Characterisation of nematode symbiotic bacteria and the in vitro liquid culture of Heterorhabditis zealandica and Steinernema yirgalemense

Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Entomopathogenic nematodes have the potential to be outstanding biocontrol agents against agricultural pest insects. Combined with their bacterial symbionts, these biocontrol agents have proven to be very effective against numerous pests. The nematodes belong to the families Steinernematidae and Heterorhabditidae, and are ideal to be used in, and integrated with, pest management systems. There is a dire need for new and innovative methods to control agricultural pests, as numerous pest insects have developed resistance against broad-spectrum insecticides. Together with the environmental impact of these insecticides and the safety aspect regarding humans and animals, the need to develop new technologies, including entomopathogenic nematodes for pest management, is high. In this study, the associated symbiotic bacteria of three entomopathogenic nematodes species were isolated, and the potential of two nematode species to be successfully mass cultured in liquid medium was evaluated. Regarding the symbiotic bacteria, results from the study showed that bacteria species from all three nematode species, Heterorhabditis noenieputensis, Steinernema khoisanae and Heterorhabditis zealandica, were novel. Heterorhabditis noenieputensis was isolated in the Mpumalanga province during a previous survey conducted in citrus orchards. The bacterium isolated from this nematode belongs to the genus Photorhabdus, and bear closest similarity (98.6%) to the type strain of P. luminescens subsp laumondii (TT01T). Photorhabdus luminescens subsp. noenieputensis subsp. nov., derives its name from the area where the nematode was sourced, namely the farm Springbokvlei, near the settlement Noenieput close to the Namibian border. Thus far, 85 Steinernema spp. have been described worldwide, including S. khoisanae which was isolated in the Western Cape province of South Africa. Four S. khoisanae strains, namely SF87, SF80, SF362 and 106-C, were used for characterisating the new bacteria from different localities in South Africa. Using the neighbor-joining method, all the strains were aligned with 97% homology to the 16S rRNA sequences of several Xenorhabdus- type strains, indicating that they belonged to the same genus. The multigene approach was used to distinguish between the Xenorhabdus spp. and partial recA, dnaN, gltX, gyrB and infB gene sequences of the various strains were analysed. The bacterium species was named Xenorhabdus khoisanae sp. nov. after the nematode from which it was isolated. The results showed that the third bacterium species, which was isolated from H. zealandica, was new. The sequence of the bacteria strain clustered with the type strains of P. temperata and P. asymbiotica, indicate that it belonged to the genus Photorhabdus. This is the first study to show that H. zealandica associates with a luminescent Photorhabdus species, rather than with the known non-luminescent P. temperata. The potential of H. zealandica and Steinernema yirgalemense mass culture in liquid was investigated. Results illustrated that H. zealandica and its P. luminescens symbiont can be successfully cultured in liquid. However, two generations occurred during the process time, instead of the desirable one-generation. The growth curve of the symbiotic bacteria during the process time was measured, in order to determine when the stationary phase was reached, with the results showing this to occur after 36 h. Therefore, the optimum amount of time required for inoculating the IJs and for aiding in maximum infective juvenile (IJ) recovery is 36 h for adding the nematodes post pre-culturing of the bacteria. Future research goals should be to increase the percentage recovery in liquid culture, which would increase the number of nematodes produced per ml, which would, therefore, reduce the processing time significantly. The results from mass culturing the second nematode species, S. yirgalemense, indicated an asynchronous nematode development in the first generation. Growth curves were performed with the symbiotic bacteria that showed the exponential phase of Xenorhabdus started after 15 h, and that, after 42 h, the stationary phase was reached, with an average of 51 × 107 cfu·ml-1. Bioassays were performed to compare the virulence between in vitro- and in vivo-produced nematodes, with the results showing that the in vitro-produced nematodes were significantly less virulent than were the nematodes produced in vivo. The success obtained with the production of S. yirgalemense in liquid culture can serve as the first step in the optimising and upscaling of the commercial production of nematodes in industrial fermenters. The last aim of the current study was to determine when Xenorhabdus reached the stationary phase, when it is grown in a 20-L fermenter, as this would be the optimum time at which to add the IJs of S. yirgalemense. Such characteristics as the effect of stationary phase conditions on the bacterial cell density and on the DO2 rate in the fermenter were investigated. The results showed that the stationary phase of Xenorhabdus was reached after 36 h at 30˚C, which took 6 h less than did the same procedures followed with the Xenorhabdus sp. cultured in Erlenmeyer flasks on orbital shakers. This is the first step toward the liquid mass culturing of S. yirgalemense in industrial-size fermenters. Data from this study indicated the optimum amount of time that is required for adding nematodes to the bacterial culture in the fermenter, and for ensuring the optimum recovery of IJs, as well as a subsequent high yield of nematodes within a minimum processing time. This is the first report of its kind to investigate comprehensively the successful liquid culture of two South African entomopathogenic nematode species for the sole purpose of evaluating potential commercialisation. Results emanating from this study could be used as groundwork in future, in combination with similar research such as culturing nematodes intensively in large fermenters.AFRIKAANSE OPSOMMING: Entomopatogeniese nematodes het die potensiaal om as doeltreffende biologiese beheeragente teen sleutelplaaginsekte gebruik te word. Elke nematood werk interaktief met ‘n spesifieke bakterium. Entomopatogeniese nematodes, behorende tot die families Steinernematidae en Heterorhabditidae, is ideale kandidate vir gebruik in ‘n geïntegreerde plaagbestuurprogram. Tans is daar ʼn behoefte vir nuwe metodes vir die beheer van plaaginsekte, omdat meeste insekte reeds weerstand opgebou het teen bestaande plaagdoders. As gevolg van die negatiewe impak van plaagdoders op die omgewing, asook kommer oor veiligheid vir die mens en diere, is die ontwikkeling en gebruik van alternatiewe plaagbeheermiddels noodsaaklik. In die eerste deel van die studie word drie nuwe bakterie spesies geïsoleer en beskryf. Resultate van hierdie studie het aangetoon dat die bakterië spesies vanuit die nematode spesies, Heterorhabditis noenieputensis, Steinernema khoisanae, en Heterorhabditis zealandica, tot dusver onbeskryf was. Eersgenoemde, H. noenieputensis, is afkomstig van ʼn sitrusboord in die Mpumalanga Provinsie. Die bakterie hieruit geïsoleer behoort tot die genus Photorhabdus en is biologies verwant (98.6%) aan P. luminescens subsp laumondii (TT01T). Die bakterie is benaam as Photorhabdus luminescens subsp. noenieputensis nov. en is na die nematood waaruit dit geïsoleer is vernoem. Tot dusver is wêreldwyd 82 spesies van Steinernema spp. beskryf, insluitende S. khoisanae van die Weskaap provinsie. Vier bakterie isolate is van S. khoisanae, SF87, SF80, SF362 en 106-C geïsoleer. Die buur-koppeling metode was gebruik om te bepaal dat hierdie bakterie isolate tot 97% ooreenstem met verskeie isolate van Xenorhabdus se 16S rRNA DNS volgordebepalings. Om tussen Xenorhabdus spp. te onderskei is ʼn multi-geen benadering gebruik deur gedeeltelike recA, dnaN, gltX, gyrB en infB DNS basispaar volgordebepalings van die verskeie isolate te bepaal. Hierdie bakterie isolaat is soortgelyk ook vernoem as, Xenorhabdus khoisanae sp. nov., na die nematood waaruit dit geïsoleer is. Die derde onbekende bakteriële spesie is uit H. zealandica geïsoleer. Die DNS basispaar volgordebepaling van die 16S geen van SF41 toon aan dat dit in dieselfde groep as P. temperata en P. asymbiotica val en sodoende aan die genus Photorhabdus behoort. Hierdie is die eerste studie met die bevinding dat H. zealandica ook met ʼn ander bakterie spesie geassosieer kan word buiten die normale P. temperata spesie. Die tweede deel van die studie gaan oor die teling van twee nematood spesies, H. zealandica en Steinernema yirgalemense, en hulle is geëvalueer vir hulle potensiaal om geteel te word in ʼn vloeibare medium. Die resultate het gewys dat H. zealandica met sy P. luminescens simbiont suksesvol in vloeistof aangeteel kan word, ten spyte van die feit dat daar twee generasies ontwikkel het, in plaas van die meer ideale enkel generasie. Die groeikurwe van die simbiotiese bakterie was gemonitor om te bepaal wanneer die stasionêre fase bereik word. Die resultate toon dat hierdie fase na 36 uur bereik was. Dus was die infektiewe nematode larwes eers na 36 uur tot die vloeibare medium waarin die bakterie geteel was bygevoeg. Navorsing in die toekoms moet dus gefokus wees om die persentasie herwinning van die infektiewe larwes te verhoog. Dit sal daartoe lei dat meer nematodes per ml geproduseer kan word en ook die prosesseringstyd van die nematodes verminder. ʼn Tweede nematode spesie, S. yirgalemense, was ook in vloeistof geteel. Hier het ʼn asinkroniese ontwikkeling in die eerste generasie plaasgevind wat problematies is. Groeikurwes is bepaal van die bakteriële simbiont en die resultate het gewys dat die groeifase van Xenorhabdus na 15 uur in aanvang geneem het en dat die stasionêre fase bereik was na 42 uur met ʼn gemiddelde van 51 × 107 selle·ml-1. Die virulensie van nematodes wat in vitro geteel is, is vergelyk met die virulensie van nematodes wat in vivo geteel is en die resultate het getoon dat die in vitro geteelde nematodes minder virulent was. Die teling van S. yirgalemense in vloeistof was oor die algemeen meer suksesvol as die teling van H. zealandica in dieselfde medium. Die doelwit van die laaste gedeelte van hierdie studie was om te bepaal wanneer Xenorhabdus die stasionêre fase bereik wanneer dit in ʼn 20-L fermenter gekweek word. Dit bepaal sodoende die optimale tyd wanneer die infektiewe larwes van S. yirgalemense bygevoeg behoort te word. Die uitwerking van die stasionêre fase op die bakteriële selle, asook die DO2-konsentrasie in die fermenter, was geëvalueer. Resultate het gewys dat die stasionêre fase van Xenorhabdus na 36 uur bereik was, wat 6 uur korter is as toe dit gekweek is in Erlenmeyer flesse. Hierdie studie is die eerste stap om die massa teling van S. yirgalemense in industriële fermenters suksesvol te bemeester. Die data wat verkry was, het aangedui wat die ideale tydsduur sal wees om die bakteriegetalle te vermeerder voordat die nematode bygevoeg word. Hierdie is die eerste studie wat die teling van twee Suid-Afrikaanse nematode spesies omvattend in vloeistof evalueer het. Die hoof doelwit is om die potensiaal van hierdie nematode spesies, met die oog op kommersiële gebruik, te meet. Die resultate van hierdie studie kan gekombineer word met toekomstige studies in hierdie spesifieke navorsingsveld

Rearing of the banded fruit weevil, Phlyctinus callosus (Schonherr) (Coleoptera: Curculionidae) and control with entomopathogenic nematodes

Thesis (MScAgric (Conservation Ecology and Entomology))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: The banded fruit weevil, Phlyctinus callosus (Schönherr), is a key pest of apples, nectarines and grapevines in the southern areas of the Western Cape. The control of P. callosus is not satisfactory and the insecticides used to control this insect have not proved to be effective since the development of tolerance to pyrethroids and acephate. A control method that can be used, despite it being very labour-intensive, is that of tree trunk barriers. The use of such a method will prevent the weevils from reaching the fruit, as they are unable to fly. Alternative control options, such as the use of entomopathogenic nematodes, are urgently needed for the control of P. callosus. Entomopathogenic nematodes belonging to the Steinernematidae and Heterorhabditidae are ideal biocontrol agents for incorporation into an integrated pest management programme. In order to develop control strategies for P. callosus, large numbers and a predictable quantity of different weevil stages are needed. Especially large numbers of larvae are needed, as this is the stage that will be targeted with nematodes. One of the aims of the current study was to assess various artificial diets for rearing larvae of P. callosus. Though adult weevils were easily collected from orchards, it was very difficult to obtain large numbers of larvae. Modified versions of an agar diet, as well as different carrot based diets, were tested at 21°C. The highest percentage survival obtained for the agar diet was 50% and 60% for one type of carrot diet. A better rearing method proved to be that of planting full-grown carrots in pots, kept at 25°C, resulting in the attainment of the highest percentage survival rate of 90%. A study was undertaken to assess how long, and at what temperature, P. callosus eggs could be stored. A mean percentage hatch of 45.7% was obtained when eggs were stored at 4°C for 70 days. Eggs started hatching after 47 days and 10 days, when stored at temperatures of 11°C and 14°C, respectively. If the aim of the employment of such a method is only to delay egg hatching, the two temperatures (11°C and 14°C) will be suitable. For the following part of the study, several entomopathogenic nematode isolates were evaluated for their potential use as biological control agents against P. callosus. The susceptibility of P. callosus larvae and adults to nematode infection was assessed in the laboratory by screening for their mortality, using different nematode isolates. Larvae were found to be more susceptible to nematode infection than adults. Heterorhabditis isolates were found to cause higher levels of mortality than the Steinernema isolates during screening, when a concentration of 400 infective juveniles (IJ) per insect V was used. Biological characteristics, such as the effect of different temperatures on nematode activity and the minimum concentration of nematodes needed to obtain acceptable levels of control for P. callosus, were also investigated. The percentage mortality ranged from no infection to 75% after four days for the larvae, and the SF41 isolate of Heterohabditis zealandica was selected as the most promising isolate for further laboratory experiments. The vertical movement of nematodes in sand, compared with such movement in sandy loam soil, and the biology of H. zealandica in P. callosus larvae was also investigated in laboratory bioassays. After four days, the LD50 and LD90 values were 96 IJ/50 μl and 278 IJ/50 μl, respectively. Nematodes were found to be inactive at 11°C, with the highest mortality rate of P. callosus resulting from nematode infection being recorded at 25°C. A higher percentage mortality rate was obtained with the sandy loam soil (95.2%) than with the sand (77.5%). Heterorhabditis zealandica could successfully complete its life cycle in 6th instar P. callosus larvae. The study showed that P. callosus larvae are suitable hosts for H. zealandica, and that the control of P. callosus in the field by the selected isolate holds promise. The persistence of the SF41 isolate of H. zealandica at different concentrations was investigated in the last part of the study. The experiment took place in a blueberry orchard, subject to a high rate of infestation by P. callosus. Concentrations of 0, 20, 30 and 45 IJ/cm2 were topically applied, with persistence being evaluated for days 1, 35 and 84. Percentage persistence for 30 IJ/cm2 was calculated as 87.5% for days 35 and 84. The persistence of soil samples taken on day one, and kept in plastic containers at room temperature, was again evaluated on day 128, with the finding that both 30 IJ/cm2 and 45 IJ/cm2 caused 100% mortality of Tenebrio molitor (L.). Results indicated good persistence of H. zealandica after 84 days in field conditions, with a high maintenance of P. callosus populations. The study indicated the potential use of H. zealandica for the control of P. callosus, with the possibility of persistence for at least three months. Future research into the control of P. callosus with nematodes should aim to investigate the technical aspects of field application. The current study shows that entomopathogenic nematodes have potential for controlling the soil stages of P. callosus. The capacity to rear large numbers of P. callosus larvae in the laboratory, for later use in laboratory and field trials, is of key importance.AFRIKAANSE OPSOMMING: Die gebande vrugtekalander, Phlyctinus callosus (Schönherr), is ʼn groot plaag in appel- en nektarienboorde sowel as wingerde in die suidelike gebiede van die Wes-Kaap. Phlyctinus callosus word nie voldoende beheer nie, en plaagdoders wat voorheen gebruik is om dié insek in toom te hou, het doeltreffendheid ingeboet weens weerstandontwikkeling teen piretroϊede en asefaat. ʼn Alternatiewe beheermetode is stamsperbande. Omdat die kalanders nie kan vlieg nie, moet hulle teen stamme uitklim om die vrugte te bereik. Stamsperbande versper dus die insekte se toegang tot die vrugte, maar is baie arbeidsintensief. Meer haalbare metodes vir die beheer van P. callosus is daarom dringend nodig, en die gebruik van entomopatogeniese nematodes blyk ʼn besliste moontlikheid te wees. Entomopatogeniese nematodes, wat tot die Steinernematidae en Heterorhabditidae behoort, is uitstekende biobeheermiddels vir insluiting by geϊntegreerde plaagbeheerprogramme. Om doeltreffende beheerstrategieë vir P. callosus te bedink, is groot en voorspelbare hoeveelhede kalanders nodig veral groot hoeveelhede larwes, aangesien nematodes op hierdie ontwikkelingstadium gemik sal wees. Die eerste doel met die studie was dus om ʼn kunsmatige dieet vir die teling van P. callosus larwes te ontwikkel. Volwasse kalanders kon maklik in vrugteboorde ingesamel word, maar groot hoeveelhede larwes was moeiliker bekombaar. Aangepaste weergawes van ʼn agardieet sowel as verskillende worteldiëte is by 21°C beproef. Die hoogste persentasie larwale groei en -oorlewing op die agardieet was 50%, en 60% op een bepaalde soort worteldieet. Die beste teelmetode blyk egter volgroeide wortels te wees wat in potte geplant is en by 25°C gehou word. Dié metode het ʼn oorlewingspersentasie van 90% opgelewer. ʼn Studie is onderneem om te bepaal hoe lank en by watter temperature P. callosus eiers vir toekomstige gebruik geberg kan word. ʼn Gemiddelde uitbroeipersentasie van 45.7% is verkry toe eiers vir 70 dae by 4°C geberg is. Eiers wat onderskeidelik by 11°C en 14°C geberg is, het ná 47 en 10 dae onderskeidelik begin uitbroei. Indien die doel is om die eiers slegs stadiger te laat uitbroei, sal hierdie twee temperature dus geskik wees. VII Hierna is verskeie entomopatogeniese nematode-isolate vir moontlike gebruik as biologiese beheermiddels vir P. callosus beoordeel. Phlyctinus callosus larwes en volwassenes se vatbaarheid vir nematode infeksie is in die laboratorium bepaal deur dit met behulp van verskillende nematodeisolate vir mortaliteit te toets. Dié toetse het getoon dat larwes meer vatbaar is vir nematode infeksie as volwassenes. In die proefnemings het die Heterorhabditis-isolate hoër mortaliteit as die Steinernema-isolate veroorsaak teen ʼn konsentrasie van 400 infektiewe larwes (IJ) per insek. Biologiese eienskappe, soos die uitwerking van verskillende temperature op nematode aktiwiteit, sowel as die minimum konsentrasie nematodes om aanvaarbare vlakke van beheer uit te oefen, is ondersoek. Die persentasie mortaliteit vir die larwes het ná vier dae tussen 0% en 75% gewissel, en die SF41-isolaat van Heterohabditis zealandica is as die belowendste isolaat vir die res van die proefnemings gekies. Die vertikale beweging van nematodes in sand teenoor leemgrond, sowel as die biologie van H. zealandica in P. callosus larwes, is ook bestudeer. Ná vier dae was die LD50- en LD90-waardes onderskeidelik 96 en 278 IJ/50 μl. Wat temperatuur betref, is daar bevind dat nematodes onaktief is by 15°C, terwyl die hoogste mortaliteit van P. callosus larwes as gevolg van nematode infeksie by 25°C aangeteken is. Die mortaliteit was hoër in die leemgrond (95.2%) as in die sandgrond (77.5%). Heterorhabditis zealandica kon sy lewensiklus suksesvol in 6de instar P. callosus larwes voltooi. Die studie het derhalwe getoon dat P. callosus larwes geskikte gashere is vir H. zealandica, en dat hierdie isolaat dus in die praktyk ʼn doeltreffende beheermiddel vir P. callosus kan wees. Die oorlewing van verskillende konsentrasies H. zealandica is ten slotte bestudeer. Die proefneming is in ʼn bloubessieboord met ʼn groot populasie P. callosus uitgevoer. Konsentrasies van 0, 20, 30 en 45 IJ/cm2 is op die grond (uitwendig) toegedien, en oorlewing is op dag 1, 35 en 84 gemeet. Die persentasie oorlewing vir die 30 IJ/ cm2 konsentrasie was 87.5% op sowel dag 35 as 84. Oorlewing in grondmonsters wat op dag een ingesamel en by kamertemperatuur in plastiekhouers geberg is, is weer op dag 128 beoordeel. Daar is bevind dat sowel die 30 IJ/cm2 as die 45 IJ/cm2 konsentrasie 100% mortaliteit by T. molitor veroorsaak het. Heterorhabditis zealandica blyk ʼn goeie oorlewing te hê ná 84 dae in veld kondisies wat erg met P. callosus besmet is, en is dus ʼn moontlike beheermiddel vir P. callosus, met potensiële oorlewing vir minstens drie maande

Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica

The original publication is available at http://ijs.sgmjournals.org/content/63/Pt_5/1853.fullThe bacterial symbiont AM7T, isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7T within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7T revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29T, P. luminescens subsp. akhurstii FRG04T and P. luminescens subsp. hainanensis C8404T, respectively.South African Apple and Pear Producers Association (SAAPPA), Citrus Research International (CRI) and the Technology and Human Resources for Industry Programme (THRIP)Post-prin

Description of Xenorhabdus khoisanae sp. nov., the symbiont of the entomopathogenic nematode Steinernema khoisanae

The original publication is available at http://ijs.sgmjournals.org/content/63/Pt_9/3220.fullBacterial strain SF87T, and additional strains SF80, SF362 and 106-C, isolated from the nematode Steinernema khoisanae, are non-bioluminescent Gram-reaction-negative bacteria that share many of the carbohydrate fermentation reactions recorded for the type strains of recognized Xenorhabdus species. Based on 16S rRNA gene sequence data, strain SF87T is shown to be closely related (98 % similarity) to Xenorhabdus hominickii DSM 17903T. Nucleotide sequences of strain SF87 obtained from the recA, dnaN, gltX, gyrB and infB genes showed 96–97 % similarity with Xenorhabdus miraniensis DSM 17902T. However, strain SF87 shares only 52.7 % DNA–DNA relatedness with the type strain of X. miraniensis, confirming that it belongs to a different species. Strains SF87T, SF80, SF362 and 106-C are phenotypically similar to X. miraniensis and X. beddingii, except that they do not produce acid from aesculin. These strains are thus considered to represent a novel species of the genus Xenorhabdus, for which the name Xenorhabdus khoisanae sp. nov. is proposed. The type strain is SF87T ( = DSM 25463T = ATCC BAA-2406T).Post-prin

Photorhabdus luminescens subsp noenieputensis subsp nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica

The bacterial symbiont AM7(T), isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7(T) within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7(T) revealed 95.8, 95.4 and 94.9% nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29(T), P. luminescens subsp. akhurstii FRG04(T) and P. luminescens subsp. hainanensis C8404(T), respectively. These identity values are less than the threshold of 97% proposed for classification within one of the existing subspecies of P. luminescens. Unlike other strains described for P. luminescens, strain AM7(T) produces acid from adonitol, sorbitol and xylitol, assimilates xylitol and has no lipase activity on medium containing Tween 20 or 60. Strain AM7(T) is differentiated from P. luminescens subsp. caribbeanensis by the assimilation of N-acetylglucosamine and the absence of haemolytic activity. Unlike P. luminescens subsp. akhurstii, strain AM7(T) does not assimilate mannitol, and it is distinguished from P. luminescens subsp. hainanensis by the assimilation of trehalose and citrate, the inability to produce indole from tryptophan and the presence of acetoin production and urease activity. Strain AM7(T) (=ATCC BAA-2407(T) =DSM 25462(T)) belongs to a novel subspecies, and is proposed as the type strain of Photorhabdus luminescens subsp. noenieputensis sp. nov

Photorhabdus heterorhabditis sp nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica

The bacterial symbionts SF41(T) and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41(T), SF783 and 0614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724(T) initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41(T)., SF783 and 0614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41(T)=ATCC BAA-2479(T)=DSM 25263(T))