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。     

不同引物PCR检测向日葵黑茎病菌的灵敏度比较及应用

为准确快速筛查进境油葵种子中携带的向日葵黑茎病菌Plenodomus lindquistii,采用常规PCR方法比较了3对向日葵黑茎病菌PCR检测引物320FOR/320RVE、LEPB/LEPF和LLF/LLR的灵敏度,对哈萨克斯坦进境120批油葵种子样品进行PCR检测,并对检测为阳性的样品进行产物序列测定及病原菌分离。结果显示,引物320FOR/320RVE的检测灵敏度最高,可达10-5ng/μL,引物LEPB/LEPF和LLF/LLR分别为10-4ng/μL和10-3ng/μL。利用引物320FOR/320RVE、LEPB/LEPF和LLF/LLR对120批进境油葵种子样品进行检测,阳性检出率分别为65%、50%和45%,阳性检出率高低与引物的检测灵敏度相一致。阳性样品扩增的产物序列与Gen Bank中向日葵黑茎病菌的序列同源性最高;分离的病原菌菌落为乳白色至灰白色,分生孢子器黑褐色、球形并产生透明状分生孢子液,分生孢子单胞、无色、肾型、两端有油球,与已报道的向日葵黑茎病菌的病原特征描述一致,初步鉴定哈萨克斯坦进境油葵种子中存在向日葵黑茎病菌。     

瓜拟多隔孢引起的砂田西瓜叶疫病

 近年来,甘肃省白银市靖远县的一些砂田西瓜中出现了严重的叶疫病,严重田块病株率超过70%。2018年7月,从靖远县高湾乡罹病西瓜叶片上分离得到拟多隔孢属Stagonosporopsis真菌,病叶检出率达100%。采用离体叶片和植株接种法评价单孢分离菌株XG-3对西瓜和甜瓜的致病性,所有接种处理在24 h内均发病显症而对照未发病,其中发病叶片原接种菌的检出率为100%。菌株XG-3在PDA和OA平板培养基上20和25 ℃培养20 d,未见产孢。在自然感病的西瓜叶片上观察到少量分生孢子器,在人工接种发病的西瓜和甜瓜茎、叶上产生大量分生孢子器和分生孢子:分生孢子器球形至亚球形,大小为(82.4~243.3) μm×(82.4~188.4)μm,分生孢子器具1~2个孔口,孔口直径15.7~27.5 μm;分生孢子无色,0~3个隔膜,杆状、柱状、长椭圆形、花生形及不规则形,直或稍弯曲,分隔处不缢缩或缢缩,大小为(5.2~28.3)μm×(2.2~6.0)μm,分生孢子形态和大小依基质和环境条件的不同而有较大差异。BLASTn分析结果显示,菌株XG-3(GenBank登录号:MW282128)的rDNA-ITS序列与瓜拟多隔孢Sta. cucurbitacearum分离物287ITS(GenBank登录号:AY293804.1)的序列相似性达99.80%。在基于rDNA-ITS构建的系统发育树中,菌株XG-3与Sta. cucurbitacearum聚为一组,与西瓜拟多隔孢Sta. citrulli和木瓜拟多隔孢Sta. caricae区分开来。依据病菌形态学和分子生物学特征,将菌株XG-3鉴定为瓜拟多隔孢Sta. cucurbitacearum [Basionym: Sphaeria cucurbitacearum]。     

进境美国大豆夹杂向日葵黑茎病菌的检疫鉴定

正大豆是一种重要的粮食作物和食用油原料,国内的市场需求量日益增长,目前我国已成为大豆净进口国。进口大豆虽能满足国内市场需求,但加大了外来有害生物传入的风险。向日葵黑茎病是一种毁灭性的向日葵病害,其病原菌向日葵黑茎病菌Plenodomus lindquistii属于我国进境检疫性植物病原真菌(段维军等,2015;2017)。2017年12月,宁波口岸自进境美国大豆夹杂的向日葵种子上分离获得1株菌株72298-10,为明确其分类地位,本研究通过形态学观察、ITS序列分析和致病性测定对其进行鉴定,以期为口岸疫情检测、防控和后续处理等工作提供参考。     

矮化核桃枝枯病病原菌的形态与分子鉴定

为了明确河南省洛阳地区核桃枝条一种新病害的病原种类,对采自田间的典型症状枝条进行了常规组织分离,对单孢菌株进行了形态鉴定、rDNA ITS序列分析及致病性测定。结果表明,病原菌在PDA培养基上菌落初为白色,随着色素的积累,由中心向边缘变为灰绿色,再变成墨绿色,最终全部变成黑色;气生菌丝棉絮毛状,菌落边缘不整齐,近圆形或不规则形。分生孢子器散生或聚生,呈黑色小粒状,分生孢子器球形或近球形,深褐色,具孔口;分生孢子梗短缺,无色,杆状,末端产生分生孢子;分生孢子梭形、纺锤形,无色,无隔,基部钝圆,顶部稍尖,单孢(24.5～29.0)μm×(4.8～7.5)μm。离体枝条接种结果表明,该菌能侵染核桃枝条引起与田间相同的症状。据此将分离物鉴定为葡萄座腔菌Botryosphaeria dothidea。     

河南省小麦全蚀病菌的分离及变种类型鉴定

 从河南省10个地市采集的小麦病根样品中分离得到82株病原分离株, 通过形态特征观察、回接致病性测定、以及分子生物学的方法对所分离菌株进行鉴定。其中47个菌株菌丝分支处都形成典型的“∧”状;子囊壳埋生或半埋生, 子囊棍棒状, 子囊孢子线形, 稍弯曲, 无色, 具有5~10个分隔, 大小为68~94 μm×2~4 μm;对82个分离株rDNA-ITS区进行PCR扩增和序列测定, BLAST序列比对结果表明, 47株菌株与小麦全蚀病菌的ITS序列同源性达97%～99%, 据此确定47株菌株为小麦全蚀病菌。应用小麦全蚀病菌4个变种的特异性引物进行PCR扩增都得到禾顶囊壳小麦变种(870 bp)的特异性片段, 鉴定47株菌株均为禾顶囊壳小麦变种 (Gaeumannomyces graminis var.tritici)。     

甘肃省马铃薯枯萎病（Fusarium avenaceum）鉴定及其病原生物学特性

对甘肃省马铃薯枯萎病菌进行了分离、鉴定及培养特性的研究。结果表明,该病原菌大型分生孢子镰刀形,无色,多细胞,大小为（12.5~30）μm×（2.5~5）μm;小型分生孢子数量较多,无色,卵圆形或椭圆形,大小为（4.70~12.94）μm×（1.76~2.35）μm;ITS序列分析表明,病原菌与Fusarium avenaceum（JN6317483）亲缘关系最近,相似性达到100%,结合形态特征鉴定该病原菌为燕麦镰刀菌(Fusarium avenaceum)。该病原菌在5~40 ℃范围内均可生长,最适温度为20 ℃;供试的碳、氮源中,果糖和亮氨酸对该菌菌丝生长有显著的促进作用（P<0.05）,而显著抑制菌丝生长的是氯醛糖和碳酸铵（P<0.05）;不同类型培养基中,燕麦片培养基对菌丝生长有显著促进作用（P<0.05）。     

进口智利李子上核果褐腐病菌鉴定

从进口智利李子上分离到一种引起李子果实腐烂的病原真菌,接种李子能引起果实变褐和腐烂。该真菌菌丝在PDA培养基上的生长最适温度为25℃,产孢少,菌落初始为浅灰色,边缘浅裂状,多次传代后逐渐变为浅白色,边缘整齐。分生孢子无色,单孢,柠檬形或卵圆形,大小11．5（9．5～14．7）μm&#215;7．4（5．3～9．2）μm。ITS序列分析结果表明该分离物ITS区序列与GenBank中登录的16株舱laxa序列相似性分别为99．8％～100％,碱基的差异为0～1bp;与21株旭frueticola序列相似性为99．0％～99．2％,碱基的差异为4～5bp;与10株M.fructigena的序列相似性为97．5％～97．7％,碱基的差异为13～14bp。基于ITS序列的系统发育关系分析表明分离物sh675和GenBank中登录的16个M.laxa菌株属于同一个聚类群,相关的Mfructieola和M.fructigena属于另一个聚类群。M.laxa特异性引物ITS1Mix／ITS4Mlx能扩增分离物sh675的菌丝DNA,得到预期的356bp扩增产物。根据该真菌的菌落形态特征、分生孢子形态特征、ITS序列和PCR检测结果,将其鉴定为核果褐腐病菌（Monilinia laxa）。     

桑里白粉病菌重寄生真菌的分离与鉴定

为了解桑里白粉病菌的重寄生真菌种类,通过分离和寄生性验证,获得2个桑里白粉病菌重寄生真菌菌株HP8和HP9,这两个菌株寄生闭囊壳后,子囊及子囊孢子溃解。菌株HP8菌落绿色,菌丝体无色至褐色,菌丝有隔膜,分生孢子梗结节状膨大,分生孢子椭圆形、单胞、褐色或无色,ITS序列（MT463536）与尖孢枝孢Cladosporium oxysporum（MF135506）同源性为100%,系统进化树分析发现菌株HP8与尖孢枝孢（MF135506）聚在一起;菌株HP9菌落白色,菌丝无色,分生孢子月牙形、单胞、无色,ITS序列（MT463537）与刀孢蜡蚧菌Lecanicillium psalliotae（KC881072）同源性为99%,在系统进化树中,菌株HP9与刀孢蜡蚧菌（KC881072）聚在一起。结合形态特征和ITS序列分析,鉴定出菌株HP8和HP9分别为尖孢枝孢和刀孢蜡蚧菌。高通量测序结果表明,病桑叶在室内保存4个月后,桑里白粉病菌的重寄生真菌为刀孢蜡蚧菌。     

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。     

Notes on pistachio seed wasps from two locations in the east Mediterranean

Two seed wasps, Eurytoma plotnikovi Nikol’skaya. (Hymenoptera: Eurytomidae) and Megastigmus pistaciae Walker (Hymenoptera: Torymidae), were collected from pistachio nuts, Pistacia vera (Anacardiaceae), in Gaziantep province in Turkey and in El Harub, Judean Hills, Palestinian Authority. E. plotnikovi was clearly identified for the first time in Turkey among the reared materials, but in the past it had been recorded there by several authors under different names. The percentages of fruit infestations by these wasps in untreated pistachio orchards in Gaziantep province and in one orchard in El Harub were 69% and 81%, respectively. Gugolzia karadagae Doğanlar (Hymenoptera: Pteromalidae) was responsible for 2.2–17.5% parasitism of E. plotnikovi in Gaziantep province. So far, G. karadagae was not found in any other part of Turkey, nor in El Harub or Israel.     

Cloning, Sequencing and Expression of a Xylanase Gene from the Maize Pathogen Helminthosporium Turcicum

A gene encoding an endoxylanase from the phytopathogenic fungus Helminthosporium turcicum Pass. was cloned and sequenced. The entire nucleotide sequence of a 1991 bp genomic fragment containing an endoxylanase gene was determined. The xylanase gene of 795 bp, interrupted by two introns of 52 and 62 bp, encoded a protein of 227 amino acids showing up to 95% amino acid homology with other fungal xylanases. The precise splicing site of the introns was identified by sequencing the corresponding cDNA. A northern blot showed that the gene is expressed when the fungus is grown in a medium containing xylan as a sole carbon source. The cloned xylanase gene was expressed in maize plants during infection.     

芥菜乙醇提取物对西瓜枯萎病菌的抑菌机制

为探究芥菜乙醇提取物对植物病原菌的抑制机理,以西瓜枯萎病菌Fusarium oxysporum f.sp. niveum为指示菌,以最低抑菌浓度(minimum inhibitory concentration,MIC)0.5 g/mL为基准,设置0、0.25、0.5和1.0 g/mL共4种处理浓度,通过测定菌丝形态结构、菌液电导率、菌体丙二醛含量、细胞壁相关水解酶活性、呼吸代谢途径酶活性、可溶性蛋白含量及还原糖含量等指标的变化分析其抑菌机理。结果显示,1.0 g/mL芥菜乙醇提取物对西瓜枯萎病菌的处理效果最好,其中,处理后病菌菌液的电导率明显增大,最大可达1.45 ms/cm,是对照组的1.57倍;丙二醛含量达3.16 mmol/g,为对照组的2.24倍;几丁质酶和β-1,3-葡聚糖酶的活性最高,分别为10.57 U/g和14.16 U/g,较对照组分别显著升高了48.04%和17.80%,同时细胞壁降解,使细胞完整性受损;琥珀酸脱氢酶和苹果酸脱氢酶的活性最低,分别为7.33 U/mg和0.28 U/mg,较对照组分别显著下降了73.02%和47.17%;可溶性蛋白和还原糖的含量显著降低,分别为0.44 mg/g和5.34 mg/g,较对照组分别下降了60.71%和56.30%,呼吸能量代谢受阻,抑制菌体生长。表明芥菜乙醇提取物可有效防治西瓜枯萎病。     

杠柳杀虫活性成分的分离

采用柱层析分离、高效液相色谱切分和生物活性追踪法,从杠柳 Periploca sepium 根皮甲醇提取物中分离出2个具有杀虫活性的化合物(G1和G2),经鉴定其分别为已知物杠柳新苷D和F。生物活性测定结果表明,化合物G1和G2 对3龄粘虫 Mythimna separata 48 h的胃毒致死中浓度(LC50)分别为0.39和0.34 mg/mL, 对小菜蛾 Plutella xyllostella 48 h的胃毒LC50值分别为1.21和1.39 mg/mL。     

广西油茶炭疽病病原菌鉴定及生物学特性

为明确广西油茶炭疽病病原菌种类及其生物学特性,通过形态学方法对分离获得的病原菌进行初步鉴定,以核糖体内转录间隔区、微管蛋白基因、几丁质合成酶基因、肌动蛋白基因、3-磷酸甘油醛脱氢酶基因进行多基因系统分析鉴定;并用十字交叉法、菌丝干重法和血球计数法对其生物学特性进行研究。结果表明,经形态学和多基因联合系统结合分析鉴定,广西油茶炭疽病病原菌为胶孢炭疽菌Colletotrichum gloeosporioides Penz.。该病原菌生长温度范围为10~35℃,最适生长温度为28℃;产孢温度范围为15~32℃,最适产孢温度为32℃;生长p H范围为3~11,最适生长p H为4;可促进菌丝生长并适合产孢的碳源为菊糖,阿拉伯树胶粉抑制菌丝生长但可促进产孢;对菌丝生长及产孢均有促进作用的氮源为酵母粉和牛肉膏,而硫酸铵和硝酸铵则表现抑制作用。     

Differentiation of three whitefly-transmitted geminiviruses from the Republic of Yemen

Three viruses collected in southern Yemen in 1990, infecting watermelon, tobacco and tomato were shown to be transmitted by the whiteflyBemisia tabaci and to have particle morphologies typical of geminiviruses. Colonies ofB. tabaci collected from different locations and from different hosts were used in virus transmission tests with the same host range of plants. Colonies established from both watermelon and cotton in the Yemen were identified as the squash silverleaf-inducing B biotype. The culture host of the colony did not influence virus acquisition and transmission efficiencies to and from other hosts. The tobacco and tomato geminiviruses had a similar host range, but differed in their severity in some hosts. Both these viruses differed from the watermelon geminivirus in host range and symptoms.Datura stramonium, an alternative host for all three viruses, could be co-infected by the watermelon and tobacco viruses.B. tabaci was able to acquire both viruses from the co-infectedD. stramonium and infect seedlings of either original host plant species with their respective viruses orD. stramonium with both. The viruses were identified as watermelon chlorotic stunt virus, tobacco leaf curl virus and tomato yellow leaf curl virus and were distinguished by cross hybridisation.     

Note: Molecular identification of three entomopathogenic nematodes from turkey by PCR-RFLP of the ITS regions

Molecular identification methods are widely used for the classification of organisms worldwide. Entomopathogenic nematodes are the most often isolated insect parasitic nematodes in the tropical and subtropical regions. In our investigation, PCR-RFLP (Polymerase Chain Reaction — Restriction Fragment Length Polymorphism) of the ITS region (Internal Transcribed Spacer) on the ribosomal (r) DNA of three entomopathogenic nematodes isolated from Ankara, Turkey, was analyzed for identification. The ITS region of rDNA was amplified by PCR and then digested with the following nine restriction enzymes: Alu I, Dde I, Hae III, Hha I, Hind III, Hinf I, Hpa II, Rsa I and Sau 3AI. The amplified and restricted sequences of the ITS regions were separated by agarose gel electrophoresis and the RFLP patterns of these three species were shown in this study. According to our results, these species were identified asSteinernema feltiae, Steinernema carpocapsae andHeterorhabditis bacteriophora. http://www.phytoparasitica.org posting Nov. 4, 2005.     

Virulence structure of theMagnaporthe grisea rice population from the northwestern Himalayas

The virulence structure of theMagnaporthe grisea rice population from the northwestern Himalayan region of India was deciphered on 24 rice genotypes harboring different blast resistance genes. Matching virulences appropriate to all the rice genotypes, except Fukunishiki (Pi-z, Pi-sh) and Zenith (Pi-z, Pi-a, Pi-i), were present in the pathogen population. Moreover, a very low percentage of isolates were virulent on Tetep (Pi-ta, Pi-k ^h, Pi-4^b) and Tadukan (Pi-ta/Pi-ta ²). Although virulence was recorded on most of the lines tested, none was susceptible to all of the isolates. Three pairs of genotypes, namely, C101LAC:C101A51; K-1: Dular; and Dular: HPU-741, exhibited complementary resistance spectra as no isolate combined virulence to both the members of each of the three pairs of genotypes despite the fact that individual members were susceptible to a major portion of the pathogen population. The blast resistance genesPi-z, Pi-k ^h, Pi-l andPi-2 and their various combinations were construed to provide broad spectrum and durable blast resistance in Himachal Pradesh. Pathotype analysis revealed the existence of extremely high pathotypic diversity in the pathogen population. Based on the observed population structure forM. grisea, it was not possible to designate a minimum set of pathogen isolates that could be used in blast resistance screens to identify effective sources of blast resistance. The overall results suggested that the pathotype analysis alone is insufficient to describe the existing pathogenic variability, especially when this information has to be used for guiding the breeding programs aimed at developing durable blast resistance. However, population genetics approach of studying pathogenic specialization by monitoring the frequency of individual virulence genes and analyzing virulence gene combinations for their association or dissociation might generate useful information for developing durable blast resistance. http://www.phytoparasitica.org posting May 14, 2006.     

A new plant pathogenic sterile white basidiomycete from Australia

A sterile white fungus was isolated from the healthy looking roots of buffalo grass (Stenotaphrum secundatum) grown on cleared bush land in Perth, Western Australia. The fungal strain was pathogenic on 12 plant species screened under the greenhouse conditions. The clamp connections and dolipore septa indicated that the isolate was a Basidiomycete. Mycelial features, growth rate at different temperatures, as well as pathogenicity patterns of this sterile white basidiomycete (SWB) were distinctly different from those of a strain with a similar morphology, ATCC 28344, previously described as a pathogen in Florida and Georgia (USA). All attempts to induce sporulation failed. The isolates were also compared using the nucleotide sequence analysis of the ribosomal DNA array. Approximately 1 kbp of the 5 end of the large subunit ribosomal RNA gene, complete sequences of the small subunit ribosomal RNA gene and the entire ITS region (including ITS1, ITS2 and 5.8S gene) were sequenced for the purpose. The obtained sequences were compared with the homologous regions of other genera of Agaricales available in GenBank. Relatively low sequence similarities between the American and Australian strains, as well as the phylogenetic analysis of the studied regions has suggested that these two fungi belong to different genera. Interesting results were achieved in the case of the large subunit ribosomal DNA since this region has been widely studied for taxonomy of Basidiomycetes. The Australian strain 3034 appeared to be closely related to the genus Campanella and the American SWB was identified as belonging to the genus Marasmius, possibly to M. graminum. Our data suggest that the Australian strain is a novel pathogen, and is different from the American SWB isolates described to date.     

Cucumber mosaic virus isolated from Aconitum spp. in Japan

Viruses were isolated from leaves of plants of Aconitum species with symptoms such as mottling and yellowing in Hokkaido and Gunma prefectures in Japan. These viruses were identified as Cucumber mosaic virus (subgroup II) based on particle morphology, host range, aphid transmission, and serology.