Diversity of pathogenic Pseudomonas isolated from citrus in Tunisia

Abstract

The damages observed in Tunisian citrus orchards have prompted studies on the Pseudomonas spp. responsible for blast and black pit. Prospective orchards between 2015 and 2017 showed that the diseases rapidly spread geographically and to new cultivars. A screening of Pseudomonas spp. isolated from symptomatic trees revealed their wide diversity according to phylogenetic analysis of their housekeeping rpoD and cts genes. The majority of strains were affiliated to Pseudomonas syringae pv. syringae (Phylogroup PG02b), previously described in Tunisia. However, they exhibited various BOX-PCR fingerprints and were not clonal. This work demonstrated, for the first time in Tunisia, the involvement of Pseudomonas cerasi (PG02a) and Pseudomonas congelans (PG02c). The latter did not show significant pathogenicity on citrus, but was pathogenic on cantaloupe and active for ice nucleation that could play a role in the disease. A comparative phylogenetic study of citrus pathogens from Iran, Montenegro and Tunisia revealed that P. syringae (PG02b) strains are closely related but again not clonal. Interestingly P. cerasi (PG02a) was isolated in two countries and seems to outspread. However, its role in the diseases is not fully understood and it should be monitored in future studies. The diversity of pathogenic Pseudomonas spp. and the extension of the diseases highlight that they have become complex and synergistic. It opens questions about which factors favor diseases and how to fight against them efficiently and with sustainable means.

Keywords: Citrus; Cultivars; Pathogenicity; Pseudomonas syringae; rpoD.

Fig.1

Geographical localization and distribution of symptoms in Tunisian governates sampled. No pathogen detection: Presence of symptoms but no isolation of P. syringae

Fig.2

Aspect of symptoms on different plant organs and in field a A tree infected with blast, leaf dryness b Beginning of blast in petiole c Black pit: necrotic spots occasionally surrounded by a chlorotic halo on lemons and oranges d leaves dry and roll, while still firmly attached to the tree, before eventually dropping without petioles e Lesions and black areas on twigs

Fig.3

Phylogenetic tree of isolated strains and 203 Pseudomonas species type strains, based on the partial rpoD sequence analysis. Distance matrices were calculated by the Jukes-Cantor method (Jukes and Cantor 1969). Dendrograms were generated by the neighbour-joining method. The Cellvibrio japonicus Ueda107 rpoD sequence was used as the outgroup. The bar indicates sequence divergence. Percentage bootstrap values of more than 50% (from 1000 replicates) are indicated at the nodes. GenBank accession numbers are given in parentheses. Colors indicate the sampling locations

Fig.4

Phylogenetic tree of representative strains of the P. syringae group isolated in this study from citrus plants with symptoms. Concatenated 16S rRNA, gyrB and rpoD gene partial sequences were used and reference strains of the P. syringae phylogenetic group were included in the analysis. Distance matrices were calculated by the Jukes-Cantor method (Jukes and Cantor 1969). The tree was generated by the neighbour-joining method. P. aeruginosa ATCC 10145 ^T was used as the outgroup. The bar indicates sequence divergence. Percentage bootstrap values of more than 50% (from 1000 replicates) are indicated at the nodes

Fig.5

Detail of necrotic spots on leaves and fruits of Citrus limon cv. ‘Eurêka’ induced by artificial inoculation with P. syringae pv. syringae. Black pit on fruits 5 days after inoculation and blast on leaves, 24 h later as compared with negative control (treated with sterile distilled water)