Reproduction de : Thèse de doctorat : Génie des procédés et de l'environnement : Toulouse, INPT : 2007. Reproduction de : Thèse de doctorat : Sciences de la Vie : Beyrouth, USJ Beyrouth : 2007. / Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. 175 réf.

Reproduction de : Thèse de doctorat : Microbiologie et Biocatalyse industrielles : Toulouse, INPT : 2008. Reproduction de : Thèse de doctorat : Microbiologie et Biocatalyse industrielles : Suranaree, SUT : 2008. / Thèse soutenue en cotutelle. Titre provenant de l'écran-titre.

Thesis--Wisconsin. / Includes bibliographical references (leaves 72-79).

Maize (Zea mays L.) is one of the world’s largest food crops and thus any pathogens of maize are of great importance. Aspergillus flavus is one of these pathogens and it produces a carcinogenic metabolite called aflatoxin. Efforts to reduce infection by A. flavus and subsequent aflatoxin accumulation include the development of maize lines resistant to aflatoxin accumulation. However, resistant lines that have been developed contain agronomically unfavorable traits. Gene-based markers would allow for easier transfer of resistance from resistant inbred lines into maize lines with good agronomic traits. The focus of this research was the development of gene-based markers for resistance to aflatoxin accumulation. To this end, two genes were characterized for their association with reduced aflatoxin accumulation in maize. A gene coding for a photosytem II3 protein shown to be differentially regulated between maize lines Mp313E (resistant) and Va35 (susceptible) was used to develop the marker MpM1. This marker was shown to be associated with resistance to aflatoxin accumulation in three F2:3 mapping populations derived from Mp313E x B73, Mp313E x Va35, and Mp715 x T173 and identified a new quantitative trait locus (QTL) on chromosome 4. The second gene chosen was the chitinase A gene (chiA), which has been shown to inhibit fungal growth and is differentially regulated between resistant and susceptible lines of maize. ChiA also had an association with reduced aflatoxin accumulation in the three F2:3 mapping populations and identified a new QTL in the Mp313E x Va35 population. Together, MpM1 and chiA were associated with 27% of the phenotypic variation in one environment of the Mp313E x B73 population. These markers represent the first two gene-based markers developed for resistance to aflatoxin accumulation, and the methodology developed in this study can be used to screen other candidate genes for potential use as gene-based makers.

The fungus Aspergillus flavus L. is an agricultural threat, particularly for maize (Zea mays L.). Its invasive growth and contamination before and after harvest can lead to the loss of the commodity and cause life threating consequences to humans and livestock that consume a contaminated product. The differential expression profile of two resistant maize inbred lines, Mp313E and Mp719, and two susceptible maize inbred lines, B73 and Va35, were evaluated in the mRNA and protein levels. The experimental designed used allowed to observe the responses of these maize lines to A. flavus inoculation and to the stress caused by wounding on kernels. Candidate genes were selected from a prior published GWAS and pathway analysis for the expression analysis at the mRNA level. Seventeen candidate genes were selected, and gene expression analysis via RT-qPCR was performed for nine of them. Two of characterized candidate genes that showed an upregulation above 2olds in the resistant lines. These genes are involved in oxidation responses and had an associated positive allele effect, e.g. contribute to aflatoxin accumulation. The results indicate that their role is not necessarily to make the plant more susceptible to the accumulation of aflatoxin but rather to alleviate the oxidative stress caused by the fungus. Susceptible lines, in general, did not show any difference in the expression of the selected candidate genes. At the protein level, an in-gel analysis identified a variety of stress-related proteins that were upregulated in response to A. flavus inoculation and to wounding stress in the resistant lines. A list of genes associated to the proteins identified was compiled for further characterizations and possible use as molecular markers in marker-assisted selection of commercial maize lines resistant to A. flavus.

Screening for resistance to Aspergillus flavus and aflatoxin contamination in maize is an ongoing effort by universities, state and federal agencies. We evaluated two techniques to screen for resistance; quantitative polymerase reaction (QPCR) and solid-phase microextraction (SPME). Methods were adapted to accurately detect and quantify the fungus in culture and in the vegetative stage of plant tissues. These assays can eliminate microbiological techniques. The primary objectives of the study were to utilize 1) QPCR to detect and quantify fungal biomass in maize stem tissues to evaluate resistance in maize genotypes to A. flavus colonization in situ and in vivo and 2) SPME to identify key MVOC’s to differentiate aflatoxigenic and non-aflatoxigenic strains of A. flavus in situ. A novel QPCR TaqMan probe (OMG3) was designed to detect a region in the aflP gene. The OMG3 probe detected 98.3% of the aflatoxigenic strains. The predominant MVOC’s extracted from both aflatoxigenic and non-aflatoxigenic strains were alcohols, ketones and hydrocarbons. The aflatoxigenic strain produced 39 compounds and the non-aflatoxigenic strain produced 41 compounds. Dimethylsulfide and 2-heptanol were key MVOC biomarkers produced only by the aflatoxigenic strain of A. flavus. Accuracy of the QPCR OMG3 probe, in vivo and in situ procedures were developed. A toothpick inoculation method was used to artificially inoculate maize stems in the vegetative stage five (V5). Plants were harvested at V7 and sampled at predetermined sites. This method was 91% consistent for infecting maize plants. The OMG3 probe was evaluated in in vivo and in situ studies conducted in the greenhouse, growth chamber, and field. Lesion length was greater in susceptible lines in 4 of 7 greenhouse trials. Based on inoculation data, subsequent research should focus on refining tissue-sampling methods and increasing length of plant growth time for tissue sampling post-inoculation.

This study aimed to understand the mechanism of maize inbreds resistance to A. flavus by exploring the proteins that are differentially regulated in presence of pathogen. Silk has been hypothesized as one of the entry routes of fungal growth and so the proteome of silks was investigated by 1) performing a comparative proteomic study to identify silk proteins that are abundant in resistant maize inbreds and down-regulated or absent in susceptible inbreds, 2) identifying the up-regulated proteins in maize resistant and susceptible inbreds when challenged by A. flavus 3) by mapping the proteome of silk proteins in a A. flavus resistant inbred and 4) performing an antiungal assay to test antiungal activity of silk proteins extracted from resistant and susceptible maize inbreds. Using comparative proteomics, proteins that are contributing to the resistance phenotype and could be used for marker-assisted selection in breeding programs were identified from silks collected from resistant (Mp313E, Mp420) and susceptible (SC212m, Mp339) maize inbred 21 and 25 days after silk emergence (DAS) and also, from the silks of ears inoculated at 15 DAS and collected 6 days after inoculation (DAI). Silk proteins were extracted and analyzed by 2-dimensional gel electrophoresis (2-DE). Gel images were analyzed by PD Quest software (Bio-Rad) and proteins that were consistently different were identified using MALDI-TOF-TOF. Two candidate genes that were up-regulated in 21 and 25 DAS in resistant tissues were investigated for polymorphisms and their RNA expression was also studied. Nine proteins from all the differentially regulated proteins were mapped to chromosomes 1, 2, 4 and 6 which are known to have aflatoxin resistance QTLs. Proteome map of Mp313E silks was developed using 2-DE and multi dimensional identification technology (MudPIT) and approximately 971 identified proteins were functionally annotated from the sequences available at AgBase website. The reference map of Mp313E silks could also be used to link proteomics with trancriptomics, metabolic mechanisms and genomics. Antifungal assays using GFP-tagged A.flavus and chitinase assay on silk proteins from resistant and susceptible corn inbreds showed significant activity in the resistant line compared to the susceptible line (p<0.01). A model describing the role of silk proteins in fungal resistance is proposed.

Identification of fungal isolates is critical in studying Aspergillus flavus ecology and for developing methods to reduce aflatoxin contamination. In our efforts to track biocontrol applications of the atoxigenic A. flavus K49 (NRRL 30797), we have developed a rapid and accurate classification system for A. flavus based on PCR product melting temperatures (Tm). Using 18 primers and a total of 59 Aspergilli strains, including all 49 representatives of the Georgian peanut Vegetative Compatibility Groups (VCGs), a decision tree Tm flowchart was generated. The decision tree can classify all 59 strains using only 9 of the SSR primers and an average of 3.4 primers for each definitive classification. To confirm the effectiveness of the decision tree for strain identification, unknown samples isolated from experimental fields inoculated with various A. flavus strains in Stoneville, MS were analyzed. Ninety-six percent of the samples could be placed into a VCG using Tm(s) coupled with the decision tree. This dynamic system is an excellent tool for researchers studying biodiversity of A. flavus.

<p style="text-align: justify;">Prisustvo plesni i mikotoksina u hrani predstavlja vi&scaron;estruku opasnost, kako sa aspekta bezbednosti hrane, tako i sa aspekta globalne trgovine. Učestalost i intenzitet pojave plesni na uzorcima zrna hlebne p&scaron;enice i spelte prikupljenih u regionu Vojvodine određeni su nakon žetve tokom trogodi&scaron;njeg perioda (2015-2017). Istraživanja su obuhvatila identifikaciju i karakterizaciju 38 izolata A. flavus primenom polifaznog pristupa koji uključuje klasične mikrobiolo&scaron;ke i molekularne metode. Ispitivanjem potencijala biosinteze AFB₁ izolata A. flavus utvrđeno je da su dva izolata poreklom sa zrna hlebne p&scaron;enice pokazala aflatoksigeni potencijal. Ve&scaron;tačka inokulacija različitih Triticum vrsta: hlebne p&scaron;enice, spelte, korasan i hibrida p&scaron;enice toksigenim izolatama izvr&scaron;ena je u fazi cvetanja u cilju poređenja otpornosti ovih vrsta na pojavu A. flavus i produkciju AFB₁. Visok nivo AFB₁ (256 &mu;g/kg) je kvantifikovan samo u zrnu spelte, dok kod drugih Triticum vrstama, zrno nije bilo kontaminirano AFB₁ (&lt;LOD). Određivanjem fizičko-hemijskih karakteristika plevičastih omotača Triticum vrsta potvrđen je njihov potencijalni uticaj na rast i razvoj A. flavus i biosintezu AFB₁. Efekti različitih temperatura (15, 23, 30 i 37&deg;C) i aktivnosti vode (0,85; 0,90; 0,95 i 0,99) na biosintezu AFB₁ ispitani su na ve&scaron;tački inokulisanim uzorcima spelte sa plevičastim omotačima kao i prethodno olju&scaron;tenim zrnima. Optimalni uslovi za biosintezu tj. uslovi pri kojima je ostvaren najveći prinos AFB₁ bili su temperatura 30 &deg;C i aw 0,99 u svim tipovima uzoraka (zrna spelte inkubirana bez plevičastih omotača - ZBPO, plevičasti omotači - PO i zrna nakon lju&scaron;tenja tj. olju&scaron;tena zrna - OZ). Rezultati su pokazali da je prisustvo plevičastih omotača bilo za&scaron;titna barijera za razvoj infekcije i akumulaciju AFB₁ u zrnu. Matematički modeli, razvijeni primenom faktora sa visokom značajno&scaron;ću kao &scaron;to su temperatura skladi&scaron;tenja i aktivnost vode, mogu biti kori&scaron;ćeni u predviđanju akumulacije AFB₁ u zrnu spelte &scaron;to predstavlja ključni korak u proceni rizika. Ispitivanjem uticaja različitih nivoa kontaminacije spelte AFB₁ u poređenju sa kontrolnim nekontaminiranim uzorkom ukazano je na smanjenje određenih parametara tehnolo&scaron;kog kvaliteta i potencijalne gubitke pecivnih svojstava speltinog bra&scaron;na pri sadržaju AFB₁ od&nbsp;&nbsp; 50 &mu;g/kg i 250 &mu;g/kg.</p> / <p>The presence of fungi and mycotoxins in food presents a multiple risk, both from the aspect of food safety and from the aspect of global trade. The frequency and incidence of mycobiota on common wheat and spelt grains samples collected in the region of Vojvodina were determined after harvest during the three-year period (2015-2017). The research covered the identification and characterization of 38 A. flavus isolates using a polyphase approach including classical microbiological and molecular methods. Testing the A. flavus isolates for AFB₁ biosynthesis, it was found that two isolates originating from wheat grains possess the aflatoxigenic potential. Artificial inoculation of different Triticum species: common wheat, spelt, khorasan and hybrid wheat with toxigenic isolates was carried out in the flowering stage in order to compare the resistance of these species to the occurrence of A. flavus and the production of AFB₁. The highest AFB₁ level (256 &mu;g/kg) was determined only in the dehulled spelt grains, in comparison to other species where AFB₁ was not detected in dehulled grains. In order to investigate the impact of wheat hulls on development of A. flavus, including the biosynthesis of toxic fungal metabolites, physico-chemical and structural properties of different Triticum spp. hulls were characterized. The effects of different temperatures (15, 23, 30 and 37 &deg; C) and water activity (0.85; 0.90; 0.95 and 0.99) on AFB₁ biosynthesis were examined on artificially inoculated hull-less as well as hulled spelt grains. The optimal conditions for AFB₁ biosynthesis (the conditions in which the highest AFB₁ yield was achieved) were temperature 30 &deg;C and 0.99 aw in the all tested spelt samples (hull-less grain, dehulled grains and hulls). Accumulation of AFB₁ was significantly higher in hull-less than in dehulled grains that implicate the protective effect of spelt hulls. Mathematical models, developed using high-significance factors such as storage temperature and water activity, can be used to predict the accumulation of AFB₁ in spelt grains, which is a key step in risk assessment. By examining the influence of different levels contamination levels of spelt grain with AFB₁ and comparing to the control (uncontaminated) sample, the reduction in certain technological quality parameters and the potential loss of dough properties of spelt flour with AFB₁ content of 50 &mu;g/kg and 250 &mu;g/kg was pointed out.</p>

Os objetivos foram: identificar a microbiota fúngica e a ocorrência de aflatoxinas em cultivar de amendoim, identificar a microbiota fúngica do solo e correlacionar os resultados obtidos com os níveis de atividade de água encontrados. As amostras (solo e amendoim) foram provenientes de: Jaboticabal, Rosália, Tupã e Cafelândia (SP). A microbiota fúngica do solo revelou que o fungo do gênero Penicillium foi o mais frequente (52,1 %) nas quatro regiões durante as duas coletas (após a emergência das plantas e duas semanas antes do arranquio). Dentro do gênero Aspergillus, A. flavus foi a espécie mais frequente (13,5 %). A microbiota fúngica dos grãos e das cascas, das quatro regiões, nas duas coletas (duas semanas antes do arranquio e após o arranquio), demonstrou maior frequência de isolamento do fungo do gênero Fusarium (70,2 %). Do gênero Aspergillus, a espécie A. flavus foi a mais frequente (9,8 %). A análise de aflatoxinas revelou a presença de aflatoxinas em 5 % das amostras de grãos analisadas. Nas cascas, 13,75 % das amostras apresentaram contaminação por aflatoxinas. / The objectives were: to identify the mycoflora and the occurrence of aflatoxins in a peanut variety, identify the soil mycoflora and compare the acquired results with the levels of water activity found. The samples (peanut and soil) were collected from: Jaboticabal, Rosália, Tupã and Cafelândia (SP/Brazil). The mycoflora of the soil revealed that the genus Penicillium was the most frequent (52.1 %) in the four regions during the two trials (after the emergence of the plants and two weeks before the uprooting). Within the genus Aspergillus, A. flavus was the most frequent specie (13.5 %). The mycoflora of the kernels and peanut hulls, from the four regions in the two trials (two weeks before the uprooting and after the uprooting), demonstrated greater frequency of isolation of the genus Fusarium (70.2 %). Within the genus Aspergillus, A. flavus was the most frequent specie (9.8 %). The analysis of aflatoxins revealed the presence of aflatoxins in 5 % of the kernels samples analyzed. In peanut hulls, 13.75 % of the samples presented aflatoxin contamination.