Comparison of whole-tissue maceration and sap extraction for isolation of Candidatus Liberibacter asiaticus

Abstract

Candidatus Liberibacter spp.' are fastidious alphaproteobacteria associated with Huanglongbing (HLB), a highly destructive disease for citrus production around the world. So far, no axenic culture of these bacteria have been established, limiting its study. However, the first step to cultivate plant pathogenic bacteria is to obtain viable cells. So, the aim of this study was to compare two methods to extract viable cells of 'Ca. L. asiaticus' from diseased plant tissue of infected citrus, periwinkle and dodder by using the PMA-qPCR technique. We applied two methods for extraction of bacterial cells, the whole-tissue maceration procedure for citrus, periwinkle and dodder and the sap extraction method by using the Scholander pressure pump for citrus and periwinkle. The extraction of 'Ca. L. asiaticus' cells by tissue maceration method provided a larger number of viable and non-viable bacterial cells when compared to the Scholander pressure pump method in both, citrus and periwinkle. The largest amount of bacteria extracted by the Scholander pressure pump was in periwinkle plants. However, the amount of cells obtained from dodder plants using maceration was higher than those obtained from the other plants by the same method, pointing out that dodder could be an excellent inoculum source for 'Ca. L. asiaticus' isolation tests. In this study, the two methods provided sufficient amounts of 'Ca. L. asiaticus' to perform bacterial isolation tests, and may be used in further studies on HLB bacteria and its disease. Index terms: propidium monoazide, greening, extraction method, pressure bomb.Comparação da maceração de tecido inteiro e da extração de seiva para isolamento de 'Candidatus Liberibacter asiaticus' RESUMO 'Candidatus Liberibacter spp.' são alfaproteobactérias fastidiosas associadas ao huanglongbing (HLB), uma doença destrutiva para a citricultura mundial. Até o momento, não foi possível o estabelecimento de culturas axênicas dessas bactérias, o que limita seu estudo. Entretanto, o primeiro passo para o cultivo de bactérias fitopatogênicas é a obtenção de células viáveis. Assim, o objetivo desse estudo foi comparar dois métodos de extração de células viáveis de 'Ca. L. asiaticus' de plantas de citros, vinca e cuscuta infectadas utilizando a técnica de PMA-qPCR. Os métodos utilizados foram por bomba de pressão de Scholander para extração da seiva das plantas de citros e de vinca, e