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（0.1MPa),低热（30℃）抽干20－24h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（15.86Pa)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干燥,在35℃下烘干24h所获孢子粉含水量,24h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≤24h的孢子粉。     

真空干燥球孢白僵菌纯孢粉的活孢率、毒力与贮存期*

采用真空冷冻干燥法 (冻干 )和真空室温干燥法 (抽干 )对球孢白僵菌 (_{Beauveriabassiana})原菌粉进行了干燥工艺的比较。在固体产孢基质上生产的新鲜纯孢粉 (原菌粉 )含水量达 58.65 % ,经 36h冻干或抽干 ,孢子粉的含水量分别降至 3.97%和 4.26% ,含孢量分别为 1.29×10¹¹ 孢子 /g和 1.25×10¹¹ 孢子 /g ,活孢率分别为 99.0 %和 97.9%。冻干粉和抽干粉之间的活孢率无显著     

真空干燥球孢白僵菌纯孢粉的活孢率、毒力与贮存期

采用真空冷冻干燥法 (冻干 )和真空室温干燥法 (抽干 )对球孢白僵菌 (Beauveriabassiana)原菌粉进行了干燥工艺的比较。在固体产孢基质上生产的新鲜纯孢粉 (原菌粉 )含水量达 5 8 65 % ,经 36h冻干或抽干 ,孢子粉的含水量分别降至 3 97%和 4 2 6% ,含孢量分别为 1 2 9× 1 0 1 1 孢子 /g和 1 2 5× 1 0 1 1 孢子 /g ,活孢率分别为 99 0 %和 97 9%。冻干粉和抽干粉之间的活孢率无显著差异 ,但萌发速度不同。原菌粉、抽干粉及冻干粉对桃蚜(Myzuspersicae)在接种后第 7d的LC50 分别为 1 1 5 ,5 89和 2 95× 1 0 4 个孢子 /mL ;在 1 0 6 个孢子 /mL的剂量下对桃蚜的LT50 分别为 3 6d、 4 4d和 3 9d。从生产成本低得多 ,抽干法比冻干法在白僵菌纯孢粉的生产中更有应用前景 ,尽管冻干粉的质量略优于抽干粉。在4℃和 2 0℃下贮存冻干粉并进行为期一年的孢子萌发率和毒力检测。结果显示 ,冻干粉在2 0℃下贮存至 2 5 5d时完全失活而丧失染虫能力 ,而在 4℃下贮存一年后的活孢率仍达90 2 % ,LT50 仅从初始的 3 9d延长至 4 7d。这说明仅通过降低含水量以延长白僵菌孢子粉在常温下的贮存期仍是不可能的。     

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     

含水量与温度对球孢白僵菌孢子粉贮存寿命的影响及模拟分析

将含水量 1 12 %、4 73%、7 2 3%、9 84 %及 14 11%的球孢白僵菌BeauveriabassianaSG870 2分生孢子粉在4℃和 2 5℃下黑暗贮存 18个月 ,定期检测活孢率 ,以确定孢子粉的贮存寿命。结果显示 ,各温度处理中含水量显著(P <0 0 5 )影响孢子贮存期间的活孢率。在 4℃下 ,含水量为 1 12 %～ 9 84 %处理的活孢率在头 16个月均稳定在91%以上 ,且相互间差异不显著 ;次高含水量处理的活孢率在第 18个月才显著低于其它较低含水量处理 ;高含水量处理的活孢率则从第 6个月起显著低于其余较低含水量处理 ,第 12个月时降至 2 4 2 %。而在 2 5℃下 ,第 3和第 6个月的活孢率在不同含水量处理间均呈极显著差异 (P <0 0 1) ,即随含水量升高而显著下降 ,次高含水量处理在第6个月的活孢率仅剩 17 6 %。贮存期间活孢率对贮存时间和孢子粉含水量的依赖关系很符合改进的存活衰变模型(r2 >0 85 )。根据拟合的模型预测 ,在 4℃下贮存 ,若保证活孢率 90 % ,12 %含水量的孢子粉可贮存 7 3个月 ,10 %为 11 2个月 ,9%为 14 9个月 ,8%为 2 1 0个月 ,7%为 33 0个月 ,6 %达 6 5 5个月 ,故孢子粉冷贮的含水量应控制在8%以下。若在 2 5℃下贮存并保证活孢率 80 % ,含水量 10 %的孢子粉仅可贮存 1 7个月 ,8%为 2 3个月 ,6 %为 3 0个     

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.     

白僵菌孢子悬乳剂田间控蚜效果与常温货架寿命测定

将高纯度球孢白僵菌分生孢子粉兑入一种含悬浮稳定剂及紫外保护剂的矿物油中,配成含孢量为100亿/ml的孢子悬乳剂(纯菌剂),再添加3%的吡虫啉10%可湿剂而得菌药混配剂,于2003年5月在浙江余姚进行了防治蔬菜蚜虫的田间药效试验和农户示范。在两块夏熟白菜地中,纯菌剂和菌药混配剂均用1000倍稀释液(107个孢子/ml)常规喷雾2次,并设清水对照和低用量吡虫啉对照。结果表明,混配剂在两块菜地的相对防效分别达94.5%和86.2%,纯菌剂分别达86.4%和73.1%,而低用量吡虫啉处理的最高防效仅为50%左右。在入夏豇豆、甘蓝、白菜、萝卜及梨树上用菌药混配剂1000倍稀释液对多种蚜虫进行的农户示范试验中,一次性喷雾后5~10d内对菜蚜防效为48%~68%,尤对梨二叉蚜的防效高达99%。在对含有和不含添加剂的两配方孢子悬乳剂进行的常温避光18个月贮存中,活孢率在头7个月均稳定在95%左右且未受配方影响,第10个月活孢率仍有75%左右。而后,含添加剂配方的活孢率下降加速,至16个月时基本失活;而不含添加剂配方的活孢率在第16、17及18个月时还有57%、51%和42%。     

白僵菌固态发酵试验研究*

以产孢量为指标,筛选出白僵菌固态发酵的培养基为:90%麦麸+5%玉米粉+5%黄豆粉,原孢粉的产孢量可达244.7亿/g。在25m³发酵罐内进行浅盘固态发酵,培养基的最适起始含水率为60%~65%;最佳通气条件为:前12h不通气,12~48h通气量为1:2,48~144h的通气量为1:1;而温度只需在12~48±8h内进行控制。常温干燥能最大限度地维持白僵菌的活孢率。     

甲醇毕赤酵母表达木质素过氧化物酶的研究

将含有黄孢原毛平革菌(Phanerochaetechrysosprium)木质素过氧化物酶基因的甲醇毕赤酵母工程菌进行了鉴定和筛选,筛选得到木质素过氧化物酶活力高的菌株PMLIP08。确定了一步法发酵的最优葡萄糖浓度,优化其发酵培养条件,结果表明葡萄糖的添加量为10g/L时,发酵条件为pH3.0,诱导温度24℃,培养时间12h,甲醇添加量1.1%,诱导时间72h后发酵液中酶活可达4888U/L。     

淡紫拟青霉固体发酵工艺研究

淡紫拟青霉（Paecilomyces lilacinus）是控制植物病原线虫的优良生防菌,同时对多种土传尖孢镰刀菌引起的枯萎病等病害具有防病作用。固态发酵技术为分生孢子的大规模、低成本生产提供了许多有利条件。为获得淡紫拟青霉固体发酵的最佳条件,采用单因素实验和响应面试验对淡紫拟青霉固体发酵培养基的组成、料液比、种子液接种量和烘干条件进行了优化,结果表明淡紫拟青霉固体发酵最佳的培养基组成为麸皮玉米粉为 0.9：1（体积比）、蔗糖添加量4%（质量分数,下同）、尿素添加量 0.16%、硫酸铵添加量 0.17%、料水比为1：0.5（体积比）,培养条件为接种量10%、培养温度28 ℃、培养时间6 d、固体菌剂最适烘干条件为35 ℃烘干24 h,在此条件下淡紫拟青霉固体菌剂的有效活菌数为 2.49×10¹⁰ cfu/g。     

Psychrophilic and Mesophilic Fungi in Fruit-Filled Pastries

Surveys of the mold flora of frozen blueberry and cherry pastries were undertaken. Molds were enumerated by preparing pour plates of the blended product and incubating the plates at 0, 5, 10, and 20 C. In this manner, the total fungal content of the product could be ascertained from the 10 and 20 C plates, and the psychrophilic fungal population was represented by those fungi which grew at 0 and 5 C. The pastry portion, or crust, of the blueberry material was sampled separately from the filling portion. Certain differences in fungal flora were apparent. Aureobasidium pullulans was the dominant fungus in crust at all temperatures of isolation. However, Penicillium thomii proved to be the most common mesophilic fungus in the filling portion, and A. pullulans was the most common psychrophile in the filling. Aspergilli were quite common in the crust, but, in general, were absent from the fruit filling. Cherry pastries had a much smaller total fungal flora than did the blueberry product. However, A. pullulans again was the most prevalent fungus in cherry pastries at all temperatures of isolation. Certain differences in fungal flora were apparent in the two fruit products. Phoma spp. were almost completely absent in blueberries, but represented the second most common fungus in cherry pastries. Blueberry filling had 440 psychrophilic fungi per gram of sample (at 0 C), blueberry crust had 65 per gram, and cherry pastries had 77 per gram.     

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.     

Experimental determination of viability loss of Penicillium bilaiae conidia during convective air-drying

A study was conducted on the drying of Penicillium bilaiae, a fungal micro-organism used to promote soil-bound phosphorous uptake in several crop species, such as wheat, canola and pulse crops. A wet pellet formed from a mixture of the inoculant and a starch-based carrier was air-dried to the appropriate water activity to extend the shelf-life of the viable fungal conidia. Convective air-drying was examined as a low-energy alternative to the more expensive freeze-drying technology that is currently in use. Experiments were conducted to measure the loss of conidia viability during drying in a fixed-bed, thin-layer convective dryer. The dryer air inlet temperature and relative humidity were controlled in experiments to determine the effect of thermal and dessicative stresses on conidial viability. The measured survivor fraction was determined to be dependent on solids temperature, moisture content and drying rate. Thermal stresses became significant for process temperatures above 30°C, while the survivor fraction fell sharply below a dry basis moisture ratio of 30%. Slower drying kinetics associated with high inlet air relative humidity were found to significantly improve the recovery of viable conidia. By minimising environmental stresses, survivor fractions of up to 75% could be achieved, but this result fell dramatically with the introduction of more severe conditions. A general linear statistical model is used to quantify experimental error and the significance level of each factor.     

Effects of wetting on the persistence of quiescent conidia of Isaria fumosorosea

Quiescent conidia of Isaria fumosorosea were submitted to various wetting–drying cycles under different regimes of temperature and air humidity. Germination and viability of conidia collected on cultures freshly host-passed (P2) were not affected at 25 °C during five cycles at increasing wet phase duration (2–12 h per daily cycle) under any moisture conditions (13–86% RH). Infectivity levels remained stable, but mortality was slightly postponed. In vitro-cultured inocula (P5) were significantly affected after only one cycle at higher air humidity (75 and 86% RH) and temperature (35 and 40 °C). The persistence of I. fumosorosea conidia suspended in water soluble extracts of leaf surfaces (corn and cabbage) confirmed the better persistence of P2 conidia and the relatively higher detrimental effect of lower air humidity conditions when combined with moderate temperatures. Quiescent conidia deposited in situ on potted plants of cabbage showed a higher persistence on wet foliage and on foliage submitted to wetting–drying cycles, than on dry foliage. These results underline that constraints prevailing in targeted environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

A novel strain of Pseudozyma aphidis from mulberry parasitises the conidia of mulberry powdery mildew fungus Phyllactinia sp. and its biocontrol effect in the fields

Plant surface is colonised with a vast community of non-pathogenic epiphytic microorganisms which play an important role in host defence. In the present study, we reported a fungus from mulberry leaf surface that showed an antagonistic effect against mulberry powdery mildew fungal pathogen Phyllactinia sp. This novel isolate is a yeast-like fungus that was identified as Pseudozyma aphidis CNm2012 based on morphologic and phylogenetic analysis. According to our research, P. aphidis CNm2012 directly acted on the powdery mildew conidia via parasitism which caused conidial atrophy, collapse and eventually, cleavage and death. During the parasitic process, we found the isolate could gather around the conidia of Phyllactinia sp. and form hyphae that grew on the conidial surface and utilise the conidia as a nutrient source. Field studies revealed that P. aphidis CNm2012 could suppress the disease incidence of mulberry powdery mildew caused by Phyllactinia sp., and reduce the disease severity. To our knowledge, it is the first report of P. aphidis directly against powdery mildew fungus Phyllactinia spp. by parasitism. Our results indicated that P. aphidis CNm2012 could be served as an environmentally friendly alternative of chemical pesticides to manage mulberry powdery mildew disease.     

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.     

State transitions and physicochemical aspects of cryoprotection and stabilization in freeze-drying of Lactobacillus rhamnosus GG (LGG)

Aims: The frozen and dehydrated state transitions of lactose and trehalose were determined and studied as factors affecting the stability of probiotic bacteria to understand physicochemical aspects of protection against freezing and dehydration of probiotic cultures. Methods and Results: Lactobacillus rhamnosus GG was frozen (–22 or –43°C), freeze‐dried and stored under controlled water vapour pressure (0%, 11%, 23% and 33% relative vapour pressure) conditions. Lactose, trehalose and their mixture (1 : 1) were used as protective media. These systems were confirmed to exhibit relatively similar state transition and water plasticization behaviour in freeze‐concentrated and dehydrated states as determined by differential scanning calorimetry. Ice formation and dehydrated materials were studied using cold‐stage microscopy and scanning electron microscopy. Trehalose and lactose–trehalose gave the most effective protection of cell viability as observed from colony forming units after freezing, dehydration and storage. Enhanced cell viability was observed when the freezing temperature was ?43°C. Conclusions: State transitions of protective media affect ice formation and cell viability in freeze‐drying and storage. Formation of a maximally freeze‐concentrated matrix with entrapped microbial cells is essential in freezing prior to freeze‐drying. Freeze‐drying must retain a solid amorphous state of protectant matrices. Freeze‐dried matrices contain cells entrapped in the protective matrices in the freezing process. The retention of viability during storage seems to be controlled by water plasticization of the protectant matrix and possibly interactions of water with the dehydrated cells. Highest cell viability was obtained in glassy protective media. Significance and Impact of the Study: This study shows that physicochemical properties of protective media affect the stability of dehydrated cultures. Trehalose and lactose may be used in combination, which is particularly important for the stabilization of probiotic bacteria in dairy systems.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

Development of a user-friendly delivery method for the fungus Metarhiziumanisopliae to control the ectoparasitic mite Varroa destructor in honey bee, Apis mellifera, colonies

A user-friendly method to deliver Metarhizium spores to honey bee colonies for control of Varroa mites was developed and tested. Patty blend formulations protected the fungal spores at brood nest temperatures and served as an improved delivery system of the fungus to bee hives. Field trials conducted in 2006 in Texas using freshly harvested spores indicated that patty blend formulations of 10 g of conidia per hive (applied twice) significantly reduced the numbers of mites per adult bee, mites in sealed brood cells, and residual mites at the end of the 47-day experimental period. Colony development in terms of adult bee populations and brood production also improved. Field trials conducted in 2007 in Florida using less virulent spores produced mixed results. Patty blends of 10 g of conidia per hive (applied twice) were less successful in significantly reducing the number of mites per adult bee. However, hive survivorship and colony strength were improved, and the numbers of residual mites were significantly reduced at the end of the 42-day experimental period. The overall results from 2003 to 2008 field trials indicated that it was critical to have fungal spores with good germination, pathogenicity and virulence. We determined that fungal spores (1 × 10¹⁰ viable spores per gram) with 98% germination and high pathogenicity (95% mite mortality at day 7) provided successful control of mite populations in established honey bee colonies at 10 g of conidia per hive (applied twice). Overall, microbial control of Varroa mite with M. anisopliae is feasible and could be a useful component of an integrated pest management program.