CN113337405B - Mycorrhizal fungi of genus ascomycete and application thereof in cultivation of apple continuous cropping resistant barrier rootstock - Google Patents
Mycorrhizal fungi of genus ascomycete and application thereof in cultivation of apple continuous cropping resistant barrier rootstock Download PDFInfo
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Abstract
The invention discloses a strain of mycorrhizal fungi of a genus of coccidioidomycosis and application thereof in cultivation of apple continuous cropping resistant barrier rootstocks. The mycorrhizal fungi of the genus coccidioidomycosis are obtained by separation and identification. The mycorrhizal fungi of the dominant bacteria species of the coccidiodes obtained by the invention are inoculated to the root of the apple rootstock to form the rootstock with continuous cropping resistance obstacle, so that the continuous cropping obstacle can be effectively reduced. Compared with the traditional resistant rootstock cultivation method, the cultivation efficiency is improved, and the cultivation time can be shortened by 7-10 years.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to mycorrhizal fungi of a strain of ascomycete and application of the mycorrhizal fungi in cultivation of apple continuous cropping resistant barrier rootstocks.
Background
Continuous cropping obstacles are a phenomenon commonly existing in the plant world, wherein the continuous cropping obstacles of apples are particularly serious. Limited by land resources, in the process of updating and rebuilding the aged orchards in the main apple production area in China, 40-90% of orchards need to be subjected to continuous cropping cultivation, and the continuous cropping orchards have the phenomena of serious plant diseases and insect pests, short and small plants, irregular orchards, low yield, poor quality and the like, and have serious economic loss.
Research shows that the main pathogenic bacteria causing continuous cropping obstacle of apples in China are fusarium. The traditional prevention measures are difficult to be applied in production because of difficult popularization (such as soil dressing) or poor effect or long time consumption (such as crop rotation). In the early foreign countries, the effect of preventing and treating apple continuous cropping obstacles by adopting methyl bromide is good, but the application is forbidden due to the harmfulness to the environment and the human body. Other soil disinfectants have far lower effect than methyl bromide and are complex in method, and the biological control method has the problems of difficult colonization, unstable effect and the like. Recently, more CG series apple successive cropping obstacle resistant rootstocks bred by the American cornell university have good test results, but verification on aspects such as grafting affinity, regional adaptability and the like is still needed, and if the method is applied in China, long-time and large-scale experimental work is needed besides the problem of intellectual property.
Mycorrhiza refers to a reciprocal symbiont formed by fungi and plant root systems, and fungi capable of infecting the plant root systems to form mycorrhiza are mycorrhizal fungi. The mycorrhizal fungi can expand the effective absorption range of host in soil, promote plant's absorption of phosphorus and other mineral elements, raise the disease resistance, stress resistance and water utilization of host and promote plant growth. The continuous cropping problem of apples is solved by using VA mycorrhizal fungi such as poplar flood, and the like, and VAM fungi are inoculated after formalin is used for disinfecting soil for treatment, so that the effects of increasing the planting survival rate and promoting growth are good. However, no report is found about the cultivation of apple continuous cropping resistant barrier rootstocks by utilizing mycorrhizal fungi.
Disclosure of Invention
Aiming at the prior art, the invention separates and identifies the dominant strain (ascomycete-like) of arbuscular mycorrhizal fungi from the apple rhizosphere soil. The mycorrhizal fungi of the apple rootstock is inoculated to the root of the apple rootstock to form a continuous cropping resistant rootstock, so that continuous cropping obstacles can be effectively reduced.
Specifically, the invention relates to the following technical scheme:
the invention provides a continuous cropping resistant mycorrhizal fungi (Paraglomus sp.) SW1 of the genus Coccomyia, which is preserved in China general microbiological culture Collection center (CGMCC for short, address: Beijing West Lu No. 1 Hospital, Taiyang district, Beijing) 10.16 days in 2020, and the biological preservation number is as follows: CGMCC NO. 20744.
The mycorrhizal fungi (Paraglomus sp.) of the genus coccidioidomycosis were isolated from rhizosphere soil of old orchards and had the following characteristics: spores are either solitary or clustered in the soil. The spores are transparent and light yellow. Spherical, nearly spherical, 60 μm-100 μm. Spore wall 3 layer, wall easily separated, Melzer's reagent appears bright yellow, easily cracks when crushed. The inclusion is in the form of transparent oil drops. The hyphae of Neurospora were bent to one side and were pale yellow.
The fungicide of the mycorrhiza fungi (Paraglomus sp.) of the genus Gliocladium also belongs to the protection scope of the invention.
In a second aspect of the present invention, there is provided a use of the above-mentioned mycorrhizal fungus (paramlomus sp.) SW1 or mycorrhizal fungus (paramlomus sp.) of the genus coccidioidomyia for at least one of the following (1) to (3):
(1) cultivating rootstocks resistant to apple continuous cropping obstacles;
(2) cultivating apple plants resistant to apple continuous cropping obstacles;
(3) preventing and controlling continuous cropping obstacle of apples.
The third aspect of the invention provides a cultivation method of apple continuous cropping obstacle-resistant rootstocks, which comprises the following steps:
inoculating the mycorrhizal fungi (Paraglomus sp.) of the genus of the coccidioidomycosis or the fungicide of the mycorrhizal fungi (Paraglomus sp.) of the genus of the coccidioidomycosis to a root system of a stock in a tissue culture, breeding and hardening stage of the apple stock, and culturing to obtain the apple continuous cropping resistant barrier stock.
Preferably, the inoculation is specifically as follows: after the tissue culture seedling of the apple rootstock takes root, transplanting the apple rootstock into a mixed matrix containing mycorrhizal fungi (Paraglomus sp.) of the genus of the coccidioidomycosis, and culturing for 60-70 days.
Preferably, in the mixed matrix, the mass ratio of the matrix to the mycorrhizal fungi (Paraglomus sp.) inoculum of the genus coccidioidomycosis is 3: 1.
In a fourth aspect of the present invention, there is provided a method for cultivating apple plants resistant to apple replant obstacle, comprising the steps of:
and (3) moving the apple continuous cropping obstacle resistant rootstock obtained by cultivation to a field, grafting an apple variety when the diameter of the apple continuous cropping obstacle resistant rootstock plant reaches 0.7-0.8cm at the height of 20-25cm, and further growing and developing to form an apple plant resistant to apple continuous cropping obstacles.
In a fifth aspect of the invention, the application of the stock or the apple plant obtained by the cultivation method in the prevention and control of apple continuous cropping obstacles is provided.
The invention provides a method for preventing and controlling apple continuous cropping obstacles, which comprises the following steps:
and (3) excavating planting holes on the apple continuous cropping soil, and planting the apple plants which are obtained by cultivation and are resistant to apple continuous cropping obstacles in the planting holes.
The invention has the beneficial effects that:
(1) the invention separates and obtains a strain of mycorrhizal fungi (Paraglomus sp.) of the genus coccidioidomycosis, and the apple rootstock cultivated by the mycorrhizal fungi has the characteristics of fast root growth, continuous cropping resistance and the like, and can better reduce the continuous cropping obstacle of apples.
(2) The mycorrhizal fungi (Paraglomus sp.) of the mycorrhizal fungi of the ascomycete-like genus can be used for cultivating the stock resistant to apple continuous cropping obstacles, and compared with the traditional resistant stock cultivating method, the cultivation efficiency is improved, and the cultivation period can be shortened by 7-10 years.
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FIG. 1: and (4) analyzing the composition of the AM fungal community of the soil to be tested.
FIG. 2: SW1 spore morphology (A) and Meler's staining (B).
FIG. 3: phylogenetic tree of SW1 strain 18S rDNA sequence.
FIG. 4: mycorrhiza infection rate after SW1 inoculation is dynamic.
FIG. 5: influence of different treatments on the growth of the Malus hupehensis Rehd seedlings; in the figure, CK1 represents a continuous control; SW1 indicates SW1 vaccination treatment.
FIG. 6: effect of different treatments on the copy number of the fusarium venenatum (a) and fusarium moniliforme (B) genes.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified.
Example 1: efficient dominant strain screening
Collecting a Laizhou Shahezhen gulf head village apple rhizosphere soil sample, quickly freezing and returning, sending to Meiji organisms for high-throughput sequencing, directly screening the local arbuscular mycorrhizal fungi dominant strain from the apple rhizosphere soil by a second-generation high-throughput sequencing technology, and displaying that the soil sample dominant strain belongs to the genus Strystoma (Paraglomus sp.) at the genus level (figure 1A) and the species level (figure 1B) as shown in figure 1.
Example 2: isolation and identification of highly effective dominant strains
1. A sample of rhizosphere soil of Malus of Bay Tokamura in Shahe town of Laizhou was collected.
2. Uniformly mixing the collected soil sample with sterilized river sand according to the weight ratio of 1:1, potting, dividing into three parts, sowing corn in the first part, sowing clover in the second part, planting Malus hupehensis seedling in the third part (each part is repeated by 3), carrying out enrichment culture on the dominant bacteria of the genus Gliocladium, and uniformly mixing three parts of soil for sowing the corn, the clover and the Malus hupehensis seedling after culturing for 4 months in a greenhouse to separate the dominant bacteria of the genus Gliocladium.
3. And (3) taking 20g of a soil sample from the uniformly mixed soil, putting the soil sample into a 1L beaker, adding 500mL of sterile water, uniformly stirring, and sequentially sieving by using a 50-mesh and 400-mesh sample separation sieve. Transferring the residues on the 400-mesh sieve into a 50mL centrifuge tube, centrifuging at 4000rpm for 3min to remove the supernatant, adding 15-20mL of sucrose solution with the mass concentration of 60%, stirring uniformly, centrifuging at 3000rpm for 2min to obtain spores, sucking all the supernatant into a new culture dish, sucking single spores by using a 10 microliter gun under a stereoscopic microscope, and placing the spores in a 1cm culture dish3The morphological identification is carried out on the spores on the filter paper sheet, and the result shows that: the spores are transparent and light yellow. Spherical, nearly spherical, 60 μm-100 μm. Spore wall 3 layer, wall easily separated, Melzer's reagent appears bright yellow, easily cracks when crushed. The inclusion is in the form of transparent oil drops. The hyphae of Neurospora were bent to one side and pale yellow (FIG. 2). It is named SW 1.
4. The molecular identification is further carried out on the basis of morphological identification: SW1 spores were aspirated, washed with sterile water at least 5 times, the water surrounding the spores was removed, a small amount of Tap polymerase was added, the mixture was transferred to another new 1.5mL centrifuge tube (under a stereoscope) by washing the hair, the spores were crushed with a tip, 14. mu.L of Tap polymerase PCR buffer was quickly added, and the mixture was placed in a 94 ℃ water bath for 4 min. After 4min, the cells were placed on ice for PCR amplification. Specific primers NS1-NS4 and NS31-AM1 were used for amplification by nested PCR.
First round PCR: 20 μ L system, ddH2O13. mu.L, 10 XE XTqq Buffer 2. mu.L, EX Taq 0.2. mu.L, 2mm dNTP 2. mu.L, primer NS 10.4. mu.L/NS 40.4. mu.L, template DNA 2. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 40 ℃ for 1min, and extension at 72 ℃ for 1min (35 cycles); extension at 72 ℃ for 10min and holding at 4 ℃.
Second round PCR: the first round PCR product was diluted 1:100 and used as template with primers NS1 and NS4 for PCR amplification. The reaction system was 20. mu.L total volume, ddH2O13. mu.L, 10 XBuffer 2. mu.L, EX Taq 0.2. mu.L, dNTP 2. mu.L, primer NS 310.4. mu.L/SW 10.4. mu.L, template DNA 2. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 1min (35 cycles); extension at 72 ℃ for 10min and holding at 4 ℃.
Table 1: primer sequences
The PCR product was purified and recovered for sequencing. The SW1 strain 18S rDNA sequence length amplified by using the nested-PCR technique was 521bp (shown in SEQ ID NO: 5), the sequence was aligned with that on MaarjAM (https:// www.maarjam.botany.ut.ee) and NCBI, and phylogenetic analysis was performed by the maximum likelihood method in MEGA 5.1, and the SW1 strain 18S rDNA sequence had 99% homology with Paraglomus IH1 VTX00444 (FIG. 3), indicating that the strain is of the genus Synechocystis (Paraglomus sp.). Species were not identified due to imperfections in the GenBank data.
The strain of the gloriopsis globulosa SW1 is subjected to biological preservation, and the preservation information is as follows:
the preservation date is as follows: year 2020, 10 and 16.
The storage place: china general microbiological culture Collection center (CGMCC, address: No. 3 Xilu-1 of Beijing, Chaoyang, North Chen, China).
The biological preservation number is as follows: CGMCC NO. 20744.
Example 3: preparation of SW1 microbial inoculum
The SW1 strain was inoculated to the roots of clover growing for one week, and the clover was subjected to single spore culture in a greenhouse for 1 month, and further propagated, and the clover roots were pulverized to obtain powder containing SW 1.
Mixing a sterilized substrate (a common seedling substrate, which is commercially available) and a powder containing SW1 in a mass ratio of 3:1, mixing evenly, sowing clover, and expanding propagation in greenhouse and pot culture. After three months, the infection rate is detected, the overground part is removed, the roots of the clovers and the matrix are crushed together to obtain the SW1 microbial inoculum for subsequent tests, and the SW1 microbial inoculum is dried in the shade and stored at 4 ℃.
Example 4: apple continuous cropping resistant barrier rootstock cultivation
1. And (3) experimental design:
the experiment is carried out in the national apple engineering experiment center and the apple continuous cropping and microorganism laboratory in the southern school district of Shandong agricultural university, the plant to be tested in the pot culture experiment is the seedlings of Malus hupehensis (Malus hupeheus Rehd.), the seeds of the Malus hupehensis are laminated for about 40 days at 4 ℃, and the seeds are sowed in a sterilization substrate and a microbial inoculum (SW 1 microbial inoculum prepared in example 3) according to the mass ratio of 3:1, culturing seedlings in a mixed culture pot; the SW1 inoculum was sterilized and then mixed with a sterilized substrate to prepare a non-mycorrhizal control. When the seedlings grow to 6 main leaves, selecting plants which have consistent growth vigor and do not have plant diseases and insect pests, transplanting the plants into a clay tile pot (the upper diameter is 25cm, the lower diameter is 17cm and the height is 18cm) filled with 6.5kg of different treated soil in 1 day in 5 months, sampling in the middle ten days of 8 months, and measuring related experimental indexes.
Soil for pot culture test is taken from 32-year old apple orchard in Manzhuantan Qingwan, Taian, Shandong, and is randomly taken at multiple points in an area 80.00cm from a trunk and 5.00-40.00 cm deep, and is uniformly mixed. The soil type is sandy soil, the content of organic matters is 13.97g/kg, the content of quick-acting nitrogen is 35.76mg/kg, the content of quick-acting potassium is 116.64mg/kg, the content of quick-acting phosphorus is 20.42mg/kg, and the pH value of the soil is 5.06.
The experiment was set up with 3 treatments, respectively: planting the non-mycorrhizal seedlings into continuous cropping soil (CK1), planting the mycorrhizal seedlings into continuous cropping soil (T), planting the non-mycorrhizal seedlings into bromomethane fumigating continuous cropping soil (CK2), planting 2 seedlings in each pot every 20 pots of treatment, and managing fertilizer and water uniformly.
2. Measurement indexes are as follows:
the height, the ground diameter and the dry fresh weight of the seedlings are determined by a conventional method.
0.5g of the sieved fresh soil was taken as E.Z.N.A.
Extracting DNA by the soil DNA extraction kit operation steps, carrying out real-time fluorescence quantitative analysis on gene copy numbers of rhizoctonia solani and rhizoctonia solani in soil by adopting CFX96TMthermal Cycler (Bio-Rad), verifying the inhibition effect of SW1 apple rhizosphere seedlings on rhizosphere soil layer outgrowth and rhizoctonia solani, and referring to Wangongshai (circular Bohai sea continuous cropping soil fungus community structure analysis and mixed cropping shallot to reduce apple continuous cropping obstacle research [ D)]Wangongshuai, Shandong university of agriculture 2018).
Mycorrhiza infection rate after SW1 inoculation
After SW1 inoculation, the mycorrhiza infection rate of the Malus hupehensis Rehd seedling can be obviously improved, the mycorrhiza infection rate can reach 65.38 percent (figure 4) after two months of cultivation before plant transplantation, and the sterile root of the un-inoculated SW1 is infected. The mycorrhizal infection rate of mycorrhizal seedlings is slightly increased after 1 month of transplanting, but the infection rate of non-mycorrhizal seedlings is obviously increased, which shows that the indigenous AMF existing in the continuous cropping soil can infect the Malus hupehensis Rehd seedlings, but the infection rate is still lower than that of SW1 mycorrhizal seedlings.
Effect of SW1 on growth amount
As can be seen from Table 2, the SW1 post-treatment (T) significantly promoted the plant height, ground diameter, fresh weight and dry weight of the continuous cropping Malus hupehensis seedling compared with the non-mycorrhizal seedling (CK1), and the indexes of the SW1 post-treatment (T) such as the plant height, the ground diameter and the like are even similar to those of the SW 2 fumigated seedling. It can be seen that SW1 inoculation can significantly promote the growth of Malus hupehensis seedling (FIG. 5).
Table 2: effect of SW1 inoculation on Malus hupehensis seedling Biomass
Note: different lower case letters indicate significant differences at the 0.05 level (Duncan's test), as follows.
Influence of SW1 inoculation treatment on protective enzyme of Malus hupehensis seedling root system
As can be seen from Table 3, the SW1 inoculation treatment (T) improved the respiration rate of the roots of the seedlings of Malus hupehensis and the activity of root protective enzymes, which are significantly higher than that of the continuous cropping control (CK 1). Compared with the continuous control (CK1), the SW1 inoculation treatment has the advantages of obviously increased root respiration rate, CAT activity, SOD activity and POD activity. It can be seen that the SW1 inoculation treatment can significantly promote the activity of protective enzymes of the roots of the seedlings of the cognac rubus hupehensis chun (table 3).
Table 3: influence of SW1 inoculation treatment on protective enzyme of Malus hupehensis seedling root system
Effect of SW1 inoculation on plant nutrient content:
as shown in Table 4, the mycorrhizal seedlings can obviously improve the total nitrogen and total phosphorus content of plants, and compared with non-mycorrhizal seedlings (CK1), the total nitrogen and total phosphorus content of the plants after SW1 inoculation treatment (T) is respectively increased by 6.56% and 6.52%, but the total nitrogen and total phosphorus content of the plants can be obviously improved.
Table 4: effect of SW1 inoculation on plant nutrient content
Inhibition effect of the SW1 strain on Fusarium:
the numbers of gene copies of the emerged and fusarium moniliforme among the 3 treatments, which were continuously used as a control (CK1), were the highest and the SW1 inoculation treatment (T) was reduced by 4.58 times and 5.04 times, respectively, as compared with the control (CK1), as determined by real-time fluorescent quantitative PCR (fig. 6). It can be seen that SW1 inoculation treatment (T) reduced the number of spores and Fusarium moniliforme in the soil.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture, Shandong Huimei farming-grazing development Co., Ltd
<120> one strain of mycorrhizal fungi of genus coccidioidomycosis and application thereof in cultivation of apple continuous cropping resistant barrier rootstock
<130> 2021
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cgggggcatc agtattcaat tgtcagaggt gaaattcttg gatttattga agactaacta 360
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gacgatcaga taccgtcgta gtcttaacca taaactatgc cgactaggga tcggacgatg 480
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Claims (9)
1. A strain of mycorrhizal fungi (Paraglomus sp.) of the genus Gliocladium, the biological preservation number of which is: CGMCC NO. 20744.
2. A microbial agent comprising the mycorrhizal fungi (Paraglomus sp.) of genus Mycosphaerellis according to claim 1.
3. Use of the mycorrhiza fungi of genus coccidioidomycosis (Paraglomus sp.) according to claim 1 or the microbial agent according to claim 2 in at least one of the following (1) to (3):
(1) cultivating rootstocks resistant to apple continuous cropping obstacles;
(2) cultivating apple plants resistant to apple continuous cropping obstacles;
(3) reducing apple continuous cropping obstacle.
4. The cultivation method of the apple continuous cropping resistant barrier rootstock is characterized by comprising the following steps:
inoculating the mycorrhizal fungi (Paraglomus sp.) of the genus Phycomyces of claim 1 or the microbial inoculum of claim 2 to root systems of rootstocks in the tissue culture, breeding and hardening-seedling stage of apple rootstocks, and cultivating to obtain the apple continuous cropping resistant barrier rootstocks.
5. Cultivation method according to claim 4, characterized in that said inoculation is in particular: after the apple rootstocks root, transplanting the apple rootstocks into a mixed matrix containing mycorrhizal fungi (Paraglomus sp.) of the genus thylakocystoid, and culturing for 60-70 days.
6. The cultivation method as claimed in claim 5, wherein the mass ratio of the substrate to the mycorrhiza fungi (Paraglomus sp.) inoculum of the genus Gliocladium in the mixed substrate is 3: 1.
7. A cultivation method of apple plants resistant to apple continuous cropping obstacles is characterized by comprising the following steps:
transplanting the apple continuous cropping obstacle-resistant rootstock cultivated according to the claim 4 into a field, grafting an apple variety when the diameter of the apple continuous cropping obstacle-resistant rootstock plant reaches 0.7-0.8cm at the height of 20-25cm, and further growing and developing to form an apple plant resistant to apple continuous cropping obstacle.
8. Use of the rootstock bred in claim 4 or the apple plant bred in claim 7 for the prevention and control of apple continuous cropping obstacles.
9. A method for alleviating apple continuous cropping obstacles, comprising the steps of:
digging a planting hole on the apple continuous cropping soil, and planting the apple plants resistant to apple continuous cropping obstacles, which are obtained by the cultivation of claim 7, in the planting hole.
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| MYCORRHIZAL INOCULATION OF APPLE IN REPLANT SOILS - ENHANCED TREE GROWTH AND MINERAL NUTRIENT STATUS;Gastol, M等;《ACTA SCIENTIARUM POLONORUM-HORTORUM CULTUS》;20151231;第14卷(第4期);第17-37页 * |
| 苹果连作障碍研究进展;尹承苗等;《园艺学报》;20171125(第11期);第2215-2230页 * |
| 草酸青霉A1菌株的鉴定及对苹果4种镰孢病菌的拮抗作用;张先富等;《园艺学报》;20160525(第05期);第841-852页 * |
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