CN115093983A

CN115093983A - Growth-promoting disease-resistant biological agent

Info

Publication number: CN115093983A
Application number: CN202210422883.XA
Authority: CN
Inventors: 刘涛; 王宪菊; 杨汝军; 字淑慧
Original assignee: Yulong Wusheng Plantation And Cultivation Co ltd; Yunnan Agricultural University
Current assignee: Yulong Wusheng Plantation And Cultivation Co ltd; Yunnan Agricultural University
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2022-09-23

Abstract

The invention discloses a growth-promoting disease-resistant biological agent, which is prepared by one or more of bacillus cereus (Bacillus cereus), bacillus thuringiensis (Bacillus thuringiensis), fusarium oxysporum (Fusariumoxysporum) and aspergillus flavus (Aspergillus amarii). The invention utilizes the symbiotic relationship of microorganisms and plants to promote the growth of medicinal plants, improve the disease resistance of the plants, relieve the contradiction between large demand of the market for the paris polyphylla and shortage of natural resources, promote the remarkable improvement of each growth index of tobacco and effectively improve the yield and the quality of the tobacco.

Description

Growth-promoting disease-resistant biological agent

Technical Field

The invention relates to the technical field of microbial agents, in particular to a growth-promoting disease-resistant biological agent.

Background

Rhizoma paridis is a perennial herb plant of Paris (trillaeaceae) of trilobate, takes rhizomes as a medicine, has the curative effects of promoting blood circulation, removing blood stasis, relieving swelling and pain, cooling liver, arresting convulsion, clearing heat, detoxicating, resisting cancer, inhibiting bacteria and the like, is a main component of some important Chinese patent medicines such as Yunnan white drug powder, Gongxuening capsules, Sichuan white drug powder and the like at present, and steroidal saponins are main medicinal active components of rhizoma paridis.

Tobacco Black Shank (Tobacco Black Shank) is caused by an infestation of the soil-borne fungus Phytophthora nicotianae (Phytophthora nicotiana). Soil-borne diseases are diseases which occur when a part or most of pathogens existing in soil in life history invade the roots or stems of plants under appropriate conditions. The tobacco black shank mainly occurs in a field stage, and can occur in both a tobacco seedling stage and a adult stage, typical symptoms are withered, leaf yellowing, dwarfing and root and stem base necrosis, the whole plant dies in the late stage of infection, pathogenic bacteria can survive in soil for years, and the harm to tobacco planting is great. In the field, the tobacco black shank affects the base of the stem, and the chemical bactericide metalaxyl is generally used for preventing and treating diseases. However, the method is not beneficial to the development of tobacco sustainable agriculture, excessively depends on chemical agents, leaves bactericide residues and causes environmental pollution.

Disclosure of Invention

The invention aims to provide a growth-promoting disease-resistant biological agent, which utilizes the symbiotic relationship of microorganisms and plants to improve the accumulation of active ingredients of medicinal plants, promote the growth of plants, improve the disease resistance of the plants, relieve the contradiction between large demand of high-quality paris polyphylla and shortage of natural resources in the market, promote the remarkable improvement of various growth indexes of tobacco and effectively improve the yield and the quality of the tobacco.

The technical purpose of the invention is realized by the following technical scheme:

the biological agent is prepared by one or more of Bacillus cereus, Bacillus thuringiensis (Bacillus thuringiensis), fusarium oxysporum (Fusarium oxysporum) and Aspergillus tamarii (Aspergillus tamarii) in combination.

Further preferably, the biological agent is a biological agent prepared from Bacillus cereus.

Further preferably, the biological agent is made of Bacillus thuringiensis (Bacillus thuringiensis).

Further preferably, the biological agent is a biological agent prepared from fusarium oxysporum (fusarium oxysporum).

Further preferably, the biological agent is made of Aspergillus tamarii (Aspergillus tamarii).

Further preferably, the biological agent is used for promoting the growth and resisting diseases of the paris polyphylla.

Further preferably, the biological agent is used for promoting the growth and resisting diseases of tobacco.

Compared with the prior art, the invention has the following beneficial effects:

firstly, the four symbiotic bacteria have different promoting effects on the growth of paris polyphylla seedlings, and the fusarium oxysporum has the most remarkable promoting effects on plant height, root length, root weight, leaf length, leaf width and fresh weight on the ground; the bacillus thuringiensis has the most remarkable promotion effect on chlorophyll a, chlorophyll b and total chlorophyll; fusarium oxysporum has the most remarkable improvement on net photosynthetic rate (Pn), stomatal conductance (Cond) and transpiration rate (Tr). The single backcrossed 4 growth-promoting bacteria can effectively stimulate the paris polyphylla seedlings to synthesize the paris polyphylla saponin, and the total saponin content of the paris polyphylla is obviously increased. The expression of saponin synthesis pathway genes is improved to different degrees by all treatments, the expression levels of HMGS, SE2 and CAS are highest under the treatment of the bacillus thuringiensis strain, the expression levels of GGPS and FPPS are highest under the treatment of the fusarium oxysporum strain, and the expression level of SE1 is highest under the treatment of the bacillus cereus.

The physiological indexes of plants in the growth promoting group and the antagonistic group are increased to different degrees, and the treatment of the bacillus cereus strain is most obvious in improvement of the height, the crown width, the leaf length and the leaf width of the tobacco plant; the 4 strains of growth-promoting antagonistic bacteria have promoting effects of different degrees on the enzymatic activity of tobacco plants, the growth-promoting group inoculated with the bacillus thuringiensis alone has the most obvious effect on the enzymatic activity of SOD, the growth-promoting group inoculated with the strain of bacillus cereus alone has the most obvious effect on the promoting effect on the enzymatic activity of POD, the growth-promoting group inoculated with the strains of fusarium oxysporum and bacillus thuringiensis has the most obvious effect on the enzymatic activity of PPO, and the PAL enzymatic activity has the highest content under the treatment of the growth-promoting group inoculated with the strain of bacillus thuringiensis alone and the antagonistic group inoculated with the sum of fusarium oxysporum and Y. And analyzing the disease index and the inhibition rate of the tobacco black shank pathogenic bacteria inoculated with the growth-promoting antagonistic bacteria, and inoculating 4 strains of the growth-promoting antagonistic bacteria to play an antagonistic role on phytophthora nicotianae. The gene analysis result shows that the genes related to the growth and the resistance of the tobacco treated by the fusarium oxysporum and the bacillus thuringiensis strains are up-regulated, and the genes are not expressed in other treatments.

Drawings

FIG. 1 is a plate confrontation test of different strains and phytophthora nicotianae pathogenic bacteria;

FIG. 2 is a phenotype diagram of two-year old seedlings after symbiosis;

FIG. 3 shows the effect of symbiotic bacteria on chloroplast pigment content of two-year old seedling leaves;

FIG. 4 shows the effect of symbiotic bacteria on steroid saponin content of two-year-old Paris polyphylla;

FIG. 5 shows the effect of symbiotic bacteria on the expression of each relevant gene in Paris polyphylla rhizome;

FIG. 6 shows tobacco SOD enzyme activity under different bacterial treatments;

FIG. 7 shows tobacco POD enzyme activity under different bacterial treatments;

FIG. 8 shows tobacco PPO enzyme activity under different bacterial treatments;

FIG. 9 shows tobacco PAL enzyme activity under different bacterial treatments;

FIG. 10 shows the pathogenesis of inoculated Fusarium oxysporum and bacterial tobacco;

FIG. 11 shows the expression levels of tobacco epidemic prevention enzyme genes in different bacterial treatments;

FIG. 12 shows the expression levels of tobacco growth-related resistance genes under different bacterial treatments;

FIG. 13 shows the expression levels of tobacco black shank associated resistance genes under different bacterial treatments.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described in detail with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.

Example 1

A biological agent for promoting growth and resisting diseases is prepared from Bacillus cereus (Bacillus cereus) and is used for promoting growth and resisting diseases of rhizoma paridis and tobacco.

Example 2

A biological agent for promoting growth and resisting disease is prepared from Bacillus thuringiensis (Bacillus thuringiensis) and can be used for promoting growth and resisting disease of rhizoma paridis and tobacco.

Example 3

A biological agent for promoting growth and resisting diseases is prepared from Fusarium oxysporum (Fusarium oxysporum), and can be used for promoting growth and resisting diseases of rhizoma paridis and tobacco.

Example 4

A biological agent for promoting growth and resisting diseases is prepared from Aspergillus tamarii (Aspergillus tamarii), and can be used for promoting growth and resisting diseases of rhizoma paridis and tobacco.

An experiment is set to verify the growth promoting and disease resisting effects of the 4 strains, and the four strains are numbered: bacillus cereus (LgD2), Bacillus thuringiensis (LgD10), Fusarium oxysporum (TPB), and Aspergillus flavus (TPD 11).

1. Setting experiments verify the inhibiting effect of the four strains on the tobacco black shank

LgD2, LgD10, TPB and TPD11 were cultured in the presence of pathogenic bacteria, and control CK and control diameter of only phytophthora nicotianae pathogenic bacteria were measured after 7 days, as shown in FIG. 1 (FIG. A: TPB; FIG. B: TPD 11; FIG. C: LgD 10; FIG. D: LgD 2; and FIG. E: only phytophthora nicotianae pathogenic bacteria). According to the formula: the inhibition rate (100%) (control colony radius-treatment colony radius)/control colony radius x 100% to calculate the inhibition rate of the bacteria on pathogenic bacteria. As shown in Table 1, the inhibition rate of LgD2 and LgD10 on phytophthora nicotianae is 88 percent and 90 percent; the inhibition rate of TPB and TPD11 on phytophthora nicotianae by two strains of symbiotic fungi is 92% and 81%.

TABLE 1 inhibition of pathogenic bacteria by different strains

2. Symbiotic culture of growth-promoting bacteria and Paris polyphylla seedling

2.1 Effect of growth-promoting bacteria on the Biomass of Paris polyphylla

After 4 symbiotic bacteria LgD2, LgD10, TPB and TPD11 and the paris polyphylla seedlings are subjected to symbiotic culture, the appearance of the seedlings after the coculture is observed, the growth vigor of each treatment room is good, and the disease infection character does not appear, referring to fig. 2. And (3) counting the agronomic characters of the processed paris polyphylla part, wherein the agronomic characters are shown in a table 2. The heights of the four TPB, LgD10, TPD11 and LgD2 treated strains inoculated with the growth-promoting bacteria are all obviously higher than that of a control CK and are respectively 55.23 percent, 47.08 percent, 43.56 percent and 43.27 percent higher than that of the control CK. The growth amounts of the five treatments in the plant height are TPB > LgD10> TPD11> LgD2> CK from large to small. The largest effect on plant height was the TPB strain followed by LgD10, whereas the difference between TPD11 and LgD2 was not significant. The fresh weight, the leaf length and the leaf width of the overground part inoculated with the TPB strain are obviously higher than those of a control, and are respectively 65 percent, 46 percent and 42 percent higher than those of the control; the treatment by inoculating the TPD11 strain is 53%, 45.94% and 40.84% higher respectively; the inoculated LgD10 strain treatment is 52.38%, 42.19% and 37.93% higher respectively; the inoculated LgD2 strain treatment was 48.27, 43.34 and 37% higher, respectively. The main medicinal part of the paris polyphylla is rhizome, so the root weight and the root length of the underground part of the paris polyphylla are also measured independently. The results show that: the root weight and root length of the underground part inoculated with the growth-promoting bacteria are obviously higher than those of the control. The underground weight of the treatment inoculated with the TPB strain is 45.91 percent higher than that of the control, while the underground weight of the treatment inoculated with the TPB strain is not significantly different from that of the treatment inoculated with the TPD11, LgD2 and LgD 10. The highest root length was 49.92% higher for treatment with the strain TPB inoculated than the control, followed by 42.03% higher for treatment with the strain LgD2 than the control, while the difference between TPD11 and LgD10 was not significant.

TABLE 2 influence of different strains on the biomass of seedlings of Paris polyphylla grown in two years

Note: data in the table are mean ± sem, with different letters indicating significant differences between treatments (P <0.05)

In conclusion, the 4 symbiotic bacteria can improve the plant height, the leaf length and the leaf width of the paris polyphylla, promote the fresh weight of the upper part and the lower part of the paris polyphylla, stimulate the extension of roots and promote the development of the root system of the paris polyphylla.

2.2, influence of growth-promoting bacteria on chloroplast pigment content of Paris polyphylla

Referring to fig. 3, the contents of chlorophyll a, chlorophyll b and total chlorophyll in the paris polyphylla seedlings inoculated with the growth-promoting bacteria treatment are significantly higher than those of the control, wherein LgD10 strain treatment reaches the peak value, and the contents are respectively 0.31, 0.57 and 0.89 (mg/gFW); secondly, the treatment contents of the LgD2 strains are respectively 0.31, 0.56 and 0.86 (mg/gFW); the TPB strain treatment contents were 0.26, 0.55 and 0.82(mg/gFW), respectively; the treated levels of TPD11 strain were 0.24, 0.54, and 0.79(mg/gFW), respectively. The chlorophyll a, chlorophyll b and total chlorophyll contents of the 5 treatment rooms are LgD10> LgD2> TPB > TPD11> CK from high to low in sequence. Treatment without growth-promoting bacteria resulted in a significantly higher CK chlorophyll a/b than the other treatments, whereas the difference between the LgD10 and LgD2 strain treatments was not significant.

2.3, influence of growth-promoting bacteria on Yunnan rhizoma paridis gas exchange parameters

After the co-culture of 4 symbiotic bacteria and two-year-old paris polyphylla seedlings is finished, the gas exchange parameters of the paris polyphylla plants are measured and subjected to statistical analysis, and the results are shown in table 3. After 4 growth-promoting bacteria are inoculated, the net photosynthetic rate (Pn), the stomatal conductance (Cond), the transpiration rate (Tr) and the intercellular CO2 concentration (Ci) of the leaves of the paris polyphylla are all remarkably improved compared with CK. Statistically, the net photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration of TPD11 strain were the highest of all treatments; followed by TPB, LgD10 and LgD2 strain treatments. In general, the inoculation of growth-promoting bacteria helps the leaves of Paris polyphylla undergo photosynthesis.

TABLE 3 influence of growth-promoting bacteria on the gas exchange parameters of seedling leaves of rhizoma paridis Yunnanensis grown in two years

Note: data in the table are mean ± sem, with different letters indicating significant differences between treatments (P < 0.05).

2.4 influence of growth-promoting bacteria on the content of saponin of Paris polyphylla

After 4 symbiotic bacteria are inoculated in a single loop, the processed rhizoma paridis rhizome is subjected to determination of the contents of rhizoma paridis saponin I, rhizoma paridis saponin II, rhizoma paridis saponin VI, rhizoma paridis saponin VII, rhizoma paridis saponin D, rhizoma paridis saponin H and rhizoma paridis total saponin, the contents are calculated by contrasting with rhizoma paridis saponin standard substances, and the statistical result is shown in figure 4. After single loop grafting, significant influence is caused on the saponin of the paris polyphylla.

The content of the paris polyphylla saponin I is the highest after LgD2 strain treatment, and is 0.049%, and then LgD10, TPD11 and TPB are respectively 0.034, 0.038 and 0.019%. LgD2 the content of the processed rhizoma paridis saponin I is remarkably higher than that of LgD10, TPD11 and TPB strains; LgD10 and TPD11 strains showed no significant difference between treatments, and no chiretta saponin I was detected in CK treatment. The contents of the paris saponin II treated by 4 strains inoculated with LgD2, LgD10, TPD11 and TPB are LgD2> LgD10> TPD11> TPB > CK from high to low, and are respectively 0.613, 0.422, 0.264, 0.155 and 0.056 percent. The content of the paris polyphylla saponin VI is the highest under LgD10 strains, the content of the paris polyphylla saponin VI after inoculation is LgD10> LgD2> TPB > TPD11> CK from large to small, the content of the paris polyphylla saponin VI after inoculation is 0.071, 0.039, 0.032, 0.031 and 0.016 respectively, the content of LgD2 is obviously higher than that of TPD11 and TPB strains, and the difference between TPD11 and TPB strains is not obvious. The content of the paris saponin VII treated by the inoculated growth-promoting bacteria is obviously higher than that of a control, and is LgD10, LgD2, TPD11, CK and TPB from high to low, which are respectively 0.396, 0.347, 0.208, 0.149 and 0.142 percent. Paris saponin D was detected only under CK and TPB strain treatment, 0.022 and 0.016%, respectively, and was not detected by the rest of the treatments. Paris saponin H treated with strains LgD10, TPD11 and TPB were all higher than the control, 0.017, 0.025, 0.022 and 0.025% respectively, while no treatment with LgD2 was detected. The content of the total saponins of the paris polyphylla treated by singly inoculating the growth-promoting bacteria is obviously higher than that of a control group, and the total saponins are LgD2, LgD10, TPD11, TPB and CK from high to low, and are respectively 1.049, 0.951, 0.563, 0.387 and 0.262 percent. In short, the treatment of four strains increases the content of the paris polyphylla saponin in the rhizome of paris polyphylla seedling to different degrees, and the change of the content of the total saponin is particularly obvious.

2.5 expression analysis of growth-promoting bacteria induced Paris polyphylla steroid saponin synthetic pathway gene

2.5.1 detection of total RNA quality of rhizoma paridis Yunnanensis and cDNA Synthesis

RNA was extracted from each of the 5 treated Paris polyphylla, resulting in RNA concentrations ranging from 230-400 ng/ul and OD 260/280 between 1.8-2.0. The extraction results were checked by 1% agarose gel electrophoresis, and it was observed that each lane had a clear and clean band of interest at the 28S and 18S rRNA bands, and the brightness ratio of 28S to 18S was about 2: 1. The extracted RNA is relatively complete and high in quality, meets the requirements of subsequent experiments, and can be used for the next reverse transcription experiment reaction. After reverse transcription, the cDNA concentration is about 2300ng/ul, and the cDNA is stored at-20 ℃ for later use.

2.5.2 fluorescent quantitative analysis of the expression of the synthetic pathway gene of the steroid saponins of the paris polyphylla by the symbiotic bacteria

In order to discuss the influence of symbiotic bacteria on the expression of genes related to the synthetic pathway of steroid saponin of paris polyphylla, quantitative fluorescence PCR analysis is carried out on key genes (HMGS, GGPS, FPPS, SS, SE1, CAS and SE2) of the synthetic pathway of steroid saponin, which are presumed and reported by the prior art, beta-Actin is used as an internal reference gene, and the expression result is calculated, wherein the specific result is shown in figure 5. The expression of saponin synthesis pathway genes is improved to different degrees by all treatments, the expression levels of HMGS, SE2 and CAS are the highest under the treatment of LgD10 strain, the expression levels of GGPS and FPPS are the highest under the treatment of TPD11 strain, and the expression level of SE1 is the highest under the treatment of LgD2 strain. The HMGS expression levels of the processed paris polyphylla inoculated with the four growth-promoting bacteria TPB, LgD10, TPD11 and LgD2 are all up-regulated and are respectively 0.3, 1.9, 2.5 and 2.2 times of that of a control, and the difference of TPD11 and the control is not significant; the expression quantity of the paris polyphylla GGPS genes inoculated with four growth-promoting bacteria is obviously increased and is respectively 7.8 times, 1.6 times, 4.6 times and 3.1 times of that of a control. The expression level of the paris polyphylla FPPS gene inoculated with four growth-promoting bacteria is respectively up-regulated, and is respectively 10.1, 1.4, 8.3 and 9.8 times of that of the control, the difference between TPD11, LgD10 and LgD2 is not obvious, and the difference between TPB and the control is not obvious although the TPB is up-regulated; the SS gene expression levels of the paris polyphylla inoculated with the four growth-promoting bacteria are obviously increased and are respectively 3.0, 3.1, 2.3 and 3.1 times of those of the control, and the difference between the four growth-promoting bacteria is not obvious; SE1 gene expression levels of strains inoculated with TPD11, LgD10 and LgD2 were all significantly up-regulated and were 4.0, 6.8 and 8.4 times higher than those of the control, respectively, and SE1 gene expression was inhibited by the treatment of strains inoculated with TPB. The expressions of the SE2 genes of the paris polyphylla inoculated with the four growth-promoting bacteria are respectively up-regulated, which are respectively 3.0 times, 2.9 times, 9.2 times and 7.3 times of those of a control, and the difference between TPB and TPD11 is not obvious; the CAS gene expression of the paris polyphylla inoculated with four growth-promoting bacteria is respectively up-regulated, which is 1.8 times, 0.5 times, 12.1 times and 4.7 times of that of a control, and TPB and TPD11 have no significant difference compared with the control.

3. Influence of growth-promoting bacteria and pathogenic bacteria inoculated tobacco on tobacco physiology

3.1 Effect of growth-promoting bacteria on tobacco Biomass

The method comprises the following steps of treating 4 strains, singly connecting the strains to tobacco in a root irrigation mode through a bacterial suspension, using the strains as growth promoting group tests, connecting pathogenic bacteria to the tobacco in a stem inoculation mode, using the strains as a pathogenic bacteria infection test group, respectively co-inoculating the 4 strains with corresponding pathogenic bacteria, using the strains as an antagonistic group test, pouring sterile water into a control group, respectively co-culturing for 21 days, and then carrying out statistical analysis on the growth condition of the biomass of the tobacco, wherein the results are shown in table 4. After the co-culture is finished, the biomass of the tobacco which is independently inoculated with the growth-promoting antagonistic bacteria and inoculated with the pathogenic bacteria and the growth-promoting bacteria is obviously higher than that of a control. In the independent inoculation of the growth-promoting bacteria, the plant height, the crown width, the leaf length and the leaf width of the LgD2 strain are respectively increased by 20 percent, 8 percent, 24 percent and 20.59 percent compared with the control; LgD10 strain treatment increased 19.31, 10.11, 27.14 and 23.95% respectively; TPD11 strain treatment increased by 15.82, 5.52, 20.31 and 16.93%, respectively; TPB strain treatment increased by 7.86, 8.25, 23.31 and 20.59% respectively; compared with CK, LgD2 and LgD10 strains have the largest influence on the height, the crown width and the leaf length of the tobacco plants, and the difference between the two strains is not obvious; the leaf width of the inoculated growth-promoting bacteria treatment is obviously improved compared with the control, but the difference between the treatments is not obvious. The plant height, crown width, leaf length and leaf width of the tobacco plant which is singly inoculated with phytophthora pathogenic bacteria to treat YM are obviously lower than those of the tobacco plant which is jointly inoculated with four strains of growth promoting bacteria and phytophthora, wherein LgD2+ Y is respectively increased by 11.34, 16.80, 12.96 and 12.13 percent compared with that of the tobacco plant which is singly inoculated with the phytophthora pathogenic bacteria to treat YM; LgD10+ Y is respectively increased by 11.95%, 17.17%, 18.75% and 19.46% compared with single-inoculated YM; TPB + Y is respectively improved by 10.71, 17.61, 15.21 and 12.13 percent compared with single-inoculated YM; TPD11+ Y increased 7.4, 19.31, 13.33, 15.95% respectively over YM inoculated alone. Compared with CK, the difference of plant height and leaf width of YM treated by singly inoculating phytophthora pathogenic bacteria is not significant.

TABLE 4 growth-promoting antagonistic strains on tobacco growth

Note: the data in the table are mean ± sem (n ═ 3). Different lower case letters after the same column of data indicate that the level of difference was significantly different (p <0.05) under different strain treatments.

3.2 Effect of growth-promoting bacteria on the Activity of tobacco enzymes

3.2.1 Effect of growth-promoting bacteria on tobacco SOD enzyme activity

After the co-culture of the growth-promoting antagonistic strain, the pathogenic bacteria and tobacco was completed, the SOD enzyme activities thereof were measured, respectively, and the results are shown in FIG. 6. The SOD activity of the 3 strains of growth-promoting antagonist bacteria inoculated with TPD11, LgD10 and LgD2 independently is higher than that of a control CK by 4.63 percent, 24.37 percent and 4.08 percent respectively, but the difference between LgD2 and the control is not obvious, and the SOD activity of the inoculated TPB treated bacteria is lower than that of the control by 17.88 percent; the SOD enzyme activities of the 3 treatments of TPD11+ Y, LgD10+ Y and LgD2+ Y inoculated with growth-promoting bacteria and phytophthora nicotianae pathogenic bacteria are obviously higher than those of YM treated by singly inoculated phytophthora nicotianae pathogenic bacteria, and are respectively 8.05, 5.85 and 2.68 percent higher than that of YM, and the differences between the 3 treatments of the growth-promoting bacteria and the pathogenic bacteria are not obvious. The enzyme activity of the inoculated TPB + Y treatment is 18.14 percent lower than that of the control. The SOD enzyme activity of YM treated by singly inoculating phytophthora nicotianae pathogenic bacteria is obviously lower than that of CK compared with the control.

3.2.2 Effect of growth-promoting bacteria on the Activity of tobacco POD enzyme

After the growth-promoting antagonistic strain, pathogenic bacteria and tobacco were co-cultured, their POD enzyme activities were measured, and the results are shown in FIG. 7. The 4 growth-promoting antagonistic bacteria are independently inoculated with TPD11, TPB, LgD10 and LgD2, the POD enzyme activity of the growth-promoting antagonistic bacteria is obviously higher than that of a control CK and is respectively 18.36, 7.48, 31.57 and 42.17 percent higher than that of the control CK, and the POD enzyme activity of the 4 growth-promoting antagonistic bacteria is LgD2> LgD10> TPD11> TPB from large to small. The POD enzyme activities of the 4 treatments of TPD11+ Y, TPB + Y, LgD10+ Y and LgD2+ Y inoculated with growth-promoting bacteria and phytophthora nicotianae pathogenic bacteria are obviously higher than that of YM treated by singly inoculated phytophthora nicotianae pathogenic bacteria, and are respectively 12.99, 7.62, 25.67 and 21.21 percent higher than that of YM, and the enzyme activity sizes of the POD treatments are respectively LgD10+ Y > LgD2+ Y > TPD11+ Y > TPB + Y. POD enzyme activity of YM treated by singly inoculating phytophthora nicotianae pathogenic bacteria is obviously higher than that of a control CK.

3.2.3 Effect of growth-promoting bacteria on the Activity of PPO enzyme in tobacco

After the growth-promoting antagonistic strain, pathogenic bacteria and tobacco were co-cultured, the PPO enzyme activities were measured, respectively, and the results are shown in fig. 8. After the 3 strains of growth-promoting antagonistic bacteria TPD11, LgD10 and LgD2 are independently inoculated for treatment, the PPO enzyme activity is obviously higher than that of a control CK and is respectively 30.43 percent, 33.54 percent and 11.18 percent higher than that of the control CK, the PPO enzyme activity of the 3 strains of growth-promoting antagonistic bacteria is LgD10 more than that of TPD11 more than that of LgD2 in sequence from large to small, and the PPO enzyme activity of the inoculated TPB treatment is 10.56 percent lower than that of the control CK; in the treatment of inoculating growth-promoting bacteria and phytophthora nicotianae pathogenic bacteria, only the PPO enzyme activity treated by inoculating LgD10+ Y is obviously 8.67 percent higher than that treated by YM, while the difference between the TPD11+ Y and LgD2+ Y treatment and the YM treatment is not obvious, and the PPO enzyme activity treated by inoculating TPB + Y is obviously 12.67 percent lower than that treated by YM. PPO enzyme activity of YM treated by single inoculation of phytophthora nicotianae pathogenic bacteria is obviously lower than that of a control CK.

3.2.4 Effect of growth-promoting bacteria on tobacco PAL enzyme Activity

After the co-culture of the growth-promoting antagonistic strain, pathogenic bacteria and tobacco was completed, the PAL enzyme activities were measured, and the results are shown in FIG. 9. Treatment with TPD11 and LgD10 growth-promoting antagonist bacteria alone, had PAL enzyme activities significantly 12.09 and 13.19% higher than control CK; compared with a control, the PAL enzyme activity of the inoculated LgD2 treated is not significantly different, and the inoculated TPB treated is 8.79 percent lower than that of the control CK; the PAL enzyme activity of 4 strains of growth-promoting antagonistic bacteria is LgD10> TPD11> LgD2> TPB from large to small. The PAL enzyme activity of the 4 treatments of TPD11+ Y, TPB + Y, LgD10+ Y and LgD2+ Y inoculated with growth-promoting bacteria and phytophthora nicotianae pathogenic bacteria is higher than that of YM inoculated with phytophthora nicotianae pathogenic bacteria alone, and is respectively 17.78, 6.67, 10.00 and 2.22 percent higher than that of YM, only the enzyme activity of TPD11+ Y is obviously higher than that of YM treated with phytophthora nicotianae pathogenic bacteria, and the difference of the treatment of TPB + Y, LgD10+ Y and LgD2+ Y is not obvious compared with that of YM treatment. PAL enzyme activity of YM treated with P.nicotianae pathogen alone was not significantly different from that of the control CK.

3.3 inhibiting action of growth-promoting bacteria on tobacco pathogenic bacteria

After co-culturing the growth-promoting antagonistic strain and tobacco, the number of diseased plants and the disease grade were measured, and according to the formula for calculating the tobacco disease, the disease incidence (%) was the number of diseased plants/the total number of investigated plants x 100%, the disease index (%) ∑ (number of diseased plants at each stage x corresponding to the number of investigated plants)/the highest-order value x 100% of the total number of investigated plants x, and the relative inhibition (%) was (1-disease index in treatment area/disease index in control area) x 100%, the disease incidence, disease index and relative inhibition of each treatment were calculated, and the results are shown in fig. 10. The incidence of the treatment by singly inoculating the phytophthora nicotianae pathogenic bacteria YM reaches 100 percent, compared with the treatment by singly inoculating the YM, the incidence of the TPD11+ Y, TPB + Y, LgD10+ Y and LgD2+ Y inoculated with the growth-promoting bacteria and the phytophthora nicotianae pathogenic bacteria is respectively reduced by 33.33, 13.89, 30.56 and 16.67 percent, and the incidence of the TPD11+ Y and LgD10+ Y inoculated with the phytophthora nicotianae pathogenic bacteria YM is obviously lower than that of the TPB + Y and LgD2+ Y treatments. The disease index of the treatments inoculated with TPD11+ Y, TPB + Y, LgD10+ Y and LgD2+ Y were significantly reduced, respectively by 47.92, 35.42, 45.83 and 39.58% compared with the control, and the disease index of the treatment inoculated with TPD11+ Y was significantly lower than that of the other treatments. Compared with the control, the relative inhibition rates of the treatments inoculated with TPD11+ Y, TPB + Y, LgD10+ Y and LgD2+ Y are remarkably reduced and respectively reach 47.92%, 35.42%, 45.83% and 39.58%, and the relative inhibition rate reaches the lowest value under the treatment inoculated with TPD11+ Y.

3.4 expression of defensive enzymes and resistance related genes by growth-promoting bacteria

3.4.1 detection of total RNA extraction quality of tobacco leaves

RNA was extracted from 10 treated tobacco leaves, respectively, with an OD 260/280 of between 1.8 and 2.2. The extraction results were checked by 1% agarose gel electrophoresis, and it was observed that each lane had a clear and clean band of interest at 28S and 18S rRNA bands, and the ratio of 28S to 18S brightness was about 2: 1. The extracted RNA is relatively complete and high in quality, meets the requirements of subsequent experiments, and can be used for the next reverse transcription experiment reaction. After reverse transcription, the cells are stored at-20 ℃ for later use.

3.4.2 influence of growth-promoting antagonistic bacteria on genes related to defense enzymes

PPO, PAL, SOD and POD are enzymes playing a role in tobacco disease resistance, and NtPO, PAL, NtSOD and NbDADIR genes are genes controlling the synthesis of the four enzymes, respectively. In this study, the ntpp gene expression levels of TPD11 and TPB + Y treatments were significantly up-regulated compared to the control (fig. 11), 92 and 73% higher than the control, respectively, and then both LgD10 and LgD2+ Y treatments were up-regulated, 73.02 and 69.38% higher than the control, respectively. Compared with the control, PAL genes of plants inoculated with TPD11 and LgD10 are up-regulated, which is 94.36 and 58.25% higher than that of the control respectively, and PAL genes treated by other strains are not significantly different from that of the control. Compared with a control, the NtSOD genes of the plants inoculated with TPD11, LgD10 and LgD10+ Y are all obviously up-regulated, which are respectively 97.70%, 88.61% and 65.12% higher than the control, and the NtSOD genes of the other treated plants are not expressed. The expression level of NbDADIR genes inoculated with TPD11, LgD10 and TPB + Y for treatment is up-regulated, which is respectively 98.73, 93.49 and 2.80 percent higher than that of a control, and the NbDADIR genes of other treated plants are not expressed. After inoculating TPD11 strain, the expression quantities of NtPO, PAL, NtSOD and NbDADIR in tobacco leaf all reach peak value.

3.4.3 expression of tobacco growth-related resistance genes

The genes NtHSR203J, NtTTG2 and NtPR1a were involved in tobacco growth and disease resistance, and in this study, the expression of NtHSR203J gene inoculated with TPD11 and LgD10 treatments was significantly up-regulated (FIG. 12), 95.20 and 85.12% higher than the control, respectively, and the expression level was the highest in TPD11, and none was expressed in the other strains. The expression of the NtTTG2 gene inoculated with TPD11 and LgD10 treatments was up-regulated, 90.32 and 37.84% higher than the control, respectively, and none of the other strains were expressed, which is consistent with the expression result of the NtHSR203J gene. The NtPR1a gene was not expressed in all strains treated for inoculation.

3.4.4 expression of tobacco Black shank related resistance Gene

The tobacco NbRbohA gene and the NbRbohB gene play an important role in the process of resisting Phytophthora infestans (Phytophthora infestans) of tobacco, and in the research, compared with a control, the expression levels of the NbRbohA genes inoculated with TPD11 and LgD10 are both up-regulated (figure 13), are respectively 80.43 and 88.71% higher than the control, the NbRbohA gene inoculated with LgD10+ Y is also up-regulated, but the difference is not obvious, and the NbRbohA genes of other treated tobacco plants are not expressed. The expression level of NbRbohB genes inoculated with TPD11 and LgD10 is obviously up-regulated, which is 91.81% and 91.76% higher than that of the control, the NbRbohB genes inoculated with LgD2, LgD10 and Y are up-regulated, but the difference is not obvious, and the NbRbohA genes of other treated tobacco plants are not expressed.

The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as required after reading the present specification, but all of them are protected by patent law within the scope of the present invention.

Claims

1. A growth-promoting disease-resistant biological agent is characterized in that: the biological agent is prepared by one or a plurality of combinations of Bacillus cereus, Bacillus thuringiensis, fusarium oxysporum and Aspergillus tamarii.

2. The growth-promoting anti-disease biological agent according to claim 1, wherein: the biological agent is prepared from Bacillus cereus (Bacillus cereus).

3. The growth-promoting anti-disease biological agent according to claim 1, wherein: the biological agent is prepared from Bacillus thuringiensis (Bacillus thuringiensis).

4. The growth-promoting anti-disease biological agent according to claim 1, wherein: the biological agent is prepared from fusarium oxysporum (fusarium oxysporum).

5. The growth-promoting anti-disease biological agent according to claim 1, wherein: the biological agent is prepared from Aspergillus tamarii (Aspergillus tamarii).

6. The growth promoting and disease resisting biological agent as claimed in any one of claims 1-5, wherein: the biological agent is used for promoting growth and resisting diseases of the paris polyphylla.

7. The growth-promoting anti-disease biological agent according to any one of claims 1 to 5, wherein: the biological agent is used for promoting the growth and resisting diseases of tobacco.