Phytopathogenic Fungus Botrytis Cinerea Research Articles

Farnesol induces apoptosis-like phenotype in the phytopathogenic fungus Botrytis cinerea

This study demonstrates that the isoprenoid farnesol produces a toxic effect on the phytopathogenic fungus Botrytis cinerea in solid and liquid media. In solid media farnesol retarded 72 h the beginning of mycelial growth. Also, it was demonstrated that the toxic effect is due to farnesol triggers apoptosis in B. cinerea because ROS accumulation, DNA fragmentation and phosphatidylserine externalization were detected in farnesol-treated mycelium. Therefore, compounds that increase the intracellular farnesol or induce apoptosis could have a potential application as fungicide against B. cinerea.

EVALUATION OF ANTIFUNGAL PROPERTIES AGAINST THE PHYTOPATHOGENIC FUNGUS BOTRYTIS CINEREAOF ANTHOCYANIN RICH-EXTRACTS OBTAINED FROM GRAPE POMACES

ABSTRACT The anthocyanin content of grape pomace from different Chilean grape varieties ( Vitis vinifera var. Cabernet Sauvignon, Carmenere and Syrah) from two vineyards (Misiones de Rengo and Miguel Torres) was determined by the differential pH method and the anthocyanin perfile was characterized by HPLC. In all chromatograms, a major peak corresponding to malvidin-3-O-glucoside was observed. Also, the in vitro antifungal activity of these extracts against mycelia growth of the phytopathogenic fungus Botrytis cinerea was evaluated. The anthocyanin containing extract from Cabernet Sauvignon grape pomace of Misiones de Rengo vineyard was the most active. The extract from the same variety form Miguel Torres vineyard did not inhibit mycelia growth of B. cinerea . In general, the other extracts were less active against the fungus. Keywords: Anthocyanins, antifungal activity, Botrytis cinerea , grape pomace. e-mail: leonora.mendoza@usach.cl; milena.cotoras@usach.cl INTRODUCTION In the wine industry, a large amount of grape residues (grape pomace) are obtained and this residue consists mainly on skin residues, broken cells with pulp remains, stalks, and seeds

Antibacterial and antifungal activities of Otanthus maritimus (L.) Hoffmanns. & Link essential oil from Sicily

The chemical composition of the essential oil obtained from the flowers of Otanthus maritimus L., a perennial plant growing wild in maritime sands in the Mediterranean region, was investigated by GC and GC–MS analyses. Totally 66 were identified. The oil was dominated by the high content of monoterpene compounds, especially oxygenated monoterpenes which accounted for 73.1%. The most abundant components were yomogi alcohol (20.8%), camphor (15.8%), artemisyl acetate (15.3%) and artemisia alcohol (13.7%). The oil was tested against two Gram (+) and six Gram (−) bacterial strains, both American Type Culture Collection standard strains and clinically isolated (CI), one potentially pathogenic yeast (Candida albicans CI) and two filamentous phytopathogenic fungi (Botrytis cinerea and Rhizoctonia solani). The results show that the oil from O. maritimus exerts strong antibacterial and antifungal activities.

The Homeobox BcHOX8 Gene in Botrytis Cinerea Regulates Vegetative Growth and Morphology

Filamentous growth and the capacity at producing conidia are two critical aspects of most fungal life cycles, including that of many plant or animal pathogens. Here, we report on the identification of a homeobox transcription factor encoding gene that plays a role in these two particular aspects of the development of the phytopathogenic fungus Botrytis cinerea. Deletion of the BcHOX8 gene in both the B. cinerea B05-10 and T4 strains causes similar phenotypes, among which a curved, arabesque-like, hyphal growth on hydrophobic surfaces; the mutants were hence named Arabesque. Expression of the BcHOX8 gene is higher in conidia and infection cushions than in developing appressorium or mycelium. In the Arabesque mutants, colony growth rate is reduced and abnormal infection cushions are produced. Asexual reproduction is also affected with abnormal conidiophore being formed, strongly reduced conidia production and dramatic changes in conidial morphology. Finally, the mutation affects the fungus ability to efficiently colonize different host plants. Analysis of the B. cinerea genome shows that BcHOX8 is one member of a nine putative homeobox genes family. Available gene expression data suggest that these genes are functional and sequence comparisons indicate that two of them would be specific to B. cinerea and its close relative Sclerotinia sclerotiorum.

Characterization of extracts from winery by-products with antifungal activity against Botrytis cinerea

Phenolic compound profiles of grape pomace extracts from a mixture of Chilean grape varieties (Cabernet Sauvignon, Carmènere and Syrah) were analyzed. Two extraction methods were used: solvent and soxhlet extraction, followed by fractionation with hexane, chloroform and ethyl acetate. Phenolic compounds in different fractions were identified by HPLC. Ethyl acetate phase of solvent extraction contained a wide variety of phenolic compounds. Hexane phase of the extracts presented lowest diversity. Quercetin was found in almost all fractions.Also, the in vitro antifungal activity of these extracts against the phytopathogenic fungus Botrytis cinerea was evaluated. Hexane or chloroform fractions from extracts obtained by solvent extraction showed the highest inhibitory effect on mycelia growth of this fungus, with IC50 value of 40ppm. In general, ethyl acetate fractions were less active against B. cinerea. Therefore, it can be concluded that grape pomace are a good low cost source to obtain antifungal extracts.

In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit

Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense-related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins-specific inhibitors of cysteine proteases. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno-tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp-sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L(-1)) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L(-1)) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules.

Metabolites from Aspergillus fumigatus, an Endophytic Fungus Associated with Melia azedarach, and Their Antifungal, Antifeedant, and Toxic Activities

Thirty-nine fungal metabolites 1-39, including two new alkaloids, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6) and 3-hydroxyfumiquinazoline A (16), were isolated from the fermentation broth of Aspergillus fumigatus LN-4, an endophytic fungus isolated from the stem bark of Melia azedarach. Their structures were elucidated on the basis of detailed spectroscopic analysis (mass spectrometry and one- and two-dimensional NMR experiments) and by comparison of their NMR data with those reported in the literature. These isolated compounds were evaluated for in vitro antifungal activities against some phytopathogenic fungi, toxicity against brine shrimps, and antifeedant activities against armyworm larvae (Mythimna separata Walker). Among them, sixteen compounds showed potent antifungal activities against phytopathogenic fungi (Botrytis cinerea, Alternaria solani, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. vasinfectum, and Gibberella saubinettii), and four of them, 12β-hydroxy-13α-methoxyverruculogen TR-2 (6), fumitremorgin B (7), verruculogen (8), and helvolic acid (39), exhibited antifungal activities with MIC values of 6.25-50 μg/mL, which were comparable to the two positive controls carbendazim and hymexazol. In addition, of eighteen that exerted moderate lethality toward brine shrimps, compounds 7 and 8 both showed significant toxicities with median lethal concentration (LC(50)) values of 13.6 and 15.8 μg/mL, respectively. Furthermore, among nine metabolites that were found to possess antifeedant activity against armyworm larvae, compounds 7 and 8 gave the best activity with antifeedant indexes (AFI) of 50.0% and 55.0%, respectively. Structure-activity relationships of the metabolites were also discussed.

Growth rate inhibition of phytopathogenic fungi by characterized chitosans.

The inhibitory effects of fifteen chitosans with different degrees of polymerization (DP) and different degrees of acetylation (FA) on the growth rates (GR) of four phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer) were examined using a 96-well microtiter plate and a microplate reader. The minimum inhibitory concentrations (MICs) of the chitosans ranged from 100 μg ×mL-1 to 1,000 μg ×mL-1 depending on the fungus tested and the DP and FA of the chitosan. The antifungal activity of the chitosans increased with decreasing FA. Chitosans with low FA and high DP showed the highest inhibitory activity against all four fungi. P. expansum and B. cinerea were relatively less susceptible while A. alternata and R. stolonifer were relatively more sensitive to the chitosan polymers. Scanning electron microscopy of fungi grown on culture media amended with chitosan revealed morphological changes.

Synthesis, characterization, and crystal structure of Ni(II) and Cu(II) complexes with N-furoyl-N′,N′-diethylthiourea: antifungal activity

The complexes cis-bis(N-furoyl-N′,N′-diethylthioureato-k 2O,S)nickel(II) and cis-bis(N-furoyl-N′,N′-diethylthioureato-k 2O,S)copper(II) were prepared by the reaction of metal acetate with the corresponding acylthiourea derivative. The complexes were characterized by IR, 1H-NMR, 13C-NMR, and single-crystal X-ray diffraction. Both complexes show two furoylthiourea ligands bonded to metal to form a four-coordinate complex with square-planar geometry. The antifungal activity of the prepared complexes was studied against the phytopathogenic fungi Botrytis cinerea and Colletotrichum gloeosporioides, responsible for important plant diseases.

Biocontrol with Myxococcus sp. KYC 1126 Against Anthracnose in Hot Pepper

Antifungal activity of three Myxococcus spp., KYC 1126, 1136, and 2001, was tested in vitro against three phytopathogenic fungi (Botrytis cinerea, Colletotrichum acutatum, and Pyricularia grisea). Spore germination and mycelial growth of the three pathogenic fungi were completely inhibited by bioactive substances from a myxobacterium KYC 1126. In addition, the activity of KYC 1126 was fungicidal, but liquid culture filtrate of KYC 1126 did not affect protoplast reversion in C. acutatum. A bioassay of KYC 1126 filtrate against anthracnose in hot pepper was conducted in the greenhouse and field at 2009 and 2010. The incidence of anthracnose in control seedlings was 74%, but was reduced to 29% after KYC 1126 treatment. The control value with KYC 1126 was 60% while that with the fungicide dithianon was 42%. In the greenhouse, disease incidence with KYC 1126 was consistentely 10-35% lower than with fungicide as a positive control. The control value with KYC 1126 was 13.4% and 41.0%, whereas that with the fungicide was 52.3% and 63% in 2009 and 2010, respectively. Although anti-anthracnose activity of KYC 1126 was not maintained for long time in the field, the bacteriolytic myxobacterium KYC 1126 could be a prospective biocontrol agent.

Detoxification of cruciferous phytoalexins in Botrytis cinerea: Spontaneous dimerization of a camalexin metabolite

Phytopathogenic fungi are able to overcome plant chemical defenses through detoxification reactions that are enzyme mediated. As a result of such detoxifications, the plant is quickly depleted of its most important antifungal metabolites and can succumb to pathogen attack. Understanding and predicting such detoxification pathways utilized by phytopathogenic fungi could lead to approaches to control plant pathogens. Towards this end, the inhibitory activities and metabolism of the cruciferous phytoalexins camalexin, brassinin, cyclobrassinin, and brassilexin by the phytopathogenic fungus Botrytis cinerea Pers. (teleomorph: Botryotinia fuckeliana) was investigated. Brassilexin was the most antifungal of the phytoalexins, followed by camalexin, cyclobrassinin and brassinin. Although B. cinerea is a species phylogenetically related to the phytopathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary, contrary to S. sclerotiorum, detoxification of strongly antifungal phytoalexins occurred via either oxidative degradation or hydrolysis but not through glucosylation, suggesting that glucosyl transferases are not involved. A strongly antifungal bisindolylthiadiazole that B. cinerea could not detoxify was discovered, which resulted from spontaneous oxidative dimerization of 3-indolethiocarboxamide, a camalexin detoxification product.

Disruption of the Bcchs3a Chitin Synthase Gene in Botrytis cinerea Is Responsible for Altered Adhesion and Overstimulation of Host Plant Immunity

The fungal cell wall is a dynamic structure that protects the cell from different environmental stresses suggesting that wall synthesizing enzymes are of great importance for fungal virulence. Previously, we reported the isolation and characterization of a mutant in class III chitin synthase, Bcchs3a, in the phytopathogenic fungus Botrytis cinerea. We demonstrated that virulence of this mutant is severely impaired. Here, we describe the virulence phenotype of the cell-wall mutant Bcchs3a on the model plant Arabidopsis thaliana and analyze its virulence properties, using a variety of A. thaliana mutants. We found that mutant Bcchs3a is virulent on pad2 and pad3 mutant leaves defective in camalexin. Mutant Bcchs3a was not more susceptible towards camalexin than the wild-type strain but induced phytoalexin accumulation at the infection site on Col-0 plants. Moreover, this increase in camalexin was correlated with overexpression of the PAD3 gene observed as early as 18 h postinoculation. The infection process of the mutant mycelium was always delayed by 48 h, even on pad3 plants, probably because of lack of mycelium adhesion. No loss in virulence was found when Bcchs3a conidia were used as the inoculum source. Collectively, these data led us to assign a critical role to the BcCHS3a chitin synthase isoform, both in fungal virulence and plant defense response.

Genome characterization of a debilitation-associated mitovirus infecting the phytopathogenic fungus Botrytis cinerea

The full-length sequences of B otrytis c inerea mito virus 1 (BcMV1) and an associated RNA (BcMV1-S) in strain CanBc-1c-78 of Botrytis cinerea were determined. Sequence analysis showed that BcMV1 is 2804 nt long and AU-rich (66.8%). BcMV1 shares 95% nucleotide sequence identity with O phiostoma n ovo- u lmi mito virus 3b (OnuMV3b). However, it is 472 nt longer than OnuMV3b. Mitochondrial codon usage revealed that BcMV1 contains one open reading frame encoding RdRp, which is 96% identical to the RdRp of OnuMV3b. These findings confirm that BcMV1 belongs to the genus Mitovirus and is a strain of OnuMV3b. BcMV1-S is 2171 nt long and derived from BcMV1 through a single internal in-frame deletion of 633 nt, suggesting that it is a defective RNA of BcMV1. BcMV1-S was found to suppress the replication of BcMV1 and to be co-transmissible with BcMV1 through hyphal anastomosis. Its presence, however, did not alleviate the BcMV1-associated debilitation phenotypes of B. cinerea.

Gene disruption and characterization of a class V chitin synthase in Botrytis cinerea

Cell-wall chitin biosynthesis in the phytopathogenic fungus Botrytis cinerea involves 7 classes of chitin synthases. In this study, we disrupted a gene encoding a chitin synthase with a myosin-like motor domain (BcchsV) through Agrobacterium tumefaciens mediated transformation. The resulting mutant was not significantly affected in either growth characteristics or pathogenicity on tomato leaves. Surprisingly, the BcchsV mutant exhibited a 31% (m/m) increase in its chitin content compared with the wild-type strain. In addition, the BcchsV mutant showed increased sensitivity to Calcofluor White and slightly enhanced tolerance to cell-wall disturbing substances and osmosis regulators, including SDS, sorbitol, and NaCl. These results suggest that Bcchs does not play an essential role in the synthesis of cell-wall chitin in B. cinerea. However, disruption of this gene provoked a compensatory mechanism regulating the cellular response to cell-wall damage.

PH controls both transcription and post-translational processing of the protease BcACP1 in the phytopathogenic fungus Botrytis cinerea

During pathogenesis, the ascomycete Botrytis cinerea secretes a range of cell-wall-degrading enzymes such as polygalacturonases, glucanases and proteases. We report the identification of a new member of the G1 family of proteases, BcACP1, which is secreted by B. cinerea during infection. The production of BcACP1 correlates with the acidification of the plant tissue, and transcriptional analysis of the Bcacp1 gene showed that it is only expressed under acidic growth conditions. Using a transcriptional reporter system, we showed that pH regulation of Bcacp1 is not mediated by the canonical PacC transcription factor binding site. Like other G1 proteases, BcACP1 is produced as a pro-enzyme. Trapping of the zymogen form allowed investigation of its maturation process. Evidence is presented for an autocatalytic proteolysis of the enzyme that is triggered by acidic pH. Environmental pH therefore controls Bcacp1 production at both the transcriptional and post-translational level.

Global Antifungal Profile Optimization of Chlorophenyl Derivatives against Botrytis cinerea and Colletotrichum gloeosporioides

Twenty-two aromatic derivatives bearing a chlorine atom and a different chain in the para or meta position were prepared and evaluated for their in vitro antifungal activity against the phytopathogenic fungi Botrytis cinerea and Colletotrichum gloeosporioides. The results showed that maximum inhibition of the growth of these fungi was exhibited for enantiomers S and R of 1-(4'-chlorophenyl)-2-phenylethanol (3 and 4). Furthermore, their antifungal activity showed a clear structure-activity relationship (SAR) trend confirming the importance of the benzyl hydroxyl group in the inhibitory mechanism of the compounds studied. Additionally, a multiobjective optimization study of the global antifungal profile of chlorophenyl derivatives was conducted in order to establish a rational strategy for the filtering of new fungicide candidates from combinatorial libraries. The MOOP-DESIRE methodology was used for this purpose providing reliable ranking models that can be used later.

Synthesis and Quantitative Structure−Antifungal Activity Relationships of Clovane Derivatives against Botrytis cinerea

Twenty-three clovane derivatives, nine described here for the first time, bearing substituents on carbon C-2, have been synthesized and evaluated for their in vitro antifungal activity against the phytopathogenic fungus Botrytis cinerea. The results showed that compounds 9, 14, 16, and 18 bearing nitrogen atoms in the chain attached at C-2 displayed potent antifungal activity, whereas mercapto derivatives 13, 19, and 22 displayed low activity. The antifungal activity showed a clear structure-activity relationship (SAR) trend, which confirmed the importance of the nature of the C-2 chain on the antifungal activity. On the basis of these observations, the metabolism of compounds 8 and 14 by the fungus B. cinerea, and the metabolism of other clovanes by this fungus, described previously, a pro-drug action mechanism for 2-alkoxyclovane compounds is proposed. Quantitative structure-activity relationship (QSAR) studies were performed to rationalize the results and to suggest further optimization, using a topological sub-structural molecular design (TOPS-MODE) approach. The model displayed good fit and predictive capability, describing 85.5% of the experimental variance, with a standard deviation of 9.502 and yielding high values of cross-validation determination coefficients (q2CV-LOO = 0.784 and q2boot = 0.673). The most significant variables were the spectral moments weighted by bond dipole moment (Dip), hydrophobicity (Hyd), and the combined dipolarity/polarizability Abraham molecular descriptor (Ab-pi2H).

Role of the Botrytis cinerea FKBP12 ortholog in pathogenic development and in sulfur regulation

The functional characterization of the FKBP12 encoding gene from the phytopathogenic fungus Botrytis cinerea was carried out. B. cinerea genome sequence owns a single ortholog, named BcFKBP12, encoding a FK506-binding protein of 12 kDa. BcFKBP12 mediates rapamycin sensitivity both in B. cinerea and in Saccharomyces cerevisiae, a property unique to FKBP12 proteins, probably via the inhibition of the protein kinase TOR (target of rapamycin). The relative abundance of the prolyl isomerase appeared to be regulated and increased in response to the presence of extracellular nutrients. Surprisingly, the BcFKBP12 deletion did not affect the pathogenic development of the strain B05.10, while it was reported to cause a reduction of the virulence of the strain T4. We report for the first time the BcFKBP12 involvement in the sulfur repression of the synthesis of a secreted serine protease. Rapamycin treatment did not relieve the sulfur repression of the reporter system in the wild-type strain. Thus BcFKBP12 may participate in sulfur regulation and its contribution seems to be independent of TOR.

Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins

In this study, for the first time, functionally active, recombinant, cysteine-rich plant proteins snakin-1 (SN1) and defensin (PTH1) were expressed and purified using a prokaryotic expression system. The overall level of antimicrobial activities of SN1 and PTH1 produced in Escherichia coli was commensurate with that of the same proteins previously obtained from plant tissues. Both proteins exhibited strong antibacterial activity against the phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus (50% inhibitory concentration (IC 50) 1.5–8 μM) and antifungal activity against the phytopathogenic fungi Colletotrichum coccoides and Botrytis cinerea (IC 50 5–14 μM). Significantly weaker activity was observed against Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. tabaci. A pronounced synergistic antimicrobial effect against P. syringae pv. syringae and an additive effect against P. syringae pv. tabaci occurred with a combination of SN1 and PTH1. Aggregation of C. michiganensis subsp. sepedonicus bacterial cells at all protein concentrations tested was observed with the combination of SN1 and PTH1 and with SN1 alone. Our results demonstrate the use of a cost effective prokaryotic expression system for generation and in vitro characterization of plant cysteine-rich proteins with potential antimicrobial activities against a wide range of phytopathogenic microorganisms in order to select the most effective agents for future in vivo studies.

Effect of extracts from in vitro-grown shoots of Quillaja saponaria Mol. on Botrytis cinerea Pers.

The ethanolic and aqueous extracts from in vitro shoots of Quillaja saponaria Mol. (Quillay) were studied for their antifungal activity against the phytopathogenic fungus Botrytis cinerea Pers. These extracts reduced conidial germination and mycelial growth of B. cinerea, ethanolic extracts being more active than aqueous extracts. In addition, the damage areas produced by this fungus on tomato leaves and strawberry fruits pre-treated with quillay extracts were diminished. The fungitoxic effect of in vitro-grown quillay extract was similar to those obtained with commercial fungicides of both natural (BC-1000) and synthetic (iprodione–dicarboximide) origin. On the other hand, the antifungal action of quillay extracts obtained from adult trees naturally grown was only slightly superior to the fungitoxic activity of the extract from in vitro plants. HPLC analysis of the extract showed that it contained saponins and some phenolic compounds such as chlorogenic, caffeic, vanillic, and salicylic acids, and scopoletin, which have been identified as antifungal agents on phytopathogenic fungi. The results obtained in this work, suggests that extracts of in vitro-grown quillay have an important protective effect against B. cinerea and support the use of an in vitro culture system as a biotechnological alternative to obtain environmental safe antifungal quillay extracts to control B. cinerea, contributing to the preservation of this indigenous Chilean species.