Doctor of Philosophy / Department of Plant Pathology / Christopher R. Little / Fusarium spp. are among the most important pathogen groups on soybeans. However, information regarding this genus on soybean seeds in the state of Kansas remains underexplored. Therefore, the goal of this study was to characterize the identity, frequency, and pathogenicity of soybean seedborne Fusarium spp. in the state of Kansas. For the identification and frequency of seedborne Fusarium spp., culture-dependent (i.e. semi-selective medium) and -independent (i.e. DNA metabarcoding) approaches were used. Also, information regarding the pathogenicity of the most common seedborne Fusarium spp. from soybeans was assessed to better understand their role as soybean pathogens. Overall, eleven Fusarium spp. were identified in this study. Semi-selective media showed that approximately 33% of soybean seed samples were infected with Fusarium spp. Moreover, Fusarium spp. were isolated from seed sampled from 80% of the locations in Kansas. Furthermore, a low incidence of Fusarium spp. was observed within infected seed samples and averaged 2%. Nine Fusarium spp. were found in soybean seeds using the culture-dependent approach. Fusarium semitectum was the most frequent, followed by F. proliferatum and F. verticillioides. Fusarium acuminatum, F. equiseti, F. fujikuroi, F. graminearum, F. oxysporum, and F. thapsinum were found in lower frequencies among naturally infected seeds. DNA metabarcoding experiments showed that Fusarium spp. are more frequent in soybean seeds than previously known. All asymptomatic soybean seeds analyzed, using Illumina MiSeq platform, showed the presence of the genus Fusarium including two pathogenic species, F. proliferatum and F. thapsinum. Fusarium acuminatum, F. merismoides, F. solani, F. semitectum, and Fusarium sp. were also identified using the culture-independent approach. Preliminary results also showed that F. proliferatum and F. thapsinum were observed in all three major soybean seed tissues: seed coat, cotyledons, and the embryo axis. Depending on the soybean genotype, inoculum potential and aggressiveness, F. proliferatum, F. graminearum, F. fujikuroi, F. oxysporum, F. semitectum, F. thapsinum, and F. verticillioides were pathogenic to soybean and negatively affect soybean seed quality, at different levels, in controlled conditions. Moreover, F. equiseti and F. acuminatum did not cause significant damage to soybean seeds and seedlings. Understanding seedborne Fusarium spp. and their influence on soybean seed and seedling diseases is critical for the development of effective disease control strategies, especially regarding early detection of pathogenic strains in seeds (i.e., seed health testing), ensuring the crop productivity, quality, and safety.

The taxonomy of Fusarium species has been controversial. The controversy surrounding the morphology-based classification, as well as the limitations and advantages of the various tools used to circumvent this problem are discussed as an introduction to the research presented in this thesis. Emphasis in this case was on the F. oxysporum species complex. The body of the research focuses on Fusarium species in Ethiopian agriculture about which little has been published. Results from this study suggest that F. oxysporum and F. solani are the two species most commonly isolated from agricultural soils and plant tissues from Ethiopia. The Ethiopian F. solani isolates were further characterized using AFLPs which showed that these isolates group into two separate clusters. Additional characterization using EF-1á sequence analyses showed that all these isolates belong to one of the three clades previously designated for the F. solani-Nectria haematococca species complex. Nine SSR markers were developed for studying the F. oxysporum complex. These markers are highly polymorphic and should be useful for many different population genetic studies of the F. oxysporum species complex. Ethiopian F. oxysporum isolates, and F. oxysporum isolates obtained from other fungal collections, were characterized using AFLPs, SSRs and DNA sequence analyses. These studies resolved the isolates into three concordant groups that corresponded to the three clades of F. oxysporum previously designated for this species complex. Most of the Ethiopian isolates grouped in one of these three clades. This and the high degree of AFLP- and SSR-based genetic similarity observed among the Ethiopian isolates suggested that these isolates are largely clonal. In addition, a PCR-based diagnostic was developed to allow for the accurate differentiation between F. oxysporum and F. redolens which has proved to be difficult using morphological characters. The studies presented in this thesis contribute to a better understanding of Fusarium species commonly found in Ethiopia. Clearly, there is much that will still need to be done. The research presented in this thesis establishes a foundation for other studies and also provides a stimulus for further studies of these important fungi in Ethiopia. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Bogale, MA 2006, Molecular characterization of Fusarium isolates from Ethiopia, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-11072006-172206 / > D87/ag / Thesis (PhD)--University of Pretoria, 2007. / Genetics / Unrestricted

Fusarium oxysporum is a ubiquitous soil-borne fungus that includes pathogenic and non-pathogenic members. The pathogenic members are best known for causing Fusarium wilt diseases of many economically important agricultural crops. One such a crop is banana (Musa spp.), which is affected by a special form of the fungus known as F. oxysporum f.sp. cubense (Foc). Fusarium wilt was responsible for devastating losses of Gros Michel export bananas in Central America during the first half of the 20th century, and is now, once again, threatening world banana production that is primarily based on the sweet Cavendish varieties, both in the tropics and subtropics. To effectively manage Fusarium wilt, adequate knowledge of the pathogen, plant and environment is required. With this thesis I hope to contribute to the current knowledge available on the pathogen. Previous studies investigated the phenotypic and genotypic diversity, the spread and distribution, and the phylogeny of Foc. Some aspects related to the biology, physiology, diversity and pathogenicity of Foc, however, appeared to be unresolved. These aspects are important in order to develop a sustainable management strategy for Fusarium wilt to ensure continued banana production. Chapter 1 depicts a general review on F. oxysporum as the causal agent of Fusarium wilt of various fundamental crops, and gives a broad overview of the biology, taxonomy, physiology and pathogenicity of the pathogen. Through the application of modern molecular genetic techniques, a lot of progress has been made in the identification of genes and processes involved in the biology and pathogenesis of Fusarium wilt pathogens. The review concludes that some work, however, still needs to be conducted before topics such as race designation and pathogenesis in Foc are fully understood. Temperature, pH and nutrition are all factors contributing to the pathogenesis of F. oxysporum. The different factors can either favour or suppress the pathogen, or they can have a stimulating or inhibiting effect on the host plant. In Chapter 2 the pathogenicity and phenotypic characteristics of a genotypically uniform population of Foc was investigated. Physiological studies included determining the minimum, maximum and optimum temperatures and pH at which Foc grows in vitro, and what nitrogen sources stimulate and inhibit growth of Foc. Knowledge on these aspects could contribute to the management of the pathogen in the field. Differentiation among species of Fusarium can be problematic. To resolve questions related to the nomenclature in Fusarium, our research focus has shifted to the use of molecular tools for identification and determination of evolutionary relationships among and within species. In the past, phylogenetic studies on Foc were conducted using molecular tools such as sequencing, Restriction Fragment Length Polymorphisms, Random Amplified Polymorhic DNA and DNA Amplification Fingerprinting, with varying amounts of success. In Chapter 3 the usefulness of Amplified Fragment Length Polymorhism (AFLP) analysis to study diversity inFoc isolates was investigated. Of the 21 vegetative compatibility groups (VCGs) of Foc identified around the world, only VCG 0120 is found in South Africa. Chapter 4 aimed to identify an AFLP polymorphic DNA fragment unique to VCG 0120, and to develop a molecular marker of this fragment. Such a marker would be extremely valuable to distinguish between VCG 0120 and other isolates of F. oxysporum in terms of identification and confirmation of Fusarium wilt of banana in South Africa. Several pathogenicity-related genes have been identified in F. oxysporum. In Chapter 5, the presence of three pathogenicity-related genes (fmk1, pg1 and xyl3) in F. oxysporum isolates pathogenic and non-pathogenic to banana were verified by means of PCR amplification. The value of pathogenicity genes such as fmk1 and pg1 in comparative phylogenetic analysis was further substantiated. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Three independent studies were conducted for spot blotch (Bipolaris sorokiniana), Fusarium head blight (FHB) (Fusarium graminearum), and root rot diseases (Fusarium species and B. sorokiniana). Histopathology of compatible and incompatible interactions between different pathotypes of B. sorokiniana and different genotypes of barley was examined with red fluorescent protein-tagged fungal isolates. The fungus penetrated the host cell wall and developed multicellular globular infection hyphae (IH) in the lumen of epidermal cells, but infected epidermal cells appeared to be alive till 16 hours post-inoculation (HPI). In the susceptible plants, the tip of IH was found to grow ahead of the dead tissue and invade the surrounding live mesophyll cells, whereas growth of IH in the resistant plants was restricted to the dead tissue after 20 HPI. The amount of H2O2 accumulation and the fungal biomass were also significantly higher in the susceptible hosts than in the resistant hosts. To map resistance to FHB, two populations consisting 130 doubled haploid lines from the cross Grandin × PI277012 and 237 recombinant inbred lines from the cross Bobwhite × ND2710 were phenotyped and genotyped. QTL for Type I resistance were identified on chromosomes 1A, 2B, 4B, 5B and 6B in the GP population. These QTL explained 10.7-19 % of the total phenotypic variation. With the BN population, QTL for Type I resistance were identified on chromosomes 2A, 5A and 6B, explaining 6.2-13.7% of the total phenotypic variation. To assess the prevalence, incidence and severity of wheat crown rot (CR) and common root rot (CRR) in ND, wheat root samples were collected from fields across the state in 2012, 2013, and 2014. Fungal isolations indicated that B. sorokiniana was most frequently recovered in all sampled years. Seedling tests on ten spring wheat lines showed that Glenn was the least susceptible while Steele-ND was the most susceptible to one F. culmorum isolate and one B. sorokiniana isolate tested. Evaluation of 20 spring wheat genotypes for reaction to CRR at the adult plant stage showed that Freyr and RB07 were more resistant while Len and Briggs were more susceptible to CRR compared to other wheat genotypes evaluated. / North Dakota Wheat Commission, / Minnesota Wheat Research and Promotion Council / ND State Board of Agricultural Research and Education / Triticeae-CAP project (2011-68002-30029) of the US Department of Agriculture National Institute of Food and Agriculture / U.S. Wheat and Barley Scab Initiative (USWBSI)

Dry Pea or field pea (Pisum sativum L.) is an important cool season legume crop grown in the United States. Field peas are vulnerable to many diseases of which, soil borne diseases including wilt and root rot are of major economic importance and can cause significant reduction in yield. There is a dearth of satisfactory methods for control of root rot and no varieties with complete resistance to Fusarium root rot are currently available. Root rot disease was found to be prevalent in all the major pea growing counties of North Dakota surveyed in 2004, 2005, 2010 and 2011. Fusarium species were the most frequently isolated fungal species from the infected pea roots of which, F. oxysporum and F. avenaceum were the most common. 21 Field pea varieties were screened for resistance against F. avenaceum and F. solani f. sp. pisi, the Fusarium species traditionally associated with root rots of field pea in growth chamber experiments and field trials. Low levels of resistance were detected in a few cultivars but no variety was found to be completely resistant to any of the pathogens tested. Efficiency of precipitated calcium carbonate (PCC) in controlling Fusarium species most commonly associated with root rots was evaluated under in vitro and field conditions. Significant reduction in spore production, spore germination, and dry mycelial weight of Fusarium spp. were detected on PCC amended media in laboratory studies. In greenhouse and field experiments significant reduction in root rot disease severity was observed with PCC application compared to control. Fungal gene expression in artificially infected field pea roots and F. graminearum grown in culture was assessed using the Illumina mRNA-Seq technology. A total of 613 F. graminearum genes were found to be differentially expressed in planta on pea. Functional classes associated with amino acid metabolism, nitrogen metabolism, extracellular polysaccharide degradation, detoxification by degradation and defense related proteins were found to be significantly enriched in the up-regulated gene set as determined using FunCatDB. Expression of four up-regulated genes was confirmed by RT-PCR to validate the inferences from the sequencing results.

This diploma thesis concentrates on testing the efficiency of antifungal and biological agents against fungal pathogens Colletotrichum acutatum and Fusarium culmorum. Five single-component antifungal agents were used for the testing and the sizes of the inhibition zones were evaluated. It was found out that the sensitivity of the pathogens to the agents varies. Most effective against Colletotrichum acutatum was the azoxystrobin compound in the Ortiva agent, whereas against Fusarium culmorum it was the Horizon agent with the active compound tebuconazole. With the biological agents, the zone of mycoparasitism was evaluated and the effect of the agents on the growth of the phytopathogenic fungi mycelia was analysed. Trichoderma harzianum exhibited better ability to colonize and suppress the growth of the pathogens‘ mycelia than Pythium oligandrum.

<p>  </p> <p>Adenosine-to-Inosine (A-to-I) mRNA editing is a post-transcriptional modification of specific sites within the mRNA that has only recently been observed in filamentous fungi. In the wheat scab fungus <em>Fusarium graminearum,</em> this phenomenon has shown to be facilitated by FgTad2 and FgTad3, homologs of Adenine Deaminase Acting on tRNA (ADAT). Interestingly, these two proteins are constitutively expressed in all different life stages<em>, </em>in contrast to only the sexual stage-specific nature of A-to-I mRNA editing in <em>F. graminearum</em>. To understand the molecular mechanisms regulating this process, six candidate co-factor genes were identified which interact with FgTad2 and/or FgTad3, specifically during sexual reproduction. Deletion mutants of four candidate co-factor genes were successfully generated. All four mutants displayed normal asexual development of <em>F. graminearum</em>, but four mutants also altered sexual function. Those four mutant led to formation of morphologically normal perithecia and ascospores, but the perithecia failed to discharge ascospores. More interestingly, in <em>FGSG_10943 </em>deletion mutant, most of these ascospores germinated precociously within the perithecium. I also observed, that among the candidate co-factor genes which are specifically expressed during sexual reproduction, <em>FGSG_10943</em> was significantly upregulated during the later stage of sexual development. This gene is restricted in nature to only a few orders of fungi in the class Sordariomycetes that form dark pigmented ascocarps, particularly Hypocreales and Glomerellales. Taken together, these results indicate that the four candidate co-factor genes are dispensable for vegetative growth of the fungus and involved in ascospore discharge. <em>FGSG_10943</em> appears to be involved in autoinhibition of ascospores inside the perithecia and interact with FgTad2 during sexual reproduction to mediate A-to-I mRNA editing in <em>F. graminearum</em>.</p>

Challenges to growing winter wheat in North Dakota include winter temperature and disease pressure. Fusarium head blight (FHB) is a devastating disease that necessitates breeding for resistance. In the NDSU breeding program FHB resistance genes are often associated with a decrease in performance. This study used single seed descent to advance lines while maintaining a near random population. Early generation (F4) selection focused heavily on yield and the presence of FHB resistance quantitative trait loci to develop winter wheat lines with FHB resistance and high yield. Stripe rust is a fungal disease that is becoming increasingly problematic in North Dakota. To assess the available stripe rust resistance in the NDSU winter wheat germplasm, two sets of diverse breeding lines were used for stripe rust resistance phenotyping and genotyping by sequencing. The phenotype and genotype data were then used to locate resistance genes through genome wide association study.

Fusarium graminearum (Schwabe), causal organism of fusarium head blight (FHB), has become a major pathogen of wheat (Triticum aestivum L.) throughout North America. Since its discovery in the United States, the disease has spread south and east until at present it is an annual threat for growers of winter wheat in the Mid-Atlantic region. Yield losses for soft red winter (SRW) wheat averaged 908 kg ha-1 in the FHB outbreak of 1998 (Griffey et al., 1999). The economic loss from this single FHB epidemic was an estimated 8.5 million dollars. Environmental conditions favorable for FHB development, including above average rainfall and temperatures during anthesis, have become more common in the Upper-Midwestern wheat-growing region over the past decade, leading to substantial losses in wheat and barley crops. This, coupled with low prices being paid for wheat, has prompted research toward solving the problem of FHB across the nation. The majority of labor and financial resources devoted to FHB research are dedicated to incorporating FHB resistance into adapted wheat lines. While this is a prudent method of combating this disease, this process will take many years to complete. We have examined all FHB assessment parameters, which include FHB incidence, FHB severity, FHB index, percentage fusarium damaged kernels (percentage FDK), and 15-acetyl deoxynivalenol toxin (DON toxin) accumulation, to ascertain which assessment parameters best quantify FHB resistance levels in addition to grain yield and grain volume weight (GVW) losses. FHB index provides the most reliable in-field assessment of a genotype's resistance level, whereas percentage FDK provides a reliable measure of a genotype's resistance level post-harvest. FHB index and percentage FDK are also the most predictive assessment parameters with regard to grain yield and GVW loss. A wide range in both level and type of resistance was observed among genotypes examined in this study. The cultivars Agripro Patton, Ernie, INW9824, Roane, and the experimental line NY87048W-7388 consistently had lower scores for FHB assessment parameters and lower losses of grain yield and GVW. / Master of Science

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