Effects of Soil Sterilization on the Formation and Function of Two Strains of Pisolithus Tinctorius on Eucalyptus Urophylla*)

To examine the effects of soil microbial population on mycorrhizal development and function, Eucalyptus urophylla seedlings were inoculated with two Pisolithus tinctorius isolates and grown in sterile, partly sterile and non-sterile soil. The two isolates of Pisolithus were an effective isolate (H445) collected from under eucalypts in Australia and an isolate (H615) collected from under eucalypts in the Philippines. Soils used were infertile acid soils collected from field sites in Pangasinan, Luzon and Surigao, Mindanao. In both soils, the Australian Pisolithus H445 improved the growth of E. urophylla seedlings more than Philippine isolate H615. The uninoculated seedlings exhibited stunted growth typical of P deficiency. Height at 8 weeks was significantly taller in sterile than in non-sterile soil. A significant interaction effect of inoculation and soil sterilization on height at harvest was observed only in Surigao soil. Soil sterilization had a varied effect on mycorrhizal formation. In Pangasinan soil, root colonization by H445 was significantly greater in non-sterile soil than in sterile soil. Whereas in Surigao soil, root colonization was significantly reduced by 54% from partly sterile to non-sterile soil. On the other hand, H615 showed significant mycorrhizal colonization in non-sterile soil compared from those in partly sterile and sterile soils. The degree of infection did not necessarily correspond to growth promotion in E. urophylla seedlings. These results indicate that the performance of the H445 was markedly affected by the microbial flora of the two soils. Thus, its potential use in the Philippines needs to be thoroughly tested in the field before its widespread use in any inoculation program

EFFECTS OF SOIL STERILIZATION ON THE FORMATION AND FUNCTION OF TWO STRAINS OF PISOLITHUS TINCTORIUS ON EUCALYPTUS UROPHYLLA*)

To  examine  the  effects  of  soil  microbial  population  on  mycorrhizal  development  and  function, Eucalyptus urophylla seedlings were inoculated with two Pisolithus tinctorius isolates and grown in sterile, partly sterile and non-sterile soil. The two isolates of Pisolithus were an effective isolate (H445) collected from under eucalypts in Australia and an isolate (H615) collected  from under eucalypts in the Philippines. Soils used were infertile acid soils collected from field sites in Pangasinan, Luzon and Surigao, Mindanao. In both soils, the Australian Pisolithus H445 improved the growth of E. urophylla seedlings more  than Philippine isolate H615. The uninoculated seedlings exhibited stunted growth typical of P deficiency. Height at 8 weeks was significantly taller in sterile than in non-sterile soil. A significant interaction effect of inoculation and soil sterilization on height at harvest was observed only in Surigao soil. Soil sterilization had a varied effect on mycorrhizal formation. In Pangasinan soil, root colonization by H445 was significantly greater in non-sterile soil than in sterile soil. Whereas in Surigao soil, root colonization was significantly reduced by 54% from partly sterile to non-sterile soil. On the other hand, H615 showed significant mycorrhizal colonization in non-sterile soil compared from those in partly sterile and sterile soils. The degree of infection did not necessarily correspond to growth promotion in E. urophylla seedlings. These results indicate that the performance of the H445 was markedly affected by the microbial flora of the two soils. Thus, its potential use in the Philippines needs to be thoroughly tested in the  field before its widespread use in any inoculation program.Key words: Pisolithus tinctorius/Laccaria fraterna/Pinus patula/Inoculum/Seedlings/Growth

Research and Development Priorities for Smallholder Forestry in the Philippines

The Philippine Council for Agriculture, Forestry and Natural Resources Research and Development (PCARRD) corporate plan - CORPLAN 2004-2010 - sets out strategic science and technology activities for the agriculture, forestry and natural resources sectors for the the six years 2004-2010. The four banner programs embody PCARRD's strategic S&T directions. These are based on the principles of technology-based productivity and competitiveness; effective science technology-adoption link; conducive R&D environment; and good leadership, collaboration, and coordination. The PCARRD Forestry and Natural Resources concerns include forestry, environment, soil and water sectors. The specific areas of responsibility under forestry are wood-based resources, non-timber resources, agroforestry, protected areas and wildlife, bamboo and rattan, and environment. A major area of research interest involves agroforestry systems. Priority is being placed on: evaluating and documenting indigenous agroforestry systems, the suitability of agriculture commodity recommendations for agroforestry systems in multiple-use and buffer zones of watersheds and protected areas; the social, environmental and economic performance of agroforestry systems; and capacity building for the promotion of agroforestry systems. A number of research and development interventions are in operation with respect to timber utilisation and socio-economics, marketing and policy, including innovative and efficient wood processing and manufacturing technologies

Field performance of Eucalyptus urophylla inoculated with an introduced and idigenous strains of Pisolithus at three sites in the Philippines

The effectiveness of an isolate of Pisolithus from Australia was compared with a Philippine Pisolithus isolate in promoting the growth of Eucalyptus urophylla on three acid (pH 4.1-5.9, 0.005M CaCl2) sites in the Philippines (Pangasinan, Bukidnon and Surigao). Isolates of Pisolithus were taken from basidiocarps collected under eucalypts growing in Western Australia and from the Philippines. Generally, the introduced Pisolithus promoted greater wood volume of E. urophylla planted in dry marginal land (Pangasinan) and in moist logged-over area (Surigao) in the Philippines than the Philippine Pisolithus isolate. Root colonization by the two fungi did not vary but there was a difference in the root colonization levels between sites implying that the prevailing microclimatic conditions on each site had affected the performance of the ECM inoculants. In this study, the number of isolates tested was limited, thus, future field trials should include a wider range of ectomycorrhizal fungi. Further work is required to determine whether the growth responses measured at the two sites (Pangasinan and Surigao) are maintained until the trees are harvested

Assessing the Effect of Disturbances on Ectomycorrhiza Diversity

Ectomycorrhiza (ECM) communities can be described on a species level or on a larger scale at an ecosystem level. Here we show that the species level approach of successional processes in ECM communities is not appropriate for understanding the diversity patterns of ECM communities at contaminated sites. An ecosystem based approach improves predictability since different biotic and abiotic factors are included. However, it still does not take into account the hierarchical structure of the ecosystem. We suggest that diversity patterns of ECMs communities in forests can best be investigated at three levels. This hypothetical approach for investigation can be tested at sites of secondary succession in areas contaminated with metals. Once the diversity patterns are appropriately described by a hierarchical ecosystem approach, to the species level is used to explain these patterns by populational and ecotoxicological mechanisms

Ecto- and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings

In common with many plants native to low P soils, jarrah (Eucalyptus marginata) develops toxicity symptoms upon exposure to elevated phosphorus (P). Jarrah plants can establish arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations, along with a non-colonizing symbiosis described recently. AM colonization is known to influence the pattern of expression of genes required for P uptake of host plants and our aim was to investigate this phenomenon in relation to P sensitivity. Therefore, we examined the effect on hosts of the presence of AM and ECM fungi in combination with toxic pulses of P and assessed possible correlations between the induced tolerance and the shoot P concentration. The P transport dynamics of AM (Rhizophagus irregularis and Scutellospora calospora), ECM (Scleroderma sp.), non-colonizing symbiosis (Austroboletus occidentalis), dual mycorrhizal (R. irregularis and Scleroderma sp.), and non-mycorrhizal (NM) seedlings were monitored following two pulses of P. The ECM and A. occidentalis associations significantly enhanced the shoot P content of jarrah plants growing under P-deficient conditions. In addition, S. calospora, A. occidentalis, and Scleroderma sp. all stimulated plant growth significantly. All inoculated plants had significantly lower phytotoxicity symptoms compared to NM controls 7 days after addition of an elevated P dose (30 mg P kg−1 soil). Following exposure to toxicity-inducing levels of P, the shoot P concentration was significantly lower in R. irregularis-inoculated and dually inoculated plants compared to NM controls. Although all inoculated plants had reduced toxicity symptoms and there was a positive linear relationship between rank and shoot P concentration, the protective effect was not necessarily explained by the type of fungal association or the extent of mycorrhizal colonization

Effects of soil pH on the ectomycorrhizal response of Eucalyptus urophylla seedlings

To examine the effects of soil pH on ectomycorrhizal formation and function on Eucalyptus urophylla S. T. Blake, seedlings inoculated with nine ectomycorrhizal fungi (seven isolates of Pisolithus spp., Scleroderma cepa and Laccaria laccata collected under eucalypt stands in Australia and the Philippines) were transplanted into pots containing a non-sterile acid (pH 4·6) sandy loam amended with four levels of CaCO3 that raised the soil pH from 4·6 to 6·6 (5 mM CaCl2). Pots were placed in temperature-controlled water baths (28 ± 2 °C) inside an evaporatively cooled glasshouse for 9 wk. Increase in soil pH from 4·6 to 6·6 significantly decreased plant d. wt and shoot nutrient content of uninoculated and inoculated seedlings. Inoculation with four Pisolithus spp. (H445, H2144, M56 and H4003) significantly increased the growth of E. urophylla seedlings at pH 4·6. At pH 6·6, eight ectomycorrhizal isolates significantly improved total d. wt compared with those of the uninoculated seedlings. Pisolithus isolates stimulated seedling growth more than L. laccata whereas S. cepa was ineffective at all pH levels. Total d. wt of H445 inoculated plants grown in P-deficient (8 mg P kg-1 soil) soil was 147% more than that of uninoculated plants given the same P rate and was 70% that of plants fertilized with 64 mg P kg-1 soil (P64) at pH 46. At soil pH 5·8 and 6·6, M56 was the best growth-promoter for E. urophylla. These results indicate that soil pH can significantly alter the development and function of ectomycorrhizal fungi. Soil pH did not significantly affect mycorrhizal formation by the different ectomycorrhizal fungi. However, the percentages of mycorrhizal root tips formed by the different ectomycorrhizal fungi differed significantly. Pisolithus isolate H445 formed the highest percentage of colonized roots and highest total d. wt at pH 4·6 and 5·2, implying its potential for commercial use in acidic conditions

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

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