Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Evaluation of phenotypic stability of resistance to Phytophthora spp. in cacao clones

Resistance of fourteen Theobroma cacao clones to Phytophthora spp. was evaluated using stem inoculations on grafted seedlings. Concepts of phenotypic stability were used to interpret the results and to express horizontality of the resistance. The linear regression coefficient 'b', the determination coefficient (R²) and average lesion size were used to determine the level of horizontal resistance, the phenotypic stability and the predictability of all clones. The results indicated that clones P 7 and MA 15 present highest levels of horizontal resistance and stability, but with moderate predictability. Clones CAS 1 and CEPEC 13 were classified as those with high horizontal resistance, stability and predictability, while clones PA 30, UF 650 and SIAL 88 and EET 59 showed intermediate resistance and stability and high predictability. Clones SPA 17, OC 61, PA 150, SIAL 505, ICS 1 and R 41 presented high susceptibility and intermediate or low stability and moderate or high predictability.

Efeito da cianogênese na incompatibilidade entre clones de copa de seringueira e o clone de painel IPA 1

O objetivo deste trabalho foi verificar o envolvimento dos glicosídeos cianogênicos na incompatibilidade, por translocação, do clone de seringueira IPA 1 com copas enxertadas com clones de outras espécies de Hevea. Determinou-se, na casca dos clones de painel IPA 1, Fx 4098 e Fx 3864, a atividade das enzimas beta-glicosidase, beta-diglicosidase e beta-cianoalaninassintase (beta-CAS) e o desenvolvimento de combinações de enxertos, tendo como clone de painel o IPA 1 e como copa enxertada, clones de baixo ou alto potencial cianogênico (HCNp). Dosou-se o HCN-p de folhas jovens e maduras de clones de copa já conhecidos como compatíveis ou incompatíveis. A atividade de beta-CAS no IPA 1 foi baixa, sendo alta no Fx 3864 e intermediária no Fx 4098. Esse fato, associado à maior atividade da beta-glicosidase e beta-diglicosidase encontradas no IPA 1, confere a este clone extrema sensibilidade ao cianeto. Os enxertos com clones de baixo HCN-p nas folhas jovens mostraram-se compatíveis, bem como com os clones em que o alto HCN-p nas folhas jovens permanece alto nas folhas maduras. Os clones incompatíveis apresentaram alto HCN-p nas folhas jovens e significativa redução nas maduras. Essas evidências reforçam a hipótese de a incompatibilidade ser causada pela translocação de linustatina, das folhas dos clones enxertados, para o caule do IPA 1. Os sintomas se assemelham aos de secamento do painel de sangria, indicando o envolvimento da cianogênese na sua evolução.