Arbuscular mycorrhizae and growth enhancement of micropropagated Prunus rootstock in different soilless potting mixes

Propagation of fruit rootstocks by in vitro techniques is done widely and has become a standard procedure in many nurseries. In these circunstances, the plantlets are transferred from sterile in vitro media to potting mixes prepared with organic substrates and inorganic conditioners which lack arbuscular mycorrhizal fungi (AMF) propagules. Fruit tree crops, the prunus species in particular (Gilmore 1971), develop arbuscular mycorrhizae (AM). The early inoculation of plantlets with AM fungi in the nursery could enhance plant growth and increase field trasplant survival (Gianinazzi et al. 1989). The receptivity of the organic substrates used in the potting mixes to the AMF and the effects of the inoculation on plant growth have been little studied. Calvet et al. (1992) found that certain types ofpeat and composted substrates had a negative effect on the establishment of the AM symbiosis, although the AMF germination and early mycelial growth were not affected, and suggest a biological cause for the inhibition. Vidal et al. (1992) found that the symbiosis could be established in peat:sand mixes although soil:sand mixes were more conducive to AM root colonisation of micropropagated avocado plants. Schubert et al. (1990) working on micropropagated grapevine found that the AMF colonised the roots in all peat based media used, but only when soil was added to the mixes there was a significant response of the plant to the inoculation. Vestberg (1992) found that sand fertilised with bone meal was superior to rich based peat substrates in initiating rapid AM colonisation and sporulation of the AMF used. Biermann and Linderman (1983) have shown also that the addition of soil to peat induces growth responses to AMF inoculation. Nonetheless commercial growers are reluctant to add soil to theirpotting mixes due to the increase in weight and the risk of introducing soil borne pathogens. This work has studied the receptivity of two different peat based mixes, without added soil, to four AM fungi and the effects of the inoculation on plant growth.


Introduction
Propagation of fruit rootstocks by in vitro tech- niques is done widely and has become a standard procedure in many nurseries.In these circunstanc- es, the plantlets are transferred from sterile in vitro media to potting mixes prepared with or- ganic substrates and inorganic conditioners which lack arbuscular mycorrhizal fungi (AMF) propagules.Fruit tree crops, the prunus species in par- ticular (Gilmore 1971), develop arbuscular mycorrhizae (AM).The early inoculation of plantlets with AM fungi in the nursery could enhance plant growth and increase field trasplant survival (Gianinazzi et al. 1989).The receptivity of the organic substrates used in the potting mixes to the AMF and the effects of the inoculation on plant growth have been little studied.Calvet et al. (1992) found that certain types ofpeat and composted substrates had a negative effect on the establishment of the AM symbiosis, although the AMF germination and early mycelial growth were not affected, and suggest a biological cause for the inhibition.Vi-dal et al. (1992) found that the symbiosis could be established in peat:sand mixes although soil:sand mixes were more conducive to AM root colonisation of micropropagated avocado plants.Schubert et al. (1990) working on micropropagated grapevine found that the AMF colonised the roots in all peat based media used, but only when soil was added to the mixes there was a significant response of the plant to the inocula- tion.Vestberg (1992) found that sand fertilised with bone meal was superior to rich based peat substrates in initiating rapid AM colonisation and sporulation of the AMF used.Biermann and Linderman (1983) have shown also that the addition of soil to peat induces growth responses to AMF inoculation.Nonetheless commercial growers are reluctant to add soil to their potting mixes due to the increase in weight and the risk of introducing soil borne pathogens.
This work has studied the receptivity of two different peat based mixes, without added soil, to four AM fungi and the effects of the inoculation on plant growth.

Material and methods
The transfer from the weaning media to the sub- strate used in the growth and hardening phase of the plantlets was targeted as the easiest step to perform the arbuscular-mycorrhizal inoculation in the nursery.

Plant material and conditions of growth
The plant used was a Prunus persica x Prunus amygdalus clone; GF677 widely used as both peach and almond rootstock.Rooted plantlets were obtained from Agromillora Catalana S.A. and were inoculated and grown in pots of 12 cm diameter.There were 5 treatments per potting mix: Con- trol, inoculated with G. mosseae, inoculated with G. intraradices, inoculated with Glomus sp.(E3) and inoculated with A. laevis.Pots were ran- domised and grown under greenhouse conditions.After 14 weeks the plants were harvested.Plant height, weight and stem diameter were measured.Root colonisation was assessed after staining (Phillips and Hayman 1970) using the grid-line intersect method (Giovanetti and Mosse 1980).Results from both mixes were analysed separately using ANOVA and Tuckey's test for multiple range comparisons.

Arbuscular mycorrhizal fungi inoculation
The fungi employed were: Glomus mosseae (Nicol.& Gerd.)Gerdemann & Trappe, Glomus intraradices Schenck & Smith, Glomus sp (E3) and Acaulospora laevis Gerdemann & Trappe.To ensure sufficient AM propagules, 20 g of soil containing spores, mycelia and mycorrhizal roots from heavily colonised pot cultures were used as inoculum and placed under the plantlet roots at the moment of the transfer from the weaning me- dia.

Potting mixes
Two sphagnum peat types were selected : Flora- torf and TKS-1 both are widely used in nurseries and have similar physical properties (Table I).TKS-I comes as a ready to use peat, neutralised and amended while Floratorf has a pH of 3.9 and had to be neutralised with calcium carbonate to reach a pH of 6.5 before using it.Both organic substrates were mixed (50:50, v;v) with perlite and used without prior sterilisation.

Results and discussion
The four AMF studied colonised the plant roots in both substrate mixes, although there were dif- ferences in the percentage colonisation achieved (Table 2 and 3).These differences can be attrib- uted to either a certain host preference (Estaun et al. 1987) or to the characteristics of the sub- strate (Table 1) that might be more suitable de- pending on the fungus studied (Guttay 1982,  Nemec 1987, Calvet 1989).In the TKS-I mix G. intraradices achieved the highest level of root colonisation.In the Floratorf mix the maximum colonisation levels were reached by G. intraradi- ces and Glomus sp (E3).G. intraradices showed a higher level of root colonisation in TKS-I than in Floratorf, whilst Glomus sp (E3) was favoured by the latter.The other fungi assayed: G. mosse- ae and A. laevis maintained a low level of colonisation in both substrates.G. intraradices is con- sistently the more successful strain in colonising plant roots in both susbtrates.This indicates a higher specificity of this fungus for the host plant.Mean of 10replicates.Values in the same column followed by different letters are significantly different (p=o.os).Tuckey's test.
Table 3. Effects of AM fungi on GF677 plant growth in Floratorf; perlite mix.Glomus sp (E3) root colonisation was substantially different from one substrate to the other.In TKS-1 Glomus sp (E3) colonisation percentage was almost negligible whilst in Floratorf it reached the maximum level of all the fungi assayed, Glomus sp (E3) is clearly favoured by the Flora- torf mix stressing the importance of the growing media as the site of specific interactions with the fungus which is a continuous entity with mycelia in the roots and in the substrate (Mosse 1972).
The two mixes studied differ very little in their physical properties (Table I), the original differ- ence in their pH was compensated with the addi- tion of calcium carbonate to the Floratorf mix.Many results have correlated low colonisation rates with high nutrient levels of the growing media (Sieverding and Howeler 1985, Miranda et al. 1989, Cooke et al. 1992), unexpectedly the substrate with the highest nutrient content was best for one of the fungi studied, although for the other fungi studied it decreased or it did not affect the root colonisation.No general state- ments can be made and each different combina-tion of fungi-substrate and host plant deserves to be studied.
When considering plant growth, the effects of the substrate mixes overcame those of the inoculation treatments.TKS-1 was better than Floratorf in all treatments and for all parameters meas- ured.The inoculation in this substrate mix increased shoot weight (G.mosseae, G. intraradi- ces and Glomus sp ( E 3)) and the stem diameter (G.intraradices).The root/shoot ratio was lower for all the inoculated treatments when compared to the control.In the Floratorf mix although all the inoculated treatments had a significantly lower root/ shoot ratio than the control treatment only the plants colonised by A. laevis showed a significant growth response to the inoculation.The decrease of the R/S ratio is a parameter that is associated to the mycorrhizal symbiosis and our results show that, although no increase of growth was found, the mycorrhiza was functional in all AM treatments.The beneficial effects of the sym- biosis might appear in later stages of the plant development, especially in woody host plants grown in rich substrates.In the inoculated TKS-1 mixes this functionality was only translated into a significant growth increase for one of the fungi assayed: G. intraradices, which is the one that showed a highest colonisation of the plant roots.Sanders et al. (1977) found that the most effec- tive fungal strains were those that produced a more rapid and extensive root colonisation, Ab- bot and Robson (1981) also found a correlation between infectivity of the AMF and effectivity of the symbiosis.However, in the Floratorf mix, there was no correlation between fungal colonisation and plant growth, supporting the findings of Smith  and Smith (1981), Abbot and Robson (1985)  and Graham and Fardelmann (1986).These dis- crepancies between experiments and the results we have found for the same combination of host plant-fungus but different growing media might be due to initial differences in the symbiosis de- velopment rate that can not be seen at the time of harvest.Furthermore the performance of particular fungi in their ability to enhance plant growth lies in the extraradical mycelia development, in the efficiency of this mycelia to absorb and trans- locate nutrients and how this is translated into an increase of biomass production (Smith and Gi- aninazzi-Pearson 1988).The performance of a given combination of host plant-AM fungus can be modified by factors such as the growth sub- strate used (Guttay 1982, Calvet 1989), pH (Graw 1979) and nutrient levels (Chambers et  al. 1980) which might have a direct or indirect effect on the endophyte.
From our work it appears that the growing me- dia is one of the major factors not only in deter- mining the AMF infectivity and symbiosis estab- lishment but also in the regulation of its func- tionality.
From these results we conclude that even in reach peat based, unsterilised potting mixes the inoculation with a previously selected fungus can enhance plant growth.
Aknowledgemenls.This work was partially founded by grant no.AGF92-0428 of the Comisiön Interministerial de Ciencia y Tecnologfa (Spanish Ministery of Education and Science).

Table 1 .
Physicochemical properties of the two types of peat used for the potting mixes (afterAldrufeu et al. -220

Table 2 .
Effects of AM fungi on GF677 plant growth in TKS-1: perlite mix.