Mycorrhization of micropropagated mature wild cherry ( Primus avium L . ) and common ash ( Fraxinus excelsior L . )

Lovato, P.E. , 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: 297302. (' 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 Kingdom. Present address: Paulo E. Lovato, Universidade Federal de Santa Catarina, Florianöpolis, Brazil.)


Introduction
Micropropagation is an important method for rapidly 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 phosphorus (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 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 mycorrhizal fungi during the weaning phase.The performance of substratum-fertiliser combinations, similar to those used in nurseries, in the produc- tion of mycorrhizal micropropagated wild cherry was also assessed.
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).

Data analysis
Data were analysed with the Stat-ITCF Program and differences were calculated by the Newman- Keuls test at P<0.05.

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 significantly 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<o.l),which was richer in peat.

Comparison of fungi
Plants were inoculated with G. intraradices, G. deserticola or G. rosea, and transferred only to substratum 1 with the lower fertiliser level (Fert.1).At the end of the weaning period, roots were well infected by the Glomus species, showing about 75% of the cortex occupied by mycorrhizal fungal structures, whilst the infection rate was about 45% with G. rosea.Shoot growth was bet- ter with the Glomus species than with the control plants, whilst G. rosea had a depressive effect on plant height.After four weeks in substratum 1, there were some differences (P<0.05) between fungal treatments, with a positive effect of G. deserticola.At 9 weeks, however, the differences diminished, and plant growth was similar in all treatments at 13 weeks (Fig 2 a, b).

Comparison of substrata and fertilisers
The effect of substrata was evaluated at the lower fertiliser level, using control plants and plants inoculated with G. intraradices or G. deserticola.The early beneficial effects of inoculation with G. deserticola were observed again, and, as in the first experiment, they disappeared with time, since at 9 and 13 weeks, only the type of substra- tum had significant effects on growth (P<0.05).At 13 weeks, plants in substratum 1 were 100 cm Fig. 1.Plant height (a) and stem diameter (b) of common ash plants non inoculated (NM) or inoculated with Glomus intraradices (Gi) and growing in substrata with 20% peat (SI) or with 40% peat (S 2) in a greenhouse.Mean of five replicates.Bars represent standard errors of the mean.Fig. 2. Plant height (a) and stem diameter (b) of wild cherry plants non inoculated (NM) or inoculated with Glomus intraradices (Gi), Glomus deserlicola (Gd) or Gigaspora rosea (Gro), in a greenhouse.Bars represent stand- ard errors of the mean.
in height, with a stem diameter of 7.0 mm, and those in substratum 2 were 107 cm in height, with a stem diameter of 7.9 mm [LSD (5%) were 6.3 cm for height and 0.75 mm for diameter].
In a third experiment, interactions between in- oculation, substratum and fertiliser level were evaluated on wild cherry plants inoculated with G. intraradices.Plant growth was affected by the substratum (P<0.05),growing better in the medi- um with a higher peat content, but not by fertiliser or inoculation with the mycorrhizal fungus.

Discussion
Micropropagated ash proved to be highly responsive to mycorrhizal inoculation, as demonstrated previously with conventionally propagated plants of other Fraxinus spp. (Clark 1969, Douds and  Chaney 1986, Ponder 1984).The latter studies used substrata containing at least one-third soil and no peat.In the current experiments, howev- er, we achieved beneficial mycorrhizal effects us- ing a substratum with a high level (60%) of inert or low density materials which are not normally conducive to mycorrhiza formation (Ravolanirina 1990, Wood 1991).Our results using rela- tively high P fertilisation, equivalent to at least 360 kg P ha -1 , are similar to those of Douds and  Chaney (1986) and Le Tacon and Bouchard  (1988), who also showed mycorrhizal effects with Fraxinus spp. in the presence of high soil P.
Although we did not observe a positive myc- orrhizal effect with the clone of Prunus avium used, the mycorrhizal dependence of this woody plant has been demonstrated with other species including peach (Lambert et al. 1979) and Pru- nus cerasifera (Fortuna et al. 1992); the latter authors also recommended early inoculation with rapidly infecting fungal strains.Better results with wild cherry might be achieved using other fungal strains, since mycorrhizal benefits depend on the fungus used, as observed for Prunus dulcis (Roldan-Fajardo et al. 1982) or for the almond x peach clone rootstock GF677 (Estaun et al.  1994).The latter authors obtained lower root/shoot (R/S) ratios with mycorrhizal fungal inoculation whatever the fungus used, showing that irrespective of the growth of aerial part, inoculated plants had an underground organ more efficient in the promotion of growth.The poor response to G. rosea may be due to the fungus needing longer to infect wild cherry roots.The results also suggest that micropropagated wild cherry should be grown in a substratum with a high proportion of low- density materials, such as peat and wood chips.In fact, it should be stressed that, under these conditions, the clones of Prunus used attained two to three times the size of plants reported in the literature for a comparable period of growth (Estaun et al. 1994).Furthermore, it took the plants only a few months to achieve the height usually obtained for wild cherry seedlings after two years in nurseries.However, the fertilizer dose should be re-evaluated in order to obtain similar development at lower cost; this might be possible with the introduction of efficient mycorrhizal fungi in the substratum.
The ash and wild cherry plants produced in our study will be transferred to the field to assess whether establishment and disease resistance are improved in mycorrhizal plants.It will also be necessary to evaluate the agronomic and economic benefits of mycorrhization, especially the scope for reducing fertilisation requirements.This will require further screening and comparisons offun- gal strains to select those best adapted to each species.