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1.
ABSTRACT As a first step toward analysis of genetic variation and population structure in Peronospora tabacina, we used a collection of random genomic DNA fragments to survey for restriction fragment length polymorphisms (RFLPs) in DNA from a collection of isolates from Kentucky and other tobacco-growing regions of the United States. Also included in the study were isolates from the wild tobacco species, Nicotiana repanda, and from ornamental tobacco, N. alata. In a preliminary survey using DNA from 10 pathogen isolates, no polymorphisms were detected at six single-copy DNA loci using 22 probe-enzyme combinations. Moderately repetitive and highly repetitive regions of the genome were also remarkably similar between isolates, with only 6 of 15 different probes identifying genetic differences. Some of the polymorphic probes were then used to analyze a larger collection of isolates, most of which were from Kentucky. This resulted in the identification of very few additional polymorphisms, indicating that the population of P. tabacina that infects the Kentucky tobacco crop is genetically very homogeneous. The low level of polymorphism detected in this study overall, suggests that genetic variability may be lacking in P. tabacina populations throughout the United States. Two of the RFLP markers gave hybridization patterns that were consistent with P. tabacina being diploid. Frequencies of alleles at these loci and linkage disequilibrium between different marker loci indicated that genetic recombination does not occur frequently in the pathogen population. DNA polymorphisms that were identified in this study enabled us to differentiate the pathogen population into at least 10 haplotypes. One isolate was analyzed in detail and was shown to be genetically stable through several rounds of single-spore isolation and through several pathogenic cycles. 相似文献
2.
Several accessions of Nicotiana langsdorffii, a wild tobacco relative native to South America, express an incompatible interaction in response to infection by Peronospora tabacina, an o?mycete that causes blue mold disease of tobacco (N. tabacum) and many other species of Nicotiana. In resistant accessions of N. langsdorffii, such as S-4-4, incompatibility takes the form of necrotic lesions that appear 48 h after pathogen inoculation, restricting pathogen growth, and suppressing subsequent asexual sporulation. Significantly elevated levels of salicylic acid and expression of a defense-related gene (HSR203J) were observed in S-4-4 leaves following blue mold infection. Genetic segregation analysis in F(2) and modified backcross populations showed that blue mold resistance is determined by a single dominant gene (NlRPT) present in S-4-4. Further characterization of this unique host-pathogen interaction has revealed that (i) necrotic lesion resistance is due to the hypersensitive response (HR), (ii) HR-mediated resistance is present in 7 of 10 N. langsdorffii accessions examined, but not in closely related species, (iii) in some accessions of N. langsdorffii, resistance is expressed in cotyledon tissue and seedling leaves as well as in adult plants, and (iv) several resistant accessions including S-4-4 express an unregulated ("runaway") HR in response to P. tabacina infection. 相似文献
3.
ABSTRACT Long-term cocultures of the tobacco blue mold pathogen, Peronospora tabacina, with Nicotiana tabacum and N. repanda callus were derived from infected host plant tissue. In this apparently contaminant-free system, sporulation occurred under similar conditions as in intact plants. Sporangia were collected from cocultures and used to complete Koch's postulates. The cocultures were grown under two light regimes. One consisted of 23 h of light followed by 1 h of darkness and the second comprised total darkness. Sporulation occurred frequently in the 23 h light-grown cocultures but resulted in production of abnormal sporangiophores and sporangia. Production of normal sporangiophores and sporangia was achieved by transferring light-grown cocultures to overnight darkness and resulted in necrosis of the callus. Cocultures of Peronospora tabacina with either host species, grown in total darkness, frequently sporulated with minimal necrosis over the course of 1 year. Thus, cocultures should prove useful as a source of Peronospora tabacina over extended periods of time at low risk of pathogen release, for studying the physiology of Peronospora tabacina- Nicotiana interactions, maintaining Peronospora tabacina lines for genetic studies, and providing a reliable source of axenic inoculum for research. 相似文献
4.
5.
稻瘟菌TAC文库的构建与评价 总被引：5，自引：2，他引：5
本文构建了一个包含16 128个克隆的稻瘟菌基因组的TAC文库,74%的克隆含有外源插入片段,其插入片段大小平均为59 kb,该库相当于稻瘟菌基因组的18倍。本文并对文库的代表性进行了评价,采用3个单拷贝的标记作为探针筛库,分别得到16,17和16个阳性克隆,这与估算的该库的覆盖率是一致的;从MH 18S1为探针所筛选的阳性克隆中,随机挑取8个并对其限制性酶切图谱进行分析,结果表明:其组成的重叠群跨度为153 kb,未发现嵌合和缺失现象。以上结果表明,此TAC文库适合用于稻瘟菌基因组的分析。 相似文献
6.
Sugar accumulation in tobacco plants systemically protected against blue mold (Peronospora tabacina)
A two- to threefold increase in total soluble sugar content was detected in the leaves of tobacco plants(Nicotiana tabacum L.) 24 days after stem inoculation with conidia ofPeronospora tabacina (=Peronospora hyoscyami f. sp.tabacina), relative to leaves of non-inoculated control plants. Glucose and fructose accounted for most of this increase. The leaves of the inoculated plants were also protected against blue mold incited byP. tabacina. The increased sugar content may be due to the three- to fourfold increase in activity of (β-1,3 glucanase in the leaves and/or to the threefold increase in activities of β-1,3 glucanase, invertase and amylase in the inoculated sems. Tobacco leaf discs floated on glucose or other mono- and disaccharides at 55.5 mM, but not on polyethyleneglycol at a similar osmotic potential, reacted hypersensitively upon challenge withP. tabacina. 相似文献
7.
During recent years, Peronospora tabacina has frequently been intercepted on leaves in lots of imported Greek oriental tobacco at ports of entry in China. The question arises whether such infected leaf debris can be a source of primary inoculum. The present study has examined the pre-export viability of P. tabacina on stored Greek oriental tobacco. Firstly, basic experiments were done: (1) to find a suitable medium (2% Bacto (Difco) agar) for in vitro germination of P. tabacina sporangia; (2) to determine the optimum (15–21 °C), maximum (30°C) and minimum (2°C) temperatures for sporangial germination; (3) to devise a suitable method of inoculation for an infectivity test. Secondly, viability tests were done on stored Greek oriental tobacco harvested in 1986 and 1987. In vitro germination tests and infectivity tests were conducted separately, according to microscopic grading of the apparent turgidity of sporangia and sporangiophores collected from lesions of infected tobacco leaves of different cultivars and crop years. The tests were maintained for a much prolonged incubation period as compared with the controls. Neither in vitro germination nor infectivity tests showed any sporangia to be viable. Further work is needed to determine what period of storage is rationally acceptable for both plant quarantine and commercial trade. 相似文献
8.
烟草赤星病拮抗细菌的筛选及其控病作用 总被引：12，自引：0，他引：12
从烟草叶片上分离到236株非病原细菌,通过平板对峙培养,筛选出对不同致病力的烟草赤星病菌Alternaria alternata(Fr.)Keissl均有拮抗作用的菌株:Ata28、Ata81、Ata124和Ata160.室内测定其对赤星病菌抑菌带的宽度达6.1～10.5mm;室内离体叶片悬滴法测定,提前喷施拮抗菌其防治效果为53.9%～73.0%;接赤星病菌后喷施的防治效果为44.0%～60.0%;在温室中进行盆栽防病试验,防治效果为52.3%～75.8%.无菌滤液试验表明,拮抗菌Ata28无菌滤液在一定浓度范围内均能有效地抑制菌丝生长,减少孢子萌发,且浓度越高,抑制能力越强. 相似文献
9.
Moshe Reuveni Sadik Tuzun James S. Cole Malcolm R. Siegel William C. Nesmith Joseph Ku 《Physiological and Molecular Plant Pathology》1987,30(3)
Dipping leaf strips of greenhouse or field-grown tobacco (Nicotiana tabacum L., cv. Ky-14) plants into acetone for 1 s, prior to inoculation with sporangia of Peronospora tabacina Adam, increased their susceptibility to blue mold. Disease severity and sporangial production on leaf discs from acetone-treated leaves were markedly increased compared to those on discs from untreated leaves. Treatment with acetone also decreased variation in susceptibility of leaves from plants of various ages. Disease severity on discs obtained from attached leaves which were dipped in acetone for 1 s was three times greater up to 15 days after dipping than on discs from leaves that were not dipped. TLC and GLC analyses of the acetone extracts indicated that 95% or more of the major cuticular diterpenoids, α- and β-4,8,13-duvatriene-1,3-diols (DVT), were removed from the surface by dipping for 1 s. These compounds had not reappeared on the leaf surface 15 days after leaves were dipped in acetone for 1 s. Aqueous suspensions of the acetone-soluble constituents as well as authentic DVT, inhibited sporangial germination of P. tabacina (ED100 = 25 ppm) and the antifungal activity was accounted for by DVT. When DVT was removed from a leaf surface and added back to the same leaf strip, the resistance of the leaf tissue was restored. As tobacco plants aged, their susceptibility to blue mold decreased and the quantity of DVT on the leaf surface increased. The data support a role for DVT in the resistance of tobacco against blue mold. 相似文献
10.
大麦黄矮病毒(BYDV) cDNA的合成、克隆及初步应用 总被引：6，自引：0，他引：6
以大麦黄矮病毒二叉蚜和麦长管蚜专化株的病毒核酸为模板,以小牛胸腺DNA为引物,合成cDNA的第一条链,再用缺口翻译法合成第二条链,然后采用加装BamH1人工接头的方法将ds-cDNA插入到质粒载体pUC8中,重组质粒于大肠杆菌JM—83中进行克隆,以克隆的颜色变化选择含有外源DNA的克隆,再用病毒核酸制备的探针筛选真正病毒cDNA插入的克隆。重组质粒中ds-DNA的插入长度在300—1600bp之间。用缺口翻译法制备质粒DNA分子探针检测同源病毒液,反应灵敏度在100pg-1ng之间。应用cDNA探针检测不同病毒和病毒株系,从中筛选出黄矮病毒株系专化克隆系,黄矮病毒专化克隆系和黄矮病毒组专化克隆系. 相似文献
11.
ABSTRACT Peronospora tabacina is an obligate plant pathogen that causes downy mildew disease on several species of Nicotiana, including N. tabacum (tobacco). The primary objective of this study was to use gnotobiotic associations to describe interactions between the pathogen and roots of either N. tabacum (cv. KY14) or N. repanda. We found that the pathogen was capable of moving systemically from shoots to roots of both host species and emerged from the root tissues as hyphae. We also demonstrated that root-associated hyphae were infectious on roots of nearby plants and resulted in new systemic infections. Following overnight darkness, sporulation of the pathogen was observed on infected roots exposed to air on both host species. We also found that within 2 months in culture, structures resembling resting stages of Peronospora tabacina were produced on hyphae emerging from roots of N. repanda but not N. tabacum. These findings appear relevant to both the epidemiology of the disease and to future studies of this and other downy mildew pathosystems. 相似文献
12.
为克隆和研究链孢粘帚霉Gliocladium catenulatum寄生核盘菌菌核的相关基因,应用抑制消减杂交技术构建了cDNA消减文库并进行了筛选。通过PCR技术从文库中共筛选到1315个阳性克隆,克隆中插入片段大小主要集中于300～600bp之间。随机挑取120个克隆,经测序和同源性分析,获得60条有效序列,其中部分序列所编码的血红素加氧酶、核糖体蛋白L11、细胞色素P450及热激蛋白等均参与机体对胁迫条件的应答反应。11条序列在NCBI数据库中未找到显著匹配的序列,可能为新基因片段。分别将寄生于核盘菌菌核上的粘帚霉cDNA和粘帚霉与核盘菌纯培养的cDNA混合物经RasⅠ酶切后进行标记作为探针,利用反向Northern杂交技术验证了所选取的25条序列全部为差异表达基因片段。 相似文献
13.
Genetic and pathogenic variation of Xanthomonas axonopodis pv. manihotis in Venezuela 总被引：1，自引：0，他引：1
DNA polymorphism and variation in virulence of Xanthomonas axonopodis pv . manihotis (Xam), the causal agent of cassava bacterial blight, were studied within a pathogen population from Venezuela. Collections were made in several fields at different sites within an edaphoclimatic zone where cassava is a major crop. DNA polymorphism was assessed by RFLP analysis, using an Xam plasmidic DNA sequence ( pth B) as a probe to determine the relatedness of 91 Venezuelan isolates. A high degree of polymorphism existed among the isolates, whether collected from the same or different fields. Based on a multiple correspondence analysis, the Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin. One set of haplotype strains representing the range of variability detected in Venezuela was further characterized by another RFLP analysis using two repetitive genomic probes (pBS6 and pBS8) to establish the usefulness of these probes and their complementarity with the pth B probe. Variation for virulence was observed in the Xam Venezuelan collection by inoculating a set of cassava cultivars with 28 isolates of the pathogen, each representing a haplotype. Understanding the genetic and pathogenic variation in the pathogen population is useful for designing cassava bacterial blight management strategies. 相似文献
14.
Miaw-Fan Chen Chan-Pin Lin 《European journal of plant pathology / European Foundation for Plant Pathology》1997,103(2):137-145
EcoRI restriction fragments of genomic DNA from the phytoplasma associated with peanut witches'-broom (PNWB) were cloned in plasmid pGEM-3Zf(+). Cloned inserts from seven PNWB-phytoplasma-specific recombinant plasmids and two subcloned plasmids were excised with restriction enzymes, labeled with digoxigenin, and used as probes. Probe PNWB281 and its derivative subclones PNWB281-4 and PNWB281-5 hybridized with DNA from PNWB-phytoplasma infected peanut and periwinkle specifically but not with DNA from healthy plants or plants infected with phytoplasmas associated with sweetpotato witches'-broom (SPWB), loofah, Ipomoea obscura, and paulownia witches'-broom, elm and aster yellows, rice yellow dwarf, and bamboo little leaf disease. Six other probes hybridized with DNA derived from PNWB and SPWB-phytoplasma-affected periwinkle but not with DNA from healthy plants or plants infected with other phytoplasmas mentioned. In Southern hybridizations, four of the nine cloned and subcloned probes could differentiate the PNWB-phytoplasma from SPWB-phytoplasma. Three primer pairs for PCR were synthesized according to the partial sequences at both ends of the cloned inserts and were able to distinguish PNWB-phytoplasma from SPWB-phytoplasma by using PCR for the first time. A minimum of 1 pg and 10 pg of total DNA from diseased periwinkle and peanut, respectively, was sufficient to amplify the specific PNWB-phytoplasma PCR fragments, allowing the detection of PNWB-phytoplasma DNA from healthy-looking periwinkle plants two weeks after graft inoculation. 相似文献
15.
PSR(Puccinia Striiformis Repeat)序列的基因组特异性和指纹遗传稳定性研究 总被引：6，自引：0，他引：6
以PSR331S3(Puccinia striiformis repeat)为探针,对感染小麦条锈菌P. striiformis f. sp. tritici模式菌系的叶片和常用繁殖寄主健康叶片总DNA进行Southern分析,结果显示PSR331S3具有良好的指纹分辨力和基因组特异性。对系列单孢系的指纹分析表明,PSR位点在有丝分裂中是稳定遗传的,可以用于条锈菌的遗传分析。 相似文献
16.
A sensitive method for detecting bamboo mosaic virus (BaMV) and establishment of BaMV-free meristem-tip cultures 总被引：7，自引：0，他引：7
A sensitive method was used to detect bamboo mosaic virus (BaMV) and its associated satellite RNA (satBaMV) by 32 P- and digoxigenin (Dig)-labelled probes synthesized from cDNA clones of BaMV genomic (L probe) and satBaMV (S probe) RNA. Both the 32 P- and Dig-labelled L and S probes could detect as little as 490 pg of BaMV viral RNA by slot- and dot-blot hybridization. In infected leaf extracts, 32 P-labelled L and S probes detected virus at 25-fold higher dilutions than Dig-labelled probes, which were also successfully used to detect BaMV infection in plants derived from meristem-tip culture. However, immunoassays failed to detect BaMV in meristem culture. By dot-blot hybridization assays, 25% of the seedlings were shown to be virus-free. These results suggest that a highly sensitive method for the detection of BaMV infection is required for the establishment of BaMV-free cultures. Meristem-tip culture also provides an efficient method for obtaining virus-free bamboo plants. 相似文献
17.
地高辛标记的cDNA探针检测烟草花叶病毒、黄瓜花叶病毒及马铃薯Y病毒 总被引：8，自引：0，他引：8
根据已发表的烟草花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和马铃薯Y病毒(Potato virus Y,PVY)的外壳蛋白基因序列,设计特异引物,分别以提取的TMV、CMV和PVY侵染的病叶总RNA为模板,反转录PCR进行体外扩增,分别得到长度为0.44、0.77、0.80 kb的目的片段,并克隆到pGEM-T easy质粒载体上,以构建的重组质粒为模板,用PCR方法合成了相应的地高辛标记的双链DNA探针。以合成的探针通过斑点杂交技术检测烟草病叶总RNA和烟草病叶汁液。TMV、CMV和PVY的3种地高辛探针检测各自感染的烟草病叶总RNA的稀释低限分别为1:1000、1:10000、1:320,检测各自侵染烟草病汁液的最大稀释倍数分别为1:100、1:100、1:10,而每种探针与健康烟草和其它2种病毒的反应均为阴性。 相似文献
18.
Reinhard Zipper Timo R. Hammer Otmar Spring 《European journal of plant pathology / European Foundation for Plant Pathology》2009,123(3):367-375
Bioassays testing the fungicide sensitivity against metalaxyl of Peronospora tabacina isolates collected in German tobacco fields in 2005 revealed the presence of two phenotypes, resistant and sensitive. DNA
fingerprints using SSR and minisatellite primers allowed separation of the samples into two groups. The differences in amplification
patterns coincided with the sensitive and resistant reaction of the isolates in metalaxyl bioassays. New primers were developed
which allowed PCR-based detection of P. tabacina and differentiation of the metalaxyl-sensitive and the metalaxyl-resistant phenotype, respectively. Screening of recent blue
mold isolates from Germany and other European countries for metalaxyl sensitivity with leaf disk bioassays coincided completely
with the PCR-based identification of the two phenotypes. In Germany, exclusively resistant isolates were found between 2002
and 2004. These still dominate. Since 2005 the co-occurrence of sensitive isolates has been shown. No similar monitoring has
been done in any other European country. However, we found the resistant phenotype reaction in a French isolate of 2004 and
in two out of three Italian isolates in 2007. Two isolates from Poland and Bulgaria in 2007 were sensitive to metalaxyl. For
all 58 isolates tested since 2002 the metalaxyl bioassay-based resistance type and the PCR-based tests for sensitive and resistant
genotype coincided. Using genotype-specific primers for future population studies may help to trace the sources of the pathogen
from which yearly propagation starts. 相似文献
19.
ABSTRACT We have developed a piezoelectric DNA-sensor based on DNA-RNA hybridization for the detection of two orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Specific oligonucleotide probes modified with a mercaptohexyl group at the 5'-phosphate end were directly immobilized onto 10-MHz AT-cut quartz crystal microbalance (QCM). QCMs coated with such oligonucleotide probes were exposed to test solutions containing viral RNA for hybridization. Various experimental conditions evaluated were (i) DNA probe coating concentration, (ii) sensitivity and specificity of the probes at different hybridization temperatures, and (iii) effects of incubation temperature on the hybridization time. The specific nucleotide probe-coated QCM-based DNA sensors were able to detect both CymMV and ORSV in quantities as low as approximately 1 ng in purified RNA preparations and 10 ng in the crude sap of infected orchids. This is the first application of a DNA biosensor for the detection of plant viruses. 相似文献
20.
In recent studies, we found that Apll (a PthA homologue) bound to three citrus proteins. Amino acid sequence analysis revealed
that one of the target proteins was homologous to that of S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT), an enzyme which is specific for the substrate trans-caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA. From the consensus nucleotide sequences of CCoAMT genes, primers were chosen for PCR amplification of this
gene from citrus total DNA. Two selected DNA fragments of 1.0 kb and 2.0 kb were obtained. These fragments were used as the
probe to screen a citrus library. One clone, pCCl00, contained a 1.0-kb SalI fragment that hybridized to the probes. The nucleotide sequence of this fragment was determined in both directions. In this
fragment, there was an open reading frame of 232 amino acids interrupted by an intron of 106 nt, and the deduced amino acid
sequence had 95.9% homology to tobacco CCoAMT. Southern blot analysis of total citrus DNA showed that four EcoRI fragments hybridized to the probes, suggesting the presence of more than one copy of CCoAMT in the citrus DNA.
Received 4 November 1999/ Accepted in revised form 26 November 1999 相似文献