CN117844655B

CN117844655B - Panus funiculosus and application thereof in prevention and treatment of plant fungal diseases

Info

Publication number: CN117844655B
Application number: CN202410256901.0A
Authority: CN
Inventors: 王秦虎; 姜佳琦; 田凯
Original assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Current assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Priority date: 2024-03-07
Filing date: 2024-03-07
Publication date: 2024-05-24
Anticipated expiration: 2044-03-07
Also published as: CN117844655A

Abstract

The invention belongs to the technical field of microorganisms, and discloses a panus funiculosus and application thereof in preventing and treating plant fungal diseases. The invention provides a basket-shaped fungus (Talaromyces funiculosus) GE44 with a preservation number of CGMCC No.41019. The panus funiculosus GE44 has broad-spectrum antagonism to pathogenic bacteria of plant fungal diseases, and can effectively inhibit hypha growth, conidium generation, toxin accumulation and sexual reproduction of pathogenic bacteria related to plant fungal diseases. The string-shaped basket fungus GE44 provided by the invention has good application prospect as a biocontrol fungus agent.

Description

Panus funiculosus and application thereof in prevention and treatment of plant fungal diseases

Technical Field

The invention belongs to the technical field of microorganisms, and discloses a panus funiculosus and application thereof in preventing and treating plant fungal diseases.

Background

Fusarium fungi are various in species and widely distributed in nature, and can parasitize or rot for survival, and plants infected by Fusarium are mostly withered, root rot, spike rot and other symptoms. Disease caused by Fusarium has wide distribution and large harm, and can often cause serious reduction of crop yield, thereby causing great economic loss. For example, wheat scab caused by fungi such as fusarium graminearum (Fusarium graminearum, sexual state: gibberella zeae) is a plant fungal disease that causes not only serious yield-reduction and quality-reduction of wheat, but also mycotoxins remaining in the grain of diseased wheat can cause poisoning of humans and animals, posing a great threat to grain production and food safety.

In recent years, due to the strict implementation of cultivation measures such as wheat straw returning, the number of disease residues is increased, the field bacterial source is increased, and the plant fungal diseases are frequently generated, so that a new challenge is provided for agricultural production. At present, chemical pesticides are mainly adopted by farmers for preventing and treating plant fungal diseases in the market, but long-term application of the chemical pesticides can cause pathogenic bacteria to generate drug resistance, so that environmental pollution is caused, and the ecological environment is seriously influenced. The biological control has the advantages of environmental friendliness, safety to people and animals, lasting control effect, easy coordination with other plant protection measures, and the like, and provides an effective way for reducing the use of chemical pesticides.

Fungi of the genus talaromyces (talaromycessepp.) family generally have good natural environment and stress adaptability. The fungus plays an important role in production practice, and some strains have the capabilities of phosphate dissolving and cellulase producing, and some strains can be used as pigment producing bacteria. In addition, some of the yellow basket bacteria, the purple basket bacteria and the like have application potential in plant disease control.

Disclosure of Invention

The invention aims to provide strain basket-shaped fungus GE44 with biological control effect and application thereof. The basket-shaped fungus GE44 provided by the invention has broad-spectrum inhibition on plant pathogenic fungi, and particularly has remarkable effect on inhibiting fusarium fungi and the formation of wheat gibberella.

The invention provides a basket rope (Talaromyces funiculosus) which is identified as basket rope GE44. Experiments prove that the strain GE44 provided by the invention can effectively inhibit growth of fusarium graminearum hyphae, generation of conidium, accumulation of deoxynivalenol (Deoxynivalenol, DON) toxin and formation of wheat gibberella graminearum sources during co-culture of the strain GE44 and the fusarium graminearum. In addition, the basket-shaped strain GE44 has general inhibition on other fusarium and broad-spectrum antibacterial effect on other plant pathogenic fungi.

The basket-shaped fungus GE44 is separated from wild elymus capense, and the preservation information is as follows.

Preservation name: string basket GE44 (Talaromyces funiculosusstrain GE 44);

Classification naming: talaromyces funiculosus;

Preservation time: 2023, 12, 28;

Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);

Deposit unit address: the dynasty district North Star, department 1, hospital 3 in Beijing;

preservation number: CGMCC No.41019.

The ITS sequence of the basket-shaped strain GE44 is shown as SEQ ID NO. 1.

The BenA sequence of the basket-shaped fungus GE44 is shown as SEQ ID NO. 2.

The PRB2 sequence of the basket-shaped fungus GE44 is shown as SEQ ID NO. 3.

The invention also claims the application of the panus funiculosus in the prevention and treatment of plant diseases.

Preferably, the use of the invention is carried out by controlling plant diseases.

As the preference of the application of the invention, the application of the panus funiculosus in preparing a plant fungal disease prevention and treatment drug is provided.

Further, pathogenic bacteria of the plant disease include fusarium spp, rhizoctonia cerealis Rhizoctonia cerealis, apple anthracis Collectotrichum fructicola, apple rot fungi Cytospora mandshurica, apple ring rot fungi Botryospuaeria berengeriana, sclerotium rolfsii Sclerotinia sclerotiorum, phytophthora capsici Phytophthora capsici.

Preferably, the use of the invention is such that the basket is used to inhibit the formation of a source of gibberella wheat.

Preferably, the basket is used for inhibiting fusarium graminearum hyphae growth, conidium production, toxin accumulation and sexual reproduction.

Compared with the prior art, the technical scheme provided by the invention has at least the following beneficial effects or advantages:

(1) The panus funiculosus GE44 provided by the invention has broad-spectrum antibacterial effect on pathogenic bacteria of plant diseases, such as rhizoctonia cerealis Rhizoctonia cerealis, apple anthracnose Collectotrichum fructicola, apple rot fungi Cytospora mandshurica, apple ring rot fungi Botryospuaeria berengeriana, sclerotium rolfsii Sclerotinia sclerotiorum and phytophthora capsici Phytophthora capsici, and has good antibacterial effect. The panus funiculosus GE44 can be used for preventing and treating plant diseases, and has excellent preventing and treating effect on plant fungal diseases.

(2) In particular, the rope basket fungus GE44 provided by the invention has a good inhibition effect on Fusarium (Fusarium spp.), and can effectively inhibit hypha growth, conidium generation, toxin accumulation and sexual reproduction of Fusarium fungi.

(3) When the rope-shaped basket-shaped fungus GE44 and the fusarium graminearum are co-cultured, the invention can effectively inhibit the growth of fusarium graminearum hyphae, the generation of conidium, the accumulation of deoxynivalenol (Deoxynivalenol, DON) toxin and the formation of a wheat fusarium graminearum source.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings that are used in the embodiments will be briefly described below. It is evident that the drawings in the description are only some preferred embodiments of the invention and that other similar results can be obtained from these drawings by a person skilled in the art without inventive effort.

FIG. 1 is a diagram showing colony morphology of basket-like fungus GE 44. Wherein A is the front surface of a strain of the panus string GE 44; b is the back of the strain of basket-like strain GE 44.

FIG. 2 is a diagram showing microscopic morphological characteristics of the basket-like strain GE 44. Wherein A is conidiophore; b is conidium.

FIG. 3 is a graph showing the results of coculture of basket-like strain GE44 with Fusarium graminearum. Wherein PH-1 represents Fusarium graminearum PH-1 cultured alone, and PH-1+GE44 represents coculture of basket-like GE44 with Fusarium graminearum PH-1.

FIG. 4 is a graph showing the results of inhibition of F.graminearum conidium by basket-like GE 44. Wherein A is a phenotype diagram when separately culturing fusarium graminearum PH-1; b is a phenotype diagram of the coculture of the fusarium graminearum PH-1 and the basket-like strain GE 44; c is the amount of conidia when F.graminearum PH-1 is cultured alone or when F.graminearum PH-1 is co-cultured with basket-like strain GE 44.

FIG. 5 is a bar graph of the inhibition of Fusarium graminearum DON toxin production by basket rope GE 44.

FIG. 6 is a schematic representation of the inhibition of fusarium graminearum exine shell production by basket-like GE 44. Wherein PH-1 represents Fusarium graminearum PH-1 cultured alone, and PH-1+GE44 represents coculture of basket-like GE44 with Fusarium graminearum PH-1.

FIG. 7 is a microstructure of F.graminearum of basket rope GE44 heavy parasitic. Wherein the arrows point to the "grommet-like" and "antenna-like" structures formed by GE 44.

FIG. 8 is a phylogenetic tree of basket GE 44.

Detailed Description

The following describes the technical scheme of the present invention with reference to examples. The experimental methods and the detection methods in each embodiment are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

Example 1

This example describes the acquisition and identification process of basket string GE 44.

Acquisition and isolation of strains: in 2022, 5 months, wild grass (elymus capense, elymus dahuricus) with better stress resistance is collected in Yang Ling fields of Shaanxi province, the wild grass plant tissues are sterilized with 75% ethanol and 10% NaClO for 1min respectively, and then placed on PDA culture medium (38 g/L of potato dextrose agar powder and the same below) containing 1% ampicillin and 1% streptomycin sulfate, and cultured under dark condition at 25 ℃, and mycelium growth is observed at the cut position of the plant tissues during the period. If so, cutting the edge of the colony to the PDA culture medium for culturing, repeating for 2-3 times to achieve the purification purpose, and moving to the PDA inclined plane for preservation.

Screening of strains: and (3) determining the inhibition effect of different isolated strains on the growth of Fusarium graminearum by using a plate facing method, and selecting the strain with the best inhibition effect for subsequent experiments. And (3) taking a plate which is not inoculated with endophytes to be detected as a control, inversely culturing for 4d at the temperature of 25 ℃ in a dark condition for 3 times in each group, calculating the bacteriostasis rate of different isolated strains on Fusarium graminearum, and selecting a strain GE44 with the highest bacteriostasis rate for subsequent experiments.

The DNA of the strain to be tested was extracted by CTAB method, and ITS ITS, benA and RPB2 sequences were amplified with 3 pairs of primers in Table 1, respectively. The PCR reaction system and amplification procedure were as follows.

PCR reaction system: PCR buffer 1.5. Mu.L, dNTP 1.2. Mu.L, upstream primer 0.3. Mu.L, downstream primer 0.3. Mu.L, taq enzyme 0.18. Mu.L, genomic DNA 0.6. Mu.L, DDW 10.92. Mu.L.

PCR amplification procedure: 3 min at 94 ℃;94℃for 30 s,58℃ (ITS/BenA) or 50℃for 40 s (RPB 2) and 72℃for 1min cycles; 10min at 72 ℃; preserving at 4 ℃.

The PCR products were sequenced and the resulting sequences were searched for homology at NCBI and their taxonomic status was confirmed by phylogenetic analysis. The phylogenetic tree is shown in FIG. 8, and the analysis result shows that the strain GE44 to be tested is the panus funiculosus (Talaromyces funiculosus).

TABLE 1 primers for identification of basket-like molecules

Morphological identification of strains: the strain GE44 cake was inoculated to PDA medium, and cultured in the dark at 25℃for 7 days, and the colony growth was observed. As shown in figure 1, the bacterial colony has neat edge, grey green front surface and pale yellow back surface, dense and short aerial hyphae and a large number of conidia attached to the surface. The conidiophores were branched in the form of double-round broom, smooth, round or oval, and about 4.01.+ -. 0.48. Mu.m, by 3.40.+ -. 0.38. Mu.m, when observed under a 40-fold microscope (FIG. 2).

Example 2

This example illustrates the hyphal growth and spore production inhibition of Fusarium graminearum by basket in rope form GE 44.

Inhibition of fusarium graminearum growth: bacterial cakes are respectively picked up at the colony edges of the fusarium graminearum PH-1 (wild type) and the rope-shaped basket-shaped bacterial GE44 by an 8 mm puncher, inoculated on a PDA flat plate (phi=90 mm), and the inhibition rate of the rope-shaped basket-shaped bacterial GE44 on the fusarium graminearum PH-1 is measured after the two bacterial cakes are oppositely cultivated for 5 days at a distance of 4 cm. As shown in FIG. 3, the measurement result shows that the basket-shaped fungus GE44 has a remarkable inhibition effect on the hypha growth of the Fusarium graminearum PH-1.

Inhibition of fusarium graminearum spore production: the cakes were removed with an 8mm punch on Fusarium graminearum PH-1, the edge of the string basket GE44 colony, and the blank PDA dish, respectively. 5 Fusarium graminearum cakes were picked up and placed in a conical flask containing 50mL of CMC liquid medium (ammonium nitrate 1g, potassium dihydrogen phosphate 1g, acid hydrolyzed casein 1g, magnesium sulfate heptahydrate 0.5g, yeast extract 1g, 15g of sodium carboxymethylcellulose was added after water was added to 1L, autoclave sterilization was carried out at 121 ℃ C. For 20min; the same applies below), and then 5 string basket GE44 cakes were picked up and placed in the conical flask, and 3 replicates were set as an experimental group. Similarly, 5 Fusarium graminearum cakes were picked up and placed in a conical flask containing 50mL CMC liquid medium, and then 5 blank PDA cakes were picked up and placed in the conical flask, and 3 replicates were set as a control group. After shaking culture at 25deg.C and 180rpm in the dark for 5d, all treatments were filtered with sterile filter cloth, centrifuged at 3500rpm for 7min, and resuspended in 30mL of sterile water. The number of conidia was counted under a microscope with a hemocytometer, and the analysis was performed in combination with the counted results. As shown in FIG. 4, when Fusarium graminearum PH-1 was co-cultured with basket-like strain GE44, the spore yield of Fusarium graminearum PH-1 was significantly reduced by about 50%.

Example 3

This example illustrates the inhibition of DON production by Fusarium graminearum by basket rope GE 44.

Preparation of spore suspension: shaking culture of Fusarium graminearum PH-1 conidium by CMC liquid culture medium, filtering and centrifuging after 4-6 d, and re-suspending by TBI liquid culture medium, and adjusting the spore concentration to 10 ⁶ spores/mL. In addition, a little TBI culture medium is added into a culture dish for culturing the string-shaped basket-shaped fungus GE44, a sterile gun head is used for scraping surface conidia, a mixture of the TBI culture medium and the string-shaped basket-shaped fungus GE44 is filtered by a sterile filter cloth, the conidia are left after mycelium is removed, a spore suspension is prepared, and the concentration is adjusted to 3.5 multiplied by 10 ⁶、2.5×10⁶、1.5×10⁶ spores/mL.

The preparation of TBI medium was as follows:

After TBI preparation, 40. Mu.L of trace elements was added to 1L of TBI medium, pH was adjusted to 5.5, 0.3g of plant gel was added, and the mixture was sterilized at 121℃for 20 minutes.

Induction of DON toxin: DON toxin production was induced by TBI medium on 24-well plates. The experimental design is as follows: (1) control group: 2.01mL of TBI medium and 10. Mu.L of Fusarium graminearum PH-1 spore solution (10 ⁶ spores/mL) were added to one well of a 24-well plate; (2) experimental group: to one well of the 24-well plate, 2mL of TBI medium was added, and 10. Mu.L of Fusarium graminearum PH-1 spore liquid (10 ⁶ spores/mL) and 10. Mu.L of basket-like fungus GE44 spore liquid (3.5X10 ⁶,2.5×10⁶,1.5×10⁶ spores/mL) at different concentrations were added, respectively. Each of the above treatments was repeated 3 times, covered with tinfoil and then transferred to a dark culture at 25℃for 7d.

Determination of DON toxin: after the induction is finished, DON toxin is extracted. After filtration through PE cartridge, vacuum concentration and evaporation to dryness and derivatization with silylating reagent TMS (TMSI/TMCS, v/v=100/1). Finally, the DON content is determined by GC/MS-MS. As shown in FIG. 5, the results demonstrate that Fusarium graminearum PH-1 co-cultured with basket-like GE44 produced less toxin than Fusarium graminearum PH-1 alone under conditions that induce toxigenesis. Thus, basket-like bacteria can significantly reduce DON production by Fusarium graminearum.

Example 4

This example illustrates the inhibition of the wheat red fungus source by basket rope GE 44.

Preparing straw: cutting mature wheat straw into small sections of about 5cm, ensuring that each small section contains one nodule, and sterilizing at 121deg.C for 20min. 15mL of 2% WA medium (agar 20g/L, the same applies below) WAs poured into the dish, and 3 stalks were placed before the medium solidified.

Inoculating straw: first, F.graminearum PH-1 and GE44 spore suspensions of basket-like bacteria were prepared and their concentrations were adjusted to 2X 10 ⁶ spores/mL and 2X 10 ⁷ spores/mL, respectively (the preparation was the same as in example 3, and the spore suspension concentration was adjusted with 0.1% Tween). Next, whether the strain was applied by basket-like strain GE44 or not was judged to be able to suppress the formation of gibberella source on wheat straw. The experimental design is as follows: (1) control group: only 500. Mu.L of Fusarium graminearum spore liquid (2X 10 ⁶ spores/mL) was added; (2) experimental group: 500. Mu.L of the liquid of basket-like spores (2X 10 ⁷ spores/mL) was added first, and after 3d, the liquid of Fusarium graminearum (2X 10 ⁶ spores/mL) was added. Each treatment was set up with 3 biological replicates.

Induction of ascus shells: after the spore liquid on the surface of the culture medium is dried, transferring the culture medium into a 25 ℃ incubator for dark culture for 7-10 days, adding 0.1% Tween, scraping hypha on the surface of the culture medium, transferring the culture medium into a 25 ℃ black light incubator for continuous culture for 1 week, and inducing the fusarium graminearum to generate ascus shells on wheat straw.

Inhibition result observation: microscopic observation is carried out on the formation of bacterial sources on wheat straws in a dish, as shown in fig. 6, a large number of fusarium graminearum shells are generated on the straws in a control dish, and in the dish with the application of the string-shaped basket-shaped bacteria, only one layer of basket-shaped hyphae is attached to the straws, and no fusarium graminearum shells are formed, namely, the formation of a sterile source is carried out, so that the string-shaped basket-shaped bacteria can inhibit the formation of the wheat gibberella graminearum sources on the straws.

Example 5

This example illustrates the action of basket rope GE44 on F.graminearum.

To investigate the mode of action of basket GE44 against Fusarium graminearum PH-1, microscopic observations were made of mycelia in which PH-1 and GE44 were in contact with each other. First, a sterile filter paper was laid on a petri dish, soaked with sterile water, and a sterile slide was placed in the petri dish, on which a 1mm thick layer of PDA medium was laid. Secondly, the string basket fungus GE44 and the fusarium graminearum PH-1 are respectively arranged at the two ends of the glass slide, and the two fungus blocks are separated by about 1 cm. Finally, culturing in dark at 25 ℃ until the mycelia come into contact, and observing the mycelia morphology under a 40-fold microscope. The results of fig. 7 show that a portion of the basket cord forms an "antennal" like structure adjacent to the fusarium graminearum hypha, and a portion of the basket cord wraps around the fusarium graminearum hypha in a "grommet" like manner, indicating that the basket cord has a re-mailing effect on the fusarium graminearum.

Example 6

This example illustrates the inhibition of Fusarium species and pathogenic bacteria of common plant fungal diseases by basket in rope form GE 44.

Inhibition of different Fusarium species by basket string GE 44: antagonism of Fusarium species such as Fusarium graminearum, fusarium asiaticum, fusarium flavum, fusarium pseudograminearum, etc. by the plate-facing method was determined. The inhibition rate of the strain of the basket GE44 was calculated by measuring the radius of the antagonistic colony by culturing the strain in the dark at 25℃for 5 days in 3 replicates per group using a plate without inoculating the strain of the basket GE44 as a control. As shown in Table 2, the basket-like fungus GE44 has a good inhibitory effect on various pathogenic bacteria of Fusarium genus.

TABLE 2 antibacterial Rate of basket string GE44 against Fusarium species

Inhibition of pathogenic bacteria of other common plant fungal diseases by basket rope GE 44: antagonism of the panzer GE44 against pathogenic bacteria of various plant fungal diseases was measured by the plate-facing method, and the results showed that it also had excellent inhibitory effects against these pathogenic bacteria (Table 3). Therefore, the panus funiculosus GE44 is a biocontrol bacterium with broad-spectrum bacteriostasis.

TABLE 3 antibacterial Rate of basket-like rope GE44 against other plant pathogens

As can be seen from the above examples, the basket-like fungus GE44 provided by the present invention has excellent antagonistic effect against wheat scab pathogenic fungus Fusarium graminearum and the like, and can inhibit hypha growth, conidium production, DON toxin production and the like. More importantly, the wheat scab inhibitor can inhibit the formation of wheat scab bacterial sources on the straw, so that the primary infection source of the wheat scab is reduced, and the effect of controlling the wheat scab from the source is achieved.

The embodiments described above are only some of the embodiments of the present invention and their description is not intended to limit the scope of the claimed invention. All other embodiments, which can be derived and substituted by a person skilled in the art from the idea of the invention without any inventive effort, are within the scope of the protection of the invention.

Claims

1. The string-shaped basket bacteria is characterized in that the string-shaped basket bacteria are preserved in China general microbiological culture collection center (CGMCC NO): 41019.

2. The basket rope according to claim 1, wherein ITS, benA, RPB of the basket rope has the sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

3. The basket rope of claim 1, wherein the basket rope has antagonism against pathogenic bacteria of fungal plant diseases.

4. The basket in accordance with claim 3 wherein the pathogenic bacteria of the plant fungal disease comprises Fusarium spp.

5. The use of the panus funiculosus as claimed in claim 1 for controlling plant diseases, wherein the panus funiculosus is used for controlling plant fungal diseases;

Pathogenic bacteria of the plant disease include Fusarium spp, rhizoctonia cerealis Rhizoctonia cerealis, apple anthracis Collectotrichum fructicola, apple rot fungi Cytospora mandshurica, apple ring rot fungi Botryospuaeria berengeriana, rape sclerotium Sclerotinia sclerotiorum, phytophthora capsici Phytophthora capsici.

6. The use according to claim 5, wherein the use of basket-like bacteria for the preparation of a medicament for controlling fungal diseases of plants.

7. The use according to claim 5, wherein the panus funiculosus is used for inhibiting the formation of a source of gibberella wheat, the pathogenic bacteria of which is fusarium graminearum Fusarium graminearum.

8. The use according to claim 5, wherein the basket is for inhibiting hyphal growth, conidiogenesis, toxin accumulation and sexual reproduction of fusarium graminearum Fusarium graminearum.