CN113801807B

CN113801807B - Growth-promoting microbial inoculum for inducing systemic resistance of processed tomatoes and application thereof

Info

Publication number: CN113801807B
Application number: CN202110765918.5A
Authority: CN
Inventors: 林青; 娄恺; 史应武; 霍向东; 杨红梅; 楚敏; 高雁; 曾军; 包慧芳; 王宁
Original assignee: Institute Of Microbial Applications Xinjiang Academy Of Agricultural Sciences (china Xinjiang-Armenia Bioengineering Research And Development Center)
Current assignee: Institute Of Microbial Applications Xinjiang Academy Of Agricultural Sciences (china Xinjiang-Armenia Bioengineering Research And Development Center)
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2023-03-10
Anticipated expiration: 2041-07-06
Also published as: CN113801807A

Abstract

The invention discloses a growth-promoting bacterium Paenarthrobacter nitroguajacolicus D8 capable of mediating processed tomatoes to generate induction system resistance, which has obvious control effect on 3 diseases under the condition of no antagonism with tomato early blight, gray mold and bacterial wilt pathogenic bacteria. The strain is separated from rhizosphere soil of a green bean plant in a farm green bean field in Xishan Wulu wood Qi of Xinjiang, is a P.nitroguajacolicus-like new strain, is prepared into a P.nitroguajacolicus D8 microbial growth promoting microbial inoculum by liquid fermentation, treats and processes tomato seeds, obviously increases the root length, the germination potential and the germination rate of the seeds, and obviously improves the root length, the fresh weight and the plant height of the processed tomato seedlings. The induced inoculation of the strain D8 bacterial liquid can obviously increase the content of endogenous jasmonic acid in the processed tomatoes and maintain the content for a certain time, so that the processed tomatoes have systemic resistance, and the disease resistance of the processed tomatoes to 3 pathogenic bacteria is enhanced. The invention provides a novel growth-promoting microbial inoculum, which is used for processing tomato disease prevention and control, is environment-friendly, has low cost and has wide application value in the technical field of expanding the application of microbial growth-promoting microbial strains.

Description

Growth-promoting microbial inoculum for inducing systemic resistance of processed tomatoes and application thereof

Technical Field

The invention relates to the technical field of microbial agents and application thereof, in particular to a Paenarthrobacter nitroguajacolicus D8 microbial growth promoting microbial agent for inducing and processing tomatoes to generate system resistance and the technical field of application thereof.

Background

The tomato processing is the dominant industry of Xinjiang-style economy, is one of the three major industries of Xinjiang, accounts for 18 percent of the total world production, and has important position in China and even the world. Early blight, bacterial wilt and gray mold are 3 serious diseases in the growth process of processed tomatoes, and the yield of the tomatoes is reduced by 10-30% every year. In recent years, diseases are increasingly generated with the increase of planting area of processed tomatoes, however, continuous cropping phenomena are generally generated in the aspects of regional layout and industrial production, and in recent years, the diseases tend to be generated in an increasing variety and an increasing incidence. Severely restricting the sustainable development of the local processing tomato planting industry.

However, no immune or highly resistant varieties are currently found against early blight, phytophthora, viral diseases or bacterial diseases of processed tomatoes. The disease control is mainly a method of applying chemical pesticides, the chemical pesticides need to adopt a control mode of applying different types of protective agents and therapeutic agents for multiple times before and during the onset of diseases, so that the production cost is increased, the problems of environmental pollution, pesticide residue and the like are easily caused, beneficial bacteria in soil are even harmed, and the ecological balance of planting soil is damaged. The identification of pathogenic bacteria and the biological control of antagonistic bacteria have been the focus of research in recent years. Many scholars have already clarified pathogenic bacteria of Xinjiang processing tomato various diseases, and successfully screen antagonistic bacteria with better antibacterial performance, and have certain prevention effect on the diseases. However, the antagonistic bacteria depends on stable and long-term colonization, is influenced by factors such as plant varieties, cultivation modes, pesticide application, field ecological environment and the like, and has unstable prevention and treatment effects.

The induction of plant resistance is one of the important means for biological control of certain endophytic bacillus, and some microbes can be used as plant activators to induce plants to generate systemic resistance, so that the plant growth regulator has a good disease prevention effect. After the biocontrol bacteria are used for treating plants, a series of physiological reaction phenomena beneficial to enhancing the disease resistance of the plants are induced in the plants, which has important significance for the healthy growth of crops and the prevention and treatment of plant diseases. It is generally considered as a new disease control strategy for exploiting the intrinsic resistance mechanism of plants.

At present, the research aiming at the resistance of tomato systems mainly focuses on the research of growth-promoting bacteria such as bacillus, photosynthetic bacteria and the like in inducing the resistance of tomatoes to gray mold or early blight systems, the application effect needs to be improved, few growth-promoting bacteria suitable for inducing the resistance of processed tomatoes to gray mold, early blight and bacterial wilt systems exist, the selection of growth-promoting bacteria with obvious appropriate effect is urgently needed, and the application of Paenarthrobacter nitirguajacolicus strains in inducing the resistance of processed tomatoes to gray mold, early blight and bacterial wilt systems is not reported in documents and patents, so that the screening of growth-promoting bacteria capable of inducing the resistance of processed tomatoes to gray mold, early blight and bacterial wilt systems has wide application prospect in eco-friendliness of processed tomatoes and cost reduction.

Disclosure of Invention

Aiming at the problems that the prior processed tomatoes have various diseases, are mainly controlled by chemical pesticides, have unstable application effect of antagonistic bacteria for biological control and the like, and no literature and patent report is found about the application of the Paenarthrobacter nitroguajacolicus strain in inducing the systemic resistance of the tomatoes to gray mold, early blight and bacterial wilt. The invention aims to provide a growth-promoting bacterium Paenarthrobacter nitroguajacolicus D8 which can mediate processed tomatoes to generate induction system resistance and has better prevention effect on early blight, gray mold and bacterial wilt. The influence of the strain on the systemic resistance of the tomato to gray mold, early blight and bacterial wilt in induced processing of the tomato is further considered, and the result shows that the Paenarthrobacter nitirguajacolicus D8 bacterial solution can obviously improve the jasmonic acid content of the processed tomato, so that the processed tomato plant generates induced systemic resistance, and the mechanism of the strain is verified to find that the Paenarthrobacter nitirguacolicus D8 bacterial strain has no antagonistic resistance to tomato gray mold pathogenic bacteria Botrytis cinerea (Botrytis cinerea), tomato early blight pathogenic bacteria Alternaria solanacearum (Alternaria solani) and tomato bacterial wilt pathogenic bacteria Ralstonia solanacearum, so that the effect of inhibiting the tomato pathogenic bacteria by the antagonistic strain D8 is not achieved through the action among the strains, but the induced systemic resistance is generated by induced processing of the tomato, and the control effect on the tomato three pathogenic bacteria is achieved. The strain D8 is applied to the control of tomato epidemic situation, is green and environment-friendly, has low cost, provides a new growth-promoting bacterium for the development of an intrinsic resistance mechanism of tomato processing, and has wide application value in the technical field of expanding the application of microbial growth-promoting bacterium strains.

In order to achieve the technical effects, the invention adopts the following technical scheme:

the invention provides a paenrothbacteria nitroguajacolius D8 microbial agent for inducing and processing systemic resistance of tomatoes, which is obtained by fermenting the paenrothbacteria nitroguajacolius D8.

In the invention, the strain Paenarthromobacter nitroguajacolius D8 is screened and separated from the rhizosphere soil of a vigna unguiculata plant in a field vigna unguiculata farm in Xinjiang Ulu wood Qishan mountain, and is identified by well-known and accepted molecular level identification of a strain system in the field, so that the obtained category of the strain Paenarthromobacter is proved, wherein the strain Paenarthromobacter nitroguajacolius D8 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC No.21584, preservation date: 2021, 01 month 04.

The gene sequence of the Paenarthrobacter nitroguajacolicus D8 CGMCC No.21584 strain is shown in SEQ ID NO. 1.

In the invention, the Paenarthrobacter nitroguajacolicus D8 in the microbial agent is separated and purified in a culture medium and culture conditions: 3g of beef extract powder, 10g of peptone, 5g of sodium chloride, 15g of agar, 1L of distilled water, pH7.3 +/-0.1 and sterilizing at 121 ℃ for 15min.

The Paenarthrobacter nitroguajacolicus D8 isolation and purification medium provided above is suitably used for preparing a corresponding plate medium, a slant medium or a liquid medium.

Meanwhile, the invention provides a preparation method of the Paenarthrobacter nitroguajacolicus D8 microbial growth promoting microbial inoculum for inducing and processing systemic resistance of tomatoes, which comprises the following steps:

(1) Activating the Paenarthrobacter nitroguajacolicus D8 which is preserved at low temperature on a plate culture medium, selecting a single colony on a slant culture medium, and culturing for 24 hours in an incubator at 37 ℃ to obtain the activated Paenarthrobacter nitroguajacolicus D8 strain.

(2) And (2) scraping a ring of the activated Paenarthrobacter nitroguajacolius D8 strain obtained in the step (1) by using a sterile inoculating ring, inoculating the strain into 100mL of liquid culture medium, and performing shake culture for 24h at 37 ℃ and at the rotating speed of a shaking table of 180r/min to obtain the Paenarthrobacter nitroguajacolius D8 microbial growth promoting bacteria liquid.

Further, the invention provides an application of the microorganism of Paenarthrobacter nitroguajacolius D8 for inducing and processing systemic resistance of tomatoes in preparing a medicament for preventing and treating gray mold, early blight and bacterial wilt of tomatoes. By using the Paenarthrobacter nitroguajacolicus D8 bacterial solution prepared by the invention to treat and process the tomato seeds, the bud length, the germination rate and the germination vigor of the tomato seeds are all obviously increased, and the root length, the fresh weight and the plant height of the tomatoes are also obviously improved.

Further, the invention provides an application of the microbial growth promoting agent of Paenarthrobacter nitroguajacolicus D8 for inducing and processing systemic resistance of tomatoes in preparing a medicament for preventing and treating botrytis cinerea, early blight and bacterial wilt of tomatoes. The Paenarthrobacter nitroguajacolius D8 bacterial liquid can obviously improve the jasmonic acid content of the processed tomatoes, so that the processed tomato plants generate induced system resistance, the anti-induction effect on three germs can reach more than 28 percent, and the epidemic situation index of the three tomato germs is reduced by 15 to 43 percent

By implementing the specific technical scheme provided by the invention, the following beneficial effects can be achieved:

(1) The invention provides a strain Paenarthhromobacter nitroguajacolicus D8, which is proved to belong to a new application of the Paenarthhromobacter nitroguajacolicus with a strain number D8 in the category of the obtained Paenarthhromobacter through the molecular level identification of a well-known and accepted strain system in the field, and further needs to be preserved according to legal requirements.

(2) The invention provides a Paenarthrobacter nitroguajacolicus D8 strain which has no antagonistic resistance to tomato gray mold pathogen botryococcus griseus (Botrytis cinerea), tomato early blight pathogen Alternaria solani (Alternaria solani) and tomato bacterial wilt pathogen Ralstonia solani (Ralstonia solanacearum), and shows that the inhibition of the strain D8 on tomato pathogenic bacteria is not through antagonistic action among strains, but through induced processing of tomato to generate systemic resistance on three tomato pathogenic bacteria, the prevention and treatment effect is achieved. The strain D8 is applied to the epidemic situation prevention and control of processed tomatoes, is green and environment-friendly, has low cost, provides a new growth-promoting bacterium for the discovery of an intrinsic resistance mechanism of the processed tomatoes, and has wide application value in the technical field of expanding the application of microbial growth-promoting bacterium strains.

(2) The application of the Paenarthrobacter nitroguajacolicus D8 in the prevention and treatment of the gray mold, the early blight and the bacterial wilt of the tomatoes, the prepared Paenarthrobacter nitroguajacolicus D8 bacterial liquid is used for treating and processing the tomato seeds, the bud length, the germination rate and the germination vigor of the tomato seeds are obviously increased, and the root length, the fresh weight and the plant height of the tomatoes are also obviously improved. The strain D8 does not inhibit the tomato pathogenic bacteria through antagonism among strains, the Paenarthrobacter nitroguajacolicus D8 bacterial solution can improve the jasmonic acid content of the processed tomato, so that the processed tomato plant generates induction system resistance, the anti-induction effect on the three pathogenic bacteria can reach more than 28%, and the epidemic situation index of the three tomato pathogenic bacteria is reduced by 15-43%. The invention provides a new growth-promoting bacterium for discovering and processing the internal resistance mechanism of tomatoes, and the strain D8 is applied to the control of tomato epidemic situation, so that the method is green and environment-friendly, has low cost, and has wide application value in the technical field of expanding the application of microbial growth-promoting bacterium strains.

Drawings

FIG. 1 is a diagram of a phylogenetic tree constructed by strain D8 based on the 16Sr DNA sequence.

FIG. 2 is a colony morphology of strain D8.

FIG. 3 is a graph showing the change of jasmonic acid content of processed tomato leaves with time after D8 bacterial liquid root irrigation treatment.

FIG. 4 is the experimental chart of the confrontation of the strain D8 with tomato gray mold, early blight and bacterial wilt pathogens, wherein FIG. 4A is the antagonistic chart of the strain D8 against tomato gray mold pathogen Botrytis cinerea (Botrytis cinerea); FIG. 4B is a diagram of the antagonism of strain D8 against the early blight pathogen Alternaria solani (Alternaria solani); FIG. 4C is a graph showing the antagonism of strain D8 against the bacterial wilt pathogen Ralstonia solanacearum.

Detailed Description

The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All the raw and auxiliary materials selected in the present invention, as well as the selected culture method of the strain and the selected DNA extraction kit are well known and selected in the art, and the% referred to in the present invention are weight percentages unless otherwise specified. DNA extraction kit: biometrics (Shanghai) Ltd.

Example 1: separation, screening and identification of Paenarthrobacter nitroguajacolicus D8 CGMCC No.21584

(I) separation and purification

In 2020, the rhizosphere soil of the vigna unguiculata field vigna unguiculata plants collected from farm vigna unguiculata in Xinjiang, and the root surface soil refers to a small amount of soil adhered to the surface of roots after a large amount of soil adhered to the root system is shaken off. Cutting off roots, weighing the roots, placing the roots into a triangular flask filled with 45mL of sterile normal saline, shaking for 1-2 min by hand, taking out the roots after the soil adhered to the surfaces of the roots is washed, sucking water on the surfaces of the roots by using absorbent paper, and weighing the roots, wherein the weight of W1-W2 is the weight of the surface soil of the roots. Diluting root surface soil to 10-4 times, taking 10-1,10-2,10-3 and 10-4, sucking 100 μ L of diluent, respectively coating on NA culture medium plate, and culturing at 30 deg.C. Single colonies were picked from the plates, inoculated into new separation plates for purification, and repeated 3-4 times for purification. The purified strain is inoculated on a culture medium, cultured for 24 hours at a constant temperature of an incubator at 37 ℃, and stored at 4 ℃ for later use, see figure 2.

(II) Classification and identification

Sequencing and analysis of 16Sr RNA Gene of Paenarthrobacter nitroguajacolicus D8 (hereinafter referred to as "Strain D8"):

(1) Extraction of PCR template DNA

Inoculating the strain D8 into a culture medium, carrying out shake culture at 37 ℃ for 24h at the rotation speed of 180r/min, centrifugally collecting thalli, and extracting the genome DNA by adopting a genome DNA extraction kit of an industrial Ezup column type bacterium.

(2) PCR amplification

Inoculating the bacterial strain BJT-7 into a culture medium, shaking and culturing at the rotation speed of 3000r/min and the temperature of 37 ℃ for 24h, centrifugally collecting thalli, and extracting genome DNA by adopting a novel plant genome DNA rapid extraction kit. The total DNA of the extracted strain D8 is taken as a template for PCR amplification, and the primers are 27F (5-. Carrying out PCR amplification on a target fragment by adopting a 16S rDNA specific primer, wherein the total volume of a PCR reaction system is 25 mu L, and the PCR amplification condition is 94 ℃ for 5min; 30s at 94 deg.C, 30s at 54 deg.C, 1min30s at 72 deg.C, 35 cycles, 10min at 72 deg.C, and preserving at 4 deg.C.

(3) Sequence determination

The PCR amplification product is detected by electrophoresis and purified and then sequenced, the 16SrDNA sequence of the strain D8 is sequenced, the 16SrDNA partial ordered sequence is spliced by DNAstar software, the length is 1344bp, the sequence is shown as SEQ ID NO:1, BLAST homologous sequence retrieval is carried out in GenBank, MEGA 5.0 software commonly adopted in the field is utilized to establish phylogenetic tree (repeated sampling is carried out for 1000 times) by a Neighbor-Joining method, and the result is shown in figure 1. Through comparison analysis, the 16Sr DNA gene sequence of the strain D8 has the highest homology with the Paenarthrobacter nitroguajacolius, the similarity is 100 percent, in a phylogenetic tree constructed by the 16Sr DNA gene sequence, the 16SrDNA sequence of the strain D8 has the closest genetic relationship with the Paenarthrobacter nitroguajacolius (the accession number is: NR _ 027199), the confidence is 99.93 percent, and the strain D8 can be determined to be a strain in the category of the Paenarthrobacter through the molecular level identification of series strains, and is temporarily named as the Paenarthrobacter nitroguajacolius D8 from the taxonomic angle.

The above-mentioned strain, paenarthrobacter nitroguajacolius D8, has been deposited with the International Collection of microorganisms under the Budapest treaty: china general microbiological culture Collection center (CGMCC). Address: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, preservation date of 2021, 01 month, 04 days, preservation number of CGMCC No.21584.

Example 2: preparation of microbial growth promoting microbial inoculum of Paenarthrobacter nitroguajacolius D8 CGMCC No.21584

The embodiment provides a preparation method of a Paenarthrobacter nitroguajacolicus D8 microbial agent for inducing systemic resistance of processed tomatoes, which comprises the following specific steps:

The culture medium for separating and purifying Paenarthrobacter nitroguajacolicus D8 and the culture conditions are as follows: 3g of beef extract powder, 10g of peptone, 5g of sodium chloride, 15g of agar and 1L of distilled water, wherein the pH value is 7.3 +/-0.1, and the beef extract powder is sterilized at 121 ℃ for 15min.

Example 3: application of Paenarthrobacter nitroguajacolius D8 CGMCC No.21584 (1) Effect test of Paenarthrobacter nitroguajacolius D8 strain on germination of processed tomato seeds

Carrying out shake flask culture on the strain D8 for 24h under the conditions of 37 ℃ of liquid culture medium and 180rpm, and diluting the strain D8 to 10 percent by using normal saline ⁴ Multiple, bacterial liquid concentration 5.9X 10 ⁴ cfu/ml, used for processing the seed dip of tomato seeds. Treating with clear water as blank control, soaking the seeds in 2% sodium hypochlorite for 10min, rinsing with clear water for 5 times, soaking in 75% alcohol for 30s, rinsing with clear water for 5 times, and sterilizing the surface. The disinfected seeds are respectively soaked in diluted bacterial liquid and clear water, the temperature is kept at 37 ℃ for 4 hours, 10 seeds are sown on a culture dish paved with sterile water-wet filter paper in a spot mode, each treatment is repeated for 3 times, the culture dish is placed in a 28 ℃ incubator to be cultured in a dark place, the germination rate of the seeds is investigated after 3 days, the bud length is measured, the germination potential of the seeds is investigated after 5 days, and specific results are shown in table 1.

Table 1: the present invention provides the effect of strains on processing tomato seeds

	Bud length (cm)	Germination rate	Vigor of germination
				Control of	1.73±0.68a	0.60±0.12a	0.70±0.13a
D8	3.09±0.56b	0.83±0.06b	0.97±0.06b

Note: the data in the same column are marked with different letters to indicate that the new repolarization test is remarkable, and the lower case letter is P =0.05.

As can be seen from the data in Table 1, the bacterial liquid of the strain D8 provided by the invention is used for treating and processing tomato seeds, the bud length, the germination rate and the germination potential of the processed tomato seeds are all obviously improved, wherein the bud length is increased by nearly two times, the germination rate is improved by 20%, and the germination potential is also improved by nearly 30%, so that the strain provided by the invention has a growth promoting effect on the processed tomato seeds, and the effect is obvious.

(2) Test of influence of different treatment modes of Paenarthrobacter nitroguajacolicus D8 bacterial liquid on tomato processing

In the experiment, on the basis of the test (1), the strain D8 is placed in a liquid culture medium to be subjected to shake flask culture for 24 hours at 37 ℃ and 180 rpm. The bacterial liquid is subjected to two treatments: a is diluted to 10 with normal saline ⁴ Multiple, bacterial liquid concentration of 10 ⁴ cfu/ml, soaking the processed tomato seeds; b, centrifuging the bacterial liquid of the strain D8 for 10min at the rotating speed of 9000rpm, discarding the supernatant, and resuspending the precipitated thallus into 10-degree bacteria by using clear water ⁹ And (5) the cfu/ml bacterial solution is used for performing root irrigation treatment on the processed tomato seeds. The control group and the root irrigation treatment group are soaked in normal saline, and the seeds are soakedThe treatment group is soaked with diluted bacterial liquid and is insulated for 4 hours at 37 ℃. Using clear water treatment as a blank control, and respectively adding the following components in percentage by weight: and (3) dibbling the seeds after the seed soaking in a small flowerpot with vermiculite =2 medium 1, pouring 100ml of clear water in each pot of the control group and the seed soaking group, adding 15ml of heavy suspension bacteria liquid in each pot of the root irrigation group, and adding 75ml of clear water. Each treatment was performed in 10 pots, and repeated 3 times. The clear water is poured once every 5 days, and each time, the water is poured into a basin with 100 ml. When the tomato grows to 3 leaves and 1 heart, the root length, plant height and fresh weight are measured, and specific results are shown in table 2.

Table 2: the invention provides the influence of different treatment modes of bacterial liquid of a strain D8 on tomato processing

	Root length	Plant height	Fresh weight of
				Control	10.82±3.30a	18.38±2.10a	0.41±0.29a
D8 irrigated root	13.72±2.79b	20.99±2.91b	0.75±0.33b
				D8 seed soaking	13.79±2.12b	20.50±2.70b	1.21±0.53c

Note: the data in the same column are marked with different letters to indicate that the new repolarization test is remarkable, and the lower case letter is P =0.05

As can be seen from the data in Table 2, the bacterial liquid of the strain D8 provided by the invention has a promoting effect on the growth of processed tomatoes regardless of seed soaking treatment or root irrigation treatment. The root length of the processed tomatoes treated by the strain D8 bacterial liquid provided by the invention is increased by nearly 3cm compared with a control group, the plant height of the processed tomatoes is increased by 3cm, and the fresh weight of the processed tomatoes is increased by 0.5g in the seed soaking treatment compared with the root irrigating treatment and 0.8g in the control fresh weight. Therefore, the bacterial liquid of the strain D8 provided by the invention has obvious growth promotion effect on the germination of processed tomatoes and the later growth process.

(3) Influence of bacterial liquid of strain D8 on resistance of tomato processing system

In the experiment, based on the experiment 1-2, when the processed tomato seedlings grow to 3 leaves and 1 heart, the tomato seedlings with the same growth vigor and plant height are selected for root irrigation. The strain D8 is placed in a liquid culture medium to be cultured for 24 hours in a shaking way under the conditions of 37 ℃ and 180 rpm. Centrifuging the bacterial solution at 9000rpm for 10min, discarding the supernatant, and resuspending the precipitated thallus with clear water to a concentration of 10 ⁹ cfu/ml, 15ml of the resuspended bacterial liquid is added to each pot of the processed tomatoes, and 75ml of clear water is added. Each treatment was repeated 3 times with 10 pots. The substrate is kept moist by treating with clear water as a blank control and watering in the same amount. Collecting tomato leaves 5 days after root irrigation, measuring the jasmonic acid content of the tomato leaves by using a plant Jasmonic Acid (JA) enzyme-linked immunosorbent assay (ELISA) kit according to the operation of the instruction, and the specific result is shown in figure 3.

As can be seen from the data in FIG. 3, the jasmonic acid content of the tomato leaves processed by the control group shows regular fluctuation along with watering, and the jasmonic acid content of the tomato leaves processed by the treatment group obviously increases on the 3 rd day after the root is irrigated by the D8 bacterial liquid and is maintained for a period of time. The fact that the bacterium liquid of the strain D8 can obviously improve the jasmonic acid content of the processed tomatoes is shown.

(4) Influence of bacterial liquid of strain D8 on disease resistance of processed tomatoes

Preparation of pathogenic bacteria: inoculating Botrytis cinerea (Botrytis cinerea) and Alternaria solani (Alternaria solani) with Botrytis cinerea and Alternaria solani, culturing at 28 deg.C for 7-10 days until spores grow, washing with sterile water under aseptic condition to remove spores, counting with a blood counting plate, and adjusting concentration to 10 ⁶ cfu/ml spore suspension. Culturing Ralstonia solanacearum in NB liquid medium at 37 deg.C under 180rpm for 24 hr to obtain a culture medium with a concentration of 10 ⁹ cfu/ml of bacterial liquid.

Induction and inoculation: when the processed tomato seedlings grow to 3 leaves and 1 heart, selecting the tomato seedlings with consistent growth vigor and plant height, irrigating roots of the tomato seedlings with the heavy suspension D8 bacterial liquid (the method is the same as the test (2)), and carrying out induced inoculation.

Challenge inoculation: after the induction treatment for 5 days, respectively spraying the prepared 3 pathogenic bacteria spore suspension/bacterial liquid on the leaf surfaces by using a spraying method, and preserving moisture for 24-48h after inoculation, wherein the temperature of a culture room is 25 ℃, and the humidity RH is more than or equal to 90%. The epidemic index and the anti-induction effect are counted on the 5 th day and the 14 th day of challenge inoculation, and the results are shown in tables 3-4.

Table 3: the invention provides the influence of the bacterial liquid of the strain D8 on the epidemic situation index of the processed tomatoes

Table 4: the invention provides the influence of the bacterial liquid of the strain D8 on the anti-trapping effect of processed tomatoes

	Gray mold	Early blight	Bacterial wilt
				Anti-trap effect (%)	37.9±5.06	32.97±6.59	28.77±4.03

As can be seen from the data in tables 3-4, after tomato is processed by the bacterial liquid of the strain D8, the disease resistance of the plant to 3 pathogenic bacteria can be effectively enhanced, and the disease degree is obviously lower than that of a control. When the strain D8 bacterial liquid is treated for 5 days, the epidemic index of the tomato gray mold is reduced by 15% compared with that of a control group, the epidemic index is further reduced along with the prolonging of the treatment time, and when the strain D8 bacterial liquid is treated for 14 days, the epidemic index of the tomato gray mold is reduced by 22% compared with that of the control group; when the strain D8 bacterial liquid is used for treating the tomato early blight on day 5, the epidemic index of the tomato early blight is reduced by 14% compared with that of a control group, and when the strain D8 bacterial liquid is used for treating the tomato early blight on day 14, the epidemic index of the tomato early blight is reduced by 16% compared with that of the control group; when the bacterial strain D8 is used for treating the tomato bacterial wilt disease on the 5 th day, the epidemic index of the tomato bacterial wilt disease is reduced by 43 percent compared with that of a control group, and when the bacterial strain D8 is used for treating the tomato bacterial wilt disease on the 14 th day, the epidemic index of the tomato early blight disease is reduced by 25 percent compared with that of the control group. As can be seen from the data of the anti-inducing effect of the strain D8 on the processed tomatoes in the table 4, the anti-inducing effect of the strain D8 on the processed tomatoes is over 28 percent, the bacterial solution of the strain D8 has significant growth promotion effect on seed germination and late growth of the processed tomatoes, and the epidemic situation index of the processed tomatoes is significantly reduced by the treatment of the strain D8. The invention provides a new growth-promoting bacterium for discovering and processing the intrinsic resistance mechanism of tomatoes, and the strain D8 is applied to the control of tomato epidemic situation, so that the method is green and environment-friendly, has low cost, and has wide application value in the technical field of expanding the application of microbial growth-promoting bacterium strains.

(4) Antagonistic test of Paenarthrobacter nitroguajacolicus D8 strain against tomato pathogens

In order to further verify the inhibition mechanism of the strain D8 on tomato pathogenic bacteria, the test inspects the influence of the strain D8 on main tomato processing pathogenic bacteria, the test selects tomato gray mold pathogenic bacteria Botrytis cinerea (Botrytis cinerea), tomato early blight pathogenic bacteria Alternaria solani (Alternaria solani) and tomato bacterial wilt pathogenic bacteria Ralstonia solanacearum to carry out the test, and the specific test result is shown in figure 4.

As can be seen from the results in FIG. 4, FIG. 4A shows the antagonistic effect of the strain D8 against Botrytis cinerea (Botrytis cinerea), a pathogen of tomato Botrytis cinerea; 4B is the antagonistic effect of the strain D8 on tomato early blight pathogenic bacteria Alternaria solani (Alternaria solani); and 4C is the antagonistic effect of the strain D8 on the pathogenic bacteria of tomato bacterial wilt, namely Ralstonia solanacearum. As can be seen, the strain Paenarthrobacter nitroguajacolicus D8 has no antagonism to tomato Botrytis cinerea (Botrytis cinerea), tomato early blight pathogen Alternaria solani (Alternaria solani) and tomato bacterial wilt pathogen Ralstonia solani (Ralstonia solanacearum), and the strain D8 has the effect of enhancing the disease resistance to the three tomato pathogens by inducing and processing the tomato instead of the antagonism among the strains. The strain Paenarthrobacter nitroguajacolicus D8 is applied to the control of tomato epidemic situation, is green and environment-friendly, has low cost, provides a new growth-promoting bacterium for the development of an internal resistance mechanism of processed tomatoes, and has wide application value in the technical field of application of expanding microbial growth-promoting bacterium strains.

The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.

Sequence listing

<110> institute of microorganism application of academy of agricultural sciences in Xinjiang (Xinjiang-Yameiya bioengineering research and development center, china)

<120> growth promoting microbial inoculum for inducing systemic resistance of processed tomatoes and application thereof

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aatggcctac caaggcgacg acgggtagcc ggcctgagag ggtgaccggc cacactggga 240

ctgagacacg gcccagactc ctacgggagg cagcagtggg gaatattgca caatgggcgc 300

aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa acctctttca 360

gtagggaaga agcgtaagtg acggtacctg cagaagaagc gccggctaac tacgtgccag 420

cagccgcggt aatacgtagg gcgcaagcgt tatccggaat tattgggcgt aaagagctcg 480

taggcggttt gtcgcgtctg ctgtgaaaga ccggggctca actccggttc tgcagtgggt 540

acgggcagac tagagtgcag taggggagac tggaattcct ggtgtagcgg tgaaatgcgc 600

agatatcagg aggaacaccg atggcgaagg caggtctctg ggctgtaact gacgctgagg 660

agcgaaagca tggggagcga acaggattag ataccctggt agtccatgcc gtaaacgttg 720

ggcactaggt gtgggggaca ttccacgttt tccgcgccgt agctaacgca ttaagtgccc 780

cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag 840

cggcggagca tgcggattaa ttcgatgcaa cgcgaagaac cttaccaagg cttgacatga 900

accggaaaga cctggaaaca ggtgccccgc ttgcggtcgg tttacaggtg gtgcatggtt 960

gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca accctcgttc 1020

tatgttgcca gcgcgttatg gcggggactc ataggagact gccggggtca actcggagga 1080

aggtggggac gacgtcaaat catcatgccc cttatgtctt gggcttcacg catgctacaa 1140

tggccggtac aaagggttgc gatactgtga ggtggagcta atcccaaaaa gccggtctca 1200

gttcggattg gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta atcgcagatc 1260

agcaacgctg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca agtcacgaaa 1320

gttggtaaca cccgaagccg gtgg 1344

Claims

1. A microbial growth promoting agent of Paenarthrobacter nitroguajacolicus D8 for inducing systemic resistance of processed tomatoes is characterized in that the microbial growth promoting agent is obtained by activated culture of the Paenarthrobacter nitroguajacolicus D8, and the preservation number of the Paenarthrobacter nitroguajacolicus D8 is CGMCC No.21584.

2. The method for preparing the paenrobacter nitroguajacolicus D8 microbial growth promoting agent for inducing systemic resistance of processed tomatoes as claimed in claim 1, wherein the preparation method comprises the following steps:

(1) Activating the Paenarthrobacter nitroguajacolicus D8 stored at low temperature on a plate culture medium, selecting a single colony on a slant culture medium, and culturing for 24 hours in an incubator at 37 ℃ to obtain an activated Paenarthrobacter nitroguajacolicus D8 strain;

3. The use of the microorganism of Paenarthrobacter nitroguajacolius D8 for inducing systemic resistance in tomato as claimed in claim 1 for preparing a medicament for preventing and treating gray mold, early blight and bacterial wilt of tomato.

4. The use of the microorganism growth promoting microbial agent of paenrhromobacter nitroguajacolicus D8 for inducing systemic resistance of tomato as claimed in claim 1 in the preparation of a medicament for preventing and treating tomato gray mold, early blight and bacterial wilt.