WO2014149324A1 - Chemical and biological agents for the control of molluscs - Google Patents

Abstract

Compositions and methods for controlling molluscs, such as members of the Gastropoda and Bivalvia classes, including compounds obtainable from Pseudomonas.

Description

CHEMICAL AND BIOLOGICAL AGENTS FOR THE CONTROL OF MOLLUSCS

FIELD OF THE INVE TION

The present invention provides compositions and methods for controlling molluscs, such as mussels and/or snails and/or slugs and includes the compounds pyoluteorin and 2,4- diacetylphloroglucinol for controlling molluscs. Also provided are methods and

compositions for increasing the efficacy of chemical and biological control for molluscs.

BACKGROUND OF THE INVENTION

The Zebra mussel, Dreissena poiymorpha, was originally native to the Caspian Sea and the Ural River in Asia. In the nineteenth century, it spread west and now occurs in most of Europe, the western portion of the Commonwealth of Independent States (formally the Soviet Union), and Turkey. Over two decades ago, the mussels, such as zebra mussel, Dreissena poiymorpha, and quagga mussel, Dreissena hugensis, were introduced into North America. Their wide spread through inland waters has led to the coverage of most of eastern of US (U.S. Army Engineer Waterways Experiment Station. 1995. Zebra mussels: Biology, Ecology, and Recommended Control Strategies. Technical Note. ZMR-1-01. Zebra Mussel Research Program, Vicksburg, MS). Similarly, Golden Mussel, Limnoperna fortune, affected Asian and Southern American countries (Golden Mussel - Limnoperna fortune). Asian Clam, Corbicula fluminea, almost spread in all Asian countries and the US. Other mussels such as unionid mussels exist in US and other countries.

The ability of the mussels to quickly colonize new areas, rapidly achieve high densities and attach to any hard substratum (e.g., rocks, logs, aquatic plants, shells of native mussels, and exoskeleions of crayfish, plastic, concrete, wood, fiberglass, pipes made of iron and polyvinyl chloride and surfaces covered with conventional paints) result in serious adverse consequences. These consequences include damages of water-dependent infrastructure, increased millions of dollars in ihe operating expense and significant damage of the ecological systems (O'Neill, C.R., Jr. 1997, Gt. Lakes Res. Rev. 3, 35-44; Karatayev, A.Y., Journal Shellfish Research, 16, 187-203; MacTsaac, H.J., 1996. American Zoology-, 36, 289-299; D.P. Moiloy, Journal of Shellfish Research, 1998 (17) 177-183), as well as productivity reduction which costs billions of dollars in lost revenue (Connelly, N. A,, et al. (2007), Environmental Management 40: 105-1 12). Additionally, rapid invasion of aquatic ecosystems by these invasive mussels has caused decline in the richness and abundance of endemic unionid mussels, an important part of biodiversity ( icciardi, A, 1998. Journal of Animal Ecology 67: 613-619,).

Management of mussels is very important for protecting water-dependent infrastructure and water ecological systems. There are many ways to reduce the populations of mussels. These methods include pre-active and reactive methods. Reactive removal includes the mechanical removal, predator removal, and chemical and biochemical removal. For example, fish, birds, crayfish, crabs, leeches and mammals have shown to predate mussels. However, it is unlikely that mussel population will be controlled by natural predation, especially in man-made structures such as pipes or pumping plants.

Application of molluscieides is another effective ways to reduce the mussel population. For example, sodium hypochlorite is a commonly used control agent in Europe, US, and Canada. However, mussels can withstand this treatment for several days by closmg their shells and chlorine can be only used in pipes or ducts that contain pressure sensing or other equipment due to environmental toxicity of chlorine (L^!.S, Army Engineer Waterways Experiment Station. 1995. Zebra mussels: Biology, Ecology, and Recommended Control Strategies. Technical Note. ZMR-1-01. Zebra Mussel Research Program, Vicksburg, MS). In addition, there are many other commercialized molluscieides such as surfactant ammonium salts, Butylated hydroxytoluene (BUT) in paints, N-triplienylmethyl-morphofine and so on. These chemicals either have low selectivity or affect the water ecosystems. For example, a 4-trifluroethyl-4-nitrophenol marketed as Bayluscide® (Bayer) is a possible candidate for control such invasive exotic species. However, the toxic mechanism of such a chemical is to affect mussel cellular respiration, which in nature will limit its selectivity between mussel and other aquatic species such as fish (Karen Perry, livdrobiologia (2009) 628:153-164; 1 Takougang, Mem Inst Oswaldo Cruz, (2006) 101(4): 355-358).

It is crucial to manage the invasive mussels in a safe, environmental friendly and cheap manner. In order to find less harmful methods to control these invasive mussels, New- York State Museum's (NYSM) Field Research Laborator screened more than 700 bacterial isolates as potential biological control agents to be used against zebra and quagga mussels. As a result, they found an isolate, strain CL145A of Pse domonas fluorescens, to be lethal to these mussels (see Molloy, D. P. US Patent No. 6,194,194, issued February 27, 2001). This bacterium is worldwide in distribution and is present in all North American waterbodies. In nature it is a harmless bacterial species that is found protecting the roots of plants from rot and mildew. It is so ubiquitous that it is a common food spoilage organism in the average household refrigerator (Daniel P. Molloy and Denise A. Mayer, Overview of a Novel Green Technology: Biological Control of Zebra and Quagga Mussels with Pseudomonas flwrescens, Version 6: Updated August 24, 2007). BRIEF SUMMARY OF THE INVENTION

This invention is directed to the compounds, compositions and methods for controlling molluscs, particularly members of the Gastropoda and/or Bivalvia classes and more particularly mussels, snails and slugs. The invention includes the isolated compounds pyoluteorin and 2,4-diacetylphioroglucinol (DAPG) obtainable from (a) microorganism, particularly, Pseudomonas species, more particularly, Pseudomonas fluorescens or alternatively, Pseudomonas ATCC 55799; and (b) is toxic to a member of a class of molluscs selected from Bivalvia, particularly, mussels (e.g., Dreissana sp.) and/or Gastropoda., particularly, snails, which includes but is not limited to aquatic snails (e.g., Biomphalaria sp.) and garden snails, including but not limited to brown garden snails, white garden snails (e.g., Cantareus sp., Cornu sp., Theba sp.), and/or slugs, including but not limited to gray garden slug ( e.g., Deroceras sp.}, the banded or three-band slug (e.g., Lehmannia sp,.), the tawny- slug (e.g., Limacus sp.), and the greenhouse slug (e.g., Milax sp.). These compounds may be formulated into compositions which may be used to control molluscs, particularly members of the Gastropoda and/or Bi valvia classes and more particularly mussels, snails and slugs. In one embodiment, the compound is pyoluteorin. In another embodiment, the compound is DA PG. Control of molluscs may be achieved by inducing death in one or more molluscs comprising contacting said molluscs with the compounds set forth above. The molluscs may be contacted in a body of water or solid surface.

The invention is further directed to a composition comprising at least one or more compounds for controlling one or more molluscs and an inert material. The inert material may be a clay mineral (kaolinite, smectite, attapulgite, or a combination thereof). The invention is further directed to a method for controlling one or more molluscs in a location where control is desired comprising introducing in said location a substance effective for controlling said molluscs and one or more inert materials in amounts effective to control said molluscs in said location containing said molluscs. The inert material may be a clay mineral (kaolinite, smectite, attapulgite, or a combination thereof). In particular, the substance for controlling said molluscs is present in an amount effective to result in at least about a 2.0% mortality relative to untreated control, typically about 50-95% and said inert material is present in an amount sufficient or effective to increase mortality rate of said substance for controlling said molluscs at least about 20%, typically 25-40%. In a particular embodiment, the inert material is introduced into said location prior to introduction of the substance for controlling said molluscs; in a more particular embodiment, the inert material is introduced at least about one hour prior to the introduction of the substance. In another particular embodiment, the inert material is introduced into the location simultaneously with the substance for controlling molluscs set forth above.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the evolutionary relationships of taxa - A Neighbor- Joining free to visualize the relationship of CL.145A (MBI-401 ) to the type strains of the genus

Pseudomonas,

Fig. 2. shows the evolutionary relationships of taxa - A Neighbor- J oining Tree to visualize the relationship of CL145 (MBI-401) to the top matches to Pseudomonas fiiiorescens and Pseudomonas protegens from an NCBT BLAST search.

Fig. 3 shows development of mortality over time for mussels treated with clay and P. fiiiorescens biopesticide product in a biobox.

DETAILED DESCRIPTION OF THE INVENTION

Where a range of values is provided, it is underst ood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. It must be rioted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

As defined herein, "controlling mussels" means controlling the eggs, larvae, veiigers and post-veligers of the mussel by killing or disabling them so that they cannot colonize in a give location.

As defined herein, "derived from" and "obtainable from" means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source. These terms are used interchangeably throughout the specification.

As defined herein, an "isolated compound" is essentially free of other compounds or substances, e.g., at feast about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by analytical methods, including but not limited to chromatographic methods, electrophoretic methods.

The compounds used in the compositions and methods of the present in v ention include pyoluteorin and 2,4-diacetyfphlorogluciiiol (DAPG). The compounds have the followin structures :

Pyoluteorin DAPG

Methods of Production

As noted above, the compounds and compositions of the present invention may be obtained, is obtainable or derived from an organism having the identifying characteristics of a Pseudomonas species. The Pseudomonas species includes but is not limited to

Pseudomonas protegens, Pseudomonas saponiphila, Pseudomonas ficuserectae,

Pseudomonas congelans, Pseudomonas tremae, Pseudomonas caricapapayae, Pseudomonas mandelii, Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas chlororaphis subsp, piscium, Pseudomonas cannabina, Pseudomonas marginalis, Pseudomonas simiae, Pseudomonas avelianae, Pseudomonas chlororaphis subsp. aurantiaca, Pseudomonas chlororaphis subsp. chlororaphis, Pseudomonas frederikshergensis, Pseudomonas amygdali, Pseudomonas exiremaiistralis, Pseudomonas kilonensis, Pseudomonas lini, Pseudomonas Antarctica, Pseudomonas corrugata, Pseudomonas poae, Pseudomonas grimontii,

Pseudomonas brassicacearum subsp. Neoaurantiaca, Pseudomonas meridian, Pseudomonas trivialis, Pseudomonas veronii, Pseudomonas lundensis, Pseudomonas salomonii,

Pseudomonas rhodesiae, Pseudomonas arsenicoxydans, Pseudomonas ihivervalensis, Pseudomonas deceptionensis, Pseudomonas palleroniana, Pseudomonas chlororaphis subsp. aureofaciens, Ps beudomonas costantinii, Pseudomonas lurida, Pseudomonas migulae, Pseudomonas orientalis, Pseudomonas extremorientalis, Pseudomonas mediterranean Pseudomonas brassicacearum subsp. brassicacearum, Pseudomonas abietaniphila,

Pseudomonas baetica, Pseudomonas brenneri, Pseudomonas psychrophila, Pseudomonas jessenii, Pseudomonas fragi, Pseudomonas tolaasii, Pseudomonas proieolytica,

Pseudomonas taetrolens, Pseudomonas mohnii, Pseudomonas moorei, Pseudomonas moraviensis, Pseudomonas gessardii, Pseudomonas cichorii, Pseudomonas libanensis, Pseudomonas benzenivorans, Pseudomonas panacis, Pseudomonas umsongensis,

Pseudomonas reinekei, Pseudomonas fluorescens, Pseudomonas agarici, Pseudomonas lutea, Pseudomonas mucidolens, Pseudomonas azoioformans, Pseudomonas viridiflava,

Pseudomonas koreensis, Pseudomonas kuykendallii, Pseudomonas synxantha, Pseudomonas segetis, Pseudomonas marincola, Pseudomonas cedrina subsp. cedrina, Pseudomonas graminis, Pseudomonas vancouverensis, Pseudomonas cedrina subsp. fulgida, Pseudomonas plecoglossicida, Pseudomonas cuatrocienegasensis, Pseudomonas iaiwanensis,

Pseudomonas puiida, Pseudomonas rhizosphaerae, , Pseudomonas anguilliseptica,

Pseudomonas monteilii, Pseudomonas fuscovaginae, Pseudomonas mosselii, Pseudomonas taeanensis, Pseudomonas asplenii, Pseudomonas entomophila, Pseudomonas

cremoricolorata, Pseudomonas parafulva, Pseudomonas alcaliphila, Pseudomonas oleovorans subsp. lubricantis, Pseudomonas borbori, Pseudomonas composti, Pseudomonas toyotomiensis, Pseudomonas batumici, Pseudomonas flavescens, Pseudomonas vranovensis, Pseudomonas punonensis, Pseudomonas balearica, Pseudomonas indoloxydans,

Pseudomonas guineae, Pseudomonas japonica, Pseudomonas siutzeri, Pseudomonas seleniipraecipitans, Pseudomonas peli, Pseudomonas fulva, Pseudomonas argentinensis. Pseudomonas xanthomarina, Pseudomonas pohangensis, , Pseudomonas oleovorans, Pseudomonas mendocina, Pseudomonas luleola, Pseudomonas straminea, Pseudomonas caeni, Pseudomonas aeruginosa, Pseudomonas tuomuerensis, Pseudomonas azotifigens, Pseudomonas indica, Pseudomonas oryzihahitansPseudomonas otitidis, Pseudomonas psychrotolerans, Pseudomonas zeshuii, Pseudomonas resinovorans, Pseudomonas oleovorans subsp. oleovorans, Pseudomonas therm oiolerans, Pseudomonas bauzanensis, Pseudomonas duriflava, Pseudomonas pachastreliae, Pseudomonas citronellolis,

Pseudomonas alcaligenes, Pseudomonas xinjiangensis, Pseudomonas delhiensis,

Pseudomonas sabulinigri, Pseudomonas liioralis, Pseudomonas pelagia, Pseudomonas linyingensis, Pseudomonas knackmussii, Pseudomonas panipatensis, Pseudomonas nitroreducens, Pseudomonas nitritireducens, Pseudomonas jinjuensis, Pseudomonas pertucinogena, Pseudomonas xiamenensis, Pseudomonas cissicola, Pseudomonas halophile, Pseudomonas boreopolis, Pseudomonas geniculate, Pseudomonas beieli, Pseudomonas hibiscicola, Pseudomonas pictorum, and Pseudomonas carboxydohydrogena. In particular, the Pseudomonas organism may be a strain of Pseudomonas protegens or fluorescens or may be the Pseudomonas fluorescens or protegens isolate, ATCC 55799 as set forth in US Patent No. 6,194,194, The methods comprise cultivating these organisms and obtaining the compounds and/or compositions of the present invention by isolating these compounds from the cells of these organisms.

In particular, the organisms are cultivated in a nutrient medium using methods known in the art. The organisms may be cultivated by shake flask cultivation, small scale or large scale fermentation (including but not limited to continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in suitable medium and under conditions allowing cell growth. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available may be available from commercial sources or prepared according to published compositions. A particular embodiment is disclosed in the examples infra and in US Patent No. 6, 194, 194.

After cultivation, the cells may be concentrated and subsequently suspended in a buffer to obtain a cell suspension. The compounds and/or compositions of the present invention may be extracted from the suspension. The extract may be fractionated by chromatography. Chromatography may be assayed for toxic activity against molluscs, such as mussels, snails (e.g., aquatic and/or garden snails) and/or slugs, using methods known in the art; one particular embodiment is disclosed in the examples, infra. This process may be repeated one or more times using the same or different chromatographic methods.

The compounds of the present invention may also be obtained by synthetic methods, or ma be obtained from commercial sources.

The composition of the present invention may comprise a chemical or biopesticide product that is useful in controlling molluscs, particularly members of the Gastropoda and'or Bivalvia classes and more particularly mussels, snails and slugs, such as pyoluteorin and 2,4- diacetylphloroglucinol.

The compounds disclosed above and used in the invention can be made into compositions (also alternatively referred to as ''formulations") and can be formulated in any form. Non-limiting formulation examples include emulsifiable concentrates (EC), wettabie powders (WP), soluble liquids (SL), aerosols, ultra-low volume concentrate solutions (ULV), soluble powders (SP), microencapsulation, water dispersed granules, flowables (FL), microemulsions (ME), nano-emulsions (NE), etc. In any formulation described herein, percent of active ingredient is within a range of 0.01% to 99.99%. In a particular embodiment, the formulations may be free of surfactants.

The compositions may include an inert solid material Examples of the inert material that may be used in the compositions of the present invention include, but are not limited to, inorganic minerals such as kaolin, mica, gypsum, phyllosilicates, carbonates, sulfates, or phosphates; or botanical materials such as wood products, cork, powdered com cobs, rice hulls, peanut hulls and walnut shells. In a particular embodiment, the inert material can be obtained or derived from a clay mineral (kaoiinite, smectite, attapulgite, or a combination thereof) suspended in water at a rate of about I to 20 mg/liter corresponding to approximately 1 to 20 NTU (normalized turbidity units). The inert materials used to enhance mussel siphoning can be applied in solid form or as a suspension in aqueous solution, preferably water, directly to the water or the location (e.g., solid surface) where the mussels are treated. In a particular embodiment, to enhance product efficacy, an inert material such as clay, silt, sediment or any other material with no nutritional value and with a small enough particle size can be suspended in water prior to the treatment with a chemical or a biopesticide product.

Methods of Use The compounds and compositions of the present invention may be used to control molluscs, particularly, a member of the Gastropoda and/or Bivalvia class, more particularly mussels (e.g., Dreissana species) and/or Gastropoda, particularly, snails, which includes but is not limited to aquatic snails (e.g., Biomphalaria species) and garden snails, including but not limited to brown garden snails, white garden snails (e.g., Cantareus species, Cornu species, Theba species), and/or slugs, including but not limited to gray garden slug ( e.g., Deroceras sp,), the banded or three-band slug (e.g., Lehmannia sp.), the tawny slug (e.g., Limacus sp,.), and the greenhouse slug (e.g., Milax in a location where control is desired, such as a body of water or on surfaces where molluscs such as mussels, snails and/or slugs gather. Surfaces where molluscs such as mussels, snails and/or slugs gather include but are not limited to plastic, concrete, wood, fiberglass, pipes made of iron and polyvinyl chloride and surfaces covered with paints and/or coatings. The end product (which contains the active compound) may be used at 10-200 mg/L, more specifically at 25- 100 mg/L (ppm) or 25 - 10000 mg/kg. it will be applied either as a dry product or suspended in water into pipes, dam structures, holding tanks, and open waters such as streams, rivers, lakes, irrigation canals, ponds and lakes through specific application pumps and mixing systems.

In a particular embodiment, the present invention is directed to a method for improving biopesticidal and pesticidal activity of materials used to control invasive molluscs, particularly mussels, comprising the steps of:

1. suspension of inert material such as clay into the water to trigger the siphoning

activity for about 1 - 24 hours before the chemical or biopesticide treatment

2. addition of a chemical or a biopesticide into the water at a desired level

The invention is also directed to a method comprising a step of administering a compound having nioiiuscicidai activity in combination wiih an inert material such as clay to enhance the uptake of the compound and hence, increase the mortality of mussels.

To activate the mussel siphoning, this clay (turbidity) treatment should be carried on for about 1 to 6 hours, usually about 3-4 hours, and for about 1 to 24 hours, typically about 14-18 hours before the treatment with a chemical/pesticide. Alternatively, the turbidity treatment can be applied simultaneously with the chemical or biopesticide treatment.

According to an embodiment of this invention, treatment of molluscs, such as mussels, snails and slugs can be carried out in 500-mL glass jars or in a biobox constructed of acrylic sheets. In the glass jars, aeration during treatment is provided by airflow through aquarium air stones connected to nylon tubing. In the biobox, water is constantly flowing at a rate of 1 gallon per minute.

The materials for the turbidity treatment as well as for the ehemical/biopesticide product can be mixed in the %^rater by pipetting or via a peristaltic pump, in bioboxes, a more uniform mixing is achieved using a paddle mixer at the point of injection. The compositions of the present invention can be in a suitable form for direct application or as a concentrate or primary composition, which requires dilution with a suitable quantity of water or other diluent before application.

The effective amount of the turbidity materials will depend upon the application, water temperature, if applied to water, and duration of the treatment. In general, the composition may be applied at a rate of from about 1 to about 20 mg per liter; preferably at a rate of from about 5 to about 10 mg per liter so that the measured turbidity does not rise above 20 NTU. EXAMPLES

The examples below are presented to describe preferred embodiments and utilities of the invention and is not meant to limit the invention unless otherwise stated in the claims appended hereto. EXAMPLE 1: ANALYSIS OF PSEUDOMONAS STRAIN CL145A (ATCC 55799} The Pseiidomonas strain CL145A (ATCC 55799) has been further characterized through a pofyphasic approach by investigating its phenotypic and geiiotypic characteristics. The results from the studies disclosed herein indicate that CL145A is a better match to Pseiidomonas protegens, rather than Pseudomonas fluoresce ns.

Recent developments in the taxonomical study of the genus Pseudomonas resulted in the creation of a new species Pseudomonas protegens, Pseudomonas protegens includes several strains formerly identified as Pseudomonas fluorescens including strains CHA0, PF, PGNL1 , PGNR 1, PGNR. 2, PGNR 3, PGNR 4, PINR 3 and Pfl . The basis of the reclassification is described by Ramette et ah, (201 1 ) (Systematic and Applied Microbiology. 34, pages 180 - 188), and was officially validated in 2012. Based on this new information, the best match for CL145A is Pseudomonas protegens, MBI-401 will now be identified as Pseudomonas protegens strain CL145A ("CL145 "). CL145A was characterized through a polyphasic approach investigating phenotypic and genotypic characteristics, as well as confirmation of the presence of two metabolites; 2,4- Diacetylphloroglueinol and Pyoluteorin, Pyoluteorin and 2,4-Diacetylphloroglucinol are central characteristics to the differentiation of Pseudomonas fluorescens and Pseudomonas protegens. Isolates that do not produce these two compounds, or that only produce one of them, remain known a s Pseudomonas fluorescens , while Pseudomonas fluorescens that produce both compounds have been reclassified as Pseudomonas protegens as described by Ramette et al, 2Ql l(supra).

1,1 Analysis of biochemical, physiological and metabolic characteristics

Pseudomonas protegens strain CL145A (ATCC 55799) was subjected to biochemical testing to characterize the isolate and create a baseline for further tracking. As part of this characterization strategy, the growth of CL145 A was tested at temperatures of 16°C and 37°C, and API ZYM and API 20NE assays were performed, which allowed for

semiquantitation of enzymatic activities (API ZYM) and identification of Gram-negative non-Enterohacteriaceae (API 20NE). Fatty Acid Profiles and MALDI-TOF profiles were performed as well.

1.1.1 Growth at 16°C and 37°C

Pseudomonas species are known to grow- at a wide range of temperatures, with 28"C reported as the optimal for many species, and ranging from 4°C to 45°C for some species. Pseudomonas fluorescens does not grow at 41 °C, but some strains show growth as low as 4°C.

A dilute cell suspension of CL145A was prepared in phosphate buffer. The suspension was inoculated onto agar plates and incubated overnight at 16°C and 37°C.

Incubators were set at the proper temperatures and allowed to equilibrate overnight before incubations took place. CL 145 A. grew well at both temperatures. 1.1.2. API ZYM

API ZYM provides a platform for rapid semi-quantitation of enzymatic activity. The assay was performed following the manufacturer's directions (Biomerieux). A 1 day PDA plate was inoculated from a glycerol stock of CLI45A (ATCC 55799), and mcubated overnight at 25°C. Colonies growing on the plate were used to inoculate the API ZYM strip according to manuiaciurer's instructions, and incubated at 30°C for 48,5 hours. Results are own in Table 1 below.

ible 1 : API ZYM test results for CL145A (ATCC 55799)

The results indicated thai CL145A (ATCC 55799) has strong enzymatic activity for acid and alkaline phosphatase, leucine arylamidase and naphtol-AS-BI-phosphohydrolase. Negative results were recorded for all other enzyme tests. 1.1.3. API 20NE

API 20NE allow s for semiquantitation of enzymatic activities and identification of Gram-negative mm-Enterobacteriaceae. The assay was performed following manufacturer's directions (Biomerieux). A 1 day PDA plaie was inoculated from a glycerol stock of CL145A (ATCC 55799), and incubated overnight at 25°C. Colonies growing on the plate were used to inoculate the API. 2 ONE strip according to manufacturer's instructions, and incubated at 30°C for 48.5 hours. Results are shown in Table 2.

Table 2: API 20NE test results for CL145A (ATCC 55799)

Transparent O aque

|MAL| D-maltose Assimilation of maltose

Potassium Assimilation of Transparent Opaque "i- jGNTl

gluconate potassium gluconate

Assimilation of capric Transparent Opaque -r jCAPj Capric acid

acid

Assimilation of adipic Transparent Opaque + jADIl Adipic acid

acid

Transparent O aque

jMLTj Malic acid Assimilation of malate

Assimilation of Transparent Opaque -i-

|CIT| Tri sodium citrate

trisodium citrate

Assimilation of Transparent Opaque

jPAC! Phenylacetic acid

phenylacetic acid

Key: + (Positive), - (Negative), ± (weak) Pseudomonas protegens does not reduce nitrate. Additionally, Ramette el at. (2011)

(supra) report that P. protegens can assimilate N-acetyl-D-glucosatnine, while P. fluorescens cannot. CL145A can assimilate N-acetyl-D-glucosamine according to API 20 NE results. CL145A and P. protegens also share the ability to assimilate phenyl acetate (P. fluorescens cannot). CL145A displayed negative glucuronidase activity in the API ZYM test, P.

protegens cannot assimilate D-glucuronate, while P. fluorescens can assimilate D- glucuronate.

In summary, CLI45A (also referred to as ATCC 55799) shares many phenotypic traits that differentiate it from P. fluorescens and indicate closer similarity to P. protegens. However, Pseudomonas identification based on phenotypic characteristics can be difficult and an ultimate identification always requires a DNA- based approach.

1.1.4. Antibiotic resistance profile

One glycerol stock vial of CL145A was equally distributed onto Mueller-Hinton Agar plates ( 100 μΐ per plate) and spread on the plate using a sterile cell spreader. Antibiotic discs were then placed onto the plates along with a blank sterile disc. Plates were incubated at 25°C in the dark for 72 hours. Results are shown, in Table 3. Antibiotic resistance profile for CL145A (ATCC 55799)

The antibiotic profile results indicated that CL145A is resistant to tetracycline, erythromycin, streptomycin, penicillin, ampicillin, chloramphemcol and cefuroxime as shown in Table 3 as growth of CL145A was not inhibited by the antibiotic disc; and shown to be sensitive to kanamycin, oxytetracycline, ciprofloxacin, gentamiein, piperacillin, imipenem and sulphaniethoxazole-treimethoprim as growth of CL145A was inhibited following 72 hours incubation.

1.1.5. Analysis of fatty acid methyl ester composition (FAME analysis)

An agar plate with 24-hour old colonies of CL 145 A was submitted for Fatty Acid Methyl Ester (FAME) profiling to Microbial ID, Inc. (Newark, NJ). The main fatty acids found are described below in Table 4.

Table 4: FAME analysis for CL145A (ATCC 55799)

Lipid name % of total Lipid name % of total

Sum In Feature 2 0.45 12:0 30I-I 5.54

10:0 0.36 16: 1 w5c 0.1 1

10:0 3OH 4.74 17:0 iso 0.10

14:0 0.46 16:0 3 OH 4.41 12:0 1.91 Sum In 18.69

Feature 8

Sum In Feature 3 32.23 18:0 0.65

16:0 26.89 18: 1 w7c 0.18

1 l-meihyi

12:0 20H 5.15 17:0 0.10

12: 1 30H 1.01 17:0 cyclo 1.42

The FAME profile for CL145A appears to match with a Pseudomonas putida biotype A strain in the database, showing the highest similarity index (0.730). The next three best matches were with Pseudomonas fluorescens biotype A, biotype B and biotype G, all with similarity indices below 0.700.

1.2. 16S rR A gene amplification and sequencing

1.2.1 DNA extraction of CL145A (ATCC 55799)

Pseudomonas protegens strain CLI45A (ATCC 55799) was streaked on fresh potato dextrose plates and allowed to grow for 2-3 days or until enough biomass was evident. A loopful of the bacterium was suspended in DNA extraction buffer (included in the MoBio Ultra Clean Microbial DNA Extraction Kit, Cat No. 12224-50, Carlsbad, CA, USA) using a sterile loop. DNA was extracted using the MoBio Ultra Clean Microbial DNA extraction kit using the manufacturer's protocol. DNA extract was checked for quality and quantity by running a 5 μΐ. aliquot on a 1% agarose gel.

1.2.2 PGR amplification of the US rRNA gene from CL145A (ATCC 55799)

PGR reactions for the amplification of the 16s rRNA gene were performed by combining 1.5 ul of DNA extract CL145A (ATCC 55799) with 20 μΐ- nuclease-free sterile water, 25 _,uL OoTaq Green Mastermix (Promega), μΐ. forward primer (SEQ TD NO: !), and 1.5 μΕ reverse primer (SEQ ID NO:2). The PGR reaction was performed using a thermocycler PGR machine under the following conditions: 10 minutes at 95°C (initial denaturing), 30 cycles of 45 seconds at 94°C, 45 seconds at 55°C and 2 minutes at 72°C, followed by 5 mmutes at 72°C (final extension) and a final hold temperature of 10 °C. The size, quality and quantity of the PGR product was evaluated by running a. 5uL aliquot on a. 1% agarose gel and comparing the product band to a mass ladder (Hi-Lo mass ladder, Bionexus, Oakland, CA).

1.2.3 16S rRNA sequencing

Excess primers, nucleotides, enzyme and template were removed from the PCR product using the MoBio PCR clean up Kit (Cat No. 12500-50) following the manufacturer's instructions. The cleaned PCR product was subjected to direct sequencing using the primers described above. 1.2.4 Data analysis

The forward and reverse sequences were aligned using the BioEdit software

(http://www.mbio.ncsu.edu/BioEdit/bioedit.html), and a consensus sequence was generated for further comparison to sequence databases. The identification of phylogeneiic neighbors was initially carried out by the BLASTN (Aitschul, S. F., (1997). Nucleic Acids Res 25, 3389-3402) program against the database containing type strains with validly published prokaryotic names and representatives of uncultured phylotypes (Kim, O.S., (2012). Int JSyst Evoi Microbiol 62, 716-721). The top thirty sequences with the highest scores were then selected for the calculation of pairwise sequence similarity using global alignment algorithm (Myers, E. W. & Miller, W. (1988). Appl Biosci 4, 1 1 - 17.), which was implemented at the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net ; Kim, O.S., (2012) (supra)).

1,2.5 Results

The forward (SEQ ID NO:3) and reverse (SEQ ID NO:4) sequences were used to generate a 1445 base pair consensus sequence (SEQ ID NO:5).

The 1 6S rRNA gene consensus sequence of CL145A (ATCC 55799) was compared to those available sequences of type strains using EzTaxon-e server.

The search and comparison implements on Ex-Taxon-e server indicated that CL145A (also referred to as ATCC 55799 or MB1-401) was most similar to Pseudomonas protegens CHAO¹ and in comparison, more distantly related to Pseudomonas fluorescens. Pseudomonas protegens CHAO¹ is the type strain as described by Ramette, A., (201 1 ) (supra).

Sequences were downloaded into MEGA5, and aligned using MUSCLE. A Neighbor- Joining tree was built to visualize the relationship of CL145 A to the type strain of the genus Pseudomonas (Figure 1). The tree clearly illustrates that CL 145A is a strain of Pseudomonas protegens, and that Pseudotnonas fluorescens falls in a more distant and separate branch of ihe tree.

The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987. Mol. Biol Evol. 4:406-425). The bootstrap consensus tree inferred from 2000 replicates (Felsenstein J. (1985). Evolution. 39, pages 783-791) is taken to represent the evolutionary' history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are coll apsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to ihe branches (Felsenstein, 1985, supra). The iree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method (Juices and Candor, 1969. Evolution of Protein Molecules. New York: Academic Press, p. 21-132) and are in the units of the number of base substitutions per site. The analysis involved 21 nucleotide sequences. Codon positions included were 1 st+2nd+3rd+Noncoding. Ail ambiguous positions were removed for each sequence pair. There were a total of 1505 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura K., (201 1).

MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood,

Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and

Evolution).

Additionally, comparisons were done with representatives of the bacterial domain using NCB1 BLA ST and limiting the search to the Reference Sequence Database. In order to confirm that the top matches from NCBI BLAST were actually due to misnaming of Pseudotnonas isolates as Pseudotnonas fluorescens, the top matches to Pseudomonas fluorescens (strains LMG 5167, Pf-101, Pf-68, CPF- 10, 7- 1, and LC-G-2) were compared to Pseudomonas protegens in Ez-Taxon to confirm that they were not incorrectly named in the NCBI BLAST database. The sequences were imported into MEGA5, aligned by MUSCLE against CL145A (MBI-401) and Pseudomonas protegens CHA0^T and Pseudomonas fluorescens DSM 50080 '^" A. phylogenetic tree was constructed to evaluate taxonomy (Figure 2). The phylogenetic tree shown in Figure 2 illustrates that Pseudomonas strains LMG 5167, Pf- 01 , Pf-68, CPF- 10, 7- 1 , and LC-G-2 were found to all match to Pseudomonas protegens type strain (CHA0^T), and ihai ihese strains were grouped together with Pseudomonas protegens strains in the phylogenetic tree. In contrast, the Pseudomonas fluorescens type strain (DSM¹) is not in the same group as the Pseudomonas protegens type strain (CHA0¹), as both strains are in different branches of the phylogenetic tree. The evolutionary history was inferred using the Neighbor- Joining method (Saitou and Nei, 1987, supra). The bootstrap consensus tree inferred from 2000 replicates (Felsenstein J. (1985), supra) is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method (Jukes and Cantor, 1969, supra) and are in the units of the number of base substitutions per site. The analysis involved 14 nucleotide sequences. Codon positions included were 1 st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 1515 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura K., (201 1), supra).

1.3 Production of Pyoluteorin and 2,4-Diacetylphologlucinol

Ramette et al. (201 1, supra) describe the production of two secondary metabolites, pyoluteorin and 2,4-Diacetyfphloroglucmol (DAPO), as a major characteristic differentiating Pseudomonas protegens from Pseudomonas fluorescens strains alongside genotypic and phenotypic characterization data. The fluorescent Pseudomonas type strain Pf-5 is known to produce both secondary metabolites, pyoluteorin and DAPG, and this strain was used as the positive control.

CL145A and Pf-5 strains were grown on pyoluteorin production broth enriched with 2% glycerol (PhGiy), as described by Wang et al, (201 1), The media contains per liter: 3g NH4NO₃, I g yeast extract, lg KH2PO4, 2g NaCl, 0.5g MgSC and 1ml of trace minerals solution. Fermentations were performed in 250 ml Erlenmever flasks with 50 ml of media. Incubation was performed at 25 °C and 200 rpm for 48 hours. Fermentations were done side- by-side with CL145A and Pf-5 (NRRL B-23932) and harvested after 48 hours. Due to the lack of commercially available standards for pyoluteorin, strain Pf-5 was used as an internal standard.

The fermentation broths were extracted using Amberlite XAD-7 resin (Asoikar et al., 2006) by shaking the Whole Cell Broth ( WCB) with resin at 225 rpm for two hours at room temperature. The resin and cell mass were collected by filtration through cheesecloth and washed with deionized (DI) water to remove salts. The resin, cell mass, and cheesecloth w^rere then soaked for 1 hour in acetone/methanol (1 : 1) after which the solvent was filtered and dried under vacuum using rotary evaporator to give the crude extract. The crude extracts obtained from the above were dissolved in methanol to get a known concentration (10 mg/mL) which were later analyzed using Liquid ehromatography-mass spectrometry (LCMS).

Mass spectroscopy analysis of crude extract samples were performed on a Thermo Finnigan LCQ Deca XP Plus eiec rospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m/z 100- 1500 Da) and on a LCQ DECA XPplus Mass Spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (HPLC) instrument equipped with a Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4,6 mm x 100 mm Luna C18 5 um column (Phenomenex). The solvent system consisted of w^rater (solvent A) and acetonitrile (solvent B). The mobile phase began at 10% solvent B and is linearly increased to 100% solvent B over 20 min and then kept for 4 min, and finally returned to 10% solvent B over 3 min and kept for 3 min. The flow rate was 0.5 mL/min. The injection volume was 10 μΕ and the samples were kept at room temperature in an auto sampler. Mass spectroscopy analyses of compounds of interest present in the samples were performed under the following conditions: The flow rate of the nitrogen gas was fixed at 30 and 15 arb for the sheath and aux/sweep gas flow rate, respectively. Eiectrospray ionization was performed with a. spray voltage set at 5000 V and a capillary voltage at 35.0 V. The capillary temperature was set at 400°C. The data was analyzed on Xcalibur software. The compounds of interest in this analysis were pyoluteorin and DAPG, which were characterized by comparing the UV absorbance profile, retention time (RT) and molecular weight with those of the internal reference standard compounds.

As a standard sample of pyoluteorin was not available, the crude extract of Pf-5 was used to identify the production of pyoluteorin in a crude extract of CL145A. Pyoluteorin has a RT of 1 1 :30 min, a molecular mass of 272.02 in positive ionization mode and UV absorption max at 206, 254 & 308 nm. The isotopie spitting pattern in the MS confirms the presence of the two chlorine atoms in the molecule. The standard sample of 2,4- diacetylphloroglucinol (2) was purchased from Santa Cruz Biotechnology (CAS 2161-86-6) and has a RT of 13.99 min, molecular weight of 2.10, 14 and UV absorption max at 204, 268 & 320 nm. The production of both the compounds 1 & 2. were detected with RT 1 1 :30 and 13:96 min respectively in the crude extract of CL 145 A grown in the fermentation medium containing glycerol, with identical UV and mass spitting pattern to that of standard compounds. The structures for pyoluteorin and DAPG are shown below.

The production of both secondary metabolites pyoluteorin and DAPG by CL145A when grown under specific media, temperature and agitation conditions designed to optimize for the production of said metabolites. Presence of pyoluteorin was confirmed by identification of peaks according to mass spectrum patterns, retention times and UV spectra that are specific to pyoluteorin. The presence of DAPG was further confirmed by comparison to commercial standard.

Batches were produced in media FM3 and DM7 and analyzed as described above. Pyoluteorin and DAPG were not detected in these media under fermentation conditions typical of commercial manufacturin .

Pyoleuteorin DAPG 1,4 Conclusion

MBI-4Q1 (CL145A) was conclusively identified as Pseudomonas proiegens strain CL145A. An earlier effort to characterize the microorganism had yielded an identification of Pseudomonas fluoresceins (Pj). The change in the species identity is the outcome of recent revisions to the taxonomy of Pseudomonas fluorescens that grouped several strains pre v ious known as P into a new species characterized by divergence of 16S rRNA gene sequences and the production of pyoluteorin and DAPG, as well as other biochemical traits. Therefore, CL 145A is now classified as a strain of the newly formed Pseudomonas proiegens grouping.

EXAMPLE 2: MOLLUSCICIDE STUDIES OF PYOLUTEORIN AND DAPG

Tests were conducted in controlled temperature conditions, targeted at 20 °C, and using U.S. Environmental Proiection Agency (USEPA) siandard dilution water as a control. Quagga mussels were collected from Lake Havasu on January 14, 2014. The mussels were acclimatized to lab conditions for ten days in standard mussel hard water. Twenty four hours before the tests, ten healthy mussels were placed in each test plate, with three extra plates of mussels available. Unhealthy mussels were defined as mussels that were dead or gaping open, damaged, empty, or cracked. After 24 hours, the health of each mussel was reassessed and any unhealthy ones were replaced with mussels from the extra plates. The w^rater was poured out from each plate and each plate was treated with 15 mL of the desired treatment: test samples, DMSO control, and untreated control. Each sample was tested in triplicate. The samples were held in a dark cabinet for 2.4 hours without disruption. After 24 hours, the plates were poured out and rinsed twice with hard water, dead mussels removed, and the plates were filled with fresh water. The water in each plate was changed ever day. Mortality was monitored every day for up to 7 days, removing any dead mussels as they were found. % mortality was calculated by 1 00 x (cumulative dead mussels m the treatment - cumulative dead mussels in the blank)/total mussels in the treatment. Mean mortality was determined from the average of 3 plates per treatment.

2, 4-diacetylphoroglucinol (DAPG) and pyoluteorin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Final concentrations of 22.2, 1 1 .1 & 2.2 ug/^'mL were prepared by using 1 , 0.5 & 0.1 mg of compound and dissolving in 100 uL of DMSO and then diluting with hard water to 45 mL. 15 mL of the test sample was used to dose each plate as described above.

The results are shown in Table 5.

Table 5.

N/A = not applicable because no live mussels remaining EXAMPLE 5: KAOLIN EFFECTS

The effect of kaolin clay on the efficacy of a microbial biopesticide based on P. fluorescens bacteria is tested in a biobox study conducted at 1 1.8°C. On the first day of the experiment, kaolin clay is applied to the biobox from a concentrated stock solution via a peristaltic pump so that the final turbidity in the biobox is approximately 20 NTU

(normalized turbidity units). Fifty quagga mussels are placed in 1 -foot long acrylic tubes closed with a nylon mesh at both ends, and the tubes are placed in the botto m of the biobox for the treatment. The duration of the clay application is 6 hours, after which the mussels in the acrylic tubes were exposed to fresh running water in the biobox for 18 hours. The following day, extra clay is cleaned off from the bottom of the biobox, and the biopesticide in aqueous suspension is applied via a peristaltic pump to a final concentration of 200 ppm. After the 6-hour biopesticide treatment, mussels in tubes are incubated in the biobox with fresh running water at a rate of 1 gallon per minute. Mussels are observed and counted weekly for 5 weeks for determination of % mortality. The control treatments included an untreated control, a treatment with only kaolin clay (with no biopesticide) and a treatment with only biopesticide (with no clay pre- treatment). All treatments are run in three replicates.

Results presented in Table 6 and Figure 3 show a significant increase in mortality for mussels exposed to kaolin clay 18 hours before the biopesticide treatment compared with mussels with no clay pre-treatment. This phenomenon can be explained by increased siphoning activity of mussels harvested and treated in cold (1 1.8°C) water during the period of low biological activity. This increased siphoning results in greater uptake of pesticide product, which in turn results in increased mussel mortality.

Table 6. Efficacy of biopesl tkide £ expressed ; as % mortality ^• measure* d at ea ch time point

% mortal ity

1 9 16 21 27 34

Untreated control 0 0 0 0 0 0

Kaolin 20 NTU 0 0 0 0 0 1

Kaolin 20 NTU + 200 ppm 0 35 64 71 77 83

200 ppm 0 22 46 55 58 63 Although this invention has been described with reference to specific embodiments, the details thereof are not to be construed as limiting, as it is ob vious that one can use various equivalents, changes and modifications and still be within the scope of the present invention.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. A method for controlling one or more molluscs in a location where control is desired comprising introducing into said location a composition comprising an isolated compound in an amount effective to control said one or more molluscs, wherein the compound is selected from pyoluteorin and 2,4-diaeetylphloroglucinoL

2. The method according to claim 1, wherein said molluscs species are controlled by inducing death in said molluscs species.

3. The method of claim 1 or 2, wherein the compound is pyoluteorin.

4. The method of claim 1 or 2, wherein the compound is 2,4-diaeetylphlorogiucinol.

5. The method of any one of claims 1-4, wherein the mollusc is a member of a Gastropoda or Bivalvia class.

6. The method of any one of claims 1 -5, wherein the mollusc is a mussel.

7. The method of any one of claims 1-5, wherein the mollusc is a snail.

8. The method of any one of claims 1-7, wherein the composition further comprises a clay mineral.

9. ^'The method of claim 8, wherein said clay mineral is selected from kaolinite, smectite, and attapulgite, or a combination thereof.