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1.
Powdery mildew is the most common disease on oaks in Europe where it was first recorded at the beginning of the 20th century. Yet, little is known about the origin of the causal agent. In this study, we analysed the variability of the ribosomal DNA (rDNA) of the pathogen. The internal transcribed spacer (ITS) region, the 5.8S rRNA coding gene and the intergenic spacer (IGS) of the rDNA of 33 European (mostly French) samples of oak powdery mildew were amplified using polymerase chain reaction (PCR) and PCR products were subsequently sequenced. Four different haplotypes were obtained for ITS among the various samples (ITSA, ITSB, ITSC and ITSD). Each ITS sequence corresponded to a different IGS sequence. The comparison of ITS sequences obtained with sequences accessible in the GenBank database revealed very high homologies with different taxa. Of these, three taxa had already been described on oaks in Europe, i.e. Erysiphe alphitoides (100% homology with ITSA), Erysiphe hypophylla (99.4% homology with ITSC) and Phyllactinia guttata (97.64% homology with ITSD). Our data also confirmed the 100% homology between ITSA and the sequence described for Oïdium mangiferae, the agent of mango powdery mildew. The fourth haplotype, i.e. ITSB, represented by nearly 25% samples, showed 100% homology with the recently described Erysiphe quercicola from Quercus spp. in Asia, and several tropical and sub‐tropical powdery mildew species, including Oïdium heveae, a major pathogen of rubber trees worldwide. Our results suggest that oak powdery mildew might originate from host shifts of tropical Erysiphe species introduced to Europe through infected exotic host plants. 相似文献
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Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species‐specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries. 相似文献
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A polymerase chain reaction (PCR)‐based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base‐pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralis DNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200 P. lateralis zoospores in water. 相似文献
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ITS sequences of ten kinds of plants of Lespedeza and an out group were obtained by primer design PCR, sequencing and cluster analysis. The results show that ITS1 section length was 228–243 bp, 5.8S sequence length was 165 bp which was very conservative; ITS2 section lengths varied from 215 to 220 bp and the conservative sites occupied 88.1%. Phylogenetic analysis of ITS sequences using PAUP 4.0 software and their genetic relationship were discussed. 相似文献
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Real‐time PCR assays based on the TaqMan system and using ITS sequences were developed for the identification of Phytophthora species, including P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina, all of which are currently causing significant damage to roots of forest trees in both managed stands and natural ecosystems. Total genomic DNA was extracted from mycelia of aforementioned Phytophthora isolates. Species‐specific primers for P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina were designed based on ITS sequences of rDNA. The amplification efficiency of target DNA varied from 93.1% (P. pseudosyringae) to 106.8% (P. quercina). The limit of the detection was calculated as 100 – 1,000 fg DNA, depending on the Phytophthora species. In mixed soil samples, all Phytophthora species were detected for Ct values shifted by 0.7 – 2.1 cycles. Based on these real‐time PCR assays we were able to identify the five Phytophthora species. These techniques will be of value in the identification of these pathogens, which may cause up to 80 – 90% fine root loss in oak stands. 相似文献
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Samad Jamali 《Agroforestry Systems》2017,91(2):335-343
During June 2013 to March 2014, several visits were made to the truffle-bearing areas of Kermanshah province, Iran. In this study, two specimens associated with roots of oak (Quercus brantii Lindl.) were identified as Tuber aestivum Vittad based on morphological and cytological characteristics. Internal transcribed spacer (ITS) region was amplified by PCR using primer pair ITS1/ITS4 and the sequences were analyzed. Phylogenetic trees constructed based on ITS sequences revealed that all Iranian specimens were in the same branch in a clade with T. aestivum reported from others. All T. aestivum sequences, including Iranian specimen, showed an average of 97 % similarity (ranged from 96 to 100 %). The results of physico-chemical analyses on soil samples collected from oak forest indicated that T. aestivum was prevalent in the sandy soil with rather low phosphorus concentration, low in organic matter, and high CaCo3. To our knowledge, this is the first report of T. aestivum and its host plant from Iran. 相似文献
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A nested and real‐time PCR assay was developed for the rapid and accurate detection of Ceratocystis fagacearum, which is the causal agent of oak wilt in stained wood and soil. Based on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species‐specific primers, CF01/CF02, was designed. Whereas a 280‐bp product was amplified using the purified DNA from three isolates of C. fagacearum as the template, no PCR product was obtained from template of other 18 fungi. The detection sensitivity was 10 pg genomic DNA per 25‐μl PCR reaction volume. To increase detection sensitivity, a nested PCR was developed by using ITS1/ITS4 as the first‐round primers and CF01/CF02 in the second round, as it can detect 1 pg genomic DNA per 25‐μl PCR reaction volume. More importantly, CF01/CF02 primers were successfully adapted to real‐time PCR with a detection limit of 0.1 pg genomic DNA per 20‐μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil. 相似文献
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Hiroto Suhara Nitaro Maekawa Takashi Kubayashi Ryuichiro Kondo 《Journal of Wood Science》2005,51(1):83-88
In order to monitor the basidiomycetous fungus Phlebia brevispora isolated from butt rot of Chamaecyparis obtusa (Japanese cypress) in 1997 in Nagasaki Prefecture, a sensitive polymerase chain reaction (PCR)-based assay was developed to specifically detect the fungus on-site. A species-specific primer for P. brevispora was derived from the internal transcribed spacer (ITS) region (containing 5.8S ribosomal DNA, ITS1 and ITS2) sequences of the fungus. The PCR assay was able to detect down to 1fg DNA (per 1µl PCR reaction mixture) and down to 0.2mg mycelium of P. brevispora (per 1g of decayed wood). The samples for on-site monitoring were collected in 2002 from the decayed tree stump in which P. brevispora had first been isolated. From the decayed tree tissue, P. brevispora could be detected by PCR assay even when its mycelium could not isolated from the tree tissue by culturing. This indicates that the PCR amplification using the specific primer developed here is a useful method for monitoring P. brevispora on-site. 相似文献
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R. Schubert G. Bahnweg J. Nechwatal T. Jung D. E. L. Cooke J. M. Duncan G. Müller-Starck C. Langebartels H. Sandermann Jr W. Oßwald 《Forest Pathology》1999,29(3):169-188
Oligonucleotide primers were developed for the polymerase chain reaction (PCR)-based detection of selected Phytophthora species which are known to cause root-rot diseases in European forest trees. The primer pair CITR1/CITR2, complementing both internal transcribed spacer regions of the ribosomal RNA genes, gave a 711 bp amplicon with Phytophthora citricola. The Phytophthora cambivora specific primer pair CAMB3/CAMB4, producing a 1105bp amplicon, as well as the Phytophthora quercina specific primer pair QUERC1/QUERC2, producing a 842 bp amplicon, were derived from randomly amplified polymorphic DNA (RAPD)-fragments presented in this paper. All three primer pairs revealed no undesirable cross-reaction with a diverse test collection of isolates including other Phytophthora species, Pythium, Xerocomus, Hebeloma, Russula, and Armillaria. Under the PCR conditions described the detection of a well discernable amplicon was possible down to 100 pg (P. cambivora), 4pg (P. quercina), and 2pg (P. citricola) target DNA. This diagnostic PCR system was able to detect P. citricola, P. quercina, and P. cambivora in seedlings of pendunculate oak (Quercus robur) and European beech (Fagus sylvatica) which were artificially infected under controlled conditions. 相似文献
11.
Deividas Valiunas Rasa Jomantiene Algirdas Ivanauskas Donatas Sneideris Marija Zizyte‐Eidetiene Jonathan Shao Zhao Yan Stefano Costanzo Robert E. Davis 《Forest Pathology》2019,49(6)
In order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini ’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma . 相似文献
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For diagnosis of pine wilt disease, a simple PCR‐RFLP method was developed to identify and to differentiate two similar nematode species, based on a living or preserved single specimen. Pinewood nematodes, Bursaphelenchus xylophilus, and Bursaphelenchus mucronatus were examined. A single nematode in 1 µl of distilled water was put on a glass slide. When the water had almost dried the nematode was crushed with a filter paper chip, 1.5 mm × 1.5 mm, with the aid of forceps. The filter paper chip containing nematode remains was immediately placed into PCR buffer as the DNA template. The primer set used was to amplify ribosomal DNA containing the inter‐transcribed spacer (ITS) 1, 5.8S and ITS2 regions. The PCR product was consistently obtained from a single nematode, and digesting the product with restriction endonuclease, Hinf I, enabled discrimination between B. xylophilus and B. mucronatus. This method was simple, convenient and definitive, and could successfully determine the pathogen in the diagnosis of pine wilt disease. This method was applicable also to nematode specimens preserved under various conditions except in the case of those preserved in aldehyde‐containing fixatives. 相似文献
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【目的】通过测序法分析兰考泡桐与白花泡桐和毛泡桐在叶绿体rps16序列上的遗传差异,旨在分析三者之间在叶绿体基因上的变化特点和规律,探讨其种间的遗传关系。【方法】选取兰考泡桐、白花泡桐和毛泡桐各15个样本,对其提取的DNA用PCR扩增获得特异片段,并将其纯化与测序。利用软件Clustal X 2.0对所得序列进行排序;运行MEGA 4软件,进行多序列比对,分析其序列特征,并计算出K2P遗传距离。【结果】(1)对获得的rps16序列进行测定分析,得兰考泡桐序列长度分别为932 933 bp;白花泡桐序列长度为932 bp;毛泡桐序列长度分别为916918 bp。对所得rps16序列进行排序后的长度为938 bp,平均GC含量为34.31%。3个种所代表的个体之间共有10个变异位点,占整个序列长度的1.07%。其中有9个变异位点属于碱基插入或缺失类型,占变异位点总数的90%,占整个序列长度的0.96%。有1个变异位点属于碱基替换类型,占整个变异位点总数的10%,占整个序列长度的0.11%。(2)整个rps16片段的序列共有10个变异位点,其中兰考泡桐与白花泡桐在总的变异位点上,具有一致的碱基位点9个,占总变异的90%。而兰考泡桐与毛泡桐相比,没有相同的碱基。【结论】根据三种泡桐的rps16序列的序列特征和变异位点的分析,表明在叶绿体遗传方面,兰考泡桐具有与白花泡桐更多相似的遗传物质,其亲缘关系较近。综上所述,推测兰考泡桐与白花泡桐可能来自同一母系遗传。 相似文献
15.
Genetic diversity of pine‐parasitic nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus in China 
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下载免费PDF全文 The internal transcribed spacer (ITS) regions of rDNA have been routinely employed for identification and phylogenetic analysis of many nematode species. In this study, the intra‐ and interspecies ITS genetic diversity of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus was evaluated. Ninety‐one isolates of the two nematode species collected from 14 Chinese provinces, Japan and Korea were used for ITS‐PCR and sequencing. An unweighted pair group cluster analysis dendrogram clustered them as two B. mucronatus and one B. xylophilus independent clades. Principal component analysis showed the phylogenetic relationship of the two nematode species more clearly; B. mucronatus isolates were separated into more than four groups, whereas B. xylophilus isolates still clustered into a group. The results of the Mantel test indicated the correlation of genetic distance matrices and geographic distance matrices was significant for both nematode species. The genetic differentiation coefficient (Gst) and gene flow (Nm) of B. mucronatus were 0.341 and 1.091, respectively, suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B. mucronatus isolates in China. However, Gst and Nm of B. xylophilus were 0.188 and 2.151, respectively, very different compared to B. mucronatus. This could be owing to the short‐term introduction of B. xylophilus into China and a rapid spread through anthropogenic pathways. Our work adds to the understanding of the genetic diversity and genetic relationship of the two pine‐parasitic nematode species, and will aid in controlling them in the future. 相似文献
16.
Hamed Yousefzadeh Abbas Saidi Somayeh Tayebi Davoud Kartoolinejad Reza Naghdi 《林业研究》2017,28(4):661-670
Castanea sativa is a valuable tree species in Hyrcanian forests, an evolutionary relict ecosystem whose communities suffer from overexploitation and fungal diseases. In the current study, three species delimitation methods were utilized with ITS regions sequencing to determine the taxonomic status of Septoria causing leaf blotch of C. sativa in Hyrcanian forests. The results indicated that the length of ITS region in the genus Septoria (extracted from GenBank) varied from 650 to 680 bp. There were almost three times more variable sites in ITS1 than in ITS2. The ITS2 secondary structure of Hyrcanian Septoria community had the highest similarity with Septoria castaneicola, except for some differences in helix II and III. Also, Hyrcanian samples had minimum genetic distances with S. castaneicola and maximum with Septoria quercicola. The maximum parsimony method divided the studied Septoria genus into three distinct clades, mostly located in clade I. Clade II consisted entirely of Septoria aciculosa, while clade III contained S. castaneicola as well as Hyrcanian samples. 相似文献
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Pyrofomes demidoffii is a basidiomycetous fungus that causes a white heart rot in living junipers (Juniperus spp.). Morphological characters and genetic variation in species of Pyrofomes were investigated for specimens from Ethiopia, Tanzania, Macedonia, USA and Costa Rica. In addition, the behaviour of isolates from Ethiopia was studied in vitro. The Ethiopian specimens had relatively larger basidiospores than collections from other countries and showed a high sequence similarity in the ITS region of rDNA sequences with most of strains evaluated in this study (95–100%), but only 89–92% with P. demidoffii from the USA. Phylogenetic analysis of ITS‐5.8S rDNA sequences resulted in two well‐supported (99–100% bootstrap value), separate clades of P. demidoffii. Deeper branches were less well supported (bootstrap < 90%), but the two geographically and phylogenetically separated clades within P. demidoffii may still represent sister groups. For Ethiopian strains, the temperature optimum was between 20 and 25 °C and the pH optimum was around 5.0. A phylogenetic study with additional genetic markers and with a more representative collection of samples is needed. 相似文献
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Poplar rusts due to Melampsora larici‐populina (Mlp), M. allii‐populina (Map) and M. medusae f. sp. deltoidae (Mmd) are the most serious disease in Europe on cultivated poplars, that is, Populus × euramericana and P. × interamericana hybrids. These pathogenic species can be identified by the observation of morphological characteristics of urediniospores but this method is not appropriate for high‐throughput analysis and cannot be used on other spore stages, such as aeciospores or teliospores, that are morphologically similar. The aim of this study was to develop a rapid and sensitive molecular method based on PCR amplification that was able to specifically detect these species on various hosts for routine analysis. Three primer pairs ITS‐MLP‐F/ITS‐MLP‐R, ITS‐MAP‐F/ITS‐MAP‐R and ITS‐MMD‐F/ITS‐MMD‐R were designed within the internal transcribed spacer (ITS) sequences of ribosomal DNA to target Mlp, Map and Mmd, respectively, and their specificity were confirmed on a wide range of isolates and species. ITS‐MLP‐F/ITS‐MLP‐R and ITS‐MAP‐F/ITS‐MAP‐R primers proved to be highly specific to Mlp and Map, respectively, whereas ITS‐MMD‐F/ITS‐MMD‐R cross‐reacted with DNA from M. larici‐tremulae and M. pinitorqua. However, these species are not pathogenic on cultivated poplars that all belong to sections Aigeiros and Tacamahaca of the genus Populus. Specific Mmd primers proved to be very sensitive as a positive signal could be obtained with DNA extracts from 6 target urediniospores mixed with 800 000 urediniospores of Mlp. An internal amplification control (IAC) was included to discriminate false negative results due to the potential presence of inhibitory compounds in DNA extracts. ITS‐MMD‐F/ITS‐MMD‐R primers are therefore efficient for the detection of the quarantine pathogen Mmd on samples collected on poplar or larch and are fit for use in official tests. This new PCR assay has been used in routine for ten years, and Mmd has hitherto never been detected in commercial poplar nurseries in France. 相似文献
20.
J. A. Hoff N. B. Klopfenstein G. I. McDonald J. R. Tonn M.‐S. Kim P. J. Zambino P. F. Hessburg J. D. Rogers T. L. Peever L. M. Carris 《Forest Pathology》2004,34(4):255-271
The fungal community inhabiting large woody roots of healthy conifers has not been well documented. To provide more information about such communities, a survey was conducted using increment cores from the woody roots of symptomless Douglas‐fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) growing in dry forests on the eastern slope of the Cascade Mountains in Washington state, USA. Fungal isolates were cultured on standard media, and then were identified using a combination of molecular and morphological methods. Fungal genera and species identified in this study will provide baseline data for future surveys of fungal endophytes. Examination of internal transcribed spacer (ITS1 and ITS2) and 5.8S rDNA sequences and morphology of cultured fungi identified 27 fungal genera. Two groups predominated: Byssochlamys nivea Westling (20.4% of isolations) and Umbelopsis species (10.4% of isolations). This is the first report of B. nivea within large woody roots of conifers. Both taxa have been previously identified as potential biological control agents. Although some trends were noted, this study found no significant evidence of host species or plant association effects on total recovery of fungal endophytes or recovery of specific fungal taxa. 相似文献