Method of preparation of microsclerotia of <i>Beauveria bassiana </i>

Abstract

Disclosed is a bioinsecticide formulation comprising highly resistant Beauveria bassiana microsclerotia and to its use for insect control, in particular for control of insects in grain silos, and to a method of preparation of said formulation. The microsclerotia are obtained in a culture medium comprising an iron (II) compound at a concentration of approximately 0.2 g/L.

Claims

1. A method to prepare an isolated microsclerotium of Beauveria bassiana, comprising: incubating the microsclerotium at 26° C. for 4 days with an agitation rate of 250 rpm in a culture medium comprising 0.4 g KH.sub.2PO.sub.4, 1.4 g Na.sub.2HPO.sub.4, 0.6 g MgSO.sub.4.7H.sub.2O, 1.0 g KCl, 0.7 g NH.sub.4NO.sub.3.7H.sub.2O, 0.2 g FeSO.sub.4.7H.sub.2O, 10 g glucose, and 5 g yeast extract in 1 L of distilled water.

2. The method according to claim 1, further comprising: culturing Beauveria bassiana fungi to obtain a conidium, harvesting the conidium, preparing conidial suspensions, and isolating the microsclerotium.

3. The method according to claim 2, wherein the carbon:nitrogen ratio of said culture medium is equal to or greater than 12.5:1.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention will be described in further detail below.

(2) The term “approximately” as used herein when referring to a measurable value means that it comprises variations of ±10% from the specified amount.

(3) As used herein, the terms “comprises”, “has” and “includes” and their conjugations mean “including but not limited to”.

(4) Preparation of Beauveria bassiana Microsclerotia

(5) Beauveria bassiana was routinely cultured in potato dextrose agar plates at 26° C. for 14 days.

(6) Conidia were harvested, suspended in 0.01% (v/v) Tween 80 in sterile distilled water, vortexed for approximately 3 min, and filtered through a 75 mm sieve to remove debris. The conidial suspensions were adjusted with a Neubauer chamber to a final concentration of 1×10.sup.7 conidia mL.sup.−1, and were used to inoculate in 500-mL liquid media with different carbon:nitrogen ratios (C:N) amended with ampicillin (50 μg mL.sup.−1) in 1-L Erlenmeyer shaken flasks and then incubated at 26° C. for 4 days with orbital agitation at 250 rpm to obtain microsclerotia. The culture medium was composed of 0.4 g KH.sub.2PO.sub.4, 1.4 g Na.sub.2HPO.sub.4, 0.6 g MgSO.sub.4.7H.sub.2O, 1.0 g KCl, 0.7 g NH.sub.4NO.sub.3.7H.sub.2O, 0.2 g FeSO.sub.4.7H.sub.2O, 10 g glucose, and 5 g yeast extract in 1 L of distilled water. The addition of Fe.sup.2+ is crucial for the microsclerotial formation (Table 1). The fungus cultured in this medium with a lower agitation rate of 180 rpm and without iron supplementation produced only mycelium but not microsclerotia.

(7) Microsclerotia production was assayed also in media with different C:N ratios (Table 2). Carbon to nitrogen ratios were determined by considering 40% C in glucose, 45% C and 8% N in yeast extract, and 13.6% N in NH.sub.4NO.sub.3.7H.sub.2O. All cultures (500 mL of each medium supplemented with ampicillin in 1-L Erlenmeyer shaken flasks) were inoculated with 1 mL of 1×10.sup.7 conidia mL.sup.−1 and incubated during 4 days at 26° C. and 250 rpm.

(8) At the end of the fermentation process, fungal biomass from the culture broth was retrieved by centrifugation for 20 min at 7600×g and washed with sterile distiller water to remove media components.

(9) Fresh biomass samples containing microsclerotia were placed in desiccators with activated silica gel at 4° C. for 2-3 days until moisture content was reduced to 5% (w/w). Fungal cells harvested from 1 mL of culture broth was dried until reaching constant weight, and dried biomass production was further calculated as mg of dry mass mL.sup.−1 of culture broth. Additionally, 25 mL of each microsclerotia culture medium was similarly dried and 30 mg of that dried biomass were plated on nutrient-free water-agar medium (2% w/v) with ampicillin for 14 days at 26° C. Fungal growth was monitored daily and conidia produced by microsclerotia were harvested and suspended in sterile 0.01% Tween 80. Conidial production from the suspension was determined with a Neubauer chamber at 400× magnification with a phase contrast, and then expressed as total conidia per gram of dry biomass.

(10) To assess conidial viability from sporulated microsclerotia on water-agar plates, the suspension was also used to inoculate PDA plates and germination was recorded by microscopic examination at 400× after 24 h of incubation at 26° C. in total darkness. For microsclerotia concentration measurements, 1 mL of culture broth from each test liquid media was diluted to 1/10 and 100 μl were placed on glass slides (26 mm×76 mm) and covered with a coverslip (20 mm×20 mm) and then observed on the microscope to count microsclerotia. Only discrete pigmented aggregates larger than 100 μm were counted as microsclerotia.

(11) TABLE-US-00001 TABLE 1 Comparison of culture media with different carbon:nitrogen ratio (C:N) and the addition of Fe.sup.2+ to the media in Beauveria bassiana microsclerotia production (+). Microsclerotia producing media C:N Without Fe.sup.2+ With Fe.sup.2+  5:1 − − 12.5:1.sup.  − + 30:1 − + 50:1 − +

(12) TABLE-US-00002 TABLE 2 Glucose concentration modified from Complete Minimal (CM) medium to obtain different C:N ratios to study microsclerotia production in liquid cultures of Beauveria bassiana. Media Glucose (g L.sup.−1) Total carbon (g L.sup.−1) C:N 1 0.625 10  5:1 2 10 25 12.5:1.sup.  3 31.9 60 30:1 4 56.9 100 50:1

(13) The conidial suspensions used to obtain the microsclerotia were incubated for periods longer than 4 days and up to 10 days, without significant changes in the assessed microsclerotia production. The method of the present invention is thus more efficient, in that incubation times are reduced to 4 days with respect to the methods of the prior art.

(14) Preparation of Bioinsecticide Formulation

(15) A granular formulation was prepared mixing 100 mL of culture broth containing microsclerotia with 5 g of sterile diatomaceous earth and dried in a desiccator with activated silica or vacuum chamber. Then, granules were disrupted into a powder with mortar and pestle to assay their insecticidal activity.

(16) Insecticidal activity was assessed against Tribolium castaneum adult insects and larvae and Tenebrio molitor adult insects.

(17) The obtained bioinsecticide formulation can be successfully used to control insects, in particular wheat weevils. The formulation can be used in silos of stored grains for storing wheat and what flour.

(18) The bioinsecticide formulations of the present invention based on Beauveria bassiana microsclerotia have a controlled released with respect to conidia-based formulations of the prior art.