Field management of Rotylenchulus reniformis on pineapple [Ananas comosus (L.) Merr] in eco-friendly cropping system including rotation with sunn hemp (Crotalaria juncea) and systemic resistances induced by chemical elicitors or endophytic bacteria. Pineapple Endophytes

A collection of 51 bacterial endophyte strains were isolated from the three pineapple cultivars, Victoria (Queen), MD-2 and Smooth Cayenne, in Martinique. The isolates were identified by phylogenetic analysis of partial 16S rRNA gene sequences according the following protocol. Genomic DNA from individual isolates was obtained by suspending a colony in 100 μl sterile water heating at 95 °C for 5 min and chilled on ice. Crude DNA extracts were stored at -20° C prior to polymerase chain reaction (PCR) amplification. PCR amplification were performed using the primers FGPS1509 (5’-AAGGAGGGGATCCAGCCGCA-3’) and FGPS6 (5’-GGAGAGTTAGATCTTGGCTCAG-3’) as follows (25 µl reaction): 1X Green GoTaq® Reaction Buffer (Promega, Charbonnieres, France), 0.6 units of GoTaq® DNA Polymerase (Promega), 0.4 µM of each primer, 1 µl crude DNA extract, 160 µM of each dNTP, with the following cycling conditions: 94°C for 3min; 30 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 90 s; a final elongation step at 72°C for 10 min. PCR products were then sequenced using the primer 16S-1080r (5’-GGGACTTAACCCAACATCT-3’) (Genoscreen, Lille, France).Phylogenetic analysis of 16S rRNA sequences obtained from the isolates was performed in the MEGA X software package (Kumar et al. 2018), using the multiple alignment software MUSCLE, the maximum likelihood method, and bayesian estimation of the best-fitting model of molecular evolution. Nodal robustness of the tree was assessed by bootstrapping (500 replicates). The strain GVS2 affiliated to Bacillus cereus group was pre-selected for its ISR capacities against Rotylenchulus reniformis on pineapple vitroplants in controlled conditions, and was used for greenhouse and field experiments of SR induction on pineapple cv MD-2 and Victoria (Queen).