A Pathogen of Pinellia tuber rot and Its Application

Abstract

The invention discloses a strain of a pathogenic bacterium of tuber rot of pinellia and its application, and belongs to the field of microbial technology. Serratia marcescens is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, Beijing, with a deposit date of January 12, 2024, and a deposit number of CGMCC NO.29585. Application of a strain of Serratia marcescens, a pathogenic bacterium of tuber rot of pinellia, in screening disease-resistant strains of pinellia. The invention mainly solves the problem of pathogens of tuber rot of pinellia and screening of disease-resistant strains in disease-resistant breeding of pinellia, and provides a new pathogen and screening method for disease-resistant strains for breeding of tuber rot resistance of pinellia. The invention discovers a new pathogen of tuber rot of pinellia - Serratia marcescens, and establishes a method for identifying tuber rot resistance of pinellia breeding strains using the pathogen of tuber rot of pinellia - Serratia marcescens.

Description

Technical Field

[0001] The invention relates to a pinellia tuber rot pathogen and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Pinellia ternate (Thunb.) Breit. It is a plant of the genus Pinellia of the Araceae family. It is a medicinal underground tuber and a traditional Chinese medicinal material. It has the effects of relieving cough and expectorant, relieving adverse reactions and stopping vomiting, eliminating lumps and dispersing nodules, anti-arrhythmic, lowering blood lipids, anti-tumor, anti-early pregnancy, sedative and hypnotic, detoxification and anti-inflammatory, and lowering blood pressure. The market demand for pinellia is huge, with an average annual demand of more than 4,000 tons. Artificial cultivation of pinellia has become the main source of this medicinal material, but the cultivation of pinellia is seriously affected by tuber rot, with an incidence rate of up to 15% to 30%. It is the main disease in the pinellia production area and also the most important disease. In the affected plots, 80% of the mild ones rot in one week, and in the seriously affected plots, all rot in 3 to 5 days, with a yield loss of up to 30% or even a total crop failure, causing significant losses to the production of pinellia. Traditional prevention and control measures for this disease include crop rotation and chemical pesticides, but the effect is poor and it is difficult to control. Tuber rot has seriously restricted the development of the pinellia planting industry and has become a bottleneck in the cultivation and production of pinellia. Conducting disease-resistant breeding research and providing rot-resistant varieties for pinellia production is an economical and effective way to solve the harm of tuber rot in pinellia. The discovery of pathogens can provide breeding targets for disease-resistant breeding. The present invention provides a new pathogen for the breeding of tuber rot resistance in Pinellia ternata and provides a technical method for the screening of tuber rot resistance strains in disease-resistant breeding of Pinellia ternata.

[0003] The most similar technical solution: In the paper published by Shi Jianlong, "[1] Shi Jianlong, Li Yuquan, Hu Kunmin, et al. Isolation and identification of the pathogenic bacteria of tuber rot of Pinellia ternata in Guizhou [J]. Bulletin of Microbiology, 2015, 42(02): 289-299. DOI: 10.13344 / j.microbiol.china.140391.", 12 pathogenic bacteria and 5 pathogenic fungi that can cause tuber rot of Pinellia ternata were isolated, namely, Pectinobacterium carotovora, Fusarium oxysporum and Fusarium solani. Shi Jianlong isolated the pathogenic bacteria of tuber rot of Pinellia ternata by tissue separation method, and determined the pathogenicity according to Koch's postulate, and identified the pathogenic bacteria of Pinellia ternata by combining morphological, physiological and biochemical characteristics and molecular biological methods. However, the experiment did not isolate the pathogen used in the present invention, and did not conduct pathogenicity testing on the selected pathogens, nor did it use the pathogens to screen the disease resistance of Pinellia ternata breeding lines.

[0004] Among the currently known diseases of tuber rot in Pinellia ternata, the pathogens isolated and screened are mostly Fusarium oxysporum and Fusarium solani in the fungi, and Pectobacterium carotovora and Erwinia in the bacteria. There are almost no related reports on other genera, as described in paper [2] "Li Weizhong, Cao Hailu, Liang Chunbo, Tan Longyan. Research progress on tuber rot in Pinellia ternata [J]. Tropical Agricultural Sciences, 2024, 44(01): 135-139." After screening the pathogenic fungus Fusarium oxysporum, morphological observations and pathogenicity tests were carried out, but its pathogenicity was not tested, as described in paper [3] "Sun Xinrong , Hu Liping, Liu Yanmei, et al. Identification of the pathogen of tuber rot of Pinellia ternata and comparison of drug efficacy [J]. Chinese Journal of Traditional Chinese Medicine, 2010, 35(07): 837-841. "In addition, Wang Hailing et al. [4] "Wang Hailing, Liu Hongmei, Ruan Peijun, et al. Effects of tuber rot pathogens on different populations of Pinellia ternata [J]. Lishizhen Traditional Chinese Medicine, 2016, 27(05): 1222-1226.", 16 disease-resistant Pinellia ternata germplasm resources were screened out through potted plant experiments, and the pathogens used were respectively Pectinobacterium carotovora, Fusarium oxysporum and Fusarium solani, which did not include the pathogens used in the present invention.

[0005] Pinellia is widely cultivated. As described in the paper [5] "Wang Jialu, Wang Yu, Mou Lan, Hou Mengmeng, Li Xiwen. Analysis of global ecological suitability and quality ecology of Pinellia [J]. Modern Chinese Medicine, 2021, 23(11): 1864-1868", Pinellia has high economic value. However, in the process of planting Pinellia, it was found that tuber rot disease is a serious hazard, which can cause tuber rot and a large number of seedlings to die, causing serious losses to Pinellia production. Compared with other regions, the tuber rot disease of Pinellia in Hebei is particularly serious, as described in the paper [6] "Zhang Xuehui. Occurrence and prevention of root rot of Pinellia [J]. Modern Horticulture, 2010(02): 41-42" and the paper [7] "Wang Caiyun, Hou Jun, Wang Yong, Cha Qin, Zhang Xiangyu, Ruan Peijun. Screening of high-quality Pinellia provenance in Northwest Guizhou [J]. Northern Horticulture, 2019(07): 127-132".Among the pathogens of tuber rot of Pinellia ternata isolated by Shi Jianlong, the pathogen is Pectobacterium carotovora, as described in [8] Shi Jianlong. Isolation and identification of pathogens of tuber rot of Pinellia ternata in Guizhou and screening of antagonistic bacteria [D]. Guiyang Medical College, 2015. This is consistent with the pathogens isolated by He Yubo et al. [9] He Yubo, Hu Xiufang, Chen Haimin, Chen Jishuang, Gao Bi Da, Ying Feixiang. Isolation and identification of bacterial soft rot pathogens of Pinellia ternata [J]. Acta Phytopathologica Sinica, 2007, (04): 337-342. It is consistent with the paper published by Wang Fanfan et al.

[10] Wang Fanfan, Tang Tao, Mao Ting et al. Identification, biological characteristics and control of pathogens of Pinellia ternata soft rot in Hubei Province [J]. China Journal of Traditional Chinese Medicine, 2022, 47(04): 889-896. The pathogen isolated is Dickeya Fangzhonggdai)", and the paper published by Yao Tianming et al.

[11] "Yao Tianming, Fu Jianzeng, Nan Jianjun, et al. Study on Pinellia rot caused by combined infection of Fusarium oxysporum and Erwinia [J]. Gansu Agricultural Science and Technology, 2020, (07): 54-58." The Erwinia found is not a species of bacteria, but the pathogenic bacteria isolated above are not the Serratia marcescens found in the present invention, and are not pathogens found in the Hebei production area; and in the above experiments, only Koch's postulates were used to identify the pathogens. In the previous study, the pathogens were screened, but the pathogenicity of the selected pathogens was not determined, and the degree of pathogenicity of the pathogens was unclear. In addition, Wang Hailing et al. screened the disease resistance of different populations of Pinellia ternata, as described in the paper [4] "Wang Hailing, Liu Hongmei, Ruan Peijun et al. Effects of tuber rot pathogens on different populations of Pinellia ternata [J]. Lishizhen Traditional Chinese Medicine, 2016, 27(05): 1222-1226." However, the strains used were Pectobacterium carotovorum subsp. Pectcarotovorum; the fungus Fusarium solani and the fungus Fusarium oxysporum, which are different from those in this study.

[0006] At present, there is no effective means of preventing and treating the tuber rot of Pinellia ternata. The selection of disease-resistant Pinellia ternata varieties is an effective and most economical prevention and treatment method. Therefore, the breeding of disease-resistant Pinellia ternata varieties has become an important problem that needs to be solved in the current production of Pinellia ternata. In view of this, the purpose of the present invention is to separate the pathogenic bacteria of the tuber rot of Pinellia ternata in Hebei Province, and apply it to the disease resistance screening of Pinellia ternata breeding lines, so as to provide improved varieties for field production, improve the yield and quality of Pinellia ternata produced in the field, and realize the green production of Pinellia ternata cultivation. Summary of the invention

[0007] In view of the above-mentioned problems such as the serious tuber rot of Pinellia ternata, the purpose of the present invention is to provide a new pathogen, and the present invention provides a new pathogen for the breeding of Pinellia ternata resistant to tuber rot.

[0008] At the same time, the invention provides a method for screening a new pathogenic bacteria.

[0009] At the same time, the invention provides a new method for screening disease-resistant strains of Pinellia ternata.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0011] A strain of Serratia marcescens, the pathogen of tuber rot of Pinellia ternata, was deposited in the General Microbiology Center of China Culture Collection Administration, Beijing, with a deposit date of January 12, 2024 and a deposit number of CGMCC NO.29585.

[0012] A method for screening a strain of Serratia marcescens, a pathogen of tuber rot of pinellia, comprises the following steps:

[0013] S01, collecting rotten pinellia tubers and performing surface disinfection to obtain disinfected rotten pinellia tubers;

[0014] S02, isolation, purification and preservation of pathogenic bacteria: Use a sterilized surgical blade to cut the diseased and healthy junctions of the disinfected rotten Pinellia tubers into pieces, put them into a sterilized mortar, add 5 mL of physiological saline, and grind them thoroughly to obtain a pathogenic bacteria sample homogenate; dilute the homogenate with physiological saline to 10 times, 20 times, and 40 times; use the partitioning and streaking method to streak the homogenate of different dilution multiples on the surface of the KB culture medium using an inoculation loop, set 3 replicates for each gradient, and culture in a constant temperature incubator at 25°C;

[0015] After culturing in a constant temperature incubator for 2 to 3 days, a single colony was picked up with an inoculation needle and purified on a new NA medium, and then streaked and isolated and cultured, and repeated several times until a single colony was obtained; a total of 60 bacterial strains were isolated; the purified strains were inoculated into LB liquid culture medium with an inoculation needle, and after culturing for 24 hours in a shaker at 25°C and 180 r / min, 500 μL of bacterial suspension was taken out with a pipette and mixed with 500 μL of sterilized glycerol, and evenly stored in a cryopreservation tube, which was numbered and stored in a -80°C ultra-low temperature refrigerator;

[0016] S03, Pathogen Screening:

[0017] Preparation of bacterial culture: The stored bacteria were placed on NA medium for activation, and a single colony was picked and inoculated into NB liquid culture medium for culture. The culture was cultured in a shaker at 25°C and 180 r / min for 48 h, and the bacterial culture was diluted with sterile water to 1×10 8 cfu / mL bacterial suspension, for later use;

[0018] Treatment of Pinellia plants: First, sterilize the breeding Pinellia tubers, then sterilize the flower pots and soil; finally, plant the sterilized Pinellia tubers in flower pots filled with sterilized soil and transfer them to an indoor environment with a temperature of 35°C, a relative humidity of 75%, and a light period of 16h / darkness of 8h;

[0019] Inoculation of Pinellia ternata plants: When the Pinellia ternata plants grow to the 6th week and the three Pinellia ternata leaves are fully unfolded, pierce the stem of Pinellia ternata with an inoculation needle at 1.0-2.0 mm from the ground and inoculate the prepared 1×10 8 30 mL of bacterial suspension with cfu / mL was inoculated into potted Pinellia ternata; as a control, sterile water was inoculated into potted Pinellia ternata in the same way; 5 plants were placed in each pot, and each treatment was repeated three times;

[0020] 48 hours after inoculation with the bacterial suspension of Pt-43, the pinellia plants rotted and a foul odor was produced; thus, a pinellia tuber rot pathogen was obtained, which was Serratia marcescens Pt-43.

[0021] Preferably, in S01, the collected rotten pinellia tuber samples are wrapped with sterile gauze, rinsed under tap water, the surface soil is removed, and the moisture is absorbed with sterile filter paper; the rotten pinellia tuber samples are disinfected in an ultra-clean workbench and the surface is sterilized; first, the surface is disinfected with 70% ethanol solution for 30 seconds and washed with sterile distilled water for 3 times; secondly, the rotten pinellia tubers are completely immersed in 1% sodium hypochlorite solution for 3 to 5 minutes, stirred with sterile tweezers during the immersion process to ensure that the tubers are in full contact with the disinfectant, and washed with sterile distilled water until the sterile distilled water after washing is clear; the washed rotten tubers are placed on sterile filter paper to absorb the moisture and set aside.

[0022] Preferably, in S03, the method for sterilizing the pinellia tubers for breeding is: disinfecting with 70% ethanol solution for 30 to 60 seconds, and washing with sterile water for 3 times; secondly, soaking the pinellia tubers in 1% sodium hypochlorite solution for 5 minutes, and washing with sterile water until the sterile water is clear.

[0023] Preferably, in S03, the method for sterilizing the flower pots and soil is: sterilizing the flower pots and soil under high temperature and high pressure conditions of 121° C. for 20 minutes.

[0024] A method for screening disease-resistant strains of pinellia ternata comprises the following steps: planting different strains of pinellia ternata in an experimental field, and after the pinellia ternata plants have grown for 2 months and three leaves of the pinellia ternata planted in the experimental field are fully unfolded, before preparing to inoculate bacterial solution, lightly sweeping the pinellia ternata leaves with a bamboo broom to cause slight wounds on the pinellia ternata leaves, and then inoculating 1×10 8 ~1×10 9 cfu / mL was inoculated into the test field, 10L was inoculated per mu, and it was sprayed evenly with a common sprayer. The mortality rate of Pinellia ternata was counted 48 hours after spraying, and the death of the aboveground parts was taken as the death standard of Pinellia ternata plants. The varieties with a mortality rate of <5% were regarded as disease-resistant varieties of Pinellia ternata.

[0025] Mortality rate = number of deaths / total number of Pinellia ternata × 100%.

[0026] Application of a strain of Serratia marcescens, the pathogen of tuber rot of Pinellia ternata, in screening disease-resistant strains of Pinellia ternata.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The invention mainly solves the problem of pathogenic bacteria of tuber rot of pinellia and screening of disease-resistant strains in disease-resistant breeding of pinellia, and provides a new pathogenic bacteria and screening method of disease-resistant strains for breeding of tuber rot-resistant pinellia.

[0029] The invention discovers a new pinellia tuber rot pathogen, Serratia marcescens, and determines the identification sequence of the bacteria; the invention also establishes a method for identifying the resistance of pinellia tuber rot to the pinellia breeding lines by using the pinellia tuber rot pathogen, Serratia marcescens.

[0030] The present invention determines the pathogenicity of the pathogenic bacteria of the tuber rot of Pinellia ternata and finds that the pathogenic bacteria concentration is 1×10 8 ~1×10 9 cfu / mL, the pathogenicity of the pathogen is strong; for potted experiments, the concentration of the pathogen fermentation liquid was 1×10 8 ~1×10 9 cfu / mL, the Pinellia plants were wounded and then the bacterial solution was applied. The disease-resistant strains of Pinellia were screened according to the mortality rate. For the field experiment, the concentration of the pathogen fermentation liquid was 1×10 8 ~1×10 9 cfu / mL, use a bamboo broom to gently sweep the leaves of Pinellia once to cause a slight wound on the Pinellia, then apply the bacterial solution, and screen the disease-resistant strains of Pinellia based on the mortality rate.

[0031] The invention provides a new pinellia tuber rot pathogen - Serratia marcescens, which is found in pinellia for the first time; the invention provides a method for identifying a pinellia tuber rot-resistant breeding strain using the pathogen, the method is convenient and quick, and can be used for pinellia tuber rot-resistant breeding. There is no report on using the pathogen for pinellia tuber rot-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 For rotten Pinellia plants;

[0033] Figure 2 This is a sample of rotten Pinellia tuber;

[0034] Figure 3 Bacterial colonies (partial) isolated and purified from rotten Pinellia tubers;

[0035] Figure 4 The disease index of Pinellia ternata after being treated with different concentrations of pathogenic bacteria Pt-43;

[0036] Figure 5 The colony morphology and microscopic observation of pathogenic bacteria Pt-43;

[0037] Figure 6 The phylogenetic tree constructed for the pathogen Pt-43 based on 16s rRNA sequence and gyrB sequence;

[0038] Figure 7 This is the growth curve of the pathogen Pt-43 strain at different temperatures;

[0039] Figure 8 This is the growth curve of the pathogen Pt-43 strain under different NaCl concentrations;

[0040] Fig. 9 It is a test for the phosphorus solubilization of the pathogenic bacteria Pt-43 strain;

[0041] Fig.10 This is an experiment on the production of iron carriers by the pathogenic bacteria Pt-43 strain (a control group; b experimental group). DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0043] Glossary of technical terms related to the present invention:

[0044] 1) Pathogen: A microorganism that causes disease, also known as pathogenic microorganism, refers to a microorganism that can invade the host and cause infection, including bacteria, fungi, viruses, etc. They can produce pathogenic substances and cause host infection.

[0045] 2) NA medium: beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, agar 15-20 g / L, water 1000 mL, pH 7.4±0.2.

[0046] 3) NB liquid culture medium: beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, water 1000 mL, pH 7.4±0.2.

[0047] 4) Phylogenetic evolutionary tree: It is also generally called phylogenetic tree or evolutionary tree. It can use tree-like branching diagrams to represent the relationship between species or genes.

[0048] 5) Disease index: It is a comprehensive indicator that takes into account the incidence and severity. When the severity is expressed by graded representative values, the disease index = 100×∑(number of diseased leaves at each level×representative value at each level) / (total number of leaves surveyed×highest representative value).

[0049] 6) Pathogenicity: the ability of a pathogen to cause disease in the host.

[0050] 7) 16S rRNA: 16S rRNA is a subunit of ribosomal RNA, and 16S rDNA is the gene encoding this subunit. 16S rDNA is the DNA sequence corresponding to the rRNA encoded on the bacterial chromosome and exists in all bacterial chromosomal genes.

[0051] 8) gyrB: The gyrB gene is a gene commonly found in bacteria that encodes the B subunit protein of DNA gyrase.

[0052] 9) KB medium: King's medium (KB): 20.0 g of peptone, 10.0 mL of glycerol, 1.5 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, and 16.0 g of agar were added to a conical flask containing 1000 mL of distilled water and the pH was adjusted to 7.2-7.4.

[0053] 10) LB liquid medium: Broth solid medium (LB): 5.0 g of yeast extract, 10.0 g of peptone, 5.0 g of sodium chloride, and 16.0 g of agar were added to a conical flask containing 1000 mL of distilled water and the pH was adjusted to 7.4-7.6.

[0054] Example 1

[0055] 1 Experimental Materials

[0056] The experimental samples were collected on September 11, 2021 in Shifu Town (38°40′56.8”, 115°45′22.2”), Chengnan Village (38°73′78.16”, 114°59′05.12”) and Zhangdongzhuang (38°42′48.28”, 115°33′34.08”) in Anguo City, Hebei Province. Associate Professor Fang Huiyong and Professor Zhao Yunsheng of Hebei University of Traditional Chinese Medicine identified them as tubers of Pinellia ternata. The Pinellia plants collected in this experiment were water-soaked and rotten, and the underground tubers were partially rotten, accompanied by a foul odor, such as Figure 1 Use a sterile shovel to dig underground tubers and put them into sterile bags, then take them to the laboratory. The collected rotten tubers are briefly cleaned and stored in an ultra-low temperature refrigerator until the next step. The sample collection information is as follows Figure 2 shown.

[0057] The experimental samples consisted of 34 Pinellia ternata breeding lines obtained by mixed selection, named P1-P34 respectively, and healthy Pinellia ternata tubers with uniform size and diameter not exceeding 1.0 cm were selected as samples.

[0058] 2 Experimental methods

[0059] 2.1 Surface disinfection of rotten pinellia tubers

[0060] Wrap the collected rotten pinellia tuber samples with sterile gauze, rinse them under running water, remove the surface soil, and dry them with sterile filter paper. Disinfect the rotten pinellia tuber samples in a clean bench and sterilize the surface. First, disinfect the surface with 70% ethanol solution for 30 seconds and wash with sterile distilled water three times; secondly, immerse the rotten pinellia tubers completely in 1% sodium hypochlorite solution for 3 to 5 minutes, stir with sterile tweezers during the immersion process to ensure that the tubers are in full contact with the disinfectant, and wash with sterile distilled water until the sterile distilled water after washing is clear. Place the washed rotten tubers on sterile filter paper to dry the water and set aside.

[0061] 2.2 Isolation, purification and preservation of pathogens

[0062] Use a sterilized surgical blade to cut the diseased and healthy junctions of the disinfected rotten tubers into small pieces, put them into a sterilized mortar, add 5 mL of physiological saline, and grind them thoroughly to obtain a pathogenic bacteria sample homogenate. Use physiological saline to dilute the homogenate in a gradient manner to 10 times, 20 times, and 40 times. Use the partition streaking method to use an inoculation loop to streak the homogenate of different dilution multiples onto the surface of the KB culture medium. Set 3 replicates for each gradient and place it in a constant temperature incubator at 25°C for culture.

[0063] After culturing in a constant temperature incubator for 2 to 3 days, check the growth morphology of the strain on the culture medium. According to the differences in colony size, color, edge smoothness, glossiness, morphology, etc., use an inoculation needle to pick up a single colony and purify it on a new NA culture medium, then streak and separate the culture, and repeat several times until a single colony is obtained. The purified strain is inoculated into LB liquid culture medium with an inoculation needle. After culturing for 24 hours in a shaker at 25°C and 180r / min, use a pipette to draw 500μL of bacterial suspension and 500μL of sterilized glycerol, mix them evenly and store them in a cryopreservation tube. After writing the number, place it in an ultra-low temperature refrigerator for subsequent experiments.

[0064] 2.3 Pathogen screening

[0065] Preparation of bacterial culture: The stored bacteria were placed on NA medium for activation, and a single colony was picked and inoculated into NB liquid culture medium for culture. The culture was cultured in a shaker at 25°C and 180 r / min for 48 h, and the bacterial culture was diluted with sterile water to 1×10 8 cfu / mL bacterial suspension for later use.

[0066] Treatment of Pinellia plants: Screening was carried out according to Koch's postulates. First, the Pinellia tubers were sterilized with 70% ethanol solution for 30-60 seconds and washed with sterile water three times. Second, the Pinellia tubers were soaked in 1% sodium hypochlorite solution for 5 minutes and washed with sterile water until the sterile water was clear. Then, the flower pots and soil were sterilized at 121°C for 20 minutes under high temperature and high pressure. Finally, the sterilized tubers were planted in pots filled with sterilized soil and transferred to an indoor environment at 35°C, relative humidity of 75%, and light 16h / dark 8h.

[0067] Inoculation of Pinellia ternata plants: When the Pinellia ternata plants grow to the 6th week and the three Pinellia ternata leaves are fully unfolded, pierce the stem of Pinellia ternata 1.0-2.0 mm from the ground with an inoculation needle and inoculate the prepared 1×10 8 The bacterial suspension with cfu / mL was inoculated into the potted Pinellia ternata at a volume of 30 mL / pot. As a control, 30 mL of sterile water was inoculated into the potted Pinellia ternata in the same way. There were 5 plants per pot and each treatment was repeated three times.

[0068] 2.4 Determination of pathogenicity of pathogens

[0069] The Pinellia plants were treated according to the method in 2.3 Pathogen Screening and Pathogenicity Determination, and a single colony of the pathogen was inoculated into NB liquid culture medium for cultivation. The culture was cultured in a shaker at 25°C and 180 r / min for 48 h, and the pathogen solution was diluted to 1×10 8 cfu / mL, 1×10 6 cfu / mL, 1×10 4cfu / mL and 0 cfu / mL. The Pinellia ternata plant inoculation test was carried out according to the method in 2.3 Pathogen screening and pathogenicity determination, with 10 plants per pot and three replicates for each treatment.

[0070] Disease grading standards: (0) normal (no damage to the whole plant); (1) wilted (stem base broken); (2) severely wilted (stem base broken, and water-soaked rot); (3) almost dead (stem base broken, stem base and leaves both water-soaked rot); (4) dead (all above-ground parts dead, tubers water-soaked rot). Based on the above conditions, this is used as the plant disease degree standard, and the Pinellia disease index is calculated based on this standard.

[0071]

[0072] Statistical analysis: This study used IBM SPSS25.0 and Graphpad Prism 8 software for data statistics and visualization. Duncan's method was used for significance test. Different letters represent differences among treatment groups, and P<0.05 indicates significant differences among different treatments.

[0073] 2.5 Molecular Biological Identification

[0074] The pathogenic bacteria Pt-43 strain was inoculated into NB liquid culture medium and cultured at 25°C and 180 r / min for 12 h. The bacteria were collected by high-speed refrigerated centrifuge at 12000 rpm for 10 min, and 100 μL ddH 2 O, and then the pathogen Pt-43 strain DNA was extracted by thermal lysis. Using pathogen DNA as a template, bacterial universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-TACCTTGTTACGACTT-3' and gyrB-F: 5'-GCGAAGTGGCGTTCTTT-ATTGC-3', gyrB-R5'-CAGGCTGTGGTTGGTGAGTTC-3' were used to amplify the 16S rRNA gene and gyrB gene of the strain. The PCR reaction system (25 μL) was as follows: MasterMix 12.5 μL, Primer upstream primer 1.0 μL, Primer downstream primer 1.0 μL, template (pathogen solution) 1.0 μL, ddH 2O 9.5 μL. The PCR reaction parameters were: 94°C pre-denaturation for 10 min, 95°C cycle denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 70 s, 35 cycles, 72°C overall extension for 10 min, and storage at 4°C. 1% agarose gel electrophoresis was used to detect the specific bands of the PCR amplification products, and samples containing the target fragments in the PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0075] The sequences of 16S rRNA and gyrB genes were compared for homology in the NCBI gene library using the BLAST tool, and standard strain sequences with a homology greater than 97.0% with the target strain were screened out. Strains with both 16S rRNA and gyrB sequences available were selected for comparison. The 16S rRNA and gyrB gene sequences were compared using BioEdit, and the irregular sequences at both ends were removed. After format conversion using ClustalX, the valid intervals were spliced, and the spliced ​​sequence evolution tree was constructed using Paup*4.0 based on the maximum parsimony algorithm to identify the isolated strain Pt-43.

[0076] 2.6 Physiological and biochemical identification

[0077] The specific operations for the determination of bacterial physiological and biochemical indicators are as follows: Gram staining of endophytic bacteria, VP determination test, gelatin liquefaction test, ammonia production test, hydrogen peroxide test, methyl red test, starch hydrolysis test, nitrate test, CAS test, and salt tolerance test are performed according to the "Handbook of Common Bacterial System Identification".

[0078] 2.7 Potted experiment to screen the disease resistance of different strains of Pinellia ternata

[0079] Potting and soil treatment: Mix the soil and substrate (organic nutrient soil: nitrogen, phosphorus, potassium ≥6%, organic matter ≥45%, pH 5.5-6.5) in a volume ratio of 3:1, put it into a high-pressure steam sterilizer for sterilization for 20 minutes, and mix it after cooling and put it into a pot (outer diameter 19.0×14.5cm, height 11.0cm).

[0080] Treatment of different varieties of tubers of Pinellia: First, disinfect the tubers of different varieties of Pinellia with 70% ethanol solution for 30 to 60 seconds, and wash them with sterile water three times; secondly, soak the tubers of Pinellia with 1% sodium hypochlorite solution for 5 minutes, and wash them with sterile water until the sterile water is clear; finally, plant the sterilized tubers of different varieties in pots with sterilized soil, 15 plants per pot, and replicate three times for each Pinellia variety. After sowing, transfer them to an indoor environment at 28°C, 75% relative humidity, and 16h light / 8h dark.

[0081] Pathogen inoculation treatment: When the three leaves of different strains of Pinellia ternata were fully expanded after growing to the sixth week, the stems of Pinellia ternata were pierced with an inoculation needle at 1.0-2.0 mm from the ground and 1×10 8 ~1×10 9 cfu / mL of bacterial suspension was inoculated into potted Pinellia ternata of different strains at a volume of 30mL / pot, with 15 plants per pot and three replicates for each Pinellia ternata strain. The death of all aboveground parts was taken as the death standard of Pinellia ternata plants. The mortality of different strains of Pinellia ternata was observed and recorded every 2 days for a total of 10 days.

[0082] The mortality rate of Pinellia ternata = number of deaths / total number of Pinellia ternata × 100%

[0083] 2.8 Field trials to screen different strains of Pinellia for disease resistance

[0084] Different strains of Pinellia ternata were planted in the experimental field of Qizhou Campus of Hebei University of Traditional Chinese Medicine. The planting area of ​​each strain was 4m×1.8m. After the Pinellia ternata plants grew for 2 months, the three leaves of different strains of Pinellia ternata in the experimental field were fully unfolded. The Pinellia ternata to be inoculated was swept lightly with a bamboo broom to cause slight wounds on the Pinellia ternata. The prepared bacterial suspension was inoculated at a rate of 1×10 8 ~1×10 9 cfu / mL concentration, spray evenly with an agricultural sprayer, use 10L / mu of bacterial solution, inoculate the pathogen into different strains of Pinellia ternata, and use the death of all above-ground parts as the death standard of Pinellia ternata plants. Observe and record the mortality rate of Pinellia ternata every 2 days for a total of 10 days.

[0085] The mortality rate of Pinellia ternata = number of deaths / total number of Pinellia ternata × 100%

[0086] 3 Results

[0087] 3.1 Separation and purification

[0088] Pathogens were isolated from rotten tubers of Pinellia ternata. After cultivation, the traditional isolation method was used to observe the morphological characteristics of the colonies, such as size, color, edge smoothness, and glossiness. The morphology of the bacterial isolates was studied macroscopically, and single colonies were selected for purification. In this experiment, 60 bacterial strains were isolated from rotten tubers of Pinellia ternata. Most of them were round, milky white, translucent or opaque, with a shiny surface, no wrinkles, smooth and neat edges, and accompanied by a foul odor. Some colonies were pink and lemon yellow, transparent, without gloss, with wrinkles on the surface and uneven edges, such as Figure 3 shown.

[0089] 3.2 Pathogen screening

[0090] The pathogenicity of the isolated strains was tested according to Koch's postulates, and the results of the Pinellia plant inoculation test showed that 48 hours after inoculation with the Pt-43 bacterial solution, the whole plant of Pinellia ternata rotted and a foul odor was produced.

[0091] 3.3 Determination of pathogenicity of pathogens

[0092] Different concentrations of the pathogen Pt-43 were used to inoculate Pinellia ternata, and the disease index was calculated to determine the pathogenicity of the pathogen. Figure 4 As shown, after being treated with different concentrations of pathogenic bacteria Pt-43 (1×10 8 cfu / mL, 1×10 6 cfu / mL, 1×10 4 cfu / mL, 0 cfu / mL), and the disease index of Pinellia ternata (value ± standard deviation) was 42.5 ± 5.40, 9.17 ± 4.25, 1.67 ± 2.36, and 0, respectively. In the three repeated experiments, the concentration of Pt-43 bacterial solution was 1×10 8 cfu / mL, the number of Pinellia ternata plants with grade 4 disease was the largest, with a total of 5 plants, and there were 9 plants with grade 3 disease and 2 plants with grade 2 disease, with the pathogenicity reaching the medium level; the concentration of Pt-43 bacterial solution was 1×10 6 cfu / mL, the highest disease level of Pinellia ternata was level 4, with only one plant, one plant with level 3 disease and two plants with level 2 disease. The concentration of Pt-43 bacterial solution was 1×10 4 When the concentration of Pt-43 bacterial solution was 0 cfu / mL, only one plant was diseased and its grade was 2; when the concentration of Pt-43 bacterial solution was 0 cfu / mL, no Pinellia ternata plant was diseased and there were no symptoms. Figure 4 If there are no identical letters between the two groups, it means that the difference between the two groups is statistically significant (P<0.05).

[0093] 3.4 Morphological characteristics and physicochemical properties of pathogenic bacteria strain Pt-43

[0094] The morphological characteristics of the pathogenic bacteria Pt-43 strain were observed on NA medium, such as Figure 5 As shown, the colonies on the plate were found to be moist and sticky, nearly round, with a raised, smooth surface, milky white, slightly yellowish, opaque, shiny, with neat edges, and a foul odor. The specific bacterial morphology is short club-shaped, about 0.5×(0.5~1.0)μm in size, without capsules, spores, and flagella, which can be determined to be Gram-negative bacteria.

[0095] The results of the physiological and biochemical characteristics experiments are shown in Table 1-1. The results of the sugar fermentation test, VP test, gelatin liquefaction test, ammonia production test, and catalase test were all positive, while the results of the methyl red test, starch hydrolysis test, and nitric acid reduction test were negative.

[0096] Table 1-1 Results of physiological and biochemical tests on pathogenic bacteria Pt-43

[0097]

[0098] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0099] 3.5 Molecular biological identification of pathogen Pt-43

[0100] The DNA of the pathogenic bacteria strain Pt-43 was extracted and used as a template. The 16S rRNA and gyrB genes were PCR amplified and sequenced according to the primer sequences, and two DNA fragments of about 1400 bp and 300 bp were obtained. The NCBI gene numbers were KF557597.1 and OX291654.1, respectively. The two were compared by BLAST sequence comparison on the NCBI database and a phylogenetic tree was constructed. The results showed that the strain and Serratia marcescens were clustered in the same branch and were bacteria of the same species. They were more distantly related to Aeromonas hydrophila. The genes used to construct the phylogenetic tree of Serratia marcescens are shown in Table 1-2, and the phylogenetic tree is shown in Table 1-3. Figure 6 Based on the morphological characteristics of the colonies and combined with the results of physiological and biochemical tests, the pathogen Pt-43 was identified as Serratia marcescens.

[0101] Table 1-2 Gene sequence information used to construct the phylogenetic tree of pathogens

[0102]

[0103] 3.6 Growth curves of pathogens at different temperatures

[0104] By detecting the OD of pathogenic bacteria Pt-43 at different growth times 600 The results showed that the strain grew best and fastest at a culture temperature of 25°C, and grew worst and slowest at a culture temperature of 15°C. The growth curve is shown in Figure 7 As shown. The pathogenic bacteria Pt-43 strain had no obvious growth changes within 3 hours after inoculation and was in the stage of environmental adaptation. It was in the logarithmic growth phase 4 to 12 hours after inoculation and entered the stable growth phase after 12 hours. After the culture time exceeded 20 hours, the absorbance value of the strain fermentation broth remained unchanged, and even showed a downward trend. The bacteria began to age and autolyzed. Under the culture conditions of 25℃ to 35℃, the growth of the pathogenic bacteria Pt-43 strain was not affected by temperature changes. It is a mesophilic bacterium with the most suitable growth temperature of 25℃.

[0105] 3.7 Growth curves of pathogens at different NaCl concentrations

[0106] By detecting the OD of pathogenic bacteria Pt-43 at different growth times 600 Value, analyze the effect of different NaCl concentrations on its growth. The pathogenic bacteria Pt-43 strain can grow in high concentration NaCl solution and has the characteristics of salt stress resistance. When the NaCl concentration is in the range of 1% to 3%, the growth rate of the pathogenic bacteria Pt-43 strain is almost the same, and they can all grow normally; when the NaCl concentration is 4-5%, its growth rate is affected to a certain extent, the growth rate slows down, and the growth rate slows down, but it can still grow. The growth curve of the pathogen Pt-43 under different NaCl concentrations is shown in the figure below. Figure 8 As shown, the bacteria have a certain ability to resist salt.

[0107] 3.8 Determination of phosphorus solubilization characteristics of pathogenic bacteria

[0108] The qualitative test on inorganic phosphorus plates proved that the pathogenic bacteria Pt-43 strain has the ability to dissolve inorganic phosphorus, and a large transparent zone is produced around its colonies. Fig. 9 shown.

[0109] 3.9 Qualitative determination of siderophore production by pathogenic bacteria

[0110] The qualitative test on inorganic phosphorus plates proved that the pathogenic bacteria Pt-43 strain has the ability to secrete iron carriers, and an orange-red transparent circle is produced around its colonies. Fig.10 shown.

[0111] 3.10 Pot experiment to screen the disease resistance of different Pinellia ternata breeding lines

[0112] This experiment screened the disease resistance of 34 Pinellia ternata breeding lines, and the results are shown in Tables 1-3. From the mortality rates of Pinellia ternata of different lines, it can be seen that the Pinellia ternata of the P3 line has the strongest disease resistance, and no diseased plants were found, followed by the Pinellia ternata of the P25 line, with mortality rates of 0 and 4.44% respectively; the Pinellia ternata of the P15 line has the worst disease resistance, with more than half of the Pinellia ternata plants dead, with a mortality rate of 55.56%, and the mortality rates of the other Pinellia ternata lines did not exceed 40%; among the five Pinellia ternata breeding lines selected from Hebei farmers (P10, P17, P29, P33 and P34), the highest mortality rate was in the line P34, reaching 35.56%, while the mortality rates of P33 and P10 were relatively low, at 8.89% and 6.67% respectively. Among the 34 Pinellia ternata lines, the P3 line has the strongest disease resistance.

[0113] Table 1-3 Disease resistance screening of 34 Pinellia strains

[0114]

[0115]

[0116] 3.11 Field trials to screen for disease resistance in different Pinellia ternata breeding lines

[0117] The pathogen Pt-43 was inoculated into different strains of Pinellia ternata grown in the field and their disease resistance was screened. The results were basically consistent with the results of the potted experiment. The mortality rate of the P3 strain was 4.44%, the mortality rate of the P34 Pinellia strain was 33.16%, and the mortality rate of the P15 Pinellia ternata was 44.54%.

[0118] Example 2

[0119] The difference between this embodiment and embodiment 1 is that:

[0120] Pot experiment to screen the disease resistance of Pinellia ternata strain P34

[0121] When the three leaves of the Pinellia ternata plant were fully expanded in the sixth week of growth, the stem of the Pinellia ternata was pierced with an inoculation needle at 1.0-2.0 mm from the ground, and the prepared bacterial suspension was inoculated with a concentration of 1×10 8 cfu / mL were inoculated into potted Pinellia ternata. There were 15 plants in each pot, and each treatment was repeated three times. The death of all aboveground parts was regarded as the death standard of Pinellia ternata plants. The experiment found that the concentration of pathogens was 1×10 8 cfu / mL, the plant mortality rate of the Pinellia ternata breeding line P34 was 35.56%, and its disease resistance was poor.

[0122] Identification of disease resistance of Pinellia ternata breeding line P34 in field experiments

[0123] After 2 months of emergence of the Pinellia plants, when the three leaves of the Pinellia plants planted in the experimental field were fully unfolded, the leaves were gently swept with a bamboo broom to make slight wounds before inoculation of the bacterial solution. The prepared bacterial suspension was then inoculated at a concentration of 1×10 8 cfu / mL was inoculated into the test field, 10L per mu was inoculated, and the death of all the above-ground parts was taken as the death standard of the Pinellia ternata plants. After the pathogen was applied to the test field, the mortality rate of the P34 Pinellia ternata strain was 33.16%, and the disease resistance was poor.

[0124] Example 3

[0125] The difference between this embodiment and embodiment 1 is that:

[0126] Identification of disease resistance of Pinellia ternata breeding line P3 by pot experiment

[0127] When the three leaves of the Pinellia ternata plant were fully expanded in the sixth week of growth, the stem of the Pinellia ternata was pierced with an inoculation needle at 1.0-2.0 mm from the ground, and the prepared bacterial suspension was inoculated with a concentration of 1×108 cfu / mL were inoculated into potted Pinellia ternata. There were 15 plants in each pot, and each treatment was repeated three times. The death of all aboveground parts was regarded as the death standard of Pinellia ternata plants. The experiment found that the concentration of pathogens was 1×10 8 cfu / mL, the mortality rate of the Pinellia breeding line P3 was 0, and its disease resistance was strong.

[0128] Identification of disease resistance of P3 Pinellia ternata strain in field experiments

[0129] After 2 months of emergence of the Pinellia plants, when the three leaves of the Pinellia plants planted in the experimental field were fully unfolded, the leaves were gently swept with a bamboo broom to make slight wounds before inoculation of the bacterial solution. The prepared bacterial suspension was then inoculated at a concentration of 1×10 8 cfu / mL was inoculated into the test field, 10L per mu was inoculated, and the death of all the above-ground parts was taken as the death standard of the Pinellia plants. After the pathogen was applied to the test field, different strains of Pinellia were screened and it was found that the mortality rate of the Pinellia breeding strain P3 was 4.44%, which was relatively strong in disease resistance.

[0130] It should be understood that in order to streamline the present disclosure and aid in understanding one or more of the various inventive aspects, in the above description of exemplary embodiments of the present invention, various features of the present invention are sometimes grouped together into a single embodiment, or description thereof. However, this disclosed method should not be interpreted as reflecting the intention that the claimed invention requires more features than those expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in less than all of the features of the previously disclosed embodiments. Therefore, the claims that follow the detailed description are hereby expressly incorporated into the detailed description, with each claim itself serving as a separate embodiment of the present invention.

[0131] Although the present invention has been described according to a limited number of embodiments, it will be apparent to those skilled in the art, with the benefit of the above description, that other embodiments may be envisioned within the scope of the invention thus described. In addition, it should be noted that the language used in this specification is selected primarily for readability and teaching purposes, rather than for explaining or defining the subject matter of the present invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the present invention is illustrative, not restrictive, with respect to the scope of the present invention, which is defined by the appended claims.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Serratia marcescens, the pathogen of tuber rot in Pinellia ternata Serratia marcescens ), characterized in that, Serratia marcescens is deposited in the General Microbiology Center of China Culture Collection Administration, Beijing, with a deposit date of January 12, 2024 and a deposit number of CGMCC NO.29585.

2. The bacterial suspension prepared from the pathogenic bacteria of tuber rot of Pinellia ternata according to claim 1, characterized in that: The following steps are involved: The Serratia marcescens in claim 1 ( Serratia marcescens ) were placed on NA medium for activation, and a single colony was picked and inoculated into NB liquid medium for culture. The culture was cultured in a shaker at 25°C and 180 r / min for 48 h, and the bacterial solution was diluted with sterile water to 1×10 8 cfu / mL bacterial suspension for later use.

3. A method for screening disease-resistant strains of Pinellia ternata, characterized in that: The following steps are involved: Different strains of Pinellia ternata were planted in the test field. After the Pinellia ternata plants grew for 2 months, the three leaves of the Pinellia ternata planted in the test field were fully unfolded. Before preparing to inoculate the bacterial solution, the Pinellia ternata leaves were lightly swept with a bamboo broom to cause slight wounds on the Pinellia ternata leaves. Then, 1×10 8 ~1×10 9 cfu / mL was inoculated into the experimental field, 10 L was inoculated per mu, and it was sprayed evenly with a common sprayer. The mortality rate of Pinellia ternata was counted 48 hours after spraying, and the death of the aboveground parts was taken as the death standard of Pinellia ternata plants. The varieties with a mortality rate of <5% were regarded as disease-resistant varieties of Pinellia ternata.

4. The method for screening disease-resistant strains of Pinellia ternata according to claim 3, characterized in that: Mortality rate = number of deaths / total number of Pinellia ternata × 100%.

5. Use of the strain of Serratia marcescens, the pathogen of tuber rot of Pinellia ternata according to claim 1, in screening disease-resistant strains of Pinellia ternata.

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