Cellulolytic Activity of Botrytis cinerea in vitro and in vivo

Experiments were carried out to determine whether stepwise breakdown of native cellulose is carried out by B. cinereain vitro and in vivo. Protein fractions were obtained from ungerminated conidia, from culture filtrates 24, 48 and 96 h after inoculation with conidia, and from culture filtrates of 12 day-old cultures growing on cotton wool as the carbon source. In addition, petioles and fruits of tomato plants were inoculated and the protein fraction of the colonized tissues were tested. Using filter paper, carboxymethylcellulose and cellobiose as substrates, all fraction showed c₁, glucanase and cellobiase activity respectively.     

Endo- and Exochitinase Activity in Kiwifruit Infected with Botrytis cinerea

Endo- and exochitinase activities were determined in autoclaved leaves, live leaves, and fruit of kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward [A. Chev.]Liang and Ferguson) following infection with Botrytis cinerea (Pers.). Appreciable endochitinase activity was found in healthy and diseased leaves and fruit at harvest and after cool-storage, but was absent in plant tissue killed by autoclaving. Later harvested fruit produced more endochitinase activity in storage than did early harvested fruit. Exochitinase activity was only associated with diseased tissue, and was present in diseased autoclaved leaves. The results suggest that endochitinase activity originates from the plant whilst exochitinase is associated with the pathogen. The significance of these findings is discussed.     

Isolation,X-Ray Structure and Biological Activity of Deacetyl-dihydrobotrydial Produced by Botrytis squamosa

Mitogenic substances on human peripheral blood mononuclear leukocytes were screened from culture filtrates of microorganisms newly isolated from soil and sea water by measuring [³H]- thymidine incorporation into the cells. Strong mitogenic activity was found in marine bacteria, particularly in marine vibrios. These mitogen samples exhibited neither hemagglutinating activity nor leukoagglutinating activity. They could scarcely stimulate murine lymphocytes.

Cell-cell interaction among leukocyte subsets in response to a bacterial mitogen was investigated using the most powerfully mitogenic sample (culture filtrate of strain H 52–2). A slight decrease in the mitogen response was observed on depletion of plastic surface adherent cells. Separation of T and non-T cells from each other by erythrocyte-rosette sedimentation resulted in a markedly diminished mitogen response. Considerable restoration of the mitogen response was obtained when T cells were mixed with mitomycin C-treated adherent cells or mitomycin C-treated non-T lymphocytes, or when non-T lymphocytes were mixed with mitomycin C-treated T cells.     

The Influence of o-hydroxyethylorutin on Botrytis cinerea Oxidant–Antioxidant Activity

The objective of this study was to evaluate the effects of o‐hydroxyethylorutin on Botrytis cinerea mycelium growth and metabolism. Hydrogen peroxide concentration, superoxide dismutase, catalase and peroxidase activities were compared in the pathogens’ mycelium grown on control and o‐hydroxyethylorutin containing medium. Transfer of B. cinerea mycelium to medium supplemented with 5 mm o‐hydroxyethylorutin resulted in a large decrease in catalase activity. No changes in mycelium growth, hydrogen peroxide concentration and superoxide dismutase activity were observed. Guaiacol and ascorbate peroxidases were not detected in mycelia. The data are consistent with previous findings that o‐hydroxyethylorutin treatment of tomato plants restricts the development of B. cinerea infection due to the induction of higher active oxygen species (AOS) generation in plants by this compound. Being poor in catalase, the pathogen may not be able to cope with increasing AOS formation. The results indicate that catalase is an infective agent of B. cinerea.     

Biological control of Botrytis cinerea on stem wounds with moderately halophilic bacteria

Infection of tomato stem wounds by Botrytis cinerea is an important problem which can cause severe economic losses in greenhouse tomato crops. Three moderately halophilic bacteria were tested for their ability to protect pruning wounds from attacks by B. cinerea under growth chamber conditions. The severity of the disease estimated by the length of the rotted stem was used to calculate the area under the disease progress curves (AUDPC). Bacterial antagonists (B1, B2 and B3) were very effective in controlling Botrytis-infection on the tomato stems during the first 6 days and later by the end of the experiment. Plants treated with Bacillus subtilis (B1) had the lowest AUDPC (0). It was followed by B. subtilis (B3) and Halomonas sp. (B2) with AUDPC of 9.8 and 17.02, respectively. While the B1 strain best inhibited grey mold development when applied as young culture (24 h), the B3 strain performed better as an older culture (48 h). In contrast to the results obtained with Bacillus species, the efficacy of the bacterial treatment B2 seems to be independent of the growth phase. The co-cultures with fungal spores and either B. subtilis (B1) or Halomonas sp. (B2) applied as a 24 h bacterial culture completely inhibited the germination of B. cinerea after 24 h at 21°C.     

Composition and Enzymatic Activity of the Extracellular Matrix Secreted by Germlings of Botrytis cinerea

Germlings of Botrytis cinerea, an important fungal pathogen of plants, produce an extracellular matrix (ECM), or ensheathing film, that serves, in part, in their attachment (R. P. Doss, et al., Appl. Environ. Microbiol. 61:260–265, 1995). The composition of this film has been ascertained by using samples obtained by growing germlings on a glass surface, removing the fungal mycelium by vigorous washing, and collecting the tightly attached film by scraping the substratum with a razor blade. Slightly over half of the dry weight of the ECM was found to be carbohydrates (about 20%), proteins (about 28%), and lipids (about 6%). Hydrolysis of the carbohydrate portion of the ECM revealed that glucose was the most prominent monosaccharide present, comprising about 60% of the total monosaccharides. Also present were mannose (about 35%) and myo-inositol (about 5%). The proteinaceous fraction of the ECM was made up of a number of polypeptides separable by polyacrylamide gel electrophoresis. The lipid fraction of the ECM, analyzed by thin-layer chromatography, was made up of several simple lipid components, including free fatty acid, mono- and triacylglycerol, wax ester, fatty alcohol, and several unidentified components. No complex lipids were detected. Isolated ECM exhibited polygalacturonase and laccase activity and was able to catalyze the hydrolysis of p-nitrophenyl butyrate, a model substrate for assessing cutinase activity. Cellulase, pectin lyase, and pectin methyl esterase activities were noted with both heated and unheated ECM preparations. Proteinase activity was not detected.     

Evaluation of the Effects of Biological Preparations on Phytopathogenic Fungi Didymella applanata and Botrytis cinerea

Fungicidal and fungistatic effects of biological preparations involving bacteria of the genera Pseudomonas and Bacillusand fungi of the genusChaetomium on phytopathogenic fungi Didymella applanata and Botrytis cinereawere evaluated. All the biological preparations under study inhibited the growth of colonies of the fungi; however, the degree of the inhibition depended on the nature of each particular microorganism and the concentration of each particular preparation. A preparation containing Bacillus subtilis at a concentration of 0.2% effected maximum suppression ofB. cinerea (the diameter of the colonies decreased sevenfold). Preparations containing bacteria of the genus Pseudomonas and fungi of the genus Chaetomium were most efficient in suppressing D. applanata.Preparations containingB. subtilis and Chaetomium spp. showed promise as agents against simultaneous development of spur blight and Botrytis blight.     

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract

In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.     

Novel Hypovirulence-Associated RNA Mycovirus in the Plant-Pathogenic Fungus Botrytis cinerea: Molecular and Biological Characterization

Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A −1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The −1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.     

Antifungal Vapour‐phase Activity of a Combination of Allyl Isothiocyanate and Ethyl Isothiocyanate Against Botrytis cinerea and Penicillium expansum Infection on Apples

The potential use of allyl isothiocyanate (AITC) and ethyl isothiocyanate (EITC), singly and in combination, was tested in in vitro and in vivo trials for their effect on Penicillium expansum Link and Botrytis cinerea Persl. infection on apple when used as a fumigant. A 3 : 1 ratio of AITC : EITC was more efficient at reducing in vitro spore germination of P. expansum and B. cinerea than were other combinations or either AITC or EITC alone. The optimized combination showed the lowest EC₅₀ values, at 0.08 and 0.14 μg/ml air, for P. expansum conidial germination and mycelial growth, respectively, and 0.07 and 0.12 μg/ml air for B. cinerea conidial germination and mycelial growth, respectively. In in vivo trials, artificially infected apples were exposed for 4 days to an ITC‐enriched atmosphere. Among the ITCs tested, AITC, EITC and their combinations reduced incidence by more than 85% after 3–4 days of apple incubation at 20°C. Although further studies are necessary to evaluate any detrimental effects on apple quality, the evidence from this study supports the use of fumigation based on ITCs, and in particular a 3 : 1 combination of AITC and EITC, for control of postharvest mildew in apple fruit.     

Preventive and Curative Biological Treatments for Control of Botrytis cinerea Stem Canker of Greenhouse Tomatoes

The effects of biological treatments with PlantShield^®, Prestop^®, Quadra 136, RootShield^®, and S33 (Rhodosporidium diobovatum) and chemical treatment with Decree^® applied as a preventive or curative sprays on stem canker caused by Botrytis cinerea on tomato plants grown in sawdust were studied under near-commercial greenhouse conditions. Prestop^® and Decree^®, applied as preventive or curative sprays, PlantShield^® applied as curative spray, and S33 and Q-136 applied as preventive or preventive plus one spray to wounded surface provided season-long protection from B. cinerea stem canker. These treatments also increased fruit yield and decreased the number of dead plants compared with the inoculated control.     

Bioconversion of alpha-Damascone by Botrytis cinerea

Bioconversion of alpha-damascone (compound 1) was studied with four strains of Botrytis cinerea in grape must (pH 3.2). As biotransformation products of compound 1, 3-oxo-alpha-damascone, cis- and trans-3-hydroxy-alpha-damascone, gamma-damascenone, 3-oxo-8, 9-dihydro-alpha-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-alpha-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-alpha-damascone was observed. Identifications were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e., on-line HRGC-mass spectrometry and HRGC-Fourier transform infrared spectroscopy, after extractive sample preparation.     

Endogenous Elicitor Activity from Damaged Carrot Root Tissue and Induced Resistance to Botrytis cinerea

A peptide-containing fraction (mol. wt. c 5000 Da) extractedfrom carrot root tissues damaged by slicing or freeze-thawing,induced active defence mechanisms in carrot slices against Botrytiscinerea. Endogenous elicitor activity was present in homogenatesof fresh and freeze-thawed tissues and those treated with germinationfluid, but was absent in autoclaved tissue. The detection ofthe elicitor in homogenates within 2h of treatment, suggeststhat it is released or activated during the early stages ofcell damage. Botrytis cinerea, carrot, induced resistance, endogenous elicitor     

Bioconversion of citronellol by Botrytis cinerea

Summary Bioconversion of citronellol 1 was studied with four strains of Botrytis cinerea. Using grape must predominant transformation of 1 to 2,6-dimethyl-1,8-octandiol 2 and (E)-2,6-dimethyl-2-octen-1,8-diol 3 was observed. In minor amounts 2,6-dimethyl-2,8-octandiol 4, two p-menthan-3,8-diol isomers 5a, 5b, (Z)-2,6-dimethyl-2-octen-1,8-diol 6, isopulegol 7, 2-methyl-2-hepten-6-one-1-ol 8 and 2-methyl--butyrolactone 9 were found. Using a small amount of grape must in a synthetic medium (1:700) the bioconversion products 2, 4, 5a and 5b were absent, but additionally 2-methyl-2-hepten-6-one 10, 2-methyl-2-hepten-6-ol 11 and citronellic acid 12 were detected. The results obtained were strongly dependent on the strains used; one strain did not show any metabolic activity against 1. The bioconversion products were identified by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e. on-line — mass spectrometry (HRGC-MS) and — Fourier transform infrared spectroscopy (HRGC-FTIR).     

Oxidation of Alcohols by Botrytis cinerea

Crude cell-free preparations of Botrytis cinerea were found to oxidize straight-chain primary alcohols (except methanol), aromatic primary alcohols, and unsaturated primary alcohols. The resulting products were the corresponding aldehydes and an equal molar quantity of hydrogen peroxide.     

Activity of fungicide mixtures against Botrytis cinerea isolates resistant to benzimidazoles,strobilurins and dicarboximides

Botrytis cinerea, the causal agent of grey mould in a broad range of crops, is considered a high‐risk plant pathogen for fungicide resistance development. The use of fungicide mixtures, particularly combinations with synergistic activity, can be a useful tactic to counteract resistance build‐up in pathogen populations. The present study aimed to investigate the effects of different ratios of two‐way mixtures of carbendazim, iprodione, kresoxim‐methyl, tebuconazole and penconazole on four B. cinerea isolates that were sensitive or resistant to benzimidazoles, dicarboximides and strobilurins. The isolates that were resistant to benzimidazoles and strobilurins had E198A and G143A mutations in β‐tubulin and cytochrome b genes, respectively. The mixtures had different effects on each of the isolates in vitro but, in 13 combinations, the synergistic effect was observed against all or three isolates. In greenhouse experiments, 11 fungicide combinations used in decreased (EC₇₅) concentrations showed the maximum control efficiency. The two follow‐up greenhouse experiments using six selected combinations revealed they were highly effective against additional isolates with various fungicide resistance profiles. The identified mixtures‐ratios have potential for use in grey mould management programs in the greenhouse.