Biocontrol of Postharvest Grey Mould on Tomato by Yeasts

The fungal pathogen Botrytis cinerea causes severe rots on tomato fruit during storage and shelf life. Biological control of postharvest diseases of fruit may be an effective alternative to chemical control. Yeasts are particularly suitable for postharvest use, proving to be highly effective in reducing the incidence of fungal pathogens. Yeast fungi isolated from the surface of solanaceous plants were evaluated for their activity in reducing the postharvest decay of tomato caused by B. cinerea. Of 300 isolates, 14 strains of Rhodotorula rubra and Candida pelliculosa were found to be strongly antagonistic to the pathogen in vitro and were selected for further storage experiment. The antagonists were evaluated for their effect on the biological control of postharvest grey mould. Artificially wounded fruits were treated by means of a novel technique: small sterile discs of filter paper imbibed separately in suspensions of each yeast and the pathogen were superposed onto each wound. After 1‐week, 11 isolates were significantly effective in reducing the diameter of lesions by more than 60% compared to the control treated with B. cinerea alone. Total protection was obtained with the strain 231 of R. rubra on fruits challenged with pathogen spores. To our knowledge, R. rubra and C. pelliculosa have not been described as biocontrol agents against grey mould caused by B. cinerea. Our data demonstrate that the application of antagonistic yeasts represents a promising and environmentally friendly alternative to fungicide treatments to control postharvest grey mould of tomato.     

Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum

Antifungal activities of zinc oxide nanoparticles (ZnO NPs) and their mode of action against two postharvest pathogenic fungi (Botrytis cinerea and Penicillium expansum) were investigated in this study. ZnO NPs with sizes of 70 ± 15 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ were used. Traditional microbiological plating, scanning electron microscopy (SEM), and Raman spectroscopy were used to study antifungal activities of ZnO NPs and to characterize the changes in morphology and cellular compositions of fungal hyphae treated with ZnO NPs. Results show that ZnO NPs at concentrations greater than 3 mmol l⁻¹ can significantly inhibit the growth of B. cinerea and P. expansum. P. expansum was more sensitive to the treatment with ZnO NPs than B. cinerea. SEM images and Raman spectra indicate two different antifungal activities of ZnO NPs against B. cinerea and P. expansum. ZnO NPs inhibited the growth of B. cinerea by affecting cellular functions, which caused deformation in fungal hyphae. In comparison, ZnO NPs prevented the development of conidiophores and conidia of P. expansum, which eventually led to the death of fungal hyphae. These results suggest that ZnO NPs could be used as an effective fungicide in agricultural and food safety applications.     

In vitro and in vivo antifungal activates of the essential oils of various plants against strawberry grey mould disease agent Botrytis cinerea

In order to investigate the effects of antifungal essential oils on postharvest decay and some quality factors of strawberry fruit, experiments were conducted under in vitro and in vivo conditions. The antifungal activates of essential oils obtained from fennel, anis, peppermint and cinnamon at concentrations 0, 200, 400, 600 and 800 μL L^?1 were investigated against Botrytis cinerea with four replications. In vitro results showed that the growth of B. cinerea was completely inhibited by fennel, cinnamon and anis essential oils at relatively low concentrations (400–800 μL L^?1). In vivo results showed that all the used essential oils at all applied concentrations caused an increase in the shelf life and inhibited of B. cinerea growth on strawberry fruits completely in comparison to the controls. The results of this study confirmed the antifungal effect of four essential oils in both in vitro and on fruit postharvest.     

Geographic distribution and sequence diversity of the mycovirus Botrytis virus F

Mycoviruses are obligate species that are found throughout all subdivisions of the fungal kingdom, with more constantly being discovered. However, only limited information is available about their mode of transmission and distribution. This research describes the distribution and sequence diversity of the Botrytis virus F (BotV-F) mycovirus from a survey of 84 Botrytis cinerea isolates collected from New Zealand and around the world. Using an RT-PCR approach, 12 BotV-F positive isolates were discovered, but there was no correlation to either plant host or geographic region from which the fungus was isolated. Subsequent phylogenetic analysis of BotV-F sequences suggest that this mycovirus has had a long association with B. cinerea, and has been co-distributed worldwide as B. cinerea has spread. In addition, these results suggest that the B. cinerea vegetative incompatibility mechanism may not completely prevent transmission of mycoviruses like BotV-F between fungal isolates from different compatibility groups. The potential utility of mycovirus sequence analysis to studies of fungal populations is discussed.     

Inhibitory effect of chitinases isolated from Semillon grapes (Vitis vinifera) on growth of grapevine pathogens

Characteristics and antifungal activity of chitinases in Semillon grapes were investigated. Chitinases were isolated from the juice of Semillon grapes by chitin affinity chromatography. Native and SDS-PAGE analyses of the fraction showing chitin affinity (active fraction) demonstrated only the presence of protein bands of chitinases. Three types of class IV chitinases (chi-1a, chi-1b and chi-2) were purified from the active fraction. These chitinases actively hydrolyzed chitin under acidic conditions (pH 4.0–4.5). The isoelectric points and the molecular weights of chi-1a, chi-1b and chi-2 were 4.73, 4.60, and 7.87, and 32.1 kDa, 31.6 kDa, and 29.0 kDa, respectively. The active fraction was found to inhibit Botrytis cinerea mycelial growth and the inhibitory effect was due to the activity of chitinases. The active fraction inhibited twenty strains of B. cinerea collected from the experimental vineyard. The effect of chitinases was enhanced in media containing more than 20% sugar. When the active fraction was tested on Glomerella cingulata, the growth inhibitory effect observed was markedly less than that seen on B. cinerea.     

Overexpression of mannitol dehydrogenase in zonal geranium confers increased resistance to the mannitol secreting fungal pathogen Botrytis cinerea

The sugar alcohol mannitol is a carbohydrate with well-documented roles in both metabolism and osmoprotection in plants and fungi. In addition, however, mannitol is an antioxidant, and current research suggests that pathogenic fungi can secrete mannitol into the plant’s extracellular spaces during infection to suppress reactive oxygen-mediated host defenses. In response to pathogen attack, plants have been shown to secrete the normally symplastic enzyme, mannitol dehydrogenase (MTD). Given that MTD converts mannitol to the sugar mannose, extracellular MTD may be an important defense against mannitol-secreting fungal pathogens. Previous work demonstrated that overexpression of MTD in tobacco did, in fact, provide increased resistance to the mannitol-secreting fungal pathogen Alternaria alternata. In the present work we demonstrate that the fungal pathogen Botrytis cinerea also can secrete mannitol, and that overexpression of MTD in zonal geranium (Pelargonium × hortorum) in turn provides increased resistance to B. cinerea. These results are not only an important validation of previous work, but support the idea that MTD-overexpression might be used to engineer a broad variety of plants for resistance to mannitol-secreting fungal pathogens like B. cinerea for which specific resistance is lacking.     

In vitro antifungal activity and mode of action of selected polyphenolic antioxidants on Botrytis cinerea

Six selected antioxidants (catechin, quercetin-3-galactoside, cyanidin-3-glucoside, pelargonidin-3-glucoside, ellagic and gallic acids) were evaluated in vitro for their antifungal activities and mode of action on Botrytis cinerea Pers., one of the most important pathogens of strawberries. Inhibitory effects were found for all the tested antioxidants, but varied at different fungal developmental stages. Catechin and quercetin-3-galactoside showed linear inhibitory effects on germ tube elongation, with the highest suppression ratios of 54.8% and 58.8% respectively. No significant effect was found on spore germination between treatments and control. Gallic acid showed very strong and linear inhibition on spore germination (r = ?0.95), but the effect diminished after spore germination. Cyanidin-3-glucoside and pelargonidin-3-glucoside provided effective control on the fungi as concentrations increased. The arresting effect of ellagic acid on development of B. cinerea was quadratic. Ellagic acid inhibited germ tube elongation and mycelial growth at its highest and lowest concentrations, while no effects were observed at its medium concentration used in this study.     

Inhibitory Effect of Selenium Against Penicillium expansum and Its Possible Mechanisms of Action

Some organic and inorganic salts could inhibit the growth of many pathogens. Selenium (Se), as an essential micronutrient, was effective in improving the plant resistance and antioxidant capacity at a low concentration. Penicillium expansum is one of the most important postharvest fungal pathogens, which can cause blue mold rot in various fruits and vegetables. In this study, the inhibitory effect of Se against P. expansum was evaluated. The result showed that Se strongly inhibited spore germination, germ tube elongation, and mycelial spread of P. expansum in the culture medium. The inhibitory effect was positively related to the concentration of Se used. Fluorescence microscopy observation of P. expansum conidia stained with propidium iodide (PI) indicated that the membrane integrity decreased to 37 % after the conidia were treated with Se (20 mg/l) for 9 h. With the use of an oxidant-sensitive probe 2,7-dichlorofluorescin (DCHF-DA), we found that Se at 15 mg/l could induce the generation of intracellular reactive oxygen species (ROS). Furthermore, methane dicarboxylic aldehyde (MDA) content, hydrogen peroxide (H₂O₂), and superoxide anion (O₂ ^?) production rate in P. expansum spores exposed to Se increased markedly. Compared with the control, the activities of superoxide dismutase (SOD) and the content of glutathione (GSH) were reduced, confirming that damage of Se to cellular oxygen-eliminating system is the main reason. These results suggest that Se might serve as a potential alternative to synthetic fungicides for the control of the postharvest disease of fruit and vegetables caused by P. expansum.     

Biofortification with potassium: antioxidant responses during postharvest of cherry tomato fruits in cold storage

Tomato fruits are sensitive to storage at low temperatures after harvest. Under these conditions, the main mechanism induced in fruits is oxidative stress, which can translate as lipid peroxidation and in turn deteriorate fruit quality. The aim of the present work was to investigate whether the effect of a biofortification program with potassium (K) improves the postharvest storage of cherry tomato fruits at 4 °C, through a better antioxidant response. Three K treatments were applied during the crop cycle of the plants: 5, 10, and 15 mM of KCl. The parameters in fruits on the day of harvest and after 21 days of postharvest cold storage at 4 °C, such as activity of lipoxygenase, malondialdehyde, catalase, superoxide dismutase, and the enzymes involved in the AsA–GSH cycle as well as the forms of ascorbate (AsA) and glutathione (GSH), were analyzed. The tomato fruits harvested from plants treated with 15 mM of KCl after 21 days of postharvest at 4 °C showed a lower degree of lipid peroxidation, an effective regeneration of AsA, and the highest pool of this compound in comparison with the other treatments. This response was because it presented the highest ascorbate peroxidase and monodehydroascorbate reductase activity. In addition, the treatments of 10 and 15 mM KCl presented the highest GSH pool, as well as a satisfactory regeneration of this tripeptide. All these results lead to the conclusion that the rate of 15 mM of KCl applied to this tomato variety (Solanum lycopersicum L. cv AsHiari grafted on cv. Maxifort rootstock) is adequate to mitigate the negative effects of postharvest chilling.     

Acclimation-induced changes in cell membrane composition and influence on cryotolerance of in vitro shoots of native plant species

Cell membranes are the primary sites of cryopreservation injury and measuring changes to membrane composition arising from cold acclimation may assist with providing a rationale for optimising cryopreservation methods. Shoot tips from two south-west Western Australian species, Grevillea scapigera and Loxocarya cinerea, and Arabidopsis thaliana (reference species) were subjected to cryopreservation using the droplet vitrification protocol. Two pre-conditioning regimes involving a constant temperature (23 °C, CT with a 12 h light/dark cycle) or an alternating temperature (AT) regime (20/10 °C with a 12 h light/dark cycle) were compared. Soluble sugars, sterols and phospholipids present in the shoot tips were analysed. Use of AT pre-conditioning (acclimation) resulted in a modest decrease in cryotolerance in A. thaliana, increased cryotolerance in G. scapigera, and increased survival in the non-frozen control explants of L. cinerea in comparison to CT pre-conditioning. Increased cryotolerance was accompanied by a higher total sugar sterol and phospholipid content, as well as an increase in strong hydrating phospholipid classes such as phosphatidylcholine. The double bond index of bound fatty acyl chains of phospholipids was greater after AT pre-conditioning, mostly due to a higher amount of monoenes in A. thaliana and trienes in G. scapigera and L. cinerea. These findings suggest that AT pre-conditioning treatments for in vitro plants can have a positive influence on cryotolerance for some plant species and this may be related to observed changes in the overall composition of cell membranes. However, alternative factors (e.g. oxidative stress) may be equally important with other species (e.g. L. cinerea).     

Fungicide resistance of Botrytis cinerea in tomato greenhouses in the Canary Islands and effectiveness of non-chemical treatments against gray mold

Tomato greenhouses in the Canary Islands, Spain, were surveyed to estimate frequencies of resistance to benzimidazoles, dicarboximides, anilinopyrimidines and N-phenylcarbamates in Botrytis cinerea. Resistance to carbendazim, iprodione, pyrimethanil and diethofencarb was found in 74.2, 86.4, 28.8 and 31.8 % of isolates, respectively. Benzimidazole- and anilinopyrimide-resistant isolates were highly resistant, showing EC₅₀ values above 500 µg/ml carbendazim and a mean EC₅₀ value of 28.42 µg/ml pyrimethanil, respectively. By contrast, a low level of resistance was observed among dicarboximide-resistant isolates (mean EC₅₀ value of 1.81 µg/ml iprodione). Phenotypes with double resistance to carbendazim and iprodione, and triple resistance to carbendazim, iprodione and pyrimethanil were the most common, occurring in 36.4 and 28.8 % of isolates. The surveyed greenhouses had never been treated with fenhexamid and Signum? (pre-packed mixture of boscalid and pyraclostrobin), and baseline sensitivities of B. cinerea isolates to these fungicides were determined. The EC₅₀ values were within the range of 0.009–0.795 µg/ml fenhexamid and of 0.014–0.48 µg/ml Signum. In addition, available formulations based on elicitors of plant defense response and biocontrol agents were evaluated against B. cinerea in tomato plants under semi-controlled greenhouse conditions, the yeast Candida sake CPA-1 being able to reduce gray mold significantly when it was applied on petiole wounds and the plants were inoculated 24 h later. Likewise, C. sake was effective against B. cinerea in harvested tomato fruits, yeast-treated tomatoes showed a 70.66 and 30.31 % reduction in the diameters of decay lesions compared with controls after 10 days of storage at 20 and 9 °C, respectively.     

Biocontrol of strawberry fruit infected by Botrytis cinerea: Effects on the microbial communities on fruit assessed by next‐generation sequencing

Fruit grey mould, caused by the fungus Botrytis cinerea, is known to be a harmful disease of strawberry at postharvest stage. However, effects of an application of biological control agents (BCAs) on strawberry fruit in terms of shift in the microbial community are still unknown. The present research aimed to investigate the effects of an application of BCAs on postharvest microbial populations present on strawberry fruits. Strawberry plants were sprayed with three kinds of BCA, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum‐P (Trichoderma harzianum T22) and Naturalis (Beauveria bassiana ATCC 74040), targeting Botrytis cinerea fungus. Control plots were composed of water and fungicide treatments. Microbial communities (bacteria and fungi) were analysed via next‐generation sequencing on an Illumina MiSeq. Analysis of 16S RNA and ITS rRNA sequences indicated that the BCAs application modified both bacterial and fungal community compositions and diversity. An application of two BCAs together had more effects on microbial community composition than a single application. These results suggest that BCAs can modify bacterial and fungal community composition and diversity on strawberry fruits, which may consequently improve the efficiency and establishment of these products on control of postharvest diseases of fruits, such as grey mould.     

Effects of ethylene on the susceptibility to Botrytis cinerea infection of different tomato genotypes

Ethylene at 10 and 100 μl/litre stimulated germ-tube elongation of Botrytis cinerea spores incubated within normal and non-ripening nor tomato fruits, but had little influence on the total percent of germination. Values of germ-tube length within the mature-green normal fruits and the mature-green or mature nor fruits were similar to those recorded within the normal mature fruits when held in air. Exposure of the normal and the mutant fruits to 100 μl/litre ethylene immediately after inoculation with B. cinerea insignificantly increased lesion development, but resulted in increased sporulation. When tomato fruits were exposed to ethylene for 3 days before inoculation a marked stimulatory effect on rot development was exhibited on the mature-green normal fruits but not on the nor mutant fruits. The results indicate that exogenous ethylene may directly stimulate germ tube growth of B. cinerea in both normal and mutant fruit, but that it may affect subsequent fungal growth indirectly, via stimulation of the ripening process, only in preclimacteric normal tomato fruit.     

Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach,apple and plum and its modes of action

The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 10⁸ cells ml⁻¹ and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2 °C and 95% relative humidity (RH) for 28 days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1 °C and 95% RH for 21 days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4 °C and 95% RH for 45 days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 10⁸ cells ml⁻¹, whereas at 10⁶ cells ml⁻¹ the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.     

Synthesis and QSAR study of novel α-methylene-γ-butyrolactone derivatives as antifungal agents

Thirty-six new α-benzylidene-γ-lactone compounds based α-methylene-γ-butyrolactone substructure were prepared and characterized by spectroscopic analysis. All compounds were evaluated for antifungal activities in vitro against six plant pathogenic fungi and the half maximal inhibitory concentration (IC₅₀) against Botrytis cinerea and Colletotrichum lagenarium were investigated. Compounds 5c-3 and 5c-5 with the halogen atom exhibited excellent fungicidal activity against B. cinerea (IC₅₀ = 22.91, 18.89 μM). The structure-activity relationships (SARs) analysis indicated that the derivatives with electron-withdrawing substituents at the meta- or para-positions improves the activity. Via the heuristic method, the generated quantitative structure-activity relationship (QSAR) model (R² = 0.961) revealed a strong correlation of antifungal activity against B. cinerea with molecular structures of these compounds. Meanwhile, the cytotoxicity of 20 representative derivatives was tested in the human tumor cells line (HepG2) and the hepatic L02 cells line, the result indicated that the synthesized compounds showed significant inhibitory activity and limited selectivity. Compound 5c-5 has the highest fungicidal activity with IC₅₀ = 18.89 μM (against B. cinerea.) but low cytotoxicity with IC₅₀ = 35.4 μM (against HepG2 cell line) and IC₅₀ = 68.8 μM (against Hepatic L02 cell line). These encouraging results can be providing an alternative, promising use of α-benzylidene-γ-lactone through the design and exploration of eco-friendly fungicides with low toxicity and high efficiency.     

Cloning and Partial Characterization of Endopolygalacturonase Genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Influence of antagonist, host fruit and pathogen on the biological control of postharvest fungal diseases by yeasts

The yeasts Rhodotorula glutinis (LS-11), Cryptococcus laurentii (LS-28), Candida famata (21-D) and Pichia guilliermondii (29-A) and the yeast-like fungus Aureobasidium pullulans (LS-30), previously selected and characterized for mechanisms of action and antagonistic activity against postharvest pathogens in small and large-scale experiments, were used in this study in order to assess interrelationships among the main factors (antagonist, host fruit and fungal pathogen) involved in biological control of postharvest diseases. The antagonists were evaluated for their inhibitory activity (IA) against six common postharvest fungal pathogens on six different host fruits. Artificially wounded fruits were first inoculated with the antagonist and 2 h later with the pathogen; subsequently they were kept at 20°C for 4–6 days. The IA of each antagonist was evaluated and data were submitted to factorial analysis of variance. The populations of antagonists were also monitored on wounded and unwounded fruits kept at 20°C for 7 days. Each factor examined (antagonist, host fruit and fungal pathogen) as well as their interactions significantly affected the IA. However, among the antagonists, isolates LS-28 and LS-30 were only slightly affected by both host and pathogen, showing a wide range of activity, whereas isolate LS-11 had a variable IA. All the antagonists rapidly colonized the wounds, while their population remained substantially unchanged on unwounded fruits. These results suggest that in order to select yeasts with a broad spectrum of action, more suitable for commercial development, it would be advantageous to perform preliminary assays against several pathogens and in particular on different fruit species. Received 23 February 1999/ Accepted in revised form 09 July 1999