GB/T 43160-2023Detection and identification of Erwinia amylovora (English PDF)

1 Scope

The document describes methods for the detection of the fire blight bacterium by isolation and culture, by immunological means and by molecular biology.

It applies to the quarantine detection and identification of the fire blight bacterium carried by plants, rootstocks, scions and young fruit of pear, apple and other rosaceous species, and to field survey and monitoring of the disease.

2 Normative references

The document has no normative references.

3 Terms and definitions

No terms or definitions need to be defined for this document.

4 Basic information on the fire blight bacterium

Scientific name: Erwinia amylovora (Burrill, 1883) Winslow et al., 1920.

Routes of spread: diseased plant propagating material, including seedlings, rootstocks and scions, insects, wind, rain, and contaminated packaging materials and means of transport, together with other natural and human factors, can all cause the spread of fire blight. Among these, diseased plant propagating material is the main route of long-distance spread.

Further information on the fire blight bacterium is given in Annex A.

5 Principle of the methods

Immunological detection is carried out on the basis of the specific reaction between the fire blight bacterium and antibodies; molecular biological detection is carried out on the basis of specific DNA sequences of the bacterium; and isolation, culture, identification and pathogenicity determination are carried out on the basis of the cultural characteristics, biological properties, host range and damage symptoms of the pathogen.

6 Instruments, equipment and main reagents

Instruments, equipment and utensils: clean bench, constant-temperature incubator, shaker, ultra-low-temperature freezer, ordinary refrigerator, autoclave, constant-temperature water bath, small centrifuge, high-speed centrifuge, PCR instrument, real-time fluorescence PCR instrument, electrophoresis apparatus, horizontal electrophoresis tank, gel imaging system, Biolog microplates, automatic microbial identification system (Biolog), ice maker, electronic balance, vortex mixer and micro-injectors.

Main reagents: two times concentrated PCR reaction premix and two times concentrated fluorescence PCR reaction premix; buffers and culture media are prepared according to Annex B; unless otherwise specified, all reagents are of analytical grade.

7 Quarantine detection and identification methods

Symptom inspection. Plants are inspected for the typical symptoms of fire blight described in Annex A, early spring being the key monitoring period. The shoots, flowers, fruit and trunk are the main parts to inspect: whether shoots show wilting in the shape of a shepherd's crook and blacken and die; whether flowers and young fruit blacken and die; whether the leaf veins blacken upward from the base of the petiole; and whether the trunk bears canker patches and whether bacterial ooze is present on the diseased tissue. Plants showing typical or suspect symptoms are photographed and recorded, and symptomatic shoots, flower clusters, branches and fruit are collected and sent to the laboratory for detection and identification. On young shoots that do not show typical symptoms, sampling focuses on the tip and the base.

Preparation of samples from symptomatic plants. Samples are tested as soon as possible after collection; if immediate testing is not possible they are kept at 4 °C to 8 °C, if possible for no more than a week, and cross-contamination is avoided during collection, transport and handling. Sample processing uses a general procedure valid for isolation and culture, immunological detection and molecular biological detection: the sample is treated with sterile distilled water or with phosphate buffered saline at pH 7.2 and used directly. Plant parts showing the most typical symptoms and bearing bacterial ooze, such as flowers, shoots, young branches, leaves or fruit, are chosen; tissue is picked at the boundary between diseased and healthy parts, cut into pieces of 0.1 g to 1.0 g, placed in a centrifuge tube or Petri dish containing an appropriate amount of phosphate buffered saline or sterile distilled water, ground or squeezed, and left to stand for at least 5 min to give a sample suspension for the subsequent tests.

Preparation of samples from symptomless plants. Symptomless samples are processed singly or in groups of at most ten, and cross-contamination is avoided during processing and extraction. From the beginning of flowering to the mid-stage of fruit swelling, flower parts, young fruit and young shoot segments are collected into sterile bags or containers; shoots of about 20 cm in length or flower parts at flowering are cut from the plants to be tested. For winter sampling, five to ten flower buds are collected from each plant. In the laboratory, flower parts at flowering, flower stalks and the petiole bases or stem segments of several leaves at the lower part of the shoots are cut from the selected plants; 0.1 g to 1.0 g of plant material is weighed and soaked in the sterile distilled water or phosphate buffer described above to give a sample suspension. Where a sample consists of several plant materials, it can be placed in a sterile conical flask with an appropriate amount of phosphate buffered saline or sterile distilled water and shaken vigorously on a shaker at room temperature for 1 h to give a sample suspension for direct analysis; alternatively the suspension is transferred to a 50 mL centrifuge tube and centrifuged at 12 000 r/min for 20 min, the supernatant is discarded, and 2 mL of sterile distilled water or phosphate buffer is added to resuspend the pellet, giving an enriched sample for the subsequent tests.

Immunological detection. For samples with suspect symptoms, such as fresh leaves and young fruit, a commercial immuno-colloidal gold test strip can be used for on-site preliminary screening. A commercial enzyme-linked immunosorbent assay (ELISA) is used for detection, carried out and interpreted according to the instructions supplied with it. If the result is negative, the sample is judged as not carrying the fire blight bacterium. If the result is positive, isolation, culture, identification and pathogenicity determination are required. Immunological detection may also be omitted and molecular biological detection carried out directly.

Template preparation. For plant samples, after a sample suspension or enriched sample has been prepared as in 7.2, total DNA is extracted directly with a commercial plant genomic DNA extraction kit according to its instructions and kept at minus 20 °C for later use. Suspect strains obtained by isolation and culture are used directly or after lysis, an appropriate amount of bacterial suspension being placed in a 1.5 mL centrifuge tube, held in a boiling water bath for 10 min and centrifuged at 12 000 r/min for 10 min, the supernatant being kept at minus 20 °C as the template for molecular biological detection.

PCR and gel electrophoresis. A standard strain of E. amylovora serves as the positive control, a plant bacterium other than E. amylovora as the negative control, and double distilled water in place of template as the blank control. Two pairs of specific amplification primers targeting the plasmid pEA29 of the fire blight bacterium and its chromosomal genes are used for PCR amplification of the samples under test, followed by gel electrophoresis; the detailed steps follow Annex C.

Real-time fluorescence PCR. The detailed steps follow Annex D, with the same control arrangements as for PCR and gel electrophoresis.

Isolation, culture and identification. After a sample suspension or enriched sample has been prepared as in 7.2, a sterile inoculating loop is used to streak the suspension on LB agar plates in a clean bench, or the suspension is diluted tenfold in series with sterile water and 100 µL of each dilution is spread on plates, each in three replicates. The plates are placed in a constant-temperature incubator at 25 °C for 24 h to 48 h, suspect single colonies are picked and purified, and are subcultured two to three times before pathogenicity determination, Biolog identification, and immunological or molecular biological identification. On LB medium the fire blight bacterium forms milky white colonies that are relatively large and raised, smooth and lens-shaped, whereas the common contaminant Erwinia herbicola forms yellow colonies on LB medium.

Biolog identification. After culture, the isolate is transferred to BUG medium and grown at 30 °C for 24 h; following the Biolog instructions, the turbidity of the isolate suspension is adjusted and 100 µL is added to each well of a Biolog GN microplate or GEN III microplate. The microplates are incubated at 30 °C for 18 h and then read with the Biolog instrument to obtain the test result.

Pathogenicity determination, young fruit inoculation test. Young pear fruit of about 3 cm to 5 cm in diameter are washed and air-dried, cut transversely with a sterile blade and placed on a Petri dish lined with sterile filter paper with a small amount of sterile water. Colonies to be identified, grown on LB medium for 24 h to 36 h, are picked up with an inoculating needle and inoculated into the flesh of the young fruit by pricking, three to four points on one cut face. After inoculation the fruit are kept moist at 26 °C to induce disease and the formation of bacterial ooze, and after 24 h the inoculation sites are examined for necrosis and ooze. Sterile water serves as the control.

Pathogenicity determination, shoot inoculation test. Young current-year shoots of pear trees are cut and placed in a conical flask of tap water; colonies to be identified, grown on LB medium for 24 h to 36 h, are picked up with an inoculating needle and inoculated by wounding at a point 5 cm to 10 cm below the apical growing point; alternatively, after wounding, one drop of 3 µL to 5 µL of a bacterial suspension at a concentration of ten to the seventh power CFU/mL is applied to the wound. After inoculation the shoots are bagged and kept moist at about 25 °C, and after 2 d to 3 d the inoculation points are examined for blackening, tissue shrivelling, shoot wilting and the appearance of bacterial ooze.

8 Interpretation of results

For plant samples with typical symptoms, a positive result from immunological or molecular biological detection is taken as detection of the fire blight bacterium.

For symptomless samples, a positive result from immunological or molecular biological detection gives a preliminary determination that the fire blight bacterium has been detected; if isolation and culture then produce typical colonies and Biolog identification is positive, or pathogenicity determination produces typical symptoms, detection of the fire blight bacterium is confirmed.

Molecular biological detection can be carried out by PCR with gel electrophoresis or by real-time fluorescence PCR according to laboratory conditions, and a positive result from either method makes the molecular biological result positive. Pathogenicity determination can likewise be carried out by the young fruit test or the shoot test according to laboratory conditions, and the appearance of typical symptoms in either test makes the pathogenicity result positive.

9 Preservation of samples and records

Preservation of samples and strains. Samples are properly stored after registration and signature by the person handling them. Samples in which the fire blight bacterium has been detected are kept in a refrigerator at 4 °C for possible re-examination, and after the storage period such samples are autoclaved before further handling. Strains isolated from test samples and identified as the fire blight bacterium are properly preserved, either in glycerol in an ultra-low-temperature freezer or by freeze-drying; strains that do not need long-term preservation are sterilised promptly.

Recording of results and preservation of records. A complete experimental record includes the source and kind of sample, the time, place, method and result of the test, and carries the signatures of the person performing the test and of the reviewer. Suspect symptoms found during field quarantine, colony characteristics on the culture medium and typical symptoms from artificial inoculation are photographed and kept; the enzyme-linked immunosorbent method has to have the raw data of the enzyme reaction, PCR with gel electrophoresis has to have a photograph of the electrophoresis result, and fluorescence PCR has to have the raw data.

Annex A Information related to the fire blight bacterium

Name and taxonomic position. Chinese name: the pear fire blight bacterium. English name as printed: Fire Blight of Pear. Taxonomic position: kingdom Bacteria, phylum Proteobacteria, class Gammaproteobacteria, order Enterobacteriales, family Enterobacteriaceae, genus Erwinia.

Geographical distribution. The annex lists the countries and regions where the organism has been reported, grouped by continent: in Asia, Armenia, Azerbaijan, China (mainland and the Taiwan region), Georgia, India (Maharashtra), Iran, Israel, Japan (Hokkaido and Honshu), Jordan, Kazakhstan, Kyrgyzstan, Lebanon, Saudi Arabia, Syria, Turkey and Viet Nam; in Europe, some thirty-five countries and territories from Albania to the United Kingdom, including the Channel Islands and Northern Ireland; in Africa, Algeria, Egypt, Morocco, Nigeria and Tunisia; in Oceania, Australia and New Zealand; in North America, Bermuda, Canada with its named provinces and territories, Mexico and a long list of states of the United States; in Central America and the Caribbean, Guatemala and Haiti; and in South America, Chile, Colombia and Venezuela.

Host range. The annex gives an extensive host list by genus and species, covering Amelanchier, Amygdalus triloba, Armeniaca dasycarpa, Aronia arbutifolia, Arracacia xanthorrhiza, Aruncus sylvester, Chaenomeles, Cotoneaster with some thirty species, Crataegus, Cydonia oblonga, Dichotomanthus tristaniaecarpa, Diospyros kaki and Diospyros lotus, Docynia delavayi, Eriobotrya japonica, Fragaria, Juglans, Malus with some fifteen entries, Mespilus, Osteomeles anthyllidifolia, Peraphyllum ramosissimum, Photinia, Prunus with some fourteen entries, Pyracantha, Pyrus with some sixteen entries, Raphiolepis, Rosa, Rubus idaeus, Sorbus, Spiraea vanhouttei and Stranvaesia davidiana.

Damage symptoms. The bacterium mainly infects the inflorescences, leaves, branches, young fruit and trunk of the plant; the disease develops rapidly and the damage is heavy. Diseased trees usually begin to show disease on the flowers, leaves and young shoots; brown to black lesions appear on the leaves and black to brown streaks form on the midrib, the lesions can spread to the whole branch, and diseased branches change colour and die within a few days, causing heavy loss of yield.

Blossom blight. The bacterium first infects the flower parts, producing water-soaked spots, and the flower parts soon wilt, blacken and die. The bacterium spreads down the pedicel of the diseased flower to the flower cluster, then to the other flowers or young fruit of the cluster and to the petioles and leaf veins, until the whole leaf blackens and dies, and finally the whole flower cluster dies and blackens. Under humid conditions bacterial ooze exudes from tissues such as the pedicel, the fruit stalk and the petiole.

Shoot and branch blight. The bacterium infects current-year shoots, mainly spring shoots; the point of entry first shows blackening of the bark, and the lesion spreads rapidly upward and downward, quickly causing the shoot to wilt and die in the shape of a shepherd's crook, with the dead leaves remaining attached. Shoot wilting develops very fast and under favourable conditions can extend 15 cm to 30 cm or more within a few days, killing the whole branch. Diseased branches usually blacken and, in humid weather, bacterial ooze appears on the branches and petioles. Sometimes the disease is confined to a cluster bud or a new shoot and a canker patch forms at the junction between the cluster bud or new shoot and the two-year-old branch. Sometimes the bacterium spreads downward along the new shoot as far as the scaffold branch and kills the whole scaffold branch.

Canker patches. The bacterium spreads along the flower cluster or new shoot downward to branches and trunk of the next order; when unfavourable conditions are met the lesion stops spreading, the bark dies and shrinks and becomes sunken while the cambium continues to grow, so that cracks appear at the boundary between diseased and healthy tissue and canker patches of varying size are formed. The necrosis of the canker patches is usually not deep and is confined to the bark. These canker patches can serve as overwintering sites for the bacterium; in the following year, when spring warmth brings flowering, the bacterium starts to multiply and is spread by wind, rain, insects and other vectors, causing new infections.

The damage symptoms of fire blight on pear trees do not differ markedly from those of Asian pear blight; the only symptomatic difference is that in typical fire blight the tissue under the bark of the diseased part is brown, whereas in Asian pear blight it remains green.

Figures A.1 to A.4 show, respectively, blackening of the midrib of a pear leaf, bacterial ooze symptoms on the fruit stalk, drying and withering symptoms of the leaves on a young branch, and wilting and dieback symptoms of a branch.

Annex B Preparation of buffers and culture media

Phosphate buffered saline at pH 7.2: sodium chloride 8.0 g, potassium chloride 0.2 g, disodium hydrogen phosphate dodecahydrate 2.9 g, potassium dihydrogen phosphate 0.2 g, distilled water added to 1.0 L, stirred and mixed until dissolved.

LB medium: tryptone 10.0 g, yeast extract 5.0 g, sodium chloride 10.0 g, agar 15.0 g, distilled water added to 1.0 L, pH adjusted to 7.5, sterilised by moist heat at 121 °C for 20 min.

BUG agar medium: BUG agar medium 57.0 g, distilled water added to 1.0 L, pH adjusted to 7.4, sterilised by moist heat at 121 °C for 20 min.