GB/T 40135-2021 PDF in English
GB/T 40135-2021 (GB/T40135-2021, GBT 40135-2021, GBT40135-2021)
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Detection and identification of Xylophilus ampelinus (Panagopoulos) Willems et al.
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GB/T 40135-2021: PDF in English (GBT 40135-2021) 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
1 109 CFU/mL. Use a sterile knife to cut off the topmost part of the vine stem;
inoculate 1 drop of the bacterial suspension onto the wound; 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 a humid
environment.
9 Result Determination
9.1 Plant Tissues with Typical Symptoms
In accordance with 8.3 and 8.4, test the sample extracts or suspected strains. If the
test results of both methods are positive, then, it can be determined that xylophilus
ampelinus (panagopoulos) Willems et al. is detected. If the test results of both methods
are negative, then, it can be determined that xylophilus ampelinus (panagopoulos)
Willems et al. is not detected. If only one of the test results is positive, conduct
separation of pathogenic bacteria to obtain typical or suspected strains, which are
tested through one of the methods in 8.3 or 8.4 (a different method than the initial
screening test that obtains positive result). If the result is positive, then, it can be
determined that xylophilus ampelinus (panagopoulos) Willems et al. is detected. If
typical or suspected strains are not separated, or the strain identification result is
negative, then, it can be determined that xylophilus ampelinus (panagopoulos) Willems
et al. is not detected.
9.2 Plant Tissues without Typical Symptoms
Adopt the methods in 8.3 and 8.4 to conduct detection and screening of the sample
extracts. In the screening results, if there is 1 positive result or both are positive results,
then, conduct separation of pathogenic bacteria to obtain typical or suspected bacterial
colonies, which are tested through 8.3 and 8.4. If necessary, in combination of the
results of the pathogenicity experiments, if there are 2 positive results, then, it can be
determined that xylophilus ampelinus (panagopoulos) Willems et al. is detected; if
there are 2 negative results, then, it can be determined that xylophilus ampelinus
(panagopoulos) Willems et al. is not detected; if there is 1 positive result, then, the
PCR method in 8.4 that is different from the method that obtains positive result or the
pathogenicity experiments may be adopted for further identification. If the result of the
further identification is negative, then, it can be determined that xylophilus ampelinus
Appendix A
(informative)
Xylophilus Ampelinus (Panagopoulos) Willems et al.
A.1 Distribution
This pathogen is currently distributed in: South Africa, Tunisia, Japan, Turkey, Austria,
Belgium, Bulgaria, France, Greece, Italy, Moldova, Netherlands, Portugal, Serbia,
Slovenia, Spain, Switzerland, United Kingdom (UK), Argentina and Uruguay.
A.2 Host
Natural host: grape (Vitis vinifera Linn.) is currently the only known host.
A.3 Onset Characteristics
From spring to June each year, the symptoms can be observed on the vines. On 12
cm ~ 30 cm long rattan canes, the lower 2 or 3 segments are often the first to be
attacked, which is then gradually expanded upward. In the early stage, reddish-brown
streaks appear, extending from the stem of the cane to the top, and gradually develop
into lenticular cracks or ulcers, sometimes reaching deep into the pith, and the cane is
finally wilted and withered (see Figure A.1). On some young branches, there is less
discoloration; the whole branch is withered and the diseased branch is shorter, which
leads dwarf phenomenon of the grapes. On the cross section of the stem, the tissue is
brown. The symptoms of the diseased main branches and branches are the same as
the symptoms generated by the tip of the branches. On the leaves, pathogens invade
the leaves through the petiole and vascular bundles, and the leaves are often withered
(see Figure A.2). In addition, the pathogens directly infect the leaves through the
stomata. This type of infection causes reddish-brown horn spots on the leaves. When
the pathogens infect from the stomata, it turns reddish-brown. When the temperature
is relatively high, yellowish bacterial ooze can be seen flowing out of the diseased
leaves. After the flowers are infected, they turn black and wither before maturity. The
pathogens can also infect the roots of the grapes, whether it is grafted plants or plants
on their own rootstocks, they will decelerate the growth of the tip of the branches. The
life cycle of xylophilus ampelinus (panagopoulos) Willems et al. has not been
thoroughly elucidated yet, and the initial infection mainly occurs on 1 ~ 2 year-old
branches, and invades the plants through leaves, flowers and fruits. The pathogens
propagate with plants, especially in humid and windy weather, which makes it easier
for the propagation. Then, in early summer, they spread to other new buds. The
occurrence of the disease requires warm and humid conditions.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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