Patent Publication Number: US-2022220432-A1

Title: Novel bacteria and uses thereof

Description:
STATEMENT OF CORRESPONDING APPLICATIONS 
     This application is based on the Provisional specification filed in relation to Australian Patent Application Number 2006903111, the entire contents of which are incorporated herein by reference. 
     TECHNICAL FIELD 
     The present invention relates to a novel bacterium and uses thereof. In particular, the present invention relates to applications concerned with the biopesticide activity of the novel bacterium and of its mutant or variant strains. The present invention also concerns substances obtained or derived from the aforesaid bacteria. 
     BACKGROUND ART 
     The invention describes a pesticidal strain of a new species to be called  Yersinia  entomaphaga that is active against a wide range of insect species. The novel bacterium also produces a toxic filtrate component that is also useful as a biopesticide agent in the control of insect species. 
     A novel insecticidal bacterium isolated from a New Zealand insect is described. The bacterium is a new species residing within the genus  Yersinia  and has been named  Yersinia entomophaga  MH96 . Y. entomophaga  has a broad host range towards members of the coleopteran and lepidopteran species amongst others. Death occurs within 72 hours post inoculation. The infection process appears to be due to a rapid build up in the bacterial population followed by a rapid invasion of the haemocoel leading to the cadaver taking on a deliquescing black appearance. Data are provided on biochemical utilisation tests (API), DNA sequences relating to phylogenetic analysis encompassing 16s ribosomal RNA sequencing and MLST sequence analysis of known  Yersinia  genes is given. In addition the DNA sequence of ˜132 short random  Y. entomophaga  genomic sequences are given. 
     A gram-negative bacterium was isolated from an infected grass grub field collected from New Zealand soils. Inoculation of grass grub larvae with the bacterium showed that death occurred within 2-3 days at 15° C. Standard biochemical identification using API20E and API50CH test strips indicated the bacterium is a member of the Enterobacteriaceae most similar to  E. sakazakii , but subsequent molecular characterisation placed it in the genus  Yersinia.    
     The continued use of  B. thuringiensis  and derivatives as a biopesticide over many years can lead to an increase in resistant insects. There is, therefore, a need for novel biopesticides to control insects. 
     There is also a need for biological control agents such as biopesticides to provide an alternative to chemical pesticides which can be toxic to non-target organisms in the environment. 
     All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country. 
     It is acknowledged that the term ‘comprise’ may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term ‘comprise’ shall have an inclusive meaning—i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term ‘comprised’ or ‘comprising’ is used in relation to one or more steps in a method or process. 
     It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. 
     Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only. 
     DISCLOSURE OF INVENTION 
     The original organism name  Yersinia  entomophagous MH-1 has been amended herein to refer to  Yersinia entomophaga  MH96, to conform to the official nomenclature of this organism. Thus, it should be clear that both names refer to the same organism as originally described in the provisional specification and the subject of the biological deposit at DSMZ on 4 May 2006 and designated accession no. DSM 18238. 
     It should be appreciated by those skilled in the art that unless the context clearly relays otherwise use of the terms  Yersinia entomophaga  MH96 or bacteria in this specification should also be taken to include mutant and variant strains of  Yersinia entomophaga  MH96 which retain the biopesticide activity. 
     According to a first aspect of the present invention there is provided an isolated  Yersinia entomophaga  MH96 bacterium deposited at DSMZ on 4 May 2006 and designated accession no. DSM 18238. 
     According to a second aspect of the present invention there is provided the use of  Yersinia entomophaga  MH96 to directly or indirectly obtain a biopesticide. 
     According to a third aspect of the present invention there is provided the use of  Yersinia entomophaga  MH96 as a biopesticide. 
     According to a fourth aspect of the present invention there is provided the use of  Yersinia entomophaga  MH96 in the manufacture of a composition suitable as a biopesticide. 
     According to a fifth aspect of the present invention there is provided a culture of  Yersinia entomophaga  MH96 as deposited at DSMZ on 4 May 2006 and designated accession no. DSM 18238. 
     According to a sixth aspect of the present invention there is provided the use of a culture of  Yersinia entomophaga  MH96 to directly or indirectly obtain a biopesticide 
     According to a seventh aspect of the present invention there is provided the use of a culture of  Yersinia entomophaga  MH96 as a biopesticide. 
     According to a eighth aspect of the present invention there is provided the use of a culture of  Yersinia entomophaga  MH96 in the manufacture of a composition suitable as a biopesticide. 
     According to a ninth aspect of the present invention there is provided a cellular extract obtained from  Yersinia entomophaga  MH96 as deposited at DSMZ on 4 May 2006 and designated accession no. DSM 18238 or a culture thereof. 
     According to a tenth aspect of the present invention there is provided the use of a cellular extract of  Yersinia entomophaga  MH96 to directly or indirectly obtain a biopesticide. 
     According to an eleventh aspect of the present invention there is provided the use of a cellular extract of  Yersinia entomophaga  MH96 as a biopesticide. 
     According to a twelfth aspect of the present invention there is provided the use of a cellular extract of  Yersinia entomophaga  MH96 in the manufacture of a composition suitable as a biopesticide. 
     According to a thirteenth aspect of the present invention there is provided a sonicated cell filtrate of  Yersinia entomophaga  MH96 as deposited at DSMZ on 4 May 2006 and designated accession no. DSM 18238 which has a biopesticide activity. 
     According to a fourteenth aspect of the present invention there is provided the use of a sonicated cell filtrate of  Yersinia entomophaga  MH96 to directly or indirectly obtain a biopesticide. 
     According to a fifteenth aspect of the present invention there is provided the use of a sonicated cell filtrate of  Yersinia entomophaga  MH96 as a biopesticide. 
     According to a sixteenth aspect of the present invention there is provided the use of a sonicated cell filtrate of  Yersinia entomophaga  MH96 in the manufacture of a composition suitable as a biopesticide. 
     According to a further aspect of the present invention there is provided a supernatant of a whole broth culture of  Yersinia entomophaga  MH96 as deposited at DSMZ on 4 May 2006 and designated accession no. DSM 18238. 
     According to a further aspect of the present invention there is provided the use of the supernatant of a whole broth culture of  Yersinia entomophaga  MH96 as a biopesticide. 
     According to a further aspect of the present invention there is provided the use of the supernatant of a whole broth culture of  Yersinia entomophaga  MH96 to directly or indirectly obtain a biopesticide. 
     According to a further aspect of the present invention there is provided the use of the supernatant of a whole broth culture of  Yersinia entomophaga  MH96 in the manufacture of a composition suitable as a biopesticide. 
     According to a further aspect of the present invention there is provided a composition that includes an effective amount of  Yersinia entomophaga  MH96 wherein said bacteria exhibits a biopesticide activity. 
     According to a further aspect of the present invention there is provided a composition formulated from an effective amount of a culture of  Yersinia entomophaga  MH96 wherein said culture exhibits a biopesticide activity. 
     According to a further aspect of the present invention there is provided a composition included an effective amount  Yersinia entomophaga  MH96, wherein  Yersinia entomophaga  MH96 has been killed as an intact form and maintains a biopesticide activity. 
     According to a further aspect of the present invention there is provided a composition formulated from an effective amount of a whole broth culture of  Yersinia entomophaga  MH96 wherein said whole broth culture exhibits a biopesticide activity. 
     According to a further aspect of the present invention there is provided a composition formulated from an effective amount of a supernatant of a whole broth culture of  Yersinia entomophaga  MH96 wherein the supernatant from the culture exhibits a biopesticide activity. 
     According to a further aspect of the present invention there is provided a composition formulated from an effective amount of a cellular extract of  Yersinia entomophaga  MH96 wherein said extract exhibits a biopesticide activity. 
     According to a further aspect of the present invention there is provided a composition formulated from an effective amount of a sonicated cell filtrate of  Yersinia entomophaga  MH96, wherein said extract exhibits a biopesticide activity. 
     Preferably, the composition may be formulated with at least one biopolymer compound. Preferably, at least one biopolymer compound is at least one type of gum compound. 
     Preferably, the composition may be formulated as a gel composition. 
     Preferably, the composition may be formulated with at least one biopolymer compound and at least one desiccating agent. 
     Preferably, the composition may be formulated with at least one type of gum compound and the at least one desiccating agent is at least one inert clay compound. 
     Preferably, the composition may be formulated as a dough or granular material. 
     Preferably, the composition may be formed into a prill or granule shape. 
     Preferably, the composition may be mixed with an aqueous liquid and sprayed onto a substrate. Other embodiments, the composition may be coated onto a substrate. Preferably, the substrate may be a seed. 
     According to a further aspect of the present invention there is provided a method of treating or protecting a plant and/or plant derived materials from pest infestation wherein the method comprises applying to the plant or its environment an effective amount of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method substantially as described above wherein the effective amount of  Yersinia entomophaga  MH96 is obtained from a culture of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method substantially as described above wherein the effective amount of  Yersinia entomophaga  MH96 is obtained from a supernatant from a whole broth culture of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method of treating or protecting a plant or plant derived materials from pest infestation wherein the method comprises applying to the plant or its environment an effective amount of a cellular extract of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method of treating or protecting a plant and/or plant derived materials from pest infestation wherein the method comprises applying to the plant or its environment an effective amount of a sonicated cell filtrate of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method of treating or protecting a plant and/or plant derived from pest infestation wherein the method comprises applying to the plant or its environment a composition comprising an effective amount of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method substantially as described above wherein the effective amount of  Yersinia entomophaga  MH96 is obtained from a culture of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method substantially as described above wherein the effective amount of  Yersinia entomophaga  MH96 is obtained from a supernatant from a whole broth culture  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method of treating or protecting a plant and/or plant derived materials from pest infestation wherein the method comprises applying to the plant or its environment a composition comprising an effective amount of a cellular extract of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method of treating or protecting a plant and/or plant derived materials from pest infestation wherein the method comprises applying to the plant or its environment a composition comprising an effective amount of a sonicated cell filtrate of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method of controlling and/or preventing a pest infestation characterised by the step of applying a composition comprising an effective amount of  Yersinia entomophaga  MH96 to a surface. 
     According to a further aspect of the present invention there is provided a method as claimed in claim  48 , wherein the effective amount of  Yersinia entomophaga  MH96 is obtained from a culture of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method, as claimed in claim  48 , wherein the effective amount of  Yersinia entomophaga  MH96 is obtained from a supernatant from a whole broth culture of  Yersinia entomophaga  MH96. 
     According to a further aspect of the present invention there is provided a method of controlling and/or preventing a pest infestation characterised by the step of applying a composition comprising an effective amount of a cellular extract of  Yersinia entomophaga  MH96 to a surface. 
     According to a further aspect of the present invention there is provided a method of controlling and/or preventing a pest infestation characterised by the step of applying a composition comprising an effective amount of a sonicated cell filtrate of  Yersinia entomophaga  MH96 to a surface. 
     According to a further aspect of the present invention there is provided a method of controlling and/or preventing a pest infestation characterised by the step of applying a composition as substantially described above, to a surface. 
     According to a further aspect of the present invention there is provided the use of an isolated  Yersinia entomophaga  MH96 bacterium deposited at DSMZ on 4 May 2006 and designated accession no. DSM 18238, or culture thereof, for use in the biopesticide activity against the insect species listed in Table 13 and/or the larvae thereof. 
     It should be appreciated by those skilled in the art that discovery by the inventors that  Yersinia entomophaga  MH96 has a biopesticide activity is of broad application. 
     Preferably, the biopesticide activity may be for the application against insect species listed in Table 13 and/or the larvae thereof. 
     As used herein the term “isolated” means removed from the natural environment in which the bacteria naturally occurs and is separated from some or all of the co-existing materials in the natural system from which the bacteria has been obtained. 
     As used herein the term “biopesticide” refers to a biologically derived substance having the ability to kill, or retard the growth of, insects and/or the larvae thereof. In particular, a biopesticide of the present invention should be capable of retarding growth, or killing, one or more of the insect species listed in Table 13 and/or the larvae thereof. Most preferably, a biopesticide of the present invention should be capable of retarding the growth, or killing, at least one, but preferably all or most of the species listed in Table 13 and/or the larvae thereof. 
     As used herein the term “culture” refers to a population of bacteria together with the media in or on which the population was propagated (i.e. grown). 
     As used herein the term “whole broth culture” refers to a liquid media and the population of bacteria therein. 
     In preferred embodiments the broth may be Luria-Bertani broth. However, it will be appreciated by a person skilled in the art that other suitable broths may be used. 
     As used herein the term “cellular extract” refers to a substance or mixture of substances obtained from a bacterial cell. 
     As used herein the term “sonicate” or grammatical variants thereof refers to subjecting a cell to ultrasonic vibrations in order to fragment the cell wall to release the contents of the cell. 
     It should be appreciated that the ‘cellular extract’ may be obtained in a variety of different ways, and may come in a variety of different forms without departing from the scope of the present invention. 
     In some embodiments the cellular extract may be a crude extract of the contents of the cell. In general the crude extract may be obtained via centrifugation of a whole broth culture re-suspended in a suitable buffer. 
     Such an extract may have been derived from Sonication; French press; Mantin gaulin press, bead basher, bead mill mincer osmotic lysis and enzyme related lysis as outlined in Scopes (1993); Doonan (1996) and Sambrook et al. (1989). 
     In other embodiments the cellular extract may be a freeze dried or a spray dried extract. In general, the freeze or spray dried extract may be obtained via any cellular extract which has also been subjected to a freeze- or spray drying process or alternate processes as outlined in Maa and Prestrelski (2000). 
     In preferred embodiments the cellular extract may be derived from the aforementioned methods via sonication; French press; Mantin gaulin press, bead basher, bead mill mincer osmotic lysis or enzyme related lysis. 
     In general the inventors have found that a supernatant having a biopesticide activity will be obtained when the organism is grown in Luria-Bertani broth at 25° C. 
     The term ‘plant’ refers to the plant in it&#39;s entirety or a part thereof including selected portions of the plant during the plant life cycle such as the plant seeds, shoots, leaves, bark, pods, roots, flowers, stems and the like, including crop food and plant derived materials or parts thereof. 
     The term ‘plant derived materials’ refers to products that may be produced from a plant or part thereof. It will be appreciated that a person skilled in the art will know of various examples of plant derived products, such as hay, silage or other types of feed or products. 
     Compositions of the present invention may be formulated in a variety of different ways without departing from the scope of the present invention. In general the formulation chosen will be dependent on the end application. For example, possible formulations include, but should not be limited to:
         Vectors such as the Trojan vector;   Matrixes;   Soluble powders;   Granules;   Micro encapsulation in a suitable medicine;   Aqueous suspensions;   Non-aqueous suspensions;   Emulsions;   Pastes;   Emulsifiable concentrations; or   Baits.       

     The present invention may preferably include formulations suitable for:
         direct application to insect affected areas e.g. drench, spray form;   suspended in a bait matrix;   slow release prills for subterranean applications; or   hydrophobic matrixes facilitating buoyancy for aquatic surface filter feeders.       

     It will be appreciated that other suitable formulations and/or methods of preparing the formulations and/or compositions will be known to those skilled in the art. Examples of other such methods to stabiles or prepare a composition include the methods described in patent applications WO 02/15702 or WO 02/15703. 
     The term bait as used herein refers to any foodstuff or other attractant to an insect or larvae thereof which includes an effective amount of:
         a)  Yersinia entomophaga  MH96; or   b) a mutant or variant strain of  Yersinia entomophaga  MH96; or   c) a derivative of a) or b).       

     The term ‘effective amount’ as used herein refers to a suitable quantity for a biopesticide activity to be exhibited. 
     The  Yersinia entomophaga  MH96 of the present invention produces a biopesticide which can be applied directly to surfaces where insects may contact such as artificial/cultural surfaces (e.g. milled wood, concrete, and urban dwellings); as well as in agricultural systems such as plant surfaces seed coats or matter of plant origin. 
     The present invention has application in both terrestrial and aquatic environments and may be applied in or on both soil and phylloplane or rhizospere systems. 
     Thus, preferred embodiments of the present invention may have a number of advantages over the prior art which can include:
         providing a new biopesticide which has a broad efficacy across a range of insects;   providing a new method for controlling insects; and   providing a new biopesticide which has a range of different forms.       

    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: 
         FIG. 1  shows a phylogenetic comparison of 16s ribosomal DNA of species within the genus  Yersinia , using the program DNAML (Phylip suite) with default values, and with randomised input order (data from Kotetishvili et. al 2005; except for  Yersinia entomophaga  MH96); 
         FIG. 2  shows a phylogenetic comparison of 16s ribosomal DNA based on amplification of 190 bp from 16s ribosomal DNA; based on 190 bp region analysed using the programme DNAman, tree created using the Neighbor-Joining method (Saitou and Nei, 1987, Mol. Biol. Ecol. 4:406-425); 
         FIG. 3  shows a phylogenetic comparison based on data from 428 bp of Y-HSP60 amplification, analysed using the programme DNAman, tree created using the Neighbor-Joining method (Saitou and Nei, 1987, Mol. Biol. Ecol. 4:406-425) (data from Kotetishvili et. al 2005; except for  Yersinia entomophaga  MH96); 
         FIG. 4  shows a phylogenetic comparison based on data from glyA amplification, analysed using the programme DNAman, tree created using the Neighbor-Joining method (Saitou and Nei, 1987, Mol. Biol. Ecol, 4:406-425) (data from Kotetishvili et. al 2005; except for  Yersinia entomophaga  MH96); 
         FIG. 5  shows a phylogenetic comparison based on data from amplification of 394 bp from recA, analysed using the programme DNAman, tree created using the Neighbor-Joining method (Saitou and Nei, 1987, Mol. Biol. Ecol. 4:406-425) (data from Kotetishvili et. al 2005; except for  Yersinia entomophaga  MH96); 
         FIG. 6  shows a phylogenetic comparison based on data from gyrB amplification, analysed using the programme DNAman, tree created using the Neighbor-Joining method (Saitou and Nei, 1987, Mol. Biol. Ecol. 4:406-425) (data from Kotetishvili et, al 2005; except for  Yersinia entomophaga  MH96); 
         FIG. 7  shows a phylogenetic comparison based on data from amplification of 1765 bp assembled fragments, using the programme MrBayes v3.1.2 and trees viewed using TreeView (data from Kotetishvili et. al 2005; except for  Yersinia entomophaga  MH96); 
         FIG. 8  shows a phylogenetic comparison based on data from amplification of GlnA, GyrB, RecA, Y-hsp60 (1,525 base) assembled fragments, analysed using a maximum likelihood tree using DNAML from the Phylip package (data from Kotetishvili et. al 2005; except for  Yersinia entomophaga  MH96); 
         FIG. 9  shows clover seedlings cut to approximately 10 mm, and Kibbled wheat baits can be seen on the surface of the potting mix; 
         FIG. 10  shows clover plants at day 12, container A had four plants destroyed by  Wiseana  spp. larvae feeding, while all the plants survived in the container B on the right; and 
         FIG. 11  shows Tunnel house temperature over the duration of the bioassay. 
     
    
    
     BEST MODES FOR CARRYING OUT THE INVENTION 
     Discovery 
     During routine prefeeding assays of grass grub larvae that had been collected from various field locations throughout the South Island of New Zealand, larvae that appeared diseased were put aside and assessed for the presence of a causative bacterial agent. 
     Larvae were surface sterilized by submerging in 70% methanol. The larvae were then shaken in sterile DH 2 O, removed and blotted dry. A 10 μl pipette tip was inserted through the back of the larvae breeching the haemocoelic cavity, an aliquot withdrawn and serial diluted in Luria Bertini broth. The diluent was plated on non-selective Luria Bertina media and incubated at 30° C. Morphologically different isolates were purified, and accessed for virulence by standard bioassay. 
     Example 1: PHYSIOLOGICAL AND METABOLIC CHARACTERISATION 
     Gram Negative Rod 
     Growth in LB media with subsequent plating, shows that two colony forms are apparent these are:
         i) convex circular   ii) dimpled circular       

     However, if the colonies are allowed to grow to over 3-4 days all colonies have exhibit a convex circular form indicative that the dimpled circular form is growth stage dependent 
     The bacteria exhibits growth retardation if grown at 37° C. 
     The bacteria form large flocs of bacterial cells in the RSYE culture grown at 37° C. just prior to 6 hours at 250 rpm. 
     Dilution plated samples taken at 48 and 72 hours required longer incubation at 30° C. before colonies were visible and able to be counted. 
     The colonies are positive on DNAase plates within 24 hours (O&#39;Callaghan and Jackson 1993). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 ASSILMIATION TESTS AND RESULTS 
               
            
           
           
               
               
               
            
               
                   
                 Test 
                 Result 
               
               
                   
                   
               
               
                   
                 Gram stain 
                 negative 
               
               
                   
                 Oxidase 
                 negative 
               
               
                   
                 Glucose acid 
                 positive 
               
               
                   
                 API 20E 
                 1-307-160 No match 
               
               
                   
                 DNAase 
                 weak positive 
               
               
                   
                   
               
            
           
         
       
     
     The strains were tested using a commercial bacterial identification system API, bio-Merieux. Results are shown in Table 2 below. 
     Carbon source utilisation tests were done by using API strips (API system, La Balme les Grottes, France) 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Testing results from API bacterial identification system 
               
            
           
           
               
               
               
            
               
                   
                 API 
                 
                   Y. entomophaga 
                 
               
               
                   
                   
               
               
                   
                 ONPG (beta-galactosidase) 
                 + 
               
               
                   
                 ADH (arginine 
                 − (weak) 
               
               
                   
                 dehydrolase) 
                   
               
               
                   
                 LDC (lysine 
                 − 
               
               
                   
                 decarboxylase) 
                   
               
               
                   
                 ODC (orthinine 
                 + 
               
               
                   
                 decarboxylase) 
                   
               
               
                   
                 CIT (citrate utilisation) 
                 + 
               
               
                   
                 H 2 S (H 2 S production) 
                 − 
               
               
                   
                 URE (urease) 
                 − 
               
               
                   
                 TDA (tryptophane 
                 − 
               
               
                   
                 desaminase) 
                   
               
               
                   
                 IND (indole production) 
                 − 
               
               
                   
                 VP (acetoin production) 
                 + 
               
               
                   
                 GEL (gelatinase) 
                 + 
               
               
                   
                 GLU (glucose 
                 + 
               
               
                   
                 fermentation) 
                   
               
               
                   
                 MAN (mannitol 
                 + 
               
               
                   
                 fermentation) 
                   
               
               
                   
                 INO (inositol fermentation) 
                 − 
               
               
                   
                 SOR (sorbitol fermentation) 
                 − 
               
               
                   
                 RHA (rhamnose 
                 − 
               
               
                   
                 fermentation) 
                   
               
               
                   
                 SAC (sucrose 
                 + 
               
               
                   
                 fermentation) 
                   
               
               
                   
                 MEL (melibiose 
                 + 
               
               
                   
                 fermentation) 
                   
               
               
                   
                 AMY (amygdalin 
                 − 
               
               
                   
                 fermentation) 
                   
               
               
                   
                 ARA (arabinose 
                 − 
               
               
                   
                 fermentation) 
                   
               
               
                   
                 OX (oxidase) 
                 − 
               
               
                   
                 DNAse 
                 + 
               
               
                   
                 glycerol 
                 + 
               
               
                   
                 erythritol 
                 − 
               
               
                   
                 D-arabinose 
                 − 
               
               
                   
                 L-arabinose 
                 − 
               
               
                   
                 ribose 
                 + 
               
               
                   
                 D-xylose 
                 − 
               
               
                   
                 L-xylose 
                 − 
               
               
                   
                 adonitol 
                 − 
               
               
                   
                 β methyl-xyloside 
                 − 
               
               
                   
                 galactose 
                 + 
               
               
                   
                 D-glucose 
                 + 
               
               
                   
                 D-fructose 
                 + 
               
               
                   
                 D-mannose 
                 + 
               
               
                   
                 L-sorbose 
                 − 
               
               
                   
                 Rhamnose 
                 − 
               
               
                   
                 Dulcitol 
                 − 
               
               
                   
                 inositol 
                 − 
               
               
                   
                 mannitol 
                 + 
               
               
                   
                 Sorbitol 
                 − 
               
               
                   
                 α methyl-D-mannoside 
                 − 
               
               
                   
                 α methyl-D-glucoside 
                 − 
               
               
                   
                 N acetyl glucosamine 
                 + 
               
               
                   
                 amygdaline 
                 − 
               
               
                   
                 arbutine 
                 − 
               
               
                   
                 esculine 
                 − 
               
               
                   
                 salicine 
                 − 
               
               
                   
                 cellobiose 
                 ? 
               
               
                   
                 maltose 
                 + 
               
               
                   
                 lactose 
                 ? 
               
               
                   
                 melibiose 
                 + 
               
               
                   
                 saccharose 
                 + 
               
               
                   
                 trehalose 
                 + 
               
               
                   
                 inuline 
                 − 
               
               
                   
                 melezitose 
                 − 
               
               
                   
                 D-raffinose 
                 + 
               
               
                   
                 amidon 
                 − 
               
               
                   
                 glycogene 
                 − 
               
               
                   
                 xylitol 
                 − 
               
               
                   
                 β gentiobiose 
                 − 
               
               
                   
                 D-turanose 
                 − 
               
               
                   
                 D-lyxose 
                 − 
               
               
                   
                 D-tagatose 
                 − 
               
               
                   
                 D-fucose 
                 − 
               
               
                   
                 D-arabitol 
                 − 
               
               
                   
                 L-arabitol 
                 − 
               
               
                   
                 gluconate 
                 ? 
               
               
                   
                 2 ceto-gluconate 
                 − 
               
               
                   
                 5 ceto-gluconate 
                 ? 
               
               
                   
                   
               
               
                   
                 score api 50 = +24 hours (ATCC cultures &gt; 48 hrs). ? denotes inconclusive result. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 AGAR phenotypes of  Yersinia entomophaga  MH96 grown  
               
               
                 on various commercially supplied agar (Fort Richards)  
               
               
                 bacteria grown for 24 hours at 30° C. 
               
            
           
           
               
               
            
               
                 Plate media 
                 Phenology 
               
               
                   
               
               
                 Orientation agar 
                 purple 
               
               
                 XLD agar 
                 orange slight yellow halo 
               
               
                 Col sheep blood 
                 no lysis 
               
               
                 Col horse blood 
                 no lysis 
               
               
                 Violet red bile agar 
                 growth 
               
               
                 Brilliant green agar modified 
                 yellow 
               
               
                 PFA agar 
                 no growth 
               
               
                 Dermatophte test medium 
                 turquoise 
               
               
                 Chocolate sens 
                 matt rhizoid morphology 
               
               
                 Cetrimide agar 
                 no growth but very slight after 
               
               
                   
                 48 hours 
               
               
                 Bismuth sulphite agar 
                 dark green 
               
               
                 Macconkey agar w/d cv 
                 orangly matt rhizoid morphology 
               
               
                 Haemin agar 
                 no lysis 
               
               
                 Brilliant green agar 
                 light green tinge rhizoid growth 
               
               
                 Thayer martin agar 
                 no growth 
               
               
                   
               
            
           
         
       
     
     Example 2: Genetic Identification 
     DNA-DNA Hybridization 
     DNA-DNA hybridization was determined at the Deutsche Sammlung von Mikroorganismen und. Zellkulturen, Braunschweig, Germany and carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6×6 multicell changer and a temperature controller with in-situ temperature probe (Varian). DNA-DNA relatedness was tested at 70° C. in 2×SSC plus 10% (v/v) DMSO 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 % DNA-DNA similarity (in 2 X SSC at 70° C.) 
               
            
           
           
               
               
               
            
               
                   
                   
                 ″ Y. entomophaga  MH96″ DSM 
               
               
                   
                   
                 18238 (ID 06-840) 
               
               
                   
                   
               
               
                   
                 
                   Yersinia pseudotuberculosis 
                 
                 23.0 (19.7) 
               
               
                   
                 DSM 8992 T  (ID 06-841) 
                   
               
               
                   
                   Yersinia ruckeri  DSM 18506 T    
                 45.6 (52.1) 
               
               
                   
                 (ID 06-842) 
                   
               
               
                   
                   Yersinia intermedia  DSM 18517 T    
                 0.82 (2.15) 
               
               
                   
                 (ID 06-843) 
               
               
                   
                   
               
            
           
         
       
     
     Results derived indicate that  Y. entomophaga  MH96 DSM 18238 (ID 06-840) does neither belong to the species  Y. pseudotuberculosis  DSM 8992 T  (ID 06-841) nor to the species  Y. ruckeri  DSM 18506 T  (ID 06-842) and nor to the species  Y. intermedia  DSM 18517 T  (ID 06-843) when the recommendations of a threshold value of 70% DNA-DNA similarity for the definition of bacterial species by the ad hoc committee (Wayne et al., 1987) are considered. 
     DNA G+C Content 
     The DNA G+C content of  Yersinia entomophaga  MH96 DSM 18238 (ID 06-840): was determined at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany according to the method of Mesbah et al. (1989) and as three independent measurements. Cells were disrupted using a French pressure cell and the DNA was purified according to the procedure of Cashion et al. (1977) and degraded as described by Mesbah et al. (1989) and assessed by HPLC system (Shimadzu Corp. Japan). Using this method the The DNA G+C content of  Y. entomophaga ” MH96 DSM 18238 (ID 06-840): was at 49.3 mol % G+C These estimates are within the accepted limits for the genus  Yersinia  of 46-50 mol % (Bercovier &amp; Mollaret, 1984.). 
     Plasmid Visualisation 
     Plasmid visualisation by method of Kado and Lui (1981) showed that no extra-chromosomal elements such as plasmids were present. 
     Purification of Genomic DNA 
     Genomic DNA for rRNA sequencing was isolated by a modified method of Cathala G et al, (1983). A 1.5 ml O/N culture was pelletted, and resuspended in 500 μl of lysis solution (5M guanidine isothiocyanate, 10 mM EDTA, 50 mM Tris-HCl (pH 7.5), 8% mercaptoethanol). An equal volume of phenol:chloroform was added, the tube inverted several times and centrifuged at 13000 g for 15 minutes. The upper layer removed to a new eppendorf and ethanol precipitated. To remove residual Guandium and other inhibitory compounds, the resultant pellet was air dried and resuspended in 500 μl of DH 2 O, placed in a 37° C. water bath, and intermittently agitated for 1-2 hours. The solution was then re-ethanol precipitated and resuspended in 50 μl DH 2 O 
     rRNA PCR 
     Closely related strains identified by API were obtained from the American Type Culture Collection, Md., and included the  Enterobacter sakazakii  strains ATCC29004 and ATCC51329. 
     The 16S gene was amplified with primers as shown in Table 5. 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Primers used to amplify 16s rRNA 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 U16a 
                 AGA GTT TGA TCC TGG CTC 
               
               
                   
                   
               
               
                   
                 U16b 
                 TAC GGY TAC CTT GTT ACG ACT T 
               
               
                   
                   
               
               
                   
                 UB16A2 
                 GCC GCG GTA AT ACG GAG G 
               
               
                   
                   
               
               
                   
                 U16B2 
                 AGG ATA AGG GTT TGC GCT CCG 
               
               
                   
                   
               
            
           
         
       
     
     PCR 
     94oc 15 s 60° C.(30 s) 62oC(2′)×5 cycles 
     94oc 15 s 57° C.(30 s) 62oC(2′)×40 cycles 
     PCR template was sequenced using automated sequencing and an Applied Biosystem 373A or 377 autosequencer. Sequence data was assembled using SEQMAN. The database at the National Centre for Biotechnology Information was searched using BLASTN and the WWW. Nucleotide sequence accession numbers. The sequences determined in this study have been assigned the GenBank accession numbers DQ400713-DQ400845. 
     rDNA Sequence Comparison 
     Table 6 below shows 16s comparison based on 1428 bp and compared with GenBank sequences: 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 16s comparison based on 1428 bp and compared with GenBank 
               
               
                 sequences. 
               
            
           
           
               
               
               
               
            
               
                 Genbank 
                   
                   
                   
               
               
                 accession 
                 Genus, species 
                   
                 Accession 
               
               
                 number 
                 and strain 
                 Reference/author 
                 date 
               
               
                   
               
               
                 AF366385 
                 
                   Yersinia ruckeri 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                   
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
                   
               
               
                 AB004746 
                 
                   Enterobacter 
                 
                 Harada, H. 
                 25 Jul. 1997 
               
               
                   
                 
                   sakazakii 
                 
                   
                   
               
               
                   
                 (strain: JCM1233) 
                   
                   
               
               
                 RAU90757 
                 
                   Rahnella aquatilis 
                 
                 Brenner, D. J., Muller, H. E., 
                 15 Apr. 1998 
               
               
                   
                   
                 Steigerwalt, A. G., Whitney, A. M., 
                   
               
               
                   
                   
                 O&#39;Hara, C. M. and Kampfer, P. 
                   
               
               
                   
                   
                 (1998) Two new  Rahnella   
                   
               
               
                   
                   
                 genomospecies that cannot be 
                   
               
               
                   
                   
                 phenotypically differentiated 
                   
               
               
                   
                   
                 from  Rahnella aquatilis . Int. J. 
                   
               
               
                   
                   
                 Syst. Bacteriol. 48 Pt 1, 141-149 
                   
               
               
                 YEN16SA 
                 
                   Y. enterocolitica 
                 
                 Harmsen, D. 
                 27 Jun. 1996 
               
               
                   
                 (strain O:3 108 c) 
                   
                   
               
               
                 AF366384 
                   Yersinia rohdei  16S 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                   
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
                   
               
               
                 S000001663 
                 
                   Yersinia 
                 
                 Harmsen, D. W., Schmelz, J. F. 
                 30 Jul. 1996 
               
               
                   
                   pseudotuberculosis ; 
                 and Heesemann, J. 
                   
               
               
                   
                 Serotype III 1B1 
                   
                   
               
               
                   
                 B28 (W.W.) 
                   
                   
               
               
                 S000001661 
                 
                   Yersinia 
                 
                 Ibrahim, A., Goebel, B. M., 
                 12 Jun. 1995 
               
               
                   
                   enterocolitica ; ER- 
                 Liesack, W., Griffiths, M. and 
                   
               
               
                   
                 26036-92; serotype 
                 Stackebrandt, E. 
                   
               
               
                   
                 O:3 
                 (1993) The phylogeny of the 
                   
               
               
                   
                   
                 genus  Yersinia  based on 16S 
                   
               
               
                   
                   
                 rDNA sequences. FEMS 
                   
               
               
                   
                   
                 Microbiol. Lett. 114 (2), 173-177 
                   
               
               
                 S000004821 
                 
                   Yersinia 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 17 May 2001 
               
               
                   
                 
                   pseudotuberculosis 
                 
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
                   
               
               
                 S000004821 
                 
                   Yersinia 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 17 May 2001 
               
               
                   
                 
                   pseudotuberculosis 
                 
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                 83 
                 Park, Y.-H. 
                   
               
               
                 S000003234 
                   Yersinia rohdei  (T);  
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                 ATCC 43380 
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H, 
                   
               
               
                 YS17B16S 
                   Yersinia  sp. (isolate 
                 Ibrahlm, A., Liesack, W., 
                 17 Feb. 1997 
               
               
                   
                 YEM17B 
                 Steigerwalt, A. G., Brenner, D. J., 
                   
               
               
                   
                   
                 Stackebrandt, E. and Robins- 
                   
               
               
                   
                   
                 Browne, R. M. 
                   
               
               
                   
                   
                 (1997) A cluster of atypical 
                   
               
               
                   
                   
                   Yersinia  strains with a distinctive 
                   
               
               
                   
                   
                 16S rRNA signature 
                   
               
               
                   
                   
                 FEMS Microbiol. Lett. 146 (1), 
                   
               
               
                   
                   
                 73-78 
                   
               
               
                 YPD16SRN 
                   Yersinia pestis  (D- 
                 Ibrahim, A., Goebel, B. M., 
                 27 May 2000 
               
               
                   
                 28) 
                 Liesack, W., Griffiths, M. and 
                   
               
               
                   
                   
                 Stackebrandt, E. (1993) The 
                   
               
               
                   
                   
                 phylogeny of the genus  Yersinia   
                   
               
               
                   
                   
                 based on 16S rDNA sequences. 
                   
               
               
                   
                   
                 FEMS Microbiol. Lett. 114 (2), 
                   
               
               
                   
                   
                 173-177 
                   
               
               
                 AJ414156 
                   Yersinia pestis  CO9 
                   
                   
               
               
                 YPE16SA 
                   Y. pestis  (strain EV 
                 Harmsen, D. 
                   
               
               
                   
                 pst+ c) 
                   
                   
               
               
                 YEPRGD 
                 
                   Yersinia pestis 
                 
                 Wilson, K. H. and Hills, H. G. 
                 19 Jan. 1995 
               
               
                 AF365949 
                 
                   Yersinia 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 17 May 2001 
               
               
                   
                 
                   pseudotuberculosis 
                 
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                 strain 6088 
                 Park, Y.-H. 
                   
               
               
                 YR16SRN 
                   Yersinia rohdei  (ER- 
                 Ibrahim, A., Goebel, B. M., 
                 27 May 2000 
               
               
                   
                 2935) 
                 Liesack, W., Griffiths, M. and 
                   
               
               
                   
                   
                 Stackebrandt, E. (1993) The 
                   
               
               
                   
                   
                 phylogeny of the genus  Yersinia   
                   
               
               
                   
                   
                 based on 16S rDNA sequences. 
                   
               
               
                   
                   
                 FEMS Microbiol. Lett. 114 (2), 
                   
               
               
                   
                   
                 173-177 
                   
               
               
                 YK16SRRN 
                 
                   Yersinia kristensenii 
                 
                 Ibrahim, A., Goebel, B. M., 
                 27 May 2000 
               
               
                   
                 (ER-2812) 
                 Liesack, W., Griffiths, M. and 
                   
               
               
                   
                   kristensenii  2 
                 Stackebrandt, E. (1993) The 
                   
               
               
                   
                   
                 phylogeny of the genus  Yersinia   
                   
               
               
                   
                   
                 based on 16S rDNA sequences. 
                   
               
               
                   
                   
                 FEMS Microbiol. Lett. 114 (2), 
                   
               
               
                   
                   
                 173-177 
                   
               
               
                 AF366381 
                 
                   Yersinia kristensenii 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                   
                 Chung, S.-L, Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
                   
               
               
                 AF366382 
                 
                   Yersinia mollaretii 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                   Yersinia mollaretii 2 
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
                   
               
               
                 YM16SRRN 
                 
                   Yersinia mollaretii 
                 
                 Ibrahim, A., Goebel, B. M.,  
                 27 May 2000 
               
               
                   
                 (ER-2975) 
                 Liesack, W., Griffiths, M. and 
                   
               
               
                   
                   
                 Stackebrandt, E. (1993) The 
                   
               
               
                   
                   
                 phylogeny of the genus  Yersinia   
                   
               
               
                   
                   
                 based on 16S rDNA sequences. 
                   
               
               
                   
                   
                 FEMS Microbiol. Lett. 114 (2), 
                   
               
               
                   
                   
                 173-177 
                   
               
               
                 AF366379 
                 
                   Yersinia 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                 
                   frederiksenii 
                 
                 Chung, S.-L, Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
                   
               
               
                 AF366380 
                 
                   Yersinia intermedia 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                   
                 Chung, S.-L, Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
                   
               
               
                 AF366376 
                 
                   Yersinia aldovae 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                   
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H, 
                   
               
               
                 YB16SRRN 
                 
                   Yersinia bercovieri 
                 
                 Ibrahim, A., Goebel, B. M., 
                 27 May 2000 
               
               
                   
                 
                   Yersinia bercovieri 
                 
                 Liesack, W., Griffiths, M. and 
                   
               
               
                   
                 1 
                 Stackebrandt, E. (1993) The 
                   
               
               
                   
                   
                 phylogeny of the genus  Yersinia   
                   
               
               
                   
                   
                 based on 16S rDNA sequences. 
                   
               
               
                   
                   
                 FEMS Microbiol. Lett. 114 (2), 
                   
               
               
                   
                   
                 173-177 
                   
               
               
                 AF366377 
                 
                   Yersinia bercovieri 
                 
                 Kim, W., Song, M.-O., Song, W., 
                 8 May 2001 
               
               
                   
                   
                 Chung, S.-I., Choi, C.-S. and 
                   
               
               
                   
                   
                 Park, Y.-H. 
               
               
                   
               
            
           
         
       
     
     As shown in  FIGS. 1 and 2 , using 16s rRNA sequences  Y. entomophaga  aligns with atypical strains of  Y. frederiksenii  ( FIG. 1 ) or  Y. kristensenii  ( FIG. 2 —190 bp). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Multi locus sequence tagging (MLST) Primers 
               
               
                 used for MLST of the  Yersinia  species 
               
               
                 (Derived from Kotetishvili et al. 2005), 
               
            
           
           
               
               
               
            
               
                   
                 Primers 
                 Accession number 
               
               
                 Gene 
                 (5′→3′) 
                 and Results 
               
               
                   
               
               
                 16S rDNA 
                 AGTTTGATC 
                 DQ400782 
               
               
                   
                 ATGGCTCAG 
                 FIG. 1 and 2 
               
               
                   
                 TTACCGCGG 
                   
               
               
                   
                 CTGCTGGCA 
                   
               
               
                   
               
               
                 GlnA 
                 CGATTGGTG 
                 DQ400780 
               
               
                   
                 GCTGGAAAG 
                 FIG. 4 
               
               
                   
                 GCTTGGTCA 
                   
               
               
                   
                 TRGTRTTGA 
                   
               
               
                   
                 AGCG 
                   
               
               
                   
               
               
                 GyrB 
                 CGGCGGTTT 
                 DQ400781 
               
               
                   
                 GCAYGGYGT 
                 FIG. 6 
               
               
                   
                 RGGCAGSGT 
                   
               
               
                   
                 RCGRGTCAT 
                   
               
               
                   
                 YGCCG 
                   
               
               
                   
               
               
                 recA 
                 GGGCCAAAT 
                 DQ400835 
               
               
                   
                 TGAAAARCA 
                 FIG. 5 
               
               
                   
                 RTTCGGCGC 
                   
               
               
                   
                 CRATYTTCA 
                   
               
               
                   
                 TRCGRATYT 
                   
               
               
                   
                 GGT 
                   
               
               
                   
               
               
                 Y-HSP60 
                 GACGTNGTA 
                 DQ400829 
               
               
                   
                 GAAGGTATG 
                 FIG. 3 
               
               
                   
                 YAGCGCCGC 
                   
               
               
                   
                 CAGCCAGTT 
                   
               
               
                   
                 TAGC 
               
               
                   
               
            
           
         
       
     
     MLST analysis, based on primer sequences as above in Table 7, in conjunction and analysis of random genomic sequence analysis (Results shown in  FIGS. 3-8  of phylogenetic comparison of sequences from the above genes), indicates that  Yersinia entomophaga  MH96 is a new species residing within the genera  Yersinia. A presumptive name foe the new species would be Yersinia entomophaga  MH96 (as it eats insects) 
     Random Genomic Sequencing of  Yersinia entomophaga  MH96 
     To further help define what species  Yersinia entomophaga  MH96 is, genomic DNA of  Yersinia entomophaga  MH96 was made and digested using the restriction enzymes HindIII; EcoRI and PstI in independent reactions. The digested DNA was then ligated to the vector DNA (pUC19) digested with the aforementioned enzymes. Using this method approximately 132 independent random HindIII; PstI; or EcoRI; clones were constructed. Using the pUC19 M13F and M13R based primers DNA from the clones was sequenced. The DNA sequence data has been deposited under the GenBank accession number (DQ400713-DQ400845). This data have enabled the generation of random snap shots of the  Yersinia entomophaga  MH96 genome and shown that many genes have greater than 90% DNA similarity to the DNA of  Yersinia pestis . While other DNA remains at this point in time novel scoring no apparent similarity to DNA I the current database 
     The DNA nucleotide sequence of 132 random Y  entomophaga  sequences have been submitted to GenBank and assigned the numbers DQ400713-DQ400845 
     Example 3: Culture Conditions 
       Yersinia entomophaga  MH96 can be grown in LB broth or on LB agar (Sambrook and Russell, 2001) or any alternate common laboratory media as yet no defined media for the isolation of  Yersinia entomophaga  MH96 has been defined, optimum growth for  Yersinia entomophaga  MH96 is 25° C.-30° C. Cultures were incubated at 200 rpm in a Raytek orbital mixer incubator. 
     Crude Toxin Isolation Using Cell Lysis Such as Sonication 
     From a 3 ml overnight culture pellet by centrifugation (8,000 g 3 minutes) resuspended in 1.0 ml of 1.5 ml phosphate buffer (10 mM phosphate buffer, pH 7.4; 2.7 mM KCl; 137 mM NaCl), two 0.7 ml samples were transferred to an eppendorf and subjected to three 30 s rounds of sonication on wet ice using a Sanyo soniprep 150 sonicator (18Ω). The sonicated samples were centrifuged (16,000 g) and the supernatant filter sterilised through a 0.2 μm filter to a sterile eppendorf. The filtrate&#39;s were placed on wet ice and used immediately for bioassay analysis. The efficacy of the lysate was assessed by the oral injection of 5 μl of filtrate sample through the larval mouth parts or the application of 5 μl of filtrate sample to the surface of a 3 mm 3  carrot from which the grass grub larvae would feed. Under these conditions toxins can be visualised on a standard Laemmli SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The toxins have activity only if the bacterium is subjected to sonication. Bioassays of  Yersinia entomophaga  MH96 culture supernatant show no effect (refer Table 8-12). 
     Crude Toxin Isolation Via  Y. entomophaga  MH96 Grown at 25° C. 
     Induction and Purification of  Y. entomophaga  MH96 Toxin 
     From an overnight culture grown at 25° C. Bacterial debris was removed by centrifugation (30 min; 12000 g; 4° C.) and the supernatant filter sterilised through a 0.2 μm filter to a sterile eppendorf. 
     Standard Bioassay 
     Healthy, feeding larvae, collected from the field, were individually fed squares of carrot which had been rolled in colonies of putative pathogenic bacteria that had grown overnight on solid media. Twelve second or third instar larvae were used for each treatment. Inoculated larvae were maintained at 15° C., in ice-cube trays. Larvae were left feeding on treated carrot for 3-4 days, then transferred to fresh trays and re-fed with untreated carrot for up to 10-14 days and signs of disease noted. 
     Dose Response Assay 
     An overnight culture of bacteria was grown, and a dilution series set up in phosphate buffer. Five μl of each dilution were inoculated onto pre air dried carrot cubes measuring approximately 3 mm 3 . Grass grub were placed into each of the trays cubicles, and results monitored as previously described under standard bioassay. 
     Experimental Protocols 
     Testing of  Yersinia entomophaga  MH96 on the Diamond Backed Moth (DBM). 
     The bioassay of efficacy of  Yersinia entomophaga  MH96 live cells and the toxic proteins from  Yersinia entomophaga  MH96 was tested on the Diamond Backed Moth (DBM). 
     Five fractions of the bacterial culture tested:
         1. Live cell broth; 1 ml freshly cultured broth used.   2. Concentrated live cells. 10 ml of broth centrifuged at 8000 rpm for 8 min, the resulting pellet harvested, and resupended in 1 ml of PB; 3. Resuspended live cells. 1 ml broth centrifuged at 8000 rpm for 8 min, the resulting pellet harvested and cells resuspended in equal volume of PB;   4. Heat killed broth. 1 ml broth subjected to boiling water for 10 min; broth plated out in LB plate to confirm if any live cells.   5. Sterile filtrate broth. 10 ml broth centrifuged at 8000 rpm for 8 min, the resulting pellet harvested, resuspended in 1 ml PB, then sonicated and centrifuged at 1300 rpm, 5 min; the supernatant harvested. Supernatant plated out in LB plate to confirm if any live cells.       

     All fractions mixed with 0.2% Tween 80 as emulsifier. LB broth and PB plus 0.2% Tween 80 used as controls. 
     Assessment: Leaf Disc Method.
         1. The 2 nd  to 4 th  instars collected from plants and place in a container supplied with cabbage leaves. If not enough for an experiment, larvae stored in fridge for an extended 2 to 3 day period until further collections.   2. Larvae transferred to clean or sterile Petri dishes containing no cabbage leaves by a sterile fine art brush at least 4 h prior to being exposed to treatments to ensure sufficient uptake of bacteria and the fractions tested.   3. Leaf discs (1.0 cm in diameter) punctured from tender leaves of cabbage seedlings, and stored in a Petri dish containing a small piece of wet tissue   4. Using freshly flame sterilized soft tweezers transfer the leaf discs individually into the wells of plates, with the upper surface of the leaves upward.   5. 5 μl of test suspension pipetted onto the upper surface of the leaf disc and spread with a sterile glass rob or homogenizer.   6. Larvae transferred individually onto a leaf disc with alcohol sterilized fine art brushes carefully. All larvae used for a treatment pooled in a plate covered by parafilm to prevent larval escaping from wells.   7. Recode the developmental stage of each larva.   8. Plates sealed in plastic bags and held at 15 C under 14:10 (L:D) h photoperiod.   9. Leaf discs renewed daily using the method above. Mortality monitored within 5 d post-inoculation.   8-12 larvae tested for each treatment, Experiments carried out three replications.       

     SUMMARY 
     Pathogenicity of Bacteria  Yersinia entomophaga  MH96 to Diamond Back Moth,  Plutella xylostella  (L.) 
     Laboratory bioassay of  Yersinia entomophaga  MH96 toxicity to DBM larvae 
     Determination of Active Fractions 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Effect of the culture broth fractions of  Yersinia entomophaga   
               
               
                 MH96 on the mortality of diamond back moth larvae. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 No. larvae 
                 No. dead 
                 Mortality 
                   
               
               
                 Fraction 
                 Rep* 
                 tested 
                 larvae 
                 (%) 
                 Mean (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Live cell broth 
                 1 
                 10 
                 10 
                 100.0 
                   
               
               
                   
                 2 
                 12 
                 12 
                 100.0 
                   
               
               
                   
                 3 
                 12 
                 12 
                 100.0 
                   
               
               
                   
                 4 
                 12 
                 12 
                 100.0 
                 100.0 
               
               
                 Resuspended live 
                 1 
                 10 
                 10 
                 100.0 
                   
               
               
                 cells 
                 2 
                 12 
                 11 
                 91.7 
                   
               
               
                   
                 3 
                 12 
                 12 
                 100.0 
                   
               
               
                   
                 4 
                 12 
                 11 
                 91.7 
                 95.8 
               
               
                 Heat killed broth 
                 1 
                 10 
                 2 
                 20.0 
                   
               
               
                   
                 2 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 4 
                 12 
                 1 
                 8.3 
                 7.1 
               
               
                 Sonicated cell 
                 1 
                 10 
                 10 
                 100.0 
                   
               
               
                 filtrate 
                 2 
                 12 
                 12 
                 100.0 
                   
               
               
                   
                 3 
                 12 
                 11 
                 91.7 
                   
               
               
                   
                 4 
                 12 
                 11 
                 91.7 
                 95.8 
               
               
                 Broth supernatant 
                 1 
                 10 
                 0 
                 0.0 
                   
               
               
                   
                 2 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 4 
                 12 
                 0 
                 0.0 
                 0.0 
               
               
                 Control 1 (PBS) 
                 1 
                 10 
                 2 
                 20.0 
                   
               
               
                   
                 2 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 4 
                 12 
                 0 
                 0.0 
                 5.0 
               
               
                 Control 2 (LB 
                 1 
                 10 
                 1 
                 10.0 
                   
               
               
                 broth) 
                 2 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 4 
                 12 
                 0 
                 0.0 
                 2.5 
               
               
                   
               
            
           
         
       
     
     Screenings of LD 50  of Active Fractions 
     Live Cell Broth 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Effect of  Yersinia entomophaga  MH96 dose on the mortality of  
               
               
                 diamond back moth larvae. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Dilution 
                   
                   
                 No. 
                 No. 
                 No. 
                   
                   
               
               
                 series 
                   
                 Dose 
                 larvae 
                 dead 
                 dead 
                 Mortality 
                 Mean 
               
               
                 tested 
                 Rep 
                 (cells/cm 2 ) 
                 tested 
                 larvae 
                 pupae 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 10 0   
                 1 
                 28000000 
                 12 
                 12 
                 0 
                 100.0 
                 94.4 
               
               
                   
                 2 
                 21000000 
                 12 
                 11 
                 0 
                 91.7 
                   
               
               
                   
                 3 
                 31000000 
                 12 
                 11 
                 0 
                 91.7 
                   
               
               
                 10 −1   
                 1 
                 2800000 
                 12 
                 8 
                 2 
                 83.3 
                 88.9 
               
               
                   
                 2 
                 2100000 
                 12 
                 10 
                 0 
                 83.3 
                   
               
               
                   
                 3 
                 3100000 
                 12 
                 12 
                 0 
                 100.0 
                   
               
               
                 10 −2   
                 1 
                 280000 
                 12 
                 9 
                 0 
                 75.0 
                 83.3 
               
               
                   
                 2 
                 210000 
                 12 
                 7 
                 3 
                 83.3 
                   
               
               
                   
                 3 
                 310000 
                 12 
                 9 
                 2 
                 91.7 
                   
               
               
                 10 −3   
                 1 
                 28000 
                 12 
                 7 
                 0 
                 58.3 
                 58.3 
               
               
                   
                 2 
                 21000 
                 12 
                 4 
                 1 
                 41.7 
                   
               
               
                   
                 3 
                 31000 
                 12 
                 6 
                 3 
                 75.0 
                   
               
               
                 10 −4   
                 1 
                 2800 
                 12 
                 5 
                 3 
                 66.7 
                 52.8 
               
               
                   
                 2 
                 2100 
                 12 
                 5 
                 2 
                 58.3 
                   
               
               
                   
                 3 
                 3100 
                 12 
                 2 
                 2 
                 33.3 
                   
               
               
                 10 −5   
                 1 
                 280 
                 12 
                 6 
                 1 
                 58.3 
                 38.9 
               
               
                   
                 2 
                 210 
                 12 
                 2 
                 2 
                 33.3 
                   
               
               
                   
                 3 
                 310 
                 12 
                 1 
                 2 
                 25.0 
                   
               
               
                 Control 
                 1 
                 0 
                 12 
                 0 
                 0 
                 0.0 
                 0.0 
               
               
                   
                 2 
                 0 
                 12 
                 0 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 0 
                 12 
                 0 
                 0 
                 0.0 
               
               
                   
               
            
           
         
       
     
     Sonicated Cell Filtrate 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Effect of the sonicated cell filtrate concentration of  Yersinia   
               
               
                   entomophaga  MH96 on mortality of diamond back moth larvae. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 No  
                 No 
                 No 
                   
                   
               
               
                   
                   
                 larvae 
                 dead 
                 dead 
                 Mortality 
                 Mean 
               
               
                 Concentration 
                 Rep 
                 tested 
                 larvae 
                 pupae 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 100% 
                 1 
                 12 
                 11 
                 0 
                 91.7 
                 91.7 
               
               
                   
                 2 
                 12 
                 11 
                 0 
                 91.7 
                   
               
               
                   
                 3 
                 12 
                 10 
                 1 
                 91.7 
                   
               
               
                  50% 
                 1 
                 12 
                 6 
                 1 
                 58.3 
                 80.6 
               
               
                   
                 2 
                 12 
                 11 
                 0 
                 91.7 
                   
               
               
                   
                 3 
                 12 
                 11 
                 0 
                 91.7 
                   
               
               
                  20% 
                 1 
                 12 
                 9 
                 0 
                 75.0 
                 75.0 
               
               
                   
                 2 
                 12 
                 9 
                 0 
                 75.0 
                   
               
               
                   
                 3 
                 12 
                 7 
                 2 
                 75.0 
                   
               
               
                  10% 
                 1 
                 12 
                 5 
                 0 
                 41.7 
                 58.3 
               
               
                   
                 2 
                 12 
                 7 
                 0 
                 58.3 
                   
               
               
                   
                 3 
                 12 
                 8 
                 1 
                 75.0 
                   
               
               
                  2% 
                 1 
                 12 
                 2 
                 0 
                 16.7 
                 30.6 
               
               
                   
                 2 
                 12 
                 3 
                 0 
                 25.0 
                   
               
               
                   
                 3 
                 12 
                 5 
                 1 
                 50.0 
                   
               
               
                  1% 
                 1 
                 12 
                 0 
                 0 
                 0.0 
                 2.8 
               
               
                   
                 2 
                 12 
                 0 . 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 12 
                 1 
                 0 
                 8.3 
                   
               
               
                 Control 
                 1 
                 12 
                 0 
                 0 
                 0.0 
                 0.0 
               
               
                   
                 2 
                 12 
                 0 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 12 
                 0 
                 0 
                 0.0 
               
               
                   
               
            
           
         
       
     
     Screenings of Stability of Active Fractions 
     Live Cell Broth 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Effect of ambient temperature and length of storage period on  
               
               
                 toxicity of  Yersinia entomophaga  MH96 live cells to DBM larvae 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 No 
                   
                   
               
               
                   
                   
                 No larvae 
                 dead 
                 Mortality 
                 Mean 
               
               
                 Treatment 
                 Rep 
                 tested 
                 larvae 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 0 d (Fresh 
                 1 
                 12 
                 11 
                 91.7 
                   
               
               
                 culture) 
                 2 
                 12 
                 10 
                 83.3 
                   
               
               
                   
                 3 
                 12 
                 9 
                 75.0 
                 83.3 
               
               
                 1 d, 20° C. 
                 1 
                 12 
                 12 
                 100.0 
                   
               
               
                   
                 2 
                 12 
                 10 
                 83.3 
                   
               
               
                   
                 3 
                 12 
                 9 
                 75.0 
                 86.1 
               
               
                 7 d, 20° C. 
                 1 
                 12 
                 10 
                 83.3 
                   
               
               
                   
                 2 
                 12 
                 11 
                 91.7 
                   
               
               
                   
                 3 
                 12 
                 7 
                 58.3 
                 77.8 
               
               
                 1 d, 4° C. 
                 1 
                 12 
                 8 
                 66.7 
                   
               
               
                   
                 2 
                 12 
                 7 
                 58.3 
                   
               
               
                   
                 3 
                 12 
                 8 
                 66.7 
                 63.9 
               
               
                 7 d, 4° C. 
                 1 
                 12 
                 10 
                 83.3 
                   
               
               
                   
                 2 
                 12 
                 11 
                 91.7 
                   
               
               
                   
                 3 
                 12 
                 11 
                 91.7 
                 88.9 
               
               
                 Control 
                 1 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 2 
                 12 
                 1 
                 8.3 
                   
               
               
                   
                 3 
                 12 
                 1 
                 8.3 
                 5.6 
               
               
                   
               
            
           
         
       
     
     Sonicated Cell Filtrate 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Effect of temperature and length of storage period on toxicity of  
               
               
                   Yersinia entomophaga  MH96 sonicated cell filtrate to DBM larvae 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 No 
                   
                   
               
               
                   
                   
                 No larvae 
                 dead 
                 Mortality 
                 Mean 
               
               
                 Treatment 
                 Rep 
                 tested 
                 larvae 
                 (%) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 0 d (Fresh 
                 1 
                 12 
                 12 
                 100.0 
                   
               
               
                 culture) 
                 2 
                 12 
                 9 
                 75.0 
                   
               
               
                   
                 3 
                 12 
                 11 
                 91.7 
                 88.9 
               
               
                 1 d, 20° C. 
                 1 
                 12 
                 11 
                 91.7 
                   
               
               
                   
                 2 
                 12 
                 9 
                 75.0 
                   
               
               
                   
                 3 
                 12 
                 11 
                 91.7 
                 86.1 
               
               
                 7 d, 20° C. 
                 1 
                 12 
                 12 
                 100.0 
                   
               
               
                   
                 2 
                 12 
                 10 
                 83.3 
                   
               
               
                   
                 3 
                 12 
                 11 
                 91.7 
                 91.7 
               
               
                 1 d, 4° C. 
                 1 
                 12 
                 8 
                 66.7 
                   
               
               
                   
                 2 
                 12 
                 9 
                 75.0 
                   
               
               
                   
                 3 
                 12 
                 7 
                 58.3 
                 66.7 
               
               
                 7 d, 4° C. 
                 1 
                 12 
                 11 
                 91.7 
                   
               
               
                   
                 2 
                 12 
                 8 
                 66.7 
                   
               
               
                   
                 3 
                 12 
                 9 
                 75.0 
                 77.8 
               
               
                 Control 
                 1 
                 12 
                 1 
                 8.3 
                   
               
               
                   
                 2 
                 12 
                 0 
                 0.0 
                   
               
               
                   
                 3 
                 12 
                 1 
                 8.3 
                 5.6 
               
               
                   
               
            
           
         
       
     
     Bait Formulation of  Yersinia entomophaga  MH96 Against 7 th -8 th  Instar  Wiseana  Sp Larvae 
     Experiment 1 
     Method 
       Wiseana  spp. larvae (most likely  W. copularis  based on size and flight times of moths in January) were collected from pasture on Taieri Plain. The moths were housed in 60 ml specimen containers three quarters filled with ground pine bark (&lt;2 mm) to which were added white clover ( Trifolium repens  var. Huia) leaves as food. This food was changed every 3-4 days and the larvae moved to fresh containers after three weeks and again one day prior to the commencement of the bioassay. 
     For the bioassay, ten larvae were randomly allocated to be given  Yersinia entomophaga  MH96 kibbled wheat baits and ten allocated as controls. Those larvae in the  Yersinia entomophaga  MH96 treatment were given approximately ½ teaspoon of kibbled wheat (8-12 grains) while the control larvae continued to be given clover leaves. Larval survival and feeding was assessed after five days and again at ten days. Surviving larvae were fed again after five days according to treatment. 
     Results 
     After five days, six of the  Wiseana  spp. larvae given  Yersinia entomophaga  MH96 were dead while all the control larvae were alive and apparently healthy. After ten days all larvae given  Yersinia entomophaga  MH96 had died and all control larvae were alive. On both occasions the larvae given kibbled wheat had taken it into their burrows and signs of feeding were evident. 
     Conclusion 
       Yersinia entomophaga  MH96 treated kibbled wheat was associated with the deaths of  Wiseana  spp. larvae. 
     Experiment 2 
     Introduction 
     The earlier laboratory bioassay in Experiment 1 above showed that the  Yersinia entomophaga  MH96 treatment caused mortality of large and small  Wiseana  spp larvae. However these bioassays were carried out using treated kibbled wheat where no alternative food source for the larvae was available, as the larvae were exposed to the baits and in small 60 ml specimen containers. Therefore, the current experiment was aimed to test the effectiveness of  Yersinia entomophaga  MH96 treatment under a more realistic situation where the larvae had an alternative food supply and could more easily avoid contact with the baits. 
     Method 
     Ten containers with transparent acrylic sides and measuring 500(l)×300(w)×300(h) mm ( FIG. 9 ) were filled to a depth of 150 mm with fine (&lt;3 mm) pine bark. A 30-40 mm layer of Yates™ potting mix was applied over the bark surface. Twelve  Trifolium repens  seedlings were planted into each of the ten containers and allowed to establish. The seedlings were held in a white shade-cloth covered tunnel house at ambient air temperature which was measured by two ‘Tiny Tag” temperature data loggers. Five days after planting, ten final instar stage  Wiseana  spp. larvae collected and placed in each container. 
     At 14 days after planting, the seedlings were cut to approximately 10 mm high ( FIG. 9 ).  Yersinia entomophaga  MH96 broadcast kibbled wheat baits where applied to the surface of five of the containers at a rate equivalent to 50 kg baits/ha (0.83 g/container). The remaining five containers were untreated (controls). 
     The clover plants were assessed for survival 12 days after the application of  Yersinia entomophaga  MH96 broadcast kibbled wheat baits. A second application of bait was made 13 days after the first application and plant survival assessed again 25 days after the initial application. The plants were harvested (cut to “ground” level) two days later and dried at 80° C. overnight to assess dry matter production over the duration of the experiment. The containers were also broken down at this time and the potting mix/bark searched for  Wiseana  spp. larvae. The data were analysed by one way analyses of variance with no blocking (Genstat version 8). 
     Results 
     Although  Wiseana  spp. larvae destroyed some plants (Table 13, and  FIG. 10 ) overall there were few differences in plant survival between treatments or assessment times. Plant survival was higher on both occasions in the containers treated with the bait, but this difference was not significant (Table 13 (P&lt;0.13, P&lt;0.08, first and second assessments respectively)). 
     There was no difference in clover production between the baited treatment and the control treatments (Table 13 (P&lt;0.54)). Although survival of larvae was significantly higher in the control containers compared to those treated with the 1.0 bait. (Table 13 (P&lt;0.001)) it is probable that the warm temperatures and high nutrient status of the potting mix allowed the clover plants in those containers with high numbers of larvae to outgrow and compensate for the affects of larvae feeding. 
     The  Wiseana  spp larvae survival in the control containers was approximately 46%, and is considered to be satisfactory for field collected larvae and average density in these containers equated to 31 larvae/m 2 . This would be a moderate field density but the vegetation within the containers was sparse relative to pasture. The reduction in larvae numbers associated with the  Yersinia entomophaga  MH96 application was approximately 78%. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Plant survival and production and  Wiseana  spp larvae  
               
               
                 survival (mean) over the duration of the bioassay. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 N° plants 
                 N° plants 
                 Dry Matter (g) 
                 Live Larvae 
               
               
                   
                 Day 12 
                 Day 25 
                 Day 27 
                 Day 27 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Bait 
                 11.6 
                 11.6 
                 11.2 
                 1.0 
               
               
                 Treatment 
                   
                   
                   
                   
               
               
                 Control 
                 10.2 
                 10.2 
                 10.3 
                 4.6 
               
               
                 SED 
                 0.8 
                 0.7 
                 1.3 
                 0.5 
               
               
                   
               
            
           
         
       
     
     The average temperature within the tunnel house during the bioassay was 10° C. but ranged from 0 to 32° C. (see  FIG. 11 ). This was higher than usual outside air temperatures for this time of year and may have affected larvae activity and plant growth. 
     Examples of Other Susceptible Invertebrate Species 
     Table 13 below summaries a list of various other invertebrate species, including the DBM and  Wiseana  spp tested for susceptibility to whole  Yersinia entomophaga  MH96 cells, 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Summary of the susceptibility of invertebrates to  
               
               
                   Yersinia entomophaga  MH96. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Develop- 
                 Patho- 
               
               
                 Insect 
                 Class: Family 
                 mental stage 
                 genic? 
               
               
                   
               
               
                 Lepidoptera 
                   
                   
                   
               
               
                 Diamondback moth 
                 Lepidoptera: 
                 1st-4 th  instar 
                 yes 
               
               
                 
                   Plutella xylostella 
                 
                   
                 larvae 
                   
               
               
                 Porina 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 
                   Wiseana copularis 
                 
                 Heplidae 
                   
                   
               
               
                 Cotton bollworm  
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 
                   Helicoyerpa amjgera 
                 
                   
                   
                   
               
               
                 Greater wax moth 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 
                   Galleria mellonella 
                 
                 Galleriidae 
                   
                   
               
               
                 Painted apple moth 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 
                   Teia anartoides 
                 
                 Lymantrlidae 
                   
                   
               
               
                 Greenheaded leafroller 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 
                   Planotortrix notophaea 
                 
                 Tortricidae 
                   
                   
               
               
                 Greenheaded leafroller 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 
                   Planotortrix excessana 
                 
                 Tortricidae 
                   
                   
               
               
                 Lightbrown apple moth 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 
                   Epiphyas postvittana 
                 
                 Tortricidae 
                   
                   
               
               
                 Brownheaded leafroller 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                   Ctenoptusis  spp. 
                 Tortricidae 
                   
                   
               
               
                 
                   Pieris rapae 
                 
                 Lepidoptera: 
                 larvae 
                 yes 
               
               
                 white butterfly 
                 ?Pieridae 
                   
                   
               
               
                 Coleoptera 
                   
                   
                   
               
               
                 New Zealand grass grub 
                 Coleoptera: 
                 larvae 
                 yes 
               
               
                 
                   Costelytra zealandica 
                 
                 Scarabaeidae 
                   
                   
               
               
                 Red headed cockchafer 
                 Coleoptera: 
                 larvae 
                 yes 
               
               
                 
                   Adoryphorus couloni 
                 
                 Scarabaeidae 
                   
                   
               
               
                 Tasmania grass grub 
                 Coleoptera: 
                 larvae 
                 yes 
               
               
                 
                   Acrossidius tasmaniae 
                 
                 Scarabaeidae 
                   
                   
               
               
                 
                   Pericoptus truncatus 
                 
                 Coleoptera: 
                 larvae 
                 (yes) 
               
               
                 Sand scarab 
                 Scarabaeidae 
                   
                   
               
               
                 Chafer beetles? 
                 Coleoptera: 
                 larvae 
                 (yes) 
               
               
                   Odontria  sp. 
                 Scarabaeidae 
                   
                   
               
               
                 Bark beetle 
                 Coleoptera: 
                 adults 
                 partial 
               
               
                 
                   Hylastes ater 
                 
                 Scolytidae 
                   
                   
               
               
                 Black vine weevil 
                 Coleoptera: 
                 larvae 
                 yes 
               
               
                 
                   Otiorhynchus sulcatus 
                 
                 Curculionidae 
                   
                   
               
               
                 Clover root weevil (CRW) 
                 Coleoptera: 
                 adult 
                 yes 
               
               
                 
                   Sitona lepidus 
                 
                 Curculionidae 
                   
                   
               
               
                 Argentine stem weevil (ASW) 
                 Coleoptera: 
                 adult 
                 adult- 
               
               
                 
                   Listronotus bonariensis 
                 
                 Curculionidae 
                   
                 partial 
               
               
                 Hymenoptera 
                   
                   
                   
               
               
                 Darwin&#39;s ant 
                 Hymenoptera: 
                 nest 
                 yes 
               
               
                 
                   Doleromyrma darwiniana 
                 
                 Formicidae 
                   
                   
               
               
                 
                   Vespula vulgaris 
                 
                 Hymenoptera: 
                 larvae 
                 yes 
               
               
                 Common wasps 
                 Vespidae 
                   
                   
               
               
                 Orthoptera 
                   
                   
                   
               
               
                 Locusts 
                 Orthoptera: 
                 neonates 
                 yes 
               
               
                 
                   Locusta migratoria 
                 
                   
                 older instar 
                 yes 
               
               
                 Diptera 
                   
                   
                   
               
               
                 root lesion nematode 
                 Nematoda 
                   
                 slight 
               
               
                 
                   Pratylenchus penetrans 
                 
               
               
                   
               
            
           
         
       
     
     It would be appreciated that the present invention provides a new biopesticide or method for controlling insects which has a broad efficacy across a range of insects, and providing a new biopesticide in a range of different forms. 
     Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 
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