Rozprawy doktorskie na temat „PLANT DISEASE DETECTION”

Sclerotinia sclerotiorum, a fungal pathogen of over 400 plant species has been estimated to cost UK based farmers approximately £20 million per year during severe outbreak (Oerke and Dehne 2004). S. sclerotiorum disease incidence is difficult to predict as outbreaks are often sporadic. Ascospores released from the fruiting bodies or apothecia can be dispersed for tens of kilometres. This makes disease control problematic and with no S. sclerotiorum resistant varieties available, growers are forced to spray fungicides up to three times per flowering season in anticipation of the arrival of this devastating disease. This thesis reports the development of the first infield S. sclerotiorum biosensor which aims to enable rapid detection of airborne ascospores, promoting a more accurate disease risk assessment and fungicide spraying regime. The sensor is designed to detect the presence of oxalic acid, the main pathogenicity factor secreted during early S. sclerotiorum ascospore germination. Upon electrochemical detection of this analyte in the biosensor, a binary output is relayed to farmer to warm him of a disease risk. This project focused on the development of a nutrient matrix which was designed to be contained within the biosensor. The role of this matrix was to promote the growth of captured airborne S. sclerotiorum ascospores and induce high levels of oxalic acid secretion. The use of the designed biological matrix to promote oxalic acid production was tested during three field trials in S. sclerotiorum artificially inoculated fields. This thesis describes the use of contemporary pathogenomics technologies to further investigate candidate genes involved in pathogenicity alongside the secretion of oxalic acid. A pre-described bioinformatics pipeline was used to predict the S. sclerotiorum secretome to identify potential effector proteins as well as explore proteins which are unique to S. sclerotiorum to be used as other novel targets for detection. GFP tagged constructs were designed to investigate the expression of the putative targets for S. sclerotiorum detection. The transcriptomes of wild type and oxalic acid deficient S. sclerotiorum strains during infection as well as during a saprotrophic stage were investigated. This study provided expression support for not only some of the unannotated genes identified in the putative secretome, but some candidate genes speculated to be involved in infection.

Laurel wilt disease is a vascular wilt affecting the xylem and water conductivity in trees belonging to the family Lauraceae. The disease was introduced by an invasive species of ambrosia beetle, Xyleborus glabratus. The beetle, together with its newly described fungal symbiont Raffaelea lauricola (pathogenic to host trees), has lead to the devastation and destruction of over 300 million wild redbay trees in southeastern forests. Ambrosia beetles make up a very unique clade of beetle and share a co-evolved obligatory mutualistic relationship with their partner fungi. Rather than consuming host tree material, the beetles excavate galleries or canals within them. These galleries serve two purposes: reproduction and fungal gardening. The beetles house fungal spores within specialized sacs, mycangia, and essentially inoculate host trees with the pathogenic agent. They actively grow and cultivate gardens of the fungus in galleries to serve as their sole food source. Once the fungus reaches the xylem vessels of the host tree, it thrives and leads to the blockage of water flow, both because of fungal accumulation and to the host response of secreting gels, gums and tyloses to occlude vessels in an attempt to quarantine the fungus. This disease spreads rapidly, and as a result, once symptoms become visible to the naked eye, it is already too late to save the tree, and it has likely already spread to adjacent ones. The present study presents the first documented study involving the early detection of disease from deep within a tree through the use of scent-discriminating canines. In addition, the present study has lead to the development of a novel sample collection device enabling the non-destructive sampling of beetle galleries. Finally, a metabolomics approach revealed key biochemical pathway modifications in the disease state, as well as potential clues to disease development.

Bud rot disease has been considered as a devastating disease of oil palm in Latin America. Severe outbreaks of this disease have been reported in Colombia, Brazil, Ecuador, Panama and Suriname. The causal agent of bud rot disease in Colombia has been identified as Phytophthora palmivora. This pathogen is known to be responsible for several tropical diseases such as black pod and stem canker disease of cocoa, especially during the rainy season. Phytophthora palmivora has also been reported to attack durian, rubber, pepper and jackfruit causing diseases in various parts of the plant such as fruit, leaves and stems. However, no outbreaks of the disease have been reported in oil palm in Malaysia or other Southeast Asian countries. Several aspects of research need to be conducted to understand why this pathogen causes problems in oil palm in South America but not in Southeast Asia. This study aimed to analyze variation between the Colombian P. palmivora isolates that cause bud rot disease in comparison with Malaysian isolates and other isolates gathered from different hosts and regions. Our hypothesis was that P. palmivora isolates from the different regions and/or hosts have different molecular characteristics and have dissimilar levels of pathogenicity. Sequence alignments of several genetic markers, the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) gene cluster, beta-tubulin (β-tubulin), translation elongation factor 1 alpha (EF-1α), cytochrome c oxidase subunit I (CoxI) and subunit II (CoxII) genes failed to distinguish between Colombian oil palm isolates and P. palmivora from different hosts and regions. It was concluded that these markers are more suitable for inter-specific studies between species but not for intra-specific evaluation within species of P. palmivora. However, a new marker named as P. palmivora hypothetical avirulence effector protein (PpHPAVR) along with analyses of amplified fragment length polymorphisms (AFLPs), separated the Malaysian and Colombian isolates into distinct clades. This indicates that there is genomic variation within P. palmivora isolates. The zoospores of P. palmivora from various hosts and demographic origin were shown to have the ability to cause infection to oil palm seedlings, durian and rubber. However, not enough evidence has been collected to confirm that pathogenicity correlates with the distinct clades observed with AFLPs and PpHPAVR. Phytophthora palmivora species-specific diagnostic using PCR and loop-mediated isothermal amplification (LAMP) have been developed based on the PpHPAVR region.

The main objective of this thesis dissertation is functionally characterizing the genes involved in biotic and abiotic stresses of plants at molecular level. Previously, upon pathogen attack Rad6 gene expression was found to be changed in wheat and barley plants. To functionally characterize the Rad6 gene, VIGS (Virus induced gene silencing) system was used. HR (Hypersensitive response) like symptoms was detected in every silenced barley and wheat plants. To figure out, transcriptomes and proteomes of Rad6 silenced plants were analyzed. 2-D PAGE analysis was also performed on silenced and control wheat plants. No pathogen growth was observed in Rad6 silenced barley lines. Additionally, the susceptible wild type Arabidopsis plants showed resistant phenotype when any of the Rad6 gene copies is mutated. This suggests that Rad6 gene has a negative regulatory role in plant disease resistance which was proved for the first time. Yeast two hybrid protein interaction study suggests that RAD6 carrying out its function by interacting with SGT1 protein and regulating resistance related genes. It has been first time reported in this thesis that E2 (Ubiquitin conjugating enzyme) takes role in plant disease resistance. Boron which is the other consideration in the scope of thesis as an abiotic stress factor at a very limited amount is necessary for the normal development of plants. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were tested in the presence of high boron (35 mg/kg) concentrations. The transcriptomes of the plant samples treated with the excess levels of boron to that of the samples grown under normal concentration were compared using differential display PCR method. Thirty bands showing differential expression levels at varying time points were analyzed. 18 of them were confirmed via qRT-PCR.

Sclerotinia sclerotiorum is a pathogenic fungus that infects around 400 species of host    plants. Stem rot disease caused by this fungus is economically disastrous for Brassica napus cultivators in Sweden. Due to the lack of disease resistant cultivars, disease management has been solely dependent on fungicide application. The current disease  prediction models are not scientifically accurate and take into account factors such as   weather, previous disease incidence, and conomic effects which often result in unnecessary and excessive use of fungicides by cultivators. Real-Time Polymerase Chain Reaction has proven to be the fastest, most accurate and reliable technique for detecting plant pathogens as it gives an idea about disease severity by measuring pathogen concentration in environmental samples. Reproducible and able qPCR assays have the potential of being the main principle on which more scientifically accurate plant disease prediction and management models an be developed. The aim of this study was to validate a previously established qPCR assay to detect S. sclerotiorum. An absolute quantification experiment     was performed by using plasmid DNA cloned with a target gene as template. Further,   three different qPCR machines  were compared  to make a plausible conclusion regarding    their sensitivity and efficiency in detecting minuscule amounts of DNA from the   environment. While a solid conclusion could not be reached regarding the sensitivity of    each of these machines, this study pointed out some basic trends about each machine    that may help researchers in selecting the most efficient qPCR system when working with detection of plant pathogens.

Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

The detection of plant diseases is an important part of commercial greenhouse crop production and can enable continued disease and pest control which will ultimately lead to the economical benefit as well as the significant reduction in use of chemical and biochemical treatments. A plant subject to infection typically releases exclusive volatile organic compounds (VOCs) which may be detected by appropriate sensors. A set of experiments were designed, constructed and conducted at University of Warwick in which the state-of-the-art gas sensors namely Electronic Nose (EN) and Field Asymmetric Ion Mobility Spectrometry (FAIMS) were employed to sample the VOC profiles in order to detect powdery mildew-infected and spider mite-infested tomato plants in a non-destructive manner. The data acquired from the EN and FAIMS devices was analysed using Principal Component Analysis, Linear Discriminant Analysis, Support Vector Machines and Artificial Neural Networks. Both EN and FAIMS proved to be able to distinguish between healthy and infected tomato plants with desirable accuracy when coupled with an appropriate data analysis technique. A review of the literature on plant diseases, destructive and non-destructive plant's disease detection tools as well as VOC sampling procedures and instrumentation will be given throughout this thesis. Moreover, the latter part of this thesis presents the master-slave synchronisation of identical chaotic dynamical systems using the open-plus close-loop (OPCL) control method. The study is mainly concerned with the behaviour of the synchronisation of chaotic dynamical systems in respect to an added bias and in the case of mismatch of parameters of master and slave systems. The link between the external bias and the synchronisation error generated as well as between the value of parameters mismatch and the synchronisation error is examined and discussed. The usability of the newly proposed approaches is assessed by the aid of two applications. The first application demonstrates that a weak bias acting on Nano-mechanical resonator shows the linear correlation with the synchronisation error and, consequently, the bias can be estimated via this error. The second application is related to the synchronisation of the cantilevers commonly found in ENs and Atomic Force Microscopy (AFM). It is suggested to use a novel scheme of coupled master{slave cantilevers and, estimate the difference in cantilever-surface interactions in master oscillator and in slave oscillator via measuring the synchronisation error. The scheme is particularly useful for using the master cantilever as a control and the slave cantilever as a unit device which measures surface properties. The study shows that by calculating the error of synchronisation, a precise measurements can be conducted when two cantilevers leave the synchronous region, that is when they de-synchronise. Thus, this thesis also contributes to the understanding of de-synchronisation of nano-scale chaotic systems (Nano electromechanical Systems) in respect to the addition of an external bias and/or parameters mismatch by outlining the possible applications.

Thesis (MScAgric)--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood for infection by other pathogens. Knowledge about the epidemiology and especially inoculum sources of this disease is imperative for subsequent development of management strategies. Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through infected propagation material in South Africa. However, the infection pathways and inoculum sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen in various media is by means of isolation onto artificial growth media. This has proven to be problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it can be identified. The aim of this study was (i) to develop a protocol for the molecular detection of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora. A protocol was developed and validated for the molecular detection of Pa. chlamydospora in grapevine wood. Firstly, several previously published protocols were used to develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic DNA from grapevine wood. The protocol was validated using various grapevine material from 3 different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3 different nurseries, including grapevines that were subjected to hot water treatment. The basal end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial medium and molecular detection. The identity of PCR products obtained from a subset of samples, that only tested positive for Pa. chlamydospora based on molecular detection, was confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1% of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora was not isolated from hot water treated samples. The results confirm the importance of hot water treatment for proactive management of Petri disease in grapevine nurseries. However, Pa. chlamydospora DNA was molecularly detected in hot water treated samples in frequencies similar to that detected in non-hot water treated samples. As expected, the DNA in hot water treated plants was not destroyed and could be detected by the developed molecular detection protocol. This is an important consideration when using molecular detection for disease diagnosis or pathogen detection and shows that these methods should be used in conjunction with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10 to 15 times cheaper than commercial DNA extraction kits. Preliminary studies showed that the aforementioned molecular detection technique was not specific and sensitive enough for detection of Pa. chlamydospora in soil and water (unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. Rootstock cane sections and soil samples were taking from the mother blocks from several nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage and grafting. Scion and rootstock cuttings were also collected during grafting and soil were collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were Pa. chlamydospora specific, except for five bands obtained from callusing media and one band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12- hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the callusing medium samples. These media should therefore be considered as potential inoculum sources or infection points of the pathogen during the nursery stages. The results furthermore confirmed previous findings that Pa. chlamydospora is mainly distributed through infected rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or chemical amendments in the hydration water and callusing medium and wound protection from soil borne infections.
AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora is die hoof veroorsakende organisme van Petri se siekte wat lei tot die agteruitgang en terugsterwing van jong wingerdplante (1-7 jaar oud) en veroorsaak verhoogde vatbaarheid van hout vir infeksie deur ander patogene. Kennis oor die epidemiologie en veral die inokulumbronne van die siekte is noodsaaklik vir die daaropvolgende ontwikkeling van beheerstrategieë. Isolasies het getoon dat Pa. chlamydospora meestal versprei deur middel van geïnfekteerde voortplantingsmateriaal in Suid-Afrika. Die infeksieweë en inokulumbronne in wingerdkwekerye is egter steeds onbekend. Die enigste bestaande metode vir die opsporing van die patogeen, in verskeie mediums, is deur middel van isolasie op kunsmatige groeimediums. Dit is egter gevind om problematies te wees aangesien die swam uiters stadig groei (dit vat tot 4 weke vanaf isolasie tot identifikasie) en die kulture is telkens oorgroei deur ander organismes voordat identifikasie kan plaasvind. Die doel van die studie was (i) om ‘n protokol te ontwikkel vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout, en (ii) om die protokol te gebruik, saam met ander, om verskillende monsters (water, grond, onderstok- en bostok-ente en kallusmedium) te toets, wat versamel is van kwekerye in Suid- Afrika, tydens verskillende kwekerystadiums, vir die teenwoordigheid van Pa. chlamydospora. ‘n Protokol is ontwikkel en geverifieer vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout. Eerstens is verskeie protokols wat voorheen gepubliseer is, is as grondslag gebruik vir die ontwikkeling van ‘n ekonomiese en tydbesparende DNA ekstraksie protokol. Hierna is PKR (polimerase ketting reaksie) amplifikasie met spesie-spesifieke inleiers (Pch1 en Pch2) gevind om sensitief genoeg te wees om so min as 1 pg van Pa. chlamydospora genomiese DNA van wingerdhout op te spoor. Die protokol is geverifieer deur verskeie wingerdhoutmateriaal van 3 verskillende onderstokkultivars (101-14 Mgt, Ramsey en Richter 99) te gebruik, wat elk versamel is van 3 verskillende kwekerye. ‘n Aantal van die wingerstokke is ook onderwerp aan warmwaterbehandeling. Die basale kant van die onderstok is parallel geanaliseer vir Pa. chlamydospora deur gebruik te maak van isolasies op kunsmatige groeimedium asook molekulêre opsporing. Die identiteit van ‘n submonster van PKR produkte van verskeie monsters, wat slegs positief getoets het vir Pa. chlamydospora met die molekulêre opsporing, is bevestig om Pa. chlamydospora spesifiek te wees. Dit is gedoen deur middel van restriksie ensiem analise met AatII. Molekulêre opsporing is gevind om aansienlik meer sensitief te wees as isolasies, deurdat Pa. chlamydospora opgespoor is van positiewe sowel as negatiewe isolasies. Die molekulêre tegniek het Pa. chlamydospora in ‘n gemiddeld van 80.9% van die monsters opgespoor, terwyl slegs ‘n gemiddeld van 24.1% van die monsters postief getoets het vir Pa. chlamydospora, deur middel van isolasies. Pa. chlamydospora is nie geïsoleer van die monsters wat warmwaterbehandeling ondergaan het nie. Die resultate bevestig hoe belangrik warmwaterbehandeling is vir die proaktiewe beheer van Petri se siekte in wingerdkwekerye. Pa. chlamydospora DNA is met die molekulêre tegniek opgespoor, in warmwaterbehandelde monsters, in getalle wat ooreenstemmend is met die van niewarmwaterbehandelde monsters. Soos verwag, is DNA in warmwaterbehandelde plante nie vernietig nie en kon dit telke male opgespoor word deur die ontwikkelde molekulêre opsporing protokol. Dit is ‘n belangrike feit wat in ag geneem moet word wanneer molekulêre opsporing gebruik word in siekte diagnose en opsporing van patogene en dit is ‘n aanduiding dat die metodes gebruik moet word in samewerking met ander diagnostiese tegnieke. Die DNA ekstraksie protokol het getoon om tot en met 10 tot 15 kere goedkoper te wees as kommersiële DNA ekstraksie pakkette. Voorlopige studies het getoon dat die bogenoemde molekulêre opsporings tegniek nie spesifiek en sensitief genoeg is vir die opsporing van Pa. chlamydospora uit grond en water nie (ongepubliseerde data). Daarom is ‘n enkel-buis geneste-PKR tegniek geoptimiseer vir die opsporing van Pa. chlamydospora DNA wat geëkstraheer is vanaf grond, water, kallusmedium en wingerdhout. Dele van onderstokke en grond monsters is geneem vanaf moederblokke van verskeie kwekerye. Gedurende die voor-opberging en enting periodes is watermonsters versamel vanaf hidrasie en fungisied tenke. Bostok- en onderstokente is ook versamel gedurende enting en grond is versamel vanaf kwekerybeddens net voor uitplanting. Die enkelbuis geneste-PKR was sensitief genoeg om so min as 1 fg van Pa. chlamydospora genomiese DNA vanaf water en 10 fg vanaf hout, kallusmedium en grond op te spoor. PKR analise van die verskillende kwekerymonsters het getoon dat daar ‘n teenwoordigheid is van verskeie putatiewe Pa. chlamydospora spesifieke bande (360 bp). Daaropvolgende analise deur middel van DNA volgordebepaling en restriksie ensiem analise het bevestig dat al die 360 bp PKR bande wel Pa. chlamydospora spesifiek is, behalwe vir vyf bande wat verkry is vanaf kallusmedium en een band verkry vanaf water. As slegs Pa. chlamydospora spesifieke bande in ag geneem word, is daar met molekulêre opsporing die teenwoordigheid van Pa. chlamydospora gevind in 25% van die onderstokke, 17 % van die grond versamel vanaf moederblokke, 42% van die onderstokente versamel tydens enting, 16% van die bostokente, 40% van die watermonsters versamel voor die 12-uur hidrasie periode, 67% van die watermonsters versamel gedurende enting en 8% van die kallusmediummonsters. Hierdie mediums moet dus beskou word as potensiële inokulumbronne of infeksiepunte van die patogeen gedurende die kwekerystadiums. Die resultate bevestig ook verdere bevindinge wat aandui dat Pa. chlamydospora meestal versprei word deur geïnfekteerde onderstokke en ente. Geïnfekteerde bostokente is ook aangedui om potensiële draers van die patogeen te wees. Beheerstrategieë moet dus wondbeskerming van onderstok moederplante insluit, asook uitwissing van die patogeen vanaf onderstokente (bv. warmwaterbehandeling), toediening van biologiese of chemiese stowwe in die hidrasie water en kallusmedium en wondbeskerming teen grondgedraagde infeksies.

La bacteriose a e. Chrysanthemi pv. Dianthicola (echr) facteur limitant de la production du dahlia est transmise par la multiplication vegetative. Afin de proposer une methode de diagnostic plus precise que la detection visuelle, les methodes immunoenzymatiques ont ete etudiees et adaptees pour la detection d'echr dans les tissus du dahlia. La methode das-elisa (double antibody sandwich) est evaluee par rapport aux methodes de diagnostic de reference (isolement et immunofluorescence). Son utilisation, pour l'analyse sanitaire du materiel de propagation vis-a-vis d'echr seul et associe eventuellemnt a la mosaique du dahlia (damy), est etudiee en vue d'une selection sanitaire. Les etudes effectuees pour optimiser les reactifs, pour determiner les parametres pouvant modifier la reaction antigene-anticorps

A cana-de-açúcar, é atualmente a cultura mais plantada no estado de São Paulo, apresentando grande importância no setor agrícola. Assim como qualquer outra cultura, é hospedeira de uma série de patógenos que podem limitar sua produção. A bactéria fastidiosa Leifsonia xyli subsp. xyli (Lxx) é o agente causal do raquistismo-das-soqueiras (RSD) em canade- açúcar cujo o principal sintoma é a redução acentuada do crescimento observada em plantas adultas. Essa doença é de difícil diagnose pois a evolução dos sintomas é lenta devido à natureza fastidiosa da bactéria. Lxx pode ser considerada como um endófíto obrigatório que cresce a níveis patogênicos nos tecidos da planta dependendo de estímulos bióticos e abióticos. Devido à importância da cultura e aos danosoCasionados pela Lxx, este trabalho apresentou dois enfoques principais: o primeiro foi o desenvolvimento de um protocolo para quantificação de Lxx em tecido de cana-de-açúcar por meio da técnica de PCR quantitativo em tempo real; o segundo foi o estudo da interação entre cana-de-açúcar e Lxx, em busca de uma melhor compreensão da evolução desse processo através da identificação de proteínas que apresentaram alteração em abundância ao longo do tempo em função da colonização pela bactéria em duas variedades de cana (RB835486 e SP80-3280) e em seguida enfatizando o sistema antioxidante nesta relação. Para isso, foram desenvolvidos primers específicos para detecção de Lxx que permitiram quantificar baixos níveis bacterianos em tecido foliar, revelando diferenças entre variedades segundo a cinética do crescimento bacteriano. Plantas com diferentes títulos bacterianos obtidas mediante inoculação artificial ou não foram submetidas à análise proteômica por meio da técnica de 2D-DIGE, uma vez que para o RSD, os danos estão relacionados à alta colonização de Lxx em seus tecidos. Os resultados alcançados com o sequenciamento de proteínas que apresentaram alteração em abundância revelaram que, em plantas da variedade RB835486 observou-se repressão de proteínas da via de estresse oxidativo e metabolismo primário, em contraste com a variedade SP80-3280, que apresentou aumento da abundância de proteínas relacionadas à via de estresse oxidativo, ambas para o mesmo tratamento, em plantas não inoculadas artificialmente. Já em plantas inoculadas com Lxx foram identificadas alteração na abundância de proteínas relacionadas ao crescimento da planta, ciclo celular, vias de sinalização celular e hormonal, esses resultados são consistentes com o principal sintoma da doença, o raquitismo, pois indica que as alterações temporais observadas na expressão de proteínas relacionadas com o aumento do título de Lxx in planta podem resultar em alterações do equilíbrio hormonal e menor crescimento da planta. Alguns resultados observados com a análise bioquímica corroboram com os dados acima descritos, pois a variedade RB835486 apresentou uma resposta precoce ao estresse oxidativo e mostrou maior controle do crescimento bacteriano, o que pode estar relacionado ao balanço de ERO\'s (espécies reativas de oxigênio) utilizadas pelo metabolismo como sinalizador celular na interação planta-patógeno. Em contraste, a variedade SP80-3280, em sua maioria, apresentou indução da atividade de enzimas antioxidantes mais tardiamente, momento em que a bactéria Lxx apresentou os maiores títulos de crescimento.
Sugarcane is currently the most grown crop in the state of São Paulo, with great importance in the agricultural sector. Like every crop, sugarcane is host to a number of pathogens that may limit its production. The fastidious bacterium Leifsonia xyli subsp. xyli (Lxx) is the causal agent of ratoon stunting in sugarcane (RSD), which the main symptom is a sharp reduction in growth observed in adult plants, this disease is difficult to diagnose because the evolution is slow due to nature of fastidious bacteria. Lxx can be considered as an obligatory endophyte that grows at pathogen levels in plant tissues depending on biotic and abiotic stimuli. Due to the importance of culture and the damage caused by the Lxx, this work presents two main approaches: the first one was the development of a protocol for the quantification of Lxx in sugarcane tissue by quantitative real time PCR; the second one was to study the interaction between sugarcane and Leifsonia xyli subsp. xyli aiming to a better understanding of the evolution of this process, identifying alterations in abundance of proteins over time depending on the bacterial colonization in the two varieties of sugarcane (RB835486 and SP80-3280) and then, emphasizing the antioxidant system in this relationship. Thus, we developed specific primers that enabled Lxx quantification at low bacterial levels in leaf tissue. The assay showed differences among sugarcane varieties according to the kinetics of bacterial growth. Plants that presented different titers of bacteria were obtained by artificial inoculum or not were selected to proteomic analysis by 2D-DIGE technique, since for RSD, the damage is related to high colonization in plant tissues. The identification of sugarcane proteins revealed that for variety RB835486 were observed a repression of stress proteins and proteins related to primary metabolism, in contrast with SP80-3280 variety, which it was identified high abundance of proteins of oxidative stress pathway, in not inoculated plants, for both varieties. However in inoculated plants it was identified a change in the abundance of proteins related to plant growth, cell cycle, cell signaling and hormonal pathways. These results corroborate with the main symptom of the disease, ratton, and suggest that temporal changes in expression of sugarcane cited proteins by increasing title of Lxx can result in hormonal imbalance and the decrease of plant growth. The major part of biochemistry analyzes corroborate with previous data obtained in proteomic approach. The RB835486 variety showed an early response to oxidative stress and suggests a greater control of bacterial growth in its tissues, which may be related to the balance of ERO\'s in signaling metabolism in plant pathogen interaction. While the SP80-3280, showed a later induction of antioxidant enzymes, when the bacterium Lxx had the highest titer of bacteria.

International trade in plants, especially with potting substrates, is recognised as the main pathway of plant pathogen dissemination on a global scale. In the last 20 years, the wide use of internet commerce has become common in the nursery sector and, due to the nature of online sales, may be aggravating this risk. Oomycetes in the genera Phytophthora, Pythium and Phytopythium, cause a range of important plant diseases, responsible for serious economic and biological losses. This research focused on the detection of Oomycetes in imported potted ornamental plants in the UK and The Netherlands, including internet sales and asymptomatic plants. Isolation techniques and molecular protocols were developed to quantify pathogen load in ornamental plants, using TaqMan PCR and Next Generation Sequencing (NGS) to assess Oomycete diversity using a multi-locus approach. Survival of Phytophthora cinnamomi and Fusarium verticillioides was estimated in two commercial potting mixes used in ornamental plant production. Oomycetes were detected in all samples analysed with the NGS approach, with 38 Phytophthora spp. and 48 Pythium/Phytopythium spp. identified. Phytophthora ramorum, P. alni subsp. alni and P. cryptogea were common. TaqMan PCR quantification showed high numbers of Oomycetes in all samples, especially in substrates, followed by roots and baiting waters. During sampling by isolation, Pythium kashmirense was recovered from Viburnum plicatum, the first record of this species in the UK. The survival experiment showed that Fusarium verticillioides remained viable after 17 months, whereas Phytophthora cinnamomi was viable up to 7 months after inoculation. This work clearly demonstrated the widespread presence of Oomycete pathogens in the plants for planting pathway. Moreover, the protocols developed and findings of this work contribute greatly to the understanding of the potential for pathogens to spread in the international horticultural trade and may help to improve plant biosecurity protocols in the UK and Europe.

Polyclonal antisera produced against spores, soluble protein and the whole mycelium fractions of Didymella lycopersici reacted with the homologous and heterologous antigens. The most sensitive antiserum was that raised against the whole mycelium, the soluble protein and the spore, in decreasing order of sensitivity. Using the antiserum raised against the whole mycelium it was possible to detect D. lycopersici on diseased plants and infested seeds. Cross reactivity was observed between the antisera produced to D. lycopersici and D. applanata, D. bryoniae and other tomato fungal pathogens including Fusarium spp. and B. cinerea. ELISA was most sensitive and reliable compared to double immunodiffusion, and latex tests. No reactions were obtained using the latex agglutination procedure and no antiserum detected spores in double diffusion tests. Protein profiles on SDS-PAGE revealed that the total number of protein bands decreased with increased age of cultures of D. lycopersici incubated in liquid media. Western blots probed with the antiserum raised against the whole mycelium showed that protein bands from extracts of both D. lycopersici and D. applanata were antigenic.
Land and Food Systems, Faculty of
Graduate

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The aim of this study was to combine two common diagnostic tools: serological kits and genetic fingerprinting to identify cherry leaf-roll nepovirus (CLRV), and to establish a marker system to characterize walnut germplasm. The detection of plant viruses is difficult. Restrictions are imposed for quarantine purposes on the importation of plant material from foreign countries. Modern techniques such as a PCR based screening method for CLRV are required to ensure material do not harbour viruses. A primer pair was designed to amplify a 430 bp non-coding homologous region. For the choice of primers, consensus sequences were considered and areas where the sequence data shared 98.5% homology, were chosen. The sensitivity of this detection method was 100-fold higher when compared to the ELISA. The PCR fragment was verified by nucleotide sequencing. AFLP technology was used to identify polymorphic fragments for 6 walnut cultivars and a rootstock, and SCARs were developed from AFLP specific bands. The AFLP technique distinguished all the walnut cultivars and the rootstock. However, conversion of AFLP fragments to SCAR markers for the development of a simple robust technique for cultivar discrimination, was not successful. Using 27 AFLP primer combinations, polymorphic fragments as high as 47.8% were scored. The reason for the lack of efficient conversion was as the result of the AFLP technique. The SCAR primers were generated from sequences internal to the AFLP primers but the specificity of the markers was in the AFLP primers not the internal sequence. In this study using AFLP, walnut cultivars were found to be closely related. The AFLP primer pairs used, provided polymorphic fragments. From these fragments, 7 SCAR markers were developed. It was expected that these SCARs derived from the AFLP markers would detect slight differences between cultivars. The Paradox SCAR marker was the only one that could divide the cultivars into two groups. When Chandler SCAR products were digested with the restriction enzyme Rsal, the same banding pattern as that of Paradox SCAR products was observed.
AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om twee algemene opsporingstegnieke te kombineer: serologiese toetsstelle en genetiese vingerafdrukke om cherry leaf-roll nepovirus (CLRV) te eien en om In merkersisteem te ontwikkel wat okkerneut kiemplasma kan karakteriseer. Die opsporing van plant virusse is baie moeilik. As gevolg van kwarantyn vereistes, word daar beperkinge geplaas word op die invoer van plant materiaal vanuit die buiteland. Moderne tegnieke soos hierdie een wat op PKR berus, word benodig om te verseker dat CLRV nie in plantmateriaal teenwoordig is nie. In Stel inleiers is ontwerp wat In 430 bp nie-koderende homoloë area amplifiseer. Hiervoor is konsensus volgordes bestudeer en slegs die volgordes wat 98,5% homologie getoon het, is gekies. In vergelyking met ELISA was die sensitiwiteit van hierdie deteksie metode 100 maal beter. DNA volgordebepaling is op die resulterende fragment gedoen om die PKR produk te verifieer. AFLP tegnologie is gebruik om polimorfiese fraqmente vir 6 okkerneut kultivars en 'n onderstok te identifiseer en SCARs is uit hierdie fragmente ontwikkel. Die AFLP tegniek kon tussen al die okkerneut kultivars en die onderstok onderskei. Die omskakeling van die AFLP fragmente in SCAR merkers om sodoende In eenvoudige kragtige tegniek vir kultivar onderskeiding te ontwikkel, was egter nie suksesvol nie. Met die gebruik van 27 AFLP inleier kombinasies, kon polimorfiese fragmente van so hoog as 47.8% verkry word. Die rede hoekom omskakeling onsuksesvol was lê by die aard van die AFLP tegniek. Die SCAR inleiers is ontwikkel uit volyordes intern tot die AFLP inleiers, maar die spesifisiteit van die merkers het juis in die AFLP inleiers gelê en nie in die interne volgordes nie. In hierdie studie, met die gebruik van AFLP, is gevind dat okkerneut kultivars baie naby verwant is. Die AFLP inleierstelle wat gebruik is, het polimorfiese fragmente gelewer. Uit hierdie fragmente is 7 SCAR merkers ontwikkel. Daar is verwag dat die SCARs wat uit die AFLP merkers ontwikkel is, klein verskille tussen kultivars sou opspoor. Dit was egter net die Paradox SCAR merker wat die kultivars in twee groepe kon verdeel. Restriksie ensiem vertering met Rsalop die Chandler SCAR produkte het dieselfde bandpatrone as die van die Paradox SCAR produkte gelewer.

The grape industry in Oklahoma was valued at $98 million in 2010. In 2015, symptoms resembling Grapevine Leafroll disease were observed, but Grapevine Leafroll-associated Viruses were not detected using enzyme-linked immunosorbent assay (ELISA). A 2-year Cooperative Agricultural Pest Survey was initiated to determine the etiology of the red leaf symptoms in Oklahoma vineyards. In 2016, a total of 121 symptomatic grapevines from 13 counties were sampled and 96 symptomatic grapevines from 14 counties were sampled in 2017. Each sample was tested for Grapevine Red Blotch Virus (GRBV), Xylella fastidiosa (Pierce’s Disease), and ‘Candidatus Phytoplasma spp,’ by polymerase chain reaction (PCR). ELISA was used to test for Grapevine Leafroll associated Virus (GLRaV) strains 1,3 and 4 strains. Rotbrenner, caused by Pseudopezicula traceiphila, (2017 only), can be found in xylem from petioles and the xylem was examined morphologically for signs of fungal structures. In 2016, GRBV was detected in 38% of 121 symptomatic samples, GLRaV-1 and -3 were detected in 16%, GLRaV 4 strains were detected in 2%, and X. fastidiosa was detected in 2%. There were no detections of ‘Ca Phytoplasma spp’ in 2016 or 2017. In 2017, GRBV was detected in 34% of the 96 samples, GLRaV-1 and -3 were detected in 17%, GLRaV 4 strains were detected in 3%, and X. fastidiosa was detected in 3%. Rotbrenner was not detected in any of the samples in 2017. The findings of this survey provide information to Oklahoma grape growers and extension personnel about the cause of red leaf diseases affecting grapevines so that appropriate management strategies can be implemented in the near future.

Lethal disease (LD), a phytoplasma lethal yellowing-type disease of coconut palms, is the major threat to coconut cultivation in the coastal areas of Tanzania. Two molecular approaches have been developed for early and accurate disease diagnosis. Random fragments of LD phytoplasma DNA were generated as probes for pathogen detection. LD DNA extracted from infected coconut tissue was randomly fragmented and cloned into pUC 18. Selected recombinants were labelled with DIG-dUTP and used as probes in dot-hybridizations with total DNA from LD infected palms. The probes hybridized strongly to DNA from infected palms, but there was also a significant level of background hybridization to DNA from healthy palms. The second technique used oligonucleotide primers for conserved regions of the 16S rRNA gene and variable spacer regions between 16S and 23S rRNA genes in the polymerase chain reaction (PCR). Amplification of phytoplasma rDNA was primed from LD-infected palms in Tanzania, Kenya and Mozambique, and no amplification products were obtained from healthy coconut tissue. By use of these techniques infection could be reliably detected in the spear leaves and root tips of affected palms thereby avoiding destructive palm sampling. The pathogen was found in all meristematic tissues with highest concentrations of phytoplasmas in the petioles of young unemerged leaves, the area below the growing point and the root tips in palms with moderately advanced diseases symptoms. Root tips proved reliable for sampling when compared to spears, and are now recommendet do be sampled together with the spears in routine, non-destructive sampling. Phytoplasmas could be detected in symptomless palms one month before the onset of disease symptoms by use of DNA probes and two months before by PCR, when spear leaves were sampled monthly from 180 randomly selected palms for a year. Of the 24 palms which subsequently developed disease LD was detectable in 25% prior to the onset of disease and in 46% at the time disease symptoms were visible. In 29% of these palms, phytoplasmas were not detected at all. No phytoplasmas were detected in any of the palms which remained healthy. The genetic relatedness of the LD phytoplasma to twelve different non-coconut infecting phytoplasmas, two spiroplasmas and phytoplasmas causing LYD in Kenya, Mozambique, Ghana, Florida and Jamaica were investigated. The LD DNA probe did not hybridize to any of the non-coconut infecting phytoplasmas and spiroplasmas. However, the probes detected a strong genetic relationship to all the LYD phytoplasmas. By use of PCR analyses, the phytoplasma causing LYD in Kenya was not found to differ from LD, but the pathogen causing LYD in Mozambique was found to be different. This appeared to be more closely related to the LYD phytoplasmas in West Africa. Studies on auchenorrhinchous insects in LD infected coconut fields revealed a strong relationship between seasons and insect flight into the fields. They also showed that local environmental conditions have a strong influence on vector populations, and may be indirectly responsible for the differences in disease incidence observed in different regions of the country. The flight pattern of auchenorynchous insects in general and of Diartrombus mkurangai in particular , coincided with the pattern of disease spread implying that this species is the most probable vector of LD. A good correlation obtained when the disease incidence data was regressed on the numbers of Diastrombus mkurangai and Meenoplus spp, but not on the total number of trapped auchenorynchous insects provided additional evidence or implicating these species as vectors of LD. More than 5000 individual insects were analysed by PCR in attempts to identify the insect vector or vectors for LD. PCR products of the right size were amplified from a few individuals of the species Diastrombus mkurangai and Meenoplus spp, and were shown to be LD phytoplasma by RFLP analysis. The techniques have provided a quicker and more reliable means of detecting LD phytoplasmas in coconut tissues and in putative insect vectors than the conventional methods. Possible improvements on the techniques are suggested and the prospects of utilising them to find a sustainable method of disease control discussed.

The yearly losses incurred by bacterial blackspot disease are high. Often trees are asymptomatic, with the pathogen either in the resident phase or latent stage of infection. Detection of the pathogen in these asymptomatic trees is one of the most important means of controlling the disease. Isolates which consistently differed in virulence were isolated from symptomatic mango plants. These isolates could be categorised into four groups based upon differences in virulence. Monoclonal antibodies (mAbs) were successfully raised using separate and pooled isolates for immunisation. MAbsraised were of the lgG class and reacted with a proteinaceous epitope. These monoclonal antibodies could distinguish between different virulence groups of Xanthomonas campestris pv. mangiferaeindicae by means of Western Blot analysis. These antibodies were used along with a selective medium, BVGA for detection of epiphytic populations as well as latent infections in mango. An enrichment step prior to the enzyme- linked immunosorbent assay (ELISA) is important, since bacterial counts on trees with latent infections are too low to result in a positive signal. These techniques in combination are thus useful for detection and monitoring of the pathogen, which may play an important role in controlling the spread of the disease.
Dissertation (MSc Agric)--University of Pretoria, 1993.
gm2014
Microbiology and Plant Pathology
Unrestricted

Rapid blight is a new disease of cool season turf grasses caused by Labyrinthula terrestris. It is problematic in Arizona and ten other states in cool season turfgrasses at sites with elevated salinity of soil and/or irrigation water. L. terrestris colonizes Tifgreen bermudagrasses in the field, but causes no apparent disease. Laboratory trials have shown that as concentrations of sodium chloride in irrigation water increase, disease severity increases, and when calcium and potassium salts are used to increase salinity, disease is greatly reduced or not observed. In preliminary field assays of cool-season turfgrasses irrigated with effluent, L. terrestris was observed in laboratory cultures from non-symptomatic turfgrass. To further substantiate if L. terrestris and/or other Labyrinthula species were present in non-symptomatic turfgrass in the field and to determine if disease could be induced by increased salinity, a trial was conducted at the Karsten Turfgrass Research Facility of The University of Arizona. In August 2006, field plots were established in bermudagrass "Tifway 419" and overseeded with Poa trivialis "Laser" in October. Plots were treated with potassium chloride, potassium sulfate or sodium chloride salts to increase soil salinity. Other plots treated with fungicides that are ineffective in controlling rapid blight as well as a sulfur treatment also were included in the assays. Poa trivialis was sampled in December 2006 and April 200. In laboratory assays using a semi-selective medium, Labyrinthula was detected in all treatments. Incidence was significantly higher in the untreated control and fungicide treated plots than in the salt treated plots. Results show that increasing soil salinity did not induce disease or result in an increase in detection of Labyrinthula at this site. Results of this study on Poa trivialis and previous studies on Tifgreen bermudagrass suggest that Labyrinthula may be widespread in non-symptomatic turfgrasses.

Soap Lake, located in Washington State, was the subject of an NSF funded Microbial Observatory and is a naturally occurring saline and alkaline lake. Several organisms inhabiting this lake have been identified as producers of siderophores that are unique in structure. Two isolates SL01 & SL28 were the focus of this study of siderophore production, structure elucidation and vesicle self-assembly. Bacterial isolates, enriched from Soap Lake sediment and water samples, were screened for siderophore production. Siderophore production was confirmed through the chrome azurol S (CAS) agar plate method. Isolates SL01 and SL28 were found to produce relatively high concentrations of siderophores in liquid medium. Extraction was performed by the methanol/water protocol in Varian cartridges and siderophore purification was done on HPLC with a 0-70% acetonitrile gradient. Lyophilization or in vacuo evaporation followed in order to store siderophores. Siderophore structure was determined using liquid chromatography and tandem mass spectrometry (LC/MS/MS) with fatty acid methyl ester (FAME) analysis. Vesicle self-assembly studies were performed using dynamic light scattering (DLS) and epifluorescence microscopy (employing cryoembedding and cryosectioning). Three new amphiphilic siderophore families (two from SL01 and one from SL28) were produced by the bacterial isolates, found to be most closely related to Halomonas variablis and Halomonas pantelleriensis, respectively. These siderophores resemble the amphiphilic aquachelin siderophores produced by Halomonas aquamarina strain DS40M3, a marine bacterium. Addition of ferric iron (Fe⁺³) at different equivalents demonstrated vesicle formation and this was confirmed by both DLS and epifluorescence microscopy. Bacteria thriving under saline and alkaline conditions are capable of producing unique siderophores resembling those produced by microbes inhabiting marine environments. Vesicle self-assembly was confirmed quantitatively and qualitatively. Amphiphilic siderophores may have different applications in medical and environmental fields.

The transmission of diseases from unhealthy to healthy plants is one of the most disastrous threats to the agriculture industry. Diseases transferred spread like wild fire and have the potential to infest the whole farm if not detected early. Plant disease detection methods aid in identifying infected plants in their very early stages and also help the user in scaling the identification of plant diseases to a variety of plants in a cost-effective manner. The aim of this thesis is to implement two different machine learning models, namely, Convolution Neural Networks (CNN) and K-nearest Neighbors (KNN) for the application of plant disease detection in tomato leaves.The two machine learning models were evaluated on four different metrics in order to find the best performing model among the two. The four different metrics were, Accuracy, Precision, Recall and F1-Score. Other than identifying the diseases using the aforementioned machine learning models, this study also focused on providing explainability to the predictions made by the respective models using the Explainable Artificial Intelligence technique, Local Interpretable Model-agnostic Explanations (LIME). In vein of collecting domain specific expertise, a user study was implemented in which the user trust of the AI and XAI models were evaluated and feedback from farmers were collected in order to provide recommendations for future research.The results on implementing the machine learning models showed that the CNN model performed better than the KNN model in all of the four evaluation metrics and the results from the user study signify that the farmers do not trust the AI and XAI models, however, the user study through the feedback collected from the farmers helps identify areas in which the trust of the farmers can be grown and strengthened.

Intelligent sensing for production of high-value crops Scientific and technical quality This thesis has been realized within the CROPS project. CROPS will develop scientific know-how for a highly configurable, modular and clever carrier platform that includes modular parallel manipulators and intelligent tools (sensors, algorithms, sprayers, grippers) that can be easily installed onto the carrier and are capable of adapting to new tasks and conditions. Several technological demonstrators will be developed for high value crops like greenhouse vegetables, fruits in orchards, and grapes for premium wines. The CROPS robotic platform will be capable of site-specific spraying (targets spray only towards foliage and selective targets) and selective harvesting of fruit (detects the fruit, determines its ripeness, moves towards the fruit, grasps it and softly detaches it). Another objective of CROPS is to develop techniques for reliable detection and classification of obstacles and other objects to enable successful autonomous navigation and operation in plantations and forests. The agricultural and forestry applications share many research areas, primarily regarding sensing and learning capabilities. The project started in October 2010 and will run for 48 month. The aim of this thesis is to lay the foundations, suggesting the guidelines, of one task addressed by the CROPS project, in particular, the aim of this work is to study the application of a VIS-NIR imaging approach (intelligent sensing), based on a relatively simple algorithm, to detect symptoms of powdery mildew and downy mildew disease at early stages of infection (sustainable production of high-value crops). Also a preliminary work for botrytis detection will be shown. Concept and objectives Many site-specific agricultural and forestry tasks, such as cultivating, transplanting, spraying, trimming, selective harvesting, and transportation, could be performed more efficiently if carried out by robotic systems. However, to date, agriculture and forestry robots are still not available, partly due to the complex, and often contradictory, demands for developing such systems. On the one hand, agro-forestry robots must be of reasonable cost, but on the other, they must be able to deal with complex, dynamic, and partly changing tasks. Addressing problems such as continuously changing conditions (e.g., rain and illumination), high variability in both the products (size, and shape) and the environment (location and soil properties), the delicate nature of the products, and hostile environmental conditions (e.g. dust, dirt, extreme temperature and humidity) requires advanced sensing, manipulation, and control. Since it is impossible to model a-priori all environments and task conditions, the robot must be able to learn new tasks and new working conditions. The solution to these demands lies in a modular and configurable design that will keep costs to a minimum by applying a basic configuration to a range of agricultural applications. At least a 95% yield rate is necessary for economical feasibility of an agro-forestry robotic system. Objectives An objective of CROPS project is to develop an “intelligent tools” (sensors, algorithms, sprayers) that can easily be installed onto a modular and clever carrier platform. The CROPS robotic platform will be capable of site-specific spraying (targeted spraying only on foliage and selected targets). Research efforts To achieve the novel systems described above, we will focus on intelligent sensing of disease detection on crop canopy (investigating different types and/or multiple sensors with decision making models). Technology evaluation Technology evaluation of the developed systems will include the performance evaluation of the different components (e.g., capacities, success rates/misses). Progress beyond the state-of-the-art Despite the extensive research conducted to date in applying robots to a variety of agriculture and forestry tasks (e.g., transplanting, spraying, trimming, selective harvesting), limited operating efficiencies (speeds, success rates) and lack of economic justification have severely limited commercialization. The few commercial autonomous agriculture and forestry robots that are available on the market include a cow milking robot, a robot for cutting roses (RomboMatic), and various remote-controlled forest harvesters. These robots either have a low level of autonomy or are able to perform only simple operations in structured and static environments (e.g. dairy farms and plant breeding facilities). Developing capabilities for robots operating in unstructured outdoor environments or dealing with the highly variable objects that exist in agriculture and forestry is still open-ended, and one of CROPS aims is to address this problem. Current state-of-the-art Field trials have routinely shown that most crop damage due to diseases and pests can be efficiently controlled when treatments are applied timely and accurately by hand to susceptible targets (i.e., by intelligent spraying). Site-specific spraying targeted solely to trees and/or to infected areas can reduce pesticide use by 20–40%. An issue of relevance to targeted agriculture is the detection of diseases in field crops. Since such events often have a visual manifestation, state-of-the-art methods for achieving this goal include fluorescence imaging or the analysis of spectral reflectance in carefully selected spectral bands. While reports of these methods used separately achieved performance at 75–90% accuracy, attempts to combine them have boosted disease discrimination accuracy to 95%. We must note here, however, that despite these promising results, very little research has been conducted on in-field disease detection. Expected progress The diseased detection approach for precision pesticide spraying will be developed investigating image processing techniques (after a laboratory spectral evaluation and greenhouse testing) for high-precision close-range targeted spraying to selectively and precisely apply chemicals solely to targets susceptible to specific diseases/pests, with a mean 90% success rate. Local changes in spectral reflection of parts of the canopy will be used as an indication of disease. “Soft-sensor” for detection of ripeness and diseases (noncontact rapid sensing system) will be developed by multispectral sensor (multispectral spectral camera). These “soft sensor” can be used as a decision model for targeted spraying.

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010.
ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs.
AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.

Early detection of disease and insect infestation within crops and precise application of pesticides can help reduce potential production losses, reduce environmental risk, and reduce the cost of farming. The goal of this study was the advanced detection of early blight (Alternaria solani) in potato (Solanum tuberosum) plants using hyperspectral remote sensing data captured with a handheld spectroradiometer. Hyperspectral reflectance spectra were captured 10 times over five weeks from plants grown to the vegetative and tuber bulking growth stages. The spectra were analyzed using principal component analysis (PCA), spectral change (ratio) analysis, partial least squares (PLS), cluster analysis, and vegetative indices. PCA successfully distinguished more heavily diseased plants from healthy and minimally diseased plants using two principal components. Spectral change (ratio) analysis provided wavelengths (490-510, 640, 665-670, 690, 740-750, and 935 nm) most sensitive to early blight infection followed by ANOVA results indicating a highly significant difference (p < 0.0001) between disease rating group means. In the majority of the experiments, comparisons of diseased plants with healthy plants using Fisher’s LSD revealed more heavily diseased plants were significantly different from healthy plants. PLS analysis demonstrated the feasibility of detecting early blight infected plants, finding four optimal factors for raw spectra with the predictor variation explained ranging from 93.4% to 94.6% and the response variation explained ranging from 42.7% to 64.7%. Cluster analysis successfully distinguished healthy plants from all diseased plants except for the most mildly diseased plants, showing clustering analysis was an effective method for detection of early blight. Analysis of the reflectance spectra using the simple ratio (SR) and the normalized difference vegetative index (NDVI) was effective at differentiating all diseased plants from healthy plants, except for the most mildly diseased plants. Of the analysis methods attempted, cluster analysis and vegetative indices were the most promising. The results show the potential of hyperspectral remote sensing for the detection of early blight in potato plants.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Infection of potatoes by viral pathogens causes reduced crop yield and subsequent economic loss. In South Africa Potato virus Y (PVY) and Potato leafroll virus (PLRV) are the two most destructive viruses infecting potatoes. Several other viral pathogens exist, including Potato virus X (PVX), Potato virus M (PVM), Potato virus A (PVA), Potato virus S (PVS), Potato mop-top virus (PMTV), Tomato spotted wilt virus (TSWV) and Potato spindle tuber viroid (PSTVd). Although the aforementioned pathogens are found infecting potatoes around the world, there are no published information pertaining to the prevalence of these viral agents in South Africa. Currently, the occurrence of PLRV infection in potatoes of South Africa has reached epidemic proportions. A previous phylogenetic investigation undertaken in our laboratory of South African PLRV isolates, using coat protein (CP) gene sequences, found large variation between native South African PLRV isolates and most other isolates from elsewhere in the world; with their nearest relatives being single isolates from Australia and North America. In this study the incidence of PVX, PVM, PVA, PVS, PMTV, TSWV and PSTVd was investigated. A large number of potato plant and tuber samples was collected and infected samples were identified with reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the CP gene or the whole genome in the case of PSTVd. The amplified nucleic acid segments were sequenced, aligned with international reference sequences and analysed phylogenetically to determine their relative relationships with these reference sequences. The CP genes of PLRV isolates were sequenced and phylogenetically investigated to determine how these new isolates compared relative to the previous findings from our laboratory. In addition, the complete genomes of two PLRV isolates were sequenced and phylogenetically investigated as a preliminary study to investigate the apparent increase of pathogenicity of certain variants of South African PLRV. Results obtained showed that only PVX and PVS were present in the samples collected and the incidences of these viruses were very low (2.0 and 1.1% respectively). The phylogenetic analyses of the CP genes, indicated that the PVX and PVS variants isolated in this study, were part of the dominant types of variants found worldwide. From the analyses of the PLRV CP and whole genome sequences, it was determined that many of the PLRV variants found in South Africa, are genetically distinctly different from those around the world. This warrants further investigation into the increased pathogenicity experienced with South African PLRV.
AFRIKAANSE OPSOMMING: Infeksie van aartappels deur virale patogene veroorsaak verlaagde opbrengs en gevolglike ekonomiese verlies. In Suid-Afrika is Aartappelvirus Y (PVY) en Aartappelrolblad virus (PLRV) die twee mees vernietigende virusse wat aartappels infekteer. Verskeie ander virale patogene, insluitend Aartappelvirus X (PVX), Aartappelvirus M (PVM), Aartappelvirus A (PVA), Aartappelvirus S (PVS), Aartappel "moptop" virus (PMTV), Kromnekvirus (TSWV) en Aartappel "spindle tuber" viroïed (PSTVd) kom ook wêreldwyd in aartappels voor. Alhoewel hierdie virusse aartappels wêreldwyd besmet, is daar geen gepubliseerde inligting met betrekking tot die voorkoms van hierdie virusse of die viroïed in Suid-Afrika nie. Tans het die voorkoms van PLRV infeksie in aartappels in Suid-Afrika epidemiese proporsies bereik. In 'n vorige filogenetiese ondersoek van die mantelproteïen (MP) nukleotiedvolgordes van Suid Afrikaanse PLRV isolate in ons laboratorium, is groot variasie tussen hierdie inheemse isolate en die meeste ander isolate van elders in die wêreld bevind. Die Suid Afrikaanse PLRV variante betree 'n unieke intermediêre posisie tussen die internasionale isolate en enkele isolate van Australië en Amerika. In hierdie studie is die voorkoms van PVX, PVM, PVA, PVS, PMTV, TSWV en PSTVd ondersoek. Groot aantal aartappelplant en -knol monsters is versamel en infeksie is getoets met tru-transkripsie polimerase kettingreaksie (RT-PCR) amplifisering van die MP geen, of die hele genoom in die geval van PSTVd. Die nukleïensuurvolgordes is bepaal en vergelyk met internasionale verwysingsvolgordes. Die relatiewe verhoudings tussen die bepaalde volgordes en die verwysingsvolgordes is geanaliseer met filogeneties ontledings. Die MP gene van PLRV isolate se volgordes is bepaal en filogeneties ontleed om hierdie nuwe isolate te vergelyk relatief tot vorige bevindinge in ons laboratorium. Die volledige genome van twee PLRV isolate se volgordes is bepaal en filogeneties ontleed as 'n voorlopige studie om die oënskynlike toename in patogenisiteit van Suid-Afrikaanse PLRV te ondersoek. Resultate het getoon dat slegs PVX en PVS teenwoordig was in die monsters wat versamel is en dat die voorkoms van hierdie virusse baie laag was (2.0% en 1.1% onderskeidelik). Die filogenetiese ontleding van die MP gene het aangedui dat die Suid Afrikaanse variante van PVX en PVS, geisoleer in hierdie studie, van die dominante tipes is wat mees gereeld internationaal voorkom. Uit die ontleding van die PLRV MP en heelgenoom volgordes, is vasgestel dat baie van die PLRV variante wat in Suid-Afrika aangetref word, geneties meer verskillend is as die van regoor die wêreld. Dus, regverdig dit, verdere ondersoek van die verhoogde patogenisiteit van Suid Afrikaanse PLRV variante.

The detection by serology of nematode-transmitted polyhedral viruses (nepoviruses) in grapevines is often unreliable. Nepoviruses were detected by enzyme-linked immunosorbent assay (ELISA) in tissue-cultured and field-grown grapevines. Nepovirus detection in in vitro plants (plantlets) was affected by virus distribution and growth room temperature. The reliability of virus detection in field-grown grapevines was improved when modified grinding buffers were used. Arabis mosaic virus (AMV) was detected by ELISA, for the first time, in in vitro grapevines initiated from field-and screenhouse-grown plants throughout the summer. The virus was not reliably and repeatedly detected in in vitro plantlets grown at 25°C. AMV and grapevine fanleaf virus (GFLV) distribution was not uniform throughout the plantlets. This distribution was affected by the culture room temperature. The best plant parts to sample for virus detection came from the zones of rapidly proliferating shoots. The viruses were sometimes not detected in samples taken from other tissues. Growth room temperature had an important effect on virus detection in plantlets. The highest virus titres were found in plants growing at 15°C. Temperature increases in 5°C steps to 30°C reduced virus titre. AMV became undetectable in nearly all plantlets growing at 30°C for as little as 30 days. Growth at 30°C reduced ELISA absorbance values by 76% after 8 days and after 21 days the values were at 4% of pre-treatment levels. The virus titre dropped below detectable levels in most plantlets. AMV could not be detected in plantlets or rooted explants after being placed in a 30°C treatment for 2 months. Tomato ringspot virus was detected by ELISA, for the first time, in in vitro grapevine plants. The virus was repeatedly detected in in vitro plants growing at 20°C. Under the typical summer conditions experienced at Sidney, B.C., modifying the standard ELISA grinding buffer (0.01 M phosphate buffered saline, pH 7.4, 0.05% Tween-20, 0.2% ovalbumin, 2% polyvinylpyrrolidone) was essential for reliable detection of AMV or GFLV. AMV was reliably detected by ELISA in foliar samples from field or screenhouse plants throughout the summer when the grinding buffer was modified by increasing the pH to at least 8.2 and adding 1% nicotine or 0.15 M phosphate buffered saline. The most reliable results with GFLV were obtained with the nicotine enhanced buffers. In comparison, because of the increased workload associated with growing plants in vitro and the unreliable detection of viruses in these plants, it remained preferable to detect nepoviruses in field plants by ELISA.
Land and Food Systems, Faculty of
Graduate

Le specie appartenenti al genere Calonectria sono state associate ad un'ampia gamma di piante ospite, causando vari sintomi di malattia e presenti in tutto il mondo. Su colture orticole, le specie di Calonectria sono state riportate per lo più nell'emisfero settentrionale, soprattutto in giardini e nei vivai di produzione di piante ornamentali. Benzimidazoli (MBC) e procloraz sono i fungicidi principali per il controllo chimico delle malattie causate da Calonectria spp. Tuttavia, l'uso di MBC è stato posto seriamente in dubbio dallo sviluppo di resistenza da parte di popolazioni di C. pauciramosa e C. morganii in Italia. In questo studio, sono stati condotti diversi monitoraggi in campo, osservando e rilevando una nuova malattia in Italia su piante di Laurus nobilis, causata da Calonectria ilicicola e nuove malattie in Tunisia su diverse piante ornamentali causate da Calonectria mexicana e Calonectria polizzii, oltre a due specie nuove: Calonectria Tunisiana e Calonectria pseudomexicana. Inoltre è stato effettuato un saggio per determinare la sensibilità al procloraz in una popolazione italiana appartenente a Calonectria scoparia spp. complex comprendente isolati raccolti in due periodi diversi (1993-1996 e 2005-2009). La sensibilità ridotta osservata in vitro è stata confermata anche in prove sperimentali effettuate in vivo. Questi rappresentano i primi dati sulla sensibilità al procloraz in popolazioni di Calonectria spp.

Quite a number of important crops have been genetically modified with genes for agronomically important traits, such as insect and viral resistance. As the numbers of genetically modified foods continue to increase on the market, the need for rapid development of GMO detection methods is indispensable. This study was carried out to detect if genetically modified potatoes exist on food market in Turkey. Thirty samples from different places were collected. Using a DNA based PCR method, potato samples were examined for the presence of 35S promoter, Nos terminator, neomycin phosphotransferase (nptII) genes, and synthetic cry3A gene which is the general transgene in all approved Newleaf transgenic potato lines. The experimental design of this study was to detect Newleaf insect resistant lines. In 11 samples at least one genetic element was detected. Sample R from Ankara has shown to be belonging to Newleaf insect resistant lines. Since 35S promoter was not detected in samples M3, 14 and F1, it is proposed that they are belonging to Newleaf virus and insect resistant lines (Newleaf plus or Newleaf Y). Although Nos terminator was not detected in samples H2, Z2 and D, cry3A fragments amplified in those samples have been verified that they are from the synthetic cry3A regions of Newleaf lines. The detected synthetic cry3A gene in GM potatoes was amplified by specific primers, which cannot amplify Bacillus thuringiensis tenebrionis natural cry3A gene. In addition, the authenticity of the synthetic cry3A PCR products were confirmed by both sequencing and restriction digestions. Our results showed that genetically modified Newleaf potatoes exist in food market in Turkey. Further studies by accredited laboratories are strongly recommended.

Brazil is the largest citrus producer in the world and has a large global citrus market share. However, several diseases affect the crop, being postbloom fruit drop (PFD) one of them. PFD has gained importance in São Paulo State for the displacement of citrus areas to regions with weather conditions more favorable for this disease. The accurate identification of the causal agent of the PFD has been performed and it was renamed as Colletotrichum abscissum. The origin of the initial inoculum is still an enigma for PFD epidemics and the hypotheses that the initial inoculum could be present in propagation material have been discussed but it has never been demonstrated. The objective of this work was to detect and quantify Colletotrichum abscissum from citrus leaves of budwood increase block and citrus nursery plants by qPCR. Four commercial citrus farms from São Paulo State, Brazil with budwood increase block and citrus nursery plants of Pera and Valencia sweet orange varieties were used for this work. C. abscissum was detected in budwood increase block and in nursery plant in both varieties (Valencia and Pera) at the four farms sampled. Out of 122 budwood increase block samples, 89 (73%) were positive for C. absicissum. From nursery plants, out of 175 samples, 129 (73%) were detected with the pathogen. The majority of the positive samples of budwood increase blocks and nursery plants contained 10 to 200 and 10 to 400 conidia of C. absicissum, respectively. With the methods used was not possible to isolate the fungus from vegetative material. This finding suggests a new long distances dispersion type of C. abscissum in the cycle of postbloom fruit drop by propagation material. Confirmation of C. abscissum in budwood increase block and nursery plants would lead to update regulations for the production of certified citrus nursery trees and searching for new control strategies of the pathogen.
O Brasil é o maior produtor de citros do mundo e possui uma grande participação no mercado global de citros. No entanto, várias doenças afetam a cultura, sendo uma delas a podridão floral dos citros (PFC). PFC ganhou importância no Estado de São Paulo pelo deslocamento de áreas de citros para regiões com condições climáticas mais favoráveis para a doença. A identificação precisa do agente causal do PFC foi realizada, tendo sido renomeado como Colletotrichum abscissum. A origem do inóculo inicial ainda é um enigma para as epidemias de PFC e as hipóteses do que o inóculo inicial poderia estar presente no material de propagação já foram discutidas, mas nunca foram demonstradas. O objetivo deste trabalho foi detectar e quantificar Colletotrichum abscissum em folhas de borbulheiras e mudas de citros por meio de qPCR. Neste trabalho, foram utilizadas quatro fazendas comerciais de citros do Estado de São Paulo, Brasil, com borbulheiras e viveiros de mudas de citros das variedades laranja Pera e Valência. C. abscissum foi detectado em borbulheiras e em mudas em ambas as variedades (Valência e Pêra) nas quatro fazendas amostradas. Das 122 amostras de folhas de borbulheiras, 89 (73%) foram positivas para C. absicissum. Das 175 amostras de folhas de mudas de citros, 129 (73%) foram detectadas com o patógeno. A maioria das amostras positivas de borbulheiras e mudas de citros continham 10 a 200 e 10 a 400 conídios de C. absicissum, respectivamente. Com os métodos utilizados, não foi possível isolar o fungo do material vegetativo. Esta descoberta sugere um novo tipo de dispersão a longas distâncias de C. abscissum no ciclo de podridão floral dos citros por meio do material de propagação. A confirmação de C. abscissum nas borbulheiras e mudas de citros levaria à atualização da regulamentação para a produção de mudas de citros certificadas e à busca de novas estratégias de controle do patógeno.

Thesis (MSc)--University of Stellenbosch, 2008.
ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past few years. Even more worrying is the variation of symptoms observed during PVY infection and the recent appearance of the more virulent PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently seed potato certification is done using ELISA kits imported from Europe. These kits were developed for the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY. In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix with international reference sequences, analyzed and grouped according to strain. Examination of the CP gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to South African strains of PVY expressing coat proteins which vary from those found overseas. This may render the currently used European ELISA method of detection less effective and subsequently result in an increase in viral prevalence. This reinforced the need for a detection method based on local viral strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and PVYO had evolved and that PVYNTN was such a recombinant. The second part of the study aimed to develop and establish detection methods based on local variants of PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed. Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix. A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY. The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and does not allow for amplicon verification through melting curve analysis, but it does add more specificity due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother material and confirm borderline cases in seed certification.
AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare. Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare, betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase chain reaction” (qRT-PCR) protokol vir die opsporing van PVY. In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is. Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk. Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes. Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde “Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te bevestig.

The management of weeds in agriculture is a time consuming and expensive activity, including in Australia where the predominant strategy is blanket spraying of herbicides. This approach wastes herbicide by applying it in areas where there are no weeds. Discrimination of different plant species can be performed based on the spectral reflectance of the leaves. This thesis describes the development of a sensor for automatic spot spraying of weeds within crop rows. The sensor records the relative intensity of reflected light in three narrow wavebands using lasers as an illumination source. A prototype weed sensor which had been previously developed was evaluated and redesigned to improve its plant discrimination performance. A line scan image sensor replacement was chosen which reduced the noise in the recorded spectral reflectance properties. The switching speed of the laser sources was increased by replacing the laser drivers. The optical properties of the light source were improved to provide a more uniform illumination across the viewing area of the sensor. A new opto-mechanical system was designed and constructed with the required robustness to operate the weed sensor in outdoor conditions. Independent operation of the sensor was made possible by the development of hardware and software for an embedded controller which operated the opto-electronic components and performed plant discrimination. The first revised prototype was capable of detecting plants at a speed of 10 km/h in outdoor conditions with the sensor attached to a quad bike. However, it was not capable of discriminating different plants. The final prototype included a line scan sensor with increased dynamic range and pixel resolution as well as improved stability of the output laser power. These changes improved the measurement of spectral reflectance properties of plants and provided reliable discrimination of three different broadleaved plants using only three narrow wavelength bands. A field trial with the final prototype demonstrated successful discrimination of these three different plants at 5 km/h when a shroud was used to block ambient light. A survey of spectral reflectance of four crops (sugarcane, cotton, wheat and sorghum) and the weeds growing amongst these crops was conducted to determine the potential for use of the prototype weed sensor to control spot-spraying of herbicides. Visible reflectance spectra were recorded from individual leaves using a fibre spectrometer throughout the growing season for each crop. A discriminant analysis was conducted based on six narrow wavebands extracted from leaf level spectral reflectance measured with a spectrometer. The analysis showed the potential to discriminate cotton and sugarcane from

Characteristics of quantitative resistance in pea (Pisum sativum L.) to Erysiphe pisi DC, the pathogen causing powdery mildew, were investigated. Cultivars and seedlines of pea expressing quantitative resistance to E. pisi were identified and evaluated, by measuring the amounts of pathogen present on plant surfaces in field and glasshouse experiments. Disease severity on cv. Quantum was intermediate when compared with that on cv. Bolero (susceptible) and cv. Resal (resistant) in a field experiment. In glasshouse experiments, two groups of cultivars, one with a high degree of resistance and the other with nil to low degrees of resistance to E. pisi, were identified. This indicated either that a different mechanism of resistance applied in the two groups, or that there has been no previous selection for intermediate resistance. Several other cultivars expressing quantitative resistance were identified in a field experiment. Quantitative resistance in Quantum did not affect germination of E. pisi conidia, but reduced infection efficiency of conidia on this cultivar compared with cv. Pania (susceptible). Other epidemiological characteristics of quantitative resistance expression in Quantum relative to Pania were a 33% reduction in total conidium production and a 16% increase in time to maximum daily conidium production, both expressed on a colony area basis. In Bolero, the total conidium production was reduced relative to Pania, but the time to maximum spore production on a colony area basis was shorter. There were no differences between the cultivars in pathogen colony size or numbers of haustoria produced by the pathogen. Electron microscope studies suggested that haustoria in Quantum plants were smaller and less lobed than those in Pania plants and the surface area to volume ratios of the lobes and haustorial bodies were larger in Pania than in Quantum. The progress in time and spread in space of E. pisi was measured in field plots of cultivars Quantum, Pania and Bolero as disease severity (proportion of leaf area infected). Division of leaves (nodes) into three different age groups (young, medium, old) was necessary because of large variability in disease severity within plants. Disease severity on leaves at young nodes was less than 4% until the final assessment at 35 days after inoculation (dai). Exponential disease progress curves were fitted for leaves at medium nodes. Mean disease severity on medium nodes 12 dai was greatest (P<0.001) on Bolero and Pania (9.3 and 6.8% of leaf area infected respectively), and least on Quantum (1.6%). The mean disease relative growth rate was greatest (P<0.001) for Quantum, but was delayed compared to Pania and Bolero. Gompertz growth curves were fitted to disease progress data for leaves at old nodes. The asymptote was 78.2% of leaf area infected on Quantum, significantly lower (P<0.001) than on Bolero or Pania, which reached 100%. The point of inflection on Quantum occurred 22.8 dai, later (P<0.001) than on Pania (18.8 dai) and Bolero (18.3 dai), and the mean disease severity at the point of inflection was 28.8% for Quantum, less (P<0.00l) than on Pania (38.9%) or Bolero (38.5%). The average daily rates of increase in disease severity did not differ between the cultivars. Disease progress on Quantum was delayed compared with Pania and Bolero. Disease gradients from inoculum foci to 12 m were detected at early stages of the epidemic but the effects of background inoculum and the rate of disease progress were greater than the focus effect. Gradients flattened with time as the disease epidemic intensified, which was evident from the large isopathic rates (between 2.2 and 4.0 m d⁻¹) Some epidemiological variables expressed in controlled environments (low infection efficiency, low maximum daily spore production and long time to maximum spore production) that characterised quantitative resistance in Quantum were correlated with disease progress and spread in the field. These findings could be utilised in pea breeding programmes to identify parent lines from which quantitatively resistant progeny could be selected.

Agriculture, also known as Farming, is the science or practice of raising crops. And the whole world is dependent on it and around 38% of the world is dependent on it. So, due to this, its productivity rate should be high. The productivity rate of a plant is affected by the disease in a plant. So that’s why plant disease should be detected at an early stage. For this, Farmers generally hire an agricultural expert who detects the disease using the naked eye and also they use instruments as well which are very expensive and which is not possible for all the farmers to afford it. There’s another way to detect a plant disease, by using Artificial Intelligence(AI). Machine Learning(ML), Deep Learning(DL) which is a sub-branch of AI, is used in agriculture in order to detect disease in a plant. So, in this work, a Dense-INC model is proposed which is based on Convolutional Neural Network(CNN) and it’s inspired by DenseNet and InceptionNet. This model is trained on the “Plant Pathology 2020: FGVC7 dataset” and “Plant Pathology 2021: FGVC8 dataset”. The proposed model is first trained with 4 optimizers: Adam, Adadelta, Adagrad, and SGD with momentum and when I compare results then shows that Adagrad gives better results than other optimizers. To further evaluate the performance of the proposed model, the proposed model is further compared with two CNN-based models with Adagrad optimizer which are already been proposed. And the results show that the proposed model gives better results than two other CNN-based models and it’s able to detect the disease with a low error rate.

碩士
國立臺灣大學
生物產業機電工程學研究所
104
In recent years, the climate change has significantly affected the agricultural production. Maintaining the crop production is one of main concerns in agriculture. High temperature and changes of rainfall patterns enhance the spread of plant diseases. Hence it is desirable to seek for early detection of plant disease, and thus to control the spread of plant disease. Hyperspectral imaging has been proved to be an efficient tool for early detection of strawberry Anthracnose. To improve the efficiency of plant disease detection, this research aims to build a handheld multispectral imaging device for strawberry Anthracnose detection. This device uses an embedded system as the controller of the device. By placing filters in front of four miniature cameras, the images of four characteristic wavelengths are acquired. After capturing images using the handheld multispectral imaging device, images are processed to correct the effect of uneven lighting. Then by further processing the multispectral images and incorporating the RGB image of inoculated strawberry leaves, we are able to analyze the status of strawberry leaves at various infection stages. In this research, we first used the multispectral imaging device to classify the healthy and symptomatic areas in strawberry leaves. Then we further attempted to classify the status into three categories: healthy, incubation and symptomatic. SVM model was applied for classification of infection stages. For classification of healthy and symptomatic status, detection accuracy is above 90%. For classification between healthy, incubation, and symptomatic status, the accuracies are 92.2%, 68.6%, and 97.9%, respectively. The classification result of strawberry Anthracnose infection is further displayed on the handheld device as pseudo-color image so the user can easily observe the plant health condition, and so the disease management can be applied if necessary. Since the detection accuracy can be affected by lighting and shadow due to uneven surface of strawberry leaves. We propose a method to amend the effect of shadow on status classification. Through observations of the original four images and their association, a new set of images derived from the original four images was selected and tested to rectify the shadow effect. Using this new set of derived images and trained with SVM, classification accuracy for healthy status increased from 71.3% to 95.7% and the classification accuracy for symptomatic status also increased from 82.3% to 88.9%.

Identifying regions in an image and labeling them to class is called image segmentation. Automatic image segmentation has been one of the major research areas which is in trend nowadays. Every other day a new model is being discovered to do better image segmentation for the task of computer vision. As the better a computer is able to see, the better we can automate the tasks around our daily life. In this survey we are comparing various image segmentation techniques and on the basis of our research we are applying the best approach to an application i.e. developing a model to identify diseased plants and to give an idea to the people what kind disease is present in a plant. The detailed analysis of the methodology is done with the help of various analysis techniques, which are used in reference to the context of the work. Our focus is on the techniques which we are able to optimize and make them better than the one which are present before. This survey emphasizes on the importance of application of image segmentation techniques and to make them more useful for the common public in daily life. So that they get benefits of this technology in the monitoring of any activity occurring around that can’t be done manually.

Agriculture is the process of growing crops and raising livestock and cultivating other forms of food or fiber. It has been a fundamental activity for human societies through out history, providing food and other resources necessary for survival. It also provides employment, economic growth and environmental conservation. Some farming practices, such as sustainable agriculture, can promote environmental conservation and biodiver sity. Hence, plant illness can have a substantial effect on the economy, particularly in agricultural-dependent countries. Crop yield loss, trade restrictions, increased production costs, reduced agricultural productivity, and research and development costs are some of the ways plant diseases can affect the economy. It is essential to prevent and manage plant diseases to ensure food security and maintain a healthy agricultural sector. Farmers visually inspect their crops for symptoms of diseases, such as discoloration, spots, wilting, and deformities. Farmers can also use their sense of touch and smell to detect diseases, such as the sticky or slimy feel of plant leaves infected with fungal diseases and the foul smell of rotting or decaying plant material. However, traditional methods of plant disease detection do have limitations. Visual inspection and other traditional methods may not always detect diseases at an early stage, and there is a risk of misdiagnosis. Additionally, traditional methods may not be able to detect diseases that are not visible to the naked eye. The other alternatives are the use of Artificial Intelligence (AI) which includes training computers to detect plant diseases using image recognition technology. AI can analyze thousands of images to detect subtle changes in plant health that may indicate the pres ence of diseases. Some of the regular AI techniques used for plant disease detection are Convolutional Neural Networks (CNNs), Support Vector Machines (SVMs), Random Forest (RF), Deep Belief Networks (DBNs), Transfer Learning. These AI methods are increasingly being used for plant disease detection because they offer a fast, accurate, and cost-effective way to diagnose plant diseases, which can help prevent crop losses and iv increase yields. Here in this research work, a Multi-layered CNN model is introduced which is inspired from InceptionNet. The proposed model is trained on the “Plant Pathology 2020: FGVC7 dataset” and “Plant Pathology 2021: FGVC8 dataset”. This proposed model is compared with pertained models: InceptionV3. According to the findings, the suggested model outperforms other pre-existing models in terms of accuracy or performance and it’s able to detect the disease with a low error rate.

碩士
國立中興大學
高階經理人碩士在職專班
103
The objectives of this thesis are on markets of detectors for the diseases of economic animals and plants. Typically, the production values and production volumes of the aquacultures, livestock, companion animals and crops are collected and analyzed to estimate which is the niche market for the disease detectors. Statistic data show that production values of Asia fisheries, livestock and agricultural are top of the world. Asia has the development potential in the disease detector markets. Europe and North America have the greatest market of companion animals. We study two companies as cases, they are IDEXX Laboratories, Inc. (in America) and GeneReach Biotechnology Corp. (in Taiwan). They are focused on specific areas of product developments and sales. Meanwhile, both companies have the same features as follows. First, focus on the product development of economic animals. Second, aim at launching the international market. Third, concentrate on the diseases of animals or aquaculture which are bred or cultured on large scale. We also find that the margin of detector products is considerable and can generate synergy in business performance, once the company is dedicated to the series diseases on a specific animal. This thesis proposes the following suggestions to the manufacturers of economic animal and plant diseases detectors: 1. Developing the detector products with features of low cost, short detection time, accuracy and portable can increase the acceptance of markets. 2. Pets are potential for the market. 3. Aiming at high unit prices can approach the niche market. 4. Expand and spread the markets globally.

碩士
亞洲大學
資訊工程學系碩士班
99
This study presents a method for detecting plant diseases using color Local Binary Patterns (LBP). LBP provides highly discriminative texture information and is invariant to any monotonic changes in gray level. The proposed method uses pixel color and the LBP features extracted from a region surrounding a pixel to segment the diseased regions. Experimental results show that color LBP texture features combining with Support Vector Machine (SVM) classifier are effective for segmenting diseased regions in plant color images.

The limited specificity, sensitivity and multiplex capacity of detection techniques currently available for important seed-borne pathogens of tomato is a significant risk for the global tomato trade and production industry. These pathogens can be associated with seed at low concentrations but, due to their highly virulent nature, these low levels can be sufficient to infect germinating seedlings and spread to neighbouring plants and fields, potentially causing epidemics and economic losses. In this study, detection techniques currently available for phytodiagnostics were evaluated for the capacity to accurately detect and identify five agronomically important seed-borne pathogens of tomato: Pepino mosaic virus (PepMV), Tomato mosaic virus (ToMV), Clavibacter michiganensis subsp. michiganensis (Cmm), Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato. A prototype diagnostic microarray was also designed in an attempt to develop a tool that could simultaneously detect these five seed-borne pathogens from a single sample. Viral detection based on serological techniques was rapid, accurate and reliable but only detected a single pathogen per assay and required supplementary bioassays to indicate the viability of detected viral pathogens. Selective media plating for bacterial detection demonstrated unreliable recovery of targeted bacteria from infected seed and leaf samples and required supplementary tests to validate the identity of presumptive positives. Assays were lengthy, laborious and sometimes too ambiguous for accurate diagnosis of bacterial pathogens. Nucleic acid-based technologies demonstrated improved sensitivity and specificity for detection of targets from pure culture, leaf and seed extracts, compared to conventional and serological methods, yet also required supplementary bioassays or media assays to validate the viability of detected pathogens. Amplification efficiency however, was affected by the presence of PCR inhibitors and despite positive detection, variable banding intensity in electrophoretic analysis of amplified products necessitated the use of reference cultures to validate diagnosis. The developed microarray incorporated 152 pathogen-specific and control probes to facilitate diagnosis and taxonomic classification of detected pathogens. The array was challenged with pure culture extracts of the five target pathogens, selected related and non-target, unrelated pathogens of tomato. Positive detection of each of the pathogens was demonstrated but the production of hybridisation signals was highly variable and extremely sensitive to minor technical differences. Each of the five pathogens were successfully detected in combination proving that different classes of seed-borne pathogens could be detected from a single sample using the developed microarray. This prototype microarray has good potential for phytodiagnostic screening of the five targeted pathogens, and further validation, optimisation and extension for testing tomato seed samples may facilitate incorporation of this array into standard diagnostic protocols.

Orchid fleck virus (OFV) is one of three commonly found viruses infecting orchids. Infected plants have reduced flower quality and often unsightly leaf markings and are unsaleable. Effective disease control relies on routine detection of the virus, but foliar symptoms can vary markedly and are often not reliable for diagnosis. Current laboratory tests use examination of leaf sap by electron microscopy. This is time consuming and costly. The preferred immunological tests are not available as OFV has an unstable virion and attempts to purify virus particles for antibody production have failed. However, direct isolation of viral nucleic acid from infected plants may enable development of alternative detection systems. In this project OFV was characterised by mechanical inoculation to alternate hosts. Sap inoculation was found to be difficult and affected by glasshouse temperatures, however, was successful at temperatures significantly lower than previously reported. The host range of a Tasmanian isolate of OFV was found to be different to that previously reported for isolates of OFV from Japan. Attempts to purify OFV and clone the viral RNA were unsuccessful, however primers specific to OFV were obtained. OFV was detected using RT-PCR with a primer complementary to a region of its nucleoprotein gene together with a polydT/SP6 primer complementary to the 3' terminus of the genomic segment. The resulting DNA fragments were 0.8kb long and their sequences were determined directly. The sequences of DNA fragments obtained from 33 OFV isolates from Australia, Brazil, Germany and South Africa were shown to be closely related (<2.5% difference), but a single German isolate was clearly a distinct strain and the . sequence of the targeted region of its nucleoprotein gene differed from that of the others by about 16%. Failure of RT-PCR using a second primer set complimentary to part of the phosphoprotein gene of the Japanese OFV isolate with all OFV isolates tested suggests that the Japanese isolate may represent a third distinct strain of OFV or a different virus. A search of the international nucleotide database with the OFV sequences showed them to be related, but distantly, to regions of the genomes of three plant rhabdoviruses. Isolates of coffee ringspot virus (CoRSV), citrus leprosis virus (CiL V), a common violet (Viola sp.), schefflera, hibiscus, ivy and ligustrum leaves showing ringspot symptoms and containing small bacilliform virus particles were tested using RT-PCR and the OFV specific primers. A single product of 800bp was amplified from one isolate of coffee ringspot virus and the violet sample using the primer complementary to a region of the OFV nucleoprotein gene together with a polydT/SP6 primer. The DNA products were shown to be identical to OFV when sequenced. No other sample gave an amplified product. These results suggest citrus leprosis disease, ligustrum ringspot and the ringspots on schefflera, hibiscus and ivy are caused by viruses different to OFV. However, this study was completed with a limited number of samples and the results are not conclusive, the relationship between these viruses should be further investigated.

Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns similar to those observed on western blotting, where anti-D. pinea, anti-F. circinatum and anti-F. oxysporum antibodies showed the same cross-reactivity relationships. Anti-F. circinatum and anti-F. oxysporumantibodies showed a significant difference when reacting with antigens isolated from other pathogens including D. pinea, F. circinatum, F. oxysporum, F. solani, F. graminearum and F. culmorum (P = 0.001). No significant difference was observed when the antigens were detected with anti-D. pinea antibodies. Reactivity of anti-F. circinatum-exo and anti-D. pinea34 kDa antibodies was mostly similar to that of non-immune antibodies and showed no significant difference between detection of different antigens. Pine seedlings were artificially infected with the three fungal pathogens using a spore concentration of 1 – 1 x 106conidiaml-1.Infection was monitored using scanning electron microscopy. Results showed increased levels of mycelium growth on the stem and roots of the F. circinatum and F. oxysporum infected seedlings and on the leaves and stem in the case of D. pinea infected seedlings. These plant parts were used in ELISA tests for the detection of antigens. Isolation of antigens from the plant materials involved crushing plant parts in buffer and centrifugation of the suspension. The supernatant obtained was directly used in the assay. ELISA tests prepared in this study were sensitive enough to detect infection caused by 1 conidium ml-1at two weeks post inoculation. A positive reaction for detection of F. circinatum and F. oxysporum was indicated by an ELISA reading above an optical density at 405 nm. The plant material used in ELISA tests were further analysed using PCR. Results indicated that there was no cross-infection between seedlings and served as a confirmation of the disease-causing pathogen. This indicated that cross-reactivity observed was due to other factors such as common epitopes on the major antigens. Use of an ELISA dip-stick or ELISA using these antibodies should provide an easy, fast field test to identify infections of pine, discriminating between F. circinatum, F. oxysporum and D. pinea.
M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.