94 The Philippine Agricultural Scientist Vol. 91 No. 1 (March 2008) Hypervirulent Isolate from Race 1 Ralstonia solanacearum M.E. Orlina-Villareal et al.
THE PHILIPPINE AGRICULTURAL SCIENTIST ISSN 0031-7454
Vol. 91 No. 1, 94-98 March 2008
Research Note
A Hypervirulent Isolate Identified from a Race 1 Ralstonia solanacearum Strain from Tomato (Lycopersicon esculentum Mill. cv. L-180)
M. E. Orlina-Villareal
1,*, N. L. Opina
2and A. K. Raymundo
1,**1Institute of Biological Sciences, University of the Philippines Los Baños, College, Laguna 4031, Philippines
2Institute of Plant Breeding, University of the Philippines Los Baños, College, Laguna 4031, Philippines
*Current Address: Graduate School of Life and Environmental Sciences, University of Tsukuba,Tsukuba City, Japan
**Author for correspondence; e-mail: akraymundo@uplb.edu.ph; asuncionraymundo@yahoo.com
Results of inoculation of 10 Ralstonia solanacearum strains to resistant (Hawaii 7996, C108, 508), moderately resistant (Improved Pope, L-180-1) and susceptible (Yellow Plum, L390) tomato cultivars revealed four of the strains to be the most virulent based on their wilting percentages. The four strains were then compared by reisolation from the inoculated plants, DNA extraction and repetitive extragenic palindromic - polymerase chain reaction (REP-PCR) fingerprinting. Out of the 542 fingerprinted DNA samples from reisolations, two of the strains, one of which was T523, had isolates which manifested variable REP fingerprints. An isolate, T523-731, had a missing 0.4 kb band and additional bands of about 0.7 kb and 0.45 kb. Reinoculation of T523-731 on the same set of tomato cultivars resulted in an average wilting of 96% on two resistant cultivars (C108 and 508) and 17% on the highly resistant cultivar Hawaii 7996. In contrast, reinoculation of an isolate (T523-726) that exhibited the fingerprint of the parent strain, resulted in 16.7% wilting in cv. 508 but did not cause wilting in cvs. C-108 and Hawaii 7996. The parent strain T523 caused 3% wilting in cv. 508, and similarly did not cause wilting in cvs. C- 108 and Hawaii 7996. Similar results were obtained with a subsequent virulence test. The isolate T523- 731 was considered a hypervirulent isolate as it was able to overcome the resistance of the tomato cultivars that were earlier found to be highly resistant to bacterial wilt.
Key Words: bacterial wilt, hypervirulence, fingerprint, Ralstonia solanacearum
INTRODUCTION
Bacterial wilt is the most widespread and destructive dis- ease of solanaceous crops in the warm tropical areas of the world (Kelman 1954; Sequeira 1998). Losses of about 15–
55% due to bacterial wilt have been reported in tomato (Hartman et al. 1991).
Planting resistant cultivars is still considered the most practical and effective way of dealing with bacterial wilt. However, race 1 strains of R. solanacearum E. F. Smith isolated from tomato were established to be of great diver- sity (Jaunet and Wang 1999; Raymundo et al. 2005). This diversity causes problems, especially in the development and consequent breakdown of resistant varieties. The fail- ure of many attempts to breed universally resistant tomato lines has been attributed to strain diversity. Most plant pathogens have been known to respond rapidly to the se- lection pressure imposed by new crop cultivars with novel resistance genes. Recently, pathogen pressure has been
viewed as a factor in selecting functional resistance speci- ficities that result in the maintenance of resistance gene diversity.
We report the finding of a hypervirulent isolate de- rived from a wild-type strain of R. solanacearum with a different fingerprint, and which is capable of inducing wilt- ing in highly resistant cultivars of tomato (Lycopersicon esculentum Mill.).
MATERIALS AND METHODS
Plant Material Inoculation and Isolation
Isolated colonies from pure cultures of 10 strains of R.
solanacearum obtained from the Institute of Plant Breed- ing, University of the Philippines Los Baños were streaked on tryptone glucose yeast agar (TGYA) and used for plant inoculation. For DNA fingerprinting, a single colony was obtained from a TGYA plate and inoculated to TGY broth.
