CN107164234B - Fungus with strong weed suppression effect screened from passion fruit rhizosphere soil - Google Patents

Abstract

The invention belongs to the field of microbial herbicides, and particularly relates to a grass-inhibiting fungus screened from passion fruit soil. Lettuce (Compositae), barnyard grass (Gramineae) and pennisetum alopecuroides (Gramineae) are respectively used as receptors, and biological tests are carried out on the grass inhibition capacity of the fungus strain fermentation liquor, so that the fungus strain fermentation liquor has strong inhibition rates on the root length and the plant height of the two receptors. Phenolic acid with the grass inhibiting effect of protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid and cinnamic acid is detected in the fermentation liquor. The strain was named FJ-01-BXG-08. The morphological and ITS sequence analysis and identification result of the strain shows that the strain isAspergillus sydowii(Aspergillus polyvidus) is a member of the genus Aspergillus polyvidus, which is a strong weed-inhibiting fungus selected from eucalyptus soil for the first time (Aspergillus sydowii）。

Description

Fungus with strong weed suppression effect screened from passion fruit rhizosphere soil

Technical Field

The invention belongs to the field of crop disease control, and particularly relates to a fungus with strong weed suppression effect, which is screened from passion fruit rhizosphere soil.

Background

Passion fruit (A)Passiflora edulis) The passion flower and the passion fruit are lianas planted in tropical and subtropical zones, and the juice of the passion flower and the passion fruit has the functions of digestion aiding and nourishing and is popular with people. It is estimated that the planting area of the passion fruit in the world reaches more than 2 million hectares, but as the passion fruit is more and more popular, the market demand is continuously increased, and the planting area of the passion fruit is also continuously increased. Statistics show that the demand of the world in the first 80 years for passion fruit exceeds 25 ten thousand tons, but the production amount is only 12 ten thousand tons, and the demand is far greater than the supply. Am on meThe country is originally introduced in the beginning of the 20 th century, and the main planting places include Fujian, Taiwan, Hainan, Guangdong, Yunnan and the like. However, due to the long-term planting of the passion fruit, the physicochemical property and the microecology of the soil for planting the passion fruit are changed, so that seedlings and adult plants of the passion fruit are easily damaged by root rot. At present, the root rot is researched and considered that pathogenic bacteria mainly comprise fusarium, pythium and other pathogenic bacteria. Rhizosphere microorganisms are abundant and large in number, and roots and soil microorganisms are generally obligate to each other based on the pressure of co-evolution. In addition, the species of root exudates can affect the rhizosphere microflora structure.

Weeds are an important factor affecting the improvement of crop yield and have now become a worldwide problem. The main current weeding mode is to utilize chemical herbicides to control weeds in farmlands. However, long-term, high-volume, non-selective application of chemical herbicides can lead to soil and groundwater contamination, and weeds can also develop resistance. High concentrations of chemical herbicides also increase the residue of harmful substances in crops. Therefore, there is a need for a more ecologically friendly method for weed control. The biological herbicide is plant pathogenic microorganism or microbial phytotoxin for biological weeding, has the advantages of environmental safety, good compatibility, no residue in soil and the like, and is a research hotspot at present.

Allelopathy means that plants, algae, bacteria, fungi and actinomycetes produce and release secondary metabolites to influence the growth and development (inhibition or promotion effect) of other plants around and the agricultural system. These secondary metabolites mainly include: phenolic acids, terpenes, flavonoids, fatty acids, and the like. The content of these allelochemicals determines the strength of the allelochemicals.

By utilizing the plant allelopathy principle and the rhizosphere microecology theory, pathogenic bacteria harmful to plants are screened at the rhizosphere of passion fruit plants infected with root rot in orchards where passion fruits are planted for a long time, and a new thought is provided for preventing and controlling weeds and reducing the use of chemical herbicides.

Disclosure of Invention

The invention provides aspergillus polyvidus screened from passion fruit rhizosphere soil and application thereof, wherein the aspergillus polyvidus fermentation liquor contains protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, cinnamic acid and other phenolic acids with a grass inhibiting effect, so that the fermentation liquor has the grass inhibiting effect. The invention has the advantages of safe environment, good environmental compatibility, no residue in soil and the like.

In order to achieve the purpose, the invention is realized by the following technical scheme:

aspergillus sydowii screened from eucalyptus rhizosphere soil, wherein the Aspergillus sydowii isAspergillus sydowiiFJ-01-BXG-08; has been preserved in China center for type culture Collection with the preservation address: china, Wuhan and Wuhan university, the preservation date is 2017.4.19, and the preservation number is CCTCC M2017198.

The thallus and fermentation liquor of Aspergillus polytrichus FJ-01-BXG-08 have inhibitory effect on growth of lettuce, Echinochloa crusgalli of Gramineae and pennisetum alopecuroides L.

Another object of the present invention is to provide a method for using the aspergillus sydowii selected from the eucalyptus rhizosphere soil as a weed-suppressing fungus, which comprises the following steps:

(1) taking the strain FJ-01-BXG-08 for fermentation culture;

(2) diluting the fermentation liquor by 1-100 times;

(3) and pouring the diluted fermentation liquor around the seeds or plants.

The invention has the advantages that:

the fungus strain fermentation liquor contains protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, cinnamic acid and other phenolic acids with the grass inhibiting effect, and has the advantages of safe environment, good environmental compatibility, no residue in soil and the like; and has strong inhibition rate on the root length and plant height of barnyard grass and lettuce.

Drawings

FIG. 1 shows HPLC chromatograms of phenolic acid in fermentation broth of strain FJ-01-BXG-08; wherein peak 1 is protocatechuic acid; peak 2 is p-hydroxybenzoic acid; peak 3 is vanillic acid; peak 6 is salicylic acid; peak 7 is cinnamic acid.

FIG. 2 morphogram of strain FJ-01-BXG-08; FIG. 2A is a strain conimap (10X 40), and FIG. 2B is a colony map.

FIG. 3 is a band electrophoretogram of the FJ-01-BXG-08-ITS I region of the strain; FIG. 3A shows the band electrophoresis of the FJ-01-BXG-08-ITS I region of the fungal strain in lane 1, DL10000DNAmarker in lane 2, and DL10000DNAmarker in lane 3B.

FIG. 4 shows a developmental tree constructed from the strain FJ-01-BXG-08 by the NJ method (adjacent growing method).

FIG. 5 the effect of fermentation broth of strain FJ-01-BXG-08 at different concentrations on the growth of millet.

Detailed Description

Example 1

Biological test of grass inhibition capability of strain fermentation liquor

Respectively putting a piece of filter paper in a tissue culture bottle, adding 5 mL of fermentation liquor diluted by 50 times into the tissue culture bottle, sowing 5 lettuce seeds just suffering from white color into each tissue culture bottle, using distilled water as a control, and culturing the lettuce seeds in a constant-temperature incubator at 28 ℃, wherein each experiment is repeated for 3 times. And measuring the root length and the plant height of the lettuce after 3d of culture. The inhibition rate is calculated according to the formula:IR=（1-TR/CK) X 100%. Wherein the content of the first and second substances,IR: an Inhibition rate (Inhibition rate);TR: treatment (Treatment);CK: control (Control).

And respectively taking barnyard grass and pennisetum alopecuroides as experimental objects to carry out the grass suppression capability test of the fermentation liquor. Taking barnyard grass as an experimental object, and diluting fermentation liquor by 20 times; the pennisetum alopecuroides is used as an experimental object, and the bacterium solution is diluted by 50 times to test the weed suppression capability. The experimental procedures of the grass-inhibiting ability biological test are the same as above, and the results of the grass-inhibiting ability test are shown in Table 1.

TABLE 1 FJ-01-BXG-08 Strain fermentation broth diluted to effect on recipient plants

Test results show that the fermentation liquor of the FJ-01-BXG-08 strain diluted by 50 times has strong inhibition effects on the root length and the plant height of the lettuce, and the inhibition effects are 80.08% and 37.98% respectively. The inhibition rate of the root length of the pennisetum alopecuroides reaches 45.15%, but the inhibition rate of the plant height of the pennisetum alopecuroides is only 9.93%. For barnyard grass with stronger resistance, the strain fermentation liquor has strong inhibition effect on the root length and the plant height after being diluted by 20 times, and the inhibition rates respectively reach 58.41 percent and 34.43 percent.

Example 2 Strain identification

1. Morphological identification

Morphological Observation of colonies

Inoculating the fungus cake punched by a puncher with the diameter of 0.8cm into the middle of a PDA solid culture medium plate, continuously culturing at 28 ℃ for 7d, and observing morphological characteristics such as texture, color ornamentation and the like of the edges and the surface of the colony every day.

Observation of microscope morphology

Picking out a little bacterial colony from the bacterial colony cultured for 3d on a PDA solid medium plate by using a dissecting needle, placing the bacterial colony on a glass slide with sterile water dropped in the middle, then dispersing the bacterial colony by using two dissecting needles as much as possible, covering a cover glass, sucking water around the glass slide by using water absorption paper, and observing the forms of conidiophores, spores and the like under an optical microscope.

Morphological identification the strains were preliminarily identified according to characteristics of colonies, according to "fungal identification handbook" by weijing and introducory Mycology by c.j.

The conidiogram and colony morphology are shown in FIG. 2, wherein FIG. 2A is the conidiogram of strain FJ-01-BXG-08, and FIG. 2B is the colony morphology. Culturing the bacterial colony on a PDA culture medium at a constant temperature of 28 ℃ for 5 days, wherein the diameter is 3.00-4.00cm, the bacterial colony is velvet to flocculent, and has dense concentric rings and white edges; the color is grayish green in the first birth and is light brown after the aging; the back side is milky white or dark brown, and the surface has no exudation. The microconidia is like a mildew, and is loose cylindrical or scattered.

2. Molecular biological characterization of fungal strains

Extracting the purified genome DNA of the fungus by using an Ezup column type fungus genome DNA extraction kit. The following fungus universal primers were selected for PCR amplification sequencing of the ITS I region of the strain:

an upstream primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3',

the downstream primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3';

the PCR amplification system was 25. mu.L, 2 × TaKaRa TaqTMHS Perfect Mix 3.7. mu.L, upstream and downstream primers (20. mu. mol. mL)^-1) 0.5. mu.L each, 0.5. mu.L of DNA, supplemented with sterile deionized water to 25. mu.L. The reaction condition is that the pre-denaturation is carried out at 94 ℃ for 4min, then the circulation is carried out, the circulation parameters are 94 ℃ for 45s, 55 ℃ for 45s and 72 ℃ for 1min, and after 30 circulation, the final extension is carried out at 72 ℃ for 10 min. mu.L of the PCR product was electrophoresed on a 1% wt agarose gel, and the electrophoresis result is shown in FIG. 3, and the size of the target fragment FJ-01-BXG-08-ITS I region was approximately 542 bp. The PCR product FJ-01-BXG-08-ITS I region is purified by a glue recovery kit, and then the PCR product is subjected to sequence determination by committee of the biological engineering (Shanghai) corporation, and sequence information is analyzed and compared by an NCBI database.

The ITS sequencing result of the strain shows that the sequence size is 542bp, and the strain is compared with data in NCBI (blast) through homologyAspergillus sydowiiThe sequence similarity reaches more than 99 percent, and the strain is concluded to beAspergillus sydowii(Aspergillus polydorus). Phylogenetic analyses were performed using MEGA 5.05 software. The phylogenetic tree constructed by the Neighbor-Joining method (NJ) is shown in FIG. 4.

The strain is shown to be by morphology and ITS identification resultsAspergillus sydowii(Aspergillus polyvidus).

EXAMPLE 3 determination of phenolic acids in Strain broths

Taking 100mL of fermentation liquor, adjusting the pH value to 4.00 with phosphoric acid, adding 8g of NaCl, and dissolving the solution for later use. And (3) soaking the Cleanert PEP solid-phase extraction cartridge with distilled water-methanol-distilled water (8 mL-8 mL-6 mL) and leaching and activating, and removing methanol residues for later use. And (3) adding the treated fermentation liquor into the activated PEP solid phase extraction cartridge, adding 2mL of distilled water for washing after the fermentation liquor completely flows out, adding 10mL of methanol for elution, collecting the eluent, and freeze-drying. After being dissolved by 1mL of methanol (chromatographic purity), the solution is filtered in a sample loading bottle by a 0.22 mu mL filter membrane, thus obtaining a sample of the fermentation liquor.

Taking 7 kinds of phenolic acid such as protocatechuic acid, ferulic acid, cinnamic acid, vanillic acid, salicylic acid, p-hydroxybenzoic acid and the like as standard substances (all purchased from Aladdin company), and preparing into mother liquor of 1mg/mL with methanol (chromatographic purity) for later use. The mother liquor is diluted into a mixed solution with 7 concentration gradients as a standard curve, and the equation of the standard curve is shown in table 2.

TABLE 2 Standard Curve equation for phenolic acid species

The procedure of high performance liquid chromatography is slightly adjusted according to the literature Zhengxinyu and the like, and is briefly described as follows: the HPLC is Shimadzu corporation (CBM-20A, LC-20AD, SPD-20A), and the column is SunFireTW-C18 (4.6 mm. times.250 mm, ID 5 μm); mobile phase A-methanol (chromatographic purity) and B-0.1% phosphoric acid solution (analytical purity); elution conditions: 0-5min, A: b =27:73, 5-10min a: b =27:73, 10-15min a: b =50:50, detection wavelength is 280 nm; the flow rate is 1.6 mL/min; the sample volume is 10 mu L; the column temperature was 30 ℃.

Comparing the spectrogram of the test solution with the spectrogram of the mixed standard sample, determining substances according to retention time, and calculating the relative content of each substance according to a standard curve method; the test results are shown in FIG. 1, and the contents of the respective substances are shown in Table 3.

TABLE 3 bacterial suspension phenolic acid content analysis of strain FJ-01-BXG-08

The HPLC detection result of the bacterial liquid of the strain FJ-01-BXG-08 is shown in Table 2, the bacterial liquid contains 0.078mg/L protocatechuic acid, 1.05 mg/L p-hydroxybenzoic acid, 4.19 mg/L vanillic acid, 28.17 mg/L salicylic acid and the highest cinnamic acid content of 33.39 mg/L.

Example 4 verification of grass-suppressing potential of Strain-Pot culture experiment

The method is carried out in an incubator of a laboratory, the temperature is 28 ℃, and the illumination is carried out for 12 hours. Taking the paddy field soil to naturally dry, removing broken stones and impurities in the soil after grinding, and sieving. 500 g of the fine soil after sieving was weighed and placed in a plastic pot (diameter 12cm, height 6 cm). 5 particles of sprouted small barnyard grass are sowed into the surface layer of the soil. The fermentation broth of FJ-01-BXG-08 strain cultured for 7 days at 28 ℃ and 180rpm/min was diluted 3 times in gradient for 4 treatments. Treatment 1: stock, treatment 2: dilution 5-fold, treatment 3: dilution 20-fold, treatment 4: diluting by 50 times. 30mL of each gradient was separately applied around the seed. The control was distilled water. Each treatment was repeated 3 times. 20mL of distilled water was added with a sprayer every day to keep the soil wet. And (5) measuring the root length and the plant height of the receptor plant after planting for 5 d.

The effect of the strain fermentation liquid on the small barnyard grass under the soil culture condition is shown in a table 4, and the result shows that the strain fermentation liquid with different concentration gradients has an effect on the growth of small barnyard grass seedlings. The highest inhibition rate on the height of barnyard grass plants reaches 56.53 percent.

TABLE 4 influence of fermentation broths on soil-cultured barnyard grass

The influence of the strain fermentation liquor on the small barnyard grass under the soil culture condition is shown in a table 4, and the result shows that the strain fermentation liquor with different concentration gradients can inhibit the growth of small barnyard grass seedlings, and the inhibition rate is higher than 40%; can be used for inhibiting weed growth.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> a fungus with strong weed suppression effect screened from passion fruit rhizosphere soil

<130>3

<160>3

<170>PatentIn version 3.3

<210>1

<211>19

<212>DNA

<213> upstream primer ITS1

<400>1

tccgtaggtg aacctgcgg 19

<210>2

<211>20

<212>DNA

<213> downstream primer ITS4

<400>2

tcctccgctt attgatatgc 20

<210>3

<211>584

<212>DNA

<213> FJFAFU-BXG01-ITS I region

<400>3

taggtgaacc tgcggaagga tcattaccga gtgcgggtcc tttgggccca acctcccatc 60

cgtgtctatt gtaccctgtt gcttcggcgg gcccgccgct tgtcggccgc cgggggggcg 120

cctctgcccc ccgggcccgt gcccgccgga gaccccaaca cgaacactgt ctgaaagcgt 180

gcagtctgag ttgattgaat gcaatcagtt aaaactttca acaatggatc tcttggttcc 240

ggcatcgatg aagaacgcag cgaaatgcga taactaatgt gaattgcaga attcagtgaa 300

tcatcgagtc tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga 360

gcgtcattgc tgccctcaag cccggcttgt gtgttgggtc gccgtccccc tctccggggg 420

gacgggcccg aaaggcagcg gcggcaccgc gtccgatcct cgagcgtatg gggctttgtc 480

acatgctctg taggattggc cggcgcctgc cgacgttttc caaccattct ttccaggttg 540

acctcggatc aggtagggat acccgctgaa cttaagcata tcaa 584

Claims (3)

1. A aspergillus polyvidus screened from passion fruit rhizosphere soil is characterized in that aspergillus polyvidus (A)Aspergillus sydowii) Is FJ-01-BXG-08; has been preserved in China center for type culture Collection with the preservation date of 2017.4.19 and the preservation number of CCTCC NO of M2017198.

2. The Aspergillus sydowii screened from the passion fruit rhizosphere soil as claimed in claim 1, wherein the thallus and fermentation liquid of the Aspergillus sydowii FJ-01-BXG-08 have an inhibiting effect on the growth of lettuce, barnyard grass of Gramineae and pennisetum alopecuroides.

3. A method for using the Aspergillus sydowii screened from the passion fruit rhizosphere soil as the grass-inhibiting fungus in the claim 1 is characterized by comprising the following steps:

(1) taking the strain FJ-01-BXG-08 for fermentation culture;

(2) diluting the fermentation liquor by 1-50 times;

(3) and pouring the diluted fermentation liquor around the seeds or plants.

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