Molecular Distinctions Between Phytophthora capsici and Ph. tropicalis Based on ITS Sequences of Ribosomal DNA

Among four species of Phytophthora tested, only Ph. capsici and Ph. tropicalis showed the same length for DNA sequence for both internal transcribed spacer (ITS)1 and ITS2 of ribosomal DNA. Phytophthora palmivora and P. nicotianae have lengths different from each other, and from the other two species. Although A1 and A2 types of Ph. capsici differ from each other by only one nucleotide, there are 10 different nucleotides between A1 and A2 types of Ph. tropicalis. Phylogenetic analysis of combined ITS sequences identified four clades each consisting A1 and A2 mating types of same species. The neighbor‐joining and maximum parsimony trees show that Ph. tropicalis (A2) is clustered with the clade of two isolates of Ph. capsici before joining the clade of A1 and two other isolates of Ph. tropicalis from GenBank. Our results support the separation of Ph. tropicalis and demonstrate the need to sequence more than a single isolate of a species in the study of molecular phylogeny of Phytophthora. The phylogenetic trees also suggest that Ph. tropicalis (A2) may represent a transitional isolate in the process of species evolution.     

Detection of Phytophthora nicotianae in Soil with Real-time Quantitative PCR

Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora , the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pg/μl in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/μl and in TaqMan PCR 1.2 fg/μl, and real-time PCR was 10⁴–10⁵ times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/μl. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method.     

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.     

Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Molecular data reveal convergence in fruit characters used in the classification of Thlaspi s. I. (Brassicaceae)

Phylogenetic relationships of 18 Thlaspi s.l. species were inferred from nuclear ribosomal internal transcribed spacer (ITS) sequence data. These species represent all sections of the basic classification system of Schulz primarily based on fruit characters. The molecular phylogeny supported six clades that are largely congruent with species groups recognized by Meyer on the basis of differences in seed coat anatomy, i.e. Thlaspi s. s., Thlaspkeras, Moccaea {Raparia included), Microthhspi, Vania and Neurotropy. Some of these lineages include species which are morphologically diverse in fruit shape (e.g. Thlaspi s. s.: T. arvense - fruits broadly winged, T. ceratocarpum - fruits with prominent horns at apex, T. alliaceum - fruits very narrowly winged). Furthermore, the same fruit shape type is distributed among different clades. For instance, fruits with prominent horns at apex are found in Thlaspi s. s. ( T. ceratocarpum) and Thlaspiceras (T oxyceras). These results clearly indicate convergence in fruit characters previously used for sectional classification in Thlaspi s. l.     

Analysis of internal transcribed spacer (ITS) region of Phytophthora tropicalis causing quick wilt disease of black pepper in Vietnam

Phytophthora quick wilt is a devastating disease of black peppers in Vietnam. The internal transcribed spacer (ITS) region of the ribosomal DNA of four Phytophthora samples isolated from the diseased vines in Daknong province of the central highland part of Vietnam was Polymerase chain reaction-amplified, cloned, sequenced and characterised. Database search have showed that they are most closely related to an isolate of Phytophthora tropicalis from Taiwan. Sequence comparisons and phylogenetic analyses based on the ITS region of the four Vietnamese and other GenBank isolates of P. tropicalis and a closely related species, P. capsici, provide strong evidences that the Vietnamese isolates are all different isolates of P. tropicalis.     

Molecular characterization of fungal endophytes from Calotropis procera plants in Taif region (Saudi Arabia) and their antifungal activities

Fungal endophytes were isolated from the leaves of Calotropis procera (Apocynaceae) collected from Taif region (Saudi Arabia). Thirty-three different taxa were recovered. The overall foliar colonization rate was 35.1%. A total of 161 isolates were obtained and identified into 33 distinct operational taxonomic units based on the sequencing of the internal transcribed spacer regions of the rRNA gene. The most prevalent fungi were Aspergillus flavus, Chaetomium globosum, Cochliobolus lunatus, Fusarium dimerum, F. oxysporum, and Penicillium chrysogenum. A total of 161 isolates were tested for antifungal activities against four plant pathogenic fungi (Alternaria alternata, Fusarium oxysporum, Botrytis cinerea, and Pythium ultimum), of which 33 isolates showed antifungal activity against at least one plant pathogenic fungi. Four isolates of Chaetomium globosum and three isolates of Myrothecium verrucaria showed the strongest antifungal activity. This study reported the occurrence of a much wider spectrum of fungi, when compared with previous work. Also, it confirmed the variation of different isolates from the same species in terms of antifungal activity.     

Molecular appraisal of Bunium and some related arid and subarid geophilic Apiaceae–Apioideae taxa of the Ancient Mediterranean

Bunium is unusual in Apiaceae in having a variable cotyledon number and broad infrageneric dysploidy. To test the monophyly of the genus, phylogenetic relationships among 39 Bunium species were investigated with DNA sequence data from nuclear (nrITS) and plastid (psbA‐trnH intergenic spacer) regions. Several other taxa with a similar ecology and geography were also included in the analyses. Our results suggest that Bunium is not monophyletic. Bunium spp. in the eastern part of the study area are more closely related to the Central Asian genera Elaeosticta, Galagania, Hyalolaena, Mogoltavia and Oedibasis than to those in the western part. Our study revealed that molecular, morphological (cotyledon number and width of fruit commissure) and karyological data reveal similar patterns in the taxa studied. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 149–170.     

Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR

Aims: The ichthyotoxic species Prymnesium parvum (Haptophyceae) is difficult to quantify in a microscopy‐based monitoring programme, because the cells are very small, fragile and their morphology can be distorted by the use of fixatives. In the attempt to overcome these problems, a real‐time PCR‐based method for the rapid and sensitive identification and quantification of P. parvum was developed. Methods and Results: A quantitative real‐time PCR assay was optimized with primers designed on the internal transcribed spacer 2 rDNA region of P. parvum. This PCR assay was specific, showing no amplification of DNA extracted from closely related species, and sensitive. Moreover, this method was able to detect and reliably quantify P. parvum cells in preserved environmental samples artificially spiked with known amounts of cultured cells. Conclusions: Considering the specificity, sensitivity and applicability to preserved environmental samples, this method may be a useful tool for the monitoring of this toxic species. Significance and Impact of the Study: The real‐time PCR method described in this study may represent a progress towards the rapid detection and quantification of P. parvum cells in water‐monitoring programmes, allowing the early application of strategies to control bloom events, such as the use of clay minerals.     

Detection of the antimicrobial peptide gene in different Amaranthus species

Using primers to amplify the gene AMP2 in Amaranthus caudatus, we found the gene to be present in seven other species of the Amaranthus genus (A. albus, A. cruentus, A. blitum, A. hybridus, A. hypochondriacus, A. retroflexus and A. tricolor), in which it had not been described previously. The PCR products were sequenced and it was established that all the sequences were identical, except for two polymorphisms. These single nucleotide polymorphisms occurred at nucleotide positions 45 and 246. This exchange of one nucleotide for another was manifested in an amino acid change in both cases. Due to the fact that both polymorphisms lay outside the region encoding the chitin-binding peptide domain, which is crucial for antimicrobial peptide function, they will not likely affect the proper functioning of the peptide. With the exception of the above-mentioned polymorphisms, all sequences were identical to the sequence of the AMP2 gene that codes for the A. caudatus Ac-AMP2 (antimicrobial peptide isolated from Amaranthus caudatus seeds). The detection of sequences with high degree of sequence similarity to A. caudatus AMP2 gene leads us to the assumption that an antimicrobial peptide could also be produced by other amaranth species.     

Development and comparison of ELISA and PCR methods for the early detection of Ganoderma disease of coconut

Abstract

Molecular diagnosis, chemo-diagnosis and physiological parameter have been applied for detecting the Ganoderma disease of coconut. Polyclonal antibodies (PAbs) raised against mycelial protein of Ganoderma, specific mycelial protein (62 kDa) of Ganoderma isolates and basidiocarp protein of Ganoderma were used for detection. All the PAbs could detect Ganoderma in diseased coconut root tissues in early stage of the disease before symptom expression by indirect – ELISA at the antiserum dilution of 1:1000 for mycelial protein, 1:700 for specific protein and 1:3000 for basidiocarp protein. Low cross reactions were observed with saprophytic fungi occurring in coconut roots and also with other basidiomycetous fungi. For polymerase chain reaction tests, the primer was generated from the internal transcribed spacer region one (ITS 1) of rDNA of Ganoderma, which produced a PCR product of 167 bp in size. Utility of this method was confirmed at the field level.     

Molecular definition and the ubiquity of species in the genus Naegleria

To investigate the variability within species of the genus Naegleria, the ITS1,5.8S and ITS2 rDNA were sequenced of several strains of N. lovaniensis and its Western Australian variants, N. australiensis, N. fowleri, N. andersoni, N. jamiesoni, N. tihangensis, N. pringsheimi, N. pagei, N. gruberi sensu lato and a Naegleria lineage that lost a group I intron from the SSUrDNA twintron. As a result, it is possible to define a molecular species within the Naegleria genus. In addition, one strain of each different allozyme cluster was sequenced to investigate whether they belong to described species or should be treated as distinct new species. This leads to the proposal of eleven new species. The sequencing results from those Naegleria spp. of which several strains are available indicate that these species are ubiquitous. The only exception might be the species represented by the WA variants. However, there are still many Naegleria spp. for which only one strain has been isolated, hence, it is important that the search for more isolates should be continued worldwide.     

Molecular Evidence for the Existence of Two Species of Marteilia in Europe

Marteilia refringens is one of the most significant pathogens of bivalve molluscs. Previous sequencing of the small subunit ribosomal RNA gene of M. refringens isolates derived from the infected mussels (Mytilus edulis and Mytilus galloprovinciallis) and the oyster (Ostrea edulis) in Europe did not reveal genetic polymorphisms despite indications from epizootiological data that distinct types may exist. We investigated the existence of polymorphisms in the internal transcribed spacer region of the ribosomal RNA genes. The sequences of this region proved to be clearly dimorphic among Marteilia from five sampling sites. The distribution of the two genetic types, named "O" and "M", appeared to be linked to the host species, oysters and mussels, respectively. We therefore support the recognition of two species of Marteilia in Europe and propose that the "O" type corresponds to M. refringens and the "M" type to M. maurini.     

泛耐药细菌NDM-1基困Taqman PCR快速检测方法的建立

产NDM-1(New Delhi Metallo-β-lactamase 1,Ⅰ型新德里金属β-内酰胺酶)细菌是新近报道的一种泛耐药细菌,由于对绝大多数常用抗生素均耐药,又被称为超级细菌.目的:建立一种可快速检测泛耐药细菌NDM-1基因的Taqman探针实时荧光定量PCR法.方法:根据NDM-1基因序列,设计引物和Ta...     

Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S-23S rDNA internal transcribed spacer (ITS) region

AIM: Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S-23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples. METHODS AND RESULTS: Campylobacter species-specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples. CONCLUSIONS: The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples. SIGNIFICANCE AND IMPACT OF STUDY: Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.     

利用三种分子标记研究缘毛类纤毛虫的系统发育地位

为了探讨缘毛类纤毛虫的系统发育地位 ,利用RAPD方法得到了 9种缘毛类纤毛虫、 1种四膜虫和1种喇叭虫的 3个随机引物的电泳带谱 ;测定了 7种缘毛类纤毛虫rRNA基因中的间隔区 1(ITS1)和小亚基核糖体核糖核酸 (SSrRNA)基因序列 ,并构建了相应的系统树。在比较和分析RAPD、ITS1和SSrRNA基因序列在缘毛类纤毛虫系统发育研究中的适用范围的基础上 ,以SSrRNA基因序列为分子标记研究了缘毛类纤毛虫系统发育地位 ,结果表明 :①缘毛亚纲是单系的 ,作为寡膜纲中一个亚纲的分类地位是合理的 ;②缘毛类纤毛虫可能是寡膜纲中较高等的一个类群。     

An in vitro investigation of Malaysian Phytophthora palmivora isolates and pathogenicity study on oil palm

Bud rot disease affecting oil palm in South American countries is reported to be caused by Phytophthora palmivora. P. palmivora is a local pathogen affecting various crops in Malaysia, and this finding caused an alarm, which prompted an investigation of pathogenicity using Malaysian P. palmivora to assess the potentials of this Oomycete to infect oil palm in Malaysia. A total of 11 P. palmivora isolates were obtained from cocoa and durian for the study. Leaf bioassays via artificial inoculation using 50,000 zoospores/ml and mycelial agar disc showed severe necrotic lesions on the infection spot of oil palm (DxP) spear leaves. Mild infection was observed in oil palm spear leaves of OxG hybrids indicating lower susceptibility against P. palmivora infection. Phylogenetic analysis using internal transcribed spacer (ITS) data revealed that Malaysian isolates were genetically similar to Colombian isolates supported by significant bootstrap values. The leaf bioassay results revealed that Malaysian oil palm materials are susceptible towards local P. palmivora infection. The Colombian P. palmivora isolates causing bud rot incidence may have evolved over a long period of time, undergone sequential genetic shift to become more virulent towards Colombian oil palm planting materials.     

Molecular Evidence for the Hybrid Origin of Bauhinia blakeana （Caesalpinioideae）

Bauhinia blakeana Dunn is the Hong Kong Special Administrative Region emblem and a popular horticultural species in many Asian countries. It was first described as a new species from Hong Kong almost a century ago. This plant is sterile and has long been considered a hybrid, possibly from two related species, B. purpurea and B. variegata. However, not much evidence based on molecular methods was available to support this hypothesis. In this study, sequences of internal transcribed spacer I （ITS1）, rbcL and atpB-rbcL intergenic spacer for five Bauhinia species and two varieties of one of the species were determined and compared. There were two types of ITS1 sequences in B. blakeana, one indistinguishable from that of B. purpurea and the other one identical to that of B. variegata. This confirmed that B. blakeana was a hybrid of these two species. Chloroplast atpB-rbcL intergenic spacer sequence of B. blakeana was identical to that of B. purpurea, indicating that B. purpurea was the female parent. The hybridization event seemed to occur only recently and was a rare incident. Its occurrence was likely facilitated by interspecific pollen competition. It appeared that human efforts played a crucial role in the preservation and ubiquity of B. blakeana.     

Characterization of Alder Phytophthora Isolates from Wallonia and Development of SCAR Primers for their Specific Detection

Isolates of alder Phytophthora were collected in the southern part of Belgium on riverbanks planted with Alnus glutinosa and A. incana. They were compared with strains isolated in other European countries in terms of maximum temperature for growth, oogonia shape, pathogenicity on Alnus seedlings and genetic traits. Using both molecular techniques [random amplified polymorphic DNA (RAPD) and random amplified microsatellite (RAMS)], two groups of isolates were identified, the first group being further divided into two subgroups, Ia and Ib, using RAPD. Most of the Walloon alder Phytophthora isolates as well as the standard type from UK (formally designated P. alni subsp. alni) fell into group Ia. One isolate was classified in group Ib with the German and Dutch variants (P. alni subsp. multiformis), while three isolates were placed with the Swedish variant (P. alni subsp. uniformis) in group II. In terms of morphological properties, isolates from groups Ia and Ib developed colonies with a felt‐like appearance and usually produced numerous oogonia, varying from wavy to warty after 1 week (group Ia) or 2–3 weeks (Ib) in darkness. In contrast, colonies from group II isolates were generally irregular, and smooth oogonia were produced in low quantities after approximately 1 month in culture. A polymerase chain reaction (PCR) using sequence‐characterized amplification region (SCAR) primers derived from a polymorphic amplification product generated with a RAPD primer was developed for the specific detection of alder Phytophthora. The specificity and sensitivity of this test are discussed here.     

Molecular phylogeny of the genus Philodendron (Araceae): delimitation and infrageneric classification

The genus Philodendron (Araceae) is a large neotropical group whose classification remains unclear. Previous classifications are based on morphological characters, mainly from the inflorescence, flower and leaf shape. The classification by Krause, with few modifications, is still the most commonly used system. To examine phylogenetic relationships in the genus, two ribosomal DNA nuclear markers, internal transcribed spacer (ITS) and external transcribed spacer (ETS), and the chloroplast intron rpl 16, were sequenced and analysed for more than 80 species of Philodendron and its close relative Homalomena . According to the resulting phylogeny, the genus Homalomena may be paraphyletic to the genus Philodendron . The inclusion of the American Homalomena species within the genus Philodendron might resolve this taxonomic problem. All three subgenera of Philodendron were revealed as monophyletic. Below the subgeneric level, the groups obtained in our phylogeny globally correspond to sections recognized in previous classifications. Among the morphological characters used by previous taxonomists to build their classifications, and which we optimized onto one of the most parsimonious trees, most characters were found to be homoplasious. However, leaf shape, characteristics of the sterile zone on the spadix and venation patterns are useful for delimiting subgenera and sections within the genus.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 13–27.