Apple planting method and application thereof in soil improvement

Abstract

The invention relates to the technical field of agricultural planting, in particular to an apple planting method and application thereof in soil improvement. The planting method comprises the steps that goat milk diluent is sprayed to leaves, young fruits and roots of apple trees before apple bagging; root watering is conducted twice at the end of July and August every year, and goat milk diluent and ecological enzymes are applied according to the apple yield; the ecological enzyme is composed of apple enzyme and soybean protein. According to the method, the community structure of rhizosphere microorganisms is changed, the abundance of harmful bacteria is inhibited, the abundance of beneficial bacteria is increased, the soil quality is improved, apple nutrient substance accumulation is improved, the sugar-acid ratio of apples is remarkably increased, and the taste of the apples is improved.

Description

Technical Field

[0001] The present invention relates to the field of agricultural planting technology, and is a method for planting apples and its application in improving soil, a technology for improving the distribution of rhizosphere microbial communities, improving soil quality, and improving the taste of apples and the content of nutritional components. Background Art

[0002] Apple is known as the "king of fruits". It is rich in nutrients and has a long history of cultivation. my country is also the world's main apple producing area, with a planting area and output exceeding 50% of the world's total. There are 6 main apple producing areas in my country. The Bohai Bay production area represented by Dalian is one of the best apple cultivation areas in my country and even in the world. The apple industry also faces many problems in industrial upgrading, such as increasing constraints on land resources, imbalance of nutrients in orchard soil, single apple variety, aging of trees and even obstacles to continuous cropping, and invasion of pests and diseases (rot, gray mold, anthracnose, etc.), which have led to a serious decline in fruit quality. Apple has a well-developed root system and has high requirements for soil. Reasonable fertilization can improve soil nutrients to prevent nutrient deficiency from affecting apple growth, while unreasonable water and fertilizer treatment may cause soil deterioration. For example, long-term application of chemical fertilizers will cause the soil pH to drop from 6.4-7.2 to below 5.5, or even to 4.0; high-concentration chemical fertilizer application causes excessive accumulation of phosphorus and potassium in the soil, while calcium, magnesium, boron, zinc and other elements are lacking, and the organic matter level is also low. The fruit quality of fruit trees over 10 years old has declined significantly. Overcoming the obstacle of continuous cropping essentially requires soil repair, and can provide a rhizosphere microbial community structure that is more suitable for fruit tree growth, further helping fruit trees resist diseases. Therefore, it is necessary to pay attention to improving soil conditions, re-understanding and evaluating the cultivation and management technology system, promoting variety structure adjustment, including the use of soil testing, formula fertilization and nutritional diagnosis to improve soil conditions, and developing water and fertilizer integration, recycling industry, orchard grass and other technologies to promote the organic and intelligent development of the apple industry. To grow fruit, one must first cultivate the soil. If we can improve the composition of fertilizers to intervene in the structure of soil rhizosphere microbial communities in a targeted manner, allow growth-promoting bacteria to occupy favorable ecological niches and reduce the abundance of pathogenic bacteria, and further establish a good microbial-plant interaction system, we can fundamentally improve the soil and achieve "strong bacteria, nourishing the soil, and growing fruit."

[0003] The rhizosphere is the gateway for soil water and minerals to enter the root system to participate in the biological cycle. It is also the area where the root system's own life activities and metabolism have the most direct and strongest impact on the soil. The rhizosphere participates in the decomposition of organic matter in the soil, the absorption of nutrients, the transport of trace elements, and the utilization of various nutrients in the soil by host plants, affecting the stability of the ecosystem and playing an important role in plant-microorganism and microorganism-microorganism interactions. There is a strong mutual influence between plants and rhizosphere microorganisms. During the growth period, plants affect the types, quantity, dominant physiological groups, metabolism, growth and development, and diversity of rhizosphere soil microorganisms through the types and quantities of root secretions; and changes in the number and types of soil microorganisms have a greater impact on plants: beneficial rhizosphere microorganisms can promote plant growth by combining nitrogen fixation and synthesizing bioactive substances, and can also perform biological control on plants by producing siderophores, antagonism, competition, and inducing systemic resistance; while harmful rhizosphere microorganisms can affect the development of plant roots or seedlings by producing plant toxins or plant hormones. The number and types of beneficial and harmful microorganisms in the rhizosphere soil of apple trees can vary significantly depending on the origin, the age of the fruit trees, and the fertilization conditions. The physical and chemical factors also show different differences. For example, fruit farmers excessively pursue yields and apply large amounts of chemical fertilizers, which will destroy the balance of the microecology in the soil, and ultimately lead to many problems such as soil compaction and nutrient imbalance. Conversely, some types of rhizosphere microorganisms can promote plant growth and become growth-promoting bacteria. Combined with the fermentation products of apple leaves and defective fruits as bio-fertilizers, growth-promoting bacteria can greatly improve soil composition. If the structure of the rhizosphere microbial community can be changed to improve the taste of apples and the accumulation of nutrients, and improve the growth and development of fruit trees, it can provide new ideas for the improvement of apple planting. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a feasible plan of "growing strong bacteria in soil and growing fruit", which combines goat milk and ecological fertilizer irrigation with fine field management to provide "prebiotics" for rhizosphere microorganisms, thereby changing the community structure of rhizosphere microorganisms, and further improving the interaction between microorganisms, soil and plants, improving soil quality, and enhancing the accumulation of nutrients in apples, thereby improving the taste of the fruit, increasing the weight of single fruit and yield per mu, and then combining the intensive and fine management measures of "managing branches, tidying up leaves, and protecting fruits" to effectively improve the quality and competitiveness of fruits. The ordinary Fuji cultivated by this method also has a rich milky flavor.

[0005] The present invention adopts the following technical solutions:

[0006] The present invention provides a method for planting apples, the method comprising the following steps:

[0007] (1) Before bagging apples, spray the leaves, young fruits, and roots with diluted goat milk. This allows the leaves and young fruits to absorb it, improves the smoothness and cleanliness of the fruit surface, produces physiological changes, and stores the required nutrients in advance. The fruit maturity can be advanced by 7-10 days.

[0008] (2) Water the roots twice a year at the end of July and the end of August, and apply diluted goat milk and ecological enzymes according to the apple yield; the ecological enzymes are composed of apple enzymes and soy protein.

[0009] In the above technical solution, further, the goat milk diluent is goat milk and water diluted in a mass ratio of 1:30-1:100.

[0010] In the above technical solution, further, the mass ratio of apple enzyme to soy protein is 1:10-70.

[0011] In the above technical scheme, further, the apple enzyme is mixed with brown sugar, defective apples (including fallen fruits, twisted fruit stalks, bruised young fruits, etc.) and water in a mass ratio of 1:4-6:100. The defective apples are chopped, mixed and placed in a barrel and placed in a cool and ventilated place for fermentation for 3 months before use. The fermentation is closed and air is released regularly (weekly); the soy protein is soy milk, and the concentration of the soy milk is not more than 20%.

[0012] In the above technical scheme, further, in step (1), the apple yield is estimated based on the number of young fruits, and the goat milk dilution is applied according to the apple yield, with 0.5-2kg of goat milk dilution applied for every 100 jin of apple yield during the spraying operation; in step (2), 10-30 jin of goat milk is applied for every 100 jin of apple yield during the root irrigation operation; and 5-20 jin of ecological enzyme is applied for every 100 jin of apple yield during the root irrigation operation.

[0013] In the above technical scheme, further, when applying the goat milk dilution liquid and ecological enzyme in step (2), radial grooves with a depth of 8-10 cm are dug around the main trunk of the tree for 40-60 cm and the prepared liquid is evenly poured in.

[0014] The present invention also provides application of the aforementioned apple planting method in improving apple planting soil to change the rhizosphere microbial community structure.

[0015] In the above technical solution, further, for prokaryotic microorganisms, the abundance of Cytophagale, Paenibacillaceae, and Bradyrhizobium is increased.

[0016] In the above technical scheme, further, for eukaryotic microorganisms, the abundance of Neocosmospora_rubicola is increased, and the abundance of Pezizomycetes, Mortierellomycetes, Fusarium, Albifimbria, Devriesia, Colletotrichum, and Fusarium circinatum is inhibited.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a new planting method of "strong bacteria, soil and fruit cultivation", which uses diluted goat milk, enzymes and soy protein to irrigate fruit trees in a coordinated manner, provides "prebiotics" for rhizosphere microorganisms, and thus changes the community structure of rhizosphere microorganisms. Oligosaccharides in goat milk and ecological enzyme fertilizers act as prebiotics to help probiotics colonize and antagonize pathogens, thereby improving the community structure of rhizosphere microorganisms, improving soil structure through microbial-soil-plant interactions, improving the growth and development of fruit trees, better accumulating nutrients, and continuously improving the soil; for prokaryotic microorganisms, the Alpha diversity of rhizosphere microorganisms is increased, and the abundance of beneficial bacteria such as Cytophagale, Paenibacillaceae and Bradyrhizobium is increased; for eukaryotic microorganisms, several plant pathogens such as Pezizomycetes, Mortierellomycetes, Fusarium, Albifimbria, Devriesia, Colletotrichum and Fusarium are significantly inhibited. circinatum, significantly increased the abundance of the beneficial bacteria Neocosmospora_rubicola; soy protein, goat milk and enzymes worked synergistically to improve the soil, increase the content of soil organic matter, improve the soil's ability to retain water and nutrients, promote the balance of the soil ecosystem, enhance the immunity of fruit trees, make the leaves strong and shiny, significantly increase the sugar-acid ratio of apples, and make the fruit taste better. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Pictures of Fuji apples grown in Example 1; (A) morphology on the tree; (B) the "crystal heart" after cutting;

[0019] Figure 2 Example 1: Single fruit weight analysis diagram of Fuji and common red Fuji fruits grown;

[0020] Figure 3 Example 1: Analysis of water content of Fuji and common red Fuji fruits grown;

[0021] Figure 4 Total sugar content determination results; (A) Total sugar content of Fuji and common Fuji fruits grown in Example 1; (B) Standard curve;

[0022] Figure 5 Results of reducing sugar content determination; (A) reducing sugar content of Fuji and common red Fuji fruits grown in Example 1; (B) standard curve;

[0023] Figure 6 Example 1 The titratable total acid content of Fuji and common red Fuji fruits grown;

[0024] Figure 7 Results of polyphenol content determination; (A) polyphenol content of Fuji and common red Fuji fruits grown in Example 1; (B) standard curve;

[0025] Figure 8 Comparative analysis of rhizosphere microbial communities (prokaryotic cells); (A) Chao1 index; (B) Shannon index;

[0026] Fig. 9 Beta diversity diagram of rhizosphere microbial community (prokaryotic) NMDS analysis;

[0027] Fig.10 Venn diagram of rhizosphere microbial community distribution;

[0028] Fig.11 Species distribution (prokaryotic) bar graph; A. Phylum level; B. Genus level

[0029] Fig.12 Comparative analysis of rhizosphere microbial communities (eukaryotic cells); (A) Chao1 index; (B) Shannon index;

[0030] Fig.13 Beta diversity diagram of rhizosphere microbial community (eukaryotic) NMDS analysis;

[0031] Fig.14 Venn diagram of rhizosphere microbial community distribution (eukaryotic);

[0032] Fig.15 Species distribution (eukaryotic) bar graph; A. Phylum level; B. Genus level. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0034] Example 1

[0035] 1. Fruit tree treatment and fruit collection

[0036] The apple test garden is located in Dongmatoun Village, Xutun Town, Wafangdian District, Dalian City, Liaoning Province (122°17'E, 39°91'N). The orchard is in a temperate continental monsoon climate zone with an average annual temperature of 10.5℃ and a total annual solar radiation of 143kcal / cm 2 .

[0037] The main variety planted in the orchard is Red Fuji. The existing Red Fuji fruit trees are treated according to the method of the present invention, including: (1) applying farmyard manure as base fertilizer in spring, spraying fresh goat milk dilution on leaves, young fruits and roots before bagging apples every year (late May to early June); the goat milk is diluted with water, and the dilution ratio is about 1:80. The apple yield is estimated by the number of young fruits, and 1 catties of goat milk dilution is sprayed for every 100 jin of apple trees. (2) Watering the roots twice at the end of July and the end of August every year, and apply 20 jin of ecological enzymes and 20 jin of goat milk dilution in step (1) for every 100 jin of apple yield. When applying, dig 8-10 cm deep radial trenches around the trunk of the tree with a radius of 50 cm, and pour the prepared liquid evenly. The ecological enzyme is composed of apple enzyme and soy protein, the mass ratio of apple enzyme to soy protein is 1:20, the apple enzyme is mixed with brown sugar, defective apples and water in a ratio of 1:5:100, the defective apples are chopped, mixed and placed in a plastic bucket and fermented in a cool and ventilated place for 3 months before use; the soy protein can be soy milk, the mass concentration of soybeans in the soy milk is not more than 20%, and the soy milk concentration used in this embodiment is about 15%.

[0038] The fruit was collected in early November 2019. The Red Fuji apples planted by the above method of the present invention were collected at the same time as the Red Fuji apples planted by the ordinary method and the Red Fuji apples applied with goat milk and apple enzymes for comparison, and the root and / or intergenic microbial analysis was performed. Among them, the ordinary planting method is that the aforementioned biological enzymes, goat milk dilutions, and soy protein are not used throughout the process, and only farmyard manure base fertilizer is applied in spring (the utilization rate of base fertilizer is higher than that of autumn fertilization), and the bagging operation is the same as above.

[0039] Example 2 Analysis of nutritional components of fruits

[0040] 1. Fruit nutritional composition analysis method

[0041] Rhizosphere soil samples of fruit trees of the same age were collected in an ordinary Red Fuji orchard (Group A) and an apple test orchard using the method of Example 1. Samples of apples of Example 1 were collected after continuous goat milk irrigation and organic fertilizer (enzyme) treatment for 2 years (Group B) and 3 years (Group C).

[0042] Groups A and C were as described above, and Group D was set up as a control group, where only enzymes and goat milk were added when watering the roots in July and August, but no soy protein was added. The watering period was three years. The nutritional components of the fresh fruits of Groups A, C and D were analyzed immediately after picking, and the synergistic effects of enzymes, goat milk and soy protein were considered.

[0043] (1) Single fruit weight analysis: 20 samples were randomly selected from each batch, and the single fruit weight was measured. The average value of the five batches was calculated.

[0044] (2) Analysis of fruit water content: Quarter the peeled fresh fruit to about 20g, accurately weigh the mass as m1, dry in a 60℃ oven until constant weight (about 24h), weigh the mass as m2. Water content = (m2-m1) / m1*100%. Perform five parallel experiments.

[0045] (3) Determination of total sugar content in pulp: The sulfuric acid-phenol method was used to determine the total sugar content in pulp.

[0046] Preparation of standard curve: Prepare 100 mg / L glucose standard solution, take 0, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of glucose standard solution in colorimetric tubes, accurately fill to 2.0 mL with deionized water, add 1.0 mL of freshly prepared 6% (v / v) phenol solution, mix well, add 5.0 mL of concentrated sulfuric acid, mix well, place in a 90°C water bath for 30 min, immediately cool to room temperature with running water, and measure the absorbance (OD) at 490 nm. 490 ), draw the standard curve.

[0047] Accurately weigh the peeled and pitted fresh fruit, chop it up and grind it into powder using liquid nitrogen, transfer all of it to a 50 mL volumetric flask with deionized water and make up the volume. After extracting it in a microwave oven at mid-range (400W) for 1 min, place it in a water bath at 80°C for 30 min to fully release the sugar, centrifuge it at 12,000 rpm, take the supernatant and dilute it to an appropriate multiple, and determine the total sugar as above. 490 The total sugar concentration was obtained by substituting into the standard curve.

[0048] (4) Determination of reducing sugar in pulp: The DNS method was used to determine the reducing sugar in pulp.

[0049] Preparation of standard curve: Prepare 1.0 mg / mL glucose standard solution, take 0-2.0 mL of different volumes of glucose standard solution and add them to 25 mL colorimetric tubes, and accurately make up to 2.0 mL with ultrapure water. Add 1.5 mL of DNS reagent to each tube, boil it in a water bath for 5 minutes, cool it to room temperature with running water, make up to 25 mL, and mix it. Measure its absorbance at a wavelength of 540 nm with a spectrophotometer and draw a standard curve.

[0050] The reducing sugar sample to be tested in the pulp was obtained by the polysaccharide extraction method described above. The sample was appropriately diluted to make the reducing sugar concentration between 0.1-1.0 mg / mL, 2.0 mL was added to a 25 mL colorimetric tube, 1.5 mL of DNS reagent was added, and the mixture was placed in a boiling water bath for 5 minutes, cooled to room temperature with running water, and made up to 25 mL with deionized water. The absorbance was measured at a wavelength of 540 nm with a spectrophotometer, and the reducing sugar concentration was calculated from the standard curve. Five parallel tests were performed.

[0051] (5) Determination of total acidity in pulp titratable: Accurately weigh peeled and pitted fresh fruit (mass m), grind with liquid nitrogen, transfer to a 50 mL volumetric flask with deionized water (volume V3), extract with microwave (400 W) for 1 min, keep warm in a water bath at 80 °C for 30 min to fully release the acid, centrifuge at 12000 rpm, take the supernatant and dilute to measure the total acidity. Take 35.00 mL of the supernatant (volume V4), add 3 drops of phenolphthalein solution, and titrate with 0.1000 mol / L sodium hydroxide solution (concentration C) until the color turns pink and does not fade for 30 seconds, which is the titration endpoint. The volume of sodium hydroxide used is V1, and the remaining volume of sodium hydroxide is V2. Measure 5 parallel samples.

[0052] Calculation formula: X = C*(V1-V2)*K / m*V3 / V4*100, X is the total acid content in grams of apples (g / kg).

[0053] In the above formula, K is based on malic acid, which is the number of grams of 1mmol / LNaOH relative to malic acid, K=0.067, and the unit is the percentage of titratable acid mass to fresh fruit weight.

[0054] (6) Determination of the sugar-acid ratio of apple pulp: The sugar-acid ratio of apple = the average value of total sugar in apples / the average value of total acid in apples.

[0055] (7) Determination of total polyphenol content in pulp: The total phenol content was determined by the Folin-Ciocalteu method.

[0056] Preparation of standard curve: Accurately measure different volumes of 233.0 mg / L gallic acid standard solution and add them to 25 mL colorimetric tubes, dilute with 6.0 mL ultrapure water, add 1.0 mL Folin-phenol reagent, and then add 8 ml 120 g / L Na2CO3 solution, mix and dilute to 25 mL, react at room temperature for 60 min. Measure the absorbance at 760 nm with a spectrophotometer and draw a standard curve.

[0057] Accurately weigh the peeled and cored apple pulp, cut into small pieces, add liquid nitrogen and grind to powder, wash three times with 70% (v / v) acidic ethanol reagent, transfer all to a 50mL volumetric flask, mix and dilute to volume, then ultrasonically extract at 35°C for 30min, cool to room temperature, and centrifuge at 12000rpm for 10min. Take 1.0mL of supernatant and add it to a 25mL colorimetric tube, add 6.0mL ultrapure water to dilute, then add 1.0mL Folin-phenol reagent and 8.0mL 120g / L sodium carbonate solution, mix and dilute to volume. React at room temperature for 60 minutes, and measure the absorbance at a wavelength of 760nm with the help of a spectrophotometer. Calculate the total phenol content in the sample (calculated as gallic acid) from the standard curve, in mg / kg fresh fruit. Perform 5 parallel determinations.

[0058] A t-test was performed on this part of the data to examine the significance, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0059] 2. Fruit nutritional analysis results

[0060] The appearance of the apples grown in group C of the present invention and the "cold sugar core" after cutting are as follows: Figure 1 As shown, at the same time, the apples grown in Group C have a strong milky flavor.

[0061] (1) Analysis of single apple weight and water content

[0062] like Figure 2-3 As shown, for the same growth time, the average single fruit weight of apples planted in group C of the present invention is slightly higher than that of apples planted in group D and ordinary Red Fuji. The average single fruit weight of apples planted in group C of the present invention is 329.5±41.2g, the average single fruit weight of apples planted in group D is 323.4±35.2g, and that of Red Fuji is 325.7±23.6g, with no significant statistical difference; the water content of apples planted in group C of the present invention is lower than that of ordinary Red Fuji, and the difference is significant (p<0.05), while the difference between apples planted in group D and groups A and C is not significant. It is shown that the present invention promotes the accumulation of fruit nutrients by synergistic irrigation with goat milk, apple enzyme and soy protein. However, the effect of goat milk + apple enzyme + soy protein is more significant. Next, the main organic matter such as sugar and acid is measured.

[0063] (2) Determination of fruit sugar and acid accumulation

[0064] The total sugar content of the pulp was determined by the sulfuric acid-phenol method, and the reducing sugar content of the pulp was determined by the DNS method. The results are as follows: Figure 4-5As shown. The total sugar content of the apple flesh of the apples planted in group C of the present invention reached 0.1978±0.0106g / g, the total sugar content of the apples planted in group D was 0.19176±0.02001g / g, and the common Fuji apple had only 0.1417±0.0173g / g. The difference between group C and group A of common Fuji apple was extremely significant, while the difference between group D and group A of common Fuji apple was significant but not significant compared with group C. The reducing sugar content of the apples planted in group C of the present invention was 0.1442±0.0002g / g, the apples planted in group D were 0.1421±0.002g / g, and the common Fuji apple was 0.1249±0.0002g / g. The difference between group C and group A of common Fuji apple and group D and group A of common Fuji apple was very significant. This indicates that during the growth and development of the fruit, the fruits of Group C and Group D of Example 1 are more advantageous in terms of sugar accumulation, and synergistic irrigation with goat milk, apple enzyme, and soy protein can more stably enhance sugar accumulation.

[0065] (3) Determination of titratable total acidity of fruit

[0066] The results of the titratable total acidity test of the fruit are as follows: Figure 6 As shown, the titratable total acid content of the fruits of group C of Example 1 is 0.2220±0.00035(%), that of group D is 0.318±0.0005(%), and that of the common Fuji is 0.3421±0.000058(%), with extremely significant differences. The sugar-acid ratios of the apples planted in group C of the present invention, the apples planted in group D, and the common Fuji fruit are calculated to be 89.17%, 60.3%, and 41.37%, respectively, based on the total sugar and total acid determination values. The high sugar-acid ratio may be an important reason for the significant improvement in the taste of the Fuji fruit of the present invention.

[0067] (4) Determination of fruit polyphenol content

[0068] Polyphenol compounds have the effects of enhancing immune function, anti-cancer, anti-aging, and lowering blood lipids, but they have astringent and bitter taste, which affects the taste to a certain extent. The polyphenol content of Fuji fruit of the present invention is 363.96±73.95mg / kg ( Figure 7 ), slightly lower than the common Red Fuji (347.43±69.90), with no statistically significant difference: slightly higher than the apples grown in Group D (357.24±71.35), but also with no statistically significant difference. Later, the polyphenol content of the peel was also measured, and it was found that the polyphenol content of the peel was more than twice that of the flesh, but there was no significant difference between the apples grown in Group C of the present invention, the apples grown in Group D, and the common Fuji. In addition, it was found later that the polyphenol content would fluctuate with different storage time.

[0069] In summary, it can be seen that the key indicators of nutrients such as sugar, acid, and sugar-acid ratio of the apple of the present invention are significantly different from those of ordinary Red Fuji. Nutrients such as sugar, acid, and polyphenols can significantly affect the appearance and taste of apples, define the commodity grade of apples, and determine the product competitiveness of apples. In particular, the synergistic effect of group C enzymes, goat milk, and soy protein significantly improves the sugar-acid ratio of apples. The high sugar-acid ratio is an important reason for the improved taste, making the taste of apples better and the fruit sweeter.

[0070] Example 3

[0071] 1. Collection method of rhizosphere soil of fruit trees

[0072] Rhizosphere soil samples of fruit trees of the same age were collected in an ordinary Red Fuji orchard (Group A) and an apple test garden using the method of Example 1. Samples of apples in Example 1 that had been continuously irrigated with goat milk and treated with organic fertilizer for 2 years (Group B) and 3 years (Group C) were collected. Three fruit trees were randomly selected from each group, and each fruit tree was sampled using a five-point sampling method. The sampling depth was 15-20 cm from the ground surface. 1-2 mm of rhizosphere soil attached to the roots of the tree was brushed off with a sterile brush into a sterile centrifuge tube, and animal and plant residues were removed. The soil was stored at low temperature and quickly sent back to the laboratory to extract total soil DNA using FastDNASpin Kit for Soil (MP Company, USA), and transported to Beijing Biomarker Biotechnology Co., Ltd. in liquid nitrogen for subsequent biodiversity analysis.

[0073] 2. Analysis methods of rhizosphere microbial community diversity

[0074] The third-generation high-throughput sequencing method was used to determine the full-length sequence of 16S rDNA (primers 27F and 1492R) and ITS sequence to analyze the diversity of rhizosphere microbial bacteria and fungal communities, respectively. The samples were sequenced using the PacBio sequencing platform, and the CCS (Circular Consensus Sequencing) sequences were filtered and OTUs (Operational Taxonomic Units) were clustered. The sequences were clustered using USEARCH at a similarity level of 97%, and OUT was filtered out with a threshold of 0.005% of all sequence numbers. Alpha diversity analysis was performed using Mothur to study the species diversity within a single sample, and the Ace and Shannon indexes of each sample at a similarity level of 97% were calculated. Qiime was used for non-metric multi-dimensional scaling (NMDS) to characterize the beta diversity between sample groups. Taxonomic analysis was performed on each sample at each classification level to obtain community structure diagrams at the phylum, class, order, family, genus, and species taxonomic levels. Metastats was used to screen biomarkers. Metastats analysis (http: / / metastats.cbcb.umd.edu / ) compares the significance of differences between two groups at each classification level through p and q values, performs T test on species abundance data between groups to obtain p value, and corrects p value to obtain q value; finally, the species that cause the difference in composition between the two groups of samples are screened out according to p value (or q value).

[0075] 3. Results of analysis of rhizospheric bacterial diversity

[0076] Rhizosphere microorganisms, especially rhizosphere bacteria, play an important role in soil microecology, such as releasing carbon dioxide and acidic metabolites to promote mineral absorption by plants, secreting metabolites and iron carriers to inhibit the reproduction of plant pathogens, secreting plant growth hormones, and helping plants fix nitrogen. Goat milk and ecological fertilizers provide a considerable amount of "prebiotics", which may interfere with the growth and development of fruit trees by affecting the diversity of rhizosphere microorganisms. In order to explore the mechanism, the rhizosphere soil of ordinary Red Fuji (Group A), 2 years (Group B) and 3 years (Group C) of fruit trees irrigated in Example 1 were sampled, and the full-length sequence of 16S rDNA was determined by third-generation high-throughput sequencing to analyze the diversity of rhizosphere microbial communities. The CCS sequence sequenced by the PacBio sequencing platform was filtered and OTUs clustered and filtered at a similarity level of 97%.

[0077] (1) Analysis of Alpha Diversity and Beta Diversity

[0078] Alpha diversity can reflect the species richness and species diversity of a single sample. The Chao1 index measures species richness. The larger the value, the higher the species richness. The Shannon index is used to measure species diversity. It is affected by the species richness and species uniformity in the sample community. The larger the Shannon index value, the higher the species diversity. The OUT coverage of the 9 samples in the experiment was above 91.1%. The Chao1 and Shannon index statistical values ​​are as follows: Figure 8 As shown, the overall indexes showed a trend of C>B>A, but there was no significant difference among the groups of Shannon index; the Chao1 index group C and group A were significantly different (p<0.05). The present invention used goat milk, apple enzyme, and soybean protein to synergistically irrigate to improve the Alpha diversity of rhizospheric bacteria.

[0079] The non-metric multi-dimensional scaling method (NMDS) was used to perform sample Beta diversity analysis. The NMDS method simplifies the research objects (samples or variables) in the multi-dimensional space to a low-dimensional space for positioning, analysis and classification, while retaining the original relationship between objects. It can better reflect the nonlinear structure of the data and overcome the shortcomings of linear models such as PCA and PCoA. When Stress is less than 0.2, it indicates that the NMDS analysis has a certain degree of reliability. Fig. 9 As shown, the Stress value of this analysis is 0.0929, and the analysis is reliable. It can be seen from the area divided by the confidence ellipse in the figure that the boundaries between group C and groups A and B are clear, but the areas of groups A and B overlap, indicating that there is a significant difference between the rhizosphere microbial community composition of fruit trees irrigated for 3 years of the present invention and the untreated group of ordinary Red Fuji in the control group, but there is no significant difference between irrigating for 2 years and irrigating for 3 years. The following analysis is a comparative analysis of group C (irrigated for three years) and group A (control).

[0080] (2) Species distribution analysis

[0081] like Fig.10 As shown, the differences in OUTs among the three groups A, B, and C are displayed in the form of a Venn diagram. It can be seen that there are 852 OUTs in the three groups, and there are OUTs unique to each group.

[0082] At the gate level (Phylum, Fig.11 A) and genus level (Genus, Fig.11B) Compare groups A and C. As shown in the figure, the dominant phylum in each group at the phylum level is mainly Proteobacteria. Example 1 The relative abundance of Proteobacteria and Bacteroidetes in the irrigation group (C) is lower than that in the ordinary red Fuji group (A), while the relative abundance of Acidobacteria, Actinobacteria, Verrucomicrobia, etc. is increased. At the genus level, the differences between the groups are large. The relative abundance of Rhodanobacter (7.54%) and Chujaibacter (5.63%) at the genus level in group A is relatively high, while in group C, it is uncultured_bacterium_f_Gemmatimonadaceae and Candidatus Udaeobacter of the Gemmatimonadaceae family.

[0083] In order to study the indicator microorganisms between the control and experimental group samples, Metastats software was used to perform a T test on the species abundance data between the groups; the p value was obtained, and the q value was obtained by correcting the p value; finally, the species with different composition between the two groups of samples were screened out according to the p value, and the results are shown in Table 1.

[0084] Table 1 Metastats analysis (prokaryotic)

[0085]

[0086] Cytophagales belongs to the order Cytophagales, which can decompose cellulose and convert dead branches and leaves into glucose that can be absorbed by plants. It is the most active aerobic bacteria in the soil that decomposes cellulose. It has a strong ability to decompose cellulose and can effectively promote the material cycle in the soil, which is beneficial to the growth of fruit trees.

[0087] Paenibacillaceae belongs to the Paenibacillus family. It can produce antibacterial substances to prevent and control fruit tree diseases. It can also be used as an insecticide and has toxic effects on invertebrates. It acts on bacterial cell membranes, inhibits the growth of harmful bacteria, induces plants to produce disease resistance, and can also play a disease-resistant effect through interaction with plants. Moreover, it has biological activities such as phosphorus solubilization, potassium solubilization, and nitrogen fixation, which is beneficial to increasing fruit tree yields. It has good stress resistance and has great potential in the development of bacterial fertilizers, which is beneficial to the growth of trees. It can colonize at the root tips of fruit trees and accumulate in the intracellular space outside the vascular column of the plant roots, greatly accelerating the process of fruit trees absorbing nutrients. It can also degrade toxic substances in the environment, such as petroleum, polycyclic aromatic hydrocarbons, organochlorine pesticides, and organophosphorus pesticides, improve soil quality, and is beneficial to the healthy growth of fruit trees; it can also be used as a desert water retainer, which is beneficial to soil water storage. It is a species with potential for the development of microecological bacterial agents.

[0088] Bradyrhizobium belongs to the genus Bradyrhizobium. It has been reported that an analysis of the bacterial diversity in vineyard soil after the application of grape waste enzyme fertilizer also found an increase in the content of Bradyrhizobium.

[0089] It can be seen from the results in Table 1 that the abundance of the above beneficial bacteria in Group C (irrigated for three years) is higher than that in Group A (control group), indicating that the planting method of the present invention promotes the increase of the above beneficial bacteria.

[0090] 4. Results of analysis of rhizosphere fungal diversity

[0091] The rhizosphere soil of common Red Fuji (Group A) and fruit trees irrigated for 2 years (Group B) and 3 years (Group C) in Example 1 was sampled and genome extracted as before, and the ITS (ITS1+ITS2) sequence was determined by third-generation high-throughput sequencing to analyze the diversity of rhizosphere microbial community fungi. The CCS sequence sequenced by the PacBio sequencing platform was filtered and OTUs clustered and filtered at the level of 97% similarity.

[0092] (1) Analysis of Alpha Diversity and Beta Diversity

[0093] like Fig.12 As shown in the figure, the Chao1 index and Shannon index showed an overall downward trend after the application of organic fertilizer and goat milk, but there was no significant difference. This indicates that the Alpha diversity of eukaryotic microorganisms decreased after the application of organic fertilizer. Considering that the Alpha diversity of prokaryotic microorganisms in group C increased compared with group A, there may be antagonism between bacteria and fungi. Beta diversity analysis was performed using non-metric multidimensional scaling (NMDS), as shown in the figure. Fig.13 As shown, there was no significant difference between groups A and B, so groups A and C were compared in the follow-up.

[0094] (2) Species distribution analysis

[0095] Comparison of eukaryotic microbial OUT components Fig.14 As shown in the Venn diagram, there are 125 common OUTs in the three groups. The relative abundance of samples was compared at the phylum and genus levels, as shown in Fig.15As shown in the figure, Ascomycota and Basidiomycota were the main dominant phyla, and the relative abundance of Ascomycota in group A was significantly higher than that in group C, while the relative abundance of Mortierellomycota and Mucoromycota was significantly lower than that in group C. There were also significant differences in the distribution of genus levels between groups A and C. The relative abundance of Penicillium, Neocosmospora, and Staphylotrichum was higher in group A, while the relative abundance of Penicillium, Metarhizium, Mortierella, and Aspergillus was higher in group C.

[0096] Similar to the analysis of prokaryotic microorganisms, Metastats was used to analyze indicator fungi, and the results are shown in Table 2. As can be seen from Table 2, the abundance of several pathogenic fungi was significantly reduced after planting using the method of the present invention.

[0097] Pezizomycetes is a class of phytopathogens that can cause various plant diseases, produce black spots or moldy substances, often forming obvious dead spots with obvious dark edges, and produce ethylene and abscisic acid, causing leaf shedding, and overwintering in the diseased remains as ascocarps. As a source of infection, it can asexually produce a large number of conidia to infect fruit trees again; Mortierellomycetes is a class of phytopathogens that are mycorrhizal fungi of forest trees or pathogens of animals, harming the growth of fruit trees; Fusarium is a genus of Fusarium, which is an important plant pathogen or secondary invader, and many species can also produce fungal toxins and even cause diseases in humans and animals; Albifimbria is the main pathogen of many plant root diseases, with a wide range of hosts, which harms the health of fruit trees; Devriesia is a genus of Devriesia that can cause apple sooty mold; Colletotrichum is a genus of Colletotrichum, which is widely distributed and can cause anthracnose diseases in many plants. It is an important plant pathogen that harms the growth of fruit trees; Fusarium circinatum is a pine resin canker pathogen, a dangerous forest pathogenic fungus that seriously harms trees. In 1986, it caused a devastating blow to pine trees in California, USA, killing more than 10,000 pine trees. It is a serious tree fungal disease that harms the health of trees.

[0098] Table 2 Metastats analysis (eukaryotic)

[0099]

[0100] In addition, it was found that the relative abundance of Neocosmospora_rubicola in group C was significantly higher than that in group A. Neocosmospora_rubicola has an antagonistic effect on forest pathogens. Studies have shown that the fungus can achieve a 100% inhibition rate against chestnut blight pathogens, and the antagonism coefficient can reach the I level. The antagonism coefficient for some other pathogens can reach the II level, such as Colletotrichum gloeosporioides, Fusarium solani, and Fusarium oxysporum.

[0101] It can be clearly seen from the above data that there are significant differences in fungal diversity between the group irrigated with goat milk, apple enzyme and soy protein and the group not irrigated, and the relative abundance of several plant pathogens is significantly inhibited.

[0102] In summary, the planting method of the present invention uses enzymes prepared by fermentation of secondary fruits and soy protein, and then uses goat milk and other substances containing a large amount of "prebiotics" to affect the structure of the rhizosphere microbial community of fruit trees, promote the proliferation of "probiotics" of fruit trees, and antagonize pathogenic microorganisms. In addition, goat milk is rich in milk oligosaccharides, and the fallen fruits and brown sugar used to prepare enzymes also provide rich oligosaccharide substances. The main components of "prebiotics" may be these oligosaccharides. Changes in the structure of the rhizosphere microbial community of fruit trees affect the accumulation of nutrients in the fruit, especially significantly improving the sugar-acid ratio of apples, making the taste better.

[0103] For any technician familiar with the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention should still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for growing apples, characterized in that: The method comprises the following steps: (1) Before bagging apples, spray the leaves, young fruits, and roots of the apple trees with diluted goat milk; (2) Water the roots twice a year at the end of July and the end of August, and apply diluted goat milk and ecological enzymes according to the apple yield; the ecological enzymes are composed of apple enzymes and soy protein.

2. The method for growing apples according to claim 1, characterized in that: The goat milk diluent is prepared by diluting goat milk and water in a mass ratio of 1:30-1:

100.

3. The method for growing apples according to claim 1, characterized in that: The mass ratio of apple enzyme to soy protein is 1:10-70.

4. The method for growing apples according to claim 1, characterized in that: The apple enzyme is prepared by mixing brown sugar, defective apples and water in a mass ratio of 1:4-6:

100. The defective apples are chopped and mixed, and then placed in a barrel and placed in a cool and ventilated place for fermentation for 3 months before use. The soy protein is soy milk, and the concentration of the soy milk is not more than 20%.

5. The method for growing apples according to claim 1, characterized in that: In step (1), the goat milk dilution is applied according to the apple yield, and 0.5-2kg of the goat milk dilution is applied for every 100 catties of apple yield during the spraying operation; in step (2), 10-30 catties of goat milk is applied for every 100 catties of apple yield during the root irrigation operation, and 5-20 catties of ecological enzyme is applied for every 100 catties of apple yield during the root irrigation operation.

6. The method for growing apples according to claim 1, characterized in that: When applying the goat milk diluted solution and ecological enzyme in step (2), radial trenches with a depth of 8-10 cm and a diameter of 40-60 cm are made around the trunk of the tree and the prepared liquid is evenly poured in.

7. Application of the apple planting method according to claims 1-6 in improving apple planting soil to change the structure of rhizosphere microbial community.

8. The use according to claim 7, characterized in that: For prokaryotic microorganisms, the abundance of Cytophagale, Paenibacillaceae, and Bradyrhizobium was increased.

9. The use according to claim 7, characterized in that: For eukaryotic microorganisms, the abundance of Neocosmospora_rubicola was increased, and the abundance of Pezizomycetes, Mortierellomycetes, Fusarium, Albifimbria, Devriesia, Colletotrichum, and Fusarium circinatum was inhibited.

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