苹果盘二孢Marssonina coronaria的分离培养研究

苹果盘二孢Marssonina coronaria引起的褐斑病是造成我国苹果树早期落叶的主要原因,由于该病菌分离培养困难,阻碍了对其生物学特性的研究,进而影响了对其防治机理和流行规律的研究.本研究应用4种培养基质,探索了3种方法对苹果盘二孢的分离效果.结果表明,3种方法均可分离到病原菌,但组织块分离法和分生孢子团分离法成功率仅有10%左右,而单孢分离法污染少,成功率高达到90%以上,明显优于其他两种方法.不同培养基上菌落形态、大小和产孢情况差异也很大,培养1个月(25℃)后PDA上菌落黑褐色隆起,表面蚯蚓粪状,无气生菌丝,无子实体和基内菌丝;10%V8培养基上菌落中央隆起,黑褐色,表面生少量气生菌丝,边缘放射状,基内菌丝深褐色,有子实体;苹果叶片葡萄糖琼脂培养基(LDA)上菌落平坦,黄褐色,表面生茂密的金黄色气生菌丝,基内菌丝深褐色,有子实体;苹果叶片煎汁葡萄糖琼脂培养基(LEDA)上菌落有明显的不规则隆起,黄褐色至黑褐色,表面生少许气生菌丝,菌落生长缓慢,无基内菌丝,分生孢子盘菌落表面生,菌落直径仅2mm左右,而在其他培养基上的菌落直径可达6-8mm,说明培养基质、分离方法均对苹果盘二孢的分离培养和生长发育有明显的影响.     

萨克拉普奇尼亚菌串孢变种——北方根结线虫卵寄生真菌(英文)

分离自山东省牟平市塑料大棚种植的甜瓜根结线虫卵上的菌株CFCC84965,根据培养条件和形态特征鉴定为萨克拉普奇尼亚菌串孢变种Pochonia suchlasporia var.catenata,为中国新记录种。该菌生长较快,在CMA培养基培养15 d后菌落直径为27 ～43 mm,气生菌丝中等发达,白色至浅黄色,背面为黄色至淡褐色。分生孢子梗长,大多从菌丝上直立生长,多分支。分生孢子多串生,表面略粗糙。老熟菌丝上形成会聚的菌丝膨大细胞或形成少量的间生厚垣孢子。在琼脂培养基上可形成晶体。     

尖顶羊肚菌单孢菌株群体培养特性研究

目的:羊肚菌不同分离物在培养过程中形态学变化较大且极不稳定,通过同一子实体不同单孢菌株在不同培养基上的培养特性研究特别是产菌核能力的变化回答这些变化是否是由于多核菌株的不稳定性引起.方法:以不同来源尖顶羊肚菌单孢菌株为材料,并以粗柄羊肚菌为对照,研究了菌株在不同培养基上的培养特性,并对同一子实体及不同子实体产菌核和不产菌核单孢进行配对培养.结果:尖顶羊肚菌单孢菌株按培养特征可分为9类,同等条件下每一菌株的培养特性保持稳定;在综合马铃薯葡萄糖培养基(CPDA)、葡萄糖硝酸钠琼脂培养基(GN)、酵母膏胨葡萄糖琼脂培养基(YPD)进行转接培养时,可成功地将产菌核菌株转化为不产菌核菌株;尖顶羊肚菌同一子实体及不同子实体各产菌核单孢菌株产核数量及分布变化很大,交配后单孢之间性状会发生较大变化,包括菌核形态、菌丝形态、生长势,特别是产菌核能力会消失和发生转移.     

稻绿核菌无性孢子形成过程及厚垣孢子萌发率测定

对马铃薯蔗糖人工培养基(PSA)上绿核菌(稻曲病菌)不同培养时期的产孢情况进行了系统的扫描电镜观察。研究结果表明,在培养的前期(前20d),菌落表面往往形成集结状菌丝结构,其上开始产生大量分生孢子;一些分散的菌丝上也可产生少量的分生孢子。而在培养的后期,菌落表面往往形成黄色子实体,内部集生大量厚垣孢子。说明绿核菌在人工培养基上前期以形成分生孢子为主,后期则以厚垣孢子为主,且厚垣孢子的量远远大于分生孢子。萌发试验表明,成熟的厚垣孢子会随着保存时间的延长萌发率急剧下降。因此,新鲜的成熟厚垣孢子是最为理想的接种体。     

诱导茄链格孢菌分生孢子形成的新技术

本文报道诱导茄链格孢菌(Alternaria solani)分生孢子形成的一种新技术。生长在马铃薯-葡萄糖-琼脂(PDA)上两天的茄链格孢菌琼脂块移接到玉米培养基上,置于日光灯下照射,诱发分生孢子梗生长。然后,再放在18℃下黑暗培养。12小时后,在菌丝块表面有大量的分生孢子形成。成熟的茄链格孢菌分生孢子用蒸馏水洗脱。     

林生地霉血液分离株的形态学观察

目的 进一步了解林生地霉的形态学特征和产孢方式。方法受试菌株复苏后,转种于沙堡培养基,37℃和27℃温箱培养3周,每天观察菌落生长情况,并对其丝状型和酵母样型两种菌落进行光镜观察。在培养1周时,挑取菌落常规制片后行扫描电镜和透射电镜观察。结果 培养第2～3d,菌落开始生长,初为白色绒毛样、粉末样,1～2周后菌落均转变为乳酪样,其中37℃下培养的菌落转变较早。菌丝相时,光镜下可见丰富的分支、分隔菌丝,成链的关节孢子和圆形、椭圆形小分生孢子。酵母相时,光镜下可见丰富的芽生孢子,少量的厚膜孢子、外生或内生的关节孢子,菌丝稀少。扫描电镜下可见菌丝分支末端或侧面着生球形、棒状的分生孢子,末端凹陷。透射电镜下可见分生孢子与菌丝分离时全壁断裂,另一端正在出芽。结论 林生地霉包括丝状型和酵母样型两种菌落形态和链状排列的矩形关节孢子、圆形或椭圆形的小分生孢子、厚膜孢子以及分支、分隔菌丝为其形态学特征,有芽殖、外生和内生节孢子三种产孢类型。     

地生弯颈霉的培养与生长条件

从长白山北坡森林土壤中分离纯化得到1株地生弯颈霉(Tolypocladium geodes),对其培养及生长条件(如培养基、光照、温度、碳源、氮源和pH等)进行了试验。结果表明,地生弯颈霉在麦芽浸膏琼脂培养基(MEA)上菌落生长直径最大,马铃薯葡萄糖琼脂培养基(PDA)上产孢量最高;5~30℃温度范围内均能生长,25℃时生长和产孢量最佳;最适碳源和氮源分别为葡萄糖和柠檬酸铵;光暗交替条件下,最利于其生长和产生孢子;pH4~9范围内均能生长和产孢,pH=8时菌落直径最大,pH=6时产孢量最高。     

球孢白僵菌继代培养中菌落局变现象及环境影响因素的研究

球孢白僵菌野生型多孢标以及从其上分离的单孢株在不同培养基,光照,温度和湿度条件下继代培养。结果显示：无论是多孢株还是单孢株。在传代中均发生显著的菌落局变,并产生形态各异的分离子,多表现为菌苔瘠薄或气生菌丝徒长,肉眼可见产孢量减少,尤其是单孢株菌落局变产生的分离子往往伴随着棕黄色或红棕色色素的分泌。培养基、温度、湿度和光照等环境因素对菌落局变产生具明显诱导作用,以ＳＤＡＹ培养基、偏低的温湿度和全光照     

球孢白僵菌继代培养中的菌落局变现象

通过球孢白僵菌两个野生型菌株及其单孢株在不同培养基、温度、湿度和光照条件下继代培养,结果产生3种不同类型的菌落局变.局变产生的分离子多表现为菌落瘩薄或气生菌丝陡长、产孢量下降、生长速率增加等现象.培养基、温度、湿度和光照等因素对菌落局变有明显诱导作用.湿度为15℃,相对湿度较低及全光照条件下于SDAY培养基上培养,局变发生频率最低.在实验室继代培养中,野生型菌株在4-14代内即可发生局变而被局变分离子所取代.引人注意的是,单孢株局变产生的分离子同时伴随着棕黄色或红棕色素的分泌,仅以菌落形态和颜色等培养特征作为球孢白僵菌种的划分依据是不可靠的.文中提出了菌落局变发生的遗传变异可能机制.     

红色毛癣菌的生物学特性研究

目的 观察红色毛癣菌在不同温度、不同培养基上的生长和产孢情况,并对其进行分子生物学鉴定.方法 ①大培养:采用沙堡葡萄糖琼脂(SDA)和马铃薯葡萄糖琼脂(PDA)平皿,27℃、35℃黑暗培养,测量菌落直径,绘成生长曲线.②小培养(钢圈法):采用SDA、PDA、溴甲酚紫乳固体葡萄糖琼脂(BCP-MSG)、乳蜜琼脂(M)和复合维生素B(VitB)培养基,27℃、30℃黑暗培养,观察镜下菌丝生长、孢子产生情况.③进行rDNA 18S和ITS序列测定.结果 在SDA,PDA上,27℃条件下菌落生长速度较35℃快;在5种培养基上,SDA、PDA产孢较快较多,复合维生素B培养基产孢较慢,但产生大分生孢子较多.30℃产孢更丰富.对部分菌株rDNA ITS、18S PCR扩增产物纯化后直接测序,结果在GenBank中比对、分析,相似度为98%～100%,均鉴定为红色毛癣菌.结论 SDA、PDA均为鉴定和分离红色毛癣菌的合适培养基.5种培养基均可用来刺激红色毛癣菌产孢,其中SDA、PDA产孢较早、较丰富.红色毛癣菌rDNA 18S和ITS序列测定是一种快速准确的红色毛癣菌分子生物学鉴定方法.     

Development of novel Alicyclobacillus spp. isolation medium

AIM: To develop a new isolation medium with higher recovery rates of Alicyclobacillus spp. METHODS AND RESULTS: SK agar was developed with optimized incubation temperature, pH, acidulant, Tween 80 concentration and divalent cation addition. Results indicate that detection of Alicyclobacillus spp. by SK agar was significantly higher (P > 0.05) than those obtained by K agar, orange serum agar, and potato dextrose agar. CONCLUSIONS: Current media used for Alicyclobacillus spp. isolation still resulted in high numbers of false negative products. The sensitivity of SK agar to Alicyclobacillus spp. allows detection of low numbers of Alicyclobacillus spp. and also provides a more higher isolation results compared with currently used media. SIGNIFICANCE AND IMPACT OF THE STUDY: SK agar will be useful to the fruit juice industry to obtain more accurate numbers of contaminant Alicyclobacillus spp. With this media, false negative samples can be reduced, and the likelihood of exported products being rejected can be greatly reduced.     

冠盘二胞Marssonina coronaria侵染不同抗性苹果叶片的组织病理学研究

利用光学和电子显微镜,从组织细胞学水平系统研究了冠盘二胞Marssonina coronaria在苹果抗、感病品种叶片上的侵染过程及侵染后寄主细胞的超微结构特征。结果表明：冠盘二胞的侵入和定殖过程可以分为6个阶段：孢子萌发与芽管形成、附着胞形成、侵入细胞角质层、在叶肉细胞内产生吸器、菌丝在叶肉细胞间和细胞内扩展、分生孢子盘形成。随着菌丝扩展,受侵寄主细胞出现细胞壁加厚,细胞壁降解,质壁分离,叶绿体内淀粉粒、嗜饿颗粒积累,叶绿体基粒片层瓦解,线粒体空泡化等现象。在不同抗性的苹果品种上,分生孢子萌发率差别不明     

Evaluation of different growth media for the recovery of the species of Alicyclobacillus

AIMS: Five different isolation media, namely potato dextrose agar (PDA), orange serum agar (OSA), K agar, yeast-starch-glucose agar and Bacillus acidocaldarius medium were evaluated for the recovery of Alicyclobacillus spp. from inoculated diluted and undiluted fruit-juice concentrates. METHODS AND RESULTS: Plates of PDA (pH 3.7), spread with vegetative cells (3.9 x 10(6) CFU ml(-1)) of Alicyclobacillus acidoterrestris from single-strength pear juice, recovered 2.9 x 10(6 )CFU ml(-1) after 5 days at 50 degrees C (74% recovery). The recovery of endospores from single-strength pear juice, after a heat treatment at 80 degrees C for 10 min, was higher on spread plates of OSA (pH 5.5) at 50 degrees C for 5 days (97% recovery). CONCLUSIONS: PDA (pH 3.7) and OSA (pH 5.5) at 50 degrees C for 3-5 days recovered the highest numbers of vegative cells and endospores of Alicyclobacillus spp. from sterilized fruit juices and concentrates. SIGNIFICANCE AND IMPACT OF THE STUDY: The most appropriate synthetic media for the recovery of Alicyclobacillus species from inoculated fruit juices and concentrates are shown.     

Overwintering form of the causal agent of shot hole disease in Khorasan Razavi, Iran

Shot hole disease of stone fruit trees caused by some plant pathogenic fungi is a major constraint to stone fruit production worldwide where the trees are grown. Identification of the causal agents of the disease and their overwintering forms in stone fruit trees of Khorasan Razavi was necessary for disease management programs. Buds, twigs, fallen leaves and fruits were collected from the infected peach, apricot, nectarine and almond trees in winter 2007. The samples were superficially disinfested in 1% sodium hypochlorite for 2-3 min and then in 70% ethanol for 45 sec. Two to three fragments of 4x4 mm from each tissue were separately cultured on 2% water agar and potato dextrose agar (PDA), and purified on PDA. Just a pathogenic fungal species, Wilsonomyces corpophilus was isolated from the infected buds and twigs. No microorganism was isolated from the fallen leaves and fruits collected from underneath of the infested stone fruit trees. Pathogenicity of the fungus was examined on detached shoots of current year of four varieties of stone fruit trees. Fungal discs were placed under the bark of the bud base. Control shoots were similarly treated with sterile PDA discs. Inoculated shoots were placed in a humid growth chamber at 25 degrees C. Fungal hyphae appeared at 30 days post inoculation. Control shoots were asymptomatic. Pathogenicity intensities or lesion lengths were significantly different among the four varieties tested. A completely randomised design with five replicates was employed to measure the number of spores in infested buds and twigs of each variety of stone fruit tree. The samples were sliced and placed into a glass tube of centrifuge containing 3 ml of sterile distilled water. They were mixed on a vortex mixer for 30-40 min and centrifuged at 3000 rpm for 5 min. Pelleted material from each sample was suspended in 500 microl of sterile distilled water and the spores were counted using a hemocytometre. Results revealed that the fungus overwinters as hyphae and conidia in the infected buds, and as hyphae and globular chlamydospores in twig lesions.     

A method for both mass and individual rearing of fungivorous astigmatid mites (Acari)

Several species of common fungi were assessed as food for fungivorous astigmatid mites. Hypocrea nigricans, Botrytis cinerea and Flammulina velutipes were generally good food sources for most mites examined. Fungal mycelia growing on PDA (potato dextrose agar) medium were not only nutritionally adequate but the system also maintained high humidity through the water-based agar medium. Among acarid mites, most species of Rhizoglyphinae could be reared easily with the method. Although filter-feeding histiostomatid mites do not feed directly on hyphae, some species were successfully maintained with the same method through multiple generations. Presumably, these mites obtained sufficient nutrition from the agar medium and fungal metabolites leaching into it. Most species ultimately produced dispersing heteromorphic deutonymphs on these media. Individual mites were also maintained in isolation within glass rings on fungal colonies. Using this technique, we were able to compare developmental periods, fecundity and survival periods of mites reared under different conditions.     

Medium- and Light-dependence of Growth and Conidiomata Production in Phaeocytosporella zeae Pathogenic to Maize

Culture appearance, mycelium growth and pycnidial conidiomata formation in Phaeocytosporella zeae cultivated on potato dextrose agar (PDA), Leonian agar and carnation leaf agar (CLA) during permanent dark (25 C) or a 12-h photoperiod (24/18°C) are described. Different media and light conditions significantly affected fungus growth and the occurrence of conidiomata.
Dark conditions favoured more mycelium growth of the fungus than alternating light and dark. Moreover, fungus growth was more rapid on PDA and CLA media than on Leonian agar. Carnation leaf agar and a 12-h photoperiod provided excellent conditions for the promotion of rapid conidiomata formation in a number of P. zeae isolates.     

Corynespora cassiicola leaf spot of pawpaw (Carica papaya L.) in Nigeria

Leaf spot of pawpaw is hereby reported for the first time in Nigeria. The symptom is characterized by a papery center surrounded by a yellow halo. The causal organism is Corynespora cassiicola. Ripe fruits and abaxial surfaces of the leaves were significantly more susceptible to infection than unripe fruits and adaxial surfaces of leaves. Growth and sporulation of the fungus on several media was investigated. The organism grew faster on malt-extract agar (MEA) derived media and slowest on potato-dextrose agar (PDA) supplemented with thiamine. Sporulation was highest on Czapek-dox agar (CDA) plus biotin and lowest on PDA and PDA + thiamine. Reasons for increased susceptibility of ripe fruit are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

Mycophenolic Acid Production by Penicillium brevicompactum on Solid Media

When grown on Czapek-Dox agar, Penicillium brevicompactum produced mycophenolic acid after a vegetative mycelium had been formed and as aerial hyphae were developing. Nutrients were still plenteous in the agar when the synthesis began. If aerial hyphal development was prevented by placing a dialysis membrane over the growing fungus, no mycophenolic acid was produced. When the dialysis membrane was peeled back and, as a consequence, production of aerial hyphae began, mycophenolic acid biosynthesis was observed. We concluded that mycophenolic acid was produced only by P. brevicompactum colonies that possessed an aerial mycelium.