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.     

Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogen Botrytis cinerea

* Botrytis cinerea is a necrotrophic fungus that causes grey mould on a wide range of food plants, especially grapevine, tomato, soft fruits and vegetables. This disease brings about important economic losses in both pre- and postharvest crops. Successful protection of host plants against this pathogen is severely hampered by a lack of resistance genes in the hosts and the considerable phenotypic diversity of the fungus. * The aim of this study was to test whether B. cinerea manipulates the immunity-signalling pathways in plants to restore its disease. * We showed that B. cinerea caused disease in Nicotiana benthamiana through the activation of two plant signalling genes, EDS1 and SGT1, which have been shown to be essential for resistance against biotrophic pathogens; and more interestingly, virus-induced gene silencing of these two plant signalling components enhanced N. benthamiana resistance to B. cinerea. Finally, plants expressing the baculovirus antiapoptotic protein p35 were more resistant to this necrotrophic pathogen than wild-type plants. * This work highlights a new strategy used by B. cinerea to establish disease. This information is important for the design of strategies to improve plant pathogen resistance.     

Bacterial rhamnolipids are novel MAMPs conferring resistance to Botrytis cinerea in grapevine

Rhamnolipids produced by the bacteria Pseudomonas aeruginosa are known as very efficient biosurfactant molecules. They are used for a wide range of industrial applications, especially in food, cosmetics and pharmaceutical formulations as well as in bioremediation of pollutants. In this paper, the role of rhamnolipids as novel molecules triggering defence responses and protection against the fungus Botrytis cinerea in grapevine is presented. The effect of rhamnolipids was assessed in grapevine using cell suspension cultures and vitro-plantlets. Ca²⁺ influx, mitogen-activated protein kinase activation and reactive oxygen species production form part of early signalling events leading from perception of rhamnolipids to the induction of plant defences that include expression of a wide range of defence genes and a hypersensitive response (HR)-like response. In addition, rhamnolipids potentiated defence responses induced by the chitosan elicitor and by the culture filtrate of B. cinerea . We also demonstrated that rhamnolipids have direct antifungal properties by inhibiting spore germination and mycelium growth of B. cinerea . Ultimately, rhamnolipids efficiently protected grapevine against the fungus. We propose that rhamnolipids are acting as microbe-associated molecular patterns (MAMPs) in grapevine and that the combination of rhamnolipid effects could participate in grapevine protection against grey mould disease.     

Comparative analysis of defence responses induced by the endophytic plant growth-promoting rhizobacterium Burkholderia phytofirmans strain PsJN and the non-host bacterium Pseudomonas syringae pv. pisi in grapevine cell suspensions

Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms that colonize the rhizosphere of many plant species and confer beneficial effects, such as an increase in plant growth. PGPR are also well known as inducers of systemic resistance to pathogens in plants. However, the molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain largely unknown. Burkholderia phytofirmans strain PsJN is an endophytic PGPR that colonizes grapevine and protects the plant against the grey mould disease caused by Botrytis cinerea. This report focuses on local defence events induced by B. phytofirmans PsJN after perception by the grapevine cells. It is demonstrated that, after addition to cell suspension cultures, the bacteria were tightly attaching to plant cells in a way similar to the grapevine non-host bacteria Pseudomonas syringae pv. pisi. B. phytofirmans PsJN perception led to a transient and monophasic extracellular alkalinization but no accumulation of reactive oxygen species or cell death were detected. By contrast, challenge with P. syringae pv. pisi induced a sustained and biphasic extracellular alkalinization, a two phases oxidative burst, and a HR-like response. Perception of the PGPR also led to the production of salicylic acid (SA) and the expression of a battery of defence genes that was, however, weaker in intensity compared with defence gene expression triggered by the non-host bacteria. Some defence genes up-regulated after B. phytofirmans PsJN challenge are specifically induced by exogenous treatment with SA or jasmonic acid, suggesting that both signalling pathways are activated by the PGPR in grapevine.     

Characterisation of Pythium paroecandrum and its antagonism towards Botrytis cinerea, the causative agent of grey mould disease of grape

Pythium paroecandrum (B-30), an oomycete, was isolated from soil samples taken from a wheat field in Genlis in the Burgundy region of France and was found to check the growth and development of Botrytis cinerea, a serious grapevine pathogen. The oomycete is a fast-growing organism, living on vegetable debris, and can be recognised by its catenulate hyphal swellings, catenulate oogonia, and monoclinous antheridia. When grown together with B. cinerea, the causal agent of the grey mould disease of the grapevine, P. paroecandrum shows a pronounced antagonism and suppresses its growth and its aptitude to provoke the grey mould symptoms. Morphological features of this oomycete, its antagonism to B. cinerea, the sequences of the internal transcribed spacer region of its nuclear ribosomal DNA, and its comparison with related species are discussed in this article.     

Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26 degrees C) and low (4 degrees C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26 degrees C and 4 degrees C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26 degrees C and 4 degrees C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO(2) fixation and O(2) evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil

During pathogenesis, fungal pathogens are exposed to a variety of fungitoxic compounds. This may be particularly relevant to Botrytis cinerea, a plant pathogen that has a broad host range and, consequently, is subjected to exposure to many plant defense compounds. In practice, the pathogen is controlled with fungicides belonging to different chemical groups. ATP-binding cassette (ABC) transporters might provide protection against plant defense compounds and fungicides by ATP-driven efflux mechanisms. To test this hypothesis, we cloned BcatrB, an ABC transporter-encoding gene from B. cinerea. This gene encodes a 1,439 amino acid protein with nucleotide binding fold (NBF) and transmembrane (TM) domains in a [NBF-TM6]2 topology. The amino acid sequence has 31 to 67% identity with ABC transporters from various fungi. The expression of BcatrB is up regulated by treatment of B. cinerea germlings with the grapevine phytoalexin resveratrol and the fungicide fenpiclonil. BcatrB replacement mutants are not affected in saprophytic growth on different media but are more sensitive to resveratrol and fenpiclonil than the parental isolate. Furthermore, virulence of deltaBcatrB mutants on grapevine leaves was slightly reduced. These results indicate that BcatrB is a determinant in sensitivity of B. cinerea to plant defense compounds and fungicides.     

Antagonistic actinomycetes from Moroccan soil to control the grapevine gray mold

One hundred and forty-two different actinomycete strains were isolated from rhizosphere soil of Vitis vinifera L. sampled from four Moroccan areas. To evaluate the antifungal effect of the different collected actinomycete isolates, five fungi known to be phytopathogens (Pythium ultimum, Fusarium oxyysporum f. sp. albedinis, Sclerotium rolfsii, Verticillium dahliae and Botrytis cinerea) were used. Results showed that 24 isolates had an in vitro inhibitory effect toward at least 4 of the indicator fungi, but only 9 inhibited all these phytopathogens. These nine isolates were subsequently evaluated individually using in vitro grapevine plantlets for their ability to protect against plant gray mold. We demonstrate here that pre-inoculation of plantlets with these isolates allow them to withstand Botrytis cinerea. Six of these strains were shown to belong to the genus Streptomyces and three to the genus Micromonospora. These findings indicate the potential of developing effective actinomycetes from Moroccan habitats for the biological control of Botrytis cinerea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhancement of Chilling Resistance of Inoculated Grapevine Plantlets with a Plant Growth-Promoting Rhizobacterium, Burkholderia phytofirmans Strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26°C) and low (4°C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26°C and 4°C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26°C and 4°C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO₂ fixation and O₂ evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

Synthesis and fungicidal activity of new N,O-acyl chitosan derivatives

Novel N,O-acyl chitosan (NOAC) derivatives were synthesized to examine their fungicidal activity against the gray mould fungus Botrytis cinerea (Leotiales: Sclerotiniaceae) and the rice leaf blast fungus Pyricularia oryzae (Teleomorph: Magnaporth grisea). The fungicidal activity was evaluated by the radial growth bioassay. NOAC derivatives were more active against the two plant pathogens than chitosan itself, and the effect was concentration dependent. Against B. cinerea, 4-chlorobutyryl chitosan (EC50=0.043%), decanoyl chitosan (EC50=0.044%), cinnamoyl chitosan (EC50=0.045%), and p-methoxybenzoyl chitosan (EC50=0.050%) were the most active (12-13-fold more active than chitosan). (Un)-substituted benzoyl chitosan derivatives were more active against B. cinerea than most of these with N,O-alkyl derivatives. Against P. oryzae chitosan derivatives with lauroyl, methoxy acetyl, methacryloyl and decanoyl were the most active.     

Licensed to kill: the lifestyle of a necrotrophic plant pathogen

Necrotrophic plant pathogens have received an increasing amount of attention over the past decade. Initially considered to invade their hosts in a rather unsophisticated manner, necrotrophs are now known to use subtle mechanisms to subdue host plants. The gray mould pathogen Botrytis cinerea is one of the most comprehensively studied necrotrophic fungal plant pathogens. The genome sequences of two strains have been determined. Targeted mutagenesis studies are unraveling the roles played in the infection process by a variety of B. cinerea genes that are required for penetration, host cell killing, plant tissue decomposition or signaling. Our increasing understanding of the tools used by a necrotrophic fungal pathogen to invade plants will be instrumental to designing rational strategies for disease control.     

Exogenous application of a lipid transfer protein-jasmonic acid complex induces protection of grapevine towards infection by Botrytis cinerea.

Type I plant lipid transfer proteins (LTPs) are small, basic, cystein-rich proteins involved in plant defense mechanisms. Five type I LTPs isoforms, named VvLTP1, 2, 3, 4 and 5 (Vitis vinifera lipid transfer proteins 1-5) were purified to homogeneity from the culture media of 41B grapevine cell suspension. The full sequence of isoforms 1, 3, 4 and 5 could be determined from mass spectrometry measurements of the enzymatically hydrolyzed proteins and from available VvLTP sequences. Phylogenetic analysis revealed that these proteins form two subgroups, one with isoforms 1 and 4, and the second one with isoforms 3 and 5. The ability of the three most abundant ones (VvLTP1, 4 and 3) to interact with jasmonic acid (JA) was tested by fluorometric studies, showing that VvLTP4 was the most efficient to interact with this oxylipin. Exogenous application of the VvLTP4-JA complex on grapevine plantlets induced a high level (80.3+/-10.05%) of tolerance towards Botrytis cinerea, as compared with control plants (18.65+/-12.13%); whereas plants treated with JA or VvLTP4 alone exhibited a lower protection level (31.04+/-9.72% and 45.52+/-7.51% of protection, respectively). The results are discussed in the context of grapevine defense mechanisms.     

陕西省烟草灰霉病病原及云芝多糖对其防治研究

采用形态学观察和分子鉴定方法对2011年在陕西省发生的一种烟草未知病害的病原菌进行鉴定。从病叶组织分离纯化得到病原菌,通过致病性测定以及人工接种后再分离病菌,证明编号LJL007的菌株为该病的致病菌。依据病原菌的形态学和培养特征,将菌株LJL007鉴定为灰葡萄孢Botrytis cinerea Pers.,其有性型为富氏葡萄孢盘菌Botryotinia fuckeliana Whetzel。通过核糖体DNAITS序列分析,分离菌株LJL007序列(登录号:HM17900)与富氏葡萄孢盘菌序列(登录号:HM849615)同源性达100%,进一步证明该病原菌是灰葡萄孢Botrytis cinerea。云芝多糖在离体条件下,对灰葡萄孢的菌丝生长和孢子萌发均无直接抑制作用。云芝多糖对烟草灰霉病有较好预防保护作用,其预防效果可达56.29%。云芝多糖可显著提高烟草体内几丁质酶和β-1,3-葡聚糖酶活性,其活性峰值分别比对照提高56.89%和429.83%,说明云芝多糖可诱导植物产生抗病性。     

Pythium periplocum, an aggressive mycoparasite of Botrytis cinerea causing the gray mould disease of grape-vine

Pythium periplocum Dreschler has been found to be an aggressive mycoparasite of Botrytis cinerea, the causal agent of the gray mould disease of the grape-vine. When grown together, the former enters the latter's mycelium, branches freely within, coagulates its cytoplasm and finally tears its hyphae apart, bringing about widespread destruction of the grape-vine pathogen. Extensive coiling around the host, as reported in the case of other mycoparasites belonging to the genus Pythium, has not been observed here. The infected mycelium of B. cinerea fails to infect the grape-vine and does not induce the characteristic gray mould symptoms. Since P. periplocum is not a grape-vine parasite, it could be useful for the biological control of B. cinerea. A brief account of this mycoparasitism is discussed in this article.     

番茄灰霉病拮抗内生放线菌的筛选、鉴定及其活性评价

对安徽省淮北市番茄植株根、茎、叶中内生放线菌进行了分离、筛选,并测定了其抑菌活性。结果表明:番茄根、茎和叶中的内生放线菌的数量分别为5.66×104、0.67×104和0.39×104CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到93株内生放线菌,通过对峙实验,筛选到7株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生放线菌总数的7.5%。来自根组织中的菌株HNU-EA27的抑菌效果最佳,抑菌圈直径达28.3mm。根据形态特征、培养特征、生理生化特性、细胞壁组分和16SrDNA序列分析,将菌株HNU-EA27鉴定为毒三素链霉菌(Streptomyces toxytricini)。室内测定菌株HNU-EA27发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明:菌株HNU-EA27发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为30%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明:30%菌株HNU-EA27发酵滤液对番茄灰霉病的预防与治疗效果分别为80.6%和73.8%,均高于50%多菌灵可湿性粉剂600倍液。本研究表明,菌株HNU-EA27是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

芳樟醇对灰葡萄孢生长的影响及对番茄灰霉病的防控效果

通过平板抑菌试验和孢子萌发试验研究了芳樟醇对灰葡萄孢的生长抑制作用,并通过盆栽试验进一步验证了芳樟醇对番茄灰霉病的防控效果。结果表明: 芳樟醇能够显著抑制灰葡萄孢菌丝的生长,半最大效应浓度(EC₅₀)值为0.581 mL·L^-1。孢子萌发试验中,芳樟醇能够有效抑制灰葡萄孢孢子的萌发,并表现出浓度依赖性。芳樟醇处理提高了灰葡萄孢菌菌丝体的相对电导率和丙二醛(MDA)含量,说明芳樟醇可引起氧化损伤效应导致灰葡萄孢菌的膜系统被破坏;芳樟醇处理后灰葡萄孢菌中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性较对照组分别下降了27.4%、68.9%和26.0%,说明芳樟醇抑制了灰葡萄孢菌体内的抗氧化系统。盆栽试验结果显示,芳樟醇处理的病斑直径较对照组显著降低;番茄叶片中的SOD、CAT、POD、多酚氧化酶和苯丙氨酸解氨酶活性均显著高于对照组;而MDA含量下降了41.5%,说明芳樟醇可减轻灰葡萄孢菌对番茄植株造成的氧化损伤以提高植物抗病性。综上,芳樟醇对灰葡萄孢的生长有显著抑制作用并对番茄灰霉病有较好的防治效果,研究结果可为开发新型植物源抑菌剂防控番茄灰霉病提供理论依据。     

Effects of Trichosporon sp. in Combination with Calcium and Fungicide on Biocontrol of Postharvest Diseases in Apple Fruits

The capability of yeast Trichosporon sp., an antagonist isolated from peach fruit, in biological control was evaluated in apple ( Malus domestica Borkh. cv. Fuji) fruits, when inoculated with different concentrations of Botrytis cinerea Pers. and Penicillium expansum (Link) Thom, as well as in combination with calcium and fungicide. The concentrations of the yeast cells and pathogen spores obviously influenced disease incidence and lesion development in apples. There was a significant negative correlation between concentrations of the yeast cells and infectivity of the pathogens. When the yeast cell suspensions reached the concentration of 108 colony-forming units (CFU)/mL, there was no infection caused by B. cinerea and P. expansum with spore concentrations below 106 spores/mL in apple fruits. The yeast at concentrations of 106-107 CFU/mL in combination with fungicide (iprodione at 50 μL/L) provided control of decay caused by B. cinerea and P．expansum better than separate application. Effect of controlling gray mould and blue mould rots was enhanced when Trichosporon sp., even at low concentration of 105 CFU/mL, was applied in the presence of 1%-2% CaCl2 in an aqueous suspension.