No description available.

White grubs (Coleoptera: Scarabaeidae) are important turfgrass pests in Virginia. Insecticides such as the neonicotinoid imidacloprid are commonly applied to turfgrass in order to control these pests. As an alternative to synthetic insecticides, entomopathogenic fungi (EPF), including Metarhizium brunneum (Petch) and Beauveria bassiana (Balsamo) Vuillemin may also be used for white grub control. The interaction of combining these two control tactics for white grubs in Virginia merits further investigation as does their effects on other soil organisms such as earthworms, which cohabitate with white grubs in turfgrass soil ecosystems. Herein, I investigate the following: 1) the efficacy of combined applications of the EPF, M. brunneum and B. bassiana with lower rates of imidacloprid or the diamide insecticide, chlorantraniliprole against white grubs; 2) interactions of earthworms with white grubs and EPF; and 3) the effect of white grub control products on earthworms. In the laboratory, a combined application of one half the recommended rate of chlorantraniliprole plus the full recommended rate of B. bassiana caused significantly higher mortality of third instar Cyclocephala spp. grubs than the untreated control. In the field, imidacloprid applied at lower rates as a single treatment or as part of a combined treatment with EPF resulted in significantly fewer grubs when applications were made in June. In the greenhouse, Japanese beetle, Popillia japonica Newman females laid a significantly reduced number of eggs in turf treated with lower rate of imidacloprid either applied as a single treatment or as part of a combined treatment compared with untreated control. In an earthworm-white grub interaction study, the earthworms Eisenia fetida (Savingy) and E. hortenis (Michaelsen) were shown to transfer B. bassiana spores from fungus-infected soil to uninfected soil in the laboratory. However, the presence of earthworms in fungal infected soil did not enhance the mortality of Cyclocephala spp. grubs. In bioassays conducted in the laboratory, only two neonicotinoids, dinotefuran and imidacloprid, caused significantly higher mortality to adult Lumbricus terrestris L. earthworms than untreated control consistently. When applied as a drench to turfgrass in spring, summer, and fall, none of the insecticides significantly reduced the earthworm densities compared with a water control. / Ph. D.

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) is a devastating pest of pome fruit in temperate regions of the world. Control of this pest, previously involved the extensive use of broad-spectrum insecticides. However, concerns over human safety, environmental impact, widespread dispersal of resistant populations of codling moth and the sustainability of synthetic pesticides in agroecosystems, has encouraged the development and use of alternative environmentally-friendly pest management technologies including the use of entomopathogenic nematodes. These nematodes are lethal pathogens of insects and belong to the families Steinernematidae and Heterorhabditidae, and are ideal candidates for incorporation into the integrated pest management programme currently being developed for residue-free pome fruit production in South Africa. The biological and ecological factors pertaining to the successful use of these nematodes for the control of codling moth were investigated in this study. Their use for bin-disinfestations was evaluated, focusing on the optimum handling conditions to ensure the survival and subsequent efficacy of the nematodes. The study proved that the local isolate SF41 of Heterorhabditis zealandica Poinar 1990 could be used for successful bin-disinfestation. The use of the same nematode isolate was also investigated for the disinfestation of mulch layers of diapausing codling moth larvae. An insect containment device which allowed for direct trial efficacy evaluation was identified and ecological factors pertaining to the successful use of nematodes for mulch disinfestation were investigated. The biological control potential of local nematode isolates, which had previously never been tested against codling moth larvae, was investigated in the laboratory under conditions as can be expected during orchard applications. The efficacy of the selected isolates was confirmed in field experiments. Innovative insect containment methods for above-ground trial efficacy evaluation in the field were investigated. Desiccation proved to be the most limiting factor to the survival and subsequent efficacy of the nematodes during field applications in temperate regions. The effect of low moisture levels on H. zealandica’s efficacy to control diapausing codling moth larvae was subsequently investigated and a starch-based formulation was further tested to overcome the issue of desiccation. Conclusive results indicated that there were several biological and ecological factors influencing the survival of nematodes and illustrated how these factors could be manipulated to overcome these issues and thereby ensure the efficacy of treatments. This is the first report of its kind to comprehensively investigate the use of South African entomopathogenic nematodes for the control of diapausing codling moth larvae and all results emanating from the study can be integrated into a framework for the commercial use of these nematodes in this regard in future. / AFRIKAANSE OPSOMMING: Kodlingmot, Cydia pomonella (L.) is ‘n ernstige sleutelplaag in appel- en peerboorde in gematigde klimaats gebiede wêreldwyd. In die verlede is hoofsaaklik breëspektrum insektedoders gebruik vir die beheer van hierdie plaaginsek. Maar, kommer oor veiligheid vir die mens, impak op die omgewing, verspreiding van weerstandbiedende populasies van kodlingmot en beperkte volhoubaarheid van sintetiese plaagdoders het die ontwikkeling en gebruik van alternatiewe plaagbeheer tegnologieë, insluitend die gebruik van entomopatogeniese nematodes, genoodsaak. Entomopatogeniese nematodese horende tot die families Steinernematidae en Heterorhabditidae, is ideale kandidate vir insluiting in die geïntegreerde plaagbestuur programme wat huidiglik ontwikkel word vir gebruik in plaaslike boorde met die uiteindelike doel om residu-vrye vrugte te produseer. In hierdie studie word die biologiese en ekologiese faktore bestudeer wat die sukses van ‘n nematode-toediening gemik op kodlingmot beïnvloed. Hierdie studie het bewys dat die lokale SF41 isolaat van Heterorhabditis zealandica Poinar 1990 gebruik kan word om vrugtekratte te disinfesteer van kodlingmot. Die gebruik van dieselfde isolaat vir die disinfestasie van deklae is ook ondersoek. ‘n Metode van insek-inkamping is ook ontwikkel wat die evaluering van toedienings vergemaklik en meer effektief maak. Die omgewings-toestande wat ook bydrae tot die oorlewing en gevolglike sukses van ‘n toediening is ook ondersoek. Die biologiese beheer potensiaal van ‘n paar lokale isolate wat nog nooit voorheen teen kodlingmot getoets is nie, is ook bestudeer. Die isolate se effektiwiteit is ook bevestig in veldproewe en insek-bekampings metodes wat meer van toepassing is vir bogrondse plaaginsekte is ook geïdentifiseer. Resultate dui daarop dat vogverlies en gevolglike uitdroging van nematodes die grootste beperkende faktor is vir hierdie tipe toedienings in gematigde gebiede en ‘n stysel-gebaseerde formulasie is dus ondersoek om hierdie probleem te oorkom. Die uiteindelike gevolgtrekking van die studie was, dat alhoewel daar verskeie biologiese en ekologiese faktore is wat die oorlewing van nematodes beperk, daar tog verskeie maniere is om hierdie faktore te manipuleer en sodoende te oorkom, wat bydrae tot die uiteindelike sukses van ‘n toediening. Hierdie is die eerste studie wat werklik die praktiese gebruik van lokale entomopatogeniese nematodes vir die beheer van kodlingmot ondersoek en alle bevindinge kan geïntegreer word in toekomende riglyne vir die kommeriële gebruik van nematodes vir die beheer van kodlingmot.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The agricultural industry in South Africa is dominated by the use of insecticides. Producers rely heavily on chemicals that cause increased risk to health, the environment and ecology, rapid resistance development in key insect pests and pesticide residues on crops. The increased concern regarding the impact of these pest management practices on the environment and alternative pest management strategies are being investigated. Entomopathogenic nematodes (EPNs) have been identified as being promising biological control agents of key insect pests. The two EPN genera that have shown promise for use as biological control agents within an integrated pest management programme areSteinernema and Heterorhabditis. Commercialisation and the successful use of EPNs to control pests in North America, Australia, Europe and Asia have confirmed the effectiveness of these organisms as biological control agents. Unfortunately, EPNs in large enough numbers for commercial field applications are not yet available on the South African market. Large numbers of EPNs can be produced through either in vivo or in vitro culturing practices. The objective of this study was to streamline the in vivo production process by using two endemic EPN species, Heterorhabditis zealandica (SF41) and H. bacteriophora (SF351). These EPN isolates have been shown to be effective control agents of codling moth Cydia pomonella, false codling moth Thaumatotibia leucotreta, obscure mealybug Pseudococcus viburni, and the banded fruit weevil Phlyctinus callosus. A comparative study was conducted to identify suitable host insects for EPN production of local H. zealandica (SF41) and H. bacteriophora (SF351) strains. Hosts were selected according to their susceptibility to the two EPN species used, their general availability and the ease and cost of rearing. Wax moth larvae Galleria mellonella (WML) and mealworms Tenebrio molitor (MW) were selected as hosts. In order to produce nematodes of consistent quality, a continuous source of host insects reared on a standardised diet was required. WML and MW were each reared on five different diets in the dark at ±26°C. A superior diet for each host was selected according to the diet that produced, on average, the larvae with the highest body mass within a specific timeframe. The heaviest WML, at an average weight of 0.19 g per larva, were produced on a diet consisting of 118 g wheat flour, 206 g wheat bran, 118 g milk powder, 88 g brewer‟s yeast, 24 g wax powder, 175 ml honey and 175 ml glycerol. The heaviest MW larvae weighed, on average, 0.0154 g per larva, and were produced on a diet consisting of 100% wheat bran. To confirm the hypothesis that a linear relationship exists between the weight of a host and the number of nematodes produced from that host, a study was conducted to determine the number of H. zealandica and H. bacteriophora produced per g of host. WML, MW, codling moth larvae and false codling moth larvae were weighed individually and inoculated with the two nematode species respectively. In addition, nematode production in frozen MW and WML was tested. The number of nematodes harvested from each host was counted, and the average number of nematode progeny produced in each host was calculated. A significant linear correlation between the weight of WML and MW and the number of H. zealandica and H. bacteriophora respectively produced confirmed the hypothesis that nematode production within the specified host increases with an increase in host weight. WML produced the highest number of H. zealandica and H. bacteriophora per g of host (1 459 205 ± 113 670 and 1 898 512 ± 94 355), followed by MW larvae (836 690 ± 121 252 and 414 566 ± 67 017). Lower numbers of H. zealandica and H. bacteriophora per g codling moth (57 582 ± 10 026 and 39 653 ± 8 276) and per g false codling moth (192 867 ± 13 488 and 97 652 ± 23 404) were produced. Successful infection of a suitable insect host is one of the key factors in an efficient in vivo nematode production process. Three inoculation techniques were compared using H. zealandica and H. bacteriophora: inoculation with a pipette; shaking of hosts in the nematode inoculum; and immersion of hosts in the nematode suspension. With each inoculation technique, WML and MW were used as host larvae and were inoculated with nematodes at a concentration of 200 infective juveniles (IJs) / larva. The percentage mortality of insect hosts was determined after two days, and EPN infectivity, confirmed by colour change and dissection, after seven days. The highest percentage EPN infection was obtained using pipetting for both nematode isolates and hosts. Nematode infection rates for all nematode-host combinations obtained with pipetting were above 90%, with the exception of MW inoculation with H. bacteriophora, where the percentage of infection obtained was 76%. The current study conclusively demonstrated that variations in infection levels occur, depending on the inoculation technique used. In an additional effort to enhance infectivity during inoculation, H. zealandica, H. bacteriophora and MW were subjected to host-stressor regimes and to nematode- infectivity-enhancing additives. Three treatments, plus a control treatment, were compared. Exposing MW to 70°C tap water prior to inoculation did not increase infection levels. On the contrary, reduced infection levels were observed with host immersion in 70°C tap water followed by inoculation with H. bacteriophora, compared to the control. Only 12% infection was obtained compared to the 48% infection achieved in the control. Infection obtained using H. zealandica was 21%. Treating H. zealandica and H. bacteriophora IJs withMn2+SO4.H20 in a suspension, prior to inoculating MW, did not significantly enhance nematode virulence. Inoculation of MW with treated H. zealandica IJs led to an infection rate of 81%, compared to the control, with which 80% infection rate was obtained. Heterorhabditis bacteriophora caused 47% MW infection, compared to the control, which was subject to 48% infection. A combination of the two above-mentioned treatments did not enhance the infection levels either. Immersing MW into 70°C tap water prior to inoculation with nematodes treated with Mn2+SO4.H20 led to infection levels of 13% and 9% respectively when H. bacteriophora and H. zealandica were used. Future research is required to optimise the protocol used in this study of subjecting MW and local nematode isolates to stressor regimes. The ability of two formulations to maintain biological activity and virulence of H. zealandica was investigated. A quality standard control measure was used to measure the percentage survival and virulence of formulated H. zealandica over a period of 21 days. IJs were formulated into Pesta granules and coconut fibres, while nematodes stored in tap water served as the control. The numbers of live H. zealandica in Pesta granules and coconut fibres decreased drastically after seven days of storage. The survival of nematodes in Pesta granules dropped to 9.79% after 21 days compared to the control, where the survival rate was 79.79%. Nematode survival in coconut fibres was even lower, at 25.84% after seven days and 2.25% after 21 days. After 21 days in storage, 100%+of nematodes survived in the control for coconut fibres. The application of the standard quality control measure, which was used to determine the virulence of formulated H. zealandica, proved to be ineffective. Higher MW mortality rates were obtained in the control where no nematodes were added to larvae, compared to where nematodes were added in varying dosages. However, adjusting certain aspects in the protocol of this quality control measure specifically to accommodate local conditions could possibly make it a more effective tool for measuring endemic nematode virulence. / AFRIKAANSE OPSOMMING: Die landboubedryf in Suid-Afrika word oorheers deur die gebruik van insekdoders. Vervaardigers steun swaar op chemikalieë wat toenemend gesondheids-, omgewings- en ekologiese risiko's, asook die snelle ontwikkeling van weerstand in sleutelinsekteplae veroorsaak, en wat reste van plaagdoders op gewasse laat. Na aanleiding van toenemende besorgdheid oor die impak van hierdie plaagbestuurspraktyke op die omgewing, word alternatiewe plaagbestuurstrategieë ondersoek. Entomopatogeniese nematodes (EPNs) is geïdentifiseer as belowende biologiese beheeragente van sleutelinsekteplae. Die twee EPN genera wat belofte inhou vir gebruik as biologiese beheeragente binne 'n geïntegreerde plaagbestuursprogram is Steinernema en Heterorhabditis. Kommersialisering en die geslaagde gebruik van EPNs om insekplae te beheer in Noord-Amerika, Australië, Europa en Asië, het die doeltreffendheid van hierdie organismes as biologiese beheeragente bevestig. Ongelukkig is EPNs in groot genoeg getalle vir kommersiële aanwending in die veld nog nie op die Suid-Afrikaanse mark beskikbaar nie. Groot getalle EPNs kan deur in vivo en in vitro teling verkry word. Die doelwit van hierdie studie was om die in vivo produksieproses te stroomlyn deur die gebruik van twee endemiese EPN spesies, Heterorhabditis zealandica (SF41) en H. bacteriophora (SF351). Hierdie EPN isolate is deur navorsing bewys om doeltreffende beheeragente van kodlingmot Cydia pomonella, vals kodlingmot Thaumatotibia leucotreta, ligrooswitluis Pseudococcus viburni, en gebande vrugtekalanders Phlyctinus callosus te wees. 'n Vergelykende studie is gedoen om geskikte gasheerinsekte vir EPN produksie van plaaslike H. zealandica (SF41) en H. bacteriophora (SF351) isolate te vind. Gashere is geselekteer op grond van vatbaarheid vir die EPN spesie wat gebruik word, en algemene beskikbaarheid en gemak en koste van teling. Wasmotlarwes Galleria mellonella (WML) en meelwurms Tenebrio molitor (MW) is as gashere gekies. Ten einde nematodes van konsekwente kwaliteit te teel, word 'n deurlopende bron van gasheerinsekte benodig wat op 'n gestandaardiseerde dieet voed. WML en MW is onderskeidelik op vyf verskillende diëte geteel by ±26°C in die donker. Die beste dieet vir elke gasheer is gekies op grond van die dieet wat, gemiddeld, die swaarste larwes binne 'n spesifieke tydsraamwerk opgelewer het. Die swaarste WML, teen 'n gemiddelde massa van 0.19 g per larwe, is geteel op 'n dieet wat bestaan het uit 118 g koringmeel, 206 g semels, 118 g melkpoeier, 88 g brouersgis, 24 g verpoeierde was, 175 ml heuning en 175 ml gliserol. Die swaarste MW larwes het gemiddeld 0.0154 g per larwe geweeg en is geteel op 'n dieet van 100% semels. Ten einde die hipotese te bevestig dat 'n lineêre verwantskap bestaan tussen die massa van 'n insekgasheer en die aantal nematodes wat deur daardie gasheer geproduseer word, is 'n studie gedoen om die aantal H. zealandica en H. bacteriophora per gasheergram te bepaal. WML, MW, kodlingmotlarwes en vals kodlingmotlarwes is individueel geweeg en met infektiewe larwes van die twee onderskeidelike EPN spesies geïnokuleer. Daarbenewens is die vermeerdering van nematodes in bevrore MW en WML ook getoets. Die aantal nematodes wat in elke gasheer geoes is, is getel, en die gemiddelde nematode-afstammelinge in elke gasheer bereken. 'n Beduidende lineêre korrelasie tussen die massa van WML en MW en die aantal H. zealandica en H. bacteriophora wat onderskeidelik geproduseer is, het die hipotese bevestig dat nematode-vermeerdering binne hierdie gashere toeneem namate die gasheermassa toeneem. WML het die meeste H. zealandica en H. bacteriophera per gasheergram opgelewer (1 459 205± 113 670 en 1 898 512± 94 355 onderskeidelik), gevolg deur MW larwes (836 690± 121 252 en 414 566± 67 017 onderskeidelik). Laer getalle H. zealandica and H. bacteriophora per gram kodlingmot (57 582 ± 10 026 en 39 653 ± 8 276) en per gram vals kodlingmot (192 867 ± 13 488 en 97 652 ± 23 404) is egter geproduseer. Een van die sleutelfaktore vir die doeltreffendheid van die in vivo vermeerdering van nematodes is geslaagde gasheerinfeksie. Drie inokulasietegnieke is dus geëvalueer en vergelyk deur H. zealandica en H. bacteriophora te gebruik: inokulasie met 'n pipet, skud van gashere in 'n nematode-inokulum, en gasheerindompeling in 'n nematode-suspensie. WML en MW is as gashere gebruik vir elke inokulasietegniek, en is geïnokuleer met nematodes wat uit 'n konsentrasie van 200 infektiewe larwes (ILs) / insek larwe bestaan het. Die persentasie dooie insekgashere is na twee dae bepaal, en infeksie soos bevestig deur kleurverandering en disseksie, na sewe dae. Die hoogste persentasie infeksie deur sowel nematode-isolate as gashere te gebruik, was met die pipet-tegniek. Die infeksiekoerse vir alle nematode-gasheerkombinasies met die pipet-tegniek was hoër as 90%, met die uitsondering van MW-inokulasie met H. bacteriophora, waar die infeksie 76% was. Hierdie studie toon dat afwykings voorkom in die mate van gasheerinfeksie, na gelang van die inokulasietegniek wat gebruik is. In 'n bykomende poging om infeksie na inokulasie te verhoog, is H. zealandica, H. bacteriophora en MW onderwerp aan stressors en bymiddels om nematode-infeksie te bevorder. Drie behandelings, asook 'n kontrole-behandeling, is vergelyk. Infeksievlakke het nie verhoog deur MW voor inokulasie aan kraanwater van 70°C bloot te stel nie. Inteendeel, laer infeksievlakke is opgemerk waar gashere in kraanwater van 70°C gedompel is en daarna met H. bacteriophora geïnokuleer is, vergelyke met die kontrole. Gasheerinfeksie van slegs 12% is verkry, vergelyke met 48% in die kontrole. Infeksie van 21% is met H. zealandica verkry. Die virulensie van nematodes het nie beduidend toegeneem deur H. zealandica en H. bacteriophora IL in 'n suspensie met Mn2+SO4H20 te behandel voor MW geïnokuleer is nie. Inokulasie van MW met behandelde H. zealandica IL het tot 'n infeksie van 81% gelei, vergelyke met die kontrole waar 'n infeksie van 80% behaal is. H. bacteriophora het 'n MW-infeksie van 47% veroorsaak, vergelyke met die kontrole se infeksiekoers van 48%. 'n Kombinasie van die twee bogenoemde behandelings het eweneens nie gasheerinfeksievlakke verhoog nie. Die indompeling van meelwurms in kraanwater van 70°C voor inokulasie met nematodes wat met Mn2+SO4H20 behandel is, het tot gasheerinfeksie van 13% en 9% onderskeidelik gelei wanneer H. bacteriophora en H. zealandica gebruik is. Toekomstige navorsing is nodig om die protokol te verbeter wat in hierdie studie gebruik is om MW en plaaslike nematode-isolate aan stressors te onderwerp. 'n Ondersoek is gedoen na die vermoë van twee formulasies om biologiese aktiwiteit en virulensie van H. zealandica te onderhou. 'n Kwaliteitsstandaardtegniekis gebruik om weekliks die persentasie oorlewing en virulensie van geformuleerde H. zealandica oor 'n tydperk van 21 dae te meet. IL is in Pesta korrels en klappervesel geformuleer, terwyl nematodes in kraanwater gedien het as kontrole. Die aantal lewende H. zealandica in Pesta korrels en klappervesel het drasties verminder na sewe dae in die formulasie. Oorlewing van nematodes in Pesta korrels het gedaal tot 9.79% na 21 dae vergyleke met die kontrole, waar 79.79% oorleef het. Nog minder nematodes - 25.84% - het na sewe dae in die klappervesel oorleef, en slegs 2.25% na 21 dae. Na 21 dae van berging het 100%+ van nematodes oorleef in die kontrole vir klappervesel. Die toepassing van die kwaliteitsstandaardtegniek om die virulensie van geformuleerde H. zealandica te bepaal, het ondoeltreffend geblyk. Verhoogde MW sterftesyfers is verkry in die kontrole waar geen nematodes by die inseklarwes gevoeg is nie, vergelyke met die byvoeging van hoër dosisse nematodes. Nietemin, die aanpassing van sekere aspekte in die protokol van hierdie kwaliteitsbeheermeting om spesifiek plaaslike toestande in ag te neem, sou dit moontlik 'n meer doeltreffende middel kon maak om die virulensie van endemiese nematodes te bepaal.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), the vine mealybug, is of economic importance to the wine and table grape (viticulture) industries, as it characteristically causes more damage than other mealybug species. Mealybug infestations contaminate grapes with their waxy secretions, egg-sacs and honeydew production, on which sooty mould grows, resulting in the fruit being unmarketable. Many export grapes are rejected, prior to shipment, as a result of infestations and phytosanitary concerns with regard to mealybug infestations. They are also vectors for various plant viruses. Up to date, the most common method of mealybug control in South Africa has been the use of chemical insecticides. Unfortunately, mealybugs are difficult to control chemically, due to their secretive/hidden lifestyle, where chemicals do not reach them. Of great concern is the ability of mealybugs to rapidly build up resistance to insecticides as well as the negative environmental effects associated with chemical pesticide use. Alternatively, entomopathogenic nematodes (EPNs), belonging to the families Heterorhabditidae and Steinernematidae, have been identified as lethal insect pathogens and their insecticidal action, towards a variety of insect pests, has proven them to be valuable and effective biocontrol agents. Laboratory bio-assays, to determine the ability of eight different EPN isolates to infect and kill P. ficus, were conducted. Six of the isolates were indigenous species and the other two, Heterorhabditis bacteriophora and Steinernema feltiae, were produced in Germany and are commercially available in South Africa. Planococcus ficus was highly susceptible to two indigenous species, Heterorhabditis zealandica and Steinernema yirgalemense; responsible for 96% ± 2% and 65% ± 10% mealybug mortalities, respectively. Biological studies illustrated that both H. zealandica and S. yirgalemense are able to complete their life cycles within adult female P. ficus. There was no significant difference in the pathogenicity of commercially produced H. bacteriophora, recycled through an insect host, and those from the formulated commercial product. However, commercially produced S. feltiae individuals, that were recycled through an insect host, were statistically more effective than those that were not. The LC50 and LC90 values for H. zealandica, in the current study, were 19 and 82 infective juveniles (IJs) respectively, which were similar to the LC50 and LC90 values for S. yirgalemense at 13 and 80, respectively. The LC50 and LC90, for commercially available H. bacteriophora, were greater than they were for both H. zealandica and S. yirgalemense, with values of 36 and 555, respectively. Such results indicate that there is a definite positive relationship which exists between the concentration of IJs of all three nematode species, used for inoculation, and the percentage mortality of P. ficus. Sand column tests resulted in S. yirgalemense outperforming H. zealandica significantly, with average mortalities of 95% ± 1.4% and 82% ± 4.1%, respectively. As a result S. yirgalemense was chosen for further studies in the field. IJs of commercially produced H. bacteriophora and S. feltiae were exposed to imidacloprid in laboratory bioassays to determine the effect on survival and infectivity. This study established the fact that these two EPN species can be applied, in combination with imidacloprid, in an integrated pest management scheme. Soil application field trials at Welgevallen and Nietvoorbij, using S. yirgalemense and mealybugs in Eppendorf tubes, buried 15 cm in the soil, resulted in 50% ± 10% and 52% ± 12% mealybug mortalities, respectively, when applying IJs at a concentration of 80 IJs/cm2. No significant difference was found between mealybug mortalities as a result of the three IJ concentrations applied (20, 40 and 80 IJs/cm2) for both vineyards. Persistence trials indicated that after four months post application, Cydia pomonella larval mortalities showed no significant reduction in infectivity on the Welgevallen vineyard, while on the Nietvoorbij vineyard there were no larval mortalities. Tests to establish whether or not S. yirgalemense and H. zealandica produced ant deterrent factors, showed no significant differences between the number of intact cadavers for both nematode species and for cadavers that were either four or six days old. There is, however, indication that deterrent factors may be in action in cadavers that were used six days after inoculation with 60% and 49% remaining intact for H. zealandica and S. yirgalemense infected cadavers respectively. All freeze killed cadavers were consumed by Linepithema humile (Mayr) (Argentine ant). The effects of low temperatures on EPN movement and infectivity were tested for H. zealandica and S. yirgalemense in the laboratory. The mortality of P. ficus at 14˚C, as opposed to 25˚C, for S. yirgalemense and H. zealandica were found to be 9.1% ± 2.6% and 2.5% ± 1.2% respectively. Vertical sand column tests were also conducted at 14˚C for S. yirgalemense and H. zealandica, which produced low mealybug mortalities of 3.5% ± 2.4% and 8.5% ± 1.4% respectively. This illustrates the low infectivity of the two local species at low temperatures. Laboratory persistence trials, conducted over a period of four months with S. yirgalemense, showed steady persistence of 100%, while H. zealandica had a statistically significant decrease of codling moth mortalities to 44% ± 5%. A three armed olfactometer was designed to establish if S. yirgalemense responds and moves towards chemical cues in the soil. A significant greater average number of IJs moved towards the grape vine roots (246 ± 0.124 IJs), than to the mealybugs (133 ± 0.168 IJs) and to the control (4 ± 1.02 IJs). This demonstrates that S. yirgalemense does actively seek out its hosts and that volatile cues produced by damaged grape vine roots, are more attractive to the EPN than cues produced by P. ficus. This study illustrates that S. yirgalemense has great potential as a biopesticide for controlling P. ficus in the soil of South African grape vineyards. Emphasis was placed on soil application of S. yirgalemense in the field, which produced good results, while laboratory tests indicate the potential for further aerial field application trials on grape vines. As the EPNs are not negatively affected by the agrochemical imidacloprid, the simultaneous use and combined action of both agents will potentially provide the farmer with excellent control against P. ficus. Further field- and aerial application studies will complement the current study and hopefully provide positive results for the efficient control of P. ficus found on grape vines. / AFRIKAANSE OPSOMMING: Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), die wingerd witluis, is van groot ekonomiese belang vir die wyn en tafeldruif industrieë, aangesien dit kenmerkend meer skade veroorsaak as enige ander witluis spesies. Witluis infestasies besmet druiwe met hulle wasagtige afskeidings, eierssakke en heuningdou produksie, waarop swamme groei, wat tot gevolg het dat die druiwe onbemarkbaar is. Baie besendings druiwe, bestem vir uitvoer, word afgekeur weens witluis besmettings en ook as gevolg van fitosanitêre oorwegings. Hulle tree ook op as vektore van verskeie plantvirusse. Die mees algemene manier waarmee witluis in Suid-Afrika beheer word, is chemiese behandeling. Ongelukkig is witluis baie moeilik om chemies te beheer vanweë hulle verskuilde lewenswyse wat dit moeilik maak vir chemikalieë om hulle te bereik. Die vermoë van witluis om vinnig weerstand op te bou teen insekdoders, asook die negatiewe effek van chemiese middels op die omgewing, is kommerwekkend. Alternatiewelik, kan entomopatogeniese nematodes (EPNs) van die families Heterorhabditidae en Steinernematidae gebruik word vir die beheer van witluis. Hierdie nematodes is geïdentifiseer is as dodelike insek patogene, vir ʼn groot verskeidenheid van pes insekte en daar is bewys dat hulle as waardevolle en effektiewe biologiese beheer agente kan optree. Laboratorium biotoetse is gedoen om die vermoë van agt EPN isolate te evalueer om P. ficus te beheer. Ses van die EPN isolate is inheems, terwyl die ander twee, Heterorhabditis bacteriophora en Steinernema feltiae, in Duitsland produseer is en kommersieel beskikbaar is in Suid-Afrika. Planococcus ficus is hoogs vatbaar vir die twee inheemse EPN spesies, naamlik Heterorhabditis zealandica en Steinernema yirgalemense en hulle is verantwoordelik vir 96% ± 2% en 65% ± 10% van witluis mortaliteit. Biologiese studies het aangetoon dat beide H. zealandica en S. yirgalemense in staat is om hul lewensiklus te voltooi in volwasse wyfies van P. ficus. Daar is geen beduidende verskil gevind in die patogenisiteit van die geformuleerde kommersiële produk H. bacteriophora en dié wat in vivo geproduseer is nie. Daar is egter in die geval van S. feltiae, gevind dat die nematodes, wat in insekte produseer is, statisties beduidend meer effektief was, as dié wat kommersieel beskikbaar was. Die LC50 en die LC90 waardes van H. zealandica, in die huidige studie, was 19 en 82 infektiewe larwes (IJs), wat baie naby die LC50 en LC90 waarders van S. yirgalemense van 13 en 80 was. Die LC50 en LC90 vir die kommersieel beskikbare H. bacteriophora was groter as vir beide H. zealandica en S. yirgalemense, met waardes van 36 en 555 onderskeidelik. Hierdie resultate dui daarop dat daar ʼn positiewe verwantskap bestaan tussen die konsentrasie van die IJs van drie EPN spesies en die persentasie mortaliteit van P. ficus. Sand kolomtoetse dui daarop dat S. yirgalemense baie beter vaar as H. zealandica met gemiddelde mortaliteite van 95% ± 1.4% en 82% ± 4.1% onderskeidelik. Op grond van hierdie resultate is S. yirgalemense gebruik vir verdere veld studies. IJs van kommersieel geproduseerde H. bacteriophora en S. feltiae is in laboratorium biotoetse blootgestel aan imidacloprid om die effek op die oorlewing en infektiewe vermoë vas te stel. Hierdie studie het aangetoon dat die twee EPN spesies aangewend kan word saam met imidacloprid in ʼn geïntegreerde plaagbestuur opset. Grond aanwendings is in veld proewe by Welgevallen en Nietvoorbij gedoen deur gebruik te maak van S. yirgalemense en P. ficus volwasse wyfies in Eppendorf buisies, 15 cm in die grond begrawe, het albei 50% ± 10% en 52% ± 10% witluis mortaliteit, respektiewelik, tot gevolg gehad, met die toediening van nematodes teen ʼn konsentrasie van 80 IJs/cm2. Geen beduidende verskille is gevind tussen die witluismortaliteit en die resultate van die verskillende EPN konsentrasies (20, 40 en 80 IJs/cm2) wat op beide wingerde toegedien is nie. Oorlewings toetse het aangedui dat, drie maande na toediening, met Cydia pomonella as indikator, geen beduidende verskille in die infeksie potensiaal van die Welgevallen wingerd to gevolg gehad het nie, terwyl daar op die Nietvoorbij wingerd geen verdere larvale mortaliteit gevind is was nie. Toetse om vas te stel of S. yirgalemense en H. zealandica afkrikmiddels vir miere in besmette insek kadawers produseer het aangetoon dat daar geen beduidende verskil is tussen die getal kadawers wat intakt is vir beide EPN spesies en kadawers wat vier of ses dae oud is nie. Daar is egter aangetoon dat die afskrikmiddels wel ses dae na infeksie deur insek kadawers afgeskei word; aangesien 60% en 49% van die oorblywende kadawers nog volledig was toe dit geïnfekteer was met H. zealandica en S. yirgalemense, onderskeidelik. Al die insek kadawers, wat deur bevriesing doodgemaak is, was deur Linepithema humile (Mayr) (Argentynse mier) verorber.

Les Nématodes Entomopathogènes (NEP) de la famille des Steinernematidae et Heterorhabditidae sont des parasites pour les insectes. Ils hébergent dans leur intestin un symbiote bactérien (genres Xenorhabdus et Photorhabdus) essentiel au succès parasitaire. Les NEP sont présents dans les sols des cinq continents de la planète. Leur diversité génétique et leurs propriétés biologiques constituent une intéressante ressource biologique. Grâce à leur entomotoxicité, les NEP sont de bons outils de lutte biologique en agriculture et en culture ornementale un peu partout dans le monde. La diversité et la biogéographie des NEP dans les cinq continents de la Terre (à l'exception de l'Antarctique) ont été étudiées partout dans le monde mais le Liban est parmi les rares pays du moyen orient où aucune prospection de ces nématodes n'a été réalisée alors que des NEP en Turquie, Syrie, Jordanie, Palestine et Egypte ont déjà été trouvés et caractérisés. L'objet de la thèse est de procéder à une étude biogéographique dans le but de connaître la diversité des NEP au Liban. L'enjeu scientifique est donc de combler un « vide » dans la connaissance de la répartition et de la biodiversité mondiale des NEP. Pour cela, un échantillonnage à l'échelle des étages de végétation est mené au Liban. Des échantillons de sol sont ainsi prélevés, mis en contact avec des larves de Galleria mellonella pour isoler les nématodes entomopathogènes et leurs symbiotes. Les nématodes et leurs symbiotes sont par la suite identifiés morphologiquement et moléculairement. Par la suite, une approche à l'échelle de l'habitat fait l'objet de cette thèse également pour étudier les interactions biotiques et abiotiques influençant la présence des nématodes entomopathogènes dans le sol. Les enjeux technologiques, exposés au second volet de la thèse, sont liés aux propriétés biologiques des nématodes et de leurs symbiotes afin de valoriser leur entomotoxicité en lutte biologique. Dans ce cadre, la sensibilité des Cephalcia tannourinensis, ravageur des cédraies au Liban, par rapport aux nématodes entomopathogènes est exploitée in vitro ; différentes espèces de nématodes entomopathogènes sont testées pour suivre leur cycle à l'intérieur des Cephalcia. / Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. Thanks to their entomotoxicity, EPNs are good tools for biological control in agriculture almost everywhere in the world. They are ubiquitous, having been isolated from every inhabited continent (except Antartica) from a wide range of ecologically diverse soil habitats including cultivated fields, forests, grasslands, deserts, and even ocean beaches. Biogeographic assessments of EPNs in the Eastern Mediterranean basin have been conducted in several countries such as Turkey, Syria, Jordan, Israel, Palestine and Egypt. Lebanon is among the few countries of the Middle East for which no survey of EPNs has been done. The scientific stake is thus to fill a gap in our knowledge of EPNs distribution in the Mediterranean basin. Survey of EPNs was conducted in this framework to cover the different vegetation levels defined in Lebanon. Soil samples were removed placed in contact with Galleria mellonella to isolate entomopathogenic nematode and their symbiotic bacteria. EPNs and their bacteria were then identified morphologically and molecularly. On the other hand, despite the different national surveys conducted on EPNs distribution around the world, habitat preferences remain inadequately known for entomopathogenic nematodes. Therefore, a comprehensive understanding of their distribution and the various biotic and abiotic factors influencing their presence is also a second object of our work. Beside a technological approach related to the biological properties of the nematodes and their symbiotics: valorisation of the entomotoxicity in biological control will be part of the third shutter of the thesis. In this framework, the sensibility of cedar pests, Cephalcia tannourinensis against entomopathogenic nematodes is exploited in vitro; different EPNs species were tested to study their life cycle inside Cephalcia larvae.

Bacteria of the genus Xenorhabdus live as the natural symbionts of the entomopathogenic nematodes of the family Steinernematidae. They produce a wide range of toxic secondary metabolites of different chemical structure and effect. The synthesis of particular products is specific for each strain of Xenorhabdus. The thesis is focused on evaluating the impact of bacterial metabolites on facultatively entomoparasitic nematodes Oscheius myriophila, the free-living nematode Caenorhabditis elegans and the phytophagous potato cyst nematode Globodera rostochiensis. Target species of nematodes were exposed to 37 strains of Xenorhabdus bacteria, isolated from various species of Steinernematidae. Testing the impact of bacterial metabolites on O. myriophila and C. elegans nematodes was performed both by direct cultivation of target species on solid medium with live bacterial cultures as well as in sterilized liquid bacteria cultures. The effect of toxic substances on G. rostochiensis was evaluated only in selected sterilized bacteria cultures.

O controle de Hypothenemus hampei é realizado basicamente com uso de inseticidas químicos, especialmente aqueles a base de endossulfan, cuja comercialização será proibida no Brasil a partir de julho de 2013. Diante disso, outras estratégias devem ser integradas buscando-se o controle desta praga com menor impacto ao ambiente. Nesse contexto, o fungo Beauveria bassiana destacase como um dos agentes de controle biológicos mais promissores. Neste estudo, buscou-se a associação da técnica de impregnação de tecido têxtil com B. bassiana associado à armadilha contendo a mistura de alcoóis metanol: etanol (1:1 v/v) como atraentes químicos para H. hampei, visando a auto-inoculação e disseminação do fungo pelo inseto, visando o seu controle. Inicialmente foi determinado em laboratório, a virulência de nove isolados de B. bassiana, através da pulverização de suspensões de 107 conídios.mL-1, diretamente sobre adultos de H. hampei. Todos os isolados apresentaram baixa virulência ao inseto, com mortalidade máxima de 38,5%. O isolado ESALQ-PL63 foi escolhido para dar continuidade aos estudos por possuir registro de comercialização. Posteriormente, sete tecidos têxteis sintéticos foram avaliados quanto à produção do isolado selecionado sobre sua superfície em duas condições de luz (fotofase de 0 e 12 horas). O tecido sintético Lã \"Sherpa\" foi selecionado por permitir uma maior produção de conídios nas duas condições testadas, atingindo até 5,44 × 108 conídios.cm-2. A exposição de H. hampei ao tecido com o fungo por apenas 5 segundos foi suficiente para causar 88,5% de mortalidade dos insetos. Em área de café sombreado foram conduzidos dois experimentos para avaliar uma armadilha de auto-inoculação contendo o patógeno produzido sobre o tecido. Foram usadas duas armadilhas controle, sendo uma armadilha de autoinoculação sem fungo e uma armadilha de eficiência reconhecida (modelo IAPAR). A eficiência de coleta da armadilha de auto-inoculação com o fungo foi menor do que a armadilha IAPAR, mas esta se mostrou eficiente na contaminação e mortalidade dos insetos pelo fungo. No primeiro experimento, a mortalidade confirmada pelo patógeno no último dia de avaliação (151 dias) foi de 64,7%. A viabilidade dos conídios passou de 98,3%, logo após a montagem das armadilhas, para 86,9% após 65 dias. Após160 dias a viabilidade reduziu para 44,0%. No segundo experimento, a última avaliação foi realizada após 40 dias, sendo nesta data observada mortalidade confirmada de 89,9% e viabilidade dos conídios de 78,1%. Em ambos os experimentos a concentração de conídios reduziu-se ao longo do tempo. O sistema de auto-inoculação apresentou resultados promissores, mas sendo necessárias alterações na armadilha para aumentar a captura de insetos e estudos epizootiológicos para avaliar a capacidade de disseminação da doença no campo pelos insetos que passaram pela armadilha. / The control of Hypothenemus hampei is accomplished primarily with the use of chemical insecticides, mainly endossulfan, whose commercialization will be prohibited in Brazil in July 2013. Therefore, other strategies must be integrated seeking control of this pest with less impact on the environment. In this context, the fungus Beauveria bassiana stands out as one of the most promising biological control agents. In this study, we associated the technique of fabric impregnated with B. bassiana with a trap containing a mixture methanol: ethanol (1:1 v/v) as an attractive chemical for H. hampei, aiming the self-inoculation and fungus dissemination by the insect, therefore their control. Initially, we determined in the laboratory, the virulence of nine isolates of B. bassiana using a spray suspension of 107 conidia.mL-1 directly on adults of H. hampei. All isolates showed low virulence to insects, with maximum mortality of 38.5%. The isolate ESALQ-PL63 was chosen to continue the studies for having a market register. Subsequently, seven synthetic fabrics were evaluated for the production of the selected isolate on its surface under two light conditions (photofase of 0 and 12 hours). The synthetic fabric Lã \"Sherpa\" was selected because it allows greater conidia production under the two conditions tested, reaching 5.44 × 108 conidia.cm-2. Exposure of H. hampei to the fabric impregnated with the fungus for only 5 seconds was enough to cause 88.5% of insect mortality. In an area of shaded coffee, two experiments were conducted to evaluate a self-inoculation trap containing the pathogen. We used two control traps without fungus: one self-inoculation trap without the fungus and a widely recognized efficient trap (IAPAR model). The collection efficiency of the self-inoculation trap with the fungus was lower than that of the IAPAR trap, however, it proved effective in contamination and insect mortality caused by the fungus. In the first experiment, mortality caused by the pathogen at the last evaluation day (151 days) was 64.7%. The viability of the conidia surpassed 98.3% after the assembly of the traps and 86.9%, after 65 days. After 160 days, the viability decreased to 44.0%. In the second experiment, the last evaluation was performed after 40 days, when we observed mortality of 89.9% and conidia viability of 78.1%. In both experiments, the conidia concentration reduced over time. The self-inoculation system showed promising results, but changes are needed to increase insect catches and further epizootiological studies to assess the possibility of disease spread in the field by insects that crossed the trap.

Os estudos da epizootiologia foram realizados em Pindorama-SP e em Itiquira-MT durante dois anos. No primeiro local, constatou-se em agosto de 1998 uma epizootia de Sporothrix sp. em ninfas do percevejo. Em Itiquira, quando a infestação atingiu 56 ninfas e 18 adultos/folha, verificou-se a ocorrência natural de Sporothrix sp., reduzindo significativamente a população. Na seleção de fungos entomopatogênicos, testou-se sessenta isolados obtidos de diferentes hospedeiros e localidades sobre ninfas do 4º ínstar. Os melhores isolados foram B. bassiana (619 e 1196) com 56 e 84% de mortalidade com 3 dias após a inoculação, M. anisopliae (1144 e 1104) ambos com 90%, S. insectorum (1229) com 92% e P. fumosoroseus (1200) com 100%. Trichoderma sp., após quatro dias da inoculação causou 78% de mortalidade. Na avaliação de campo, as concentrações superiores a 10 12 e 10 13 conídios/ha foram eficientes para controle do percevejo sendo que os fungos P. fumosoroseus (1200), B. bassiana (447) e M. anisopliae (1175 e 1144) foram eficientes para controle de L. heveae. Nos estudos de toxicidade, as formulações Decis e Nuvacron foram compatíveis com B. brongniartii (619). A maioria das combinações, com exceção da formulação de Decis foi compatível com V. lecanii (972). M. anisopliae (1144) foi muito sensível para todas as formulações. Nuvacron e Stron foram compatíveis com B. bassiana (1196), P. fumosoroseus (1200) e S. insectorum (1229). Marshal foi compatível com B. bassiana (1196) e S. insectorum (1229) e Dipterex foi compatível com P. fumosoroseus (1200). Avaliou-se dois métodos de produção e dois meios de cultura para os fungos B. bassiana (1196), M. anisopliae (1189), Sporothrix insectorum (1229), Paecilomyces fumosoroseus (1200) e Verticillium lecanii (1200). Os fungos P. fumosoroseus e B. bassiana foram os que mais produziram pelo método da caixa. No método da bandeja, B. bassiana foi o fungo mais produtivo. Entre os métodos de produção testados V. lecanii produziu mais no método de bandeja e P. fumosoroseus no método da caixa. Em observações em Microscópio Eletrônico de Varredura LEO 435VP (MEV) verificou-se que em geral, todos os hifomicetos apresentaram ciclos biológicos semelhantes sobre L. heveae, tendo um período de adesão, germinação e início da penetração de 24 h, crescimento vegetativo entre 48 e 72 h, e a extrusão e conidiogênse dos patógenos em 96 h. / Studies on epizootiology were carried out in Pindorama-SP and Itiquira-MT, Brazil during two years. In Pindorama, in August 1998 an epizootic of Sporothrix sp. was observed in lacebug nymphs. In Itiquira, when the infestation reached 56 nymphs and 18 adults/leaf, a natural occurrence of Sporothrix sp. was observed, reducing significantly the pest population. Entomopathogenic fungi for use in lacebug control were selected from sixty isolates obtained from different hosts and applied to 4th instar nymphs. The best isolates were Beauveria bassiana (619 and 1196) with 56 and 84% mortality 3 days after the inoculation, Metarhizium anisopliae (1144 and 1104) both with 90%, S. insectorum (1229) with 92%, and Paecilomyces fumosoroseus (1200) with 100%. Trichoderma sp. caused 78% of mortality four days after inoculation. In field evaluations, the concentrations above 10 12 conidia/ha were efficient for control of the lacebug and the fungal isolates P. fumosoroseus (1200), B. bassiana (447) and M. anisopliae (1175 and 1144) were efficient for control of L. heveae. In the toxicity studies, the formulations Decis and Nuvacron were compatible with B. brongniartii (619). Most of the combinations, except for Decis, were compatible with Verticillium lecanii (972). M. anisopliae (1144) was very sensitive to all the formulations. Nuvacron and Stron were compatible with B. bassiana (1196), P. fumosoroseus (1200) and S. insectorum (1229). Marshal was compatible with B. bassiana (1196) and S. insectorum (1229), and Dipterex was compatible with P. fumosoroseus (1200). Two production methods and two culture media were evaluated for production of the fungi B. bassiana (1196), M. anisopliae (1189), S. insectorum (1229), P. fumosoroseus (1200) and V. lecanii (1200). The fungi P. fumosoroseus and B. bassiana were the most productive using the box method. In the tray method, B. bassiana was the most productive fungus. Between the production methods tested, V. lecanii was more productive in the tray method and P. fumosoroseus in the box method. Observations in Scanning Electronic Microscope LEO 435VP (SEM) revealed that, in general, all of the hyphomycetes had similar biological cycles on L. heveae, with adhesion, germination and beginning of the penetration within 24 h, vegetative growth between 48 and 72 h, and the extrusion and conidiogenesis of the pathogens by 96 h.

Os isolados de nematóides entomopatogênicos provenientes do solo de Monte Negro (RO) receberam o prefixo LPP seguido de um número sequencial. As linhagens de bactérias simbiontes receberam o prefixo MN como referência ao local de isolamento. As culturas dos isolados de nematóides entomopatogênicos LPP foram estabelecidas em nosso laboratório, sendo necessário cultivar também o lepidóptero Galleria mellonella para as infeções visando manter os nematóides em cultura. Os isolados, tanto de nematóides como das bactérias, foram identificados através do sequenciamento de genes ribossômicos (ITS 1 e 2 além do 5.8S para os nematóides e 16S para as bactérias). Análises integradas, abrangendo morfologia (em colaboração com a Dra. Claudia Dolinski e Inês Machado) e análises filogenéticas permitiram a identificação dos nematóides. Essas análises morfomoleculares mostram que as linhagens não podem ser descritas como novas espécies, baseadas apenas no local de isolamento, mas como pertencentes à espécies já descritas e bem caracterizadas. As linhagens de nematóides foram identificadas como pertencentes a duas espécies do gênero Heterorhabditis (Rhabditida): Heterorhabditis baujardi (LPP5, 7, 8, 10 e 11) Heterorhabditis indica (LPP1, 2, 3, 4, 9). A bactéria entomopatogênica simbionte isolada a partir do nematoide Heterorhabditis baujardi linhagem LPP7 foi identificada como pertencente à espécie Photorhabdus luminescens subsp. luminescens linhagem MN7. Análises filogenéticas mostram que há similaridade entre o isolado bacteriano e as outras linhagens já descritas de Photorhabdus luminescens subsp. luminescens. Ao mesmo tempo em que eram isoladas as bactérias simbiontes, ocorreu o isolamento de outra espécie de bactéria forética dos nematóides entomopatogênicos, identificada após sequenciamento parcial do 16S, como pertencentes ao gênero Ochrobactrum. As linhagens isoladas foram denominadas OMN2, OMN3, OMN4. / The entomopathogenic nematodes isolates from Monte Negro (RO) soils received the prefix LPP followed by a sequential number. The strains of bacterial symbionts received the prefix MN as a reference to its isolation site. Cultures of LPP entomopathogenic nematodes were established in our laboratory and the need for suitable hosts were supplied by cultivating the lepidopteran Galleria mellonella. Nematode and bacterial isolates were identified through ribosomal genes (ITS 1 and 2, 5.8S for nematodes and 16S for bacteria) sequencing. Integrated analysis, including morphology (in collaboration with Dr. Claudia Dolinski and Inês Machado) and a phylogenetic approach allowed the identification of nematodes. These morpho-molecular analysis clearly showed that our strains can not be described as new species, solely based on the isolation site critheria. It should be described as belonging to previously described and well characterized species. The nematode strains were identified as belonging to two species of the genus Heterorhabditis (Rhabditida): Heterorhabditis baujardi (LPP5, 7, 8, 10 and 11) Heterorhabditis indica (LPP1, 2, 3, 4, 9). The entomopathogenic bacterial symbiont isolated from the nematode Heterorhabditis baujardi strain LPP7 was identified as belonging to the species Photorhabdus luminescens subsp. luminescens strain MN7. Phylogenetic analysis showed similarities between isolated bacterial strains and other previously described Photorhabdus luminescens subsp. luminescens strains. In paralel work, along with bacterial symbionts isolation, other entomopathogenic nematodes foretic bacteria were isolated and identified after partial sequencing of 16S, as belonging to the genus Ochrobactrum. The isolated strains were named OMN2, OMN3, OMN4.