Highly efficient inoculation method of apple viruses to apple seedlings

Abstract

Virus inoculation to original plants is an important step in research for many reasons. For example, it is used to satisfy Koch’s postulates, to test resistance to viruses in breeding programs, and to analyze gene function by virus vectors etc. However, it is generally difficult to inoculate viruses to woody fruit trees like apple, and an efficient inoculation method has not been developed thus far. In this study, we showed that a biolistic inoculation of total RNAs from infected tissues or virus RNAs resulted in a high infection rate in apple seedlings. Total RNAs extracted from Chenopodium quinoa leaves infected with Apple latent spherical virus (ALSV) or Apple chlorotic leaf spot virus (ACLSV) and ALSV- RNAs from purified virus were biolistically inoculated to the cotyledons of apple seedlings by a Helios Gene Gun system (BIO-RAD) or a PDS-1000/He Particle Delivery System (BIO-RAD). Analysis of true leaves 2-4 weeks after inoculation by Northern blot hybridization, RT-PCR, or ELISA showed that 36 out of 38 plants (95%) inoculated with total RNAs from ALSV-infected tissues, 39 out of 41 plants (95%) inoculated with ALSV-RNAs, and 6 out of 7 plants (86%) inoculated with total RNAs from ACLSV-infected tissues were infected with each virus. Thus, the biolistic inoculation of total RNAs from infected tissues or virus RNAs from purified virus to apple seedlings is found to be an efficient inoculation method of apple viruses. We think that the method can be applied to other virus-fruit tree combinations.

Keywords: ALSV, ACLSV, biolistic inoculation, apple, cotyledon