Method for promoting growth of anoectochilus formosanus seedlings

Abstract

The invention provides a method for promoting growth of anoectochilus formosanus seedlings. According to the method for promoting the growth of the anoectochilus formosanus seedlings, the anoectochilus formosanus seedlings are sprayed with the mycorrhizal fungi culture solution of the orange-yellow magnolia denudate, so that stems and leaves of the anoectochilus formosanus seedlings are infiltrated with the mycorrhizal fungi culture solution. The method for promoting the growth of the anoectochilus formosanus seedlings can obviously antagonize the stem rot pathogenic bacteria of the anoectochilus formosanus seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, in particular to a method for promoting the growth of roxburghii seedlings. Background Art

[0002] Anoectochilus roxburghii is an orchid with high medicinal and ornamental value. Due to excessive exploitation, the wild resources of Anoectochilus roxburghii have been depleted. It is now in urgent need of artificial mass breeding. However, with the expansion of the Anoectochilus roxburghii industry, the problem of pathogenic bacteria contamination of tissue culture seedlings has become prominent, such as stem rot. For example, the Chinese invention patent application with application number 201210430120.6 uses wood substrate instead of leaf mold for cultivation, thereby reducing leaf rot of Anoectochilus roxburghii tissue culture seedlings. However, due to the presence of countless pathogenic bacteria in the environment, it is not effective to reduce the contamination of Anoectochilus roxburghii tissue culture seedlings by simply reducing the content of pathogenic bacteria in the cultivation soil. Bacterial contamination is relatively limited; some researchers have screened out beneficial strains that have significant antagonism against stem rot pathogens of A. roxburghii from the perspective of mycorrhizal fungi, such as the Chinese invention patent application with application number 201710702306.5, which isolated endophytic fungi that can significantly antagonize stem rot pathogens from A. roxburghii, which can effectively promote the large-scale reproduction of A. roxburghii, but there are not many reports on the discovery of fungi that have significant antagonism against pathogens of stem rot of A. roxburghii seedlings. Therefore, the discovery of fungi that have significant antagonism against pathogens of stem rot of A. roxburghii seedlings is still an unremitting pursuit of technicians in this field. Summary of the invention

[0003] The purpose of the invention is to overcome the deficiencies in the prior art and provide a method for promoting the growth of roxburghii seedlings which can significantly antagonize the pathogenic bacteria of roxburghii seedling stem rot.

[0004] The objective of the present invention is achieved through the following technical solutions:

[0005] A method for promoting the growth of roxburghii seedlings comprises the following steps: watering the roxburghii seedlings with mycorrhizal fungi culture solution of rueba rueba, so that the mycorrhizal fungi culture solution infiltrates the stems and leaves of the roxburghii seedlings.

[0006] In one embodiment, the mycorrhizal fungus culture solution of the orange-yellow hibiscus is used to water the roxburghii seedlings, comprising the following steps:

[0007] Obtaining the roxburghii roxburghii seedlings;

[0008] The mycorrhizal fungus culture solution is used to spray the roxburghii seedlings, so that the mycorrhizal fungus culture solution adheres to the stems and leaves of the roxburghii.

[0009] In one embodiment, the usage of the mycorrhizal fungus culture solution is 5 ml to 15 ml.

[0010] In one embodiment, the mycorrhizal fungus culture solution is obtained by the following steps:

[0011] Pick wild orange-yellow hibiscus flowers;

[0012] Performing rhizome treatment on the orange-yellow jade flower to clean the surface of the rhizome of the orange-yellow jade flower and obtain new nutrient roots that form mycorrhizae;

[0013] Scraping the bacterial mass from the newly-born nutrient root to absorb the bacterial mass on the newly-born nutrient root to obtain the orange-yellow jade flower bacterial community;

[0014] The mycorrhizal fungus community is subjected to a proliferation and culture operation to obtain the mycorrhizal fungus culture solution.

[0015] In one embodiment, the bacterial mass scraping operation is performed on the newly formed nutrient root, and the specific operation is as follows:

[0016] The newly-born nutrient root is placed in a culture dish containing sterile water, and a sterilized inoculation needle is used to scrape a single hyphae mass of the newly-born nutrient root.

[0017] In one embodiment, the proliferation and cultivation operation of the orange-yellow hibiscus flora is carried out, and the specific operation is:

[0018] The orange-yellow hibiscus bacterial community is inoculated into a PDB culture medium, and then the PDB culture medium is placed in a shaking incubator at a temperature of 25° C. to 28° C. for culture.

[0019] In one embodiment, the rotation speed of the shaking table is 110 r / min to 120 r / min.

[0020] In one embodiment, the specification of the PDB culture medium is 100ml to 150ml.

[0021] In one embodiment, the PDB culture medium is placed in a shaking incubator at 25° C. to 28° C. and cultured for at least 3 days.

[0022] In one embodiment, the PDB culture medium is placed in a shaker at a temperature of 25°C to 28°C for culture, and then the PDB culture medium is broken and mixed so that the clumping hyphae in the PDB culture medium are evenly dispersed in the PDB culture medium to obtain the mycorrhizal fungal culture solution.

[0023] Compared with the prior art, the present invention has at least the following advantages:

[0024] The method for promoting the growth of roxburghii seedlings of the present invention comprises the following steps: using the mycorrhizal fungus culture solution of the roxburghii flower to water the stems and leaves of the roxburghii seedlings, so that the mycorrhizal fungus culture solution infiltrates the stems and leaves of the roxburghii seedlings, namely, the mycorrhizal fungus culture solution directly acts on the stems and leaves of the roxburghii seedlings, thereby promoting the endophytic fungi of the roxburghii flower to enter the stems and leaves of the roxburghii seedlings, effectively enhancing the stress resistance of the roxburghii seedlings, especially having a good antagonistic effect on the pathogenic bacteria of stem rot, thereby better improving the survival rate of the roxburghii seedlings. DETAILED DESCRIPTION

[0025] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only and do not represent the only implementation method.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0027] The present application provides a method for promoting the growth of golden thread vine seedlings. In order to better understand the method for promoting the growth of golden thread vine seedlings of the present application, the method for promoting the growth of golden thread vine seedlings of the present application is further explained below:

[0028] In one embodiment of the method for promoting the growth of roxburghii seedlings, the roxburghii seedlings are watered with the mycorrhizal fungus culture solution of the jacaranda, so that the mycorrhizal fungus culture solution infiltrates the stems and leaves of the roxburghii seedlings.

[0029] The above-mentioned method for promoting the growth of roxburghii seedlings uses the mycorrhizal fungus culture solution of the orange-yellow jade flower to water the stems and leaves of the roxburghii seedlings, so that the mycorrhizal fungus culture solution infiltrates the stems and leaves of the roxburghii seedlings, that is, the mycorrhizal fungus culture solution directly acts on the stems and leaves of the roxburghii seedlings, prompting the endophytic fungi of the orange-yellow jade flower to enter the stems and leaves of the roxburghii seedlings, effectively enhancing the stress resistance of the roxburghii seedlings, especially having a good antagonistic effect on the pathogenic bacteria of stem rot, thereby better improving the survival rate of the roxburghii seedlings.

[0030] In one embodiment, the orange-yellow Jade Flower was collected from Xiangtoushan Nature Reserve. Furthermore, the orange-yellow Jade Flower was collected from a location at 114° east longitude, 23° north latitude, and 460 m above sea level.

[0031] In one embodiment, the mycorrhizal fungus culture solution of the orange-yellow hibiscus is used to water the roxburghii seedlings, comprising the following steps:

[0032] S110, obtaining roxburghii seedlings. Further, the roxburghii seedlings come from a roxburghii planting and breeding base of Hujinyuan Ecological Agriculture Development Co., Ltd. in Longmen County, Huizhou City, Guangdong Province.

[0033] S120, spraying the roxburghii seedlings with a mycorrhizal fungus culture solution, so that the mycorrhizal fungus culture solution adheres to the stems and leaves of the roxburghii.

[0034] It can be understood that the mycorrhizal fungi culture solution is directly sprayed on the stems and leaves of the obtained golden thread vine seedlings, so that the mycorrhizal fungi culture solution can effectively contact and infiltrate the stems and leaves of the golden thread vine seedlings, thereby facilitating the endophytic fungi of the orange-yellow jade flower to enter the stems and leaves of the golden thread vine seedlings. It can also be understood that in the process of directly spraying the stems and leaves of the golden thread vine seedlings, part of the mycorrhizal fungi culture solution will flow down to the roots of the golden thread vine seedlings along with the stems and leaves of the golden thread vine seedlings, and at the same time promote the endophytic fungi of the orange-yellow jade flower and the root system of the golden thread vine seedlings to form a symbiotic state, which has a good enhancement effect on the overall stress resistance of the golden thread vine seedlings.

[0035] In one of the embodiments, the obtained roxburghii seedlings are planted in a planting matrix and then sprayed with a mycorrhizal fungus culture solution.

[0036] In one of the embodiments, the planting matrix is ​​peat soil, which is conducive to the growth of golden thread vine seedlings. In one of the embodiments, after the step of obtaining golden thread vine seedlings and before the step of spraying the golden thread vine seedlings with mycorrhizal fungal culture solution, the method for promoting the growth of golden thread vine seedlings also includes the following steps: performing a penetration-promoting treatment on the mycorrhizal fungal culture solution or the golden thread vine seedlings to promote the attachment and penetration of the mycorrhizal fungal culture solution on the stem and leaf surfaces of the golden thread vine seedlings. Further, the mycorrhizal fungal culture solution is subjected to a penetration-promoting treatment, and the specific operation is: adding tetrahydropiperine to the mycorrhizal fungal culture solution. Further, the mass percentage of tetrahydropiperine in the mycorrhizal fungal culture solution is 0.02% to 0.1%. It can be understood that the use of tetrahydropiperine effectively promotes the invasion of endophytic fungi of orange-yellow jade flower into the stems and leaves of golden thread vine seedlings, that is, it better promotes the endophytic fungi of orange-yellow jade flower to enter the stems and leaves of golden thread vine seedlings, better ensures the enhancement of the stress resistance of golden thread vine seedlings, and thus better ensures the survival rate of golden thread vine seedlings.

[0037] In one embodiment, the seedlings of Anoectochilus roxburghii are treated to promote penetration, and the specific operation is: the surface of the Anoectochilus roxburghii seedlings is locally rubbed to form wounds on the cortical layer of the local stems and leaves of the Anoectochilus roxburghii seedlings. Further, no more than 0.1% of the stems and leaves of the Anoectochilus roxburghii seedlings have wounds formed on the cortical layer. It can be understood that controlling the formation of wounds on the cortical layer of the stems and leaves of no more than 0.1% of the Anoectochilus roxburghii seedlings will not affect the growth of the Anoectochilus roxburghii seedlings. Further, the formation of wounds on the cortical layer of the stems and leaves of 0.1% of the Anoectochilus roxburghii seedlings can effectively promote the invasion of the endophytic fungi of the orange-yellow jade flower into the stems and leaves of the Anoectochilus roxburghii seedlings, that is, better promote the endophytic fungi of the orange-yellow jade flower to enter the stems and leaves of the Anoectochilus roxburghii seedlings, better ensure the enhancement of the stress resistance of the Anoectochilus roxburghii seedlings, and thus better ensure the survival rate of the Anoectochilus roxburghii seedlings.

[0038] In one embodiment, the amount of mycorrhizal fungi culture solution used is 5ml to 15ml. It can be understood that when the stems and leaves of the golden thread vine seedlings are fully infiltrated with the mycorrhizal fungi culture solution by spraying, the antagonistic effect on the stem rot pathogens of the golden thread vine seedlings is better when the amount of mycorrhizal fungi culture solution used is 5ml to 15ml, and if the amount used is less, the advantage of the endophytic fungi of the orange-yellow jade flower is poor, and it is difficult to enhance the stress resistance of the golden thread vine seedlings; and if the amount used is more, the advantage of the endophytic fungi of the orange-yellow jade flower is stronger, which will affect the advantage of the native fungi of the golden thread vine seedlings, and then affect the growth of the golden thread vine seedlings.

[0039] In one embodiment, the mycorrhizal fungus culture solution is obtained by the following steps:

[0040] S210, picking wild orange-yellow hibiscus flowers;

[0041] S220, treating the rhizomes of the orange-yellow jade flower to clean the surface of the rhizomes of the orange-yellow jade flower to obtain new nutrient roots that form mycorrhizae;

[0042] S230 performs a bacterial mass scraping operation on the newly-born nutrient root to absorb the bacterial mass on the newly-born nutrient root to obtain the orange-yellow jade flower bacterial population;

[0043] S240, performing proliferation and culture operations on the fungus community of the orange-yellow jade flower to obtain mycorrhizal fungal culture solution.

[0044] It can be understood that the endophytic fungi of the orange-yellow jade flower are proliferated and cultured together to form a mycorrhizal fungal culture solution, among which the presence of at least one endophytic fungus of the orange-yellow jade flower can effectively enhance the stress resistance of the golden thread vine seedlings. The present application does not purify and separate the specific types of endophytic fungi, but only protects the endophytic fungi of the orange-yellow jade flower to form a mycorrhizal fungal culture solution after proliferating and cultured together, which plays a good role in enhancing the stress resistance of the golden thread vine seedlings. It is worth mentioning that although there are multiple endophytic fungi in the mycorrhizal fungal culture solution, it will not affect the growth of the golden thread vine seedlings if the usage is controlled.

[0045] In one embodiment, the rhizome of the orange-yellow hibiscus is processed, which specifically comprises the following steps:

[0046] S310, washing and cutting the rhizome of the orange-yellow jasmine to remove particles on the surface of the rhizome of the orange-yellow jasmine to obtain rhizome segments of the orange-yellow jasmine. Further, there are a plurality of rhizome segments of the orange-yellow jasmine.

[0047] S320, performing deep cleaning on the rhizome segments of the orange-yellow jade flower to remove bacteria and harmful substances on the surface of the rhizome segments of the orange-yellow jade flower;

[0048] S330, cleaning the rhizome segments of the orange-yellow Jade Flower that have been deeply cleaned to remove liquid on the surface of the rhizome segments of the orange-yellow Jade Flower to obtain new nutrient roots.

[0049] It can be understood that by cleaning the particles, bacteria and harmful substances on the surface of the rhizome of the orange-yellow jade flower separately, it is easier to obtain the endophytic fungal flora of the orange-yellow jade flower with higher purity at the newly formed nutrient roots, which is beneficial to reduce the influence of miscellaneous bacteria or other substances on the growth of the flora at the newly formed nutrient roots during the proliferation culture process.

[0050] In one of the embodiments, the rhizomes of the orange-yellow jade flower are rinsed and cut, and the specific operation is: the rhizomes of the orange-yellow jade flower are rinsed under tap water, and then the rhizomes of the orange-yellow jade flower are cut into segments to obtain orange-yellow jade flower rhizome segments, which better ensures the effective removal of particulate matter on the surface of the rhizomes of the orange-yellow jade flower, and the formation of the rhizome segments of the orange-yellow jade flower is conducive to the scraping of the bacterial mass of the newly formed nutrient roots in the later stage.

[0051] In one embodiment, the length of the rhizome segment of the orange-yellow Jade Phoenix flower is 1 cm to 2 cm, which is conducive to scraping the bacterial mass of the newly formed nutrient roots in the later stage.

[0052] In one of the embodiments, the rhizome segments of the orange-yellow jasmine flower are subjected to a deep cleaning treatment, and the specific operation is: the rhizome segments of the orange-yellow jasmine flower are soaked in a sodium hypochlorite solution, and then taken out and soaked in an ethanol solution, thereby effectively removing bacteria and harmful substances on the surface of the rhizome segments of the orange-yellow jasmine flower.

[0053] In one embodiment, the mass percentage of the sodium hypochlorite solution is 2.5%. Further, the rhizome segments of the orange-yellow jasmine flower are immersed in the sodium hypochlorite solution for 0.5 to 1 minute, which further effectively removes bacteria and harmful substances on the surface of the rhizome segments of the orange-yellow jasmine flower.

[0054] In one embodiment, the mass percentage of the ethanol solution is 70%. Further, the rhizome segments of the orange-yellow jasmine flower are immersed in the ethanol solution for 0.8 min to 1 min, which further effectively removes bacteria and harmful substances on the surface of the rhizome segments of the orange-yellow jasmine flower.

[0055] In one of the embodiments, the rhizome segments of the orange-yellow jade flower after deep cleaning are cleaned, and the specific operation is: the rhizome segments of the orange-yellow jade flower are rinsed with sterile water at least three times, and then the liquid on the surface of the rhizome segments of the orange-yellow jade flower is absorbed with sterile absorbent paper, thereby further reducing the residues of various reagents or miscellaneous bacteria on the surface of the orange-yellow jade flower.

[0056] In one of the embodiments, a mycelium scraping operation is performed on the newly-born nutrient roots. Specifically, the newly-born nutrient roots are placed in a culture dish containing sterile water, and a sterilized inoculation needle is used to scrape a single mycelium mass of the newly-born nutrient roots, thereby effectively obtaining the endophytic fungi on the newly-born nutrient roots.

[0057] In one embodiment, before the step of performing proliferation and cultivation operations on the orange-yellow jade flower fungus community and after the step of performing a fungus scraping operation on the newly formed nutrient roots, the mycorrhizal fungus culture solution also includes the following acquisition step: selectively culturing the orange-yellow jade flower fungus community to inhibit the excessive growth of miscellaneous bacteria in the orange-yellow jade flower fungus community.

[0058] In one embodiment, the selective culture of the Euphorbia cerasifera flora is carried out by adding fluconazole to the PDB medium. Furthermore, the amount of fluconazole used is 0.1 g / L to 0.5 g / L.

[0059] It can be understood that since there are many types of endophytic fungi in the orange-yellow jade flower, and at least one of the endophytic fungi in the orange-yellow jade flower has an enhancing effect on the stress resistance of the golden thread vine seedlings, therefore, in order to better achieve the effective growth of the endophytic fungi in the orange-yellow jade flower that have an enhancing effect on the stress resistance of the golden thread vine seedlings, fluconazole was used to carry out selective proliferation culture of the endophytic fungi in the orange-yellow jade flower. In this way, the obtained mycorrhizal fungal culture solution has a better enhancing effect on the stress resistance of the golden thread vine seedlings.

[0060] In one embodiment, the preparation of PDB culture medium is specifically performed as follows: 200 g of peeled potatoes are boiled in boiling water for 30 min, filtered through gauze, 20 g of glucose is added, water is added to make the volume to 1 L, and sterilized at 121° C. for 30 min.

[0061] In one of the embodiments, the orange-yellow jade flower flora is subjected to a proliferation and cultivation operation, and the specific operation is: the orange-yellow jade flower flora is inoculated into a PDB medium, and then the PDB medium is placed in a shaker at a temperature of 25°C to 28°C for cultivation. Further, the rotation speed of the shaker is 110r / min to 120r / min. Further, the specification of the PDB medium is 100ml to 150ml. Further, the PDB medium is placed in a 25°C to 28°C shaker for at least 3 days, which effectively realizes the rapid proliferation of the orange-yellow jade flower flora, reduces the mutual interference of the orange-yellow jade flower flora, and thus ensures that the mycorrhizal fungal culture solution formed after the proliferation and cultivation of the orange-yellow jade flower flora has an enhancing effect on the stress resistance of the golden thread vine seedlings. It can be understood that if the Orange-Yellow Jade flora flora grows excessively, it will affect the mutual interference among the Orange-Yellow Jade flora flora, and affect the effect of the formed mycorrhizal fungal culture solution on enhancing the stress resistance of the Golden Thread Lotus seedlings; if the Orange-Yellow Jade flora flora does not grow fully, the concentration of endophytic fungi of the Orange-Yellow Jade in the mycorrhizal fungal culture solution will be low, which will make it difficult to meet the needs of enhancing the stress resistance of the Golden Thread Lotus seedlings.

[0062] In one embodiment, the PDB culture medium is placed in a shaker at a temperature of 25°C to 28°C for culture, and then the PDB culture medium is broken and mixed so that the clumping hyphae in the PDB culture medium are evenly dispersed in the PDB culture medium to obtain the mycorrhizal fungus culture solution.

[0063] Compared with the prior art, the present invention has at least the following advantages:

[0064] The method for promoting the growth of roxburghii seedlings of the present invention comprises the following steps: using the mycorrhizal fungus culture solution of the roxburghii flower to water the stems and leaves of the roxburghii seedlings, so that the mycorrhizal fungus culture solution infiltrates the stems and leaves of the roxburghii seedlings, namely, the mycorrhizal fungus culture solution directly acts on the stems and leaves of the roxburghii seedlings, thereby promoting the endophytic fungi of the roxburghii flower to enter the stems and leaves of the roxburghii seedlings, effectively enhancing the stress resistance of the roxburghii seedlings, especially having a good antagonistic effect on the pathogenic bacteria of stem rot, thereby better improving the survival rate of the roxburghii seedlings.

[0065] Some specific examples are listed below, and if % is mentioned, it means percentage by weight. It should be noted that the following examples do not exhaust all possible situations, and the materials used in the following examples can be obtained from commercial sources unless otherwise specified.

[0066] Example 1

[0067] Sampling: Orange-yellow Jade Phoenix, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m above sea level);

[0068] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30min, filter with gauze, add 20g of glucose, add water to make up to 1L, put into electric pressure steam sterilizer at 121℃ for 30min, divide into conical flasks and set aside for later use. The capacity of the conical flask is 100ml.

[0069] The collected roots of the orange-yellow Jade were rinsed with running water to clean the sand and other particles on the surface of the rhizomes of the orange-yellow Jade. The rhizomes of the orange-yellow Jade were selected and cut with scissors and placed in a fixed cup, and then moved to a clean bench. The rhizomes of the orange-yellow Jade were cut into small sections of 1.5±0.5 cm each, and then immersed in a 2.5% sodium hypochlorite solution for 0.5 min, and then each sample plant was immersed in a 70% ethanol solution for 0.8 min, washed with sterile water (rinsed 3 times), and the surface of the rhizomes was gently scraped clean with a sterilized inoculation needle, rinsed with sterile water 5 times, and placed on sterile absorbent paper to dry, to obtain new nutrient roots;

[0070] Place the newly grown nutrient roots in a culture dish filled with sterile water, and use a sterilized inoculation needle to scrape out a single hyphae mass from the newly grown nutrient roots;

[0071] On the clean bench, use a sterilized pipette and bamboo stick to pick up the single mycelium mass of the newly formed nutrient root into the PDB medium, and culture it on a constant temperature shaker at 25°C and 110 r / min for 3 days. Use sterilized pliers and scissors to cut the mycelium ball in the PDB medium into pieces, and use a vortex oscillator to evenly mix the mycelium and the bacterial solution to make the mycorrhizal fungal culture solution.

[0072] Example 2

[0073] Sampling: Orange-yellow Jade Phoenix, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m above sea level);

[0074] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30min, filter with gauze, add 20g of glucose, add water to make up to 1L, put into electric pressure steam sterilizer at 121℃ for 30min, divide into conical flasks for later use, the capacity of the conical flask is 150ml;

[0075] The collected roots of the orange-yellow Jade were rinsed with running water to clean the sand and other particles on the surface of the rhizomes of the orange-yellow Jade. The rhizomes of the orange-yellow Jade were selected and cut with scissors and placed in a fixed cup, and then moved to a clean bench. The rhizomes of the orange-yellow Jade were cut into small sections of 1.5±0.5 cm each, and then immersed in a 2.5% sodium hypochlorite solution for 0.8 min, and then each sample plant was immersed in a 70% ethanol solution for 0.9 min, washed with sterile water (rinsed three times), and the surface of the rhizomes was gently scraped clean with a sterilized inoculation needle, rinsed with sterile water for 6 times, and placed on sterile absorbent paper to dry, to obtain new nutrient roots;

[0076] Place the newly grown nutrient roots in a culture dish filled with sterile water, and use a sterilized inoculation needle to scrape out a single hyphae mass from the newly grown nutrient roots;

[0077] On the clean bench, use a sterilized pipette and bamboo stick to pick up the single mycelium mass of the newly formed nutrient root into the PDB medium, and culture it on a constant temperature shaker at 26°C and 116 r / min for 4 days. Cut the mycelium ball in the PDB medium into pieces with sterilized pliers and scissors, and use a vortex oscillator to evenly mix the mycelium and the bacterial solution to make the mycorrhizal fungal culture solution.

[0078] Example 3

[0079] Sampling: Orange-yellow Jade Phoenix, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m above sea level);

[0080] Preparation of PDB medium: Boil 200g of potatoes (peeled) in boiling water for 30min, filter with gauze, add 20g of glucose, add water to make up to 1L, put into electric pressure steam sterilizer at 121℃ for 30min, divide into conical flasks for later use, the capacity of the conical flask is 150ml;

[0081] The collected roots of the orange-yellow Jade were rinsed with running water to clean the sand and other particles on the surface of the rhizomes of the orange-yellow Jade. The rhizomes of the orange-yellow Jade were selected and cut with scissors and placed in a fixed cup, and then moved to a clean bench. The rhizomes of the orange-yellow Jade were cut into small sections of 1.5±0.5 cm each, and then immersed in a 2.5% sodium hypochlorite solution for 1 minute, and then each sample plant was immersed in a 70% ethanol solution for 1 minute, washed with sterile water (rinsed three times), and the surface of the rhizomes was gently scraped clean with a sterilized inoculation needle, rinsed with sterile water for 6 times, and placed on sterile absorbent paper to dry, to obtain new nutrient roots;

[0082] Place the newly grown nutrient roots in a culture dish filled with sterile water, and use a sterilized inoculation needle to scrape out a single hyphae mass from the newly grown nutrient roots;

[0083] On the clean bench, use a sterilized pipette and bamboo stick to pick up the single mycelium mass of the newly formed nutrient root into the PDB medium, and culture it on a constant temperature shaker at 28°C and 120r / min for 5 days. Use sterilized pliers and scissors to cut the mycelium ball in the PDB medium into pieces, and use a vortex oscillator to evenly mix the mycelium and the bacterial solution to make the mycorrhizal fungal culture solution.

[0084] Example 4

[0085] Sampling: Orange-yellow Jade Phoenix, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m above sea level);

[0086] Preparation of PDB medium: Boil 200 g of potatoes (peeled) in boiling water for 30 min, filter with gauze, add 20 g of glucose and 0.1 g / L of fluconazole, add water to make up to 1 L, put it in an electric pressure steam sterilizer at 121°C for 30 min, divide it into conical flasks and set aside for later use. The capacity of the conical flask is 150 ml.

[0087] The collected roots of the orange-yellow Jade were rinsed with running water to clean the sand and other particles on the surface of the rhizomes of the orange-yellow Jade. The rhizomes of the orange-yellow Jade were selected and cut with scissors and placed in a fixed cup, and then moved to a clean bench. The rhizomes of the orange-yellow Jade were cut into small sections of 1.5±0.5 cm each, and then immersed in a 2.5% sodium hypochlorite solution for 1 minute, and then each sample plant was immersed in a 70% ethanol solution for 1 minute, washed with sterile water (rinsed three times), and the surface of the rhizomes was gently scraped clean with a sterilized inoculation needle, rinsed with sterile water for 6 times, and placed on sterile absorbent paper to dry, to obtain new nutrient roots;

[0088] Place the newly grown nutrient roots in a culture dish filled with sterile water, and use a sterilized inoculation needle to scrape out a single hyphae mass from the newly grown nutrient roots;

[0089] On the clean bench, use a sterilized pipette and bamboo stick to pick up the single mycelium mass of the newly formed nutrient root into the PDB medium, and culture it on a constant temperature shaker at 28°C and 120r / min for 5 days. Use sterilized pliers and scissors to cut the mycelium ball in the PDB medium into pieces, and use a vortex oscillator to evenly mix the mycelium and the bacterial solution to make the mycorrhizal fungal culture solution.

[0090] Example 5

[0091] Sampling: Orange-yellow Jade Phoenix, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m above sea level);

[0092] Preparation of PDB medium: Boil 200 g of potatoes (peeled) in boiling water for 30 min, filter with gauze, add 20 g of glucose and 0.2 g / L of fluconazole, add water to make up to 1 L, put it in an electric pressure steam sterilizer at 121°C for 30 min, divide it into conical flasks for later use, and the capacity of the conical flask is 150 ml;

[0093] The collected roots of the orange-yellow Jade were rinsed with running water to clean the sand and other particles on the surface of the rhizomes of the orange-yellow Jade. The rhizomes of the orange-yellow Jade were selected and cut with scissors and placed in a fixed cup, and then moved to a clean bench. The rhizomes of the orange-yellow Jade were cut into small sections of 1.5±0.5 cm each, and then immersed in a 2.5% sodium hypochlorite solution for 1 minute, and then each sample plant was immersed in a 70% ethanol solution for 1 minute, washed with sterile water (rinsed three times), and the surface of the rhizomes was gently scraped clean with a sterilized inoculation needle, rinsed with sterile water for 6 times, and placed on sterile absorbent paper to dry, to obtain new nutrient roots;

[0094] Place the newly grown nutrient roots in a culture dish filled with sterile water, and use a sterilized inoculation needle to scrape out a single hyphae mass from the newly grown nutrient roots;

[0095] On the clean bench, use a sterilized pipette and bamboo stick to pick up the single mycelium mass of the newly formed nutrient root into the PDB medium, and culture it on a constant temperature shaker at 28°C and 120r / min for 5 days. Use sterilized pliers and scissors to cut the mycelium ball in the PDB medium into pieces, and use a vortex oscillator to evenly mix the mycelium and the bacterial solution to make the mycorrhizal fungal culture solution.

[0096] Example 6

[0097] Sampling: Orange-yellow Jade Phoenix, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m above sea level);

[0098] Preparation of PDB medium: Boil 200 g of potatoes (peeled) in boiling water for 30 min, filter with gauze, add 20 g of glucose and 0.3 g / L of fluconazole, add water to make up to 1 L, put it into an electric pressure steam sterilizer and sterilize it at 121°C for 30 min, divide it into conical flasks and set aside for later use. The capacity of the conical flask is 150 ml.

[0099] The collected roots of the orange-yellow Jade were rinsed with running water to clean the sand and other particles on the surface of the rhizomes of the orange-yellow Jade. The rhizomes of the orange-yellow Jade were selected and cut with scissors and placed in a fixed cup, and then moved to a clean bench. The rhizomes of the orange-yellow Jade were cut into small sections of 1.5±0.5 cm each, and then immersed in a 2.5% sodium hypochlorite solution for 1 minute, and then each sample plant was immersed in a 70% ethanol solution for 1 minute, washed with sterile water (rinsed three times), and the surface of the rhizomes was gently scraped clean with a sterilized inoculation needle, rinsed with sterile water for 6 times, and placed on sterile absorbent paper to dry, to obtain new nutrient roots;

[0100] Place the newly grown nutrient roots in a culture dish filled with sterile water, and use a sterilized inoculation needle to scrape out a single hyphae mass from the newly grown nutrient roots;

[0101] On the clean bench, use a sterilized pipette and bamboo stick to pick up the single mycelium mass of the newly formed nutrient root into the PDB medium, and culture it on a constant temperature shaker at 28°C and 120r / min for 5 days. Use sterilized pliers and scissors to cut the mycelium ball in the PDB medium into pieces, and use a vortex oscillator to evenly mix the mycelium and the bacterial solution to make the mycorrhizal fungal culture solution.

[0102] Example 7

[0103] Sampling: Orange-yellow Jade Phoenix, collected from Xiangtoushan Nature Reserve (114°E, 23°N, 460m above sea level);

[0104] Preparation of PDB medium: Boil 200 g of potatoes (peeled) in boiling water for 30 min, filter with gauze, add 20 g of glucose and 0.5 g / L of fluconazole, add water to make up to 1 L, put it in an electric pressure steam sterilizer at 121°C for 30 min, divide it into conical flasks for later use, and the capacity of the conical flask is 150 ml;

[0105] The collected roots of the orange-yellow Jade were rinsed with running water to clean the sand and other particles on the surface of the rhizomes of the orange-yellow Jade. The rhizomes of the orange-yellow Jade were selected and cut with scissors and placed in a fixed cup, and then moved to a clean bench. The rhizomes of the orange-yellow Jade were cut into small sections of 1.5±0.5 cm each, and then immersed in a 2.5% sodium hypochlorite solution for 1 minute, and then each sample plant was immersed in a 70% ethanol solution for 1 minute, washed with sterile water (rinsed three times), and the surface of the rhizomes was gently scraped clean with a sterilized inoculation needle, rinsed with sterile water for 6 times, and placed on sterile absorbent paper to dry, to obtain new nutrient roots;

[0106] Place the newly grown nutrient roots in a culture dish filled with sterile water, and use a sterilized inoculation needle to scrape out a single hyphae mass from the newly grown nutrient roots;

[0107] On the clean bench, use a sterilized pipette and bamboo stick to pick up the single mycelium mass of the newly formed nutrient root into the PDB medium, and culture it on a constant temperature shaker at 28°C and 120r / min for 5 days. Use sterilized pliers and scissors to cut the mycelium ball in the PDB medium into pieces, and use a vortex oscillator to evenly mix the mycelium and the bacterial solution to make the mycorrhizal fungal culture solution.

[0108] The mycorrhizal fungi culture solution obtained in Examples 1 to 7 was used to cultivate roxburghii seedlings.

[0109] 1. Materials:

[0110] 1.1 The golden thread vine seedlings with the same growth potential come from the golden thread vine planting and breeding base of Hujinyuan Ecological Agriculture Development Co., Ltd. in Longmen County, Huizhou City, Guangdong Province;

[0111] 1.2 Cultivation medium: peat soil;

[0112] 2. Culture conditions:

[0113] The culture experiment of Anoectochilus roxburghii was conducted in the School of Life Sciences of Huizhou University, Huizhou City, Guangdong Province, with the geographical coordinates of 114.42°E, 23.03°N, and a subtropical marine monsoon climate with an average annual temperature of 22°C and an annual precipitation of 2200 mm, concentrated from April to September;

[0114] 3. Test methods:

[0115] 3.1 A completely randomized design was adopted, with 10 roxburghii seedlings forming a small group, and 3 small groups of roxburghii seedlings forming a large group, which were numbered for a total of 8 large groups, with the 8th large group being the blank control group;

[0116] 3.2 The usage of mycorrhizal fungus culture solution is as follows:

[0117] The 1st to 7th groups of Anoectochilus roxburghii seedlings were cultured with the mycorrhizal fungi culture solutions of Examples 1 to 7, respectively;

[0118] For each of the first to seventh groups: the amount of the mycorrhizal fungi culture solution used for the roxburghii seedlings in the first group was 5 ml; the amount of the mycorrhizal fungi culture solution used for the roxburghii seedlings in the second group was 10 ml; the amount of the mycorrhizal fungi culture solution used for the roxburghii seedlings in the third group was 15 ml;

[0119] Group 8: Each group of golden thread vine seedlings uses the same amount of sterile water as each group of golden thread vine seedlings in other groups;

[0120] 3.3 Spray the corresponding amount of mycorrhizal fungi culture solution for each roxburghii seedling on the stems and leaves of the corresponding roxburghii seedlings, and collect the roxburghii seedlings after they grow 150 meters;

[0121] 4. Measurement method:

[0122] The survival rate and plant height of each group of A. roxburghii seedlings were recorded; and the biomass of each group of A. roxburghii seedlings was recorded: the drying method was used, and the temperature was 105°C for 2 hours to constant weight. After drying, the dry weight of the A. roxburghii seedlings was weighed, and the biomass and water content of the A. roxburghii seedlings were calculated using the formula.

[0123] 5. Measurement results:

[0124] 5.1 Table 1: Plant height growth of Anoectochilus roxburghii seedlings (unit: cm)

[0125]

[0126]

[0127] 5.2 Table 2: Survival rate of Anoectochilus roxburghii seedlings (unit: %)

[0128]

[0129]

[0130] 5.3 Table 3: Biomass of Anoectochilus roxburghii seedlings (total chlorophyll and flavonoids in mg / g; reducing sugars and polyphenols in μg / g)

[0131]

[0132]

[0133] As can be seen from Table 1, the plant height growth of the golden thread vine seedlings using the mycorrhizal fungal culture solution is significantly faster than that of the golden thread vine seedlings not using the mycorrhizal fungal culture solution, indicating that the mycorrhizal fungal culture solution has a positive promoting effect on the growth of the stems and leaves of the golden thread vine seedlings; As can be seen from Table 2, the survival rate of the golden thread vine seedlings using the mycorrhizal fungal culture solution is significantly higher than that of the golden thread vine seedlings not using the mycorrhizal fungal culture solution, indicating that the mycorrhizal fungal culture solution has a positive promoting effect on the survival of the golden thread vine seedlings, enabling the golden thread vine seedlings to effectively Antagonistic stem rot pathogens; As can be seen from Table 3, the total chlorophyll and the contents of reducing sugars, flavonoids and polyphenols of the A. roxburghii seedlings treated with mycorrhizal fungal culture solution were significantly increased compared with those of the A. roxburghii seedlings not treated with mycorrhizal fungal culture solution. The increase in the total chlorophyll and the contents of reducing sugars, flavonoids and polyphenols of the A. roxburghii seedlings will lead to the improvement of the stress resistance of the A. roxburghii seedlings. In other words, the mycorrhizal fungal culture solution has a significant effect on improving the stress resistance of the A. roxburghii seedlings.

[0134] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for promoting the growth of golden thread vine seedlings, characterized in that: The mycorrhizal fungus culture solution of the orange-yellow hibiscus is used to water the roxburghii seedlings, so that the mycorrhizal fungus culture solution infiltrates the stems and leaves of the roxburghii seedlings.

2. The method for promoting the growth of roxburghii seedlings according to claim 1, characterized in that: The method of watering roxburghii seedlings with the mycorrhizal fungus culture solution of rosmarinus officinalis comprises the following steps: Obtaining the roxburghii roxburghii seedlings; The mycorrhizal fungus culture solution is used to spray the roxburghii seedlings, so that the mycorrhizal fungus culture solution adheres to the stems and leaves of the roxburghii.

3. The method for promoting the growth of roxburghii seedlings according to claim 1 or 2, characterized in that: The usage amount of the mycorrhizal fungus culture solution is 5ml to 15ml.

4. The method for promoting the growth of roxburghii seedlings according to claim 1, characterized in that: The mycorrhizal fungus culture solution is obtained by the following steps: Pick wild orange-yellow hibiscus flowers; Performing rhizome treatment on the orange-yellow jade flower to clean the surface of the rhizome of the orange-yellow jade flower and obtain new nutrient roots that form mycorrhizae; Scraping the bacterial mass from the newly-born nutrient root to absorb the bacterial mass on the newly-born nutrient root to obtain the orange-yellow jade flower bacterial community; The mycorrhizal fungus community is subjected to a proliferation and culture operation to obtain the mycorrhizal fungus culture solution.

5. The method for promoting the growth of roxburghii seedlings according to claim 4, characterized in that: The bacterial mass scraping operation is performed on the new nutrient roots, and the specific operation is as follows: The newly-born nutrient root is placed in a culture dish containing sterile water, and a sterilized inoculation needle is used to scrape a single hyphae mass of the newly-born nutrient root.

6. The method for promoting the growth of roxburghii seedlings according to claim 4, characterized in that: The orange-yellow hibiscus bacterial community is subjected to a proliferation and cultivation operation, and the specific operation is as follows: The orange-yellow hibiscus bacterial community is inoculated into a PDB culture medium, and then the PDB culture medium is placed in a shaking incubator at a temperature of 25° C. to 28° C. for culture.

7. The method for promoting the growth of roxburghii seedlings according to claim 6, characterized in that: The rotating speed of the shaking table is 110 r / min to 120 r / min.

8. The method for promoting the growth of roxburghii seedlings according to claim 6, characterized in that: The specification of the PDB culture medium is 100ml to 150ml.

9. The method for promoting the growth of roxburghii seedlings according to claim 8, characterized in that: The PDB culture medium was placed in a shaking incubator at 25°C to 28°C and cultured for at least 3 days.

10. The method for promoting the growth of roxburghii seedlings according to claim 6, characterized in that: The PDB culture medium is placed in a shaker at a temperature of 25° C. to 28° C. for culture, and then the PDB culture medium is broken and mixed so that the massed hyphae in the PDB culture medium are evenly dispersed in the PDB culture medium to obtain the mycorrhizal fungus culture solution.

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