No description available.

Blumeria graminis, the obligate biotrophic grass powdery mildew, is a highly pathogenic fungus capable of inflicting foliar diseases and of causing severe yield losses. There is asexual and sexual propagation in the life cycle of B. graminis. In the epidemiological processes of this pathogen, both types of spores - asexual conidia and sexual ascospores – are crucial. Conidia of B. graminis are demonstrated to perceive cuticular very-long-chain aldehydes as molecular signal substances notably promoting germination and differentiation of the infection structure (the appressorium) – the prepenetration processes – in a concentration- and chain-length-dependent manner. Conidial germination and appressorium formation are known to be dramatically impeded by the presence of free water on the host surface. However, sexually formed ascospores are reported to easily germinate immersed in water. There are abundant assays on conidial prepenetration processes. However, with respect to the stimulating effects of very-long-chain aldehydes and to the influence of the presence of free water, ascosporic prepenetration processes are still obscure. In order to study the effects of very-long-chain aldehydes on the ascosporic prepenetration processes of wheat powdery mildew fungus B. graminis f. sp. tritici, Formvar®-based in vitro systems were applied to exclude the secondary host effects (such as host resistance) and to reproducibly provide homogeneous hydrophobic substratum surfaces. By the presence of even-numbered very-long-chain aldehydes (C22 - C30), the appressorium formation of the ascospores was notably triggered in a chain-length dependent manner. N-octacosanal (C28) was the most inducing aldehyde tested. Unlike conidia, ascospores could easily differentiate immersed in water and showed a more variable differentiation pattern even with a single germ tube differentiating an appressorium. To evaluate the alternative management against barley powdery mildew fungus Blumeria graminis f. sp. hordei, the suppressing effects of UV-C irradiation on the developmental processes of conidia on artificial surfaces (in vitro) and on host leaf surfaces (in vivo) were assayed. In vitro and in vivo, a single dose of 100 J m-2 UV-C was adequate to decrease conidial germination to < 20 % and to reduce appressorium formation to values < 5 %. UV-C irradiation negatively affected colony pustule size and vegetative propagation. Under photoperiodic conditions of 2h light/16h dark, 6h dark/12h light or 6h dark/18h light, UV-C-treated conidia showed photoreactivation (photo-recovery). White light-mediated photoreactivation was most effective immediately after UV-C irradiation, suggesting that a prolonged phase of darkness after UV-C application increased the efficacy of management against B. graminis. UV-C irradiation increased transcript levels of three putative photolyase genes in B. graminis, indicating those were probably involved in photoreactivation processes. However, mere white light or blue light (wavelength peak, 475 nm) could not induce the up-regulation of these genes. To determine whether visible light directly impacted the prepenetration and penetration processes of this powdery mildew pathogen, conidia of Blumeria graminis f. sp. hordei and Blumeria graminis f. sp. tritici were inoculated onto artificial surfaces and on host leaf surfaces. Samples were analyzed after incubation periods under light conditions (white light intensity and spectral quality). Increasing white light intensities directly impaired conidial prepenetration processes in vitro but not in vivo. Applying an agar layer under the wax membrane compensated for conidial water loss as a consequence of high white light irradiation. Light stimulated in vitro and in vivo the appressorium elongation of B. graminis in a wavelength-dependent manner. Red light was more effective to trigger the elongation of appressorium than blue light or green light assayed. Taken together, the findings of this study demonstrate that 1) a host surface recognition principle based on cuticular very-long-chain aldehydes is a common feature of B. graminis f. sp. tritici ascospores and conidia; 2) the transcriptional changes of three putative photolyase genes in B. graminis are mediated in a UV-C-dependent manner; 3) light directly affected the (pre)penetration processes of B. graminis. / Blumeria graminis, der obligate biotrophe Grasmehltau, ist ein hochpathogener Pilz, der in der Lage ist, Blätter zu schädigen und in Getreidekulturen schwere Ertragsverluste zu verursachen. Im Lebenszyklus von B. graminis kommen sowohl sexuelle als auch asexuelle Reproduktion vor. In den epidemiologischen Prozessen dieses Erregers sind beide Arten von Sporen - asexuelle Konidien und sexuelle Ascosporen - von entscheidender Bedeutung. Conidien von B. graminis reagieren nachweislich auf kutikuläre, sehr langkettige Aldehyde als molekulare Signalstoffe, die insbesondere die Keimung und Differenzierung der Infektionsstruktur (das Appressorium) - die Präpenetrationsprozesse - konzentrations- und kettenlängenabhängig fördern. Es ist bekannt, dass die Konidienkeimung und Appressoriumbildung durch die Anwesenheit von freiem Wasser auf der Wirtsoberfläche dramatisch behindert wird, während sexuell gebildete Ascosporen unter diesen Bedingungen keimen. Es gibt zahlreiche Untersuchungen zu Konidien-Präpenetrationsprozessen. Im Hinblick auf die stimulierende Wirkung von langkettigen Aldehyden und den Einfluss von freiem Wasser sind die askosporischen Präpenetrationsprozesse jedoch noch unklar. Um die Effekte langkettiger Aldehydmoleküle auf die ascosporischen Präpenetrationsprozesse von Weizenmehltaupilz zu untersuchen, wurde B. graminis f. sp. tritici auf Formvar®-basierten In-vitro-Systemen angewendet. Dadurch konnten sekundäre Wirtseffekte (wie Wirtsresistenz) ausgeschlossen und homogene hydrophobe Substratoberflächen reproduzierbar bereitgestellt werden. Die Anwesenheit geradzahliger sehr langkettiger Aldehyde (C22 – C30) erhöhte deutlich die Ausbilung des Appressoriums von Ascosporen. Von den getesteten Aldehyden zeigte n-Octacosanal (C28) die größte Induktionskraft. Im Gegensatz zu Konidien konnten bei Ascosporen auch unteri Anwesenheit von freiem Wasser variable Differenzierungsmuster festgestellt werden, bei denen auch einzelne Keimröhren zur Differenzierung des Appressoriums führten. Zur Bewertung der alternativen Bekämpfung gegen Gerstenmehltaupilz Blumeria graminis f. sp. hordei wurde die Unterdrückungswirkung von UV-C-Strahlung auf die Entwicklungsprozesse von Konidien auf künstlichen Oberflächen (in vitro) und auf Wirtsblattoberflächen (in vivo) untersucht. In vitro und in vivo war eine Einzeldosis von 100 J m-2 UV-C ausreichend, um die Konidienkeimung auf < 20 % zu verringern und die Appressoriumsbildung auf Werte < 5 % zu reduzieren. Die UV-C-Bestrahlung beeinflusste die Größe der Kolonepusteln und die vegetative Vermehrung negativ. UV-C behandelte Konidien wurden unterschiedlichen Lichtverhältnissen ausgesetzt: 2 h hell / 16 h dunkel, 6 h dunkel / 12 h hell und 6 h dunkel / 18 h hell. Dabei zeigten sie unterschiedliche Photoreaktivierungsaktivitäten. Die Weißlicht-vermittelte Photoreaktivierung war unmittelbar nach der UV-C-Behandlung am effektivsten, was darauf hindeutet, dass eine verlängerte Phase der Dunkelheit nach der UV-C-Anwendung die Wirksamkeit des Managements gegen B. graminis erhöhte. UV-C-Bestrahlung erhöhte die Transkriptmengen von drei mutmaßlichen Photolyase-Genen in B. graminis, was darauf hindeutet, dass sie wahrscheinlich an Photoreaktivierungsprozessen beteiligt waren. Weißes oder blaues Licht (Wellenlängen-Peak, 475 nm) konnte die Hochregulation dieser Gene nicht induzieren. Um zu bestimmen, ob sichtbares Licht direkt auf die Präpenetrations- und Penetrationsvorgänge dieses Mehltau-Erregers einwirkt, wurden Konidien von Blumeria graminis f. sp. hordei und Blumeria graminis f. sp. tritici auf künstliche Oberflächen und auf Wirtsblattoberflächen inokuliert. Die Proben wurden nach Inkubationunter verschiedenen Lichtbedingungen (Weißlichtintensität und Spektralqualität) analysiert. Das Auftragen einer Agarschicht unter der Wachsmembran kompensierte den Wasserverlust der Konidien als Folge der Bestrahlung mit hoher Lichtintensität. Zunehmende Weißlichtintensitäten beeinträchtigten Konidienpräpenetrationsprozesse direkt in vitro, aber nicht in vivo. Eine wellenlängenabhängige Stimulation der Verlängerung des Appressoriums konnte sowohl in vitro als auch in vivo beobachtet werden. Dabei war rotes Licht wirksamer, um die Verlängerung des Appressoriums auszulösen, als blaues oder grünes Licht. Zusammengefasst zeigen die Ergebnisse dieser Studie, dass 1) ein Prinzip der Wirtsoberflächenerkennung basierend auf kutikulären, sehr langkettigen Aldehyden ein gemeinsames Merkmal von B. graminis f sp. tritici Ascosporen und Konidien ist; 2) die Transkriptionsänderungen von drei putativen Photolyase-Genen in B. graminis in einer UV-C-abhängigen Weise vermittelt werden und 3) Licht direkt die (Vor-) Penetrationsvorgänge von B. graminis beeinflusste.

No description available.

Powdery mildew caused by <i>Blumeria graminis</i> f. sp. <i>tritici</i> is one of the most devastating diseases in wheat (<i>Triticum aestivum</i>) worldwide. Hypersensitive, race specific genes primarily have been deployed to control the disease, however recent efforts have shifted to breeding for more durable resistance, such as "adult plant resistance" (APR). Molecular markers and quantitative trait loci (QTL) associated with APR to powdery mildew must first be validated and QTL effects evaluated in different genetic backgrounds and breeding populations to be useful in marker-assisted selection (MAS) programs. Eighteen simple sequence repeat (SSR) markers previously mapped near the three QTL in Massey for APR to powdery mildew were evaluated for association with APR for powdery mildew in a recombinant inbred line (RIL) breeding population derived from a cross between USG 3209 and a moderately susceptible cultivar Jaypee, wherein Massey is the resistance source for APR in USG 3209. Thirteen new SSR markers were added to the pre-existing genetic linkage maps near the three QTL associated with APR in the Becker by Massey (BM) population. Interval mapping analysis of mildew severity data collected in 2002 (F_5:6) and 2003 (F_6:7) field experiments with marker genotype data obtained in 2003 (F_6:7) confirmed the presence of three QTL for APR on chromosomes 1B, 2A, and 2B in the USG 3209 by Jaypee (UJ) population. The QTL on chromosomes 1B, 2A, and 2B explained 12% to 13%, 59% to 69%, and 22% to 48% of the phenotypic variance for powdery mildew severity in the UJ RIL populations, respectively, in the two field experiments. The efficiency of MAS was examined using powdery mildew data collected in 2002 and 2003 field experiments and also from a greenhouse experiment in 2004 (F_7:8), wherein adult plants of the 293 RILs were evaluated for disease severity using a composite of five different isolates of <i>B. graminis</i>. Selection of RILs possessing the QTL on chromosome 2A and to a lesser extent the one on chromosome 1B was effective in identifying powdery mildew resistance in both greenhouse and field experiments, whereas the effect of the QTL on chromosome 2B was insignificant in the greenhouse. Overall, selecting RILs with QTL on chromosomes 2A and 2B was most successful in identifying highly resistant RILs compared to selecting RILs having other combinations of two or three QTL combinations. The RILs possessing both QTL on chromosomes 2A and 2B had mean mildew severities of 4.4% and 3.2% in 2002 and 2003 field experiments, respectively. Breeders implementing MAS programs for APR to powdery mildew via selection of RILs containing the two QTL on chromosomes 2A and 2B likely will obtain RILs having high levels of resistance in the field. However, combining all three QTL may ensure greater durability of APR, on the basis that resistance conferred by QTL on chromosome 2A and 1B are genetically stable across all environments in this study. / Master of Science

<p>Prouzrokovač pepelnice (Blumeria graminis f. sp. tritici) je stalni pratilac proizvodnje p&scaron;enice u agroekolo&scaron;kim uslovima Srbije. Sposobnost seksualne i aseksualne reprodukcije, kao i visok genetski protok patogena, čine da je prouzrokovač pepelnice genetski veoma divergentan, značajnog potencijala za adaptabilnost i promenu u virulentnosti populacije. Do sada je utvrđen znatan broj patotipova ovog patogena, dok se veliki broj konstantno stvara, te je efikasna otpornost p&scaron;enice kratkotrajne prirode. Rad na selekciji na otpornost p&scaron;enice prema prouzrokovaču pepelnice je važan zadatak oplemenjivačkih programa &scaron;irom sveta.&nbsp; Cilj istraživanja je bio utvrđivanje strukture virulentnosti populacije patogena B. graminis f. sp. tritici koja potiče sa teritorije Srbije, kao i dinamike njene promene tokom godina. U polnoj populaciji prouzrokovača pepelnice identifikovana je virulentnost prema svim genima p&scaron;enice za otpornost prema prouzrokovaču pepelnice (Pm geni), tokom ispitivanih godina i u svim ispitivanim lokalitetima. Utvrđene su statistički značajne razlike u virulentnosti gena patogena prema Pm genima domaćina, kao i linearni trend promene frekvencije virulentnosti gena polne populacije B. graminis f. sp. tritici. Najnižu frekvenciju virulentnosti u populaciji tokom ispitivanog perioda ispoljila je kombinacija gena V-5+6, dok je frekvencija virulentnih gena V-6 i V-7 gena bila na konstantno visokom nivou. Najznačajnija promena u populaciji je karakteristična za kombinaciju gena V-2+4b+6. U polnoj populaciji patogena u ispitivanom periodu nijedan izolat nije ispoljio virulentnost prema kombinaciji gena Pm17, Pm2+, Pm2+6 i Pm5+6. Klaster analizom utvrđen je visok stepen genetičkog diverziteta izolata u zavisnosti od godine. Analizom veze između gena avirulentnih lokusa utvrđena je pozitivna avirulentna veza između parova gena virulentnih prema Pm17 i Pm2+6, Pm2 i Pm2+; Pm2+ i Pm3a; Pm2+ i Pm1+2+9; Pm2+ i Pm2+4a+6; Pm3a i Pm8; Pm3a i Pm1+2+9; Pm3a i Pm2+6; Pm8 i Pm17; Pm17 i Pm1+2+9; Pm17 i Pm2+6 i parova gena</p><p><br /><br /><br /><br />Pm1+2+9 i Pm2+6. Piramiding ovih parova gena može biti dobra strategija za produžetak perioda efikasnosti otpornosti određene sorte. Najniža frekvencija virulentnosti polne populacije utvrđena je u lokalitetu Sremska Mitrovica. Najveći koeficijent genetičke udaljenosti utvrđen je između izolata koji potiču sa datog lokaliteta i izolata koji potiču sa ostalih ispitivanih lokaliteta. Između genetičke i geografske udaljenosti izolata prouzrokovača pepelnice za ispitivane lokalitete nije utvrđena statistički značajna linearna veza.&nbsp; Analiza strukture virulentnosti bespolne populacije ukazala je da komplekstnost patotipova prouzrokovača pepelnice raste sa porastom useva. Geni patogena za virulentnost prema genima Pm3a i Pm2+ nisu utvrđeni. Najvi&scaron;u frekvenciju virulentnosti imao je gen za virulentnost prema Mld genu za otpornost p&scaron;enice prema prouzrokovaču pepelnice. Statistički značajne korelacije su utvrđene između polne i bespolne populacije ispitivanog patogena. Istraživanja su imala za cilj i određivanje otpornosti genotipova p&scaron;enice koja je uslovljena promenom u populaciji prouzrokovača pepelnice. Visok nivo parcijalne otpornosti ispoljilo je sedam genotipova koji u svojoj genealogiji imaju roditelja sa kombinacijom gena Pm5+6, kao i kombinacijom gena&nbsp; Pm5+6 i Pm2+4b+6. Na osnovu ispitivanja strukture virulentnosti populacije prouzrokovača pepelnice smatra se da je kombinacija gena Pm5+6 nosioc otpornosti datih genotipova.</p> / <p>Powdery mildew (caused by Blumeria graminis f. sp. tritici) is the common disease of wheat in the agroecological conditions of Serbia. The ability of sexual and asexual reproduction, as well as a high genetic flow of this pathogen, make B. graminis f. sp. tritici genetically very divergent, with a significant potential for adaptability and change in the virulence of its population. So far, a large number of pathotypes of this pathogen have been identified, while a large number is constantly produced and effective resistance of wheat is short-termed. Improving wheat resistance to powdery mildew is an important task of breeding programs worldwide. The objective of this research was to determine and characterize the virulence structure of the B. graminis f. sp. tritici population that originate from the territory of Serbia, as well as the dynamics of its change over the years. During the examined period virulence of sexual stage of B. graminis f. sp. tritici population was identified for all wheat resistance genes (Pm genes) and in all investigated locations. Cluster analysis showed a high degree of genetic diversity of B. graminis f. sp. tritici isolates, depending on the year. Statistically significant differences were found between the virulence genes, as well as the linear trend of change in the virulence frequency of the genes during the investigated period. The lowest frequency of virulence in the population was found for the combination of the V-5+6 gene, while the frequency of the virulent genes V-6 and V-7 was at a consistently high level. The most significant change in the population was characteristic for the combination of the V-2+4b+6.&nbsp; Data were analyzed for associations among pairs of avirulence genes, and a positive avirulent relationship was established between the pairs of genes virulence to Pm17 and Pm2+6, Pm2 and Pm2 +; Pm2 + and Pm3a; Pm2 + and Pm1+2+9; Pm2+ and Pm2+4a+ 6; Pm3a and Pm8; Pm3a and Pm1+2+9; Pm3a and Pm2+6; Pm8 and Pm17; Pm17 and Pm1+2+9; Pm17 and Pm2 6 and gene pairs Pm1+2+9 and Pm2+6.</p><p><br /><br /><br /><br />Pyramiding these gene pairs can be a good strategy for extending the period of effectiveness of the resistance of a particular variety of wheat. The lowest frequency of virulence of the sexual stage of population was determined at the location of Sremska Mitrovica. The highest coefficient of genetic distances is established between isolates originating from a given location and isolates originating from other investigated locations. Between the genetic and geographical distance of B. graminis f. sp. tritici isolates a statistically significant linear relationship was not found. Virulence structure of the asexual population indicated that the complexity of the powdery mildew pathotypes grows with the crop growth. In this part of population the genes virulence to Pm3a and Pm2 + have not been found. The highest frequency of virulence had gene virulence to Mld resistance gene. Statistically significant correlations were found between the sexual and asexual population of the examined pathogen. The research was also aimed at determining the resistance of wheat genotypes to powdery mildew. High level of partial resistance was found in seven tested wheat genotypes whose parents have combination of the Pm5+6 gene, as well as a combination of the Pm5+6 and Pm2+4b+6 genes. Based on the study of the virulence structure of the powdery mildew population in this research, it is considered that the combination of the Pm5+6 gene is the carrier of the resistance of the given genotypes</p>

L’utilisation de molécules stimulatrices des défenses des plantes (SDP), également appelées inducteurs de résistance, constitue une alternative potentielle aux traitements fongicides conventionnels pour combattre les maladies dues à des champignons phytopathogènes. Trois SDPs, le tréhalose (TR), l’acide salicylique (SA) et l’heptanoyl d’acide salicylique (HSA), un dérivé fonctionnel du SA, protègent le blé (Triticum aestivum L.) contre l’oïdium (Blumeria graminis f.sp. tritici), lorsqu’ils sont utilisés de façon préventive. La protection obtenue n’est pas liée à un effet fongistatique direct sur la germination des spores du champignon, mais à l’induction chez le blé de défenses qui diminuent le développement de la maladie. Notre travail consistait à caractériser les mécanismes de défense mis en jeu après applications foliaires de TR, de HSA et de SA chez un cultivar de blé sensible à l’oïdium. Un suivi de l’expression de gènes marqueurs de défense, réalisé par RTqPCR, a été mené en cinétique,depuis le traitement par les SDPs jusqu'à 4 jours après infection. Les activités enzymatiques correspondantes ont été également mesurées, et l’influence indirecte des SPDs sur le processus infectieux a été observé en microscopie in planta. Ainsi, les réactions de défense déclenchées par le TR, le SA et le HSA ralentissent l’évolution de l’infection, jouant respectivement sur la germination des conidies, structures infectieuses de Bgt, sur la germination du tube germinatif appressorial (AGT) et sur la proportion d’AGTs qui parviennent à pénétrer dans les tissus foliaires. Le TR est à l’origine d’une augmentation de l’expression des gènes codant pour une lipoxygénase, une protéine de transfert des lipides et une phospholipase C, impliquées dans le métabolisme lipidique et la signalisation, et de gènes codant pour des protéines PR comme les chitinases et PR1, tous connus comme marqueurs de défense. Ainsi, les réactions déclenchées par le TR correspondent à un effet inducteur de défenseplutôt qu’à une réaction de stress osmotique. Le HSA modifie particulièrement le métabolisme lipidique, en induisant fortement et pendant toute la cinétique, l’expression du gène codant pour la LOX et l’activité correspondante, aussi bien hors contexte infectieux qu’en contexte infectieux. Cette augmentation de l’activité LOX n’est pas retrouvée chez des feuilles traitées au SA et caractérise donc le HSA. L’importance des réactions observées avec le TR, le SA et le HSA, hors contexte infectieux et en présence de Bgt amène à discuter les effets éliciteurs et potentialisateurs de ces 3 SDPs. / The use of plant elicitors, also known as resistance inducers, is an alternative to conventional fungicides to control diseases caused by phytopathogenic fungi. Three resistance inducers, trehalose (TR), salicylic acid (SA) and heptanoyl salicylic acid (HSA), a functional derivative of SA, protect wheat (Triticum aestivum L.) against powdery mildew (Blumeria graminis f . sp. tritici) when applied prior to infection. The protection obtained is not linked to any direct fungistatic effect on the fungal spore germination, but to the induction of wheat defences that impair the development of the disease. Our work aimed at characterizing the defence mechanisms triggered after foliar applications of TR, HSA and SA in a wheat cultivar susceptible to powdery mildew. Monitoring of defence markers genes expression by RTqPCR was conducted during a time-course experiment from the treatment time until 4 days after infection. Corresponding enzyme activities were also measured, and the indirect influence of elicitors on the infectious process was observed by microscopy in planta. Thus, defence responses triggered by TR, SA and HSA slow the progression of the infection, respectively altering the germination of infectious structures such as conidia, the differentiation of appressorial germ tube (AGT) and the proportion of AGTs that manage to penetrate the epidermis. TR causes an increase in the expression of genes encoding a lipoxygenase, a lipid transfer protein and a phospholipase C, which are involved in lipid metabolism and signaling, and genes encoding for PR-proteins such as chitinases and PR1, all known as markers of defence. Thus, the reactions triggered by TR match with the ones triggered during induced defence rather than during osmotic stress response. HSA specifically targeted lipid metabolism, inducing strongly and throughout the time-course, the expression ofthe gene encoding LOX and the corresponding enzyme activity, both in infectious and non-infectious contexts. This increase in LOX activity was not found in leaves treated with SA and thus characterizes HSA mode of action. The importance of the reactions observed with TR, SA and HSA, in non-infectious conditions and in the presence of Bgt have to be considered regarding either elicitation or potentiation.

Exocyst is a protein complex conserved in yeast, animals and plants. It mediates tethering of a secretory vesicle to the plasma membrane in the semifinal step of exocytosis. Several roles of exocyst in the processes of cell polarization in plant cells have been implied, including polarized growth of polen tubes and root hairs, cytokinesis, deposition of seed coat pectin and possibly autophagy. One of the most recent roles of exocyst includes also a response to bacterial and fungal pathogens. Exo70B2 and Exo70H1 subunits were shown to play prominent roles in this respect, with Exo70H1 being responsible for mediating defense against bacterial (Pseudomonas syringae) and Exo70B2 defense against both bacterial and fungal (Blumeria graminis) pathogens. Recently, new data appeared indicating the interaction between Exo70B1 and RIN4 and Exo70A1 and NOI6, respectively. RPM-1 interacting protein 4 (RIN4) is a well known negative regulator of both basal and effector-triggered resistance. This thesis shows interaction between NOI6 and several exocyst subunits, confirming previous data. I show here that exocyst subunints interact specifically with N terminus of NOI6 protein and that this interaction is lost in the shorter version of NOI6 mimicking AvrRpt2 cleavage. Since AvrRpt2 is an effector protein from...

Made available in DSpace on 2016-12-08T16:44:42Z (GMT). No. of bitstreams: 1 PGPV11MA101.pdf: 1302742 bytes, checksum: b7efc06d336ca4a49c0225d1d0b03cfb (MD5) Previous issue date: 2011-02-08 / The objectives of the work were: a) to obtain the damage function equations for multiple pathosystem to calculate the EDT to time fungicides application; b) to quantify the control and damage of the yield components, and c) To relate the brown spot incidence and severity in different growth stages. The experiments were carry out at the NBN Seeds Company during the 2009 and 2010 crop seasons, located in Muitos Capões county, Rio Grande do Sul state, Brazil and in the Agro science Center at Santa Catarina State University only in 2010. The cultivar BRS Cauê was used as susceptible cultivar to brown spot and powdery mildew. The experiments design was in randomized block with four replications. The nine treatments consisting of different rates (half and recommended rates) and fungicide applications number (one, two, three and four) of mixture strobilurin and triazole fungicides, generating the disease gradients intensity. The total area of 5.0 x 2.5 m was the experimental unit in both experiments. The fungicide applications and incidence and severity assessment were done at EC 22, EC 31, EC 39, EC 45 and EC 56 plant development stages. Plants from central rows of each plot were manually harvested and grain yield (GY), one-thousand grain weight (TGW) and granulometry (G) were evaluated. In the first chapter, the damage function equations between disease intensity and GY for each plant development stages, obtained by regression analysis, in both 2009-10 crop growing seasons were significant and negative indicative that increasing disease severity lead into decreasing grain yield. The damage coefficients of these equations can be used to calculate the economic damage threshold. In the second chapter, the values of disease intensity were used to calculate the area under disease progress curve (AUDPC). The GY, TGW, G and AUDPC values were tested using the mean comparison between treatments and the percentage of damage and disease control were determinated. The largest 2009 crop damage was 45.87%, 15.47% and 25.84% for GY, G and TGW, respectively. The ultimate control was 68.11% when considered the severity and four foliar applications, independent of the used dose. In 2010, the greatest damage were 31.16%, 14.02% and 10.76% in Muitos Capões and 39.44%, 23.59% and 45.88%, in Lages, for GY, 10 TGW and G, respectively. The highest percentage of control, based on the leaf severity were 71.63% and 73.96% for Muitos Capões and Lages, respectively. The greater control, independent of used dose were obtained with three and four applications in Muitos Capões and four applications in Lages. In the third chapter, brown spot incidence and severity data were subjected to regression analysis and correlation and the obtained values were significant and positive. The brown spot and powdery mildew diseases incidence and severity recommended by the Technique Indication of crop (TIC) to initiate fungicide applications is 20% and 5% respectively. The severity average values for initiating chemical control are 0.77% and 0.34% respectively for 2009 and 2010 crop seasons when the incidence diseases were substituted in the equations. These values are lower than TIC recommended values / Os objetivos do trabalho foram: a) obter equações de função de dano para patossistema múltiplo para calcular o LDE servindo como critério indicador de aplicação de fungicidas; b) quantificar controle e dano nos componentes de rendimento; e c) relacionar incidência e severidade foliar da mancha marrom em diferentes estádios fenológicos. Os experimentos foram conduzidos nas safras agrícolas 2009 e 2010 na NBN Sementes no município de Muitos Capões, RS; e, na safra de 2010 no Centro de Ciências Agroveterinárias no município de Lages, SC. Em todos os experimentos foi utilizada a cultivar BRS Cauê suscetível à mancha marrom e oídio. O delineamento foi blocos casualisados, com quatro repetições e nove tratamentos constituídos de diferentes doses (meia dose e dose indicada) e número (uma, duas, três e quatro) de aplicações de fungicidas triazóis e estrobilurinas para gerar os gradientes de intensidade das doenças. A área de cada unidade experimental correspondeu a 5,0 x 2,5 m. As aplicações e as avaliações da incidência e severidade foliar ocorreram nos estádios de crescimento (EC) EC 22, EC 31, EC 39, EC 45 e EC 56. A colheita foi manual cortando as plantas das linhas centrais de cada parcela. Foram avaliados rendimento de grãos (RG), massa de mil grãos (MMG) e granulometria (G). No primeiro capítulo, as equações de função de dano entre intensidade de doença e RG para cada EC, obtidas por análise de regressão, em ambas as safras agrícolas, foram significativas e negativas, ou seja, à medida que aumentou a intensidade de doença, diminuiu o RG. Os coeficientes de dano obtidos podem ser utilizados no cálculo do limiar de dano econômico. No segundo capítulo, os valores de intensidade das doenças foram usados para o cálculo da área abaixo da curva de progresso da doença (AACPD). Os valores de RG, MMG, G e AACPD foram submetidos ao teste de comparação de médias. Foram determinados o percentual de dano e de controle das doenças. Na safra 2009, os maiores danos foram 45,87%, 15,47% e 25,84% para RG, MMG e G, respectivamente. O controle máximo foi 68,11% considerando a severidade foliar e quatro aplicações, independente da dose usada. Em 2010, os maiores danos foram 31,16%, 14,02% e 10,76% em Muitos Capões e 39,44%, 23,59% e 8 45,88% em Lages, respectivamente para R, MMG e G. Com base na severidade obteve-se controle de 71,63% e 73,96% em Muitos Capões e Lages, respectivamente. Em Muitos Capões três e quatro aplicações e em Lages quatro aplicações, independente da dose, apresentaram maior percentual de controle. No terceiro capítulo, os dados de incidência e severidade foliar de mancha marrom foram submetidos à análise de regressão e correlação. As equações obtidas foram significativas e positivas. A incidência e a severidade foliar recomendado pela Indicação Técnica da cultura (ITC) para iniciar as aplicações de fungicidas é de 20% e 5%, respectivamente. Substituindo esse valor de incidência nas equações obtidas têm-se valores médios de severidade para iniciar o controle químico de 0,77% e 0,34% respectivamente para 2009 e 2010, inferior ao recomendado pela ITC

La cendrosa de l'ordi, causada pel fong Blumeria graminis (D.C.) E. O. Speer f. sp. hordei Em. Marchal, sin. Erysiphe graminis D.C.: Fr. f. sp. hordei Em. Marchal, anamorf: Oidium monilioides (Nees) Link, és la malaltia aèria més important en aquest conreu a Catalunya. El 1987 es van iniciar les investigacions sobre l'etiologia de les principals malalties dels cereals. Es realitzà una prospecció per tota la l’àrea cerealícola de la geografia catalana, que va permetre estimar la distribució i importància de les malalties que afecten l'ordi, el blat, el panís i l'arròs. Els resultats del període estudiat, 1987-1990, conclogueren que per a la cendrosa de l’ordi, la probabilitat d’una epidèmia severa (severitat final=60%5% en varietats susceptibles) varià entre p=0,2 i p=0,6 (entre dos i sis anys amb epidèmia severa de cada deu anys de cultiu), segons zones de conreu. Amb aquestes premisses es va anar elaborant un sistema de suport a la presa de decisions (SSPD) propi per a la cendrosa de l’ordi a l’àrea de Catalunya anomenat CENCONT, el qual prediu la malaltia tenint en compte que la cendrosa forma part d’un ‘complex de malalties’ i considerant la severitat (i l’àrea sota la corba epidèmica) com a variable independent i el possible efecte de la resta de malalties i plagues com a covariants. Per a l’aplicació pràctica de les anàlisis epidèmiques a CENCONT, es van estimar els valors dels paràmetres del model Logist com mitjanes d’un nombre de epidèmies representatives dels distints processos: de reacció varietal i de control químic. Aquests valors van resultar útils per a la predicció d’epidèmies i l’avaluació de CENCONT va demostrar la seva utilitat, tant pel que fa a la seva precisió com per a l’anàlisi econòmica fonamentada en el càlcul de la Funció de Guany. Però les assumpcions teòriques fetes en ajustar models tipus Logist, anomenats sintètics, a les dades epidèmiques de camp, han de permetre fer prediccions amb un marge d'error acceptable, en les condicions ambientals definides per cada predicció, la qual cosa s’ha de demostrar que és certa en una eina SSPD. Per aquesta raó els objectius que s’han formulat en aquest treball s’han basat resumidament en: 1) Fer una anàlisi matemàtica dels models sintètics, principalment els Logist i Richards, veient el seu comportament i estudiant les relacions y0, r i k mitjançant simulació, 2) Contrastar els resultats teòrics amb els de les epidèmies desenvolupades en condicions naturals, aplicant els models, en ajusts a epidèmies de camp de la cendrosa de l'ordi, amb la condició d'anar fixant els paràmetres o bé deixar-los lliures en el model, principalment l'asímptota k, i 3) Discutir la validesa de les assumpcions fetes en els models per ajustar les equacions a les dades experimentals, veient les interaccions entre paràmetres i les conseqüències que derivades de l'anàlisi comparativa d'epidèmies i la seva aplicació a sistemes predictius de la malaltia. Per assolir els objectius plantejats s’han utilitzat 113 processos epidèmics observats en camp en el període 1991-2002, avaluant principalment la intensitat de malaltia en el temps i basats en dos objectius experimentals: control químic i resistència varietal Paral•lelament, s’han analitzat des del punt de vista matemàtic mitjançant el programa MAPLE els models exponencial, Logist, Gompertz, Monomolecular, Von Bertalanffy-Richards i Weibull, i s’ha valorat la seva idoneïtat en la seva aplicació a malalties vegetals, especialment a la cendrosa de l’ordi. Després de l’anàlisi matemàtica, s’han aplicat els models mitjançant el programa Table Curve 2D©. En una primera fase s’han ajustat tots els models a les epidèmies de camp, excepte el model exponencial, i en una segona, de tots els models aplicats s’han seleccionat els que donaven millor rendiment estadístic pel que fa als diversos paràmetres d’avaluació. En aquest procés d’ajust, també s’han descartat les epidèmies que no aconseguien un ajust de suficient qualitat segons els criteris establerts. A més, s’han establert comparacions de les taxes relatives epidèmiques obtingudes en els ajusts mitjançant la utilització de la taxa mitjana ponderada absoluta. S’ha fet una anàlisi especial del comportament del paràmetre de forma del model Richards i s’han elaborat models de superfície de resposta per als paràmetres epidèmics y0, r i k dels models Logist i Richards. Entre les conclusions més importants s’ha trobat que el model Richards s’ajusta millor que Logist i aquest que Gompertz al conjunt d’epidèmies de cendrosa de l’ordi a Catalunya. La resta de models analitzats es van descartar per pitjor comportament. Quant als paràmetres dels models, en primer lloc no és el mateix fixar l’asímptota que no fixar-la, com a mínim des de la perspectiva estadística. En segon lloc, la taxa relativa epidèmica disminueix de valor segons si s’ajusten les dades a Richards, Logist o Gompertz respectivament, i això passa sempre respecte qualsevol variant dels models, asímptota lliure o bé asímptota fixa, i per a qualsevol objectiu epidèmic, control químic o bé resistència varietal. Respecte a la severitat inicial, no s’ha trobat una pauta de comportament definida. Sobre la predicció, es pot fer una millora d’aquesta en el sistema d’avisos CENCONT passant del model Logist al model Richards. El model Richards es pot utilitzar amb els tres paràmetres clàssics o bé amb quatre paràmetres, incorporant el paràmetre de forma però amb unes certes restriccions. Si s’agafa el model Richards triparamètric, serà necessari fixar el paràmetre de forma a un valor que raonablement pot ser inferior a 5. Aquest tipus de modelització és la recomanada per a incloure en Sistemes de suport a la presa de decisions com és el CENCONT. La millora en la predicció no implica necessàriament una millora en la presa de decisions del sistema d’avisos de la cendrosa de l’ordi a Catalunya, perquè les implicacions econòmiques no són en la major part dels casos prou importants com per provocar un canvi de decisió. La diferència màxima calculada de pèrdues associades estimades segons el model Richards, amb asímptota lliure i les associades segons el model Logist, se situa en el 2.9%, calculant-les a partir de l’ASCPM i de la severitat final. A efectes pràctics es pot considerar el mateix haver predit segons el model Richards, millor en el treball, o el Logist, el qual fou el que realment s’havia aplicat en el programa CENCONT. L’estimació Logist podria ser suficient per a la major part de les situacions (epidèmies) i que només en un petit percentatge hauria valgut la pena canviar de model. De tota manera, per al tipus de prediccions del present treball, no es veuria malament el fet d’adoptar el model Richards en un futur, amb la condició d’haver fixat el valor característic del paràmetre m per a la zona d’estudi. / El oídio de la cebada causado por el hongo Blumeria graminis (D.C.) E. O. Speer f. sp. hordei Em. Marchal, sin. Erysiphe graminis D.C.: Fr. f. sp. hordei Em. Marchal, anamorf: Oidium monilioides (Nees) Link, es la enfermedad aérea más importante en este cultivo en Cataluña. El año 1987 se iniciaron las investigaciones sobre la etiología de las principales enfermedades de los cereales. Se realizó una prospección por toda el área cerealista de la geografía catalana que permitió estimar la distribución e importancia de las enfermedades que afectan la cebada, el trigo, el maíz y el arroz. Los resultados del período estudiado, 1987-1990, concluyeron que para el oídio de la cebada, la probabilidad de una epidemia severa (severidad final=60%5% en variedades susceptibles) osciló entre p=0,2 y p=0,6 (entre dos y seis años con epidemia severa de cada diez años de cultivo), según zonas de cultivo. Con estas premisas se elaboró un sistema de soporte a la toma de decisiones (SSTD) propio para el oídio de la cebada en el área de Cataluña, llamado CENCONT, el cual predice la enfermedad teniendo en cuenta que el oídio forma parte de un ‘complejo de enfermedades’ y considerando la severidad (y el área bajo la curva epidémica) como variable independiente y el posible efecto del resto de enfermedades y plagas como covariables. Para la aplicación práctica de los análisis epidémicos en CENCONT se estimaron los valores de los parámetros del modelo Logist como medias de un número de epidemias representativas de los distintos procesos: reacción varietal y control químico. Estos valores resultaron útiles en la predicción de epidemias y la evaluación de CENCONT demostró su utilidad, tanto en lo que se refiere a su precisión como para el análisis económico fundamentado en el cálculo de la Función de Ganancias. Pero las asunciones teóricas realizadas en el ajuste de modelos tipo Logist, llamados sintéticos, a los datos epidémicos de campo, han de permitir predicciones con un margen de error aceptable, en las condiciones ambientales definidas para cada predicción, lo cual se debe demostrar como cierto en una herramienta SSPD. Por esta razón los objetivos que se han formulado en este trabajo se han basado resumidamente en: 1) Efectuar un análisis matemático de los modelos sintéticos, principalmente Logist y Richards, viendo su comportamiento y estudiando las relaciones y0, r i k mediante simulación, 2) Contrastar los resultados teóricos con los obtenidos de las epidemias desarrolladas en condiciones naturales, aplicando los modelos en ajustes a epidemias de campo del oídio de la cebada, con la condición de ir fijando los parámetros o bien dejarlos actuar libremente en el modelo, principalmente la asíntota k, y 3) Discutir la validez de las asunciones realizadas en los modelos para ajustar las ecuaciones a los datos experimentales, viendo las interacciones entre parámetros y las consecuencias derivadas del análisis comparativo de epidemias y su aplicación a sistemas predictivos de la enfermedad. Para conseguir los objetivos propuestos se han utilizado 113 procesos epidémicos observados en campo durante el período 1991-2002, evaluando principalmente la intensidad de enfermedad en el tiempo y basados en dos objetivos experimentales: control químico y resistencia varietal. Paralelamente se han analizado los modelos exponencial, Logist, Gompertz, Monomolecular, Von Bertalanffy-Richards y Weibul desde un punto de vista matemático mediante el programa MAPLE, y se ha valorado su idoneidad en la aplicación a enfermedades vegetales, especialmente al oídio de la cebada. Después del análisis matemático se han aplicado los modelos mediante el programa Table Curve 2D©. En una primera fase se han ajustado las epidemias de campo a todos los modelos excepto el modelo exponencial, y en una segunda fase de todos los modelos aplicados se han seleccionado aquellos que resultaban en un mejor rendimiento estadístico respecto a diversos parámetros evaluados. En este proceso de ajuste también se han descartado las epidemias que no conseguían un ajuste de suficiente calidad según los criterios preestablecidos. Además se han hecho comparaciones de las tasas relativas epidémicas obtenidas en los ajustes mediante la utilización de la tasa media ponderada absoluta. Se ha realizado un análisis especial del comportamiento del parámetro de forma del modelo Richards y se han elaborado modelos de superficie de respuesta para los parámetros epidémicos y0, r i k de los modelos Logist y Richards. Entre las conclusiones más importantes se destaca que el modelo Richards ajusta mejor que Logist y este que Gompertz respecto al conjunto de epidemias de oídio de la cebada en Cataluña. El resto de modelos analizados se descartaron debido a su peor comportamiento. Respecto a los parámetros de los modelos, en primer lugar no es lo mismo fijar la asíntota que no fijarla, como mínimo desde el punto de vista estadístico. En segundo lugar, la tasa relativa epidémica disminuye de valor según si se ajustan los datos a Richards, Logist o Gompertz respectivamente, sucediendo esto siempre respecto a cualquier variante de los modelos, asíntota libre o bien asíntota fija, y para cualquier objetivo epidémico, ya sea control químico o bien resistencia varietal. Respecto a la severidad inicial, no se ha encontrado una pauta de comportamiento definida. Sobre la predicción, se puede afirmar que se realizaría una mejora en el sistema de avisos CENCONT pasando del modelo Logist a utilizar el modelo Richards. El modelo Richards se puede utilizar con los tres parámetros clásicos o bien con cuatro parámetros, incorporando el parámetro de forma pero con ciertas restricciones. Si se escoge el modelo Richards triparamétrico, será necesario fijar el parámetro de forma hasta un valor que razonablemente puede ser inferior a 5. Este tipo de modelización es la recomendada para Sistemas de Soporte a la Toma de Decisiones como CENCONT. La mejora en la predicción no implica, sin embargo, una mejora en la toma de decisiones del sistema de avisos del oídio de la cebada en Catalunya, porque las implicaciones económicas no son en la mayor parte de los casos suficientemente importantes como para provocar un cambio de decisión. La diferencia máxima calculada de pérdidas asociadas, estimadas según el modelo Richards con asíntota libre y las asociadas según el modelo Logist se sitúa en el 2.9%, calculándolas a partir de l’ABCPE (Área Bajo la Curva de Progreso de la Enfermedad) y de la severidad final. A efectos prácticos se puede considerar como lo mismo el haber predicho según el modelo Richards, mejor en el trabajo, o el Logist, el cual fue el que realmente se había aplicado en el programa CENCONT. La estimación Logist podría ser suficiente para la mayor parte de las situaciones (epidemias) ya que solamente en un pequeño porcentaje habría valido la pena cambiar de modelo. De todas formas, para el tipo de predicciones del presente trabajo, no se vería como inconveniente el adoptar el modelo Richards en un futuro, con la condición de fijar un valor característico del parámetro m para la zona de estudio. / Powdery mildew caused by the fungus Blumeria graminis (D.C.) E. O. Speer f. sp. hordei Em. Marchal, sin. Erysiphe graminis D.C.: Fr. f. sp. hordei Em. Marchal, anamorf: Oidium monilioides (Nees) Link, is the most important disease on barley crop in Catalonia (Northeast of Spain). Surveys of cereal –barley, wheat, maize and rice– diseases in Catalonia were carried out on 1987 and provided the necessary data of the relative importance and crop losses of diseases on these crops. The results sowed that, in the period of 1987 to 1990, barley Powdery mildew disease had a probability of severe epidemic of p=0,2 and p=0,6 (final severity=60%5% in susceptible cultivars) depending on the sub-area. This means that frequency of critic disease is between two and six years in a ten years period of time. Those studies gave as a result the elaboration of a Decision Support System (DSS) on Powdery mildew in Catalonia called CENCONT. This DSS gives predictions of the disease as a “disease complex”, considering severity (and AUDPC) an independent variable and the rest of the diseases and pests as covariates. To build CENCONT parameters of the Logist model were estimated as means of a diversity of numerous epidemics obtained from chemical control and resistance cultivar trials. These values had showed to be useful on predicting epidemics and consequently evaluating CENCONT in the point of view of economical analysis. But theoretical assumptions of those synthetic models, as Logist model, must be sufficient to predict in an acceptable range of error in the environmental conditions of a particular prediction. This conclusion should be demonstrated. Thus, the objectives of this work had been: 1) working out of mathematical modelling using synthetic models, especially Logist and Richards, to study their behaviour and the relationships between y0, r and k by means of program simulation, 2) to check theoretical conditions applied to epidemics developed in natural conditions, adjusting models to data and testing the parameter behaviour using different starting fitting values, and 3) to validate the adjusting conditions to experimental data, knowing the interactions between parameters and their consequences in Comparative Epidemiology and Decision Support Systems. To achieve with the objectives there have been used 113 field epidemics obtained between 1991 and 2002, assessing the disease intensity vs. time and based on two experimental objectives: Chemical Control and Cultivar Resistance. Eventually, the Exponential, Logist, Gompertz, Monomolecular, Von Bertalanffy-Richards and Weibull models have been analysed mathematically using MAPLE computer program, and assessed their fitness on applied disease epidemics, particularly to powdery mildew of barley. Furthermore, the models have been applied to field epidemic data using Table Curve 2D computer program. First, all models except Exponential were tested to know their ability to fitness and later some of the models were discarded. To eliminate models statistical yield criteria on fitting were used. As well as the models some epidemics were also discarded in this process using the criteria of poor quality results of fitting. Once more, comparisons between relative epidemic rate data were done by testing the suitability of weighted mean absolute rate, and an additional analysis to study the particular behaviour of shape parameter of Richards’ model was found necessary. Finally, a 3D model of the surface response curve of epidemic parameters y0, r and k of Logist and Richards previous 2D models was developed. Main results of this work are that behaviour of Richards’ model is better than Logist model to the fitting to barley powdery mildew epidemics in Catalonia. Yields of the rest of the models had been worst and are discarded of the normal use in our conditions. With regard to parameters, it is stated that the use of fixed asymptote is statistically different in front of free asymptote. Besides, after fitting the relative epidemic rate values decrease in the order of Richards’, Logist and Gompertz models. This happens independently of considering the use of free or fixed asymptote and both chemical control or cultivar resistance objectives. Moreover, the initial severity of disease does not show any pattern of behaviour. As a consequence of the data obtained, it is obvious that should be convenient to use Richards’ model instead of Logist in the CENCONT computer program. Richards’ model could be used with his three or four parameter form, but if the second, the use of shape parameter should be controlled and restricted in a short range of values. Within the form parameter, a value les than 5 should be recommended. This modelling pattern is suitable to include in those DSS as CENCONT is. The improvement on prediction does not means that making decisions using this DSS should be better because the economic consequences are not enough frequent and relevant to decide changing the pattern. The maximum difference of crop losses calculated comparing the use of Richards’ model instead of Logist model is about 2.9%. These estimates are of final severity or AUDPC of the epidemic. In fact, the use of Richards’ model or the Logist model does not normally bear differences in the economic recommendations of CENCONT which uses Logist model. So actually the Logist model could be sufficient to the most of environmental circumstances (epidemics) and only few of them should carry a model change. Instead of this, the use of Richards’ model in next future should be considered as a clear improvement of the prediction, but fixing the shape parameter to the particular conditions of the area should be recommended as necessary in this hypothetical case.

Cereal loss due to fungal pathogens is an ongoing setback in agriculture. Elucidating plant&rsquo / s resistance and susceptibility mechanisms against these cereal killers, promises progress in agriculture. In the way of understanding barley resistance against fungus Blumeria Graminis we silenced FAS-Associated Factor 1 (FAF1) gene in its mRNA level with Virus Induced Gene Silencing (VIGS) technique. Previous research in our lab had shown an augmentation in mRNA levels of FAF1 gene in fungus infected wheat, suggesting a role of this gene in the resistance mechanism. We hypothesized that the apoptotic role of FAF1 protein in metazoan is conserved in plants by including FAF1 as a factor in hypersensitive response. Barley lines Pallas01 and Pallas03 which are respectively resistant and susceptible against fungus Blumeria graminis hordei 103 (Bgh103) were used for fungal inoculations after FAF1 silencing, to test if the hypersensitive response against fungus Bgh103 was prevented. In this aspect the formation of death lesions on the Pallas01 leaf due to fungal resistance was not prevented demonstrating that FAF1 silencing with VIGS in the resistant Pallas01 line of barley is not sufficient to stop apoptosis. On the other hand the FAF1-silenced barley susceptible line Pallas03 became more sensitive to fungal stress based on conidia (body part of the fungus) counting after trypan blue staining of the infected leaves. In the C-terminus of FAF1 an ubiquitin like domain-X (UBX) is found, which is the cause of stress sensitivity based on the reported data obtained about this domain&rsquo / s loss of function in other proteins. These results suggest that FAF1 is a catalyst in the hypersensitive response and its loss of function makes barley more susceptible to fungal stress. On the other hand a short mRNA homology was found among FAF1 and many pathogen disease related proteins making this homology a possible target site for VIGS of FAF1 generated siRNAs, which might cause some other protein to be responsible for the barley susceptibility against the fungus.