Molecular and functional characterization of a fructose specific transporter from the gray mold fungus Botrytis cinerea

In the gray mold fungus Botrytis cinerea, spore germination and plant infection are stimulated in the presence of nutrients, in particular sugars. Applied at micromolar concentrations, fructose is a more potent inducer of germination than glucose. To test whether preferred fructose uptake is responsible for this effect, and to study the mechanism of fructose transport in B. cinerea, a gene (frt1) encoding a fructose transporter was cloned. FRT1 is highly similar to recently identified fructose transporters of yeasts, but much less to other fungal hexose transporters characterized so far. By using a hexose uptake deficient yeast strain for expression, FRT1 was found to be a high affinity proton coupled symporter specific for fructose but not for glucose. B. cinerea frt1 disruption mutants were created and showed normal vegetative growth and plant infection, but a delay in fructose-induced germination when compared to wild-type. Sugar uptake experiments with both wild-type and mutant conidia showed a higher affinity for glucose than for fructose. Thus, we propose that the specific effect of fructose on germination is not due to transport but rather to an as yet unknown intracellular sensing.     

The small GTPase BcCdc42 affects nuclear division, germination and virulence of the gray mold fungus Botrytis cinerea

The small GTPase Cdc42 plays a central role in various processes in eukaryotic cells including growth, differentiation and cytoskeleton organization. Whereas it is essential in the yeast Saccharomyces cerevisiae, its role in filamentous fungi differs, due to the complementing, partly overlapping function of Rac. We analyzed the role of the Cdc42 homologue in the necrotrophic, broad host range pathogen Botrytis cinerea. Deletion mutants of bccdc42 showed various growth abnormalities; the mutants had reduced growth rate and hyphal branching, they produced fewer conidia, which were enlarged and misshapen and had germination defects. Additionally, the mutants were impaired in sclerotia development. Cytological studies indicate that at least part of this phenotype could be attributed to disturbed control of nuclear division: conidia and hyphae of the mutant showed twofold higher nucleus/cytoplasm ratio compared to wild type cells. Apart from these effects on vegetative growth and differentiation, Δbccdc42 strains were attenuated in penetration and colonization of host tissue, confirming that BcCdc42 – though being not essential like in yeast – is involved in important developmental processes in B. cinerea.     

Mycoparasitism of Acremonium strictum BCP on Botrytis cinerea, the gray mold pathogen

A fungal strain BCP, which parasitizes Botrytis cinerea gray mold pathogen, was isolated and identified as Acremonium strictum. BCP strain overgrew the colonies of B. cinerea and caused severe lysis of the host hyphae. Frequent penetration and hyphal growth of A. strictum BCP inside the mycelia of B. cinerea were observed under light microscopy. In addition, some morphological abnormalities such as granulation and vacuolation of the cytoplasm were observed in mycelia and spores of B. cinerea. In dual culture test, A. strictum BCP strongly inhibited the mycelial growth of several plant pathogenic fungi as well as B. cinerea. To our knowledge, this is the first report on mycoparasitism of Acremonium species on B. cinerea.     

The P450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: DeltaBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene.     

Biological control of strawberry gray mold caused by Botrytis cinerea using Bacillus licheniformis N1 formulation

Bacillus licheniformis N1 is a biological control agent to control gray mold diseases caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were generated and evaluated for the activity to control strawberry gray mold. The wettable powder type formulation N1E was selected in pot experiments with remarkable disease control activity on both strawberry leaves and flowers. The N1E formulation contained 400 g of corn starch, 50 ml of olive oil, and 50 g of sucrose per a liter of bacterial fermentation culture. Optimum dilution of N1E to appropriately control the strawberry gray mold appeared to be 100-fold dilution in plastic house artificial infection experiments. The significant reduction of symptom development in the senescent leaves was apparent by the treatment of N1E at 100-fold dilution when N1E was applied before Bo. cinerea inoculation, but not after the inoculation. Both artificial infection experiments in a plastic house and natural infection experiments in the farm plastic house under production conditions revealed that the disease severity of gray mold on strawberry leaves and flowers was significantly reduced by N1E treatment. The disease control value of N1E on strawberry leaves was 81% under production conditions, as compared with the 61.5% conferred by a chemical fungicide, iprodione. This study suggests that our previously generated formulation of B. licheniformis N1 will be effective to control strawberry gray mold by its preventive activity.     

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。     

A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea

Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in ∆BcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that ∆BcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.     

Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea)

A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance. Received: 31 March 1996 / Revision received: 2 July 1997 / Accepted: 18 July 1997     

Screening of wild accessions resistant to gray mold (Botrytis cinerea Pers.) in Lycopersicon

Tomato gray mold (Botrytis cinerea Pers.) is a common disease worldwide, and often causes serious production loss by infecting leaves, stems, flowers and fruits. Presently, no resistant cultivars are available. To find new breeding materials for gray mold resistance, assessment for resistance of the leaflet and stem in six tomato cultivars, 44 wild tomato accessions and a Solanum lycopersicoides accession was performed. Although no correlation was observed (r=−0.127^ns) between resistance of the leaflet and the stem, L. peruvianum LA2745, L. hirsutum LA2314 and L. pimpinellifolium LA1246 showed high resistance both in the leaflet and in the stem. Particularly, in the leaves of LA2745, no lesions were observed even more than two weeks after the inoculation with conidia, and F1s between a cultivated tomato and LA2745 also showed high resistance as observed in LA2745. From these results, LA2745 is thought to be a promising material for breeding gray-mold resistant cultivars.     

Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.     

Endophytic microorganisms for biocontrol of the phytopathogenic fungus Botrytis cinerea

Botrytis cinerea is the most widely studied necrotrophic phytopathogenic fungus. It causes economic losses that are difficult to calculate due to the large     

Apoptosis-like programmed cell death in the grey mould fungus Botrytis cinerea: genes and their role in pathogenicity

A considerable number of fungal homologues of human apoptotic genes have been identified in recent years. Nevertheless, we are far from being able to connect the different pieces and construct a primary structure of the fungal apoptotic regulatory network. To get a better picture of the available fungal components, we generated an automatic search protocol that is based on protein sequences together with a domain-centred approach. We used this protocol to search all the available fungal databases for domains and homologues of human apoptotic proteins. Among all known apoptotic domains, only the BIR [baculovirus IAP (inhibitor of apoptosis protein) repeat] domain was found in fungi. A single protein with one or two BIR domains is present in most (but not all) fungal species. We isolated the BIR-containing protein from the grey mould fungus Botrytis cinerea and determined its role in apoptosis and pathogenicity. We also isolated and analysed BcNMA, a homologue of the yeast NMA11 gene. Partial knockout or overexpression strains of BcBIR1 confirmed that BcBir1 is anti-apoptotic and this activity was assigned to the N'-terminal part of the protein. Plant infection assays showed that the fungus undergoes massive PCD (programmed cell death) during early stages of infection. Further studies showed that fungal virulence was fully correlated with the ability of the fungus to cope with plant-induced PCD. Together, our result show that BcBir1 is a major regulator of PCD in B. cinerea and that proper regulation of the host-induced PCD is essential for pathogenesis in this and other similar fungal pathogens.     

The minisatellite MSB1, in the fungus Botrytis cinerea,probably mutates by slippage

A minisatellite was identified in the intron of the ATP synthase of the filamentous fungus Botrytis cinerea, and it was named MSB1. This is the second fungal minisatellite described to date. Its 37-bp repeat unit is AT-rich, and it is found at only one locus in the genome. The introns of 47 isolates of Botrytis species were sequenced. The number of tandem repeats varied only from 5 to 11, but there were many repeat variants. The structure of MSB1 is peculiar: the variants are in the same physical order in all individuals, and this order follows the most parsimonious tree. These original characteristics, together with a total lack of recombination between alleles of the flanking regions, suggest that MSB1 probably mutates by slippage. MSB1 was found in the intron of the ATP synthase of all of the Botrytis species analyzed, but the repeat unit was not found in any other genus examined, including Sclerotinia, which is the genus closest to Botrytis.     

Genetic analysis and relationship to pathogenicity in Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. Ten strains from different locations collected on different years have been isolated and characterized by several methods (morphological, biochemical, genetical and molecular). Results showed that clear morphological differences exist between strains, and showing a relationship between the presence of sclerotia and pathogenicity. The conidial size and the nuclear number were highly variable between different strains. Pulsed-field gel electrophoresis showed a unique karyotype for each strain, highly polymorphic between strains and with a number of bands ranging from 4 to 8. An efficient transformation system has been achieved through the plasmid pAMPF21, containing the region AMA1 of Aspergillus nidulans. Lastly, from a genomic library the gdhA gene has been cloned. This gene produces an RNAm of 1.7 Kb and complements the deficiency on glutamate dehydrogenase activity of A. nidulans.     

灰葡萄孢菌AUR1基因内含子对肌醇磷脂酰神经酰胺合成酶表达的影响及致病性

【目的】AUR1编码的肌醇磷脂酰神经酰胺(IPC)合成酶是真菌鞘脂代谢的关键酶,在转录水平和翻译水平研究AUR1内含子对其基因表达的影响,以及AUR1内含子对相关致病因子的影响,为内含子调控基因表达的分子机制提供理论依据。【方法】实时定量PCR测定野生型灰葡萄孢菌(BcAUR1)和AUR1缺失115 bp内含子突变体(BcAUR1a)的mRNA表达量,高效液相层析测定IPC合成酶活性,分别采用辣根过氧化物酶法、邻苯三酚自氧化法、愈创木酚法和紫外分光光度法测定单位菌体的H2O2含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的酶活力。【结果】突变体BcAUR1a的IPC合成酶基因cDNA测序结果表明,IPC合成酶无氨基酸突变。实时定量PCR和高效液相层析的结果表明BcAUR1a的AUR1基因mRNA表达量和IPC合成酶活力比野生型BcAUR1分别增加了50.2%和14.16%。短梗霉素A(AbA)显著刺激BcAUR1 H2O2、SOD、POD和CAT的分泌,但对BcAUR1a的这几种物质的分泌无显著影响。【结论】突变体BcAUR1a的AUR1基因在转录和翻译水平上表达上调,AbA显著增强野生型灰葡萄孢菌致病力,但对突变体影响较小。突变体产生了对AbA的抗性,推测AUR1基因内含子在AUR1基因表达调控中起转录抑制子的作用。     

The cAMP-dependent signaling pathway and its role in conidial germination, growth, and virulence of the gray mold Botrytis cinerea

In Botrytis cinerea, some components of the cAMP-dependent pathway, such as alpha subunits of heterotrimeric G proteins and the adenylate cyclase BAC, have been characterized and their impact on growth, conidiation, germination, and virulence has been demonstrated. Here, we describe the functions of more components of the cAMP cascade: the catalytic subunits BcPKA1 and BcPKA2 and the regulatory subunit BcPKAR of the cAMP-dependent protein kinase (PKA). Although Deltabcpka2 mutants showed no obvious phenotypes, growth and virulence were severely affected by deletion of both bcpka1 and bcpkaR. Similar to Deltabac, lesion development of Deltabcpka1 and DeltabcpkaR was slower than in controls and soft rot of leaves never occurred. In contrast to Deltabac, Deltabcpka1 and DeltabcpkaR mutants sporulated in planta, and growth rate, conidiation, and conidial germination were not impaired, indicating PKA-independent functions of cAMP. Unexpectedly, Deltabcpka1 and DeltabcpkaR showed identical phenotypes, suggesting the total loss of PKA activity in both mutants. The deletion of bcras2 encoding the fungal-specific Ras GTPase resulted in significantly delayed germination and decreased growth rates. Both effects could be partially restored by exogenous cAMP, suggesting that BcRAS2 activates the adenylate cyclase in addition to the Galpha subunits BCG1 and BCG3, thus influencing cAMP-dependent signal transduction.