CN106701635B - Banana endophytic streptomycete with root-knot nematode killing capability and biological seedling culture substrate developed by banana endophytic streptomycete - Google Patents
Banana endophytic streptomycete with root-knot nematode killing capability and biological seedling culture substrate developed by banana endophytic streptomycete Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention provides a strain of banana endophytic streptomycete with root-knot nematode killing capability and a biological seedling culture substrate developed by the strain. A Streptomyces capoamus NAU-SA-1 is preserved in China general microbiological culture Collection center with the preservation date of 2015, 1 month and 26 days and the preservation number of CGMCC NO. 10441. A functional vegetable seedling biological matrix is obtained by adding the streptomycete NAU-SA-1 bacterial liquid into a common seedling matrix. The product selects specific endophytic actinomycetes aiming at banana seedlings, and the endophytic actinomycetes are added into a matrix to obtain an active biological matrix product, so that the product has a good growth promoting effect on the banana seedling stage, can breed high-quality seedlings with a large number of active functional microorganisms in roots, and has the function of enhancing the resistance of the bred seedlings to root-knot nematodes.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and relates to a strain of banana endophytic streptomyces with root-knot nematode killing capability and a biological seedling culture substrate developed by the strain.
Background
Bananas are large herbal fruit trees specific to tropical and subtropical regions. Root-knot nematodes (Meloidogyne spp.) are plant root system-colonizing endoparasites, one of the major crop pathogenic nematodes in China at present, often cause crop yield loss of 10-30%, and in severe cases more than 70%, and cause annual loss of hundreds of billions of dollars to important commercial crops in the world. The fertilizer is distributed in most provinces of China and can infect hundreds of crops, wherein the damage of vegetables and fruit trees is the most serious. In the production link, the secondary seedling production technology is particularly important. Generally, the banana heeling-in process is changed from original soil cultivation to the present decomposed coconut husk substrate cultivation, and the banana heeling-in process is easy to carry pests once being contacted with soil and is unfavorable for the growth of banana tissue culture seedlings. Especially blight and soil nematodes, once infected, transplanted to the field, are extremely detrimental to later growth. The quantity of harmful nematodes in the seedling culture substrate is controlled due to large engineering quantity, the operation is complicated, and the control is not easy to realize. Therefore, it becomes important to cultivate banana seedlings that are resistant to root-knot nematodes. The endophyte with the functions of promoting growth and killing nematode is combined with the conventional banana seedling substrate to produce the banana seedling substrate of the bacteria with the colonization function, so that the resistance of the banana seedlings to the root-knot nematode is improved. At present, in domestic production, a substrate is isolated from soil, and a chemical nematicide is applied during seedling raising to prevent and control root-knot nematodes, but the chemical nematicide has short lasting time and is easy to have adverse effects on bananas and soil after being applied for many times, so the research of the patent becomes especially important. CN101463332A discloses a subspecies of Streptomyces antibioticus subspecies (Streptomyces antibioticus subspecies xi' an), the fermentation product of the strain can inhibit broad-spectrum fungi and protozoa, and field experiments can prevent and control crop fungal diseases and root-knot nematodes. However, this Streptomyces is not taxonomically related to the Streptomyces (Streptomyces capoamus) NAU-SA-1 claimed in the present invention.
The plant endophytic growth-promoting actinomycetes is a general name of actinomycetes which colonize plant roots and can promote plant growth. Can promote the growth of plants, improve the capability of preventing diseases and insects and increase the yield of crops by means of directly generating signal substances or improving the resistance of the plants, accelerating the circulation of soil nutrient elements and the like. And thus has become a hot spot for many scholars to study.
Disclosure of Invention
The invention aims to provide a strain of Streptomyces endophyticus (Streptomyces capoamus) NAU-SA-1 with the capability of killing meloidogyne hapla.
The invention also aims to provide a functional banana seedling raising biological matrix containing the strain and application thereof.
The invention also aims to provide a preparation method of the functional banana seedling raising biological matrix.
The purpose of the invention can be realized by the following technical scheme:
a Streptomyces capoamus NAU-SA-1 is preserved in China general microbiological culture Collection center with the preservation date of 2015, 1 month and 26 days and the preservation number of CGMCC NO. 10441. Bacterial colonies of the strain NAU-SA-1 on a Gao's first plate are light yellow, dry and opaque, the surface of the bacterial colonies is compact velvet, the bacterial colonies are tightly connected with a culture medium, the edges of the bacterial colonies are irregular, and the bacterial colonies are not easy to pick up (attached figure 1 in the specification). A phylogenetic tree constructed by a 16S rDNA sequence of the strain shows that the strain NAU-SA-1 is positioned on a Streptomyces capoamus branch, the similarity with Streptomyces capoamus strain Y24-25 is more than 99 percent, the similarity with the disclosed Streptomyces antibioticus is 77 percent, and the NAU-SA-1 is temporarily classified into Streptomyces carvacillatus by combining a physiological and biochemical test.
The invention relates to application of streptomycete NAU-SA-1 in preparation of a banana seedling biological matrix.
A functional banana seedling biological matrix is obtained by adding the streptomycete NAU-SA-1 fermentation broth into a common seedling matrix.
Wherein the common seedling culture medium contains organic matter (dry weight of the medium, the same below) not less than 90 percent and total nutrient (N + P)2O5+K2O) is more than or equal to 2 percent, the water content is less than or equal to 50 percent, the pH value is 5.5-6.5, and the Ec is less than or equal to 1 ms/cm.
The number of viable bacteria of streptomycete NAU-SA-1 in the functional banana seedling raising biological matrix is preferably more than or equal to 6.0 x 105CFU/g dry weight of matrix.
The streptomycete NAU-SA-1 fermentation liquor is prepared by the following method: inoculating streptomycete NAU-SA-1 with the preservation number of CGMCC NO.10441 into a Gao's first culture solution for liquid fermentation production, wherein the conditions of the fermentation production are as follows: pH7.2-7.4, fermentation temperature range is 30 ℃,stirring at 170 rpm, forming spores in middle and late stage of fermentation for 7 days to make the bacteria or spore content in the fermentation liquid not less than 3 × 107Per ml; the preparation method of the Gao's No. one culture medium comprises the following steps of preparing 1L of culture medium: soluble starch 20g, K2HPO40.5g，KNO31g，MgSO40.5g，FeSO40.01g, fixing the volume to 1000ml, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 20 min.
The preparation method of the functional banana seedling biological matrix comprises the following steps:
(1) preparing Streptomyces NAU-SA-1 fermentation liquor, and making the bacteria or spore content in the fermentation liquor be greater than or equal to 3X 107Per ml;
(2) adding the streptomycete NAU-SA-1 fermentation liquor prepared in the step (1) into a common seedling substrate, wherein the inoculation amount of the fermentation liquor is 5% (v/w).
The common seedling culture medium contains organic matters (dry weight of the medium, the same applies below) more than or equal to 90 percent, total nutrients (N + P2O5+ K2O) more than or equal to 2 percent, water less than or equal to 50 percent, pH value of 5.5-6.5 and Ec less than or equal to 1 ms/cm.
The number of beneficial viable bacteria in the obtained product is more than or equal to 6.0 multiplied by 105CFU/g (dry weight of matrix, same below), organic matter not less than 90%, total nutrient (N + P)2O5+K2O) is more than or equal to 2 percent, the water content is less than or equal to 50 percent, the pH value is 5.5-6.5, and the Ec is less than or equal to 1 ms/cm.
The functional banana seedling raising biological matrix is applied to preventing and controlling banana root-knot nematodes and/or promoting banana growth.
Has the advantages that:
after the streptomycete NAU-SA-1 bacterial liquid is inoculated into a common seedling substrate, due to the good permeability and the high-quality water-retaining property of the substrate, the survival capability of streptomycete strains can be greatly promoted after the common substrate is combined with the streptomycete NAU-SA-1, the streptomycete can be better colonized into root systems after the bagged banana seedlings are fixedly planted in the substrate, and the later-stage growth effect of the cultured seedlings is obviously superior to the growth effect without adding the streptomycete into the substrate.
The product has good growth promoting effect on banana seedlings, can obtain high-quality seedlings, and simultaneously, functional bacteria can be colonized in seedling roots in a large amount. The active microorganisms contained in the product have the effects of promoting growth, high efficiency and preventing and controlling certain root-knot nematodes, and have obvious technical advantages and functional advantages. The matrix can remarkably promote the growth of the cultivated seedlings in the later cultivation process, and can enhance the function of preventing and controlling the root-knot nematodes of the cultivated seedlings.
Drawings
FIG. 1 colony morphology of NAU-SA-1 on Gao's first plate
FIG. 2 phylogenetic tree based on 16S rRNA gene sequence of strain NAU-SA-1 and established by adjacency method
FIG. 3 is a graph showing the effect of functional bacteria inoculation on banana seedling
FIG. 4 functional mycorrhizal colonization of banana seedlings by biological matrix
FIG. 5 the prevention and cure effect of functional banana seedlings on Meloidogyne javanica
FIG. 6 is a graph showing the growth promoting effect of functional bacteria inoculation on banana seedlings
FIG. 7 control Effect of functional Banana seedlings on plant-feeding nematodes
Biological material preservation information
NAU-SA-1, which is classified and named as Streptomyces capoamus of ascending island, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing and Korean district, the preservation date is 2015, 1 month and 26 days, and the preservation number is CGMCC NO. 10441.
Detailed description of the preferred embodiments
Example 1 isolation and characterization of the strains
Cleaning banana root, cutting into small segments with length of about 3cm, weighing 10g of root sample, sterilizing with 5% NaClO for 8min, washing with sterile water for 3 times, sterilizing with 75% ethanol for 5min, washing with sterile water for 3 times, grinding in a mortar containing 10ml of sterile water, and coating after gradient dilution of the grinding liquid. Culturing in dark for 3-4 days, selecting single colony, purifying by plate, selecting 150 strains, and determining the effect of each strain fermentation liquid on killing root-knot nematode. Obtaining a strain NAU-SA-1 with excellent killing effect, and storing for further research.
Bacterial colony of the strain NAU-SA-1 on a Gao's first plate is light yellow, dry and opaque, the surface of the bacterial colony is compact velvet, the connection between the bacterial colony and a culture medium is tight, the edge of the bacterial colony is irregular, and the bacterial colony is not easy to pick up. (FIG. 1).
The phylogenetic tree constructed from the 16S rDNA sequence of the strain is shown in FIG. 2. The strain NAU-SA-1 is positioned on a Streptomyces capoamus branch, the similarity with Streptomyces capoamus strain p14C01 and Streptomyces capoamus strain Y24-25 is more than 99 percent, and the NAU-SA-1 is identified as the Streptomyces by combining a physiological and biochemical test.
EXAMPLE 2 production of bacterial solution of functional bacterium
Inoculating a strain NAU-SA-1(CGMCC NO.10441) into a Gauss I culture solution for liquid fermentation production, wherein the conditions of the fermentation production are as follows: pH7.2-7.4, fermentation temperature range of 30 deg.C, stirring speed of 170 rpm, forming spores in middle and later stages of fermentation for 7 days to make the content of bacteria or spores in the fermentation liquor more than or equal to 3 × 107Per ml; the preparation method of the Gao's No. one culture medium comprises the following steps of preparing 1L of culture medium: soluble starch 20g, K2HPO40.5g，KNO31g，MgSO40.5g，FeSO40.01g, fixing the volume to 1000ml, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 20 min.
Example 3 colonization Effect of functional microorganisms in Banana roots
Transferring the rooting aseptic bagged banana seedlings into a tissue culture bottle filled with a rooting culture medium, wherein all operations are aseptic conditions, inoculating 5% of strain NAU-SA-1 bacterial liquid into a banana root enclosure culture medium, and counting the colonization condition of NAU-SA-1 in roots by coating after surface disinfection: cleaning banana root, weighing, sterilizing with 5% NaClO for 8min, washing with sterile water for 3 times, sterilizing with 75% ethanol for 5min, washing with sterile water for 3 times, grinding root sample with sterile mortar, and spreading the grinding liquid. The preparation method of the Gao's No. one culture medium comprises the following steps of preparing 1L of culture medium: soluble starch 20g, K2HPO40.5g，KNO31g，MgSO40.5g，FeSO40.01g of agar powder and 20-30g of agar powder, metering to 1000ml, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 20 min. The number of the functional bacteria NAU-SA-1 strain is calculated by weight per gram of root.
The test treatments were as follows: 1) CK: NAU-SA-1 bacterial liquid is not inoculated; 2) and SA: 5% of NAU-SA-1 bacterial liquid is added. Each treatment was 10 replicates. After 50 days, the number of functional bacteria NAU-SA-1 colonized in roots in each treatment was counted by dilution and coating. The result shows that compared with the non-inoculated CK bacterium, the functional bacterium NAU-SA-1 bacterium liquid has obvious growth promotion effect on banana seedlings (see figure 3), and can be well colonized in roots, and the colonizing result is shown in figure 4.
Example 4 functional biomatrix development
4.1 prevention and treatment effects of functional banana seedlings on Meloidogyne javanica
Sterilizing the seedling substrate at high temperature to kill all nematodes, inoculating 5% NAU-SA-1 bacterial liquid into a common seedling substrate, grinding into a biological seedling substrate, and transplanting bagged banana seedlings into a seedling cup biological seedling substrate for seedling.
The test treatments were as follows: 1) CK: culturing banana seedlings by using a common seedling culture medium (without inoculating NAU-SA-1 bacterial liquid); 2) and SA: inoculating 5% NAU-SA-1 bacterial liquid into a common seedling substrate, and grinding into a biological seedling substrate for banana seedling cultivation. Pure meloidogyne javanica was inoculated 50 days after transplantation, 10 replicates per treatment. Counting the number of infected root knot nematodes in roots after 50 days, and collecting and counting by adopting a tray method after the surface of the roots is disinfected: cleaning banana root, sterilizing with 5% NaClO for 3min, washing with tap water, and separating nematode in root by tray method. Nematode numbers were calculated per 10 gram root weight. The result shows that the number of the meloidogyne javanica infected in the seedling roots of the bananas inoculated with the NAU-SA-1 bacterial liquid biological seedling substrate is remarkably reduced by 86 percent compared with CK, and the nematicidal effect is obvious (as shown in figure 5).
4.2 Pot culture test of Banana seedlings grown by functional seedling growing matrix
Performing pot experiment on banana seedlings raised by the functional matrix and the common matrix, and setting 2 treatments: 1) CK: banana seedlings grown on a common substrate; 2) and SA: 5% NAU-SA-1 inoculation amount of banana seedlings cultured by the biological matrix. And (3) applying 1% of common organic fertilizer to each pot of 10 pots of soil and 5 kg of soil in each pot, uniformly mixing the common organic fertilizer and the soil to serve as base fertilizer, and measuring the plant height, stem thickness, leaf area, SPAD value, number of nematodes in roots and number of phytophagous nematodes in soil of each treatment at the time of planting for 60 days.
The result shows that the functional bacteria in the active biological matrix play the most important role in promoting growth, and meanwhile, some growth promoting substances generated by fermentation play a certain role in promoting growth. After 60 days of permanent planting, the plant height, stem thickness and leaf area of the SA treated plants are obviously increased compared with CK, and only the SPAD value is reduced (see Table 1). The result of the pot experiment shows that the streptomycete can better play the function of the seedlings raised by the active biological matrix because a large number of functional bacteria are already colonized in the roots. Harmful nematodes infecting banana roots and phytophagous nematodes in the soil were significantly reduced (as shown in fig. 7). The seedling bred by the active biological matrix developed by the functional bacteria with the addition of 5 percent has better prevention effect on the posterior root-knot nematode.
In conclusion, the active biological matrix developed by the functional bacteria with the addition of 5 percent is obviously superior to the effect of the common seedling culture matrix in the aspects of the colonization condition of the functional bacteria, the prevention effect on the meloidogyne javanica and the later potting effect, and achieves obvious difference.
TABLE 1 seedling raising effect of inoculating NAU-SA-1 bacterial liquid on banana
The common seedling culture medium used in the above examples is banana seedling culture medium provided by tissue culture division of ten thousand of the Hainan company, wherein the banana seedling culture medium contains not less than 90% of organic matter and total nutrients (N + P)2O5+K2O) is more than or equal to 2 percent, the water content is less than or equal to 50 percent, the pH value is 5.5-6.5, and the Ec is less than or equal to 1 ms/cm.
Claims (8)
1. A strain of Streptomyces capoanus NAU-SA-1 is preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation date is 2015, 1 month and 26 days, and the preservation number is CGMCC NO. 10441.
2. Use of streptomyces ascendens NAU-SA-1 as claimed in claim 1 for the preparation of a biological substrate for banana seedling.
3. A functional banana seedling raising biological substrate, which is characterized in that the functional banana seedling raising biological substrate is obtained by adding the Streptomyces roseoanus NAU-SA-1 fermentation liquor in claim 1 into a common seedling raising substrate.
4. The functional banana seedling raising biological matrix according to claim 3, wherein the common seedling raising matrix contains more than or equal to 90% of organic matters and N + P2O5+K2The total nutrient of O is more than or equal to 2 percent, the water content is less than or equal to 50 percent, the pH value is 5.5-6.5, and the Ec is less than or equal to 1 ms/cm.
5. The functional banana seedling biological substrate according to claim 3, wherein the viable count of Streptomyces roseoagulae NAU-SA-1 of claim 1 in the functional banana seedling biological substrate is not less than 6.0 x 105CFU/g dry weight of matrix.
6. The functional banana seedling biological matrix according to claim 3, wherein the fermentation broth of Streptomyces ascendens NAU-SA-1 is prepared by the following method: inoculating Streptomyces ascendens NAU-SA-1 with the preservation number of CGMCC NO.10441 into a Gao's first culture solution for liquid fermentation production, wherein the conditions of the fermentation production are as follows: pH7.2-7.4, fermentation temperature range of 30 deg.C, stirring speed of 170 rpm, forming spores in middle and later stages of fermentation for 7 days to make the content of bacteria or spores in the fermentation liquor more than or equal to 3 × 107Per ml; in the case of preparing 1L of the culture medium, the preparation method of the Gao's No. one culture medium is as follows: soluble starch 20g, K2HPO40.5g，KNO31g，MgSO40.5g，FeSO40.01g, fixing the volume to 1000ml, adjusting the pH value to 7.2-7.4, and sterilizing at 121 ℃ for 20 min.
7. The method for preparing the functional banana seedling raising biological matrix as claimed in claim 3, which is characterized by comprising the following steps:
(1) preparing fermentation liquor of Streptomyces ascendens NAU-SA-1 to make the bacteria or spore content in the fermentation liquor be greater than or equal to 3X 107Per ml;
(2) adding the fermentation liquor of the Streptomyces ascendens NAU-SA-1 prepared in the step (1) into a common seedling substrate, wherein the inoculation amount of the fermentation liquor is 5% (v/w).
8. The application of the functional banana seedling raising biological matrix of claim 3 in preventing and controlling banana root-knot nematode and/or promoting banana growth.
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| CN113789288B (en) * | 2021-11-03 | 2023-01-06 | 江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心） | Streptomyces JXGZ01, biological agent and application |
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| CN101619293B (en) * | 2008-09-27 | 2012-07-04 | 河北省农林科学院遗传生理研究所 | Streptomyces vinaceusdrappus, filtering method and application |
| CN101508968B (en) * | 2009-03-06 | 2011-04-20 | 中国农业大学 | Biological medicament for preventing and treating plant parasitic nematode and uses thereof |
| CN103382449B (en) * | 2013-07-15 | 2015-01-14 | 中国农业科学院农产品加工研究所 | Streptomyces sp. T2-10 with broad-spectrum insecticidal and antibacterial activity and its application |
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| CN104357358A (en) * | 2014-11-11 | 2015-02-18 | 山东苏柯汉生物工程股份有限公司 | Compound bacterium for preventing and treating root-knot nematode and preparation method thereof |
| CN105028023A (en) * | 2015-07-24 | 2015-11-11 | 广西大学 | Seedling cultivation substrate for preventing meloidogyne incognita chitwood of bananas |
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