Penicillium radicum: studies on the mechanisms of growth promotion in wheat

The aims of this study were to investigate the P solubilising activity of Penicillium radicum and identify other possible mechanisms of plant growth promotion that were not related to P solubilisation. The plant chosen for the studies was wheat, the largest cereal commodity produced by Australian agriculture. Given the large area that is planted annually to wheat, this crop represents a large potential market for P. radicum-based inoculants. However, it is unlikely that P. radicum will be effective in all wheat growing regions and all environmental conditions seen in these areas. Research on the modes of action may help to identify the conditions where P. radicum has a good chance of being effective. The P solubilising activity of P. radicum was assessed by examining the amount of P released from rock phosphate (RP) in-vitro. The effectiveness of P. radicum to solubilise RP was compared to that of another P solubilising fungus, the isolate P. bilaiae RS7B-SD1. Both fungi were cultured in a liquid medium that contained either NO₃⁻ or NH ₄⁺ as the sole source of N. Changes in culture pH, soluble P and gluconic acid concentration were determined for both fungi in a 168 hour incubation in both buffered (100 mM Tris-HCl pH 7.8) and non-buffered medium. For P. radicum, the maximum concentration of soluble P was 6.8 fold higher in the presence of NH₄⁺ compared to NO₃⁻. In contrast, for P. bilaiae RS7B-SD1 the highest concentration of soluble P measured in the fungal culture was not significantly affected by N-source. In buffered medium, P. radicum did not appear to solubilise RP and levels of soluble P were generally <1 mg L⁻¹. In contrast, the RP solubilising activity of P. bilaiae RS7B-SD1 was not affected by buffering. Increased RP solubilisation with NH₄⁺ as the N source and lack of RP solubilisation in buffered medium suggested that acidification was the main mechanism of P solubilisation by P. radicum. RP solubilisation by P. bilaiae RS7B-SD1 was similar over the range of culture conditions tested and mechanisms of RP solubilisation are likely to be a combination of mechanisms that relate to both acidification and the production of organic anions. The effect of inoculation with P. radicum on plant growth and P nutrition was studied under glasshouse conditions using a sand culture assay that supplied defined sources of P. The plant growth and P response to P. radicum inoculation were determined in two separate experiments. In Experiment 1, plants were grown to pre-heading stage and supplied with either dibasic calcium phosphate (Ca-P), crystalline iron phosphate (Fe-P), rock phosphate (RP) or phytate (Pₒ) as the source of P. In Experiment 2, plants were harvested after 8 weeks and supplied with either NO₃⁻ or NH₄⁺ as the sole source of N and the P sources were either Ca-P or RP. In Experiment 1, the plant P response (defined as higher shoot P concentration and P uptake) to inoculation was dependent on the P source. The greatest plant P response to inoculation was observed for Ca-P and no significant P response was measured in plants that were supplied with Fe-P, RP or Pₒ. In pots that supplied Fe-P as the P source, there was an increase in shoot dry matter in response to P. radicum inoculation but this occurred without a concomitant plant P response. In Experiment 2, the plant P response to inoculation was dependent on the N source. In the presence of NH₄⁺, P. radicum significantly increased the availability of P sources RP and Ca-P. While there was no significant plant P response under NO₃⁻ supply, there was a significant increase in dry matter production due to P. radicum inoculation. When the data of Experiments 1 & 2 are taken together, results suggest that P. radicum possesses at least two mechanisms of plant growth promotion, (1) P solubilisation and (2) general growth promotion that is independent of P solubilisation. In agreement with P solubilisation in solution cultures, the P solubilisation mechanism of P. radicum in sand culture required NH₄⁺. The ability of P. radicum to increase plant growth independently of a plant P response gave further evidence of general growth promoting abilities of the fungus. While sand culture is a useful tool to elucidate the fungal mechanisms of plant growth promotion, this approach cannot fully reflect the complexity of the rhizosphere in non-sterile soil. Hence, a subsequent experiment was done to determine the effect of P. radicum on plant growth and P nutrition in a number of field soils. The P solubilising activity of P. radicum was determined in four Australian field soils using isotopic dilution. Three soils were chosen on the basis of their chemistry of P retention: (1) Minnipa soil from South Australia was chosen due to P retention associated with the highly alkaline calcareous nature of this soil; (2) Innisfail Queensland, in this soil P retention was dominated by reaction with Fe oxides; and (3) Mt Schank South Australia, a volcanic soil where P retention was predominantly associated with Al oxides. The fourth soil, from Mingenew Western Australia, was chosen due to previous reports that P. radicum inoculation increased the yield of field grown wheat (Bio-Care Technology, unpublished data). The four field soils were each labelled with KH₂ ³²PO₄ and the specific activity (³²P) of the wheat seedling tissue was measured after four weeks growth. When the data was averaged across all four soil types, inoculation with P. radicum caused a significant 11.7% increase in the shoot dry weight of these seedlings. However, P. radicum did not cause any consistent significant difference in the specific activity (³²P) of plants when compared to uninoculated control plants. These results suggested that P. radicum did not have a strong ability to solubilise P from the test soils, and mechanisms other than P solubilisation were in operation to stimulate plant growth. The production of plant growth regulators (PGR) was considered as a mechanism of plant growth promotion not related to P solubilisation. To further explore the hypothesis that the production of PGR acts as a mechanism of plant growth promotion, the ability of P. radicum to produce the auxin, indole-3-acetic acid (IAA) was investigated. Examination with thin-layer chromatography and the Avena coleoptile straight growth assay indicated that fractions of P. radicum culture medium with chemical characteristics similar to IAA (i.e. similar reaction to the Salkowski reagent and Rf as IAA) also possessed auxin-like activity. Using competitive enzyme linked immunosorbent assay (ELISA) it was found that in liquid culture amended with the precursor tryptophan, P. radicum produced IAA at concentrations up to 0.406 µM. These studies show that P. radicum can produce IAA under laboratory culture conditions. To determine the significance of IAA as a mechanism of plant growth promotion, further studies need to link effects on plant growth and development to the production of IAA by P. radicum. The ability of P. radicum to antagonise root pathogens and control root disease was considered as a further mechanism of growth promotion. Under in-vitro conditions, P. radicum produced hyphal growth patterns and enzymes (protease, β-1,3- and β-1,4-glucanase activity) that were indicative of hyperparasitism. Hyperparasitic growth was seen as hyphal coiling and branching of P. radicum against host hyphae of Rhizoctonia solani, Fusarium pseudograminearum and Pythium irregulare when these soilborne pathogens were studied in dual culture with P. radicum. The effect of P. radicum on the fungal root disease severity of take-all was studied using a seedling bioassay under glasshouse conditions. The ability of P. radicum to suppress take-all disease appeared to be related to the timing of P. radicum infection of wheat seedling roots and placement of the Ggt inoculum in relation to the roots. Compared to soils where Ggt inoculum was only distributed at distances >1 cm below the seed, uniform mixing of the Ggt inoculum throughout the soil negated the beneficial effect of P. radicum on plant growth and its ability to reduce take-all root lesion size. Conversely, early infection of wheat roots by P. radicum gave wheat seedlings some protection against take-all disease. Where treatment with P. radicum was effective, increasing the inoculum dose significantly reduced take-all lesion size. While P. radicum exhibited antagonism towards F. pseudograminearum, Py. irregulare, Bipolaris sorokiniana and R. solani cereal root pathogens in-vitro, further studies under non-sterile soil conditions are needed to evaluate the potential for P. radicum to reduce root disease caused by these fungi. In conclusion, it is unlikely that one single mechanism explains the beneficial effect of P. radicum on wheat growth. In-vitro studies showed that P. radicum has a number of attributes that could function as mechanisms of plant growth promotion. These attributes were, (1) P solubilisation, (2) production of IAA and (3) the ability to antagonise soilborne pathogens in-vitro and reduce the lesion size of the take-all disease in a seedling bioassay. Sand culture assays revealed that at least two plant growth mechanisms were in operation, (1) P solubilisation and (2) a general growth promotion mechanism that was independent of P solubilisation. In agreement with Whitelaw et al. (1999), the P solubilisation mechanism of P. radicum operates via an acidification mechanism. The effectiveness of this mechanism may be limited by the availability of NH₄⁺ in the rhizosphere. Since NH₄⁺ appears to be required for P solubilisation there may exist an interaction between P. radicum and ammoniacal fertilisers. This will have implications for its effectiveness in the field. In-vitro studies suggest that the general mechanism of growth promotion may be related to the production of PGRs such as IAA. In this aspect the known colonisation of the interior of wheat roots by P. radicum would ensure that IAA produced by the fungus is taken up by the root cell and less subject to chemical degradation and/or degradation by other soil microorganisms. Further studies are required to identify the effect of IAA production on plant growth and the effect of P. radicum inoculation on root disease severity in non-sterile soil.Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 200