一串红叶斑病的病原菌鉴定

一串红Salvia splendens叶斑病被发现在杭州市农科所栽培花卉的遮阳网大棚中,是由棒孢霉属真菌侵染引起的。在田间,病原菌侵染植株的茎和叶片,引起当年移植苗发生茎腐、落叶、植株枯萎和死亡。通过对病原菌形态学观察以及核糖体DNA ITS序列分析,证明侵染一串红的病原菌为决明生棒孢霉菌Corynespora cassiicola。致病性试验结果表明,一串红是决明生棒孢霉菌的寄主。种子带菌检验结果显示,种子带菌率仅为1%。     

The occurrence of root rot and crown rot of rice in Gilan and Zanjan provinces, Iran

Root rot and crown rot of rice is one of the important fungal diseases of rice in Gilan and Zanjan provinces, Iran. During 1999--2002, samples of plant and soil around the roots of infected rice plants were collected and used to identify the causal agent. Root and crown parts were surface sterilized with sodium hypochlorite and then cultured on PDA (potato dextrose agar), PPA (pepton pentacholoritobenzene agar) and CLA (carnation leaf agar) media. Soil samples prepared in water agar were used to isolate the pathogen. The causal agent was identified as Fusarium moniliforme. Colonies were initially white but turned violet to grey late. Microconidia were arranged in chain and macroconidia were cylindrical and long with 3-5 septa. The disease was severe in Zanjan province particularly along Ghezel Ozan river where the infection ranged from 70-80%. Root and crown rot was more prevalent in areas where Champa and Gerdeh were being cultivated continuously. On the other hand, Sadri cultivars had relatively less infection. Persistent cultivation of rice and seed sowing method intensified disease development and caused significant economic losses.     

Root and Crown Rot of Brazilian Pine (Araucaria angustifolia) Caused by Phytophthora cinnamomi

In an area reforested with Brazilian pine (Araucaria angustifolia) located in Paraná State, southern Brazil, 20‐ to 40‐year‐old trees representing 0.2% of the surveyed area had symptoms of root and crown rot, yellowing and browning of leaves from the uppermost branches and death. Three Phytophthora isolates obtained from diseased plant tissue were tested against 1‐year‐old Brazilian pine seedlings and found to display positive pathogenicity. Based on their morphological and physiological characteristics, the isolates were identified as Phytophthora cinnamomi. A GenBank BLAST search of partial sequences from the β‐tubulin and elongation factor‐1α genes, as well as the ITS regions and 5.8S gene of rDNA, confirmed the species identification. This is the first report of the involvement of this pathogen on the aetiology of Brazilian pine root and crown rot.     

Phoma glomerata as causal agent of crown rot disease of fennel in southern Italy

In the autumn seasons of the years 2005 and 2006 millions of fennel seedlings under greenhouse in nurseries where observed in Basilicata, Calabria, Campania and Apulia Regions, southern Italy, with serious symptoms of damping off and root necrosis. The basal part of seedling stem was much thinner than the uninvaded parts above it and the crown was brown and shrink. The incidence of disease reached values ut to 100% in some blocks where the humidity was higher, and seedlings attached earlier died. Almost all fennel varieties were interested by the disease, some of them more seriously. Seedlings attached later recovered to a great extent after transplanting in open field. In this work the identification of the causal agent of this disease was made applying Koch's postulates. Moreover, for a rapid and unambiguous identification of the fungus species, the internal transcribed spacers and the 5.8 rDNA gene (ITS1-5.8-ITS2) were amplified from DNA extracted from the isolated colonies. The fragment of amplified DNA was sequenced and compared with those present in Gene Bank (NCBI). The fungus isolated after the morphological and molecular characterization was ascribed to the species Phoma glomerata (Corda) Wollenweber & Hochapfel. This is the first report of P. glomerata as agent of crown rot of fennel.     

Botryosphaeria spp. isolated from apple and several deciduous fruit trees are divided into three groups based on the production of warts on twigs, size of conidia, and nucleotide sequences of nuclear ribosomal DNA ITS regions

We examined the phytopathological and biological characters ofBotryosphaeria spp. isolated from apples and other deciduous fruit trees, and determined the nucleotide sequences of their rDNA ITS regions. TheBotryosphaeria isolates from deciduous fruit trees can be divided into three groups based on their production of warts on twigs, size of the conidia, and nucleotide sequences of rDNA ITS 1, ITS 2 and 5.8S rDNA. Isolates ofBotryosphaeria in ITS group A produced conidia of intermediate size and showed warts on infected twigs prior to the development of ring rot on fruit. This group was common on deciduous fruit trees in Japan as a causal agent of ring rot and wart bark diseases of apples and pears; and it appears similar to theB. dothidea from the US that was isolated from apple exhibiting white rot. The ITS group BBotryosphaeria produced small conidia and induced shoot blight without wart development prior to the development of ring rot on fruit. This group was localized on pear, persimmon, and kiwi fruit in restricted areas of Japan. The ITS group CBotryosphaeria consisted ofB. obtusa, the causal agent of apple black rot in the US, which produced large dark brown conidia.     

Plant growth promoting potential of the fungus <Emphasis Type="Italic">Discosia</Emphasis> sp. FIHB 571 from tea rhizosphere tested on chickpea,maize and pea

The ITS region sequence of a phosphate-solubilizing fungus isolated from the rhizosphere of tea growing in Kangra valley of Himachal Pradesh showed 96% identity with Discosia sp. strain HKUCC 6626 ITS 1, 5.8S rRNA gene and ITS 2 complete sequence, and 28S rRNA gene partial sequence. The fungus exhibited the multiple plant growth promoting attributes of solubilization of inorganic phosphate substrates, production of phytase and siderophores, and biosynthesis of indole acetic acid (IAA)-like auxins. The fungal inoculum significantly increased the root length, shoot length and dry matter in the test plants of maize, pea and chickpea over the uninoculated control under the controlled environment. The plant growth promoting attributes have not been previously studied for the fungus. The fungal strain with its multiple plant growth promoting activities appears attractive towards the development of microbial inoculants.     

黄颡鱼卵水霉病病原的分离鉴定及其无性繁殖特性

【目的】对黄颡鱼卵水霉病病原进行分离鉴定,并对其无性繁殖特性进行研究。【方法】采用传统方法从患水霉病的黄颡鱼卵上进行丝状真菌的分离,然后通过人工感染实验证实分离菌株的致病性,通过形态学观察和ITS rDNA序列分析对致病菌株进行鉴定,并进一步通过单因子法研究其无性繁殖特性。【结果】从患水霉病的黄颡鱼卵上分离了4株丝状真菌,经人工感染试验证实其中一株丝状真菌HP对黄颡鱼卵具有致病性,并进一步研究了其形态与无性繁殖特性,开展了ITS rDNA序列分析。实验结果表明,菌株HP菌丝为透明管状结构,中间无横隔,分枝较少;游动孢子囊多数呈棒状,游动孢子发育成熟后从孢子囊中释放出来,并迅速游离;能够产生第二孢孢子;新孢子囊以内层出的方式产生;藏卵器呈球形,与雄器同枝或异枝。菌株HP的ITS rDNA序列与GenBank基因库中水霉属菌株自然聚类,同源性高达99%,与多子水霉菌株Arg4S(GenBank登录号GQ119935)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株HP为多子水霉(Saprolegnia ferax)。此外,菌株HP在5°C-35°C、pH 4-10范围内均能产生游动孢子,产生游动孢子的最适温度和pH分别为20°C和7,而且5-25 mg/L福尔马林和0.25 1.25 mg/L二硫氰基甲烷对菌株HP产生游动孢子具有明显的抑制作用。【结论】分离鉴定了黄颡鱼卵水霉病病原,并确定了其无性繁殖特性,可以作为该病防治用药的依据。     

Intrastrain internal transcribed spacer heterogeneity in Ganoderma species

Intrastrain internal transcribed spacer (ITS) heterogeneity is first reported from Ganoderma, a fungal genus within Basidiomycetes. ITS amplification products from 4 strains, representing 4 Ganoderma species, were cloned and sequenced. Two to five different ITS types were found within a single strain. The clone sequences were analyzed along with other sequences from Ganoderma retrieved from GenBank. The results show that sequence variation within strains varies considerably with species and the heterogeneity may occur in the 3 parts (ITS1, ITS2, and 5.8S) of the ITS region.     

香菇蛛网病病原菌树状枝葡霉生物学特性

香菇是我国产量最高的食用菌之一,因其独特浓郁的风味而广受赞誉。然而随着香菇栽培面积的不断扩大,其各种病害的发生也日趋频繁。近期在浙江省庆元县多个菇棚内发现一种新病害。为了明确致病菌,本研究通过对罹病香菇子实体进行组织分离和单孢分离获得纯化菌株QS02,随后结合形态学特征和分子生物学技术对其进行鉴定,并通过柯赫氏法则的验证,确定该病原菌为树状枝葡霉Cladobotryum dendroides。同时,本文还对该病原菌展开了生物学特性研究,结果表明:病原菌菌丝生长最适培养基为PDA培养基,最适温度为25℃,最适碳源为可溶性淀粉,最适氮源为牛肉膏,最适pH区间为5–6;光照对该菌菌丝生长有一定的抑制作用。这是由树状枝葡霉引起的香菇蛛网病在国内的首次报道。     

Occurrence of Charcoal Rot Caused by Macrophomina phaseolina,an Emerging Disease of Adzuki Bean in China

Adzuki bean (Vigna angularis) is an important legume crop in China. Soil‐borne charcoal rot caused by Macrophomina phaseolina (Tassi) Goid is an important and devastating disease of many crops including legumes. During late August and early September, 2014, symptoms similar to charcoal rot were observed on adzuki bean plants in Yulin City of Shanxi Province, and Fangshan County of Beijing, China. This study was conducted to determine the causal agent of the emerging disease on adzuki bean. Four fungal isolates were obtained and identified as M. phaseolina based on morphological and molecular characteristics, including species‐specific primer detection and sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA. The resulting sequences showed 99% identity with more than 60 M. phaseolina strains from diverse hosts. The virulence on adzuki bean was verified using pathogenicity tests, producing symptoms similar to those observed in the fields. To our knowledge, this is the first report of M. phaseolina causing charcoal rot on adzuki bean.     

基于香菇菌株rDNA-ITS序列的系统发育分析

根据真菌核糖体通用引物ITS1和ITS4扩增出13个福建袋栽香菇主要菌株的5.8S rDNA、ITS序列,对该序列进行测序后,得到完整的5.8S rDNA、ITS序列,将该序列提交NCBI并获得登录号,对该序列进行比对分析并构建了系统发育树,从分子水平对香菇菌株进行了区分鉴定,结果显示13个菌株可以明显的分成2丛,而其他菌株又可以从一丛中延伸出几个亚丛。     

进境美国大豆中值得关注的真菌Diaporthe novem

采用常规平板分离法,从一批进境的美国大豆样品中获得1株可疑的间座壳属菌株MDD57.经形态学观察发现,该菌株在PDA培养基上产生分生孢子器,且同时产生大量α型和β型分生孢子,未见有性阶段.经ITS和tef1α基因扩增、核酸序列比对分析,发现该菌株同GenBank中2株Diaporthe novem菌株的基因序列同源性达...     

黄颡鱼卵致病性绵霉的分离鉴定与药敏特性

【目的】对黄颡鱼水霉病病原进行分离鉴定,并对其药敏特性进行研究。【方法】参照传统方法对患水霉病的黄颡鱼卵上的丝状真菌进行分离,通过人工感染实验证实分离菌株的致病性,然后根据形态学特征和ITS rDNA序列分析对致病菌株进行鉴定,并进一步采用倍比稀释法研究其药敏特性。【结果】从患水霉病的黄颡鱼卵上分离到8株丝状真菌,经人工感染试验证实菌株YC对黄颡鱼卵具有致病性,并进一步研究了其形态与药敏特性,开展了ITS rDNA序列分析。结果表明,菌株YC为透明管状结构,中间无横隔;游动孢子囊多呈圆筒形、棍棒形或穗状,游动孢子发育成熟后不断从孢子囊顶端释放出来,成团聚集在游动孢子囊口,并经过一个时期的静休后,成团脱落或直接分散在水中游动;次生孢子囊具有典型的侧生现象;藏卵器呈球形或梨形,大多与雄器异枝,少数与雄器同枝,含1?15个卵孢子;成熟卵孢子中生或亚中生,偏生一个大油球。其ITS rDNA序列与GenBank基因库中绵霉属菌株自然聚类,同源性高达99%,与异丝绵霉(Achlya klebsiana)菌株CBS101.49(GenBank登录号AF119579)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株YC为异丝绵霉(Achlya klebsiana)。此外,在实验选用的中草药和消毒剂中,黄连和异噻唑啉酮分别对菌株YC的抑菌效果最好,其对菌株YC的最小抑菌浓度分别为256 mg/L和2 mg/L。【结论】首次分离了黄颡鱼卵致病性异丝绵霉菌株YC,并确定了其药敏特性,可以作为该病防治用药的依据。     

Fusarium culmorum is a single phylogenetic species based on multilocus sequence analysis

Fusarium culmorum is a major pathogen of wheat and barley causing head blight and crown rot in cooler temperate climates of Australia, Europe, West Asia and North Africa. To better understand its evolutionary history we partially sequenced single copy nuclear genes encoding translation elongation factor 1-α (TEF), reductase (RED) and phosphate permease (PHO) in 100 F. culmorum isolates with 11 isolates of Fusarium crookwellense, Fusarium graminearum and Fusarium pseudograminearum. Phylogenetic analysis of multilocus sequence (MLS) data using Bayesian inference and maximum parsimony analysis showed that F. culmorum from wheat is a single phylogenetic species with no significant linkage disequilibrium and little or no lineage development along geographic origin. Both MLS and TEF and RED gene sequence analysis separated the four Fusarium species used and delineated three to four groups within the F. culmorum clade. But the PHO gene could not completely resolve isolates into their respective species. Fixation index and gene flow suggest significant genetic exchange between the isolates from distant geographic regions. A lack of strong lineage structure despite the geographic separation of the three collections indicates a frequently recombining species and/or widespread distribution of genotypes due to international trade, tourism and long-range dispersal of macroconidia. Moreover, the two mating type genes were present in equal proportion among the F. culmorum collection used in this study, leaving open the possibility of sexual reproduction.     

1株产漆酶白腐真菌的筛选和鉴定

从不同的生境采集生物样品,利用Bavendamm反应反复筛选产漆酶的白腐真菌。利用真菌通用引物对ITS1/ITS4扩增菌株rDNAITS区序列,对扩增产物进行测序。测序结果在GenBank中进行同源性搜索,下载部分具有代表性种的ITS序列进行序列比对,利用软件MEGA4构建分子系统发育树,通过序列分析,并结合形态学鉴定出4220为香栓孔菌(Trametes suaveolens)。     

青藏高原黄绿蜜环菌纯培养菌种的分离培养及分子鉴定

首次从采自青藏高原、与高原牧草嵩草属Kobresia草本植物形成外生菌根的黄绿蜜环菌Armillarialuteo-virens子实体中分离获得一组织分离菌株,运用rDNA-ITS和rDNA-IGS-1测序技术对该组织分离菌株是否为黄绿蜜环菌的纯培养菌种进行分子鉴定,并基于黄绿蜜环菌的5.8S/ITS和IGS-1序列进行核酸序列数据库GenBank同源性检索比对、构建系统发育树。结果表明,本研究获得的黄绿蜜环菌子实体组织分离菌株即为其纯培养菌种。基于ITS的系统发育分析表明黄绿蜜环菌与口蘑科内其它属间物种的系统发育关系较远;基于IGS-1的系统发育分析表明黄绿蜜环菌与蜜环菌属内的其它种序列差异较大,系统发育关系较远,而与Lepiota属内的部分种具有较近的系统发育关系。本研究首次基于分子手段对我国青藏高原的黄绿蜜环菌种进行了分离培养、分子鉴定和系统发育分析,为黄绿蜜环菌的科学分类提供了分子依据。     

Taxonomy, phylogeny, and distribution of Puccinia graminis, the black stem rust: new insights based on rDNA sequence data

Puccinia graminis (Uredinales) is an economically important and common host-alternating rust species on Berberidaceae/Poaceae (subfamilies Pooideae and Panicoideae) that has been spread globally by human activities from an unknown center of origin. To evaluate the taxonomic implications, phylogenetic relationships, and distribution/spread of this complex species, we sequenced and cladistically analyzed the ITS1, 5.8S, and ITS2 regions from herbarium specimens on various host plants from Iran (17), Europe (1), and North America (4). The ITS region plus the 5.8S gene ranged from 686 to 701 bp, including the flanking partial sequences of the 18S and 28S rDNA. Our phylogenetic analysis included 54 bp of the 18S sequence, the entire ITS1 + 5.8S + ITS2, and 58 bp of the 28S sequence. A second analysis used only the last 42 bp of ITS1, and all the 5.8S and ITS2, to incorporate data from additional sequences downloaded from GenBank. In addition to variation in sequence length, there was variation in sequence content. The analysis does not support classical morphology-based taxonomic concepts of the P. graminis complex. Also, host range, host taxonomy, and geographic origin provide minor information on taxonomic relationships. Puccinia graminis is most probably monophyletic. Coevolutionary aspects can hardly be discussed because of lack of sequence data from alternate host specimens. The occurrence of unrelated fungal taxa on the same host species suggests that, besides coevolution with the host, host jumps and hybridization may have played an important role in the evolution of P. graminis. From rDNA data we conclude that the pathogen was introduced to North America at least twice independently. For a new taxonomic concept, we think the complex has to be split into at least two species. New morphological features and further features other than sequence data, however, must be checked for taxonomic value first and, if necessary, be considered.     

生姜共生真菌的分离及其体外抑菌效应初探

为筛选得到优良植物病害生防菌,对广西生姜Zingiber officinale种植区健康生姜根系和叶片中的共生真菌进行了组织分离,以生姜茎腐病菌群结腐霉Pythium myriotylum和香蕉枯萎病菌尖孢镰刀菌古巴专化型4号生理小种Fusarium oxysporum f. sp. cubense race 4为指示菌,通过平板对峙培养法和发酵液菌落直径法试验进行筛选评价,并结合形态学观察及ITS序列分析对筛选出的生防效果最好的共生真菌进行了鉴定。结果表明,从生姜植株共分离得到34株共生真菌,其中根系分离22株,叶片分离12株;对峙培养发现有6株共生真菌对生姜茎腐病菌和香蕉枯萎病菌均有抑制作用;其中菌株SBM-11拮抗作用最强,对生姜茎腐病菌抑制率达到93%,对香蕉枯萎病菌抑制率达到82%;SBM-11的发酵液对生姜茎腐病菌和香蕉枯萎病菌抑制率分别为82%、73%,与其他菌株发酵液抑制效果相比差异明显;结合形态和分子鉴定结果表明SBM-11菌株为绿色木霉Trichoderma viride,极具生防潜力。     

Analysis of Yeast-Like Symbiote Diversity in the Brown Planthopper (BPH), Nilaparvata lugens Stål, Using a Novel Nested PCR-DGGE Protocol

Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S–ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S–ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC–ITS2, ITS1FGC–ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.     

The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi

A unique oligonucleotide pair, GOCC56:GOCC427, was designed that correctly primed specific amplification of a approximately 370-bp sequence spanning the ITS and 5.8S rDNA regions of Glomus occultum and Glomus brasilianum. In addition, this primer pair successfully detected G. occultum and G. brasilianum DNA in nested PCR using a primary PCR product amplified from highly diluted extracts of colonized corn (Zea mays) roots using modified ITS1:ITS4 primers. A second primer pair, GBRAS86:GBRAS388, primed specific amplification of a approximately 200-bp sequence spanning the ITS and 5.8S rDNA regions present only in G. brasilianum and Glomus strain GR582. Combined use of both primer pairs provides the means to detect and differentiate two ancient endomycorrhizal species, G. occultum and G. brasilianum, undetectable by standard root staining procedures. Sequence analysis showed that the purported G. occultum strain GR582 is likely a strain of G. brasilianum.