Functional analysis of an extracellular catalase of Botrytis cinerea

There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H₂O₂ as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H₂O₂ and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H₂O₂ accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H₂O₂ in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H₂O₂in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H₂O₂. Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H₂O₂ comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.     

灰葡萄孢产孢新基因的功能分析

灰葡萄孢是一种重要的植物病原真菌,其寄主范围广泛,能危害世界上230多种双子叶植物,常给农业生产造成重大的经济损失[1-3].由灰葡萄孢引起的灰霉病是目前我国温室蔬菜生产中最主要的病害之一,一般造成全年减产20％-25％,严重时达到40％以上[4].因此,研究该病菌的致病机理对该病防治具有重要意义,并且随着灰葡萄孢基因组测序的完成,灰葡萄孢已成为发育生物学、分子植物病理学研究的模式生物之一.     

源自不同寄主的灰葡萄孢生物学特性的比较研究

本研究以分离自番茄、辣椒、草莓、葡萄的灰葡萄孢Botrytis cinerea为供试菌株,从生长温度、pH适应性、碳源、氮源营养利用等方面对不同寄主来源的灰葡萄孢菌株的生物学性状进行了比较研究。结果表明,5个不同寄主来源的灰葡萄孢菌株的菌丝生长温度范围相同,均为0-35℃;但它们的最适生长温度和分生孢子致死温度存在差异,来自和县番茄菌株HX12最适生长温度为20℃,分生孢子致死温度为47℃ 10min,其余最适生长温度均为25℃,分生孢子致死温度均为48℃ 10min;不同菌株在相同温度下的生长速率有显著差异。pH对不同寄主来源灰葡萄孢菌株菌丝生长的影响存在差异,来自长丰辣椒的菌株LJ菌丝在pH2-9的范围内均能生长,以在pH3-6.5时生长较快,pH6时最快;其余4个菌株在pH2-12的范围内均能生长,以在pH3-9时生长较快,pH6左右最快。不同碳源、氮源营养对灰葡萄孢菌株菌丝生长和分生孢子产生均有显著影响,不同寄主来源的菌株间在碳源、氮源营养利用差异均极显著。在相同碳源、氮源营养条件下,不同寄主来源的菌株的线性生长、菌丝干重和分生孢子产量均有显著差异。     

The HOG1-like MAP kinase Sak1 of Botrytis cinerea is negatively regulated by the upstream histidine kinase Bos1 and is not involved in dicarboximide- and phenylpyrrole-resistance

In filamentous ascomycetes, HOG-like signal transduction cascades are involved in the resistance to hyper-osmotic conditions and to dicarboximides and phenylpyrroles. The histidine kinase (HK) Bos1 and the mitogen-activated protein kinase (MAPK) Sak1 are important for the adaptation to hyper-osmotic and oxidative stress, development, and pathogenicity in the phytopathogenic fungus Botrytis cinerea. However, bos1Δ and sak1Δ mutants created previously, also presented different phenotypes, especially the sak1Δ mutants were not resistant to high fungicide concentrations. Since both single mutants were constructed in different parental strains, phenotypic variations due to the genetic background might be suspected. In order to establish the relationship between both protein kinases, we analyzed Sak1 phosphorylation under the control of the Bos1 HK and we realized epistasis analysis between bos1Δ and sak1Δ mutations through the construction of isogenic single and double mutants. Our results show that Bos1 negatively regulates Sak1 phosphorylation and that Bos1 regulates certain phenotypes independently of Sak1. They include fungicide susceptibility, adaptation and conidiation on high neutral osmolarity.     

Comparison of Botrytis cinerea Populations Collected from Tomato Greenhouses in Northern Algeria

To estimate the genetic diversity and population structure for a better understanding of the spread of Botrytis cinerea, we genotyped with nine microsatellite markers 174 isolates collected from four greenhouses during three growing seasons in the region of Bejaia. Four of these isolates were detected as Botrytis pseudocinerea according to the allele size at locus Bc6. For all other isolates further studied, all loci were polymorphic, with the mean number of alleles per locus ranging from 2.77 to 5.22. Considerable genetic variability was detected in all subpopulations (D* > 0.87; Hnb > 0.40). Based on the standardized index of association analysis, significant but low levels of clonality occurred, not excluding the possibility of recombination (r_D = 0.07, P < 0.001). A total of 109 haplotypes were characterized among the isolates, few of which were shared between subpopulations. This, together with moderate genetic differentiation among subpopulations according to the geographical origin (0.080 < F_ST < 0.167), suggested a low level of inoculum exchange among greenhouses and little carry‐over of inoculum from one sampling season to the next. The importance of genetic structure of B. cinerea populations is discussed and should be taken into consideration for the management of grey mould.     

A class III histidine kinase acts as a novel virulence factor in Botrytis cinerea

Filamentous ascomycetes contain large numbers of histidine kinases (HK) that belong to eleven classes. Members of class III from different species were previously shown to be involved in osmoregulation and resistance to dicarboximide and phenylpyrrole fungicides. We have inactivated the gene encoding the single group III HK, BOS1, in the economically important plant pathogen Botrytis cinerea. BOS1 inactivation had pleiotropic effects on the fungus. Besides the expected osmosensitivity and resistance to fungicides, null mutants presented additional characteristics indicating that BOS1 is necessary for normal macroconidiation and full virulence. On standard culture media, null mutants very rarely formed conidiophores and those few conidiophores failed to produce conidia. This defect could be partially restored with 1 M sorbitol, suggesting that another BOS1-independent signal cascade may be involved in macroconidiation. The mutants were not found to be hypersensitive to various oxidative stresses but were more resistant to menadione. Finally, pathogenicity tests showed that bos1-null mutants were significantly reduced in the ability to infect host plants. Appressorium morphogenesis was not altered; however, in planta growth was severely reduced. To our knowledge, this is the first class III HK characterized as a pathogenicity factor in a plant-pathogenic ascomycete.     

Comparison of Botrytis cinerea populations isolated from two open-field cultivated host plants

The necrotrophic fungus Botrytis cinerea is reported to infect more than 220 host plants worldwide. In phylogenetical–taxonomical terms, the pathogen is considered a complex of two cryptic species, group I and group II. We sampled populations of B. cinerea on sympatric strawberry and raspberry cultivars in the North-East of Hungary for three years during flowering and the harvest period. Four hundred and ninety group II B. cinerea isolates were analyzed for the current study. Three different data sets were generated: (i) PCR-RFLP patterns of the ADP-ATP translocase and nitrate reductase genes, (ii) MSB1 minisatellite sequence data, and (iii) the fragment sizes of five microsatellite loci. The structures of the different populations were similar as indicated by Nei's gene diversity and haplotype diversity. The F statistics (F_st, G_st), and the gene flow indicated ongoing differentiation within sympatric populations. The population genetic parameters were influenced by polymorphisms within the three data sets as assessed using Bayesian algorithms. Data Mining analysis pointed towards the five microsatellite loci as the most defining markers to study differentiation in the 490 isolates. The results suggest the occurrence of host-specific, sympatric divergence of generalist phytoparasites in perennial hosts.     

Bioconversion of α-Damascone by Botrytis cinerea

Bioconversion of α-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-α-damascone, cis- and trans-3-hydroxy-α-damascone, γ-damascenone, 3-oxo-8, 9-dihydro-α-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-α-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-α-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.     

多种抑制剂对灰葡萄孢菌多聚半乳糖醛酸酶和漆酶活性的影响

目的:研究EDTA、CaCl2、红酵母酸和肠菌素对Botrytis cinerea多聚半乳糖醛酸酶(PG)和漆酶(LC)的抑制作用。方法:分离得到2株具有致病性、能抵抗杀菌剂以及分泌PG和LC的灰葡萄孢菌。观察四种抑制剂对B.Cinerea感染苹果的防治效果。将菌种活化后分别给予四种抑制剂(EDTA、CaCl2、红酵母酸和肠菌素)处理7d后,测定不同组别菌体干重和总蛋白含量。利用酶动力学的方法比较四种抑制剂EDTA、CaCl2、红酵母酸和肠菌素对两株灰葡萄孢菌中PG和LC活性的影响。结果:EDTA和红酵母酸能显著提高健康苹果对B.Cinerea感染的抵抗能力,而氯化钙和肠菌素则对已感染了B.Cinerea的苹果感染面积的扩大有较好的控制作用。肠菌素对两菌株LC酶活性的抑制作用最强、抑制率达70-80%,氯化钙对PG活性的抑制作用最强、抑制率达45%。结论:EDTA和红酵母酸可预防B.Cinerea的感染,肠菌素和氯化钙对B.Cinerea的感染有治疗作用。其防治作用可能与抑制B.Cinerea中PG和LC的活性有关。     

Control of Botrytis cinerea and Botrytis squamosa in overwintered salad onions by fungicide sprays

In 1974/75, 13 sprays of 0·2% a.i. thiram applied at 14 day intervals to overwintered salad onions reduced the incidence of Botrytis cinerea and significantly increased onion yields. In 1977/78 both B. cinerea and B. squamosa occurred, and 12 iprodione sprays at 0·1% a.i. applied at 14-day intervals or 6 sprays at 0·2% a.i. applied at 28-day intervals gave good control of B. cinerea and B. squamosa and significantly increased onion yields. Benomyl (0·1% a.i. at 14-day intervals, or 0·2% a.i. at 28-day intervals) failed to control either pathogen because of the development of carbendazim-insensitive strains of the fungi. Effective control of both pathogens and increased yields were obtained with an application of 0·4% a.i. thiram in October and November followed by an application of 0·2% a.i. iprodione in December and January.     

Presence of a vanadium-dependent haloperoxidase in Botrytis cinerea

The presence of a haloperoxidase in the mycelium of Botrytis cinerea, extractable with buffer, is demonstrated. A low level of extracellular enzyme activity was also detected. The haloperoxidase from the fungus is a vanadium-dependent glycoprotein, with a pH optimum of about 5.5. Native gel electrophoresis indicates that it is a high molecular mass protein. It appears to react with antibodies against haloperoxidase from Caldariomyces fumago. Enzyme activity is increased 3.5-fold and 15-fold by culture of the fungus in the presence of NaCl or vanadium, respectively. Activity is partly reduced by removal of vanadium and activity can be restored by the addition of vanadium to the enzyme. The possible function of this haloperoxidase is discussed.     

Melanin in the extracellular matrix of germlings of Botrytis cinerea

Previous work on the composition of the extracellular matrix of germlings of the plant pathogenic fungus Botrytis cinerea demonstrated the presence of carbohydrate, protein, and simple lipids; which, together, comprised 50-60% of the dry weight. Here we show that most of the remaining mass of the extracellular matrix consists of a chemically inert dark pigment with the electron paramagnetic resonance characteristics of a melanin. Scanning electron micrographs of the purified pigment, and transmission electron micrographs of thin sections made using the pigment indicate that it has a filamentous structure. We conclude that melanin is an important component of the extracellular matrix of germlings of B. cinerea. This is the first report of a melanin present in the extracellular matrix of a plant pathogenic fungus.     

Benzyl Alcohol Oxidase and Laccase Synthesis in Botrytis cinerea

Benzyl alcohol oxidase (BAO) has been assayed for the culture medium and mycelia of Botrytis cinerea, grown in the presence of aromatic alcohols with either glucose or galactose. Veratryl and coniferyl alcohols increased the BAO activities of both fraction. Activities were highest with the combination of veratryl alcohol and galactose. The implication of BAO in the degradation of lignin-related compounds with regards to the host-parasite interaction is discussed.     

Structural and Functional Studies of the Yeast Class II Hda1 Histone Deacetylase Complex

Yeast class II Hda1 histone deacetylase (HDAC) complex is an H2B- and H3-specific HDAC in Saccharomyces cerevisiae consisting of three previously identified subunits, the catalytic subunit scHda1p and two non-catalytic structural subunits scHda2p and scHda3p. We co-expressed and co-purified recombinant yeast class II HDAC complex from bacteria as a functionally active and trichostatin-A-sensitive hetero-tetrameric complex. According to an extensive analysis of domain organization and interaction of all subunits (or domains), the N-terminal domain of scHda1p associates through the C-terminal coiled-coil domains (CCDs) of the scHda2p-scHda3p sub-complex, yielding a truncated scHda1pHDAC-scHda2pCCD2-scHda3pCCD3 complex with indistinguishable deacetylase activity compared to the full-length complex in vitro. We characterized the interaction of the HDAC complex with either single-stranded or double-stranded DNA and identified the N-terminal halves of scHda2p and scHda3p as binding modules. A high-resolution structure of the scHda3p DNA-binding domain by X-ray crystallography is presented. The crystal structure shows an unanticipated structural homology with the C-terminal helicase lobes of SWI2/SNF2 chromatin-remodeling domains of the Rad54 family enzymes. DNA binding is unspecific for nucleotide sequence and structure, similar to the Rad54 enzymes in vitro. Our structural and functional analyses of the budding yeast class II Hda1 HDAC complex provide insight into DNA recognition and deacetylation of histones in nucleosomes.