1. Study on diverse grafting techniques for their capability in rapid and efficient transmission of Apple Proliferation disease to different host plants Aldaghi M. 1,2, Massart S. 1, Roussel S. 1, Steyer S. 3, Lateur M. 3 and Jijakli M.H. 1 1 Plant Pathology Unit, Gembloux Agricultural University (FSAGx), Passage des déportés 2, 5030 Gembloux, Belgium 2 Plant Diseases Department, Plant Pests & Diseases Research Institute (PPDRI), P.O. Box 19395-1454, Tehran, Iran 3 Biological Control & Plant Genetic Resources Department, Wallon Agricultural Research Center (CRA-W), Rue de Liroux 4, 5030 Gembloux, Belgium Phytoplasmas are wall-less plant pathogenic bacteria in Mollicutes class. They inhabit phloem of hundreds of plant species, are responsible of diseases spread worldwide and, in several cases, present in the list of quarantine pests as defined by the International Plant Protection convention. Apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali’, is one of the most important apple diseases affecting both the fruits yield and quality. It belongs to the EPPO A2 list of quarantine plant pathogens. Phytoplasmas are obligatory pathogens and, up to now, they were not cultivated in axenic culture, therefore Koch postulates are only sometimes fulfilled by using alternative tools, such as graft or insect transmission (Bertaccini, 2007). So, phytoplasma graft transmission remains of outstanding importance for the accurate detection and in the phytosanitary certification schemes. Different grafting methods may be used on woody or herbaceous plants to detect AP infection. These methods include whip, bark or side grafting, budding and chip-budding. For the first time, comparisons between 4 different grafting techniques for ‘Ca. P. mali’ inoculation and detection by biological indexing are presented. Materials and Methods Background and Objective References Biological indexing is laborious, time-consuming, and skill-demanding (Di Terlizzi B. 1998). Even if the molecular and serological diagnostic protocols can replace biological indexing as a fast screening technique in certification programs, biological indexing will often remain mandatory as a second technique to fulfil certification of propagate materials. In this context, this work allowed the identification of the best suited methodology for AP indexing. The best results were obtained with whip and bark grafting on MM106 rootstocks, and we perceive apple trees are more suitable than periwinkle for biological indexing of apple materials. Finally, within a certification program, symptoms observation after biological indexing could be confirmed by phytoplasma detection using molecular methods. Conclusions Healthy MM106 apple rootstocks and AP-infected scion woods (5 isolates) were provided by CRA-W (Gembloux, Belgium) and Quarantine Station (Lempdes, France), respectively. Healthy and AP-infected (5 isolates) periwinkles were prepared by FSAGx and Institut für Pflanzenschutz (Dossenheim, Germany), respectively. All inoculation experiments were carried out in July-August in insect-proof greenhouse (14h light, high relative humidity, 20-25°C). Four different grafting methods were applied for phytoplasma transmission. These methods are described in figures 1 to 4. Additional comments are exposed: whip (tongue) graft method works best when the stock and scion are of similar diameter, preferably between 8 and 12 mm. Bark grafting may be performed on branches ranging from 12 to 25 mm in diameter. Budding is a form of grafting in which a single bud with a thin layer of bark is used as the scion rather than a section of stem. Budding is carried out in summer, when the bark of the stock slips easily and when there are well-grown buds. For chip-budding, in mid-summer, non- flowering shoots of rootstocks are selected with similar diameter to the scions from well-ripened, current season’s growth as bud. The union (rootstock-scion) was always bound tightly with tape and, for whip and bark grafting the union and scion were carefully covered with plastic bag for 1- 2 weeks. The symptom emergence was monitored on inoculated plants by time. In periwinkle plants, the symptoms as yellowing and reduced leaf size and/or vigour were surveyed. In apple trees, proliferation, chlorosis, small leaves and enlarged stipules were noticed. 1) Bertaccini A. 2007. Phytoplasma: diversity, taxonomy, and epidemiology. Frontiers in Bioscience 12, 673-689. 2) Di Terlizzi B. 1998. Biological diagnosis of virus and virus-like disease: a special reference to stone fruits certification. Option Méditerranéennes, Sér. B/n°19 - Stone fruit viruses and certification in the Mediterranean countries: problems and prospects. 151-170. AB C A B C A B C D E A B C Figure 1. Whip grafting Figure 2. Bark graftingFigure 3. BuddingFigure 4. Chip-budding Figures were borrowed from www.gartenbauvereine.org/texte/merkinfo/m_VeredelungObst.html (figures 1 & 4), www.ext.nodak.edu/county/cass/horticulture/fruit/graft/graft.htm (figure 2), and http://www.gutenberg.org/files/18189/18189-h/18189-h.htm (figure 3). Periwinkle plants: The infected periwinkle and apple plant materials were grafted on periwinkles by chip- budding, the only feasible grafting technique. Symptoms, appeared during the second month after grafting, were observed in more than 90% of periwinkles grafted by an infected periwinkle. For periwinkles grafted with apple material, scions gradually died but symptoms were observed in 60% of plants 4-6 months after grafting (Table 1). To our knowledge, this is the first report of direct AP transmission from apple to periwinkle. Apple plants: In this case, all grafting methods were performed using infected apple materials (as scion). First symptoms of phytoplasma infection appeared very sooner with whip and bark grafting than with budding and chip-budding. An efficiency of 100% of inoculations was obtained by whip and bark graft techniques (Table1). In our tests, 7 transplants were not covered by bag, so they did not grow after grafting, nevertheless, the phytoplasma transmission from them to the rootstock was sometimes recorded (28.6%). Also, the efficiency of budding and chip-budding were 38 and 54%, respectively (Table 1). Grafting method Periwinkle * inoculated by Apple * inoculated by Periwinkle materials Apple materials Whip graft--10/10 Bark graft--11/11 Budding--3/8 Chip-budding9/1012/206/11 * : symptomatic plants number per total tested plants - : not-tested (not-feasible) Table1. Biological indexing of ‘Ca. P. mali’ by grafting Results