植物油对虫霉菌液体培养与保存的作用

在萨氏营养液（SDB）中加入用0．1％蔗糖脂肪酸酯－15（SE15）乳化的0．50％芝麻油，可使在SDB中生长慢而结块的新蚜虫疠霉、飞虱虫疠霉和根虫瘟霉等四种虫霉的五个菌株形成均匀分散的菌丝体和能稳定转接的菌液。在SDB中加入乳化芝麻油（0．25％－1．5％）、菜籽油（0．50％－1．5％）和色拉油（80％花生油和20％菜籽油，0．75％－1．5％），能明显提高新蚜虫疠霉F98018的生物量；而在SDB中加入0．50％乳化芝麻油，可使液培菌丝产孢量增加近一倍；在SDB中加入的三种植物油（0．75％），在F98018生长4d后，含量分别下降了85％－94％，但其中脂肪酸相对组成基本不变，表明该菌生长能充分利用植物油中各脂肪酸。用萨氏培养基（SDAY）、加入0．50％乳化芝麻油的SDAY（OSSDAY）和牛奶蛋黄培养基（SEMA）培养安徽虫瘟霉F97028和新蚜虫疠霉F98028，两菌株在SEMA上生长最快；在OS－SDAY以较慢速度正常生长；而在SDAY上，两菌株或不能生长，或在第二次培养时出现明显退化。用SEMA和OS－SDAY上，两菌株或不能生长，或在第二次培养时出现明显退化。用SEMA和OS－SDAY在3℃下来保上述四种虫霉的七个菌株，在OS－SDAY上，所有菌株在9个月后仍保持活性，而在SEMA上有五个菌株死亡；同时比较F97028和F98028在用OS－SDAY保存前后的生长速度和菌落形态，无退化产生。     

植物油对虫霉菌液体培养与保存的作用*

在萨氏营养液（ＳＤＢ）中加入用 ０．１％蔗糖脂肪酸酯－１５（ＳＥ１５）乳化的０．５０％芝麻油,可使在ＳＤＢ中生长慢而结块的新蚜虫疠霉、安徽虫瘟霉、飞虱虫疠毒和根虫瘟霉等四种虫霉的五个菌株形成均匀分散的菌丝体和能稳定转接的菌液。在ＳＤＢ中加入乳化芝麻油 （ ０．２５％～１．５％）、菜籽油（ ０．５０％～１．５％）和色拉油（ ８０％花生油和 ２０％菜籽油,０．７５％～１．５％）,能明显提高新蚜虫疠霉Ｆ９８０１８的生物量;而在ＳＤＢ中加入０．５０％乳化芝麻油,可使液培菌丝产孢量增加近一倍;在ＳＤＢ中加入的三种植物油（０．７５％）,在Ｆ９８０１８生长４ｄ后,含量分别下降了８５％～９４％,但其中脂肪酸相对组成基本不变,表明该菌生长能充分利用植物油中各脂肪酸。用萨氏培养基（ ＳＤＡＹ）、加入 ０．５０％乳化芝麻油的 ＳＤＡＹ（ ＯＳ－ＳＤＡＹ）和牛奶蛋黄培养基（ＳＥＭＡ）培养安徽虫瘟霉Ｆ９７０２８和新蚜虫疠霉Ｆ９８０２８,两菌株在ＳＥＭＡ上生长最快;在ＯＳ－ＳＤＡＹ以较慢速度正常生长;而在ＳＤＡＹ上,两菌株或不能生长,或在第二次培养时出现明显退化。用ＳＥＭＡ和ＯＳ－ＳＤＡＹ在３℃下来保存上述四种虫霉的七个菌株,在ＯＳ－ＳＤＡＹ上?     

飞虱虫疠霉杀蚜效果及开发应用前景

通过比较飞虱虫疠霉 (Pandoradelphacis)与其它几种常见蚜虫专化性病原真菌新蚜虫疠霉(P .neoaphidis)、安徽虫瘟霉 (Zoothphoraanhuiensis)和根虫瘟霉 (Z .radicans)对桃蚜 (Myzuspersicae)毒力 ,证实了该菌的杀蚜应用潜力。时间 -剂量 -死亡率模型分析表明 ,飞虱虫疠霉杀蚜的各项毒力指标与其它几种虫霉相似或略高 ,但杀蚜速率更快。同时对培养性状、产孢特性和生长适温等的比较研究发现 ,该菌具有易发酵生产、产孢能力强和温度生长范围宽等特点 ,是一种较为理想的有望研制开发成杀蚜真菌制剂的病原菌。此外 ,根据虫霉孢子主动弹射的生物学特性 ,提出了该菌的适用剂型 ,并对它们的流行学特性及其在设栽培中的应用前景进行了讨论。     

块状耳霉的分离、鉴定、培养和寄主范围

1981年自感染真菌的扁豆蚜(Aphis craccivora Koch)虫尸上分离到一株虫毒菌,经鉴定是耳霉属的块状耳霉(couidiobolus thromboides Drechsler)。该菌易于分离和培养,在培养基上可形成大量休眠孢子,休眠孢子容易萌发,六天中萌发率达53%。经试验该种虫霉可以感染多种蚜虫,是一种有希望制成杀蚜菌剂的真菌。     

福建厦门地区蚜虫病原真菌调查及其发生规律

从2014年至2015年对厦门地区蚜虫虫霉发生和流行情况进行初步调查,共鉴定出6种虫霉目病原真菌:新蚜虫疠霉Pandora neoaphidis、努利虫疠霉Pandora nouryi、普朗肯虫霉Entomophthora planchoniana、暗孢耳霉Conidiobolus obscurus、弗雷生新接霉Neozygites fresenii、根虫瘟霉Zoophthora radicans。新蚜虫疠霉和普朗肯虫霉是春季蔬菜蚜虫种群中发生、流行的最重要病原真菌,夏季未观察到虫霉的发生。     

环境因子对努利虫疠霉产孢和孢子萌发的影响

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

近藤虫疠霉分类地位的分子证据*

对近藤虫疠霉和伊萨卡虫瘴霉的18SrRNA基因进行克隆测序(登录号分别为AF351133和AF351134),并于GenBank中的新蚜虫疠霉相应序列(登录号AF052405)进行比较。近藤虫疠霉有43个碱基差异,而伊萨卡虫瘴霉仅有38个碱基差异。这证明近藤虫疠霉作为一个独立的种存在是合理的。系统发育进化树发现近藤虫疠霉和伊萨卡虫瘴霉的亲缘关系比它和新蚜虫疠霉更近。这对Humber的新系统提出了异议。     

近藤虫疠霉分类地位的分子证据

对近藤虫疠霉和伊萨卡虫瘴霉的18SrRNA基因进行克隆测序（登录号分别为AF351133和AF351134）,并于GenBank中的新蚜虫疠霉相应序列（登录号AF052405）进行比较,近藤虫疠霉有43个碱基差异,而伊萨卡虫瘴霉仅有38个碱基差异,这证明近藤虫疠霉作为一个独立的种存在是合理的,系统发育进化树发现近藤虫疠霉和伊萨卡虫瘴霉的亲缘关系比它和新蚜虫疠霉更近,这时Humber的新系统提出了异议。     

几种病原真菌对二点叶螨、桃蚜的侵染能力研究

从兰州市刘家堡乡自然感病的菜豆蚜尸上,经分离、纯化、筛选得到5种病原真菌,即新接霉属、干尸霉属、虫瘴霉属、虫疠霉属、匍柄霉属。以室内盆栽菜豆上饲养供试桃蚜、二点叶螨为供试生物,测定了几种病原真菌的侵染力。结果表明:匍柄霉属、新接霉属、虫瘴霉属、虫疠霉属对桃蚜有较高的侵染力,接种第7 d后校正死亡率分别为74.0%、70.0%、64.0%、62.0%;对二点叶螨雌成螨的侵染力相对较低,干尸霉属、新接霉属、虫瘴霉属、匍柄霉属接种第7 d后校正死亡率分别为48%、45.9%、42.7%和38.8%。几种病原真菌孢子悬浮液处理二点叶螨螨卵后,可延长螨卵的发育历期,降低螨卵孵化率。     

黑刺粉虱8种虫生真菌培养性状及其侵染率

蚧侧链孢（Pleurodesmospora coccora）、韦伯虫座孢（Aegerita webberi）、枝孢霉（Cladosporium sp.）、顶孢霉（Acremonium sp.）、拟青霉（Paecilomyces sp.）、米曲霉（Aspergillus oryzae）、被毛孢（Hirsutella sp.）和祁门4号（学名？）是茶园黑刺粉虱（Aleurocanthus spiniferus）主要的虫生真菌。它们在马铃薯培养基、萨氏培养基和察氏培养基上生长状况较好,但韦伯虫座孢不能产生有性世代。中描述了它们在察氏平板培养基上25℃恒温培养3星期的菌落特征围 伯虫座孢是皖南山区茶园中的优势种,“霉雨”季节侵染率达90％以上。蚧侧链孢、顶孢霉和枝孢霉是皖南丘陵地区茶园中优势种,流行盛期的联合侵染率为0.4%-11%。     

病原性丝状真菌的菌种保藏方法

目的 探讨常见病原性丝状真菌的菌种保藏方法.方法将73株病原性丝状真菌经过纯化后,接种于马铃薯葡萄糖琼脂斜面分别在4℃和-80℃冷冻管保存,均用10％ (v/v)的丙三醇作为保护剂.结果经过1 a左右的保存,将菌株复活,发现4℃斜面保藏法菌株的存活率为100％,-80℃冷冻管保藏法菌株的存活率为98.6％,有些毛癣菌属...     

定期转种法和低温冷冻法保藏致病真菌活性的能力比较

定期转种法和低温冷冻保存法是临床实验室最常用的两种真菌保存方法,为比较两种方法保藏致病真菌活性的能力,本研究使用两种保藏方法对实验室689株致病真菌保藏5年后进行检测。定期转种法是将菌落接种于马铃薯斜面培养基并将其储存在4℃冰箱,每6个月转种1次。低温冷冻法是挑取马铃薯斜面培养基上生长良好的菌落于无菌10%甘油中,放置在-80℃储存。保藏5年后,将两种方法保藏的菌株转种复苏,比较菌株的复活率。对于念珠菌属Candida、新生隐球菌Cryptococcus neoformans、毛癣菌属Trichophyton、曲霉属Aspergillus和孢子丝菌属Sporothirix真菌,两种方法的菌株复活率无统计学差异;对于小孢子菌属Microsporum真菌和马尔尼菲蓝状菌Talaromyces marneffei,使用低温冷冻法保藏的菌株复活率高于定期转种法保藏的菌株复活率;对于着色霉属Fonsecaea真菌,低温冷冻法保藏的菌株复活率低于定期转种法保藏的菌株复活率。因此,我们认为对于常见致病真菌的长期保藏,使用10%甘油作为保护剂的低温冷冻法优于定期转种法,但其不适用于着色霉属Fonsecaea真菌的长期保藏。     

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.     

Comparison between conidia and blastospores of Esteya vermicola, an endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively.     

Characteristics of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The sporulation capacities of the mycelial form of Paracoccidioides brasiliensis were determined. Five different culture media were used and four human isolates studied. Conidia were produced in three agar media, namely water-agar, glucose-salts and yeast-extract. Corn meal and Sabouraud dextrose agars failed to induce sporulation. Various types of spores were characterized with peculiar bulging arthroconidia and single-celled, pear-shaped conidia predominating. The size of these conidia varied from 3.6 to 4.6 micron in length. It is concluded that the mycelial form of P. brasiliensis produces characteristic spores if the proper culture media are employed.     

Preservation of Chytridiomycota in culture collections

Methods for the preservation of fungi in the Chytridiomycota in culture collections are reviewed in this paper. The Chytridiomycota can be preserved with varying degrees of success using a number of different protocols including cryopreservation. The survival of fungi in the Chytridiomycota is sensitive to environmental factors such as lack of moisture, high temperatures, high osmotic potential, and availability of oxygen, all of which must be considered in designing preservation methods. The age of the culture at the initiation of preservation appears to be a particularly important determinant of viability. Recently, commonly used methods for preservation of other groups of fungi have been modified to improve the survival of the Chytridiomycota in culture collections. High rates of survival have been reported after cryopreservation of aerobic and anaerobic chytrids in 10 % glycerol or dimethyl sulphoxide as cryoprotectants. The rates of freezing and thawing must be carefully controlled in the methods for cryopreservation considered in this review. Further research on increasing long-term survival rates and morphological, physiological and genetic stability of Chytridiomycota at low temperatures is necessary.     

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

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

A new method for the preservation of fungus stock cultures by deep-freezing

Recovery of 66 fungus stock cultures including Oomycota, Zygomycota, Ascomycota, Basidiomycota, and mitosporic mycetes were examined after cryopreservation. Almost all the stock cultures remained viable when the mycelia that had grown over the sawdust medium containing 10% glycerol as the cryoprotectant (65% moisture content, W/W) were frozen rapidly at −85°C and then allow to thaw naturally at room temperature. Test stock cultures were preserved for more than 10 years by this preservation method without any programmed precooling and rapid thawing for their cryopreservation. Most of the test fungi could survive for 5 years in medium containing 10% glycerol even after alternate freezing and thawing at intervals of 6 months. When a strain of Flammulina velutipes was tested for mycelial growth rate and productivity of fruit-bodies after cryopreservation for 3 years, the fungus reproduced with its initial capability. These results demonstrate that the sawdust-freezing method using a cryoprotectant is expected to be a reliable and easy preservation method for fungus stock cultures. Received: December 7, 2000 / Accepted: December 19, 2001     

Antifungal Activity of Sodium Bicarbonate Against Fungal Agents Causing Superficial Infections

Although sodium bicarbonate—NaHCO₃ (SB) has many domestic and medical, traditional and empirical uses, only little scientific documentation of its activity is available. The aims of this study were to investigate the antifungal activity of SB on the three fungal groups (yeasts, dermatophytes and molds) responsible for human skin and nail infections. We first evaluated the in vitro antifungal activity of SB on 70 fungal strains isolated from skin and nail infections: 40 dermatophytes, 18 yeasts and 12 molds. A concentration of 10 g/L SB inhibited the growth of 80 % of all the fungal isolates tested on Sabouraud dextrose agar. The minimal inhibitory concentration 90 (MIC90) of SB measured on Sabouraud dextrose agar, Sabouraud dextrose broth and potato dextrose broth was 5 g/L for the yeasts, 20 g/L for the dermatophytes and 40 g/L for the molds. In a second step, we prospectively evaluated the ex vivo antifungal activity of SB on 24 infected (15 dermatophytes, 7 yeasts and 2 molds) clinical specimens (15 nails and 9 skin scrapings). The fungal growth was completely inhibited for 19 (79 %) specimens and reduced for 4 (17 %) specimens after 7 days of incubation on Sabouraud dextrose–chloramphenicol agar supplemented with 10 g/L of SB as compared to Sabouraud dextrose–chloramphenicol agar without SB. In conclusion, we documented the antifungal activity of SB on the most common agents of cutaneous fungal infection and onychomycosis, and we specified the effective concentrations for the different groups of pathogenic fungi. The mechanism of action of SB has yet to be explored.     

Maintenance and preservation of ectomycorrhizal and arbuscular mycorrhizal fungi

Short- to long-term preservation of mycorrhizal fungi is essential for their in-depth study and, in the case of culture collections, for safeguarding their biodiversity. Many different maintenance/preservation methods have been developed in the last decades, from soil- and substrate-based maintenance to preservation methods that reduce (e.g., storage under water) or arrest (e.g., cryopreservation) growth and metabolism; all have advantages and disadvantages. In this review, the principal methods developed so far for ectomycorrhizal and arbuscular mycorrhizal fungi are reported and described given their distinct biology/ecology/evolutionary history. Factors that are the most important for their storage are presented and a protocol proposed which is applicable, although not generalizable, for the long-term preservation at ultra-low temperature of a large panel of these organisms. For ECM fungi, isolates should be grown on membranes or directly in cryovials until the late stationary growth phase. The recommended cryopreservation conditions are: a cryoprotectant of 10 % glycerol, applied 1–2 h prior to cryopreservation, a slow cooling rate (1 °C min^?1) until storage below ?130 °C, and fast thawing by direct plunging in a water bath at 35–37 °C. For AMF, propagules (i.e., spores/colonized root pieces) isolated from cultures in the late or stationary phase of growth should be used and incorporated in a carrier (i.e., soil or alginate beads), preferably dried, before cryopreservation. For in vitro-cultured isolates, 0.5 M trehalose should be used as cryoprotectant, while isolates produced in vivo can be preserved in dried soil without cryoprotectant. A fast cryopreservation cooling rate should be used (direct immersion in liquid nitrogen or freezing at temperatures below ?130 °C), as well as fast thawing by direct immersion in a water bath at 35 °C.