Pseudomonas koreensis and its application in trapping and controlling southern root-knot nematode

Abstract

The present application relates to the field of the prevention of southern root-knot nematode, and discloses a Pseudomonas kribbensis ZJU1, with a preservation number of CCTCC NO: M 20221258.The present application also discloses the use of the Pseudomonas kribbensis ZJU1 in trapping and killing southern root-knot nematode.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of prevention and treatment of southern root-knot nematode, and particularly relates to application of Pseudomonas kribbensis ZJU1 in trapping and killing southern root-knot nematode. BACKGROUND

[0002] Plant parasitic nematodes are a kind of pathogenic organisms with serious harm, which parasitize in the plant body. They have the characteristics of wide parasitism, strong adaptability, easy transmission and so on, and are extremely difficult to control, and are usually called crop cancer. In China, southern root-knot nematode (Meloidogyne incognita) is the most important plant parasitic nematode and one of the most harmful parasitic nematodes.

[0003] The life cycle of southern root-knot nematode includes three stages: egg, juvenile, and adult. The juvenile stage is divided into four periods, which are first juvenile (J1), second juvenile (J2), third juvenile (J3) and fourth juvenile (J4). J1 is curled in the egg mass after oviposition, J2 enters the invasion stage, changes from linear to pod-shaped after entering the host plant, J3 is shaped like an eggplant and gradually begins to differentiate into male and female, and J4 completes female differentiation. Root-knot nematodes overwinter in the form of eggs or adults in the soil and plant residues. In the spring of the following year, when the soil temperature is above 10℃, the egg mass begins to hatch J2 which can invade the roots of plants. The range of activity of the juvenile in the soil is 1-15 cm. The second juvenile (J2) generally invades from the elongation zone of the plant root tip. At the beginning of the invasion, the juvenile first secretes pectinase, cellulase and expansin and other plant cell wall loosening substances from the salivary gland, and releases them into the middle lamella through the stylet. After the cell is loosened, the second juvenile enters the vicinity of the plant vascular tissue, injects toxic substances into the plant cells, and thus induces the host plant cells to produce various complex physiological pathologies, such as the formation of giant cells. Adult males reproduce parthenogenetically and parasitize on the surface of the plant in the form of egg mass. Generally, male worms do not participate in the reproduction process and leave the plant roots to enter the soil.

[0004] For a long time, the use of chemical nematicides is still the most commonly used control method. Although chemical control can reduce the invasion and occurrence of southern root-knot nematode, it has great side effects on the environment and soil structure, and the residues of chemical pesticides seriously affect the quality of agricultural products and human health. Therefore, it is urgent to develop green control technology for root-knot nematode to ensure the green and sustainable development of agriculture in China. At present, the development and utilization of bacteria and fungi that can inhibit the growth and invasion of nematodes have gradually become a green means of root-knot nematode control. Bacteria have better application value and broader application prospect in the non-polluting control of southern root-knot nematode due to their fast growth, short cycle and easy cultivation.

[0005] Pseudomonas kribbensis was first isolated from coastal soil, and there are few researches on it, and the effect of the bacterium on southern root-knot nematode is still unclear.

[0006] The application of CN109265461A "Pseudomonas kribbensis metabolite and its application in biocontrol" discloses that Pseudomonas kribbensis Paenibacillus kribbensis, strain TRCC82001, preservation number CGMCC No.7996, the bacterium metabolite has strong bacteriostatic effect on plant pathogenic fungi, and has significant prevention and treatment effect on rice sheath blight, corn small spot and corn large spot, and provides a good basis for the development and application of biocontrol agents.

[0007] As common sense in the industry, we know that rice sheath blight, corn small spot and corn large spot are plant diseases caused by pathogenic fungi, and mainly harm plant leaves, while southern root-knot nematode is a soil-borne disease, and is a plant parasitic animal mainly harming plant roots. Therefore, the two have no correlation. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a Pseudomonas kribbensis ZJU1 strain and its application in trapping and preventing southern root-knot nematode.

[0009] To solve the above technical problem, the present application provides a Pseudomonas kribbensis ZJU1, which has a preservation number of CCTCC NO:M 20221258.

[0010] The present application also provides the use of the above-mentioned Pseudomonas kribbensis ZJU1 for trapping southern root-knot nematode.

[0011] The present application also provides another use of the above-mentioned Pseudomonas kribbensis ZJU1 for killing southern root-knot nematode.

[0012] The strain provided by the present application is Pseudomonas kribbensis ZJU1 strain, which is isolated and purified from the rhizosphere soil of corn in the agricultural test station of Zhejiang University Zijinggang Campus in Hangzhou, Zhejiang Province. It has the following biological characteristics: after being cultured on LB medium plate at 30 DEG C for 2 days, the single colony of the strain is milky white, the surface is smooth and the edge is regular, the bacterial body is rod-shaped and arranged in a bead-like manner, and the gram staining is negative. The preservation information is as follows:

[0013] Deposit name: Pseudomonas kribbensis ZJU1, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M 20221258, deposit time: August 09, 2022.

[0014] The Pseudomonas kribbensis ZJU1 provided by the present application is a bacterium isolated from the rhizosphere soil of corn with root-knot nematode disease. The strain has good trapping and killing effects on southern root-knot nematodes. Experiments show that the strain can significantly attract southern root-knot nematodes, and the fermentation broth has a mortality rate of 61.9% on the second instar larvae of root-knot nematodes. It can be used as a root-knot nematode trapping agent and killing agent, and has high and stable effect, and has potential and practical application value in the research of southern root-knot nematodes.

[0015] The strain is a bacterium isolated from the rhizosphere soil of corn with southern root-knot nematode disease, and has good trapping and killing effects on root-knot nematodes. In the future, it can be applied to the green prevention and control of southern root-knot nematodes, and realize the safe and pollution-free production of crops. BRIEF DESCRIPTION OF DRAWINGS

[0016] The specific embodiments of the present application will be further described in combination with the drawings.

[0017] Figure 1 It is a schematic diagram of a petri dish test device.

[0018] Figure 2 It is a schematic diagram of a potting test device.

[0019] Figure 3 It is the influence of Pseudomonas kribbensis on the chemotactic behavior of southern root-knot nematodes in a petri dish.

[0020] Figure 4 It is the influence of Pseudomonas kribbensis on the chemotactic behavior of southern root-knot nematodes in a pot.

[0021] Figure 5 It is the influence of different components of Pseudomonas kribbensis on the mortality rate of southern root-knot nematodes.

[0022] Figure 6 It is the 16S rDNA gene sequence of the strain. DETAILED DESCRIPTION

[0023] The present application will be further described in combination with specific examples, but the protection scope of the present application is not limited to this:

[0024] Example 1: Isolation and identification of Pseudomonas kribbensis ZJU1

[0025] 1. Isolation of Pseudomonas kribbensis ZJU1

[0026] Pseudomonas kribbensis ZJU1 was isolated from the rhizosphere soil of corn planted in the Zijingang Campus of Zhejiang University in Hangzhou, Zhejiang Province, which had been infected with root-knot nematode disease, using soil dilution method. The isolation method is as follows:

[0027] The rhizosphere soil sample (rhizosphere soil of corn infected with root-knot nematode disease) was collected from the rhizosphere of corn planted in the Zijingang Campus of Zhejiang University in Hangzhou, Zhejiang Province. The roots were gently shaken to remove loose soil, and the remaining attached soil on the roots was collected as rhizosphere soil. 1 g of soil was weighed on a balance and placed in a 50 mL conical flask containing 10 mL of sterile water in a clean bench. The flask was placed on a shaker at 28°C and 180 r / min for 2 h to mix thoroughly. At this time, the supernatant was a soil suspension. 100 μL of the suspension was taken with a pipette and added to a centrifuge tube containing 900 μL of sterile water, and mixed thoroughly. At this time, the concentration of the obtained suspension was 10 -1 . According to this method, the soil suspension was diluted in gradient of 10 -1 -10 -10 . 100 μL of the soil suspension with gradient of 10 -5 -10 -8 was taken with a pipette and added to LB solid medium. The uniformly coated plate was cultured in an incubator at 30°C for 1-2 days. During the culture period, the growth of colonies on the plate was observed every day, and colonies with obvious differences in morphology and good growth were selected for sequencing and identification.

[0028] Thus, Pseudomonas kribbensis ZJU1 was obtained.

[0029] 2. Identification of Pseudomonas kribbensis ZJU1

[0030] (1) Microbiological characteristics:

[0031] After 2 days of culture at 30°C on LB medium plates, the single colonies of the strain were milky white, smooth in surface and regular in edge. The bacterial bodies were rod-shaped and arranged in a beaded manner, and were negative in Gram staining.

[0032] (2) Molecular biological characteristics:

[0033] The single colony was picked up with a sterilized inoculation needle in a clean bench, placed in a PCR tube containing 20 μL sterile water, lysed at 95°C for 10 min, and then the bacterial suspension was diluted to 100 μL. The universal primers 27F and 1492R were used to amplify the 16S rRNA sequence of the bacteria. The PCR amplification reaction system was 20.8 μL, containing 0.2 μL High Fidelity enzyme, 1 μL forward primer, 1 μL reverse primer, 1 μL DNA template, 10 μL GC, 1.6 μL dNTP, and 6 μL sterile water. The amplification conditions were as follows: 98°C pre-denaturation for 1 min, 98°C denaturation for 10 s, 60°C annealing for 5 s, 72°C extension for 2 min, 35 cycles; 72°C extension for 2 min. The amplification product was separated and identified by 1% agarose gel electrophoresis, and the PCR product was directly sequenced.

[0034] The 16S rRNA sequence of the strain was amplified by primers 27F and 1492R, and the PCR product was electrophoresed on 1% agarose gel. Sequencing analysis showed that the amplified 16S rRNA sequence of the strain was 1390 bp long (NO1 of the sequence listing). BLAST alignment analysis of the 16S rRNA sequence of the strain showed that the similarity of the strain to Pseudomonas kribbensis reached 100%. The strain was identified as Pseudomonas kribbensis.

[0035] It was preserved, and the preservation information is as follows:

[0036] Preservation name: Pseudomonas kribbensis ZJU1, preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M 20221258, preservation time: August 9, 2022.

[0037] Example 2: Trapping and killing effect of Pseudomonas kribbensis on southern root-knot nematode

[0038] 1. Preparation of Pseudomonas kribbensis ZJU1 strain fermentation broth

[0039] The Pseudomonas kribbensis ZJU1 strain was inoculated into LB liquid medium and cultured at 30°C at a speed of 180 rpm until the OD 600=1, incubation time approximately 24 hours; to obtain a bacterial suspension of *Pseudomonas kurstii* ZJU1. This suspension was further centrifuged (5000 rpm for 10 minutes). The supernatant was referred to as "fermentation broth," and the precipitate as "cells." Before centrifugation, the entire suspension was referred to as "bacterial suspension." Sterile water was used as a control for cell treatment, and LB liquid medium was used as a control for both the fermentation broth and the bacterial suspension. LB liquid medium consisted of: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and distilled water to a final volume of 1 L, sterilized at 121°C for 15 minutes.

[0040] 2. Preparation of experimental nematodes

[0041] Southern root-knot nematodes were collected from the greenhouse in the West Campus of Zhejiang University. The roots of maize plants infected with root-knot nematodes were removed, gently rinsed with water, and disinfected in 1% sodium hypochlorite for 1 minute. They were then rinsed three times with sterile water and placed in petri dishes containing a small amount of sterile water. The petri dishes were incubated at 25°C. After 3 days, the hatched second-instar larvae of the root-knot nematodes were collected and suspended in sterile water for experimental research. Approximately 1000 second-instar larvae were obtained per ml of sterile water.

[0042] 3. Test Methods

[0043] 3.1) Petri dish test: such as Figure 1 As shown, prepare 1% sterile agarose petri dishes, and mark them 2cm from the edge of the 9cm plate. Cut off 0.5cm from each end of the yellow pipette tip and use it to fill the Pseudomonas kuribda suspension (OD). 600 =1) and sterile water, place the pipette tip at two marked points on the petri dish, add about 80 southern root-knot nematodes between the two marked points, and place in a 25°C dark constant temperature incubator. After 3 hours, count the number of southern root-knot nematodes in the same area (about 0.5 cm) around the pipette tip on both sides to determine the effect of Pseudomonas kirchii on the chemotactic behavior of southern root-knot nematodes in the petri dish. Each treatment has 10 replicates.

[0044] 3.2) Pot experiment: such as Figure 2 As shown, prepare two identical black plastic flowerpots and fill them with sterilized sand (400g of sterilized sand in each pot). Connect the two pots with a black straw. Add 2mL of OD to the left pot. 600 =1 Pseudomonas kirchiii suspension, with an equal volume of sterile water added to the right side, followed by inoculation of 1500 second-instar southern root-knot nematodes suspension in the middle of the pipette. After 12 hours, sand was collected from both sides of the pot, and the southern root-knot nematodes were collected through a Behmann funnel and counted under a microscope to determine the effect of Pseudomonas kirchiii on the chemotactic behavior of southern root-knot nematodes in potted plants. Each treatment was replicated 10 times.

[0045] 3.3) Strain mortality: Prepare different sterile 96-well plates, respectively take 20 μL of different components of P. chrysogenum (i.e. fermentation broth, bacterial body, bacterial suspension) into the wells, add 30 second instar J. ssp. at the same time, take sterile water and LB liquid medium as control, put into 25℃ dark constant temperature incubator, observe the death of nematodes after 48 hours, and calculate the mortality, 8 replicates for each treatment. Mortality (%) = number of dead nematodes / number of added nematodes * 100%.

[0046] 4. Test results

[0047] The attraction of P. chrysogenum to J. ssp. (the number of nematodes that made a chemotactic choice) was determined in Petri dishes and pots, and the mortality of J. ssp. to different components of P. chrysogenum was determined in 96-well plates.

[0048] Figures 3-4 It can be shown that P. chrysogenum has good attraction to J. ssp.

[0049] Note: In the Petri dish test, part of the J. ssp. did not show chemotactic behavior, i.e. still in the center of the Petri dish, because the data corresponding to this part of the J. ssp. was not reflected in Figure 3 .

[0050] Similarly, in the pot test, part of the J. ssp. did not show chemotactic behavior, i.e. still in the black straw, because the data corresponding to this part of the J. ssp. was not reflected in Figure 4 .

[0051] In the Petri dish test, the number of nematodes near the tips of the two guns was significantly different, and the J. ssp. tended to be more on the side of the gun tip with P. chrysogenum added, with an increase of 66% compared to the control side. In the pot test, the number of nematodes that moved to the sand on both sides was also significantly different, and the J. ssp. tended to be more on the side of the pot with P. chrysogenum added, with an increase of 97% compared to the control side. This shows that the P. chrysogenum has good attraction to J. ssp.

[0052] Figure 5 It can be shown that P. chrysogenum significantly increased the mortality of J. ssp. after 48 hours of treatment.

[0053] In the 96-well plate, the mortality of nematodes treated with sterile water was 7.9%, while the mortality of nematodes treated with P. chrysogenum bacterial body was 21.7%, with a significant difference. Similarly, the mortality of nematodes treated with LB medium was 8.9%, and the mortality of nematodes treated with fermentation broth and bacterial suspension was 61.9% and 44.6% respectively, which was significantly different from the control of LB medium. This shows that the P. chrysogenum has good killing effect on J. ssp.

[0054] In a comparative experiment, DSM 100278 (Pseudomonas kribbensis) was tested according to the above-described "Petri dish test", and no significant difference in the number of nematodes near the two lateral gun heads was found.

[0055] Although the present application has been described in detail with general description, specific embodiments and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present application. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application.

Claims

1. Use of Pseudomonas kribbensis ZJU1, characterized in that: For trapping and killing southern root-knot nematodes; The preservation number of the Pseudomonas kribbensis ZJU1 is CCTCC NO: M20221258.

2. Pseudomonas kribbensis ZJU1, characterized in that: The preservation number is CCTCC NO: M 20221258. The preservation number is CCTCC NO: M 20221258.

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