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.     

Comparison of different PCR tests to identify Monilinia fructicola

Monilinia fructicola was until very recently a regulated pest in the European Union, and EU countries were requested to monitor its presence on their territories. As accredited laboratories should use validated tests, the mycological laboratory of CRA‐PAV carried out a validation process for the multiplex based PCR test (Coté et al., 2004 ), that is one of the most widely used tests for the identification of M. fructicola, although this test is not described in the EPPO diagnostic protocol PM 7/18 (2) because the validation data were lacking. The performance characteristics of this multiplex PCR test were established according to the EPPO Standard PM 7/98 (1) and the test was compared in a collaborative study with the end point PCR test (Ioos & Frey, 2000 ), considered as the ‘standard test’. The validation data were obtained using different isolates of M. fructicola, M. laxa, M. fructigena and Monilia polystroma, as well as different fruit tissues. Four series of the DNA target at different concentration, repeated three times, were analyzed in four Italian laboratories. The results showed that the multiplex PCR detection test (Coté et al., 2004 ) was fit for diagnostic purpose, although the analytical sensitivity was significantly lower compared to the conventional PCR ‘standard test’.     

Monilia mumecola, a new brown rot fungus on Prunus mume in Japan

In 1982, an anamorphic fungus in the genus Monilia was first isolated as the causal agent of brown rot disease of Japanese apricot or mume (Prunus mume) in Oita Prefecture, Kyushu, Japan. Inoculation of flowers, shoots, and fruit of P. mume with the fungus reproduced brown rot disease symptoms similar to those found in nature. The fungus somewhat resembled the colony appearance of Monilinia (anamorph Monilia) laxa, the apricot brown rot fungus, on PSA plates, but it differed from the latter and the other two brown rot fungi, M. fructigena and M. fructicola, in terms of growth rate, temperature optima for mycelial growth and sporulation, morphology and germination pattern of conidia, nuclear number in the conidium, and nucleotide sequences in the ITS region of ribosomal DNA. It is newly described as Monilia mumecola Y. Harada, Y. Sasaki & T. Sano. A key to anamorphic states of four brown rot fungi of fruit trees is provided.     

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.     

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.     

Frequency of brown rot fungi on blossoms and fruit in stone fruit orchards in Greece

Brown rot is a devastating disease of stone fruits caused by Monilinia spp. This study was conducted to investigate the disease aetiology on blossoms and fruit in peach, apricot, sweet cherry and plum orchards, in Greece. In total, 1433 isolates obtained from orchards located in the main stone fruit production regions of Greece were identified to species based on the presence/size of a cyt b intron. Monilinia laxa and M. fructicola were detected at frequencies of 59 and 41%, respectively, while M. fructigena was absent. Monilinia fructicola was more common on fruit whereas M. laxa occurred in similar frequency on blossoms and fruit. Monilinia laxa was replaced by M. fructicola in fruit infections of peach in both regions investigated and in fruit infections of plum in the Imathia region. Assessments of aggressiveness of 30 isolates of both species on the petals and fruits of the hosts showed that M. fructicola isolates were more aggressive. This suggests that the predominance of M. laxa on the blossoms cannot be explained by higher aggressiveness. Measurements of the effect of temperature on mycelial growth showed that M. laxa isolates had a higher growth rate than M. fructicola at the lowest temperature tested of 5°C, whereas M. fructicola isolates showed higher growth rates at higher temperatures. The observed high frequency of M. fructicola in Greece represents a major threat for stone fruit production. Furthermore, the information obtained about delineation of species and plant organ preference could be useful for the implementation of disease management strategies.     

First report of Monilia polystroma on apple in Hungary

A Monilinia fructigena-like isolate (UFT) was collected from apple shoots in northeastern Hungary (Újfehértó). Brownish dieback and buff-coloured stromata were observed on shoots and small fruits of cv. ‘Ashton Bitter’. On potato dextrose agar (PDA) the colonies were yellowish in colour and irregular black stromatal crusts occurred. Conidia (16.6?×?10.1 µm) were slightly smaller than the average of M. fructigena. The fungus caused brown rot on inoculated apple fruits, and produced numerous sporodochia. The sequences of the rDNA internal transcribed spacer regions of the UFT isolate were almost identical to that of a previously described Monilia polystroma isolate, containing all five nucleotides that distinguish it from M. fructigena. Comparison of a genomic sequence of unknown function revealed that repetitive sequence motifs occurred in different numbers as insertions in the genomes of M. fructigena, Monilia polystroma, and the UFT isolate. Classical and molecular characterisation indicated that the UFT isolate belonged to Monilia polystroma. To our knowledge this is the first report of Monilia polystroma in Europe.     

Quantification of airborne spores of <Emphasis Type="Italic">Monilinia fructicola</Emphasis> in stone fruit orchards of California using real-time PCR

The fungal pathogen Monilinia fructicola causes blossom blight and fruit brown rot of stone fruits in California. In this study, spore densities in the air were monitored in six orchard/year combinations with Burkard spore traps. A real-time PCR assay was developed to efficiently quantify the dynamics of spore density in these orchards during the growing season. Different patterns of dynamics of spore density were observed in these orchards. A linear relationship between numbers of spores counted with a compound microscope and those determined with the real-time PCR assay was obtained, using the same samples of spore traps. Spore density in five of six orchard/year combinations ranged from 0.0 to 0.05 spores l⁻¹, except for that in orchard 4, which showed much higher values of spore density in the air, as well as higher values and wider range of incidences of blossom infection and fruit rot than those in the other orchards. The results demonstrated a potential method to quantitatively determine spore inoculum potential in orchards by using a real-time PCR assay.     

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.     

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     

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self‐splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5′–3′) 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above‐mentioned 1166 bp intron, a self‐splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron. Copyright © 2010 Society of Chemical Industry     

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.     

High inoculum of Monilinia fructicola is a threat to peach production in the tropics due to fruit susceptibility at all development stages

Brown rot is the main disease of stone fruits in Brazil, but the susceptibility of peaches to brown rot at different stages of development in the field has not been studied under subtropical conditions. This information is relevant to guide the management of the disease. The objective of this research was to determine the influence of inoculating peaches with Monilinia fructicola at different stages of development on the infection and progress of brown rot at postharvest. Two experiments were carried out: one ex vivo with two cultivars and the other in the field for two seasons. Peaches were inoculated at different sizes for both experiments. In the field, peaches were bagged to avoid natural infection, and M. fructicola inoculum was monitored. The ex vivo incidence of the disease was lower at pit hardening than at other fruit stages for both cultivars. The incidence of brown rot for peaches attached to the trees increased with fruit ripening. Conversely, the time for symptom expression was reduced according to peach diameter. Peaches inoculated with a diameter smaller than 2 cm showed a lower incidence of brown rot and longer periods for disease expression than fruit inoculated near harvest. In conclusion, in areas with high inoculum in the orchard, a common condition in the subtropics, the grower must prevent infection at all stages of fruit development, thus avoiding losses during marketing.     

A review of pest surveillance techniques for detecting quarantine pests in Europe

This paper provides reviews of the most commonly used methods to detect plant pests belonging to groups of invasive organisms with high economic relevance, including Coleoptera (bark beetles, flathead borers, leaf beetles, longhorn beetles, weevils), Diptera (cone and seed flies, fruit flies), Homoptera (aphids, leafhoppers and psyllids, whiteflies), Lepidoptera (moths and butterflies), Thysanoptera (thrips), bacteria (potato brown rot Ralstonia solanacearum) and fungi (pitch canker disease Gibberella circinata, brown rot disease Monilinia fructicola). Future perspectives in detection methods are discussed, with particular reference to the considerable increase in the volume, commodity type and origins of trade in plant material from third countries, the introduction of new crops, the continuous expansion of the EU with new border countries being added, and the impact of climate change affecting the geographical boundaries of pests and their vectors.     

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.     

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.     

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.     

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.     

Species-Specific Detection of Monilinia fructicola from California Stone Fruits and Flowers

ABSTRACT A set of molecular diagnostics was developed for Monilinia fructicola, causal agent of brown rot of stone fruits, capable of sensitive detection of the pathogen in planta. Species-specific repetitive sequences were identified from a partial library of 312 recombinant clones hybridized with total DNA, followed by subsequent screening for specificity. One hundred isolates, comprising 12 fungal species common to California stone fruits, were surveyed for specificity. Three clones hybridized to 60 geographically diverse M. fructicola isolates (California, Michigan, Georgia, Oregon, and Australia) to the exclusion of all other fungi surveyed, including the closely related M. laxa (n = 12). Two clones were identical and of extrachromosomal origin (pMF73 and pMF150), whereas the third (pMF210) migrated with uncut DNA. The sensitivity of all three was comparable and capable of detecting 50 pg of fungal DNA in dot blot hybridizations. Six species-specific primer pair sets were designed. They maintained the same specificity patterns observed in the initial hybridization surveys and were sensitive enough to detect 50 fg of fungal DNA template, approximately equivalent to 10 spores. The species-specific clones were capable of detecting the pathogen in planta, specifically from infected plum flowers and nectarine fruit tissue, using both hybridization- and polymerase chain reaction-based methodologies.