Thesis (PhD(Agric))--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Postharvest decay of stone fruit in the Western Cape province of South Africa is caused primarily by Botrytis cinerea (grey mould) and Monilinia laxa (brown rot). Little is known about the relative importance and seasonal occurrence of the two pathogens in nectarine and plum orchards, the mode of penetration of fruits by M laxa, latency and subsequent disease expression by the latter pathogen. These aspects were investigated in this study. By sampling from the Unifruco Quality Evaluation Scheme and from 11 stone fruit orchards, observations were made over a 3-year period of the occurrence of grey mould and brown rot in the major stone fruit regions. Botrytis cinerea was found to be the most important pathogen causing blossom blight and postharvest decay on stone fruit. The pathogen was most prominent on early- and mid-season culti~ars. Brown rot was exclusively caused by M laxa and no evidence was found that M fructicoZa had been introduced into the region. Monilina laxa was most prominent on the later maturing cultivars. Botrytis cinerea blossom infection did not contribute directly to postharvest decay. Both surface inoculum and latent infection consistently occurred on fruit in each orchard, although at fluctuating levels. Disease expression on developing fruit was not governed by the amount of B. cinerea occurring on fruit surfaces, but by the ability of fruit to resist disease expression. The amount of B. cinerea on fruits was generally higher during spring than during summer. Monilinia laxa occurred sporadically on the blossoms of late-maturing cultivars. Immature fruit were generally pathogen-free and disease expression occurred on maturing fruit only. These findings suggest that conidia of M laxa are generally produced in orchards when fruits are approaching maturity and can penetrate and infect maturing fruit only. The behaviour of airborne M laxa conidia was subsequently studied on nectarine (cultivar Flamekist) and plum (cultivar Laetitia) fruit. For these studies, an inoculation method that simulates natural infection by airborne conidia was used. Fruit at pit hardening, 2 wk before harvest, harvest stage and after cold storage (nectarines 4 wk at -o.soC followed by 1 wk at 23°C at ±56% RH; plums 10 days at .....().5°C,18 days at 7.5°C followed by 1 wk at 23°C at ±56% RH) were dusted with dry conidia of M laxa in a settling tower. The fruits were incubated for periods ranging from 3 to 48 h at high relative humidity (2':93%, humid fruit) or covered with a film of water (wet fruit). Behaviour of the solitary conidia was examined with an epifluorescence microscope on skin segments stained in a differential stain containing fluorescein diacetate, aniline blue and blankophor. The ability of solitary conidia to colonise the fruit surface, penetrate fruit skins and to induce disease expression was determined by using a differential set of tests. For these tests, fruit were surface-sterilised (30 s in 70% ethanol) or left Unsterile. From each group, fruit were selected for isolation (skin segment test), immersed in a 3% paraquat solution (paraquat-treated fruit test) or left untreated (sound fruit test). 1be findings demonstrated that solitary conidia of M laxa behaved consistently on plum and nectarine fruit surfaces: appressorium formation and direct penetration was not observed on any of the fruit surfaces and germ tubes penetrated fruit predominantly through stomata, lenticels and microfissures in the fruit skin. The monitoring of airborne conidia revealed subtle effects of the fruits on the behaviour of solitary germlings, which could not be seen when using conidial suspensions. On both fruit types, no deleterious effect was seen on conidial and germling survival when fruit were kept humid at pit hardening, 2 wk before harvest and harvest. However, conidial and germling survival were drastically reduced by prolonged wet incubation of fruits. The findings on disease expression in the skin segment, paraquat-treated fruit and sound fruit tests clearly showed that the skin of both nectarine and plum fruits were not penetrated at the pit hardening stage, latent infections were not established and fruitsreacted resistant to disease expression. These facets on both fruit types were furthermore unaffected by wetness. The barrier capacity of the fruit skin of the two stone fruit types however differed drastically later in the season. On nectarine, fruit skins were more readily penetrated and disease expression became more pronounced when fruit approached maturity. Penetration and disease expression on ripening nectarine fruit were furthermore greatly influenced by wetness. Maturing plum fruit, on the other hand, did not display the drastic change in the barrier capacity of fruit skins as observed on nectarine. The influence of wetness on infection and disease expression was also less pronounced than on nectarine. In fact, plum fruit remained asymptomatic in the sound fruit test after inoculation and humid incubation at the 2 wk before harvest stage, harvest stage and after cold storage. Plum fruit at these stages only developed disease after a prolonged period (~12 h) of wet incubation. The paraquat fruit test revealed that these fruits became more susceptible to latent infection, but they were not as susceptible as nectarine. Collectively, these findings indicate that M. laxa fruit rot epidemics on plum and nectarine are driven by inoculum levels on fruit approaching maturity and by weather conditions prevailing during the preharvest and harvest period. However, the barrier capacity of plum skins is considerably more effective than that of nectarine fruit. Wounds would therefore play an important role in the epidemiology of M. laxa on plum fruit. Infection of fresh wounds by airborne M. laxa conidia, and by conidia and germlings that have established on fruits, was therefore investigated. Plum fruit (cultivar Laetitia) at pit hardening, 2 wk before harvest, harvest stage and after cold storage were dusted with dry conidia of M. laxa in a settling tower.- Infection of rionwounded fruit and of fresh wounds by \ the airborne conidia on dry, humid and wet plum fruit surfaces, and by conidia and germlings that have been established on fruits under the wetness regimes was then investigated. Nonwounded immature and mature fruit remained mostly asymptomatic, whereas nonwounded cold stored fruit decayed readily. Wounding drastically increased infection by airborne conidia. Immature fruits were less susceptible to wound infection by the airborne conidia than mature fruits. Conidia dispersed freshly were more successful in infecting fresh wounds than conidia that were deposited, or germlings that established, on fruit surfaces 4 days prior to wounding. This decrease in infectivity was especially pronounced on humid and even more on wet incubated fruit. This study clearly showed that in order to reduce. the incidence of brown rot, inoculum levels on fruit approaching maturity should be reduced by sanitation practices and fungicide applications. Furthermore, it is essential to protect fruits, especially. near-mature fruits, from being wounded. / AFRIKAANSE OPSOMMING: EPIDEMIOLOGIE VAN MONILINIA LAXA OP NEKTARIEN EN PRUIM: INFEKSIE VAN VRUGTE DEUR KONIDIA OPSOMMING Naoesverrotting van steenvrugte in die Wes-Kaap provinsie van Suid-Afrika word hoofsaaklik veroorsaak deur Botrytis cinerea (vaalvrot) en Monilinia laxa (bruinvrot). Min is bekend oor die relatiewe belang en seisoenale voorkoms van hierdie patogene in nektarienen pruimboorde, asook oor die infeksieweg, latensie en daaropvolgende siekte-uitdrukking van M laxa. Hierdie aspekte is in dié studie nagevors. \ \ Monsters IS oor 'n 3-jaar periode van die Unifruco Kwaliteitsevalueringskema, en ook van 11 steenvrugboorde verkry. Die voorkoms van vaalvrot en bruinvrot in die hoof steenvrugareas is so bepaal. Botrytis cinerea was die belangrikste patogeen wat betref bloeiselversenging en naoesverrotting. Verder was hierdie patogeen ook meer prominent op die vroeë- en middel-seisoen kultivars. Bruinvrot is uitsluitlik deur M Iaxa veroorsaak en geen aanduiding omtrent die moontlike voorkoms van M fructicola in Suid-Afrika is waargeneem nie. Monilinia laxa was meer prominent op die laat-seisoen kultivars. Botrytis cinerea bloeiselinfeksie het nie direk bygedra tot naoesverrotting nie. Beide oppervlakkige inokulum en latente infeksie het deurgaans, maar wel teen wisselende hoeveelhede, op vrugte in die onderskeie boorde voorgekom. Siekte-uitdrukking op ontwikkelende vrugte is egter nie beinvloed deur die hoeveelheid B. cinerea op die vrug nie, maar eerder deur die vermoë van die vrug om siekte-uitdrukking te onderdruk. Die hoeveelheid B. cinerea op vrugte was verder hoër gedurende lente as gedurende somer. Monilinia laxa het slegs sporadies op die bloeisels van laat-seisoen kultivars voorgekom. Groen vrugte was in die algemeen vry van die patogeen en siekte-uitdrukking het slegs op ryp vrugte plaasgevind. Hierdie bevindinge dui daarop dat M laxa in boorde hoofsaaklik op ryper vrugte geproduseer word. Hierdie swam infekteer ook net ryp vrugte. Die gedrag van luggedraagde M laxa conidia is bestudeer op nektarien- (kultivar Flamekist) en pruimvrugte (kultivar Laetitia). 'n Inokulasie-metode wat natuurlike infeksie deur luggedraagde konidia simuleer, is vir hierdie studies gebruik. Vrugte van die pitverharding-, twee weke voor oes-, oesstadium, asook koud-opgebergde vrugte (nektariene, 4 weke by -o.soe gevolg met 1 week by 23°C en ±56% RH; pruime, 10 dae by -O.5°e, 18 dae by 7.Soe gevolg deur 1 week by 23°C en ±56% RH), is met droë konidia in 'n inokulasietoring geïnokuleer. Die vrugte is vir periodes wat gewissel het van 3 tot 48 h geïnkubeer by hoë relatiewe humiditeit (~93% RH, vogtige vrugte), of dit is bedek met'n film water (nat vrugte). Die gedrag van die enkelspore (konidia) op die vrugoppervlak is met 'n epifluorisensiemikroskoop bestudeer. Skilsegmente is gekleur in 'n kleurstof, bevattende fluorisein diasetaat, analien-blou en blankofor. Die vermoë van die enkelspore om die vrugoppervlak te koloniseer, te penetreer en om siekte-uitdrukking te induseer, is met 'n differensiële stel toetse bepaal. Vir hierdie toetse is die vrugte oppervlakkig gesteriliseer (30 s in 70% etanol), of nie-steriel gelaat. In elke groep is vrugte geneem vir isolasie (skilsegment-to\~ts), of gedoop in "n 3% parakwat-oplossing (parakwat vrugtoets), of\, onbehandeld gelaat (onbehandelde vrugtoets ). Die. bevindinge het op die soortgelyke gedrag van M laxa enkelspore op die verskillende vrugsoorte gedui: appressoria en direkte penetrasie is nie waargeneem nie, en kiembuise het die vrugte hoofsaaklik deur huidmondjies, lentiselle en mikro-krakies .in die vrugskil gepenetreer. Deur luggedraagde spore te bestudeer, is sekere subtiele effekte van die vrug op die gedrag van enkelspore op die vrugoppervlak waargeneem. Op beide vrugtipes is geen nadelige effek op konidiurn- en kiembuisoorlewing opgemerk wanneer die vrugte onder hoë vogtoestande geïnkubeer is. Konidiurn- en kiembuisoorlewing is egter drasties verlaag hoe langer die vrugte onder nat toestande geïnkubeer is. Die bevindinge van die skilsegment-, parakwat en onbehandelde vrugtoetse het duidelik daarop gewys dat die vrugskil van nektarien en pruim nie gepenetreer is tydens die pitverhardingstadium nie, latente infeksies is nie gevorm nie, en die vrugte was bestand teen siekte-uitdrukking. Hierdie fasette op beide vrugtipes is ook nie beinvloed deur inkubasie-natheid nie. Die beskermingskapasiteit van die vrugskil van hierdie steenvrugtipes het egter drasties verskil later in die seisoen. Nektarien-vrugskille is meer geredelik gepenetreer en siekte-uitdrukking het toegeneem met rypwording. Penetrasie en siekteuitdrukking is verder in 'n groot mate deur inkubasie-natheid bevoordeel. Rypwordende pruime het egter nie so In drasties verandering in die beskermingskapasiteit van die vrugskil getoon nie. Die invloed van inkubasie-natheid op infeksie en siekte-uitdrukking was ook minder opsigtelik as op nektarien. Pruimvrugte van die twee weke voor oes-, oesstadium, en , koud-opgebergde pruime, wat onder hoë vog geïnkubeer is, het simptoomloos in die onbehandelde vrugtoets gebly. Vrugte van hierdie stadia het slegs simptome ontwikkel na periodes van langer as 12 h onder nat toestande. Die parakwat-behandelde vrugtoets het egter gewys dat die pruimvrugte meer vatbaar vir latente infeksies raak, maar steeds nie so vatbaar soos die nektarienvrugte nie. Gesamentlik dui hierdie bevindinge daarop <41tM laxa bruinvrot epidemies op pruim en nektarien afhanklik is van inokulumvlakke op rypwordende vrugte, asook die weerstoestande gedurende die vooroes- en oesstadia. Die beskermingskapasiteit van pruim vrugskille was egter aansienlik meer effektief as dié van nektarien vrugte. Wonde op vrugte sal dus 'n groter rol speel in die epidemiologic van M laxa op pruim. Infeksie van vars wonde deur luggedraagde M laxa konidia, en deur konidia en kiembuise wat reeds op die vrugoppervlak gevestig is, is gevolglik bestudeer. Pruimvrugte (kultivar Laetitia) van die pitverharding-, twee weke voor oes-, oesstadium, asook koud- \ \ opgebergde vrugte is in 'n inokulasie-toring geïnokuleer met droë M laxa konidia. .Infeksie , , van nie-gewonde vrugte en van vars wonde deur luggedraagde konidia op droë, vogtige en nat pruim vrugoppervlaktes, asook deur konidia en kiembuise wat reeds op die vrugoppervlak onder hierdie toestande gevestig is, is bepaal. Nie-gewonde groen tot ryp vrugte het meestal simptoomloos gebly, terwyl koud-opgebergde ryp vrugte wel verrot het. Wonde .het die hoeveelheid infeksie deur luggedraagde spore drasties vermeerder. Konidia wat geïnokuleer is op vrugte met vars wonde, was meer in staat om hierdie wonde te infekteer as konidia en kiembuise wat 4 dae voor wonding gevestig is. Hierdie afname in infektiwiteit was meer sigbaar op die vogtige, maar veral die nat vrugte. Hierdie studie het duidelik gewys dat inokulumvlakke op rypwordende vrugte verlaag moet word deur sanitasie-praktyke en fungisiedtoedienings. Dit is verder belangrik om vrugte, veral rypwordende vrugte, teen wonding te beskerm.

In November of 1984 and 1985, Euphorbia lathyris was planted into a field naturally infested with Macrophomina phaseolina located at the Campbell Avenue Farm in Tucson, Arizona. Plants without foliar symptoms and rhizosphere soil were sampled regularly from emergence until the following May or June. Soil rhizosphere populations ranged from 0.7-3.0 cfu/g soil in 1985 to 8.0-24.1 cfu/g soil in 1986, and did not change significantly over either growing season (P > 0.05). Both the incidence of disease and the number of infection sites per cm of root increased significantly (P < 0.05) over each growing season and were not related to rhizosphere soil populations of M. phaseolina (P > 0.05). The distribution of infection sites along the tap root over both growing seasons remained the same in that most were located in the top 0-7 cm of tap root. Infected E. lathyris without apparent symptoms were subjected to low-water and high-temperature stress treatments in growth chambers. Root infection was not found to be dependent upon any stress. Lesion development was significantly dependent upon the imposition of any stress treatment, and further root colonization was significantly dependent upon low-water stress (P < 0.05). M. phaseolina was consistently recovered from asymptomatic roots. A consistently lower leaf water potential was measured on infected E. lathyris than from non-infected controls when no stress treatment was applied. Polyclonal antisera made against hyphae and microsclerotia of M. phaseolina was not successful in detecting this pathogen in E. lathyris by I-ELISA. Antisera applied to fresh thin sections of infected plant tissue was effective in staining hyphae of M. phaseolina when used with a second antibody conjugated to fluorescence isothiocyanate or to an enzyme (to which a substrate was added to "stain" hyphae).

Phymatotrichum root rot is a fungal disease with a host range that includes many economically important crops in the southwestern United States and Mexico. While it has been studied since the late nineteenth century, ecological relationships of the disease, particularly those related to its distribution and dispersal, are not understood. Combined ground radiance sampling and aerial photographic interpretation was employed to study the distribution of Phymatotrichum root rot in cotton. Radiometric ground sampling showed that diseased cotton has a characteristic spectral signature that is significantly different from healthy cotton at visible wavelengths. Micro-scale examination of distribution within fields utilized multitemporal photography, both within season (1983) and for four seasons (1979-1982), revealed that the disease spreads during a season, but is not recurrent in many cases between years. Meso-scale mapping employed multitemporal photography to map distribution during a four-year period. When compared to mapped soil units, these data revealed a significantly non-random relationship between the diseased areas of fields and fine-textured soil units that may be based on moisture-holding potential. A yield analysis was also preformed using Thematic Mapper Simulator data and computer analysis.

No description available.

Canola/rapeseed (Brassica napus L.) is a major oilseed crop worldwide. However, its production is largely affected by the fungal disease Sclerotinia stem rot as well as seed glucosinolates. So far the genetic mechanisms controlling these two traits have been poorly understood. In the present study, three bi-parental doubled haploid B. napus populations M730, M692 and ZT were grown in either natural or artificial environments and genotyped using the Brassica 60K Infinium® SNPs and/or sequence related amplified polymorphisms. Three genetic linkage maps covered 2,597.7 cM, 2,474.1 cM and 1,731.6 cM in 19 chromosomes for M730, M692 and ZT, respectively. Plants were inoculated with Sclerotinia sclerotiorum mycelia on stems at the reproductive stage to evaluate their resistivity. Four aliphatic glucosinolates and one indolic glucosinolate were detected in the seeds using high-performance liquid chromatography. 4-hydroxy-3-indolylmethyl predominated over aliphatic glucosinolates in canola, but inversely constituted a small portion of total glucosinolate content in semi-winter rapeseed. In rapeseed, 2-hydroxy-3-butenyl predominated in 4C aliphatic glucosinolates, which in turn predominated in total aliphatic glucosinolates, which likewise predominated in total glucosinolate content. QTLs regulating major glucosinolates were located on chromosome A9 for high glucosinolate content populations M730 and ZT, and on chromosome C7 for low glucosinolate content population M692. Major QTLs for Sclerotinia stem rot resistance were located on chromosomes A7 and C6 in M730, on chromosomes A3 and A7 in ZT, while no major QTLs were found in M692. Additive genetic effect was the major factor explaining phenotypic variations of the two traits. No direct genetic relationship was observed between Sclerotinia stem rot resistance in adult plants and seed glucosinolates in B. napus. The findings in the studies could be used to formulate breeding and research strategies in B. napus and the major QTLs controlling the two traits and their closely linked SNP markers could be validated over wide germplasm and used in marker assisted selection. / October 2016

No description available.

Receptor-like cytoplasmic kinases (RLCKs) form a large family of proteins in plants. RLCKs have been found in different plant species, regulating plant immunity to different bacterial and fungal pathogens. Previous studies implicated <i>Arabidopsis</i> <i>botrytis induced kinase1 (BIK1)</i> and <i>tomato protein kinase 1b (TPK1b)</i> in plant resistance to <i>Pseudomonas syringae</i> and <i>Botrytis cinerea</i>. In this study, we classified 195 putative maize RLCKs into ten subfamilies. Based on the amino acid sequence similarity to BIK1 and TPK1b, a novel maize RLCK,<i> zea mays bik1-like kinase 1 (ZmBLK1)</i> was identified. Enzyme assays with cloned <i>ZmBLK1</i> revealed a functional kinase when expressed in planta. The recombinant protein located to the plasma membrane. Expression of <i>ZmBLK1</i> is highest in maize leaves compared to other structures at silking stage. Expression of the recombinant <i>ZmBLK1</i> significantly reduced the rate of lesion spread in maize leaves inoculated with the Goss’s wilt pathogen. In maize kernels, expression of <i>ZmBLK1</i> increases during kernel maturation. Kernels from transgenic maize overexpressing <i>ZmBLK1</i> were not resistant to <i>Aspergillus flavus</i> or to aflatoxin contamination. In addition, mutations were made in <i>ZmBLK1</i> that were hypothesized to create a constitutively active kinase. However, resulting proteins had similar activity to the wild-type ZmBLK1 and transgenic plants showed similar responses to the Goss’s wilt and Aspergillus ear rot pathogens. Overall, this research established the first characterization of RLCKs in maize and described a potential contribution of ZmBLK1 to maize immune responses.

Os polímeros sintéticos compõem cerca de 20% do lixo urbano no Brasil. Além da não biodegradabilidade, formam uma camada impermeabilizante que impede a passagem de líquidos e gases originados no apodrecimento dos detritos, retardando a estabilização da matéria orgânica. A exploração da madeira produz uma grande quantidade de rejeitos que não são inteiramente aproveitados para gerar energia ou outros produtos, e acarreta sérios problemas ambientais. A finalidade deste trabalho é a produção de compósitos biodegradáveis reunindo rejeitos de madeira e um polímero biodegradável - o poli (álcool vinílico) [PVA]. Para facilitar a degradação do PVA, este foi modificado por anidrido ftálico (AF). A modificação foi estudada através de tempo de gelatinização, calorimetria exploratória diferencial (DSC) e análise termomecânica dinãmica (DMTA). Resíduos da madeira Sugi (Criptomeria japonica) foram refinados até a obtenção de uma farinha com partículas menores que 63 &#181; m. Esta farinha foi adicionada ao meio de reação AF/PVA. As proporções de AF e PVA, bem como os parâmetros utilizados na prensagem foram determinados segundo um planejamento estatístico fatorial. Os compósitos foram moldados a quente (180 C e 50 MPa). Variando-se a proporção AF/PVA, compósitos com valores de módulo de elasticidade (MOE) de &#732;10 GPa e módulo de ruptura (MOR) de &#732;90 MPa na flexão foram obtidos. Os valores são inferiores aos apresentados pelo polímero puro, seco e sem plasticizante (acima de 152 MPa), porém superiores às placas de madeira reconstituída de MDF e OSB, disponíveis comercialmente, que apresentam valores de MOR em torno de 49 MPa. A degradação por microorganismos foi avaliada pelo ensaio de soterramento utilizando uma adaptação do método para avaliar a resistência de materiais lignocelulósicos aos fungos da podridão mole (Publicação IPT No. 1157 D5). Os fungos da podridão mole que ocorreram naturalmente nos compósitos foram isolados e identificados segundo a técnica do microcultivo. O gênero mais frequente foi o celulolítico Trichoderma spp. e o mais degradador foi o Chaetomium spp. As mudanças na estrutura e na morfologia dos compósitos foram estudadas através de espectroscopia de infravermelho (IR) e microscopia eletrônica de varredura (MEV). As perdas de massa e das propriedades mecânicas foram monitoradas em intervalos pré-estabelecidos. Compósitos com altas concentrações de AF apresentaram biodegradabilidades superiores à da madeira maciça de Pinus sp. e levemente inferiores à da madeira de Eucalyptus grandis, utilizadas como referências. A biodegradabilidade se deve à facilidade dos grupos ésteres, dos ftalatos de PVA e de celulose, de serem hidrolizados e de regenerar o ácido ftálico. Mesmo sob hidrólise enzimática, a estrutura PVA-AF-celulose, que forma ligações cruzadas entre microfibrilas de celulose, não se desintegra, conservando as propriedades mecânicas por muito tempo. Estas se mantêm superiores, ao término de 180 dias de exposição, às da madeira maciça de Sugi antes de entrar no processo de biodegradação. Uma correlação entre a estrutura, as propriedades físicas e mecânicas, e a taxa de biodegradação dos compósitos de rejeitos de madeira-PVA foi estabelecida. / Synthetic polymers constitute around 20% of urban solid waste in Brazil. Besides being non-biodegradable, they form an impermeable barrier that prevents the liberation of liquids and gas originated in the waste deterioration, retarding organic matter stabilization. The wood industry produces large amounts of waste wood which is not entirely utilized to produce energy or other products, and it brings several environmental problems. The aim of this study is the production of an environmentally friendly wood-based product composed of waste wood and poly (vinyl alcohol) [PVA]. PVA is the most widely produced water soluble and biodegradable synthetic polymer worldwide. However, PVA degradation in aqueous and soil environments has proved to be quite slow under unadapted conditions. To accelerate its degradation, the PVA has been modified by phthalic anhydride (AF). These modifications have been studied by means of gelation time, Differential Scanning Calorimetry (DSC) and (Dynamic Mechanical Thermal Analysis (DMTA). Sugi (Criptomeria japonica) flour (particles size &#8249; 63 &#181;m) has been obtained by milling waste samples. Wood flour has been added into AF/PVA reaction medium. AF and PVA ratios and pressing conditions have been set by factorial design. The final pressing temperature and pressure have been set as 180 &#176;C and 50MPa respectively. Varying AF/PVA ratios, composites presenting modulus of elasticity (MOE) values of &#732;10GPa and modulus of rupture (MOR) of &#732;90 MPa have been obtained. The MOR values are lower than that presented by the pure, dry, no plasticized PVA (higher than 152 MPa), but they are higher than commercial MDF (medium density fiberboard) and OSB (oriented strand board) of around 49 MPa. Degradation by microorganisms has been performed by soil burial test (method adapted from IPT Edition No. 1157 D5). Naturally occurring soft rot fungi have been isolated and identified according to micro cultivation techniques. Most frequent genus has been the cellulolytic Trichoderma spp. and most degrader has been Chaetomium spp. Changes in the composites microstructures and morphology throughout the biodegradation process have been studied by IR and SEM and decreasing in the mechanical properties monitored. The weight Ioss shown by composites with high AF concentration has been higher than the softwood Pinus sp. and comparable to the hardwood Eucalyptus grandis, utilized as witnesses. Even under enzymatic hydrolysis, the PVA-AF-cellulose structure has been only slightly broken, preserving considerable mechanical properties that remain superior to solid Sugi before entering any biodegradation process, even after 180 days of exposure. A correlation has been established between the structure, physical and mechanical properties and biodegradation rate of waste wood-PVA composites

O objetivo deste trabalho foi avaliar se as enzimas secretadas pelo fungo de decomposição parda Gloeophyllum trabeum podem atuar como auxiliares às celulases comerciais na hidrólise de substratos celulósicos oxidados. Para isto foi realizada a oxidação de celulose microcristalina Avicel mediante reação de Fenton (Fe+2, H2O2) e tratamento com CuSO4. O substrato oxidado foi caracterizado e posteriormente foi determinado o efeito desta modificação sobre a eficiência hidrolítica de enzimas comerciais (Celluclast/Novozym) e sobre misturas destas enzimas comerciais suplementadas com extrato enzimático obtido de G. trabeum. As reações realizadas com Avicel indicaram que ambos tratamentos químicos foram efetivos para modificar grupos funcionais presentes na celulose. A caracterização fisicoquímica dos substratos oxidados permitiu concluir que o tratamento Fenton gerou transformações mais intensas na celulose do que o tratamento com CuSO4. O análise dos hidrolisados ácidos das amostras oxidadas por Fenton mediante espectrometria de massas (GC-MS) confirmou que a oxidação ocorre principalmente no C1. As hidrólises enzimáticas utilizando preparações com diferentes cargas das enzimas comerciais apresentaram uma diminuição significativa da conversão de celulose quando as amostras são tratadas com o reagente Fenton. Por outro lado, a celulose oxidada gerada pelo tratamento com CuSO4 ainda é eficientemente hidrolisada pelas enzimas. As duas amostras de celulose Avicel oxidadas também foi hidrolisada utilizando misturas de enzimas comerciais e extratos enzimáticos de G. trabeum. Em todas as condições ensaiadas a suplementação da mistura reacional com extratos enzimáticos do fungo de decomposição parda G. trabeum diminuíram a eficiência de hidrólise enzimática da celulose oxidada. O emprego do dobro de carga enzimática de enzimas comerciais permitiu recuperar os níveis de hidrólise observados na celulose in natura. / The goal of this work was to evaluate whether enzymes produced by the brown-rot fungus Gleophyllum trabeum would be able to act as auxiliary enzymes of commercial cellulases during the enzymatic hydrolysis of oxidized cellulose. To reach this goal an oxidation of microcrystalline-cellulose (Avicel) was performed by two differente reaction systems: Fenton reaction (Fe2+, H2O2) or CuSO4 treatments. The oxidized subtracts were characterized and the effect of cellulose modification on the hydrolytic efficiency of commercial enzymes, supplemented or not with the enzymes recovered from a G. trabeum culture, was determined. The Avicel treatment by the oxidative systems resulted in cellulose structural changes as evidenced by infrared spectroscopy, viscosity measurements and chemical characterization by HPLC and GC/MS chromatography of the monomers recovered after acid hydrolysis. The physicochemical characterization of the oxidized celluloses allowed to conclude that the transformation of the cellulose was more intense when Fenton treatment was used as compared with the CuSO4 treatment. The analysis of the acid hydrolysates by GC-MS indicated that the oxidation by Fenton reaction occurred mainly at the C1 of the cellulose chain, resulting in gluconic acid in the acid hydrolysates. A meaningful decrease of the cellulose conversion was observed when Fetnon-treated samples were submitted to enzymatic hydrolysis using different amounts of commercial enzymes. On the other hand, the CuSO4-oxidized cellulose was still efficiently hydrolyzed by high enzyme dosages. The oxidized Avicel substrates were also hydrolyzed using a mixture of the commercial enzymes with enzymes recovered from G. trabeum cultures. At all tested conditions, the supplementation of the reaction mixture with enzymatic extracts from G. trabeum decreased the enzymatic hydrolysis efficiency. Nevertheless, the normal levels of enzymatic hydrolysis were recovered when the commercial enzymes dosage two-fold increased.

Fungos de podridão branca e fungos de podridrão parda são capazes de degradar a biomassa lignocelulósica utilizando diferentes mecanismos. Os fungos de podridão parda degradam a celulose e a hemicelulose enquanto modificam a lignina, sendo, portanto, as enzimas hidrolíticas ativas em substrato rico em lignina. O arsenal enzimático diferenciado desses fungos torna-os alvos para prospecção de enzimas que podem ser aplicadas em diversos processos biotecnológicos. O fungo Gloeophyllum trabeum é uma das espécies mais compreendidas deste grupo, e sabe-se que é capaz de degradar a celulose sem produzir CBHs. Além disso já foi reportado uma GH5 proveniente desse organismo com caraterísticas processivas. Nossa tentativa de clonagem dessa enzima (GtGH5) em A. nidulans A773 não foi bem sucedida, todavia nosso grupo esta repetindo os passos de clonagem e expressão. No genoma desse organismo esta presente um gene que codifica uma GH45. A GtGH45 foi a clonada e expressa com alto rendimento em A. nidulans cepa A773. A expressão, secreção e acumulaçao da GtGH45 foi positiva após 72 h de incubação em meio líquido (maltose como indutor), confirma por eletroforese SDS-PAGE do extrato enzimático concentrado e dialisado. A massa molar de 18,4 kDa foi consistente com a predição da sequência de 204 amino-ácidos obtída apartir da sequência gênica e corrobora com a caracterização por espectometria de massas (18,9 kDa). A análise filogenética é consistente com estudos anteriores, agrupando a proteína alvo com outras GH45s de basidiomicetos na subfamília C. A enzima purificada foi testada para determinação da especificidade pelo substrato apresentando maior atividade em arabinoxilana, xilana, arabinoxilana e CMC e foi capaz de gero celo-oligômeros a partir da hidrólise de PASC. O pH ótimo em CMC foi 2,5, além se demonstrar ser temoestável em ensaio de Thermoflour. A hidrólise enzimática de substratos lignocelulósicos derivados de cana-de-açúcar mostrou que a GtGH45 foi eficiente para suplementar coqueteis enzimáticos comerciais, principalmente na conversão de xilana. / White-rot and brown-rot basidiomycetes are able to transform lignocellulosic materials through different mechanisms. The brown-rot fungi are able to degrade cellulose and hemicellulose meanwhile modifies lignin. The hydrolytic enzymes of these fungi act on a lignin-enriched substrate, which makes them targets to prospect new enzymes for polysaccharides hydrolysis. Gloeophyllum trabeum is one of the best understood fungal species in this group. An interesting processive GH5-endoglucanase (EG) has been described in this species suggesting an unusual pathway for lignocellulosic degradation without cellobiohydrolases (CBH). Our first attempt to clone this GtGH5 was default but our grup is trying a new attempt of heterologous expression of this protein. Furthermore, G. trabeum genome points out for a low molar mass GH45-EG. Here we report on a recombinant high-yield G. trabeum ATCC 11539 GtGH45 production system. Expression and secretion of endoglucanase GtGH45 was positive after 72 h incubation of the transformed A. nidulans stain A773 in stationary liquid cultures (maltose as inductor). The SDS-PAGE electrophoresis of ultra-filtrated extract showed a single-band GtGH45 over expressed band. The molar mass of 18,4 KDa was consistent with the predicted 204 amino acids sequence derived from gene sequence analyses and corroborates with mass spectometry characterization (18,9 KDa). Even more, the phylogenetic analyze is consistent to previews studies, clustering the target protein with others GH45 from basidiomycetes at subfamily C. The purified protein was assayed for substrate specificity showing activity against xylan, arabinoxylan and PASC. GtGH45 was able to produce cello-oligomers from PASC. The optimum pH was 2.5 with CMC as the substrate. Xylan conversion was enhanced when GtGH45 was mixed with commercial enzymes in enzymatic hydrolysis of alkaline-sulfite pretreated sugar cane bagasse.