Effect of (V)A mycorrhiza on nutrient concentration and uptake of oil palm plantlets

plantlets.

In investigations of Azcón-Aguilar et al. (1994), Lovato et al. (1994) and Rapparini et al.

(1994), it was reported that micropropagated plantlets which were inoculated with mycorrhizal fungi had enhanced plant quality, nutrient uptake and plant growth. The inoculation of plants with (V)AM fungi increases the growth of plants by several fold, and generally most of the growth enhancement effects observed in root colonisation with (V)AM fungi are caused by increased P absorption (Bagyaraj and Varma, 1995).

6.5 Effect of (V)A mycorrhiza on nutrient concentration and uptake of oil palm plantlets

6.5.1 Phosphorus

In this study, the inoculation with (V)AMF isolates significantly increased phosphorus concentration and uptake of the oil palm plantlets compared to the non-inoculated controls.

The role of (V)AM fungi in uptake of nutrients, particularly P, is well documented (Bolan, 1991; Marschner and Dell, 1994; Smith et al., 1998). Estrada-Luna et al. (2000) reported that mycorrhizal inoculation altered the nutrient uptake of micropropagated guava plantlets, especially the leaf elemental concentration of P was significantly higher in mycorrhizal plants. It was seen in Experiment 1 and 3 that inoculated plants showed increased P uptake even when they were not fertilised.

The growth stimulation of mycorrhizal plants in this study may be explained by higher mineral P concentrations in foliar tissues. Koch and Johnson (1984) and Fitter (1988) reported that net photosynthesis of mycorrhizal plants can increase as a result of improved plant nutritional status. Plants with an optimal P concentration should be more vigorous with high net photosynthesis (A) and stomatal conductance (g_s) than plants with limiting P (Radin and Eidenbock, 1986). Low P can also reduce the concentration and activity of RubisCO, and slow down the regeneration of intermediate substances in the Calvin cycle (Lauer et al. 1989).

The interaction between inoculation and fertilisation in Experiment 2 demonstrated the high effectiveness of mycorrhizal inoculation on P uptake. The highest P uptake in this experiment was observed in the inoculated plants which were fertilised with the slowly soluble

hydroxy-apatite (HA). Especially Acaulospora appendicula significantly increased P uptake of plants compared with all inoculated plants of the non-fertilised or 'BAYFOLAN' fertilised treatments. Mycorrhizal fungi often have access to forms of nutrients which are not directly available to plants (Lange-Ness and Vlek, 2000), as well as being able to acquire nutrients which are spatially separated from roots. However, it is now evident that these associations have a greater benefit when phosphorus is present in less-soluble forms (Bolan 1991, Tarafdar

& Marschner 1994, Kahiluoto & Vestberg 1998, Lange-Ness and Vlek, 2000).

The results of Experiment 2 also indicate that mycorrhizal inoculation has a greater effect on P uptake of shoots than fertilisation. A comparison between all non-inoculated control plants shows that the fertilisation with the complete foliar ‘BAYFOLAN’, which is practised at IOPRI, or fertilisation with hydroxy-apatite, does not significantly increase the P uptake of the plants, unless accompanied by (V)AMF. The combination of both, inoculation and fertilisation, resulted in a highly improved P uptake by the plants.

According to the results of this study, the first six weeks after transplanting to soil seem to be the most critical for the survival and growth of the sensitive oil palm plantlets. Inoculated plantlets of Experiment 2 harvested after six weeks of post vitro growth showed significantly increased P concentration and uptake. At this early growth stage, the P uptake of mycorrhizal plants was 2 times higher compared to the non-inoculated control plants. A significantly increased nutrient concentration at that growth stage was only found for phosphorus, but not for potassium or nitrogen. These results indicate that P seems to be the most limiting growth factor during the early post vitro stage of the oil palm clones. Addition of fertilisers does not greatly improve the nutrient status of propagated plants in their early growing stage because of their relatively small root systems and inability to take up nutrients efficiently.

In Experiment 3, the beneficial effect of (V)AMF inoculation on P uptake of the plants was confirmed. The effectiveness of the fungal isolates was far greater with soil sterilisation. The effect of inoculation on P uptake was lower, but still significantly improved if plants grew in non-sterilised soil. The higher effectiveness of the (V)AMF isolates may be due to the mobilisation of nutrients, particularly P, and/or the decreased competition between native and introduced mycorrhizal fungi, as a result of the soil sterilisation.

The mycorrhizal dependency of oil palm plantlets on P uptake during the critical post vitro phase was illustrated in this study. The improvement of plant nutrition, especially phosphate, is a physiological factor that can be used to evaluate the functioning of the endomycorrhizal symbiosis.

According to a wide spectrum of research work documented in literature, phosphorus is recognised as being the most important plant-growth limiting factor which can be supplied by

mycorrhizal associations, because of the many abiotic and biotic factors which can restrict its mobility in soils (Harley & Smith 1983, Hayman 1983, Marschner 1986, Bolan 1991, etc.).

6.5.2 Potassium and Nitrogen

(V)AM fungi might also increase the uptake of other nutrients that move to the root surface primarily by diffusion (Abbott and Robson, 1984). This could explain the higher K uptake of the inoculated oil palms in this study.

Nevertheless, no direct mycorrhizal effect on K uptake has been demonstrated to date (Harley and Smith, 1993). Considering the importance of P and K during the early growth stages of micropropagated plants, the better growth response in mycorrhizal plants was probably due to the increased uptake of these two nutrients.

Matos et al. (1996) investigated the effect of endomycorrhizal inoculation on the growth of micropropagated pineapple plants. After 12 months of growth, the mycorrhizal plants showed significantly greater dry matter accumulation of leaves than the controls. It was also observed that in the mycorrhizal treatments, the P and K concentrations in the leaves were higher.

These data indicate that mycorrhizal plants showed a greater vigour and a better nutritional status when compared to the non-mycorrhizal plants. The better nutritional status of mycorrhizal plants is primarily due to the improved plant growth as a result of increased P uptake (Cooper, 1984; Rizardi, 1990).

Declerck et al., (1995) investigated the growth response of micropropagated banana plants to (V)AM inoculation. Inoculation with Glomus mosseae and Glomus geosporum resulted in significantly higher shoot fresh and dry weights as compared to the control plants. Shoot P and K contents were also significantly higher in inoculated plants. The authors concluded that the higher P content could be related to various mechanisms, including the larger soil volume which reduces the distance of ion diffusion to the plant roots, and rhizosphere modifications (Bolan, 1991). (V)AM fungi might also increase the uptake of other nutrients that move to the root surface, primarily by diffusion (Abbott and Robson, 1984).

Improvement of the nitrogen status of the host is one of the ecologically significant consequences of mycorrhizal associations. (V)AM fungal hyphae have the ability to extract nitrogen and transport it from the soil to the plant due to the increased absorption surface provided by the mycorrhizal fungi. Nitrogen exists in many forms, namely free nitrogen, nitrate, nitrite, ammonium ions and organic nitrogen. Ammonium constitutes a significant proportion of the inorganic nitrogen pool at a low pH level. Ammonium is less mobile in soil because it is fixed in lattice of clay minerals and absorbed to negatively charged clay

minerals. (V)A mycorrhiza hyphae transport this immobile ammonium to the plant roots (Bowen, 1987).

This study shows that inoculation with efficient (V)AMF isolates leads to an improved plant nutritional status which is probably the cause of improved plant development and consequently reduces plant mortality. Several authors report that (V)AM fungal colonisation of horticultural plants improves growth by increasing uptake of P and other minerals (Gianinazzi and Gianinazzi-Pearson, 1986; Sieverding, 1991; Pearson and Jakobsen, 1993), thus increasing transplanting uniformity which resulted in reduced plant mortality (Biermann and Linderman, 1983) and injury (Menge et al., 1978).

6.6 (V)AMF infectivity and effectiveness

In (VA)MF symbiosis, the ability of a fungal species to infect a host plant rapidly plays an important role in determining the success of the relationship. The main parameters, which can be utilised to measure this type of host affinity, are fungal infectivity and the growth-promoting ability of (V)AM fungi.

All mycorrhizal fungi tested in the initial screening of Experiment 1 had been shown to form mycorrhizas with the oil palm plantlets. Levels of infection observed after trypan blue staining differed between isolates and ranged in most cases between 17-23%. Plants inoculated with Glomus mosseae (M4) showed the highest infection rate (27%) while the lowest infection rate was observed in roots of plantlets inoculated with Glomus clarum (M8), although inoculation with both fungi improved plant growth significantly. This would suggest that Gl. clarum is a highly effective symbiont in this mycorrhiza-host association, even when root infection is weak. Despite the low percentage of root infection by the tested (V)AM fungi, the effectiveness on plant survival and on growth improvement was significant in this study. Pinochet et al. (1997) also reported that the percentage of mycorrhizal colonisation of Gl. interadices was relatively low at 27%, although plant growth in mycorrhizal treatments was significantly better than in non-mycorrhizal treatments for all growth characteristics (in terms of shoot length, number of leaves, fresh and dry weights).

Investigations of Lovato et al. (1992) showed that the growth of micropropagated grapevine plants was improved due to inoculation with different commercial arbuscular endomycorrhizal fungal inoculates. The authors reported that the infection levels were low (30%) and concluded that the intensity of infection is a relative parameter (Abbot and Robson, 1978). According to Douds et al. (1998), the physiological response of a plant is the result of the interactions between environment, plant, and fungus genotype. Increases in the growth rate are not always related to colonisation (Guillemin et al., 1992), but may be

influenced by other factors such as extension of the external mycelium and transport of nutrients from the soil to the host.

In Experiment 2, the oil palms were infected early, during the first six weeks after inoculation, by the used (V)AMF. Despite the relatively low percentage of (V)AMF infection (9-23%), which varied between the fungal isolates, the colonisation was highly effective in promoting P concentration of the inoculated clones at that early growth stage, which resulted in improved plant development. Differences in the ability of (V)AMF to colonise the root system are well known and may have played a part in the lower percentage of root colonisation observed in E.

columbiana (M2) inoculated plants after the first harvest.

Little is known about the early stages of (V)AM infection in micropropagated plants, through early root colonisation has been demonstrated to be very important during the delicate acclimatisation phase (Ravolanirina et al., 1990; Gianinazzi et al., 1990).

The infectivity of the mycorrhizal fungi was highly affected by the sterilisation of the growth substrate as could be seen in Experiment 3. The assessment of the root colonisation at this plant stage showed that the soil sterilisation influenced the degree to which the fungi colonised the root. In the sterilised soil, colonisation of the roots was 27-42%, while in the non-sterilised soil the colonisation was only 17-23%. The sterilisation of the soil reduced the indigenous fungal population of the soil, an effect that led to a decreased competition between the native and the introduced fungi.

Non-inoculated control plants of Experiment 3 grown in sterilised soil were not infected by mycorrhizal fungi. According to Munro et al. (1999) the absence of mycorrhizal infection indicates that there was no ingress of infective propagules during the experiment; this is also indicative of the negative impacts of soil sterilisation on mycorrhizal infection and growth in the absence of inoculation.

Weak mycorrhizal formation in the non-inoculated control plants, which occurred in all experiments after twelve weeks of post vitro growth, was due to indigenous fungi which were present in the native, non-sterilised soil. These colonisations indicated the natural levels of infection, which occur in plant roots under ‘normal’ nursery conditions. The low effectiveness of these fungi on plant survival and development was evident in this study. The (V)AMF isolates were able to compete with the native fungi and infected the roots earlier (see 1^st harvest) and to a higher extent than the indigenous fungi, showing that they were more effective in the development of a well functioning symbiosis.

In experiments of Vosatka et al. (2000), it was shown, as has already been stated, that the indigenous (V)AMF are not always the most effective fungi as regards either mycorrhizal

development or mycorrhizal growth response tested on strawberry plants (Koomen et al., 1988).

It can be concluded that in soils containing infective but inefficient mycorrhizal fungi, growth of micropropagated oil palms can be greatly improved by inoculation with introduced fungal isolates. The results of this study indicate that although mycorrhizal fungi are present in the soil, and that the roots are eventually colonised with the native endophytes, the early inoculation of micropropagated oil palms with selected more effective (V)AM fungi greatly improves the resistance of the plants to the stress situation after transplanting to post vitro conditions.