CN110396492B

CN110396492B - Walnut branch blight antagonistic bacterium and application thereof

Info

Publication number: CN110396492B
Application number: CN201910822493.XA
Authority: CN
Inventors: 马荣; 赵颖; 王敏; 蔡桂芳; 王雪萍
Original assignee: Xinjiang Agricultural University
Current assignee: Xinjiang Agricultural University
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2020-12-15
Anticipated expiration: 2039-09-02
Also published as: CN110396492A

Abstract

The invention relates to a walnut branch blight antagonistic bacterium and application thereof, the bacterium is Bacillus subtilis QB002, the preservation number is CGMCC NO.18218, the invention finds that the QB002 can inhibit the growth of walnut branch blight hyphae, so that the hyphae are deformed and twisted, a plurality of branches are generated, the growth inhibition rate of the QB002 on walnut branch blight pathogenic bacteria is 88.6% by plate opposite culture, and the bacterial strain has certain inhibition effect on apple rot, jujube black spot and plum brown rot, and has broad-spectrum bacteriostatic ability; the bacillus subtilis QB002 provided by the invention has good biocontrol potential on walnut branch blight, and can be used for preventing and treating the walnut branch blight.

Description

Walnut branch blight antagonistic bacterium and application thereof

Technical Field

The invention belongs to the fields of microbiology and plant disease control, and particularly relates to bacillus subtilis with a good inhibition effect on various plant pathogens and application thereof.

Background

Walnut (Juglans regia L.) is a woody oil tree species of the genus Juglans of the family Juglandaceae, the first of the four dry fruits, and has very high nutritional and medicinal values. At present, large-area cultivation is carried out in more than 20 provinces and cities in China, and the planting quantity and the yield are at the top of the world. Xinjiang is one of the original producing area and the main producing area of the walnut, the yield of the Xinjiang walnut accounts for 15% of the total yield of China, and the walnut industry becomes an important component of the economic development of Xinjiang. However, the walnut branch blight caused by the fungus Juglanconis juglandina is serious in the Xinjiang walnut producing area, so that branches are faded green and withered, and the health of trees and the yield and the quality of walnuts are seriously influenced. The germs can also damage the leaves and fruits of the walnuts, nearly circular scabs are formed on the damaged leaves, the surfaces of the leaves are covered by the scabs in the later period, and the photosynthesis of the tree body is seriously influenced; after the walnut fruit is damaged, black spots are formed on the surface of the fruit, and the yield and the quality of the fruit are influenced (figure 1). The walnut branch blight generally has the disease incidence rate of 20-30%, and the disease incidence rate can reach more than 50% in severe cases, so that a large number of branches die, the growth of trees and the quality of walnuts are directly influenced, and huge economic losses are caused to fruit growers.

At present, the walnut branch blight is mainly chemically prevented and treated in production, and the spread of the disease is controlled by applying bactericides such as mancozeb and the like, so that the ecological health of an orchard is damaged, the quality of walnuts is influenced, and the problems of pesticide residue, soil micro-ecological imbalance and the like are caused. The biological control principle reduces the occurrence and expansion of diseases from the viewpoint of ecological safety and environmental protection. Therefore, the loss caused by the branch blight is reduced to the maximum extent, and the development of a biocontrol agent is imperative. In recent years, through wide research on disease prevention and control of biocontrol strains, starfish cleaner and the like are found that walnut rhizosphere antagonistic bacteria can better inhibit peach brown rot, and the antibacterial substances of the antagonistic bacteria are proved to have the characteristics of strong antibacterial activity and good stability. And screening a strain with strong antagonistic effect on walnut rot by the tissue separation method and the plate antagonistic culture method of the Wangyang and the like, and finally identifying the strain as the Bacillus subtilis.

The antagonistic bacterium is one of biological control, has good application prospect on plant disease control, and relevant researches on the biological control of walnut diseases are carried out in China. Studies such as Gekang and the like find that the biocontrol bacterium Bacillus subtilis has antagonistic effect on pathogenic bacteria of hickory dry rot (Botryosphaeria dothidea). Zhang Jing, Lijian and so on screen 7 strains of Pseudomonas sp, Bacillus sp and Streptomyces sp, which have antagonistic walnut rot disease strains. The walnut endophyte XHE7 screened by the weight of the seeds can promote the root length and the bud length of the seeds.

Walnut branch blight (Juglancis juglandina) is a disease newly found on branches, fruits and leaves of Xinjiang walnuts in recent years, and no related research on biocontrol strains capable of antagonizing Juglancis juglandina exists at present.

The walnut has become the leading industry of economic development in rural areas in southern Xinjiang and peasants, so that aiming at walnut branch blight as a research object, development of a batch of bacterial strains with excellent biocontrol effect is urgently needed for biological control of the walnut branch blight.

Disclosure of Invention

The invention aims to provide Bacillus subtilis with good bacteriostatic ability on walnut branch blight bacteria (Juglancis juglandina) and application thereof.

In order to realize the purpose of the invention, the Bacillus subtilis is obtained by separating populus euphratica rot disease specimens collected from the Uygur autonomous region in Xinjiang and is named as QB 002. Morphological characteristics, physiological and biochemical characteristics and 16S rDNA gene sequence analysis prove that the antagonistic strain QB002 is Bacillus subtilis. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of West Lu No.1 of Beijing university Hokko Yang district, microbiological research institute of Chinese academy of sciences, zip code 100101) in 7 months and 12 days in 2019, and is named as Bacillus subtilis by classification, and the preservation number is CGMCC No. 18218.

The microbiological characteristics of the Bacillus subtilis QB002 are as follows: after the strain QB002 is cultured on the NA culture medium for 24 hours, a single colony is light yellow, irregular in shape, wrinkled in the middle, opaque, wet in texture and unsmooth in surface. Gram staining is purple, gram positive bacteria are obtained, and the bacteria are in short rod shape through microscopic observation.

The physiological and biochemical characteristics show that the strain is gram-positive anaerobic bacteria; can utilize 23 carbon sources such as D-turanose, D-stachyose and the like; the catalase test is positive; the phenylalanine ammonia enzyme test, the tyrosinase test and the nitric acid reduction test are negative;

the invention provides a microbial inoculum containing the bacillus subtilis QB 002.

The fermentation product of the bacillus subtilis QB002 belongs to the protection scope of the invention.

The invention provides a microbial inoculum containing the Bacillus subtilis QB002 or a fermentation product thereof. The microbial inoculum is a single microbial inoculum, and can also be a composite microbial inoculum.

The invention provides a medicament containing the Bacillus subtilis QB002 or a fermentation product thereof.

The medicine is used for inhibiting walnut Sclerotinia jgelandia, apple canker (Cytospora salicicola, Cytospora mali), jujube black spot pathogen (Alternaria alternata) and Monilinia fructicola.

The invention provides application of Bacillus subtilis QB002, a fermentation product thereof or a microbial inoculum and a medicament containing the Bacillus subtilis QB002 in controlling plant diseases.

Plants of the present invention include, but are not limited to, walnut, Xinjiang poplar, arrowroot poplar, willow, apple, birch, oleaster or grape.

The invention provides application of the Bacillus subtilis QB002, a fermentation product thereof or a microbial inoculum and a medicament containing the Bacillus subtilis QB002 in preventing and treating walnut branch blight bacteria (Juglancis jugandina), apple rot bacteria (Cytospora salicicola, Cytospora mali), jujube black spot bacteria (Alternaria alternata) and plum brown rot bacteria (Monilinia fructicola).

The bacillus subtilis QB002 provided by the invention can inhibit the growth of hyphae of walnut branch blight bacteria, so that hyphae are deformed and distorted, a plurality of branches are generated, the inhibition rate of the bacillus subtilis QB002 on the walnut branch blight is 88.6% as determined by a plate scribing method, and the bacillus subtilis QB has certain inhibition effects on walnut branch blight bacteria (Juglancis jugelida), apple rot disease (Cytospora mali), jujube black spot disease (Alternaria alternata) and plum brown rot disease (Monilinia uctica), and has broad-spectrum bacteriostatic ability; the bacillus subtilis QB002 provided by the invention has good biocontrol potential on walnut branch blight, can be used for preventing and controlling the walnut branch blight, has the characteristics of no pollution, no residue, biological environmental protection and the like, and has a remarkable effect in the aspect of preventing and controlling plant diseases.

Drawings

FIG. 1 shows the field hazard symptoms of walnut branch blight, wherein A shows the damaged branches, B shows the damaged leaves, and C shows the damaged fruits.

FIG. 2 shows the A pattern of the QB002 colony morphology and the B pattern of gram-stained QB002 bacteria, which is purple.

FIG. 3 is a phylogenetic tree of Bacillus subtilis QB 002.

FIG. 4 is a graph showing the bacteriostatic effect of the strain QB002 of example 1 on different pathogens, wherein the A graph shows the colony morphology of walnut Sclerotinia solanacearum (Juglanconis Juglandina) inhibited by QB 002; panel B shows the colony morphology of normally growing Sclerotinia Juglandis (Juglanconis juglandina); panel C shows the colony morphology of apple rot (Cytospora mali) inhibited by QB 002; FIG. D is a colony morphology of normally growing Malassezia furfur (Cytospora mali); panel E shows the colony morphology of Cytospora salicicola (Cytospora salicicola) inhibited by QB 002; panel F shows the colony morphology of the normally growing species of Malassezia californica (Cytospora salicicola); g is colony morphology of Sclerotinia pipiens (Alternaria alternata) inhibited by QB 002; h is colony morphology of normally growing Alternaria alternata (Alternaria alternata); FIG. I is a colony morphology of Monilinia fructicola (Monilinia fructicola) inhibited by QB 002; J-Panel shows the colony morphology of normally growing Monilinia fructicola (Monilinia fructicola).

FIG. 5 shows the effect of Bacillus subtilis QB002 on the hypha of pathogenic bacteria Juglancis juglandina under microscope observation, wherein the left picture shows the hypha of Juglancis juglandina after being inhibited by QB002, and the right picture shows the hypha of normal pathogenic bacteria Juglancis juglandina.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The walnut branch blight pathogenic bacteria (Juglancis juglandina) used in the examples described below were isolated from the injured tissue of Juglans mandshurica, Stephania hainanensis, Ohio, Xinjiang university of agriculture, supplied at the pathology laboratory of the forestry and gardening colleges, No. 1429.

In the examples, the culture and plate confrontation experiments of walnut branch blight pathogenic bacteria adopt PDA culture medium: 200g of potato, 14g of glucose, 12g of agar and 1000mL of water, and the pH value is natural.

The bacterial culture adopts NA culture medium: 3.0g of beef extract, 10g of peptone, 5g of NaCl, 15g of agar and distilled water till the volume is 1000mL and the pH value is 7.0 +/-0.1.

The antagonistic bacteria fermentation liquor adopts NB culture medium: 3.0g of beef extract, 10g of peptone, 5g of NaCl and distilled water are supplemented to 1000mL, and the pH value is 7.0 +/-0.1.

Example 1 isolation and characterization of Bacillus subtilis QB002

1. Isolation of antagonistic bacteria

Collecting disease specimens in different areas of Xinjiang, separating bacterial strains by using a tissue isolation method, selecting single colony plates with different culture shapes, streaking the single colony plates on an NA culture medium, culturing at the constant temperature of 28 ℃ for 24 hours, observing and recording the size, color, surface smoothness, edge shape and the like of colonies, and selecting single colonies to store at 4 ℃ for later use.

2. Identification of antagonistic bacteria species

2.1 Observation of cell morphology and Biolog analysis and identification

The culture morphology and gram staining of QB002 strain were observed. After the strain QB002 is cultured on the NA culture medium for 24 hours, a single colony is light yellow, irregular in shape, wrinkled in the middle, opaque, wet in texture and unsmooth in surface. Gram staining is purple, gram positive bacteria, and the thallus is short rod-shaped (figure 2).

Physiological and biochemical characteristics show that the strain QB002 is gram-positive anaerobic bacteria; can utilize 23 carbon sources such as D-turanose, D-stachyose and the like (Table 1); the catalase test is positive; the phenylalanine ammonia enzyme test, the tyrosinase test and the nitric acid reduction test are negative.

The identification of the target strain was carried out according to the procedure specified in the Biolog full-automatic identification apparatus. The data of the different reactions were compared to data of known model strains using GP plates. And screening out a species with higher Probability (Proavailability) and Similarity (Similarity) in the comparison result of the Biolog automatic identification system database, and finally identifying the strain QB002 as Bacillus subtilis.

TABLE 1 utilization of 95 carbon sources by strain QB002

Note "+" positive; negative; "b" between positive and negative.

Note:“+”.Positive；“－”.Negative；“b”.Intermediate.

2.2 identification of 16S rDNA molecules of antagonistic strains

Extracting the total genome DNA of the bacteria by a water boiling method.

Amplification and sequencing of 16S rDNA: PCR primers were designed based on the conserved region of bacterial 16S rDNA. The bacterial universal primers 63F (5'-CAGGCCTAACACATGCAAGTC-3') and 1387R (5 '-GGGCGGWGTGTACAAGGC-3', wherein W is degenerate base A or T) were used, and the primers were synthesized by Shanghai Bioengineering Co., Ltd. PCR reaction (50. mu.L): mu.L of template DNA, 2.5. mu.L of each primer, 4. mu.L of dNTP, 5. mu.L of 10 XBuffer, 0.5. mu.L of Taq polymerase, and the complement of ddH2O to 50. mu.L. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 95 deg.C for 1 min; annealing at 55 deg.C for 1 min; extension at 72 ℃ for 1.5min for 30 cycles; extension at 72 ℃ for 10 min. mu.L of the PCR product was subjected to 1% agarose electrophoresis (120V/25min) to detect the PCR product. The target fragment was sequenced by Shanghai Biotechnology engineering, Inc. And performing BLAST (BLAST-specific sequence alignment) homologous sequence alignment on a GenBank website on the sequencing result, constructing a phylogenetic tree by using an NJ (adjacent junction method) method in MEGA 6.0 software, and testing the phylogenetic tree by using a bootstrap method (bootstrap) with a bootstrap data set of 1000 times. Amphibacillus xylanus is taken as an exome.

The sequence obtained by sequencing is subjected to Blast comparison at NCBI, and the result shows that the strain QB002 has close genetic relationship with B.subtilis, the homology is 100%, and the sequence is displayed on a phylogenetic tree constructed based on an adjacency method: the strain QB002 and B.subtilis are gathered in the same branch and have higher support rate (99%) (figure 3), and finally the strain QB002 is determined to be Bacillus subtilis by integrating the morphological and culture characteristics of the strain, the carbon source utilization condition and the molecular phylogenetic tree.

The experimental result shows that the strain QB002 is Bacillus subtilis. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu No.1 of Xinyang district, Beijing, China academy of sciences, postal code 100101) in 7.12.7.2019, and is classified and named as Bacillus subtilis with the preservation number of CGMCC No. 18218.

Example 2 growth inhibitory Effect of Bacillus subtilis QB002 on walnut Branch blight

The inhibition effect of the strain QB002 on the walnut branch blight bacteria is determined by adopting a plate confronting culture method. And (3) beating a pathogen bacteria cake which is purified and cultured and has the diameter of 5mm, and placing the pathogen bacteria cake in the center of a PDA flat plate. Activating the strain QB002 on NA culture medium, culturing at constant temperature for 24 hr, selecting single colony 2cm away from pathogenic bacteria, streaking, and repeating the treatment 3 times with only pathogenic bacteria as control. Culturing at 28 deg.C, observing and measuring the width of antibacterial band and the growth diameter of pathogenic bacteria when the control bacterial colony grows over the plate, calculating the growth inhibition rate of bacterial colony, and screening out the bacteria with optimal antagonistic effect.

1 strain QB002 (figure 4) with the best antagonistic effect on walnut branch blight bacteria (Juglancis juglandina) is obtained by screening 5 antagonistic bacteria by a plate confrontation method, and the inhibition rate of the strain QB002 on the growth of pathogenic bacteria hypha reaches 88.6%.

Example 3 preliminary study on Bacillus subtilis QB002 bacteriostatic mechanism

Antagonistic strain QB002 active substance has inhibitory effect on hypha of Rhizoctonia solani (Juglanconis juglandina) by plate confronting method, inoculating pathogenic bacteria cake with diameter of 5mm into PDA plate center, placing aseptic cover glass around 1cm from plate center, streaking bacteria QB002 at 1cm from cover glass after hypha growth, and culturing at 30 deg.C for 3 d. The influence of the active product of the antagonistic bacterium QB002 on the shape of the hyphae of the pathogenic bacteria is observed under an optical microscope by taking the hyphae only inoculated with the pathogenic bacteria as a control. The result shows that the inhibition rate of the antagonistic bacteria on the growth of the walnut branch blight bacteria reaches 88.6 percent. Hyphae of a bacterium (Juglanconis Juglandina) inhibited by the antagonistic bacterium exhibited abnormalities, most typically characterized by hyphal distortion and malformation, resulting in multiple branches (left panel of FIG. 5).

Example 4 determination of the bacteriostatic Profile of Bacillus subtilis QB002

The method comprises the steps of taking apple rot, jujube black spot and plum brown rot as test germs, adopting a plate confronting method, taking non-inoculated antagonistic bacteria QB002 as a control, repeating each treatment for 3 times, culturing at a constant temperature of 28 ℃, measuring the growth diameter of a germ colony inhibited by the antagonistic bacteria when the control colony grows over a plate, and calculating the bacteriostasis percentage. The results show that the strain QB002 has a certain inhibition effect on not only Sclerotinia Juglandis bacterial 1429-1, but also apple rot, jujube black spot and plum brown rot, shows broad antibacterial spectrum (figure 4), and the growth inhibition rate of pathogenic bacteria is shown in Table 2.

TABLE 2 bacteriostasis results of the strain QB002 against different germs

The invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, but it is possible to make modifications or improvements on the basis of the invention, which will be apparent to those skilled in the art. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. Bacillus subtilis QB002 with preservation number of CGMCC NO. 18218.

2. A fermentation product of Bacillus subtilis QB002 according to claim 1.

3. A bacterial agent comprising Bacillus subtilis QB002 or a fermentation product thereof according to claim 1.

4. A medicament comprising Bacillus subtilis QB002 or a fermentation product thereof according to claim 1.

5. The medicament according to claim 4, wherein the medicament is a medicament having an inhibitory effect on Sclerotinia juglandis (Juglanconis jugladina), Cytospora canescens (Cytospora salicicola, Cytospora mali), Sclerotinia scabra (Alternaria alternata), and Monilinia fructicola (Monilinia fructicola).

6. The use of Bacillus subtilis QB002, its fermentation product or microbial inoculum containing it, medicament of claim 1 in controlling or preventing plant diseases.

7. The use of claim 6, wherein the plant is walnut, populus Sinkiangensis, populus arvensis, populus diversifolia, willow, apple, birch, oleaster or grape.

8. The use according to claim 6, wherein the plant disease is a plant disease caused by Rhizoctonia solani (Juglanconis jugladina), Cytospora pomifera (Cytospora salicina), Phytospora spinulosa (Alternaria alternata) and Monilinia fructicola (Monilinia fructicola).

9. Use of Bacillus subtilis QB002, fermentation product thereof or microbial inoculum or drug containing the same according to claim 1 for inhibiting Rhizoctonia Juglandis (Juglancis Juglandina), Cytospora canker (Cytospora mali), Pseudocercospora spinulosa (Alternaria alternata), and Monilinia fructicola (Monilinia fructicola).