WO2002087344A1

WO2002087344A1 - Biological control of soil dwelling pests

Info

Publication number: WO2002087344A1
Application number: PCT/GB2002/001940
Authority: WO
Inventors: Munoo Prasad; Tariq M. Butt
Original assignee: Uws Ventures Limited; Bord Na Mona Horticulture Limited
Priority date: 2001-04-27
Filing date: 2002-04-29
Publication date: 2002-11-07
Also published as: EP1387613A1

Abstract

A composition for substantially combating one or more pests detrimental to plant material present in a medium of growth, which composition comprises: (i) 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; and (ii) at least one agent capable of providing nutritional function to said plant material present in said medium of growth. The composition is preferably substantially free of antagonistic biota.

Description

Biological Control Of Soil Dwelling Pests

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.

Otiorhynchus sulcatus, commonly known as 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 . As with the vine weevil, 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.

It is, in principle, advantageous to use biological control agents instead of chemical control agents because of the limited impact that biological control agents have on the environment compared to chemical control agents.

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.

It is an object of the present invention, however, to provide an improved method of treating plant material present in a medium of growth and more particularly an improved method of treating plant material whereby a biological control material is employed for substantially combating soil dwelling pests to which the plant material is susceptible to suffering damage therefrom. The biological control agent used in the present invention is the fungus Metarhizium .

According to the present invention, therefore, there is provided 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, wherein 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.

According to a preferred aspect of the present invention, there is provided a composition 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.

"Medium Growth" is hereinafter referred to as "growth medium" and 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 . In the treatment of ticks, the composition according to the present invention, may be applied to the soil or vegetation in tick habitats. Alternatively, according to a further aspect of the present invention, Metarhizium anisopliae conida may be applied directly to the soil or vegetation in tick habitats.

Therefore 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. Advantageously, ectoparasites, such as ticks, falling in the bedding could become infected with the pathogen.

Accordingly, there is provided 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.

Typically, the bedding includes, peat, peat-free compost, sand, hay, straw or the like. The bedding may also include other suitable vegetation.

As well as being pathogenic to the aforementioned larvae and substantially reducing egg laying in or on the growth medium, 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.

The term "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. Examples of 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.

Preferably 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.

It is preferred that 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.

Substantially as hereinbefore described 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 .

Preferably, the compost further includes 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. Such 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) .

The control agents are preferably biocontrol agents which advantageously combat one or more of arthropod pests, weeds and diseases. Advantageously, as the different control agents do not have to be applied separately, 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.

Preferably, the biological control agents work synergistically with other components of the composition.

Advantageously, 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 ,

Lagenidium giganteum, Myrothecium verrucaria , Duddingtonia flagrans, Verticillium chlamydosporium .

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 la: Fungi developed or being developed for the biological control of diseases (Butt & Copping 2000; Butt T.M., Jackson, C. . & Magan, N..(eds). 2001. Fungi as biocontrol agents: Progress, Problems and Potential. CAB International, Wallingford, Oxon, ϋ.K)

Table lb - Fungi developed or being developed for the biological control of pests

Entomogβnous Fungi

Table lc - Fungal Agents being developed or commercially available for the Biological Control of Weeds (Butt et al . , 1999; Butt & Copping 2000; Templeton & Heiny, 1994).

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. When 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.

Different types of compost may be used in the present invention. For example, 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.

In a particularly preferred aspect of the present invention there is provided 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.

In an alternative embodiment of the present invention 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.

It is preferred that 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.

The present invention will now be illustrated with reference to the accompanying figures, which are given by way of example only.

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.

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 22 shows a graph indicating M. anisopliae control of vine weevil larvae in pottered impatiens in multipurpose compost (plants destructively assessed for number of live larvae per pot 8 weeks post inoculation - Dose a=1010 conidia/litre of compost, Dose b=108 conidia/litre of compost .

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.

Examples Method of culturing and harvesting Metarhizium and other fungi

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

(15g/L)) (spores and/or mycelial plug) and broken rice was autoclaved, inoculated with fungi (spores and/or mycelial plug) and incubated at 20-25oC for 14 days. After the fungi had sporulated, the fungi conidia were harvested by sieving.

Example 1 Identification of virulent strains of Metarhizium anisopliae against Vine Weevil First Petri dish bioassay

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. For comparison the pathogenicity of the fungus 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 .

Table 2

Conidia of Metarhizium anisopliae (strains V208, V245 and V275) and Beauveria bassiana (strain Bbl3) were cultured and harvested as described above and suspended in 0.05% v/v Aq. Tween 80 to a concentration of 107 conidia/ml. Otiorhynchus sulca tus (vine weevil) larvae at the 4th and 5th instar stage of development were used in this bioassay. The larvae were divided into groups of 5 larvae per group and each group of larvae was immersed in either 15ml of one of the above conidia suspensions or 0.05% v/v Aq. Tween 80 (control) for 10 seconds. 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. Referring to Figure 1, 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.

Second Petri dish bioassay

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. For comparison 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 .

Table 3

Results

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) .

Results from the second Petri dish bioassay can be seen in Figures 3 and 4. 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.

The following different types of compost were used in the experiment described hereafter:

1. Shamrock Multipurpose Peat Free (MP) ;

2. Shamrock Irish Peat Moss (pH 3.8 to 4.4) (IPM) ;

3. Shamrock Seed and Modular compost (SM) ;

4. Shamrock Seed and Potting compost (SP) ; and 5. Glasshouse (unsterilised) compost (GH) .

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.

Method 1

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.

Method 2 Tenebrio molitor larvae were inoculated into a Petri dish

(5 larvae per dish) which had been filled with one of the different composts mentioned above, which compost had been previously immersed in 100 ml of the conidia suspension for one hour, filtered through a Buchner funnel to remove excess moisture, and then air dried for one hour. For a control, larvae were inoculated into Petri dishes (5 larvae per dish) filled with one of the composts mentioned above which had been immersed in 0.05% v/v Aq. Tween 80 only.

Each treatment in the above described methods 1 and 2 was replicated 3 times. The Petri dishes from methods 1 and 2 were all incubated at 25°C in the dark. The Petri dishes were examined daily for larva movement, larva mycosis and larva mortality.

Results

Method 1

The percentage mortality of Tenejbrio molitor larvae treated using method 1 are shown 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 and 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.

There were no mortalities seen for larvae which had been immersed in 0.05% v/v Aq. Tween 80 (control) (results not shown in Figures 5 and 6) .

The results shown in Figures 5 and 6 indicate that none of the composts tested prevented infection of Tenebrio moli tor larvae by Metarhizium anisopliae (strains V245 and V275) .

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.

There were no mortalities seen for larvae inoculated in compost which that had been previously immersed in 0.05% v/v Aq. Tween 80 (control) (results not shown in Figures 7 and 8) . The results shown in Figures 7 and 8 indicate that Metarhizium anisopliae strain V245 is more virulent than strain V275 because there was 100% mortality of larvae inoculated in all types of compost immersed in Metarhizium anisopliae strain V245 conidia suspension by 4 DPI. By 5 DPI there was 100% mortality of larvae inoculated in all types of compost immersed in Metarhizium anisopliae (strain V275) conidia suspension. These results indicate that all types of compost tested which have been previously immersed in Metarhizium anisopliae strains V245 and V275 conidia suspension effectively kill Tenebrio molitor larvae within 5 days of introduction of the larvae into the compost.

The results from method 1 and 2 above indicate that mortality of Tenebrio moli tor larvae was high whether the larvae were directly inoculated with Metarhizium anisopliae

(strains V245 and V275) or whether the larvae were exposed to Metarhizium anisopliae (strains V245 and V275) incorporated into the compost.

Example 3 Effect of compost leachates on conidial germination

Two sets of 25 grams of each of the composts as hereinbefore described in Example 2 were suspended in 50ml

^■ of distilled water in a 250ml Conical flask and incubated in a rotary shaker (Sanyo-GallenKamp) at 120rpm and 24

C. The suspensions were filtered (using Whatmans No. 1) after 1 days suspension (Treatment A) and after 7 days suspension (Treatment B) . The filtrate was collected in small plastic tubes.

Once all the compost particles settled at the base of the small plastic tubes, 1 ml of compost leachate was transferred to 1.5 ml Eppendorf tubes and inoculated with 100 ml of 107 conidia/ml of Metarhizium anisopliae strains V275 and V245 suspension. Each treatment was replicated three times and incubated at 25°C in the dark for 24hrs.

Following incubation, lOμl of the suspension in the Eppendorf tubes was applied to glass microscope slides which were coated with a thin layer of Sabouraud Dextrose Agar (SDA) medium. All the slides were kept in closed plastic containers lined with moist tissue paper to provide humidity for fungal growth. Slides were examined under the microscope at 12, 20 and 24 hrs post inoculation (HPI) . 100 conidia from each treatment were examined with 3 replicates per treatment.

Results

The effect of compost leachates on the germination and growth of Metarhizium anisopliae V245 and V275 are summarised in Table 4. No inhibiting effect of compost leachate was observed on conidial germination, however, germ tubes and hyphae were slightly inhibited by leachates taken from 7 day-old compost suspensions (Treatment B) . Except for the GH compost, there were no significant differences between compost types. The GH compost was slightly more inhibitory than the other composts tested. Differences in growth were more obvious at 12 and 20 HPI onto the SDA-coated glass microscope slides. There were no clear differences between treatments 24 HPI onto SDA-coated slides.

Table 4: Effect of compost leachates on the germination and growth of Metarhizium anisopliae V245 and V275

Treatment A - leachates from compost suspended in water for 1 day; and

Treatment B - leachates from compost suspended in water for

7 days.

Table 4

Garden 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 Metarhiziυm 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 10⁸ conidia ml^"1 . Alternatively, 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

The percentage mortality of Tenebrio moli tor larvae treated with Metarhizium anisopliae (strain V 275) suspension, which Metarhizium anisopliae had been isolated using ODA as described above are shown in Figures 9 and 10. 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.

The percentage mortality of Tenebrio moli tor larvae treated with the suspension of Metarhizium anisopliae (strain V 275) sporulating on broken rice grain as described above is shown in Figure 11. Figure 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) . For example, 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) .

There were no mortalities seen for larvae inoculated in compost which had been previously immersed in 0.05% v/v Aq. Tween 80 (control) (results not shown in Figures 9 to 11) .

The results shown in Figures 9 to 11 indicate that both free Metarhizium anisopliae (strain V275) conidia and Metarhizium anisopliae (strain V275) conidia on broken rice caused larval mortality in all the composts tested and significant mortalities were achieved 5 DPI whatever the treatment .

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

Adult Otiorhynchus sulcatus (vine weevil) were fed Euonymus leaves and were incubated at 22-24D C and 14:10 hrs light and dark photoperiod. Eggs were collected twice weekly and immediately transferred to young potted Polyanthus ( Primula polyanthus) plants. Once the plant wilted, pots were destructively checked for young larvae. First instar larvae were transferred to young potted Polyanthus. Second instar larvae were transferred to chambers containing seed and potting compost. These larvae were fed pieces of carrot. Plants used in this example were Impatiens (F2 hybrid Safari Mixed) seedlings (ca. 14 days old) transplanted in plastic pots (11 cm top diameter, 9cm high, 7.5cm bottom diameter) containing one of the composts as hereinbefore described in example 2. Each pot held ca. 0.5 litre of moist compost.

Metarhizium anisopliae preparation

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.

To determine the amount of conidia produced on the rice, 5g of the rice grain was suspended in 50ml of 0.05%Tween 80 and stirred vigorously using a magnetic stirrer. The concentration of conidia was determined using an improved Neubauer haemocytometer (Weber Scientific Ltd. UK) .

Viability was determined by inoculating 10 μl of 1x107 conidia/ml on a thin layer of SDA (ca 200 μl 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.

Briefly, lg broken rice in 10 mis Aq Tween yielded 1.38x108 conidia/ml. Of this, only 70% were viable so the actual was 0.96 XlO⁸ conidia/ml. We worked on the basis that lg of broken rice yielded 1 X 10⁸ conidia/ml. Dry conidia had a viability of 95%.

Inoculation of pots

An equal volume of compost was used to fill the pots. One Impatiens seedling was planted per pot and kept at 18-27 C and 12:12 light and dark photoperiod in the glasshouse. After 3 weeks, ten 2nd and 3rd instar larvae were placed on the compost. Inoculum or 0.05% Aq Tween (control) was applied 2hrs later. This consisted of either:

1) 20 ml of conidia suspension (inoculum A) applied as a drench to each pot; or

2) 2 g of sporulating broken rice (inoculum B) applied to each pot then drenched with 20ml 0.05% Aq Tween (= 1x108 conidia/ml when dissolved in 20 ml of 0.05% Aq. Tween 80)

Each treatment was replicated five times.

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.

Results

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.

Conidial suspension (20ml) applied as a drench caused higher mortalities than the use of sporulating broken rice grain (Figure 12) . Application using broken grain resulted in mortalities ranging between 24 and 34%. Highest mortalities were obtained in SM (46%) and SP (42%) compost using a spore drench.

Nearly all the dead larvae were found in the top 2-5 cm of compost and might have been in direct contact with the inoculum throughout the experimental period. The rest of the larvae were found at different depths. Saprophytic fungi colonised the surface of broken rice and may explain the poor mortality rate of larvae obtained using this form of the inoculum.

Although the mortalities we report are low it should be noted that all the live larvae recovered were ultimately killed by the fungus. Sporulation usually occurred 2-3 days after death.

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

The different composts used in this example were as hereinbefore described in example 2, however glasshouse (GH) compost was used in initial experiments but was subsequently excluded because seedlings failed to establish in this compost (possibly due to "damping off") . Each of the different composts (enough to fill ten pots) were spread on a plastic sheet and air-dried. Metarhizium anisopliae inoculum (500ml of 1x108 conidia/ml) was applied as a conidial suspension (so effectively each pot received 50 ml of 1x108 conidia/ml) . The inoculum was mixed into the compost by hand as hereinbefore described in example 4 then Impatiens seedlings were potted in 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.

Two 2nd or 3rd instar 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.

Results

Figure 13 shows the percentage mortality (14 DPI) of Otiorhynchus sulca tus (vine weevil) larvae in different composts treated with Metarhizium anisopliae conidial suspension.

Increasing the volume (and dose) of the inoculum from 20ml to 50ml resulted in substantially higher mortality (figure 13) . Mortality was 70% in seed and potting (SP) and Irish peat moss (IPM), and 60 and 50 % in seed and modular (SM) and multipurpose (MP) composts, respectively. Control mortality was ca. 10%, mainly of 2nd instar larvae (Figure 13) . Example 7 Susceptibility of different instar larvae of Otiorhynchus sulcatus (vine weevil) to Afetarhizium anisopliae

The same method was used in this example as was hereinbefore described in example 6 except the assays in this example were carried out in seed and potting (SP) compost only.

Plants whose compost had been treated with Metarhizium anisopliae conidia suspension and untreated plants

(control) were exposed to Otiorhynchus sulca tus (vine weevil) eggs and Otiorhynchus sulca tus (vine weevil) 1st,

2nd, 3rd and 4th instar larvae. The number of eggs or larvae per pot depended on the developmental stage (see table 5 below) .

Table 5

Results

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) .

Because of their small size, assessment was based on the recovery of live larvae. The number of larvae recovered from the compost of control pots was significantly higher than the number of larvae recovered from the compost of pots treated with Metarhizium anisopliae conidia suspension when both the control and treated pots had been infested with Otiorhynchus sulca tus (vine weevil) eggs (Figure 14) . On average 9.4 larvae were found in each of the control composts which had been infested with eggs and only one larvae was recovered from the Metarhizium anisopliae treated compost which had been infested with eggs.

In the case of pots infested with 1st instar larvae, the number of live larvae recovered 14 dpi was 3.6 in the control compared with 0.6 in Metarhizium anisopliae treated pots (Figure 14) . No significant difference in plant or root growth was observed in the different treatments. Most of the larvae were recovered from the middle of the pot close to the young developing roots.

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) and ground castor bean seed (Ricinus Communis) was mixed into peat at a concentration of 5g or lOg per litre of peat. 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. At various time intervals, 0,2,4,8,12 and 16 weeks, 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. In comparison, when soluble fertiliser (eg calcium ammonium nitrate) is used almost all the nutrients get released in two weeks using this methodology (as shown in above mentioned reference by Prasad M & Woods M J) .

Example 9 - Identification of virulent strains of Metarhizium anisophliae against male and female ticks

Spore suspensions of Metarhizium anisopliae V245 and V275 were prepared as hereinbefore described in Example 1. Adult Ripicephal us appendiciva tus were inoculated by immersion in a solution of fungal conidia. These were transferred (15 ticks of each sex) to incubating chambers and incubated at 28°C. The chambers were checked daily for mortalities and fungal emergence. Dead ticks were removed and transferred to Petri dishes lined with moist filter paper to encourage fungal emergence and sporulation.

Results

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

The same method was used in this Example as hereinbefore described in Example 7. Results

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 .

The results determine that 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.

Results

Figure 20 shows the cumulative mortality of soft and hard ticks treated with M. anisopliae strains V245 and V275.

The results indicate that the soft tick is less susceptible to Metarhizium than hard ticks.

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.

Results

Fig 21 shows the susceptibility of starved and engorged Ixodes hexagonous to M. anisopliae .

The results indicate that engorged ticks are more susceptible than starved ticks. Furthermore, strain V245 appeared to be more aggressive to engorged ticks than V275.

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) were grown and maintained at University of Wales, Swansea. Polyanthus Pacific Giants and Cyclamen (Miracle White and Miracle Deep Salmon) were purchased from Ball Colegrave Ltd, UK. Two inoculum doses corresponding to IxlO¹⁰ and IxlO⁸ conidia/litre of compost were used.

Fungal inoculum was applied as follows:

(1) Component of compost (premixed) - dry conidia are uniformly mixed into the compost before transplanting/potting.

(2) Drench - dry conidia are suspended in 0.03% Aq. Tween 80 to a final concentration of IxlO¹¹ and IxlO⁹ conidia/ml, then 50ml applied as a drench per pot. (3) Mulch - dry conidia are uniformly mixed into compost so the mulch contains IxlO¹¹ or IxlO⁹ conidia/litre. Then 50ml of this preparation is applied to compost surface.

Each treatment was replicated 25 times for Impatiens and Polyanthus and 15 times for both varieties of cyclamen. Control plants received only 0.03% Aq. Tween. A commercial chemical pesticide (Provado, Bayer a.i. 5% w/w imidacloprid) was also included in trials. It was prepared by dissolving 2.8g Porvado in llitre of water. This was enough to treat 10 liters of compost - at 50ml pesticide per pot. Tables 13.1-13.3 summarise the different treatments. The whole experiment was repeated twice.

Once each pot had been treated with the fungus then 15- melanized vine weevil eggs were gently placed around the base of the young plant. Eggs were added 2 weeks after seedling transplantation of polyanthus and Cyclamen. Seedlings were approximately 2 weeks old at the time of transplantation. Impatiens (cuttings) was inoculated with eggs approximately 6 weeks after transplantation.

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 .

All treatment were destructively assessed for the number of live larvae, dead larvae, mycosed larvae. This was 4 weeks after application of Metarhizium in cyclamen and polyanthus but 8 weeks for Impatiens.

Live larvae from treated pots were transferred to special Petri dish chambers to see if they would ultimately die because of fungal infection. Dead larvae were placed in Petri dishes lined with moist filter paper to encourage emergence of the white mycelium of the pathogen which ultimately produces greenish conidia. Table 13.1 identifies the trials conducted on Impatiens

MP = Multipurpose Compost SP = Seed & Potting Compost

The results are given in Figure 22

Table 13.2 identifies the trials conducted on Polyanthus The experiment was repeated.

The results are given in Figures 23 & 24. Referring to figure 23, M. anisopliae was applied at two doses and three application methods. Each pot was inoculated with 15 melanized vine weevil eggs 2 weeks after seedling transplantation. Each pot contained ca.0.5 litre of Bord Na Mona (BNM) seed and potting compost. Plants were destructively assessed for number of live larvae per pot 4 weeks post inoculation. Dose a=10¹⁰ conidia/litre of compost. Dose b=10⁸ conidia/litre of compost. Referring to figure 24, M. anisopliae was applied at two doses and three application methods. 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 multipurpose compost. Plants were destructively assessed for number of live larvae per pot 4 weeks post inoculation. Dose a=10¹⁰ conidia/litre of compost. Dose b=10⁸ conidia/litre of compost.

Tables 13.3 identifies the trials conducted on two varieties of cyclamen. The experiment was repeated for each variety twice.

The results are given in Figures 25,26,27 and 28 Referring to figure 25, M. anisopliae was applied at two doses and three application methods. Each pot was inoculated with 15 melanized eggs of Vine weevil 2 weeks after seedling transplantation. Each pot contained ca.0.5 litre of BNM Seed and Potting compost. Plants were destructively assessed for number of live larvae per pot 4 weeks post inoculation. Dose a=10¹⁰ conidia/litre of compost. Dose b=10⁸ conidia/litre of compost.

Referring to figure 26, M. anisopliae was applied at two doses and three application methods. 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 multipurpose compost. Plants were destructively assessed for number of live larvae per pot 4 weeks post inoculation. Dose a=10¹⁰ conidia/litre of compost. Dose b=10⁸ conidia/litre of compost.

Referring to Figure 27, M. anisopliae was applied at two doses and three application methods. 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 Seed and Potting compost. Plants were destructively assessed for number of live larvae per pot 4 weeks post inoculation. Dose a=10¹⁰ conidia/litre of compost. Dose b=10⁸ conidia/litre of compost.

Referring to Figure 28, 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.

Dose a=10¹⁰ conidia/litre of compost. Dose b=10⁸ conidia/litre of compost. Results

Potted Impatiens :

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.

Plant appearance. Mild symptoms of damage were apparent during the early stages, however, at the end of the trail all the treated plants appeared healthy. The root system of plants treated with M. anisopliae had well-developed root systems irrespective of the method of application. Presumably, the established plants (6 week old cuttings) had an extensive fibrous root system to sustain vine weevil damage before the fungus killed the larvae. In contrast, untreated plants started wilting 2 months after inoculation with the vine weevil eggs. Damage to the root system was extensive. Larvae ate the roots then invaded the collar or base of the stem. Plants treated with IxlO⁸ conidia/litre of M. anisopliae has a slight reduced root system compared with those treated with 1x1010 conidia/litre of compost.

Polyanthus :

Effiacy of M. anisopliae against vine weevil in potted polyanthus .

Metarhizium treated polyanthus plants appeared healthier than untreated controls. However, the efficacy of M. anisopliae depended on the method of application and dose.

A dose of IxlO¹⁰ conidia/litre of compost gave 50-93% control. The drench application gave the best control (88-

93% control) . Premixed and mulch treatments gave 64-82% and 50-66% control, respectively, Imidacloprid (chemical pesticide) gave 85% control.

Metarhizium gave little to moderate protection when used at the lower dose (IxlO⁸ 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 .

Cyclamen :

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 IxlO¹⁰ 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.

A dose of IxlO⁸ conidia/litre of compost was insufficient to give adequate protection to young cyclamen plants. The highest protection achieved was 40%.

Claims

1. Use of 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, wherein 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.

2. Use according to claim 1, wherein the Metarhizium is Pathogenic to vine weevil larvae, root weevil larvae, larvae, maggots and/or eggs of mushroom flies, sciarids, phorids, fungus, gnats ticks and other pests which may be present in or on the growth medium, or in or on the plant material present in the growth medium.

3. Use according to Claim 1 or 2, wherein the Metarhizium is capable of preventing establishment of pest such as vine weevil, mushroom flies, sciarids, phorids, fungus gnats and ticks in or on the growth medium.

4. Use according to any of Claims 1 to 3, wherein at least one Metarhizium strain is of the species Metarhizium anisopliae, preferably selected from V275, V245, Biogreen, V208, ARSEF 1910, Ma 23, ARSEF 817 or ARSEF 9601 .

5. Use according to any of Claims 1 to 4, wherein the Metarhizium is cultured on a solid media, fermentation on a semi-solid media, submerged fermentation and diphasic fermentation.

6. Use according to any of Claims 1 to 5, wherein at least one agent is capable of providing nutritional function to said plant material present in said medium of growth is a compost or a mulch.

7. Use according to any of claims 1 to 6, wherein the agent capable of providing nutritional function is substantially free of soil biota (such as antagonistic biota) .

8. Use according to any of claims 1 to 7, which includes a fertilizer, such as an organic fertilizer.

9. Use according to any of Claims 1 to 8, wherein the Metarhizium identified in (i) is present in an amount in the range 0.5-5.0g per litre of the agent.

10. Use according to any of Claims 1 to 9, wherein the agent is capable of providing sustained nutritional function to plant material present in a growth medium.

11. Use of a composition according to any Claims 1 to 10, wherein the composition further includes at least one control agent, which is preferably a beneficial organism for crop and/or animal protection.

12. A composition for substantially combating one or more pests detrimental to plant material present in a medium of growth, which composition comprises: (i) 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; and

(ii) at least one agent capable of providing nutritional function to said plant material present in said medium of growth.

13. A composition according to Claim 12, wherein (i) and (ii) are intimately mixed.

14. A composition according to Claim 12 or 13, wherein the Metarhizium is Pathogenic to vine weevil larvae, root weevil larvae, larvae, maggots and/or eggs of mushroom flies, sciarids, phorids, fungus, gnats, ticks and other pests which may be present in or on the growth medium, or in or on the plant material present in the growth medium.

15. A composition according to Claim 14, wherein the Metarhizium is pathogenic to ticks selected from Amblyomma , Boophilus , Dermacentor, Haemaphysalis , Hyalomma , Ixodes and/or Rhipicephalus .

16. A composition according to any of Claims 12 to 15, wherein the Metarhizium is capable of preventing establishment of pest such as vine weevil, mushroom flies, sciarids, phorids, fungus gnats and ticks in or on the growth medium.

17. A composition according to any of Claims 12 to 16, wherein at least one Metarhizium strain is of the species Metarhizium anisopliae, preferably selected from V275, V245, Biogreen, V208, ARSEF 1910, Ma 23, ARSEF 817 or 9601.

18. A composition according to any of Claims 12 to 17, wherein the Metarhizium is cultured on a solid media, fermentation on a semi-solid media, submerged fermentation and diphasic fermentation.

19. A composition according to Claim 18, wherein the Metarhizium strain is harvested and stored as air dried conidia and/or mycelium, either free or on a suitable substrate such as grain or the like.

20. A composition according to any of Claims 12 to 19, wherein at least one agent capable of providing nutritional function to said plant material present in said medium of growth is a compost or a mulch.

21. A composition according to any of claims 12 to 20, wherein the agent capable of providing nutritional function includes peat or peat-free compost.

22. A composition according to any of claims 12 to 21, wherein the agent capable of providing nutritional function is substantially free of soil biota (such as antagonistic biota) .

23. A composition according to any of claims 12 to 22, which includes a fertilizer, such as an organic fertilizer.

24. A composition according to any of Claims 12 to 23, wherein the Metarhizium and the agent are blended by hand or using a mechanical mixing apparatus.

25. A composition according to any of Claims 12 to 24, wherein the Metarhizium identified in (i) is present in an amount in the range 0.5-5.0g per litre of the agent .

26. A composition according to any of Claims 12 to 25, wherein the agent is capable of providing sustained nutritional function to plant material present in a growth medium.

27. A composition according to any of Claims 12 to 1326, wherein the agent is capable of providing nutrients to the plant material over a period of 3 to 4 months.

28. A composition according to any of Claims 12 to 27, wherein the agent provides nutrients to the Metarhizium (preferably on a sustained basis) .

29. A composition according to any Claims of 12 to 28, wherein the agent is a biodegradable agent.

30. A composition according to any Claims 12 to 29, wherein the composition further includes at least one control agent, which is preferably a beneficial organism for crop and/or animal protection.

31. A composition according to Claim 30, wherein the beneficial organism is a nematode (suitable for slug and insect control) , bacteria (suitable for pest and disease control) and or fungi (suitable for pest, weed and disease control) .

32. A composition according to Claim 30 or 31, wherein the control agent is a biocontrol agent arranged to combat one or more of arthropod pests, weeds and/or diseases.

33. A composition according to Claim 32, wherein the control agent works synergistically with other components of the composition.

34. A composition according to any of claims 32 or 33, wherein the biocontrol agents are capable of protecting seedlings, young plants, nursery and ornamental plants against pests and/or diseases.

35. A composition according to any of Claims 32 to 34, wherein the biocontrol agent for use in the biological control of diseases is Phlebiopsis gigan tea , Gliocladiun catenula tum, Gliocladium virens,

Coniothyrium minitans , Ampelomyces quisqualis , Cryptococcus albidus, Candida oleophila , Endothia parasi tica (non-pathogenic strain) , Fusarium oxysporium, Pythium oligandrum, Trichoderma harzianum, T viride, T.polysporum and/or Trichoderma harzianum .

36. A composition according to any of Claims 32 to 35, wherein the biocontrol agent for use in the biological control of pests is selected from the group consisting Verticillium, lecanii , Metarhizium anisopliae, Beauveria bassiana , Beauveria brongniartii ,

Metarhizium flavoviride, Paecilomyces fumosoroseus, Lagenidium giganteum, Myrothecium verrucaria , Duddingtonia flagrans Paecilomyces lilicanus and/or Verticillium chlamydosporium .

37. A composition according to any of Claims 32 to 36, wherein the biocontrol agent for the use in the biological control of weeds is Acremonium diospyn , Alternaria zinniae, Alternaria eichhornia , Alternaria cassiae, Cercospora rodmanii, Colletotrichum coccodes, Colletotrichum gloeosporioides f. sp cusutae, Colletotrichum gloeosporioides f. sp aeschynomene , Colletotrichum orbiculare, Chondrosterium purpureum, and/or Phytophthara palmivora .

38. A composition according to any of Claims 12 to 37, which includes a fungi arranged for the biological control of pests, diseases and/or weeds.

39. A composition according to any of Claims 12 to 38, which is in the form of a block to be added to a growth medium.

40. A plant material growth medium aid 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.

41. An aid according to Claim 40, wherein the agent capable of providing nutritional function may be soil, a mixture of at least soil and a compost or mulch.

42. An aid according to Claim 40 or 41, which further includes one or more 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.

43. A method of using in combination at least one strain of the fungus Metarhizium, together with at least one agent capable of providing nutritional function to plant material, which method comprises applying the Metarhizi um and the at least one agent in combination to a growth medium, so as to substantially combat pests .

44. A method of using a plant material growth medium aid comprising a compost or mulch intimately mixed with at least one strain of the fungus Metarhizium, which method comprises applying the product to a growth medium for substantially combating pests.

45. 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.

46. Animal bedding according to claim 45, wherein the bedding includes peat and/or peat-free compost, sand, straw, hay or other suitable vegetation.

47. Use of at least one strain of the fungus Metarhizium in the manufacture of a treated animal bedding, wherein the Metarhizium 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.

48. Use according to claim 46, wherein the bedding includes, peat, peat-free compost, sand, hay, straw or the like.

49. 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.