Application of serratia marcescens WHArb-01 in prevention and control of taxodium bistriatum and euonymus japonicus

Abstract

The invention discloses an application of serratia marcescens WHArb-01 in prevention and treatment of taxodium bistriatum and euonymus japonicus. According to the method disclosed by the invention, the stock solution or diluent of fermentation liquor of serratia marcescens WHArb-01 is used for treating the Changlin bark beetles and the hedyotis bifida, the corrected death rate of the Changlin bark beetles treated by the stock solution of the fermentation liquor and the 25-fold diluent reaches 97.7%, and the corrected death rate of the hedyotis bifida treated by the stock solution reaches 86.9%. The biological control method is free of pesticide residues and toxic and side effects, and the use of chemical pesticides can be reduced.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to use of Serratia marcescens WHArb-01 for controlling Cynomolgus bifasciatus and Cynomolgus sylvaticus. Background Art

[0002] Semanotus bifasciatus, a member of the Cerambycidae family of the order Coleoptera, is a destructive borer of cypress trees. In 2013, the State Forestry Administration, based on a comprehensive risk analysis of forest pests, included Semanotus bifasciatus in the national list of dangerous forest pests, and it is one of the 35 quarantine objects identified by the state. The larvae mainly harm weak trees, dead standing trees, and newly felled trees, eating through the phloem and xylem, cutting off or destroying the transporting tissues and affecting the transport of water and nutrients. In mild cases, it affects growth potential, causing the needles to turn green and yellow, and in severe cases, it causes the death of the entire branch or the entire tree, posing a serious threat to forestry resources.

[0003] Hylurgus ligniperda, also known as the red-haired beetle, mainly harms Pinus plants. The spread of Hylurgus ligniperda is mainly through pine wood trade activities. It is one of the most intercepted insects in imported pine logs in countries around the world. Many international or regional organizations list it as an important quarantine pest. It has now spread to the whole of Europe and parts of Africa, Asia, Oceania, and the Americas. As a secondary pest, Hylurgus ligniperda mainly harms dying wood, weak wood, newly felled logs, and felled piles. However, when the population density of Hylurgus ligniperda is high, it may also harm living trees or seedlings.

[0004] Serratia is an important class of biocontrol bacteria with great potential in pest control. It is now widely used in the control of Lepidoptera, Orthoptera, Homoptera and Coleoptera. Studies have found that Serratia marcescens is also highly toxic to pests such as cotton bollworm, cutworm, armyworm, cabbage worm, potato tuber moth, etc. Bumblebees can use their ovipositors to transfer Serratia marcescens from infected bollworms to healthy bollworms, thereby infecting them.

[0005] At present, there are no reports on the use of Serratia marcescens to control the two-striped cedar beetle or the long forest beetle. CN2024112564070 used tissue separation method to isolate the Serratia marcescens WHArb-01 strain from the adult brown-stemmed longhorn beetle. The strain was deposited in the China Center for Type Culture Collection on May 23, 2022, with the deposit number CCTCCNO: M2022697. The strain can secrete pigments, grow and reproduce inside living trees, and coexist with trees. It can be used to dye living trees. On this basis, the applicant continues to develop new functions of Serratia marcescens WHArb-01 in the hope of obtaining broader application prospects. Summary of the invention

[0006] In view of the above problems, the present invention provides the use of Serratia marcescens WHArb-01 for controlling Serratia bifasciata and Leptosphaeria longifolia. The fermentation liquid of Serratia marcescens WHArb-01 can be used as a biological pesticide to control Serratia bifasciata and Leptosphaeria longifolia, and has good application prospects.

[0007] The invention firstly provides the use of Serratia marcescens WHArb-01 or its fermentation liquid for simultaneously preventing and controlling Cynomolgus sibiricus and Cynomolgus sibiricus.

[0008] The invention also provides a method for preventing and controlling the longwood borer by using the fermentation liquid of Serratia marcescens WHArb-01, which is characterized in that the longwood borer is treated by spraying the original liquid or the diluted liquid of the fermentation liquid of Serratia marcescens WHArb-01.

[0009] The present invention also provides a method for preventing and controlling the two-striped cedar beetle by using the fermentation liquid of Serratia marcescens WHArb-01, which is characterized in that the original liquid or the diluted liquid of the fermentation liquid of Serratia marcescens WHArb-01 is used to treat the two-striped cedar beetle.

[0010] The technical effect of the present invention is that the corrected mortality rate of the fermentation liquid stock solution and the 25-fold dilution solution of the longwood borer treated by the spray method reaches 97.7%; the corrected mortality rate of the fermentation liquid stock solution treated with the two-striped cedar longhorn beetle reaches 86.9%. Since the fermentation liquid of Serratia marcescens has no drug residues and no toxic side effects, it can reduce the use of chemical pesticides, and thus has important application prospects in biological control of two-striped cedar longhorn beetle and longwood borer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The symptoms of the two-banded longhorn beetle after being infected with the Serratia marcescens WHArb-01 strain (stock solution); Figures A, B, C, D, E, and F are the symptoms at 24h, 48h, 60h, 72h, 84h, and 96h of infection, respectively;

[0012] Figure 2These are the symptoms of the long forest beetle infected with the Serratia marcescens WHArb-01 strain (original solution) (72 hours after taking the medicine). DETAILED DESCRIPTION

[0013] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0014] Example 1: Verification of the insecticidal effect of Serratia marcescens WHArb-01 fermentation liquid on Anoplophora bifasciatus

[0015] This example provides relevant experiments on the fermentation broth of Serratia marcescens WHArb-01 against Alpinia bifasciata.

[0016] 1.1 Test reagents

[0017] The preparation method of the Serratia marcescens WHArb-01 fermentation broth prepared by the present invention is as follows:

[0018] Seed liquid culture medium: 3g beef extract, 10g peptone, 5g sodium chloride, 1000mL distilled water, pH 7.0-7.4.

[0019] The fermentation medium is: sucrose 10g / L, yeast powder 20g / L, calcium chloride 5g / L.

[0020] Preparation of seed solution: The activated Serratia WHArb-01 strain was inoculated into the seed solution culture medium, and cultured at 28°C and 180 r / min for 12 hours to prepare the seed solution.

[0021] The fermentation conditions are: inoculate the seed liquid into the fermentation medium at a volume ratio of 5%, the fermentation temperature is 28°C, the fermentation time is 48h, and the rotation speed is 240r / min. The fermentation liquid (WHArb-01 bacterial liquid stock solution) obtained by fermentation culture by this method has a viable bacterial count of ≥5 billion / mL.

[0022] 1.2 Test insects

[0023] The test insects were larvae of Diplodocus bifasciatus, which were collected from the Jinan Forest Farm.

[0024] 1.3 Test methods

[0025] The test site was located at Shandong Academy of Forestry Sciences in Jinan City, Shandong Province, and the insecticidal test was conducted on April 29, 2023.

[0026] The experiment designed four concentration gradients of Serratia marcescens WHArb-01 bacterial solution, 25-fold, 50-fold, and 100-fold, with sterile water as the control, for a total of 5 treatments (repeated 3 times), each treatment with 10 larvae of two-striped spruce beetle, a total of 150.

[0027] (1) Prepare several insect boxes, spread sterilized Platycladus orientalis sawdust and wood chips as feed in the boxes, pound them with a small wooden stick, poke a small hole, and then put the larvae of the two-striped cedar beetle in the boxes. Place the insect boxes in an artificial incubator at a temperature of 25°C and a humidity of 40% for breeding and set aside.

[0028] (2) Weigh 300 g of sterilized feed and mix it with different concentrations of Serratia marcescens WHArb-01 bacterial solution (original solution, 25 times, 50 times, 100 times, with sterile water as the control) at a bacterial solution: feed mass ratio of 1:20, and divide it into 15 portions in insect boxes. Each insect box contains 10 healthy larvae of two-striped spruce beetle and is raised at 25°C and 40% humidity.

[0029] 1.4 Experimental investigation and calculation method

[0030] 1.4.1 Drug efficacy investigation, investigation time and frequency

[0031] During the whole experiment, the pesticide was applied once, and observation was made every 24 hours for 7 consecutive days, and the number of dead insects was counted.

[0032] 1.4.2 Calculation method

[0033] The mortality rate and adjusted mortality rate were calculated according to the following formula:

[0034]

[0035]

[0036] 2. Results

[0037] 2.1 Symptoms of infection with Serratia marcescens WHArb-01 in P. bifasciatus

[0038] After the larvae of the two-striped longhorn beetle are infected with the WHArb-01 strain, the symptoms are not obvious within 24 hours. After 48 hours, the insect body turns light pink locally, and then spreads to the entire insect body, and the color deepens from light pink to red. The insect body becomes soft and rots (see Figure 1 ).

[0039] 2.2 Indoor control effect of Serratia marcescens WHArb-01 on P. bifasciata

[0040] As shown in Table 1, after the fermentation liquid of Serratia marcescens WHArb-01 was used to treat the longhorn beetle with different concentrations, the mortality rates of the original liquid and 25-fold liquid treatments on the 7th day reached 86.9% and 56.4%, respectively. The corrected mortality rates of the treatments with different concentrations were significantly different at the P<0.05 level. With the increase of the dilution multiple, the corrected mortality rate decreased. Under the same concentration treatment, with the increase of treatment time, the corrected mortality rate increased.

[0041] Table 1 The lethal effect of Serratia marcescens WHArb-01 strain on Anoplophora bifasciatus

[0042]

[0043] 3. Summary

[0044] Judging from the corrected mortality of P. bifasciata after treatment with different concentrations, the Serratia marcescens WHArb-01 strain has a good application prospect against P. bifasciata.

[0045] Example 2: Verification of the insecticidal effect of Serratia marcescens WHArb-01 fermentation liquid on the long forest beetle

[0046] This example provides relevant experiments on the fermentation broth of Serratia marcescens WHArb-01 against Microhylidae longissima.

[0047] 1.1 Test reagents

[0048] The preparation method of the Serratia marcescens WHArb-01 fermentation broth prepared by the present invention is shown in Example 1.

[0049] 1.2 Test insects

[0050] The test insects were adults of Microhylidae longissima, which were collected from Muping District, Yantai City.

[0051] 1.3 Test methods

[0052] The test site was located at Shandong Academy of Forestry Sciences in Jinan City, Shandong Province, and the insecticidal test was conducted on March 31, 2023.

[0053] The experiment designed four concentration gradients of Serratia marcescens WHArb-01 solution, 25 times, 50 times, and 100 times, with sterile water as the control, for a total of five treatments. An appropriate amount of pine wood chips was placed in a sterile culture dish, and 20 long wood beetles were placed in each culture dish. They were sprayed with different concentrations of WHArb-01 solution and placed in an artificial incubator with a temperature of 25℃ and a humidity of 40% for breeding and observation.

[0054] 1.4 Experimental investigation and calculation method

[0055] 1.4.1 Drug efficacy investigation, investigation time and frequency

[0056] During the whole experiment, the pesticide was applied once, and observation was made every 24 hours for 5 consecutive days, and the number of dead insects was counted.

[0057] 1.4.2 Calculation method

[0058] The mortality rate and adjusted mortality rate were calculated according to the following formula:

[0059]

[0060] 2. Results

[0061] 2.1 Symptoms of the longwood beetle infected with Serratia marcescens WHArb-01

[0062] After infection with the WHArb-01 strain of Serratia marcescens, the longwood beetle showed slow movement, twitching, some of the elytra opened outward, and the body became rigid and dead (see Figure 2 ).

[0063] 2.2 Indoor control effect of Serratia marcescens WHArb-01 on longwood beetles

[0064] After treatment with different concentrations of Serratia marcescens WHArb-01, some longwood beetles died after 24 hours. Under the same concentration treatment, the mortality rate increased with the increase of treatment time. After 120 hours of treatment with the original solution and 25 times solution, the corrected mortality rate of longwood beetles reached 97.7%; after 120 hours of treatment with 100 times solution, the corrected mortality rate still reached more than 50%. After 120 hours, there was no significant difference in the corrected mortality rate between the original solution and the 25 times treatment at the P<0.01 level, but there was a significant difference compared with other concentration treatments.

[0065] Table 2 The lethal effect of Serratia marcescens WHArb-01 strain on the long forest beetle

[0066]

[0067] 3. Summary

[0068] From the insecticidal effect point of view, the WHArb-01 strain of Serratia marcescens has a good insecticidal effect on the long forest borer and has good application prospects.

Claims

1. Use of Serratia marcescens WHArb-01 or its fermentation liquid for simultaneously controlling Cynomolgus sibiricus and Cynomolgus sibiricus.

2. A method for controlling the longwood borer using the fermentation liquid of Serratia marcescens WHArb-01, characterized in that: The original solution or diluted solution of the fermentation liquid of Serratia marcescens WHArb-01 was sprayed to treat the longwood borer.

3. A method for controlling Semen Longipes bifasciatus using fermentation liquid of Serratia marcescens WHArb-01, characterized in that: The original solution or diluted solution of the fermentation liquid of Serratia marcescens WHArb-01 was used to treat the longhorn beetle.

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