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WO2004052103A1 - The process of preparing a biopesticide formulation for use against coffee berry borer (cbb) - Google Patents

The process of preparing a biopesticide formulation for use against coffee berry borer (cbb) Download PDF

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Publication number
WO2004052103A1
WO2004052103A1 PCT/IN2003/000355 IN0300355W WO2004052103A1 WO 2004052103 A1 WO2004052103 A1 WO 2004052103A1 IN 0300355 W IN0300355 W IN 0300355W WO 2004052103 A1 WO2004052103 A1 WO 2004052103A1
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WO
WIPO (PCT)
Prior art keywords
mycelial
lit
preparing
bassiana
fungus
Prior art date
Application number
PCT/IN2003/000355
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French (fr)
Inventor
S. R. Niranjana
Shekar H. Shetty
Original Assignee
Department Of Biotechnology
University Of Mysore
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Filing date
Publication date
Application filed by Department Of Biotechnology, University Of Mysore filed Critical Department Of Biotechnology
Priority to AU2003276691A priority Critical patent/AU2003276691A1/en
Publication of WO2004052103A1 publication Critical patent/WO2004052103A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom

Definitions

  • This invention relates to the process of preparing a biopestieide formulation for use against coffee berry borer (CBB).
  • This method of control is not only uneconomical but also hazardous to the environment destroying most of the non-target beneficial insect populations in the plantations like Honey bees, Ladybird beetle etc., and other beneficial microorganisms.
  • Coffee berry borer develops resistance to this chemical very fast in 2 to 3 generations. Once the CBB develops resistance, only the non-target insects are killed and CBB thrives more profusely. Endosulfan residue that remains in coffee beans has deleterious effects on humans.
  • Be uveri has been reported to cause infective mortality in many terrestrial and aquatic insect pests (Bidochka and Khachatourians, 1991) and also has been successfully used against European corn borer, migratory grass hopper, green house whitefly, pine caterpillar codling moth, mosquitoes and lygus bug (Bidochkha and Khachatourians, 1991 and Khachatourians, 1992). Larval and adult stages of several insects are hosts to many Deuteromycetouos fungal species. These fungi cause mycosis to both Coleopterans and Lepidopterans. Among these pathogens, Beauveria, Metarhisium and Paecilomyces spp.
  • B. bassiana has been successfully used as foliar applications by several researchers for suppressing populations of several insects, like Bemisia tabaci (Gennadius) (Wright, 1992; Carruthers et at, 1993), Ostrinia nubilalis (Hubner) (Lewis et al., 1996), and Leptinotarsa decemlineata (Say) (Poprawski et at, 1997).
  • B. bassiana has been shown as an important entomopathogen in controlling diamondback moth, Plutella xylostella (L.) by Vandenberg et al. (1998).
  • -5. bassiana has been successfully used for control of P. xylostella with crucifer transplants by Shelton et al. (1998).
  • the effect of ento ogenous fungus B. bassiana was tested on insect predators like Coccinella sp. t C.
  • Doane (1959) reported epizootic among larvae of the small elm bark beetle, Scolytus multistriatus in the United States, caused by -5. bassiana. The report suggested the possibility of employing entomogenous fungi for the biological control of the elm bark beetles, S. scolytus and S. multistriatus, the vectors of Dutch elm disease in Germany.
  • bassiana has been successfully used as foliar applications by several researchers for suppressing populations of several insects, like Bemisia tabaci (Gennadius) (Wright, 1992; Carruthers et at, 1993), Ostrinia nubilalis (Hubner) (Lewis et al., 1996), and Leptinotarsa decemlineata (Say) (Poprawski et al., 1997).
  • Bemisia tabaci Genenadius
  • Hubner Ostrinia nubilalis
  • Say Leptinotarsa decemlineata
  • bassiana production has recently been developed by Mycotech Corporation, USA (Bradley et al., 1992).
  • a corn starch and corn starch oil formulations of B. bassiana mycelia as well as pure dry mycelial preparations was obtained by Feng et al. (1994).
  • Bidochka et al. (1997) have found out that hydrophobic interactions, appressoria formation, and mucus production by the fungus are involved in adhesion to the cuticle.
  • the technology of protoplast fusion is a valuable method for intra- and inter specific hybridization. Through this a somatic hybrid of a cross between two strains of B. bassiana has been obtained (Viaud, 1998).
  • An object of this invention is to propose a process for preparing a biopesticide formulation against coffee berry borer.
  • object of this invention is to propose a formulation containing entomopathogenic fungus which is eco-friendly.
  • Yet another object of this invention is to prepare a formulation which is economical and easy to deliver.
  • CBB coffee berry borer
  • a process for preparing talcum powder formulation comprising; harvesting mycelial mat of B. bassiana from roux bottles; sterilizing purified talcum powder; mixing sterile talcum powder with the harvested mycelial mat; mixing sterilized carboxy m thyl cellulose; spreading out the fo ⁇ nulation in trays for drying.
  • the fungus is first isolated from coffee berry borer (CBB) cadavers.
  • CBB cadavers were washed with sterile distilled water, homogenized using a pestle and mortar in sterile distilled water, serially diluted and plated on
  • PDA Potato Dextrose Agar
  • the fungus was cultured on Dextrose-Yeast extract liquid medium supplemented with peptone (YPD). The fungus formed a white mycelial mat and sporulated after 12-14 days of incubation at 25 ⁇ 2 0 C.
  • YPD peptone
  • a mycelial agar plug cut out from the actively growing seed culture on specific medium (YPD A) was inoculated into roux culture bottles containing sterile YPD broth.
  • the broth was decanted without mixing.
  • the mycelial mat was taken into a clean container and stored at 4°C for use with in 24 hours.
  • To prepare lyophilized culture after the mycelial mat is taken from the Roux bottle it is put into round bottom corning flask frozen at -40°C for 6 hours and connected to a manifold assembly for lyophilisation. After lyophilisation the dry mycelia mat is stored under-40°C for use within 60 days.
  • Formulation 1 Fresh culture in liquid broth Fresh culture is produced in specific YPD liquid medium in Roux bottles.
  • YPD broth is prepared and 100 ml is dispensed into Roux bottles.
  • Formulation of B. bassiana was prepared using purified talcum powder.
  • Mycelial mat was harvested from roux bottles blot dried and mixed with

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  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Biotechnology (AREA)
  • Agronomy & Crop Science (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

A process for preparing a biopesticide formulation comprising the steps of: Isolating the fungus from coffee berry borer (CBB) cadaver; Preparing a liquid medium; Culturing the said isolation fungus of B. bassiana in said liquid medium to form a mycelial mat; Inoculating mycelial agar plugs cut from actively growing mycelial mat into a medium broth; Incubating the broth; Harvesting the conidiated mycelial by decantation.

Description

FIELD OF THE INVENTION
This invention relates to the process of preparing a biopestieide formulation for use against coffee berry borer (CBB).
BACKGROUND OF THE INVENTION
At present coffee berry borer is being controlled using the toxic orga hlorine chemical Endosulfan 35 EC. Apart from the use of chemicals certain phytosanitary practices are also followed like cleaning the canopy of plantations, plucking all the leftover berries, using plucking mats etc., in coffee plantations.
This method of control is not only uneconomical but also hazardous to the environment destroying most of the non-target beneficial insect populations in the plantations like Honey bees, Ladybird beetle etc., and other beneficial microorganisms. Moreover Coffee berry borer develops resistance to this chemical very fast in 2 to 3 generations. Once the CBB develops resistance, only the non-target insects are killed and CBB thrives more profusely. Endosulfan residue that remains in coffee beans has deleterious effects on humans.
Be uveri has been reported to cause infective mortality in many terrestrial and aquatic insect pests (Bidochka and Khachatourians, 1991) and also has been successfully used against European corn borer, migratory grass hopper, green house whitefly, pine caterpillar codling moth, mosquitoes and lygus bug (Bidochkha and Khachatourians, 1991 and Khachatourians, 1992). Larval and adult stages of several insects are hosts to many Deuteromycetouos fungal species. These fungi cause mycosis to both Coleopterans and Lepidopterans. Among these pathogens, Beauveria, Metarhisium and Paecilomyces spp. have been proved to be the efficient biological control agents (Hegedus and Khachatourians, 1995). The selected natural enemies from the native range of a given pest are used to reduce the pest's negative effects (DeVault et al., 1996). B. bassiana has been successfully used as foliar applications by several researchers for suppressing populations of several insects, like Bemisia tabaci (Gennadius) (Wright, 1992; Carruthers et at, 1993), Ostrinia nubilalis (Hubner) (Lewis et al., 1996), and Leptinotarsa decemlineata (Say) (Poprawski et at, 1997). B. bassian has been tried as early season applications to control Colorado potato beetle by Poprawski et al. (1997). B. bassiana has been shown as an important entomopathogen in controlling diamondback moth, Plutella xylostella (L.) by Vandenberg et al. (1998). -5. bassiana has been successfully used for control of P. xylostella with crucifer transplants by Shelton et al. (1998). The effect of ento ogenous fungus B. bassiana was tested on insect predators like Coccinella sp.t C. septempu tata, Menochilus sexmaculatus, Erysyrphus altemans and Ischiodon scuteUaris and was proved that all the predator species were susceptible to the infection of B. bassiana (Haseeb and Murad, 1998).
The most frequently recorded fungus on H. kampei is -5. bassiana, though others have also been recorded from many countries (Steyaert, 1935; Pascalet, 1939; Ticheler, 1961; Villacorta 1984). The levels of incidence of this fungus vary from country to country and it is believed that among the different strains of the fungus, some are being more virulent than others (Hawskworth, 1974 and Bridge et al., 1990). Schafer (1936) described the appearance of -5. bassiana on the red locust. In United States, Charles (1941) and Mook and Wolfenbarger (1943), frequently recorded the fungus infecting insect larvae and adults. Doane (1959) reported epizootic among larvae of the small elm bark beetle, Scolytus multistriatus in the United States, caused by -5. bassiana. The report suggested the possibility of employing entomogenous fungi for the biological control of the elm bark beetles, S. scolytus and S. multistriatus, the vectors of Dutch elm disease in Britain.
Marcandier and Khachatourians (1987) have reported the susceptibility of the migratory grasshopper, Melanoplus sanguinipes to B. bassiana. There are several records of B. bassiana infecting grasshoppers and it can cause natural epizootics in these insects (Goettel, 1992; Moore and Erlandson, 1988); Prior and Greathead, 1989). B. bassiana has been successfully used as foliar applications by several researchers for suppressing populations of several insects, like Bemisia tabaci (Gennadius) (Wright, 1992; Carruthers et at, 1993), Ostrinia nubilalis (Hubner) (Lewis et al., 1996), and Leptinotarsa decemlineata (Say) (Poprawski et al., 1997). Early season applications of B. bassiana has been tried to control Colorado potato beetle by Poprawski et al. (1997) and for control of P. xylostella with crucifer transplants by Shelton et al. (1998). Haseeb and Murad (1998) have tested the effect of the entomogenous fungus B. bassiana on insect predators like Coccinella sp., C. septempumtata, Menochilus sexmaculatus, Erysyrphus altemans and Ischiodon scuteUaris and have concluded that all the predator species were susceptible to the infection of -5. bassiana. This entomopathogen -9. bassiana has been shown as an important organism in controlling diamondback moth, Plutella xylostella (L.) by Vandenberg et al. (1998). The role of entomopathogenic fungi in causing disease in insects was first recognized in the early part of the 19s1 Century from studies on silk worms. There are 700 species of entomopathogenic fungi (Robert et al., 1991). Of these Beauveria bassiana has been studied most extensively. -5. bassiana an hypomycetous fungus has no known sexual cycle. The asexual conidia attaches to the host cuticle and degrade pectin, chitin and iipids in the insect integument by various enzymatic activities (Kachatourians, 1991). The host insects are killed due to depletion of their haemolymph nutrients and or due to toxemia caused by fungal toxic metabolites (Roberts, 1981; Kachatourians, 1991).
To date, more than 200 insects species have recorded as hosts of -5. bassiana worldwide (Li, 1988). It has been used against Red Imported Fire Ant in the US (Siebeneicher et al., 1992), migratory grasshopper (Marcandies and Khachatourians, 1987), predatory insects (Donegan, 1989), Stem -borer (Maniania, 1993) and Rice hispa (Hazarik and Puzari, 1990). The virulence of the fungus and pathogenicity of isolates and temperature conditions to establish the virulence. Variation in virulence of B. bassiana may also be related to enzyme production and activities during the course of penetration of the host cuticle (Biodochka and Khachatourians, 1990). The B. bassiana production has recently been developed by Mycotech Corporation, USA (Bradley et al., 1992). A corn starch and corn starch oil formulations of B. bassiana mycelia as well as pure dry mycelial preparations was obtained by Feng et al. (1994). Bidochka et al. (1997) have found out that hydrophobic interactions, appressoria formation, and mucus production by the fungus are involved in adhesion to the cuticle. The technology of protoplast fusion is a valuable method for intra- and inter specific hybridization. Through this a somatic hybrid of a cross between two strains of B. bassiana has been obtained (Viaud, 1998).
OBJECTS OF THE INVENTION
An object of this invention is to propose a process for preparing a biopesticide formulation against coffee berry borer.
Further, object of this invention is to propose a formulation containing entomopathogenic fungus which is eco-friendly.
Yet another object of this invention is to prepare a formulation which is economical and easy to deliver.
SUMMARY OF THE INVENTION
According to this invention there is provided a process for preparing a biopesticide formulation comprising the steps of:
Isolating the fungus from coffee berry borer (CBB) cadaver;
Preparing a liquid edium;
Culturing the said isolation fungus of B. bassiana in said liquid medium to form a mycelial mat;
Inoculating mycelial agar plugs cut from actively growing mycelial mat into a medium broth; Incubating the berth;
Harvesting the conidiated mycelial by decantation,
In accordance with a further embodiment of this invention there is also provided a process for preparing talcum powder formulation comprising; harvesting mycelial mat of B. bassiana from roux bottles; sterilizing purified talcum powder; mixing sterile talcum powder with the harvested mycelial mat; mixing sterilized carboxy m thyl cellulose; spreading out the foπnulation in trays for drying.
DESCRIPTION OF THE INVENTION
In accordance with this invention, the fungus is first isolated from coffee berry borer (CBB) cadavers.
CBB cadavers were washed with sterile distilled water, homogenized using a pestle and mortar in sterile distilled water, serially diluted and plated on
Potato Dextrose Agar (PDA) plates. After incubation, fungi was isolated and identified as B. bassiana based,
PREPARATION OF MAT
The fungus was cultured on Dextrose-Yeast extract liquid medium supplemented with peptone (YPD). The fungus formed a white mycelial mat and sporulated after 12-14 days of incubation at 25±20C.
Fresh culture in Liquid broth
Medium compoaitlύϊi (Yeast extract ept ne dextrose ---tedium [YPD])." The liquid medium was developed with the following composition- Peptone - 5 to 15g/lit; Yeast extract - 5 to 15g/iit; Glucose -10 to 30g/lit; Starch - 10 to 30g/lit, Sodium chloride 2 to 8g/lit and Calcium carbonate - 2 to 8g/lit. Chloramph nic l-1 5 mg/liter.
A mycelial agar plug cut out from the actively growing seed culture on specific medium (YPD A) was inoculated into roux culture bottles containing sterile YPD broth.
Incubation conditions:
Temperature - 20±28°C
Light - 12/12 h NUV and dark conditions
Time - 10 to 20 days
Product recovery:
The broth was decanted without mixing. The mycelial mat was taken into a clean container and stored at 4°C for use with in 24 hours. To prepare lyophilized culture, after the mycelial mat is taken from the Roux bottle it is put into round bottom corning flask frozen at -40°C for 6 hours and connected to a manifold assembly for lyophilisation. After lyophilisation the dry mycelia mat is stored under-40°C for use within 60 days.
EXAMPLES:
Formulation 1 - Fresh culture in liquid broth Fresh culture is produced in specific YPD liquid medium in Roux bottles.
YPD broth is prepared and 100 ml is dispensed into Roux bottles. The
bottles are autoclaved at 15 psi for 30 min. The cooled medium is inoculated
with mycelial agar plugs cut from actively growing colonies on YPD agar
medium under aseptic conditions. The bottles are incubated at 25±2°C under
12/12h alternate cycles of NUV and darkness for 12 to 14 days. The
profusely conidiatεd mycelial mat is harvested by decantation, packed in
polythene bags and kept at 4°C for use within 24 hours or processed for
lyophilisation.
Formulation 2 - Talcum powder formulation
Formulation of B. bassiana was prepared using purified talcum powder.
Mycelial mat was harvested from roux bottles blot dried and mixed with
talcum powder (1:10) to obtain a conidia! concentration of approximately
ϊxlO8 conidiospores/g. Carboxy methyl cellulose at the rate of 1% was
added as sticker. The formulation was spread out in plastic trays and allowed
to dry under shade in a fume hood for 24 hours and packed in polythene
bags. The formulations thus prepared are stored in dark at 4°C to be used
before 60 days.

Claims

We Claim :
1. A process for preparing a biopesticide formulation comprising the steps of:
Isolating the fungus from coffee berry borer (CBB) cadaver;
Preparing a liquid medium;
Culturing the said isolation fungus of B. bassiana in said liquid medium to form a mycelial mat;
Inoculating mycelial agar plugs cut from actively growing mycelial mat into a medium broth;
Incubating the broth;
Harvesting the conidiated mycelial by decantation.
2. The process as claimed in claim 1, wherein the said step of isolation is preformed by washing the coffee berry borer with sterile distilled water, homogenized using a pestle and motar in sterile distilled water, serially diluted and plated on potato dextrose agar (PDA) plates and after incubation at 25±2°C it was isolated and identified.
3. The process as claimed in claim 1, wherein said liquid medium comprises:
5 to 15 g/lit of peptone;
5 to 15 g/lit of yeast extract;
10 to 30 g/lit of glucose;
10 to 30 g lit of starch;
2 to 8 g lit of sodium chloride;
2 to 8 g/lit of calcium carbonate and
125 mg/lit of chloramphenicol.
4. The process as claimed in claim 1 wherein optionally the mycelial mat is put into a round bottom corning flask at -40°C for 6 hrs and connected to a manifold assembly for lyophilisation.
5. A process as claimed in claim 1, wherein the said step of incubation is preformed in roux culture bottles containing yeast extract peptone dextrose medium (YPD).
6. A process as claimed in claim 1, wherein the broth containing the mycelial are incubated at a temperature of 20 to 28°C for 10 to 20 days.
7.A process for preparing a talcum powder biopesticide formulation comprising: harvesting the mycelial mat of B. bassiana as clai ed in claim 1; sterilizing purified talcum powder; mixing sterile talcum powder with the harvested mycelial mat.
PCT/IN2003/000355 2002-12-12 2003-11-05 The process of preparing a biopesticide formulation for use against coffee berry borer (cbb) WO2004052103A1 (en)

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Applications Claiming Priority (2)

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IN1244DE2002 2002-12-12
IN1244/DEL/2002 2002-12-12

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US8623420B2 (en) 2006-12-08 2014-01-07 University Of Vermont And State Agriculture College Sweet whey based biopesticide composition
US9578873B2 (en) 2011-10-11 2017-02-28 Universidad Nacional De Quilmes Methods for controlling leaf-cutting ants
WO2017151684A1 (en) * 2016-03-01 2017-09-08 Sustainable Bioproducts Holdings, Llc Filamentous fungal biomats, methods of their production and methods of their use
US10851396B2 (en) 2014-07-03 2020-12-01 The Fynder Group, Inc. Acidophilic fusarium oxysporum strains, methods of their production and methods of their use
KR20210063760A (en) * 2019-11-25 2021-06-02 영남대학교 산학협력단 Fabrication of scaffolds using fungi and tissue regeneration method using same
US11039635B2 (en) 2019-02-27 2021-06-22 The Fynder Group, Inc. Food materials comprising filamentous fungal particles
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US9578873B2 (en) 2011-10-11 2017-02-28 Universidad Nacional De Quilmes Methods for controlling leaf-cutting ants
US10851396B2 (en) 2014-07-03 2020-12-01 The Fynder Group, Inc. Acidophilic fusarium oxysporum strains, methods of their production and methods of their use
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US10533155B2 (en) 2016-03-01 2020-01-14 Sustainable Bioproducts, Inc. Filamentous fungal biomats, methods of their production and methods of their use
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US11427957B2 (en) 2019-06-18 2022-08-30 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11447913B2 (en) 2019-06-18 2022-09-20 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11414815B2 (en) 2019-06-18 2022-08-16 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11649586B2 (en) 2019-06-18 2023-05-16 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11718954B2 (en) 2019-06-18 2023-08-08 The Fynder Group, Inc. Fungal textile materials and leather analogs
US11118305B2 (en) 2019-06-18 2021-09-14 The Fynder Group, Inc. Fungal textile materials and leather analogs
KR102334114B1 (en) 2019-11-25 2021-12-02 영남대학교 산학협력단 Fabrication of scaffolds using fungi and tissue regeneration method using same
KR20210063760A (en) * 2019-11-25 2021-06-02 영남대학교 산학협력단 Fabrication of scaffolds using fungi and tissue regeneration method using same
CN114071992A (en) * 2020-02-03 2022-02-18 麦克沃克斯股份有限公司 Novel microstructures of mycelium and mycelium-based materials

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