Oligogalacturonide signal transduction,induction of defense-related responses and protection of grapevine against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H₂O₂, followed by differential expression of nine defense-related genes and stimulation of chitinase and -1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and -1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55–65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.Abbreviations AOS Active oxygen species - Chit Chitinase - DPI Diphenylene iodonium - -Glu -1,3-Glucanase - GST Glutathione-S-transferase - MAP Mitogen-activated protein - OGA Oligogalacturonides - PAL Phenylalanine ammonia-lyase - PR Pathogenesis-related - PGIP Polygalacturonase inhibiting protein - PIN Serine-proteinase inhibitor - STS Stilbene synthase     

Characterization of new bacterial biocontrol agents Acinetobacter,Bacillus, Pantoea and Pseudomonas spp. mediating grapevine resistance against Botrytis cinerea

A collection of 282 bacterial isolates from the rhizosphere and different organs of healthy field-grown grapevine plants was obtained and screened for their ability to protect grapevine leaves against Botrytis cinerea, the causal agent of gray mold. Twenty-six strains effectively controlled B. cinerea infections on leaves. After phenotypic and molecular analysis, seven strains were identified as Pseudomonas fluorescens PTA-268 and PTA-CT2, Bacillus subtilis PTA-271, Pantoea agglomerans PTA-AF1 and PTA-AF2, and Acinetobacter lwoffii PTA-113 and PTA-152. In vitro antifungal experiments showed that from these seven strains, only PTA-AF1 and PTA-CT2 exhibited a direct antagonism against B. cinerea. Furthermore, the biocontrol activity of the seven bacteria was associated with differential induction of defense-related responses lipoxygenase, phenylalanine ammonia-lyase and chitinase in grapevine leaves. Our results show that the selected bacteria can efficiently protect grapevine leaves against gray mold disease through an induction of plant resistance and in some cases by an additional antagonistic activity.     

Revisiting the Cellulosimicrobium cellulans yeast-lytic β-1,3-glucanases toolbox: A review

Cellulosimicrobium cellulans (also known with the synonyms Cellulomonas cellulans, Oerskovia xanthineolytica, and Arthrobacter luteus) is an actinomycete that excretes yeast cell wall lytic enzyme complexes containing endo-β-1,3-glucanases [EC 3.2.1.39 and 3.2.1.6] as key constituents. Three genes encoding endo-β-1,3-glucanases from two C. cellulans strains have been cloned and characterised over the past years. The βglII and βglII _A genes from strain DSM 10297 (also known as O. xanthineolytica LL G109) encoded proteins of 40.8 and 28.6 kDa, respectively, whereas the β-1,3-glucanase gene from strain ATCC 21606 (also known as A. luteus 73–14) encoded a 54.5 kDa protein. Alignment of their deduced amino acid sequences reveal that βglII and βglII _A have catalytic domains assigned to family 16 of glycosyl hydrolases, whereas the catalytic domain from the 54.5 kDa glucanase belongs to family 64. Notably, both βglII and the 54.5 kDa β-1,3-glucanase are multidomain proteins, having a lectin-like C-terminal domain that has been assigned to family 13 of carbohydrate binding modules, and that confers to β-1,3-glucanases the ability to lyse viable yeast cells. Furthermore, βglII may also undergo posttranslational proteolytic processing of its C-terminal domain, resulting in a truncated enzyme retaining its glucanase activity but with very low yeast-lytic activity. In this review, the diversity in terms of structural and functional characteristics of the C. cellulans β-1,3-glucanases has been compiled and compared.     

Gene Cloning and Heterologous Expression of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete Chrysosporium

The basidiomycete Phanerochaete chrysosporium produces several β-1,3-glucanases when grown on laminarin, a β-1,3/1,6-glucan, as the sole carbon source. To characterize one of the major unknown β-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed in the methylotrophic yeast Pichia pastoris degraded branched β-1,3/1,6-glucan as well as linear β-1,3-glucan, the kinetic features of the enzyme were compared with those of other β-1,3-glucanases.     

<Emphasis Type="Italic">Pichia anomala</Emphasis> in biocontrol for apples: 20 years of fundamental research and practical applications

Fungal pathogens such as Botrytis cinerea, Penicillium expansum and the Gloeosporioides group are mainly responsible for important economical losses of post-harvest apples. Application of biological control agents (BCAs) is an emerging alternative to synthetic fungicides. However, before becoming an economically feasible alternative to chemical control, BCAs have to satisfy different requirements related to biological, technological and toxicological properties. The different steps for a successful strategy of disease control (selection, production and formulation, study of mechanisms of action, ecological characterization, molecular monitoring, pilot efficacy trials, registration) are reviewed in this paper considering the antagonistic yeast Pichia anomala strain K. This strain was selected for its high and reliable antagonistic activity against B. cinerea and P. expansum on apples. The studies of mode of action and ecological fitness are emphasized because they can lead to a better efficacy of strain K. Recently advanced molecular techniques have contributed to improving knowledge on the modes of action. Thanks to the identification of genes involved in biocontrol properties, the genetic basis of action mechanisms can be understood. That approach was adopted for P. anomala strain K and led to the identification of genes coding for exo-β-1,3-glucanases implicated in the efficacy. Based on that identification, a formulation involving β-1,3-glucans was developed and applied with higher efficacy in controlled conditions. The importance of ecological characterisation is also highlighted in the context of pre-harvest application of P. anomala strain K. UV light, temperature and humidity were identified as major factors influencing the strain K population. A model taking into consideration temperature and humidity was developed and could be useful in deciding whether pre-harvest treatment is sufficient to allow fast colonization of wounds prior to the arrival of wound pathogens, or whether it is wise to apply further post-harvest treatment to increase the yeast population density. This summary presenting 20 years of work also paid attention to practical application of strain K and its integration with other methods of control.     

Screening tomato-associated bacteria for biological control of grey mold on tomato

Aiming at discovering effective biocontrol agents (BCAs) against grey mold on tomato caused by Botrytis cinerea Pers., we selected 819 bacterial isolates from the surface as well as the interior of the roots, stems, and leaves of tomato plants grown in B. cinerea-infested fields. In a dual-culture assay, 116 isolates (14.16%) showed antagonism against B. cinerea and fewer ones against five additional tomato-associated fungal pathogens – Pythium ultimum, Phytophthora capsici, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum and Ralstonia solanacearum. Thirty-one isolates with antagonism to B. cinerea and at least one of the five additional pathogens were assessed for their efficacy in controlling grey mold on tomato in a greenhouse test. Thirteen of them attained the efficacy over 50% and were subjected to the second greenhouse test, in which 12 isolates consistently accomplished the biocontrol efficacy over 50%, with isolates ABc28 and ABc22 achieving the efficacy of 66.71% and 64.90%, respectively. Under greenhouse conditions, the above two as well as isolates ABc2, ABc11 and ABc17 increased tomato biomass by more than 20% in comparison with the control. The 12 antagonistic isolates accomplishing the biocontrol efficacy over 50% in both greenhouse tests were considered potential BCAs against grey mold, which were identified as Pseudomonas spp., Pantoea spp., Bacillus spp. and Chryseobacterium spp. Ten of them were found to produce at least one of the three hydrolytic enzymes (protease, cellulase and chitinase) and/or siderophore, which might be involved in their mechanisms of suppressing the disease. Based on the origin of these 12 strains, the leaf tissue, especially the leaf interior, of tomato plants grown in a B. cinerea-infested field appears to be a good source of potential BCAs against grey mold.     

The extracellular system of β-1,3-glucanases ofAlternaria tenuissima andAspergillus vesicolor

During cultivation in a minimal medium with glucoseAlternaria tenuissima andAspergillus vesicolor produce constitutively α- and β-glucanases. Fractions of β-1,3-glucanases exhibiting affinity for laminarin were separated by means of gel filtration chromatography. Two neutral β-1,3-glucanases with affinity for yeast glucan were isolated by affinity chromatography and further characterized.     

Impact of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.     

Effects of yeast antagonist in combination with UV-C treatment on postharvest diseases of pear fruit

The yeast antagonist Candida guilliermondii and ultraviolet-C (UV-C) treatment were investigated for controlling infection following artificial inoculation with Penicillium expansum or Botrytis cinerea, or natural infection in pear fruit stored at 20°C. Applied separately, both C. guilliermondii and UV-C (5 kJ m⁻²) effectively inhibited decay caused by P. expansum or B. cinerea, and natural infection. The combination of C. guilliermondii and UV-C showed better control efficacy. Application of UV-C did not affect the growth of C. guilliermondii in pear fruit wounds, while UV-C induced a significant increase in the activities of chitinase, β-1,3-glucanase, catalase and peroxidase in pear fruit. The mechanism by which UV-C enhanced the biocontrol efficacy of C. guilliermondii may be related to the elicitation of defense responses in pear fruit. The combination of C. guilliermondii and UV-C radiation could be a promising method for the control of P. expansum and B. cinerea in pear fruit.     

Expression of soybean b-1,3-endoglucanase cDNA and effect on disease tolerance in kiwifruit plants

Kiwifruit was transformed with a soybean β-1,3-endoglucanase (EC 3.2.1.39) cDNA under the control of the cauliflower mosaic virus (CaMV) 35S RNA promoter. The introduced gene was expressed in young leaves of the transformants. Assays of protein extracts from young leaves showed an increase in enzyme activity in many transformants, the transformant with the highest level of enzyme activity having an about sixfold increase over the control plants. When leaves from control and three transformants were inoculated with Botrytis cinerea, which causes gray mold disease, the disease lesion areas for two transformants were smaller than on control plants. Received: 5 March 1998 / Revision received: 19 October 1998 / Accepted: 27 October 1998     

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.     

Production of lytic enzymes by<Emphasis Type="Italic">Trichoderma</Emphasis> spp. and their effect on the growth of phytopathogenic fungi

The production of β-1,3-glucanases and chitinases by three strains ofTrichoderma in submerged cultures was determined. The synthesis of enzymes was induced by cell wall biopolymers of phytopathogenic fungi (Botrytis cinerea, Fusarium culmorum andF. oxysporum). T. hamatum produced the highest β-1,3-glucanase activity; the most effective inducer of enzyme synthesis was the biomass ofF. oxysporum. All examined strains ofTrichoderma inhibited phytopathogen growth in biotic tests. The diffusion tests showed that the lytic enzymes take part in growth inhibition of phytopathogenic fungi.     

The polyphagous plant pathogenic fungus Botrytis cinerea encompasses host-specialized and generalist populations

The host plant is often the main variable explaining population structure in fungal plant pathogens, because specialization contributes to reduce gene flow between populations associated with different hosts. Previous population genetic analysis revealed that French populations of the grey mould pathogen Botrytis cinerea were structured by hosts tomato and grapevine, suggesting host specialization in this highly polyphagous pathogen. However, these findings raised questions about the magnitude of this specialization and the possibility of specialization to other hosts. Here we report specialization of B. cinerea populations to tomato and grapevine hosts but not to other tested plants. Population genetic analysis revealed two pathogen clusters associated with tomato and grapevine, while the other clusters co-occurred on hydrangea, strawberry and bramble. Measurements of quantitative pathogenicity were consistent with host specialization of populations found on tomato, and to a lesser extent, populations found on grapevine. Pathogen populations from hydrangea and strawberry appeared to be generalist, while populations from bramble may be weakly specialized. Our results suggest that the polyphagous B. cinerea is more accurately described as a collection of generalist and specialist individuals in populations. This work opens new perspectives for grey mould management, while suggesting spatial optimization of crop organization within agricultural landscapes.     

Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.     

Characterization and function of wall-bound exo-β-glucanases of Lilium longiflorum pollen tubes

Two exo-β-glucanases (LP-ExoI, 83 kDa and LP-ExoII, 71 kDa) were extracted and partially purified from the cell wall of Lilium longiflorum pollen tubes. Both LP-ExoI and LP-ExoII hydrolyzed laminarin (1,3-β-glucan). These enzymes also exhibited some activity toward 1,3:1,4-β-glucans of Hordeum vulgare and Cetraria islandica and the 1,6-β-glucan of Umbilicaria papullosa. The pH for optimum activity for both exo-β-glucanases was 5.5. Methylation analysis of the reaction products revealed that purified LP-ExoI decreased both 1,3- and 1,4-glucosyl linkages in hemicellulosic polysaccharides isolated from the cell wall of lily pollen tubes. D-gluconolactone and nojirimycin, inhibitors of glucosidase, inhibited activities of both exo-β-glucanases, as well as growth of the lily pollen tubes. These results disclosed that the wall-bound exo-β-glucanases play an important role in the regulation of lily pollen tube growth. Received: 3 January 2000 / Revision accepted: 8 March 2000     

Ability of the antagonistic yeast Cryptococcus albidus to control Botrytis cinerea in strawberry

The yeast Cryptococcus albidus, originally isolated from mature strawberry fruits, was tested for antagonistic activity against Botrytis cinerea, the causal agent of grey mould in strawberries. Conidial germination and germ tube growth of conidia of B. cinerea were inhibited by a cell suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) after 6 and 24 hours of incubation. Application of a cell suspension (1 × 10⁶ cells/ml) on detached strawberry leaf disks incubated at 10°C reduced incidence and conidiophore density of B. cinerea by 86 and 99%, respectively, but effectiveness was reduced at higher temperatures. Treatments with C. albidus during bloom of strawberries reduced incidence of grey mould on ripe strawberry fruits after harvest by 33, 28 and 21% in three years of field trials. The effectiveness of the yeast was increased when formulation substances (alginate, xanthan and cellulose) were added to the cell suspension.     

Purification and molecular characterization of exo-β-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2

The extracellular β-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific β-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40°C, respectively. The enzyme was significantly stimulated by Li⁺, Ni²⁺, and Ba²⁺. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR which is the conserved motif of the β-1,3-glucanases from other yeasts.     

Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases

A metagenomic cosmid library was prepared in Escherichia coli from DNA extracted from the contents of rabbit cecum and screened for cellulase activities. Eleven independent clones expressing cellulase activities (four endo-β-1,4-glucanases and seven β-glucosidases) were isolated. Subcloning and sequencing analysis of these clones identified 11 cellulase genes; the encoded products of which shared less than 50% identities and 70% similarities to cellulases in the databases. All four endo-β-1,4-glucanases and all seven β-glucosidases, respectively, belonged to glycosyl hydrolase family 5 (GHF 5) and family 3 (GHF 3) and formed two separate branches in the phylogenetic tree. Ten of the 11 cloned cellulases exhibited highest activities at pH 5.5 ∼ 7.0 and 40 ∼ 55°C, a condition similar to that in the rabbit cecum. All the four endo-β-1,4-glucanases could hydrolyze a wide range of β-1,4-, β-1,4/β-1,3- or β-1,3/β-1,6-linked polysaccharides. One endo-β-1, 4-glucanase gene, umcel5G, was overexpressed in E. coli, and the purified recombinant enzyme was characterized in detail. The enzymes cloned in this work represented at least some of the cellulases operating efficiently in the rabbit cecum. This work provides the first snapshot on the cellulases produced by bacteria in rabbit cecum.     

Potential biocontrol activity of a strain of Pichia guilliermondii against grey mold of apples and its possible modes of action

The efficacy of Pichia guilliermondii strain M8 against Botrytis cinerea on apples was evaluated under storage conditions, and its possible modes of action were investigated both in vitro and in vivo experiments. After storage at 1 °C for 120 days, M8 reduced grey mold incidence from 45.3% (control) to 20.0%. In apple juice medium (AJM) and in wound-inoculated apples, M8 at 10⁹ and 10⁸ cells ml⁻¹ inhibited the spore germination of B. cinerea and the grey mold development. When co-culturing B. cinerea in vitro or in vivo in the presence of the yeast, neither inactivated cells nor culture filtrate of the yeast had any effect on spore germination or germ tube elongation. In AJM, the spore germination was significantly recovered by the addition of 1% glucose, sucrose and fructose, or 0.5% and 1% of (NH₄)₂SO₄, phenylalanine and asparagine. When the pathogen and the yeast were co-incubated in apple wounds with addition of the same nutrients, the inhibition of rots was significantly reduced by the supplemental nutrients. Light microscopy revealed that the yeast strongly adhered to the hyphae and spores of B. cinerea. M8 produced hydrolytic enzymes, including β-1,3-glucanase and chitinases in minimal salt media with different carbon sources. Pretreatment with M8 at 10⁸ cells ml⁻¹ followed by washing, significantly reduced grey mold lesions, suggesting an induction of defense responses. Direct attachment, competition for nitrogen and carbon sources, secretion of hydrolytic enzymes and induction of host resistance play a role in the biocontrol mechanism of P. guilliermondii M8 against B. cinerea.     

Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.