CN114774286B - ZH-1 of zygomycetes and application thereof - Google Patents
ZH-1 of zygomycetes and application thereof Download PDFInfo
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- CN114774286B CN114774286B CN202210300907.4A CN202210300907A CN114774286B CN 114774286 B CN114774286 B CN 114774286B CN 202210300907 A CN202210300907 A CN 202210300907A CN 114774286 B CN114774286 B CN 114774286B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention discloses a zygomycete ZH-1 and application thereof, and the application of the zygomycete ZH-1 as a biological agent for preventing and treating rice blast. The invention can effectively inhibit rice blast germ and provides a new way for green and safe control of rice blast.
Description
Technical Field
The invention relates to the field of plant fungal disease control, in particular to zygomycetes ZH-1 and application thereof.
Background
Pyricularia oryzaeMagnaporthe oryzae syn. Pyricularia oryzae) The rice blast is one of the world fungal diseases and the most serious rice diseases, and the rice blast bacteria have the characteristics of strong burst, easy epidemic, wide distribution and the like, and cause about 10-30% of yield loss in the worldwide planting area each year. In some developing countries like kennia, bangladesh and african parts of the country, in recent years rice has been almost banned due to large-scale outbreaks of rice blast, which means that local farmers have little income and even the whole country will face panic caused by hunger. In addition, even though there is only 10% loss in yield, these losses can live around 6000 million people, with economic value exceeding $ 7000 hundred million. However, since Pyricularia oryzae has strong environmental adaptability and continuous variation, the conventional breeding method and the conventional chemical control technology cannot effectively inhibit the spread of Pyricularia oryzae at present. In China, rice is a first large grain crop, the planting area is the second world, and the annual incidence of rice blast in China and the yield loss caused by the annual incidence of rice blast are always high. To date, the agents for controlling rice blast mainly comprise chemical pesticides, and single agents for controlling rice blast are used for years to easily cause rice blast bacteria to generate resistant types. Once resistant strains appear, the control of rice blast will becomeAnd is more troublesome.
Biological control is a measure of inhibiting the growth and reproduction of pathogenic bacteria or regulating plant growth by using other microorganisms or microbial metabolites. In recent years, the research on biological control technology in China is relatively more, and good effect is achieved. At present, the main microbial pesticides used for preventing and treating rice blast are fungi, actinomycetes and bacteria; botanical pesticides such as extracts of fructus Gleditsiae Abnormalis, fructus Sapindi Mukouossi, and Camellia oleifera; plant protecting compounds such as Huang Lilei compound sakura extract, terpenoid rice hull ketone A and rice hull ketone B, etc. The biological control has the advantages of safety, green, high efficiency and environmental protection, and becomes a hot spot for controlling plant diseases.
Botrytis cinereaZygosporium) Belongs to the fungus of the Hypsizygus sp.gracilis, is a common parasitic fungus, is distributed on various angiosperms and some gymnosperms, and has the distribution range from temperate zone to tropical zone. Most zygosporium fungi are parasitic bacteria of plants and seriously harm the health of host plants. The use of zygomycetes for controlling rice blast has not been reported at present.
Disclosure of Invention
The invention aims to provide the zygomycete ZH-1 and the application thereof, which can effectively inhibit rice blast and provide a new way for green and safe control of rice blast.
The technical scheme adopted for solving the technical problems is as follows:
a zygomycete ZH-1 strain is namedZygosporium masoniiZH-1 was preserved in China center for type culture Collection (CCTCC NO) at 24 months of 2022: m2022148.
The application of the zygomycetes ZH-1 as a biological agent for preventing and treating rice blast.
A microbial pesticide for preventing and treating rice blast is provided, wherein the microbial source of the microbial pesticide is zygomycetes ZH-1.
A method for preventing and treating rice blast includes such steps as inoculating the zygomycete ZH-1 to liquid culture medium, culturing, amplifying, filtering, immersing rice seeds in the filtrate, sowing or spraying the filtrate on the surface of rice plant.
The beneficial effects of the invention are as follows: the strain can obviously inhibit the growth of plant pathogenic fungi such as rice blast bacteria, anthracnose bacteria and the like and the germination of conidium, and the filtrate in the liquid culture state has the activity of effectively inhibiting the rice blast bacteria, so that the control of rice blast can be realized.
Drawings
FIG. 1 shows colony morphology of ZH-1.
Fig. 2 shows the hyphae (scale 10 μm) and conidium morphology (scale 2 μm) of zygomycetes ZH-1.
FIG. 3 shows the inhibition of Pyricularia oryzae and anthrax colonies by ZH-1.
FIG. 4 shows the inhibition of germination of Magnaporthe grisea spores by the culture filtrate of ZH-1, on a scale of 50. Mu.m.
FIG. 5 shows the infection of barley leaves with Pyricularia oryzae after treatment of the culture filtrate of ZH-1.
FIG. 6 shows the bacteriostasis of seed-immersed rice by culture filtrate of ZH-1 of zygomycetes.
Detailed Description
The technical scheme of the invention is further specifically described by the following specific examples.
In the present invention, the materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1 isolation and identification of zygomycete ZH-1 Strain
1. Obtaining, separating and purifying ZH-1 strain
The rice blast bacteria are inoculated in a CM solid culture medium (a biological organism) for 7 days under the environment of a laboratory (a plant protection and microorganism research institute fungus and plant research laboratory of Zhejiang province), pink single colony appears in the culture medium, the colony can obviously inhibit the growth of the rice blast bacteria, and the strain is transferred to a new CM solid culture medium for 25 ℃ and 16/8 h light/dark alternate culture for identifying the strain. After 15 days of culture, 2 were added to the mediumSterile mL water, gently scraping the mycelium, filtering with three layers of filter paper to collect spores, and diluting the spores to 10 3 100 mu L of spore liquid is evenly coated in a CM solid culture medium, the culture is carried out for 5 days at 25 ℃, and single colony is selected and transferred to a fresh CM solid culture medium for continuous culture, and the serial number is ZH-1.
2. Identification of strains
(1) Morphological identification: colony diameter was 1.2 CM after 7 days of growth in CM medium at 25 ℃; colony expansion was slow, basal bulge aerial hyphae were sparse (fig. 1). The mycelium was observed under a microscope to have a septum and the conidiophore did not branch. Spore cells were colorless, oval, 7-10×3.5-7 μm (FIG. 2).
(2) Molecular identification: ZH-1 was grown at 25deg.C in CM solid medium for 7 days, 1 CM mycelium block was crushed with shaker, transferred into CM liquid medium (Bio-organism) for 5 days at 25deg.C at 150 rpm/min, 1 g mycelium was collected after filtration, genomic DNA was extracted by liquid nitrogen milling and ITS sequencing, and the sequencing ITS nucleotide sequence (SEQ ID. No. 1) was BLAST aligned with the genus Zygomycetes accession No. MH860771.1 and MZ568332.1 using NCBI databaseZygosporium sp) Botrytis cinereaZygosporium masonii) The sequence coverage is 98% respectively, the similarity is 99.81%, and the molecular identification and morphological analysis show that ZH-1 is a novel zygomycete mannheimia.
EXAMPLE 2 ZH-1 Strain controlling Pyricularia oryzae
(1) ZH-1 test for colony inhibition of Pyricularia oryzae and anthracnose
The ZH-1 strain and the Pyricularia oryzae are inoculated on a CM solid medium respectively by adopting a counter culture method, and the culture is carried out for 7 days by 16/8 h light/darkness alternation at 25 ℃. ZH-1 was found to have a growth inhibition rate of 37.3% for Pyricularia oryzae and 41.7% for anthrax (FIG. 3).
(2) ZH-1 strain inhibits rice blast fungus spore germination
ZH-1 is grown in CM solid culture medium at 25deg.C for 7 days, 1 square centimeter mycelium blocks are taken, and crushed by shaking with a shaker, and transferred into CM liquid culture medium for culture at 25deg.C 150 rpm/minFor 5 days, filtering with three layers of filter paper, filtering the filtrate with 0.45 μm filter membrane, mixing with Magnaporthe grisea spores (CM culture solution as control), and diluting the spore concentration to 5×10 4 Every milliliter, 25 microliters of the spores are dripped on a glass slide, cultured at 25 ℃ for 12 h, and the germination of the spores is observed under a microscope. As a result, it was found that the inhibition rate of the filtrate against Pyricularia oryzae spores was 100% (FIG. 4).
(3) ZH-1 filtrate mixed rice blast fungus spore inoculated barley leaf
Taking 1 square centimeter mycelium blocks after ZH-1 grows in CM solid culture medium at 25deg.C for 7 days, shaking with a shaker, transferring into CM liquid culture medium, culturing at 25deg.C for 5 days at 150 rpm/min, filtering with three layers of filter paper, filtering the filtrate with 0.45 μm filter membrane, mixing with Magnaporthe grisea spores (CM culture solution as control), and diluting spore concentration to 5×10 4 Every milliliter, 25 microliters of the liquid is dripped on barley leaves, the barley leaves are subjected to moisturizing culture for 4 days, and the disease condition of the leaves is observed. Leaf inoculation experiments showed that the Magnaporthe grisea after treatment with the filtrate of zygomycetes ZH-1 will not be infested (FIG. 5).
(4) Bacteriostatic experiment of ZH-1 filtrate immersed rice seeds
The ZH-1 filtrate was filtered with a 0.45 mu m filter membrane and the seed rice seeds were immersed, and after 2 days of culture at 37 ℃, the seed rice seeds not immersed were found to be significantly mildewed and grown, the germination rate was reduced, while the seed rice seeds immersed with the ZH-1 filtrate were not grown, and the germination rate was normal (FIG. 6).
The zygomycete ZH-1 capable of inhibiting rice blast germ provided by the invention can inhibit rice blast germ conidium germination, and the inhibition rate reaches 100%. Leaf inoculation experiments show that the Magnaporthe grisea after treatment with the filtrate of the zygomycetes ZH-1 will not be infested. The ZH-1 filtrate soaked rice seeds have the functions of bacteriostasis and germination acceleration. Indicating that the zygomycete ZH-1 has wide prospect and great value in the field of plant pathogenic fungi control.
The above-described embodiment is only a preferred embodiment of the present invention, and is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims.
SEQUENCE LISTING
<110> academy of agricultural sciences in Zhejiang province
<120> a zygomycete ZH-1 and its use
<130> 2022.03
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 558
<212> DNA
<213> zygote (Zygosporium masonii)
<400> 1
tccgtaggtg aacctgcggg cggagggatc attactgagt tttctaaact cccaaaccca 60
tgtgaacata ccttttgttg cctcggcagt ggtgccgtcg cccctcgcgg cggcggcagc 120
ctgccggcgg acccaataaa ctcttgttgt tacagtgtca ttctgaatta aaaaacttaa 180
ttagttaaaa ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcgaa 240
atgcgataag taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt 300
gcgcccatta gtattctagt gggcatgcct gttcgagcgt catttcaacc cttaagcccc 360
agttgctaag tgtcgggaga ctgccgtaaa acgcagctcc tcagagttag ctggcagagt 420
tggttccaat ctctgggcgt agtaaatctt tatctcgtct cttgcagttg ggccggcgct 480
cgccattaaa caccccaaat ttttctatgg ttgacctcgg atcaggtagg aatacccgct 540
gaacttaagc atatcaaa 558
Claims (4)
1. A zygomycete ZH-1 is characterized by that it is a zygomycete genusZygosporium sp) Botrytis cinereaZygosporium masonii) Named afterZygosporium masoniiZH-1 was preserved in China center for type culture Collection (CCTCC NO) at 24 months of 2022: m2022148.
2. Use of the zygomycetes ZH-1 according to claim 1 as a biological agent for controlling rice blast.
3. A microbial pesticide for preventing and treating rice blast, which is characterized in that the microbial source of the microbial pesticide is the zygomycete ZH-1 according to claim 1.
4. A method for preventing and controlling rice blast, which is characterized in that the zygomycetes ZH-1 of claim 1 are inoculated into a liquid culture medium for culture, filtered after the expansion culture, and the rice seeds are soaked in the filtrate and then sown or the filtrate is sprayed on the surfaces of rice plants.
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| JP2014533660A (en) * | 2012-11-02 | 2014-12-15 | バイエル・インテレクチユアル・プロパテイー・ゲー・エム・ベー・ハー | How to prevent disease in transplanted rice |
| WO2015067800A1 (en) * | 2013-11-11 | 2015-05-14 | Basf Se | Antifungal penicillium strains, fungicidal extrolites thereof, and their use |
| CN105557703B (en) * | 2014-11-07 | 2019-03-01 | 沈阳中化农药化工研发有限公司 | It is a kind of prevent and treat rice blast oil-suspending agent and its application |
| FR3046180B1 (en) * | 2015-12-28 | 2018-09-21 | IFP Energies Nouvelles | MUTANT STRAINS OF TRICHODERMA REESEI |
| CN109536389A (en) * | 2018-12-21 | 2019-03-29 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | A kind of trichoderma strain being located away from extreme drought desert Environment, cultural method, application method, stalk field planting method and application |
| CN113215002B (en) * | 2021-05-14 | 2022-03-15 | 浙江省农业科学院 | Endophytic fungus M-B927 and application thereof |
| CN113249229B (en) * | 2021-05-14 | 2021-11-30 | 浙江省农业科学院 | Pseudocercosporus endophytic fungus P-B313 and application thereof |
| CN114075528A (en) * | 2021-09-08 | 2022-02-22 | 浙江省农业科学院 | Conidium coating method for producing large number of sexual generations of rice blast germs |
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