Mycorrhization of micropropagated mature wild cherry (Primus avium L.) and common ash (Fraxinus excelsior L.) Paulo E. Lovato, Neil Hammatt, Vivienne Gianinazzi-Pearson and Silvio Gianinazzi Lovato, P.E. 1 , Hammatt, N. 2 , Gianinazzi-Pearson, V. 1 & Gianinazzi, S. 1 1994. Mycorrhization of micropropagated mature wild cherry (Prunus avium L.) and common ash (Fraxinus excelsior L.). Agricultural Science in Finland 3: 297- 302. (' Laboratoire de Phytoparasitologie INRA-CNRS, Station de Génétique et d’Amélioration des Plantes, INRA, BV 1540, 21034 Dijon, France; horticulture Research International, East Mailing, West Mailing, Kent ME 19 6BJ, United King- dom. Present address: Paulo E. Lovato, Universidade Federal de Santa Catarina, Florianöpolis, Brazil.) Micropropagated plants of common ash and wild cherry were inoculated with arbuscular mycorrhizal fungi during a 20-day weaning period, after which they were transferred to two different substrata supplemented with slow-release fertiliz- er. After a 13-week growth period, the stem height and diameter of the ash plants which had been inoculated with Glomus intraradices were three times greater than those of uninoculated control plants. Increasing the peat content of the substratum improved growth of ash. Four weeks after being transferred to pots, shoots of wild cherry inoculated with G. intraradices or G. deserticola were taller and stems thicker than those of control plants, whereas those inoculated with Gigaspora rosea had shorter shoots and thinner stems than the controls. These beneficial effects of fungal inoculation on plant development disappeared after 13 weeks. Increasing the peat content, but not the level of fertiliser of the substratum, improved growth of both inoculated and uninoculated wild cherry. Key words: substratum. Glomus intraradices. Glomus deserticola, Gigaspora ro- sea, arbuscular mycorrhizas, weaning, tree, forestry Introduction Micropropagation is an important method for rap- idly propagating many plants. Indeed, in some trees, including common ash (Fraxinus excelsior L.), micropropagation is the only published relia- ble means of clonal propagation (Ahuja 1993). Micropropagated plants are usually transferred to disinfested soil or artificial substrata. Thus, their access to mycorrhizal fungi is reduced or elimi- nated. Such fungi have beneficial effects on plant development, especially through improved phos- phorus (P) nutrition, increased resistance to path- ogens and better root development (Gianinazzi et al. 1990). The absence of symbiosis may ac- count for the poor development of ash plants, even in heavily fertilised soils (Douds and Chaney 1986, Le Tacon and Bouchard 1988). inoculation with mycorrhizal fungi can enhance the growth of micropropagated plants, as demon- strated by Pons et al. (1983) and Ravolanirina et al. (1989), who succeeded in obtaining mycor- rhizal infection of wild cherry and vine micro- plants under axenic conditions. The time of inoc- ulation of microplants has, however, been proved important. Pineapple (Guillemin et al. 1992), oil 297 Agricultural Science in Finland 3 (1994)Research Note https://www.c-info.fi/en/info/?token=bh7rZzuNqDyUOEXD.bR2h4AKpbiQpUtWluYv0Lg.lbVvwcN7JKXpnTBOzRP84MQHRvJwt5McOnTh3oebLz6E_vpUKxINq-Ay8OGDMq-N97kaLcIhVln7t1t-SkEyJ7_bNOISpNOTUX49XU2m_nBo6OJ2XzcpVLT3A7IS15E4MaWC249KRxZzA9qMC-BZtKkBeV2hir9vZSnYTYuJptqlXlyXwePRA2FTopdzYEOFGT7j0VH2KBQxO11AS6FPgj2Ddb9T-yrOxZUyxqVDnBp2Py2iWRQfpHoYBFuGDnFIPmC8626y1qPd_BVY6wMoShe-S9hqqYYYB8Jhf-_ztI6Pqx4oaJuU0Jzqja1ax_Yy1ZDxtS30pfPNhwDHcMHjxAf91oJ5FJ8 palm (Blal et al. 1990) and vine (Ravolanirina et al. 1989) microplants, for example, were more successfully established if they were inoculated with mycorrhizal fungi at the beginning of the weaning period, whilst avocado microplants grew better if inoculated after a period of weaning in an uninoculated substratum (Azcön-Aguilar et al. 1992). The current experiments were carried out to assess whether micropropagated ash and wild cherry would benefit from inoculation with myc- orrhizal fungi during the weaning phase. The per- formance of substratum-fertiliser combinations, similar to those used in nurseries, in the produc- tion of mycorrhizal micropropagated wild cherry was also assessed. Material and methods Plant material Common ash (Fraxinus excelsior L.) clone 71 and wild cherry ( Prunus avium L.) cv. FI2/1 were obtained by micropropagation using previously published techniques (Hammatt 1994, Hammatt and Grant 1993). Weaning and fungal inoculation Micropropagated plants with one or two root pri- mordia were transferred to seed trays containing a 2:1:1 mixture of clay loam, perlite and grit. For mycorrhizal plants, the inoculum consisted of roots of leek infected with Glomus intraradices Schenck & Smith (isolate LPA 8), or sievings of soil containing Glomus deserticola Trappe, Bloss & Menge (LPA 27) or Gigaspora rosea Nicolson & Schenck (LPA 23). Plants were weaned for 20 days in a tall lid propagator (Maxi-SerreR , Bouil- lard Fréres, Saint Germain-en-Plain, France) (12h. per day, 19-22°C, 220 mE.cm'ls' 1, 70% r.h.). After this period, root samples were taken to check for the presence of mycorrhizal infection after clearing with KOH and staining with trypan blue (Phillips and Hayman 1970). Growth in greenhouse After weaning, plants were transferred to pots (2 dm’) in a greenhouse (July-October, 19-24°C, light supplemented to 16h day). Two substrata were tested; substratum 1, which consisted of40% (v/v) clay loam, 20% peat, 20% wood chips and 20% grit; and substratum 2, which consisted of 20% clay loam. 40% peat, 20% wood chips and 20% grit. The substrata were steam disinfected and received a mixture of CaC0 4 and MgCO, (3:2) corresponding to 1.5 kg.nr 3 , and NH4NO, corresponding to 150 g.trr3 . Two levels of fertili- sation were used, corresponding to 2 kg.nr3 (Fert. 1) or to 4 kg.nr 3 (Fert. 2) of a slow release ferti- liser (Osmocote R , Sierra Chemical Europe, Her- leen, The Netherlands) with a final composition of NPK of 16:9:12, in a 1:1 mixture of 3 to 4- month and 8 to 9-month release time types. Treat- ments were arranged in randomized blocks with five replicates. Data analysis Data were analysed with the Stat-ITCF Program and differences were calculated by the Newman- Keuls test at P<0.05. Results Common Ash Common ash plants were inoculated with Glomus intraradices only and fertilised at the Fert. I lev- el. During weaning, roots of inoculatedash plants were well infected by the mycorrhizal fungus (about 70% of the root cortex), but development of the mycorrhizal plants was depressed. Howev- er, 4,9, and 13 weeks after transfer to substrata 1 and 2, stems of inoculated plants were signifi- cantly taller and thicker (P<0.05) than those of the control plants (Fig. I a, b). At 9 weeks, there were significant differences in stem height be- tween the substrata (P<0.05). Overall, there was a tendency for better growth of uninoculated plants in substratum 2 (P