EMS离体诱变及抗草莓灰霉病愈伤组织的筛选

试验用甲基磺酸乙酯（EMS）处理草莓愈伤组织后通过草莓灰霉病病原毒素（Toxin）离体筛选获得抗病愈伤组织并诱导成苗。EMS诱变处理‘幸香’草莓离体叶片产生的愈伤组织,用经毒力检测的草莓灰霉菌毒素粗提液进行离体筛选,通过在愈伤组织继代培养中附加不同浓度毒素粗提液,以确定适宜的抗病选择压,获得抗灰霉病突变体材料。结果表明,将愈伤组织直接进行EMS处理的成活率高于将愈伤组织切割成小块继代培养再经EMS处理的愈伤组织;且0.2% EMS处理1h为最适剂量。在含80%毒素粗提液的培养基培养10 d为抗性筛选的适宜选择压。诱变处理后的867块愈伤组织,在经不连续二次筛选后有195块成活,成活率22.5%,其中有82块再生出了不定芽,再生率为42.1%。不定芽经生根后移栽至田间进一步进行抗性鉴定。     

胨冻样芽孢杆菌(Bacillus mucilaginosus)对烟草灰霉病菌(Botrytis cinerea)的抑制作用

本文首次报道胨冻样芽孢杆菌XDB1和D4B1对烟草灰霉病菌(Botrytis cinerea)的抑制作用。实验表明,XDB1和D4B1的发酵原液和无细胞提取物具有相同的抑菌效果,而经121℃处理30分钟后,抑菌活性丧失。D481菌株的抑菌活性高于XDB1,钾长石作为基质时的活性高于土壤矿物。但是,XDB1和D481对立枯丝核病菌(R..solani)、西瓜尖孢镰刀菌(F.oxysporum)、腐霉(Pythium sp)、小麦全蚀病菌(G.graminis)、烟炭疽病菌(C.gloeosporides)、烟草赤星病菌(A.alternata)、烟草黑胫病菌(P.nicotianae、串珠镰刀病菌(F.moniliforme)和水稻稻瘟病菌(P.grisea)无抑制作用。     

Antifungal activity and biotransformation of diisophorone by Botrytis cinerea

Diisophorone (1) was tested against two strains of the necrotrophic plant pathogen Botrytis cinerea.Fungal sensitivity varied according to the strain. B. cinera 2100 was more sensitive than B. cinereaUCA992: its mycelial growth was significantly inhibited at concentrations of 50 ppm and above. Although diisophorone (1) showed an effective control of B. cinerea, a detoxification mechanism was present. The detoxification of racemic diisophorone (1) by B. cinerea was investigated. Incubation with two strains of B. cinerea gave one and four biotransformation products (2-5), respectively. Their structures were established as the known 8beta-hydroxydiisophorone (2), 6alpha-hydroxydiisophorone (3), 6beta-hydroxydiisophorone (4) and 8beta,14beta-dihydroxydiisophorone (5) on the basis of their spectroscopic data, including two-dimensional NMR analysis [heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser enhancement spectroscopy (NOESY)] and an X-ray crystallographic study.     

灰葡萄孢霉角质酶粗提液酶学特性研究

为探究灰葡萄孢霉角质酶的酶学性质以及角质酶对植物角质层的降解作用,以灰葡萄孢霉为材料,采用角质诱导的方法得到角质酶发酵液,上清液经过分离得到角质酶粗提液,采用分光光度法探究温度、pH值、金属离子及有机溶剂对角质酶活力的影响,并通过扫描电镜探究角质酶粗提液对葡萄角质层的作用。结果表明,灰葡萄孢霉角质酶的最适反应温度为35℃,最适反应pH值为7.5,此条件下能够保持较高的热稳定性;Na⁺、Mg²⁺、Ca²⁺对酶活有促进作用,而Zn²⁺、Cu²⁺、Fe³⁺对酶活有抑制作用;甲醇、丙酮、异丙醇对酶活具有促进作用,乙醇、乙腈和三氯甲烷对酶活具有抑制作用;角质酶粗酶液对葡萄角质层具有明显的降解作用。本研究结果为果蔬采后病害的控制提供了理论依据。     

Metabolic influence of Botrytis cinerea infection in champagne base wine

Botrytis cinerea infection of grape berries leads to changes in the chemical composition of grape and the corresponding wine and, thus, affects wine quality. The metabolic effect of Botrytis infection in Champagne base wine was investigated through a (1)H NMR-based metabolomic approach. Isoleucine, leucine, threonine, valine, arginine, proline, glutamine, γ-aminobutyric acid (GABA), succinate, malate, citrate, tartarate, fructose, glucose, oligosaccharides, amino acid derivatives, 2,3-butanediol, acetate, glycerol, tyrosine, 2-phenylethanol, trigonelline, and phenylpropanoids in a grape must and wine were identified by (1)H NMR spectroscopy and contributed to metabolic differentiations between healthy and botrytized wines by using multivariate statistical analysis such as principal component analysis (PCA). Lowered levels of glycerol, 2,3-butanediol, succinate, tyrosine, valine derivative, and phenylpropanoids but higher levels of oligosaccharides in the botrytized wines were main discriminant metabolites, demonstrating that Botrytis infection of grape caused the fermentative retardation during alcoholic fermentation because the main metabolites responsible for the differentiation are fermentative products. Moreover, higher levels of several oligosaccharides in the botrytized wines also indicated the less fermentative behavior of yeast in the botrytized wines. This study highlights a metabolomic approach for better understanding of the comprehensive metabolic influences of Botrytis infection of grape berries in Champagne wines.     

Quantitative structure-antifungal activity relationships of some benzohydrazides against Botrytis cinerea

Fourteen benzohydrazides have been synthesized and evaluated for their in vitro antifungal activity against the phytopathogenic fungus Botrytis cinerea. The best antifungal activity was observed for the N',N'-dibenzylbenzohydrazides 3b-d and for the N-aminoisoindoline-derived benzohydrazide 5. A quantitative structure-activity relationship (QSAR) study has been developed using a topological substructural molecular design (TOPS-MODE) approach to interpret the antifungal activity of these synthetic compounds. The model described 98.3% of the experimental variance, with a standard deviation of 4.02. The influence of an ortho substituent on the conformation of the benzohydrazides was investigated by X-ray crystallography and supported by QSAR study. Several aspects of the structure-activity relationships are discussed in terms of the contribution of different bonds to the antifungal activity, thereby making the relationships between structure and biological activity more transparent.     

Benzothiadiazole enhances resveratrol and anthocyanin biosynthesis in grapevine, meanwhile improving resistance to Botrytis cinerea

Pre-harvest multiple treatments of grapevine (cv. Merlot) with the plant activator benzothiadiazole (BTH, 0.3 mM) enhanced trans-resveratrol content in berries by about 40%. An even more striking effect was observed on anthocyanin synthesis, particularly on malvidine 3-glucoside, malvidine 3-(6-O-acetyl)glucoside and malvidine 3-(6-O-p-coumaroyl)glucoside, whose amounts were more than doubled. These data were obtained with a novel and time-saving HPLC method, set up for the simultaneous detection of stilbenes and anthocyanins, using an RF-10Axl fluorimetric detector instrument, with excitation at 330 nm and emission at 374 nm, and a SPD-Avp UV detector with absorption at 520 nm. Furthermore, BTH treatments induced systemic acquired resistance in grapevine, as assessed by inoculating clusters from treated and untreated plants with Botrytis cinerea. Disease severity, estimated according to the percentage of infected berries per cluster, was significantly reduced in grapes from BTH-treated plants. These results indicate that BTH treatments, besides improving the content of two important classes of nutraceuticals, with their well-known antioxidant, antitumoral, and phytoestrogenic activities, could be exploited in vineyard to protect grape against gray mould infection, thereby limiting an excessive use of fungicides     

Characterization of metabolites of fungicidal cymoxanil in a sensitive strain of Botrytis cinerea

The metabolism of cymoxanil [1-(2-cyano-2-methoxyiminoacetyl)-3-ethyl urea] by a very sensitive strain of Botrytis cinerea toward this fungicide was studied by using [2-(14)C]-cymoxanil. Labeled cymoxanil was added either in a culture of this strain or in its enzymatic extract. The main metabolites, detected in biological samples, were isolated and identified by mass and NMR spectrometry. Their identification allowed us to show that this strain quickly metabolized cymoxanil according to at least three enzymatic pathways: (i) cyclization leading, after hydrolysis, to ethyl parabanic acid, (ii) reduction giving demethoxylated cymoxanil, and (iii) hydrolysis and reduction followed by acetylation leading to N-acetylcyanoglycine. In a cell-free extract of the same strain, only the first and the second enzymatic reactions, quoted above, occurred. Biological tests showed that, among all the metabolites, only N-acetylcyanoglycine is fungitoxic toward this sensitive strain.     

伞形花内酯处理对采后徐香猕猴桃果实灰霉病抗的影响

为探讨伞形花内酯处理对猕猴桃果实采后灰霉病的控制效应,本试验研究了伞形花内酯对灰霉菌孢子和生长的影响,以及对美味猕猴桃徐香损伤接种灰霉菌后果实病害发展、抗病相关酶活性和抗性物质的影响.结果表明,离体条件下,0.5和1.0 mg·mL-1伞形花内酯能够显著抑制灰霉菌孢子的萌发和生长;0.5和1.0 mg·mL-1伞形花内...     

Evidence for protein degradation by Botrytis cinerea and relationships with alteration of synthetic wine foaming properties

Botrytis cinerea is an important fungal pathogen particularly dreaded in the cool climate vineyard. It is responsible for important damage, especially the decrease in foamability of sparkling wines, such as Champagne. Different studies have shown that proteins are largely involved in the stabilization of Champagne foam despite their low concentration. Other works demonstrated changes in the electrophoretic characteristics of must proteins originating from botrytized grapes, although the cause of such alterations was never explained. In the first part of this study, results showed the release by B. cinerea of 3.5 mg/L total proteins in a synthetic liquid medium. Among these proteins, the presence of a protease activity on bovine serum albumin (BSA) and must proteins was demonstrated by using a colorimetric method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the model wine, the Bradford method showed a BSA loss of 66% after 24 h and a loss of 96% after 120 h. In the same model wine, the soluble must protein concentration decreased by 35% after 1 week and by 53% after 2 weeks while the control showed no protein loss. B. cinerea proteases were then able to degrade BSA and must proteins and were above all active at must and wine pH and in the presence of ethanol and SO(2). The second part of this work was dedicated to the relationship between the presence of B. cinerea proteases and its effects on the synthetic wine foaming properties. The addition of a B. cinerea culture medium (1/33 v/v) to the synthetic wine containing 21 mg/L soluble grape proteins induced a decrease in foamability by 60% after 1 week. For BSA in the model wine, the foamability decreased by 32% after 24 h and by 95% after 120 h, as shown by the colorimetric method. These experiments demonstrate for the first time the relationship between B. cinerea protease activity and the decrease in wine foaming properties.     

3-Sulfanylhexanol precursor biogenesis in grapevine cells: the stimulating effect of Botrytis cinerea

Volatile thiols, compounds that contribute strongly to the varietal aroma, are present in much higher concentrations in sweet wines than in dry wines. This positive effect, due to the presence of Botrytis cinerea on the berries, in fact results from a strong enrichment of cysteine S-conjugate precursors in botrytized berries. In the present study, a convenient model was investigated to reproduce and therefore study this phenomenon. A Vitis vinifera cell culture was used as a simple model, and we focused on S-3-(hexan-1-ol)-l-cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexanol. We demonstrated that grapevine cells were able to produce P-3SH and that the presence of B. cinerea considerably increased the precursor level (up to 1000-fold). This positive result was determined to be due to metabolites secreted by the fungus. These molecules were temperature sensitive, unstable over time, and their production was activated in the presence of grapevine cells. Moreover, part of the pathway leading to P-3SH was deciphered: it was directly derived from the cleavage of S-3-(hexan-1-ol)-l-glutathione, which itself was generated after a conjugation of glutathione on (E)-2-hexenal.     

Interaction with and effects on the profile of proteins of Botrytis cinerea by C6 aldehydes

The natural volatile compounds cis-3-hexenal (c-3-H) and trans-2-hexenal (t-2-H) have significant antifungal activity with potential for use as postharvest fumigants of fruits and vegetables. However, the nature of their interaction with fungi and impact on fungal growth at the molecular level are largely unknown. The sites of interaction of these six carbon (C6) aldehydes with Botrytis cinerea, a common pathogen of many plant species, was characterized using 3H-labeled c-3-H and t-2-H. Radiolabeled C6 aldehydes were produced with lipoxygenase and hydroperoxide lyase extracts using [9,10,12,13,15,16-3H6]linolenic acid as a substrate. Following exposure of B. cinerea cultures to radiolabeled C6 aldehydes, radiolabel was recovered in protein-enriched but not lipid-enriched fractions. Radiolabel was incorporated at higher levels (6-fold per milligram of fresh weight and 4-fold per microgram of protein) into conidia than mycelia. About 95% of the radiolabeled aldehyde recovered in the protein fraction was from the surface of the fungal tissue, while 5% was from protein in internal tissue (cell wall, membrane, and cytosol). Exposure to t-2-H at both 5.4 and 85.6 micromol affected the protein profile of B. cinerea, changing the intensity of over one-third of all proteins. Both up-regulation and down-regulation of specific proteins were observed by two-dimensional gel electrophoresis, indicating a clear effect of t-2-H on changes in the protein profile of B. cinerea. This is the first evidence that fungal proteins are targets of the volatile C6 aldehydes and that sublethal levels of the aldehydes cause changes in the protein profile of a fungus.     

In vitro inhibition studies of phytoene desaturase by bleaching ketomorpholine derivatives

The herbicidal activities of homochiral steroisomeric 5-methy1-2-(3-trifluoromethybenzyl)-3-keto- morpholine derivatives were investigated in vitro as inhibitors of phytoene desaturase, a key enzyme in carotenoid biosynthesis. It was demonstrated that ketomorpholines are classical bleaching compounds which directly inhibit phytoene desaturase, accumulating phytoene at the expense of colored carotenoids. Ketomorpholines interact with phytoene desaturase in a noncompetitive manner with respect to phytoene. A structure-activity investigation for in vitro inhibition of phtoene desaturase activity revealed that the relative and absolute stereochemistry is important for optimum inhibition for the 5-methyl derivatives, and that the distance of the phenyl group from the ketomorpholine ring is critical for the inhibitory potential. The average herbicidal score on 7 weeds and the in vitro I(50) values related very well with the exception of two compounds. It was postulated that the discrepancies may possibly occur through modification in plants to compounds that are either more or less active herbicides.     

Characterization of the antifungal activity on Botrytis cinerea of the natural diterpenoids kaurenoic acid and 3beta-hydroxy-kaurenoic acid

The antifungal activity on Botrytis cinerea of the diterpenoids 3beta-hydroxy-kaurenoic acid and kaurenoic acid, obtained from the resinous exudates of Pseudognaphalium vira vira, was determined. 3beta-Hydroxy-kaurenoic acid reduced the mycelial growth of B. cinerea in solid and liquid media. Additionally, the damage produced by the fungus on the surface of tomato leaves in the presence of the diterpenoids was evaluated. A higher protective effect was observed in the presence of the hydroxylated diterpene. On the other hand, the effect of the diterpenoids on the production of enzymes that participate in the plant infection by B. cinerea was analyzed. p-Nitrophenylbutyrate esterase production was induced by both diterpenoids, whereas laccase production was only induced by the hydroxylated diterpene. In the study of the mechanism of action of these compounds, it was determined that 3beta-hydroxy-kaurenoic acid would produce permeabilization of the cell membrane of B. cinerea.     

Effect of water deficit irrigation and inoculation with Botrytis cinerea on strawberry (Fragaria x ananassa) fruit quality

Deficit irrigation (DI) detrimentally affected berry size but had a profound effect on fruit physiology and biochemistry. Strawberry cv. Elsanta fruit from DI-treated plants had higher levels of abscisic acid (ABA). Dry matter content as a proportion of fresh weight was increased by a quarter in fruit from water-stressed plants as compared to fruit harvested from plants held at or near field capacity. Concomitant to this, the concentration of some taste-related (viz. monosaccharides and sugar/acid ratios) and health-related compounds/parameters (viz. antioxidant capacity and total phenolics) were generally much greater in DI-treated fruit. The effect of inoculation with Botrytis cinerea on fruit quality was also tested. Fruit derived from inoculated plants displayed symptoms of gray mold postharvest disease earlier than noninoculated fruit and had double the concentration of ABA. Inoculation had no significant effects on all other target analytes measured. There was no interaction between water treatment and inoculation. The possible mechanisms for increased synthesis of ABA and the different effects of pathogen-induced stress versus drought stress on fruit quality are discussed.     

In vitro antioxidative effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl derivatives in green coffee beans

Seven kinds of hydroxycinnamic acid derivatives identified as 3-caffeoylquinic acid (3-CQA), 4-caffeolyquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA), 5-feruloylquinic acid (5-FQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), and 4,5-dicaffoylquinic acid (4,5-diCQA) by MS, 1H NMR, and HPLC analyses were isolated from low-quality (immature) and commercial quality green coffee beans. The quantity of chlorogenic acid isomers (10.4 g/100 g), especially 5-CQA, in commercial green coffee beans [West Indische Bereiding (West India processing beans from Sumatra Island, Indonesia, WIB)] was higher than that in low-quality beans [9.1 g/100 g, Eerste Kwaliteit (Export low-quality beans from Java Island, Indonesia, EK-1, grade 4)], whereas little difference in diCQAs was detected between the two kinds of beans. The free radical scavenging activity of these isolates was evaluated in assay systems using DPPH free radicals and superoxide anion radicals generated by xanthine-XOD. The diCQAs showed strong (1.0-1.8-fold) free radical scavenging activity compared to commonly used antioxidants such as alpha-tocopherol and ascorbic acid. The potency order of superoxide anion radical scavenging activity was diCQAs > caffeic acid, CQAs > 5-FQA. The activities of the diCQAs were twice as effective as those of CQAs and 4 times as effective as that of 5-FQA. The diCQAs also exhibited more potent (2.0-2.2-fold) tyrosinase inhibitory activities compared to CQAs, arbutin, and ascorbic acid. The isolates exhibited antiproliferation activities in four cancer cell lines, U937, KB, MCF7, and WI38-VA. Among these, KB cells were most sensitive (IC50 = 0.10-0.56 mM).     

Determination of fungicide residues in field-grown strawberries following different fungicide strategies against gray mold (Botrytis cinerea)

In a 2 year experiment, residues in field-grown strawberries were investigated from the fungicides fenhexamid, pyrimethanil, tolylfluanid, and kresoxim-methyl resulting from different strategies, as regards the dose, number, and time of fungicide applications. Kresoxim-methyl was only used the first year and in full or no dose to control powdery mildew. In the first year, the highest concentrations analyzed were 0.66 mg kg(-1) for pyrimethanil and 0.63 mg kg(-1) for fenhexamid resulting from the use of recommended dose rates and a preharvest interval (PHI) of 10 days, thus not exceeding the Danish maximum residue limit (MRL) of 1 mg kg(-1). Tolylfluanid was used no later than 21 days before harvest, which left residue contents in the berries of 0.48 mg kg(-1), a value well below the MRL of 5 mg kg(-1). In the second year, fungicide residues found in the samples were generally lower, 0.39 and 0.03 mg kg(-1) for pyrimethanil and fenhexamid, respectively. No residues of kresoxim-methyl were found in any of the samples from the field trials, indicating that kresoxim-methyl residues had declined to a level well below the detection limit within the 28 day period between the last application and the harvest.     

Differential regulation of calmodulin,phenylalanine ammonia-lyase,and salicylic acid in response to Botrytis cinerea infection in tomato with different Ca2+ concentrations

To investigate the responses to Botrytis cinerea (B. cinerea) infection and the effects of exogenous Ca²⁺ on calmodulin (CaM), phenylalanine ammonia-lyase (PAL), and salicylic acid (SA) biosynthesis and concentrations, we investigated the role of exogenous calcium in defense and resistance to the necrotrophic fungus B. cinerea in tomato (Lycopersicon esculentum Mill.). Specifically, 7.5 mM Ca²⁺ was the best treatment for plant resistance, resulting in stable and permanent resistance to the pathogen. This treatment improved plant resistance and the results also confirmed that the concentrations of CaM and SA are negatively correlated with the resistance response.     

Screening study of lead compounds for natural product-based fungicides: antifungal activity and biotransformation of 6alpha,7alpha-dihydroxy-beta-himachalene by Botrytis cinerea

Eleven beta-himachalene derivatives were tested, using the poisoning food technique, for their potential antifungal activity against the phytopathogen Botrytis cinerea. Compounds 1-11 displayed moderate activity, whereas the 6,7-diol derivative (12) produced an inhibition of 91% after 6 days. The microbial transformation of 12 was investigated and yielded four new compounds hydroxylated at positions C-5 (13), C-2 (14), C-4 (15), and C-12 (16). The structures were established on the basis of their spectroscopic data including two-dimensional NMR analysis (HMQC, HMBC, nOesy) and nOes. The results obtained from biotransformation experiments shed further light on the detoxification mechanism of the phytopathogenic fungus against this compound and give an indication of the structural modifications that may be necessary if substrates of this type are to be further developed as selective fungal control agents for B. cinerea.     

In vitro binding of germanium to proteins of rice shoots

The possibility of in vitro binding between proteins of rice shoots and germanium (Ge) was investigated. The proteins in mixtures of aqueous extracts of rice shoots and radioactive germanium (⁶⁸GeO₂) were fractionated. The binding of radioactivity to the proteins was observed even after 5 successive fractionation steps from the original mixtures. At the final fractionation step using polyacrylamide gel electrophoresis, a constant proportionality between protein concentration and associated radioactivity was found in most samples although not all. These results indicate that the binding of ⁸⁸Ge to proteins is not due to the simple adsorption by proteins.