Doctor of Philosophy / Department of Agronomy / Tesfaye Tesso / Sweet Sorghum [Sorghum bicolor (L.) Moench] is a type of cultivated sorghum grown primarily for its sugar-rich stalks. Because of its high fermentable sugar content, the crop is widely recognized as an alternative feedstock source for bio-fuel production. The extent to which stalk sugar accumulation occurs may be determined by several factors including the sink size. Grain is the most important sink in sorghum and other grain crops. Three experiments were conducted in this study to determine the extent to which the grain sink can reduce sugar accumulation in the stalks, to test and validate a genetic system that allows development of sterile sweet sorghum hybrids, and to assess the potential of sugar-rich hybrids to overcome stalk rot diseases. The first experiment, based on 22 sweet sorghum genotypes, was undertaken to study the effect of eliminating the grain sink (removing the head prior to anthesis) on stalk juice yield, sugar accumulation, and biomass. The data showed that the grain sink had a significant effect on all traits measured. Elimination of the grain sink significantly increased oBrix % (17.8%), dry biomass (27.8%), juice yield (23.9%), and total sugar yield (43.5%). The second experiment was aimed at validating the role of A3 genetic male sterility system for producing sterile sweet sorghum hybrids. Ten sweet sorghum pollinator lines of variable sugar content were selected among the entries included in the previous experiment. The lines were crossed to four A1 and A3 cytoplasmic male sterile (CMS) lines using a Design II mating scheme. The A3 females did not have effective restorers so that the hybrids were expected to be sterile. The parental lines and corresponding hybrids were evaluated for biomass production, oBrix, juice and sugar yield using a randomized complete block design. All A3 hybrids were sterile and did not produce seed when heads were covered prior to pollination. The effect of grain sink represented by the A1 vs. A3 CMS were highly significant for Brix%, biomass, juice, and sugar yield. Comparison of parents vs. crosses component was highly significant, indicating marked heterosis effect for the traits. Both general (GCA) and specific (SCA) combining ability effects were also significant for all traits, indicating the role of both additive and dominance genetic effects in the inheritance of the characters. Earlier studies have shown positive relationships between stalk sugar concentration and stalk rot disease resistance in sorghum. Thus, the objective of the third experiment was to study the effects of the CMS mediated differential accumulation of stalk sugar on severity of charcoal rot disease caused by Macrophomina phaseolina. The experiment provided an opportunity to test the effect of variable stalk sugar in the same genetic backgrounds. The data indicated that hybrids produced from A3 cytoplasm were more resistant to charcoal rot (7.1cm lesion length) compared to those produced from the A1 hybrids (9.5 cm lesion length). The enhanced resistance of hybrids with higher sugar yield could have significant agronomic advantage in sugar based bio-fuel feedstock production.

The ultimate goal of this research is to develop an efficient, reproducible and low cost method for analysis of VOCs in complex mixtures such as those in exhaled breath and in headspace of fungi cultures. In Chapter I; analytical methods for volatile biomarkers identification is reviewed In Chapter II, active SPME GCMS was employed to analyze VOCs in the breath of a single healthy male and a single female. The goal was to determine the extent of intra-individual variations in the VOC profiles. In Chapter III, a preliminary study was carried out in a greenhouse to determine the pathogenicity of different isolates of M. phaseolina on soybeans. This will allow, in future studies, the matching of VOC profiles of different isolates of M. phaseolina with their relative pathogenicity. This is a key step towards the development of an early warning system for the detection of pathogenic M. phaseolina fungus contaminations.

Macrophomina phaseolina is a generalist ascomycetic fungal pathogen, capable of infecting over 500 genera of plants and limiting yield in crops grown in Mississippi. Recent documentation of M. phaseolina on Impatiens × hybrida, a newfound host, has merited multiple experiments to quantify the exact nature of this relationship. Despite M. phaseolina being a soil-borne pathogen, disease symptoms were only reported in aboveground tissue. Mode of infection experiments revealed both above and belowground tissues are susceptible to infection. In vitro experiments identified the optimal temperature for the growth of M. phaseolina to be 26°C, where more than 10x the accumulated biomass resulted compared to samples grown at 37°C. Impatiens × hybrida hosts were particularly prone to infection at temperatures above 27°C. In vitro fungicide assays revealed Banrot and T-Bird to be suitable chemical control agents for limiting M. phaseolina growth.

The use of matrix assisted laser desorption ionization time-oflight mass spectrometry, Fourier transform infrared spectroscopy, and other analytical means of identifying and differentiating microorganisms hold much promise. These analytical tools have been extensively assessed for their ability to differentiate bacteria and fungi. Most of this research has been coordinated in medically relevant microorganisms, but the technology can work just as well with agriculturally important microorganisms. In this thesis, these technologies were reviewed and then subsequently studied for their ability to differentiate Aspergillus species (that devastate corn and other crops yearly with aflatoxin contamination), as well as Macrophomina phaseolina and Thielaviopsis basicola which limit yields on soybean and other crops yearly. With the use of these technologies, harmful plant pathogens could be identified and subsequently treated to improve crop yields and also help to protect our nation and state’s food supply.

Microbial-root associations are important to help plants cope with abiotic and biotic stressors. Managing these interactions offers an opportunity for improving the efficiency and sustainability of agricultural production. By characterizing the bacterial and archaeal community (via 16S rRNA sequencing) associated with the bulk and rhizosphere soil of sixteen strawberry cultivars in two controlled field studies, we explored the relationships between the soil microbiome and plant resistance to two soilborne fungal pathogens of strawberry (Verticillium dahliae and Macrophomina phaseolina). Overall, the plants had a distinctive rhizosphere microbiome relative to the bulk soil, with higher abundances of known beneficial bacteria such as Pseudomonads and Rhizobium. Plant genotype, biomass, leaf nutrient content and mortality were influenced differently by the rhizosphere microbiome in each of the two trials. In the V. dahliae trial, the rhizosphere microbiome was associated with plant biomass and leaf nutrient content and only indirectly to the disease resistance. In the M. phaseolina trial, the rhizosphere microbiome was associated to plant biomass, but not nutrient content; furthermore, resistant cultivars had larger abundances of Pseudomonas and Arthrobacter in their rhizosphere relative to susceptible cultivars. The mechanisms involved in these beneficial plant-microbial interactions and their plasticity in different environments should be studied further for the design of low-input disease management strategies.

Effects of nitrogen management and cultivar on strawberry production under disease pressure Kamille Garcia-Brucher California strawberry growers face increasing regulatory pressures to manage nitrogen (N) applications in their production system. Standard practice in the California strawberry industry is to apply a synthetic pre-plant controlled release fertilizer (CRF) to ensure the crop has sufficient N during winter establishment. Some research from the UC Cooperative Extension suggests this practice is not efficient at delivering N to the crop since most of the N is released from CRF before strawberry crop N uptake is significant. Another concern for California strawberry growers is loss of their crop to a myriad of soilborne pathogens. Compost is commonly applied as a soil amendment in California strawberry fields as it offers both agronomic and environmental benefits including the potential for disease suppression. In light of legislation restricting N in some California cropping systems, Ag Order 4.0, and incentives programs established to promote soil conservation practices, compost may be a viable substitute for synthetic pre-plant CRF N. In this study, we investigated the effects of pre-plant fertilizer and strawberry cultivar on fruit yield, disease incidence, soil and plant N dynamics and soil carbon (C) at the Cal Poly Strawberry Center, San Luis Obispo, CA in a field infested with Macrophomina phaseolina. Pre-plant fertilizer treatments included 100 lb N/ac Cal Poly certified organic compost, 100 lb N/ac synthetic CRF and a control treatment (0 lb N/ac). Strawberry cultivars included three UC varieties, ‘Monterey,’ ‘Albion,’ and ‘San Andreas,’ and one Driscoll’s proprietary cultivar. Fruit yield and plant mortality data were collected throughout the growing season. Soil C was measured from soil samples collected in the root zone (6 in) while soil nitrate was measured from pore water samples collected in and below the root zone (6 and 12 in, respectively). Strawberry crop N uptake was determined using destructive plant samples while fruit N concentration was determined from subsamples of harvested fruit taken in April, May, June, and July each year. Although compost application did not significantly affect C sequestration and did not reduce disease incidence, there was no significant difference in total yield between compost and CRF treatments suggesting that compost can substitute for synthetic CRF without negatively affecting yield. There was significantly less plant mortality in control treatments compared with compost and CRF treatments suggesting excessive pre-plant N impacts disease incidence by M. phaseolina but more research is needed to better understand the mechanisms of infection by this soilborne pathogen. Total yield in this experiment was lower compared with statewide averages and crop N concentration was lower compared with the literature which is likely a result of disease pressure. Fruit N concentrations for the cultivars in this study were lower than the conversion coefficient defined by the Ag Order which means growers are removing less N through harvest allowing them more room in their N budget. Based on our results, compost may be substituted for synthetic CRF without negatively affecting yield and perhaps even make desirable soil improvements in this production system. And in fields with significant levels of M. phaseolina in the soil, N applications should be considered as it was seen to impact disease incidence. Keywords: compost, controlled release fertilizer, M. phaseolina, nitrogen uptake, Ag Order 4.0

Allelopathy occurs when one organism releases a compound into the environment that affects the functioning of another organism. Scientists have long suspected that alleopathic plant compounds could offer novel, softer chemistries to the ongoing battle of controlling pests in agricultural fields. Strawberry growers rely on toxic fumigants to kill soilborne fungal pests, weeds, nematodes, and insects. Increased regulations have reduced the use of fumigants (including methyl bromide), and strawberry growers need new sustainable pest control solutions. We selected four putative allelochemicals with known fungicidal and herbicidal activity (ferulic acid, gallic acid, juglone, and p-Coumaric acid). We assessed the pesticidal activity of these plant compounds both in agar and in soil on two emerging soilborne fungal pathogens (Macrophomina phaseolina and Fusarium oxysporum f.sp. fragariae), and four annual weeds commonly found in strawberry production fields (Malva parviflora, Melilotus officinalis, Poa annua, and Senecio vulgaris). We also assayed lettuce (Lactuca sativa ‘Inferno’), which served as a positive control plant species due to its sensitivity to phytotoxic compounds. Fitted sigmoidal dose-response curves predicted EC50 and EC75 values for each combination of plant compound and pest. All plant compounds inhibited the in vitro radial mycelial growth of the two soilborne fungal pathogens in a dose-dependent manner. Fusarium oxysporum f.sp. fragariae was more sensitive to the plant compounds than Macrophomina phaseolina. Average EC50 values for the radial mycelial growth of two F. oxysporum f.sp. fragariae isolates were 75.1 parts per million by weight (ppmw) juglone, 469 ppmw p-Coumaric acid, and 687 ppmw ferulic acid. Average EC50 values for the radial mycelial growth of two M. phaseolina isolates were 196 ppmw juglone, 2869 ppmw p-Coumaric acid, and 5716 ppmw ferulic acid. The three compounds we assayed in vitro also reduced M. phaseolina colony forming unit counts in soil and the EC50 values were 476 ppmw ferulic acid, 612 ppmw juglone, and 827 ppmw p-Coumaric acid. Metconazole, the conventional fungicide control, did not inhibit M. phaseolina colony forming unit counts in soil at its label high rate. The plant compounds required similar or lower rates to inhibit colony forming units that grew from M. phaseolina overwintering structures (microsclerotia) in soil as to inhibit radial mycelial growth in vitro. Based on the EC50 value in soil assays, ferulic acid was the least expensive plant compound to apply on a per acre basis to inhibit M. phaseolina ($74,226). In F.oxysporum f.sp. fragariae soil assays, the compounds induced hormesis at lower rates and may be germination stimulant candidates. Metconazole and the high rates of every compound effectively or completely inhibited F. oxysporum f.sp. fragariae colony forming units in soil. The plant compounds were more herbicidal than fungicidal in vitro. When combining the in vitro seedling length results for L. sativa, M. parviflora, P. annua, and S. vulgaris the EC50 values differed significantly (p < .0001) and were: 47 ppmw juglone, 120 ppmw p-Coumaric acid, 189 ppmw ferulic acid, and 297 ppmw gallic acid. At least one rate of ferulic acid, juglone, and p-Coumaric acid inhibited the germination of all plant species, while gallic acid only inhibited the germination of P. annua at 1000 ppmw (p < .05). In soil, visible microbial contamination in individual wells of 24-well plates and seed dormancy made it difficult to fit curves to weed seedling length data. The soil assay L. sativa seedling length EC50 values 11 days after initial treatment were slightly higher than in vitro, although plant compounds were in the same order of phytotoxicity: 129 ppmw juglone, 616 ppmw p-Coumaric acid, 644 ppmw ferulic acid, and 1584 ppmw gallic acid. Based on the EC50 value in soil assays, the least expensive compound to inhibit L. sativa seedling length on a per acre basis was gallic acid ($21,676). Germination 26 days after initial soil treatment generally declined in a dose-dependent manner for each compound. There was a direct relationship between plant compound rate and seedling damage in soil with the higher rates of all compounds, except p-Coumaric acid, inducing damage comparable to a conventional herbicide (pendimethalin or oxyfluorfen). Contaminated treatments appeared to be due to an interaction between plant compounds and microorganisms because herbicide and water controls had almost no microbial growth 11 days after initial treatment. Further, there was a significant positive linear relationship between level of contamination in phenolic acid-treated wells (ferulic acid, gallic acid, and p-Coumaric acid, p < .0001) and the in soil rate. This relationship was slightly negative in juglone soil treatments (p = .0167), which may be due to its greater antimicrobial activity than the phenolic acids. We propose that herbicidal effects in soil were due to the joint effect of the plant compounds themselves, and the microbial growth in wells. Microbial growth was either antagonistic or additive to the inhibitory action of the plant compounds. The plant compounds we assayed were inhibitory of emerging fungal pathogens in strawberry production and common annual strawberry field weeds. Evidence presented in this thesis correlates well with past research that not only found plant compounds to be herbicidal and fungicidal, but also described their modes-of-action (such as the production of reactive oxygen species that causes necrotic lesions on roots, and inhibition of glycolytic enzyme activity that prevents germination), and implicate plant compounds as carbon sources for a variety of microorganisms. Compound prices are currently exorbitant, but may decline as demand increases. Whether or not they provide effective pest control may depend on soil texture, organic matter, microbial diversity, and other edaphic factors.

No description available.

An evaluation was made of soil solarization to control Macrophomina phaseolina and Sclerotium rolfsii under the climatic conditions of the Sonoran Desert region of Arizona. Tarping of moist soil with clear polyethylene in the summer was most effective in raising soil temperatures, while tarping during the fall and spring were less efficient. In one summer trial, the maximum temperatures achieved were 7-8 C higher than control plots at 1, 15, and 30 cm depths. In none of the tests was the application of tarp effective in reducing M. phaseolina populations to non-detectable levels. After a six-week summer solarization treatment, S. rolfsii was controlled at the 15-cm but not the 30-cm depth. During a fall treatment control of S. rolfsii was achieved only at the 1-cm depth. After solarization, seeds of Euphorbia lathyris were planted in solarized and control plots. The incidence of M. phaseolina-associated mortality among seedlings planted in solarized plots was always the same as the control plot with the highest disease incidence. The utility of this technique may be limited by the heat tolerance of the target organisms. Studies of bacterial antagonists were initiated to determine their utility in enhancing pathogen control after the solarization treatment. Screening 43 bacterial isolates showed that seven of Pseudomonas fluorescens and one of Serratia marcescens exhibited some form of antagonism toward M. phaseolina, S. rolfsii and Verticillium dahliae in vitro. Antagonism was manifested as a complete inhibition of fungal development or reduced hyphal development coupled with suppression of sclerotial development. The action of the antagonists was found to be fungitoxic or fungistatic rather than fungicidal. None of the four P. fluorescens isolates tested were effective in preventing M. phaseolina infection of E. lathyris seedlings. The ability of these bacteria to prevent sclerotial formation while still permitting hyphal growth may be a useful technique for studying the two phases of the fungal life cycle separately.

Doctor of Philosophy / Department of Agronomy / Tesfaye Tesso / Fusarium stalk rot and charcoal rot caused by Fusarium thapsinum and Macrophomina phaseolina respectively are devastating global diseases in sorghum that lead to severe quality and yield loss each year. In this study, three sets of interrelated experiments were conducted that will potentially lead to the development of resistance based control option to these diseases. The first experiment was aimed at identifying sources of resistance to infection by M. phaseolina and F. thapsinum in a diverse panel of 300 sorghum genotypes. The genotypes were evaluated in three environments following artificial inoculation. Out of a total of 300 genotypes evaluated, 95 genotypes were found to have resistance to M. phaseolina and 77 to F. thapsinum of which 53 genotypes were resistant to both pathogens. In the second experiment, a set of 79,132 single nucleotide polymorphisms (SNPs) markers were used in an association study to identify genomic regions underlying stalk rot resistance using a multi-locus mixed model association mapping approach. We identified 14 loci associated with stalk rot and a set of candidate genes that appear to be involved in connected functions controlling plant defense response to stalk rot resistance. The associated SNPs accounted for 19-30% of phenotypic variation observed within and across environments. An analysis of associated allele frequencies within the major sorghum subpopulations revealed enrichment for resistant alleles in the durra and caudatum subpopulations compared with other subpopulations. The findings suggest a complicated molecular mechanism of resistance to stalk rots. The objective of the third experiment was to determine the functional relationship between stay-green trait, leaf dhurrin and soluble sugar levels and resistance to stalk rot diseases. Fourteen genotypic groups derived from a Tx642 × Tx7000 RIL population carrying combinations of stay-green quantitative trait loci were evaluated under three environments in four replications. The stg QTL had variable effects on stalk rot disease. Genotypes carrying stg1, stg3, stg1,3 and stg1,2,3,4 expressed good levels of resistance to M. phaseolina but the combination of stg1 and stg3 was required to express the same level of resistance to F. thapsinum. Other stg QTL blocks such as stg2 and stg4 did not have any impact on stalk rot resistance caused by both pathogens. There were no significant correlations between leaf dhurrin, soluble sugar concentration, and resistance to any of the pathogens.