Pot trial analyses

Pot trials have predominantly been used to study a variety of effects and parameters first before a field trial is conducted. Frequently, generalisations are made from these trials without conducting field validation trials. In this study the use of a pot trial was conducted to determine the potential of using AM fungi in the rehabilitation of mine spoils (the MBT Site) with co-application of fertilisers. This setup was similar to the initial proposed objective for the field analysis from which comparisons would have been drawn from a field and pot trial perspective. However this comparison can now be done indirectly.

AM fungi have become a well-known bio-inoculant due to their numerous benefits to plants in agriculture and its uses in phytoremediation with no known environmental risks, unlike the use of chemical fertilisers that is a common practice. The use of fertilisers has been reported to affect AM fungal functionality (Ryan and Graham, 2002) as inorganic P fertilisers and animal manures are routinely applied to improve or meet crop P demand. The present study used Organic Tea and 3:1:5 NPK to boost the nutrient levels of the soil obtained from the mine spoil as well as to determine its effect on AM fungal root colonisation. This study demonstrated that in spite of the treatment and time period, the shoot height remained insignificantly different in some treatment pots (Table 3.2). The measurement of few numbers of plant shoot in each pot may have accounted for the insignificant difference in shoot heights among treatments. Therefore, in subsequent pot trials, thinning out of plants should be considered, especially in cases were small seedlings such as C. dactylon are difficult to count. Otherwise, attempts should be made to measure at least 50% of the shoots grown. The observed difference in shoot heights, between pots treated with Organic Tea only at 6 weeks and those treated with 3:1:5 NPK + AMF at 6 and 28 weeks (Table 3.2) is an indication that the overall significant effect of treatment obtained for shoot height was largely attributed to the presence of AM fungi. Also, the

insignificant difference between pots with each fertiliser + AM fungal treatment in terms of plant biomass (Table 3.2) indicates that the two fertilisers used are equally capable of improving plant growth and could be used in rehabilitation, if the desired outcome is to establish a vegetation cover over a short period of time. The rate of fertiliser absorption by plants can be a contributing factor to the observed treatment differences, as organic fertilisers are believed to be slow releasing compounds as opposed to inorganic fertilisers (Arden-Clarke and Hodges, 1988). Contrary to this generalisation, the Organic Tea + AMF increased shoot height at 6 weeks than the 3:1:5 NPK fertiliser, but reduced subsequently at 12 and 28 weeks period. This could mean that the Organic Tea was taken up by the plant at a higher rate than the 3:1:5 NPK fertiliser and would require more than one application for plant growth to be maintained.

The choice of fertilisers in most studies or systems is probably determined by the ease of application (Arden-Clarke and Hodges, 1988). Organic fertilisers such as the Organic Tea used in this study may not be desirable as compared to the 3:1:5 NPK due to its liquid nature, as application would require good spraying equipment or irrigation (fertigation). If no suitable irrigation is available, the Organic Tea will unlikely be used but the granular 3:1:5 NPK can be easily broadcast on Sites.

According to Rosen and Bierman (2005) some organic fertilisers such as manure or compost when not stored properly loose nutrients such as P and K through leaching.

Therefore, consideration into these factors may be the reason why inorganic fertilisers remain widely accepted. Studies have reported that different fertilisers such as superphosphate, NPK, urea, N2 and organic fertilisers increased biomass, shoot height and crop yield (Fletcher et al., 2004). Increasing attention is now being paid to organic farming practices whereby the use of compost, green manures and animal droppings as fertilisers are encouraged (Ashman and Puri, 2002).

The effects of fertilisers on AM fungi using field and pot trials have been well documented. Studies have reported that fertilisers that are high in P or N reduce AM fungal colonisation (Azcón et al., 2003; Xu et al., 2000; Kurle and Pfleger, 1994;

Thompson, 1994a). Azcón et al., (2003) demonstrated this when they evaluated how levels of N and P nutrients affected the rate of micronutrient uptake by Gl. mosseae using lettuce plants. They observed that the use of 0.1mM P increased colonisation of

Gl. mosseae, which contributed to the accumulation of micronutrients than when 0.5mM of P was applied. From the present study, the root colonisation analysis (Fig.

3.5), showed that 3:1:5 NPK fertiliser allowed 80% colonisation, while Organic Tea allowed 65% colonisation of C. dactylon roots by AM fungi. This study anticipated that the use of Organic Tea would result in a greater percentage colonisation because, if P was in a form unavailable to plants, mycorrhizal fungi could play a vital role in mineralisation of organic constituents. However, it was observed that the amount of P in the Organic Tea was 0.5% = 5000ppm (Appendix A1), which is equivalent to 125ppm when a 1:40 dilution was made. According to Swift (2006), mycorrhizal fungi exercise a greater benefit when soil P levels are at or below 50ppm (50mg/kg).

This means that the lower percentage colonisation observed with Organic Tea was as a result of high P level rather than the form of P present. Similar results were obtained by Maropula (2006) who determined the compatibility of four organic fertilisers (Organic Tea, Compost, Kelp and Nitrosol) with the same mycorrhizal inoculum used in this study. Furthermore, her study confirmed the reduced mycorrhizal colonisation (32%) caused by Organic Tea in Spinach roots, while the other organic fertilisers evaluated were non-compatible with AM fungi with the exception of Nitrosol; but were able to improved plant growth. In spite of this similarity in the reduced effect of Organic Tea, the difference between studies was that the Organic Tea was applied on a weekly basis as opposed to a once off application in this study. This could, account for a higher colonisation obtained with Organic Tea (65%) in this study compared to the 35% obtained by Maropula (2006). One of the disadvantages of using organic fertiliser is the inability to single out a particular compound that may be detrimental to AM fungal establishment. Contrary to this, the use of chemical fertilisers allows the selection of a compound that may be required in a reduced amount. An example is the 3:1:5 NPK as opposed to 2:3:2 NPK that was chosen in this study in order to reduce the P content. Besides the environmental hazards of these fertilisers such as leaching and runoffs, this study suggests that the use of a reduced P fertiliser in conjunction with an AM fungal inoculant for plant growth will be more efficient and cost effective for crop yield or establishing a vegetation cover (Khan, 2006; Xu et al., 2000).

The pot trial can be compared indirectly to the field trial at the MBT Site in terms of percentage root colonisation. It was observed that the field trial, which had 9.4% root colonisation at 9 months (Table 3.8), was approximately 7 to 8 times less than the

percentage colonisation obtained in the pot trial with the two-fertiliser treatments.

This high variation between the results could be associated to different growth systems. In a controlled environment plant growth and subsequent AM fungal colonisation will not be affected by any environmental factors such as rainfall, temperature, humidity, or wind. Therefore, the problem of inadequate irrigation encountered in this study could have affected the AM fungal colonisation in the field.

Although AM fungi are known to improve drought tolerance of plants, germination of spores and fungal propagules prior to colonisation requires moisture (Al-karaki et al., 2004). Al-karaki et al., (2004) demonstrated this by determining the colonisation of Gl. mosseae and Gl. etunicatum in a field grown wheat under well-watered and poorly watered conditions. It was observed that the colonisation of these AM fungal species was higher (41.3%) than the percentage colonisation in water stressed plant (18.8%), confirming the need for water and its influence on AM fungal colonisation. This is to say that it is only when the relationship has become established that AM fungi can improve drought tolerance (Augé, 2001).

Kurle and Pfleger (1994) stated that species of AM fungi differ in their response to chemical fertilisers. To determine this, Caravaca et al., (2006) found that among three species of AM fungi (Gl. mosseae, Gl. intraradices and Gl. deserticola), the latter two species produced an increased effect on shoot biomass of Retama sphaerocarpa seedlings in combination with medium dosage of liquid organic amendment obtained from dry olive residues. Gl. mosseae produced an increased effect on plant biomass when inoculated alone. Also Podeszfinski et al., (2002) observed the predominance of G. margarita and S. calospora at Sites that were high and low in P, respectively. This means that the higher percentage colonisation of plant roots obtained with 3:1:5 NPK rather than the Organic Tea could be as a result of AM fungal species tolerance to fertiliser types. These results highlight the potential use of AM fungal inoculant for a rehabilitation of an overburdened soil with reduced P fertiliser.