Effect of Carbohydrate Composition of the Cytosol of Aspergillus niger Conidia on Their Viability During Storage

It is shown that the rate of sporogenesis, the appearance of low-molecular-weight thiols, and the activation of carbohydrate synthesis in spores adversely affected the viability of Aspergillus niger conidia during storage. Conversely the prevalence of trehalose over mannitol and the absence of glycerol, erythritol, and glucose in the carbohydrate composition facilitated the viability of conidia. The data obtained are discussed with regard to the biochemical criteria that may be used to characterize the quiescent state of fungi and retaining the viability of the inoculum.     

Changes in the carbohydrate composition of cytosol from fungal spores in connection with environmental temperature and during storage

Differences in the carbohydrate composition were revealed among spores of fungi belonging to Zygomycetes, Ascomycota, Basidiomycota, and Oomycota, part of the novel kingdom Chromista. It was shown for the first time that Phytophthora infestans contains arabitol in addition to glucose and trehalose. Sucrose was detected in Pleurotus ostreatus basidiospores. It was established that Blakeslea trispora stylospores contain inositol. The dependence of the spore carbohydrate composition on the temperature of the habitat of the corresponding species is discussed. It was shown that the cytosol of the conidia is dominated by trehalose and inositol under hypothermic conditions and by mannitol and glucose under hyperthermic conditions. Neomycota and Eomycota were shown to differ in their responses to stress (starvation), which correlated with the differences in the carbohydrate composition of the spore cytosols. Assuming that cytosol carbohydrates perform a protective function, we explain the higher viability of conidia compared to stylo- and sporangiospores.     

Alterations in the carbohydrate composition of the cytosol of fungal spores caused by temperature variations and the storage process

Differences in the carbohydrate composition were revealed among spores of fungi belonging toZygomycetes, Ascomycota, Basidiomycota, andOomycota, part of the novel kingdomChromista. It was shown for the first time thatPhytophthora infestons contains arabitol in addition to glucose and trehalose. Sucrose was detected inPleurotus ostreatus basidiospores. It was established thatBlakeslea trispora stylospores contain inositol. The dependence of the spore carbohydrate composition on the temperature of the habitat of the corresponding species is discussed. It was shown that the cytosol of the conidia is dominated by trehalose and inositol under hypothermic conditions and by mannitol and glucose under hyperthermic conditions.Neomycota andEomycota were shown to differ in their responses to stress (starvation), which correlated with the differences in the carbohydrate composition of the spore cytosols. Assuming that cytosol carbohydrates perform a protective function, we explain the higher viability of conidia compared to stylo- and sporangiospores.     

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.     

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.     

Effects of Rehydration on the Conidial Viability of Metarhizium flavoviride Mycopesticide Formulations

The rehydration of dried conidia of Metarhizium flavoviride was investigated in an attempt to increase speed of kill of locusts and grasshoppers by formulations of this fungus. Conidia were dried to 4-5% moisture content with no apparent adverse effects on viability, but rapid rehydration (by putting dried conidia directly in free water) reduced viability. Rehydration in an atmosphere of high humidity allowed dry conidia to absorb sufficient moisture to avoid imbibition damage. Rehydrating and pre-germinating conidia prior to spraying (in an oil-based formulation) on to the desert locust, Schistocerca gregaria, did not decrease the time to death, suggesting that moisture uptake by dry conidia on the desert locust cuticle is easily achieved.     

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     

Encapsulation of Trichoderma harzianum conidia as a method of conidia preservation at room temperature and propagation in submerged culture

The objective of the present work was to develop a method for the preservation of T. harzianum conidia at room temperature and the immobilised conidia propagation in submerged culture. This was accomplished by immobilising the strain in sodium alginate capsules (white capsules) and subsequently propagating them in a column bubble reactor (green capsules). Three capsule diameters were tested (micro, medium and large capsules), which were produced by emulsion internal gelation and dripping methods. Tested variables were the immobilised conidia propagation in submerged culture for free conidia production, the immobilised conidia viability throughout the time (two years), the resistance of the encapsulated conidia to the UV irradiation of short and long wavelength, and the antagonistic effect of the encapsulated T. harzianum against four phytopathogenic fungi. It was found that the medium capsules (1.5?±?0.3?mm) favoured the massive production of released conidia in submerged culture and that the higher the density of conidia per capsule, the greater the protection against the ultraviolet irradiation. Regarding the conidia preservation in calcium alginate, a viability loss of around 30% was observed two years after storage at environmental temperature in both white and green capsules; along the two years that the viability of conidia was analyzed, the purity of the formulation was corroborated. The results presented here show the efficacy of the green and white capsules for T. harzianum preservation at room temperature for a long period of time.     

Effects of sheep fleece washings on the germination and growth of Beauveria bassiana

The development of biopesticides against ectoparasites must take into account the effect that an animal host’s secretions and host associated micro-organisms may have on the viability of the applied agent. In this study, the effects of secretions washed from the pelt of sheep on the viability and growth of the fungal pathogen Beauveria bassiana, were assessed. The fungal isolate had been obtained from the parasitic sheep scab mite Psoroptes ovis. It was added to 0.05% Tween 80 in which sheep fleece had been washed up to six times, to ascertain whether successive washings had any effect on the viability of conidia over 6 days. The effects of sterile and non-sterile washings on viability and growth were also investigated. Results indicated that substances in the sheep fleece may cause a significant reduction in the viability of conidia. Viability was linked to the number of times the sheep pelt had been washed in the Tween, with conidia incubated in the first wash from the sheep pelt showing a significantly greater decrease in viability compared to those incubated in the sixth wash. Viability was not linked to the sterility of the washes, although there was a significant difference between length of germ tube growth from viable conidia in sterile and non-sterile washings.     

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.     

Influence of temperature and humidity on the survival of Monilinia fructicola conidia on stone fruits and inert surfaces

The survival of the fungus Monilinia fructicola on fruit and inert surfaces at different temperatures (range: 0–30°C) and relative humidity (RH) (range: 60–100%) was investigated. M. fructicola conidia survived better on fruit than on inert surfaces. The viability reduction rate at 20°C and 60% RH was 1.2 and 5.8 days^?1 on fruit and inert surfaces, respectively. Overall, on fruit surfaces, conidia viability was reduced at high temperatures and was longer at higher RH than at lower RH; in contrast, on inert surfaces, conidia viability was longer at only low temperatures. On fruit surfaces, at 0°C and 100% RH, conidia survived up to 35 days, and at 30°C and 60% RH, conidia survived up to 7 days. However, on inert surfaces at 20°C and 30°C, conidia lost their viability after 48 and 24 h, respectively. These results suggest that M. fructicola can remain viable in cold rooms for over 30 days on fruit surfaces or over 25 days on inert surfaces. Furthermore, under the orchard conditions during the growing season, conidia may remain viable for only 2–3 days on immature fruit surfaces before conidia will be unable to penetrate the host.     

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.     

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.     

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.     

害虫防治用玫烟色拟青霉分生孢子粉的干燥工艺优化*

用液—固两相法生产的玫烟色拟青霉Paecilomyces fumosoroseus Pfr116菌株的分生孢子粉,在高真空冷冻干燥、高真空室温抽干、35℃下烘干和低真空低热干燥条件下进行不同程序的干燥处理,以筛选适合该菌孢子粉生产的干燥工艺条件。结果表明,低真空(0.1 MPa)低热(30℃)抽干20~24 h的干燥方法最适合用于该菌孢子粉的干燥,既能保证含水量在9.0%以下,又能保证87%以上的活孢率和1130亿~1310亿/g的含孢量,而且操作简便,成本较低,可作为高纯度孢子粉生产的首选干燥工艺。高真空(15.86 Pa)条件下无论冻干还是室温抽干,虽然孢子粉的含水量(2.2%~8.7%)和含孢量(1270亿~1360亿/g)指标符合生产要求,但活孢率仅62%,说明该菌孢子不适合在高真空条件下干燥。在35℃下烘干24 h所获孢子粉含水量、24 h萌发率和含孢量分别为9.6%、82.8%和1200亿/g,该方法也可在生产中应用,但其活孢率显著低于(P<0.05)低真空低热抽干24 h的孢子粉。     

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.     

A comparison of the virulence,stability and cell-wall-surface characteristics of three spore types produced by the entomopathogenic fungus Beauveria bassiana

Summary Beauveria bassiana can produce three spore types; aerial conidia, submerged conidia and blastospores. We have examined the spore surface characteristics (hydrophobicity and cell-wall surface lectins), thermal inactivation and the virulence towards the migratory grasshopper, Melanoplus sanguinipes, of each of the three spore types. The hydrophobicities of the aerial and submerged conidia were quite similar. Blastospores were less hydrophobic than either of the two types of conidia. Hydrophobic interactions are thought to play a significant role in attachment of the spore to the host organism. However, the less hydrophobic blastospores were slightly more virulent (LT₅₀ of 6.50 days) when compared to the aerial and submerged conidia (7.12 and 7.24 days), respectively. The lectin-binding characteristics of the aerial and submerged conidia were very similar but differed from that of blastospores. Growth of blastospores on a variety of carbohydrates did not affect their lectin-binding characteristics. Spore viability measurements showed that aerial and submerged conidia retained their viability for a longer period than blastospores. The similarity in hydrophobicity, stability, virulence and lectin-binding of aerial and submerged conidia make the latter an ideal candidate for mycoinsecticide production since they can be recovered after growth on inexpensive substrates.Offprint requests to: G. G. Khachatourians     

The potential of Beauveria bassiana inoculum formulated into a polymeric matrix for a microbial control of spruce bark beetle

Two local strains of Beauveria bassiana originally isolated from naturally infected spruce bark beetles in Slovakia were tested for their virulence to Ips typographus (IT) and for their compatibility with a polymeric matrix composed of low-molecular polyethylene. Conidia could be homogenously immobilized in the low-molecular polyethylene matrix with no adverse effect on their viability and infectivity. At constant temperature (25°C), viability of immobilized conidial decreased only by 1–2% after 7 or 14 days when compared with non-formulated conidia. In field conditions, viability of conidia formulated in the matrix was even significantly higher than non-formulated conidia 35 days after their application in traps. Conidia incorporated into the polymeric matrix were infective to IT adults in laboratory bioassays. Mean values of LC₅₀ for native conidia (0.72–2.05?×?10⁶ conidia?ml^?1) and conidia immobilized in the polymeric matrix (0.64–1.03?×?10⁵ conidia?mm^?2) demonstrated high virulence. The efficacy of the local strains was significantly higher than that of B. bassiana strains from mycoinsecticides (Boverol^®, Botanigard^® ES and Naturalis-L^®). Results showed potential of this polymeric material for its use in microbial control of IT when mixed with conidia of B. bassiana.     

Carbohydrate and lipid components of hyphae and conidia of human pathogenFonsecaea pedrosoi

The carbohydrate and lipid components of mycelium and conidia ofFonsecaea pedrosoi (Brumpt) were analysed by paper, thin-layer and gas-chromatography, mass spectrometry and ultraviolet spectroscopy. Glucose, mannose, galactofuranose, rhamnose and glucosamine were polysaccharide components identified inF. pedrosoi. Significant changes in the carbohydrate pattern occurred during the conversion of mycelium into conidia. Rhamnose was predominant in conidia whereas galactose was prominent in mycelium. Palmitic, stearic, oleic, linoleic, and arachidonic acids were the fatty acids identified in the total lipid fraction. Palmitic and oleic acids were major fatty acids. Marked alterations in the fatty acid constituents were observed between the cell types ofF. pedrosoi. Arachidonic acid was detected only in conidia and linoleic acid was preferentially identified in mycelium. Differences in the sterol composition was also associated with morphogenesis inF. pedrosoi. Two main sterols, ergosterol and another less polar sterol, not fully characterized, were found in mycelium whereas in conidia only the latter sterol was present.     

Increased heat tolerance afforded by oil-based conidial formulations of Metarhizium anisopliae and Metarhizium robertsii

The thermotolerance of oil-based conidial formulations of Metarhizium anisopliae s.l. (IP 46) and Metarhizium robertsii (ARSEF 2575) were investigated. Conidia of IP 46 or ARSEF 2575 were suspended in different adjuvants and exposed to 45?±?0.2°C for 4, 6, 8 or 24?h; their viability was then assessed after 48?h incubation at 27?±?1°C. Conidia heated in pure mineral or vegetable oil exhibited mean relative viability exceeding 70% after 8?h of heat exposure, whereas low germination (≤20%) was observed when conidia were heated in water (Tween 80^® 0.01%), carboxymethyl cellulose gel or emulsifiable oils (Graxol^® or Assist^®) and exposed to heat for 6 or 8?h. In addition, conidia of IP 46 suspended in either pure mineral or canola oil and exposed to heat for 48?h had moderate viability, 57% or 41%, respectively. Unstable oil-in-water emulsions showed a higher percentage of conidia incorporated into oil micellae, while the stable emulsions had higher percentage of conidia outside the oil micellae. The thermotolerance of conidia formulated in stable emulsions, however, did not differ from that of conidia formulated in unstable emulsions. The present study highlights possibilities to alleviate the deleterious effects of heat stress towards Metarhizium spp. conidia applied for controlling arthropod pests and vectors through oil-based formulations.