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.     

Modeling Spatial Characteristics in the Biological Control of Fungi at Leaf Scale: Competitive Substrate Colonization by Botrytis cinerea and the Saprophytic Antagonist Ulocladium atrum

ABSTRACT A spatially explicit model describing saprophytic colonization of dead cyclamen leaf tissue by the plant-pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum was constructed. Both fungi explore the leaf and utilize the resources it provides. Leaf tissue is represented by a two-dimensional grid of square grid cells. Fungal competition within grid cells is modeled using Lotka-Volterra equations. Spatial expansion into neighboring grid cells is assumed proportional to the mycelial density gradient between donor and receptor cell. Established fungal biomass is immobile. Radial growth rates of B. cinerea and U. atrum in dead cyclamen leaf tissue were measured to determine parameters describing the spatial dynamics of the fungi. At temperatures from 5 to 25 degrees C, B. cinerea colonies expanded twice as rapidly as U. atrum colonies. In practical biological control, the slower colonization of space by U. atrum thus needs to be compensated by a sufficiently dense and even distribution of conidia on the leaf. Simulation results confirm the importance of spatial expansion to the outcome of the competitive interaction between B. cinerea and U. atrum at leaf scale. A sensitivity analysis further emphasized the importance of a uniform high density cover of vital U. atrum conidia on target leaves.     

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.     

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.     

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.     

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.     

Effect of interrupted leaf wetness periods on suppression of sporulation ofBotrytis allii andB. cinerea by antagonists on dead onion leaves

Saprophytic antagonists were evaluated for suppression of sporulation ofBotrytis allii andB. cinerea on artificially killed segments of onion leaves that were pre-inoculated with the pathogens. During incubation of the antagonisttreated leaf segments in moist chambers, periods of leaf wetness and leaf dryness were alternated to simulate conditions in the field. Interruption of humid conditions with dry periods had a differential effect on antagonists.Alternaria alternata, Chaetomium globosum, Ulocladium atrum andU. chartarum suppressed sporulation ofB. allii almost completely under continuously wet conditions, and when the leaf wetness periods were interrupted with drying periods of 9h imposed 16, 40, and 64 h after the antagonists were applied. When leaf wetness was interrupted 16 h after antagonist application, the number of conidia ofB. allii produced cm^–2 leaf surface after eight days was under the detection limit of 5.2 × 10³ conidia on leaves treated with these antagonists compared to 3.7 × 10⁵ conidia on leaves that were not treated. On the other hand,Gliocladium roseum, G. catenulatum andSesquicillium candelabrum, all highly efficient under continuously wet conditions, were of low to moderate efficiency when leaf wetness periods had been interrupted 16 h after application of the antagonists. The antagonists showed the same differentiation and sensitivity to interrupted wetness periods when tested withB. cinerea.     

Universally Primed Polymerase Chain Reaction Alleles and Internal Transcribed Spacer Restriction Fragment Length Polymorphisms Distinguish Two Subgroups in Botrytis aclada Distinct from B. byssoidea

ABSTRACT Fifty-one isolates representing the four Botrytis spp. associated with onion neck rot were clustered by unweighted pair group method with arithmetic mean based on universal-primed polymerase chain reaction (UP-PCR) fingerprints. Bootstrap analysis of the consensus phenogram clearly demonstrated five strong clusters among the four Botrytis spp.: B. cinerea (C), B. squamosa (S), B. byssoidea (B), and B. aclada (AI and AII). Subdivision of the 30 B. aclada isolates, AI (14) and AII (16), from Europe, Egypt, North America, and Japan was further supported by restriction analysis of the internal transcribed spacer of the ribosomal genes and spore size measurements. Gene diversities (H) among AI and AII isolates were very low (0.007 and 0.043, respectively). A likelihood ratio chi-square test (G(2)) of Nei's coefficient of genetic differentiation (G(ST)) showed that both B. aclada subgroups, AI and AII, were significantly different from B. byssoidea (P < 0.001), and that B. aclada subgroups AI and AII were significantly different from each other (P < 0.001). No UP-PCR alleles were shared by AI and B. byssoidea isolates, whereas 10 and 12 alleles were shared by AI:AII and AII:B. byssoidea, respectively. The hypothesis that AII may be a hybrid between AI and B. byssoidea is discussed.     

Suppression of sporulation ofBotrytis spp. as a valid biocontrol strategy

In this study, the hypothesis was tested that removal of substrate for sporulation ofBotrytis spp. may lead to a retardation of an epidemic if the majority of the inoculum is produced inside the treated crop. Suppression of sporulation ofBotrytis spp. could be an attractive option for biological control ofBotrytis leaf spot in onions. In a field experiment, necrotic leaf tissue was removed to simulate the effect of a biocontrol agent. By this means, the amount of substrate on whichBotrytis spp. sporulates was reduced. In the experiment, the spore load above the onion plots was significantly reduced and the epidemic of onion leaf spot was retarded. At the end of the growing season, the number of leaf lesions in the green leaf area was lower in plots with substrate removal than in control plots (0.6 and 1.1 cm^–2, respectively). The results demonstrated that an epidemic of onion leaf spot largely depends on the rate of inoculum production inside a crop. Thus, suppression of sporulation on necrotic leaf tissue is a valid control strategy that could be applied by using sporulation suppressing antagonists.     

出芽短梗霉菌株YW1防治储藏期草莓灰霉病的研究

本文研究了出芽短梗霉Aureobasidium pullulans菌株Yw1防治储藏期草莓果实灰霉病Botrytiscinerea的效果,并对其防病机制进行了初步探索。结果表明,菌株Ywl可以有效的控制储藏期草莓灰霉病的发生,发病率可由95．1％降至30．5％-55．6％,菌株Ywl产生的挥发性物质可以抑制灰葡萄孢菌丝生长,抑制率达到52．1％。去除菌体的上清液对灰霉菌无明显抑制活性,说明其抗菌作用机制可能是活菌的竞争作用。由此可见,菌株Ywl是1株有应用潜力的生防菌。     

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.     

野生药用植物内生真菌的分离及拮抗菌株筛选

为挖掘生防新资源,采用组织分离法从21种野生药用植物不同组织部位分离纯化内生真菌,以6种植物病原菌为靶标菌筛选拮抗菌株,并根据形态学及分子生物学对菌株进行鉴定,在此基础上研究了高效拮抗菌株对靶标菌菌丝生长及孢子萌发的影响。结果表明:从分离纯化的478株内生真菌中筛选出11株高活性拮抗菌株,分属于青霉属Penicillium、平脐蠕孢属Bipolaris、棘壳孢属Pyrenochaeta、镰孢属Fusarium、粒毛盘菌属Lachnum、垫壳孢属Coniella和Neonectria 7个属,其中青霉属占比最高,达36.36%。平皿对峙试验表明, 棘壳孢菌12-R-5对灰葡萄孢表现出高效性和专一性,其对菌丝抑制率达66.67%。含药平皿试验表明,菌株12-R-5发酵滤液10倍稀释液对灰葡萄孢菌丝生长抑制率达100%,与其他处理差异显著。菌株12-R-5 10倍发酵滤液处理灰葡萄孢10 h,孢子不萌发;100倍稀释液处理对孢子萌发抑制率为91.01%。显微镜观察发现,棘壳孢菌使灰葡萄孢菌丝扭曲变形,膨大肿胀及断裂,部分菌丝发生抑缩、消融现象。说明棘壳孢菌12-R-5菌株具有很好的生防潜力及应用前景。     

Resistance of postharvest biocontrol yeasts to oxidative stress: a possible new mechanism of action

ABSTRACT We detected the generation of the reactive oxygen species (ROS) superoxide anion ( O.(-) (2)) and hydrogen peroxide (H(2)O(2)) in apple wounds 2 immediately after wounding, and assessed the relationships between (i) timely colonization of apple wounds by biocontrol yeasts, (ii) resistance of these microorganisms to oxidative stress caused by ROS, and (iii) their antagonism against postharvest wound pathogens. We analyzed a model system consisting of two yeasts with higher (Cryptococcus laurentii LS-28) or lower (Rhodotorula glutinis LS-11) antagonistic activity against the postharvest pathogens Botrytis cinerea and Penicillium expansum. LS-28 exhibited faster and greater colonization of wounds than LS-11. In contrast to LS-28, the number of LS-11 cells dropped 1 and 2 h after application, and then increased only later. In vitro, LS-28 was more resistant to ROS-generated oxidative stress. The combined application of biocontrol yeasts and ROS-deactivating enzymes in apple wounds prevented the decrease in number of LS-11 cells mentioned above, and enhanced colonization and antagonistic activity of both biocontrol yeasts against B. cinerea and P. expansum. Polar lipids of LS-11 contained the more unsaturated and oxidizable alpha-linolenic acid, which was absent in LS-28. Resistance to oxidative stress could be a key mechanism of biocontrol yeasts antagonism against postharvest wound pathogens.     

Comparison of Antagonists of Botrytis cinerea in Greenhouse-grown Cucumber and Tomato under Different Climatic Conditions

The efficacy of Trichoderma harzianum T39 and the yeasts Aureobasidium pullulans and Cryptococcus albidus against Botrytis cinerea in cucumber and tomato was compared with chemical control. Four experiments were conducted in cucumber grown under different climatic conditions in The Netherlands, and two experiments were done in tomato both in the Netherlands and in Israel. T. harzianum and A. pullulans showed the most consistent control of B. cinerea, reducing stem lesions and death of plants by 40–100% in most cases. Control of stem lesions and subsequent wilting was generally better than control of symptoms on fruits. In some cases, the biocontrol agents were more effective than the broad-spectrum fungicide tolylfluanid and the selective fungicide iprodione. The climatic conditions did not strongly influence the efficacy of the biocontrol agents, but regression analysis showed that high temperature during the day and high vapour pressure deficit during the night reduced biocontrol efficacy. From the results, prospects for biocontrol of B. cinerea in greenhouse vegetables appear good under a range of conditions.     

Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field

ABSTRACT Selected isolates of Aureobasidium pullulans, Rhodotorula glutinis, and Bacillus subtilis reduced the size and number of lesions on wounded apples caused by the postharvest pathogens Penicillium expansum, Botrytis cinerea, and Pezicula malicorticis. Combinations of the antagonistic microorganisms were applied to apple trees in the field late in the growing season of two consecutive years. The population dynamics of the introduced microorganisms and the incidence of fruit decay were determined. Population sizes of introduced antagonists on apple surfaces increased in the field following application of treatments until harvest. After transfer of the fruit from the field into cold storage, the populations of the introduced antagonists remained higher than in the control treatments. Identification of the applied isolates of A. pullulans and R. glutinis during the experiments was achieved by isolate-specific DNA probes generated from random amplified polymorphic DNA. A combination of two strains of A. pullulans and one strain of R. glutinis suppressed rotting of apple to the same extent as the commonly used fungicide Euparen. Our data demonstrate that the application of antagonistic microorganisms in the field represents a promising alternative to fungicide treatments to control post-harvest diseases of apple.     

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.     

两株贝莱斯芽胞杆菌鉴定及其对薄壳山核桃黑斑病病原菌的拮抗效果

黑斑病为薄壳山核桃重要真菌性病害,为了实现薄壳山核桃黑斑病绿色防控,本研究旨在筛选优良的内生拮抗菌株,为黑斑病的生物防控提供生防资源。通过形态鉴定、生理生化特征测定和16S r DNA与Gyr A基因分析,对薄壳山核桃内生拮抗菌进行鉴定;采用平板对峙法测定内生拮抗菌对黑斑病病原真菌（5属21种26株）菌丝生长的抑制能力,分离筛选到2株高效拮抗菌ZJJDZY和ZJJDYB,鉴定为贝莱斯芽胞杆菌Bacillus velezensis。平板对峙法测定结果表明,2株拮抗菌均对12种12株炭疽菌Colletotrichum spp.、1种4株葡萄座腔菌Botryosphaeriadothidea、2种2株链格孢菌Alternariaspp.、4种7株新拟盘多毛孢Neopestalotiopsisspp.和1种1株拟茎点霉菌Phomopsissp.均具有拮抗效果。其中,对C.henanense PCZJJD18、C. sojae PCAHFY5、N. mesopotamica PCSCLZ2、A. alternata PCAHFY6、Phomopsis PCZJJD14sp.的抑制率为最高,抑制率...     

Mechanisms involved in the biological control ofBotrytis cinerea incited diseases

Mechanisms involved in the biological suppression of infection and inoculum potential ofBotrytis cinerea are numerous and variable and the involvement of two or more mechanisms has been demonstrated in several systems. Reported combinations include antibiosis with enzyme degradation ofB. cinerea cell walls; competition for nutrients followed by interference with pathogenicity enzymes of the pathogen or with induced resistance; and alteration of plant surface wettability combined with antibiosis. Since germinatingB. cinerea conidia are dependent on the presence of nutrients, competition for nutrients is regarded as important in systems where biocontrol is involved. Conidial viability and germination capacity are also potentially affected by the presence of antibiotics produced by biocontrol agents and present in the phyllosphere. Slower in action are mechanisms involving induced resistance in the host plant and production of hydrolytic enzymes that degradeB. cinerea cell walls. The latter has been demonstrated much more convincinglyin vitro than in the phyllosphere. Biocontrol in established lesions and reduction of sporulation on necrotic plant tissues is a means to minimize the pathogen inoculum.Abbreviations BCA bio-control agent - Bc Botrytis cinerea - PG polygalacturonase - PL Pectin lyase - PME Pectin methyl esterase - PR pathogenesis related - VPD vapour pressure deficit     

Biological control ofBotrytis cinerea in residues and flowers of Rose (Rosa hybrida)

Microbial isolates from living petals, petal residues and leaf residues of rose, and from laboratory collections, were evaluated for control ofBotrytis cinerea in rose. In leaf residues artificially infested withB. cinerea, isolates of the filamentous fungiGliocladium roseum, FR136 (unidentified) andTrichoderma inhamatum reduced sporulation of the pathogen by >90%, other filamentous fungi were 25–90% effective, and those of yeasts and bacteria were <50% effective. In artificially inoculated petal residues, no microbe reduced sporulation ofB. cinerea by >75%, but isolates ofCladosporium oxysporum and four yeasts were 51–75% effective, and three filamentous fungi, eight yeasts andBacillus subtilis isolates were 26–50% effective. Isolates ofT. inhamatum, C. oxysporum andG. roseum performed best againstB. cinerea among isolates evaluated in leaf residues naturally infested with the pathogen and indigenous microorganisms. Totals of ten isolates of filamentous fungi (includingC. oxysporum andC. cladosporioides), two of yeasts and five ofBacillus subtilis completely prevented lesion production byB. cinerea in detached petals, and a further six isolates of filamentous fungi (includingG. roseum) and six yeasts were 90–99% effective. Isolates ofC. oxysporum, C. cladosporioides andB. subtilis, the most effective microorganisms againstB. cinerea in flower buds, reduced number of lesions in the range of 42–65% compared with 59–89% for à standard fungicide (vinclozolin). It is suggested that application of leading antagonists Jo living rose leaves and flowers should optimize control of inoculum production byB. cinerea when the tissues die. Optimal biocontrol of lesion production in flower buds requires a better understanding of the microenvironment of petals.     

Epidemiology of Grey Mould in Annual Waiting-bed Production of Strawberry

The epidemiology of Botrytis cinerea was studied in five annual strawberry crops using waiting-bed transplants, a system widely adopted in the Netherlands. On dead leaves of transplants the incidence of B. cinerea varied from 26.7% to 52.6%, but the leaf area with potential sporulation was low (3.5–15.6%). During each crop cycle, the availability of necrotic leaf substrate for spore production of B. cinerea was generally low and varied between seasons and with the quality of transplants. B. cinerea sporulated on a maximum of 15.5 cm² of leaf area per plant, measured as potential sporulation. The aerial concentration of B. cinerea conidia in untreated plots did not differ from the concentration in plots where all dead leaves had been removed, nor from the concentration at 25–50 m distance from the strawberry plots. B. cinerea incidence on flowers ranged from 5% to 96%, but no correlation was found with the potential spore production on necrotic leaves. Grey mould at harvest varied from 1.4% to 11.3% and was correlated with the average precipitation during the harvesting period but not with B. cinerea incidence on flowers. Post-harvest grey mould ranged from 2.1% to 32.6% and was correlated with petal colonisation by B. cinerea. The results suggest that in the annual cropping system with waiting-bed transplants, necrotic leaves are not a significant source of B. cinerea inoculum, unlike in other strawberry production systems. Therefore, control measures of grey mould in this annual system should focus on protection of flowers and young developing fruits, and not on the reduction of inoculum production on leaf debris.