Study on the Growing Competition Relationship Between Virulent and Avirulent Strains of Ralstonia solanacearum Isolated from Tomato

Study on the Growing Competition Relationship Between Virulent and Avirulent Strains of Ralstonia solanacearum Isolated from Tomato

ZHU Yu-jing, LIU Bo*, CHE Jian-mei, CAO Yi

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract：The differences between growth abilities of the virulent and avirulent strains of Ralstonia solanacearum isolated from tomato were studied. While being cultured solely, the avirulent strain grew faster than the virulent before 24 h, but slower than the virulent after 24 h. Especially at 12 h, the growth speed of avirulent strain was 5.6 times than the virulent. While being cultured mixed under room temperature for 24 h, the amounts of the virulent increased when RV:RA>1 and reduced when RV:RA=l or<1; the revised increased ration of the virulent in five mixture treatments were-119.57~-33.57. All the amounts of the avirulent strain in five mixture treatments increased, the revised increased ration were 32.03~346.09. It was concluded that the avirulent had higher growth ability than the virulent in short time under sole culture condition, and could inhibit the growth of the virulent under mixture culture condition, which could be used as an biocontrol agent for the short-term control efficiency of ABPS to bacterial wilt disease of tomato in the field.

Key words: Ralstonia solanacearum, Avirulent Strain, Growing Competition
Attenuation Characteristies of Bacterial-wilt-disease Biocontrol Strain Anti-8098A（Bacillus cereus）to Ralstonia solanaceamm

LIU Bo*, LIN Ying-Zhi, ZHU Yu- Jing, GE Ci-Bin, CAO Yi

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract：The attenuation characteristics of bacterial-wilt-disease biocontrol Bacillus cereus strain ANTI-8098A caused by culture, physical, chemical factors, and other bacteria were compared. The attenuation index to distinguish the pathogeniciyt for Ralstonia solanacearum（Rs）was based on the TTC cultural medium and the infection mortality to the host. The Rs virulent strain was recognized by the indexlesshtan 0.60 and the Rs avirulent strain by that higher than 0.80. The culture activity could not make the Rs attenuated in 120 h within 10 generations. The similar attenuation indexes and infection mortality were persisted in the Rs virulent strains after treating with ultrasonic in different times, indicating no attenuation activity happened by the physical factor. The streptomycin as a chemical factor could inhibit thte Rs strain without attenuation activity happened by the physical factors. Fitfeen bacterium strains were used in the test of attenuating the Rs virulent strain, only two of Bacillus cereus ANTI-8098A and Bacillus sphaercus showed the attenuation activity to change the Rs virulent straining to the Rs avirulent strain in 24 h. The other 13 strains had no the characteristic. It was concluded that the bioconrtol strain ANTI-8098A had the attenuation characteristics to make Rs avirulent in 24 h, of which the activity significantly differed from that caused by culture, physical and chemical factors.

Key words: Ralstonia solanaceamm, Bacillus cereus, Attenuation, Avirulent strain

Influences of the biocontrol strain ANTI-8098A, Bacillus cereus on the growth competition between the virulent and avirulent strains of Ralstonia solanacearum

GE Ci-bin, LIU Bo*, LIN Ying-zhi, ZHU Yu-jing, XIAO Rong-feng

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract：The present paper dealt with the growth competition between the virulent strain F-01-V and avirulent strain F-01-A of Ralstonia solanacearum, and the effects of the extracellular substances of bacterial-wilt-disease blocontrol strain ANTI-8098A, Bacillus cereus on the growths of strains F-01-V and F-01 -A as well as influences of strain ANTI-8098A on the growth competitions at different temperatures. The results showed that the numbers of strain F-01-V increased with the largest increasing rate of 123.7%, that of strain F-01-A decreased with the largest decreasing rate of 60.7%, when they were separately maintained at temperatures 15, 20, 25, 30 and 35 ℃ for 48 h, respectively. While they were in mixing cultures of strain F-01-V and F-01-A for 48 h, the growth of strain F-01-V was inhibited and the growth of strain F-01-A was facilitated. The extracellular substances of strain ANTI-8098A could inhibit the growth of strain F-01-V, but it could facilitate the growth of strain F-01-A. There was a positive correlation between the inhibiting（or the facilitating）rate and temperature. When the mixed cultures were maintained at temperatures at 30 ℃, the ratio values of F-01-V/F-01-A were less than 0.01, which indicated the strain F-01-A were significantly dominated.

Key words: Ralstonia solanaceamm, Bacillus cereus, Growth Competition, Avirulent strain

Study on Numerical and Pathogenic Variations of Ralstonia solanacearum Distributed Within the Tissue of Host Plants

LIU Bo*, ZHU Yu-jing, LIN Kang-mei, XIAO Rong-feng, GE Ci-bin, LAN Jiang-lin

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract: The numerical and pathogenic variations of Ralstonia solanacearum distributed within the tissue of host plants were investigated for a preliminary study. Scientists discussed the relationship between the pathogen and the plant particularly from the point view of an ecological niche. The samples were taken in the fields of tomato, eggplant, tobacco, peanut and ginger in Fujian province. R. solanacearum was separated from the different parts of tissues on TTC medium with Colony-Forming-Unit and Pathogenic Index（Attenuation Index）calculations. The results showed that the numerical averages of R. solanacearum within the infected plants of tomato and eggplant（>100 × 10⁸ cfu/g）were significantly higher than those within the infected plants of tobacco, peanut and ginger（<70× 10⁸ cfu/g）. The amounts of R. solanacearum in the tissue of tomato ranged in the ascending order of root>middle-top stem >middle-low stem. They reduced gradually from root to top stem in the tissues of eggplant and peanut and were significantly higher in the root and middle stem than low and top stems in the tissues of tobacco. They were higher in the low part of stem than in the middle-top part of stem in tissues of ginger, respectively. The averages of attenuation indexes in the infected plants of different hosts were counted in ascending order as eggplant>tobacco>peanut>ginger; the pathogen from ginger exhibited as virulent strain with attenuation index of 0.49（<0.60）, while that from eggplant was close to avirulent strain with attenuation index of 0.80. Those from tobacco, peanut and tomato belonged to strains of uncertain virulence with attenuation indices of 0.64-0.70. R. solanacearum could be found in both healthy and infected plants of peanut, whereas it could only be detected in the infected plants of eggplant and ginger. The amount and pathogenicity of R. solanacearum were tested in great differences at the different periods of disease developing stages. It was concluded that the amount and pathogenicity of R. solanacearum were significantly distributed within the host tissues in different hosts, in position parts of plants, in the periods of disease developing stages, which led to the differentiation of ecological niche in the pathogen.

Key words: Ralstonia solanaceamm, Numerical variation, Pathogenicity, Ecological niche

Polymorphism of Fatty Acid of Ralstonia solanacearum in Fujian Province

WANG Qiu-hong, LIN Ying-zhi, ZHU Yu-jing, LAN Jiang-1in, LIU Bo*

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract: The fatty acids of 40 strains of Ralstonia solanacearum isolated from diferent hosts in the fields in Fujian Province were detected by gas chromatography(GC). The polymorphism of R. solanacearum fatty acids relating to the pathogenicity was observed. The MIDI system and cluster analysis were introduced in analyzing fatty acids to display the relations among the polymorphism, race, biovartype and pathogenicity. The results showed that the patterns of fatty acids were significant diferent in R. solanacearum strains both isolated from the diferent hosts an d the diferent body parts of the same hosts. According to the fatty acids the strains were clustered into three groups, e.g. group I relating to the strains with non-pathogenicity, group II in which the strain pathogenicity was changeable with some virulent and avirulent ones, and group III respondent to high pathogenicity. It was proved that the model of fatty acids has no relations to races and biovartypes in R. solanacearum. It is the fist time to describe the polymorphism of fatty acids in R. solanacearum in this paper. The pathogenicity could be grouped by the models of fatty acids to distinguish the pathogenicity, which could be used in the identification of R. solanacearum under species differentiation.

Key words: Ralstonia solanacearum, Fatty acid, Polymorphism, Gas chromatography

Genetic Diversity Analysis of Ralstonia solanacearum Based on ITS Sequence and RAMS Marker

ZHENG Xue-fang, CHE Jian-mei, LIN Ying-zhi, LAN Jiang-lin, LIU Bo*

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract：The genetic diversity among 21 strains of from different climate zones and host crops were analyzed by ITS sequence and RAMS（random amplified micro-satellites）methods, respectively. Three types of ITS sequence were obtained, and there were only 1-2 bases or no diversity in ITS sequence compared to the standard strains GMI1000. The total genomic DNA was then examined by the sensitive technique of RAMS. The results revealed that an obvious diversity of their genomic DNA existed. The cluster analysis based on the RAMS bands showed that the tested isolates could be divided into 3 clusters. The strains came from the same host crops were clustered into the same group, and the strains came from the same climate zones were belonged to the same subgroup. The result suggested that the genetic variation of R. solanacearum was more closely related to the host crops than to the different climate zones.

Key words: Ralstonia solanacearum，genetic diversity, ITS sequence, RAMS marker

Community and diversity of endophytic bacteria inside eggplant

LAN Jiang-lin, LIU Bo*, ZHU Yu-jing, LIN Kang-mei, SU Ming-xing, SHI Huai

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract：In order to study the community structure and diversity of endophytic bacteria in the eggplant, the healthy and disease infested eggplant plants were collected from three sites in Fujian Province, PR China. The endophytic bacteria were isolated using the culture method and identified by Microorganism Identification System（MIDI system produced by USA）. The results showed that a total of 28 microbial species identified belonging to 18 genera of bacteria were colonized detected in the eggplant, of which, there were 23 microbial species belonging to 15 genera were found inside the healthy plant with a amount of 5.96×10⁵ CFU·g^-1, and 16 microbial species belonging to 14 genera were displayer in the diseased infested plant with a amount of 3.191×10⁸ CFU·g^-1, especially Ralstonia solanacearum reached to the amount of 3.187×10⁸ CFU.g^-1. The number of endophytic bacteria decreased in the turn of root, stem, leaves. The significant difference of endophytic bacteria was observed among the eggplants collected from different sites, i.e. 17 species belonging to 13 genera from Jinjiang, 9 species belonging to 7 genera from Dongyang and 4 species belonging to 3 genera from Fuqing. The indices of SIMPSON（D）and SHANNON（H1）for the endophytic bacterium community in the plant from Jinjiang county were 0.9632 and 3.5725, respectively, higher than those in the plant from Dongyang and Fuqing counties. The evenness of endophytic bacteria was 0.9708 in the plant from Dongyang county, which was higher than that of 0.9654 or 0.9464 in the plant from Jinjiang county or Fuqing county, respectively. The community structure and diversity of endophytic bacteria were significant different in the healthy conditions and the cultivated sites

Key words: eggplant plant, endophytic bacteria, community structure, diversity

Tagging of Bacterial-wilt-disease Biocontrol Bacteria ANTI-8098A（Bacillus cereus）with the Green Fluorescence Protein Gene and its Biological Characteristics

Che Jian-mei, Liu Bo^*, Lan Jiang-lin

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract: The transformation system by electroporation of the biocontrol bacteria ANTI-8098A（Bacillus cereus）was optimized in this paper. The results showed that the system was best for the electroporation when the concentration of compentent cell was 1.14×10⁸ CFU/mL and the content of plasmid was 118 ng. The percent of the green cells was 100% when the strains of ANT8098A：pCM20 was reinculated in the non-selective medium for 16 times.And the growth speed of gfp-tagged strains was almost the same to the wild type strain ANTI-8098A. The best culture conditions of wild type strain and gfp-tagged one including the culture temperature of 35℃ and rotation speed of 250 r/min were almost the same. Both strains ANTI8098A and ANTI8098A：pCM20 showed the same in vitro inhibition of Ralstonia solanacearum from different crops.

Key words: Bacterial wilt disease, Biocontrol bacteria, Bacillus cereus, Green fluorescence protein

Construction of Avirulent Mutants of Ralstonia solanacearum by Tn5 Transposon and Analysis of Its Biological Characteristics

Cheng Ben-liang, Che Jian-mei, Liu Bo*

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract: Ralstonia solanacearum is the agent of bacterial wilt. In order to provide the development of plant vaccine with avirulent mutants of R. solanacearum whose genetics are stable and insetion sites,this study constructed the avirulent mutants of R. solanacearum（strain Rs91）using EZ-Tn5 transposon mutagenesis. Through electroporation and screening, thriteen avirulent mutants were obtained. Results showed that the insertion sites of the 5 mutants were found to locate in phcA and phcS genes separately. The growth curves and the relative contents of Exopolysaccharide（EPSI）of the 5 mutants were significantly lower than that of the original strain Rs91, but their optimum temperature and pH for the mutants to grow changed a few. The supernatants of the 5 mutants cultured in the TTC liquid medium were scanned by the UV-spectrophotometer, and the result showed that the absorbance of the mutants was higher than that of Rs91. The result of scanning was analyzed by UPGMA dendrogram. The cluster was displayed that the 5 avirulent mutants and Rs91 could be separated into 3 groups, e.g. the original strain Rs91 was in groupⅠ, the mutants of phcA-were in groupⅡ, and the mutant of phcS-was in groupⅢ. The 5 mutants were determined by the bioassay of potted tomato（Lycopersicon esculentum）and they did not fall ill after 15 days. The 5 mutants were determined to be avirulent R. solanacearum. This study provides foundation data for developing plant vaccine against bacterial wilt.

Key words: Ralstonia solanacearum, TTC medium, Avirulence mutant, Inverse-PCR (IPCR), Exopolysaccharide (EPSI)

Growth and pathogenicity characteristics of Ralstonia solanacearum strain RS1100 in long-term stationary phase culture

Zhu Yu-jing, Xiao Rong-feng, Liu Bo*

（Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China）

Abstract：Nonsporulating gram-negative bacteria remain metabolically active and also develop increased resistance to a variety of environmental stresses after exponential growth has stopped and cells enter stationary phase. Ralstonia solanacearum is a nonsporulating gram-negative soil-borne pathogen that causes

lethal wilt diseases of many plants around the world. Its growth and pathogenicity characteristics for entering longterm stationary phase were investigated by a prolonged 20-day laboratory culture. The data obtained from viability trial revealed that~99% of R. solanacearum strain RS1100 (race 1) died at day 14 in SPA medium and then survivors maintained at~10⁵ cfu ml^–1 until the 20th day. Binary fission was observed and two modes of death procedures were assumed for growth of R. solanacearum under transmission electronic microscope, nucleoid condensation or nucleoid fragmentation and cytolysis. The latent period for R. solanacearum causing plant wilt prolonged while the cell density reduced gradually. The pathogen retained its aggressiveness at low density as 1.13 × 10⁵ cfu ml^–1 at 20th day. Both mucoid and nonmucoid forms of R. solanacearum were coexistent throughout the experiment with varying ratio. The results here confirm that upon entering long-term stationary phase R. solanacearum can survive the stress conditions of nutrient starvation during prolonged stationary phase.

Key words: growth, pathogenicity, Ralstonia solanacearum, RS1100, stationary phase, viability

Genetic diversity analysis of Ralstonia solanacearum based on BOX-PCR and REP-PCR

Lin Hai-yun^1,2, Che Jian-mei¹, Liu Bo¹*, Zheng Xue-fang¹

（1.Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences , Fuzhou, 350003, China; 2. Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, 350002, China）

Abstract：Genetic diversity of Ralstonia solanacearum is very important for knowing the happening and spreading of bacterial wilt diseases. Special primer was used to identify Ralstonia solanacearum strains, and 84 strains of R. solanacearum from different host plants in the Fujian province were identified, which produced a specific band in the location of 504 bp. By BOX-PCR and REP-PCR polymorphism analysis of these strains, BOX-PCR amplified 19 specific bands and REP-PCR amplified 20 specific bands. Cluster of PCR analysis showed that genetic diversity of R. solanacearum was concerned with geographic origin and host plants, and different host plants played a leading role in the genetic difference of R. solanacearum. Through further analysis, the difference of regions was mainly provided by the BOX-PCR, and the difference of host plants was mainly provided by REP-PCR. It was indicated that REP-PCR and BOX-PCR could provide an alternative way for the study of genetic diversity of R. solanacearum.

Key words：Ralstonia solanacearum, Genetic diversity, BOX-PCR, REP-PCR

GFP tagging Ralstonia solanacearum with gfp/luxAB mini-Tn5 and the differentiation in the microbiological characteristics

Che Jian-mei, Lan Jiang-lin, Liu Bo*

（Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences , Fuzhou, 350003, China）

Abstract: The Ralstonia solanacearum strain was chromosomally tagged with the marker genes gfp and luxAB by electrotransformation with a pUT mini-Tn5 transposon vector. The microbiology properties and the pathogenicity were compered. The Ralstonia solanacearum was transformed by the electroporation. The condition and processor of the invasion of the GFP-tagged one was observed in the tomato tissue culture plants. Transformants were screened for the green fluorescence phenotype of the gfp marker by fluorescence stereomicroscopy. A 3.0 Kb fragment of the gfp gene and luxAB gene was amplified from the genome DNA of the GFP-tagged strains. Morphologically the cells of the engineered strain were similar to the original strain as determined by laser scanning confocal microscopy. Luciferase activity of the GFP-tagged strain increased rapidly during log phase growth in 0.1×TSB medium, but decreased in stationary phase. The standard growth curve of the strain the GFP-tagged strain was delayed by 24% than the wild type one. And the green fluorescence could be kept until 20 times of subcultures. It was the same for the GFP-tagged one and the wild type strain on the culture temperature, pH value and rotation speed. These results confirmed that the dual marker was inserted successfully into the Ralstonia solanacearum strain. The pathogenicity of the wild type Ralstonia solanacearum strain and the GFP-tagged one was over 90% which means gfp gene had no effect on the pathogenicity. The GFP-tagged Ralstonia solanacearum was got in this paper. The microbiology propertities and the pathogenicity of the GFP-tagged one was the same to the wild type strain.

Key words: Ralstonia solanacearum，Electrotransformation，gfp/luxAB gene