Beauveria bassiana strain sl-066 and use thereof

Abstract

The application discloses a Beauveria bassiana strain SL-066 and application thereof, and relates to the technical field of biological strains. The strain provided by the application is preserved in the China General Microbiological Culture Collection Center on August 8, 2022, and the preservation number is CGMCC No. 40269. The Beauveria bassiana strain SL-066 provided by the application can effectively prevent and control various rice pests, is a Beauveria bassiana strain capable of preventing and controlling the coleopteran pest Curculionidae (rice water weevil adult), the coleopteran pest Chrysomelidae (rice water weevil larva) and the lepidopteran pest Chilo suppressalis (Chilo suppressalis larva), and has a good prevention and control effect on the three common pests on rice.

Description

Technical Field

[0001] This invention belongs to the field of biological strain technology, specifically relating to a Beauveria bassiana strain SL-066 and its applications. Background Technology

[0002] The rice water weevil (Lissorhoptrus oryzophilus Kuschel) belongs to the order Coleoptera, family Weevilsidae, subfamily Marsh Weevils, and genus Lissorhoptrus. It primarily damages rice, but also hosts wheat, barley, pasture grasses, and gramineous weeds. It is characterized by a long period of damage, rapid reproduction and spread, and strong resistance to pesticides and adverse conditions. Adults feed on rice leaves, while larvae bore into the roots, generally causing a 15%–20% reduction in rice yield, with severe cases resulting in yield losses exceeding 50% or even total crop failure. It is a significant quarantine pest of rice and is listed by the International Union for Conservation of Nature (IUCN) as one of the 100 most threatening invasive alien species globally. The rice water weevil has become a major potential threat to rice production in my country, and the country has listed it as one of the five invasive alien species that have been prioritized for control in recent years.

[0003] The rice stem borer (Chilo suppressalis Walker), belonging to the order Lepidoptera and family Pyralidae, is commonly known as the rice stem borer, heart borer, and stalk borer. It is one of the most serious and prevalent pests of rice in my country. Besides rice, the rice stem borer also damages water chestnuts, corn, sorghum, sugarcane, rapeseed, broad beans, wheat, and weeds such as reeds, barnyard grass, and Leymus chinensis. The larvae bore into the leaf sheaths, whorls, and stems of rice plants, causing damage such as withered sheaths, dead hearts, and whiteheads. At maturity, they cause semi-withered panicles, leading to severe yield reduction. The rice stem borer typically causes a 5%-10% reduction in rice yield, with severe cases resulting in 20%-30% reduction.

[0004] The rice leaf beetle (Oulema oryzae Kuwayama), commonly known as the rice leaf beetle or rice dung beetle, belongs to the order Coleoptera and the family Chrysomelidae. It is distributed throughout rice-growing areas of my country, primarily in the Northeast, and is one of the major pests in cold-climate rice-growing regions. Both adults and larvae feed on the leaf mesophyll, causing white streaks and longitudinal tearing of rice leaves, thus affecting yield. It typically causes a 15%–40% reduction in rice yield annually, severely impacting rice yield and quality, and has become one of the key pests to control during the rice seedling stage.

[0005] Currently, the control of the three major rice pests in rice production mainly relies on chemical pesticides. However, the long-term and extensive use of chemical pesticides has led to pesticide resistance in many commonly used insecticides, causing frequent outbreaks and consequently increasing pesticide usage. Furthermore, the application of chemical pesticides pollutes the environment, hindering the sustainable development of agricultural production. Therefore, finding an environmentally friendly, pollution-free, highly efficient biological control method that can simultaneously control all three rice pests is of great significance.

[0006] Beauveria bassiana is a filamentous fungus belonging to the subphylum Deuteromycetes, class Hyphomycetes, order Moniliales, family Moniliaceae, and genus Beauveria. It is a broad-spectrum insect pathogenic fungus, primarily reproducing asexually by producing conidia, but can also reproduce sexually by producing fruiting bodies. Beauveria bassiana possesses advantages such as high pathogenicity, the ability to form epidemics among hosts, non-polluting environment, and ease of large-scale production, making it widely used in agricultural pest control as a highly effective microbial insecticide. However, Beauveria bassiana exhibits some host specificity; studies have shown significant differences in pathogenicity against target pests among strains from different regions and host sources, with varying lethal times (LTs) among different strains. 50 The difference can be several times to tens of times.

[0007] Currently, when using Beauveria bassiana for field pest control, specialized strains are often screened for different target pests. Since multiple pests can infest a single crop, this presents some inconvenience in the application of Beauveria bassiana. This is especially true for integrated pest management of a single crop, which not only affects its field control efficacy but also hinders the further promotion and application of Beauveria bassiana as a microbial pesticide. In actual production, Beauveria bassiana strains effective against Coleoptera pests (such as the rice water weevil) are extremely rare. Screening for a Beauveria bassiana strain that controls Coleoptera pests (weevils, adult rice water weevils), Coleoptera pests (leaf beetles, larvae of rice leaf beetles), and Lepidoptera pests (larvae of the rice stem borer) while also demonstrating good control efficacy against all three pests of the same crop is even more challenging! Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a Beauveria bassiana strain SL-066. This strain not only has a high mortality rate against three common rice pests (rice water weevil, rice stem borer, and rice leaf beetle), but also the insecticidal fungal preparations produced by this strain have good control effects on these three rice pests in the field.

[0009] The *Beauveria bassiana* strain SL-066 of this invention has the accession number CGMCC No. 40269. This *Beauveria bassiana* strain SL-066 was collected and isolated from rice paddies in Gongzhuling City, Jilin Province. This *Beauveria bassiana* strain SL-066 was deposited on August 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40269, and classified as *Beauveria bassiana*.

[0010] The strain SL-066 described in this invention was inoculated onto a PDA plate and cultured to form colonies. Initially, the colonies were white and fluffy to cottony, turning pale yellow after conidia. The hyphae were transparent, septate, and branched, with a diameter of 3.1-3.5 μm. The conidiophores were densely clustered, whorled, or solitary, with broom-like branches, measuring 2.5-4.5 × 1.5-2.5 μm, and produced flask-shaped sterigmata at their ends, measuring 15.5-20.5 × 1.8-3.0 μm. Long chains of conidia were continuously formed from the ends of the sterigmata in a basal manner. The conidia were unicellular, spherical or nearly spherical, measuring 2.1-3.6 × 2-2.5 μm.

[0011] Another objective of this invention is to provide a method for using the aforementioned strain SL-066 as a rice insecticide. Specifically, the Beauveria bassiana strain SL-066 is inoculated onto a PDA plate culture medium and cultured for 10-15 days at 25°C and 90% relative humidity. Conidia are then collected and formulated into Beauveria bassiana wettable powders of different concentrations. The rice pests mentioned in this invention include rice water weevil, rice stem borer, and rice leaf beetle.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention provides a Beauveria bassiana strain SL-066, which can effectively control three kinds of rice pests (rice water weevil, rice stem borer and rice leaf beetle) and has a very good control effect. Detailed Implementation

[0014] To make the objectives, features, and advantages of the present invention more apparent and understandable, specific embodiments of the invention are described in detail. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present invention will be thorough and complete.

[0015] 1. Morphological identification

[0016] The Beauveria bassiana strain SL-066 of this invention was inoculated on a PDA plate and cultured at 28°C to observe the colony morphology, sporulation structure, and spore shape and size.

[0017] The results showed that the colonies on PDA medium were initially white and fluffy to cottony in appearance, turning pale yellow after conidia. The hyphae were transparent, septate and branched, with a diameter of 3.0-3.5 μm. The conidiophores were densely clustered, whorled or solitary, with broom-like branches, measuring 2.5-5.5 × 1.5-2.5 μm, and produced flask-shaped sterigmata at their ends, measuring 15.5-25.5 × 1.5-3.0 μm. Long chains of conidia were continuously formed from the ends of the sterigmata in a basal manner. The conidia were unicellular, spherical or nearly spherical, measuring 2-3 × 2-2.5 μm.

[0018] 2. ITS sequence amplification, sequencing and molecular identification of Beauveria bassiana strain SL-066

[0019] Beauveria bassiana SL-066 strain was cultured in SDAY liquid medium with shaking for 3 days (200 rpm, 28℃). Hyphae were collected by centrifugation at 10,000 rpm for 20 min at 4℃. After homogenization in liquid nitrogen, total genomic DNA was extracted from Beauveria bassiana SL-066 using a fungal genomic DNA extraction kit. Using the extracted total DNA as a template, primers designed according to White's design were used to amplify the ITS1–5.8S–ITS2 rDNA region of Beauveria bassiana. The primers used were ITS15'-TCCGTAGGTGAACCTGCGG-3' and ITS45'-TCCTCCGCTTATTGATATGC-3', producing a 550 bp amplification product. Amplification conditions: 94℃ pre-denaturation for 4 min, 1 cycle; 94℃ denaturation for 1 min, 53℃ annealing for 0.5 min, 72℃ extension for 45 s, 35 cycles; final extension at 72℃ for 10 min. The amplification products were recovered using a gel extraction kit and their sequences were determined by dideoxy sequencing at Sangon Biotech (Shanghai) Co., Ltd.

[0020] Upon testing, the ITS sequence of the Beauveria bassiana strain SL-066 described in this invention was compared with known sequences in Genbank, and the homology of its ITS sequence was 97%. The strain was identified as Beauveria bassiana (Bals.-Criv.) Vuill.

[0021] 3. Indoor insecticidal activity assay of Beauveria bassiana SL-066 against three rice pests.

[0022] Test insects: Overwintering surviving rice stem borer larvae were collected from rice stubble in spring. After diapause was relieved indoors, the larvae pupated, emerged, laid eggs, and hatched. The larvae were fed with artificial feed. When they developed to the 3rd instar, indoor biological tests of Beauveria bassiana strains were conducted. Healthy and active live rice water weevils and rice leaf beetle 3rd instar larvae were collected from the field in spring and selected as test insects.

[0023] Beauveria bassiana strains were inoculated onto PDA plates and cultured at 25°C and 90% relative humidity for 10-15 days. Conidia were then collected and dissolved in 0.1% Tween-80 sterile water to prepare a solution with a concentration of 1.5 × 10⁻⁶. 6 A conidia suspension of 1 conidia / mL was prepared. The conidia suspension was poured into a hyperbaric throat sprayer.

[0024] The tested rice stem borer and rice leaf beetle 3rd instar larvae, as well as rice water weevils, were placed in sterilized enamel dishes. Each larva was then sprayed with a throat sprayer (3 sprays per replicate), with 4 replicates per strain and 20 larvae per replicate. After spraying, each rice stem borer, rice leaf beetle, and rice water weevil 3rd instar larvae was individually placed into a finger-shaped tube containing a filter paper strip. The opening of the finger-shaped tube was plugged with a cotton ball to maintain ventilation. The control group was inoculated with an equal volume of 0.1% Tween-80 sterile water. Both treatments and controls were incubated at 25℃ and 90% relative humidity. Water was added to the finger-shaped tubes to maintain high humidity for three days. From the second day onwards, the number of infected and dead insects was investigated and recorded daily for 15 days, and the cumulative mortality rate was calculated. The laboratory's existing strain XJ8 was used as the control (CK) strain. The pathogenicity of Beauveria bassiana strains against three rice pests is shown in Table 1.

[0025] Table 1. Pathogenicity of Beauveria bassiana strains against three rice pests.

[0026]

[0027] Table 1 shows that the Beauveria bassiana strain SL-066 exhibited strong pathogenicity against the three rice pests, with mortality rates of 91.25%, 90.00%, and 72.50%, respectively. The corrected mortality rates were 90.79%, 89.47%, and 71.05%, respectively, all higher than the control (CK) strain's control efficacy against the three pests, and all showed significant differences from the control (CK) strain. The lethal time LT... 50 The values ​​for (d) were 4.94d, 5.98d, and 6.51d, respectively, all significantly lower than those of the control (CK) strain.

[0028] In summary, it can be seen that Beauveria bassiana strain SL-066 has good insecticidal effects against rice stem borer, rice water weevil, and rice leaf beetle.

[0029] 4. Field efficacy trial of Beauveria bassiana SL-066 wettable powder for controlling rice water weevil and rice leaf beetle.

[0030] Test date: June 9, 2021;

[0031] Test location: Dayushu Village, Nanwaizi Town, Gongzhuling City, Jilin Province;

[0032] Test reagents: Beauveria bassiana SL-066 strain high spore powder was prepared into a 25 billion / g Beauveria bassiana wettable powder (WP), and the control agent was the biological pesticide matrine;

[0033] Application dosage: 100g per acre, applied in two applications, with a second spraying 7 days apart;

[0034] Application method: Use a backpack sprayer to mix the pesticide with water and spray.

[0035] Investigation method: Before and after the application of pesticides, each treatment plot was sampled at 5 points every 3 days, with 20 plants at each point, to investigate the residual number of adult rice water weevils and larvae of rice leaf beetles. The mortality rates of adult rice water weevils and larvae of rice leaf beetles in each plot were counted, and the insect population reduction rate and control effect were calculated.

[0036] Insect population reduction rate (%) = (Number of live insects before treatment - Number of live insects after treatment) / Number of live insects before treatment × 100

[0037] Corrected efficacy (%) = (Reduction rate in treated area - Reduction rate in control area) / 1 - Reduction rate in control area × 100

[0038] The field control efficacy of Beauveria bassiana wettable powder against adult rice water weevils and larvae of rice leaf beetles is shown in Tables 2 and 3, respectively.

[0039] Table 2. Survey on the control efficacy of Beauveria bassiana wettable powder against rice water weevil.

[0040]

[0041] As shown in Table 2, the average population reduction rate of the three treatments against adult rice water weevils was 82.09%, while the population reduction rate of the biological pesticide matrine (CK) was only 33.82%. The corrected control efficacy of the three treatments against adult rice water weevils was 76.61%, 71.67%, and 70.51%, respectively, with an average control effect of 72.93%. This indicates that Beauveria bassiana wettable powder can effectively control rice water weevils.

[0042] Table 3. Survey on the control efficacy of Beauveria bassiana wettable powder against rice leaf beetle larvae.

[0043]

[0044] As shown in Table 3, the average reduction rate of rice leaf beetle larvae by the three treatments was 82.92%, while the reduction rate of the biological pesticide matrine (CK) was only 31.20%. The corrected control efficacy of the three treatments against rice leaf beetle larvae was 73.88%, 75.15%, and 76.49%, respectively, with an average control effect of 75.17%. This shows that Beauveria bassiana wettable powder can effectively control rice leaf beetle larvae.

[0045] 5. Field efficacy trial of Beauveria bassiana SL-066 wettable powder for controlling rice stem borer.

[0046] Test time:

[0047] The experiment to control the rice stem borer involved two applications of pesticide. The first treatment was carried out on July 2, 2020, and the second treatment was carried out 7 days later on July 9, 2020.

[0048] Experimental location: Wujia Village, Yongji County, Jilin Province; the treatment area and the blank control area were 1 hectare and 0.5 hectares, respectively.

[0049] Experimental materials:

[0050] Beauveria bassiana SL-066 strain high spore content was formulated into a 25 billion / g Beauveria bassiana wettable powder (WP), with an effective content of 25 billion spores / gram and a germination rate of 90%; per mu (667m²) 2 Apply 50g each time, divided into two applications;

[0051] One agricultural drone was used, and the operating range was set throughout the test using a GPS ground station system.

[0052] Test method:

[0053] Agricultural drones were used for spraying, with flight parameters set as follows: flight altitude 1.5m, flight speed 5m / s, and effective spray width 5m.

[0054] Survey Methodology:

[0055] In each demonstration area, the rate of sheath blight and heart blight was investigated approximately one month after pesticide application, at the end of July; the rate of white panicles was investigated in September. Sampling method: 5 sampling points were used, with continuous sampling at each point, 25 clumps / 50m2.

[0056] Method for calculating the effectiveness of prevention:

[0057] Dead heart rate (per 100 acupoints) = Number of dead hearts / 100 Prevention and control effect = (Dead heart rate in control group - Dead heart rate in treatment group) * 100 / Dead heart rate in control group

[0058] Withered sheath rate (per 100 holes) = Number of withered sheaths / 100 Control effect = (Withered sheath rate of control group - Withered sheath rate of treatment group) * 100 / Withered sheath rate of control group

[0059] White ear rate (per 100 hills) = Number of white ears / 100 Control effect = (White ear rate in control group - White ear rate in treatment group) * 100 / White ear rate in control group

[0060] Average control efficacy = (control efficacy for dead heart rate + control efficacy for dead sheath rate + control efficacy for white ear rate) / 3

[0061] Experimental results:

[0062] Table 4 shows the control efficacy of Beauveria bassiana wettable powder applied by agricultural drones against rice stem borer. As can be seen from Table 4, the average control efficacy at the three survey sites was 86.24% for dead heart rate, 83.34% for sheath dead rate, and 89.33% for whitehead rate. All three indicators exceeded 80%, with an average control efficacy of 86.30%. This demonstrates that Beauveria bassiana wettable powder is effective in controlling rice stem borer in the field.

[0063] Table 4. Survey on the control efficacy of Beauveria bassiana wettable powder sprayed by agricultural drones against rice stem borer.

[0064]

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A type of Beauveria bassiana ( Beauveria Bassiana strain SL-066, characterized in that, The strain was deposited on August 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40269.

2. The Beauveria bassiana according to claim 1 ( Beauveria Bassiana The application of strain SL-066 is characterized by, The application refers to using the strain SL-066 as a microbial insecticide to control rice pests, namely rice water weevils, rice stem borers, or rice leaf beetles.

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