Thesis (Ph.D. (Botany)) - Rhodes University, 2008.

Bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on spring-sown cereals. The aphid reduces yield and can act as a vector of barley yellow dwarf virus. R. padi does not give visible symptoms, but heavy infestation can reduce the yield substantially. R. padi is one of the most important pests of barley, which has the fourth rank among the cereals in global production. In order to overcome this problem, the plants are treated with pesticides. However, some of the pesticides are no longer permitted to use due to their negative environmental effects. Another problem is that the aphids develop resistance against them. Therefore, a new approach is to identify genetic factors that could be used in breeding host plants for resistance. There are many examples of successful plant breeding for resistance to aphids, but in the case of R. padi and barley, no resistant cultivar is commercially available. The aim of this thesis was to identify and characterise aphid resistance and susceptibility factors in barley. This was done using two major approaches. Firstly, constitutive and aphid-induced gene expression was studied in a large number of barley genotypes with known levels of resistance. Secondly, two cDNAs putatively adding to aphid resistance were transformed to Arabidopsis and barley and the effect of transformation on aphid performance and behaviour was evaluated.      The study of constitutive transcript abundance in 23 barley genotypes gave suggestive evidence that two genes might be related to aphid resistance; a thionin and a proteinase inhibitor gene, and that a lipoxygenase gene might be related to aphid susceptibility.The study of both constitutive and aphid-induced transcript abundances of three glucanase genes, gave support to the idea that two of them might be susceptibility factors. The cDNA of the proteinase inhibitor mentioned above was expressed in Arabidopsis, under control of either a constitutive or a phloem-specific promoter and the effects were evaluated using the green peach aphid, Myzus persicae Sulzer. This aphid is a generalist, feeding on many different plant species, both monocot (such as barley) and dicot (such as Arabidopsis).  The results showed lower settling and fecundity on some of the transgenic lines as compared to on controls.        In conclusion, the thesis suggests a role in resistance against R. padi in barley for two genes encoding a thionin and a proteinase inhibitor. It has also given support for a role in susceptibility against R. padi of genes encoding a lipoxygenase and two glucanases. The work further shows that a cDNA from barley, selected based on the interaction between a monocot plant and an essentially monocot specialist aphid, affected the resistance of the dicot Arabidopsis against a generalist aphid. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

In this paper, I will reflect on the challenges I faced from the first idea to the final export of 40 Meters Down, and how I overcame these.

Bacillus subtilis Stämme vom FZB - FZB24, FZB37 und FZB38 des FZB Biotechnik, Berlin - und ihre Stoffwechselprodukte wurden als Agenzien für eine Resistenzinduktion gegen Schädiger in drei Wirtspflanzen-Erreger-Systemen, Vicia faba - Uromyces appendiculatus, Vicia faba - Aphis fabae und Triticum aestivum - Rhopalosiphum padi untersucht. Prä-inokulativ wurden Blätter und Saatgut der Pflanzen mit den Testsubstanzen behandelt. Zur Testung gelangten Bakterien-Kulturfiltrat (KF), KF-Zenrifugationsüberstände, die vegetativen Zellen und Sporen der B. subtilis Stämme. Nach lokaler Applikation der KF-Überstände von B. subtilis auf die Wirtspflanzenblätter konnte eine erhebliche Breite antibiotischer (entwicklungshemmender) Wirkungen auf A. fabae und R. padi beobachtet werden. Eine Untersuchung der freien Aminosäuren im Phloemsaft von Vicia faba zeigte, daß sich nach Befall von Aphis fabae speziell bei den zuvor mit bakteriellem Kulturfiltrat und KF-Überstand behandelten Pflanzen im Vergleich zu der nur mit Wasser behandelten Kontrolle, die Konzentration von neun Aminosäuren änderte. / In topical and systemic plant treatment, in three host–parasite systems, i.e. Vicia faba - Uromyces appendiculatus, Vicia faba - Aphis fabae and Triticum aestivum - Rhopalosiphum padi the culture filtrate and supernatant of Bacillus subtilis (FZB24, FZB37 and FZB38 from FZB Biotechnik Berlin) was shown to inhibit the development of urediospores produced by Uromyces appendiculatus. The performance of Aphis fabae and Rhopalosiphum padi was evaluated using life table tests where the aphids'' development time (tD), pre-reproduction time (td), relative growth rate (RGR) and intrinsic rate of natural increase (rm) were assessed. A wide range of antibiosis effects in Aphis fabae and Rhopalosiphum padi was observed when the supernatant of Bacillus subtilis was used as foliar topical treatment. The tested aphids presented longer development and pre-reproduction time; conversely a lower relative growth rate and intrinsic rate of natural increase was observed. The investigation of the free amino acids of the phloem sap of V. faba plants, showed the concentration of the amino acid serine remained unchanged in the supernatant induced plants in this study, which was interpreted as the major reason for the observed antibiosis effect on the tested aphids.

The fields in farming landscape were united to lerge areas in the last 50 years. The balks were ploughed up and their disappearance contributed to the significant loss of biodiversity. Nowadays attepmts are being made to restore the complex agricultural landscape. The creation of territorial system of ecological stability started. Planting biocorridors is a part of this system. Biocorridor is a linear element, consisting of trees and shrubs, which divides large fields. Such non-crop habitats have large positive impact on surrounding landscape. They can also act as refuges for the insect pest. Exapmle is the bird cherry, Prunus padus, a winter host of a cereal pest aphid, Rhopalosiphum padi. In the work the positive and negative effects of TSES are analysed. The important part of the work was to explore, whether the presence of Prunus padus affects the abundance of aphid in adjacent fields. The relatively newly planted biocorridor in Sedlec u Českých Budějovic was used for the work. Based on the results, the suggestions for planting of biocorridors were made, in order to avoid negative effects of spread of this aphid pest species.

<p>Since plants can not escape their predators by walking, they use some other defense systems, like induction or repression of defense genes. A microarray experiment performed with barley attacked by the bird cherry-oat aphid (Rhopalosiphum padi), led to the hypothesis that contig 16360 (similar to ser/thr kinases) could be linked with the resistance of barley against R. padi, and contig 6519 (similar to WIR 1A) with the susceptibility. Time course experiments showed that contig16360 and AJ250283 (similar to BCI-4) are almost induced in the same way, each, by two different aphids (R. padi and Metopolophium dirhodum). Genomic PCR was used to test the hypothesis that when plants have the gene for contig 16360, they are more likely to be resistant against aphid attack, and when plants have the gene for contig 6519, they are more likely to be susceptible. This test was performed with 69 barley lines: wild, commercial or breeding lines. Results were that the presence of WIR 1A gene has no correlation with the susceptibility, while presence of ser/thr kinase seems to be correlated with resistance.</p>

Since plants can not escape their predators by walking, they use some other defense systems, like induction or repression of defense genes. A microarray experiment performed with barley attacked by the bird cherry-oat aphid (Rhopalosiphum padi), led to the hypothesis that contig 16360 (similar to ser/thr kinases) could be linked with the resistance of barley against R. padi, and contig 6519 (similar to WIR 1A) with the susceptibility. Time course experiments showed that contig16360 and AJ250283 (similar to BCI-4) are almost induced in the same way, each, by two different aphids (R. padi and Metopolophium dirhodum). Genomic PCR was used to test the hypothesis that when plants have the gene for contig 16360, they are more likely to be resistant against aphid attack, and when plants have the gene for contig 6519, they are more likely to be susceptible. This test was performed with 69 barley lines: wild, commercial or breeding lines. Results were that the presence of WIR 1A gene has no correlation with the susceptibility, while presence of ser/thr kinase seems to be correlated with resistance.

Master of Science / Entomology / C. Michael Smith / The bird cherry oat aphid, Rhopalosiphum padi, and one of the viruses it vectors, barley yellow dwarf virus (BYDV), form a very damaging pest unit on cereals. Understanding how people can better manage crops to prevent damage or recognize environmental or geographic factors that put their crops at risk for BYDV could lead to improved virus aphid management strategies. One of the most successful methods for mitigating pest damage is using pest-resistant varieties of crops. Seven candidate wheat varieties were screened for resistance to R. padi, by testing aphid population densities and aphid host choice. Results of this research identified six varieties of wheat that show resistance to R. padi. One of the major knowledge gaps in BYDV management is forecasting potential damage. Our objective was to create viral presence maps, to start building the foundation of correlations between persistent and changing frequency of viruliferous R. padi. The results of BYDV assays in R. padi in wheat fields across Kansas indicated that the amount of BYDV infection in viruliferous aphids changes rapidly from year to year, and differs considerably between geographic regions and field landscape characteristics in Kansas. Neonicotinoid wheat seed treatment is a management technique that reduces R. padi populations. However, a common question among producers is whether or not seed treatments stop viral transmission. Results of greenhouse seed treatment experiments with plants from neonicotinoid treated and untreated seed infested with viruliferous R. padi indicated no significant difference in viral transmission due to seed treatment. This new information about R. padi wheat varietal resistance, geographic distribution of BYDV in Kansas, and neonicotinoid seed treatment creates a better understanding of aphid-virus-wheat interactions. Results from this thesis directly enhance producer ability to forecast risk from BYDV, select wheat varieties that effectively reduce R. padi as a BYDV vector, and to make better decisions about the use of insecticide to reduce BYDV infection.

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.

O Processamento e Análise Digital de Imagens é utilizado cada vez mais para agilizar processos, aumentar a precisão, segurança e confiabilidade de dados extraídos de imagens nas mais diversas áreas de pesquisa. No entanto, muitas vezes é necessário que o pesquisador faça, ele próprio, o pré-processamento das imagens, mesmo não sendo um especialista nesta área. Isto coloca em risco o próprio objetivo do uso do Processamento e Análise Digital de Imagens. Este trabalho analisa a relação dos dados extraídos de uma imagem (micrografia) através do software livre ImageJ com relação ao seu processamento final desejado, avaliando assim, a necessidade ou não, de uma ou mais sequencias de pré-processamento para adequar a imagem para o processamento final, indicando ainda quais fatores de influência apresentam informações irrelevantes ou incompletas para o processamento final utilizando ferramentas da Lógica Paraconsistente Anotada. Os resultados obtidos mostram que esta abordagem carece de informações diversificadas sobre a imagem original capturada que possam subsidiar a tomada de decisão quanto aos procedimentos necessários e, para o pré-processamento adequado ao objetivo desejado. / Processing and Analysis of Digital Images is increasingly used to streamline processes, improve accuracy, safety and reliability of data extracted from images in various research areas. However, it is often necessary for the researcher to make himself, the preprocessing of images, although not an expert in this area. This puts at risk the very purpose of using the Processing and Analysis of Digital Images. This paper analyzes the relationship of the data extracted from an image (micrograph) through the free software ImageJ, with respect to its desired final processing. Thus, evaluating the necessity or not, of one or more sequences of preprocessing to adjust the image to the final processing, further indicating which factors influence presents incomplete or irrelevant information for final processing using tools of Annotaded Paraconsistent Logic. The results show that this approach lacks diversified information about the original image captured that can support decision making about procedures for appropriate preprocessing to the desired goal.