Nod factors, lipo-chitooligosaccharides (LCOs), are rhizobial signal molecules important in the establishment of nodule formation, leading to atmospheric dinitrogen fixation in legume-rhizobium symbioses. Recently, LCOs were also found to regulate other plant processes. We demonstrated that, at 10 -6 M, four LCOs produced by Bradyrhizobium japonicum enhanced soybean seed germination. Evaluation of G-protein inhibitors showed that U-73122, a phospholipase C inhibitor, also increased soybean seed germination, similar to the increase by LCO NodBj-V(C18:1 MeFuc), indicating different mechanisms for the plant perception to LCOs for nodule initiation and seed germination. This was confirmed as LCOs were not able to break dormancy of skotodormant lettuce seeds. Soybean early seedling growth was also increased by the application of LCOs. Pulse 14Ca2+ experimentation showed that the increase might also be related to an increase in Ca 2+ uptake by shoots. We confirmed this with both genistein-induced and non-induced B. japonicum 532C; however, strain 168 (a mutant unable to produce LCO) and non-host rhizobia (Rhizobium leguminosarum, Sinorhizobium meliloti), did not increase Ca2+ uptake. Addition of 1.6 g L-1 casein hydrolysate in yeast extract mannitol broth drastically increased bacterial growth and increased volume-basis LCO production, but decreased LCO production per cell. Best conditions for sterilizing and storing LCOs were determined. LCO should be sterilized by using polyestersulfone filter or autoclaving for up to 30 minutes. LCO was degraded faster when stored at room temperatures (23 +/- 2°C) than low temperature (4 +/- 1°C) and can be stored more than one year.

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), collectively termed as jasmonates, are naturally occurring in plants and are important signal molecules involved in induced disease resistance and stress responses of plants. Besides their role in-planta, they are also rhizosecreted by root cells. Germinating soybean seeds exude large quantities of jasmonic acid; however there is no knowledge regarding how jasmonates influence cells of the soybean symbiont, Bradyrhizobium japonicum, in the rhizosphere. We studied the role of jasmonates in the soybean-Bradyrhizobium symbiosis. Bradyrhizobium japonicum cultures were induced with jasmonates and the resulting Nod factors were isolated and purified. Our results showed that JA and MeJA strongly induced the production of Nod factors by the tested B. japonicum strains. When added together, genistein and jasmonates resulted in greater LCO production than either one alone. Jasmonic acid is produced from linoleic and linolenic acids via the octadecanoid pathway; we studied the effect of these two fatty acids on B. japonicum nod gene induction. Interestingly both linoleic and linolenic acids induced the nod genes and caused LCO production by B. japonicum cultures. Since jasmonates induced the nod genes and also caused LCO production in B. japonicum, I conducted experiments in the greenhouse and field to determine whether incubation of B. japonicum with JA or MeJA prior to inoculation increases soybean nodulation variables and grain yield. Both genistein and McJA increased nodule number and nodule dry weight per plant. Due to enhanced nitrogen fixation, attributed to increased nodule number and weight, soybean dry matter accumulation and grain yield were increased. These results document the discovery of jasmonates and their precursors as new signal molecules in the Bradyrhizobium - soybean nitrogen fixing symbiosis.

Examensarbete.

Submitted by MARCOS LEANDRO TEIXEIRA DE OLIVEIRA (marcosteixeira@ufv.br) on 2018-09-18T13:33:07Z No. of bitstreams: 1 texto completo.pdf: 2023955 bytes, checksum: c00bada9d7cdc721128642c40a1fc2a1 (MD5) / Made available in DSpace on 2018-09-18T13:33:07Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2023955 bytes, checksum: c00bada9d7cdc721128642c40a1fc2a1 (MD5) Previous issue date: 2017-03-31 / Na natureza, um fitopatógeno geralmente está sob influência de um complexo de microrganismos, que associados garantem o equilibrio ecológico estável. Com foco no fungo nematófago Pochonia chlamydosporia, começamos investigando sua compatibilidade com Bradyrhizobium japonicum em soja e a influência dessa interação sobre o controle do nematoide das galhas e sobre a absorção de nutrientes pela planta. Constatou-se que a aplicação conjunta desses dois organismos não prejudica a ação de controle de nematoides por P. chlamydosporia e nem a fixação de N 2 por B. japonicum. Adicionalmente, quando os dois agentes estavam juntos, ocorreu maior produção de nódulos da bactéria nas raízes e aumento do conteúdo de Fe na parte aérea das plantas de soja. No segundo capítulo, a compatibilidade dos agentes de controle biológico de nematoides, P. chlamydosporia e P. penetrans foi avaliada. A co-aplicação dos microrganismos possibilitou maior redução do número de ovos do nematoide do que a aplicação em separado e foi possível observar a associação do fungo nas raízes infectadas por nematoides colonizados por P. penetrans. Pesquisamos ainda, no terceiro capítulo, a presença de vírus em isolados de P. chlamydosporia. Dos dezoito isolados avaliados, apenas um (Pc-M4) estava infectado por dois virus, um com genoma de RNA fita dupla (dsRNA) e outro de RNA fita simples sentido negativo (-ssRNA). Os nomes propostos para os micovírus são Pochonia chlamydosporia chrysovirus 1 (PcCV1) e Pochonia chlamydosporia negative-stranded RNA virus 1 (PcNSRV1). Ensaios biológicos do isolado fúngico Pc-M4 revelaram que este foi capaz de parasitar ovos, produzir metabólitos e proteases letais aos juvenis e reduzir a reprodução do nematoide de galhas M. javanica. Os resultados apresentados indicam que o fungo P. chlamydosporia é capaz de interagir com diferentes organismos sem perder a capacidade de controlar o nematoide de galhas e de promover o crescimento vegetal, o que faz dele um excelente agente de biocontrole de nematoides. / In nature, a plant pathogen is usually under the influence of a complex of microorganisms, which guarantees stable ecological balance. Focusing on the nematophagous fungus Pochonia chlamydosporia, we started investigating its compatibility with Bradyrhizobium japonicum in soybean. We also looked at the influence of this interaction on the control of the root knot nematode and on the nutrient uptake by the plant. It was found that the joint application of these two organisms does not affect the action of control of nematodes by P. chlamydosporia nor the fixation of N 2 by B. japonicum. In addition, when the two agents were together, there was a higher production of nodules of the bacteria in the roots and an increase in the Fe content in the aerial part of the soybean plants. In the second chapter, the compatibility of biological control agents of nematodes, P. chlamydosporia and P. penetrans was evaluated. The co-application of the microorganisms allowed a greater reduction in the number of nematode eggs than the separate application. It was possible to observe the association of the fungus in the roots infected by nematodes colonized by P. penetrans. We also investigated the presence of viruses in isolates of P. chlamydosporia in the third chapter. Of the eighteen isolates evaluated, only one (Pc-M4) was infected by two viruses, one with double-stranded RNA genome (dsRNA) and the other with RNA single-stranded sense negative (-ssRNA). The proposed names for the mycoviruses are Pochonia chlamydosporia chrysovirus 1 (PcCV1) and Pochonia chlamydosporia negative-stranded RNA virus 1 (PcNSRV1). Biological assays of the fungal isolate Pc-M4 revealed that it was able to parasitize eggs, produce metabolites, lethal proteases to juveniles and reduce reproduction of M. javanica. The results indicated that the fungus P. chlamydosporia is capable of interacting with different organisms without losing the ability to control the root knot nematode and to promote plant growth. This makes it an excellent biocontrol agent for plant parasitic nematodes.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21152号 / 農博第2278号 / 新制||農||1059(附属図書館) / 学位論文||H30||N5126(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 奥本 裕, 教授 白岩 立彦, 教授 冨永 達 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Maximizing productivity and profitability are the primary reasons for double-cropping soybean with small grain in the Mid-Atlantic, USA. Reduced double-crop yield can be attributed to: delayed planting that results in a shortened growing season and less vegetative growth; later-maturing cultivars that terminate main stem growth after flowering and have less growth and nodes; less soil moisture and plant-available nutrients due to small grain uptake; greater air and soil temperatures during vegetative stages that reduce early-season growth; and more favorable conditions for disease development during pod and seed formation. Field experiments were conducted in 2012 and 2013 in eastern Virginia to 1) evaluate cultivar stem growth habit, seeding rate, seed-applied inoculant, starter nitrogen (N) applied at planting, and foliar fungicide on soybean vegetative growth, total N uptake (TNU), seed yield and quality, and yield components; 2) determine the effect of starter N rate, applied with and without inoculant, on soybean vegetative growth, TNU, seed yield and quality, and yield components; and 3) evaluate the response of maturity group (MG) IV and V soybean cultivars to foliar fungicide. Greater seeding rates, inoculant, N, and fungicide typically were not required together to increase yield. Although cultivar interacted with other factors, early-maturing indeterminate 95Y01 yielded more than late-maturing determinate 95Y20 at 4 of 6 locations. Seeding rate interacted with other factors, but the greater seeding rate increased MG IV yield at 1 of 6 locations and decreased MG V yield at 2 of 6 locations. Starter N increased seed yield by 6 kg ha-1 per kg N applied until yield plateaued at 16 kg N ha-1, which continued to 31 kg N ha-1. When N rate was increased greater than 31 kg N ha-1, yield decreased. Fungicide increased yield for MG IV and V cultivars at 4 of 6 and 3 of 6 locations, respectively and prevented yield loss via mid- to late-season disease control, delayed leaf drop, and greater seed size. Optimum fungicide timing depended on environment and disease development. These data assisted in understanding agronomic inputs' combined or individual effects on double-crop soybean growth, canopy, N uptake, seed yield, and yield components. / Ph. D.

Infecções fúngicas em humanos vem aumentando nos últimos anos e acometem principalmente pacientes imunocomprometidos, portadores do vírus HIV, transplantados ou com câncer. Os antifúngicos empregados no tratamento pertencem a poucos grupos de fármacos e o aparecimento de resistência antifúngica em muitos patógenos leva à necessidade de desenvolvimento de novos agentes antifúngicos. As cepas Trichosporon japonicum QU139 e Candida catenulata LV102 apresentam atividade killer sobre diversas leveduras patogênicas, apresentando bom potencial para desenvolvimento de novos agentes antimicrobianos. O objetivo do trabalho foi a otimização das condições para produção e detecção dos compostos antimicrobianos, para seu futuro uso terapêutico, e sua purificação. O efeito killer da cepa T. japonicum QU139 foi avaliado pelo método dos poços contra células sensíveis de Cryptococcus gattii C20 nos meios GYP, YM e Queijo em diferentes pH e temperaturas. A máxima atividade killer foi encontrada no meio GYP, pH 4,5 à 25°C após 24 horas de incubação para T.japonicum QU139 e C. catenulata LV102. Não foi possível isolar o composto antimicrobiano produzido pela levedura T.japonicum QU139 pelos métodos de isolamento de proteína/glicoproteína, corroborando a hipótese de que a toxina seja um glicolipídeo. / Human fungal infections have increased in the last years and affect mainly immunocompromised patients, carriers of HIV vírus, transplanted or with cancer. The antifungal agents used in treatment belong to a few groups of drugs and the increase of antifungal resistance in many pathogens leads to the necessity of developing new antifungal agents. Strains Trichosporon japonicum QU139 and Candida catenulata LV102 showed killer activity against several pathogenic yeasts, having a good potential for the development of new antimicrobial agents. The objective of the work was the optimization of conditions for production and detection of the antimicrobial compounds, aiming their future terapeutic use, and their purification. The killer effect of T. japonicum QU139 strain was evaluated by the well method against sensitive cells of Cryptococcus gattii C20 in media GYP, YM and Cheese in different pH and temperatures. The maximum killer activity was found in media GYP, pH 4.5, 25°C after 24 hours of incubation for T.japonicum QU139 and C. catenulata LV102. The isolation of the antimicrobial compound produced by the yeast T.japonicum QU139 was not possible by the methods for isolation of proteins/glicoproteins, corroborating the hypothesis that the toxin is a glycolipid.

Introduction Schistosomiasis is one of the most important and widespread diseases of rural poverty. Worldwide, approximately 779 million people are at risk of infection, with 207 million already infected. Infections with Ascaris lumbricoides, hookworms and Trichuris trichiura, collectively known as the soil-transmitted helminths (STHs), are also highly endemic throughout the tropics, particularly in resource-poor settings. An estimated 1 billion people worldwide are estimated to be infected with STHs. Schistosomes and STHs often co-exist in the same epidemiological settings and, given the high prevalence of concurrent multiple species infections (multiparasitism), a combined approach to prevention and control could lead to significant improvements, including reducing costs associated with single-species control programmes. In China, rigorous national schistosomiasis control efforts over the past 60 years have decreased the prevalence by over 90%; however, since 2000 the number of infected individuals has been rising, possibly indicative of a re-emergence. Fishermen, migrant communities and poor households in rural areas may be most at risk of single and multiple-species parasitic infections and the associated morbidity. Objectives This Ph.D. thesis is structured according to four main goals and a number of specific objectives: Firstly, to update estimates of S. japonicum prevalence, intensity and associated morbidity levels in Hunan province, China, according to the third national PES carried out in 2004; Secondly, to investigate existing barriers in access to preventive, diagnostic and treatment services for advanced schistosomiasis; Thirdly, to compare and evaluate direct and proxy methods of measuring household socio-economic position, according to data on income, savings and asset-based estimations of wealth; and Finally, to explore and identify behavioural, demographic, economic, environmental and social risk factors associated with the distribution of S. japonicum, STHs and multiple species infections, in two villages of the Dongting Lake region, Hunan province, China. Methods Firstly, we used data from the third national schistosomiasis periodic epidemiological survey (PES) of 2004. In Hunan province, the PES was carried out in 47 villages of the endemic Dongting lake area. A total of 47144 human serological, 7205 stool, and 3893 clinical examinations were performed. For the reservoir hosts, stools from 874 buffaloes and other domestic animals were examined for schistosomiasis by the miracidial hatching test. Secondly, we conducted an in-depth study involving interviews with 66 schistosomiasis control staff and 79 advanced schistosomiasis patient, and six focus group discussions (FGDs), in the Dongting lake region, between August 2002 and February 2003. Using the Health-Access Livelihood framework we examined availability, accessibility, affordability, adequacy and acceptability of schistosomiasis control in the Dongting lake area. Lastly, we carried out two village-wide parasitological, clinical and questionnaire-based investigations between October and December 2006. Parasitological examinations for the prevalence of S. japonicum and the STHs were performed by the Kato-Katz thick smear method, with repeated sampling of each individual. We took fingerprick blood samples to assess haemoglobin levels, using a B-haemoglobin HemoCue photometer. The household-based questionnaire focused on direct and proxy measurements of household wealth, while the individual-based questionnaire focused on demographic and behavioural factors, treatment history and self-perceived symptoms. Results Human sero-prevalence was 11.9% (range: 1.3-34.9% at village level), and the rate of egg-positive stools was 1.9% (0-10.9%) for the same population. The prevalence of infection among buffaloes was 9.5% (0-66.7%). Extrapolating to the entire population of the Dongting Lake region, an estimated 73225 people and 13973 buffaloes were infected. Most frequently reported symptoms were abdominal pain (6.2%) and bloody stools (2.7%). Accessibility and affordability were major barriers in access to schistosomiasis control. Many of staff interviewed indicated that a majority of patients who develop advanced schistosomiasis resided in mildly-endemic or non-endemic settings. None of the patients interviewed had any form of health insurance, and most of their health expenses were out-of-pocket payments. Exploratory factor analysis generated internally robust proxy wealth indices, however these were not complementary to direct measures of household wealth, as indicated by low correlation co-efficients. We found wide disparities in household ownership of durable assets, utility and sanitation, within both settings. Pooled data from the rural and peri-urban settings highlighted structural differences in socioeconomic position (SEP), more likely a result of localised urbanization and modernization. We found higher infection prevalences in rural settings, than in peri-urban settings, for schistosomiasis (6.3% and 6.7% respectively), ascariasis (8.3% and 2.2%, respectively) and trichuriasis (5.1% and 0.5%, respectively), but lower for hookworms (0.1% and 1.5% respectively). Multiple species infections (2.6% and 0.2%, respectively) were less prevalent than single species infections (14.5% and 10.4%). There were significant disparities in the prevalence of parasitic infections between poorest and least poor quintiles of the cohort population. Anaemia and other symptoms, especially headache, stomach ache and swollen stomach, were common in both rural and peri-urban village settings. Conclusion The studies conducted within the framework of this Ph.D. thesis document the current situation pertaining to schistosomiasis and the STHs in Hunan province, China. Our findings highlight the need for increased surveillance, monitoring and health education, with relation to schistosomiasis and STHs, in non-endemic or post-transmission control settings. Based upon these results, we call for improved diagnostic tools, particularly in the case of low intensity infections and for hookworm, and propose an extension of the use of available infrastructure, human resources, knowledge and technology by integrating prevention and control of schistosomiasis with that of other intestinal helminths, particularly STHs. In the future, our studies may form a base from which to further examine local needs and priorities for parasitic disease control in the area.