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1.
A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils. 相似文献
2.
Leila Eshraghi Nader Aryamanesh Jonathan P. Anderson Bryan Shearer Jen A. McComb Giles E. St. J. Hardy Philip A. O’Brien 《European journal of plant pathology / European Foundation for Plant Pathology》2011,131(3):419-430
A reliable method for measuring disease progression is important when evaluating susceptibility in host—pathogen interactions.
We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis.
Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation
of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen
biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves
the accuracy of pathogen measurement in both resistant (non-host A. thaliana–P. cinnamomi) and susceptible (host Lupinus angustifolius–P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis. 相似文献
3.
Daniela Minerdi Marino Moretti Yuan Li Laura Gaggero Angelo Garibaldi Maria Lodovica Gullino 《European journal of plant pathology / European Foundation for Plant Pathology》2008,122(2):227-237
A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed
from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR.
The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected
gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection
limit was 5 × 103
P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the
possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution
of closed soilless cultivation systems. 相似文献
4.
Kai Chen Zhonghuan Tian Lan Wang Chao-an Long 《European journal of plant pathology / European Foundation for Plant Pathology》2017,149(1):201-209
Specific primers targeting Penicillium digitatum were developed based on fungal genes RPB1 and cmd, which are conserved among the genomes of Penicillium spp. The specific primers were designed based on the mutational sites in the homologous regions of the conserved genes. The results indicated that primer pairs RPB1–1 and cmd-3 were specific enough to distinguish Penicillium digitatum (N1) from Penicillium chrysogenum (Q), Penicillium italicum (A10) and Penicillium expansum (L) when the DNA samples were diluted 100-fold. To further verify the effectiveness and specificity of the two primer pairs RPB1–1 and cmd-3, 38 strains of fungal isolates from sources related to citrus were detected using both primer pairs, and 14 candidate P. digitatum strains were identified. Then, the fourteen candidate P. digitatum strains were further identified as P. digitatum by morphological and molecular methods, which confirmed the detection accuracy and reliability of the specific primer pairs RPB1–1 and cmd-3 as molecular markers of P. digitatum. This work may significantly facilitate the rapid identification of P. digitatum in the citrus industry. 相似文献
5.
Virgilio?Balmas Barbara?Scherm Stefano?Ghignone Ali?Ould?Mohamed?Salem Santa?Olga?Cacciola Quirico?Migheli
Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence. 相似文献
6.
Keisuke Tomioka Yuuri Hirooka Takayuki Aoki Toyozo Sato 《Journal of General Plant Pathology》2008,74(3):264-266
Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium
graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium
rot of hyacinth” as a new disease because only the anamorph, F.
graminearum, was identified on the diseased host plant.
The authors contributed equally to this work.
The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute
of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively. 相似文献
7.
Federica Bini Klaus Geider Carlo Bazzi 《European journal of plant pathology / European Foundation for Plant Pathology》2008,122(3):403-411
Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted
for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and
ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine
strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For
simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was
performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from
grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets
were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells
per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA
purification kit as a step for the isolation of nucleic acids. 相似文献
8.
Leptosphaeria maculans,a fungal pathogen of Brassica napus, secretes large amounts of a 28kDa protein (SP2) in liquid culture. This protein shows high sequence similarity to secreted serine proteases from other ascomycetes and is the major component of culture filtrate with protease activity, as analysed on casein zymogels. The sp2 gene is expressed during infection of B.napuscotyledons when L. maculans hyphae are growing between mesophyll cells, as well as at later stages when the fungus invades the vascular tissue. 相似文献
9.
Mathias De Backer Hossein Alaei Erik Van Bockstaele Isabel Roldan-Ruiz Theo van der Lee Martine Maes Kurt Heungens 《European journal of plant pathology / European Foundation for Plant Pathology》2011,130(3):325-338
Puccinia horiana is the causal agent of chrysanthemum white rust or Japanese rust. This microcyclic autoecious rust has a quarantine status
and can cause major damage in the commercial production of Chrysanthemum x morifolium. Given the international and often trans-continental production of planting material and cut flowers of chrysanthemum and
the decreasing availability of registered fungicides in specific regions, breeding for resistance against P. horiana will gain importance and will need to involve the appropriate resistance genes for the pathotypes that may be present. As
pathotypes have not been well characterized in this system, the main objective was to build an international collection of
isolates and screen these on a large collection of cultivars to identify different pathotypes. Using a robust and high throughput
bioassay, we tested 36 selected cultivars with 22 individual single-pustule isolates of P. horiana. The isolates originated from three different continents over 4 different collection years and included some isolates from
cultivars previously reported as resistant. In most cases the bioassays resulted in a clear scoring of interaction phenotypes
as susceptible or resistant, while in several cases consistent intermediate phenotypes were found, often on specific cultivars.
Twenty-four of the cultivars gave a differential interaction phenotype profile. All isolates produced a unique profile, infecting
a minimum of 4 and a maximum of 19 differential cultivars. Based on the Person analysis of these profiles, this pathosystem
contains at least seven resistance genes (and seven avirulence genes), demonstrating the highly complex race structure in
this pathosystem. 相似文献
10.
David J. Luckett Raymond B. Cowley Mark F. Richards David M. Roberts 《European journal of plant pathology / European Foundation for Plant Pathology》2009,125(1):131-141
Pleiochaeta root rot (PRR) caused by Pleiochaeta setosa is a serious, widespread fungal disease in lupin crops, especially in Lupinus albus (broad-leaf lupin, or white lupin). PRR resistance is common in the gene pool of L. albus with various landraces from the Mediterranean region being the most resistant, and suitable for use in breeding new cultivars.
Heritability of resistance is sufficient to make good gains from selection but only when controlled-environment (CE) screening
is used. Field disease nurseries on loamy soil gave much lower heritability of resistance. Field disease nurseries had spatially
variable spore counts despite continuous lupin cropping, and this was partly responsible (along with climatic conditions)
for their reduced precision compared to tests conducted in a CE. Giving infected L. albus roots a single, most-severe-lesion score on a 0–9 scale was adequate for CE screening but not as precise or discriminating
as the more time-consuming method of six scores per root. Replication in CE experiments was reduced to two pots of 16 seedlings
each without sacrificing genotype discrimination. 相似文献
11.
Insect-borne viruses promote several changes in plant phenotype, which can modify plant-vector interactions in favor of virus survival and dissemination. Although co-infections commonly occur in the field, little is known about their effects on interactions with the vector. The ecological interactions between Barley Yellow Dwarf Virus (BYDV) and its aphid vector, Rhopalosiphum padi, have been investigated extensively, but the vector’s behavior in more complex scenarios has yet to be examined. We assessed olfactory response and performance of R. padi to wheat singly and doubly infected by the pathogenic fungus Giberella zeae and BYDV. Non-viruliferous aphids preferred odors of BYDV-infected wheat over healthy wheat, as previously reported in the literature, and they were still preferentially attracted to BYDV-infected plant during co-infection. However, around 35% more non-viruliferous aphids chose healthy wheat over G. zeae-infected wheat. Viruliferous aphids did not show any preference to the treatments. BYDV-infected wheat was a superior host than healthy wheat for the aphids whose population increased in 25%. We observed a synergistic effect of the co-infected wheat, which was the best host for aphids, and promoted an elevation of 42% on population growth. Our results indicate that co-infection might be beneficial for virus spread as does not interfere with aphid olfactory preference and provides greater colony growth than in singly infected plants. 相似文献
12.
Masamichi Isogai Koji Ishii Seisaku Umemoto Manabu Watanabe Nobuyuki Yoshikawa 《Journal of General Plant Pathology》2009,75(2):140-143
Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush
blueberry (Vaccinium
corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic
acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome.
This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan.
The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884
to AB469893 for BRRV isolates from Japan. 相似文献
13.
Khalid A. Hussein Mohamed A. A. Abdel-Rahman Ahmed Y. Abdel-Mallek Saad S. El-Maraghy Jin Ho Joo 《Phytoparasitica》2012,40(2):117-126
The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have
recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between
the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 106 conidia ml−1, 5.86 × 105 conidia ml−1, and 4.8 × 106 conidia ml−1, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark
and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC50 values were 1.43 × 103, 1.04 × 105 and 5.06 × 104 for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis
program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively. 相似文献
14.
From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia
of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of
Inago elements is relatively stable. 相似文献
15.
Ahmed A. Kheder Yasunori Akagi Hajime Akamatsu Konomi Yanaga Nitaro Maekawa Hiroshi Otani Takashi Tsuge Motoichiro Kodama 《Journal of General Plant Pathology》2012,78(1):30-38
The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has
been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin
biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and
disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves
after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the
tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the
wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin
confers UV tolerance. 相似文献
16.
Ying-Hong Lin Jing-Yi Chang En-Tzu Liu Chih-Ping Chao Jenn-Wen Huang Pi-Fang Linda Chang 《European journal of plant pathology / European Foundation for Plant Pathology》2009,123(3):353-365
Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management
practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race
4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02
amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters,
the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic
DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be
easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
17.
Lani E. Yakabe M. Malendia Maccree Padma Sudarshana Ali E. McClean Shane R. Parker William P. Wechter Gernot Presting Mizuri Marutani-Hert Daniel A. Kluepfel 《Journal of General Plant Pathology》2012,78(2):121-126
Novel PCR primers were developed to amplify a 243-bp fragment of an intergenic region between gene 5 and tms2 on the T-DNA of Agrobacterium tumefaciens biovar 1. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence, and did
not generate extraneous bands, thus facilitating robust real-time PCR detection. 相似文献
18.
The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His6− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected. 相似文献
19.
Shigemitsu Kimura Susumu Tokumaru Kazuhiko Kuge 《Journal of General Plant Pathology》2009,75(4):322-324
Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs
in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal
transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence
of yeast spot in azuki bean in Japan.
The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for
E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8. 相似文献
20.
Protocols for producing virus-free Allium plants require an indexing system that is more sensitive than DAS-ELISA and can detect low virus concentrations in infected plants. In the present work, degenerate primers were designed and a one-step IC-RT-PCR protocol was developed to differentiate between Leek yellow stripe virus (LYSV) and Onion yellow dwarf virus (OYDV) in single and mixed infections in several Allium spp. A 566-bp band was observed for LYSV, a 489-bp band for OYDV in single infections, and two bands of the same sizes in mixed infections in different species of Alliaceae. A 508-bp band of Shallot yellow stripe virus and a 594-bp band of Turnip mosaic virus were also amplified with the same primers. RT-nested-PCR was also conducted directly in microtitre plate wells after negative or questionable reactions were produced in an ELISA experiment. The detection limit of the DAS-ELISA for LYSV was 16.5–27.3 ng ml−1. The RT-nested-PCR done after DAS-ELISA was 102 times more sensitive than the DAS-ELISA alone. In parallel, an IC-RT-nested-PCR in microcentrifuge tubes was 104 times more sensitive than the DAS-ELISA. The DAS-ELISA-RT-nested-PCR enables the initial screening of samples by DAS-ELISA to eliminate a high percentage of virus-positive plants, considerably reducing the number of plants to analyze further by RT-PCR. 相似文献