台湾省苹果上牛眼果腐病菌的截获鉴定

为明确从宁波空港口岸入境旅客携带苹果上截获的1株明孢盘菌属菌株60017的分类地位,通过形态学特征观察、内转录间隔区(ITS)和β-微管蛋白基因(β-tubulin)序列比对分析以及致病性测定,对该菌株进行了种类鉴定。结果表明:菌株60017在PDA培养基上的菌落为圆形,边缘整齐,呈乳白色,气生菌丝较少,可产生大量分生孢子;基于ITS和β-tubulin序列构建的系统发育树中,菌株60017和苹果牛眼果腐病菌Neofabraea kienholzii处于同一分支;菌株60017接种苹果后,在接种部位能引起褐腐症状,与原发病症状一致。结合形态学特征、致病性测定结果以及分子鉴定结果,最终将该菌株鉴定为苹果牛眼果腐病菌N. kienholzii。     

进境苹果果实中苹果牛眼果腐病菌的检疫鉴定

对来自美国的苹果进行了病原菌分离,结果在果实中分离到1株疑似苹果牛眼果腐病菌的菌株Nk-2968。对其进行了病原菌形态学观察、致病性测定并结合分子生物学方法检测,实验结果表明,菌株Nk-2968在PDA培养基上生长良好,能产生大量的分生孢子;经真菌通用引物(ITS4/ITS5)及β-tubulin引物(Bt-T2m-Up/Bt-LVL-Lo)分别扩增和测序,菌株Nk-2968与Gen Bank中登录号AF281462.1、HG793110.1和HG793112.1的菌株序列同源性达到100%;病菌接种苹果8 d后开始发病。根据上述实验结果,将分离获得的菌株Nk-2968鉴定为苹果牛眼果腐病菌(Neofabraea kienholzii)。这是我国口岸首次在进境的苹果果实上截获该危险性有害生物。     

进境澳大利亚大麦上葡萄茎枯病菌的检疫鉴定

自进境澳大利亚大麦样品上,采用常规平板分离法获得1株疑似葡萄茎枯病菌菌株74919。通过形态学特征观察、β-tubulin和actin序列比对分析以及致病性测定,对该菌株进行了种类鉴定。结果表明:菌株74919在PDA培养基上生长良好,可产生大量分生孢子器和厚垣孢子;基于β-tubulin和actin序列构建的系统发育树中,菌株74919和其他葡萄茎枯病菌相关序列划分在同一分支;菌株74919接种大麦叶片,在接种部位引起叶斑症状。根据上述实验结果,将菌株74919鉴定为葡萄茎枯病菌(Didymella glomerata),这是我国口岸首次从进境澳大利亚大麦中截获葡萄茎枯病菌。     

进境阿根廷大豆中苹果壳色单隔孢溃疡病菌的检测与鉴定

从阿根廷进境的一批大豆中分离到一株疑似苹果壳色单隔孢溃疡病菌菌株。该菌株在燕麦琼脂培养基(OA)上产生大量的分生孢子器及分生孢子;分生孢子长椭圆形,单胞,无色,继续培养3 d后变褐色,产生1隔膜,个别2隔膜;采用真菌通用引物(ITS4/ITS5)扩增和测序,该菌株与GenBank中已登录的苹果壳色单隔孢溃疡病菌16个菌株的ITS序列同源性最高,均为99%;菌株接种梨、苹果和大豆幼苗不同部位后,均发病。经形态特征鉴定、分子生物学检测及致病性测定,该菌株鉴定为苹果壳色单隔孢溃疡病菌。     

猕猴桃果腐病菌PCR和实时荧光PCR检测

为了快速、准确地鉴定猕猴桃果腐病菌(Neofabraea actinidiae),根据GenBank中N. actinidiae的β-tubulin序列设计特异引物NAC-F/R和探针NAC-P,建立了常规PCR和实时荧光PCR检测方法。利用引物NAC-F/R扩增供试的4株N. actinidiae能得到389 bp的预期目标条带,但扩增其他20个非N. actinidiae供试菌株不能得到预期产物,检测灵敏度为140 pg菌丝体DNA;探针NAC-P对供试4株N. actinidiae表现为阳性扩增,而对其他菌株和空白对照均表现为阴性扩增,检测灵敏度可达14 pg菌丝体DNA,比常规PCR高10倍。样品检测试验结果表明两种PCR方法可用于口岸植物检疫中快速、准确地检测猕猴桃果腐病菌。     

应用实时荧光PCR检测苹果果实球壳孢腐烂病菌

根据苹果果实球壳孢腐烂病菌(Sphaeropsis pyriputrescens Xiao&J.D.Rogers)ITS区序列设计特异性引物和TaqMan荧光探针,建立了苹果果实球壳孢腐烂病菌的实时荧光PCR检测方法。该方法能够特异性检测苹果果实球壳孢腐烂病菌,供试的目标菌株检测结果为阳性,而苹果轮纹病菌、苹果炭疽病菌、苹果干腐病菌和苹果褐腐病菌、苹果腐烂病菌等5种对照菌株检测结果为阴性。该方法具有快速、简便、准确的优点,适合于该病害的快速诊断和口岸检验检疫运用。     

进境美国大豆上Diaporthe ueckerae的检测鉴定

挑选美国进境的黄大豆中变色、皱缩和腐烂等症状的病籽粒进行分离培养，获得1株纯化的间座壳属真菌Duc-0530，该菌株菌落初期白色平铺，气生菌丝少，培养后期背面中央呈黑褐色。病菌α型分生孢子无色透明、光滑、纺锤形至椭圆形，无隔膜，有2个油球，大小为（5～5.8）～(6.5～7.5)μm×（2～2.3）～3.0μm（平均5.86μm×2.54μm）。利用真菌5个基因片段（ITS、β-tubulin、EF-1α、HIS和CAL）的通用引物对菌株Duc-0530进行PCR扩增和序列分析，发现该菌株的5个基因的序列与GenBank中Diaporthe ueckerae的同源性高，达到98%～100%。系统发育分析结果显示菌株Duc-0530与D. ueckerae以自展支持率100%聚集在同一分支。该菌株接种大豆和甜瓜出现茎基部位溃疡，叶片呈变褐皱缩症状。根据上述检测结果，将菌株Duc-0530鉴定为D. ueckerae。该病菌是国际上大豆生产中新发危险性病菌，为我国口岸在美国大豆中的首次截获报道。     

解淀粉芽胞杆菌XJ5挥发性物质抑菌活性及对苹果褐腐病防效测定

解淀粉芽胞杆菌Bacillus amyloliquefaciens XJ5挥发性物质对苹果褐腐病菌Monilinia fructigena等多种病原真菌具有良好的拮抗活性。通过固相微萃取-气相色谱质谱联用仪（Solid-phase microextraction-Gas chromatography mass spectrometry，SPME-GC-MS）对菌株XJ5产生的挥发性物质进行检测，利用平板对扣法测定挥发性化合物的抑菌活性，并离体接种评价菌株XJ5挥发性物质对苹果褐腐病的防效。结果表明，XJ5菌株挥发性物质对9种植物病原真菌的抑菌率在47.9%~84.8%，其中对苹果褐腐病菌的抑菌率最高为84.8%。SPME-GC-MS检测及抑菌活性测定表明，菌株XJ5分泌的主要挥发性物质为十二醛（Dodecanal），具有抑菌活性的挥发性物质主要为2-壬醇（2-Nonanol）、2-乙基己醇（2-Ethyl-1-hexanol）和2-十一醇（2-Undecanol）。其中2-壬醇抑制苹果褐腐病菌的EC₅₀为3.43 μg/mL，且XJ5菌液离体熏蒸与2-壬醇离体熏蒸均可有效抑制苹果褐腐病菌丝生长及病斑扩展，其对苹果褐腐病的离体防效分别在14.4%~71.2%和66.4%~97.6%，与对照差异显著。     

进境澳大利亚大麦夹杂油菜茎基溃疡病菌的检疫鉴定

采用常规平板分离法, 从进境澳大利亚大麦中夹杂的油菜籽上获得1株疑似油菜茎基溃疡病菌的菌株01829?通过致病性测定?形态学观察?特异性引物扩增?ITS序列比对分析, 对01829进行了种类鉴定?结果表明:菌株01829在PDA培养基上生长较慢, 菌落边缘不整齐, 产生大量分生孢子器和分生孢子; 采用特异性引物对LMR1-D和Lmb分别进行PCR检测, 结果均有预期扩增片段产生; 基于ITS序列构建的系统发育树中, 菌株01829和GenBank中其他油菜茎基溃疡病菌相关序列聚在同一分支; 菌株01829接种油菜子叶和茎基部, 在子叶和茎基部接种部位分别引起叶斑和凹陷溃疡斑?根据上述试验结果, 将菌株01829鉴定为油菜茎基溃疡病菌, 这是我国口岸首次从进境澳大利亚大麦中截获油菜茎基溃疡病菌?     

进境美国柚子中冬生疫霉的分离鉴定

从美国加州进境柚子表现褐腐症状的组织中分离到1个疫霉菌株9099,对其进行形态学特征观察、PCR检测、序列分析和致病性测定。试验结果表明,该菌株在V8培养基上菌落平贴,呈花瓣状;在PDA培养基上菌落边缘不规则,白色。孢子囊卵形或椭圆形,大小为(28.4~46.8)μm×(16.3~23.8)μm(平均37.5μm×19.4μm),长宽比为1.7~2.2∶1(平均1.9∶1)。菌株为同宗配合,卵孢子黄橙色,球形,满器,大小26.3~39.3μm(平均32.7μm),壁厚1~3μm。利用冬生疫霉(Phytophthora hibernalis)特异引物对PHIB1/PHIB2和751F/752R扩增9099的基因组DNA分别得到407 bp和616 bp的预期目标条带。菌株9099的ITS序列与Gen Bank中P.hibernalis的序列相似性为99.89%~100%。基于ITS序列构建的系统进化树显示菌株9099与P.hibernalis聚在同一个分支,亲缘关系最近。创伤接种柚子果实,接种处组织出现典型褐腐症状。根据形态学特征及ITS序列分析等,将菌株9099鉴定为P.hibernalis。     

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。     

Identification of Neofabraea species causing bull's eye rot of apple in Poland and their direct detection in apple fruit using multiplex PCR

Based on partial sequence analysis of the β‐tubulin gene, 19 isolates of fungi causing bull's eye rot on apple in Poland were classified into species: Neofabraea alba, N. perennans and N. kienholzii. To the authors’ knowledge, the detection of N. kienholzii is the second in Europe and the first in Poland. Species affiliation of these fungi was confirmed by a new species‐specific multiplex PCR assay developed on the basis of previously published methods. The new protocol allowed for the specific identification of bull's eye rot‐causing species, both from pure cultures and directly from the skin of diseased or apparently healthy apples. In 550 samples of diseased fruits collected from nine cold storage rooms located in three regions of Poland, in 2011 and 2012, N. alba was detected as the predominant species causing bull's eye rot, occurring on average in 94% of the tested samples. Neofabraea perennans was found in a minority of apple samples, N. kienholzii was found only in two apple samples, while N. malicorticis was not detected in any sample tested. In tests on 120 apparently healthy fruits, only N. perennans was detected in a single sample. The results of genetic diversity analyses of bull's eye rot‐causing fungi based on the β‐tubulin gene sequence and an ISSR (inter‐simple sequence repeat) PCR assay with two primers were consistent, showing the expected segregation of tested isolates with respect to their species boundaries. However, the genetic distance between N. perennans and N. malicorticis was very low, as reported previously.     

江西番茄烂果病病原菌鉴定

In recent years since 2018, the disease of tomato fruit rot has been often noted in Jiangxi province. In order to ascertain the causal agent, common tissue isolation method was used to isolate the pathogen collected from 8 counties and cities of Jiangxi province. A total of 17 isolates was obtained, which exhibited similar phenotype on V8 agar plates with production of antheridia, oogonia and oospore indicating the characteristics of Phytophthora spp.. The pathogenicity test for the isolates showed the similar disease symptoms with that in the field and the pathogen was reisolated from the infected tomato tissues, which fulfilled the Koch’s postulate. BLAST search with rDNA-ITS, partial Ypt1 and β-tubulin gene sequences for 17 isolates showed 99%-100% of identities to Phytophthora capsici that in correspond with the clustering result of phylogenetic analysis for two represented strains. Combined with morphologic characteristic observation, pathogenicity test and sequence ana-lysis, the pathogen causing tomato fruit rot was identified as Phytophthora capsici. This is the first report of P. capsici causing fruit rot on tomato in Jiangxi province, China.     

Intraspecific diversity within avocado field isolates of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from south-east Spain

Fifty-five isolates of Rosellinia necatrix, the cause of common avocado white root rot disease, were collected from south-east Spain and characterised according to their virulence behaviour and their molecular patterns to assess broader levels of genetic diversity. Virulence properties were revealed by in vitro inoculation on avocado plants. Differences in reaction types showed variability among these isolates. No sequence differences were observed when the internal transcribed spacer 1 (ITS1) and ITS2 regions and DNA fragments of the β-tubulin, adenosine triphosphatase and translation elongation factor 1 genes were explored in representive isolates from five virulence groups. Random amplified polymorphic DNA (RAPD) amplifications were also performed for each isolate using 19 random primers. Four of these primers revealed polymorphism among isolates and repetitive and discriminative bands were used to build an unweighted pair group with arithmetic mean tree. However, RAPD clustering showed low stability, and no correlation between RAPD and virulence groups was observed, possibly indicating high levels of sexual recombination.     

红花黄萎病病原菌鉴定

番茄斑萎病毒Tomato spotted wilt virus （TSWV）是我国进境植物检疫性有害生物,近年来相继在国内一些省份发现。利用 TSWV 的通用引物 NF302／NR575对从山东烟台地区收集的15份疑似感染番茄斑萎病毒病的番茄样品进行检测;进一步对特异引物 TSWV-NF2037／TSWV-NR2825扩增的 TSWV 的 N 基因序列克隆、测序,并对 N 基因片段编码氨基酸进行遗传距离及系统发育分析。结果表明,15份疑似样品中有4个样品扩增得到 TSWV 病毒片段;基于 N 基因序列分析发现,山东 TSWV 番茄分离物与云南 TSWV 番茄分离物（AEI70836．1）的遗传距离最近,为0．8％,且与云南 TSWV 番茄分离物聚为一支。这是山东地区首次利用分子标记证实番茄斑萎病毒病的危害。     

基于Histone H3、EF-1α和β-tubulin基因部分序列的芒果畸形病菌鉴定与系统发育研究

<正>芒果畸形病是芒果生产中重要的病害之一,主要表现为花畸形和芽畸形,受害果园常因畸形花絮无法挂果而造成减产,我国攀西地区的部分果园株发病率高达100%,给当地的芒果产业带来严重经济损失。该病自1891年首次在印度发现后,陆续在世界芒果产区发生危害~([1])。引起芒果畸形病的病原菌有Fusarium mangiferae、F.mexicanum、     

进境澳大利亚油菜籽中茎基溃疡病菌的检测

 41 fungal isolates with similar morphological characteristics to Leptosphaeria maculans were obtained by the deep-freezing filter paper method from 2100 seeds of Brassica napus imported from Australia.The isolate 8129-5 showed a slower growth on PDA at 20℃with growth rate of 2.8 mm/day.The colonies on PDA at 20℃ had an irregular or regular margin with white or grayish white compact aerial mycelium.No diffusible pigment was produced on PDA at 31℃ or in liquid Czapek-Dox media at 20℃.PCR detection showed that the isolate 8129-5 could be amplified by L.maculans-specific primers LmacF/LmacR and got expected product of 331 bp.The sequence analysis revealed that the ITS sequence of isolate 8129-5 had 99.8% identity with L.maculans.Pathogenicity of the isolate 8129-5 was confirmed on cotyledons of rape seed by artificial inoculation compared with typical symptom of L.maculans.Based on the morphological characteristics, PCR detection and the result of pathogenicity test, the isolate 8129-5 was identified as L.maculans.     

广东南瓜细菌性叶枯病及其病原鉴定

 在广东省雷州市发生一种南瓜(Cucurbita moschata)叶枯病,病株叶片边缘开始出现水渍状病斑,逐步发展成大病斑,后期病斑焦枯;在叶片上也可形成近圆形水渍状病斑,伴有黄色晕圈,后期病斑联合形成不规则大枯斑;叶柄和匍匐茎被侵染后呈水渍状腐烂。从病斑上分离到一种细菌,在KB培养基上,菌落为椭圆形,乳白色,半透明,边缘参差不齐,紫外灯照射下产生荧光反应。致病性测定结果表明,该病原细菌可侵染6个南瓜品种引起与田间症状相同的叶枯病。生理生化试验结果表明,该病原细菌与丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)的特性一致。应用假单胞菌属特异引物Ps-for/Ps-rev和丁香假单胞丁香致病变种组群特异性引物Group III-F/Group III-R,可从该病原细菌中扩增出预期大小分别为1 018 bp和750 bp的目的片段。应用丁香致病变种syrB基因特异性引物B1/B2,可从该病原菌中扩增出预期大小为750 bp的丁香霉素基因片段。基于16S rDNA与gyrB基因序列系统进化分析均表明,南瓜叶枯病菌株与已报道的P. syringae pv. syringae菌株HS191(CP006256)亲缘关系最近,二者聚类在一起形成一个小分支。人工接种条件下,该病原细菌还可侵染西葫芦、丝瓜、茄子、番茄、菜豆、扁豆等植物。这些结果表明,引起广东省南瓜叶枯病的病原为丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)。这是首次在中国发现丁香假单胞丁香致病变种引起南瓜叶枯病。     

A Molecular Marker Identifying Subspecific Populations of the Soybean Brown Stem Rot Pathogen, Phialophora gregata

ABSTRACT A molecular marker was developed to separate and identify subspecific populations of Phialophora gregata, the causal agent of soybean brown stem rot. A variable DNA region in the intergenic spacer of the nuclear rDNA was identified. Two specific primers flanking the variable region were developed for easy identification of the genotypes using polymerase chain reaction (PCR). These two specific primers amplified three DNA products. The three PCR products were used to separate isolates of P. gregata into distinct genotypes: A (1,020 bp), B (830 bp), and C (660 bp). Genotype C was found in isolates obtained from Adzuki beans from Japan, whereas all 292 isolates obtained from soybean and the 8 isolates from mung bean belonged to either genotype A or B. The original nondefoliating (type II) strain ATCC 11073 (type culture of P. gregata) belonged to genotype B. The difference between genotypes A and B was due only to an 188-bp insertion or deletion; genotype C, however, differs from genotypes A and B at 58 point mutations, in addition to the length difference. Isolates of both genotypes A and B were widespread in seven Midwestern states. Genotype A was found mostly in certain susceptible soybean cultivars like Sturdy and Pioneer 9305, whereas genotype B was found predominately in brown stem rot-resistant soybean cvs. Bell, IA 3003, and Seiben SS282N. The specific primers were also used to directly detect cultivar-preferential infection by the two genotypes in infected soybean stems growing in the same field. Data from direct detection in soybean stems showed that cultivar-preferential infection by the two genotypes of P. gregata was significant.     

智利苹果中牛眼果腐病菌的分离鉴定研究

天津检验检疫局从智利进口的腐病苹果中分离了一株真菌,通过菌落、分生孢子形态特征比较,ITS序列比对分析,并通过科赫氏法则验证,确定该菌株为牛眼果腐病菌(Neofabraea alba),该病菌是导致苹果牛眼果腐病的4种病原真菌之一,该有害生物为我国首次截获。