The invention content is as follows:
the invention aims to: aiming at the problems that the root rot of panax notoginseng is seriously generated at present, the area is enlarged year by year, the prevention and treatment measures such as pesticide, crop rotation, chemical treatment of soil and the like are not ideal in effect, and environmental pollution is caused, the biocontrol bacterium for efficiently preventing and treating the root rot of panax notoginseng and the application thereof are provided.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a biocontrol endophytic fungus Z1-1031 for preventing and treating root rot of panax notoginseng, which is identified as Penicillium sp through morphological and molecular biological research.
The preparation process of the biological control endophytic fungus penicillium comprises the following steps:inoculating the strain into a PDA culture medium plate, and performing inverted culture at 28 ℃ for 7-10 days to activate the strain. The activated strain was prepared into a spore suspension using distilled water, and the spore concentration was counted under a microscope to be about 108cfu/ml. 2ml of spore suspension was inoculated into the fermentation medium PDB. Culturing at 26-28 deg.C and 120-150r/min for 7-10 days.
The application method of the biocontrol endophytic fungi in preventing and treating the root rot of the panax notoginseng comprises the following steps:
can be used by irrigating root at adult stage of Notoginseng radix, counting under microscope, and diluting spore solution to 106cfu/ml concentration, 50ml per strain. The application is continued for 2-3 times, each time with 10-15 days interval. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
The invention has the beneficial effects that:
the screened biological control endophytic fungus penicillium fermentation liquor can obviously inhibit the growth of panax notoginseng pathogenic fungi fusarium solani, cylindracea, alternaria, and the like, and promote the growth of panax notoginseng plants. Because the biological preparation is adopted, a series of problems caused by the use of chemical pesticides do not exist, and the biological preparation is greatly beneficial to reducing agricultural pollution and effectively preventing and treating the root rot of the panax notoginseng. The biocontrol endophytic fungi are separated from the roots of healthy three-year-old panax notoginseng, also exist in rhizosphere soil of panax notoginseng, are ecologically compatible with the panax notoginseng soil, and are beneficial to fully exerting the advantages of strains.
The biocontrol endophytic fungi strain has the advantages of simple culture conditions, stable heredity, easy industrial production expansion and good development and application prospects.
The Penicillium (Penicillium sp.) of the invention is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation addresses are as follows: west road No.1, north west of the morning area, beijing, institute of microbiology, china academy of sciences, date of deposit: number of deposit at 07/2017, number: CGMCC No. 14134.
The specific implementation mode is as follows:
for a better understanding of the present invention, the following figures and examples further illustrate, but are not to be construed as limiting, the experimental procedures set forth in the following examples are conventional and are not intended to be limiting.
Example 1 isolation of Panax notoginseng endophytic fungi
The specific process is as follows:
1 Collection of samples
Healthy three-year-old panax notoginseng plants are collected from a Miao Xiang planting base in Wenshan county of Yunnan province, are subpackaged and marked, and are put into freshness protection bags to be brought back to a laboratory for later use.
2 isolation of endophytic fungi
Plant washing procedure: cleaning soil on the surface of a plant under tap water, slightly drying the soil, cutting the plant into small blocks, putting the small blocks into a clean triangular flask, ultrasonically cleaning the small blocks repeatedly until the cleaned water becomes clear, inverting the cleaned water, and emptying the cleaned water.
Plant surface disinfection procedure: 0.1% Tween 20, 1min (soak, treat with beaker, same plant with a flask, beaker require sterilization) → treatment of root sample with sodium hypochlorite solution with 5% available chlorine content 6min → treatment of sterilized 2.5% sodium thiosulfate solution 10min → sterile water rinse at least 3 times → treatment of root sample with 75% ethanol (with sterile water) 6min → sterile water rinse 5 times. The surface sterilized samples were placed in sterile petri dishes (with sterile filter paper applied) and placed on a clean bench to dry (blow dry, no water visible, overnight in clean bench).
The surface disinfection effect is detected: taking 0.2mL of water for washing the sample in the last time, coating the water on a separation culture medium to be used, placing the flat plate at 28 ℃ for more than 2 weeks, and detecting whether the colonies grow out.
Crushing plants: placing the sample in a sterile stirrer (sterilization method same as biological identification plate, soaking with 75% alcohol for 5min, ultraviolet irradiation for 30min), crushing (beating 5s and stopping 5s when crushing, shaking the stirring cup to make the plant under the blade and not adhere to the wall), placing in a sterile plate, heating at 50 deg.C for 20min, picking the sample with sterile bamboo stick, and spreading on a separation plate. After incubation at 28 ℃ and observation for 7 days, different single colonies were picked and streaked for purification. Transferring the purified strain to a PDA culture medium for culture, storing at 4 ℃ for later use, and co-separating to obtain 21 endophytic fungi.
Example 2 identification of Strain Z1-1031
1 morphological feature Observation
The strain Z1-1031 cultured for 7-10 days was opened to a petri dish lid and photographed under a black background. The colony is gray green, and the edge is regular and is grayish white. The surface was velvet-like hyphae, and yellow pigment was produced in the medium (FIG. 1). A small amount of mycelium was taken out and made into a chip, and morphological characteristics of the mycelium and spores were observed under an optical microscope. The hyphae have transverse septa, the conidiophores of the hyphae are subjected to multiple branching to generate a plurality of asymmetric small stalks, the shapes of the small stalks are like brooms, the conidiophores are spherical and smooth (figure 2), and the strain Z1-1031 is preliminarily identified as the penicillium group according to the morphological characteristic analysis.
2 ITS sequence analysis
After extracting the genomic DNA of the strain Z1-1031 by a liquid nitrogen grinding method, adopting a primer: ITS 4: 5'-TCCTCCGCTTATTGATATGC-3', respectively; ITS 5: 5'-GGAAGTAAAAGTCGTAACAAGG-3', PCR amplification was performed. After gel electrophoresis, the gel was sent to bioengineering (Shanghai) Limited company for sequencing, and the total length of the sequence was 566bp (see SEQ). The obtained sequence was submitted to GenBank database for BLAST analysis and alignment, and the strain having high homology to Z1-1031 was found to belong to the genus Penicillium, and the strain sequence number KU360251 was submitted to NCBI.
Example 3 fermentation of Strain Z1-1031 and treatment of fermentation broth
Spore suspension 10 of the above strain8cfu/mL is inoculated in PDB liquid culture medium, the inoculation amount is 1-2mL, the temperature is 26-28 ℃, the fermentation amount is 100 mL/bottle after culturing for 7-10 days at 120-150 r/min. Separating the bacterial balls from the fermentation liquor by using gauze, extracting the fermentation liquor for 3 times by using ethyl acetate with the same volume, collecting extract liquor, and performing decompression and pumping drying to finally obtain the fermentation liquor with the dry weight of 0.1-0.2 g/bottle. The dried material is dissolved with a small amount of methanol and removed to obtain hairCrude extract of the yeast liquid.
Example 4 Strain Z1-1031 metabolite bacteriostasis test
Antagonism of pathogenic bacteria of panax notoginseng (Fusarium solani, Cylindrocarpon dynum, Alternaria panax) was tested with the Z1-1031 metabolite using 96-well plate turbidimetry. The final concentration of the extract of the fermentation broth is 0.1 mg/ml. Washing spores from slant to obtain spore suspension, counting with blood count plate, and diluting to spore concentration of 107cfu/ml. 0.4ml of the diluted spore suspension is sucked and added into 40ml of liquid PDB culture medium, and the mixture is mixed evenly to form suspension. Blank Control (CK): 198. mu.l of suspension; DMSO control group: 198. mu.l suspension +2ul DMSO; positive control group: 198. mu.l suspension + 2. mu. l G418; experimental groups: 198. mu.l suspension + 2. mu.l broth Extract (Extract), three replicates per group. The 96-well plate was incubated at 28 ℃ in an incubator, and OD was measured at 600nm after 48 hours. As shown in FIG. 3, the fermentation broth extract showed a killing effect on (Fusarium solani, Cylindrocarpon dynum, Alternaria panax), which is significantly different from the blank control group. The result of the activity screening experiment shows that the fermentation liquor of the strain Z1-1031 has better antagonistic action on pathogenic bacteria of the panax notoginseng root rot.
Example 5 crude extract of fermentation broth of Strain Z1-1031 and Strain replanting experiment
The replant strain Z1-1031 is inoculated to PDA culture medium and cultured for 7 days at 28 ℃. Preparing bacterial suspension with sterile water, and diluting to 10%6cfu/mL, and 50mL of root irrigation is carried out on each panax notoginseng seedling. The crude extract of the fermentation liquor is dissolved by water to prepare a solution of 1mg/L, and each notoginseng is irrigated with 50mL of roots. Each treatment is divided into three groups, and 200 panax notoginseng seedlings are inoculated in each group and planted on new soil (0 year soil). The seedling survival rate of the panax notoginseng seedlings is recorded every 1 month, and 4 months are observed in total. Is assisted by medicinal plant research institute of Wenshan pseudo-ginseng research institute and Yunnan province agricultural science research institute. The results are shown in FIG. 4. From the first month to the fourth month, the seedling survival rate of the control group is reduced from 92% to 55%, and the application fermentation liquor group and the replant bacteria group are respectively 100% to 90% and 100% to 88%. The experimental result shows that the strain Z1-1031 fermentation liquor and thalli can obviously reduce the death rate of panax notoginseng, improve the seedling survival rate and show good application potential.
Sequence listing
<110> Shenyang university of pharmacy
<120> biocontrol endophytic fungus and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 566
<212> DNA
<213> Penicillium purpureum (Penicillium janthinellum)
<220>
<221> allele
<400> 1
tgcggaggac attaccgagt gagggccctc tgggtccaac ctcccacccg tgtttatcat 60
acctagttgc ttcggcgggc ccgccgtcat ggccgccggg gggcacccgc ccccgggccc 120
gcgcccgccg aagccccccc tgaacgctgt ctgaagattg ctgtctgagc gattagctaa 180
atcagttaaa actttcaaca acggatctct tggttccggc atcgatgaag aacgcagcga 240
aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca tcgagtcttt gaacgcacat 300
tgcgccccct ggtattccgg ggggcatgcc tgtccgagcg tcattgctgc cctcaagcac 360
ggcttgtgtg ttgggccccc gccccccggc tcccgggggg cgggcccgaa aggcagcggc 420
ggcaccgcgt ccggtcctcg agcgtatggg gcttcgtcac ccgctctgta ggcccggccg 480
gcgcccgccg gcgacccccc tcaatctttc tcaggttgac ctcggatcag gtagggatac 540
ccgctgaact taagcatatc aataag 566