CN112779177A - Compound microbial agent special for saline-alkali soil and application thereof - Google Patents
Compound microbial agent special for saline-alkali soil and application thereof Download PDFInfo
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- CN112779177A CN112779177A CN201911079211.8A CN201911079211A CN112779177A CN 112779177 A CN112779177 A CN 112779177A CN 201911079211 A CN201911079211 A CN 201911079211A CN 112779177 A CN112779177 A CN 112779177A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention relates to a microbial soil conditioning and repairing technology, in particular to a compound microbial agent special for saline-alkali soil and application thereof. The microbial inoculum comprises rhizobium YIC4027 and arbuscular mycorrhizal fungi Glomus Mosseae (GM). The microbial inoculum can effectively improve the antioxidase activity of the wild soybeans so as to reduce the malonaldehyde content of the wild soybeans, so that the salt tolerance of the wild soybeans is improved.
Description
Technical Field
The invention relates to a microbial soil conditioning and repairing technology, in particular to a compound microbial agent special for saline-alkali soil and application thereof.
Background
With the increasing population of the world, the cultivated land resources are increasingly in short supply, and the saline-alkali soil resources become important reserve cultivated land resources. Saline-alkali soil is a general term for saline soil, alkaline earth and various salinized and alkalized soils. The rational improvement and exploitation of saline-alkali soil resources to reduce the decrease of soil productivity caused by soil salinization has received global attention as an available farming land resource. The area of the saline-alkali soil in China reaches 0.99l hundred million hm2The method occupies about 10 percent of the area of the saline-alkali soil in the world, and the agricultural production efficiency of China is seriously influenced by the existence of large-area saline-alkali soil in China, so that a method for effectively preventing and controlling the saline-alkali soil, an improvement measure of the saline-alkali soil and the like are searched at the present stage, and the method has important significance for the reasonable utilization of the saline-alkali soil.
The existing saline-alkali soil improvement measures mainly comprise: (1) chemical measures, namely, a modifier is mainly added into the soil to adjust the pH value of the soil and change the reaction of a soil solution, so that the nutrition condition of the soil and the composition of adsorbability cations of soil colloid are improved; (2) agricultural measures, such as tillage and fertilization, rice improvement, mulching, additional application of organic fertilizer and the like are effective measures for improving saline-alkali soil; (3) irrigation measures, namely irrigation by utilizing saline-alkali water, wherein the main modes comprise circulating irrigation and mixed irrigation; (4) biological measures including plant improvement and microbial improvement.
Among the various saline-alkali soil improvement methods, the plant microorganism combined improvement has the advantages of low cost, large scale, high economic benefit and the like. In the method, a series of life activities of plants are utilized to improve the soil structure, reduce the volume weight of the soil, increase the porosity of the soil, facilitate the water and air permeability of the soil and increase the organic matter and nutrient content of the soil. The plant transpiration can reduce the water evaporation of the soil, so that the simple soil evaporation effect is changed into the plant transpiration effect, the underground water level is effectively reduced, the salt leaching is accelerated, the salt content, the pH value and the ESP of the soil are finally reduced, and the salt accumulation and salt return of the soil are prevented. Meanwhile, microorganisms are various and can be spread in all corners of the soil, activities of the microorganisms often affect all indexes of the soil, and microbial metabolites can provide nutrition for plants so as to promote life activities of the plants; meanwhile, the secretion of the plant can provide a carbon source for the microorganism, thereby promoting the metabolism of the microorganism. Therefore, it is desired to research and explore the soil improvement effect by selecting the microorganism to act on the plant, and further by the interaction between the microorganism and the plant, the salt tolerance of the plant is finally improved.
Disclosure of Invention
The invention aims to provide a compound microbial agent special for saline-alkali soil and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a compound microbial agent special for saline-alkali soil comprises rhizobium YIC4027 and arbuscular mycorrhizal fungi (Glomus mosseae, GM).
The microbial inoculum is a fermentation product or a metabolite respectively obtained by rhizobium YIC4027 or arbuscular mycorrhizal fungi GM, the fermentation product or the metabolite obtained by each strain is respectively mixed with a carrier to respectively obtain solid substances, and the solid substances are mixed according to the mass ratio of 1: 1.
The rhizobium YIC4027 and arbuscular mycorrhizal fungi GM are respectively cultured according to a conventional mode, wherein the viable count of the rhizobium YIC4027 is about 2 multiplied by 107Per gram; the arbuscular mycorrhizal fungi GM culture solution contains about 10 spores per gram. The composite microbial agent is prepared from the following components in a weight ratio of 1:1 and mixing.
The rhizobium solid is prepared by fermenting and culturing rhizobium YIC4027, centrifuging the culture, washing, and resuspending the culture in phosphate buffer solution until the final concentration of the bacterial liquid is 108The amount of the active carbon is one/mL,then mixing the heavy suspension with a carrier to obtain a rhizobium solid; wherein the volume mass ratio of the resuspension to the carrier is 1: 4.
The sand-stone substrate of the arbuscular mycorrhizal fungi Moxisa (Glomus mosseae, GM) is used for expanding propagation by planting clovers in a container, watering is stopped when the clovers grow for about 30 days, the clovers are waited to be naturally dried to death, the substrate is completely dried, the overground part is cut off, root systems, stems and leaves of the clovers are completely cut into pieces and are fully and uniformly mixed with the substrate, and a large amount of solid microbial inoculum containing the arbuscular mycorrhizal fungi GM can be obtained.
The arbuscular mycorrhizal fungus GM is Glomus mosseae (Glomus mossea) and is provided for research by plant nutrition and resource research institute (Beijing, China).
The rhizobium YIC4027 and arbuscular mycorrhizal fungi GM are respectively cultured according to a conventional mode, wherein the viable count of the rhizobium YIC4027 is about 2 multiplied by 107Per gram; the arbuscular mycorrhizal fungi GM inoculum contains about 20 spores per gram. The composite microbial agent is prepared from the following components in a weight ratio of 1:1 and mixing.
The application of the microbial agent special for saline-alkali soil is to promote the growth of leguminous plants under the salt stress of the saline-alkali soil.
The leguminous plant growth promoting method is characterized in that the weight of a single leguminous plant is increased and the plant height of the leguminous plant is increased under the salt stress of saline and alkaline land.
The leguminous plant is wild soybean.
The microbial inoculum is applied to the soil according to the proportion that 10-20g of solid microbial inoculum is applied to 1kg of soil.
The invention has the following beneficial effects:
1. the arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 in the microbial agent can respectively promote the growth of the wild soybeans under the condition of salt stress. The microbial inoculum containing the compound bacterial strains has obviously higher growth promotion effect on the wild soybeans than the wild soybeans inoculated with a single bacterial strain.
2. The compound microbial inoculum is applied to soil, so that the nodulation number and mycorrhiza infection rate of the wild soybeans are increased.
3. Reactive oxygen species, which are the products of salt stress, accumulate to cause oxidation, damage and ultimately programmed cell death of the lipid membranes, proteins and nucleic acids of the plant cells. The malondialdehyde content of the wild soybean group adopting the composite microbial inoculum is obviously lower than that of the wild soybean group which is not inoculated, and simultaneously, the content of the antioxidant enzyme activity of the wild soybean group is also obviously higher than that of the wild soybean group which is not inoculated, so that the composite microbial inoculum can effectively improve the antioxidant enzyme activity of the wild soybean so as to reduce the malondialdehyde content of the wild soybean, and improve the salt tolerance of the wild soybean.
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
Compared with non-inoculated wild soybeans, under the salt stress of NaCl concentration of 100 and 200mmol/L, the dry weight of stems and roots is increased by about 80%, the chlorophyll content is increased by 22-44%, the nodulation number is increased by about 50%, the infection rate is increased by about 23%, the malondialdehyde content is obviously lower than that of non-inoculated wild soybean groups, and simultaneously, the content of the antioxidase activity of the compound microbial inoculum is also obviously higher than that of the non-inoculated wild soybean groups. The composite microbial inoculum can effectively improve the antioxidase activity of the wild soybeans so as to reduce the malonaldehyde content of the wild soybeans, so that the salt tolerance of the wild soybeans is improved.
Example 1
Preparation of the microbial inoculum:
obtaining a rhizobium solid bacterial fertilizer:
activating rhizobium YIC4027 overnight according to a conventional rhizobium mode, inoculating the rhizobium YIC4027 into a fermentation culture medium for culturing for 12h, centrifugally collecting bacterial liquid, washing the bacterial liquid by using a phosphate buffer solution, and suspending the bacterial liquid until the final concentration of the bacterial liquid is 108one/mL. And mixing 800g of the grass carbon carrier with 200mL of the rhizobium bacterial liquid to obtain the rhizobium solid bacterial fertilizer.
The formula of the fermentation medium (L3+ N) is as follows: KH in 1L water2PO4 1.36mg，MgSO4 100mg，NaCl 50mg，CaCl2 40mg，FeCl3 5.4mg，Na2MoO45mg, biotin 2mg, nicotinic acid 4mg, pantothenic acid 4mg, succinic acid 1.18g, NH4Cl 0.53mg, wherein CaCl2，FeCl3Biotin, nicotinic acid and pantothenic acid were filtered through a 0.22 μ M filter and the other components were sterilized at 121 ℃.
The rhizobium YIC4027 is separated from sesbania nodules growing in saline-alkali soil, belongs to aerobic gram-negative bacteria, has the optimal growth temperature of 28 ℃ and the optimal pH value of 7.0, can tolerate the concentration of 4 percent NaCl, forms white and opaque circular protruding colonies on a YMA culture medium, can form effective nodules on an original host, and cannot form effective nodules on plants such as sophora flavescens, clover, liquorice, kidney beans, alfalfa and the like. Rhizobium YIC4027 is preserved in the institute of microbiology of Chinese academy of sciences with the preservation number of CGMCC No. 10994. And is described in the document entitled Rhizobium and uses thereof, application No. 201510585141.9.
Obtaining an arbuscular mycorrhizal fungi GM solid bacterial fertilizer:
(1) sterilizing vermiculite at high temperature, placing into an expanding propagation container to about 3/5, adding sandstone matrix of arbuscular mycorrhizal fungi GM according to the inoculation amount of 1%, sowing clover seeds in the container every 2cm, covering a layer of sterilized vermiculite matrix to 4/5, and completely watering the matrix in each pot thoroughly.
(2) The container is moved into a greenhouse, a layer of preservative film is covered on the container for water retention and heat preservation, and a plurality of small holes are punched on the preservative film for ventilation. Controlling the temperature at 20-35 ℃ during the culture management period, and watering once every 3 days with a proper amount each time. When the seedling emergence of the seeds is against the preservative film, the preservative film can be removed.
(3) And (3) stopping watering when the clovers grow for about 30-40 days, waiting for the clovers to naturally dry and die, completely drying the matrix, cutting off the overground part, completely cutting root systems, stems and leaves of the clovers, and the like, and fully mixing the ground materials with the matrix to obtain a large amount of the matrix containing the arbuscular mycorrhizal fungi GM for subsequent experiments.
The arbuscular mycorrhizal fungus GM is Glomus mosseae (Glomus mossea) and is provided for research by plant nutrition and resource research institute (Beijing, China).
Mixing the obtained rhizobium solid microbial inoculum and arbuscular mycorrhizal fungi GM solid microbial inoculum according to the weight ratio of 1:1, and mixing to obtain the compound microbial inoculum.
Application example
The obtained rhizobia solid microbial inoculum, the arbuscular mycorrhizal fungi GM solid microbial inoculum obtained in the embodiment and the compound microbial inoculum obtained in the embodiment are respectively used as experimental groups, and meanwhile, the non-added microbial inoculum is used as a control; 4 different microbial inoculum inoculation treatments and 3 different salt concentration treatments are set.
Experimental setup: the method comprises the steps of planting wild soybeans under 3 different salt concentrations of 0, 100 and 200mmol/L, respectively adding rhizobia agents, arbuscular mycorrhizal fungi GM agents, compound agents and control without the agents under each salt concentration, measuring the chlorophyll content, the growth of roots, the activity of superoxide dismutase and the content of malondialdehyde of the wild soybeans under 4 experimental treatments under different salt stresses, analyzing the influence of inoculated arbuscular mycorrhizal fungi GM on soybean nodulation and the influence of inoculated rhizobia YIC4027 on the infection rate of arbuscular mycorrhizal fungi GM, and comprehensively analyzing the advantageous effects of the compound agents.
When the single strain is applied, the application amount of the rhizobium YIC4027 microbial inoculum is 5g of rhizobium microbial inoculum inoculated in each 1kg of soil; the application amount of the arbuscular mycorrhizal fungi GM microbial inoculum is 5g of microbial inoculum in 1kg of soil; when the microbial inoculum is a compound microbial inoculum, the application amount is 10g of the compound microbial inoculum inoculated in every 1kg of soil.
3.1 determination of growth index of wild Soybean
After the cotyledon falls off, the chlorophyll content of the wild soybean leaf was measured by a chlorophyll meter. After growing for a certain time, cutting off the roots of the fresh plants from the leaf marks of the first true leaves, measuring the height and fresh weight of the overground part, and simultaneously measuring physiological indexes such as root fresh weight, main root length, lateral root length, root stem thickness and the like. Deactivating enzyme in a 105 deg.C oven for 12min, baking to constant weight at 80 deg.C, and weighing to obtain dry weight. The above indexes are average values of multiple wild soybean plants.
3.1.1 chlorophyll content of wild Soybean
After the cotyledon falls off, the chlorophyll content of the wild soybean leaf in each treatment was measured by a chlorophyll meter. As can be seen from table 3, with increasing salt concentration, the chlorophyll content of both the uninoculated group of wild soybeans and the group of wild soybeans inoculated with only rhizobia YIC4027 gradually decreased, and the difference between the chlorophyll content of the two groups of treated wild soybeans was smaller at the same salt concentration. At salt concentrations of 0, 100, 200mmol/L, chlorophyll content of the wild soybean group inoculated with only arbuscular mycorrhizal fungus GM and the wild soybean group inoculated with both arbuscular mycorrhizal fungus GM and Rhizobium YIC4027 was significantly increased compared to chlorophyll content of the other two groups of treated wild soybeans.
3.1.2 root and Stem growth indices of wild Soybean
After growing for a certain time, cutting off the roots of the fresh plants from the leaf marks of the first true leaves, measuring the height and fresh weight of the overground part, and simultaneously measuring physiological indexes such as root fresh weight, main root length, lateral root length, root stem thickness and the like. Deactivating enzyme in a 105 deg.C oven for 12min, baking to constant weight at 80 deg.C, and weighing to obtain dry weight. The above indexes are average values of multiple wild soybean plants.
As can be seen from Table 1, the dry weight, fresh weight and length of the roots and stems of the respective groups of wild soybeans gradually decreased with increasing salt concentration.
In the group with 0mmol/L NaCl concentration, the fresh stem weight, dry stem weight and plant height of the wild soybean inoculated with only arbuscular mycorrhizal fungi GM are respectively about 1.37, 1.34 and 1.29 times of those of the non-inoculated wild soybean, and the fresh root weight, dry root weight and root length are respectively about 1.11, 1.19 and 1.09 times of those of the non-inoculated wild soybean group. The fresh stem weight, dry stem weight and plant height of the wild soybeans inoculated with the rhizobium YIC4027 are respectively 1.20, 1.22 and 1.09 times of those of the non-inoculated wild soybeans, and the fresh root weight, dry root weight and root length of the wild soybeans are respectively 1.40, 1.29 and 1.16 times of those of the non-inoculated wild soybeans. The fresh stem weight, dry stem weight and plant height of the wild soybeans inoculated with the arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 are respectively 1.83 times, 1.92 times and 1.51 times of those of the non-inoculated wild soybeans, and the fresh root weight, dry root weight and root length of the wild soybeans are respectively 1.71 times, 1.68 times and 2.63 times of those of the non-inoculated wild soybeans.
In the group with NaCl concentration of 100mmol/L, the stem fresh weight, stem dry weight and plant height of the wild soybean inoculated with only arbuscular mycorrhizal fungi GM are respectively 1.22 times, 1.28 times and 1.28 times of those of the non-inoculated wild soybean, and the root fresh weight, root dry weight and root length are respectively 1.15 times, 1.32 times and 1.11 times of those of the non-inoculated wild soybean group. The fresh stem weight, dry stem weight and plant height of the wild soybeans inoculated with the rhizobium YIC4027 are respectively 1.11 times, 1.21 times and 1.08 times of those of the non-inoculated wild soybeans, and the fresh root weight, dry root weight and root length of the wild soybeans are respectively 1.27 times, 1.41 times and 1.19 times of those of the non-inoculated wild soybeans. The fresh stem weight, dry stem weight and plant height of the wild soybeans inoculated with the arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 are respectively 1.63, 1.84 and 1.40 times of those of the non-inoculated wild soybeans, and the fresh root weight, dry root weight and root length of the wild soybeans are respectively 1.77, 1.57 and 1.31 times of those of the non-inoculated wild soybeans.
In the group with NaCl concentration of 200mmol/L, the stem fresh weight, stem dry weight and plant height of the wild soybean inoculated with only arbuscular mycorrhizal fungi GM are respectively 1.36, 1.20 and 1.35 times of those of the non-inoculated wild soybean, and the root fresh weight, root dry weight and root length are respectively 1.14, 1.18 and 1.07 times of those of the non-inoculated wild soybean group. The stem fresh weight, stem dry weight and plant height of the wild soybeans inoculated with the rhizobium YIC4027 are respectively 1.21, 1.19 and 1.09 times of those of the non-inoculated wild soybeans, and the root fresh weight, root dry weight and root length of the wild soybeans are respectively 1.32, 1.32 and 1.18 times of those of the non-inoculated wild soybeans. The fresh stem weight, dry stem weight and plant height of the wild soybeans inoculated with the arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 are respectively 1.64, 1.60 and 1.54 times of those of the non-inoculated wild soybeans, and the fresh root weight, dry root weight and root length of the wild soybeans are respectively 1.45, 1.60 and 1.32 times of those of the non-inoculated wild soybeans.
The dry stem weight, fresh weight, height and dry root weight, fresh weight and length of the wild soybeans inoculated with only the 3 groups of arbuscular mycorrhizal fungi GM, rhizobia YIC4027, arbuscular mycorrhizal fungi GM and rhizobia YIC4027 were superior to those of the uninoculated wild soybeans, wherein the effect of the wild soybeans inoculated with the arbuscular mycorrhizal fungi GM and rhizobia YIC4027 was the most superior. The results show that the wild soybean inoculated with the arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 can effectively promote the growth of the wild soybean and improve the salt tolerance of the wild soybean to a certain extent, and meanwhile, the arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 have a synergistic promotion relationship, and the synergistic promotion relationship between the microorganisms enables the promotion relationship of the double-inoculated arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 on the wild soybean to be better than that of a single strain.
3.1.3 root nodule count of Glycine max
As can be seen from Table 3, the number of nodules of the root of the wild soybean decreased significantly with increasing NaCl concentration, and the weight of the nodules and the size of the individual nodules also decreased with increasing salt concentration. The number of nodules of the wild soybean group inoculated with the arbuscular mycorrhizal fungus GM is higher than that of the wild soybean group inoculated with only the rhizobium YIC4027 under the condition of 3 salt concentrations. Double inoculation of arbuscular mycorrhizal fungi GM and rhizobium Y406 can increase the nodulation capacity of wild soybeans.
3.2 wild Soybean mycorrhiza infection rate
3.2.1 method for determining mycorrhiza infection rate of wild soybean
The mycorrhizal infection rate of the wild soybean is determined by adopting a trypan blue staining method
Mycorrhiza infection rate (%) - (number of infected root segments) × 100/(total studied root segments)
(1) Cutting the cleaned root system with proper thickness (the diameter of the root system is about 1-2 mm) into root sections of about 1cm, putting the root sections into a triangular flask, adding 10% KOH, and heating in a water bath at 90 ℃ for 15-30 min (the time required by young roots is about 15min, the time required by old roots is about 30min, and taking the relative transparency of the root system as a standard).
(2) Discarding the alkali liquor, washing with clear water for 3-5 times (drying to room temperature and then washing), adding 2% hydrochloric acid, and acidifying at room temperature for 5 min.
(3) 0.05% trypan blue staining solution (0.5 g trypan blue, 200mL distilled water, 200mL glycerin, 600mL lactic acid per 1L of 0.05% trypan blue staining solution) was added and heated in a water bath at 90 ℃ for 30 min.
(4) After trypan blue was removed, the solution was discarded and washed with tap water, and a lactic acid glycerol solution (lactic acid:
glycerol: water is prepared according to the volume of 1:1: 1) is decolorized for 24 hours at room temperature.
(5) The 15 strips were picked up with tweezers and arranged on glass slides, 2 pieces per sample, and microscopically examined (10X 10).
3.2.2 analysis of results on the mycorrhizal infection ratio of wild Soybean
As can be seen from table 3, in the wild soybean groups having NaCl concentrations of 0, 100, and 200mmol/L, respectively, the mycorrhizal infection rate of the wild soybean group inoculated with only arbuscular mycorrhizal fungus GM gradually decreased to some extent with the increase in salt concentration, but did not significantly differ as a whole. The mycorrhizal infection rate of the wild soybean group inoculated with the arbuscular mycorrhizal fungi GM and the rhizobia YIC4027 in a double way is slightly higher than that of the wild soybean group inoculated with the arbuscular mycorrhizal fungi GM only, and is respectively improved by 14.4%, 21.2% and 25.8% under 3 different NaCl concentrations of 0, 100 and 200mmol/L, and further, the interaction result of the rhizobia YIC4027 and the arbuscular mycorrhizal fungi GM is better than that of the two fungi in a single action mode.
3.3 determination of the antioxidase Activity of wild Soybean
3.3.1 extraction of assay samples
Weighing 0.2g of leaf, grinding into powder with liquid nitrogen, adding 5mL of 50mM phosphate buffer (pH7.8), adding 2% PVP and a small amount of quartz sand, grinding into homogenate in ice bath, centrifuging at 4 deg.C and 12000rpm for 20min, and collecting supernatant as crude enzyme extract for determination of antioxidase activity.
3.3.2 determination of superoxide dismutase
3.3.2.1 method for measuring superoxide dismutase
In this study, Superoxide Dismutase (SOD) was measured by a Nitrobluetetrazolium (NBT) photochemical reduction method.
(1) Taking 4 test tubes, marking as S1, S2, S3 and S4, wherein 2 (S1 and S2) are measuring tubes, and the other 2 (S3 and S4) are control tubes, and marking.
(2) The various solutions were added as follows
Table for preparing SOD sample for measuring Nitrogen Blue Tetrazole (NBT) photochemical reduction method
(3) The control group S4 was placed in the dark, and after the other tubes were exposed to 4000lx sunlight for 20min, the reaction was stopped in the dark, and the OD was measured immediately560。
(4) The absorbance inhibited the photochemical reduction of NBT to 50% as one unit of enzyme activity.
In the formula: total SOD activity-expressed in enzyme units of fresh mass per gram of sample (U/gFW);
ACK-the absorbance of the light control tube;
AE-absorbance of the sample tube;
v-total volume of sample fluid (mL);
VT-determining the sample volume (mL);
FW-fresh weight of sample (g)
3.3.2.2 content analysis of superoxide dismutase
As can be seen from Table 4, there was no significant difference in the superoxide dismutase activity of 4 groups of differently treated wild soybeans at a NaCl concentration of 0 mmol/L; at a NaCl concentration of 100 and 200mmol/L, the SOD activity of the ungerminated group was close to that of the group of wild soybeans inoculated with only the arbuscular mycorrhizal fungus GM, while the SOD activity of the group of wild soybeans inoculated with only the rhizobia YIC4027 and the group of wild soybeans inoculated with both the arbuscular mycorrhizal fungus GM and the rhizobia YIC4027 was slightly higher than that of the other two groups, about 1.3 times that of the other two groups.
3.3.3 determination of Catalase
3.3.3.1 method for measuring catalase
(1) 3 of 10mL test tubes were designated S1, S2, and S3, respectively, 2 (S1, S2) being sample measurement tubes and 1 (S3) being blank tubes. The reaction system contained 1.5mL of phosphate buffer, 1mL of distilled water and 0.2mL of crude enzyme solution.
(2) The tube S3 was boiled in boiling water bath for 1min to kill the enzyme solution and cooled.
(3) After all tubes were preheated at 25 deg.C, 0.3mL of 0.1mol/L H was added tube by tube2O2And timing immediately after adding 1 tube, quickly pouring into a quartz cuvette, measuring the absorbance at 240nm, reading for 1 time every 1min, and measuring for 4 min.
(4) Within 1min A240The amount of enzyme reduced by 0.1 is 1 enzyme activity unit.
In the formula: delta A240The difference between the light absorption value of the control tube and the light absorption value of the sample tube
VT-total volume of crude enzyme extract (mL);
V1-determining the volume (ml) of crude enzyme solution;
FW-sample fresh weight (g);
0.1——A240every 0.1 reduction is 1 enzyme activity unit (U);
t-time to last reading (min) of hydrogen peroxide addition.
3.3.3.2 determination of catalase content
This example was divided into a non-inoculated wild soybean group, a wild soybean group inoculated with only arbuscular mycorrhizal fungus GM, a wild soybean group inoculated with only rhizobia YIC4027, a wild soybean group inoculated with both arbuscular mycorrhizal fungus GM and rhizobia YIC 4027. The salt concentration is 0, 100 and 200 mmol/L.
As can be seen from Table 4, the catalase activity of the wild soybean group inoculated with only Rhizobium YIC4027 and the wild soybean group inoculated with both arbuscular mycorrhizal fungi GM and Rhizobium YIC4027 was higher than that of the wild soybeans treated in the other two groups, about 1.7 times that of the other two groups at a NaCl concentration of 100mmol/L and about 1.4 times that of the other two groups at a NaCl concentration of 200mmol/L, as the salt concentration increased.
3.3.4 peroxidase Activity.
3.3.4.1 method for measuring peroxidase
In this study, the guaiacol method was used to measure Peroxidase (POD).
(1) 3 10mL test tubes were sampled and labeled, 2 (S1, S2) sample tubes, and 1 (S3) control tube. The reaction system was 2.9mL of 50mM phosphate buffer, 1mL of 2% H2O21mL of 0.05M guaiacol and 0.1mL of crude enzyme solution, and the control tube was charged with the addition of the inactivated crude enzyme solution.
(2) Adding enzyme solution, immediately preserving heat in 34 deg.C water bath for 3min, and rapidly diluting to 1 time at 470nm
The absorbance A470 was recorded 1 time every 1min for a total of 5 times for wavelength colorimetry.
(3) In minute A470The change 0.01 is 1 enzyme activity unit.
In the formula: delta A470-change in absorbance over reaction time;
VT-total volume of crude enzyme extract (mL);
VS-determining the volume (mL) of crude enzyme solution;
FW-sample fresh weight (g);
0.01——A470every 0.01 reduction is 1 enzyme activity unit (U);
t-time to last reading (min) of enzyme addition.
3.3.4.2 Activity assay of peroxidase
As can be seen from Table 4, the peroxidase activity of the 4 groups of differently treated wild soybeans did not differ significantly at a NaCl concentration of 0 mmol/L; when the NaCl concentration is 100mmol/L, the peroxidase activity of the non-inoculated wild soybean group is similar to that of the wild soybean group inoculated with only arbuscular mycorrhizal fungi GM,
the catalase activity of the wild soybean group inoculated with only Rhizobium YIC4027 and the wild soybean group inoculated with the arbuscular mycorrhizal fungi GM and the Rhizobium YIC4027 was higher than that of the wild soybeans treated in the other two groups, and was about 1.3 times that of the two groups at a NaCl concentration of 100mmol/L and about 1.5 times that of the two groups at a NaCl concentration of 200 mmol/L.
3.4 malondialdehyde content of wild soybean
3.4.1 measurement of malondialdehyde content
(1) 4 dry clean test tubes were sampled and numbered, 2 (S1, S2) sample tubes, 1 (S3) standard tubes, and 1 (S4) blank tubes. And respectively taking 2mL of sample to be detected and 2mL of TBA reaction solution for reaction, adding distilled water into a blank tube as a negative control, and adding a standard substance into a standard tube as a positive control.
(2) The solution in the test tube was shaken well, then subjected to a water bath at 95 ℃ for 30min, and then immediately placed in an ice bath for cooling. After centrifugation at 1500rpm for 10min at 4 ℃ the absorbance at 530nm was measured.
(3) The MDA content per gram fresh weight is calculated according to the formula as follows.
In the formula: a. the530-absorbance of the sample tube;
Ablank space-absorbance of blank tube;
Astandard of merit-absorbance of a standard tube;
10-Standard concentration (nmol/mL);
c-sample concentration (gFW/mL).
3.4.2 malonaldehyde content analysis
As can be seen from table 4, the malondialdehyde content in the wild soybeans showed an overall rising trend with increasing salt concentration. The malondialdehyde content was lowest in the groups of wild soybeans inoculated with the arbuscular mycorrhizal fungus GM and Rhizobium YIC4027 at NaCl concentrations of 0,100 mmol/L, but overall there was no significant difference between the 4 groups of differently treated wild soybeans. When the NaCl concentration is 200mmol/L, the malondialdehyde content of the non-inoculated wild soybean group is obviously higher than that of the wild soybeans subjected to inoculation treatment of the other 3 groups, which indicates that the wild soybeans in the group are stressed most seriously.
TABLE 1 fresh Stem weight, Dry Stem weight, plant height of wild Soybean at different salt concentrations
TABLE 2 fresh root weight, Dry root weight, root length of wild soybeans at various salt concentrations
TABLE 3 chlorophyll content, nodulation number and infection rate of wild soybean at different salt concentrations
TABLE 4 Oxidation preventive Activity and malondialdehyde content of wild soybeans at different salt concentrations
Claims (8)
1. A compound microbial agent special for saline-alkali soil is characterized in that: the microbial inoculum comprises rhizobium YIC4027 and arbuscular mycorrhizal fungi Glomus Mosseae (GM).
2. The microbial agent special for saline-alkali soil according to claim 1, which is characterized in that: the microbial inoculum is a fermentation product or a metabolite respectively obtained from rhizobium YIC4027 or arbuscular mycorrhizal fungi GM, each strain and the fermentation product or the metabolite thereof are respectively mixed with a carrier to respectively obtain solid substances, and then the solid substances are mixed according to the mass ratio of 1: 1.
3. The microbial agent special for saline-alkali soil as claimed in claim 2, which is characterized in that: the rhizobium solid is prepared by fermenting and culturing rhizobium YIC4027, and culturingThe material is washed after centrifugation and is resuspended in phosphate buffer until the final concentration of the bacterial liquid is 108Mixing the resuspension with a carrier to obtain a rhizobium solid; wherein the volume mass ratio of the resuspension to the carrier is 1: 4.
4. The microbial agent special for saline-alkali soil as claimed in claim 2, which is characterized in that: the arbuscular mycorrhizal fungi GM solid is a sand-stone substrate containing 1% of arbuscular mycorrhizal fungi GM, clover is planted on the substrate for propagation, watering is stopped when the clover grows for about 30 days, when the clover naturally dries up to death and the substrate dries up completely, root systems and stems and leaves of the clover are completely cut up and are fully mixed with the substrate, and a large amount of solid microbial inoculum containing the arbuscular mycorrhizal fungi GM with the spore number of about 20/g can be obtained.
5. The application of the special microbial agent for saline-alkali soil as claimed in claim 1 is characterized in that: the microbial inoculum is applied to promoting the growth of leguminous plants under the salt stress of saline-alkali soil.
6. The application of the special microbial agent for saline-alkali soil as claimed in claim 5, is characterized in that: the leguminous plant growth promoting method is characterized in that the weight of a single leguminous plant is increased and the plant height of the leguminous plant is increased under the salt stress of saline and alkaline land.
7. The application of the special microbial agent for saline-alkali soil as claimed in claim 5 or 6, which is characterized in that: the leguminous plant is wild soybean.
8. The use method of the special microbial agent for saline-alkali soil as claimed in claim 1, which is characterized in that: the microbial inoculum is applied to the soil according to the proportion that 10-20g of solid compound microbial inoculum is applied to 1kg of soil.
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| CN1425279A (en) * | 2001-12-12 | 2003-06-25 | 中国科学院沈阳应用生态研究所 | Structural method for nitrogen-fixing plants combined symbiont |
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| CN107493896A (en) * | 2017-09-06 | 2017-12-22 | 河南科技大学 | The method grown using AMF promotion sugar grass in salt-soda soil |
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| CN1425279A (en) * | 2001-12-12 | 2003-06-25 | 中国科学院沈阳应用生态研究所 | Structural method for nitrogen-fixing plants combined symbiont |
| CN104893991A (en) * | 2015-06-26 | 2015-09-09 | 北京农学院 | Microorganism composite preparation and application thereof |
| CN105062935A (en) * | 2015-09-15 | 2015-11-18 | 中国科学院烟台海岸带研究所 | Rhizobium and application thereof |
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