Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting

被引:112
作者
Atkins, SD [1 ]
Clark, IM
Sosnowska, D
Hirsch, PR
Kerry, BR
机构
[1] Rothamsted Res, NIU Div, Harpenden AL5 2JQ, Herts, England
[2] Inst Plant Protect, Dept Biocontrol & Quarantine, Poznan, Poland
基金
英国生物技术与生命科学研究理事会;
关键词
D O I
10.1128/AEM.69.8.4788-4793.2003
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 66% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.
引用
收藏
页码:4788 / 4793
页数:6
相关论文
共 27 条
[1]   PCR-based molecular discrimination of Verticillium chlamydosporium isolates [J].
Arora, DK ;
Hirsch, PR ;
Kerry, BR .
MYCOLOGICAL RESEARCH, 1996, 100 :801-809
[2]  
ATKINS SD, 2002, IN PRESS IOBC WPRS B
[3]  
ATKINS SD, 2002, INITIAL TESTING POTE, P79
[4]   Approaches to molecular characterization of fungal biocontrol agents:: some case studies [J].
Avis, TJ ;
Hamelin, RC ;
Bélanger, RR .
CANADIAN JOURNAL OF PLANT PATHOLOGY, 2001, 23 (01) :8-12
[5]   The application of real-time PCR to the identification, detection and quantification of Pyrenophora species in barley seed [J].
Bates, J. A. ;
Taylor, E. J. A. ;
Kenyon, D. M. ;
Thomas, J. E. .
MOLECULAR PLANT PATHOLOGY, 2001, 2 (01) :49-57
[6]   Real-time quantitative PCR:: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophora infestans and Phytophthora citricola in their respective host plants [J].
Böhm, J ;
Hahn, A ;
Schubert, R ;
Bahnweg, G ;
Adler, N ;
Nechwatal, J ;
Oehlmann, R ;
Osswald, W .
JOURNAL OF PHYTOPATHOLOGY-PHYTOPATHOLOGISCHE ZEITSCHRIFT, 1999, 147 (7-8) :409-416
[7]  
CRUMP DH, 1998, ASPECTS APPL BIOL, V52, P383
[8]   Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers [J].
Cullen, DW ;
Lees, AK ;
Toth, IK ;
Duncan, JM .
EUROPEAN JOURNAL OF PLANT PATHOLOGY, 2001, 107 (04) :387-398
[9]   ORGANIZATION OF RIBOSOMAL-RNA GENES IN THE FUNGUS COCHLIOBOLUS-HETEROSTROPHUS [J].
GARBER, RC ;
TURGEON, BG ;
SELKER, EU ;
YODER, OC .
CURRENT GENETICS, 1988, 14 (06) :573-582
[10]   IDENTIFICATION OF INDIGENOUS AND INTRODUCED SYMBIOTIC FUNGI IN ECTOMYCORRHIZAE BY AMPLIFICATION OF NUCLEAR AND MITOCHONDRIAL RIBOSOMAL DNA [J].
GARDES, M ;
WHITE, TJ ;
FORTIN, JA ;
BRUNS, TD ;
TAYLOR, JW .
CANADIAN JOURNAL OF BOTANY-REVUE CANADIENNE DE BOTANIQUE, 1991, 69 (01) :180-190