First Report of Pantoea ananatis Causing Necrotic Symptoms in Mango Trees in the Canary Islands, Spain

In a recent study, a bacterial necrotic disease of mango trees (Mangifera indica L.) elicited by pathogenic strains of Pantoea agglomerans was reported in the Canary Islands (Gutiérrez-Barranquero et al. 2019). Interestingly, in that study five bacterial isolates belonging to a different species of the Pantoea genus were also isolated from necrotic symptoms. These bacterial isolates were identified as Pantoea ananatis based on biochemical and physiological tests (including Gram staining, glucose metabolism, growth on yeast dextrose carbonate agar medium, production of fluorescent pigments and levan, oxidase reaction, arginine dihydrolase, and tobacco hypersensitive response). In this study, partial nucleotide sequences of gyrB and rpoB housekeeping genes were obtained as described previously (Delétoile et al. 2009; Gutiérrez-Barranquero et al. 2019) because they have shown a satisfactory resolution to identify and phylogenetically classify different Pantoea species. This approach included the five P. ananatis strains isolated from mango (Pan UMAF3070, Pan UMAF3071, Pan UMAF3072, Pan UMAF3073, and Pan UMAF3074), P. ananatis CFBP3612, P. ananatis PA13 as reference from the database, Pantoea stewartii subsp. indologenes CFBP3614, four Pantoea agglomerans strains (Pa UMAF3067, Pab 4188, Pag 824-1, and Pa CECT850), and Pseudomonas syringae pv. syringae (Pss) UMAF0158 as an external outgroup. The nucleotide sequences of gyrB and rpoB genes and the phylogenetic tree generated with them confirmed that these five bacterial isolates belonged to P. ananatis species (GenBank accession numbers included in the supplementary material). P. ananatis has been described as a pathogenic bacterium that causes disease in a wide range of host plants (Weller-Stuart et al. 2017) and particularly causes center rot of onion, a devastating disease first described in Georgia, U.S.A., in 1997 (Gitaitis and Gay 1997). Thus, to decipher if P. ananatis strains isolated from mango could be involved in necrotic symptom development, an initial pathogenicity test using onion bulbs was performed following the protocol recently described (Gutiérrez-Barranquero et al. 2019), because it was an easier and faster approach than mango pathogenicity experiments. This experiment was repeated twice including Pan UMAF3071 and Pan UMAF3074 mango isolates, Pan CFBP3612 as a positive control, and phosphate buffer as a negative control. Onion bulbs inoculated with the control strain developed typical brown necrotic symptoms of center rot disease, whereas Pan UMAF3071 and Pan UMAF3074 showed weaker brown necrotic symptoms. To fulfill Koch’s postulates on mango trees, a pathogenicity test performed on 2-year-old mango plants was conducted as previously described (Cazorla et al. 1998; Gutiérrez-Barranquero et al. 2019), by using the same P. ananatis strains mentioned above. Pss UMAF0158 (the reference strain of Pss that causes bacterial apical necrosis [BAN] of mango trees) and phosphate buffer were included as positive and negative controls, respectively. Seven randomly selected apical buds were inoculated by each strain and the negative control. The Pss control strain displayed typical necrotic BAN symptoms 1 month after the inoculation. Pan UMAF3071 and Pan UMAF3074 showed the first necrotic symptoms 2.5 months after the inoculation and at the end of the experiment reached a disease incidence higher than 50%. P. ananatis control strain CFBP3612 also showed necrotic symptoms on mango buds. In both pathogenicity experiments, bacterial isolation and identification were conducted from symptomatic onion bulbs and apical buds of mango trees, confirming that P. ananatis strains isolated from mango were the causal agent of necrotic symptoms observed in those assays, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of P. ananatis causing necrotic symptoms in mango trees.