Virus-induced changes in the response of faba bean to infection by Botrytis

Prior infection of faba bean with the viruses bean yellow mosaic and bean leaf roll increased host susceptibility to subsequent infection by Botrytis fabae and B. cinerea. Cell necrosis beneath inoculum droplets, rate and extent of lesion spread and sporulation of B. fabae were all increased on detached leaves from virus-infected compared with healthy plants. Changes were most marked in young leaves showing conspicuous symptoms of systemic virus infection and in plants virus-infected for at least 2-4 weeks. Localized lesions produced by B. cinerea or a low concentration of B. fabae conidia (10³ spores/ ml) showed increased cell necrosis but were not transformed into aggressive, spreading lesions on virus-infected leaves.     

Fungal development and plant response in detached onion, onion bulb scales and leaves inoculated with Botrytis allii, B. cinerea, B. fabae and B. squamosa

Fungal development and plant responses were examined in detached leaves and mid-bulb scales of Allum cepa. Following inoculation with suspensions of 105 conidia/ml distilled water Botrytis squamosa consistently produced spreading lesions in leaves and bulb scales. B. allii produced spreading lesions at most sites in bulbs but was very inconsistent in its infection of leaves; lesions were often confined to inoculation sites. Limited lesions were usually produced by B. cinerea but R. fabae failed to produce symptoms at most sites. Extensive colonization by B. allii and B. tauamosa required rapid penetration and totally necrotrophic fungal growth. During development of a spreading lesion, plant cell walls became very swollen around intramural hyphae and wall swelling appeared to precede epidermal cell death. Resistance to colonization was due to poor germination, failure to produce distinct infection hyphae (associated with accumulation of deposits of granular reaction material [RM] in underlying live cells) or restriction of infection ryphae amongst small groups of dead cells (limited lesion formation). Only B. fabae germinated poorly, and germ-tubes produced often failed to attempt penetration but grew over the leaf or bulb scale surface. Reducing numbers of conidia increased the frequency of sites associated with RM accumulation; granular deposits being particularly common at sites inoculated with low numbers of B. allii conidia. Electron microscopy revealed that RM granules were osmiophilic aggregates formed between the plasma membrane and epidermal cell wall. In the absence of RM, growth of avirulent species was restricted within the swollen walls of dead epidermal cells. Results ae compared with those from studies on tulip and broad bean leaves.     

Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea

Three hybridoma cell lines secreting antibodies that specifically recognize Botrytis cinerea and B.fabae , but not B. allii, have been raised from splenocytes of mice immunized with a low molecular-weight fraction (30 kDa) from surface washings of B. cinerea. Antibodies from these cell lines have been used to develop an antigen-based elisa test that will detect B. cinerea in strawberries. This monoclonal antibody immunoassay detection assay should prove useful to both the cut-flower and wine industries. Supernatants from the three specific cell lines recognize mycelial fragments, saline extracts of mycelia and germinating conidia by both ELISA and immunofluorescence. Recognition of non-germinating spores is poor. Supernatants from the specific cell lines did not recognize other fungi normally involved in post-harvest spoilage of fruits and vegetables. Supernatants from KH4 gave the lowest background values with healthy tissue. Indirect evidence from heat, protease and periodate treatment of the antigens indicates that antibodies from all three specific cell lines are recognizing carbohydrate epitopes on a glycoprotein.     

两种蚕豆赤斑病菌对常用杀菌剂的抗药性检测及比较

对采自浙江、湖北和安徽3省的蚕豆赤斑病样品进行了病原菌的分离和鉴定,采用菌丝生长速率法检测了引起赤斑病的2种病原菌——蚕豆葡萄孢Botrytis fabae和灰葡萄孢B.cinerea 的抗药性发生情况,并在离体条件下通过抗药性诱导试验比较了二者的抗药性风险。结果共分离得到153个菌株,其中蚕豆葡萄孢122株(占79.7%),灰葡萄孢31株(占20.3%)。共检测到37株多菌灵高水平抗药性菌株(其中蚕豆葡萄孢9株)和42株异菌脲低水平抗药性菌株(其中蚕豆葡萄孢17株);嘧霉胺对153个菌株的EC50值在0.01~5.13 μg/mL之间,平均EC50值为0.72±0.15 μg /mL;表明蚕豆赤斑病菌对常见杀菌剂已表现出一定的抗药性,且灰葡萄孢的抗药性问题比蚕豆葡萄孢要严重得多。抗药性诱导试验进一步证实,灰葡萄孢的抗药性风险明显高于蚕豆葡萄孢。     

Biological Control of Botrytis cinerea in Cyclamen with Ulocladium atrum and Gliocladium roseum Under Commercial Growing Conditions

ABSTRACT The effect of treatments with conidial suspensions of Ulocladium atrum and Gliocladium roseum on leaf rot of cyclamen caused by Botrytis cinerea was investigated under commercial greenhouse conditions. Spraying U. atrum (1 x 10(6) conidia per ml) or G. roseum (2 x 10(6) conidia per ml and 1 x 10(7) conidia per ml) at intervals of 2 to 3 weeks during the production period and spraying U. atrum (1 x 10(6) conidia per ml) at intervals of 4 to 6 weeks resulted in a significant reduction of natural infections of petioles by B. cinerea. U. atrum or G. roseum (1 x 10(7)conidia per ml) was as effective as the standard fungicide program. B. cinerea colonized senesced leaves within the plant canopy and infected adjacent petioles and leaves later. The antagonists colonized senesced leaves and reduced B. cinerea development on these leaves. Thus, the inoculum potential on petioles adjacent to necrotic leaf tissues was reduced. The fate of U. atrum conidia on surfaces of green cyclamen leaves during a 70-day period after application was studied. The number of conidia per square centimeter of leaf surface remained relatively constant during the entire experiment. Sixty percent of the conidia sampled during the experiments retained the ability to germinate. When green leaves were removed from the plants to induce senescence and subsequently were incubated in a moist chamber, U. atrum colonized the dead leaves. Senesced leaves also were colonized by other naturally occurring fungi including B. cinerea. On leaves treated with U. atrum from all sampling dates, sporulation of B. cinerea was significantly less as compared with the untreated control. Our results indicate that early applications of U. atrum before canopy closure may be sufficient to achieve commercially satisfactory control of Botrytis leaf rot in cyclamen.     

Effect of Germination Initiation on Competitive Capacity of Trichoderma atroviride P1 Conidia

ABSTRACT Trichoderma biocontrol isolates are most effective as highly concentrated inocula. Their antagonism to other fungi may be a result of pregermination respiration. In a nutrient-rich medium, almost all Trichoderma atroviride P1 (P1) conidia initiated germination processes and increased respiration, even in dense suspensions. When 1 x 10(7) P1 conidia/ml were coinoculated with 1 x 10(5) Botrytis cinerea conidia/ml, dissolved oxygen fell to <1% within 2 h and the pathogen failed to germinate. More dilute P1 suspensions consumed oxygen slowly enough to allow coinoculated B. cinerea to germinate. On nutrient-poor media, fewer P1 conidia initiated germination. Oxygen consumption by the inoculum and inhibition of B. cinerea were enhanced when P1 conidia were nutrient activated before inoculation. Pregermination respiration also affected competitive capacity of the antagonist on solid substrates, where respiratory CO(2) stimulated germination rate and initial colony growth. These parameters were directly correlated with inoculum concentration (R(2) >/= 0.97, P < 0.01). After initiating germination, Trichoderma conidia became more sensitive to desiccation and were killed by drying after only 2 h of incubation on a nutrient-rich substrate at 23 degrees C. These results indicate that nutrient-induced changes preceding germination in Trichoderma conidia can either enhance or decrease their biological control potential, depending on environmental conditions in the microhabitat.     

A Laboratory Simulation for Vectoring of Trichosporon pullulans by Conidia of Botrytis cinerea

ABSTRACT A mechanism that could contribute to the suppression of Botrytis cinerea during pathogen sporulation was examined in this study. Yeasts capable of binding to B. cinerea were formulated with a cellulose carrier and applied to sporulating colonies of the pathogen. The particles from this yeast/cellulose product attached to B. cinerea conidia in the sporulating colony. Inoculum from treated colonies was harvested and applied to tomato stem tissue to test for subsequent pathogenicity. Disease development from inoculum obtained from cultures that had been treated with Trichosporon pullulans was significantly retarded (P = 0.0001) compared with cellulose-only controls. However, between 5 and 11% of conidia applied were attached to yeast cells. The removal of conidia not attached to yeast resulted in inoculum composed of >90% of conidia attached to yeast, and from this inoculum, disease development was significantly retarded (P < 0.05). When inoculum from treated B. cinerea colonies was applied to nutrient limiting agar and then incubated, the B. cinerea conidia germinated, and yeast cells infested the new hyphal growth. Constraints of the formulation of the yeast used in this study, and the implications of this vectoring approach for the suppression of B. cinerea during pathogen sporulation are discussed.     

Activity of the Antifungal Protein from Aspergillus giganteus Against Botrytis cinerea

ABSTRACT Botrytis blight (gray mold), caused by Botrytis cinerea, is one of the most widely distributed diseases of ornamental plants. In geranium plants, gray mold is responsible for important losses in production. The mold Aspergillus giganteus is known to produce and secrete a basic low-molecular-weight protein, the antifungal protein (AFP). Here, the antifungal properties of the Aspergillus AFP against various B. cinerea isolates obtained from naturally infected geranium plants were investigated. AFP strongly inhibited mycelial growth as well as conidial germination of B. cinerea. Microscopic observations of fungal cultures treated with AFP revealed reduced hyphal elongation and swollen hyphal tips. Washout experiments in which B. cinerea was incubated with AFP for different periods of time and then washed away revealed a fungicidal activity of AFP. Application of AFP on geranium plants protected leaves against Botrytis infection. Cecropin A also was active against this pathogen. An additive effect against the fungus was observed when AFP was combined with cecropin A. These results are discussed in relation to the potential of the afp gene to enhance crop protection against B. cinerea diseases.     

两种季铵盐阳离子表面活性剂对番茄灰霉病菌的毒理效应

为探讨季铵盐阳离子表面活性剂1227和C8-10对番茄灰霉病菌Botrytis cinerea的毒理效应,运用电子显微镜、氧电极、紫外-可见分光光度计等测定了1227、C8-10对番茄灰霉病菌分生孢子萌发、细胞壁结构、呼吸作用和三种细胞壁降解酶的影响,并在番茄叶片活体上接种经药剂处理的番茄灰霉病菌,观察药剂对其侵染活性的影响.结果显示,两种季铵盐阳离子表面活性剂5μg/mL处理可使分生孢子芽管变短变粗、基部明显膨大,显著抑制聚甲基半乳糖醛酸酶、多聚半乳糖醛酸酶和羧甲基纤维素酶的活性,并可显著影响番茄灰霉病菌在番茄活体叶片上的侵染活性;40μg/mL处理可导致菌体细胞壁表面出现多处破损、细胞内含物聚集等现象;100μg/mL处理显著抑制分生孢子的呼吸作用;250μg/mL和1 000μg/mL处理对病原菌菌丝呼吸产生破坏作用.说明两种季铵盐杀菌剂高剂量能够破坏菌体细胞壁结构,导致番茄灰霉病菌呼吸完全停止和死亡,而低剂量可以抑制细胞胞壁降解酶的活性,降低其致病力.     

Dispersal of conidia of Ascochyta fabae f. sp. lentis from infected lentil plants by simulated wind and rain

Splash was shown to be an effective mechanism for short-range dispersal of conidia of Ascochyta fabae f. sp. lentis , the cause of ascochyta blight of lentil. The dispersal gradients were well described by the power law model in its linear form, In y = In a-b In x. In still air the slope of the linearized dispersal gradient, b , ranged from 2.83 to 4.07 and was steeper for 4.9 mm than for 3.9 mm incident drops. Nevertheless, for all drop sizes tested, fewer than 50% of the conidia were splashed more than 15 cm from the source. The pattern of conidium dispersal was similar for both drop sizes when horizontal windspeeds were 2.5 or 5 m/s. Wind significantly decreased the value of b (range 2.35-2.43 at 2.5 m/s, 1.71–1.91 at 5 m/s) and increased by about 2 m the maximum distance that conidia in ballistic droplets were deposited. In addition, the experiments suggested two other potentially important mechanisms for dispersal of the pathogen over longer distances, namely conidia in small air-borne droplets and windblown leaflets.     

邻烯丙基苯酚对植物病原真菌抑制机理初探

用含毒介质培养法测定了邻烯丙基苯酚(银果)对番茄灰霉病菌Botrytis cinerea、苹果腐烂病菌Valsa mali和小麦纹枯病菌Rhizoctonia cerealis的影响。通过培养观察发现,邻烯丙基苯酚可抑制番茄灰霉病菌分生孢子的产生、促进灰霉病菌菌核的形成及苹果腐烂病菌子囊壳的形成。光学显微观察表明:用50μg/mL的邻烯丙基苯酚处理番茄灰霉病菌菌丝后,菌丝伸长缓慢,分支间距缩短,分生孢子形成受到抑制。电镜观察表明:经邻烯丙基苯酚处理后,番茄灰霉病菌菌丝内液泡增多,苹果腐烂病菌菌丝细胞壁增厚,小麦纹枯病菌菌丝中内质网处泡囊增多。     

The use of Trichoderma species to control strawberry fruit rots

The effect of temperature on the growth and antagonistic properties of Trichoderma species against Botrytis cinerea and Mucor mucedo (strawberry fruit pathogens) was studied. Five strongly antagonistic isolates were further used in field experiments. The incidence of pre-harvest rots caused by B. cinerea and the rate of post-harvest spoilage were similarly reduced when strawberry flowers were sprayed either with the fungicide dichlofluanid or with spores of selected Trichoderma isolates.     

五种杀菌剂胁迫下灰葡萄孢产孢所需碳源种类分析

灰葡萄孢是引起作物灰霉病的病原菌,分生孢子是其传播的主要载体.本文采用代谢技术分析了灰葡萄孢对Biolog FF板碳源的利用及其产孢情况,并测定了在多菌灵、丙环唑、嘧霉胺、异菌脲和咪鲜胺5种杀菌剂胁迫下灰葡萄孢产孢所需碳源种类.结果表明:糖类、氨基酸类等92种碳源均能被灰葡萄孢代谢,其中,杏仁苷、L-阿拉伯糖等35种碳...     

Biocontrol of seedborne Botrytis cinerea in chickpea with Gliocladium roseum

An isolate of Gliocladium roseum proved highly antagonistic to Botrytis cinerea . Sporulation of B. cinerea on chickpea seed naturally infected or inoculated with B. cinerea was suppressed by seed treatment with conidial suspensions of G. roseum at 10⁷ and 10⁸ conidia/mL, respectively. Establishment of healthy seedlings in punnets (small trays) 5 weeks after sowing with inoculated seed was increased from 29.2% to 59.7% by treatment with G. roseum at 3×10⁷ conidia/mL, and from 1.4% to 69.4% with G. roseum at 3×10⁸ conidia/mL, the latter being equivalent to disease control by Thiram. There was no significant effect of Rhizobium on disease suppression by G. roseum , and treatment with G. roseum at 10⁸ conidia/mL did not reduce nodulation. Amendment with culture filtrates of G. roseum did not affect the growth rate of B. cinerea on potato dextrose agar, indicating that constitutive production of an antibiotic is not involved in biocontrol. A selective medium was developed to enumerate propagules of G. roseum on seed recovered from soil. There was no significant change in the population of G. roseum on seed after incubation for 4 weeks in soil to which the isolate of G. roseum was indigenous.     

Botrytis cinerea Infection in Grape Flowers: Defense Reaction, Latency, and Disease Expression

ABSTRACT Inflorescences of field-grown grapevines (Vitis vinifera L. cv. Gamay) were inoculated with a Botrytis cinerea conidia suspension or dried conidia at different stages during bloom in moist weather. Approximately 10% of the conidia germinated within 72 h, resulting in two to three times more latent infections than uninoculated controls in pea-size (7 mm in diameter) berries. In surface-sterilized pea-size berries, latent B. cinerea was present predominantly in the receptacle area. After veraison, latent B. cinerea also was found in the style and, in mature berries, latent colonies were distributed throughout the pulp. Inoculation at full bloom led to the highest disease severity (66%) at harvest, compared with 38% in controls. Stilbene stress metabolites in the flowers were measured by high-performance liquid chromatography. Resveratrol accumulated mainly after pre-bloom and full-bloom inoculation, but did not prevent infection. Piceid levels did not change following inoculation, while epsilon-viniferin was found in necrotic tissues only, and pterostilbene and alphaviniferin were not detected at all. B. cinerea conidia suspensions also were applied to various locations on flowers of pot-grown cvs. Pinot Noir and Chardonnay. Inoculation of the receptacle area, but not that of the stigma and ovary, resulted in latent infections. Stilbene synthesis was similar to the field results, with resveratrol accumulating mainly in the calyptra and receptacle area. Constitutive soluble phenolic compounds (mainly derivatives of quercetin and hydroxy-cinnamic acid) were present at high concentrations in the calyptra but at low levels in the receptacle area. These experiments confirmed bloom as a critical time for B. cinerea infection in grapes and suggest that the most likely site of infection is the receptacle area or cap scar exposed at anthesis. Stilbenes may have a limited role in inhibition of flower infection and latency in susceptible grape cultivars, and epsilon-viniferin may be a by-product rather than a deterrent of infection.     

Spatial and temporal variation of reactive oxygen species and antioxidant enzymes in o-hydroxyethylorutin-treated tomato leaves inoculated with Botrytis cinerea

Pretreatment of detached tomato leaves with o -hydroxyethylorutin reduced the percentage leaf area affected, and delayed the appearance of necrosis, following inoculation with conidial suspensions in droplets of the grey mould fungus Botrytis cinerea . o -Hydroxyethylorutin delayed, but did not inhibit, in vitro germination of conidia, although overall percentage germination was reduced compared with water controls. Both the reactive oxygen species (ROS) – superoxide anions and hydrogen peroxide – increased twice as much in o -hydroxyethylorutin-treated leaf tissue 2 and 6 h postinoculation with B. cinerea conidia in tissues under inoculation drops, as well as in surrounding tissues, whereas in plants not pretreated with the compound ROS generation was noticed later, and only in tissues under inoculation drops. Compared with these compounds, changes in the levels of hydroxyl radicals, lipid peroxidation and the activity of the enzymes superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase were largely unchanged. In stimulating ROS in inoculated tomato tissue, B. cinerea appeared to be affected directly pre- and postinfection, but indirect effects increasing host resistance cannot be excluded.     

Antagonism of Nutrient-Activated Conidia of Trichoderma harzianum (atroviride) P1 Against Botrytis cinerea

ABSTRACT The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum (atroviride) P1 to suppress Botrytis cinerea was investigated in laboratory, greenhouse, and field trials. Preliminary nutrient activation at 21 degrees C accelerated subsequent germination of the antagonist at temperatures from 9 to 21 degrees C; at >/=18 degrees C, the germination time of preactivated T. harzianum P1 conidia did not differ significantly from that of B. cinerea. When coinoculated with B. cinerea, concentrated inocula of preactivated but ungerminated T. harzianum P1 conidia reduced in vitro germination of the pathogen by >/=87% at 12 to 25 degrees C; initially quiescent conidia achieved this level of suppression only at 25 degrees C. Application of quiescent T. harzianum P1 conidia to detached strawberry flowers in moist chambers reduced infection by B. cinerea by >/=85% at 24 degrees C, but only by 35% at 12 degrees C. Preactivated conidia reduced infection by >/=60% at 12 degrees C. Both quiescent and preactivated conidia significantly reduced latent infection in greenhouse-grown strawberries at a mean temperature of 19 degrees C, whereas only preactivated conidia were effective in the field at a mean temperature of 14 degrees C on the day of treatment application. An antagonistic mechanism based on initiation of germination in sufficiently concentrated inocula suggests that at suboptimal temperatures the efficacy of Trichoderma antagonists might be improved by conidia activation prior to application.     

Influence of environmental conditions in a glasshouse on conidia ofBotrytis cinerea and on post-harvest infection of rose flowers

Quantification and horizontal distribution of air-borne inoculum ofBotrytis cinerea in a rose crop in a glasshouse of 300 m² was studied in 1991 and 1992. Conidia ofB. cinerea were caught in spore traps consisting of an agar medium selective forB. cinerea in Petri dishes placed within the crop, at flower height 1 m above the ground. Spore catches were counted as colonies, after incubation. Lesions due to conidial infection were counted on petals of rose flowers, also after incubation. Relative humidity (RH) and temperature within the glasshouse and global radiation and windspeed outside were recorded during the experiments. The horizontal distribution ofB. cinerea in a rose crop grown under glass was fairly uniform in both years. In 1991 a clear seasonal pattern in the number of colonies could not be found. In 1992 the number of colonies were high in August, September and October. The number of lesions on rose flowers showed a distinct pattern in both years. In August, September and October many lesions were counted whereas in the other months few lesions appeared. In linear regression analysis, variation in numbers of colonies (spore catches) could not be explained by environmental factors recorded during the experiments. Linear regression accounted for 76 and 63% of the variation in the number of lesions on rose flowers in 1991 and 1992, in terms of relative humidity (positively correlated), global radiation outside the glasshouse (negatively correlated) and, numbers of colonies on spore traps (positively correlated). The results in the rose crop suggest that RH, global radiation and spore density in glasshouses are important variables in regulating the numbers of lesions during storage and transport. The numbers of spores in glasshouses are dependent on the production system. A glasshouse with a system resulting in wet dead tissue on the ground give higher amount of spores in the glasshouse air and through that high numbers of lesions on flowers. On roses outside the glasshouses very high numbers of lesions were counted sometimes, mostly during and after rain showers, as a result of rain-deposition of spores onto the flowers.     

Improving biological control by combining biocontrol agents each with several mechanisms of disease suppression

ABSTRACT Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves and plants. Scanning electron microscopy revealed significant inhibition of Botrytis cinerea conidial germination in the presence of Pichia guilermondii, whereas Bacillus mycoides caused breakage and destruction of conidia. When both biocontrol agents were applied in a mixture, conidial destruction was more severe. The modes of action of each of the biocontrol agents were elucidated and the relative quantitative contribution of each mechanism to suppression of Botrytis cinerea was estimated using multiple regression with dummy variables. The improvement in control efficacy achieved by introducing one or more mechanisms at a time was calculated. Pichia guilermondii competed with Botrytis cinerea for glucose, sucrose, adenine, histidine, and folic acid. Viability of the yeast cells played a crucial role in suppression of Botrytis cinerea and they secreted an inhibitory compound that had an acropetal effect and was not volatile. Bacillus mycoides did not compete for any of the sugars, amino acids, or vitamins examined at a level that would affect Botrytis cinerea development. Viable cells and the compounds secreted by them contributed similarly to Botrytis cinerea suppression. The bacteria secreted volatile and non-volatile inhibitory compounds and activated the defense systems of the host. The nonvolatile compounds had both acropetal and basipetal effects. Mixture of Pichia guilermondii and Bacillus mycoides resulted in additive activity compared with their separate application. The combined activity was due to the summation of biocontrol mechanisms of both agents. This work provides a theoretical explanation for our previous findings of reduced disease control variability with a mixture of Pichia guilermondii and Bacillus mycoides.     

Leaflet abscission and phytoalexin production during the response of two faba bean breeding lines to Botrytis infection

Comparisons were made between two breeding lines of faba bean (designated 224 and 335), following inoculation of leaf material with Botrytis cinerea and the more aggressive B. fabae . Line 335 exhibited greater initial resistance to infection, in terms of retarded lesion development and higher rates of phytoalexin production. However, in whole plant inoculation experiments line 335 abscinded its leaflets more readily after infection. The role of the gaseous plant hormone ethylene in this reaction was analysed. Both lines exhibited a similar abscission response to exogenous ethylene, applied at a concentration of 10 μL L⁻¹. However, there were significant differences in the ability of the lines to produce ethylene. In response to inoculation with B . fabae , line 335 liberated ethylene at a higher concentration and for a longer period. These results suggest that the differential levels of abscission are a consequence of differences in the biosynthesis of ethylene in the two lines rather than in their responses to ethylene.