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Title: A PCR PROTOCOL FOR THE IDENTIFICATION OF PSEUDOMONAS SYRINGAE PV TAGETIS BASED ON GENES REQUIRED FOR TAGETITOXIN PRODUCTION

Author
item Kong, Hyesuk
item Patterson, Cheryl
item ZHANG, WENMING - ALBERTA RES, CANADA
item SUZUKI, A. - SHIZUOKA UNIV. JAPAN
item TAKIKAWA, Y. - SHIZUOKA UNIV. JAPAN
item Lydon, John

Submitted to: Biological Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2003
Publication Date: 5/1/2004
Citation: Kong, H.N., Patterson, C.D., Zhang, W., Suzuki, A., Takikawa, Y., Lydon, J. 2004. A pcr protocol for the identification of pseudomonas syringae pv tagetis based on genes required for tagetitoxin production. Biological Control. 30:83-89.

Interpretive Summary: An important aspect of Intergrated Pest Management is the use of biological organisms to control pest, commonly referred to as biological control. A bacterial organism, Pseudomonas syringae pv. tagetis, has been evaluated by several research groups for its ability to control Canada thistle (Cirsium arvense). An important feature of P. syringae pv. tagetis is its ability to produce tagetitoxin, a phytotoxin that blocks the development of chloroplasts. This study is the first to attempt to characterize the genes required for tagetitoxin production. In the course of the study, two genes were mutated that resulted in the loss of tagetitoxin production. Interpretations of the proteins produced by these genes indicated that one is involved in the transport of ions and small compounds across the bacterial membranes, and the other is involved in the transfer on nitrogen groups, both features that could be involved in the production and movement of tagetitoxin in P. syringae pv. tagetis. Using the sequence data from the mutated genes, a polymerase chain reaction (PCR) protocol was developed that could distinguish P. syringae pv. tagetis from other P. syringae plant diseases. We used the protocol to demonstrate that a P. syringae species that was isolated from Canada thistle with symptoms identical to those caused by P. syringae pv. tagetis species was in fact not P. syringae pv. tagetis. The protocol was useful in detecting the presence of P. syringae pv. tagetis in plant tissue. The results of this study will be useful in identifying the presence of P. syringae in plant tissue and could be helpful in monitoring insects to determine how the disease is vectored. That information could be useful to researchers who are studying how bacterially-based biological control agents spread in nature. The ultimate benefit is an improved knowledge base from which decisions can be made on the proper use of biological control agents, which benefits farmers and the general public alike.

Technical Abstract: A PCR protocol that can be used to distinguish Pseudomonas syringae pv. tagetis from other P. syringae pathovars and other P. syringae species that induce apical chlorosis was developed based on DNA sequences of genes characterized from non-toxigenic mutants of P. syringae pv. tagetis. Following Tn5 mutatagenesis and cloning of the effected DNA from non-toxigenic isolates of P. syringae pv. tagetis, DNA sequences of two affected genes were obtained. A BLAST search revealed that one of the genes had homology to exbB, which codes for an auxiliary protein in the TonB/ExbD/ExbB membrane transport system. The other affected gene had homology to asnO, which codes for an asparagine synthase. PCR primer sets designated TAGTOX-9 and TAGTOX-10 that were designed from the sequences of the mutated genes that, when in PCRs with DNA from most strains of P. syringae pv. tagetis, produced amplicons of 507 and 733 bp, respectively. The same size amplicons were produced in PCRs containing DNA from chlorotic leaf and stem tissue obtained from sunflower plants infected with P. syringae pv. tagetis. In PCRs with DNA from 16 other P. syringae pathotypes, only DNA from P. syringae pv. helianthi produced the same size amplicons with the respective primer sets. Of significance was the low level of the 507 bp amplicon produced in PCRs with the P. syringae pv. helianthi DNA and TAGTOX-9 primers; results similar to those obtained in PCRs with the same primer set and DNA from non-toxigenic strains of P. syringae pv. tagetis. Results from PCRs with the TAGTOX-9 and TAGTOX-10 primers provide strong evidence that the newly described Pseudomonas syringae species CT99B016C isolated from Cirsium arvense displaying apical chlorosis is not a P. syringae pv. tagetis.