O gene TrCit do fungo filamentoso Trichoderma reesei codifica a proteína citrato sintase, uma enzima chave do ciclo de Krebs. Análise da região 5´ upstream de TrCit mostra que o gene está adjacente ao gene TrIcl (que codifica a proteína isocitrato liase, uma enzima do ciclo de glioxalato), em uma orientação cabeça-cabeça. A região promotora intergênica de 647 pb rica em G + C, apresenta uma ilha CpG, seqüência INR, caixas GC, caixas CAAT, sítios de ligação para diversos fatores de transcrição e é isenta de caixa TATA. O gene TrCit de 1573 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1422 pb produz uma proteína de 474 aminoácidos, com um peso molecular estimado de 52,3 kD. O gene TrIcl de 1880 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1788 pb produz uma proteína de 596 aminoácidos, com um peso molecular estimado de 65,4 kD. A atividade transcricional da região promotora foi analisada utilizando como repórter o gene de higromicina B fosfotransferase (hph). Uma região funcional necessária à transcrição de ambos os genes foi identificada na região central do promotor e contém uma caixa GC que liga o putativo fator de transcrição Sp1 de T. reesei (TrZnFSp1). O gene do putativo fator de transcrição “zinc-finger” TrZnFSp1 de 1500 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1344 pb produz uma proteína de 448 aminoácidos, com um peso molecular estimado de 48,4 kD. Os resultados mostram que ambos os genes são transcritos de forma divergente a partir de um promotor bidirecional que compartilha na região central uma caixa GC, necessária para a transcrição de ambos os genes. / The TrCit gene from the filamentous fungus Trichoderma reesei codes for the citrate synthase protein, a key enzyme in the Krebs cycle. Analysis of TrCit 5’ upstream region showed that it is adjacent to the TrIcl gene that codes for isocitrate lyase protein, an enzyme involved in the glyoxylate cycle. Both genes, on a head-to-head orientation, are separated by an intergenic GC-rich and TATA-less promoter region of 647 base pairs. This bidirectional promoter has diverse cis regulatory elements: a CpG island, two INR sequences, GC boxes, CAAT boxes and several putative interaction sites for different transcription factors. The TrCit gene, 1,573-base pair-long, has an open reading frame of 1,422 base pairs interrupted by two introns. The gene codes for a protein with an estimated molecular weight of 52.3 kD. The TrIcl gene, 1,880-base pair-long, contains 3 exons and 2 introns and a putative coding sequence of 1,788 base pairs. The estimated molecular weight of TrICL is 65.4 kD. he transcriptional activity of the intergenic promoter region was analyzed using hygromicin B phosphotransferase (hph) as a reporter gene. A functional region required for the transcription of both genes was identified in the centre of this promoter. It has a GC box that interacts with a putative transcription factor Sp1 from T. reesei (TrZnFSp1). The results presented in this work show that both genes are divergently transcribed from a bidirectional promoter that shares an essential central GC box.

O projeto visou aumentar a produção de celulases em fungos T. harzianum IPT 821 e T. reesei QM 9414. Esporos foram submetidos à radiação UV-C. Células das colônias mais capacitadas ao crescimento foram sucessivamente cultivadas em meio solidificado, contendo concentrações progressivamente reduzidas fonte de carbono, de modo a resultar pressão ambiental seletiva e crescente. Após esta etapa as linhagens isoladas foram cultivadas em meio composto por bagaço de cana/farelo de trigo na proporção 80/20, 60% de umidade, 30°C por 72h. Uma linhagem, originária da cepa IPT 821, apresentou atividade de 3,7 FP U/gms, 50% superior em relação à parental: 2,6 U/gms (p=0,001; p<0,05). Análises das frações enzimáticas indicaram uma diferença significativa (p=0,001; p<0,05) na atividade de xilanase: 4,7 U/gms (mutante) e 4 U/gms (parental). Ensaios de hidrólise, Avicel como substrato (1% de sólidos; w/v) indicaram um aumento de quase 70% na hidrólise em 48h, da mutante em comparação à parental (8,7% (mutante) e 4,8% (parental), concentração enzimática de 2,5 FP U/gms). / This project aimed to increase cellulase production in fungi T. harzianum IPT 821 and T. reesei QM 9414. Spores were exposed to UV-C radiation. Colonies were plated and those showing best growth were successively cultivated in plates containing increasingly stress conditions reduced concentrations of the carbon source thus creating a progressive selective pressure. After this pre-selection step isolated strains were cultivated in medium comprising sugar cane bagasse and wheat straw, in the proportion of 80/20 respectively, 60% of moisture, 30°C for 72h. One strain, originated from IPT 821 strain, showed a cellulolytic activity of 3,7 U/gdw; 50% superior to the parental strain: 2,6 U/gdw (p=0,001; p<0,05). Analysis of the enzymatic cocktail showed significant difference (p=0,001; for p <0,05) on xylanase activity: 4,7 U/gdw (mutant strain) and 4 U/gdw (parental strain). Hydrolysis assays, using Avicel as substrate (1% w/v) showed an increase on hydrolysis of about 70%, in 48h (8,7% (mutant strain) and 4,8% (parental strain), enzyme load of about 2,5 U/gdw).

No description available.

Supplementary material to figures contained on DVD only available with manuscript. / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences, 2005. / Bibliography: p. 221-244. / Introduction -- Materials and methods -- Isolation of the hex1 gene from Trichoderma reesei and Ophiostoma floccosum -- Expression of DsRed under the cbh1 promoter and the hex1 promoter with random integration -- Modified expression vectors containing a fusion to a portion of hex1 gene sequence -- Expression of DsRed from the hex1 locus and the phenotypic characteristics of a hex1 deletion mutant -- Summary and concluding discussion. / For Trichoderma reesei to be developed as an effiecient producer of a large variety of proteins, the expression system requires diversification. In particular, the choice of promoters available needs to be broadened to include promoters which are active in conditions other than those conducive to induction of cellulase expression. Using proteomics, the HEX1 protein was identified as an abundant protein of the cell envelope of T. reesei when grown on a range of carbon sources, suggesting that a strong constitutive promoter drives the expression of this physiologically important protein. This thesis is an exploration into the hex1 gene promoter and the role of hex1 in the maintenance of mycelium integrity in T. reesei with consideration for the application of this gene in the further development of filamentous fungi as protein expression systems. -- The single copy hex1 gene and flanking regions were isolated from T. reesei and another biotechnologically important fungus, Ophiostoma floccosum. The fluorescent reporter protein DsRed1-E5 was expressed under the T. reesei hex1 promoter and promoter activity was monitored by fluorescence CLSM and RNA analysis. During the rapid growth phase of a culture, the hex1 promoter was active in a range of carbon sources and three transcipt types with alternative tsp and splicing sites were discovered for the hex1 gene. The distribution of fluorescence throughout the mycelium suggested spatial regulation of the hex1 promoter as well as temporal regulation. The promoter was continually active in the absence of a functional hex1 gene product suggesting that the hex1 promoter is regulated in part, by negative feedback from the endogenous gene product. Interruption of the hex1 gene produced hyphae that leaked excessive volumes of cytoplasm when physically damaged which may be advantageous for the externalisation of selected protein products. The results indicate that the regulation of the hex1 hene promoter is complex and that the hex1 gene is integral to the maintenance of the integrity of the fungal mycelium. / Mode of access: World Wide Web. / xv, 244 p. ill

Thesis by publication. / "August 2006" / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences & Dept. of Chemistry & Biomolecular Sciences), 2007. / Bibliography: leaves 157-183. / 1. Introduction -- 1.1. Proteomics and two-dimensional electrophoresis -- 1.2. A proteomic approach to study the filamentous fungus Trichoderma -- 1.3. Aims of the thesis -- 2. Materials and methods -- 3. Results and discussion -- 3.1. Method development for the display and identification of fungal proteins by 2DE and mass spectrometry -- 3.2. Discovery of novel determinants in the biological control of phytopathogens by Trichoderma atroviride -- 3.3. Summary and concluding remarks. / Trichoderma harzianum and T. atroviride are filamentous fungi commonly found in soil. Both display biocontrol capabilities against a range of phytopathogenic fungi including Rhizoctonia solani and Botrytis cinerea which are known pests of hundreds of commercially important crops including tomatoes, potatoes, beans, cucumber, strawberries, cotton and grapes. These Trichoderma species secrete a combination of enzymes degrading cell walls and antibiotics to overgrow and kill fungal phytopathogens. They are seen as an environmentally friendly alternative to chemical fungicides currengly used on crops. / A proteomic approach was taken to separate and identify proteins from a strain of T. harzianum with well established biocontrol properties. Several methods were developed in this thesis to display the whole proteome content and several subcellular proteome fractions from T. harzianum. Proteins were separated by two-dimensional electrophoresis and identified by mass spectrometric methods. The resulting proteomic maps represent the first extensive array of cellular and sub-cellular proteomes for T. harzianum. / Cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls were compared by differential gel electrophoresis to identify a suite of new proteins involved in the biological control response. Twenty four T. atroviride protein spots up-regulated in the presence of the R. solani cell walls were identified by mass spectrometry and N-terminal sequencing. Proteins identified from this study included previously implicated enzymes degrading cell walls and three novel proteases, vacuolar serine protease, vacuolar protease A and trypsin-like protease. The genes encoding two of these proteases, vacuolar protease A and vacuolar serine protease have been cloned by degenerate primer PCR and genomic walking PCR and sequenced. The gene sequences and protein sequences derived from these genes have been partially characterised. / Mode of access: World Wide Web. / 194 leaves ill

Made available in DSpace on 2014-07-29T15:16:28Z (GMT). No. of bitstreams: 1 Marcelo Ramada -.pdf: 3086154 bytes, checksum: d8d600492d75495c63dd916423b6e86b (MD5) Previous issue date: 2010-03-31 / Trichoderma harzianum is a saprophytic fungus, known for its potential as a biological control agent of different phytopathogens that causes losses in crops. Its action is based on different mechanisms like volatile and non-volatile antibiotics production, competition for nutrient and space, production of hydrolytic enzymes and mycoparasitism. Fusarium solani is the agent of bean dry root rot, and is responsible for significant losses. This work sought to analyze and identify secreted proteins from T. harzianum grown in the presence of F. solani cell wall, in an attempt to obtain new insights on biocontrol.In the dual culture test, T. harzianum proved to be a potent antagonist of F. solani. Some differences in the secreted proteins profile and final medium pH were observed when T. harzianum was grown in TLE medium containing glucose and in TLE and Minimum medium containing the cell wall of the phytopathogen. 33 proteins from 18 different genes were identified. The protein Sm1 was identified and secreted in all growth conditions. Many hydrolytic enzymes related to mycoparasitism, like endochitinases, &#946;-1,3, &#946;-1,6-glucanases, proteases and &#945;-1,3-glicanases, were identified and among the proteases, a hypothetical metalocarboxipeptidase and a serine subtilisin-like never described for T. harzianum. Some enzymes with unkown roles in the mycoparasitism, such as &#946;-1,3-glucanosyltransferase, secreted only in MM growth medium supernatant, were also identified. / Trichoderma harzianum é um fungo saprofítico, conhecido devido ao seu grande potencial como agente de controle biológico de diferentes fitopatógenos. Sua ação é baseada em diferentes mecanismos como a produção de antibióticos voláteis e não-voláteis, competição por espaço e nutrientes, produção de enzimas hidrolíticas e o micoparasitismo. Fusarium solani causa a podridão radicular seca no feijoeiro, resultando em perdas significativas. Este trabalho buscou analisar e identificar as proteínas secretadas por T. harzianum quando crescido na presença de parede celular de F. solani, em uma tentativa de obter novas informações sobre o biocontrole. Nos testes de pareamento, T. harzianum provou ser um potente antagonista de F. solani. Foi observada uma variação tanto no perfil de proteínas secretadas quanto no pH final, quando T. harzianum foi crescido em meio TLE contendo glicose e em meio TLE e Mínimo (M.M.) contendo parede celular do fitopatógeno. No total, 33 proteínas de 18 genes diferentes foram identificadas neste trabalho. A proteína Sm1 foi identificada e secretada em todas as condições de crescimento. Várias enzimas hidrolíticas relacionadas ao micoparasitismo, como endoquitinases, &#946;-1,3, &#946;-1,6-glicanases, proteases e &#945;- 1,3-glicanases, foram identificadas e dentre as proteases, uma hipotética metalocarboxipeptidase e uma serina subtilisin-like nunca descritas em T. harzianum. Foram identificadas, também, enzimas que não possuem função conhecida no micoparasitismo, como a &#946;-1,3-glicanosiltransferase, presente somente no sobrenadante do crescimento realizado em MM.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Grapevine trunk diseases are a cause of decline and loss of productivity in grapevines at all stages of growth. These diseases are caused by a complex of wood-inhabiting fungi that infect mainly through pruning wounds. The management of these diseases relies on wound protection to prevent infection since there are no eradicative control measures to cure infected vines. There are few or no fungicides registered for grapevine pruning wound protection in most countries, while Trichoderma biocontrol agents are often available. This study aimed at improving grapevine wound protection by Trichoderma (T.) spp. and to gain a better understanding of the factors and mechanisms involved in biocontrol. The effect of pruning time (early or late) and five timings of application of the biocontrol agent after pruning on pruning wound colonisation by T. atroviride and T. harzianum were determined. Chenin blanc and Cabernet Sauvignon vineyards were pruned in July (early) and August (late) of 2011 and 2012, and pruning wounds were treated with suspensions of the Trichoderma spp. at various times (0, 6, 24, 48 and 96 hours) after pruning. Wound colonisation was depended on the physiological state of the vine at pruning for both cultivars. However, for the 2012 season in Chenin blanc, wound colonisation was similarly high for both pruning times, which was attributed to high rainfall and humidity. Application of the biocontrol agents 6 hours after pruning consistently resulted in high wound colonisation by the Trichoderma spp. in both cultivars and pruning times. In both cultivars, pruning wound infection due to natural inoculum was higher in wounds made in late winter than those made earlier. The effect of conidial formulation in nutritional (glucose, yeast extract and urea) and bio-enhancing (chitin and cell free culture filtrates) additives, on pruning wound colonisation by T. atroviride was also investigated. Nutritional additives increased the extent of pruning wound colonisation by T. atroviride compared to the un-amended conidial suspensions in a glass house study. The additives as well as Garrison, a fungicide containing pruning wound paint, and Eco77®, a registered T. harzianum biocontrol product, were tested in field trials for wound protection from infection by Phaeomoniella (Pa.) chlamydospora. In 2011, the pathogen was inoculated a day after pruning and all the Trichoderma spp. treatments similarly reduced Pa. chlamydospora infection by 75% to 90% in Thompson Seedless, while control was less in Chenin blanc and ranged from 40% to 74%. In 2012, the trial was carried out on Chenin blanc only and the pathogen was inoculated at intervals of 1, 3 and 7 days after pruning. Wound protection by the Trichoderma treatments was highest when wounds were inoculated with Pa. chlamydospora seven days after pruning. Two conidial formulations, a culture filtrate made from a chitin based medium and a combination of yeast extract, urea and glucose, consistently enhanced biocontrol efficacy. These formulations reduced Pa. chlamydospora infection to levels similar to those of Garrison. The integration of chemical and biological wound protection could provide both immediate and long term wound protection, but is limited by the sensitivity of the biocontrol agent to fungicides. Benzimidazole resistant Trichoderma strains were generated by gamma irradiation from the wild type isolates of T. atroviride (UST1 and UST2) and T. harzianum (T77). Mutants from UST1 and UST2 were of similar biological fitness as the wild type isolates and retained their in vitro antagonistic activity against grapevine trunk pathogens, while the mutant from T77 had reduced fitness and was not antagonistic to the pathogens. The wild type, UST1, and its mutant were tested alone and in combination with thiophanate methyl and carbendazim, respectively, for their ability to prevent pruning wound infection by Pa. chlamydospora. The combination of the UST1 mutant and carbendazim was the most effective treatment and gave the highest reduction in Pa. chlamydospora infection (70% to 93% control). Grapevine cell cultures were used to compare the response of grapevines to T. atroviride and Eutypa (E.) lata as a first step to determining the importance of Trichoderma-grapevine interactions in pruning wound bio-protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis related (PR) proteins was profiled over a 48-hour period using quantitative reverse transcriptase PCR. The cell cultures responded to fungal elicitors in a hypersensitive-like response that lead to a decrease in cell viability. Fungal elicitors from both fungi triggered the same genes and caused up-regulation of phenylalanine ammonia-lyase (PAL), 4 coumaroyl Co-A ligase (CCo-A), stilbene synthase (STS), chitinase class IV (CHIT IV), PR 3 and PR 4, and a down regulation of chalcone synthase (CHS) genes. Higher expression of PAL and CHIT IV in cell cultures treated with the T. atroviride elicitor led to a significantly higher (P < 0.05) total phenolic content and chitinolytic enzyme activity of the cell cultures compared to cell cultures treated with the E. lata elicitor. The response of the cell cultures to the T. atroviride elicitor signifies that the induction of grapevine resistance may be involved in wound bio-protection. The role of secondary metabolites produced by Trichoderma spp. used in pruning wound protection was also investigated. A volatile antimicrobial compound, 6-pentyl α-pyrone (6PP), was isolated and found to be the major secondary metabolite from the T. atroviride (UST1 and UST2) and T. harzianum (T77) isolates. This metabolite was found to inhibit mycelial growth, spore and conidia germination of E. lata, Neofussicocum (N.) australe, N. parvum and Pa. chlamydospora. The production of 6PP was induced when the T. atroviride isolates were grown in a grapevine wood extract medium while for UST1, the 6PP concentration was further doubled when it was co-cultured with N. parvum. Results therefore, indicate that 6PP is involved in the Trichoderma-pathogen interactions on pruning wounds. The results of this study have provided new information in regards to the application of Trichoderma-based pruning wound products. The best time of application proved to be 6 hours post pruning. The formulation of conidial suspensions of Trichoderma spp. with nutritional additives and in protein extracts of the biocontrol agent showed potential in reducing variability of wound bio-protection. However, further research would be necessary to develop commercial products. The application of a fungicide together with Trichoderma spp. in the field holds promise to improve control, but would require further trials for possible commercialisation. This study is the first to report on grapevine host defence genes that are activated by the Trichoderma spp. used in pruning wound protection. Together with the characterisation of the major secondary metabolite produced by these Trichoderma spp., this information aids in understanding the mechanisms involved in the complex interaction between the biocontrol agent, the host and the pathogen. / AFRIKAANSE OPSOMMING: Wingerdstamsiektes veroorsaak terugsterwing en verlies aan produktiwiteit in wingerdstokke gedurende alle groeifases. Hierdie siektes word veroorsaak deur „n verskeidenheid van hout-koloniserende swamme wat die wingerdstok meestal deur snoeiwonde infekteer. Die bestuur van hierdie siektes is afhanklik van wondbeskerming om infeksie te verhoed, omdat daar geen uitwissende beheermetodes na infeksie bestaan nie. In meeste lande is daar min of geen swamdoders geregistreer vir snoeiwond beskerming, terwyl Trichoderma biobeheer agente gereëld beskikbaar is. Hierdie studie poog om wingerd wondbeskerming deur Trichoderma (T.) spp. te verbeter en „n meer volledige begrip van die faktore en meganismes betrokke by biologiese beheer te ontwikkel. Die effek van die tydsberekening van snoei (vroeg of laat) en vyf behandelingstye van die biobeheer agent na snoei op die kolonisering van snoeiwonde deur T. atroviride en T. harzianum is bepaal. Chenin blanc en Cabernet Sauvignon wingerde is gesnoei gedurende Julie (vroeg) en Augustus (laat) in 2011 en 2012, en snoeiwonde is behandel met Trichoderma spp. suspensies op verskillende tydspunte (0, 6, 24, 48 en 96 ure) na snoei. Wond-kolonisering was afhanklik van die fisiologiese toestand van die wingerdstok gedurende snoei vir albei kultivars. Gedurende die 2012 seisoen was wond-kolonisering ewe hoog vir albei snoeitye op Chenin blanc. Dit is verklaar deur hoë reënval en humiditeit gedurende daardie seisoen. Die aanwending van biobeheer agente 6 ure na snoei het konsekwent hoë kolonisering deur Trichoderma spp. tot gevolg gehad op albei kultivars en albei snoeitye. In albei kultivars is wondinfeksie as gevolg van natuurlike inokulum hoër gewees in wonde gemaak gedurende laat winter as in wonde wat vroeër in die seisoen gemaak is. Die effek van konidia formulasie in voeding (glukose, gisekstrak en urea) en bioverbetering (chitien en sel-vrye kultuurfiltraat) toevoegings op snoeiwond-kolonisering deur T. atroviride is ook ondersoek. Voeding toevoegings het die omvangs van snoeiwond-kolonisering deur T. atroviride vergroot in vergelyking met ongewysigde konidia suspensies gedurende „n glashuis studie. Die toevoegings, sowel as Garrison, „n snoeiwond verf wat „n swamdoder bevat, en Eco77®, „n geregistreerde T. harzianum biobeheer produk, is getoets in veldproewe vir wondbeskerming teen infeksie deur Phaeomoniella (Pa.) chlamydospora. In 2011 is die patogeen geïnokuleer „n dag na snoei en al die Trichoderma spp. behandelings het infeksie verminder met 75% tot 90% op Thompson Seedless. Beheer was minder suksesvol op Chenin blanc, waar slegs 40% tot 74% beheer behaal is. In 2012 is die proef uitgevoer slegs op Chenin blanc en die patogeen is geïnokuleer teen intervalle van 1, 3 en 7 dae na snoei. Wondbeskerming by die Trichoderma behandelinge was die hoogste wanneer wonde sewe dae na snoei geïnokuleer is met Pa. chlamydospora. Twee konidia formulasies, „n kultuurfiltraat wat bestaan het uit „n chitien-gebaseerde medium en „n kombinasie van gisekstrak, urea en glukose het deurlopend die effektiwiteit van biobeheer verbeter. Hierdie formulasies het Pa. chlamydospora infeksie verminder tot soortgelyke vlakke behaal deur Garrison. Die integrasie van chemiese- en biobeheer in wondbeskerming kan onmiddelike en langtermyn wondbeskerming bied, maar is beperk deur die sensitiwiteit van die biobeheer agent teen swamdoders. Benzimidazole-weerstandbiedende Trichoderma isolate is ontwikkel deur gamma-bestraling van die wilde-tipe isolate van T. atroviride (UST1 en UST2) en T. harzianum (T77). Mutante van UST1 en UST2 het soortgelyke biologiese fiksheid getoon as die wilde-tipe en het hul in vitro antagonistiese aktiwiteit teen wingerd stampatogene behou, terwyl die mutant van T77 verminderde fiksheid getoon het en nie meer antagonisties teen patogene was nie. Die wilde-tipe, UST1, en sy mutant is apart en in kombinasie met thiofanaatmetiel en carbendazim, respektiewelik, getoets vir die vermoë om snoeiwonde te beskerm teen Pa. chlamydospora. Die kombinasie van die UST1 mutant met carbendazim was die mees effektiewe behandeling en het die hoogste vermindering in Pa. chlamydospora infeksie gelewer (70 tot 93% beheer). As „n beginpunt om die belang van Trichoderma-wingerd interaksies in snoiewondbeheer te bepaal, is die invloed van T. atroviride en Eutypa (E.) lata op somatiese selkulture van wingerd vergelyk. Die effek van dié behandelings op ensieme in die fenielpropanoïedweg en patogenese-verwante (PR) proteïene is bepaal deur intydse PKR (real time PCR) van die korresponderende gene oor „n 48 uur tydperk. Die swam-afkomstige ontlokkers het „n hipersensitiewe-tipe reaksie in die selkulture ontlok, wat tot „n afname in sellewensvatbaarheid gelei het. Ontlokkers afkomstig van beide swamme het dieselfde gene aangeskakel en het induksie van fenielalanien ammoniak-liase (PAL), 4 kumaroïel Ko-A ligase (CCo-A), stilbeen sintase (STS), chitienase klas IV (CHIT IV), PR 3 en PR 4 veroorsaak en „n onderdrukking in chalkoon sintase (CHS) gene tot gevolg gehad. Hoër uitdrukking van PAL en CHIT IV in selkulture behandel met die T. atroviride ontlokker het gelei tot „n beduidende hoër (P < 0.05) totale fenoolinhoud en chitienolitiese aktiwiteit in selkulture in vergelyking met selkulture wat behandel is met die E. lata ontlokker. Die reaksie van die selkulture op die T. atroviride ontlokker dui daarop dat die induksie van wingerd weerstandbiedenheid betrokke mag wees in wond biobeheer. Die rol van sekondêre metaboliete geproduseer deur Trichoderma spp. wat gebruik word in snoeiwond beheer is ook ondersoek. „n Vlugtige antimikrobiese verbinding, 6-pentiel α-pyroon (6PP) is geïsoleer en bepaal om die hoof sekondêre metaboliet afkomstig vanuit die T. atroviride (UST1 en UST2) en T. harzianum (T77) isolate te wees. Hierdie metaboliet is betrokke by inhibisie van miselium groei, spoor en konidium ontkieming van E. lata, Neofusicoccum (N.) australe, N. parvum en Pa. chlamydospora. Die produksie van 6PP is geïnduseer deur die T. atroviride in wingerd hout ekstrak te kweek. In die geval van UST1, is die 6PP konsenstrasie verdubbel deur die isolaat met saam met N. parvum te kweek. Hierdie resultaat is „n aanduiding dat 6PP betrokke is in die Trichoderma-patogeen interaksie op snoeiwonde. Die resultate van hierdie studie het nuwe inligting met betrekking tot die aanwending van Trichoderma-gebaseerde snoeiwond produkte verskaf. Die beste tyd vir aanwending van sulke produkte was 6 ure na snoei. Die formulasie van konidia suspensies van Trichoderma spp. met voeding toevoegings en in proteïen ekstrakte van die biobeheer agent het potensiaal getoon in die vermindering van variasie in wondbeskerming deur biobeheer agente. Verdere navorsing sal nodig wees om kommersiële produkte te ontwikkel. Die aanwending van „n swamdoder saam met Trichoderma spp. in die wingerd is belowend om beheer te verbeter, maar het meer proewe nodig voor kommersialisering. Hierdie studie is die eerste om wingerd beskerming gene wat deur Trichoderma spp. geaktiveer word aan te meld. Laasgenoemde, saam met die beskrywing van die hoof sekondêre metaboliete wat deur hierdie Trichoderma spp. geproduseer word, dra by tot „n meer volledige begrip van die meganismes betrokke by die komplekse interaksie tussen die biobeheer agent, die gasheer en die patogeen.

Com o objetivo de avaliar o efeito de isolados de Trichoderma em cultivares de alface foram conduzidos dois experimentos divididos em dois capítulos. O primeiro com a finalidade de avaliar o efeito do Trichoderma na qualidade de mudas de alface e o segundo a sua potencialidade no controle de Meloidogyne enterolobii em plantas adultas de alface. O experimento I foi conduzido em casa de vegetação na Universidade Federal do Tocantins, em delineamento inteiramente casualizado em esquema fatorial, com 5 repetições. Foram utilizados três isolados de Trichoderma (UFT201, UFT202 e UFT205) e duas cultivares comerciais de alface (Elba e Solaris). Para este experimento foram avaliadas as seguintes características: Altura de plantas, diâmetro do caule, comprimento da raiz, número de folhas, massa seca da parte aérea e raiz, massa seca total, índice de qualidade de Dickson e eficiência relativa. Para o capítulo II foram conduzidos dois experimentos com a utilização das cultivares Elba e Solaris, dois isolados de Trichoderma UFT201 e UFT205, um isolado de nematoide M. enterolobii e a cultivar de tomate Santa Clara, usada como testemunha hospedeira padrão dos nematoides. As variáveis avaliadas foram: Comprimento da raiz, diâmetro da cabeça, massa fresca da raiz, massa fresca total, massa seca da parte aérea, eficiência relativa, número de galhas, tamanho médio de galhas, posicionamento de galhas, índice de massas de ovos e índice de reprodução. No experimento I, em geral a presença de Trichoderma não resultou em mudas de melhor qualidade, quando comparados à testemunha. O fator tempo pode ter influência, visto que o trabalho foi realizado em condição de mudas. Para o experimento II, houve redução no número de galhas e massas de ovos de nematoides nos tratamentos inoculados com Trichoderma. Através dos parâmetros que determinam a presença do nematoide na planta, foi possível constatar para as duas cultivares o potencial antagônico do Trichoderma no controle de M. enterolobii. / In order to evaluate the effect of Trichoderma isolates in lettuce cultivars two experiments were conducted, divided into two chapters. The first, in order to evaluate the effect of Trichoderma in lettuce seedlings, and the second, its potential in control of Meloidogyne enterolobii in lettuce adult plants. The first experiment was conducted in a greenhouse at the Federal University of Tocantins, in a factorial randomized design, with five repetitions. Three Trichoderma strains were used (UFT201, UFT202, and UFT205), and two commercial cultivars of lettuce (Elba and Solaris). For this experiment the following characteristics were evaluated: plant height, stem diameter, root length, number of leaves, dry weight of shoot and root, total dry weight, quality index Dickson and relative efficiency. For Chapter II two experiments were conducted with the use of Elba cultivars and Solaris, two isolates of Trichoderma UFT201 and UFT205, an isolate of nematode M. enterolobii and tomato cultivar Santa Clara, used as the default host a witness of the nematodes. The variables evaluated were: root length, diameter of the head, fresh root mass, fresh mass total shoot dry mass, relative efficiency, number of galls, average size of galls, positioning of galls, egg mass index and reproduction index. In the first experiment, in general the presence of Trichoderma did not result in better quality seedlings compared to the control. The time factor may play a role, since the work was done on seedlings. For the second experiment, there was a reduction in the number of galls and egg masses of nematodes in treatments inoculated with Trichoderma. By parameters that determine the presence of nematodes in the plant, it was established for both cultivars the antagonistic potential of Trichoderma to control M. enterolobii.

Orientador: Luciana Francisco Fleuri / Resumo: O Eucalyptus é um importante gênero florestal plantado mundialmente para fins comerciais. O Brasil é líder global em produtividade de Eucalyptus, devido às condições climáticas favoráveis e a eficientes programas de melhoramento baseados em plantações clonais, porém alguns clones de Eucalyptus possuem dificuldades de propagação, principalmente relacionadas ao enraizamento. A inoculação fúngica e bacteriana pode auxiliar o enraizamento e desenvolvimento vegetal pela produção de metabólitos tais como ácido indol-3-acético (AIA) e fitases. O objetivo deste trabalho foi produzir AIA e fitases por fermentação em estado sólido (FES) utilizando Aspergillus spp., Trichoderma spp. e Bacillus spp. e a inoculação das cepas de maior produtividade em mudas de Eucalyptus grandis x E. urophylla (clone IPB2) visando otimizar o desenvolvimento vegetal. A FES ocorreu utilizando bagaço de mandioca, farelos de soja e trigo e grãos secos de destilaria com solúveis (DDGS) de sorgo e milho como substratos. Nos filtrados da FES foram determinados a concentração de AIA e a atividade de fitase. As características físicas e químicas dos substratos [macroporosidade (%), microporosidade (%), retenção de água (mL/cm3), condutividade elétrica (mS/cm3), pH, proteína bruta (%), lipídeos (%), hemicelulose (%), celulose (%) e lignina (%)] foram determinadas e correlacionadas à produtividade das biomoléculas, a fim de esclarecer a influência na alteração do metabolismo microbiano para maior ou menor produtivida... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Eucalyptus is an important forest genus planted worldwide for commercial purposes. Brazil is the global leader in Eucalyptus productivity due to favorable climatic conditions and efficient breeding programs based on clonal plantations, but some Eucalyptus clones have difficulties of propagation, mainly related to rooting. Fungal and bacterial inoculation can positively influence rooting and plant development by producing metabolites such as indole-3-acetic acid (IAA) and phytases. This work aimed to produce IAA and phytases under solid state fermentation (SSF) using Aspergillus spp., Trichoderma spp. and Bacillus spp. and inoculate the best producers in cuttings of Eucalyptus grandis x E. urophylla (clone IPB2), aiming to optimize plant development. The SSF technique was performed using cassava bagasse, soybean and wheat bran and distillers dried grains with solubles (DDGS) of sorghum and maize as substrates. The concentration of IAA and phytase activity were determined in SSF filtrates. The physical and chemical characteristics of the substrates [macroporosity (%), microporosity (%), water retention (mL/cm3 ), electrical conductivity (mS/cm3 ), pH, brute protein (%), lipids (%), hemicellulose %), cellulose (%) and lignin (%)] were determined and correlated to the biomolecules productivity in order to clarify their influence on the alteration of microbial metabolism for higher or lower productivity of IAA and phytases. The microorganisms with higher productivity of IAA and ph... (Complete abstract click electronic access below) / Doutor

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-03-14T13:04:27Z No. of bitstreams: 1 Jose Aldo Teixeira da Silva.pdf: 972040 bytes, checksum: d6bba973ec0295e20860c1178753f8e3 (MD5) / Made available in DSpace on 2017-03-14T13:04:27Z (GMT). No. of bitstreams: 1 Jose Aldo Teixeira da Silva.pdf: 972040 bytes, checksum: d6bba973ec0295e20860c1178753f8e3 (MD5) Previous issue date: 2015-10-26 / Cassava has economic importance in Brazil and abroad, for their food importance. The Northeast is the region that most increased this crop in Brazil, but farmers use few technologies, resulting in diseases and decline in production. The crop is attacked for numerous pathogens as Fusarium solani and Scytalidium lignicola, that causes diseases such as cassava root rot and cassava black root, affecting the commercial part of plant, the tubera. Several research, support the use of practices that enable the production and support the environment, so the use of biocontrol has been used, particularly with the use of Trichoderma spp. However, little research reporting the physiological mechanisms that are activated by plants when exposed to this interaction, pathogen-plant-antagonist such as plant response to infection diseases. Thus, the objective this work was to verify the potential antagonism of Trichoderma spp. against F. solani and S. lignicola, and evaluating the physiological response of plants exposed to the pathosystem. We analyzed in vitro inhibition of mycelial growth of F. solani and S. lignicola to select the best Trichoderma antagonist with potential direct these pathogens. By the method of use of basal medium with colloidal chitin as the only carbon source, it was used to evaluate the best Trichoderma production of chitinase. In greenhouse, the selected Trichoderma was compared against the use of plant resistance inducer in vivo inhibition assessing the severity of disease infested plants after 92 days of growth. Then we evaluated the enzyme of antioxidative complex (peroxidase ascorbate, catalase, peroxidase and polyphenyl oxidase). All strains were inhibited the growth of pathogens. However, the best strain against F. solani was T. hamatum (6656), and has been for S. lignicola was T. harzianum (3086), with values of 88.91 and 80.78% growth mycelial respectively, being designated as the best candidates for treatments in the greenhouse. For chitinase production evaluation, all Trichoderma tested were positive, highlighting the T. aureoviride (5158) to produce 6.70 U mL-1, being selected candidate for inhibiting the severity of pathogens in greenhouse. All Trichoderma selected for in vivo testing against the severities of diseases, presented efficiency compared to the control with the presence of the pathogen. However, the T. aureoviride (5158), showed the blunt values for both pathogens. The results of the enzymes of the plants, treatments that stopped inoculation of Trichoderma showed satisfactory results, collaborating with the answer in a greenhouse, however, again treating with the strain (5158), showed the best values, especially the production of the enzymes peroxidase and ascorbate peroxidase. Therefore, concludes the efficiency of Trichoderma aureoviride (5158), for use as biocontrol of root rot and black rot of cassava as induced resistance to plant pathogens, contributing to the production of enzymes antioxidatives. / A mandioca tem grande expressão econômica no Brasil e no mundo, pela sua importância alimentar. O Nordeste é a região que mais aumentou a produção dessa cultura no Brasil, porém, os produtores usam poucas tecnologias, acarretando em possíveis desenvolvimentos de doenças e queda na produção. A cultura sofre muitas perdas na produção pela ação de inúmeros fitopatógenos, sendo os do solo os mais severos, o Fusarium solani e Scytalidium lignicola, ganham ênfase por causarem doenças como a podridão radicular e negra da mandioca, respectivamente, afetando diretamente a parte comercial da planta, a tubera. Muitas pesquisas, apoiam o uso de práticas que viabilizem a produção e favoreçam o ambiente, assim, a utilização de biocontroladores vem ganhando destaque, principalmente fungos do gênero Trichoderma spp., por suas características como antagonistas de uma gama de fitopatógenos. Todavia, há poucas pesquisas que relatam os mecanismos fisiológicos que são ativados pelas plantas, quando expostos a essa interação, patógeno-planta-antagonista, como resposta vegetal a infecção de doenças. Desse modo, objetivou-se verificar o potencial antagônico de dez Trichoderma spp. aos patógenos F. solani e S. lignicola, e avaliar a resposta fisiológica das plantas exposta a esse patossistema. Foi analisado, in vitro, a inibição do crescimento micelial do F. solani e S. lignicola em meio batata-dextrose-ágar, para selecionar o melhor Trichoderma com potencial antagonista direto a esses patógenos. Através do método da utilização de meio basal com quitina coloidal, sendo a única fonte de carbono, foi utilizada para avaliar o melhor Trichoderma para produção de quitinase. Em casa de vegetação, foi comparado os Trichoderma selecionados, contra o indutor de resistência, in vivo, avaliando a inibição das doenças infestadas nas plantas, após 92 dias de crescimento. Em seguida, avaliou-se a produção de enzimas das plantas dos tratamentos in vivo, do complexo oxidativo (ascorbato peroxidase, catalase, peroxidase e polifeniloxidase). Todas as estirpes apresentaram inibição do crescimento dos fitopatógenos. Porém, a melhor estirpe contra o F. solani foi o T. hamatum (6656), e para o S. lignicola foi o T. harzianum (3086), com valores de 88,91 e 80,78% de inbiçao de crescimento micelial, respectivamente, sendo designados como os melhores candidatos para tratamentos em casa de vegetação. Para a avaliação de produção de quitinase, todos os Trichoderma testados foram positivos, destaque dado ao T. aureoviride (5158) por produzir 6,70 U mL-1, sendo selecionado para candidato da inibição da severidade dos patógenos em casa de vegetação. Todas os Trichoderma selecionados para teste in vivo contra as severidades das doenças, apresentaram eficiência em comparação ao controle com a presença do patógeno. No entanto, o T. aureoviride (5158), foi a que apresentou valores contundentes para os dois patógenos. Dos resultados das enzimas das plantas, os tratamentos que detiveram a inoculação dos Trichoderma apresentaram resultados satisfatórios, colaborando com a resposta em casa de vegetação, contudo, novamente o tratamento com a estirpe (5158), foi a que apresentou melhores valores, com destaque a produção das enzimas ascorbato peroxidase e peroxidase. Portanto, conclui-se a efiência do Trichoderma aureoviride (5158), para uso como biocontrolador da podridão radicular e da podridão negra da mandioca, pois induziu resistência a planta aos fitopatógenos, colaborando com a produção das enzimas antioxidativas.