Real-time PCR for the detection of latent infections and the assessment of inocula viability of Monilinia spp.

Success story of the Euphresco project 'Development and validation of molecular tools for detection and identification of European Monilinia species

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.info:eu-repo/semantics/publishedVersio

Development of brown rot epidemics in Spanish peach orchards

A new approach to modelling epidemics of brown rot caused by Monilinia spp. in Ebro Valley peach orchards has been developed. This compartmental model was subdivided according to the phenological stages in which the disease can develop (blossom, immature fruit, and ripe fruit). Information host susceptibility, primary and secondary inoculum sources and latent infections in immature fruit was taken into account. The compartmental model is described by a system of differential equations, and is simple enough to allow an analytical study of the main epidemiological factors that determine the rate of disease progress during a single growing season. The proposed model fits well to the epidemic pattern of brown rot observed in northeastern Spain. The transmission of the disease as a nonlinear term implied that small changes in the infection rate had a large effect on the development of the disease. The model has confirmed the usefulness of removing mummies (infected fruit that remains in the crop during winter) from the field to reduce the final incidence of the disease. In addition, all control measures that reduce the rate of secondary infection in ripe fruit, either through the use of more resistant varieties or the use of fungicides, are effective in reducing brown rot incidence. The proposed epidemic model is flexible and allows to add complexities to the system and evaluate the effectiveness of different control strategies.info:eu-repo/semantics/publishedVersio

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 applesinfo:eu-repo/semantics/publishedVersio

Comparative Genomics Used to Predict Virulence Factors and Metabolic Genes among Monilinia Species

Brown rot, caused by Monilinia spp., is among the most important diseases in stonefruits, and some pome fruits (mainly apples). This disease is responsible for significant yield losses,particularly in stone fruits, when weather conditions favorable for disease development appear.To achieve future sustainable strategies to control brown rot on fruit, one potential approach will be to characterize genomic variation among Monilinia spp. to define, among others, the capacity to infect fruit in this genus. In the present work, we performed genomic and phylogenomic comparisons of five Monilinia species and inferred differences in numbers of secreted proteins, including CAZy proteins and other proteins important for virulence. Duplications specific to Monilinia were sparse and, overall, more genes have been lost than gained. Among Monilinia spp., low variability in the CAZome was observed. Interestingly, we identified several secondary metabolism clusters based on similarity to known clusters, and among them was a cluster with homology to pyriculol that could be responsible for the synthesis of chloromonilicin. Furthermore, we compared sequences of all strains available from NCBI of these species to assess their MAT loci and heterokaryon Compatibility systems. Our comparative analyses provide the basis for future studies into understanding how these genomic differences underlie common or differential abilities to interact with the host plant.info:eu-repo/semantics/publishedVersio

Depicting the battle between nectarine and Monilinia laxa: the fruit developmental stage dictates the effectiveness of the host defenses and the pathogen’s infection strategies

Infections by the fungus Monilinia laxa, the main cause of brown rot in Europe, result in considerable losses of stone fruit. Herein, we present a comprehensive transcriptomic approach to unravel strategies deployed by nectarine fruit and M. laxa during their interaction. We used M. laxa-inoculated immature and mature fruit, which was resistant and susceptible to brown rot, respectively, to perform a dual RNA-Seq analysis. In immature fruit, host responses, pathogen biomass, and pathogen transcriptional activity peaked at 14–24 h post inoculation (hpi), at which point M. laxa appeared to switch its transcriptional response to either quiescence or death. Mature fruit experienced an exponential increase in host and pathogen activity beginning at 6 hpi. Functional analyses in both host and pathogen highlighted differences in stage-dependent strategies. For example, in immature fruit, M. laxa unsuccessfully employed carbohydrate-active enzymes (CAZymes) for penetration, which the fruit was able to combat with tightly regulated hormone responses and an oxidative burst that challenged the pathogen’s survival at later time points. In contrast, in mature fruit, M. laxa was more dependent on proteolytic effectors than CAZymes, and was able to invest in filamentous growth early during the interaction. Hormone analyses of mature fruit infected with M. laxa indicated that, while jasmonic acid activity was likely useful for defense, high ethylene activity may have promoted susceptibility through the induction of ripening processes. Lastly, we identified M. laxa genes that were highly induced in both quiescent and active infections and may serve as targets for control of brown rot.info:eu-repo/semantics/publishedVersio

Proficiency of real-time PCR detection of latent Monilinia spp. infection in nectarine flowers and fruit

Rapid and reliable detection of Monilinia latent infections is needed to prevent and control dispersion of Monilinia spp. in infected localities and non-infected countries. A fast multiplex quantitative real-time PCR method (qPCR) for the detection and identification of Monilinia spp. latent infections in blossoms and fruit of nectarine trees (Prunus persica var. nucipersica) was tested in an inter-laboratory trial. The test performance study involving five laboratories was conducted to validate the sensitivity and specificity of several real-time PCR platforms for the detection of low amounts of Monilinia DNA (latent infections), using a common protocol, and to identify possible difficulties when these tests were implemented by diagnostic laboratories or national reference centres. The method has two hydrolysis probes distinguishing between Monilinia fructicola and M. fructigena/M. laxa. Validation included test performance accuracy, analytical specificity and sensitivity, repeatability, and reproducibility, as defined by standard PM7/98 of the European Plant Protection Organization (EPPO). All qPCR platforms detected Monilinia latent infections and mycelium samples with both hydrolysis probes, and healthy flowers and fruit samples gave negative results. The method specificity was consistent between different laboratories, despite different equipment used, and there were no laboratories with z-scores in the unacceptable region. Monilinia fructicola latent infection samples were correctly detected by all laboratories, but some M. laxa samples were cross-detected as if they were M. fructicola. Monilinia laxa cross-detection could be compensated by including the allelic discrimination step in qPCR runs, which permitted differentiating between M. fructicola and M. laxa samples. The inter-laboratory comparison demonstrated the robustness of the developed method and confirmed in-house validation data. This method could be used to detect latent infections of Monilinia in asymptomatic nectarine fruit and flowers

Interactions of pesticides and mycoflora of peach twigs

The effects of pesticides applied singly (copper oxychloride, thiram) or in combination (captan, dinocap, benomyl and methomyl) on peach twig mycoflora were studied under orchard, glasshouse and laboratory conditions in 1986 and 1987. Fungal populations, which were determined by two indirect methods using agar media and a direct method (scanning electron microscopy), were lowest on untreated twigs in the orchard in April and June, increasing as the season progressed to reach maxima in September and February. Following copper oxychloride and thiram applications, the fungal population in both years, was either slightly reduced or not reduced, but with captan + dinocap applications in both, the flora was appreciably reduced. Other applications (captan + benomyl + methomyl and dinocap + benomyl + methomyl) also significantly reduced fungal populations. Hyphomycetes were the most abundant fungi on both untreated and treated twigs, especially Alternaria spp.;Cladosporium spp.;Pencillium spp. Populations of Cladosporium spp.;Alternaria spp. were considerably depressed by the pesticides. However, numbers of Penicillium spp. (especially P. frequentans, a powerful antagonist of the peach pathogen Monilinia laxa), were not reduced in 1986, and reductions in 1987 were less than for Alternaria spp.;Cladosporium spp. Penicillium spp. were more frequent on treated twigs than Alternaria spp.;Cladosporium spp. from the end of May to August. The saprotrophic colonisation of peach twigs by selected fungi in the glasshouse was also found to be reduced by the application of fungicides. The results which can be partially explained by the in vitro sensitivity of the isolates to the fungicides have potential practical implications in the development of an integrated control programme against M. laxa, combining biological and chemical methods. Although natural populations of P. frequentans found in this study during the infection period were insufficient to control the disease, P. frequentans is a good candidate for biological control since it readily colonizes twigs and is highly competitive with other components of the mycoflora. © 1992, British Mycological Society. All rights reserved