Laboratory investigations on the potential of entomopathogenic fungi for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae and pupae

The susceptibility of third instar Helicoverpa armigera to seven strains of three entomopathogenic fungal species, i.e. Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus, was tested under laboratory conditions using the larval immersion method. High efficacies ranging from 68 to 100% corrected mortality were recorded with more profound effects in treatments with B. bassiana and P. fumosoroseus strains. The median lethal concentration (LC₅₀) for L3 was 6.0×10⁵ in M. anisopliae 79, 1.5×10⁵ in B. bassiana 124 and 4.2×10⁴ in P. fumosoroseus 14. These three strains were further used to characterize the age-dependent mortality of different larval stages (L2-L5) and the effect against pupae of H. armigera. Larval stages did not differ in their mortality but differed i in median lethal time, with shorter values recorded in the second instar. Tested fungi also caused a high reduction between 74.4 and 100% in the emergence of pupae using the soil inoculation method and the pupal immersion technique. All three fungal species, especially P. fumosoroseus, have a high potential for biocontrol of H. armigera larvae and also as a soil treatment targeting the pupae.     

Influence of Agricultural By-products in Liquid Culture on Chlamydospore Production by the Potential Mycoherbicide Fusarium oxysporum Foxy 2

Economically feasible inoculum mass production methods are required for successful application of Fusarium oxysporum Foxy 2 as a potential mycoherbicide. Therefore, different substrates (agricultural by-products) and the factors that influence the production of spores, especially chlamydospores, of Foxy 2 were investigated in liquid cultures. The substrates tested were cotton seed cake, maize stover, wheat and triticale stillage. The presence of plant fibers in the medium of unfiltered cotton seed cake (2.5%, w/v) significantly enhanced chlamydospore, micro- and macroconidia production by 150, 185, and 300%, respectively, compared to the filtered (fiber-free) medium. Regardless of the type of substrate tested, Foxy 2 was able to produce abundant chlamydospores (0.14-2.7×10⁷ mL^-1) in all growth media. Generally, increasing the concentrations of cotton seed cake and maize stover in the medium significantly increased chlamydospore formation; however, this was not the case for wheat-based stillage. To optimize conditions required for chlamydospore production of Foxy 2, the effect of near ultra-violet (NUV) light, substrate combinations (synergism), level of agitation, incubation time and their interaction were studied. A liquid culture of 2.5% (w/v) filtered cotton seed cake, exposed to continuous NUV for 15 days, doubled the yield of chlamydospores (4.7×10⁶ mL^-1) and macroconidia (5×10⁵ mL^-1), and increased microconidial production by one-third (1×10⁸ mL^-1) compared to natural light. An apparent synergistic effect of substrate combination was observed, since the addition of 20% (v/v) of either wheat or triticale-based stillage to maize stover medium (1%, w/v) increased the number of chlamydospores produced by 16 or 12 times, respectively, compared to maize stover alone (1.4×10⁶ mL^-1), and 2 times more than either of the stillages. A significantly positive effect between a high level of agitation tested during incubation and chlamydospore production of Foxy 2 was recorded. All in all, substantial chlamydospore production of Foxy 2 (4.3×10⁷ mL^-1) was successfully achieved within 12 days of incubation in a single-step liquid fermentation through the combination of 0.5% (w/v) maize stover plus 20% (v/v) wheat-based stillage and a high level of agitation (200 rpm).     

Biological Control and Use of Adjuvants against Multiple Seeded Cocklebur (Xanthium strumarium) in Comparison with Several Other Cocklebur Types

Common cocklebur has several biotypes including multiple seeded cocklebur (MSC), NCC-TX, and NCC-MS. Alternaria helianthi applied at 2.5×10⁴ conidia mL^-1 in a 50% micro-emulsion of unrefined corn oil (MESUCO) or 0.2% Silwet L 77 caused 60-75% mortality on NCC-TX and MSC. Increasing the conidial concentration to 5×10⁴ mL^-1 increased mortality to 100% on MSC and NCC-TX, and 75% on NCC-MS. At 10×10⁴ conidia mL^-1, A. helianthi caused 100% mortality in all three biotypes. No mortality occurred in any biotype at inoculation rates of 2.5 and 5×10⁴ conidia mL^-1 when applied in water. Increasing the dew period from 0 to 12 h increased mortality from 0 to 100% on all three biotypes at a rate of 2.5×10⁴ conidia mL^-1 in Silwet and MESUCO. MSC appears to be the most sensitive biotype.     

Evaluation of the Entomopathogenic Fungi Metarhizium anisopliae and Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) for Control of the Green Leafhopper Empoasca decipiens (Homoptera: Cicadellidae) and Potential Side Effects on the Egg Parasitoid Anagrus atomus (Hymenoptera: Mymaridae)

The efficacy of Metarhizium anisopliae strain Ma43 and Paecilomyces fumosoroseus strain Pfr12 (both Deuteromycotina: Hyphomycetes) against adults of Empoasca decipiens (Homoptera: Cicadellidae) and potential side effects on the egg parasitoid Anagrus atomus (Hymenoptera: Mymaridae), were investigated in greenhouse cage and laboratory experiments. Treating leafhopper-infested faba bean plants at a dose rate of 1×10⁷ conidia mL^-1 resulted in up to 97% mortality 7 days after application and a 100% infection rate. Experiments on the residual effects revealed a significant decrease in adult E. decipiens mortality with increasing time from application to insect release. The decrease in mortality over time corresponded well with data from conidia germination tests. The germination of conidia on agar medium after washing them from the surafce of sprayed plants declined significantly from 95 and 96% immediately after application for M. anisopliae Ma43 and P. fumosoroseus Pfr12, respectively, to 29 and 27% 5 days later. Experiments on potential side effects of the entomopathogenic fungi on A. atomus showed that the tested isolates had no influence on adult emergence and longevity; however, the rates of parasitism were significantly reduced by the fungal treatments. The latter might be due to either density effects and/or could indicate that A. atomus avoids fungal-treated plants. However, the parasitoid is substantially less susceptible to the fungal strains tested than the host itself.     

Factors Affecting Disease Development by Rhynchosporium alismatis in Starfruit (Damasonium minus), a Weed of Rice

The fungal pathogen Rhynchosporium alismatis is being developed for biological control of starfruit (Damasonium minus), an important aquatic weed in Australian rice fields. The development of R. alismatis in starfruit differs between juvenile and adult plants. Juvenile starfruit plants are stunted as a result of fungal infection, while in adult plants, the main effect is necrosis and chlorosis of floating leaves. A conidial concentration of 1×10⁴ conidia mL^-1 was adequate to cause disease symptoms on floating leaves, but the stunting effect on juveniles was caused by concentrations of at least 1×10⁵ conidia mL^-1. To successfully inoculate juvenile plants, the water must be drained before inoculation to expose plants to the inoculum. The artificial addition of dew periods did not enhance disease development in plants. The stunting of juvenile starfruit plants caused by the infection of R. alismatis may give rice plants a competitive advantage over the weed at the seedling stage     

Whey for mass production of Beauveria bassiana and Metarhizium anisopliae

Spore production of Beauveria bassiana and Metarhizium anisopliae was studied in a novel whey-based culture media. Spore yield and viability were determined for two B. bassiana (GHA-726 and CA-603) and two M. anisopliae (CA-1 and IMI 330189) isolates following production in three whey-based systems: solid, liquid, and a diphasic production system. Our study indicated that whey permeate can be used effectively for production of spores of entomopathogenic fungi. However, spore yield and viability were significantly influenced by fungal isolate, whey concentration, and the type of production process used. Under the conditions defined in the present study, spore yields ranging from 1.3 × 10⁹–10 × 10¹¹ spores l⁻¹ of whey medium could be obtained depending on the strain and production process used. Our study revealed that spores produced by all strains in whey-based solid and liquid media showed between 73–99 % viability; germination rates were comparable with those obtained using the standard SDA medium. In the two-stage production process, the viabilities of conidia produced by GHA-726, CA-603, and CA-1 were 35–86, 32–98, and 6–29 %, respectively; viability was correlated with whey concentration and isolates. Whey permeate can be used as a growth substrate for mass production of biocontrol fungi. We hypothesize that spore yield and viability could be improved by careful selection of whey content in the medium, incorporation of critical additives and optimization of culture conditions.     

Selection of an Isolate of the Insect Pathogenic Fungus Metarhizium anisopliae Virulent to the Lettuce Root Aphid, Pemphigus bursarius

The virulence of hyphomycete entomopathogenic fungi was measured in laboratory bioassays against the lettuce root aphid, Pemphigus bursarius, a serious pest of field lettuce grown in the UK. Of 25 isolates of fungi examined, only one isolate, Metarhizium anisopliae 391.93, killed lettuce root aphids consistently. This fungus was isolated originally from the closely related saltmarsh aphid, P. trehernei. The median lethal concentration of conidia at 10 days post6- 1 inoculation estimated from five independent bioassays was 2.45 × 10⁶ conidia ml^-1. The fungus had no significant effect on the mean number of offspring/aphid produced, but it sporulated 6 profusely on host cadavers, producing approximately 4 × 10⁶ conidia/cadaver 14 days after treatment, and diseased aphids died attached to plant roots. It thus has the potential to spread through densely packed colonies of P. bursarius feeding on the roots of susceptible or partially resistant plants.     

Isolation, Pathogenicity and Safety of Curvularia eragrostidis Isolate QZ-2000 as a Bioherbicide Agent for Large Crabgrass (Digitaria sanguinalis)

A pathogen isolated from lesions on blighted leaves of crabgrass in three different locations of China was identified as Curvularia eragrostidis. Isolate QZ-2000 was the most virulent of six isolates tested. Experiments on morphology, pathogenicity, effect of environmental factors, and host-range of isolate QZ-2000 were conducted in the laboratory, greenhouse and field to assess the potential of this isolate as a biocontrol agent for grassy weeds. Pathogenicity was quantitatively determined based on mortality and dry-weight reduction of infected large crabgrass. Inoculum concentration, rapeseed oil concentration in formulaton, post-inoculation dew temperature and duration, and plant growth age all significantly influenced the efficacy of the isolate. A total of 85-100% control of large crabgrass was obtained when inoculum concentrations were ≥1×10⁶ conidia mL^-1, oil concentrations ≥0.9% (v/v), dew period ≥24 h and air temperatures 20-30°C in the greenhouse. A total of 51 plant species in 20 families were screened against isolate QZ-2000 in host-range studies. Six other species of grassy weeds were susceptible to isolate QZ-2000, but no mortality or significant dry-weight reduction was observed for maize (Zea mays), rice (Oryza sativa), soya bean (Glycine max), cotton (Gossypium hirsutum), or any other economically important crops and plants. In field trials, with 5×10⁶ conidia mL^-1 inoculum density, 60-7% reduction in dry weight was achieved for large crabgrass seedlings under natural dew-free conditions. These results indicate that isolate QZ-2000 is a potential microbial bioherbicide for control of large crabgrass in crops such as corn, soybean, cotton, water-melon, and peanut.     

A Multiple-Pathogen System for Bioherbicidal Control of Several Weeds

A novel system using four host-specific fungal plant pathogens applied in a single, postemergent spray to control pigweed, sicklepod, and showy crotalaria was tested under greenhouse conditions. The four pathogens were Phomopsis amaranthicola (a pathogen of pigweed species), Alternaria cassiae (a pathogen of sicklepod and showy crotalaria), Colletotrichum dematium f. sp. crotalariae and Fusarium udum f. sp. crotalariae (pathogens of showy crotalaria). Spore suspensions of each pathogen alone (10⁶ spores mL^-1) or a mixture of the four pathogens (1:1:1:1, v/v, 2.5×10⁵ spores mL^-1 of each pathogen, total 10⁶ spores mL^-1) were tested on four- to six-leaf stage seedlings of the three weed species grown together in pots. One week after inoculation (WAI), all sicklepod and showy crotalaria seedlings were killed, and all pigweed seedlings were killed by 6 WAI when inoculated with their respective pathogen(s) alone or a mixture of the pathogens. None of the weeds inoculated with the root-infecting pathogen F. udum developed wilt disease by the time the experiment was completed (6WAI). The results demonstrate the feasibility to control three weeds simultaneously with different fungi without loss of efficacy or alterations in host-specificity of each fungus in the given mixture. Scanning electron microscopy showed visual differences in the appearance or germination and further development of conidia of each pathogen on its respective host leaf surface compared to nonhost leaf surfaces, whether the pathogen was applied alone or in a mixture with the other pathogens studied. Application of several host-specific fungal pathogens in a bioherbicide mixture as a multi-component bioherbicide system may be advantageous for further development of simultaneous, broad-spectrum weed control.     

碳源对玫烟色虫草菌丝生长、芽生孢子产生及其耐热性的影响

提高虫生真菌孢子应对热胁迫的能力是生防菌应用研究的关键,为研究菌丝培养阶段碳源对玫烟色虫草Cordyceps fumosorosea IF-1106耐热性的影响,选择了麦芽糖、可溶性淀粉、蔗糖、葡萄糖、果糖、海藻糖为碳源的培养基对玫烟色虫草IF-1106进行液体培养,评估了不同碳源条件下菌丝的生长、产孢及所产芽生孢子的耐热性。结果表明,在菌株培养阶段,培养基中碳源的种类及浓度对菌丝产量、产孢量及所产芽生孢子的耐热性有显著影响,其中蔗糖为碳源时,所产芽生孢子的耐热性强,45 ℃热胁迫条件下LT₅₀为1.65 h;蔗糖浓度为40 g/L时,可产生大量耐热芽生孢子,液体培养3 d后产孢量可达3.43×10⁷个孢子/mL。为探索不同培养条件下所产芽生孢子耐热性不同的原因,提取了孢子内的海藻糖并采用离子色谱法对其进行了定量分析,发现耐热性高的芽生孢子胞内海藻糖含量普遍较低,可见海藻糖是与芽生孢子耐热性密切相关的内源物质。综上所述,选择适宜的培养基是调控孢子耐热性的有效途径,本研究为生产高耐热的玫烟色虫草生防制剂提供了有益的指导。     

Characterisation of optimum cultural environmental conditions for the production of high numbers of Metarhizium anisopliae blastospores with enhanced ecological fitness

The aim of this study was the production of high numbers of M. anisopliae blastospores with enhanced germination efficiency under conditions of water-stress (ecological fitness). Different nitrogen sources and concentrations were screened for their ability to induce blastospore production while keeping pH and water activity (a_w) at fixed levels (6.8, and 0.98 a_w, respectively). After optimum nitrogen status was determined (cornsteep solid (CS) + yeast extract (YE); or cottonseed flour (CF) + YE), the effect of interaction of nitrogen source, pH (3.5, 5, 6.8, 8, 9 and 10) and solutes for a_w adjustment (KCl, NaCl, PEG 200) on blastospore production, endogenous polyol content (glycerol, erythritol, arabitol and mannitol) and total protein, were determined. For both ionic (NaCl, KCl) and non-ionic solutes (PEG 200), optimum blastospore production (between 4×10⁷ and 2×10⁸ blastospores ml^-1) and growth occurred in the pH range 6.8-8, with the CF + YE nitrogen profile giving higher yields than CS + YE. Optimum conditions for high erythritol and total protein endogenous concentrations (40.37-73.44 and 14.33-18.90 mg g^-1 fresh weight, respectively) occurred between pH 6.8 and 8 by ionic a_w modification (KCl, NaCl) and with the CF + YE nitrogen profile. Germination of blastospores produced under these cultural conditions was between 62 and 89% under conditions of water-stress (0.96 a_w). On the other hand, blastospores with lower amounts of erythritol and total protein content had decreased germination (8-67%). These results could have significant implications for developing a liquid fermentation medium for the production of high numbers of fungal propagules with enhanced efficacy under non-optimum environmental conditions.     

Effect of volume and concentration of conidial suspensions of Coniothyrium minitans on infection of Sclerotinia sclerotiorum sclerotia

White mould, caused by Sclerotinia sclerotiorum, is a serious disease affecting a wide range of agricultural and horticultural crops. Biological control is one option available to limit its damage. Field experiments to evaluate various concentrations and volumes of Coniothyrium minitans spore suspensions applied to S. sclerotiorum-infected bean crops were conducted in 1997 and 1998. Percentage sclerotia infected by C. minitans were scored. Three replicate experiments were performed in time in 1997 with 21 combinations of isolates, volumes and concentrations, including two controls. In 1998, 22 combinations of isolates, volumes and concentrations plus two controls were used, combined with the absence or presence of a maize buffer, with two replicates for each. Isolates as well as concentration and volume had no effect on infection by C. minitans, but there was a significant effect of total dose (volume×concentration) of inoculum applied over the full range from 100 L ha^-1 at 10⁴ conidia mL^-1 to 1000 L ha^-1 at 10⁷ conidia mL^-1. Percentage infected sclerotia increased linearly with log (dose) as well as from 1 to 4 weeks after application of C. minitans, and reached a level of about 100% at high doses under the humid conditions of 1998. Apothecia of S. sclerotiorum developing from sclerotia in collected soil samples from the 1997 experiment showed no significant effect of C. minitans inoculum dose, but there was a significant effect of the replicate experiments. The influence of weather conditions is highlighted, and the implications of the results for cost-effective biocontrol of S. sclerotiorum are discussed.     

Production of a Mixed Inoculum of Fusarium oxysporum Fo47 and Pseudomonas fluorescens C7 to Control Fusarium Diseases

Strains of non-pathogenic Fusarium oxysporum and Pseudomonas fluorescens are effective biocontrol agents against Fusarium diseases. Use of strain Fo47 of F. oxysporum in combination with strain C7 of P. fluorescens improves disease control in comparison to application of Fo47 alone. To develop a product based on a combination of these two strains, it would be beneficial to produce and formulate both organisms together. When an irradiated peat was inoculated with these strains either alone or in combination, they grew actively and reached similar maximum population densities irrespective of the initial inoculum concentration (1×10³ and 1×10⁶ g^-1 of peat) and mode of inoculation. These populations survived well with only a slight decrease in population densities after 11 months storage at 25°C. A bioassay was performed to evaluate peat and microbial suspension inocula of C7 and Fo47 alone or in combination against the flax pathogen, F. oxysporum f. sp. lini. Biocontrol activity of the peat-produced inocula was as effective as the suspensions. There was no dose-response relationship for C7, and when C7 was used as a mixed inoculum with Fo47, the biocontrol activity was always greater than that provided by Fo47 alone. This process could be used to produce an effective mixed inoculum with a shelf life of one year or more.     

Pectinase and cellulase enhance the control of Abutilon theophrasti by Colletotrichum coccodes

Inundative mycoherbicidal biocontrol agents are typically insufficiently virulent to be commercially competitive with herbicides in row crop agriculture, and require enhancement. Pectinase and cellulase are typically used by pathogens during infection. Thus, it was hypothesized that adding exogenous cell wall degrading enzymes might enhance fungal infection. Pectinase or cellulase was added to inocula of aqueous chopped mycelial suspensions of a strain of Colletotrichum coccodes for control of Abutilon theophrasti. Plants treated with 5.3×10⁶ C. coccodes propagules mL^-1 and 1.65 U mL^-1 pectinase had more rapid and complete disease development. Similar trend was achieved when 10 U mL^-1 of cellulase were added to 2.2×10⁶ C. coccodes propagules mL^-1. Adding pectinase or cellulase did not increase the host range of the wild-type fungus. The results suggest that there might be value to transforming biocontrol agents to overproduce these enzymes.     

Liquid-culture production of blastospores of the bioinsecticidal fungus<Emphasis Type="Italic"> Paecilomyces fumosoroseus</Emphasis> using portable fermentation equipment

The production of fungal spores using on-site, non-sterile, portable fermentation equipment is technically constrained. Very little information is available on the production requirements, such as medium concentration, inoculum stabilization, required fermentation times, and maintenance of axenic growth. In this study, we developed a two-part, liquid concentrate of the production medium that remains stable and soluble at room temperature. We also examined inoculum stability and showed that freeze- or air-dried blastospore preparations were stable for 7 days after rehydration when stored at 4 °C. The use of a low-pH (pH 4), relatively rich complex medium provided a growth environment deleterious to bacterial growth yet conducive to rapid sporulation by Paecilomyces fumosoroseus. High concentrations of blastospores (7.9×10⁸/ml) of P. fumosoroseus were produced in a 40-h fermentation with very low levels of bacterial contamination when the fermentor was charged with a blastospore production medium with a starting pH of 4 and inoculated with blastospore concentrations greater than 1×10⁶ spores/ml. These studies demonstrate that the use of disinfected, portable fermentation equipment has potential for on-site production of high concentrations of blastospores of the bioinsecticidal fungus P. fumosoroseus.     

Screening the physical properties of novel Pseudomonas exopolysaccharides by HPSEC with multi-angle light scattering and viscosity detection

The physical properties of three novel acidic exopolysaccharides obtained from P. marginalis types A, B and C, one from P. ‘gingen’, one from P. andropogenis and one from P. fluorescens have been partially characterized. These EPSs were chromatographed on three serially placed SE Shodex OH pak columns covering a molar mass range for pullulans from about 4 × 10⁷ to 1 × 10³. The mobile phase was 0.05 M NaNO₃. Physical measurements were performed on about 30 mg of sample for each EPS. The weight average molar mass of these EPSs ranged from about 0.71 to 2.85 × 10⁶, the weight average intrinsic viscosity from 7.15 to 35.3 dl/ g and the radius of gyration from 62 to 123nm. The polydispersities of these EPSs ranged from 1.01 to 1.37. The large molar mass, size and viscosities of these EPSs may indicate that they have potential for use as thickeners, stabilizers, emulsifiers, and gelling agents in the food and non-food industries.     

Cultivation and preservation of vinegar bacteria

Ten strains of acetic acid bacteria were investigated for their characteristics of growth and metabolism. The strains were identified as those presently in use for industrial vinegar production in southern Germany. At the time of isolation from industrial acetators the total concentrations, i.e. acetic acid (w/v) plus ethanol (v/v), of the fermenting vinegars were 6.1–14.9%. The applicability of a previously described method for starter preparation was examined for the various isolates as well as for the type strains of species of the genera Gluconobacter and Acetobacter. Isolates from cider or wine vinegar fermentations grew readily in RAE-medium to total counts of >1×10⁹ cells ml⁻¹. For the cultivation of strains isolated from spirit vinegar fermentations AE-medium proved most suitable. Cultures of these strains exhibited lag phases of 2–5 days and grew up to total counts of <1×10⁹ cells ml⁻¹. All type strains could be grown on RAE-agar. The use of 20% malt extract as cryo-protectant was effective for the preservation of all strains. Upon revitalization the cultures were suitable as inoculum for starting fermentations in pilot acetators. 16S rRNA-targeted oligonucleotide probes were constructed which were species specific for Gluconobacter oxydans or Acetobacter aceti or group specific for Acetobacter europaeus/Acetobacter xylinum. The probes hybridized with the DNA of the respective type strains. Four isolates were allotted to A. europaeus/A. xylinum applying the group specific probe. The DNA of six of the Acetobacter sp. hybridized with none of the probes.     

Augmentation of Soil with Sporangia of Actinoplanes spp. for Biological Control of Pythium Damping-off

A method was developed for applying strains of Actinoplanes spp. that are hyper-parasites of oospores of Pythium ultimum to soil for reducing Pythium damping-off of plants. The method is based on the augmentation of soil with sporangia of a strain of Actinoplanes spp. borne on clay granules. In vitro sporulation of strains K30, W57, W257 and 25844 was: (1) greater for most strains on dilute Czapek-Dox agar than on four other agar media; (2) inhibited by continuous exposure to fluorescent light of intensity 4-150 μEm^-2s^-1, but not by exposure to 1 μEm^-2s^-1 or darkness; (3) greater at 20-307deg;C than at 10°C;and (4) greater at pH 6-7 than at pH 5 or 8. On solid carriers treated with dilute Czapek-Dox broth (pH 7) and incubated in the dark at 30°C for 3 weeks, strains sporulated poorly or not at all on vermiculite, perlite and rice hulls, but sporulated abundantly (10⁷-10⁹ colony-forming units (CFU) g^-1 of granules) on montmorillonite clay granules. When strains 25844, W57 and W257 were applied as granules (4 10⁷ - 4 × 10⁸ CFU g^-1) at 5% (w/w) to field plots infested with 750-1000 oospores of P. ultimum g^-1 of soil, only strain 25844 consistently increased emergence and reduced root rot of table beets 8- 1 at 24-28 days after planting compared with controls. Strain 25844 (10⁸ CFU g^-1 of granules) at 1% (w/w) also increased the emergence of bush beans at 28 days after planting in P. ultimum-infested plots, but lower rates were ineffective. The inoculum viability of strain 25844 on clay granules declined 100-fold during 2 months of storage at 5-35°C, but thereafter remained stable for another 4 months. Strain 25844 on 6-month-old granules retained a high degree of hyper-parasitic activity toward oospores of P. ultimum. Augmentation of field soil with sporangia of Actinoplanes spp. is a valid approach to the biological control of pythium damping-off.     

两株链霉菌对非洲菊生长和生理生化指标的影响

本研究以非洲菊(Gerbera jamesonii)为试材,采用不同浓度的脱叶链霉菌FT05W和深蓝链霉菌ZEA17I孢子悬浮液浇灌盆栽非洲菊,筛选出非洲菊的最佳施用浓度,探索两株链霉菌对非洲菊生长和生理生化指标的影响,为非洲菊生产上科学合理施用链霉菌提供理论依据。结果表明: 脱叶链霉菌FT05W和深蓝链霉菌ZEA17I不同浓度孢子悬浮液均能有效促进非洲菊的生长,前者对非洲菊的作用效果优于后者,以浓度为1×10⁹ CFU·mL^-1的脱叶链霉菌FT05W效果最好。与蒸馏水对照相比,该处理显著促进了非洲菊株高(30.2%)和冠幅(41.5%)的生长,并在一定程度上促进了花茎的增长和增粗;能显著提高非洲菊植株叶片的叶绿素含量(65.2%)、根系活力(103.3%)和超氧化物歧化酶活性(84.4%),使丙二醛含量维持在较低的水平。因此在一定的浓度范围内,脱叶链霉菌FT05W和深蓝链霉菌ZEA17I均能有效促进非洲菊的生长,有利于非洲菊植株体内同化物的转运和积累,增强非洲菊植株抗逆性,特别是脱叶链霉菌FT05W具有在未来作为生物肥料解决非洲菊连作障碍的应用潜力。     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.