The prevailing use of chemical pesticides has generated several problems including insecticide resistance, outbreak of secondary pests, safety risks for humans and domestic animals, contamination of ground water and decrease in biodiversity among other environmental concerns (Webster, 1982). These problems and the nonsustainability of control programs based mainly on conventional insecticides have stimulated increased interest in the development and implementation of costeffective, environmentally safe alternatives to chemical pesticides for insect pest control. One of the most promising strategies to help minimize dependence on chemical pesticides has been the recent application of entomopathogenic nematodes (EPNs) as biocontrol agents. EPNs in the families Steinernematidae and Heterorhabdidae have been shown to have considerable potential as biological control agents. As a natural process, biological control has the potential to play an important role in the suppression of field crop pests in agriculture. EPNs as biocontrol agents have the following advantages: high virulence, safety to non target organisms, ability to search for hosts, high efficacy in favourable habitats, high reproductive potential, ease of mass production, ease of application (Ferron & Deguine, 1996). To isolate the EPNs in South African soil, 200 soil samples were randomly collected from 5 locations in the agricultural research council (ARC) Pretoria, Gauteng province in April 2006; and 5 locations in Brits, North West province in March, 2006. At the different collection sites, soil samples were obtained from soils associated with various types of vegetation. The nematodes were collected from sandy soil by the insect-baiting technique and maintained successfully in vivo for 12 months on Galleria mellonella (G. mellonella), 4 months on Tenebrio molitor (T.molitor); 2 months Pupae and in vitro (lipid agar) for 2 weeks in the laboratory. Out of a total of 200 soil samples that were baited, 2 were found to be positive for EPNs.EPNs. IV In addition to completing Koch’s postulates, the colour of cadavers infected by the putative EPNs were also used as a diagnostic characteristic for categorizing the nematode isolates. Characterization and identification of the EPN isolates were based on morphological characters, as well as on a molecular marker (18S rDNA). On the basis of the morphological and molecular data that was obtained both of the EPNs isolates were placed in the family Heterorhabdidae: Heterorhabditis bacteriophora (H. bacteriophora) and Heterorhabditis zealandica (H. zealandica). Also from the phylogenetic trees generated from the 18S rDNA sequence, the indigenous putative H. bacteriophora was shown to be closely related to H. bacteriophora (accession number EF690469) and indigenous putative H. zealandica to H. zealandica (accession number AY321481). The two EPNs were found associated with Gram negative rod-shaped bacteria. The bacterial symbionts of the two isolates were isolated and a region of the 16S rDNA gene was sequenced. National Center for Biotechnology Information (NCBI-BLAST) results of the 16S rDNA sequence obtained showed the endosybiotic bacteria to be Photorhabdus luminescens laumondii (P. laumondii) (H. bacteriophora) and Photorhabdus sp (H. zealandica). Results of the tree showed that isolates from H. bacteriophora appeared to be closely related to P. luminescens subsp laumondii strain TT01 Ay 278646. The isolates from H. zealandica appeared to be most closely related to Photorhabdus sp Accession number: Q 614 Ay 216500). Bioassays were used to determine the infectivity of the two EPNs. In this experiment different infective juvenile (IJs) concentrations (5, 10, 25, 50, 100,200 400 and 500) of the two EPNs were applied per G. mellonella; T. molitor larva and pupae. The bioassay was carried out in two parts. In the first part, mortality data was collected for H. bacteriophora and H. zealandica. The results showed that the degree of susceptibility of G. mellonella, T. molitor larvae and pupae to each nematode species was different. When 24 h post-exposure mortality data for larvae exposed to the IJs of H. bacteriophora and H. zealandica were analyzed, ANOVA showed no differences V in mortality between insects exposed to different H. bacteriophora IJ doses (Fig: 8.1 ABC). However, there were significant differences in mortality between insects exposed to different IJ doses of H. zealandica such as 5 and 500 IJs/insect (Fig: 8.2 ABC) Therefore, no differences were noted when mortality data was compared between IJ doses at both 72 h and 96 h following IJ application to the insects. The highest susceptibility was observed with G. mellonella followed by T. molitor pupae and then T. molitor larvae. According to Caroli et al., (1996), the total mortality of insect such as G. mellonella and other lepidopterans, was reached within 24-72 h of exposure to nematodes at concentrations such as those tested here. In this study similar results were observed with high concentration of nematodes (100, 200 and 500). In the second part of the dose response bioassay, the number of progeny IJs emerging from EPN-infected cadavers was determined for all two EPNs. The results indicate that IJ progeny production differed among the three insect hosts used, the IJ doses they were exposed to, as well as the EPN species (Figs 8.3 & 8.4). The highest number of emerged IJs of H. zealandica was produced by G. mellonella (mean ± SEM: 220500 ± 133933 IJs), followed by T. molitor larvae (mean ± SEM: 152133 ± 45466 IJs) and the lowest then T. molitor pupae (mean ± SEM: 103366 ± 56933 IJs).

Entomopathogenic nematodes (EPN) (Heterorhabditis bacteriophora and H. megidis) and entomopathogenic fungi (EPF) (Metarhizium anisopliae and Beauveria bassiana) were evaluated for efficacy against masked chafer white grub, Cyclocephala spp., under laboratory and greenhouse conditions, as well as their efficacy against various grub stages in the field.  Under both laboratory and greenhouse conditions, additive interactions were found between EPN and EPF in their combined application against Cyclocephala spp., except a few observations that showed antagonism or synergism.  Significantly greater control occurred from the combination of a nematode and a fungus compared with a fungus alone, but not compared with a nematode alone.  The combined effect did not differ significantly for nematode and fungi applied simultaneously or at different times.  EPF had no significant impact on EPN infection and production of infective juveniles (IJs) in grub carcasses.  Nematodes alone or in combination with fungi were comparable to the insecticide Merit 75 WP (imidacloprid) against 3rd instar Cyclocephala spp in the greenhouse. Efficacy of EPF and EPN varied dramatically between field sites and conditions; EPN and EPF applied alone or in combination were less effective than Merit 75 WP in >50% field trials, but some EPN + EPF treatments were more effective than the insecticide in reducing grub numbers.  EPN and EPF showed better potential than insecticides for providing extended control of white grubs in the subsequent generation.  In addition, the sub-lethal effects of EPF on southern masked chafer, C. lurida, were investigated.  Neither M. anisopliae nor B. bassiana had a sub-lethal effect on grub weight gain, adult longevity, oviposition, pupation and eclosion.  Finally, interaction between H. bacteriophora and M. anisopliae was examined to determine the potential of the nematode in improving fungal distribution in soil.  H. bacteriophora enhanced fungal distribution in sandy loam soil without grass thatch, but not in sandy soil with thatch.  In both soil types, soil depths significantly affected nematode and fungal distribution. In water profile, M. anisopliae conidia germinated hyphae that attached to sheath of H. bacteriophora IJs, which molted to detach from the fungus.  IJs mortality and virulence were not affected by the presence of M. anisopliae. / Ph. D.

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.

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-12Bitstream added on 2014-06-13T21:06:59Z : No. of bitstreams: 1 tavares_fm_dr_botfca.pdf: 538754 bytes, checksum: da1ec50d10839c5269a5a1390715eb52 (MD5) / Fundação de Apoio à Pesquisa Agrícola (FUNDAG) / Nematóides entomopatogênicos (NEPs) dos gêneros Heterorhabditis e Steinernema vem sendo utilizados para o controle de mosca-dos-fungos em diversos países. Esse estudo teve por objetivo avaliar diferentes isolados de NEPs, contra larvas de Bradysia mabiusi, em condições de laboratório e casa de vegetação. Nos testes de seleção de isolados, o nematóide Heterorhabditis indica apresentou melhor desempenho comparado a todos os demais isolados, proporcionando mortalidade do inseto de até 98%. Para o estudo em casa de vegetação, avaliou-se o efeito dos nematóides entomopatogênicos Heterorhabditis indica e Steinernema feltiae, nas dosagens de 10, 50 e 100 JI/cm2, em cultivo protegido de Chrysanthemum sp. Foram realizados 3 experimentos. Houve diferença significativa entre os tratamentos somente no terceiro teste, entretanto, nos três testes os nematóides demonstraram uma tendência de redução na população do inseto, em função do aumento das dosagens, o que sugere o efeito dos agentes no controle do inseto. Da mesma forma, em todos os testes, o nematóide H. indica apresentou maior redução na população da mosca-dos-fungos que o S. feltiae, confirmando sua maior eficiência com níveis de controle variando de 6 a 67,5% no primeiro ensaio, de 17 a 77,5% no segundo, e de 62,5 a 78,5% no terceiro. Os resultados revelam que H. indica apresenta potencial de uso para o controle da mosca-dos-fungos. / Entomopathogenic nematodes (EPNs) of the genus Heterorhabditis and Steinernema have been use against the fungus gnat in several countries. This study aimed to evaluate different strains of EPNs against larvae of Bradysia mabiusi, in laboratory and greenhouse conditions. At the screening of EPNs, the nematode H. indica showed better performance compared to the other nematodes, providing up to 98% mortality of the insect. To the study in greenhouse, evaluated the effects of the entomopathogenic nematodes Heterorhabditis indica e Steinernema feltiae, at dosage of 10, 50 and 100 IJ/cm2 in commercial growth of Chrysanthemum sp., in greenhouse conditions. Three experiments were accomplished. There were significative difference among the treatments only for the third experiment, mean while, in the three tests, the nematodes showed a tendency of reduction on the insect population according to the dosage increase, which suggest the effect of the agents on the insect control. In addition, in all the tests, the nematode H. indica showed higher reduction on the insect population compared to the S. feltiae, confirming its higher efficiency with control levels ranging from 6 to 67,5% in the first test, from 17 to 77,5% in the second, and from 62,5 to 78,5% in the third. The data show that H. indica has potential use for the controlling of the fungus gnat.

Orientador: Antonio Batista Filho / Banca: Carlos Frederico Wilcken / Banca: Silvia Renata Siciliano Wilcken / Banca: Laerte Antonio Machado / Banca: Inajá Marchizeli Wenzel / Resumo: Nematóides entomopatogênicos (NEPs) dos gêneros Heterorhabditis e Steinernema vem sendo utilizados para o controle de mosca-dos-fungos em diversos países. Esse estudo teve por objetivo avaliar diferentes isolados de NEPs, contra larvas de Bradysia mabiusi, em condições de laboratório e casa de vegetação. Nos testes de seleção de isolados, o nematóide Heterorhabditis indica apresentou melhor desempenho comparado a todos os demais isolados, proporcionando mortalidade do inseto de até 98%. Para o estudo em casa de vegetação, avaliou-se o efeito dos nematóides entomopatogênicos Heterorhabditis indica e Steinernema feltiae, nas dosagens de 10, 50 e 100 JI/cm2, em cultivo protegido de Chrysanthemum sp. Foram realizados 3 experimentos. Houve diferença significativa entre os tratamentos somente no terceiro teste, entretanto, nos três testes os nematóides demonstraram uma tendência de redução na população do inseto, em função do aumento das dosagens, o que sugere o efeito dos agentes no controle do inseto. Da mesma forma, em todos os testes, o nematóide H. indica apresentou maior redução na população da mosca-dos-fungos que o S. feltiae, confirmando sua maior eficiência com níveis de controle variando de 6 a 67,5% no primeiro ensaio, de 17 a 77,5% no segundo, e de 62,5 a 78,5% no terceiro. Os resultados revelam que H. indica apresenta potencial de uso para o controle da mosca-dos-fungos. / Abstract: Entomopathogenic nematodes (EPNs) of the genus Heterorhabditis and Steinernema have been use against the fungus gnat in several countries. This study aimed to evaluate different strains of EPNs against larvae of Bradysia mabiusi, in laboratory and greenhouse conditions. At the screening of EPNs, the nematode H. indica showed better performance compared to the other nematodes, providing up to 98% mortality of the insect. To the study in greenhouse, evaluated the effects of the entomopathogenic nematodes Heterorhabditis indica e Steinernema feltiae, at dosage of 10, 50 and 100 IJ/cm2 in commercial growth of Chrysanthemum sp., in greenhouse conditions. Three experiments were accomplished. There were significative difference among the treatments only for the third experiment, mean while, in the three tests, the nematodes showed a tendency of reduction on the insect population according to the dosage increase, which suggest the effect of the agents on the insect control. In addition, in all the tests, the nematode H. indica showed higher reduction on the insect population compared to the S. feltiae, confirming its higher efficiency with control levels ranging from 6 to 67,5% in the first test, from 17 to 77,5% in the second, and from 62,5 to 78,5% in the third. The data show that H. indica has potential use for the controlling of the fungus gnat. / Doutor

In this thesis, the effect of infection elicited by entomopathogenic nematodes (EPNs) Steinernema carpocapsae on Pyrrhocoris apterus and Drosophila melanogaster models were evaluated, and a role of adipokinetic hormones (AKHs) during the infection was characterized. These were monitored by determination of mortality, and various biochemical and physiological characteristics such as AKH levels both in the central nervous system (CNS) and in hemolymph, AKH gene expression in CNS, level of anti-oxidative stress markers, general metabolism and level of nutrients in normal and genetically modified insects. At P. apterus the mortality tests revealed that application of AKH increases the efficacy of EPN treatment. This result was confirmed using the firebugs with AKH receptor deficiency. Further, the increase of AKH expression and AKH levels in CNS and hemolymph seemed to be coordinated after the nematode treatment. At the D. melanogaster model also, the effect of adenosine into the above-mentioned characteristics was included. For this, mutants in AKH (AHK1), adenosine receptor (AdoR1) genes, and in both these genes together (AHK1 AdoR1 double mutant) were employed. Altogether, the results confirmed the involvement of AKH, and partially also adenosine into the antistress defense reactions elicited by the nematobacterial infection. Finally, the last part of the study was focused on examination of the vitellogenin (Vg) role in the defense reaction in firebug body P. apterus affected by two entomopathogenic organisms, the nematode S. carpocapsae and the fungus Isaria fumosorosea. The results revealed that Vg proteins play an important role in the defense against both types of the infections and are also able to kill entomopathogenic bacteria Xenorhabdus nematophila, that are symbionts of S. carpocapsae and that increase toxicity of this nematode.

Orientador: Antonio Batista Filho / Banca: Carlos Frederico Wilcken / Banca: Luis Carrigos Leite / Resumo: Buscando-se novas alternativas para o controle do bicudo da cana-de-açúcar, Sphenophorus levis Vaurie, 1978, foi testado em laboratório, em estufa e no campo, a ação dos nematóides entomopatogênicos Heterorhabditis indica Poinar, 1992 e Steinernema sp., e o efeito da associação desses agentes com subdosagens dos inseticidas químicos fipronil (Regent 800WG), thiamethoxam (Actara 250WG) e imidacloprid (Confidor 700WG), no controle de larvas e adultos do inseto. Inicialmente foram avaliadas diferentes dosagens dos nematóides contra larvas do inseto. O nematóide Steinernema sp. apresentou-se mais eficiente no controle de larvas, podendo ser utilizada na menor dosagem avaliada (2,4 JI/cm2), já que ocasionou 69% de mortalidade corrigida do inseto em estufa, não diferindo significamente das maiores dosagens. Esse nematóide apresentou-se mais eficiente também contra adultos do inseto, em teste de laboratório, proporcionando níveis de mortalidade do inseto igual ou superior a 70% nas dosagens de 2,4, 12 e 60 JI/cm2, quando avaliado em associação com o inseticida thiamethoxam na subdosagem de 250 g p.c./ha. No teste de campo foi possível verificar nas parcelas tratadas com Steinernema sp., na dosagem de l,0xl08 JI/ha, associado ao thiamethoxam na dose de 500 g p.c./ha, ganho superior a 28 toneladas de cana crua quando 2 comparada a testemunha. Quanto ao efeito de fipronil e thiamethoxam sobre a viabilidade dos nematóides em laboratório, esses produtos químicos apresentaram-se compatíveis a esses agentes, não afetando a viabilidade dos juvenis infectivos. O nematóide Steinernema sp. é uma alternativa viável para o controle de S. levis, podendo ser usado isoladamente ou associado com subdosagem de thiamethoxam. Palavras-chave: controle microbiano, controle biológico, bicudo da cana de açúcar, inseticida químico, nematóide entomopatogênico. / Abstract: With the purpose to find new alternatives to control the sugarcane billbug Sphenophorus levis, this study aimed to evaluate in laboratory, green house and field conditions, the effect of the nematodes Heterorhabditis indica and Steinernema sp., in combinations with sub-dosages of the chemical insecticides fipronil (Regent 800WG), thiametoxam (Actara 250WG) and imidacloprid (Confidor700WG), against larva and adults of this pest. Firstly, several dosages of the nematodes were evaluated against larvae. The nematode Steinernema sp. was the most efficient against larva, with its dosage of 2,4 IJ/cm2 showing to be the best one for the insect control since it provide 69% larva mortality, not significantly differing from the other dosages. This nematode was also the most efficient against adults in laboratory test, providing insect mortality levels equal or higher than 70% when evaluated at the dosages of 2,4; 12 and 60 IJ/ha, in combination with thiametoxam at the sub-dosage 250 g c.p./ha. The insecticides fipronil and thiamethoxam did not affected the viability of the infect juveniles. For the field test, the combination of Steinernema sp. (108 IJ/ha) plus thiamethoxam (500 g c.p./ha) provided an increasing to the sugarcane production higher than 28 ton/ha. The nematode Steinernema sp. seems to be a viable alternative for the 4 controlling of S. levis, providing expressive increasing to the sugarcane production when used alone or in combination with thiamethoxam. / Mestre

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-11-01Bitstream added on 2014-06-13T18:45:54Z : No. of bitstreams: 1 wenzel_im_dr_botfca.pdf: 544393 bytes, checksum: f67161337e2bde1fdbfde14613c06462 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Secretaria Agricultura / Este trabalho teve como objetivo avaliar a patogenicidade do isolado JAB 02 de Lecanicillium lecanii (Zimm.) Zare & Gams ao ácaro rajado Tetranychus urticae e a sua compatibilidade com agrotóxicos, nematóides entomopatogênicos e ácaros predadores. Os conídios do fungo, produzidos em arroz, foram avaliados nas concentrações de 5 x 106, 1 x 107, 5 x 107, 1 x 108 e 5 x 108 conídios/mL. A partir das porcentagens de mortalidade de T. urticae, que variaram entre 49 e 90% no décimo dia de avaliação, foi determinada a CL50 de 5,25 x 106 con./mL. A compatibilidade em laboratório foi verificada misturando-se os agrotóxicos em meio de cultura BDA e os parâmetros utilizados para a avaliação foram crescimento vegetativo, esporulação e viabilidade do entomopatógeno. Em condições de estufa, as suspensões dos produtos químicos e, posteriormente, a do fungo foram pulverizadas em plantas de crisântemo. Após a aplicação, quatro folhas foram coletadas e lavadas obtendo-se uma suspensão que foi plaqueada em BDA, sendo então avaliado o crescimento das colônias do fungo. Verificou-se, em laboratório, que a maioria dos inseticidas e os acaricidas foram compatíveis ao fungo, com exceção do inseticida Thiodan® classificado como muito tóxico. Todos os fungicidas testados foram classificados como tóxico e muito tóxico. Em condições de estufa, no tratamento com o fungicida Rovral® foi observado um número de colônias fúngicas formadas compatível com a testemunha. Essa compatibilidade repetiu-se para os produtos Alto 100® e Thiodan® em alguns dos tempos avaliados. No teste de patogenicidade, os conídios produzidos em meio de cultura com inseticidas e acaricidas, a partir da compatibilidade em laboratório, não tiveram sua viabilidade afetada e foram patogênicos ao ácaro rajado Tetranychus urticae. Além disso, foram realizados... / The objective of this study was to evaluate the pathogenicity of Lecanicillium lecanii (Zare & Gams), isolate JAB 02, to the two-spotted mite Tetranychus urticae (Koch), its compatibility with agrochemical products and its selectivity to entomopathogenic nematodes and predatory mites. The pathogenicity of the fungus produced in rice to T. urticae was evaluated at the concentrations of 5 x 106, 1 x 107, 5 x107, 1 x 108 and 5 x 108 conidia/mL. The mortality rates of T. urticae ranged from 49 and 90 % in the tenth evaluation day and the LC50 was 5,25 x 106 conidia/mL. The compatibility in laboratory was verified by adding chemical products to the PDA media and the evaluated parameters were the vegetative growth, sporulation and viability of the entomopathogen. In greenhouse, L. lecanii were sprayed on chrysanthemum plants after the pulverization of the chemical products; subsequently, four leaves of each treatment were collected and washed to obtain a suspension that were transferred to PDA medium to evaluate the growth of the fungus colonies in laboratory conditions. The acaricides and the most of insecticides were compatible to the isolate JAB 02, except the insecticide Thiodan®, which was classified as very toxic. All the evaluated fungicides were classified as toxic or very toxic. In greenhouse assays, the fungicide Rovral® produced a number of grown colonies similar to those of the control and this compatibility was also showed by the products Alto 100® and Thiodan® in some of the evaluated periods. In another pathogenicity test, when L. lecanii were grown in culture media containing the insecticides, the conidia viability were not affected and the fungus were pathogenic to T. urticae, with mortality rates superior to the control in some cases. Also, compatibility bioassays involving L. lecanii at 5 x 106, 1 x 107 and 5 x 107 con./mL and the entomopathogenic nematodes Steinernema sp. (CB n6) and Heterorhabditis indica (CB n5) were conducted.

Orientador: Antonio Batista Filho / Banca: Carlos Frederico Wilcken / Banca: Antonio Carlos Monteiro / Banca: Jose Eduardo Marcondes Almeida / Banca: Luis Garrigos Leite / Resumo: Este trabalho teve como objetivo avaliar a patogenicidade do isolado JAB 02 de Lecanicillium lecanii (Zimm.) Zare & Gams ao ácaro rajado Tetranychus urticae e a sua compatibilidade com agrotóxicos, nematóides entomopatogênicos e ácaros predadores. Os conídios do fungo, produzidos em arroz, foram avaliados nas concentrações de 5 x 106, 1 x 107, 5 x 107, 1 x 108 e 5 x 108 conídios/mL. A partir das porcentagens de mortalidade de T. urticae, que variaram entre 49 e 90% no décimo dia de avaliação, foi determinada a CL50 de 5,25 x 106 con./mL. A compatibilidade em laboratório foi verificada misturando-se os agrotóxicos em meio de cultura BDA e os parâmetros utilizados para a avaliação foram crescimento vegetativo, esporulação e viabilidade do entomopatógeno. Em condições de estufa, as suspensões dos produtos químicos e, posteriormente, a do fungo foram pulverizadas em plantas de crisântemo. Após a aplicação, quatro folhas foram coletadas e lavadas obtendo-se uma suspensão que foi plaqueada em BDA, sendo então avaliado o crescimento das colônias do fungo. Verificou-se, em laboratório, que a maioria dos inseticidas e os acaricidas foram compatíveis ao fungo, com exceção do inseticida Thiodan® classificado como muito tóxico. Todos os fungicidas testados foram classificados como tóxico e muito tóxico. Em condições de estufa, no tratamento com o fungicida Rovral® foi observado um número de colônias fúngicas formadas compatível com a testemunha. Essa compatibilidade repetiu-se para os produtos Alto 100® e Thiodan® em alguns dos tempos avaliados. No teste de patogenicidade, os conídios produzidos em meio de cultura com inseticidas e acaricidas, a partir da compatibilidade em laboratório, não tiveram sua viabilidade afetada e foram patogênicos ao ácaro rajado Tetranychus urticae. Além disso, foram realizados...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the pathogenicity of Lecanicillium lecanii (Zare & Gams), isolate JAB 02, to the two-spotted mite Tetranychus urticae (Koch), its compatibility with agrochemical products and its selectivity to entomopathogenic nematodes and predatory mites. The pathogenicity of the fungus produced in rice to T. urticae was evaluated at the concentrations of 5 x 106, 1 x 107, 5 x107, 1 x 108 and 5 x 108 conidia/mL. The mortality rates of T. urticae ranged from 49 and 90 % in the tenth evaluation day and the LC50 was 5,25 x 106 conidia/mL. The compatibility in laboratory was verified by adding chemical products to the PDA media and the evaluated parameters were the vegetative growth, sporulation and viability of the entomopathogen. In greenhouse, L. lecanii were sprayed on chrysanthemum plants after the pulverization of the chemical products; subsequently, four leaves of each treatment were collected and washed to obtain a suspension that were transferred to PDA medium to evaluate the growth of the fungus colonies in laboratory conditions. The acaricides and the most of insecticides were compatible to the isolate JAB 02, except the insecticide Thiodan®, which was classified as very toxic. All the evaluated fungicides were classified as toxic or very toxic. In greenhouse assays, the fungicide Rovral® produced a number of grown colonies similar to those of the control and this compatibility was also showed by the products Alto 100® and Thiodan® in some of the evaluated periods. In another pathogenicity test, when L. lecanii were grown in culture media containing the insecticides, the conidia viability were not affected and the fungus were pathogenic to T. urticae, with mortality rates superior to the control in some cases. Also, compatibility bioassays involving L. lecanii at 5 x 106, 1 x 107 and 5 x 107 con./mL and the entomopathogenic nematodes Steinernema sp. (CB n6) and Heterorhabditis indica (CB n5) were conducted. / Doutor

Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-02Bitstream added on 2014-06-13T20:37:49Z : No. of bitstreams: 1 tavares_fm_me_botfca.pdf: 1326049 bytes, checksum: 86d24916183c6adc243be54cbe86a695 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundação de Apoio à Pesquisa Agrícola (FUNDAG) / Secretaria Agricultura / Buscando-se novas alternativas para o controle do bicudo da cana-de-açúcar, Sphenophorus levis Vaurie, 1978, foi testado em laboratório, em estufa e no campo, a ação dos nematóides entomopatogênicos Heterorhabditis indica Poinar, 1992 e Steinernema sp., e o efeito da associação desses agentes com subdosagens dos inseticidas químicos fipronil (Regent 800WG), thiamethoxam (Actara 250WG) e imidacloprid (Confidor 700WG), no controle de larvas e adultos do inseto. Inicialmente foram avaliadas diferentes dosagens dos nematóides contra larvas do inseto. O nematóide Steinernema sp. apresentou-se mais eficiente no controle de larvas, podendo ser utilizada na menor dosagem avaliada (2,4 JI/cm2), já que ocasionou 69% de mortalidade corrigida do inseto em estufa, não diferindo significamente das maiores dosagens. Esse nematóide apresentou-se mais eficiente também contra adultos do inseto, em teste de laboratório, proporcionando níveis de mortalidade do inseto igual ou superior a 70% nas dosagens de 2,4, 12 e 60 JI/cm2, quando avaliado em associação com o inseticida thiamethoxam na subdosagem de 250 g p.c./ha. No teste de campo foi possível verificar nas parcelas tratadas com Steinernema sp., na dosagem de l,0xl08 JI/ha, associado ao thiamethoxam na dose de 500 g p.c./ha, ganho superior a 28 toneladas de cana crua quando 2 comparada a testemunha. Quanto ao efeito de fipronil e thiamethoxam sobre a viabilidade dos nematóides em laboratório, esses produtos químicos apresentaram-se compatíveis a esses agentes, não afetando a viabilidade dos juvenis infectivos. O nematóide Steinernema sp. é uma alternativa viável para o controle de S. levis, podendo ser usado isoladamente ou associado com subdosagem de thiamethoxam. Palavras-chave: controle microbiano, controle biológico, bicudo da cana de açúcar, inseticida químico, nematóide entomopatogênico. / With the purpose to find new alternatives to control the sugarcane billbug Sphenophorus levis, this study aimed to evaluate in laboratory, green house and field conditions, the effect of the nematodes Heterorhabditis indica and Steinernema sp., in combinations with sub-dosages of the chemical insecticides fipronil (Regent 800WG), thiametoxam (Actara 250WG) and imidacloprid (Confidor700WG), against larva and adults of this pest. Firstly, several dosages of the nematodes were evaluated against larvae. The nematode Steinernema sp. was the most efficient against larva, with its dosage of 2,4 IJ/cm2 showing to be the best one for the insect control since it provide 69% larva mortality, not significantly differing from the other dosages. This nematode was also the most efficient against adults in laboratory test, providing insect mortality levels equal or higher than 70% when evaluated at the dosages of 2,4; 12 and 60 IJ/ha, in combination with thiametoxam at the sub-dosage 250 g c.p./ha. The insecticides fipronil and thiamethoxam did not affected the viability of the infect juveniles. For the field test, the combination of Steinernema sp. (108 IJ/ha) plus thiamethoxam (500 g c.p./ha) provided an increasing to the sugarcane production higher than 28 ton/ha. The nematode Steinernema sp. seems to be a viable alternative for the 4 controlling of S. levis, providing expressive increasing to the sugarcane production when used alone or in combination with thiamethoxam.