Characterization of two plant pathogenic Arsenophonus bacteria responsible for strawberry marginal chlorosis disease in Italy.

Strawberry marginal chlorosis (SMC) is a disease that was observed in nurseries and production fields in France, Japan and Italy. Affected plants show pronounced stunting and a poor development of the root system. Older leaves are rolled upward and display a marked premature purple coloration while new leaves are reduced in size, chlorotic, with short petioles and generally cupped. This disease can be caused by two different phloem-limited plant pathogenic prokaryotes: the stolbur phytoplasma and the gamma-3 proteobacterium "Candidatus Phlomobacter fragrariae". During the fall season from 2005 to 2013, surveys were conducted in Emilia-Romagna region collecting and analysing symptomatic strawberry plants. Twenty-eight out of 168 samples tested positive by PCR analysis using the previously published primers Fra-4/Fra-5, indicated for tha amplification of a 550-bp fragment of the 16Sr RNA region of "Candidatus Phlomobacter fragrariae". The proteobacterium was also visualized on ultrathin sections of SMC-diseased strawberry leaf veins and petioles, by transmission electron microscopy. Sequence analysis of the 16Sr RNA and spoT-spoU-recG genes of selected isolates, surprisingly, revealed the presence of two Arsenophonus bacteria: "Candidatus Phlomobacter fragrariae" and "Candidatus Arsenophonus phytopathogenicus", reported to be the casual agent of the "basses richesses" syndrome of sugar beet plants in eastern France. To our knowledge this is the first detection of two different protobacteria associated to SMC and the first visualization in Italy of the proteobacterium by electron microscopy. Further investigations will be necessary to better understand the etiology and the epidemiology of this disease in Italy.