Tesis sobre el tema "Plant microbiology"

Aspergillus flavus infection of agriculturally important crops such as tree nuts, maize, peanuts, and cotton has decreased crop value. Researchers have identified three major approaches to combat A. flavus growth and aflatoxin accumulation: identifying natural resistance in crops, genetically engineering crops for enhanced resistance, and introducing an atoxigenic fungal strain as a competitor. In this dissertation, I investigated two of the three means to control A. flavus growth and infection: genetically engineered crops and identification of natural resistance. My studies of natural resistance in cotton crop show that Sa 1595, a Gossypium hirsutum cultivar, is significantly more susceptible to A. flavus infection; however, no significantly resistant cultivars were observed, but I did observe a trend of diminished susceptibility in A2 186 and Tamcot Sp 23. I then examined synthetic antimicrobial peptide, D4E1, as a means to increase resistance in crops. My research shows that D4E1 effectively increases reactive oxygen species (ROS), an apoptosis precursor at concentrations as low as 1 µM. Breaches in the membrane that allow infiltration and subsequent fluorescence from Sytox^® green occur at higher concentrations. Finally, genetically engineered tobacco plants were examined for D4E1 localization. My research shows that the HA-D4E1 construct was present in the most abundance in the chloroplast of plastid transformed plants, while nuclear transformed plants had nuclear localization. All of my findings suggest that cotton crops do not exhibit any significant enhanced natural resistance to A. flavus infection and growth; however, engineering crops with D4E1 will exhibit enhanced crop resistance.

Legume crops are significant agriculturally and environmentally for their ability to form symbiosis with specific soil bacteria capable of nitrogen fixation. Nitrogen fixation for a given legume in a given soil is limited by the availability of the plant’s bacterial partners, and by variation in the effectiveness of those symbionts. We used a global-level hierarchical sampling scheme to comprehensively characterize the evolutionary relationships and distributional limitations of nitrogen-fixing bacterial symbionts of the legume crop chickpea. This has been accomplished using culture-dependent and independent approaches to generate over 1,200 draft whole-genome assemblies at the level of bacterial populations, as well as 14 finished-quality genomes using the Pacific Biosciences platform. These strategies reveal that chickpea’s symbionts across the globe are confined to the genus Mesorhizobium , but a diversity of taxa within the genus (chapter 1 and 3). Comparative phylogenomic analysis reveals that despite chickpea’s symbionts within and across regions coming from different taxa, all share almost identical genes for symbiosis. PacBio genome-assemblies reveal that this is due to the horizontal transfer of a 500 kb chromosomal island known as a symbiosis island, between unrelated strains of the genus Mesorhizobium . Analyzing the symbiosis island at the population level reveals that the symbiosis island spreads repeatedly once introduced to a region, suggesting that strains well-adapted to a particular soil climate continue to dominate once the new host (chickpea) has been introduced, through repeated acquisition of the symbiosis island. This dataset provides additional insights into the functional and taxonomic diversity of other bacteria associated with chickpea nodules (chapter 2).

Developing maize kernels are vulnerable to colonization by microbes. When colonization allows proliferation of the microbe at the expense of the host, disease occurs. The ascomycete fungal pathogens Aspergillus flavus and Fusarium verticillioides are capable of colonizing maize kernels, causing ear rots and contamination of the kernel with mycotoxins. These diseases lead to significant losses of crop yield and quality, and constitute a threat to food safety and human health. Thus, the significance of these diseases has prompted extensive research efforts to understand these plant-parasite interactions. However, pathogenesis and resistance mechanisms remain poorly characterized, hampering the development of effective control strategies. No commercial maize lines are completely resistant to these fungi. We applied an integrated approach consisting of histology, in situ gene expression and transcriptional profiling to better understand the nature of the interactions that occur between maize kernels and these fungi. Maize inbred line B73 was hand pollinated and inoculated with either A. flavus or F. verticillioides by wounding the kernel with a needle bearing conidia. Histological staining of the kernel sections revealed fungal mycelium in kernels adjacent to the inoculation site by 48 hours post inoculation (hpi), and in all tissues at 96 hpi. Compared with F. verticillioides, A. flavus more aggressively colonized kernel tissue and formed a unique biofilm-like structure around the scutellum. Transcriptome profiling using RNA-sequencing (RNA-seq) coupled with pathway analysis showed that these fungi were recognized by the kernel tissues prior to visible colonization. Infection of the kernel by these fungi induced transcriptional changes in defense-related genes, hormone signaling networks, as well as primary and secondary metabolism pathways. To dissect tissue-specific responses of the kernel, RNA in situ hybridization and histological staining were carried out in adjacent serial sections. We found that two maize genes, pathogenesis related protein, maize seeds (PRms) and shrunken-1 (Sh1) , were expressed in the aleurone and scutellum during infection by these fungi. By staining the adjacent sections, we found that these genes were induced in the tissue before the establishment of fungal colonization. Integration of histology, in situ gene expression and transcriptional profiling to study pathogenesis of maize kernels by these two fungi revealed distinctive and common features between the two pathosystems, and provided information that will facilitate the development of resistance genotypes in maize.

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

Fusarium wilt of cotton, caused by the soilborne fungus Fusarium oxysporum f. sp. vasinfectum, is a widespread disease occurring in most cotton-growing regions of the world. Fusarium wilt occurs in all domesticated cotton. Currently, six nominal races are recognized: 1, 2, 3, 4, 6, and 8, as well as many un-named genotypes worldwide. Many are widespread in the U.S., but race 4, which is highly virulent, is apparently restricted to California. Race 4 is found in an increasing number of fields in California due in part to seed-borne dissemination. The first aim of this study was to evaluate the efficacy of hot water treatments alone or in conjunction with fungicides and other treatments to reduce the viability of FOV race 4 in infected cotton seed. The second aim was to develop and evaluate a rapid and reliable molecular diagnostic assay, the AmplifyRP^® Acceler8™, for the direct detection of FOV race 4 in cotton tissue. In the seed treatment assay, a 1 hour immersion of seed in water or sterile 30% potato dextrose broth (PDB) at 24°C followed by a 20 minute immersion in a 60°C solution containing four fungicides (azoxystrobin, fludioxonil, thiabendazole, and thiophanate) or thiophanate alone were the most effective pretreatment-treatment combinations in reducing FOV in seed and avoiding loss of seed germination and vigor. The incidence of FOV in the seed was reduced by approximately 86% without reducing seed germination and vigor based on recovery of the fungus on petri plates and greenhouse grow-out assays. FOV was completely eliminated from infected seed when the seed was pretreated in water at 24°C followed by a 20 minute immersion in a solution of thiophanate heated to 70°C. With this treatment, seed germination was reduced by 36% and vigor was reduced by 38%. The AmplifyRP^® Acceler8™ diagnostic assay consistently detected FOV race 4 from all infected tissue samples. The test is rapid, simple and more sensitive than conventional PCR. The AmplifyRP^® Acceler8™ diagnostic assay detected DNA from FOV race 4 at concentrations of 1 ng/µL and above. In addition, it did not amplify DNA from other known FOV races (races 1, 2, 3, 6, and 8). The whole process from sample preparation to reading the results was completed in as little as 30 minutes. The test detected FOV race 4 in cotton taproots, petioles, and stems.

Pediomelum esculentum (commonly prairie turnip) is a perennial legume of the Great Plains, consisting of a deep taproot and large edible tuber, and has served as a nutritious staple in Native American diets. The tuber is capable of storing up to 20 percent protein by weight. P. esculentum is a legume, but not a prominent nodule former; instead, it grows in nitrogen-limited soils and produces large amounts of protein. This suggests the involvement of biological nitrogen fixation. We have investigated the presence of diazotrophic endophytes in P. esculentum. Bacteria were isolated from wild plants on nitrogen free media, identified with their partial 16S rRNA gene sequences, and screened for the presence of the nitrogen fixation gene nifH. Select isolates were applied as a co-inoculum to seedlings grown under gnotobiotic conditions in a growth chamber with no nitrogen source. Seedlings in both the inoculated and uninoculated group developed nodules and showed no signs of nitrogen stress. Bacteria isolated from the nodules and tubers of both groups were closely related to the same Bacillus bacterium isolated from seeds germinated under sterile conditions, according to partial 16S rRNA sequences. Bright field and fluorescence imaging revealed bacteria present in the intercellular space of four-week-old tubers and in the sterile germinated seeds. Sectioning and imaging of the nodules show a central nodule vasculature and infected cells extending inwards to the main root vasculature. Nitrogen fixation in the plants was indirectly confirmed by acetylene reduction. Our results suggest P. esculentum has formed a unique symbiosis with a nitrogen fixing Bacillus bacterium that transmits vertically in the seeds and induces nodules.