Dissertationen zum Thema „Fusarium Rot“

This thesis had three objectives: i) Determining distribution of FCR and common root rot (CRR) of wheat in Montana; ii) Determining population dynamics between F. pseudograminearum and Bipolaris sorokiniana at different wheat development stages, and iii) Development of an integrated disease management program for Fusarium crown rot (FCR) using biological and fungicide seed treatments, cultivar resistance, and induced systemic resistance (SAR). Surveys of 91 fields over two years using qPCR identified FCR in 57% and CRR in 93% of the fields surveyed. Bipolaris sorokiniana, F. culmorum and F. pseudograminearum were isolated from 15, 13 and 8% of tillers respectively. FCR distribution was highly clustered while CRR was uniformly distributed with soil type, elevation and growing degree days influencing distribution. Data from intensively sampled fields estimated yield losses caused by FCR and CRR at 3.2 to 34.9% with losses influenced by pathogen population. This study is the first time qPCR was used to survey the distribution of FCR and CRR and to study the interaction of the respective pathogens. The effect of F. pseudograminearum and B. sorokiniana inoculum applied singly or in combination at three rates showed high and low rates of F. pseudograminearum inoculum reduced Bipolaris populations, while B. sorokiniana inoculations did not affect Fusarium populations in stems. Populations of both pathogens increased from heading until harvest with Fusarium colonizing stems earlier than Bipolaris. Mixed inoculations increased incidence of infection and co-infection relative to that observed in production fields. Both fungi alone or combined reduced the seedling counts. Grain yield was inversely correlated with Fusarium populations. Difenoconazole-mefenoxam seed treatment reduced FCR severity between 29.3-50% and fungal and bacterial seed treatments were ineffective. The cv. Volt was identified as partially resistant and had the highest levels of chitinase and beta-1, 3-glucanase activity of cultivars evaluated. Induction of SAR by Bacillus mycoides isolate BmJ or acibenzolar Smethyl significantly reduced the severity of FCR compared to water controls. Integration of cultivar resistance plus fungicide seed treatment or SAR induction provided equal control in greenhouse and irrigated trials. In a dryland field trial, integration of all management tools reduced FCR more than individual tools.

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.

Fusarium root rot is one of the most important diseases of pulse crops, with numerous Fusarium spp. comprising the disease complex. Fusarium solani and F. avenaceum have been reported to be major pathogens in the pea root rot complex, and all commonly grown varieties are susceptible. Greenhouse methods to evaluate peas for resistance to Fusarium root rot resulted in inconsistent disease severity across varieties. In 2015, F. avenaceum infested field plots were more heavily damaged based on emergence and yield than F. solani infested plots, and opposite trends were observed in 2016. Differences in root rot severity between years could be due to F. solani infestation causing more damage under warmer temperatures, while plots infested with F. avenaceum caused more damage under cooler temperatures. These results highlight the difficulties observed when screening for soil-borne pathogens, and the increased difficulties when a pathogen complex and changing environmental conditions are involved.

Dry bean and field pea root rots have resulted in substantial yield losses in North Dakota. Root rot symptoms range from small lesions to complete root destruction. Traditional management practices such as seed treatment fungicides and crop rotation have proven insufficient under high disease pressure. The objective of this research was to determine the efficacy of in-furrow fungicide applications for management of dry bean and field pea root rot under field and greenhouse conditions. Fungicides were applied in-furrow at planting on dry beans and field peas. In most trials, the inoculated/non-treated control displayed significantly higher levels of root rot than the non-inoculated/non-treated control. In-furrow fungicides generally reduced root rot severity, sometimes significantly over the seed treatment; however, the level of control varied among hosts and pathogens. The results of these studies indicate that the use of in-furrow fungicides, along with cultural practices, may improve the overall management of root rot.

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.

Thesis (MSc)--Stellenbosch University, 2015
ENGLISH ABSTRACT: Maize (Zea mays L.) is an important crop worldwide and forms the staple diet of many African countries including South Africa. Fusarium ear rot (FER) of maize is caused by a fungus, Fusarium verticillioides, which also produces the fumonisin mycotoxin group. The consumption of fumonisin contaminated maize grain has been associated with serious human and animal health complications. Several South African maize inbred lines exhibiting resistance to FER and fumonisin contamination have been identified. These locally adapted inbred lines could be used to generate mapping populations to identify QTLs associated with resistance to FER and fumonisin contamination. The corresponding markers could be utilised in breeding programmes through marker-assisted selection to ensure the development of commercial cultivars with resistance to FER and fumonisin contamination. In this study, resistant and susceptible maize inbred lines were utilised to commence the development of recombinant inbred line (RIL) populations for the mapping and validation of QTLs associated with FER and fumonisin resistance. One F2 population was phenotypically and genotypically analysed to produce a linkage map for the preliminary identification of QTLs associated with resistance to F. verticillioides infection and fumonisin deposition. A potential QTL for resistance to FER was detected and should be validated across several locations and years in the subsequent RIL population. Additionally, potential resistance barriers of maize to infection by F. verticillioides were investigated by histological studies. The importance of a closed stylar canal in determining resistance to FER was established for nine South African maize inbred lines by means of scanning electron microscopy (SEM). No significant association was observed between a closed stylar canal and the resistance/susceptible status of maize inbred lines before pollination, while the canals appeared closed in all inbred lines following pollination. The results suggest that the stylar canal architecture is not an essential factor determining resistance to F. verticillioides ingress in the maize inbred lines selected for this study. Furthermore, the possibility of resistance to FER and fumonisin contamination being initiated during the seedlings phase of a resistant and susceptible maize inbred line was investigated by means of confocal laser scanning microscopy (CLSM). Fusarium verticillioides growth originating from soil-borne or seed-borne contamination was monitored in various above and below soil plant tissues but no significant difference in the colonisation could be determined between resistant and susceptible maize seedlings. No fumonisin was produced regardless of the inoculation method or resistance status of the plant. These results suggests that the resistant and susceptible maize seedlings used in this study may not be resistant to systemic fungal ingress but may resist the deposition of fumonisins. The resistance associated with the resistant inbred line is not mediated during the seedling phase but potentially through structural and biochemical defence mechanisms during later plant developmental stages.
AFRIKAANSE OPSOMMING: Mielies (Zea mays L.) is ‘n belangrike graangewas wat wêreldwyd geproduseer word en dien as stapelvoedsel in talle Afrika-lande, insluitend Suid-Afrika. Fusarium kopvrot (FKV) in mielies word veroorsaak deur die swam, Fusarium verticillioides, wat ook die fumonisien mikotoksien groepe produseer. Die inname van fumonisien-geïnfekteerde mielies gaan gepaard met ernstige gesondheidsprobleme in mense en diere. Verskeie Suid-Afrikaanse ingeteelde mielielyne, wat weerstandbiedend is teen FKV en fumonisien kontaminasie, is voorheen identifiseer. Hierdie plaaslik-aangepaste teellyne kan gebruik word om kartering populasies te genereer om kwantitatiewe eienskap loci (KEL) te identifiseer wat verband hou met weerstandbiedenheid teen FKV en fumonisien kontaminasie. Die ooreenstemmende merkers kan gebruik word in teelprogramme deur gebruik te maak van merker-geassisteerde seleksie om kommersieële kultivars, wat weerstandbiedend is teenoor FKV en fumonisien kontaminasie, te ontwikkel. In hierdie studie is weerstandbiedende en vatbare mielie inteellyne gebruik om rekombinante inteellyn (RIL) populasies te begin ontwikkel vir die kartering en validasie van KEL’e geassosieer met FKV en fumonisien weerstandbiedenheid. Een F2 populasie was fenotipies en genotipies geanaliseer om ‘n koppeling-kaart te verwek vir die voorlopige identifikasie van KEL’e geassosieer met weerstandigheid tot F. verticillioides infeksie en fumonisein afsetting. ‘n Potensiële KEL vir weerstandbiedenheid is geïdentifiseer, wat verdere bevestiging in die daaropvolgende RIL populasie in verskeie geografiese areas en oor addisionele seisoene, benodig. Potensiële fisiese versperrings teen F. verticillioides tydens mieliesaad infeksie is ook ondersoek met behulp van histologiese studies. Die belangrikheid van ‘n geslote styl-kanaal vir weerstandbiedendheid teenoor FKV is bevestig in nege Suid-Afrikaanse inteellyne deur middel van skandeer elektron mikroskopie (SEM). Geen beduidende verwandskap tussen ‘n geslote styl-kanaal en die weerstandbiedenheid/vatbaarheid van die inteellyne voor bestuiwing is gevind nie, terwyl die kanaal in alle inteellyne gesluit was na bestuiwing. Die resultate dui daarop dat die styl-kanaal argitektuur nie ‘n noodsaaklike faktor is in die bepaling van weestand tot F. verticillioides besmetting in die suiwer mielielyne wat geselekteer was in hierdie studie nie. Verder is die moontlikheid dat weestand tot FKV en fumonisien kontaminasie geïnisieer kan word gedurende die saailing-fase ondersoek in beide ‘n weerstandbiedende en vatbare mielie inteellyn met behulp van konfokale laser skandering mikroskopie (CLSM). Die groei van F. verticillioides afkomstig vanuit die grond of saad is gemonitor in verskeie bo- en ondergrondse plantweefsels, maar geen beduidende verskille in kolonisasie kon opgespoor word tussen weerstandbiedende en vatbare mielie saailinge nie. Geen fumonisien produksie is waargeneem nie, ongeag die innokulasie metode of weerstand-status van die plant. Hierdie resultate dui daarop dat die weerstandbiedende en vatbare mielie saailinge wat in hierdie studie gebruik is moontlik nie weerstandbiedend is teen sistemiese swaminfeksie nie, maar wel weerstand kan bied tot afsetting van fumonisiene. Die weerstand geassosieër met die weerstandbiedende inteellyn word nie bemiddel gedurende die saailingfase nie maar waarskynlik deur strukturele en biochemiese verdedigingsmeganismes tydens latere plant ontwikkelings-stadia.
National Research Foundation (NRF)

Foot rot disease of wheat is caused by the pathogens Fusarium cuImorum, F.avenaceum and Microdochium nivale. Symptoms of foot rot are a general browning of the stem base and leaf sheath. There is a discrepancy between the ability of fungicides to control these pathogens in vivo and in vitro, and no relationship between disease symptom severity and yield loss has been established in wheat. The identification of the causal agents of foot rot disease is not possible from examination of disease symptoms alone. This work showed that the azole fungicides flusilazole and prochloraz inhibited the germination of conidia and mycelial growth of F. culmonon, F. avenaceum and M. nivale in vitro to a varying extent. However, no consistent control of these pathogens in wheat was observed in the field using the same fungicides. Further studies employing a semicontrolled outdoor experiment showed a relationship between density and timing of inoculum application, disease symptom severity and yield loss in wheat artificially inoculated with F. culmorum and M. nivale. Molecular marker systems were used to address the problem of pathogen detection and identification. A Random Amplified Polymorphic DNA (RAPD) assay was developed to differentiate F.culmorum, F.avenaceum and two types of M.nivale (M.nivale var.nivale and M. nivale var .majus) in vitro. Selected RAPD products were cloned and sequenced and species specific primers constructed from this sequence infonnation. These primers were used in the polymerase chain reaction (peR) and were shown to detect the pathogens in host tissue. This technique was adapted by addition of a competitor fragment to the peR reaction resulting in a quantifiable competitive peR assay. Using this method the fungal biomass of each pathogen present in the host tissue could be estimated. The development of these techniques for the identification, detection and quantification of F. cuimorum, F.avenaceum, M.nivale var.nivale and M.nivale var.majus in plant tissue will allow more extensive studies of the epidemiology of these species, the competition between species and the effect of fungicides on these pathogens can be carried out.

La fusariosi della spiga e il marciume al colletto in frumento sono malattie causate da Fusarium graminearum, F. culmorum e F. pseudograminearum. Essi determinano ingenti perdite di raccolto oltre a contaminare il prodotto con micotossine appartenenti alla famiglia dei tricoteceni. I batteri Gram-positivi appartenenti al genere Streptomyces sono ubiquitari nel suolo ed endofiti dei tessuti interni delle radici. Essi producono una vasta gamma di metaboliti secondari con proprietà antimicrobiche e possono essere utilizzati come agenti promotori della crescita delle piante, limitando lo sviluppo dei patogeni. L’obiettivo del presente dottorato di ricerca è stato quello di selezionare ceppi di streptomiceti attivi contro Fusarium spp. in grano. La prima fase dello studio ha permesso di conoscere lo stato dell’arte sull’utilizzo di streptomiceti contro specie micotossigene appartenenti al genere Fusarium (Chapter 1). Successivamente sono state sviluppate strategie innovative per la selezione degli stessi (Chapter 2) e l’efficacia dei ceppi più promettenti è stata poi saggiata in diverse condizioni (Chapter 3). Inoltre, i metaboliti secondari responsabili dell’attività antifungina sono stati caratterizzati (Chapter 4). L’influenza della variabilità dei ceppi di Fusarium spp. e dei substrati colturali sull’attività di biocontrollo è stata valutata tramite saggi di antibiosi. Questi fattori hanno avuto un’influenza significativa nel determinare il livello di attività in vitro. I mezzi standard utilizzati in laboratorio hanno diminuito infatti tale parametro. Inoltre è stata riscontrata un’assenza di correlazione con il livello di biocontrollo ottenuto in pianta. Unica eccezione è per i risultati ottenuti utilizzando un terreno a base di frumento, che ha permesso di osservare un valore di correlazione più elevato con il livello di biocontrollo riscontrato contro marciume radicale in frumento (r = 0.5). Successivamente, al fine di saggiare metaboliti limitanti la produzione di tricoteceni, è stato sviluppato un sistema in micropiastra che misura la fluorescenza emessa da un ceppo di F. graminearum trasformato con il costrutto TRI5::GFP. TRI5 è il primo gene essenziale coinvolto nella via metabolica di produzione di tricoteceni. Da questa prova si è potuto selezionare il ceppo di streptomicete DEF39 che riduce significativamente la produzione di DON. Le potenziali attività di promozione della crescita e di biocontrollo di una selezione dei ceppi più promettenti (N = 21) sono state saggiate in pianta. Gli streptomiceti testati non hanno esibito la capacità di aumentare i parametri di sviluppo dei germinelli di frumento, ma il ceppo DEF09 ha ridotto significativamente il marciume al colletto e la fusariosi della spiga ottenendo livelli di protezioni sopra al 40% e del 60% rispettivamente. Basandosi sui risultati ottenuti, quattro ceppi (DEF09, DEF20, DEF39, DEF48) sono stati applicati su grano sterilizzato testando due tempistiche di trattamento per osservarne la capacità di riduzione della biomassa fungina e della produzione di DON. Inoltre tramite qPCR si è osservata la fitness degli agenti di biocontrollo nelle condizioni testate. Gli streptomiceti, abili colonizzatori del substrato testato, sono stati efficaci nel ridurre la produzione di micotossine, limitando -quando co-inoculati con il patogeno- lo sviluppo dello stesso. I ceppi selezionati agiscono perciò sia contro lo sviluppo fugino e/o contro la produzione di DON. Ulteriori studi saranno necessari per confermarne l’attività in pianta, così come per permettere lo sviluppo di formulati efficaci per limitare la contaminazione da tricoteceni.
Fusarium head blight (FHB), root rot (FRR) and foot rot (FFR) cause important yield losses in wheat. The harvested product is often contaminated with mycotoxins, belonging to the group of trichothecenes. The main causal agents are Fusarium graminearum, F. culmorum and F. pseudograminearum. The biocontrol approach is a feasible option in order to reduce disease severity, as well as trichothecene contamination in grains. Streptomyces spp. are Gram-positive bacteria, ubiquitous in soil and endophytes of inner tissues of plant roots. They produce a wide range of secondary metabolites able to limit pathogen development and disease severity in planta, as well as to enhance plant growth. This PhD project aimed to select Streptomyces strains active within the wheat-Fusarium spp. pathosystem. To achieve this, a detailed literature and patents analysis focused on biocontrol of toxigenic Fusarium spp. was carried out (Chapter 1) and new methodological approaches for antagonist screening have been developed (Chapter 2). Furthermore, the biocontrol efficacy of a selected subset of strains obtained from the culture collection maintained at the Plant Pathology laboratory (DeFENS, University of Milan, Italy) was evaluated in different conditions (Chapter 3) and bioactive metabolites were isolated (Chapter 4). The influence of growth media and Fusarium strain diversity on streptomycete antifungal activity was assessed in dual culture assays. All the factors influenced the level of antifungal activity. The media commonly used for in vitro screening reduced the inhibitory activity of streptomycetes. Overall, results from dual culture assays and level of disease protection observed in planta did not correlate, except for those recorded on a medium based on wheat grains. Indeed, it was the most effective in eliciting antifungal activity and showed the highest correlation (r = 0.5) with FRR inhibition. Subsequently, being TRI5 the first and essential gene involved in trichothecene biosynthetic pathway in Fusarium spp., a microplate bioassay using a TRI5::GFP transformed F. graminearum strain was developed and validated in order to screen the effect of natural products on GFP fluorescence and consequently on trichothecene production. Surprisingly, culture filtrate from DEF39 strain completely suppressed deoxynivalenol (DON) production without affecting fungal growth. The most promising isolates (N = 21) were further characterized for their potential plant growth promotion ability, as well as for their activity against FRR and FFR in wheat seedlings. None of them was able to increase plant growth. However, DEF09 strain exhibited consistent efficacy to limit FRR-FFR symptom severity (protection level > 40%) in soil and soilless conditions. Therefore, a field trial was performed to test its ability to reduce FHB severity, obtaining up to 60% protection. Based on the activity observed from the previous screenings, four promising streptomycetes (DEF09, DEF20, DEF39, DEF48) were applied on sterilized wheat grains (microsilage) at two timepoints of application, in order to evaluate their ability to suppress fungal growth and DON production. Moreover, the fitness of streptomycetes in microsilage conditions was assessed by qPCR analysis. Streptomycetes were able to efficiently colonize the substrate, which resulted in reducing fungal biomass and DON accumulation only when co-inoculated with the pathogen. A pool of promising biocontrol agents has been selected against fungal development and/or DON production. This research highlighted the complexity of finding an efficient screening procedure due to multiple interactions occurring in wheat-Fusarium spp. pathosystem. Further studies will be needed to confirm the activity of the strains in planta. The identification of the mechanisms of action and the molecules involved in the bioactivity of the strains will possibly allow to develop effective treatments limiting trichothecene accumulation in wheat.

Basal rot of Narcissus caused by Fusarium oxysporum f.sp. narcissi is the major limiting factor for the increased export of ornamental bulbs from the United Kingdom. There is no known resistance to the disease amongst commercial cultivars. The purpose of this study was to develop techniques for the rapid detection and identification of the pathogen in bulbs, culture and soil, and to develop serological and biochemical tests to investigate differences amongst isolates of this forma specialis from a range of countries. Significant differences in virulence were observed between the isolates tested, two being identified as avirulent. Results suggest that there is wider morphological variation within this forma specialis than previously recorded. Isolates stored at -80°C have remained viable over a three year period. Thirty three species and varieties of Narcissus and seventeen cultivars were screened for resistance to the pathogen. Accessions were either wholly resistant, partially resistant (a variable portion of the bulbs infected) or susceptible. Accessions identified as resistant are now being used as parents in a resistance breeding programme. F. oxysporum Esp. narcissi isolates of low virulence and avirulence showed greater agglutination of microconidia with Narcissus pseudonarcissus lectin. In addition increased lectin levels were found in cultivars showing greater disease resistance; this suggests a possible host pathogen interaction. Polyclonal antisera were elicited against extracts of isolates and a specific and sensitive enzyme-linked immunosorbent assay was developed for the detection of the pathogen in bulbs. Little cross-reactivity was shown by other bulb-rotting fungi. A direct correlation was observed between positive results in the ELISA and recovery of the pathogen on selective media. The ELISA which was developed will be field evaluated with growers during the coming season. In indirect ELISA and Western blotting of fungal extracts, all fungi outside the genus Fusariwn showed consistently low binding. Isolates of F. oxysporum f.sp. narcissi could be discriminated from other Fusarium species and further split into a series of sub-groups. Avirulent isolates or isolates of low virulence showed differential binding to the PAbs. Other sub-groups were not related to virulence. Western blotting allowed greater differentiation of isolates and bands specific to F. oxysporwn f.sp. narcissi were identified. Serological detection of the pathogen in soil proved possible through the inclusion of a soil enrichment stage with a selective medium.

This study evaluated commercially available seed treatment products for their ability to control Fusarium solani and F. avenaceum, causal agents of root rot in dry edible bean and field peas, respectively, through in-vitro, growth chamber and field trials. Disease severity was assessed using a 0 to 5 scale, and root health parameters were recorded. The in-vitro tests conducted were not considered good predictors of fungicide performance in growth chamber or field trials in case of dry beans for management of F. solani. In case of field peas, in-vitro and growth chamber studies provided consistent results and allowed the identification of fludioxonil, trifloxystrobin and pyraclostrobin as the most effective products to manage F. avenaceum. Overall, integration of chemical seed treatments along with cultural practices; crop varieties partially resistant to root rot, and drench application is necessary to effectively manage Fusarium root rot of dry beans and field peas in field conditions.

Fusarium oxysporum Schl. var. vanillae (Tucker) Gondon is known to cause root rot in Vanilla planifolia Andrews in most regions where it is grown, including the major plantations in Xishuangbanna, Yunnan Province of China. This is of serious economic concern to the Province since the vanilla flavouring extractable from the beans of the plant is a valuable food product and an important export commodity. There are no fungicides registered for the control of Fusarium root rot and the only available chemical control methods are ineffective and cause serious contamination of the soil. Breeding for resistance is difficult when no dominant gene is known or where little information is available on fungal pathogenicity. Biocontrol is the main alternative for disease control in this crop, an attractive approach because of increasing concerns for environmental protection. The investigation considers two biocontrol strategies: first the introduction of virulent, antagonistic, non-pathogenic strains, closely-related to the pathogen, to overcome pathogenic populations in infected soils; second the use of essential oils with antimicrobial properties when applied to infected soils. Pathogenicity tests have been done on 81 out of 87 F. oxysporum isolates collected in Yunnan Province. Among these, 32 isolates were non-pathogenic and 49 were pathogenic. The pathogenicity results showed the complexity of F. oxysporum in Yunnan. Seventeen isolates were recovered from the Daluo plantation, of which 14 were pathogenic isolates and 3 non-pathogenic isolates; 26 from the Menglun plantation, in which 12 were pathogenic and 14 were non-pathogenic; 18 isolates from the Manjingdai plantation, in which 12 isolates were pathogenic, whilst the other 6 were non-pathogenic and 20 were obtained from the plantation in Hekou i County, of which 11 were pathogenic isolates and 9 were non-pathogenic. Genetic diversity within this population of F. oxysporum has been investigated with respect to vegetative compatibility and to determine the relationship between VCGs and virulence. The VCG results showed that the 87 strains of Fusarium oxysporum f.sp vanillae isolated from Yunnan Province were complex. They could be distributed into 12 different VCGs and that a direct relationship between VCGs group and virulence could not be drawn. Two non-pathogenic strains, ML-5-2 and HK-5b-4-1, have been screened from 87 strains as candidate biocontrol agents by pathogenicity and VCG, which are self-incompatible and closely related to the pathogens. These two strains were effective in vanilla root rot control in controlled environments, but their effects in field experiments were less conclusive. Seven essential oils, which have long been regarded as having inhibitory effects on pathogens in nature, have also been investigated as biocontrol agents. Three oils, cinnamon oil, thyme oil and clove oil, were effective in inhibiting the growth of pathogen in vitro. These oils may develop into useful components of different management strategies with non-pathogenic strains. For the future, consideration will need to be given to the mechanism(s) of the interaction of the antagonistic components with the soil microbe population and host plant and also to appropriate formulation, to take account of soil type, crop status, cultural practices, environmental and economic factors. Biocontrol methods have considerable potential but must be acceptable to farmers as part of an overall crop management programme.

[Abstract]Crown rot, caused by Fusarium pseudograminearum (Fpg), is an important soilborne disease of wheat and barley. The degree of crop damage depends on seasonal conditions. Typically, high moisture conditions early in the season encourage seedling infection from stubble residues. Moisture stress later in the season leads to the production of unfilled “whiteheads”. Current control relies on cultural practices and sowing of partially resistant varieties. In order to understand the nature of partial resistance, I have examined the patterns of disease symptom development and pathogen spread in susceptible and partially resistant tissues of both pot-grown wheat, barley and oat seedlings and field-grown inoculated wheat trials. Further research was conducted to determine whether differences in pathogenicity occur amongst a small subset of Australian Fpg isolates. Seedling experiments confirmed that differences in disease ratings between susceptible and partially resistant genotypes are detected in younger leaf sheaths of older seedlings. At later harvest times differences between these genotypes are not significant in older leaf sheaths. Re-isolation of Fpg from inoculated seedlings has shown that each tissue was infected later in partially resistant genotypes compared to susceptible ones with a significantly lower number of isolations recorded at each harvest time in 42 day old seedlings. Barley cultivars were rapidly infected by the pathogen and exhibited high levels of disease symptoms. By comparison levels of infection in oats were low compared to all other genotypes. No significant differences between genotypes were observed in coleoptile tissues, either in fungal colonisation or development of disease symptoms. Disease development in the subcrown internode varied between lines/cultivars but was not representative of the relative susceptibility of each genotype. The pathogen did not appear to invade plant tissue via the vascular system but rather spread directly across the stem from leaf sheath to leaf sheath. Field trials were designed to study disease symptom development and localisation of Fpg hyphae in all expanded tissues (excluding head and roots) in wheat genotypes of known susceptibility to crown rot. Plants were harvested at approximately fortnightly intervals throughout the growing season. The main effects and interactions of harvest, genotype and tiller on each plant part were examined with a detailed statistical analysis of differences seen in these factors between susceptible and partially resistant wheat genotypes, in two inoculated field trials. While differences between genotypes were mostly not significant at each harvest when disease rating or isolations from leaf sheath tissues were examined, important differences between susceptible and resistant genotypes were seen in disease developments and Fpg infections of stem tissue in field trials. Restriction of pathogen growth and symptom development was more pronounced in the tissues of 2-49 (possesses seedling resistance) than in the field resistant Sunco. At present, the mechanisms that lead to these resistance responses are unknown. The pathogenicity study aimed to determine whether 7 Fpg isolates and a mixed inoculum differed in ability to cause crown rot in 9 wheat genotypes ranging in susceptibility to this disease. Although a genotype*inoculum interaction was significant, there is no evidence of stable pathogenic races in the isolates examined in these experiments. The growth of all isolates was partially inhibited in a consistent manner on resistant genotypes when compared to very susceptible genotypes. These results confirm significant differences in the aggressiveness of Fpg isolates on wheat, evidenced by variation in mean disease severity between isolates growing on a range of host genotypes.

Stink bugs, including native brown stink bug (Euschistus servus) and invasive brown marmorated stink bug (Halyomorpha halys), cause damage to a variety of crops including field corn (Zea mays). Frequency and size of stink bug infestations have increased in corn fields in the Mid-Atlantic U.S., and there are growing concerns that these infestations may contribute to reductions in grain quality including increased mycotoxin concentrations. Prior research on native and invasive stink bugs has focused on understanding their biology, the damage they cause, and elucidating effective and economic management strategies. However, few studies examined the potential for stink bugs to facilitate fungal infection and mycotoxin contamination of corn grain. Thus, the objectives of this research were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxigenic Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. A correlation between H. halys feeding injury and fumonisin concentrations was identified, and the ability of H. halys to increase F. verticillioides infection and fumonisin concentrations in corn was demonstrated in field experiments. Fusarium species including fumonisin-producing F. verticillioides and F. proliferatum were isolated from field-collected stink bugs, and in laboratory experiments, E. servus was able to transmit F. verticillioides to non-infected corn ears after feeding on F. verticillioides-infected corn. In field studies, both fungicide and insecticide reduced stink bug-associated mycotoxin concentrations in corn, but levels of control were inconsistent. Thus, additional tactics that target both the stink bug and Fusarium should be implemented to mitigate risks of mycotoxin contamination in corn.
Doctor of Philosophy
Native and invasive stink bugs can severely damage crops including field corn. Frequency and size of stink bug infestations in Mid-Atlantic U.S. corn fields have increased, and there is growing concern that this may contribute to reductions in grain quality. Insect feeding injury is a risk factor for fungal infection and mycotoxin contamination in corn. Mycotoxins are toxic chemicals produced by certain fungi that have detrimental health effects on animals including livestock and humans. The relationship between stink bug feeding injuries and mycotoxin contamination in corn grain is not well understood, and management strategies to minimize the risk of mycotoxin contamination in corn need to be identified. The main goal of this research was to characterize interactions between stink bugs and mycotoxin-producing fungi and identify tactics for controlling both the insect pest and pathogen. Specific objectives were to: 1) assess the relationship between invasive brown marmorated stink bug (H. halys) feeding injuries and fumonisin contamination of field corn in the Mid-Atlantic U.S., 2) determine if stink bugs are a vector for mycotoxin-producing Fusarium spp. in corn, and 3) evaluate the efficacy of pesticides for mitigating stink bug feeding injury and associated mycotoxin contamination in field corn. Results from this work indicated that stink bugs have the ability to cause feeding injuries which facilitate invasion of mycotoxin-producing Fusarium species, leading to increases in mycotoxin concentrations in corn grain. Studies also demonstrated that stink bugs can vector Fusarium species during feeding and increase Fusarium infection of corn resulting in subsequent mycotoxin contamination. Field studies indicated that pesticide applications targeting both the stink bugs and mycotoxigenic fungi may be needed to minimize risk of mycotoxin contamination in corn. However, under low pest pressure, application of pesticides is unlikely to be profitable.

Depuis le début des années 2000 et ce, malgré un haut niveau d’exigence sanitaire, la filière française de l’ail subit des pertes de plus en plus importantes liées à l’émergence d’une maladie fongique entraînant le pourrissement des bulbes lors du stockage. Les pertes subies par certains producteurs peuvent atteindre les 90%. En 2016, une collaboration naît entre INRAE et la filière française afin de comprendre les tenants et aboutissants de cette maladie. Dans le but de développer une méthode de lutte efficace et durable contre la pourriture brune de l’ail il a fallu 1) établir les causes de la maladie, 2) caractériser les agents responsables, 3) élucider le processus de pénétration des gousses et 4) mettre au point une méthode de détection des microorganismes. La première étape a été de constituer une collection de souches à partir d’un échantillonnage de 5493 gousses provenant de 354 têtes collectées pendant 3 saisons, dans les deux bassins principaux de production et sur le plus de variétés d’ail possibles conduisant à la mise en collection de plus de 1000 souches fongiques. Grâce à l’identification moléculaire, deux espèces fongiques isolées de symptômes ont été identifiées : F. proliferatum, majoritaire, et F. oxysporum. Grâce aux séquences d’ADN du gène tef-1α dit de ménage (translation elongation factor 1 alpha) un haplotype majoritaire au sein de l’espèce F. proliferatum a pu être décrit en plus d’une vingtaine d’autres haplotypes appartenant aux deux espèces identifiées. Un test d’agressivité sur ail a été développé afin de valider les postulats de Koch et de caractériser le pouvoir pathogène des souches de F. proliferatum et F. oxysporum. Un panel représentatif de souches a été formé. L’ensemble des souches sélectionnées ont recréé des symptômes sur ail avec des niveaux d’agressivité variables. La diversité d’agressivité des souches a été mise en parallèle avec la diversité génétique observée précédemment : certains haplotypes ont montré une agressivité statistiquement supérieure aux autres. Afin de comprendre quand et comment F. proliferatum arrivait à pénétrer au sein des gousses, des observations histologiques du plateau racinaire (zone de croissance des racines) ont été réalisées en partenariat avec Avignon Université et la plateforme de microscopie 3A. Les résultats montrent que, lorsque le champignon est inoculé à la base de la gousse, il peut pénétrer au niveau de blessures naturelles (ex : sortie des racines), que le plateau racinaire peut constituer un environnement favorable à son développement et que la dégradation des tissus par le mycélium se fait à distance et dans toutes les directions. Enfin, grâce aux séquences d’ADN obtenues lors de l’identification moléculaire, des amorces LAMP ont pu être conçues et testées in vitro, première étape de la méthode de détection de F. proliferatum. Ces amorces ont montré une spécificité vis-à-vis de l’espèce F. proliferatum. Les tests in vivo en cours viendront dessiner la forme finale de la méthode de détection. Cette méthode sera transmise aux acteurs de la filière française et permettra d’élucider le cycle biologique de F. proliferatum sur l’ail et à l’avenir de prévenir le risque sur gousses
Since the early 2000s and despite a high level of sanitary requirements, the French garlic sector has suffered increasingly significant losses linked to the emergence of a fungal disease causing rotting of the bulbs during storage. The losses endured by some producers can reach 90%. In 2016, a collaboration was established between INRAE and the French garlic industry in order to clarify the etiology of this disease and to develop diagnostic methods in view of the long terms goal of developing effective and sustainable methods of control. The specific objectives of this collaboration were to 1) establish the causes of the disease, 2) characterize the causative agents, 3) elucidate the process of clove penetration and 4) develop a method for detecting the pathogen(s) involved.The first step was to build a collection of strains from a sample of 5,493 cloves from 354 heads collected over 3 seasons, in the two main production basins and on as many varieties of garlic as possible leading to a collection of> 1000 fungal strains. Through molecular identification, two fungal species associated with symptoms were identified: F. proliferatum, the dominant pathogen, and F. oxysporum. Based on the DNA sequences of the so-called housekeeping gene (translation elongation factor 1 alpha) a predominant haplotype within the strains of F. proliferatum was described in addition to twenty other minor haplotypes among the two identified species.A test of aggressiveness on garlic was developed to validate Koch's postulates and to characterize the pathogenicity of strains of F. proliferatum and F. oxysporum. A representative panel of strains was chosen. All of the selected strains recreated garlic symptoms with varying levels of disease intensity. The variability of the aggressiveness of the strains was compared with the genetic diversity observed previously: some haplotypes showed statistically superior aggressiveness than others.In order to understand when and how F. proliferatum penetrates into cloves, histological observations of the root plateau (root growth zone) were carried out in partnership with Avignon University and the 3A microscopy platform. The results showed that, when the fungus is inoculated at the base of the clove, it can penetrate at the level of natural wounds (at the exit point of roots, for example). The root plate can constitute an environment favorable to fungal development and degradation of the tissue by the mycelium happens at a distance from the mycelial front and in all directions.Finally, based on DNA sequences obtained during molecular identification, LAMP primers were designed and tested in vitro as the first step in the method for detecting F. proliferatum. These primers showed specificity to F. proliferatum. The in vivo tests in progress will delimit the final form of the detection method. This method will be passed on to stakeholders in the French garlic sector and will make it possible to elucidate the biological cycle of F. proliferatum on garlic and, in the future, to limit the occurrence of this disease

Three cultivators, Rabewerk, Kongskilde and Hiniker, and three intercrops, soybean, lupin and red clover + rye grass were investigated for their impact on the incidence of ear rot of corn, a common disease of maize in eastern Canada. Wheat was seeded in the corn rows to serve as an additional indicator of cultivation and intercropping effects on the pathogen. An artificial inoculum of F. graminearum that produced perithecia and ascospores was used to mimic natural inoculum.
In 1993 and 1994, the infection in the corn was not very severe and there were no differences between the treatments and the controls.
In 1993 and 1994, at both sites, wheat seeds from cultivation trials showed a tendency for greater disease incidence in the non-cultivated herbicide treatment when compared to any of the other cultivator treatments. Cultivators till the soil and bury corn residues, this action led to the destruction of some of the inoculum and a subsequent reduction of the disease incidence in the cultivated plots.
In the intercrop trial of 1993 and 1994, wheat infection was moderate to severe, except at L'Assomption in 1993, but no significant differences were observed among the treatments. It is believed that interplot interference, due to ascospores moving from one plot to the next, masked differences.
The results indicated that weed cultivation would have a negligible or no effect on the development of fusarium ear rot of corn in Quebec. (Abstract shortened by UMI.)

Fusarium verticillioides è l’agente responsabile della Fusariosi della Spiga del mais, contamina la granella con fumonisine, micotossine responsabili di diverse patologie umane e animali. Per la resistenza alla fusariosi e all’accumulo di fumonisine esiste variabilità tra genotipi diversi ma non sono ancora disponibili ibridi immuni. L’obiettivo di questo lavoro è stato quello di individuare marcatori associati alla resistenza a F. verticillioides. Mediante un bioassay è stato testato un association panel per la resistenza a F. verticillioides. Al fine di identificare i marcatori di resistenza sono stati applicati un approccio GWAS e uno per geni candidati. L’analisi GWAS è stata eseguita con 227K SNPs restituendo 206 marcatori significativi. Da un lavoro di RNASequencing sono stati individuati i geni coinvolti nella risposta a F. verticillioides mentre i geni R sono stati recuperati della letteratura scientifica. Genotipi resistenti (CO433 e CO441) e suscettibili (CO354 e CO389) sono stati scelti per individuare polimorfismi nei geni candidati da associare ai fenotipi rilevati mediante il bioassay. Quattro marcatori sono risultati significativi. Infine, la correlazione tra l’incidenza della fusariosi rilevata in campo e mediante bioassay è stata analizzata in una popolazione di 172 RIL derivanti da CO441 x CO354, tuttavia, non è stata individuata alcuna corrispondenza.
Fusarium verticillioides is the causal agent of Fusarium ear rot (FER) in maize and contaminates grains with fumonisin, a family of mycotoxins involved in several human and animal diseases. Quantitative genetic variation exists for resistance to FER and fumonisin contamination among genotypes, however, resistant maize hybrids are currently not available. The aim of this work was the identification of genetic markers associated to resistance against F. verticillioides. A bioassay was used to screen inbred lines of the maize association population for FER resistance, GWAS and candidate gene approaches were applied to identify markers. GWAS was performed using a 227K SNP matrix and resulting in 206 significant markers. Genes involved in F. verticillioides response in developing maize kernels were retrieved from a previous RNASequencing study while maize R genes were retrieved from scientific literature. Resistant (CO433 and CO441) and susceptible genotypes (CO389 and CO354) were selected to amplify and sequence candidate genes. Polymorphisms detected were used to find association with phenotypes scored using the bioassay. Four significant markers were found. Finally, the correlation between FER phenotypes scored in field experiments and bioassay phenotypes was investigated. A population of 172 RILs (CO441 x CO354), was tested. No correlation was found.

Fusarium verticillioides è l’agente responsabile della Fusariosi della Spiga del mais, contamina la granella con fumonisine, micotossine responsabili di diverse patologie umane e animali. Per la resistenza alla fusariosi e all’accumulo di fumonisine esiste variabilità tra genotipi diversi ma non sono ancora disponibili ibridi immuni. L’obiettivo di questo lavoro è stato quello di individuare marcatori associati alla resistenza a F. verticillioides. Mediante un bioassay è stato testato un association panel per la resistenza a F. verticillioides. Al fine di identificare i marcatori di resistenza sono stati applicati un approccio GWAS e uno per geni candidati. L’analisi GWAS è stata eseguita con 227K SNPs restituendo 206 marcatori significativi. Da un lavoro di RNASequencing sono stati individuati i geni coinvolti nella risposta a F. verticillioides mentre i geni R sono stati recuperati della letteratura scientifica. Genotipi resistenti (CO433 e CO441) e suscettibili (CO354 e CO389) sono stati scelti per individuare polimorfismi nei geni candidati da associare ai fenotipi rilevati mediante il bioassay. Quattro marcatori sono risultati significativi. Infine, la correlazione tra l’incidenza della fusariosi rilevata in campo e mediante bioassay è stata analizzata in una popolazione di 172 RIL derivanti da CO441 x CO354, tuttavia, non è stata individuata alcuna corrispondenza.
Fusarium verticillioides is the causal agent of Fusarium ear rot (FER) in maize and contaminates grains with fumonisin, a family of mycotoxins involved in several human and animal diseases. Quantitative genetic variation exists for resistance to FER and fumonisin contamination among genotypes, however, resistant maize hybrids are currently not available. The aim of this work was the identification of genetic markers associated to resistance against F. verticillioides. A bioassay was used to screen inbred lines of the maize association population for FER resistance, GWAS and candidate gene approaches were applied to identify markers. GWAS was performed using a 227K SNP matrix and resulting in 206 significant markers. Genes involved in F. verticillioides response in developing maize kernels were retrieved from a previous RNASequencing study while maize R genes were retrieved from scientific literature. Resistant (CO433 and CO441) and susceptible genotypes (CO389 and CO354) were selected to amplify and sequence candidate genes. Polymorphisms detected were used to find association with phenotypes scored using the bioassay. Four significant markers were found. Finally, the correlation between FER phenotypes scored in field experiments and bioassay phenotypes was investigated. A population of 172 RILs (CO441 x CO354), was tested. No correlation was found.

Fusarium verticillz'oz'des was the dominant Fusarium species isolated in surveys undertaken of both sweet corn and maize in various regions of New South Wales, Australia. The findings are based on the plating of 4105 pieces of corn tissue from which 2500 isolates of Fusarium were produced. Cob rot epidemics occurred in sweet corn in 2002 and maize in 2003 in New South Wales. Consequently surveys were undertaken to establish the species of Fusarium associated with both corn types. The main Fusarium species isolated in the sweet corn survey in 2003 were F. verticillioides, F. proliferatum and F. subglutinans all of which have been reported as causing cob and stalk rot of corn. Fusarium species were commonly isolated fiom stalks, peduncles and kernels of sweet corn irrespective of the presence of cob rot symptoms. F. subglutinans was the dominant species isolated from the Bathurst region, a cooler region at higher altitude than the other com growing areas. In contrast F. verticillz'oides and F. proliferatum were dominant in the hot western flat plains country. There was no reported cob rot on sweet corn in the year of the survey (2003) but it was severe in maize in the Murrumbidgee Irrigation Area. F. verticz'llioz'des, F. proliferatum and F. subglutinans were the dominant Fusarz'um species isolated from maize grains, stalks and peduncles. The season was characterised by above average temperatures and high evaporation rates during crop growth and silking and heavy rainfall close to crop maturity. F. verticillioides was the dominant species isolated from the two areas (Dubbosweet corn and Griffith-maize) where cob rot was observed. These areas are characterised by high temperatures during the growing season and both cob rot incidents were considered to be instigated by this endophytic fungus. F. proliferatum was the second most dominant species identified in the same regions as F. verticillioides and presumably contributed to cob rot symptoms in the maize cob rot event in 2003.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Common bean (Phaseolus vulgaris L.) is grown in Brazil in three different cropping seasons, and in diverse agroecosystems. In such different environments, the crop is exposed to several constraints responsible for yield losses, such as pathogenic organisms. Among common bean relevant diseases, fusarium wilt (Fusarium oxysporum f. sp. phaseoli), dry root-rot (Fusarium solani) and Curtobacterium wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens) have similar symptoms, hindering diagnosis in the field, and whose identification in seed health testing is also limited. In both cases, identification at species level is an important step to manage this root pathogen complex, whose detection can be improved by molecular biology tools. Therefore, this study aimed to: 1) to develop and validate a multiplex PCR (m-PCR) method for simultaneous identification of three common bean pathogens, F. oxysporum f. sp. phaseoli, F. solani and C. flaccumfaciens pv. flaccumfaciens; and 2) develop an isothermal amplification of DNA (LAMP) method to detect of F. oxysporum f. sp. phaseoli on seeds. M-PCR method was developed for identification of isolated colonies, as well as infected seeds. In seeds, total DNA was obtained by alkaline lysis method, which inactivates nucleases during the extraction process. M-PCR allowed the identification of all pathogens, with detection of C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli and F. solani amplicons in agarose gel with respectively 306, 609 and 143 base pairs. Furthermore, m-PCR also reduced costs and time to detect Fusarium oxysporum f. sp. phaseoli from 10 days to three hours. It was not possible to develop an optimized protocol for detection of F. oxysporum f. sp. phaseoli by the LAMP method, using only the tf1 gene for design of primers, since such primers were functional only for amplifying large amounts of target DNA. Based on the negative results with LAMP, it is suggested that further studies should be performed using other DNA sequences available in GenBank database.
O feijoeiro-comum (Phaseolus vulgaris L.) é cultivado durante todo o ano no território brasileiro, em três épocas distintas e em vários agroecosistemas. Nestes ambientes distintos, a cultura está exposta a diversos fatores que causam perdas de rendimento, como o ataque de patógenos. Dentre as doenças do feijoeiro-comum encontram-se a murcha-de-fusarium (Fusarium oxysporum f. sp. phaseoli), a podridão-radicular-seca (Fusarium solani) e a murcha-de-curtobacterium (Curtobacterium flaccumfaciens pv. flaccumfaciens) que apresentam sintomas semelhantes, dificultando seu diagnóstico no campo, e cuja identificação em testes de sanidade de sementes também é limitada. Em ambos os casos, a identificação em nível de espécie é uma importante etapa do manejo deste complexo de patógenos, cuja detecção pode ser aperfeiçoada com a adoção de ferramentas de biologia molecular. Portanto, este estudo teve como objetivos: 1) Desenvolver e validar um método de multiplex PCR (m-PCR) para identificação simultânea de três espécies de patógenos do feijoeiro-comum, F. oxysporum f. sp. phaseoli, F. solani e C. flaccumfaciens pv. flaccumfaciens; e 2) desenvolver a técnica de amplificação isotérmica de DNA (LAMP) para detecção de F. oxysporum f. sp. phaseoli em sementes. O método de m-PCR foi desenvolvido para identificação de colônias isoladas bem como sementes infectadas. Nas sementes, o DNA total foi obtido pela lise alcalina, método que inativa nucleases durante o processo de extração. A m-PCR possibilitou a identificação de todos os patógenos, com detecção de C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli e F. solani em bandas formadas em gel de agarose respectivamente com 306, 609 e 143 pares de base. Além disso, a extração do DNA total das sementes pela lise alcalina em combinação com a m-PCR também possibilitou redução de custos e tempo de realização do diagnóstico de Fusarium oxysporum f. sp. phaseoli, de 10 dias para três horas. Não foi possível estabelecer um protocolo otimizado para detecção de F. oxysporum f. sp. phaseoli pelo método LAMP, utilizando somente o gene tf1 para desenho dos iniciadores, uma vez que, os iniciadores revelaram-se funcionais apenas para a amplificação com grandes quantidades de DNA alvo. Diante dos resultados obtidos com LAMP, sugere-se que estudos posteriores sejam realizados empregando outras sequências de DNA disponíveis no banco de dados GenBank.

Common bean (Phaseolus vulgaris L.) is the most produced legume in the U.S. and worldwide. Fusarium root rot (FRR) is a widespread soil-borne diseases causing up to 86% yield reduction in beans. Large-seeded cultivars are usually susceptible to root rot. Finding FRR resistant genotypes under naturally infected soil and mapping genomic regions involved in its resistance were the main objectives in this research. In addition, halo blight, days to flower, growth habit, plant survival, seed weight, and seed yield were studied. Fusarium root rot and halo blight diseases were highly epidemic during the research period. The objectives were accomplished through two consecutive steps. First, phenotyping a set of genotypes from Andean diversity panel (ADP) under field conditions during three years starting with 310 genotypes in 2013. A Randomized Incomplete Block Design with two replications was used as the experimental design. From three years phenotypic data, ADP462-PI527540B, ADP48-W6_6534, ADP624-Dolly, ADP68-Soya, and ADP438-46_1 genotypes were resistant to FRR and ADP73-Masusu, ADP601-Camelot, ADP636-Montcalm, and ADP511-Canario were susceptible. In addition, ADP84-Kablanketi-defu, ADP55-Kabuku, ADP122-Kranskop, ADP454-INIAP429, and ADP50-Salunde were among the most resistant to halo blight and ADP638-Redhawk, ADP676-CELRK, ADP677-Etna, ADP242-G9013, and ADP269-G13092 were among the most susceptible. Genotypes ADP48-W6_6534, ADP624-Dolly, ADP438-46_1, and VAX3 (check) were resistant to both diseases. These genotypes can be used as parents in the bean breeding programs. Second, for GWAS, 3525 filtered single nucleotide polymorphism (SNP) markers of 246 Andean genotypes were used to find significant (P?0.001) trait-marker associations. After correcting for population structure and relatedness, genomic regions on three chromosomes were associated with five traits. The study provided insights into the genetic architecture for FRR, halo blight, days to flower, growth habit and plant survival. Resistant genotypes can be used in the breeding programs, genomic regions should be validated before using as molecular markers to accelerate the breeding process.
Senescyt, Ecuador
Northarvest Bean Growers Association, ND/MN

Crown rot is one of the most important postharvest diseases with a great negative impact on banana fruit quality. Bananas are harvested green and many packaging processes are carried out before coming on the market. The infections occur at harvest, but the symptoms appear after overseas transportation. Different pathogens are involved in crown rot, varying according to farming area. Little is known about its etiology and causal agents in organic farming. Dominican Republic is one of the leading exporters of organic bananas, and therefore, in this PhD thesis, five organic farms and their corresponding packing stations located in Valverde were investigated. To the best of our knowledge this is the first study covering Dominican Republic and it focused in particular on: the disease etiology, conditions, infection time and mechanisms that determine its development. Over a period of three years, 558 banana hands were collected and a total of 5000 fungal colonies were obtained from the crown tissues and 1750 representative colonies were purified. The identification of mycoflora associated with crown tissues was carried out with the final aim to search for disease management strategies compatible with organic production. Fungi were found in all the analyzed samples collected from various processing stages: from field to packing houses, and obtained in high rate starting from field from flowers as well as crown parts. The diffusion of the pathogen inoculum occurs principally during the banana processing, especially during the dehanding and in washing tanks. The final crown trimming followed by washing and quality of water used in the application of protective products were the critical points of crown infections. Five hundred and eighteen representative colonies were characterized and identified using morphological and molecular methods. The fungal community was dominated by Fusarium, the most frequent genus (55%) found in more than 80% of all analyzed samples. It was represented by nine species; F. incarnatum 53%, F. verticillioides 12%, F. sacchari 12%, F. proliferatum 7%, and F. solani 6%. Strains belonging to eight less frequent genera were represented by Colletotrichum musae 7% and found in 13% of all samples; Lasiodiplodia theobromae 4% and L. pseudotheobromae 1%, both found in 7% of all samples; Nigrospora sp. 11%, Alternaria spp. 6%, Phoma spp. 2%, Pestalotiopsis sp. 2%, Curvularia spp. 1% and Microdochium sp. 1%. Considering the main genera, the results based on morphological and molecular aspects showed a high variability among strains. By conducting the experimental inoculation trials, C. musae strains resulted from the most virulent among different species, followed by F. sacchari, L. theobromae, L. pseudotheobromae and F. verticillioides. The remaining strains had low pathogenicity, and their role could be ancillary in the crown rot development, or could be considered saprophytic. Summarizing the isolation frequency and pathogenicity tests, F. incarnatum strains played the main role in crown rot disease of organic bananas in the investigated areas.

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)

Fusarium verticillioides è responsabile della fusariosi della spiga in mais e della contaminazione della granella con micotossine. Sono state individuate le regioni geniche e i geni candidati per la resistenza a Fusarium dal confronto tra una linea di mais resistente (CO441) e una suscettibile (CO354), impiegando tre diversi approcci: analisi QTL, analisi trascrittomica (RNASeq) e analisi metabolomica. 184 famiglie F2:3 (CO441xCO354) sono state valutate in due diversi ambienti nell’anno 2011 e inoculate artificialmente con due diverse tecniche (forchetta e stuzzicadente). E’ stata rilevata una significativa variazione genotipica in risposta all’infezione. Sulla base di una mappa preliminare di linkage molecolare contenente 74 marcatori microsatelliti polimorfici, sono stati determinati 8 QTLs comuni alla resistenza alla fusariosi della spiga e alla riduzione della contaminazione da fumonisine. Sono stati considerati geni candidati per la resistenza i geni differenzialmente espressi, risultanti dall’ RNASeq, in semi di mais CO441 prima e 72 ore dopo l’infezione. I metaboliti putativi correlati alla resistenza sono stati rilevati tramite high resolution LC-MS in entrambe le linee di mais. I geni candidati e i metaboliti mappano in pathway coinvolti nei meccanismi di difesa: fenilalanina, tirosina e triptofano biosintesi, fenilpropanoidi e flavonoidi biosintesi, metabolismo dell’acido linoleico e α-linolenico. Abbondanti trascritti derivano dalla biosintesi dei terpenoidi e dei diterpenoidi. Nei geni candidati verranno ricercati polimorfismi fra le due linee di mais e che andranno ad arricchire la mappa di linkage molecolare.
Fusarium verticillioides is responsible for Fusarium ear rot in maize and mycotoxin contamination of grain. Genomic regions and candidate genes for resistance to Fusarium were detected through the comparison of resistant (CO441) and susceptible (CO354) maize lines, by following three different approaches: Quantitative Trait Locus (QTL), transcriptomic (RNASeq) and metabolomic analyses. 184 F2:3 families (CO441xCO354) were evaluated in two different environments in 2011 and artificially infected with two side-needle inoculation methods (pin-bar and toothpick). Significant genotypic variation in response to infection was detected. On the basis of a genetic draft map containing 74 polymorphic SSRs markers, 8 common QTLs for Fusarium ear rot resistance and fumonisin contamination reduction were revealed. Candidate genes for resistance resulted from differentially expressed genes before and 72 hours post infection of CO441 kernels through RNASeq technology. Putative metabolites related to resistance were detected through high resolution LC-MS in both maize lines. Candidate genes and metabolites mapped in pathways involved in defense mechanism: phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid and flavonoid biosynthesis, linoleic and α-linolenic acid metabolism. Abundant genic transcripts derived from terpenoid and diterpenoid biosynthesis. Candidate genes will be screened for polymorphisms between the two maize lines in order to enrich the linkage map.

Fusarium verticillioides è responsabile della fusariosi della spiga in mais e della contaminazione della granella con micotossine. Sono state individuate le regioni geniche e i geni candidati per la resistenza a Fusarium dal confronto tra una linea di mais resistente (CO441) e una suscettibile (CO354), impiegando tre diversi approcci: analisi QTL, analisi trascrittomica (RNASeq) e analisi metabolomica. 184 famiglie F2:3 (CO441xCO354) sono state valutate in due diversi ambienti nell’anno 2011 e inoculate artificialmente con due diverse tecniche (forchetta e stuzzicadente). E’ stata rilevata una significativa variazione genotipica in risposta all’infezione. Sulla base di una mappa preliminare di linkage molecolare contenente 74 marcatori microsatelliti polimorfici, sono stati determinati 8 QTLs comuni alla resistenza alla fusariosi della spiga e alla riduzione della contaminazione da fumonisine. Sono stati considerati geni candidati per la resistenza i geni differenzialmente espressi, risultanti dall’ RNASeq, in semi di mais CO441 prima e 72 ore dopo l’infezione. I metaboliti putativi correlati alla resistenza sono stati rilevati tramite high resolution LC-MS in entrambe le linee di mais. I geni candidati e i metaboliti mappano in pathway coinvolti nei meccanismi di difesa: fenilalanina, tirosina e triptofano biosintesi, fenilpropanoidi e flavonoidi biosintesi, metabolismo dell’acido linoleico e α-linolenico. Abbondanti trascritti derivano dalla biosintesi dei terpenoidi e dei diterpenoidi. Nei geni candidati verranno ricercati polimorfismi fra le due linee di mais e che andranno ad arricchire la mappa di linkage molecolare.
Fusarium verticillioides is responsible for Fusarium ear rot in maize and mycotoxin contamination of grain. Genomic regions and candidate genes for resistance to Fusarium were detected through the comparison of resistant (CO441) and susceptible (CO354) maize lines, by following three different approaches: Quantitative Trait Locus (QTL), transcriptomic (RNASeq) and metabolomic analyses. 184 F2:3 families (CO441xCO354) were evaluated in two different environments in 2011 and artificially infected with two side-needle inoculation methods (pin-bar and toothpick). Significant genotypic variation in response to infection was detected. On the basis of a genetic draft map containing 74 polymorphic SSRs markers, 8 common QTLs for Fusarium ear rot resistance and fumonisin contamination reduction were revealed. Candidate genes for resistance resulted from differentially expressed genes before and 72 hours post infection of CO441 kernels through RNASeq technology. Putative metabolites related to resistance were detected through high resolution LC-MS in both maize lines. Candidate genes and metabolites mapped in pathways involved in defense mechanism: phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid and flavonoid biosynthesis, linoleic and α-linolenic acid metabolism. Abundant genic transcripts derived from terpenoid and diterpenoid biosynthesis. Candidate genes will be screened for polymorphisms between the two maize lines in order to enrich the linkage map.

A cultura do milho é responsável por 40% dos grãos produzidos no país, sendo considerada um dos segmentos mais importantes para a economia do agronegócio brasileiro. Devido ao seu alto valor nutritivo, o milho possui grande suscetibilidade ao crescimento de fungos, com destaque para Fusarium verticillioides, que além de causar danos nas raízes, colmos e espigas, é responsável por produzir fumonisinas, um grupo de micotoxinas termoestáveis e possivelmente carcinogênico, ao homem e animais. É sabido que apesar da importância não existem muitas informações associando a influência de diferentes fatores sobre a sua produção, principalmente nas condições tropicais brasileiras. Desta forma, estudos foram realizados com o objetivo geral de avaliar a influência de fatores ambientais, genótipos e métodos de inoculação de F. verticillioides na severidade da podridão de espigas, qualidade sanitária das sementes, produtividade do milho, na produção de fumonisinas e histopatologia em frangos de corte. As investigações aqui relatadas foram divididas em quatro capítulos. No primeiro capítulo, avaliou-se o efeito de fatores ambientais, genótipos e métodos de inoculação de F. verticillioides na produção de fumonisinas em grãos de milho. No segundo capítulo, comparou- se em locais diferentes métodos de inoculação artificial (spray, injetável) e natural de F. verticillioides em dez genótipos de milho pela quantificação da severidade da doença e produtividade. No terceiro capítulo, realizou-se um levantamento da população de F. verticillioides através de análise sanitária e transmissão do fungo à campo das plantas para as sementes e das sementes para as plântulas inoculados com diferentes métodos de inoculação. Por fim, o quarto capítulo, avaliou-se na safra de 2015/2016, procedentes de lavouras experimentais contendo cultura de milho, híbridos comerciais inoculados com F. verticillioides e determinou-se os parâmetros zootécnicos e histopatológicos em frangos de corte. O delineamento experimental para o primeiro e segundo capítulos, desenvolvidos em campo, foi igualmente realizado nos quatros locais estudados, sendo o de blocos ao acaso em fatorial de 10x3, com três repetições, sendo dez genótipos de milho e três métodos de inoculação. No terceiro capítulo, desenvolvido em laboratório, os experimentos tiveram o delineamento inteiramente casualizado, em fatorial de 10x3 com quatro repetições, sendo dez genótipos de milho e três métodos de inoculação. Utilizou-se as sementes provenientes dos híbridos que foram inoculados pelos três métodos de inoculação. Cada unidade experimental consistiu de um Gerbox contendo 25 sementes. No quarto capítulo, o experimento foi conduzido no aviário experimental da Escola de Medicina Veterinária e Zootecnia da Universidade Federal do Tocantins, no campus de Araguaína-TO. Foram utilizados 160 pintos de um dia, frangos de corte da linhagem Cobb 500, distribuídos em delineamento experimental inteiramente casualizado, com dois tratamentos (ração basal de milho com ausência de fumonisina e a ração de grãos de milho com fumonisina), com 10 repetições contendo 8 aves cada. Todas as análises da concentração fumonisinas foram realizadas no Laboratório de Micotoxologia (LAMIC), da Universidade Federal de Santa Maria (UFSM), e utilizou-se a metodologia de quantificação denominada LC-MS/MS (Liquid Chromatography Mass Spectrometry) POP 45. Com relação aos resultados obtidos, para o primeiro capítulo, concluiu-se que não foi possível assegurar que grãos sintomáticos e assintomáticos quanto à infecção de Fusarium verticillioides, estejam livres de contaminação por fumonisinas e os métodos de inoculação por aspersão e o inoculado no centro da espiga não influenciaram em diferentes concentrações de fumonisinas. A produção de fumonisinas nos grãos de milho é potencializada por condições ambientais de temperatura elevada e pela susceptibilidade do genótipo e alta severidade de F. verticillioides. Nestas condições, houve tendência de produzir maiores teores de fumonisinas para a maioria dos genótipos avaliados. A produtividade de grãos foi reduzida pela incidência de F. verticillioides. Para o segundo capítulo, verificou-se que o método de inoculação artificial através de aspersão no estilo-estigma e o injetável no centro da espiga, proporcionaram maior intensidade da podridão da espiga, possibilitando diferenciar do método natural. Em Planaltina, verificou-se que o método de inoculação por aspersão resultou em maior severidade da doença, causada por Fusarium verticillioides do que o método de inoculação injetável no centro da espiga. Os híbridos de milho P4285, DKB390PRO2 e DKB310PRO2 foram os mais resistentes à podridão da espiga, nos quatro ambientes, enquanto que o híbrido 32R48YH apresentou a maior severidade da doença. O método de inoculação artificial foi o mais eficiente em ambientes de climas amenos e esta informação é importante na padronização do método para seleção e caracterização de genótipos de milho em programas de melhoramento. Para o terceiro capítulo, concluiu-se que as sementes oriundas de espigas inoculadas com spray apresentaram maior grau de incidência do patógeno, sendo este, o método mais eficiente de inoculação artificial de F. verticillioides na fase R1 para a avaliação de emergência de sementes. Os híbridos 32R48YH e o DKB240PRO2 tiveram o maior nível de infecção por F. verticillioides pois obtiveram a maior incidência do fungo. Enquanto que os híbridos P3630H e P4285H demostraram ser os mais resistentes, pois obtiveram as menores incidência do patógeno. Os híbridos P3250, 32R48YH e 30K75Y apresentaram menor percentagem de emergência quando a inoculação foi realizada pelo método injetável enquanto que os híbridos P4285H, 30K75Y e DOW30A37PW apresentaram menor percentagem de emergência quando a inoculação foi realizada pelo método spray. A transmissão da semente para a planta foi confirmada pela análise molecular. Verificou- se que, apesar das plantas não terem apresentado sintomas visíveis, pôde-se confirmar que houve infecção latente. A técnica de PCR foi capaz de revelar a presença do patógeno nas sementes de milho em menor nível de incidência avaliado (1%). No quarto capítulo, foi concluído que a concentração de 2,78μg/g de fumonisina determinada na ração não foi suficiente para provocar alterações nos parâmetros zootécnicos (peso das aves e no consumo de ração) e histopatológico (tecidos do intestino, fígado e coração) de aves da linhagem COBB 500, alimentadas até o 21° dia. As análises dos parâmetros zootécnicos e histopatológicos permitiram revelar concentrações mínimas e seguras de fumonisina que não provocaram danos toxicológicos em aves da linhagem COBB 500. Este é um importante parâmetro que pode contribuir nas etapas de produção de carnes e de produtos avícolas.
The corn crop is responsible for 40% of the grains produced in the country, being considered one of the most important segments for the Brazilian agribusiness economy. Due to its high nutritional value, maize is highly susceptible to fungus growth, especially Fusarium verticillioides, which besides causing damage to the roots, stalks and ears, is responsible for producing fumonisins, a group of thermostable mycotoxins and possibly carcinogenic, to man and animals. It is known that despite the importance, there is not much information associating the influence of different factors on its production, mainly in Brazilian tropical conditions. Studies were carried out with the general objective of evaluating the influence of environmental factors, genotypes and methods of inoculation of F. verticillioides on the severity of ear rot, seed quality, maize productivity, fumonisin production and histopathology in broilers. The investigations reported here were divided into four chapters. In the first chapter we evaluated the effect of environmental factors, genotypes and methods of inoculation of F. verticillioides on the production of fumonisins in corn grains. In the second chapter, was compared in different locations, methods of artificial (spray and injectable) and natural inoculation of F. verticillioides in ten maize genotypes by quantification of disease severity and productivity. In the third chapter a population of F. verticillioides was carried out through sanitary analysis and transmission of the fungus from plants to the seeds and from seeds to the seedlings inoculated with different methods of inoculation. Finally, the fourth chapter evaluated the harvest of 2015/2016 coming from crops containing commercial hybrids corn inoculated with F. verticillioides and the zootechnical and histopathological parameters were determined in broilers. The experimental design for the first and second chapters, conducted in the field, was also performed in the four localities studied, being a randomized block design in a 10x3 factorial scheme, with three replications, being ten maize genotypes and three inoculation methods. In the third chapter the experiments had a completely randomized design, in a 10x3 factorial scheme with four replications, being ten maize genotypes and three inoculation methods. Were used seeds from the hybrids that were inoculated by the three methods of inoculation. Each replicate consisted of a Gerbox containing 25 seeds. In the fourth chapter, the experiment was conducted in the experimental aviary of the School of Veterinary Medicine and Animal Science of the Federal University of Tocantins, on the campus of Araguaína-TO. A total of 160 broilers of Cobb 500 lineage (of one day old), were used in a completely randomized experimental design with two treatments (basal corn meal with no fumonisin and corn grain feed with fumonisin), with 10 replicates containing 8 broiler chicks each. All fumonisin concentration analyzes were performed at the Laboratory of Mycotoxology (LAMIC), Federal University of Santa Maria (UFSM), and the quantitative methodology called LC-MS / MS (Liquid Chromatography Mass Spectrometry) POP 45 was used. Regarding the results obtained, for the first chapter, it was concluded that it was not possible to assure that symptomatic and asymptomatic grains for Fusarium verticillioides infection are free from fumonisin contamination and the methods of inoculation by spraying and inoculation in the center of the ear did not influence different concentrations of fumonisins. Fumonisin production in maize grains is potentiated by high temperature environmental conditions and the susceptibility of the genotype and high severity of F. verticillioides. Under these conditions, there was a tendency to produce higher levels of fumonisins for most genotypes evaluated. The grain yield was reduced by the incidence of F. verticillioides. For the second chapter, it was verified that the method of artificial inoculation by sprinkling in the style-stigma and the injectable in the center of the spike, provided a greater intensity of the rot of the ear, allowing to differentiate of the natural method. In Planaltina, it was verified that the spray inoculation method resulted in a greater severity of the disease, caused by F. verticillioides than the injectable inoculation method in the center of the spike. Hybrids of maize P4285, DKB390PRO2 and DKB310PRO2 were the most resistant to ear rot in the four environments, while the hybrid 32R48YH showed the highest severity of the disease. The artificial inoculation method was the most efficient in mild climate environments and this information may be important in the standardization of the method for selection and characterization of maize genotypes in breeding programs. For the third chapter, it was concluded that the seeds from ears inoculated with spray presented a higher level of incidence of the pathogen, being this the most efficient method of artificial inoculation of F. verticillioides in the R1 phase for seed emergence evaluation. The hybrids 32R48YH and DKB240PRO2 had the highest level of infection by F. verticillioides because they had the highest incidence of the fungus. While the hybrids P3630H and P4285H proved to be the most resistant because they obtained the lowest incidence of the pathogen. Hybrids P3250, 32R48YH and 30K75Y presented a lower percentage of emergence when the inoculation was performed by the injectable method, while the hybrids P4285H, 30K75Y and DOW30A37PW showed a lower percentage of emergence when the inoculation was performed by the spray method. The transmission of the seed to the plant was confirmed by molecular analysis. It was found that, although the plants showed no visible symptoms, it was possible to confirm that there was a latent infection. The PCR technique was able to reveal the presence of the pathogen in maize seeds at a lower incidence level (1%). In the fourth chapter, it was concluded that the concentration of 2.78 μg/g of fumonisin determined in the diet was not sufficient to cause alterations in the zootechnical parameters (broilers weight and feed consumption) and histopathological parameters (intestinal, liver and heart tissues) of broilers of the COBB 500 lineage, fed until the 21st day. Analyzes of the zootechnical and histopathological parameters allowed to reveal minimum and safe concentrations of fumonisin that did not cause toxicological damages in broilers of Cobb 500 lineage. This is an important parameter that can contribute in the stages of production of meat and broiler chicks products.

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010.
ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs.
AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.

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PROMOP
Gibberella ear rot (GER) caused by Fusarium graminearum reduces the yield and quality of maize grain, interfering in the presence of damaged grains (DG). The disease control is hampered by unavailability of resistance hybrids and effectiveness of cultural practices to reduce fungal inoculum sources. The objective of this study was to evaluate the effect of preventative and curative fungicide application during maize silking on the GER control and the effect on grain yield (GY) and incidence of DG. The study was conducted in experimental area of Santa Catarina State University, in Lages, Brazil in two sowing dates in 2014/15 growing seasons, using the hybrid P32R48YH. Two F. graminearum isolates, one from Minas Gerais (MEMR0004) and other from Paraná state (MEMR0005) were inoculated in the maize ears five days after silking. Eight fungicides belonging to the chemical group of triazoles, triazolinthione, strobilurins, and benzimidazole, individually or in combination were tested. Maize plants inoculated with fungus without fungicide applicaton were used as experiment control. An application with different fungicides was carried out on tassels with pressurized spray with flow rate of 200 L ha-1, 48 hours before inoculation (preventive action) or 48 hours after inoculation (curative action). There was homoscedasticity between sowing dates and the data were analyzed together. Differences statistics were not found between treatments to GER severity and DG. Preventative application of fungicides (mean values) to MG isolate reduce GER severity and increased GY differing from curative application. Isolated PR showed higher percentages of DG differing from the isolated MG. In PR isolated curative application of fungicides affected the GY with a significant difference between treatments. The fungicides trifloxystrobin + prothioconazol and azoxystrobin + cyproconazol + carbendazim showed stability in control of GER keeping the lower severity to 46%. These fungicidal beyond carbendazim used alone reduced the DG incidence. The tebuconazol + trifloxystrobin and azoxystrobin + cyproconazol + carbendazim in preventive application maintained GY higher than control, in curative were tebuconazol, trifloxystrobin + prothioconazol and methyl thiophanate. The fungicides application on corn silking is a strategy control that can be used in the integrated management of GER to maintain hybrid potential production and grain quality due to reduction in the percentage of DG
A podridão da espiga de giberela (PEG), causada pelo fungo Fusarium graminearum, reduz o rendimento e a qualidade de grãos de milho, interferindo na presença de grãos avariados (GA). O controle da doença é dificultado pela indisponibilidade de híbridos resistentes e pela baixa eficácia de práticas culturais em reduzir as fontes de inóculo do fungo. O objetivo deste trabalho foi avaliar o efeito da aplicação preventiva e curativa de fungicidas no espigamento do milho no controle da PEG e na influência sobre rendimento de grãos (RG) e incidência de GA. O estudo foi conduzido na área experimental da Universidade do Estado de Santa Catarina, no município de Lages, Brasil em duas épocas de semeadura na safra 2014/15, utilizando o híbrido P32R48YH. Dois isolados de F. graminearum um proveniente de Minas Gerais (MEMR0004) e outro do Paraná (MEMR0005) foram inoculados nas espigas cinco dias após emissão dos estilos-estigmas. Oito fungicidas pertencentes aos grupos químicos dos triazois, triazolintiona, estrobilurinas e benzimidazóis isolados ou em mistura foram testados. Plantas de milho inoculadas sem aplicação de fungicidas foram utilizadas como controle experimental. Uma aplicação dos diferentes fungicidas foi realizada sobre os pendões com auxílio de pulverizador pressurizado com vazão de 200 L ha-1, 48 horas antes da inoculação (ação preventiva) e 48 horas depois da inoculação (ação curativa). Houve homocedasticidade entre as épocas de semeadura sendo os dados analisado de forma conjunta. Não houve diferença estatística na severidade da PEG e GA entre os tratamentos. Aplicação preventiva de fungicidas (valores médios) no isolado MG reduziu a severidade da PEG e aumentou o RG diferindo da aplicação curativa. O isolado PR apresentou maiores percentuais de GA diferindo do isolado MG. No isolado PR a aplicação curativa de fungicidas afetou o RG havendo diferença significativa entre os tratamentos. Os fungicidas trifloxistrobina + protioconazole e azoxistrobina + ciproconazole + carbendazim apresentaram estabilidade no controle da PEG mantendo a severidade inferior a 46%. Esses fungicida além do carbendazim utilizado isoladamente reduziram a incidência de GA. Os fungicidas tebuconazole + trifloxistrobina e azoxistrobina + ciproconazole + carbendazim aplicados de forma preventiva mantiveram o RG superior ao controle, em aplicações curativas foram tebuconazol, trifloxistrobina + protioconazole e tiofanato metílico. A aplicação de fungicidas no espigamento do milho é uma estratégia de controle que pode ser utilizada no manejo integrado da podridão da espiga de giberela com a finalidade de manter o potencial produtivo do hibrido e a qualidade de grãos devido a redução do percentual de GA

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Universidade Estadual Paulista (UNESP)
O trabalho objetivou verificar os fatores químicos e microbiológicos do lodo de esgoto e do solo responsáveis pela incidência da podridão do colmo do milho, causada por Fusarium em solo fertilizado com lodo, e foi desenvolvido em condições de campo, numa área que vem sendo tratada com lodo e cultivada com milho desde 1999 em latossolo vermelho distróférrico (textura argilosa), localizada no município de Jaguariúna/SP. Os dois lodos utilizados foram obtidos nas Estações de Tratamento de Esgoto de Franca e de Barueri/SP. Os lodos foram incorporados ao solo nas concentrações de 0, 1, 2, 4 e 8 vezes a dose recomendada com base no teor de nitrogênio, em dois cultivos sucessivos de milho (Savana 133S) nas safras 2001/2002 e 2002/2003, em parcelas de 200 m2. As doses de lodo foram comparadas com a adubação mineral recomendada para a cultura do milho. Os tratamentos foram distribuídos em delineamento experimental casualizado em blocos e com três repetições. Foram verificadas, nas safras de 2001/2002 e 2002/2003, diferenças significativas entre as doses de lodo de esgoto, sobre a variação de Fusarium spp. no solo, na rizosfera, na raiz, no colmo e na semente, bem como sobre a incidência de plantas com podridão no colmo e nas espigas. As análises de regressão mostraram que a porcentagem de plantas doentes foi positivamente correlacionada com a concentração dos lodos incorporados ao solo. Os coeficientes de determinação para o cultivo de 2001/2002 foram de R2=0,61 e R2=0,32, enquanto para o cultivo de 2002/2003 foram de R2=0,76 e R2=0,45, para os lodos de Franca e Barueri, respectivamente. As concentrações de lodos também apresentaram correlação positiva com a comunidade de Fusarium do solo e da rizosfera e com a incidência de Fusarium na raiz, no colmo e na semente. As correlações entre a comunidade de Fusarium do solo e da rizosfera e o percentual de plantas... .
This work aimed at verifying the chemical and microbiological sewage sludge and soil factors re 0sponsible for the incidence of the corn stalk rot caused by Fusarium in soil fertilized with sludge and has been developed in field conditions, in an area that has been treated with sludge and cultivated with corn since 1999, in red dystroferric latosol (clayey texture), in Jaguariúna/SP. Both sludges used were obtained in the sewage sludge treatment stations of Franca and Barueri/SP. Sludges were incorporated into the soil in concentrations of 0, 1, 2, 4 and 8 times in relation to the recommended rate based on the nitrogen contents, in two successive corn cultures (Savana 133S), in 2001/2002 and 2002/2003, in plots of 200 m2. Sludge doses were compared to the mineral fertilization recommended to the corn culture. The treatments were distributed in a randomized design experimental drawing in blocks and with three repetitions. Significant differences were verified in both 2001/2002 and the 2002/2003 crops among the sewage sludge doses about the variation of Fusarium spp. in soil, root, stalk and seed, as well as about the incidence of plants with stalk and corn rot. The regression analyses have showed that the percentage of diseased plants was positively correlated to the concentration of the sludges incorporated to the soil. The determination coefficients to the 2001/2002 culture were R2=0.61 and R2=0.32, while the 2001/2003 culture showed R2=0.76 and R2=0.45, to Franca and Barueri sludges, respectively. The sludges concentrations also presented positive correlation with the Fusarium community of the soil and the rhizosphere, and with the incidence of Fusarium in the root, stalk and seed. The correlations between the Fusarium community of the soil and rhizosphere and the percentage of diseased plants with chemical soil attributes were significant and positive, for both kinds of sludges, in the level of 1 and 5%, to phosphorus and calcium contents.

Герасименко, Т. В. Дія різних систем удобрення на фітосанітарний стан посівів гороху : випускна кваліфікаційна робота : 201 "Агрономія" / Т. В. Герасименко ; керівник роботи О. П. Тимошенко ; НУ "Чернігівська політехніка", кафедра аграрних технологій та лісового господарства. – Чернігів, 2021. – 58 с.
Об'єктом дослідження виступає технологія вирощування та система удобрення гороху. Предметом дослідження в дипломній роботі є вплив удобрення та мікробного препарату Ризогуміну на фітосанітарний стан посівів гороху. Застосування органічної системи та різних доз мінерального удобрення сприяє зниженню ураження кореневими гнилями рослин гороху відносно контролю без добрив. При вирощуванні гороху на дерново-підзолистому ґрунті можна рекомендувати середні дози мінерального удобрення у поєднанні з мікробним препаратом комплексної дії Ризогуміном, як економічно доцільні для оптимального живлення рослин та пригнічення фузаріозної кореневої гнилі, і як результат, загального покращення фітосанітарного стану посівів культури. Пропозиції по поліпшенню технології вирощування гороху в даному господарстві заключаються в наступному: для підвищення пропоную провести сортозаміну і використати такі сорти гороху, як середньоранній сорт Мадонна та середньостиглий сорт Отаман. Рекомендовано проводити вапнування - завдяки чому відбувається посилення мікробіологічних та біохімічних процесів у грунті, збільшується кількість доступних поживних речовин, зокрема нітратів, легкозасвоюваних форм фосфору і калію. При цьому кількість засвоюваного азоту зростає у рік вапнування, а фосфору і калію – у наступні роки.
The object of study is the technology of cultivation and fertilization system of peas. The subject of research in the thesis is the effect of fertilizer and microbial preparation Rhizohumin on the phytosanitary status of pea crops. The use of an organic system and different doses of mineral fertilizers helps to reduce the damage of root rot of pea plants in relation to control without fertilizers. When growing peas on sod-podzolic soil, we can recommend medium doses of mineral fertilizers in combination with microbial preparation of complex action Rhizohumin, as economically feasible for optimal plant nutrition and suppression of fusarium root rot, and as a result, overall improvement of phytosanitary condition. Proposals to improve the technology of growing peas in this farm are as follows: to increase the proposal to carry out sortosamina and use such varieties of peas as medium-early variety Madonna and medium-ripe variety Otaman. It is recommended to carry out liming - due to which there is an increase in microbiological and biochemical processes in the soil, increasing the amount of available nutrients, including nitrates, easily digestible forms of phosphorus and potassium. The amount of assimilated nitrogen increases in the year of liming, and phosphorus and potassium - in subsequent years.

Este trabalho teve dois objetivos distintos, o primeiro foi realizar um levantamento da micoflora associada a grãos ardidos de híbridos comerciais de milho pertencentes à empresa Dow AgroSciences e o outro foi avaliar linhagens de milho tropical para a resistência a podridão da espiga, incidência de grãos ardidos e produção de fumonisinas por Fusarium verticillioides. Para o primeiro objetivo, coletou-se grãos de dois híbridos (2B587 e 2B710) cultivados em diferentes localidades do Brasil durante a safra verão e safrinha, sob as zonas macro-climáticas SA (subtropical alta), SB (subtropical baixa), TA (tropical alta), TB (tropical baixa) e TT (tropical de transição) afim de, realizar a determinação da incidência fúngica através do método do papel de filtro com congelamento. O delineamento experimental foi inteiramente casualizado, com quatro repetições de 50 grãos de cada amostra. Os resultados indicaram Fusarium spp. e Penicillium spp. como os fungos de maior incidência nos grãos, sendo que na safra verão o comportamento dos híbridos em relação a incidência desses fungos variou de acordo com a zona climática. Na safrinha, o híbrido 2B710 foi o mais resistente a Fusarium spp. e Penicillium spp. independente da zona climática. Para o segundo objetivo, seis linhagens tropicais de milho, previamente classificadas como resistentes (R1, R2 e R3) e suscetíveis (S1, S2 e S3) a F. verticillioides pela Dow AgroSciences foram cultivadas na estação experimental da companhia, localizada no município de Jardinópolis/SP durante a safra verão e safrinha. O ensaio consistiu de dois tratamentos (inoculados e não inoculados), sendo que a inoculação foi feita através da pulverização 2 mL de uma suspensão de 106 conídios/ml no estigma da espiga e nas espigas não inoculadas a doença ocorreu por meio de infecção natural. A avaliação de severidade foi feita por uma escala de notas de 0 a 7, a incidência foi avaliada através da porcentagem de grãos ardidos em sub - amostras de 200 grãos de cada tratamento por repetição e a detecção dos níveis de fumonisinas foi realizada pelo método de Elisa. O delineamento experimental foi em blocos ao acaso com três repetições. Os resultados obtidos na safra verão indicaram uma variação na severidade de 1,2 a 4,3, na incidência de grãos sintomáticos de 13,7 a 46% e os níveis de fumonisinas detectados entre as linhagens variaram de 5,5 µg/g a 41 µg/g. Na safrinha, não houve diferenças significativas comparada à safra verão para a severidade, mas houve uma redução na incidência de grãos sintomáticos que variaram de 4,2% a 22,7% e redução nos níveis de fumonisina, os quais ficaram entre 1,5 µg/g a 22,7 µg/g. Nas duas safras, as linhagens R2 e R3 comportaram-se como mais resistentes a produção de fumonisinas e a R1 comportou-se como mais resistente a podridão da espiga e incidência de grãos ardidos, demonstrando que não houve correlação entre severidade da doença e incidência de grãos ardidos com níveis de fumonisinas.
This work aimed to survey the mycoflora associated to damaged grains of commercial hybrid maize provided by the company Dow AgroScience and to assess the resistance levels of six tropical inbred lines to ear rot, incidence of damaged grains and of fumonisins production by Fusarium veticillioides. For the first objective, two hybrids (2B587 and 2B710) were collected and cultivated in different regions in Brazil, during the normal season and the late-summer plantings, known as safrinha, on macro-climatic zones SA (high subtropical), SB (low subtropical), TA (high tropical), TB (low tropical) and TT (tropical transition). The fungal incidence determination was made through the deep - freezing blotter test. The experimental design was totally randomized with four replicates of 50 grains each sample. The results showed Fusarium spp. and Penicillium spp. as the most prevalent fungi, and the incidence of such fungi in each hybrid varied according to the climatic zone. In the late-summer plantings, the 2B710 hybrid was the most resistant to Fusarium spp. and Penicillium spp., independent of the climatic zone. For the second aim, six inbreed lines of tropical maize, previously classified as resistant (R1,R2 and R3) and susceptible (S1,S2 and S3) to F. verticillioides by Dow AgroSciences were cultivated in the experimental station of the company, localized in Jardinópolis / SP during the normal season and the late-summer plantings. The tests were made by two kinds of treatment (inoculated and non inoculated), and the inoculated treatment was made by spraying 2 mL of a 106 conidia/mL suspension onto the silk channel of the corn ear and, in the no inoculated ones, the disease occurred by natural infection. Severity was evaluated using a scale of 0 to 7, whereas the incidence was evaluated by counting the damaged grains in sub samples of 200 grains. Fumonisins levels detection were determined by the ELISA method. The experimental design was made in random blocks with three replicates. The results, in the normal season indicated a severity variation of 1.2 to 4.3, and in the symptomatic grains, the incidence was from 13.7 to 46% and the detected fumonisins levels among the inbreed lines ranged from 5.5 µg/g to 41 µg/g. No statistical differences between the normal season and the late-summer plantings were observed, concerning severity. However, there was a decrease in symptomatic grains, ranging from 4.2% to 22.7%, as well as a decrease in fumonisins levels, from 1.5 µg to 22.7 µg. The R2 and R3 inbreed lines showed more resistance to fumonisins production and R1 showed higher resistance to corn ear rot and damaged corn grains incidence. It was concluded that there was no relation between the disease severity and the of incidence damaged grains with high fumonisins levels.

This study evaluated the effect of Pratlenchus brachyurus and Fusarium verticillioides, and their relationship, on growth and development of two mayze hybrids (30F80 and 30K73). The experiment was conducted in greenhouse conditions at Pioneer Hi-bred Research Center in Itumbiara, GO, Brazil. The experimental design was randomized blocks with 15 treatments and 10 replications. Treatments included a non-inoculated control; inoculation of Fusarium in the seed and in the soil; three populations levels of Pratylenchus at sowing and at 30 days after planting (50, 100 and 500 nematodes/plot), with and without the inoculation of Fusarium in the soil; and three populations leves of Pratylenchus inoculated 30 days after sowing with Fusarium inoculated in seed at sowing. At plant maturity the following variables were evaluated: plant height and ear length, stalk diameter, root fresh and dry matter, stalk fresh and dry matter, reproduction factor and fusariosis severity on the stalk. Fusarium stalk colonization was evaluated in a scale from 1 to 9. The 30F80 hybrid had lower plant height and ear length, stalk diameter, stalk and root fresh matter when the seeds were inoculated with the suspension of Fusarium and sowing immediately after inoculation. The maize hybrids 30F80 and 30K73, showed the lowest root length, stalk fresh and dry matter, and root fresh and dry matter mainly when the soil was inoculated with Fusarium and sown in the same day, and the inoculation with 500 juveniles and/or adults of Pratylenchus, was done 30 days after sowing. The maize hybrid 30K73 obtained lower plant height and ear length when seeds were inoculated with the suspension of Fusarium and sown in the same day. Maize seeds inoculated with Fusarium verticillioides at planting decreased plant height, ear length, and root and shoot fresh weight on both hybrids. Stalk diameter of 30K73 hybrid and root length of hybrid 30K73 were also reduced. Soil inoculation of Fusarium verticillioides at planting and Pratylenchus brachyurus inoculated 30 days after planting (500 nematodes/plot) decreased root elongation and stalk, and root dry weights on both hybrids. However, plant height was only reduced on 30K73 hybrid. Future research is needed to better understand the relationship between Pratylenchus brachyurus and Fusarium verticillioides on maize growth and development.
O objetivo deste estudo foi avaliar o efeito de Pratylenchus brachyurus e/ou Fusarium verticillioides e sua relação no desenvolvimento de dois híbridos de milho (30F80 e 30K73). O experimento foi conduzido em casa de vegetação no Centro de Pesquisa da Pioneer Sementes de Itumbiara-GO. O delineamento experimental foi inteiramente ao acaso com 15 tratamentos/por híbrido (totalizando 30 tratamentos) e 10 repetições. Os tratamentos foram: testemunha (sem inoculações), inoculação de fungo nas sementes e no solo; inoculação de três populações de nematóide na semeadura e 30 dias após a semeadura (50, 100 e 500 nematóides/vaso), com e sem inoculação do fungo no solo; e três níveis populacionais de nematóides inoculados 30 dias após a semeadura, com o fungo inoculado na semente. Após a maturação (estádio R7), foram avaliadas as características: altura de planta e de espiga, diâmetro de colmo, matéria fresca e seca de raiz, matéria fresca e seca de colmo, fator de reprodução do nematóide e severidade de fusariose no colmo do milho. Observou-se que o híbrido de milho 30F80 obteve uma menor altura de planta e de espiga, diâmetro de colmo, matéria fresca de colmo e de raíz, quando as sementes foram inoculadas com a suspensão de conídios de Fusarium verticillioides e semeadura realizada no mesmo dia dessa inoculação. Os híbridos de milho 30F80 e 30K73 apresentaram o menor comprimento de raiz, matéria fresca e seca de colmo, e matéria fresca e seca de raiz, principalmente quando o solo foi inoculado com Fusarium verticillioides, semeou-se no mesmo dia e a inoculação do nematóide na população de 500 juvenis e/ou adultos de Pratylenchus brachyurus realizada 30 dias após a semeadura. O híbrido de milho 30K73 obteve uma menor altura de planta e de espiga quando as sementes foram inoculadas com a suspensão de conídios de Fusarium verticillioides e semeadura realizada no mesmo dia dessa inoculação. Sementes de milho inoculadas com o fungo na semeadura diminuiram a altura de planta e de espiga e peso fresco de raiz e de parte aérea nos dois híbridos de milho. Diâmetro de colmo no híbrido 30F80 e comprimento de raiz no híbrido 30K73 também foram reduzidos. A inoculação do solo com o fungo na semeadura e nematóide inoculado 30 dias após (500) proporcionou menor comprimento de raiz e peso seco e fresco de raiz e colmo nos dois híbridos. Contudo, altura de planta foi reduzida somente no híbrido 30K73. Outros estudos de interações entre microrganismos, em especial de Pratylenchus brachyurus e Fusarium verticilliodes, deverão ser realizados, a fim de melhor entender o efeito desta combinação no prejuízo das plantas.
Mestre em Agronomia

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The objective of this study was to evaluate the incidence and damage of stalk rot (SR) in corn hybrids grown in tillage rotation with beans and succession to oats; quantify the efficiency of detection and control of Fusarium verticillioides in seed and seedlings health test according to the time of incubation. The first study was carried out at Muitos Capões municipal district on Rio Grande do Sul State, during the agricultural seasons of 2012/13 and 2013/14.The hybrids tested were P30F53 Hx, P32R22 YHR, DKB 250 VTPRO2, AS1555 VTPRO2, AS 1656 VTPRO2, AG8025 VTPRO e AG 9045 VTPRO. A randomized block design with four replications was employed. The incidence of SR was determined during harvesting and the damage by threshing operation analyzed separately in healthy and unhealthy plants. The means were compared by Scott-Knott test with a significance level of 5% (P <0.05). Statistical interaction between corn hybrid and harvesting season was detected. For this reason, the data was analyzed by harvesting season. In the 2012/13 growing season the prevailing diseases were diplodia blight, Anthracnosis and Fusarium, and as opposed to 2013/14, when Gibberella, Diplodia blight and Fusarium wilting were the prevailing in 2013/14. The incidence of SR in 2012/13 and 2013/14 ranged from 7.3% to 100% (46.2% in average), and from 3.14% to 72.6% (34.8% in average). The average of damage in the yield of hybrids were 6,48% and 8,75%, ranging from 197 kg ha-1 to 1205 kg ha-1 and from 113 kg ha-1 to 2536 kg ha-1, with an average of 640 kg ha-1 and 768 kg ha-1, respectively in 2012/2013 and 2013/14. In both growing seasons the hybrids P32R22 YHR and AG9045 VTPRO showed the highest SR incidence. The lowest incidence of this disease was detected in the P30F53 Hx and AG8025 VTPRO hybrids in 2012/13 and AG8025 VTPRO and DKB 250 VTPRO2 in 2013/14. The highest damages were found in AS1555 VTPRO2 (14%) and AG9045 VTPRO (11.2%) in 2012/13, and AG9045 VTPRO (26.3%) in 2013/14. The yield ranged from 8.430 kg ha-1 to 10.667 kg ha-1 in 2012/13 and 7.175 kg ha-1 and 8.790 kg ha-1 in the 2013/14. Secondly, hybrid corn seeds Status TL TG treated with metalaxyl-m + fludioxonil fungicides and seeds without seed treatment were cultured in PDA Culture Media to obtain symptomatic seedlings (infected by F. verticullioides) and asymptomatic, with 7 days of age. The samples were separated and transferred to test tubes containing agar-water culture media and maintained in growth chamber at 25 °C under 12 hours of photoperiod until the completion of 20 days. The treatments were T1 - uninfected seedlings without TS; T2 - infected seedlings without TS; T3 - uninfected seedlings with TS; T4 - infected seedlings with TS. The fungus incidence was quantified in the mesocotyl, crown and remaining seed and shoot and root length and dry matter weight were measured. Randomized block experimental design with eight replications was used. The data was submitted to variance analysis and Tukey’s test at 5% of significance level. The incidence of F. verticillioides in the remaining seeds was 96% for T1 and T3, and 100% for T2 and T4, with no difference between treatments. The incidence of F. verticillioides was 88% and 93% for mesocotyl, and 88% and 95% in the crown in the T1 and T3 seedlings, respectively. The root length showed 14.1 cm and 10.7 cm for T3 and T4 respectively, which are higher than T1 (9.6 cm) and T2 (5.8 cm). Higher root weight was detected in T3 (0.25 g) and T4 (0.18 g) cultivars compared to T1 (0.12 g) and T2 (0.09 g). T3 showed the highest shoot length (14.2 cm) when compared to the other treatments. The corn seed treatment with metalaxyl-m + fludioxonil fungicides is not effective in controlling F. verticillioides. The detection of F. verticillioides in BDA media at the seventh day of incubation does not represent the real incidence of the fungus, once the pathogen was recovered from asymptomatic seedlings when they were re-evaluated at 20 days. In all treatments, the seed treatment enhanced the seedlings development
Os objetivos deste trabalho foram avaliar a incidência e os danos causados por podridões do colmo (PC) em híbridos de milho cultivados em sistema de semeadura direta com rotação ao feijão e sucessão à aveia; e quantificar a eficiência de detecção e controle de Fusarium verticillioides em meio batata-dextrose-ágar em sementes e plântulas de milho em função do tempo de incubação. Foram realizados dois estudos, sendo estes conduzidos no município de Muitos Capões, estado do Rio Grande do Sul, nas safras agrícolas 2012/13 e 2013/14. Foram avaliados os híbridos P30F53 Hx, P32R22 YHR, DKB250 VTPRO2, AS1555 VTPRO2, AS1656 VTPRO2, AG8025 VTPRO e AG9045 VTPRO. O delineamento experimental foi de blocos casualizados com quatro repetições. A incidência de PC foi determinada na colheita do milho, sendo o dano mensurado pela trilha em separado de espigas das plantas sadias e plantas doentes. A comparação das médias foi realizada pelo teste de Scott-Knott, em nível de significância de 5% (p<0,05). Houve interação entre híbridos e safras, sendo os dados analisados separadamente por safra. Em 2012/13, as doenças predominantes foram diplodia, antracnose e fusariose; em 2013/14, houve predomínio de giberela, diplodia e fusariose. A incidência de PC em 2012/13 e 2013/14 variou de 7,3% a 100% (média de 46,2%) e de 3,14% a 72,6% (média de 34,8%), respectivamente. O dano médio no rendimento de grãos dos híbridos foi de 6,8% e 8,7%, respectivamente para 2012/13 e 2013/14. Nas duas safras os híbridos P32R22 YHR e AG9045 VTPRO apresentaram maior incidência de PC. As menores incidências foram detectadas nos híbridos P30F53 Hx e AG8025 VTPRO na safra de 2012/13, e AG8025 VTPRO e DKB250 VTPRO2 na safra de 2013/14. Os maiores danos foram encontrados nos híbridos AS1555 VTPRO2 (14%) e AG9045 VTPRO (11,2%) em 2012/13, e AG9045 VTPRO (26,3%) em 2013/14. No segundo estudo, sementes de milho tratadas com fungicida metalaxil-m + fludioxonil e sem tratamento foram semeadas em meio de cultura de batata-dextrose-ágar para obtenção de plântulas sintomáticas (infectadas por F. verticillioides) e assintomáticas. Aos sete dias as plântulas foram separadas e transferidas para tubos de ensaio contendo meio de cultura ágar-água e mantidas em câmara de crescimento com temperatura de 25ºC e fotoperíodo de 12 h, onde permaneceram até completarem 20 dias. Obtiveram-se os tratamentos: T1 - plântulas assintomáticas sem fungicida; T2 - plântulas sintomáticas sem fungicida; T3 – plântulas assintomáticas com fungicida; T4 – plântulas sintomáticas com fungicida. A incidência foi quantificada no mesocótilo, coroa e semente remanescente das plântulas e o comprimento e peso seco da parte aérea e raiz. Os dados foram submetidos à análise de variância e teste de Tukey a 5%. A incidência de F. verticillioides em sementes remanescentes foi de 96% para T1 e T3, e 100% para T2 e T4, não diferindo os tratamentos entre si. Foi observada incidência de F. verticillioides de 88 e 93 % para mesocótilo, e 88 e 95% para coroa em plântulas provenientes de T1 e T3, respectivamente. O comprimento de raiz foi de 14,1 cm e 10,7 cm para T3 e T4 respectivamente, sendo estes superiores ao T1 (9,6 cm) e T2 (5,8 cm). Foram detectados maiores pesos de raiz nos tratamentos T3 (0,25 g) e T4 (0,18 g), quando comparados a T1 (0,12 g) e T2 (0,09 g). O T3 apresentou o maior comprimento de parte aérea,14,2 cm, em relação aos demais tratamentos. O tratamento de sementes com metalaxil-m + fludioxonil não foi eficiente no controle de F. verticillioides. A detecção de F. verticillioides em meio BDA aos sete dias de incubação não representou a real incidência do fungo, pois o patógeno foi recuperado de plântulas até então assintomáticas quando essas foram reavaliadas aos 20 dias. De qualquer forma o tratamento proporcionou maior comprimento e peso de raiz e parte área em plântulas de milho

O gênero Fusarium é um grupo de fungos de importância mundial, não só por causar patologias em plantas, mas também porque abriga espécies toxigênicas. Dentre estas, Fusarium verticillioides, Fusarium proliferatum e Fusarium subglutinans, cujos teleomorfos se agrupam no complexo Gibberella fujikuroi, estão associadas a patologias em milho. O presente estudo caracterizou uma coleção de 100 isolados de Fusarium spp. tanto do ponto de vista morfológico como molecular com vistas a identificá-los em nível de espécie. Para isto, foram usados como marcadores morfológicos a presença/ausência de clamidósporos, o tipo de célula conidiogênica e a disposição dos microconídios sobre a célula conidiogênica. Os isolados foram também identificados em espécie baseado em informações disponíveis na literatura acerca de marcadores moleculares desenvolvidos a partir da reação de PCR e primers espécie-específicos. A ausência de clamidósporo permitiu alocar a totalidade dos isolados no complexo G. fujikuroi. Os demais critérios morfológicos permitiram identificar 77 isolados como F. verticillioides, 20 como F. proliferatum, 2 como F. subglutinans. Apenas 1 isolado não foi possível identificar em espécie. Análises moleculares concordaram em 100% dos casos em que os isolados foram identificados como F. verticillioides e F. subglutinans. Porem, no caso dos 20 isolados identificados morfologicamente como F. proliferatum apenas 4 foram confirmados na análise molecular; os demais foram identificados como F. verticillioides. A diversidade genética estudada por AFLP ratificou a separação das espécies F. verticillioides e F. proliferatum, com um índice de similaridade de 0,40. Marcadores AFLP também evidenciaram alta diversidade genética de F. verticillioides. Todos os isolados causaram podridão do colmo em três híbridos comerciais de milho e não variaram em agressividade, independente do nível de resistência dos híbridos.
The genus Fusarium is an important group of fungi worldwide, not only for its capability to cause disease but because it also contains species which produce toxins. Among these, Fusarium verticillioides, Fusarium proliferatum and Fusarium subglutinans, which teleomorphs are grouped in the Gibberella fujikuroi group are associated with diseases in maize. This study characterized a collection of 100 isolates of Fusarium spp. under morphological and molecular criteria to identify them at the species level. For this purpose, morphological markers such as presence/absence of chlamydospores, conidiogenou cell type and microconidia arrangement on conidiogenou cell were assessed. The isolates were also identified at the species level based upon available information about molecular markers developed from the PCR reaction using speciesspecific primers. The absence of chlamydospores in all of the isolates placed them within the G. fujikuroi complex, and based on the other morphological criteria, 77 isolates were identified as F. verticillioides, 20 as F. proliferatum, 2 as F. subglutinans and one isolate remained unidentified at the species level. The molecular analyses agreed with the morphological identification of all F. verticillioides and F. subglutinans. However, in the case of 20 isolates identified morphologically as F. proliferatum only 4 were confirmed, the rest being identified as F. verticillioides. The genetic diversity based on AFLP confirmed the separation of F. verticillioides and F. proliferatum in two groups, with a similarity index of 0.40. AFLP markers also showed high genetic diversity within F. verticillioides. All isolates were caused stalk rot on three commercial hybrids and did not vary in aggressiveness regardless of the resistance level of the hybrids.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The gibberella ear rot caused by fungus Fusarium graminearum, causes damage in productivity and quality of maize kernel being disease of difficult to control due to the unavailability of resistant hybrids and low efficacy of cultural practices. Three studies were conducted, the aim of the first was to compare four methods of inoculation (injection silkchannel, sprinkling on silk, kernel- wounding and deposition in the ear leaf sheath) and a control treatment in four hybrids (P30R50H, P30B30H, P3989, P32R48H) based on disease severity and kernel yield; the second study determined the reaction of 20 hybrids to gibberella ear rot by quantifying the incidence of the disease, rot kernels and F. graminearum in the kernels and their correlations; and the third study characterized the genetic resistance of hybrids to gibberella ear rot through preparation of a reaction scale involving classes and disease intensity and their correlation with kernel yield and inoculation methods. The experiments of first study were conducted in 2011/12 harvest in Cruz Alta, RS and Lages. The experiments of the second and third studies were conducted in two environments in the season 2012/13 in Cruz Alta. The design of the experiments was a randomized complete blocks design with treatments arranged in a factorial with four replications. In the first study there was no difference in disease severity between hybrids and inoculation methods. The hybrid P32R48H presented higher disease severity, distinguishing from the others. Inoculations in silk-channel and wounding the kernels provided higher disease severity and lower kernel yield. In the second study, the method of the wounding the kernels provided increases above 40% in the incidence of gibberella ear rot, rot kernels and occurrence of F. graminearum in the grain when compared to the injection silk-channel method. There was an effect of hybrids and the values were 37.2% and 95.6% for the incident of gibberella ear rot, 2.1% to 17.0% for rot kernels and 4.0% to 29.3% for incidence of F. graminearum in the kernels. Significant and positive correlations (r = 0.70) were found between incidence of F. graminearum in the kernels and rots kernels. In the third study it was watched that the kernel yield was affected by hybrids, regardless of the inoculation method. The P30F35YH and BG7049H hybrids obtained better yields, regardless of the environment and inoculation method, being classified as moderately susceptible to gibberella ear rot. The AG8025PRO, DKB250PRO2, AS1555PRO, Status, 30K64HNSR and P4285 were classified as moderately resistant considering the intensity of gibberella ear rot and the involvement of resistance to colonization of the stigma (tip of the ear) and kernel resistance. There were significant and positive relationship between productivity with injection in the silk-channel (r = 0.93) and wounding the kernels with productivity (r = 0.94) in environment 1 and 2. It was found that although there are no resistant hybrids there is genetic variability and the involvement resistance of stigma mechanisms and the kernels, and the giberella ear rot affected more kernel quality than quantity
A podridão de giberela causada pelo fungo Fusarium graminearum, provoca danos na produtividade e qualidade de grãos de milho sendo doença de difícil controle devido à indisponibilidade de híbridos resistentes e baixa eficácia de práticas culturais. Foram realizados três estudos, o objetivo do primeiro foi comparar quatro métodos de inoculação (injeção no canal do estilo-estigma, aspersão nos estigmas, ferimento dos grãos no centro da espiga e deposição na bainha da folha da espiga) e um tratamento controle em quatro híbridos (P30R50H, P30B30H, P3989, P32R48H) com base na severidade da doença e produtividade de grãos; o segundo estudo determinou à reação de 20 híbridos a podridão de giberela pela quantificação da incidência da doença, grãos ardidos e F. graminearum nos grãos e suas correlações; e o terceiro estudo caracterizou a resistência genética de híbridos à podridão de giberela pela elaboração de uma escala de reação envolvendo classes e intensidade de doença e suas correlações com produtividade de grãos e métodos de inoculação. Os experimentos do primeiro estudo foram conduzidos na safra 2011/12 em Cruz Alta, RS e Lages, SC. Os experimentos do segundo e terceiro estudos foram realizados em dois ambientes na safra agrícola 2012/13, em Cruz Alta. O delineamento dos experimentos foi em blocos casualizados com tratamentos arranjados num fatorial com quatro repetições. No primeiro estudo houve diferença na severidade da doença entre híbridos e métodos de inoculação. O híbrido P32R48H apresentou maior severidade da doença, diferindo dos demais. Inoculações no canal do estilo-estigma e ferimento dos grãos proporcionaram maior severidade da doença e menores produtividades de grãos. No segundo estudo o método de ferimento dos grãos proporcionou acréscimos superiores a 40% na incidência de espigas giberelas, incidência de grãos ardidos e incidência de F. graminearum nos grãos quando comparado ao método de injeção no canal do estilo estigma. Houve efeito de híbridos e os valores foram de 37,2% a 95,6% para incidência de espigas giberelas, 2,1% a 17,0% para grãos ardidos e 4,0% a 29,3% para incidência de F. graminearum nos grãos. Correlações significativas e positivas (r = 0,70) foram obtidas entre incidência de F. graminearum nos grãos e grãos ardidos. No terceiro estudo foi observado que a produtividade de grãos foi afetada pelos híbridos, independentemente do método de inoculação. Os híbridos P30F35YH e BG7049H obtiveram melhores produtividades, independente do ambiente e método de inoculação, sendo classificados como moderadamente suscetível à podridão de giberela. Os híbridos AG8025PRO, DKB250PRO2, AS1555PRO, Status, 30K64HNSR e P4285 foram classificados como moderadamente resistente, considerando a intensidade de giberela e o envolvimento da resistência à colonização do estigma (ponta da espiga) e resistência do grão. Houve relações significativas e positivas entre produtividade com injeção no canal do estilo-estigma (r = 0,93) e produtividade com ferimento dos grãos (r = 0,94) nos ambientes 1 e 2. Foi constatado que apesar de não haver hibridos resistentes existe variabilidade genética e o envolvimento de mecanismos de resistência do estigma e dos grãos, e que as podridões de giberela afetaram mais a qualidade dos grãos do que a quantidade

E’ stata approfondita l’espressione genica complessiva in spighe di mais, in seguito all’ infezione fungina. Nella prima parte del lavoro, sono stati valutati un genotipo di mais resistente ed uno suscettibile a F. verticillioides, campionando le cariossidi 48 ore dopo l’infezione. Sono state identificate circa 800 sequenze differenzialmente espresse e circa il 10% è stato assegnato alla categoria della difesa. Nel genotipo resistente, i geni coinvolti nella difesa hanno mostrato un tipo di risposta basale, mentre in quello suscettibile tali geni rispondevano specificamente all’infezione. Nella seconda parte del lavoro, l’analisi di espressione è stata estesa a fasi precoci e tardive dell’infezione utilizzando un ceppo normale ed uno mutante di F. verticillioides. Numerosi geni risultavano differenzialmente regolati 48 ore dopo l’infezione con entrambi i ceppi. Il ceppo normale era in grado di attivare i meccanismi di difesa prima del mutante. Nella terza parte del lavoro, 10 linee resistenti e suscettibili sono state infettate con 4 specie fungine. In tutti i genotipi l’espressione dei geni coinvolti nella difesa era indotta in seguito all’infezione, ma le linee resistenti presentavano una risposta basale di difesa.
We investigated global gene expression in maize ears at several time points after fungal infection. In the first part of the work, resistant and susceptible genotypes were tested in kernels sampled 48 h after infection with a wild type strain of F. verticillioides. About 800 differentially expressed sequences were identified and nearly 10% assigned to the category cell rescue, defense and virulence. In the resistant genotype, defense-related genes provided basic defense against the fungus, while in the susceptible genotype defense genes responded specifically to pathogen infection. In the second part of the work the expression analysis was extended to early and late phases of infection with a wild type and a mutant strains of F. verticillioides. Kernels were sampled in the area around the point of infection. Most of genes were differentially regulated 48 h after infection with both fungal strains. The wild type strain was able to activate host defense genes before the mutant strain. In the third part of the work, ten resistant and susceptible lines were infected by different fungal species. All genotypes were able to induce the expression of defense genes upon infection, but the resistant lines showed a basal defense response.

E’ stata approfondita l’espressione genica complessiva in spighe di mais, in seguito all’ infezione fungina. Nella prima parte del lavoro, sono stati valutati un genotipo di mais resistente ed uno suscettibile a F. verticillioides, campionando le cariossidi 48 ore dopo l’infezione. Sono state identificate circa 800 sequenze differenzialmente espresse e circa il 10% è stato assegnato alla categoria della difesa. Nel genotipo resistente, i geni coinvolti nella difesa hanno mostrato un tipo di risposta basale, mentre in quello suscettibile tali geni rispondevano specificamente all’infezione. Nella seconda parte del lavoro, l’analisi di espressione è stata estesa a fasi precoci e tardive dell’infezione utilizzando un ceppo normale ed uno mutante di F. verticillioides. Numerosi geni risultavano differenzialmente regolati 48 ore dopo l’infezione con entrambi i ceppi. Il ceppo normale era in grado di attivare i meccanismi di difesa prima del mutante. Nella terza parte del lavoro, 10 linee resistenti e suscettibili sono state infettate con 4 specie fungine. In tutti i genotipi l’espressione dei geni coinvolti nella difesa era indotta in seguito all’infezione, ma le linee resistenti presentavano una risposta basale di difesa.
We investigated global gene expression in maize ears at several time points after fungal infection. In the first part of the work, resistant and susceptible genotypes were tested in kernels sampled 48 h after infection with a wild type strain of F. verticillioides. About 800 differentially expressed sequences were identified and nearly 10% assigned to the category cell rescue, defense and virulence. In the resistant genotype, defense-related genes provided basic defense against the fungus, while in the susceptible genotype defense genes responded specifically to pathogen infection. In the second part of the work the expression analysis was extended to early and late phases of infection with a wild type and a mutant strains of F. verticillioides. Kernels were sampled in the area around the point of infection. Most of genes were differentially regulated 48 h after infection with both fungal strains. The wild type strain was able to activate host defense genes before the mutant strain. In the third part of the work, ten resistant and susceptible lines were infected by different fungal species. All genotypes were able to induce the expression of defense genes upon infection, but the resistant lines showed a basal defense response.

Orientador : Raquel Ghini
Banca: Raquel Ghini
Banca: Wagner Bettiol
Banca: Nilton Luiz de Souza
Banca: Idalmir dos Santos
Banca: Gisela Maria Fantin
Resumo: O trabalho objetivou verificar os fatores químicos e microbiológicos do lodo de esgoto e do solo responsáveis pela incidência da podridão do colmo do milho, causada por Fusarium em solo fertilizado com lodo, e foi desenvolvido em condições de campo, numa área que vem sendo tratada com lodo e cultivada com milho desde 1999 em latossolo vermelho distróférrico (textura argilosa), localizada no município de Jaguariúna/SP. Os dois lodos utilizados foram obtidos nas Estações de Tratamento de Esgoto de Franca e de Barueri/SP. Os lodos foram incorporados ao solo nas concentrações de 0, 1, 2, 4 e 8 vezes a dose recomendada com base no teor de nitrogênio, em dois cultivos sucessivos de milho (Savana 133S) nas safras 2001/2002 e 2002/2003, em parcelas de 200 m2. As doses de lodo foram comparadas com a adubação mineral recomendada para a cultura do milho. Os tratamentos foram distribuídos em delineamento experimental casualizado em blocos e com três repetições. Foram verificadas, nas safras de 2001/2002 e 2002/2003, diferenças significativas entre as doses de lodo de esgoto, sobre a variação de Fusarium spp. no solo, na rizosfera, na raiz, no colmo e na semente, bem como sobre a incidência de plantas com podridão no colmo e nas espigas. As análises de regressão mostraram que a porcentagem de plantas doentes foi positivamente correlacionada com a concentração dos lodos incorporados ao solo. Os coeficientes de determinação para o cultivo de 2001/2002 foram de R2=0,61 e R2=0,32, enquanto para o cultivo de 2002/2003 foram de R2=0,76 e R2=0,45, para os lodos de Franca e Barueri, respectivamente. As concentrações de lodos também apresentaram correlação positiva com a comunidade de Fusarium do solo e da rizosfera e com a incidência de Fusarium na raiz, no colmo e na semente. As correlações entre a comunidade de Fusarium do solo e da rizosfera e o percentual de plantas... (Resumo completo, clicar acesso eletrônico abaixo).
Abstract: This work aimed at verifying the chemical and microbiological sewage sludge and soil factors re 0sponsible for the incidence of the corn stalk rot caused by Fusarium in soil fertilized with sludge and has been developed in field conditions, in an area that has been treated with sludge and cultivated with corn since 1999, in red dystroferric latosol (clayey texture), in Jaguariúna/SP. Both sludges used were obtained in the sewage sludge treatment stations of Franca and Barueri/SP. Sludges were incorporated into the soil in concentrations of 0, 1, 2, 4 and 8 times in relation to the recommended rate based on the nitrogen contents, in two successive corn cultures (Savana 133S), in 2001/2002 and 2002/2003, in plots of 200 m2. Sludge doses were compared to the mineral fertilization recommended to the corn culture. The treatments were distributed in a randomized design experimental drawing in blocks and with three repetitions. Significant differences were verified in both 2001/2002 and the 2002/2003 crops among the sewage sludge doses about the variation of Fusarium spp. in soil, root, stalk and seed, as well as about the incidence of plants with stalk and corn rot. The regression analyses have showed that the percentage of diseased plants was positively correlated to the concentration of the sludges incorporated to the soil. The determination coefficients to the 2001/2002 culture were R2=0.61 and R2=0.32, while the 2001/2003 culture showed R2=0.76 and R2=0.45, to Franca and Barueri sludges, respectively. The sludges concentrations also presented positive correlation with the Fusarium community of the soil and the rhizosphere, and with the incidence of Fusarium in the root, stalk and seed. The correlations between the Fusarium community of the soil and rhizosphere and the percentage of diseased plants with chemical soil attributes were significant and positive, for both kinds of sludges, in the level of 1 and 5%, to phosphorus and calcium contents.
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