CN119081894B - Co-culture fermentation liquor of trichoderma and burkholderia, microbial inoculum and application of microbial inoculum in preventing and treating ginger root rot - Google Patents
Co-culture fermentation liquor of trichoderma and burkholderia, microbial inoculum and application of microbial inoculum in preventing and treating ginger root rot Download PDFInfo
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Abstract
The invention relates to the technical field of biological control, in particular to a co-culture fermentation liquor of trichoderma and burkholderia, a microbial inoculum and application of the microbial inoculum in controlling ginger root rot. The preparation method of the co-culture fermentation liquid comprises the steps of co-culture fermentation of trichoderma atroviride (Trichoderma atroviride) HB20111 and Burkholderia vietnaese (Burkholderia vietnamiensis) P418 to obtain the co-culture fermentation liquid, wherein the co-culture fermentation liquid comprises fermentation thalli, fermentation supernatant and produced metabolites through co-culture. Compared with a single biocontrol strain, the trichoderma atroviride HB20111 and the Vietnam Burkholderia P418 can generate wide antibacterial and growth-promoting secondary metabolites through co-culture fermentation, prevent and control the ginger root rot, are not easy to generate drug resistance, have obvious effects and have high application value.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to a co-culture fermentation liquor of trichoderma and burkholderia, a microbial inoculum and application of the microbial inoculum in controlling ginger root rot.
Background
In recent years, biological control has received increasing attention in controlling plant diseases, playing a key role. The secondary metabolites are low molecular weight compounds produced by microorganisms, and the secondary metabolites such as antibiotics, alkaloids, terpenoids, flavonoids, lipopeptides, polyketides and the like can not only regulate the reactions of individuals and populations, but also shape the whole microbial community. With the development of genome sequencing technology, a large number of biosynthetic gene clusters have been identified in the microbial genome that infer the synthesis of secondary metabolites. However, there is a large gap between the genomic capacity of biosynthetic gene clusters and secondary metabolites found in microorganisms, since many biosynthetic gene clusters remain silent or low expression levels under normal laboratory culture conditions.
Co-cultivation of microorganisms of different species has become an effective method for the discovery of new secondary metabolites. Co-cultivation utilizes complex ecological relationships among co-cultivated members, including synergistic metabolism, reciprocal symbiosis, competitive antagonism, interactions between signal molecules, etc., by simulating the natural microbial community growth environment to activate expression of silent biosynthetic gene clusters and induce synthesis of new secondary metabolites. Velmourougane et al observed that the biofilm produced by co-cultivation of Trichoderma viride ITCC 2211 and azotobacter chroococcum W5 helped better colonize the rhizosphere of chickpea, and co-cultivation improved chickpea root biomass, plant enzyme activity and soil available nutrients compared with single inoculation, and could save 25% of nitrogen fertilizer application. Li Tingting and the like optimize the co-culture condition of the trichoderma atroviride SG3403 and the bacillus subtilis 22 for secreting antifungal secondary metabolites, and find that the inhibition rate of the co-culture fermentation filtrate on fusarium graminearum is improved by 54.22 percent, and the secondary metabolites such as mevastatin and Koninginin A in the co-culture are identified through LC-MS analysis. Yang Kai and the like analyze the growth promoting effect of the co-culture fermentation filtrate of trichoderma harzianum LTR-2 and Arthrobacter urealyticum DnL-1 on wheat by using metabonomics, and find that secondary metabolites such as amino acid and the like produced by co-culture have remarkable promoting effect on wheat seed germination.
Trichoderma and Burkholderia are used as important biocontrol strains, and have good control effects on plant diseases caused by various soil-borne pathogenic fungi. However, there is no study on co-culturing trichoderma with burkholderia.
Disclosure of Invention
Aiming at the technical problem of lack of related research on co-culture of trichoderma and burkholderia, the invention provides co-culture fermentation liquor and microbial inoculum of trichoderma and burkholderia and application of the co-culture fermentation liquor and microbial inoculum in preventing and treating ginger root rot. Compared with single biological prevention strain, the bacterial inhibition and growth promotion secondary metabolite produced by co-culture fermentation is wide, the prevention and treatment of ginger root rot is not easy to produce drug resistance, the effect is obvious, and the application value is very high.
In a first aspect, the present invention provides a co-cultured broth of trichoderma and burkholderia, the preparation method comprising:
Trichoderma atroviride (Trichoderma atroviride) HB20111 and Vietnam Burkholderia (Burkholderia vietnamiensis) P418 are subjected to co-culture fermentation to obtain a co-culture fermentation broth, wherein the co-culture fermentation conditions are as follows:
28-30℃ shaking culture at 180-200 rpm for 5-7 days;
Trichoderma atroviride HB20111 has been preserved in 2018, 12 months and 5 days to China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of number 3 of West Song No. 1, north Star, kogyo area of Beijing, and a preservation number of CGMCC No. 16963, and is classified and named as Trichoderma atroviride Trichoderma atroviride;
Burkholderia vietnamese P418 has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 8 months and 30 days in 2004, and has a deposit address of Guangcun North 13 in the sea lake area of Beijing, china academy of sciences microbiological culture Collection, a deposit number of CGMCC No. 1212, and a classification designation of Burkholderia vietnamese Burkholderia vietnamiensis.
Further, trichoderma atroviride HB20111 and Burkholderia Vietnam P418 were inoculated into MKB fermentation medium according to the viable count of 1:1 for co-cultivation fermentation.
Further, the co-culture fermentation method is as follows:
(1) Bacterial strain activation, namely inoculating trichoderma atroviride HB20111 on a PDA culture medium, and placing the culture medium in a constant temperature incubator for activation culture at 28 ℃ for 5 days;
picking out a P418 colony of Burkholderia yunnanensis, streaking on a TY culture medium, and placing the TY culture medium in a constant-temperature incubator at 30 ℃ in a dark place for culturing until a single colony grows out (generally 24-48 h);
(2) Preparing seed liquid, namely scraping trichoderma atroviride HB20111 spores by using sterile water, and diluting the spores to the concentration of 1.0X10 8 cfu/mL to obtain seed liquid A;
Transferring single bacterial colony of Vietnam Burkholderia P418 into TY liquid culture medium, placing in a shaking table, shaking and culturing at 30 ℃ and 180rpm until logarithmic growth phase, wherein OD 660 value is about 0.6-0.8, and concentration is 1.0X10- 8 cfu/mL, and taking as seed liquid B;
(3) And (3) co-culture fermentation, namely inoculating the seed liquid A and the seed liquid B obtained in the step (2) into an MKB fermentation medium respectively in an inoculum size of 10% (volume percentage), and performing co-culture fermentation under the culture conditions of 28-30 ℃ and 180-200 rpm for 5-7 days to obtain a co-culture fermentation liquid.
Further, the co-culture broth comprises fermentation cells by co-culture, fermentation supernatant and metabolites produced, including the following major secondary metabolites:
cyclic (L-pro-L-casein) dipeptide, cyclic (L-pro-L-valyl) dipeptide, cyclic (D-leu-L-pro) dipeptide, indoleacetic acid, indole-2-acetic acid, DL-indole-3-lactic acid, natamycin, amikacin, ofloxacin and baveromycin B1.
In a second aspect, the invention provides an application of the co-culture fermentation liquor of trichoderma and burkholderia for preventing and treating ginger root rot, wherein the pathogenic bacteria of the ginger root rot are Fusarium oxysporum and ginger specialization (Fusarium oxysporum f. Sp. Zingiberi, foz).
In a third aspect, the present invention provides a co-culture microbial inoculum of trichoderma and burkholderia, wherein the co-culture fermentation broth of trichoderma and burkholderia is absorbed by a carrier to form a solid microbial inoculum, and the solid microbial inoculum is mixed with a binder to prepare the co-culture microbial inoculum.
Further, the carrier is selected from diatomite, medical stone or turfy soil, and the mass ratio of the fermentation liquor to the carrier is 5:1.
Further, the binder is selected from sodium hydroxymethyl cellulose and/or sodium alginate, and the mass ratio of the solid microbial inoculum to the binder is 100:6.
In a fourth aspect, the invention provides an application of the co-culture microbial inoculum of trichoderma and burkholderia for preventing and treating ginger root rot, wherein pathogenic bacteria of the ginger root rot are fusarium oxysporum ginger specialization.
Furthermore, the co-culture microbial inoculum also has the effects of promoting the growth of the ginger and improving the yield of the ginger.
The invention has the beneficial effects that:
Compared with most of composite microbial inoculum products obtained by single-fungus culture and mixing, the trichoderma atroviride HB20111 and the Vietnam Burkholderia P418 in the co-culture fermentation liquor and the co-culture microbial inoculum can coexist in a symbiotic mode, have mutually overlapped effects, and can not antagonize and inhibit each other and influence the respective growth.
Compared with single-strain culture, the trichoderma atroviride HB20111 and the Burkholderia Vietnam P418 are subjected to co-culture fermentation, so that the yield of secondary metabolic substances for inhibiting pathogenic fungi and promoting plant growth is improved. Wherein natamycin, amikacin, ofloxacin and bafexomycin B1 and cyclic dipeptides such as cyclic (L-pro-L-valyl) dipeptide, cyclic (L-pro-L-tyr) dipeptide and cyclic (D-leu-L-pro) dipeptide show strong inhibition effect on ginger root rot pathogen, namely fusarium oxysporum ginger specialization, so as to achieve the effect of preventing and treating ginger root rot.
Meanwhile, the trichoderma atroviride HB20111 and the Vietnam Burkholderia P418 have rhizosphere growth promoting effects, can colonize and occupy sites in the root of crops, the inside of plants or soil ecology, can directionally secrete secondary metabolites such as indoleacetic acid, indole-2-carboxylic acid DL-indole-3-lactic acid and the like from the rhizosphere of plants, can induce the ginger to generate systematic defense reaction and disease resistance, has nutrition superposition effects, thereby improving the microecological environment of the ginger planting soil, improving the soil structure and physicochemical properties, and improving the biological yield and quality of the ginger.
The co-culture fermentation liquor of trichoderma and burkholderia and the preparation process of the co-culture microbial inoculum of the invention have the advantages of cost saving, stable fermentation method and simple use mode, and belong to the environment-friendly type.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 shows the results of culturing Trichoderma atroviride HB20111 and Burkholderia Vietnam P418, wherein in FIG. 1, A is the result of culturing P418 single bacteria, B is the result of culturing HB20111 single bacteria, and C is the result of co-culturing HB 20111+P418.
Fig. 2 shows the results of bacteriostasis of trichoderma atroviride HB20111 and Vietnam Burkholderia P418 on a plate of fusarium oxysporum ginger specialization Foz, wherein in fig. 2, A is the result of bacteriostasis of co-culture fermentation filtrate on Foz, B is the result of bacteriostasis of HB20111 single-bacteria fermentation filtrate on Foz, C is the result of bacteriostasis of P418 single-bacteria fermentation filtrate on Foz, and D is Foz blank plate control.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Trichoderma atroviride HB20111 used in the invention is preserved in 2018 and 12 months 5 to China general microbiological culture Collection center with a preservation address of North Star XILU No. 1 and 3 of the Korean area North Star of Beijing city, and the preservation number is CGMCC No. 16963, and the classification is named Trichoderma atroviride Trichoderma atroviride, and the strain is disclosed in China patent No. CN 110564627B. Burkholderia vietnamese P418 was deposited in 8 months and 30 days 2004 to China general microbiological center, the deposit address is 13 in Guangcun North in the sea lake area of Beijing, the deposit number is CGMCC No. 1212, the classification name is Burkholderia vietnamiensis, and the strain is disclosed in Chinese invention patent CN 103478147B.
Each of the media used in the present invention may be obtained using a self-made process or purchased directly from commercial media products. PDA culture medium comprises peeled potato 20%, glucose 2%, agar powder 1.5%, distilled water 1L, and natural pH. The TY culture medium comprises 1% of tryptone, 0.5% of yeast powder, 0.02% of calcium chloride, 1.5% of agar powder, 1L of distilled water and pH 7.2-7.4. The TY liquid culture medium comprises 1% of tryptone, 0.5% of yeast powder, 0.02% of calcium chloride, 1L of distilled water and pH 7.2-7.4. The MKB fermentation medium comprises casein hydrolysate 2%, glycerol 1%, dipotassium hydrogen phosphate 0.15%, magnesium sulfate 0.15%, distilled water 1L, and pH 7.2+ -0.2.
EXAMPLE 1 preparation of Trichoderma and Burkholderia Co-culture broth, fermentation filtrate
(1) Strain activation Trichoderma atroviride HB20111 was inoculated onto PDA medium and placed in a constant temperature incubator at 28℃for 5 days of activation culture.
The Boehmeria Vietnam P418 colony is picked and streaked on TY culture medium, and the Boehmeria Vietnam P418 colony is placed in a constant temperature incubator at 30 ℃ to be cultivated in a dark place until a single colony grows out.
(2) The seed liquid is prepared by scraping Trichoderma atroviride HB20111 spores with sterile water, and diluting to 1.0X10 8 cfu/mL to obtain seed liquid A.
The single colony of Vietnam Burkholderia P418 is transferred into TY liquid culture medium, and is placed in a shaking table for shaking culture at 30 ℃ and 180rpm until the logarithmic growth phase, the OD 660 value is about 0.6-0.8, and the concentration is 1.0X10- 8 cfu/mL, and the culture medium is used as seed liquid B.
(3) Preparing three PDA culture mediums, inoculating the seed liquid A and the seed liquid B obtained in the step (2) to obtain a trichoderma atroviride HB20111 single-strain plate, a Vietnam Burkholderia P418 single-strain plate and a trichoderma atroviride HB 20111+Vietnam Burkholderia P418 co-culture plate. The results of the three PDA cultures are shown in FIG. 1, demonstrating that there is no antagonism between Trichoderma and Burkholderia, and symbiotic coexistence is possible.
(4) And (3) co-culturing and fermenting, namely inoculating the seed liquid A and the seed liquid B obtained in the step (2) into the same MKB fermentation medium according to 10% (volume percentage) of inoculum size, and carrying out shaking co-culturing and fermenting for 7 days at 28-30 ℃ and 180-200 rpm to obtain co-culturing fermentation liquor of trichoderma and Burkholderia, wherein the co-culturing fermentation liquor comprises co-cultured trichoderma atroviride HB20111 and fermentation thalli of Burkholderia P418, fermentation supernatant and metabolites produced by the fermentation thalli.
(5) Filtering and sterilizing, namely filtering and sterilizing the co-culture fermentation liquor of the trichoderma and the burkholderia by using a 0.22 mu m microporous filter membrane to obtain co-culture fermentation filtrate of the trichoderma and the burkholderia, wherein the co-culture fermentation filtrate comprises co-culture fermentation supernatant, produced metabolites and the like.
Simultaneously, seed liquid A and seed liquid B are respectively inoculated into two MKB fermentation media according to 10% (volume percentage) of inoculum size for single-strain culture, fermentation conditions are the same as those of the co-culture fermentation in the step (3), and the shaking co-culture fermentation is carried out for 7 days at 28-30 ℃ and 180-200 rpm, so that HB20111 single-strain fermentation liquid and P418 single-strain fermentation liquid are obtained. Filtering and sterilizing HB20111 single-fungus fermentation liquor and P418 single-fungus fermentation liquor by using a 0.22 mu m microporous filter membrane to obtain HB20111 single-fungus fermentation filtrate and P418 single-fungus fermentation filtrate.
(6) The analysis and identification of co-culture metabolites comprise the steps of freeze-drying co-culture fermentation filtrate, HB20111 single-fungus fermentation filtrate and P418 single-fungus fermentation filtrate in a freeze dryer, adding 100 mu L of 80% methanol aqueous solution, vortex shaking for 30s, standing in an ice bath for 5min, centrifuging at 4 ℃ and 12000rpm for 15min, diluting the supernatant with mass spectrum grade water until the methanol content is 53%, centrifuging at 4 ℃ and 12000rpm for 15min, and taking a supernatant sample for LC-MS analysis and identification.
The differential metabolites produced by co-culture fermentation of Trichoderma and Burkholderia are shown in Table 1 below, and it can be seen that the co-culture fermentation significantly increases the content of various substances such as natamycin, amikacin, ofloxacin, bafexorubicin B1, cyclo (L-pro-L-casein) dipeptide, cyclo (L-pro-L-valin) dipeptide, cyclo (D-leu-L-pro-dipeptide), indoleacetic acid (IAA), indole-2-carboxylic acid (ICA) and DL-indole-3-lactic acid (ILA). Wherein, natamycin, amikacin, ofloxacin and bafilomycin B1 have antifungal effect, IAA, ICA and ILA have plant growth promoting effect, which lays a foundation for the development of co-culture microbial inoculum.
TABLE 1 differential metabolite produced by Co-culture fermentation of Trichoderma and Burkholderia
(7) The activity of the co-cultured metabolite antagonistic fusarium oxysporum is determined by uniformly mixing 10 percent (volume percent) of co-cultured fermentation filtrate, 10 percent of HB20111 single-bacterial fermentation filtrate and 10 percent of P418 single-bacterial fermentation filtrate with a PDA culture medium, respectively pouring the mixture into a plate to serve as three treatment groups, taking the PDA culture medium added with 10 percent of sterile water as a blank plate control, inoculating a fusarium oxysporum ginger specialized Foz bacterial cake in the center of each plate, and culturing for 5 days at 28 ℃ to obtain the result shown in a figure 2.
The distance between the center and the edge hypha of the fusarium oxysporum ginger specialized colony is measured by adopting a crisscross method, and the inhibition rate of the fusarium oxysporum ginger specialized is calculated by the following calculation formula:
。
The calculated inhibition rate of the co-culture fermentation filtrate on the fusarium oxysporum ginger specialization type is 94.1 percent, which is obviously higher than 88.02 percent of HB20111 single-fungus fermentation filtrate and 18.19 percent of P418 single-fungus fermentation filtrate. The results show that in the co-culture fermentation mode, trichoderma atroviride HB20111 and Burkholderia Vietnamii P418 can generate more secondary metabolites with antibacterial effect, and the antagonistic activity on fusarium oxysporum ginger specialization Foz is improved.
EXAMPLE 2 preparation of Co-culture inoculant for Trichoderma and Burkholderia
The co-culture fermentation broth of the example 1 is adsorbed by taking diatomite as a carrier, the mass ratio of the co-culture fermentation broth to the diatomite is 5:1, a solid microbial inoculum is formed, 3wt% of sodium carboxymethyl cellulose and 3wt% of sodium alginate are added into the solid microbial inoculum, and then the mixture is uniformly mixed by a mixer, so that a co-culture microbial inoculum A of trichoderma and Burkholderia is prepared, wherein the co-culture microbial inoculum A contains co-cultured trichoderma atroviride HB20111 and fermentation thalli, fermentation supernatant and metabolites produced by the P418.
The method comprises the steps of taking turfy soil as a carrier, adsorbing co-culture fermentation liquor of the embodiment 1, wherein the mass ratio of co-culture fermentation filtrate to diatomite is 5:1, forming a solid microbial inoculum, adding 6wt% of sodium carboxymethyl cellulose into the solid microbial inoculum, and uniformly mixing by a stirrer to prepare co-culture microbial inoculum B of trichoderma and Burkholderia, wherein the co-culture microbial inoculum B contains fermentation thalli of trichoderma atroviride HB20111 and Burkholderia Vietnamii P418, fermentation supernatant and the like.
The medical stone is used as a carrier to adsorb the co-culture fermentation liquid in the embodiment 1, the mass ratio of the co-culture fermentation filtrate to diatomite is 5:1, a solid microbial inoculum is formed, 6wt% of sodium alginate is added into the solid microbial inoculum, and then the solid microbial inoculum is uniformly mixed by a mixer, so that a co-culture microbial inoculum C of trichoderma and burkholderia is prepared, wherein the co-culture microbial inoculum C contains components such as fermentation thalli of trichoderma atroviride HB20111 and burkholderia Vietnamii P418, fermentation supernatant and metabolites produced by the fermentation thalli.
EXAMPLE 3 application of Trichoderma and Burkholderia co-culture microbial inoculum in preventing and treating ginger root rot
Experiment site, namely, the area of experiment in the ginger planting area with high incidence of early ginger root rot in the Lizhen area of Laiwu district, jinan, shandong province is 100 square meters.
The test crop is Laiwu ginger.
The experiments were divided into three treatment groups, a blank control group, a carbendazim treatment group (positive control) and a co-cultivation microbial inoculum treatment group.
The blank group was not pretreated.
Carbendazim treatment group, namely, at the sowing period of the ginger, applying 50% carbendazim wettable powder by mixing soil according to the dosage of 2 kg/mu, and planting ginger sprouts.
And (3) uniformly applying the co-culture microbial inoculum A prepared in the example 2 into soil in a soil mixing manner at the sowing time of the ginger, and planting ginger buds.
The planting and management of the three treatment groups are consistent with the local cultivation conditions, and the prevention effect and the growth promotion effect of the co-culture microbial inoculum on the ginger field district are investigated and detected after 180 days of field planting.
The ginger root rot is investigated plant by plant according to a 7-level grading method, and the severity grading standard is as follows:
Grade 0, that is, the whole plant is free from diseases;
grade 1, the basal part of the stem is slightly discolored, or leaves below 1/4 are slightly discolored or withered;
Stage 3, the color change of the basal part of the stem is obvious, or 1/4 to 1/2 of the leaf is slightly withered;
grade 5, leaf withering of 1/2 or more than 2/3 of the stem base browning around the whole stem;
And 7, completely withering or dying the disease plants.
The disease index and the disease prevention effect are calculated according to the following formula:
;
。
The field plot control results of the ginger root rot are shown in the following table 2, and it can be seen that the co-culture microbial inoculum can utilize trichoderma atroviride HB20111 and Vietnam Burkholderia P418 to construct microecological balance of soil, and antibacterial substances produced by the trichoderma atroviride HB20111 and the Vietnam Burkholderia P418 can also antagonize the growth of pathogenic bacteria, so that the incidence rate of the ginger root rot is effectively reduced, the control effect of the ginger root rot reaches 77.05%, and the microbial inoculum is remarkably higher than that of chemical pesticide carbendazim treatment 63.88%. The co-culture microbial inoculum can also obviously improve various growth indexes of the ginger, the plant height and the single plant yield are obviously different from those of a blank control (p is less than 0.05), the promotion effect on the plant height and the single plant yield of the ginger is respectively improved by 23.87 percent and 28.30 percent compared with that of the control group, and the plant height and the single plant yield are obviously higher than that of carbendazim treatment (18.83 percent and 24.76 percent respectively). The application of the co-culture microbial inoculum can not only effectively prevent and treat the root rot of the ginger, but also play an obvious role in promoting the yield of the ginger, thereby reducing the use amount of pesticides and fertilizers and having great application value, economy and social benefit.
Table 2 statistical table of ginger field cell tests
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
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