Master of Science / Department of Agronomy / Ramasamy Perumal / Tesfaye Tesso / Grain mold (GM) is an important biotic constraint limiting yield and market value of sorghum grains. It results in kernel discoloration and deterioration. Such kernels have reduced seed viability, low food and feed quality. Breeding for grain mold resistance is challenging because of the complex nature of host-pathogen-environment interactions. This complex task could be made simpler by utilizing molecular markers. Utilization of marker resources may help to find genomic regions associated with grain mold resistance. In this study, three sets of field and laboratory based experiments were performed which will help in finding potential grain mold pathogens responsible for kernel deterioration in the studied environment and search for genotypes with better kernel quality and grain mold resistance. In the first part of the study, in vitro screening of 44 grain mold resistant sorghum genotypes developed and released by Texas A & M AgriLife Research. This study was aimed at identifying sources resistance to grain mold infection through laboratory screening. The result revealed that genotypes Tx3371, Tx3373, Tx3374, Tx3376, Tx3407, Tx3400, and Tx3402 were have high level of resistance and were identified as potential sources of grain mold resistance as each showed minimal fungal infection and higher grain quality traits. The second experiment was performed to optimize surface sterilization protocol for the extraction of fungal pathogens from the kernel surface (pericarp) and to study the effect of bleach percentage and time period on pathogen extraction. Seven treatments using sterilized double distilled water (0 % bleach (v/v)) and different bleach (NaOCl) concentrations (2.5, 5, 7.5, 10, 12.5 and 15 %) were used with a time interval of 2.5, 5, 7.5 and 10 min. Optimized surface sterilization in the range of 7.5 to 15 % bleach (v/v) for 7.5 to 10 min resulted least contamination and fungal genera isolation from the surface of the kernel. The third study was aimed at characterizing genotypes (sorghum association panel) for grain mold pathogen F. thapsinum and by using genome wide association (GWA) tool in order to find genomic regions associated with grain mold resistance. We studied the effect of different agronomic and panicle architecture traits on grain mold incidence and severity. Effects of grain mold on kernel quality traits were also studied. We reported two loci associated with grain mold resistance. Based on first year field screening results, 46 genotypes having grain mold ratings 1-5 (1 = < 1% panicle kernel molded; 5 = > 50% panicle kernel molded) were selected for a detailed study aimed at understanding grain mold x fungal pathogen interactions to physical and chemical kernel traits. Seed germination test, vigor index, and tetrazolium viability test were performed to study effect of grain mold infection on kernel viability and vigor. Alternaria, Fusarium thapsinum, F. verticillioides and F. proliferatum were the main fungal genera isolated from bisected kernels. Based on two year screening, SC623, SC67, SC621, SC947 and SC1494 were most resistant based on both PGMR and TGMR rating while SC370, SC833, SC1484, and SC1077 showed the most susceptible reaction and this was consistent for individual location analysis. SC309, SC213, SC833, SC971 and SC1047 are genotypes having identified loci for grain mold resistance.

Five breeding populations were created by crossing elite U.S. sorghum parental lines (RTx430, RTx436, BTx631, BTx635, and Tx2903) with 'Sureño', a dual purpose grain mold resistant sorghum cultivar. Molecular markers associated with five previously-reported quantitative trait loci (QTL) for grain mold resistance originating in 'Sureño' were used to determine if their presence enhanced selection for grain mold resistance in these populations. The allelic status of 87 F4 lines, with respect to these QTL, was determined using both simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) markers. All 87 F4:5 lines and their parental lines, were evaluated for grain mold resistance in replicated trials in eight diverse environments in South and Central Texas during the summer of 2002. The effects of each allele from the grain mold resistant parent 'Sureño' were determined across and within all five populations, within individual environments, and in each population x environment combination. With a few exceptions, the QTL were effective in reducing grain mold susceptibility only within the RTx430/Sureño progeny, the identical cross that was used in the original mapping study. The results indicate that while that these alleles do confer additional grain mold resistance, they are only selectable in the original mapping population. This fact limits their potential usefulness in an applied breeding program.

Five breeding populations were created by crossing elite U.S. sorghum parental lines (RTx430, RTx436, BTx631, BTx635, and Tx2903) with 'Sureño', a dual purpose grain mold resistant sorghum cultivar. Molecular markers associated with five previously-reported quantitative trait loci (QTL) for grain mold resistance originating in 'Sureño' were used to determine if their presence enhanced selection for grain mold resistance in these populations. The allelic status of 87 F4 lines, with respect to these QTL, was determined using both simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) markers. All 87 F4:5 lines and their parental lines, were evaluated for grain mold resistance in replicated trials in eight diverse environments in South and Central Texas during the summer of 2002. The effects of each allele from the grain mold resistant parent 'Sureño' were determined across and within all five populations, within individual environments, and in each population x environment combination. With a few exceptions, the QTL were effective in reducing grain mold susceptibility only within the RTx430/Sureño progeny, the identical cross that was used in the original mapping study. The results indicate that while that these alleles do confer additional grain mold resistance, they are only selectable in the original mapping population. This fact limits their potential usefulness in an applied breeding program.

Sorghum grain mold and anthracnose are two major diseases of sorghum (Sorghum bicolor) that constrain sorghum production worldwide. Grain mold is caused by several species of fungi, but the two most common are Curvularia lunata and Fusarium thapsinum. Isolates of these two species were used to inoculate panicles of selected sorghum cultivars in green house and field experimentations. Panicles were sprayed at the time of anthesis with conidial suspensions of the two fungal species individually or in a mixture and with water to serve as a control. Samples were collected 48 hours after inoculation for RNA extraction. In greenhouse studies, four cultivars (Tx2911, Sureno, SC170 and RTx430) were used while thirteen cultivars were grown in the field experiments. Gene expression was measured for the following genes using real time polymerase chain reactions (rt-PCR): PR10, β-glucanase, chitinase, thaumatin, sormatin, phenyalanine ammonia lyase (PAL), obtusifoliol 14α-demethylase (Obtus), antifungal protein (AFP), apoptosis related protein (Apop) and leucine rich repeat (LRR). Seed germination tests in field grown cultivars indicated that germination rates for SC279-14E, SC660 and Sureno were not greatly influenced by grain mold. Covering the panicles with bags served to protect them against grain mold pathogens. The seed mycoflora test showed that Fusarium thapsinum was the most frequently recovered species and there were more species present in non-covered panicles. The response of sorghum cultivars to grain mold infection involves multiple defense genes. Real time PCR used to study the expression of sorghum defense in greenhouse grown plants showed that mRNA encoding PR-10, a small 10 kDa protein, was highly expressed in the glumes and spikelets of resistant cultivars Tx2911 and Sureno and constitutively in leaves. The expression of some other defense genes like beta-glucanase, chitinase and AFP was variable. Sormatin was not expressed. Expression of β-glucanase, chitinase, and PR10 was higher in field than in greenhouse experiments. A second area of research involved tagging of a resistance gene for sorghum anthracnose. Three AFLP markers (Xtxa607, Xtxa3181 and Xtxa4327) and three SSRs (Xtxp3, Xtxp55 and Xtxp72) were identified. These markers were loosely linked to the resistance genes. The markers are located on linkage group B. The results suggest that markers located 20-30 cM on one side or the other of those tested should provide useful tags for the resistance gene.

Sorghum grain mold and anthracnose are two major diseases of sorghum (Sorghum bicolor) that constrain sorghum production worldwide. Grain mold is caused by several species of fungi, but the two most common are Curvularia lunata and Fusarium thapsinum. Isolates of these two species were used to inoculate panicles of selected sorghum cultivars in green house and field experimentations. Panicles were sprayed at the time of anthesis with conidial suspensions of the two fungal species individually or in a mixture and with water to serve as a control. Samples were collected 48 hours after inoculation for RNA extraction. In greenhouse studies, four cultivars (Tx2911, Sureno, SC170 and RTx430) were used while thirteen cultivars were grown in the field experiments. Gene expression was measured for the following genes using real time polymerase chain reactions (rt-PCR): PR10, β-glucanase, chitinase, thaumatin, sormatin, phenyalanine ammonia lyase (PAL), obtusifoliol 14α-demethylase (Obtus), antifungal protein (AFP), apoptosis related protein (Apop) and leucine rich repeat (LRR). Seed germination tests in field grown cultivars indicated that germination rates for SC279-14E, SC660 and Sureno were not greatly influenced by grain mold. Covering the panicles with bags served to protect them against grain mold pathogens. The seed mycoflora test showed that Fusarium thapsinum was the most frequently recovered species and there were more species present in non-covered panicles. The response of sorghum cultivars to grain mold infection involves multiple defense genes. Real time PCR used to study the expression of sorghum defense in greenhouse grown plants showed that mRNA encoding PR-10, a small 10 kDa protein, was highly expressed in the glumes and spikelets of resistant cultivars Tx2911 and Sureno and constitutively in leaves. The expression of some other defense genes like beta-glucanase, chitinase and AFP was variable. Sormatin was not expressed. Expression of β-glucanase, chitinase, and PR10 was higher in field than in greenhouse experiments. A second area of research involved tagging of a resistance gene for sorghum anthracnose. Three AFLP markers (Xtxa607, Xtxa3181 and Xtxa4327) and three SSRs (Xtxp3, Xtxp55 and Xtxp72) were identified. These markers were loosely linked to the resistance genes. The markers are located on linkage group B. The results suggest that markers located 20-30 cM on one side or the other of those tested should provide useful tags for the resistance gene.

Industry demand for local sources of grain for animal feed has increased sorghum production in the mid-Atlantic region of the U.S. Sorghum anthracnose (causal agent Colletotrichum sublineola) and the grain mold complex, which includes mycotoxin-producing Fusarium spp., limit the yield and quality of grain sorghum in humid climates worldwide. A majority of U.S. grain sorghum production is in arid regions, and management strategies have not been developed for the mid-Atlantic U.S. where warm, wet conditions favor disease. The specific objectives of this research were to: (1) determine the effectiveness of fungicides and their application timing for the management of sorghum foliar anthracnose, (2) compare five grain sorghum hybrids for their susceptibility to foliar anthracnose, grain mold and mycotoxin contamination under field conditions, (3) integrate host resistance and fungicide application to manage anthracnose and grain mold, and (4) identify Fusarium spp. associated with grain mold and mycotoxin contamination of sorghum in the mid-Atlantic U.S. For Objective 1, it was determined that a single application of pyraclostrobin-containing fungicide no later than flowering reduced anthrancose, protected yield and maximized farm income. Objective 2 focused on sorghum hybrid selection as a disease management tactic, and it was determined that hybrids with high yield potential and moderate disease resistance should be selected for mid-Atlantic sorghum production in order to maximize grain yield and quality while minimizing the need for fungicide inputs. Objective 3 focused on integrated management and demonstrated that under moderate disease pressure, a high-yielding susceptible hybrid required a single application of pyraclostrobin-based fungicide to minimize fungal diseases and maintain acceptable yields, whereas under high disease pressure it was necessary to integrate hybrid resistance and judicous applications of fungicides. The aim of Objective 4 was to characterize potential causal agents of mycotoxin contamination in mid-Atlantic sorghum, and thirteen phylogenetically distinct Fusarium species (F. lacertarum, F. graminearum. F. armeniacum, F. proliferatum, F. fujikuroi, F. verticillioides, F. thapsinum and several in Fusarium incarnatum-equiseti species complex) were found to be associated with grain mold and fumonisin and/or deoxynivalenol contamination of sorghum grain. This work has provided insights into the impacts of fungal diseases on grain sorghum yield and quality in the mid-Atlantic and has aided in development of best management practices for the region. / Doctor of Philosophy / Sorghum is grown in tropics, sub-tropics and semi-arid region worldwide for food, feed, forage and fuel. Sorghum acreage in the mid-Atlantic is increasing due the demand for locally grown grain by poultry and swine industries. During the growing season, warm and humid conditions are common in the southeastern and mid-Atlantic states favoring fungal diseases development that reduce the grain yield and quality. Anthracnose and grain mold, which includes toxic mycotoxin-producing Fusarium species, are the two major constraints in sorghum production in the region. However, management alternatives have not been developed. The main goal of this research was to develop management strategies to protect yield and maximize farm profitability by controlling anthracnose and grain mold of sorghum using chemicals and/or host resistance. The specific objectives were to: (1) determine the effectiveness of fungicides and their application timing for the management of sorghum foliar anthracnose, (2) compare grain sorghum hybrids for their susceptibility to foliar anthracnose, grain mold and mycotoxin contamination under field conditions, (3) assess the value of integrating host resistance and judicious use of fungicides to manage sorghum anthracnose and grain mold, and (4) identify Fusarium spp. associated with grain mold and mycotoxin contamination of sorghum in the mid-Atlantic U.S. Results from this research indicate that a single application of pyraclostrobin-containing fungicides no later than flowering reduces anthrancose, protects yield, and increases farm income. Sorghum hybrids varied in susceptibility to anthrancnose and grain mold, and planting a moderately resistant hybrid and applying a fungicide under high disease risk conditions provided the greatest return on investment. Both fumonisin and deoxynivalenol were frequently detected from sorghum grain, and mycotoxin contamination was associated with 13 different Fusarium species from three distinct species complexes. Based on the results of this work, best management practices for minimizing sorghum disease losses were developed for the mid-Atlantic region.