Corsaccharomyces cohnii and its application in preventing and controlling plant diseases and promoting growth

Abstract

The present invention discloses Cossackia cohnii and its application in preventing and controlling plant diseases and promoting growth. The present invention separates endophytes from soybean plants, and finally screens out an endophyte strain SH2 that can promote plant growth, prevent and control plant diseases and improve plant drought resistance. The strain SH2 is identified as Cossackia cohnii ( Kosakonia cowania ), and its microbial preservation number is CGMCC No. 30737. Experiments have shown that the Corsaccharomyces cohnii SH2 can produce IAA and promote the increase of soybean plant height, root length or fresh weight, prevent and control soybean root rot or improve soybean drought resistance, and has application prospects in promoting plant growth, preventing and controlling plant diseases or improving plant drought resistance.

Description

Technical Field

[0001] The present invention relates to endophytes isolated from plants, and in particular to Cossackia corynaeus isolated from soybean plants. Kosakonia cowanii ) and its application in promoting plant growth, preventing and controlling plant diseases or improving plant drought resistance, belonging to the field of Cossackia coli and its application. Background Art

[0002] Studies have shown that plant endophytes play an important role in the drought resistance of host plants and enhance their drought resistance by regulating the growth, metabolic activities and genetic traits of the host in various ways. Endophytes improve host resistance by regulating plant growth and physiological activities, such as increasing the number of stems and branches and biomass of plants, reducing the relative water content and chlorophyll content of leaves, inducing changes in stomatal conductance and net photosynthetic rate, etc. At present, there are many studies on the use of microorganisms to improve plant stress resistance, but there are few reports on the use of Cossackia coli to improve plant drought resistance and improve soybean plant resistance to root rot.

[0003] With the rapid development of agriculture, related environmental problems have become increasingly prominent, especially desertification and salinization of cultivated land soil, which have become urgent ecological protection issues to be solved. Therefore, an efficient, convenient and environmentally friendly method to improve plant drought tolerance is of great significance to promote the green and sustainable development of agriculture. Summary of the invention

[0004] One of the objects of the present invention is to provide a strain of Cossackia coli ( Kosakonia cowanii ) strain;

[0005] The second object of the present invention is to provide a fermentation broth, a microbial preparation or a compost prepared by the Cossackia coli strain;

[0006] The third object of the present invention is to apply the Cossackia coli strain to promote plant growth, prevent and treat plant diseases or improve plant drought resistance.

[0007] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0008] One aspect of the present invention is to provide a strain of Cossackia coli ( Kosakonia cowanii )SH2, its microbial preservation number is CGMCC No.30737; its classification name is: Cossackia coli Kosakonia cowanii ; The preservation time is: May 22, 2024; the preservation unit is: General Microbiology Center of China Culture Collection Administration; the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The colony and bacterial morphology of Cossackia coli described in the present invention are as follows: after culture on LB medium, the colony is observed to be nearly circular in shape, with a smooth and moist surface, and a yellow, opaque color.

[0010] The isolated Cossackia coli SH2 produced by the present invention can promote the growth of soybeans by producing IAA; the Cossackia coli SH2 has a significant inhibitory effect on fungi including Fusarium oxysporum, and can play a clear preventive and therapeutic role on soybean root rot; the Cossackia coli SH2 has good drought resistance and can improve the resistance of soybeans to drought environments.

[0011] Another aspect of the present invention is to provide a fermentation liquid prepared from the Cossackia coli SH2, or a microbial preparation or compost containing the Cossackia coli SH2; wherein the effective viable count of Cossackia coli SH2 in the microbial preparation or compost is 1×10 6~8 cfu / mL.

[0012] In a preferred specific embodiment, the present invention provides a method for preparing the fermentation broth, comprising: activating the strain of Cossackia coli SH2 and inoculating it into a liquid culture medium for fermentation to obtain a fermentation broth; wherein the composition of the liquid culture medium is, by mass percentage, 1.5% tryptone, 0.58% yeast extract, 0.54% NaCl, and the remainder is distilled water, and the pH value is 6.8; the fermentation culture conditions include: the fermentation culture temperature is 27°C, the fermentation culture is carried out in a shaker, wherein the shaker speed is 150 r / min, and the fermentation time is 3 d.

[0013] Those skilled in the art can prepare the Cossackia coli SH2 provided by the present invention into various conventional microbial preparations or composts according to conventional preparation methods of microbial preparations, which are all technical means well known to those skilled in the art.

[0014] Another aspect of the present invention is to use the Cossackia coli SH2, the fermentation liquid prepared by the Cossackia coli SH2, or the microbial preparation or compost containing the Cossackia coli SH2 to promote plant growth, prevent and control plant fungal diseases or improve plant drought resistance.

[0015] In a preferred embodiment of the present invention, the Cossackia coli SH2, the fermentation broth prepared by the Cossackia coli SH2, or the microbial preparation containing the Cossackia coli SH2 is used to produce IAA; wherein the yield of IAA is 47.89 μg / mL.

[0016] In a preferred specific embodiment of the present invention, the Cossackia coli SH2, the fermentation broth prepared by the Cossackia coli SH2, or the microbial preparation containing the Cossackia coli SH2 is used to promote plant growth; wherein promoting plant growth includes increasing the plant height, root length or fresh weight of the plant; wherein the fresh weight refers to the weight of the whole plant; and the plant is soybean.

[0017] In a preferred specific embodiment of the present invention, the Cossackia schrenkiana SH2, the fermentation broth prepared by the Cossackia schrenkiana SH2, or the microbial preparation containing the Cossackia schrenkiana SH2 is used to prevent and control plant diseases; wherein the pathogens of the plant diseases include Fusarium oxysporum; wherein, more preferably, the plant disease is soybean root rot.

[0018] In a preferred embodiment of the present invention, the Cossackia cohnii SH2 is used to improve the drought resistance of plants; wherein the plant can be a monocot or a dicot, preferably soybean.

[0019] The present invention isolates endophytes from soybean plants and finally screens out a strain SH2 that can promote plant growth, prevent and treat plant diseases or improve plant drought resistance. The strain SH2 is identified as Cossackia cohnii ( Kosakonia cowanii ). Experiments have shown that the Cossackia cohnii SH2 can produce IAA to promote the increase of soybean plant height, root length or fresh weight, has a significant preventive and therapeutic effect on fungal plant diseases such as soybean root rot, and can also significantly improve soybean's resistance to drought environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the colony morphology of Coxsackia coli SH2;

[0021] Figure 2 is the SH2 phylogenetic tree of Coxsackia coli;

[0022] Figure 3 This is a graph showing the results of the IAA production capacity test of Coxsackia coli SH2;

[0023] Figure 4 This figure shows the results of promoting plant growth in soybean plants inoculated with Coxsackieae SH2 under drought stress. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0025] Example 1 Isolation and identification of strain SH2

[0026] 1. Strain Isolation

[0027] Using the tissue separation method, the soybean plants collected from the field were rinsed with water several times to keep the surface clean. The plant tissues were cut and soaked in 75% ethanol for 3 minutes and 2% NaClO for 15 minutes to remove the surface bacteria. The surface ethanol and NaClO were rinsed clean with sterile water three times. The chopped plant tissue blocks were placed in sterile water and shaken overnight to obtain a solution containing endophytes. The solution containing endophytes was diluted and spread on LB solid culture medium and cultured at 25°C. After the colonies grew, they were streaked several times according to the color, morphology, size, etc. of the colonies, and a single strain was obtained after separation and purification. The endophyte finally screened out was named SH2.

[0028] 2 Identification of strain SH2

[0029] After the strain SH2 was cultured on LB solid medium at 25℃ for 48 h, the colony morphology was as follows Figure 1 As shown. The colony is nearly round in shape, smooth and moist on the surface, yellow in color, and opaque. Pick a single colony of the strain to be identified in a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 min to collect the bacteria. Bacterial DNA was extracted using a bacterial genomic DNA extraction kit purchased from Tiangen Biochemical Technology Co., Ltd. For detailed steps, please refer to the product manual. Using 16S rDNA universal primers and yrA The primers gyrA-F and gyrA-R were used to amplify the 16S rDNA and yrA The gene fragments were amplified by PCR using conventional methods. After the PCR products were tested by 1% agarose gel electrophoresis, they were sequenced to obtain the sequences, which were then analyzed by NCBI online BLAST. Figure 2 As shown, a phylogenetic tree was constructed. Combined with morphological characteristics, it was identified as Cossackia coli ( Kosakonia cowani ).

[0030] Example 2 Preparation of Cossackia coli SH2 fermentation broth

[0031] 1. Plate activation

[0032] Cossackia coli SH2 was inoculated on LB plates and cultured at 28°C for 3 days.

[0033] 2 Liquid fermentation

[0034] The culture medium formula is calculated by weight percentage, specifically: 1.5% tryptone, 0.58% yeast extract, 0.54% NaCl, and the rest is distilled water, and the pH value is 6.8.

[0035] The activated strain was inoculated into a 250 mL triangular flask (100 mL per flask) and fermented for 3 days at 27 °C with a shaking speed of 150 r / min to obtain the fermentation liquid of Cossackia coli SH2. The effective viable count of Cossackia coli SH2 was 1×10 8 cfu / mL.

[0036] Test Example 1 Test on IAA production by Coxsackie SH2

[0037] 1 Test method

[0038] King'B medium: 20 g peptone, 1.5 g magnesium sulfate heptahydrate, 1.5 g potassium hydrogen phosphate, distilled water to 1000 mL, pH 7.0-7.2.

[0039] Salkowski colorimetric solution: Weigh 12 g of ferric sulfate and dissolve it in distilled water. Then slowly add 429.7 mL of concentrated sulfuric acid. After the solution cools, dilute to 1 L with distilled water. The measurement range is 0.3~20 mg / mL.

[0040] The ability of K. cohnii SH2 to produce IAA was determined using the classic Salkowski reagent colorimetric method. A single colony was picked from the pure bacterial plate and inoculated into LB medium for activation. After a single colony grew out, a single colony was picked and inoculated into liquid LB medium to prepare a bacterial solution (OD 660 =1). The bacterial solution was inoculated into King'B medium without tryptophan at a 1% inoculation volume, and sterile water was used as a control. The above-mentioned conical flask was placed in a shaking incubator at 28°C and 160 rpm for 7 days. 1 mL of bacterial solution was added dropwise to the test tubes, and IAA was added to one of the test tubes as a control. The same amount of Salkowski colorimetric solution was added respectively. The test tubes were placed at room temperature for color development for 15 minutes, and the color change was observed. It was compared with IAA and King'B medium without tryptophan with sterile water. The redder the solution color, the stronger the ability to produce IAA. If it does not change color, it is negative and does not produce IAA.

[0041] The IAA production capacity of Cossackia coli SH2 was quantitatively determined under the same culture conditions as above. Three replicates were set up in each group. The OD of the bacterial solution cultured for 24 h was first determined. 600 Then centrifuge the bacterial solution at 10000 r / min for 10 min, take the supernatant and add an equal volume of Salkowski colorimetric solution, keep it in the dark for 30 min, and measure the OD immediately after taking it out. 530 The value was adjusted to zero using CK added with colorimetric solution to calculate the IAA production; the bacterial concentration OD was calculated 600 When the value is 1, it is the content of IAA in the fermentation broth per unit volume.

[0042] 2 Test results

[0043] The preliminary results of IAA production by Coxsackie SH2 are as follows Figure 3 As shown, after the Salkowski colorimetric solution was added to the bacterial solution of Cossackia coli SH2, the color of the fermentation solution turned light red, indicating that Cossackia coli SH2 has a certain ability to produce IAA.

[0044] The quantitative determination of the IAA production capacity of Cossackia coli SH2 showed that the IAA production of Cossackia coli SH2 was 47.89 μg / mL.

[0045] Test Example 2 Test on the ability of Cossackia coli SH2 to promote plant growth

[0046] 1 Test method

[0047] Preparation of fermentation broth of Cossackia coli SH2: Use a 5-mm diameter puncher to punch out 5 bacterial cakes of Cossackia coli SH2, transfer them to a conical flask containing 200 mL LB liquid culture medium, and culture them at 27 °C and 180 r / min for 3 days. Then, filter through double-layer gauze to remove excess bacterial cakes and agar blocks, and dilute the fermentation broth 100 times with sterile water for later use.

[0048] Soybean seeds (Dongsheng 719) were soaked in 75% ethanol for 30 s and 5% NaClO for 45 s to remove the bacteria on the surface of the seeds. After rinsing with sterile water three times to clean the surface ethanol and NaClO, they were transplanted into sterilized nutrient soil pots with a size of 11 cm × 9 cm. The sterile soil and sterilized vermiculite were mixed in a volume ratio of 1:1. When the seedlings grew to the 3-5 leaf stage, they were treated. The prepared fermentation liquid of Cossackia SH2 was inoculated into the roots of the plants by the root injury inoculation method, 10 mL per plant, and the root irrigation treatment with an equal amount of blank LB liquid culture medium was used as a control. Twelve pots were treated each time. The temperature was maintained at 23°C and the relative humidity was 60%. The LED light source in the climate chamber was used for supplementary lighting. The light and dark conditions were alternating for 12 hours, and watering was performed every 5 days. After 30 days, the survey was conducted, the growth indicators were recorded, and the growth promotion effect was calculated.

[0049] 2 Test results

[0050] The growth-promoting ability test of Cossackia coli SH2 is shown in Table 1 and Figure 4 As shown in the figure, the height, root length and fresh weight of soybean plants increased by 27.72%, 45.24% and 122.05% respectively after inoculation with Cossackia coli SH2, indicating that Cossackia coli SH2 has a significant growth-promoting effect on soybean plants.

[0051] Table 1 Growth-promoting effect of Cossackia cohnii SH2 on soybean plants

[0052]

[0053] Test Example 3 Test of Cossackia cohnii SH2 in preventing and controlling plant diseases

[0054] 1 Test method

[0055] The pathogen used in the present invention is Fusarium oxysporum ( Fusarium oxysporum ), which was preserved and purified by the Institute of Plant Protection, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences.

[0056] The fermentation broth of Cossackia cohnii SH2 was prepared according to the method of Example 2, and the fermentation broth was diluted 100 times with sterile water for later use; Fusarium oxysporum was inoculated into PDA liquid culture medium and cultured at 25°C for 7 days to obtain a spore suspension, and the spore concentration was adjusted to 10 7 spores / mL.

[0057] Soybean seeds (Dongsheng 719) were soaked in 75% ethanol for 30 s and 5% NaClO for 45 s to remove the bacteria on the surface of the seeds. The surface ethanol and NaClO were rinsed with sterile water three times. The seeds were first placed on a culture dish for 3 days. After germination, the seedlings were transplanted into pots, where sterile soil and sterilized vermiculite were mixed in a 1:1 volume ratio. Twelve pots were treated for each treatment. When the seedlings grew to the 3-5 leaf stage, the prepared diluted fermentation liquid of Cossackia coli SH2 (bacterial content of 1×10 6 cfu / mL) was inoculated at the root of the plant, 20 mL per plant; no fermentation solution was set as the control (CK). After 3 days, the spore suspension of Fusarium oxysporum was inoculated by root injury inoculation method, 20 mL per plant. The plants were maintained at 25 ℃, relative humidity 70%, supplemented with LED light source in the climate chamber, and cultured under alternating light and dark conditions for 12 hours, and watered every 2 days. After 28 days, the disease was investigated, the disease level was recorded, and the disease index and control effect were calculated.

[0058] The disease classification standard and the calculation of disease index are shown in Table 2:

[0059] Table 2 Grading standards for soybean root rot disease index

[0060]

[0061]

[0062] 2 Test results

[0063] The test results of Cossaccharomyces cerevisiae SH2 in preventing and controlling plant diseases are shown in Table 3. Cossaccharomyces cerevisiae SH2 can significantly improve the resistance of soybean plants to root rot, and its prevention efficiency reaches 64.40%.

[0064] Table 3 Effect of Corsaccharomyces cohnii SH2 on improving soybean plant resistance to root rot

[0065]

[0066] Test Example 4 Test on the use of Cossackia cohnii SH2 in improving plant drought resistance

[0067] 1 Test method

[0068] The indoor drought stress detection method uses PEG-6000 to artificially simulate drought conditions. The activated Cossackia coli SH2 was made into bacterial cakes using a 5 mm diameter puncher, and inoculated into LB liquid culture medium with a concentration of 9%, 11%, and 14% PEG-6000, respectively. The culture was placed in a constant temperature shaker at 27°C and 180 r / min for 12 h to prepare the fermentation liquid, and the OD was measured using a spectrophotometer.600 The absorbance value of the fermentation broth was measured with LB liquid culture medium without PEG-6000 as the control, and the drought resistance of Cossackia coli SH2 was calculated.

[0069] The purified Cossackia SH2 was inoculated into LB liquid medium and cultured in a shaker at 180 r / min and 27°C for 3 days. The substrate was potted for standby use. Soybean seeds (Dongsheng 719) were soaked in 75% ethanol for 30 s and 5% NaClO for 45 s to remove the bacteria on the surface of the seeds. The surface ethanol and NaClO were rinsed with sterile water three times. The seeds were first cultured on a culture dish for 3 days. After germination, the seedlings were transplanted into pots. Two plants were transplanted into each pot, and the surface was covered with soil. For the transplanted plants, 50 mL of PEG-6000 solution with a mass concentration of 10% was irrigated. The experimental group added 10 mL of effective viable bacteria in the pot to 1×10 8 cfu / mL of Cossackia coli SH2 bacterial solution, and the control group was irrigated with 10 mL of distilled water. Each group was repeated 5 times for 3 consecutive days. After two weeks of growth, the root length, plant height, and fresh weight of soybean plants were measured to analyze the effect of Cossackia coli SH2 on the growth of soybean plants under drought stress.

[0070] 2 Test results

[0071] The results of indoor drought stress test showed that Cossackia coli SH2 could grow in LB liquid culture medium with concentrations of 9%, 11%, and 14% PEG-6000, and had strong drought resistance.

[0072] Table 4 Growth-promoting effect of Cossaccharomyces cohnii SH2 on soybean plants under drought stress

[0073]

[0074] The results of the test on the growth-promoting effect of Cossacella cohnii SH2 on soybean plants under drought stress are shown in Table 4. After inoculation with Cossacella cohnii SH2 under drought stress, the plant height, root length and fresh weight of soybean plants increased by 62.45%, 9.96% and 141.92%, respectively. This shows that Cossacella cohnii SH2 has a significant growth-promoting effect on soybean plants under drought stress and can significantly improve the drought resistance of soybean plants.

Claims

1. A strain of Coxsackieae Kosakonia cowanii ) SH2, characterized in that Its microbial preservation number is: CGMCC NO.30737.

2. A fermentation broth, characterized in that: The method is prepared by fermentation of the Cossackia coli SH2 according to claim 1.

3. A method for preparing the fermentation broth according to claim 2, characterized in that: include: The strain of Cossackia cohnii SH2 described in claim 1 is activated and inoculated into a liquid culture medium for fermentation to obtain a fermentation broth; wherein the liquid culture medium is composed of: 1.5% tryptone, 0.58% yeast extract, 0.54% NaCl, and the remainder is distilled water, and the pH value is 6.

8.

4. Use of the Cossackia coli SH2 according to claim 1 or the fermentation broth according to claim 2 in producing IAA.

5. Use of the Cossackia coli SH2 according to claim 1 or the fermentation liquid according to claim 2 in promoting plant growth; wherein, The plant is soybean.

6. The use according to claim 5, characterized in that: The promoting plant growth includes increasing the plant height, root length or fresh weight of the plant; wherein the fresh weight refers to the weight of the entire plant.

7. Use of the Cossackia cohnii SH2 according to claim 1 or the fermentation liquid according to claim 2 in preventing and controlling plant diseases; wherein the plant disease is soybean root rot caused by Fusarium oxysporum.

8. Use of the Cossackia cohnii SH2 according to claim 1 or the fermentation liquid according to claim 2 for improving drought resistance of plants; the plant is soybean.

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