Tesis sobre el tema "Biology, Molecular|Biology, Microbiology|Agriculture, Plant Pathology"

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.

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.

Plant endogenous small RNA pathways generate non-coding regulatory RNAs that regulate gene expression through target mRNA cleavage, translation inhibition or chromosomal modifications. Regulation of small RNAs and their targets during pathogen infection is tightly controlled to promote defensive mechanisms against disease progression. The oomycete pathogen, Phytophthora sojae is a principal infectious agent of soybean. To date, there is limited information on small RNAs that regulate defense responsive genes against P. sojae .

Infection response in plants is evidently regulated in part by small RNAs. High-throughput sequencing of small RNA libraries constructed from P. sojae-infected and mock-infected soybean roots and subsequent computational analysis revealed approximately 324 known soybean miRNAs and 109 potential novel soybean miRNAs that differentially accumulate between the P. sojae-infected and mock-infected samples. Of these, 8 conserved miRNAs and 2 novel miRNAs were verified by Northern blot analysis. Targets of the miRNAs displayed abundance changes respective to their complementary miRNA's levels.

The down-regulation of the conserved miR393 by target mimicry points to a positive regulatory role for miR393 during pathogen response. In addition, we noted the induction of miRNA-directed expression of phasiRNAs from multiple NB-LRR loci. These results indicate a pool of miRNAs specific in responding to P. sojae infection. Our study identified multiple conserved and novel soybean miRNAs with potential defensive roles against P. sojae. Our data demonstrates that plant response to pathogen infection is complex and multi-layered. Further study of small RNAs involved in defense regulation may contribute to combating Phytophthora diseases.

The ascomycete fungus Magnaporthe oryzae, causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and M. oryzae is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made M. oryzae an ideal model system for understanding general plant-pathogen interactions as well.

The objective of this dissertation was to characterize the M. oryzae homologs of Histoplasma capsulatum RYP ( Required for Yeast Phase ) genes that are required for transition to the parasitic phase. H. capsulatum is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37°C, the host body temperature. FourH. capsulatum RYP genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. RYP1 is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. RYP2 and RYP3 are homologs of vosA and velB, members of the Velvet family, best characterized in Aspergillus nidulans, where they coordinate morphological differentiation with secondary metabolism. RYP4 is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the M. oryzae RYP1 homolog, RIG1 ( Required for Infectious Growth ), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. Δrig1 was blocked in the transition to infectious hyphal growth, similar to H. capsulatum ryp1, which could not transition to the yeast phase. Deletion mutants of M. oryzae RYP2, RYP3, and RYP4 homologs were similar to the wild type in somatic growth and pathogenicity indicating that although RIG1 is a pathogenicity factor conserved in plant and animal pathogens, such conservation does not apply to all of the RYP pathogenicity genes identified in H. capsulatum.

Δrig1 is the first M. oryzae mutant known to be blocked in production of primary infection hyphae. Overall, the study suggests limited parallels exist in phase transition of fungal pathogens of plants and animals.

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.

Butternut (Juglans cinerea L.), or white walnut, has suffered large population declines in the past half-century due to poor regeneration and mortality caused by an introduced fungus, Ophiognomonia clavigignenti-juglandacearum (Nair, Kostichka & Kuntz) Broders & Boland. This fungus causes branch and trunk cankers that can coalesce to girdle adult trees. Chapter 1 provides background information on butternut and butternut canker. We used next-generation sequencing to identify new nuclear DNA markers for butternut and Japanese walnut, a congener with which butternut readily hybridizes. We also examined the alignment of SSR repeat sequences in butternut and Japanese walnut with similar sequences from other angiosperms in public sequence databases. The methods used and results obtained in this process are detailed in Chapter 2. Chapter 3 summarizes an investigation of the environmental and genetic factors contributing to canker disease incidence, severity, and mortality in a large (n=113) population of butternut in southern Wisconsin and two other populations of butternut, one near the main study site in southern Wisconsin and another in the Great Smoky Mountains National Park. We present evidence for weak correlations of genetic similarity and phenotypic similarity for several disease traits, parentage analysis of regeneration in the smaller Wisconsin population, and evidence for significant microsite influences on butternut mortality over an 11-year period in the large Slocum's Woods butternut population.