Separation and identification method of German iris soft rot pathogenic bacteria

Abstract

The invention discloses a separation and identification method of German iris soft rot pathogenic bacteria, and belongs to the technical field of plant pathogenic bacteria detection. The method comprises the following steps: collecting diseased plants which are poor in growth and have typical soft rot symptoms from an iris tectorum planting base as materials in a German iris tectorum soft rot full-emergence period, and carrying out separation and purification of pathogenic bacteria and PCR amplification and identification of the pathogenic bacteria; according to the method, the German iris soft rot pathogenic bacteria are identified by utilizing a molecular biology means for the first time, compared with a traditional separation and identification method, the identification time of the pathogenic bacteria is greatly shortened, the accuracy of an identification result is also greatly improved, disease-resistant breeding and comprehensive prevention and control of German iris are promoted, and the method has a wide application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of plant pathogen detection, and in particular to a method for separating and identifying pathogens of German iris soft rot. Background Art

[0002] Iris Iris germanica L.) is a perennial herbaceous plant of the genus Iris, belonging to the rhizomatous iris. It has peculiar flower shapes, rich colors, fragrant flowers, and is evergreen all year round. It has broad application prospects in garden planting, ground cover greening, and potted flower viewing. Iris is susceptible to various pathogens during cultivation, which seriously affects its quality and ornamental value. Common diseases of irises include black spot, root rot and soft rot, and soft rot is the main disease of rhizomatous irises such as German irises. German iris soft rot is a devastating bacterial disease. It develops and spreads fastest during the rainy season. In severe cases, the susceptibility rate of varieties with weak resistance to soft rot is as high as 100%, and once infected, the mortality rate exceeds 80%. Soft rot has become an important bottleneck restricting the industrialization development of German irises in the middle and lower reaches of the Yangtze River in my country.

[0003] Symptoms of German iris soft rot usually first appear at the base of new leaves of the plant, which are water-soaked and translucent. Since all leaves of German iris are basal, once the plant is infected, the symptoms spread from the new leaves at the base to the old leaves, and also spread rapidly from the infected area to the surrounding plant stem tissues. In severe cases, the whole plant will experience tissue infiltration and soft rot from the base upward within a few days, and eventually fall over and die. Soft rot is very harmful to German iris. After infection, the incidence rate is high, the mortality rate is high, and it is highly contagious. It often breaks out over a large area and in patches, and can even cause the species or population to be on the verge of extinction.

[0004] In 1997, Horita et al. first reported that the pathogen causing rhizome disease of German iris in Hokkaido, Japan was Erwinia spp. Erwinia carotovora subsp. Carotovora). In 2006, Han Yulin isolated two pathogenic bacteria from plants infected with soft rot of German iris cultivar. Through morphological observation and physiological and biochemical reactions, the study showed that the pathogen was different from that reported by Horita et al. and was another pathogenic species of Erwinia. According to Bergey's Bacterial Identification Manual, it was finally identified as Erwinia chrysanthemi ( Erwinia chrysanthemi Burkhdder, McFadden & Dimock) soft rot pathogenic type. Due to the complexity of the pathogens that cause soft rot, relying on traditional morphological characteristics for identification is not only time-consuming and laborious, but also has certain inaccuracies. Molecular biological identification can greatly improve the accuracy and efficiency of identification. However, there is no report on the method of using molecular biological methods to detect the pathogens of German iris soft rot. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for separating and identifying the pathogenic bacteria of German iris soft rot.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is: Provided is a method for isolating and identifying the pathogen of German iris soft rot, comprising the following steps: (1) Collection of diseased samples: During the peak period of soft rot in German iris during the summer rainy season, we selected diseased fields with severe soft rot from the Iris Germplasm Resource Garden of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province, and took diseased plants with typical symptoms of soft rot, placed them in fresh-keeping bags, and brought them back to the laboratory for later use; (2) Isolation of pathogens: Take the diseased leaves of German iris, rinse the surface soil with running water, sterilize the surface, and then dry the water with sterile filter paper. Cut the tissue at the junction of the diseased and healthy leaves and grind it into a homogenate in a sterile mortar. Dilute the leaf homogenate 20 times and then spread it on LB solid culture medium. Finally, place the plate in a constant temperature incubator for incubation; (3) Purification of pathogens: After culturing the LB plate in a constant temperature incubator for 24 h, observe the growth of the colonies and pick a single colony to transfer to a new LB medium for purification. After 5-6 transfers, a pure strain is obtained and stored in 20% glycerol for later use. (4) Extraction of genomic DNA of pathogens: scrape the purified pathogen colonies into a 2 mL centrifuge tube, add an appropriate amount of vibration beads, use a homogenizer to fully break up the pathogen colonies, and use the CTAB plant DNA extraction kit to extract the strain genomic DNA; (5) PCR amplification and identification of pathogens: Using pathogen genomic DNA as a template, PCR amplification was performed using bacterial 16s universal primers 27F / 1492R and specific primers gapA326F / gapA845R. The amplified products were sequenced after 1.5% agarose gel electrophoresis to obtain the pathogen sequence. The sequencing results were subjected to BLAST comparison analysis in GenBank, and then the phylogenetic tree was constructed using MEGA software to identify the species of the pathogen.

[0007] Furthermore, in step (2), the plate is placed upside down in the constant temperature incubator.

[0008] Furthermore, in step (2), the surface sterilization method is: soaking in 75% alcohol for 10 seconds, then treating with 1% sodium hypochlorite solution for 3 minutes, and finally taking out and rinsing with sterile water for 4-6 times.

[0009] Furthermore, in step (3), the temperature of the constant temperature incubator is 30°C, the culture condition is dark culture, and the storage temperature is -20°C.

[0010] Furthermore, in step (5), the primer sequence of 27F is 5'-AGTTTGATCMTGGCTCAG-3', the primer sequence of 1492R is 5'-GGTTACCTTGTTACGACTT-3'; the primer sequence of gapA326F is 5'-ATCTTCCTGACCGACGAAACTGC-3', and the primer sequence of gapA845R is 5'-ACGTCATCTTCGGTGTAACCCAG-3'.

[0011] Furthermore, in step (5), the PCR reaction system is 50 μL: 2 μL of DNA template, 2 μL of each of 10 mmol / L upstream and downstream primers, 45 μL of 1×Taq PCR Master Mix (containing dye), and the PCR amplification program is: 98°C pre-denaturation for 2 min, amplification cycle including 98°C denaturation for 10 s, 56°C annealing for 10 s, 72°C extension for 30 s, 32 amplification cycles, and finally 72°C extension for 5 min.

[0012] Furthermore, in step (5), the identified pathogen is Erwinia persica ( Erwinia persicina ).

[0013] The beneficial effects of the present invention are: 1. The present invention, through separation, cultivation and screening, finally identified a new pathogenic bacteria of German iris soft rot as Erwinia persica ( Erwinia persicina ); 2. The present invention realizes the rapid separation and accurate identification of the pathogen of German iris soft rot for the first time through PCR amplification and molecular sequencing, as well as BLAST comparison analysis of the sequencing results. Compared with traditional separation and identification methods, the method is more efficient and the identification results are more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure is a morphological diagram of the pathogen separated and purified in the present invention on LB medium.

[0015] Figure 2 This is a gel image of the colony PCR amplification product of the pathogen isolated in the present invention, wherein M: Marker; 1: 16s; 2: gap.

[0016] Figure 3 This is the phylogenetic tree of the pathogen of German iris soft rot constructed in the present invention. DETAILED DESCRIPTION

[0017] The specific implementation of the present invention is further described in detail below, but the examples of the present invention are not limited to the scope represented by the examples. The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention. Example

[0018] A method for isolating and identifying the pathogen of German iris soft rot, comprising the following steps: 1. Sample collection: During the peak period of soft rot in German iris during the summer rainy season, we selected fields with severe soft rot from the Iris Germplasm Resource Garden of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province, and took 3-5 diseased plants with typical symptoms of soft rot, placed them in fresh-keeping bags, and brought them back to the laboratory for later use; 2. Isolation of pathogens: Take the diseased leaves of German iris, rinse the surface soil with running water, place them on a clean bench for surface sterilization (soak in 75% alcohol for 1 min, then treat with 1% sodium hypochlorite solution for 3 min, and finally take them out and rinse them with sterile water for 4-6 times), then use sterile filter paper to absorb the water, cut the tissue at the junction of the diseased and healthy parts and grind them into a homogenate in a sterile mortar, dilute the leaf homogenate 20 times, then spread it on LB solid culture medium, and finally invert it in a constant temperature incubator for culture; culture conditions: 30℃, dark culture; 3. Purification of pathogens: After 24 h of LB plate culture in a constant temperature incubator, observe the growth of the colonies and pick a single colony to transfer to a new LB medium for purification. After 5-6 transfer cultures, a pure strain is obtained. The strain appears as a round, smooth, grayish-white bulge on the LB medium ( Figure 1 ), and finally the strain was stored in 20% glycerol for later use; 4. Extraction of genomic DNA of pathogens: scrape the purified pathogen colonies into a 2 mL centrifuge tube, add an appropriate amount of vibration beads, use a homogenizer to fully break up the pathogen colonies, and use the CTAB plant DNA extraction kit (Beijing Qingke Biotechnology Co., Ltd.) to extract the strain genomic DNA; 5. PCR amplification and identification of pathogens: Using the genomic DNA of pathogens as template, PCR amplification was performed using two primers: 27F / 1492R and gapA326F / gapA845R. The amplified products were detected by 1.5% agarose gel electrophoresis ( Figure 2 ), and commissioned Qingke Biotechnology (Nanjing) Co., Ltd. to sequence. The sequencing results were compared with the NCBI database by BLAST, and sequences were selected from GenBank to construct a phylogenetic tree using MEGA software ( Figure 3 ), identify the species of pathogens.

[0019] The sequence of the primer 27F is 5'-AGTTTGATCMTGGCTCAG-3', the sequence of the primer 1492R is 5'-GGTTACCTTGTTACGACTT-3'; the sequence of the primer gapA326F is 5'-ATCTTCCTGACCGACGAAACTGC-3', and the sequence of the primer gapA845R is 5'-ACGTCATCTTCGGTGTAACCCAG-3'.

[0020] The reaction system for PCR identification was 50 μL: 2 μL DNA template, 2 μL each of 10 mmol / L upstream and downstream primers, 45 μL 1× TaqPCR Master Mix (containing dye), and PCR amplification program: 98°C pre-denaturation for 2 min, amplification cycle including 98°C denaturation for 10 s, 56°C annealing for 10 s, and 72°C extension for 30 s, 32 cycles of amplification, and finally extension at 72°C for 5 min.

[0021] Finally, it was determined that the pathogen of German iris soft rot isolated and purified in the present invention was Erwinia persica ( Erwinia persicina ).

Claims

1. A method for isolating and identifying the pathogen of German iris soft rot, characterized in that: The steps include: (1) Collection of diseased samples: During the peak period of soft rot in German iris during the summer rainy season, we selected diseased fields with severe soft rot from the Iris Germplasm Resource Garden of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province, and took diseased plants with typical symptoms of soft rot, placed them in fresh-keeping bags, and brought them back to the laboratory for later use; (2) Isolation of pathogens: Take the diseased leaves of German iris, rinse the surface soil with running water, sterilize the surface, and then dry the water with sterile filter paper. Cut the tissue at the junction of the diseased and healthy leaves and grind it into a homogenate in a sterile mortar. Dilute the leaf homogenate 20 times and then spread it on LB solid culture medium. Finally, place the plate in a constant temperature incubator for incubation; (3) Purification of pathogens: After culturing the LB plate in a constant temperature incubator for 24 h, observe the growth of the colonies and pick a single colony to transfer to a new LB medium for purification. After 5-6 transfers, a pure strain is obtained and stored in 20% glycerol for later use. (4) Extraction of genomic DNA of pathogens: scrape the purified pathogen colonies into a 2 mL centrifuge tube, add an appropriate amount of vibration beads, use a homogenizer to fully break up the pathogen colonies, and use the CTAB plant DNA extraction kit to extract the strain genomic DNA; (5) PCR amplification and identification of pathogens: Using pathogen genomic DNA as a template, PCR amplification was performed using bacterial 16s universal primers 27F / 1492R and specific primers gapA326F / gapA845R. The amplified products were sequenced after 1.5% agarose gel electrophoresis to obtain the pathogen sequence. The sequencing results were subjected to BLAST comparison analysis in GenBank, and then the phylogenetic tree was constructed using MEGA software to identify the species of the pathogen.

2. The method for isolating and identifying the pathogen of German iris soft rot according to claim 1, characterized in that: The surface sterilization method in step (2) is: soak in 75% alcohol for 10 seconds, then treat with 1% sodium hypochlorite solution for 3 minutes, and finally take out and rinse with sterile water for 4-6 times.

3. The method for isolating and identifying the pathogen of German iris soft rot according to claim 1, characterized in that: The LB plate in step (2) is placed upside down in the constant temperature incubator.

4. The method for isolating and identifying the pathogen of German iris soft rot according to claim 1, characterized in that: The temperature of the constant temperature incubator in step (3) is 30°C, and the culture condition is dark culture; the storage temperature is -20°C.

5. The method for isolating and identifying the pathogen of German iris soft rot according to claim 1, characterized in that: In step (5), the primer sequence of 27F is 5'-AGTTTGATCMTGGCTCAG-3', and the primer sequence of 1492R is 5'-GGTTACCTTGTTACGACTT-3'; the primer sequence of gapA326F is 5'-ATCTTCCTGACCGACGAAACTGC-3', and the primer sequence of gapA845R is 5'-ACGTCATCTTCGGTGTAACCCAG-3'.

6. The method for isolating and identifying the pathogen of German iris soft rot according to claim 1, characterized in that: The PCR reaction system in step (5) is 50 μL: 2 μL of DNA template, 2 μL of each of 10 mmol / L upstream and downstream primers, 45 μL of 1× TaqPCR Master Mix (containing dye), and the PCR amplification program is as follows: pre-denaturation at 98°C for 2 min, amplification cycle including denaturation at 98°C for 10 s, annealing at 56°C for 10 s, and extension at 72°C for 30 s, amplification for 32 cycles, and finally extension at 72°C for 5 min.

7. The method for isolating and identifying the pathogen of German iris soft rot according to claim 1, characterized in that: The pathogen identified in step (5) is Erwinia persica ( Erwinia persicina ).

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