Brown Rot Of Stone Fruit Research Articles

Novel mandelic acid derivatives containing piperazinyls as potential candidate fungicides against Monilinia fructicola: Design, synthesis and mechanism study

Brown rot of stone fruit, a disease caused by the ascomycete fungus Monilinia fructicola, has caused significant losses to the agricultural industry. In order to explore and discover potential fungicides against M. fructicola, thirty-one novel mandelic acid derivatives containing piperazine moieties were designed and synthesized based on the amide skeleton. Among them, target compound Z31 exhibited obvious in vitro antifungal activity with the EC50 value of 11.8 mg/L, and significant effects for the postharvest pears (79.4 % protective activity and 70.5 % curative activity) at a concentration of 200 mg/L. Antifungal activity for the target compounds was found to be significantly improved by the large steric hindrance of the R1 groups and the electronegative of the piperazines in the molecular structure, according to a three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis. Further mechanism studies have demonstrated that the compound Z31 can disrupt cell membrane integrity, resulting in increased membrane permeability, release of intracellular electrolytes, and affect the normal growth of hyphae. Additional, morphological study also indicated that Z31 may disrupt the integrity of the membrane by inducing generate excess endogenous reactive oxygen species (ROS) and resulting in the peroxidation of cellular lipids, which was further verified by the detection of malondialdehyde (MDA) content. These studies have provided the basis for the creation of novel fungicides to prevent brown rot in stone fruits.

Identification of pathogens causing brown rot of stone fruit in Cuneo province (Italy) and assessment of sensitivity to azoxystrobin, cyprodinil, fenhexamid, fludioxonil, and tebuconazole

Monilinia spp. cause brown rot and blossom blight of stone fruit. This study characterized the diversity of Monilinia spp. associated with stone fruit rots in the Cuneo province, the major fruit production area in Piedmont, and assessed their sensitivity to azoxystrobin, cyprodinil, fenhexamid, fludioxonil and tebuconazole. Species diversity was determined by PCR amplification and sequencing of isolate internal transcribed spacer (ITS) regions. Sensitivity to fungicides was determined by measuring in vitro mycelium growth on fungicide-amended media. Fifty isolates were obtained from apricot, cherry, or peach fruits with typical brown rot symptoms. Thirteen isolates were identified as M. fructicola, and 37 as M. laxa. Nine isolates of Monilinia laxa and two of M. fructicola had resistance factor (RF) values greater than 10 for different fungicides. The greatest (RF) value (48.96) was measured for azoxystrobin against the M. fructicola isolate CVG 1514. Among the M. laxa isolates, isolate CVG 1547 had the greatest RF value to cyprodinil, while isolate CVG 1709 had RF values greater than 10 for cyprodinil and tebuconazole. A systematic and wider sampling should be carried out in the Piedmont region to determine the distribution of fungicide resistant Monilinia spp. in stone fruit crops. The use of site-specific fungicides remains the most effective strategy for control brown rot, and continued monitoring for fungicide resistance within Monilinia spp. populations is recommended.

Control of Brown Rot Produced by Monilinia fructicola in Peaches Using a Full-Spectrum Extract of Zuccagnia punctata Cav.

Brown rot of stone fruit, caused by Monilinia spp., is one of the most important diseases worldwide, causing significant production losses. Currently, the standard practices for controlling this infection consist of repetitive use of synthetic fungicides. The global tendency encourages the demand for high-quality food products harmless to health and the environment, leading to a reduction in the use of these types of substances. Zuccagnia punctata (Fabaceae) is a perennial shrub extensively used for the treatment of fungal and bacterial infections in Argentinean traditional medicine. In this study, we isolated and characterized (morphologically and molecularly) a pathogenic and virulent strain of Monilinia fructicola, which is the most hostile species of the genus. Consequently, we explored the in vitro antifungal activity of the ethanolic extract of Z. punctata against this phytopathogen. The chalcones 2′,4′-dihydroxy-3′-methoxychalcone and 2′,4′-dihydroxychalcone were isolated from the extract and evaluated against M. fructicola demonstrating that they were responsible for this activity. To promote full-spectrum extract rather than pure compounds, we performed ex-vivo assays using fresh peaches inoculated with the pathogen, and then treated by immersion in an extract solution of 250 µg/mL concentration. Treatment with Z. punctata extract did not show a statistically significant difference from commercial fungicides in the control of fruit rot. In addition, Huh7 cell cytotoxicity evaluation showed that Z. punctata extract was less cytotoxic than commercial fungicides at the assayed concentrations. Based on our research, this plant extract could potentially offer a safer alternative to commercial fungicides for treating peach brown rot.

A Review on Bio-Based Control of Post-Harvest Diseases

Bacillus subtilis non-pathogenic beneficial bacteria, promotes plant growth, disease resistance and tolerance to abiotic stresses. It produces bioactive substances with antibiotic properties and induces physiological features in plant metabolism without adverse effects on the environment or human health. Bacillus subtilis has been used to treat various postharvest diseases during handling, transportation and storage of various fresh fruits and vegetables. It is the first microorganism patented as a postharvest bio control agent for Brown rot of stone fruits, improving the post-harvest physiology of various fruit/vegetables. Bacillus strains AG1 and H110 have been shown to be effective against Vine wood fungal pathogens and post-harvest pathogens. They have been shown to reduce symptoms of Anthracnose in fruit caused by fungal pathogens Colletotrichum gloeosporioides and C. acutatum and White rot caused by Botryosphaeria dothidea. Endophytic Bacillus strains have been developed that can colonize plant tissues and live in the same ecological niches as pathogens, thus preventing post-harvest diseases and improving preservation during storage. Bacillus strains induce auxins, cytokinins, gibberellins, ABA, JA and SA in plants, which can stimulate plant growth under stressful conditions. Endophytic bacteria can induce ISR against pathogens and abiotic stressors, extending the shelf life of stored fruits and vegetables. Microbial antagonists can be applied after harvest to control fruit and vegetable diseases, but a single microbial strain cannot prevent all fruits/vegetables from decaying during storage. Combining diverse antagonistic microorganisms with diverse microbial activity and combining various bio-controlling characteristics can prevent post-harvest decay on fruits/vegetables.

Postharvest application of natural compounds and biocontrol agents to manage brown rot of stone fruits

Postharvest application of natural compounds and biocontrol agents to manage brown rot of stone fruits

Identification and Biosynthesis of DHN-melanin Related Pigments in the Pathogenic Fungi Monilinia laxa, M. fructicola, and M. fructigena

Monilinia is the causal agent of brown rot in stone fruit. The three main species that cause this disease are Monilinia laxa, M. fructicola, and M. fructigena, and their infection capacity is influenced by environmental factors (i.e., light, temperature, and humidity). To tolerate stressful environmental conditions, fungi can produce secondary metabolites. Particularly, melanin-like pigments can contribute to survival in unfavorable conditions. In many fungi, this pigment is due to the accumulation of 1,8-dihydroxynaphthalene melanin (DHN). In this study, we have identified for the first time the genes involved in the DHN pathway in the three main Monilinia spp. and we have proved their capacity to synthetize melanin-like pigments, both in synthetic medium and in nectarines at three stages of brown rot development. The expression of all the biosynthetic and regulatory genes of the DHN-melanin pathway has also been determined under both in vitro and in vivo conditions. Finally, we have analyzed the role of three genes involved in fungi survival and detoxification, and we have proved that there exists a close relationship between the synthesis of these pigments and the activation of the SSP1 gene. Overall, these results deeply describe the importance of DHN-melanin in the three main species of Monilinia: M. laxa, M. fructicola, and M. fructigena.

Transcriptional profiling of the terpenoid biosynthesis pathway and in vitro tests reveal putative roles of linalool and farnesal in nectarine resistance against brown rot

The most devastating fungal disease of peaches and nectarines is brown rot, caused by Monilinia spp. Among the many plant responses against biotic stress, plant terpenoids play essential protective functions, including antioxidant activities and inhibition of pathogen growth. Herein, we aimed to characterize the expression of terpenoid biosynthetic genes in fruit tissues that presented different susceptibility to brown rot. For that, we performed artificial inoculations with Monilinia laxa at two developmental stages (immature and mature fruit) of two nectarine cultivars (‘Venus’ –mid-early season cultivar - and ‘Albared’ –late season cultivar-) and in vitro tests of the key compounds observed in the transcriptional results. All fruit were susceptible to M. laxa except for immature ‘Venus’ nectarines. In response to the pathogen, the mevalonic acid (MVA) pathway of the ‘Venus’ cultivar was highly induced in both stages rather than the methylerythritol phosphate (MEP) pathway, being the expression of some MEP-related biosynthetic genes [e.g., PROTEIN FARNESYLTRANSFERASE (PpPFT), and 3S-LINALOOL SYNTHASE (PpLIS)] different between stages. In ‘Albared’, both stages presented similar responses to M. laxa for both pathways. Comparisons between cultivars showed that HYDROXYMETHYLGLUTARYL-CoA REDUCTASE (PpHMGR1) expression levels were common in susceptible tissues. Within all the terpenoid biosynthetic pathway, linalool- and farnesal-related pathways stood out for being upregulated only in resistant tissues, which suggest their role in mediating the resistance to M. laxa. The in vitro antifungal activity of linalool and farnesol (precursor of farnesal) revealed fungicidal and fungistatic activities against M. laxa, respectively, depending on the concentration tested. Understanding the different responses between resistant and susceptible tissues could be further considered for breeding or developing new strategies to control brown rot in stone fruit.

Genetic variability in the susceptibility of immature peach fruit to Monilinia laxa is associated with surface conductance but not stomatal density

Monilinia laxa is a fungus that causes brown rot in stone fruit. Immature green fruits in the first stage of fruit development (stage I) are generally susceptible. To investigate the relationship between the physical characteristics of immature fruit and susceptibility to M. laxa, we characterized the progeny, derived from a clone of wild peach (Prunus davidiana) crossed with two commercial nectarines (Prunus persica) varieties, through laboratory infection, transpiration monitoring and stomata counting. Two types of fruit infections were observed - ‘classic’ brown rot and ‘clear spot’ symptoms - which have not previously been described in the literature. The number and density of stomata did not explain the observed variability of infection in the progeny. However, surface conductance was positively correlated with infection level. This study provides experimental evidence partially linking physical fruit characteristics to brown rot infection at the immature fruit stage. The role of delayed cuticle deposition in susceptibility to brown rot of immature fruit is discussed.

Phenotypic plasticity of Monilinia spp. in response to light wavelengths: From in vitro development to virulence on nectarines

The development of brown rot in stone fruit caused by the necrotrophic fungus Monilinia spp. is influenced by many abiotic factors, such as temperature, humidity, and light. Specifically, filamentous fungi perceive light as a signal for ecophysiological and adaptive responses. We have explored how specific light wavelengths affect the in vitro development, the regulation of putative development genes and the virulence of the main species of Monilinia (M. laxa, M. fructicola and M. fructigena). After subjecting Monilinia spp. to different light wavelengths (white, black, blue, red, far-red) for 7 days, several differences in their phenotype were observed among light conditions, but also among species. These species of Monilinia exhibited a different phenotypic plasticity in response to light regarding pigmentation, growth, and specially conidiation of colonies. In this sense, we observed that the conidial production was higher in M. laxa than M. fructicola, while M. fructigena showed an inability to produce conidia under the tested conditions. Growth rate among species was significantly lower in M. fructicola under red light wavelength while among light conditions it was increased under far-red light wavelength for M. laxa and under black light for M. fructicola; in contrast, no statistical differences were observed for M. fructigena. Gene expression analysis of 13 genes involved in fungal development of Monilinia spp. revealed a significant difference among the three species of Monilinia, and especially depended on light wavelengths. Among them, a high expression of OPT1, RGS2, RGS3 and SPP1 genes was observed in M. laxa, and LTF1 and STE12 in M. fructicola under black light. In contrast, a high expression of REG1 and C6TF1 genes occurred in both M. fructicola and M. laxa subject to red and far-red light wavelength, respectively. When nectarines were artificially infected with M. laxa and M. fructicola subjected to black light, the virulence was clearly reduced, but not in M. fructigena. Overall, results presented herein demonstrate that light wavelengths are a key abiotic factor for the biology of Monilinia spp., specially modulating its capacity to form conidia, and thus, influencing its spreading and the onset of the disease on nectarines during postharvest.

Survey and Genetic Analysis of Demethylation Inhibitor Fungicide Resistance in Monilinia fructicola From Michigan Orchards.

Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern United States, is associated with a sequence element termed "Mona" that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009 to 2011 and in 2019, and we determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays, but the Mona insert was not uniformly detected and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated with DMI resistance in M. fructicola isolates from Michigan, and there was no obvious correlation between the presence of the Mona element and elevated expression of MfCYP51. However, sequence analysis of MfCYP51 from 25 DMI-resistant isolates did not reveal any point mutations that could be correlated with resistance. Amplification and sequencing upstream of MfCYP51 resulted in detection of DNA insertions in a wide range of isolates typed by DMI phenotype and the presence of Mona or other unique sequences. The function of these unique sequences or their presence upstream of MfCYP51 cannot be correlated to a DMI-resistant genotype at this time. Our results indicate that DMI resistance was established in Michigan populations of M. fructicola by 2009 to 2011, and that relative resistance levels have continued to increase to the point that practical resistance is present in most orchards. In addition, the presence of the Mona insert is not a marker for identifying DMI-resistant isolates of M. fructicola in Michigan.

Polyketide Synthase Gene Expression in Relation to Chloromonilicin and Melanin Production in Monilinia fructicola.

Monilinia fructicola is a fungal pathogen of worldwide significance that causes brown rot of stone fruits. There are only few reports related to the production of biologically active polyketides by this pathogen. In this study, we examined an atypical M. fructicola strain TW5-4 that shows strong antimicrobial activity against various plant pathogens. TW5-4 also displays sparse growth in culture, low virulence, and higher levels of melanin compared with its albino mutant, TW5-4WM, and a wild-type strain Mf13-81. Antifungal compounds were extracted from TW5-4 and purified by thin-layer chromatography following visualization with an on-the-chromatogram inhibition assay. The principal antifungal compound was identified by linear ion trap mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and proton nuclear magnetic resonance analyses as the polyketide chloromonilicin. Multiple M. fructicola polyketide synthase (PKS) sequences were then cloned by degenerate PCR and inverse PCR. Sequence analyses support presence of a 10-member PKS gene family in the M. fructicola genome. Analyses of PKS gene expression found no strong correlation between chloromonilicin production in culture and transcript levels of any of the PKS gene family members in mycelium of strains TW5-4, TW5-4WM, and Mf13-81. However, MfPKS12, a homolog of BcPKS12 involved in biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in Botrytis cinerea, was strongly expressed in mycelia of TW5-4 and Mf13-81. An MfPKS12-silenced mutant accumulated significantly less melanin in mycelia, had lower resistance to polyethylene glycol-induced osmotic stress, and displayed reduced virulence on nectarine fruit. The results suggest that DHN-melanin is required for tolerance to osmotic stress and full virulence in M. fructicola.

Comparisons between genetic diversity, virulence and colony morphology of Monilinia fructicola and Monilinia laxa isolates

Monilinia fructicola and M. laxa, causing brown rot in stone fruit (Prunus species), were first described from Western Australia in 1997. Our previous work indicated that original invasive isolates of both species were each of a single genotype. This research aims to compare isolates from the region in the present day in terms of genetic diversity, colony morphology, and virulence on fruit. Genetic diversity of 68 M. laxa and 66 M. fructicola isolates collected in 2016-2017 from five populations was measured using ISSR markers. The genetic diversity within M. fructicola was greater than that of M. laxa, having Shannon’s diversity indices of 0.50 and 0.41, respectively. Most genetic variation was present within populations for both M. fructicola (77%) and M. laxa (76%), with the rest of the variation between geographically separated populations. Genotype did not correlate closely with virulence of isolates as measured by symptom severity index (SSI) on inoculated plum fruit and colony morphology on potato dextrose agar (PDA) medium. M. fructicola isolates with wide genetic variation exhibited closely similar SSIs and one morphotype on PDA, whereas M. laxa isolates exhibited a broad range of SSIs and four different morphotypes.

Aureobasidium pullulans volatile organic compounds as alternative postharvest method to control brown rot of stone fruits

Volatile compounds produced by L1 and L8 strains were assayed against mycelia and conidia growth of Monilinia laxa, M. fructicola, M. polystroma, and M. fructigena of stone fruits. Results showed that volatile metabolites inhibited significantly pathogens growth, in particular M. fructigena mycelium growth (70% by L1 and 50% by L8) and M. fructicola conidia germination (85% by L1 and 70% by L8) compared to the control. Moreover, the antagonistic activity was enhanced by the addition of asparagine (120 mg L−1) in the culture media composition. Synthetic pure compounds were tested in vitro on pathogens mycelial and conidia growth and their EC50 values were estimated, confirming 2-phenethyl as the most active compound. For this reason 2-phenethyl and VOCs of both yeast strains were assayed in vivo on cherry, peach, and apricot fruits. Regarding peach fruit, both treatments, yeasts and pure compounds, displayed the best inhibiting action against all the pathogens especially against M. laxa (100% by L1, 84% by L8 and 2-phenethyl). ATR/IR spectroscopy analysis showed how VOCs produced by both strains increase the fruit waxes complexity reducing the pathogens attack so playing an essential role in the antagonistic activity of both yeast strains and on fruit structural composition.

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.

Integration of biological and chemical control of brown rot of stone fruits to reduce disease incidence on fruits and minimize fungicide residues in juice

Brown rot is a major disease of stone fruits caused by Monilinia spp. It can reach high incidence that leads to considerable losses during storage. Our work aimed at the evaluation of innovative biological and integrated approaches to control brown rot of apricots and peaches, and of their influence on the presence of fungicide residues both in the harvested fruits and derived peach juices. In this work, we report the results of a two-year field investigation carried out in 5 different farms in Southern Italy. Plant health management strategy was based on two treatments performed in the last thirty days before harvest. Two biocontrol agents (BCAs), the yeast Rhodotorula kratochvilovae strain LS11 and the commercial biofungicide Serenade Max® (based on Bacillus subtilis strain QST 713), were applied alone (biocontrol) or in combination with low doses of the fungicides (25% of full dosage, integrated control) cyprodinil (30 days before harvest), and boscalid (7 days before harvest). The highest protection was recorded in conventional (fungicides applied at full dosage) and integrated control treatments, both on apricots and peaches. In the integrated control, residues of fungicides in fruits and in peach juice were significantly lower compared to those of the conventional control in both fruit species. Our data show that biological and integrated control based on two key treatments at thirty and seven days before harvest can be considered as effective and safer control strategies of brown rot of stone fruits. Furthermore, the applied strategies can dramatically reduce and even set to zero the level of fungicide residues in derived fruit juices.

Postharvest biocontrol ability of Pseudomonas synxantha against Monilinia fructicola and Monilinia fructigena on stone fruit

The biocontrol properties of the endophyte Pseudomonas synxantha DLS65 were tested in vitro and in vivo against Monilinia fructicola and Monilinia fructigena, causal agents of postharvest brown rot of stone fruit. P. synxantha cells significantly reduced the mycelial growth of both pathogens on Potato Dextrose Agar (PDA), and strongly inhibited the Monilinia fructicola growth on Peach Extract Agar (PEA). Cell-free culture filtrates inhibited the pathogens on PDA and PEA to lesser extent. The production of volatile organic compounds (VOCs), with in vitro inhibitory effects on mycelial growth, was also observed. P. synxantha significantly reduced brown rot incidence and severity on peach fruit artificially inoculated with M. fructicola after 5 d at 25 °C. Moreover, P. synxantha more significantly reduced incidence and severity after 10 d at 10 °C and after 20 d in cold storage at 0 °C in comparison to control fruit, even if its activity was never comparable to that of the synthetic fungicide Scholar® (fludioxonil). Similarly, P. synxantha exhibited an excellent antagonistic activity against M. fructigena on fruit at 10 and at 0 °C, and a weak biocontrol activity at 25 °C. Competition for nutrients and space, production of diffusible toxic metabolites and VOCs may play a role in the antagonism of P. synxantha toward M. fructicola and M. fructigena, especially at the lowest temperatures of storage. For that reason, this strain of P. synxantha could be suggested as active ingredient for the setting up of bioformulates against Monilinia species representing a limiting factor for stone fruit production.

Characterization of Monilinia spp. Populations on Stone Fruit in South Italy.

Monilinia spp. are responsible for brown rot decay of stone and pome fruit in the field as well as in postharvest. Monilinia laxa and M. fructigena are considered indigenous to Europe, while M. fructicola is a quarantine pathogen in the European and Mediterranean Plant Protection Organization area included in the A2 List. In Italy, it was first reported in 2009 in Piedmont (northern Italy) and rapidly spread to central Italy. We carried out a monitoring program on the occurrence of Monilinia spp. in southern Italy and a comparative characterization of the three main fungal pathogens. Molecular assays based on direct polymerase chain reaction (PCR) and real-time quantitative PCR for molecular identification of Monilinia spp. from rotted fruit were set up, validated, and applied in a monitoring program. Of the tested 519 isolates from 26 orchards, 388 (74.8%) were identified as M. fructicola, 118 (22.7%) as M. laxa, 10 (1.9%) as M. fructigena, and 3 (0.6%) were M. polystroma. M. fructicola colonies grew faster and had a higher optimal temperature for growth (26°C) than M. laxa (23°C) and M. fructigena (20°C). No relevant difference in virulence could be observed on artificially inoculated apricot, cherry, and peach fruit. The fungal species showed different responses to fungicides, because M. fructicola was more sensitive than M. laxa, especially to cyflufenamid, and M. fructigena revealed a lower sensitivity to succinate dehydrogenase inhibitors (boscalid, fluopyram, and fluxapyroxad) and quinone outside inhibitors (mandestrobin). In summary, the two species M. fructicola and M. polystroma were first detected in southern Italy where M. fructicola has largely displaced the two indigenous pathogens M. laxa and M. fructigena; the relative proportions of the three pathogens in orchards should be considered when defining the management of brown rot of stone fruit due to differences in their responses to fungicides.

Identyfikacja morfologiczna grzybów rodzaju Monilinia występujących na owocach jabłoni w sadach województwa lubelskiego

Among the pathogens occurring on apple trees, Monilinia fungi have a great importance. It causes brown rot of stone fruit. It is a disease that occurs in apple orchards throughout the country. The purpose of the research was monitoring Monilinia fungi in apple orchards in the Lublin Province. Research conducted in the years 2013–2014 included a collection of mummies (mummified apples with visible sporodochia) from the ecological orchards in the spring and autumn. Morphological characteristics of the colonies were evaluated, such as growth rate, color and surface of culture, as well as spore size. Colonies of Monilinia spp. obtained in laboratory were assigned to the species. According to the research, four species of Monilinia fungi, namely M. fructigena, M. laxa, M. fructicola and M. polystroma, were found in apple orchards located in the Lublin Province. M. fructigena fungus was predominant on apple fruits. This species was isolated more often in spring than in autumn. There was also a significant number of M. fructicola and M. laxa in the mummies whose colonies were isolated more often in autumn than in spring. Morphological identification of Monilinia spp. should be confirmed by genetic analysis because the macroscopic and microscopic features of Monilinia spp. are very similar. The conducted studies indicate the need for systematic monitoring of fruit orchards.

Environmental fate and behaviour of the biocontrol agent Bacillus amyloliquefaciens CPA-8 after preharvest application to stone fruit.

Bacillus amyloliquefaciens strain CPA-8 has been described as an effective biocontrol agent to control brown rot in stone fruit for both preharvest and postharvest applications. However, no information about the environmental fate and behaviour of this strain under field conditions is available. The dispersion of the CPA-8 application was evaluated using water-sensitive papers, and complete coverage was observed on the leaves of treated trees, while <1% of non-treated tree leaves had CPA-8. CPA-8 persisted on the fruit of treated trees during preharvest and postharvest conditions, while a significant decrease on leaves and weeds was observed 21 days after treatment. On non-treated trees, CPA-8 was detected on leaves until 180 days after treatment, and on weeds, the CPA-8 population was dependent on the distance from the treated trees. A high persistence of CPA-8 was detected on inert materials, such as clothes and gloves worn by handlers and plastic harvesting boxes. More than 99% of the samples with a CPA-8 phenotype were confirmed as CPA-8 using polymerase chain reaction (PCR). This work demonstrated a good distribution, persistence and adaptation of the CPA-8 strain to field and postharvest conditions. Monitoring of dispersion and persistence is an excellent tool to determine the time of application and provides valuable information for registering issues. © 2017 Society of Chemical Industry.

The Point Mutation G461S in the MfCYP51 Gene is Associated with Tebuconazole Resistance in Monilinia fructicola Populations in Brazil.

The ascomycete Monilinia fructicola is the causal agent of brown rot of stone fruit in Brazil, causing major pre- and postharvest losses. For many years, the demethylation inhibitor (DMI) fungicide tebuconazole has been used as the most effective active ingredient for controlling brown rot and, as a result, strains of M. fructicola resistant to this ingredient have emerged in many Brazilian states producing stone fruit. The aim of this study was to investigate the mechanisms associated with the resistance of M. fructicola to DMI tebuconazole. By sequencing the M. fructicola CYP51 (MfCYP51) gene, encoding the azole target sterol 14α-demethylase, a mutation was identified at the nucleotide position 1,492, causing the amino acid substitution from glycine to serine at the codon position 461, associated with reduced tebuconazole sensitivity. In addition, it was observed that MfCYP51 gene expression could play a secondary role in DMI fungicide resistance of M. fructicola strains in Brazil. However, for the specific isolate found to exhibit elevated expression levels of MfCYP51, no insertions that would trigger gene expression were found. Based on the point mutation associated with tebuconazole resistance, an allele-specific polymerase chain reaction method was developed to quickly identify resistant genotypes within the Brazilian population. This is the first report determining molecular mechanisms for DMI resistance identification for M. fructicola isolates from Brazil. This information provides an important advancement for risk assessment of DMI fungicides used to manage brown rot of stone fruit.