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Gray mold and brown rot, caused respectively by Botrytis cinerea and Monilinia spp., are fungal diseases responsible for significant losses during the storage of fruit and vegetables. Nowadays, the control of postharvest diseases is shifting towards more sustainable strategies, including the use of plant secondary metabolites. In this study, the antifungal activity of Origanum vulgare , Thymus vulgaris , Thymus serpyllum , Melaleuca alternifolia , Lavandula officinalis , Lavandula hybrida , Citrus bergamia , Rosmarinus officinalis , Cinnamomum zeylanicum essential oils (EOs) in vapor phase was tested in vitro against B. cinerea, Monilinia fructicola , Monilinia fructigena , and Monilinia laxa . For the experiments, a protocol using a volatile organic compounds (VOC) chamber was designed. Results indicate a dose-dependent inhibitory activity of all the tested EOs, with O. vulgare , T. vulgaris , and T. serpyllum being the most active ones, with minimum inhibitory concentrations (MIC) of 22.73, 45.45, and 22.73 µl/L, respectively, against B. cinerea and a range between 5.64 and 22.73 µl/L against the three Monilinia spp. Overall, B. cinerea presented lower sensitivity to vapor-phase EOs than any of the Monilinia strains, except for the C. zeylanicum EO, which consistently showed higher inhibition against B. cinerea . Among the three Monilinia spp., M. fructicola was the least sensitive, while M. fructigena was the most sensitive. The use of VOC chambers proved to be a reliable protocol for the assessment of antimicrobial activities of EOs. These results suggest that the VOC emitted by the tested EOs are effective towards important decay-causing fungi, and that they could be used for the control of gray mold and brown rot in in vivo trials.

Background Brown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola , M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses. Results Illumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola , 31,103 for M. laxa and 28,890 for M. fructigena . Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea , Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola , 30 in M. laxa and 31 in M. fructigena . Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements. Conclusions This is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola , M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi.

Cyanobacteria synthesize secondary metabolites with antifungal activity, making them potential biopesticide agents for sustainable, eco-friendly agriculture. Programmes to identify Cyanobacterial strains with effective bioactivity generally screen strains maintained in culture collections. These strains are often monoclonal but non-axenic and this may potentially influence the bioactivity of the generated biomass. The present study investigated in vitro antifungal activity of Nostoc muscorum MACC-189 and N. linckia MACC-612 strains co-isolated with fungal co-partners and maintained in the Mosonmagyaróvár Algal Culture Collection (MACC). The fungal co-partners were isolated from the Cyanobacterial stock cultures and identified as Purpureocillium lilacinum and Sarocladium sp., respectively. The cultures were tested against seven phytopathogens. The phytopathogenic fungi were grown on potato dextrose agar plates and suspension cultures of the Cyanobacteria-fungi and isolated fungal co-partners were placed in the centre of the plate. Antifungal effects were assessed semi-quantitatively after 10 days of incubation. The Cyanobacteria-fungal co-cultures had antifungal activity against Monilinia fructigena and Aspergillus sp. with the N. muscorum/P. lilacinum culture being the most effective. The fungal isolates inhibited M. fructigena with P. lilacinum having a dose-dependent response but did not inhibit Aspergillus sp. This suggested that the antifungal effect of the Cyanobacterial cultures on M. fructigena was due to the fungal partner rather than the cyanobacterium while the antifungal effect on Aspergillus sp. was due to the cyanobacterium partner. As it was not possible to maintain living axenic N. muscorum and N. linckia cultures, this could not be conclusively confirmed. These results highlight the importance of either using axenic cultures or identifying the co-isolates when testing Cyanobacteria cultures for antifungal bioactivity.

Necrotrophic phytopathogenic fungi secrete degrading enzymes and phytotoxins to penetrate and colonise plant tissues. Since knowledge on the pathogenic processes of brown rot in fruit which becomes infected by Monilinia spp. is limited, we undertook an investigation whose aims were to detect: (i) the degrading enzymes of Monilinia spp. when cultured on media amended with different substrates, and (ii) the phytotoxins which are secreted by Monilinia fructicola, M. fructigena and M. laxa in brown rot-infected fruit. The sporulation and growth of 10 M. fructicola isolates, 9 M. fructigena isolates, and 10 M. laxa isolates plates, which contained 25 mL 2% (w/v) bacteriological agar, 0.67% (w/v) yeast nitrogen base, and 1% (w/v) of one of the following substrates: (1) β1–3 glucan (callose), (2) casein hydrolysate, (3) cutin, (4) methylcellulose, (5) apple pectin, (6) polygalacturonic acid sodium salt, (7) D(+)-sucrose, and (8) xylan were determined in order to detect the degrading enzymes. To look for the phytotoxins, the juice of nectarines that were inoculated with conidial suspensions M. fructicola, M. fructigena and M. laxa were subjected to liquid-liquid extraction with different organic solvents. The extracts were tested for their ability to cause necrosis on nectarine discs and the ones with a positive response were analysed by high performance liquid chromatography with DAD and MS spectrometer detectors and different fractions were collected to delimit metabolites. We found that Monilinia spp. secrete phytotoxins with a molar mass between 329 and 387 g mol−1 and degrading enzymes (cutinases, α-glucosidases, pectin lyases, polygalacturonases, proteases, and xylanases) for penetrating the fruit surface and invading and colonising fruit tissues.

   Plant diseases cost the global economy billions of US dollars every year. The problem has mainly been addressed by using chemical pesticides, but recently, the use of ants has shown promising effects against plant pathogens. However, the mechanisms accounting for these effects have not yet been determined. One possible explanation is antimicrobial microorganisms associated with ants. Through controlled laboratory experiments, we investigated the inhibitory effects of wood ants ( Formica polyctena ) and their associated microorganisms against economically important plant pathogenic fungi. All live ants, extracts from crushed ants, and extracts from washed ants significantly inhibited the apple brown rot ( Monilinia fructigena ) while yielding the growth of other microbes. Furthermore, all investigated wood ants transferred microorganisms to their surroundings within 10 s when walking across a surface. We isolated the most dominant microorganisms deposited by walking ants and from washed ant extracts (i.e., strains likely found on the surface of ants), resulting in four bacterial cultures and one yeast. Two of these isolates, strain I3 (most closely related to Pseudomonas sichuanensis and P. entomophila ) and strain I1b (most closely related to Bacillus mycoides ), showed inhibitory effects against apple brown rot and apple scab ( Venturia inaequalis ), while strain I3 also inhibited gray mold ( Botrytis cinerea ) and Fusarium head blight ( Fusarium graminearum ). These results suggest that wood ants have potential as biological control agents against commercially relevant plant pathogens, and that their inhibitory effect might be at least partially caused by antibiotic compounds produced by their associated microorganisms.

Monilinia fructigena is one of the most important fungal pathogens causing brown rot on apple and is heavily affecting fruit production. The main objective of this study was to screen for potential bacterial isolates with higher antagonistic activity against M. fructigena. Our study focused on the identification of potential bacterial isolates capable of reducing both the mycelial growth of M. fructigena and the disease severity using in vitro and in planta trials, respectively. To achieve this goal, thirteen bacteria, isolated from natural soil, were evaluated for their abilities to produce lytic enzymes (amylase, cellulase and protease), hydrocyanic acid (HCN) and lipopeptides (bacillomycin, fengycin, iturin and surfactin). Further, results from the dual culture method, volatile and bacterial free-cell filtrate bioassays indicated that tested isolates showed a fungicidal activity against the mycelial growth of M. fructigena. Thus, out of the 13 isolates tested, 12 exhibited significant mycelial inhibition (more than 70%) against M. fructigena, while remaining the last isolate displayed only a partial inhibition (up to 43%). Further, 12 of the bacteria isolates displayed an amylase production, 10 showed cellulase production, 11 revealed protease production, while only 2 displayed HCN production. In addition, most bacterial isolates were found to have genes encoding for different lipopeptides: bacillomycin (10), fengycin (3), iturin (11) and surfactin (1). Interestingly, two bacterial isolates, Bacillus amyloliquefaciens B10W10 and Pseudomonas sp. B11W11 were found to be the most effective and displayed the lowest disease severity in planta trial. These two bacteria reduced the brown rot incidence compared to the synthetic fungicide in a semi-commercial large-scale trial. Therefore, our findings suggest that these two later bacterial isolates provide apple protection against M. fructigena via direct and indirect mechanisms. These isolates may be used, therefore, as potential biological control agents (BCAs) in preventive treatment to control brown rot disease on apple fruits.

Monilinia fructigena, a causal agent of brown rot in apple and other fruit crops, poses a significant threat to fruit production and postharvest quality in temperate regions. This study reports on the molecular and morphological identification of M. fructigena isolates obtained from symptomatic apple fruits in the Almaty region of Kazakhstan. Nine isolates were characterized through a combination of morphological assessment, real-time PCR, target locus (ITS and TEF1-α gene) sequencing, and whole genome sequencing using nanopore sequencings. Morphological analysis revealed typical features of M. fructigena, including blastoconidia and microconidia. Pathogenicity tests on ‘Idared’ and ‘Golden Delicious’ apples confirmed the high aggressiveness of the isolates, with lesion development observed within 24–48 h post-inoculation. Molecular identification via real-time PCR and target sequencing confirmed all isolates as M. fructigena with high mapping quality and sequence identity. The whole genome sequencing of a representative isolate further validated the species identity based on comparative alignment with Monilinia reference genomes. Thus, the combination of the used traditional and molecular methods allowed us to unambiguously identify the isolated fungus as M. fructigena. This integrative approach enhances the understanding of Monilinia species in Central Asia and supports the implementation of modern molecular tools for phytopathogen surveillance and agricultural biosecurity.

The main objective of this study was to evaluate the ability of both antagonistic bacteria Bacillus amyloliquefaciens (SF14) and Alcaligenes faecalis (ACBC1) used in combination with salicylic acid (SA) to effectively control brown rot disease caused by Monilinia fructigena. Four concentrations of salicylic acid (0.5%, 2%, 3.5%, and 5%) were tested under in vitro and in vivo conditions. Furthermore, the impact of biological treatments on nectarine fruit parameters’ quality, in particular, weight loss, titratable acidity, and soluble solids content, was evaluated. Regardless of the bacterium, the results indicated that all combined treatments displayed a strong inhibitory effect on the mycelial growth of M. fructigena and disease severity. Interestingly, all SA concentrations significantly improved the biocontrol activity of each antagonist. The mycelial growth inhibition rate ranged from 9.79% to 88.02% with the highest reduction rate recorded for bacterial antagonists in combination with SA at both concentrations of 0.5% and 3.5%. The in vivo results confirmed the in vitro results with a disease severity varying from 0.00% to 51.91%. A significant biocontrol improvement was obtained with both antagonistic bacteria when used in combination with SA at concentrations of 0.5% and 2%. The lowest disease severity observed with ACBC1 compared with SF14 is likely due to a rapid adaptation and increase of antagonistic bacteria population in wounded sites. The impact of all biological treatments revealed moderate significant changes in the fruit quality parameters with weight loss for several treatments. These results suggest that the improved disease control of both antagonistic bacteria was more likely directly linked to both the inhibitory effects of SA on pathogen growth and induced fruit resistance.

Simultaneous treatment with antagonistic bacteria Bacillus amylolquefaciens (SF14), Alcaligenes faecalis (ACBC1), and the food additive sodium bicarbonate (SBC) to control post-harvest brown rot disease caused by Monilinia fructigena, and their effect on the post-harvest quality of nectarines were evaluated. Four concentrations of SBC (0.5, 2, 3.5, and 5%) were tested. Results showed that bacterial antagonists displayed remarkable compatibility with different concentrations of SBC and that their viability was not affected. The results obtained in vitro and in vivo bioassays showed a strong inhibitory effect of all treatments. The combination of each bacterial antagonist with SBC revealed a significant improvement in their biocontrol efficacies. The inhibition rates of mycelial growth ranged from 60.97 to 100%. These results also indicated that bacterial antagonists (SF14 or ACBC1) used at 1 × 108 CFU/ mL in combination with 2, 3.5, or 5% SBC significantly improved the control of M. fructigina by inhibiting the germination of spores. Interestingly, disease incidence and lesion diameter in fruits treated with SF14, ACBC1 alone, or in combination with SBC were significantly lower than those in the untreated fruits. In vivo results showed a significant reduction in disease severity ranging from 9.27 to 64.83% compared to the untreated control, while maintaining the appearance, firmness, total soluble solids (TSS), and titratable acidity (TA) of fruits. These results suggested that the improved disease control by the two antagonistic bacteria was more likely due to the additional inhibitory effects of SBC on the mycelial growth and spore germination of the pathogenic fungus. Overall, the combination of both bacteria with SBC provided better control of brown rot disease. Therefore, a mixture of different management strategies can effectively control brown rot decay on fruits.

Use of novel alternative compounds in agriculture is being promoted to reduce synthetic pesticides. An in vitro study was conducted to evaluate antimicrobial and antioxidant activities of chitosan hydrochloride (CH) and COS (chito-oligosaccharides)-OGA (oligo-galacturonides) at concentrations of 1%, 0.5%, 0.25%, 0.1%, 0.05%, and 0.025%. COS-OGA at 1% and 0.5% concentrations completely inhibited mycelial growth of Alternaria alternata, Alternaria brassicicola, Botrytis cinerea, Monilinia laxa, Monilinia fructigena, and Monilinia fructicola. Further, complete inhibition was observed with 0.25% COS-OGA for M. fructigena and M. laxa. Inhibition for B. cinerea, M. fructicola, A. alternata, and A. brassicicola at 0.25% COS-OGA was 86.75%, 76.31%, 69.73%, and 60.45%, respectively. M. laxa and M. fructigena were completely inhibited by CH concentrations of 1–0.25% and M. fructicola by concentrations of 1–0.5%. At CH 0.25%, inhibition for M. fructicola, A. brassicicola, A. alternata, and B. cinerea was 93.99%, 80.99%, 69.73%, and 57.23%, respectively. CH showed effective antibacterial activity against foodborne Escherichia coli. COS-OGA had higher antioxidant activity than CH when assessed by DPPH and hydroxyl radical scavenging assays. Our findings offer insights into the antimicrobial efficacy and mechanisms of action of these novel compounds, which have the potential to serve as alternatives to synthetic pesticides. In vivo investigations are required to validate the prospective application of these treatments for pre- and postharvest disease management.