Dissertations / Theses on the topic 'Disease suppression; Rhizoctonia solani'

Compost can suppress soilborne plant pathogens that cause significant damage on globally important food crops. However, reports of plant pathogen suppression are inconsistent likely because there are no established standards for feedstock material, application rate, and maturity age upon application. Excellent results can be achieved in greenhouse trials, but field applications are much less reliable. Disease suppression occurs through the activity of biocontrol organisms (direct antagonism), and general microbial competition. Biocontrol species are hypothesized to colonize the pile during the curing phase, but single species may not be as important as microbial consortia. Substrate composition during maturation may give rise to a suppressive microbial community. More research is needed to understand the relationships between feedstock, maturity, and production process on compost microbial ecology. The thesis had two main objectives: 1) identify biological indicators in compost that could (a) characterize maturity, process, and feedstock, and (b) predict disease suppression against R. solani, and 2) identify bacterial and fungal community composition and/or structure that is associated with suppression of soilborne disease. Rhizoctonia solani is a facultative saprophytic fungus and soilborne plant pathogen that attacks many globally important food crops and turfgrass. Prior research suggests that managing carbon quality and compost maturity will alter relative competition between biological control microbes and the R. solani pathogen. The pathogen is responsible for economic losses to organic vegetable production in Vermont and there are no available methods to manage the disease that meet organic certification. R. solani on radish was chosen as a model system given its global importance, competitiveness affected by carbon quality, and lack of disease management options for organic production. Compost samples were most abundant in the bacterial phyla Proteobacteria and Bacteroidetes, and known biocontrol species were not detected in abundance. Compost samples did not differ significantly in fungal community composition, suggesting a dominance effect from the native soil fungal community. Overall, anaerobic digestate and vermicompost were most suppressive against R. solani. Thermophilic composts were not very suppressive overall, though a specially made hardwood bark compost was comparable to the suppressiveness of vermicompost application. Ecoenzyme analysis was able to integrate information on environmental substrate composition, microbial nutrient acquisition, and microbial community metabolism, offering the best view of current ecological conditions in compost. Ecoenzyme analysis showed that the most suppressive composts, anaerobic digestate and vermicompost, were most nutrient limited. All compost samples were severely nitrogen (N) limited, and anaerobic digestate and vermicompost were severely limited in both N and phosphorus (P). The additional P limitation may support non-pathogenic species to outcompete R. solani. The key to disease suppression may lie in matching up the ecology of the plant pathogen with the ecology of biocontrol, which may be engineered in compost.

Use of known antagonists and antagonistic, endophytic bacteria was investigated to screen a successful antagonist for the fungal strains R. solani AG 2-1, R. solani AG 4 and R. solani AGSL01.   Reputed antagonists used in the study; Bacillus subtilis MBI 205,  B. subtilis MBI 600, Pseudomonas fluorescens B5 and P. corrugate R. 117 were capable of suppressing the fungus in dual cultures. Manipulation of soil factors to reduce pre-plant density of R. solani sclerotia involved a laboratory experiment followed by two field experiments.  In the laboratory study, the effect of constant soil temperatures was investigated by incubating sclerotia at 30, 35, 40, 45 and 50°C.  Sclerotia were counted into lots of 100, placed in polyester mesh bags (85 μm pore size, 10 x 10 cm dimension) at depths of 5 and 10 cm in rice field soils contained in plastic containers (20x20x18 cm).  Total loss of viability was observed on day 1, day 8 and day 28 at 45°C, 40°C and 35°C, respectively.  Loss was even detected within 6.00 h at 50oC. Field experiments of soil solarization (carried out at The Regional Agricultural Research Institute (RARI), Bombuwela, Sri Lanka) were conducted during the fallow periods between the two main cropping seasons in 2003.  During the trials, the effect of polythene mulching, straw incorporation and their combined effect on the viability of sclerotia were investigated.  Sclerotia (lots of 100) were buried at depths of 5 and 10 cm and the soil temperature was recorded at 8.00, 11.00, 14.00 and 17.00h daily at both depths.  During the field trials, at both depths, the effect of treatment over time was noted on percentage recovery and viability of sclerotia (p<0.001).  The results showed a drop in mean percentage recovery of less than 50% during the first week of both trials.  Germination was markedly reduced to less than 10% by the first week in all the treatments.  Depth of burial and straw incorporation had no effect.  In solarized plots, a significant increase in soil temperature at both soil depths was observed between 14.00 and 17.00h.  Average temperatures of 40°C and 34°C were observed for solarized and non-solarized plots, respectively.  The study therefore has identified a practical, low cost and environmentally friendly method of control, and the use of polythene five weeks prior to rice seed sowing is recommended to minimise sheath blight and other diseases caused by similar soil-borne fungal pathogens.

Rhizoctonia solani (AG-3), the causal agent of Rhizoctonia disease of potato, overwinters as sclerotia on potato tubers. To develop a biocontrol strategy based on the prevention of the sclerotial germination, an isolation of microorganisms colonizing sclerotia of infected potato tubers (cultivars Norland, Atlantic and Souris), was conducted. In vitro screening was used to select effective antagonistic fungi against Rhizoctonia solani. Fifty fungal isolates were selected in order to cover all identified genera and potato variety and examined for their ability to inhibit germination of sclerotia which were incubated with the test fungus for 14 days. Twenty-four (24) fungal isolates were retained based on their ability to reduce sclerotial viability by more than 50% as compared with 100% viability of untreated sclerotia. These 24 isolates were further examined for their ability to protect Table beet seedlings against the pathogen in greenhouse soils. Based on their ability to protect Table beet seedlings from Rhizoctonia infections and to increase the number of secondary roots and root length isolates, F2, F11, F132, F158, and F258 were screened and test their efficacy to increase beet seed germination in field soils. (Abstract shortened by UMI.)

Brown patch, caused by Rhizoctonia solani Kuhn, is an important disease on tall fescue (TF, Festuca arundinacea Schreb, synonym Schedonorus phoenix (Scop.) Holub). Rhizoctonia zeae Voorhees, a related pathogen, causes similar symptoms. Confusion over which Rhizoctonia species is causing symptoms and subjective visual evaluations of disease severity may contribute to variability in observed BP resistance of TF cultivars at multiple locations. The objectives of this study were to develop an objective digital image analysis (DIA) method for evaluating disease and to use DIA to screen tall fescue plant introductions (PIs) for resistance to R. solani and R. zeae. There was a strong correlation (r² = 0.97) between actual disease severity, measured by applying lesioned tissue of a known area to healthy leaves, and DIA calculated disease severity using scanned images of individual leaves (DIA-IL). The accuracy and precision of visual evaluations and DIA evaluations of entire plants (DIA-WP) were evaluated using DIA-IL as a standard of accuracy. Accuracy of DIA-WP was not significantly different from visual evaluation accuracy. Precision was significantly higher for DIA-WP. Evaluation of PIs and putatively BP resistant TF cultivars for resistance to R. solani and R. zeae using DIA-WP identified clones within each PI that ranked high for resistance to R. solani or R. zeae. No clones were identified with high resistance to both R. solani and R. zeae. Improved precision of DIA evaluation methods and inclusion of R. zeae in BP resistance breeding may decrease variability of TF cultivar performance across locations.
Master of Science

The chemicals inducers SA, BABA, and BTH were tested as seed treatment and soil drench on a partial-resistant cultivar of sugar beet grown in sand infested with the Rhizoctonia solani AG 2-2IIIB. In another series of experiments, BTH was applied as soil drench on sugar beet plants inoculated with R. solani. The chemical inducers were ineffective in reducing pre-emergence damping-off and post-emergence plant mortality. Despite these results, treatment with BTH altered the levels of expression ratios of four defence encoding genes associated with systemic resistance: chitinase, peroxidase, chalcone isomerase, and chalcone synthase. BTH sensitised sugar beet plants without the necessity of R. solani infection to up-regulate substantially the transcript level ratios of chalcS and chit3, while levels of chalcI were down-regulated levels below 1. Of interest, was the significant increase of transcript levels of chit3 in sugar beet plants infected with R. solani and treated with BTH. In conclusion, sugar beet plants were capable of over expressing selected genes in response to a chemical inducer, but contrary to what had been reported, gene activation in sugar beet as a result of BTH treatment does not confer disease resistance against R. solani.

Rhizoctonia solani is the most damaging pathogen on sugarbeet (Beta vulgaris L.) in North Dakota and Minnesota. Research was conducted to evaluate penthiopyrad for controlling R. solani and determine when the plants are most susceptible to infection. Penthiopyrad applied in-furrow and as a soil drench resulted in significantly higher percent survival than the positive control whereas penthiopyrad applied in a band was ineffective at controlling R. solani. Penthiopyrad can also be used as a seed treatment at the 14 g rate to provide effective control of R. solani. Sugarbeet plants, irrespective of their inherent level of resistance, were easily infected by R. solani up to three weeks after planting, even longer for susceptible varieties, highlighting the need for additional protection in the form of seed treatment or fungicide application that may be required to protect vulnerable sugar beet planted in fields with a history of the disease.

A time course study was conducted to monitor disease development and expression of the defense-related genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and hydroxyproline-rich glycoprotein (HRGP) in bean (Phaseolus vulgaris L.) plants colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices , and post-infected with the soil-borne pathogen Rhizoctonia solani. Pre-colonization of bean plants by the AM fungus did not significantly reduce the severity of rot symptoms. RNA blot analysis revealed a systemic increase in transcript levels of the four defense-related genes in response to R. solani infection. On the other hand, pre-colonization of bean plants with G. intraradices elicited no change in PAL, CHS and CHI transcripts, but an increase of HRGP transcripts in leaves was detected. A differential and systemic alteration in the expression of all four defense genes was observed in AM beans post-infected with R. solani. Depending on the time after infection with R. solani and the tissue examined, varying responses from stimulation, suppression, to no change in transcript levels were detected.

In this study, we have demonstrated that inoculation of bean seeds with non-pathogenic binucleate Rhizoctonia (np-BNR) at sowing protected bean seedlings from infection of R. solani. Using quantitative real-time RT-PCR (QRT-PCR), transcript levels of defense genes encoding 1,3-beta-glucanase (GLUC), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) in one-week old bean seedlings was monitored during np-BNR and R. solani interaction. The results revealed that protection effect of np-BNR correspond to a systemic suppression of these three defense genes' expression from significant higher level elicited by R. solani to the level of non-infected plants. This indicates that bio-protection by np-BNR isolates is not correlated to activation of these three defense genes' expression. Similar suppression was achieved for pre-colonization of bean seedlings with arbuscular mycorrhizal (AM) Glomus introradices on GLUC gene expression, although the AM fungus did not significantly reduce rot symptoms. Possible mechanisms implicated in down-regulation during plant-pathogen and np-BNR or AM interaction are discussed.

Afin d'identifier des mécanismes impliqués dans le développement des foyers causés par Rhizoctonia solani AG2-2 sur betteraves, des échantillons de sol ont été prélevés le long de transects traversant des zones infectieuses et des zones saines dans un champ cultivé en betteraves sucrières. Le potentiel infectieux et la résistance du sol aux maladies dues à R. Solani ont été mesurés et comparés. Le sol devient résistant au sein des foyers. L'analyse des structures de communautés bactériennes et fongiques par des méthodes génétiques et biochimiques ont mis en évidence l'émergence de Trichoderma spp. Au sein des foyers. Des isolements et une caractérisation génotypique, phénotypique et fonctionnelle de ces Trichoderma spp. Ont révélé qu'au sein d'une même espèce (T. Gamsii) des populations antagonistes de R. Solani s'étaient développé dans les foyers. En milieu contrôlé, la population de T. Gamsii testée inhibe fortement le développement et la croissance de celle de R. Solani AG 2-2 dès que celle dernière atteint une densité seuil. Cela expliquerait que le sol devienne résistant au coeur du foyer mais que l'apparition et le développement initial du foyer ne soient pas contrôlés. Des prélèvements réalisés au cours du temps montrent l'évolution de la structure des communautés microbiennes aboutissant au développement du foyer et révèlent que l'incidence d'une maladie aérienne causée par Cercospora beticola sur le feuillage de la betterave n'est pas neutre vis-à-vis de la microflore tellurique
To assess the mechanisms involved in the development of disease patches caused by Rhizoctonia solani AG 2-2 in sugar beet field, transects were done across the sugar beet plots and soil was sampled within disease patches and healthy areas. Soil suppressiveness and soil inoculum potential were measured and compared. The soil became less conducive within the patches. The analyses of the structures of the microbial communities (bacteria and fungi, use of both genetic and physiological approaches) indicated changes leading to the isolation of various Trichoderma sp. They have been characterized, including the functional diversity i. E. Antagonistic activity (and the mechanisms involved) towards R. Solani. Those from the inside of the patches were more antagonistic towards R. Solani than those from healthy areas, although they could belong to the same species (T. Gamsii). Population dynamics were conducted to study the interactions between a strain of Trichoderma and R. Solani AG 2-2. They revealed that indeed T. Gamsii inhibits significantly the development and the density of R. Solani population once its population is above a threshold that may explain why the soil became less conducive within a patch but could not prevent the occurrence of the patch. Samplings were also performed along the development of the patches to study the temporal dynamics. The relative influence of an aerial disease of sugar beet caused by Cercospora beticola and the disease caused by R. Solani was investigated too, revealing that the effect of C. Beticola on the soil microflora was not neutral

One of the motivating reasons for the development of hydroponics was avoidance of root pathogens. Hydroponics involves growing crops in relatively sterile media, isolated from the underlying soil which may have disease pressure. However, even when hydroponics is coupled with controlled environments such as high tunnels and climate-controlled greenhouses, soil-borne pathogens can enter the growing area and proliferate due to optimal environmental conditions for pathogen growth. Control of root pathogens is difficult and usually achieved through synthetic fungicides since few biocontrol options are available. Compost water extracts (CWE) have recently been gaining the attention of greenhouse growers because they may be a low-cost, environmentally friendly approach to control root disease. CWE are mixtures of compost and water incubated for a defined period of time, either with or without aeration, and with or without additives intended to increase microbial populations, which in turn suppress disease. Much anecdotal, but very little scientific, evidence exists describing CWE effect on suppressing soil-borne pathogens. The present study 1) examined the effect of an aerated CWE on disease suppression at the laboratory scale and in container studies using different soilless substrates, 2) investigated a phenotypic change at the root level caused by CWE that may be associated with disease suppression, and 3) isolated some factors in the production of CWE that affect the ability of a CWE to suppress disease. The common model pathogen-host system of Fusarium solani f.sp. pisi and pea was used to examine CWE-induced disease suppression, with information then being translatable to similar patho-systems involved in greenhouse crop production. In the first study, laboratory-based root growth and infection assays resulted in 100% suppression of F. solani when roots were drenched in CWE. These protected seedlings were then taken to a greenhouse and transplanted into fine coconut coir, watered with hydroponic nutrient solution, and grown for five weeks. At the end of the experiment, 23% of the shoots of the pathogen-inoculated, CWE-drenched seedlings remained healthy while only 2% of the inoculated seedlings without CWE drench remained healthy. All of the roots of the inoculated seedlings developed lesions, even those drenched in CWE. However, 29% of the CWE drenched roots were able to recover from disease, growing white healthy roots past the lesion, while only 2% recovered naturally. A shorter-term container study was conducted in the laboratory to determine the effects of CWE-induced suppression when peas were grown in different substrates and to determine if the hydroponic nutrient solution had an effect on the suppression. Peas were grown in sterilized fine and coarse coconut coir fiber and sand irrigated with water, with a second set of fine coir irrigated with hydroponic nutrient solution. Pea seeds with 20-25mm radicles were inoculated with pathogen and sown directly into CWE-drenched substrate and grown for three weeks. At the end of the experiment, 80%, 60%, 90%, and 50% of the shoots of the inoculated, CWE-drenched seedlings remained healthy when grown in fine coir, coarse coir, sand, and fine coir irrigated with hydroponic nutrient solution, respectively. Nearly 100% of the roots grown in coconut coir substrates again developed necrotic lesions but 83%, 87%, 100%, and 87% grew healthy roots beyond the disease region. The hydroponic nutrient solution had a negative effect on suppression, with a reduction of at least 30 percentage points. Sand demonstrated a natural ability to suppress F. solani. Only 23% of inoculated seedlings had dead or dying shoots by the end of the experiment (compared to 77-80% in coir substrates) and although all but one of the roots developed lesions, all were able to recover on their own with CWE. CWE further increased shoot health and also prevented 57% of the roots from developing lesions. In a second study, two different CWE were used to examine the effect on root border cell dispersion and dynamics in pea, maize, cotton, and cucumber and its relation to disease suppression. Dispersal of border cells after immersion of roots into water or CWE was measured by direct observation over time using a compound microscope and stereoscope. Pictures were taken and the number of border cells released into suspension were enumerated by counting the total number of cells in aliquots taken from the suspension. Border cells formed a mass surrounding root tips within seconds after exposure to water, and most cells dispersed into suspension spontaneously. In CWE, >90% of the border cell population instead remained appressed to the root surface, even after vigorous agitation. This altered border cell phenomena was consistent for pea, maize, and cotton and for both CWE tested. For most cucumber roots (n=86/95), inhibition of border cell dispersal in both CWE was similar to that observed in pea, maize, and cotton. However, some individual cucumber roots (8±5%) exhibited a distinct phenotype. For example, border cells of one root immersed into CWE remained tightly adhered to the root tip even after 30 minutes while border cells of another root immersed at the same time in the same sample of CWE expanded significantly within 5 minutes and continued to expand over time. In a previous study, sheath development over time in growth pouches also was distinct in cucumber compared with pea, with detachment of the sheaths over time, and root infection was reduced by only 38% in cucumber compared with 100% protection in pea (Curlango-Rivera et al. 2013). Further research is needed to evaluate whether this difference in retention of border cell sheaths plays a role in the observed difference in inhibition of root infection. In the third study, a series of investigations were conducted to isolate different factors that contribute to the suppression ability of a CWE by changing incrementally changing some aspect of the CWE production process. The basic aerated CWE recipe (with molasses, kelp, humic acid, rock phosphate, and silica) provided 100% protection of pea from root disease while the non-aerated basic recipe CWE provided 72% protection. Aerated CWE made of only compost and water resulted in 58% protection. It was found that molasses did not contribute to the suppression ability of the ACWE, while kelp contributed strongly. When soluble kelp was added by itself to the compost and water, the CWE provided 80% suppression. However, when all additives were included except molasses and kelp, suppression remained high (93%) indicating that humic acids, rock phosphate, and/or silica were also major contributors toward the suppression effect. Optimal fermentation time for ACWE was 24 hr to achieve 100% suppression, with increased time resulting in inconsistent suppression results. Optimal fermentation time for NCWE was 3 days or 8 days. These studies are important contributions to understanding the differences that might be expected in CWE suppression when growing in different substrates, some of the factors in the production of CWE that affects the ability of a CWE to suppress disease, and the phenotypic effect CWE has on the root zone of plants and the possible relationship between that effect and disease suppression.

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Rhizoctonia canker, caused by Rhizoctonia solani, is an important cowpea disease in Northeastern region of Brazil. This thesis aimed: a) to evaluate the potential of crop rotation and green manures in inducing suppressiveness to Rhizoctonia canker in cowpea; b) to identify the biotic factors related to suppression; and c) to analysis the stability of the disease suppression by selected crop rotation in relation to different isolates and inoculum density of the pathogen, and soils from different locations. The experiments were conducted in microplots in the field. In relation to crop rotations, Initially, 14 treatments were compared in four growing seasons, including different combinations of cowpea (CA) in rotation with cotton (AL), sunflower (AL), castor bean (MA), corn (MI) and sorghum (SO), and in combination with fallow (PO) and cowpea monoculture. The rotations CA-MI-SO-CA, CA-GI-SO-CA and CA-MI-PO-CA provided reductions of 77.6%, 74.0% and 73.9% in the disease severity, respectively. When the stability of disease control by CA-MI-SO-CA rotation was evaluated in relation to different pathogen isolates, inoculum densities and soils, in all situations the disease severity was significantly (P0,0001) lower under crop rotation as compared to cowpea monoculture, indicating the potential of the crop rotation for controlling the Rhizoctonia canker. In relation to green manures, 13 treatments were compared in five growing seasons, considering different combinations of cowpea and green manures (sunn hemp, breviflora crotalaria, showy crotalaria, jack bean, velvet bean, dwarf mucuna, lablab bean, dwarf pigeon pea, forage pigeon pea, corn, millet and sorghum). For the two evaluations after green manures cultivation, the incorporation of sorghum into the soil led to smallest levels of severity of cowpea Rhizoctonia canker, reducing by 81.3% and 83.3% the disease severity. There was no significant correlation from levels of disease severity with microbiological variables.
A rizoctoniose, causada por Rhizoctonia solani, é uma importante doença do feijão-caupi na região Nordeste do Brasil. Essa tese teve como objetivos: a) avaliar o potencial da utilização de rotações de culturas e adubos verdes na indução da supressividade à rizoctoniose do feijão-caupi; b) identificar os possíveis fatores bióticos dos solos responsáveis pela supressividade; e c) analisar a estabilidade do controle pela rotação de cultura selecionada em relação a diferentes isolados e densidades de inóculo do patógeno, bem como solos de diferentes localidades. Os experimentos foram realizados em microparcelas no campo. Em relação à rotação de culturas, inicialmente foram comparados 14 tratamentos em quatro ciclos de cultivo, incluindo diferentes combinações de feijão-caupi (CA) em rotação com as culturas de algodão (AL), girassol (GI), mamona (MA), milho (MI) e sorgo (SO), bem como em combinação com pousio (PO) e feijão-caupi em monocultura. As rotações CA-MI-SO-CA, CA-GI-SO-CA e CA-MI-PO-CA propiciaram reduções na severidade da doença de 77,6%, 74,0% e 73,9%, respectivamente. Quando avaliada a estabilidade do controle da doença pela rotação CA-MI-SO-CA a diferentes isolados, densidades de inóculo e solos, em todas as situações a severidade foi significativamente inferior (P0,0001) sob a rotação de culturas comparada à monocultura, indicando o potencial de utilização da rotação no controle da rizoctoniose. Em relação aos adubos verdes, foram comparados 13 tratamentos, considerando diferentes combinações de feijão-caupi e adubos verdes (crotalária breviflora, crotalária juncea, crotalária spectabilis, feijão-de-porco, guandu anão, guandu forrageiro, labe-labe, mucuna anã, mucuna preta, milho e sorgo), em cinco ciclos de cultivo. Nas duas avaliações realizadas após o cultivo de adubos verdes, a incorporação de sorgo ao solo propiciou os maiores níveis de controle da doença, reduzindo em 81,3% e 83,3% a severidade. Não houve correlação significativa dos níveis de severidade da rizoctoniose com as variáveis microbiológicas avaliadas nos solos.

O controle de doenças em plantas geralmente é realizado com o uso de agroquímicos, que podem desencadear efeitos maléficos ao ser humano ou ao ambiente como um todo. Diversos trabalhos mostram o potencial de plantas medicinais no controle de fitopatógenos, tanto por sua ação fungitóxica direta, inibindo o crescimento micelial e a germinação de esporos, quanto pela capacidade de ativar o metabolismo de defesa das plantas, como o acúmulo de fitoalexinas, indicando a presença de moléculas com características elicitoras. Foram desenvolvidos dois experimentos na Universidade Tecnológica Federal do Paraná – Câmpus de Dois Vizinhos, com objetivos de avaliar o pontencial de preparados de Cavalinha (Equisetum sp.) na síntese de metabólitos de defesa em cotilédones de soja (Glycine max L.) e o efeito sobre o crescimento de Rhizoctonia solani, in vitro. O delineamento experimental utilizado para os experimentos foi inteiramente casualizado em esquema fatorial 3x5 resultante da combinação de três formas de extração (extrato alcoólico, infusão e maceração) e cinco concentrações (zero; 1; 10, 20 e 40%), com 4 repetições. No primeiro experimento foi avaliada a indução de fitoalexinas em cotilédones de soja em resposta aos derivados a base de cavalinha. Sementes de soja foram semeadas em areia autoclavada e mantidas em temperatura ambiente por 10 dias. Em seguida os cotilédones das plântulas foram removidos e na face abaxial destes foram aplicados os tratamentos. Após seguir os procedimentos metodológicos da técnica de extração, obteve-se via espectofotometria a quantificação da fitoalexina gliceolina, sendo então utilizados os cotilédones para quantificação da atividade da enzima fenilalanina amônia-liase (FAL) e o teor de fenóis totais. No segundo experimento foi avaliado o potencial fungistático dos preparados sobre Rhizoctonia solani. Em placas de Petri foram adicionados no meio de cultura BDA os preparados nas diferentes concentrações. Após a solidificação foi transferido para o centro de cada placa, um disco de micélio com Rhizoctonia solani. As placas foram incubadas sob alternância de fotoperíodo de 12 horas, em temperatura de 25oC. Os preparados de extrato alcoólico, infusão e maceração de cavalinha apresentam capacidade de indução das fitoalexinas gliceolinas em cotilédones de soja, bem como, ativam o metabolismo de compostos fenólicos. Entre os preparados, o extrato alcoólico e a maceração, se sobressaem sobre a infusão. Os preparados de extrato alcoólico, infusão e maceração de cavalinha em todas as suas concentrações inibem o crescimento do fungo Rhizoctonia solani, in vitro, sendo que o extrato alcoólico apresenta maior capacidade de supressão do crescimento micelial em comparação as demais formas de obtenção dos preparados.
Usually, the plant diseases control is accomplished with the use of chemicals products that can trigger harmful effects for the people or the environment. Several studies showed the potential of medicinal plants in the control of pathogens, either by their direct fungitoxic action, inhibiting the mycelial growth and spore germination, and by ability to activate the metabolism of plant defense, such as phytoalexin accumulation, indicating the presence of molecules with characteristics elicitoras. Two experiments were carried out in the Universidade Tecnológica Federal do Paraná - Câmpus Dois Vizinhos. The aims of these works were to evaluate the potential of "cavalinha" (Equisetum sp.) prepared in the defense metabolites synthesis of soybean cotyledons (Glycine max L.) and the effect on Rhizoctonia solani growth in vitro. The experimental design used for the experiments was completely randomized in factorial 3 x 5 (extract form x concentration), with four replications. The extract forms were alcoholic extract, infusion and maceration and the concentrations tested were zero, 1, 10, 20 and 40%. In the first experiment, the phytoalexins induction in the soybean cotyledons the according with "cavalinha" prepared was evaluated. Soybean seeds were sown in autoclaved sand and kept at room temperature for 10 days. After, the plantlets cotyledons were removed and on the surface of these the treatments were applied. Then, it followed the methodological procedures of extraction technique and it was obtained via spectrophotometry the phytoalexin glyceollin quantification. The cotyledons were then used to quantify of the enzyme phenylalanine ammonia lyase (PAL) activity and total phenols contend. In the second experiment was evaluated the potential fungistatic preparations on Rhizoctonia solani. The prepared with different concentrations was added in Petri dishes with PDA culture medium. After solidification was transferred to the center of each plate, a disc with Rhizoctonia solani mycelia. The plates were incubated alternating the photoperiod in 12 hour, at 25 °C temperature. The alcoholic extract, infusion and maceration "cavalinha" prepared present glyceolin induction of phytoalexins in soybean cotyledons, as well as it to activate the metabolism phenolic compounds. Among the prepared, the alcoholic extract and maceration forms were superior in relation the infusion form. The alcoholic extract, infusion and maceration "cavalinha" prepared using all over the concentrations inhibit Rhizoctonia solani growth in vitro. The alcoholic extract had a higher capacity to suppress mycelial growth compared to other forms of production preparations.

Rhizoctonia solani AG8 is a significant soil-borne pathogen of cereal roots in semi-arid Mediterranean regions of Australia and the Pacific North West region in the United States of America, causing severe productivity and economic losses to farmers. During the past twenty years the conversion of many farming systems to conservation tillage has meant that the mycelial network of the pathogen is no longer seasonally disturbed by cultivation which has subsequently increased the potential for greater incidence of Rhizoctonia root rot. There has been some success in reducing incidence by using modifications to direct drill seeding equipment enabling some disturbance to Rhizoctonia at sowing. However, a long term sustainable solution with both economic and environmental benefit, as concluded from a review of the literature (Chapter 1) is to harness the potential for biological control of the disease via natural or induced suppression in soils. Biological suppression to specific disease organisms in soil has been reported worldwide from a range of environments. Further, the development of biological soil-borne suppression to Rhizoctonia root rot has been described for one specific agricultural location (Avon) in South Australia following a decade of stubble retention together with higher than average nutrient inputs (Chapter 1). The studies in this thesis investigate soilborne suppression to Rhizoctonia in agricultural soils from a semi-arid region of South Australia called Eyre Peninsula (EP) that produces 40% of the State’s grain. The context is that historically in Eyre Peninsula farming systems crop residue inputs to soil are inherently low, as are fertiliser N and P inputs. However, recent intensification of these systems with the implementation of continuous cereals and minimum or zero tillage has resulted in greater inputs of stubbles and fertilisers. Rhizoctonia root disease is prevalent in the mainly coarse textured alkaline soils of the region, and the reduction in cultural control associated with adoption of reduced till systems has highlighted a need for alternative control measures. In a broad context, the key question addressed in this thesis is whether the soil ecology to suppress Rhizoctonia is present or can develop in these soils from a region considered an extremely harsh environment climatically as well as edaphically. Specific key questions will be addressed in the discussion section of each chapter. The thesis, through a series of controlled environment studies, examines abiotic-biotic interactions between the soil, the Rhizoctonia solani pathogen and wheat seedlings. The work assesses how the soil organisms involved in disease suppression (both the pathogen Rhizoctonia solani AG8 organism and other antagonists or competitors) are influenced by cereal stubbles and fertilizer inputs to the system. Through a series of preliminary experiments (Chapter 3) the important variables of soil moisture and amount of pathogen inoculum (e.g. number of pathogen infected agar plugs) suitable for a bioassay method were standardised, and used throughout the rest of the work described in this thesis. Two controlled environment bioassay experiments (Chapter 4) were undertaken surveying soils from six sites across the region differing in physico-chemical and biological properties to elucidate the influence of abiotic and biotic factors on plant-soil-pathogen interactions and the potential for suppression of Rhizoctonia. A comparison was made with soil from the long term study site in SA (Avon) reported to be suppressive to Rhizoctonia. Studies growing wheat seedlings in sterilised soils demonstrated that the soils assessed were intrinsically different in terms of the growth supported by the abiotic matrix. Greatest shoot and root dry weight was observed in the soil from a region outside the EP (i.e. Avon) and the least was in an EP soil with extremely high calcium carbonate content (e.g. Streaky Bay) – a clear example of plant-soil abiotic interaction. Avon soil was confirmed as suppressive to Rhizoctonia root rot since the Avon soil inoculated with its own biotic component reduced root infection to 50% from more than 70% in the sterilised abiotic control. Whereas, for plants in the two EP soils with low calcium carbonate root infection was similar in the sterilised abiotic matrix to that in the soils inoculated with their biotic component, suggesting they were not biologically suppressive. Further evidence of the suppressive capability of the biotic component of Avon soil was obtained where it was inoculated into the two EP soils with higher carbonate and reduced root infection in plants grown in these two soils, although not in the lower carbonate content abiotic matrix of Minnipa, another EP soil. Surprisingly, considering the hostile edaphic conditions, root infection was reduced in the high calcium carbonate soil inoculated with its own biotic component, suggesting it was suppressive but not to the same extent as Avon. It was hypothesised this was possibly related to the organic C content in that soil being similar to Avon and higher than the other two EP soils. Shifts in soil organism community structure were observed when plants were grown in sterilised soils inoculated with the biotic component from another soil (i.e. rhizosphere soil from plants grown in another non-sterile matrix). Overall this work suggested there was some biotic potential for suppression in EP soils but low organic C was likely to be a constraint. EP soils were not as suppressive as Avon and abiotic constraints were highly likely, for example, the high carbonate reducing availability of P due to chemical fixation. A long term glasshouse study (Chapter 5) was undertaken to measure the effect of carbon addition to two EP soils, as stubble or young root residues, on the potential to suppress Rhizoctonia. Other measurements in this experiment were microbial biomass carbon and quantitative PCR for DNA of pathogen and other specific micro-organisms implicated as contributing to disease suppression. C input to EP soils suppressed Rhizoctonia infection in wheat seedlings (despite abiotic constraints). C input as young roots increased DNA of Rhizoctonia solani and beneficial soil organisms Microbacterium spp. and Pantoea agglomerans. C input as stubble increased the populations of the beneficial soil organism, Trichoderma spp. A further bioassay experiment (Chapter 6) investigated the effect of N and P fertiliser inputs on plant growth and Rhizoctonia suppression in two EP soils. The bioassay further investigated the interaction of these fertiliser nutrients with added available C in these two EP soils, one of which was highly calcareous. There was a positive plant growth response to added ammonium–N in both soils but no effect on Rhizoctonia infection. Addition of fertilizer P to the highly calcareous soil increased shoot and root growth and also Rhizoctonia infection without compromising effects on plant growth. Addition of available C (sucrose) with P fertiliser in the highly calcareous soil markedly suppressed Rhizoctonia infection. Two final experiments focussed on measuring the changes in pathogen and other microbial communities in response to inputs of fertiliser and C in a highly calcareous EP soil, since Rhizoctonia root rot impacts are considered a particularly big issue in this soil type. In the first experiment (Chapter 7) it was hypothesised that fertiliser P may affect suppression of Rhizoctonia root rot not only via increasing plant growth but also by altering microbial community composition. Results showed that virulence of Rhizoctonia solani was unaltered by P addition although pathogen DNA in soil and plant root infection increased. The effect of P fertiliser on plant growth compensated for the effect of P on increased pathogen population and root infection. Whilst fertiliser P increased microbial activity no shifts were detected in communities so the effects of P on soil organisms involved in suppression of Rhizoctonia root rot were not conclusive. However, in the last experiment (Chapter 8) there were measured shifts in populations of organisms resulting from addition of fertiliser P in conjunction with stubble. The known suppressive soil organisms Pantoea agglomerans and Microbacterium spp. increased whereas Rhizoctonia solani (DNA) remained constant and hence Rhizoctonia infection decreased. In summary, some soils from the EP region of South Australia expressed a degree of suppression to Rhizoctonia root rot via their biotic component in pot culture experiments. Furthermore, some of the soils, although not necessarily the same ones, contained soil micro-organisms implicated by other studies in suppression of Rhizoctonia root rot. The biotic component from some of the EP soils, whilst not suppressive in the soil matrix it was extracted from did demonstrate the potential to suppress Rhizoctonia root rot when transferred into another soil matrix, indicating an abiotic constraint to suppression. It is postulated that important abiotic properties in these EP soils were calcium carbonate content, with organic carbon and to a lesser extent mineral N and P also important since these latter properties bridge the abiotic to biotic divide. Important biotic properties are likely to be microbial activity, microbial community structure and the population of the pathogen, Rhizoctonia solani AG8. Results from this thesis work suggest that suppression to Rhizoctonia root rot can occur in EP soils despite abiotic and biotic constraints of limited C and P. Improvement and maintenance of a high suppressive capacity in soils in this semi-arid environment will require integrated agronomy aimed at maintaining a healthy crop using fertilisers, particularly P. Available carbon appears to be the most limiting constraint to microbe based biological disease suppression of Rhizoctonia root rot in these soils. Therefore it is essential that adequate available C is supplied via stubble input to develop and maintain a highly functioning soil biota. Although these results highlight that disease suppression to Rhizoctonia root rot is indeed possible in the constrained soils of the EP, the time required to develop this suppressive capacity in a field situation remains to be investigated.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013

碩士
大葉大學
分子生物科技學系碩士班
98
Taiwan is located at the tropics and the subtropics, its climate is suited for the melons cultivation, various watermelon species were cultivated in large area. The watermelon is a cucurbitaceae, dicotyledons flowering plant. Watermelon is one of economically important agricultural products in Taiwan. The plant fungal pathogens which cause severe yield losses, still can’t be prevented and controlled without enviromentel pollution. We tried to introduce the anti-fungal protein genes into watermelons to control watermelon diseases. There is still no paper about transgenic watermelon related to anti-fungi ability. This inverstigation were tried to set up an approach of Agrobacterium-mediated transformation of watermelon carrying with Bo-AFP3 or Cp-AFP3. The anti-fungal protein genes, Cp-AFP3 and Bo-AFP3, were kindly supplied by Dr. Jei-Fu Shaw, Academia Sinica. Night putative Bo-AFP3 transgenic lines and six putative Cp-AFP3 transgenic lines were obtained. PCR and Southern blot analysis confirmed that the foreign genes were incorporated into the genic DNA of the regenatants. The transgenic lines were challenge with Rhizoctonia solani by inoculation in vitro, they showed different levels of resistance, ranging from delaying of symptom development to complete resistance. Three transgenic lines were conferred with better resistance against R. solani.

Cotton seedling disease complex is caused by a number of pathogens inducing similar symptoms and can lead to thin, uneven stands that grow slowly and yield poorly. Preliminary work indicated that a set of high tannin cotton lines developed and released in 1989 by Texas A&M AgriLife Research, (Smith et al., 1990a, Smith et al., 1990b, Schuster et al., 1990) may possess increased resistance to disease. This evidence, along with additional studies that show a clear role of tannin in disease resistance, suggest that these high tannin cotton lines may prove useful in breeding for increased resistance to cotton seedling disease complex. High tannin cotton lines were screened for their resistance to Rhizoctonia solani and Pythium aphanidermatum. While no high tannin germplasm line was more resistant to R. solani than the resistant control, Tamcot SP 21, the potential for significant gains from selection was demonstrated. Fifteen high tannin lines expressed resistance to P. aphanidermatum equal to the resistant control, Tamcot Sphinx (El-Zik and Thaxton, 1996). This resistance was not shown to be correlated with tannin content, though it is still unclear whether or not this lack of correlation is real or due to limitations in measuring tannin in infected seedlings.

Wheat is one of the three major cereal crops, alongside rice and maize, which together supply half of the world population's food energy requirement. Wheat production is affected by climatic conditions and biotic factors including weed competition and pathogen attack. Around 18% of yield loss in Australian wheat is attributable to root diseases. Plant defence against root-invading pathogens often involves multiple quantitative resistance genes. Rhizoctonia root rot, caused by 'Rhizoctonia solani' AG 8 (teleomorph 'Thanatephorus cucumeris'), costs Australian farmers around $60 million per annum. The necrotrophic fungus attacks seedling roots, resulting in patches of severely stunted plants. During root invasion R. solani AG 8 secretes a range of enzymes; however specific requirements for pathogenesis are not yet understood. Effective resistance to Rhizoctonia is not available in wheat cultivars, so farmers must rely on management techniques to control the disease. This research aimed to discover genetic resistance to Rhizoctonia root rot in 'Brachypodium distachyon', a grass developed in recent years as a model for cereals. The species has shown strong potential as a model for wheat shoot and crown diseases, as well as cereal root architecture. Two 'B. distachyon' germplasm resources were used in this project: natural accessions collected from Turkey and Spain, and a T DNA insertional mutant collection. 'Rhizoctonia solani' AG 8 produced similar disease phenotypes and severity in 'Brachypodium distachyon' and wheat. A method developed to screen for disease resistance in the 'B. distachyon' collections incorporated toothpick baits to check for the presence of inoculum and contamination in pots. The major indicator of disease severity was reduced root length, with leaf lengths and plant development rate being secondary symptoms. Resistance of different lines was ranked based on the ability of plants grown in infested soil to maintain root and shoot measurements similar to uninfested control plants. Twenty-six genetically diverse natural accessions and 25 selected T-DNA lines of 'B. distachyon' were included in replicated experiments to screen for variation in resistance to 'R. solani' AG 8. Greatest variation in resistance to was identified in the natural accession collection. Root length of the least resistant line was reduced to 19% of the control in 'R. solani' infested soil, while the most resistant line maintained 53% of control root length. This difference is similar to quantitative resistance levels in other grasses and wheat mutant lines described in the literature. Exploring potential resistance mechanisms, nodal root emergence in response to infection correlated with greater resistance in an experiment with natural accessions. Increased endogenous seedling vigour was linked with lower resistance, but this factor alone did not explain all variation in resistance. Further work is required to validate increased resistance associated with a T-DNA tagged gene, Bradi3g14370, that encodes a putative beta-1,3-galactosyltransferase. 'Brachypodium distachyon' was found to be a useful model pathosystem for wheat root diseases. The variation in genetic resistance to 'R. solani' AG 8 described in the natural accession collection provides a basis for further work to discover genes involved in resistance to this pathogen in 'B. distachyon' and subsequently in wheat.

Xanthomonas axonopodis pv. malvacearum(Smith), Pythium aphanidermatum(Edson) and Rhizoctonia solani(Kuhn) have all been shown to cause significant yield losses in cotton. Previous work has demonstrated that a set of high tannin cotton germplasm lines developed and released in 1989 by Texas A&M AgriLife Research may possess resistance to these three diseases. In this research, the usefulness of these high tannin lines in breeding for resistance to these pathogens as well as the role of tannin in conferring this resistance were examined. The high tannin lines were screened for their resistance to Xanthomonas axonopodis pv. malvacearum and five highly resistant lines were subjected to generation means and diallel analysis in order to determine the relative importance of different types of gene action in conferring resistance as well as which lines would be most useful in breeding for resistance. The effect of selection for R. solani resistance was measured and selected lines were subjected to diallel analysis. Lines showing elevated resistant to P. aphanidermatum were subjected to a single cycle of selection and the effect of selection was measured. Lines possessing high degrees of P. aphanidermatum resistance were subjected to generation means and diallel analysis. The importance of tannin content in conferring resistance to X. axonopodis and R. solani was also measured. Four high tannin lines were found to be not different from Tamcot Sphinx in their resistance to Xam. Generation means analysis for Xam resistance showed that in the five high tannin lines tested, additive gene effects were of the greatest importance. No correlation between tannin content and Xam resistance was detected. Under the conditions of this study tannin content was also shown to be unimportant in R. solani resistance. Two rounds of recurrent phenotypic selection were effective in increasing the resistance of selected HT lines to R. solani. One cycle of phenotypic selection for P. aphanidermatum resistance was found to produce significant improvements in seven of the most resistant HT lines. Generation means analysis for P. aphanidermatum resistance showed that in the HT lines tested, additive genetic effects were by far the most important.

碩士
國立中興大學
植物病理學系
93
柒、英文摘要 The non-sterile propagation and mode of action of Streptomyces griseobrunneus S3 for controlling diseases caused by Pythium aphanidermatum and Rhizoctonia solani AG4. Chen Tai-Yuan The main objectives of this investigation were to establish a non-sterile propagation system (NSPS) for biological control application of Streptomyces griseobrunneus S3 (SGS3) and to explore the mode of action of the disease control observed. The tested biological control agent SGS3, originally isolated by Lai (2003) from citrus rhizosphere, has been shown having great potential for biofungicide application. The main attributes known pertaining to its effectiveness as biocontrol agent included the excellence in chitin and β-1,3-glucan degradation, and as well the antibiotic and sporulating activities. And for the biofungicide application, a pilot scale liquid fermentation technique platform was established; the yield of spore suspension of SGS3 reached 5x1011 cfu/ml. The establishment of a NSPS discussed in this investigation was aimed to provide a grower do-it-yourself (DIY) system for the mass propagation and reactivation of the fermenter produced spore biomass right before application and thus to reduce the cost of the biofungicide application. In a preliminary screening trial where that sterile culture system was applied, the provision of wheat bran at 3% was found the best among the tested grain varieties as natural substrate for supporting the spore production of SGS3 in a broth culture system. Urea at 0.2% was found to be an appropriate nitrogen supplement for boosting up the antibiotic activity of the produced broth culture. For the serial trials conducted to establish the NSPS technique, the test bacterium was cultured with the test growth medium without autoclave sterilization. In a NSPS broth culture using wheat bran as the major growth substrate, the growth of SGS3 appeared to fluctuate during the culturing period mainly because the presence of certain microbial contaminants. A 5-minute boiling treatment of the wheat bran broth medium was found helpful in reducing the interference of the contaminating microbes thus provided significantly increased yield. However, with the optimization of the cultural substrate formulation, the applied inoculants, and the buffer capacity of the growth medium, the interference of the contaminating microbes became non-significant and the boiling treatment appeared not necessary. In an optimized cultural condition using wheat bran broth as major nutrient constituent, the growth of SGS3 was greatly improved by the addition of 100 mM K2HPO4 and 0.1% oyster shell powder; the spore yield of SGS3 reached approximately 2x1012 cfu/ml 3 days after incubation. The stimulatory effect of K2HPO4 addition appeared to be associated with the buffer capacity provided. In addition to SGS3, the optimized NSPS established appeared to work for Streptomyces sp. S1, Streptomyces sp. S4, Streptomyces saraceticus S31; the spore yield all reached 1012 cfu/ml level except that of S31 which yielded only to 1010 cfu/ml. The usefulness of the established system in Streptomyces sp. biofungicide application was clearly indicated further by the fact that the disease control effectiveness provided by the NSPS broth culture was equibalent to that by the original fermenter produced culture broth. For seedling damping offs caused by Pythium aphanidermatum and Rhizoctonia solani AG4, effective disease control was demonstrated. By drenching treatment with SGS3 broth cultures at 100X in dilution 2 days (for P. aphanidermatum) to 4 days (for R. solani AG4) before seeding, the percent infection of the two diseases was reduced by 77 to 64%, respectively, as compared to the water treated control. A comparable disease control effectiveness was also demonstrated when SGS3 was applied by seed coating. What worth to mention is that when the pre-seeding drenching treatment was combined with seed coating, the percent infection of both targeted diseases was reduced over 81%; the protective effect was equivalent to that by the compared chemical treatment- metalaxyl (applied at 350 g a.i./ml for P. aphanidermatum) and pencycuron (applied at 250 g a.i./ml for R. solani AG4). The effectiveness of disease control appeared to be dosage dependent which relies primarily on the presence of SGS3 spore propagules. The role of metabolites present in the broth culture (i.e. cultural filtrate) appeared to be minor as regards to the disease control demonstrated although its presence did contribute some additive effect in counteracting the fungal invasion. Upon artificial inoculation of SGS3, the microscopic examination revealed that the applied bacterial spores germinate readily, grew on, coiled up and even penetrated the mycelia of P. aphanidermatum and R. solani AG4. The mycoparasitism led to rapid increase of electrolytes from the host fungal mycelia, and the parasitized mycelia appeared to be killed within 24 hours. The mycoparasitic effect was manifested by the formation of eroded concave lesion reflecting the functioning of chitin/glucan degrading enzymes; the activity was greatly stimulated by the addition of chitin whereas was repressed by the addition of glucose. The killing of target fungi, as revealed by fluorescen diacetate/propidium iodide vital staining, was detected before dismantling of the fungal cell became apparent indicating the involvement of antibiotics during the process. The fast and lethal mycoparasitic activity well illustrated how SGS3 helps plants fight with the fungal pathogens. As for the disease control, the effect of SGS3 application on the disease resistance was investigated using tomato as a model. An enhanced expression of PR-1 (pathogenesis related protein 1) gene, indicating the induction of resistance gene expression, was detected consistently from foliar tissue after drenching treatment of SGS3. As the induced PR-1 gene expression was detected from the culture broth treated rather than the culture filtrate treated plants, the existence of SGS3 propagules again appeared to be critical. The data herein presented indicates clearly the disease control by SGS3 is a result of multiple mode of action including mycoparasitism, antibiotic activity, and induced resistance. The effective disease control equivalent to that by chemical fungicides was demonstrated. The success of NSPS development warrant the innundative application of the viable, active SGS3 propagules in practical application.