CN110669702B - Biocontrol strain JK17 for preventing and treating walnut anthracnose as well as screening method and application thereof - Google Patents
Biocontrol strain JK17 for preventing and treating walnut anthracnose as well as screening method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a biocontrol strain JK17 for preventing and treating walnut anthracnose, and a screening method and application thereof. The preservation number of the biocontrol strain JK17 is as follows: CGMCC No. 18726. According to the invention, the pseudomonas fluorescens in the pseudomonas is a biocontrol strain obtained by separating and purifying walnut leaves without plant diseases and insect pests, and can prevent and treat anthracnose of walnut, the prevention and treatment effect can reach more than 84%, and the bacteriostatic effect is good; the biocontrol strain provided by the invention can reduce the use of pesticides, is safe and nontoxic, and realizes the dual purposes of walnut protection and treatment.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a biocontrol strain JK17 for preventing and treating walnut anthracnose, and a screening method and application thereof.
Background
Walnut (Juglans regia L.) and walnut, which has a long cultivation history in China and is widely planted, is an important strategic tree species of woody grain and oil in China. Because the walnuts have high economic, ecological and social benefits, the demand of the walnuts in domestic and international markets is continuously increased. With the continuous expansion of walnut cultivation area, the disease also becomes more and more serious. The anthracnose is one of main diseases damaging walnut production, and the incidence rate of the anthracnose in major walnut producing areas such as Taian, Jinan and Jining in Shandong province is up to 90 percent, and the disease rate is more than 30 percent. Anthracnose mainly damages fruits of walnuts, leaves, buds and young tips, easily causes early fruit drop, rotten fruits or nut shriveling after being infected with diseases, and seriously influences yield and quality. There are two common causes of anthracnose of peach, Colletotrichum gloeosporioides (Penz.) Penz.Sacc. and Colletotrichum acutum J.H.Simmonds ex J.H.Simmonds ].
The anthracnose of walnut becomes a main factor for restricting the development of walnut industry. In order to prevent anthracnose, chemical bactericides are mainly used in production, so that a large amount of pesticides, capital and labor are required to be spent each year. The pesticide resistance of walnut anthracnose pathogen is caused by long-term use of the bactericide in an orchard, so that the control difficulty of anthracnose is increased. And the long-term use of a large amount of chemical pesticide will cause environmental pollution and serious harm to human health. Therefore, it is urgent to find a new method for safely, non-toxic and effectively controlling the anthracnose of the walnut. At present, research reports of biological control bacteria as substitutes of chemical agents in academic circles are increased year by year, and biological control of diseases by using the biological control bacteria is an inevitable trend.
Disclosure of Invention
The invention aims to provide a biocontrol strain JK17 for preventing and treating walnut anthracnose.
The invention also provides a screening method of the biocontrol strain for preventing and treating the walnut anthracnose.
The invention further aims to provide application of the biocontrol strain in preventing and treating the anthracnose of the walnut.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a biocontrol strain JK17 for preventing and treating walnut anthracnose, which has the preservation number as follows: CGMCC No. 18726.
The invention also provides a screening method of the biocontrol strain JK17 for preventing and treating the walnut anthracnose, which comprises the following steps:
(1) collecting and separating a sample: collecting walnut leaves without damage of diseases and insects from a walnut garden, taking 0.5-1.0 cm of the walnut leaves, disinfecting the walnut leaves with 75% alcohol for 30s, treating the walnut leaves with 1.0% NaClO for 10min, and finally washing the walnut leaves with sterile water for three times; adding 5mL of sterile water into the surface-sterilized leaves, grinding, and standing for 10 min;
(2) after standing, the mixture is subjected to gradient concentration of 1 × 102、1×103And 1X 104Sequentially diluting with sterile water, respectively coating 100 μ L onto NA plate, and culturing in 28 deg.C constant temperature incubator; after the plate grows out of the colonies, selecting a single colony according to the morphological characteristics of the colony, and obtaining a bacterial strain JK17 after scribing and purification, wherein the bacterial strain JK17 is stored on an NA inclined plane in a refrigerator at 4 ℃.
The invention also provides application of the biocontrol strain JK17 in preventing and treating walnut anthracnose.
Furthermore, when the biocontrol strain is used for preventing and treating the walnut anthracnose, the activated strain or strain fermentation liquor is adopted.
The strain fermentation liquor is prepared by the following method: placing JK17 strain in 100 mL beef extract peptone liquid medium (NB), and shake-culturing at 28 deg.C for more than 2 daysThe concentration of JK17 bacterial liquid reaches 105～106CFU/mL, centrifuging zymocyte solution 8000rmp for 20min, collecting supernatant, and filtering with 0.22 μ filter membrane to obtain sterile filtrate.
Further, the frequency of the oscillation is 160 r/min.
Information on strain preservation
Preservation time: the year 2019, the day 10, the day 23,
the preservation unit: china general microbiological culture Collection center,
the preservation number is: the number of the CGMCC number 18726,
the address of the depository: the microbiological research institute of western road 1, 3, national academy of sciences, north-kyo, chaoyang, the postal code: 100101
And (3) classification and naming:Pseudomonas fluorescens
the invention has the beneficial effects that:
(1) according to the invention, the pseudomonas fluorescens in the pseudomonas is a biocontrol strain obtained by separating and purifying walnut leaves without plant diseases and insect pests, and can prevent and treat anthracnose of walnut, the prevention and treatment effect can reach more than 84%, and the bacteriostatic effect is good;
(2) the biocontrol strain provided by the invention can reduce the use of pesticides, is safe and nontoxic, and realizes the dual purposes of walnut protection and treatment.
Drawings
FIG. 1 shows the colony morphology of JK17 strain.
FIG. 2 is a diagram showing the bacteriostatic action of the test bacterial strain JK17 on walnut anthrax.
FIG. 3 is a phylogenetic tree of strain JK17 and other Pseudomonas strains constructed by the adjacency approach based on the 16S rDNA sequence.
FIG. 4 is a diagram showing the bacteriostatic action of the fermentation liquor of the test bacterial strain JK17 on walnut anthrax.
FIG. 5 is a bacteriostasis diagram of indoor in vitro inoculation of walnut anthracnose.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
Example 1
(1) Collecting and separating a sample: collecting walnut leaves without damage of diseases and insects from a walnut garden, taking 0.5 cm-sized leaves, disinfecting the leaves with 75% alcohol for 30s, treating the leaves with 1.0% NaClO for 10min, and finally washing the leaves with sterile water for three times; adding 5mL of sterile water into the surface-sterilized leaves, grinding, and standing for 10 min;
(2) after standing, the mixture is subjected to gradient concentration of 1 × 102、1×103And 1X 104Sequentially diluting, respectively coating 100 μ L of the diluted solution on an NA plate, and culturing in a constant-temperature incubator at 28 ℃; after the plate grows out of the colonies, selecting a single colony according to the morphological characteristics of the colony, and obtaining a bacterial strain JK17 after scribing and purification, wherein the bacterial strain JK17 is stored on an NA inclined plane in a refrigerator at 4 ℃ as shown in figure 1;
the strain fermentation liquor is prepared by the following method: placing the JK17 strain in 100 mL beef extract peptone liquid medium (NB) (the formula of the medium is 10g of peptone, 3g of beef extract, 5g of sodium chloride, 1000mL of distilled water and the pH value is 7.0-7.2), culturing for more than 2d at 28 ℃ in a shaking mode (160 r/min), wherein the concentration of the JK17 strain liquid reaches 105～106CFU/mL, centrifuging zymocyte solution 8000rmp for 20min, collecting supernatant, and filtering with 0.22 μ filter membrane to obtain sterile filtrate.
(3) Preliminary identification of biocontrol bacteria
The antagonism of the separated strain to the walnut anthracnose is determined by adopting a plate antagonism method. Activating walnut anthrax bacteria preserved at 4 deg.C on PDA plate, and making into desired shape with puncherd=7 mm), a mycelium block is punched at the edge of a colony of activated walnut anthracnose, the mycelium block is placed in the center of a PDA (personal digital assistant) plate, the activated bacteria strain to be tested is inoculated at a point 2cm away from the mycelium block, and walnut anthracnose without the inoculated bacteria strain is used as a blank control. After culturing at the constant temperature of 26 ℃ for 7d, measuring the radius of the inhibition zone. As can be seen from FIG. 2, the JK17 strain has strong inhibitory effect on the growth of hypha of anthracnose of walnut.
(4) 16S rDNA sequence analysis and identification of biocontrol bacterial strain
Collecting 4mL of bacterial liquid cultured to logarithmic phase, centrifuging and collecting thalli, extracting bacterial genome DNA by using a bacterial DNA extraction kit, performing 16SrDNA amplification by using the genomic DNA as a template and universal primers F27 (5'-AGAGTTTGATCATGGCTCAG-3') and R1492 (5'-GGCTACCTTGTTACGACTT-3'), performing gel recovery on agarose gel containing target DNA by using a gel recovery kit, handing over a recovered PCR product to a company Limited in the biological engineering (Shanghai) for sequencing, and performing homology analysis on a sequence obtained by sequencing in an NCBI database by using Blast. The homology of the JK17 strain and the 16S rDNA of the pseudomonas reaches more than 98 percent. Phylogenetic trees were constructed from 39 strains of pseudomonas using MEGA6.0 software based on the neighbor joining method (fig. 3). The phylogenetic tree results show that the JK17 strain is on the same branch as Pseudomonas fluorescens (KJ511771.1), indicating that the JK17 strain has the closest relationship with Pseudomonas fluorescens. The JK17 strain was identified as P.fluorescens in the genus Pseudomonas.
Effect verification
Preparation of JK17 strain fermentation liquor and antagonistic effect thereof on walnut anthrax
Placing JK17 strain in 100 mL beef extract peptone liquid medium (NB), culturing at 28 deg.C under shaking (160 r/min) for more than 2 days until the concentration of JK17 strain liquid reaches 105～106CFU/mL. Centrifuging the zymocyte solution at 8000rmp for 20min, collecting supernatant, and filtering with 0.22 μ filter membrane to obtain sterile filtrate. Then using a punch (d=7 mm), hypha blocks are punched at the edges of activated colony of walnut anthracnose, the hypha blocks are placed in the center of a PDA flat plate, after 48 hours of culture, 1 aseptic filter paper disc is respectively placed at the positions, 2cm away from the hypha blocks, of the upper part, the lower part, the left part and the right part of a culture dish, and 10 mu L of JK17 bacterial liquid fermentation liquor stock solution and sterile filtrate are respectively injected into each filter paper disc. The inoculated dishes were incubated at 26 ℃ and each treatment was repeated 3 times, and sterile water was injected onto the filter paper sheets as a control. When the control hypha grows over the plate, the colony diameter is measured by a cross method, and the result shows that the colony diameter after the fermentation liquor stock solution treatment is 17.81mm, the control colony diameter is 89.25mm, and the inhibition rate is 86.85%. The sterile filtrate does not inhibit the growth of pathogenic bacteria. As can be seen from FIG. 4, the JK17 strain fermentation stock solution has strong inhibitory effect on the growth of walnut anthracnose hypha.
(II) JK17 indoor in-vitro prevention and effect determination for walnut anthracnose
Sterilizing the surface of healthy and disease-free walnut fruits, perforating the surface of the walnut fruits by using a sterilized perforator to perform wound inoculation, and then performing tests according to the following 4 treatment methods: treating 1, inoculating only walnut anthracnose fungus cakes; processing 2, inoculating JK17 bacterial liquid, and then inoculating a walnut anthracnose bacterial cake; treating 3, inoculating JK17 bacterial liquid only; treatment 4, inoculation of sterile water only. 15 walnut fruits were selected for each treatment and repeated 3 times. Placing the treated walnut in a sterilized beaker, covering with a preservative film, maintaining a certain humidity, placing in a constant-temperature incubator at 26 ℃, checking the walnut disease condition after 7 days, and calculating the disease index and the prevention and treatment effect according to the following formula. Walnut anthracnose grade division standard: level 0: the disease is not developed; level 1: the lesion area accounts for less than 25% of the whole fruit surface area; and 2, stage: the lesion area accounts for 25% -50% of the whole fruit surface area; and 3, level: the lesion area accounts for 50% -75% of the whole fruit surface area; 4, level: the lesion area accounts for more than 75% of the whole fruit surface area. Disease index = Σ (number of disease stages × number of corresponding disease stage fruits)/(highest number of disease stages × total number of fruits) × 100; the prevention and treatment effect = (blank control disease index-treatment disease index)/blank control disease index x 100%. The result shows that the scab area of the walnut after the inoculation of the biocontrol bacteria and then the inoculation of the anthrax is obviously smaller than the scab area of the walnut after the inoculation of the anthrax (figure 5), and the control effect of the biocontrol bacteria JK17 is 89.02%. The wound withered lesion inoculated with the biocontrol bacteria only does not expand. The JK17 strain fermentation liquor has good effect on the in vitro prevention and treatment of the walnut anthracnose, and the JK17 has double effects of protection and treatment of the walnut anthracnose.
(III) determination of field control effect of JK17 strain on walnut anthracnose
A test for preventing and controlling the walnut anthracnose in the field is carried out in a walnut plantation. Selecting walnut fruits without disease spots randomly in all directions of the crown, washing with sterile water, and drying in the air for later use. After the walnut is punched, biocontrol strain fermentation liquor is smeared on the walnut, then the walnut anthracnose strain cake is inoculated, after the treatment, the walnut is bagged by a plastic bag for moisturizing, and meanwhile, only the inoculated anthracnose is used as a control. Repeating the treatment for 10 fruits 3 times, investigating and recording the disease incidence at 7d, and calculating the disease prevention effect. The morbidity of only inoculating the anthrax is 100% at 7d after inoculating the anthrax, and the control effect of inoculating JK17 is 83.99%. The JK17 can be used for preventing and controlling the anthracnose of the walnut in the field.
Claims (5)
1. A biocontrol strain JK17 for preventing and treating walnut anthracnose is characterized in that the preservation number is as follows: CGMCC No.18726, and the classification and designation of the biocontrol strain are as follows: pseudomonas fluorescens (Pseudomonas fluorescens）。
2. The application of the biocontrol strain JK17 as defined in claim 1 in preventing and treating anthracnose of walnut.
3. The use of claim 2, wherein the biocontrol strain is activated strain or strain fermentation broth for preventing and treating anthracnose of walnut.
4. The use of claim 3, wherein the strain fermentation broth is prepared by the following method: placing JK17 strain in 100 mL beef extract peptone liquid medium, performing shake culture at 28 deg.C for more than 2 days until the concentration of JK17 strain liquid reaches 105～106CFU/mL, centrifuging zymocyte solution 8000rmp for 20min, collecting supernatant, and filtering with 0.22 μ filter membrane to obtain sterile filtrate.
5. Use according to claim 4, wherein the frequency of the oscillations is 160 r/min.
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