Dissertation (PhD(Agric)) -- University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most important diseases of this cereal in the south Western Cape Province of South Africa. This fungus exists as two different types (forms), namely a nettype and a spot-type that are distinguished by differential symptom expression on barley leaves. Based on this specific plant pathological difference a series of studies of agricultural importance were executed to investigate the effects of sexual recombination between these two types. In addition, studies were done to determine the difference between local net- and spot-type populations with regards to population structure and fungicide sensitivity. This dissertation therefore, consists of a collection of separate publications and as a result a certain degree of redundancy has been unavoidable. Recombination is one of the most important evolutionary forces involved with sexual reproduction. In plant-fungal agricultural ecosystems this may result in pathogenic fungal populations adapting more rapidly to control programs such as fungicide applications. The first section of the review in part 1 of this dissertation covers different aspects of sexual reproduction in ascomycetes, specifically focussing on mating-type genes, vegetative incompatibility and recombination. The major part of the review is then dedicated to various plant pathological aspects of P.teres, specifically addressing the differences between the two types, and in various cases highlighting the significance of sexual recombination within and between the net- and spot-type. Using morphological criteria for identification purposes there have been many conflicting reports concerning the identity of leaf spot isolates in the Western Cape Province of South Africa. In part 2, the correct identity was eventually achieved employing mating studies and molecular markers .: This was accomplished after single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on the differential cultivars susceptible to the net-blotch and leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. Fifteen of the net-spot hybrid progeny (F1) produced from the mating study in Part 2 were screened in Part 3 to assess their viability and genetic stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and Clipper (both differentially susceptible to spot-type isolates) produced intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a healthy set of seedlings produced similar intermediate symptoms. RAPDs conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny revealed profiles similar to those obtained for F1 isolates. RAPD molecular data, therefore, indicated that hybrid progeny of this net x spot mating were genetically stable after having been subjected to two repetitive inoculation and reisolation cycles. Phylogenetic analysis of DNA sequences of the internal transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA gene and the 5' end partial histone-3 gene confirmed the genetic stability of the hybrid progeny. These results also indicated that the hybrid progeny produced consistent symptoms throughout the series of experiments, and maintained their virulence to the differential cultivars screened. Both types of P. teres are prevalent in the south Western Cape Province of South Africa, found on susceptible cultivars often grown within close proximity of each other. In Part 4, a net- and spot-type population were characterised in terms of their population structure using RAPD markers. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. A total of 65 loci were produced of which 54 were polymorphic. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spottype populations. A coefficient of genetic differentiation (Gs) of 0.0149 was obtained between sites within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis done on the two populations together with six progeny from the mating between a netand spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the nettype population also contained a unique spot-type RAPD fragment. This suggested that sexual recombination may be taking place between isolates of the net- and spot-type under field conditions. Fungicide application is the most important method used in the control of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values) of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol demethylation inhibiting fungicides were determined, based on the inhibitory effect on radial mycelial growth. The fungicides evaluated were triadimenol, bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and spot-type isolates revealed strong resistance to triadimenol while flusilazole was shown to be the strongest inhibitor of fungal growth. Spot-type isolates showed a higher resistance than net-type isolates to all five fungicides screened. The ICsD values indicated significant differences between four of the fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The ICsD values between propiconazole and bromuconazole were not significant. This study suggested that spot-type isolates showed a higher degree of resistance to commercially used fungicides than net-type isolates. The overall conclusion of this study is that the spot-type of P. teres is the pathogen associated with leaf spots of barley in the south western Cape province of South Africa and not P. japonica as earlier reported. Together with the net-type, both types exist as genetically variable populations in this barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to one another in which either net- or spot-type susceptible cultivars are being cultivated may lead to sexual progeny being produced. This in turn may lead to an increased rate at which fungal populations may become resistant to commercially used fungicides. It is furthermore suggested that an alternative fungicide seed treatment is used instead of triadimenol due to high resistance of P. teres to this fungicide. / AFRIKAANSE OPSOMMING: Netvlek op gars is een van die belangrikste siektes van hierdie graansoort in die suidelike deel van die Westelike Kaapprovinsie. Dié siekte word veroorsaak deur die swam Pyrenophora teres. Hierdie swam kom voor as twee verskillende tipes, naamlik 'n net-tipe en 'n kol-tipe wat onderskei word op grand van die voorkoms van hulle simptome op garsblare. Hierdie planpatologiese verskil in ag genome, is 'n reeks studies van landboukundige waarde uitgevoer om die effek van geslagtelike rekombinasie tussen die twee tipes te ondersoek. Daarbenewens is ook studies uitgevoer om om die verskil te bepaal tussen plaaslike net- en koltipe populasies ten opsigte van populasiestruktuur en fungisiedsensitiwiteit. Hierdie verhandeling bestaan dus uit 'n versameling afsonderlike publikasies en as gevolg daarvan is daar onvermydelik'n mate van oorvleueling. Rekombinasie is een van die belangrikste evolusionêre kragte betrokke by geslagtelike voortplanting. In plant-swam landboukundige ekostelsels kan dit veroorsaak dat patogene swampopulasies vinniger aanpas by beheerpragramme soos fungisiedtoediening. Die eerste gedeelte in deel 1 van hierdie verhandeling dek die verskillende aspekte van geslagtelike voortplanting van ascomycetes, met spesifieke verwysing na paringstipe gene, vegetatiewe onverenigbaarheid en rekombinasie. Die grootste gedeelte van die oorsig word gewyaan verskeie plantpatologiese aspekte van P. teres,en wys veralop die verskille tussen die twee tipes. In verskeie gevalle word die betekenis van geslagsrekombinasie binne en tussen die net- en koltipe uitgelig. Deur morfologiese kenmerke vir identifikasiedoeleindes te gebruik, is daar baie teenstrydige verslae rakende die identifikasie van blaarvlekisolate in die Westlike Kaapprovinsie van Suid-Afrika. In deel 2 is die korrekte identifikasie eventueel verkry deur gebruik te maak van paringstudies en molekulêre merkers. Dit is bereik nadat enkel ascospore verkry is uit pseudothecia gevorm na in vitro paring plaasgevind het tussen 'n bevestigde P. teres netvlek isolaat uit Denemarke en 'n verteenwoordigende Pyrenophora blaarvlekisolaat van Suid- Afrika. Deur gebruik te maak van versterkte fragmentlengte polimorfisme [AFLP] en RAPD merkers, is rekombinasie gedemonstreer in die nasate wat DNA bandpatrone gehad het wat verskil het van dié van die "ouer" isolate. Patogenisiteitstoetse het ook bevestig dat rekombinasie tydens paring plaasgevind het. Inokulasies is uitgevoer op die verskillende cultivars wat vatbaar is vir die netvlek en blaarvlek vorme. Die twee ouers het tipiese netvlek of blaarvlek simptome veroorsaak, terwyl die nasate hoekige vlekke veroorsaak het op elke cultivar. Toetse vir fungisiedsensitiwiteit deur gebruik van die ergosterol biosintese inhibeerders het gewys dat a.g.v. rekombinasie sekere nasate verhoogde weerstand teen hierdie fungisiedes het. As gevolg van paring en daaropvolgende rekombinasie tussen 'n netvlek isolaat van P. teres en 'n verteenwoordigende blaarvlek isolaat is afgelei dat laasgenoemde P. teres f. maculata is. Vyftien van die netvlek hibried nakomelinge (F1) verkry van die paringstudie in deel 2 is ondersoek in deel 3 om hul lewensvatbaarheid en genetiese stabiliteit te bepaal. Hibried nasate (F1) geïnokuleer op garssaailinge bestaande uit die volgende cultivars: Stirling (soms vatbaar vir net-tipe isolate) , B87/14 en Clipper (albei soms vatbaar vir kol-tipe isolate) het intermediêre simptome op al die cultivars veroorsaak. Akseniese kulture (F1-1) geïsoleer uit blaarletsels gevolg deur herhaalde inokulasie en isolasie (F1-2) op 'n gesonde stel saailinge het dieselfde intermediêre simptome veroorsaak. RAPDs uitgevoer met twee 10-mer inleiers op al die isolate van F1-1 en F1-2 nasate het profiele opgelewer soortgelyk aan dié wat vir F1 isolate verkry is. RAPD molekulêre data het dus gewys dat die hibried nasate van hierdie net x kol paring geneties stabiel was nadat dit onderwerp is aan twee inokulasie en reïsolasie siklusse. Genetiese stabiliteit van die hibried nageslag is bevestig deur filogenetiese analise van die DNA volgorde van die interne getranskribeerde spasieerders (ITS1 en ITS2) reg langs die 5.8S nukluêre ribosomale RNA geen en die 5' end gedeeltelike histoon-3 geen. Hierdie resultate het ook gewys dat die hibried nasate konstante simptome getoon het tydens die hele reeks eksperimente en hulle virulensie behou het vir die kultivars wat getoets is. Beide tipes van P. teres kom algemeen voor in die suidelike deel van die Westelike Kaapprovinsie en word gevind op vatbare cultivars wat dikwels naby mekaar groei. In deel 4 is 'n net- en kol-tipe populasie gekarakteriseer in terme van hulle populasiestruktuur deur gebruik van RAPD merkers. Monsters is versamel van geïnfekteerde garsblare van twee aparte kwadrante in elke saailand. Die twee kwadrante is geplaas in die hoeke van die saailand, diagonaal tot mekaar. 'n Totaal van 65 lokusse is gevorm, waarvan 54 polimorfies was. Die algehele genetiese verskeidenheid bepaal vir alle lokusse, het gelei tot gemiddelde indekse van 0.063 en 0.082 soos gevind vir die net- en kol-tipe populasies. 'n Koëffisiënt van genetiese differensiasie (Gs ) van 0.0149 is gevind tussen gebiede tussen populasies, terwyl 'n koëffisiënt (GT) van 0.63 gevind is tussen die twee populasies. Genotipiese variasie het 13 duidelike multilokus genotipes (haplotipes) getoon in die net-tipe populasie, terwyl daar twaalf was in die kol-tipe populasie. UPGMA groeperingsanalises wat gedoen is op die twee populasies tesame met ses nasate van die paring van 'n net- en koltipe isolaat het tot gevolg gehad dat drie hoof groepe gevorm is, een vir elke populasie en een vir die nasate. Een isolaat wat versamel is, van die net-tipe populasie het 'n unieke kol-tipe RAPD fragment bevat. Dit wys daarop dat geslagtelike rekombinasie in veldomstandighede mag voorkom tussen isolate van die net- en kol-tipe. Fungisiedtoediening is die belangrikste metode wat gebruik word om netvlek in Suid-Afrika te beheer. In deel 5 is die fungisiedsensitiwteit (Ieso waardes) van 89 enkelkonidiale isolate (46 net-tipe en 43 kol-tipe) van P. teres teen sterol demetielasie inhiberende fungisiedes bepaal, op die basis van die onderdrukkende effek op die radiale groei van die miselium. Die volgende fungisiedes is geëvalueer: triadimenol, bromuconazole, flusilazole, propiconazole en tebuconazole. Beide net- en kol-tipe isolate het 'n sterk weerstand teen triadimenol openbaar, terwyl flusilazole gevind is as die sterkste onderdrukker van swamgroei. Kol-tipe isolate het 'n hoër weerstand as die net-tipe isolate teen al vyf fungisiedes wat getoets is, gehad. Die lesowaardes het aangedui dat daar beduidende verskille tussen vier van die fungisiedes IS (triadimenol, tebuconazole, flusilazole en propiconazole). Die leso waardes tussen propiconazole en bromuconazole was nie beduidend nie. Die gevolgtrekking van hierdie studie is dus dat die kol-tipe isolate 'n hoër graad van weerstand teen kommersiëel gebruikte fungisiedes as die net-tipe isolate gehad het. Die algehele gevolgtrekking van hierdie studie is dat die kol-tipe van P. teres, die patogeen is wat geassosieer word met blaarvlekke op gars in die suidwestelike Kaapprovinsie van Suid-Afrika, en nie P. japonica soos voorheen gerapporteer nie. Tesame met die net-tipe, kom altwee tipes voor as geneties veranderlike populasies in hierdie gars verbouingstreek. Paring tussen die twee tipes lei tot geslagtelike nasate wat geneties stabiel is. Dit impliseer dat aangrensende garsvelde waarop net- óf kol-tipe vatbare kultivars verbou word, mag lei tot die produksie van geslagtelike nasate. Dit kan weer lei tot 'n verhoogde tempo waarteen swampopulasies weerstandbiedend teenoor kommersiële fungisiedes raak. Daar word verder ook voorgestel dat alternatiewe fungisied saadbehandelings gebruik word in plaas van triadimenol as gevolg van verhoogde weerstand van P. teres teenoor laasgenoemde.

Made available in DSpace on 2017-07-25T19:29:57Z (GMT). No. of bitstreams: 1 Monica G Harms.pdf: 1165275 bytes, checksum: cc8059801e06e439fa7ed83ee82ad8db (MD5) Previous issue date: 2013-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The ideal plants density consists in an extremely important condition in the determining of yield and in the commercial production of onion bulbs. The producers have sought new spatial distributions of plants, aiming harvest bulbs in accord with the size required by the consumer. The plants population have also influence on the development of diseases, because it is associated with the infective units spread of the pathogen and microclimate in culture. The experiment was conducted in the Capão da Onça School Farm aiming to evaluate the influence of plants density and the fungicide use in the occurrence of foliar diseases and the productive characteristics of Bola Precoce cultivar and Bella Dura hybrid. The experimental design was complete randomized blocks, with four replications for the Bola Precoce cultivar and three replications for the Bella Dura hybrid. The treatments were arranged in a split plot design, where the plots were either the presence or the absence of fungicide (metiram + piraclostrobina - 2,5 kg p.c. ha-1) and plant densities (12, 14, 16, 18, 20 and 22 plants m-1) were the split plots. Parameters evaluated were: severity of downy mildew (Peronospora destructor) and purple blotch (Alternaria porri), number of harvest bulbs, bulb mass, bulb diameter, bulb classification and yield. With the data of severity, the area under disease progress curve (AUDPC) was calculated. For Bola Precoce cultivar, no differences were observed between the densities for the parameters AUDPC (downy mildew and purple blotch), purple blotch severity, bulb mass, bulb diameter and bulbs class 0, 2, 3 and 3C. There was difference between densities for downy mildew severity in the evaluation made at 111 days after transplantation (DAT), and the maximum value (34,65%) was found in 15,3 plants m-1 density, in presence of fungicide. The fungicide was effective on downy mildew control in the evaluation made at 97 DAT and on the purple blotch control in the evaluation made at 97 and 104 DAT. The maximum yield was obtained in 22 plants m-1 density, reaching 18,21 and 17,70 t ha-1 in presence and absence of fungicide, respectively. For Bella Dura hybrid, no differences were observed between densities for AUDPC (downy mildew and purple blotch), bulbs class 0, downy mildew severity at 90, 97 and 104 DAT, and purple blotch severity at 90, 97 and 111 DAT. The fungicide was effective in controlling downy mildew in the evaluations made at 97 and 104 DAT. In absence of fungicide, the yield increased with the plant density increasing. In presence of fungicide, the maximum yield (22,09 t ha-1) was obtained in 17,45 plants m-1 density. There was a reduction of the mass and diameter of bulb with the plant density increment, when no fungicide was applied. In presence of fungicide, the percentage of bulbs class 3C decreased with the plant density increase. / A densidade ideal de plantas é extremamente importante na determinação da produtividade e da produção comercial de bulbos de cebola. Produtores têm buscado novas distribuições espaciais de plantas, objetivando colheita de bulbos de acordo com o tamanho exigido pelo consumidor. A densidade de plantas também influencia o desenvolvimento de doenças, pois tem relação com a disseminação das unidades infectivas do patógeno e o microclima na cultura. O experimento foi conduzido na Fazenda Escola Capão da Onça com o objetivo de avaliar a influência da densidade de plantas e o uso de fungicida na ocorrência de doenças foliares e nas características produtivas do cultivar Bola Precoce e do híbrido Bella Dura. O delineamento experimental utilizado foi o de blocos ao acaso com quatro repetições para o cultivar Bola Precoce e três repetições para o híbrido Bella Dura. Os tratamentos foram arranjados em parcelas subdivididas, sendo que as parcelas constituíram a presença ou ausência de fungicida (metiram + piraclostrobina - 2,5 kg p.c. ha-1) e as densidades de plantas (12, 14, 16, 18, 20 e 22 plantas m-1) constituíram as subparcelas. As características avaliadas foram: severidade de míldio (Peronospora destructor) e de mancha púrpura (Alternaria porri), número de bulbos, massa média do bulbo, diâmetro médio do bulbo, classificação dos bulbos e produtividade total. Com os dados de severidade calculou-se a área abaixo da curva do progresso das doenças (AACPD). Para o cultivar Bola Precoce, não foram observadas diferenças entre as densidades para os parâmetros AACPD (míldio e mancha púrpura), severidade de mancha púrpura, massa média do bulbo, diâmetro do bulbo e bulbos classe 0, 2, 3 e 3C. Houve diferença entre densidades para a severidade do míldio na avaliação realizada aos 111 dias após o transplante (DAT), sendo o maior valor (34,65%) encontrado na densidade de 15,3 plantas m-1 na presença de fungicida. O fungicida foi eficiente no controle do míldio na avaliação realizada aos 97 DAT e no controle da mancha púrpura nas avaliações realizadas aos 97 e 104 DAT. A produtividade total máxima foi obtida na densidade de 22 plantas m-1, alcançando 18,21 e 17,70 t ha-1 na presença e ausência de fungicida, respectivamente. Para o híbrido Bella Dura, não foram observadas diferenças entre as densidades para os parâmetros AACPD (míldio e mancha púrpura), bulbos classe 0, severidade de míldio aos 90, 97 e 104 DAT e severidade de mancha púrpura aos 90, 97 e 111 DAT. O fungicida foi eficiente no controle do míldio nas avaliações realizadas aos 97 e 104 DAT. Na ausência do fungicida a produtividade total aumentou com o incremento da densidade de plantas. Na presença do fungicida, maior produtividade (22,09 t ha-1) foi obtida na densidade de 17,45 plantas m-1. Houve redução da massa média dos bulbos e do diâmetro do bulbo com o incremento da densidade de plantas quando o fungicida não foi aplicado. Na presença do fungicida, a porcentagem de bulbos pertencentes à classe 3C diminuiu com o aumento da densidade de plantas.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-16T14:20:21Z No. of bitstreams: 1 Aldenir de Oliveira.pdf: 717930 bytes, checksum: eb22d1cac453b579b74b66da5fd0bc67 (MD5) / Made available in DSpace on 2017-02-16T14:20:21Z (GMT). No. of bitstreams: 1 Aldenir de Oliveira.pdf: 717930 bytes, checksum: eb22d1cac453b579b74b66da5fd0bc67 (MD5) Previous issue date: 2008-02-29 / This dissertation aimed to study: colonization of Acidovorax avenae subsp. citrulli in melons after inoculation of the first pair of true leaves, seeds and hermaphrodite flowers; bacterial survival in fruit and leaf residues incorporated to the soil at 0, 5, 10 and 15 cm depth and in soils without the host plant, under the influence of different soil type, temperature (10, 15, 20, 25, 30 and 35ºC) and humidity (50 and 100% of field capacity). In all studies a mutant resistant to 100 ppm of rifampicin (Aac1Rif) was utilized. Bacterial colonization was detected until 30 days after inoculation in the 10th pair of true leaves, with populations of 3.1 log UFC g-1 of leaf. In the same period, the shoot segment between the 10th and 11th leaf pair showed population of 3.52 log UFC g-1 of shoot. After seed inoculation the pathogen colonized the hypocotyl, roots, cotyledonary leaves, true leaves and shoots, until reach undetectable levels at 27 days after inoculation. Flower colonization by the bacteria was not verified. Aac1Rif was found in fruit and leaf residues at 0, 5 and 10 cm during 21 days and at 15 cm during 14 days. Highest population relative extinction rates (TERP)were presented by fruits on soil surface [0.1464 log (UFC) day -1] and on leaves at 10cm [0.084 log (UFC) day -1]. Aac1Rif survived on seven soil types only during three days and the soil C showed the highest TERP [0.9062 log (UFC) day-1]. Higher concentrations of Na+ and silt as well as higher populations of actinomycetes and Trichoderma correlated to faster extinction of Aac1Rif populations in soil. Generally for all soils the lowers TERP were found at 10 or 15ºC and the higher, at 30 or 35ºC. There was no significant (P=0.05) interaction between soil and humidity, however the T test showed significant difference (P=0.05) between the TERP at 100% [0.6685 log (UFC) day -1] and 50% [0.504591 log (UFC) day -1] of field capacity. Independent of temperature and humidity, Aac1Rif also survived in soil only during three days. / Esta dissertação teve como objetivos estudar: colonização de A. avenae subsp. citrulli em meloeiro a partir da inoculação no primeiro par de folhas verdadeiras, sementes e flores hermafroditas; sobrevivência da bactéria em restos de folhas e frutos incorporados ao solo a diferentes profundidades (0, 5, 10 e 15 cm) e em solos na ausência da planta hospedeira, sob a influência de diferentes tipos de solo (sete solos), temperaturas (10, 15, 20, 25, 30 e 35ºC) e umidades (50 e 100% da capacidade de campo). Para os estudos foi utilizado um mutante resistente a 100 ppm de rifampicina (Aac1Rif). Quando a bactéria foi inoculada nas folhas, a colonização foi detectada até os 30 dias, no 10º par de folhas verdadeiras, com população de 3,1 log UFC g-1 de folha. Nesse mesmo período, observou-se a colonização no segmento de ramo compreendido entre o 10º e 11º par de folhas com população de 3,52 log UFC g-1 de ramo. A partir de sementes, a bactéria colonizou o hipocótilo, raízes, folhas cotiledonares, folhas verdadeiras e ramos, até atingir níveis não detectáveis aos 27 dias após a inoculação. Não foi verificada colonização das flores pela bactéria. Aac1Rif foi encontrada em restosde frutos e folhas de meloeiro a 0, 5 e 10 cm durante 21 dias e a 15 cm por 14 dias. As maiores taxas de extinção relativa da população (TERP) ocorreram nos frutos na superfície do solo [0,1464 log (UFC) dia-1] e nas folhas a 10 cm [0,084 log (UFC) dia-1]. Aac1Rif sobreviveu nos sete tipos de solo apenas durante três dias e o solo C apresentou a maior TERP [0,9062 log (UFC) dia-1]. Maiores concentrações de Na+ e silte bem como maiores populações de actinomicetos e Trichoderma estiveram correlacionadas a mais rápida extinção da população de Aac1Rif no solo. Para a maioria dos solos, as menores TERP foram atingidas a 10 ou 15ºC e as maiores, a 30 ou 35ºC. Não houve interação significativa (P=0,05) entre solos e umidade, contudo o teste de T evidenciou diferença significativa (P=0,1) entre as TERP a 100% [0,6685 log (UFC) dia-1] e 50% [0,504591 log (UFC) dia-1] da capacidade de campo. Independente da temperatura e umidade, Aac1Rif também sobreviveu nos solos apenas por três dias.

The occurrence of spot type net blotch (Pyrenophora teres f. maculata) and net type net blotch (P. teres f. teres) as well as the occurrence of net blotch mating types has been investigated in Lithuania, Latvia and Estonia. Genetic diversity of barley net blotch isolates from various locations in Lithuania was investigated using ISSR and AFLP markers. The net blotch resistance of 150 spring barley varieties was investigated under artificial and natural infection conditions in the field. / Panaudojant molekulinius žymeklius ištirta tinkliškosios dryžligės patogeno Pyrenophora teres populiacijos genetinė įvairovė, nustatyti P. teres formų ir lytinio dauginimosi tipų sutinkamumas Lietuvoje, Latvijoje ir Estijoje. Lietuvos sąlygomis įvertintas Vakarų Europos ekotipo 150 vasarinių miežių veislių ir linijų jaurumas tinkliškajai dryžligei esant skirtingai pradinei infekcijai.

Accroître la diversité végétale au sein d'une culture par l'utilisation d'associations variétales est une stratégie qui permet de limiter la sévérité des maladies à dispersion éolienne. Les potentialités de cette pratique culturale restent à être caractérisées de manière précise dans le cas des maladies à dispersion pluviale, telle que la septoriose du blé durant sa phase épidémique. Cette maladie foliaire, due au champignon pathogène Mycosphaerella graminicola, est prédominante sur blé et est capable de causer des pertes substantielles de rendement, allant jusqu'à -40 %. Des expérimentations au champ ont été menées durant cinq années, de 2008 à 2012, sur le site INRA de Grignon (Yvelines, France), avec une association constituée de deux variétés de blé ayant des niveaux contrastés de résistance à M. graminicola, et dont les proportions étaient de une plante sensible pour trois assez résistantes. Par rapport à leur culture monovariétale respective, nous avons observé une diminution de la sévérité de la septoriose sur la plus sensible des variétés (en moyenne, 45 % de surface pycnidiale foliaire en moins sur les trois dernières feuilles), sans affecter significativement la variété plus résistante. Une méthodologie originale semi-automatisée a été développée pour quantifier le flux de spores dispersés par la pluie en conditions naturelles. Les mesures expérimentales ont permis de corréler l'intensité de plusieurs épisodes pluvieux avec la dispersion de spores au sein de différents couverts incluant des associations variétales. Un modèle mécaniste et stochastique a été développé afin de décrire la progression du potentiel de maladie au sein d'un couvert végétal hétérogène en trois dimensions. Cette approche théorique combine physique et épidémiologie, d'une part, (i) pour calculer l'interception des gouttes de pluie avec les organes végétaux et la trajectoire des gouttelettes d'éclaboussement au sein du couvert et, d'autre part, (ii) pour prendre en compte les niveaux de résistance variétale et la nature polycyclique de l'épidémie. À partir de ce modèle, il a été mis en évidence pour des associations de deux variétés que les proportions, ainsi que le différentiel de résistance entre les variétés à associer, pouvaient être optimisées pour réduire la sévérité de la maladie. Par ailleurs, ce modèle permet d'évaluer et d'identifier les distributions spatiales des variétés les plus propices à une réduction de la progression d'une maladie à dispersion pluviale. Parmi les précédents travaux traitant des potentialités des associations de variétés pour lutter contre des pathogènes dispersés par l'action mécanique de la pluie, certains avançaient des conclusions souvent contradictoires et peu en faveur de cette pratique. Nous avons montré ici qu'il est possible sous certaines conditions d'association de variétés (proportions, agencement spatial, différentiel de résistance globale) et de pluviométrie d'obtenir un effet significatif en termes de réduction de la maladie. / Increasing plant diversity within a crop by the use of cultivar mixtures is a strategy which allows to reduce severity of windborne diseases. Potentialities of this cropping practice have still to be precisely characterized in the case of rain-borne diseases, such as septoria tritici blotch during its spring epidemiological stage. This disease, due to the pathogen fungus Mycosphaerella graminicola, is prevalent on wheat crops and it may be result in substantial yield losses, up to -40%. Field experiments were carried out during five years, from 2008 to 2012, at the Grignon location (Yvelines, France), with a mixture consisting of two wheat cultivars with contrasted resistance to M. graminicola in a 1/3 susceptible/resistant ratio. In comparison with their pure stands, we observed a severity decrease of septoria tritici blotch for the most susceptible cultivar (on average, less 45% of leaf pycnidial leaf surface on the three upper leaf levels), without significantly affect the more resistant cultivar. An original semi-automated methodology was developed to quantify the splash-dispersed spore flux in outdoor conditions. Experimental measurements allowed to correlate intensity of several rainfall events with spore dispersal within different canopies including a cultivar mixture. A mechanistic and stochastic model was implemented in order to describe disease potential progression within a heterogeneous three-dimensional plant canopy. This theoretical approach combines physics and epidemiology in order to, on one hand, (i) compute interception of raindrops with plant organs and the pathway of splash droplets within the canopy and, on the other hand, (ii) take into account cultivar resistance levels and the polycyclism of epidemics. From this model, we highlighted for two-component cultivar mixtures that the proportions and the difference between resistance levels of cultivars to mix together could be optimized in order to reduce disease severity. Furthermore, this modelling approach makes it possible to assess and identify the cultivar spatial distributions the most favourable to a decrease of progression of a splash-dispered disease. Previous studies about potentialities of cultivar mixtures to control splash-dispersed pathogen agents, led in some cases to conclusions with inconsistent and not in favour of this cropping practice. We showed here that it was possible under certain cultivar designing conditions (proportions, spatial arrangement, difference between resistance levels) and rainfall properties to obtain a consistent significant effect in terms of disease reduction.

Resumo: Com o objetivo da redução da aplicação de fungicidas na produção de alho, foram estudados os efeitos dos Microrganismos Eficazes (E.M.-4 e E.M.-5) sobre o controle da mancha púrpura, a produção da cultura e as propriedades físicas, químicas e biológicas do solo. Os experimentos foram conduzidos, em condições de campo, por dois e três anos, na Fazenda de Ensino, Pesquisa e Produção da Unesp em São Manuel, SP. O E.M. foi aplicado nos bulbilhos de alho antes do plantio, adicionado ao material orgânico incorporado ao solo e pulverizado nas plantas após incubação ou não com melaço. A utilização do E.M. + melaço (não incubado) não proporcionou controle da doença nos experimentos. No entanto, com a incubação do E.M. + melaço houve redução na severidade da mancha púrpura em um experimento e incremento na emergência e número de folhas verdes por planta em ambos os experimentos. A altura das plantas, superbrotamento, produção, bulbos de maior valor comercial (classes 5+6+7) e as propriedades físicas (densidade do solo, condutividade hidráulica, estabilidade dos agregados, resistência à penetração e infiltração de tinta), químicas e biológicas do solo (conteúdo de polissacarídeos, carbono da biomassa microbiana do solo e atividade da desidrogenase) não foram alteradas pela utilização do E.M. A adição de material orgânico ao solo promoveu maior agregação do solo (estabilidade dos agregados), independentemente dos tratamentos empregados. No entanto, a densidade do solo, a condutividade hidráulica e a resistência à penetração não sofreram alterações com a adição do material orgânico. / Abstract: With the purpose to reduce fungicides application for garlic production, the effects of Effective Microorganisms (E.M.-4 and E.M.-5) were studied on the control of garlic purple blotch, garlic yield and physical, chemical and biological properties of the utilized planting soil. Experiments were carried out under field conditions, for two and three years, an experimental farm, belonging to São Paulo State University, located in São Manuel, São Paulo, Brazil. E.M. was applied on garlic cloves before planting, additioned to the incorporated organic matter and sprayed on garlic aerial part after incubation or not with molasse. When E.M. plus molasse (not incubated) was utilized the control of purple blotch was not observed in two experiments. However, with the incubation of E.M. with molasse, the results showed a reduction of purple blotch severity in one experiment as well as an increment of seedling emergency and number of green leaves per plant in two experiments. Plant height, bulbil sprouting, yield, bulbs with higher market grades (classes 5+6+7) and soil physical (bulk density, hidraulic conductivity, stable soil aggregates, resistance to soil penetration and tint infiltration) chemical and biological (polyssacharides, carbon of microbial biomass and dehydrogenase content) proprieties were not affected by E.M. utilization. Organic matter addition promoted soil agreggation in all treatments, however, soil bulk density, hidraulic conductivity and resistance to soil penetration was not changed by organic matter addition. / Orientador: Chukichi Kurozawa / Coorientador: Roberto Lyra Villas-Bôas / Banca: Chukichi Kurozawa / Banca: Julio Nakagawa / Banca: Rumy Goto / João Batista Vida / Hasime Tokeshi / Doutor

Melon (Cucumis melo L.) and watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) belong to the family Cucurbitaceae. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies are very limited and include: seed production field sanitation, greenhouse transplant sanitation, copper-based bactericide sprays, crop rotation, disease-free healthy seeds, isolating diseased plants, and peroxyacetic acid seed treatments. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. No nondestructive assays are commercially available to detect BFB in seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate NB-LRR genes for disease resistance, and optimization of virus induced gene silencing for melon and watermelon crops. The potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds were evaluated. It was possible to detect BFB in seeds that were pistil inoculated via x-ray imaging and pericarp inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea (Camellia sinensis) and tea polyphenols as seed treatments to sanitize seeds infected with A. citrulli. Green tea unlike black tea inhibited growth of A. citrulli because of polyphenols. Eighty one melon and forty four watermelon NB-LRR genes were reidentified, and genes that have potential resistance against A. citrulli on melon plants were screened based on host selectivity of the pathogen. Finally, the virus-induced, gene-silencing method was optimized for melon and watermelon for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate R genes were identified that might confer stable resistance in the right genetic background. / Doctor of Philosophy / Melon and watermelon crops both belong to the gourd family. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies and detection methods are very limited. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate disease resistance genes, and optimization of virus induced gene silencing methodology for melon and watermelon crops. There are currently no nondestructive assays available to detect BFB in seeds. We evaluated the potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds. It was possible to detect BFB inside layers of seeds that were naturally inoculated through the flowers via x-ray imaging and seedcoat inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea and tea constituents as seed treatments to sanitize seeds infected with BFB. Green tea unlike black tea inhibited growth of BFB. Eighty one melon and forty four watermelon disease resistance genes were reidentified and genes that have potential resistance against BFB on melon plants were screened based on host selectivity of the pathogen. Finally, the virus induced gene silencing method was optimized for melon and watermelon plants for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate resistance genes were identified that might confer stable resistance in the right genetic background.

L'utilisation de molécules stimulatrices de défense des plantes (SDP), également appelées inducteurs de résistance, constitute une alternative possible aux traitements fongicides conventionnels pour contrôler les maladies dues aux chanpignons phytopathogènes. Dans cette étude, nous avons mis en évidence que trois produits à caractère SDP (FSOV2, FSOV7 et FSOV10) protègent le blé (Triticum aestivum L.) contre la septoriose (Mycosphaerella graminicola, anamorphe Zymoseptoria tritici) lorsqu'ils sont utilisés de façon préventive, mais cette protection dépend fortement du cultivar considéré. Les cultivars Alixan, Premio et Altigo testés ici présentaient au départ des niveaux de résistance distincts à la septoriose. Les protections obtenues ne sont pas liées à un effet direct sur la germination des spores du champignon, mais à l'induction des mécanismes de défense chez le blé qui réduisent la nécrose foliaire et la sporulation du champignon. Ainsi, l'observation des différents stades du processus infectieux de M. graminicola en microscopie et le dosage des activités enzymatiques fongiques de dégradation des parois (CWDE) in planta révèlent que le niveau de protection induite varie avec le SDP appliqué et avec le cultivar traité. L'expression de neuf gènes impliqués dans différentes voies de défense, suivi par RTqPCR, et les activités enzymatiques peroxydase et phénylalanine ammonia lyase ont été mesurée au cours du temps, depuis le traitement par les SDP jusqu'à 5 jours après infection. Les résultats obtenus montrent, que les mécanismes de défense sont induits différemment en fonction du cultivar et en fonction du SDP appliqué. Ces résultats suggèrent que la réussite au champ des SDP est conditionnée de façon déterminante par le choix du couple SDP-cultivar. / The use of resistance inducers (RI) is a potential alternative to conventional fungicide treatments against plant fungal diseases. In the present study, we revealed that preventive applications of three RI conferred protection efficacies against M. graminicola, with protection levels varying with the wheat cultivar. Alixan, Premio and Altigo cultivars were previously known to exhibit distinct resistance levels to M. graminicola. The observed protections did not result from a direct effect on spore germination, but were related to the induction of wheat defense mechanisms. The induced resistances reduced foliar necrosis, as well as the sporulation level of the fungus. Microscopic observations of the infection process of M. graminicola and cell wall degrading enzymes (CWDE) activities measured in planta showed that the applied RI as well as the considered treated wheat cultivar influences the impact on the infection process and the protection efficacy. We investigated from the time of treatment until 5 days after inoculation plant peroxidase and phenylalanine ammonia lyase activities and the expression of nine genes involved in distinct defense pathways. Our results indicated that defense mechanisms are differently induced according both to the wheat cultivar and the RI. Therefore, the successful use of RI at the field level strongly depends on the RI-cultivar combination.

Um dos principais fatores limitantes da produção do melão é a ocorrência de doenças. Entre os patógenos mais importantes estão as bactérias, que causam perdas significativas na produção. Causada pela bactéria Acidovorax avenae subsp. citrulli (Aac), a mancha-aquosa-do-melão, também conhecida como mancha-bacteriana-dofruto, é uma doença grave, que tem preocupado produtores do nordeste, sendo que todos os tipos de melão apresentam suscetibilidade ao patógeno. A principal fonte de inóculo para esta bactéria é a semente infectada. Este trabalho teve como objetivo o desenvolvimento de um meio semi-seletivo para detecção e identificação de Acidovorax avenae subsp. citrulli em sementes de melão, para testes de rotina em laboratórios de patologia de sementes. Por meio de testes de fungitoxicidade, antibiogramas qualitativos e quantitativos e testes bioquímicos foi desenvolvido o seguinte meio semiseletivo para a detecção de Aac em sementes de melão: dextrose (5 g/L), NaCl (5 g/L), peptona (5 g/L), KH2PO4 (2 g/L), MgSO4.7H2O (0,2 g/L), vermelho de fenol (0,012 g/L), uréia (25 g/L), agar (17 g/L), benomil (100 mg/L), nistatina (200 mg/L) e amoxicilina (15 mg/L). / One of the main limiting factors of the melon production is the occurrence of diseases. The bacteria are among the most important pathogens causing significant losses in the production. Caused by the bacterium Acidovorax avenae subsp. citrulli (Aac), the bacterial-fruit-blotch is a serious disease that affects all types of melon and has worried northeast producers. The main source of inoculum for this bacterium is the infected seed. This work had as objective the development of a semi-selective medium to detect and identify Acidovorax avenae subsp. citrulli in melon seeds, for routine tests in seed pathology laboratories. By means of fungitoxicity tests, qualitative and quantitative antibiograms and biochemical tests the following semi-selective medium to detect Aac in melon seeds was developed: dextrose (5 g/L), NaCl (5 g/L), peptone (5 g/L), KH2PO4 (2 g/L), MgSO4.7H2O (0,2 g/L), phenol red (0,012 g/L), urea (25 g/L), agar (17 g/L), benomil (100 mg/L), nistatina (200 mg/L) and amoxicilin (15 mg/L).

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-07-04T13:48:44Z No. of bitstreams: 1 JoseMC_TESE.pdf: 3634440 bytes, checksum: 6ac32b0fb28add3a21ce28608edd3cdf (MD5) / Approved for entry into archive by Vanessa Christiane (referencia@ufersa.edu.br) on 2017-07-04T16:47:51Z (GMT) No. of bitstreams: 1 JoseMC_TESE.pdf: 3634440 bytes, checksum: 6ac32b0fb28add3a21ce28608edd3cdf (MD5) / Made available in DSpace on 2017-07-04T16:57:57Z (GMT). No. of bitstreams: 1 JoseMC_TESE.pdf: 3634440 bytes, checksum: 6ac32b0fb28add3a21ce28608edd3cdf (MD5) Previous issue date: 2017-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of the present work was to identify sources of resistance in melon accessions to Liriomyza sativae; Alternaria spp., and Acidovorax avenae. Two experiments were performed in a greenhouse and in the Laboratory of Applied Entomology (UFERSA) in a completely randomized design, with ten replicates, each plant being a plot. In the first experiment 38 accessions and two hybridswere evaluated. The infestation was held in cages coated with antiophidic screen. The second experiment was performed with the accessions that revealed to be promising and some were susceptible in the first trial. In the evaluations, the number of mines, larvae, pulps and adult flies were determined. In some materials, leaf colorimetry, number, size of stomata and trichomes were analyzed. The accessions AM 55 and Nantais Oblong as well as the Ruthenium hybrid are resistant to Liriomyza sativa. The number of mines, larvae and adult flies correlated negatively with the number of trichomes and positively with the size of the stomata. Regarding the Alternaria leaf blight, fifty-eight accessions were initially evaluated in a randomized block design (DBC) with three replications under field conditions. In a second experiment, twelve accessions were also evaluated under field conditions, in both tests the evaluations were accomplished 50 days after the transplanting, as far as the severity and incidence of plants with Alternaria. It was concluded that there is variability in the germplasm of melon for reaction to the Alternaria leaf blight, being the accessions C-AC-04, C-AC-08, C-AC-26, C-AC-31, I 162, I 173 e MR-1 with lower severity averages and incidence of diseased plants, being evaluated as promising for use in breeding programs of melon for resistance to the pathogen. For the reaction to A. avenae, fifty accessions were evaluated in greenhouse and in field conditions. There has been accomplished inoculations with bacterial suspension in the concentration os 1,0 x 108 UFC ml-1. There were evaluated leaves (in a controlled environment), leaves and fruits (in field) with bacteriose symptoms. The C-AC-12 accession has intermediate resistance Acidovorax avenae / Os objetivos do presente trabalho foram: identificar fontes de resistência em acessos de meloeiro a Liriomyza sativae, Alternaria spp. e Acidovorax avenae. Dois experimentos foram realizados em casa de vegetação e no Laboratório de Entomologia Aplicada da (UFERSA) em delineamento inteiramente casualizado, com dez repetições, sendo cada planta uma parcela. No primeiro experimento foram avaliados 38 acessos e dois híbridos de meloeiro. A infestação foi realizada em gaiolas revestidas com tela antiafídica. O segundo experimento foi realizado com os acessos que se mostraram promissores e alguns suscetíveis no primeiro ensaio. Nas avaliações, determinou-se o número de minas, larvas, pupários e moscas adultas, em alguns materiais fez-se analise de colorimetria das folhas, número, tamanho de estômatos e tricomas. Os acessos AM 55 e Nantais Oblong bem como o híbrido Rutênio são resistentes a Liriomyza sativae. O número de minas, larvas e moscas adultas correlacionaram negativamente com o número de tricomas e positivamente com o tamanho dos estômatos. Com relação a mancha de alternaria, inicialmente foram avaliados 58 acesso sem delineamento em blocos casualizados (DBC) com três repetições em condições de campo. Em um segundo experimento, foram avaliados 12 acessos também em condições de campo, em ambos os ensaios as avaliações foram realizadas aos 50 dias pós o transplantio, quanto a severidade e incidência de plantas com alternariose.Concluiu-se que, existe variabilidade no germoplasma de meloeiro para reação a mancha de alternaria, sendo os acessos C-AC-04, C-AC-08, C-AC-26, C-AC-31, I 162, I 173 e MR-1 com menores médias de severidade e incidência de plantas doentes, sendo avaliados como promissores para uso em programas de melhoramento genético do meloeiro visando resistência ao patógeno. Para a reação a A. avenae, foram avaliados 50 acessos de meloeiro em estufa e em condições de campo. Realizou-se inoculações com suspensão bacteriana na concentração de1, 0 x 108 UFC ml-1.Avaliou-se folhas (em ambiente controlado), folhas e frutos (em campo) com os sintomas da bacteriose. O acesso C-AC-12 possui resistência intermediaria a Acidovorax avenae / 2017-07-04