Method to produce a biological control product and product for the control of locusts and a method for the control of locusts

Abstract

The invention provides a method for the preparation of an entomopathogenic product for the control of Schistocerca gregaria, which method includes the steps of spreading watery suspension having a concentration of between 2x1010 - 13 conidia / ml of a selected strain of Metarhizium conidia on an agar substrate and incubating at 25°C ± 1 °C for 7 to 12 days to germinate and grow conidia, followed by harvesting the resulting conidia.

Description

[0001] Method to Produce a Biological Control Product and Product for the Control of Locusts and a Method for the Control of Locusts.

[0002] Technical field of the invention

[0003] This invention relates to a method to produce an entomopathogenic product for the control of locusts and a method for the control of locusts and the use of a fungus preparation to control locusts, in particular the Desert Locust Schistocerca gregaria.

[0004] Background to the invention

[0005] The outbreak of the Desert Locust when it occurs in plague numbers, can infest vast areas. Such outbreak or upsurges are characterized by the presence of many hopper bands and swarms that cause considerable economic losses and lead to piercing reduction in subsistence crops and grazing pasture. The Desert Locust is a catastrophic pest for about 50 countries in Africa, the Middle East and Southwest Asia.

[0006] The Desert Locust is a significant threat to the Saharan, sub-Saharan, Arabic economy, agriculture and food security. When the conditions are right, usually after a period of drought, the Desert Locust can change from the harmless solitary type to the swarming and destructive gregarious type which flies with the wind up to 200km a day. Up to 60 countries can be effected within an area of about 32 million km2. The Desert Locust Information Service (DLIS) of the Food and Agricultural Organisation of the United Nation (FAO) monitors and predict swarming activity. Insecticides is used to try and control such swarms, which required precise application by vehicle or air and which can be harmful to the environment other species and is normally only used in emergency situations. The environmental concerns led to the development of biological control methods based on entomopathogens. There are different methods and technique for spraying the chemical insecticides during the campaign operations against Desert Locust, these may include aerial application, vehicle mounted sprayer, handheld or motorized knapsack sprayers. The genera of entomopathogenic fungi Metarhizium, have been shown to be a potentially effective (mortality of less than 70%) biological control agent for locust and grasshoppers.

[0007] It is an object of the invention to provide an improved method to produce an entomopathogenic product for the control of locusts and a method for the control of locusts and the use of an improved fungus preparation to control locusts, in particular the Desert Locust Schistocerca gregaria with a mortality rate of more than 70%, up to 85% mortality.

[0008] General description of the invention

[0009] According to one aspect of the invention there is provided a method for the preparation of an entomopathogenic product for the control of Schistocerca gregaria, which method includes the steps of: spreading watery suspension having a concentration of 2x1010, 2x1011, 2x1012, 2x1013conidia / ml and a preferred concentration 2X1012conidia / ml of a selected strain of Metarhizium conidia on an agar substrate and incubating at 25°C ± 1 °C for 7 to 12 days, preferably 10 days to germinate and grow conidia; harvesting the resulting conidia. The conidia are harvested by adding tween 80 (0.5%) in distilled water onto the culture plates. A metallic spatula is used to scrape the conidia onto polysorbate 80, polyoxyethylene sorbitan monooleate (tween 80) suspension. The suspension is then sieved through a methylene cloth to remove hyphen fragments and then vortexed stirred in a test tube and shaken for six minutes to avoid clumping of spores; preparing inoculative suspensions by mixing the harvested conidia with water and a non-ionic emulsifier selected from polysorbate 80, polyoxyethylene Sorbitan monooleate at Different concentrations e.g. 2.5x101°, 2.5X1011, 2.5x1012, and 2.5x1013were tested, the most effective is 2.5x1012; and wherein the selected strain of Metarhizium is selected from Metarhizium spp (DLCO 91) and Metarhizium spp (DLCO 26)]. It is to be appreciated that all the tested isolates, were given code number e.g. DLCO. The most effective and virulent strain of Metarhizium which were selected are DLCO 91 and DLCO 26.

[0010] The active ingredient (Conidia / spores of the fungus) is produced in solid or powder form. The product is then formulated in oil and this is compatible with existing ULV spraying for locust control. The product is applied in the field using known ULV spray equipment and techniques for spraying ULV chemical pesticides.

[0011] The product may be suspended in oil for spray application and the oils may be selected from diesel oil, kerosene +vegetable oil and mineral oil oils (Ondina shell Sol-T).

[0012] Detailed description of the invention

[0013] DLCO-EA Metarhizium Isolates (Green invention):

[0014] About ninety -one local strain of entomopathogenic fungi were isolated in More than hundred samples were identified as Beauveria and Metarhizium spp. Twelve of the Metarhizium spp., were isolated from hosts or substrates other than grasshoppers. Seven were isolated from the Scarabid beetle, Pachnoda interrupta, a sorghum chafer, and five from soil samples. The isolates were screened against laboratory reared 3rd and 4th hopper instars of Desert Locust, Schistocerca gregaria and Migratory Locust; Locusta migratoria migratorioides.

[0015] Laboratory Bioassay:

[0016] Materials and Methods:

[0017] Some the local strain of Metarhizium were selected based on pathogenicity studies. The Metarhizium strains IMI 330189 was used as Standard. Preparation of the inoculum:

[0018] A 10 ml suspension of 2x1010, 2x1011and 2x1012and 1 O10, 1011and 1012conidia / ml of each strain was spread plated on Sabouraud Dextrose agar plates and placed in the incubator at 25°C ± 1 °C for 10 days after which conidia were harvested. Inoculative suspensions were prepared by mixing conidia with distilled water and tween 80. Tween 80 (polysorbate 80, polyoxyethylene sorbitan monooleate) is a non-ionic surfactant that is widely used as an emulsifier). Appropriate concentrations were estimated using a Neubauer haemocytometer and serial dilution. Standard concentration of 1.2x107conidia / ml were used for all the treatments. A control treatment of distilled water and tween 80 was used.

[0019] Germination assessment:

[0020] A malt agar media was prepared to assess the percentage of germination of conidia of each strain including the strain IMI 330189, to assess the viability before the bioassay. A suspension of 106conidia / ml was prepared and 0.1 ml was spread plated on malt-agar and placed in the incubator for 16 h. 4 cover slips on each plate and the germination percentages were estimated.

[0021] Inoculation and bioassay:

[0022] A topical inoculation was applied on each insect. A quantity of 2pL was applied on the insect pronotum using micropipette. Each treatment was replicated 3 times and each replicate having 5 insects. Maize leave were provided as food source. Insects were kept in room 12:12 D:L conditions at 25°C ± 1 °C. Bioassay boxes cleaned daily and mortality was monitored for 3 weeks. Dead insects were removed and kept in pert dishes with wet blotter paper and observed for mycosis.

[0023] Laboratory bioassay Results:

[0024] At standard concentration, the conidia germination percentage was over 90% (Table. All the Metarhizium treatments were pathogenic to Desert Locust and Locusta migratoria. There was no difference in term of mortality among the treatments and control in the first three days. In general, after 3 weeks, the DLCO-EA Isolates (Green invention) showed remarkably higher performances comparative to the standard (strain IMI 330189) which gave 70% mortality whereas three of DLCO-EA isolates achieved mortality ranging from 70 - 85%, on Desert Locust and Migratory locust (Figure-1 ). DLCO-26 & DLCO-91 isolates have showed better performance compared to the standard strain IMI 330189. Moreover, the advantage of those isolates are indigenous, this feature may help harmonize the registration within the member countries of DLCO-EA.

[0025] Figure 1 shows the percent accumulated mortality of DLCO 26 & 91 in comparison with GM.

[0026] Advantages of the invention:

[0027] The chemical pesticides used widely in the world to control the locusts are being strongly opposed and discouraged by scientists, public health experts, governments’ officials. The main reason is because those chemical pesticides give rise social costs: high toxicity on the food-chains, water and land ecosystems and environmental health. This situation has created a huge market demand for alternative pesticides or safe and sustainable benign agents for the management of locusts.

[0028] What the invention consists of and how it works:

[0029] ■ The invention consists of conidia of the fungus Metarhizium spp.

[0030] ■ Spores get attached to the cuticle of the insect and start to germinate.

[0031] ■ The germinated hypha penetrates the cuticle by pressure and enzymatic action to reach the body cavity.

[0032] ■ There it breaks in short pieces and multiply by living on the liquid contents of the body cavity.

[0033] ■ At some point, the fungus spread in the locust body and invade the tissues. This process takes a number of days (3-5) after which the insect becomes starved of nutrients and dies.

[0034] ■ If the ambient humidity is high enough, the fungus breaks through the cuticle and form new spores.

[0035] It shall be understood that the examples are provided for illustrating the invention further and to assist a person skilled in the art with understanding the invention and are not meant to be construed as unduly limiting the reasonable scope of the invention.

Claims

AMENDED CLAIMS received by the International Bureau on 19 December 20241 . A method for the preparation of an entomopathogenic product for the control of Schistocerca gregaria, which method includes the steps of: spreading watery suspension having a concentration of between 2x1010'13conidia / ml of a selected strain of Metarhizium conidia on an agar substrate and incubating at 25°C ± 1 °C for 7 to 12 days to germinate and grow conidia; wherein the selected strain of Metarhizium is selected from Metarhizium spp (DLCO 91) and Metarhizium spp (DLCO 26)] and harvesting the resulting conidia.

2. A method as claimed in claim 1 , wherein the conidia are harvested by adding tween 80 (05%) in distilled water onto the culture plates.

3. A method as claimed in claim 2, wherein a metallic spatula is used to scrape the conidia onto polysorbate 80, polyoxyethylene sorbitan monooleate (tween 80) suspension.

4. A method as claimed in claim 3, including the step of sieving the suspension through a methylene cloth to remove hyphen fragments and then vortexed stirring in a test tube and shaken for six minutes to avoid clumping of spores.

5. A method as claimed in claim 4, including preparing inoculative suspensions by mixing the harvested conidia with water and a non-ionic emulsifier selected from polysorbate 80, polyoxyethylene sorbitan monooleate at a selected concentration between 2.5x101° and 2.5x1013.

6. A method as claimed in claim 1 , wherein the active ingredient (Conidia / spores of the fungus) is in solid or powder form and then formulated in oil and this is compatible with existing ULV spraying for locust control.AMENDED SHEET (ARTICLE 19)7. A method as claimed in claim 5 wherein the oil is selected from diesel oil, kerosene +vegetable oil and mineral oil oils (Ondina shell Sol-T)8. A entomopathogenic product for the control of Schistocerca gregaria prepared by the method as claimed in any one of claims 1 to 7.

9. Use of the product as claimed in claim 8 to control locust pests.AMENDED SHEET (ARTICLE 19)

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