Pseudomonas compound microbial agent and application thereof in disease-resistant, yield-increasing and quality-improving of angelica sinensis
1. The compound microbial agent is characterized by comprising pseudomonas fluorescens, pseudomonas alcaligenes and pseudomonas psychrophile.
2. The complex microbial inoculant according to claim 1, which consists of pseudomonas fluorescens CBS5, pseudomonas alcaligenes CBS7 and pseudomonas psychrophila CBSB; the 16S rDNA sequence of the pseudomonas fluorescens CBS5 is shown in SEQ ID NO. 1; the 16S rDNA sequence of the pseudomonas alcaligenes CBS7 is shown in SEQ ID NO.2, and the 16S rDNA sequence of the pseudomonas psychrophila CBSB is shown in SEQ ID NO. 3.
3. Use of the complex microbial inoculant of claim 1 or 2 for promoting plant growth.
4. The use of claim 3, wherein the plant is Angelica sinensis.
5. The use of the complex microbial inoculant according to claim 1 or 2 for controlling plant diseases.
6. The use according to claim 5, wherein the plant is Angelica sinensis, and the disease comprises Fusarium pathogenic fungi causing rot and blight.
7. The use of the complex microbial inoculant as defined in claim 1 or 2 for promoting yield increase and quality improvement of angelica sinensis.
8. The use of claim 7, wherein said complex microbial agent is used for increasing the content of marker components and/or endogenous hormones in angelica sinensis, wherein said marker components comprise ferulic acid, Z-ligustilide, senkyunolide A; the endogenous hormones comprise salicylic acid and jasmonic acid.
9. A pesticide for controlling plant diseases, which comprises the complex microbial agent of claim 1 or 2.
10. A microbial fertilizer specially used for angelica sinensis, which is characterized by comprising the compound microbial agent of claim 1 or 2.
Background
The mode of 'ecological planting of traditional Chinese medicinal materials' proposed by the research team of the guo lan duckweed, the Ministry of traditional Chinese medicine science, Huang Lu Qi Yao, not only emphasizes the field ecological conditions, but also more emphasizes the input of agricultural material input products, and is the main mode of traditional Chinese medicinal material production at present. The method pays attention to the recycling of wastes and the diversity of species, does not use chemically synthesized chemical fertilizers, pesticides and plant growth regulators, and advocates the use of products of animal sources, plant sources, microbial sources, natural minerals and the like as basic requirements.
Plant growth-promoting rhizobacteria (PGPR) refers to a beneficial bacterium that lives in the rhizosphere of plants to promote plant growth or antagonize pathogenic bacteria. Can improve the soil environment of plant rhizosphere, improve the content of available nutrients, degrade toxic substances, secrete disease-resistant growth-promoting substances, induce the interaction of the bacteria and the plants, and have obvious influence on the growth and development of the whole plant, disease resistance and growth promotion. The production of the traditional Chinese medicinal materials not only pays attention to the yield, but also pays attention to the quality, so that the problems of growth promotion, disease resistance and quality improvement need to be comprehensively considered for developing the microbial inoculum special for the traditional Chinese medicinal materials.
Angelica sinensis (Oliv.) Diels, a dried root of Angelica sinensis (Oliv.) Diels, a plant belonging to the family Umbelliferae, is a traditional bulk Chinese medicine, and is listed in the homology of medicine and food. Gansu is the region of Angelica sinensis production in the field, and the planting area of the whole province reaches 400km2Yield 1.2X 105kg, which accounts for more than 80% of the total supply quantity of the national market, and plays a decisive role in the national market price of Chinese angelica. The problems of heavy input of chemical fertilizers and pesticides, poor stability of a biological system, low diversity of soil microorganisms, continuous cropping obstacles and the like generally exist in the artificial cultivation traditional Chinese medicines such as angelica and the like, and the development of the functional microbial agent for the angelica is imperative. The prior art already discloses that the growth-promoting bacterium Bacillus spKTS-1-1 can promote the activity enhancement of the antioxidant enzyme system of the radix pseudostellariae and the increase of the hormone content, so as to promote the growth (see the influence of the growth-promoting bacterium Bacillus sp. KTS-1-1 and a nitrogen-phosphorus-potassium compound fertilizer on the growth and metabolism of the radix pseudostellariae in 2020); the growth promoting effect of the Pseudomonas R28NF 1-17 and brevibacterium frigidum NF2-4 composite microbial inoculum on the sophora flavescens is remarkable (see the separation of the high-efficiency azotobacter at the rhizosphere of the sophora flavescens and the growth promoting effect of composite bacterial fertilizer on seedlings, 2020); pseudomonas fluorescens N21.4 is inoculated on blackberry plants, so that the flavonoid metabolism is enhanced, and the content of catechin in fruits is increased by 1.1-1.8 times (see "Metabolic organisms of Pseudomonas fluorescens N21.4 elicti vitamin in blackberry front", 2021); bacillus subtilis XG-2 and Bacillus velezensis XG-3 separated from rhizosphere soil can promote growth of angelica sinensis and biomass accumulation, and increase content of butenyl phthalide (see' Impact of Bacillus on phenols Accu)Immunization in Angelica sinensis (Oliv.) by Stoichimetry and microbiological university Analysis, 2021). Therefore, different PGPR have different mechanisms on the growth, yield and quality of medicinal plants, the final yield amplification result is different, and different quality indexes have different influences on different components.
In addition, the study in the earlier stage of the subject group finds that after angelica sinensis is treated by the microorganisms disclosed in the prior art, the phenomena of increasing adventitious roots generally exist, and the processing loss of decoction pieces is increased. However, the appearance of the commercial Chinese angelica is good in terms of few rootlets, thick reed rhizome and long reed rhizome. Based on the problems, the development of the fertilizer special for angelica needs to consider not only the yield, the disease resistance and the quality, but also the appearance and the character of the root of angelica. In addition, the existing literature reports that the contents of endogenous salicylic acid and jasmonic acid in plants are in negative correlation, namely, salicylic acid and jasmonic acid in angelica can not be simultaneously improved. At present, no microbial agent capable of promoting the growth of angelica, improving disease resistance, improving the appearance quality of angelica and the content of various marking components is available.
Disclosure of Invention
Aiming at the technical problem, the invention provides a compound microbial agent of pseudomonas, which is prepared by compounding 3 bacteria (pseudomonas fluorescens, pseudomonas alcaligenes and pseudomonas psychrophile) of pseudomonas of proteobacteria; the compound microbial agent has ACC deaminase activity, SOD activity, POD activity, NEX neutral xylanase activity, chitinase activity and laccase activity; the compound microbial agent promotes the growth of medicinal organ roots of underground parts of the angelica, increases the diameter of the reed head, the length of the main root and the average individual plant weight, not only increases the yield, but also improves the appearance of the commercial angelica; the compound microbial agent improves the content of marked components of ferulic acid, Z-ligustilide and senkyunolide A of the angelica, and the content of endogenous hormones such as jasmonic acid, salicylic acid and the like, can obviously improve the quality of the angelica, and can be used for preparing a special microbial fertilizer for the angelica; the compound microbial agent can prevent and treat plant diseases caused by fusarium pathogenic fungi, and can be used for preparing pesticides for preventing and treating the plant diseases. The method specifically comprises the following steps:
in a first aspect, the invention provides a complex microbial inoculant comprising pseudomonas fluorescens, pseudomonas alcaligenes and pseudomonas psychrophila.
Preferably, the compound microbial agent consists of pseudomonas fluorescens CBS5, pseudomonas alcaligenes CBS7 and pseudomonas psychrophila CBSB; the 16S rDNA sequence of the pseudomonas fluorescens CBS5 is shown in SEQ ID NO. 1; the 16S rDNA sequence of the pseudomonas alcaligenes CBS7 is shown in SEQ ID NO.2, and the 16S rDNA sequence of the pseudomonas psychrophila CBSB is shown in SEQ ID NO. 3. The pseudomonas fluorescens CBS5, the pseudomonas alcaligenes CBS7 and the pseudomonas psychrophila CBSB are purchased from Gansu division center of China center for preservation and management of industrial microorganism strains.
In a second aspect, the present invention provides the use of the complex microbial inoculant described in the first aspect for promoting plant growth.
Preferably, the plant is angelica; the compound microbial agent not only promotes the growth of medicinal organ roots of underground parts of the angelica, but also increases the diameter of the reed head, the length of the main root and the average individual plant weight, and improves the appearance of the commercial angelica while increasing the yield.
In a third aspect, the invention provides the use of the complex microbial agent of the first aspect in controlling plant diseases.
Preferably, the plant is angelica.
Preferably, the disease comprises a rot disease or a blight disease caused by a pathogenic fungus of the genus fusarium.
In a fourth aspect, the invention provides an application of the compound microbial agent of the first aspect in promoting yield increase and quality improvement of angelica sinensis.
Preferably, the compound microbial agent is used for increasing the content of a marked component and/or endogenous hormone in angelica, wherein the marked component comprises ferulic acid, Z-ligustilide and senkyunolide A; the endogenous hormones comprise salicylic acid and jasmonic acid; the compound microbial agent can improve salicylic acid in angelica sinensis and the content of jasmonic acid in angelica sinensis, and has unexpected effects compared with the negative correlation of salicylic acid and jasmonic acid disclosed by the prior art; the composite microbial agent can obviously improve the content of endogenous salicylic acid and the content of jasmonic acid in plants, reduce the content of auxin and simultaneously does not influence the content of gibberellin.
In a fifth aspect, the present invention provides a pesticide for controlling plant diseases, which comprises the complex microbial agent of the first aspect.
Preferably, the pesticide contains the microbial strain with the CFU number of 106～109/ml。
In a sixth aspect, the invention provides a special microbial fertilizer for angelica sinensis, which comprises the compound microbial agent of the first aspect.
The invention has the beneficial effects that:
(1) the invention provides a compound microbial agent consisting of pseudomonas microorganisms, which has ACC deaminase activity, SOD activity, POD activity, NEX neutral xylanase activity, chitinase activity and laccase activity, wherein the ACC deaminase, SOD, POD and laccase can remove substances inhibiting growth such as ethylene, active oxygen, phenol amines and the like, the chitinase can degrade fungal cell walls and improve the resistance to pathogenic fungi, and the xylanase can degrade xylan in hemicellulose of the plant cell walls and stimulate plants to produce endogenous jasmonic acid;
(2) the field test shows that the compound microbial agent can promote the growth of angelica, the overground part is shown to increase stem thickness, promote plant height and enlarge crown width, and the underground part is mainly shown to increase the diameter of reed heads, the length of main roots and the average single plant weight;
(3) the compound microbial agent can reduce the incidence rate of root rot of angelica by producing chitinase and antibacterial substances, and can be used for preparing pesticides for preventing and treating plant diseases
(4) The compound microbial agent achieves the effects of improving the content of ferulic acid and improving the quality by promoting the accumulation of salicylic acid and jasmonic acid in angelica leaves. And the content of salicylic acid and jasmonic acid in the angelica leaves is increased, the content of auxin is reduced, and the gibberellin content is not obviously changed just after the compound microbial agent is treated, so that the special endogenous hormone response mode can be used for preparing the special microbial fertilizer for the angelica by increasing the diameter of the reed head, the length of the reed head and the length of the main root to increase the average single plant weight.
Drawings
FIG. 1 shows the results of the enzyme activity and the content detection of hydrogen peroxide, malondialdehyde, and chlorophyll after the angelica is treated with the composite microbial agent;
FIG. 2 shows the analysis results of the hormone and quality index principal components after the angelica is processed by the compound microbial inoculum;
FIG. 3 is a diagram showing the analysis results of the principal components of physiological and biochemical indicators such as hormone and enzyme activity of the leaves of angelica after the angelica is treated by the compound microbial inoculum.
Detailed Description
The present invention will be further described with reference to the following examples, but the scope of the present invention is not limited to the following examples, and modifications or substitutions of the methods, steps or conditions of the present invention can be made without departing from the spirit and substance of the present invention.
The tested plants are 1-year-old angelica seedlings, the variety is Mingui No.1, and the test plants are provided by Min county Chinese medicinal material production technology guide stations. The test is carried out in the demonstration base for ecologically planting the Chinese medicinal materials in Gansu Tianzhu in 2019 in 4-10 months. The soil type is millet calcium soil, the pH value is 7.85, and the previous crop is rape; preparing soil at the bottom of 4 months, applying organic fertilizer (organic matter is more than or equal to 45 percent, and total nutrient N + P)2O5+K2O≥5％)3000kg·hm-2. The reagents and culture medium used in the test are all chemically pure. The hormone and the quality detection related reagent are chromatographically pure. Malondialdehyde, chlorophyll, hydrogen peroxide, and enzyme activity detection related kit were purchased from Suzhou Keming Biotechnology Ltd.
Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, Pseudomonas psychrophila CBSB, Bacillus subtilis GSICC 32822, Bacillus amyloliquefaciens GSICC 32823 and Bacillus belgii 30287 described in the following examples were all purchased from the Gansu division center (biological research institute of scientific college, Gansu province) of the China center for the preservation and management of industrial microbial cultures.
The media components used in the following examples: PDB culture medium: potato 200g/L, glucose 20g/L, natural pH.
Preparation and experimental design of the microbial inoculum in the following examples:
experimental group complex microbial inoculant (T1): mixing Pseudomonas fluorescens CBS5(NCBI No. MW981369.1), Pseudomonas alcaligenes CBS7(NCBI No. MW981370.1) and Pseudomonas psychrophila CBSB (NCBI No. MW981371.1) which are respectively cultured by PDB culture medium in equal proportion; the CFU number of the microbial strain is 3-4 multiplied by 107/ml；
Positive control group bacillus complex (T2): respectively mixing Bacillus subtilis GSICC 32822, Bacillus amyloliquefaciens GSICC 32823 and Bacillus belgii 30287 which are cultured by PDB culture medium in equal proportion; the CFU number of the microbial strain is 3-4 multiplied by 107/ml；
Positive control group pseudomonas fluorescens inoculum (T3): pseudomonas fluorescens CBS5(NCBI No. MW981369.1) cultured in PDB medium; the CFU number of the microbial strain is 3-4 multiplied by 107/ml；
Negative control group (CK): PDB medium was not inoculated.
The test adopts single-factor completely random design, and is provided with T1, T2, T3 and CK treatment groups, the treatment is repeated for 3 times, and the area of a cell is 30m2(4 m.times.7.5 m). And 7-8 months in 2019, spraying the Chinese angelica (non-film-mulching planting) leaf surfaces once every 2 weeks for 4 times, wherein the spraying amount and the spraying volume of different cells are equal. The field management is carried out according to the conventional measures. And 7 days after the 4 th treatment, randomly selecting 30 Chinese angelica plants in each cell, shearing off functional leaves at the 3 rd to 5 th positions at the same positions, putting the mixed samples into a sample bag, quickly freezing by using liquid nitrogen, storing at-80 ℃, and measuring related indexes. Digging roots in the harvesting period of the angelica, cleaning, drying in the shade and storing to be measured for relevant indexes.
Example 1 detection of enzyme Activity related to Complex microbial Agents
Respectively inoculating the bacteria into 20mL PDB culture medium, and performing shaking culture (30 ℃, 180r/min) for 24 h; centrifugally collecting thalli at 5000r/min, re-suspending the thalli by sterilized distilled water until the turbidity of the thalli is about 1, and adding the kit into a 1.5mL centrifugal tubeAdding 1mL of extracting solution of corresponding enzyme, adding 100 mu L of bacterial solution to be detected, carrying out ice bath ultrasonic wave to break bacteria (power is 200W), carrying out ultrasonic wave for 3s, carrying out interval of 10s, and repeating for 30 times; centrifuging the ultrasonic bacterial liquid for 10min (4 ℃, 9000r/min), sucking the supernatant into a new centrifugal tube, and placing on ice to be tested; respectively measuring enzyme activity according to the method of corresponding measuring kit, and dividing the calculated result by the turbidity (OD) of the corresponding bacterial liquid to be measured600) And standardizing the enzyme activity of the bacterial liquid to be detected in unit turbidity.
The relative enzyme activities of the experimental group composite microbial agent T1, the positive control group bacillus composite microbial agent T2 and the positive control group pseudomonas fluorescens microbial agent T3 are shown in Table 1, and in general, the absolute enzyme activities of two microbial agents POD, laccase and SOD of the experimental group composite microbial agent T1 and the positive control group bacillus composite microbial agent T2 are larger than that of the positive control group pseudomonas fluorescens microbial agent T3, so that the effects of eliminating active oxygen, polyphenol and other growth inhibiting substances and promoting plant growth are strong. The enzymatic activity differences of the two bacterial agents T1 and T2 are obvious, except for ACC deaminase, the enzymatic activities of SOD, POD, NEX, chitinase and laccase of the compound microbial agent T1 are all higher than those of a positive control group bacillus compound bacterial agent T2; compared with the bacillus composite microbial inoculum T2, the activities of SOD, POD, NEX, chitinase and laccase in the composite microbial inoculum T1 are respectively 21.8%, 27.8%, 74.8%, 56.7% and 125.1%. The results show that the composite microbial agent has stronger effects on growth promotion and disease defense than the bacillus composite microbial agent, and especially the laccase activity is 1.25 times of that of the bacillus composite microbial agent.
TABLE 1 related enzyme activities of different complex microbial inoculum: (n＝3)
Enzyme activity
T1
T2
T3
ACC deaminase/μmol min-1
0.734±0.011b
1.146±0.014a
0.131±0.002c
SOD activity/U.mg-1
35.882±1.162a
29.466±1.009b
12.180±0.134c
POD Activity/U.mg-1
142.283±8.756a
111.31±7.284b
68.154±1.249c
NEX Activity/U.mg-1
0.535±0.018a
0.306±0.013b
0.229±0.007c
Chitinase Activity/mg.h-2·mg-1
6.536±0.058a
4.171±0.062b
2.921±0.107c
Laccase Activity/U. mg-1
80.257±1.936a
35.652±0.614b
30.145±0.801c
Example 2 Effect of different microbial inoculum treatments on physiological and biochemical characteristics of Angelica sinensis leaf
Measuring chlorophyll, MDA and H of different treated angelica leaves according to the method of the kit2O2Content, SOD, POD, CAT, PAO, DAO, PPO activity. Each sample was repeated 3 times and the average was calculated.
As shown in FIG. 1, the activities of CAT, SOD, POD, PAO, DAO and PPO and H in the leaves of Angelica sinensis are significantly improved by the complex microbial agent T1 and the positive control group Bacillus complex microbial agent T2 compared with the negative control group CK, and the activities of H, SOD, POD, PAO, DAO and PPO in the leaves of Angelica sinensis are significantly improved by the complex microbial agent T1 and the positive control group Bacillus complex2O2Chlorophyll content, obviously reduces the content of malondialdehyde (P) in the leaves<0.05), the two treatments have better stress protection effect; compared with the positive control group bacillus composite microbial inoculum T2, after the treatment of the composite microbial inoculum T1, the activities of CAT, POD and PAO in the angelica leaves and the chlorophyll content are respectively increased by 7.7%, 17.3%, 6.4% and 9.1%; the activities of SOD and PPO are respectively reduced by 7.2 percent and 11.8 percent. The SOD, POD and laccase activities of the compound microbial agent T1 are greater than those of a positive control group bacillus compound microbial agent T2, and after the compound microbial agent T1 is sprayed on the angelica, the SOD and PPO activities of the leaves of the angelica are reduced, which shows that the compound microbial agent T1 has a stronger stress protection effect on the angelica; the CAT and POD activities of the angelica leaves treated by the compound microbial agent T1 are higher than those of the leaves treated by the positive control group bacillus compound agent T2, and compared with the positive control group bacillus compound agent T2The ratios of POD/SOD, CAT/SOD and POD/CAT of the angelica sinensis leaves treated by the compound microbial agent T1 are respectively improved by 28.6%, 15.7% and 8.7%, which shows that the compound microbial agent T1 obviously activates the POD and CAT activities of plants compared with a positive control group bacillus compound agent T2, and the POD expression is stronger than that of CAT.
Example 3 Effect of different treatments on endogenous hormones in Angelica sinensis
According to a method reported in a literature of 'the influence of nano-iron and melatonin on the yield and quality of fritillaria kansuensis under domesticated cultivation conditions' (journal of Chinese experimental and prescriptions, 2021), the contents of different endogenous hormones in angelica sinensis leaves are determined by UPLC-MS, each sample is repeated for 3 times, and an average value is calculated.
The results are shown in table 2, except for salicylic acid, auxin and gibberellin, the compound microbial agent T1 and the positive control group bacillus compound agent T2 show consistent change trends of other hormones compared with the negative control group CK, the jasmonic acid content is remarkably improved, and the jasmonic acid content is respectively increased by 257.91% and 99.94%; the content of abscisic acid is reduced by 64.69 percent and 66.91 percent respectively; the content of cytokinin is increased by 37.27 percent and 42.64 percent respectively; the reduction of the content of abscisic acid and the increase of the content of jasmonic acid and cytokinin are common factors for promoting the improvement of the yield and the quality of angelica by the compound microbial agent T1 and the positive control group bacillus compound agent T2; compared with a negative control group CK, the compound microbial agent T1 can obviously improve the content of salicylic acid in angelica sinensis, and the improvement rate is 30.59%; the content of auxin is reduced, and the reduction rate is 42.85 percent; the gibberellin content has no significant difference; compared with a negative control group CK, the positive control group bacillus composite inoculant T2 reduces the content of salicylic acid in the angelica sinensis, and the reduction rate is 5.94%; the contents of auxin and gibberellin are increased by 102.61% and 15.37%, respectively.
The data, root and yield related indexes are subjected to correlation analysis, and the results are shown in tables 3-4. The auxin is in negative correlation with salicylic acid and CAT/SOD, and is in positive correlation with gibberellin and indefinite roots; gibberellin is positively correlated with the number of adventitious roots, so that the positive control group T2 has the advantage that the number of adventitious roots is increased mainly by promoting auxin and gibberellin compared with the compound microbial agent T1. Salicylic acid is positively correlated with jasmonic acid, CAT/SOD, POD/SOD, long reed rhizome and fresh weight of single plant; the jasmonic acid is positively correlated with CAT/SOD, POD/SOD, reed head diameter, reed head length, main root length and single plant fresh weight, so that compared with a positive control group T2, the compound microbial agent T1 provided by the invention mainly promotes salicylic acid, jasmonic acid, CAT/SOD and POD/SOD, increases the reed head diameter, reed head length and main root length, and finally the single plant fresh weight is higher than T2.
Generally speaking, the salicylic acid and jasmonic acid content in a plant body have a negative correlation (see 'conversation mechanism of salicylic acid and jasmonic acid in plant defense reaction', journal of cell biology, 2002), while the salicylic acid and jasmonic acid content have a positive correlation after angelica is treated by the compound microbial agent T1, the compound microbial agent T1 has increased salicylic acid content of 38.84% and jasmonic acid content of 79.01% compared with a positive control group bacillus compound agent T2, and the compound microbial agent T1 has increased salicylic acid content of 30.59% and jasmonic acid content of 257.91% compared with a negative control group CK; after the positive control group bacillus composite inoculant T2 is treated, compared with the negative control group CK, angelica salicylic acid and jasmonic acid show a negative correlation; after the compound microbial agent T1 is treated, angelica salicylic acid and jasmonic acid show a positive correlation relationship, and an unexpected effect is achieved.
TABLE 2 Effect of different treatments on endoglin of Angelica sinensis leaves (ng g)-1，n＝3)
T1
T2
CK
Salicylic acid
31.427±0.091a
22.635±0.045c
24.065±0.084b
Jasmonic acid
296.369±1.347a
165.558±1.038b
82.805±0.272c
Abscisic acid
12.988±0.076b
12.172±0.045b
36.780±0.102a
Gibberellins
17.383±0.103ab
19.595±0.075a
16.985±0.066b
Growth hormone
15.004±0.118c
53.192±0.211a
26.254±0.124b
Cytokinins
16.275±0.076a
16.911±0.061a
11.856±0.017b
TABLE 3 analysis of the root of Angelica and yield-related indices Pearson correlation 1
Salicylic acid
Jasmonic acid
Growth hormone
Gibberellins
CAT/SOD
POD/SOD
Salicylic acid
1
0.854**
-0.823**
-0.507
0.912**
0.829**
Jasmonic acid
0.854**
1
-0.408
0.015
0.777*
0.933**
Growth hormone
-0.823**
-0.408
1
0.907**
-0.752*
-0.436
Gibberellins
-0.507
0.015
0.907**
1
-0.466
-0.047
CAT/SOD
0.912**
0.777*
-0.752*
-0.466
1
0.795*
POD/SOD
0.829**
0.933**
-0.436
-0.047
0.795*
1
Indefinite number of roots
-0.232
0.299
0.735*
0.943**
-0.175
0.22
Diameter of reed head
0.645
0.921**
-0.125
0.288
0.571
0.870**
Long reed root
0.938**
0.922**
-0.641
-0.276
0.853**
0.875**
Length of main root
0.652
0.928**
-0.13
0.285
0.59
0.817**
Fresh weight of single plant
0.780*
0.988**
-0.291
0.137
0.730*
0.919**
TABLE 4 analysis of the root and yield indices of Angelica gigas Pierson correlation 2
Indefinite number of roots
Diameter of reed head
Long reed root
Length of main root
Fresh weight of single plant
Salicylic acid
-0.232
0.645
0.938**
0.652
0.780*
Jasmonic acid
0.299
0.921**
0.922**
0.928**
0.988**
Growth hormone
0.735*
-0.125
-0.641
-0.13
-0.291
Gibberellins
0.943**
0.288
-0.276
0.285
0.137
CAT/SOD
-0.175
0.571
0.853**
0.59
0.730*
POD/SOD
0.22
0.870**
0.875**
0.817**
0.919**
Indefinite number of roots
1
0.513
0.035
0.513
0.407
Diameter of reed head
0.513
1
0.726*
0.895**
0.939**
Long reed root
0.035
0.726*
1
0.754*
0.866**
Length of main root
0.513
0.895**
0.754*
1
0.965**
Fresh weight of single plant
0.407
0.939**
0.866**
0.965**
1
Example 4 Effect of different treatments on growth and yield of Angelica plants
The stem thickness, the plant height and the crown breadth are measured in the growth period by a conventional method. And measuring the diameter of the reed head, the length of the main root and the fresh weight of a single plant after harvesting. The mean value was calculated for 30 strains per cell.
The statistical results of the related indexes of the aerial parts of the angelica sinensis are shown in table 5, the statistical results of the related indexes of the roots of the angelica sinensis and the yield of the angelica sinensis are shown in table 6, compared with a positive control group pseudomonas fluorescens agent T3 and a negative control group CK, the compound microbial agent T1 and the positive control group bacillus compound agent T2 increase the stem thickness, the plant height and the crown width of the angelica sinensis, the occurrence rate of root rot is reduced, and the difference between T1 and T2 is not obvious; compared with a negative control group CK, the compound microbial agent T1 provided by the invention has the advantages that the number of indefinite roots, the diameter of the reed head, the length of the main root and the fresh weight of a single plant are increased, and the growth rates are respectively 13.40%, 18.64%, 11.21%, 11.43% and 41.43% (P is less than 0.05); compared with a negative control group CK, the positive control group bacillus composite inoculant T2 has the advantages that the number of adventitious roots, the diameter of the reed heads and the fresh weight of a single plant are increased, and the growth rates are respectively 28.35%, 12.39% and 20.29% (P is less than 0.05); compared with a negative control group CK, the positive control group pseudomonas fluorescens agent T3 has no obvious influence on the indefinite number of roots, the diameter of the reed head, the length of the main root, the fresh weight of a single plant and the like; compared with a positive control group bacillus composite microbial agent T2, the composite microbial agent T1 provided by the invention has the advantages that the length of the reed rhizome and the fresh weight of a single plant are obviously increased, the growth rate is respectively 10.13% and 17.58%, the number of adventitious roots is reduced, and the reduction rate is 11.65%.
The results show that the root phenotype and the yield of the compound microbial agent T1 are superior to those of the positive control group bacillus compound agent T2.
TABLE 5 statistics of relevant indexes of aerial parts of Dang Gui: (n＝30)
Number of stems/number
Thickness of stem/mm
Plant height/cm
Crown width/cm
Root rot incidence/%)
T1
5.93±0.83a
9.86±0.72a
44.90±5.13a
65.37±8.26a
3.93b
T2
6.13±0.97a
9.95±0.89a
44.63±5.58a
66.84±7.35a
4.08b
T3
5.99±0.75a
9.28±0.36b
41.23±4.46b
62.99±5.14b
17.82a
CK
5.93±0.91a
9.14±0.58b
40.40±4.72b
61.69±4.17b
19.62a
TABLE 6 relevant index of the root of Angelica and yield statistics: (n＝30)
Indefinite number of pieces/piece
Diameter of reed head/mm
Length of reed rhizome/mm
Length of main root/mm
Fresh weight per gram of individual plant
T1
16.08±3.06b
30.74±3.16a
48.60±8.91a
260.58±21.66a
115.73±19.37a
T2
18.20±4.57a
29.12±3.42a
44.13±10.20b
250.90±31.42ab
98.43±20.06b
T3
14.87±3.02c
27.97±3.01b
43.54±8.24b
239.86±27.69b
87.32±23.94c
CK
14.18±2.89c
25.91±3.34b
43.70±8.71b
233.86±23.24b
81.83±15.12c
Example 5 Effect of different treatments on Angelica sinensis quality
The quality control evaluation indexes of the whole drug effect of the angelica are ferulic acid and Z-ligustilide. According to the method of the literature 'influence of different phytohormones and microorganisms on the yield and quality of angelica in stubble fields' (Chinese medicinal materials, 2021 years), the contents of ferulic acid, senkyunolide I, coniferyl ferulate and senkyunolide A, Z-ligustilide are determined, repeated for 3 times, and the average value is calculated.
As shown in Table 7, the absolute values of ferulic acid, coniferyl ferulate and Z-ligustilide were large, and the absolute value of senkyunolide A, I was small. Compared with a negative control group CK, ferulic acid and Z-ligustilide of the compound microbial agent T1 are respectively increased by 72.82 percent and 10.32 percent; compared with CK, the positive control group Bacillus complex inoculant T2 has increased ferulic acid and Z-ligustilide by 38.64% and 21.78%, respectively; compared with CK, the positive control group pseudomonas fluorescens agent T3 has no obvious difference in ferulic acid and Z-ligustilide. The compound microbial agent T1 provided by the invention obviously improves the overall quality control index of angelica.
TABLE 7 quality index content (mg. g) of Chinese angelica treated differently-1，n＝3)
Ferulic acid
Senkyunolide I
Coniferous ferulateEsters
Senkyunolide A
Z-ligustilide
T1
0.890±0.08a
0.016±0.00b
1.306±0.09c
0.142±0.00a
14.109±0.15b
T2
0.714±0.06b
0.018±0.00a
1.892±0.12a
0.133±0.01c
15.574±0.18a
T3
0.586±0.05c
0.018±0.00a
1.435±0.06b
0.138±0.02b
12.945±0.16c
CK
0.515±0.11c
0.018±0.00a
1.453±0.07b
0.095±0.00d
12.789±0.08c
Example 6 different Process data analysis and summary
The analysis of hormone content and quality index of radix Angelicae sinensis in different processed leaves is shown in FIG. 2, and the load of principal component 1 is 54.06%, and the load of principal component 2 is 44.25%. Senkyunolide A, ferulic acid, jasmonic acid, cytokinin and abscisic acid have great contribution to the main component 1; coniferyl ferulate, auxin, gibberellin, salicylic acid make a great contribution to the main component 2; the contribution of senkyunolide I and Z-ligustilide to main components 1 and 2 is equivalent. Compared with a negative control group CK, the composite microbial agent T1 and the positive control group Bacillus composite microbial agent T2 are mainly distinguished by related indexes of a main component 1, and have certain promotion effects on other quality indexes except the senkyunolide I. The compound microbial agent T1 and the positive control group bacillus compound agent T2 are mainly distinguished by related indexes of a main component 2, and the compound microbial agent T1 promotes the increase of the content of ferulic acid by promoting the accumulation of salicylic acid and jasmonic acid in leaves; the positive control group bacillus composite bacterial agent T2 promotes the increase of contents of coniferyl ferulate and Z-ligustilide by promoting the accumulation of auxin and gibberellin in leaves.
The analysis of the main components of physiological and biochemical indexes such as hormone and enzyme activity of the angelica leaf is shown in figure 3, the load of the main component 1 is 74.23%, and the load of the main component 2 is 22.09%. Chlorophyll, cytokinin, abscisic acid, MDA, an active oxygen scavenging enzyme system, polyamine oxidase, PPO and the like have great contribution to the main component 1; auxin, gibberellin, salicylic acid, jasmonic acid, CAT/SOD and POD/SOD have a large contribution to the main component 2. Compared with a negative control group CK, the composite microbial agent T1 and the positive control group Bacillus composite microbial agent T2 are mainly distinguished by related indexes of a main component 1, so that the contents of abscisic acid and Malondialdehyde (MDA) are reduced, the contents of chlorophyll and cytokinin are increased, and the activities of an active oxygen scavenging enzyme system and a polyphenol polyamine oxidase system are combined. Compared with a positive control group bacillus composite microbial agent T2, the composite microbial agent T1 is mainly distinguished by related indexes of a main component 2, and the composite microbial agent T1 improves the contents of salicylic acid and jasmonic acid and the ratio of CAT/SOD to POD/SOD; the positive control group bacillus composite inoculum T2 improves the content of auxin and gibberellin. The compound microbial agent T1 and the positive control group bacillus compound agent T2 have different action mechanisms, and the angelica has different yield increasing amplitude and root appearance properties. After the compound microbial agent T1 is treated, the content of salicylic acid and jasmonic acid in the angelica leaves is increased, the content of auxin is reduced, and the content of gibberellin is not obviously changed.
The comprehensive consideration of yield, quality, disease resistance and root appearance properties shows that the treatment effect of the compound microbial agent T1 is better than that of a positive control group bacillus compound microbial agent T2 and better than that of a positive control group T3 and a negative control group CK.
Sequence listing
<110> institute of biological research of science institute of Gansu province
Xinjiang Academy of agricultural reclamation Sciences
<120> pseudomonas compound microbial agent and application thereof in disease-resistant, yield-increasing and quality-improving of angelica sinensis
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 886
<212> DNA
<213> Pseudomonas sp (Pseudomonas sp)
<400> 1
cgctaactgc actctagcgg tcagaaactt gcttctcttg aagcggcgga cgggtgaata 60
atgcctacga atctgcctgg tagtggggga taacgttccg aaaccgacgc taataccgca 120
tacgtcctac gggagaaagc aggggacctt cgggccttgc gctatcagat gagcctaggt 180
cggattagct aggtggtgag gtaatggctc accaaggcga cgatccgtaa ctggtctgat 240
aggatgatca gtcacgctgg aactgagaca cggtccagac tcctacagga ggcaccagtg 300
gggaatattg gacgatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc 360
ttcggattgt aaagcacttt aagttgggag gaagggcagt tacctaatac gtgattgttt 420
tgacgttacc gacagaataa tcaccggcta actctgtgcc agcaaccgcg gtaatacaga 480
gggtgcgagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt ttgttaagtt 540
ggatgtgaaa tccccgggct caacctggga actgcattcg aaactgactg actacagtat 600
ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc ggaaatatat gaaggagcac 660
aagtggcaaa agcgagcacc tggactgata ctgacactga ggtgcgaaag cgtggggagc 720
aaactggatt agataccgtg gaagtccacg ccgtaaacga tgtcgactag gacgttggga 780
gccgtgagct cttagtggcg catctaacgc attgagttga cggcctgcgg agaacgggcc 840
gcaaggctag aaatcgaatg aattgactgg ggccggcaca agcgga 886
<210> 2
<211> 894
<212> DNA
<213> Pseudomonas sp (Pseudomonas sp)
<400> 2
gggtgtcacg ctacctgcag tcgagcggat gagtggagct tgctccatga ttcagcggcg 60
gacgggtgag taatgcctag gaatctgcct ggtagtgggg gacaacgttt cgaaaggaac 120
gctaataccg catacgtcct acgggagaaa gcaggggacc ttcgggcctt gcgctatcag 180
atgagcctag gtcggattag ctagttggtg aggtaaaggc tcaccaaggc gacgatccgt 240
aactggtctg agaggatgat cagtcacact ggaactgaga cacggtccag actcctacgg 300
gaggcagcag tggggaatat tggacaatgg gcgaaagcct gatccagcca tgccgcgtgt 360
gtgaagaagg tcttcggatt gtaaagcact ttaagttggg aggaagggca gtaagttaat 420
accttgctgt tttgacgtta ccgacagaat aagcaccggc taacttcgtg ccagcagccg 480
cggtaatacg aagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cgcgtaggtg 540
gttcagcaag ttggatgtga aagccccggg ctcaacctgg gaactgcatc caaaactact 600
gagctagagt acggtagagg gtggtggaat ttcctgtgta gcggtgaaat gcgtagatat 660
aggaaggaac accagtggcg aaggcgacca cctggactga tactgacact gaggtgcgaa 720
agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgtcgact 780
agccgttggg atccttgaga tcttagtggc gaagctaacg cgataagtcg accgcctggg 840
gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcg 894
<210> 3
<211> 939
<212> DNA
<213> Pseudomonas sp (Pseudomonas sp)
<400> 3
ggggttcacg cttcctgcac tcgagcggta gagagaagct tgcttctctt gagagcggcg 60
gacgggtgag taatgcctac gaatctgcct ggtagtgggg gataacgttc ggaaacggac 120
gctaataccg catacgtcct acgggagaaa gcaggggacc ttcgggcctt gcgctatcag 180
atgagcctag gtcggattag ctagttggtg aggtaatggc tcaccaaggc gacgatccgt 240
aactggtctg agaggatgat cagtcacgct ggaactgaga cacggtccaa actcctacgg 300
gaggcaccag tggggaatat tggacgatgg gcgaaagcct gatccagcca tgccgcgtgt 360
gtgaagaagg tcttcggatt gtaaagcact ttaagttggg aggaagggca gttacctaat 420
acgtgattgt tttgacgtta ccgacagaat aatcaccggc taactctgtg ccagcagccg 480
cggtaataca gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cgcgtaggtg 540
gtttgttaag ttggatgtga aatccccggg ctcaacctgg gaactgcatt cgaaactgac 600
tgactacagt atggtagagg gtgatggaat ttcctgtgta ccggtgaaat gcgtacatat 660
atgaaggagc accagtggcg aaggcgacca cctggactga tactgacact gaggtgcgaa 720
agcgtgggga gcaacacgat tagataccgt ggaagtccac gccgtaaacg atgccaacta 780
cccggtggga gccgtgagct cttagtggcg cagctaacgc attatctcac cgcctgggga 840
gaacggccgc aggctagaac tcacatgaat tgacggggcc cgcacaagcg atgtaggatg 900
ggggtagttc taagcacgcg aagaccttac cggggttga 939
