The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

Ocorrência de Meloidogyne enterolobii Yang & Eisenback, no município de Uberlândia, Minas Gerais, Brasil

Meloidogyne enterolobii Yang & Eisenback (Sin.: M. mayaguensis Rammah & Hirschmann), poliphagous plant parasitic nematode, has been reported causing hard damage in several plant species in Brazil. This communication represents the first occurrence of this nematode in the municipality of Uberlandia, Minas Gerais State, Brazil.

Reaction of sugarcane genotypes to Pratylenchus brachyurus and P. zeae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Reprodução de Meloidogyne javanica em olerícolas e em plantas utilizadas na adubação verde

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo Comparativo da Biologia de Meloidogyne enterolobii (= M. mayaguensis) e Meloidogyne javanica em tomateiros com gene Mi

Visando conhecer a relação parasito hospedeiro de M. enterolobii em plantas com resistência a nematoides das galhas, estudos comparativos da biologia de M. enterolobii e M. javanica em tomateiros com o gene Mi foram conduzidos. O experimento foi conduzido em esquema fatorial 2x2, composto de dois porta-enxertos de tomateiro ('Magnet' e 'Helper M') e duas espécies de nematoides das galhas (M. enterolobii e M. javanica) com cinco repetições. As plantas foram inoculadas com 500 juvenis infectantes (J2) de M. enterolobii ou M. javanica. As raízes foram coletadas aos 3, 10, 17, 24 e 31 dias após a inoculação, coloridas com fucsina ácida, e dissecadas sob microscópio estereoscópico para a localização e contagem dos diferentes estádios de desenvolvimento dos nematoides. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Tukey a 5% de probabilidade. Os resultados obtidos mostraram que, embora ambas as espécies tenham sido capazes de penetrar as raízes dos porta-enxertos de tomateiro, somente M. enterolobii conseguiu desenvolver-se normalmente, com as fêmeas maduras realizando suas posturas, a partir de 24 dias após a inoculação.

Reação de porta-enxertos de videira a Pratylenchus brachyurus e Pratylenchus zeae

Nesse trabalho, o objetivo foi avaliar a reação de oito porta-enxertos de videira aos nematoides das lesões radiculares. As estacas dos porta-enxertos Kober, SO4, 101-14, R99, 420-A, Rupestris du Lot, Riparia do Traviú e Telek 5C, cedidas pelo Centro APTA de Frutas/IAC, foram plantadas em vasos contendo mistura de solo:areia na proporção 2:1 (v:v) e mantidas em casa de vegetação. Após quatro meses, os porta-enxertos foram inoculados com 1.200 espécimes de Pratylenchus brachyurus ou P. zeae, e o milho foi usado para comprovar a viabilidade do inóculo. Aos 180 dias após a inoculação, avaliou-se o número de nematoides por sistema radicular e em 100 cm³ de solo. O milho foi avaliado aos 90 dias após a inoculação e cada vaso recebeu uma nova planta, que foi avaliada juntamente com os porta-enxertos. No milho, as populações finais de P. brachyurus e P. zeae foram respectivamente iguais a 8.040 e 6.940 indivíduos. Todos os porta-enxertos comportaram-se como imunes a P. brachyurus e P. zeae, isto é, a população final dos nematoides e o fator de reprodução foram iguais a zero. Recuperaram-se seis e 11 espécimes de P. zeae nas amostras de solo cultivadas com os porta-enxertos Kober e 420-A, respectivamente. Conclui-se que os porta-enxertos estudados apresentam potencial para serem usados em áreas infestadas com esses nematoides das lesões.

Agentes microbianos no controle de nematóides e fungos fitopatogênicos de soja e sua compatibilidade com agroquímicos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Agressividade comparada de Pratylenchus brachyurus com P. zeae e eficácia de métodos de controle de nematoides em cana-de-açúcar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Seleção de porta-enxertos resistentes a Didymella bryoniae, Meloidogyne incognita e M. javanica e efeitos no desempenho do meloeiro rendilhado

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Eficiência do controle de nematóides, ferrugem e bicho mineiro em cafeeiros

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

Patogenicidade de Meloidogyne incognita e Meloidogyne javanica a bananeira cv. Prata Anã em diferentes substratos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Espécies de Meloidogyne Goeldi em cafeeiro no município de Araguari-MG

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Reação de genótipos e híbridos de tomateiro à Meloidogyne enterolobii

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reação de maracujazeiro amarelo a Meloidogyne Incognita Raça 3

This study concerned the reaction of yellow passion fruit ‘Maguary’ and ‘Afruvec’ to the phytonematode Meloidogyne incognita race 3 in greenhouse conditions. An entirely randomized experimental design with 3 treatments (‘Maguary’, ‘Afruvec’, and tomato cv. ‘Rutgers’) and 4 repetitions was used, each plot consisting of 1 vase containing 1 plant. After 6 months, an evaluation was made of the index of galls and egg mass in the yellow passion fruit varieties and in the tomato cv. ‘Rutgers’. The classification of resistance to the phytonematode was made by criterion of the reproduction factor (RF). ‘Maguary’ presented a zero index of galls and egg mass, while ‘Afruvec’ showed a low index of galls and egg mass in relation to the tomato cv. Rutgers. According to the RF, ‘Maguary’ was characterized as immune to the phytonematode, while ‘Afruvec’ was resistant, and the tomato cv. ‘Rutgers’ was susceptible.

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V, hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.