Apple Proliferation disease: insights on the phenomenon of ‘recovery’ and use of fungal endophytes for phytoplasma control

Phytoplasmas are insect-transmitted, phloem-restricted pathogens that can cause devastating losses in crops of economical importance such as fruits, vegetables, ornamental or weeds. Apple proliferation (AP) disease is considered as one of the most important disease in all apple production areas generally and in Italy especially. AP disease is associated with the phytoplasma ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’), which belongs to the 16SrX group. Spontaneous remission of AP symptoms in plants that previously were symptomatic is called ‘recovery’. Recovery of apple trees from AP were studied by gene expression analyses of several plant defense-related genes. Real Time-PCR analyses demonstrated that genes were differentially expressed in apple leaf tissue according to the plants’ state of health. Since phytoplasma diseases are not curable, the fungal endophyte Epicoccum nigrum, previously reported as biocontrol agent or resistance inducer against different pathogens, inoculated in apple plants, maintained in semi-field conditions, as a possible strategy for the control of AP disease. In this study, the elucidation of plant–phytoplasma–endophyte relationships performed by defense-related gene expression analyses and ultrastructural observations on leaf tissues. Real Time-PCR analyses demonstrated that tested genes were differentially expressed in apple leaf tissue following endophyte inoculation in the shoot. Phytoplasma concentration was quantified by real-time PCR in endophyte-treated and untreated plants: preliminary results revealed that ‘Ca. P. mali’ was less concentrated in the treated plants. Ultrastructural observations revealed that in endophyte-treated plants, cytological changes, such as abundant callose depositions and P-protein aggregations in the sieve elements, occurred. Thirteen endophytic strains of E. nigrum were cultivated (submerged culture) in two liquid media (YM and ZM ½) and the crude extracts were then analyzed by HPLC-DAD/MS. The molecular phylogenetic affinities among the different strains were also analyzed using ITS and partial ß-tubulin gene sequences. HPLC-DAD/MS metabolite profiles for the thirteen isolates grown the two different culture media revealed a variation in the secondary metabolites profiles. Using the ZM ½ medium resulted in more complex metabolite profile and more variability of the produced metabolites. Preliminary ultramicroscopical observations performed on leaf tissues from AP-infected cuttings of the model plant Catharanthus roseus, treated with the most active fungal secondary metabolites, revealed ultrastructural modifications in some phytoplasma cells and cytological defense reactions in the host plant tissues. The results of this study shown that E. nigrum is a highly diverse fungal endophyte, producing a broad range of bioactive secondary metabolites. Moreover, this study might represent a first step in the clarification of plant–phytoplasma–endophyte relationships to find possible strategies for phytoplasma diseases control.