AU665398B2 - Preparations containing entomopathogenic nematodes for biological control of the banana weevil - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION (Original) Name of Applicant/Nominated Person: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION Actual Inventors: NEIL LEONARD TREVERROW ROBIN ANTHONY BEDDING Address for Service: DAVIES COLLISON CAVE, Patent Attorneys 1 Little Collins Street, Melbourne, Victoria 3000 Invention Title: "PREPARATIONS CONTAINING ENTOMOPATHOGENIC NEMATODES FOR BIOLOGICAL CONTROL OF THE BANANA WEEVIL" Details of Associated Provisional Application: PROVISIONAL PATENT APPLICATION No PL 1162 a 0 9 0 0 ft

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II The following statement is a full description invention, including the best method of performing to us: of this it known s 1

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2 Technical Field This invention concerns preparations which include entomopathogenic nematodes, for use in the biological control of banana weevils. More particularly, it concerns preparations containing third stage infective juvenile (J3) entomopathogenic nematodes which kill adult banana weevils when used in a bait comprising banana corms with holes therein, containing the preparation.

Background The banana borer or banana weevil, Cosmopolites sordidus, is a major pest of bananas throughout the world. Larvae of C. sordidus tunnel through banana corms (sometimes called banana rhizomes) and interfere with root initiation and weaken the plant. At low population levels, C. sordidus usually confines its attack to the corm that is left after the plant has been cut down (the residual corm). However, populations of the weevil can gradually increase to such an I 0 extent that the weevils attack the corms of the growing plants soon after flowering, thus causing a significant reduction in fruit yield.

S, C. sordidus is curreiatly controlled with insecticides (mainly organophosphates and carbamates). There has been considerable pressure to develop alternative means of r 2 control because C. sordidus has developed a significant resistance to organochiorides, and also to Sorganophosphates, and (ii) because there is increasing public concern about the use of such insecticides, particularly in banana growing areas which are close to urban development.

it 9 i 13 i-I t 3 It has been suggested by several workers that it may be possible to control C. sordidus by use of entomopathogenic nematodes.

The third stage juveniles (J3) of entomopathogenic nematodes are able to infect and kill a wide range of insects. They have been used to control various stem borers, including banana weevil (as reported by C Laumond et al, in their paper entitled "New data on the range of hosts and the parasitism of Neoaplectana carpocapsae", published in Entomophaqa, volume 24, 1979, pages 13 to 27, and by N Treverrow et al, in their paper entitled "Evaluation of entomopathogenic nematodes for control of Cosmopolites sordidus Germar (Coleoptera; Curculionidae), a pest of banana in Australia", published in Annal; of 15 Applied Biology, volume 119, 1991, pages 139 to 145). To destroy C. sordidus, third stage infective juveniles of Steinernema species enter the insect's haemocoel via natural orifices. Heterorhabditis species J3 nematodes also enter the insect through interskeletal membranes.

Once in the insect's blood, the J3 nematodes of each species release symbiotic bacteria, Xenorhabdus species, causing septicaemia and rapid death of the insect.

Subsequently, the nematodes multiply in the cadaver and escape back into the environment.

25 Large numbers of these infective third stage juvenile entomopathogenic nematodes can be reared on artificial media and adequately processed and stored (for example, using the techniques described in the specification of eo a 6 a a a a a 0 *0 9 o a c e 1 t _1 International patent application No PCT/AU88/00127) for use in the commercial control of insect pests.

Preliminary investigations with several species and strains of entomopathogenic nematodes have shown that, in laboratory experiments, J3 nematodes of the species Steinernema carpocapsae are able to infect larvae of C.

sordidus within excised residual corms in the laboratory.

However, a similar infection does not occur in the field when the J3 nematodes of this species are sprayed around the residual corm/soil interface. It has also been found that introducing such nematodes, in a water thickener, into the residual corm, via cuts or holes in the corm, gives greater access to the larval tunnels within the corm. More importantly, it has been found that adult weevils are 15 attracted to the cuts or holes, and some are parasitised by S* nematodes placed therein. P F Parniski et al, in their paper entitled "Screening of 31 Heterorhabditis pp and Steinernema spp (previously Neoaplectana) for the control of adults of Cosmopolites sordidus in laboratory trials in Tonga and Australia", to be published soon in Fundamentals of Applied Nematology, have reported the testing of the effectiveness against C. sordidus of 31 different strains/species of the entomopathogenic nematodes Heterorhabditis and Steinernema. Parniski et al introduced 25 J3 nematodes into holes drilled in corms, and found that 4 4 while all species were able to kill some adult Cosmopolites S sordidus, some species gave a higher mortality than others.

iThe nematode strains which were consistently most effective were Steinernema carpocapsae, BW strain, and a strain of Heterorhabditis zealandica, NZH. Nevertheless, it was 1 1 1 r i- 5 rarely possible to kill more than about 60 per cent of the adult weevils attracted to a corm. In addition, because sap from the corm tended to weep into many of the drilled holes, the water thickener in the nematode preparation became so diluted that the nematodes were less able to attack the weevils (almost certainly because the nematodes had less of a purchase on the medium and they tended to sink to the bottom of the hole).

An additional difficulty in using J3 entomopathogenic nematodes to control adult banana weevils is that the adult banana weevil is one of the most resistant insects to entomopathogenic nematodes. The reasons for this are the rostrum of the banana weevil is too narrow for nematode entry; (ii) the anus of the banana weevil is kept tightly closed, thus preventing nematode entry; and (iii) the elytra (wing coverings) of the banana weevil provide such a tight seal over the abdomen that it is a, It almost impossible for nematodes to crawl beneath them and then enter the spiracles.

Disclosure of the Present Invention S' The object of the present invention is to provide a formulation of entomopathogenic J3 nematodes which, when placed in holes or cuts in the corms of banana plants, or S0" in pieces of corm used as baits, significantly increases the mortality of adult banana weevils attracted to the corms or pieces of corm, without making such corms or baits less attractive to the weevils. I 6 This objective is achieved by providing a preparation which includes particles of an absorbent material (such as polyacrylamide gel) to absorb fluid that might accumulate in drill holes in banana corms or pieces of corm; and an additive which is a quantity of an oil, or a surfactant, or a non-lethal dose of an insecticide, or a mixture of such additives, or any other additive which causes the banana weevil to move the elytra or otherwise make it more vulnerable to infection by entomopathogenic nematodes.

The present inventors have found that, when using such preparations or formulations, it is possible to get the 15 banana weevil to move the elytra, thus enabling nematodes S: to penetrate readily into the insect and kill it. No other physical damage to the insect is known to occur with the use of these formulations.

The benefits of using particles of polyacrylamide gel or other absorbent in these formulations are fourfold.

4 4 4 Firstly, they absorb fluid which might otherwise accumulate in the drill holes (this fluid has a viscosity lower than i that which is required by the nematodes to swim and obtain S' a purchase on the insect, so that the fluid effectively stops the nematodes from readily penetrating the insect; in addition, the nematodes tend to fall to the bottom of the drill hole and there is inadequate aeration for optimal nematode activity). Secondly, they provide a large surface area for nematode/insect contact. Thirdly, they allow V Ii -7sufficient aeration for nematode activity; and fourthly, they provide a large surface area for establishing contact between the weevil and the oil, surfactant or insecticide in the preparation.

The presence of oil, and/or a surfactant and/or a non lethal dosage of insecticide in these formulations is particularly important. It is believed that this additive, by forcing the insect to lift the elytra to allow air to more readily pass to the spiracles, increases the susceptibility of the banana weevil adults to parasitism by the nematodes. If oil is used in the nematode preparation, Sit is believed that the oil forms an almost airtight seal between the elytra and abdomen of the adult banana weevil.

Thus, in order to allow air to pass to the spiracles, the ratr S, 15 insect must temporarily lift the elytra, and this allows nematodes to crawl beneath them. The presence of a surfactant in the nematode preparation is believed to reduce air penetration beneath the elytra by promoting wetting of the seal between the elytra and abdomen, so that the insect must lift the elytra to breath. Sub-lethal doses of insecticide are believed to increase the l respiratory requirements of the weevils, which also leads i to a lifting of the elytra, although an alternative 1 explanation of the effectiveness of this additive is that i 25 sub-lethal doses of insecticide may weaken the insect o sufficiently to make it more susceptible to nematodes in a manner not yet ascertained. It should be noted that the present inventors have discovered that lethal doses of insecticide applied around banana corms, and in holes in the corms, actually repel the adult banana weevils.

1 -8 However, use of preparations of the present invention which contain non-lethal doses of insecticide has resulted in no reduction of the attractiveness of the corm to banana weevils.

As noted above, a preparation formulated in accordance with the present invention may contain two, or all three, of the additives oil, a surfactant, and a non-lethal dose of insecticide.

.oo Thus, according to the present invention, a preparation 10 for use in the control of the banana weevil Cosmopolites sordidus comprises: partially hydrated particles of a liquid absorbing medium; about, or in excess of, 25,000 infective third stage juvenile entomopathogenic nematodes per gram of the preparation; and a(c) an additive present in sufficient quantity to cause a banana weevil which comes into contact with the t preparation to move its elytra, said additive being an oil; or (ii) a surfactant; or (iii) a nonlethal (to the weevil) dose of an insecticide, or (iv) i a mixture of two or all three of the additives I (ii) and (iii); or any other additive which causes a banana weevil to move its elytra.

The present invention also encompasses a bait comprising a banana corm, or a piece of a banana corm, with at least one hole formed therein, said (or each) hole containing a preparation as defined in the last preceding paragraph.

Embodiments of, and examples of the use of embodiments of, the present invention will now be described.

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9 Examples of Implementations of the Invention The present invention has been successfully tested in the field using the nematode species Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, Heterorhabditis bacteriophora, Heterorhabditis zealandica and one undescribed species of Heterorhabditis (Dl) from Australia.

Preparations in accordance with the present invention have also been tested in the laboratory with most of the known species of entomopathogenic nematodes, including: Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, Steinernema anomali, Heterorhabditis Ibacteriophora, Heterorhabditis meqidis, Heterorhabditis zealandica and three further as yet undescribed new species of Steinernema from (respectively) Australia, The People's 15 Republic of China and the United States of America, and with one as yet undescribed species of Heterorhabditis from Australia. All species of entomopathogenic nematodes which were tested had an improved infectivity to banana weevil Swhen used in preparations in accordance with the present invention. Thus, since these species belong to two distinct families of nematodes (Steinernematidae and Heterorhabditidae) and include nearly all the species found S" therein, it is clear that all such nematodes can have their infectivity to the banana weevil increased using the 25 present invention.

A- All the nematodes used in the trials cinducted to confirm the efficacy of the present invention were reared and harvested by the methods of R A Bedding, M A Stanfield and G W Crompton, which have been described in the r 1 10 specification of International patent application No PCT/AU91/00136. However, the nematodes may be reared on insects in vivo or in liquid culture, or by any other suitable method, preferably ensuring that the nematodes so produced are free from appreciable amounts of extraneous matter remaining from the culture medium and are relatively free from nematode stages other than J3.

The nematodes reared by the present inventors were stored using attapulgite clay in accordance with the methods of R A Bedding which have been described in the specification of International patent application No PCT/AU88/00127, or by a new storage technique which involves polyacrylamide gel crystals at a specific range of water activities. Using the second of these two 15 techniques, approximately 250,000 entomopathogenic S'nematodes per gram (dry weight) of the gel were stored.

Significantly greater numbers of nematodes could have been stored per gram of the gel, had the inventors felt it desirable to do so.

When stored by the first of these methods, the nematodes were extracted from the attapulgite clay carrier by mixing with water, sedimenting out most of the attapulgite clay j and then adding the nematode suspension in water to polyacrylamide gel particles (or other absorbent particles) 25 so that all the water is absorbed into the particles, (ii) the nematodes cover the swollen absorbent particles, and (iii) the absorbent particles are only partially hydrated, so that they can absorb additional liquid. After absorption of the water of the nematode suspension, a small i I r

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4 0 4 4 44 C 4 04 4 444 4 4444 44 44 444444 insecticide, were thoroughly mixed with the nematode-coated particles to form a thin covering of oil, surfactant or insecticide on the particles of absorbent.

When stored by the second method mentioned above, water is simply added to the polyacrylamide gel on which nematodes and oil are already distributed. In this way, the nematodes are activated but the polyacrylamide gel is not fully hydrated and thus retains adequate capacity to absorb additional liquid. (1 gm of anhydrous polyacrylamide gel 10 can absorb 400 ml of water. The partially hydrated absorbent particles, coated w: nematodes and oil, surfactant or insecticide, were t added to holes or cuts in banana corms (allowing ample room for expansion) and left until the corms were no longer attractive to banana weevil adults (a period of from two to four weeks).

The nematode preparation, when used in the fifals.d, should contain a relatively large number of J3 nematodes, to ensure that when the weevil lifts the elytra, there is a high probability of invasion of the weevil by nematodes.

Thus it is preferred that approximately 250,000 J3 nematodes should be present in a typical 10 gm sample of the preparation which is placed in a hole in a banana corm.

Preferably, the additive content of the sample of the preparation which is placed in the hole in the corin is about 1 per cent.

Significantly higher concentrations of additive can 1

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<~4 12 be used, but a concentration of about 1 per, cent of oil and about 0.1 per cent of surfactant have been found to be sufficient for good control of the banana weevil, and increasing the content of oil and/or surfactant in the nematode preparation simply increases its cost.

In practice, a "concentrated" preparation will normally be prepared first. The concentrated preparation will be diluted with water to produce (after thorough mixing) the preparation placed in a hole in a corm.

In principle, any number of holes may be made in a corm.

However, in practice, because damage of the corm is undesirable, only one, two or three conical holes to I receive the preparation will be made in a banana corm.

Having broadly described the techniques used to formulate preparations in accordance with the present invention, particular examples of the use of the invention will now be described, by way of illustration only.

J 'Example 1 A suspension of approximately 25 million infective juvenile 20 Steinernema carpocapsae BW strain nematodes in 1 litre of 'tap water was added to 100 gm of anhydrous polyacrylamide gel particles marketed under the trade name "ALCOSORB AB3C" (each gel particle .vighed approximately 10 mg). This mixture was stirred until all the water had been absorbed and the nematodes were fairly evenly distributed over each gel particle. Then 8 ml of paraffin oil were sprinkled over the surface of the gel particles. The gel particles Pr -13 were then stirred thoroughly so that small droplts of oil were also well distributed over all of the gel particles.

This preparation was transported to the field in a bucket.

100 residual banana corms were inoculated with this formulation in the following manner. Two conical holes, each about 10 cm deep and 5 cm diameter at the mouth, were made on opposite sides of each residual corm, at an angle of about 450, using a desuckering gouge of the type that is commonly used by banana growers for removing suckers from corms. (The gouge is about 1 metre long, with a T bar at one end and a flattened, tapered spoon-shaped bowl at the other end. The bowl is 15 cm long and 5 cm wide at its widest point, with sharpened edges at its narrow end.) a o0 About 10 gm of the formulation (containing approximately 15 250,000 nematodes) was added to the bottom of each hole using an applicator. The applicator consisted of a plastic cylinder of appropriate size with about one third of its wall cut away along its length and a handle attached. This '"cylinder was used to scoop up the 10 gm of formulation and was then placed in the hole, where its contents were pushed out with a plastic rod. The cone of corm, removed to create the hole, was then replaced loosely. The holes I containing the formulation from the cylinder were then left Suncaged for three days to allow adult banana weevils to 25 enter. 15 holes were randomly selected and caged to oprevent the removal of dead weevils by predators, while weevils were placed in each of a further 5 holes and caged.

14 days later, all weevi.ls were removed from the caged 14 holes and assessed for mortality. Mortality ranged from per cent to 100 per cent in all holes.

Example 2 A formulation containing entomopathogenic nematodes was prepared in the same manner as in Example 1, except that ml of "Agridex Summer Oil" (trade mark) were added to 1000 gm of the mixture of gel particles, water and nematodes instead of paraffin oil. This new formulation was used in the same manner as the formulation of Example 1, except that only 10 corms (20 holes) were inoculated with the formulation. The mortality results were essentially the same as those of Example 1.

Example 3 A formulation containing entomopathogenic nematodes was prepared in the same manner as in Example 1, except that I 10 ml of a solution of 0.1 per cent synthetic pyrethrum were added to 200 gm of the mixture of gel particles, water i and nematodes, instead of oil. This new formulation was used in the same manner as the formulation of Example 1, I* 20 except that only 10 corms were inoculated with the formulation. Mortality after 14 days, in this case, ranged from 50 per cent to 80 per cent.

Example 4 A formulation containing entomopathogenic nematodes was prepared in the same manner as in Example 2, except that instead of 10 ml of oil, 10 ml of 0.1 per cent "Tween (trade mark) surfactant were added to the mixture of nematodes and partially hydrated gel particles. This new if 15 formulation was used in the same manner as the formulation of Example 1. In this case, 60 to 72 per cent of the adult weevils were killed in each hole.

Example A formulation containing entomopathogenic nematodes was prepared in the same manner as in Example 1, except that the nematode used was Heterorhabditis zealandica NZH strain. This new formulation was used in the same manner as the formulation of Example 1, except that only 20 corms were inoculated with the formulation. The banana weevil mortality ranged from 70 to 85 per cent after 14 days.

'Example 6 A suspension of 20 million infective juveniles of Steinernema carpocapsae BW strain in 240 ml of water was 15 added to 80 gm of polyacrylamide gel ("Alcosorb AB3C") particles and thoroughly mixed until all the water had been absorbed into the gel. 4.5 ml of paraffin oil were sprinkled over the surface of the gel particles. The mixture was then stirred thoroughly so that small droplets of oil were well distributed over all of the gel particles.

This preparation was then placed in a plastic food t container with a sealable lid which had 10 air holes around its rim, and was stored at 5°C for two months.

Immediately before its use, 480 ml of water were added to S 25 this preparation to produce a total volume of 800 ml, which was stirred until all surplus water had been absorbed by the gel particles. Approximately 10 gm portions of the resulting formulation were added to each one of a number of r T 16 drill holes in banana corms, in the same manner as in Example 1. 10 banana weevil adults were placed in each of drill holes and caged for 14 days. At the end of that period, it was found that the mortality of the weevils was between 90 per cent and 100 per cent.

Example 7 A series of laboratory tests conducted with formulations in accordance with the present invention, comprising nematodes, oil and polyacrylamide gel, were carried out for each of the following nematode species: Steinernema 513; Steinernema carpocapsae All strain; Heterorhabditis meqidis; Heteorhabditis Species Dl; 15 Steinernema o Steinernema anomali; Steinernema Mew; and Steinernema carpocapsae BW (in order to compare laboratory results and field results).

zt Ten samples of each formulation were placed in respective plastic tubes. As a control, some plastic tubes, each containing a preparation comprising nematodes with polyacrylamide gel, but no oil or other additive of the present invention, were also used in the series of tests.

Five adult banana weevils were introduced into each plastic S" tube. The tubes were then closed with a cotton wool plug and stored at 23 0 C for ten days. At the end of this period, there was a mortality of weevils of from 60 per cent to 100 per cent in the tubes containing a preparation I I; -17in accordance with the present invention, and an average mortality of 20 per cent in the "control" tubes.

Those skilled in this field will appreciate that although specific examples of the implementation of the present invention have been described above, variations and modifications to the described implementations can be made without departing from the present inventive concept.

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Claims (9)

1. A preparation for use in the control of the banana weevil Cosmopolites sordidus, said preparation comprising partially hydrated particles of a liquid absorbing medium; about 25,000 infective third stage juvenile entomopathogenic nematodes per gram of the preparation; and an additive present in sufficient quantity to 0 9 cause a banana weevil which comes into contact 0 9 with the preparation to move its elytra, said additive being an oil; or (ii) a surfactant; or (iii) a non-lethal (to the weevil) dose of an o insecticide, or (iv) a mixture of two or all three of the additives (ii) and (iii); or any other additive which causes a banana weevil to move its elytra.

S2. A preparation for use in the control of the banana weevil Cosmopolites sordidus, said preparation comprising partially hydrated particles of a liquid absorbing medium; in excess of 25,000 infective third stage juvenile entomopathogenic nematodes per gram of Sthe preparation; and 0 an additive present in sufficient quantity to cause a banana weevil which comes into contact with the preparation to move its elytra, said 18a additive being an oil; or (ii) a surfactant; or (iii) a non-lethal (to the weevil) dose of an insecticide, or (iv) a mixture of two or all three of the additives (ii) and (iii); or any other additive which causes a banana weevil to move its elytra.

3. A preparation as defined in claim 1 or claim 2, in which said absorbing medium is polyacrylamide gel. t

4. A preparation as defined in claim 1 or claim 2, in which the additive comprises about 1 per cent of the .I preparation. S:

5. A preparation as defined in claim 2, claim 3 when S' i appended to claim 2, or claim 4 when appended directly Sor indirectly to claim 2, for transportation to a site at which the preparation is to be used, said preparation being such that, when diluted for use at the site, the additive comprises -19 about 1 per cent of the diluted preparation, and a gm sample of the diluted preparation contains about 250,000 nematodes.

6. A bait for use in the control of the banana weevil Cosmopolites sordidus, said bait comprising a banana corm or a piece of banana corm with at least one hole therein, said or each hole containing a preparation as defined in any preceding claim.

7. A bait as defined in claim 6, in which the corm or piece of corm has two or three conical holes in the i upper region thereof, and each conical hole contains about 10 gm of the preparation. 4 r

8. A bait as defined in claim 6 or claim 7, in which the or each hole is created by the removal of a conical Ssegment of the corm, and the or each conical segment is replaced loosely in its respective hole after the preparation has been placed therein. S"

9. A preparation, or a bait, for use in the control of the banana weevil Cosmopolites sordidus, substantially as hereinbefore described with reference to the Examples. DATED this twenty-fifth day of February 1993 "E COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION by its Patent Attorneys DAVIES COLLISON CAVE ABSTRACT Conventional methods for the control of the banana weevil Cosmopolites sordidus are becoming increasingly ineffective and environmentally unacceptable. Previous attempts to control C. sordidus with entomopathogenic nematodes have been unsuccessful. The present invention provides preparations containing third stage juvenile (J3) entomopathogenic nematodes which can be used to control C. sordidus. The preparations comprise the J3 nematodes, partially hydrated particles of an absorbent material, and an additive which is believed to cause the banana weevil to lift its elytra and thus make the weevil susceptible to attack by the nematodes. The additive is an oil, (ii) a surfactant, (iii) a non-lethal dose of insecticide, or (iv) a combination of two or all three of these additives. The preparation is placed in a hole in a banana corm or a piece of banana corm to form a bait for C. sordidus. i 4 4I 4 i 4 i *t ,1