CN117946948B - Pseudomonas strain SCMHT-110 and application thereof - Google Patents
Pseudomonas strain SCMHT-110 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and discloses a pseudomonas strain SCMHT-110 and application thereof, wherein the pseudomonas strain SCMHT-110 is identified as Pseudomonas extremorientalis and is preserved in China center for type culture Collection (China, with the preservation number of 7 th year and 10 th year of 2023): the strain is a new pseudomonas strain with low toxicity, environment friendliness, remarkable prevention and control effects and broad-spectrum antibacterial activity, provides an effective way for preventing and treating kiwi fruit canker, and has good prevention and control potential for pathogenic fungi of kiwi fruit rot and postharvest fruit diseases such as botrytis cinerea, phomopsis and viticola.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a pseudomonas strain SCMHT-110 and application thereof.
Background
The kiwi fruit is a perennial vine native to China, and is popular with consumers due to unique fruit flavor and rich nutritional value, and the research report of the Chinese kiwi fruit industry in 2021 indicates that: the planting area of the kiwi fruits in China exceeds 18 ten thousand hm2, the annual output reaches 219 ten thousand tons, the world is first, and the main production areas are provinces of Shaanxi, sichuan, guizhou and the like. However, with the continuous development of industry, various diseases of kiwi fruits are more serious.
The kiwi canker is one of serious bacterial diseases in world kiwi production caused by pseudomonas syringae kiwi pathogenicity (Pseudomonas syringae pv. Actinidae, PSA). The disease is expressed as 'one disease and multiple symptoms', infects the trunk, branches, leaves, flower buds and roots of the kiwi fruits, and is extremely easy to cause death of plants. After the outbreak in japan since the 80 s of the 19 th century, the main areas of kiwi fruits such as china, korea, italy, new zealand, portugal, spanish, turkish and the like were sequentially dug. PSA is strongly pathogenic and infectious, not only can survive in fallen leaves and pruned shoots, but also can attach in asymptomatic leaves, posing a serious threat to the world's kiwi fruit industry development. In recent years, prevention and treatment of kiwi canker mainly depend on copper preparations (copper hydroxide, copper sulfate and the like) and antibiotics (kasugamycin and the like), but long-term application of the medicaments can not only cause drug resistance of pathogenic bacteria, but also cause toxicity and pollution to plant and planting environment microbial communities.
The bacterial flower rot of the kiwi fruits is also called as bacterial flower rot of the kiwi fruits, pathogenic bacteria of the bacterial flower rot are mainly Pseudomonas sp, and the bacterial flower rot mainly comprises Pseudomonas aeruginosa P. viridiflava, pseudomonas syringae P. syringae and the like, the flower buds and flowers of the kiwi fruits are damaged, young fruits and leaves are damaged, a large number of flowers and fruits fall off, small fruits and abnormal fruits can be caused, the yield and quality of the kiwi fruits are seriously influenced, the occurrence of the kiwi flowers rot is serious in recent years, and the pathogenic bacteria have generated certain drug resistance to traditional applied chemical pesticides, antibiotics and other control agents, so that the control difficulty of the kiwi flowers rot is increased, and the development of the kiwi fruits industry in China is limited.
The fruit fungus rot disease of the kiwi fruits after picking is one of important diseases of the kiwi fruits, serious economic losses can be caused in the post-ripening period of the kiwi fruits, the storage, transportation and sales periods of the fruits, pathogenic fungi which can cause the fruit rot of the kiwi fruits at present mainly comprise Botrytis cinerea (Botrytis cinerea), botrytis cinerea (Botryosphaeriaceae sp.) and phomopsis (Phomopsis sp.), the chemical prevention and treatment mainly using fungicides are mainly carried out in the present stage, and the method has the defects that great food safety hidden hazards exist for the kiwi fruits eaten, meanwhile, the fruit fungus rot disease after picking also generates drug resistance, and the development of industry is restrained.
Therefore, there is a need to find novel efficient, environment-friendly and broad-spectrum biological invention strains for preventing and treating diseases such as kiwi fruit canker, flower rot and postharvest fruit fungus rot so as to promote the healthy development of kiwi fruit industry.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a pseudomonas strain SCMHT-110 and application thereof, is a novel pseudomonas strain which is low in toxicity, environment-friendly, remarkable in prevention and control effects and has broad-spectrum antibacterial activity, particularly provides an effective way for preventing and treating kiwi fruit canker, and has good prevention and control potential for pathogenic fungi of kiwi fruit diseases such as botrytis cinerea, phomopsis and viticola, and a novel broad-spectrum antibacterial activity strain which can be used for preventing and treating various kiwi fruit diseases is developed as a novel source of biological pesticides.
The aim of the invention is realized by the following technical scheme:
The primary object of the present invention is to provide a Pseudomonas sp. SCMHT 110. SCMHT-110, molecular identification Pseudomonas extremorientalis, deposited with China center for type culture Collection, accession number: CCTCC NO: M20231249.
Further, the 16S rDNA gene sequence of the Pseudomonas strain SCMHT-110 is SEQ ID NO. 1.
Still further, the single colony of the Pseudomonas strain SCMHT-110 cultured on KB solid medium is round, light yellow, regular and regular in edge, and moist and smooth in surface.
In a second aspect of the invention, there is provided a biological agent comprising a Pseudomonas strain SCMHT-110 as described above.
In a third aspect of the present invention, there are provided a stock solution, a supernatant and a filtrate of the above Pseudomonas strains SCMHT-110.
Further, the preparation method of the stock solution, the supernatant and the filtrate comprises the following steps:
picking SCMHT-110 single colonies in KB liquid culture medium, placing the single colonies in a constant-temperature shaking table for culturing for 72 hours at 28 ℃ and 150rpm/min to obtain stock solution; centrifuging the stock solution at 4000rpm for 20min to obtain supernatant; the supernatant was filtered through a 0.2 μm filter to obtain a filtrate.
In a fourth aspect, the invention provides application of the pseudomonas strains SCMHT-110 and/or the biological agent and/or the stock solution, the supernatant and the filtrate in preventing and treating kiwi canker, flower rot and postharvest fruit fungus rot pathogenic bacteria of kiwi fruits.
Further, the pathogenic bacteria of the fruit fungus rot disease of the picked kiwi fruits are one or two or three of staphylococcus cinerea, phomopsis and staphylococcus aureus.
The beneficial effects of the invention are as follows:
The scheme provides the pseudomonas strain SCMHT-110, the 16S rDNA gene sequence of which is SEQ ID NO:1, the pseudomonas strain is a novel pseudomonas strain which has low toxicity, environment friendliness, remarkable prevention and control effects and broad-spectrum antibacterial activity, can provide an effective way for preventing and treating kiwi fruit canker, and has good prevention and control potential on pathogenic fungi of kiwi fruit rot and fruit diseases after kiwi fruit picking, namely botrytis cinerea, phomopsis and viticola.
Drawings
FIG. 1 is a strain morphology of Pseudomonas SCMHT-110 of the present invention on a culture medium;
FIG. 2 is a scanning electron microscope morphology of Pseudomonas SCMHT-110 of the present invention;
FIG. 3 phylogenetic tree based on 16S rDNA and whole genome;
FIG. 4 is a graphical representation of an antibacterial test of Pseudomonas SCMHT-110 of the present invention against different kiwi fruit disease pathogenic bacteria; wherein A is negative control, B is positive control streptomycin sulfate, C is stock solution cultured by the strain of the invention, D is supernatant fluid after the stock solution of the strain of the invention is centrifuged, E is filtrate after the supernatant fluid of the strain of the invention is filtered;
FIG. 5 is a graphical representation of the control experiment of Pseudomonas SCMHT-110 of the present invention on kiwi plants;
FIG. 6 is a graphical representation of the condition of lesions of kiwi leaves under different treatment groups of experimental conditions;
FIG. 7 is a graphical representation of test results of antagonism of fungi by Pseudomonas SCMHT-110 of the present invention; wherein A is a negative control; b is positive control prochloraz; c is stock solution cultured by the strain; d is supernatant fluid after the strain stock solution is centrifuged; e is the filtrate of the strain supernatant of the invention after filtration.
Detailed Description
The technical scheme of the present invention is described in further detail below with reference to specific embodiments, but the scope of the present invention is not limited to the following description.
Example 1
Isolation, purification and identification of Pseudomonas SCMHT-110
The strain Pseudomonas SCMHT-110 is a wild Chinese goosebeery male plant branch from a five-finger peak scenic spot (N26 DEG 32'85, E114 DEG 09'25;875 m) in Jinggang mountain city of Jiangxi province, and the sampling time is 2014 and 4 months.
1. Separation and purification method
Collecting wild kiwi fruit branches, filling into a sterile bag, and storing in an ice bag to a laboratory. The tissue of the bud eye part of the branch is taken by a scalpel, put into a sterile bag with a small amount of sterile normal saline (0.9%), knocked into a hammer, added into one third of the sterile bag, and soaked for more than 30 minutes. The pipettor took 100. Mu.L of its supernatant and placed on a prepared KB solid medium and streaked with an inoculating loop. After sealing the culture dish with the sealing film, the culture dish is placed in a constant temperature incubator at 28 ℃ in an inverted manner. Picking colonies with different colors and shapes by using an inoculating loop, scribing on KB solid culture medium, repeatedly purifying until SCMHT-110 single colonies with uniform size and consistent color are obtained, then sealing in a freezing tube by using a glycerol preservation method, and storing strains in an ultralow temperature refrigerator at-80 ℃.
2. Observation of culture State
The Pseudomonas SCMHT-110 strain is activated on KB solid culture medium, placed in a constant temperature incubator at 28 ℃ for culture, and colony growth is observed. After 24 hours of culture, the colony is round, the edge is neat and regular, the surface is moist and smooth, and the colony is light yellow; as shown in fig. 1. The strain morphology of Pseudomonas SCMHT-110 was observed and photographed at a magnification of 10000X using a scanning electron microscope and is shown in FIG. 2.
3. Strain biochemical index determination
SCMHT-110 single colonies were streaked on KB solid medium, and biochemical index measurement was performed according to the steps described in the bacterial trace biochemical identification tube (Qingdao high-tech Industrial park, haibo biotechnology Co., ltd.) and the reaction was shown in Table 1.
TABLE 1 SCMHT-110 Biochemical Properties
4. Molecular biological identification
Extracting DNA of the strain SCMHT-110 by using a OMGA bacterial genome DNA extraction kit, and sending the extracted DNA to Beijing qing biological science and technology Co-Ltd for sequencing and identification; the thalli are sent to the Beijing NodeB source technology and technology Co., ltd. For completing the sequencing of the chart, the molecular identification result is shown in a sequence table SEQ ID NO:1, and the 16S rDNA sequence length of the strain SCMHT-110 is 1401bp. Phylogenetic trees based on 16S rDNA and whole genome both showed SCMHT-110 clustered on the same branch as P. extremorientalisLMG19695T (FIG. 3). SCMHT-110 the computer-simulated DNA hybridization with the genome of P. extremorientalisLMG19695T was 96.4, far exceeding the new seed threshold (70%), a different strain belonging to the same species of bacteria as P. extremorientalisLMG 19695T.
Example 2
Screening of kiwi canker inventive strains
The invention uses oxford cup method to screen bacterial strain of pathogenic bacteria of kiwi fruit bacterial diseases (canker and flower rot). The method comprises the following specific steps:
1. Activation of target pathogenic bacteria: the strain was removed from the-80℃ultra-low temperature refrigerator, activated using the plate streaking method, and detailed information of the strain is shown in Table 2. Single colonies were picked up in KB liquid medium and placed in a constant temperature shaker at 28℃and 150rpm/min for 72h. 200. Mu.L of the original bacterial liquid (the bacterial liquid concentration is about 10 8 CFU/mL) is sucked by a liquid-transferring gun, the original bacterial liquid is respectively coated on KB culture dishes by a coater, oxford cups are placed on the culture medium coated with the bacterial liquid, and three groups of bacteria are repeated in parallel.
TABLE 2 information table of activated strains
2. Preparation of stock solution, supernatant and filtrate of the inventive strain (Pseudomonas SCMHT-110): SCMHT-110, picking single colony, culturing in KB liquid culture medium at 28deg.C in a constant temperature shaking table at 150rpm/min for 72 hr to obtain stock solution with concentration of 8.33X10 9 CFU/mL, centrifuging at 4000rpm for 20min to obtain supernatant, filtering the supernatant with 0.2 μm filter membrane to obtain filtrate, respectively sucking 200 μl of the filtrate with a pipette, and adding into oxford cup. The negative control is clear water (ddH 2 O), the positive control is 60mg/L streptomycin sulfate solution, after culturing for 48 hours in a constant temperature incubator at 28 ℃, observing whether a bacteriostasis ring exists in a culture dish, measuring the diameter of the bacteriostasis ring, and performing LSD variance analysis.
Strains SCMHT-110 have inhibitory effects on kiwi fruit canker strains 155, 540 and Pythium gracile 325 of different sources (shown in FIG. 4). Analysis of the data showed (table 3): for kiwi canker, the diameters of the inhibition zones of the antagonistic strain filtrate are larger than 30mm, the antibacterial effect is best, and compared with the positive control streptomycin sulfate, the strain stock solution and the strain supernatant, the antagonistic strain filtrate has obvious difference; for kiwi fruit flower rot, the strain stock solution and the supernatant have the best effect, are obviously different from filtrate and streptomycin sulfate, and have no obvious difference from the filtrate and the streptomycin sulfate. The indoor culture dish oxford cup test of the strain shows that the strain has good antibacterial activity and antagonism on pathogenic bacteria of kiwi canker and flower rot.
TABLE 3 antagonistic experiment antibacterial circle diameter (mm)
Note that: oxford cup diameter 7.5mm, LSD analysis of variance, with distinct letters in each column of the table indicating significant differences (p=0.05)
Example 3
SCMHT-110 control effect on kiwi fruit plants
1. Selecting healthy young seedlings of 'red sun' kiwi fruits with more than or equal to 6 leaves, marking the first leaves of each seedling with knitting wool, and treating 4 seedlings each. Activating strains SCMHT-110 (bacterial liquid concentration is 3×10 9 CFU/mL), preparing streptomycin sulfate with concentration of 100mg/kg, taking clear water (ddH 2 O) as a positive control, adding Tween 80 (final concentration is 0.5%) in each treatment, spraying on the front and back sides of leaves of seedlings, and experimental treatment is shown in Table 4.
2. The ulcer bacteria strains 155 and 540 were activated, diluted and mixed with ddH 2 O1:1, and the bacterial liquid concentration was 1.5X10. 10 8 CFU/mL. After 48 hours of spraying the strain of the invention, the back of the leaf was sprayed with ddH 2 O as a control according to the treatment of Table 4. Randomly and randomly placing the seedlings in a culture room (shown in figure 5), controlling the temperature to be 17-19 ℃ and the humidity to be 83-97% RH, and carrying out temperature and humidity detection by a temperature and humidity recorder. After 4 weeks, all leaves under the marker leaf were trimmed, the leaves were scanned one by one with a scanner (shown in FIG. 6), the leaf spot area percentage was determined with software leaf doctor, and the differences in the disease areas of each treatment were analyzed by LSD variance.
TABLE 4 evaluation of seedlings of the inventive strains
Note that: LSD analysis of variance, the different letters in each column of the table indicate that there is a significant difference (p=0.05)
The experimental results are as follows: the ulcerative bacteria cause serious disease of the leaves, and the area ratio of leaf spots is 14.61%; the strain SCMHT-110 has good control effect on canker with the concentration of 3 multiplied by 10 9 CFU/mL, and the area ratio of the lesion is only 4.57%; has no obvious difference with 100mg/kg of streptomycin sulfate, and has obvious difference with positive control canker. The leaf spot ratio of the single-application strain SCMHT-110 is not obviously different from that of the negative control water spraying, which proves that the single-application strain is safe to kiwi fruit leaves.
Example 4
Safety experiment
1. Blade safety experiment: the stock solution (8.33X10 9 CFU/mL), supernatant and filtrate of the strain of the invention obtained in example 2 were sprayed on leaves of healthy young seedlings of red-yang kiwi fruits, ddH 2 O was used as a negative control, and the mixed bacteria (1.5X10 8 CFU/mL) of ulcer bacteria 155 and 540 were used as a positive control, and 4 strains were repeated.
2. Stem safety test: the uppermost mature leaf (next leaf of immature leaf) of the 'red sun' plant was marked with a knitting wool, the stem was pierced with a toothpick below, the stock solution (8.33X10 9 CFU/mL), supernatant and filtrate of the strain of the invention obtained in example 2 were transferred to the toothpick piercing site with a pipette, ddH 2 O was used as a negative control, and the mixed bacteria (1.5X10 8 CFU/mL) of ulcer bacteria 155, 540 was used as a positive control, and 4 strains were repeated.
The temperature is controlled to be 17-19 ℃, the humidity is 83-97% RH, a temperature and humidity recorder is used for carrying out temperature and humidity detection, and the effect on the leaves and stems of the kiwi fruits is observed. The results show (Table 5) that the plants inoculated with the canker were found to have obvious leaf spots on the leaves and brown spot at the stem grafting sites, while the stock solution, supernatant and filtrate of the inventive strain were found to have no obvious symptoms, and the inventive strain was safe to kiwi fruits.
Table 5 safety experiments on kiwi plants
Note that: asymptomatic, + symptomatic, the++ symptoms are obvious
Example 5
Disease control effect of fungus fruit rot disease after kiwi fruit harvest
The bacterial cake method comprises inoculating the dominant fungus of fructus Actinidiae chinensis fruit disease, staphylococcus aureus (Botrytis cinerea) 755, phomopsis (no morphology Phomopsis sp.) 758, and Phyllostachys Pubescens (Botryosphaeria dothidea) 766 on PDA solid culture medium, culturing in constant temperature incubator at 28deg.C, and placing oxford cup around after the fungus has grown 1/3 of the diameter of the plate. The strain concentration of the invention is 8.33X10 9 CFU/mL of stock solution, the supernatant after the centrifugation of the stock solution at 4000rpm for 20min, and the filtrate after the filtration of the supernatant by a 0.2 μm filter membrane are added into an oxford cup. The positive control was 10mg/L prochloraz solution and three groups were repeated in parallel. After the fungus strain was cultured in a constant temperature incubator at 28℃for 72 hours, a photograph was taken (shown in FIG. 7). The results show (Table 6) that SCMHT-110 strains (stock solution, supernatant and filtrate) of the invention have good inhibition effect on staphylococcus griseus and phomopsis, and the inhibition effect is equivalent to the effect of 10mg/L prochloraz; the stock solution and the supernatant have certain inhibition effect on the viticola, but the effect is lower than prochloraz.
Table 6 antagonistic experiment inhibition zone effect
Note that: -no effect, + + effect, ++ effect is evident
Example 6
Disease control effect of fungus fruit rot disease after kiwi fruit harvest
Taking the 'red sun' well stored in a cold storage to room temperature, randomly taking 3 fruits, measuring the initial hardness of the fruits, respectively using two treatments of 0.5% Tween 80+ SCMHT-110 (concentration is 3X 10 8 CFU/ml) and 0.5% Tween 80+ clear water to soak for 20 minutes, airing, taking the non-soaked fruits as a control, putting 108 fruits in each treatment, and recording by a temperature and humidity recorder at the room temperature of 20-22 ℃. The number of rotten fruits is investigated every 2 days, rotten fruits are picked out, and the hardness of the fruits is measured until the hardness is reduced to 0.4 (the edible state of the red-yang kiwi fruits is between 1 and 0.4kfg in hardness, and the fruits below 0.4kfg are too soft and lose use value). The data show (Table 7) that kiwi fruits soften rapidly at room temperature, rapidly decreasing from the initial 2.29kfg to below 1kfg, reaching the edible state, and the strain SCMHT-110+0.5% tween treated by the invention prolonged the edible state for 2 days at room temperature. Compared with the blank treatment without soaking, the 0.5% Tween soaking can aggravate the rot of the fruits, but the treatment of SCMHT-110+0.5% Tween of the strain obviously reduces the rot quantity of the fruits, and the prevention effect reaches 61.5% (Table 8).
TABLE 7 fruit hardness variation at room temperature for different treatments
Remarks: hardness units kfg
TABLE 8 incidence and control of fruit after soaking fruit with different treatments
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (6)
1. A pseudomonas strain SCMHT-110, having a molecular identity of Pseudomonas extremorientalis, wherein said pseudomonas strain has been deposited in the chinese collection of typical cultures, accession number: CCTCC NO: M20231249.
2. A biological agent comprising pseudomonas strain SCMHT-110 of claim 1.
3. The stock solution, supernatant, filtrate of pseudomonas strains SCMHT-110 of claim 1.
4. The stock solution, supernatant and filtrate according to claim 3, wherein the method for preparing the stock solution, supernatant and filtrate comprises the steps of:
Picking SCMHT-110 single colonies in KB liquid culture medium, placing the single colonies in a constant-temperature shaking table for culturing for 72 hours at 28 ℃ and 150rpm/min to obtain stock solution;
centrifuging the stock solution at 4000rpm for 20min to obtain supernatant;
the supernatant was filtered through a 0.2 μm filter to obtain a filtrate.
5. Use of the pseudomonas strain SCMHT-110 of claim 1 and/or the biological agent of claim 2 and/or the stock solution, supernatant, filtrate of claim 3 for controlling fruit fungus rot pathogens of kiwi fruit canker, flower rot, post-harvest kiwi fruit;
The actinidae and the actinidia deliciosa pathogenic bacteria are Pseudomonas syringae pv.actinidae and the flower rot pathogenic bacteria are Pseudononas syringae pv.syringae;
the pathogenic bacteria of the fungus rot disease of the fruit of the picked kiwi fruits are one or two of Botrytis cinerea and Phomopsis sp.
6. Use of the pseudomonas strain SCMHT-110 of claim 1 and/or the biological agent of claim 2 and/or the stock solution of claim 3, supernatant for controlling post harvest fruit fungal rot pathogens of kiwi fruits;
the pathogenic bacteria of the fungal rot disease of the fruit of the picked kiwi fruits are Botryosphaeria dothidea.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080089324A (en) * | 2008-09-11 | 2008-10-06 | 조정일 | Growing methods of kiwi using streptomyces cultivation mixture |
| CN107083344A (en) * | 2017-06-02 | 2017-08-22 | 四川省自然资源科学研究院 | A kind of pseudomonad strain for preventing and treating Prospect on Kiwifruit Bacterial Canker and its application |
| CN109609402A (en) * | 2018-12-21 | 2019-04-12 | 湖北工程学院 | Te Jila bacillus Bacillus tequilensis XG18 and application |
| CN114304146A (en) * | 2021-12-07 | 2022-04-12 | 西北农林科技大学 | Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof |
| CN116970521A (en) * | 2023-06-21 | 2023-10-31 | 贵州大学 | Bacillus bailii GUMHT p116 and application thereof |
| CN117448189A (en) * | 2022-07-20 | 2024-01-26 | 浙江大学 | Bacillus and application thereof |
| CN117965404A (en) * | 2024-04-02 | 2024-05-03 | 西北农林科技大学深圳研究院 | Pseudomonas orientalis and application thereof in plant disease control |
| CN118006511A (en) * | 2024-04-08 | 2024-05-10 | 西北农林科技大学深圳研究院 | Pseudomonas aeruginosa subspecies orange strain and application thereof |
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080089324A (en) * | 2008-09-11 | 2008-10-06 | 조정일 | Growing methods of kiwi using streptomyces cultivation mixture |
| CN107083344A (en) * | 2017-06-02 | 2017-08-22 | 四川省自然资源科学研究院 | A kind of pseudomonad strain for preventing and treating Prospect on Kiwifruit Bacterial Canker and its application |
| CN109609402A (en) * | 2018-12-21 | 2019-04-12 | 湖北工程学院 | Te Jila bacillus Bacillus tequilensis XG18 and application |
| CN114304146A (en) * | 2021-12-07 | 2022-04-12 | 西北农林科技大学 | Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof |
| CN117448189A (en) * | 2022-07-20 | 2024-01-26 | 浙江大学 | Bacillus and application thereof |
| CN116970521A (en) * | 2023-06-21 | 2023-10-31 | 贵州大学 | Bacillus bailii GUMHT p116 and application thereof |
| CN117965404A (en) * | 2024-04-02 | 2024-05-03 | 西北农林科技大学深圳研究院 | Pseudomonas orientalis and application thereof in plant disease control |
| CN118006511A (en) * | 2024-04-08 | 2024-05-10 | 西北农林科技大学深圳研究院 | Pseudomonas aeruginosa subspecies orange strain and application thereof |
Non-Patent Citations (10)
| Title |
|---|
| 四川省猕猴桃溃疡病调查及病原菌株型鉴定;何利钦等;中国南方果树;20190718;第48卷;第73-78, 90页 * |
| 患溃疡病或正常猕猴桃内生及土壤细菌多样性及群落结构研究(;张钟月;中国优秀硕士学位论文全文数据库（电子期刊）;20210715(第07期);第1-68页 * |
| 我国主产区猕猴桃采后腐烂病病原多样性研究;封露等;植物病理学报;20210321;第52卷;第1-8页 * |
| 梨灰霉病菌拮抗细菌Pseudomonas extremorientalis的鉴定、发酵条件优化及防效评价;王燕等;中国生物防治学报;20190608(03);第90-96页 * |
| 猕猴桃溃疡病生防芽孢杆菌B2的鉴定及应用;邵宝林等;中国农学通报;20230824;第31卷;第103-108页 * |
| 猕猴桃溃疡病防治策略的研究现状与展望;游雨欣等;浙江农业科学;20220421;第63卷;第1322-1328页 * |
| 猕猴桃细菌性溃疡病生防菌的筛选、鉴定及其防效初探;朱海云等;微生物学杂志;20161015(第05期);第33-36页 * |
| 猕猴桃细菌性溃疡病的发生及其防治;陈兴涛等;四川农业科技;20160915(第09期);第53-54页 * |
| 猕猴桃细菌性病害的发生及抗性研究进展;张弛等;南方农业(园林花卉版);20110415(第02期);第90-96页 * |
| 红阳猕猴桃叶斑病病原鉴定及室内药剂筛选;郭睿文等;资源开发与市场;20160615;第32卷;第741-744, 753页 * |
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