广西防城港首次截获苜蓿黄萎病菌

从一批进境的美国苜蓿草样品中保湿分离得到了3株与苜蓿黄萎病菌(Verticillium albo-atrum)相似的目标菌VA1、VA2、VA3.该目标菌在PDA上生长迅速,菌丝生长0.8～1.0 mm/d,2～3 d即可生成肉眼可见的菌落斑点;菌落为白色圆形,边缘规则呈圆形,菌丝较致密,气生菌丝较少,菌落中心乳白色奶冻状;培养4～5 d即后产生气生典型轮枝状分生孢子梗,7～8 d后菌落中央表面因产生休眠菌丝开始变成黑褐色至黑色,20 d后菌落的表面和背面大部分均变黑色,仍不产生微菌核和厚垣孢子.该目标菌的纯培养用V.albo-atrum特异引物Vaa1/Vaa2进行检测,PCR 扩增后得到预期330 bp的产物片段,产物序列与V.albo-atrum相应序列的相似性为100％.从而鉴定确认该目标菌为苜蓿黄萎病菌V.albo-atrum.     

辽宁局全国首次在进境荷兰马铃薯微型薯中检出苜蓿黄萎病菌

正2017年3月,大连机场出入境检验检疫局将首次从荷兰进境的马铃薯微型薯送至国家级重点实验室北方隔离苗圃进行隔离检疫,经查验发现一表面干腐,切开后有浅褐色病变组织、无凹陷症状种薯,采用PDA培养后进行形态观察,初步判断为轮枝孢属(Verticillium)真菌,提取核酸采用苜蓿黄萎病菌特异性引物(ITS1/4和Vaa1/2)进行巢氏PCR扩增测序比对后,明确该病菌为苜蓿黄萎病菌(V.albo-atrum),这是我国口岸首次在马铃薯中检出该检疫性病原真菌。     

进境油菜籽中黑胫病菌和茎基溃疡病菌的检测

为准确鉴定从进境澳大利亚油菜籽样品中分离的真菌分离物,利用形态学特征、PCR检测、序列分析以及致病性测试等方法对分离物6382-43和6382-51进行了鉴定试验。结果表明,分离物6382-43的形态特征和油菜茎基溃疡病菌Leptosphaeria maculans相似,菌丝生长较慢,菌落边缘不规则,不产生色素。油菜茎基溃疡病菌特异性引物LmacF/LmacR检测为PCR阳性;ITS区序列和油菜茎基溃疡病菌的序列相似性为99.8%;接种幼嫩油菜子叶产生油菜茎基溃疡病的典型症状。分离物6382-51的形态特征和油菜黑胫病菌L.biglobosa相似,菌丝生长较快,菌落边缘规则,产生色素;油菜黑胫病菌特异性引物LbigF/LmacR检测为PCR阳性;ITS区序列和油菜黑胫病菌的序列相似性为100%;接种幼嫩油菜子叶产生油菜黑胫病的典型症状。根据分离物的形态特征、PCR检测结果、序列分析以及致病性测试结果,将进境澳大利亚油菜籽样品中的真菌分离物6382-43和6382-51分别鉴定为油菜茎基溃疡病菌Leptosphaeria maculans和油菜黑胫病菌L.biglobosa。     

苜蓿黄萎病菌实时荧光PCR检测方法

苜蓿黄萎病菌(Verticillium albo-atrum)能够危害多种重要的农艺和园艺作物,是我国重要检疫性有害生物。本研究根据V.albo-atrum的ITS基因序列,结合张正光设计的引物Vaa-1和Vaa-2,设计一条TaqMan探针Vaa-probe,研究实时荧光PCR的检测方法,以更加快速、灵敏的检测出V.albo-atrum。利用该方法可检测到含V.albo-atrumDNA浓度0.0005 ng/μL以上的样品,大大提高了检测的灵敏度。     

进境美国苜蓿草中值得关注的真菌帚状刺壳霉

从进境美国苜蓿干草中发现带有褐色至黑色斑点的苜蓿草茎杆,经保湿培养,在病部表面形成分生孢子器,球形或近球形,褐色至黑色,孔口边缘着生刚毛。分生孢子透明,光滑,单细胞,近圆柱形至卵圆形。在PDA上,菌落初为黄褐色,后转为橄榄绿色,气生菌丝体稀疏,边缘不规则,表面呈绒状,有褶皱,背面呈橄榄绿色。培养5 d后开始产生分生孢子器。培养过程中有厚垣孢子产生和微循环产孢现象,菌丝束浅棕色。Blast分析表明,从分离物基因组中扩增到的ITS基因与Gen Bank中已知的帚状刺壳霉(Chaetopyrena penicillata(Fuckel)Hhn.1918)菌株的ITS序列同源性达99%~100%,基于ITS基因序列的系统发育分析显示分离物与帚状刺壳霉(C.penicillata)聚在同一簇。综合形态学特征、培养性状及ITS序列分析,将分离物鉴定为帚状刺壳霉(Chaetopyrena penicillata(Fuckel)Hhn.1918),此为我国口岸首次截获。迄今为止,除伊朗和南非外,世界其他国家和地区尚未有该病菌在苜蓿草上发生为害的报道。     

进境柠檬样品上柑桔溃疡病菌的检测

从进境柠檬样品上分离到一株疑似柑桔溃疡病菌的分离物X206,对分离物进行了菌落形态学特征观察、16S r RNA序列测定、PCR检测、Biolog测定及致病性测试。试验结果表明分离物X206在NA培养基上形成黄色,有光泽,圆形,全缘,微隆起,粘稠状的菌落。特异引物Xac01/Xac02扩增分离物X206的DNA得到582bp的预期产物,产物序列与柑桔溃疡病菌序列的相似性为100%。其16S r RNA序列与柑桔溃疡病菌的序列完全一致。Biolog测定将分离物X206和柑桔溃疡病菌阳性菌株FZ01均鉴定为Xanthomonas campestris pv.dieffenbachiae。致病性测试显示分离物X206能导致柑桔叶片产生明显的溃疡病斑。根据试验结果将分离物X206鉴定为柑桔溃疡病菌(Xanthomonas axonopodis pv.citri)。     

进境澳大利亚绿豆中菜豆细菌性萎蔫病菌的检测

利用NA培养基从澳大利亚进境绿豆样品中分离到一株疑似菜豆细菌性萎蔫病菌(Curtobac-terium flaccumfaciens pv.flaccumfaciens,Cff)的细菌分离物2000-1,对该分离物进行革兰染色试验、PCR检测、多位点序列分析和致病性测试.分离物在NA平板上菌落浅黄色,圆形,隆起,黏性,...     

进境菜豆种子中普通细菌性疫病菌的分离和鉴定

对经甘肃口岸进境的30批菜豆Phaseolus vulgaris种子进行了普通细菌性疫病菌的检测,利用选择性培养基MT从波兰进境菜豆种子上分离到1株细菌597,对该分离物进行菌落形态特征观察、致病性测定、16S rDNA及16S-23S rDNA ITS序列分析和特异性PCR检测。结果表明,该分离物在MT培养基上菌落呈黄色、圆形、黏稠、表面光滑向外隆起、菌落周围有水解圈。分离物597接种菜豆幼苗后导致叶片枯萎,接种点干枯。结合菌落形态、16S-23S rDNA ITS序列、特异性PCR检测结果,将分离物597鉴定为地毯草黄单胞杆菌菜豆致病变种Xanthomonas axonopodis pv.phaseoli。     

进境美国大豆中大豆茎褐腐病菌的截获

 从进境的美国大豆豆秆样品中分离到2株疑似大豆茎褐腐病菌Phialophora gregata的分离物247-8和8300-5,2株分离物在PGM培养基上菌落圆形,边缘规则,白色至暗褐色,表面隆起,粗糙,轮纹状。分生孢子卵形至椭圆形,无色,平滑,单胞,平均大小4.3 μm×1.9 μm。分生孢子梗具有瓶梗状结构,无色,无隔膜或有隔膜,大小(5~16)μm×(2~3) μm,呈桶型或长颈瓶型。特异性引物BSRIGS1/2、BSR1/2和Pgl/4分别扩增分离物247-8的DNA得到预期1 022 bp、483 bp和499 bp的产物;分离物8300-5的DNA经PCR扩增分别得到834 bp、483 bp和499 bp的预期条带。2株分离物的ITS区序列完全一致,与GenBank中大豆茎褐腐病菌(登录号AB190396、DQ459387、DQ459386和AF132804)的序列相似性为100%。人工接种大豆幼嫩植株茎基部均产生大豆茎褐腐病菌的典型症状。根据分离物形态特征、PCR检测、序列分析以及致病性测试结果,将进境美国大豆样品中的分离物247-8和8300-5鉴定为大豆茎褐腐病菌P. gregata。     

法国向日葵种子中向日葵黑茎病菌的首次截获与检测

从法国进境向日葵种子中分离到3株疑似向日葵黑茎病的菌株,对所有菌株进行形态学、致病性测定和分子序列比对分析。分离菌菌落乳白色或象牙色至灰白色,有大量黑褐色分生孢子器产生,分生孢子器球形至扁球形,内含无色单胞、卵圆形分生孢子,有明显或不明显油球;针刺接种4片真叶向日葵幼苗的下胚轴,7～9d后茎部产生典型黑茎病黑色椭圆形病斑,病斑上着生黑色分生孢子器;菌丝DNA用ActF1/R1和ITS1/ITS4扩增,序列与NCBI基因库中P.macdonaldii序列相似性为98%～100%。形态学、分子生物学及致病性检测结果显示,截获的3株菌均为向日葵黑茎病菌。     

丹皮酚对植物病原真菌的体外抑制作用

为揭示丹皮酚在牡丹体内的抗菌机制,采用生长速率法测定了丹皮酚对供试病菌菌丝生长的抑制作用;用涂布平板法测定了丹皮酚对供试病菌孢子萌发及形成的影响。结果表明,丹皮酚含量与病菌菌落直径线性增长及菌丝生物学产量的相对抑制率呈正相关,在丹皮酚含量0.08、0.4mg/mL培养液中培养禾谷丝核菌和茄病镰刀菌5天,对菌丝干重的抑制率分别为79.81%和55.08%。丹皮酚对供试病菌的孢子萌发无作用,但孢子萌发后,其芽管基部膨大、顶端膨大及中间局部膨大。丹皮酚推迟病菌分生孢子梗的形成时间,降低分生孢子的形成数量,在0.20、0.24mg/mLPSA培养基上培养玉蜀黍赤霉和茄病镰刀菌10天,对分生孢子形成的抑制率分别为100%和33.33%。     

韭菜提取物及其主要活性物质防控苹果轮纹病发生的研究

本文研究了韭菜提取液对苹果轮纹病菌菌丝生长及其形态的影响;以苹果枝条和果实为材料,研究了韭菜提取液对苹果轮纹病的抑制作用;并进一步探索了韭菜中4种主要活性成分对苹果轮纹病菌菌丝生长的抑制作用。结果表明,在离体条件下韭菜提取液对苹果轮纹病菌菌丝生长具有显著的抑制作用,在韭菜提取液为1:250(1 g韭菜/250 mL水)和1:100(1 g韭菜/100 mL水)的平板上生长5 d后,苹果轮纹病菌斑直径分别为对照的43.25%和4.62%;在提取液浓度为1:20(1 g韭菜/20 mL水)的平板上,苹果轮纹病菌的生长被100%抑制。韭菜提取液对菌丝形态有明显的破坏作用。韭菜提取液对苹果轮纹病的发生也存在显著的抑制作用,1:100的韭菜提取液处理的枝条和果实上病斑直径分别为相应对照菌斑直径的46.25%和42.35%;而浓度为1:20韭菜提取液处理则100%抑制了苹果轮纹病的发生。韭菜所含的4种主要活性成分中,二甲基三硫和甲基烯丙基三硫醚对苹果轮纹病菌生长具有最强的抑制效果,在浓度达1:4000(v/v)时即可100%抑制菌丝生长。以上结果表明韭菜提取液及其主要成分对苹果轮纹病菌丝生长具有显著的抑制作用,对由该病原菌引起的苹果轮纹病的发生具有显著的防控效果,为苹果轮纹病的防控提供新的思路。     

辣木枝枯病病原菌鉴定及其生物学特性

为明确海南省辣木枝枯病病原菌及其生物学特性,采用组织分离法获得菌株MO157,通过柯赫氏法则、形态学特征及分子生物学技术对该菌株进行鉴定,并测定了其菌丝生长的最佳条件。结果表明,菌株MO157回接10 d后,辣木茎干表面周围呈黄褐色,随后发病部位表现出水渍状病斑,与自然发病辣木的病状相符;菌落近圆形,菌丝初期呈白色绒毛状,后期呈浅黄色,分生孢子顶胞钩状,3~5个隔膜,厚垣孢子呈球形,在菌丝间串生;菌株MO157的ITS序列与Gen Bank中木贼镰刀菌Fusarium equiseti的相似性为100%,结合形态特征与分子鉴定最终将其确定为木贼镰刀菌(Gen Bank登陆号:KX197955)。该菌菌丝生长适宜温度25~30℃,最适温度28℃;pH 6~8时菌丝生长速率增快,pH 7时最适菌丝生长;PDA培养基和PSA培养基最适合该菌生长,以蔗糖为碳源时利用率最高;以牛肉浸膏为氮源时利用率最高;该菌致死温度为65℃,10 min;光照对菌丝生长影响较小,不同处理间菌落直径无显著差异。     

The Course of Colonization of Two Different Vitis Genotypes by Plasmopara viticola Indicates Compatible and Incompatible Host-Pathogen Interactions

ABSTRACT The course of colonization of leaf mesophyll by the causal agent of grapevine downy mildew, Plasmopara viticola, in a susceptible and a resistant grapevine genotype was examined in order to characterize the development of the pathogen in compatible and incompatible host-pathogen interactions. Within a few hours after inoculation, the pathogen was established in the susceptible Vitis vinifera cv. Müller-Thurgau and formed primary hyphae with a first haustorium. No further development occurred in the following 10 to 18 h. The next step, in which the hyphae grew and branched to colonize the intercellular space of the host tissue, was observed 1.5 days after inoculation. After 3 days, the intercostal fields were entirely filled with mycelium and sporulation was abundant under favorable environmental conditions. The first infection steps were essentially the same in the resistant V. rupestris. However, the invasive growth of P. viticola was delayed, and further development ceased before the intercostal fields were filled with mycelium.     

Effects of the fungus Crinipellis perniciosa, causal agent of witches' broom disease, on cell and tissue cultures of cocoa (Theobroma cacao L.)

The symptoms of witches' broom disease in cocoa, caused by the Basidiomycete fungus Crinipellis perniciosa , are pronounced swelling of the terminal and axillary buds followed in the long term by necrosis of this tissue. The direct effect of C. perniciosa on cocoa cells was examined under controlled conditions by growing primary and secondary phase cultures of the fungus separately and also with callus cultures and with cell suspensions. Both primary and secondary phase mycelium reduced growth of callus cultures by about 47% after one week compared with the controls. However, cell suspensions containing primary phase mycelium showed initial growth double that of the uninfected controls after 5 days, but then growth was reduced below that of the control and particularly when the primary phase became secondary phase mycelium. This change in fungal development coincided with the time that the cell culture reached the stationary growth stage. Cell cultures inoculated with stationary phase mycelium showed the same growth as the control after 5 days but then growth was reduced to 50% of the control after 19 days incubation and remained at this low level subsequently. The inhibitory effect of secondary phase mycelium was examined by incubating callus and cell suspensions with culture filtrate from liquid cultures of the secondary phase. Inclusion of 50% by volume of culture filtrate from the secondary phase in the growth medium for callus and cell suspensions, respectively, resulted in a reduction in growth of the plant tissue cultures. Addition of fungal culture filtrates also led to loss in potassium and loss of viability of cell suspensions and of isolated cells as represented by protoplasts. The necrotrophic mode of the secondary phase may be achieved through the production of phytotoxins acting on the host cell membrane.     

来源于中药的稻瘟病菌拮抗解淀粉芽胞杆菌鉴定及其活性成分特性

为寻找新的水稻稻瘟病拮抗微生物资源,本研究从中药提取液中分离纯化到一株性能优良的拮抗细菌CWJ2。平板对峙试验显示该菌株对水稻稻瘟病菌丝生长抑制率为69.51%。根据菌落、菌株形态、16S rDNA和gyrB基因序列,鉴定该菌株为解淀粉芽胞杆菌Bacillus amyloliquefaciens。该菌株发酵滤液能明显抑制稻瘟病菌菌丝生长和孢子萌发,10%的发酵滤液能100%抑制菌丝生长;2%的发酵滤液能导致菌丝分支明显减少,菌丝变短、膨大或畸变。菌株CWJ2的发酵液在高温、蛋白酶K、紫外和酸碱处理下,抑菌活性无显著变化。试验结果表明,菌株CWJ2对稻瘟病菌拮抗效果好,且抗菌活性成分稳定,有望作为生防菌株开发防治稻瘟病制剂。     

Colonization of rose by Sphaerotheca pannosa (Wallr.) Lév. var. rosae Wor.

Colonization of rose by powdery mildew (Sphaerotheca pannosa) is described in terms of mycelium growth, conidiophore production and sporulation in time. The data used are gathered during different years, put together and treated by means of graphic models. Colonies could be separated into fast and slow growing colonies. Colonies initiated on leaves of increasing age showed a decreasing growth rate. Production of conidiophores and conidia started on the same day, and the relative activity of conidiophore production reached its maximum 6 days after the end of the latency period, followed 1 day later by the maximum activity of conidium production. Both conidiophore and conidium production continued for a long time at a low level. The effect of leaf age on conidiophore production found expression in differences in production rate during the first days of colony development and in final production levels. Observations on naturally infected leaves in an outdoor experiment showed a rapid decrease of sporulation on leaves of 10 days and older. Highest percentages of sporulating leaf area were observed on leaves between 7 and 10 days old.     

培养条件对柑橘黑斑病菌丝生长的影响

以南丰蜜桔黑斑病菌为实验材料,研究了不同生长环境下病原菌生物学特性。结果表明,病菌在5~35℃的范围内均能生长,25℃为菌丝生长的最适温度。适宜pH值范围为5~7,最适pH值为6;在不同培养基中,菌丝在PDA培养基上生长最快,在OA培养基上菌落周围产生一定量的黄色素;光照时间长短对菌丝生长几乎无影响。     

灰枣红点软腐病病原菌的鉴定

 The damage and symptoms of jujube fruit soft rot in Xinzheng, Henan Province, were investigated from 2006 to 2008, and the pathogen was identified based on the morphological characteristics and the ITS se-quence of ribosome DNA. The results showed that the aerial mycelium of colony was white in color in the first four days, then turned gray after incubation on PDA for 5-6 d at 25℃ and became black two weeks later. The mycelia grew luxuriantly with velvet character. The pycnidium was flask-shaped with a height of 196.9μm and a width of 213.3μm on the avereage. The conidium was colorless with single cell and had the shape of spindle, its size was (15.0-20.0)μm× (4.5-6.5)μm. The conidiophore was fastigiate. The homology of ITS sequence of ribosome DNA between the tested strain NXK and Botryosphaeria dothidea (GenBank ac-cession number:AJ938005) reached 99.87%, with a difference of only two base pairs. Based on the results of both morphological characters and molecular identification, the pathogen of jujube fruit soft rot in Xinzheng was identified as B. dothidea (Moug. ex Fr.) Ces. et de Not..