Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-08-30T13:40:23Z No. of bitstreams: 1 Ana Caroline de Azevedo Teixeira.pdf: 500119 bytes, checksum: c6f5c469790bac6fbe0bfe816ac29466 (MD5) / Made available in DSpace on 2016-08-30T13:40:23Z (GMT). No. of bitstreams: 1 Ana Caroline de Azevedo Teixeira.pdf: 500119 bytes, checksum: c6f5c469790bac6fbe0bfe816ac29466 (MD5) Previous issue date: 2015-02-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Cucumber crop has its production affected by the attack of the aphid Aphis gossypii Glover (Hemiptera: Aphididae), which colonizes the plant throughout all developmental stages. The use of chemical insecticides to control it has generated serious problems such as the emergence of resistant populations to the active ingredients used. This work aimed to evaluate fungus preparations based on entomopathogenic to control this insect pest in cucumber plants, and develop an insecticide the base of Beauveria bassiana (Bals.) Vuill. Under laboratory conditions, the three most promising isolates were tested, CG 864, PL 63 and IBCB 66,with five concentrations of conidia (1x105, 1x106, 1x107, 1x108 and 1x109 conidia / ml) and control (sterile water + Tween 80) . In the third stage of the experiments studies were conducted involving two preparations of biological insecticide: a) an aqueous suspension [pure conidia of B. bassiana 864 CG + 0.01% Tween 80; used as a standard] b) formulation of oil dispersion [conidia pure vegetable oil emulsifiable +] CG 864 isolated, and diluted with water for spraying the concentrations of 0.5; 1; 2 and 4% oil. The fungus preparations were standardized to have 1.0 x 107 spores / mL in applied spray. Isolates of the fungus B. bassiana were more virulent aphid A. gossypii that isolates Metarhizium anisopliae (Metschn.) Sorok. and Lecanicillium longisporum (Zimmerman) Zare & Gams. The isolates CG 864, IBCB 66 and PL 63 of the B. bassiana were the most virulent. B. bassiana preparations reduced the aphid population A. gossypii, with control efficiencies of 52.3% to 83.8%, however, there was no difference in the pest population density between the oil dispersion and the aqueous suspension conidia. / A cultura do pepino tem sua produção afetada pelo ataque do pulgão Aphis gossypii Glover (Hemiptera: Aphididae), que coloniza a planta durante todo seu estágio fenológico. O uso de inseticidas químicos para seu controle tem gerado sérios problemas, como o surgimento de populações resistentes aos princípios ativos utilizados. Este trabalho teve por objetivo avaliar preparações à base de fungo entomopatogênico para controle desse inseto-praga em plantas de pepino e desenvolver um inseticida a base de Beauveria bassiana (Bals.) Vuill. Em condições de laboratório, os três isolados mais promissores CG 864, PL 63 e IBCB 66, foram testados, com cinco concentrações de conídios (1x105; 1x106; 1x107; 1x108 e 1x109 conídios/mL) e a testemunha (água esterilizada +Tween 80). Foram realizados estudos envolvendo duas preparações do inseticida biológico: a) suspensão aquosa (conídios puros de B. bassiana CG 864 + Tween 80 a 0,01%), utilizada como padrão e b) formulação em dispersão oleosa [conídios puros + óleo vegetal emulsionável], do isolado CG 864, sendo diluída em água para pulverização nas concentrações de 0,5, 1; 2; 4; 8 e 16% de óleo. As preparações do fungo foram padronizadas para 1,0 x 107 conídios/mL na calda aplicada. Isolados do fungo B. bassiana foram mais virulentos ao pulgão A. gossypii que os isolados de Metarhizium anisopliae (Metschn.) Sorok. e Lecanicillium longisporum (Zimmerman) Zare & Gams. Os isolados CG 864, IBCB 66 e PL 63 de B. bassiana foram os mais promissores. Preparações de B. bassiana reduziram a população do pulgão A. gossypii, com níveis de eficiência de controle de 52,3% a 83,8%, porém, não houve diferença na densidade populacional da praga entre a dispersão oleosa e a suspensão aquosa de conídios.

As cucurbitáceas no Brasil podem ser infectadas por diferentes vírus, tais como o Papaya ringspot virus - type W (PRSV-W); o Zucchini yellow mosaic virus (ZYMV) e o Cucumber mosaic virus (CMV). Os dois primeiros pertencem ao gênero Potyvirus e no geral ocorrem com maior freqüência do que o CMV, que é uma espécie do gênero Cucumovirus. Os dois potyvirus e o cucumovirus são transmitidos por afídeos de maneira não persistente. O principal objetivo desse trabalho foi o de obter subsídios que possam explicar a menor incidência do CMV em espécies de cucurbitáceas, estudando: (a) a interferência dos potyvirus PRSV-W e ZYMV na transmissão do CMV por Aphis gossypii e Myzus persicae para plantas de abobrinha de moita (Cucurbita pepo ‘Caserta’) e (b) o efeito de isolados do CMV provenientes de maracujazeiro (Passiflora edulis f. flavicarpa), de pimentão (Capsicum annuum), de pepineiro (Cucumis sativus), de meloeiro (Cucumis melo) e de trapoeraba (Commelina virginica) na infectividade de plantas de abobrinha de moita por meio da transmissão por afídeos. Para avaliar a possível interferência dos potyvirus na transmissão do CMV, as plantas de abobrinha de moita foram inoculadas com afídeos que adquiriram cada um dos vírus isoladamente; o CMV simultaneamente com cada um dos potyvirus; um dos potyvirus seguido pelo CMV e vice-versa. Os resultados mostraram, na maioria das vezes, que a transmissão dos vírus isoladamente foi mais eficiente do que em mistura, tanto através de aquisição simultânea como seqüencial. Os potyvirus no geral foram mais eficientemente transmitidos por ambas espécies de afídeos. Quando em mistura (aquisição simultânea ou sequencial), de uma maneira geral, houve uma redução na taxa de transmissão do CMV e do potyvirus presente na mistura. As avaliações sobre o efeito da origem dos isolados do CMV na infectividade de abobrinha de moita mostraram que apenas o isolado de pimentão não infectou plantas de abobrinha de moita quando transmitido por meio dos afídeos A. gossypii e M. persicae. Também não houve infecção quando inoculado mecanicamente. Os demais isolados infectaram abobrinha de moita através da transmissão por ambas espécies de afídeos. Análise da proteína capsidial dos diferentes isolados do CMV indicaram que todas apresentaram a mesma mobilidade em gel de SDS-PAGE. A origem do isolado o CMV, a eficiência da espécie de afídeo na sua transmissão e a interferência dos potyvirus PRSV-W e ZYMV podem explicar em parte a menor incidência desse cucumovirus em cucurbitáceas no país. / The cucurbits in Brazil can be infected by different viruses, such as Papaya ringspot virus - type W (PRSV-W); Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV). The first two belong to the genus Potyvirus and in general they occur more frequently than CMV, which is a species of the genus Cucumovirus. The two potyviruses and the cucumovirus are transmitted by means of aphids in a non persistent way. The main objective of this work was to obtain subsidies that can explain the lower incidence of CMV in cucurbit species, studying: (a) the interference of the potyviruses PRSV-W and ZYMV in the transmission of CMV by means of Aphis gossypii and Myzus persicae to zucchini squash plants (Cucurbita pepo 'Caserta') and (b) the effect of isolates of CMV from passion flower (Passiflora edulis f. flavicarpa), bell pepper (Capsicum annuum), cucumber (Cucumis sativus), melon (Cucumis melo) and Commelina virginica in the infectividade of zucchini squash plants through the transmission by aphids. To evaluate the possible interference of the potyvirus in the transmission of CMV, zucchini squash plants were inoculated with aphids that acquired each one of the viruses separately; CMV simultaneously with each one of the potyvirus; one of the potyvirus follow by CMV and vice-versa. The results showed that the transmission of PRSV-W, ZYMV and CMV separately was more efficient than in mixture. The potyviruses in general were more efficiently transmitted by both species of aphids than CMV. When in mixture (simultaneous or sequential acquisition), there was a reduction in the rate of transmission of CMV as well as that of the potyvirus present in the mixture. The evaluation on the effect of the origin of the isolate of CMV in the infectivity of zucchini squash showed that only the isolate from bell pepper did not infected the plants when inoculated by means of A. gossypii and M. persicae. This isolate also did not infecte zucchini squash when inoculated mechanically. The others isolate infected zucchini squash when transmitted by both species of aphids. Analysis of the capsidial protein of the different isolates of CMV indicated that all presented the same mobility in SDS-PAGE. The origin of the isolate of CMV, the efficiency of the species of aphid and the interference of the potyviruses PRSV-W and ZYMV on its transmission can partly explain the lower incidence of this cucumovirus in cucurbits species in Brazil.

The rosy apple aphid, Dysaphis plantaginea, (Homoptera: Aphididae) is a serious pest of apples in British Columbia (BC), Canada and especially in organic orchards where conventional controls cannot be used. The goals of this study were to determine the environmental or management factors of an orchard that lead to high aphid populations, to conduct an economic assessment of the damage, to determine the timing of autumn migration, and to test several autumn and spring chemical control methods and two novel autumn mechanical control methods targeting the aphids while on their alternate host, Plantago major. To explain the variation among orchards, I evaluated several potential correlates of aphid density: abundance of the alternate host (plantain, Plantago major), foliar tree nitrogen, tree age, tree planting density, and the application of an oil treatment in spring. Stepwise regression indicated that foliar nitrogen and tree age explain 27% of the variation. Orchards receiving a spring oil application had a 53% lower average aphid infestation level. Plantain abundance was not related to aphid population on apple. However, experimental manipulation of leaf angle from the ground and size showed that significantly more alate and apterous aphids occurred on large, low angle leaves. Mowing prior to spring aphid migration was associated with 75% fewer alatae and apterae on the plantain. The loss in harvest resulting from aphid damage ranged from 3% to 76% of the crop. Effective autumn control depends on accurate timing of aphid flight. The peak of female flight occurred on the 27th of September, 2007 at 11:56 hours daylength (sunrise to sunset) and the peak of male flight occurred on the12th of October, 2007 at 11:02 hours daylength. Aphid densities in the spring of 2008 were very low, making comparisons between treatments and controls difficult. Autumn applications of Superior dormant oil and kaolin clay were not effective. The PureSpray Green treatments of two October applications and one April application showed a significant reduction in rosy apple aphid infested clusters compared with the untreated control. Mowing and rotavating did not result in a significant reduction in aphid infestation level. / Land and Food Systems, Faculty of / Graduate

Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.

The infestation of the cultivated grain crops by phloem feeding aphids has generated a great deal of interest over the years, due to the serious damage they cause to the crops and yield losses that result. The mechanism of the interaction between aphids and host plants remains largely unknown in spite of efforts to understand the basis of aphid feeding on grain crops. Greater efforts are required to explain the mechanism(s) of this interaction in order to achieve sustainable agriculture. This thesis focused on an investigation of the mechanism of feeding by the Russian wheat aphid, Diuraphis noxia Mordvilko (RWA) and the bird cherry-oat aphid, Rhopalosiphum padi L. (BCA) on barley and wheat cultivars. These two aphids co-occur naturally, but they inflict very different feeding effects on host plants. Structural and functional approaches were employed to investigate their feeding habits and these were then related to the observed differences in their host plants. Transmission electron microscopy (TEM) techniques were used to study the ultrastructural damage, while fluorescence microscopy techniques – using aniline blue fluorochrome (a specific stain for callose) and 5, 6-CFDA (a phloem-mobile fluorophore) – were employed to investigate the functional response to damage via wound callose formation and phloem transport capacity respectively. RT-PCR and quantitative real-time RT-PCR techniques were used to investigate the regulation of the genes involved in callose synthesis and degradation at the transcriptional level. Morphological observation of the damage caused by the aphids show that infestation by RWA results in extensive leaf chlorosis, necrosis and rolling, while infestation by BCA does not lead to any observable symptoms within the same period. Interestingly, the population study shows that BCA breeds faster than RWA within the two-week experimental period. The ultrastructural study of feeding damage caused by the two aphids on the vascular bundles of susceptible barley cv Clipper, shows a different patterns of damage. Probing the vascular bundles results in the puncturing of vascular parenchyma by both aphids, but severe damage occurs in sieve tubes-companion cell complex during sustained feeding by RWA. In contrast, less damage occurs when BCA feeds on the phloem. Drinking from the xylem by RWA results in deposition of a large quantity of electron-dense watery saliva, which apparently seals the xylem vessels completely, by blocking all the pit membrane fields between the xylem vessels and associated parenchyma cells. In contrast, drinking from xylem by BCA results in deposition of a dense, granular saliva into the xylem vessels only, which does not appear to totally occlude the pit membrane fields. This is the first known report in which ultrastructural evidence of aphids’ drinking in xylem is provided. The comparative effects of RWA feeding on a susceptible Betta and resistant Betta-Dn1 wheat cultivars showed that after two weeks, the Betta cultivar expressed damage symptoms such as chlorosis, necrosis and leaf roll, while few chlorotic patches and necrotic spots occur in resistant Betta-Dn1 cultivars. An ultrastructural investigation of the feeding damage caused to all leaf tissues revealed, for the first time, that RWA is capable of both intra- and inter-cellular probing within mesophyll cells. Probing in the mesophyll cells induces a more severe damage in susceptible Betta than in the resistant Betta-Dn1 counterpart. Similar differences in damage occurred during feeding in the thin-walled sieve tubes of the phloem, with the sieve tubes of the Betta showing more damage than that of the resistant Betta-Dn1. However, drinking from xylem resulted in the characteristic occlusion of metaxylem vessels by copious deposition of saliva by RWA in both Betta and Betta-Dn1 cultivars. In all cases of probing, feeding, and drinking by RWA in both cultivars, all probed cells with evidence of salivary material deposit and those cells adjacent to salivary material deposit, exhibit significant damage in susceptible Betta cultivar, whereas similar cells in Betta-Dn1 cultivars do not show as damage as severe. Investigation of the functional response of the plants to feeding by aphids through the deposition of wound-induced callose shows that formation and deposition of wound callose occurs in both longitudinal and cross veins within 24h of feeding by RWA. This deposition increases through short-term feeding (72h) and prolonged feeding (14d). This is in sharp contrast to the observations with BCA feeding,where little or no callose formation occurs within the same time frame. Callose formation and deposition occurs only when a higher population of BCA feeds on barley leaves. This is the first report of aphid-induced wound callose by BCA. In all cases of callose deposition, aphid stylet tracks were associated with callose and the deposition of callose appears to be a permanent feature, because wound callose remained in the leaf tissues even after 120h of the aphids’ removal. Wound callose signals (defence and anti-defence) are discovered to be transported in the phloem tissues and are dependent on the direction of assimilate flow. Examination of the possible regulation of wound callose genes at the transcriptional level shows that the two expressed glucan synthase gene sequences (GSL – genes involved in callose formation) analysed did not show any significant increase or regulation upon aphid infestation. Contrary to expectation, all three aphid-induced β-1, 3-glucanases (genes which are thought to be involved in callose degradation) showed higher expression in RWA-infested tissue than in BCA-infested tissue. The results of the feeding damage on the transport capacity of the phloem shows that BCA infestation does not lead to a significant reduction in the phloem transport capacity during short-term feeding (72h), while RWA-infested leaves showed considerable reduction in the transport capacity of the phloem within the same period. However, prolonged feeding (14d) by BCA induces a considerable reduction on the transport capacity of the phloem on the infested tissues. In contrast, a marked reduction in the transport capacity of the phloem occurs in RWA-infested leaves and in most cases, complete cessation of transport ensues. In conclusion, these data collectively suggest that RWA is a serious and most destructive phloem feeder in comparison to the BCA. RWA causes severe damage to all cellular tissues of the host plants, which result in apoplasmic and symplasmic isolation of xylem and phloem tissues, while BCA infestation does not result in such isolation within the same time and population levels. Resistance genes appear to function by conferring resistance to cell damage on the resistant cultivars during aphid feeding. Responses by plants to aphid infestation via wound callose deposition are again shown to be species-specific. A quick response results when RWA feeds, even at a very low population level, while a response occurs only at a higher infestation level by BCA, and this response was shown as not regulated at the transcriptional level. Differences in the damage to leaf tissues and wound callose deposition eventually lead to varying degrees of damage to the transport capacity of the phloem. These differences in the damage signatures are hereby suggested to be the cause of the diversity in the observed damage symptoms and the yield losses upon infestation by the two aphid species.

The Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is a major pest of cultivated small grains. It is particularly devastating because of is high reproductive rate which results in the growth of large populations which become damaging to its host plants. Development of resistant barley (Hordeum vulgare L.) cultivars is complicated as resistance is polygenic. As a result, the industry remains at risk now that the RWA has spread throughout South Africa. It has, as recently as, 2013, been identified in the SW Cape, which was previously geographically isolated. This is South Africa‟s principle barley growing region. Now a potentially huge problem exists. Therefore, it is imperative that an alternative to pesticide use is found. Testing potential innate resistance in barley cultivars is thus, critical. In this thesis, I present data on four barely cultivars where I have examined their resistance/ lack of resistance to three known RWA biotypes, RWASA1, RWASA2 and RWASA3. The barley varieties used were two economically important South African malt barley cultivars (S5 and SSG 564) along with two potentially RWA resistant Afghan accessions (CIho 4125 and CIho 4159). The RWA biotype population growth rates on each of the plants were determined over a 14 day period. The aim was to establish baseline data of the effects of RWA population growth on the host plants under ambient CO2 (380 – 400 ppm) conditions. The extent of RWA feeding damage was investigated at the cell level by examining saliva deposition and cell disruption using Transmission Electron Microscopy; at the tissue/vascular level using fluorescence microscopy, to determine the extent of callose formation; at a whole leaf level by recording percent chlorosis and leaf roll; and finally, at a whole plant level by measuring biomass loss.The experiments were repeated under elevated CO2 (450 ppm) to model any changes in RWA/plant interaction with respect to future climate change. The effects of an elevated CO2 environment and RWA feeding on host plant foliar N and C:N ratio were compared to ambient CO2 conditions, to provide a clearer picture of the potential nutrient drain that a feeding RWA colony exacts on its host. Of the varieties tested, the CIho accessions performed better than the two SA barley cultivars as the CIho accessions appeared to express a mild antibiosis resistance response as RWA populations, particularly those of RWASA1, were smaller than those observed on either S5 or SSG 564. In addition, less damage was evident in the two CIho accessions due to RWA feeding. II RWASA2 was the most virulent of the three RWA biotypes tested, followed by RWASA3 while RWASA1 was the least virulent. Under elevated CO2 conditions, RWA feeding damage was exacerbated but the trend of biotype virulence remained the same. Higher aphid population sizes were recorded under elevated CO2, meant that even the more resistant CIho accessions were overcome by the increased demand made by the larger aphid colonies on the host plants. The % foliar N data showed that under elevated CO2 aphid-free control plants had increased N levels in their leaves. Increased “food” supply (as shown by the increased N levels) therefore allowed significantly larger aphid populations to develop on the plants exposed to elevated CO2, due to improved nutrient status of the phloem sap taken up by RWA. The knock-on effect of a higher aphid population was increased cell disruption as a result of extensive probing, extensive formations of wound callose, with the result that phloem damage impeded nutrient flow through the vascular tissues which contributed to chlorosis and (eventually plant) death. The major conclusion from this study is that even a mild CO2 elevation resulted in an increase aphid population which may pose a severe and very real threat to a barley crop. Therefore, without effort to identify and deploy resistant barley cultivars, it could well be possible that future barley cultivation in South Africa may no longer be viable.

Cereal crop productivity is hampered when these plants are infested by phloem feeding aphids. A great deal of research has been carried out with the direct aim of a clearer understanding of the mechanism involved in the interaction between aphids and their host plants. Research has directly or indirectly been geared towards enhanced plant productivity and achieving sustainable agriculture. Barley (Hordeum vulgare L.) is an important small grain crop in South Africa, whose crop performance is negatively affected by fluctuations in weather patterns as well as by agricultural pests. One of the insect pests infesting barley is the Russian wheat aphid, Diuraphis noxia Kurdjumov (RWA), of which the two South African biotypes, codenamed RWASA1 and RWASA2, were studied in this thesis. During dry spells, RWA infestation becomes a more serious threat to barley productivity. Resistant plants have been used to combat RWA infestation of small grains. In South Africa, 27 RWA-resistant wheat cultivars are currently used in commercial cultivation, but no resistant barley lines have, unfortunately, been developed, in spite of this grain’s significant economic importance. This informed the study in this thesis, and this interest particularly focussed on three RWA-resistant lines developed by the USDA, testing their performance against South African RWA biotypes, for possible adaptation to South Africa. The aim was thus to examine the plant-aphid interactions, aphid breeding rates, plant damage and sustainability, evidence of resistance or tolerance and finally potential performance under elevated CO2 (a very real climate change threat). Two major avenues of research were undertaken. The first aspect involved examination of structural and functional damage caused by RWASA1 and RWASA2 on the three resistant and a non-resistant line. Aphid population growth and damage symptoms (chlorosis and leaf roll) of infestation by these aphid biotypes were evaluated. This was followed by a structural and functional approach in which the effects of feeding on the transport systems (phloem and xylem) of barley were investigated. Fluorescence microscopy techniques (using aniline blue fluorochrome, a specific stain for callose and 5,6-CFDA, a phloem-mobile probe) were applied to investigate the feeding-related damage caused by the aphids, through an examination of wound callose formation and related to this, the resultant reduction in phloem transport capacity. Transmission electron microscopy (TEM) techniques provided evidence of the extent of the feeding-related cell damage. The second aspect involved a study of the effect of changing CO2 concentrations ([CO2]) on the resistant and susceptible barley cultivars to feeding by the two RWA biotypes. Leaves of plants grown at ambient and two elevated levels of [CO2] were analysed to investigate the effect of changing [CO2] on biomass, leaf nitrogen content and C:N ratio of control (uninfested) and infested plants. The population growth studies showed that the populations of the two RWA biotypes as well as bird cherry-oat aphid (BCA, Rhopalosiphum padi L.) increased substantially on the four barley lines. BCA was included here, as it had been the subject of several previous studies. RWASA2 bred faster than RWASA1 on all lines. The breeding rates of the two RWA biotypes were both suppressed and at near-equivalent levels on the three resistant lines, compared to the non-resistant PUMA. This suggests that the resistant lines possessed an antibiosis resistance mechanism against the feeding aphids. Feeding by the aphids manifested in morphological damage symptoms of chlorosis and leaf roll. The two biotypes inflicted severe chlorosis and leaf roll on the non-resistant PUMA. In the resistant plants, leaf rolling was more severe because of RWASA2 feeding compared to RWASA1 feeding. In contrast, chlorosis symptoms were more severe during RWASA1 feeding than was the case with RWASA2 feeding. Investigation of the effect of aphid feeding on the plants showed that callose was deposited within 24h and that this increased with longer feeding exposure. Wound callose distribution is more extensive in the non-resistant PUMA than in the resistant plants. RWASA2 feeding on the resistant lines caused deposition of more callose than was evident with RWASA1 feeding. During long-term feeding, it was evident that variation in the intensity and amount of wound callose was visible in the longitudinal and transverse veins of the resistant plants. Of the three STARS plants, STARS-9301B had the least callose. Interestingly, wound callose occurred in both resistant and non-resistant plants, in sharp contrast to what has been reported on resistant wheat cultivars that were developed in South Africa. The relative reduction in the wound callose deposited in the resistant line, when compared to the non-resistant lines, suggests the presence of a mechanism in the resistant lines, which may prevent excessive callose formation. Alternatively, the mechanism may stimulate callose breakdown. RWASA2 feeding on the resistant lines deposited more wound callose than RWASA1 feeding. This evidence supports the hypothesis that RWASA2 is a resistance breaking and more aggressive feeder than RWASA1 is; and further underscores the urgent need for development of RWA-resistant barley cultivars. The ultrastructural investigation of the feeding damage showed that the two biotypes caused extensive vascular damage in non-resistant plants. There was extensive and severe cell disruption and often obliteration of cell structure of the vascular parenchyma, xylem and phloem elements. In sharp contrast, among the resistant plants, feeding-related cell damage appeared to be substantially reduced compared to the non-resistant PUMA. Low frequency of damaged cells indicated that majority of the cell components of the vascular tissues were intact and presumed functional. There was evidence of salivary material lining the secondary walls of the vascular tissue, which resulted in severe damage. Within xylem vessels, saliva material impregnated half-bordered pit pairs between the vessels and adjacent xylem parenchyma. This is believed to prevent solute exchange through this interface, thereby inducing leaf stress and vi leaf roll. A notable finding is that RWASA2 effectively induced more cell damage to vascular tissues in the resistant lines than did RWASA1. In general the experimental evidence (see Chapter 5) suggests that the resistant lines are possibly more tolerant (or able to cope with) to RWA feeding. Evidence for this is the reduction of wound callose and at the TEM level, a comparatively less obvious cell damage in the resistant lines, which suggests that they possess antibiosis and tolerance capacity. The apparent reduction of feeding-related cell damage from the TEM study confirmed the disruptive action of the feeding aphids in experiments using the phloem-mobile probe, 5,6-CF. Results showed that feeding by RWASA1 and RWASA2 reducedthe transport functionality of the phloem in all cases, but that RWASA2 feeding caused a more obvious reduction in the rate and distance that 5,6-carboxyfluorescein was transported, than did RWASA1. Investigation of the effect of changing [CO2] on the barley cultivars showed that in the absence of aphids and under elevated CO2 conditions, the plants grew more vigorously. In this series of experiments, the infested plants suffered significant reduction in biomass under ambient (as was expected) and under the two elevated CO2 regimes. Biomass loss was greater at elevated CO2 than under ambient [CO2]. The infested nonresistant PUMA plants showed a more significant biomass loss than did the resistant cultivars. Clearly, the benefits derived from elevated CO2 enrichment was thus redirected to the now-advantaged aphids. Uninfested vii plants showed an increase in leaf nitrogen under the experimental conditions. However, feeding aphids depleted leaf nitrogen content and this was more apparent on plants exposed to RWASA2 than was the case with RWASA1. The end result of this was that C:N ratio of infested plants were higher than uninfested plants. Clearly, the faster breeding rates of the aphids at elevated CO2 caused depletion of N and a resultant deficiency exacerbated chlorosis as well as leaf rolling due to the higher aphid population density under elevated CO2 than at ambient. By 28 days after infestation (DAI), majority of the plants exposed to enriched CO2 treatments had died. A major finding here was thus that although this study demonstrated that elevated CO2 resulted in an increase in biomass, this was detrimentally offset in plants infested by the aphids, with a decline in biomass and loss of functionality leading to plant death at 28DAI. The overriding conclusion from this study is a clear signal that the twin effects of CO2 enrichment (a feature of current climate change) and aphid infestations may precipitate potential grain shortages. A disastrous food security threat looms.

Doctor of Philosophy / Department of Biology / Jyoti Shah / Phloem-feeding insects like aphids feed on a variety of crop plants and limit plant productivity. In addition they are vectors for important plant viruses. Efforts to enhance plant resistance to aphids have been hampered by lack of sufficient understanding of mechanisms of plant defense against aphids. I have utilized a plant-aphid system consisting of the model plant Arabidopsis thaliana and the generalist aphid, Myzus persicae Sulzer (green peach aphid [GPA]), to study plant response to aphids. These studies have demonstrated an important role of premature leaf senescence in controlling aphid growth in Arabidopsis. Molecular and physiological studies suggest that the Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT 4) gene modulates the GPA feeding-induced senescence process. Furthermore, in comparison to the wild type plants, GPA growth was higher on pad4 mutant plants, suggesting an important role for PAD4 in plant defense against GPA. In contrast, constitutive expression of PAD4 in transgenic Arabidopsis enhanced basal resistance against GPA. Unlike its involvement in plant defense against pathogens, the role of PAD4 in Arabidopsis resistance to GPA is independent of its involvement in phytoalexin biosynthesis and of its interaction with EDS1, a PAD4-interacting protein. Instead, the heightened resistance to GPA in these PAD4 constitutively expressing plants was associated with the rapid activation of leaf senescence. The association of premature leaf senescence in basal defense against GPA is supported by our observation that in comparison to the wild type plant, GPA growth was restricted on the Arabidopsis hypersenescence mutants, ssi2 and cpr5. Gene expression studies suggested some overlap between plant responses to pathogens and aphids, for example, activation of genes associated with the salicylic acid (SA) signaling pathway. However, the characterization of aphid performance on Arabidopsis SA biosynthesis and signaling mutants have ruled out the involvement of SA signaling in controlling aphid growth.

Doctor of Philosophy / Department of Entomology / Gerald R. Reeck / John C. Reese / Salivary secretions are a key component of aphid-plant interactions. Aphids’ salivary proteins interact with plant tissues, gaining access to phloem sap and eliciting responses which may benefit the insect. In an effort to isolate and identify key components in salivary secretions, we created a salivary gland cDNA library. Several thousand randomly selected cDNA clones were sequenced. We grouped these sequences into 1769 sets of essentially identical sequences, or clusters. About 22% of the clusters matched clearly to (non-aphid) proteins of known function. Among our cDNAs, we have identified putative oxido-reductases and hydrolases that may be involved in the insect's attack on plant tissue. C002 represents an abundant transcript among the genes expressed in the salivary glands. This cDNA encodes a novel protein that fails to match to proteins outside of aphids and is of unknown function. In situ hybridization and immunohistochemistry localized C002 in the same sub-set of cells within the principal salivary gland. C002 protein was detected in fava beans that were exposed to aphids, verifying that C002 protein is a secreted protein. Injection of siC002-RNA caused depletion of C002 transcript levels dramatically over a 3 day period after injection. With a lag of 1 – 2 days, the siC002-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections of adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands.

M.Sc. / Wheat is one of the most extensively grown small grain crops in Southern Africa. It is a staple food source used by humans, animals and other living organisms around the globe (Feldman, 2001). One of the major threats of wheat production in South Africa is the Russian wheat aphid (RWA), (Kurdjumov), (Diuraphis noxia, Homoptera: Aphididae) (Brooks et al., 1994; Du Toit & Walters, 1984). The RWA was first described as a pest in South Africa in 1978 and in the US in 1986. Thereafter it was rapidly reported as becoming a major pest of cereals in these countries (Brooks et al., 1994; Du Toit & Walters 1984). A short life span, asexual reproduction, the development of resistance towards insecticides and rapid colonisation of the host plant are all factors that have made the management of the RWA a difficult task (Dogimont et al., 2010; Hein et al., 1998). As a result of complications such as these, host plant resistance is the most viable option to counter the RWA (Dogimont et al., 2010). The method of feeding of the RWA is detrimental to the plant as they insert their stylets into, and feed primarily from, the phloem sieve elements. This damages the plants through nutrient drainage (Dixon, 1985; Klingler et al., 2009) and results in a variety of symptoms, the most common being chlorosis, necrosis, wilting, stunting, curling of the leaves (which provides the pest with a sheltered environment protected from predators and pesticides), misshapen or nonappearance of new growth, and localised cell death at the site of aphid feeding. The RWA elicits an increase in essential amino acids in the phloem sap, by triggering the breakdown of proteins in infested wheat leaves (Burd & Burton, 1992; Du Toit, 1986; Haley et al., 2004; Ma et al., 1998; Miller et al., 2001; Walters et al., 1980). It also reduces transport of labelled tracers (amino acids) from the feeding site to the roots and other sinks in the plant. This increases the nutrient concentration at the site of aphid feeding by increasing the import of resources from other sites in the plant, mobilising local resources and blocking their export to other organs. The damage of the foliar tissue that occurs as a result of the RWA feeding is thought to play a role in the pest’s ability to increase the nutritional quality of the host plant (Botha et al., 2006; Goggin, 2007; Shea et al., 2000).