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Strawberry fruit is highly susceptible to decay by fungi. The objective of this study was to determine if essential oils (EOs) or nano-emulsions (Nano-EM) of EOs from Thymus vulgaris (Th), Matricaria chamomilla (Mc), Pistacia atlantica (Pa), or Mentha longifolia (Me) could inhibit growth of strawberry spoilage fungi Botrytis cinerea and their effect, if any, on strawberry quality parameters. An In vitro study showed that Th and Me EOs had the same minimal inhibitory concentrations (MIC) of 0.021 % while the MICs for Mc and Pa EOs were 0.9 % and 1.5 %, respectively. Th and Me EOs were used for subsequent experiments. In the second experiment, the application of Th and Me EOs and their nano-EM at 0.021, 0.1, 0.5 and 1 % were studied to control B. cinerea growth on the fruit surface. Nano-EM of EOs had higher antifungal activity in the control of B. cinerea than EOs on fruit surface. Generally, antifungal activity was increased at higher concentrations of Nano-EM, but in the case of EOs, their antifungal activity was not increased by increasing concentration. Nano-EM of EOs with 0.5 % was selected for further study. Finally, the quality changes and postharvest losses of fruit treated with Nano-EM of EOs of Th and Me at 4 °C were studied. The results of third experiment showed that Nano-EM of both EOs reduced microbial load, decay index, weight loss and induced greater firmness, vitamin C, total flavonoid and antioxidant activity in strawberry during storage. NanoEM-ThEO 0.5 % was more effective than NanoEM-MeEO 0.5 % to retain strawberry firmness, vitamin C and total flavonoid.

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An antagonistic yeast strain was isolated from the strawberry fruit cv. Paros, and its antifungal properties against Botrytis cinerea causal agent of strawberry grey mold disease were evaluated under in vitro and in vivo experiments. The isolate was tentatively identified as Tetrapisispora sp. strain 111A-NL1 based on phenotypic characteristics and sequence analysis of D1/D2 domains of the 26S rRNA gene. Volatile organic compounds (VOCs) produced by the 111A-NL1 strain inhibited the mycelial growth of fungal pathogen (75.19%) and conidial germination (63.34%); however, inhibition percentage of mycelial growth of pathogen by dual culture test was less (19.49%). Also, the strain produced pectinase, siderophore, chitinase, IAA, as well as gibberellin, and could solubilize phosphate. Additionally, the disease severity of strawberry grey mold was decreased by employing living cells and volatile metabolites methods by 47.61% and 74.05%, respectively, in comparison with untreated control seven days after inoculation. Therefore, its mode of action might consist of antibiosis and VOCs production by yeast strain 111A-NL1 against B. cinerea. The VOCs released by strain 111A-NL1 were analyzed, and thirty-three chemical compounds were determined by gas chromatography-mass spectroscopy (GC-MS). Out of them, Decane (12.79%), Squalene (9.60%), Undecane (7.98%), Benzene, 1,2,3-trimethyl- (7.67%), Nonane, 2,6-dimethyl- (5.69%), Benzene, 1-ethyl-3-methyl- (5.55%), Mesitylene (4.17%), and Phenylethyl Alcohol (3.33%) were the major components. In addition, the selected strain reduced natural decay incidence and weight loss of fruit, and preserved quality parameters of strawberry fruit including firmness, soluble solids content, and titratable acidity. This research averred, for the first time, that the creation of VOCs by Tetrapisispora sp. strain 111A-NL1 could play an essential role as a biofumigant in the antifungal activity against strawberry grey mold.

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Several bacterial strains belonging to Serratia spp. possess biocontrol capability, both against phytopathogens and human pathogenic species, thanks to the production of secondary metabolites, including as a red-pink, non-diffusible pigment, 2-methyl-3-pentyl-6-methoxyprodiginine (prodigiosin). Botrytis cinerea is the causal agent of gray mold, which is an economically relevant disease of many crops worldwide. Gray mold is normally controlled by chemical fungicides, but the environmental and health concerns about the overuse of pesticides call for environmentally friendly approaches, such as the use of biocontrol agents. In this study, the efficacy of a specific strain of Serratia rubidaea (Mar61-01) and its metabolite prodigiosin were assessed against B. cinerea under in vitro and in vivo conditions. This strain was effective against B. cinerea, and the effect of prodigiosin was confirmed under in vitro and in vivo conditions. The strain suppressed mycelial growth of B. cinerea (71.72%) in the dual-culture method. The volatile compounds produced by the strain inhibited mycelial growth and conidia germination of B. cinerea by 65.01% and 71.63%, respectively. Efficacy of prodigiosin produced by S. rubidaea Mar61-01 on mycelial biomass of B. cinerea was 94.15% at the highest concentration tested (420 µg/mL). The effect of prodigiosin on plant enzymes associated with induction of resistance was also studied, indicating that the activity of polyphenol oxidase (PPO), superoxide dismutase (SOD) and phenylalanine ammonia lyase (PAL) were increased when prodigiosin was added to the B. cinerea inoculum on strawberry fruits, while catalase (CAT) and peroxidase (POD) did not change. In addition, the volatile organic compounds (VOCs) produced by S. rubidaea Mar61-01 reduced mycelial growth and inhibited conidial germination of B. cinerea in vitro. The findings confirmed the relevant role of prodigiosin produced by S. rubidaea Mar61-01 in the biocontrol of B. cinerea of strawberries, but also indicate that there are multiple mechanisms of action, where the VOCs produced by the bacterium and the plant-defense reaction may contribute to the control of the phytopathogen. Serratia rubidaea Mar61-01 could be a suitable strain, both to enlarge our knowledge about the potential of Serratia as a biocontrol agent of B. cinerea and to develop new biofungicides to protect strawberries in post-harvest biocontrol.

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Didymella glomerata and Truncatella angustata associated with grapevine trunk diseases (GTDs) in Iran, were grown in vitro to evaluate the production of phytotoxic metabolites as potential pathogenicity determinants. 2,5-Dihydroxymethylfuran and (+)-6-hydroxyramulosin were isolated from the culture filtrates of D. glomerata and T. angustata, respectively. They were identified by physical and spectroscopic (essentially 1 D and 2 D 1H and 13C NMR and ESIMS) methods and X ray analysis. Both compounds induced significant necrosis and curling on the leaves of the host plant Vitis vinifera L. and the effects were concentration dependent. No effect was observed on the leaves of the non-host Solanum lycopersicum L.. plant.

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The phytopathogenic fungus Truncatella angustata, associated with grapevine trunk diseases (GTDs) in Iran, produces the well-known secondary metabolite isocoumumarin (+)-6-hyroxyramulosin and surprisingly also phenazine-1-carboxylic acid (PCA). PCA, identified by spectroscopic (essentially 1H NMR and ESI MS) spectra, is a bacterial metabolite well known for its antifungal activity and was found for the first time in T. angustata culture filtrates. The antifungal activity of PCA was assayed against four different fungi responsible for GTDs, Phaeoacremonium minimum, Phaeoacremonium italicum, Fomitiporia mediterranea, involved in grapevine esca disease, and Neofusicoccum parvum, responsible for Botryosphaeria dieback. The activity was compared with that of the known commercial fungicide, pentachloronitrobenzene, and the close phenazine. PCA and phenazine exhibited strong antifungal activity against all phytopathogenic fungi, inhibiting the fungal growth by about 90-100% and 80-100%, respectively. These results suggested that T. angustata could use PCA to compete with other phytopathogenic fungi that attack grapevine and thus PCA could be proposed as a biofungicide against the fungi responsible for grapevine esca and Botryosphaeria dieback diseases.

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Grapevine trunk diseases (GTDs) are well-known and significant fungal diseases of Vitis vinifera with a worldwide distribution. During August to November 2016 in a survey to characterize fungi associated with grapevine trunk diseases in Kermanshah Province (west of Iran) vineyards, 286 fungal isolates were obtained. Based on morphology and DNA sequences data eight species were identified, of which Biscogniauxia rosacearum, Neoscytalidium hyalinum and Phaeoacremonium minimum were the most aggressive fungal pathogenic species characterized in this research. N. hyalinum was the most prevalent species. N. hyalinum and Ph. minimum have previously been reported from Vitis vinifera. Thus far, there are two records of Biscogniauxia mediterranea and Biscogniauxia capnodes on grapevine in the world with no data on pathology aspects. To our knowledge, it is the first time B. rosacearum is reported from grapevine across the globe. Pathogenicity test with three strains of B. rosacearum on 2-year-old potted grapevines confirmed the pathogenicity of B. rosacearum on grapevine. The proximity of vineyards to the oak trees in Zagros forests as one of the plant hosts of Biscogniauxia spp. further highlights the need for extensive studies on B. rosacearum as a new fungal pathogen.

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A strain of Kalmusia variispora associated with grapevine trunk diseases (GTDs) was identified in Iran and induced disease symptoms on the host in greenhouse conditions. The grapevine pathogens are able to produce a plethora of toxic metabolites belonging to different classes of naturally occurring compounds. Two homogeneous compounds were isolated from the organic extract of K. variispora culture filtrates. They were identified by physic (specific optical rotation), and spectroscopic (essentially 1D 1H and 13C NMR and HR ESIMS) methods as the fungal polyketides massarilactones D and H (1 and 2). The unassigned absolute configuration of massarilactone D was unambiguously determined by X-ray diffractometric analysis. Massarilactones D and H showed phytotoxic activity on Vitis vinifera L. at two concentrations used and depending from the days of inoculation. Phytotoxicity is also increased when the 3,4,7-O,O',O"-triacetyl derivative of massarilactone D (3) was assayed on the host plant. This is the first report on the investigation of phytotoxic metabolites produced by K. variispora isolated from infected grapevine in Iran and they seem to be involved in the development of disease symptoms.

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This study assessed the feasibility of using essential oils (EOs) against Colletotrichum nymphaeae inciting strawberry anthracnose. Two EOs, extracted from Allium sativum (garlic) and Rosmarinus officinalis (rosemary), were selected because their fungicide efficacy was already well characterized under laboratory and greenhouse conditions. We characterized both EOs in terms of efficacy and impact on qualitative traits and sensory quality of strawberry fruit. The gas chromatography-mass spectrometry analysis confirmed the Diallyl trisulfide (29.08%) and (α)-pinene (15.779%) as the main components of A. sativum and R. officinalis EOs, respectively. Both A. sativum and R. officinalis EOs significantly inhibited the mycelial growth and conidial germination of C. nymphaeae in contact and vapor assays compared with untreated control. However, EC50 assay indicated A. sativum EO was more effective than R. officinalis EO against the pathogen. Malformations of the vegetative structures of the pathogen exposed to both EOs were revealed as shriveled, collapsed, and swelling mycelia in the cultures. Both EOs confirmed their efficacy under in vivo and greenhouse conditions; in fact, they significantly reduced the development of fruit decay and anthracnose disease incidence and severity, compared with untreated controls. Both EOs preserved sensory attributes and quality parameters of strawberry fruit including firmness, total soluble solids, ascorbic acid, antioxidant activity, and anthocyanin, but may leave unwanted smells. These findings suggest that two EOs can potentially represent an alternative to synthetic chemical fungicides against C. nymphaeae preserving fruit quality factors, although their cost and the impact on the fruit odor must be carefully taken into consideration before developing a commercial product.

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Combined application of plant essential oils (EOs) with known antimicrobial effects and silica nanocapsules with high loading capacity and protection capability of the EOs make them proper candidates for creating environmentally friendly fungicides. In this study, EOs of the Lemongrass (LGO) and Clove (CO) were used against Gaeumannomyces graminis var. tritici (Ggt), a causal agent of take-all disease of wheat. To provide controlled delivery of the EOs, they were encapsulated into mesoporous silica nanoparticles (MSNPs) and then compared to the effects of pure EOs both in- vitro and in- vivo. MSNPs were synthesized via the sol-gel process. Various techniques such as Fourier transform infrared spectroscopy (FTIR), the Brunauer-Emmett-Teller (BET), thermogravimetric analysis (TGA), and UV-Vis spectroscopy were used to evaluate the successful loading of the EOs into the pore of MSNPs. The encapsulation efficiency (EE) was calculated as high as 84.24% for LGO and 80.69% for CO, while loading efficiency (LE) was determined 36% and 29% for LGO and CO, respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) displayed spherical shapes and porous structures with average diameters of 50-70 nm. Recognition of the main components of the EOs via gas chromatographic-mass spectrometry (GC-MS) before and after the EO loading, detected eugenol and citral as the most frequent compounds in LGO and CO, respectively. For antifungal test in- vitro, selected concentrations of the pure EOs, EOs loaded in MSNPs (EOs- MSNPs) and Mancozeb ® fungicide based on pre-tests were mixed using potato dextrose agar (PDA). The inhibition percentage (IP) of fungal growth at each concentration, as well as minimum inhibition concentration (MIC) and minimum fungicidal concentrations (MFC) were obtained. The results indicated that antifungal effects in the encapsulated form increased by up to three times. In- vivo, the sterile wheat seeds were treated with pure EOs, EOs-MSNPs, and mancozeb at MFC concentration. Also, in order to keep on the EOs-MSNPs around the seeds, sodium alginate was used. The consequences of in- vivo experiments indicated that rate of disease control in presence of EOs-MSNPs and mancozeb was the same (~70%) and higher than pure EOs (LGO: 57.44%, CO: 49%). Also, improving the growth parameters in wheat plant, the covering of the EOs-MSNPs in alginate, had better control (84%) than that of EOs-MSNPs alone. Further, the release kinetics studies showed a gradual release of LGO and CO from MSNPs for four weeks in water and for five weeks in the soil-plant system. To the best of our knowledge, this is the first report of the control effect of LGO, CO, and their nanocapsule in MSNPs against the take-all disease of wheat. These results showed that the EOs-MSNPs can be a safe product for the efficient control of take-all disease in wheat crop.

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Colletotrichum nymphaeae is the causal agent of strawberry anthracnose, which is one of the most important disease affecting strawberry plant in Iran. This research aimed to apply the selected plant essential oils (EOs) such as Achillea millefolium, Mentha longifolia, and Ferula kuma to the management of strawberry anthracnose disease under in vitro, in vivo, and greenhouse conditions. In vitro tests indicated that all the EOs and fungicide were able to inhibit mycelial growth and conidial germination of the pathogen. Scanning electron microscopy (SEM) revealed that EOs significantly suppressed the mycelia growth and caused a change in morphology of fungal mycelia. The severity of strawberry anthracnose disease was significantly (p ≤ 0.05) reduced by all EOs under in vivo and greenhouse conditions. Results of all experiments showed that M. longifolia EO was the best EO to control C. nymphaeae. Also, EOs almost reduced weight loss and preserved firmness, ascorbic acid, total phenol, antioxidant activity (DPPH), and enzyme peroxidase activity in treated fruit. Moreover, EOs preserved the sensory quality of strawberry fruit during the storage period so that there were no significant differences between treatments (EOs) in their appearance, flavor, odor attributes, and overall evaluation compared to the control. Our results indicate that EOs are excellent bio-fungicides for the management of strawberry anthracnose.

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A nonpathogenic endophytic bacterial isolate, recovered from Fragaria × ananassa stolon, and its antifungal activity against Colletotrichum nymphaeae was evaluated under in vitro, in vivo, and greenhouse conditions. Bacterial isolate was identified as Staphylococcus sciuri MarR44 (Strain ID: WDCM 891 = CCSM-B 00640) using phenotypic and biochemical properties and molecular phylogenetic analysis of the 16S rDNA gene sequences. The living cells of strain MarR44 inhibited mycelial growth of C. nymphaeae (52.46%) using dual-culture method. The volatile compounds (VOCs) produced by MarR44 inhibited mycelial growth and conidial germination of C. nymphaeae by 34.52% and 82.81%, respectively. However, inhibition percentage of mycelial growth of pathogen by culture filtrates of the strain MarR44 was lower (23.07%) than that for the two dual culture and volatile compounds assay tests. Moreover, the cell-free-culture filtrates of this strain reduced the biomass and conidial germination of pathogen by 91.89% and 41.10%, respectively. Also, the strain MarR44 was capable of producing protease, chitinase, HCN, siderophore, IAA, gibberellin, and biofilm. The living cells and volatile compounds of the strain MarR44 reduced anthracnose disease at post-harvest on fruit by 52.45% and 72.17%, respectively. Furthermore, disease severity of strawberry anthracnose was reduced using drenching soil and inoculated plants methods by 77.77 and 72.22%, respectively, 60 days after inoculation. The VOCs released by strain MarR44 were analyzed by Gas chromatography-mass spectroscopy (GC-MS). Out of 24 identified VOCs, Mesityl oxide (81.436%), Acetic acid, 2-methylpropyl ester (3.442%), 4-Methyldecane (1.837%), 4-Penten-2-one,4-methyl- (1.739%), Toluene (1.248%), and o-Xylene (1.24%) were the major components. The mode of action of S. sciuri MarR44 on the C. nymphaeae was through the production of antifungal volatile compounds (Antibiosis), which inhibited mycelial growth and conidial germination of pathogen in vitro and fruit decay development in vivo. To the best of our knowledge, this is the first report of S. sciuri having antifungal activity against causal agent strawberry anthracnose. These results indicated that the VOCs of S. sciuri strain MarR44 are promising biofumigant for management of strawberry anthracnose.

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40 isoeugenol-tolerant yeasts were isolated from the rhizosphere soil samples which in turn were collected from aromatic plants in different regions of Iran, and further tested for their ability to grow on a minimal medium containing isoeugenol as the sole carbon and energy source. Nine isolates which were able to grow on isoeugenol were examined for their ability to convert isoeugenol into vanillin under growing cell experiments. Of the tested yeasts, the highest conversion efficiency was observed in isolate MP24. The isolate was identified as Trichosporon asahii based on morphological, biochemical and molecular (ITS region) characters and tested to effectively convert isoeugenol into vanillin under resting cell system. A comparative analysis of thin layer chromatography (TLC), UV-Vis spectrometry, and high-performance liquid chromatography (HPLC) verified that vanillin and vanillic acid are accumulated as two major metabolites using T. asahii strain MP24 resting cells. In the presence of 7.5 g/l of wet weight cells of the strain MP24 pre-grown on isoeugenol and harvested at the end of the exponential growth phase, the optimal concentration of vanillin reached 2.4 g/l with a molar conversion of 52.5% in the potassium phosphate buffer (100 mM, pH 5.8) supplemented with 5 g/l of isoeugenol and 2% (v/v) N,N-dimethylformamide (DMF). The total concentration of vanillin and vanillic acid obtained from the bioconversion process was 4.2 g/l (total molar yield of 88.3%). Until now, no data has been published on the conversion of isoeugenol into vanillin by the strains of the genus Trichosporon.

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In this study, antifungal activity of essential oils of Cymbopogon citratus and Ocimum basilicum and two fungicides Mancozeb and Metalaxyl-Mancozeb in six different concentrations were investigated for controlling three species of Phytophthora, including P. capsici, P. drechsleri and P. melonis on pepper, cucumber and melon under in vitro and greenhouse conditions, respectively. Under the in vitro condition, the median effective concen- tration (EC50) values (ppm) of plant essential oils and fungicides were measured. In greenhouse, soil infested with Phytophthora species was treated by adding 50 ml of essential oils and fungicides (100 ppm). Disease severity was determined after 28 days. Among two tested plant essential oils, C. citratus had the lowest EC50 values for inhibition of the mycelial growth of P. capsici (31.473), P. melonis (33.097) and P. drechsleri (69.112), respectively. The mean EC50 values for Metalaxyl-Mancozeb on these pathogens were 20.87, 20.06 and 17.70, respectively. Chemical analysis of plant essential oils by GC-MS showed that, among 42 compounds identified from C. citratus, two compounds ß-geranial (α-citral) (39.16%) and z-citral (30.95%) were the most abundant. Under the greenhouse condition, Metalaxyl-Mancozeb caused the greatest reduction in disease severity, 84.2%, 86.8% and 92.1% on melon, cucumber, and pepper, respectively. The C. citratus essential oil reduced disease severity from 47.4% to 60.5% compared to the untreated control (p≤0.05). Essential oils of O. basilicum had the lowest effects on the pathogens under in vitro and greenhouse conditions. These results show that essential oils may contribute to the development of new antifungal agents to protect the crops from Phytophthora diseases.

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For all industrial processes, modelling, optimisation and control are the keys to enhance productivity and ensure product quality. In the current study, the optimization of process parameters for improving the conversion of isoeugenol to vanillin by Psychrobacter sp. CSW4 was investigated by means of Taguchi approach and Box-Behnken statistical design under resting cell conditions. Taguchi design was employed for screening the significant variables in the bioconversion medium. Sequentially, Box-Behnken design experiments under Response Surface Methodology (RSM) was used for further optimization. Four factors (isoeugenol, NaCl, biomass and tween 80 initial concentrations), which have significant effects on vanillin yield, were selected from ten variables by Taguchi experimental design. With the regression coefficient analysis in the Box-Behnken design, a relationship between vanillin production and four significant variables was obtained, and the optimum levels of the four variables were as follows: initial isoeugenol concentration 6.5 g/L, initial tween 80 concentration 0.89 g/L, initial NaCl concentration 113.2 g/L and initial biomass concentration 6.27 g/L. Under these optimized conditions, the maximum predicted concentration of vanillin was 2.25 g/L. These optimized values of the factors were validated in a triplicate shaking flask study and an average of 2.19 g/L for vanillin, which corresponded to a molar yield 36.3%, after a 24 h bioconversion was obtained. The present work is the first one reporting the application of Taguchi design and Response surface methodology for optimizing bioconversion of isoeugenol into vanillin under resting cell conditions.