CN112625918B - Separation and identification of heavy parasitic fungus for preventing and treating wheat stripe rust and microbial inoculum - Google Patents

Separation and identification of heavy parasitic fungus for preventing and treating wheat stripe rust and microbial inoculum Download PDF

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CN112625918B
CN112625918B CN202011531153.0A CN202011531153A CN112625918B CN 112625918 B CN112625918 B CN 112625918B CN 202011531153 A CN202011531153 A CN 202011531153A CN 112625918 B CN112625918 B CN 112625918B
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stripe rust
wheat stripe
fungus
wheat
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CN112625918A (en
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王晓杰
王宁
王建锋
张珊
胡泽宇
樊昕
汤春蕾
康振生
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Northwest A&F University
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Abstract

The invention belongs to the technical field of agricultural biological control and microbial biological preparations, and discloses a heavy parasitic fungus separation, identification and microbial inoculum for preventing and treating wheat stripe rust.A heavy parasitic strain identified and used in the invention is a strain system found under natural conditions, and the heavy parasitic fungus is obtained by separating white parasitic fungus and grey brown parasitic fungus on the surface of wheat stripe rust, so that the infection and harm of wheat stripe rust to wheat plants can be effectively inhibited, and the production and activity of wheat stripe rust are reduced by using the isolate; has high application value in production; the parasitic fungus can obviously inhibit the growth and development of wheat stripe rust and the generation of spores, and the culture mode of the heavy parasitic fungus is simple and easy to operate, so that the parasitic fungus is a novel fungus line which can be potentially widely applied to control wheat stripe rust.

Description

Separation and identification of heavy parasitic fungus for preventing and treating wheat stripe rust and microbial inoculum
Technical Field
The invention belongs to the technical field of agricultural biological control and microbial biological preparations, and particularly relates to a heavy parasitic fungus for controlling wheat stripe rust, a separation and identification method and a microbial inoculum.
Background
Currently, the closest prior art: wheat stripe rust is a major disease causing damage to global wheat producing areas caused by wheat stripe Puccinia striiformis f.sp.tritici, Pst, and wheat stripe rust is a low-temperature gas-borne disease capable of damaging the whole growth cycle of wheat. The control of wheat stripe rust mainly depends on the use of chemical pesticides, but the excessive dependence on chemical pesticides comes at the cost of environment sacrifice.
In summary, the problems of the prior art are as follows:
(1) the creation speed of disease-resistant varieties is limited, and the utilization rate of the disease-resistant varieties is reduced.
(2) The excessive use of chemical pesticides seriously affects the food production safety in China.
(3) Wheat stripe rust develops resistance, and a novel chemical or biological agent needs to be developed.
The difficulty of solving the technical problems is as follows: the development of a more environmentally friendly biological agent for the control of wheat stripe rust.
The significance of solving the technical problems is as follows: the development and development of a novel biological agent are important for the control of the wheat stripe rust.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a heavy parasitic fungus for preventing and treating wheat stripe rust, a separation and identification method and a microbial inoculum.
The invention is realized in such a way that the heavy parasitic fungus paecilomyces for preventing and treating the wheat stripe rust has the preservation number of CGMCC No.20276, the classification name of the strain is Silplicium obclavatum, the strain name is RV26, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the address is as follows: western road No. 1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101. Thirty years of storage from 9/30 of 2020; the preservation center tests at 9 months and 30 days in 2020, and the result is survival; the request depositor: northwest university of agriculture and forestry.
The severe parasitic fungi for preventing and treating the wheat stripe rust are cladosporium cladosporioides and pseudo-blueAnd (3) mildew, which is sprayed on the surface seedlings of the wheat leaves by preparing a spore suspension. The isolate is from yellow rust (grown under natural conditions) with spore concentration of 1.0 × 106Spore, mL-1。
The invention also aims to provide a biological agent prepared from the heavy parasitic fungus for preventing and treating the wheat stripe rust.
The invention also aims to provide application of the biological agent in preventing and treating wheat stripe rust.
Another object of the present invention is to provide a bacterial line for controlling wheat stripe rust, which is established by the said heavy parasitic fungus for controlling wheat stripe rust.
Another object of the present invention is to provide a method for separating a heavy parasitic fungus for controlling wheat stripe rust, which comprises the following steps:
firstly, collecting the diseased leaves of wheat stripe rust in an incubator, wherein when stripe rust is inoculated for 16 days, the stripe rust is represented as orange-yellow spore heap;
secondly, the surface of the sporophyte is found to be grayish brown in the incubator, the leaf section of the diseased part is taken and cut into small sections of 5mm by scissors and then the small sections are treated by ddH2Rinsing with O, performing surface disinfection treatment with 70% alcohol, performing surface disinfection with 1% NaClO, and washing with sterile water for 3-5 times;
thirdly, inoculating the strain on a freshly prepared PDA culture medium, separating and culturing the strain at 25 ℃ for one week, selecting the single-growth strain, beating the cultured strain into a strain block with the diameter of 5mm by using a perforating method, transferring the strain block to the new PDA culture medium, and culturing the strain at 25 ℃ for 1 week to obtain the purified strain.
Further, the solid culture medium of the separation method of the heavy parasitic fungus for preventing and controlling the wheat stripe rust comprises the following steps: the separation and culture of the heavy parasitic bacteria adopt a solid potato glucose agar PDA culture medium, and the diseased leaves are inoculated on the PDA solid plate culture medium to obtain the heavy parasitic bacteria through separation and purification.
The invention also aims to provide a molecular identification method of the heavy parasitic fungus for preventing and treating wheat stripe rust, which comprises the following steps: separating R23Bo mycelium from the colony cultured in dark at 25 deg.C for 5 days; extracting DNA from the mycelium by a hexadecyl trimethyl ammonium bromide method; PCR amplification is carried out by using a primer of eukaryotic ribosome DNA; the PCR product was separated on 1.5% agarose gel and collected and purified using agarose gel DNA extraction purification kit.
In summary, the advantages and positive effects of the invention are: the invention is based on the discovery that the growth and development of the wheat stripe rust and the generation of spores can be obviously inhibited in the process of naturally propagating the wheat stripe rust, and has potential application value for biological control of the wheat stripe rust. The two heavy parasitic fungi discovered by the identification of the invention can effectively inhibit the growth and development of wheat stripe rust and the generation of spores, and the two germs are easy to culture and obtain.
The two strains which can be obtained by identification and can be heavily parasitic on the wheat stripe rust are utilized to carry out large-scale propagation spraying or injection on the wheat infected by the wheat stripe rust, and the wheat stripe rust can be effectively prevented and treated. The two heavy parasitic bacteria are identified and found to be cladosporides C.cladosporioides and paecilomyces S.obclavatum, and have obvious inhibition effect on the growth and development of the wheat stripe rust bacteria in the process of carrying out bacteriostasis effect experiments, can complete growth and generate conidia on a PDA culture medium, has no adverse effect on the growth and development of wheat, and can be used as a potential biological preparation for preventing and treating the wheat stripe rust bacteria.
Wheat stripe rust is an obligate biotrophic fungus caused by Puccinia striiformis f.sp.tritici transformed from Puccinia striiformis of Puccinia striiformis. Two types of heavy parasitic bacteria were isolated and identified from Puccinia triticina, and they were subjected to morphological identification and molecular marker multiposition analysis to find that they were Cladosporium cladosporioides and Paecilomyces mobilis Obclavatum. The regenerated bacterium is separated from a taupe parasitic bacterium on the surface of puccinia triticina koch, can effectively inhibit the infection and harm of puccinia triticina koch to wheat plants, and reduces the generation and activity of puccinia triticina koch. Therefore, the two isolates, C.cladosporioides and S.obclavatum, have potential application value in the biological control of wheat stripe rust.
The heavy parasitic strain identified and used in the invention is a strain system found in an incubator under natural conditions, and has high application value in production; the two parasitic fungi can obviously inhibit the growth and development of wheat stripe rust and the generation of spores, and the culture mode of the fungi is simple and easy to operate, so that the two parasitic fungi are a novel fungus system which can be potentially widely applied to control wheat stripe rust.
Drawings
FIG. 1 is a flow chart of a separation method of a heavy parasitic fungus for preventing and controlling wheat stripe rust, which is provided by the embodiment of the invention.
FIG. 2 is a schematic diagram of the growth morphology of Cladosporioides C.cladosporioides obtained by isolation and identification according to the embodiment of the present invention; A-B are the cultured hypha morphology, wherein A is the positive photograph of the culture plate; b, positive photograph of the culture plate.
Fig. 3 is a schematic diagram of the micro-morphological characteristics of cladosporides c.
Fig. 4 is a schematic diagram of the efficacy of a cladosporium c.cladosporioides spore suspension on puccinia striiformis in isolation and identification provided by the embodiment of the present invention.
FIG. 5 is a schematic diagram of the growth morphology of Paecilomyces S.obclavatum obtained by separation and identification according to the embodiment of the present invention; A-B are the cultured hypha morphology, wherein A is the positive photograph of the culture plate; b, positive photograph of the culture plate.
Fig. 6 is a schematic diagram of the micro-morphological characteristics of the paecilomyces sp.
Fig. 7 is a schematic diagram of the effect of the suspension of the spore of the paecilomyces sp.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a heavy parasitic fungus for preventing and treating wheat stripe rust, a separation and identification method and a microbial inoculum, and the invention is described in detail by combining the attached drawings.
The heavy parasitic fungi for preventing and treating the wheat stripe rust provided by the embodiment of the invention are Cladosporium cladosporioides and paecilomyces camplicium obclavatum. The preservation number is: CGMCC 20276.
As shown in fig. 1, the separation method of the heavy parasitic fungus for controlling wheat stripe rust provided by the embodiment of the invention comprises the following steps:
s101: collecting the diseased leaves of wheat stripe rust in an incubator, wherein when stripe rust is inoculated for 16 days, the stripe rust is expressed as orange-yellow spore heap;
s102: the surface of the sporophyte is found to be grayish brown in the incubator, leaf sections of the diseased parts are taken and cut into small sections of 5mm by scissors and then are subjected to ddH2Rinsing with O, performing surface disinfection treatment with 70% alcohol, performing surface disinfection with 1% NaClO, and washing with sterile water for 3-5 times;
s103: inoculating on freshly prepared PDA culture medium, separating, culturing at 25 deg.C for one week, selecting single-growth thallus, beating the cultured thallus into blocks with diameter of 5mm by punching method, transferring to new PDA culture medium, and culturing at 25 deg.C for 1 week to obtain purified thallus.
The separation and identification method of the heavy parasitic fungus for preventing and treating the wheat stripe rust, provided by the embodiment of the invention, comprises the following steps of:
the method comprises the following steps: separation and purification of heavy parasitic bacteria
Collecting the leaf of wheat stripe rust in the incubator, wherein the stripe rust is represented as orange-yellow spore pile when stripe rust is inoculated for 16 days, and accidental emergence occurs in the incubatorThe surface of the sporangium appears grayish brown, and the leaf segment of the diseased part is cut into 5mm small segments by scissors and then passes through ddH2Rinsing with O, sterilizing with 70% alcohol, sterilizing with 1% NaClO, washing with sterile water for 3-5 times, inoculating to freshly prepared PDA culture medium, separating and culturing at 25 deg.C for one week, selecting single-growth thallus, making the thallus into blocks with diameter of 5mm by punching, transferring to new PDA culture medium, and culturing at 25 deg.C for 1 week to obtain purified thallus.
Step two: morphological observation of heavy parasitic bacteria
The isolated pure cultured heavy parasitic bacterium R23Bo was cultured under the same conditions for 10 days by placing a 5mm diameter block on a new PDA medium plate in a triangular shape. The cultured thalli is photographed and recorded on the front surface and the back surface respectively, and the shapes of bacterial colonies, hyphae, conidia, conidiophores and other bacteria are observed and measured under a light microscope.
The samples were prepared as described previously (Zheng et al, 2017) by first fixing the samples in glutaraldehyde solution at 4 ℃, washing 4 times for 10 minutes with PBS buffer, dehydration for 15-20 minutes, 5 concentration gradients (30%, 50%, 70%, 80%, 90%) of ethanol. Soaking in isoamyl acetate for 10-20 min, and treating the dehydrated sample with a carbon dioxide drier. Finally, the samples were treated by the paint bomb method. The microscopic morphology of the bacteria was observed in the sample under SEM (scanning electron microscope).
Step three: molecular characterization of heavy parasitic bacteria
The R23Bo mycelia were isolated from colonies cultured in the dark at 25 ℃ for 5 days. DNA was extracted from the mycelia by the cetyltrimethylammonium bromide (CTAB) method (Wang et al.2015). PCR amplification was performed using primers for ITS (eukaryotic ribosomal DNA). The PCR product was separated on a 1.5% agarose gel and collected and purified using an agarose gel DNA extraction and purification kit (Takara, Japan). The amplified fragment was sequenced by AuGCT corporation (Beijing, China).
Step four: bacteriostatic analysis of heavy parasitic bacteria
Wheat Fielder was grown in an incubator for 20 days (16 ℃, 16h light; 12 ℃ C.)8h in darkness), firstly, inoculating puccinia striiformis CYR31 summer spores, carrying out moisture-preserving culture at 16 ℃ in darkness for 24h, and then placing in a growth incubator under the same culture conditions. Three, five, seven, ten days after inoculation with Puccinia striiformis, the plants had spore suspensions (1.0X 10) of different isolates6Spores/ml), dark moisturized at 16 ℃ for 24h, then returned to the growth incubator under the same conditions. The plants inoculated with Puccinia striiformis only were used as a control, symptoms and phenotypes were recorded, and spores on the leaf surface were counted using Image J number counting software. Meanwhile, in order to further evaluate the control effect of the separated heavy parasitic fungus on the puccinia striiformis, leaf tissues at different inoculation times are respectively adopted to extract DNA, and the fungal biomass (Pst-DNA/wheat DNA ratio) is determined. Scanning Electron Microscopy (SEM) is used for carrying out ultrastructural analysis on infected leaf samples which are treated differently after wheat stripe rust inoculation, and therefore the influence of the heavy parasitic bacteria on the growth and development of wheat stripe rust is revealed.
The culture conditions of the present invention include:
solid medium: the separation and culture of the heavy parasitic bacteria adopt a solid potato glucose agar PDA culture medium, and diseased leaves are inoculated to the PDA solid plate culture medium to obtain the heavy parasitic bacteria through separation and purification;
liquid culture medium: in the research of the control effect of the heavy parasitic bacteria on the wheat stripe rust, the cultured fungus cakes are inoculated into a PDA liquid culture medium, the PDA liquid culture medium is subjected to shake culture at 25 ℃ for 3 to 4 days to obtain a suspension of the heavy parasitic bacteria, and the suspension is treated and counted for inoculation.
The technical effects of the present invention will be described in detail below with reference to the accompanying drawings.
Two parasitic bacteria which can be parasitized on the puccinia striiformis in the incubator are obtained through separation and identification, the two parasitic bacteria are discovered to be cladosporium cladosporioides C.cladosporioides strain R23Bo and paecilomyces S.obclavatum strain RV26 through molecular identification and microscopic observation, and spore suspensions of the two bacteria are injected into wheat leaves infected by the puccinia striiformis, so that the morbidity of the puccinia striiformis is obviously weakened, the spore production quantity is also obviously reduced, and the diseases are obviously inhibited.
Research on the heavy parasitic bacteria on the wheat stripsThe rust disease preventing and treating effect is that cultured fungus cake is inoculated into PDA liquid culture medium, shaking culture is carried out at 25 ℃ for 3-4 days to obtain suspension of heavy parasitic fungus, the suspension is treated and counted for inoculation, the number of spore suspension is 1.0 multiplied by 106one/mL. And inoculating wheat infected by the puccinia striiformis, collecting wheat leaves after inoculation, carrying out scanning electron microscope observation and phenotype observation, and counting the number of summer spore piles on the surface of the published leaves. The results prove that the growth and development of the wheat stripe rust fungus are obviously inhibited after two kinds of heavy parasitic fungi are inoculated, and the sporulation quantity is obviously reduced.
Cladosporium cladosporioides strain R23Bo and paecilomyces sp obclavatum strain RV26 can successfully invade wheat stripe rust fungus spores, the wheat stripe rust fungus surface is planted through a bud tube, nutrition is absorbed to complete growth and development of the wheat stripe rust fungus, a large number of conidia are generated to infect the wheat stripe rust fungus again, and the wheat stripe rust fungus is completely destroyed.
Scanning electron microscope observation further shows that the two separated strains R23Bo and RV26 can effectively parasitize the puccinia striiformis. C. cladosporioides conidia, which generated the germ tube and started to invade the puccinia striiformis spores at R23Bo 36h inoculation. Cladosporioides produced large numbers of spores at 120h inoculation completely destroyed the summer spores of rust streak. It is further clarified that RV26 strain s.obclavatum can effectively parasitize puccinia striiformis and has an effect on Pst. Observing appearance morphological characteristics of Pst inoculated with S.obclavavatum under a scanning electron microscope, wherein the S.obclavavatum invades Pst summer spores through germ tubes and gradually surrounds the Pst until the ends of the S.obclavavatum completely colonize the Pst summer spores at 120h and 168 h.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (3)

1. The heavy parasitic fungus for preventing and treating the wheat stripe rust is characterized by being paecilomyces (Simplicillium obclavatum) with the preservation number of CGMCC No. 20276.
2. A biological agent prepared from the heavy parasitic fungus for controlling wheat stripe rust of claim 1.
3. The application of the biological agent as claimed in claim 2 in preventing and treating wheat stripe rust.
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Inventor before: Kang Zhensheng

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