Development of a Polymerase Chain Reaction Protocol for Detection of Xylella fastidiosa in Plant Tissue.

IF 3.1 2区农林科学 Q2 PLANT SCIENCES

Phytopathology Pub Date : 2025-08-01 DOI:10.1094/Phyto-84-456

G V Minsavage, C M Thompson, D L Hopkins, R M V B C Leite, R E Stall

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引用次数: 0

Abstract

A 7.4-kb EcoRI fragment of genomic DNA of Xylella fastidiosa strain PCE-RR (ATCC 35879) was used as a probe and was conserved in 18 strains of Xylella. The nucleotide sequence of a 1.0-kb internal EcoRV portion of the fragment was determined, and oligonucleotides were selected for primers that amplified genomic DNA specific to X. fastidiosa in 33 strains tested by the polymerase chain reaction (PCR). Plant extracts for PCR and enzyme-linked immunosorbent assay (ELISA) were obtained by maceration of grape petioles and by vacuum extraction of citrus stems. Known cell numbers of X. fastidiosa were added to the plant extracts contained in a succinate-citrate-phosphate buffer prior to assay. Amplification of DNA by PCR was inhibited in the presence of plant extracts unless sodium ascorbate and acid-washed polyvinylpyrrolidone were added to the extraction buffer. Detection of Xylella by PCR was 100-fold more sensitive than by ELISA; the limits of detection were 1 × 10² cfu/ml for PCR and 2 × 10⁴ cfu/ml for ELISA. Restriction endonuclease digestion of PCR amplification products with RsaI differentiated two pathotypes of X. fastidiosa.

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植物组织中苛养木藻聚合酶链反应检测方法的建立。

以苛养木杆菌PCE-RR （ATCC 35879）基因组DNA的7.4 kb EcoRI片段为探针，该片段保存在18株木杆菌中。测定了该片段1.0 kb EcoRV内部片段的核苷酸序列，并选择了引物的寡核苷酸，通过聚合酶链反应（PCR）扩增了33个菌株的苛养螺旋体特异性基因组DNA。采用葡萄叶柄浸渍法和柑桔茎真空提取法获得了用于PCR和酶联免疫吸附试验（ELISA）的植物提取物。已知的苛养X.的细胞数被添加到含有琥珀酸-柠檬酸盐-磷酸盐缓冲液的植物提取物中，在测定之前。除非在提取缓冲液中加入抗坏血酸钠和酸洗聚乙烯吡咯烷酮，否则存在植物提取物时，DNA的PCR扩增受到抑制。PCR检测木杆菌的灵敏度是ELISA的100倍；PCR检测限为1 × 102 cfu/ml， ELISA检测限为2 × 104 cfu/ml。用RsaI酶切PCR扩增产物可区分两种病型。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Phytopathology 生物-植物科学

CiteScore

5.90

自引率

9.40%

发文量

505

审稿时长

4-8 weeks

期刊介绍： Phytopathology publishes articles on fundamental research that advances understanding of the nature of plant diseases, the agents that cause them, their spread, the losses they cause, and measures that can be used to control them. Phytopathology considers manuscripts covering all aspects of plant diseases including bacteriology, host-parasite biochemistry and cell biology, biological control, disease control and pest management, description of new pathogen species description of new pathogen species, ecology and population biology, epidemiology, disease etiology, host genetics and resistance, mycology, nematology, plant stress and abiotic disorders, postharvest pathology and mycotoxins, and virology. Papers dealing mainly with taxonomy, such as descriptions of new plant pathogen taxa are acceptable if they include plant disease research results such as pathogenicity, host range, etc. Taxonomic papers that focus on classification, identification, and nomenclature below the subspecies level may also be submitted to Phytopathology.