Characterization of Monilinia species associated with brown rot in stone fruit in Brazil

Fungi of the Monilinia genus occur worldwide and affect a wide range of economically important stone fruits. Several Monilinia species are responsible for brown rot. Although this disease is common in Brazil, Monilinia sp. genetic variability in Brazilian orchards has generally been poorly characterized. The present study represents the first report on the genetic diversity of Monilinia sp. from Brazilian orchards. The genetic structure of the Brazilian population was also compared to isolates from other countries, together with some morphological characteristics and aggressiveness. Sixty‐one isolates belonging to the Monilinia genus were obtained from different orchards in Brazilian states. Ten Monilinia fructicola isolates from the United States and one isolate from a fruit imported into Brazil were also evaluated. Phylogenetic analysis of the ITS1‐5.8S‐ITS2 region (internal transcribed spacer) clustered most Brazilian and American isolates with M. fructicola authentic strains from Q‐Bank. Two isolates (one from an imported fruit) clustered as Monilinia laxa. The results revealed M. fructicola as the prevalent species associated with brown rot in Brazilian orchards. To evaluate the intraspecific diversity of M. fructicola and M. laxa, multigene sequence analysis was performed using ITS1‐5.8S‐ITS2 and TEF1 (elongation factor 1). Whilst TEF1 is the most phylogenetically informative gene for intraspecific studies of M. fructicola, RPB2 (RNA polymerase II gene) displayed low variation in intraspecific analysis, but was an informative locus for assigning isolates to M. fructicola or M. laxa species. The amova suggests that Brazilian isolates from the States of the main producing regions belong to a single genetic population, which is genetically distinct from the US (Californian) population of M. fructicola.     

Discontinuance of tebuconazole in the field restores sensitivity of Monilinia fructicola in stone fruit orchards

Demethylation inhibitor (DMI) fungicides are used to control brown rot in stone fruit worldwide. However, their specific mode of action can select resistant isolates of Monilinia fructicola. Monilinia fructicola resistant to DMI fungicides are associated with a fitness cost in the absence of selective pressure, indicating that the sensitive population can be re-established when discontinuing the fungicide in the field. This work aimed to build up the sensitive population of M. fructicola after discontinuing the use of tebuconazole for successive crop seasons. The sensitivity of M. fructicola to tebuconazole was assessed in four commercial peach orchards in Paraná and São Paulo States from 2012/13 to 2015/16. Different fungicide programmes were used and DMI fungicides were discontinued from 2013/14. The sensitivity of M. fructicola to tebuconazole was assessed by a mycelial growth assay in vitro and by determining the frequency of the G461S mutation in the MfCYP51 gene. The isolates from Paraná had high sensitivity to the fungicide across all seasons and the frequency of the G461S mutation remained below 5%. The isolates from São Paulo were highly resistant in the 2012/13 season; however, there was a gradual decline until 2015/16. In addition, the G461S mutation frequency in Sao Paulo State was about 80% in the 2012/13 season, but reduced until it was completely undetectable in 2015/16. These results provide evidence that resistance can be managed in orchards with high selective pressure to tebuconazole after discontinuing the use of the fungicide for at least 3 years.     

Monilinia species identified on peach and nectarine in Croatia,with the first record of Monilinia fructicola

During 2012, an official survey was conducted for Monilinia species present on peach and nectarine in Croatia. In total, 169 Monilinia spp. isolates were collected from 24 commercial orchards and identified according to morphology in culture and PCR. Eighty of the isolates were identified as Monilinia laxa, 70 as M. fructigena and 19 as M. fructicola. M. fructicola was found only at one location in the Mediterranean part of the country, and this is the first record of this quarantine fungus in Croatia. PCR diagnostic tests using M. fructicola‐specific primer pair MO368‐5/MO368‐10R repeatedly gave false negatives for some isolates. PCR tests using primer pair ITS1‐Mfc1/ITS4‐Mfc1 amplified M. fructicola‐specific product in all isolates and was therefore shown to be more suitable for diagnostic purposes.     

Fungicide sensitivity,growth rate,aggressiveness and frost hardiness of <Emphasis Type="Italic">Monilinia fructicola</Emphasis> and <Emphasis Type="Italic">Monilinia laxa</Emphasis> isolates

Monilinia fructicola, the most destructive pathogen of the genus Monilinia, has recently been introduced into Serbia and many other European countries. Since then, many studies have been conducted to evaluate the characteristics of Monilinia species that have a role in the establishment and survival of the pathogen in new areas. The present study assessed the capacity of M. fructicola to repress and replace Monilinia laxa in Serbia based on: fungicide sensitivity, growth rate and aggressiveness at different temperatures, as well as frost hardiness of the isolates of both species. The results showed that the isolates of M. fructicola, compared to M. laxa, were significantly less sensitive to the following fungicides: iprodione, tebucanozole, chlorothalonil, azoxystrobin, fluopyram, and boscalid. In addition, M. laxa isolates exhibited little variation in sensitivity to all of the tested fungicides, whereas M. fructicola isolates displayed a wide range of sensitivity. The temperature of 5°C favored M. laxa growth and aggressiveness, while at 30°C M. fructicola grew faster and had higher lesion expansion rate. These results support an assumption that M. fructicola will continue to spread in Serbian orchards in coming years, particularly on stone fruits harvested during hot summer weather.     

Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance

BACKGROUND: Quinone outside inhibitor (QoI) resistance as a consequence of point mutations in the cytochrome b (cyt b) gene has been reported in numerous plant pathogenic fungi. To examine the potential for QoI resistance development in those Monilinia species causing brown rot of stone and pome fruits [Monilinia fructicola (G Winter) Honey, M. laxa (Aderhold & Ruhland) Honey and M. fructigena (Aderhold & Ruhland) Honey], an examination was made of the sequence and exon/intron structure of their cyt b genes for the presence of any point mutations and/or introns commonly associated with resistance to QoIs in fungal plant pathogens. RESULTS: None of the point mutations typically linked to QoI resistance was present in any of the Monilinia isolates examined. Furthermore, the cyt b genes from M. fructicola and M. laxa, but not M. fructigena, possessed a group‐I‐like intron directly after codon 143. Based on the results obtained, a simple PCR assay using a single primer pair was developed, allowing discrimination between the three Monilinia species without the need for culturing. CONCLUSIONS: Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143‐associated intron. Copyright © 2010 Society of Chemical Industry     

A real‐time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real‐time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus‐specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena/M. laxa/Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No cross‐reactions were observed. The assay was found to have a detection limit of 0·6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real‐time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.     

Effect of hot water treatment on peach volatile emission and Monilinia fructicola development

The effect of hot water treatment (HWT) to control peach brown rot was investigated. Peaches were dipped in water at 60°C for 60 s and artificially inoculated with Monilinia fructicola conidia. HWT failed to control brown rot if applied before inoculation and microscopic observations revealed a stimulatory effect on germ tube elongation of M. fructicola conidia placed immediately after HWT on the fruit surface, compared to the control. The influence of fruit volatile emission due to HWT was performed on the pathogen conidia exposed to the headspace surrounding peaches. The results showed an increase of M. fructicola conidial germination ranging from 33 to 64% for cultivars Lucie Tardibelle and Red Haven heat‐treated peaches, respectively, compared to the control. The volatile blend emitted from heat‐treated fruit was analysed by solid‐phase microextraction/gas chromatography‐mass spectrometry (SPME/GC‐MS) and proton transfer reaction‐time of flight‐mass spectrometry (PTR‐ToF‐MS). Fifty compounds were detected by SPME/GC‐MS in volatile blends of cv. Lucie Tardibelle peaches and significant differences in volatile emission were observed among heated and control fruit. Using PTR‐ToF‐MS analysis, acetaldehyde and ethanol were detected at levels 15‐ and 28‐fold higher in heated fruit compared to unheated ones, respectively. In vitro assays confirmed the stimulatory effect (60 and 15%) of acetaldehyde (0·6 μL L^?1) and ethanol (0·2 μL L^?1) on M. fructicola conidial germination and mycelial growth, respectively. The results showed that volatile organic compounds (VOCs) emitted from heat‐treated peaches could stimulate M. fructicola conidial germination, increasing brown rot incidence in treated peaches when the inoculation occurs immediately after HWT.     

Genetic diversity and pathogenicity of Monilinia polystroma – the new pathogen of cherries

Brown rot caused by fungi belonging to the genus Monilinia is one of the major limiting factors of sour and sweet cherry production. Up to now, three species, M. fructigena, M. laxa and M. fructicola, have been identified as causal agents of brown rot on cherries worldwide. From 2010 to 2013, during the monitoring of cherry orchards in different areas of Poland, a fourth species, M. polystroma, was isolated from brown rot symptoms on sour and sweet cherry fruits. To the best of the authors’ knowledge, this is the first time M. polystroma has been reported as the causal agent of brown rot on cherries. The genetic diversity of M. polystroma isolates from cherries and other hosts was analysed using PCR MP, ISSR and RAPD techniques and showed its clear distinctness from other Monilinia spp. tested. The cluster analysis of fingerprinting data revealed a high similarity of M. polystroma isolates from Poland and their close relationship with the reference strain from Japan, indicating that this species is a recently introduced pathogen. The highest genetic distance between the examined isolates and the highest number of different genotypes was observed in an ISSR assay. Detailed genetic diversity characteristics revealed that M. polystroma isolates from cherries did not create a distinct group but were intermingled with M. polystroma isolates from other hosts. The results of the pathogenicity test conducted on different fruit species indicated a lack of host specificity for M. polystroma isolates.     

Genomic Variation within Monilinia laxa, M. fructigena and M. fructicola, and Application to Species Identification by PCR

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.     

Epidemiology and management of brown rot on stone fruit caused by Monilinia laxa

Stone fruit is attacked by various pathogens, of which brown rot disease is one of the important diseases. There are three Monilinia species mainly responsible for the brown rot disease: Monilinia fructicola is mainly found in North America and Australasia, and M. laxa and M. fructigena mainly in Europe. Both M. fructicola and M. laxa can infect flowers, resulting in blossom blight, as well as both healthy and wounded fruit, resulting in brown rot. On the other hand, M. fructigena can only infect wounded fruit. Compared to the two other species, M. fructicola has been extensively studied, whereas the equally important M. laxa has had less attention. This paper addresses this imbalance and reviews research on the biology, epidemiology and management of M. laxa on stone fruits. Due to EU regulations, the number of fungicides available for controlling plant diseases has been steadily decreasing, particularly in the post-harvest environment. This has placed much more emphasis on alternative control methods, a focus of the present review. Numerous physical and biological approaches to control have achieved successful outcomes but often in small-scale trials and in isolation from integrated strategies. Promising physical control methods include removal of mummified fruit in orchards and post-harvest hot-water treatment. Many micro-organisms have been shown to have biocontrol potential against brown rot but only a few have been commercially formulated. It is generally agreed that the use of biocontrol agents needs to be integrated with other measures. Current research focuses on disease management from flowering to post-harvest period. Recent results have suggested that reducing overwintering inoculum should be considered as one of key aspects of integrated management of brown rot on stone fruit. Finally, we make recommendations about future research and development on integrated pest management strategies for control of M. laxa, especially on strategic deployment of biocontrol agents and interactions among brown rot pathogens.     

Temporal development and relationship amongst brown rot blossom blight,fruit blight and fruit rot in integrated and organic sour cherry orchards

The aim of this 4‐year study was to characterize temporal development of brown rot blossom blight and fruit blight (caused by Monilinia spp.) and their sporulating areas in sour cherry orchards; and to determine the relationships amongst incidence and sporulating area of blossom blight, fruit blight and fruit rot. The study was performed in integrated and organic orchard blocks on two cultivars (Újfehértói fürtös and Érdi b?term?). On both cultivars, disease progress on flowers and fruits was 2–10 times slower in the integrated than in the organic management system. The peak incidence values were 9 and 31 days after petal fall for blossom blight and fruit blight, respectively. After these dates, no new blight symptoms on flowers and/or fruits appeared and the disease was levelling off. Final blossom blight incidence ranged from 1 to 5% and from 12 to 34%, and fruit rot incidence from 2 to 6% and from 11 to 26% in the integrated and the organic orchards, respectively. The sum of fruit blight incidence ranged from 9 to 22% for the organic system, but was below 5% for the integrated system, while the final sporulating area was 5–16 mm² and <3 mm², respectively. Among the five highest Pearson's correlation coefficients, relationships between blossom blight and early fruit blight stage (r = 0·845, P = 0·0087 integrated; r = 0·901, P = 0·0015 organic), and between sporulating area and fruit rot (r = 0791, P = 0·0199 integrated; r = 0·874, P = 0·0039 organic) were the most significant relationships from an epidemic standpoint as they indicated a connection between different brown rot symptom types.     

Assessment of viable conidia of Monilinia fructicola in flower and stone fruit combining propidium monoazide (PMA) and qPCR

Brown rot is the most economically important fungal disease of stone fruits and is primarily caused by Monilinia laxa and M. fructicola. Conventional methods used to identify M. fructicola are mainly based on phenotypic characteristics and pathogen quantification is not always accurate. In contrast, methodologies based on molecular tools improve pathogen characterization and identification but are not able to differentiate between live and dead conidia. In this study the PMA‐qPCR methodology was optimized, validated and applied to quantify viable cells of M. fructicola in artificially and naturally infected samples. qPCR methodology showed good primer efficiency and sensitivity with quantification limits lower than obtained using a plate count method. The conditions of propidium monoazide (PMA) pretreatment were 60 μm PMA for 20 min incubation and 30 min of light‐emitting diode (LED) exposure that, combined with qPCR, measured live cells accurately without overestimation of dead cells. Using this methodology in naturally infected samples, M. fructicola live cells were quantified specifically, in contrast to other traditional methodologies that cannot distinguish among Monilinia spp. The developed methodology based on combined PMA‐qPCR will be a new tool to quantify viable M. fructicola in further epidemiological and ecological studies of this fungus.     

Pectin methyl esterases and rhamnogalacturonan hydrolases: weapons for successful Monilinia laxa infection in stone fruit?

The secretion of cell wall-degrading enzymes is one of the mechanisms used by necrotrophic fungi to colonize host tissues. However, information about virulence factors of Monilinia spp., the causal agents of brown rot in stone fruit, is scarce. Plant cell walls have three main components that are broken down by fungal enzymes: cellulose, hemicellulose and pectin. In order to identify Monilinia laxa candidate proteins involved in pectin hydrolysis, two in vitro approaches were conducted: (i) phenotypic and ecophysiological characterization of growth of the pathogen at different pHs, in glucose- and pectin-containing solid media for 7 days' incubation; and (ii) expression analysis of genes encoding M. laxa pectin methyl esterases (MlPMEs) and rhamnogalacturonan hydrolases (MlRG-HYDs) after incubation for 0.5, 2, 6, 24 and 48 h in glucose- and pectin-containing liquid media. Phenotypic tests showed the role of carbon source on M. laxa growth rate and aggressiveness, and indicated that pectinases were greatly affected by pH. Gene expression analyses uncovered differences among members of each family of pectinases and between the two families, defining sets of genes expressed at earlier (0.5–6 h) and later (48 h) phases. Notably, the up- or down-regulation of these target genes was carbon source-dependent. Finally, an in vivo study confirmed the synergistic and complementary role that these genes play in the M. laxa–stone fruit pathosystem. Based on these results, it is hypothesized that MlPME2, MlRG-HYD1 and MlRG-HYD2 may be potential virulence factors of M. laxa in the process from infection to colonization.     

Characteristic of Monilinia spp. fungi causing brown rot of pome and stone fruits in Poland

Brown rot, caused by fungi belonging to the genus Monilinia, is one of the most important diseases of stone and pome trees in the world. During the summers of 2010 and 2011, a total of 670 Monilinia spp. isolates were obtained from infected fruits. They were collected from different commercial stone and pome fruit orchards, located in northern, southern and central Poland. All isolates were identified using multiplex PCR. Twenty isolates obtained from plum, peach and apple fruits were identified as M. polystroma and 5 isolates from plums as M. fructicola. The remaining isolates were identified as M. fructigena or M. laxa. The identification of the isolates was also confirmed on the basis of growth characteristics in culture according to the EPPO standard PM 7/18. A comparison of morphological features of four Monilinia spp. growing on two selective growth media, APDA-F500 and CHA, indicated significant differences between these species. In artificial inoculation of fruits, all the examined Monilinia spp. isolates were pathogenic. The species affiliation of M. polystroma and M. fructicola isolates collected from orchards in Poland was confirmed on the base of phylogenetic and sequence analysis of the internal transcribed spacer (ITS1/5.8S rDNA/ITS2) region of ribosomal DNA.     

Production of hydrogen peroxide and expression of ROS‐generating genes in peach flower petals in response to host and non‐host fungal pathogens

Reactive oxygen species (ROS) play dual roles in plant–microbe interactions in that they can either stimulate host resistance or enhance pathogen virulence. Innate resistance in peach (Prunus persica) to the brown rot fungal pathogen Monilinia fructicola is very limited, and knowledge of the mechanism of virulence is rudimentary. In this study, production of hydrogen peroxide, a major component of ROS, was determined in peach flower petals in response to M. fructicola (a host pathogen) and Penicillium digitatum (a non‐host pathogen). Monilinia fructicola was able to infect flower petals while P. digitatum was not. During the host‐specific interaction, M. fructicola induced hydrogen peroxide accumulation in flower petals. Application of exogenous antioxidants significantly reduced hydrogen peroxide accumulation as well as the incidence of brown rot disease. Application of M. fructicola spores to the surface of intact flower petals induced gene expression and increased enzyme activity of NADPH oxidase and cell wall peroxidase in host tissues, resulting in the production of hydrogen peroxide. Petals inoculated with M. fructicola exhibited high levels of protein carbonylation and lipid peroxidation. No significant response in gene expression, enzyme activity or hydrogen peroxide levels was observed in peach flower petals treated with P. digitatum. These results suggest that M. fructicola, as with other necrotrophic fungi, uses the strong oxidative response as part of a virulence mechanism.     

桃褐腐病菌(Monilinia fructicola)对3种杀菌剂的敏感性

采用生长速率法测定了采自北京平谷区3个桃园的125株桃褐腐病菌对甲基硫菌灵、戊唑醇和异菌脲3种杀菌剂的敏感性,发现甲基硫菌灵对桃褐腐病菌的EC50主要分布在1.0×10^-5～0.2μg/mL,戊唑醇对桃褐腐病菌的EC₅₀主要分布在0.006～0.022μg/mL之间。异菌脲对桃褐腐病菌的EC₅₀主要分布在0.15～0.55μg/mL之间。研究结果表明,北京地区的桃褐腐病菌对这3种杀菌剂都比较敏感,未产生明显的抗药群体。建立了褐腐病菌对异菌脲抗药性的敏感基线。而且,数据分析表明:甲基硫菌灵、戊唑醇和异菌脲之间均不存在交互抗性。     

Relationship between the incidence of latent infections caused by <Emphasis Type="Italic">Monilinia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> and the incidence of brown rot of peach fruit: factors affecting latent infection

Five field experiments were performed in commercial orchards located in Lleida (Spain) over three growing seasons, 2000–2002, in order to estimate the relationship between the incidence of latent infection caused by Monilinia spp. in peaches and the incidence of post-harvest brown rot. No latent infection was recorded at popcorn and the maximum incidence occurred pre-harvest; in some orchards a second peak was detected during the pit hardening period. Monilinia laxa is the most prevalent species isolated from peaches with brown rot. There was a positive correlation between the incidence of latent infection and that of post-harvest brown rot. The average incidence of latent infection during the crop season explained 55% of the total variation in the incidence of post-harvest brown rot. The effect of temperature (T) and duration of wetness (W) on the incidence of latent infection in peach and nectarine orchards was analysed using multiple regression. The regression analysis indicated that T and W jointly explained 83% of the total variation in the incidence of latent infection. The model predicts no latent infections when T < 8°C, and >22 h wetness are required when T = 8°C but only 5 h at 25°C are necessary for latent infection to occur. The incidence of brown rot and latent infection of peaches caused by M. laxa under controlled experimental conditions were also affected by T and W, as well as by fruit maturity and inoculum concentration. Latent infections were produced in fruit when T was not suitable for the development of brown rot symptoms. In these experiments more than 4–5 h of daily wetness were required after embryo growth in fruit sprayed to run-off with an inoculum concentration higher than 10⁴ conidia ml⁻¹ of M. laxa for brown rot and latent infections to develop. The fitted model obtained from the field data was able to predict the observed data obtained under controlled environmental conditions.     

Development and implementation of cost-effective strategies to manage brown rot of peach trees in Imathia,Greece

Development of cost-effective strategies to manage brown rot caused by Monilinia laxa of peach implies a better understanding of the susceptibility of different cultivars and pre-harvest contamination. This study investigated the susceptibility of 24 peach and nectarine cultivars to shoot blight caused by Monilinia spp. and found various levels of susceptibility, with the nectarine cultivar Tasty Free scored as the most susceptible. Studies on the the existence and detection of latent infections by Monilinia spp. in three peach (‘A37’, ‘Andross’, and ‘E-45’) and three nectarine (‘Venus’, ‘Fantasia’, and ‘Tasty Free’) cultivars were also conducted. The results showed that latent infections were detected only in nectarine cultivars when fruit were collected on 23 May and 22 June. In contrast, nectarine fruit collected on 7 June and all peach cultivars tested had no detectable latent infection. This study also indicated that the fungicide thiophanate methyl applied at the pit hardening stage reduced significantly the percentage of latent infection and subsequently preharvest fruit rots. Finally, a disease forecast model to predict blossom blight, caused by M. laxa, was evaluated in the Prefecture of Imathia, Greece. Trees, sprayed according to the model predictions, showed a statistically lower percentage of blighted shoots than those of unsprayed trees.     

SSR marker analysis of Monilinia fructicola from Swiss apricots suggests introduction of the pathogen from neighbouring countries and the United States

Monilinia fructicola is a quarantine fungal pathogen in Europe, but many major stone fruit growing countries in Europe have reported its presence recently. In Switzerland, the fungus was first found in a single apricot orchard in 2008. This study confirms the presence of M. fructicola in nine out of 22 commercial orchards in Canton Valais, Switzerland. Five simple sequence repeat markers (SSRs) were developed for M. fructicola and samples from Switzerland, Spain, Italy, France and the United States were analysed and compared in order to assess the genetic diversity of the pathogen, identify the origin of the disease, and verify if the fungus reproduces sexually in Europe. In the 119 European samples analysed, 12 different haplotypes were found, indicating a relatively high genetic diversity of the pathogen considering that the first report in Europe was 10 years ago. Three haplotypes found in Europe were identical to those found in the American samples (two from the east coast and one from the west coast). Population structure analysis suggests that the European population is derived from at least two ‘invasion’ events probably originating from the US (one from the east coast, the other from the west coast). Preliminary evidence of sexual reproduction of M. fructicola in Europe is reported.