The use of entomopathogenic fungi to control agriculturally important pests, both in greenhouses and in the field, has been demonstrated by various authors for a number of years. This has been brought about by the development of resistance in certain pest species to chemical applications and a growing public awareness of the safety implications of residual insecticides. Several entomopathogenic fungi were tested against four insect pests found in the Republic of South Africa (RSA), the greenhouse whitefly, Trialeurodes vaporariorum, the rice weevil, Sitophilus oryzae, the grapevine mealybug, Planococcusficus and the sugarcane stem borer, Eldana saccharina. Further concentration, temperature and humidity studies were conducted with selected isolates on the rice weevil, S. oryzae. Sitophilus oryzae is considered one ofthe most important pests of stored grain. Several fungal isolates were tested against the rice weevil, four of which, B1, PPRI 6690, PPRI 6864 and PPRI 7067, were selected for further testing based on the mortality results over a 21 d period. Varying conidial concentrations were applied and at high doses of 1x10 -6 conidia ml -1 with mortality rates of to 84% achieved. LT 50 values ranged from 6 - 68d. Increased spore concentration resulted in an increase in overall mortality. Temperature and humidity was found to affect the infection potential of the four isolates tested. Four temperatures ranging from 15 - 30°C were tested. The highest mortality rates were obtained at 25°C where mortality ranged from 46 - 65% in 14d. Mortality rates decreased with decreasing temperature, and no mortality was recorded at 30°C. Temperature was found to significantly alter the LT 50 values, increasing the LT 50 with decreasing temperatures. Decreasing the humidity resulted in an increased LT 50 and a reduction in the overall mortality rates. The mortality of S. oryzae ranged according to the RH and isolate. Isolates Bland PPRI 6690 resulted in the highest mortalities of 80 and 83% at 92.5% RH, with LT 50's of 6.3d and 6.4d, respectively. Several entomopathogenic fungi were tested against T vaporariorum, P. ficus and E. saccharina, three key pests of South African crops. Nine fungal isolates were tested against the greenhouse whitefly, T vaporariorum, with mortalities ranging from 26.7 - 74.7% over 14d. Beauveria bassiana Isolates Bl and PPRl 6690 produced the highest mortality rates and were recommended for further pathogenicity testing against T. vaporariorum. Planococcus ficus is a common pest ofvineyards in the Western Cape Province, South Africa. Nine entomopathogenic fungi were screened against P.ficus, only two of which produced mortality. Eldana saccharina is a stalk borer, which infests sugarcane in large areas of Southern Africa. Five isolates were tested against second and third instar larvae, three of which, B1, PPRl 6864 and PPRl 6690 resulted in mortalities. Mean percentage mortality was low for all three isolates. From the study it was evident that two of the isolates tested, Bland PPRI 6690 (B. bassiana), showed potential against three of the four pests, and two isolates of Lecanicillium lecanii caused mortality in P. ficus. Further research and understanding of the effect of environmental conditions, spore concentration and epizootic potential would result in the further development of these isolates as future biological control agents. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os begomovÃrus, vÃrus da famÃlia Geminiviridae transmitidos por mosca branca, tÃm emergido como sÃrios patÃgenos de culturas agronÃmicas e hortÃcolas em regiÃes tropicais e subtropicais de muitos paÃses em todo o mundo. A partir da dÃcada de 80, tÃm aumentado os relatos da disseminaÃÃo da mosca branca, Bemisia argentifolii, e de begomovÃrus provocando impacto devastador nas regiÃes em que ocorrem. No Brasil, estes patÃgenos tÃm sido limitantes para a produÃÃo de tomate (Lycopersicon esculentum) em vÃrias Ãreas de cultivo com incidÃncia crescente desde 1994. No presente trabalho, plantas de tomate exibindo sintomas de infecÃÃo provocada por vÃrus como mosaico, clorose internerval, enrolamento do limbo foliar e reduÃÃo do crescimento, foram coletadas em lavouras de tomate indÃstria em AnÃpolis-GO. O vÃrus foi identificado como pertencente ao gÃnero Begomovirus mediante tÃcnica de PCR usando oligonucleotÃdeos especÃficos que amplificaram fragmentos dos componentes A e B do genoma viral. No capÃtulo I sÃo apresentados os resultados da caracterizaÃÃo molecular e no capÃtulo II, os dados da determinaÃÃo do cÃrculo de hospedeiros e da investigaÃÃo da relaÃÃo do begomovÃrus com o vetor Bemisia argentifolii. O isolado denominado GOANPL, foi clonado e parcialmente seqÃenciado tendo sido obtidas as seqÃÃncias nucleotÃdicas dos genes da capa proteica, Rep e de toda a regiÃo intergÃnica, em um total de 2.130 nucleotÃdeos. A anÃlise comparativa das seqÃÃncias revelou que, em geral, o GOANPL possui relacionamento genÃtico distante com begomovÃrus da Ãsia, Europa e Ãfrica sendo mais prÃximo das espÃcies do Brasil, particularmente, com os begomovÃrus identificados em Minas Gerais (TRMV) e no Distrito Federal (DF-BR2). Com este Ãltimo, apresentou alta homologia em todo o genoma podendo vir a constituir, com o mesmo, uma nova espÃcie. A determinaÃÃo do cÃrculo de hospedeiros do GO-ANPL foi realizada inoculando-se 46 espÃcies vegetais pertencentes a nove famÃlias botÃnicas, sob duas modalidades de inoculaÃÃo: mecÃnica e com a mosca branca. Constatou-se que o GO-ANPL infecta, preferencialmente, plantas da famÃlia Solanaceae como Nicotiana benthamiana, Datura stramonium e Nicandra physalodes. O nÃmero de espÃcies infectadas com o inseto vetor foi superior ao obtido pela inoculaÃÃo mecÃnica e diferiu dos resultados obtidos para outros isolados de begomovÃrus de tomate no Brasil. Os testes foram todos confirmados com hibridizaÃÃo com sondas moleculares, em \"dot blot\"No estudo da relaÃÃo vÃrus-vetor, foram investigados o perÃodo de acesso de aquisiÃÃo do vÃrus (PAA), o perÃodo de acesso de inoculaÃÃo do vÃrus (PAI), e o perÃodo de latÃncia do vÃrus na fase adulta do vetor, empregando-se cinco moscas/planta de tomate \'Santa Clara\' em todos os tratamentos. Para a definiÃÃo do PAA e do PAI, foram testados nove diferentes perÃodos de tempo: 0,25, 0,5, 1, 2, 4, 8, 16, 20 e 24 horas. Nos testes para determinaÃÃo do PAA, apÃs cada um desses perÃodos seguiu-se uma inoculaÃÃo de 48 horas e para definiÃÃo do PAI, antes de cada perÃodo antecedeu-se um perÃodo de acesso de aquisiÃÃo fixo de 72 horas. Constatou-se que o PAA mÃnimo da mosca branca foi de apenas 0,25 hora, com o qual foram obtidas 6% de plantas infectadas. O percentual de plantas aumentou de 6 para 65% com a extensÃo do PAA de 0,25 para 24 horas. Com relaÃÃo ao perÃodo de acesso de inoculaÃÃo do vÃrus, foram registrados 18% de plantas infectadas com o PAI de 0,5 hora. O percentual elevou-se para 67% quando 24 horas de PAI foram concedidos. Valores isolados de 90 e 100% na transmissÃo viral, tambÃm foram observados. O tÃrmino do perÃodo latente do vÃrus no vetor ocorreu 16h apÃs a aquisiÃÃo do mesmo em planta infectada, considerando os 3% de infecÃÃo observados nas plantas inoculadas. Os dados obtidos indicam que a interaÃÃo vÃrus-vetor à estabelecida desde a fase inicial de desenvolvimento do inseto. Como parte do estudo dessa interaÃÃo, avaliou-se a presenÃa do begomovÃrus GO-ANPL em todas as fases de desenvolvimento do inseto vetor (ovo, 1 ao 4 Ãnstar e adulto) na planta infectada, em adultos com diferentes PAA, na progÃnie de fÃmeas virulÃferas e em adultos cujos estÃgios ninfais desenvolveram-se em tomateiro infectado. A tÃcnica PCR foi empregada para a detecÃÃo do GO-ANPL em mais de 2.500 espÃcimens testados. O vÃrus foi detectado em ninfas do 1 ao 4 Ãnstar que se alimentaram em plantas de tomate infectada, contudo, em ovos provenientes de avirulÃferas, os quais foram ovipositados em planta infectada e coletados apÃs sete dias, o vÃrus nÃo foi detectado. A transmissÃo à progÃnie foi constatada pela detecÃÃo do vÃrus em ovos, ninfas e adultos que se desenvolveram em planta nÃo hospedeira do vÃrus. A transmissÃo transestadial ocorreu com Ãndice elevado e, ao lado da transmissÃo à progÃnie, indica que a retenÃÃo do vÃrus à uma etapa importante da interaÃÃo vÃrusâvetor. A transmissÃo do vÃrus para mudas de tomate, a partir de adultos da progÃnie de fÃmeas virulÃferas, nÃo foi constatada. Contudo, transmissÃo para tomateiro em um percentual de 33% foi verificado nos casos em que a inoculaÃÃo das plantas foi realizada pelos adultos que retiveram o vÃrus da sua fase imatura (transestadial) / The whitefly-transmitted viruses from the family Geminiviridae, genus Begomovirus, have been reported as an economically important pathogen group that affect important crops in tropical and subtropical countries. Since the beginning of the 1980 decade, the occurrence of the whitefly associated to Begomovirus infection has drastically increased worldwide. In Brazil, these pathogens have been responsable for severe economical losses in tomato (Lycopersicon esculentum) orchards and the production has hampered since 1994. In this work, infected tomato plants showing symptoms, such as mosaic, intervein clearing, leaf curling and growth reduction were collected in tomato orchards in AnÃpolis, State of GoiÃs. The virus was identified as a member of the genus Begomovirus by PCR reaction, using specific primers to amplify fragments of A and B components of the virus DNA genome. The Chapter I of this thesis presents the results of the molecular characterization of the virus and the Chapter II shows the determination of its host range and the relationship with its natural vector Bemisia argentifolii. The virus isolate denoted GO-ANPL was cloned and partially sequenced. Part of the sequenced genome (2.180 nucleotides long) corresponded to the coat protein and Rep genes and comprised the entire intergenic region. Sequence comparison revealed that the GO-ANPL isolate is distantly related to the begomoviruses found in Asia, Europe and Africa, and it is related to other begomoviruses reported in Brazil. The virus isolate showed to be more closely related to viruses found in the State of Minas Gerais (TRMV isolate) and in the Federal District (isolate DF-Br2). The highest homology was observed with the isolate DF-Br2 and it may represent a new specie of the genus Begomovirus. In order to determine the virus host range, 46 plant species from nine different botanical families were mechanically and using the virus vector inoculated. The GO-ANPL isolate preferentially infected plants of the family Solanaceae as Nicotiana benthamiana, Datura stramonium and Nicandra physalodes. The number of infected plants was higher when they were inoculated by the virus vector, and the results were distinct from those obtained for other begomoviruses reported in Brazil. Viruses infections were all confirmed by dot blot hybridization using specific molecular probes to the virus. 4 To study virus/vector interaction, the acquisition access period (AAP), inoculation access period (IAP), and the latent period were determined transfering five whiteflies per plant and using tomato cv. Santa Clara as the host. For the AAP and IAP, nine different time periods were tested: 0.25, 0.5, 1, 2, 4, 8, 16, 20 and 24 h. The minimal AAP determined was 0.25 h, after which, 6% of the tested plants became infected. The number of infected plants increased to 65% with an AAP of24h.Afteran IAP of 0.5 h, 18% of the plants were infected and their number increased to 67% after an IAP of 24 hours. The latent period was considered to be 16 h, after which, 3% of the inoculated plants became infected. The results of AAP, IAP and latent period seem to indicate an early interaction between virus and vector starting at early stages of vector development. The presence of the GOANPL was determined in all stages of the vector (eggs, 1st to 4th instar and adults) in infected plants, in adults under different AAPs, in the progenies of viruliferous females, and in adults originated from nymphs developed from infected plants. More than 2.500 insects were tested by PCR to detect the GO-ANPL isolate. The virus was detected in nymphs from the 1st to 4th instar that had fed in infected plants and no virus was found in eggs from aviruliferous female that had been laid in infected plants. Transmission to the progenies was observed, since the virus was detected in all stages of insect development from eggs to adults. High level transmission was also observed in newly emerged adults that had acess to virus-infected plants as immatures. This fact, in addition to the transmission to the progenies, suggests that virus retention is an important part of virus/vector interaction. No transmission was observed from adults originated from viruliferous females. However, 33% of virus transmission was obtained when adults that retained virus from their early larval stages were employed