The potential synergism between chosen species of entomopathogenic nematodes (Steinernema arenarium, Steinernema feltiae) and entomopathogenic fungi (Beauveria bassiana, Isaria fumosorosea, Lecanicillium lecanii) is investigated in this study. It is theoretically possible to expect increasing of their efficiency at the collective introduction into environment. Creating of uniform laboratory method which will be possible to use at the research of this interaction system is a part of this study.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-07T19:23:55Z No. of bitstreams: 1 eldersimoesdepaulabatista.pdf: 1322510 bytes, checksum: 06eb43efa3e850761ef16081f42484f6 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-11T11:34:00Z (GMT) No. of bitstreams: 1 eldersimoesdepaulabatista.pdf: 1322510 bytes, checksum: 06eb43efa3e850761ef16081f42484f6 (MD5) / Made available in DSpace on 2017-04-11T11:34:00Z (GMT). No. of bitstreams: 1 eldersimoesdepaulabatista.pdf: 1322510 bytes, checksum: 06eb43efa3e850761ef16081f42484f6 (MD5) Previous issue date: 2010-02-25 / As cigarrinhas do gênero Mahanarva causam danos às culturas da família Poaceae, como braquiária, capim-elefante e cana-de-açúcar, em função da sucção de seiva das raízes (fase imatura) e folhas (fase adulta). Com a crescente demanda de redução do uso de inseticidas químicos para o controle de insetos praga, surge a necessidade do desenvolvimento de novos métodos de controle. Nesse cenário, o controle biológico recebe destaque e no caso das cigarrinhas, os nematóides entomopatogênicos (NEPs) são potenciais agentes de controle, pois exploram a superfície do solo, mesmo ambiente da praga enquanto esta se alimenta nas raízes. Portanto, com o objetivo de conhecer a ação de NEPs e selecionar isolados desses patógenos contra a cigarrinha-das-pastagens, Mahanarva spectabilis, e a cigarrinha-dasraízes, Mahanarva fimbriolata, experimentos foram desenvolvidos com diversos isolados de NEPs sobre diferentes fases de desenvolvimento do inseto. Em dois experimentos ninfas de M. spectabilis e M. fimbriolata foram expostos aos patógenos, em condições de laboratório e casa-de-vegetação e a partir daí foram selecionados os isolados mais patogênicos para cada espécie de praga. Em seguida, avaliou-se a eficiência de quatro métodos de aplicação de NEPs (pipetador, pulverização sobre ninfas com e sem espuma e inseto-cadáver) sobre a cigarrinha-das-pastagens, em casa-de-vegetação. Por último, avaliou-se a eficiência de uma espécie de NEP sobre ovos e adultos da cigarrinha-das-pastagens. Todos os isolados testados são patogênicos às ninfas das cigarrinhas causando mortalidade de 40 a 92% e 38 a 90% em condições de laboratório e 14 a 71% e 48 a 72% em casa-de-vegetação, para M. spectabilis e M. fimbriolata, respectivamente. Os métodos de aplicação mais eficientes foram o de pulverização sobre ninfas com espuma e inseto-cadáver, não havendo diferença na eficiência provocada pelas concentrações e isolados utilizados. Observou-se que não ocorre infecção de NEPs sobre ovos de cigarrinha-das-pastagens. Os adultos da cigarrinha não foram infectados pelos NEPs, e não houve redução no número de ovos em função da presença do patógeno. Dos isolados testados, os mais eficientes foram Steinernema riobravis, S.feltiae e Heterorhabditis amazonensis RSC1 para M. spectabilis e S. feltiae, S. riobravis, H. baujardi LPP7 e S. carpocapsae para M. fimbriolata. Conclui-se portanto que nematóides entomopatogênicos podem ser utilizados no controle de cigarrinhas do gênero Mahanarva, sendo incluídos em programas de manejo integrado, devendo ser empregados contra as ninfas, pulverizados sobre a espuma ou através de inseto-cadáver. / The spittlebugs belonging to the genus Mahanarva cause damage to the Poaceae family cultures, like signal grass, elephant grass and sugar cane, by the sap feeding on roots (immature phase) and leaves (adult phase). With the increase demand to reduce use of chemical insecticides for the insect pests’ control, comes the need to develop new control methods. In this scenario, the biological control gains highlight and in the spittlebugs case, the entomopathogenic nematodes (EPNs) are potential control agents, exploring the soil surface, the same pest environment while it feeds on roots. Thus, with the aim to know the EPNs’ action and to screening strains of these pathogens against the leaf spittlebug, Mahanarva spectabilis, and the root spittlebug, Mahanarva fimbriolata, experiments were conducted with various EPNs strains over different insect development phases. In two experiments M. spectabilis and M. fimbriolata nymphs were exposed to the pathogens, in laboratory and greenhouse conditions, and then were screened the more pathogenic strains to each pest species. Following, were evaluated the efficiency of four application methods of EPNs (pippeting, spray over nymphs with and without froth and infected host cadaver) over the leaf spittlebug, at greenhouse. At last, were evaluated the efficiency of an EPN species over eggs and adults of the leaf spittlebug. All tested strains are pathogenic to the spittlebugs nymphs causing 40-92% and 38-90% mortality at laboratory and 14-71% and 48-72% at greenhouse, to M. spectabilis and M. fimbriolata, respectively. The more efficient application methods were spray over nymphs with froth and infected host cadaver, with no difference because the concentrations and strains. Observations showed that don’t occurs eggs infection by EPNs in leaf spittlebugs. The leaf spittlebug adults not were infected by the EPNs, and no egg reduction were observed. The most pathogenic EPNs strains were Steinernema riobravis, S.feltiae and Heterorhabditis amazonensis RSC1 to M. spectabilis and S. feltiae, S. riobravis, H. baujardi LPP7 and S. carpocapsae to M. fimbriolata. Thus I conclude that entomopathogenic nematodes can be utilized to control Mahanarva genus spittlebugs, and can be inserted in integrated pest management programs, should to be applied over nymphs exclusively, sprayed over the froth or through infected host cadevers.

The primary objectives of this research are to determine effective biological and alternative control strategies of insect and disease pests in order to reduce harsh chemical use during greenhouse crop production and transport s. This research includes two separate studies: 1) testing the practical viability of rearing and storing four species of entomopathogenic nematode (EPN), Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica; and, 2) the efficacy of UV-C radiation applied, pre-transport, as a preventative disease control strategy against Botrytis cinerea. A study was conducted testing EPN infectious juvenile (IJ) rearing production counts and IJ viability after a six-day storage period. When all four species are compared, S. feltiae had a greater number of infectious juveniles emerge from the wax moth cadavers and S. carpocasae had the least. All four species survived the six day storage period but EPN infectious juvenile counts were significantly different among species. Our second study tested the efficacy of UV-C radiation as an alternative control to traditional fungicides to deactivate B. cinerea in vitro and to determine plant tolerance to UV-C. The crops tested were poinsettia (Euphorbia pulcherrima) and primula (Primula vulgaris). All the UV-C doses, 1.0, 2.8, 3.7 or 4 W/m2, significantly decreased B. cinerea conidial germination in vitro and resulted in zero percent damage on poinsettia bracts. However, all UV-C doses during both replications caused minor damage, 15% or less, to primula flowers. / Master of Science in Life Sciences / Entomopathogenic nematodes (EPN) shows promise in being non-chemical and environmentally friendly solution for greenhouse pest and disease control. These can also be referred to as Biological Controls (Biocontrols). Entomopathogenic nematodes are used widely to control multiple greenhouse plant pests which include both Lycoriella spp., Fungus Gnats, and Frankliniella spp., Western Flower Thrips. However, there are challenges with EPN viability and storage from the manufacture to the greenhouse producer. We studied four EPN species, Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica, which were reared and stored to determine differences in production viability between species. Results show that the EPN species do not respond the same to storage and produce different amounts of infectious juveniles during rearing when conditions are the same. Separate from, but just as concerning as greenhouses plant pests are plant diseases. Ultraviolet radiation in the C spectra is known to be germicidal due to its narrow wavelengths. Because of this, UV-C has been shown to deactivate many different plant pathogens on contact and is being considered as a possible Biocontrol alternative to harsh traditional fungicides and bactericides. One disease that is known to contribute to the highest volume of annual crop losses is Botrytis cinerea. Botrytis cinerea is a plant disease that impacts floricultural crops to vegetables during propagation through the production supply chain to shipping and storage. We evaluated UV-C radiation at different doses, to determine if it could be used to replace a traditional fungicide before plants are shipped to reduce B. cinerea infection during transport. We found that UV-C successfully deactivated B. cinerea in vitro, but the viability of the application to plant tissue before transport has yet to be proven successful as a practical method of reducing B. cinerea during transport.

Nematodes are highly diverse animals capable of interacting with almost every other form of life on Earth from general trophic interactions to intimate and persistent symbiotic associations. Much of their recognition originates from their various parasitic lifestyles. From an agricultural standpoint, plant parasitic nematodes are widely known for the destruction they can cause to crop plants, such as the case of the root-knot nematode Meloidogyne incognita, or livestock animals, like the Trichinella spiralis, which infects pigs and other animals. From a human health perspective, nematodes can cause many debilitating diseases, for example Wuchereria bancrofti, which is a causative agent of lymphatic filariasis or elephantiasis. However, not all parasitic nematodes have bad implications for human health. For instance, the diverse interactions of insect parasitic nematodes can be used to our benefit. Many of these species have been considered as biological control alternatives to different insect pests that wreak havoc on human, animal, and plant health. There still remain many questions surrounding their evolution, ecology, and physiological capabilities. Many of these taxa are hard to cultivate in the lab due to their complex and intimate lifestyles. Entomopathogenic nematodes (EPNs) are of great interest in agriculture because they vector insect pathogenic bacteria, which are capable of causing death to an insect host within 48 hours post-infection. Much of the molecular underpinnings in this system still remain to be discovered, from understanding the basic ability of these two organisms to associate with one another to genetically engineering more robust and host specific pathogens for application in the field. The focus of the research presented herein is on Steinernematidae nematodes and their bacterial symbionts. Specifically, it focused on the relationship between Xenorhabdus bovienii and its Steinernema hosts. Bioassays were designed to investigate insect virulence of X. bovienii alone in two Lepidoptera insect species with known differential susceptibility to Steinernema-Xenorhabdus pairs. A comparative genomic analysis was performed to compare different Xenorhabdus bovienii strains with observed variation in insect virulence. Results from this analysis demonstrated that virulent strains possess a type VI secretion system (T6SS) locus that is completely absent in strains with attenuated virulence. Bacterial competition assays between T6SS+ and T6SS- strains suggest this locus is involved in bacterial competition. Additionally, symbiont preference assays were carried out to investigate whether Steinernema hosts are able to discern between virulent and attenuated X. bovienii strains. Results from these assays revealed that Steinernema nematodes are able to distinguish between cognate and non-cognate X. bovienii symbionts, giving preference to virulent strains over those with attenuated virulence. Altogether these results provide further evidence that supports the notion that symbiont-switching events have occurred over the Steinernema-Xenorhabdus co-evolutionary history. Specifically, the competitive virulence of certain X. bovienii strains may have conferred them the ability to be selected by different Steinernema hosts, therefore contributing to the success of the nematode-bacterium partnership in being pathogenic to diverse insect hosts.

This study investigated a system comprised of entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae), their symbiotic bacteria (Xenorhabdus, Photorhabdus) and an insect host, M. sexta. The focus of this investigation was to examine the effects insect host condition on the nematode-bacterium mutualistic partnership as a whole. The EPN used in the study had varying host-searching strategies: an ambusher, Steinernema carpocapsae, and a cruiser, Heterorhabditis sonorensis. Insect host age and diet were evaluated as factors that affect insect host condition. Wandering 5th instar M. sexta were much less susceptible to EPN infection compared to non-wandering 5th instar larvae, regardless of EPN species. Insect host diet had a significant impact on H. sonorensis susceptibility to EPN infection, and a non-significant trend was observed for S. carpocapsae. EPN and bacterial symbiont fitness were unaffected by insect host condition. This is the first record of the effect of insect host condition on both EPN and their symbiotic bacteria.

Les nématodes entomopathogènes Steinernema sont associés symbiotiquement à des bactéries du genre Xenorhabdus. Leur cycle de vie comprend deux phases : une phase libre, dans le sol, où les nématodes portent leurs symbiotes dans le tube digestif, et une phase parasite, dans l'insecte, où les deux partenaires se multiplient côte à côte. Le bilan fin de l'interaction, globalement bénéfique pour les deux partenaires, a été peu étudié pour le moment. Dans un premier temps, nous avons abordé ces symbioses d'un point de vue coûts-bénéfices pour le nématode afin d'identifier les pressions de sélection agissant sur son investissement dans la symbiose. Nous montrons que l'association est à la fois bénéfique (reproduction en phase parasitaire) et coûteuse (mortalité en phase libre) pour le nématode, en proportion de sa charge symbiotique. Ces corrélations engendrent un compromis survie-reproduction pour le nématode, médié par ses symbiotes. Selon les conditions environnementales, elles pourraient éventuellement déstabiliser l'association, et, notamment, altérer sa spécificité. Dans un second temps, nous avons donc exploré la spécificité de ces associations dans la nature et au laboratoire. Lors d'un échantillonage de terrain, nous avons retrouvé la spécificité de ces associations largement décrite par ailleurs. Au laboratoire, la réassociation expérimentale entre nématodes et bactéries non natives montre que le spectre des bactéries retenues est plus étroit que le spectre des bactéries bénéfiques. De plus, des différences de modalités d'association apparaissent entre espèces de nématodes, qui suggèrent que la correspondance nématode - bactérie ne serait pas aussi stricte que prévu.

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-21Bitstream added on 2014-06-13T19:44:46Z : No. of bitstreams: 1 garcia_lc_dr_botfca.pdf: 577642 bytes, checksum: 5156a8041539b0ed81f6f941b21fc230 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A tecnologia de aplicação desenvolvida para produtos químicos tem sido utilizada para organismos entomopatogênicos sem o estudo prévio das necessidades de adaptações que visem à manutenção da viabilidade desses agentes de controle biológico. Assim, o objetivo dessa pesquisa foi avaliar o efeito de diferentes tecnologias de aplicação sobre a concentração, viabilidade e eficácia dos juvenis infectantes dos nematóides Heterorhabditis indica e Steinernema sp. no controle da lagartas-do-cartucho (Spodoptera frugiperda), em terceiro instar, na cultura do milho. Testaram-se as dosagens de zero a 400 juvenis infectantes em placa de Petri para controle da lagarta-do-cartucho. As tecnologias de aplicação dos entomopatógenos, em ambiente controlado, abrangeram as pulverizações com pontas hidráulicas e centrífuga, com e sem fornecimento de carga elétrica à calda. Na pulverização do Steinernema sp. em plantas de milho sob condição ambiental controlada, com e sem emprego de tensoativo e lâmina de água, as dosagens variaram entre zero e 288 milhões.ha-1. Conclui-se que para matar as lagartas em terceiro ínstar de S. frugiperda em laboratório foram necessários 280 juvenis infectantes de Steinernema sp. Pode-se pulverizar os entomopatógenos, sem que haja perda significativa na concentração e viabilidade desses organismos, com equipamentos que forneçam carga elétrica à calda, ponta centrífuga e pontas hidráulicas, exceto àquelas que requerem elementos filtrantes com malha igual a 100. Os tensoativos Break-Thru®, Extravon® e Iharaguen- S® não afetaram a viabilidade dos entomopatógenos. A pulverização em plantas de milho (V6) com até 288 milhões de juvenis infectantes de Steinernema sp. por hectare, diluídos em volume de calda de até 800 L.ha-1 com 0,01 % de tensoativo ou nesse volume seguido de exposição à chuva artificial (lâmina de água de 6 mm) não proporcionaram adequado controle da praga. / Spraying technologies has been developed to work with agrochemicals, but it has been utilized with entomopatogenic organisms without a prior study of adaptation that will help maintain the viability of the biological control agents. In this way, the objective of this research was to evaluate the effect of different spraying technologies on the concentration, viability and efficiency of the infective juveniles of Heterorhabditis indica and Steinernema sp. nematodes to control fall armyworm (Spodotera frugiperda) at the third instar on corn plants. Rates between zero and 400 infective juveniles were tested in a Petri dish against fall armyworm. The study to evaluation spraying technologies stable environment, involved hydraulic nozzles and rotary spray nozzles with and without electrical charge added to the spraying mix. The dosages of Steinernema sp. sprayed on corn plants in controlled environment with or without adjuvant agent and water blade, varied of zero at 288 millions ha-1. For killing 100% the fall armyworm in laboratory, it was necessary 280 juvenile infectants of Steinernema sp. It is possible to spray entomopatogenic organisms without an significative loss in the concentration and viability of these organisms, with equipment that produces electrical charges to the spraying mix, and with those using hydraulic and rotary nozzles tips, but without mash 100 filtrating elements. Tensoactive agents Break-Thru®, Extravon® and Iharaguen-S® did not affect the viability of the entomopatogens. Spraying corn plants with up to 288 millions of juvenile infectants of Steinernema sp. per hectare, diluted in the spraying mix up to 800 L.ha-1, with 0,01% of tensoactive agent or in this volume followed by artificial rain (water blade of 6 mm), was not enough to control the infestation.

Orientador: Carlos Gilberto Raetano / Banca: Wilson Badiali Crocomo / Banca: Antonio Batista Filho / Banca: Luis Garrigós Leite / Banca: Hamilton Humberto Ramos / Resumo: A tecnologia de aplicação desenvolvida para produtos químicos tem sido utilizada para organismos entomopatogênicos sem o estudo prévio das necessidades de adaptações que visem à manutenção da viabilidade desses agentes de controle biológico. Assim, o objetivo dessa pesquisa foi avaliar o efeito de diferentes tecnologias de aplicação sobre a concentração, viabilidade e eficácia dos juvenis infectantes dos nematóides Heterorhabditis indica e Steinernema sp. no controle da lagartas-do-cartucho (Spodoptera frugiperda), em terceiro instar, na cultura do milho. Testaram-se as dosagens de zero a 400 juvenis infectantes em placa de Petri para controle da lagarta-do-cartucho. As tecnologias de aplicação dos entomopatógenos, em ambiente controlado, abrangeram as pulverizações com pontas hidráulicas e centrífuga, com e sem fornecimento de carga elétrica à calda. Na pulverização do Steinernema sp. em plantas de milho sob condição ambiental controlada, com e sem emprego de tensoativo e lâmina de água, as dosagens variaram entre zero e 288 milhões.ha-1. Conclui-se que para matar as lagartas em terceiro ínstar de S. frugiperda em laboratório foram necessários 280 juvenis infectantes de Steinernema sp. Pode-se pulverizar os entomopatógenos, sem que haja perda significativa na concentração e viabilidade desses organismos, com equipamentos que forneçam carga elétrica à calda, ponta centrífuga e pontas hidráulicas, exceto àquelas que requerem elementos filtrantes com malha igual a 100. Os tensoativos Break-Thru®, Extravon® e Iharaguen- S® não afetaram a viabilidade dos entomopatógenos. A pulverização em plantas de milho (V6) com até 288 milhões de juvenis infectantes de Steinernema sp. por hectare, diluídos em volume de calda de até 800 L.ha-1 com 0,01 % de tensoativo ou nesse volume seguido de exposição à chuva artificial (lâmina de água de 6 mm) não proporcionaram adequado controle da praga. / Abstract: Spraying technologies has been developed to work with agrochemicals, but it has been utilized with entomopatogenic organisms without a prior study of adaptation that will help maintain the viability of the biological control agents. In this way, the objective of this research was to evaluate the effect of different spraying technologies on the concentration, viability and efficiency of the infective juveniles of Heterorhabditis indica and Steinernema sp. nematodes to control fall armyworm (Spodotera frugiperda) at the third instar on corn plants. Rates between zero and 400 infective juveniles were tested in a Petri dish against fall armyworm. The study to evaluation spraying technologies stable environment, involved hydraulic nozzles and rotary spray nozzles with and without electrical charge added to the spraying mix. The dosages of Steinernema sp. sprayed on corn plants in controlled environment with or without adjuvant agent and water blade, varied of zero at 288 millions ha-1. For killing 100% the fall armyworm in laboratory, it was necessary 280 juvenile infectants of Steinernema sp. It is possible to spray entomopatogenic organisms without an significative loss in the concentration and viability of these organisms, with equipment that produces electrical charges to the spraying mix, and with those using hydraulic and rotary nozzles tips, but without mash 100 filtrating elements. Tensoactive agents Break-Thru®, Extravon® and Iharaguen-S® did not affect the viability of the entomopatogens. Spraying corn plants with up to 288 millions of juvenile infectants of Steinernema sp. per hectare, diluted in the spraying mix up to 800 L.ha-1, with 0,01% of tensoactive agent or in this volume followed by artificial rain (water blade of 6 mm), was not enough to control the infestation. / Doutor

It is known that entomopathogenic nematodes rarely live in fields where pesticides and fertilizers are used. Because they are organisms with a high potential for biological and integrated pest control the thesis tried to determine the influence of pesticides on the infectivity and mortality of selected nematodes: Steinernema feltiae, S. arenarium and S. kraussei in water solution of some pesticides. The results are that preparations Vydate (a. i. oxamyl) and Sulka (a. i. sulphur) have the highest negative influence on both characteristics. These pesticides cause a mortality of 94.3 - 99.9 % and reduce infectivity by about 50 {--} 75 % (level 3). The following preparations may be identified as having higher potential for infectivity reduction: above all, Starane 250 EC (fluroxypyr), Sumithion super (fenithrothion), and Novozir MN 80 (mancozeb); on the other hand, Treflan 48 EC (trifluralin) significantly increases mortality. Another important observation is a fact that infectivity is significantly influenced only by concentration of pesticides, not by exposure time. During an experiment with enhacement of resistance to oxamyl (Vydate) I falling mortality and increasing infectivity of entomopathogenic nematodes were observed after five rounds.

The presented thesis deals with interspecific competition and host range of entomopathogenic nematodes and assess the mechanisms enabling coexistence of multiple EPN species. The thesis further studied the scavenging ability of EPNs and their defense mechanisms against invertebrate scavengers. The results revealed that coexisting EPN species share the same niche and their interaction are complex and may be asymmetric. EPNs were found to be able to colonise and multiply in cadavers of different insects and scavenging seems to be an important alternative to normal infection. The defence of EPNs against scavengers seems to be an adaptation of the nematode-bacteria complex.