Aims Phase I

Interim Report Phase I
  1. Characterization of Ralstonia
       solanacearum(Rs) strains
  2. Resistance of tomato genotypes
  3. Cultural control of bacterial
       wilt: effect of silicon on
       bacterial wilt development
  4. Biological control
  5.Cooperative studies

SUMMARY PHASE 1
ZUSAMMENFASSUNG

Aims and planned studies Phase II

Publications

Scientists:

Dr. K. Wydra
N. Thaveechai
H. Wadhawan (Beri)

last update:
July 2007

An integrated approach to control bacterial wilt, focussing on host plant resistance - with major emphasis on screening under protected cultivation and investigations on mechanisms of resistance and host-pathogen interactions - supported by characterisation of strains of Ralstonia solanacearum, collected from different origins in Thailand, is proposed for the first phase of the present project. The aspects of interaction with nematodes - tritrophic interactions of plant pathogenic bacteria/nematodes/host plant - and investigations on agronomic and cultural control measures (soil amendments and treatments) and biological control will be covered in preliminary studies in close collaboration with Thai scientists (from Kasetsart University) and subprojects 3 and 6 in year 3. These studies shall be extended in the second phase of the project.







Back to main page

Overview

In project phase I, a focus of research was laid on host plant resistance including (i) the analysis of possible virulence factors of the pathogen and selection of strains suitable for screening for resistance, (ii) monitoring of disease development of various genotypes simulating Thai conditions in controlled environments and under protected cultivation in Thailand, (iii) determination of the level of latent infections of resistant genotypes and the state of the pathogen in planta, and (iv) investigations on resistance mechanisms and the interaction between host and pathogen on molecular level in planta. Additionally, two more practically oriented subjects on control of R. solanacearum were studied, the effect of (v) silicon amendment on symptom and bacterial population development as well as on the state of the pathogen and of (vi) antagonists on bacterial wilt development.

1. Characterization of Ralstonia solanacearum (Rs) strains

1.1. Virulence and virulence factors
Thirty-one strains from Thailand and 3 reference strains from Taiwan revealed high variability in virulence (highly to non virulent) on tomato genotype L390. Among ten additionally tested Rs strains under conditions of protected cultivation in Thailand six were highly virulent, two moderately virulent, one lowly virulent and one avirulent on cultivar Seeda.

1.1.2. Lipopolysaccharides (LPS)
Strains Pss190 (Taiwan) (highly virulent), Pss 216 (Taiwan) (medium virulent) and Pe104 (Thailand) (lowly virulent) were characterized for sugar, amino acid and fatty acid composition. LPS were typical for Rs. Structural analysis of O-chain, Lipid A and core region are ongoing. Rare sugars were detected in some strains.

1.1.3. Extracellular polysaccharides (EPS)
Positive correlation between EPS production (colony morphology) and virulence was established. Some exceptions were observed indicating phenotypic conversion of the pathogen.

1.2. Genetic characterization
RFLP patterns of thirty-nine Rs strains worldwide origin including 14 Rs strains from tomato from Thailand and Taiwan showed high diversity. Phylogenetic distances of 22 Rs-strains from ginger from worldwide origin (including 5 strains from Thailand) were established by sequencing of the endoglucanase-gene. Ginger strains seemed distinct from strains from other hosts. Virulence tests on tomato and ginger are ongoing.

Conclusions:
Strains from Thailand were highly diverse in virulence, EPS production and genetic characteristics.

2. Resistance of tomato genotypes

2.1. Wilt incidence under protected cultivation
Thirteen tomato genotypes, among them commercial Tahi cultivars and resistant genotypes from AVRDC, were tested against 5 newly isolated strains from Thailand under the conditions of protected cultivation. Eight genotypes showed no symptoms, while genotype King Kong2 showed 10-30% plant death.

2.2. Wilt incidence of ‘resistant‘ genotypes under controlled conditions
Fifteen genotypes selected as resistant by AVRDC showed various degrees of wilt incidence. Only two genotypes differed significantly in the percentage of wilted plants. Genotype King Kong2 revealed moderately resistant to highly susceptible (to a strain from Thailand) depending on the strain inoculated (3 strains from Thailand and 2 from Taiwan inoculated).

2.3. Determination of latent infection

2.3.1. Optimization of method for detection of latent infection: PCR, NCM ELISA, culturing of bacteria
Culturing of bacteria revealed as most exact and reliable detection method. In spiked stem tissue material, the detection limit was 100 CFU/ml.

2.3.2. Bacterial numbers in upper stem part, midstem and collar of ‘resistant‘ genotypes
Bacterial numbers were most divergent between the resistant genotypes in the midstem and allowed the formation of 3 significantly different groups of genotypes.

2.4. Identification of resistance mechanisms: analysis of cell wall polysaccharides of genotypes with different resistance
Changes on cell wall level were observed after infection in susceptible genotypes and differences between genotypes were identified.

2.5. Rheological interaction between LPS from Rs and pectin
No synergistic interaction (gel formation) between LPS and pectin from the susceptible genotype, and not interaction with pectin from the resistant genotype were observed.

Conclusions:
Most ‘resistant’ genotypes showed wilt incidence. Only the quantification of latent infection allowed differentiation of genotypes in three, significantly different groups. Highest differences in bacterial numbers were observed in midstems where resistance mechanisms are located. Genotype Hawaii 7996 showed no wilt and low colonization. Therefore, latent infection should be considered in screening for resistance. Genotype King Kong2 revealed susceptible to a strain from Thailand.
Cell wall characteristics of genotypes may be useful for screening and breeding for resistance.

3. Cultural control of bacterial wilt: effect of silicon on bacterial wilt development

The effect of silicon on symptom development was tested in hydroponic culture. Symptom development was retarded in the susceptible genotype, and retarded and reduced in the moderately resistant genotype. Bacterial numbers were reduced in the moderately resistant and resistant genotype, while shoot weight in inoculated treatments increased in the resistant genotype after silicon amendment. Silicon content in roots was significantly increased, while leaves and stems increased generally not significantly in silicon content. A significant correlation between silicon content in roots and bacterial numbers in stems was observed, indicating the effects of induced resistance. In the susceptible genotype effects of induced tolerance were observed when bacterial numbers were not reduced, but symptom development was retarded.

Conclusions:
Silicon amendment reduced symptom development and bacterial populations and increased the host plant tolerance.

4. Biological control

Antagonistic bacteria were isolated from Thai soils or tomato rhizosphere and showed effective in vitro and ad planta under conditions of protected cultivation. In a susceptible genotype survival increased by 30-60% four weeks after inoculation, but plant death occurred 8 weeks after inoculation, while in a moderately resistant plant survival increased by 0-50% 8 weeks after inoculation, depending on antagonistic strain inoculated.

Conclusions:
Promising antagonists were identified. Application has to be optimised and combined with other control measures.

5. Cooperative studies

Genotype screening and biological control experiments under conditions of protected cultivation were conducted by and in collaboration with the Thai partner, Dr. N. Thaveechai, Kasetsart University, Bangkok.


SUMMARY PHASE 1
Based on the results of project phase I, control measures of bacterial wilt will be optimized and combined in phase II. Furthermore, mechanisms which lead to increased host plant resistance will be studied in detail.

Silicon amendment to hydroponic cultures of tomato increased resistance of tomato genotypes against Ralstonia solanacearum. Tomato genotypes with desired traits and an optimal resistance induction towards R. solanacearum after silicon supplementation will be selected and tested in a suitable substrate (collaboration with Thai partner). The resistance induction and differences in the reaction of the genotypes will be studied by biochemical, immunohistochemical and partly molecular genetic methods (collaboration with P3 and P6). Immuno-histological methods which were elaborated in the first project phase will be used for structural analysis of cell wall components.

Microorganisms with already identified effect against nematodes and soilborne pathogens will be tested for activity against R. solanacearum, and rhizosphere-bacteria with activity against R. solanacearum will be tested for effect against nematodes and soilborne pathogens (collaboration with P6, Thai partner and P4). Successful antagonists will be studied for their mode of action and involved mechanisms, with special emphasis on induced resistance (collaboration with P6). The Thai partner will complement the studies with trials on the effect of plant extracts for suppression of bacterial wilt. Finally, a novel combination of an optimal genotype, soil amendment and biological control will be developed.

ZUSAMMENFASSUNG PHASE 1
Aufbauend auf den Ergebnissen der Projektphase I werden in Phase II einerseits Bekämpfungsmaßnahmen der bakteriellen Welke optimiert und kombiniert, andererseits sollen zugrundeliegende Mechanismen, die zur Erhöhung der Wirtspflanzenresistenz führen, tiefergehend untersucht werden. Eine Siliziumgabe erhöhte die Resistenz von Tomatensorten gegen Ralstonia solanacearum in hydroponischer Kultur. Tomatensorten mit gewünschten Eigenschaften und einer optimalen Resistenzinduktion gegen R. solanacearum nach Siliziumgabe sollen selektiert und im geeigneten Substrat getestet werden (Zusammenarbeit mit KU). Eine Resistenzinduktion und Unterschiede in der Reaktion der Sorten sollen mit biochemischen, immun-histologischen und teilweise molekulargenetischen Methoden untersucht werden (Zusammenarbeit mit P3 und P6), wobei u.a. in der ersten Projektphase erarbeitete immunhistologische Methoden der Zellwandstrukturanalyse angewandt werden.

Mikroorganismen mit nachgewiesener Wirkung gegen Nematoden und/oder bodenbürtige Pilze sollen gegen R. solanacearum, und Rhizosphärenbakterien mit in Vorversuchen nachgewiesener antagonistischer Wirkung gegen R. solanacearum gegen Nematoden und bodenbürtige Pilze getestet (Zusammenarbeit mit P6, P4, KU,) und auf ihren Wirkungsmechanismus hin, insbesondere hinsichtlich induzierter Resistenz, untersucht werden (Zusammenarbeit mit P6). Der Thailändische Partner wird die Untersuchungen mit Studien zum unterdrückenden Effekt von Pflanzenextrakten ergänzen. Letztendlich ist eine bisher nicht beschriebene Kombination von optimaler Sorte, Bodenbeimengung und biologischer Bekämpfung angestrebt.


Back to content

In phase II, promising results of phase I will be further followed up, with a focus on the practical aspects of control of R. solanacearum investigating the effects of silicon and of antagonists as well as of an optimal genotype, single and in combination, with the aim to develop an integrated control system. These studies will be accompanied by basic research on the mode of action of the cultural and biological measures, based on methods developed and insights achieved on resistance mechanisms of the host plant and the variability of the pathogen in phase I. This combination of applied and basic research should result in recommendations for an applicable integrated control of R. solanacearum and also elucidate to date unknown resistance mechanisms, possibly including induced resistance through a chemical element and through rhizobacteria, and, thus, reveal novel insights into the highly variable and complicated network of interaction of R. solanacearum with its host on molecular level. This can be achieved by close collaboration of project P7 with the Thai partner (2 MSc students) and P6/Bonn (methodological exchange, laboratory stays, common supervision of diploma student) as well as P3/Pant Nutrition and P4/Epidemiology.


Back to content