Visual detection of fungicide resistance by combining RPA and CRISPR/Cas12a in peach Brown rot fungus Monilinia fructicola.

BACKGROUND: Peach brown rot caused by Monilinia fructicola severely affects the quality and yield of peach, resulting in large economic losses worldwide. Methyl benzimidazole carbamate (MBC) fungicides and sterol demethylation inhibitor (DMI) fungicides are among the most applied chemical classes used to control the disease but resistance in the target pathogen has made them risky choices. Timely monitoring of resistance to these fungicides in orchards could prevent control failure in practice. RESULTS: In the current study, we developed methods based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems to detect MBC and DMI resistance based on the E198A mutation in the ß-tubulin (MfTub2) gene and the presence of the Mona element in the upstream region of the MfCYP51, respectively. For MBC resistance, RPA primers were designed that artificially incorporated PAM sites to facilitate the CRISPR/Cas12a reaction. Subsequently, specific tcrRNAs were designed based on the E198A mutation site. For the detection of the Mona element, we designed RPA primers M-DMI-F2/M-DMI-R1 that in combination with crRNA1 detected 'Mona' and distinguished resistant from sensitive strains. CONCLUSION: Both methods exhibited high sensitivity and specificity, requiring only a simple isothermal device to obtain results within 1 h at 37 °C. The FQ-reporter enabled visualization with a handheld UV or white light flashlight. This method was successfully used with purified DNA from lab cultures and crude DNA from symptomatic fruit tissue, highlighting its potential for on-site detection of resistant strains in orchards. © 2024 Society of Chemical Industry.

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.

Complete genome analysis of a novel hypovirus in the phytopathogenic fungus Monilinia fructicola.

Monilinia fructicola is one of the most devastating fungal diseases of rosaceous fruit crops, both in the field and postharvest, causing significant yield losses. Here, we report the discovery of a novel positive single-stranded RNA virus, Monilinia fructicola hypovirus 3 (MfHV3), in a strain (hf-1) of the phytopathogenic fungus Monilinia fructicola. The complete genome of MfHV3 is 9259 nucleotides (nt) in length and contains a single large open reading frame (ORF) from nt position 462 to 8411. This ORF encodes a polyprotein with three conserved domains, namely UDP-glycosyltransferase, RNA-dependent RNA polymerase (RdRp), and DEAD-like helicase. The MfHV3 polyprotein shares the highest similarity with Colletotrichum camelliae hypovirus 1. Phylogenetic analysis indicated that MfHV3 clustered with members of the genus Betahypovirus within the family Hypoviridae. Taken together, the results of genomic organization comparisons, amino acid sequence alignments, and phylogenetic analysis convincingly show that MfHV3 is a new member of the genus Betahypovirus, family Hypoviridae.

Inhibition of Monilinia fructicola sporulation and pathogenicity through eucalyptol-mediated targeting of MfCat2 by Streptomyces lincolnensis strain JCP1-7.

Peach brown rot, attributed to Monilinia fructicola, presents a significant threat to postharvest peach cultivation, causing losses of up to 80%. With an increasing number of countries, spearheaded by the European Union, imposing bans on chemical agents in fruit production, there is a growing interest in mining highly active antibacterial compounds from biological control strains for postharvest disease management. In this study, we highlight the unique ability of Streptomyces lincolnensis strain JCP1-7 to inhibit M. fructicola sporulation, despite its limited antimicrobial efficacy. Through GC-MS analysis, eucalyptol was identified as the key compound. Fumigation of diseased fruits with eucalyptol at a concentration of 0.0335 µg cm-3 demonstrated an in vivo inhibition rate against M. fructicola of 93.13%, completely suppressing spore formation. Transcriptome analysis revealed the impact of eucalyptol on multiple pathogenesis-related pathways, particularly through the inhibition of catalase 2 (Cat2) expression. Experiments with a MfCat2 knockout strain (ΔMfCat2) showed reduced pathogenicity and sensitivity to JCP1-7 and eucalyptol, suggesting MfCat2 as a potential target of JCP1-7 and eucalyptol against M. fructicola. Our findings elucidate that eucalyptol produced by S. lincolnensis JCP1-7 inhibits M. fructicola sporulation by regulating MfCat2, thereby effectively reducing postharvest peach brown rot occurrence. The use of fumigation of eucalyptol offers insights into peach brown rot management on a large scale, thus making a significant contribution to agricultural research.

Pseudomonas protegens volatile organic compounds inhibited brown rot of postharvest peach fruit by repressing the pathogenesis-related genes in Monilinia fructicola.

Brown rot, caused by Monilinia fructicola, is considered one of the devasting diseases of pre-harvest and post-harvest peach fruits, restricting the yield and quality of peach fruits and causing great economic losses to the peach industry every year. Presently, the management of the disease relies heavily on chemical control. In the study, we demonstrated that the volatile organic compounds (VOCs) of endophyte bacterial Pseudomonas protegens QNF1 inhibited the mycelial growth of M. fructicola by 95.35% compared to the control, thereby reducing the brown rot on postharvest fruits by 98.76%. Additionally, QNF1 VOCs severely damaged the mycelia of M. fructicola. RNA-seq analysis revealed that QNF1 VOCs significantly repressed the expressions of most of the genes related to pathogenesis (GO:0009405) and integral component of plasma membrane (GO:0005887), and further analysis revealed that QNF1 VOCs significantly altered the expressions of the genes involved in various metabolism pathways including Amino acid metabolism, Carbohydrate metabolism, and Lipid metabolism. The findings of the study indicated that QNF1 VOCs displayed substantial control efficacy by disrupting the mycelial morphology of M. fructicola, weakening its pathogenesis, and causing its metabolic disorders. The study provided a potential way and theoretical support for the management of the brown rot of peach fruits.

Streptomyces virginiae XDS1-5, an antagonistic actinomycete, as a biocontrol to peach brown rot caused by Monilinia fructicola.

BACKGROUND: Peach brown rot, caused by the pathogen Monilinia fructicola, represents a significant postharvest infectious disease affecting peach fruit. This disease is responsible for a substantial increase in fruit decay rates, leading to significant economic losses, often exceeding 50%. Currently, there is a growing interest in identifying biocontrol agents to mitigate peach brown rot, with a predominant interest in Bacillus species. RESULTS: In this investigation, we isolated 410 isolates of actinomycetes from non-farmland ecosystem soil samples. Subsequently, 27 isolates exhibiting superior inhibitory capabilities were selected. Among these, strain XDS1-5 demonstrated the most robust fungistatic effect against brown rot disease, achieving an 80% inhibition rate in vitro and a 66% inhibition rate in vivo. XDS1-5 was identified as belonging to the Streptomyces virginiae species. Furthermore, a fermentation filtrate of XDS1-5 exhibited the ability to metabolize 34.21% of the tested carbon sources and 7.37% of the tested nitrogen sources. Particularly noteworthy was its capacity to disrupt the cell membrane structure directly, leading to increased cell membrane permeability and cytoplasmic leakage. Additionally, our investigation indicated that indoline, a metabolite produced by XDS1-5, played a pivotal role in inhibiting the growth of M. fructicola. CONCLUSION: In summary, our study has identified a biocontrol actinomycete, XDS1-5, with the potential to effectively inhibit postharvest brown rot disease in peaches. This finding holds great significance for the biological control of peach brown rot, offering promising prospects for mitigating the economic losses associated with this devastating disease. © 2024 Society of Chemical Industry.

Global Transcriptome Analysis of the Peach (Prunus persica) in the Interaction System of Fruit-Chitosan-Monilinia fructicola.

The peach (Prunus persica L.) is one of the most important stone-fruit crops worldwide. Nevertheless, successful peach fruit production is seriously reduced by losses due to Monilinia fructicola the causal agent of brown rot. Chitosan has a broad spectrum of antimicrobial properties and may also act as an elicitor that activate defense responses in plants. As little is known about the elicitation potential of chitosan in peach fruits and its impact at their transcriptional-level profiles, the aim of this study was to uncover using RNA-seq the induced responses regulated by the action of chitosan in fruit-chitosan-M. fructicola interaction. Samples were obtained from fruits treated with chitosan or inoculated with M. fructicola, as well from fruits pre-treated with chitosan and thereafter inoculated with the fungus. Chitosan was found to delay the postharvest decay of fruits, and expression profiles showed that its defense-priming effects were mainly evident after the pathogen challenge, driven particularly by modulations of differentially expressed genes (DEGs) related to cell-wall modifications, pathogen perception, and signal transduction, preventing the spread of fungus. In contrast, as the compatible interaction of fruits with M. fructicola was challenged, a shift towards defense responses was triggered with a delay, which was insufficient to limit fungal expansion, whereas DEGs involved in particular processes have facilitated early pathogen colonization. Physiological indicators of peach fruits were also measured. Additionally, expression profiles of particular M. fructicola genes highlight the direct antimicrobial activity of chitosan against the fungus. Overall, the results clarify the possible mechanisms of chitosan-mediated tolerance to M. fructicola and set new foundations for the potential employment of chitosan in the control of brown rot in peaches.

Resistance to the DMI fungicide mefentrifluconazole in Monilinia fructicola: risk assessment and resistance basis analysis.

BACKGROUND: Brown rot disease, caused by Monilinia fructicola, poses a significant challenge to peach production in China. The efficacy of mefentrifluconazole, a new triazole fungicide, in controlling brown rot in peaches has been remarkable. However, the resistance risk and mechanism associated with this fungicide remain unclear. This study was designed to assess the resistance risk of M. fructicola to mefentrifluconazole and reveal the potential resistance mechanism. RESULTS: The mean median effective concentration (EC50 ) of 101 M. fructicola isolates to mefentrifluconazole was 0.003 µg mL-1 , and the sensitivity exhibited a unimodal distribution. Seven mefentrifluconazole-resistant mutants were generated from three parental isolates in the laboratory through fungicide adaption. The biological characteristics of the resistant mutants revealed that three of them exhibited enhanced survival fitness compared to the parental isolates, whereas the remaining four mutants displayed reduced survival fitness. Mefentrifluconazole showed strong positive cross-resistance with fenbuconazole, whereas no cross-resistance was observed with pyrimethanil, procymidone or pydiflumetofen. No overexpression of MfCYP51 gene was detected in the resistant mutants. Multiple sequence alignment revealed that three resistant mutants (MXSB2-2, Mf12-1 and Mf12-2) had a point mutation (G461S) in MfCYP51 protein. Molecular docking techniques confirmed the contribution of this point mutation to mefentrifluconazole resistance. CONCLUSION: The risk of M. fructicola developing resistance to mefentrifluconazole is relatively low-to-medium and point mutation G461S in MfCYP51 could confer mefentrifluconazole resistance in M. fructicola. This study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for mefentrifluconazole. © 2023 Society of Chemical Industry.

Development of a novel 1-octen-3-ol-loaded agar/curdlan hydrogel for inhibiting peach fruit diseases.

Our previous study found that 1-octen-3-ol fumigation treatment could effectively induce the resistance of peach fruit diseases. However, 1-octen-3-ol is a liquid fumigant, which is not conducive to storage and application. Herein, the gel of 1 % agar compound with 1 % curdlan was used as a novel material for covering 1-octen-3-ol. The interaction of agar and curdlan was promoted by adding 1-octen-3-ol, leading to a higher thermostability compared to single-component antibacterial gels. Moreover, 1-octen-3-ol resulted in changes in the internal structure and mechanical properties of gel to form a pore-like structure, which is beneficial to the retention and release of 1-octen-3-ol. Additionally, the 2 % agar gel containing 1-octen-3-ol had the best inhibitory effect on the mycelial growth of Monilinia fructicola and Rhizopus stolonifer in vitro, and the compound hydrogel of 1 % agar and 1 % curdlan with 1-octen-3-ol could most effectively inhibit brown rot and soft rot caused by these two pathogens in vivo. Overall, the data indicated that the novel 1-octen-3-ol-loaded agar/curdlan hydrogels could effectively retain and release 1-octen-3-ol, and induce the resistance of peach fruit diseases.

Hydroxypropyl Methylcellulose and Gum Arabic Composite Edible Coatings Amended with Geraniol to Control Postharvest Brown Rot and Maintain Quality of Cold-Stored Plums.

In this study, the effect of hydroxypropyl methylcellulose (HPMC) and gum Arabic (GA) edible coatings amended with 0.2% geraniol (GE) were evaluated for the control of brown rot, caused by Monilinia fructicola, on artificially inoculated plums (Prunus salicina Lindl., cv. Angeleno) stored for 5 weeks at 1 °C. Brown rot is the most important pre- and postharvest fungal disease of stone fruits, causing severe economic losses worldwide. Geraniol is an important constituent of many essential oils that can be obtained as a byproduct from different industrial procedures, such as those of the juice industry. Fruit postharvest quality was also evaluated after 5 and 8 weeks of storage at 1 °C, followed by 3 days at 7 °C plus 5 days at 20 °C, simulating packinghouse, transport, and retail shelf-life conditions, respectively. HPMC coatings containing 0.2% GE reduced the incidence and severity of brown rot by 37.5 and 64.8%, respectively, compared to uncoated fruit after 5 weeks of storage at 1 °C. HPMC-coated plums, with and without GE, showed the highest level of firmness, the lowest change in external peel color parameters (L*, a*, b*, C*, hue), and the lowest flesh bleeding compared to uncoated control and GA-coated samples throughout the entire storage period, which correlated with a higher gas barrier of these coatings without negatively affecting sensory quality. Furthermore, the HPMC-0.2% GE coating provided the highest gloss to coated plums, showing the potential of this coating as a safe and environmentally friendly alternative to conventional fungicides and waxes for brown rot control and quality maintenance of cold-stored plums.

Stone fruit phenolic and triterpenoid compounds modulate gene expression of Monilinia spp. in culture media.

Phenolic and triterpenoid compounds are essential components in stone fruit skin and flesh tissues. They are thought to possess general antimicrobial activity. However, regarding brown rot disease, investigations were only confined to a limited number of phenolics, especially chlorogenic acid. The activity of triterpenoids against Monilinia spp., as an essential part of the peach cuticular wax, has not been studied before. In this work, the anti-fungal effect of some phenolics, triterpenoids, and fruit surface compound (FSC) extracts of peach fruit at two developmental stages were investigated on Monilinia fructicola and Monilinia laxa characteristics during in vitro growth. A new procedure for assaying anti-fungal activity of triterpenoids, which are notoriously difficult to assess in vitro because of their hydrophobicity, has been developed. Measurements of colony diameter, sporulation, and germination of second-generation conidia were recorded. Furthermore, the expression of twelve genes of M. fructicola associated with germination and/or appressorium formation and virulence-related genes was studied relative to the presence of the compounds. The study revealed that certain phenolics and triterpenoids showed modest anti-fungal activity while dramatically modulating gene expression in mycelium of M. fructicola on culture medium. MfRGAE1 gene was overexpressed by chlorogenic and ferulic acids and MfCUT1 by betulinic acid, at 4- and 7- days of mycelium incubation. The stage II FSC extract, corresponding to the period when the fruit is resistant to Monilinia spp., considerably up-regulated the MfLAE1 gene. These findings effectively contribute to the knowledge of biochemical compounds effects on fungi on in vitro conditions.

Effect of peach trichome removal on post-harvest brown rot and on the fruit surface microbiome.

Postharvest peaches undergo rapid soft ripening and are susceptible to fungal diseases, which often result in severe losses during storage. The peach epidermis contains trichomes that form a specific structure on the peach surface. However, the relationship between trichomes and postharvest disease and involved mechanisms has not been well studied. In this study, the removal of trichomes reduced the disease incidence of peach brown rot caused by Monilinia fructicola. Cryo-scanning electron microscope observations showed that the fungal hyphae were found attached to the surface of trichomes. The fungal and bacterial communities on the peach surface at 0 d and 6 d were obtained by amplicon sequencing technology. Fungal communities on the peach surface contained a total of 1089 amplicon sequence variants (ASVs), which were demarcated into eight fungal phyla, 25 classes, 66 orders, 137 families, and 228 genera. The bacterial communities contained 10,821 ASVs assigned to 25 phyla, 50 classes, 114 orders, 220 families, and 507 genera. Higher bacterial diversity than fungal diversity was recorded on the peach epidermis. Trichome removal changed the microbial diversity and community on the peach surface. Compared with peach epidermis samples, the peach epidermis excluded trichomes samples contained similar fungal alpha diversity but significantly lower bacterial diversity. Seventeen different fungal genera and twenty-eight different bacterial genera were identified between peach trichome and peach epidermis excluded trichomes samples. The fungal and bacterial diversity on the peach epidermis showed a decreasing trend during storage. Beta diversity analysis revealed that the microbial communities of the peach epidermis and trichomes show different change trends between 0 d and 6 d. Trichome removal decreased relative abundance of Monilinia spp. and increased relative abundance of potential yeast and bacterial biocontrol agents. This study suggested that trichomes might modulate the microbial communities on fruit surfaces, and trichome removal technology after harvest might be developed to control peach postharvest decay.

NADES blend for bioactive coating design as a sustainable strategy for postharvest control.

Bioactive functional coatings constitute a trendy topic due to they reduce postharvest fruit losses worldwide. Also, they could be carriers of biocompounds providing health benefits to the consumer. In this work, an innovative natural bioactive coating based on Natural Deep Eutectic Solvents (NADES) and Larrea divaricata extract was optimized by mixture-mixture design for the management of postharvest diseases caused by Monilinia fructicola. A NADES composed of lactic acid-glucose-water (LGH) for phenolic extraction from L. divaricata was optimized by a Simplex Lattice design and response surface methodology (RSM).Then, a d-optimal mixture-mixture design was carried out in order to optimize the bioactive coating composition, being the optimal proportion of 0.7 L. divaricata-LGH extract and 0.3 NADES plasticizer (composed by glycerol, citric acid and water). The optimal biocoating achieved an in vitro antimicrobial activity of 72 % against M. fructicola. Interestingly, NADES plasticizer improves the biocoating functionality, creating a smooth and uniform surface.

Development of natamycin-loaded zein-casein composite nanoparticles by a pH-driven method and application to postharvest fungal control on peach against Monilinia fructicola.

This work fabricated natamycin-loaded zein-casein nanoparticles (N-Z/C NPs) by a pH-driven approach and applied to control postharvest peach brown rot caused by Monilinia fructicola. When casein and phosphoric acid were used as a stabilizer and neutralizing acid, respectively, NPs with mean particle sizes < 100 nm and zeta-potentials < -30 mV could be obtained. The NPs could increase the aqueous dispersibility of natamycin and showed high stability against environmental changes, which could be attributed to both hydrophobic stacking and hydrogen bonds between natamycin and zein. Besides, the effects of N-Z/C NPs on the storage of peach were assessed in vitro and in vivo. Nanoencapsulation did not affect the antifungal activities of natamycin. The NPs with 20 and 80 µg/mL of natamycin could basically inhibit the spore germination and mycelial growth of M. fructicola, respectively. The N-Z/C NPs coatings exhibited better results than natamycin in controlling of peach brown rot.

Prevalence of H6Y mutation in ß-tubulin causing thiophanate-methyl resistant in Monilinia fructicola from Fujian, China.

Brown rot disease broke out in stone fruit orchards of Fujian, China in 2019, despite pre-harvest application of methyl benzimidazole carbamate (MBC). To determine the reason, a total of 44 Monilinia fructicola strains were collected from nectarine, plum and peach fruits in this study, among which 79.5% strains were resistant to thiophanate-methyl, indicated by discriminatory dose of 5 µg/mL. The resistance of these strains was confirmed by treating detached peach fruit with label rates of formulated thiophanate-methyl which only completely inhibit infection of the sensitive strains, but not the resistant strains. Further analysis of the mechanism of MBC resistance revealed that all resistant strains carry a H6Y mutation in ß-tubulin protein Tub2, which was only reported previously in the M. fructicola strains from California, USA, and do not display obvious fitness penalties, as no significant defects in mycelial growth rate, sporulation, conidia germination, aggressiveness on detached peach fruit and temperature sensitivity was detected. In addition, we found that diethofencarb, the agent for managing MBC-resistance strains, was unable to inhibit growth of the H6Y strains. Taken together, our study, for the first time, identified a mutation form of H6Y in the ß-tubulin protein of M. fructicola in China, rendering the strains wide resistance to thiophanate-methyl. This mechanism of M. fructicola gaining resistance to MBC fungicides needs to be fully considered, when designing management strategies to control brown rot disease in stone fruit orchards.

Postharvest Treatments with Sulfur-Containing Food Additives to Control Major Fungal Pathogens of Stone Fruits.

The sulfur-containing salts, classified as food additives, sodium metabisulfite (SMBS), potassium metabisulfite (PMBS), aluminum sulfate (AlS), and aluminum potassium sulfate (AlPS), were evaluated for their activity against Monilinia fructicola, Rhizopus stolonifer, and Geotrichum candidum, the most economically important fungal pathogens causing postharvest disease of stone fruit. In in vitro tests with potato dextrose agar (PDA) Petri dishes amended with different concentrations of the salts (0, 10, 20, 30, 50, and 100 mM), SMBS and PMBS at all concentrations, AlS above 20 mM, and AlPS above 30 mM, completely inhibited the mycelial growth of the three fungi after incubation at 25 °C for up to 10 days. In in vivo primary screenings with artificially inoculated nectarines, aqueous solutions of the four salts reduced the incidence and severity of brown rot (BR) at concentrations of 10 and 50 mM, whereas only AlS and AlPS reduced Rhizopus rot (RR), and none of the salts was effective against sour rot (SR). Solutions at 100 mM were phytotoxic and injured the fruit peel. In small-scale trials, 1 min dip treatments at 20 °C in SMBS or PMBS at 10 mM significantly reduced the incidence and severity of BR after incubation at 20 °C for up to 8 days. Conversely, dips in AlS and AlPS reduced neither BR nor RR. Results highlight the potential of SMBS and PMBS as new nonpolluting tools for the integrated control of BR, but not RR and SR, on stone fruit.

In Vitro Antifungal Activity and Toxicity of Dihydrocarvone-Hybrid Derivatives against Monilinia fructicola.

The aim of this study was to synthesize a series of novel and known dihydrocarvone-hybrid derivatives (2-9) and to evaluate mycelial growth activity of hybrid molecules against two strains of Monilinia fructicola, as well as their toxicity. Dihydrocarvone-hybrid derivatives have been synthesized under sonication conditions and characterized by FTIR, NMR, and HRMS. Antifungal efficacy against both strains of M. fructicola was determined by half maximal effective concentration (EC50) and toxicity using the brine shrimp lethality test (BSLT). Among the synthesized compounds, 7 and 8 showed the best activity against both strains of M. fructicola with EC50 values of 148.1 and 145.9 µg/mL for strain 1 and 18.1 and 15.7 µg/mL for strain 2, respectively, compared to BC 1000® (commercial organic fungicide) but lower than Mystic® 520 SC. However, these compounds showed low toxicity values, 910 and 890 µg/mL, respectively, compared to Mystic® 520 SC, which was highly toxic. Based on the results, these hybrid compounds could be considered for the development of more active, less toxic, and environmentally friendly antifungal agents against phytopathogenic fungi.

Antifungal mechanisms of lavender essential oil in the inhibition of rot disease caused by Monilinia fructicola in postharvest flat peaches.

Lavender essential oil (LEO), a natural antimicrobial agent, is generally recognized as safe and effective in the inhibition of phytopathogenic fungi. Direct contact and fumigation (in vivo and in vitro) were used to study the fungistatic effect of LEO on Monilinia fructicola. Additionally, the effect on the ultrastructure of cells and the degree of destruction of the cell membrane of M. fructicola were revealed. In addition, the effects of LEO on the expression levels of apoptosis-related genes in M. fructicola cells were detected, and GC-MS was used to analyze the main components of LEO. LEO had a good inhibitory efficacy against M. fructicola in flat peaches, with almost complete growth inhibition at 800 µL/L. These effects were associated with the leakage of cytoplasmic contents, hyphal distortion, and spore disruption. Moreover, the expression of apoptosis RTG1 and RLM1 genes increased with LEO treatment. These results demonstrate that LEO can inhibit M. fructicola by inducing cytoplasmic membrane damage and cell apoptosis in fungi, and that the major ingredients of LEO are monoterpenes and sesquiterpenes, which are presumed to contribute to the inhibitory effects.

First Multi-Target Application of Exclusion Net in Nectarine Orchards: Effectiveness against Pests and Impact on Beneficial Arthropods, Postharvest Rots and Fruit Quality.

Over the past few years, there has been an increasing interest in the development of alternative pest control strategies to reduce environmental impact. In this contest, exclusion nets have been evaluated as a sustainable alternative to pesticides. In this study, the use of a photoselective exclusion net was investigated in semi-field conditions as a potential strategy to protect nectarine orchards from different pests (i.e., fruit moths, Halyomorpha halys and Drosophila suzukii) in NW Italy. The presence and abundance of pest populations inside and outside the net, as well as the damage they caused on fruits, were evaluated. Moreover, any possible effects of the net on beneficial arthropods, postharvest rots and fruit quality and nutraceutical parameters were considered. The exclusion net significantly reduced pest populations. At harvest, fruit damage caused by Grapholita molesta and H. halys in netted plots was reduced up to 90% and to 78%, respectively, compared with insecticide-treated plots. The exclusion net allowed the production of healthier fruits with a strong reduction of insecticide treatments (up to seven less) and of their related costs without any negative impact on postharvest rots, neither fruit quality nor nutraceutical properties.

Extracellular laccase from Monilinia fructicola: isolation, primary characterization and application.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.