Detección y caracterización de bacterias endofíticas en árboles de cafeto (Coffea arabica L.) y de sombra en Puerto Rico

Abstract

Among bacterial diseases affecting coffee (Coffea arabica L.), coffee leaf scorch (CLS) caused by the bacterium Xylella fastidiosa (Xf) Wells et al., limits production in many countries. The importance of endophytic bacteria is related to potential antagonistic or synergistic interactions with Xf. The objective of this research was to characterize bacterial endophytes of coffee and shade trees in Puerto Rico (PR). Three types of shade treatments were studied: coffee shaded with trees of high species diversity, coffee shaded with trees of low species diversity and sun coffee, in four localities in the dry and rainy seasons during years 2010-2012. In this study ANOVA in a complete randomized design was used to determine the effect of the factors. Endophytic bacteria were isolated on Periwinkle medium (semi selective to Xf) and characterized phenotypically. Endophytic Gram negative bacteria and tree samples with CLS disease symptons, were tested with a double antibody sandwich enzyme linked immune absorbent assay (DAS-ELISA for Xf). A subset of bacteria and tree samples suspected to have CLS and bacteria were analyzed with polymerase chain reaction (PCR) with Xf primers 272 -1-2, COX Xf and Xf-16S rRNA. Cluster analysis was carried out using Gower method for fastidious bacteria and spatial cluster analysis using SatScan for symptomatic trees. Few bacterial strains were of fastidious type. There were no significant differences in the percentage of fastidious bacteria, in the number of colony forming units (CFU/ml) and bacterial diversity per tree in symptomatic versus asymptomatic trees. CFU/ml and bacterial diversity per tree were higher in the rainy season compared to the dry season (α=0.05). The effects of location and shade treatment were more variable, with a tendency of lower diversity in sun versus shaded coffee. Spatial clustering showed more than 14 times greater relative risk of encountering diseased coffee trees at higher altitudes such as Adjuntas, Jayuya and Yauco (p<0.001). Some DNA products were amplified with PCR (272 - 1-2); but they were not related to Xf, neither with COX Xf primers. DNA sequencing was negative for Xf. Ambiguous results in DNA amplification and sequencing do not allow us to confirm that these samples are Xf. Therefore, the causal agent of the disease observed in PR remains to be identified according to Koch’s postulates.