Biological and molecular proof of phytoplasma seed transmission in corn

Phytoplasma seed transmission is still a poorly investigated topic inspite of growing experimental evidences. Corn seeds deriving from 5 symptomatic and phytoplasma-infected cobs were sown and the germinated plants were analyzed to verify the presence of infected progenies by nested PCR on 16S ribosomal DNA, and by attempts of phytoplasma cultivation in chemically defined media to verify their viability. After sterilization, seeds were in vitro germinated and the plantlets were transferred in sterile soil in insect-proof cages and tested when they were 30 to 100 days old. Preliminary trials were carried out on 9 seedlings, 6 of which, positive to aster yellows and “stolbur” phytoplasmas, were used as plant sources for isolation trials in PivL® medium. From three plants the isolation allowed to detect aster yellows and “stolbur” phytoplasma DNAs after chloroform/phenol extraction from 1 ml of liquid medium from isolation tubes and from tubes obtained after serial dilution that have shown color change. Further tests on 79 seedlings resulted in 17 of them positive for aster yellows and “stolbur” phytoplasmas. Isolation from three of these plants after 30 days from germination resulted positive for phytoplasma DNA in tubes deriving after 2 to 10 serial dilution in fresh medium that have all shown colour change.Reisolation carried out at 90 days from germination confirmed these results only from one plants. Plating carried out together with DNAs extraction from liquid medium produced colonies of different size and shapes. The same type of colonies were obtained from plating tubes maintained for seven month at 25°C. Single colonies were picked and transferred in broth for purification steps at several times and small colonies were obtained resulting to consistently contain aster yellows DNAs; moreover this type of colony growth was observed for at least 3 subsequent passages liquid/solid media carried out about every 5 days. These preliminary results are indicating the viability of phytoplasmas isolated in corn seedlings grown in insect-proof environment.