CN110663709B

CN110663709B - Preparation method and application of paris polyphylla extractive solution

Info

Publication number: CN110663709B
Application number: CN201911120879.2A
Authority: CN
Inventors: 刘新琼; 张佑宏; 杨小林; 张向明; 王春台
Original assignee: Wuhan Runtu Biotechnology Co ltd; South Central University for Nationalities
Current assignee: Wuhan Runtu Biotechnology Co ltd; South Central Minzu University
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2021-06-29
Anticipated expiration: 2039-11-15
Also published as: CN110663709A

Abstract

The invention relates to the field of biology, and particularly relates to a preparation method and application of a paris polyphylla extracting solution. The technical scheme has the beneficial effects that the paris polyphylla extracting solution capable of inhibiting rice blast germs and the preparation method thereof are provided, and the extracting solution can induce the expression of rice defense genes WRKY45 gene, PBZ gene and PR1b gene.

Description

Preparation method and application of paris polyphylla extractive solution

Technical Field

The invention relates to the field of biology, and in particular relates to a preparation method and application of a paris polyphylla extractive solution.

Background

The rice blast is a worldwide disease caused by rice blast germs and is one of three main diseases in rice production [1 ]. The data show that the annual loss of rice caused by rice blast is about 10-15% of total yield, the loss is 10 hundred million yuan 2, and the yield is reduced by half in serious cases. The disease is mainly caused by the leaves, and large or small disease spots are formed. At present, the cultivation of rice blast resistant varieties and the popularization and planting of the varieties are one of the most economic and effective methods for preventing and treating rice blast. However, the rice blast fungi are divided into many different physiological races, and the physiological races are complex and variable, and are easily subject to variation [3], and are easily resistant to a single chemical agent. Since the sixties, the resistance of the bred disease-resistant variety is continuously lost in the process of popularization and planting, so that the disease-resistant level is lower and lower. In addition, producers also control the occurrence of diseases by chemical pesticides. However, chemical control has disadvantages of environmental pollution, alteration of soil structure and composition, and high harmfulness to the body of the producer. Therefore, the botanical pesticide has the advantages of broad-spectrum antibacterial activity, low residue, no pollution and the like. According to the method, various characteristic plant extracting solutions in Wuling mountain areas are selected, repeated screening is carried out, the paris polyphylla is found to have a good bacteriostatic action on rice blast germs, different tissue parts of the paris polyphylla are further separated, corresponding extracting solutions are respectively prepared, antibacterial activity screening is carried out, and a foundation is laid for developing plant source bactericides which are safe to people and livestock, free of pollution, low in toxicity and low in residue.

Disclosure of Invention

The invention aims to provide a paris polyphylla extract capable of inhibiting rice blast germs and a preparation method thereof.

The technical scheme provided by the invention is that a paris polyphylla extractive solution is prepared by the following steps:

step 1: drying Paris polyphylla peel, and grinding into powder;

step 2: dissolving the powder in the step 1 by using methanol for the first time, and taking supernatant after carrying out ultrasonic treatment and centrifugation;

and step 3: drying the supernatant obtained in the step 2 under reduced pressure, and fully dissolving the supernatant with distilled water;

and 4, step 4: and (4) extracting the solution obtained in the step (3) by using n-butyl alcohol, drying under reduced pressure again, and adding methanol for secondary dissolution to obtain a paris polyphylla extracting solution.

Wherein, the root of Paris polyphylla is used for replacing the pericarp of Paris polyphylla in the step 1.

Wherein the Paris polyphylla peel in step 1 is replaced by a mixture of its root and peel.

Further, the methanol is anhydrous methanol.

And the paris polyphylla extract has the inhibiting effect on rice blast germs.

And the paris polyphylla extract has the effect of promoting the expression of rice defense genes.

Furthermore, the defense genes are WRKY45 gene, PBZ gene and PR1b gene.

Compared with the prior art, the technical scheme has the beneficial effects that: the paris polyphylla extractive solution can effectively inhibit rice blast germs and promote rice defense gene expression. The extract is a botanical fungicide which is safe to human and livestock, has no pollution, low toxicity and low residue.

Drawings

FIG. 1 is a graph showing the effect of an extract solution (n-butanol phase) of Paris polyphylla on the inhibition of Magnaporthe oryzae spores;

FIG. 2 is a graph showing the bacteriostatic effect of Paris polyphylla peel extract (n-butanol phase) on Magnaporthe oryzae spores;

FIG. 3 is a graph showing the effect of isoprothiolane pesticide on the inhibition of rice blast fungus spores;

FIG. 4 is a graph showing the bacteriostatic effect of absolute methanol on Magnaporthe grisea spores;

FIG. 5 is a relative expression analysis of WRKY45 gene;

FIG. 6 is a relative expression analysis of PBZ gene;

FIG. 7 is a relative expression analysis of PR1b gene.

Detailed Description

Collecting Paris polyphylla in Wuling mountain area, separating different tissue parts of Paris polyphylla, i.e. root, stem, leaf, flower, pericarp and seed, and preparing corresponding extractive solution. Rice blast germs: race 007. Rice variety: nipponbare.

The preparation method of the paris polyphylla peel extracting solution comprises the following steps:

(1) oven drying 0.5g of Paris polyphylla pericarp to constant weight, grinding into powder with a pulverizer, and sieving with 40 mesh sieve;

(2) ultrasonically treating the powder for 1h by using 50mL of methanol, centrifuging the powder for 20min at the temperature of 4 ℃ and the speed of 4000rpm, and taking supernatant into a round-bottom flask;

(3) drying the mixed solution at 58 ℃ under reduced pressure by using a rotary evaporator, and washing the wall of the round-bottom flask by using distilled water for full dissolution;

(4) extracting the solution with n-butanol for 2-3 times, and transferring to a new round-bottom flask;

(5) and drying again at 70 ℃ by using a rotary evaporator under reduced pressure, concentrating, and then diluting to 10mL by using methanol.

The preparation method of extractive solution of other parts of rhizoma Helminthostachydis Zeylanicae is the same as above.

Preparation of target pathogen spore suspension

Culturing Magnaporthe grisea in oat culture medium (30 g of oat, 15g of agar and 7g of agar, and adding distilled water to a constant volume of 1L) at 26 deg.C in dark, scraping surface hyphae with cotton swab after thallus grows over the surface of the culture medium (about 10 d), culturing (3-4d) under ultraviolet lamp for sporulation, and observing that the surface turns grey. Washing spores on the surface with sterile water and a brush, filtering with double-layer gauze, taking a drop onto a glass slide, and adjusting the concentration of the spores under a 10-fold microscope to obtain 50-80 spores in each visual field.

Determination of antibacterial Activity

(1) A method for inhibiting spore germination: the plant extracts and the target pathogen spore suspension were mixed in different volumes (see Table 1), spread on water agar medium (7g agar to 1L volume), and treated with 1X 10-3 isoprothiolane as a control agent for 5 replicates per sample per concentration. And (2) performing microscopic examination on spore germination conditions after culturing for 24 hours in an incubator at 26 ℃, taking the spore germ tube length larger than the short radius of the spore as germination, ensuring that 30-70 spores exist under each visual field, calculating the total number of the spores and the number of the germinated spores in each visual field, finding 5 visual fields for observing one glass slide each time in each treatment, and calculating the average germination inhibition rate, wherein the result shows that the root and pericarp extracting solution has obvious inhibition on spore germination of rice blast germs, and the average inhibition rates of the root and pericarp extracting solution on spore germination are respectively 78.00% and 77.00%.

Table 1 shows the determination of the antibacterial activity of the extract solution of different parts of Paris polyphylla on Pyricularia oryzae

Therefore, the difference of the average inhibition rates of the extract liquid of different parts of the paris polyphylla on the spore germination of the pyricularia grisea is obvious. In microscopic examination, most of spores germinate after the stem extract is added, wherein spore silks are long and the inhibition effect is poor.

The paris polyphylla peel extract (n-butyl alcohol phase) and the paris polyphylla root extract (n-butyl alcohol phase) have obvious effect of inhibiting spore germination, most spores do not germinate, and the shape of the spores is similar to a more regular ellipse. In two groups of control experiments, the isoprothiolane pesticide almost completely inhibited the germination of spores; in the case of absolute methanol, most of the spores germinated, and relatively thin spore filaments appeared at both ends, as shown in FIGS. 1 to 4.

(2) Pot live inoculation assay: and (3) selecting rice seedlings with 3-5 leaf stages and consistent growth vigor to perform pot living prevention and control experiments. Spore suspension is prepared according to the method, and then 0.02% tween is added and mixed evenly for standby. Experimental groups: spraying the extractive solution of the pericarp of Paris polyphylla, inoculating the spore suspension of Magnaporthe grisea by spraying after 24h, and treating in dark for 24h while keeping the air humidity above 90%. Experimental groups: inoculating rice blast germ spore suspension, performing dark treatment for 24h, and spraying Paris polyphylla pericarp extractive solution under the same culture conditions. A control group was also set: namely spraying water firstly and spraying the rice blast germ spore suspension after 24 hours. Ensuring the consistency of the inoculation time of the rice blast germs. And after inoculating and culturing for 7d, observing the disease condition of the rice leaves, and counting the number of disease spots. Then respectively collecting rice leaves at the same position, processing each rice leaf to about 100mg, wrapping with tinfoil paper, marking, quick freezing with liquid nitrogen, and placing in a refrigerator at-80 deg.C for use.

The number of rice leaf blast germs scab is counted by an experimental group (spraying first), an experimental group (spraying spores first) and a control group (spraying water first). Experiments show that the average disease index of the liquid extract of the pericarp after the liquid extract of the rice blast fungus is inoculated is the lowest, the average disease index of the liquid extract of the pericarp after the liquid extract of the rice blast fungus is inoculated is the highest, and the liquid extract of the pericarp after the liquid extract of the rice blast fungus is sprayed and the liquid extract of the rice blast fungus after the liquid extract of the pericarp after. Therefore, the control effect of the pericarp extract on rice blast germs is higher than the control effect.

(3) Detection and analysis of relative expression of defense gene

Extracting total RNA of the rice leaf according to an Axyrep total RNA miniprep kit, and synthesizing cDNA by using a reverse transcription kit PrimeScriptRTreagentKitwithDNAeraser. Taking rice leaves from a control group and an experimental group respectively, extracting RNA, reversing cDNA, then carrying out expression analysis, and detecting the relative expression of the defense gene WRRY45 gene, the PBZ gene and the PR1b gene respectively.

Expression analysis was performed using a real-time fluorescence quantification kit SYBRPremixExTaq using applied biosystems7500 FastFasal-TimePCRSystem.

The experiment adopts a method for quantitative analysis of relative expression quantity, and obtains the relative expression quantity of target genes WRRY45, PBZ and PR1b by taking housekeeping gene Rubq with constant expression as a reference gene, thereby reflecting the change condition of the expression quantity of 3 groups of experimental genes. The relative expression amount is calculated by a 2-delta Ct method: relative expression amount 2- Δ Δ Ct 2- [ (E-F) - (a-B) ], so: the relative expression amount was 2(F-B) - (E-a). Wherein A is Ct value of gene to be detected before intervention; b is Ct value of reference gene before intervention; e is the Ct value of the gene to be detected after intervention; f is the Ct value of the reference gene after intervention.

The relative expression quantity of the defense gene is detected, and the relative expression quantity shows a trend of increasing in sequence from the experiment of spraying water first and then spraying spores, spraying the spores first and then spraying the spores, and spraying the spores first and then spraying the spores. WRKY45, PBZ and PR1b have the highest relative expression in rice leaves which are sprayed with the spore before and then are sprayed with the peel extracting solution; and the expression quantity of 3 genes in the rice leaf blade sprayed with the spore suspension liquid after spraying water is the lowest. Therefore, the extractive solution of the pericarp of paris polyphylla can directly inhibit the germination of the spore of the rice blast fungus, and simultaneously induce the expression of the defense gene to activate the basic resistance of the rice to achieve good control effect, as shown in fig. 5 to 7.

In conclusion, in vitro bacteriostasis experiments and in vivo prevention and treatment experiments show that the paris polyphylla extractive solution (especially the paris polyphylla peel extractive solution) has strong bacteriostasis activity on rice blast germs and can induce or cause the expression of defense genes WRKY45, PR1b and PZ1 in plants.

Claims

1. A paris polyphylla extract has an inhibition effect on rice blast germs, and the preparation method of the paris polyphylla extract comprises the following steps:

step 1: drying Paris polyphylla peel, and grinding into powder;

2. The inhibitory effect of the paris polyphylla extractive solution on pyricularia oryzae according to claim 1, characterized in that: replacing the pericarp of Paris polyphylla in step 1 with the root of Paris polyphylla.

3. The inhibitory effect of the paris polyphylla extractive solution on pyricularia oryzae according to claim 1, characterized in that: replacing the Paris polyphylla pericarp in step 1 with the Paris polyphylla root and pericarp mixture.

4. The inhibitory effect of the paris polyphylla extractive solution on pyricularia oryzae according to any one of claims 1 to 3, characterized in that: the methanol is anhydrous methanol.

5. An induction effect of paris polyphylla extract on rice defense gene expression is disclosed, wherein the preparation method of the paris polyphylla extract comprises the following steps:

step 1: drying Paris polyphylla peel, and grinding into powder;

6. The inducing effect of the paris polyphylla extractive solution on rice defense gene expression as claimed in claim 5, wherein: replacing the pericarp of Paris polyphylla in step 1 with the root of Paris polyphylla.

7. The inducing effect of the paris polyphylla extractive solution on rice defense gene expression as claimed in claim 5, wherein: replacing the Paris polyphylla pericarp in step 1 with the Paris polyphylla root and pericarp mixture.

8. The induction of rice defense gene expression by paris polyphylla extractive solution according to any one of claims 5-7, wherein: the methanol is anhydrous methanol.

9. The inducing effect of the paris polyphylla extractive solution on rice defense gene expression as claimed in claim 5, wherein: the defense genes are WRKY45 gene, PBZ gene and PR1b gene.