Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:39:32Z No. of bitstreams: 2 Dissertação - André Luís Elias Moreira - 2014.pdf: 2397469 bytes, checksum: 4a631b1ca63986a489d7e4bec3dfe90c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:41:32Z (GMT) No. of bitstreams: 2 Dissertação - André Luís Elias Moreira - 2014.pdf: 2397469 bytes, checksum: 4a631b1ca63986a489d7e4bec3dfe90c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T13:41:32Z (GMT). No. of bitstreams: 2 Dissertação - André Luís Elias Moreira - 2014.pdf: 2397469 bytes, checksum: 4a631b1ca63986a489d7e4bec3dfe90c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The paracoccidioidomycosis (PCM) is a systemic disease caused by the thermo dimorphic fungus Paracoccidioides spp.. The route of infection of the PCM occurs by the inhalation of conidia or mycelia fragments. Until now, no proteomic studies were performed with conidia of Paracoccidioides sp. In this sense, characterize the proteome of the conidia, may contribute to the detailed knowledge of the proteins expressed during the propagation phase and their potential roles in virulence and pathogenicity, providing possible targets for antifungal strategies. For the conidia production, the mycelia of isolate Pb01 (ATCC MYA-826) were cultured in potato agar medium during 90 days at 18 ºC. After production, the conidias were collected and purified. The proteins were extracted and subjected to tryptic digestion with subsequent identification by NanoUPLC-MSE. We identified a total of 242 proteins of conidia of Paracoccidioides. The in silico analysis were used to characterize the proteins present in Paracoccidioides conidia. Proteins as GAPDH, enolase, triosephosphate isomerase, and some glycoproteins like ECM33 and β-1,3-glucanosyltransferase gel2 were identified during analysis. Some of these have been described as adhesins in Paracoccidioides and in other pathogenic fungi. Were also identified proteins related to signal transduction pathways, such as: Ras GTPase, RhoA GTPase and calmodulin, described in other fungi involved in morphologic changes. Proteins related to evasion, defense and virulence of the fungus, such as HSP90, catalase, mitochondrial peroxiredoxin Prx1, have been described with functions related to temperature shifts or oxidative stress provided by the host environment, were also identified. These results highlight that Paracoccidioides conidia contain proteins that can contribute to its maintenance in the environment and have molecules related to important processes necessary for the initial steps of infection in the host. / A paracoccidioidomicose (PCM) é uma micose sistêmica causada pelo fungo termodimórfico Paracoccidioides spp.. A principal rota de infecção da PCM ocorre por inalação de conídios ou fragmentos de micélio. Até o momento, estudos proteômicos não foram realizados com conídios de Paracoccidoides sp. Neste sentido, caracterizar o proteoma do conídio poderá contribuir para o conhecimento detalhado das proteínas expressas durante a fase de propagação e seus potenciais papéis na virulência e patogenicidade, fornecendo prováveis alvos para estratégias antifúngicas. Para a produção de conídios, o micélio do isolado Pb01 (ATCC MYA-826) foi cultivado em meio ágar batata durante 90 dias a 18 ºC. Após a produção, os conídios foram coletados e purificados. As proteínas foram extraídas e submetidas a digestão tríptica com subsequente identificação por NanoUPLC-MSE. Foram identificados um total de 242 proteínas de conídios de Paracoccidioides. As análises in silico foram utilizadas para caracterizar as proteínas presentes em conídios de Paracoccidioides. Proteínas como a GAPDH, enolase, triosefosfato isomerase e algumas glicoproteínas como ECM33 e β-1,3-glicanosiltransferase Gel2 foram identificadas durante as análises. Algumas destas já foram descritas como adesinas em Paracoccidioides e em outros fungos patogênicos. Também foram identificadas proteínas relacionadas as vias de transdução de sinais, tais como: Ras GTPase, RhoA GTPase e calmodulina, descritas em outros fungos envolvidas em alterações morfológicas. Proteínas relacionadas à evasão, defesa e virulência do fungo, tais como HSP90, catalase B, peroxiredoxina mitocondrial Prx1, foram descritos com funções relacionadas a mudanças de temperatura ou estresse oxidativo proporcionado pelo ambiente hospedeiro, também foram identificados. Esses resultados demonstram que o conídio de Paracoccidioides contem proteínas que podem contribuir para sua manutenção no meio ambiente e possuem moléculas relacionadas a processos importantes necessários para os passos iniciais da infecção no hospedeiro.

The recently identified causative agent of White-Nose Syndrome (WNS), Geomyces destructans, has been responsible for the mortality of an estimated 5.7 million North American bats since its emergence in 2006. A primary focus of the National Response Plan, established by US Fish and Wildlife in 2011, was the identification of biological and chemical control options. In an effort to identify potential biological and chemical control options for WNS, six previously described bacterially produced volatile organic compounds (VOCs) and multiply induced Rhodococcus rhodochrous DAP96253 were screened for anti-Geomyces destructans activity. Geomyces destructans conidia and mycelial plugs were exposed to the VOCs and induced Rhodococcus in a closed air space at 15°C and 4°C and evaluated for inhibition of conidia germination and mycelial extension. Additionally, in situ application methods for induced Rhodococcus, such as fixed cell catalyst and fermentation cell paste in non-growth conditions, were screened with positive results. Rhodococcus was assayed for ex vivo activity via exposure to bat tissue ex-plants inoculated with G. destructans conidia. All VOCs inhibited radial growth of mycelial plugs and growth from conidia at both temperatures, with the greatest effect at low temperature (4°C). Induced Rhodococcus completely inhibited growth from conidia at 15°C and had a strong fungistatic effect at 4°C. Induced Rhodococcus inhibited Geomyces destructans growth from conidia when cultured in a shared air space with bat tissue explants inoculated with Geomyces destructans conidia. During the evaluation diffusible brown pigment was observed in G. destructans cultures exposed to induced Rhodococcus or select VOCs. The pigment was induced by light and oxidative challenge and hypothesized to be melanin. Traditional microbiological methods, as well as copper sulfide-silver staining and ultraviolet-visible spectroscopy, were utilized to confirm this hypothesis. This was a noteworthy result as melanin is a known virulence factor in other pathogenic fungi and may play a significant role in WNS. The identification of bacterially produced VOCs and inducible biological agents with anti-Geomyces destructans activity expands the pool of potential biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

Aflatoxin contamination accounts for millions of dollars worth of losses for corn and cotton in Texas. Two atoxigenic strains of Aspergillus flavus, AF36 and Afla-Guard, are labeled for its management. The purpose of this study was to measure differences in the ability of these strains to sporulate and to track movement of their conidia in corn and cotton fields. Sporulation was evaluated by incubating the two strains on their commercial formulations (inoculated on cereal grains) at six constant humidity levels ranging from 0-100%, using closed chambers with saturated salt solutions. Conidial production by Afla-Guard was 3-fold greater than that of AF36 at 100% humidity. Sporulation of the two strains was also evaluated on one substrate by inoculating their conidia on sterile, hulled barley. After 3 days, there was a 234-fold increase in conidia recovered from the barley inoculated with Afla-Guard, compared with a 21-fold increase in conidia recovered from the AF36-inoculated barley. These data suggest that the Afla-Guard strain sporulates better than the AF36 strain, which may be a factor in effectiveness for biological control. An in vitro de-Wit competition experiment showed that sporulation by the Afla-Guard strain was not affected by co-inoculation with either AF36 or the wildtype NRRL3357 toxin producing strain. To measure conidial movement, an Afla-Guard nitrate non-utilizing mutant colonizing autoclaved corn seed, was placed at one point in a field of cotton and corn. For detection, aliquots washed from leaf samples were plated onto a medium containing potassium chlorate. The mutant was recovered at a maximum distance of 6.4 m in corn fields along the same row and as far as 10.2 m across rows from the point source. In cotton fields, the mutant was recovered at 9.1 meters along the same row and 6.1 m across rows from the point source. There was no recovery at 24.3 m from the point source - the maximum distance evaluated. The experiment was repeated in a second year with similar results. These data suggest that plots in field trials may not need wide separation in order to avoid cross contamination. To assess the viability of a toxigenic and atoxigenic strain of A. flavus over time, polycarbonate packets containing conidia and sclerotia of both strains were buried in Ships Clay soil with the matric potential held constant at -24 kPa or -154 kPa. After 10 months, viable conidia were recovered in all treatments. After 14 months, viability of the atoxigenic strain incubated at -154 kPa ψm was lost, while other treatments remained viable. Ears of corn were inoculated via silk channel at different stages of silk senescence. Sclerotia were enumerated from the same plants following harvest of the crop. Sclerotial production by A. flavus was greatest from ears with silks inoculated at senescence, compared with inoculation when silks were green. The isolation frequency of Penicillium sp. from surface-sterilized kernels at harvest was the highest from ears that were inoculated with A. flavus when silks were fresh, as compared with A. flavus inoculation of ears with senescent silks. A Fusarium and Penicillium species was isolated from harvested kernels, and their sterile Czapek-Dox broth culture filtrates were tested for their effect on development of three strains of A. flavus on agar. The Penicillium broth filtrate greatly reduced sclerotial numbers relative to the control and the Fusarium filtrate (P<0.05). When A. flavus was grown in the presence of autoclaved Penicillium culture filtrate, there was no effect on sclerotial production. The Penicillium filtrate increased the rate of radial hyphal growth of the A. flavus isolates on agar compared to the control and the Fusarium culture filtrate.

Alternaria solani, cause of early blight, threatens potato yields. Fungicide resistance has made control of early blight difficult and there are concerns that in-season fungicide use results in resistance to boscalid, fluopyram, and chlorothalonil. Concern of high levels of resistance to boscalid a group 7 fungicide may confer cross-resistance to fungicides of the same group such as fluopyram. From 2014 to 2015, A. solani isolates were collected from field plots treated with boscalid, fluopyram, and chlorothalonil to test resistance levels. Isolates were determined resistant if EC50 values were higher than 5 µg ml-1. Boscalid and chlorothalonil mean EC50 values decreased two fold from 2014 to 2015, while fluopyram values increased two fold. A negative correlation between fluopyram and boscalid indicate no cross-resistance. Higher resistance levels to fluopyram (17.1 µg ml-1) were observed in the treatment C-14 where only fluopyram was applied in 2014. Treatments D-14 and D-15, only treated with chlorothalonil, had the highest mean EC50 values to chlorothalonil (2.3 and 1.1 µg ml-1, respectively). Field trials show fluopyram+chlorothalonil had lowest disease severity of 6.6 to 6.8%. Leaf residues of boscalid fluopyram, and chlorothalonil measured an average of 10.2, 4.9, and 55.0 ppm on leaves throughout the canopy. After 14 days average residues diminished to 0.74, 0.39, and 16.9 ppm for boscalid, fluopyram and chlorothalonil, respectively. Boscalid is not effective for early blight control because of high resistance; fluopyram resistance is increasing as treatments of fluopyram are applied; and chlorothalonil does not seem to be affected by continued fungicide application.

Development of early leafspot, caused by Cercospora arachidicola Hori, was monitored on' Florigiant' peanut (Arachis hypogaea L.) at two field sites in Suffolk, Virginia. In one study, plants in 27-cm-diameter plots were inoculated with 20,000 conidia and inoculation dates were replicated in five randomized complete blocks. At location one in 1985 and 1986, lesions/leaf at two weeks after inoculation correlated significantly (P ≤ 0.05) with infection indices (IND) developed by the Virginia leafs pot advisory and hours of relative humidity (RH) ≥ 95%. At location two, correlations between lesions/leaf and IND as well as hours of RH ≥ 95% were significant in 1986, but not in 1985. Certain site specific factors were believed to have altered plant susceptibility to leafspot at this site in 1985. In another study, pots with greenhouse-grown peanut were placed between unsprayed rows of field plants, heavily colonized by C. arachidicola. Plants were removed after 3, 5, and 7 days of field exposure for six consecutive weeks in 1986 and returned to the greenhouse. Lesions/leaf at two weeks after initial exposure were correlated with IND values computed by five versions of the leaf spot advisory. Significant correlations were found between lesions/leaf on plants with field exposures of 5 and 7 days and cumulative IND values and hours of RH ≥ 90% and 95%. The low incidence of lesions resulting with field exposures of only 3 days coupled with a lack of significant correlations between disease and cumulative IND values for 3 days after inoculation in both studies suggests that infection processes require several days, and that fungicides may be applied to achieve disease control during this time. / M.S.

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.

Metarhizium rileyi é um importante fungo patogênico a várias espécies de pragas desfolhadoras da família Noctuidae. Existe uma grande demanda para a produção em escala deste fungo, mas ainda não há produtos comerciais no mercado brasileiro. Os objetivos do presente estudo foram avaliar a produção de conídios de M. rileyi por fermentação sólida usando diferentes grãos e cereais e a produção de blastosporos por fermentação líquida em meios com diferentes fontes de carbono e nitrogênio. Na produção de conídios em substrato sólido, foi empregado o isolado ESALQ 1305 em oito tratamentos com grãos/cereais incubados por 15 dias. Para os experimentos de produção de blastosporos em substrato líquido, foram conduzidos quatro experimentos sequenciais onde foram testados diferentes meios e concentrações de células oriundas do pré-cultivo e diferentes fontes e concentrações de carbono, nitrogênio, relação C:N e tempo de cultivo. Os dados foram submetidos a análise de variância e as médias comparadas pelo teste de Tukey a 5%. Os substratos sólidos que resultaram em maior produção de conídios foram o feijão fradinho moído sem peneirar (7,4 ± 2,8 x 108 conídios.g-1) e trigo moído peneirado (3,3 ± 1,2 x 108 conídios.g-1). Nos experimentos de fermentação líquida a 300 R.P.M e 25 °C observou-se que os meios desenvolvidos por Jaronski e Jackson (2012) com relação C:N de 50:1, proporcionam as maiores concentrações de blastosporos de M. rileyi. Os melhores resultados foram obtidos no último experimento com o isolado ESALQ 1305 quando o pré-cultivo foi realizado com meio contendo 36 g.L-1 de concentração de carbono e relação C:N de 50:1, posteriormente inoculado na razão de 105 blastosporos.mL-1 nos meios de cultivo contendo 40 g.L-1 e 80 g.L-1 de carbono, tendo extrato de levedura como fonte proteica, obtendo-se concentrações de 3,6 ± 1,6 x 109 blastosporos.mL-1 e 4,0 ± 0,6 x 109 blastosporos.mL-1, respectivamente, após quatro dias. Os resultados demonstram a viabilidade da produção de blastosporos de M. rileyi, por fermentação líquida. / Metarhizium rileyi is an important fungus pathogenic to various species of defoliating pests of the family Noctuidae. There is a great demand for mass production of this fungus, but there is still no commercial products in the Brazilian market. The objectives of this study were to evaluate the conidial production of M. rileyi by solid fermentation using different grains and cereals and the production of blastospores by liquid fermentation in media with different sources of carbon and nitrogen. In the production of conidia in solid substrate, the isolated ESALQ 1305 was used in eight treatments with grains/cereals incubated for 15 days. For the production of blastospores in liquid substrate, four sequential experiments were conducted using different media, concentrations of pre-cultivation, sources of carbon, nitrogen, C:N ratio and cultivation time where tested. Data were analyzed by the use of ANOVA and post hoc comparisons of means using the Tukey\'s test to 5%. The solid substrates resulting in higher production of conidia were black-eyed beans milled without sieving (7.4 ± 2.8 x 108 conidia.g-1) and wheat milled and sieved (3.3 ± 1.2 x 108 conidia.g-1). In the liquid fermentation experiments to 300 R.P.M. and 25 °C it was observed that the media developed by Jaronski and Jackson (2012) with C:N ratio of 50:1, provides the greatest concentration of blastospores of M. rileyi. The best results were obtained in the last experiment with the isolate ESALQ 1305, when the pre-cultivation was conducted in media containing concentration of carbon of 36 g.L-1 and C:N ratio of 50:1, inoculated in the ratio of 105 blastosporos.mL-1 in culture media containing 40 g.L-1 and 80 g.L-1 of carbon, using yeast extract as protein source, obtaining the concentrations of 3.6 ± 1.6 x 109 blastospores.mL-1 and 4.0 ± 0.6 x 109 blastospores.mL-1, respectively, after four days. The results demonstrate the viability of high-yield production of blastospores of M. rileyi by liquid fermentation.

O Brasil é o único produtor, em escala comercial, de guaraná do mundo e o Estado do Amazonas destaca-se como o segundo maior produtor do Brasil, sendo superado apenas pelo Estado da Bahia. O principal fator limitante à expansão da cultura do guaranazeiro na região amazônica é a antracnose, causada por Colletotrichum guaranicola Alburq. Este trabalho teve como objetivo caracterizar isolados do fungo visando sua correta identificação. Foram realizadas as caracterizações cultural, morfológica, fisiológica, enzimática, patogênica e molecular (filogenia multilocus). Na caracterização morfo-cultural houve variação no tamanho de conídios e apressórios entre os isolados, porém todos ficaram dentro da faixa de amplitude descrita para a espécie C. guaranicola e para duas outras espéciess (C. gloeosporioides e C. boninense). Predominaram conídios de formato cilindro a oblongo e apressórios arredondados. Com exceção de um grupo de isolados de Maués (série C), todos os demais exibiram hilo na base do conídio. Comprimento e largura de conídios e apressórios não foram características importantes na diferenciação dos isolados de C. guaranicola devido à sobreposição no tamanho. A taxa de crescimento micelial foi determinada em meio BDA e permitiu diferenciar dois grupos de isolados: com taxa de crescimento acima de 10 mm/dia e abaixo dessa taxa. Com exceção de C-12 e C-14, todos os isolados da série C cresceram acima dos demais, comparando-se aos isolados padrões das espécies C. gloeosporioides, C. boninense e C. acutatum. Todos os demais cresceram abaixo desse valor. O maior crescimento foi registrado para C-09 (12,25 mm/dia) e o menor para 2601 (2,71 mm/dia). Os demais ficaram na faixa intermediária. Diferença na coloração da colônia também permitiu separar os isolados em duas categorias: colônia branca a creme amarelada e colônias cinza a cinza escuro, semelhante aos padrões de C. boninense e C. gloesoporioides, respectivamente. Todos os isolados cresceram melhor na faixa de 25 ºC, porém os isolados C-18 e C-19 foram capazes de crescer em temperaturas mais altas, em relação aos demais isolados. A caracterização enzimática revelou que C. guaranicola produz várias enzimas, destacando-se a pectinase. Por outro lado, houve baixa produção de amilase. Somente dois isolados de C. guaranicola (C-18 e C-19) e os padrões de C. gloeosporioides e C. boninense foram capazes de utilizar o ácido cítrico como fonte de carbono. Com exceção dos isolados 2361, 2419 e 2554, os demais isolados testados foram capazes de utilizar o tartarato de amônio. A caracterização filogenética foi realizada com base na amplificação e sequenciamento parcial dos genes Gliceraldeído-3-Fosfato Desidrogenase (GAPDH), Actina, Quitina Sintase, Calmodulina e região transcrita interna (ITS). Os isolados testados formaram dois clados distintos, o primeiro (25 isolados) agrupou no complexo C. boninense próximo a C. petchii e o outro no complexo C. gloeosporioides próximo a C. siamense. A caracterização patogênica foi realizada com base no diâmetro da lesão produzida por oito isolados de C. guaranicola em dois genótipos de guaranazeiro (suscetível e resistente). O isolado C-18 expressou maior severidade quando inoculado nos dois genótipos, enquanto o isolado 2512 demonstrou a menor severidade. / Brazil is the world\'s sole producer of guaraná on a commercial scale, and the Amazonas State stands out as Brazil\'s the second largest producer, only after the Bahia State. The main limiting factor for the expansion of guaraná culture in the Amazon region is anthracnose caused by Colletotrichum guaranicola Alburq. This study aimed to characterize isolates of the fungus for its correct identification. Cultural, morphological, physiological, enzymatic, pathogenic, and molecular characterizations were performed (multilocus phylogeny). In the morpho-cultural characterization, there was variation in the size of conidia and appressoria among the isolates, but all remained within the range of amplitude described for the species C. guaranicola and for two other species (C. gloeosporioides and C. boninense). The cylinder-shaped conidia were oblong and the appressoria were round, except for a group of isolates of Maués (C series), all other isolates exhibited a hilo at the base of the conidium. Length and width of conidia and appressoria were not important features in the differentiation of isolates of C. guaranicola due to the overlap in size. The mycelial growth rate was determined in BDA medium and allowed to differentiate two groups of isolates: growth rate above 10 mm/day and growth rate below 10 mm/day. With the exception of C-12 and C-14, all C-series isolates grew above the rest, comparing to standard isolates of species C. gloeosporioides, C. boninense and C. acutatum. All others grew below the standard values. The highest growth was registered for C-09 (12.25 mm/day) and the lowest for 2601 (2.71 mm/day). The others remained within an intermediate range. Color difference of the colony also allowed to separate the isolates into two categories: white to yellowish cream colony and grey to dark grey colonies, similar to standards of C. boninense and C. gloesoporioides, respectively. All isolates grew best at the range of 25°C, but the isolates C-18 and C-19 were able to grow at higher temperatures, compared to other isolates. The enzymatic characterization showed that C. guaranicola produces various enzymes, mainly pectinase. On the other hand, there was low production of amylase. Only two isolates of C. guaranicola (C-18 and C-19) and the standards of C. gloeosporioides and C. boninense were able to use citric acid as a carbon source. With the exception of isolates 2361, 2419 and 2554, the other isolates tested were able to use the ammonium tartrate. The phylogenetic characterization was performed based on amplification and partial sequencing of genes Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Actin, Chitin Synthase, Calmodulin and internal transcribed section (ITS). The isolates tested formed two distinct clads. The first (25 isolates) grouped in the complex C. boninense near C. petchii and the other in the complex C. gloeosporioides near C. siamense. The pathogenic characterization was performed based on the lesion diameter caused by eight isolates of C. guaranicola in two guaraná genotypes (susceptible and resistant). The isolate C-18 expressed greater severity when inoculated into both genotypes, while isolate 2512 showed least severity.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.