Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.     

Preventative and Curative Applications of Verticillium lecanii for Biological Control of Cucumber Powdery Mildew

The effect of timing of the application of the mycoparasite , Verticillium lecanii, on cucumber powdery mildew , Sphaerotheca fuliginea, was studied in a rooted cucumber leaf bioassay . The mycoparasite was applied at different times before and after mildew inoculation . At near - to maximum humidity ( 95% relative humidity) , early preventative (9 and 5 days before mildew inoculation) and early curative control treatments (2 days after mildew inoculation) gave considerable reduction in mildewed leaf areas , while late curative treatments resulted in greater mildewed leaf area but ultimately a reduced amount of healthy mildewed leaf area ( 20 % ) . Appropriate timing of biocontrol treatment application of V. lecanii is important to achieve good control .     

Pathogenicity of the Fungus Verticillium lecanii to Aphids and Powdery Mildew

Aspects of the host specificity and pathogenicity of the hyphomycete, Verticillium lecanii , were investigated under laboratory conditions. DiVerences were observed in the pathogenicities of three strains of V. lecanii (DAOM 198499, DAOM 216596 and Vertalec) to the potato aphid Macrosiphum euphorbiae and Sphaerotheca fuliginea , the causal agent of cucumber powdery mildew. The estimated median lethal concentration required to achieve 50% mortality (LC ), 50 median lethal time leading to 50% mortality (LT ) and aphid net reproductive rate ( R ) 50 0 indicated that Vertalec and strain 198499 were more virulent to aphids than strain 216596. The estimated median colonization time for 50% of fungal colonies (CT ) showed that strain 50 198499 was the best antagonist of cucumber powdery mildew. Further comparison suggested that the mean pathogenicity of V. lecanii strain 198499 to cucumber powdery mildew was almost equivalent to that of Sporothrix flocculosa , a biological control agent of greenhouse fungal pathogens. These observations provide convincing experimental evidence that V. lecanii is biologically active against both arthropods and fungi. The potential of using V. lecanii strain 198499 in biological control is discussed.     

Glasshouse Experiments on Biocontrol of Cucumber Powdery Mildew (Sphaerotheca fuliginea) by the MycoparasitesVerticillium lecaniiandSporothrix rugulosa

Two potential biological control agents of cucumber powdery mildew (Sphaerotheca fuliginea),Verticillium lecaniiandSporothrix rugulosa,were tested under glasshouse conditions. Two experiments were carried out. In the first experiment, two cucumber varieties with different levels of resistance, cv Corona (susceptible) and cv Flamingo (partially resistant), were used.Verticillium lecaniicontrolled the mildew better thanS. rugulosa.On cv Flamingo,V. lecaniicould keep the mildew severity below 15% infected leaf area for 9 weeks after inoculation withS. fuliginea.Treatment by Hora Oleo 11E, alone or as an additive toV. lecanii,was as good as a fungicide treatment. In the second experiment, weekly and biweekly treatments withV. lecaniiwere compared on cv Flamingo. Weekly treatments withV. lecaniikept mildew severity at a level below 20% infected leaf area during 10 weeks after inoculation withS. fuliginea.If applied to a partially resistant cucumber cultivar,V. lecaniiis an effective candidate for biological control ofS. fuliginea.     

Dry-Heat Destruction of Bacillus subtilis Spores on Surfaces: Effect of Humidity in an Open System

Bacillus subtilis var. niger spores were tested for dry-heat resistance on stainless-steel strips hung in an oven. Heat resistance was dependent on the relative humidity before and during treatment, which in turn affected the water content of the spores. Higher humidities increased the heat resistance of the spores. D-values ranged from 16.1 min for spores conditioned at <2% relative humidity (RH) and treated at 0.34% RH to 37.6 min for spores conditioned at 89% RH and treated at 1.1% RH. The y-intercept of the regression line ranged from 6.94 x 10(4) for spores conditioned and treated at the low humidities to 2.00 x 10(5) for spores conditioned at 89% RH and treated at 0.34% RH. For a constant value of N(0), the y-intercept appears to be lowered by low-humidity conditions. The statistic log y(0)/log N(0) is used to measure the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2% RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments.     

氨基寡糖素对黄瓜白粉病菌侵染的抑制作用*

用透射电子显微镜,研究了寡聚糖类药剂中科6号(2%氨基寡糖素)处理黄瓜Cucumis sativus 植株叶片后对白粉病菌Sphaerotheca fuliginea侵染的抑制作用。超微结构观察表明,病菌发育受到明显抑制,表现为白粉菌菌丝细胞质凝聚,细胞器解体和细胞组织崩解;吸器内的原生质电子致密度加深,吸器畸形,吸器壁增厚,细胞器解体,最终吸器坏死。     

The Interaction of Humidity and Resistance Elicitors on Expression of Polygenic Resistance of Barley to Mildew

Elicitor-active glucan was applied to leaves of genotypes of barley expressing different levels of resistance to powdery mildew ( Erysiphe graminis f. sp. hordei )which was assessed by measuring mildew colonies per leaf or speed of papilla induction response to mildew challenge. The influence of humidity was also assessed by treating leaves with glucan, inoculating with mildew and immediately sealing the leaves in Perspex containers in an apparatus which enabled temperature, air speed and humidity to be controlled accurately. In water-treated controls low humidity alone reduced colonies per leaf, and at 60% r.h. genotype 7204 showed significantly less effect of humidity compared with other genotypes, although at 40% it responded as other genotypes. At low humidity glucan stimulated resistance for genotypes Proctor and 7204 but not for Golden Promise or 9855; the effect of humidity or elicitor alone was only significant on genotype 7204, Chitin also proved an effective elicitor, but at high and not at low humidity. Time of pre-treatment before mildew challenge, leaf stage, type of elicitor, concentration of elicitor, post-inoculation light regime, mildew isolate and barley genotype all had major influences on papilla response. Other factors were less important. Based on papilla formation, the barley cultivar Proctor was most and Golden Promise the least responsive to glucan as an elicitor. The potential for exploiting the genetic basis of inducible resistance is discussed.     

Enhanced ovicidal activity of an oil formulation of the fungus Metarhizium anisopliae on the mosquito Aedes aegypti

Abstract.  The effect of humidity on the activity of Metarhizium anisopliae IP 46 (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) formulated in sunflower oil against Aedes aegypti (L.) (Diptera: Culicidae) eggs was examined. After exposure of eggs at 75% relative humidity (RH) for ≤ 25 days, ovicidal activity was not increased by oil-in-water formulated conidia, hyphal bodies or pure-oil formulated conidia, compared with conidia or hyphal bodies prepared in water only. At optimal > 98% RH, eclosion was ≤ 13.7% after treatment with oil-in-water formulated propagules in ≤ 10% oil, and it was completely inhibited when conidia were applied in pure oil. At 86–100% RH, new conidia were found on eggs treated with oil-formulated conidia and incubated down to 91% RH. Ovicidal activity was still detected at 93% RH and was augmented with increasing humidity and time of exposure of eggs. Eclosion of larvae was distinctly reduced by IP 46 pure-oil formulated conidia after a minimal initial exposure of 3 days at > 98% RH, followed by: (a) a 12-day exposure at 75% RH before submersion in water; (b) a minimal 5-day exposure at > 98% RH and direct subsequent transfer of treated eggs to water, or (c) a minimal daily 20-h exposure at > 98% RH alternating with 4 h at 75% RH for 10 days. We demonstrate that oil-based formulations of conidia of M. anisopliae enhance ovicidal activity at high humidities and conclude that these formulations have potential in the integrated control of Ae. aegypti .     

The Potential of Stagonospora sp. as a Mycoherbicide for Field Bindweed

Field bindweed ( Convolvulus arvensis L.) is one of the 12 most important weeds worldwide. Stagonospora sp. Isolate LA39 was isolated from diseased field bindweed plants collected in Europe. No crop tested was susceptible to the fungus, but disease symptoms were observed on other Convolvulaceae species. On field bindweed, the fungus induces disease symptoms (i.e. lesions) mainly on leaves and less severely on stems. The application of spores in an oil emulsion (10% oil in water) enhanced the disease on field bindweed plants compared with spores suspended in a 0.1% aqueous solution of the surfactant agent, Tween 80. The necrotic leaf area of inoculated plants increased as the length of exposure to 100% relative humidity (RH) and the spore density applied increased. Severe disease developed on plants inoculated with 1 107 spores/ml in oil emulsion, even in the absence of exposure to 100% RH. A delay of exposure to 100% RH (up to 8 h) did not have a significant eVect on disease severity. Field bindweed was susceptible to the fungus at all growth stages tested, but older plants were more susceptible than younger ones. It was concluded that isolate LA39 has potential as a biocontrol agent of field bindweed, especially when applied in an oil emulsion. The oil emulsion maintains the aggressiveness of the pathogen during a dew-free period and provides a favourable microenvironment during the infection process.     

Control of glasshouse whitefly, Trialeurodes vaporariorum, by an 'aphid' strain of the fungus Verticillium lecanii

Heavy infestations of whitefly on glasshouse cucumber plants were controlled below the level of economic crop damage by fortnightly or monthly sprays of Verticillium lecanii spores. The fungus did not spread from glasshouse to glasshouse, or from plant to plant, and often not from diseased whitefly scales bearing fungal spore heads to nearby healthy scales. Some scales survived and the resulting adults laid eggs on new leaves bearing no infected scales, creating another, healthy, generation. This makes regular spraying of new leaves essential. Blastospores were as effective as conidia in controlling scales when sprayed to ‘run off’ at concentrations near 10⁷ spores ml^-1 sprayed on to the undersurfaces of leaves. A fivefold increase in spore concentration at levels near 10⁷ spores ml^-1 usually caused significant improvement in mortality, but increase above this concentration is likely to be unrewarding. Thorough coverage of leaves was found to be vital. Control was impaired by dry conditions and by prolonged air temperatures above 25 °C. The fungicide dimethirimol, used against cucumber mildew, did not impair whitefly control by V. lecanii.     

Effects of Heat Shock on Photosynthetic Properties,Antioxidant Enzyme Activity,and Downy Mildew of Cucumber (Cucumis sativus L.)

Heat shock is considered an abiotic stress for plant growth, but the effects of heat shock on physiological responses of cucumber plant leaves with and without downy mildew disease are still not clear. In this study, cucumber seedlings were exposed to heat shock in greenhouses, and the responses of photosynthetic properties, carbohydrate metabolism, antioxidant enzyme activity, osmolytes, and disease severity index of leaves with or without the downy mildew disease were measured. Results showed that heat shock significantly decreased the net photosynthetic rate, actual photochemical efficiency, photochemical quenching coefficient, and starch content. Heat shock caused an increase in the stomatal conductance, transpiration rate, antioxidant enzyme activities, total soluble sugar content, sucrose content, soluble protein content and proline content for both healthy leaves and downy mildew infected leaves. These results demonstrate that heat shock activated the transpiration pathway to protect the photosystem from damage due to excess energy in cucumber leaves. Potential resistance mechanisms of plants exposed to heat stress may involve higher osmotic regulation capacity related to an increase of total accumulations of soluble sugar, proline and soluble protein, as well as higher antioxidant enzymes activity in stressed leaves. Heat shock reduced downy mildew disease severity index by more than 50%, and clearly alleviated downy mildew development in the greenhouses. These findings indicate that cucumber may have a complex physiological change to resist short-term heat shock, and suppress the development of the downy mildew disease.     

Survival of Airborne Bacteria in a High Urban Concentration of Carbon Monoxide

Vegetative cells of Serratia marcescens 8UK, Sarcina lutea, and spores of Bacillus subtilus var. niger were held in aerosols, with and without an urban concentration of CO (85 muliters per liter or ppm), for up to 6 hr at 15 C and a relative humidity (RH) of approximately 0, 25, 50, 75, and 95%. It was found that CO enhanced the death rate of S. marcescens 8UK at least four- to sevenfold at low RH (ca. 1 to 25%), but protected the cells at high RH (ca. 90%). Death rates of S. lutea, with or without added CO, were comparatively low over the entire RH range. However, in the first hour, airborne S. lutea held in CO-containing air were more stable than those in air without added CO (i.e., CO protection). A marked increase in the death rate (up to 70-fold) occurred in the subsequent 5 hr within the RH range of approximately 0 to 75%. Statistical analysis indicated that aerosol decay rates of B. subtilus var. niger spores decreased significantly, when held in a CO-containing as compared to a non-CO-containing atmosphere, in the 0 to 85% RH range. Thus, the data presented indicate that CO in the urban environment may have a protective or lethal effect on airborne bacteria, dependent upon at least the microbial species, aerosol age, and relative humidity. A mechanism for CO death enhancement and protection of airborne S. marcescens 8UK is suggested to involve CO uncoupling of an energy-requiring death mechanism and an energy-requiring maintenance mechanism at high and low RH, respectively.     

Fungitoxic action of dimethirimol and ethirimol

The systemic fungicides dimethirimol and ethirimol were shown to be toxic to spores of Erysiphe graminis and Sphaerotheca fuliginea, in germination tests in vitro. Toxicity of dimethirimol to spores of Botrytis fabae, Phytophthora infestans, Puccinia recondita, Uncinula necator and Venturia inaequalis was relatively feeble or absent. When applied in foliage sprays or in root treatments, both compounds inhibited the emergence of germ tubes from powdery mildew spores on cucumber and barley plants. The specific disease control shown by these fungicides can be explained by their direct fungitoxic action. The effects of dimethirimol and ethirimol on powdery mildew infections and on spore germination were greatly decreased by the presence of riboflavin in the light. A rapid photo-chemical interaction between riboflavin and dimethirimol was demonstrated. Marked reversals both of disease control and of fungitoxicity were also given by folic acid, and it is suggested that folic acid overcomes a metabolic block induced in powdery mildew fungi by the fungicides.     

Oleozon: A novel control strategy against powdery mildew in cucumber

Ozonized sunflower oil (oleozon) is an effective agent for controlling powdery mildew in cucumber. In this study, the mechanisms of oleozon in the control of powdery mildew were determined. The development of Podosphaera xanthii on cucumber leaves treated with oleozon (2%) and water was investigated at different times after inoculation. The germinating conidia, hyphae and conidiophores of the pathogen were severely damaged by oleozon. No visible phytotoxic effect was observed on cucumber after the application of oleozon. This compound had highly preventive effects as well as curative effects against powdery mildew based on in vivo potted seedling assays. The control effects of oleozon were further confirmed in a greenhouse trial. These results may provide a basis for further development of a natural fungicide against cucumber powdery mildew.     

Management of powdery mildew and gray mold of cucumber by Trichoderma harzianum T39 and Ampelomyces quisqualis AQ10

Two biocontrol preparations were tested for their ability to control Sphaerotheca fusca and Botrytis cinerea on greenhouse cucumber. Trichoderma harzianum T39 (TRICHODEX) spray reduced powdery mildew severity by up to 97% but its efficacy declined to 18–55% control as the epidemic progressed. Unlike on young leaves, on older leaves the control of powdery mildew by T. harzianum T39 was poor. Ampelomyces quisqualis (AQ10) was very effective against powdery mildew, achieving up to 98% of control. Its effectiveness declined with the progress of the epidemic but unlike the other biocontrol agent it retained significant control capability on older leaves. Two aliphatic petroleum distillate oil products improved the efficacy of both biocontrol agents. The co-application of T. harzianum T39 and A. quisqualis AQ10 was tested on cucumber plants infected with powdery mildew followed by fruit gray mold infection. It resulted in no improvement of the control of powdery mildew, and in an improvement of gray mold control, the latter probably because of the use of additive oil (ADDQ) along with the second biocontrol preparation. There was no significant interference between the biocontrol agents in the co-application treatment as compared with the application of each agent alone; the level of population of T. harzianum T39 remained similar and the parasitism of S. fusca by A. quisqualis was not nullified. The application of T. harzianum T39 to soil instead of spraying it resulted in 75–90% lower powdery mildew coverage on the leaves. It was concluded that the mode of action of T. harzianum T39 in powdery mildew control is induced resistance, not mycoparasitism or antibiotic action.     

Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores

Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0 °C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable, temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1% in 14 days.     

Survival of Phytophthora ramorum hyphae after exposure to temperature extremes and various humidities

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorum hyphae. Spore-free hyphal colonies were grown on dialysis squares atop V8 medium. Colonies were transferred to water agar plates positioned at 27.5-50 C on a thermal gradient plate and incubated 2.5-480 min. For low temperature trials colonies were transferred to vials of distilled water and incubated in a water bath at -5 to -25 C for 1-24 h. In the relative humidity trials hyphal colonies were transferred to sealed humidity chambers containing various concentrations of glycerin for 1-8 h. Relative humidity was 41-93% at 20 C and 43-86% at 28 C. Survival in all trials was characterized by growth from dialysis squares into V8 medium. Temperatures of 37.5-40 C were lethal to P. ramorum hyphae within several hours, and temperatures of 42.5-50 C were lethal within minutes. Exposure to 32.5 and 35 C resulted in reduced survival over 8 h, while 30 C had no effect on three of four isolates. Hyphal colonies demonstrated considerable tolerance to cold, with all isolates surviving a 24 h exposure to -5 C. Survival diminished over time at lower temperatures, however a few colonies survived 24 h exposure to -25 C. Temperature also affected the ability of hyphal colonies to withstand reduced humidity. A RH of 41-43% was lethal in 2 h at 28 C compared to 8 h at 20 C. Three of four isolates were unaffected by an 8 h exposure to 81 and 95% RH at 20 C, and 73 and 86% RH at 28 C. Isolate differences were apparent in tolerance to freezing temperatures and reduced humidity. From these results it is apparent that the cold temperatures found in the northeastern USA are not likely to prevent the establishment of P. ramorum. There is also the potential for hyphae, and presumably spores, to survive periods of high humidity on the leaf surface in the absence of free water.     

Thermal imaging of cucumber leaves affected by downy mildew and environmental conditions

Pathogenesis of Pseudoperonospora cubensis causing downy mildew of cucumber resulted in changes in the metabolic processes within cucumber leaves including the transpiration rate. Due to the negative correlation between transpiration rate and leaf temperature, digital infrared thermography permitted a non-invasive monitoring and an indirect visualization of downy mildew development. Depending on the stage of pathogenesis and the topology of chloroses and necroses, infection resulted in a typical temperature pattern. Spatial heterogeneity of the leaf temperature could be quantified by the maximum temperature difference (MTD) within a leaf. The MTD increased during pathogenesis with the formation of necrotic tissue and was related to disease severity as described by linear and quadratic regression curves. Under controlled conditions, changes in temperature of infected leaves allowed the discrimination between healthy and infected areas in thermograms, even before visible symptoms of downy mildew appeared. Environmental conditions during thermographic measurement, in particular air temperature and humidity, as well as water content and age of the leaf influenced the temperature of its surface. Conditions enhancing the transpiration rate facilitated the detection of changes in leaf temperature of infected leaves at early stages of infection. As modified by environmental conditions, MTD alone is not suitable for the quantification of downy mildew severity in the field.     

Efficacy of iodine-treated biocidal filter media against bacterial spore aerosols

Aims: To assess the effectiveness of iodine-treated biocidal filter media against bacterial spore aerosols. Methods and Results: Bacillus subtilis spores were aerosolized and introduced into a filtration system. Both treated and untreated filters exhibited high viable removal efficiency (>99·996%) with negligible variation in pressure drop during the entire experiment. The viability of collected spores on the filter was investigated by enumeration of spores extracted from the filter by vortexing. At room temperature and low relative humidity (RH), the survival fraction of the treated filter was significantly lower than that of the untreated filter (P-value < 0·05). Meanwhile, at room temperature and high RH and at high temperature and high RH, the survival fractions on the treated medium were statistically the same as the untreated control at room temperature and low RH. Conclusions: Both treated and untreated filters achieved excellent viable removal efficiency for spores. The pressure drop of the treated filter was not affected by the iodine treatment. The viability of collected bacterial spores was decreased because of the exertion of iodine disinfectant. Significance and Impact of the Study: The evaluation demonstrates that the iodine-treated filter is a viable medium for respiratory protection against infectious spore aerosols. The results warrant further evaluation of smaller biological agents, which exhibit higher penetration.     

The mechanism of the mycoinsecticide diluent on the efficacy of the oil formulation of insecticidal fungus

Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M. anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT₅₀ for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M. anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.