论文部分内容阅读
Abstract [Objectives] This study was conducted to analyze the infection ability of Rhizobium radiobacter from Rosa chinensis to peach tree， so as to provide a theoretical basis for the identification and control of crown gall of peach. [Methods] With peach variety "Chiyohime" as an experimental material， R. radiobacter GOU1 was extracted from crown gall tissue of R. chinensis susceptible to crown gall， and its infection ability to peach tree was studied by in-vitro dip method. [Results] Stem segments of peach tree were inoculated with R. radiobacter GOU1 by in-vitro dip method. Calli grew rapidly after inoculation， and 28 d later， the infection rate of R. radiobacter GOU1 was 50%， indicating that R. radiobacter GOU1 could infect peach tree easier. The infection on stem segments of peach tree could be detected in the fourth week by in-vitro dip method. Culturing peach stem segments in MS medium containing 10 μM/L NAA and 10 μM/L BAP in vitro is beneficial to survival and could promote growth of callus. [Conclusions] The detection rate of R. radiobacter GOU1 infection on stem segments of peach tree did not change from the fourth week to the sixth week. Therefore， in future studies， the detection of R. radiobacter infection on inoculated stem segments of peach tree could be performed after 4 weeks of culture rather than 6 weeks of culture.
Key words Peach； Rhizobium radiobacter； Strain GOU1； Infection ability
Crown gall， also known as crown gall and root knot， is a kind of common bacterial disease in plants， which could infect fruit trees including peach， pear and fig， and flowers including Rosa chinensis and Rosa multiflora， and once infected， plants are difficult to be cured[1-4]. The disease has various pathogens， which differ in infectivity and pathogenicity. The pathogen is realized through integrating T-DNA of its own Ti plasmid into chromosomal DNA of plant host to induce plant tumorigenesis. Genes controlling synthesis of cytokinin， auxin， and nopaline in T-DNA are duplicated and expressed accompanied by the replication and expression of new plant endogenous hormone systems， multiplication is realized with the help of fertility of plant cells， and large quantities of auxin and cytokinin are produced at the inoculation part of plant， giving rise to milk white or light green soft gall with smooth surface at the crown of R. chinensis at early stage of infection； and cells enter an uncontrollable fission state， finally forming crown gall[5]. Crown gall of R. chinensis often occurs at the wound of crown of R. chinensis or graft union， and with the growth and propagation of pathogens in plant， the gall darkens to brown， and has its surface gradually hardened， fractured and finally festered. Once a gall forms， it would continuously absorb nutrition and water in plant like parasites to satisfy its growth and propagation， and as a result， R. chinensis plant cannot flower normally or even die due to nutritional deficiency[6]. The R. radiobacter "G-Ag-27" extracted from grape and R. radiobacter strains ‘A208’ and ‘C58clrif-R’ extracted from cherry all could infect R. chinensis and produce galls[7]. Strain GOU1[8] is the pathogen causing crown gall of R. chinensis. It is a kind of R. radiobacter with short-bar-shaped bipolar flagellation carrying crown gall gene[9]. R. radiobacter strain GOU1 of R. chinensis could infect a variety of R. chinensis， including R. ‘Ducat’ and R. ‘Matsushima No.3’， while F1 of R. hybrida×multiflora， No.1 has stronger resistance against crown gall[10]. R. PEKcougel also has stronger resistance against crown gall[11]. However， there were few studies on the infection mechanism of R. radiobacter strain GOU1 of R. chinensis to fruit tree， and among studies on crown gall of fruit trees， there were also fewer studies on rapid identification of crown gall infection condition of fruit trees using in-vitro culture. In this study， stem segments of peach tree were inoculated by in-vitro dip method and cultured， and the browning of stem segments and formation of callus were observed， so as to analyze the infection ability of R. radiobacter to peach tree. This study will provide a theoretical basis for the identification and control of crown gall of peach tree. Materials and Methods
Tested materials
The tested material was peach cultivar， ‘Chiyohime’， which was planted in the experimental orchard field of Gifu University in Japan.
The tested strain was R. radiobacter strain GOU1， which was isolated and extracted from the root knot tissue of R. chinensis susceptible to crown gall by Research Laboratory of Horticulture， Department of Applied Biological Sciences， Gifu University. The original bacteria were activated using YEB medium （beef extract 5 g/L+yeast 1 g/L+MgSO4·7H2O 0.493 g/L+peptone 5 g/L+sucrose 5 g/L） for 24 h， and the activated bacterial liquid was 2×107 ind./ml.
Experimental design
Sterilization of material
New shoots of peach tree with uniform growth and diameter and a length of 15-20 cm having young leaves which were at the status from non-completely expanded to completely expanded， were selected as culture materials. The culture materials were washed with neutral abluent and flushed with tap water for 20 min. The stem segments were sterilized with 70% ethanol for 30-60 s， and then with the solution of 1% NaClO+2-3 drops of Tween-20 for 20 min. The materials were then transversely cut on a clean bench into small internode segments with a thickness of （5±1） mm.
Inoculation and culture of explants
The explants were subjected to different treatments and inoculated to different media. There were three treatments： ① stem segments free of inoculation with strain GOU1， inoculated to MS medium without the addition of any plant hormone （MS-No inoculation for short）， ② stem segments free of inoculation with strain GOU1， inoculated to MS medium containing 10 μM/L NAA+10 μM/L BAP （HMS-No inoculation for short）， and ③ stem segments inoculated with strain GOU1， inoculated to MS medium without the addition of any plant hormone containing 100 mg/L antibiotic （MS-inoculation for short）. In treatment MS-inoculation， inoculation was performed by out-test-tube dipping method developed by Gifu University. The internode stem segments were put into 2×107 bacteria/ml bacterial liquid and infected for 5 min. The infected stem segments were subjected to coexistence culture in MS medium without any plant hormone for 72 h， then taken out and cleaned in 100， 200 and 500 μg/ml antibiotic solutions sequentially for 1 min. The stem segments were finally cultured in MS medium without the addition of any plant hormone containing 100 mg/L antibiotic. The inoculation and culture time was the same for each treatment， and media were not changed midway. The experiment adopted randomized block design. Into each plot， 20 internode segments were inoculated， with three replicates， and each culture dish contained five internode stem segments. The culture was performed at 25 ℃ under 3 000 lx for 16 h.
Determination and analysis of experimental indexes
Identification of infection with R. radiobacter
Whether stem segments were infected with R. radiobacter was identified by Otten paper electrophoresis[12]. The cultured stem segments were taken out， added into 1.5 ml centrifuge tubes and crushed with plastic rods， and the stem segment liquids were preserved. The upper end of electrophoretic filter paper had spotting holes at interval of 1 cm on the same horizontal line. Mark solution （methylene green 10 mg/ml， 2 μl）， nopaline purification solution （2 μl） and differently treated stem segment liquid （3 μl） were sequentially added into spotting hole from left to right with a pipette. After air-drying of all solutions， filter paper was completely soaked and wetted with electrophoretic liquid （formic acid∶acetic acid∶water=10∶30∶100， ml）， and placed into the electrophoretic tank added with the electrophoretic liquid. After setting the voltage at 250 V， electrophoresis was performed for 20 min. After ensuring that the mark had moved， the filter paper was taken out and dried with a hair dryer. Then， staining liquid A （phenanthrenquinone∶anhydrous ethanol=20∶100， ml） and liquid B （solution of anhydrous NaOH particles 10 g and 60% ethanol 100 ml） were mixed at a ratio of 1∶1， and uniformly sprayed onto the filter paper with a sprayer. The filter paper was re-dried and detected with a UV lamp detector at 365 nm to observe whether fluorescent spots appeared， so as to judge whether the stem segments were infected with R. radiobacter. The infection rate was calculated according to following equation： Infection rate=Number of infected stem segments/Total number of stem segments×100%.
Analytical methods of survival rate， callus formation rate and callus area
After 4 weeks of culture， survival rate， callus formation rate and callus area were investigated. Survival rate was calculated according to Survival rate=Number of stem segments maintaining green/Total number of stem segments×100%. Callus formation rate was calculated according to Callus formation rate=Number of stem segments forming callus/Total number of survived stem segments×100%. And callus area was calculated according to Callus area=Length×Width of the part forming callus. Relationship between detection rate of R. radiobacter and culture time
The method was the same as "Identification of infection with R. radiobacter". Each group had 20 stem segments， and there were six groups. From the first to the sixth week， one group was selected for electrophoresis detection every week. The test was designed with three replicates.
Methods of statistic analysis
Statistical analysis and plotting were performed in SPSS18.0， and multiple comparisons were performed by Duncan method.
Results and Analysis
Comparison of survival rate between different treatments of peach stem segments after 28 d of in-vitro culture
After 28 d of in-intro culture， there were significant differences in survival rate of stem segments between different treatments. The stem segments free of inoculation with the strain cultured in MS medium added with 10 μM/L NAA amd 10 μM/L BAP exhibited the highest survival rate of 85%. The stem segments inoculated with R. radiobacter GOU1 showed the second highest survival rate of 55%. The stem segments free of inoculation cultured in MS showed the lowest survival rate of 40% （Fig. 1）. It indicates that the addition of 10 μM/L NAA and 10 μM/L BAP into MS medium is beneficial to the survival of stem segments of peach tree.
Comparison of callus formation rate between different treatments of peach stem segments
After 28 d of in-vitro culture， there were significant differences in callus formation rate between different treatments of peach stem segments. The stem segments free of inoculation added with 10 μM/L NAA and 10 μM/L BAP showed the highest callus formation rate of 86.67%， but there were no significant difference in callus formation rate between the stem segments inoculated and not inoculated with R. radiobacter GOU1 （Fig. 2）.
Comparison of callus/gall area between different treatments of peach stem segments
After 28 d of in-vitro culture， there were significant differences in callus or gall area between different treatments of stem segments. The stem segments inoculated with R. radiobacter GOU1 exhibited the largest callus area， followed by the stem segments free of inoculation with GOU1 cultured in MS medium added with 10 μM/L NAA and 10 μM/L BAP and the ones free of inoculation with GOU1 cultured in MS medium sequentially. The addition of 10 μM/L NAA and 10 μM/L BAP in MS medium could promote the formation and growth of calli from peach stem segments （Fig. 3）. Agricultural Biotechnology2018
Comparison of R. radiobacter infection condition between different treatments
After 28 d of in-vitro culture， 50% of the stem segments inoculated with R. radiobacter GOU1 were detected to be infected with R. radiobacter， while other treatments were not infected （Fig. 4-Fig. 6）.
Relationship between detection rate of R. radiobacter GOU1 in inoculated peach stem segments and culture time
After 2 weeks of culture， 10% of stem segments inoculated with R. radiobacter GOU1 were detected to be infected with the strain， the detection rate reached 35% to the third week and 50% to the fourth week， and in the fifth week and the sixth week， the detection rate of R. radiobacter infection did not change over time （Fig. 7）.
Discussion and Conclusions
Zhuang et al.[10] has demonstrated that in-vitro dip method could be used for identifying crown gall of R. chinensis. By this method， the infection rate of stem segments of peach variety "Chiyohime" inoculated with R. radiobacter GOU1 and cultured in vitro was detected to be 50%， and the non-inoculated stem segments were detected to be not infected. It indicates that R. radiobacter GOU1 could infect peach variety "Chiyohime" easier， and the in-vitro dip method for detecting R. radiobacter is accurate， reliable and rapid. In future， validation could be performed using the in-vitro dip method and field infection tests.
After 28 d of culture， the survival rate of stem segments inoculated with R. radiobacter GOU1 and cultured in MS medium without plant hormone （MS-Inoculation） was higher than that of stem segments free of inoculation cultured in MS medium without plant hormone （MS-No inoculation）， but lower than that of stem segments free of inoculation cultured in MS medium containing NAA 10 μM/L+BAP 10 μM/L （HMS-No inoculation）. It indicates that R. radiobacter GOU1 could improve survival rate during in-vitro culture， which might be due to that there are plant hormones promoting plant survival produced after infection of stem segments.
The calli formed on the stem segments inoculated with R. radiobacter GOU1 were bigger than those on stem segments free of inoculation， indicating that R. radiobacter GOU1 could promote rapid growth of callus， which might be due to that large quantities of plant hormones are produced after the infection of stem segments.
It could be seen from the results of this study that the detection rate of R. radiobacter GOU1 infection on stem segments of peach tree did not change from the fourth week to the sixth week. Therefore， in future studies， the detection of R. radiobacter infection on inoculated stem segments of peach tree could be performed after 4 weeks of culture rather than 6 weeks of culture. References
[1] TAO WQ， CHEN FW， GUO SX， et al. Occurrence and prevention of crown gall of peach[J]. Greening and Life， 2001， （1）： 24-25. （in Chinese）
[2] JIAO SP， ZHANG XP， ZHANG JW， et al. Analysis of risk of fruit tree root cancer invading Xinjiang[J]. Jiangsu Agricultural Sciences， 2013， 06： 114-116. （in Chinese）
[3] AEINI M， MIRZAEE H， TAGHAVI SM， et al. Occurrence of crown gall disease on Ficus benjamina in Fars and Isfahan provinces of Iran[J]. Archives of Phytopathology & Plant Protection， 2014， 47（18）： 2257-2262.
[4] LUO ZJ， HUI WX， ZHAO WX， et al. Thinking on the problem of inspection and control of woody plant root cancer[J]. China Forestry Science and Technology， 2011， 04： 6-11. （in Chinese）
[5] SHI WX， WANG HK， GUO QY. Grape root cancer research[J]. Journal of Zhejiang Agricultural Sciences， 2013， （11）： 1418-1421. （in Chinese）
[6] OHTA K. Studies on crown gall and hairy root of flower crops[J]. Shizuoka Agricultural Experiment Station Technical Bulletin， 1993， 16： 1-63.
[7] ZHOU LIN， SUZUKI K， NARUSE T， et al. In Vitro Testing of Rose Rootstocks Resistance to Crown Gall Disease[J]. The Japanese Society for Horticultural Science， 2000， 69（2）： 171-175.
[8] YOUNG JM， KUYKEDALL LD， MARTINEZ-ROMERO E， et al. A revision of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combination： Rhizobium radiobacter， R. rhizogenes， R. rubi， R. undicola and R. vitis[J]. Int J Syst and Evol Microbiol， 2001， 51： 89-103.
[9] BRISBANE PG， KERR A. Elective media for three biovars of Agrobacterium[J]. J. Appl. Bacteriol.， 1983， 52： 425-431.
[10] ZHUANG DF， MA C， FUNAHASHI SHINGO， et al. Pre-Selection of Rootstock with Resistance Against Crown Gall Disease and Root Rot Disease in Chinese Rose[J]. Hubei Agricultural Sciences， 2016（19）： 4960-4963. （in Chinese）
[11] ZHOU L， SUZUKI K， FUKUI H， et al. In vitro inoculation test for resistance to crown all disease on rose[J]. Co，. Proc. Inter. Plant Prop. Soc.， 1996， 46： 750-754.
[12] OTTEN LM， SCHILPEROORT RA. A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities[J]. Biochim Biophys Acta， 1978， 527： 497-500.
Key words Peach； Rhizobium radiobacter； Strain GOU1； Infection ability
Crown gall， also known as crown gall and root knot， is a kind of common bacterial disease in plants， which could infect fruit trees including peach， pear and fig， and flowers including Rosa chinensis and Rosa multiflora， and once infected， plants are difficult to be cured[1-4]. The disease has various pathogens， which differ in infectivity and pathogenicity. The pathogen is realized through integrating T-DNA of its own Ti plasmid into chromosomal DNA of plant host to induce plant tumorigenesis. Genes controlling synthesis of cytokinin， auxin， and nopaline in T-DNA are duplicated and expressed accompanied by the replication and expression of new plant endogenous hormone systems， multiplication is realized with the help of fertility of plant cells， and large quantities of auxin and cytokinin are produced at the inoculation part of plant， giving rise to milk white or light green soft gall with smooth surface at the crown of R. chinensis at early stage of infection； and cells enter an uncontrollable fission state， finally forming crown gall[5]. Crown gall of R. chinensis often occurs at the wound of crown of R. chinensis or graft union， and with the growth and propagation of pathogens in plant， the gall darkens to brown， and has its surface gradually hardened， fractured and finally festered. Once a gall forms， it would continuously absorb nutrition and water in plant like parasites to satisfy its growth and propagation， and as a result， R. chinensis plant cannot flower normally or even die due to nutritional deficiency[6]. The R. radiobacter "G-Ag-27" extracted from grape and R. radiobacter strains ‘A208’ and ‘C58clrif-R’ extracted from cherry all could infect R. chinensis and produce galls[7]. Strain GOU1[8] is the pathogen causing crown gall of R. chinensis. It is a kind of R. radiobacter with short-bar-shaped bipolar flagellation carrying crown gall gene[9]. R. radiobacter strain GOU1 of R. chinensis could infect a variety of R. chinensis， including R. ‘Ducat’ and R. ‘Matsushima No.3’， while F1 of R. hybrida×multiflora， No.1 has stronger resistance against crown gall[10]. R. PEKcougel also has stronger resistance against crown gall[11]. However， there were few studies on the infection mechanism of R. radiobacter strain GOU1 of R. chinensis to fruit tree， and among studies on crown gall of fruit trees， there were also fewer studies on rapid identification of crown gall infection condition of fruit trees using in-vitro culture. In this study， stem segments of peach tree were inoculated by in-vitro dip method and cultured， and the browning of stem segments and formation of callus were observed， so as to analyze the infection ability of R. radiobacter to peach tree. This study will provide a theoretical basis for the identification and control of crown gall of peach tree. Materials and Methods
Tested materials
The tested material was peach cultivar， ‘Chiyohime’， which was planted in the experimental orchard field of Gifu University in Japan.
The tested strain was R. radiobacter strain GOU1， which was isolated and extracted from the root knot tissue of R. chinensis susceptible to crown gall by Research Laboratory of Horticulture， Department of Applied Biological Sciences， Gifu University. The original bacteria were activated using YEB medium （beef extract 5 g/L+yeast 1 g/L+MgSO4·7H2O 0.493 g/L+peptone 5 g/L+sucrose 5 g/L） for 24 h， and the activated bacterial liquid was 2×107 ind./ml.
Experimental design
Sterilization of material
New shoots of peach tree with uniform growth and diameter and a length of 15-20 cm having young leaves which were at the status from non-completely expanded to completely expanded， were selected as culture materials. The culture materials were washed with neutral abluent and flushed with tap water for 20 min. The stem segments were sterilized with 70% ethanol for 30-60 s， and then with the solution of 1% NaClO+2-3 drops of Tween-20 for 20 min. The materials were then transversely cut on a clean bench into small internode segments with a thickness of （5±1） mm.
Inoculation and culture of explants
The explants were subjected to different treatments and inoculated to different media. There were three treatments： ① stem segments free of inoculation with strain GOU1， inoculated to MS medium without the addition of any plant hormone （MS-No inoculation for short）， ② stem segments free of inoculation with strain GOU1， inoculated to MS medium containing 10 μM/L NAA+10 μM/L BAP （HMS-No inoculation for short）， and ③ stem segments inoculated with strain GOU1， inoculated to MS medium without the addition of any plant hormone containing 100 mg/L antibiotic （MS-inoculation for short）. In treatment MS-inoculation， inoculation was performed by out-test-tube dipping method developed by Gifu University. The internode stem segments were put into 2×107 bacteria/ml bacterial liquid and infected for 5 min. The infected stem segments were subjected to coexistence culture in MS medium without any plant hormone for 72 h， then taken out and cleaned in 100， 200 and 500 μg/ml antibiotic solutions sequentially for 1 min. The stem segments were finally cultured in MS medium without the addition of any plant hormone containing 100 mg/L antibiotic. The inoculation and culture time was the same for each treatment， and media were not changed midway. The experiment adopted randomized block design. Into each plot， 20 internode segments were inoculated， with three replicates， and each culture dish contained five internode stem segments. The culture was performed at 25 ℃ under 3 000 lx for 16 h.
Determination and analysis of experimental indexes
Identification of infection with R. radiobacter
Whether stem segments were infected with R. radiobacter was identified by Otten paper electrophoresis[12]. The cultured stem segments were taken out， added into 1.5 ml centrifuge tubes and crushed with plastic rods， and the stem segment liquids were preserved. The upper end of electrophoretic filter paper had spotting holes at interval of 1 cm on the same horizontal line. Mark solution （methylene green 10 mg/ml， 2 μl）， nopaline purification solution （2 μl） and differently treated stem segment liquid （3 μl） were sequentially added into spotting hole from left to right with a pipette. After air-drying of all solutions， filter paper was completely soaked and wetted with electrophoretic liquid （formic acid∶acetic acid∶water=10∶30∶100， ml）， and placed into the electrophoretic tank added with the electrophoretic liquid. After setting the voltage at 250 V， electrophoresis was performed for 20 min. After ensuring that the mark had moved， the filter paper was taken out and dried with a hair dryer. Then， staining liquid A （phenanthrenquinone∶anhydrous ethanol=20∶100， ml） and liquid B （solution of anhydrous NaOH particles 10 g and 60% ethanol 100 ml） were mixed at a ratio of 1∶1， and uniformly sprayed onto the filter paper with a sprayer. The filter paper was re-dried and detected with a UV lamp detector at 365 nm to observe whether fluorescent spots appeared， so as to judge whether the stem segments were infected with R. radiobacter. The infection rate was calculated according to following equation： Infection rate=Number of infected stem segments/Total number of stem segments×100%.
Analytical methods of survival rate， callus formation rate and callus area
After 4 weeks of culture， survival rate， callus formation rate and callus area were investigated. Survival rate was calculated according to Survival rate=Number of stem segments maintaining green/Total number of stem segments×100%. Callus formation rate was calculated according to Callus formation rate=Number of stem segments forming callus/Total number of survived stem segments×100%. And callus area was calculated according to Callus area=Length×Width of the part forming callus. Relationship between detection rate of R. radiobacter and culture time
The method was the same as "Identification of infection with R. radiobacter". Each group had 20 stem segments， and there were six groups. From the first to the sixth week， one group was selected for electrophoresis detection every week. The test was designed with three replicates.
Methods of statistic analysis
Statistical analysis and plotting were performed in SPSS18.0， and multiple comparisons were performed by Duncan method.
Results and Analysis
Comparison of survival rate between different treatments of peach stem segments after 28 d of in-vitro culture
After 28 d of in-intro culture， there were significant differences in survival rate of stem segments between different treatments. The stem segments free of inoculation with the strain cultured in MS medium added with 10 μM/L NAA amd 10 μM/L BAP exhibited the highest survival rate of 85%. The stem segments inoculated with R. radiobacter GOU1 showed the second highest survival rate of 55%. The stem segments free of inoculation cultured in MS showed the lowest survival rate of 40% （Fig. 1）. It indicates that the addition of 10 μM/L NAA and 10 μM/L BAP into MS medium is beneficial to the survival of stem segments of peach tree.
Comparison of callus formation rate between different treatments of peach stem segments
After 28 d of in-vitro culture， there were significant differences in callus formation rate between different treatments of peach stem segments. The stem segments free of inoculation added with 10 μM/L NAA and 10 μM/L BAP showed the highest callus formation rate of 86.67%， but there were no significant difference in callus formation rate between the stem segments inoculated and not inoculated with R. radiobacter GOU1 （Fig. 2）.
Comparison of callus/gall area between different treatments of peach stem segments
After 28 d of in-vitro culture， there were significant differences in callus or gall area between different treatments of stem segments. The stem segments inoculated with R. radiobacter GOU1 exhibited the largest callus area， followed by the stem segments free of inoculation with GOU1 cultured in MS medium added with 10 μM/L NAA and 10 μM/L BAP and the ones free of inoculation with GOU1 cultured in MS medium sequentially. The addition of 10 μM/L NAA and 10 μM/L BAP in MS medium could promote the formation and growth of calli from peach stem segments （Fig. 3）. Agricultural Biotechnology2018
Comparison of R. radiobacter infection condition between different treatments
After 28 d of in-vitro culture， 50% of the stem segments inoculated with R. radiobacter GOU1 were detected to be infected with R. radiobacter， while other treatments were not infected （Fig. 4-Fig. 6）.
Relationship between detection rate of R. radiobacter GOU1 in inoculated peach stem segments and culture time
After 2 weeks of culture， 10% of stem segments inoculated with R. radiobacter GOU1 were detected to be infected with the strain， the detection rate reached 35% to the third week and 50% to the fourth week， and in the fifth week and the sixth week， the detection rate of R. radiobacter infection did not change over time （Fig. 7）.
Discussion and Conclusions
Zhuang et al.[10] has demonstrated that in-vitro dip method could be used for identifying crown gall of R. chinensis. By this method， the infection rate of stem segments of peach variety "Chiyohime" inoculated with R. radiobacter GOU1 and cultured in vitro was detected to be 50%， and the non-inoculated stem segments were detected to be not infected. It indicates that R. radiobacter GOU1 could infect peach variety "Chiyohime" easier， and the in-vitro dip method for detecting R. radiobacter is accurate， reliable and rapid. In future， validation could be performed using the in-vitro dip method and field infection tests.
After 28 d of culture， the survival rate of stem segments inoculated with R. radiobacter GOU1 and cultured in MS medium without plant hormone （MS-Inoculation） was higher than that of stem segments free of inoculation cultured in MS medium without plant hormone （MS-No inoculation）， but lower than that of stem segments free of inoculation cultured in MS medium containing NAA 10 μM/L+BAP 10 μM/L （HMS-No inoculation）. It indicates that R. radiobacter GOU1 could improve survival rate during in-vitro culture， which might be due to that there are plant hormones promoting plant survival produced after infection of stem segments.
The calli formed on the stem segments inoculated with R. radiobacter GOU1 were bigger than those on stem segments free of inoculation， indicating that R. radiobacter GOU1 could promote rapid growth of callus， which might be due to that large quantities of plant hormones are produced after the infection of stem segments.
It could be seen from the results of this study that the detection rate of R. radiobacter GOU1 infection on stem segments of peach tree did not change from the fourth week to the sixth week. Therefore， in future studies， the detection of R. radiobacter infection on inoculated stem segments of peach tree could be performed after 4 weeks of culture rather than 6 weeks of culture. References
[1] TAO WQ， CHEN FW， GUO SX， et al. Occurrence and prevention of crown gall of peach[J]. Greening and Life， 2001， （1）： 24-25. （in Chinese）
[2] JIAO SP， ZHANG XP， ZHANG JW， et al. Analysis of risk of fruit tree root cancer invading Xinjiang[J]. Jiangsu Agricultural Sciences， 2013， 06： 114-116. （in Chinese）
[3] AEINI M， MIRZAEE H， TAGHAVI SM， et al. Occurrence of crown gall disease on Ficus benjamina in Fars and Isfahan provinces of Iran[J]. Archives of Phytopathology & Plant Protection， 2014， 47（18）： 2257-2262.
[4] LUO ZJ， HUI WX， ZHAO WX， et al. Thinking on the problem of inspection and control of woody plant root cancer[J]. China Forestry Science and Technology， 2011， 04： 6-11. （in Chinese）
[5] SHI WX， WANG HK， GUO QY. Grape root cancer research[J]. Journal of Zhejiang Agricultural Sciences， 2013， （11）： 1418-1421. （in Chinese）
[6] OHTA K. Studies on crown gall and hairy root of flower crops[J]. Shizuoka Agricultural Experiment Station Technical Bulletin， 1993， 16： 1-63.
[7] ZHOU LIN， SUZUKI K， NARUSE T， et al. In Vitro Testing of Rose Rootstocks Resistance to Crown Gall Disease[J]. The Japanese Society for Horticultural Science， 2000， 69（2）： 171-175.
[8] YOUNG JM， KUYKEDALL LD， MARTINEZ-ROMERO E， et al. A revision of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combination： Rhizobium radiobacter， R. rhizogenes， R. rubi， R. undicola and R. vitis[J]. Int J Syst and Evol Microbiol， 2001， 51： 89-103.
[9] BRISBANE PG， KERR A. Elective media for three biovars of Agrobacterium[J]. J. Appl. Bacteriol.， 1983， 52： 425-431.
[10] ZHUANG DF， MA C， FUNAHASHI SHINGO， et al. Pre-Selection of Rootstock with Resistance Against Crown Gall Disease and Root Rot Disease in Chinese Rose[J]. Hubei Agricultural Sciences， 2016（19）： 4960-4963. （in Chinese）
[11] ZHOU L， SUZUKI K， FUKUI H， et al. In vitro inoculation test for resistance to crown all disease on rose[J]. Co，. Proc. Inter. Plant Prop. Soc.， 1996， 46： 750-754.
[12] OTTEN LM， SCHILPEROORT RA. A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities[J]. Biochim Biophys Acta， 1978， 527： 497-500.