New use of broomcorn millets for production of granular cultures of aphid-pathogenic fungus Pandora neoaphidis for high sporulation potential and infectivity to Myzus persicae

Glutinous broomcorn millets from the crop Panicum miliaceum were first used as substrate to produce granular cultures of Pandora neoaphidis, an obligate fungal pathogen specific to aphids. Carrying a water content of 36.5% after being steamed in a regular autoclaving procedure, millet grains of each 15 g (dry weight) in a 100-ml flask were mixed with 3 ml modified Sabouraud dextrose broth containing half a mashed colony of P. neoaphidis grown on egg yolk milk agar and then incubated at 20 degrees C and a light/dark cycle of 12 h/12 h for 21 days. Based on individually monitoring conidial production potential of 20 millet grains sampled from an arbitrarily taken flask at 3-day intervals, the millet cultures incubated for 6-15 days were capable of producing 16.8-23.4 x 10(4) conidia per millet grain with conidial ejection lasting for up to 6 days. The cultured millet grains individually produced significantly more conidia than apterous adults of Myzus persicae killed by P. neoaphidis (8.4 x 10(4) conidia per cadaver) and sporulated twice longer. The modeling of time-dose-mortality data from bioassays on M. persicae apterae exposed to conidial showers from the cultured millet grains and the mycelial mats produced in liquid culture resulted in similar estimates of LC(50) (millets: 21.4, 7.3, and 4.9 conidia mm(-2) on days 5-7 after exposure; mycelial mats: 22.1, 10.6, and 7.7 conidia mm(-2)) although the LT(50) estimated at a given conidial concentration was slightly smaller for the millet cultures than for the mycelial mats. This indicates that the millet grains cultured with P. neoaphidis produced conidia as infective as or slightly more infective to M. persicae than those from the mycelial mats. Based on the sporulation potential, infectivity, and ease and cost of the millet cultures, the method developed in this study highly improved in vitro cultures of P. neoaphidis and may adapt to culturing other entomophthoralean fungi for microbial control of insect pests.     

Effects of long-term storage at -14 degrees C on the survival of Neozygites fresenii (Entomophthorales: Neozygitaceae) in cotton aphids (Homoptera: Aphididae)

Neozygites fresenii-infected Aphis gossypii cadavers, containing dormant hyphal bodies of N. fresenii, were stored in 4 ml glass vials at -14 degrees C in a standard consumer-type refrigerator/freezer for 1, 21, 30, 43, 51, and 68 months to determine the effect of storage on fungal survival. When the cadavers were removed from the freezer and placed in 25+/-1 degrees C, 100% relative humidity, and 12:12 (L:D) conditions, N. fresenii survival, as shown by fungal sporulation from the cadavers, was high at all storage periods. The average percentage of cadavers from which the fungus sporulated were 93, 47, 100, 100, 80, and 60% from 1, 21, 30, 43, 51, and 68 months storage periods, respectively. The number of primary conidia discharged from each sporulating cadaver was estimated using a scale of 1 (low, ca. 1000 primary conidia), 2 (medium, ca. 2000 primary conidia) and 3 (high, ca. 3000 primary conidia). The median scores for the number of primary conidia produced per sporulating cadaver were 3, 2, 3, 3, 2.5, and 1 for 1, 21, 30, 43, 51, and 68 months, respectively. Therefore, except for the longest storage period, most cadavers produced medium to high numbers of primary conidia. Mean germination of primary conidia produced from N. fresenii-infected-aphid cadavers from each time period varied significantly from 66.3 to 86.1% in the 21 and 43 months categories, respectively. Infectivity of capilliconidia, produced from frozen N. fresenii, to live healthy cotton aphids varied significantly from 16.7 to 68.7% from cadavers stored 68 months and 1 month, respectively. Overall N. fresenii survived well in dried frozen cotton aphid cadavers for up to 6 years with little reduction in sporulation, numbers of spores produced, germination of primary conidia, or infectivity.     

Granulation of subtilisin by internal gelation of alginate microspheres for application in detergent formulation

Subtilisin was encapsulated within impact-resistant alginate granules produced by emulsification, internal gelation, and acetone extractive drying. The mechanical and controlled release properties of the granules were modified by adding to the alginate varying levels of formulation excipients, including titanium dioxide, polyvinyl alcohol, microcrystalline cellulose, starch and sucrose. Optimum protease activity and mass yields of 83 and 88%, respectively (mg active subtilisin/g granules), occurred for granules formulated with 3% alginate, 10% starch, 10% titanium dioxide, and 3% subtilisin. Mass losses occurred primarily during the gelation step. Maximum encapsulation efficiency is achieved by using higher molecular weight alginate, increasing the alginate concentration, and carefully controlling process temperature and pH. The strongest granules were obtained at the higher concentrations of medium-G or high-G alginate, while fastest granule dissolution was achieved when a lower concentration of alginate was used in combination with polyvinyl alcohol or microcrystalline cellulose as dispersants. Mechanical properties of alginate granules were found to be unaffected by the different cations employed in matrix gel formation.     

A novel computerised image analysis method for the measurement of production of conidia from the aphid pathogenic fungus Erynia neoaphidis

A semi-automated method has been developed for the quantification and measurement of conidia discharged by the aphid pathogen Erynia neoaphidis. This was used to compare conidiation by E. neoaphidis-mycosed pea aphid cadavers, mycelial plugs cut from agar plates, mycelial pellets from shake flasks and by mycelial pellets from different phases of liquid batch fermenter culture. Aphid cadavers discharged significantly more and significantly smaller conidia than plugs or pellets. The volume of conidia discharged was stable over the period of discharge (80 h), but more detailed analysis of the size frequency distribution showed that more very small and very large conidia were discharged after 5 h incubation than after 75 h incubation. Biomass harvested at the end of the exponential growth phase in batch fermenter culture produced significantly more conidia than biomass from any other growth phase. The implications of these findings for the development of production and formulation processes for E. neoaphidis as a biological control agent are discussed.     

Use of a Novel Sporulation Monitor to Quantify the EVects of Formulation and Storage on Conidiation by Dried Mycelia of the Entomopathogenic Fungus Zoophthora radicans

A novel piece of equipment, the sporulation monitor, is described for the comparison of conidia production from mycelia receiving diVerent formulation and storage treatments. This equipment was used to compare the viability of Zoophthora radicans mycelial samples treated either with 10% maltose solution or with distilled water before drying and storage for 0-12 weeks at 4oC. Freshly dried maltose-treated mycelial mat samples produced significantly more conidia for a significantly longer time than distilled water-treated mat samples of the same age. Very few conidia were produced from mats in either treatment after storage for 4 weeks or longer. There were great diVerences in conidia production from mycelial mat samples produced in diVerent fermenter runs. These results are discussed in relation to the potential for the use of dried mycelia in biological control programmes.     

环境因子对努利虫疠霉产孢和孢子萌发的影响

探讨努利虫疠霉Pandoranouryi(Remaudi埁re&Hennebert)H櫣mber在田间蚜虫种群中发生与流行的规律,研究外界环境因子对感菌虫尸产孢和孢子萌发的影响。结果表明,处于水琼脂培养平板上感染努利虫疠霉的桃蚜虫尸在8~25℃的温度范围内均能产生大量的初生分生孢子,在30℃下,仅弹射极少量孢子。8℃下,孢子弹射可以持续120h,当温度高于15℃,大部分的孢子会在48h内完成弹射。相对湿度小于95%,虫尸停止产孢。20℃下,光照条件不会影响虫尸弹射孢子的总量。在8℃和30℃时,24h后处于水琼脂培养平板上的孢子萌发率分别为45.23%和61.74%,显著低于15~25℃温度范围内的孢子萌发率(95%以上)。处于叶片上的真菌孢子,当相对湿度大于74%时出现萌发,但在盖玻片的表面,当湿度低于100%时未发现孢子萌发。     

Use of chitin to improve a Beauveria bassiana alginate-pellet formulation

A study was undertaken to evaluate optimum concentrations of chitin in sodium alginate pellet formulations to enhance conidia production. Chitin concentrations tested were 0, 0.5, 1, 2, 3 and 4% (w/v), with (2%, w/v) or without wheat bran. The different chitin-wheat bran pellet combinations were prepared with Beauveria bassiana isolate Qu-B306 at 10⁸ conidia mL^-1. After 21 days of incubation in a humid chamber at 28°C, conidia production was assessed. Improvements up to three times the initial conidia number were achieved using 2% chitin and 2% wheat bran. Higher levels of chitin decreased the number of conidia per pellet. For all chitin concentrations, conidia number increased with the addition of wheat bran (P≤0.05). Contamination by saprophytic fungi was reduced by the incorporation of chitin in the pellet formulation.     

飞虱虫疠霉液培菌丝的凝胶化及其杀蚜活性*

用丙烯酰胺淀粉凝胶包埋飞虱虫疠霉(Pandora delphacis)液培菌丝,制成含营养和不含营养的虫霉凝胶,并测试其产孢能力及对桃蚜(Myzus persicae)的侵染力。经持续9 d产孢观察,相同菌丝量条件下,含营养的虫霉凝胶的累计产孢量(1910.9 个/mm2）为不含营养的虫霉凝胶的3.14倍（608.5个/mm2）和对照（纯液培菌丝）的4.69倍（387.5个/mm2）。用虫霉凝胶的“孢子浴”接种桃蚜居群（成蚜与不同龄期若蚜的混合）,在2.23个/mm2的剂量下,接种后第7 d的死亡率达82.3%。结果表明,丙烯酰胺淀粉凝胶作为载体材料,与飞虱虫疠霉的基本生物学特性相容,对虫霉的剂型化具有重要意义     

Response of the entomopathogenic fungus Pandora neoaphidis to aphid-induced plant volatiles

We used a model plant-aphid system to investigate whether the aphid-specific entomopathogenic fungus Pandora neoaphidis responds to aphid-induced defence by the broad-bean plant, Vicia faba. Laboratory experiments indicated that neither in vivo sporulation, conidia size nor the in vitro growth of P. neoaphidis was affected by Acyrthosiphon pisum-induced V. faba volatiles. The proportion of conidia germinating on A. pisum feeding on previously damaged plants was significantly greater than on aphids feeding on undamaged plants, suggesting a direct functional effect of the plant volatiles on the fungus. However, there were no significant differences in the infectivity of P. neoaphidis towards A. pisum feeding on either undamaged V. faba plants or plants previously infested with A. pisum. Therefore, these results provide no evidence to suggest that P. neoaphidis contributes to plant indirect defence strategies.     

The effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of Valdensinia heterodoxa

Valdensinia heterodoxa (Sclerotiniacae) is a potential fungal bioherbicide for control of salal (Gaultheria shallon). The effect of culture media, substrates and relative humidity (RH) on growth, sporulation and conidial discharge of V. heterodoxa was determined for two isolates PFC2761 and PFC3027 in vitro. Culture media significantly affected the growth, sporulation, and conidial discharge of V. heterodoxa. Of eight agar media used, colony radial growth was optimal on salal oatmeal agar and salal potato dextrose agar for isolates PFC2761 and PFC3027, respectively; whereas sporulation was at an optimum on salal oatmeal agar for both isolates. Of the eight liquid media tested, mycelial production was highest on wheat bran–salal–potato dextrose broth. Growth on solid substrates greatly stimulated sporulation and conidial discharge of V. heterodoxa. Of the 12 solid substrates used, the greatest numbers of discharged conidia were observed from wheat bran and wheat bran–salal within 14 d of sporulation. Sporulation on solid substrates continued for 42 d. RH significantly affected the sporulation and conidial discharge for both isolates across all solid substrates tested. No conidia were produced or discharged below 93 % RH on wheat bran–salal and millet. With an increase of the RH from 93 to 97 %, sporulation and the number of discharged conidia increased significantly for both isolates on wheat bran–salal, but not on millet.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.     

Improved Medium for Sporulation of Clostridium perfringens

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.     

Effects of hydrophilic and hydrophobic kaolin-based particle films on pea aphid (Homoptera: Aphididae) and its entomopathogen Pandora neoaphidis (Entomophthorales: Entomophthoraceae)

Hydrophobic and hydrophilic kaolin-based particle films are effective for control of insect pests in certain agricultural crops. How these products interact with potential biological control agents is not well documented. This study was conducted to evaluate the effects of the hydrophobic (M96-018) and hydrophilic (Surround WP) kaolin-based particle films (Engelhard Corporation, Iselin, NJ) on pea aphid, Acyrthosiphon pisum (Harris), on peas (Pisum spp.), and on the fungal aphid pathogen Pandora neoaphidis (Remaudière and Hennebert) Humber. Over two field seasons (2001 and 2002) in northern Idaho, applications of M96-018 significantly reduced the rate of pea aphid increase on pea, but Surround WP, tested only in 2001, did not reduce aphid population growth rate. Neither particle film treatment was as effective as a standard application of esfenvalerate (DuPont Asana). In the laboratory, particle films suppressed pea aphid populations by up to 30%. M96-018 seemed to have some repellent activity based on aphid distributions after treating plants. When applied along with P. neoaphidis conidia, M96-018 but not Surround WP caused higher percentage of infection mortality of pea aphids by P. neoaphidis than occurred on controls treated only with P. neoaphidis conidia. P. neoaphidis conidia deposited on glass slides coated with M96-018, produced more germ tubes and secondary conidia than those deposited on untreated glass slides or slides treated with Surround WP. This result suggests that greater infection of pea aphids on plants treated with M96-018 is in part a result of a direct enhancement of fungal germination by the particle film.     

新蚜虫疠霉连续液体培养的菌丝生物量、产孢量及杀蚜毒力

继代培养常被疑为虫霉菌种毒力下降或某些生物学性状发生改变的原因之一。从研究新蚜虫疠霉 (Pandoraneoaphidis)固体平板菌落的液体培养获得的初始菌液出发 ,连续 6次继代液培 ,测定了其在萨氏营养液中继代培养生产的菌丝生物量、产孢量及其对桃蚜 (Myzuspersicae)的毒力。在初始菌液的生物量 8 8mg mL和产孢量 7 2× 1 0 5孢子 mg的条件下 ,以三种转接比 (种液 营养液 ,v v)连续 6次继代培养 ,在 1 2 0转接比下的菌丝生物量和产孢量分别为 6 4～ 1 0 0mg mL和 7 3× 1 0 5～ 1 0 8× 1 0 5孢子 mg,在 2 2 0下为 5 7～ 8 5mg mL和 1 0 0× 1 0 5～ 1 2 1× 1 0 5孢子 mg ,在 4 2 0下为 5 5～ 1 0 9mg mL和 6 4× 1 0 5～ 1 0 9× 1 0 5孢子 mg。方差分析表明 ,继代培养对生物量和产孢量均无显著影响 (P <0 0 5)。用初始菌液和 1 2 0转接比下继代培养的菌液制备而成的接种体对桃蚜进行两组毒力测定 ,每一组测定的接种…     

Effect of relative humidity and origin of isolates of Neozygites tanajoae (Zygomycetes: Entomophthorales) on production of conidia from cassava green mite, Mononychellus tanajoa (Acari: Tetranychidae), cadavers

Neozygites tanajoae is a very specialized fungus pathogenic to the cassava green mite (CGM), Mononychellus tanajoa, an important cassava pest introduced to Africa from the Neotropics. Conidial discharge from cadavers of CGM that died from infections with 14 isolates of N. tanajoae collected from diverse climates of Brazil was quantified to help select potential candidate strains for introduction to Africa. Studies aimed to identify isolates with lower requirements for relative humidity for sporulation and isolates that discharged more conidia during short periods of moisture. At 96 ± 0.5% RH, production of conidia was variable and even isolates from the Brazilian semi-arid region, e.g., Petrolina and Itaberaba, produced few conidia. Significant differences in the numbers of conidia produced by diverse Brazilian isolates were observed after 6, 9 and 12 h at 100% RH. At 100% RH, production of primary conidia increased considerably from an average of 57 ± 4 conidia at 6 h to 509 ± 37 conidia at 12 h. The isolate sporulating least (BIN21) discharged only 45.7% of the number of conidia produced by isolate BIN1, one of the isolates producing the most spores. Results from this study demonstrate that differences in production of conidia among isolates should be considered when selecting Neozygites isolates for new biological control introductions.     

Factors affecting sporulation and germination of Pandora nouryi (Entomophthorales: Entomophthoraecae), a pathogen of Myzus persicae (Homoptera: Aphididae)

Pandora nouryi discharged large numbers of primary conidia between 8 and 25°C from cadavers on the surface of water-agar. At 8°C conidial discharge lasted for 120 h, but most conidia were produced within 48 h when temperature was >15°C. Saturated humidity alone was not enough to allow for sporulation to occur freely and where RH?<?95%, no conidia were discharged. Light did not affect the pattern of conidial production nor the total number of conidia. Germination percentages of conidia on the surface of water-agar were 40 and 66% at 8 and 30°C, respectively, and were significantly lower than that at 15–25°C where germination was >95%. Conidia on leaves germinated well when RH?>?74%, while no germination occurred when RH?<?100% on cover slips. All eight insecticides tested entirely inhibited conidial germination at recommended doses (R), in particular, both the organophosphorus pesticides Lorsben (chlorpyrifos) and the organochlorine pesticides Thiodan (endosulfan) completely inhibited conidial germination even at 0.2R dose.     

Sporulation,storage and infectivity of obligate aphid pathogen Pandora nouryi grown on novel granules of broomcorn millet and polymer gel

Aims: Producing granular cultures of obligate aphid pathogen Pandora nouryi for improved sporulation and storage. Methods and Results: Small millet–gel granules were made of the mixtures of 80–95% millet powder with 5–20% polymer gel (polyacrylamide, polyacrylate or acrylate‐acrylamide copolymer) and inoculated with mycelia at 30 mg biomass g^?1 dry granules plus 87·5% water, followed by static incubation at 20°C for 4–12 days. The fungus grew well on 12 preparations but best on that including 10% copolymer. An 8‐day culture of this preparation discharged maximally 58·5 × 10⁴ conidia mg^?1 granule at 100% RH and was capable of ejecting conidia at the nonsaturated regimes of 86–97% RH. During storage at 6°C, granular cultures with >85% water content had twofold longevity (120 days) and half‐decline period (34–36 days) of those stored at room temperature. The steadily high water content preserved the cultures better than that decreasing at 6°C. However, conidia from 70‐day‐stored granules were less infective to Myzus persicae nymphs than those from fresh ones based on their LC₅₀s. Conclusions: The millet–gel granules had higher sporulation capacity than reported Pandora cultures and a capability of spore discharge at nonsaturated humidity. Significance and Impact of the Study: The granular cultures are more useful for aphid control.     

Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

Azotobacter vinelandii lacking the Na+-NQR activity: a potential source for producing alginates with improved properties and at high yield

The mutant ATCN4 strain of Azotobacter vinelandii, which lacks the Na(+)-NQR activity and results in an alginate overproduction (highly mucoid phenotype), was cultured in shake flasks in minimal and rich medium, and the chemical composition and rheological properties of the alginate were determined. Mutant ATCN4 exhibited a high efficiency for sucrose conversion to alginate and PHB accumulation, reaching yields that were 3.6- and 1.6-fold higher than those obtained from the wildtype cultures in minimal medium (Burk's sucrose, BS). The alginate produced by ATCN4 in the minimal medium had a high degree of acetylation (≥4 %) and a low G/M ratio (=2) with respect to the polymer synthesised in the rich medium (BS with yeast extract) (degree of acetylation = 0 % and G/M ratio of 4.5). The alginate produced in the minimal medium exhibited a pronounced pseudoplastic behaviour and a higher G* module in comparison to that observed in the alginate obtained in the cultures using a rich medium. The ATCN4 mutant culture in the minimal medium promoted the synthesis of a polymer of improved rheological quality in terms of its mechanical properties. These characteristics make this mutant a valuable source for producing alginates with improved or special properties.