Identification of <Emphasis Type="Italic">Ditylenchus</Emphasis> species associated with Fabaceae seeds based on a specific polymerase chain reaction of ribosomal DNA-ITS regions

A technique based on the use of specific primers for polymerase chain reaction (PCR) was developed for the identification of the stem and bulb nematode belonging to the Ditylenchus dipsaci species complex. The internal transcribed spacer region ITS1 and ITS2, the gene 5.8 S and part of genes 18 S and 26 S of twenty populations of the D. dipsaci species complex belonging to both D. dipsaci sensu stricto and Ditylenchus sp. B (corresponding to populations of giant individuals associated to Vicia faba) and three congeneric species were amplified with two universal ribosomal primers. PCR-amplified DNA samples were digested with five restriction enzymes in order to reveal some polymorphism allowing the identification of D. dipsaci populations associated with Fabaceae seeds. The polymorphism among species was confirmed by the sequencing of the PCR products. A primer (DdpS2) was designed in a region conserved in all populations of both D. dipsaci sensu stricto and D. sp. B studied in the present work. The other Anguinidae species (except a few species from Central Asia associated to Astereaceae and D. sp. G associated to Plantago maritima) differ in two to four nucleotides at the 3′ extremity of this region. This sequence portion coincides with a TspEI restriction site. In combination with a primer located in the ribosomal region, this first primer is a good candidate for identification by PCR of populations of the D. dipsaci species complex found in Fabaceae seeds. A second primer (DdpS1) was designed in a similar way and was specific to D. dipsaci sensu stricto. The utility of these two sets of primers is discussed against the background of quarantine regulation.     

Evidence of Two Formae Speciales in Venturia inaequalis, Responsible for Apple and Pyracantha Scab

ABSTRACT Genetic relationships, mating crosses, and host specificity of Venturia inaequalis isolates from Malus spp. and of Spilocaea pyracanthae isolates from Pyracantha spp. were evaluated. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS1-5.8S to ITS2) revealed a total similarity between these two putative species. ITS restriction fragment length polymorphism carried out with five restriction enzymes on a collection of 28 isolates confirmed a lack of diversity in this region between and within these two populations. Additional isolates from three related species (V. pirina, V. nashicola, and S. eriobotryae) were divided into two distinct monophyletic groups in a phylogenetic tree using ITS sequence comparison. These groups were related to their anamorph (i.e., Spilocaea or Fusicladium). When inoculated on their host of origin, fields isolates caused typical symptoms of scab disease, and a host specificity was demonstrated by cross pathogenicity of isolates from Malus x domestica and Pyracantha spp. Mating on dried leaves in vitro between one isolate of each putative species led to production of numerous perithecia. Ninety-six sporulating monoascosporic progenies were isolated from this cross. Based on these genetic and pathogenic data, we proposed that pathogens responsible for scab on Malus spp. and Pyracantha spp. are considered as two formae speciales belonging to V. inaequalis.     

Molecular and morphological characterization of Colletotrichum gloeosporioides from native Mexican Stylosanthes species

A total of 264 Stylosanthes spp. plants collected from 78 Stylosanthes spp. populations in seven southern Mexican states were analysed for the presence of Colletotrichum spp. Isolates were obtained from 64 plants collected from 36 Stylosanthes populations; 198 isolates produced straight conidia, while 72 isolates produced falcate conidia. Molecular identification was performed to confirm the identity of C. gloeosporioides for the straight-spored isolates. PCR amplifications using the primer CgInt, synthesized from an ITS1 fragment specific to C. gloeosporioides , and the universal primer ITS4 generated the target fragment for 120 Mexican isolates with straight conidia. The endonucleases Ava II and Sma I were used for restriction of the entire amplified ITS1 region of these 120 isolates. The tree constructed from the restriction data grouped 118 Mexican C. gloeosporioides isolates into three clusters containing reference isolates from Africa and Australia, and generated two additional clusters for two Mexican isolates. Conidial shape and growth rate on solid medium were used as the major morphological criteria for distinguishing types A and B. On the basis of 32 other morphological characteristics, a phenogram grouped the colonies into three main clusters. These clusters were partially related to the Stylosanthes species from which they were isolated, and to the molecular groups.     

Conidia as a Substrate for Internal Transcribed Spacer-Based PCR Identification of Members of the Leptosphaeria maculans Species Complex

ABSTRACT The blackleg disease of oilseed rape is caused by an ascomycete species complex termed Leptosphaeria maculans (anamorph Phoma lingam). L. maculans isolates collected worldwide were gathered in the International Blackleg of Crucifers Network (IBCN) collection. Representative IBCN isolates, along with one P. nigrificans isolate, were further analyzed using polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region. ITS size polymorphism discriminated three groups: (i) P. nigrificans, (ii) Tox(+) and 'Lepidium' isolates, and (iii) NA1, NA2, NA3, 'Thlaspi', and 'Erysimum' isolates. Digestion of the ITS region with 19 selected endonucleases showed restriction site polymorphism between the different subgroups: digestion with RsaI could discriminate Tox(+) from 'Lepidium' isolates, whereas digestion with four enzymes, i.e., HaeIII, EcoRII, RsaI, and AluI, was needed to discriminate between NA1, NA2, NA3, 'Thlaspi', and 'Erysimum' isolates. No restriction site polymorphism was observed between isolates within the 'Thlaspi', Tox(+), NA1, and NA2 subgroups. Direct amplification of the ITS region could be achieved using intact conidia, collected either in axenic cultures or on leaf lesions, with only a 4-min 95 degrees C denaturation step prior to PCR reaction. A routine identification protocol requiring no DNA extraction and a sequential use of a few restriction enzymes following PCR has been used successfully for large-scale identification of French field isolates.     

Characterization of Ribosomal DNA from Venturia inaequalis and Its Phylogenetic Relationship to rDNA from Other Tree-Fruit Venturia Species

ABSTRACT A portion of the 18S ribosomal DNA (rDNA) gene, the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA gene were polymerase chain reaction-amplified from strains and field populations of Venturia inaequalis and assessed for genetic variation. A previously reported optional group I intron in the 18S rDNA gene of V. inaequalis was detected in 75.0% of 92 strains collected worldwide and in 61.1 and 71.2% of 54 and 59 strains from two Michigan orchards, respectively. Sequence and restriction analysis of rDNA revealed four intron alleles, three of which were present both in worldwide strains and in each field population. Two ITS1 alleles were detected and found to be linked to specific intron alleles. The ITS1-5.8S-ITS2 sequences from V. asperata V. carpophila, V. cerasi, V. inaequalis, V. nashicola, V. pyrina, and Cladosporium caryigenum were compared using phylogenetic analysis. Strains of the Venturia species were placed in three distinct monophyletic groups in a phylogenetic tree. The first group comprised V. inaequalis; the second, V. pyrina and V. nashicola; and the third, V. cerasi, V. carpophila, and V. asperata. The described intron and ITS1 alleles in V. inaequalis provide genetic markers for subdividing populations of V. inaequalis, and the ITS1-5.8S-ITS2 sequences are valuable in determining the relationship of the species from tree-fruit crops with other Venturia species.     

Molecular Characterisation of Alternaria linicola and its Detection in Linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.     

Genotyping Cephalosporium gramineum and development of a marker for molecular diagnosis

This study investigated the genetic variation of 40 isolates of Cephalosporium gramineum, the causal agent of cephalosporium stripe disease of wheat, based on variations in internal transcribed spacers (ITS) and intergenic spacers (IGS) of rDNA. Of the isolates, 29 were from Japan and the rest from the USA and Europe. The ITS region was about 600 bp and almost identical among these isolates. In the IGS region (～5 kbp), restriction fragment length polymorphism analysis detected four genotypes among the 40 isolates. One representative isolate was selected from each of the four genotypes, and the IGS region was sequenced. Attempts to design a genotype‐specific marker based on the size of PCR products amplified with selected primers failed to differentiate among the four genotypes. Alternatively, a species‐specific primer set (CGIGS1 and CGIGS2) was developed that annealed within the conserved region, producing a DNA fragment of about 1·8 kbp. Tests of this primer set on a wide range of other fungi from 11 genera confirmed that it was specific to C. gramineum. This primer set could serve as an effective tool in the molecular diagnosis of C. gramineum and has the potential to assist in a better understanding of the host–pathogen interaction.     

Detection and quantification of Pratylenchus thornei in DNA extracted from soil using real-time PCR

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.     

PCR-based detection of Colletotrichum acutatum on strawberry

An oligonucleotide primer ( Ca Int 2) was synthesized from the variable internal transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) from Colletotrichum acutatum . PCR with primers Ca Int2 and ITS4 (from a conserved sequence of the rDNA) amplified a 490 bp fragment from several isolates of C. acutatum but not from other members of the genus Colletotrichum . Amplification of this fragment was achieved from 100 fg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from strawberry tissues infected by C. acutatum . Southern hybridization analysis confirmed the 490 bp fragment from C. acutatum DNA and infected strawberry to be identical. The species-specific primer ( Ca Int2) developed in this work could be used for the accurate identification of C. acutatum and its detection on other host plants.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

基于ISSR标记开发的一种用于小麦矮腥黑穗病菌的分子鉴定方法

 小麦矮腥黑穗病菌(Tilletia controversa Kühn, 简称TCK)是小麦上的一种重要检疫性真菌。本研究利用内部简单重复序列(Inter-simple sequence repeat, ISSR)技术研究TCK及其近缘种的DNA多态性,开发了一种可靠而简单的方法用于TCK的分子鉴定。用ISSR引物P4从TCK中扩增出一条1 113 bp的特异性条带,据此设计了一对特异性引物TCKF/TCKR,在12个TCK菌株中均能扩增得到一条882 bp的特异性条带,而其他近缘种包括小麦网腥黑穗病菌(T. caries)和小麦光腥黑穗病菌(T. foetida)及相关黑粉菌的14个菌株均无扩增条带。用该特异性引物检测TCK的下限为25 μL反应体系中可检测到1 ng DNA模板。本研究开发的种特异性引物,可将TCK与其形态上相似的近缘种尤其是小麦网腥黑穗病菌准确区分开,本研究基于ISSR标记建立的小麦矮腥黑穗病菌的分子鉴定方法为腥黑粉菌的检疫提供了一种便捷的方法,是对现有分子鉴定方法的一个补充。     

甘蔗赤条病菌巢式PCR检测

甘蔗赤条病是由燕麦食酸菌燕麦亚种(Acidovorax avenae subsp.avenae,Aaa)引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术,根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物,建立Aaa巢式PCR检测方法。结果表明,建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A.oryzae具有特异性,可扩增出454 bp目的条带,对近缘种德氏食酸菌A.delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L.xyli subsp.xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板,巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL,是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测,阳性检出率分别为100.0%和28.6%,表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。     

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.     

A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

ABSTRACT This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.     

A quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds

Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR‐based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5^.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.     

利用DNA条形码对10种苍耳属杂草的鉴定

使用ITS、ITS2、psbA-trnH、rbcL和matK等序列对截获的10种苍耳属(Xanthium)杂草进行扩增测序,比较各序列的扩增和测序效率,采用最近距离法和相似性搜索算法评价不同序列的鉴定效率。采用最佳鉴定序列计算种间K2P距离并构建系统进化树。结果表明,ITS、psbA-trnH2个序列的扩增效率和鉴定效率最高;ITS序列的种间变异最大,其次是psbA-trnH序列,matK序列的种间变异最小;ITS序列的NeighborJoining(NJ)树可明显地将不同苍耳属杂草种分开;psbA-trnH序列可明显地将国内苍耳种和检疫性苍耳种分开。说明利用DNA条形码能够准确地鉴别苍耳属杂草,ITS和psbA-trnH序列是鉴别苍耳属杂草较理想的条形码组合。该研究结果为苍耳属杂草的分子鉴别提供了科学依据与新的思路。     

松材线虫rDNA的测序和PCR-SSCP分析

 本文为克服形态鉴定的不足,用分子生物学的方法鉴别松材线虫。线虫核糖体DNA(r DNA)的内部转录间隔区(ITS1)区(约308bp)的测序结果显示:松材线虫种内区别很小,不超过1bp;拟松材线虫种内区别较大,最大达7bp;这2种线虫的种间区别为32~39bp。根据以上测序结果,本文结合单条线虫DNA的提取技术,对14个松材线虫和拟松材线虫样本进行了单链构象多态性(PCR-SSCP)分析,结果表明PCR-SSCP分析技术可明确区分这2种线虫,该技术可为单条松材线虫的鉴定提供一套灵敏而可靠的方法。     

十字花科蔬菜黑斑病菌的PCR鉴定

 在对十字花科蔬菜黑斑病菌(Alternaria sp.)3个种及相近种的5.8SrDNA和其侧翼ITS区进行测序的基础上,分别设计合成了鉴定白菜黑斑病菌3个种的特异性引物。PCR扩增结果表明:Abre1和Abre2引物对能特异性扩增芸苔链格孢(A.brassicae)371bp的片段,Abra1和Abra2引物对能特异性扩增甘蓝链格孢(A.brassicicola)457bp的片段,Ajap1和Ajap2引物对能特异性扩增萝卜链格孢(A.japonica)411bp的片段,而且其它近源种未扩增出目标片段,说明这3个引物对可以作为十字花科蔬菜黑斑病菌3个种快速检测鉴定的分子特征标记。     

A PCR-based assay for the detection and identification of <Emphasis Type="Italic">Pyrenochaeta lycopersici</Emphasis>

The isolation of Pyrenochaeta lycopersici, causal agent of corky root of tomato, is difficult because of its slow growth and poor sporulation. Identification is complicated due the existence of two morphologically similar forms, Types 1 and 2, that differ in several physiological and molecular features. For the rapid and unambiguous identification of isolates, two oligonucleotide primer pairs were designed using ITS region sequences. Specific PCR products of 147 and 209 bp were obtained for isolates of Type 1 and Type 2, respectively. Specificity of both primer pairs was verified using several fungal and bacterial species. As little as 0.7 pg of target DNA could be detected with the protocol. A nested PCR procedure was necessary for the detection of the fungus in plant tissue. This technique will be of use in epidemiological studies and in the implementation of control strategies.     

Genetic Diversity Within Colletotrichum acutatum sensu Simmonds

ABSTRACT Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1-5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.