 s by UV-C lamps with an output of 20.6 µW cm−2 followed by a 4-h dark period resulted in a significant decrease (P = 0.05) in P. aphanis as determined by the percentage of the leaf disc quadrants colonized by the fungus. An increase in irradiation exposure to 60 s followed by 4-h dark period resulted in complete control of the powdery mildew in most cases. Such treatment of strawberry plants once a week for 3 weeks resulted in more than a four-fold reduction (P = 0.01) of conidial production on adaxial leaf surfaces exposed to the UV-C irradiation, and did not affect leaf photosynthesis (P = 0.05). The UV-C treatment of plants over 15 weeks reduced the amount of diseased fruit and increased fruit yield and quality (P = 0.05).

Cellulose is the most abundant form of carbon on the planet. Breakdown of cellulose microfibrils in the plant cell wall is a means by which microbes gain ingress into their respective hosts. Cellulose degradation is also important for global carbon recycling and is the primary substrate for production of biofuels. In this study, we developed a cellulase assay method that rivals the commonly used dinitrosalicylic acid (DNS) assay. It was shown that the 2-cyanoacetamide method is capable of detecting D-glucose in a linear fashion, can function in various buffers at pH ranging from 4.0 to 8.0, and is as sensitive as the DNS test at detecting fungal cellulase activity using carboxymethylcellulose as a substrate. This method will be useful to others who desire to accurately and efficiently assay microbial cellulase activity without the use of phenol and other highly toxic and corrosive chemicals.

The influence of carbon, nitrogen and pH on polygalacturonase activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized 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 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 P. 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 signaling pathways that modulate nutrient acquisition, uptake and metabolism.

Biological control of fruit decays originating from wound infections after harvest has made great progress during the past two decades and several products are commercially available. However, this is not the case for postharvest decays originating from latent infections which occur in the orchard. This is mainly due to the lack of methodology used to screen and evaluate microbes for biocontrol activity against this type infection. We hypothesized that biological control of latent infections occurs in nature on fruit and that microorganisms involved could be isolated from the fruit and used for control of this type of infection after harvest. We found that direct interactions between the pathogen structure involved in latent infection (appressorium) and the test microorganism can be studied in vitro and then tested in situ on fruit. Appressoria of the brown rot pathogen, Monilinia fructicola , were produced on parafilm or wax membranes after depositing drops containing conidia in a 0.25 mM sucrose solution containing 10 mM cAMP and incubating at 18 °C for 16 h. This was followed by the addition of the test organism, additional incubation at 24 °C for 72 h, and microscopic observations. Microorganisms colonizing appressoria and mycelium were further tested for biocontrol activity on fruit containing latent infections induced artificially under laboratory conditions. Among 904 microorganisms tested, using this approach, we found several effective antagonists against brown rot originating from latent infections on stone fruits. The next step is to select those antagonists that are best adapted to conditions occurring during storage and handling of the fruit. This approach may also be adapted to develop biocontrol of latent infections in other fruit/pathogen systems.

Penicillium expansum and Colletotrichum acutatum cause blue mold and bitter rot of apples during storage which results in significant economic losses. Resistance to these pathogens in commercial apple cultivars has not been documented in the literature. An apple germplasm collection, from the center of origin in Kazakhstan, is maintained in Geneva, New York. This collection represents a more diverse apple gene pool than commercial cultivars which was evaluated for resistance to blue mold and bitter rot. Resistance reactions were skewed toward susceptibility for both fungi and comprised the majority of accessions examined. However, resistance to P. expansum was confirmed in select accessions over multiple years. Maturation patterns and quality indices for soluble solids and acidity, which also may affect pathogenicity, were highly variable and represent the genetic diversity of the germplasm collection. Resistance in four accessions to C. acutatum and two accessions resistant to both P. expansum and C. acutatum, are reported here for the first time. Data from this study will serve as a foundation for conventional apple breeding programs and molecular genetics investigations to provide resistance against blue mold and bitter rot in commercial apple varieties.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, has the potential to be an economic threat to U.S. soybean production after its arrival to the continental United States in 2004. The use of fungicides to control SBR may be problematic due to the large acreage that needs to be protected, the high costs of fungicides, and the cost of application. Cultural practices such as the use of reduced seed rates, increased row widths, and row orientation to the sun have been prescribed as environmental modifications that create a microclimate less conducive to foliar disease development. Therefore, our objective was to determine the influence of different periods of leaf wetness and respective microenvironments on infection and rust development on soybean plants in the field. A misting irrigation system was developed and applied on MGV soybean for 1 min every 30 min for 0-, 6-, 12-, and 18-h periods. This study indicates that extended periods of leaf wetness (18 h) increase disease severity and the rate of spread of the disease in the upper canopy. These results, in combination with spore monitoring, may be used to refine models of pathogen reproduction, prediction, and risk in a certain regions.

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 approximate to 30% reduction in mass of pear plugs compared with approximate to 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.

Jurick, W. M., II, Vico, I., McEvoy, J. L., Whitaker, B. D., Janisiewicz, W., and Conway, W. S. 2009. Isolation, purification, and characterization of a polygalacturonase produced in Penicillium solitum-decayed ‘Golden Delicious’ apple fruit. Phytopathology 99:636-641. Polygalacturonase (PG) was extracted and purified from decayed ‘Golden Delicious’ apple fruit inoculated with Penicillium solitum. Ammonium sulfate, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. The purified PG most likely originates from the fungus because PG activity from healthy and wounded apple tissue was undetectable. Analysis of cation exchangepurified material using sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 50-kDa band. The enzyme was active over a broad pH range (3 to 7), with optimal activity between pH 4 and 5. PG was highly active at 20 and 37°C but was also detectable at 2, 50, and 75°C. Divalent cations affected PG enzyme activity; Mg and Fe increased, whereas Ca and Mn reduced activity in vitro. Thin-layer chromatographic separation of hydrolysis products and data from a PG plate activity assay based on staining with ruthenium red showed that the enzyme exhibits both exo and endo activity. Purified PG incubated with intact apple fruit tissue in vitro caused a 30% reduction in mass after 48 h, suggesting a role in P. solitum-mediated decay of apple fruit.

Strawberries are available throughout the year either from production in the field or from high and low tunnel culture. Diversity of production conditions results in new challenges in controlling diseases before and after harvest. Fungicides have traditionally been used to control these diseases; however, their limitations necessitate a search for new approaches. We found that UV-C irradiation of Botrytis cinerea, a major pathogen of strawberry, can effectively kill this fungus if a dark period follows the treatment. The inclusion of a 4-h dark period resulted in almost complete kill of B. cinerea conidia on agar media at a dose of 12.36 J/m2. The UV-C dose did not cause a reduction in photosynthesis in strawberry leaves or discoloration of sepals, even after exposing plants repeatedly (twice a week) for 7 weeks. Although irradiation of dry conidia of B. cinerea with this dose resulted in some survival, the conidia were not infective and not able to cause decay even when inoculated onto a highly susceptible mature apple fruit. Irradiation of strawberry pollen at 12.36 J/m2 did not affect pollen germination, tube growth and length in vitro, or germination and tube growth in the style of hand-pollinated emasculated strawberry flowers. No negative effect of the UV-C treatment was observed on fruit yield and quality in high tunnel culture. In the fruit and flower petal inoculation tests, the UV-C treatment was highly effective in reducing fruit decay and petal infection. This UV-C treatment with an exposure time of 60 s may be useful in controlling gray mold in tunnel production of strawberries and may also have the potential for use in intensive field and indoor production of other fruits and vegetables providing that a 4-h dark period follows the irradiation.

Many monocot plants grown in soils amended with silicon (Si) exhibit increased levels of resistance to fungal diseases. Silicon-mediated resistance to rice blast disease, caused by Magnaporthe grisea, has been hypothesized to be the result of a mechanical barrier produced from Si polymerization in planta. The present study examined the cytological and molecular influences of Si amendment during genetically defined incompatible (resistant) and compatible (susceptible with no major resistance genes) interactions of rice with M. grisea. Differential accumulation of glucanase, peroxidase, and PR-1 transcripts were associated with limited colonization by M. grisea in epidermal cells of Si+ plants of the susceptible cultivar M201. Katy, a resistant cultivar, responded to an avirulent race of M. grisea through the development of a hypersensitive response along with a strong induction of PR-1 and peroxidase transcripts independent of Si amendment. These findings support an active participation, distinct from single-gene-defined resistance, for Si in the defense of rice against M. grisea. While not discounting a physical barrier role for Si, new insights into a potential active role for this ubiquitous element during non-genetically defined rice-blast resistance is suggested.

Increased cAMP levels inhibit sclerotial development and raise oxalate production in Sclerotinia sclerotiorum. These effects were hypothesized to be mediated by protein kinase A (PKA). To test this hypothesis, a pka1 catalytic subunit gene was cloned and disrupted. Southern hybridization confirmed disruption, and northern analysis revealed loss of a wild-type pka1 transcript. Mutant strains were cAMP-responsive, pathogenic, and had wild-type levels of PKA activity. Phylogenetic analyses of pka1 with other fungal pka genes and encoded peptides suggest that filamentous fungi possess two pka paralogs. Multiple lines of evidence presented in this work suggest that a second pka catalytic subunit gene, pka2, exists and contributes the majority of PKA activity in S. sclerotiorum.

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.

Bacterial and yeast antagonists isolated from fruit surfaces have been effective in controlling various post-harvest diseases, and several microbial antagonists have been developed into commercial products. Our knowledge of the fruit microbial community, with the exception of grapes, apples and some citrus fruit, is rudimentary and the potential of the resident yeasts for biocontrol remains largely unknown. We determined the occurrence of yeasts on plum surfaces during fruit development from the pre-hardening stage until harvest for 2 years. A total of 16 species from 13 genera were isolated. Species from three genera, basidiomycetes Rhodotorula (29.5%) and Sporidiobolus (24.7%) and the dimorphic ascomycete genus Aureobasidium (24.7%), constituted 78.7% of all isolations and were recovered throughout fruit development, while Cryptococcus spp. constituted only 6.2% of the total plum isolates. The yeast community in the final sampling was significantly different from the first three samplings, reflecting a rapidly changing fruit habitat during the maturation of fruit. For example, Hanseniaspora, Pichia, Zygosaccharomyces and Wickerhamomyces occurred only on the most mature fruit. Screening of the yeasts for antagonistic activity against Monilinia fructicola, a fungus that causes brown rot, revealed a range of biocontrol activities. Several isolates provided complete control of the decay on plums, challenged with a pathogen suspension of 10(3) conidia/ml and90% of control on fruit inoculated with the pathogen at a concentration 10 times higher. Some of the best antagonists included A. pullulans and R. phylloplana. Populations of both of these antagonists increased rapidly by several orders of magnitude in wounds of plums incubated at 24ºC and 4ºC. Our results indicate that plum surfaces harbour several yeast species, with excellent potential for use in biological control of brown rot of stone fruits.

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.

PP-235, a 4-page illustrated fact sheet by Wayne M. Jurick II, Dario F. Narvaez, Carrie L. Harmon, James J. Marois, David L. Wright, and Philip F. Harmon, describes this fungal plant disease new to the US since 2004, its symptoms, causal organism, disease cycle and epidemiology, diagnosis, and management. Includes links to web-based resources and references. Published by the UF Department of Plant Pathology, July 2007.

Plant diagnosticians routinely process soybean rust samples with one or more of the following diagnostic protocols: visual assessment using a dissecting microscope, an enzyme-linked immunosorbant assay (ELISA), and pathogen-specific PCR (conventional and real-time). Results of these three diagnostic protocols and time requirements for diagnosis were collected for samples with signs (sporulation) of soybean rust (presumed positive) and samples without sporulating rust pustules (presumed negative). Data were collected for samples processed in triplicate at six university plant disease clinics. The effects of sample treatment and storage on results of two diagnostic protocols also were examined. Visual diagnosis required the least amount of time (4 min), ELISA and PCR took approximately six-and-a-half times longer per sample (multiple samples being processed at one time) than a visual diagnosis. ELISA could not detect the pathogen in positive samples after desiccation followed by long-term storage at room temperature and following one or more autoclave treatments for 30 min. However, conventional PCR was capable of detecting P. pachyrhizi-infected plant material following all sample storage regimes. Accepted for publication 9 February 2007. Published 31 May 2007.

Sclerotinia sclerotiorum, a broad host range plant pathogen, produces pigmented, multihyphal sclerotia that are capable of long-term survival. Under favorable conditions, sclerotia carpogenically germinate to give rise to apothecia and forcibly discharged ascospores which serve as the primary source of inoculum in the disease cycle. The molecular regulator(s) of sclerotial development in filamentous fungi are largely unknown; however, pharmacological data has revealed that cyclic AMP (cAMP) negatively regulates sclerotial biogenesis in S. sclerotiorum. Based on this observation, we analyzed the role of cAMP by deleting the single copy adenylate cyclase (AC) sac1 gene from S. sclerotiorum. In culture, cyclic AMP levels in the knock-out (KO1) strain were greatly reduced compared to wild type, the hyphal branching pattern was altered, microconidia (spermatia) were more abundant, and aberrant sclerotia were produced in a concentric pattern. The KO1 strain was pathogenic on mechanically wounded tissues; however, virulence was severely attenuated. The pathogenicity defect on unwounded leaves is attributed to the absence of infection cushions and the attenuated virulence on wounded leaves correlates with the slow growth rate observed in culture. This study presents the first description of an adenylate cyclase mutant that affects both pathogenicity and sclerotial development in a broad host range necrotroph.