Ectomycorrhizal inoculation of Anisoptera thurifera (Blanco) Blume and Shorea guiso (Blanco) Blume with ectomycorrhizal fungi in Philippine red soil

May 5, 2006 by

Ectomycorrhizal inoculation of Anisoptera thurifera (Blanco) Blume and Shorea guiso (Blanco) Blume with ectomycorrhizal fungi in Philippine red soil

NS Aggangan1, JS Aggangan2, JCO Bulan2 and CAS, Limos2

1National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baños, College, Laguna 4031, Philippines: Email nea@laguna.net; 2Former Students, Philippine Science High School, Diliman, Quezon City, Philippines

 

Abstract

 

Rooted cuttings of Anisoptera thurifera (Blanco) Blume and Shorea guiso (Blanco) Blume were prepared and inoculated with mycelial beads containing vegetative mycelia of ectomycorrhizal fungus: Pisolithus sp. 1 (PTG),Pisolithus sp.2 (H6394), or Astreus sp. and grown under nursery conditions. Pisolithus sp.1 was collected from an Acacia mangium stand, Pisolithus sp.2 from Eucalyptus stand while Astreus sp. from dipterocarp forest. After four months, Astreus sp. promoted the highest root colonization (38%), height increment (86%), fine root (51%), coarse root (27%), shoot (43%) and total (40%) dry weight of A. thurifera over the control treatment. It also promoted the highest shoot P uptake (41%) while Pisolithus sp.2 promoted the highest fine root P uptake (0.273 mg/root). Pisolithus sp.2 promoted the highest height increment (100%) in S. guiso. Uninoculated cuttings had the lowest height increment, dry weight and P uptake. A longer nursery and field experiments should be conducted to achieve a complete evaluation of the physiological functions of ECM association. More ECM strains associated with dipterocarps should be tested for selection of host compatibility.

Keywords: Ectomycorrhiza, Shorea contorta, Anisoptera thurifera, Pisolithus, Astreus

Introduction

Philippine dipterocarps such as Anisoptera thurifera (Blanco) Blume and Shorea guiso (Blanco) Blume are considered at present as endangered species. In the sixteenth century, 90% of the land was covered by 27.5 million ha of forest, having commercially valuable species (Westoby 1989). As of 1990, only about 6.5 million ha (21.5% of the total land area) has old-growth forest of which less than 1 million ha are classified as virgin dipterocarp forests (DENR 1990).

In order to compensate for the loss of dipterocarp forests, forest plantation establishment is one of the top priorities of the Philippine government to ensure an adequate and sustained supply of timber and other wood products for local and export market, and to conserve endangered Philippine dipterocarps (Aggangan et al. 1997). Attempts have been unsuccessful due to soil acidity, most species require partial shade (50—70%) for the survival and growth of dipterocarp seedlings to pole stage (Mauricio 1987, Adjers et al. 1995, Tuomela et al. 1996, Aggangan 1996), and the irregularity of dipterocarp seed production.

Due to irregularity in the seed production of dipterocarps, macropropagation by cuttings is usually done to produce planting materials in spite of the danger of genetic erosion. The growth of dipterocarp cuttings is normally slower than from seeds and this requires longer rearing period in the nursery before they can be out planted in the field.Thus, there is an urgent need to develop techniques that ensure high survival and fast growth of dipterocarps to rehabilitate degraded lands.

Another constraint to plantation establishment is the soil properties.These soils are generally acidic with low nutrient availability especially nitrogen (N) and phosphorus (P) and lack other essential nutrients (Fernandez et al. 1980, Maglinao 1988).About half of the total land area of the Philippines is classified as acidic (below pH 5.5, Maglinao 1985, 1988). These soils geographically occur throughout the country (Atienza 1989, Badayos 1990).

Establishment of exotic plantations usually requires selection of fast growing genotype, nutrient application (particularly nitrogen and phosphorus), and mycorrhizal inoculation (Malacjzuk et al. 1994, Garbage et al. 1988, Aggangan 1996). Ectomycorrhizal (ECM) fungi have shown a pivotal role in the growth and survival of dipterocarp seedlings in degraded sites (Dela Cruz et al. 1988).ECM fungi mobilize plant water and nutrient uptake via hyphae and increase plant resistance to environmental stresses (Harley and Smith 1983, De la Cruz and Aggangan 1990, Mejstrick 1989).Fast growth ensures a quick supply of raw materials for the increasing market for wood (Aggangan 1996).

This research was done to: determine the effect of three ECM fungi on the early growth and development of A. thurifera and S. guiso grown in a nutrient deficient red soil, quantify phosphorus levels present in the mycorrhizal plant system and select ECM fungi that best sustain nutrient accumulation in the said dipterocarp species.

Materials and methods

Experimental design

Two separate experiments (A. thurifera and S. guiso) were conducted using three strains of ECM fungi in a screen house at the College of Forestry and Natural Resources (CFNR) and at the National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines at Los Baños, College, Laguna, following a Randomized Complete Block Design (RCBD) with 12 replicates. The inoculation treatments were: Uninoculated (Control), inoculated with Pisolithus sp. 1 (PTG), inoculated with Pisolithus sp.2 (H6394) and Astreus sp. (99-4).Three fungi were used in A. thurifera and only two (Pisolithus spp.) in S. guiso.The experiments were established in May 2002.

Soil collection and preparation

The soil (0 — 15 cm depth) used was collected in a grassland area in Caliraya, Laguna in April 2002 and was brought to BIOTECH, UPLB.Caliraya soil was chosen because it is one of the problem soils in the Philippines.The soil was air-dried, pulverized and passed through a 5 mm screen and oven-dried for 3 days at 100 oC.Two hundred fifty grams of dry soil were dispensed into 4” x 8” PE bags. The soil is bright red; texture is clay loam with an acidic pH ranging from 4.2 to 5.0 (1:2 soil: 0.005 M CaCl2).The chemical analysis are as follows:1.12% organic matter (Walkey-Black Method by Walkey and Black, 1947), and 0.03% N (Modified Kjeldahl Method by Black, 1965), 0.33 me/100 exchangeable K and 3.51 ppm available P (Bray No. 2 by Dewis and Freitas, 1970).

Ectomycorrhizal inoculants

The species of ECM fungi were Astreus sp., and two strains of Pisolithus (Pisolithus sp.1 coded PTG and Pisolithus sp.2 coded H6394).These isolates are collections in the Mycorrhiza Laboratory, BIOTECH, UPLB, College, Laguna.Pisolithus sp.1 was collected under Acacia mangium stand in Malaysia, Pisolithus sp. 2 from Eucalyptus trees growing in a nickel site in New Caledonia and Astreus sp. from dipterocarp forest in Mt. Makiling, Philippines.Astreus sp. produces dark colored hyphae while Pisolithus spp. produce golden yellow mycelia on Modified Melin Norkrans medium (MMN, Marx 1969). ECM fungi were initially grown in MMN agar medium.After three weeks, mycelial disks (1cm diameter) were taken from the actively growing portion of the culture and were transferred in MMN liquid medium.Mycelial beads were produced from macerated four-week old culture.

Dipterocarp species and propagation

Anisoptera thurifera (common name: palosapis) and S. guiso (common name: guiho) were chosen because of their high commercial value. Stem cuttings were obtained from selected parent representatives maintained at the CFNR, UPLB, College, Laguna.The cuttings were cut into 2 nodal length (approximately 2-4 in), rinsed with water, the leaves were cut into halves to reduce respiration which can facilitate faster rooting and recovery, treated with rooting hormone (100 ppm IAA) and grown in trays containing sterilized river sand.The trays were placed inside plastic bags (Figure 1A).

Ectomycorrhizal inoculation

Inoculation was done during transplanting of rooted cuttings (two months)into 4”x 8” PE bags filled with red soil.The rooted cuttings were inoculated with vegetative mycelia of Pisolithus and Astreus sp., entrapped in alginate bead at a rate of one bead per seedling. The seedlings were watered to field capacity.The inoculants were placed approximately 4 cm depth, beneath the root system of the plant.The inoculated rooted cuttings were placed inside plastic bags to maintain relative humidity and reduce desiccation, which can foster faster seedling recovery.One plastic bag contained cuttings with similar fungus to reduce cross contamination.After two weeks, the plastic bags were partially opened and were gradually exposed from low (25-50%) to higher light intensity (75-85%) for another two weeks (Figure 1B-D).

Parameters measured

1. Height and height increment.Initial height of the sprouted shoot (not the original planting stock) was measured during transplanting. Height of the sprout was measured from the node where the sprout originated up to the apical shoot tip. Measurement was done once a month for 4 months. Height increment was calculated as the difference between the monthly height measurement and the initial height.

2. Diameter.Initial diameter was measured during transplanting and the final diameter was measured after four months. Diameter increment was calculated similar to that of height increment. Diameter of the sprout was taken 1 cm above the node where it originated.

3. Plant biomass.Five replicates from each of the dipterocarps were harvested at the end of the fourth month. The sprouted shoot was cut at the base of the node of the original parent material while the roots were also cut where they originated. The fine roots (diameter less than 0.5 mm) and the coarse roots were separated. Fine roots, coarse roots and the shoots were wrapped separately with tissue paper, oven-dried at 70oC for two days and dry weights were obtained.

4. Mycorrhizal infection.Fine root samples were chopped into 2-3 mm length and evenly spread in petri dishes to assess mycorrhizal colonization under a dissecting microscope. Mycorrhizal colonization was assessed using the gridline intersection method (Giovannetti and Mosse 1980).

5. Phosphorus concentration and uptake.Powdered (0.5 g) dried fine roots and shoots from each treatment was digested with concentrated H2SO4 and 30% H2O2. P concentration was obtained by colorimetric method (Cunniff 1995).

Root sectioning and micrography

The microtechnique work was done at the Electron Microscopy Laboratory BIOTECH, UPLB. Root samples underwent gluteraldehyde fixation, buffer washing, series of dehydration and infiltration and flat embedded in LR (London Resin) white. Semi-thin sections were obtained using Latta and Hartmann triangular glass knifes after which cut sections were stained with toluidine blue or methylene blue. The samples were observed and photographed under a light microscope.

Statistical Analysis

Data on height increment, diameter, P concentration, P uptake, mycorrhizal colonization and shoot, fine root, coarse root and total dry weight were analyzed using analysis of variance (ANOVA) of raw data and treatment means were compared using Duncan’s new Multiple Range Test (DMRT) and Least Significant Difference (LSD) at p<0.05 (Duncan, 1955). The relationship between plant growth and plant P concentration and uptake with mycorrhizal infection was evaluated using regression analysis. Statistical Analyses were done using MSTATC statistical computer program.

Sources: 8th Round-Table Conference on Dipterocarps

Latest news

Oldest news

Filed Under: Sivilculture
[logo-slider]