Olfactory and gustatory behaviour by larvae of shape Lobesia botrana in response to shape Botrytis cinerea

In vineyards, larvae of Lobesia botrana (Denis and Schiffermüller) (Lepidoptera: Tortricidae) and Botrytis cinerea (Persoon: Fries) often occur together. This study was carried out to establish whether first instar larvae are attracted by the fungus. Olfactory and gustatory responses of larvae to the fungus were investigated on grape berry clusters (Vitis vinifera) and in two types of olfactometer. In clusters infected partly by B. cinerea, 82% of first instar larvae settled where berries had been infected. In the olfactometers, first instar larvae detected and discriminated between two tested foods. They significantly preferred synthetic media or grape berries (cv., 'Alphonse Lavallée' or 'Cabernet Sauvignon') infected with B. cinerea to non-infected controls. The olfactory response led to 62.2% to 72.6% of the first instar larvae attracted by the fungus and the association of olfactory with gustatory responses resulted in similar rates (64.1% to 81.6%). So, the larvae were most attracted by synthetic media or grape berries infected by the fungus as compared to the controls. Olfaction appears to be the main sense involved in this attraction process.     

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species

A range of isolation procedures including washing, sonication and incubation in nutrient broth were used separately and in combination to obtain potential bacterial antagonists to Botrytis cinerea and Pythium mamillatum from the testae and cotyledons of peas and dwarf French beans. Heat treatment was also used to bias this selection towards spore-forming bacteria. Ninety-two bacterial isolates were obtained, 72 of which were provisionally characterized as species of Bacillus . Four of these Bacillus isolates (B3, C1, D4 and J7) displayed distinct antagonism in vitro against Botrytis cinerea and P. mamillatum when screened using dual culture analysis. Further characterization of these antagonists using API 50CHB biochemical profiling identified isolate D4 as Bacillus polymyxa and isolates B3, C1 and J7 as strains of B. subtilis . In vitro screening techniques, using cell-free and heat-killed extracts of liquid cultures against Botrytis cinerea , demonstrated the production of antifungal compounds by these four Bacillus antagonists. With each isolate the antifungal activity was found not to be either exclusively spore-bound nor released entirely into the medium but present in both fractions. The antifungal compounds produced by these isolates were shown to be heat-stable. Their identification, production and release require further study for exploitation as biocontrol systems.     

番茄内生菌St24的鉴定及其对灰霉病的生防作用

从番茄植株根茎部分离到1株有抑菌活性的植物内生放线菌菌株St24,对其分类地位以及对灰霉病菌的防治效果进行研究.结果表明: 菌株St24为酒红链霉菌.St24发酵液经石油醚萃取得到的粗提物对多种病原菌有抑制作用,其中对灰霉病菌的抑制作用最强,抑制菌丝生长的EC₅₀为11.78 mg·L^-1.粗提物处理灰霉菌后,菌丝量减少,菌丝体皱缩、断裂、原生质外渗,菌体细胞表面有许多瘤状畸形.处理后的灰霉病菌培养液在260 nm处比对照多出现一吸收峰,说明粗提物对病原菌的细胞膜透性有影响.经盆栽试验, St24发酵液对番茄灰霉病有保护和治疗的作用,100 mg·L^-1粗提物叶面喷雾的保护作用效果最好,24 h后防效达到94.3%,120 h后为85.4%.     

Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.     

Volatiles from Botrytis cinerea‐infected and healthy berries of Vitis vinifera influence the oviposition behaviour of Epiphyas postvittana

Interactions among plants, plant‐feeding insects, and plant – pathogenic fungi are partially mediated by volatile compounds. Herbivorous insects use sensory cues to choose host plants for feeding and/or oviposition that are likely to support survival and development of progeny. It is known that some fungus‐induced alterations in plants can modify plant volatiles, which are recognized by the olfactory receptors of the insect, either as an attractant or as a deterrent. We tested for the presence of behaviour‐modifying volatiles emanating from the berries of Vitis vinifera L. (Vitaceae) infected with Botrytis cinerea Pers. (Helotiales). We tested the olfactory behaviour of adults of Epiphyas postvittana Walker (Lepidoptera: Tortricidae) to these volatiles using two‐choice and wind‐tunnel experiments. We hypothesized that olfactory cues influence E. postvittana's oviposition behaviour. We found that volatiles emanating from B. cinerea‐infected berries did not significantly attract the gravid females of E. postvittana; consequently, they laid significantly fewer eggs on infected berries. Furthermore, significantly fewer females of E. postvittana were found attracted to infected berries in the wind tunnel assay. Ethanol and 3‐methyl‐1‐butanol were abundant in B. cinerea‐infected berries. Oviposition assays made with laboratory standards of ethanol and 3‐methyl‐1‐butanol confirmed their role in regulating the olfactory behaviour of E. postvittana site selection.     

Histochemical and genetic analysis of host and non-host interactions of Arabidopsis with three Botrytis species: an important role for cell death control

Susceptibility was evaluated of host and non-host plants to three pathogenic Botrytis species: the generalist B. cinerea and the specialists B. elliptica (lily) and B. tulipae (tulip). B. tulipae was, unexpectedly, able to infect plant species other than tulip, and to a similar extent as B. cinerea. To study host and non-host interactions in more detail, the three Botrytis species were inoculated on Arabidopsis wild-types and 23 mutant genotypes. Disease development was monitored macroscopically by quantifying the lesion area and microscopically by bright-field and fluorescence microscopy following histochemical staining. B. cinerea and B. tulipae were very similar in their ability to infect the tested Arabidopsis genotypes, whereas B. elliptica caused disease only on a few Arabidopsis mutant genotypes. Arabidopsis mutants with a delayed or reduced cell death response were generally more resistant to Botrytis infection, whereas mutants in which cell death was accelerated were more susceptible. Differences in susceptibility between genotypes were generally gradual. Only the camalexin-deficient mutant pad 3 was fully susceptible to all three Botrytis species. Cellular changes were monitored during compatible and incompatible interactions. The formation of papillae, the presence of lysosome-like vesicles and the intracellular accumulation of H₂O₂ and nitric oxide were visualized in the infection zones using fluorescent probes. Based on histology and responses of Arabidopsis mutants, a model is proposed in which resistance against Botrytis , besides the production of camalexin, depends on the balance between cell death and survival.     

橄榄果渣乙醇提取物成分分析及抗氧化和抑菌性能研究

橄榄果渣中生物活性成分丰富。本文主要研究橄榄果渣提取物成分及其对灰葡萄孢的抑菌性能。首先以橄榄果渣为原料,采用加热回流法,分别以H₂O、50%乙醇、70%乙醇和90%乙醇为提取剂,得到四种提取物并采用HPLC和GC-MS对提取物进行成分分析。其次测定了提取物的总酚含量、抗氧化性和对灰葡萄孢的抑菌性能。研究表明,90%乙醇提取物的酚类含量较高,抗氧化水平优良,并且对灰葡萄孢有明显的抑制作用。该研究为橄榄果渣活性成分在抑菌性能方面的研究提供了一定的理论依据。     

菊芋叶片提取物抑菌活性与化学成分的研究

为了开发新型植物源杀菌剂和充分利用菊芋(Helianthus tuberosus L.)资源,本文尝试用石油醚、乙醚、乙酸乙酯和水等4种溶剂对菊芋叶片进行平行提取,采用生长速率法测定菊芋叶片提取物对水稻纹枯菌(Rhizoc-tonia solaniKühn)、小麦赤霉菌[Gibberella zeae(Schw.)Petch]、番茄早疫菌[Alternaria solani(Ellis et Martin)Jones et Grour]和番茄灰霉菌(Botrytis cinerea Pers.)生长量的抑制活性;并通过试管法和滤纸法对菊芋叶片内化学成分进行初步预试。抑菌实验结果表明:(1)菊芋叶片各溶剂提取物处理与对照处理相比差异显著;(2)菊芋叶片水提取物处理与各有机溶剂提取物处理差异也基本显著;(3)菊芋叶片各溶剂提取物同浓度处理对水稻纹枯菌、番茄早疫菌和番茄灰霉菌抑制效果较好;(4)菊芋叶片乙酸乙酯提取物抑菌效果最为显著,浓度为20mg/mL时对番茄早疫菌和番茄灰霉菌已达到完全抑制,对水稻纹枯菌抑制率也达到77.91%。初步化学预试结果说明,菊芋叶片中含有蛋白质、氨基酸、还原糖类、有机酸、酚类和鞣质、黄酮类、内酯类、强心甙以及油脂等化学成分。     

The nature of tobacco resistance against Botrytis cinerea depends on the infection structures of the pathogen

To protect themselves, plants have evolved an armoury of defences in response to pathogens and other stress situations. These include the production of pathogenesis-related (PR) proteins and the accumulation of antimicrobial molecules such as phytoalexins. Here we report that resistance of tobacco to Botrytis cinerea is cultivar specific. Nicotiana tabacum cv. Petit Havana but not N. tabacum cv. Xanthi or cv. samsun is resistant to B. cinerea . This resistance is correlated with the accumulation of the phytoalexin scopoletin and PR proteins. We also show that this resistance depends on the type of B. cinerea stage. Nicotiana tabacum cv. Petit Havana is more resistant to spores than to mycelium of B. cinerea . This reduced resistance of N. tabacum cv. Petit Havana to the mycelium compared with spores is correlated with the suppression of PR proteins accumulation and the capacity of the mycelium, not the spores, to metabolize scopoletin. These data present an important advance in understanding the strategies used by B. cinerea to establish its disease on tobacco plants.     

Botrytis cinerea: Activité Antifongique dans les Jeunes Grappes de Vitis vinifera, Varieté Gamay

Botrytis cinerea: fungicidal activity in young grape berries of Vitis vinifera (Gamay) Extracts of young grape berries of Vitis vinifera (Gamay) inhibit the germination of conidia of Botrytis cinerea. This inhibitory activity is present in extracts obtained from clusters at bloom stage and is maintained until a developmental stage close to the maturity. These extracts ruptured the cellular membranes.     

Antifungal activity of valinomycin, a peptide antibiotic produced by Streptomyces sp. Strain M10 antagonistic to Botrytis cinerea

A strain of Streptomyces sp. (M10) antagonistic to Botrytis cinerea was isolated from orchard soil obtained from Jeju Island, Korea. An antifungal substance (CN1) was purified from the culture extracts of the strain, and then identified as valinomycin through extensive spectroscopic analyses. Valinomycin showed potent in vitro antifungal activity against Botrytis cinerea and also in vivo control efficacy against Botrytis blight development in cucumber plants. Overall, the disease control efficacy of valinomycin was similar to that of vinclozolin, a commercial fungicide. This study provides the first report on the disease control efficacy of valinomycin against Botrytis blight.     

Botrytis fabiopsis, a new species causing chocolate spot of broad bean in central China

The current study was conducted to identify Botrytis spp. isolated from symptomatic broad bean plants grown in Hubei Province, China. Among 184 Botrytis strains, three distinct species, B. cinerea, B. fabae and a previously undescribed Botrytis sp., were identified based on morphology of colonies, sclerotia and conidia. The novel Botrytis sp. is described herein as a new species, Botrytis fabiopsis sp. nov. At 20 C B. fabiopsis grew on potato dextrose agar (PDA) at 12-13 mm d(-1), similar to B. fabae (13 mm d(-1)), but slower than B. cinerea (17-19 mm d(-1)). It formed pale gray colonies with short aerial mycelia and produced gray to black sclerotia in concentric rings on PDA. B. fabiopsis produced greater numbers of sclerotia than B. cinerea but fewer than B. fabae. Conidia produced by B. fabiopsis on broad bean leaves are hyaline to pale brown, elliptical to ovoid, wrinkled on the surface and are larger than conidia of B. fabae and B. cinerea. Phylogenetic analysis based on combined DNA sequence data of three nuclear genes (G3PDH, HSP60 and RPB2) showed that B. fabiopsis is closely related to B. galanthina, the causal agent of gray mold disease of Galanthus sp., but distantly related to B. fabae and B. cinerea. Sequence analysis of genes encoding necrosis and ethylene-inducing proteins (NEPs) indicated that B. fabiopsis is distinct from B. galanthina. Inoculation of broad bean leaves with conidia of B. fabiopsis caused typical chocolate spot symptoms with a similar disease severity to that caused by B. fabae but significantly greater than that caused by B. cinerea. This study suggests that B. fabiopsis is a new causal agent for chocolate spot of broad bean.     

Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F

Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.     

Proteomic approach to identify champagne wine proteins as modified by Botrytis cinerea infection

The presence of the fungal pathogen, Botrytis cinerea, in the vineyard causes reductions in both quality and quantity of grapes and wine. Because proteins are involved in the foam stabilization of sparkling wines, we have undertaken, for the first time, a thorough proteomic analysis of two champagne base wines prepared with either healthy or botrytized Chardonnay grapes, using two-dimensional electrophoresis (2DE) coupled with immunodetection and tandem mass spectrometry. Most of the identified proteins were from grape origin: invertase and pathogenesis-related (PR) proteins. The disappearance of numerous grape proteins was observed in the botrytized wine, suggesting that they were probably degraded or even repressed or the result of a differential expression of grape proteins upon fungal infection. On the other hand, two pectinolytic enzymes secreted by B. cinerea were found in the botrytized wine.     

Exo-β-1,3-Glucanase from Yeast Inhibits Colletotrichum lupini and Botrytis cinerea Spore Germination

Yeast exo-β-1,3-glucanase (EXG1) was evaluated as an inhibitory agent of Colletotrichum lupini and Botrytis cinerea. Extracts obtained from yeast transformed with the exg1 gene, expressing high levels of EXG1 activity, or control untransformed yeast cultures that lacked EXG1 activity, were added to different starting concentrations of C. lupini fungal spore suspensions (2.5 × 10³ to 80 × 10³ spores per flask), and mycelial dry weight was measured after 5 days. Inhibition of C. lupini mycelial growth by EXG1 compared with control extracts ranged from 41 to 20% when added to starting fungal spore concentrations of 2.5 × 10³ to 80 × 10³, respectively. EXG1 activity in the extracts from the transformed yeast remained high over the 5-day incubation period. Addition of the EXG1 extract after C. lupini spore germination resulted in lower inhibition, indicating that the EXG1 targets the β-glucan in the cell walls of the fungal spores at an early stage of germination. Furthermore, the yeast EXG1 extracts were also shown to inhibit Botrytis cinerea spore germination and growth. Thus, the use of the yeast exg1 gene for protection of crops, such as lupin and pear in transgenic strategies against C. lupini and B. cinerea , respectively, could be considered.     

Characterisation of acetaldehyde effects on Rhizopus stolonifer and Botrytis cinerea

Acetaldehyde (AA) vapour reduced the in vitro growth of the fruit-infecting fungi Botrytis cinerea and Rhizopus stolonifer, and decreased the respiration of R. stolonifer. Spore germination, germ tube elongation and sporulation were inhibited in both fungi. AA induced the leakage of electrolytes from B. cinerea and R. stolonifer membranes. The conductivity found in the fungal extracts was positively correlated with the AA concentration applied. The leakage of reducing sugars and amino acids suggest that cell membranes are irreversibly disrupted by AA as a first step in inhibition of the fungi in vitro.     

Characterization, induction by wounding and salicylic acid, and activity against Botrytis cinerea of chitinases and β‐1,3‐glucanases of ripening grape berries

In order to better understand the defense strategy of grape berries ( Vitis vinifera L. cv. Pinot noir) as they mature, the activities of the defense‐related proteins, chitinase (CHV, EC 3.2.1.14) and β‐1,3‐glucanase (laminarinase, EC 3.2.1.39) were first estimated in berries at different maturation stages. Chitinase levels rose proportionally to the berry reducing sugar content, an indicator of the berry ripening degree, up to values 10 times higher than the ones seen in resting grapevine leaves. This rise in activity was due to the accumulation of two isoforms, CHV 5 and CHV 11. One more chitinase isoform, CHV 12, appeared in senescent berries. Conversely, no glucanase activity could be detected in berries at any maturation stage. Accumulation of chitinases and (β‐1,3‐glucanases could be stimulated by wounding the berry peduncle. Adding salicylic acid to the wounded berries only potentiated the wounding effect on the berry chitinase activity. The most active chitinase isoform, CHV 5, was purified to homogeneity. It represented about 40% of the total extractable protein content of a ripe berry. Its molecular mass was estimated to be 31 kDa. The peptide sequencing of four of its tryptic fragments revealed strong homologies to several class IV chitinases. Finally, it was shown to inhibit the germination of conidia of Botrytis cinerea by 50% at a concentration of 7.5 µg ml⁻¹.     

Selective media for the specific isolation and enumeration of Botrytis cinerea conidia

AIMS: To develop selective media for the enumeration of Botrytis cinerea. METHODS AND RESULTS: Two new media, Botrytis Selective Medium (BSM) and Botrytis Spore Trap Medium (BSTM), were developed and compared with currently available media for the enumeration of B. cinerea conidia from the environment. The new Botrytis media proved advantageous over previous media because they were easier to prepare, had greater selectivity and allowed enumeration when a greater number of colony-forming units were present on individual plates. CONCLUSION: BSM and BSTM were shown to be suitable selective media for the enumeration of B. cinerea conidia from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The media developed can be used to monitor the population of the pathogen B. cinerea and will allow detailed studies within the crop environments.     

Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection

Physical injury inflicted on living tissue makes it vulnerable to invasion by pathogens. Wounding of Arabidopsis thaliana leaves, however, does not conform to this concept and leads to immunity to Botrytis cinerea , the causal agent of grey mould. In wounded leaves, hyphal growth was strongly inhibited compared to unwounded controls. Wound-induced resistance was not associated with salicylic acid-, jasmonic acid- or ethylene-dependent defence responses. The phytoalexin camalexin was found to be involved in this defence response as camalexin-deficient mutants were not protected after wounding and the B. cinerea strains used here were sensitive to this compound. Wounding alone did not lead to camalexin production but primed its accumulation after inoculation with B. cinerea , further supporting the role of camalexin in wound-induced resistance. In parallel with increased camalexin production, genes involved in the biosynthesis of camalexin were induced faster in wounded and infected plants in comparison with unwounded and infected plants. Glutathione was also found to be required for resistance, as mutants deficient in γ-glutamylcysteine synthetase showed susceptibility to B. cinerea after wounding, indicating that wild-type basal levels of glutathione are required for the wound-induced resistance. Furthermore, expression of the gene encoding glutathione- S -transferase 1 was primed by wounding in leaves inoculated with B. cinerea . In addition, the priming of MAP kinase activity was observed after inoculation of wounded leaves with B . cinerea compared to unwounded inoculated controls. Our results demonstrate how abiotic stress can induce immunity to virulent strains of B. cinerea , a process that involves camalexin and glutathione.     

The Purification and Characterization of Soluble Acid Invertase from Coleoptiles of Wheat (Triticum aestivum L. cv. Avalon)

Soluble acid invertase from wheat coleoptiles was purified toelectrophoretic homogeneity. A comparison of molecular weightby SDS-PAGE and gel filtration suggested that the enzyme wasa monomer of M_r50 000. The enzyme was a glycoprotein and, afterchemical deglycosylation, possessed a M_rof 48000. A polyclonalantiserum was raised against the deglycosylated protein. Thiscross-reacted specifically with acid invertase. A putative precursorof invertase synthesized in a cell-free translation system wasdetected by SDS-PAGE and fluorography of the immunoprecipitatedpolypeptides. The distribution of acid invertase in wheat seedlingshoots was investigated both by visualizing invertase activityafter starch gel electrophoresis and by immunoblotting. Bothtechniques identified two forms of invertase in extracts ofthe primary leaf and only one form in extracts of coleoptiles.The low pH optimum and the glycoprotein nature of wheat coleoptileinvertase are consistent with a vacuolar location. Fructoseinhibited its activity, suggesting that enzyme activity couldbe modulated by end-product inhibition. Key words: Acid invertase, purification, antiserum, glycoprotein, Triticum aestivum, wheat, coleoptiles