Cultivated tomato plants are extremely susceptible to the two-spotted spider mite Tetranychus urticae Koch. Selection for pest resistance is usually a crucial step required to achieve successful genetic resistance transfer from wild into cultivated tomato genotypes. S. habrochaites LA2329, a wild relative of tomato, is highly resistant to arthropods. Its resistance has been attributed to the presence of a high density of type IV and type VI trichomes and abundant production of 7-epi-zingiberene, a sesquiterpene hydrocarbon. The interspecific backcross hybrids used in this research were derived from the cross between the wild relative tomato, S. habrochaites LA2329, and the cultivated tomato, S. lycopersicum ‘Zaofen 2’ (ZH2). This population has been directly selected for type IV trichome density and zingiberene. The arthropod resistance status of the backcross hybrids was unknown when this research was initiated. Thus, the main objective of the research was to verify the transfer of arthropod resistance from S. habrochaites to cultivated tomato. The effects of glandular trichome densities and leaf zingiberene contents on spider mite behavior and biology were also explored. Also, the chemical composition of the trichome secretions in the wild tomato donor is segregating for presence and abundance of sesquiterpenoids related to zingiberene. The bioactivity of these sesquiterpenoids was explored in this research. To evaluate the relative bioactivities of zingiberene alcohol and 7-epizingiberene, extracted from glandular trichomes of Solanum habrochaites accession LA2329, as well as alpha-zingiberene obtained from ginger oil, these were purified by silica gel chromatography and bioassayed with two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae) using a bean leaf disc bioassay. Zingiberene alcohol was most efficacious and alpha-zingiberene, was least efficacious, while the efficacy of 7-epizingiberene was intermediate. Thus, tomato breeders should consider introgression of the genes responsible for the oxidation of 7-epizingiberene into zingiberene alcohol to potentially improve the spider mite resistance of cultivated tomato. Also, it is possible that this compound may be exploited as eco-biopesticide approach for integrated pest management against a broad spectrum of herbivorous pests. To verify transfer of arthropod resistance, a bioassay utilizing whole leaves was employed. Nine hybrids (BC3F3 and BC3F4) were chosen for this bioassay, based on variation of type IV trichome density and zingiberene concentration among the hybrids. The experiment also included three susceptible and three resistant control plants. Mite responses on some of the hybrids were similar to those on the resistant wild donor parent, S. habrochaites, as indicated by number of leaflet surfaces infested by mites, degree of mite webbing and feeding damage. Egg density on four backcross hybrids was similar to that on the S. habrochaites resistant controls. Based these results, we concluded that resistance had been successfully transferred from the wild accessions to the hybrids by deployment of backcrossing and indirect selection. There was a significant negative correlation of almost all mite behavioral and biological responses with Type IV trichome density and zingiberene content. This bioassay illuminated behavioral variations of mites associated with presence or absence of leaf compounds and glandular trichome densities. Also, the results support the idea that introgression of type IV trichomes and zingiberene has led to effective spider mite resistance. In another bioassay-based experiment to verify transfer of resistance, seven interspecific backcross hybrids (BC3F2), the resistant parent LA2329, and two susceptible cultivated tomato lines, the recurrent parent ZH2 and ‘Small Roma’, were used in thumbtack bioassays. Mite movement was measured by imaging bioassayed leaves at 15, 20, 30, 45, and 60 min intervals. In addition to confirming transfer of spider mite resistance, other objectives included determination of the relative contributions of type IV and VI trichome densities and leaf compounds to mite behavior over time intervals. Our findings confirmed the transfer of mite repellency from the wild resistant parent to advanced backcross hybrids. Several backcross hybrids performed similarly to the wild donor parent, displaying shorter distances traveled on the leaves after 15 and 30 min. The type IV and type VI trichome densities as well as zingiberene contents had a significant positive correlation with the number of spider mites remaining on tack. There was a significant negative correlation of type IV density and zingiberene concentration with the total distance travelled by mites for both the abaxial and adaxial surfaces across most time intervals. Stepwise multiple regression analysis showed that the type IV trichome density was the most critical factor, and zingiberene content was a secondary factor across over most time intervals. T. urticae remained longer on the thumbtack heads and traveled shorter distances on the leaf surface of the wild donor parent LA2329 and the interspecific hybrids compared to S. lycopersicum leaves. These results indicated that introgression of genetic resistance, especially repellence, against spider mite from the wild relative into cultivated tomato varieties has been successfully achieved. In conclusion, trichome type IV and/or zingiberene content has been successfully transferred from the wild relative into interspecific tomato hybrids, and the hybrids show significant adverse impact on spider mite behavior and/or biology in whole leaf and thumbtack bioassays. Type IV trichome density is the most crucial factor in mite deterrence while zingiberene seemed to be a second key factor across most of time durations for both surfaces. Collectively, several backcross hybrids had similar leaf characteristics to the S. habrochaites LA2329, also may be a potential source of resistance to other insect pests.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-02-15T13:45:43Z No. of bitstreams: 1 texto completo.pdf: 419660 bytes, checksum: e5b57e1bf91b05cd94f22bff2b654578 (MD5) / Made available in DSpace on 2016-02-15T13:45:43Z (GMT). No. of bitstreams: 1 texto completo.pdf: 419660 bytes, checksum: e5b57e1bf91b05cd94f22bff2b654578 (MD5) Previous issue date: 2015-07-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Anticarsia gemmatalis Hubner (Lepidoptera: Erebidae), com ampla distribuição geográfica, pode causar desfolhas extremas em oleaginosas. Parasitoides são agentes de controle, que regulam populações de insetos-praga. Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) tem sido alvo de estudos devido à sua capacidade de parasitismo e de busca. Compostos naturais com atividade inseticidas são estratégias promissoras para o controle de pragas, serem menos nocivos a humanos e mais biodegradáveis ao ambiente. O objetivo deste trabalho foi avaliar a toxicidade de extratos de Solanum habrochaites Knapp & Spooner (Solanaceae) para ovos e lagartas de A. gemmatalis e a seletividade ao parasitoide P. elaeisis. A pesquisa foi realizada no Laboratório de Análise e Síntese de Agroquímicos do Departamento de Química e no Laboratório de Controle Biológico de Insetos (LCBI) na Universidade Federal de Viçosa em Viçosa, Minas Gerais, Brasil. Indivíduos de A. gemmatalis e P. elaeisis foram obtidos das criações massal do LCBI. Folhas e ramos de S. habrochaites foram secas em estufa ventilada a 40 °C e a extração foi realizada por maceração a frio utilizando-se etanol 98%. Extratos brutos de S. habrochaites foram particionados em diclorometano (EDSH), acetato de etila (EASH) e metanol (EMSH) e submetidos ao fracionamento por cromatografia em coluna de sílica-gel. Ovos e lagartas de terceiro estádio de A. gemmatalis e adultos de P. elaeisis foram expostos aos concentrados de 1, 2, 10, 15 e 20% (v/v) dos extratos para obtenção das CL 50 e CL 90 . Análises do extrato de S. habrochaites em diclorometano por cromatografia a gás acoplada a espetrometria de massas permitiram identificar 14 moléculas, entre elas, hidrocarbonetos de cadeia longa, ésteres de ácidos graxos de cadeia longa, metil cetonas e aldeídos. O extrato bruto e as frações em diclorometano de S. habrochaites, foram tóxicos para ovos de A. gemmatalis, CL 50 e CL 90 de 1,67% e 7,27%; 2,83% e 6,95%, respectivamente. Extrato em acetato de etila foi toxico para P. elaeisis, com CL 50 15,07% e CL 90 37,05%, os demais extratos foram seletivos ao parasitoide. O extrato em diclorometano foi letal para imaturos de A. gemmatalis com CL 50 de 5,70% e CL 90 28,52% e seletivo para P. elaeisis. A eficiência do extrato de S. habrochaites em diclorometano para ovos e imaturos de A. gemmatalis e sua seletividade a P. elaeisis mostram seu potencial para programas de controle de pragas. / Anticarsia gemmatalis Hubner (Lepidoptera: Erebidae), with wide distribution, can cause extreme defoliation in soybean plants. Parasitoids are biocontrol agents that regulate populations of insect pests. Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) has been investigated due to its searching and parasitism capacity. Natural compounds with insecticidal activity are promising strategies for pest control, being less harmful to humans and the environment and more biodegradable. The objective of this study was to evaluate the toxicity of Solanum habrochaites Knapp & Spooner (Solanaceae) extracts to eggs and caterpillars of A. gemmatalis and selectivity to the parasitoid P. elaeisis. The survey was conducted in the Laboratory of Analysis and Agrochemical Synthesis of the Department of Chemical and at the Laboratory of Biological Control of Insects (LCBI) in Viçosa Universidade Federal de Viçosa in Viçosa, Minas Gerais, Brazil. Individuals of A. gemmatalis and P. elaeisis were obtained from mass rearing of the LCBI. Leaves and stems of S. habrochaites were dried in a ventilated oven at 40 °C and the extraction performed by cold maceration using 98% ethanol. Crude extracts of S. habrochaites was partitioned in dichloromethane (EDSH), ethyl acetate (EASH) and methanol (EMSH) and subjected to fractioning by silica gel column chromatography. Eggs and third instar A. gemmatalis larvae and adults P. elaeisis were exposed to the concentrations of 1, 2, 10, 15 and 20% (v/v) to obtain the CL 50 and CL 90 for S. habrochaites extracts. Analysis of S. habrochaites in dichloromethane extract by gas chromatography coupled to mass spectrometry permitted the identification of 14 molecules, including long chain hydrocarbons, esters of long chain fatty acids, methyl ketones and aldehydes. The crude extract and fractions in dichloromethane of S. habrochaites were toxic to A. gemmatalis eggs with LC 50 and LC 90 of 1.67% and 7.27%; 2.83% and 6.95%, respectively. Extract in ethyl acetate was toxic to P. elaeisis with CL 50 CL 90 15.07% and 37.05%, the remaining extracts were selective to the parasitoid. The dichloromethane extract was lethal to immature A. gemmatalis with CL 50 CL 90 of 5.70% and 28.52% and selective for P. elaeisis. The efficiency of S. habrochaites extract in dichloromethane to eggs and immature A. gemmatalis and its selectivity to P. elaeisis show its potential for pest control programs.

Se determinó la respuesta de resistencia de 10 patrones de tomate a una población avirulenta de Meloidogyne javanica en maceta. Los ensayos se realizaron en primavera, cuando las temperaturas permitían la expresión fenotípica de la resistencia del gen Mi-1 (28˚C), en verano sometidos a altas temperaturas y en campo, exponiéndolos a altas densidades poblacionales del nematodo. A temperaturas inferiores a 28˚C los patrones mostraron gran variabilidad en la respuesta de resistencia que osciló entre alta y moderadamente resistente (PG-76, Gladiator, MKT-410; Brigeor, 42851, 43965, Big Power y He-man), hasta susceptible (Beaufort, Maxifort). Por encima de 28˚C, sólo dos patrones (PG-76 y He-man) inhibieron la reproducción del nematodo. Frente a distintas poblaciones de M. arenaria, M. incognita y M. javanica, el patrón PG-76, fue altamente resistente a todas las poblaciones, Brigeor osciló entre altamente resistente y moderadamente resistente, mientras que Beaufort y Maxifort mostraron menor resistencia o fueron totalmente susceptibles; además ésta varió en función de la población analizada. Se caracterizó molecularmente el locus Mi-1 en los patrones híbridos y cultivares de tomate estudiados. Se emplearon los marcadores moleculares PM3, PMi y Mi23, específicos para la caracterización del locus Mi en patrones híbridos de tomate (S. lycopersicum × S. habrochaites; S. lycopersicum × S. chilense), mediante PCR. También se realizaron análisis bioinformáticos con marcadores específicos (Mint-up/do, C172, C2S4, IMO-F1/R1, y VIGS) para determinar la presencia del gen Mi-1.2 en dichos patrones. Los resultados mostraron que los marcadores PMi y Mi23 amplifican homólogos del gen Mi-1 en S. chilense, S. habrochaites y S. peruvianum y también en S. lycopersicum (marcador Mi23). El marcador PM3 amplificó el gen Mi-1.2 en Beaufort y Maxifort (S. lycopersicum × S. habrochaites) pero no fue efectivo para los híbridos de S. chilense. El marcador molecular PM3, no pudo determinar la expresión de del gen Mi-1.2 en Beaufort y Maxifort por hallarse fuera de la secuencia codificadora (CDS) del gen. Análisis bioinformáticos indicaron que ningún marcador específico diseñado para el gen Mi-1.2, podía este gen de otros homólogos presentes en S. lypcopersicum y S. peruvianum. El nuevo marcador Pau-Do en combinación con el primer C2S4, amplificaron un fragmento de 1.494 pb en la CDS del gen Mi-1.2 en raíces y hojas de Beaufort y Maxifort. La durabilidad de la resistencia del gen Mi-1 después del cultivo reiterado de patrones de tomate se determinó en ensayos de campo durante tres años consecutivos, empleando PG-76 y Brigeor. El patrón PG-76 fue muy resistente después del 1er ciclo de cultivo, pero mostró resistencia intermedia y suscetibilidad al finalizar el 2o y el 3er año de cultivo, respectivamente. El patrón Brigeor y el cultivar de tomate resistente Monika (control) mantuvieron un nivel de resistencia intermedio al final del 3er cultivo, aunque ensayos posteriores confirmaron la aparición de virulencia. Los resultados mostraron que el cultivo reiterado de patrones de tomate resistentes seleccionó rápidamente aislados virulentos de M. javanica. El fenotipo virulento de estas poblaciones se analizó molecularmente con el marcador MVC, diseñado para distinguir las poblaciones virulentas seleccionadas de Meloidogyne de los aislados naturalmente virulentos. Se analizaron dos poblaciones japonesas seleccionadas de M. incognita y M. javanica, tres poblaciones españolas virulentas seleccionadas, una población naturalmente virulenta y una avirulenta (todas M. javanica). Las muestras de ADN se obtuvieron de individuos juveniles o de hembras adultas y se incluyeron muestras de agua sin nematodos (5 µm filtrada) procedentes del drenaje de una maceta con una planta infectada por una población virulenta japonesa. El marcador MVC amplificó ADN en las muestras de agua pero no en las que sólo contenían ADN de nematodos. Las secuencias de ADN mostraron una estrecha correlación con diversas proteínas de especies de betaproteobacterias. Los experimentos revelaron que el marcador de MVC no está relacionado con un gen de virulencia del nematodo (avr) sino con betaproteobacterias. Finalmente, se estudió la existencia de homólogos del gen Mi en las especies de tomate silvestre Solanum chilense, S. habrochaites, S. peruvianum y S. huaylasense. La respuesta de resistencia de la variedad LA-1358 de S. huaylasense varió en función de la especie del nematodo estudiada: fue resistente frente a M. arenaria y susceptible frente a M. javanica. La reproducción de M. incognita fue muy variable y no difirió de la reproducción alcanzada en los dos cultivares empleados como controles. / The response of 10 Mi-1 tomato rootstocks to a Mi-avirulent population of M. javanica was determined in pot tests conducted in a greenhouse in spring when temperatures remained below the Mi-1 functionality resistance threshold (28 ˚C), and in summer when daily temperatures exceeded the Mi-1 expression threshold. Rootstocks were also evaluated in the field exposing them to high population densities of the nematode. Results on infectivity and reproduction below 28 ˚C indicated a wide variability in the resistance response of the rootstocks ranging from highly or intermediate resistance (PG-76, Gladiator, MKT-410; Brigeor, 42851, 43965, Big Power and He-man) to fully susceptible (Beaufort and Maxifort). At high temperature conditions, only PG-76 and He-man inhibited the reproduction of M. javanica. Rootstocks PG-76, Brigeor, Beaufort and Maxifort were challenged to different populations of M. arenaria, M. incognita and M. javanica. Rootstock PG-76 was highly resistant to all the populations tested, whereas the response of Brigeor ranged from highly to moderate resistance; the resistance response of rootstocks Beaufort and Maxifort varied according to the population tested. Molecular characterization of the resistance phenotype was performed for all the tomato hybrid rootstocks and cultivars tested. The markers PM3, PMi, Mi23, for the characterization of the Mi-locus of hybrid tomato rootstocks (S. lycopersicum × S. habrochaites and S. lycopersicum × S. chilense) were used for PCR reactions. In silico analyses were done with specific markers for the Mi-1.2 gene (Mint-up/do, C1/2, C2S4, IMO-F1/R1, and VIGS). Markers PMi and Mi23 were polymorphic for the Mi-1 locus in wild Solanum species (S. chilense, S. habrochaites, and S. peruvianum) and for S. lycopersicum (marker Mi23). Marker PM3 detected the Mi-1.2 gene in S. lycopersicum × S. habrochaites hybrid rootstocks, but not in the S. chilense hybrids. As marker PM3 is located outside the coding sequence of the Mi-1.2 gene, expression of this homolog could not be determined in Beaufort and Maxifort. In silico results indicated that none of the available markers for the Mi-1.2 gene could distinguish this homolog from the other Mi-homologs from S. lypcopersicum and S. peruvianum species. A new marker Pau-Do, in combination with C2S4, was designed to amplify in CDS of the Mi-1.2 gene. Amplification with these primers of cDNA from Beaufort and Maxifort indicated that the Mi-1.2 gene was expressed in both rootstocks, despite their susceptible phenotypic response to some Meloidogyne populations. The durability of the Mi-1 gene after repeated cultivation of resistant tomato rootstocks (PG-76 and Brigeor) was determined through field trials during three consecutive years. Rootstock PG-76 responded as highly resistant after the first cropping cycle, although it became fully susceptible after the second and the third cropping cycles. Rootstock Brigeor and the resistant tomato cultivar Monika (control), retained intermediate resistance levels at the end of the third year. Bioassays confirmed that selection of virulence occurred more rapidly in plots with rootstock PG-76 followed by Brigeor and the resistant tomato cultivar Monika. The virulent phenotype of the selected M. javanica populations in the field experiments was determined with MVC molecular marker, designed to distinguish selected from naturally virulent populations of Meloidogyne spp. The populations analyzed included two Japanese selected virulent populations, and the three virulent populations selected in the field trials, and one naturally virulent population and one avirulent population from Spanish. DNA samples were obtained from individual juveniles (J2) or adult females from all the selected virulent populations. Experiments included water samples free of nematodes (5-µm filtered), obtained from the draining-water of a plant infected by a Japanese selected virulent population. Amplification of DNA only occurred in samples of filtered water, but not in those containing only nematode genetic material. Sequencing and BLAST of the DNA fragments amplified by the MVC molecular marker, established a strong correlation of the amplified bands with proteins from betaproteobacteria species Overall, these results showed that the MVC marker is not related to a nematode virulence gene (avr) but to betaproteobacteria. New root-knot nematode resistant Mi-homologs were searched in accessions of the wild Solanum species. The S. huaylasense accession LA-1358 reduced reproduction of a population of M. arenaria to similar levels than the resistant tomato cultivar Anairis. Nevertheless, the resistance response of S. huaylasense accession LA-1358 was also nematode-species specific.