73 results about "Phytoplasma" patented technology

The invention relates to a phytoplasma probe, and comprises a gene chip of the probe and a method for detecting phytoplasma with the gene chip. The phytoplasma probe is a specific probe for 11 types of phytoplasma, which is point sampled and manufactured into the gene chip, and the method for detecting the phytoplasma comprises the following steps that: a plant DNA sample is extracted; a sample specific fragment is amplified through a universal primer and a product is marked; hybridization; the sample to be detected and to be marked is added into hybrid buffer fluid, and mixed uniformly into hybrid mixture; the mixture is dripped into a hybrid area of the gene chip, and put into a hybrid box for hybridization; and the hybridized chip is taken out and dried, scanning and data analysis are carried out, and whether phytoplasma exists in the sample is judged. The gene chip manufactured from the probe of the invention can quickly detect 11 types of phytoplasma with high efficiency and highflux.

The present invention discloses a LAMP primer set for detecting jujube witches disease, locust tree witches broom disease and cherry lethal yellowing phytoplasma, and its detection kit and application. The LAMP primer set is designed for detecting jujube witches disease, locust tree wilt disease and cherry lethal yellowing phytoplasma for 16SrV-B group phytoplasma tuf gene conservative sequence. The invention also discloses a LAMP detection kit for jujube witches disease, locus tree witches broom disease and cherry lethal yellowing phytoplasma. The LAMP primer set is used to establish the LAMP detection method for jujube witches disease, locust tree witches broom disease and cherry lethal yellow phytoplasma, and carries out ring-mediated isothermal amplification on the sample DNA to be tested, and determines whether the tested sample is infected with phytoplasma according to the amplification result samples. The detection method established in the invention can be used for detection of jujube witches disease, locust tree witches broom and cherry lethal yellow phytoplasma, and can better distinguish phytoplasma between different groups and inside groups, and has the advantages of good stability, specificity and high sensitivity.

The present invention discloses a method for detecting betelnut yellow phytoplasma pathogen and its special kit. The kit includes a probe whose nucleotide sequence is the sequence 1 of the sequence table, and a primer pair composed of an DNA fragment whose nucleotide sequence is the sequence 2 of the sequence table and an DNA fragment whose nucleotide sequence is the sequence 3 of the sequence table. The method includes: extracting the DNA of the betelnut to be tested as a template, using the above kit to perform real-time fluorescence PCR, if the fluorescence signal is enhanced in the PCR process, the plant infects phytoplasma. Proved by experiment, the sensitivity of the inventive method detecting the betelnut phytoplasma infection is same with that of the traditional nested PCR electrophoresis detection, while the invention has simple operation and closed tube operation, processes automation control in the entire testing, eliminates the PCR false-positive contamination opportunities, does not need post-PCR handling, is suitable for bulk samples, and has simple, rapid, and accurate features.

The invention belongs to the technical field of plant quarantine, and relates to a primer, a probe and a method for qualitatively and quantitatively detecting witches' broom phytoplasmas. The method comprises the following steps: according to difference between 16S rDNA gene sequences of the witches' broom phytoplasmas and other phytoplasmas, designing a primer pair JWB Primer-F: 5'-TGGTGAGGTAAAGGCTTA-3'/ JWB Primer-R: 5'-CTCCCGTAGGAGTTT GG-3' and a TapMan probe JWB-Probe: 5'-FAM-AATGTGGCTGTTCAACCTCTCA-TAMRA-3' for specifically detecting the witches' broom phytoplasmas; measuring a Ct value by an established real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of the witches' broom phytoplasmas; according to a positive decision criteria of a real-time fluorescent quantitative PCR detection result, qualitatively detecting the witches' broom phytoplasmas; calculating the copy concentration of a 16S rDNA gene segment of the witches' broom phytoplasmas in a sample y a fluorescent quantitative PCR standard curve equation established in advance; and obtaining the number of the witches' broom phytoplasmas in the sample so as to achieve quantitative detection. The primer, the probe and the method have the advantages of high sensitivity, strong specificity, good repeatability, high throughput and the like, and can be widely used in plant quarantine, plant protection, scientific research and other fields.

The invention discloses a method for purifying mulberry phytoplasma genome. By using the characteristic that a phytoplasma has a cell membrane and the genome of the phytoplasma is semi-autonomously copied, and adopting a DNA methylation conjugated protein, the mulberry genome is removed from purified mulberry whole genomes infected by phytoplasma, and semi-quantitative and quantitative PCRs (polymerase chain reactions) prove that the obtained DNA is relatively high in purity and is a mulberry phytoplasma genome DNA relatively high in concentration, so that the problems of low phytoplasma content and difficult pulse electrophoresis purification of woody plants are solved, the effect of the method disclosed by the invention is tested by using quantitative PCR, and the obtained phytoplasma genome is relatively high in concentration and proportion and can be used for PCR experiments and high-throughput sequencing.

Disclosed is a method utilizing sprout inhibitor to control the phytoplasma jujube withes broom disease and the phytoplasma paulownia withes broom disease of plants. The method includes: selecting the sprout inhibitor in a plant inhibitor or adding one or more organic acids which can induce the plants to develop a resistance to the sprout inhibitor to prepare an agent and injecting the agent into the plants. When the total weight of the sprout inhibitor and the organic acid is counted as 100, in the prepared agent, the weight percentage of the sprout inhibitor is 0-100% but not equal to 0 and the weight percentage of the organic acid is 0-100% but not equal to 100%. The method utilizing sprout inhibitor to control the phytoplasma jujube withes broom disease and the phytoplasma paulownia withes broom disease of the plants expands the application range of the sprout inhibitor in the plant growth inhibitor, provides a new control method for controlling plant phytoplasma diseases, is obvious in effects in the controlling of the jujube withes broom disease and the paulownia withes broom disease, and can be used for controlling other phytoplasma diseases. The method utilizing sprout inhibitor to control the phytoplasma jujube withes broom disease and the phytoplasma paulownia withes broom disease of plants is wide in agent source, low in price, simple in preparation, convenient to use, harmless to people and livestock and capable of being widely used and popularized.

The method of using a clay suspension to prevent viral and phytoplasma diseases in plants includes administering a clay suspension to the plant, such as through spraying the plant's leaves or soaking the plant's nursery shoots with the clay suspension. The clay suspension is applied to the plant to prevent the viral or phytoplasma disease prior to planting the plant in its nursery shoot stage. The clay suspension is preferably formed from natural clay suspended in water (1% w/v). The clay may be in either its natural form, or may be in the form of clay nanoparticles suspended in water. For a suspension formed from clay nanoparticles, the clay nanoparticles may be separated by sedimentation in distilled water.

The invention discloses a chip for screening 16SrI group phytoplasmas and application thereof. The nucleotide sequences of five probes for screening 16SrI group phytoplasmas are sequence 1 to sequence 5 in order in a sequence table. The invention also provides a gene chip for screening 16SrI group phytoplasmas, and the probes are fixed on the surface of the chip, so that the gene chip is formed. An experiment proves that the invention adopts a bioinformatic method to analyze the 16S rDNA, 16S-23S rDNA spacer, imp and tuf nucleotide sequences of 16SrI group phytoplasmas and designs the compatible probe for the group, and a standard phytoplasma sample test result proves that the effect of the probe is good. The chip for screening 16SrI group phytoplasmas can be used in the quarantine and identification of 16SrI group phytoplasmas.

The invention belongs to the technical field of plant quarantine and relates to a primer and a probe for a real-time fluorescence polymerase chain reaction (PCR) detection of pear decline phytoplasma. The primer and the probe for the real-time fluorescence PCR detection of the pear decline phytoplasma contain a specific cycling probe and a pair of primers: (1) probe LSTProbe: 5'-(FAM) ATTCTGACTGTA (Eclipse)-3'; and (2) primer LSTPrimer-F:5'-ACTCTGACCGAGCAACGCC-3'; LSTPrimer-R:5'-GATAACGCTTGCCCCCTATG-3', wherein a ribonucleic acid (RNA) alkali base exists in a square frame. Through sequence comparison of a large number of phytoplasmas and bacteria ITS, a set of real-time fluorescence PCR cycling probe and primer is designed, wherein the sensitivity is up to 0.5pgl-1; and the pear decline phytoplasma can be rapidly and correctly detected from a sample. The method has the advantages of simple operation, easy grasping and wide application to daily detection work of laboratories.

The invention provides a method for accurately identifying disease resistance of newly-planted sugarcane white leaf. The method comprises: cutting sugarcane stalks infected with sugarcane white leaf phytoplasma, performing juicing, adding 10 times amount of sterile water to prepare an inoculating liquid; cutting the sugarcane stalks which are a sugarcane material to be tested into stalk sections with buds, soaking the sections in running tap water for 48h, performing hot water treatment at 50 DEG C for 2h, and performing soaking with an insecticidal and sterilizing agent for 10min; performingcoating inoculation with a plastic film, and performing moisture preservation at 25 DEG C for 24h; planting an inoculating material in a plastic barrel, and placing the barrel in a 20-30 DEG C insecticidal greenhouse for culture; and investigating infected plant rate, and performing disease resistance evaluation according to 1-5 grade standards. The method of the invention firstly establishes a set of accurate and efficient method for identifying disease resistance of newly-planted sugarcane white leaf, and provides technical support for sugarcane white leaf resistance breeding. The pathogen inoculating liquid is sprayed onto the sugarcane seed and the coating inoculation with a plastic film is performed, so that a disease habitat condition is provided, and the result is objective, true and reliable. The inoculation is quick and simple, the operability is strong, and the work efficiency is high. After the inoculation, the sugarcane white leaf has obvious incidence, high sensitivity andgood reproducibility.

The invention discloses a method for sequencing plant phytoplasma genome, which comprises the following steps: obtaining Paulownia tomentosa tissue culture seedling stem rich in phytoplasma; Isolationand Extraction of Genomic DNA Mixture from Paulownia Alba and Phytoplasma; DNA Sequencing of Paulownia Alba and Phytoplasma Gene Mixture; Phytoplasma genome sequencing data processing, statistics andassembly and Paulownia tomentosa and phytoplasma transcriptome sequencing to verify the reliability of the genome. The invention obtains the genome of Paulownia arbuscular phytoplasma for the first time, and analyzes the information of interaction between Paulownia arbuscular pathogen and host through the genome. The invention is helpful to increase the information of basic metabolic pathway of phytoplasma, and lays a foundation for revealing the genome information of plant phytoplasma comprehensively.

The invention discloses a LAMP primer group and kit for detecting 16SrI-group phytoplasma leading to betel nut yellow leaf diseases in China, and application thereof. The LAMP primer group is a primergroup 16SrDNA-2 or a primer group 16SrDNA-3. The LAMP primer group disclosed by the invention has very good specificity and stability, high detection sensitivity and the lowest detection limit of 200ag/[mu] L. Phytoplasma are detected in betel nut yellow leaf disease samples from Baoting, Tunchang, Wanning and the like in Hainan Province in China by using the two sets of LAMP primers, i.e., 16SrDNA-2 and 16SrDNA-3; and no phytoplasma is detected in negative control. The LAMP primer group and kit provided by the invention are rapid and efficient in detection, simple and convenient to operateand visual in result, play a great role in methods for early detection and field diagnosis of the betel nut yellow leaf diseases, bacteria-carrying detection of betel nut seedlings, breeding of resistant betel nut varieties and the like, and provide technical support and reference basis for pathogen detection, disease prevalence, scientific prevention and control and the like of the betel nut yellow leaf diseases.

The invention discloses a cow phytoplasma substrate and preparation method thereof, and belongs to the technical field of veterinary biology. The cow phytoplasma substrate constitutes a basic substrate and an assistant substrate, wherein, the substrate is constituted from PPLO broth, sodium pyruvate, glucose, fresh yeast extract, L-glutamine, L-cysteine, water solvent milk protein, horse serum, penicillin, phenol red, and deionized water. The substrate can reduce the use level of serum, and increase evidently the active bacterium titer of cow phytoplasma, thus establishing a foundation for studying quality cow phytoplasma vaccine.

The invention relates to a PCR-RFLP identification method for different subgroups of sugarcane white leaf disease phytoplasma. A sugarcane white leaf disease phytoplasma tuf gene is taken as an objectto design a specific primer, a tuf gene fragment is obtained through PCR amplification, then restriction endonuclease Msp I is adopted for digesting the tuf gene fragment obtained through amplification, and the different subgroups of the sugarcane white leaf disease phytoplasma are distinguished through a differentiated RFLP restriction map. The PCR-RFLP identification method for the different subgroups of the sugarcane white leaf disease phytoplasma has the advantages that a sugarcane white leaf disease phytoplasma 16SrXI-B subgroup and a sugarcane white leaf disease phytoplasma 16SrXI-D subgroup can be quickly, accurately and efficiently identified without sequencing, and is important for the pathogen identification and prevention of sugarcane white leaf diseases.

The invention relates to a method for detecting a transmission medium of Sugarcane white leaf phytoplasma. The method includes the steps that a piece of field in a sugarcane white leaf occurrence areaand with crop rotation for more than 1 year is selected; susceptible varieties are used for screening out phytoplasma infected with white leaf disease and phytoplasma not infected with the white leafdisease to be sugarcane species carried with virus and sugarcane species not carried with virus; the piece of field is divided into three field ditches, one of the field ditches is equipped with a transmission medium measurement field device in which the sugarcane species not carried with virus are planted, the sugarcane species carried with virus and the sugarcane species not carried with virusare planted in the other two field ditches separately, and morbidity situation is investigated; after newly planted sugarcane detection is completed, and biennial roots are cut and harvested; and according to the disease condition of the sugarcane species carried with virus, the sugarcane species not carried with virus, and newly planted and biennial root sugarcane inside and outside the device, asystematic analyzes to determine the transmission medium of Sugarcane white leaf phytoplasma. According to the method, a set of simple, convenient and systematic effective methods for determination of the transmission medium of Sugarcane white leaf phytoplasma is firstly established, theoretical guidance and scientific basis are provided for the effective prevention and control of Sugarcane whiteleaf, and the measurement results are objective, true and reliable.

The subject matter of the invention is a hydroxyapatite carrier, corresponding to the formula: Ca_(10-x)M_x(PO₄)_(6-y)(CO₃)_y(OH)₂ in which: M is chosen from Cu, Mg, Zn, Pb, Cd, Ba, K, Mn, Mo, Fe, Ag, B, Se or a combination of at least two of such elements; —x is comprised between 0 and 2, preferably between 0 and 1, more preferably between 0 and 0.8, even more preferably between 0.02 and 0.05; —y is comprised between 0 and 2, preferably between 0 and 1, more preferably between 0.002 and 0.030, The hydroxyapatite is a carrier of a bioactive substance chosen among a metal ion selected from Cu, Zn, S, Mn, Mg, K, Fe, B, Mo, Se and/or an extract of vegetal origin. The bioactive substances are active in the treatment of vascular diseases of plants, in particular: grapevine yellows (caused by grapevine phytoplasma), bacterial canker in kiwifruit (caused by Pseudomonas syringae), olive quick decline syndrome (caused by Xylella fastidiosa) and Citrus bacterial canker disease (caused by Xanthomonas axonopodis).

The invention discloses a PCR detection method of sugarcane white leaf disease phytoplasma. The PCR detection method of sugarcane white leaf disease phytoplasma is established by designing a specificprimer SecA-F/SecA-R according to the SecA gene sequence of the sugarcane white leaf disease phytoplasma and optimizing the reaction system and program. By adopting the specific primer and method disclosed by the invention, PCR amplification is needed only once, whether the detected sample is infected by the sugarcane white leaf disease phytoplasma can be determined according to whether the electrophoresis result has a specific band, and the amplification product does not need sequencing determination; therefore, the detection specificity and accuracy are improved, the detection time is saved,and the detection cost is lowered.

The invention belongs to the technical field of plant quarantine, and relates to a qualitative and quantitative detection method for Xinjiang isolates of apricot chlorotic leaf roll (ACLR) phytoplasma. According to the qualitative and quantitative detection method, according to the difference of the ACLR phytoplasma and other phytoplasma in sequence of a 16S rDNA gene, a primer pair ACLR-F1/ACLR-R1 and a probe ACLR-Probe which are used for specific detection of the ACLR phytoplasma are designed, a cycle threshold (Ct) is determined through an established real-time fluorescent quantitative polymerase chain reaction (PCR) detection method for the ACLR phytoplasma, and qualitative detection of the ACLR phytoplasma can be realized according to the positive judgment standard of a real-time fluorescent quantitative PCR detection result; then, the segment copy concentration of the 16S rDNA genes of the ACLR phytoplasma in detected samples is worked out by a pre-established fluorescent quantitative PCR standard curve equation, and thus, the number of the ACLR phytoplasma in the samples is obtained, and thereby, quantitative detection is realized. The qualitative and quantitative detectionmethod has the advantages of high sensitivity, high specificity, high repeatability, high throughput, and the like, and can be widely applied to the fields of plant quarantine, plant protection, scientific research, and the like.

The invention discloses a jujube witches broom phytoplasma effector gene Zaofeng8 and application. The nucleotide sequence of the jujube witches broom phytoplasma effector gene Zaofeng8 is shown as SEQ ID NO.1, the gene has the functions of dwarfing plants, enabling the plants to form arbuscular phenotypes and enabling the plants to generate leaflet symptoms, and the Zaofeng8 is obtained through PCR technology cloning. The gene is transferred into Columbia type arabidopsis thaliana for functional verification by using an agrobacterium-mediated method, so that the infection mechanism of phytoplasma to hosts in the occurrence process of the jujube witches broom symptom can be clarified from a molecular mechanism, and the gene has a positive guiding effect on further prevention and treatmentof the jujube witches broom.

The invention discloses a method for establishing influence of interacting protein on phytoplasma propagation through RNAi (Ribonucleic Acid Interference). The phytoplasma is wheat blue dwarf phytoplasma, the mediator is heterosachalina fulica, and the interaction protein is heterosachalina fulica microtubulin; the method comprises the following steps: synthesizing interaction protein genes dsRNA and dsGFP, respectively feeding a mediator with the interaction protein genes dsRNA and dsGFP, detecting the gene expression of the wheat blue dwarf phytoplasma by using qRT-PCR, infecting healthy wheat with the contaminated mediator to determine the virus transmission efficiency, and determining the influence of the interaction protein on phytoplasma transmission. According to the invention, the propagation influence of the heterosacharide microtubulin on the wheat blue dwarf phytoplasma is proved, and a foundation is laid for subsequent research on interaction of the phytoplasma and a mediator.