CN103614312B - Pseudomonas chloroaphis with effects of effective zea mays sheath blight control and zea mays growth promotion - Google Patents

Abstract

The invention relates to a strain of Pseudomonas aeruginosa which effectively controls corn sheath blight and promotes corn growth, and belongs to the technical field of microorganisms. The strain is Pseudomonas chloroaphis (Pseudomonas chloroaphis) PSJ1, which is isolated from wheat rhizosphere, and its preservation number is CGMCC NO.7932. The fungus not only promotes the growth of corn plants, but also inhibits the growth of corn sheath blight and reduces the occurrence of sheath blight.

Description

A Strain of Pseudomonas chlorous that Effectively Controls Maize Sheath Blight and Promotes Maize Growth

technical field

The invention belongs to the field of microorganisms, and relates to a kind of Pseudomonas chlororaphis and its application, in particular to a kind of Pseudomonas chlororaphis which can effectively inhibit Rhizoctonia solani (Rhizoctonia solani) and can promote the growth of corn .

Background technique

Corn (Zea mays) is the main grain, feed crop and important industrial raw material in my country. It occupies an important position in my country's national economy and agricultural production. Corn production is directly related to my country's food strategic security. In recent years, diseases such as corn sheath blight, small spot and Curvularia leaf spot have become more and more common, and the damage is increasing, which seriously restricts the development of corn production.

In the comprehensive control of agricultural pests and diseases, chemical control plays an important role, but the long-term and irrational use of chemical pesticides not only pollutes the environment, destroys the ecological balance, but also has adverse effects on the health of humans and animals. With the progress of society and the improvement of living standards, people have higher and higher requirements for environmental quality and green and pollution-free food.

There are a variety of microorganisms in the soil, including bacteria, fungi, actinomycetes, protozoa and algae. Some of these microorganisms have a good inhibitory effect on plant pathogenic bacteria and have important application value in plant disease control. Many strains in the genus Pseudomonas, especially the Pseudomonas fluorescens group, are widely distributed and numerous, have simple nutritional requirements, fast reproduction, and strong competitive colonization ability. They can produce a variety of antibiotics and active substances, and can induce certain Plants produce systemic and much attention.

The present invention isolates a strain of Pseudomonas choloeaphis from the wheat rhizosphere, and names it as PSJ1. The bacterial strain can effectively inhibit the growth of Rhizoctonia solani, reduce the occurrence of corn sheath blight, and can promote the growth of corn plants.

Contents of the invention

The object of the present invention is to provide a Pseudomonas chloroaphis PSJ1 strain antagonizing corn sheath blight, which has very good effects in biological control of corn sheath blight and in promoting crop yield increase.

The present invention is realized by adopting the following technical scheme: bacterial strain PSJ1 is isolated from a wheat field in Tai'an, Shandong. An antagonistic strain with good disease control effect was screened out through the indoor plate confrontation experiment and the control effect test in the greenhouse, and it was named PSJ1. The bacterium was identified as Pseudomonas choloeaphis by 16SrRNA sequence determination and phylogenetic analysis.

The bacterial strain involved in the invention has antagonistic effect on various corn pathogenic fungi. Indoor antagonism experiments showed that the strain had good inhibitory effect on Rhizoctonia solani, Bipolaris maydis, Curvularia lunata Boedijn and so on.

When sowing, water the PSJ1 bacterial suspension with a concentration of 10 ⁸ CFU/ml. After the corn grows to the 2-3 leaf stage, spray the above-mentioned bacterial suspension on the leaves of the plants, and measure the growth-promoting effect of PSJ1 on the corn after 14 days. Effect. Compared with the control, the treatment with PSJ1 can significantly promote the growth of maize plants, and the above-ground and below-ground fresh weights of plants increased by 54% and 43%, respectively.

Greenhouse control experiments showed that the corn plants treated with biocontrol bacterium PSJ1, compared with the control after inoculation with corn sheath blight pathogen, had inhibited the expansion of disease spots to a significant level (p<0.05), and the disease prevention effect was obvious.

deposit information

Preservation time: July 16, 2013

Name of depository unit: CGMCC, General Microorganism Center of China Committee for Microorganism Culture Collection

Deposit number: CGMCC NO.7932

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Classification name: Pseudomonas chloroaphis (pseudomonas chloroaphis)

Description of drawings

Figure 1 Antagonistic effect of Pseudomonas chloropinus PSJ1 on pathogenic fungi

A is sheath blight of maize, B is head blight of wheat, and C is small spot of maize. On the left is a piece of filter paper dipped in PSJ1 bacterial solution, and on the right is clear water.

Figure 2 Amplification product of strain PSJ116S rDNA gene

Fig. 3 Phylogenetic tree constructed according to 16S rDNA sequence by NJ method

Fig. 4 Plant disease state after 7 days after inoculation of sheath blight of corn leaf sheaths

A is Agricultural University 108, B is Zhengdan 958

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually carried out according to conventional conditions.

Example 1: Isolation and preliminary screening of rhizosphere growth-promoting bacteria

The method of Fang Zhongda (1979) was used to isolate the growth-promoting bacteria in the rhizosphere. Take 10 g of the collected soil sample, add it to a conical flask filled with 90 mL of sterile water, place the conical flask on a shaker, and vibrate at 180 r/min for 20 min to make a soil suspension. Let the prepared soil suspension stand for 5 minutes, take the supernatant, and use the 10-fold serial dilution method to dilute to 10 ^-2 , 10 ^-3 , 10 ^-4 and 10 ^-5 in sequence, and pipette 200 μL of each concentration on the NA plate On, spread evenly, repeat three times for each concentration. Then the coated NA plates were incubated in a constant temperature incubator at 28°C for 24h.

When bacterial colonies appeared on the NA plate, the spore suspension (10 ⁵ spores/mL) of Geotrichum candidum was evenly sprayed on the NA plate that had grown colonies. Spray distribution should be uniform, about 200μL per dish. The amount of spraying should not be too much, so as to prevent the formation of water flow and disperse the long colonies.

Select the colonies with obvious inhibition zone around them, pick up the bacteria with a transfer ring, streak on the NA plate, and put the plate in a 28°C incubator for 12h. After the growth of a single colony, the colony was picked and streaked again, and repeated three times to obtain pure bacteria. Use a toothpick to pick and screen the colonies and put them into a test tube containing 5 mL of liquid LB medium, and culture them with shaking at 28°C for 14-16h. Take 1mL of the bacterial solution, add it to a 2mL centrifuge tube, then add 15% glycerol to mix, and store in a -80°C refrigerator.

Embodiment 2: the mensuration of antibacterial spectrum of rhizosphere growth-promoting bacteria

Use the confrontation culture method (Sijam et al., 2005). In the center of a PDA plate with a diameter of 90mm, connect bacteria cakes with a diameter of 9mm of corn sheath blight Rhizoctonia solani, wheat head blight Gibberella zeae and corn small spot fungus Bipolaris maydis, then seal the plate with a sealing film, and place it at 28°C Cultured in the incubator for 2 days. Dip the sterilized filter paper with a diameter of 6mm into the prepared bacterial solution (10 ⁸ cfu/mL), move it to a place 15mm away from the edge of the pathogenic bacteria, and place it symmetrically. The control only receives the pathogenic bacteria cake, seals it, and finally puts it in 28°C cultured in an incubator. Each treatment was repeated 3 times. After 3 days, measure the colony diameter of the pathogenic bacteria at the place where the bacteria were inoculated and the colony diameter of the control pathogenic bacteria, and calculate the inhibition rate.

The results showed that PSJ1 had a very good inhibitory effect on the tested pathogenic bacteria (Figure 1). The inhibitory rates of PSJ1 to Rhizoctonia solani, Gibberella zeae and Bipolaris maydis were 71.1%, 82.4% and 85.5%, respectively.

Embodiment 3: the acquisition of bacterial strain 16S rRNA sequence

Using the genome of Pseudomonas choloaphis as a template, its 16S rDNA was amplified by PCR. The upstream and downstream primers used were 16S-F:5'-AGAGTTTGATYMTGGCTCAG-3', 16S-R:

5'-AGGGYTACCTTGTTACGACTT-3'. The PCR reaction program was 94°C for 3min; 30 cycles of 94°C for 30s, 54°C for 45s, and 72°C for 2min; extension at 72°C for 10min; storage at 4°C. After the reaction, the PCR products were placed on a 1.0% agarose gel for electrophoresis analysis. The PCR product was recovered with reference to the PCR Purification and Recovery Kit of AXYGEN, USA, and ligated with pMD18-T overnight in a water bath at 16°C. The ligation reaction system is: 10×T4DNA Ligase buffer 1μL, pMD18-T (50ng/μL) 0.3μL, target DNA 7.7μL, T4DNA Ligase (350U/μL) 1μL.

The ligation product was transformed into competent cells by heat shock method. The specific method is: put 100 μL of E. coli competent cells on ice to melt, add 10 μL of the ligation product, stir gently to mix, place the mixture on ice for 20 minutes, then heat shock in a water bath at 42°C for 90 seconds, and quickly place it on ice for 5 minutes , then add 800 μL LB liquid medium (without antibiotics), mix well, shake at 220r/min for 45min at 37°C. Centrifuge at 6000rpm for 5 minutes, take out the supernatant to the remaining 200 μL, resuspend the bacteria, spread the bacteria evenly on the antibiotic plate containing ampicillin, then place the plate forward for 30 minutes, and incubate it upside down at 37°C for 16-20 hours until single cells grow out. colony.

Plasmid DNA was extracted by alkaline lysis (Han Zhiyong et al., 2000). The extracted plasmid was placed on a 1.0% agarose gel for electrophoresis, and the empty pUC18 plasmid was used as a control. If the electrophoretic movement rate is the same as that of pUC19, it means that no foreign fragment has been inserted; if it is slower than that of pUC19, it means that the foreign fragment has been inserted into pMD18-T. Plasmids with slow swimming rates were selected for PCR identification. The PCR program used: 94°C for 3min; 30 cycles of 94°C for 30s, 54°C for 45s, 72°C for 2min; 72°C for 10min. After the reaction, the PCR products were placed on a 1.0% agarose gel for electrophoresis analysis. The identified strains containing recombinant plasmids were sent to Shanghai Boshang Company for sequencing in the form of bacterial liquid. 2 clones per bacterial sample. The sequencing results were compared with the 16S rDNA sequences of related genera and species in GenBank using BLAST software, and the phylogenetic tree was constructed using Mega software.

Electrophoresis detection showed that a fragment of about 1.3kb was amplified from the PSJ1 genome (Figure 2). After determination, the 16SrDNA of the PSJ1 strain contains 1287bp, and the specific sequence is shown in SEQ.NO.1.

In the phylogenetic tree constructed based on the 16SrDNA sequence, PSJ1 was clustered with Pseudomonas spp., indicating that PSJ1 belonged to Pseudomonas chlorospinosa (Figure 3).

Embodiment 4: Indoor growth-promoting experiment of PSJ1 bacterial strain to corn

Select Zhengdan 958 corn seeds of the same size for surface disinfection, soak the sterilized corn seeds in sterile water for 10 hours, and change the water every 2-3 hours. The pre-germinated seeds were subjected to a greenhouse pot experiment, and 4 seeds were sown in each pot (190mm×160mm) filled with sterilized soil, 6 pots for each treatment, and repeated three times.

Use sterile water to adjust the concentration of PSJ1 bacteria to 10 ⁸ CFU/ml, and water 40 ml per pot; when the corn grows to the 2-3 leaf stage, evenly spray PSJ1 bacterial suspension with a bacterial concentration of 10 ⁸ CFU/ml on the leaves, and control The groups were sprayed with the same amount of water. After 14 days of treatment, the plant height (from above the soil surface to the highest point of the plant), root number, root length, and fresh weight of aboveground and underground parts were measured. The results are shown in Table 1.

Table 1 Effect of PSJ1 on maize growth traits

It can be seen from Table 1 that the treatment with PSJ1 bacterial suspension can significantly promote the growth of maize plants and the growth of roots. At the same time, PSJ1 treatment promoted the increase of plant fresh weight, compared with the control, the aboveground and belowground fresh weight increased by 54% and 43%, respectively.

Example 5: Control effect of PSJ1 strain on corn sheath blight

Select corn seeds of the same size for surface disinfection, soak the fully sterilized corn seeds in sterile water for 10 hours, and change the water every 2-3 hours. The pre-germinated seeds were subjected to a greenhouse pot experiment, and 4 seeds were sown in each pot (190mm×160mm) filled with sterilized soil, 6 pots for each treatment, and repeated three times. Use sterile water to adjust the concentration of PSJ1 bacteria to 10 ⁸ CFU/ml, and water each pot with 40ml. When the corn grows to the stage of two leaves and one heart, the roots are irrigated with bacterial suspension, 40ml per tray (10 ⁸ CFU/ml) once every two days, for three consecutive times, and the water treatment is used as the control. One week after the third root irrigation, the rhizosphere was inoculated with strong pathogenic corn sheath blight (YWK-100), and the rhizosphere was properly moisturized. The incidence of corn disease and the expansion of disease spots were observed and counted. The results are shown in Figure 4 and Table 2.

Table 2 Lesion extension length and plant disease incidence of plants inoculated with YWK-100 in the rhizosphere

It can be seen from the table that the extended length of lesion on the treated plants is significantly lower than that of the control, and PSJ1 treatment of Zhengdan 958 and Nongda 108 can significantly improve plant disease resistance.

The above are only individual embodiments of the present invention, and are not intended to limit the present invention in any form. All simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still belong to the present invention. within the scope of the technical program.

Claims (2)

1. a strain Pseudomonas chlororaphis, is characterized in that called after Pseudomonas chlororaphis PSJ1, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, and depositary institution is called for short: CGMCC; Preserving number: CGMCC NO.7932; Its 16SrRNA nucleotide sequence is as shown in SEQ.NO.1.

2. the application of strain Pseudomonas chlororaphis PSJ1 in control of maize banded sclerotial blight as claimed in claim 1, wherein pathogenic bacteria is Rhizoctonia solani.