EP1387613A1 - Lutte biologique contre des ravageurs endoges - Google Patents

Lutte biologique contre des ravageurs endoges

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Publication number
EP1387613A1
EP1387613A1 EP02722455A EP02722455A EP1387613A1 EP 1387613 A1 EP1387613 A1 EP 1387613A1 EP 02722455 A EP02722455 A EP 02722455A EP 02722455 A EP02722455 A EP 02722455A EP 1387613 A1 EP1387613 A1 EP 1387613A1
Authority
EP
European Patent Office
Prior art keywords
metarhizium
agent
present
composition according
compost
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02722455A
Other languages
German (de)
English (en)
Inventor
Munoo Prasad
Tariq M. School of Biological Sciences BUTT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UWS Ventures Ltd
Bord Na Mona Horticulture Ltd
Original Assignee
UWS Ventures Ltd
Bord Na Mona Horticulture Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0110314A external-priority patent/GB0110314D0/en
Application filed by UWS Ventures Ltd, Bord Na Mona Horticulture Ltd filed Critical UWS Ventures Ltd
Publication of EP1387613A1 publication Critical patent/EP1387613A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • 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

  • the present invention relates to the biological control of soil dwelling pests, in particular the subterranean pests such as vine weevil, mushroom flies, sciarids, phorids and fungus gnats.
  • subterranean pests such as vine weevil, mushroom flies, sciarids, phorids and fungus gnats.
  • Otiorhynchus sulcatus commonly known as vine weevil
  • vine weevil is an insect that can cause substantial damage to plants.
  • the vine weevil multiplies rapidly with a single adult laying between 500 to 1200 eggs.
  • the adults may feed on plant foliage whilst the larvae generally feed on the roots of plants, thereby causing the plants to wilt or be stunted and eventually killed.
  • Plants are also susceptible to infection by opportunistic plant pathogenic fungi that gain entry through wounds made by feeding insects. Killing the pests before they cause considerable damage to plant tissue reduces the risk of plants becoming infected with diseases such as Fusarium and Botrytis .
  • the weevils can feed on over 150 species of plants in particular strawberry and blackcurrant plants, as well as protected and hardy ornamentals. Many household plants are also at risk from damage by feeding weevils, for example the plant Cyclamen, Impatiens. The estimated cost of damage caused by vine weevils is hundreds of millions of dollars per annum.
  • Mushroom fly, sciarid, phorid and fungus gnat are common names for insects of the species Sciara , Lycoriella , Bradysia , Sciaridae, Boletina , Macrocera , Mycetophila , Symmerus annula tus, M Mycetophilidea .
  • the larvae or maggots of mushroom flies, sciarids, phorids and fungus gnats generally feed on the roots of numerous plants including African violet, alfalfa, carnations, clover, corn, cucumbers, cyclamen, Easter lilies, geraniums, lettuce, nasturtium, peppers, poinsettias, potatoes, soybeans, and wheat.
  • the feeding larvae or maggots can cause plants on which they are feeding to wilt, be stunted, yellow, lose foliage and eventually killed.
  • US5512280 discloses use of Metarhizium anisopliae for the control of insects.
  • the Metarhizium conidia are stored in an aqueous suspension which is administered to the insects.
  • US5418164 also discloses use of Metarhizium for combating pests and for protecting plants.
  • the Metarhizium disclosed in US5418164 are carrier-free cell granulates which are essentially bead-shaped structures which are composed of Metarhizium cells fused like tissue and containing no carrier material.
  • the biological control agent used in the present invention is the fungus Metarhizium .
  • At least one strain of the fungus Metarhizium together with at least one agent capable of providing nutritional function to plant material present in a medium of growth
  • said Metarhizium strain and said agent are provided for simultaneous, separate or sequential administration whereby (i) said agent can be administered so as to be capable of providing nutrients to said plant material present in said medium of growth and (ii) said Metarhizium strain can be administered in an amount effective for substantially combating one or more pests present in or on said medium of growth, or in or on said plant material present in said medium of growth, which pests at least when present in or on said plant material can be detrimental thereto.
  • Metarhizium is more efficacious for pest control in peat and peat free composts than garden or other non-sterile soils. It is therefore particularly preferred that the medium of growth is substantially free of antagonistic biota.
  • compositions for substantially combating one or more pests detrimental to plant material present in a medium of growth which composition comprises (ii) at least one strain of the fungus Metarhizium present in an amount effective for substantially combating pests present in or on said medium of growth, or in or on said plant material present in said medium of growth; intimately mixed with (ii) at least one agent capable of providing nutritional function to said plant material present in said medium of growth.
  • Growth medium denotes a medium such as soil, compost, or the like, through which nutrients can reach the roots of a plant, or plants, present in that medium.
  • Metarhizium is preferably employed according to the present invention so as to be highly pathogenic at least to vine weevil larvae, which may be present in or on the growth medium, or in or on the plant material present in the growth medium.
  • the Metarhizium may suitably be employed according to the present invention so as to be also pathogenic to other pests, including the larvae, maggots and/or eggs of root weevil, mushroom flies, sciarids, phorids, fungus gnats and ticks, which may also be present in or on the growth medium or in or on the plant material present in the growth medium. All of the pests, with the exception of ticks, are found in composts of horticultural plants (ie ornamentals etc) .
  • Ticks are found in the wild or in pastures where livestock graze. The engorged female ticks fall off their respective hosts and lay eggs close to where the ruminant lies down to chew the cud. These habitats may also be treated with the composition according to the present invention.
  • the ticks may include, but are not limited to, Amblyomma (Bont tick) , Boophil us, Dermacen tor, Haemaphysalis , Hyalomma r Ixodes and Rhipicepha l us .
  • the composition according to the present invention may be applied to the soil or vegetation in tick habitats.
  • Metarhizium anisopliae conida may be applied directly to the soil or vegetation in tick habitats.
  • the present invention extends to animal bedding including a composition comprising at least one strain of the fungus Metarhizium present in or on said bedding in an amount effective for substantially combating pests present in or on said bedding.
  • the present invention further extends to use of at least one strain of the fungus Metarhizium in the manufacture of a treated animal bedding, wherein the Metarhizi um is present an amount effective for substantially combating one or more pests present in or on said treated bedding, which pests at least when present in or on said bedding can be detrimental to animals in contact with said bedding material.
  • ectoparasites, such as ticks, falling in the bedding could become infected with the pathogen.
  • a method of treating animal bedding which method includes applying Metarhizium in an amount effective for substantially combating one or more pests (such as ticks) present in or on said bedding, which pests at least when present in or on said bedding can be detrimental to animals in contact with untreated bedding.
  • pests such as ticks
  • the bedding includes, peat, peat-free compost, sand, hay, straw or the like.
  • the bedding may also include other suitable vegetation.
  • the Metarhizium may also advantageously prevent establishment of pests such as adult vine weevils, mushroom flies, sciarids, phhorids, fungus gnats and ticks in or on the growth medium.
  • pests includes any organism which is detrimental to a plant, or plants, which plant is present, or is intended to be present, in the growth medium.
  • pests include animals, such as harmful arthropods, nematodes or the like, and microbial pests, such as harmful bacteria, fungi, or the like.
  • the present invention is, however, particularly suitable for use in substantially combating one or more insects, selected from the group consisting of vine weevil, root weevil, mushroom fly, sciarid, phorid, fungus gnat, tick or the like. In a particularly preferred aspect, the present invention is suitable for use in combating vine weevil and/or ticks.
  • the present invention is advantageous in controlling arthropod vectors of diseases of medical and veterinary importance such as ticks.
  • At least one Metarhizium strain as employed according to the present invention is a strain of the species Metarhizium anisopliae, and for example it is preferred that at least one strain of Metarhizium employed according to the present invention is selected from the group consisting of the strains V275, V245, Biogreen, V208, ARSEF 1910, Ma 23, ARSEF 817, ARSEF 9601, ARSEF 689, ARSEF 3297, ARSEF 4556, or ARSEF 686.
  • a most preferred Metarhizium anisopliae strain for use according to the present invention is strain V275 or V245 as described above. It is particularly preferred that the strains of Metarhizium anisopliae may be selected from ARSEF 689, ARSEF 3297, ARSEF 4556 and ARSEF 686 when the pest includes ticks .
  • At least one strain of the fungus Metarhizium used in the present invention may be cultured using any general method for the production of fungal propagules on artificial media. Examples of suitable methods include surface culture on a solid media, fermentation on a semi-solid media, submerged fermentation and diphasic fermentation.
  • the cultured Metarhizium strain or strains may then be harvested and stored as air dried conidia and/or mycelium, either free or on a suitable substrate such as grain or the like.
  • the harvested Metarhizium strain or strains may also be stored in a suitable carrier, for example, oil, water or water containing a surfactant such as Tween. Drying Metarhizium conidia in the presence of desiccating agents such as silica gel or calcium chloride may improve the viability of the conidia, however, direct contact of the conidia with the some desiccating agents (but not all) can be detrimental.
  • the Metarhizium conidia for use according to the present invention may be mixed into a compost or mulch, as employed according to the present invention substantially as hereinafter described in greater detail, by hand or using a mechanical mixing apparatus.
  • the amount of conidia incorporated into a compost or mulch depends on the virulence and shelf life of the particular species and strain of Metarhizium being used, however, the usual dose may be in the range 0.5 - 5.0g conidia per litre of compost or mulch.
  • an agent capable of providing nutritional function substantially as hereinbefore described suitable for use according to the present invention is capable of providing sustained nutritional function to plant material present in a growth medium substantially as hereinbefore described and preferably the agent is capable of providing nutrients to the plant material over a period of 3 to 4 months.
  • the agent providing nutritional function may provide nutrients to a plant or plants (preferably on a sustained basis) , which plant is present, or is intended to be present, in the growth medium. Additionally, the agent may provide nutrients to the Metarhizium (preferably on a sustained basis) employed according to the present invention.
  • the agent providing nutritional function can generally comprise a biodegradable agent, of the type suitable for addition to the growth medium. It is a preferred feature of the present invention that the agent comprises a compost, mulch or the like (most preferably a compost or mulch) . It is particularly preferred that the growth medium is substantially free of antagonistic biota.
  • a particularly preferred agent providing nutritional function includes peat and peat-free composts.
  • the agent providing nutritional function is typically free of soil biota which may suppress the activity of the introduced biological control agent, namely Metarhizium .
  • the compost further includes a fertilizer, such as an organic fertilizer.
  • a fertilizer such as an organic fertilizer.
  • the composition preferably further includes one or more control agents.
  • the control agent may be a beneficial organism for crop and/or animal protection.
  • beneficial organisms may include nematodes (suitable for slug and insect control), bacteria (suitable for pest and disease control) and fungi (suitable for pest, weed and disease control) .
  • control agents are preferably biocontrol agents which advantageously combat one or more of arthropod pests, weeds and diseases.
  • the addition of such control agents to the composition reduce application and labour costs.
  • the inclusion of control agents in the composition has a further advantage in that plant disturbance is minimised.
  • the biological control agents work synergistically with other components of the composition.
  • biocontrol agents incorporated into the composition protect seedlings, young plants, nursery and ornamental plants against a wide range of pests and diseases.
  • Mycoherbicides would prevent establishment of selective weeds thereby reducing competition with crops (ornamental, vegetable etc) and reduce weeding costs.
  • Preferred control agents for use in the biological control of diseases include (but are not limited to) Phlebiopsis gigantea , Gliocladiun ca tenula tum, Gliocladi um virens , Coniothyrium minitans , Ampelomyces quisqualis, Cryptococcus albidus, Candida oleophila , Endothia parasi tica (non- pathogenic strain) , Fusarium oxyspori um , Pythium oligandrum, Trichoderma harzianum or T viride .
  • Preferred control agents for use in the biological control of pests include (but are not limited to) Verticillium, lecanii , Mmetarhizium anisoplide, Beauveria bassiana , Beauveria brongniartii , Metarhizium flavoviride,
  • Paecilomyces fumosoroseus Paecilomyces lilicanus .
  • Preferred control agents for use in the biological control of weeds include (but are not limited to) Acremonium diospyn , Al ternaria zinniae, Al ternaria eichhornia , Al ternaria cassiae, Cercospora rodmanii , Colletotrichum coccodes, Colletoa trichum gloeosporioides f. sp cuscutae, Colletotrichum gloeasporioides f. sp aeschynomene, Colletotrichum orbiculare, Chondrosterium purpureum, Phytophthara palmivora .
  • the preferred fungal control agents (together with their target and commercial name) are listed in Tables la, lb and lc.
  • Table lb - Fungi developed or being developed for the biological control of pests
  • the compost or mulch may be provided as a block which can be added to the growth medium (either on or below the surface of said growth medium) .
  • the nutrients and Metarhizi um in or on the block typically slowly release or seep out into the growth medium, advantageously combating pests in the growth medium and providing nutrients to the growth medium.
  • the nutrients are preferably provided to the growth medium for a sustained period of time, substantially as hereinbefore described, generally 3 to 4 months.
  • a beneficial material substantially as hereinafter described in greater detail is employed according to the present invention, such a beneficial material can also be included in or on the block of compost or mulch, and this beneficial material may also seep out into the growth medium.
  • the compost may be a peat based compost such as peat moss, peat free compost, or an organic compost such as coconut fibre, bark or the like. Such compost may be for seeding or for propagating cuttings or the like.
  • a product for substantially combating one or more pests detrimental to plant material present in a growth medium which product comprises (i) at least one strain of the fungus Metarhizium present in an amount effective for substantially combating pests present in or on said growth medium, or in or on said plant material present in said growth medium; intimately mixed with (ii) at least one agent comprising (preferably consisting essentially of) at least one compost or mulch capable of providing nutritional function to said plant material present in said growth medium.
  • the agent capable of providing nutritional function may be soil, a mixture of at least soil and a compost or mulch substantially as hereinbefore described.
  • the present invention further employs a material that is beneficial to plant material substantially as hereinbefore described and also at least one strain of Metarhizium substantially as hereinbefore described.
  • Preferred materials which may be employed include fertilisers based on soya and/or castor or the like capable of providing sources of nitrogen, phosphorous and/or potassium or the like, as well as trace elements such as iron, boron or the like.
  • the present invention further provides a method of using, in combination at least one strain of the Metarhizium, together with at least one agent capable of providing nutritional function to plant material according to any aspect of the present invention substantially as hereinbefore described, which method comprises applying the combination to a growth medium so as to substantially combat pests.
  • the method of application of the combination may be any known horticultural, forestry or agricultural method such as, for example, top dressing or admixing to the growth medium.
  • the present invention further provides a preferred method of using a product comprising a compost or mulch intimately mixed with at least one strain of the fungus Metarhizium substantially as hereinbefore described according to the present invention, which method comprises applying the product to a growth medium for substantially combating pests substantially as hereinbefore described.
  • Suitable species and strains of Metarhizium employed in a method according to the present invention and the preferred pests substantially combated by the Metarhizi um are substantially as hereinbefore described.
  • Figure 1 shows a diagrammatic representation of the key components of a Petri dish bioassay.
  • Figure 2 shows a graph representing the progressive percentage mortality of Otiorhynchus sulca tus (vine weevil) larvae following inoculation with Metarhizium anisopliae conidia (strains V275,V245 and V208) and Beauveria bassiana conidia (strain Bbl3) from day 4 to day 10 post inoculation (DPI) of the larvae with the aforementioned conidia.
  • Otiorhynchus sulca tus (vine weevil) larvae following inoculation with Metarhizium anisopliae conidia (strains V275,V245 and V208) and Beauveria bassiana conidia (strain Bbl3) from day 4 to day 10 post inoculation (DPI) of the larvae with the aforementioned conidia.
  • Figure 3 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 5 days post inoculation (DPI) with different strains of Metarhizium anisopliae, Bea uveria bassiana and Beauveria brongniartii .
  • Figure 4 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 7 days post inoculation (DPI) with different strains of Metarhizium anisopliae, Beauveria bassiana and Beauveria brongniartii .
  • Figure 5 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 2 to 6 days post inoculation (DPI) in Metarhizium anisopliae (strain V245) conidia suspension, which larvae had been introduced into different types of compost.
  • Figure 6 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 2 to 6 days post inoculation (DPI) in Metarhizi um anisopliae (strain V275) conidia suspension, which larvae had been introduced into different types of compost.
  • Figure 7 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 2 to 4 days post inoculation (DPI) in each of the different compost tested, which composts had been previously immersed in Metarhizium anisopliae (strain V245) conidia suspension.
  • Figure 8 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 2 to 5 days post inoculation (DPI) in each of the different compost tested, which composts had been previously immersed in Metarhizium anisopliae (strain V275) conidia suspension.
  • Figure 9 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 3 to 5 days post inoculation (DPI) in each of the different compost tested and incubated at a constant temperature of 25oC, which composts had been previously soaked in Metarhizium anisopliae (strain V275) conidia suspension.
  • Figure 10 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 3 to 6 days post inoculation (DPI) in each of the different compost tested and incubated under fluctuating temperatures of 18 to 27oC, which composts had been previously soaked in Metarhizium anisopliae (strain V275) conidia suspension.
  • Figure 11 shows a graph representing the percentage mortality of Tenebrio moli tor larvae 3 to 7 days post inoculation (DPI) in each of the different compost tested and incubated at 25oC, which composts had been previously soaked in Metarhizium anisopliae (strain V275) conidia suspension.
  • Figure 12 shows a graph representing the percentage mortality (10 DPI) of Otiorhynchus sulca tus (vine weevil) larvae treated with either Metarhizium anisopliae conidial suspension or Metarhizium anisopliae conidia sporulating on broken rice, both applied as a drench to the surface of different composts.
  • Figure 13 shows a graph representing the percentage mortality (14 DPI) of Otiorhynchus sulca tus (vine weevil) larvae in different composts treated with Metarhizium anisopliae conidial suspension.
  • Figure 14 shows a graph representing the number of Otiorhynchus sulca tus (vine weevil) larvae recovered 14 days post inoculation (DPI) from compost of plants whose compost had been treated with Metarhizium anisopliae conidia suspension and from compost of untreated plants (control) .
  • the compost of the treated and untreated plants had been infested with Otiorhynchus sulca tus (vine weevil) eggs and Otiorhynchus sulca tus (vine weevil) 1st instar larvae.
  • Figure 15 shows a graph representing the mortality of Otiorhynchus sulca tus (vine weevil) 2nd/3rd and 4th instar larvae exposed to compost treated with Metarhizium anisopliae conidia suspension and untreated compost (control) .
  • Figure 16 shows graphs representing the progressive release of nitrogen, phosphorus and potassium from peat treated with different concentrations of soya bean seed and castor bean seed.
  • Figure 17 shows a graph indicating the % mortality of R . appendicula tus when exposed to M. anisopliae V245 or V275.
  • Figure 18 shows a graph indicating susceptibility of adults and nymphs exposed to strains V245 and V275.
  • Figure 19 shows a graph indicating emergence of V245/V275 from infected adult and nymphs of R . appendicula tus .
  • Figure 20 shows a graph of cumulative mortality of soft and hard ticks treated with M. anisopliae strains V245 and V275.
  • Figure 21 shows a graph indicating susceptibility of starved and engorged sp Ixodes to M. anisopliae.
  • Figure 23 shows a graph Efficacy of M. anisopliae and imidacloprid for control of vine weevil larvae in potted polyanthus in seed and potting compost.
  • Figure 24 shows a graph Efficacy of M. anisopliae for control of vine weevil larvae in potted polyanthus in multipurpose compost.
  • Figure 25 shows a graph Efficacy of M. anisopliae and imidacloprid for control of vine weevil larvae in potted cyclamen "Miracle White” in BNM seed and potting compost.
  • Figure 26 shows a graph Efficacy of M. anisopliae for control of vine weevil larvae in potted Cyclamen Miracle White.
  • Figure 27 show a graph Efficacy of M. anisopliae and imidacloprid for control of vine weevil larvae in potted cyclamen "Deep Salmon”. in BNM seed and potting compost.
  • Figure 28 shows a graph Efficacy of M. anisopliae for control of vine weevil larvae in potted Cyclamen Deep Salmon. M. anisopliae was applied at two doses and three application methods.
  • Metarhizium and other fungi used in the following methods were cultured on the surface of a solid medium. Briefly, an autoclavable plastic bag containing Sabouraud dextrose agar (SDA) (mycopeptone (lOg/L), dextrose (40g/L) and agar
  • SDA Sabouraud dextrose agar
  • the pathogenicity of Metarhizium anisopliae strains V208, V245 and V275 to Otiorhynchus sulca tus (vine weevil) larvae was tested using the following Petri dish bioassay.
  • Beauveria bassiana strain Bbl3 to vine weevil larvae was also tested.
  • Table 2 shows the original host or source and the country of origin for each of the above strains of Metarhizium anisopliae and Beauveria bassiana .
  • each larvae was then transferred to a 9cm diameter Petri dish partially filled with moist compost (acidic, Irish Peat moss compost - Bord Na Mona, Kildare, Ireland) and a slice of carrot or potato was provided to each dish as food.
  • the Petri dish 1 therefore contained a single larva 2 (in a cell it had created) surrounded by moist compost 3 as shown in Figure 1 of the accompanying drawings.
  • the Petri dish also contained slices of carrot or potato 4.
  • Each treatment of the larvae with the conidia suspension or the control was therefore replicated five times as there were 5 larvae in each group immersed in either conidia suspension or the control as described above.
  • the Petri dishes were then sealed with Parafilm except for small slits at the top and bottom to allow ventilation and drainage, respectively.
  • the Petri dishes were stored vertically in a cardboard box and incubated at 25oC in the dark to simulate subterranean conditions.
  • the compost in the Petri dishes was kept moist throughout the experiment.
  • the Petri dishes were examined daily for larva movement, larva mycosis and mortality, and the formation of cells by the larvae. The above described Petri dish bioassay was repeated three times.
  • a second petri dish bioassay was carried out using a similar method as described above in connection with the first bioassay, however TeneJrio moli tor mealworm larvae at the 4th and 5th instar stage of development where used instead of Otiorhynchus sulca tus (vine weevil) larvae. Tenejrio moli tor (mealworm) is a pest of flour and stored grain. Both Tenebrio and Otiorhynchus belong to the insect order Coleoptera .
  • the virulence of Metarhizium anisopliae (strains V275, V245, V208, ARSEF 1910, ARSEF 817, Ma23, 9601, Biogreen and 9609) against Tenebrio moli tor mealworm larvae was tested.
  • the virulence of Beauveria bassiana (strains 97011, Bbl3, ARSEF 813, ARSEF 1073, ARSEF 1074 and ARSEF 1075) and Beauveria brongniartii (strains BIPESCO NOl, BIPESCO N02, BIPESCO N03, and BIPESCO N04) against Tenejrio moli tor larvae was also tested.
  • Table 3 shows the original host or source for each of the aforementioned strains of Metarhizium anisopliae, Beauveria bassiana and Beauveria brongniartii .
  • Results from the first Petri dish bioassay are shown in the graph of Figure 2 which shows the progressive percentage mortality of Otiorhynchus sulca tus (vine weevil) larvae following inoculation with Metarhizium anisopliae (strains V275,V245 and V208) and Beauveria bassiana (strain Bbl3) from 4 to 10 days post inoculation (DPI) of the larvae with the conidia suspension as described above.
  • Figure 2 indicates that the most virulent strain of fungus tested was Metarhizium anisopliae strain V275 which caused 50% mortality of larvae at 4 DPI. All the Metarhizium anisopliae strains tested caused 100% mortality of larvae at 6 DPI.
  • Beauveria bassiana (strain Bbl3) was the least virulent fungi tested and caused only 75% mortality of larvae at 10 DPI. There was no mortality of larvae in the control Petri dishes (results not shown on Figure 2) .
  • Figure 3 is a graph which shows the percentage mortality of Tenebrio moli tor larvae 5 days post inoculation (DPI) with different strains of Metarhizium anisopliae, Beauveria bassiana and Beauveria brongniartii .
  • Figure 4 is a graph which shows the percentage mortality of Tenebrio moli tor larvae 7 days post inoculation (DPI) with different strains of Metarhizium anisopliae, Beauveria bassiana and Beauveria brongniartii .
  • Figure 3 indicates that the most virulent strain of fungi tested was Metarhizium anisopliae strains V275 and V245 which cause 100% mortality of Tenebrio moli tor larvae 5 DPI.
  • Metarhizium anisopliae strains V208, ARSEF 1910 and Biogreen all cause 100% mortality of Tenebrio moli tor larvae 7 DPI.
  • Beauveria bassiana and Beauveria brongniartii do not appear to be as virulent as Metarhizium anisopliae as none of the strains of Beauveria bassiana and Beauveria brongniartii caused 100% mortality of Tenebrio moli tor larvae 7 DPI. There was no mortality of larvae in the control Petri dishes (results not shown on Figures 3 and 4) .
  • Example 2 Effect of different potting composts on the efficacy of Metarhizium anisopliae against subterranean insect pests .
  • Metarhizium anisopliae strains V245 and V275 were used in the following experiment. Strains V245 and V275 were shown to be highly virulent against both Tenebrio moli tor larvae and Otiorhynchus sulca tus (vine weevil) larvae in the above described first and second Petri dish assay.
  • Composts 1 to 4 above were provided by Bord Na Mona Company (Kildare, Ireland) and compost 5 was provided by University of Wales Swansea.
  • Conidia of Metarhizium anisopliae (strains V245 and V275) were cultured and harvested as described above and suspended in 0.05% v/v Aq. Tween 80 to a concentration of 109 conidia/ml. Tenejbrio molitor larvae were incubated in one of the above mentioned composts using either method 1 or 2 as described hereafter.
  • Tenebrio moli tor larvae were immersed in the conidia suspension or 0.05% v/v Aq. Tween 80 (control) for 20 seconds and then transferred to a Petri dish (3 larvae per dish) . Each Petri dish was then partially filled with one of the composts mentioned above. As a further control Tenebrio moli tor larvae which had been immersed in the conidia suspension or 0.05% v/v Aq. Tween 80 as described above were incubated in Petri dishes lined with moist filter paper only.
  • Figures 5 and 6 show the percentage mortality of Tenejbrio molitor larvae treated using method 1 in Figures 5 and 6.
  • Figure 5 shows the percentage mortality of TeneJbrio molitor larvae 2 to 6 days post inoculation (DPI) in Metarhizium anisopliae (strain V245) conidia suspension
  • Figure 6 shows the percentage mortality of Tenejbrio moli tor larvae 2 to 6 days post inoculation (DPI) in Metarhizium anisopliae (strain V275) conidia suspension.
  • the graphs of Figures 5 and 6 show mortality of larvae incubated in each of the compost tested and incubated in Petri dishes lined with moist filter paper (FP) only.
  • FP moist filter paper
  • Method 2 The percentage mortality of Tenebrio moli tor larvae treated using method 2 are shown in Figures 7 and 8.
  • Figure 7 shows the percentage mortality of TeneJbrio molitor larvae 2 to 4 days post inoculation (DPI) in each of the different compost tested, which composts had been previously immersed in Metarhizium anisopliae (strain V245) conidia suspension.
  • Figure 8 shows the percentage mortality of Tenebrio moli tor larvae 2 to 5 days post inoculation (DPI) in each of the different compost tested, which composts had been previously immersed in Metarhizium anisopliae (strain V275) conidia suspension.
  • strains V245 and V275 or whether the larvae were exposed to Metarhizium anisopliae (strains V245 and V275) incorporated into the compost.
  • Plant soils may contain antibiotics which interfere with the efficacy of fungal biocontrol agents such as Metarhizium anisopliae .
  • Table 4 indicates that glasshouse soil (GH) , which are often modified by addition of sterile peat composts, only slightly interfere with Metarhizium anisopliae.
  • Example 4 Effect of temperature on the efficacy of Metarhizi ⁇ m anisopliae against Tenebrio molitor larvae - Petri dish assay.
  • the composts used in example 4 were as hereinbefore described in connection with example 2.
  • the insect larvae used in example 4 were Tenebrio moli tor larvae at the 4th and 5th instar stage of development. The larvae were cultured and harvested as described above and maintained on bran flakes at 25 ⁇ 2°C and 16:8 hours (light:dark) photoperiod.
  • Metarhizium anisopliae (strain V 275) was passaged through Teneibrio moli tor larvae and isolated using oatmeal dodine agar (ODA) , then individual colonies/ conidia were transferred to SDA (Difco) . Conidia were harvested from sporulating cultures and suspended in 0.05%v/v Aq. Tween to a final concentration of 10 8 conidia ml "1 .
  • Metarhizium anisopliae (strain V 275) sporulating on broken rice grain (lOg) was suspended in 0.05% Aq. Tween.
  • Compost (enough to fill 6 X 9cm diameter Petri dishes) was soaked in one of the above prepared Metarhizium anisopliae suspension in a rectangular plastic container (17 x 17 X 9 cm depth), with intermittent hand mixing, for one hour. Excess moisture was removed by filtration through a Buchner funnel and the compost air dried (at laboratory temperature) for one hour. Six (9cm diameter) Petri dishes were then filled with the treated compost. Five 4th and 5th instar larvae were transferred to each Petri dish. One half were incubated in the dark at 25°C and the other half kept in the glasshouse where the temperature fluctuated between 18 and 27°C. The whole procedure was repeated for all 5 composts tested. Control larvae were treated as above except the compost was treated with 0.05% Aq. Tween only. The whole experiment was repeated twice. Results
  • Figure 9 shows the percentage mortality of Tenebrio moli tor larvae 3 to 5 days post inoculation (DPI) in each of the different compost tested and incubated at a constant temperature of 25oC, which composts had been previously soaked in the Metarhizium anisopliae (strain V275) conidia suspension.
  • Figure 10 shows the percentage mortality of Tenebrio moli tor larvae 3 to 6 days post inoculation (DPI) in each of the different compost tested and incubated under fluctuating temperatures of 18 to 27oC, which composts had been previously soaked in the Metarhizium anisopliae (strain V275) conidia suspension.
  • FIG. 11 shows the percentage mortality of Teneibrio moli tor larvae 3 to 7 days post inoculation (DPI) in each of the different compost tested and incubated at 25°C, which composts had been previously soaked in the Metarhizium anisopliae (strain V275) suspension.
  • Mortalities were significantly higher at constant 25°C than under glasshouse conditions where the temperature fluctuated between 18 and 27°°C (see Figures 9 and 10) .
  • Tenebrio larval mortality in seed and potting compost (SP) 5 DPI was 100% and 73% at 25oC and 18-27°, respectively. More larvae were killed if the conidia had been mixed into the soil as opposed to conidia on broken rice (compare Figures 9 and 11) . This may have been because of better distribution of conidia suspension through the soil profile.
  • the broken rice at the soil surface was often colonised by saprophytic fungi (possibly Mucor or Rhizopus) .
  • Example 5 Effect of different potting composts on the efficacy of Metarhizium anisopliae against vine weevil larvae - Pot trials where inoculum is applied to surface
  • Metarhizium anisopliae inoculum used consisted of either : A) Air-dried conidia suspended in 0.05%Aq. Tween 80 to a concentration of 1x108 conidia/ml; or B) Conidia produced on broken rice.
  • Viability was determined by inoculating 10 ⁇ l of 1x107 conidia/ml on a thin layer of SDA (ca 200 ⁇ l media on one slide) .
  • the inoculated slides were incubated in a plastic box lined with moist filter tissue paper at 25DC for 20 hrs in the dark.
  • the slide was examined using a microscope (X40 objective) and conidia were considered to have germinated if they produced a germ tube half the length of the spore.
  • Plants were checked daily and when required irrigated with 30 ml of water. After ten days incubation, the plants were removed from the pot and larvae removed from the soil. The number of live, dead and mycosed larvae calculated. Live larvae were placed in Petri dishes containing moist compost to see if these were killed by the fungus. Dead larvae were placed in Petri dishes lined with moist filter paper to encourage fungal emergence and external sporulation.
  • Figure 12 shows the percentage mortality (10 DPI) of Otiorhynchus sulca tus (vine weevil) larvae treated with either Metarhizium anisopliae conidial suspension or Metarhizium anisopliae conidia sporulating on broken rice, both applied as a drench to the surface of different composts.
  • Control mortality was less than 10% except in Irish moss peat where it reached 18%. Most of these were 2nd instar larvae.
  • Example 6 Effect of different potting composts on the efficacy of Metarhizium anisopliae against vine weevil larvae - Pot trials where inoculum is mixed into the compost
  • Control composts were treated with 0.05% Aq Tween only. An additional 20 ml of 1x108 conidia/ml inoculum was applied to each pot (except controls which received 0.05% Aq. Tween) . This was to ensure that the soil around the root ball also contained inoculum. Plants were checked daily and if required they were irrigated with 30 ml of water. Watering was kept to a minimum to make sure inoculum was not washed out of the compost.
  • Otiorhynchus sulca tus (vine weevil) larvae were placed on the soil surface at the base of the seedling, 3 days after transplanting. Plants were kept in the glasshouse where temperatures fluctuated between 18- 25°C. Plants were irrigated with 30 ml of water on alternate days. Plant health was monitored daily. Fourteen days after the larvae were exposed to the various treatments, larval mortality was determined as hereinbefore described in example 5.
  • Figure 13 shows the percentage mortality (14 DPI) of Otiorhynchus sulca tus (vine weevil) larvae in different composts treated with Metarhizium anisopliae conidial suspension.
  • control were exposed to Otiorhynchus sulca tus (vine weevil) eggs and Otiorhynchus sulca tus (vine weevil) 1st,
  • Figure 14 shows the number of Otiorhynchus sulca tus (vine weevil) larvae recovered 14 DPI from compost of plants whose compost had been treated with Metarhizium anisopliae conidia suspension and from compost of untreated plants (control) .
  • the compost of the treated and untreated plants had been infested with Otiorhynchus sulcatus (vine weevil) eggs and Otiorhynchus sulca tus (vine weevil) 1st instar larvae.
  • Figure 15 shows the mortality of Otiorhynchus sulca tus (vine weevil) 2nd/3rd and 4th instar larvae exposed to compost which had been treated with Metarhizium anisopliae conidia suspension and untreated compost (control) .
  • Mortality of 2nd/3rd versus 4th instar larvae in pots treated with Metarhizium anisopliae was 70% and 80%, respectively. Control mortalities were ca. 10% and 20% for 2-3rd and 4th instar larvae, respectively ( Figure 15) . Two control plants were severely damaged when exposed to 4th instar larvae. These plants were wilting. Close examination showed feeding damage to the stem. Growth of plants whose compost had been treated with Metarhizium anisopliae was vigorous and the root ball showed little or no sign of damage. In control plants, root growth was approximately half of the Metarhizium anisopliae treated plants .
  • Example 8 Release characteristics of components of organic fertilisers for sustained nitrogen, phosphorus and potassium nutrition.
  • Ground soya bean seed ⁇ Glycine max Ground soya bean seed ⁇ Glycine max
  • ground castor bean seed Ricinus Communis
  • Dolomitic limestone was also added to the peat.
  • One litre of peat was packed into a cylinder (leaching column) with two replicates for each of the different treatments.
  • the packed peat was leached with distilled water and one litre of leachate of the liquid was collected.
  • the leachate was then analysed for total nitrogen (NH4-N, + N03-N) , phosphorus and potassium concentration. Details of the methodology for these leaching columns are as described in Prasad M & Woods M.J. 1971, J.Agr.Food Chem. 19:96-98.
  • Results Figure 16 shows the progressive release of nitrogen, phosphorus and potassium from peat treated with different concentrations of soya bean seed and castor bean seed.
  • Figure 16 indicates that both ground soya bean and ground castor bean have slow release properties for nitrogen, phosphorus and potassium, which when applied to plants will give sustained nutrition.
  • the release rate of nutrients from soya is faster than from castor.
  • soluble fertiliser eg calcium ammonium nitrate
  • Figure 17 shows the percentage mortality (0-14 DPI) of R. appendicula tus when exposed to M. anisopliae V245 or V275.
  • the LT50 for male ticks exposed to V275 is ca 6.5 days and for females ca. 7-5 days which suggests that females are slightly more susceptible than males. Females were also more susceptible than males when exposed to V245. However, V275 appears to be more aggressive than V245. The LT50 of males exposed to V245 is 9 days whereas that for females was ca 10 days.
  • Example 10 Susceptibility of adult Riphicephalus apendiculatus and nymphs Riphicephalus appendiculatus to the entomopathogenic fungus Metarhizium anisopliae
  • Figure 18 shows the susceptibility of adults and nymphs exposed to strain V245 and V275.
  • Figure 18 shows the emergence of V245/V275 from infected adults and nymphs of R . appendicula tus t .
  • V275 was more aggressive than V245. There was not much difference in the susceptibility of adults and nymphs. The pathogen emerged from dead ticks 3-5 days after death.
  • Example 11 Determination whether soft ticks are more susceptible than hard ticks to entomogenous fungi
  • the soft tick Orni thodorous moutaba and the hard tick Ixodes ricinus were immersed in spore suspension of 108 conidia/ml. Isolate V245 and V275 were tested.
  • Figure 20 shows the cumulative mortality of soft and hard ticks treated with M. anisopliae strains V245 and V275.
  • Example 12 Determination of whether engorged sp Ixodes are more susceptible to M. anisopliae than starved ticks
  • the ticks were prepared and inoculated by immersion.
  • Fig 21 shows the susceptibility of starved and engorged Ixodes hexagonous to M. anisopliae .
  • Example 13 Effect of different doses of M. anisopliae and three different application methods on the control of vine weevil .
  • Impatiens F2 hybrid Safari Mixed
  • Polyanthus Pacific Giants and Cyclamen (Miracle White and Miracle Deep Salmon) were purchased from Ball Colegrave Ltd, UK.
  • Two inoculum doses corresponding to IxlO 10 and IxlO 8 conidia/litre of compost were used.
  • Drench - dry conidia are suspended in 0.03% Aq. Tween 80 to a final concentration of IxlO 11 and IxlO 9 conidia/ml, then 50ml applied as a drench per pot.
  • Mulch - dry conidia are uniformly mixed into compost so the mulch contains IxlO 11 or IxlO 9 conidia/litre. Then 50ml of this preparation is applied to compost surface.
  • Plants were kept in glasshouses where temperature varied from 13°C to 30°C during the experimental period. However, average day temperature was 22-27°C and night temperature remained at 14-18°C. Plants received an average of 14 hours daylight (14:10 hours light and dark photoperiod) . Frequent "damping down” was necessary during warm, sunny spells .
  • Tables 13.3 identifies the trials conducted on two varieties of cyclamen. The experiment was repeated for each variety twice.
  • Each pot was inoculated with 15 melanized eggs of Vine weevil 2 weeks after the seedling transplantation.
  • Each pot contained ca.0.5 litre of BNM
  • Multipurpose compost Plants were destructively assessed for number of live larvae per pot 4 weeks post inoculation.
  • Control M. anisopliae gave 100% and ca. 90% control at 1x1010 conidia/litre of compost, respectively. At the higher dose, 100% control was obtained irrespective of the application method. Minor differences were noted at the lower dose; premixed, drench and mulch gave 98%, 95% and 92% control, respectively. On average 7 healthy, larvae were recovered from untreated control pots.
  • M. anisopliae depended on the method of application and dose.
  • Metarhizium gave little to moderate protection when used at the lower dose (IxlO 8 conidia/litre of compost) . Approximately 20-40% control was achieved irrespective of the method of application. There were no significant differences using BNM Seed & Potting and Multipurpose composts .
  • Metarhizium anisopliae gave 53-86% control of vine weevil larvae in the young cyclamen plants ( ⁇ 2 months old) but control was slightly influenced by the cultivar. Better control was obtained in Cyclamen "Miracle White” than "Deep Salmon". Best control was achieved using a dose of IxlO 10 conidia/litre of compost applied as a drench; 79-86% control in Miracle White and 80-81% control in Deep Salmon (Figs 20 & 22) . Premixed applications of M. anisopliae were moderately effective resulting in 62-74% control in Miracle White and 61-75% control in Deep Salmon. Mulch treatments were least effective, although significantly better than untreated controls. Mulch applications resulted in 53-55% control in Miracle White and 59-71% control in Deep Salmon plants. Imidacloprid applied as drench provided 90-92% control in Miracle White and Deep Salmon, respectively.

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Abstract

Cette invention concerne une composition permettant de combattre un ou plusieurs ravageurs nocifs pour la matière végétale présente dans un milieu de croissance. Cette composition comprend: (i) au moins une souche du champignon Metarhizium présente en quantité suffisante pour combattre les ravageurs présents dans ou sur le milieu de croissance ; et (ii) au moins un agent capable d'assurer une fonction de nutrition pour ledit matériau végétal présent dans ledit support de croissance. Cette composition est de préférence exempte de biote antagoniste.
EP02722455A 2001-04-27 2002-04-29 Lutte biologique contre des ravageurs endoges Withdrawn EP1387613A1 (fr)

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GB0110314A GB0110314D0 (en) 2001-04-27 2001-04-27 Biological control of soil dwelling pests
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GB0128962 2001-12-03
GB0128962A GB0128962D0 (en) 2001-04-27 2001-12-03 Biological control of soil dwelling pests
PCT/GB2002/001940 WO2002087344A1 (fr) 2001-04-27 2002-04-29 Lutte biologique contre des ravageurs endoges

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WO2008062413A2 (fr) * 2006-11-21 2008-05-29 Mitam Ltd. Formulations de champignons entomopathogènes pour la lutte contre les insectes
WO2010091337A1 (fr) 2009-02-06 2010-08-12 Cornell University Souches de trichoderma qui induisent une résistance à des maladies végétales et/ou augmentent la croissance des plantes
EP2561757A1 (fr) * 2009-03-25 2013-02-27 Bayer CropScience AG Combinaisons de matière active nématicide comprenant du fluopyram et de l'ethiprole
CN102260103A (zh) * 2011-04-29 2011-11-30 山东理工大学 一种抗菌蝇食用菌生物培养基
CN102234619B (zh) * 2011-06-16 2013-02-27 四川省农业科学院植物保护研究所 一种防治菌核病的生防菌盾壳霉Cm2004菌株及其制备方法和应用
UA119331C2 (uk) 2013-11-08 2019-06-10 Новозімес Біоаґ А/С Композиції та способи для обробки від шкідників
CN107446955A (zh) * 2017-08-25 2017-12-08 陕西省微生物研究所 以中药废弃物为基质的抗虫土壤调节剂及其生产方法
CN108675868A (zh) * 2018-07-07 2018-10-19 安徽爱能洁生物科技有限公司 一种基于植物秸秆可防害虫的有机复合肥料
GB2577250A (en) * 2018-09-14 2020-03-25 Bionema Ltd Insect-pathogenic fungus, spores, composition and use of same
CN110256168A (zh) * 2019-07-23 2019-09-20 甘玮 杀虫真菌有机肥

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CZ292347B6 (cs) * 1999-04-27 2003-09-17 Lovochemie, A. S. Průmyslové hnojivo zajišťující současně výživu i ochranu rostlin

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