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 3.191×10 8 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-yun1,2, 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