Enterobacter capable of inhibiting strawberry anthracnose by generating holes

Abstract

According to the application of the enterobacter to treatment of strawberry anthracnose, the endophyte enterobacter of the enterobacter is found to be capable of generating holes in strawberry stem bases to inhibit germination of anthrax spores by exploring reasons of strawberry voids, so that the disease resistance of strawberries can be effectively improved, and the disease resistance of the strawberries can be improved. The antibacterial effects of different enterobacter and the protection of strawberries are compared, and a method capable of more effectively inhibiting strawberry anthracnose and improving the survival rate of strawberries is researched. The enterobacter reagent provided by the invention has good biocompatibility, is simple to use, is not easy to generate resistance, and has broad prospects in the fields of microorganisms and biological control.

Description

Technical Field

[0001] This invention relates to the field of microorganisms and biological control, specifically to the use of Enterobacteriaceae to inhibit the germination of anthracnose spores by creating cavities at the base of strawberry stems. Background Technology

[0002] Strawberries are a popular berry, but as a disease-prone plant, the emergence of new pests and diseases during intensive cultivation has become a bottleneck restricting the development of the industry.

[0003] Since the promotion of intensive strawberry cultivation in the early 20th century, anthracnose has become the "number one killer" of the strawberry industry. Caused by *Colletotrichum gloeosporioides* and *Colletotrichum strawii*, this disease can infect strawberries throughout their entire growth cycle, causing significant seedling death, especially during the seedling stage and early transplanting. Typical symptoms include: sunken ulcers on stolons and petioles, leading to "black stem"; dark brown necrotic spots on leaves; acute wilting and wilt of roots; and sunken soft rot lesions on fruits. The pathogen spreads through diseased plant debris, soil, and infected seedlings, and is highly prevalent under hot and humid conditions. Currently, commonly used protective agents for strawberry anthracnose include mancozeb and copper-based formulations, while therapeutic agents include difenoconazole, imazalil, or pyraclostrobin. However, chemical control has significant side effects. Under high temperatures and strong sunlight, it can easily cause yellowing of leaves and poor taste of fruit. Excessive use can pollute soil and water sources, harm beneficial organisms such as bees, and long-term use can lead to drug resistance in pathogens, resulting in reduced control effectiveness.

[0004] In recent years, a new type of "hollow disease" has appeared in several strawberry producing areas in my country, characterized by rotting at the base of the strawberry stem and the formation of cavities. It is currently known that the occurrence of strawberry stem base rot is related to the genus *Fusarium* (…). Fusarium Phytophthora ( ) Phytophthora Related to pathogens such as ) and Beijing suburbs Dactylonectria alcacerensis and D. torresenisis Strawberry root rot caused by fungi can also lead to browning and rotting of the strawberry stem base, resulting in necrosis. In Baoding, Hebei Province, strawberry plants were stunted, withered older leaves, and browning of the rootstock; in severe cases, the entire plant died. Identification revealed that the disease was caused by *Neoplasmosis rubrum* (…). Neopestalotiopsis clavispora The pathogen causing hollow heart disease in strawberries in Anhui, Liaoning, and Hebei provinces is *Fusarium solani* (also known as solanum spp.). Fusarium salani This disease manifests as necrosis and disintegration of the fruit pulp, forming cavities, accompanied by browning and loose texture of the pulp. In severe cases, sunken lesions appear on the fruit surface, reducing its commercial value. However, these symptoms are significantly different from the new "cavity disease," whose core characteristic is the formation of cavities at the base of the strawberry stem. After infection, growth is hindered, new leaves turn yellow and are easily broken, and fruit setting is difficult. This disease breaks out 1-3 months after planting, is highly contagious, and overlaps with the flowering and fruit setting period, leading to severe yield reduction. The cause is still unclear.

[0005] Therefore, it is urgent to find the pathogen of the new "cavitation disease" in strawberries and to find a green, healthy biological agent that is not prone to developing resistance and can effectively prevent strawberry anthracnose and / or cavitation disease. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an application of Enterobacteriaceae in the treatment of strawberry anthracnose. By investigating the causes of strawberry cavitation, it was discovered that the endophytic Enterobacteriaceae can create cavities at the base of the strawberry stem to inhibit the germination of anthracnose spores, effectively improving the disease resistance of strawberries. The antibacterial effects of different proportions of Enterobacteriaceae were compared to explore a method that can more effectively inhibit strawberry anthracnose and reduce cavities.

[0007] On the one hand, the present invention provides the use of Enterobacteriaceae in the preparation of a reagent that promotes the formation of cavities at the base of strawberry stems, thereby improving the disease resistance of strawberries.

[0008] The core characteristic of the "hollow disease" at the base of the strawberry stem described in this invention is that cavities form at the base of the strawberry stem. After infection, growth is hindered, new leaves turn yellow and turn pale green, are easily broken, and fruit setting is difficult.

[0009] This invention simultaneously isolates a large number of bacterial and fungal pathogens from the stem base of strawberry anthracnose and strawberry stem base "cavitation disease".

[0010] Among the bacterial isolates, Enterobacteriaceae ( Enterobacter sp. The sample isolation rate was 100%, and it accounted for approximately 30% of the colonies. Other bacteria included Pseudomonas (…). Pseudomonas Lauterii spp. Raoultella Acinetobacter ( ) Acinetobacter sp. ), Klebsiella sp. , Yokenella sp. The fungal isolates were dominated by *Anthracnose*, with an isolation rate of approximately 63.90%, followed by *Fusarium*, with an isolation rate of 37.45%, and also included small amounts of other fungi such as *Pythium*. Each "cavitation disease" sample contained *Enterobacter*, while the fungi could be one or more of *Anthracnose*, *Fusarium*, *Pythium*, or other fungi. *Enterobacter* was not isolated from healthy strawberry plants, strawberry plants with anthracnose (without cavities), or strawberry plants with *Fusarium wilt* (without cavities).

[0011] Research has found that the strains detected in strawberry anthracnose samples were similar to those in samples with "cavitation disease" at the base of strawberry stems, but no Enterobacteriaceae were found. This means that when Enterobacteriaceae are present, even if anthracnose bacteria are present in strawberries, anthracnose symptoms will not appear, but cavitation disease symptoms will appear. This indicates that Enterobacteriaceae can form cavities in strawberries to inhibit fungi such as anthracnose bacteria, thereby improving the disease resistance of strawberries.

[0012] The possible cause of the stem base cavity of the strawberry is that the infection of anthracnose hinders the circulation of carbon, sulfur and phosphorus elements of the strawberry plant, and the enterobacter, as an endophytic rhizosphere biocontrol agent of the strawberry, preferentially utilizes the accumulated carbon, sulfur and phosphorus elements in the stem base for metabolic activities to resist the harm of the anthracnose fungus, and the CO2 gas produced in the process of aerobic respiration or fermentation of the enterobacter is accumulated in the tissue gap due to the structural damage of the stem base tissue after the infection of the fungus, and the continuous gas pressure leads to further disintegration of the tissue and formation of a cavity, and finally develops into stem base cavity disease.

[0013] As a beneficial microbial elicitor, the enterobacter can be recognized by the strawberry plant and activate its own systemic defense system, which is similar to “vaccinating” the plant, and this process usually involves the initiation of defense signal pathways such as salicylic acid or jasmonic acid, which promotes the thickening of the cell wall of the strawberry and the production of antibacterial substances such as plant hormones, so that the whole strawberry is in a highly alert “pre-immune” state, and when the plant encounters a real pathogenic fungus in large numbers, the strawberry can make a more rapid and strong defense response, greatly enhancing its overall disease resistance. Further, the enterobacter includes any one or more of E. ludwigii 、 E. cloacae subsp. Dissolvens and E. cancerogenus .

[0014] Through molecular identification system analysis of the enterobacter, it is determined that the enterobacter strain of the present application is composed of different enterobacter strains, including any one or more of E. ludwigii 、 E. cloacae subsp. Dissolvens and E. cancerogenus .

[0015] It can be understood that any kind of enterobacter has the effect of forming a cavity in the strawberry to inhibit fungi and improve the disease resistance of the strawberry. However, research has shown that different types of enterobacter have certain differences in inhibiting fungi and forming cavities in the strawberry.

[0016] Further, the enterobacter improves the disease resistance of the strawberry, including improving the ability of the strawberry to inhibit the infection of the pathogenic fungus.

[0017] Further, the pathogenic fungus includes any one or more of Colletotrichum , fusarium oxysporum ( Fusarium oxysporum ).

[0018] In some ways, the enterobacter can be directly prepared by using a live bacterial solution of the enterobacter.

[0019] In some ways, the enterobacter can be an endophytic bacterial isolation culture from the strawberry.

[0020] Further, the enterobacterium is E. ludwigii .

[0021] A large number of experiments prove that the enterobacterium E. ludwigii The effect of inhibiting fungi in strawberries to improve the disease resistance of strawberries is more obvious.

[0022] In some ways, the isolation and purification method of the enterobacterium in the endophyte isolated culture from strawberries includes the following steps: After single spore isolation of the fungal isolate, culture on PDA for 5 days; Use a 5 mm puncher to take the colony edge block and place it in PD liquid medium for 5 days of shaking culture at 25°C; Centrifugal collection of conidia, dilution with sterile water to 10000 spores / mL; Add 1 mL of spore solution to each hydroponic bottle, and after inoculation, place the plant in a light incubator at 20-28°C with 14 h light / 10 h darkness, observe and record the disease condition every day.

[0023] In some ways, the isolation and purification method of the pathogenic fungus isolate in step a) is: after removing the epidermis of the stem base of the diseased strawberry, healthy strawberry, disinfecting with 70% alcohol for 1 min, washing with sterile water for 3 times, using tissue isolation and streaking method to isolate the pathogenic fungus, using potato sucrose medium (PSA) and Waksman potato semi-synthetic medium (W medium) to purify single colony for several times, and then store for use.

[0024] Studies have shown that the fungal pathogen isolate obtained by isolation is inoculated into strawberry seedlings in vivo. After inoculation of anthracnose and fusarium wilt, the incidence rate reaches 100%, and the stem base of all diseased plants is hollow.

[0025] And the enterobacterium obtained by isolation is inoculated into strawberry seedlings in vivo, and after 4 weeks of inoculation, the strawberry plants are still disease-free. The enterobacterium does not infect strawberry seedlings in different ways, so the enterobacterium is safe for healthy strawberry seedlings.

[0026] In some ways, the inoculation method of the bacterial isolate (enterobacterium) includes: (1) Ex vivo inoculation: single colony of bacterial pathogen is placed in W liquid medium for overnight culture, OD 600 =0.5, respectively, take the unfolded leaves of healthy strawberry plants, prick with a sterile needle, drop the bacterial solution to the hole (about 20 μL), use sterile W liquid medium as control, and place in a 14 h light / 10 h dark incubator for humidification culture.

[0027] (2) In vivo needle inoculation method: the bacterial pathogen single colony is placed in W liquid medium for overnight culture, then centrifuged at 5℃ with a centrifuge at rcf 8000 for 10 min, and the supernatant is removed. Adjust the concentration to OD 600 =0.5 with sterile water, and a small amount of bacterial solution is injected into the strawberry seedling stem with a sterile syringe. The inoculated plants are placed in a glass greenhouse with a natural temperature of 20-28℃, and the field management is as usual.

[0028] (3) In vivo water culture inoculation: the strawberry seedlings are planted with commercially available nutrient solution, and after survival, the bacterial isolates are prepared according to the "in vitro inoculation" method, diluted with sterile water to OD 600 =0.5, and 5 mL of bacterial solution is irrigated per strawberry seedling. Sterile water is used as a control.

[0029] The three bacterial isolates can achieve the effect of proving the invention.

[0030] The application provides a use of enterobacter for preparing a reagent for promoting the formation of cavities in strawberries and improving the disease resistance of strawberries, the enterobacter can inhibit the spore germination of strawberry anthracnose fungus, wherein E. ludwigii has the best inhibitory effect.

[0031] The pathogenic isolates are inoculated in combination, and the effect of enterobacter on the spore germination of anthracnose fungus is analyzed. Research proves that the enterobacter has a certain inhibitory effect on the spore germination of anthracnose fungus, wherein E. ludwigii has the highest inhibition rate of 32.07% on the spore germination of anthracnose fungus, E. cloacae subsp . Dissolvens has an inhibition rate of 23.44% on the spore germination of anthracnose fungus, E. cancerogenus and 13.64% on the spore germination of anthracnose fungus.

[0032] Further, the enterobacter is used for preparing a reagent for inhibiting or treating strawberry anthracnose. The reagent is an enterobacter bacterial solution, and the preparation method comprises the following steps: preparing a viable bacterial solution by using enterobacter and water, wherein the viable bacterial concentration of the enterobacter is 10 5 cfu / mL~10 7 cfu / mL.

[0033] Preferably, the optimal viable bacterial concentration of the enterobacter viable bacterial solution is 10 6 cfu / mL.

[0034] After 7 days of enterobacter treatment, anthracnose fungus is inoculated, the spore concentration of the anthracnose fungus spore suspension is 10000 / mL, and the inoculation method comprises the following steps: pouring 5 mL of spore suspension along the wall of the pot.

[0035] As a rhizosphere growth-promoting bacterium, the enterobacter can form a protective layer after colonizing the roots, which can protect the strawberries from the invasion of xanthomonas, fusarium, botrytis cinerea and other pathogens, but does not cause the formation of "hollows". However, after being infected with the anthracnose fungus, the enterobacter can promote the formation of "hollows" at the base of the strawberry stems, thereby inhibiting the germination of the anthracnose fungus spores and improving the disease resistance of the strawberries.

[0036] In another aspect, the enterobacter improves the disease resistance of the strawberries by promoting the formation of "hollows" at the base of the strawberry stems, and the enterobacter bacterial liquid is poured onto the substrate of the strawberry plants, and the treatment method is as follows: the bacterial liquid is poured along the pot wall, and the base of the plant stem is avoided, 5 mL of bacterial liquid is inoculated every week, and the inoculation is continuously performed four times.

[0037] Researches show that the fungal isolates cannot cause "hollows" at the base of the strawberry stems, and the single inoculation of the enterobacter cannot cause diseases and "hollows", but promotes the growth of the strawberry plants, so that the enterobacter is not a pathogenic bacterium causing "hollows", but an endophyte. That is to say, only when the enterobacter is used against harmful bacteria such as anthracnose, the enterobacter can form "hollows" at the base of the strawberry stems, and under normal circumstances, the enterobacter does not form "hollows".

[0038] The biochemical properties of the enterobacter are determined in the application, and it is found that E. cloacae subsp . Dissolvens Under aerobic or anaerobic conditions, the enterobacter can utilize carbon sources such as glucose, xylose, mannose and mannitol and grow rapidly, and at the same time, can utilize most of the carbon sources and produce a large amount of carbon dioxide gas. E.ludwigii At low temperature (about 10-15℃), the enterobacter can utilize arabinose and slowly produce a small amount of carbon dioxide. The gas produced by the strain pushes the sealed paraffin oil or forms a large amount of gas bubbles and hollows in the solid culture medium, which is very similar to the hollows of plant tissues.

[0039] When the carbon source is mannitol, mannose and trehalose, the enterobacter produces the largest amount of gas, and the maximum amount of gas produced by the enterobacter is 3.65 cm 3 after 48 h. When the carbon source is xylose, the enterobacter produces the smallest amount of gas. Among the three enterobacters, E. cloacae subsp . Dissolvens produces the largest amount of gas, E. ludwigii produces the smallest amount of gas.

[0040] E. ludwigii When using lysine, arginine and ornithine, the enterobacter can produce a small amount of gas, E. cancerogenus When using arginine and ornithine, the enterobacter produces a small amount of gas, E. cloacae subsp . Dissolvens When using arginine, the enterobacter can produce a large amount of gas.

[0041] Therefore, it is speculated that the reason for the hollow of the base of the strawberry stem is that the anthracnose competes for the necessary carbon, sulfur, phosphorus and other elements in the strawberry plant, and the enterobacter produces CO2 and other gases in the process of utilizing the carbon, sulfur and phosphorus cycle of the strawberry, which cannot be timely removed due to the damage of the stem base tissue structure, resulting in the hollow disease of the stem base, and at the same time, the enterobacter competes with the anthracnose for nutrients and space, and inhibits the spore germination of the anthracnose. Among them, the strain E. ludwigii has the best inhibitory effect on the germination of the anthracnose and produces the least gas, so the hollow formed is also the least.

[0042] Further, the enterobacter promotes the formation of hollows in the strawberry to improve the disease resistance of the strawberry, and the optimal viable concentration of the enterobacter live bacteria solution is 10 6 cfu / mL.

[0043] Further, the enterobacter bacteria solution is poured onto the substrate of the strawberry plant, and the hollows are generated at the base of the strawberry stem to achieve the effect of inhibiting or treating the anthracnose of the strawberry.

[0044] On the other hand, the present application provides a use of a mixture of enterobacter and bacillus for preparing a reagent for improving the disease resistance of the strawberry.

[0045] When the enterobacter exists, even if the strawberry contains the anthracnose, no symptoms of anthracnose will appear, but symptoms of hollow disease will appear, which indicates that the enterobacter can form hollows in the strawberry to inhibit fungi such as anthracnose, thereby improving the disease resistance of the strawberry.

[0046] As a kind of endophytic bacteria of plants, bacillus can colonize in the rhizosphere, body surface or body of plants, compete with the nutrients and living space of pathogenic bacteria, and secrete active substances such as antibacterial peptides, chitinase and lipopeptides, thereby destroying the cell wall of pathogenic bacteria or inhibiting the spore germination of pathogenic bacteria, and effectively preventing and treating the anthracnose of the strawberry. Therefore, the present application believes that the combination of enterobacter and bacillus can further improve the disease resistance of the strawberry.

[0047] Further, the mixture can reduce the formation of hollows at the base of the strawberry stem and improve the disease resistance of the strawberry.

[0048] As a rhizosphere growth-promoting bacterium, enterobacter can form a protective layer after colonizing in the root, which can protect the strawberry from the invasion of xanthomonas, fusarium and botrytis, but will not cause the production of "hollows". However, after being infected with the anthracnose, the enterobacter can promote the strawberry to form "hollows", thereby inhibiting the spore germination of the anthracnose and improving the disease resistance of the strawberry. Under the combined action of the enterobacter and bacillus subtilis, the spore germination of the anthracnose is inhibited, the production of "hollows" at the base of the strawberry stem is reduced, and the survival rate of the strawberry is increased. Further, the enterobacter includes E. ludwigii , E. cloacae subsp. Dissolvensand E. cancerogenus Any one or more of the following:

[0049] The enterobacterium strain of the present application can be any of the following enterobacterium strains: E. ludwigii , E. cloacae subsp. Dissolvens and E. cancerogenus , etc.

[0050] It can be understood that any kind of enterobacterium has the effect of forming cavities in strawberries to inhibit fungi and improve the disease resistance of strawberries. However, research has shown that different kinds of enterobacterium have certain differences in inhibiting fungi and forming cavities in strawberries. Among them, the strain E. ludwigii has the best inhibitory effect on the germination of anthracnose fungus.

[0051] Further, the bacillus is Bacillus subtilis. Bacillus subtilis .

[0052] In some ways, the enterobacterium and bacillus subtilis mixture can be directly prepared by mixing enterobacterium and bacillus subtilis live bacterial liquid.

[0053] Further, the mixture can effectively resist strawberry anthracnose fungus, gray mold fungus, and root rot fungus, etc.

[0054] Further, the enterobacterium and bacillus subtilis mixture is used for preparing a reagent for inhibiting or treating strawberry anthracnose. The reagent is an enterobacterium liquid and a bacillus subtilis liquid, and the preparation method comprises: mixing enterobacterium, bacillus subtilis, and water to prepare a live bacterial liquid mixture, and the concentration of the live bacterial liquid in the mixture is 10 6 cfu / mL.

[0055] In another aspect, the enterobacterium and bacillus subtilis mixture is poured onto the strawberry plant to inhibit strawberry anthracnose and reduce the formation of "cavities", and the treatment method is: pouring the mixture along the pot wall, avoiding the plant stem base, inoculating 5 mL per week, and continuously for four times.

[0056] After 7 days of treatment of the mixture, the anthracnose fungus is inoculated, the spore concentration of the anthracnose spore suspension is 10000 / mL, and the inoculation method comprises: pouring 5 mL of the spore suspension along the pot wall into the substrate.

[0057] The research shows that the fungal isolates (such as anthracnose) cannot cause the "hollow" of the stem base of strawberry, and the single inoculation of enterobacter and bacillus subtilis cannot cause the "hollow" and can promote the growth of strawberry plants. However, under the action of anthracnose, enterobacter can cause the "hollow" of the stem base of strawberry, but anthracnose is inhibited; the combination of enterobacter and bacillus subtilis reduces the "hollow" caused by enterobacter for inhibiting strawberry anthracnose, and still has a good inhibitory effect on anthracnose.

[0058] The enterobacter produces air holes with similar size and shape. By determining the biochemical properties of enterobacter, the causes of the "hollow" are analyzed: in the process of promoting the carbon cycle of strawberry, enterobacter produces CO2 gas, which cannot be timely removed due to the damage of the tissue structure of the stem base, resulting in the "hollow" disease of the stem base, and competes with the nutrients and space of the growth of anthracnose, thereby inhibiting the spore germination of anthracnose. Under the action of bacillus subtilis, the "hollow" of the stem base of strawberry is reduced, and the spore germination rate of anthracnose is reduced, further protecting strawberry from the damage of anthracnose and "hollow" disease.

[0059] Further, the ratio of enterobacter and bacillus subtilis in the mixed agent is 1:1.

[0060] Further, the enterobacter and bacillus subtilis mixed agent inhibits strawberry anthracnose and reduces the "hollow" generation, and the concentration of live bacteria in the mixed agent is 10 6 cfu / mL.

[0061] Further, the enterobacter and bacillus subtilis mixed agent is poured onto the substrate of the strawberry plant, so as to inhibit or treat strawberry anthracnose and the new "hollow" disease.

[0062] The present application has the following beneficial effects: The preparation method of the reagent for protecting strawberry seedlings from anthracnose by enterobacter is provided, E. ludwigii In the process of promoting the carbon cycle of strawberry, gas is produced, which cannot be timely removed due to the damage of the tissue structure of the stem base, resulting in the "hollow" disease of the stem base, and competes with the nutrients and space of the growth of anthracnose, thereby inhibiting the spore germination of anthracnose. The control effect is equivalent to that of the commonly used chemical agent, but it is significantly better than the chemical agent in promoting the recovery of plants.

[0063] Enterobacter can invade the healthy tissue inside strawberry and become endophytic bacteria, which can provide continuous protection from the inside of the plant without affecting the growth of the plant, which cannot be achieved by chemical agents. At the same time, anthracnose is difficult to produce drug resistance, which ensures the durability and stability of the control effect.

[0064] Enterobacter and Bacillus subtilis as endophytic bacteria of strawberry, effectively prevent and control anthracnose, at the same time, through the characteristics of plant growth promotion such as nitrogen fixation, phosphorus dissolution and auxin secretion, significantly improve the growth of strawberry plants under the stress of anthracnose, promote the germination of new roots and new leaves, restore the vitality of plants, finally reduce the yield loss caused by diseases to the minimum, and improve the fruit quality, realize the double goal of "reducing disaster" and "increasing yield".

[0065] The preparation method of the reagent for protecting strawberry seedlings from the invasion of anthracnose bacteria by the mixed agent of Enterobacter and Bacillus subtilis is provided, E. ludwigii In the process of promoting the carbon cycle of strawberry, gas is produced, and the stem base tissue structure is damaged, which cannot be discharged in time, resulting in stem base cavity disease, at the same time, it competes with the nutrients and space of anthracnose bacteria growth, inhibits the germination of anthracnose bacteria spores, Enterobacter and Bacillus subtilis combined to inhibit strawberry anthracnose and reduce the "cavity" production, thereby improving the disease resistance and yield of strawberry, and the control effect is equivalent to that of the commonly used chemical agent, but it is significantly better than the chemical agent in promoting the recovery of plants.

[0066] The mixed agent can invade the inside of healthy strawberry tissue to become endophytic bacteria, provide continuous protection from the inside of the plant without affecting plant growth, which is not achieved by chemical agents. At the same time, under the action of the mixed agent, it is difficult for anthracnose bacteria to produce drug resistance, which ensures the durability and stability of the control effect.

[0067] Enterobacter and Bacillus subtilis as endophytic bacteria of strawberry, effectively prevent and control anthracnose, at the same time, reduce the production of cavities, promote the carbon cycle in strawberry plants, finally reduce the yield loss caused by diseases to the minimum, and improve the fruit quality, realize the double goal of "reducing disaster" and "increasing yield". BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 It is the field symptom and stem section graph of "cavity disease" of strawberry in example 1; Figure 2 It is the inoculation picture of different pathogen isolates in example 1 (A: Enterobacter; B: Enterobacter inoculation in strawberry water culture; C: stem base cross section after inoculation of Enterobacter; D: strawberry anthracnose bacteria; E: strawberry water culture inoculation of anthracnose bacteria for 5 days; F: strawberry stem base cross section after suffering from anthracnose; G: strawberry fusarium wilt bacteria; H: strawberry water culture inoculation of fusarium wilt bacteria for 5 days; I: strawberry stem base cross section after suffering from fusarium wilt; J: strawberry seedlings survived after mixed inoculation for 1 month; K: cross section of strawberry stem base after mixed inoculation); Figure 3 It is the bacterial species in the stem base of strawberry suffering from "cavity disease" in example 2 (A: bacterial species in the stem base of strawberry plant with anthracnose (no cavity), B: bacterial species in the stem base of strawberry plant with cavity disease); Figure 4 It is the phylogenetic tree result of Enterobacter in example 2; Figure 5 The morphological characteristics of Enterobacteriaceae in Example 3; Figure 6 The symptoms of salt damage observed in strawberry seedlings after exposure to 40mM sodium chloride solution in Example 3; Figure 7 This refers to the amount of gas produced by Enterobacteriaceae using different carbon sources at 48 h in Example 3; Figure 8 In Example 3 E. ludwigii Gas production under different nutritional conditions, with carbon sources from left to right being ornithine, arginine, mannose, mannitol, fructose, glucose, trehalose, sorbitol, maltose, and xylose. Detailed Implementation

[0069] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The details of the invention will become clearer from the following description and claims.

[0070] Example 1: Enterobacteriaceae inhibit anthracnose by promoting the formation of cavities at the base of strawberry stems. 1. Isolation and purification of strawberry pathogens Strawberry stem base "hollow disease," also known as "hollow heart disease" or "broken head disease," initially presents with no obvious symptoms. Symptoms typically include dull leaves, new leaves failing to unfold or turning slightly yellow, uneven leaf size, slow growth, yellowing and stunted growth, and easy breakage. Pulling up an infected plant and longitudinally cutting the shortened stem reveals water-soaked lesions on the vascular bundles at the leaf petiole attachment point. These lesions expand from the outside in. A longitudinal cut of the strawberry stem reveals water-soaked, light reddish-brown lesions with clear, dark reddish-brown edges. As the lesions continue to enlarge, one or more cavities of varying sizes appear within the shortened stem. Figure 1 As shown. This disease develops slowly and mainly occurs from August to October, primarily from the time strawberries are planted until they flower and bear fruit. Low temperatures and high humidity can easily lead to a high incidence and outbreak of this disease.

[0071] Currently, many types of endophytic bacteria have been reported in strawberries, including both bacteria and fungi. Bacillus sp. and Pantoea sp. Isolated from the leaves and roots of strawberry plants, it effectively inhibits the growth of Botrytis cinerea mycelium, while endophytic fungi... Bacillus velezensis , Bacillus megaterium and Herpaspirillum huttiense The combined action of these fungi can achieve an inhibition rate of 79.4% against Rhizoctonia solani. Many endophytic fungi, such as..., have also been found on strawberries. Cladophialophora chaetospira SK51 , Trichoderma spp. Streptomyces Antagonistic against Fusarium oxysporum ( Fusarium oxysporum ), Armillaria fungi ( Armillaria mellea ) and peritrichous tetroma ( Glomerella cingulataThe role of ) . De Melo Pereira discovered 92 endophytic bacteria in strawberry fruit, among which Enterobacter sp. and Pantoea punctata They accounted for 15.5% and 13.3% respectively, and it was confirmed that they promoted plant growth. Other endophytic bacteria from strawberry plants, such as... Brevibacillus fluminis MET12M2 It also has a growth-promoting effect.

[0072] Strawberry stem base samples of "hollow bud disease," anthracnose, and healthy strawberries were collected from multiple strawberry growing bases in Hangzhou's Fuyang, Jiande, and Linping; Jinhua; Taizhou's Xianju; Wenzhou's Yueqing; Anhui's Changfeng; and Yunnan's Qujing. Various strawberry pathogens were isolated and identified. The specific isolation and identification implementation plan is as follows: (1) Remove the epidermis from the base of the stems of diseased and healthy strawberries, disinfect with 70% alcohol for 1 minute, and wash with sterile water 3 times. (2) Pathogens were isolated by tissue isolation and streak plating, and purified by single colony purification multiple times on potato sucrose agar (PSA) and potato semi-synthetic agar (W agar), and then stored for later use. (3) The disinfected diseased strawberry stem base tissue was sent to Beijing Qingke Biotechnology for 16S bacterial genome diversity sequencing, with anthracnose (without cavities) and healthy strawberry stem base as controls.

[0073] SPSS software was used for statistical analysis of experimental data under different treatments, and Excel 2010 and MEGA 7.0 software were used for data processing and analysis. Table 1 shows that Enterobacteriaceae (…) were present in the bacterial isolates. Enterobacter sp. The sample isolation rate was 100%, and it accounted for approximately 30% of the colonies. Other bacteria included Pseudomonas (…). Pseudomonas Lauterii spp. Raoultella Acinetobacter ( ) Acinetobacter sp. ), Klebsiella sp .、 Yokenella sp. While *Xanthomonas* was isolated, no *Xanthomonas* was found. Anthracnose was the predominant fungal isolate, with an isolation rate of approximately 63.90%, followed by *Fusarium* at 37.45%, and a small amount of other fungi such as *Pythium*. Each *cavitary* sample contained *Enterobacter*, and the fungi could be one or more of *Anthracnose*, *Fusarium*, or *Pythium*. *Enterobacter* was not isolated from healthy strawberry plants, anthracnose-infected strawberry plants (without cavities), or *Fusarium wilt*-infected strawberry plants (without cavities).

[0074] Table 1. Isolation rate of pathogens of "hollowing disease" at the base of strawberry stems

[0075] Note: Fungal isolation rate is based on the number of isolated samples, with 3-5 small samples isolated from each sample. Bacterial isolation rate is based on the number of sequencing samples, with 3-5 colonies sequenced from each diseased sample.

[0076] Enterobacteriaceae ( Enterobacteriaceae Enterobacteriaceae is a widely distributed family of bacteria that commonly resides in soil, water, decaying matter, and the intestines of humans and animals. They can cause various diseases in humans, livestock, and plants. Common medical pathogens such as Salmonella, Helicobacter pylori, Shigella, Escherichia coli, and Vibrio cholerae belong to this family. In recent decades, through comprehensive analysis of genetic studies including DNA comparison and mol% G+C content, as well as standardized biochemical and phenotypic data, the number and types of bacteria within the Enterobacteriaceae family have been continuously increasing and changing. Currently, there are 42 genera, of which 29 are medically relevant, and the genera primarily associated with plants include Pectinobacterium (…). Pectobacterium ), Dickeya genus, Erwinia genus, Brennera genus, Pantoea Ten genera, including the genus *Fusarium*, can cause a variety of crop diseases, such as black shank of potato, stem base rot of rice, fire blight of pear, bacterial wilt of cucumber, black rot of papaya, and bacterial wilt of maize.

[0077] Enterobacter These bacteria are well-known for causing various symptoms in humans and animals, including diarrhea, vomiting, inflammation, and organ infections. They are mainly found in plants, seeds, fruits, and the environment. As opportunistic pathogens, Enterobacter These can also be endophytic fungi in plants, and in special cases, they can be pathogens. West reported the presence of endophytic fungi in grape roots. Enterobacter cloacae This bacterium has also been found in rice, tomatoes, and bananas, demonstrating that it can absorb labeled nitrogen from the soil and transport it to the host plant, bananas, promoting their growth; it also mitigates the toxicity of heavy metals such as cadmium and nickel in the soil to the host plant. Several nitrogen-fixing endophytic bacteria of the genus *Enterobacter* have been found in sugarcane, such as... E. arachidis , E. oryzae , E. radicincitans , E. roggenkampii At the same time, Enterobacteriaceae can also interact with other endophytic bacteria such as Klebsiella sp. By dissolving phosphorus, it produces iron carriers, IAA, and aminocyclopropanecarboxylic acid (ACC), which promote the growth of the host plant, pepper. This was isolated from ryegrass. E. ludwigii It possesses the characteristic of producing HCN. Under salt stress, endophytic bacteria... Enterobacter sp. SA187 It participates in the host plant's metabolism and can repair phosphorus starvation in Arabidopsis thaliana. Furthermore, it has been found in several salt-sensitive Arabidopsis thaliana sulfur metabolism mutants to have added [phosphorus]. Enterobacter sp. SA187 Similar to adding sulfur, L-cysteine, or L-methionine, it can partially or completely repair mutant defects.

[0078] 2. Analysis of strawberry status after inoculation with pathogenic isolates of strawberry The inoculated plants were healthy tissue culture strawberry seedlings with 3-6 leaves, and the strawberry variety was 'Zhang Ji'. The plants were grown in pots using a substrate. Different inoculation methods are described below. Three replicates were set up for each strain. Symptoms were observed and recorded every day after inoculation.

[0079] The specific steps for isolating and purifying the strains are as follows: (1) After single spore isolation of the strains, culture on PDA for 5 days; (2) Take the edge of the colony with a 5 mm puncher and place it in PD liquid medium. Shake culture at 25°C for 5 days; (3) Centrifuge to collect conidia, and dilute with sterile water to 10,000 spores / mL; (4) Add 1 mL of the spore solution to each water culture bottle. After inoculation, place the plants in a light incubator at 20-28°C with 14 h light / 10 h darkness. Observe and record the disease every day.

[0080] The specific inoculation method for bacterial isolates is ex vivo inoculation, with the following steps: (1) Incubate the bacterial pathogen with a single colony in W liquid medium overnight, with OD 600 = 0.5; (2) Take the unfolded leaves of healthy strawberry plants, prick with a sterile needle, and drop the bacterial solution (about 20 μL) into the hole; The inoculation method for fungal isolates includes single strain live inoculation and complex inoculation, with the following specific inoculation methods: Single strain live inoculation: After single spore isolation of the fungal isolates, culture on PDA for 5 days. Take the edge of the colony with a 5 mm puncher and place it in PD liquid medium. Shake culture at 25°C for 5 days. Centrifuge to collect conidia, and dilute with sterile water to 10,000 spores / mL. Add 1 mL of the fungal spore solution to each water culture bottle. After inoculation, place the plants in a light incubator at 20-28°C with 14 h light / 10 h darkness. Observe and record the disease every day.

[0081] Complex inoculation: Inoculate Enterobacter and anthracnose bacteria in the substrate of 'Zhang Ji' strawberry seedlings (tissue culture seedlings grown in substrate, 3 leaves and 1 heart) in the order of first Enterobacter 5 mL (OD 600=0.5, the same as above), along the wall of the pot, the control is water. 7 days later, again inoculated with 5 mL of anthracnose (10000 spores / mL, the same as above), the spore suspension is poured into the substrate along the wall of the pot. 24 strawberry seedlings per treatment. After inoculation, the plants are placed in a light incubator at 20-28°C with 14h light / 10h darkness, and the disease is observed and recorded every day.

[0082] The results are shown in Figure 2 After 20 days of inoculation with anthracnose, the old leaf blades have round or irregular brown spots, the new leaves are twisted and not unfolded, and the white new roots are few, the root system is brown, and part of the root system is black and rotten. The stem is cut longitudinally, and the inside of the entire shortened stem is water-like reddish brown and rotten, without cavities. After inoculation with Fusarium, the plants appear weak, the newly born leaves are significantly smaller, the petioles are shortened, and the root system is brown or black. After cutting the shortened stem longitudinally, the inside of the stem is dry reddish brown without cavities. After inoculation with anthracnose and fusarium, the incidence rate reaches 100%. The stem base of all diseased plants has no cavities.

[0083] In order to determine the pathogenicity of the isolated enterobacter, the leaf blade method was used for inoculation, and the results showed that whether the young leaves or other leaves had no symptoms; or a small amount of enterobacter was injected into the stem base of healthy strawberries, and no symptoms were found after 4 weeks of inoculation; or the in vivo hydroponics method was used, and the strawberry plants still had no disease symptoms after 4 weeks of inoculation. And using different ways of inoculation, enterobacter cannot infect strawberry seedlings.

[0084] Strawberry is a widely planted economic crop around the world, and its yield, quality and safety have always been of concern. The "cavity disease" of strawberry stem base is a strawberry disease that has occurred in recent years, and has occurred in different regions from south to north, with a wide range of harm, a large area, and serious harm. This disease has the characteristics of special symptoms, slow occurrence, low mortality, and difficult pathogen identification. The present application finds that a large number of bacterial isolates - enterobacter and fungal isolates - anthracnose or fusarium can be isolated from the "cavity disease" sample. It is known that these fungal isolates cannot cause "cavity" in the stem base of strawberry, and the inoculation of enterobacter alone cannot cause disease and "cavity", but can promote the growth of strawberry plants, so enterobacter is not the pathogenic bacteria that cause "cavity", but an endophytic bacteria that mainly colonizes in the shortened stem and petiole of strawberry, with a small amount in the root and no presence on the leaf blade. The enterobacter does not have cellulase, which is essential for pathogenic bacteria, in its physiological and biochemical characteristics, which also confirms this point.

[0085] 3. Analysis of the colonization site of endophytic enterobacter in strawberry and its growth-promoting effect In this embodiment, bacteria were cultured using an in vitro inoculation method based on bacterial isolates. Enterobacter bacillus culture was poured onto the substrate of the strawberry plants (pouring the culture along the pot wall, avoiding the base of the plant stem). One week later, anthracnose spores were inoculated using the same method. The viable Enterobacter bacillus culture was isolated from the strawberry plants at a concentration of 10. 6 CFU / mL, 5 mL per week for 4 consecutive weeks, with water as a control. Five plants of each strain were inoculated. After rinsing the inoculated and control strawberry plants thoroughly with water, the entire plant was divided into four parts: roots, stems, petioles, and leaves. Each part was disinfected with 5% silver nitrate for 20 seconds, rinsed three times, and then disinfected with 75% alcohol for 1 minute, rinsed three times. The tissue was then ground with sterile quartz sand and sterile water, and the tissue fluid was streaked onto W solid medium containing Enterobacteriaceae. Incubation was carried out at 30℃ in the dark for 1-2 days. Single colonies were picked for culture and gene amplification and sequencing. Subsequently, strawberry seedlings were inoculated with Enterobacteriaceae + Anthracnose, while the control group consisted of strawberry seedlings treated with water + Anthracnose (CK). After inoculation, the plants were placed in a light incubator with 14 h light / 10 h dark at 20℃-28℃, and disease incidence was observed and recorded daily.

[0086] Twenty-four hours after inoculation with anthracnose, most leaves of both the treated and control strawberry seedlings drooped while petioles remained upright. The number of drooping leaves in the Enterobacter-treated strawberry plants was significantly greater than in the control. Three to four days after inoculation, sporadic wilting appeared on the strawberry leaves, petioles softened and collapsed, and older leaves developed necrotic spots. Six to seven days after inoculation, approximately 5% of the Enterobacter-treated strawberry seedlings developed anthracnose symptoms and died, while the mortality rate in the water control was 12.5%, significantly higher than the treated strawberries. Ten days after inoculation, the control strawberry seedlings began to show widespread anthracnose symptoms, reaching a mortality rate of 50% by day 14, while the mortality rate in the Enterobacter-treated strawberry seedlings was 21.67%. Forty days after inoculation, all control strawberry seedlings died, while the survival rate of the Enterobacter-treated strawberry plants was 50.1%. Cutting open the base of the surviving strawberry plant stems revealed cavities, consistent with the field symptoms of strawberry "cavity disease," and Enterobacteriaceae were re-isolated from the base of the surviving strawberry stems. As shown in Table 2, after inoculation with Enterobacter, the cavitation rate of strawberry seedlings infected with anthracnose was 54.6%, and the survival rate was 50.1%. All surviving strawberry plants developed cavities, indicating that Enterobacter inhibited anthracnose by promoting the formation of cavities at the base of the strawberry stem. Among the plants with cavities, 91.76% survived. In contrast, no Enterobacter was present in the control plants.

[0087] Table 2. Relationship between Enterobacterium presence in plants and plant health after mixed inoculation.

[0088] In conclusion, Enterobacteriaceae, as endophytic bacteria in strawberries, enhance the disease resistance of strawberries by promoting the formation of cavities at the base of the strawberry stem.

[0089] Example 2, determination of enterobacteriaceae strains 1. Bacterial analysis of the stem base of "cave disease" plants The strains were sent to the China Agricultural Microbial Culture Collection Center for Biolog™ biological carbon source analysis determination. DNA was extracted using the Ezup column bacterial genomic DNA extraction kit, and specific primer sequences atpD01F / atpD02R, infB01F / infB02R, RpoB-Fb / RpoB-Rb, and UP-1 / UP-2r were used to amplify the ATP synthase beta subunit gene (atpD), translation initiation factor 2 gene (infB), RNA polymerase beta subunit (rpoB), and gyrase B subunit (gyrB), respectively. Gene sequencing and primer synthesis were completed by Zhejiang Shangya Biotechnology Co., Ltd.

[0090] As shown in Figure 3 , after sending the "cave disease" samples from the Fuyang strawberry and the anthracnose samples without cavities for examination, it was found that there were a large number of bacteria in the stem base of the strawberry plants, among which the Enterobacteriaceae (Enterobacteriaceae) bacteria accounted for 42% in the stem base of the strawberry plants with "cave disease", the Enterobacteriaceae accounted for 8%, and the rest were Romboutsia (Romboutsia) in the Bacteroidetes (Bacteroidetes), Eubacterium (Eubacterium), Parabacteroides (Parabacteroides), and high-temperature desulfurization Arcobacter (Arcobacter) and other nitrite oxidizing bacteria (Nitrosomonas) and other bacteria. In the stem base of the anthracnose sample (without cavities), the types of strains detected were similar to those of "cave disease", but the proportions were different, and no Enterobacteriaceae was found. Enterobacteriaceae Firmicutes Romboutsia Coprostanoligenes Parabaceroides Thermodesulfouibrio Nitrospirota In the stem base of the anthracnose sample (without cavities), the types of strains detected were similar to those of "cave disease", but the proportions were different, and no Enterobacteriaceae was found.

[0091] Specific primers were used to amplify the ATP synthase beta subunit gene (atpD), translation initiation factor 2 gene (infB), RNA polymerase beta subunit (rpoB), and gyrase B subunit (gyrB), and fragments of 650bp, 750bp, 600bp, and 1000bp were obtained, respectively. As shown in Figure 4 , the nucleotide sequences were arranged in the order of atpD + infB + gyrB + rpoB, and alignment and homology analysis was performed, and the phylogenetic tree results showed that different test strains formed groups with different published model strains of Enterobacteriaceae, and formed obvious branches, among which strain 2 had 100% homology with E. ludwigii , strain 18 had 99% homology with E. cloacae subsp. dissolvens , and strain 22 was E. cancerogenus . Therefore, it was determined that the Enterobacteriaceae strains in this study were composed of different Enterobacteriaceae strains, including​​​​​​E. ludwigii 、 E. cloacae subsp. Dissolvens and E. cancerogenus .

[0092] 2. Effects of Enterobacter on strawberry plants This example analyzed the changes in plant height, leaf number, leaf length, and fresh weight of strawberry plants treated with Enterobacter for 4 weeks compared to control plants, and isolated strains from different parts of the strawberry plants for comparison, to analyze the colonization sites and growth-promoting effects of Enterobacter in strawberry plants, according to the method provided in Example 1.

[0093] As shown in Table 3, the growth of strawberry seedlings was more vigorous after Enterobacter inoculation than the water control, with plants being erect, leaves being large, and leaf number and fresh weight increasing. The plant height, leaf number, leaf length and width, and fresh weight of strawberry plants treated with Enterobacter for 4 weeks were increased by 3%, 12.33%, 4.74%, 4.18%, and 6.06%, respectively, compared to control plants, and a large number of Enterobacter strains were isolated from the short stems and petioles of the treated plants, with a small amount of Enterobacter present in the roots, but no Enterobacter in the leaves. Gene amplification of the re-isolated Enterobacter strains showed that the types of the re-isolated Enterobacter strains were the same as the inoculated strains. No Enterobacter was present in the control plants.

[0094] Table 3. Biomass of strawberry plants after Enterobacter treatment

[0095] 3. Effects of Enterobacter on spore germination of Colletotrichum gloeosporioides Enterobacter single colonies were placed in W liquid medium for overnight culture, then centrifuged at 8000 rcf for 10 min at 5°C twice, the precipitate was removed, and the supernatant was filtered twice with a 0.22 µm bacterial filter and used as needed. Colletotrichum gloeosporioides spores were collected and diluted with the bacterial filtrate to adjust the concentration to 1×10 5 spores / mL of the spore suspension. The spore suspension was spread on water agar medium, with 3 replicates. Sterile W medium was used as a control. After incubation at 25°C for 5-6 h, the number of spores germinated and the length of hyphae were observed and recorded every 4 h under a microscope.

[0096] The results showed that the fermentation broth of different Enterobacter strains had a certain inhibitory effect on the spore germination of Colletotrichum gloeosporioides. The filtrate of strain 2 had the highest inhibition rate of 32.07%, and the filtrate of strain 22 had the lowest inhibition rate of 13.64%.

[0097] Table 4. Inhibitory effects of Enterobacter secretions on spore germination of Colletotrichum gloeosporioides

[0098] 4. Protection of Enterobacter to strawberry seedlings The strawberry seedlings were inoculated with three different Enterobacter strains and anthracnose spores, and the control group was treated with water and anthracnose spores (CK). In the four groups of strawberry plants, all the surviving strawberry stem bases showed cavities, which further indicated that Enterobacter protected strawberry seedlings by promoting the formation of cavities in the strawberry stem base. In this example, the survival rate of strawberry plants with cavities in the stem base was analyzed to analyze the protection of different Enterobacter strains to strawberry seedlings.

[0099] As shown in Table 5, the survival rate of strawberry plants with cavities in the stem base was the highest under strain 2 treatment, that is, strain 2 had the best protection effect on strawberry plants.

[0100] Table 5. Protection of different Enterobacter strains to strawberry

[0101] In summary, strain 2 E. ludwigii has the highest inhibition rate on anthracnose spore germination and the best protection effect on strawberry plants.

[0102] Example 3. Biochemical characterization of Enterobacter The physiological and biochemical properties of the isolated bacterial isolates were determined, including Gram staining, gelatin liquefaction, sugar fermentation, phenylalanine deaminase, tryptophan deaminase, tryptophan decarboxylase, arginine decarboxylase, and ornithine decarboxylase determination. The strains were sent to the China Agricultural Microbial Culture Collection Center for BiologTM biological carbon source analysis determination.

[0103] 1. Physiological and biochemical characterization of Enterobacter As shown in Figure 2 A, after 24 h growth of Enterobacter strains at 23°C on solid medium, the single colonies were raised and semicircular, light cream in color, uniform in texture, smooth on the surface, and shiny. As shown in Figure 5 , transmission electron microscopy showed that the bacterial cells were straight and short rod-shaped, with peritrichous flagella. Gram staining was red and negative.

[0104] 2. Effect of salt stress on colonization of Enterobacter As shown in Figure 6 , after treatment with 40 mM sodium chloride solution, the strawberry plants showed obvious salt damage, with old leaves showing leaf margin scorching and new leaves showing leaf margin rolling. After 7 days of adding Enterobacter, the new leaves of the plants were flat, and the salt damage symptoms of the plants were significantly reduced. The old leaves that were not dried were removed, and the Enterobacter at the base of the petiole was determined. It was found that Enterobacter colonized each salt-treated plant, while the colonization rate of Enterobacter in the control group treated with water was 46.67%.

[0105] 3. Utilization of different carbon sources by enterobacter The physiological and biochemical indicators of different enterobacter were determined. It was found that strains 18 and 22 could utilize glucose, xylose, mannitol, mannose, trehalose, fructose, maltose, sorbitol, inositol, dextran, arabinose and other carbon sources and grow rapidly under aerobic or anaerobic conditions. They could also utilize most of the carbon sources and produce a large amount of carbon dioxide gas. Strains 2 and 18 did not produce gas when utilizing inositol, xylitol, dextran and arabinose, while strain 22 could not utilize inositol and could utilize arabinose at low temperature (about 10-15°C) to produce a small amount of carbon dioxide slowly. All strains could utilize xylitol under aerobic conditions but could not utilize it under anaerobic conditions. The gas produced by the strains pushed the sealed paraffin oil or formed a large number of bubbles and cavities in the solid culture medium, which were very similar to the cavities in plant tissues.

[0106] The volume of gas produced by different enterobacter under different carbon sources was measured. The enterobacter produced the largest amount of gas when the carbon source was mannitol, mannose and trehalose, and the maximum amount of gas produced was 3.65 cm 3 after 48 h. The smallest amount of gas was produced when the carbon source was xylose. As shown in Table 2, among the four enterobacter, strain 18 produced the largest amount of gas, while strain 2 produced the smallest amount of gas. Figure 7

[0107] 4. Utilization of different amino acids by enterobacter Different enterobacter could produce lysine decarboxylase, arginine decarboxylase and ornithine decarboxylase under aerobic or anaerobic conditions, making the medium containing lysine, arginine and ornithine turn bright purple and produce a small amount of carbon dioxide gas. The amount of gas produced by different strains was different. As shown in Table 6, strain 2 could produce a small amount of gas when utilizing the three amino acids, while strain 22 could produce a small amount of gas when utilizing arginine and ornithine, and strain 18 could produce gas when utilizing arginine. All enterobacter could not produce phenylalanine deaminase and tryptophan deaminase, and could not produce urease. The control medium (without adding amino acids) inoculated with enterobacter was yellow under anaerobic conditions and purple under aerobic conditions, which was probably because the bacteria utilized the glucose in the medium to produce a large amount of gas. Figure 8 E. ludwigii Gas production under different nutritional conditions

[0108] Table 6 Gas production of different enterobacter under different nutritional conditions

[0109] Note: "+" and "-" represent positive and negative reactions, respectively; Y: yellow; BP: blue purple ​​In summary, the enterobacterium has the characteristics of endophyte. When the strawberry seedlings are not stressed by pathogenic microorganisms (such as anthracnose and fusarium), the enterobacterium has the effect of promoting the growth of the plants, and no hollows are formed at the base of the strawberry stems. However, after being infected by the anthracnose for one month, the strawberry seedlings with the enterobacterium mutant strain have a small number of survival, while the strawberry seedlings in the water control group all die. After cutting the shortened stems of the surviving strawberry plants, it is found that the inside of the stems all have hollows of different sizes, which are consistent with the field symptoms. The enterobacterium is re-isolated from the base of the stems of the surviving strawberry plants, and the Koch's postulates are verified. However, the enterobacterium not only protects the host plants, but also forms new problems, i.e. the hollows at the base of the stems.

[0110] The enterobacterium that inhibits the strawberry anthracnose by forming the hollows at the base of the strawberry stems mainly includes three kinds of enterobacterium, wherein E. cancerogenus and E. cloacae subsp. Dissolvens At most, it also includes a small amount of E. ludgiwii . E. cancerogenus and E. cloacae subsp. Dissolvens can utilize most of the carbon sources and part of the amino acids to produce a large amount of carbon dioxide or other types of gas, E. ludgiwii can utilize the relatively rare sugar such as arabinose and produce a small amount of gas. Therefore, it can be concluded that the enterobacterium as a facultative anaerobic gas-producing bacterium produces a large amount of gas in the process of competitive growth and reproduction, and the gas accumulates at the base of the strawberry stem to form a hollow. When there is no pathogenic fungus stress, the strawberry converts CO2 into organic carbon through photosynthesis, as the "energy core" of the whole plant, and at the same time, the root system absorbs the soil small molecular organic carbon to supplement the insufficient photosynthesis and realize the energy supply balance. The anthracnose inhibits the utilization of carbon sources by destroying the key link of carbon source metabolism: destroying the photosynthetic structure, hindering the absorption of CO2, and reducing the generation of organic carbon by infecting the leaves; blocking the carbon source transport by infecting the stem base; reducing the soil organic carbon absorption and nutrient acquisition ability by infecting the roots, ultimately leading to insufficient carbon source supply and distribution disorder, affecting the growth of the plants and the quality of the fruits. The enterobacterium promotes the formation of the hollow at the base of the strawberry stem, on the one hand, inhibits the spore germination of the anthracnose, and on the other hand, promotes the utilization of carbon sources by the strawberry, so as to achieve the effect of improving the disease resistance and yield of the strawberry.

[0111] Therefore, the "hollow disease at the base of the stem" of the strawberry is a symptom produced by the synergistic endophyte against the pathogenic bacteria, rather than a new disease. The enterobacterium produces CO2 when utilizing the carbon source, and the enterobacterium, E. ludgiwiiThe inhibition rate of spore germination of anthracnose bacteria is the highest, and the cavity is the smallest. For the host plant, the stem base cavity affects the strawberry to absorb more nutrients and water; for the pathogenic bacteria, the cavity inhibits the further infection of the pathogenic bacteria to a certain extent. The occurrence of this symptom means that the interaction between strawberry and environmental microorganisms is complex and diverse. In the decades of intensive production of facility agriculture, strawberry and environmental microorganisms are constantly coevolving and developing into a new way. As an important plant companion, enterobacteriaceae promotes plant growth by increasing phosphorus, potassium and zinc nutrients, biological nitrogen fixation (BNF), and producing iron carriers, ammonia, hydrogen cyanide (HCF); at the same time, when subjected to biological stress, it activates plant cell signaling pathways, opens plant pattern-triggered immunity (PTI) or effector protein-triggered immunity (ETI) and other antibacterial defense mechanisms to help plants resist pathogenic microbial infection.

[0112] Example 4, enterobacteriaceae and bacillus combined to inhibit anthracnose and cavity formation According to the research contents of examples 1, 2 and 3, it can be concluded that enterobacteriaceae can improve the ability of strawberry to resist anthracnose by promoting the formation of cavity at the base of strawberry stem, but at the same time, it also leads to a new problem, that is, the formation of cavity at the base of strawberry stem. Among them E. ludwigii The inhibition effect on spores of anthracnose bacteria is the best and the protection effect on strawberry is the best, so the present application selects E. ludwigii Further explore how to inhibit the spore germination of anthracnose bacteria while reducing the formation of cavity.

[0113] Bacillus ( Bacillus ) is a kind of gram-positive, endogenous bacillus that can form resistance, which belongs to bacillus family ( Bacillaceae ), and is widely distributed in soil, water, air, plant surface and intestinal tract. Bacillus subtilis ( Bacillus subtilis ) and bacillus velezensis ( Bacillus velezensis ) belong to bacillus family bacillus, both of which can promote the growth of strawberry root system and plant, and reduce the opportunity of pathogenic bacteria infection.

[0114] Therefore, the present application selects E. ludwigii (isolated from strawberry plant) and Bacillus subtilis (bought from microbial preservation center) or E. ludwigii and Bacillus velezensis (bought from microbial preservation center) to explore whether the mixed agent has the effect of inhibiting the spore germination of anthracnose bacteria and reducing the formation of cavity. The viable bacterial concentration in the two mixed agents is 10 6 cfu / mL, E. ludwigii and Bacillus subtilis and E. ludwigii andBacillus velezensis The ratio was 1:1. One week after inoculation with single-strain bacterial suspension or mixture, anthrax spores were inoculated.

[0115] In this embodiment, the strain was inoculated according to the method provided in Example 1. After inoculation, the plants were placed in a light incubator with 14 hours of light / 10 hours of darkness at 20℃-28℃, and the disease incidence was observed and recorded daily. As shown in Table 7, after 40 days of inoculation, all strawberry seedlings in the control group died, compared to those inoculated alone. E. ludwigii , E. ludwigii When used in combination with two different Bacillus species, the cavity rate of strawberry plants decreased and the survival rate increased. Among them, E. ludwigii With Bacillus subtilis ( Bacillus subtilis The combined use of these two agents yields better results. When used together, the germination rate and cavity rate of anthracnose spores in strawberry plants are close to 0%, and the survival rate is close to 100%. This not only reduces the formation of cavities at the base of strawberry stems but also helps to control anthracnose.

[0116] Table 7. Control effects of different bacterial strains on anthrax bacteria.

[0117] In summary, when Enterobacter and Bacillus subtilis are used in combination, the anthracnose spore germination rate is the lowest, the cavity rate is significantly reduced, and the strawberry survival rate is the highest. Therefore, E. ludwigii and Bacillus subtilis The combined use helps reduce the formation of cavities while preventing strawberry anthracnose.

[0118] While the present invention has been disclosed above, it is not limited thereto. Its application scope in the field of microfluidics can be expanded accordingly. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. Use of Enterobacteriaceae for preparing an agent for promoting the hollowing of the strawberry stem base, thereby improving the disease resistance of the strawberry.

2. Use as claimed in claim 1, characterized in that, The enterobacteriaceae include any one or more of: E. ludwigii , E. cloacae subsp. Dissolvens , E. cancerogenus .

3. Use as claimed in claim 2, characterized in that, The disease resistance of the strawberry improved by the Enterobacteriaceae includes the ability of the strawberry to inhibit the infection of pathogenic bacteria.

4. Use as claimed in claim 3, characterized in that, The pathogenic bacteria include anthracnose bacteria, gray mold bacteria, and rhizoctonia.

5. Use as claimed in claim 4, characterized in that, The Enterobacteriaceae can inhibit the spore germination of the anthracnose bacteria of the strawberry.

6. Use as claimed in claim 5, characterized in that, The enterobacterium is E. ludwigii .

7. Use of Enterobacteriaceae for preparing an agent for inhibiting or treating the anthracnose of the strawberry.

8. A method of promoting the formation of cavities in strawberries to increase the disease resistance of the strawberries, characterized by, The Enterobacteriaceae bacterial solution is poured onto the substrate of the strawberry plant.

9. The method of claim 8, wherein, The amount of the enteric bacilli is 10 5 cfu / mL ~ 10 7 cfu / mL.

10. A method of inhibiting or treating anthracnose of strawberry, characterized by, The Enterobacteriaceae bacterial solution is poured onto the substrate of the strawberry plant.

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