Tesis sobre el tema "Bacterial wilt disease management"

Ralstonia solanacearum, the causal agent of bacterial wilt disease, has been found to affect numerous economically important plants. Understanding the molecular basis of resistance, tolerance and susceptibility of plants to pathogens such as R. solanacearum is a major goal of molecular plant pathologists. Prior to this study it was thought that Arabidopsis accession Kil-0 shows gene-for-gene “resistance” to an African Eucalyptus isolate of R. solanacearum, BCCF402. However, a subsequent preliminary study indicated that Kil-0 may exhibit “tolerance” which is defined as the plant’s ability to support high pathogen numbers without displaying disease symptoms or a reduction in host fitness. The aim of this study was to determine if Kil-0 was tolerant to R. solanacearum BCCF402. The bacterial load of R. solanacearum was quantified in accessions Kil-0 and Be-0 using dilution plating and quantitative PCR methods. The cytC gene region was used to quantify R. solanacearum in Arabidopsis plants and the amount of bacterial DNA was normalized to “alien” DNA that was spiked into each sample. High bacterial concentrations of BCCF402 were found in Kil-0 but plants exhibited no wilting symptoms. Additionally, Kil-0 plants inoculated with BCCF402 showed no significant reduction in fitness compared to control Kil-0 plants. In contrast, high bacterial numbers and severe disease symptoms were observed in the susceptible Be-0 plants, whereas Nd1 plants contained a low number of bacteria and no disease symptoms indicative of a resistance response. These results illustrated that Kil-0 is tolerant to R. solanacearum isolate BCCF402. A tool for the visualization of R. solanacearum in Arabidopsis plants was designed. R. solanacearum isolate BCCF402 was tagged with two mCherry-containing plasmids under the constitutive expression of the tac promoter. The expression levels of mCherry were suitable for successful visualization in planta. BCCF402 cells transformed with the mCherry-containing plasmids were not affected in terms of virulence or disease progression compared to wildtype BCCF402 cells. A plasmid loss of 30-35% was observed in mCherry-tagged BCCF402 cells at later stages of Arabidopsis infection. mCherry-tagged BCCF402 was successfully visualized in Kil-0 leaves at early infection stages.
Dissertation (MSc)--University of Pretoria, 2014.
gm2014
Plant Science
unrestricted

In 2007 and 2008 more than 100 million dollars of fresh market tomatoes were grown in Virginia, with the majority of production occurring on the Eastern Shore of Virginia (ESV), according to the National Agricultural Statistics Service. Bacterial wilt of tomato, caused by Ralstonia solanacearum (Smith) and Yabucchi et al., is the most devastating disease of tomato on the ESV. Four â observational trials' were conducted on the ESV over three growing seasons to determine the temporal and spatial distribution of this disease in commercial tomato fields. Plants were assessed at approximately one-week intervals throughout the growing seasons and the incidence of bacterial wilt for each individual plant was recorded. A steady increase in both disease incidence and clustered distribution of the disease within rows was observed as the growing season progressed. Positive correlations between disease incidence and percentage of rows exhibiting a significant clustered distribution occurred in all trials, which indicated an increase in clustered distribution as disease incidence increased. Research trials were conducted over three years, beginning in the summer of 2007, to investigate the effects of tomato bacterial wilt resistant cultivars on the ESV. In 2008 and 2009, the selective, systemic compound which induces host plant resistance, acibenzolar-S-methyl (ASM) was incorporated into resistant cultivar trials. Results from the 2007 trial revealed significant resistance in some of the breeding lines, CRA 66 and PI 126408. The 2008 and 2009 trials revealed that ASM was not effective at reducing levels of bacterial wilt. Grafted transplants in the spring trials of 2008 and 2009 had varied results in resistance and yield. Results revealed the tomato cultivar BHN 669 was an excellent resistant cultivar with promising yield potential and fruit quality.
Master of Science

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This study evaluated the use of silicon (Si) in micropropagation of banana 'Silk' and 'Pacovan Ken' aiming to reduce the severity of fusarium wilt caused by Fusarium oxysporum f. sp. cubense and moko disease caused by Ralstonia solanacearum race 2. The banana plantlets were produced in vitro by adding calcium silicate and potassium silicate (0, 0.25, 0.5, 0.75 and 1 g L-1) to MS medium in the phases of multiplication and rooting. After in vitro culture, the plants were transferred to plastic tubes containing substrate plus the same sources of Si, and maintained in a greenhouse for 45 days, when they were inoculated with the pathogens. With respect to fusarium wilt in cultivars Silk and Pacovan Ken, the elevation of Si increased the incubation period (IP) and reduced the disease index (DI) and area under the disease progress curve (AUDPC). In cultivar Silk but not in Pacovan Ken calcium silicate was significantly more effective than potassium silicate. In shoots and roots of both cultivars in both sources, before and after acclimatization Si concentration was greater at a dose of 1.0 g L-1 compared to the control without Si. Before acclimatization, calcium silicate provided higher Si concentration in the shoots than potassium silicate. The opposite happened with the Si concentration in the roots. After acclimatization, there was no difference between the calcium silicate and potassium silicate, for both cultivars. In general, for both cultivars and sources of Si there were positive correlations with the concentration of Si and IP, and negative correlations with DI and AUDPC. Before and after acclimatization, the anatomical variable of roots: thickness of the root epidermis, cortex, endodermis and central cylinder of banana 'Silk' and 'Ken Pacovan' were influenced by Si sources. Calcium silicate was more efficient in increasing the thickness of the root epidermis, cortex and central cylinder, while potassium silicate was more efficient in thickening of the endodermis. In general, there were positive correlations among anatomical variable of roots with PI and negative correlations with DI and AUDPC, except for potassium silicate in cultivar Silk. The research conducted with moko disease showed that increase of Si in Silk and Pacovan Ken cultivars caused increase in IP and decreases the DI and AUDPC. At the dosage of 1.0 g L-1 AUCPD was reduced by 27.3%. In cultivar Silk, calcium silicate was more effective than potassium silicate (P≤0.05), while in „Pacovan Ken‟ there was no difference. In both cultivars, plants treated with Si showed, in general, concentrations of chlorophylls a, b and total higher than plants Si- up to six days after inoculation, which may have influenced the disease IP. In general, both the enzymes related to oxidative stress (CAT, SOD and APX), as the plant defense (POX, PPO, CHI and GLU), had increased its activities in plants treated with Si, especially those with calcium silicate, indicating a possible role in reducing the severity of the disease. The supply of Si in micropropagation of banana 'Silk' and 'Pacovan Ken' promoted reduction of Fusarium wilt and moko disease, and therefore can be used as a new technology in the management of these diseases.
Este estudo avaliou o uso do silício (Si) na micropropagação de bananeira „Maçã‟ e „Pacovan Ken‟ visando a redução da severidade da murcha-de-fusário, causada pelo Fusarium oxysporum f. sp. cubense e do moko da bananeira causado por Ralstonia solanacearum raça 2. As mudas de bananeira foram produzidas in vitro com adição de silicato de cálcio e silicato de potássio (0; 0,25; 0,5; 0,75 e 1 g L-1) ao meio de cultivo MS nas fases de multiplicação e enraizamento. Após o cultivo in vitro, as plantas foram transferidas para tubetes contendo substrato acrescido das mesmas fontes de Si, e mantidas em casa de vegetação por 45 dias, quando foram inoculadas com os patógenos. Com relação à murcha-de-fusário, nas cultivares Maçã e Pacovan Ken, a elevação das doses de Si aumentou o período de incubação (PI) e reduziu o índice de doença (IDO) e a área abaixo da curva de progresso da doença (AACPD). Em „Maçã‟ o silicato de cálcio foi significativamente mais eficiente que o silicato de potássio, o que não ocorreu na „Pacovan Ken‟. A concentração de Si na parte aérea e raízes das cultivares, em ambas as fontes, antes e após a aclimatização foi maior na dose de 1,0 g L-1 em relação à testemunha sem Si. Antes da aclimatização, o silicato de cálcio proporcionou maior concentração de Si na parte aérea que o silicato de potássio. O contrário aconteceu com a concentração de Si nas raízes. Após aclimatização, não houve diferença entre o silicato de cálcio e o silicato de potássio, para as duas cultivares. Em geral, para ambas as cultivares e fontes de Si foram observadas correlações positivas da concentração de Si com PI e correlações negativas com IDO e AACPD. Antes e após a aclimatização das plantas, as espessuras da epiderme radicular, córtex, endoderme e cilindro central das bananeiras „Maçã‟ e „Pacovan Ken‟ foram influenciadas pelas fontes de Si. O silicato de cálcio foi mais eficiente no aumento da espessura da epiderme radicular, córtex e cilindro central, enquanto o silicato de potássio foi mais eficiente no aumento da espessura da endoderme. Em geral, foram observadas correlações positivas das variáveis anatômicas das raízes com PI e correlações negativas com IDO e AACPD, exceto para silicato de potássio em bananeira „Maçã‟. Na pesquisa desenvolvida com o moko da bananeira, a elevação das doses de Si nas cultivares Maçã e Pacovan Ken causou aumento no PI e reduções do IDO e AACPD. Na dosagem de 1,0 g L-1, a AACPD foi reduzida em até 27,3%. Em bananeira „Maçã‟ o silicato de cálcio foi mais eficiente que o silicato de potássio (P≤0,05), enquanto na „Pacovan Ken‟ não houve diferença. Nas duas cultivares, plantas tratadas com Si apresentaram, de maneira geral, concentrações de clorofilas a, b e total maiores que as plantas Si- até os seis dias após inoculação, o que pode ter influenciado o PI da doença. Em geral, tanto as enzimas relacionadas ao estresse oxidativo (CAT, SOD e APX), quanto as de defesa da planta (POX, PFO, GLU e QUI), tiveram suas atividades aumentadas nos tratamentos com silício, especialmente naqueles com silicato de cálcio, indicando uma possível participação na redução da severidade da doença. O fornecimento de Si na micropropagação de bananeiras „Maçã‟ e „Pacovan Ken‟ promoveu redução da murcha-de-fusário e moko da bananeira, podendo ser utilizado como uma nova tecnologia no manejo dessas doenças.

Bacterial spot disease of stone fruits, caused by Xanthomonas arboricola pv. pruni, is of high economic importance in the major stone-fruit-producing areas worldwide. Disease control is mainly based on preventive measures, such as quarantine regulation, breeding for resistance or preventive copper spray applications, since no effective chemical control is available. Therefore, a better understanding of disease epidemiology can be valuable in developing disease management strategies. This thesis was aimed at developing a mechanistic forecasting system for bacterial spot disease of stone fruits, which is based on three components: i) epiphytic inoculum potential, ii) weather conditions conducive to infections, and iii) disease symptom appearance. The effects of environmental parameters and inoculum populations on different steps of the disease cycle were quantified and modeled. The results contributed with new knowledge on the epidemiology of bacterial spot disease of stone fruits and offer new possibilities in its management
La taca bacteriana dels fruiters de pinyol, causada per Xanthomonas arboricola pv. pruni, té un gran impacte econòmic a les principals zones productores de tot el món. El control de la malaltia es basa principalment en mesures preventives, com ara una regulació de quarantena, la selecció de varietats d’hostes resistents o aplicacions preventives de coure, ja que no es disposa de cap mètode de control químic curatiu i efectiu. Per tant, l’estudi de l'epidemiologia de la malaltia pot ser un factor valuós en el desenvolupament d'estratègies per al seu maneig. L’objectiu d’aquesta tesi va ser el desenvolupament d'un sistema de predicció del desenvolupament de la taca bacteriana dels fruiters de pinyol, el qual es basa en tres components: i) el potencial d'inòcul epífit, ii) les condicions meteorològiques favorables en el procés d’infecció, i iii) l’aparició dels símptomes de la malaltia. Els efectes dels paràmetres ambientals i del potencial d'inòcul es van quantificar i modelar en diferents processos clau del cicle de la malaltia. Els resultats obtinguts aporten nous coneixements sobre l'epidemiologia de la taca bacteriana dels fruiters de pinyol que ofereixen noves possibilitats en el seu maneig

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Universidade Estadual Paulista (UNESP)
Este trabalho visou avaliar a ocorrência da bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens em lavouras de feijoeiro localizadas em alguns municípios do Estado de Santa Catarina nas safras 2002/03 e 2003/04; a reação de genótipos de feijoeiro à murcha-de-curtobacterium; e o efeito de doses de nitrogênio (N) e potássio (K) sobre a severidade da doença, peso da matéria seca e o conteúdo de diversos nutrientes na parte aérea das cultivares IAC Carioca Pyatã, IPR 88 - Uirapuru e SCS 202 - Guará. Foram coletadas plantas com sintoma de murcha em lavouras de feijoeiro em 12 municípios. Posteriormente, procedeu-se ao isolamento, à purificação e à caracterização da bactéria e aos testes de patogenicidade. Aos 10 dias após a semeadura, plântulas de 24 genótipos de feijoeiro, conduzidas em casa-de-vegetação, foram inoculadas com o isolado FJ 36. A severidade da murcha-de-curtobacterium foi avaliada, a cada cinco dias, até aos 25 dias após a inoculação. Tanto no experimento que avaliou o efeito do N (uréia) quanto do K (cloreto de potássio) na severidade da doença, empregou-se a dose recomendada pela análise de solo e variações de 25 e 50 % abaixo e acima da mesma. Foi coletada a parte aérea das plantas antes e após as adubações, aos 25 DAS, para se aferir o peso da matéria seca e o conteúdo de N, fósforo (P), K, cálcio (Ca) e magnésio (Mg). Constatou-se que C. f. pv. flaccumfaciens esteve presente em plantas cultivadas nos municípios de Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada e Tigrinhos. Dos genótipos avaliados, somente as cultivares IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considerados como padrões de resistência à doença, apresentaram-se com as menores notas de severidade média e valores de área abaixo da curva do progresso da murcha-de-curtobacterium (AACPMC)... .
This work aimed evaluate the occurrence of the bacteria Curtobacterium flaccumfaciens pv. flaccumfaciens in common bean fields of Santa Catarina State, during the harvest of 2002/03 and 2003/04; the reaction of bean genotypes to the bacterial wilt; and the effect of nitrogen (N) and potassium (K) levels on the severity of the disease, the weight of the dry mass and the content of nutrients I nthe aerial part of the cultivars IAC Carioca Pyatã, IPR 88 - Uirapuru and SCS 202 - Guará cultivars. Plants with symptoms of wilt were collected in bean fields of 12 cities. It was made the isolation, purification and cultural characterization of the bacteria and the procedures to fulfill the Kochþs postulates. The inoculation with the strain FJ 36 was done in the 10th day after the sow of 24 common bean genotypes, under greenhouse conditions. The bacterial wilt severity was evaluated, in each five days, until the 25th day after inoculation. It was adopted as treatments the recommended level of N (urea) and K (potassium chloride), by the soil analysis, as well levels 25% and 50% under and below it. The aerial part of the plants was collected before and after the fertilizations, to determine the weight of the dry mass and the content of N, phosphorus (P), K, calcium (Ca) and magnesium (Mg). Curtobacterium flaccumfaciens pv. flaccumfaciens was detected in plants cultivated in Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada and Tigrinhos. Regarding the evaluated genotypes, only the cultivars IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considered as patterns of resistance, had the lower values of average severity and area under the bacterial wilt progress curve (AUBWPC). However, although to have been considered susceptible, the cultivars SCS 202 - Guará and IPR Graúna showed relatively low values of AUBWPC... (Complete abstract, click electronic address below).

Orientador: Antonio Carlos Maringoni
Banca: Antonio Carlos Maringoni
Banca: Roberto Lyra Villas Boas
Banca: Julio Rodrigues Neto
Banca: Silvania Furlan de Oliveira
Banca: Ivan Paulo Bedendo
Resumo: Este trabalho visou avaliar a ocorrência da bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens em lavouras de feijoeiro localizadas em alguns municípios do Estado de Santa Catarina nas safras 2002/03 e 2003/04; a reação de genótipos de feijoeiro à murcha-de-curtobacterium; e o efeito de doses de nitrogênio (N) e potássio (K) sobre a severidade da doença, peso da matéria seca e o conteúdo de diversos nutrientes na parte aérea das cultivares IAC Carioca Pyatã, IPR 88 - Uirapuru e SCS 202 - Guará. Foram coletadas plantas com sintoma de murcha em lavouras de feijoeiro em 12 municípios. Posteriormente, procedeu-se ao isolamento, à purificação e à caracterização da bactéria e aos testes de patogenicidade. Aos 10 dias após a semeadura, plântulas de 24 genótipos de feijoeiro, conduzidas em casa-de-vegetação, foram inoculadas com o isolado FJ 36. A severidade da murcha-de-curtobacterium foi avaliada, a cada cinco dias, até aos 25 dias após a inoculação. Tanto no experimento que avaliou o efeito do N (uréia) quanto do K (cloreto de potássio) na severidade da doença, empregou-se a dose recomendada pela análise de solo e variações de 25 e 50 % abaixo e acima da mesma. Foi coletada a parte aérea das plantas antes e após as adubações, aos 25 DAS, para se aferir o peso da matéria seca e o conteúdo de N, fósforo (P), K, cálcio (Ca) e magnésio (Mg). Constatou-se que C. f. pv. flaccumfaciens esteve presente em plantas cultivadas nos municípios de Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada e Tigrinhos. Dos genótipos avaliados, somente as cultivares IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considerados como padrões de resistência à doença, apresentaram-se com as menores notas de severidade média e valores de área abaixo da curva do progresso da murcha-de-curtobacterium (AACPMC)... (Resumo completo, clicar acesso eletrônico abaixo).
Abstract: This work aimed evaluate the occurrence of the bacteria Curtobacterium flaccumfaciens pv. flaccumfaciens in common bean fields of Santa Catarina State, during the harvest of 2002/03 and 2003/04; the reaction of bean genotypes to the bacterial wilt; and the effect of nitrogen (N) and potassium (K) levels on the severity of the disease, the weight of the dry mass and the content of nutrients I nthe aerial part of the cultivars IAC Carioca Pyatã, IPR 88 - Uirapuru and SCS 202 - Guará cultivars. Plants with symptoms of wilt were collected in bean fields of 12 cities. It was made the isolation, purification and cultural characterization of the bacteria and the procedures to fulfill the Kochþs postulates. The inoculation with the strain FJ 36 was done in the 10th day after the sow of 24 common bean genotypes, under greenhouse conditions. The bacterial wilt severity was evaluated, in each five days, until the 25th day after inoculation. It was adopted as treatments the recommended level of N (urea) and K (potassium chloride), by the soil analysis, as well levels 25% and 50% under and below it. The aerial part of the plants was collected before and after the fertilizations, to determine the weight of the dry mass and the content of N, phosphorus (P), K, calcium (Ca) and magnesium (Mg). Curtobacterium flaccumfaciens pv. flaccumfaciens was detected in plants cultivated in Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada and Tigrinhos. Regarding the evaluated genotypes, only the cultivars IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considered as patterns of resistance, had the lower values of average severity and area under the bacterial wilt progress curve (AUBWPC). However, although to have been considered susceptible, the cultivars SCS 202 - Guará and IPR Graúna showed relatively low values of AUBWPC... (Complete abstract, click electronic address below).
Doutor

Acacia species are exotic hardwood trees extensively planted in SE Asia. Due to their rapid growth, good wood quality and tolerance to a wide spectrum of environmental conditions, they are important economically, not only for timber products, but also as raw material for significant pulp and paper industries in Indonesia. A devastating disease of plantation Acacia mangium in SE Asia is caused by a fungal pathogen belonging to the genus Ceratocystis. This fungal pathogen, Ceratocystis manginecans, causes stem cankers and rapid wilting of trees. At the start of my doctoral studies very little was known about the disease and the search for resistance in A. mangium was proving challenging requiring a large number of different sources of germplasm to be screened and for alternative management strategies to be considered. The aim of my first investigation was to improve screening procedures for resistance to C. manginecans in Acacia species by comparing the current screening protocol (inoculation of potted plants) with two potential rapid screening procedures; inoculation of detached acacia phyllodes (using a spore suspension) and stem segments (using mycelial plugs). Inoculation procedures were tested on three species of Acacia; A. mangium, A. crassicarpa and an Acacia hybrid (A. mangium x A. auriculiformis clones). Two cultures of C. manginecans obtained from different geographic regions were used in the experiments. Lesion length on stems and necrosis length on phyllodes were assessed and used as a measure of the susceptibility of the tested plant. The relative levels of susceptibility in each protocol were compared as a basis to assess the utility of the rapid screening protocols. The stem segments were prone to contamination by other fungi and to desiccation, while results from the potted plant and phyllode protocols showed similar trends of susceptibility among the Acacia clones and species. The ease, rapidity and reproducibility of the phyllode inoculation protocol makes this a potential replacement for inoculation of potted plants as a preliminary screening protocol to identify A. mangium germplasm that is less susceptible to ceratocystis wilt and canker disease. We also evaluated whether the expression of resistance is influenced by the type of inoculum (mycelium or spore suspension). Both mycelial plugs and spore suspensions produced a similar level of disease incidence, so either can be used for inoculation assays permitting the testing of plants with spores of different isolates in the same inoculum. Acacia crassicarpa and A. auriculiformis are considered by the industry as promising sources for resistance against ceratocystis wilt and canker disease. This was also demonstrated in my study; the A. crassicarpa clones used were demonstrably more tolerant to Ceratocystis infection than A. mangium, with much smaller lesions. The disease indices for the Acacia hybrid were intermediate between A. crassicarpa and A. mangium. Bacterial endophytes living in roots, stems and phyllodes of A. mangium were cultured to investigate their biodiversity and to obtain a library of isolates to test for biological control activity against C. manginecans. Samples from trees between one and five years old were collected and their culturable endophytic bacteria were identified by sequence analysis of their 16S rDNA. In total, 278 bacterial isolates were derived from 270 samples representing 90 trees. Most bacteria were isolated from roots and more isolates were obtained from young acacia trees than from the older trees. Analysis of 16S rDNA sequences grouped the endophytic bacteria into five clusters: Firmicutes, Alphaproteobacteria, Betaproteobacteria, Gammaproteo-bacteria and Actinobacteria. Firmicutes were predominant with 66.2% of the isolates belonging to this group. Among 25 genera which were isolated successfully, Bacillus and Burkholderia were the most frequently isolated endophytic bacteria in A. mangium. Several of the genera isolated from A. mangium are those previously demonstrated to include species with potential as biological control agents (BCAs) against plant pathogens and/or the ability to enhance plant growth. In-vitro assays of the 278 isolated endophytic bacteria against C. manginecans revealed 157 isolates with an ability to inhibit the growth of this fungal pathogen. Nine isolates demonstrated an ability to produce metabolites that inhibit the growth of C. manginecans in the absence of viable bacteria. The 16S rDNA analysis of these nine isolates placed them in the genera Paenibacillus, Lysinibacillus, Staphylococcus, Pantoea, Ralstonia, Cupriavidus and Ochrobactrum. Several methods for inoculating the host tissue of A. mangium with these endophytic bacteria were evaluated. Two selected endophytic bacteria, Paenibacillus (EB-05/EB-93) and Lysinibacillus (EB-232) were introduced into A. mangium through the different methods and were positively detected in acacia tissues using specific primers. Applying liquid bacterial cultures to the root zone of germinating acacia seedlings was the most efficient inoculation method when compared with inoculation by seed treatment, or by dipping or spraying of acacia stem cuttings. The deployment of any management strategy in the field will require monitoring to assess effectiveness. At the start of this thesis there was only anecdotal knowledge as to the timing of symptom development (e.g. reduction in leaf area index and yellowing) and the appearance of signs (e.g. insect damage) associated with C. manginecans. Healthy trees neighbouring diseased trees were monitored over 19 weeks. This data (especially LAI data) will be invaluable for carrying out monitoring activities, especially early detection through aerial observation. In summary, the research in this thesis demonstrates the potential of phyllode inoculation as a rapid and low-cost method to extensively test and replicate resistance screening. Selected bacterial endophytes inhibit C. manginecans isolates in vitro and these endophytes can be effectively delivered into a germinating Acacia seedling. The application of bacterial endophytes as biocontrol agents could be part of an alternative management strategy for C. manginecans integrated with the deployment of resistance. My study is the first attempt to establish a description and potential timeline associated with the disease life cycle. Such information can be used to improve monitoring programs for screening resistance and/or alternative management strategies in field experiments or the detection of ceratocystis wilt and canker disease for inventory purposes.

碩士
中興大學
植物病理學系所
99
Diverse endophytic bacteria that grow in the internal tissues of plants are found in a wide variety of plant species. Previous studies showed that some endophytic bacteria have unique metabolic activities that can alter the physiological conditions of the host plants, and some produce antibiotic substances and degradative enzymes that protect host plants from microbial infection. To test if endophytic bacteria have the potential in protecting tomato plants against Ralstonia solanacearum, fourteen endophytic bacteria were isolated from different cultivars of disinfested tomato seedlings in this study. All isolates were identified by FAME and 16S rRNA gene sequences, and one strain was identified as Bacillus cereus (Bce1) that harbors aiiA coding for the quorum quenching enzyme AHL (acyl homoserine lactone) lactonase. The endophytic growth of Bce1 in tomato seedlings was measured by hypocotyl cutting method to show that the bacterium can grow in the vascular tissues to reach a population density of 104 CFU/g at 30 days post inoculation (dpi). In comparison with the untreated plants, tomato seedlings inoculated with Bce1 showed at least one fold of increase in the total weight at 30 dpi, indicating that Bce1 can promote tomato growth. Bce1 had an inhibitory effect on the growth of R. solanacearum strain PS64 that can be readily observed by a dual culture method. When tomato seedlings were inoculated with Bce1 for 30 days and subsequently challenged with R. solanacearum strain PS152 by soil drench, the disease incidence was reduced by 75% in comparing with the 100% wilting symptom of the untreated plants. Knowing the expression of virulence factors in many plant pathogenic bacteria, including R. solanacearum, depends on the quorum sensing (QS) signaling pathways, Bce1 was genetically modified to increase the expression of the AHL-degrading enzyme AiiA to test if the disease severity of tomato bacterial wilt can be further reduced by the removal of QS signaling molecules in planta. Surprisingly, Bce1 with elevated AiiA lost the inhibitory capability on the in vitro growth of R. solanacearum PS64, the beneficial effect on tomato growth, and the protection against tomato bacterial wilt disease, suggesting the bioactive compounds of Bce1 that are involved in promoting plant growth and health may be directly or indirectly regulated by the AHL autoinducers. Genetic studies demonstrated that the extracellular polysaccharide (EPS) of R. solanacearum is an essential virulence factor whose synthesis depends on the other QS signal known as 3-hydroxy palmitic acid methyl ester (3-OH PAME). The feasibility of using genetically engineered endophytic bacteria in the management of tomato bacterial wilt disease will be tested again by expressing the 3-OH PAME degradative enzymes in the endophytic and growth-promoting bacterium Bce1.

Bacterial wilt caused by Ralstonia solanacearum has affected the potato industry in South Africa since 1914. Control of bacterial wilt is difficult and depends greatly on integrated management strategies. Little information is available on the longevity of R. solanacearum in soil under South African conditions and how it is influenced by cultural practices. Information regarding local weeds which can serve as alternative hosts is limited. Soil survival of R. solanacearum biovar 2 (race 3) was investigated in an artificially infested field managed by either one of four cultural practices, namely maize and potato monoculture, bare and weed-fallow. After a five-year period wilting of indicator plants was observed in all treatments with potato the highest, followed by bare-fallow, maize monoculture and lastly weed-fallow. Results demonstrated a greater ability of biovar 2 to survive in soil than generally accepted. Subsequently, the susceptibility of 22 weed and three grass species was investigated in the greenhouse. Five species were susceptible to biovar 2 and 13 to biovar 3. Preliminary in vitro studies conducted to determine suppressiveness of some weeds/grasses, indicate that microbial activity associated with some weeds could be involved in suppression of the wilt organism. Further studies are however required. The effect of maize on Ralstonia solanacearum populations was evaluated in a pot trial as well as in hydroponic culture. Results indicated that microbial populations present in the maize plant, could play a role in the susceptibility of maize to bacterial wilt infection. Antagonistic bacteria associated with some maize plants or with the maize rhizosphere could be partly responsible for suppression of wilt.
Dissertation (MSc Agric (Agronomy))--University of Pretoria, 2006.
Plant Production and Soil Science
unrestricted

博士
國立中興大學
植物病理學系所
103
Abstract Sweet potato [Ipomoea batatas L. (Lam)], the dicotyledonous plant of the family Convolvulaceae, is the seventh most important food crop in the world after wheat, rice, maize, potato, barley and cassava. Besides food supply, sweet potato has diverse uses in green source, ornamental, feed, starch and liquor manufacture, human consumption, biofuel and bioplastic production, etc. Many cultivars have been currently developed for different uses through the world. Among these cultivars, vegetable sweet potato (VSP) has been bred for edible leafy vegetable and can produce a lot of leaves, tender shoots and small/non tubers constantly throughout the growth period in whole year period, especially, summer and rainy season. The VSP is considered as an important green source in summer or rainy season in Taiwan, and similar cultivars of VSP have been bred and grew in Japan. A new disease, Bacterial wilt (BW) caused by Ralstonia solanacearum (RS) broke out during 2000’s and reduced 30 to 80% yield of VSP last decade in Taiwan. The R. solanacearum isolates obtained from diseased VSP were identified as Ralstonia solanacearum phylotype I race 1 biovar 4 (R1bv4) based on physical and molecular analyses. Moreover, these isolates also caused wilting in convolvulaceous, solanaceaous and cruciferous plants. Field investigation indicated that R1bv4 was generally distributed in soil of VSP fields with 1.3×102 to 9.5×105 cfu/g soil. Further detection showed R1bv4 could latently infect healthy VSP cuttings with 2 to 98% isolation frequency. The severity of BW was closely related to R1bv4-carried VSP cuttings (R=0.913); however, the severity of BW did not show significant correlation with the R1bv4 density in soil (R=0.086). Similar phenomenon was observed in greenhouse test. Thus, the cuttings carried R1bv4 were more important inocula source than the R1bv4 residing in soil. The distribution of R1bv4 in VSP indicated that the terminal shoots or erect stems had low R1bv4 containation perecentage (<31%) and creeping stem had high R1bv4 containation percentage (45 to 100%) 8 wks after the VSP planted in infested soil (106 cfu/g soil). Results demonstrated that R1bv4 did not consistently move to the part of erect stem cuttings. For confirming the efficacy of the erect stem cutting on control BW of VSP in the fields, the erect stem cuttings were collected from VSP field. The results revealed that the erect stem cuttings used as new plants could decrease the BW in field. Moreover, companied with early R1bv4 detection in erect stems could increase the control efficient of BW in VSP production area. In this study, a bacterial endophyte, Bacillus amyloliquefaciens SPX1 from healthy VSP in Dali, had good ability to inhibit the growth of R1bv4 on TTC medium. The further experiment showed that the SPX1 isolate could decrease the wilting development of VSP in greenhouse and field conditions by soaking treatment. In addition, a resistance line of VSP, VSPSL-1, showed high reistance to bacterial wilt in field. Thus, the VSPSL-1 line is a promising cultivar resistant to R1bv4 in the future.

博士
國立中興大學
植物病理學系所
96
Bacterial wilt caused by the soil-borne plant pathogenic bacterium, Ralstonia solanacearum, is a globally important disease. The disease has been recorded on 28 plant species as well as several weed species in Taiwan. This pathogen is a species complex with significant phenotypic and genetic diversity. In Taiwan, all reported strains of R. solanacearum are race 1 and biovar 3 or 4, but they are highly variable in virulence on different plant species including tomatoes. Recently, race 3 biovar 2 strains were detected from infected potatoes. The purposes of this study were to (i) determine the phylotype of Taiwanese strains using the new phylotyping classification scheme, determine the phylogenic origins of strains collected from different locations and sources in Taiwan using endoglucanase (egl) and mutase (mutS) partial gene sequences, and examine associations of the classification results with biovar, virulence, strain origins, and geographical origins; (ii) develop a simple BIO-PCR protocol using the semi-selective medium, MSM-1, and species-specific primer pairs, AU759/760, and evaluate the effectiveness of this protocol with field samples; and (iii) develop a preliminary screen to select locally adapted resistant rootstock and soil amendment for integrated management of tomato bacterial wilt in the field. First, the diversity among 157 strains of R. solanacearum isolated from 22 host, 11 weed, 2 water, and 4 soil origins in Taiwan was evaluated on their biovar, virulence, and phylogenic origin. All strains tested were biovar 3 or 4 except for two strains isolated from potato were biovar 2. The biovar 3 strains are predominant. The phylotype-specific multiplex PCR identified all tested strains as phylotype I, except the two biovar 2 potato strains as phylotype II. The 58 out of 157 strains were selected and inoculated on three tomato varieties with different resistance to bacterial wilt. Seven groups with various profiles of virulence were detected. Phylogenetic analysis of partial egl and mutS gene sequences of the same 157 strains revealed a major branch within phylotype I. This is the first evidence to imply the possible existence of an evolution divergence within phylotype I strains in Asia and at least two subgroups could be distinguished. Based on the chisquare test, the distribution of the two subgroups of phylotype I defined by egl or mutS gene sequence over biovars or strain origins was not independent. Only distribution of strains of egl phylogenic groups, but not mutS, was associated with virulence phenotypes. For detection of R. solanacearum, a BIO-PCR method was established to detect R. solanacearum from field soil, weed, and water samples. The method involved incubating field samples in liquid MSM-1 medium at 30oC for 3 days and then detecting with species-specific primer pair AU759/760 without DNA purification. Samples of 320 field soil, 91 weed, and 85 water samples were collected from eight fields with different disease histories and cropping systems located in major tomato production areas. The frequency of positive detections by BIO-PCR was higher than that by plating on MSM-1 medium. Results indicated that weed rhizosphere soils could be good sampling targets to monitor the pathogen in the field. Use of the BIO-PCR method confirmed the uneven distribution of the pathogen in the field. Five composite samples per 1,000-m2 field each consisting of 36 sub-samples at a sampling density of one sub-sample per 5.5 m2 were sufficient to determine the presence of R. solanacearum in the field. For integrated control of bacterial wilt, a preliminary screen was developed to increase the chances of identifying successful control measures over locations. Pre-incubation tests indicated that the mixture of urea and slaked lime was most suppressive to R. solanacearum in three tested soils, and was used in subsequent field experiments. Resistant eggplant (EG203) and tomato (Hawaii 7996) rootstocks were selected based on stable resistance against representative strains of R. solanacearum at the seedling stage. Integrated use of Hawaii 7996 as the rootstock and mixture of urea and slaked lime as the soil amendment provided significantly greater control of wilt than use of Hawaii 7996 as rootstock alone in only one of the four locations, whereas the soil amendment did not provide significant control effect when EG203 was used as the rootstock. Understanding strain variations of R. solanacearum in Taiwan, use of the BIO-PCR detection method to monitor pathogen distribution, and application of the preliminary screen scheme to select disease control strategy can contribute greatly to sustainable management of tomato bacterial wilt.

Tomato spotted wilt virus (TSVVV) occurs sporadically in potato (Solanum tuberosum) crops in Australia, sometimes causing severe losses. Very little is known about the disease in potato as evidenced from the literature review (Chapter 1). Part of the problem stems from the sporadic nature of the disease in potato, which makes it hard to find a consistent data set at the desired level of aggregation, both for disease incidence and intensity as the variables to be explained and for explanatory variables. This makes it difficult to formulate empirical descriptions of risk factors and statistical exploration of interactions of different variables that develop into functional complexes causing the epidemics. Surveys were conducted from June 2001 to March 2004 in commercial potato fields in the southern States of Australia. Data sets from these surveys were used to develop dynamic mathematical models, defining spatio-temporal oscillations, in both population structure and demography, of the only two thrips vector species, Thdps tabad and Frankliniella schulei, found in potato crops (Chapter 2). The Gompertz and Exponential curves best described the relationships between the vector and the regional weather variables. In general, the optimum daily minimum air temperature (°C) required by T. tabaci population to start rising within potato crops ranged between 10°C and 12.0°C and daily maximum air temperature (°C) range was 14.5°C and 22°C. The observed versus predicted estimates for the daily relative humidity at 3pm in Tasmania had low percentage variance (r2) values for T. taboo: The general optimum daily minimum air temperature (°C) range for F schultzei populations to start rising was between 4°C and 6°C and daily maximum air temperature (°C) range of 20°C and 22°C. Less than favourable daily relative humidity at 3pm of more than 40% depresses F. schultzei populations in both Victoria and South Australia. Both T. tabaci and F. schultzei populations are depressed by precipitation as indicated by three models across the sampling sites. The models can be used as a hazard prediction to orient integrated TSWV disease management. To test the significance of the potato cultivars being grown in affecting the epidemiology of TSVVV, 27 cultivars were evaluated for two seasons in both glasshouse and natural field conditions. Variations in susceptibility to infection were exhibited by potato cultivars and were conditioned by the age of the plant at the time of infection as measured by symptom expression, shoot and tuber infections in both mechanical and thrips transmissions (Chapter 3). For both foliar and tuber infections, more cultivars exhibited symptoms when infected late than early. TSVVV infections between cultivars were not significantly different (P>0.05), except in tubers from mechanically inoculated plants during 2002/2003. Foliar infections in a number of cultivars did not translate into virus translocation to tubers except in Atlantic, Bintje, Fontenot, Kennebec, Kipfler, McCains 1 and Shepody. Thrips had a high feeding preference for the cultivar Royal Blue, but this did not translate into high TSVVV infections. The cultivar Bismarck, which despite being highly susceptible to foliar and tuber TSVVV infections through mechanical inoculations, exhibited strikingly robust field resistance to thrips feeding and consequently TSVVV infection in the field. The study suggests that the risk of TSVVV epidemics depends on when and to what extent the plants develop resistance in relation to the age of the crop. Certification guidelines for tolerance levels of TSVVV infection in seed potatoes in Australia should reflect this on a cultivar specific basis. The independent and interactive effects of temperature with plant age at the time of inoculation on viral movement restriction, symptom expression and foliar and tuber infections were assessed and quantified in potato cv. Shepody (susceptible) and Russet Burbank (tolerant) under controlled conditions (Chapter 4). Plants exposed immediately after inoculation or after 72 hours to four different temperature regimes at two different stages of growth (early versus late) showed variations in symptom expressions and virus movement patterns. In inoculated plants immediately exposed to different temperature treatments, foliar infections and symptoms were generally restricted to lower and middle leaves of the canopies and auxiliary shoots. More leaves of early-inoculated plants expressed symptoms than in late inoculations. In early-inoculated cv. Shepody plants continuously exposed to 22°C, virus migration reached top canopies two days earlier (5 days after inoculation) and induced TSVVV symptoms than similar plants under continuous 16 °C. In both cultivars, plants were more resistant to TSVVV infections when inoculated late and immediately exposed to 22 °C for 48 hours, inoculated and then transferred to 16°C. Under these conditions, some infections were detected in lower inoculated leaves of cv. Russet Burbank plants at 12 days after inoculation. When plants had a 72 hours delayed exposure to different temperature regimes, the number of plants with detectable TSVVV in shoots, number of leaves on each plant and total leaf areas with symptoms were in the middle and top canopies. The virus was detected only in early-inoculated plants. Most plants did not exhibit TSVVV symptoms and the virus was not detectable by ELISA, except in Shepody plants exposed to 16°C continuously and 22°C for 48 hours, inoculated and then transferred to 16°C. In these plants the virus reached the middle and top canopies where it was detected in cvs. Russet Burbank and Shepody at 28 and 43 days after inoculation, respectively. The underlying mechanism by which temperature influences these virus movements and symptom expressions in TSVVV infections is still unclear. Results from this study would be useful in the rational selection of suitable conditions when screening potato cultivars for TSVVV resistance by breeding programs and production in appropriate climatic conditions. In summary, the results from the above studies, collectively, contribute to uncover some ecological relationships and patterns of both TSWV and its vector thrips that can be integrated with plausible mechanisms to explain the epidemiology of the virus in potato crops and advance the rationale for future research in this direction.

Plants regulate responses to their environment through complex hormone signaling; these hormones can be categorized broadly into two categories: growth and defense, though many have roles in both. Much remains to be understood about the complexity of hormone signaling in relation to environmental responses, especially species- and genotype-specific differences. Unraveling this complexity of hormone signaling will lead to the development of resilient crops that are able to respond appropriately to their environment. In this dissertation, I hypothesize novel roles for growth and defense hormones in Solanum spp. responses to 1) biochar, a black carbon soil amendment (Chapter 2), 2) infection with Ralstonia solanacearum, an economically important soilborne pathogen causing bacterial wilt (Chapter 3), and 3) endophytic colonization by the soil bacterial community (Chapter 4). In Chapter 2, I showed that biochar upregulates GA signaling and affects GA-related traits in a species- and cultivar-specific manner. Biochar amendment also downregulates defense signaling. In Chapter 3, I demonstrated a novel role for auxin in resistance against R. solanacearum, including differential expression of auxin signaling genes in resistant genotype H7996 compared to susceptible WV in response to R. solanacearum infection. In addition, I observed stronger and faster upregulation of defense hormone marker genes for SA and ET in H7996 compared to WV. In Chapter 4, I showed that SA and ET are required for normal tomato root microbial community assembly, affecting the colonization of a few key taxa in order to promote alpha diversity. H7996 and WV root communities differ in alpha diversity, and a panel of H7996 x WV RILs showed quantitative variation in alpha diversity that correlated negatively with the abundance of these key taxa. In conclusion, I elucidated novel roles for hormones in responses to the soil environment, pathogen infection, and root community colonization. These findings are important for developing resilient, sustainable crops.