Preparation method of <i>Beauveria bassiana </i>microsclerotium and formulation thereof, application of formulation thereof

Abstract

The present invention provides a preparation method of Beauveria bassiana (Bals.) Vuill microsclerotium and a formulation thereof, and an application of the formulation thereof, belonging to the technical field of microsclerotium formulation preparation; the Beauveria bassiana microsclerotium formulation is prepared by the following steps of: 1) mixing conidia of Beauveria bassiana with Tween® 20 aqueous solution to obtain spore suspension; 2) inoculating the prepared spore suspension into a liquid induction medium for induction culture to obtain microsclerotium; 3) mixing the prepared microsclerotium with a padding for drying to obtain dried microsclerotium; 4) mixing the prepared dried microsclerotium with an auxiliary to obtain a microsclerotium formulation. The Beauveria bassiana microsclerotium formulation produced by the method has low production cost, strong product tolerance and good insecticidal activity, moreover, after rehydrated, the formulation may rapidly germinate hyphae and produce spores to infect pests.

Claims

1. A method for preparation of Beauveria bassiana microsclerotium, comprising the following steps of: 1) mixing conidia of Beauveria bassiana with an aqueous solution comprising polyoxyethylene (20) sorbitan monolaurate to obtain a spore suspension; and 2) inoculating the spore suspension obtained in step 1) into a liquid induction medium for induction culture to obtain the microsclerotium; wherein, the volume percent of polyoxyethylene (20) sorbitan monolaurate in the aqueous solution in step 1) is 0.03%-0.1%; with water as solvent, per liter of the liquid induction medium in step 2) having a pH of 6.0-7.5 comprises the following ingredients: 2-6 g KH.sub.2PO.sub.4, 0.3-0.9 g MgS0.sub.4.7H.sub.20, 0.4-1.2 g NaNO3, 0.2-0.6 g CuSO.sub.4, 1.5-4.5 g yeast powder, 1-3 g peptone; 10-30 g sucrose, and 2.5 mL polyoxyethylene (20) sorbitan monolaurate; the Beauveria bassiana strain is CQBb119 and the accession number is CGMCC NO: 15987; the spore content of the spore suspension in step 1) is 2.8×10.sup.6-3.2×10.sup.6 spores/mL; the volume ratio of the inoculated spore suspension in step 2) to the liquid induction medium is 1:10 to 1:20; and the induction culture temperature in step 2) is 23-31° C.; the induction culture time period is 5-6 days; and an oscillation or stirring frequency of the induction culture is 50-250 rpm.

2. A method for preparing a formulation comprising Beauveria bassiana microsclerotium, comprising the following steps of: A) mixing the microsclerotium prepared by the method as set forth in claim 1 with a padding for drying to obtain dried microsclerotium; and B) mixing the dried microsclerotium obtained in step A) with an auxiliary to obtain the microsclerotium formulation.

3. The method according to claim 2, wherein a dosage form of the microsclerotium formulation is granule.

4. The method according to claim 2, wherein the padding in step A) is one or more selected from the group consisting of diatomite, starch and cyclodextrin; and the volume ratio of the microsclerotium to the padding is 1:2 to 1:4.

5. The method according to claim 2, wherein the auxiliary in step B) is a sucrose ester; and the mass ratio of the dried microsclerotium to the sucrose ester is 1000:1-200:1.

Description

DETAILED DESCRIPTION

(1) The present invention provides a preparation method of a Beauveria bassiana microsclerotium, and the strain is CQBb119 and the accession number: CGMCC NO:15987.

(2) In the present invention, the Beauveria bassiana strain is inoculated into a PDA solid medium for culture and collected to obtain conidia; the diameter of the PDA plate is preferably 90 mm; the culture time of the strain is preferably 10-12 d; the culture temperature of the strain is preferably 23-25° C.; the collection method is to scrape conidia on the surface of the plate after hyphae grow all over the plate and produce spores.

(3) In the present invention, the collected conidia are mixed with Tweenx 20 aqueous solution to obtain a spore suspension; content of Tween® 20 in the Tween® 20 aqueous solution is 0.03%-0.1%, preferably 0.05%; water in the Tween® 20 aqueous solution is sterile water; the final concentration of the spore suspension is 2.8×10.sup.6-3.2×10.sup.6 spores/mL, preferably 3.0×10.sup.6 spores/mL, and the mixing is implemented in aseptic conditions.

(4) In the present invention, the obtained spore suspension is inoculated into a liquid induction medium for induction culture to obtain microsclerotium; the liquid induction medium includes the following components: counted by 1 L, 2-6 g KH.sub.2PO.sub.4, 0.3-0.9 g MgSO.sub.4.7H.sub.2O, 0.4-1.2 g NaNO.sub.3, 0.2-0.6 g CuSO.sub.4; 1.5-4.5 g yeast powder, 1-3 g peptone, 10-30 g sucrose, 0.25-0.75 mL Tween® 20 and 1000 mL water, pH=6.0-7.5; preferably, counted by 1 L, 3-5 g KH.sub.2PO.sub.4, 0.5-0.7 g MgSO.sub.4.7H.sub.2O, 0.8-1.0 g NaNO.sub.3, 0.3-0.5 g CuSO.sub.4; 2.5-3.5 g yeast powder, 1.5-2.5 g peptone, 15-25 g sucrose, 0.35-0.65 mL Tween® 20 and 1000 mL water, pH=6.5-7.2; more preferably, counted by 1 L, 4 g KH.sub.2PO.sub.4, 0.6 g MgSO.sub.4.7H.sub.2O, 0.9 g NaNO.sub.3, 0.4 g CuSO.sub.4; 3 g yeast powder, 2 g peptone, 20 g sucrose, 0.5 mL Tween® 20 and 1000 mL water, pH=7.0. In the present invention, the volume ratio of the inoculated spore suspension to the liquid induction medium is 1: (10-20), preferably 1:(12-18), more preferably 1: 15; the final content of the inoculated spores is preferably ≥1.25×105 spores/L. In the present invention, the temperature of the induction culture is 23-31° C., preferably 25-29° C., more preferably 28° C.; the time of the induction culture is 5-6 d, preferably 5.5 d; the oscillation frequency of the induction culture is 50-250 rpm, in embodiments of the present invention, preferably, the oscillation frequency after inoculated within 24 h is preferably 50-100 rpm, more preferably 100 rpm; the oscillation frequency after inoculated within 24 h is preferably 110-200 rpm, more preferably 180 rpm.

(5) The present invention provides a preparation method of the Beauveria bassiana microsclerotium formulation, and the Beauveria bassiana microsclerotium is prepared by the above solution.

(6) In the present invention, the microsclerotium prepared by the above solution is mixed with a padding for drying to obtain a dried microsclerotium; in the present invention, the padding is one or more of diatomite, starch and cyclodextrin; the mass ratio of the microsclerotium to the padding is 1: (2-4), preferably 1:3; the drying mode is preferably hot air drying; the drying temperature is preferably 30-40° C., more preferably 32° C.; the drying time is preferably 24-48 h, more preferably 36 h. In the present invention, water content of the dried microsclerotium is preferably ≤8%.

(7) In the present invention, the prepared dried microsclerotium is mixed with an auxiliary to obtain the microsclerotium formulation; the auxiliary is sucrose ester; the mass ratio of the dried microsclerotium to sucrose ester is 1000:1-200:1, preferably 800:1-400:1, more preferably 600:1; the mixing mode is preferably stirring for mixing; the rotation speed of the mixing is 15-25 rpm, preferably 20 rpm; the stirring time is preferably 20-40 min, more preferably 30 min. In the present invention, the dosage form of the Beauveria bassiana microsclerotium formulation is granule.

(8) The present invention provides an application of the Beauveria bassiana microsclerotium formulation prepared by the above solution in various peasts control. Beauveria bassiana is a kind of broad-spectrum pathogenic fungus and has good insecticidal activity to various kinds of Lepidoptera, Coleoptera and Hemiptera peasts, and it is widely applied in the biological control of various domestic and overseas pests, such as agrotis ypsilon and pine moth. In the present invention, the controlled pests are preferably grub, flatheaded borer, cutworm and Diptera larvae.

(9) In the present invention, the application process of the Beauveria bassiana microsclerotium formulation is soil broadcasting, seed dressing or root irrigation mixed with water, preferably root irrigation mixed with water, the mixing ratio of the Beauveria bassiana microsclerotium formulation to water is 1:(200-1000), more preferably 1:500. In detailed implementation process of the present invention, the mixing ratio of the Beauveria bassiana microsclerotium formulation to water is determined according to the occurrence quantity and age of pests.

(10) A preparation method of a Beauveria bassiana microsclerotium and formulation thereof, an application of the formulation thereof provided by the present invention will be described in detail with reference to embodiments below, but these embodiments should be construed as limiting the protection scope of the present invention.

Embodiment 1 a Preparation Method of Beauveria bassiana Microsclerotium

(11) 1) Beauveria bassiana Vuill strain with the accession number of CGMCC NO 15987 was inoculated into a PDA medium plate with 90 mm diameter for culture for 12 d at 23° C., conidia on the surface of the plate were scrapped after hyphae grow all over the plate and produce spores as initial inoculants.

(12) 2) The collected initial inoculants were added to 0.03% Tween® 20 sterile water (volume percent) for sterile operation and even dispersion to be prepared into a spore suspension with 3.0×10.sup.6 spores/mL spore content for further use.

(13) 3) The prepared spore suspension was inoculated into a liquid induction medium by 1:20 volume ratio so that the final concentration of spores was up to 1.5×10.sup.5 spores/mL, then put into a constant temperature shaker for culture. It was cultured for 6 d at 23° C., moreover, the rotation speed of the shaker after inoculation within 24 h was 50 rpm, and after 24 h, the rotation speed was regulated to 150 rpm, finally, the Beauveria bassiana microsclerotium was obtained by the above culture.

(14) The liquid induction medium includes the following ingredients: counted by 1 L, basal salt: 2 g KH.sub.2PO.sub.4, 0.3 g MgSO.sub.4.7H.sub.2O, 0.4 g NaNO.sub.3, trace elements: 0.2 g CuSO.sub.4, nitrogen source: 1.5 g yeast powder, 1 g peptone; carbon source: 10 g sucrose, 0.25 mL Tween® 20 and 1000 mL water, pH=6.0.

Embodiment 2 a Preparation Method of Beauveria bassiana Microsclerotium

(15) 1)Beauveria bassiana strain with the accession number of CGMCC NO 15987 was inoculated into a PDA solid medium plate with 90 mm diameter for culture for 10 d at 27° C., conidia on the surface of the plate were scrapped after hyphae grow all over the plate and produce spores as initial inoculants.

(16) 2) The collected initial inoculants were added to 0.1% Tween® 20 sterile water (volume percent) for sterile operation and even dispersion to be prepared into a spore suspension with 3.0×10.sup.6 spores/mL spore content for further use.

(17) 3) The prepared spore suspension was inoculated into a liquid induction medium by 1:20 volume ratio so that the final concentration of spores was up to 1.5×10.sup.5 spores/mL, then put into a constant temperature shaker for culture. It was cultured for 5.5 d at 31° C., moreover, the rotation speed of the shaker after inoculation within 24 h was 100 rpm, and after 24 h, the rotation speed was regulated to 200 rpm, finally, the Beauveria bassiana Vuill microsclerotium was obtained by the above culture.

(18) The liquid induction medium includes the following ingredients: counted by 1 L, basal salt: 6 g KH.sub.2PO.sub.4, 0.9 g MgSO.sub.4.7H.sub.2O, 1.2 g NaNO.sub.3, trace elements: 0.6 g CuSO.sub.4, nitrogen source: 4.5 g yeast powder, 3 g peptone; carbon source: 30 g sucrose, 0.75 mL Tween® 20 and 1000 mL water, pH=7.0.

Embodiment 3 a Preparation Method of Beauveria bassiana Microsclerotium

(19) 1)Beauveria bassiana strain with the accession number of CGMCC NO 15987 was inoculated into a PDA solid medium plate with 90 mm diameter for culture for 11 d at 25° C., conidia on the surface of the plate were scrapped after hyphae grow all over the plate and produce spores as initial inoculants.

(20) 2) The collected initial inoculants were added to 0.05% Tween® 20 sterile water (volume percent) for sterile operation and even dispersion to be prepared into a spore suspension with 3.0×10.sup.6 spores/mL spore content for further use.

(21) 3) The prepared spore suspension was inoculated into a liquid induction medium by 1:15 volume ratio so that the final concentration of spores was up to 2.0×10.sup.5 spores/mL, then put into a constant temperature shaker for culture. It was cultured for 5.5 d at 28° C., moreover, the rotation speed of the shaker after inoculation within 24 h was 50 rpm, and after 24 h, the rotation speed was regulated to 140 rpm, finally, the Beauveria bassiana microsclerotium was obtained by the above culture.

(22) The liquid induction medium includes the following ingredients: counted by 1 L, basal salt: 6 g KH.sub.2PO.sub.4, 0.9 g MgSO.sub.4.7H.sub.2O, 1.2 g NaNO.sub.3, trace elements: 0.6 g CuSO.sub.4, nitrogen source: 4.5 g yeast powder, 3 g peptone; carbon source: 30 g sucrose, 0.75 mL Tween® 20 and 1000 mL water, pH=7.2.

Embodiment 4 a Preparation Method of Beauveria bassiana Microsclerotium Formulation

(23) 1) The fermentation liquor of the Beauveria bassiana microsclerotium in Embodiment 3 was put into a 50 L plastic bucket, added corn starch which was 3 times of Beauveria bassiana microsclerotium by mass ratio for stirring evenly, added to a fluidized bed, then dried by hot air at 32° C. to water content≤8% to prepare dried microsclerotium.

(24) The dried microsclerotium was added to sucrose ester according to 1000: 1 for stirring for 30 min to be prepared into microsclerotium granule.

Embodiment 5 a Preparation Method of Beauveria bassiana Microsclerotium Formulation

(25) 1) The fermentation liquor of the Beauveria bassiana microsclerotium in Embodiment 3 was put into a 50 L plastic bucket, added diatomite which was 4 times of Beauveria bassiana microsclerotium by mass ratio for stirring evenly, added to a fluidized bed, then dried by hot air at 35° C. to water content≤8% to prepare dried microsclerotium.

(26) 2) The dried microsclerotium was added to sucrose ester according to 800: 1 for stirring for 30 min to be prepared into microsclerotium granule.

Embodiment 6 Measurement on the Storage Tolerance of Beauveria bassiana Microsclerotium Formulation Obtained by Embodiment 5

(27) Microsclerotium formulation obtained by Embodiment 5 was stored at room temperature (≤30° C.) in drying conditions, 0.05 g formulation were inoculated on a water agar plate every month to detect the germination rate of microsclerotium; it can be seen from the results that the rehydrated germination rate of the fungicide after drying is ≥99%, the rehydrated germination rate of the microsclerotium after stored for 12 months at room temperature is ≥95%.

(28) TABLE-US-00001 TABLE 1 Germination rate of the Beauveria bassiana microsclerotium formulation after stored Storage period Germination rate 0 month 1 months 3 months 5 months 7 months 9 months 12 months Repetition 1 99% 99% 98% 98% 97% 96% 96% Repetition 2 99% 99% 98% 98% 97% 96% 95% Repetition 3 99% 99% 98% 97% 97% 96% 95%

Embodiment 7 Test on the High Temperature Resistance of Beauveria bassiana Microsclerotium Formulation Obtained by Embodiment 5

(29) 0.02 g dried Beauveria bassiana microsclerotium obtained by Embodiment 5 were placed into a plastic centrifuge tube, and respectively processed for 0 min, 10 min, 20 min, 30 min at 35° C., 40° C., 45° C., 50° C., then taken out and respectively inoculated into a water agar plate medium for culture for 24 h at 25° C., thus measuring the germination rate of microsclerotium by an optical microscope. Meanwhile, fresh conidium spore suspension of 1×10.sup.7 spores/mL was processed the same as the above as a control, all steps were repeated for 3 times.

(30) It can be seen from Table 2 that for the treatment at 40° C. above, the germination rate of Beauveria bassiana microsclerotium is significantly higher than that of conidia under conditions of same temperature and processing time.

(31) TABLE-US-00002 TABLE 2 Contrast of the germination rate of microsclerotium and conidium after heat treatment Temperature Germination rate (%) (° C.) 0 min 10 min 20 min 30 min 35 Microsclerotium 92.2 a 91.6 a 91.2 a 91.3 a Conidium 93.4 a 92.6 a 91.4 a 91.1 a 40 Microsclerotium 92.6 a 92.0 a 90.3 ab 88.2 b Conidium 93.3 a 88.6 b 82.4 c 79.1 c 45 Microsclerotium 92.3 a 87.8 b 70.4 d 54.6 d Conidium 92.8 a 79.2 c 55.4 e 33.8 e 50 Microsclerotium 92.4 a 78.6 c 57.5 e 23.5 f.sup.  Conidium 93.1 a 32.5 d 13.5 f  4.8 g

(32) Data in the table are average values after 3 repetitions, and different letters behind of the data in each list show significant difference (P<0.05).

Embodiment 8 Test on the UV Tolerance of Dried Beauveria bassiana Microsclerotium Obtained by Embodiment 5

(33) 0.02 g dried Beauveria bassiana microsclerotium obtained by Embodiment 5 were evenly smeared on a water agar plate with 9 cm diameter, and put into a 60 cm square of irradiation chamber and irradiated by a 40 W UV lamp (UV-B wavelength: 308), and the UV radiation quantity was measured by an ultraviolet spectrophotometer. It was irradiated for 0, 1, 2, 3, 4 h respectively, and the UV radiation quantity of each treatment was 0, 4.86, 9.72, 14.58 and 19.44 kJ m.sup.−2. Microsclerotium was taken out of the irradiation chamber, put into a 25° C. incubator for cultivation for 24 h, then examined by an optical microscope to measure the germination rate of microsclerotium. Meanwhile, fresh conidium spore suspension of 1×10.sup.7 Beauveria bassiana was processed the same as the above as a control, all steps were repeated for 3 times. The results are shown in the table below, it can be seen from the table that the germination rate of Beauveria bassiana microsclerotium is significantly higher than that of conidia under same dosage of UV irradiation.

(34) TABLE-US-00003 TABLE 3 Test results of the microsclerotium UV tolerance Microsclerotium Conidium germination UV-B exposure germination rate (%) rate (%) 0 93.5 a  92.9 a  4.86 88.97 b 75.27 c 9.72 78.83 c 62.23 d 14.58 46.23 e 31.07 f.sup.  19.44 19.47 g 12.24 h

(35) Data in the table are average values after 3 repetitions, and different letters behind of the data in each list show significant difference (P<0.05).

Embodiment 9 Test on the Control of Delia antiqua by the Beauveria bassiana Microsclerotium Formulation Obtained in Embodiment 5

(36) Adult Delia antiqua was fed by sterile water, white granulated sugar and yeast powder in a 30×30×30 cm sarong, and then put into an automatically-controlled light and temperature incubator (23±1.0° C., LD16:8, 50-70% relative humidity). A 8 cm×5 cm-diameter plastic dish containing wet pebbles and pieces of onion was put to the sarong meanwhile for the nourishment supply and oviposition of the adult Delia antiqua. The ovi were incubated into larvae in the incubator under same conditions, and fed by sliced onion bulbs for further use.

(37) 3 small onion bulbs purchased from the market were respectively planted in each 40 cm-diameter plastic flowerpot, and inoculated with 20 3-day-old Delia antiqua larvae after the leaves of the onion grew to 10 cm in height. Then the inoculated flowerpots were put into an illumination incubator for routine culture (23±1.0° C., LD16:8, 50-70% relative humidity), the microsclerotium formulation obtained from step 5 was evenly mixed with water as inoculants according to a ratio of 1:20, and roots were irrigated by 100 mL inoculants per pot; and in a control group, roots were irrigated by fresh water. Survival number of Delia antiqua larvae in soil was respectively examined on the 5th and 10th day, and corrected mortality was calculated based upon a formula: corrected mortality (%)=(treatment mortality-control mortality)/(1-control mortality)×100%. All treatment was repeated for 10 times. Experiment results indicate that the corrected mortality of larvae 5 days later is 39.13% and it is up to 81.99% 10 days later; the dried larvae were put into a moist culture dish, 15 days later, it was be found that the Delia antiqua larvae were ossified, white hyphae and conidia grew on the surface of the majority of the polypides, indicating that Delia antiqua was infected to death by Beauveria bassiana. In the control group, there was a few of dead larvae, and no ossified larvae.

(38) TABLE-US-00004 TABLE 4 Pot experiment on the control of Delia antiqua by Beauveria bassiana microsclerotium Number of the survival larvae 0 d 5 d 10 d 15 d Treatment 200 98 29 13 Control 200 178 161 150 Corrected mortality (%) — 35.75 81.99 91.4

(39) It can be seen from the above embodiments that the present invention provides a Beauveria bassiana microsclerotium formulation and a preparation method of a formulation thereof; the Beauveria bassiana microsclerotium formulation prepared by the method has long shelf life at room temperature, high rehydration germination rate and has good effect in pest control.

(40) The foregoing descriptions are only preferred implementation manners of the present invention. It should be noted that for a person of ordinary skill in the art, several improvements and modifications may further be made without departing from the principle of the present invention. These improvements and modifications should also be deemed as falling within the protection scope of the present invention.