Application of Paenibacillus polymyxa in controlling citrus Huanglongbing disease on citrus seedlings

Abstract

The present application discloses the application of Paenibacillus polymyxa on citrus seedlings to control citrus Huanglongbing disease, and relates to the technical field of citrus virus-free seedling breeding. The Paenibacillus polymyxa is named as KN‑O3 bacterial agent, and the deposit number is CCTCC NO: M 2012078. The application method is: dilute the Paenibacillus polymyxa and then perform root irrigation treatment on citrus seedlings. The present application uses Paenibacillus polymyxa to pre-treat the diseased seedlings, and performs multiple, high-intensity pretreatments on the citrus detoxified mother seedlings to explore the control effect of applying Paenibacillus polymyxa on Huanglongbing disease. It can reduce or eliminate pathogens by adjusting the types of endophytes, and provide the most favorable stem tip material for subsequent stem tip micro-bud grafting and detoxification.

Description

Technical Field

[0001] The present application relates to the technical field of citrus virus-free seedling breeding, and to the application of Paenibacillus polymyxa on citrus seedlings to control citrus Huanglongbing disease. Background Art

[0002] Citrus Huanglongbing is a devastating disease of citrus caused by Bacillus phloem. It infects the main cultivated varieties to varying degrees. The pathogen is still difficult to cultivate artificially. Huanglongbing has caused significant losses to the citrus industry worldwide. Citrus psyllid is the main vector of the pathogen. When citrus is infected by the pathogen, it will cause yellowing of the top branches and leaves, and the fruit will appear asymmetrical or "red nose fruit". The nutrient metabolism of the infected tree becomes disordered, starch accumulates in the phloem, nutrient transport is blocked, the tree is weak, and even causes the tree to wither and die. At present, my country mainly controls Huanglongbing through three countermeasures: digging out diseased citrus trees, applying pesticides to kill citrus psyllids, and cultivating virus-free seedlings. There is no effective control method.

[0003] Paenibacillus polymyxa is a Gram-positive bacterium in the genus Paenibacillus. It is widely distributed and has a wide variety of species in plants and soil. It is a common endophyte in the root system of plants (Baker et al 1987). Polymyxa is an important potential biological agent. Through genomic analysis of polymyxa strains, it was found that the total number of gene clusters producing secondary metabolites in the bacteria accounted for 12% of the genome, indicating that the bacteria have a strong ability to synthesize natural products (Zhou et al 2020). Many polymyxa can produce active and antagonistic substances. Therefore, polymyxa often exhibit broad-spectrum antibacterial activity (Rybakova et al 2014). For example, fusaricide is a cyclohexapeptide antibacterial compound produced by polymyxa (Vatter et al 2015). Polymyxin can also produce antibacterial cyclic peptides (Tambadou et al 2015). Polymyxin has significant antibacterial activity against Gram-negative bacteria, especially Pseudomonas spp. and Escherichia coli Coliform spp. (Raza et al 2008). Antibacterial cyclic peptides can change the structure of Gram-negative bacterial membranes and rupture bacterial membranes. In addition to producing fusaricide and polymyxin, Paenibacillus can also produce substances such as tridecapeptidin and lipopeptidin (Lohans et al 2012, Cochrane et al 2014).

[0004] According to reports, polymyxa can have good effects on a variety of plant pathogens, such as bacteria, fungi, oomycetes and nematodes (Haggag & Timmassk et al 2008; Khan et al 2008; Timmusk et al 2009; Cheng et al 2017). Strain KM2501-1 can secrete toxic volatile compounds to protect the host from fungal long-horned nematodes (Rybakova et al 2017). Previous studies have shown that polymyxa strains are biological control agents for crown rot, hairy root disease, grape gas disease, pepper blight, watermelon wilt and rapeseed black leg disease (Cao et al 2011). Polymyxa E681 can also enhance the tolerance of related resistance proteins such as GST, GFX6, APX1, PER43 and CML42 to stress (Kwon et al 2016; Jeong et al 2019).

[0005] Polymyxa have been identified as plant growth promoting rhizobacteria (PGPR) in a variety of plants (Baeyen et al 2003). Paenibacillus has been reported to be present in the endosperm and embryo of ungerminated wheat and maize, with higher abundance in the endosperm (Kuzniar et al 2020). The main way PGPR helps plant growth is by increasing root access to nutrients such as nitrogen, phosphate, or iron. Some strains of Polymyxa contain nitrogen fixation genes that convert atmospheric nitrogen into ammonia that can be absorbed by plants (Erturk et al 2010). Polymyxa can solubilize phosphate by producing glucose-1-dehydrogenase and gluconate dehydrogenase to produce gluconic acid (Xie et al 2016). Strain BFKC01 can directly act on plants by activating the transcription of the plant gene MYB72 involved in the iron deficiency response (Zhou et al 2016). Bacillus can produce IAA which directly affects plant growth (Yang et al 2019).

[0006] However, there is still no report on the use of Bacillus polymyxa in the fight against citrus Huanglongbing. Summary of the invention

[0007] In view of this, the purpose of the present application is to provide the application of Paenibacillus polymyxa on citrus seedlings to control citrus Huanglongbing disease, aiming to solve the problem of the lack of effective method for controlling citrus Huanglongbing disease in the above-mentioned background technology. By using Paenibacillus polymyxa to pre-treat diseased seedlings, multiple, high-intensity pretreatments are carried out on citrus detoxified mother seedlings to explore the control effect of applying Paenibacillus polymyxa on Huanglongbing disease. By adjusting the types of endophytes, the pathogens can be reduced or eliminated, providing the most favorable stem tip material for subsequent stem tip micro-bud grafting and detoxification.

[0008] In order to achieve the above technical objectives, this application mainly adopts the following technical solutions:

[0009] The present application provides the use of Paenibacillus polymyxa for controlling citrus Huanglongbing disease on citrus seedlings. The Paenibacillus polymyxa is named as KN-03 bacterial agent, and its deposit number is CCTCC NO: M 2012078.

[0010] In an embodiment of the present application, the application includes: diluting the polymyxa bacillus and performing root irrigation treatment on citrus seedlings.

[0011] In an embodiment of the present application, the application includes using Bacillus polymyxa to reduce the titer of citrus Huanglongbing pathogen.

[0012] In an embodiment of the present application, the application includes diluting the polymyxa bacillus and performing root irrigation treatment on citrus seedlings.

[0013] In the examples of the present application, the application includes: diluting 6 g of Bacillus polymyxa stock solution with 1 L of water, using 500 mL per citrus seedling, and irrigating the roots once every 10 days.

[0014] In the examples of the present application, the Bacillus polymyxa stock solution is used to treat each citrus seedling 12 to 24 times.

[0015] In the embodiment of the present application, the application method also includes a method for performing real-time quantitative QPCR detection on citrus seedlings treated with the polymyxa bacillus stock solution to determine whether the citrus seedlings contain the Huanglongbing pathogen, specifically comprising:

[0016] DNA was extracted from citrus leaf veins, and PCR amplification was performed on the DNA of citrus leaf veins using specific primers of citrus Huanglongbing Bacillus. The Huanglongbing pathogen plasmid vector was used as a positive control, and sterilized distilled water was used as a negative control. The samples with the target bands in PCR amplification were positive plants. The relative expression of genes measured by QPCR was 2 -△△CT Samples with a value greater than 1 are considered to be positive plants containing the Huanglongbing pathogen; the pathogen status of the samples is comprehensively evaluated using PCR and QPCR detection methods.

[0017] The present application also provides the use of Bacillus polymyxa in cultivating virus-free citrus seedlings.

[0018] Compared with the prior art, this application has at least the following beneficial effects:

[0019] The citrus seedlings infected with Huanglongbing were treated with Paenibacillus polymyxa KN-03 (CCTCC NO:M 2012078). 6g of the stock solution was diluted with 1L of water, and the dosage for each plant was 500ml / plant. The roots were irrigated once every 10 days, and the molecular detection of the Huanglongbing pathogen was carried out by PCR and QPCR. The results showed that by comparing the detection results of Huanglongbing pathogens before and after treatment with Paenibacillus polymyxa KN-03, it was found that the active Paenibacillus polymyxa KN-03 bacterial agent could reduce the relative titer of the vein Huanglongbing pathogen. After treatment with KN-03, 50% of the plants turned negative; and it caused changes in multiple phenotypes of the root flora. Compared with the control samples, after treatment with Paenibacillus polymyxa, the root flora of citrus seedlings changed in aerobic, anaerobic, facultatively, and Gram-positive. After the polymyxin treatment, the abundance of aerobic bacteria, biofilm-forming bacteria, Gram-negative bacteria, and stress tolerance-related bacteria in citrus roots was higher than that in the control samples, which is of great significance for improving the detoxification efficiency of subsequent citrus stem tip micro-bud grafting. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The standard curves of the housekeeping gene COX and the target gene RNR provided in the examples of the present application;

[0021] Figure 2 This is a graph showing the detection results of OI1 / OI2c primers in the vein tissue of Yura mandarin orange before treatment with KN-03 bacterial agent provided in the examples of the present application; M: DNA marker; 1-4: live bacteria treatment group; 5-8: inactivated treatment group; 9-12: clear water control group; +: positive control; -: negative control;

[0022] Figure 3The detection result of OI1 / OI2c primers in the vein tissue of Yura mandarin orange after 12 times of treatment with KN-03 bacterial agent provided in the embodiment of the present application; M: DNA marker; 1-4: live bacteria treatment group; 5-8: inactivated treatment group; 9-12: clear water control group; +: positive control; -: negative control;

[0023] Figure 4 The detection result of OI1 / OI2c primers in the vein tissue of Yura mandarin orange after 24 treatments with KN-03 bacterial agent provided in the embodiment of the present application; M: DNA marker; 1-4: live bacteria treatment group; 5-8: inactivated treatment group; 9-12: clear water control group; +: positive control; -: negative control;

[0024] Figure 5 Photos of citrus plants before and after treatment with the bacterial agent provided in the examples of the present application; (A): after treatment with live bacteria (B): after treatment with inactivated bacteria (C): clean water control (D): before treatment with live bacteria (E): before treatment with inactivated bacteria (F): control before treatment with clean water;

[0025] FIG6 is a BugBase species bar chart provided in the embodiment of the present application, showing the difference in relative abundance percentage of species in roots caused by polymyxomycete treatment and control, wherein the horizontal axis is the group name and the vertical axis is the relative abundance percentage of species. The picture shows 9 phenotypes at the family level, which are Aerobic ( Figure 6a )、Anaerobic( Figure 6b )、Contains_Mobile_Elements( Figure 6c )、Facultatively_Anaerobic( Figure 6d )、Forms_Biofilms( Figure 6e )、Gram_Negative( Figure 6f )、Gram_Positive( Figure 6g )、Potentially_Pathogenic( Figure 6h ) and Stress_Tolerant( Figure 6i );

[0026] FIG7 is a BugBase species bar chart provided in the embodiment of the present application, showing the difference in relative abundance percentage of species in roots caused by inactivated Bacillus polymyxa treatment and control, wherein the horizontal axis is the group name and the vertical axis is the relative abundance percentage of species. The picture shows 9 phenotypes at the family level, which are Aerobic ( Figure 7a )、Anaerobic( Figure 7b )、Contains_Mobile_Elements( Figure 7c)、Facultatively_Anaerobic( Figure 7d )、Forms_Biofilms( Figure 7e )、Gram_Negative( Figure 7f )、Gram_Positive( Figure 7g )、Potentially_Pathogenic( Figure 7h ) and Stress_Tolerant( Figure 7i ). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0028] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence, nor do they play a substantial limiting role on the subsequent technical features. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0029] The present application embodiment provides an application of Paenibacillus polymyxa in resisting citrus Huanglongbing disease. The Paenibacillus polymyxa is named as KN-03 bacterial agent, and its deposit number is CCTCC NO: M 2012078.

[0030] In some embodiments, the use includes using Paenibacillus polymyxa to reduce the titer of citrus Huanglongbing pathogen.

[0031] In some embodiments, the application includes diluting the Paenibacillus polymyxa and then performing root irrigation treatment on citrus seedlings.

[0032] In some embodiments, the application method is specifically as follows: dilute 6 g of Bacillus polymyxa stock solution with 1 L of water, use 500 mL per citrus seedling, and irrigate the roots once every 10 days.

[0033] In the examples of the present application, the Bacillus polymyxa stock solution is used to treat each citrus seedling 12 to 24 times.

[0034] The embodiment of the present application provides an application method of the above-mentioned Bacillus polymyxa in resisting citrus Huanglongbing disease and its application in cultivating citrus virus-free seedlings.

[0035] In the embodiment of the present application, the application method also includes a method of performing PCR and real-time quantitative PCR detection on citrus seedlings treated with the polymyxa bacillus stock solution to determine whether the citrus seedlings contain the Huanglongbing pathogen, specifically including:

[0036] Citrus leaf vein DNA was extracted and PCR amplified using primers specific to citrus Huanglongbing pathogen. The plasmid vector of Huanglongbing pathogen was used as a positive control, and sterilized distilled water was used as a negative control. Samples with target bands were considered to be positive plants containing Huanglongbing pathogens. The relative gene expression measured by QPCR was 2 -△△CT Samples with a value greater than 1 are considered to be positive plants containing the Huanglongbing pathogen; the pathogen status of the samples is comprehensively evaluated using PCR and QPCR detection methods.

[0037] The application principle of the present invention is further described in detail below with reference to the accompanying drawings.

[0038] 1. Materials and Methods

[0039] 1.1 Plant materials and microbial agents

[0040] The plant materials used in this experiment were 2-year-old seedlings of the citrus variety 'Youliang' grafted on trifoliate orange rootstock, all of which were isolated and planted in the greenhouse of the Microbial Engineering Center. All test samples were sterilized with high temperature and high pressure after the experiment.

[0041] The bacterial agent used in this experiment was Bacillus polymyxa KN-03 (500 million CFU / g), which was provided by Wuhan Kono Biotechnology Co., Ltd.

[0042] 1.2 Solution preparation

[0043] (1) Homogenisation buffer (HB)

[0044] 500 mM sodium chloride, 100 mM Tris (pH=7.4), 50 mM EDTA, 52 mM sodium sulfite, 0.7% (w / v) sodium dodecyl sulfate. Store at room temperature.

[0045] (2) Precipitation buffer PB

[0046] 3.6M potassium acetate, 2.4M acetic acid. The buffer can be stored at room temperature for several months.

[0047] (3) Binding buffer BB

[0048] When preparing 1000 mL, add 95.5 g of 1 M guanidine hydrochloride, adjust to 166.6 mL with TE (Tris-EDTA=10:1, pH=8), add 833.3 mL of anhydrous ethanol, and store at room temperature.

[0049] (4) Wash buffer (WB)

[0050] When preparing 1000 mL, add 4 mL of 5 M sodium chloride, 2 mL of 1 M Tris-HCl (pH = 8), 194 mL of water, and 800 mL of anhydrous ethanol and store at room temperature.

[0051] 1.3 Main instruments

[0052] Table 1 Experimental instruments

[0053] Main instruments Company Fluorescence quantitative PCR instrument Corbett RG 6000 Conventional PCR instrument Bio-Rad Corporation Nucleic acid protein analyzer Amersham DYY-5 electrophoresis apparatus Beijing Liuyi Instrument Factory Gel Imager Segrate Autoclave German systec company Clean bench Shanghai Weixing Furnace Industry Co., Ltd. Digital constant temperature water bath HH-Z Guohua Electric Co., Ltd. Light incubator Ningbo Haishu Saifu Experimental Instrument Factory GNP-9080 Constant Temperature Incubator Shanghai Jinghong Experimental Equipment Co., Ltd. Electronic balance Shanghai Huachao Electric Co., Ltd. Full temperature oscillator HZQ-Q Harbin Donglian Electronics Co., Ltd. System Biological Microscope NIKON Instruments (Shanghai) Co., Ltd. Multifunctional ELISA reader PerkinElmer Benchtop centrifuge Beckman Coulter

[0054] 1.4 Bacterial agent treatment method

[0055] The Huanglongbing pathogen of citrus seedlings was detected, and the positive seedlings were used for the experiment. 12 Huanglongbing positive seedlings were planted in black plastic pots (30 cm high × 32 cm in diameter) containing a substrate of 1 / 3 of vermiculite, peat and garden soil, and the basic fertilizer and water management conditions of the seedlings were the same. Active polymyxa bacillus KN-03 (500 million CFU / g), the above seedlings were used for 4 biological replicates in the polymyxa live bacteria treatment group (live bacteria treatment group), polymyxa inactivated treatment group (inactivated treatment group) and clear water control group. Live bacteria treatment group: irrigate the roots once every 10 days, dilute 6g of the original solution with 1L of water, and use 500mL / plant per plant, inactivated treatment group: sterilize the KN-03 bacterial agent with high pressure, irrigate the roots once every 10 days, dilute 6g of the original solution with 1L of water, and use 500mL / plant per plant; the clear water treatment group applies an equal amount of clear water to each plant every 10 days. This experiment will run from July to December 2021.

[0056] 1.5 Molecular Detection of Citrus Huanglongbing

[0057] In this study, the Corbett RG 6000 (Germany) real-time quantitative PCR instrument and the Bio-Rad PCR instrument (USA) were used for molecular detection of Huanglongbing. The detection primers and instruments for Huanglongbing used are shown in Tables 1 and 2.

[0058] The DNA of each tissue was extracted by CTAB method (Wang Guanlin et al., 1998), and PCR amplification was performed on the DNA of each tissue. Citrus Huanglongbing primers OI1 / OI2c were used to amplify the DNA of citrus veins by PCR. The reaction procedure is shown in Table 3:

[0059] Table 2 Primers used for PCR and QPCR detection of Huanglongbing

[0060]

[0061]

[0062] Table 3 PCR reaction system and reaction procedure

[0063]

[0064] 1.6 Commonly used QPCR primers for citrus Huanglongbing detection and drawing standard curves

[0065] The standard plasmid was constructed using the COX gene fragment of the citrus cytochrome oxidase gene and the RNR gene fragment of the primer in the Huanglongbing genome. The two standard plasmids were diluted to a concentration of 1×10 7 copy / μL, 1×10 6 copy / μL, 1×10 5 copy / μL, 1×10 4 copy / μL, 1×10 3 copy / μL, 1×10 2 copy / μL, 1×10 1 Real-time fluorescence quantitative PCR was performed on the corresponding plasmids with primers COX+ / COX- and RNR+ / RNR-, and the standard curve was drawn and the efficiency and detection line of each primer were calculated. The results are shown in Figure 1 The reaction procedures are shown in Table 4. The data were analyzed by variance analysis and significance level test using Excel 2016 software and SPSS 25.0 software.

[0066] Table 4 Real-time fluorescence quantitative PCR reaction system and reaction procedure

[0067]

[0068] 1.7 Detection of Huanglongbing pathogens in plants before and after polymyxozoites treatment

[0069] Take the 3rd to 5th leaves from the first fully expanded leaf on healthy branches in the southeast, northwest, northeast and southeast directions of the plant that are free of pests and diseases and mechanical damage, and take the leaf veins of the same plant as samples. Extract the genomic DNA of citrus leaves, and then amplify it with OI1 / OI2c primers. The amplification procedure is shown in Table 2. Sterile distilled water is used as a negative control, and the plasmid vector of the Huanglongbing pathogen retained in the laboratory is used as a positive control. Samples with the target band are considered to be positive plants. The real-time fluorescence quantitative PCR detection reaction procedure for the Huanglongbing pathogen is shown in Table 2. The highly sensitive RNR+ / RNR- primers were selected, and 3 biological replicates were set for each sample. The callus DNA of a healthy 'Newhall' was used as a negative control, and 2 -△△CT The value was set to 1. The leaf vein DNA of the 'Sandangju' plant tested positive in the laboratory was used as the positive control for qPCR. -△△CT If the sample is 2 -△△CT A value greater than 1 was considered a positive plant (Livak et al., 2001). The data were analyzed by variance analysis and significance level test using Excel 2016 software and SPSS 25.0 software.

[0070] 1.8 Metagenomic analysis of citrus roots treated with Polymyxozoites and controls

[0071] Microbial diversity is based on the Illumina Novaseq sequencing platform, using the paired-end sequencing method to construct a small fragment library for sequencing. The species composition of the sample can be revealed by splicing and filtering the reads, clustering or denoising, and performing species annotation and abundance analysis.

[0072] Experimental procedure:

[0073] Library construction and sequencing: After extracting the total DNA of the sample, primers are designed according to the conserved region, and sequencing adapters are added to the ends of the primers. PCR amplification is performed and the products are purified, quantified and normalized to form a sequencing library. The constructed library is first subjected to library quality inspection, and the qualified library is sequenced using Illumina Novaseq 6000. The original image data file obtained by high-throughput sequencing (such as Illumina Novaseq and other sequencing platforms) is converted into original sequencing sequences (Sequenced Reads) through base calling analysis, and the results are stored in FASTQ (abbreviated as fq) file format, which contains the sequence information of the sequencing sequence (Reads) and its corresponding sequencing quality information.

[0074] Experimental principle:

[0075] Extract genomic DNA; purify PCR amplified products; quantify and normalize PCR products; construct sequencing libraries;

[0076] Paired-end sequencing.

[0077] Information Analysis:

[0078] Data preprocessing: There are mainly three steps:

[0079] 1) Quality detection: Use Trimmomatic v0.33 software to filter the Raw Reads obtained by sequencing; then use cutadapt 1.9.1 software to identify and remove primer sequences to obtain Clean Reads;

[0080] 2) Double-end sequence splicing: Using Usearch v10 software, the Clean Reads of each sample were spliced ​​through overlap, and then the length of the spliced ​​data was filtered according to the length range of different regions;

[0081] 3) Removal of chimeras: UCHIME v4.2 software was used to remove chimera sequences and obtain effective data (Effective Reads).

[0082] Information analysis content: Diversity analysis, difference analysis, correlation analysis and functional prediction analysis were performed. BugBase prediction was performed, which is a method for predicting biological level coverage of functional pathways within complex microbiomes and biologically explainable phenotypes. BugBase first normalizes OTUs by the predicted 16S copy number, and then uses the provided pre-calculated file to predict microbial phenotypes (Ward et al., 2017) for each sample in the biological dataset, estimating the relative abundance of traits in the entire range of coverage thresholds (0 to 1, in increments of 0.01). BugBase then selects the coverage threshold with the highest variance among all samples for each feature in the user data. After setting the threshold, BugBase generates a final organism-level trait prediction table containing the predicted trait relative abundance for each sample. Automatic hypothesis testing and visualization of differential traits are selectively performed based on user-specified metadata, and a group contribution map describing the relative abundance of groups with characteristic traits is generated, and the output includes a non-parametric differential test (Mann-Whitney U or Kruskal Wallis) statistical summary file.

[0083] 2 Results

[0084] 2.1 Analysis of standard curve for qPCR detection of Huanglongbing

[0085] This experiment used the Corbett RG 6000 (Germany) fluorescence quantitative PCR instrument, and used qPCR to draw a standard curve for the constructed target gene and housekeeping gene plasmids. The results are shown in Figure 1 As shown: The primer efficiency of the citrus internal reference gene primers COX+ / COX- is 99.42%, and the minimum detection line is 251.06 copies. The primer efficiency of the Huanglongbing primer RNR+ / RNR- is 95.17%, and the minimum detection line is 91.34 copies. Under the conditions of the instruments and reagents used in this laboratory, it was found that the primer RNR was used for Huanglongbing detection, and the minimum detection line had a high sensitivity, and the Huanglongbing pathogen could be detected at a minimum of 91 copies. And the primer efficiency can reach more than 90%. The Huanglongbing detection achieved a good sensitivity, and this detection method was used for the titer tracking and identification of Huanglongbing in this experiment.

[0086] 2.2 Changes in the pathogen of Huanglongbing before and after treatment with Bacillus polymyxa

[0087] After 12 treatments, the leaf veins of the plants were tested for Huanglongbing pathogens, and after 24 treatments, the leaf veins, roots, and leaf buds of the treated plants were tested for Huanglongbing pathogens. By comparing the results of Huanglongbing pathogen detection in the leaf veins before and after treatment, it was found that the relative concentration of Huanglongbing pathogens in the tree body after polymyxa treatment had a certain downward trend (Table 5). The PCR test of Huanglongbing conventional primers OI1 / OI2c showed that after 12 treatments, 1 / 4 of the live bacteria treatment group and the inactivated bacteria treatment group tested negative after treatment. After 12 treatments, the qPCR test results showed that there was no negative conversion in the plants of each treatment group, but it showed that after the live bacteria treatment, the pathogen concentration in the leaf veins of the plants was lower than the relative concentration of the pathogen in the leaf veins of the plants before treatment. The relative concentration of pathogens in the leaf veins of 3 / 4 of the plants in the inactivated treatment group decreased. There was no significant change in the pathogen concentration in the leaf veins of the plants in the clean water treatment group. After 24 treatments with the bacterial agent, the PCR molecular detection of the leaf veins of the plants in each treatment group using the conventional Huanglongbing primers OI1 / OI2c showed that 3 out of 4 plants treated with live bacteria tested negative; 3 out of 4 plants treated with sterilized bacteria tested negative; and only 1 out of 4 plants treated with clean water tested negative. The results of qPCR detection of leaf veins showed that after 24 treatments, 2 out of 4 plants treated with live bacteria tested negative, and the other 2 plants tested extremely low levels of Huanglong pathogen in the samples; both the inactivated bacteria treatment and the clean water treatment were positive after qPCR detection, and the Huanglong pathogen in the leaf veins of the 4 plants treated with inactivated bacteria decreased compared to before treatment. The gel electrophoresis diagram of PCR detection is attached. Figure 2-4 . See attached for photos of citrus plants before and after treatment with the fungicide Figure 5 .

[0088] Table 5 Detection of the pathogen of yellow dragon disease on leaf veins of plants before and after treatment with Paenibacillus polymyxa

[0089]

[0090] Note: Sampling time 1 - July 6, 2021, 2 - September 22, 2021, 3 - March 24, 2022

[0091] 2.3 BugBase phenotype prediction of Paenibacillus polymyxa treatment compared with control

[0092] Sequencing data quality assessment Expression quality requirements (Table 6). Clean Reads are the number of high-quality reads obtained after quality control of the original sequence.

[0093] Compared with the control samples, after the polymyxin treatment, the characteristics of the root flora of citrus seedlings showed obvious differences in aerobic, anaerobic, facultative, Gram positive, potential pathogen, stress tolerant, biofilm forming, and Gram negative phenotypes. Among them, Oxlobacteraceae, Comamonadaceae, Hyphomicrobiaceae, and Enterobacteriaceae were expressed in the roots of the polymyxin treatment group, while the control group did not show the above-mentioned bacteria (attached Figure 5 After the polymyxin treatment, the abundance of aerobic bacteria, biofilm-forming bacteria, Gram-negative bacteria, bacteria containing mobility factors, stress tolerance-related bacteria, and potential pathogens in the roots was higher than that in the control samples (Appendix Figure 6a-6i After the inactivated bacteria treatment, the abundance of aerobic bacteria, biofilm-forming bacteria, Gram-negative bacteria, stress tolerance-related bacteria and potential pathogens in the roots was higher than that in the control samples (Appendix Figure 7a-7i ).

[0094] Table 6 Statistics of sample sequencing data processing results

[0095]

[0096] Note: Sample ID is the sample name; Raw Reads is the number of raw reads obtained by sequencing; Clean Reads is the number of high-quality reads obtained after quality control of the raw sequence; Effective Reads is the number of effective sequences of Clean Reads after splicing (double-end), filtering length and chimeras; AvgLen (bp) is the average sequence length of the sample; GC (%) is the GC content of the sample, that is, the percentage of G and C type bases in the total bases; Q20 (%) is the percentage of bases with a quality value greater than or equal to 20 in the total number of bases; Q30 (%) is the percentage of bases with a quality value greater than or equal to 30 in the total number of bases; Effective (%) is the percentage of Effective Reads in Raw Reads.

Claims

1. Paenibacillus polymyxa ( BaciIllus polymyxa ) is used to control citrus Huanglongbing on citrus seedlings, among which, The polymyxa bacillus is named KN-03 bacterial agent, and its deposit number is CCTCC NO:M 2012077; The application is to use Paenibacillus polymyxa to reduce the pathogen titer of citrus Huanglongbing; The specific method of using the application is as follows: 6 g of the Bacillus polymyxa stock solution containing 500 million CFU / g was diluted with 1 L of water, and the dosage for each citrus seedling was 500 mL. The roots were irrigated once every 10 days, and the number of treatments was 12 to 24 times.

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