CN108949583B

CN108949583B - Trichoderma asperellum strain Sanju-19 and application thereof

Info

Publication number: CN108949583B
Application number: CN201810849198.9A
Authority: CN
Inventors: 仲乃琴; 钞亚鹏; 杨敬; 刘宁; 赵盼; 骆毛喜; 林克明; 余劲聪; 宋双伟; 郝辽洋; 熊悯梓
Original assignee: FUJIAN SANJU BIOLOGICAL SCIENCE & TECHNOLOGY CO LTD
Current assignee: FUJIAN SANJU BIOLOGICAL SCIENCE & TECHNOLOGY CO LTD
Priority date: 2018-07-28
Filing date: 2018-07-28
Publication date: 2021-05-18
Anticipated expiration: 2038-07-28
Also published as: CN108949583A

Abstract

The invention discloses a Trichoderma asperellum (Trichoderma asperellum) strain Sanju-19 which is preserved in China general microbiological culture Collection center (CGMCC for short) in 6, 14 and 2018, with the preservation number of CGMCC No.15870 and the preservation unit address of: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The strain can inhibit the growth of streptomyces pyocutaneous infection, streptomyces acidocaldarius and streptomyces albidoflavus, and has effective prevention and treatment effects on potato scab.

Description

Trichoderma asperellum strain Sanju-19 and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an application of trichoderma asperellum in a microbial inoculum for preventing and treating potato scab.

Background

The potato scab is a plant disease damaging potato tubers. The appearance of the disease is that the surface of the tuber is nearly round to irregular, suberized scab-like light brown scab or plaque, and the hand feel is rough. Generally, two types of disease symptoms are developed, including a reticulated lesion and a lacerated lesion. Generally, although the disease spots are only limited to the skin layer, the quality and the yield of the damaged potato blocks can be reduced, the damaged potato blocks are not storage-resistant, the appearance of the damaged potato blocks is not elegant, the commodity grade is greatly reduced, and certain economic loss is caused. In China, the incidence of diseases is low, the area of damage is small, and therefore people do not pay attention to the disease. However, in recent years, the disease incidence area in inner Mongolia, Hebei, Shandong and other areas is increasing, and the economic loss caused by the disease incidence area is also considerable. Therefore, scientific control of such diseases is urgently required.

According to the long-term separation and identification research on pathogenic microorganisms, pathogenic bacteria causing potato scab are mainly streptomyces scabicus and the like. And the pathogenic bacteria belong to conditional pathogenic bacteria, namely the pathogenic bacteria easily grow and cause potato diseases under the alkaline condition of the soil environment, and rarely appear in the acidic soil environment.

The control method mainly comprises the following steps: (1) selecting seed potatoes without diseases; (2) applying organic fertilizer or green manure more times can partially inhibit diseases; (3) performing rotation with Cucurbitaceae, Leguminosae, and Liliaceae vegetables for more than 5 years; (4) selecting a vegetable field with good water retention for planting, and watering in time when meeting drought in a potato bearing period; (5) and chemical prevention: some chemical pesticides can be used for preventing and treating scab; (6) biological prevention and treatment method: the method screens microorganisms with good inhibition effect on scab pathogenic bacteria and strong planting capability, and is applied in the potato seed production process and the farmland planting process to achieve the effect of inhibiting the growth of the pathogenic bacteria.

Disclosure of Invention

In order to effectively prevent and control potato scab, the invention provides a Trichoderma asperellum (Trichoderma asperellum) strain Sanju-19 which is preserved in China general microbiological culture Collection center (CGMCC for short) in 2018, 6 months and 14 days, and the preservation number is as follows: CGMCC No.15870, the preservation unit address is: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

The second purpose of the invention is to provide a microbial agent, the active component of which is trichoderma asperellum strain Sanju-19.

It is a third object of the present invention to provide a method for preparing a microbial inoculant,

the fourth purpose of the invention is to provide the application of the strain Sanju-19 and the microbial inoculum thereof, which can inhibit the growth of Streptomyces scabies (Streptomyces scabeii), Streptomyces acidosis and Streptomyces albidoflavus (Streptomyces albicans) and have effective prevention and treatment effects on potato scabies.

Description of the drawings:

FIG. 1 is a graph showing the bacteriostatic effect of the strain Sanju-19 on Streptomyces scabies 4.0076.

FIG. 2 is a graph showing the bacteriostatic effect of the strain Sanju-19 on Streptomyces scabies 4.1765.

FIG. 3 is a graph showing the bacteriostatic effect of the strain Sanju-19 on Streptomyces scabies 4.1789.

FIG. 4 is a graph showing the bacteriostatic effect of the strain Sanju-19 on Streptomyces albidoflauvs 4.3598.

FIG. 5 is a diagram showing the bacteriostatic effect of the three strains, namely, the three-torch strain 19, on three mixed strains of Streptomyces scabies 4.0076, Streptomyces scabies acidovorans 4.1789 and Streptomyces albidoflavus 4.3598.

FIG. 6 shows the bacteriostatic effect of the three strains of Sanju-19, Streptomyces scabies 4.0076, Streptomyces scabies acidovorus 4.1789 and Streptomyces albidoflavus 4.3598 in co-culture.

FIG. 7 strain three torch-19 growth scheme.

The specific implementation mode is as follows:

1. obtaining, separating and identifying strain

The strain is obtained by collecting soil samples from Fujian banana diseased soil, separating and identifying.

Colony morphology: the culture medium is fast in growth in a wort culture medium, the culture is carried out for 5 days under the dark condition at the temperature of 25 ℃, the diameter of a bacterial colony is 60-65mm, the bacterial colony is white, and the bacterial colony is light grey green and flocculent due to spore production at the later stage; the back is light brown without water soluble pigment. Conidium is produced in large quantity, and the conidium producing area is distributed uniformly. Conidiophores are tall, smooth or slightly rough in wall, usually produce branches in opposite positions, 2.5-5.0um wide; the produced phialides are few, a plurality of phialides cluster on the phialides or the branches thereof, the flask-shaped neck is bent, and the diameter is 5.0-16.7 x 2.5-3.5 um; conidia are wide oval or nearly spherical, the wall is smooth or slightly rough, and the diameter is 2.1-4.5 um; chlamydospores are common.

The molecular identification method comprises the following steps: the complete sequence of ITS1/5.8SrRNA, ITS2 and 28S region sequence fragment analysis identify Trichoderma asperellum.

2. The fungus culture method comprises the following steps:

trichoderma asperellum strain Sanju-19 was activated 3 times on potato solid medium. Then, the 1-ring culture was performed on potato solid plate medium at 28 ℃ for 5 days by shaking.

3. The application and the effect of the strain are as follows:

(1) the scab antagonistic bacteria screening material and method:

the tested pathogens are four kinds of Streptomyces scabies (Streptomyces scabeii) 4.0076 and 4.1765, Streptomyces acidosis scabies (Streptomyces acidiscabis) 4.1789 and Streptomyces albidoflavus (Streptomyces albicans) 4.3598.

Potato juice Medium (g/L): 1L of potato extract, 20g of glucose and KH₂PO₄ 3g，MgSO₄.7H₂O1.5 g, vitamin B1 in trace amount, pH6.0, and agar 15 g.

The method for culturing pathogenic bacteria comprises the following steps: culturing the four pathogenic bacteria on potato culture medium for 4-5 days at 28 deg.C; scraping spores and mycelia with 0.2ml sterile water to obtain pathogen suspension with a certain concentration; the plate surface was covered with hyphae after growth when the plate was coated in a volume of 100 ul.

(2) The bacteriostatic test method comprises the following steps:

100ul of the four pathogenic bacteria are respectively coated on different Plates (PDA), and trichoderma spores are locally inoculated on the plates. Culturing at 28 ℃. Observed after 3 days. Through four tests of Streptomyces scabies (Streptomyces scabeii) 4.0076 and 4.1765, Streptomyces acidiferus (Streptomyces acidiscabis) 4.1789 and Streptomyces albidoflavus (Streptomyces albidoflavus)4.3598, the fungus is found to be capable of effectively inhibiting the growth of pathogenic microorganisms in the growth process. As a result: the fungus is effective on 4 pathogenic bacteria (FIG. 1, FIG. 2, FIG. 3, FIG. 4)

And (3) mixed bacterium growth inhibition test: streptomyces scabies (Streptomyces scabies)4.0076, 4.1765, Streptomyces acidocaldarius (Streptomyces acidiscaibes) 4.1789, and Streptomyces albidoflavus (Streptomyces albidoflavus)4.3598 were each 100ul spread on the same PDA plate, and 10ul of Trichoderma asperellum was spotted locally on the plate. Culturing at 28 ℃. Observed after 2 days. (FIG. 5) results: trichoderma asperellum has an inhibitory effect on mixed pathogenic bacteria.

The co-culture bacteriostasis test method comprises the following steps: streptomyces scabies (Streptomyces scabies)4.0076, 4.1765, Streptomyces scabies (Streptomyces acidiscabis) 4.1789, Streptomyces albidoflavus (Streptomyces albidoflavus)4.3598 each 100ul and 10ul of Trichoderma asperellum were mixed and plated on PDA plates and cultured at 28 ℃. Observed after 2 days. As a result: trichoderma asperellum and four pathogenic bacteria can grow preferentially in the symbiotic process, and the other four bacteria are inhibited obviously (figure 6).

4. The preparation method of the microbial agent comprises the following steps:

1) potato liquid Medium (g/L) preparation: 1L of potato extract and 20g of glucose; adding 15g of agar into the solid culture medium;

2) activating Trichoderma asperellum strain Sanju-19 on potato solid culture medium for 3 times;

3) culturing 1 ring in 50ml potato liquid culture medium at 28 deg.C for 2 days;

4) after the fermentation broth was centrifuged at 12,000r/min for 1min, the supernatant was discarded, and the pellet was suspended in the same volume of sterile water to prepare a cell suspension.

Sequence listing

<110> Fujian three-torch Biotechnology Ltd

<120> Trichoderma asperellum strain Sanju-19 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 600

<212> DNA

<213> Trichoderma asperellum (Trichoderma asperellum)

<400> 1

ccttcggggt ggctcctaaa ggttacctca ccgacttcgg gtgttacaaa ctctcgtggt 60

gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat 120

tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga 180

tttgtgggat tggcttaacc tcgcggtttc gctgcccttt gttctgtcca ttgtagcacg 240

tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300

tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg 360

cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420

cctgtcactc tgcccccgaa ggggacgtcc tatctctagg attgtcagag gatgtcaaga 480

cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540

ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg agtgcttaat 600

Claims

1. Trichoderma asperellum (Trichoderma asperellum) strain Sanju-19 with preservation number of CGMCC number 15870.

2. A microbial agent characterized by comprising the trichoderma asperellum strain trichoderma trintorch-19 according to claim 1 as an active ingredient.

3. A method for preparing the microbial agent according to claim 2, wherein:

1) the strain III is activated for 3 times on a potato solid culture medium by a torch-19;

2) culturing 1 ring in 50ml potato liquid culture medium at 28 deg.C for 2 days;

3) after the fermentation liquor is centrifuged for 1min at 12,000r/min, the supernatant and the precipitate are respectively collected and suspended in sterile water with the same volume to prepare cell suspension.

4. The trichoderma asperellum strain Sanju-19 according to claim 1 for use in a medicament for preventing and treating potato scab.

5. The use according to claim 4, wherein the potato scab is caused by Streptomyces scab, Streptomyces acidocaldarius or Streptomyces albidoflavus.