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.     

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.     

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.     

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.     

Tolypocladium cylindrosporum (Deuteromycotina:Moniliales), a fungal pathogen of the mosquito Aedes australis

A laboratory fermenter was used to produce up to 12 l of infective Tolypocladium cylindrosporum blastoconidia in Sabouraud dextrose broth. Two media derived from coconuts were also demonstrated as suitable alternative systems for the production of viable blastoconidia. T. cylindrosporum conidia when dried at 37 degrees C and stored at 4 degrees C retained their viability for 10 months, but, when stored at 25 degrees C, the conidia lost viability after 2 months and blastoconidia did not survive the drying process. Distilled water suspensions were a simple, economic technique for the long-term storage of spores at both 4 and 25 degrees C. The adsorption of conidia onto silica gel crystals was a very suitable technique for the storage of stock culture material at 4 degrees C. The virulence, production and storage capabilities of both spore types were examined.     

Survival of Conidia of Clonostachys rosea on Stored Barley Seeds and Their Biocontrol Efficacy Against Seed-borne Bipolaris sorokiniana

Survival and biocontrol activity of Clonostachys rosea (isolate IK726) conidia during storage on barley seeds were investigated. The initial density of colony forming conidia on seed was 4 &#50 10 3 to 9 &#50 10 4 colony forming units (cfu)/seed. After 5 months storage at 4&#176;C, the density decreased by less than one order of magnitude and the biocontrol efficacy against seedling blight caused by seed-borne Bipolaris sorokiniana was maintained at a significantly high level ( > 80% disease reduction) for > 5 months. Conidial survival on seeds stored at 20&#176;C declined more rapidly than at 4&#176;C, and biocontrol efficacy was significantly reduced after 3-5 months. However, conidia produced on solid media over 20 days survived better than conidia produced in liquid culture and conidia from solid media produced over 12 days. In contrast, when seeds treated with conidia were packed with silica gel and stored at 20&#176;C, the cfu density decreased by less than one order of magnitude after 5 months and the biocontrol efficacy was still high after 6 months. A dose-response curve revealed that 103 cfu/seed were needed for 80% control of seedling blight. Similar control was obtained in storage experiments when approximately 103 cfu/seed were recovered from seed, indicating that conidia which survived also retained a high ability to control disease.     

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.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

Delayed Germination of Beauveria bassiana Conidia after Prolonged Storage at Low, Above-freezing Temperatures

Powder formulations were prepared with conidia of the entomopathogenic fungus Beauveria bassiana strain 447 originally isolated in Brazil from the red imported fire ant, Solenopsis invicta. The carrier materials used in formulations included talc hydrous magnesium silicate , silica gel, powdered rice and cornstarch. The formulations were stored in plastic containers under three conditions: a ambient temperature 15-38 C ; b refrigerator 6 2 C ; and c freezer 10- 7 C . Formulations stored under ambient temperature conditions completely lost viability after 1-8 months. Unformulated conidia stored under ambient conditions were totally unviable after just 2 months. All formulations stored under refrigerator and freezer conditions maintained 100 viability for 7 years. The virulences of the conidial preparations against adult workers of the fire ant, S. saevissima, and the sugarcane borer, Diatraea saccharalis, were higher for fresh conidia or conidia stored in the freezer than for conidia stored in the refrigerator. After 30 months of storage, the unformulated conidia and the formulations stored in the refrigerator showed slow germination and had low virulence. There was a strong correlation between the rate of germination and the virulence toward D. saccharalis, but not toward S. saevissima.     

Fluctuating Temperature and the Longevity of Conidia of Metarhizium flavoviride in Storage

Conidia of Metarhizium flavoviride were hermetically stored at 13.7% moisture content with four constant (20, 30, 40 and 50 C) and six fluctuating temperature regimes (20:30, 20:40, 20:50, 30:40, 30:50 and 40 C:50 C, all 24 h:24 h) for up to 119 days. Survival of conidia stored at both constant and fluctuating temperatures conformed to cumulative negative normal distributions and all 10 survival curves could be constrained to a common origin. The effect of constant storage temperature on conidia longevity was quantified satisfactorily by a negative curvilinear semi-logarithmic relation developed previously, in which the estimates of the constants CH and CQ were 0.0176 (SE 0.0013) and 0.000703 (SE 0.000019), respectively. The fitted relation at constant temperatures showed that Q10 for loss in conidia viability increased the warmer the temperature regime. The effect of the cooler temperature of each fluctuating temperature regime on conidia longevity was small, since the effective temperature of each regime for loss in conidia viability was always much warmer than the mean. Conidia were also stored in two further regimes: at 30 C for 21 or 35 days before transfer to 50 C. The standard deviations of the conidia survival curves at 50 C were unaffected by the duration of previous storage at 30 C. Thus change in temperature per se had no effect on conidia survival: conidia survival curve slopes were solely dependent upon the contemporary storage environment. Approaches are developed in order to predict loss of conidia viability in fluctuating temperature storage environments, and the predictions compared against independent observations.     

Studies of the survival ofFusarium oxysporum conidia produced by submerged culture

Summary To study the survival of conidia ofFusarium oxysporum produced by submerged culture on malt extract, a harvesting process and different packaging and storage conditions have been tested. Conidia dried with talc and stored at +4°C preserve their viability after about 4.5 months.     

不同含水量和温度下贮存中球孢白僵菌分生孢子活力与内贮营养的衰变

将球孢白僵菌(Beauveriabassiana)BBSG8702的未干燥孢子粉(含水量58.9±1.6%)和真空冷冻干燥孢子粉(含水量7.4±0.9%)置于4℃和20℃下贮存1个月,每隔5d取样测定活孢率和孢子内贮总糖和蛋白含量,发现含水量和贮存温度交互影响孢子的活力以及内贮总糖和蛋白质的代谢水平,各组合中的活孢率一般与内贮总糖和蛋白质代谢水平均存在显著或极显著相关性.在1个月的贮存期间,4℃下冻干粉总糖含量下降13.4%,蛋白质含量下降39.2%,清水中的萌发率下降32.0%,营养液中的萌发率仅下降6.7%,而未干燥孢子粉的相同指标分别下降42.4%、66.3%、96.4%和99%;在20℃下,冻干粉的上述指标分别下降了14.1%、38.2%、55.8%和 10.4%,而未干燥孢子粉则分别下降了 43.2%、65.4%、99.4%和98.4%. 显然,含水量影响活孢率和内贮营养衰变的幅度,而温度影响衰变的速度,但内贮营养的耗尽并不立即引起孢子失活,在供给外源营养之后孢子仍能萌发.将含水量降至4.0±0.9%的冻干粉贮存1年,4℃下活孢率由初始的99.0%下降至90.2%,而20℃下贮存的前165d活孢率下降较为缓慢,但此后急剧下降,至第240d时几乎全部失活.模拟分析表明,低含水量冻干粉在4℃和20℃下贮存的半衰期(即活孢率减少一半所需的时间)分别为1006d和197d.这些结果说明,白僵菌纯孢粉的含水量     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

Influence of Culture Conditions on Growth and Survival of Conidia of Trichoderma spp. Coated on Seeds

Five Trichoderma strains were grown on rice, on vermiculite plus potato-dextrose broth (PDB), on potato-dextrose agar (PDA) or in liquid cultures supplemented with glycerol, KCl or polyethylene glycol (PEG) at -1 MPa or - 2 MPa. Conidia were coated on seeds through a methyl cellulose coating or through an industrial film-coating process. The conidial yield decreased with glycerol, KCl or PEG compared with PDB alone. The percentage viability was from 23 to 44% after methyl cellulose coating, regardless of the culture conditions for conidial production. In general, the industrial coating resulted in lower numbers of living conidia. The viability during storage was enhanced when vermiculite, rice or PDA were used as substrates for fungal growth. Nevertheless, temperature of storage was found to be more critical to spore survival than the substrate used for spore production; conidial viability on seeds did not exceed 4 months at 15 C. Solid and liquid cultures produced conidia able to control R. solani and P. ultimum when applied to seeds through industrial film coating. The level of disease suppression varied with the number of viable conidia/seed and with the culture medium used for conidial production. The three main conditions for further industrial application-high yields, longevity and biocontrol effectiveness-might be optimized by selecting the appropriate medium (liquid or solid), water potential and solutes used.     

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.     

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

Effect of formulation on the virulence of Metarhizium anisopliae conidia against mosquito larvae

Metarhizium anisopliae conidia were formulated with three granular carriers and nine dust diluents and stored over an 8- to 12-month period at 4° or 20°C. The virulence of formulations, with the exception of two dust preparations, was reduced significantly compared to unformulated conidia against Culex pipiens pipiens larvae. The formulation components most detrimental to conidial virulence were corn cob granules, diatomaceous earth, and two Kaolinite diluents. This was exampled by a decline in virulence from ca. 100% for unformulated conidia to 36% or below for these formulations. LT₅₀ values also increased from 2.4–2.6 days for unformulated conidia to above 6 days. In contrast, a diluent derived from dried castor oil (Thixcin R) significantly enhanced conidial virulence at several doses above that of unformulated conidia against C. pipiens larvae. Enhancement occurred whether conidia were formulated prior to storage or stored separate from the diluent and mixed prior to application. The Thixcin R formulation was more effective against Anopheles stephensi larvae, but virulence was reduced against Aedes aegypti larvae. A bentonite formulation (Bentone-38) also maintained conidial virulence effectively, but Thixcin R was a superior diluent. It was shown that conidial virulence of formulations was not correlated with differences in conidial viability. The preparations that were applied dry by a surface method were more virulent than when an aqueous suspension containing a surfactant was used. The results demonstrate the need to assess efficacy of mycoinsecticidal formulations in a virulence bioassay prior to field testing.     

Solid Substrate Production of Alternaria alternata f. sp. sphenocleae Conidia

Sphenoclea zeylanica (gooseweed), a major weed of paddy rice in Southeast Asia, is one of the targets in a biological weed control research program in the Philippines. A fungal pathogen, Alternaria alternata f. sp. sphenocleae , is being evaluated as a biological control agent for this weed. The feasibility of solid substrate fermentation for the mass production of A. alternata f. sp. sphenocleae has been examined. Conidia production and virulence of A. alternata f. sp. sphenocleae were affected by temperature, light, and incubation period. Abundant conidia were produced under continuous light on seeds of sorghum, hard red spring wheat, and barley at 28 o C. The greatest number of conidia was produced on sorghum seed followed by barley and oats seeds at 28 o C exposed to near-ultraviolet (NUV). More conidia were produced at 28 o C under NUV light on sorghum, barley, oats, and hard red spring wheat seeds, cornmeal, and polished rice grains than on the other substrates. Less conidia were produced on these substrates under light. At 28 o C, large numbers of virulent conidia were produced on sorghum seeds after 4 weeks of incubation under either constant light or dark. A mix of equal quantities of sorghum seeds and water (w/v) maximized conidial production. Conidia produced on sorghum seeds had a shelf life of at least 12 months when stored in production flasks under room conditions (24 ±2 o C). The use of sorghum seeds as a solid substrate for production of A. alternata f. sp. sphenocleae could be a feasible method to produce conidia in a village co-operative or cottage industry type scenario in Southeast Asia.     

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.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.