Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A soja (Glycine max) à uma espÃcie de grande valor econÃmico para o Brasil dada a multiplicidade de uso de seus grÃos na alimentaÃÃo animal e na indÃstria. Embora o Brasil seja o segundo maior produtor mundial dos grÃos, as perdas na produtividade em campo ainda sÃo considerÃveis, principalmente Ãquelas causadas por nematÃides do gÃnero Meloidogyne e por fungos fitopatogÃnicos. Mesmo com a existÃncia de alternativas quÃmicas para o controle dessas espÃcies, bem como com a existÃncia de genÃtipos resistentes, as perdas agrÃcolas ainda sÃo considerÃveis, mostrando que a busca por mecanismos naturais de resistÃncia ambientalmente seguros sÃo prÃticas necessÃrias para o controle de pragas e patÃgenos e para a melhoria na produtividade. Este trabalho objetivou caracterizar bioquÃmica e funcionalmente duas toxinas protÃicas isoladas de sementes de soja, bem como avaliar os seus papÃis na defesa contra patÃgenos de importÃncia agronÃmica para essa espÃcie. Foi mostrado experimentalmente que SYTX-2 (28 kDa) à uma proteÃna Ãcida encontrada em duas isoformas (27,3 e 27,2 kDa) de pIâs 5,11 e 5,24, as quais apresentam a mesma extremidade NH2-Terminal (KTISSEDSPFFNCREK). A anÃlise por dicroÃsmo circular mostrou que a SYTX-2 apresenta um espectro tÃpico de proteÃnas que apresentam α-hÃlice e folhas-β, sendo essa estrutura semelhante Ãquela jà descrita para a SBTX. Esses padrÃes sÃo gradualmente perdidos quando a proteÃna à aquecida de 25 a 95 ÂC. Os espectros de emissÃo em 280 e 295 nm (323 e 313 nm, mÃximo) mostraram padrÃes tÃpicos de resÃduos de triptofano presentes no interior da estrutura terciÃria. SYTX-2 à uma hemilectina capaz de aglutinar indiretamente eritrÃcitos de coelho em presenÃa de anticorpos policlonais anti-SYTX-2, sendo essa atividade inibida por D-manose. AlÃm disso, in vitro, SYTX-2 apresentou atividade ribonucleÃsica, cuja atividade especÃfica (1821,42  3,34 UA. h-1 mgP) foi semelhante Ãquela descrita para a ribonuclease de raÃzes de V. unguiculata. Foi observado que SYTX-2 està presente na casca das sementes em teores menores do que os observados para os cotilÃdones, alÃm de se distribuir tambÃm em raÃzes, caules e folhas. As raÃzes jovens apresentam os maiores teores de SYTX-2 (62,62  10,10 Âg de SYTX-2/g de tecido) sendo essa expressÃo triplicada em tecidos adultos (195,12  35,54 Âg/g de tecido). Em pH 5,0 essa proteÃna à exsudada das sementes ao longo de 24 h, sendo o pico de exsudaÃÃo mostrado 18 h apÃs o contato com o tampÃo (6,16  0,08 ÂgP de SYTX-2/semente ). Tal como descrito para muitas proteÃnas de defesa, SYTX-2 foi induzida 6 h apÃs a injÃria mecÃnica de folhas (de 6,7 para 10,46 Âg de SYTX-2/ g de tecido), retornando aos valores normais 24 h apÃs a lesÃo. In vitro SYTX-2 apresentou uma potente atividade nematicida contra M. incognita RaÃa 4, induzindo a mortalidade de 85% dos J2 6h apÃs incubaÃÃo com a proteÃna, e de 100% apÃs 24 h. Essa toxina tambÃm foi capaz de inibir (20%) o crescimento de C. albicans, embora nÃo tenha sido efetiva em inibir a germinaÃÃo de esporos de fungos fitopatogÃnicos (R. solani, Phomopsis sp. e F. solani f.sp glycines). Este trabalho tambÃm descreve o isolamento, a clonagem e a caracterizaÃÃo do cDNA da subunidade de 27 kDa da SBTX (44 kDa). O cDNA foi isolado a partir de um pool de RNA extraÃdo de sementes 15, 25 e 35 dias apÃs a antese, utilizando iniciadores desenhados a partir do NH2-terminal das duas subunidades da proteÃna (27 e 17 kDa). EvidÃncias experimentais sugerem fortemente que as duas subunidades da proteÃna sÃo codificadas por genes diferentes. A subunidade de 27 kDa da SBTX apresenta um cDNA de 815 pb, composto por uma ORF de 660 nucleotÃdeos, codificante para uma proteÃna com 219 resÃduos de aminoÃcidos. A sequÃncia do cDNA da SBTX foi detectada em dois cromossomos (04 e 06) e a busca por ESTâs para essa proteÃna, mostrou que alÃm de ser expressa em todo o vegetal, nÃveis elevados de transcritos sÃo observados apÃs a infecÃÃo contra P. sojae e F. solani f. sp. glycines, evidenciando seu importante papel na defesa contra fungos fitopatogÃnicos. A sequÃncia deduzida de aminoÃcidos da subunidade de 27 kDa apresenta um peptÃdeo sinal de 26 resÃduos de aminoÃcidos, clivado para a produÃÃo da proteÃna madura, que apresenta, portanto, massa molecular de 21,7 kDa e pI 9,3, sendo uma proteÃna bÃsica. Na sequÃncia de aminoÃcidos da subunidade de 27 kDa tambÃm foram identificados: um resÃduo de cisteÃna, envolvido na formaÃÃo de uma ponte dissulfeto com a subunidade de 17 kDa, 11 sÃtios de fosforilaÃÃo em Ser, Thr ou Tyr, 8 sÃtios de glicosilaÃÃo para GlcNAc e um sÃtio para adiÃÃo de oligossacarÃdeos tipo mucina (GalNAc). A toxina tambÃm apresenta sÃtios de clivagem para pepsina, tripsina e quimiotripsina que podem justificar a ausÃncia de toxicidade observada em camundongos apÃs administraÃÃo oral. SYTX-2 e SBTX foram mostradas atravÃs de uma caracterizaÃÃo estrutural ainda mais completa que as descritas por Sousa (2006) e Siebra (2004) e as informaÃÃes obtidas permitiram definir que essas proteÃnas sÃo parte importante da defesa da soja contra fungos fitopatogÃnicos e nematÃides. AlÃm de inÃditos e de extrema relevÃncia, todos esses dados darÃo subsÃdios para estudos posteriores que objetivem, para SYTX-2, determinar sua microestrutura protÃica e isolamento gÃnico e, para SBTX, realizaÃÃo de projetos futuros, visando o desenvolvimento de plantas transgÃnicas com uma maior resistÃncia a fungos / Soybean provides significant sources of fatty acids and proteins for human and animal nutrition and also has non-food uses. Conditions in almost all cultivated land are sub-optimal for plant growth as a result of the increasing incidence of diseases, even in developed agricultural systems. To meet these challenges, genes and proteins that control their resistance to a wide range of pathogens need to be identified and characterized to facilitate improvements in crop productivity. The main focus in this thesis has been to characterize (providing basic information about biochemical characteristics) and study the functional role of SYTX-2 (28 kDa) and SBTX (44 kDa), two toxic proteins isolated from soybean seeds, in plant defense against pathogens. The SYTX-2 was purified by a combination of ammonium sulphate fractionation and two chromatographic steps. Bidimensional electrophoresis of this protein revealed the presence of two spots (27.3 e 27.2 kDa), with isoeletric points values corresponding to 5.11 and 5.24, respectively, exhibiting the same N-terminal sequence (KTISSEDSPFFNCREK). SYTX-2 has also ribonuclease activity (1821.42  3.34 UA. h-1 mgP), similar to that described in Vigna unguiculata leaves. The CD spectrum of SYTX-2 presents an alpha-beta profile spectrum, similar to the structure described to SBTX. Regarding to the temperature exposure, monitored by CD, it was observed that the structure of SYTX-2 is vulnerable to the temperatures above 40 ÂC. The fluorescence spectra of Soyatoxin-2 marked a maximum emission of fluorescence at 323-333 nm and confirmed that the tertiary structure of this protein was correctly folded. SYTX-2 behaves as a hemilectin: it does not directly promote agglutination of red blood cells, but toxin-treated erythrocytes are readily agglutinated in the presence of anti-SYTX-2 antibodies. ELISA assays showed that SYTX-2 was exuded during seed imbibition, the maximum level of exuded toxin (6.16  0.08 Âg/seed) detected being at 18 h after the start of imbibition. The expression profiles of SYTX-2 in various soybean tissues were investigated with ELISA assay or Dot Blot analysis. The expression analysis suggested that SYTX-2 was clearly detected in seed coat, leaves, roots and also in stems. However, expression of SYTX-2 in roots is higher than that in leaves and stems. A strong induction of SYTX-2 expression was also observed in wounded leaves 6 h after treatment and it decreased thereafter. In vitro, antifungal activity of SYTX-2 was not detected against R. solani, Phomopsis sp. and F. solani f.sp glycines, but this protein inhibits C. albicans growth. Nematicidal effects of SYTX-2 were studied in vitro against Meloidogyne incognita nematode and the toxin (11Âg/nematode) showed a high nematicidal activity, with the mortality of 85%, after six hours contact and of 100%, after 24 h of incubation. This work also describes the isolation, sequencing and functional analysis of cDNA (815 pb) encoding 27 kDa subunit of soybean toxin (SBTX). CDNA was amplified using a forward primer designed based on the N-terminal sequence of the toxin in combination of primer AP. The genomic location of the 27 kDa SBTX subunit SBTX was preliminarily determined with the mapped soybean ESTs database (www.phytozome.net) at Gm04 and Gm06 chromosome of soybean and thus may have two copies per genome. The deduced protein sequence of 219 amino acids (MW of mature protein 21.7 kDa, pI 9.3) included an N-terminal signal peptide. ESTâs encoding 27 kDa subunit SBTX were present in cotyledons, leaves, and seedlings and the expression of 27 kDa subunit SBTX was also induced in tissues by P. sojae and F. solani f. sp. glycines infection and by abiotic stress. In addition to these blocks, the 27 kDa deduced protein sequence contains a putative Ser/Tyr/Thr phosphorylation and also contains eight potential N-linked glycosylation sites and a threonine/serine-rich region which is a potential site for attachment of O-linked carbohydrate. Potential sites for pepsin, trypsin and chymotrypsin hydrolysis were also detected. The results add a new dimension to toxins SBTX and SYTX functionalities and support the concept that these proteins act protecting soybean against pathogens

O feijão-comum (Phaseolus vulgaris) é atacado por uma gama de patógenos que afetam a produtividade das lavouras e a qualidade dos grãos. Dentre os patógenos de importância econômica para a cultura no Brasil, destaca-se o nematoide das galhas (Meloidogyne incognita). Embora haja relatos sobre a avaliação de cultivares na presença de M. incognita, as fontes de resistência tem se mostrado pouco efetivas. Por isso, pesquisas que possibilitem um melhor entendimento sobre a interação planta-nematoide são de extrema valia e devem nortear novas estratégias para o melhoramento do feijoeiro. Assim, no presente estudo, 18 cultivares de P. vulgaris foram avaliadas quanto à resistência a M. incognita raça 3, sendo que quatro comportaram-se como pouco suscetíveis, 11 como moderadamente suscetíveis e três altamente suscetíveis. A cultivar IPR Saracura mostrou menor grau de suscetibilidade e foi, então, usada na construção de 12 bibliotecas de RNAseq, visando à identificação dos genes envolvidos na reposta à infecção pelo nematoide. Foram adotados dois tratamentos, 4 e 10 DAI (dias após inoculação), compostos de plantas inoculadas e controles. Primeiramente, realizou-se o mapeamento dos transcritos de cada biblioteca, tomando como referência o genoma de P. vulgaris (G19833), o que resultou na identificação de 27.195 unigenes. Em seguida, foi realizada a quantificação da expressão dos transcritos mapeados e genes diferencialmente expressos foram identificados. No total, 191 genes do hospedeiro apresentaram expressão diferencial, considerando-se: i) o tratamento inoculado em relação ao controle; ii) a razão de expressão (Fold Change - FC) mínima absoluta igual a 4; iii) o nível de significância ? = 0,05. Do total, 120 genes foram identificados aos 4 DAI e 71 aos 10 DAI. As sequências mapeadas foram contrastadas àquelas dos bancos de dados NCBI e TAIR, usando a ferramenta BLASTx e, posteriormente, anotadas usando os softwares Blast2GO e MapMan. Detectou-se similaridade com genes codificadores de proteínas conhecidas para 90% (24.604/27.195) dos unigenes, sendo que 69% (16.991/24.604) deles foram anotados. Quanto à expressão diferencial, 98% (188/191) dos transcritos mostraram similaridade com proteínas conhecidas e 67% (127/188) puderam ser anotados. Os transcritos foram atribuídos a diferentes categorias funcionais putativas, predominando o termo ontológico \'processos metabólicos\', em ambas as plataformas. A anotação dos genes na plataforma MapMan mostrou abundância das categorias da via de resposta a estresse, com predominância de genes de defesa superexpressos aos 4 DAI e reprimidos aos 10 DAI. Por fim, 10 genes mostraram expressão diferencial tanto aos 4 como aos 10 DAI: sete deles foram estáveis, sendo superexpressos nas plantas inoculadas, e três apresentaram comportamentos opostos nos momentos avaliados. Ênfase foi dada a um gene que codifica uma \'probable inactive ADP-ribosyltransferase\' e a quatro genes de resposta a ferimento. / The common bean (Phaseolus vulgaris) is attacked by a range of pathogens, which affect crop yield and the quality of grains. Among the pathogens of economic significance to the crop in Brazil, the root-knot nematodes (Meloidogyne incognita) deserve attention. Though there are some reports on cultivar evaluation in presence of M. incognita, the resistance sources have not being effective. Therefore, it is of valuable importance research projects that could lead to a better understanding of plant-nematode interaction and to indicate new strategies for common bean breeding. In the present study, 18 cultivars of P. vulgaris were evaluated in regard to their resistance to M. incognita race 3; four were less susceptible, 11 moderately susceptible, and three were highly susceptible. \'IPR Saracura\' behaved as the less susceptible cultivar and then was selected for the construction of 12 RNAseq libraries, aiming at the identification of genes differentially expressed in response to nematode infection. Two treatments were adopted, 4 and 10 days after inoculation (DAI), each comprised of inoculated and control plants. Firstly, the transcripts were mapped to the reference genome of P. vulgaris (G19833), resulting in the identification of 27,195 unigenes. Then, the mapped transcript\'s expression was quantified and differentially expressed genes were identified. In total, 191 genes of the host plant showed differential expression taking into consideration: i) the inoculated treatments in relation to their control; ii) an absolute fold change (FC) >= 4; iii) a level of significance ? = 0,05. Of the total, 120 genes were detected at 4 DAI and 71 at 10 DAI. The mapped sequences were compared against those deposited in NCBI and TAIR databanks using BLASTx and subsequently annotated using Blast2GO and MapMan softwares. Similarity to known proteins was detected for 90% of the unigenes (24,604/27,195) and 69% (16,991/24,604) of them were annotated. Regarding assessing differential expression, 98% (188/191) of the transcripts showed similarity to known proteins and 67% (127/188) were annotated. Transcripts were attributed to different putative functional categories and the ontological term \'metabolic process\' was predominant within both platforms. Gene annotation within MapMan platform showed predominance of stress-related pathway categories, with prevalence of defense genes overexpressed at 4 DAI and repressed at 10 DAI. Finally, 10 genes showed differential expression at both 4 and 10 DAI: seven were stably overexpressed in the inoculated plants, and three showed an opposite behavior regarding the evaluation periods. Attention was given to a gene encoding a probable inactive ADP-ribosyltransferase and four genes related to wound response.

Meloidogyne incognita (Kofoid and White) is a major pest of soybean in South Africa and due to its high level of pathogenicity to the crop it is quintessential that research in this regard should receive priority. Root-knot nematode control has in the past mostly included the use of nematicides, while crop rotation and inclusion of cultivars with genetic host plant resistance (henceforth referred to as resistance only) to these pests were also used. Since no synthetically-derived and/or biological agents are registered locally as nematicides on soybean, the use of resistant cultivars represents one of the most viable and environmentally-friendly strategies to protect local soybean crops against damage resulting from parasitism by M. incognita. Although numerous exotic soybean cultivars have been identified with resistance to M. incognita, only a few locally adapted ones have proved to exhibit resistance to the latter species. Moreover, at present Egret is the only cultivar still available for commercial use in South Africa. Little and fragmented information is, however, available on the use of plant enzymes, that are interrelated in biochemical pathways that are expressed in root-knot nematode resistant cultivars, for its use as an additional parameter to exploit such a trait. Therefore, the present study was undertaken to identify M. incognita resistance in selected, locally adapted soybean cultivars by quantifying and exploiting the latter trait by using enzyme activities as an additional parameter. In addition, resistance to M. incognita in selected resistant soybean cultivars was also verified by means of histopathological studies to identify cellular changes associated with the trait. In the first part of the present study, 31 locally adapted soybean cultivars of which 23 were commercially available in the 2012 growing season were evaluated for resistance to M. incognita. The latter was done by means of traditional screening protocols for which M. incognita-gall rating, egg and second-stage juvenile as well as the reproductive factor data per root system for each cultivar screened were recorded. Two greenhouse experiments were subsequently conducted concurrently, one of which the abovementioned nematode parameters were recorded 30 and the other 56 days after inoculation. Reproduction factor values were used as the main criterium to identify M. incognita resistance in local soybean cultivars since it is considered as a more reliable parameter for this specific type of evaluations. Reproduction factor values equal to and lower than one, indicating resistance to the M. incognita population used in this study, were recorded only for cultivar LS5995, as well as seven pre-released GCI cultivars. These eight cultivars also had very low egg, as well as egg and second-stage juvenile counts per root system, all of which differed significantly from the susceptible control, as well as a number of other cultivars. Root gall indices, on the other hand, did not show consistent results in terms of the identification of the host status of the 31 cultivar screened during this study. Using reproduction factor values, local farmers can thus be supplied with information on the resistance of commercially-available soybean cultivars. Eventually, such M. incognita-resistant cultivars can be used to reduce population levels of this nematode pest in fields of producers and also as valuable germplasm sources in breeding programs to introgress/stack this trait in newly-developed soybean cultivars. The second part of the study aimed to verify and exploit M. incognita-resistance in soybean either identified as resistant or susceptible during the screenings experiments, using enzymatic activity as biochemical markers. Cultivar LS5995 was included as the resistant and Dundee as the susceptible standard. The activity of three enzymes, namely guaiacol peroxidase, lipoxygenase and catalase were recorded at different time intervals in roots and leaf samples of the latter cultivars, of both nematode-inoculated and nematode-free plants of each cultivar. Significant (P ≤ 0.05) increases in guaiacol peroxidase activity in leaf and root samples of the M. incognita-resistant cultivars GCI7 and LS5995 (inoculated with J2) were recorded 24 hours (h) after onset of the experiment. Use of this enzyme thus emanated as a useful parameter to identify soybean cultivars that exhibit resistance against M. incognita, especially in leaves, which could substantially reduce the time needed to screen cultivars. In terms of lipoxygenase activity recorded, substantial variation existed between the cultivars tested. The M. incognita-susceptible cultivar Egret was the only cultivar for which a significant (P ≤ 0.05) increase in lipoxygenase activity in the roots was evident 24 h after inoculation. However, during the 48 h sampling time, significant (P ≤ 0.05) differences in lipoxygenase activity were also recorded for the two M. incognita resistant cultivars GCI7 and LS5995. Although the increase in lipoxygenase activity for the susceptible cultivar Egret was unexpected, it may indicate that some level of resistance is present in the latter cultivar, which has in previous studies been identified as resistant to M. incognita. Other factors such as a different M. incognita populations used and temperature differences in greenhouse conditions that applied in this study compared to that for an earlier study may, however, serve as explanations for the latter differences in host status identification of cultivar Egret. In terms of catalase activity recorded in leaf samples of the M. incognita-resistant cultivar LS5995, substantial reductions of as much as 35.6 % were recorded for J2-inoculated plants compared to those of the J2-free control plants. In leaf samples of the susceptible cultivars, Egret and Dundee, catalase was also reduced, but to a lesser extent and ranged from 6 to 26 %. Conversely, catalase activity in the leaves of J2-inoculated plants of the highly susceptible cultivar LS6248R was substantially increased by as much as 29.3 %. Enzyme data obtained as a result of the current study thus generally complemented those of traditional screening assays in which resistance in locally adapted cultivars were identified to a certain degree. It is, however, recommended that enzyme activity, to be used as bio-markers, still needs further refinement and more investigation to optimise their use in identification, verification and exploitation of M. incognita resistance in soybean cultivars. The third and final part of the study encompassed a comparison of cellular changes induced by M. incognita in resistant and susceptible soybean cultivars to verify the resistant reactions expressed in the enzyme data. According to light- and transmission electron microscope observations, distinct differences in the appearance and development of giant cells in roots of the M. incognita-resistant cultivars LS5995 and GCI7 existed when compared to those in roots of the susceptible cultivars Dundee and LS6248R. In the latter cultivars, giant cells that formed were characteristically large and contained a dense cytoplasm, with thick irregularly surfaced cell walls. Cell walls also displayed thick aggregations that appeared to be cell-wall ingrowths. These giant cells are optimal to facilitate M. incognita development and reproduction. In contrast, giant cells that were associated with the resistant cultivars LS5995 and GCI7 were small, irregularly shaped and contained increased amounts of deposited cell-wall material in the cytoplasm known as cell wall inclusions. Necrosis was also present in M. incognita-infected root cells of both cultivars. Such giant cells have been associated with retarded feeding, development and reproduction of the latter root-knot nematode species. However, it was evident that neither GCI7 nor LS5995 are immune to M. incognita since J2 survived and developed to third- and fourth and ultimately mature females that reproduced in their roots. Optimal giant cells that were formed in the roots of the M. incognitasusceptible cultivars Dundee and LS6248R thus supported the nutritional needs of the developing M. incognita individuals and led to significant increases in M. incognita populations 56 days after inoculation as was evident from the high reproduction factor values that were obtained for such cultivars during host status assessments that represented the first part of this study. The opposite was recorded the M. incognita-resistant cultivars LS5995 and GCI7 since sub-optimal giant cells in their roots could not sustain high offspring from such mature females. The presence of necrotic root tissue adjacent to giant cells, furthermore, indicated that hypersensitive reactions occurred in the latter resistant cultivars. Enzyme data obtained in the second part of this study supported the presence of hypersensitive reactions in root cells of the latter resistant cultivars. Guaiacol peroxidase and lipoxygenase inductions in particular in plant tissues have been reported to play integral roles in hypersensitive reactions that are exhibited by cultivars that are resistant to pests and diseases. Finally, results obtained from the different parts of this study complemented each other. It resulted in the resistance that was identified in the GCI7 pre-released cultivar being verified and exploited against that of the resistant standard LS5995. Research that was done during this study also represented the first investigations into the use of enzymes as biochemical markers of resistance against M. incognita in soybean in South Africa. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Meloidogyne incognita (Kofoid and White) is a major pest of soybean in South Africa and due to its high level of pathogenicity to the crop it is quintessential that research in this regard should receive priority. Root-knot nematode control has in the past mostly included the use of nematicides, while crop rotation and inclusion of cultivars with genetic host plant resistance (henceforth referred to as resistance only) to these pests were also used. Since no synthetically-derived and/or biological agents are registered locally as nematicides on soybean, the use of resistant cultivars represents one of the most viable and environmentally-friendly strategies to protect local soybean crops against damage resulting from parasitism by M. incognita. Although numerous exotic soybean cultivars have been identified with resistance to M. incognita, only a few locally adapted ones have proved to exhibit resistance to the latter species. Moreover, at present Egret is the only cultivar still available for commercial use in South Africa. Little and fragmented information is, however, available on the use of plant enzymes, that are interrelated in biochemical pathways that are expressed in root-knot nematode resistant cultivars, for its use as an additional parameter to exploit such a trait. Therefore, the present study was undertaken to identify M. incognita resistance in selected, locally adapted soybean cultivars by quantifying and exploiting the latter trait by using enzyme activities as an additional parameter. In addition, resistance to M. incognita in selected resistant soybean cultivars was also verified by means of histopathological studies to identify cellular changes associated with the trait. In the first part of the present study, 31 locally adapted soybean cultivars of which 23 were commercially available in the 2012 growing season were evaluated for resistance to M. incognita. The latter was done by means of traditional screening protocols for which M. incognita-gall rating, egg and second-stage juvenile as well as the reproductive factor data per root system for each cultivar screened were recorded. Two greenhouse experiments were subsequently conducted concurrently, one of which the abovementioned nematode parameters were recorded 30 and the other 56 days after inoculation. Reproduction factor values were used as the main criterium to identify M. incognita resistance in local soybean cultivars since it is considered as a more reliable parameter for this specific type of evaluations. Reproduction factor values equal to and lower than one, indicating resistance to the M. incognita population used in this study, were recorded only for cultivar LS5995, as well as seven pre-released GCI cultivars. These eight cultivars also had very low egg, as well as egg and second-stage juvenile counts per root system, all of which differed significantly from the susceptible control, as well as a number of other cultivars. Root gall indices, on the other hand, did not show consistent results in terms of the identification of the host status of the 31 cultivar screened during this study. Using reproduction factor values, local farmers can thus be supplied with information on the resistance of commercially-available soybean cultivars. Eventually, such M. incognita-resistant cultivars can be used to reduce population levels of this nematode pest in fields of producers and also as valuable germplasm sources in breeding programs to introgress/stack this trait in newly-developed soybean cultivars. The second part of the study aimed to verify and exploit M. incognita-resistance in soybean either identified as resistant or susceptible during the screenings experiments, using enzymatic activity as biochemical markers. Cultivar LS5995 was included as the resistant and Dundee as the susceptible standard. The activity of three enzymes, namely guaiacol peroxidase, lipoxygenase and catalase were recorded at different time intervals in roots and leaf samples of the latter cultivars, of both nematode-inoculated and nematode-free plants of each cultivar. Significant (P ≤ 0.05) increases in guaiacol peroxidase activity in leaf and root samples of the M. incognita-resistant cultivars GCI7 and LS5995 (inoculated with J2) were recorded 24 hours (h) after onset of the experiment. Use of this enzyme thus emanated as a useful parameter to identify soybean cultivars that exhibit resistance against M. incognita, especially in leaves, which could substantially reduce the time needed to screen cultivars. In terms of lipoxygenase activity recorded, substantial variation existed between the cultivars tested. The M. incognita-susceptible cultivar Egret was the only cultivar for which a significant (P ≤ 0.05) increase in lipoxygenase activity in the roots was evident 24 h after inoculation. However, during the 48 h sampling time, significant (P ≤ 0.05) differences in lipoxygenase activity were also recorded for the two M. incognita resistant cultivars GCI7 and LS5995. Although the increase in lipoxygenase activity for the susceptible cultivar Egret was unexpected, it may indicate that some level of resistance is present in the latter cultivar, which has in previous studies been identified as resistant to M. incognita. Other factors such as a different M. incognita populations used and temperature differences in greenhouse conditions that applied in this study compared to that for an earlier study may, however, serve as explanations for the latter differences in host status identification of cultivar Egret. In terms of catalase activity recorded in leaf samples of the M. incognita-resistant cultivar LS5995, substantial reductions of as much as 35.6 % were recorded for J2-inoculated plants compared to those of the J2-free control plants. In leaf samples of the susceptible cultivars, Egret and Dundee, catalase was also reduced, but to a lesser extent and ranged from 6 to 26 %. Conversely, catalase activity in the leaves of J2-inoculated plants of the highly susceptible cultivar LS6248R was substantially increased by as much as 29.3 %. Enzyme data obtained as a result of the current study thus generally complemented those of traditional screening assays in which resistance in locally adapted cultivars were identified to a certain degree. It is, however, recommended that enzyme activity, to be used as bio-markers, still needs further refinement and more investigation to optimise their use in identification, verification and exploitation of M. incognita resistance in soybean cultivars. The third and final part of the study encompassed a comparison of cellular changes induced by M. incognita in resistant and susceptible soybean cultivars to verify the resistant reactions expressed in the enzyme data. According to light- and transmission electron microscope observations, distinct differences in the appearance and development of giant cells in roots of the M. incognita-resistant cultivars LS5995 and GCI7 existed when compared to those in roots of the susceptible cultivars Dundee and LS6248R. In the latter cultivars, giant cells that formed were characteristically large and contained a dense cytoplasm, with thick irregularly surfaced cell walls. Cell walls also displayed thick aggregations that appeared to be cell-wall ingrowths. These giant cells are optimal to facilitate M. incognita development and reproduction. In contrast, giant cells that were associated with the resistant cultivars LS5995 and GCI7 were small, irregularly shaped and contained increased amounts of deposited cell-wall material in the cytoplasm known as cell wall inclusions. Necrosis was also present in M. incognita-infected root cells of both cultivars. Such giant cells have been associated with retarded feeding, development and reproduction of the latter root-knot nematode species. However, it was evident that neither GCI7 nor LS5995 are immune to M. incognita since J2 survived and developed to third- and fourth and ultimately mature females that reproduced in their roots. Optimal giant cells that were formed in the roots of the M. incognitasusceptible cultivars Dundee and LS6248R thus supported the nutritional needs of the developing M. incognita individuals and led to significant increases in M. incognita populations 56 days after inoculation as was evident from the high reproduction factor values that were obtained for such cultivars during host status assessments that represented the first part of this study. The opposite was recorded the M. incognita-resistant cultivars LS5995 and GCI7 since sub-optimal giant cells in their roots could not sustain high offspring from such mature females. The presence of necrotic root tissue adjacent to giant cells, furthermore, indicated that hypersensitive reactions occurred in the latter resistant cultivars. Enzyme data obtained in the second part of this study supported the presence of hypersensitive reactions in root cells of the latter resistant cultivars. Guaiacol peroxidase and lipoxygenase inductions in particular in plant tissues have been reported to play integral roles in hypersensitive reactions that are exhibited by cultivars that are resistant to pests and diseases. Finally, results obtained from the different parts of this study complemented each other. It resulted in the resistance that was identified in the GCI7 pre-released cultivar being verified and exploited against that of the resistant standard LS5995. Research that was done during this study also represented the first investigations into the use of enzymes as biochemical markers of resistance against M. incognita in soybean in South Africa. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Orientador: Leila Trevisan Braz / Coorientador: Pedro Luiz Martins Soares / Banca: João Ademir de Oliveira / Banca: Pablo Forlan Vargas / Resumo: Este estudo foi desenvolvido na Faculdade de Ciências Agrárias e Veterinárias (UNESP-FCAV), Câmpus de Jaboticabal- SP, com o objetivo de avaliar o modo de herança da resistência do melão 'Gaúcho Redondo' a Meloidogyne incognita. Para tanto, foi conduzido um experimento em delineamento em blocos casualizados, com três blocos e seis tratamentos, os quais envolveram as linhas parentais 'Gaúcho Redondo' (P1 resistente à M. incognita) e JAB 20 (P2 suscetível, resultado do programa de Melhoramento Genético da Cultura do Melão Rendilhado da UNESP-FCAV), assim como as gerações F1 (P1xP2), F2 (F1xF1), e retrocruzamentos (RC1P1 e RC1P2). Foram avaliadas plantas individuais após 70 dias da inoculação com o patógeno, por meio do fator de reprodução do nematoide (FR), calculado pela contagem do número de ovos e juvenis de segundo estádio, contido na suspensão de cada raiz processada, dividido pela população inicial. A hipótese de herança monogênica foi rejeitada ao nível de significância de 1% de probabilidade no teste do qui-quadrado (χ2), indicando que a resistência está sob controle de mais de um lócus gênico, o que foi confirmado pela análise quantitativa, que evidenciou a existência de seis genes envolvidos na herança da resistência ao nematoide. Houve predominância dos efeitos aditivos no controle da característica em estudo, o que permite resposta aos processos de seleção / Abstract: This study was conducted at the College of Agriculture and Veterinary Sciences (UNESP-FCAV) in Jaboticabal-SP, in order to evaluate the mode of inheritance of resistance of melon 'Gaucho Round' to Meloidogyne incognita. Therefore, an experiment was conducted in randomized block design with three blocks and six treatments, which involved the parental lines 'Gaucho Round' (P1 resistant to M. incognita) and JAB 20 (P2 susceptible result of Breeding Program Culture Melon Tracery UNESP-FCAV), as well as the F1 (P1xP2), F2 (F1xF1) and backcross (RC1P1 and RC1P2). Individual plants were evaluated 70 days after inoculation with the pathogen through the nematode reproduction factor (FR), calculated by counting the number of eggs and second stage juveniles contained in the suspension of each root processed, divided by the initial population . The hypothesis of monogenic inheritance was rejected at a significance level of 1% probability in the chi-square (χ2), indicating that resistance is under the control of more than one gene locus, which was confirmed by quantitative analysis, which showed the existence of six genes involved in inheritance of resistance to nematodes. Predominance of additive effects in controlling the trait under study, allowing response to selection processes / Mestre

O gênero Eucalyptus possui diversas espécies que apresentam grande valor comercial, sendo utilizado principalmente para produção de energia (carvão e lenha) e celulose-papel. Na literatura, entretanto, existem poucas informações acerca de nematoses nessa cultura. Além dessa carência e de sua importância econômica, o eucalipto é comumente cultivado em locais de alta incidência de Pratylenchus brachyurus e Meloidogyne incognita, além de ser consorciado com diversas outras culturas intolerantes, como por exemplo a soja. Dessa forma há uma necessidade de conhecer a reação das espécies de eucalipto a estes nematoides. Dito isso, o presente trabalho teve por objetivo testar a reação de algumas espécies de eucalipto a P. brachyurus e M.incognita. Foram realizados 6 experimentos, 5 envolvendo o nematoide das lesões e 1 com o nematoide de galhas. Os tratamentos foram inoculados com uma população inicial de nematoides e, após 90 dias, estimava-se o fator de reprodução (FR) e nematoides por grama de raiz (Nem/g). Adotou-se o critério baseado em Oostenbrink (1966), onde a espécie foi considerada suscetível quando apresenta FR >= 1 e resistente quando FR < 1. Dos eucaliptos testados, 6 apresentaram reação suscetível e 4 reação resistente para P. brachyurus. As espécies com maiores FR estão entre as mais utilizadas no Brasil, exceto E. saligna. Ressalta-se que estas não necessariamente são resistentes, necessitando apenas de um período maior para o estabelecimento do patógeno. Observou-se indícios de que E. saligna e E. camaldulensis sejam intolerantes a P.brachyurus. Para M. incognita raça 3 todas as espécies testadas foram resistentes. Aconselha-se a realização de experimentos com períodos superiores a 100 dias para uma correta classificação da reação de hospedeiro. O eucalipto é uma opção para aproveitamento de áreas infestadas com M. incognita raça 3, porém seu uso mostra-se um risco em áreas infestadas com P. brachyurus. / The Eucalyptus has several species with high commercial value and is used mainly for energy purpose (Charcoal and firewood) and paper. In the literature, however, there is little information about plant parasitic nematodes in this woody specie. In addition to this lack and its economic importance, eucalypts is also commonly cultivated in areas with high incidence of Pratylenchus brachyurus and Meloidogyne incognita, and consorted with several intolerant crops, such as soybean. Thus, there is a need to know the host status of the eucalypts species to these nematodes. With that, this work aimed to test the reaction of some Eucalyptus species to P.brachyurus and M. incognita. For this purpose six experiments were performed, five to test the lesion nematode and one to test the root-knot nematode. The treatments were inoculated and, after 90 days, it was estimated the reproduction factor (FR) and nematodes per gram of root (Nem/g). The present work adopted the criteria based on Oostenbrink (1966), where the species was considered susceptible when presenting FR >= 1 and resistant when FR < 1. Of species tested, six were susceptible hosts and four resistant host to P. brachyurus. The species with highest FR are among the most used eucalypts in Brazil, except E. saligna. It is emphasized that those species with FR < 1 are not necessarily resistant, requiring only a longer period for the pathogen establishment. There was some evidence that E. saligna and E. camaldulensis are intolerant hosts to P. brachyurus. To M. incognita Race 3 all tested species were resistant. It is advisable to perform experiments for periods longer than 100 days to a correct classification of host status. Eucalyptus is an option for utilization of areas infested with M. incognita race 3, but its use appears to be a risk in Pratylenchus brachyurus infested areas.

Orientador: Silvia Renata Siciliano Wilcken / Banca: Antonio Carlos Maringoni / Banca: Mario Massayuki Inomoto / Banca: Roberto Kazuhiro Kubo / Banca: Claudio Marcelo Gonçalves de Oliveira / Resumo: A bananeira (Musa spp.) é uma planta herbácia e sua fruta é uma das mais consumidas no mundo, principalmente nos países tropicais. Apesar da alta produtividade, o Brasil tem pequena participação no mercado internacional, devido ao elevado consumo interno e pela baixa qualidade dos frutos, que se deve a vários fatores como: genética da cultivar, tipo de solo, manejos agronômicos e sanitários. Dentre os problemas fitossanitários destacam-se os nematóides. Várias espécies de nematóides representam problemas para esta cultura. Radopholus similis, Meloidogyne spp., Pratylenchus coffeae, Helicotylenchus multicinctus e Rotylenchulus reniformis estão amplamente distribuídos nas principais regiões produtoras, causando perdas expressivas à bananicultura. Na presente pesquisa visou-se estudar a patogenicidade de M. incognita raça 2 e M. javanica em bananeira ‘Prata An㒠em substratos com diferentes fertilidades, utilizando vários níveis de população inicial de M. incognita raça 2 ou M. javanica (0, 2.000, 10.000 e 50.000 nematóides) por planta. Para isso, foram conduzidos dois experimentos em delineamento inteiramente casualizado. Cada parcela foi constituída de uma planta por vaso de 10L de capacidade, no experimento com M. incognita raça 2 e, vasos de 5L, para M. javanica. Os substratos utilizados em ambos experimentos foram: Substrato 1: contendo uma mistura de areia-solo-esterco na proporção 1:1:1 com textura arenosa e pH 7,0; substrato 2: (padrão) com textura média, com pH 5,6, sem adição de NPK; substrato 3: substrato 2 com pH ajustado para 6,4; substrato 4: substrato 3 com adição de NPK e substrato 5: substrato 2 com adição de NPK. A inoculação foi realizada uma semana após o transplantio das mudas. A avaliação final foi efetuada aos 135 dias da inoculação, quando foram determinados a altura (HP) e diâmetro do pseudocaule (DP), 2 número de... (Resumo completo, clicar acesso eletrônio abaixo) / Abstract: The banana (Musa spp.) is a herbal plant and its fruit is one of most consumed in the world mainly in tropical countries, including Brazil. Banana crops are affected by many phytosanitary problems, caused by phytopathogenic fungi, insect pests and nematodes. Radopholus similis, Meloidogyne spp., Pratylenchus coffeae, Helicotylenchus, multicinctus and Rotylenchulus reniformis are widely distributed in the main producer regions causing expressive economic losses in bananas production. This work aimed to study the reaction of banana cv. Prata Anã to Meloidogyne incognita and M. javanica in soils with different fertilities. Artificial infestation was accomplished using initial different population levels (0, 2,000, 10,000 and 50,000 nematodes / plant) of M. incognita or M. javanica. For this, two experiments were carried out in a totally random design. Each plot was constituted of one plant / pot. The soils used in both experiments were: Soil 1: with a mix of sand-soilmanure (1:1:1), sandy texture and pH 7,0; soil 2: (standard), medium texture, pH 5,6 without NPK fertilizer; soil 3: soil 2 with pH fitted to 6,4; soil 4: soil 3 with NPK fertilizer and soil 5: soil 2 with NPK fertilizer. The inoculation was proceded one week after plants set up. The final evaluation was made at 135 days after inoculation, when the height plant (HP) and pseudosterm diameter (DP), leaves number (NF), nematodes number for root gram (NºN/gR), soil and root total nematode number (NTSR), reproductive factor (FR), root fresh weight (PFR) and dry shoot (PSA) were determined. However, with the purpose of determine the best evaluation time, the parameters plant height, pseudostem leaves number and pseudostem diameter were evaluated also at 27, 56, 89 and 119 days after the inoculation. There were not verified significant interactions between M. incognita population initial levels and soil fertility for the parameters... (Complete abstract, click electronic access below) / Doutor

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-14T12:33:28Z No. of bitstreams: 1 Jeane Emili de Medeiros.pdf: 394630 bytes, checksum: 1eb59fc3e8a4ae913b395a21c71fbe51 (MD5) / Made available in DSpace on 2017-03-14T12:33:28Z (GMT). No. of bitstreams: 1 Jeane Emili de Medeiros.pdf: 394630 bytes, checksum: 1eb59fc3e8a4ae913b395a21c71fbe51 (MD5) Previous issue date: 2007-02-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The objective of this work was to isolate and select bacteria for the control of Meloidogyne incognita and to evaluate the isozymatic activity of melon plants parasitized. Sixty-one rhizobacterium isolates obtained from rhizosphere soil and 56 endophytic obtained from the Culture Collection of Plant Bacteriology Laboratory - Federal Rural University of Pernambuco were tested. Melon seedlings yellow type 10 days old had their soil infested with 1000 eggs of Meloidogyne incognita race 2. Two days before soil infestation 20 mL of bacterial suspension (0,7 OD) were deposited in each pot. After 60 days fresh biomass of shoot and root , gall index, egg mass index and nematode reproduction factor were determined. Among 117 isolates the endophytic ENM7, ENM10 and ENM51 were selected because they significantly reduced egg mass index and/or gall index. However when tested again, separately or in mixtures, these isolates did not maintain their efficiency and besides they did not affect egg hatching in vitro. In order to study the activity of the isozymes α and β-esterase, peroxidase, acid phosphatase and malate dehydrogenase, plants of melon with 10 days old, had their soil infested with 500 nematode eggs per plant, and maintained in greenhouse. At 2, 4, 6, 8, 16, 24 e 32 days after soil infestation the third leaf of each plant was collected and processed for electrophoresis of isozymes in polyacrilamide gel. Only β-esterase and malate dehydrogenase showed polymorphism between bands expressed by plants parasitized by nematodes when compared to the control plants. The nematode inhibited the expression of some β-esterase genes and activated the expression of other malate dehydrogenase genes. / O objetivo dessa pesquisa foi isolar e selecionar bactérias para o controle da meloidoginose e avaliar a atividade isoenzimática de plantas de meloeiro parasitadas por Meloidogyne incognita raça 2. A partir de solo rizosférico de meloeiro foram obtidos 61 isolados de rizobactérias que juntamente com 56 isolados endofíticos pertencentes à Coleção de Culturas do Laboratório de Fitobacteriologia da UFRPE foram testados. Plântulas de meloeiro Amarelo com 10 dias tiveram o solo infestado com 1000 ovos de M. incognita raça 2. Dois dias antes da infestação do solo foram depositados 20 mL da suspensão bacteriana (0,7 A) por vaso. Decorridos 60 dias, foram determinadas as biomassas frescas da parte aérea e das raízes, os índices de galhas e de massa de ovos e o fator de reprodução do nematóide. Dos 117 isolados testados, foram selecionados inicialmente os endofíticos ENM7, ENM10 e ENM51 que reduziram significativamente o índice de massa de ovos e/ou o índice de galhas. Contudo, quando testados novamente, separadamente ou em misturas, esses isolados não mantiveram a eficiência na redução dessas variáveis e in vitro também não afetaram a eclosão dos juvenis. Para o estudo da atividade das isoenzimas α e β-esterase, peroxidase, fosfatase ácida e malato desidrogenase, plantas de meloeiro com dez dias de idade, tiveram o solo infestado com 500 ovos do nematóide por planta, sendo mantidas em casa de vegetação. Aos 2, 4, 6, 8, 16, 24 e 32 dias após a infestação do solo a terceira folha das plantas foi coletada e submetida à eletroforese de isoenzimas em gel de poliacrilamida. Apenas a β-esterase e a malato desidrogenase mostraram polimorfismo entre as bandas expressas pelas amostras das plantas parasitadas por nematóides, quando comparadas às plantas testemunhas. A presença do nematóide inibiu a expressão de alguns genes β-esterase e ativou a expressão de outros da malato desidrogenase.

Thesis (M.Agric. (Plant Production)) -- University of Limpopo, 2018 / Meloidogyne incognita race 2 is internationally recognised as one of the most aggressive Meloidogyne species and it is also widely distributed in Limpopo Province, where it occurs alone or as mixed populations with other Meloidogyne species. Traditionally, Meloidogyne species had been managed using synthetic chemical nematicides, most of these products had been withdrawn from agro-chemical markets due to their environment-unfriendliness. Following the withdrawal of synthetic chemical nematicides, nematode resistance had been the most preferred strategy for managing high nematode population densities. The availability of nematode resistant genotypes in sweet potato (Ipomoea batatas) would enhance the use of resistance in managing Meloidogyne species and races in Limpopo Province. Generally, should post-infectional nematode resistance be available in the test sweet potato cultivars, the information would be relayed to plant breeders for use as source of introgression in various commercial cultivars where nematode-resistant genotypes do not exist. The objectives of the study, were to determine: (1) Host-status and host-sensitivity in sweet potato cv. ʹBopheloʹ, ʹBosbokʹ and ʹMvuvheloʹ to M. incognita race 2. (2) the existing nematode resistance mechanism in any of the test cultivars that had resistance to M. incognita race 2. For achieving Objective 1, eight treatments namely, 0, 25, 50, 125, 250, 625, 1250 and 3125 eggs and second stage-juveniles (J2) M. incognita race 2 were used under greenhouse trials for each cultivar. To achieve Objective 2, sweet potato plants were inoculated with 100 J2 with four plants harvested every other day for 30 days counting to 15 harvesting times. At 56 days after inoculation, cv. ʹBopheloʹ had reproductive factor (RF) values above unity for M. incognita race 2 and plant growth variables were reduced. Therefore, the cultivar was a susceptible host to M. incognita race 2 and mechanism trial was not conducted for this cultivar. Meloidogyne incognita race 2 failed to reproduce on cultivars ʹBosbokʹ and ʹMvuvheloʹ whereas nematode infection did not affect plant growth and therefore, the two cultivars were resistant to M. incognita race 2. Mechanisms of resistance to M. incognita race 2 on cultivars ʹBosbokʹ and ʹMvuvheloʹ demonstrated significance existence of (1) necrotic spots, (2) poorly developed giant cells, (3) formation of rootlet interferences (4) absence of root galls and (5) non-detectable J2 in roots. All these features suggested the existence of post-infectional nematode resistance in the two cultivars to M. incognita race 2. In conclusion, cultivar ʹBopheloʹ was susceptible to M. incognita race 2, whereas cultivars ʹBosbokʹ and ʹMvuvheloʹ were resistant to M. incognita race 2, with the evidence of post-infectional nematode resistance to the nematode species

Abamectin is a blend of B1a and B1b avermectins that is being used as a seed treatment to control plant-parasitic nematodes on cotton. Data on the toxicity of abamectin and its effectiveness as a seed treatment to control Meloidogyne incognita or Rotylenchulus reniformis on cotton are lacking. The toxicity of abamectin was based on an assay of nematode mobility, LD50 values of 1.56 µg/ml and 32.9 µg/ml were calculated based on 2 hr exposure for M. incognita and R. reniformis, respectively. There was no recovery of either nematode after exposure for 1 hr to its LD50 concentration. Sublethal concentrations greater than 0.39 µg/ml for M. incognita and 8.2 µg/ml for R. reniformis reduced (P = 0.05) infectivity on tomato. In field trials, suppression (P = 0.05) of M. incognita was observed 32 DAP by abamectin seed treatment whereas no suppression of R. reniformis was observed. No suppression of M. incognita was perceived by abamectin seed treatment in microplots. Suppression of M. incognita was observed in microplots by harpinEA and harpingαβ as a seed treatment and foliar spray, respectively. Seed cotton yields were variable for abamectin-treated seed, but numerically positive for harpin-treated cotton. Initial gall formation on developing taproots was suppressed (P = 0.001), and penetration of 5-cm long taproots by M. incognita and R. reniformis was numerically suppressed by abamectin-treated compared to non-treated seed, but infection increased with root development. Using an assay of nematode mobility, the proportion of dead second-stage juveniles (J2) was higher (P = 0.05) following exposure to an excised radicle from abamectin-treated seed than non-treated seed, but lower (P = 0.05) than J2 exposed to the abamectin-treated seed coat. Thus a higher concentration of abamectin remained on the seed coat than emerging radicle. The concentration of abamectin transferred from the seed coat to the developing roots was limited, which contributed to the variability in suppression of plant-parasitic nematodes on cotton.