E. Knapp

Localization of fruit tree viruses by immuno-tissue printing in infected shoots of Malus sp. and Prunus sp.

Immuno-tissue printing protocols for the localization of apple chlorotic leaf spot virus (ACLSV), stem grooving virus (SGV) and plum pox virus (PPV) in shoots of Prunus and Malus in vitro have been established for routine diagnosis in a virus elimination program. Since these viruses belong to different virus genera, the protocols were adapted according to the properties of the virus under investigation. Accumulation of ACLSV was highest in the base of the stem and decreased towards the apex of the shoots. ACLSV was found in the epidermis, the cortex, in the vascular bundles, but seldom in the pith tissue of in vitro apple shoots. ACLSV immuno-tissue printing was as sensitive as ELISA and the intensity of color signals in immuno-tissue prints correlated with absorbance values by two-step ELISA. SGV could be detected by immuno-tissue prints at infectivity levels, where it reacted negative in ELISA. SGV accumulated in the vascular bundles, occurred locally in the parenchymatic tissue, was found in high amounts in young leaves near the meristem, but not within the meristem. PPV was detected in all tissue types of stem sections with an irregular pattern reflecting the in vivo situation causing problems with detection. Discrimination of poorly and heavily infected shoots was possible with the naked eye.

Improved Virus Detection in Rosaceous Fruit Trees in vitro

In vitro cultivation techniques are widely used for micropropagation and germplasm storage [1,2]. Moreover, in vitro culture techniques are the only effective tools for eliminating certain pathogens from elite germplasm. Many fruit tree viruses are well identified and characterised [3,4]. Conventional laboratory diagnostics, based on serology, have been developed and are routinely used for orchard surveys [5–8]. Drawbacks in the certification of fruit plants are, however, encountered since many viruses are either latent, irregularly distributed or highly localised. Thus, the risk of false results is increased especially with field plants [9,10]. Little has been published on the virus screening of in vitro cultures; however, optimisation of sampling of in vitro plants may be achieved if carried out in combination with studies on virus localisation using immuno-tissue printing [11–13].

Characteristics of Large-Scale Production of Phyto-Sanitary Improved In-Vitro Fruit Tree Cultivars Using In-Vitro Elimination Treatments and Serodiagnostics

We present results of five years of testing in-vitro cultures of over 30 apple and 20 stone fruit cultivars in a virus elimination programme. Our aim was to develop efficient large-scale screening designed for the special features of in-vitro plants to improve reliability in selection of truly negative in-vitro elite material following elimination treatment by thermotherapy and meristem preparation.