猕猴桃软腐病致病菌的分离与鉴定

猕猴桃软腐病是一种真菌性病害,其致病菌种类较多.为明确贵州省修文县猕猴桃果实软腐病的致病菌种类,本研究对病果进行了病原菌分离和纯化,得到8个真菌;经过形态学特征和分子生物学鉴定,确定了引起修文县猕猴桃软腐病的病原菌为拟茎点霉菌(Phomopsis sp.)、黑附球菌(Epicoccum nigrum)、帚状弯孢聚壳菌(Eutypella scoparia)、镰刀菌(Fusarium sambucinum)和交链格孢(Alternaria alternata);通过致病性测定,发现拟茎点霉菌致病力最强,是猕猴桃软腐病的主要致病菌.     

甘肃省临夏州小麦脚腐病病原鉴定

对甘肃省临夏州小麦脚腐病病原进行了分离鉴定,从3块病田的37株罹病小麦上分离得到56个真菌菌株,分别被鉴定为雪腐镰刀菌[Fusarium nivale(Fr.)Ces.]、燕麦镰刀菌[Fusarium avenaceum(Corda et Fr.)Sacc.]、麦斑点附球霉[Epicoccum triticiP.Henn.]、小壳色单隔孢菌[Diplodiellasp.]、交链孢菌[Alternariaspp.]、芽枝霉[Cladosporiumsp.]、黑孢霉[Nigrosporasp.]。其中,雪腐镰刀菌致病性最强,燕麦镰刀菌致病性中强,麦斑点附球霉致病性弱,其余菌不致病。     

柠檬桉内生真菌的分离及其提取物的抗细菌活性

采用组织块分离法分离柠檬桉中的内生真菌,通过菌落观察和分子生物学相结合的方法对分离到的内生真菌进行鉴定;进一步采用薄层层析-生物自显影法测定了柠檬桉内生真菌次生代谢产物的抗细菌活性。从柠檬桉中共分离鉴定得到10株内生真菌,主要分布于附球菌属Epicoccum、镰刀菌属Fusarium、炭疽菌属Colletotrichum、Rhytidhysteron、半壳霉属Leptostroma、Campylocarpon、Neofusicoccum、白腐菌属Phlebia和Dinemasporium等9个属中。抗细菌活性的测定结果表明,内生真菌Ecf-4(Rhytidhysteron sp.,MK211261)和Ecf-1(Epicoccum sp.,MK211258)表现出最强的抑菌活性,两菌对桉树青枯病菌的抑菌斑最大直径均大于10 mm;依据其R_f值的范围和抑菌斑的最大直径,内生真菌Ecf-4的抗菌活性高于Ecf-1,表现出更强的抗细菌活性,可作为候选菌株,进一步分离和鉴定其中的活性成分。     

枯草芽孢杆菌BL03对苹果霉心病和棉苗病害田间防治效果

枯草芽孢杆菌 (Bacillussubtilis) BL 0 3菌株 ,是作者从苹果树体上分离得到的拮抗菌株之一。室内测定对苹果霉心病菌 (Alternaria alternata)、炭疽病菌 (Colltotrichum gossypii)、红霉病菌 (Trichothecium roseum)和镰刀菌 (Fusarium sp.)等 1 0种病原菌的抑制效果均为1 0 0     

加拿大进境燕麦种子中大豆北方茎溃疡病病菌的检疫鉴定

从来自加拿大的燕麦种子中分离到3株可疑真菌,经形态特征鉴定、分子生物学鉴定及致病性测定,确定截获的3株真菌均为大豆北方茎溃疡病病菌(Diaporthe phaseolorum var.caulivora)。这是我国口岸首次从进境的燕麦种子中截获该病菌。     

广东口岸进境蔬菜种子的病菌检测

对2006年1月～2007年6月广东口岸1000多批进境的蔬菜种子进行了检验和病害鉴定,共鉴定出18属8种真菌病害,其中包括茄腐镰孢(Fusarium solani)、串珠镰孢(Fusarium moniliforme)、半裸镰刀菌(Fusarium semitectum)和芸苔生链格孢(Alternaria brassicola)等重要的植物致病菌。     

大豆南方茎溃疡病菌的分离与鉴定

在对美国进境大豆进行检疫时，从混杂的豆秆上发现并分离到了大豆南方茎溃疡病菌，通过形态学和分子生物学鉴定，确定该菌为Diaporthe phaseolorum（Cke．＆ Ell.）Sacc．var．meridionalis Morgan-jones。该病原菌是国外近年来在大豆生产中新发生的危险性病原真菌，国内未见报道。     

猕猴桃储藏期病原真菌的分离与鉴定

为明确猕猴桃采后病害的病原种类,本研究对猕猴桃储藏期病果进行了病原真菌的分离培养,观察了病原的培养性状和形态特征,并进行了分子生物学鉴定及其致病性测定。结果表明,引起猕猴桃储藏期病害的病原真菌有7种(类),分别是链格孢菌(Alternaria spp.)、葡萄座腔菌(Botryosphaeria dothidea)、间座壳菌(Diaporthe spp.),无性态为拟茎点霉菌(Phomopsis spp.)、拟盘多毛孢菌(Pestalotiopsis sp.)、假尾孢菌(Pseudocercospora sp.)、刺盘孢(Colletotrichum sp.)、灰葡萄孢(Botrytis cinerea),研究结果可为后期猕猴桃采后病害的防控提供科学依据。     

外来杂草在海南的危害及病害调查

对海南13种外来杂草展开调查,其中分布广泛、危害最为严重的是飞机草和假臭草。在9种外来杂草上共分离获得17种真菌,分别是侵染假臭草的链隔孢（Alternaria sp.）、镰刀菌（Fusarium sp.）,侵染飞机草的飞机草泽兰尾孢（Cerospora eupatorii Peck.）、炭疽菌（Colletotrichum sp.）和链格孢（Alternaria sp.）,侵染蟛蜞菊的链隔孢（Alternaria sp.）和炭疽菌（Colletotrichum sp.）,侵染空心莲子草的假隔链格孢（Nimbya alternantherae）和镰刀菌（Fusarium sp.）,侵染小飞蓬的弯孢（Curvularia sp.）和链格孢（Alternaria sp.）,侵染阔叶丰花草和胜红蓟的弯孢（Curvularia sp.）,侵染三叶鬼针草的弯孢（Curvularia sp.）和黑孢霉（Nigrospora sphaerica）,侵染凤眼莲的链格孢（Alternaria sp.）和炭疽菌（Colletotrichum sp.）。     

宁波口岸连续检出大豆北方茎溃疡病菌

2005年7月,宁波检验检疫局技术中心植检实验室从北仑局送检的美国进境大豆下脚料中检测到大豆北方茎溃疡病菌（Diaporthe phaseolorum var.cauliuora）,并经浙江大学张敬泽教授复核确认.这是我国首次从进境植物中检出该病菌.不久,该实验室又从阿根廷进境的大豆中检出该病菌.这证实该病菌可以通过种子进行远距离传播.     

Molecular Identification and Phylogenetic Grouping of Diaporthe phaseolorum and Phomopsis longicolla Isolates from Soybean

ABSTRACT Diaporthe phaseolorum and Phomopsis longicolla isolates from soybean were examined using traditional mycological characteristics and molecular methods. Cultural characteristics including types of fruiting bodies and conidia were assessed for isolates collected from soybean stems and seeds. Cultures were identified as P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, or D. phaseolorum var. sojae. Molecular markers for these groups were developed and analyzed using polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLP) and DNA sequencing in the internal transcribed spacer (ITS) and the 5.8S ribosomal DNA. The ITS(4) and ITS(5) primers amplified PCR products for all isolates studied. Gel electrophoresis of undigested PCR products and DNA sequencing produced various fragment lengths including 604 bp for P. longicolla, 602 and 603 bp for D. phaseolorum var. caulivora, 603 bp for D. phaseolorum var. meridionalis, and from 597 to 609 bp for D. phaseolorum var. sojae. Digestion of these PCR products with enzymes AluI, HhaI, MseI, RsaI, and ScrFI resulted in distinct bands for identification of P. longicolla and the varieties of D. phaseolorum I. All P. longicolla, D. phaseolorum var. caulivora, and D. phaseolorum var. meridionalis isolates were distinguished using AluI and HhaI with RsaI or ScrFI. The banding patterns of D. phaseolorum var. sojae isolates were complex and were separated into 11 subgroups after digestion with AluI, HhaI, MseI, RsaI, and ScrFI. Phylogenetic analysis of 20 isolates of D. phaseolorum and P. longicolla based on the DNA sequence of the ITS region resolved six clades termed A, B, C, D, E, and F. Clade A included all sequenced D. phaseolorum var. caulivora isolates, two from Italy and one from the United States. Isolates in clade B were exclusively associated with D. phaseolorum var. meridionalis. Clades A and B formed a well-supported monophyletic group. Isolates in clades C, D, E, and F were morphologically defined as isolates of P. longicolla, D. phaseolorum var. sojae, and Diaporthe spp. The ITS sequences similarity of seven geographically diverse P. longi-colla isolates illustrated that P. longicolla isolates have a similar genetic background, with some affiliations to some D. phaseolorum var. sojae isolates. Morphological characteristics of the isolates along with the terminal clades of the ITS phylogeny suggest that P. longicolla is an individual species, D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis are varieties of D. phaseolorum, and D. phaseolorum var. sojae is either several varieties of D. phaseolorum or possibly several distinct species.     

Molecular Detection of Diaporthe phaseolorum and Phomopsis longicolla from Soybean Seeds

ABSTRACT Species-specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break the cells. DNA fragment lengths ranged from 200 to 1,200 base pairs (bp), with the majority of fragments <500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA, three TaqMan primer/probe sets were designed. Primer/probe set PL-5 amplified a 96-bp fragment within the ITS1 region of P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, and D. phaseolorum var. sojae. Set PL-3 amplified a 86-bp DNA fragment within the ITS2 region of P. longicolla. Set DPC-3 amplified a 151-bp DNA fragment within the ITS2 region of D. phaseolorum var. caulivora. TaqMan primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA. When using PCR-RFLP for Diaporthe and Phomopsis detection, the sensitivity was as low as 100 pg of pure DNA. Among 13 soybean seed lots from Italy and the United States, the total Diaporthe and Phomopsis detected using a traditional seed-plating technique ranged from 0 to 32%. P. longicolla was most prevalent, followed by D. phaseolorum var. sojae. D. phaseolorum var. caulivora, which only occurred in 0.5% of the Italian seed lots, was not detected in the U.S. seed lots. D. phaseolorum var. meridionalis was not detected in either the U.S. or Italian seed lots. Using TaqMan primer/probe set PL-3, the frequency of P. longicolla was 18% in seed lot I3, similar to the frequency obtained from PCR-RFLP and potato dextrose agar plating detection. The frequencies of D. phaseolorum and P. longicolla in each seed lot obtained by the different detection methods were comparable with respect to total infection and individual species detection. However, TaqMan detection provided the fastest results of all the methods tested.     

Seed-borne fungi detected in consignments of soybean seeds (Glycine max) imported into India

Inspection of 16 017 samples of soybean seeds imported into India from 1978 to 2004 resulted in the detection of 21 pathogens, including Peronospora manshurica which is not present in India. Seed-borne fungi of high economic significance included: Ascochyta sojicola , Botryotinia fuckeliana , Cercospora kikuchii , Colletotrichum dematium , Corynespora cassicola , Diaporthe phaseolorum var . sojae , Fusarium oxysporum, Glomerella cingulata , Glomerella glycines , Macrophomina phaseolina , Nectria haematococca , Passalora sojina , Thanatephorus cucumeris as well as other fungal pathogens for which soybean is not a host such as Alternaria padwickii , Cochliobolus sativus , Fusarium culmorum , Fusarium poae , Glomerella graminicola , Setosphaeria rostrata, Verticillium albo-atrum , etc. Some of the fungi detected have very wide host range. Details are presented on the fungi detected, the countries from which the imported consignments originated, and phytosanitary significance.     

A Preliminary Investigation of Pathogenic Fungi from Lotus (Nelumbo nucifera Gaertn)in Nanchang City

[Objectives] Lotus (Nelumbo nucifera Gaertn) is an economically important aquatic plant in China. Fungaldisease is a serious problem in lotus cultivation. In this study, the pathogenic fungi on lotus in Nanchang City were investigated to lay the foundation for the disease control. [Methods] Lotus leaves and stems in ponds of Nanchang City were collected, the fungi on leave/stem spots were isolated and purified. Colonies morphological characters and ITS sequences were used to identify the strains. [Results] 49 strains were isolated and identified to 20 species, belonging to 12 genera. [Discussion] 15 species may firstly be reported on lotus in this study, i.e., Alternaria angustiovoidea, Alternaria compacta, Alternaria ricini, Alternaria tenuissima, Arthrinium arundinis, Botryosphaeria dothidea, Curvularia spicifera, Diaporthe australiana, Diaporthe eres, Diaporthe tectonae, Epicoccum nigrum, Fusarium fujikuroi, Neofusicoccum parvum, Nigrospora sphaerica, and Phomopsis eucommii.     

Morphologic, Molecular, and Pathogenic Characterization of Diaporthe phaseolorum Variability in the Core Soybean-Producing Area of Argentina

ABSTRACT Isolates of the Diaporthe/Phomopsis (D/P) complex were collected in the main soybean producing area of Argentina during the 1996-97, 1997-98, and 1998-99 growing seasons. Twenty-three morphologic characters related to type of colonies, stroma, pycnidia and conidia, presence of perithecia, and asci length were studied by principal component analysis (PCA). Genomic DNA were analyzed by the random amplified polymorphic DNA (RAPD) technique. From both studies, 18 isolates were identified as D/P complex and grouped in four major taxa: (i) Diaporthe phaseolorum var. meridionalis, (ii) D. phaseolorum var. caulivora, (iii) D. phaseolorum var. sojae, and (iv) Phomopsis longicolla. In addition to distinguishing interspecific and intraspecific variability, molecular markers allowed the detection of differences among isolates within the same variety. Pathogenicity was assayed in the greenhouse, by the toothpick method, inoculating the D/P isolates to soybean genotypes carrying different resistance genes (Rdc1, Rdc2, Rdc3, and Rdc4) against soybean stem canker (SSC). Pathogenic analysis distinguished two main groups: (i) the SSC-producing isolates, including D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora, and (ii) the non-SSC-producing isolates, including D. phaseolorum var. sojae and P. longicolla. Cultivar RA-702 (susceptible control) was compatible with both D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora isolates; meanwhile, Tracy-M (Rdc1 and Rdc 2 genes) was incompatible with D. phaseolorum var. meridionalis but compatible with D. phaseolorum var. caulivora isolates. The fact that Rdc1 and Rdc2 together (as in Tracy-M) confer an almost immune reaction to all assayed isolates of D. phaseolorum var. meridionalis but were ineffective against the D. phaseolorum var. caulivora isolates evaluated suggests that the virulence or avirulence genes in D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora are different. Moreover, physiological races of D. phaseolorum var. meridionalis were detected by using differential soybean genotypes carrying distinct single Rdc genes. As far as we know, this is the first report on the existence of physiological races of D. phaseolorum var. meridionalis in South America. Selective pressure due to deployment of resistant host cultivars may have changed the frequency of the virulence or avirulence genes within the population of D. phaseolorum var. meridionalis. On the whole, our results show that pathogenic variability of D. phaseolorum in the core soybean-producing area of Argentina is higher than previously recognized.     

美国进境大豆北方茎溃疡病菌的分离与鉴定

在对美国进境大豆检疫过程中,对大豆籽粒和夹带茎秆进行病原真菌的分离培养,使用形态学和分子生物学相结合的方法对可疑菌株进行鉴定,确定该菌为Diaporthe phaseolorum(Cke.&; Ell.)Sacc.var.caulivora Athow &; Caldwell。该病原菌是进境植物检疫潜在危险性病原真菌,在国内属首次截获。     

CLIMEX-GIS预测大豆北方茎溃疡病菌在中国的潜在分布

大豆北方茎溃疡病菌是大豆的重要病原菌,广泛分布于世界主要大豆产区,造成严重的产量和品质损失。本文应用生物模型CLIMEX结合GIS软件预测大豆北方茎溃疡病菌在中国的适生区,并根据EI值划分相应的适生等级。结果表明,大豆北方茎溃疡病菌在我国绝大部分地区适合生长,其中东北地区、华北地区和云贵高原地区处于中适生区或高适生区。该菌在我国还未报道,通过分析其在我国潜在分布区对于防止病菌的传入、传播和蔓延有重要的检疫意义。     

大豆北方茎溃疡病菌分离鉴定技术的改进

本文对产生大豆北方茎溃疡病菌子囊壳的豆秆分别进行冷冻和水浸处理,发现2种处理均能促进成熟的子囊壳产孢,并且同一子囊壳经冷冻和水浸处理能够多次喷出子囊,释放子囊孢子,利用此特性有利于在不损失鉴定材料的前提下,多次获得分离物以完成病菌的分离鉴定,特别利于伴生其他真菌和子囊壳数量很少情况下,分离纯化大豆北方茎溃疡病菌。