As pollinators bumble bees play the crucial role of contributing to propagation of flowering plants in favour of food production as well as biodiversity. Over the course of a few decades bumble bees have seen a remarkable decline, with contributing factors being climate change, pesticides and pathogens such as viruses and parasites. In Sweden, commercially reared bumble bees are bought for the purpose of pollination in fruit and berry plantations. However, these reared bumble bees are a suspected contributor to a spillover of pathogens to wild bees in the same area. The aim of the study was to determine the prevalence of five viruses and five parasites in wild bumble bees nearby commercially reared bumble bees and to determine seasonal variation in pathogen distribution. qPCR was used for analysis of Acute bee paralysis virus, Deformed wing virus, Slow bee paralysis virus, Black queen cell virus and Sacbrood virus as well as the parasites Crithidia bombi, Apicystis bombi, Nosema bombi, Sphaerularia bombi and Locustacarus buchneri. The results showed a statistically significant, 4,8 times higher prevalence of A. bombi nearby commercially reared bumble bees in greenhouses compared to control landscapes. The results were also compared to pathogen prevalences in bumble bees caught in June the same year, showing a significantly higher prevalence in a majority of the parasites. It also showed a decrease in all viruses except Black queen cell virus, where the decrease might be explained by RNA degradation.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-20T13:02:10Z No. of bitstreams: 1 Daniela Silva Salgues de Matos.pdf: 463245 bytes, checksum: 6041b12480d1db2b27dea971c15113a0 (MD5) / Made available in DSpace on 2017-02-20T13:02:10Z (GMT). No. of bitstreams: 1 Daniela Silva Salgues de Matos.pdf: 463245 bytes, checksum: 6041b12480d1db2b27dea971c15113a0 (MD5) Previous issue date: 2006-02-21 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / More than 35 species of plant parasitic nematodes are reported associated with papaya (Carica papaya) being the root-knot nematodes one of the most important. These populations occur in polyspecific communities and different densities depending on cultivars, host stress and physical, chemical and biological soil properties. The present study had the objectives of describing nematode community structure associated with papaya croping in soil infested with Meloidogyne sp. in Mamanguape, Paraíba, and evaluating number of samples and pattern for soil colllection in order to monitore Meloidogyne sp. and Rotylenchulus sp. population density in field. Samplings were carried out in three areas with different population levels of root-knot and reniform nematodes. In each area, 5,776 m2 was split in 400 (20 × 20) squares of 14.44 m2 (3.8 × 3.8 m), containing three plants. Evaluations for nematode community structure based upon nematode community indexes: maturity index (MI), plant parasitic index (PPI) and modified maturityindex (MMI), and nematode trophic group ratios. It was simulated collecting samples in “X”, diamond, parallel lines, parallel rows, inverted “V”, zig-zag, inverted “W”, “degree”, “S”, randomly and in ordinary walking in order to select diagram pattern for sampling. For sample size evaluation, all diagram patterns were used changing the number of samples (40, 30 e 20) in each area. Population density (specimes/300cm3 soil) of both nematodes were predicted according to size and diagram of sampling and compared through Kruskal-Wallis test at 5% probability level. In areas 1 and 3 there was predominance of plant parasitic nematodes, 43.84 and 39.95 % respectively, especially Rotylenchulus sp. In area 2, Meloidogyne was the taxon of highest dominance among the plant parasitic nematodes. Inthe three areas MI and MMI were high (> 30%) and PPI ranged from 20.95 to 32.88%. Twenty samples per area were indicated for both nematode genus, being recommended to collect the samples randomly, in parallel bars, or in diamond for monitoring Meloidogyne sp. and in parallel bars or parallel lines for Rotylenchulus sp / Mais de 35 espécies de fitonematóides são relatadas na rizosfera de mamoeiro (Carica papaya), com destaque para os nematóides das galhas. Essas populações ocorrem em comunidades constituídas por diferentes espécies e densidades populacionais que variam com a cultivar, estresse sobre a hospedeira e propriedades física, química e biológica do solo. O presente estudo teve como objetivos: descrever a estrutura da comunidade de nematóides associada ao cultivo comercial do mamoeiro em áreas com diferentes níveis populacionais de Meloidogyne sp. em Mamanguape, Paraíba, e determinar padrão de caminhamento e número de amostra, para monitoramento de populações de Meloidogyne sp. e Rotylenchulus sp. no campo. Foram avaliadas três áreas, com diferentes níveis populacionais de nematóide das galhas e reniforme. Em cada área, tomou-se 5.776 m2 que foi dividido em 400 (20 × 20) quadriláteros contíguos de 14,44 m2 (3,8 × 3,8 m),com três plantas por quadrilátero. Para descrição da estrutura da nematofauna, foram usados índices de comunidade de nematóides: índice de maturidade (MI), índice de parasitos de plantas (PPI) e índice de maturidade modificada (MMI), e razões entre grupos tróficos de nematóides. Para seleção do diagrama para coleta de amostras foram simulados padrões de caminhamento em “X”, diamante, linhas paralelas, barras paralelas, “V” invertido, zigue-zague, “W” invertido, “degrau”, “S”, aleatório e caminhamento geral. Para determinação do tamanho das amostras utilizou-se todos os padrões de caminhamento testados e variou-se o número de amostras (40, 30 e 20) dentro de cada padrão em cada área. As densidades populacionais (espécimes/300cm3 de solo) de ambas espécies, estimadas em função do número de amostras e padrão de caminhamento, foramcomparadas pelo teste de Kruskal-Wallis a 5% de probabilidade. Nas áreas 1 e 3 houve predominância de fitoparasitos, respectivamente 43,84 e 39,95 % do total de nematóides encontrados, destacando-se o gênero Rotylenchulus sp. Na área 2, Meloidogyne foi o taxon de maior dominância entre os fitoparasitos. Nas três áreas, MI e MMI foram altos (> 30%) e PPI variou de 20,95 a 32,88%. Vinte amostras por área foi indicado para monitoramento dos dois gêneros de nematóides, recomendando-se caminhamento aleatório, em diamante ou em barras paralelas para coleta de amostras de Meloidogyne sp. e caminhamento em linhas ou barras paralelas para Rotylenchulus sp.

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.

Platinum mining activities contribute substantially to South Africa's economy since it exceeded gold as economical contributor in 2001. Mining activities contribute to large amounts of waste production in the form of tailings and rock waste, deposited in the surrounding environment of the mine premises. Mining companies are held responsible for damages caused to the surrounding environment. These companies are required to introduce the cost of ecological rehabilitation in their operation costs as well as compile an environmental management plan. Numerous attempts to rehabilitate mine waste have proven unsuccessful. New and improved rehabilitation techniques are required to facilitate in the rehabilitation of these mine spoils. Woodchip-vermicompost produced from platinum mining wastes (woodchips and sewage sludge) was used as an alternative amendment to inorganic fertilisers during the rehabilitation of platinum mine tailings. The effectiveness of the woodchip-vermicompost as an alternative amendment during the platinum mine tailings rehabilitation were monitored using different ecosystem components. A natural veldt in the vicinity of the mine area was randomly selected to serve as a reference site. These ecosystem components selected have previously been shown to be effective as indicators of ecosystem quality. The components selected for this study includes the use of microbial enzymatic activity, microbial community structure, nematode trophic structures, and other mesofaunal groups such as micro-arthropods. The physical and chemical properties of the platinum mine tailings and reference area as well as the vegetation cover of the platinum mine tailings were determined. Statistical and multivariate analyses were use to determine the correlation between the dependent microbial components and dominate independent chemical properties. Nematode trophic structure, Maturity Index, and Plant-Parasitic nematode Index were used to compare the two rehabilitation techniques in terms of nematodes as indicators. Microarthropods family structures were used to compare the two amendments in terms of diversity and abundance. Enzymatic activity was positively affected by the addition of woodchip-vermicompost, than in the sites treated with inorganic fertilisers. The microbial community structure showed no statistically significant (p < 0.05) differences between the two amendments. A higher abundance of nematodes especially plant-parasitic nematodes and bacterivorous nematodes were observed in the woodchip-vermicompost sites than in the inorganic fertilised sites. According to the Maturity Index, both amendments became more enriched during the study period, while the Plant-Parasitic nematode Index showed that the carrying capacity for plantparasitic nematodes on the woodchip-vermicompost sites increased while it decreased in the inorganic fertilised sites, which can be related to the decrease in vegetation cover on the inorganic fertilised sites. Both coloniser (Prostigmata) and persister (Cryptostigmata and Mesostigmata) groups of the micro-arthropods, as well as a higher diversity of micro-arthropods, were present on the woodchip-vermicompost sites whereas the inorganic fertilised sites showed only the presence of colonisers, with a decrease in diversity and abundance of micro-arthropods over the study. The colonisation of micro-arthropods may have been affected by the addition of woodchip-vermicompost and vegetation cover, which contribute to the establishment of suitable microhabitats for these soil biota. By intercorrelating the results, it may be concluded that the addition of woodchip-vermicompost may be an essential part of the rehabilitation process, by contributing to soil organic material to the ecosystem system, which may improve the recolonisation of soil biota and ecosystem processes. However further studies need to be conducted in order to determine the long-term sustainability of the woodchip-vermicompost in providing organic material and sustaining the ecosystem processes. The study also showed the necessity to integrate various ecosystem components when evaluating ecosystem development due to the unique role each component plays and the impact it may have on other components. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.

Nematodes of the genus Phasmarhabditis (Nematoda: Rhabditidae) are world-wide distributed molluscs´ parasites. Species P. hermaphrodita is the only commercially produced bio-agent for slug control. The aim of this thesis is to evaluate some ecologic characteristics of three newly described species of the genus Phasmarhabditis (P. bohemica, P. bonaquaense a P. apuliae) in polyxenic and monoxenic cultures. Three bacterial isolates, Pseudomonas sp., Acinetobacter sp. and Flavobacterium sp. were chosen for testing the monoxenic cultures. The growing ability of tested nematodes, the effect on the quality of progeny and the rate of development were examined on various organic substrates (compost, faeces of Deroceras slugs, homogenized Deroceras slugs, homogenized larvae of Galleria mellonella and homogenized pig kidney). Subsequently, the growth of nematodes was tested in solid and liquid media. The last experiment was focused on evaluating the impact of polyxenic and monoxenic nematode cultures on the mortality and feeding activity of Deroceras reticulatum and Cepaea hortensis. As is assumed in the results chapter, all examined species of nematodes are lethal facultative parasites of Deroceras spp. They are able to grow in broad spectre of organic substrates and bacteria which influence the production of the progeny, but the qualitative parameters of dauer juveniles remain more or less unaffected.

Orientador: Sandra Helena Unêda-Trevisoli / Coorientador: Ana Cristina Pinto Juhász / Coorientador: Luciany Favoreto / Banca: João Antônio da Costa Andrade / Banca: Ivana Marino Barbaro / Banca: Jaime Maia dos Santos / Banca: Pedro luiz Martins Soares / Resumo: A soja [Glycine max (L.) Merrill] é uma leguminosa de ciclo anual que apresenta distintas finalidades, dentre elas, uma crescente vertente relaciona-se ao uso na alimentação humana. Há demanda de cultivares de soja com esta destinação com resistência a patógenos e pragas. Em especial destaca-se o nematoide de cisto da soja (Heterodera glycines) que causa expressiva perda na produtividade. Em virtude do exposto, utilizando topocruzamentos de soja tipo alimento x tipo grão, o objetivo do presente trabalho é: a. Determinar a herança da cor do tegumento, da cor do hilo e da expressão do caráter derramamento de hilo em populações F2:3; b. Determinação do estudo da herança da resistência ao nematoide de cisto da soja (NCS), bem como estimativa do número e modo de ação dos genes envolvidos a fim de contribuir no delineamento de cruzamentos, que envolvem os genitores em estudo, com maior possibilidade de acumular genes que conferem resistência à raça 3 do NCS e; c. Verificar com uma abordagem quantitativa, a adequação das populações a um modelo aditivo-dominante, assim como estimar parâmetros genéticos nos cruzamentos gerados. Utilizaram-se genitores contrastantes, um resistente ao NCS raça 3 ('BRSMG 810C') e dois suscetíveis, porém aptos para utilização na alimentação humana ('BRSMG 790A' e 'BRSMG 800A'). Obtiveram-se as gerações F1, RCr, RCs e F2 que foram utilizadas para o estudo da herança da resistência, por meio da segregação do caráter. As avaliações de nematoide foram realizadas utilizando-se como estimador o Índice de Fêmeas (IF) e analisadas pelo teste de Qui-Quadrado (χ2) para testar o ajuste ao modelo proposto para a herança. As sementes F3 provenientes de plantas F2 foram caracterizadas para elucidar a herança para caracteres morfológicos de interesse (cor de tegumento, cor e aspecto de hilo). Para os caracteres morfológicos dos... / Abstract: Soybean [Glycine max (L.) Merrill] is an annual leguminous that has distinct purposes, among them, a growing amount is related to the human feed. There is a demand of cultivars aiming this destination, which are resistant to pathogens and plagues. A special plague is highlighted, the soybean nematode of cyst (Heterodera glycines) (NCS). It causes expressive loss of productivity. Facing this, using soybean topcrosses feed-like x grain-like this work aims: a. determine the tegument color, hilum color, and hilum spread character heritance in F2 populations; b. determine the NCS resistance heritage, as well the involved genes number and action mode estimative in order to contribute in the crosses design, which involve the parents studied, with a higher possibility of accumulation of genes which provide resistance to the NCS race 3; and c. Verify in a quantitative approach the adequacy population in an additive-dominant model, as well estimate the genetic parameter into the crossings. We used contrasting parents, being resistant to the race 3 ('BRSMG 810C') and two susceptible, however suitable to the human feed ('BRSMG 790A' and 'BRSMG 800A'). Thus, the generations F1, RCr, RCs, and F2 were created. They were used to study heritage resistance through the character segregation. The nematode evaluations were done using as estimator the Female Index (IF) and they were analyzed by the chi-square test (χ2) to test the adjustment to the suggested model to the heritage. The F3 seeds from the F2 plants were characterized to elucidate the heritage to interest morphologic traits (seed coat color, hilum color and hilum aspect). For the morphological character of grain, the results observed of descendent were adjusted to the expected proportion, considering that there were four genes (R/I/W/T) controlling the expression coat and hilum color to both crossings. At crossing ... / Doutor

Este projeto objetivou desenvolver um marcador molecular ligado ao gene de resistência a Tobacco mosaic virus (TMV), em vista da necessidade de aprimorar os métodos de melhoramento de plantas para atender crescentes demandas de produtividade. O outro objetivo deste trabalho foi a avaliação de uma população segregante F2 e de retrocruzamento (RC1F1) a Meloidogyne incognita raça 3, oriunda do cruzamento das cultivares comerciais Coker 176 (C176) e Coker 371 Gold (C371G). Para o desenvolvimento do marcador ligado ao gene de resistência a TMV, o gene N, foram desenvolvidos iniciadores específicos para regiões conservadas (TIR, NBS e LRR) deste gene com base em sua seqüência. Estes iniciadores foram utilizados para amplificar um marcador cuja ligação ao referido gene foi confirmada em 200 indivíduos de população segregante F2 oriunda do cruzamento entre uma linhagem resistente (Coker176) e outra suscetível ao vírus (Kentucky326). A proporção entre o número de plantas resistentes e suscetíveis (154:46) não diferiu estatisticamente daquela esperada no caso de segregação de um gene dominante de resistência, que seria de 3:1. Os resultados indicaram que o marcador e o gene estão proximamente ligados segundo taxa de recombinação, que foi de 1%. Na avaliação da hereditariedade a M.incognita utilizou-se 141 indivíduos da população segregante F2 oriunda do cruzamento entre uma linhagem resistente (Coker176) e suscetível (Coker 371G) e 138 indivíduos de RC1F1 ([C176 X C371G] X C371G). Os resultados obtidos entre a proporção entre o número de plantas resistentes e suscetíveis da população F2 (102:39) e da RC1F1 (67:71) não diferiu estatisticamente daquela esperada no caso de segregação de um gene dominante de resistência. / The aim of this study is to develop a molecular marker linked to the resistant gene to Tobacco mosaic virus (TMV) considering the necessity to improve plant breeding to meet growing demands of productivity. The other goal of this study is to evaluate the mode of inheritance of an F2 segregating population and backcross (BCF1) population of Meloidogyne incognita race 3 originated from cross breeding between commercial cultivars Coker 176 and Coker 371 Gold. For the development of the marker linked to the TMV resistant gene, the N gene, specific primers of this gene were developed for conserved regions (TIR, NBS and LRR) based on their sequence. These primers were used to amplify a marker whose connection with the aforementioned gene was confirmed in 200 individuals in a segregating F2 population originated from the cross breeding between a resistant cultivar (Coker176) and another cultivar which is susceptible to the virus (Kentucky326). The proportion between the number of resistant and susceptible plants (154:46) did not statistically differ from the one expected in the segregation of one dominant resistance, which would be a 3:1 segregation ratio. The results indicated that the marker and the gene are narrowly linked according to recombination ratio 1%. In the heredity evaluation of resistance to M.incognita 141 plants of the segregating F2 population originated from the cross breeding of a resistant (Coker176) and susceptible cultivar (Coker 371G) and 138 plants of backcross BC1F1 ([C176 X C371G) X C3371G) were used. The outcome of the proportion between the number of resistant and susceptible plants of segregating F2 (102:39) and BC1F1 population (67:71) were not statistically different from the ones expected for a monogenic dominant resistance.

Animals acquire immunity against gastrointestinal (GI) nematode infection depending on their age and continual exposure to larvae, however, expression of this acquired immunity is often penalized during pregnancy and lactating period. This is described as periparturient relaxation in immunity (PPRI), and suggested to have nutritional basis. Although dietary protein has positive effect on immunity against GI nematode infection in mammalian host, we have not fully achieved to characterise the detailed interaction between PPRI and dietary protein. Therefore, this PhD aimed to further investigate this interaction in a well-established Nippostrongylus brasiliensis re-infected lactating rat model. Feeding high protein diet (HP) as opposed to low protein diet (LP) during pregnancy was necessary in maintaining sufficient maternal performances and systemic immune response (Chapter 2 and 3). Accumulation of host’s body protein reserve during pregnancy was significantly higher in HP fed animals compared to LP fed counterparts, which led to improvement in both maternal performances and immunity during the early stage of lactation. However, as lactation period progressed and re-infection of N. brasiliensis took place, importance of current dietary status, rather than the accumulated protein reserve, became evident for maternal performances and immunity. Indeed, animals fed HP during lactation showed significantly heavier pup weight compared to LP fed animals; HP animals showed higher serum immunoglobulin levels and reduced worm burden compared to LP. N. brasiliensis, however, goes through systemic migration, entering host’s skin and migrating to lung parenchyma through blood vessels before reaching the intestine. In Chapter 4, a detailed lung pathology study following N. brasiliensis infection was performed to generate data on the effects of the nematode in the lung of the rat host as such data were scarce. Rat host showed similar lung pathology to that of mice; up-regulation of genes related to type 2 immunity and development of emphysema-like pathology were observed following N. brasiliensis re-infection. In Chapter 5, the effect of dietary protein supplementation on lung and intestinal histology and gene expression analysis was investigated. It was shown that HP fed animals showed higher expression of genes related to type 2 immunity compared to LP in the lung. This effect of protein supplementation in the lung may have contributed to fewer worm burdens in HP fed rats compared to LP in the intestine. Dietary protein supplementation significantly affected the expression of genes related to goblet cells; it resulted in up-regulation of the expression of Retnlb and down-regulation of Agr2 and Tff3 in HP fed animals compared to LP. It is evident that dietary protein is modulating intestinal immunity, and this may be targeted towards specific pathways. In addition, the effect of dietary protein supplementation on immune cell populations of secondary lymphoid organs was analysed. Marked increase in the percentage of macrophage in the spleen and T cell in the mesenteric lymph node was observed following protein supplementation, highlighting the importance of dietary protein on systemic immunity during the parasite infection. These results demonstrate that dietary protein supplementation is effective for improving both maternal performances and immune responses, not only at the intestinal phase but also at the migrating phase, when animal is undergoing PPRI during N. brasiliensis infection. Such information is expected to define strategic utilisation of nutrient supply and to result in development of sustainable parasite control strategies in mammals.

In many developing countries, farmers and pastoralists still rely on their indigenous knowledge, practices and locally available plants to control nematode parasitic infections, both in livestock and humans. The overall aim of my thesis was to undertake bioassay-guided phyto-chemical study of extracts and their constituents from Ethiopian anti-parasitic plants used by healers to control gastrointestinal nematode parasites in livestock to validate their ethno-medicinal use and to characterise and identify their active ingredients. As a first experiment (Chapter Three), four types of crude extracts (water, 70% methyl-alcohol, absolute methanol and acetone) of four indigenous Ethiopian medicinal plants (Adenia species, Cissus ruspolii, Ipomoea eriocarpa and Euphorbia thymifolia) were screened against Teladorsagia circumcincta egg hatching in vitro, not only as a first step to validate the traditional healers claim but also to choose the most promising plant extract(s) for further phyto-chemical studies. The egg hatching inhibition (EHI) test results revealed that the anti-parasitic properties of these plants depended on plant species, dose, and solvent polarity. The water extracts of both C. ruspolii and Adenia sp. exhibited largest, up to 100% EHI but also larval migration inhibition activities, and were selected for further studies. The second experiment (Chapter Four) assessed the nature of active constituents in these extracts by physico-chemical methods. It was observed that the major constituents of both plant extracts responsible for the EHI activities are likely highly polar, water-soluble, small and moderately heat-labile molecules. The third and fourth experiments (Chapters Five and Six) consisted of separating Cissus ruspolii and Adenia sp. water extracts into discrete fractions by gel-permeation chromatography, EHI tests of Bio-Gel P-2 fractions followed by thin layer chromatography (TLC) profiling of these fractions to detect separated spots (in day light, under UV-light or after staining with various staining reagents) and also to see how elution patterns of separated spots affected by column parameters. The EHI tests on the fractions obtained revealed that the active constituents of C. ruspolii and Adenia sp. water crude extracts were eluted into few fractions based on their molecular sizes. The TLC profilings of these fractions identified spot patterns of active and inactive fractions, which allowed pooling of active constituents based on their EHI and TLC profiling into three pools for each plant. The fifth experiment (Chapter Seven) was to isolate and purify compounds from these pools using various preparative planar and column chromatographic methods. Sequential applications of column chromatography followed by preparative thin layer chromatography isolated and purified five active compounds from C. ruspolii and two active compounds from Adenia sp. The sixth experiment (Chapter Eight) was to characterize and propose/elucidate structures of compounds from the active fractions using chromatographic, analytical and spectroscopic methods. In this regard, the structures of two oleanane type triterpenoid saponins isolated from one of active fractions of Adenia sp. were proposed based on their mass spectrometry (MS) and nuclear magnetic resonance (NMR) data with support of compounds property, TLC and literature. Similar outcomes for C. ruspolii were not achieved due to lack of sufficient sample to run 13C-nuclear magnetic resonance spectroscopy and distortionless enhancement by polarization transfer (DEPT), contamination of some purified compounds with ill-characterised substance from the preparative TLC matrix and in some cases mass spectrometry (MS) and nuclear magnetic resonance (NMR) data did not support each other. The last experiment (Chapter Nine) was to assess anthelmintic efficacy and safety of C. ruspolii and Adenia sp. crude water extracts in Heligmosomoides bakeri infected mice. This in vivo test revealed that both plant extracts exhibited significant reduction in worm burdens and worm egg excretion, with moderate effects on haematology and organ weights at tolerated dosages. In conclusion, both in vitro and in vivo data revealed that Adenia sp. and C. ruspolii have anthelmintic properties, thus validating traditional healer claims and supporting ethno-medicinal use. The bioassay-guided phytochemical study resulted in the isolation of a number of active compounds from these plants, for some of which a structure has been proposed.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-14T16:11:22Z No. of bitstreams: 1 Larissa de Brito Caixeta.pdf: 1251344 bytes, checksum: f836a0d0c30f9c764b4a7d186a485112 (MD5) / Made available in DSpace on 2017-03-14T16:11:22Z (GMT). No. of bitstreams: 1 Larissa de Brito Caixeta.pdf: 1251344 bytes, checksum: f836a0d0c30f9c764b4a7d186a485112 (MD5) Previous issue date: 2011-02-18 / The application of organic matter has been recognized as an efficient strategy for plant parasite nematode control, contributing for reduction of chemical products and their environmental impacts. In this context, the vinasse presents a high potential for use in alternative management of these important parasites due to the high volume of organic matter. The objectives of the present study were to evaluate nematode communities in soil and roots, correlating changes in physical and chemical soil attributes and nematode communities in function of irrigation with vinasse; evaluate the fertirrigation effect in vertical distribution of nematode communities in soil and in the spatial variability of the plant parasitic nematodes. Evaluations based on physical (soil humidity, density, porosity, granulometry, penetration resistance (MPa) and particle density) and chemical (pH, H, Al, P, Ca, Mg, Na, K and C-CO2 evolution) soil attributes and nematode community in soil and roots, carried out 30 days before and 30 and 90 days after sugarcane cut and vinasse application. There was reduction in total amount of nematode after vinasse irrigation. Soil density and Ca++ level correlated positively with the total amount of nematode and, in particular, the plant parasitic nematodes. Inversely, the organic matter correlated negatively with the free-living nematodes, plant parasitic nematodes and consequently the total amount of nematodes in soil, but presented positive correlation with endoparasites in roots. Changes in time on C-CO2 evolution were not significant neither the correlation with the taxa or trophic groups. The vertical irrigation effect on soil was signalized on Pratylenchus and Dorilamidae distribution and soil density. The higher changes in nematode densities in soil were attributed to depth, being layers of 10 and 20 cm the most crowded. According to semivariograms, endoparasite distribution fitted to spherical before and 30 days after and before vinasse application. However, ectoparasite distribution, fitted to spherical model before irrigation, shifted to exponential 30 days before vinasse application. Spatial dependence of ectoparasites was moderate after and before irrigation. Endoparasites presented moderate dependence before and weak after irrigation. At 90 days after irrigation, endo and ectoparasites did not present correlation among points sampled showing pure nugget effect. / O emprego de matéria orgânica tem sido preconizado com destacada eficiência para controle de fitonematóides, contribuindo para a redução do uso de produtos químicos e os conseqüentes impactos ao meio ambiente. Nesse contexto, a vinhaça apresenta grande potencial no manejo alternativo destes fitopatógenos, devido ao grande aporte de matéria orgânica em sua composição. Os objetivos do presente estudo foram avaliar as comunidades de nematóides no solo e raízes, correlacionando variações nos atributos físicos e químicos do solo e nas comunidades de nematóides em função da fertirrigação com vinhaça; avaliar o efeito da fertirrigação na distribuição vertical da nematofauna do solo e na variabilidade espacial dos fitonematóides. As avaliações fundamentaram-se em atributos físicos (umidade, densidade, porosidade, granulometria, resistência do solo a penetração e densidade de partícula) e químicos (pH, H, Al, P, Ca, Mg, Na, K e evolução C-CO2) do solo e caracterização da nematofauna, efetuadas 30 dias antes e 30 e 90 dias após o corte da cana e aplicação de vinhaça. Houve redução no número total de nematóides após a fertirrigação com vinhaça. A densidade do solo e o nível de Ca++ correlacionaram-se significativamente com o total de nematóides e, em particular, com os fitoparasitos. A matéria orgânica correlacionou-se negativamente com nematóides de vida livre, fitoparasitos e nematóides totais do solo, mas apresentou correlação positiva com os endoparasitos encontrados nas raízes. A evolução de C-CO2 não variou significativamente entre os períodos nem apresentou correlações com os taxa ou grupos tróficos. O efeito vertical da fertirrigação no solo foi constatado apenas nas distribuições de Pratylenchus e Dorylaimidae e na densidade do solo. As maiores variações nas densidades populacionais de nematóides no solo ocorreram nas camadas de 10 e 20 cm. A distribuição dos endoparasitos ajustou-se ao modelo esférico 30 dias antes e após a aplicação de vinhaça. Já os ectoparasitos, ajustado ao modelo esférico antes da fertirrigação, mudou para exponencial 30 dias após a aplicação de vinhaça. Os ectoparasitos apresentaram dependência espacial moderada antes e após a irrigação com vinhaça e, os endoparasitos, moderada antes e fraca 30 dias após a irrigação. Aos 90 dias após a irrigação, os endoparasitos e ectoparasitos não apresentaram correlações entre os pontos amostrados, revelando efeito pepita puro.