Monilinia fructicola genes involved in the cell wall-degrading process in early nectarine infection.

Brown rot symptoms may be linked to alterations in the gene expression pattern of genes associated with cell wall degradation. In this study, we identify key carbohydrate-active enzymes (CAZymes) involved in cell wall degradation by Monilinia fructicola, including pme2 and pme3 (pectin methylesterases), cut1 (cutinase) and nep2 (necrosis-inducing factor). The expression of these genes is significantly modulated by red and blue light during early nectarine infection. The polygalacturonase gene pg1 and the cellulase gene cel1 also exhibit photoinduction albeit to a lesser extent. Red and blue light cause an acceleration in the initial stages of brown rot development caused by M. fructicola on nectarines. Disease symptoms like tissue maceration were evident after an incubation period of 24 h followed by 14 h of light exposition, in contrast to the usual incubation period of 48 to 72 h. Furthermore, the culture media exerts an impact on gene regulation, suggesting a complex interplay between light and nutrient signalling pathways in M. fructicola. In addition, we observe that red light promotes colony growth on a 12 h photoperiod and consistently reduces conidiation. In contrast, blue light hampers growth rate on both the 12 h and the 8 h photoperiod but only diminishes conidiation on the 12 h photoperiod. These findings enhance our comprehension of genes associated with cell wall degradation and the environmental factors influencing brown rot development.

Monilinia fructicola Response to White Light.

Light represents a powerful signal for the regulation of virulence in many microbial pathogens. Monilinia fructicola is the most virulent species causing brown rot in stone fruit crops. To understand the influence of light on M. fructicola, we measured the effect of white light and photoperiods on the colonial growth and sporulation of the model M. fructicola strain 38C on solid cultures. Searches in the M. fructicola 38C genome predicted a complete set of genes coding for photoreceptors possibly involved in the perception of all ranges of wavelengths. Since white light had an obvious negative effect on vegetative growth and the asexual development of M. fructicola 38C on potato dextrose agar, we studied how light influences photoresponse genes in M. fructicola during early peach infection and in liquid culture. The transcriptomes were analyzed in "Red Jim" nectarines infected by M. fructicola 38C and subjected to light pulses for 5 min and 14 h after 24 h of incubation in darkness. Specific light-induced genes were identified. Among these, we confirmed in samples from infected fruit or synthetic media that blue light photoreceptor vvd1 was among the highest expressed genes. An unknown gene, far1, coding for a small protein conserved in many families of Ascomycota phylum, was also highly induced by light. In contrast, a range of well-known photoreceptors displayed a low transcriptional response to light in M. fructicola from nectarines but not on the pathogen mycelium growing in liquid culture media for 6 days.

A Secondary Metabolism Pathway Involved in the Production of a Putative Toxin Is Expressed at Early Stage of Monilinia laxa Infection.

The necrotrophic pathogenic fungus Monilinia laxa causes brown rot disease on stone fruit generating significant yield losses. So far, a limited number of pathogenesis-related virulence factors, such as cell wall degrading enzymes and potential phytotoxins, have been described in Monilinia spp. Using RNA-sequencing data from highly virulent M. laxa ML8L strain at early stages of the infection process (6, 14, 24, and 48 h post-inoculation, hpi) on nectarine and the Pathogen-Host-Interactions (PHI) database, we selected a number of genes for further study and ranked them according to their transcription levels. We identified a class of genes highly expressed at 6 hpi and that their expression decreased to almost undetectable levels at 14 to 48 hpi. Among these genes we found Monilinia__061040 encoding a non-ribosomal peptide synthase (NRPS). Monilinia__061040 together with other five co-regulated genes, forms a secondary metabolism cluster potentially involved in the production of epipolythiodioxopiperazine (ETP) toxin. Quantitative-PCR data confirmed previous RNA sequencing results from the virulent ML8L strain. Interestingly, in a less virulent M. laxa ML5L strain the expression levels of this pathway were reduced compared to the ML8L strain during nectarine infection. In vitro experiments showed that liquid medium containing peach extract mimicked the results observed using nectarines. In fact, upregulation of the NRPS coding gene was also observed in minimal medium suggesting the existence of a fruit-independent mechanism of regulation for this putative toxin biosynthetic pathway that is also downregulated in the less virulent strain. These results emphasize the role of this secondary metabolism pathway during the early stage of brown rot disease development and show alternative models to study the induction of virulence genes in this fungus.

Comparative analysis of Penicillium genomes reveals the absence of a specific genetic basis for biocontrol in Penicillium rubens strain 212.

Penicillium rubens strain 212 (PO212) is a filamentous fungus belonging to the division Ascomycete. PO212 acts as an effective biocontrol agent against several pathogens in a variety of horticultural crops including Fusarium oxysporum f.sp. lycopersici, causing vascular wilt disease in tomato plants. We assembled draft genomes of two P. rubens strains, the biocontrol agent PO212 and the soil isolate S27, which lacks biocontrol activity. We also performed comparative analyses of the genomic sequence of PO212 with that of the other P. rubens and P. chrysogenum strains. This is the first Penicillium strain with biocontrol activity whose genome has been sequenced and compared. PO212 genome size is 2,982 Mb, which is currently organized into 65 scaffolds and a total of 10,164 predicted Open Reading Frames (ORFs). Sequencing confirmed that PO212 belongs to P. rubens clade. The comparative analysis of the PO212 genome with the genomes of other P. rubens and Penicillium chrysogenum strains available in databases showed strong conservation among genomes, but a correlation was not found between these genomic data and the biocontrol phenotype displayed by PO212. Finally, the comparative analysis between PO212 and S27 genomes showed high sequence conservation and a low number of variations mainly located in ORF regions. These differences found in coding regions between PO212 and S27 genomes can explain neither the biocontrol activity of PO212 nor the absence of such activity in S27, opening a possible avenue toward transcriptomic and epigenetic studies that may shed light on this mechanism for fighting plant diseases caused by fungal pathogens. The genome sequences described in this study provide a useful novel resource for future research into the biology, ecology, and evolution of biological control agents.

Epidemiological Studies of Brown Rot in Spanish Cherry Orchards in the Jerte Valley.

Cherry brown rot caused by Monilinialaxa was observed and estimated in organic cherry orchard located in the Jerte Valley between 2013 and 2018 (Cáceres, Spain). Climatic variables were collected from this orchard and also from a nearby weather station. The primary inoculum of the pathogen recorded in March was detected in overwintered mummified fruits, ground mummies, and necrotic twigs and was a function of the average temperature of the previous three months (December, January, and February). The first symptoms of brown rot could be observed on flowers until fruit set in April. The months of March and April were identified as the critical period for cherry brown-rot development. A significant positive correlation was identified between brown rot observed at harvest and the mean number of consecutive days in each fortnight of March and April when the percent relative humidity was above 80%. Brown-rot incidence observed over the 6 years ranged from 0 to 38%. More than 11 days with relative humidity >80% in each fortnight of critical period would mean 100% of cherry brown rot at harvest. A forecasting model could be used to predict brown rot infection in Jerte Valley cherries.

Light-Photoreceptors and Proteins Related to Monilinia laxa Photoresponses.

Light represents a ubiquitous source of information for organisms to evaluate their environment. The influence of light on colony growth and conidiation was determined for three Monilinia laxa isolates. The highest mycelial growth rate was observed under red light for the three M. laxa isolates, followed by green light, daylight or darkness. However, reduced sporulation levels were observed in darkness and red light, but conidiation enhancement was found under daylight, black and green light with more hours of exposure to light. Putative photoreceptors for blue (white-collar and cryptochromes), green (opsins), and red light (phytochromes) were identified, and the photoresponse-related regulatory family of velvet proteins. A unique ortholog for each photoreceptor was found, and their respective domain architecture was highly conserved. Transcriptional analyses of uncovered sets of genes were performed under daylight or specific color light, and both in time course illumination, finding light-dependent triggered gene expression of MlVEL2, MlPHY2, MlOPS2, and MlCRY2, and color light as a positive inductor of MlVEL3, MlVEL4, MlPHY1, and MlCRY1 expression. M. laxa has a highly conserved set of photoreceptors with other light-responsive fungi. Our phenotypic analyses and the existence of this light-sensing machinery suggest transcriptional regulatory systems dedicated to modulating the development and dispersion of this pathogen.

Effect of chemical alternatives to methyl bromide on soil-borne disease incidence and fungal populations in Spanish strawberry nurseries: A long-term study.

BACKGROUND: Chloropicrin (PIC) mixtures of 1,3-dichloropropene and chloropicrin (DD:PIC), dazomet, and metam sodium (MS) have been applied as chemical alternatives to methyl bromide (MB) in Spanish strawberry nurseries since MB was banned as a soil fumigant in 2005. These chemical alternatives were applied to soil in two Spanish strawberry nurseries between 2003 and 2017 to test their efficacy against the main crown and root disease and soil fungal populations in comparison with the use of MB and PIC (MB:PIC). These chemicals were applied at several doses with different application methods under plastic films. Crown and root disease incidence was calculated as the percentage of plants with symptoms caused by soil-borne pathogens. Soil fungal populations were estimated as colony forming units per gram of dry soil. RESULTS: All chemicals significantly reduced soil-borne fungal disease incidence and fungal population in both nurseries over the years. Phytophthora cactorum and Fusarium spp. were the main pathogens causing soil-borne diseases, followed by Verticillium spp. MB:PIC remained the treatment that best controlled P. cactorum. MS and DD:PIC controlled Fusarium disease to a lesser extent than MB:PIC and dazomet in both nurseries. MB:PIC and PIC were the two treatments that most reduced Verticillium spp. The population of Verticillium spp. declined and the presence of other species such as Colletotrichum spp. and Rhizoctonia spp. was minimal during the study. CONCLUSION: Chemicals are necessary to obtain healthy strawberry plants. The use of chemical alternatives to MB has resulted in changes in the incidence of soil-borne diseases and soil fungal populations in strawberry nurseries. Dazomet was an effective alternative to MB as a soil-borne disease control, except against Verticillium spp. MB alternatives in strawberry nursery soils have caused Fusarium spp. to displace Verticillium spp.

Impact of Postharvest Handling on Preharvest Latent Infections Caused by Monilinia spp. in Nectarines.

Latent infections caused by Monilinia spp. in nectarines cause great economic losses since they are not detected and rejected at harvest and can appear at any time post-harvest, even at the consumer's home. The effect of a pre-cooling chamber, water dump operation, and cold-storage chamber on the activation and/or development of preharvest latent infections caused by Monilinia spp. on nectarines were studied under different postharvest conditions: (a) cold storage for 0, 1, or 3 d at 4 °C at either 75% relative humidity (RH) or 100% RH before water dumping, (b) water dumping for 10 minutes at 15 °C, and (c) cold storage for 0, 3, or 10 d at 4 °C at either 75% RH or 100% RH after water dumping. These storage conditions were transformed to fungal physiological time. For visualization of the latent infections caused by Monilinia spp., the nectarines were placed in sterile paper bags and frozen at -20 °C for 48 h in order to damage the epidermis. To compare different handling scenarios, the incidence of latent infection was modelled for physiological time description by a modified Gompertz model. The activation and/or development of preharvest natural latent infections caused by Monilinia spp. at postharvest was mainly related to temperature and incubation time at postharvest. Storing nectarines with any postharvest handling less than 11 days at 4 °C avoids brown rot symptoms and reduced the activation and/or development of pre-harvest latent infections caused by Monilinia spp., while more cold days caused the exponential phase of latent infection activation and/or development. The Gompertz model employed could be used for predicting the activation and/or development of latent infection caused by Monilinia spp. at postharvest conditions and looks at the postharvest life. To our knowledge, this is the first time that the effects of post-harvest handling on latent infections in fruit have been studied.

Proteomic Studies to Understand the Mechanisms of Peach Tissue Degradation by Monilinia laxa.

Monilinia laxa is a necrotrophic plant pathogen able to infect and produce substantial losses on stone fruit. Three different isolates of M. laxa were characterized according to their aggressiveness on nectarines. M. laxa 8L isolate was the most aggressive on fruit, 33L isolate displayed intermediated virulence level, and 5L was classified as a weak aggressive isolate. Nectarine colonization process by the weak isolate 5L was strongly delayed. nLC-MS/MS proteomic studies using in vitro peach cultures provided data on exoproteomes of the three isolates at equivalent stages of brown rot colonization; 3 days for 8L and 33L, and 7 days for 5L. A total of 181 proteins were identified from 8L exoproteome and 289 proteins from 33L at 3 dpi, and 206 proteins were identified in 5L exoproteome at 7 dpi. Although an elevated number of proteins lacked a predicted function, the vast majority of proteins belong to OG group "metabolism", composed of categories such as "carbohydrate transport and metabolism" in 5L, and "energy production and conversion" most represented in 8L and 33L. Among identified proteins, 157 that carried a signal peptide were further examined and classified. Carbohydrate-active enzymes and peptidases were the main groups revealing different protein alternatives with the same function among isolates. Our data suggested a subset of secreted proteins as possible markers of differential virulence in more aggressive isolates, MlPG1 MlPME3, NEP-like, or endoglucanase proteins. A core-exoproteome among isolates independently of their virulence but time-dependent was also described. This core included several well-known virulence factors involved in host-tissue factors like cutinase, pectin lyases, and acid proteases. The secretion patterns supported the assumption that M. laxa deploys an extensive repertoire of proteins to facilitate the host infection and colonization and provided information for further characterization of M. laxa pathogenesis.

Exploring the Extracellular Macromolecular Composition of Crude Extracts of Penicillium rubens Strain 212 for Elucidation Its Mode of Action as a Biocontrol Agent.

Penicillium rubens strain 212 (PO212) acts as an inducer of systemic resistance in tomato plants. The effect of crude extracellular extracts of PO212 on the soil-borne pathogen Fusarium oxysporum f. sp. lycopersici has been evaluated. Evidence of the involvement of soluble, thermo-labile, and proteinase-inactivated macromolecules present in PO212 crude extracts in the control of Fusarium vascular disease in tomato plants was found. Proteomic techniques and the availability of the access to the PO212 genome database have allowed the identification of glycosyl hydrolases, oxidases, and peptidases in these extracellular extracts. Furthermore, a bioassay-guided fractionation of PO212 crude extracellular extracts using an integrated membrane/solid phase extraction process was set up. This method enabled the separation of a PO212 crude extracellular extract of seven days of growth into four fractions of different molecular sizes and polarities: high molecular mass protein fraction >5 kDa, middle molecular mass protein fraction 5-1 kDa, low molecular mass metabolite fraction, and nutrients from culture medium (mainly glucose and minerals). The high and middle molecular mass protein fractions retained disease control activity in a way similar to that of the control extracts. Proteomic techniques have allowed the identification of nine putatively secreted proteins in the high molecular mass protein fraction matching those identified in the total crude extracts. Therefore, these enzymes are considered to be potentially responsible of the crude extracellular extract-induced resistance in tomato plants against F. oxysporum f. sp. lycopersici. Further studies are required to establish which of the identified proteins participate in the PO212's action mode as a biocontrol agent.

Balance between resilient fruit surface microbial community and population of Monilinia spp. after biopesticide field applications of Penicillium frequentans.

The microbial variability on the host plant surface must be maintained because population diversity and quantity are essential to avoid disease development. It would be necessary to examine the patterns and mechanisms associated with the massive and reiterative introduction of a microbial pest control agent. The effect of inundative releases of biopesticide formulations containing Penicillium frequentans for the control of Monilinia spp. populations, and the effect on fruit surface microbiota on 18 stone fruit field experiments located in four European countries for more than two crop seasons against brown rot were studied. P. frequentans was monitored after application in order to assess whether it was persistent or not in the environment. Hydrolysis of fluorescein diacetate and denaturing gradient gel electrophoresis were used to study the effects of P. frequentans on fungal and bacterial non-target populations on fruit surface. The effect of P. frequentans formulations on the populations of Monilinia spp. on fruit was also assessed in different orchards. P. frequentans population on stone fruit surfaces showed ranged from 100 to 10,000 CFU cm-2, and postharvest recovered populations were more than 10-100-fold higher than preharvest recovered populations. The population of P. frequentans varied among orchards and years, rather than by the type of formulation. P. frequentans formulation reduced Monilinia spp. population and brown rot and latent infections caused by this pathogen both before and at harvest, while stabilizing or increasing antagonist populations and avoiding non-target microorganisms. However, fungicides reduced significantly the microbial activity on nectarine surfaces.

Pectin as Carbon Source for Monilinia laxa Exoproteome and Expression Profiles of Related Genes.

Pectin, as part of the fruit cell wall, can be degraded by brown rot fungi by coordinating the production, secretion, and action of extracellular enzymes. In this study, pectin utilization by the necrotroph Monilinia laxa 8L was studied by in vitro and in silico approaches. A total of 403 genes encoding carbohydrate-active enzymes (CAZymes) were identified, including 38 coding a predicted pectin-degrading activity. Analyzing the differences between M. laxa 8L exoproteomes in media containing glucose and pectin as sole carbon sources, we identified 107 pectin-specific proteins, among them, 64.48% harbor a classical secretory activity, including 42 CAZymes and six pectin-degrading proteins. Analyzing the gene-expression patterns of some pectinase families revealed their possible sequential action in pectin disassembly. We found, in vitro, an early pectin-dependent induction of MlRGAE1, MlPG1, and three members of the rhamnosidase family (MlαRHA2, MlαRHA3, and MlαRHA6) and late response of MlPG2 and MlPNL3. M. laxa 8L has the ability to use both pectin and byproducts as carbon sources, based on a functional pectinolytic machinery encoded in its genome, subjected to pectin-dependent regulation and appropriate secretion mechanisms of these pectinolytic enzymes. Differences in the secretion and transcription profile of M. laxa 8L provided insights into the different mechanisms that contribute to brown rot development.

Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit.

To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples.

Dispersion, persistence, and stability of the biocontrol agent Penicillium frequentans strain 909 after stone fruit tree applications.

The capacity of dispersion, persistence, and stability from biocontrol agents is essential before these organisms can be developed into a commercial product. Interactions that microorganisms establish with stone fruit trees may be beneficial in the exploitation of trees in agriculture as crop production. The natural background levels of Penicillium frequentans strain 909 dispersion, persistence, and stability were assessed after tree applications and postharvest conditions. A fingerprinting-based approach to trace genetic stability of P. frequentans along stored time and its release in the field was developed. P. frequentans was successfully traced and discriminated. This strain was dispersed well in treated trees, persisting in the ecosystem up to 2 weeks and staying genetically stable after 36 months of storage. However, the dispersal of P. frequentans was very limited on around untreated trees and soil. P. frequentans dispersed randomly into the air, and its presence reduced from the first day to disappear completely at 15-21 days after application. Great losses of P. frequentans and its increased dispersal in open field conditions probably resulted from rainfall. Biological control strategies with Pf909 were discussed.

Transformation of Penicillium rubens 212 and Expression of GFP and DsRED Coding Genes for Visualization of Plant-Biocontrol Agent Interaction.

Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant-fungus interactions and tomato root colonization.

Surfactant effects on wettability of Penicillium frequentans formulations to improve brown rot biocontrol.

BACKGROUND: Penicillium frequentans can be used in the management of brown rot caused by Monilinia spp. Competition is the primary mode of biocontrol activity of P. frequentans, which must therefore cover most of fruit surface to avoid pathogen infection. Our objective was to optimize the efficacy of P. frequentans by maximizing fruit surface coverage and retention with the antagonist formulation by surfactant incorporation. RESULTS: Sixteen surfactants were assessed for the management of brown rot at 3-5 different concentrations. Nine surfactants increased the droplet surface up to 2.5 times compared with water on an inert surface, with or without the presence of P. frequentans in each drop. Eight surfactants increased P. frequentans on the fruit surface, enhancing colony forming units after run off or lateral spray application uptake by 50% compared to the control without surfactants. But only some doses of sodium carboxymethyl cellulose, gelatin, Tween 20, sorbitan alkyl esters, synthetic latex, polyethylene glycol isotridecyl ether, and hydroxypropyl methylcellulose could show the same covered fruit surface after run off or lateral spray application. There were also no phytotoxic side-effects on five different species of stone fruit. CONCLUSIONS: The efficacy of P. frequentans dry conidia can be enhanced by optimizing the composition of the formulation with surfactants. © 2018 Society of Chemical Industry.

Adaptive conditions and safety of the application of Penicillium frequentans as a biocontrol agent on stone fruit.

Penicillium frequentans (Pf909) reduces brown rot caused by Monilinia spp. in stone fruit. The registration of a microbial biocontrol agent in Europe requires information on the risks and safety of a biological product. This study focused on the impact of the physical environment on Pf909 survival and growth, Pf909 mycotoxin production on fruit surface, and the Pf909 resistance to commercial antifungal compounds used in animal and human medicine. The effect of temperature (4 to 37°C), water activity (0.999 to 0.900), pH (3 to 11), light intensity (0, 90 and 180lm) and photoperiod (0/24, 12/12, 16/8, 24/0; light/dark) on mycelial growth and sporulation of Pf909 were monitored for 10days in vitro on culture medium. Antifungal activity of antifungal compounds on mycelial growth of Pf909 was also measured by a quantitative micro spectrophotometric assay after 72h of incubation. The presence or absence of four non-volatile mycotoxins (patulin, penicillic acid, ochratoxin A and citrinin) on nectarine surfaces treated with Pf909 was determined by HPLC. Growth rate was significantly influenced by water activity, temperature and light exposure conditions. Pf909 showed maximum growth and sporulation at 22°C and 25°C, in wet conditions (0.999 water activity), with a pH5.6 to 9, and in darkness or a short light photoperiod. Our results showed all antifungal compounds used reduced significantly Pf909 mycelial growth at tested commercial doses. HPLC data analysis showed that 7days after biofungicide (Pf909) application there were no mycotoxin products on the surface of nectarine. Finally, no phylogenetic relationship has been shown among Pf909 and other species of Penicillium that produce mycotoxins. In conclusion, from an ecological point of view, Pf909 would establish and survive actively over a broad range of climatic conditions. The probability of risks to human and animal health is considered very low.

Microscopic Analyses of Latent and Visible Monilinia fructicola Infections in Nectarines.

Little is known about the histologic features of a latent Monilinia fructicola infection and brown rot in infected fruit. This report informs on the results of an investigation whose aim was to analyze the microanatomy of nectarines with a latent and visible M. fructicola infection. Mature nectarines were inoculated with an M. fructicola isolate and incubated at 25°C for 0, 24, 48, 72, or 96 hours in the dark. For investigating the latent infection process, the inoculated nectarines were first incubated at 25°C for 24 hours in the dark and then incubated at 4°C for 72, 144, 216, and 288 hours in the dark. At the end of the incubation, samples of nectarine tissue were excised from the inoculation points and prepared for light and transmission electron microscopic examinations. No signs of disease were seen on the surface of nectarines with a latent infection over the 288-hour incubation period. When the tissue samples were microscopically examined, M. fructicola colonized the stomata and this stomatal colonization progressively increased over time and was associated with gradual collapse of the epidermal cells and colonization of the subepidermis. In nectarines with visible brown rot, the disease usually appeared after 24 hours on the surface and in the uppermost layers of epidermal cells, which began to collapse after 48 hours. Subsequently, the diseased tissues of the nectarines displayed (a) colonization of the epidermis and mesocarp by M. fructicola with thin and thick hyphae, (b) collapse and disruption of epidermal and mesocarpic cells, (c) lysogenic cavities in the subepidermis and mesocarp, (d) degradation of the cuticle and epidermis, and (e) M. fructicola sporulation. M. fructicola is active during latent infections because slow and progressive colonization of nectarine subcuticular cells by the fungus occurs.

The development of genetic and molecular markers to register and commercialize Penicillium rubens (formerly Penicillium oxalicum) strain 212 as a biocontrol agent.

Penicillium oxalicum strain 212 (PO212) is an effective biocontrol agent (BCA) against a large number of economically important fungal plant pathogens. For successful registration as a BCA in Europe, PO212 must be accurately identified. In this report, we describe the use of classical genetic and molecular markers to characterize and identify PO212 in order to understand its ecological role in the environment or host. We successfully generated pyrimidine (pyr-) auxotrophic mutants. In addition we also designed specific oligonucleotides for the pyrF gene at their untranslated regions for rapid and reliable identification and classification of strains of P. oxalicum and P. rubens, formerly P. chrysogenum. Using these DNA-based technologies, we found that PO212 is a strain of P. rubens, and is not a strain of P. oxalicum. This work presents PO212 as the unique P. rubens strain to be described as a BCA and the information contained here serves for its registration and commercialization in Europe.