CN103756913B - A kind of rose dark brown Isaria bacterial strain - Google Patents

Abstract

The invention discloses a fumosorosea strain (<i>Isaria</i><i>?fumosorosea</i>). The bacterial strain is a wild bacterial strain isolated from the body of the termite worm naturally infected by entomogenic fungi in Jiulianshan National Nature Reserve in Jiangxi Province, and the wild strain is grafted back on the worm body of the domestic termite for rejuvenation to obtain the rose smoke color of the present invention. A strain of I. clavulata, for which a purified strain is obtained by isolation of single spores. The purified strain is I. fumigatus strain SCAU-IFCF02, which was preserved in the China Center for Type Culture Collection on November 1, 2013, with the preservation number CCTCC? NO: M? 2013526. The long-term infection biology research and indoor bioassay show that the strain has a very strong application potential in biological control of diamondback moth and house termite.

Description

A strain of I. fumigatus

technical field

The invention belongs to the technical field of microorganisms. More specifically, it relates to a strain of I. fumosmosus.

Background technique

Entomopathogenic fungi are an important part of insecticidal microorganisms, accounting for more than 60% of entomopathogenic microorganisms. The extensive development and utilization of microbial pesticides can effectively reduce and eliminate many negative effects such as residues, toxicity and drug resistance, which are common in current chemical pesticides. Isariafumosorosea is an important entomopathogenic fungus, widely distributed in tropical, subtropical and temperate regions, and can parasitize including Homoptera, Diptera, Lepidoptera, Hemiptera, Coleoptera and Hymenoptera and other vegetables, fruit trees and tea tree pests. I. fumigatus has the advantages of high efficiency to pests, environmental friendliness, and less resistance to pesticides. Therefore, it has become a hot spot in biological control research at home and abroad. The use of fungal and microbial pesticides to control plant diseases and insect pests is one of the important means in the sustainable production of pollution-free agricultural products, and has broad application prospects.

As a common entomopathogenic fungus, I. fumigatus is used in pest control. Its growth and development are affected by a variety of ecological factors. The pathogenic process of host insects is complex and involves the regulation of a variety of genes and their products. Practice The application prevention effect is unstable, which limits its application. Although strain improvement can improve virulence and expand its application range, there are still many unresolved or unknown problems.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and technical deficiencies such as poor pest control effect and unstable control effect of the existing strain of I. fumigatus, and provide a strain of I. fumigatus.

The object of the present invention is to provide the above-mentioned I. fumigatus strain and its culture method.

The above object of the present invention is achieved through the following technical solutions:

The invention provides a strain of Isaria fumosorosa (Wize) Brown&Smith, which is the Isariafumosorosa (Wize) Brown&Smith strain SCAU-IFCF02, which was preserved in China Type Culture on November 7, 2013. Collection Center, the deposit number is CCTCCNO: M2013526, and the deposit address is Wuhan University, Wuhan, China.

The fumigatus fumigatus strain SCAU-IFCF02 belongs to the order Hyphosporium and the genus Isyariae.

The morphological characteristics of the fume-colored I. fumigatus strain SCAU-IFCF02 are described as follows:

Colony morphology: the front of the colony is fluffy, initially white, and after sporulation is light rose smoke color, with a concave-convex fluffy powder layer; the back is milky yellow, with multiple mound-like bulges in a circle with a radius of 15 mm from the center to the edge.

Morphology of hyphae and spores: through microscopic observation, it can be seen that conidiophores grow on vegetative hyphae. Hyphae separated, transparent, smooth, 1.5-2.5 μm wide. Conidiophores are solitary or aggregated into spore bundles, 100×1.5～100×2.0μm, with smooth and transparent walls, mostly composed of whorled branches with 4 to 6 phialides forming whorls. The base of the bottle stem is spherical, or nearly elliptical and enlarged, and the upper part is slender, 5.7～8.0×1.0～2.0μm. Conidia spherical to nearly fusiform, smooth, transparent to light red, 3.0-4.0×1.0-2.0 μm, without chlamydospores.

The steps for the present invention to obtain I. fumigatus strain SCAU-IFCF02 are as follows:

S1. Sampling: House termites naturally infested by entomogenic fungi were collected from Jiulianshan National Nature Reserve in Jiangxi;

S2. Isolation: Isolate the pathogenic bacteria from the collected carcass samples of house termites infected by entomogenic fungi. Utilize 5% sodium hypochlorite solution to carry out surface disinfection to sample, the sample after disinfection is washed 3 times in sterilized water, and put into potato dextrose agar medium (PDA) plate, place 25 ± 1 ℃ in constant temperature incubator and cultivate, After the colony is formed, transfer it to a PDA slant, and then transfer it to a 4°C refrigerator for storage;

S3. Rejuvenation: the isolated bacterial strains were cultivated on the PDA plate for 15 days, after the formation of colonies, the spores were washed with sterile water containing 0.03% Tween-80, then moved into a beaker, and the spore suspension was placed on a magnetic stirrer Stir for 30 minutes, dilute to a spore suspension of 1×10 ⁷ spores/mL, spray evenly on the body surface of house termite workers with a small manual sprayer, moisturize 90% to 100%, pick out infected house termites after 7 days, and follow the above Described method isolates and obtains A isolate;

S4. Purification: isolate strain A was cultured on a PDA plate for 15 days, and after forming rose-colored spores, pick conidia to make a spore suspension of 1×10 ³ spores/mL, and drop the suspension on a lidded On the glass slide of the glass slide, observe under a biological microscope, insert a slide with only one conidia in a droplet into the PDA medium, place it in an incubator and cultivate it, and obtain a total of B, C, D, E, and F 5 isolates;

S5. Identification: Preliminary identification is carried out according to the cultured shape of the pathogenic bacteria, the morphology of mycelia, conidia and sporogenes;

S6. Screening: Measure the pathogenicity, spore production, spore germination rate and colony growth rate of the 5 isolates to target pests respectively, and compare the results to screen out the rose-colored sticks with strong activity and the best control effect on pests spore strains. Isariafumosorosea (Wize) Brown&Smith was identified as Isariafumosorosea (Wize) Brown&Smith by Guy Mecardier, an insect pathology expert at the European Biocontrol Laboratory of the United States Department of Agriculture. M2013526, deposited at Wuhan University, Wuhan, China.

The potato dextrose agar medium (PDA) formula used in the present invention is: 200g potatoes, 17-20g agar, 20g glucose, 1000mL water, sub-package, and sterilize in an autoclave at 121° C. for 30 minutes.

Entomogenic fungi are diverse in terms of genetic, ecological, and biological characteristics. Different strains of the same fungus have significant differences in pathogenicity to target pests. Different strains have different LD ₅₀ and LT ₅₀ differences of several times or even several times. ten times. Screening and obtaining high-quality strains with high spore yield, high spore germination rate and strong pathogenicity are the prerequisite and necessary link to achieve good control effect. Among the pathogenic fungi that have been reported to be more virulent to Plutella xylostella, Beauveria bassiana CS-1 has a LT ₅₀ effect on the 2nd instar larvae of Plutella xylostella under greenhouse conditions when the treatment concentration is 1×10 ⁵ spores/ml. It was 3.61 days (Yone et al., 1999); Metarhizium anisopliae was ¹ × 108 spores/ml when the treatment concentration, the LT ₅₀ of the 2nd instar larvae of Plutella xylostella xylostella was 4.97 days, and the LC ₅₀ to the 2nd instar larvae of Plutella xylostella was 2.03 × 10 ⁴ spores/ml (Ma, 2000).

The four indicators commonly used in strain screening are pathogenicity of strains to target pests, sporulation yield, spore germination rate, and colony growth rate. Based on the above indicators, the present invention screens and obtains an excellent bacterial strain SCAU-IFCF02 of I. fumigatus. On the 2nd day after inoculation of I. fumigatus strain SCAU-IFCF02, the 2nd instar larvae in each treatment all began to become ill and die, indicating that the strain had very strong pathogenicity to diamondback moth. At the highest concentration of 1×10 ⁷ spores/mL, the cumulative corrected mortality of the 2nd, 3rd and 4th instar larvae of Plutella xylostella were 100%, 98.93%, 87.23%, respectively. On the 7th day after inoculation of the 2nd, 3rd and 4th instar larvae of Plutella xylostella, the lethal medium concentration LC50 was 7.63×10 ³ spores/mL, 1.05×10 ⁴ spores/mL and 2.40×10 ⁴ spores/mL, respectively. The LC50 values of the 2nd, 3rd and 4th instar larvae of Plutella xylostella after inoculation with I. fumigatus strain SCAU-IFCF02 were 7.63×10 ³ , 1.05×10 ⁴ , 2.40×10 ⁴ spores/mL, respectively. When the treatment concentration was 1×10 ⁷ spores/mL, the LT50 of 2nd, 3rd and 4th instar larvae were 1.61 days, 1.66 days and 1.80 days, respectively. Compared with the currently commonly used strain of I. fumigatus, I. fumigatus SCAU-IFCF02 strain of the present invention has significant high pathogenicity to diamondback moth larvae, which is from its lethal concentration to diamondback moth. Both aspects can be manifested during and during death. The strain IFCAU-IFCF02 is the entomopathogenic fungus with the lowest lethal concentration and the fastest lethal rate to Plutella xylostella larvae reported so far. application potential.

The present invention has the following beneficial effects:

The present invention provides a strain SCAU-IFCF02 of I. fumigatus, which can prevent and control cruciferous vegetable pests, especially the control effect on house termites and diamondback moths. The entomopathogenic fungus with the lowest larval lethal concentration and the fastest lethal rate has strong application potential in the biological control of domestic termites and diamondback moths.

In addition, the strain SCAU-IFCF02 of Fusarium fumigatus is an entomopathogenic fungus. As a living biopesticide, it has a new mechanism of action different from existing chemical insecticides. It has no pollution to the environment and no residue. It is safe for human beings and livestock, and adapts to the requirements of organic food production; and the strain is isolated from the infested domestic termites in Jiulianshan National Nature Reserve, Jiangxi. It is a local strain of China, not imported from abroad. Can adapt to the local natural environment. It has a strong promotion and application value in the biological control of cruciferous vegetable pests such as house termites and diamondback moths.

detailed description

The present invention will be further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents used in the present invention were prepared by conventional methods or purchased from the market, and the culture of I. fumigatus strains was carried out according to conventional methods in the art.

The sterile water containing 0.03% Tween-80 used in the examples, the 0.03% refers to the volume ratio.

The acquisition of embodiment 1 fume-colored I. spp. strain SCAU-IFCF02

1. Experimental materials and conditions

(1) The carcass of a house termite (Coptotermes formosanus) infested by entomogenic fungi was collected in Jiulianshan National Nature Reserve, Jiangxi.

(2) Potato dextrose agar medium (PDA): 200g potatoes, 17-20g agar, 20g glucose, 1000mL water, aliquoted, and sterilized in an autoclave at 121°C for 30min.

(3) Sterile operating conditions: All utensils and utensils must be sterilized in an autoclave at 121°C for 30 minutes, and operations such as inoculation are performed in an ultra-clean workbench.

(4) Conditions for culturing strains: culture in a light incubator at 25±1°C. The photoperiod of the light incubator is 14L:10D. After the colony is formed, transfer it to a PDA slope, and then transfer it to a 4°C refrigerator for storage.

2. Isolation and identification of pathogenic bacteria

(1) Isolation: Isolate the pathogenic bacteria from the collected carcasses of house termites infected by entomogenic fungi. Use 5% sodium hypochlorite solution to sterilize the surface of the sample, wash the sterilized sample 3 times in sterilized water, put it on a PDA plate, and place it in a constant temperature incubator at 25±1°C for cultivation. After the colony is formed, transfer it to PDA slant, and then transferred to 4 ℃ refrigerator for storage.

(2) Rejuvenation: Culture the isolated bacterial strains on a PDA plate for 15 days, wash the spores with sterile water containing 0.03% Tween-80 after the formation of colonies, obtain a spore suspension, move it into a beaker, and dissolve the spore suspension Stir on a magnetic stirrer for 30 minutes, dilute to a spore suspension of 1×10 ⁷ spores/mL, spray evenly on the body surface of house termite workers with a small manual sprayer, moisturize 90% to 100%, and pick infected ants after 7 days House termites, according to the method described in (1) isolate and obtain A isolate.

(3) Purification: isolate strain A was cultured on a PDA plate for 15 days, and after the formation of rose-colored spores, the conidia were picked to make a spore suspension of 1×10 ³ spores/mL, and the suspension was dropped on Place the cover glass on the glass slide, observe under the biological microscope, insert the glass slide with only one conidia in a droplet into the PDA medium, and culture in the incubator to obtain B, C, D, E , F a total of 5 isolates.

(4) Identification: Preliminary identification is carried out according to the cultured shape of the pathogenic bacteria, the morphology of mycelia, conidia and sporogenes.

Colony morphology description: The front of the colony is fluffy, initially white, and after spore production, it is light rose smoke color, with a concave-convex fluffy powder layer; the back is milky yellow, with multiple mound-like bulges in a circle with a radius of 15mm from the center to the edge.

Morphological description of hyphae and spores: through microscopic observation, it can be seen that conidiophores grow on vegetative hyphae. Hyphae separated, transparent, smooth, 1.5-2.5 μm wide. Conidiophores are solitary or aggregated into spore bundles, 100×1.5～100×2.0μm, with smooth and transparent walls, mostly composed of whorled branches with 4 to 6 phialides forming whorls. The base of the bottle stem is spherical, or nearly elliptical and enlarged, and the upper part is slender, 5.7～8.0×1.0～2.0μm. Conidia spherical to nearly fusiform, smooth, transparent to light red, 3.0-4.0×1.0-2.0 μm, without chlamydospores.

3. Screening of I. fumigatus strain SCAU-IFCF02

Entomogenic fungi are diverse in terms of genetic, ecological, and biological characteristics. Different strains of the same fungus have significant differences in pathogenicity to target pests. Different strains have different LD ₅₀ and LT ₅₀ differences of several times or even several times. ten times. Screening and obtaining high-quality strains with high spore yield, high spore germination rate and strong pathogenicity are the prerequisite and necessary link to achieve good control effect. The four indicators commonly used in strain screening are pathogenicity of strains to target pests, sporulation yield, spore germination rate, and colony growth rate. Based on the above indicators, the present invention screens excellent strains of I. fumigatus.

(1) Treatment of test strains

Five isolates of I. fumigatus B, C, D, E, and F obtained after purification were placed on a PDA plate in an incubator at 25±1°C (light cycle: light:dark=14:10) cultivated in.

(2) Source of tested insects

House termites were collected from the campus of South China Agricultural University. Workers and termites were raised indoors with pine blocks, and healthy worker ants were selected for indoor virulence testing.

(3) Determination of bacterial colony growth rate and spore production

Prepare the conidia suspension of 1×10 ⁷ spores/mL from 5 isolates respectively, drop 500 μL of the suspension into PDA medium, spread it evenly with a triangular glass rod, and wait for 2 to 3 days to grow mycelia Finally, a fresh colony was drilled with a hole punch with a diameter of 5 mm, inoculated on a PDA plate, and then placed in an incubator for cultivation. The photoperiod of the light incubator was 14L:10D. 5 replicates per strain. Every 2 days, the growth amount of the bacterial colony diameter was measured regularly and in a fixed direction, and a total of 4 measurements were taken, and the growth rate was calculated, and the unit of the growth rate was mm/day. The culture was continued until the 15th day, and the sporulation of each strain was measured. The specific operation is as follows: use a sterilized puncher with a diameter of 13 mm to punch a hole from the center of the colony to the edge 1/2 and take a sample, put the sample into 10 mL of sterile water containing 0.03% Tween-80, and stir on a magnetic stirrer for 20 minutes , so that the spores are evenly dispersed, and the spore suspension is made, and the amount of sporulation is determined by counting with a hemocytometer. The results are shown in Table 1.

(4) Determination of spore germination rate

After the five isolates were cultured for 15 days, the spores were collected with sterile water containing 0.03% Tween-80 and made into a suspension. The concentration of the spores in the suspension was: about 100 conidia per field of view. Test with glass slide germination method, drop the spore suspension directly on a sterile glass slide, place it in a petri dish with sterilized filter paper at the bottom, add 4 to 5 drops of sterile water in the dish to keep 100% relative humidity (RH), microscopic examination after culturing for 18 hours. 5 replicates per treatment. The results are shown in Table 1.

(5) Determination of the pathogenicity of the isolates to house termite workers.

Prepare the spore suspension of each test strain: inoculate each test strain on a PDA plate and cultivate it for 15 days, wash the spores with sterile water containing 0.03% Tween-80, stir with a magnetic stirrer for 30 min, and double-layer sterile gauze Filter out the hyphae, make a spore suspension, and adjust the spore concentration to ¹ ×107 spores/mL with a hemocytometer.

Insect test treatment: The pathogenicity of each isolate to worker termites of house termites was determined by the insect dipping method. Select healthy worker ants, use soft tweezers to pick the worker ants for testing into the dilution of the spore suspension of each isolate, the dilution is obtained by diluting with sterile water containing 0.03% Tween-80, and pick after dipping for 10s. After the worker ants were placed on sterilized filter paper to absorb excess water, they were moved to a 9cm-diameter petri dish lined with moist filter paper at the bottom, the petri dish was sealed with plastic wrap, and holes were pricked on the film to ventilate, 20 for each treatment Worker ants, repeated 5 times, a total of 100 worker ants. In addition, sterile water containing 0.03% Tween-80 was used as a control, and the operation method was the same as above, repeated 5 times. After inoculation, each treatment was placed in an artificial intelligence box, 25±1°C, relative humidity 90%, and the photoperiod was light:dark=14:10. Observation began after 12 hours, and the treated worker ants were reared with filter paper, and the sight dish If the inner filter paper is eaten, replace it with fresh filter paper. The symptoms of worker ants' infection and the number of dead insects were recorded for a total of 7 days. Statistically adjusted mortality rates. The results are shown in Table 1.

(6) Screening of I. fumigatus isolates

When screening excellent isolates, pathogenicity and spore production are important reference indicators, followed by spore germination rate and average colony growth rate. In the present invention, as can be seen from Table 1, the average bacterial colony growth rate of bacterial strain B is significantly higher than the remaining bacterial strains, and the bacterial colony growth rate of bacterial strain B is the fastest, being 15.79mm/day, and the sporulation of bacterial strain B is between 5 It is also the highest among sub-strains, reaching 2.71×10 ⁷ spores/mL. Strain D was next at 2.32×10 ⁷ spores/mL. In terms of spore germination rate, the spore germination rate of each isolate was significantly different, and the spore germination rates of strains B and C were the highest at 92.11% and 90.39%. Bioassay results showed that strain B had the highest corrected mortality rate of 100% infecting house termites, showing super pathogenicity.

Table 1 The biological characteristics of the strains and their corrected mortality rates against domestic termite workers

The data in the table are Mean±S.E., different lowercase letters after the data in the same column indicate significant differences, Duncan method, P<0.05, all the above test data were processed on the data processing software DPS system.

Comprehensive comparison of various factors in Table 1, isolate B is the best strain, identified as Isariafumosorosea (Wize) Brown&Smith by Guy Mecardier, an insect pathologist in the European Biocontrol Laboratory of the United States Department of Agriculture, and the strain is named SCAU-IFCF02, which was released in 2013 It was deposited in the China Center for Type Culture Collection on November 7, with the deposit number CCTCCNO: M2013526, and the deposit address is Wuhan University, Wuhan, China.

The smoke-colored Isariafumosorosea (Wize) Brown&Smith strain SCAU-IFCF02 belongs to the order Hypomycetes and the genus Isariafumosorosea. The colony of this strain is fluffy on the front, white at first, light rose smoke color after sporulation, with a concave-convex fluffy powder layer; the back is milky yellow, with multiple mound-like bulges in a circle with a radius of 15mm from the center to the edge. Hyphae separated, transparent, smooth, 1.5-2.5 μm wide. Conidiophores are solitary or aggregated into spore bundles, 100×1.5～100×2.0μm, with smooth and transparent walls, mostly composed of whorled branches with 4 to 6 phialides forming whorls. The base of the bottle stem is spherical, or nearly elliptical and enlarged, and the upper part is slender, 5.7～8.0×1.0～2.0μm. Conidia spherical to nearly fusiform, smooth, transparent to light red, 3.0-4.0×1.0-2.0 μm, without chlamydospores.

Example 2 Determination of the pathogenicity of the rose-colored Isospora SCAU-IFCF02 strain to Plutella xylostella

Bioassay is one of the most important techniques in the theoretical and applied research of insect mycology and entomopathology, and it is one of the important means to detect the degree and rate of lethality of entomogenic fungi to target pests. It provides an important reference for the biocontrol potential of sporospora SCAU-IFCF02 strain. In the present invention, the pathogenicity of the strain I. fumigatus SCAU-IFCF02 to Plutella xylostella is measured to determine the optimal concentration and optimal age of the strain infecting Plutella xylostella, so as to guide the use of the bacterial strain in preventing and controlling field pests. in the application.

1. Test materials

(1) Tested insects: mature larvae of the diamondback moth Plutellaxylostella (L.) were collected from the vegetable kale field near South China Agricultural University in the suburbs of Guangzhou, and the kale seedlings were subcultured indoors to the fifth generation. (L.) 2nd, 3rd and 4th instar larvae were used for biotoxicity determination.

(2) Tested crop: Kale (B.alboglabraBailey)

(3) Treatment of test strains

The purified I. fumoscens SCAU-IFCF02 strain was spread on a PDA plate and cultured in an artificial intelligence box at 25±1° C. for 15 days. The photoperiod of the artificial intelligence box was 14L:10D. Wash the spores with sterile water containing 0.03% Tween-80, and stir on a magnetic stirrer for 30 minutes. After the spores are evenly dispersed, filter them with sterilized double-layer medical gauze to obtain a spore suspension, and use a hemocytometer to determine the spores. The concentration of the suspension was diluted with sterile water to 5 concentrations of 1×10 ⁷ , 1×10 ⁶ , 1×10 ⁵ , 1×10 ⁴ , and 1×10 ³ spores/mL for use.

(4) Determination of the pathogenicity of Isyriatium rosea SCAU-IFCF02 strain to diamondback moth larvae

The pathogenicity of the strain I. fumoscens SCAU-IFCF02 to the 2nd, 3rd and 4th instar larvae of Plutella xylostella was determined by insect dipping method. Select Plutella xylostella larvae of all instars with the same individual size and vitality, pick the larvae to be tested into the above-mentioned spore dilution with a soft brush, pick out after dipping for 10 seconds, place the larvae on sterilized filter paper to absorb excess water, and then The larvae were moved to the leaves, and put into a 9cm-diameter petri dish lined with moist filter paper at the bottom. The petri dish was sealed with plastic wrap to keep it moist, and holes were pricked on the film for ventilation. There were 20 larvae in each treatment, repeated 5 times, a total of 100 larvae. In addition, sterile water containing 0.03% Tween-80 was used as a control, and the operation method was the same as above, repeated 5 times. After inoculation, each treatment was placed in an artificial intelligence box at 25±1°C, relative humidity 90%, and the photoperiod was light:dark=14:10. Observation began after 24 hours, and fresh cabbage leaves were replaced as appropriate, and the sensitivity was recorded every day. The disease symptoms and the number of dead insects were observed continuously for 7 days, and the dead insects were picked into a petri dish with wet filter paper at the bottom of the dish, and kept moist at 25±1°C to observe the growth of mycelium on the dead insects, so as to distinguish whether it was a rose or not. I. fumigatus SCAU-IFCF02 strain is lethal to insects.

Mortality (%) = number of dead insects/total number of test insects treated at each concentration × 100

Corrected mortality rate (%)=(treatment mortality rate-control mortality rate)/(1-control mortality rate)×100

All data analyzes were performed using DPS software.

Pick dead larvae with rose-colored mycelia, soak them in 0.1% mercuric chloride solution for 1-2 minutes, then rinse them with sterile water for 3 times, transfer them to SDAY medium plates, 25±1 After culturing at ℃ for 3 days, the worms on the medium plate grew hyphae, and finally made slices for observation to identify whether the picked hyphae were I. fumigatus.

The preparation method of the SDAY solid medium is as follows: glucose 40g/L, peptone 10g/L, yeast extract 20g/L, agar 15-20g/L, H ₂ O 1L, stir and mix well, then subpackage, sterilize, and set aside .

2. Results and Analysis

(1) Comparison of accumulative corrected mortality of Plutella xylostella at different ages

Table 2 shows the cumulative corrected mortality of larvae of each instar on the seventh day after being treated with different concentrations of I. On the second day after inoculation, the larvae of all instars in each treatment began to develop disease and die, which indicated that the I. fumigatus strain SCAU-IFCF02 strain had a strong lethal effect on Plutella xylostella. Within the measured concentration range of 1×10 ³ to 1×10 ⁷ , the pathogenicity increased significantly with the increase of the concentration. At the highest concentration of 1×10 ⁷ spores/mL, the cumulative corrected mortality of the 2nd, 3rd and 4th instar larvae of Plutella xylostella were 100%, 98.93% and 87.23%, respectively. The infected and dead larvae showed typical lethal symptoms of entomogenous fungi. Mycelium began to grow from the body surface on the second day after the corpse was moistened and cultured, and then produced a large number of rose-colored smoky spores. Although less than 10% of the larvae died in the control group, none of them showed fungal lethal symptoms because the mortality rate was too low.

Table 2 Cumulative corrected mortality of Plutella xylostella at different ages infected with I. fumoscens for 7 days

(2) Toxicity determination results of Isora fumoscens SCAU-IFCF02 strain to various instar larvae of Plutella xylostella

The results of the lethal concentration of the strain I. fumigatus SCAU-IFCF02 on the larvae of Plutella xylostella xylostella are shown in Table 3. The LC ₅₀ of the 2nd, 3rd and 4th instar larvae on the 7th day after inoculation were 7.63× ¹⁰³ spores respectively /mL, 1.05×10 ⁴ spores/mL and 2.40×10 ⁴ spores/mL.

The virulence of I. rosea fumoscens SCAU-IFCF02 strain to each instar larvae of Plutella xylostella within 37 days

The lethal time of the strain I. fumigatus SCAU-IFCF02 to each instar larvae of Plutella xylostella xylostella is shown in Table 4. With the increase of the concentration of the fungal spore suspension, the LT ₅₀ value of each instar larvae tested decreased gradually, and at 1× In the range of 10 ⁵ to 1×10 ⁷ spores/mL, the LT ₅₀ of the 2nd instar larvae decreased from 2.86 days to 1.61 days, the LT ₅₀ of the 3rd instar larvae decreased from 3.21 days to 1.66 days, and the LT ₅₀ of the 4th instar larvae decreased from 3.4 days down to 1.80 days. When the 2nd instar larvae were at a high concentration of 1×10 ⁷ spores/mL, the LT ₉₀ was 3.24 days. It can be seen that the time required for the death of the 2nd instar larvae is the shortest, followed by the 3rd instar larvae, and the time required for the death of the 4th instar larvae is the longest.

Table 4 The LT ₅₀ and LT ₉₀ of the strain I. fumigatus SCAU-IFCF02 to the larvae of Plutella xylostella xylostella xylostella xylostella xylostella

The results of this experiment showed that the strain I. fumoscens SCAU-IFCF02 had a strong pathogenicity to the larvae of diamondback moth, which could be manifested in both its lethal concentration and lethal time to diamondback moth. The LC ₅₀ of the 2nd, 3rd and 4th instar larvae of Plutella xylostella after inoculation with I. fumigatus strain SCAU-IFCF02 were 7.63×10 ³ , 1.05×10 ⁴ , 2.40×10 ⁴ spores/mL, respectively. When the treatment concentration was 1×10 ⁷ spores/mL, the LT ₅₀ of the 2nd, 3rd and 4th instar larvae were 1.61 days, 1.66 days and 1.80 days, respectively. The strain of I. fumigatus rosea SCAU-IFCF02 of the present invention has significant high pathogenicity to diamondback moth larvae.

Claims (3)

1. a rose dark brown Isaria bacterial strain (Isariafumosorosea), is characterized in that, described bacterial strain is rose dark brown rodBundle spore bacterial strain SCAU-IFCF02, this bacterial strain is preserved in Chinese Typical Representative culture collection center on November 1st, 2013, and preservation is compiledNumber be CCTCCNO:M2013526.

2. rose dark brown Isaria bacterial strain according to claim 1 (Isariafumosorosea), is characterized in that, described inRose dark brown Isaria bacterial strain SCAU-IFCF02 belong to hyphomycetales, Isaria and belong to.

3. rose dark brown Isaria bacterial strain according to claim 1 (Isariafumosorosea), is characterized in that, described inThe morphological feature of rose dark brown Isaria bacterial strain SCAU-IFCF02 be described below:

Bacterium colony front fine hair shape, just white, is light rose dark brown, concavo-convex fluffy bisque after product spore; Back side milk yellow, center is to edgeIn the circle of radius 15mm, there are many mound shape protuberances;

Mycelia is separated, transparent, smooth, wide 1.5～2.5 μ m;

Conidiophore list is raw or be gathered into coremium, 100 × 1.5～100 × 2.0 μ m, wall is smooth, transparent, mostly byThe colyliform branch composition of raw 4～6 bottles stalk composition verticil; Bottle metulae portion is spherical, or intends ellipse and expand, top elongated, 5.7～8.0×1.0～2.0μm；

Conidium is spherical to nearly fusiformis, smooth, is clear to micro-pale red, and 3.0～4.0 × 1.0～2.0 μ m, without chlamydospore.