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GB/T 40135-2021: Detection and identification of Xylophilus ampelinus (Panagopoulos) Willems et al.
GB/T 40135-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.020.01
CCS B 16
Detection and Identification of Xylophilus Ampelinus
(Panagopoulos) Willems et al.
ISSUED ON: MAY 21, 2021
IMPLEMENTED ON: DECEMBER 1, 2021
Issued by: State Administration for Market Regulation;
Standardization Administration of the People’s Republic of
China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Terms and Definitions ... 4
4 Classification Information of Xylophilus Ampelinus (Panagopoulos) Willems
et al... 4
5 Method Principle ... 5
6 Reagents, Materials and Culture Medium ... 5
7 Instruments and Utensils ... 5
8 Detection and Identification Methods ... 6
9 Result Determination ... 9
10 Sample Preservation ... 10
11 Strain Preservation ... 10
Appendix A (informative) Xylophilus Ampelinus (Panagopoulos) Willems et al.
... 11
Appendix B (informative) Reagents ... 14
Appendix C (informative) Culture Medium ... 15
Appendix D (normative) Molecular Biological Detection Method for Xylophilus
Ampelinus (Panagopoulos) Willems et al... 16
Bibliography ... 21
Detection and Identification of Xylophilus Ampelinus
(Panagopoulos) Willems et al.
1 Scope
This Standard specifies the detection and identification methods (separation culture,
immunology and molecular biology, etc.) for xylophilus ampelinus (panagopoulos)
Willems et al.
This Standard is applicable to the detection and identification of xylophilus ampelinus
(panagopoulos) Willems et al. in grapes and the propagating materials.
2 Normative References
The content of the following documents constitutes indispensable clauses of this
document through normative references in the text. In terms of references with a
specified date, only versions with a specified date are applicable to this document. In
terms of references without a specified date, the latest version (including all the
modifications) is applicable to this document.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods
SN/T 2122 Sampling Methods for the Quarantine of Plants and Their Products for
Import and Export
3 Terms and Definitions
There are no terms and definitions that need to be defined in this document.
4 Classification Information of Xylophilus Ampelinus
(Panagopoulos) Willems et al.
Chinese name: 葡萄细菌性疫病菌, 葡萄嗜木质菌
Scientific name: Xylophilus ampelinus (Panagopoulos) Willems et al.,1987
Synonym: Xanthomonas ampelina Panagopoulos,1969
English name: canker of grapevine, bacterial blight of grapevine
Taxonomic status: Bacteria, Eubacteria, Proteobacteria, Betaproteobacteria,
Burkholderiales, Comamonadaceae, Xylophilus
See other information in Appendix A.
5 Method Principle
In accordance with the specific reaction between Xylophilus ampelinus (Panagopoulos)
Willems et al. and the antibody, conduct immunological detection on the test object. In
accordance with the specific DNA sequence of Xylophilus ampelinus (Panagopoulos)
Willems et al., conduct molecular biological detection. In accordance with the cultural
characteristics, biological characteristics and hazard symptoms of Xylophilus
ampelinus (Panagopoulos) Willems et al., conduct separation culture on the
pathogenic bacteria, if necessary, conduct pathogenicity detection.
6 Reagents, Materials and Culture Medium
6.1 Reagents and Materials
Unless it is otherwise specified, all reagents used for the tests are analytically pure or
biochemical reagents.
Reagents: 75% alcohol, glucose, agarose, sterile double distilled water, sodium
dodecyl sulfate (SDS), chloroform, isoamyl alcohol, phenol, isopropanol, absolute
ethanol, proteinase K, nucleic acid dye, DNA molecular mass reference substance, 50
 TAE electrophoresis buffer.
See Appendix B for information on the reagents and their preparation.
Materials: plant genomic extraction kit, bacterial genomic extraction kit, common PCR
reaction kit and fluorescent PCR reaction kit, etc.
6.2 Culture Medium
See Appendix C for information on YPGA culture medium, nutrient agar medium (NA),
culture medium and its preparation. YPGA is recommended for the separation culture
medium, and NA may also be used as a substitute.
7 Instruments and Utensils
7.1 Instruments
Biological microscope (magnification by more than 400 times), constant-temperature
incubator, electronic balance (one thousandth), plant light incubator, refrigerator,
centrifuge, microplate reader, electrophoresis instrument, PCR amplification
instrument and real-time fluorescence quantitative PCR instrument, etc.
molecular biological detection. In accordance with laboratory conditions, one of the
following methods (8.4.2 or 8.4.3) may be selected for the detection.
8.4.2 Common PCR and nested PCR
DNA extracted from the extract or other separated objects is used as a template for
the common PCR and nested PCR detection. The detection steps and conditions shall
comply with D.2.
8.4.3 Real-time florescence PCR
DNA extracted from the extract or other separated objects is used as a template for
the real-time fluorescence PCR detection. The detection steps and conditions shall
comply with D.3.
8.5 Separation of Pathogenic Bacteria
8.5.1 Plant materials with symptoms
Take the liquid obtained in 8.2.1 and directly apply it to the YPGA plate; apply 5 ~ 10
petri dishes for each sample. At 25 °C, cultivate them; from the third day, observe the
growth of colonies on the plate every day. Typical colony, which grows slowly on the
culture medium, is smooth and non-sticky, shiny, slightly convex, light yellow, circular
and with regular edges. After 7 d ~ 12 d of culture, the size is about 2 mm. If necessary,
conduct purification culture once on the colony.
8.5.2 Plant materials without symptoms
Take 1.0 mL of the liquid obtained in 8.2.2, use sterile water or phosphate buffer
solution to dilute it by 10 times, 100 times and 1,000 times. Respectively take 200 μL
of the diluent for coating separation on the YPGA plate. For each dilution degree, apply
5 ~ 10 petri dishes. The cultivation method is the same as 8.5.1.
8.6 Pathogenicity Detection
If necessary, pathogenicity determination may be carried out.
The pathogenicity experiments are carried out on potted susceptible vine stems or new
cutting seedlings. Collect the suspected bacteria freshly cultured on YPGA culture
medium and use sterile water to prepare a bacterial suspension of about 1  108
CFU/mL. On the seedling cane, use a sterile knife to cut a fresh wound of 2 cm ~ 4 cm
long, which reaches the depth of the vascular bundle. Inoculate 1 drop ~ 2 drops of the
bacterial suspension to the wound or fresh wound of the leaf; use sterile cotton
moistened by sterile water to cover the wound and use tin foil sheet to wrap it; repeat
for 5 ~ 10 times. In the same way, use the standard strain of xylophilus ampelinus
(panagopoulos) Willems et al. as a positive control; use sterile water as a negative
control; repeat for at least 2 times. Place the inoculated grape seedlings at 20 °C ~
27 °C; the sunshine duration is 14 h ~ 18 h; cultivate for 3 ~ 4 weeks in an environment
with sufficient moisture and fertilizer.
The pathogenicity experiments may also be carried out on the susceptible vine stems
that have rooted in the test tube. Collect the suspected bacteria freshly cultured on
YPGA culture medium and use sterile water to prepare a bacterial suspension of about
1 ...