CN108823104B - Fungus with function of improving content of notoginsenoside and application thereof - Google Patents
Fungus with function of improving content of notoginsenoside and application thereof Download PDFInfo
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- 241000233866 Fungi Species 0.000 title description 7
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- 235000003143 Panax notoginseng Nutrition 0.000 claims abstract description 52
- 239000000725 suspension Substances 0.000 claims abstract description 40
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 32
- 241000222290 Cladosporium Species 0.000 claims abstract description 27
- 241001222769 Cladosporium tenuissimum Species 0.000 claims abstract description 26
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- 238000000855 fermentation Methods 0.000 claims description 35
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Abstract
The invention relates to a Cladosporium tenuissimum (Cladosporium tenuissimum) which has beneficial effect on panax notoginseng and can improve the content of panax notoginseng saponins and a microbial inoculum thereof, belonging to the technical field of plant biology. The strain is separated from the stem of a biennial healthy panax notoginseng, is identified as cladosporium, is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 14126. The preparation of the microbial inoculum mainly comprises pure culture of microorganisms, preparation of spore suspension, production of the microbial inoculum and application of the microbial inoculum, and the microbial inoculum can also be mixed with an organic fertilizer to prepare a bacterial fertilizer for use. The microbial inoculum prepared by the invention not only has beneficial effect on panax notoginseng, but also can promote the increase of the content of panax notoginseng saponins to a certain extent, and shows good ecological application prospect.
Description
The technical field is as follows:
the invention relates to a biocontrol Cladosporium fungus (Cladosporium tenuissimum) and application thereof, in particular to a Cladosporium fungus with a probiotic effect on growth of panax notoginseng and application thereof in improving content of panax notoginseng saponins, and belongs to the technical field of plant biology.
Background art:
pseudo-ginseng (Panax notoginseng (Burk.) F.H.Chen), which is a perennial herb with homology of crude drugs and food in Panax of Araliaceae, is named as Panax pseudo-ginseng (pseudo-ginseng) and Cinchonas forbesii, and is originally recorded in compendium of materia medica, the root and stem of the Panax pseudo-ginseng have the effects of promoting blood circulation to remove blood stasis, and relieving swelling and pain, and the Panax pseudo-ginseng is a traditional and large common medicinal material in the medicinal material market of China, and has large market demand and high development and utilization values. Notoginseng radix mainly contains saponin, flavone, dencichine (amino acid), volatile oil, polysaccharide, etc., wherein dammarane type saponin is the main component and has strong bioactivity. The saponin components of the various batroxobin are separated from the pseudo-ginseng and the overground part thereof, and the ginsenoside Rb is required to be detected by clearly requiring the identification items in the national standard established by the Chinese pharmacopoeia1，Re，Rg1And notoginsenoside R1(ii) a The content detection items are calculated according to the dry product, and the ginsenoside Rg is containedlGinsenoside Rb1And notoginsenoside R1The total amount of (A) should not be less than 5.0%. With the improvement of living standard and the enhancement of health care consciousness of people, the use and the use amount of the pseudo-ginseng in the industries of clinic, pharmacy, health care, daily chemical industry and the like are continuously amplified, so that the supply and demand of the pseudo-ginseng are gradually and greatly increased, the annual demand of the domestic pseudo-ginseng raw material market is more than 8000 tons, and the quality of the medicinal materials is required to be ensured while the yield of the medicinal materials is ensured.
The method has the advantages that chemical fertilizers, hormones, pesticides and the like are not scientifically applied in the existing pseudo-ginseng production to promote the high yield of medicinal materials, so that the properties of the medicinal materials are obviously changed, particularly, the properties of the pseudo-ginseng are increased, the planting life is shortened, the risk of environmental pollution is increased, and even the harm of pesticide residue, heavy metal pollution and the like is brought to the pseudo-ginseng, so that the use of the pesticides is reasonably reduced, a green and environment-friendly mode for promoting the growth of the pseudo-ginseng is found, and the improvement of the saponin content has important significance for improving the quality of the pseudo-ginseng medicinal materials. The pseudo-ginseng plants are internally inhabited with a large number of microorganism groups closely related to the growth of the plants, and the nutrition absorption, the immune system regulation and the anti-infection capability of the plants are influenced, so that the biological means of reasonably regulating and controlling the rhizosphere micro-ecological environment, improving the rhizosphere environment of the pseudo-ginseng, promoting the growth of the plants and synthesizing the drug effect substances has huge application potential in the aspects of the safety of medicinal materials and the sustainability of the planting of the medicinal materials by applying the probiotic microorganisms.
The invention content is as follows:
the invention aims to: aiming at the problems that the properties of medicinal materials are obviously changed and the risk of environmental pollution is increased due to the fact that chemical fertilizers, hormones, pesticides and the like are not scientifically applied to the production of panax notoginseng at present to promote the high yield of the medicinal materials, the growth of panax notoginseng is promoted, and the content of panax notoginseng saponins is increased, a Cladosporium fungus (Cladosporium tenuissimum) and application thereof are provided.
The specific technical scheme is as follows:
the invention provides a Cladosporium tenuissimum fungus, which is identified as Cladosporium tenuissimum through morphological and molecular biological research. The bacterial colony cultured on a glucose potato agar culture medium PDA is flocculent, black brown, unsmooth edge and black back, and the diameter of the bacterial colony growing on the PDA for 10 days can reach 8-10 cm; the PDA culture medium comprises the following components: 100-200 g of potatoes, 5-20 g of glucose and 10-20 g of agar, wherein the volume is determined to be 1L of distilled water, the pH is natural, and the potatoes are sterilized for 30min at 121 ℃.
The invention also provides application of the Cladosporium fungus (Cladosporium tenuissimum) in promoting the growth of panax notoginseng and improving the content of panax notoginseng saponins.
The application of the cladosporium fungus in promoting the growth of notoginseng or ginseng and improving the saponin content is to perform root irrigation treatment on notoginseng or ginseng by using a conidium suspension of a cladosporium fungus strain, a strain fermentation liquor extract, a solid microbial inoculum or a combination of the four.
The strain conidium suspension is prepared by the following method: inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL.
The strain fermentation liquor is prepared by the following method: inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL. Inoculating the spore suspension into a fermentation culture medium PDB, culturing at 25-28 deg.C and 120-150r/min for 5-8 days.
The strain fermentation liquor extract is prepared by the following method: inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL. Inoculating the spore suspension into a fermentation culture medium PDB, culturing at 25-28 deg.C and 120-150r/min for 5-8 days. And extracting the fermentation liquor for 3 times by using equal volume of ethyl acetate, and collecting concentrated extract liquor for later use.
The bacterial strain solid microbial inoculum is prepared by the following method: inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL. 50mL of the above spore suspension was inoculated into 400g of a solid fermentation medium. Culturing at 25-28 deg.C, stirring under aseptic condition after culturing for 3-4 days, and culturing for 4-5 days to obtain solid zymophyte agent. Placing the obtained solid microbial inoculum in a drying oven at 30-35 deg.C, drying by blowing for 25-30h until the water content is 10-20%, and the viable count of the microbial inoculum is within the range ofTo achieve 1.0-5.0X 108More than one per gram. Packaging, and placing in dry place.
The PDA culture medium is as follows: 100-200 g of potatoes, 5-20 g of glucose and 10-20 g of agar, wherein the volume is determined to be 1L of distilled water, the pH is natural, and the potatoes are sterilized for 30min at 121 ℃.
The PDB culture medium is as follows: 100 g-200 g of potatoes and 5 g-20 g of glucose, diluting the mixture to 1L of distilled water with a constant volume, naturally adjusting the pH value, and sterilizing the mixture for 30min at 121 ℃.
The solid fermentation medium comprises: 350g of wheat bran 300-350g, 50-100g of corn flour, 5-10g of bean cake powder and KNO30.5g，K2HPO4 1.5g，NaCl 1.5g，CaCO34g, adding 400ml of water, and sterilizing at 121 ℃ for 30 min.
The application method of the biocontrol cladosporium fungus in promoting the growth of panax notoginseng and improving the content of panax notoginseng saponins comprises the following steps:
can be used by irrigating root at adult plant stage and seed stage of Notoginseng radix, counting under microscope, and diluting spore liquid to 1.0 × 104-1.0×108The concentration of each strain was 50 mL. The application is continued for 2-3 times, each time at 3 months interval. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
Can be used by irrigating root at adult plant stage and seed stage of Notoginseng radix, and diluting fermentation broth or fermentation broth extractive solution 102-104For double application, 50ml of each strain was used. The application is continued for 2-3 times, each time at 3 months interval. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
The solid microbial inoculum can be directly spread in the adult plant stage and the seed stage of the panax notoginseng, the solid microbial inoculum is spread on a panax notoginseng seedling bed, the using amount of each seedling is 0.5-1.0g, or the microbial inoculum is uniformly mixed by fine sand and then is scattered at the rhizosphere of the plant. The application is continued for 2-3 times, each time at 3 months interval. The microbial inoculum can also be mixed with organic fertilizer to prepare bacterial fertilizer for use.
The invention has the beneficial effects that:
the conidium suspension or fermentation liquor extracting solution or solid microbial inoculum of the biocontrol fungal Cladosporium fungus (Cladosporium tenuissimum) strain screened by the invention can obviously promote the growth of panax notoginseng and improve the content of panax notoginseng root saponin. Because the biological preparation is adopted, a series of problems caused by the use of chemical pesticides do not exist, and the biological preparation is beneficial to reducing agricultural pollution and improving the quality of medicinal materials. The biocontrol mycosporidium is separated from the stem of a biennial healthy panax notoginseng, exists in rhizosphere soil of the panax notoginseng, is harmoniously compatible with the soil ecology of the panax notoginseng and panax notoginseng plants, and is beneficial to fully exerting the advantages of the strains.
The biocontrol fungus strain has simple culture conditions, stable heredity, easy industrial production expansion and good development and application prospects.
The Cladosporium fungus (Cladosporium tenuissimum) of the invention is preserved in the China Committee for culture Collection of microorganisms with the address: west road No.1 hospital No. 3, north jing, chaoyang district, preservation time: 27 months 05 and 2017, and the preservation number is CGMCC No. 14126.
Description of the drawings:
FIG. 1 shows the colony morphology of Cladosporium tenuissimum.
FIG. 2 shows the effect of Cladosporium tenuissimum fermentation broth on the promotion of enzyme activity of root systems of Panax notoginseng.
FIG. 3 shows the effect of the fermentation broth of Cladosporium fungus (Cladosporium tenuissimum) on the promotion of the agronomic index of Panax notoginseng.
FIG. 4 shows the effect of fermentation broth of Cladosporium tenuissimum on the improvement of notoginsenoside content.
FIG. 5 shows the effect of Cladosporium tenuissimum (Cladosporium tenuissimum) solid microbial inoculum on the agronomic index promotion of Panax notoginseng.
The specific implementation mode is as follows:
for a better understanding of the present invention, the following figures and examples further illustrate, but are not to be construed as limiting, the experimental procedures set forth in the following examples are conventional and are not intended to be limiting.
Example 1 promoting action of Cladosporium fungus (Cladosporium tenuissimum) fermentation broth on enzymatic activity of root system of Panax notoginseng
Inoculating the strain into PDA culture medium plate, performing inverted culture at 25-28 deg.C for 5-8 days, activating the strain, preparing the activated strain into spore suspension with sterile water, and allowing the spore suspension to showCounting the spore concentration under a microscope, wherein the concentration of the spore suspension is as follows: 1.0X 104-1.0×108one/mL. Inoculating the spore suspension into a fermentation culture medium PDB, culturing at 25-28 ℃ for 5-8 days at 120-150r/min, and diluting the fermentation liquid by 100 times for later use. The field biennial Notoginseng radix plant is provided by medicinal plant research institute of agricultural academy of sciences of Yunnan province. Using distilled water as a control, repeating each group for three times, inoculating 100 panax notoginseng seedlings for each time, and irrigating 50mL of roots of each panax notoginseng seedling. Collecting Notoginseng radix samples 1, 2, and 3 months after fungus irrigation, respectively, and determining CAT enzyme, POD enzyme, and SOD enzyme activity. The methods for measuring the CAT enzyme activity, the SOD enzyme activity and the POD enzyme activity refer to a CAT kit, an SOD kit and POD kit specifications (Suzhou Keming Biotechnology Co., Ltd.).
Through the measurement indexes, the Graphpad software is used for significant difference analysis, the results are shown in fig. 2 and table 1, and the results show that the fermentation broth of the strain Cladosporium tenuissimum can improve the activities of enzymes CAT, POD and SOD of panax notoginseng root resistance and improve the disease resistance of panax notoginseng compared with a control group (CK).
TABLE 1 promoting action of Cladosporium fungus fermentation broth on enzyme activity of root system of Panax notoginseng
Note: 1, one month; 2, two months; -3, three months.
Example 2 Cladosporium fungus (Cladosporium tenuissimum) fermentation broth extract has an effect of promoting the agronomic index of Panax notoginseng
Inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL. Inoculating the spore suspension into a fermentation culture medium PDB, culturing at 25-28 ℃ for 5-8 days at the speed of 120-. The field biennial Notoginseng radix is prepared by medicinal planting of Yunnan academy of agricultural sciencesProvided by institute of medicine. Using distilled water as a control, repeating each group for three times, inoculating 100 panax notoginseng seedlings for each time, and irrigating 50mL of roots of each panax notoginseng seedling. Respectively collecting pseudo-ginseng samples 3 months after fungus irrigation for agronomic index determination.
Compared with a control group (CK), the plant height and the fresh root weight of the fermentation liquor extracting solution group of the Cladosporium tenuissimum are obviously improved, as shown in figure 3, the data with obvious difference are shown in a table 2, and the good effect of promoting the growth of the pseudo-ginseng is shown.
TABLE 2 promoting effect of Cladosporium fungus fermentation broth on Panax notoginseng agronomic index
Example 3 Effect of Cladosporium fungus (Cladosporium tenuissimum) fermentation broth on the improvement of notoginsenoside content
Inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL. Inoculating the spore suspension into a fermentation culture medium PDB, culturing at 25-28 ℃ for 5-8 days at 120-150r/min, and diluting the fermentation liquid by 100 times for later use. The field biennial Notoginseng radix plant is provided by medicinal plant research institute of agricultural academy of sciences of Yunnan province. Using distilled water as a control, repeating each group for three times, inoculating 100 panax notoginseng seedlings for each time, and irrigating 50mL of roots of each panax notoginseng seedling. Collecting Notoginseng radix samples 3 months after fungus irrigation, and measuring saponin content. The method for measuring the content of the panax notoginseng saponins and five monomeric saponins refers to the first part of the 'Chinese pharmacopoeia' 2015 edition.
The total saponin content, R, of the fermentation broth group was compared with that of the control group (CK)1、Rb1And a higher content of Rd, wherein Rb1And the Rd content is obviously improved, as shown in figure 4, the data with obvious difference is shown in a table 3, and the effect of promoting the improvement of the content of the notoginsenoside is shown.
TABLE 3 Effect of Cladosporium fungus fermentation broth on the improvement of notoginsenoside content
Example 4 Germination-promoting action of a conidia suspension of Cladosporium fungus (Cladosporium tenuissimum) on seeds of Panax notoginseng
Inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL for use. The Notoginseng radix seed is provided by medicinal plant research institute of academy of agricultural sciences of Yunnan province. And (4) taking distilled water as a control, repeating the steps for each group, repeatedly inoculating 100 pseudo-ginseng seeds, repeatedly irrigating 100mL for each group, and respectively counting the emergence rate 2 months after fungus irrigation. Calculated as follows: the germination rate of the conidia suspension group was: 85%, germination rate of control group (CK) is: 71 percent, showing good effect of promoting the germination of the pseudo-ginseng seeds.
Example 5 Cladosporium fungus (Cladosporium tenuissimum) solid microbial inoculum for promoting the agronomic index of Panax notoginseng
Inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108one/mL. 50mL of the above spore suspension was inoculated into 400g of a solid fermentation medium. Culturing at 25-28 deg.C, stirring under aseptic condition after culturing for 3-4 days, and culturing for 4-5 days to obtain solid zymophyte agent. And (3) putting the obtained solid microbial inoculum in a drying oven at the temperature of 30-35 ℃, and drying for 25-30h by blowing for later use. Using distilled water as control, repeating every group for 100 seedlings of Notoginseng radix at an application rate of 0.5-1.0 g/plant, wherein the field biennial Notoginseng radix plant is provided by pharmaceutical plant research institute of agriculture academy of sciences of Yunnan province. Adding the microbial inoculum for 3 monthsThe addition amount is the same as above; the growth of panax notoginseng was observed after 6 months of administration.
Compared with a control group (CK), the root weight and the main root length of the Cladosporium tenuissimum solid microbial inoculum group are remarkably improved, as shown in figure 5, the data with remarkable difference are shown in a table 4, and the good effect of promoting the growth of the panax notoginseng is shown.
TABLE 4 promoting action of solid mycoides of cladosporium fungus on agronomic index of notoginseng
Example 6 Effect of Cladosporium fungus (Cladosporium tenuissimum) solid microbial inoculum on the improvement of notoginsenoside content
The preparation and application of the microbial inoculum are the same as those in example 5, and the content of the panax notoginseng saponins is detected after 6 months of application. The measurement method is referred to the first part of "Chinese pharmacopoeia" of 2015 edition.
Compared with a control group (CK), the Cladosporium tenuissimum solid microbial inoculum group has the advantages that the total saponin content of the solid microbial inoculum group is increased by 16.3 percent compared with the control group (CK), and the effect of promoting the increase of the notoginsenoside content is good.
Claims (10)
1. Cladosporium species (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C)Cladosporium tenuissimum) The application of the cladosporium (A) in improving the content of notoginsenosideCladosporium tenuissimum) The deposit number is: CGMCC number 14126.
2. The use of claim 1, wherein the root irrigation or broadcast application treatment is carried out on the notoginseng by using conidium suspension of the cladosporium strain or strain fermentation liquor or ethyl acetate extract of the strain fermentation liquor or solid microbial inoculum, so as to promote the growth of the notoginseng and improve the content of notoginsenoside.
3. The use according to claim 2, wherein the strain conidia suspension is prepared by: inoculating the cladosporium fungus into a PDA culture medium plate, and performing inverted culture at 25-28 DEG CAnd (3) activating the strain, preparing the activated strain into a spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension has the following concentration: 1.0X 104-1.0×108one/mL.
4. The use according to claim 2, wherein the strain broth is prepared by: inoculating the strain into a PDA culture medium plate, carrying out inverted culture at 25-28 ℃ for 5-8 days, activating the strain, preparing the activated strain into spore suspension by using sterile water, and counting the spore concentration under a microscope, wherein the spore suspension concentration is as follows: 1.0X 104-1.0×108Taking spore suspension to inoculate in a fermentation culture medium PDB, culturing for 5-8 days at 25-28 ℃ and 120-150 r/min.
5. The use according to claim 2, wherein the ethyl acetate extract of the strain broth is prepared by: the broth obtained in claim 4 was extracted with an equal volume of ethyl acetate and the concentrated extract was collected.
6. The use as claimed in claim 2, wherein the strain solid microbial inoculum is prepared by the following method: inoculating the spore suspension obtained in claim 3 into a solid fermentation culture medium, culturing at 25-28 deg.C for 3-4 days, stirring under aseptic condition, stirring, culturing for 4-5 days to obtain solid fermentation microbial inoculum, placing the solid microbial inoculum in a drying oven at 30-35 deg.C, air-drying for 25-30h, drying the solid microbial inoculum to water content of 10-20%, wherein the viable count of the microbial inoculum can reach 1.0-5.0 × 108More than one per gram.
7. The use as claimed in claim 4, wherein the PDA medium is: 100-200 g of potatoes, 5-20 g of glucose and 10-20 g of agar, diluting the mixture to a constant volume of 1L of distilled water, keeping the pH natural, and sterilizing the mixture for 30min at 121 ℃; the PDB culture medium is as follows: 100-200 g of potatoes and 5-20 g of glucose, diluting the mixture to 1L of distilled water with a constant volume, naturally keeping the pH value, and sterilizing the mixture for 30min at 121 ℃.
8. The use according to claim 6, wherein the solid fermentation medium is: 350g of wheat bran 300-350g, 50-100g of corn flour, 5-10g of bean cake powder and KNO3 0.5g，K2HPO4 1.5g，NaCl 1.5g，CaCO34g, adding 400ml of water, and sterilizing at 121 ℃ for 30 min.
9. The use as claimed in claim 2, wherein cladosporium is applied by root irrigation at seedling stage or adult stage of notoginseng; the application is continued for 2-3 times, each time at 3 months interval.
10. The use according to any one of claims 1 to 9, wherein the cladosporium fungus is used alone or in combination with an organic fertilizer.
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| Endophytic fungi harbored in Panax notoginseng: diversity and potential as biological control agents against host plant pathogens of root-rot disease;You-KunZheng等;《Journal of Ginseng Research》;20170731;第41卷(第3期);第353-360页 * |
| Unveiling of Dominant Fungal Pathogens Associated With Rusty Root Rot of Panax notoginseng Based on Multiple Methods;Mi CY等;《PLANT DISEASE》;20171231;第101卷(第12期);第2046-2052页 * |
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