Characterization of Ralstonia solanacearum strains infecting Bolivian potatoes Jose A. Castillo 1, 2 , Joanna Jelenska and Jean T. Greenberg1 1Department
of Molecular Genetics and Cell Biology, The University of Chicago, U.S. 2PROINPA Foundation, Bolivia.
Introduction The most important crop in the agrarian sector of Bolivia is the potato1. Potato is the basic food security crop for the Bolivian population, being the per capita consumption 140 Kg/year, one of the Bolivia highest in the world. Nearly 300,000 families of small farmers, which constitutes about 60% of the farm families in the country, depend on potato as their main daily source of food and income2. In Bolivia, the potato crop is severely affected by a variety of pathogens. However, bacterial wilt caused by Ralstonia solanacearum (R.s.) is one of the most aggressive diseases, causing up to 75% losses in potato production, as well as the production of other valuable Bolivian crops, such as tomato and peanut3. Currently the only approach to control R. s. in Bolivia is to promote agricultural practices that minimize the dispersal of bacteria from infected plants. To develop robust disease control measures requires a multifaceted approach. Recently, it has become clear that R.s. can secrete, via the type III secretion pathway, many virulence proteins called effectors; these proteins can alternatively act as avirulence (Avr) factors to trigger defense responses in plants. The goal of this work is to characterize Bolivian isolates of R.s. and compare their effector repertoires with that of type strains GMI1000 (race2 and UW551 (race 3). Potato plant wilted by Ralstonia solanacearum
Results • Sampling
Sampling and Rs.s. identification. In October 2004, wilt symptoms were observed on potato plants cultivated in main potato areas in central region of Bolivia. Bacteria were isolated from infected stems and tubers of the host plants, and were verified as R.s. through PCR using Rsol_fliC 4 primers and 759-760 primers5. Cultural and physiological tests. Bacteria isolates were grown on Kellman medium with triphenyltetrazolium chloride. To determine biovar status, test for oxidation of sugars and sugar alcohols were carried out in Hayward’s medium supplemented with lactose, maltose, sorbitol or mannitol. Characterization of strains. Strains that tested positive for being R.s. were further analyzed to determine their phylogenetic relationship with other R.s. strains. Nucleotide sequence of egl gene (endoglucanase) was obtained to construct trees that help to establish the affiliation in phylotype scheme of Bolivian strains. Results obtained from egl sequence were confirmed using Multiplex PCR6 that allows quickly phylotype determination of R.s. strains.
• Efficient, specific and easy PCR detection of R.s. in potato plants (stems) and tubers that uses primers targeting flagellum subunit gene fliC 1
2
345
Detection of R.s. using primers Rsol_fliC-F and Rsol_fliC-R to amplify by PCR a subunit from flagellum. Lanes: 1. M.W. ladder, 2. Various samples come from Chuquisaca area, 3. Positive control (R. s. GMI1000 genomic DNA), 4. Negative control (Pseudomonas syringae B728 genomic DNA), 5. Negative control (water).
• Multiplex PCR applied to determine phylotype association. 1
Agarose gel of PCR products amplified from genomic DNA of six representative strains from Bolivia and respective controls. Lanes: 1 negative control (water), 2 to 7 Bolivian strains (BS024, BS025, BS048, BS075, BS094, BS095), 8 to 11 Phylotype controls [UW386 (Phylotype III), UW363 (Phylotype I), UW443 (Phylotype IV), K60 (Phylotype II)], 12 M.W. Ladder
2
3
4
5
6
7
8
9 10 11 12 bp 506 396 344 298
• Table summarizing Bolivian isolates characteristics Representative sample
Potato variety
Race
Biovar
Phylotype
Genomic DNA pattern
Tapial
BS024
Clon C5
3
2
II
1
Lampasillos
BS075
Clon C3
3
2
II
1
Astillero
BS094
Desireé
1
1
II
2
Astillero
BS095
Desireé
1
1
II
2
Chiriwañanay
BS01
Desireé
3
2
II
1
Estancia Waykho
BS025
Desireé
3
2
II
1
San José del Barrial
BS046
Desireé
3
2
II
1
San José del Barrial
BS048
Desireé
3
2
II
1 Chuquisaca (Highlands 2500-3500 m) 2 Santa Cruz (Inter-Andean valleys 15002500 m)
Village
• PCR of repetitive bacterial sequences (rep-PCR) analysis to assess the relatedness between Bolivian strains
1
1
2
3
4
5
6
Representative DNA patterns of repetitive sequences (BoxA) carried out in genomic DNA of different Bolivian strains of R.s. Lanes: 1. M.W. ladder, 2. BS075 (race 3 biovar 2), 3. BS085 (race 3 biovar 2), 4. BS094 (race 1 biovar 1), 5. BS095 (race 1 biovar 1), 6. GMI1000 (control strain, race 1 biovar 3).
• Tracing clonality
U W 4 4 3 U W 4 4 5 U W 4 4 6 W P2 0 U W 5 1 9 G M I1 0 0 0 U W 5 0 5 U W 2 9 8 U W 3 6 3
To study the global relatedness of Bolivian strains, PCR of repetitive bacterial sequences (repPCR) was applied analyzing the similarity in the banding patterns using BoxA primers7.
C C K 2 7 B C K U W 1 5 2 U W 2 9 6 U W 5 0 3 U W 5 1
We used sequence typing to compare 6 Bolivian and 6 additional strains from other parts of the world. Specifically, we compared the sequence of core fragments of three housekeeping loci (gyrB, gdhA and adkA) and two genes (egl and fliC) involved in virulence processes to determine the clonal descent amongst the isolates.
Neighbor-joining tree of gdhA gene sequence to determine clonality of Bolivian strains during their genome evolution. Sequences of representative strains of the four major phylotype groups were used to compare with Bolivian strains (arrows).
M A FF3 0 1 5 5 6 6 2 9 U W 3 8 6 R 2 9 2 PD 1 6 1 0 8 5 5 TK 1 3 1
• Effector repertoires determination.
U W 2 2 0 U W 3 4 4 PD 4 4 1 U W 1 3 4 U W 4 2 0 U W 2 2 4
Effector repertoire comparison. To assess R.s. isolates from Bolivia for their overlap in the effector repertoire with GMI1000, dot blot experiments were carried out. Briefly, PCR products of each effector gene from GMI1000 were attach to nylon membranes in an arranged order. Then P32-labeled fragments of genomic DNA of Bolivian strains were hybridized with the membrane.
U W 7 2 U W 3 6 5 B S 0 4 8 U W 4 4 8 U W 4 7 7 B S 0 2 5 PD 1 9 3 9 B S 0 2 4 B S 0 7 5 U W 5 5 1 1 1 3 U W 5 0 4 U W 2 7 6 PD 1 1 0 0 B S 0 9 4 B S 0 9 5 5 7 4
.
1
Ralstonia solanacearum distribution: 1. Chuquisaca 2. Santa Cruz
Bolivian main region
Materials and methods
2
• About 80 samples of R.s were obtained from potato lands with farmer participation. These samples and a collection of 100 isolates already existing in our lab were analyzed to confirm R.s. occurrence.
U W 4 6 9
Filter (macroarrays) result showing the degree of overlap in the effector repertoires between the BS048 (Bolivian) strain and GMI1000 strain. Each black spot means different effector gene homolog present in BS048
K 6 0
Literature cited
0 .0 0 5
1. Bojanic, A., 1995 Sondeo sobre la demanda nacional de semilla de papa para el sector formal y su pertinencia para la UPS/SEPA, Informe Cooperación Técnica Suiza., La Paz, Bolivia. 2. Horton, D., 1992 La papa: producción, comercialización y programas, CIP Lima y hemisferio sur, Montevideo, Uruguay. 3. Fernandez-Northcote, E. N., and V. Alvarez, 1993 Situación actual de la marchites bacteriana causada por P. solanacearum en Bolivia., Brasilia, Brasil. 4. Schönfeld J, Heder H, van Elsas J.D., and Smalla K. 2003 Specific and Sensitive Detection of Ralstonia solanacearum the Basis of PCR Amplification of fliC Fragments. Applied Environ. Microbiol. 69:7248–56 5. Opina, N., F. Tavner, G. Holloway, J.-F. Wang, T.-H. Li, R. Maghirang, M. Fegan, A.C. Hayward, V. Krishnapillai, W. F. Hong, B.W. Holloway, and J.N. Timmis. 1997. A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum). As. Pac. J. Mol. Biol. Biotechnol. 5: 19-33. 6. Prior, P. and M. Fegan. 2005. Recent developments in the phylogeny and classification of Ralstonia solanacearum. Acta Hort., in press. 7. Versalovic, J., Schneider, M., de Bruijn, F.J., and Lupski, J.R. 1994). Genomic fingerprinting of bacteria using repetitive sequence based PCR (rep-PCR). Meth.Cell. Mol. Biol. 5: 25-40
Acknowledgments
We thank the NSF (DCC-PGR) for supporting this project. We thank Dr. Caitlyn Allen for assistance with phylotyping.
For more information Please contact Jose A Castillo,
[email protected]. Further information on this and related projects can be obtained at Department of Molecular Genetics and Cell Biology, The University of Chicago, 1103 East 57th Street EBC410, Chicago, IL 60637, USA Phone 1 773 834 1906
He lives his life joyfully since Ralstonia go away !!
Conclusions • R.s. race 3/biovar 2/phylotype II is extensively spread in Bolivian potato crops, enabling its identification in all sampled areas. On the other hand, the distribution of Race 1/biovar 1/phylotype II strain is confined to a small area of the Chuquisaca mountains at the center of country (Astillero village, see map). • Two main patterns of rep-PCR analysis were found in genomic DNA isolated from Bolivian strains of R.s. These patterns match with race/biovar status, respectively. This result suggest that the epidemics occurring in Bolivia are likely due to spread of one or only a few clones of R.s. • The analysis of the core DNA sequences of three housekeeping loci (gyrB, gdhA and adkA) and two ‘pathogenicity’ gene (egl and fliC) indicates a narrow diversity amongst the Bolivian strains compared with other R.s. strains. This finding indicates that at the core genome level, Bolivian strains are highly clonal and gives clues about the low importance of recombination in driving the evolution of these strains. • Effector repertoire comparisons show that there is low overlap in the pathogenicity gene repertoires of Bolivian strains with GMI1000 (about 20%). However, there may be sequence divergence in some of the pathogenicity genes.
