Interannual dynamics of Tuber melanosporum and fungal communities in productive black truffle orchards amended with truffle nests.

Truffle growers devote great efforts to improve black truffle productivity, developing agronomic practices such as 'truffle nests' (peat amendments that are supplemented with truffle spore inoculum). It has been hypothesized that improved fruiting associated with nests is linked to stimulation of truffle mycelia previously established in soil or to changes generated in soil fungal community. To assess this, we used real-time PCR to quantify black truffle extraradical mycelium during 2 years after nests installation. We also characterized the fungal community via high-throughput amplicon sequencing of the ITS region of rRNA genes. We found that neither the abundance of truffle mycelium in nests nor in the soil-nest interphase was higher than in the bulk soil, which indicates that nests do not improve mycelial growth. The fungal community in nests showed lower richness and Shannon index and was compositionally different from that of soil, which suggests that nests may act as an open niche for fungal colonization that facilitates truffle fruiting. The ectomycorrhizal fungal community showed lower richness in nests. However, no negative relationships between amount of truffle mycelium and reads of other ectomycorrhizal fungi were found, thus countering the hypothesis that ectomycorrhizal competition plays a role in the nest effect.

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.

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.

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.

Genetic Diversity and Vegetative Compatibility of Fusarium solani Species Complex of Strawberry in Spain.

Fusarium solani is a soilborne fungus that is a pathogen to >100 plant species. It is the causal agent of crown and root rot in strawberry. We collected 100 F. solani isolates from diseased plants and soils from two distinct geographic areas of strawberry production in Spain: plant nurseries located in the north-central region of the country and fruit production fields located in the southwestern region. The aims of this study were to accurately identify the isolates within the Fusarium solani species complex (FSSC) based on multilocus sequence typing, determine the genetic diversity and population structure of strawberry-associated FSSC based on phylogenetic analysis, and determine the vegetative compatibility among isolates in both strawberry production areas. Seven phylogenetic species, restricted to clade 3 of FSSC, were defined in the Spanish strawberry crops, showing a regional variation of species composition. Isolates from nurseries were composed of four phylogenetic species (i.e., FSSC 2, FSSC 5, FSSC 9, and an unknown FSSC species) that matched with five vegetative compatibility groups (VCGs). Isolates from fruit production fields included five phylogenetic species (i.e., FSSC 2, FSSC 3 + 4, FSSC 5, FSSC 6, and FSSC 11) distributed into 29 VCGs not correlated with phylogenetic groups. FSSC 5 and FSSC 2 were the most abundant species in nurseries and fruit production fields, respectively, and they were the only species present in both production areas. Of the 47 sequence-based haplotypes defined, no haplotypes were shared between nurseries and fruit production fields. Pathogenic isolates were present in all but FSSC 6 and FSSC 9 species, and FSSC 3 + 4 contained the higher percentage of pathogenic isolates. No relationship was observed between pathogenicity and the source of isolates (plant or soil). Generally, species present in fruit production fields showed higher genetic diversity than those present in nurseries. This work can contribute to understanding the diversity of this species complex in Spanish strawberry production areas, which will be useful for developing integrated disease management strategies.

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.

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.