Effect of drying on conidial viability of Penicillium frequentans, a biological control agent against peach brown rot disease caused by Moniliniaspp

The effects of drying methods (freeze-, spray-, and fluid bed-drying) on viability of Penicillium frequentans conidia were compared. Viability, estimated by germination of fluid bed- and freeze-dried conidia, was similar to that of fresh conidia. Skimmed milk alone, or in combination with other protectants, was added to conidia before freeze-drying. After the freeze-drying process, all protectants used, except glycerol improved conidial viability. Freeze-dried P. frequentans conidia did not maintain viability after 30 days of storage at room temperature, while conidia dried by fluid bed-drying showed 28% viability following 180 days after drying. This work also demonstrated a relationship between conidial viability after 1 year of storage at room temperature, moisture content after fluid bed-drying and initial weight of sample. Conidial moisture contents must be reduced to 5-15% for optimal storage at room temperature. P. frequentans conidia dried by fluid bed-drying were as effective as fresh conidia in controlling brown rot of peaches.     

The effects of some storage conditions on viability of Lecanicillium lecanii conidia to whitefly (Homoptera: Trialeurodes vaporariorum)

A study on the survival of Lecanicillium lecanii conidia in storage at room temperature was carried out. Firstly, drying methods of conidia powder were compared. Vacuum-freeze drying (VFD) was more suitable for drying conidia as compared to vacuum drying (VD) at room temperature. Vacuum-freeze drying for 24-h resulted in a water content of 5.4%, and a viability, determined as germination of conidia in 2% glucose solution after16 h, was 90.3% and the infection in greenhouse whitefly, Trialeurodes vaporariorum was about 94.7% at a dose of 1×10⁸ conidia/mL. Secondly, the factors influencing viability of conidia stored at room temperature were evaluated in the laboratory. Temperature was the most critical factor influencing conidial storage stability, among the tested factors affecting survival of conidia stored at room temperature for 6 months. Both conidial germination and infection of hosts decreased with storage temperature increasing from 15 to 35°C, and at 35°C the survival of stored conidia for 6 months was near zero. The moisture content of the conidial powder was another major factor influencing viability of stored conidia at room temperature. Conidial powder dried to about 5% moisture content showed higher viability than non-dried conidial powder. For the carriers, clay and charcoal were more suitable for storage of L. lecanii conidia at room temperature. At a room temperature of 25°C, L. lecanii conidia which were dried to 5% water content and mixed with clay or charcoal could retain about 50% survival after 6 months' storage.     

Influence of Moisture Content on Temperature Tolerance and Storage of Metarhizium flavoviride Conidia in an Oil Formulation

Investigations of the temperature tolerances of Metarhizium flavoviride conidia, of different moisture contents, in an oil formulation were carried out. The conidia were dried to 5, 9, 15 or 19% moisture contents and exposed to a range of temperatures, -15, 12, 25, 38, 48 and 55 C. Viability declined due to high temperatures and high moisture contents. After 4 months of storage, the conidia were at over 80% viability in all but the 19% moisture content group of the 25 C treatment and all the samples stored at lower temperatures. In a second experiment, conidia dried to 4-5% moisture content showed greater temperature tolerance than conidia with higher moisture contents. Short-term storage under adverse conditions could be feasible with conidia dried to a suitable degree.     

Characterization of Growth and Conidia Production of Exserohilum monoceras on Different Substrates

On agar media the maximum conidia production of Exserohilum monoceras occurred on V-8 juice agar (VA) or centrifuged V-8 juice agar, whereas the optimal radial mycelial growth occurred on Czapek-Dox agar. The optimal temperatures for radial mycelial growth and conidia production were 28 and 27°C respectively. Light prohibited E. monoceras conidia production. The best sporulation occurred under continuous dark conditions. Echinochloa leaf decoction significantly increased conidia production on potato dextrose agar (PDA) and VA, and significantly increased germ tube length on PDA, lima bean agar and VA, but did not affect conidia germination. No conidia were produced in liquid media. Of 22 agricultural-based products evaluated as solid substrates, the most abundant sporulation (1.8 × 10⁶ conidia g^-1 of dry weight) occurred on corn leaves. The conidia production of E. monoceras on corn leaves was affected by incubation period, moisture content and substrate quantity. There were no differences in germination rate, germ tube length and virulence of conidia produced on agar media or corn leaves.     

Wheat Seed Colonized with Atoxigenic Aspergillus flavus: Characterization and Production of a Biopesticide for Aflatoxin Control

Biocontrol of aflatoxin contamination using atoxigenic Aspergillus flavus to competitively exclude aflatoxin-producing strains has previously been reported, and is currently in the third year of commercial-scale tests (treating 50-200 ha per annum). Wheat seed colonized with atoxigenic A. flavus has been used in the commercial trials. Requirements for production of this colonized wheat seed are described and the spore yield of wheat is compared to other substrates. The study suggests that the most cost-effective inoculum production would require colonization of wheat (106 conidia kg -1 of wheat seed) at 25% (w/w) moisture for 18 h at 31 C. To prevent fungal growth and associated wheat aggregation in storage, seed had to be dried below 15% (w/w) moisture, although a moisture content of 35% (w/w) did not reduce viability in sealed containers stored at 18-25 C over an 8-month period. The dry biopesticide had multi-year stability without refrigeration and withstood temperatures of 70 C for 20 min. Sporulation of the product occurred within 3 days at 31 C and 100% relative humidity with yields averaging 4.9 X 109 conidia g -1 by day 7.     

Long-term Storage of Metarhizium flavoviride Conidia in Oil Formulations for the Control of Locusts and Grasshoppers

Freshly harvested conidia of Metarhizium flavoviride (Gams & Rozsypal) were stored in two vegetable oils, groundnut or soya, or a mineral oil, Edelex. They were diluted with either Shellsol K or deodorized kerosene, and antioxidants were added to half of the vegetable oil formulations. Dried non-indicating silica gel was added to half of the formulations before storage at 8 or 17 C. Undried conidia, those without silica gel, lost viability rapidly, with germination dropping below 40% after 9 and 32 weeks at 17 and 8 C respectively. After 127 weeks (ca. 30 months) in storage, germination remained at over 60 and 80% for the dried formulations at 17 and 8 C respectively (after an unexplained drop in germination after 16-18 months in storage). Comparable figures for 160 weeks (ca. 37 months) were 47 and 68%. These figures represented germination after 24 h of incubation; after 48 h of incubation, germination was 79 and 89% from samples stored for 160 weeks at 17 and 8 C respectively. Representative formulations of the stored conidia were tested in bioassays against the desert locust Schistocerca gregaria (Forskal) up to 30 months into the experiment, and were found to have retained full virulence compared with freshly prepared formulations.     

Slow rehydration improves the recovery of dried bacterial populations.

Slow rehydration of bacteria from dried inoculant formulations provided higher viable counts than did rapid rehydration. Estimates were higher when clay and peat powder formulations of Rhizobium meliloti, Rhizobium leguminosarum biovar trifolii, and Pseudomonas putida, with water activities between 0.280 and 0.650, were slowly rehydrated to water activities of approximately 0.992 before continuing the dilution plating sequence. Rhizobium meliloti populations averaged 6.8 x 10(8) cfu/g and 1328 cfu/alfalfa seed greater when slowly rehydrated from bulk powder and preinoculated seeds, respectively. Bulk powder samples were slowly rehydrated to 0.992 water activity by the gradual addition of diluent, followed by a 10-min period for moisture equilibration. Preinoculated seed samples were placed in an environmental chamber at 24 degrees C with relative humidity greater than 80% for 1 h to allow moisture absorption. "Upshock," osmotic cellular stresses that occur during rehydration, was reduced when dried microbial formulations were slowly rehydrated and equilibrated before becoming fully hydrated in the dilution plating sequence. These procedures may also be applicable when estimating total viable bacterial populations from dried soil or other dry formulations.     

Effects of stabilizers on shelf-life of Epicoccum nigrum formulations and their relationship with biocontrol of postharvest brown rot by Monilinia of peaches

AIM: To find a formulation of Epicoccum nigrum conidia that maintains a high viability over time and which proves efficient to biocontrol peach rot caused by Monilinia spp. METHODS AND RESULTS: We tested the effect of stabilizers and desiccants on the shelf-life of Epicoccum nigrum conidia. Conidial samples were dried for 40 min at 40 degrees C in a fluidized bed-dryer to obtain moisture contents <15%. The toxicity of additives was tested by assaying production of conidia in fermentations and germinability of the produced conidia: 50% PEG300, 10%-5% KCl (stabilizers) and 95.24% Cl(2)Ca (desiccant) significantly (P = 0.05) reduced conidial germination. To enhance shelf-life of dried conidia, nontoxic stabilizers were added at the following different stages of the production-drying process: (i) to substrate contained in bags before production, (ii) to conidial centrifuge pellets obtained after production, before filtering and drying, (iii) to conidial centrifuge pellets obtained after production, before adding talc and drying, and (iv) to conidial centrifuge pellets obtained after production, before adding silica powder and drying. Conidial germinability was tested at 0, 180 and 365 days after storage at room temperature. Shelf-life of formulations retaining the highest viability were conidia produced with 1% KCl or 50% PEG 8000, conidia dried with 2.5% methylcellulose, and conidia dried with 1% KCl + silica powder. All these formulations improved the shelf-life of E. nigrum conidia and significantly reduced brown rot on peaches. CONCLUSIONS: Our results show that additives improve the shelf-life of E. nigrum and assist controlling brown rot on peaches. SIGNIFICANCE AND IMPACT OF THE STUDY: New improved formulations of a biocontrol agent have been obtained which will improve the control of Monilinia on peach.     

Production ofBeauveria bassiana [Deuteromycotina: Hyphomycetes] in different liquid media and subsequent conidiation of dry mycelium

Mycelium ofBeauveria bassiana can be grown in liquid culture, filtered, and the mycelium dried. After rehydration the mycelium sporulates. Two carbohydrate sources (sucrose and maltose), and one nitrogen/vitamin source (yeast extract) were tested for mycelium growth and subsequent conidial production. Maximum mycelium growth (12.31 mg/ml), in liquid culture, was in the sucrose (3.5%)/yeast extract (3.5%) medium, but mycelium from a maltose (2%)/yeast extract (0.75%) medium produced the maximum of 4.62×10⁶ conidia/mg dry mycelium after incubation in moist Petri dishes. Using the data on mycelium yield (in liquid culture) and conidial production (by dry mycelium) it is calculated that the sucrose (3.5%)/yeast extract (3.5%) and the maltose (2%)/yeast extract (0.75%) media produce most conidia per media volume (an equivalent of 3.52–3.72×10⁷ conidia/ml).      

Drying of Epicoccum nigrum conidia for obtaining a shelf-stable biological product against brown rot disease

AIMS: The effects of freeze-drying, spray-drying and fluidized bed-drying on survival of Epicoccum nigrum conidia were compared. METHODS AND RESULTS: Viability of E. nigrum conidia (estimated by measuring its germination) was 100% after fluidized bed-drying and freeze-drying, but it was determined that skimmed milk must be added in the case of freeze-drying conidia. Addition of other protectants (Tween-20, peptone, sucrose, glucose, starch and peptone + starch) to skimmed milk before freeze-drying did not improve the conidial viability which was obtained with skimmed milk alone. Glycerol had a negative effect on the lyophilization of E. nigrum conidia. Epicoccum nigrum conidia freeze-dried with skimmed milk, or fluidized bed-dried alone maintained an initial viability for 30 and 90 days, respectively, for storage at room temperature. Epicoccum nigrum conidial viability after spray-drying was lower than 10%. CONCLUSIONS: The best method to dry E. nigrum conidia was fluidized bed-drying. Conidia without protectants dried by this method had 100% viability and survived for 90 days at room temperature. SIGNIFICANCE AND IMPACT OF STUDY: This paper deals with methods for the potential formulation of a biocontrol agent which is being tested for eventual commercialization.     

Debilitation in conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and implication with respect to viability determinations and mycopesticide quality assessments

Germination of Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) conidia determined from a fast-rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) by holding under high humidity conditions prior to aqueous suspension. Differences in viability estimates obtained using the FR vs. SR protocols increased markedly after conidia were exposed to various stress factors in storage (high a_w, temperature, and O₂ concentrations), with the SR protocol producing higher estimates of viability in all cases. After Bb conidia were stored under moist conditions for 21 days at 25 °C, the SR estimate of viability was >21% greater than the FR estimate. In jars flushed with different O₂ concentrations and stored at 50 °C for 34 days, proportional differences between protocols varied, depending on water activity, from 18-44% in jars flushed with 0% O₂ (100% N₂) to as high as 63-93% when treated with 21-22% O₂. For conidia stored over a broad range of moderate to high temperatures in the absence of O₂, SR-FR differences were ?9% at 25-40 °C but 30% at 50 °C. Germination of stressed Bb and Ma conidia increased substantially when incubation time on the germination substrate was increased from 24 to 72 h, whereas germination of non-stressed conidia showed little change. Conidia debilitated by stress were characterized by hypersensitivity to lethal imbibitional damage (damage that is mitigated by slow rehydration) and slow germination. Viability protocols that may provide more reliable assessments of overall mycopesticide quality are discussed.     

Viability of Stored Conidia of Metarhizium flavoviride Gams and Rozsypal, Produced under Differing Culture Regimes and Stored with Clays

Conidia of Metarhizium flavoviride were cultured under a range of conditions and stored , with or without clays and silica gel , as powder or in oil . Fungal biomass was produced in shake flasks in liquid media containing nutrients before being added to sterilized rice for conidial production . Conidia pre - cultured under a low C:N ratio , or dried over a 9 - day period compared with 2 days before being placed in storage , showed greatest loss of viability . Conidia stored at 10 o C survived better than those stored at + 30 o C . A range of clays had no significant effect on storage of dried conidia but montmorillonite K10 clay was often harmful in terms of conidial viability . Conidia survived better when stored as dry powder than when stored in a mixture of mineral and vegetable oils . The addition of silica gel proved beneficial even when dried conidia were being stored .     

Effects of Moisture Content and Temperature on Storage of Metarhizium flavoviride Conidia

The effects of moisture content and temperature on the medium-term (3-4 months) storage of conidia of Metarhizium flavoviride were investigated. Conidia harvested after 24 days of culturing on rice showed greater tolerance to long storage than conidia from 12-day cultures. The moisture content of the conidia was of greatest importance; at harvest from the culture, conidial moisture contents could be 40%, while the optimal moisture content for storage was found to be 4-5%. Dried conidia stored in oil benefited from the addition of dried silica gel, as did conidia stored as powder. A range of mineral oils proved satisfactory for storage, and when dried silica gel was added to suspensions, germination levels were 79.8% after 105 days at 28-32 C. Dried conidia stored in oil maintained germination levels of up to 96 and 85% after 80 days at 10-14 C and 28-32 C respectively. Dried conidia stored as powder retained germination levels of 95% at 10-14 C, but only up to 27% at 28-32 C. In another experiment, dried conidia maintained greater than 90% germination over 128 days, with or without silica gel at 10 - 14 C or -15 - -18 C.     

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.     

Effects of temperature and relative humidity on sporulation of Metarhizium anisopliae var. acridum in mycosed cadavers of Schistocerca gregaria.

The effects of relative humidity (RH) and temperature on the sporulation of Metarhizium anisopliae var. acridum on mycosed cadavers of desert locust, Schistocerca gregaria, were assessed in the laboratory. Quantitative assessments of conidial production over 10 days under constant conditions showed that sporulation was optimized at RH > 96% and at temperatures between 20 and 30 degrees C. Under both these conditions >10(9) conidia/cadaver were produced. At 25 degrees C, conidial yield was maximized under conditions in which cadavers remained in contact with damp substrate. Relatively little sporulation occurred at 15 degrees C (< 3 x 10(7) conidia/cadaver) and 40 degrees C (< 4 x 10(6) conidia/cadaver) and no sporulation occurred at 10 or 45 degrees C. Following incubation, conidial yield was closely related to the water content of locust cadavers. In separate tests, locust cadavers were incubated for 10 days under diurnally fluctuating temperature and RH that comprised favorable (25 degrees C/100% RH) alternating with unfavorable (40 degrees C/80% RH) conditions for sporulation. In this case, fewer conidia were produced compared with cadavers that were incubated under the favorable conditions for an equal period cumulatively but were not periodically exposed to unfavorable conditions. However, this reduced sporulation observed with the fluctuating condition was not observed when cadavers were similarly incubated under favorable/unfavorable conditions of temperature but were not periodically exposed to the low RH condition. This result implies that sporulation is a dynamic process, dependent not only on periodic exposure to favorable RH but also on the interrelation of this with low RH. Associated tests and the monitoring of changes in cadaver weights imply that the mechanism driving the reduced sporulation under fluctuating RH is the net water balance of cadavers, i.e. the cumulative ability of the fungus/cadaver to adsorb water necessary for sporulation at high RH is restricted by water loss associated with intermittent exposure to a low RH. The duration of daily exposure to high humidity appears to be a crucial constraint to the recycling ability of M. anisopliae var. acridum.     

Effects of Temperature, Humidity and Inoculation Method on Susceptibility of Schistocerca gregaria to Metarhizium flavoviride

The effects of temperature on conidial germination and susceptibility of adults of the desert locust , Schistocerca gregaria, to four isolates of Metarhizium flavoviride were determined . In addition , the effects of inoculation method (topical or spray) , spore carrier (oil or water) and ambient relative humidity (RH) on susceptibility of the locust to the most thermotolerant isolate (Mf324) were investigated . There were differences among the isolates in the effects of temperature on germination of conidia after a 24 - h incubation period . Over 90% of conidia of all isolates germinated after 24 h at 30 o C . In contrast , at 40 o C , none of the isolates germinated for up to 72 h . However , there were differences in germination between the isolates at 35 o C . Locust mortality and disease progression were significantly affected by temperature . At both 25 and 30 o C , all isolates induced 98 - 100% mortality within 8 days; however , there were differences between isolates at 35 o C . None of the isolates caused significant mortality at 40 o C . Humidity and inoculation method had no influence on levels of mortaility in fungus treated locusts . In contrast , carrier type significantly affected cumulative mortality . Topical oil treatment resulted in higher overall mycosis than the three other treatments . Control mortality on the other hand was strongly affected by inoculation method and to a lesser extent by humidity and carrier . In these conditions , application of oil by spray was generally toxic at all humidities whereas topical application of water was most toxic at near saturation . The results of these laboratory studies demonstrate the importance of strain selection , formulation and application method in the development of a microbial control agent against the desert locust . Low RH should not impede use of this fungus under dry conditions .     

不同温度、气体微环境对红托竹荪干品储藏品质的影响

本研究针对红托竹荪干品在储藏过程中易发生褐变、降低商品性问题,探究了不同储藏条件(温度、气体微环境)对红托竹荪干品储藏品质的影响。以红托竹荪干品为原材料,考察了在气体微环境(空气、N₂、CO₂和脱氧)和不同储藏温度(5、25和45 ℃)下红托竹荪干品储藏品质的动态变化。在60 d的储藏期内,所有样品的褐变指数、剪切力、多酚氧化酶、过氧化氢酶、总酚、还原糖和5-羟甲基糠醛含量均增加,游离氨基酸、白度值、复水比均降低。与25、45 ℃相比,以上指标在5 ℃条件下均表现最优,5 ℃储藏条件下呈味氨基酸和挥发性成分指标更接近于0 d;在不同气体微环境比较下,CO₂储藏环境下干品品质保持最好,通过综合评分得出5 ℃低温结合CO₂充气条件下干品品质最优,其次为N₂结合5 ℃低温。结合经济成本,5 ℃低温结合CO₂或N₂充气可以作为红托竹荪干品延长货架期的推荐储藏技术。     

Survival of the postharvest biocontrol yeast Candida sake CPA-1 after dehydration by spray-drying

Spray drying was evaluated as a dehydrating method to preserve the postharvest biocontrol agent Candida sake CPA-1. The effect of drying temperature, carrier, growth and rehydrating medium on the survival of the yeast was studied. Outlet temperature had more influence on the death of the cells than inlet temperature, and survival decreased with increasing temperature. Spray drying at an inlet temperature of 150°C was optimum in terms of viability, powder recovery and moisture content of the product. Use of 10% (v/v) skimmed milk as a carrier gave the highest survival and percentage of powder recovery (34-47%). Rich rehydration media were found to be better than water or phosphate buffer, with slight differences on survival. Spray-dried cells were less effective than fresh ones in controlling Penicillium expansum rot on apples. Spray drying of C. sake was not a good dehydration method as it gave low cell survival, poor recovery of product, and low efficacy.     

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.     

Effects of chemical and botanical insecticides used for locust control on Metarhizium anisopliae var. acridum conidia after short- to medium-term storage at 30°C

The short- to medium-term viability and growth of Metarhizium anisopliae var. acridum conidia were investigated when combined with six insecticides, at three different concentrations. All of the insecticides used in this study were suitable for immediate spraying with M. anisopliae var. acridum conidia except for fenitrothion. Fipronil, teflubenzuron, and fenitrothion formulations significantly reduced conidial viability over time. The 10% teflubenzuron treatment caused loss of viability relatively quickly with 9.9% germination after 28 days. Mycelial growth was affected by all the treatments except fenitrothion.