Inhibition Of Spore Germination Research Articles

Purification and evaluation of 2, 4-di-tert butylphenol (DTBP) as a biocontrol agent against phyto-pathogenic fungi

A fungal strain, Marasmiellus sp (PUK64), isolated from the mangrove forests in Muthupet, Tamil Nadu, East coast of India, along with others were screened for the search of potent bioactive compounds. A phenolic compound, 2,4-di-tert-butylphenol (DTBP), was isolated from the most promising strain PUK64 and its chemical structure was ascertained. DTBP demonstrated remarkable antifungal activity against the phytopathogenic fungi Aspergillus oryzae, Curvularia lunata and Fusarium verticillioides. In an in-vitro experimental setup, DTBP suppressed the growth of all three fungi, among which F. verticillioides was found to be highly susceptible. This effect relates with the inhibition of spore germination and hyphal growth that we observed. DTBP showed high affinity with the F. verticillioides's β-tubulin protein (determined by ligand-protein docking) as compared to the standard fungicide carbendazim (CBZ). Molecular docking and simulation studies of DTBP with target β-tubulin further confirmed the potential of β-tubulin binding in F. verticillioides. To our knowledge, this is the first report on DTBP-mediated biocontrol of phytopathogenic fungi, produced by Marasmiellus sp. PUK64 that can be potent inhibitor of β-tubulin protein of F. verticillioides.

Characterisation of an edible chitosan film incorporating tea seed oil/montmorillonite and antifungal activity against Botrytis cinerea

SummaryChitosan films enhanced with tea seed oil (TO) and montmorillonite were solution cast and the influence of montmorillonite additives on the physicochemical and antifungal properties was evaluated. The addition of TO and montmorillonite significantly increased both the pH and viscosity of the coating solutions. SEM and AFM showed montmorillonite addition increased membrane structure homogeneity. In addition, chitosan films incorporating montmorillonite exhibited significantly improved tensile strength, at 43.87% in chitosan with montmorillonite and 44.23% in chitosan with montmorillonite and TO. Antifungal activity of the chitosan coating increased sharply when TO was added, inhibiting spore germination and germ tube length by 65.05% and 51.95% respectively. The decay inhibition rates for strawberries treated with chitosan and TO, or chitosan, TO and montmorillonite were also high at 32.77% and 28.79% respectively. This demonstrates that chitosan‐based TO/montmorillonite films are potential materials for packaging fruits or vegetables, to eventually replace synthetic polymers.

Inhibitory Effect and Control Efficacy of Picoxystrobin against Neopestalotiopsis clavispora, Causing Vine Tea Leaf Blight

Vine tea (Ampelopsis grossedentata) is a traditional herb widely consumed in southwestern China that possesses paramount potential for human health. In 2021, the outbreak of typical leaf blight disease was observed in almost all vine tea plantations in Zhangjiajie of Hunan province, resulting in significant economic losses of vine tea production. In this study, we identified Neopestalotiopsis clavispora as the causal agent of vine tea leaf blight via its morphological characteristics and molecular identification. The sensitivity distribution of N. clavispora isolates to picoxystrobin were determined based on mycelial growth and spore germination inhibition assays. The EC50 values for mycelial growth ranged from 0.0062 to 0.0658 µg/mL, with a mean of 0.0282 ± 0.0148 µg/mL. The EC50 values for spore germination ranged from 0.0014 to 0.0099 µg/mL, and the mean value was 0.0048 ± 0.0022 µg/mL. Picoxystrobin increased fungal cell membrane permeability, but inhibited fungal ATP biosynthesis. Moreover, picoxystrobin exhibited good in planta control efficacy on vine tea leaves. Three picoxystrobin-resistant mutants were obtained in the current study, but no mutations were detected in the N. clavispora Cytb gene. Competitive ability assays showed that the conidium production and pathogenicity of all picoxystrobin-resistant mutants decreased as compared to their progenitors, indicating that picoxystrobin-resistant mutants suffer fitness penalty. These findings provide important evidence for picoxystrobin in vine tea leaf blight management and increase understanding of the resistance mechanism of picoxystrobin against N. clavispora.

A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threatsin the United States, 2019 (2019),www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont,N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

Inactivation of Group I and Group II Clostridium botulinum spores by ultraviolet irradiation in water

Spores of Clostridium botulinum are widely distributed in the environment, including in foods. Prevention of foodborne botulism relies on the inhibition of spore germination and subsequent growth and toxin production, or the destruction of viable spores in food and beverages. This study examined the lethality of 254 nm UV radiation (UV-C) to spores of Group I and Group II C. botulinum. Spores of C. botulinum were inactivated by UV-C, with doses required for incremental log reduction (D10) values calculated using linear regression ranging from 2.87 to 3.70 mJ/cm2 for Group I strains and 4.46 to 6.15 mJ/cm2 for Group II strains. The measured D10 value for spores of C. sporogenes ATCC 19404 was 8.27 mJ/cm2 indicating it was more resistant than the strains of C. botulinum used in this study. Calculation of dose per log using a Weibull model resulted in higher D10 values of 6.67 to 8.81 mJ/cm2 for Group I strains and 9.24 to 10.7 mJ/cm2 for Group II strains. Spores of C. sporogenes possessed a D10 value of 14.4 mJ/cm2. The higher values for the Weibull model indicate the Weibull model to be more conservative as a result as it factors in the lag prior to inactivation and the tailing observed with very low numbers of survivors. Spores of both Group I and Group II C. botulinum strains tended to form large aggregates, visible with phase contrast microscopy, that resulted in severe tailing. Disruption of aggregates by ultrasonication was necessary to obtain linear destruction curves extending beyond 5 log reduction. All strains from Group I and Group II required <55 mJ/cm2 to achieve 5 log inactivation. The strain of C. sporogenes used in this work can therefore be a conservative non-pathogenic surrogate, having higher UV-C resistance than the C. botulinum strains used in this study. Overall, this study is the first detailed study to demonstrate UV-C as an effective treatment method to inactivate C. botulinum spores in a suspending medium. In addition, the study paves the way for further studies towards the applications of this technology to inactivate C. botulinum spores in beverages or other liquids.

Biological Control of Brassicales Ring Spot Disease Caused by Alternaria brassicicola Using Antagonistic Yeasts

T his research investigated the biological control of Ring Spot Disease of Brassicales caused by Alternaria brassicicola, which is an agriculturally important pathogen, using antagonistic yeasts. One hundred fifteen yeasts were isolated from 38 fruits and vegetable leaves. A total of twenty-nine yeast isolates showed the inhibitory activity of A. brassicicola more than a value of 50% by the dual culture method. The result showed that three antagonistic yeast isolates Y107, Y123, and Y16 showed high inhibitory percentages at 75.00%, 73.52%, and 70.33%, respectively. Interestingly, these yeasts inhibited spore germination of A. brassicicola after 12 h within the range of 80.90 to 90.26%. Subsequently, the efficacy of the selected antagonistic yeast against Ring Spots Disease on cabbage seedlings (Brassica oleracea L. var. capitata) and Pak choi (Brassica chinensis L. var. chinensis) was tested in the greenhouse. The result indicated that antagonistic yeast isolate Y107 inhibited the disease at 72.75% and 71.42% in Cabbage seedlings and Pak choi, respectively. However, the efficacy of isolates Y123 and Y16 were not significant in disease inhibition in both Cabbage seedlings (66.67% and 66.08%) and Pak choi (69.83% and 68.92%). Additionally, the antagonistic yeast isolates were identified by phylogenetic analysis of the D1/D2 domain of the large subunit ribosomal RNA (LSU) gene. Yeast isolates Y16 and Y123 were identified to Kurtzmaniella quercitrusa, while isolate Y107 was Hanseniaspora thailandica.

Transcriptome reveals the molecular mechanism of chaetoviridin A inhibiting the spore germination of Verticillium dahliae

The pathogen Verticillium dahliae causes Verticillium wilt in a number of crops, including cotton Verticillium wilt. Chaetoviridin A, a secondary metabolite of Chaetomium globosum, significantly inhibits the growth of V. dahliae. Spore germination is a major part of the disease cycle. However, the molecular mechanism of chaetoviridin A inhibiting spore germination of V. dahliae is unknown. In this work, we found that chaetoviridin A significantly inhibited spore germination of V. dahliae. Transcriptome analysis showed that DEGs were enriched in linolenic acid metabolism, alpha-Linolenic acid metabolism, Arachidonic acid metabolism and Purine metabolism pathways at 1 h, which were related to cell membrane. At 3 h, DEGs were enriched in the pathways of galactose metabolism, diterpenoid biosynthesis, cysteine and methionine metabolism, and starch and sucrose metabolism, which were mainly related to amino acid metabolism and sugar metabolism. Several genes related to glucose metabolism were identified, mainly including Glucagon endo-1,3-alpha-glucosidase agn1, glucoamylase, maltose O-acetyltransferase, and beta-galactosidase. Stress resistance gene PAL, detoxification gene P450 2C31 and ABA receptor were also down- regulated. These genes may be related to spore germination. These results provide a theoretical basis for chaetoviridin A to control fungal diseases.

Mechanisms of antifungal and mycotoxin inhibitory properties of Thymus vulgaris L. essential oil and their major chemical constituents in emulsion-based delivery system

The antifungal and mycotoxin inhibitory activity of emulsions formed with Thymus vulgaris L. essential oil (thyme oil) and four major components (thymol, p-cymene, γ-terpinene, and linalool) on Fusarium graminearum, and their corresponding mode of action (MOA) were evaluated in this study. Among the five emulsions, thymol emulsion presented the strongest antifungal activity followed by thyme oil and linalool emulsion. The spore morphology changes and cell membrane destruction were the main reasons attributed to it supported by the SEM and CLSM observation. Moreover, significant (p< 0.05) suppression on mycotoxin inhibition were achieved by all five emulsions. We also noticed that the antifungal activity and the mycotoxin inhibitory were not always correlated. Linalool appeared to be a more effective spore germination inhibitor, while p-cymene and γ-terpinene decreased mycotoxin production more effectively. We further investigated the MOA of thyme oil and thymol emulsions on suppression of mycotoxin production on generic level. We observed that thyme oil significantly (p< 0.05) up-regulated the expression of the key encoding genes in mycotoxin biosynthesis pathway that were studied (Tri3, Tri4, and Tri5), while thymol did not up-regulate any gene expression.

Biocontrol potential of wine yeasts against four grape phytopathogenic fungi disclosed by time-course monitoring of inhibitory activities.

Grapes' infection by phytopathogenic fungi may often lead to rot and impair the quality and safety of the final product. Due to the concerns associated with the extensive use of chemicals to control these fungi, including their toxicity for environment and human health, bio-based products are being highly preferred, as eco-friendlier and safer alternatives. Specifically, yeasts have shown to possess antagonistic activity against fungi, being promising for the formulation of new biocontrol products.In this work 397 wine yeasts, isolated from Portuguese wine regions, were studied for their biocontrol potential against common grapes phytopathogenic fungal genera: Aspergillus, Botrytis, Mucor and Penicillium. This set comprised strains affiliated to 32 species distributed among 20 genera. Time-course monitoring of mold growth was performed to assess the inhibitory activity resulting from either diffusible or volatile compounds produced by each yeast strain. All yeasts displayed antagonistic activity against at least one of the mold targets. Mucor was the most affected being strongly inhibited by 68% of the tested strains, followed by Botrytis (20%), Aspergillus (19%) and Penicillium (7%). More notably, the approach used allowed the detection of a wide array of yeast-induced mold response profiles encompassing, besides the decrease of mold growth, the inhibition or delay of spore germination and the complete arrest of mycelial extension, and even its stimulation at different phases. Each factor considered (taxonomic affiliation, mode of action and fungal target) as well as their interactions significantly affected the antagonistic activity of the yeast isolates. The highest inhibitions were mediated by volatile compounds. Total inhibition of Penicillium was achieved by a strain of Metschnikowia pulcherrima, while the best performing yeasts against Mucor, Aspergillus and Botrytis, belong to Lachancea thermotolerans, Hanseniaspora uvarum and Starmerella bacillaris, respectively. Notwithstanding the wide diversity of yeasts tested, only three strains were found to possess a broad spectrum of antagonistic activity, displaying strong or very strong inhibition against the four fungal targets tested. Our results confirm the potential of wine yeasts as biocontrol agents, while highlighting the need for the establishment of fit-for-purpose selection programs depending on the mold target, the timing, and the mode of application.

Enhancing bioefficacy of Bacillus amyloliquefaciens SF14 with salicylic acid for the control of the postharvest citrus green mould

Penicillium digitatum is a major post-harvest pathogen of citrus fruit causing tremendous economic losses. In this study, salicylic acid (SA) and antagonistic bacteria Bacillus amyloliquefaciens (SF14) were tested in controlling P. digitatum (green mould) infections as an alternative to chemical controls. Three concentrations of SA (0.5, 1, and 2%) were tested for their antifungal activity in both in vitro and in vivo conditions alone and in combination with SF14. Fruit quality parameters including fruit firmness, weight loss, titratable acidity, and soluble solids content were evaluated. The impact of these biological treatments was also investigated by measuring the changes in organic and mineral properties of orange fruits using Fourier transform infrared spectroscopy (FT-IR). Enzymatic activity including phenylalanine ammonia-lyase (PAL), total polyphenols and flavonoids was also tested. In vitro assays showed that SF14 combined with 0.5% SA provided the highest inhibition of green mould growth reaching 72.98%. Inhibition of spore germination was also highest in the same treatment od reaching 63.40%. FTIR of fungal biomass confirmed an alteration of fungal well wall following biological treatments. Moreover, during in vivo assays, the combination of SF14 and 0.5% SA produced the lowest disease severity (5.43%) without considerably affecting the fruit quality. Further investigation of the effect of these treatments on the mineral composition of treated oranges showed that Ca, Fe, K, Mg, and Na concentrations were significantly affected. Changes in these elements may show a defence reaction induced by the biological treatments against the pathogen, which was also confirmed with the measurement of PAL activity, total polyphenols, and flavonoids. Interestingly, FTIR of citrus peel indicated significant changes in the content of bands denoting for phenolic and aromatic rings, suggesting an induced resistance against the pathogenic fungus. Overall, our findings suggested that the combination of both bacterial antagonist SF14 and 0.5% SA can control the green mould of citrus, and as such constitutes a safer alternative to chemicals during postharvest storage.

Seaweed Extracts to Control Postharvest Phytopathogenic Fungi in Rocha Pear.

Fungal infections cause losses amounting to between 20 and 25% of the fruit industry's total outcome, with an escalating impact on agriculture in the last decades. As seaweeds have long demonstrated relevant antimicrobial properties against a wide variety of microorganisms, extracts from Asparagopsis armata, Codium sp., Fucus vesiculosus, and Sargassum muticum were used to find sustainable, ecofriendly, and safe solutions against Rocha pear postharvest fungal infections. Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, and Penicillium expansum mycelial growth and spore germination inhibition activities were tested in vitro with five different extracts of each seaweed (n-hexane, ethyl acetate, aqueous, ethanolic, and hydroethanolic). An in vivo assay was then performed using the aqueous extracts against B. cinerea and F. oxysporum in Rocha pear. The n-hexane, ethyl acetate, and ethanolic extracts from A. armata showed the best in vitro inhibitory activity against B. cinerea, F. oxysporum, and P. expansum, and promising in vivo results against B. cinerea using S. muticum aqueous extract were also found. The present work highlights the contribution of seaweeds to tackle agricultural problems, namely postharvest phytopathogenic fungal diseases, contributing to a greener and more sustainable bioeconomy from the sea to the farm.

Green Biosynthesis of Silver Nanoparticles Using Vaccinium oxycoccos (Cranberry) Extract and Evaluation of Their Biomedical Potential

Eco-friendly preparation of metallic nanoparticles (NPs) is a greatly evolving field of scientific research. These types of NPs have gained substantial recognition from scientists, including chemists, chemical biologists and technologists, who have successfully exploited them for the fabrication of a variety of advanced nanodevices. Herein, silver (Ag) NPs were synthesized by a green approach using the aqueous extract of Vaccinium oxycoccos (cranberry), which not only reduced the silver ions but also stabilized the surface of the resultant Ag NPs. The formation of Ag NPs is confirmed by different analytical techniques, including powder X-ray diffraction, UV analysis, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FT-IR). The UV analysis of the aqueous solution of the reaction mixture demonstrated an absorption band at ~450 nm, which is the typical peak of Ag NPs, leading to the confirmation of product formation. While the XRD confirmed the crystallinity of the sample and the formation of a face-centered cubic (fcc) structure, on the other hand, TEM revealed the presence of spherical NPs with an approximate size range between 5–30 nm. Furthermore, the as-obtained Ag NPs were subjected to thorough investigations to explore the biomedical potential of the sample. In this case, the Ag NPs demonstrated considerable antioxidant and antifungal properties towards various pathogens. In addition, Ag NPs also showed substantial inhibition of spore germination.

Efficacy of Saccharothrix algeriensis NRRL B-24137 to suppress Fusarium oxysporum f.sp. vasinfectum induced wilt disease in cotton.

Fusarium cotton wilt is a devastating disease of the cotton crop throughout the world, caused by Fusarium oxysporum f.sp. vasinfectum (FOV). Chemical control has many side effects, so, biological controls have been widely used for the management of Fusarium wilt. This study aimed to investigate the possible use of an actinomycetes Saccharothrix algeriensis (SA) NRRL B-24137 to control FOV. To access in-vitro anti-Fusarium ability of SA NRRL B-24137, dual culture assay, spore germination and seed germination tests were carried out. Following in-vitro investigations, several pot tests in a greenhouse environment were used to evaluate the biological control potential of SA NRRL B-24137 against FOV. Dual culture assay and spore germination revealed that SA NRRL B-24137 showed significant anti-Fusarium activity.During spore germination 87.77% inhibition of spore germination were observed. In pot experiments, SA NRRL B-24137 primed cotton seeds resulted in a 74.0% reduction in disease incidence. In soil there was a significant reduction in FOV spores in the presence of SA NRRL B-24137. Positive correlation was also observed on different concentrations of SA NRRL B-24137 towards FOV reduction. The results of this study showed that SA NRRL B-24137 has the potential to be employed as a biocontrol agent against Fusarium cotton wilt, improving cotton growth characteristics and yield.

Modelling the effect of pH and H2S on the germination of F. graminearum spores under different temperature conditions

Fusarium graminearum (F. graminearum) is a vital pathogen of Fusarium head blight (FHB). Hydrogen sulfide (H2S), which can be induced by stress in plants as a fungal inhibitor, affects the germination of spores through the detection of environmental factors such as temperature and pH. Based on mathematical models, F. graminearum was treated with different temperatures, pH, and H2S concentration to investigate spore germination events. This paper considered the synergistic effects of temperature and pH on spores in order to determine the optimal germination environment. Under these circumstances, the inhibition of spore germination with different concentrations of H2S was quantified. The results suggested that the germination time negatively correlates with the germinating rate. An increase in temperature altered the weak alkali preference of F. graminearum spores. At 28 °C, spores were more sensitive to the changes in pH. H2S gas could delay the germinating process significantly, however the required H2S concentration varied with environmental changes. This paper provides a perspective on the synergistic effect of temperature and pH on spore germination and contributes to determining the concentration of H2S as a management of FHB.

Structural Simplification of Cryptolepine to Obtain Novel Antifungal Quinoline Derivatives against Phytopathogenic Fungi.

A series of quinoline derivatives were designed and synthesized by the structural simplification of cryptolepine and evaluated for their fungicidal activity against six phytopathogenic fungi. Most of these compounds exhibited remarkable activities against Botrytis cinereain vitro. Among them, compounds A18 and L01 showed superior antifungal activity. Significantly, compared to cryptolepine, compound A18 exhibited broad-spectrum inhibitory activities against B. cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Phytophthora capsica, Magnaporthe oryzae, and Fusarium graminearum with the respective EC50 values of 0.249, 1.569, 3.915, 0.505, 0.246, and 4.999 μg/mL. Compound L01 displayed the best antifungal activity against B. cinerea with an EC50 value of 0.156 μg/mL. Preliminary mechanistic studies showed that compound A18 could inhibit spore germination, affect the permeability of the cell membrane, increase the content of reactive oxygen species, and affect the morphology of hyphae and cells. Moreover, compound A18 showed excellent in vivo protective effect against B. cinerea, which was more potent than pyrimethanil and equitant to cryptolepine. These results evidenced that compound A18 displayed superior fungicidal activities and could be a potential fungicidal candidate against plant fungal diseases.

Phytic acid is a new substitutable plant-derived antifungal agent for the seedling blight of Pinus sylvestris var. mongolica caused by Fusarium oxysporum

Phytic acid (PA) is a new substitutable plant-derived antifungal agent; however, few reports have been published regarding its antifungal effects on pathogenic fungi. The present study explored the in vitro antifungal activity of PA against four phytopathogenic fungi and found that PA was the most effective at inhibiting the growth of Fusarium oxysporum. This study aimed to investigate the in vivo and in vitro antifungal activities of PA against the seedling blight of Pinus sylvestris var. mongolica caused by F. oxysporum and to determine its possible mechanism of action. The results showed that PA inhibited spore germination and mycelial growth of F. oxysporum in a concentration-dependent manner and exhibited strong inhibition when its concentration exceeded 1000 mg/L. It mainly destroyed the integrity of the cell membrane, increasing its cell membrane permeability, causing the cell contents to spill out, and impairing fungal growth. In addition, the leakage of intercellular electrolytes and soluble proteins indicated that PA used at its EC20 and EC50 increased the membrane permeability of F. oxysporum. The increase in malondialdehyde and hydrogen peroxide content confirmed that PA treatment at its EC20 and EC50 damaged the cell membrane of the pathogen. Scanning electron microscopy revealed that PA affected the morphology of mycelia, causing them to shrivel, distort, and break. Furthermore, PA significantly reduced the activities of the antioxidant-related enzymes superoxide dismutase and catalase, as well as that of the pathogenicity-related enzymes polygalacturonase, pectin lyase, and endoglucanase (EG) in F. oxysporum (P < 0.05). In particular, EG enzyme activity was maximally inhibited in F. oxysporum treated with PA at its EC50. Moreover, PA significantly inhibited the incidence of disease, and growth indices in Pinus sylvestris var. mongolica seedling blight was determined. In summary, PA has a substantial inhibitory effect on F. oxysporum. Therefore, PA could serve as a new substitutable plant-derived antifungal agent for the seedling blight of P. sylvestris var. mongolica caused by F. oxysporum.

The antifungal potential of the chelating agent EDTA against postharvest plant pathogen Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungus that causes gray mold, a major postharvest disease of fruits and vegetables. Chemical fungicides remain the main solution to control Botrytis disease, but concerns have raised about their safety to environment and human health, and there is an increasing need for development of more effective and less toxic treatments. In this study the divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) exhibited marked antifungal activity against B. cinerea, including inhibition of spore germination, mycelial growth, infection cushion formation, stimulation of cell death, and impairment of fungal virulence. These adverse effects of EDTA could be reversed by the addition of calcium ion, implying that metal ion chelation is involved in the fungicidal mechanism. Bean leaf and tomato fruit protection assay indicated that EDTA treatment led to a significant reduction of infection by B. cinerea. Furthermore, the antifungal activity of EDTA was significantly enhanced when used in combination with fenhexamid. These findings suggest that EDTA could be a promising tool to control B. cinerea, and application of EDTA may reduce the use of conventional chemical fungicides.

Identification of the main proteins secreted byKluyveromyces marxianusand their possible roles in antagonistic activity against fungi

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.

Effects of linalool on Botrytis cinerea growth and control of tomato gray mold.

We examined the antifungal characteristics of linalool against Botrytis cinerea using plate inhibition assay and spore germination assay, and assessed the capacity of linalool in controlling tomato gray mold disease via tomato pot inoculation assay. The results showed that linalool exhibited strong inhibitive effects on mycelial growth of B. cinerea, with an EC50 value of 0.581 mL·L-1. In the spore germination test, linalool treatment inhibited spore germination in a dose-dependent manner. The electric conductivity and the malondialdehyde (MDA) contents were significantly increased in linalool-treated B. cinerea than that of the control, indicating that linalool induced oxidative damage and destroyed the cell membrane integrity in B. cinerea. The activities of the superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) in the linalool-treated B. cinerea were decreased significantly by 27.4%, 68.9% and 26.0%, respectively, suggesting that linalool inhibited the antioxidant activity of B. cinerea. In the pot experiment, the diameter of lesions in linalool-treated tomatoes was significantly smaller than that of the control. The activities of SOD, POD, CAT, polyphenol oxidase, and phenylalnine ammonialyase in the linalool-treated tomatoes increased, while the MDA content decreased, suggesting that linalool could alleviate the oxidative damage caused by B. cinerea and promote plant disease resistance. In summary, linalool had inhibitory effect on the growth of B. cinerea and could control gray mold disease in tomatoes. These findings could lay the foundation for developing bota-nical antifungal agents for management of tomato gray mold disease.

Antifungal Activity of Rhamnolipid Biosurfactant Produced by Pseudomonas aeruginosa A4 against Plant Pathogenic Fungi

Biosurfactants have been shown to have a variety of other agricultural applications. Antimicrobial activity is a desirable property in a variety of biosurfactants. Several biosurfactants produced from bacteria have antibacterial activity against plant diseases, making them a promising biocontrol molecule for ensuring agricultural sustainability in the long run. The purpose of this research was to characterize biosurfactant produced at different carbon source by biosurfactant-producing bacterial strains isolated from contaminated soapstock sediment, as well as its antifungal properties. Biosurfactant-producing bacteria were isolated from soapstock-contaminated soil which bacteria were selected by using drop collapse and oil displacement tests. Twelve isolates reduced surface tension of culture broth from 50 to 31 - 43 mN/m. According to 16S rRNA sequence analysis, these isolates belong to 8 different genera (Acinetobacter, Citrobacter, Enterobacter, Klebsiella, Pantoea, Pseudomonas, Stenotrophomonas and Xanthomonas). The supernatant of Pseudomonas aeruginosa A4 grown in MSM supplemented with soapstock was the most effective biosurfactant against Aspergillus flavus F2, Aspergillus niger F14, Cunninghamella bertholletiae F1 and Rhizopus oryzae F5, inhibiting mycelium growth by 54, 61 59 and 50 %, respectively. The extract substance inhibited spore germination against A. flavus F2 and R. oryzae F5 with a minimum inhibitory concentration of 2.75 mg/mL. TLC, FT-IR, ESI-MS and GC-MS analysis demonstrated that the mono-rhamnolipids and di-rhamnolipids had the same 3-hydroxy fatty acid composition of C8, C10 and C12. Overall, the biosurfactant-producing strains identified in this investigation show promise for future development and application in cost-effective industrial-scale biotechnological processes.&#x0D; HIGHLIGHTS&#x0D; &#x0D; Biosurfactant producing bacteria were isolated from soapstock contaminated soil&#x0D; The supernatant of Pseudomonas aeruginosa A4 grown in MSM supplemented with soapstock was the most effective biosurfactant against plant pathogenic fungi&#x0D; Biosurfactant from Pseudomonas aeruginosa A4 was identified as rhamnolipid comprising of both mono and di-rhamnolipid congeners&#x0D; &#x0D; GRAPHICAL ABSTRACT