Soil factors affecting the formation and function of Pisolithus-Eucalyptus urophylla ectomycorrhizas in acid soils in the Philippines

Abstract

With the present dwindling area of forest cover in the Philippines, the government is faced with reforestation of the vast marginal grasslands to meet the ever increasing demand for raw materials for the pulp and paper industries. Success in reforestation is low due to the inherent infertility status and acidic nature of Philippine soils. In addition, some reforestation sites have high concentrations of heavy metals such as chromium (Cr) and nickel (Ni) contributing to low survival and poor growth of trees in plantations. Eucalypts are favoured reforestation species because of their fast growth, multiple uses and adaptability to a wide range of sites. Survival of eucalypts in inhospitable sites may be due to their mycorrhizal associations. Mycorrhizas, symbiotic associations between plant roots and higher fungi, can increase tree growth in adverse sites mainly through increased nutrient uptake particularly phosphorus. Pisolithus is an ectomycorrhizal fungus that has been identified as a potential growth promoter for eucalypts in plantations in soils deficient in P and with acidic pH. Initial field trials in the Philipines have shown that Pisolithus can increase growth of pines and eucalypts and the technology has been adapted in some nurseries. Formation of ectomycorrhizas and the survival of the symbiosis depend on three main components: the host plant, the soil and the fungus. This thesis investigates the effect of key soil factors (pH, soil microorganisms and heavy metals) that affect the formation of Pisolithus ectomycorrhizas on the roots of Eucalyptus urophylla S. T. Blake seedlings on acid soils in the Philippines. In chapters relating to soil pH (Chapter 2), soil microbes (Chapters 3 and 4) and field trials (Chapter 7), Pisolithus isolates collected from under eucalypts in Western Australia and in the Philippines were used while an isolate from under eucalypts growing in a nickel mining residue in New Caledonia was included in chapters relating to heavy metals (Chapters 5 and 6). Experiments were conducted in the laboratory, glasshouse, nursery and in the Field in the Philippines as follows: a). A glasshouse experiment was established to determine the effect of liming (with CaCO3) an acidic (pH 4.6, 0.005 M CaCl2) non-sterile sandy loam from Western Australia (Bodallin) on the formation of ectomycorrhizas by nine ectomycorrhizal fungi (seven Pisolithus spp., a Laccaria laccata and a Scleroderma cepa) on E. urophylla seedlings. Soil pH did not affect mycorrhizal formation by the different ectomycorrhizal fungi. However, percent mycorrhizal root tips colonized by the different ectomycorrhizal fungi varied greatly. A Pisolithus from Western Australia colonized more roots and promoted the highest dry weight at pH 4.6 than the other ectomycorrhizal fungi studied. Generally, Pisolithus spp. stimulated seedling growth more than L. laccata while S. cepa was ineffective at all pH levels. b). A nursery experiment was conducted in the Philippines to examine the effect of soil microbes present in soils collected from three field sites (Pangasinan, Bukidnon and Surigao) in the Philippines on the formation of ectomycorrhizas by isolates of Pisolithus from Western Australia (H445) and from the Philippines (H615) on￡. urophylla seedlings. Soils were either fumigated with methyl bromide, reinfested (1% unfumigated soil added into 99% fumigated field soils, w/w) or unfumigated. The percentage of mycorrhizal root tips colonized by the Western Australian isolate was markedly affected by biological factors in unfumigated soil; highest infection was observed in reinfested soils while for the Philippine isolate it was highest in unfumigated soils. Seedlings transplanted into Bukidnon soil were affected by heavy metals resulting in very poor growth, toxicity symptoms and plant death. A follow-up experiment on the effect of soil microbes on the formation of ectomycorrhizas by an Australian and a Philippine Pisolithus isolates was conducted in a glasshouse in Western Australia. Soils collected from Western Australia (Bodallin) and from two field sites in the Philippines (Pangasinan and Surigao) were either autoclaved or left non-sterile and were added (1% w/w) into pasteurized yellow sand. The Australian Pisolithus was more effective in colonizing root tips of E. urophylla with added nonsterile Pangasinan and Surigao soils and in promoting plant dry weight than the Philippine isolate. Percentage of root tips colonized by the two isolates in unamended pasteurized yellow sand was similar. However, the addition of sterile Bodaliin, Pangasinan and Surigao soils decreased the percentage of lateral root tips colonized by the Australian Pisolithus by 28%, 40% and 43%, respectively. Percentages of mycorrhizal root tips were higher with added non-sterile Pangasinan (55%) and Surigao (60%) soils than those of the Philippine isolate (45% in Pangasinan and 15% in Surigao). The addition of sterile or non-sterile Bodaliin soil decreased the percentage of root tips colonized by either isolates. c). The effect of chromium and nickel cations on the rate of mycelial growth and dry weight of three isolates of Pisolithus and on the formation of ectomycorrhizas on E. urophylla were investigated in vitro. Nickel was more toxic to fungal growth and ectomycorrhiza formation than Cr. The fungi differed in their tolerance to the heavy metals and isolates from the Philippines and New Caledonia grew at higher Ni levels than the Australian isolate. Media concentrations of 50 mg Cr L-1 and 4 mg Ni L-1 reduced the percentage of root tips colonized by the fungi. d). The effect of Ni on the formation of ectomycorrhizas by three isolates of Pisolithus on E. urophylla was studied in two separate experiments in a glasshouse. Nickel rates in one experiment were: 0, 1.5 and 3 mg Ni kg-1 pasteurized yellow sand while in the other experiment, Ni rates were: 0, 6, 12, 24 and 48 mg Ni kg-1 sand. The Pisolithus isolate collected from a Ni contaminated site in New Caledonia was more tolerant to Ni than the other two isolates. In both experiments, the New Caledonian isolate produced the highest percentage of mycorrhizal root tips (82% to 87%) and root colonization was not affected by the addition of 12 mg Ni kg-1 sand. Plant dry weight of seedlings inoculated with this isolate was not affected by the addition of 6 mg Ni kg-1 sand but it was reduced with the addition of 12 mg Ni kg-1. Inoculation with Pisolithus did not prevent the uptake of Ni into the shoots but Ni toxicity was minimized through a dilution effect brought about by the increase in plant biomass. e). Field trials were established in three sites (Pangasinan, Luzon; Bukidnon and Surigao, Mindanao) in the Philippines to determine the effectiveness of isolates of Pisolithus from Western Australia and from the Philippines in promoting early growth (26 months) of E. urophylla trees. After 2 years, root infection levels in Pangasinan by the two isolates of Pisolithus were higher (30% and 36%, respectively) than in Bukidnon (15% and 5%) and in Surigao (5% and 2%). The Australian isolate generally promoted greater wood volume (37% at 26 months in Pangasinan and by 237% at 18 months in Surigao, relative to the uninoculated treatment) than the Philippine isolate (16% in Pangasinan and by 153% in Surigao). The two isolates promoted similar wood volume (63% and 69%) of trees 26 months after outplanting in Bukidnon. Pending further research with a wider range of fungal isolates, it is concluded that the Australian Pisolithus isolate can be used to inoculate eucalypts for reforestation on problem soils in the Philippines similar to those in Pangasinan, Bukidnon and in Surigao. Pisolithus isolates collected from under eucalypts growing in heavy metal contaminated sites, including the New Caledonian isolate used in this study can be further tested in Bukidnon and other areas in the Philippines with soils of ultramafic origin. Future work should include: i) the introduction of a wider range of ectomycorrhizal fungi from Australia and comparison with endemic strains in a range of soils in order to identify those beneficial ectomycorrhizal fungi that can survive and persist for a long time in the field under a wide range of climatic conditions, ii) isolation and identification of soil microbes present in different soils which are beneficial to ectomycorrhizal symbiosis, iii) monitoring of responses of eucalypts to inoculation over a longer period in the field and iv) the development of markers for easier monitoring of persistence of introduced fungi in the field. Thus, the work described in this thesis will act as a firm foundation for the future development of eucalypt plantation forestry in the Philippines