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.     

Possible Role of Colonization and Cell Wall-Degrading Enzymes in the Differential Ability of Three Ulocladium atrum Strains to Control Botrytis cinerea on Necrotic Strawberry Leaves

ABSTRACT Ulocladium atrum (strain 385) consistently reduced Botrytis cinerea sporulation on necrotic fragments of strawberry leaves. On these tissues, two strains of U. atrum (isolates 18558 and 18559) showed lower antagonistic activities than the reference strain 385. Colonization of strawberry leaflets by the three U. atrum strains appeared similar in the absence of B. cinerea, whether quantified by chitin or immunological assays. The second method (based on anti-U. atrum antibodies) revealed that strawberry leaflet colonization by U. atrum 385 was better than by the other U. atrum strains in the presence of B. cinerea. An immunoassay using anti-B. cinerea antibodies revealed that the colonization of B. cinerea in tissues was lower in the presence of U. atrum 385 than with the two other U. atrum strains. The enzymatic activities produced by U. atrum 385 during the colonization phases of necrotic tissues were compared to B. cinerea and U. atrum strains 18558 and 18559. U. atrum 385 had the highest lipase, pectate lyase, and cellobiase activities while B. cinerea had the highest endo-beta-1,4-glucanase activity. The study of lytic activities hydrolyzing the fungal cell wall revealed higher beta-1,3-glucanase activity with U. atrum 385, which was stimulated by B. cinerea on necrotic strawberry leaflets. These results suggest that plant and fungal cell wall-degrading enzymes produced by U. atrum 385 may play a complementary role in the competitive colonization of dead strawberry leaves against B. cinerea.     

Effect of Water Potential on Conidial Germination and Antagonism of Ulocladium atrum Against Botrytis cinerea

ABSTRACT The saprophytic fungus Ulocladium atrum was selected for its ability to competitively exclude Botrytis spp. from aboveground necrotic plant tissues which can play a crucial role in the epidemiology of diseases caused by necrotrophic Botrytis spp. Fungal growth in necrotic aboveground tissues can be hampered by fluctuating water availability. Adaptation to these adverse conditions is a key factor for the successful establishment of an antagonist population in this niche. Conidia of U. atrum germinated at water potentials between -1 and -7 MPa on water agar. Germinated conidia survived one to three interruptions of moist incubation by periods at -10 or -42 MPa. The speed of germination was significantly slower with interruption of the moist period as compared with the control, which had continuously moist incubation. However, the maximum germination percentage was the same for conidia incubated with or without interruption of the moist incubation. In bioassays with necrotic cyclamen leaves at -1, -3, and -7 MPa, U. atrum significantly reduced the sporulation of B. cinerea by more than 80%. The results demonstrate that U. atrum tolerates water stress during competitive substrate colonization with B. cinerea. The antagonist is, therefore, an attractive candidate for field applications on aboveground tissues.     

Quantification of Mycelium of Botrytis spp. and the Antagonist Ulocladium atrum in Necrotic Leaf Tissue of Cyclamen and Lily by Fluorescence Microscopy and Image Analysis

ABSTRACT A technique was developed to localize and quantify the internal mycelial colonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. This technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a monoclonal antibody specific for Botrytis spp. And an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was used to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hyphae of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume of each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and allowed the design of efficient sampling strategies for future experiments.     

Interaction of Four Antagonistic Fungi with Botrytis aclada in Dead Onion Leaves: A Comparative Microscopic and Ultrastructural Study

ABSTRACT The colonization of dead onion leaves by Botrytis aclada and the fungal antagonists Aureobasidium pullulans, Chaetomium globosum, Glio-cladium catenulatum, and Ulocladium atrum and the interactions between B. aclada and each of the four antagonists were studied at the microscopic and ultrastructural level. This approach was used in an attempt to understand the colonization pattern of these fungi and the nature of the biocontrol activity of the antagonists that have shown a potential to suppress spore production of Botrytis spp. on necrotic plant tissues. When applied alone, B. aclada and U. atrum were found throughout the leaf tissues in high densities after an incubation period of 6 days at 18 degrees C in a moist chamber. C. globosum and G. catenulatum colonized only the outer portions of the leaf, whereas A. pullulans appeared to be concentrated in the leaf stomata. When pathogen and antagonists were applied together, ultrastructural observations revealed that cells of B. aclada were plasmolyzed in the presence of G. catenulatum, suggesting a reaction to antifungal molecules. Antibiosis also seemed to be involved, albeit to a lesser extent, in the antagonistic interactions between B. aclada and A. pullulans or C. globosum. No evidence of direct parasitism was recorded. On the other hand, U. atrum appeared to completely exclude B. aclada from dead onion tissues when both fungi competed for the substrate. Ultrastructural observations of the in vitro interaction between the two fungi did not reveal parasitism or antibiosis by either fungus. Based on previous records of its biocontrol potential and observations of its colonizing properties, it appears that U. atrum can compete for and utilize necrotic tissues rapidly and extensively, thus, excluding competitors without any other antagonistic action.     

Comparative Analysis of the Role of Substrate Specificity in Biological Control of Botrytis Elliptica in Lily and B. Cinerea in Cyclamen with Ulocladium Atrum

Biological control of Botrytis spp. by the fungal antagonist Ulocladium atrum is based on their interaction in plant tissue. U. atrum is effective against B. cinerea in commercial cyclamen crops but not effective against B. elliptica in lily crops. Based on the necrotrophic nature of the Botrytis spp. and the saprophytic nature of U. atrum it is hypothesised, and experimentally confirmed, that the interaction between Botrytis spp. and U. atrum, resulting in a biocontrol effect, only takes place in necrotic plant tissue. The role of necrotic tissue in the epidemiology of B. cinerea in cyclamen and B. elliptica in lily was found to be different. Removal of symptomless senescing leaves resulted in a significant reduction of the area under the disease severity progress curve (AUDPC) for B. cinerea in cyclamen but had no effect on the disease severity in lily. U. atrum applications significantly reduced B. cinerea AUDPC values in cyclamen but were less efficient than the removal of senescing leaves. In lily, disease severity was not affected by applications of U. atrum. It is concluded that necrotic cyclamen tissue, not killed by B. cinerea, plays an important role in the onset of disease. Colonisation of this tissue by U. atrum prevents saprophytic colonisation of those leaves by B. cinerea. In contrast, conidia of B. elliptica directly infect healthy lily leaf tissue. U. atrum applications aimed at blocking the infection pathway from a saprophytic base are therefore not effective against B. elliptica. Control options based on competitive interactions in and around B. elliptica lesions resulted in a reduced production of conidia by B. elliptica but proved ineffective against disease development. The potential of U. atrum as a biocontrol agent against Botrytis spp. and possibly against other necrotrophs appears to be determined by the competitive saprophytic ability of the antagonist in mutual substrates of pathogen and antagonist and by the role of these substrates in disease epidemiology.     

The survival and saprophytic competitive ability of the Botrytis spp. antagonist Ulocladium atrum in lily canopies

In 1995, conidia of Ulocladium atrum were applied to a canopy of green lily (Lillium spp.) leaves in order to investigate its survival, colonisation of artificially induced necrotic leaf tissues and competitive ability against Botrytis spp. and naturally occurring saprophytes. U. atrum conidia density cm^-2 at the top and middle canopy levels was not significantly different following application of the antagonist with a propane powered backpack sprayer. In repeat experiments, conidia density on leaves at the lower canopy level was 18% to 20% of that deposited onto leaves at the top of the lily canopy. There was a significant (P < 0.001) linear decline of U. atrum conidia over time and after 21 days conidia density had declined by up to 73%. Germination of U. atrum on green leaves in the field reached a maximum of 81%, seven days after antagonist application. Conidial viability, measured as germination potential, declined slightly (100% to 88%) after seven days exposure to field conditions but there were no further changes in the germination potential even after 21 days of field exposure. The germination potential was not affected by canopy level. The ability of surviving U. atrum conidia to colonise necrotic tissues, artificially induced with paraquat, was measured. U. atrum colonisation was consistently highest on necrotic leaves at the top level of the canopy and consistently lower on leaves from the bottom canopy level. Necrotic leaf colonisation by U. atrum decreased over time from 51% (necrosis induced immediately after antagonist application) to 21% when necrosis was induced 21 days after antagonist application. A significant (P < 0.001) linear relationship (R² = 0.713) between colonisation of necrotic tissues and conidia density prior to induction of necrosis was detected. When necrosis was induced immediately after antagonist application, U. atrum outcompeted commonly occurring saprophytic Alternaria spp. and Cladosporium spp. The ability of U. atrum to significantly reduce colonisation by Alternaria spp. was maintained for up to 21 days. Botrytis spp. did not occur in these field experiments. It was concluded that U. atrum had the ability to survive and persist in the phyllosphere for up to 21 days in the field and provided further evidence that U. atrum has the necessary survival characteristics to be a successful biological control agent of Botrytis spp.     

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.     

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.     

Ultrastructural and Cytochemical Aspects of the Biological Control of Botrytis cinerea by Candida saitoana in Apple Fruit

ABSTRACT Biocontrol activity of Candida saitoana and its interaction with Botrytis cinerea in apple wounds were investigated. When cultured together, yeast attached to Botrytis sp. hyphal walls. In wounded apple tissue, C. saitoana restricted the proliferation of B. cinerea, multiplied, and suppressed disease caused by either B. cinerea or Penicillium expansum. In inoculated apple tissue without the yeast, fungal colonization caused an extensive degradation of host walls and altered cellulose labeling patterns. Hyphae in close proximity to the antagonistic yeast exhibited severe cytological injury, such as cell wall swelling and protoplasm degeneration. Colonization of the wound site by C. saitoana did not cause degradation of host cell walls. Host cell walls in close contact with C. saitoana cells and B. cinerea hyphae were well preserved and displayed an intense and regular cellulose labeling pattern. In addition to restricting fungal colonization, C. saitoana induced the formation of structural defense responses in apple tissue. The ability of C. saitoana to prevent the necrotrophic growth of the pathogen and stimulate structural defense responses may be the basis of its biocontrol activity.     

Effects of the saprophytic leaf mycoflora on growth and productivity of winter wheat

The effects of the saprophytic mycoflora and its interference with cereal aphids on growth and yield of winter and spring wheat was studied in field experiments in 1980, 1981 and 1982.Yields varied between 5000 and 8000 kg dry matter of kernels per ha. The effect of the saprophytic mycoflora on yield was determined in different treatments: A) no control measures against cereal aphids and saprophytic and necrotrophic fungi, B) no control of cereal aphids, control of saprophytic and necrotrophic fungi, C) control of cereal aphids and control of saprophytic and necrotrophic fungi, D) control of cereal aphids and stimulation of saprophytic mycoflora and E) control of cereal aphids, no control of saprophytic and necrotrophic fungi nor stimulation of saprophytic mycoflora.Considerable differences in top densities of saprophytic mycoflora (10 times as large in A and D as in B and C) were determined. The consequences of these differences for the growth and productivity of wheat were minor. A negative effect of saprophytic mycoflora on the yield could not be detected in 1981 and 1982, whereas a small positive significant effect was found in 1980. This stimulation may have been due to competition between necrotrophic fungal pathogens and saprophytic mycoflora. As a result of favourable weather conditions necrotrophic fungal pathogens were very numerous in 1980 and could form an important yield reducing factor. Yield levels may effect the importance of the necrotrophic and saprophytic mycoflora as yield reducing factors. Additionally, in the presence of aphid honeydew captafol was found to be relatively ineffective against saprophytic fungi.     

Control of infection and sporulation ofBotrytis cinerea on bean and tomato by saprophytic bacteria and fungi

Sixty isolates of saprophytic microorganisms were screened for their ability to reduce the severity of grey mould (Botrytis cinerea) infection and sporulation. Isolates of the bacteriaXanthomonas maltophilia, Bacillus pumilus, Lactobacillus sp., andPseudomonas sp. and the fungusGliocladium catenulatum reduced germination of conidia of the pathogen and controlled disease on bean and tomato plants. Their activity under growth room conditions was good, consistent, and similar to the activity of the known biocontrol agent,Trichoderma harzianum T39 (non-formulated). Although the tested isolates may for nutrients with the germinating conidia ofB. cinerea, resistance induced in the host by live or dead cells were also found to be involved. Inhibitory compounds were not detected on treated leaves. Sporulation ofB. cinerea after its establishment on leaves was also reduced by the above mentioned isolates and byPenicillium sp.,Arthrinium montagnei, Ar. phaeospermum, Sesquicillium candelabrum, Chaetomium globosum, Alternaria alternata, Ulocladium atrum, andT. viride. These sporulation-inhibiting fungi did not reduce the infection of leaves byB. cinerea. Most of these selected fungi and bacteria were capable of reducing lesion expansion.     

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.     

Wind dispersal of conidia of Botrytis spp. pathogenic to Vicia faba

Increasing numbers of conidia were blown from sporulating cultures of Botrytis fabae and B. cinerea as windspeed increased up to 10 m/s. Partial drying of cultures increased the number of spores blown away at low and intermediate windspeeds. Release of spores at a constant windspeed was sustained over a prolonged period. Different patterns of release from colonies of the two species when windspeed was gradually increased, or at a constant windspeed. may be related to differences in spore size affecting the drying rate. Many conidia of both species were released as clumps. A higher proportion of B. cinerea than B. fabae conidia were clumped, partly because the mean number of spores per clump was greater. Individual conidia fell more slowly in still air than did clumps. The humidity in a bean crop was more favourable to development of Botrytis lesions than that above the crop. Low windspeeds measured within crops may restrict dispersal of conidia and may result in uneven distribution of chocolate spot lesions.     

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.     

Fusarium mangiferae localization in planta during initiation and development of mango malformation disease

Mango malformation disease (MMD), caused by Fusarium mangiferae, is a major constraint to mango production, causing significant yield reduction resulting in severe economic impact. The present study characterizes fungal localization in planta during initiation and development of vegetative and floral malformation. Young mango trees were artificially inoculated with a green fluorescent protein (GFP)‐expressing strain of F. mangiferae. Shoots and buds were sampled periodically over a period of more than a year and localization of the GFP‐expressing fungi was determined using confocal microscopy. Fungal localization appears to be epiphytic: mycelia remained in close contact with the plant surface but did not penetrate the tissue. In vegetative malformation and in young inflorescences, the fungus was confined to protected regions between scales, young leaf bases and buds. Fungal colonization was only very rarely detected on open leaves or on exposed shoot sections. In developed flowers, mycelia were localized mainly to protected regions at the base of the flower organs. Upon development of the inner flower organs, specific mycelial growth occurred around the anthers and the style. Mycelial penetration through the stylar tract into aborting carpels was observed. For several months, mycelia were confined to the surface of the organs and were not detected within plant tissues. Only at later stages, transient saprophytic growth of the fungus was detected causing the malformed inflorescences to senesce and collapse, concurrent with dispersion of conidia. Implications of the present study on MMD in natural field infections are discussed.     

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.     

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development of Colletotrichum acutatum on Almond

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.     

真菌源几丁质酶在植物抗真菌病害中的应用

几丁质酶可以降解大多数真菌细胞壁,阻止或中断真菌在植物体内的侵染、定殖和扩展,在抗真菌病害的研究方面受到广泛关注。本文综述了真菌源几丁质酶的特性,对真菌的抑制作用机制,转几丁质酶基因植株的抗性评价,产几丁质酶菌株和相关制剂在生物防治方面的应用,并对其在生产上的发展前景进行了展望。     

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.