阅读说明：本技术 一种会理链霉菌及其应用 (Streptomyces corviensis and application thereof ) 是由 起登凤 邹良平 王尉 张妙宜 周登博 谢江辉 陈宇丰 于 2021-06-08 设计创作，主要内容包括：本发明提供了一种链霉菌,其为链霉菌属的一个新种,命名为：Streptomyceshuiliensis sp.nov.,于2021年3月30日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2021298T。该菌株具有稳定广谱抑菌活性,对尖孢镰刀菌4号生理小种、尖孢镰刀菌1号生理小种、香蕉长形斑病菌、香蕉大灰斑病菌、草莓炭疽病菌、和香蕉炭疽病菌等多种常见作物病原真菌有较强的抑菌作用,其发酵粗提物可以有效抑制Foc TR4真菌的菌丝生长和孢子萌发,最低抑菌浓度为6.25μg/ml,具有潜在的应用价值,为枯萎病等多种植物病害的防治拓展新领域,具有良好的开发应用前景。(The invention provides a streptomycete, which is a new species of streptomycete and is named as: streptomyces huiliensis sp. nov.was deposited at the China center for type culture Collection at 30/3 in 2021 with the deposit number of CCTCC M2021298T. The strain has stable broad-spectrum antibacterial activity, has strong antibacterial effect on a plurality of common crop pathogenic fungi such as fusarium oxysporum No. 4 physiological race, fusarium oxysporum No. 1 physiological race, banana physalospora piricola, banana gray leaf spot pathogen, strawberry anthracnose pathogen, banana anthracnose pathogen and the like, can effectively inhibit hypha growth and spore germination of Foc TR4 fungus by a fermentation crude extract, has a minimum antibacterial concentration of 6.25 mug/ml, has potential application value, expands a new field for prevention and treatment of a plurality of plant diseases such as fusarium wilt and the like, and has good development and application prospects.)

1. A streptomycete is a new species of streptomycete, and is named as: streptomyces huiliensis sp. nov.was deposited at the China center for type culture Collection at 30/3 in 2021 with the deposit number of CCTCC M2021298T.

2. The use of the Streptomyces of claim 1 for antagonizing Fusarium oxysporum No. 4 physiological race, and/or Fusarium oxysporum No. 1 physiological race, and/or Leptosphaeria plantarii, and/or Fraptosphaeria strawberrii, and/or Muptosphaeria plantarii.

3. Use of the Streptomyces according to claim 1 for the preparation of a biocontrol agent for controlling diseases caused by Fusarium oxysporum No. 4 physiological race, and/or Fusarium oxysporum No. 1 physiological race, and/or Alternaria bananas, and/or Alternaria cinerea, and/or strawberry anthracnose, and/or banana anthracnose.

4. Fermentation broth of the streptomyces of claim 1, or ethyl acetate crude extract of the fermentation broth.

5. The fermentation broth or ethyl acetate crude extract of the fermentation broth as claimed in claim 4, wherein the fermentation broth or the ethyl acetate crude extract of the fermentation broth is used for antagonizing fusarium oxysporum f.sp.4 physiological race, and/or fusarium oxysporum f.1 physiological race, and/or alternaria bananas, and/or alternaria barbata, and/or anthracnose of strawberry, and/or anthracnose of banana.

6. The fermentation liquid according to claim 4, or the ethyl acetate crude extract of the fermentation liquid, is used for preparing a biocontrol agent for preventing and treating diseases caused by fusarium oxysporum f.sp.4 and/or fusarium oxysporum f.sp.1 and/or physalospora piricola and/or physalospora cinerea and/or anthracnose of strawberry and/or banana.

7. A fungicide characterized by comprising the Streptomyces coruscus of claim 1, or comprising the fermentation broth of claim 4, or an ethyl acetate crude extract of the fermentation broth.

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a streptomyces conopsis and application thereof.

Background

The plant fungal diseases seriously threaten the safety and stability of crop production. Worldwide, 10% to 16% of agricultural yield losses are caused by plant diseases annually, of which 70% to 80% are caused by infection with pathogenic fungi (wushiming et al, 2018). Although chemical pesticide has ideal control effect on various diseases, the control effect on some diseases, especially soil-borne fungal diseases is poor, and long-term use of chemical pesticide can induce the drug resistance of pathogenic bacteria, cause pollution to the environment and generate toxic and side effects on human and animals. Biocontrol of plant diseases using highly effective, low toxicity microbial biocontrol agents is considered a viable alternative (Asghar and Mohammad, 2010).

Streptomycete is an important biological control resource, and can produce a great amount of bioactive secondary metabolites with different functions and structures, thereby improving the resistance of plants to biological and non-biological stress. Streptomyces is currently known to produce over 9000 bioactive substances, of which over 120 are used, accounting for 75% of the number of bioactive substances derived from the applied microorganisms (cinnabaris et al, 2019). However, according to predictions, more than 90% of the bioactive metabolites of streptomyces remain to be discovered, and the ability of streptomyces to produce new types of secondary metabolism appears to be endless (Berdy, 2005). The discovery of new antagonistic streptomyces strains provides the necessary conditions for the development of novel microbial biocontrol agents.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides a new Streptomyces species, Streptomyces huiliensis sp. nov, which has stable broad-spectrum antagonistic activity and strong bacteriostatic action on various common crop pathogenic fungi such as fusarium oxysporum.

In a first aspect of the present invention, there is provided a streptomyces, which is a new species of streptomyces, named: streptomyces huiliensis sp. nov. (Chinese translation name is Streptomyces corviensis, laboratory number is SCA 2-4)^THereinafter abbreviated to "SCA 2-4^T") was preserved in China center for type culture Collection at 30.3.2021 with the preservation number of CCTCC M2021298T.

The second aspect of the invention provides the use of the streptomycete of the first aspect of the invention in antagonizing fusarium oxysporum f.sp.4 physiological race, and/or fusarium oxysporum f.sp.1 physiological race, and/or alternaria bananas, and/or alternaria cinerea, and/or anthracnose of strawberry, and/or anthracnose of banana.

The third aspect of the invention provides the application of the streptomycete in the first aspect of the invention in preparing a biocontrol preparation for preventing and treating diseases caused by fusarium oxysporum f.sp.4 and/or fusarium oxysporum f.sp.1 and/or physalospora piricola and/or physalospora cinerea and/or anthracnose of strawberry and/or banana.

In a fourth aspect of the present invention, there is provided a fermentation broth of Streptomyces according to the first aspect of the present invention, or a crude ethyl acetate extract of the fermentation broth.

The fifth aspect of the invention provides the fermentation liquid of the fourth aspect of the invention, or the ethyl acetate crude extract of the fermentation liquid, for antagonizing fusarium oxysporum No. 4 physiological race, and/or fusarium oxysporum No. 1 physiological race, and/or banana alternaria leaf spot, and/or banana macrobotrya, and/or strawberry anthracnose, and/or banana anthracnose.

The sixth aspect of the invention provides the fermentation liquid of the fourth aspect of the invention, or the ethyl acetate crude extract of the fermentation liquid, in the preparation of a biocontrol agent for controlling diseases caused by fusarium oxysporum f.4 physiological race, and/or fusarium oxysporum f.1 physiological race, and/or physalospora piricola, and/or strawberry anthracnose, and/or banana anthracnose.

In a seventh aspect, the invention provides a bactericide, which comprises the streptomyces exiguus of the first aspect, or comprises the fermentation broth of the fourth aspect, or the ethyl acetate crude extract of the fermentation broth.

SCA2-4 of the invention^TThe strain has stable broad-spectrum bacteriostatic activity, has strong bacteriostatic action on a plurality of common crop fungi such as Fusarium oxysporum f.sp.cubense loopray 4, Foc TR4, Fusarium oxysporum f.sp.cubense loopray 1, Foc TR1, banana physalospora (fallax), banana macrobotrys (Curvularia lunata), strawberry anthracnose (Colletotrichum fragaria) and banana anthracnose (Colletotrichum musae), and a fermented crude extract of the strain can effectively inhibit the growth and spores of Foc TR4 fungi, has the Minimum Inhibitory Concentration (MIC) of 6.25 mu g/ml, and has a potential application value, good application prospect in preventing and controlling plant diseases such as Fusarium oxysporum and the like.

Drawings

FIG. 1 shows that the contiguous method is constructed based on SCA2-4 of complete 16SrRNA gene sequence^TThe strain phylogenetic tree, the accession number of the gene in brackets, the node value of the evolutionary tree is the percentage of self-expansion (based on 1000 replications), and the scale indicates that the branches of this length represent a degree of genetic variation of the genome of 0.002.

FIG. 2 shows the strain SCA2-4 based on 5 tandem housekeeping gene sequences atpD, gyrB, recA, rpoB and trpB constructed by the neighbor joining method^TPhylogenetic tree showing strain SCA2-4^TAt positions in the phylogenetic neighborhood, the evolutionary tree node value is the percentage of introversion (based on 1000 replicates) and the scale indicates that the branches of this length represent a genetic variation of 0.05 in the genome.

FIG. 3 shows strain SCA2-4^TScanning electron micrographs of 21 days of incubation at 28 ℃ on YE medium. A: aerial hyphae are well developed, have more branches and cannot be differentiated into spore chains; b: branched substrate hyphae.

FIG. 4 shows strain SCA2-4^TOn 6 ISP mediaAnd (5) culture characteristics.

FIG. 5 shows strain SCA2-4^TInhibiting the growth of hypha of different plant pathogenic fungi.

FIG. 6 shows strain SCA2-4^TInhibition of mycelial growth of Foc TR4 by crude extract. A: treatment with 10% DMSO; b: strain SCA2-4^TCrude extract (16 × MIC) treatment.

FIG. 7 shows strain SCA2-4^TEffect of crude extract on spore germination of Foc TR 4. A: treatment with 10% DMSO; b: strain SCA2-4^TCrude extract (16 × MIC) treatment.

Detailed Description

The invention will be better understood by reference to the following examples.

The invention provides a streptomycete, which is a new species of streptomycete and is named as: streptomyces huiliensis sp. nov., (Chinese translation name is Streptomyces corviensis, laboratory number is SCA2-4^THereinafter abbreviated to "SCA 2-4^T") was deposited in the China center for type culture Collection at 30/3/2021 at the deposition address: wuhan, China, the preservation number is CCTCC M2021298. The streptomyces collinitus SCA2-4^TIs obtained by separating and screening the rhizosphere soil of cactus collected from dry and hot valley areas of Richijie county of Sichuan province.

Strain 1 SCA2-4^TSeparation of

In 1 month in 2014, the research collects cactus rhizosphere soil samples in dry and hot valley areas of Richicounty of Sichuan province. Drying the soil sample at room temperature, sieving with 0.425mm mesh sieve, mixing 1 g of the soil sample with 9ml of sterile water, heating in 55 deg.C water bath for 20 min, diluting with continuous dilution technique, and spreading 100 μ L onto starch casein agar plate (SCA medium: 10 g of soluble starch, 0.3 g of casein, 2.0g of KNO)₃2.0g NaCl,2.0 g K₂HPO₄0.05 g MgSO₄·7H₂O, 0.02 g CaCO₃0.01 g of FeSO₄·H₂O, and 18 g agar, 1000 ml sterile water, pH 7.0-7.4), adding K to the medium₂Cr₂O₇(50mg/L) and nystatin (nysta)tin) (50mg/L) to inhibit fungal and bacterial growth. The plates were incubated at 28 ℃ for 7 days. The strain SCA2-4 was isolated from these plates^TAnd purified on YE agar medium (4 g yeast powder, 10 g malt flour, 4 g glucose, 20 g agar, 1000 ml sterile water, pH 7.3). Purified strain SCA2-4^TStreaking on YE agar slant and storing at 4 deg.C while preserving with 20% (v/v) glycerol at-80 deg.C for a long period of time.

2 Strain SCA2-4^TPhylogenetic and genomic analysis of

Genomic DNA was extracted according to the method of Ahmad et al (2017). Strain SCA2-4^TThe whole genome sequencing and assembly of (a) was accomplished by Shanghai Meiji biomedical science and technology, Inc. From strain SCA2-4^TThe complete 16S rRNA gene was extracted from the whole genome sequence and compared for similarity to the corresponding sequences in EzTaxon server (Yoon et al, 2017) and NCBI databases. Phylogenetic trees were constructed using the MEGA 7.0 program and using three methods, namely neighbor-Joining Algorithms (NJ), Maximum reduction method (Maximum-parsimony Algorithms, MP) and Maximum Likelihood Method (ML), and evolution distance and confidence of NJ phylogenetic trees were calculated using a two-parameter model of Kimura (1980) and 1000-fold auto-unfolding analysis (Felsenstein,1985), respectively. From strain SCA2-4^T5 standard housekeeping genes such as atpD (ATP synthetase, beta subunit), gyrB (DNA gyrase B subunit), recA (recombinase A), rpoB (RNA polymerase, beta subunit) and trpB (tryptophan synthetase, beta subunit) were extracted from the whole genome sequence of (9) and subjected to multi-site sequence analysis (MLSA) (Brady et al, 2008). 5 housekeeping genes of 16 related strains were downloaded from the GenBank database. The 5 housekeeping gene sequences of each strain were connected end to end in order and named in fasta format. And constructing the MLSA phylogenetic tree by using the MEGA 7.0 program and adopting the three methods. The Average Nucleotide Identity (ANI) between genomes was calculated using online OrthoANI (Yoon et al, 2017 b). The genomes of the reference strains were downloaded from the EzBioCloud public genome database (https:// www.ezbiocloud.net/searchtn ═ Nocardioides).

SCA2-4^TOf the complete 16S rRNA gene sequence of the strainThe ezbiocoud alignment indicates that the strain belongs to the genus streptomyces. It has the highest sequence similarity (99.17%) to s.mobaraensis NBRC 13819T and less than 99% similarity to other streptomyces strains. While NJ phylogenetic tree based on 16S rRNA gene sequence showed that strain SCA2-4^TAnd s.orinoci NBRC 13466^TA sub-branch (similarity 98.89%) was formed with a bootstrap value of 54% (fig. 1). MP phylogenetic trees support the results of NJ, however, in ML phylogenetic trees, strain SCA2-4^TForming a separate branch. For further research on SCA2-4^TPhylogenetic status of the strains, multi-site sequence analysis based on atpD, gyrB, recA, rpoB and trpB genes was performed, and Table 1 lists GenBank accession numbers of the 16 Streptomyces housekeeping gene sequences. MLSA phylogenetic tree of the three algorithms shows that the strain SCA2-4^TAnd s.mobaraensis NBRC 13819^TSignificant branches were formed with 100% bootstrap values (see fig. 2 for MLSA phylogenetic tree for NJ method). Strain SCA2-4^TAnd s.orinoci NBRC 13466^T、S. mobaraensis NBRC 13819^TThe MLSA distance for the strains of the related models of Streptomyces is 0.024-1.020 (Table 2), well above the species boundary line for Streptomyces recommended by Rong and Huang (2012) of 0.007. Therefore, S.orinoci NBRC 13466 was selected based on the results of EzBioCloud comparison and phylogenetic tree analysis of the 16S rRNA gene sequence with the 5 housekeeping genes described above^T(＝NRRL B-3379^TGenBank accession No. NZ _ PHNC00000000.1) and S.m abaraensis NBRC 13819^T(＝DSM 40847^TGenBank accession No. NZ _ VOKX00000000.1) genome of two strains and SCA2-4^TThe comparison was performed and the ANI value was calculated. Strain SCA2-4^TAnd the ANI values between the genomes of the two strains are 80.44% and 90.76%, respectively, and are also well below the 95-96% threshold for distinguishing new species (Richter and Rossell Lolo-M Lo ra, 2009; Chun et al, 2018). Thus, these genotype data indicate that the strain SCA2-4^TIs a new species of streptomyces.

TABLE 1 accession numbers for the allelic sequences of Streptomyces used in this study

TABLE 2 MLSA distances between Streptomyces strains used in this study

The strain is as follows: streptomyces huiliensis SCA2-4^T，2.Streptomyces mobaraensis NBRC 13819^T，3. Streptomyces abikoensis NBRC 13860^T，4.Streptomyces orinoci NBRC 13466^T，5.Streptomyces angustmyceticus NRRL B-2347^T，6.Streptomyces griseocarneus(Streptomyces alboverticillatus) NRRL B-24281^T，7.Streptomyces catenulae NRRL B-2342^T，8.Streptomyces ochraceiscleroticus NRRL ISP-5594^T，9.Streptomyces varsoviensis NRRL ISP-5346^T，10.Streptomyces caniferus NBRC 15389^T，11.Streptomyces sioyaensis NRRL B-5408^T，12.Streptomyces rimosus subsp.rimosus ATCC 10970^T，13.Streptomyces libani subsp.libani NBRC 13452^T，14. Streptomyces xinghaiensis S187 ^T，15.Streptomyces pseudoechinosporeus NBRC 12518(NRRL B-16931)^T，16.Streptomyces nigrescens NBRC AS 4.1410(12894)^T，17.Streptomyces hiroshimensis NRRL B-1823(NBRC 3839)^T

3 strain SCA2-4^TPhenotypic feature analysis

Strain SCA2-4^TThe inoculated YE medium was cultured at 28 ℃ for 21 days, and its microscopic morphology was observed using a scanning electron microscope (ZEISS, Germany) as described by Kumar et al (2014). Meanwhile, the strain SCA 2-4T was inoculated on 6 standard ISP media (Shirling and Gottlieb,1966) and cultured at 28 ℃ for 14 days, and the culture characteristics thereof were observed. ISP culture medium comprises yeast extract-wheatBud extract agar (ISP 2 or YE), oat extract agar (ISP 3), inorganic salt-starch agar (ISP 4), glycerol-asparagine agar (ISP 5), peptone-yeast agar (ISP 6), and tyrosine agar (ISP 7). Melanin formation was observed on ISP 6 and ISP 7. The color of the substrate hyphae, aerial hyphae and soluble pigment was compared with the ISCC-NBS color chart (Kelly, 1964). SCA2-4^TThe strains were inoculated on YE plates containing NaCl (0-9%, at 1% interval, w/v) and incubated at 28 ℃ for 14 days, and the strains were tested for their salt tolerance during growth. The strain was inoculated on YE plates and SCA 2-4T was incubated at different temperatures (16-46 ℃ C., 1 ℃ C. interval) for 14 days to test the temperature tolerance of the strain growth. SCA 2-4T was inoculated in NB broth (1.0g yeast powder, 0.8g beef extract, 2.0g casein, 10.0g glycerol) at pH 4-10 (1 time interval), cultured at 28 ℃ for 7 days, and the effect of pH on the growth of the strain was evaluated. The physiological and biochemical characteristics were determined by methods of Shirling and Gottlieb (1966) and Williams et al (1983).

Strain SCA2-4^TIs aerobic, gram-positive bacteria. Scanning electron microscopy analysis showed that the strain produced well-developed branch substrate hyphae and aerial hyphae that failed to differentiate into spore chains (fig. 3). Strain SCA2-4^TGrowth was good on all ISP media. Aerial hyphae were formed on ISP2-4 medium, which were off-white, red-brown, light yellow, and earthy yellow, respectively, and no aerial hyphae were produced on the other two media. The matrix hyphae of the hyphae on the ISP2-7 culture medium are dark brown, light yellow, gold, iron gray and dark brown in sequence. The strain SCA 2-4T did not produce soluble pigments on ISP3-5 medium, while brown or light brown soluble pigments were formed on the remaining ISP medium (FIG. 4). These characteristics are consistent with those of Streptomyces. Strain SCA2-4^TCan grow under the conditions of pH of 5.0-8.0 (optimum pH of 7.0), temperature of 21-45 ℃ (optimum temperature of 28 ℃) and NaCl of 0-6% (w/v). Strain SCA2-4^TCan degrade Tween 80, tyrosine, gelatin and nitrate, but can not degrade urea, Tween 20, starch and cellulose. Strain SCA2-4^TCan produce melanin, but not H₂And S. It can utilize L-arabinose, D-fructose,Carbon sources such as D-glucose, inositol, D-raffinose, L-rhamnose, and sucrose, but cellulose, D-mannitol, and D-xylose cannot be used. Strain SCA2-4^TL-phenylalanine, ammonium sulfate, L-hydroxyproline, L (+) -cysteine, histidine, glycine, valine and ammonium oxalate can be used as nitrogen sources, and ammonium acetate, ammonium nitrate, ammonium molybdate tetrahydrate, L-arginine and glutamic acid cannot be used as nitrogen sources. Strain SCA2-4^TSensitive to rifampicin, resistant to ampicillin, chloramphenicol, streptomycin, penicillin-g, gentamicin, nystatin, tetracycline, neomycin sulfate, and kanamycin sulfate.

4 Strain SCA2-4^TDetermination of broad-spectrum antifungal Activity

The strain SCA2-4 was evaluated on Potato Dextrose Agar (PDA) plates using the conventional dot inoculation method (Qi et al, 2019)^TBroad spectrum antifungal activity against 6 plant pathogenic fungi such as Fusarium oxysporum No. 4 (sp. Cubense fungal Race 4, Foc TR4), Fusarium oxysporum No. 1 (Fusarium oxysporum f.sp. Cubense fungal Race 1, Foc TR1), Musa paradisiaca (Falvularia villax), Musa paradisiaca (Curvularia lunata), Musa paradisiaca (Colletotrichum fragrans) and Musa plantaginea (Colletotrichum musae), which are provided by the environmental and plant protection research institute of the Chinese academy of agricultural sciences. Inoculating one and four test fungi with the diameter of 5mm and SCA2-4 at the central point and four symmetrical points of the PDA plate respectively^TThe hyphal mass of the strain. Three replicates were set for each pathogen tested. Plates inoculated with only the pathogen to be tested served as controls. The culture dish is placed at 28 ℃, and the diameter of the target pathogenic bacteria colony is measured by a cross method after the control culture dish is covered with hyphae. The bacteriostatic zone and the bacteriostatic rate are calculated according to the following formula: inhibition zone is C-T, growth inhibition rate is [ (C-T)/C]X 100%, where C and T represent the average diameter of the tested pathogen colonies in the control and treated plates, respectively (Aghighi et al, 2004).

The results show that strain SCA2-4^TFor Fusarium oxysporum No. 4 physiological race, Fusarium oxysporum No. 1 physiological race, Banana Leptosphaeria maculata, Banana Botrytis cinerea, strawberry anthraxThe bacteria, banana anthracnose bacteria and the like have good bacteriostatic activity. Strain SCA2-4 compared to control test pathogen growth diameter (85.00 mm. + -. 0.00)^TThe bacteriostatic bands for Fusarium oxysporum No. 4 physiological race, Fusarium oxysporum No. 1 physiological race, Musa paradisiaca, Musa cinerea, strawberry colletotrichum and Musa paradisiaca are 53.17 + -2.84 mm, 37.83 + -0.29 mm, 58.83 + -2.02 mm, 52.83 + -1.26 mm, 55.67 + -3.79 mm and 53.83 + -1.76 mm respectively. The hypha inhibition rates were 62.55%, 44.51%, 69.21%, 62.15%, 65.49%, and 63.33%, respectively (fig. 5, table 3). The results show that the strain SCA2-4^THas potential biocontrol value.

TABLE 3 Strain SCA2-4^TBacteriostatic activity against plant pathogenic fungi

5 Strain SCA2-4^TExtraction of crude extract and evaluation of antifungal Activity

SCA2-4^TThe strain was cultured in fermentation medium (15 g corn flour, 10 g glucose, 0.5 g K)₂HPO₄0.5 g NaCl, 0.5 g MgSO₄3 g of beef extract, 10 g of yeast powder, 10 g of soluble starch and 2 g of CaCO₃pH 7.2-7.4) at 150rpm, 28 ℃ for 7 days. Extracting the secondary metabolite in the fermentation liquor by using ethyl acetate (v/v ═ 1: 1). After removing the cells using Whatman No. 1 filter paper, the organic solvent containing the secondary metabolite was collected using a separatory funnel and evaporated using a rotary vacuum evaporator (N-1300, EYLEA, japan). The crude extract was dissolved in 10% dimethyl sulfoxide (DMSO) to a final concentration of 20 mg/ml. The bacteriostatic activity of the crude extract on Foc TR4 was evaluated using the agar well diffusion method described by Tepe et al (2005) after filtration of the crude extract solution through a 0.22- μm sterile filter (Millipore, Bedford, MA, United States). Using a sterilized pipette tip round end (inner diameter 5mm) and piercingFour wells were drilled at four points 26mm from the center on two perpendicular lines across the center of the plate, and 100 μ L of crude extract solution was added to each well, using the same volume of 10% DMSO as a control. A5 mm diameter Foc TR4 mycelium pellet was placed in the center of the plate. The diameter of the growth of Foc TR was measured after incubation at 28 ℃ for 7 days. The experiment was set up in triplicate and the zone of inhibition and the growth inhibition of Foc TR were calculated as described above.

Strain SCA2-4^TThe crude extract has strong bacteriostatic activity on Foc TR4 (figure 6), and the bacteriostatic zone and hypha inhibition rates are respectively 31.83 +/-2.36 mm and 42.47%.

6 Strain SCA2-4^TAnalysis of Minimum Inhibitory Concentration (MIC) of crude extract

The crude extract solution was diluted to a concentration range of 100-0.781. mu.g/ml using a two-fold serial dilution method and analyzed for Minimum Inhibitory Concentration (MIC) of Foc-TR4 using 96-well plates (Nunc MicroWell, untrained; Roskilde, Denmark) as described by Wang et al (2013). Mu.l of fungal culture medium (RPMI) was added to each well, 100. mu.l 1.0X 10⁵CFU/ml Foc-TR4 fungal suspension and 20. mu.l of crude extract dilution. As a negative control, 100. mu.l of 10% DMSO was used instead of the crude extract dilution. Mu.l of linezolid or nystatin as a positive control instead of the crude extract diluent. The culture was carried out at 28 ℃ at 150rpm in a 96-well plate closed with a plastic lid. The growth of Foc-TR4 was visually observed after 24 h. The lowest concentration of the crude extract dilution (clear due to inhibition of visible growth of Foc-TR 4) was recorded as MIC. The MIC of the standard antibiotic was recorded in the same way and the negative control should be turbid. The results show that MIC of crude extract and two standard antibiotics of cycloheximide and nystatin are 6.25. mu.g/ml, 1.563. mu.g/ml and 6.25. mu.g/ml, respectively.

7 Strain SCA2-4^TEffect of crude extracts on spore germination of Foc TR4

Strain SCA2-4 was examined using an optical microscope (Axio Scope A1, Carl ZEISS, Germany) according to the method described in Chen et al (2018)^TEffect of crude extract on spore germination of Foc TR 4. Foc TR4 spores 7 days after inoculation were collected using sterile L-shaped coating rods and sterile distilled water and adjusted to a concentration of 1.0X 10⁵CFU/ml. Diluting 10. mu.l of the crude extractThe release (16 MIC) and an equal amount of spore suspension were dropped into the slide well and mixed well. Three replicates were set up for the experiment. The slide was placed in a wet petri dish and incubated at 28 ℃ for 20 hours. 100 spores were observed per slide and the number of spores germinated (i.e., number of spore germ tubes) was counted. Calculating the germination rate and germination inhibition rate of the spores according to the following formulas:

germination rate is (A or B)/100 x 100%,

the spore germination inhibition rate is (A-B)/A x 100 percent,

wherein A and B are the numbers of germinated spores of the control group and the treated group, respectively.

The results are shown in FIG. 7, strain SCA2-4^TThe crude extract has obvious inhibition effect on the germination of Foc TR4 spores, and compared with the control group, the spore germination rate is 94.33 +/-2.08%, the spore germination rate of the treatment group is 10.33 +/-2.52%, and the inhibition rate is about 89.08%.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.