Oxidation of Alcohols by Botrytis cinerea

Crude cell-free preparations of Botrytis cinerea were found to oxidize straight-chain primary alcohols (except methanol), aromatic primary alcohols, and unsaturated primary alcohols. The resulting products were the corresponding aldehydes and an equal molar quantity of hydrogen peroxide.     

Bioconversion of alpha-Damascone by Botrytis cinerea

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

Bioconversion of citronellol by Botrytis cinerea

Summary Bioconversion of citronellol 1 was studied with four strains of Botrytis cinerea. Using grape must predominant transformation of 1 to 2,6-dimethyl-1,8-octandiol 2 and (E)-2,6-dimethyl-2-octen-1,8-diol 3 was observed. In minor amounts 2,6-dimethyl-2,8-octandiol 4, two p-menthan-3,8-diol isomers 5a, 5b, (Z)-2,6-dimethyl-2-octen-1,8-diol 6, isopulegol 7, 2-methyl-2-hepten-6-one-1-ol 8 and 2-methyl--butyrolactone 9 were found. Using a small amount of grape must in a synthetic medium (1:700) the bioconversion products 2, 4, 5a and 5b were absent, but additionally 2-methyl-2-hepten-6-one 10, 2-methyl-2-hepten-6-ol 11 and citronellic acid 12 were detected. The results obtained were strongly dependent on the strains used; one strain did not show any metabolic activity against 1. The bioconversion products were identified by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e. on-line — mass spectrometry (HRGC-MS) and — Fourier transform infrared spectroscopy (HRGC-FTIR).     

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.     

Changes in pH and the Production of Organic Acids During Colonization of Tomato Petioles by Botrytis cinerea

After inoculation of petiole stumps of tomato plants with a tomato isolate of B. cinerea, a transition zone between water-soaked and apparently healthy tissue became clearly visible. Hyphal colonization occurred up to approximately 2 mm beyond this zone. In the colonized tissue the pH values were lower than in the healthy tissue. In the region with most of the tips of the advancing hyphae, however, pH values were slightly, but consistently, higher. In the colonized tissue concentrations of oxalic, citric and succinic acid were higher than in the tissue of healthy, non-inoculated petioles. In vitro this isolate of B. cinerea produced citric, malic and succinic acid. Oxalic acid, however, could not be detected. In media enriched, with citric or malic acid, mycelial production was higher than in media without this enrichment.     

Reduced Development of Grey Mould (Botrytis cinerea) in Bean and Tomato Plants by Calcium Nutrition

Fertilization of bean plants grown in perlite with 1 and 3 mM CaCl₂ or Ca(NO₃)₂ reduced severity of grey mould as compared with control plants or plants fertilized with 5 mM of the compounds. Fertilization with Ca(NO₃)₂ reduced severity leaf grey mould and fruit ghost spots of tomato plants grown in perlite by 70 and 45%, respectively. The rate of decrease varied with the position of the fruits on the plants. Leaves from plants treated with calcium or otherwise [KNO₃, (NH₄)₂SO₄] produced less ethylene than leaves of nontreated plants. Rate of growth of B. cinerea was lower on growth medium prepared from washings from leaves of calcium fertilized plants than from leaves from other treatments. The fertilizer combination Ca(H₂PO₄)₂+ CaSO₄ (1 and 3 g/kg soil) applied once to tomato plants grown in soil reduced severity of leaf grey mould by 80 % (significant at P = 0.05) but 1–3 g CaSO₄/kg soil only tended to reduce disease severity (30–40 %, not significant) as compared with the control. The compounds CaCl₂ and Ca(NO₃)₂ increased significantly ( P = 0.05) the growth of B. cinerea on synthetic medium when applied at rates of 1 0–10.0 mM whereas reduction of growth was observed with 0.1 mM of the compounds and of CaSO₄.     

Microbial Production of Abscisic Acid by Botrytis cinerea

The effect of oryzalexin D, which has been isolated as a group of novel phytoalexins of rice plant, on DNA, RNA, protein, lipid and chitin biosyntheses, respiration and cell membrane permeability was investigated in Pyricularia oryzae. The concentration for 50% inhibition (ED₅₀) by oryzalexin D of the mycelial growth of P. oryzae was 230 ppm. At this concentration, oryzalexin D inhibited equally the incorporation of [2–¹⁴C]thymidine, [2–¹⁴C]uridine, l-[U-¹⁴C]amino acid mixture, l-[methyl-¹⁴C]methionine and d-[l-¹⁴C]glucosamine into DNA, RNA, protein, lipid and chitin in intact cells, but did not inhibit these systems in a homogenate of the mycelia of P. oryzae. Oryzalexin D scarcely inhibited the respiration of the homogenate and mitochondria at ED₅₀. On the other hand, oryzalexin D at ED₅₀ caused leakage of potassium and inhibited the uptake of glutamate by mycelial cells of P. oryzae. These results suggest that interference with the cell membrane function is responsible for the primary mode of action.of oryzalexin D against P. oryzae.     

Detection of Botrytis cinerea by loop-mediated isothermal amplification

Aims: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. Methods and Results: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross‐reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real‐time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. Conclusions: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. Significance and Impact of the Study: The LAMP method combines the sensitivity and specificity of nucleic acid‐based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on‐site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.     

灰葡萄孢菌（Botrytis cinerea）基因组中T-DNA整合模式分析

摘要：【目的】研究灰葡萄孢菌（Botrytis cinerea）基因组中T-DNA插入位点的整合模式特征。【方法】利用农杆菌（Agrobactirium tumfacience）介导法构建灰葡萄孢菌T-DNA插入突变体库。利用热不对称交错PCR（TAIL-PCR）技术对转化子中T-DNA的旁侧序列进行扩增和克隆,对获得的旁侧序列进行比对分析。【结果】T-DNA插入在灰葡萄孢菌基因组非编码区的占69%,插入在外显子的占30%。T-DNA在插入的过程中发生了碱基缺失、增加等重组现象,其中左边界（left border,LB）整合到基因组碱基缺失较少,有的保持完整,而右边界（right border,RB）及其近邻的T-DNA区域缺失碱基较多。T-DNA的插入位点还发现有额外的序列插入。【结论】对灰葡萄孢菌中插入T-DNA的整合模式的分析为开展该菌的功能基因组学奠定了基础。     

The Botrytis cinerea early secretome

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2‐D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC‐MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.     

Biotransformation of (-)-a-pinene by Botrytis cinerea

(-)-alpha-Pinene (1), a major constituent of many aromatic plants was biotransformed by the plant pathogenic fungus, Botrytis cinerea to afford three new metabolites, characterized as 3beta-hydroxy-(-)-beta-pinene (10%) (3), 9-hydroxy-(-)-a-pinene (12%) (4), 4beta-hydroxy-(-)-alpha-pinene-6-one (16%) (5) by physical and spectroscopic methods. A known metabolite verbenone (2) was also obtained.     

Microbial transformation of geraniol and nerol by Botrytis cinerea

Summary Biotransformation of geraniol 1A and nerol 1B was studied with four strains of Botrytis cinerea and three growth media. Using grape must predominant conversion of 1A/1B to E-3,7-dimethyl-2-octen-1,8-diol 5 and 2Z,6E-3,7-dimethyl-2,6-octadien-1,8-diol 16B was observed. However, with one strain and 1A, E-2-methyl-2-hepten-6-one-1-ol 2B, 7-hydroxy-6-methyl-2-heptanone 3 and p-menth-1-ene-9-ol 7 were identified as major metabolites. As further fungal bioconversion products of 1A/1B were detected: Z-2-methyl-2-hepten-6-one-1-ol 2A, 2E,6Z-, 2E,6E-and 2Z,6Z-3,7-dimethyl-2,6-octadien-1,8-diol 4A/4B/16A, Z-3,7-dimethyl-2-octen-1,8-diol 17, 3,7-dimethyl-1,8-octandiol 6, 2E,6E-8-hydroxy-2,6-dimethyl-2,6-octadienal 8, geranial and neral 9, 18, citronellol 10, Z- and E-2,6-dimethyl-2,7-octadien-1,6-diol 13A/13B, 6-hydroxy-2,6-dimethyl-2,7-octadienal 14 as well as 2,6-dimethyl-7-octen-1,6-diol 15. Using synthetic growth medium again -hydroxylation reactions were observed, but 2-methyl-2-hepten-6-one 11 and 7 were also identified as major bioconversion products of 1A and 1B, respectively. Additionally, 2-methyl-2-hepten-6-ol 12 was detected and, using 1B, also traces of 2Z,6E-8-hydroxy-2,6-dimethyl-2,6-octadienal 19 and two 3,9-epoxy-p-menth-1-ene isomers 20A/20B were found. Addition of small amounts of grape must to the synthetic medium (1:700 to 5:700) influenced both the yields of metabolites and their qualitative and quantitative distribution. Identifications of biotransformation products of 1A/1B were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.2. on-line-mass spectrometry (HRGC-MS) and-Fourier transform infrared spectroscopy (HRGC-FTIR) after extractive sample preparation.     

Oxidative metabolism of ambrox and sclareolide by Botrytis cinerea

Ambrox (1), a perfumery diterpene, was oxidatively metabolised by a plant pathogenic fungus Botrytis cinerea in a xenobiotic fashion to afford a major product, i.e., 1beta-hydroxy-8-epiambrox (13) (60%) along with three minor metabolites 3beta-hydroxyambrox (2), sclareolide (5) and 3beta-hydroxysclareolide (7). Sclareolide (5), a cytotoxic diterpenoidal lactone was fermented with the same fungus to yield 3beta-hydroxysclareolide (7) (59%) as a major metabolite together with two minor metabolites characterised as 1-ketosclareolide (15), and 3beta,14-dihydroxysclareolide (16).     

Transformations of testosterone and related steroids by Botrytis cinerea

Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7 alpha-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14 alpha-position.     

Detoxification of terpinolene by plant pathogenic fungus Botrytis cinerea

Detoxification of an antifungal monoterpene terpinolene (1) by the plant pathogenic fungus Botrytis cinerea afforded hydroxlyated metabolites 2,3-dihydro-3beta,6beta-dihydroxy-terpinolene (2) (39%) and 2,3-dihydro-1alpha,3alpha-dihydroxy-terpinolene (3) (20%), respectively. Terpinolene showed good levels of antifungal activity while both the metabolites were inactive against another plant pathogenic fungus Cladosporium herbarun.