A reliable qPCR technique for detecting viable Xanthomonas arboricola pv. pruni cells.

Xanthomonas arboricola pv. pruni (Xap) is the causal agent of bacterial spot of stone fruits and almond (Prunus spp). Detection of Xap is typically carried out using quantitative real-time PCR (qPCR) combined with culture-based isolation. However, qPCR does not differentiate between viable and dead cells, potentially leading to an overestimation of the infective population in a sample. Such overestimation could result in unnecessary phytosanitary measures. The present study aims to develop a specific protocol ideally targeting to detection of only live Xap bacterial cells. To address this challenge, the viable quantitative PCR (v-qPCR) method was evaluated using three nucleic acid-binding dyes: propidium monoazide (PMA), a combination of PMA and ethidium monoazide (EMA), and PMAxx™, an improved version of PMA. PMAxx™ proved to be the most suitable dye for the detection and quantification of living bacterial cells. This methodology was also evaluated in infected plant material over time and can be considered a rapid and reliable alternative to PCR methods for detecting only those putative infective Xap that may pose a risk for Prunus crops. KEY POINTS: • Protocol to detect biofilm and planktonic viable X. arboricola pv. pruni cells. • Host validated protocol. • Benefits, reduction of chemicals in disease control.

Putting 'X' into Context: The Diversity of 'Candidatus Phytoplasma pruni' Strains Associated with the Induction of X-Disease.

Recurrent epiphytotics of X-disease, caused by 'Candidatus Phytoplasma pruni,' have inflicted significant losses on commercial cherry and peach production across North America in the last century. During this period, there have been multiple studies reporting different disease phenotypes and, more recently, identifying different strains through sequencing core genes, but the symptoms have not, to date, been linked with genotype. Therefore, in this study we collected and assessed differing disease phenotypes from multiple U.S. states and conducted multilocus sequence analysis on these strains. We identified a total of five lineages associated with the induction of X-disease on commercial Prunus species and two lineages that were associated with wild P. virginiana. Despite a century of interstate plant movement, there were regional trends in terms of lineages present, and lineage-specific symptoms were observed on P. avium, P. cerasus, and P. virginiana, but not on P. persica. Cumulatively, these data have allowed us to define "true" X-disease-inducing strains of concern to the stone fruit industry across North America, as well as potential sources of infection that exist in the extraorchard environment.

Effect of Different Sterilization Methods on the Microbial and Physicochemical Changes in Prunus mume Juice during Storage.

This study evaluated the pasteurization (P), ozone (O3), ultrasonic (US), and high-hydrostatic-pressure (HHP) sterilization approaches for processing of Prunus mume regarding browning factors and microorganisms, compared with non-sterilization (control check, CK) treatment. The microorganisms (total bacterial count and fungi and yeast count) in the juice were identified after different sterilization techniques, while the quality parameter changes (degree of browning, color measurements, total phenolic content, reducing sugar, ascorbic acid, 5-hydroxymethyl furaldehyde (5-HMF), amino acid nitrogen, total soluble solids (TSS), pH value) were investigated. The results indicate that P and HHP treatment reduced non-enzymatic browning while substantially impacting the color measurements, TSS, and pH, while the sterilization effect was remarkable, with a rate exceeding 90%. Furthermore, the Prunus mume juices treated with P and HHP sterilization were used as the objects, and the CK group was used as the control group. They were placed at 4 °C, 25 °C and 37 °C, respectively, and stored in dark for 15 d. Sampling and determination were carried out on 0, 3, 6, 9, 12, and 15 d, respectively. M-&-Y (molds and yeasts) were not detected in the late storage period, and no obvious microbial growth was observed during storage, indicating that P and HHP treatments could ensure the microbial safety of Prunus mume juice. P- and HHP- treated Prunus mume juice has better quality and low temperature storage is beneficial for maintaining the quality of Prunus mume juice. Therefore, P treatment or HHP treatment combined with low temperature storage could achieve a more ideal storage effect. Overall, this study conclusively established that P and HHP methods were suitable for sterilizing Prunus mume juice. These techniques minimally affected overall product quality while better maintaining the quality parameters than the untreated juice samples and those exposed to O3 and US treatment.

Rhizosphere microorganisms enhance in vitro root and plantlet development of Pyrus and Prunus rootstocks.

MAIN CONCLUSION: The in vitro application of rhizosphere microorganisms led to a higher rooting percentage in Pyrus Py12 rootstocks and increased plant growth of Pyrus Py170 and Prunus RP-20. The rooting of fruit tree rootstocks is the most challenging step of the in vitro propagation process. The use of rhizosphere microorganisms to promote in vitro rooting and plant growth as an alternative to the addition of chemical hormones to culture media is proposed in the present study. Explants from two Pyrus (Py170 and Py12) rootstocks and the Prunus RP-20 rootstock were inoculated with Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 and Phoma sp. PGP03 following two different methods to determine their effects on in vitro rooting and plantlet growth. The effects of the microorganisms on the growth of fully developed Py170 and RP-20 plantlets were also studied in vitro. All experiments were conducted using vermiculite to simulate a soil system in vitro. When applied to Py12 shoots, which is a hard-to-root plant material, both C. ramotenellum PGP02 and Phoma sp. PGP03 fungi were able to increase the rooting percentage from 56.25% to 100% following auxin indole-3-butyric acid (IBA) treatment. Thus, the presence of these microorganisms clearly improved root development, inducing a higher number of roots and causing shorter roots. Better overall growth and improved stem growth of treated plants was observed when auxin treatment was replaced by co-culture with microorganisms. A root growth-promoting effect was observed on RP-20 plantlets after inoculation with C. ramotenellum PGP02, while P. oryzihabitans PGP01 increased root numbers for both Py170 and RP-20 and increased root growth over stem growth for RP-20. It was also shown that the three microorganisms P. oryzihabitans PGP01, C. ramotenellum PGP02 and Phoma sp. PGP03 were able to naturally produce auxin, including indole-3-acetic acid (IAA), at different levels. Overall, our results demonstrate that the microorganisms P. oryzihabitans PGP01 and C. ramotenellum PGP02 had beneficial effects on in vitro rooting and plantlet growth and could be applied to in vitro tissue culture as a substitute for IBA.

Evaluation of Dissipation Behavior, Residues, and Dietary Risk Assessment of Fludioxonil in Cherry via QuEChERS Using HPLC-MS/MS Technique.

The chemical fungicide fludioxonil is widely used to control post-harvest fungal disease in cherries. This study was implemented to investigate the dissipation behaviours and residues of fludioxonil on cherries. A reliable and efficient analytical method was established. Cherry samples from four product areas were analyzed by QuEChERS and HPLC-MS/MS methods with acceptable linearity (R2 > 0.99), accuracy (recoveries of 81-94%), and precision (relative standard deviation of 2.5-11.9%). The limits of quantification (LOQs) and limits of detection (LODs) of cherries were 0.01 mg/kg and 0.005 mg/kg. The dissipation of fludioxonil on cherries followed first order kinetics with half-lives of 33.7-44.7 days. The terminal residues of fludioxonil were all lower than 5.00 mg/kg, which is the MRL recommended by the European Commission. According to Chinese dietary patterns and terminal residue distributions, the risk quotient (RQs) of fludioxonil was 0.61%, revealing that the evaluated cherries exhibited an acceptably low dietary risk to consumers.

Resistance analysis of cherry rootstock 'CDR-1' (Prunus mahaleb) to crown gall disease.

BACKGROUND: Crown gall disease, caused by the pathogenic bacterium Agrobacterium tumefaciens, is responsible for extensive economic losses in orchards. Cherry rootstock 'CDR-1' (Prunus mahaleb) shows high resistance but the mechanism remains unclear. Here, we examined the morphology of pathogen-infected root neck surface, determined the activity of 10 defense-related enzymes and the content of salicylic acid (SA) and jasmonic acid (JA), and also applied transcriptome analysis, transient expression and transgenic verification to explore the crown gall resistance genes in 'CDR-1' plants. RESULTS: In our study, peroxidase increased in the first 10 days, while phenylalanine ammonialyase and lipoxygenase increased in the first 15 days post-infection. Four key enzymes in the AsA-GSH cycle also responded, to a certain extent; although JA content increased significantly after the treatment, the SA content did not. In a follow-up transcriptome analysis, the differentially expressed genes Pm4CL2, PmCYP450, PmHCT1, PmHCT2, and PmCAD were up-regulated. Based on the above results, we focused on the lignin biosynthetic pathway, and further measured lignin content, and found it increased significantly. The Pm4CL2 gene was used to conduct transient expression and transgenic experiments to verify its function in crown gall disease resistance. It showed the relative expression of the treatment group was almost 14-fold that of the control group at 12 h post-treatment. After the infection treatment, clear signs of resistance were found in the transgenic lines; this indicated that under the higher expression level and earlier activation of Pm4CL2, plant resistance was enhanced. CONCLUSIONS: The crown gall resistance of 'CDR-1' is likely related to the lignin biosynthetic pathway, in which Pm4CL2 functions crucially during the plant defense response to the pathogen A. tumefaciens. The results thus offer novel insights into the defense responses and resistance mechanism of cherry rootstock 'CDR-1' against crown gall disease.

The complete genome sequence of Rahnella aquatilis ZF7 reveals potential beneficial properties and stress tolerance capabilities.

Rahnella aquatilis ZF7 is a plant beneficial strain isolated from Sakura tree soil with potential for biocontrol. Here, we present the complete genome sequence of R. aquatilis ZF7, which consists of one 4.49 Mb circular chromosome and a 54-kb plasmid named pRAZF7. Phylogenetic analyses revealed that R. aquatilis ZF7 is much similar to the strains Rahnella sp. Y9602 and R. aquatilis HX2 than others evaluated. In this study, multiple genes encoding functions that likely contribute to plant growth promotion, biocontrol and stress tolerance were identified by comparative genome analyses, including IAA production, phosphate solubilization, antibiotic resistance and formation of Se nanoparticles (SeNPs). In addition, these functions were also confirmed by in vitro experiments. Considering its ability to form SeNPs, strain R. aquatilis ZF7 will contribute to nano-agriculture. Overall, the features of R. aquatilis ZF7 make it a high potential and competitive strain in biocontrol, and the genome data will help further studies on the mechanisms of plant growth promotion and biocontrol.

Ornithinimicrobium cerasi sp. nov., isolated from the fruit of Cerasus pseudocerasus and emended description of the genus Ornithinimicrobium.

Strain CPCC 203383T, isolated from the surface-sterilized fruit of Cerasus pseudocerasus (Lindl.) G. Don, was taxonomically characterized based on a polyphasic investigation. It had the highest 16S rRNA gene sequence similarities with Ornithinimicrobium pekingense DSM 21552 (97.2 %) and O. kibberense DSM 17687T (97.2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain formed a distinct phyletic branch within the genus Ornithinimicrobium and the whole genome sequence data analyses supported that strain CPCC 203383T was phylogenetically related to the Ornithinimicrobium species. The isolate shared a range of phenotypic patterns reported for members of the genus Ornithinimicrobium, but also had a range of cultural, physiological and biochemical characteristics that separated it from related Ornithinimicrobium species. The menaquinone was MK-8(H4). The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI) and unidentified lipids (ULs). The major fatty acids (>5 %) were iso-C15 : 0, anteiso-C15 : 0, iso-C16:0, 9-methyl C16 : 0, iso-C17 : 0 and anteiso-C17 : 0. The cell wall peptidoglycan contains l-ornithine as diagnostic diamino acid and an interpeptide bridge consisting of L-Orn←L-Ala←Gly←D-Asp. The combined genotypic and phenotypic data indicated that the isolate represents a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cerasi sp. nov. is proposed, with CPCC 203383T(=NBRC 113522T=KCTC 49200T) as the type strain. The DNA G+C composition is 72.3 mol%. The availability of new data allows for an emended description of the genus Ornithinimicrobium.

Development of three duplex real-time RT-PCR assays for the sensitive and rapid detection of a phytoplasma and five viral pathogens affecting stone fruit trees.

Three duplex real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) assays based on TaqMan chemistry, were developed for the simultaneous detection and specific quantification of apple chlorotic leafspot virus (ACLSV), plum pox virus (PPV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), peach latent mosaic viroid (PLMVd) and the European stone fruit yellows (ESFY) phytoplasma, which are considered among the most important pathogens affecting stone fruit trees. The quantitative RT-PCR (RT-qPCR) assays were optimized using RNA transcripts (linearized plasmid was used for the assay optimization of the ESFY phytoplasma) of known concentrations. No differences in sensitivity were recorded between the duplex and singleplex RT-qPCR assays. The amplification efficiency of the duplex assays reached 91.1-95.8%, while the linear range of quantification was from 20 to 2 × 107 RNA/linearized plasmid transcripts for PLMVd and ESFY phytoplasma, 40 to 4 × 107 RNA transcripts for ACLSV, PPV and PDV, and 102 to 108 RNA transcripts for PNRSV, respectively. The duplex RT-qPCR assays, which were validated using both characterized isolates from all pathogens and field samples from Prunus species in Northern Greece, exhibited a broad detection range. Overall, the developed methods comprise useful tools that could be applied for the simultaneous and reliable detection of graft-transmissible pathogens in certification programs of Prunus spp.

Inoculum quantification of canker-causing pathogens in prune and walnut orchards using real-time PCR.

AIMS: A real-time quantitative PCR (qPCR) assay was established to quantify the inoculum densities in the air and rainwater for six canker-causing pathogen groups in prune and walnut orchards in California. METHODS AND RESULTS: The previously published DNA primers to target six pathogen groups including Botryosphaeria dothidea, Cytospora spp., Diplodia spp., Lasiodiplodia spp., Neofusicoccum spp. and Phomopsis spp. were used in a qPCR assay. Air samples from Burkard spore traps and rain samples from special rain collector devices were collected periodically from various prune and walnut orchards. Using the qPCR approach, we were able to quantify the concentrations of these pathogen groups in rainwater and air samples and study the dynamics of pathogen inoculum in orchards showing severe canker potential. Phomopsis spp. and Diplodia spp. were not found in all rain samples in prune orchards, although they were detected in the 2016 in the walnut orchard. The other four pathogen groups were quantified at varying concentrations in the prune and walnut orchards. Cytospora spp. in some cases showed higher concentrations in the rainwater in prune orchards. CONCLUSIONS: The rainy season during winter and early spring is a highly risky period of time for infection by the pathogens when the inoculum of these pathogens can easily spread by air and rain water, thus serving as an important inoculum source for disease initiation. The different studied pathogen groups showed different concentrations during the growing season, indicating the complexity of the components of canker-causing species in various tree crops. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed the applicability of the qPCR assay in the quantification of inoculum in tree orchards to help reveal the mechanisms of canker disease epidemics and to help design disease management strategies.

Induced resistance in nectarine fruit by Bacillus licheniformis W10 for the control of brown rot caused by Monilinia fructicola.

Brown rot caused by Monilinia fructicola has led to considerable preharvest and postharvest losses in all major nectarine fruit-growing areas. In our previous study, we successfully identified a biocontrol strain of bacteria, Bacillus licheniformis W10, that can be used to control brown rot. However, the possible mechanism of the control of brown rot by B. licheniformis W10 is still unclear. Therefore, the objectives of this study were to determine whether B. licheniformis W10 induces resistance by activating defense-related enzymes including antioxidant enzymes in nectarine. Treatment of nectarine fruit with B. licheniformis W10 reduced both M. fructicola-induced oxidative damage and reactive oxygen species (ROS) production. Furthermore, application of B. licheniformis to nectarine fruit resulted in a significant increase in the activity of antioxidant and defense-related enzymes and increase in the expression of the corresponding genes. Overall, our results verified the proposed mechanism of B. licheniformis W10 in controlling M. fructicola via regulation of ROS levels and activation of antioxidant and defense-related enzymes.

Pathogenicity, Phylogenetic relationship and NGS based identification and assembly of tumorigenic Agrobacterium radiabacter plasmidic and chromosomic reads isolated from Prunus duclcis.

Although many Agrobacterium radiobacter strains have already been identified, only a few genomes of strains belonging to genomovar G4 have been sequenced so far. In this study, we report the first virulent genome sequence of Agrobacterium radiobacter strain tun 183, which is highly virulent to almond specie. The genome size was estimated to be 5.53 Mb, with 57.9%GC content. In total, 6486 genes encoding proteins and 61 genes encoding RNAs were identified in this genome. Comparisons with the available sequenced genomes of genomovar G4 as well as with other A. sp. were conducted, revealing a hexapartite genome containing circular and linear chromosomes in addition to two accessory plasmids and a tumor inducing plasmid (pTi) in strain tun 183. The phylogenetic analysis of recA gene clearly showed the clustering of tun 183 strain within genomovar G4, supporting the monophyly within this genomovar.

Comparative genomics and pathogenicity potential of members of the Pseudomonas syringae species complex on Prunus spp.

BACKGROUND: Diseases on Prunus spp. have been associated with a large number of phylogenetically different pathovars and species within the P. syringae species complex. Despite their economic significance, there is a severe lack of genomic information of these pathogens. The high phylogenetic diversity observed within strains causing disease on Prunus spp. in nature, raised the question whether other strains or species within the P. syringae species complex were potentially pathogenic on Prunus spp. RESULTS: To gain insight into the genomic potential of adaptation and virulence in Prunus spp., a total of twelve de novo whole genome sequences of P. syringae pathovars and species found in association with diseases on cherry (sweet, sour and ornamental-cherry) and peach were sequenced. Strains sequenced in this study covered three phylogroups and four clades. These strains were screened in vitro for pathogenicity on Prunus spp. together with additional genome sequenced strains thus covering nine out of thirteen of the currently defined P. syringae phylogroups. Pathogenicity tests revealed that most of the strains caused symptoms in vitro and no obvious link was found between presence of known virulence factors and the observed pathogenicity pattern based on comparative genomics. Non-pathogenic strains were displaying a two to three times higher generation time when grown in rich medium. CONCLUSION: In this study, the first set of complete genomes of cherry associated P. syringae strains as well as the draft genome of the quarantine peach pathogen P. syringae pv. persicae were generated. The obtained genomic data were matched with phenotypic data in order to determine factors related to pathogenicity to Prunus spp. Results of this study suggest that the inability to cause disease on Prunus spp. in vitro is not the result of host specialization but rather linked to metabolic impairments of individual strains.

Some strains that have converged to infect Prunus spp. trees are members of distinct Pseudomonas syringae genomospecies and ecotypes as revealed by in silico genomic comparison.

A complementary taxonomic and population genetic study was performed to delineate genetically and ecologically distinct species within the Pseudomonas syringae complex by assessing 16 strains including pathovar strains that have converged to infect Prunus spp. trees, and two outgroups. Both average nucleotide identity and genome-to-genome distance comparison methods revealed the occurrence of distinct genomospecies, namely 1, 2, 3 and 8 (sensu Gardan et al.), with the latter two being closely related. Strains classified as P. s. pv. morsprunorum clustered into two distinct genomospecies, namely 2 and 8. Both the AdaptML and hierarchical Bayesian analysis of population structure methods highlighted the presence of three ecotypes, and the taxonomically related genomospecies 3 and 8 strains were members of the same ecotype. The distribution of pathogenic and virulence-associated genetic traits among Pseudomonas strains did not reveal any distinct type III secretion system effector or phytotoxin distribution pattern that characterized single genomospecies and strains that infect Prunus spp. The complete WHOP (Woody HOst and Pseudomonas spp.) genomic region and the entire ß-ketoadipate gene cluster, including the catBCA operon, were found only in the members of genomospecies 2 and in the two P. s. pv. morsprunorum strains of genomospecies 8. A reduced gene flow between the three ecotypes suggested that point mutations played a larger role during the evolution of the strains than recombination. Our data support the idea that Prunus trees can be infected by different strains of distinct Pseudomonas genomospecies/ecotypes through diverse mechanisms of host colonization and infection. Such strains may represent particular lineages that emerged from environments other than that of the infected plant upon acquiring genetic traits that gave them the ability to cause plant diseases. The complementary assessment of bacterial strains using both taxonomic approaches and methods that reveal ecologically homogeneous populations has proven useful in confirming the cohesion of bacterial clusters.

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by "Candidatus Phytoplasma prunorum".

"Candidatus Phytoplasma prunorum" (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.

Immuno-detection and differentiation of Listeria monocytogenes and Listeria ivanovii in stone fruits.

AIMS: The aim of this study was to develop a rapid detection and differentiation method for pathogenic Listeria species in stone fruits. METHODS AND RESULTS: We utilized activated charcoal enrichment media (ACM) to induce overexpression and hypersecretion of pathogenic Listeria virulence proteins which can subsequently be detected via immunoblot analysis. Plum and nectarine slices spiked with either L. monocytogenes or L. ivanovii were incubated in pre-enrichment broth followed by enrichment in ACM. Secreted proteins were precipitated and subjected to SDS-PAGE and immunoblot analysis using a combination of L. monocytogenes-specific antibody (α-listeriolysin O) and antibody specific for both L. monocytogenes and L. ivanovii (α-Internalin C). As low as 1 CFU per gram of L. monocytogenes in plum and nectarine was detected, whereas a detection limit of 10 CFU per gram was achieved for L. ivanovii in each food tested following a 20-h enrichment period. Nonpathogenic Listeria species and non-Listeria bacterial pathogens tested were negative. CONCLUSIONS: These results demonstrate the highly sensitive and specific nature of the detection method for pathogenic Listeria in stone fruits using activated charcoal enrichment as well as the capability to discriminate between L. monocytogenes and L. ivanovii. SIGNIFICANCE AND IMPACT OF THE STUDY: This method is the first to identify and differentiate L. monocytogenes and L. ivanovii in select stone fruit enrichments within 24 h using immunological techniques. The rapidity and sensitivity of the method could aid in the reduction of exposure to the public in the event of an outbreak and expedite the administration of appropriate antibiotics to infected individuals.

Exploitation of Prunus mahaleb fruit by fermentation with selected strains of Lactobacillus plantarum and Saccharomyces cerevisiae.

The organoleptic attributes of Prunus mahaleb, a fruit representing a new source of bioactive compounds, are so pronounced that it can be consider non-edible. This study was designed to evaluate the acceptance of P. mahaleb fruits after fermentation with different Saccharomyces cerevisiae and Lactobacillus plantarum protechnological strains. Four different bacterial and one yeast strains, as single or mixed starter formulation, were used to inoculate an aqueous suspension of P. mahaleb fruits. The fermented fruits and fermentation broths were subjected to physico-chemical characterization and the organoleptic properties of both samples were also assessed by a hedonic panel. The obtained results indicated that all the employed strains were able to grow and to ferment the matrix. However, the mixed starter FG69 + Li180-7 (L. plantarum/S. cerevisiae) had the best impact on sensory characteristics of P. mahaleb fruit and fermented medium. The adopted protocol allowed us to attain edible fruits and a new fermented non-dairy drink with valuable probiotic health-promoting properties. In our knowledge, this is the first study concerning the exploitation of P. mahaleb fruits. This investigation confirmed the potential of yeasts and lactic acid bacteria co-inoculation in the design of starter tailored for this kind of food applications.

Boscalid Resistance in Blumeriella jaapii: Distribution, Effect on Field Efficacy, and Molecular Characterization.

Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii, is a major disease of tart cherry (Prunus cerasus L.) trees, leading to early defoliation that results in uneven ripening and poor fruit quality in the current season, reduced fruit set in the following season, and increased potential for winter injury and tree death. Pristine (BASF Corporation, Research Triangle Park, NC), a commonly used fungicide for CLS management in Michigan, is a premix of boscalid, a succinate dehydrogenase inhibitor, and pyraclostrobin, a quinone outside inhibitor. Reduced efficacy of Pristine for CLS control was observed in field trials and commercial orchards and highlights the importance of fungicide resistance monitoring. A total of 1,189 isolates from 31 commercial orchards in Michigan, 111 isolates from nontreated trees (four locations in Michigan and two locations in Ohio), and 133 isolates from a research orchard were collected during 2010, 2011, and 2012 and assayed on boscalid-amended media at concentrations ranging from 0 to 25 µg ml-1. Because of the very slow growth rate of B. jaapii in culture, we determined the minimum inhibitory concentration (MIC) of boscalid as opposed to the effective concentration that inhibits mycelial growth to 50% of the control. Isolates from nontreated trees had MIC values ranging from 0.1 to 0.5 µg ml-1; the MIC of isolates from commercial orchards ranged from 0.1 to >25 µg ml-1, and isolates from the research orchard ranged from 2.5 to >25 µg ml-1. Isolates with MIC values ≥25 µg ml-1 were considered boscalid resistant and comprised 0% of the nontreated isolates, 30.4% of the commercial isolates, and 42.1% of the research orchard isolates. Sequencing of the sdhB gene of resistant isolates led to the detection of the amino acid mutation H260R, which is known to confer boscalid resistance in other phytopathogenic fungi. Our results indicate that the occurrence of the H260R mutation in Michigan populations of B. jaapii is correlated with the reduction in sensitivity to boscalid observed in commercial orchards.

Mating System in the Brown Rot Pathogens Monilinia fructicola, M. laxa, and M. fructigena.

Monilinia fructicola, M. laxa, and M. fructigena are the most important pathogens responsible for brown rot disease of stone and pome fruits. Information on their mating system and sexual behavior is scant. A mating-type-specific PCR-based assay was developed and applied to 155 Monilinia isolates from 10 countries and 10 different host plants. We showed that single isolates carry only one of two opposite idiomorphs at the MAT1 locus consistent with a heterothallic mating system for all three species. MAT1-1 and MAT1-2 mating types were detected in similar proportions in samples of isolates of each species and hence there do not appear to be genetic obstacles to the occurrence of sexual reproduction in their populations. Inter simple sequence repeat markers suggested that asexual reproduction is prevalent, but that sexual recombination occurs in M. fructicola populations in Italy. The genetic architectures of the MAT1 loci of the three pathogens were analyzed. MAT1-1 and MAT1-2 idiomorphs are flanked upstream and downstream by the APN2 and SLA2 genes and resemble those of Botrytis cinerea and other heterothallic fungi in the family Sclerotiniaceae. Each idiomorph contains a specific couple of genes, MAT1-1-1 (with alpha-box domain) and MAT1-1-5 in MAT1-1, and MAT1-2-1 (with HMG-box domain) and MAT1-2-10 in MAT1-2. Small gene fragments (dMAT1-1-1 and dMAT1-2-1) from the opposite idiomorph were detected close to their flanking regions. Constitutive expression of the four MAT1 genes during vegetative growth was ascertained by transcriptomic analysis (RNA-Seq). Antisense transcription of the MAT1-1-1 and MAT1-2-1 genes and intergenic transcribed regions of the MAT1 locus were detected. These results represent new insights into the mating systems of these three economically-important pathogens which could contribute to improve the knowledge on their population biology.

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the "Ohanami Project", to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.