Jamaican farmers are experiencing constraints to hot pepper (Capsicum chinense) export production due to a quarantine pest -- the gall midge (Contarinia lycopersici; Prodiplosis longifila). There is a threat of gall midge introduction into the United States, where the insect pest is not known to occur. This research tests the significance of a range of variables to gall midge incidence. The purpose was to explain the spatial patterns that result from the relationships between gall midge incidence in hot pepper production and production methods and/or environmental conditions. There were three components to the sample of 47 farm visits: the interview, the hot pepper sampling, and the measurements of physical and locational attributes. Producers responded to questions about production methods, marketing, and quarantine issues. The percent of infested fruits per plot was calculated. GPS was used to record farm location. Using ArcView, environmental and climatic datasets were overlaid with farm locations and their attributes. Multiple regression was used to measure significance of variables to gall midge incidence. Cluster analyses were used to demonstrate the spatial patterns of the variability of gall midge incidence and its associated variables. There was significant effect on incidence by farm elevation, observance of pesticide-use recommendations, producer awareness of pre-clearance fumigation, and the use of intercropping in hot pepper production. / Master of Science

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / H13, a dominant resistance gene transferred from Aegilops tauschii into wheat (Triticum aestivum), confers a high level of antibiosis against a wide range of Hessian fly (HF, Mayetiola destructor) biotypes. Previously, H13 was mapped to the distal arm of chromosome 6DS, where it is flanked by markers Xcfd132 and Xgdm36. A mapping population of 1,368 F2 individuals derived from the cross: PI372129 (h13h13) / PI562619 (Molly, H13H13) was genotyped and H13 was flanked by Xcfd132 at 0.4cM and by Xgdm36 at 1.8cM. Screening of BAC-based physical maps of chromosome 6D of Chinese Spring wheat and Ae. tauschii coupled with high resolution genetic and Radiation Hybrid mapping identified nine candidate genes co-segregating with H13. Candidate gene validation was done on an EMS-mutagenized TILLING population of 2,296 M₃ lines in Molly. Twenty seeds per line were screened for susceptibility to the H13-virulent HF GP biotype. Sequencing of candidate genes from twenty-eight independent susceptible mutants identified three nonsense, and 24 missense mutants for CNL-1 whereas only silent and intronic mutations were found in other candidate genes. 5’ and 3’ RACE was performed to identify gene structure and CDS of CNL-1 from Molly (H13H13) and Newton (h13h13). Increased transcript levels were observed for H13 gene during incompatible interactions at larval feeding stages of GP biotype. The predicted coding sequence of H13 gene is 3,192 bp consisting of two exons with 618 bp 5’UTR and 2,260 bp 3’UTR. It translates into a protein of 1063 amino acids with an N-terminal Coiled-Coil (CC), a central Nucleotide-Binding adapter shared by APAF-1, plant R and CED-4 (NB-ARC) and a C-terminal Leucine-Rich Repeat (LRR) domain. Conserved domain analysis revealed shared domains in Molly and Newton, except for differences in sequence, organization and number of LRR repeat in Newton. Also, the presence of a transposable element towards the C terminal of h13 was indicative of interallelic recombination, recent tandem duplications and gene conversions in the CNL rich region near H13 locus. Comparative analysis of candidate genes in the H13 region indicated that gene duplications in CNL encoding genes during divergence of wheat and barley led to clustering and diversity. This diversity among CNL genes may have a role in defining differences in the recognition specificities of NB-LRR encoding genes. Allele mining for the H13 gene in the core collection of Ae. tauschii and hexaploid wheat cultivars identified different functional haplotypes. Screening of these haplotypes using different HF biotypes would help in the identification of the new sources of resistance to control evolving biotypes of HF. Cloning of H13 will provide perfect markers to breeders for HF resistance breeding programs. It will also provide an opportunity to study R-Avr interactions in the hitherto unexplored field of insect-host interaction.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / C. Michael Smith / The tools for arthropods with sucking-mouth parts to attack hosts are mainly in the saliva. For plant-sucking insects, these salivary secretions are primarily produced in the salivary glands. Secreted proteins (also referred to as salivary gland secretomes) are among the important components in the saliva of sucking insects. Gall midges (Cecidomyiidae), a large family of plant-sucking insects, apparently secrete proteins (some of them are effector proteins) into host tissues, inducing various forms of plant outgrowth (galls). Three major insect pest species in the genera Mayetiola, the stem gall midges, are known to produce saliva that can reprogram plant cells and manipulate the host plant growth, causing serious damage to the plants of small grains. The three pest species are the Hessian fly (Mayetiola destructor), the barley midge (Mayetiola hordei), and the oat midge (Mayetiola avenae). Another economically important species of this gall midge family is the wheat midge (Sitodiplosis mosellana). It is a major insect pest of spring wheat and feeds on wheat heads, causing damage to the developing wheat seeds. A global analysis of the salivary gland secretome of first instar larvae of the Hessian fly, (a member of Mayetiola and) a model species for studying insect-plant interactions, has previously revealed a large number of genes encoding Secreted Salivary Gland Proteins, so called SSGPs. For comparison, we conducted analyses on transcripts encoding SSGPs from salivary glands of the first instar larvae of the wheat midge, barley midge, and oat midge. In the first chapter, a transcriptomic analysis of wheat midge has been conducted. In this analysis, a total of 3,500 cDNA clones were sequenced, and 1,301 high quality sequences were obtained and approximately 25% of the cDNAs (with high quality sequences) encoded SSGPs. The SSGPs were grouped into 97 groups based on sequence homology. Among the SSGP-encoding transcripts, 206 encoded unique proteins with no sequence similarity to any known protein and 29 encoded proteins similar to known proteins including proteases, serpines, thioesterases, ankryins, and feritins. The compositions of SSGP transcripts from the wheat midge were then compared with that of Hessian fly. The analyses have identified many common characteristics between the species. Despite these commonalities, no sequence similarity was found between SSGPs from wheat midge and those from Hessian fly, suggesting that SSGPs from these two insect species perform different functions to manipulate host plants. The second chapter contains results of comparative transcriptomic analyses on the barley and oat midges. A total of 2570 cDNA clones were sequenced from the barley midge, and 743 were high quality cDNA sequences, and the analysis identified 458 cDNA clones encoding SSGPs, of these, 178 encoded unique proteins (also called unigenes). Transcripts encoding SSGPs were grouped into 51 groups based on sequence homology. A total of 3226 cDNA clones were sequenced from oat midge, and 718 cDNA sequences were high quality and used for further analysis. The analysis identified 450 cDNA clones encoding SSGPs. Among the SSGP-encoding transcripts, 194 are unigenes, which were placed into 50 groups. The compositions of SSGP transcripts from the barley and oat midges were then compared with that of Hessian fly. The analysis identified five groups containing 102 (57.3%) unigenes from barley midges and seven groups containing 107 (55.1%) unigenes from oat midges which encode SSGPs that are conserved among the three species. The SSGPs conserved among the three midges are from family one (SSGP-1), family 4 (SSGP-4), family 11 (SSGP-11), and family 71 (SSGP-71). The SSGPs conserved among the three species indicate conserved functions such as a role in plant manipulation. Some SSGP unigenes were found to be conserved between only two species. Specifically, there were eight gene groups which are conserved between two species. Within these eight groups 19 (10.7%) unigenes from the barley midge and 25 (12.9%) unigenes from the oat midge were found to be conserved between only the barley and oat midges, whereas no homologues have been found in the Hessian fly. The remaining unigenes encode SSGPs that are unique to different midge species. The highly divergent SSGP groups that have been identified with no homology among the three midges indicate potential roles of these SSGPs in host specification. Due to the important roles of effector proteins in insect-plant interactions for gall midge species and since no insect effector protein have been identified directly from infested plant tissues so far, I have chosen one of the SSGP family, SSGP-1, which are conserved among all three gall midge species, for further analysis in chapter 4. Members in family SSGP-1 are also the most abundantly expressed at the transcript level. Based on Hessian fly data, family 1 contains seven genes and are named SSGP-1A1, SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1C2, SSGP-1D1, and SSGP-1E1. To detect the presence of these proteins in the infested wheat tissues, and to identify probable targets from wheat that interact with the SSGPs in the feeding site, we have generated and purified recombinant proteins for five of the seven proteins, namely SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1D1, and SSGP-1E1 (since SSGP-1A1 and SSGP-1C2 are very similar to SSGP-1A2 and SSGP-1C1, respectively). Antibodies were produced for the recombinant proteins for western blot analyses and indirect immunostaining. Immunostaining on dissected tissues including salivary glands, guts, and Malpighian tubules from 3-day old larvae, was conducted with antibodies against the five SSGPs, and detected a specific localization of all proteins in salivary glands except SSGP-1E1, which exhibited a weak signal in the foregut, in addition to localization in salivary glands. Western blot analyses demonstrated that these five proteins were expressed in larvae at all stages. The continuous production of these proteins suggests that they play roles in initiation and maintenance in Hessian fly infestation. Consistent with their effector functions, these five proteins were detected for the first time in infested wheat tissues based on western blot analyses. To identify possible target proteins from host plants that interact with SSGP-1 family proteins, in vitro pull-down assays were performed. Putative interacting targets for SSGP-1A2, SSGP-1B1, and SSGP-1C1 have been identified by LC-MS/MS. These putative interaction target proteins included uncharacterized proteins, ribosomal proteins, a lipoxygenase, and a tubulin. Identification of these putative targets provided a base for further confirmation of their interaction with Hessian fly effectors in the future.

The African Rice Gall Midge (AfRGM), Orseolia oryzivora Harris and Gagné (Diptera: Cecidomyiidae), is an endemic rice pest found throughout Africa. The failure of most other control methods imposes the need to use crop resistance. This study was initiated: (1) to develop an accurate method for assessing damage caused by AfRGM; (2) to determine AfRGM resistance genes’ modes of action, the heritability estimates of their resistance to AfRGM and the behavioural pattern of progenies with resistance to AfRGM attack; (3) to reveal convergent evolution of same or similar resistance gene(s) in geographically distinct landraces, or divergent evolution of genotypes carrying the same gene, by analysing the genetic diversity among five AfRGM parental lines; (4) to build a core sample of progenies to be used as a reduced mapping population, largely reflecting the entire genome of the whole population, after an estimate of the heritability of 15 agro-morphological descriptors and; (5) determine Simple Sequence Repeat (SSR) markers flanking genes or quantitative trait loci (QTLs) linked to resistance to AfRGM. A method of accurately assessing damage caused by AfRGM was determined by comparing four methods of assessment including the International Rice Research Institute’s (IRRI) Standard Evaluation System (SES) for rice and three methods based on resistance index (RI) assessments differing in the computing of the percentage of tillers with galls on a resistant check variety. The RI-based assessment (RI-BA) methods consistently provided a better evaluation of AfRGM damage than the SES, regardless of the trial size. Within RI-BA methods, RI-BA2 was always more accurate than RI-BA1 and RI-BA3 when the plot was large. RI-BA2 and RI-BA3 were equally accurate when the plot size was small, and they provided better estimates than RI-BA1. When the plot was of medium size, RI-BA2 was more accurate than RI-BA3; RI-BA3 also surpassed RI-BA1. Overall, the best method of assessing AfRGM damage was RI-BA2, regardless of the plot size. Five rice populations including F1, F2 and F3 generations involving ITA306, a susceptible variety of Oryza sativa subsp. indica, and four varieties having different reactions against AfRGM were used to determine the genetic basis of resistance and estimate the heritability of resistance to AfRGM. All the F1s were susceptible, suggesting recessive gene inheritance. The F2 generations’ segregation pattern of 1R:15S in both ITA306-TOS14519 and ITA306-TOG7106 crosses as well as the segregation of 1R:8Seg:7S in ITA306-TOS7106 F3 families indicated that the AfRGM resistance expression being studied is governed by two genes. The deviation of the segregation patterns of crosses involving ITA306 and the tolerant parental lines from Mendelian segregation ratios suggests that the tolerance to AfRGM shown by BW348-1 and Cisadane is under complex mechanisms of control rather than under simple genetic control. The narrow-sense heritability estimates of resistance to AfRGM were low in populations involving tolerant varieties and were high in populations involving resistant varieties. They ranged from 0.086 in the ITA306-Cisadane population, to 0.4 in the ITA306-TOG7106 population. Conversely, the broad-sense heritability estimates ranged from 0.23 (ITA306-Cisadane) to 0.63 (ITA306-TOS14519). The behavioural patterns of progenies against AfRGM attack were evaluated for 532, 413 and 479 F2 progenies from ITA306-BW348-1, ITA306-Cisadane and ITA306-TOS14519 crosses, respectively, in addition to 90 BC1F2 progenies from the ITA306 and TOG7106 cross. One F3 generation of 649 families from a cross between ITA306 and TOS14519 was also tested. Four types of behavioural pattern categories were observed: (1) progenies were more resistant than the resistant check entry at 45 DAT and 70 DAT; (2) progenies were more resistant at 45 DAT and became susceptible at 70 DAT; (3) progenies were susceptible at both 45 DAT and 70 DAT; (4) progenies were susceptible at 45 DAT but reverted to resistant at 70 DAT. The first three categories were the most frequently observed and occurred in all cross combinations. The last category was observed only for a few progenies from the ITA306-TOS14519 F2 and F3 generations and, surprisingly, many from the ITA306 and BW348-1 cross. Heritability estimates were calculated for 15 major traits in an F3 population in order to predict the genetic gain associated with each trait, together with the resistance to AfRGM and to estimate the influence of the environment on phenotypic values. Broad-sense heritability (H2) estimates were high for the penultimate leaf length (PLL) - 0.99, penultimate leaf width (PLW) – 1.0, flag leaf length (FLL) - 0.99, flag leaf width (FLW) – 1.0, ligule length (LigL) - 0.99, tillering ability (Til) - 0.99, number of days to booting (DB) - 0.95, number of days to first heading (DFH) - 0.96, number of days to heading (DH) - 0.89, number of days to maturity (DM) - 0.98, culm length (CL) - 0.99, plant height (PH) - 0.99, panicle length (PanL) - 0.95, secondary branching (SB) - 0.95 and the thousand grains weight (TGW) - 0.71. Conversely, narrow-sense heritability estimates were very low (nearly 0) in PLL, FLL, Lig, DB, DFH, DM and SB or low (at most 0.267) in PLW, FLW, DH and PH, with a high value of 0.727 for TGW. Inheritance of the traits studied was therefore under non-additive gene effects rather than additive genetic effects and can therefore be improved using pedigree breeding schemes along with breeding for AfRGM resistance. Fine genetic evaluation of five AfRGM parental lines was studied in terms of polymorphisms using 303 SSR primers covering the rice genome. Of the 178 polymorphic primers identified, 60 were highly polymorphic and informative. The number of alleles amplified by these primers ranged from one to five for a total of 1,041 alleles. The polymorphism rate was globally high, ranging from 45.2% to 66.8%. The mean of the polymorphism information content (PIC) was 0.553. Factorial analysis, based on the allelic diversity, demarcated the parental lines into Oryza glaberrima Steud, Oryza sativa subsp. japonica and O. sativa subsp. indica groups, while a cluster analysis distinguished them into four groups: AfRGM resistant, susceptible, moderately resistant and tolerant. BW348-1 and Cisadane showed the least diversity, despite their distant geographical origins. TOS14519 and TOG7106 showed more divergence to ITA306 despite their common West African origin. This variability amongst the genotypes tested is the result of farmer-based selection for AfRGM resistance rather than direct breeding efforts through breeder intervention. A method of selecting individuals for a mapping population, based on a core sample, was developed in order to speed up the mapping procedure. A diversity study amongst F2 and F3 generations involving 15 quantitative and 26 qualitative agro-morphological characters was carried out and led to the dropping of seven non-discriminant descriptors. The diversity index (H) was calculated for each remaining character and the discriminant descriptors were selected based on a diversity index threshold value above 0.4. Four descriptors of H values less than 0.35 were therefore dropped. The sizing of the core collection of 64 individuals and the selection of these individuals were done using MSTRAT version 4.1 package in redundancy mode, a construction run of 100 times with an iteration number of 500. The core sample was similar to the whole population for clustering pattern, minimum and maximum quantitative values and diversity index, while mean values and coefficient of variation distinguished them. The core sample, which represents 10% of the whole population, also revealed the same phenotypic variation and the same genotypic segregation according to two SSR markers. It can therefore efficiently reflect the whole population as a mapping population. Finally, a study was undertaken to identify flanking markers to the gene/QTL involved in the resistance against AfRGM using bulked segregant analysis (BSA). A polymorphism study between ITA306 and TOS14519 displayed 145 polymorphic SSR markers, which were used to screen the bulks that originated from the two tails, and depicted only two SSRs as candidate markers linked to gall midge resistance. These markers included RM317 and RM17303 which displayed strong significance after an analysis of variance using an F test, meaning that they were segregating with the resistant alleles. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

La cécidomyie orange du blé, Sitodiplosis mosellana (Géhin), est un ravageur commun du froment. Présente sur les trois continents de l’hémisphère Nord, cette espèce est connue depuis deux siècles mais son contrôle reste difficile tant par sa présence discrète que par ses effectifs hautement variables. En Wallonie, les niveaux d’infestations sont globalement faibles mais atteignent localement des seuils inquiétants.<p>Au début des années 2000, la phéromone sexuelle de S. mosellana a été identifiée au Canada. Cette découverte a permis la fabrication de pièges qui ont considérablement amélioré la détection et la mesure des vols de cet insecte minuscule. De 2007 à 2010, les captures de S. mosellana ont été mesurées quotidiennement dans plusieurs dizaines de champs de Wallonie, aux historiques et aux couverts variés.<p>Les volumes de captures au piège à phéromone sexuelle ont été très importants. Il a fallu en étudier la signification, notamment en termes de mesure du risque. En effet, si les mâles sont efficacement capturés, seules les femelles constituent un risque de dégâts. L’interprétation correcte des captures à l’aide de ce type de piège, a été rendue possible par l’observation de différences fondamentales concernant la mobilité et la distribution spatiale des mâles et des femelles de S. mosellana. Même s’ils ne mesurent pas directement l’émergence proprement dite, les pièges à phéromone ont permis, grâce à leur très grande sensibilité, de préciser les connaissances sur l’émergence des adultes et de révéler que plusieurs vagues d’émergence pouvaient se succéder au cours d’une même année.<p>La prévision des émergences de la cécidomyie orange du blé, constitue la clé de voûte de la lutte contre ce ravageur dont un contrôle efficace par des insecticides ne se justifie éventuellement que lorsque la courte saison des pontes coïncide avec l’épiaison des froments. Les patrons d’émergence obtenus par les pièges ont été confrontés aux prévisions de différents modèles conçus en Europe ou en Amérique du Nord, et appliqués aux conditions météorologiques observées de 2007 à 2010. Aucun de ces modèles n’a prévu correctement les émergences sur l’ensemble des quatre années.<p>Les données d’émergence obtenues à l’aide des pièges à phéromone (effectifs élevés et relevés quotidiens) ont fait apparaître une relation de cause à effet entre, d’une part les vagues d’émergences et, d’autre part les épisodes pluvieux observés trois à six semaines plus tôt. L’écart entre une &61618;pluie inductrice&61618; et la vague d’émergence induite correspondante s’est avéré constant en termes d’accumulation de température :il équivaut à 160 degrés-jours en base 7°C. Partant de ce constat et des acquis des modèles antérieurs, un modèle prévisionnel original des émergences a été développé et validé sur le terrain. Allié à une meilleure connaissance de la biologie du ravageur, il constitue un outil majeur de la lutte intégrée.<p>Par ailleurs, les travaux menés ont également révélé l’existence d’un biais fréquent dans les essais d’évaluation des variétés, entraîné par la concentration des pontes de cécidomyie orange sur les premières parcelles atteignant le stade épiaison. Dans le système d’évaluation en vigueur, notamment pour l’inscription dans les catalogues nationaux, ce biais conduit à une sous-estimation du potentiel de rendement des variétés de blé les plus précoces.<p>Enfin, la découverte du rôle inducteur des pluies sur l’émergence des adultes a été exploitée en conditions contrôlées pour planifier des émergences échelonnées, et pour disposer, pendant une longue période, de jeunes adultes prêts à pondre. Cette application permet dès à présent de mesurer en serre le niveau de résistance des variétés exposées de façon homogène à l’insecte, quel que soit leur degré de précocité.<p>De diverses façons, cette étude contribue à une meilleure connaissance de la cécidomyie orange du blé et offre de nouveaux outils pour la lutte intégrée contre ce ravageur.<p><p>--------------------------------------------------<p><p>The orange wheat blossom midge, Sitodiplosis mosellana (Géhin), a common pest of wheat throughout the northern hemisphere, is known for two centuries but remains difficult to control due to its discrete behavior and its highly variable population level.<p>In general, the infestation levels in Wallonia (Belgium) are low, although levels could locally exceed worrying thresholds. <p>In the early 2000s, the sexual pheromone of S. mosellana has been identified in Canada. This discovery has led to the manufacturing of traps which have greatly improved the detection of this tiny insect. From 2007 to 2010 in Wallonia, S. mosellana captures have been daily registered in about 20 fields with different cropping histories and grown with different crops.<p>Insect captures by pheromone traps were numerous. Relation between amount of captures and risk measurement has been studied. As expected, only the males are attracted by the pheromone and the risk of ears infestation is mainly related to the presence of females. The correct interpretation of captures in pheromone traps has been established by the observation of fundamental differences between males and females concerning their mobility and their spatial distribution. <p>Despite the fact that pheromone traps are not real emergence traps, they have led to new information on adult emergence indicating that several emergence waves can be consecutive during the same year. <p>Forecasting the emergence of the adult orange wheat blossom midge is a key element on pest management. Insecticides treatments are sometimes justified when the egg laying period of the insect coincides with ear emergence of wheat. Emergence patterns established from captures of pheromone traps have been compared with the forecast of several models built in Europe or North America. These forecasting models were used with the meteorological data observed from 2007 until 2010. None of the six tested models provided a reliable forecast across the four years of our study. <p>Emergence data from catches in pheromone traps were very accurate because the number of catches were high and were taken each day. This emergence data showed a relation between emergence waves and rainfalls occurring during the preceding 3 to 6 weeks. The lag between inductive rain and emergence wave is constant in terms of temperature accumulation: it is equivalent to 160 degree–days above 7°C. This discovery, combined with experience from previous models, was incorporated into a new forecasting model.<p>In addition, the present work has also revealed the existence of a common bias in variety evaluation trials leading sometime to the concentration of the eggs in the earliest earing variety. In the current evaluation system, this bias leads to a sub-evaluation of the yield for the most precocious varieties. <p>Finally, the discovery of the inductive rain for adult emergence has been used in the screening for resistant varieties to S. mosellana by providing adults during the complete duration of the test. This application of the model allows to measure, under controlled conditions, the level of resistance of all varieties (early and late heading varieties) which are exposed homogeneously to the insect.<p>In total, this study has contributed to a better understanding of the orange wheat blossom midge and provides some new tools in the management of this pest.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished