Discrimination of phytoplasmas using an oligonucleotide microarray targeting rps3, rpl22, and rps19 genes

• Microarray targeting phytoplasmas' genes rps3, rpl22, and rps19 was developed.
• Single infections of 16Sr groups I, II, III, V, VI, VII, IX, X and XII were detected.
• Six out of eight artificially mixed infections were distinguished.
• Field sample infected with 16Sr groups I, III, and I + X were recognized.
• Comparison with similar methods for routine phytoplasma screening is made.

Phytoplasmas are economically important pathogens of various fruit trees. Reliable techniques therefore are needed for their detection and discrimination. For this purpose, an oligonucleotide microarray targeting the ribosomal protein genes rps3, rpl22, and rps19 of phytoplasmas was tested. Following PCR (35 cycles) of total DNA from phytoplasma-infected plants with Cy5-labelled primer, the microarray reliably detected 16Sr groups I, II, III, V, VI, VII, IX, X, and XII in single infections as well as six different mixed infections (I + II, I + III, I + V, I + VII, I + IX, and I + X) prepared artificially by mixing DNA prior to PCR. It also successfully classified 16Sr groups from field samples collected in the Czech Republic (16Sr groups I, III, and mixed infection I + X). Despite that it did not succeed in distinguishing another two artificial mixed infections (I + VI and I + XII), the microarray developed here provides a suitable alternative to rRNA-based microarrays published previously and where only single infections from experimental hosts were tested. It also advances the technique by targeting single-copy genes with higher inter-group variability than those of 16S rRNA or ribosomal spacer. The usability of the microarray in comparison with DNA barcoding and terminal restriction fragment length polymorphism techniques is discussed.