CN110951645B - Streptomyces hygroscopicus angustmycin subspecies and application thereof in preventing and treating plant oomycetes and fungal diseases and promoting plant growth - Google Patents
Streptomyces hygroscopicus angustmycin subspecies and application thereof in preventing and treating plant oomycetes and fungal diseases and promoting plant growth Download PDFInfo
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Abstract
The invention discloses a streptomyces hygroscopicus angustmycin subspecies strain CQUSa03 with the preservation number: CGMCC No. 18715. Also discloses a biological agent with the strain CQUSa03 as an active ingredient. Also discloses application of the strain CQUSa03 in preventing and controlling plant oomycetes and plant fungal diseases and application in promoting plant growth. Also discloses a preparation method of the liquid microbial inoculum containing the strain CQUSa03, which comprises the following steps: (1) activating strains; (2) seed culture; (3) liquid fermentation: adding the seed culture medium into a fermentation culture medium for fermentation to obtain a liquid microbial inoculum, wherein the formula of the fermentation culture medium is as follows: glucose 4%, soybean cake powder 1%, amino acid powder 0.2%, K2HPO40.5％、KH2PO40.5 percent, NaCl 0.5 percent, ferrous sulfate 0.15 percent, antifoaming agent 0.2 percent and pH 7.2-7.4.
Description
Technical Field
The invention relates to a microorganism and application thereof, in particular to a streptomyces hygroscopicus angustmycin subspecies and application thereof in preventing and treating plant oomycetes and fungal diseases and promoting plant growth.
Background
Potato is a dicotyledonous plant belonging to the genus solanum of the family solanaceae, and is the fourth major grain crop worldwide after rice, wheat, and corn. However, during the growth and development of the potatoes, the potatoes are threatened by more than 30 pests and diseases, which cause serious economic loss, wherein the potato late blight, early blight, blight and dry rot are the most typical diseases.
The potato late blight is an oomycete disease caused by stem and leaf death and tuber rot of potatoes and caused by Phytophthora infestans (Phytophthora infestans), is one of the most destructive diseases in potato production, and can cause large-area morbidity in fields in a short time and cause dead production due to rapid expansion of disease spots under the conditions of low temperature and high humidity. According to the comprehensive evaluation of Food and Agricultural Organization (FAO) of the United nations, the potato late blight is the epidemic plant disease which has the greatest harm, the widest range and the most difficult prevention and control in the world, and causes about 200 hundred million economic losses each year. This disease is the major killer of potato production and is therefore named potato cancer. The potato early blight is mainly the second major potato disease caused by Alternaria solani (Alternaria solani) and occurring on leaves and tubers, which causes leaf withering and tuber browning. The disease is generated and harmed increasingly in north Hebei, inner Mongolia, Heilongjiang, Gansu and Shandong provinces of China, and the yield is reduced to 30%. The potato wilt is a soil-borne fungal disease on potatoes, is widely distributed and commonly occurs in various planting areas throughout the country, and the pathogenic bacteria of the potato wilt mainly comprise Fusarium oxysporum (Fusarium oxysporum) and Verticillium dahliae (Verticillium dahliae). Besides potato wilt, fusarium oxysporum can cause the blight of more than 100 plants such as melons, solanaceae, bananas, cottons, leguminosae, flowers and the like. The host range of the verticillium dahliae is also extremely wide, and 660 plants of 38 families can be harmed, wherein 184 crops and 153 weeds are contained. The potato dry rot is a postharvest disease of potatoes, and the whole tuber is shriveled or becomes dry rot due to pathogenic bacteria infecting the tuber, so that the potato is not edible. There are 9 pathogenic bacteria, of which Fusarium solani (Fusarium solani) is the dominant population and is highly pathogenic.
At present, chemical control is a main means for controlling plant diseases and insect pests, has low cost and quick response, but has great harm to the environment, easily causes drug resistance of pathogenic bacteria and mutation into new physiological races, increases pesticide residue in agricultural products, seriously damages soil and agricultural ecological environment, and hinders implementation of 'potato staple food strategy' and 'fertilizer and pesticide reduction and synergism strategy'. How to control potato diseases and improve the yield and quality of potatoes is always a global challenge. Biological control can significantly overcome the disadvantages of chemical control. However, biological control, as an emerging science, also has many disadvantages, such as: compared with chemical bactericides, the bactericide has lower stability and slow effect. At present, biological control medicines or measures in the market are not widely popularized yet. Therefore, it is necessary to find or discover an ideal biocontrol agent or measure.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a streptomyces hygroscopicus angustmycin subspecies and application thereof in preventing and controlling plant oomycetes and fungal diseases and promoting plant growth.
In order to achieve the above purpose, the invention provides the following technical scheme:
a streptomyces hygroscopicus angustmycin subspecies strain CQUSa03 with the preservation number: CGMCC No. 18715.
A biological agent contains strain CQUSa03 as active component.
The application of the strain CQUSa03 in preventing and treating plant oomycetes and plant fungal diseases.
The plant oomycete disease refers to a plant disease caused by phytophthora infestans, and the plant fungal disease refers to a plant disease caused by alternaria solani or fusarium oxysporum or verticillium dahliae or fusarium solani.
The plant oomycete disease refers to late blight of solanaceae plants, and the plant fungal disease refers to early blight of solanaceae plants, wilt of solanaceae plants or dry rot of solanaceae plants.
Application of the strain CQUSa03 in promoting plant growth.
In the above technical scheme, the plant is a solanaceae plant.
A preparation method of a liquid microbial inoculum containing a strain CQUSa03 comprises the following steps:
(1) activating strains: inoculating the strain CQUSa03 to an oat solid culture medium, and culturing and activating to obtain an activated strain;
(2) seed culture: inoculating the activated strain obtained in the step (1) into an ISP liquid culture medium for culture to obtain a seed culture medium;
(3) liquid fermentation: adding the seed culture medium obtained in the step (2) into a fermentation culture medium for fermentation to obtain a liquid microbial inoculum, wherein the formula of the fermentation culture medium is as follows: glucose 4%, soybean cake powder 1%, amino acid powder 0.2%, K2HPO40.5％、KH2PO40.5 percent, NaCl 0.5 percent, ferrous sulfate 0.15 percent, antifoaming agent 0.2 percent and pH 7.2-7.4.
The specific conditions of the liquid fermentation in the step (3) are as follows: and (3) adding the seed culture medium obtained in the step (2) into a fermentation culture medium according to the volume percentage of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.07Mpa, the stirring speed is 100rpm, the oxygen introduction amount is more than 60%, and the fermentation time is 7 d.
The formula of the oat solid culture medium in the step (1) is as follows: 2% of oat and 2% of agar powder; the ISP liquid culture medium formula in the step (2) is as follows: maltose extract 1%, yeast extract 0.4%, D-glucose 0.4%, and pH 7.0-7.4.
The invention has the beneficial effects that: the screened streptomyces hygroscopicus strain CQUSa03 has extremely strong inhibition effect on potato late blight and has good control effect on potato late blight; has strong inhibiting effect on four pathogenic fungi of the potato; has obvious growth promoting effect on potato and tomato seedlings. The strain CQUSa03 has simple culture conditions, easy storage and easy industrial production, and has good development and application prospects; the preparation method of the liquid microbial inoculum of the strain CQUSa03 provided by the invention has the advantages of simple process, no need of valuable raw materials for a fermentation medium, low cost and easy popularization.
Drawings
FIG. 1 is a graph showing colony growth and phylogenetic tree analysis of strain CQUSa03, wherein A is colony growth and B is phylogenetic tree analysis.
FIG. 2 shows the bacteriostatic effect of strain CQUSa03 in the opposite culture of different physiological races of potato late blight pathogenic bacteria on indoor plates.
FIG. 3 shows the bacteriostatic effect of CQUSa03 strain cultured on indoor plates of four pathogenic fungi of potato.
FIG. 4 is an observation picture of the inhibition effect of CQUSa03 liquid microbial inoculum on potato leaves and potato blocks infected by potato late blight pathogenic bacteria.
FIG. 5 is a graph showing the effect of CQUSa03 liquid fungicide on the growth process promotion of potato and tomato plants.
Detailed Description
Example 1 isolation and characterization of the strains
First, separation and screening of strain CQUSa03
In a potato planting area in Wuxi county, Chongqing, proper places are selected to collect rhizosphere soil at 5-20mm according to the occurrence condition of late blight, and the sampling time, the sampling places, the vegetation and the disease occurrence condition are marked. Separating on a Gao's first culture medium by a soil dilution method, culturing at a constant temperature of 28 ℃ for 48h, and selecting and storing a single colony as a strain to be detected. Inoculating pathogenic phytophthora in the center of rye culture medium by using a plate confronting method, culturing pathogenic bacteria at 20 ℃ for 10 days, and symmetrically inoculating the bacteria to be tested around the center, repeating the steps for 3 times for each strain, and observing whether an inhibition zone appears. The strain with the best antagonistic effect is named CQUSa 03.
II, identification of strain CQUSa03
Firstly, 16S rDNA sequence analysis is carried out on a strain CQUSa03, the 16S rDNA sequence of the strain is submitted to GeneBank for Blast comparison on line, and the strain is found to be in the same evolutionary branch with Streptomyces hygroscopicus (Streptomyces angustmyces) and have homology of more than 99 percent (a phylogenetic tree is shown in figure 1B). The 16S rDNA nucleotide sequence of the strain is shown in SEQ ID No. 1. The CQUSa03 strain was then subjected to morphological (colony morphology is shown in FIG. 1A) and physiological and biochemical analyses. By combining the sequencing result and physiological and biochemical analysis, the bacterial sample CQUSa03 separated from soil preserved in the experiment is Streptomyces angustmyces subsp.
The strain CQUSa03 is preserved in China general microbiological culture Collection center (CGMCC for short, address No. 3 Xilu No.1 of the North Chen Yang district in Beijing), the preservation registration number is CGMCC No.18715, the preservation date is 2019, 10 and 21 days, the classification is Streptomyces angustmyceticus, and the detection result of CQUSa03 is survival when CGMCC is handled.
Example 2 CQUSa03 experiments on the confrontation of potato late blight pathogen and other pathogenic fungi
Preparation of a rye culture medium: 60g of rye flour is weighed and soaked in 500mL of sterile water for 36 hours. Filtering to obtain filtrate 1, and storing for later use; filtering the filter residue in water bath at 55 ℃ for 3 hours to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2 uniformly, sterilizing at 121 ℃ for 10 minutes, and filtering to obtain a filtrate 3; adding 20g of sucrose and 12g of agar into the filtrate 3, dissolving and mixing uniformly, metering to 1L with sterile water, and sterilizing for 20 minutes at 121 ℃.
Preparing a PDA culture medium: adding 20g of glucose and 15g of agar powder into 200g of the boiled filtrate of the fresh potatoes, then fixing the volume to 1L, and sterilizing for 15 minutes at 121 ℃.
There are 3 types of phytophthora pathogenic bacteria physiological races: a1, A2 and auto-breeding. The international standard strain T30-4 used in the patent belongs to A1 and is derived from the American late blight; 88069 and 3928A were of the self-bred type, pg002 was of the A2 type, and these 3 strains were isolated from the field. The physiological races of different potato late blight pathogenic bacteria are inoculated to the center of a rye culture medium plate, round agar blocks of streptomyces hygroscopicus CQUSa03 are symmetrically inoculated around the physiological races, and the plate which is not inoculated with the streptomyces hygroscopicus is a blank control group. After culturing at 20 ℃ in the dark for 10 days, the colony diameter and the inhibition rate were measured, and the inhibition rate was (control colony diameter-treated colony diameter)/(control colony diameter-0.6). The diameter of the mass of pathogenic bacteria used for inoculation was 0.6 cm. The bacteriostatic effect of CQUSa03 on different physiological races of potato late blight pathogenic bacteria is shown in figure 2. Results of bacteriostatic rate are shown in table 1: compared with the control group, the potato late blight pathogenic bacteria T30-4, 88069, 3928A and pg002 are inhibited by CQUSa03 strain very significantly (p <0.001 ×), with the inhibition rate reaching 100%.
TABLE 1 inhibition rate of CQUSa03 on different physiological races of potato late blight pathogenic bacteria
p<0.001***。
Streptomyces hygroscopicus CQUSa03, potato early blight pathogen Alternaria solani (Alternaria solani), potato blight pathogen Fusarium oxysporum (Fusarium oxysporum), Verticillium dahliae (Verticillium dahliae) and potato dry rot pathogen Fusarium solani (Fusarium solani) are subjected to opposite culture for 5 days at 28 ℃ on a potato glucose medium (PDA medium), the growth conditions of the pathogenic fungi in a control group and a treatment group are observed, and the colony diameter and the bacteriostasis rate are measured, wherein the bacteriostasis rate is (the control colony diameter-the treatment colony diameter)/(the control colony diameter-0.6). The diameter of the mass of pathogenic bacteria used for inoculation was 0.6 cm. As shown in FIG. 3, the colony growth of potato early blight pathogenic bacteria Alternaria solani, potato blight pathogenic bacteria Fusarium oxysporum and Verticillium dahliae, and potato dry rot pathogenic bacteria Fusarium solani were significantly inhibited by CQUSa03 strain. The results of the statistical bacteriostasis rate are shown in table 2.
TABLE 2 inhibition ratio of CQUSa03 to different pathogenic fungi of potato
p<0.01**，p<0.001***。
Example 3 liquid microbial inoculum containing strain CQUSa03 and preparation method thereof
The liquid microbial inoculum containing the strain CQUSa03 provided by the embodiment is prepared by the following method:
(1) activating strains: the strain CQUSa03 is inoculated on an oat solid culture medium and cultured for 7 days at 28 ℃. The formula of the oat solid culture medium is as follows: 2% of oat and 2% of agar powder.
(2) Seed culture: inoculating the activated strain into ISP liquid culture medium, culturing at 28 deg.C and 200rpm for 5d to obtain seed culture medium. The ISP liquid culture medium comprises the following components: maltose extract 1%, yeast extract 0.4%, D-glucose 0.4%, and pH 7.0-7.4.
(3) Liquid fermentation: adding the seed culture medium obtained in the step (2) into a fermentation culture medium according to the volume percentage of 10 percent, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.07Mpa, the stirring speed is 100rpm, and the oxygen introduction amount is>60% and fermentation time 7 d. The formula of the fermentation medium is as follows: glucose 4%, soybean cake powder 1%, amino acid powder 0.2%, K2HPO40.5％、KH2PO40.5 percent, NaCl 0.5 percent, ferrous sulfate 0.15 percent, antifoaming agent 0.2 percent and pH 7.2-7.4.
Example 4 indoor control Effect of liquid microbial inoculum on Potato late blight and growth promotion Effect on plants
The microbial inoculum for experiments: liquid inoculum containing streptomyces hygroscopicus CQUSa03 prepared in example 3, a clear water control.
Soaking potato leaves and tubers in a liquid microbial inoculum containing CQUSa03 for half an hour, wiping the liquid microbial inoculum, and inoculating strong pathogenic bacteria T30-4 of late blight on the potato leaves and the tubers. The inoculated leaves and potato blocks are cultured for 5 days under the conditions of 20 ℃ and 75% relative humidity, and the leaves and potato blocks treated by clear water are blank control, so that the indoor control effect of the liquid microbial inoculum on the potato late blight is judged. After 5 days of culture, the result is shown in figure 4, and the potato blocks and leaves soaked by the CQUSa03 liquid microbial inoculum are not infected with diseases, which indicates that the streptomyces hygroscopicus CQUSa03 can effectively inhibit the potato blocks and leaves from being infected by the potato late blight bacteria.
In addition, the liquid microbial inoculum containing CQUSa03 and nutrient soil are mixed uniformly according to the ratio of 1:3 to plant potato tubers and tomato seeds (treatment group), and the blank control group (CK) is mixed with clear water. Each treatment was repeated 3 times for 10 plants. The treated plants are placed in a light greenhouse, the growth conditions of the plants are investigated after the treated plants are cultured for 4 weeks at 25 ℃ (shown in figure 5), the average plant height, the average leaf length and the average leaf width of potato seedlings and tomato seedlings are measured, the statistical result is shown in table 3, and the result shows that the liquid microbial inoculum containing CQUSa03 has a remarkable promoting effect on the growth of potatoes and tomatoes.
TABLE 3 growth promoting effect of CQUSa03 liquid microbial inoculum on potato and tomato plants
p<0.01**，p<0.05*。
Sequence listing
<110> university of Chongqing; institute of urban agriculture
<120> streptomyces hygroscopicus angustmycin subspecies and application thereof in preventing and treating plant oomycetes and fungal diseases and promoting plant growth
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1427
<212> DNA
<213> Artificial sequence ()
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tgacgtgacc ttcgacgatt ccctcccaca aggggttggg ccaccggctt cgggtgttac 60
cgactttcgt gacgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcagcaa 120
tgctgatctg cgattactag caactccgac ttcatggggt cgagttgcag accccaatcc 180
gaactgagac cggctttttg agattcgctc cacctcgcgg tatcgcagct cattgtaccg 240
gccattgtag cacgtgtgca gcccaagaca taaggggcat gatgacttga cgtcgtcccc 300
accttcctcc gagttgaccc cggcagtctc ctgtgagtcc ccatcacccc gaagggcatg 360
ctggcaacac agaacaaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac 420
gagctgacga cagccatgca ccacctgtac accgaccaca agggggaccc tgtctccagg 480
gttttccggt gtatgtcaag ccttggtaag gttcttcgcg ttgcgtcgaa ttaagccaca 540
tgctccgctg cttgtgcggg cccccgtcaa ttcctttgag ttttagcctt gcggccgtac 600
tccccaggcg gggaacttaa tgcgttagct gcggcacgga cgacgtggaa tgtcgcccac 660
acctagttcc caacgtttac ggcgtggact accagggtat ctaatcctgt tcgctcccca 720
cgctttcgct cctcagcgtc agtatcggcc cagagatccg ccttcgccac cggtgttcct 780
cctgatatct gcgcatttca ccgctacacc aggaattccg atctccccta ccgaactcta 840
gcctgcccgt atcgaatgca gacccggggt taagccccgg gctttcacat ccgacgtgac 900
aagccgccta cgagctcttt acgcccmata attccggaca acgcttgcgc cctacgtatt 960
accgcggctg ctggcacgta gttagccggc gcttcttctg caggtaccgt cactctcgct 1020
tcttccctgc tgaaagaggt ttacaacccg aaggccgtca tccctcacgc ggcgtcgctg 1080
catcaggctt tcgcccattg tgcaatattc cccactgctg cctcccgtag gagtctgggc 1140
cgtgtctcag tcccagtgtg gccggtcgcc ctctcaggcc ggctacccgt cgtcgccttg 1200
gtaggccatc accccaccaa caagctgata ggccgcgggc tcatccttca ccgccggagc 1260
tttccaccac cagaccatgc ggtcggaggt cgtatccggt attagacccc gtttccaggg 1320
cttgtcccag agtgaagggc agattgccca cgtgttactc acccgttcgc cactaatccc 1380
ctcccgaagg aggttcatcg ttcgactgca tggtaagcac tcgtact 1427
Claims (10)
1. A Streptomyces hygroscopicus subspecies angustmyces strain CQUSa03, characterized by the accession number: CGMCC No. 18715.
2. A biological agent comprising the strain CQUSa03 of claim 1 as an active ingredient.
3. The use of the strain cqussa 03 according to claim 1 for controlling plant oomycetes and plant fungal diseases.
4. The use of the strain CQUSa03 according to claim 3 for controlling plant oomycetes diseases and plant fungal diseases, wherein the plant oomycetes diseases refer to plant diseases caused by phytophthora infestans, and the plant fungal diseases refer to plant diseases caused by alternaria solani or fusarium oxysporum or verticillium dahliae or fusarium solani.
5. The application of the strain CQUSa03 in controlling plant oomycete diseases and plant fungal diseases according to claim 3, wherein the plant oomycete diseases refer to late blight of solanaceae plants, and the plant fungal diseases refer to early blight of solanaceae plants, blight of solanaceae plants or dry rot of solanaceae plants.
6. Use of the strain cqussa 03 according to claim 1 for promoting plant growth.
7. Use of the strain cqussa 03 according to claim 6 for promoting plant growth, wherein the plant is a solanaceous plant.
8. A method for preparing a liquid microbial inoculum containing the strain CQUSa03 of claim 1, comprising the steps of:
(1) activating strains: inoculating the strain CQUSa03 to an oat solid culture medium, and culturing and activating to obtain an activated strain;
(2) seed culture: inoculating the activated strain obtained in the step (1) into an ISP liquid culture medium for culture to obtain a seed culture medium;
(3) liquid fermentation: adding the seed culture medium obtained in the step (2) into a fermentation culture medium for fermentation to obtain a liquid microbial inoculum, wherein the formula of the fermentation culture medium is as follows: glucose 4%, soybean cake powder 1%, amino acid powder 0.2%, K2HPO40.5％、KH2PO40.5 percent, NaCl 0.5 percent, ferrous sulfate 0.15 percent, antifoaming agent 0.2 percent and pH 7.2-7.4.
9. The method according to claim 8, wherein the specific conditions for the liquid fermentation in step (3) are: and (3) adding the seed culture medium obtained in the step (2) into a fermentation culture medium according to the volume percentage of 10%, wherein the fermentation temperature is 28 ℃, the tank pressure is 0.07Mpa, the stirring speed is 100rpm, the oxygen introduction amount is more than 60%, and the fermentation time is 7 d.
10. The method according to claim 8, wherein the formulation of the oat solid medium in step (1) is: 2% of oat and 2% of agar powder; the ISP liquid culture medium formula in the step (2) is as follows: maltose extract 1%, yeast extract 0.4%, D-glucose 0.4%, and pH 7.0-7.4.
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| 紫黑吸水链霉菌Streptomyces violaceoniger hygroscopicus 发酵代谢产物对几种常见果蔬病原菌的拮抗活性;段学辉等;《江西农业大学学报》;20040430;第26卷(第2期);第266-269页 * |
| 链霉菌( Streptomyces spp . ) 对几种蔬菜病原菌的拮抗作用;魏艳敏等;《河北农业大学学报》;20000730;第23卷(第3期);第65-68页 * |
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