Effect of arbuscular mycorrhiza on the growth and development of micropropagated Annotta cherimola plants

Annana cherimola Mill., cherimoya, is a tropical plantation crop of interest in fruit culture. Micropropagation techniques have been developed due to the need to increase productivity through clonal selection. Because of the mycorrhizal dependence exhibited by this crop for optimal growth and the recognized role of mycorrhiza establishment for the survival and development of most of the plants produced in vitro, the effect of mycorrhiza inoculation on the development of micropropagated plants of Annana cherimola was investigated. Mycorrhizal inoculation was assayed at two different stages of the micropropagation process: (i) immediately after the in vitro phase, before starting the acclimatization period, and (ii) after the acclimatization phase, before starting the postacclimatization period under greenhouse conditions. Plantlet survival was about 50 % after the acclimatization period. Plant growth and development profited remarkably from mycorrhiza establishment. Most of the arbuscular mycorrhizal fungi (AMF) assayed greatly increased shoot and root biomass and leaf area. Micropropagated Annana plants seem to be more dependent on mycorrhiza formation for optimal growth than plants derived from seeds. The greatest effects of AMF on plant growth were observed when they were introduced after the acclimatization period.


Introduction
Annana cherimola Mill., cherimoya, a tropical tree native to South America, is of particular interest in fruit culture.It is well adapted to the subtropical conditions of Southern Spain where it is successfully cropped in the Granada and Ma- laga provinces (Morton 1987).It belongs to the Annonaceae family of the Magnoliales (Gaussen et al. 1982) and, as most members of this order, it has a relatively unbranched root system, with thick roots lacking root hairs.This suggests the mycotrophic nature of this species.This fact has been corroborated in a preliminary study in which Annana showed a strong dependence on arbuscu- lar mycorrhiza formation for optimal growth and development (Azcön-Aguilar et al. 1994).
The mycorrhiza formed by Annana plants show an exclusively intracellular hyphal development, with a cell-to-cell colonization pattern, and abundance of arbuscules and coiled hyphae within the cells (Azcön-Aguilar et al. 1994).These char- acteristics correspond to those described for the Paris species (Brundrett and Kendrick 1990 a).
Although it has been argued that this type of associations may be less efficient with respect to plant growth (Brundrett and Kendrick 1990 b), the results obtained for Annona do not support this assumption (Azcön-Aguilar et al. 1994).
Research programmes for the improvement of Annona productivity include clonal selection involving micropropagation techniques.Because of the mycorrhizal dependence exhibited by this plant for optimal growth (Azcön-Aguilar et al. 1994)  and the importance of mycorrhiza establishment for the survival and development of most of the plants produced in vitro (Gianinazzi et al. 1990), the role of mycorrhiza in the development of micropropagated Annona plants was further investi- gated.
The shoots selected were then placed in 25 x 150-mm test tubes containing 25 ml of the auto- claved basic medium, to which 1 g/1 activated charcoal was added.They were incubated in light for three days.Afterwards, they were subcultured in a root induction medium similar to the basic one but supplemented with 15 g/1 sucrose, 100 mg/1 indolebutyric acid and 200 mg/1 citric acid.The cultures were then incubated for seven days in darkness and a further three-day period in light.Finally, shoots were subcultured in a root initia- tion/elongation medium which differed from the basic one in that the MS macroelement amounts were halved and the medium was supplemented with 20 g/1 sucrose and 200 mg/1 citric acid.Cul- tures were maintained for approximately two weeks in light.
During rooting, cultures were grown at 26± I°C, with 16-h light exposure to 2000 lux illumination (Sylvania regular spectrum Gro-Lux  lamps).All media were adjusted to pH 5.7 prior to autoclaving at I2l°C for 15 minutes.
At the end of the root initiation-elongation phase, most plantlets had some roots about I cm long.At this stage, they were brought into accli- matization.Plantlets were individually transplanted to 100 ml open pots containing a mixture of sterile soil-sand (1/1, v/v) and placed in a mist- ing tunnel (100% relative humidity) for four weeks.Afterwards they were transferred to an- other tunnel without mist for another two weeks.At the end of this period, plants were transplanted to 250 ml pots and transferred to normal green- house conditions (25/19°C day/night temperatures, 16/8 photoperiod, 75/90 % relative humidity).Ho- agland nutrient solution (Hoagland and Arnon  1983) at 25 % strength was used to feed the plants at the rate needed to maintain a suitable soil water content.
Mycorrhizal inoculation was assayed at two different stages: (i) immediatelly after the in vit- ro phase, before starting the acclimatization period, with the aim of establishing the mycorrhizal symbiosis as soon as possible in the ex vitro phase (Experiment I), and (ii) after the acclimatization phase, before starting the post-acclimatization period under greenhouse conditions (Experiment 2).Mycorrhizal inoculum consisted of thoroughly mixed rhizosphere samples of stock cultures, con- taining spores, hyphae and mycorrhizal root fragments of the corresponding arbuscular mycorrhizal fungi (AMF).
In Experiment 1, the AMF tested were Glomus mosseae (Nicol.& Gerd.)Gerd.& Trappe from Rothamsted Experimental Station (England), Glomus deserticola (Trappe, Bloss & Menge) from Santiago de Compostela (Spain) and a Glomus sp. from Dijon (France).Inoculation was done by mixing the mycorrhizal inoculum (10 g) with the soil-sand mixture used to fill the 100 mL pots.Twenty plantlets per treatment were assayed and those that survived after the acclimatization period were allowed to grow for ten weeks.
In Experiment 2, inoculation was done on al- ready acclimatized plantlets (51.7 % survival rate after acclimatization).Glomus deserticola and Glomus intraradices (Schenck & Smith) were tested and mycorrhizal inoculation was done when transplanting the plants to the 250 mL pots.The corresponding inoculum (10 g/pot) was mixed with the soil-sand mixture used as substrate.Ten replicate plantlets per treatment were prepared and allowed to grow for 12 weeks.
During the post-acclimatization phase, time- course records were kept on the height of the plants and on the number of leaves produced.At harvest, the fresh and dry weights of leaves, stem and roots and the leaf area were recorded and data analysed by ANOVA and Tukey's test.Mycorrhizal colonization was measured on represent- ative stained samples (Phillips and Hayman 1970) by the gridline intersect technique (Giovannetti and Mosse 1980).

Results
Inoculation with G. deserticola and Glomus sp.immediately after the in vitro phase (Exp.I) significantly increased plant height, shoot and root biomass, number of leaves and leaf area (Figs. 1  and 2).However, it did not significantly improve survival of the plantlets ex-vitro.The mycorrhizal effect on leaf number, observed at the end of the acclimatization period (six weeks), appeared before its effect on plant height (Fig. 1).Glomus mosseae, however, did not significantly stimu- late plant development (Figs. 1 and 2).
The percentage of mycorrhizal colonization was quite similar for both G. deserticola and G. mos- seae (Fig. 2).However, in some plants G. mosse- ae induced abnormal infections characterized by many aborted entry points, which consisted of apparently normal appressoria, but without hyphal penetration of the root tissues.Sometimes few short infective hyphae were produced from the appressoria, but they rapidly aborted, and re- tracted their cytoplasm, showing septa and emp- ty hyphal tips.These infective hyphae were una- ble to spread in the root cortex and to establish arbuscules.In some other cases, a very intense, and disorganized fungal colonization of the root was observed.This infection seemed to be outside host control because some meristematic and vascular tissues appeared to be colonized by the fungus.Not all plants colonized by G. mosseae exhibited these abnormal patterns of colonization.In some cases, as in those of mycorrhizal associations with G. deserticola and Glomus sp., the colonization pattern showed a morphology similar to the one described for plants derived from seeds, with the cell-to-cell passage, and the abundance of arbuscules and coiled hyphae with- in cells, which are typical of this host species.
The influence of G. deserticola when increasing the leaf area/leaf fresh weight ratio (Fig. 2) is noteworthy.This effect was not significantly induced by Glomus sp., although the effects of both fungi on plant growth were quite similar.
The mycorrhiza-induced increases in plant growth and development were even higher when mycorrhizal endophytes were inoculated after the acclimatization period (Exp. 2,Figs. 3 and 4).As in Experiment 1, the mycorrhizal effect on the Fig. 1.Shoot height and number of leaves of mycorrhiza- inoculated (Gm = Glomus mosseae, Gd = Glomus deserticola and Gsp = Glomus sp.) and non-inoculated (C) mi- cropropagated Annana cherimola plants throughout the growth period.Inoculation was done at the beginning of the acclimatization period.* Indicates a significant differ- ence (P<0.05) according to Tukey's test.
leaf number was detected before its effect on plant height (Fig. 3).All of the growth parameters meas- ured significantly increased with mycorrhizal inoculation in all of the assayed endophytes (Figs. 3 and 4).As in Experiment 1, G. deserlicola increased the leaf area/leaf fresh weight ratio.If all the data from these experiments and from others carried out on the same plant (Azcön-Agui- lar et al. 1994 and unpublished results), were pooled, it would be observed that the effect of mycorrhizal inoculation on plant height, given as percentage increase over control, is much higher Fig. 2. Biomass production and distribution, leaf area and mycorrhi/.alinfection of inoculated (Gm = Glomus mosseae, Gd = Glomus deserticola and Gsp = Glomus sp.) and non-inoculated (C) micropropagated Annona cherimola plants.
Inoculation was done at the beginning of the acclimatization period.For each parameter, values sharing the same letter did not differ significantly (P<0.05) according to Tukey's test.
for micropropagated plants than for those propagated from seeds (Fig. 5).This suggests that micropropagated Annona plants are even more de- pendent on mycorrhiza formation for optimal growth than plants derived from seeds.

Discussion
Most of the AMF assayed substantially increased shoot and root biomass and leaf area, corroborating the strong dependence of Annona cherimola on mycorrhiza for optimal growth (Azcön-Agui- lar et al. 1994).In addition to the general effect of improving plant growth, G. deserticola con- sistently enhanced the leaf area/leaf fresh weight ratio.This suggests that other mechanisms, apart from the increased nutrient uptake, could be involved in the effect that G. deserticola exerts on plant development.Plant hormone production (Barea and Azcön-Aguilar 1982) or changes in-duced in the hormonal balance of the plant (Dix- on 1990) may underly some of these mechanisms.
The fact that mycorrhizal dependence is greater for micropropagated plants than for those prop- agated from seeds can be explained on the basis of the different types of stresses (nutritional,  drought, etc.) to which a micropropagated plant is subjected.In order to cope with these stresses, they depend to a greater extent on mycorrhiza during the post-vitro growth.
In relation to the inoculation time, plants ben- efited more from mycorrhiza inoculation when the AMF were introduced after the acclimatiza- tion period.When AMF were inoculated imme- diately after the in vitro phase, mycorrizal colo- nization showed, in some cases, either aborted infection or very intense colonization, apparently outside the control of the host.It seems as if some roots (or some plantlets) were not yet ma- ture enough to control the growth of the AMF in the root tissues and, consequently, to establish the symbiosis.Thus, they react against the fun- gus (abortive infection) and, if they fail to avoid infection, it appears rather disorganized and un- controlled.Under these conditions, mycorrhiza formation could not induce beneficial effects on plant development, or it might even provoke det- rimental ones.This reaction was found for G. mosseae -inoculated plants in some cases, al- though, in others, plants showed normal coloni- zation patterns and growth increases.This suggests that this effect appears as a consequence of the physiological status of the plantlets and not as a specific reaction against G. mosseae.In fact, G. mosseae improved Annona cherimola growth in other circumstances (AzcÖn-Aguilar et al.   1994).
Studies are now underway to confirm these facts and to determine the physiological status required for the root system to be able to estab- lish a functional mycorrhizal symbiosis in micro- propagated plants.Fig. 3. Effect of different mycorrhizal fungi (Gd = Glomus deserticola, Gi = Glomus intraradices and C = non-inoc- ulated control) on shoot height and number of leaves in micropropagated plants of Annona cherimola throughout the growth period.Inoculation was done at the end of the acclimatization period.* Indicates a significant differ- ence (P<0.05) according to Tukey's test.