CN112998032A - Crop leaf surface spraying type bacillus inoculant and preparation method thereof - Google Patents

Abstract

The invention relates to a crop leaf surface spraying type bacillus inoculant and a preparation method thereof, wherein the crop leaf surface spraying type bacillus inoculant is prepared from the following raw materials: bacillus (Bacillus sp.) GBIF-16, bean cake leaching solution, rapeseed cake leaching solution, sucrose ester, and cane molasses. The microbial inoculum can promote the foliar nutrition of economic crops and the quality improvement of crops, provides technical support for the popularization and utilization of the synergy of high-efficiency liquid fertilizers, promotes the sustainable development of green agriculture, and has better application prospect.

Description

Crop leaf surface spraying type bacillus inoculant and preparation method thereof

Technical Field

The invention belongs to the technical field of application of agricultural microbial agents, and relates to a crop leaf surface spraying type bacillus agent and application thereof.

Background

Bacillus (Bacillus sp.) has the advantages of abundant diversity, wide distribution, wide antibacterial spectrum and the like, and is widely applied to biological control of diseases, including Bacillus subtilis, Bacillus cereus, Bacillus licheniformis and Bacillus pumilus. At present, under the background of 'double reduction' of fertilizer reduction and pesticide reduction in agricultural development in China, more and more agricultural scientific research technologies focus on researching and applying bacillus as a living biocontrol microbial inoculum, mainly utilize fermentation liquor of biocontrol strains and secondary metabolites thereof to prevent and treat diseases caused by fungi or bacteria of various economic crops, and have obvious effect to a certain extent; meanwhile, the bacillus serving as a biocontrol microbial inoculum is large in root irrigation use amount, the labor cost and the product promotion cost are relatively higher than those of leaf surface spraying by about 5-10 times, the promotion cost of single use is high, and the comprehensive cost in the root irrigation use process can be reduced to a great extent by spraying the bacillus microbial inoculum on the leaf surfaces.

Researches show that the bacillus can utilize a substrate target carbon source, particularly a plant carbon source, to quickly propagate and amplify in the using process, can generate a certain amount of soluble micromolecular carbon and other active substances in the fermentation process, can improve the nutrient absorption efficiency of the leaf surfaces of crops by spraying the leaves, is favorable for improving the yield of the crops and stably improving the quality, can form dominant floras on the leaf surfaces, maintains and protects the favorable micro-ecological environment of the leaf surfaces of the crops, and is more favorable for photosynthesis of the leaf surfaces of the crops, absorption of nutrient substances and defense of diseases and insect pests.

However, in the process of using bacillus singly, because of the lack of good protective agents and composite carbon sources, microorganisms are difficult to truly play a role in functional activity, and often can form spores through dormancy in a short time, and cannot play a role in promoting growth and related functions, so that the effect of applying bacillus is limited, which is an important factor for instability of the application effect of bacillus. Therefore, in the process of using the bacillus, from the aspects of cost control, strain activity maintenance and crop application range, the bacillus protective agent, the synergist and the composite carbon source are particularly important in the preparation process of the bacillus microbial inoculum, otherwise, the bacillus protective agent, the synergist and the composite carbon source are difficult to achieve a good using effect.

Disclosure of Invention

One of the purposes of the invention is to separate dominant bacteria of different crop rhizosphere and further compound the dominant bacteria with a compound carbon source and a synergist, and provide a microbial inoculum which can be applied to foliage spraying of crops, has stable effect and can well promote the improvement of the nutrition quality of the crop foliage.

The technical scheme for achieving the purpose is as follows.

A crop leaf surface spraying type bacillus agent is prepared from the following raw materials: bacillus (Bacillus sp.) GBIF-16, bean cake leaching solution, rapeseed cake leaching solution, sucrose ester, and cane molasses.

In some embodiments, the Bacillus (Bacillus sp.) GBIF-16 has biological accession number GDMCC No. 61024.

In some embodiments, the crop foliage spray type bacillus inoculant comprises the following raw materials in percentage by mass: 85-90 wt% of bacterial liquid of Bacillus (Bacillus sp.) GBIF-16 with the bacterial OD value of 0.4-0.6, 1-5 wt% of bean cake leaching liquor, 1-5 wt% of rapeseed cake leaching liquor, 0.1-0.5 wt% of sucrose ester and 1-5 wt% of cane molasses.

In some embodiments, the crop foliage spray type bacillus inoculant comprises the following raw materials in percentage by mass: 85-86 wt% of bacterial liquid of Bacillus (Bacillus sp.) GBIF-16 with the bacterial OD value of 0.4-0.6, 4-5 wt% of bean cake leaching liquor, 4-5 wt% of rapeseed cake leaching liquor, 0.3-0.5 wt% of sucrose ester and 4-5 wt% of cane molasses.

In some embodiments, the crop foliage spray type bacillus inoculant comprises the following raw materials in percentage by mass: 85 wt% of bacterial liquid of bacillus GBIF-16 with a bacterial OD value of 0.5, 5 wt% of bean cake leaching liquor, 5 wt% of rapeseed cake leaching liquor, 0.5 wt% of sucrose ester and 4.5 wt% of cane molasses.

In some embodiments, the bacterial liquid concentration of the Bacillus (Bacillus sp.) GBIF-16 is 2.0 +/-0.2 x 10⁷CFU/mL, preferably, the bacterial liquid concentration of the Bacillus (Bacillus sp.) GBIF-16 is 2.0 × 10⁷CFU/mL。

In some of these embodiments, the crop plant is a cash crop plant, the cash crop plant is oilseeds, peanuts, flax, sunflowers, sugarcane, tomatoes, or tea leaves, preferably the cash crop plant is a tomato, sugarcane, or tea leaves.

In some embodiments, the preparation method of the bacterial liquid of the Bacillus (Bacillus sp.) GBIF-16 comprises the following steps: inoculating 0.9-1.1-5-10 wt% of cane molasses and 2-5 wt% of bean cake leaching culture solution to a bacillus strain GBIF-16 with a bacterium OD value of 0.4-0.6, and culturing at 30-50 ℃ for 18-24 h to obtain a bacterium solution.

In some of these embodiments, the method for preparing the soybean cake leach solution comprises the steps of: soaking bean cake in water at 60-80 deg.C for 2 + -0.05 hr; the preparation method of the rapeseed cake leaching solution comprises the following steps: the rapeseed cake is leached by water at 60-80 ℃ for 2 plus or minus 0.05 hours, thus obtaining the rapeseed cake.

The invention also aims to provide a preparation method of the crop foliage spray type bacillus inoculant.

The technical scheme for achieving the purpose is as follows.

The preparation method of the crop foliage spray type bacillus inoculant comprises the following steps:

(1) inoculating 0.9-1.1-5-10 wt% of cane molasses and 2-5 wt% of bean cake leaching culture solution to a bacillus strain GBIF-16, and culturing at the temperature of 30-50 ℃ for 18-24 h to obtain bacterial liquid;

(2) and mixing the bacterial liquid, the bean cake leaching solution, the rapeseed cake leaching solution, the sucrose ester and the cane molasses according to a ratio to obtain the bacillus agent.

In some embodiments, the culture is performed at 30-50 ℃ and 170-220 rpm for 18-24 h under shaking.

The invention relates to a Bacillus strain (Bacillus sp.) GBIF-16, which is a dominant strain separated from a sugarcane soil rhizosphere, a tea soil rhizosphere and a tomato soil rhizosphere, and the inventor of the invention finds that a bean cake leaching solution, a rapeseed cake leaching solution, sucrose ester and cane molasses are used as nutrition synergistic carriers to be compounded with the bacterial strain GBIF-16 screened by the inventor to form a microbial inoculum, and through repeated experiments in the field, the compounded Bacillus microbial inoculum can better promote the foliar nutrition of economic crops and the improvement of the quality of crops, has stable effect and wide application range, provides technical support for the popularization and utilization of high-efficiency liquid fertilizer synergism, promotes the continuous development of green agriculture, and has better application prospect.

The Bacillus GBIF-16 is classified and named as Bacillus (Bacillus sp.), is preserved in Guangdong province microorganism strain preservation center in 5, 9 months in 2020 at the preservation address: no. 59 building No. 5 of Dazhou No. 100 of the first Liehuo of Guangzhou city, the strain number is GDMCC NO. 61024.

Drawings

FIG. 1 is a plate diagram of Bacillus strain GBIF-16.

Fig. 2, 3 and 4 show part of green tea plantation in group 1, group 2 and group 3 of example 4, respectively.

Detailed Description

The following examples of the present invention are experimental methods without specifying specific conditions, generally according to conventional conditions, or according to conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device that comprises a list of steps is not limited to only those steps or components listed, but may alternatively include other steps or components not listed, or inherent to such process, method, product, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

As used herein, unless otherwise specified or defined, "first" and "second" … are used merely for name differentiation and do not denote any particular quantity or order.

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The soybean cake leaching solution and the rapeseed cake leaching solution described in the following examples are respectively: the bean cake and the rapeseed cake are prepared by water leaching for 2 hours at the temperature of 60-80 ℃, and 20-30 g of bean cake or rapeseed soluble extract is filled in every 100mL of water; the cane molasses is a byproduct in the sugar extraction process of a cane sugar factory, comprises a soluble carbon source, a nitrogen source and medium trace elements, and is mainly used as a nutrient source of the strain GBIF-16.

Example 1

Screening and identification of bacillus strains:

the strain samples are from sugarcane planting area, tomato planting area and Guangdong river source in south sand area of Guangzhou city of Guangdong provinceMixed soil in tea growing area of Tailongmen, Nyjin county, Limited. Weighing 10g of soil mixed sample, continuously enriching by taking cane molasses, rapeseed cake leaching liquor and bean cake leaching liquor as nutrient sources for one week in a liquid enrichment culture medium (the liquid enrichment culture medium is 20g of cane molasses, rapeseed cake leaching liquor and bean cake leaching liquor in total, and 180mL of sterile water is added), after enriching for 3 days, sucking 10mL of enrichment liquid, inoculating into a new liquid enrichment culture medium, continuously culturing for the second generation, and repeating the culture until the third generation, respectively sucking 0.1mL of diluted 10^-4～10^-6Coating the strain on an LB culture medium, culturing for 3-7 days at 30 ℃, selecting the strain with fast growth, especially the dominant strain, separating and purifying to finally obtain the dominant strain with higher biological activity, which is named as GBIF-16.

Biological characteristics of Strain GBIF-16: on an LB flat plate, a colony grows after 24 hours, the colony is gram-positive G +, the colony grows fast, the surface of the colony is relatively smooth, and a single strain is rod-shaped and has the size of 1-2 mu m. According to the manual of bacteria identification, the bacterial strain GBIF-16 is preliminarily identified as bacillus (shown in FIG. 1).

Bacillus GBIF-16, taxonomic nomenclature: bacillus (Bacillus sp.), 5.9.2020, deposited in Guangdong province collection center of microorganism strains, with the deposition address: no. 59 building No. 5 of Dazhou No. 100 of the first Liehuo of Guangzhou city, the strain number is GDMCC NO. 61024. The biological characteristics are as follows: on an LB flat plate, a colony grows after 24 hours, the colony is gram-positive G +, the colony grows fast, the surface of the colony is relatively smooth, and a single strain is rod-shaped and has the size of 1-2 mu m.

LB culture medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 20g of agar and 1000mL of water, and the pH value is natural.

The 16s rDNA sequence of the Bacillus strain GBIF-16 is as follows:

TTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAGCCTCGCGGCTTCGCTGCCCTTTGTTCTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTTGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGTTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCTAAAAGCCACCTTTTATGATTGAAACATGGGGTTCAATCAAGCATTCGGTATTAACCCCGGTTTCCCGGAATTATCCCAGTCTTACCGGGAAGTTAACCACGTGGTACTCACCCGTCCGCCGCTGAACTAAGGGAACCAGCTTCCGTCGGTTCGCTCGAACTTGCATGTAATTAAGCACGCCCGCCAGCCGTTCGTTCTGGAGCAAGGA(SEQ ID NO.1)。

example 2

Preparation of crop leaf surface spraying type bacillus agent

Bacterial liquid of bacillus GBIF-16: inoculating 1 to 5 to 10 percent of cane molasses and 2 to 5 percent of bean cake leaching culture solution into a bacillus strain GBIF-16, and carrying out shaking culture for 18 to 24 hours at the temperature of 30 to 50 ℃ and the rotating speed of 170 to 220rpm to obtain the bacillus strain GBIF-16 with the concentration of 2.0 +/-0.2 multiplied by 10⁷CFU/mL of the bacterial liquid of the Bacillus GBIF-16.

Spraying a bacillus fungicide on the leaf surfaces of crops: mixing 85 wt% of the bacterial liquid of the bacillus GBIF-16, 5 wt% of the bean cake leaching solution, 5 wt% of the rapeseed cake leaching solution, 0.5 wt% of sucrose ester and 4.5 wt% of cane molasses to obtain the crop leaf surface spraying type bacillus fungicide.

Example 3

Application effect of crop leaf surface spraying type bacillus inoculant-sugarcane

Test site: east-gush township homer cooperation demonstration base for south sand areas of Guangzhou, Guangdong province

Test time: 15 days 4 and 15 months in 2018-15 days 12 and 15 months in 2018

Test treatment, 3 groups were set up: group 1. conventional fertilization by farmers (three land with each land being about 1 mu); group 2. conventional fertilization by farmers + foliar increase application of the fungicide described in example 2 (three plots are provided, each plot is about 1 mu, the amount of fertilizer application is reduced by 5% on the basis of conventional fertilization by farmers, foliar application of the fungicide described in example 2 is performed 10 times in total, 200 mL/mu each time); and group 3. conventional farmer fertilization + foliar application of the bacterial liquid of the bacillus GBIF-16 described in example 2 (three land blocks are provided, each land block is about 1 mu. the fertilizing amount is reduced by 5% on the basis of the conventional farmer fertilization, and the bacterial liquid of the bacillus GBIF-16 described in example 2 is sprayed on the leaf surface 10 times, wherein each time is 200 mL/mu).

The results show (table 1): the nutrient quality indexes of the sugarcane processed by the microbial inoculum in the embodiment 2 of the foliar application are compared with the nutrient quality indexes of the sugarcane processed by the conventional fertilization processing of farmers, and the microbial inoculum in the embodiment 2 of the foliar application is found out, so that the healthy growth of the sugarcane is promoted, the healthy and continuous growth of the sugarcane leaves and the synthesis of stable photosynthetic carbohydrates are ensured, and the absorption and distribution of the plant root system to the nutrient elements are promoted. Further as can be seen from table 1: the total amino acid, glucose content and sucrose content of the sugarcane treated by the microbial inoculum of the foliar application example 2 are respectively increased by 14.3 percent, 19.3 percent and 14.8 percent compared with the total amino acid, glucose content and sucrose content of the sugarcane treated by the conventional fertilizer application of farmers; in addition, the total amino acid content, the glucose content and the sucrose content of the sugarcane of the bacillus GBIF-16 bacterial liquid in the foliar spraying example 2 are respectively reduced by 7.5%, 6.6% and 7.2% compared with those of the microbial inoculum in the foliar application example 2; meanwhile, the fructose and vitamin C contents of the three groups of treatments have no significant difference.

Table 1. influence of foliar-spray type bacillus fungicide on sugarcane quality

Example 4

Application effect of crop leaf surface spraying type bacillus inoculant-green tea

Test site: planting base for Taiwan mountains-Taiwan green tea

Test time: 11/15/2019-8/25/2020

And (3) test treatment: the total number of the devices is 3: group 1. conventional fertilization management (three plots, each about 1 mu); group 2. conventional fertilization management + foliar application of the fungicide described in example 2 (three land sets, each land set having about 1 mu. reduced fertilizing amount by 5% on the basis of conventional fertilization by farmers, foliar application of the fungicide described in example 2 for 6 times in total, each time 500 mL/mu); group 3. conventional fertilization by farmers + increased foliar application of the Bacillus GBIF-16 bacterial liquid 85 wt% + Bean cake leach solution 7.5 wt% + rapeseed cake leach solution 7.5 wt% (three plots were provided, each about 1 mu. the amount of fertilizer applied was reduced by 5% based on the conventional fertilization by farmers, and foliar application of the Bacillus GBIF-16 bacterial liquid 85 wt% + Bean cake leach solution 7.5 wt% + rapeseed cake leach solution 7.5 wt% described in example 2 for 6 times, 500 mL/mu each).

Fig. 2-4, which are green tea plantation of group 1, group 2 and group 3, respectively.

The results show (table 2): the nutritional quality indexes of the tea leaves treated by comparing the conventional fertilization management treatment and the foliar application of the microbial inoculum described in example 2 are found as follows: the addition of the microbial inoculum described in example 2 to the leaf surface promotes the formation of good quality tea, and it can be seen from table 2 that: the contents of amino acid, caffeine, tea polyphenol and tea extract in the tea leaves treated by the microbial inoculum prepared in the embodiment 2 are respectively increased by 10.3%, reduced by 4.8%, reduced by 9.6% and increased by 5.3% compared with the contents of amino acid, caffeine, tea polyphenol and tea extract in the treated tea leaves subjected to conventional fertilization management; meanwhile, compared with the conventional fertilization management, the phenol-ammonia ratio of the tea can be reduced by 18.1% by the aid of the microbial inoculum treatment in the foliar application example 2; in addition, the contents of 85 wt% of bacillus GBIF-16 bacterial liquid, 1.5 wt% of sucrose ester and 13.5 wt% of cane molasses in the foliar spraying example 2 are respectively reduced by 5.1%, increased by 1.9%, increased by 3.3% and reduced by 1.0% compared with the contents of amino acid, caffeine, tea polyphenol and tea water extract in the microbial inoculum in the foliar application example 2; meanwhile, the phenol-ammonia ratio of the tea can be increased by 9.4 percent.

TABLE 2 influence of foliar-spray bacillus inocula on tea quality

Example 5

Application effect of crop leaf surface spraying type bacillus inoculant-tomato

Test site: planting base of Guangzhou agriculture Co Ltd in Guangzhou city, white cloud area, Guangdong province

Test time: 9/month 5/2019-11/month 5/2020

And (3) test treatment: the total number of the devices is 3: group 1. conventional fertilization management (three plots, each about 1 mu); conventional fertilization management + foliar application of the fungicide described in example 2 (three plots are provided in total, each plot is about 1 mu, the amount of fertilizer applied is reduced by 5% on the basis of conventional fertilization of farmers, and foliar application of the fungicide described in example 2 is performed 6 times in total, each time 400 mL/mu); group 3. regular fertilization by farmers + foliar application of Bacillus GBIF-16 bacterial liquid 85 wt% + sucrose ester 1.5 wt% + cane molasses 13.5 wt% (three land sets, each about 1 mu. each, reduced fertilizing amount by 5% on the basis of regular fertilization by farmers, foliar application of Bacillus GBIF-16 bacterial liquid 85 wt% + sucrose ester 1.5 wt% + cane molasses 13.5 wt% of example 2 for 6 times, 400 mL/mu each).

The results show (table 3): the nutritional quality indexes of tomatoes treated by comparing the conventional fertilization management treatment with the fungicide treatment in the foliage application example 2 are found as follows: the foliar application of the fungicide treatment described in example 2 promotes the formation of tomato nutritional quality, and further as can be seen from table 3: the lycopene, the soluble sugar, the vitamin C and the sugar-acid ratio of the microbial inoculum treatment in the embodiment 2 are respectively increased by 9.8%, 18.7%, 14.5% and 10.3% compared with the lycopene, the soluble sugar, the vitamin C and the sugar-acid ratio of the treatment of the conventional fertilization management; in addition, the ratio of lycopene, soluble sugar, vitamin C and sugar to acid of the microbial inoculum described in the embodiment 2 is respectively reduced by 1.6%, 7.3%, 5.8% and 2.3% when the bacterial solution of the bacillus GBIF-16 is sprayed on the leaf surface, the sucrose ester is sprayed by 85 wt%, the cane sugar is sprayed by 1.5 wt% and the cane molasses is sprayed by 13.5 wt%.

TABLE 3 influence of foliar-sprayed Bacillus inoculants on tomato quality

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> institute of bioengineering of academy of sciences of Guangdong province

<120> crop leaf surface spraying type bacillus inoculant and preparation method thereof

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1466

<212> DNA

<213> Artificial Sequence

<400> 1

ttcggcggct ggctccaaaa ggttacctca ccgacttcgg gtgttacaaa ctctcgtggt 60

gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat 120

tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga 180

tttgtgggat tggcttagcc tcgcggcttc gctgcccttt gttctgccca ttgtagcacg 240

tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300

tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg 360

cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420

cctgtcactc tgcccccgaa ggggaagccc tatctctagg gttgtcagag gatgtcaaga 480

cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540

ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg agtgcttaat 600

gcgtttgctg cagcactaaa gggcggaaac cctctaacac ttagcactca tcgtttacgg 660

cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgcgcc tcagcgtcag 720

ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac gcatttcacc 780

gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt ccaatgaccc 840

tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg cgcgctttac 900

gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct ggcacgtagt 960

tagccgtggc tttctggtta ggtaccgtca aggtaccgcc ctattcgaac ggtacttgtt 1020

cttccctaac aacagagttt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 1080

gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 1140

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 1200

tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag tggtagctaa 1260

aagccacctt ttatgattga aacatggggt tcaatcaagc attcggtatt aaccccggtt 1320

tcccggaatt atcccagtct taccgggaag ttaaccacgt ggtactcacc cgtccgccgc 1380

tgaactaagg gaaccagctt ccgtcggttc gctcgaactt gcatgtaatt aagcacgccc 1440

gccagccgtt cgttctggag caagga 1466

Claims (10)

1. The crop leaf surface spraying type bacillus inoculant is characterized by being prepared from the following raw materials: bacillus (Bacillus sp.) GBIF-16, bean cake leaching solution, rapeseed cake leaching solution, sucrose ester, and cane molasses.

2. The crop foliage spray type Bacillus inoculant according to claim 1, wherein the Bacillus (Bacillus sp.) GBIF-16 has a biological deposit number GDMCC No. 61024.

3. The crop foliage spray type bacillus inoculant according to claim 1, which is prepared from the following raw materials in percentage by mass: 85-90 wt% of bacterial liquid of Bacillus (Bacillus sp.) GBIF-16 with the bacterial OD value of 0.4-0.6, 1-5 wt% of bean cake leaching liquor, 1-5 wt% of rapeseed cake leaching liquor, 0.1-0.5 wt% of sucrose ester and 1-5 wt% of cane molasses.

4. The crop foliage spray type bacillus inoculant according to claim 3, which is prepared from the following raw materials in percentage by mass: 85-86 wt% of bacterial liquid of Bacillus (Bacillus sp.) GBIF-16 with the bacterial OD value of 0.4-0.6, 4-5 wt% of bean cake leaching liquor, 4-5 wt% of rapeseed cake leaching liquor, 0.3-0.5 wt% of sucrose ester and 4-5 wt% of cane molasses.

5. The crop foliage spray type bacillus inoculant according to claim 4, which is prepared from the following raw materials in percentage by mass: 85 wt% of bacterial liquid of bacillus GBIF-16 with a bacterial OD value of 0.5, 5 wt% of bean cake leaching liquor, 5 wt% of rapeseed cake leaching liquor, 0.5 wt% of sucrose ester and 4.5 wt% of cane molasses.

6. The crop foliage spray type Bacillus microbial inoculum according to claim 3, 4 or 5, wherein the concentration of the bacterial liquid of the Bacillus (Bacillus sp.) GBIF-16 is 2.0 +/-0.2 x 10⁷CFU/mL, preferably, the bacterial liquid concentration of the Bacillus (Bacillus sp.) GBIF-16 is 2.0 × 10⁷CFU/mL; the crops are cash crops, the cash crops are oilseeds, peanuts, flax, sunflowers, sugarcane, tomatoes or tea leaves, and preferably the cash crops are tomatoes, sugarcane or tea leaves.

7. The crop foliage spray type Bacillus inoculant according to claim 6, wherein the preparation method of the Bacillus (Bacillus sp.) GBIF-16 bacterial liquid comprises the following steps: inoculating 0.9-1.1-5-10 wt% of cane molasses and 2-5 wt% of bean cake leaching culture solution to the bacillus strain GBIF-16, and culturing at 30-50 ℃ for 18-24 h to obtain bacterial liquid.

8. The crop foliage spray type bacillus inoculant according to claim 1, wherein the preparation method of the soybean cake leaching solution comprises the following steps: soaking bean cake in water at 60-80 deg.C for 2 + -0.05 hr; the preparation method of the rapeseed cake leaching solution comprises the following steps: the rapeseed cake is leached by water at 60-80 ℃ for 2 plus or minus 0.05 hours, thus obtaining the rapeseed cake.

9. The method for preparing the crop foliage spray type bacillus agent as claimed in any one of claims 1 to 8, characterized by comprising the steps of:

(1) inoculating 0.9-1.1-5-10 wt% of cane molasses and 2-5 wt% of bean cake leaching culture solution to a bacillus strain GBIF-16, and culturing at the temperature of 30-50 ℃ for 18-24 h to obtain bacterial liquid;

(2) and mixing the bacterial liquid, the bean cake leaching solution, the rapeseed cake leaching solution, the sucrose ester and the cane molasses according to a ratio to obtain the bacillus agent.

10. The method according to claim 9, wherein the culturing is performed at a temperature of 30 to 50 ℃ and a rotation speed of 170 to 220rpm for 18 to 24 hours with shaking.

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