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Studies of vesicular-arbuscular mycorrhiza in Wanagama I Forest Research Center, Yogyakarta, Indonesia Sancayaningsih, Retno Peni 1991

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STUDIES OF VESICULAR-ARBUSCULAR MYCORRHIZA IN WANAGAMA I FOREST RESEARCH CENTER, YOGYAKARTA, INDONESIA by RETNO PENI SANCAYANINGSIH Sarjana, Gadjah Mada University, 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DECREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF SOIL SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1991 (c) Retno Peni Sancayaningsih, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of £o / l _ SC/crAiCe The University of British Columbia Vancouver, Canada Date r e W M / 3 . y / y , '99/ DE-6 (2/88) ABSTRACT Three studies were conducted on VA mycorrhiza in Wanagama Forest Research Center, Yogyakarta, Indonesia. The first was on VA mycorrhizal status of four forest species (Acacia mangium, Acacia holosericea, Tectona grandis, and Swietenia macrophylla) from plantations and nurseries of Acacia mangium and Tectona grandis. Samples from the field were only taken during the dry season, June 1988. These four six-year old forestry species were mycorrhizal. Nursery plants had higher VAM colonization than the plantation roots and both Acacia species have higher percent colonization than the other two species. Available phosphorus in calcareous soils is low and seems not to be a major contribution to the variation of VAM colonization. Potassium and sodium were more important in this case even though their role could not be determined in this study. The second study was conducted to determine VAM fungal species associated with the plant species. There were 16 different spore types belonging to the genera Glomus, (the most common found), Sclerocystis, Scutellispora, and probably Acaulospora. Type of inoculum and host compatibility were suggested as important factors in the success of pot culture study. The third study was carried out in a growth chamber to determine Acacia spp. response to single VAM fungal species and mixed species inoculum. Single species inoculum in both Acacia was observed to improve biomass and plant growth better than the mixed inoculum. Acacia mangium performed better with Glomus versiforme than did A. holosericea. Host compatibility, effectiveness of VAM spore inoculant, infectivity and environmental factors have major effects on plant growth responses. Study of tropical VAM requires further basic research, including taxonomy. Experimental procedures such as pot culture technique, type of inoculum, growth media and host plant specificity along with evaluation of appropriate soil chemical analysis also require i i i further elaboration. These types of studies are needed to understand the relationship between VAM and the environment and in the application studies in agriculture and forestry. This information is especially important in tropical countries, where little research results and limited resources, such as for fertilizers, are available. •* iv AKNOWLEDGEMENTS My deepest thanks are adressed to Dr Shannon Berch who patiently supported and guided me since I started from zero in UBC; to EMDI, CBIE agencies of CIDA and KLH (Ministry of Population and Environment) of Indonesia which gave me the opportunity to achieve a better education. Also to my institutions, Gadjah Mada University, Dept. of Biology and Center for Environmental Studies which supported me to study abroad. My grateful thanks also go to my committee members : Drs Ballard, Berch, Bomke, Lavkulich who helped me through the hard times. Thanks for the assistance during field work to P. Suyanto, and all staff in Wanagama Forest Research Center. Sincere thanks also go to the Dept. of Soil Science, UBC, for providing me a warm environment, to nice friends in the soil biology lab, Aaron, Barb, Bill, Buffy, Eliz, Cuoping, Jeff, Marcia, Sharmin, Silvia and all Indonesian students in UBC who cared for me while I was in Vancouver. Special thanks to Cursharan, who cared about me in his special way. Deep appreciation to Surantoko and Aji, 'suami dan anak tercinta' to my mom and mom-in-law, brothers and sister who support me with love. My daddy who taught me with an endless spirit in his past life. This is the way I could repay what I got from all of you. V TABLE OF CONTENTS Title Page i Abstract Aknowledgements >v Table of Contents v List of Tables vii List of Figures viii Chapter I : General Introduction 1 1. STUDY SITE 1 2. FORESTRY IN JAVA.. 3 3. PLANTATION HISTORY IN WANAGAMA 3 4. MYCORRHIZA 6 Chapter II : Mycorrhizal status of four Indonesian forestry species 8 1. INTRODUCTION 8 2. MATERIALS AND METHODS 10 2.1. Study site 10 2.2. Sampling procedure 14 2.2.a. Root distribution .' 14 2.2.b. Mycorrhiza study 15 2.2.b.1. Plantation study 15 2.2.b.2. Nursery study 18 2.2.c. VAM assessment 18 2.2.d. Soil analysis 18 2.2.e. Data analysis 19 3. RESULTS 20 3.1. Fine root distribution 20 3.2. VAM colonization 23 3.3. Soil chemical properties 26 4. DISCUSSION 32 4.1. Fine root distribution 32 4.2. Percent VAM colonization 32 4.3. Selected soil nutrients 35 5. CONCLUSIONS 37 Chapter III : VAM occurence on four Indonesian forestry species 38 1. INTRODUCTION 38 2. MATERIALS AND METHODS 42 3. RESULTS AND DISCUSSION 45 3.a. Sclerocystis sp 46 3.b. Sporocarpic Glomus sp 50 3.c. Nonsporocarpic Glomus sp 57 3.d. Scutellispora sp 74 3.e. Unidentified genera 77 4. CONCLUSIONS 82 VI Table of Contents (cont'd) Chapter IV : Response of Acacia mangium and A. holosericea to single and mixed species of VAM fungal inoculum 83 1. INTRODUCTION 83 2. MATERIALS AND METHODS 85 3. RESULTS 87 3.a. Leaf morphology and plant growth 87 3.b. Root and shoot dry weight 91 3. c. VAM colonization 94 4. DISCUSSION 95 4. a. Shoot height and stem diameter 95 4.b. Root and shoot dry weight 96 5. CONCLUSIONS 99 Chapter V : General Conclusions 100 Literature cited 102 Appendix 109 1. Rainfall and Anova Tables 109 2. Calculation 119 LIST OF TABLES Table 1 Root characteristics of four studied species 21 Table 2a Distribution of fine roots based on mean fresh weight of 9 samples at each location 22 Table 2b Mean root fresh weight 23 Table 3 Mean percent VAM colonization 26 Table 4 Mean value of some selected soil nutrients 28 Table 5 Correlation between percent VAM colonization and selected soil nutrients 29 Table 6 Result of backward stepwise of multiple regression analysis for selected soil nutrients which contribute to the variation of percent VAM colonization 30 Table 7a. Height and stem diameter of A. mangium and A. holosericea inoculated by C. versiforme and local soil inoculant in 2 month growing period 89 Table 7b. Height and stem diameter of A. mangium and A. holosericea inoculated by C. versiforme and local soil inoculant in 4 month growing period 89 Table 8a. Mean root and shoot dry weight of Acacia mangium and A. holosericea inoculated with local soil inoculant and Glomus versiforme within 2 month growing period 92 Table 8b. Mean root and shoot dry weight of Acacia mangium and A. holosericea inoculated with Canadian soil inoculant and Glomus versiforme within 4 month growing period 92 Table 9 Plant responses (percent change relative to control) based on shoot and root dry weight of A. holosericea and A. mangium with Canadian soil and Glomus versiforme inoculation 97 LIST OF FIGURES Figure 1 Map of Java island (1 : 5,000,000) 11 Figure 2 Map of Cunungkidul subdistrict (1 : 500,000) 12 Figure 3 Map of Wanagama I as the study site (1 : 25,000) 13 Figure 4 View of Wanagama Forest Research Center 16 a) Reforestation area b) Part of nursery c) Soil profile Figure 5 Comparison between 6 year old plantation of Acacia mangium (Am) and A. holosericea (Ah) 17 Figure 6 Sampling plots of root and soil samples in fine root distribution study 18 Figure 7 a) Coiled hypha of VAM fungus (x 2,625) 24 b) Arbuscules of VAM fungus (x 6,250) 24 Figure 8 a) Lobed vesicle shape found in 7". grandis (x 2,625) 25 b) Common vesicle types (x2,625) 25 Figure 9 VAM spore collection using wet sieving method 44 Figure 10 Murograph and pictures of U type spores (Sclerocystis sp.) 47 Figure 11 Murograph and picture of spore L/L-U type (Sclerocystis sp.) 51 Figure 12 Murograph and pictures of L/LU type spores (Glomus sp.) 52 Figure 13 Murograph and picture of L type spores (Glomus sp.) 54 Figure 14 Murograph and pictures of U spore type {Glomus sp.) 56 Figure 15 Murograph and pictures of EL Flex, spore type (Glomus sp.) 58 ix List of Figures (cont'd) Figure 16 Murograph and picture of OT21-type spores (Glomus sp.) 61 Figure 17 Murograph and spore picture of L/LU type (Glomus sp.) 64 Figure 18 Murograph and picture of E-L-U type spores (Glomus sp.) 66 Figure 19 Murograph and picture of L or EL spore type (Glomus sp.) 68 Figure 20 Murograph and pictures of OAm10 spore type (Glomus sp.) 69 Figure 21 Murograph and picture of T06 spore type (Glomus sp.) 71 Figure 22 Murograph and pictures of OT3 spore type (Glomus sp.) 73 Figure 23 Murograph and picture of U-2M spore type (Scutellispora sp.) 75 Figure 24 Murograph and picture of U-M spore type (Scutellispora sp.) 76 Figure 25 Murograph and picture of U-LFlex spore type (Acaulospora ?) 78 Figure 26 Murograph and picture of U-M-C spore type (Acaulospora ?) 80 Figure 27 a) Leaf transition from juvenile into mature leaf type after 4 week growing period. b) Comparison between A. mangium and A. holosericea leaf transition within the same growing period 88 Figure 28 a) Acacia mangium grown in 2 months 90 b) Acacia holosericea grown in 2 months 90 1 Chapter I : GENERAL INTRODUCTION 7. STUDY SITE ' This study was conducted in blocks number 17 and 5 in a 79.9 ha region of the Wanagama Forest Research Center, Cunung Kidul Subdistrict, Yogyakarta, Indonesia, which is located at 3°42'7" - 3°42'57" E and 7°52 '47" - 7°53 '20" S. This region has a complex topography from flat, to hilly (Fig. 4a), up to 600 m elevation in the north with a slope of 15 - 25%. Geologically, Gunung Kidul Subdistrict is composed of mainly limestone parent material. According to Khan (1961 in Anonymous 1988), Gunung Kidul area is divided into 4 geographical units (see diagram). These are : a. Baturagung hilly group b. Wonosari basin c. Panggung zone d. Karst zone 300 m 200 m 600 m G. sewu C^) Wonosari (b) Baturagung 9 The location of the Wanagama Forest Research Station is at the northern central part of Wonosari which is flat in the south and hummocky at the north with 6 summits of less than 300 m elevation. With a comparatively short (2 - 3 months) period of erosive rainfall, about 2116 mm annually, the soil development of this region is slow and according to Soepraptohardjo et al. in Anonymous (1988), the soil of Wanagama I forest can be categorized into 3 types : a. Lithosol (FAO/Unesco) or Lithic Troporthent (USDA). Yellowish grey to dark grey color, very thin solum without horizon differentiation. This soil type is found on the hill slopes with high erosive rainfall. b. Rendzina (FAO/Unesco) or Rendoll (USDA). Dark grey to black color, high clay content and solum thickness less than 0.5 m. Soil pH neutral to alkaline, high Ca and Mg content. c. Red Yellow Mediterrane or Luvisol (FAO/Unesco) or Tropudalf (USDA). Vertic, lithic and calcic luvisol are found. Generally these types of soils are associated in this area with a C, D, or F climates, with an annual rainfall of 800 - 2,000 mm and a 3 - 5 month dry period (Anonymous, 1988). Soil of the research site is red or yellowish red, with a high clay content, high Ca and Mg and in dry periods, shows cracking. Solum thickness is 50 - 100 cm. The climate at the Research Center is categorized into climates C and D (Schmidt and Ferguson class) with only 5 - 15% of annual rainfall collected during a 4 month dry period (Anonymous, 1988). As in most calcareous soils, free calcium carbonate and magnesium create problems of land and water use for crop production (Kadry, 1973). These two elements control the availability of phosphorus through immobilizing and fixing as calcium phosphate minerals (Cole and Olsen, 1953; Olsen, 1973). The minerals commonly found are hydroxyapatite, fluoroapatite, tricalcium phosphate, octacalcium phosphate, dicalcium phosphate and 3 dicalcium phosphate dihydrate (DCPD) (Barber, 1984). Therefore phosphorus availability in this soil depends on soil pH and the solubilty of phosphate compounds (Vickar et al., 1963). 2. FORESTRY IN JAVA Indonesia with its characteristic geographical nature and physical factors has, as its most important natural resource, the tropical rainforest. The forest industry is smaller than the oil or mining sector. Indonesia's rainforest is the third largest in the world after Brazil and Zaire, covering around 144 million hectares. Based on forest land use, this forest is divided into: protected forests (20.8%), nature reserve and recreation forests (13.2%), limited production forests (23.7%), fixed production forests (20.8%), and converted forests (21.5%) (Anonymous, 1990). Java is the most populated island in Indonesia, and forest management in Java is carried out by the state-owned company "Perhutani" which manages only 2 million hectares of plantations. This forest is mostly focused on commercial species with high economic value. One of the main species is teak (Tectona grandis) (Anonymous, 1990). During the fifth five year plan which starts in 1990, Indonesia is planning to develop industrial timber estates covering 6.2 million hectares of its area to stimulate better preparation in forestry aspects. This includes silviculture intensification, species selection and seedling preparation for the directed planting area. One of the tasks of the Wanagama I Forest Research Center is testing some tree species to be selected for industrial timber estates, especially for infertile soils such as calcareous soils. Southeastern Indonesian provinces, Lombok and the lesser Sunda islands are known as dry and calcareous regions. 3. PLANTATION HISTORY IN WANAGAMA According to previous research, Gunung Kidul Subdistrict was an evergreen mixed forest (Bailey, 1960 in Anonymous 1988). In the 1800s, this area was hardly logged at all and the government used it for game hunting. In the early 1900s, local people used the area for 4 agroforestry purposes. Under Dutch colonialism at the beginning of this century the area was converted into a coffee plantation, but ecologically, the study site was found to be unsuitable for coffee, and it failed after only a short period of time. The second serious deforestation occured in the 1940s until Japanese colonialism ended in 1945, when the remaining forest mixture of teak and mahogany was completely logged. Efforts in 1927 and in 1948 to replant block 5 with teak plantation failed. In 1963, Morus albus (mulberry) was introduced to the site in order to prevent expanding soil erosion, and to provide leaves for silk worms and silk production and to keep the continuance of the Wanagama forest management. From 1963 to 1982 under Wanagama Research Center management, this site was being reforested using Morus albus. The site was then prepared to be a pilot project of the industrial forest plantation in a calcareous region. Thirty six species in total, some of which are quick yielding, were grown. They are listed below : Scientific name local name 1. Pinus merkusii pinus 2. Tectona grandis jati 3. Swietenia macrophylla mahoni 4. Acacia auriculiformis acacia 5. Albizia falcataria albizia 6. Calliandra callothyrsus kaliandra 7. Adenantera pavonina soga 8. Dalbergia latifolia flamboyan 9. Dialium indum asem londo 10. Enterolibium saman (Samania saman) saman 11. Melaleuca leucadendron kayu putih 12. Anthocephallus cadamba jabon 13. Eucalyptus sp. eucalyptus 14. Acacia caphyllacanta acacia 5 15. Santalum album cendana 16. Podocarpus sp. podocarpus 17. Fruit stands 18. Acacia mangium mangium 19. Acacia holosericea silver 20. Cinnamomum burmanii kayu man is 21. Morus sp. murbei 22. Ochroma sp. balsa 23. Aleuritus moluccana kemiri 24. Acac/'a orar/a oraria 25. Bauhinia purpurea tayuman/kupu-kupu 26. Schleichera oleosa kesambi 27. Anacardium occidentale jambu mete/monyet 28. V/rex pubescens laban 29. Gliricidea maculata gleriside 30. MJmosop e/mg; tanjung 31. Manilkara kauki sawo kecik 32. Caesalpinnia sappan secang 33. Gmelina arborea gmelina 34. Leucaena leucocephala petai cina /kemlandingan 35. Eucalyptus sp. (18 varieties) for new provenance test 36. Calliandra callothyrsus new provenance test plant. From these available stands, four plant species were selected based on the highest economical value of commercial wood, Tectona grandis Linn. (Verbenaceae) and Swietenia macrophylla King (Meliaceae), and the fastest growing of leguminous trees, Acacia mangium Willd. and Acacia holosericea A. Cunn. ex G. Don (Leguminosae) 4. MYCORRHiZA The term 'mycorrhiza' means 'myco' or fungus and 'rhiza' or root. This term was introduced by Frank in 1885 (Harley and Smith, 1983) to describe the association between roots and symbiotic fungi. It can be interpreted more specifically as a mutual symbiosis (an association which both sides obtain benefits) between a plant and a non-parasitic fungus, in which the host plant benefits from nutrient uptake, (P, N, S, Zn, Br, CI, Mg and K), by the fungus. In return, the mycorrhizal fungus receives carbon from the host plant (Kendrick and Berch, 1985; Safir, 1987; Harley and Smith, 1983). Host plants may also be more drought tolerant if they are mycorrhizal (Safir, 1987). Based on the morphological characteristics of the fungus and the structural association, mycorrhizal classification can be grouped into seven kinds: Vesicular-arbuscular mycorrhiza, Ectomycorrhiza, Ectendomycorrhiza, Arbutoid, Monotropoid, Ericoid, and Orchidaceous. Fungal components of these mycorrhizal groups are Zygomycetes for the first group and generally Basidiomycetes and Ascomycetes for the the following groups (Harley and Smith, 1983; Powell and Bagyaraj, 1984). Vesicular-arbuscular mycorrhiza (VAM) differs from other types in the ability of the hyphae to penetrate and grow within cortical cells of the host roots, branching to form arbuscules (tree or bush-like structures) and some also form vesicles (hyphal swelling) intercalary or terminally (Bonfante-Fasolo, 1984). Arbuscules are believed to be a nutrient exchanging interface from fungal hyphae to root cells, and this is a physiologically active site compared to vesicles which are formed for lipid storage (Harley and Smith, 1983; Sanders et al., 1975; Safir, 1987). VA mycorrhiza are the most common type found in the tropics (Mikola, 1980). Some studies involving the manipulation of VAM have been carried out in agriculture and forestry (Mosse, 1981; Kormanik et al., 1982; Janos, 1980; Fogel, 1980) or for revegetation purposes (Jasper et al., 1989a). Results of many studies show that proper mycorrhiza will produce better 7 (Mosse, 1981; Kormanik et al., 1982; Janos, 1980; Fogel, 1980) or for revegetation purposes (Jasper et al., 1989a). Results of many studies show that proper rnycorrhiza will produce better plant performance, at least in seedlings (Dela-Cruz et al., 1988; Lapeyrie and Chilvers, 1985; Borges, 1988; Pope et al., 1982; Reddel and Warren, 1986). VA mycorrhizal fungi belong to the order Clomales in Zygomycotina, with three families: Clomaceae, Acaulosporaceae and Cigasporaceae. Genera of these families are Glomus and Sclerocystis in the first family, Acaulospora and Enthrophospora in the second and Gigaspora and Scutellispora in the last family (Morton and Benny, 1990). Studies about VA rnycorrhiza in the tropics, especially Indonesia, for reforestation purposes have not been applied yet. Considering that the indigenous association has a potential benefit for forestry plantations in calcareous soils especially and in developing countries as a whole, this study was addressed to these directions. Basically, this study aimed to answer why some leguminous trees survive better than other trees in P-deficient soils from a mycorrhizal point of view. This study consists of three sub-studies broken into chapters covering mycorrhizal status of selected tree species and comparison between nursery seedlings and plantations; VAM fungi occurrence in the Wanagama Research Station; and response of Acacia spp. to single species and mixed VAM fungal inoculation, as chapter II, III, and IV respectively. I should also mention here that originally, the study on the plant response to VAM inoculation was to use selected indigenous VAM fungal isolates retrieved from the original soil samples (Indonesian soils), in order to obtain more meaningful results of potential indigenous VAM fungal usage. The pot culture study, however, did not produce spores. Hopefully, from these mistakes, we can learn how to deal with VAM fungi from the tropics and I still believe that it will be possible to obtain indigenous VAM fungi which will likely have the highest potential to enhance the performance of forestry plantations. 8 Chapter II : MYCORRHIZAL STATUS OF FOUR INDONESIAN FORESTRY SPECIES 7. INTRODUCTION The occurence of mycorrhizae is widespread (Fogel, 1980) from temperate to tropical regions and from natural ecosystems to those modified by human activity. This has been documented in many reviews and studies on systems ranging from sand dunes, and semi-arid rangelands to lowland rain forests and agricultural fields (Read et al., 1976; Janos, 1980; Mikola, 1980; Gemma et al., 1989; Rajapakse et al., 1989). The most common rnycorrhiza in the tropics is VA-mycorrhiza (Readhead, 1980; Mikola, 1980; Harley and Smith, 1983). Research on VAM and nutrient uptake has expanded rapidly during the last three decades. Quick-yielding trees like legumes are now popular for reforestation on difficult to regenerate land, due to ability in fixing nitrogen. Besides its good prospects for reforestation, Acacia also has potential in commercial-scale for lumber, firewood, high heating value charcoal, good quality paper pulp, and plywood (Yantasath, 1986 in Turnbull, 1986). Acacia is one of the leguminous trees which commonly grows on dry to semi-dry (700-1200 mm annual rainfall) calcareous soils at low to medium elevation (Loigier, 1985 in Borges, 1988). Because of its fast growth, it is a natural pioneer in revegetating disturbed sites such as mines (Jasper et al., 1989a). The success of self-sustaining ecosystems in most adverse regions cannot be separated from the successional association between microorganisms and the plant community. In mine sites where phosphorus is deficient, the establishment and long-term stability of Acacia communities likely relates to the formation of VA rnycorrhiza (Jasper et al., 1989a). VA mycorrhizae play an important role in P-cycling and in plant growth in neutral calcareous soils through better exploitation of the soil phosphate pool (Azcon-Aguilar et al., 1986; Lesica and Antibus, 1986). They may also have great importance for plant growth in mineral-poor lowland tropical soils (Janos, 1980). In low P soils, specifically, mycorrhizae are a prerequisite for legume nodulation (Munns & Mosse, 1980 in Hoffman and Mitchell, 1986). 9 The only internationally available study on VA mycorrhiza done in Java was conducted by Janse who observed 56 families of woody plant and herbs and found that 69 out of 75 species are colonized by VA mycorrhiza (Janse, 1897). For the four selected species in the present studies which are all known to form VA mycorrhiza (Mikola, 1980), VAM percent colonization has never been determined. Mohankumar and Mahadevan (1988) studied VAM distribution in a tropical forest, Tamil Nadu, India, and found percent colonization varied from 24 to 78% in many different forest ecosystems with teak forest at about 50% colonization. Many studies of Acacia mycorrhizal dependency only reported the percent colonization of seedlings which was 22 - 58 % with Glomus fasciculatum and Gigaspora margarita on Acacia scleroxyla and 15 to 34% on Acacia concurrens with Scutellispora calospora and Glomus sp. (Jasper et al., 1989a). Since only a few studies regarding VAM and Acacia in the forest ecosystem have been done in the tropics and most research emphasized either plantation or seedling, therefore the purposes of this study are to determine VA-mycorrhizal status of 6 year-old plantations of four forestry species (Acacia mangium, Tectona grandis, A. holosericea, and Swietenia macrophylla) and to compare VA-mycorrhizal status of plantations to nurseries of the first two observed species. Previous studies (Nelsen et al., 1981; Rajapakse et al., 1989) done on soil nutrients found correlation between percent VAM colonization and phosphorus is negative and no correlation has been reported with other soil nutrients. However, one study on interaction of VAM with erosion in an Oxisol done by Habte, et al. (1988) found that there were relationships between selected soil properties and VAM effectiveness. They suggested to consider not only P status but also other nutrients in establishing VAM in marginal land. This raises the third purpose of this study which is to examine the relationship between percent VAM colonization and some selected soil nutrients. Those were N, P, K as macronutrients in soil, Ca, Mg which are in high concentrations in calcareous soils, and Na which is analysed at the same time as other exchangeable cations. 10 2. MATERIALS AND METHODS 2.1. Study site This study was carried out in blocks 17 and 5 of Wanagama I Forest Research Center, Gunung Kidul, Yogyakarta, Indonesia (Figures 1 and 2). Soil type on this site is categorized as red yellow mediterrane (FAO/Unesco) with mainly limestone parent material (Soepraptohardjo, 1966 in Anonymous, 1988). Topsoil is thin, (about 30 cm) and limestone concretion is found below 40 cm. Soil texture is silty clay with composition of 10 - 25% sand, 15 - 30% silt, and 50 - 70% clay, soil pH varies from 5.5 to 7.8 (Anonymous, 1988). JAVA ISLAND MAP S C A L E 1 : 5 0 0 0 0 0 0 N Figure 1 : Map of Java island (1:5,000,000) y \ GUNUNGKIDUL SUBDISTRICT Kedu \ Figure 2 : Map of Cunungkidul subdistrict (1:500,000) to STUDY SITE MAP SCALE 1:25000 LEGEND SITE BOUNDARY = ROAD RIVER Figure 3 : Map of Wanagama I as the study site (1:25,000) 14 Physical properties of this soil are sticky, plastic, low permeability with high water retention. During the dry season (May - October) it cracks quite deeply (Fig. 4c). Soil pH ranges from 6 to neutral. Mean annual rainfall recorded from three adjacent stations in 1988 was 2,041 mm with erosive rainfall during the rainy season (November - April) and about 4 dry months (Appendix 1, Table I). . Block 17 has been used over the past 7 years as the experimental site of provenance tests for about 36 forestry species , including quick yielding plants such as Acacia mangium, A. holosericea, A. auriculiformis, and Leucaena leucocephala. Almost pure stands of each species were found in this block with plant distance about 4 x 4 m and uniform in terms of stand age. At 6-year old plantations, A. mangium is taller and more robust than A. holosericea (Figure 5). Tectona grandis with the same age was selected from outside block 17, at a plantation in block 5 mixed with cassava (Manihot utilissima). There was no confusion of the two species however because M. utilissima has different root morphology. The S. macrophylla site is close to block 5 where the nursery of both Acacia is. Generally on the forest floor of the 6-year old plantation, the leaf litter was thin especially under T. grandis. Decomposition seems relatively slow under both Acacia (personal communication with local people) but leaf litter is washed away during rainy season. 2.2. Sampling procedure 2.2.a. Root distribution Four species of 6 year old forestry plants have been studied: Acacia mangium, Acacia holosericea, Tectona grandis, and Swietenia macrophylla. Sampling was carried out from June to July, 1988. To determine the sampling locations where the highest density of fine root occured, the root distribution sampling was carried out. 15 Root distribution and root characteristics of each species were determined by measuring the root length, and studying the branching and the fine root (<2mm) distribution surrounding the trunk. Three trees of each species were selected randomly and 3 transects were established along the root length of each tree, with several sample plots in each transect line. Plot size was 15 x 15 x 15 cm spaced 50 cm apart (Figure 6). Soil and roots were excavated from the sampling plot and fine roots were separated from soil by wet sieving, then roots were air dried and weighed. Since the root branching, length, and fine root abundance of each species is different, total root and soil samples for A. mangium, A. holosericea, T. grandis and S. macrophylla were 90, 54, 45 and 36 respectively (Table 1) to determine the distance of sampling plot for each species of these selected trees. 2.2.b. Mycorrhiza study 2.2.b.1. Plantation study Based on the results of the preliminary study on fine root distribution mentioned above, root sampling for the mycorhiza study was done at 4.75 m, 1.50 m, 0.5 m and 0.5 m away from the trunk for A. mangium, A. holosericea, T. grandis, and S. macrophylla. Three samples were collected equidistant from each of ten trees that were chosen randomly for every plant species. Therefore, there were 30 samples for each species. Root samples were separated from the soil by wet sieving method and were preserved in half strength 90:5:5 FAA (formaldehyde : acetic acid : alcohol) solution in water (Kormanik and McCraw, 1984 in Schenck, 1984). Soil samples were air dried, and composited from three sample plots per tree. Thus there were 10 composite soil samples for each site. Fig. 4 : View of Wanagama Forest Research Center; Fig. 4c : Soil profile in Wanagama Forest Research Center a) reforestation area; b) part of nursery. 18 2.2.D.2. Nursery study Soil and root sampling for the 10 month old nursery seedlings were carried out by choosing 20 bagged seedlings randomly from 4 nursery beds (Fig. 4b). Roots were removed from the plastic bag and roots which protruded from the bag were also retrieved. Sampling in the nursery was done only for A. mangium and T. grandis which were available at that time. Soil samples were collected by compositing from 5 samples. Therefore there were 4 composite soil samples for each species. 2.2.c. VAM assessment Root samples were washed free of FAA, cleared with 10% KOH, and, when roots were quite dark, cleared with a mixture of hydrogen peroxide and ammonium, and then stained with tryphan blue (Phillips and Hayman, 1970). After staining, roots were cut into 1 cm segments and percent VAM colonization was determined under the dissecting scope using the gridline intersect method (Kormanik and McGraw, 1984 in Schenck, 1984) or using the slide method (Giovannetti & Mosse, 1980) for roots which were satined too dark and therefore difficult to be observed under the dissecting scope. One hundred segments were counted with 3 replicates for each root sample. 2.2.6. Soil analysis Air-dried soil samples were analyzed for pH using 1:1 soihwater and a pH meter. Macronutrients analyzed in soil samples were total N, available P, K, Na, Ca and Mg content. Total N was determined using an Auto Analyzer II by Technicon. Available P content was analyzed using the Mehlich method (Lavkulich, 1981) and measured by absorbance with a Spectronic 20 (Bausch & Lomb) spectrophotometer. Major cations (K, Na, Ca and Mg) were determined after ammonium acetate extraction using an atomic absorption spectrometer (Perkin Elmer model 306 apparatus). 19 2.2.e. Data analysis All collected data were analyzed using one way analysis of variance after the normality of the data was verified and the Tukey's multiple range test was chosen to compare among mean values (Zar, 1984). Transformation of data was done when a zero value was found in the data. Correlations between percent colonization and some major nutrients were calculated using linear correlation analysis. In order to see the relationship between VAM percent colonization and soil nutrients, a multivariate regression analysis was applied. 20 3. RESULTS 3.1. Fine root distribution Root characteristics of the four studied species are very different one from the other (Table 1) in that the root branching pattern and the fine root abundance in relation to the tree trunk is specific for each species. For plants of the same age, root development of A. mangium is the greatest with root length reaching 9 m, branching starting at 3 m, and the greatest density of fine roots at about 4 m from the trunk. The least root development was found in 7. grandis with 2.5 m length, branching at about 0.5 m away from the trunk and the fine roots forming close to the trunk. The fine root abundance is presented in Table 2a which shows mean fresh weight of fine roots from 9 samples (3 transects each for three trees) at each sample distance. The highest mean of every species was obtained in a different plot number depending on the root branching characteristics. These are 4.75 m, 1.5 m, 0.5 m, and 0.5 m for A. mangium, A. holosericea, T. grandis, and S. macrophylla, respectively. Mean fine root density per m^ was significantly different for each species (Table 2b). Compared to the other species, fine root density for A. mangium is the highest at 17.2 kg/m^, followed by S. macrophylla, A. holosericea and T. grandis with 14.4, 12.5, and 8.7 kg/m^, respectively. Table 1 : Root characteristics Characteristics A. holosericea A. mangium S. macrophylla T. grandis Root length 9 m 4 m 2.5 m 2.5 m First branch 3 m 0.5 m 0.5 m 0.6 m First fine roots 4 m 1 m 0.1 m 0.1 m Fine root color brown pale brown cream dark brown Distance between plant 4 x 4 m 4 x 4 m 4 x 4 m 0.75 x 0.75 m First pit 3 m 1 m 0.5 m 0.5 m Number of pit per transect 10 6 5 4 Total pits 90 54 45 36 22 Table 2a : Distribution of fine roots based on mean fresh weight of 9 samples at each location Plot.# Distn. Root density ( kg / m )^ (m) Am Ah Sm 1 0.5 na na 13.2* 3.5* 2 1.0 na na 11.2 1.2 3 1.5 na 17.5* 10.7 2.0 4 2.0 na 16.2 9.9 2.3 5 2.5 na 15.0 10.5 6 3.0 16.2 15.5 7 3.5 15.3 14.4 8 4.0 16.0 15.3 9 4.5 18.5 10 5.0 19.2* 11 5.5 17.4 12 6.0 16.0 13 6.5 14.0 14 7.0 15.0 * 15 7.5 13.7 Note : Am = Acacia mangium Tg = Tectona grandis Ah = Acacia holosericea Sm = Swietenia macrophylla * the highest mean determines the sampling location na : not available due to root branching pattern. 23 Table 2b : Mean root fresh weight Species Fresh Weight (kg/m^) Acacia holosericea 12.5 ± 4.1 Acacia mangium 17.2 ± 3 . 8 Tectona grandis 8.7 ± 4.5 Swietenia macrophylla 14.4 ± 2.8 3.2. VAM colonization Root staining enabled us to differentiate clearly between the VAM colonization inside the root tissue and the root cells. VAM characteristics such as appressoria, coiled hyphae, arbuscles and vesicles were detected under the light microscope at 400 X and 1,000 X (Figures 7a and 7b). Lobed, pyriform, and globose vesicles were observed in T. grandis, A. mangium and A. holosericea specimens. However, T. grandis had more variation in vesicle shape than the other species, (Figure 8). Coarse VAM hyphae predominated in both Acacia species and Tectona while fine VAM hyphae were observed in a few Swietenia specimens. Determination of percent VAM colonization in fine root of these four tree species based on the presence of hyphae, and arbuscles, and/or vesicles using the gridline method proved difficult when 6 year old Acacia spp. fine roots were observed because the root tissue adsorbed stain so effectively that VAM hyphae could not be distinguished. The slide method was carried out instead. The results of the slide method using 100 or 150 root segments were not different, so 100 segments were used. Figure 7 : a) Coiled hypha of VAM fungus (x 2,625); b) Arbuscules of VAM fungi (x6,250). Figure 8 :a) Lobed vesicle shape found in T. grandis (x 2,625); b) Common vesicle shape found (X 2,625). 2 6 Comparing the gridline and the slide method with the same root sample produced different results but similar tendency. The results of the slide method were always slightly (up to 1.2 times) higher in well stained roots and up to 3 times higher in darkly stained roots. Percent VAM colonization of the roots from all four 6 year old plantations and from two nurseries is presented in Table 3. This shows that A. mangium from the nursery was the highest value with 89 %, followed by plantation A. mangium (70 %), T. grandis from the nursery (66 %), plantation A. holosericea (60 %), S. macrophylla (47 %), and T. grandis (38 %). Variability of the colonization is quite high with standard deviations from 12 % to 54 % (Table 3). Table 3 : Mean percent VAM colonization S p e c i e s 10 month old 6 year old Acacia holosericea na 60 a (16.7) Acacia mangium 89 c 70 a (12.0) (23.2) Swietenia macrophylla na 47 b (10.3) Tectona grandis 66 a 38 b (12.0) (20.2) Note : na = not available in the nursery. Value in brackets is standard deviation. Values with similar letter are not significantly different. » 3.3. Soil chemical properties Available phosphorus was significantly different among sites (P<0.05) (Appendix I: Table VII). The mean available soil phosphorus at each site can be listed respectively from the highest to the lowest value: nursery T. grandis (17.8 ppm), nursery A. mangium (2.4 ppm), 27 plantation T. grandis (2.1 ppm), plantation S. macrophylla (1.4 ppm), plantation A. mangium (0.1 ppm) and plantation A. holosericea (0.03 ppm). The two Acacia plantation soils did not differ. Tectona grandis, S. macrophylla and A. mangium nursery are also similar value (Table 4). The mean total nitrogen among sites is non significantly different and coefficient variability ranged from 8.5% to 20.8%. The highest mean, value of total nitrogen was found in T. grandis plantation by 0.24% and the lowest was found in both Acacia plantations and S. macrophylla by 0.21% (Table 4). The correlation between percent VAM colonization and available soil phosphorus is slightly negative except in the 6 year old A. mangium (Table 5). The negative correlation means that the higher the available phosphorus in the soil the lower the percent colonization. Only in T. grandis and S. macrophylla plantations did phosphorus account for the variation of percent VAM colonization by 14 and 15% respectively (Table 6). Coefficient variability of exchangeable sodium ranged from 2.6 to 12.7 % and it was significantly different among sites (P<0.05) (Appendix I: Tables 111). The mean values from the highest to the lowest were found in A. holosericea (0.49 cmolc/kg), A. mangium (0.44 cmolc/kg), T. grandis nursery (0.34 cmolc/kg), A. mangium nursery (0.32 cmolc/kg), 5. macrophylla (0.29 cmolc/kg),and T. grandis (0.28 cmolc/kg) (Table 4). Both Acacia plantations and T. grandis nursery soils have similar, high sodium contents. Percent VAM colonization in almost all species was positively correlated to exchangeable sodium except A. mangium and S. macrophylla. Coefficient variability of exchangeable potassium in the study site was quite high, ranged from 7.26 to 27.15 % yet content was significantly different among sites (P<0.05) (Appendix I: Table IV). Table 4 : Mean soil chemical properties (major nutrient only) of plant environment Species exch.Na exch.K exch.Ca exch.Mg avail. P N-tOt major cation ( cmol charge / kg ) ppm % Ah 0.49b 0.07b 12.89b 3.41 0.03b 0.21 (0.06) (0.02) (1.89) (0.69) (0.09) (0.04) Am 0.04ab 0.08b 11.86b 2.85 0.10b ' 0.21 (0.05) (0.01) (2.76) (0.45) (0.23) (0.04) AmN 0.32a 0.03a 43.61a 2.40 2.40a 0.23 (0.01) (0.01) (5.19) (0.59) (1.82) (0.02) Sm 0.29a 0.05c 44.81a 2.88 1.40a 0.21 (0.02) (0.03) (18.27) (0.89) (1.28) (0.03) Tg 0.28a 0.12d 16.82^  2.45 2.10ac 0.24 (0.04) (0.02) (2.27) (0.91) (1.46) (0.04) TgN 0.34ab 0.03a 37.74ac 2.40 17.80d 0.22 (0.02) (0.01) (13.51) (0.15) (8.13) (0.03) Note : Ah = Acacia holosericea Sm = Swietenia macrophylla Am = Acacia mangium Tg = Tectona grandis AmN = Acacia mangium nursery TgN = Tectona grandis nursery Second number is standard deviation Values with similar letter are not significantly different Table 5 : Correlation between percent VAM colonization and selected soil nutrients Nutrients Ah Am AmN Tg TgN Sm Na 0.552 -0.194 -0.036 0.278 0.048 0.366 K -0.597 -0.157 -0.406 -0.366 0.169 0.287 Ca -0.516 -0.025 -0.252 -0.647 -0.072 -0.147 Mg -0.246 0.189 0.392 0.666 -0.183 0.045 P -0.066 0.104 -0.358 -0.160 -0.077 -0.393 N -0.368 0.087 0.006 0.270 -0.211 0.075 Note : Ah = Acacia holosericea Tg = Tectona grandis Am = Acacia mangium TgN= Tectona grandis nursery AmN= Acacia mangium nursery Sm = Swietenia macrophylla to CO 30 Table 6 : Result of backward stepwise of multiple regression analysis for selected soil nutrients which contribute to the variation of percent VAM colonization S p e c i e s Main nutrients' contribution All species K Na Ntot 17% 7% 5% A. holosericea Na K 30% 18% A. mangium K Na Ca 23% ns ns T. grandis Mg P K 45% 14% ns S. macrophylla P N 15% ns A. mangium nursery * • T. grandis nursery * Note: ns= the contribution is not significant * = no significant pattern of any nutrients Percentage value shows the magnitude of variable contribution to the variation of VAM colonization. Mean value of exchangeable potassium is presented in Table 4 and the highest value was found in T. grandis plantations (0.12 cmolc/kg) followed respectively by A. mangium (0.08 cmolc/kg), A. holosericea (0.07 cmolc/kg), S. macrophylla (0.05 cmolc/kg), A. mangium in the nursery (0.03 cmolc/kg), and T. grandis from the nursery with the value of 0.03 cmolc/kg. Except T. grandis nursery and S. macrophylla, the percent VAM colonization was negatively correlated with exchangeable potassium (Table 5). The two nursery soils were not significantly different with relatively very low potassium content (Appendix I: Table IV). Variation of percentage VAM colonization in general was affected by available potassium, sodium, and total N content in soil by factor of 17%, 7%, and 5% respectively (Table 6). Potassium and sodium are the soil nutrients contributing most frequently to the variation of percent VAM colonization for all species as general and within each species (Table 6). 31 Exchangeable calcium differed significantly from one site to the other (P<0.05) (Appendix I: Table V) and the coefficient variability varied widely from 11.89 to 35.77% (Table 4). The mean calcium content from the highest to the lowest values were found in S. macrophylla plantations (44.81 cmolc/kg), A. mangium nursery (43.61 cmolc/kg), T. grandis nursery (37.74 cmolc/kg), plantations T. grandis (16.82 cmoljVkg), A. holosericea (12.89 cmolc/kg), and A. mangium (11.86 cmolc/kg) (Table 4). Tukey's multiple range test shows that the nursery soils were not significantly different with comparatively high values of calcium, and S. macrophylla and both old Acacia were similar with low calcium content (Table 4). None of the percent colonization correlated positively with the exchangeable calcium (Table 5), and calcium seems to contribute to the variation of colonization only in A. mangium plantation (Table 6). Mean value of exchangeable magnesium of each site (Table 4), from highest to lowest were found in A. holosericea (3.41 cmolc/kg), S. macrophylla (2.88 cmoIc/kg), A. mangium (2.85 cmolc/kg), 7. grandis (2.45 cmolc/kg), and both nurseries of T. grandis and A. mangium with the same value of 2.40 cmolc/kg. The exchangeable magnesium was not significantly different from site to site (Appendix I: Table VI). Percent colonizations in both nurseries and A. holosericea were negatively correlated to magnesium content and positively correlated in T. grandis, A. mangium, and S. macrophylla (Table 5). Table 6 shows that Mg contributes 45% to variation of percent colonization in T. grandis but not at all in other species. 32 4. DISCUSSION 4.1. Fine root distribution Considering that Acacia spp. are the quick yielding plants selected for provenance testing it is not surprising that in root length and fine root density they were superior to the other trees examined. Their good growth may be explained simply in that nutrient requirements for growing Acacia may be lower than for other species, since some studies done recently in mining reclamation sites (Jasper et al., 1989a) found that Acacia is naturally found as a pioneer species when there is an adequate P supply (Jasper et al., 1989a). Acacia may have an internal strategy for using nutrients or energy more effectively than other species. As a member of the Leguminosae, it benefits from association with nitrogen-fixing Rhizobium. High variability of fine root density within site can be influenced by many factors, including soil moisture, texture, compaction and fertility (Jenik, 1971 in Berish and Ewel, 1988 ; Berish and Ewel, 1988) as well as root development patterns inherent in each species (Jenik, 1978 in Berish and Ewel, 1988) or root development pattern related to plant maturity (Candar and Hughes, 1988). Seasonal and sucessional patterns of natural ecosystems also play an important role in fine-root density (Berish and Ewell, 1988; Singh and Singh, 1981). The failure to obtain clearly stained roots in both Acacia species from the plantations likely relates to the suberization process that frequently occurs in radial cell walls in the hypodermis of many vascular plants (Shiskoff, 1986 in Brundrett & Kendrick, 1987; Berish and Ewel, 1988) and leads to the absorption of more stain than nonsuberized tissue. 4.2. Percent VAM colonization Percent colonization determination relies on the presence of VAM hyphae, arbuscules, and/or vesicles. Presence of these structures depends on the physiological stage of the VAM since most arbuscles are very short-lived and can only be observed in new roots (Harley and Smith, 1983). Formation of VAM is also a seasonal phenomenon and percent colonization changes with the flux of short-lived fine roots. Brundrett and Kendrick, (1987) found that during faster fine-root growth, mycorrhizal levels rose rapidly and declined when roots senesced. In the present study site, monsoon seasonal patterns divide the year into two different conditions, dry and rainy seasons, which will probably contribute to the VAM status. This study was only done in June within dry season (March-August). This needs further study in order to determine the VAM status properly. Variability of percent colonization within site is quite high. According to Cook et al. (1988), percent colonization is influenced by soil moisture, nutrient supply, fine root distribution, growth of the host, growth and biomass allocation of the fungus. Different mycorrhizal status among sites may also be due to soil microbes that can modify the host response to colonization (Cerligione et al. , 1987; Cook et al., 1988). Plant and VAM history of the site affect VAM propagules qualitatively and quantitatively (Janos, 1980a). Thus the higher percent VAM colonization of some observed trees and differences in VAM fungal taxa found from the various study sites perhaps also relate to the vegetational history of the site. Mycorrhizal status in the nursery was higher than in the plantation, even though root density was higher in the older plants. This study was not designed to observe VAM status and root density relationship, however, it has previously been suggested that the more fine roots found the greater the proportion of colonizable roots would be. Cook et al. (1988) studied root and VAM development in a chronosequence of tallgrass prairie and found that root density was negatively correlated with VAM status and the need for rnycorrhiza becomes less with increasing root density. Our results might be explained in a number of ways. First, in the older plants, the relatively high root development with many fine roots and root hairs may replace mycorrhizal functioning in nutrient uptake (Cook et al. 1988), and therefore, colonization was low in plantation. Second, the percentage colonization in community level reaches a steady state, even though more individual roots are found the mycorrhizal status is the same, but it will likely vary among species (Van Nuffelen and Schenck, 1984) or life forms such as herbs, shrubs and trees (Cook et al., 1988). Those two reasons are applicable to the 34 tallgrass prairie in which the root system is different from those of forest trees. Most root systems of Acacia are xerophytic or mesophytic (Hoffman and Mitchell, 1986) as are those of tallgrass prairie but the tree roots penetrate more deeply. Therefore, they cannot be compared directly since the proportion of the fine root to the whole root system in most forest ecosystems is small (Vogt et al., 1986 in Berish and Ewel, 1988). Mycorrhizal association can be detected from VAM percent colonization of total fine roots(Cook et al., 1988). In the field, however, it would be more meaningful to assess mycorrhizal association based on total fine roots susceptible to colonization. Third, season might play a role in this case where sampling was done only during the dry season when most roots in the plantation were slow-growing, while the nursery was kept well watered. Berish and Ewel (1988) in their study of root development in simple and complex tropical successional ecosystems found that fine roots decreased up to 40% during the dry season. In the plantation, both Acacia were more highly colonized than the two other species. There are two possible explanations for this. First, Acacia spp. are legumes and therefore able to access nitrogen from N-fixing Rhizobium associations. Some experiments have shown that this combination of VAM and N-fixing plants is superior to VAM/ non- ^-fixing plants (Sanni, 1976), since better host growth resulting from easy N access provides more carbon source for VAM fungi. Second, compared to the other plant species, Acacia roots explore more soil volume and, with more fine roots, both Acacia spp. raise their chance to be colonized by VAM propagules from the soil. Van-Nuffelen & Schenck (1984) found in greenhouse studies that the number of root penetrations by VAM fungi was not correlated with percentage of root colonization, even though penetration was correlated with root length. However, greenhouse studies with time and space limited might not reflect the situation in the field. Soil nutrient levels, especially available P content, in the Acacia sites were lower than in the other sites (Table 4) and many studies have indicated that high VAM colonization is generally associated with low soil P (Harley and Smith, 1983). The critical level of soil P for plant 35 growth, however, is species dependent (Menge et al., 1978) due to the differences in P requirement and uptake capacity of soil P in each species (Rajapakse et al., 1989) 4.3. Selected soil nutrients A negative correlation between percent VAM colonization and available P was observed in almost all species except plantation Acacia mangium (Table 5). A similar trend was also found in a study of the influence of phosphorus level on VAM colonization using cowpea (Vigna unguilata) cultivars inoculated with Glomus fasciculatum and Bradyrhizobium. These plants were subjected to P addition ranging from 0 to 30 ppm on top of the initial P content of 13 ppm (Rajapakse et al., 1989). The maximum VAM root colonization was found at 13 ppm of available soil P. ln the present study, the maximum available soil P content found in T. grandis nursery is 17.8 ppm where the percent colonization is only 66%. The highest colonization (89%), however, was observed in A. mangium in the nursery where the available P was only 2.4 ppm. Of this phenomenon seems that at the certain P level there is a trade off function between P and N which both are beneficial for VAM colonization. N amendment in this case, is only represented by the legume species, which has better N supply than non-legume plants. Furlan and Cardou (1989) found that N fertilization alone stimulated VAM colonization by 9% in onion since after adding N and P fertilizer VAM colonization was 77%, while after N fertilization without P amendment colonization was 87%. Also with N, P and K in their study, there was no difference from N and P. One should notice that in that study the initial soil P content was 45 ppm. The positive linear correlation found between percent VAM colonization and available P content in A. mangium plantation may be due to an interaction among soil nutrients in relation to mycorrhiza which still needs to be studied further. A negative correlation was found between percent VAM colonization and exchangeable soil calcium in all plants and for Mg and K in the nursery. One study iooking at the effect and interaction of erosion and nutrient decrease on VAM effectiveness found the 36 opposite for Ca, Mg, and K (Habte et al., 1988). According to Abbott and Robson (1982) effectiveness of the VAM association was closely correlated with rate and extent of colonization. Then it can be suggested that the correlation trend of VAM effectiveness or VAM colonization with soil nutrients would be similar. The different results between Habte et al. (1988) and the present study , I believe are because there is a threshold level of soil nutrient concentration above or below which different correlations exist. The form and the initial soil calcium in Habte's study was not reported, however, and the soil type and plant species are different. Therefore, no true comparison can be made of these two studies. In calcareous soil, calcium and magnesium are the dominant cations which at neutral and alkaline pH will bind available phosphorus (Wild, 1988). Many studies have been done regarding the effect of P or the effect of N, P, K fertilization on VAM colonization (Rajapakse et al., 1989; Furlan et al., 1989), usually under controlled conditions. So far, however, there is no study regarding the relation between soil cations and VAM colonization. This causes difficulties in deciding which and at what level a soil nutrient may influence VAM colonization. Stepwise multivariate regression pattern (Table 6) shows that potassium is the most significant factor contributing to the variation of percent VAM colonization both for all species as treated generally or within species, except in Swietenia macrophylla. This may be explained by the role of potassium in carbon transfer (Furlan and Cardou, 1989) from the host plant to endophyte. Root exudates decrease when the level of potassium in soil is optimum which causes an accumulation of readily available carbohydrates in the root cortex and favors the fungal symbiont (Trolldenier, 1972 m Furlan and Cardou, 1989; Furlan and Cardou, 1989). 37 5. CONCLUSIONS The results of this study indicate that some hardwood trees in calcareous soils in the tropics are VA mycorrhizal. Colonization is highly variable from one sample to another within species. In the nursery the VAM colonization is higher than the plantation. Plant species, physiological state, season and also edaphic factors interactively contribute to the variation of VAM colonization. In calcareous soil where the level of available soil phosphorus is very low, available soil P appears not to be a major influence on mycorrhizal status. Potassium and sodium instead may be suggested to be important factors but their roles could not be determined in this study. This research also suggests further study in relation to the benefits of mycorrhizae in nursery and plantations. In comparing mycorrhizal status between nursery and plantation, we need a clear concept of which plantation roots are physiologically equal to the nursery roots in order to determine whether plantation roots are or are not as dependant as nursery roots. This kind of study would be verified by nutrient uptake analysis of plantations and of nurseries. 38 Chapter III : VAM OCCURENCE ON FOUR INDONESIAN FORESTRY SPECIES 7. INTRODUCTION VA mycorrhizal fungi form the dominant type of rnycorrhiza in the tropics (Mosse, 1981; Mikola, 1980) and have mostly been studied in relation to anatomy and morphology, host and symbiont interactions, physiology and nutrient uptake (Sanders et al. 1974; Hariey and Smith, 1983; Powell and Bagyaraj, 1984). One old study done in Java, Indonesia, found that most plant species within 56 observed families were mycorrhizal (Janse, 1897). However, Janse did not study the fungal species. Only a few studies in VAM done in the tropics have concerned fungal species identification such as a study on VAM occurence in Cuba (Herrera and Ferrer, 1980). They found 18 VAM spore types, of which 9 spore types were in Glomus, 2 types were comparable to Gigaspora calospora and Gigaspora heterogama, and one type resembled Sclerocystis coremioides Berk. & Broome. VAM fungi associated with cacao and oil palm in Malaysia were studied by Nadarajah (1980), and she found that Glomus was the dominant type and Gigaspora, Acaulospora and Sclerocystis were the minor types observed within the 22 observed spore types (pers. comm.). VAM fungi associated with woody species in the Phillipines (Reynaldo E. Dela Cruz, professor at Univ. of the Phillipinnes at Los Banos, personal comm.) found 20 types of endomycorrhiza. A study done in New Zealand (Hall, 1977) found 19 identified species with four new ones : Glomus pallidus Hall, G. magnicaulis Hall, C. invermaius Hall, C. infrequens Hall, and one new combination of C. tenuis. He found two Sclerocystis species: S. rubiformis Gerdemann & Trappe and S. coremioides Berk. & Broome; two Gigaspora : C. aurigloba Hall and G. margarita Becker & Hall; and three Acaulospora species : Acaulospora laevis Gerdemann & Trappe and two unidentified spores. New VAM fungal species have been found in tropical Colombia (Glomus glomerulatum Sieverding and Toro, Sieverding and Toro, 1987), Acaulospora splendida Sieverding was found 39 in Costa Rica (Sieverding, 1988), and Glomus callosum Sieverding and Acaulospora undulata Sieverding were found in Africa (Sieverding, 1988). The number of described species of VA mycorrhizal fungi has increased every year starting from the work of Gerdemann and Trappe (1974) which reported 31 species. In 1988, Morton reported that there were 126 VAM species which had been published in various papers. Recently, 150 described species were listed (Almeida, 1989). VA mycorrhiza fungal classification underwent a revision proposed by Morton and Benny (1990). These fungi now belong to the Glomales in the Zygomycotina. There are two suborders, Glomineae and Gigasporineae with families of Glomaceae and Acaulosporaceae in the first and the family Gigasporaceae in the second. The Glomaceae consists of genera Glomus and Sclerocystis. The Acaulosporaceae contains Acaulospora and Entrophospora, while genera Gigaspora and Scutellispora belong to family Gigasporaceae (Morton and Benny, 1990). Species identification in VA mycorrhizal fungi is based on selected morphological characteristics of vegetative (hyphae, vesicles, and arbuscules) and reproductive (spores) stages (Morton, 1990). Therefore producing a large number of healthy spores is very useful to identify VA mycorrhizal fungi since characteristics of spore development can be obtained. To identify VA mycorrhizal fungi, pot culture methods should be incorporated in order to produce enough spores (Ferguson and Woodhead, 1984). The most popular method for retrieving spores either from field soil or pot culture soil samples is wet sieving and decanting (Gerdemann and Nicolson, 1963 in Daniels and Skipper, 1984). Other methods such as sucrose centrifugation (Jenkins* 1964 in Daniels and Skipper, 1984), flotation bubbling method (Furlan and Fortin, 1975), and density gradient centrifugation (Ohms, 1957 in Daniels and Skipper, 1984) have been suggested. Identification of collected spores from the field might lead to mistakes owing to the variance of character states which are affected by time of sampling, physiological age, hyperparasitism, host, and other environmental effects (Morton, 1988). 40 To differentiate among genera, stable characteristics such as spore wall structure (Walker, 1986a), presence of auxilliary cells\ germination shields^ and bulbous suspensors^, are used as well as spore development in regard to the position relative to sporiferous saccule4, sporogenous hypha^, and fungal thallus (Walker, 1986b; Schenck and Perez, 1988; Morton, 1988; Berch, 1988). More detailed stable characteristics are needed in identifying species within a genus. Spore wall structure (Schenck and Perez, 1988; Gerdemann and Trappe, 1974; Berch and Trappe, 1988 ; Berch, 1988; Morton, 1988) seems to be the key to species identification. This inludes : number of walls and wall groups, spore wall types, and wall ornamentation (Walker, 1986a ; Morton, 1988), as well as reaction of spore walls to certain mountants and Melzer's reagent (Morton, 1988). Walker (1983) proposed to simplify and standardize the terminology of spore wall structure. He introduced two terms : muronym, an artificial code that consists of capital letters designated for wall groups (U = unit, L = laminated, M= membraneous, C = coriacious, E = evanescent, X = expanding, and A = amorphous) and small letters that show other characteristics observed on spore wall such as o = ornamented, b = beaded; and muroRraph, a computerized symbol with special patterns to designate particular wall types in a diagramatic manner (Schenck and Perez, 1988). According to Walker (1983), there were four spore wall types : 1. Unit wall (U) : a single layer, rigid, distinctive, and consistent at spore maturity. 1. auxilliary cell : singled or clustered ornamented vesicles formed on spiraled hyphae in the soil by Gigaspora or Scutellispora species (Berch, 1988). 2. germination shield : wall and membrane bound cytoplasmic intrusion between spore wall layer groups that may be two- to many-lobed and that gives rise to germ tubes (adapted from Walker and Sanders 1986 in Berch, 1988). 3. bulbous suspensor : a hyphal swelling on which spores of Gigaspora and Scutellispora species are borne (synonymous : sporogenous cell) (Schenck and Perez, 1988). 4. sporiferous saccule : terminal hyphal swelling forming before spore which then may be lateral (Acaulospora sp.) or intercalary (Entrophospora sp.) on the subtending hypha (Berch, 1988). 5. sporogenous hypha :(subtending hypha = sporophore) a spore-bearing structure, usually of branched or unbranched hyphae on which spores develop (Schenk and Perez, 1988). 41 2. Laminated wall (L) : multiple layers laid down as the spore matures. 3. Evanescent wall (E) : a unit or laminated wall layer that sloughs off from the spore during maturation. 4. Membranous wall (M) : thin, nonrigid, frequently wrinkled wall that collapses in hypertonic solution. Since then, three additional wall types have been introduced. These are : 5. Coriaceous wall (C) : flexible and leather-like wall in hypertonic solution, robust, tough (Walker, 1986a) 6. Amorphous wall (A) : formless and elastic wall affected by type of mountant (Morton, 1986). 7. Expanding wall (X) : outer unit wall which expands when treated by lactic acid or PVA (Berch and Koske, 1986 in Schenck and Perez, 1988). Melzer's reagent has the greatest potential for identifying VAM fungi even though cotton blue and Congo red have also been used for a limited number of VA mycorrhizal fungi (Morton, 1988). Two basic reactions are produced with this reagent : amyloid reaction or blue color; and dextrinoid reaction or red color (Morton, 1988). Reaction with VAM spore walis tends more to be dextrinoid or other reactions between red and blue, such as light purple, dark or red purple depending on the chemical composition of the spore walls and their degree of polymerization. For example : the red reaction was observed for the innermost membranous wall of some species of Glomus, Acaulospora, and Scutellispora; the orange reaction was reported for Glomus albidum Walker & Rhodes and Scutellispora fulgida Koske & Walker; the purple reaction was observed in some species of Acaulospora (Morton, 1988). Species identification based on available descriptions is difficult since many have mentioned that this relates to lack of uniformity in descriptions. Efforts to create a synoptic key for this fungus have been published (Gerdemann and Trappe, 1974), or worked on (Berch pers. comm.). A compilation of the Endogonaceae done by Berch (1988) proposed a 42 formatted description to unify spore information which would be beneficial for taxonomical studies. Being aware of only a few studies in which VA mycorrhizal fungal species have been identified from the tropics, especially Indonesia, the objective of this present study is to determine the occurence of VA rnycorrhiza fungi associated with four forestry species : Tectona grandis, Acacia holosericea, Acacia mangium, and Swietenia macrophylla. 2. MATERIALS AND METHODS The sampling sites were four plantations (7. grandis, A. holosericea, A. mangium, and S. macrophylla) in Wanagama I Research Station, Yogyakarta, Indonesia (See Section 11.1). To identify mycorrhizal fungi, pot culture methods were applied (Ferguson and Woodhead, 1984) in order to produce enough spores. Ten grams of each soil sample was inoculated into 200 g of autoclaved "turface" (Aimcor) by mixing them up homogeneously. Then this mixture was placed into a 15 cm plastic pot and covered with sterile turface. This was the medium for growing test plants. Onion plants (Allium sp.) were germinated directly from 10 seeds in each pot. These experimental pots were placed in a growth chamber where the environment was set to artificial tropical conditions with 60% humidity, temperature 2 8 ° - 3 0 ° C, and 14 hours light per day with an average light source of 217.5 M mol m"^ sec"1 (measured by Radio quantum meter, Li Cor type Li 185). After germinating, the plants were thinned to 3 plants in each pot. Plants were watered with distilled water every 2 days and rotated every week in order to reduce bias in environmental conditions within the growth chamber. Plants were grown for 5 months, and 3 weeks before termination the plants were kept dry to induce sporulation. Then plants were harvested by separating shoots from roots and soil. Onion roots were stained with trypan blue (Phillips and Hayman, 1970) and examined for the presence of VAM colonization (See Section II.2.2.c), and soil samples were air dried. 43 In addition, spores were collected directly from field-collected soils that had been air-dried. Spore collection was done using wet sieving and decanting method (Gerdemann and Nicolson, 1963 in Schenck, 1982) with series of 5 sieves of 1 mm, 600 n m, 212 jum, 106 M m, and 53 nm openings (Figure 9a). Spores were selected manually under the dissecting scope with a Pasteur pipette (Figure 9b) and slide preparation was carried out to group spores with similar morphological characteristics. Lactic acid was used as a mountant for temporary slides and PVL solution (15% polyvinyl alcohol : lactic acid : glycerol with ratio of 56 : 22 : 22, Trappe and Schenck, 1984) was used for the permanent slides which were then sealed with clear nail polish. Spores were described using a microscope with medium and high (400 -1000 x) magnification. Colours were determined using International standard color chart (ISCC-NBS Color-Name Charts (Inter-Society Color Council-National Bureau of Standards) a supplement to NBS cicular 553) and Melzer's reagent applied to detect spore wall structure. The spores obtained from the original soils were not healthy and a limited number of similar spores were found. Because of this, the descriptions were incomplete. Figure 9 : VAM spore collection using wet sieving method 45 3. RESULTS AND DISCUSSION Results from pot culture studies did not produce enough spores for identification. Only 25% of test plants using nontropical onions with 44 soil samples as inoculant were colonized. Those were 2.5% each from both T. grandis plantation and nursery sites, 5% from A. mangium site, and 15% from A. holosericea . None of the test plants were colonized in soil samples derived from the S. macrophylla. This failure can be accounted for by various means. First, soils as inoculants for tropical VAM fungi are less effective than colonized roots inoculants due to low density of viable spores in the tropics (lan Alexander, Aberdeen Univ., pers. comm.). In temperate soils, spores have been successfully kept for years for inoculant (Menge and Timmer, 1984). Parasitism of spores in tropical soils, however, is common (Nadarajah, 1980). Second, spore germination is affected by environmental factors which, in Vancouver, may not be appropriate for tropical VAM fungi. Third, host plants should be well adapted to the environmental conditions, acceptable as host for the VAM species in the inoculant, grow rapidly and have no pathogens (Menge and Timmer, 1984). Ian Alexander and Lee Su See (pers. comm) have found that all these criteria are critical, but the use of an inappropriate host may have been most problematic in this case. From the spore collection study using original soil samples, 79 slide preparations were selected in which there were 16 spore types based on spore wall structure and other spore characteristics, such as spore color (observed under transmitted light), size, and hyphal characteristics. Since the pot cultures failed, none of the following fungi is known to be mycorrhizal. All collections were obtained from plantation or nursery of selected plant species from the Wanagama I Research Station, Yogyakarta, Indonesia. The complete description of each type is as follows : 46 3.a. Sclerocystis sp. I. U type Sporocarp : globose or irregular, approximately 400 x 400 M m, reddish brown, surface roughened by projecting ends of spores; focal plane cross-sections of spores in intact sporocarps appear pentagonal. Peridium : none observed. Gieba : spores radially arranged around central plexus of hyphae. Spores : subglobose, obovoid to clavate, 30 - 55 x 18 - 30 M m (average of 12 spores), yellowish brown, borne in sporocarp. Spore wall: 1 group of a single unit wall, yellow, 2 j im (average of 12 spores) at sides and thickening to 6 - 8M rn at the flat end of the spore. In the other slide, thickening at both apex and base (about 4 Mm) are observed, continuous with subtending hypha, 3 - 4 Mm diameter, yellow. Collection examined : OAs 8. Original soil under six-year old Acacia holosericea plantation. June 1988. Murograph and spore photographs are presented in Figure 10 In respect to spore wall structure, overall wall thickness, spore size, shape, and sporocarp size, this collection resembles Sclerocystis sinuosa Gerdemann & Bakshi (Gerdemann & Bakshi, 1976). However the present collections lack a peridium and have spore walls that are thickest at the apex rather than the base as described in the original publication. Fig. 10 : a) Murograph; b) Sporocarp (bar = 80 u m); c) Cross section spore, arrow shows pentagonal shape (bar = 20 u m); d) Spores, arrow shows thickened wall at the spore tip (bar = 4 am). 4 8 The present specimen is similar in appearance to a spore in slide #7 of Morton's slide set (Morton, 1989) identified there as S. sinuosa. However, they both differ from the original description (Gerdemann & Bakshi, 1976), which is clearly stated as having a sinous network of hyphae as the peridium. Determining how similar the present specimens are to Morton's material would require comparison with a description of that material, but this does not seem to exist. Compared to Sclerocystis microcarpus lqbal & Bushra (lqbal & Bushra, 1980), the present specimens are similar in lacking a peridium and having wall thickening at the spore apex. However, spore shape and size differ. Spore color and thickening of spore wall at the base are the main characters of Sclerocystis rubiformis Gerdemann & Trappe (Gerdemann & Trappe, 1974) that do not fit this specimen. However, spore size, lack of peridium, spore wall, and shape are similar to the present specimen. Despite some similarities to S. sinuosa, S. microcarpus and S. rubiformis, the present specimens cannot be identified as any described species. Ila. L / LU type Sporocarp: globose , oblong to irregular, 320 - 360 M m, yellow (68 s.oy or 66 v.oy). Surface of the sporocarp irregular due to projection of spores. Peridium: absent. Gleba : spores are loosely arranged radially from the central plexus; central plexus of hyphae yellow, network of interwoven hyphae with average diameter 6 - 7 M m. Spores: obpyriform, subglobose, irregular, yellow, (35-) 37-45 (-50) x (25-) 29-31 (-34) M rn (based on 51 spores), borne in a sporocarp; supported by a straight subtending hypha, yellow, open pore, infundibuliform, with relatively thick hyphal wall (2 - 2.5 M m). Hyphal diameter 5 - 7M m at the point of attachment. Some swelling laterally at hypha observed. Spore wall structure: there are two possibilities: 49 a) 1 group of 2 walls, the outermost wall laminate, yellow, 2 - 2.5 M m; and the inner wall, unit, hyaline, 0.5 - 1 Mm. b) 1 group of single wall, laminate, yellow, 2.5 - 3.5u m, with the innermost layer very distinctive from other layers within laminated wall. Collections examined : 1) OAs2, 2) OAs4. Both from six-year old A. holosericea plantation. June 1988. lib. L/LU type Sporocarp : brownish yellow, irregular, 490 x 280 Mm. Peridium : absent. Cleba : spores loosely and radially arranged from central plexus of hypha. Spores : globose, subglobose, brownish yellow, (24-) 27 - 36 (-38) Mm. Borne on single subtending hypha. Subtending hypha : straight, brownish yellow, open pore, 4 - 6 M m diameter, 30 - 45 M m length from the nearest branch. Spore wall structure: one group, brownish yellow, laminate with smooth layers and a distinctive layer in the innermost, 2 M m. Collections examined : 1) TP5 ; 2) TP6. Six-year old Tectona grandis. June 1988. Murograph and spore photographs are presented in Figure 11. Based on the spore wall structure, these two taxa (Ila and lib) cannot be categorized into any species of Sclerocystis because of the presence of a thick laminate wall with distinctive innermost layer. If spore arrangement can no longer be used to separate Glomus and Sclerocystis (Berch, pers.comm.), then these taxa resemble Glomus vesiculifer (Thaxter) Gerdemann & Trappe (Gerdemann and Trappe, 1974) in spore wall structure but not size, shape, or presence of a peridium-like layer. When Gerdemann and Trappe (1974) erected the new combination Glomus vesiculifer (Thaxter) Gerdemann & Trappe, they suggested that £. 50 tjibodensis from Java was a later synonym (Gerdemann and Trappe, 1974), so it seems that similar material has been described from Java. Cited description of Sclerocystis pachycaulis Wu & Chen (Schenck and Perez, 1988) was not clear in the spore wall structure, and therefore cannot be compared with the present collection. However, spore size and color are similar to the present species. 3.b. Sporocarpic Glomus sp. III. L / L-U type Sporocarps : oblong or irregular, yellow, 110-240 x 160 - 300 M m . Spores arranged on loose network of hyphae. Peridium : absent. Spores : globose to subglobose, yellow, 25 - 37 x 23 - 29 M m (based on 14 spores), except in Slide PAs6 where spores are 40 - 60 x 40 -55 Mm. Spores borne on single hypha, straight, open pore, 3 - 5 M m diameter, with hyphal wall decreasing from 2 to 1 M m within 1.5 M m from point of attachment. Spore wall : 1 group of 2 walls, wall one laminate, brownish yellow, 0.75 - 1 Mm, and wall two unit, yellow, 0.5 - 0.75 M m. Spore walls brownish yellow ,1 -1 .5 M m thick. Collections examined : 1) PAm 2. Six-year old Acacia mangium. June 1988. 2) PAs 6, OAs 3, OAs 9. Six-year old A. holosericea. June 1988. 3) OT 19a. Six-year old Tectona grandis. June 1988. Murograph and spore photographs are presented in Figure 12. Fig. 11 : a) Murograph; b) Sporocarp arrangement with subglobose spores (bar =16 u m); c) Sporocarp with obpyriformic spores (bar =16 u m). Fig. 12 : a) Murograph; b) Crushed spore with swollen subtending hypha (bar = Sporocarp arrangement (bar = 10 Mm). 10 Mm); c) 53 IV. L type Sporocarps: irregular, yellowish brown, 300 - 420 x 225 - 385 n m. Spore arrangement generally loose though one specimen shows compact arrangement. Spores : globose, subglobose or broadly ellipsoidal, yellowish brown, 40 - 51 x 22 -41 J i m (based on 23 spores). Spores borne on single subtending hypha with some swelling observed on hypha, yellow, 4 - 7 nm diam., straight, open pore. Hyphal wall thickness 2 - 3 H m with some thickening of hyphal walls observed. Spore wall structure: one group of two possible interpretations: 1) single layer, laminate, brownish yellow, 1 - 2(i m, with distinct lamination in the innermost part, 2) two layers of outer thin laminate (1 n m) and inner unit (1 M m). Spore wall 2 - 3 U m thick. Collections examined : 1)PAs4. 2) PAs19. 3) PAs20. All from six-year old A. holosericea. )une 1988. Murograph and spore photograph are presented in Figure 13. The two spore types above cannot be separated in terms of major characteristics of sporocarp and spores. Spore wall structure more or less differs, however these differences could be only the thickness of each wall or the interpretation of wall structure as a unit wall or innermost layer of laminate wall. Cited description of Glomus microcarpum Tul. & Tul. suppl. Berch & Fortin (Berch and Fortin, 1984) resembles the present specimen in spore size, shape, and wall structure, but differs in wall thickness, sporocarp size, and nature of sporocarp of C. microcarpum which has a peridium. However, description of the same species by Gerdemann & Trappe (1974) suggests that this species sometimes forms aggregations or small clusters not enclosed in a peridium. Fig. 13 : a) Murograph; b) Sporocarp arrangement with crushed spores with laminate wall (bar = 10y m). 55 Compared to Glomus pallidum Hall (Hall, 1977), spore shape, approximate spore size, wall structure and thickness, hyphal wall thickening and lack of a peridium are similar. Differences include subtending hyphae of C. pallidum which are bigger, have a closed pore and lack lateral swellings. The description of Glomus aggregatum Schenck & Smith emend. Koske (Koske, 1985) indicates similarities to the present specimen in the following characteristics : spore wall structure, thickness, shape and color; hyphal diameter, wall thickness, open pore, straight position; and lack of a peridium on the sporocarp. But comparatively bigger spores and sporocarps, and curved, infundibuliform or constricted subtending hyphae are present in C. aggregatum. Small dissimilarities could happen in descriptions since the apparent characteristics of species depend on the spore condition and the number of observed spores. G. aggregatum, G. microcarpum, G. pallidum are the most similar species to the present specimen, even though none of these are exactly the same. This specimen needs verification using healthier spores. V. U type Sporocarps: irregular, brownish yellow or yellowish brown, 100 - 665 x 93 - 525 M m. Spore arrangement loose but one sporocarp found with dense spore arrangement. Spores : globose, subglobose, (26-) 32 - 47 (-57) x (21-) 25 - 47 (-54) M m (based on 38 spores), brownish yellow. Spores borne on single subtending hypha, brownish yellow, open pore, straight, 3 - 5 M rn diameter, with some lateral swellings observed. Hyphal wall thickness decreasing from 2 to 1 Mm within 5 - 15 M rn from the point of attachment. Spore wall structure: single group, single wall, unit, pale yellow, relatively thin (1 - 2 M m). Collections examined : 1) PAm1. 2) PAm5. Six-year old A. mangium. June 1988. 3) T04. 4) OT11. 5) OT19b. 6) TP7. Six-year old T. grandis. June 1988. Murograph and spore photographs are presented in Figure 14. Fig. 14 : a) Murograph; b) Crushed spore with unit wall type (1)(bar -loose arrangement (bar = 30M m). 10M rn); c) Sporocarp, 57 Glomus microaggregatum Koske, Gemma & Oiexia (Koske et al. 1986) most resembles the present specimens in spore shape, color, size, wall structure, and hyphal characteristics including hyphal diameter, pore, position relative to spores, wall thickness and thickening at the spore base. However, the present specimens form sporocarps and laterally swelling hyphae which were not described in G. microaggregatum. Compared to the description of Glomus pulvinatum (P. Henn.) Trappe & Gerdemann (Tandy, 1975), the present specimens are similar in spore color, spore wall thickness and probably structure; but not spore size and the presence of a peridium which differ from the present specimens. Glomus microaggregatum and G. pulvinatum, are close to the present description, however, it cannot be identified as either of these two species, because of basic dissimilarities. 3.c. Nonsporocarpic Glomus sp. Via. E-L-Flexible Type Sporocarps: unknown Spores : globose, yellowish brown, 135 x 135 Mm (one spore only). Spore borne singly on single subtending hypha. Subtending hypha : paler color than spore, open pore, 12M m diameter. Hyphal wall as continuation of spore wall decreasing from 4 M m to 2 M m within 30 M m from the point of attachment. Spore wall structure : 1 group of 3 walls: wall 1, hyaline, 4 M m; wall 2, laminate, yellow, 3 Mm thick; wall 3, some thin flexible layers, hyaline. Collection examined : TO 17. Six-year old T. grandis. June, 1988. Murograph and slide photographs are presented in Figure 15. Fig. 15 : a) Murograph; b) Spore wall structure with 2 layers (1,2), arrow shows subtending hypha (bar = 5 M rn); c) Spore wall with unclear membraneous wall (bar = 6 Mm); d) Crushed spore with hyphal attachment (arrow) (bar = 15 M rn). 59 Compared to Glomus ambisporum Smith & Schenck (Smith & Schenck, 1985), spore wall structure, shape, size and lack of sporocarp are similar to this present specimen. Subtending hyphal characteristics, such as bigger diameter and occluded pore, are different. Description of Glomus ambisporum Smith and Schenck (Smith and Schenck, 1985) has similarities to this present specimen in spore size, shape, hyphal diameter, and one possibility of the spore wall structure. In the description, it says that C. ambisporum can form in sporocarps or singly in soil, and spores of the second morph have a single group of three spore walls. These are evanescent, laminate, and membraneous respectively from outermost. The evanescent wall in old spores sometimes cannot be detected anymore since it is gradually degraded as spore mature. Based on these consideration, the spore wall structure is similar to the present specimen. However, since only one spore was observed, we cannot compared it to the second spore morph which forms dark brown sporocarp. Compared to Glomus diaphanum Morton and Walker, spore size, shape, wall structure, lack of sporocarp, hyphal diameter and hyphal wall thickness resemble the present specimen (Morton and Walker, 1984). Tendency to form cluster of spores and consistent hyphal wall thickness in C. diaphanum differ. VIb. E-L-Flexible Type Sporocarp : unknown Spores : globose, yellow, 83-86 x 78-82 M m (based on three spores), borne on a single subtending hypha. Subtending hypha : yellow , 5 ju m diam., closed pore, hyphal wall decreasing from 1.5 to 1 M m within 2 jum from attachment. Spore wall structure: 1 group with 2 walls: wall 1, laminate, yellow, 3M m thick; wall 2, flexible, hyaline, 1 M m thick. Collection examined : TO 16 (Glomus sp). Six-year old T. grandis. June 1988 Murograph and spore photograph are presented in Figure 15. 60 Compared to description of Glomus diaphanum Morton and Walker (Morton and Walker, 1984), the following characteristics are similar to the present specimens : lack of sporocarp, spore size, shape, spore wall structure and thickness, hyphal diameter and hyphal wall. But the extention of the second wall into the subtending hypha was not observed and spore color in the present specimen is darker. Decreasing of hyphal wall thickness from the point of attachment is not comparable. In respect to spore wall structure, hyphal diameter and hyphal wall at the point of attachment, description of Glomus cerebriforme McGee (McCee, P. 1986) closely resembles present specimens. The tendency to form sporocarps, smaller spore size, white spores, and hyphal wall decreasing within 50M m from attachment in C. cerebriforme are different. The second spore type in Glomus ambisporum Smith and Schenck (1985) was described having a broad range of spore size, globose to subglobose shape, single wall group, 5 - 10 M m hyphal diameter. These characteristics largely resemble the present specimens, except for the presence of an evanescent outermost wall and hyaline spores in C. ambisporum. VII. OT 21 type. Sporocarps : unknown but spores tend to make aggregates, 400 x 550 Mm. Spores : globose to subglobose, brown, 85 - 97 x 80 - 90 M m (based on 10 spores only). Spores borne on single hypha, yellowish brown, straight, open pore, 5-7 M m with constriction at the point of attachment. Hyphal wall 1 . 5 - 2 M m at the attachment and decreasing within 4 Mm from the attachment. Thin-walled vesicle like structures observed on some branches of hyphae, irregular shape, size equal to the smallest spore. Spore wall structure: darker color than the spore, 1 group of 2 wall layers or only 1 layer: wall 1, laminate (6 M m thick), wall 2, unit (0.5 M m); or only laminate (6.5 Mm thick) with the innermost lamina distinctive from other laminae. Collection examined : OT 21 (Glomus sp j. Six-year old T. grandis. June 1988 Fig. 16 : a) Murograph; b) Spore aggregate (bar = 25 Mm); c) Spore wall and vesicle-like structure (arrow) (bar = 10 u m). 62 A few characteristics in original description of Glomus aggregatum Koske (Koske, 1985), spore size, color, spore wall structure, and hyphal characteristics including diameter, wall thickness, position, pore, and constricted hypha are the most similar to present specimen. However, the present specimens have thicker spore wall, form in a loose aggregate, and vesicle like structures as big as the smallest spore are observed. This present specimens resemble Glomus deserticola Trappe & Bloss (Trappe and Bloss, 1984) in spore shape, wall structure, and hyphal diameter, but differs in spore color, spore wall thickness and the presence of vesicle like structures. Villa. L + U type Sporocarp : unknown. Spores : globose , yellowish brown or brown, 85 - 90 x 75 - 85 jum (based on 11 spores). Most spores borne on single subtending hypha which is sometimes paler in color than the spores, straight, open pore, 4-12 n m diameter at the point of attachment. Two subtending hyphae observed once, and also hyphal constriction was found at the point of attachment especially on spores which have small subtending hypha. Hyphal wall thickness 3 U m decreasing to1/i m within 10-15 nm from the attachment. Spore wall structure: 1 group of 2 distintive walls: wall 1, yellow, laminate, 3 - 7 n m; wall 2, mostly very thin, 0.75 - 1 JU m, unit, lighter color than wall 1. Collections examined : 1) PAsI (3). 2) PAs2. 3) PAs3. 4) PAs10. 5) PAs12. 6) PAs13. 7) PAs16. 8) OAs6. All from six-year old A. holosericea. June 1988. Vlllb. L + U type Sporocarp : unknown. Spores : shape globose or subglobose, yellowish brown or brown under transmitted light, 64 - 91 x 55 - 85 jum (based on 5 spores). Spores borne on a single hypha, straight or 63 curved, open pore, yellow, 6-11 ju m diam. at point of attachment, hyphal wall thickness 3 M m decreasing to 1 M m within 10 - 1 5M m from attachment. Spore wall : 1 group of 2 walls. Outer wall, laminate, dark yellow, 2.5 - 4.5 Mm. Inner wall unit, yellow, 0.75 - 1.5 M m . Roughness of the innermost wall surface was observed in one spore only. Overall wall thickness 4 - 6 M m. Collections examined : 1) PAm4; 2) PAm6; 3) PAm8; 4) PAm9. Six-year old A. mangium June 1988. Vlllc. L + U type Sporocarp : unknown. Spores : globose to subglobose, brownish yellow (68 SOY; or 66 VOY), 65 - 107 x 65 - 100 M m (based on 3 spores). Spores borne on single subtending hypha, straight, brownish yellow, 5 -10 Mm diameter. Subtending hyphal wall continuous with the spore wall, pore of hypha occluded by wall material. The hyphal wall decreases from 4 M m to 1 Mm within 7 M m from the point of attachment. Spore wall structure : 1 group of 2 walls: wall 1, laminate, yellow, 4 - 6 M m thick; wall 2, unit, yellow, 0.75-1 Mm. Overall wall thickness 5 - 7 Mm. Collection examined : 1) TP2 (3). Six-year old T. grandis. June 1988. Murograph and spore photographs are presented in Figure 17. Compared to Glomus delhiense Mukerji, Bhattacharjee & Tewari (Mukerji et al., 1983), spore wall structure, spore color, and subtending hyphae are similar but spore size is bigger in C. delhiense. Fig. 17 : a) Murograph: b) Spore wall structure (bar = 6 n m); c) Hyphal attachment (arrow)(bar = 6 u m), both spores are parasitized. 65 IX. E-L-U Type Sporocarps : unknown. Spores : globose, 80-97 x 80-95 Mm (based on 4 spores), yellowish brown ( 68 s OY and 66 v OY ). Spores borne on a single subtending hypha, straight, yellowish brown, 5 -15 M m diameter, hyphal constriction observed occasionally. Hyphal wall continuous to the spore wall, pore occluded by spore material, wall decreasing from 5 to 2 M m within 7 - 1 0 Mm from the point of attachment. Spore wall structure : one group of three walls: wall 1, evanesent, hyaline, remnants only seen on the surface of middle wall; wall 2, laminate, yellow , 6 - 7 Mm; wall 3, unit , hyaline, 0.75 - 2 M m. Overall wall thickness 6 - 7 Mm, brown (darker color than the spore content). Collections examined : 1) PAs8; 2) PAs9; 3) PAs15; 4) OAs9. Six-year old A. holosericea. June 1988. Murograph and spore photographs are presented in Figure 18. Description of Glomus etunicatum, Becker & Gerdemann (Becker and Gerdemann, 1977) resembles the present specimen in spore wall structure but not in spore wall thickness and spore size. Xa. L type or E + L type Sporocarps : unknown Spores : brownish-yellow, globose or subglobose, 98 - 200 x 75 - 188 Mm (based on 5 spores). Spores borne on a single hypha sometimes with constriction at the point of attachment. Subtending hypha : brownish yellow, 6 - 12 M m, straight, hyphal wall quite thick and decreasing from 5 to 1 M m within 20 M m. Fig. 18 : a) Murograph; b) Hyphal attachment (big arrow) and evanescent walKsmall arrow) (bar = 6 nm); c) Crushed spore shows spore wall structure of E, L and U walls (1,2, and 3)(bar = 6 (im). 67 Spore wall structure: 1 group of 1 wall, laminate, brownish yellow (darker color than the subtending hypha), 3 - 7 . 5 Mm. Possible evanescent wall observed on one spore as remnant on the laminate wall. Collections examined:1) OAm5, 2) OAm7. Six-year old A. mangium. June 1988. 4) TO10. Six-year old T. grandis. June 1988. Murograph and picture of ELU spore type are presented in Figure 19. Compared to description of Glomus monosporum Gerdemann and Trappe (Gerdemann and Trappe, 1974), spore shape, size, spore wall structure and hyphal diameter are similar to the present specimen. Tendency to form sporocarp, the presence of peridium, double hyphae and curved hypha in G. monosporum are different from these specimens. Xb. OAm10 type. Sporocarp : unknown Spores: globose, brownish yellow, 40 x 40 M m, borne on two or three subtending hyphae. Subtending hyphae : yellow, straight, open pore, diam. 5 M m. Spore wall structure : one group of single wall, yellow, laminate, 6 M m. Collection examined : 1) OAm10. Six years old Acacia mangium. June 1988 Murograph and spore pictures are presented in Figure 20. Description of Glomus multisubtensum Mukerji, Bhattacharjee & Tewari (Mukerji et al. 1983) spore size, shape, color, and phenomena of having more than one hypha resembles this present specimens. However, G. multisubstensum has two separable walls and the spores tend to make aggregates of 5 to 8 spores. Fig. 19 : a) Murograph; b) Spore wall structure, big arrow shows remnant of evanescent wall and small arrow shows a thick laminate wall (bar = 5 Mm). Fig. 20 : a) Murograph; b) Crushed spore shows spore wall structure (bar = 10 M m); c) Subtending hyphae (arrows) (bar = 6 n m); d) Remnant of evanescent wall shown in inset picture of spore wall structure (bar = Su m). 70 Xla. E-L-M Type Sporocarps : unknown Spores : brownish yellow , globose, 160 x 160 M rn, borne on single subtending hypha. Spore contents become crystallized in mountant (PVA). Subtending hypha : yellow, straight, pore unclear, diameter 15 Mm. Circular thickening at the point of attachment observed, and constriction. Hyphal diameter decreasing from 16 to 7 M m within 35M m of attachment, hyphal wall 1 Mm. Spore wall structure: one group of three walls: wall 1, hyaline, evanescent, remnant only; wall 2, hyaline, laminate, 6 M m; wall 3, hyaline, membraneous, less than 1 M rn. Collection examined : T05a. Six-year old T. grandis. June 1988. Murograph and spore pictures are presented in Figure 21. The description of Glomus geosporum (Nicol. & Gerd.) Walker (Walker, 1982) is much like the present specimen in spore shape, color, size, wall structure, lack of sporocarp, and characteristics of subtending hypha including hyphal diameter and length. However, hyphal constriction, hyphal wall thickening at the spore base, and the spore content which become crystallized in the present collection differ from G. geosporum. Xlb. E-L-M Type Sporocarps : unknown. Spores : globose, brownish-yellow, 156 x 156 Mm. Spore borne on a single subtending hypha, brownish yellow, 11 M m, open pore, the hyphal wall decreasing from 4 to 1 M m within 25 M m from the attachment. Spore wall structure : 1 group of 2 walls: wall 1, brownish- yellow, laminate, 3 M m; wall 2, hyaline, membraneous (might be 1 or 2 layers), 1 Mm. Collection examined : OT 3. Six-year old T. grandis. June 1988. Murograph and spore photographs are presented in Figure 21. Fig. 21 : a) Murograph; b) Spore wall structure with 3 distinctive layers (1, 2, and 3) (bar = 7 M m); c) Crushed spore with spesific spore content, arrow shows hyphal attachment (bar = 15 u m). 72 Description of Glomus claroideum Schenck and Smith (Schenck and Smith, 1982), resembles the present specimen in spore shape, size, color, wall structure and the subtending hypha characteristics. Both do not form sporocarps as well. In the present specimen the subtending hyphal wall thickness decreasing within 25 n m from point of attachment which was not described in Glomus claroideum. Compared to Glomus diaphanum Morton & Walker (Morton and Walker, 1984) the present specimen has bigger spore size, and bigger hyphal diameter. XII. T06 type Sporocarp : irregular, hyaline with loose arrangement of spores. Spores : globose, subglobose to irregular, hyaline, 31 x 45 M m (9 spores only), borne on single subtending hypha. Subtending hypha : hyaline, recurved, open pore, 4 - 5 Mm diameter. Few septa and some swelling observed. The subtending hyphae are more curvy than straight. Spore wall structure : one group, unidentified, less than 1 fx m. Collection examined : TP6. Six-year old T. grandis. June 1988. Spore picture is presented in Figure 22. Fig. 22 : a) Irregular spore (bar = 6.5 n m); b) Irregular sporocarp with loose spore arrangement (bar = 10 u m). 74 3.d. Scutellispora sp. XIII. U-2M Type. Sporocarp : unknown Spores: globose, brownish yellow ,110x110 /um, spore borne terminally on single subtending hypha with bulbous suspensor, brown, obpyriform, 45 x 40 M m. Subtending hypha : brownish yellow, straight, 13 M rn diameter, hyphal wall 1 M m. Hyphal septum at the base of bulbous suspensor observed. Spore wall structure : 6 M rn thick, consists of two groups that separate widely when spore broken : Group A with single wall 1, yellow, britle, unit (?), 5M m. Group B with three walls: wall 2, unit and wall 3 and 4, membraneous (?), hyaline, 1 M rn each; or three membraneous walls (wall 2, 3, 4). Wall 3 is wavy, wall 4 turns red in Melzer's reagent. Collections examined : 1. T06a. 2. T06b. Both from six-year old T. grandis. June 1988. Murograph and spore pictures are presented in Figure 23. XIV. U-M Type Sporocarp : unknown Spores : globose, yellow, 95 x 95 Mm, borne terminally on subtending hypha with bulbous suspensor. Bulbous suspensor obpyriform, yellow, 36 x 27M m. Spore wall structure : one group of two or maybe three walls: wall 1, hyaline, evanescent, remnant only; wall 2, yellow, unit, 5 Mm; wall 3, hyaline, membraneous or coriaceous (?), less than 1 n m. Spore materials become crystallized in hypertonic mountant (PVA). Collections examined : 1. PAs14a. 2. PAs14b. Six-year old Acacia holosericea, collected June 1988. Murograph and spore pictures are presented in Figure 24. Fig. 24 : a) Murograph; b) Bulbous suspensor (bar = 6 y m); c) Spore wall structure with radial foided (arrow) (bar = 18 y m). 77 Within Gigaspora the available descriptions cited by Schenck and Perez (1988) seem not to agree with ours since both present specimens have two clearly separated wall groups rather than a single group. Germination shields might not have developed yet. The presence of membraneous walls in the inner group of both specimens is similar to Scutellispora spore wall structure. Comparison to species in Scutellispora cannot be carried out, because mature spores detected by the presence a germination shield would be the appropriate specimens to compare to available descriptions. 3.e. Unidentified genera XV. U-L/Flexible Type (Acaulospora sp.?) Sporocarps : unknown Spores : globose, yellow, 120 - 125 x 120 - 125 n m (only 2 spores). Spores borne on single subtending hypha, yellow, straight, open pore, and relatively small (4 - 5 u m) compare to the spore size. Spore wall structure : one group with two or more walls : wall 1, possibly unit, brownish yellow , rough surface,.! - 2 n m, wall 2, laminate or some flexible layers, hyaline, smooth with many sublayers. Collection examined : T08b. Six-year old T. grandis June 1988 Murograph and spore pictures are presented in Figure 25. When spore was broken, there was a tendency of walls 1 and 2 to fold into a radiate pattern which may be because of the consistency of the wall. The spore contents are very crystalline, thick and tend to stick to the spore wall (PVA as a mountant). The nature of the spore contents is different from common spore contents which is usually thick and oily. Fig. 25 : a) Murograph; b) Crushed spore shows spore walls of unit and laminate (1 and 2) (bar =10 ym); c) Spore wall in other spore shows unit and membraneous (1 and 2)(bar = lOy m); d) Hyphal attachment (arrow) (bar = 12.5 y m). 79 This type is separated from the previous type because of the spore wall characteristics, the way the wall folds when spore was broken, and the distinctive spore contents, even though the subtending hypha attachment in the slide is not clear enough to differentiate between Glomus and Acaulospora. Compared to the slide prepared by Morton (1989), even though the appearance is similar to A. laevigatum nom.ined. in the way the wall is folded and spore contents, the spore wall structure in A. laevigatum appears more complex than this spore type and the original description is not available. The spore collected from the field may be old and, when the spore was broken, the flexible / membranous / or coriaceous layers stuck together in such a way to look like a laminate wall. XVI. U-M-C Type (Acaulospora sp ?) Sporocarp : unknown Spores : globose, yellow (101 I g Y), 140 x 140 M m (one spore only). Subtending hypha not observed, only scar of hyphal attachment, brownish yellow, 16x16 Mm, opening 2 M m. Germination shield : hyaline, approximately 68 x 68 M m (boundary is not clear), formed between spore wall groups B and C. Spore wall structure : consists of three groups: Group A : wall 1, unit or two unseparated unit walls (1 and 2), yellow, 2.25 Mm, outer surface ornamented with rounded warts, brownish color, closely packed (12 - 15 warts/10 M m), each wart less than 1 M m in diameter. Group B : walls 2 and 3, hyaline, membraneous (?), 1 Mm each. Group C : walls 4, 5 and 6, with the outermost wall (4) coriaceous (?), hyaline, 2 M m with patchy roughening of surface; middle wall 5 and the innermost wall 6, membraneous or coriaceous , hyaline, 2 and 1 M m respectively. Collection examined: TP1. Six-year old T. grandis. June 1988. Murograph and spore pictures are presented in Figure 26 Fig. 26 : a) Murograph; b) Crushed spore, arrow pointing at scar (bar = 8 n m); c) Spore wall structure with 6 layers (1 to 6) separated into three groups (a,b, and c), arrows show ornamentation (at wall 1) and roughened layer (at wall 4) (bar = 8 M m). 8 1 The presence of a germination shield in this spore first suggested the genus Scutellispora even though the bulbous suspensor was not observed. There are four described Scutellispora species with three separable wall groups : S. pellucida (Nicolson & Schenck) Walker & Sanders (Koske and Walker, 1986) S. savannicola (Herrera & Ferrer) Walker & Sanders (cited from Schenck and Perez, 1982), S weresubiae Koske & Walker (Koske and Walker, 1986), and S. tricalypta (Herrera & Ferrer) Walker & Sanders (cited from Schenck and Perez, 1982). The specimen cannot be compared to S. tricalypta because structure and thickness of wall layers in every wall group, size and the color of the spore are different. Similarly, S. weresubiae differs except for spore size and the appearance of laminate wall type at the second layer. Comparing to S. pellucida does not work because of the appearance of laminate type in the second layer and unit type in the innermost of wall group B. The closest description to this spore type is S. savannicola except for the ornamentation on the outer layer and the fourth layer found in the specimen. The evidence of having ornamentation on the outer layer and the presence of a delicate germination shield between wall group A and B can also suggest that this specimen belongs to genus Acaulospora. 82 4. CONCLUSIONS The results . of pot culture study suggest that source of inoculum, and host compatibility are important factors in spore production. From original soil of plantation and nursery, 16 different spore types were described based primarilly on spore wall structure. From these grouped spore types, Glomus is the most common genus, with some examples of Sclerocystis, Scutellispora and possibly Acaulospora. There was also a type that could not be categorized into any genus. Our inability to identify any collection to species was based on the poor condition of the spores collected and the small number of spores. Another factor may be that the VAM fungi of Java are virtually unknown, and the fungi we saw may have been unknown to science. To succeed in pot culturing these fungi will require following these steps : 1) . Quantity and type of inoculum : soils or root should be considered. 2) . Pot culture medium : turface or mixture of sand and turface as the medium. 3) . Host plants : tropical plants with wide variety of species should be tested in terms of greater spore and plant roots production. 4) . Need an adequate fertilizer for plant growth with limited phosphorus contents and tropical environment should be used. Chapter IV : RESPONSE OF ACACIA MANGIUM AND A. HOLOSERICEA TO SINGLE AND MIXED SPECIES OF VAM FUNGAL INOCULUM 7. INTRODUCTION A revegetation research project on calcareous soils in Gunung Kidul region, Yogyakarta, is being carried out by Wanagama I Forest Research Center by planting 36 different species ranging from legumes to commercial forest species, including some fast growing trees such as Eucalyptus urophylla, E. deglupta and Acacia mangium (See Section I.1.). Even though superior seeds among those selected species had been chosen and planted, heterogenous growth of plants was still obtained (Anonymous,. 1988). Calcareous soils are associated with a higher content of calcium and magnesium than regular soils and most have relatively high pH which results in less availability of phosphorus. In addition, soil organic matter as a source of phosphorus is low in most calcareous soils. However, some plants survived on soils derived from calcareous parent material in a study done by Lesica and Antibus (1986) and most of those plants (84%) were VA mycorrhizal. Very poor growth of Eucalyptus dumosa seedlings from the same parent tree and grown in calcareous soil was observed unless they had formed abundant mycorrhizae (Lapeyrie & Chilvers, 1985). Evidence that mycorrhizae are more effective at mobilizing and accumulating phosphate from insoluble calcium phosphate compared to uncolonized plants has been presented (Lapeyrie & Chilvers, 1985). Mycorrhizal plants show an increased uptake of immobile nutrients in the soils, especially phosphorus through their extension of absorbing structure by extramatrica! hyphal network (Abbott and Robson, 1982; Mosse, 1981; Stribley et al., 1980). In nonmycorrhizal plants, phosphorus supply to plant growth depends on its diffusion to roots which is related to the soil moisture content. It seems likely that mycorrhizae may be more advantageous to plant growth in arid regions (Fitter, 1985 in Michelsen and Rosendahl, 1990). 84 Most plants grown on highly weathered soiis of the tropics are phosphorus deficient (Fox, 1978 in Manjunath et al., 1989). Many legumes grown on these soils need higher P for growth, nodule formation and nitrogen fixation and are rnycorrhiza dependent (Mosse, 1977 in Dodd et al., 1990). Many studies with respect to exploitation of mycorrhizal plants growing in nutrient poor regions or mining sites obtained positive responses (Cornet et al., 1982; Jasper et al., 1989a). In addition, Jasper et al., (1989b) stated that the important factor in successful establishment of rnycorrhiza dependent plants such as Acacia spp. in P-deficient soils is likely the formation of VA rnycorrhiza. Promoting growth by VAM fungal inoculation on legume trees, Acacia spp. and Leucaena spp., either with Rhizobium or not was reported (Cornet et al., 1982; Dela Cruz et al., 1988; Habte and Manjunath, 1987; Jasper et al., 1989a). The effect of VAM fungus on plant growth is very dependent on compatible symbionts and a study found that dual inoculation of Leucaena leucocephala with Glomus fasciculatum and Rhizobium improved nodulation and dry weight (Manjunath et al., 1989). Similar results were shown by Dela Cruz et al. (1988) in Acacia mangium; and in Acacia holosericea with Glomus mosseae (Cornet et al., 1982) This study aims to compare the response of Acacia mangium and A. holosericea in two and four month growing periods, inoculated with single species inoculant (Glomus versiforme (Karsten) Berch) and mixed spore inoculant from Canadian soil. 85 2. MATERIALS AND METHODS A mycorrhizal dependency experiment was done on two plant species: A. holosericea and A. mangium using two different inoculants, the pure C. versiforme (in dried root and turface form) and garden soil taken from Abbotsford, containing a mixture of VAM spores. Experimental design was a complete randomized design with 6 replications for each control and two different test plants with two different inoculants. Crowing period effect (2 months and 4 months) was also considered in this experiment. There were 96 experimental pots (2 species x (2inoculants + 2 controls) x 2 growing periods x 6 replicates). Inoculation was carried out using a 150 ml mixture of inoculant and sterile turface (1:5 v/v) and this mixture was placed in a layer in the middle of a pot containing about 250 ml sterilized turface as the medium to grow test plants. Hydrated Acacia seeds were germinated in a sterilized turface tray and after a two week germination period, each plant was transplanted into a 10 x 10 x 9 cm experiment pot. Similar height and healthy plant appearance were considered in selecting transplants. Control pots were prepared by filling them with 250 ml of sterile turface inoculated with 150 ml of solution which was taken from a supernatant of a mixture of inoculants and sterile distilled water (1:2 v/v) after sieving it through a 15 M m sieve. This was carried out to keep the control plants in a natural condition whith the soil microorganisms except VAM fungi (Furlan and Cardou, 1989). This experiment was conducted in a growth chamber with these conditions : temperature 3 0 ° C daytime and 2 8 ° C nighttime, humidity 60% and 14 hours light per day with average light intensity of 217.5 MITIOI rrf^ sec"1 (measured by Radio quantum meter of Li Cor type Li 185). Pot rotation was done every two weeks to reduce physical condition bias. Long Ashton nutrient solution (Hewitt, 1966 modified in Furlan and Cardou, 1989) was applied once a week, 50 ml per pot (Furlan and Cardou, 1989). This solution contains macro nutrients : KNO3 400 ppm, K 2 S 0 4 350 ppm, Ca(N0 3) 2 .4H 20 900 ppm, NaH 2 P0 4 .H 2 0 50 ppm, M g S 0 4 . 7 H 2 0 500 ppm; micro nutrients: M n S 0 4 . 4 H 2 0 2.25 ppm, CuSo 4 .5H 2 0 0.25 86 ppm, ZnS0 4 .7H20 0.3 ppm, H2BO3 3 ppm, NaCl 5 ppm; and trace elements: (NH4)6Mo7C>24.4H2C) 0.5 ppm and EDTA.Fe (13%) 4 ppm. This solution was used in order to maintain normal plant development and in order to encourage VAM colonization 25% of sodium monophosphate was used. An equal amount of distilled water was used to water the plants every two days in the initial growing period and then even,' day. The experiment was conducted over a 4 month period. Plant height and stem diameter were measured every week, starting with the first 2 weeks, then shoot and root dry weight of the 2 month and 4 month growing periods were also taken. In this study, the mycorrhizal dependency assessment was represented by the difference of the root and shoot dry weight between mycorrhizal and nonmycorrhizal plants. The collected data were analyzed with a Systat package program version 4.1 using Anova for two way analysis and post hoc test to compare among significant treatments. 87 3. RESULTS 3.a. Leaf morphology and plant growth The seeds of Acacia germinated after 10 days on moist turface. When seedlings were transplanted into pots, the leaf form was compound bipinnate with a large number of small leaflets arranged along the central rachis. However, 4 weeks after planting, the leaf turned lanceolate (cymbiform) (Figure 27a) as its mature form. The transition into mature leaf form seems different and faster in A. holosericea (Figure 27b) than A. mangium. The first two month growing period does not seem to show any difference morphologically between treated plants and control, either in Acacia mangium or A. holosericea (Fig. 28). However, after the 4 month growing period, the treated plants were likely to have more roots compared to control. Plant growth results are measured as plant height and stem diameter (at 1 cm above soil surface) and are presented in Tables 7a and 7b below. Plant height in the two month growing period is significantly different in treatment and interaction between treatment and species (Appendix I table XV). The Canadian soil inoculant has a significantly lower effect on plant height compared to control and single VAM species inoculant either with A. mangium or A. holosericea (Table 7a). Figure 27 : a) Leaf transition from juvenile into mature leaf type after 4 week growing period, b) Comparison between A. mangium and A. holosericea leaf transition within the same growing period 89 Table 7a. : Height and stem diameter of A. mangium and A. holosericea inoculated by C. versiforme and local soil inoculant in 2 month growing period. Treatment plant height (cm) stem diameter (mm) A.m. A.h. A.m. A.h. Control 5.53ai 6.13ai 1.62ai 1.30ai Cnd. soil 4.22b i 5.02b i 1.23ai 1.51ai C. vers. 6.83ai 6.13al 1.68ai 1.30ai Note : Am : Acacia mangium Ah : Acacia holosericea value with the same character means not significantly different within species, i = indicates there is a significant interaction between treatment and species. Table 7b. : Height and stem diameter of A. mangium and A. holosericea inoculated by C. versiforme and local soil inoculant in 4 month growing period. Treatment plant height (cm) stem diameter (mm) A.m. A.h. A.m. A.h. Control 12.833 13.473 2.46a 2.35a Cndn. soil 10.04b 9.28b 1.70b 1.60b C. vers. 15.65a 12.053 2.20a 2.17a Note : Am : Acacia mangium Ah : Acacia holosericea value with the same character means not significantly different within species. 90 Figure 28 b : Acacia holosericea grown in 2 months 91 Treatment and species do not significantly affect stem diameter, but there is a significant interaction between species and treatment (Appendix I table XVI). During the 4 month growing period, both plant height and stem diameter have been significantly affected by treatment, and it seems similar to the 2 month growing period where the Canadian soil inoculant has the greatest effect but its negative plant growth (Appendix I, Table XVII and XVI11). Interestingly, in the 4 month growing period, the treatment effect is the only significant factor while in the 2 month growing period, besides treatment, the effect of species is also significant. It seems that in the 2 month growing period there is an adjustment period for the mycorrhizal plant. 3.b. Root and shoot dry weight The results of the mycorrhizal dependency experiment are presented in Table 8a and 8b below. In the two month root dry weight, both treatment and species are significantly different (Appendix I Table X). It can be inferred that inoculation using the pure single species of VA mycorrhizal fungus resulted in higher root biomass in both A. mangium and A. holosericea than inoculation using spore mixture, but it is not significantly different from the control. The effect of pure inoculum of VAM fungi on shoot dry weight within the 2 month growing period is greater than both mixed spore inoculum and control either in A. mangium or A. holosericea. 92 Table 8a. : Mean root and shoot dry weight of Acacia mangium and A. holosericea inoculated with local soil inoculant and Glomus versiforme within 2 month growing period. treatment root dwt (g) shoot dwt (g) s/r ratio Am Ah Am Ah Am Ah Control 0.18463* 0.1431a 0.45463 0.29883 2.46 * 2.09 Cdn. soil 0.1322b* 0.1104b 0.2789b 0.40733 4.57 * 3.69 C. vers. 0.23603* 0.1527a 0.5687a 0.4661b 2.41 * 3.05 Note : Am : Acacia mangium Ah : Acacia holosericea value with the same character means not significantly different within species. * = indicates there is a significant difference between species Table 8b.: Mean root and shoot dry weight of Acacia mangium and A. holosericea inoculated with Canadian soil inoculant and Glomus versiforme within 4 month growing period. Treatment root dry weight(g) shoot dry weight(g) s/r ratio Am Ah Am Ah Am Ah Control 0.5675a 0.58673 1.5983*3 1.66643 2.82 2.84 Cdn.soil 0.3056b 0.2664b 0.8104*b 0.5733b 2.65 2.15 G.vers. 1.1181c 0.52653 2.1800*3 1.37283 1.95 2.61 Note : Am : Acacia mangium Ah : Acacia holosericea value with the same character means not significantly different within species. * = indicates there is a significant difference between species 93 Effect of the Canadian soil was significant on shoot and root dry weight in both species and it seems that inoculation with Glomus versiforme is higher than either the mixture of spore from soil inoculant or control (Table 7a and 7b; Appendix I. table IX - XIV). Interaction between treatment and species occurs significantly in the 2 month shoot dry weight where the treatment is significantly different. However, the Canadian soil inoculant behaves differently on the two species due to this significant interaction (Figure 31 ; Appendix I table IX). Analysis of the S/R ratio shows that inoculation with spore mixture increases the S/R ratio compared to control or single VAM species inoculation; and species factor is significant (Table 8a and Appendix l table XII). It seems that during the first two month growing period, the treatment effect either on root or shoot dry weight is dependent more on the symbiont compatibility. ln the 4 month growing period, root dry weight of both Acacia inoculated by Canadian soil demonstrate the lowest value followed by control and Glomus sp. as the highest value (Table 8b). A similar trend is also shown in shoot dry weight results, even though Glomus inoculant is not significantly different from control but it does differ from local soil inoculant (Table 8b and Appendix I table XIII). In shoot dry weight, the species factor is significant. From table , it can be inferred that both root and shoot dry weight in Acacia mangium with single VAM species spore inoculation is the most positively effective treatment within the same plant species and also more effective compared to Acacia holosericea with the same inoculant. S/R ratio analysis within the 4 month growing period shows a decrease from control, Canadian soil and Glomus inoculant for A. mangium but not for A. holosericea where inoculation with Glomus versiforme increases S/R ratio. Neither treatment nor species is significantly different and also there is no significant interaction (Table 8b and Appendix I table XIV). 94 3.c. VAM colonization Percent VAM colonization assessment was done only in the four month growing period. The highest colonization was found in A. holosericea inoculated by Canadian soil inoculant (26%), followed by A. mangium with C. versiforme (17%), A. holosericea with C. versiforme (16%) and the lowest was A. mangium inoculated by Canadian soil (6%). Both control studies were 0% colonization. 95 4. DISCUSSION 4.a. Shoot height and stem diameter According to New (1984), transition from juvenile compound foliage into the adult leaf form is common in phyllodinous Acacia, the group in which both studied Acacia belong. The duration of juvenile foliage stage depends on the species, and environmental conditions such as temperature and moisture (Carr & Bourdon, 1975 in New, 1984). In the first 2 month growing period, the interaction between species and treatment is significant. Plant growth difference in Acacia mangium and A. holosericea with treatments can be explained by possible fungal species preference in symbiosis and the nature of growing pattern of both test plants. These account for the different growth response of A. holosericea with Canadian soil inoculant between the 2 month and 4 month growing periods. The lowest plant growth was found in local soil compared to control and C. versiforme in both Acacia in the 4 month growing period.. One possible explanation for this is that in Canadian soil inoculum, besides VAM fungal species, there are some microorganisms that might have a negative effect on the VAM fungal species and directly or indirectly on plant growth. In addition if the soil inoculant contains more than one VAM species these may compete for space in the roots and thereby change the effectiveness that might be expressed by a single potential VAM species for Acacia. Another possible explanation is that the Canadian soil may not have had C. versiforme in it; and the effectiveness and infectivity of VAM species found in the soil differ from C. versiforme. Shoot growth and stem diameter of both Acacia inoculated with Glomus versiforme within the 4 month growing period are not significantly different from the control. It may be that the 4 month growing period is still a lag phase for plant symbionts to grow better than control plants. These results are contradictory to the results of a study on the response of tree legumes to VAM fungi and Rhizobium (Dela Cruz et al., 1988) where it was observed that Acacia auriculiformis and Acacia mangium both inoculated by Glomus fasciculatum and 96 Rhizobium have significantly better growth (more than 100%) than the control in a 4 month growing period. Perhaps dual inoculation of VAM and Rhizobium improved nodulation compared to single inoculation (Manjunath, 1984), thus giving more nitrogen supply to the plants for better growth. 4.b. Root and shoot dry weight Within the 2 month growing period, Glomus inoculation on both Acacia resulted in higher root or shoot dry weight than control or Canadian soil inoculum. Root dry weight in A. mangium and A. holosericea increased by 27% and 7% , and shoot dry weight increased by 25% and 56% from control respectively (Table 9). It is likely that a plant species has its own strategy in allocating energy for shoot or root growth, when the data shows that A. mangium gained more root dry weight and A. holosericea gained more shoot biomass. Compared to control, inoculation with Canadian soil inoculum decreased the root dry weight by 28% and 23%, and decreased shoot dry weight by 39% and increased it by 36% in A. mangium and A. holosericea (Table 9). A study using inoculation with three different Glomus resulted in a 66% increase in Acacia nilotica in total plant biomass (Michelsen and Rosendahl, 1990). It can be inferred from this comparison that inoculation with a mixture of \VAM species in which the effectiveness of its fungi is unknown does not show any benefits for the plants. Shoot/root (S/R) ratio does not show any special pattern. Canadian soil inoculation has the highest S/R ratio compared to the two other treatments. With Glomus inoculation, the S/R ratio seems similar or slightly higher than control. These results agree with the study done by Michelsen and Rosendahl (1990) who reported higher S/R ratio in VAM inoculated seedlings, Acacia nilotica and Leucaena leucocephala, grown 3 months. 97 Table 9 : Plant responses (percent change relative to control) based on shoot and root dry weight of A. inoculation. holosericea and A. mangium with Canadian soil and Glomus versiforme Treatment Root biomass shoot biomass Species A.m. A.h. A.m. A.h. 2 Months Canadian soil -28% -23% -39% + 36% C. versiforme + 27% + 7% + 25% + 56% 4 Months Canadian soil -46% -55% -49% -66% C. versiforme + 97% -10% + 36% -18% In the 4 month growing period, the differences in root and shoot dry weight between treatments and species are more obvious. In root dry weight, inoculation with Canadian soil results in 46% and 55% decreases compared to control in A. mangium and A. holosericea; inoculation with Glomus increases root dry weight by 97% for A. mangium and decreases by 10% for A. holosericea. Shoot dry weight in A. mangium and A. holosericea inoculated by Canadian soil decreases by 49% and 66%; and with Glomus, increases by 36% and decreases by 18% respectively. From these two data, it can be suggested that the association of A. mangium with Glomus versiforme is more efficient than other associations in root and shoot biomass, especially root dry weight. Overall comparison of the growth responses between the two Acacia show that in the first two months, A. holosericea has a higher response; in four months, however, A. mangium is catching up with the growth response and inoculated A. holosericea cannot even compete with control anymore. It seems that plant sensitivity to inoculan is different. S/R ratio in the 4 month growing period shows a decrease from control to Canadian soil to C. versiforme inoculum in A. mangium but not in A. holosericea: The highest VAM colonization was found in A. holosericea with soil inoculant followed by both Acacia with G. 98 versiforme and then A. mangium with Canadian soi! inoculum. These suggest that C. versiforme is likely more compatible to A. mangium than A. holosericea in either root or shoot responses, even though the infectivity of this VAM fungus is similar in both Acacia. Even though Canadian soil inoculum has the highest infectivity in A. holosericea, it does not have a positive effect on plant growth and is, in fact, lower than control. This perhaps that soil microorganisms in Canadian soil supressed plant growth. The different percentage of colonization between single VAM species and a mixture of species in Canadian soil inoculum in both Acacia plants is likely dependent on the infectivity of every single fungus, and also on types of substances produced by plants which is perhaps not the same between these two test plants, though they are both in the same family. The ability of VAM species to establish mycorrhizae depends on these substances (Harley and Smith, 1983). 99 5. CONCLUSIONS Responses of both Acacia to single VAM species and mixture of VAM species are different and in this case the Canadian soil inoculum generally supressed biomass and plant growth. Acacia mangium with Glomus versiforme inoculation apparently has better performance. Compatibility between symbionts, effectiveness of VAM spore inoculant, infectivity and environmental factors have major effects on the plant growth responses. Plant species have their own strategy in allocating biomass in their growing pattern. This study also indicates that A. mangium has more root than shoot structure compared to A. holosericea. It is necessary to investigate further whether the root development has or has not been activated by VAM inoculation since A. mangium is the most adaptable to calcareous soils in terms of fast growth and survival. 100 Chapter V : GENERAL CONCLUSIONS This study was conducted in Wanagama Forest Research Center, Yogyakarta, Indonesia where the pilot project of provenance test of 36 different tree species prepared for forest estate industry has been carried out. Attempts for reforestation of calcareous soils in this region have succeeded with some plantation species, including quick-yielding legumes. Six-year old and ten-month old seedlings of four selected plantation species: Tectona grandis, Swietenia macrophylla, Acacia holosericea and Acacia mangium were chosen to study their VAM status. Of these chosen species, all are mycorrhizal and VAM percent colonization of plants in two nurseries was higher than in the plantations. Both Acacia have greater colonization than the other two species. Indigenous tropical VAM species associated with these plants are not known to science. Only 16 different VAM spore types were found from our original soils. These are from the genera Glomus, Sclerocystis, Scutellispora and perhaps Acaulospora. Better pot culture studies for spore identification of tropical VAM species will produce more spores and also give more information for species identification. Among these considerations are : a) source of inoculum : plant roots should be used to determine host specificity, and soils can be used for more general purposes; b) pot culture medium : combination of soil and sand or just sand medium; c) host plant : broader range of host plants should be tested to determine which are able to produce more spores and d) fertilizer application : to optimize host plant growth while encouraging the formation of VAM, only phosphorus should be limited. Improvement in pot culture for forestry application should consider the similar items above, except pot culture medium is the sterilized original soil from where the VAM will be applied, and the host plants are the species that will be used in forestry practice. Response of two selected Acacia (A. holosericea and A. mangium ) to single VAM species (Glomus versiforme (Karsten) Berch) and to mixed spore inoculation was determined. Plant response to single VAM species in this case was better than to mixed spore inoculant. 101 Acacia mangium with C. versiforme has better growth performance in 4 month period compared to mixed Canadian soil inoculant and control. Root and shoot dry weight increased by 97% and 36% respectively. Host compatibility, infectivity, and effectiveness of VAM species are important factors in plant growth responses besides the environmental factors. Ideally, this study was designed to obtain the indigenous VAM species and use them for inoculation of the plant response study. Even though the indigenous spore retrieval failed, the original purpose is still able to be pursued in other studies with the indigenous tropical VAM and legumes. Our experiences will help other researchers of the same area in the tropics. This study leads to further research in VAM for forestry application through selecting the potential indigenous VAM species for better plant growth in this region. 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Prentice-Hall, Inc., Englewood Cliffs, New Yersey. 718 pp. 109 Appendix 1.RAINFALL AND ANOVA TABLES Table 1 : Monthly rainfall table from three adjacent stations from period 1987 to June 1988 monthly rainfall (mm) month station I station I! station III average January 921 854 948 908 February 245 "152 135 177 March 289 122 168 193 April 132 98 86 1054 May 8 38 17 21 June 51 5 45 34 July 1 4 2 2 August - - - -September - - 1 -October - 1 1 1 November 68 89 95 84 December 621 488 435 515 Total annual rainfall in 1987 2041 110 January 311 282 256 283 February 537 515 568 540 March 427 373 460 420 April 149 16 1 69 78 May 371 208 213 264 June 97 36 50 61 Total rainfall 1646 I l l Table II : ANOVA table of VAM percent colonization Source of var sum of square df mean square Fcal Total 113992.524 159 716.934 group 73510.598 5 14702.120 55.93* error 40481.925 154 262.870 Note : F critical (5,154),0.05 = 3.89 Table III : ANOVA table of sodium content Source of var sum of square df mean square Fcal Total 0.398 43 0.065 group 0.315 5 0.063 28.80* error 0.083 38 0.002 Note : F critical (5,38), 0.05 = 2.90 Table IV: ANOVA table of soil potassium content Source of var Sum of square df Mean square Fcal Total 0.057 43 0.0085 group 0.038 5 0.008 15.71* error 0.019 38 0.0005 Note F critical (5,38), 0.05 = 2.90 112 Table V : ANOVA table of soil calcium content Source of var Sum of square df Mean square Fcal Total 11073.667 43 257.52693 group 8068.958 5 1613.792 20.41* error 3004.709 38 79.071 Note : F critical (5,38), 0.05 = 2.90 Table VI : ANOVA table of soil magnesium content Source of var Sum of square df Mean square Fcal Total 117.58312 43 2.734491 group 96.36159 5 19.272318 34.51* error 21.22152 38 0.558461 Note : Fcritical (5,38),0.05 = 2.90 Table VII : ANOVA table of soil phosphorus content Source of var Sum of square df Mean square Fcal Total 3.7648138 43 0.08755381 group 3.0770414 5 0.61540828 34.0* error 0.687724 38 0.01809927 Note : Fcritical (5,38), 0.05 = 2.90 113 Table VIII : ANOVA table of soil nitrogen-total Source of var Sum of square df Mean square Fcal Total 442.616 43 Group 52.218 5 10.444 1.02 Error 390.398 38 10.274 Note : F critical (5,38), 0.05 = 2.90 Table IX : ANOVA table of shoot dry weight of A. mangium and A. holosericea inoculated with G. versiforme and Canadian soil within 2 month growing period. Source of var Sum of square df Mean square Fcal Treatment 0.205 2 0.103 * 5.81 Species 0.017 1 0.017 0.96 Treat x Species 0.137 2 0.069 3.88 Error 0.530 30 0.018 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 114 Table X : ANOVA table of root dry weight of A. mangium and A. holosericea with C. versiforme and Canadian soil inoculants within 2 month growing period. Source of var Sum of square df Mean square Fcal Treatment 0.032 2 0.016 * 7.54 Species 0.021 1 0.021 10.03* Treat x species 0.006 2 0.003 1.38 Error 0.064 30 0.002 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 Table XI : ANOVA table of shoot/root ratio of A. mangium and A. holosericea inoculated with C. versiforme and Canadian soil within 2 month growing period. Source of var Sum of square df Mean square Fcal Treatment 1.757 2 0.879 0.88 Species 6.384 1 6.384 * 6.40 Treat x species 5.963 2 2.982 2.99 Error 29.913 30 0.997 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 1 1 5 Table XII : ANOVA table of shoot dry weight of A. mangium and A. holosericea inoculated with G. versiforme and Canadian soil within 4 month growing period. Source of var . Sum of square df Mean square Fcal Treatment 7.814 2 3.907 28.69* Species 0.922 1 0.922 * 6.77 Treat X species 1.182 2 0.591 4.34 Error 3.949 29 0.136 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 Table XIII : ANOVA table of root dry weight of A. mangium and A. holosericea inoculated by C. versiforme and Canadian soil within 4 month growing period. Source of var Sum of square df Mean square Fcal Treatment 1.919 2 0.959 13.44* Species 0.268 1 0.268 3.75 Treat X speciels 0.491 2 0.246 3.44 Error 2.070 29 0.071 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 116 Table XIV : ANOVA table of shoot/root ratio of A. mangium and A. holosericea inoculated by . C. versiforme and Canadian soil within 4 month growing period. Source of var Sum of square df Mean square Fcal Treatment 2.299 2 1.150 2.59 Species 0.698 1 0.698 1.57 Treat. X species 1.854 2 0.927 2.09 Error 12.878 29 0.444 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 Table XV : Anova table of plant height of Acacia mangium and A. holosericea inoculated by Glomus versiforme and Canadian soil within 2 month growing period Source of var Sum of square df Mean square Fcal Treatment 21.549 2 10.774 27.99* Species 0.490 1 0.490 1.27 Treat, x Sp. 3.980 2 1.990 5.17* Error 11.550 30 0.385 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 Table XVI : Anova table of stem diameter of Acacia mangium and A. holosericea inoculated by Glomus versiforme and Canadian soil within 2 month growing period. Source of var Sum of square df Mean of square Fcal Treatment 0.0093 2 0.047 0.73 Species 0.181 1 0.181 2.81 Treat.x sp. 0.788 2 0.394 6.12* Error 1.932 30 0.064 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 Table XVII : Anova table of plant height of Acacia mangium and A. holosericea inoculated by Glomus versiforme and Canadian soil within 4 month growing period Source of var Sum of square df Mean square Fcal Treatment 177.785 2 88.893 15.64* Species 4.203 1 4.203 0.74 Treat, x Sp. 38.402 2 3.378 0.05 Error 170.538 30 5.685 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 118 Table XVIII : Anova table of stem diameter of Acacia mangium and A. holosericea inoculated by Glomus versiforme and Canadian soil within 4 month growing period Source of var Sum of square df Mean of square Fcal Treatment 5.379 2 2.689 12.66* Species 0.006 1 0.006 0.03 Treat, x sp. 0.115 2 0.058 0.27 Error 6.372 30 0.212 Note : F-table (0.01,2,30) = 5.39 (0.05,2,30) = 4.18 F-table (0.01,1,30) = 7.56 (0.05,1,30) = 5.57 119 2. CALCULATION Regression Outputrtotal species Constant 39.265 (bO) Std Err of Y Est 19.448 R Squared 0.326 R A 2 R = 0.571 No. of Observations 160.000 Degrees of Freedom 153.000 X Coefficient(s) b1 b2 b3 b4 b5 b6 52.261 -404.747 -0.199 1.548 -8.068 1.892 Std Err of Coef. 23.794 61.692 0.139 2.284 5.823 0.588 t = b/s(b) 2.196 -6.561 -1.437 0.678 -1.386 3.220 t A 2 = F 4.824 43.044 2.066 0.459 1.920 10.365 * * * F0.05 (1,153) (0.05) 3.900 (0.01) 6.800 t = r/s(r) t= 0.029 t A 2 = 0.001 F= 0.001 F(6,153)= 2.490 3.240 ns ns ns Regression Output:minus Mg Constant 40.722 Std Err of Y Est 19.414 R Squared 0.324 No. of Observations 160.000 Degrees of Freedom 154.000 X Coefficient(s) b1 b2 b3 b5 b6 56.033 -405.293 -0.208 8.734 2.012 Std Err of Coef. 23.093 61.578 ' 0.138 5.729 0.560 t = b/s(b) 2.426 -6.582 -1.513 -1.524 3.596 t A 2 5.887 43.320 2.288 2.324 12.928 * ** ns ns * Ftab (0.05)3.900 (0.01)6.800 Regression Output:minus Ca,Mg Constant 23.246 Std Err of Y Est 19.494 R Squared 0.314 No. of Observations 160.000 Degrees of Freedom 155.000 b1 b2 b5 b6 X Coefficient(s) 75.539 -349.132 -8.967 2.087 Std Err of Coef. 19.236 49.328 5.751 0.560 t = b/s(b) 3.927 -7.078 -1.559 3.728 t A 2 15.420 50.094 2.431 13.902 t tab .900 6.800 Regression Output:minus Ca,Mg,P Constant 13.223 Std Err of Y Est 19.584 R Squared 0.303 No. of Observations 160.000 Degrees of Freedom 156.000 b1 b2 b6 X Coefficient(s) 86.440 -315.625 2.023 Std Err of Coef. 18.003 44.605 0.561 t = b/s(b) 4.801 -7.076 3.607 t A 2 23.053 50.070 13.013 y= 13.22314 +86.43976X1-315.624X2 +2.022945X6 Regression Output:on ily X1 Regression Outputonly X2 Constant 34.355 Constant 76.875 Std Err of Y Est 22.455 Std Err of Y Est 21.146 R Squared 0.072 R Squared 0.177 No. of Observations 160.000 No. of Observations 160.000 Degrees of Freedom 158.000 Degrees of Freedom 158.000 b1 b2 X Coefficient(s) 68.989 X Coefficient(s) -264.369 Std Err of Coef. 19.689 Std Err of Coef. 45.322 t(0.05) 3.504 (0.01) 12.278 R A 2 increment= Na 0.072 K 0.177 Ntot 0.054 F(1,156) (0.05) 3.900 (0.01) 6.800 Crit. value of R A 2 (0.05) 0.024.L. 121 Regression Output: Ah site general Constant 28.665 Std Err of Y Est 13.539 R Squared 0.497 r No. of Observations 30.000 Degrees of Freedom 23.000 X Coefficient(s) b1 b2 84.449 -384.467 Std Err of Coef. 69.719 195.727 t = b/s(b) 1.211 -1.964 1.467 3.858 t A 2 ns * 0.247 b3 -2.244 3.549 -0.632 0.400 ns t = 0.018 ns b4 3.474 5.762 0.603 0.364 ns h5 85.920 206.445 0.416 0.173 ns b6 0.543 1.253 0.433 0.188 ns t(0.05) 2.530 (0.01) 3.710 Regression Output:Ah site minus P Constant 57.226 Std Err of Y Est 13.304 R Squared 0.493 No. of Observations 30.000 Degrees of Freedom 24.000 XCoefficient(s) b1 b2 77.512 -416.954 Std Err of Coef. 66.521 176.369 t = b/s(b)1.165 -2.364 t A 2 1.358 5.589 ns ** t(0.05) 2.620 (0.01) 3.900 b3 b4 b6 -1.691 3.015 0.424 3.234 5.557 1.199 -0.523 0.543 0.353 0.274 0.294 0.125 ns ns ns Regression Output:Ah site minus P,N Constant 48.964 Std Err of Y Est 13.069 R Squared 0.490 No. of Observations 30.000 Degrees of Freedom 25.000 b1 b2 b3 b4 X Coefficient(s) 89.663 -381.489 -0.970 2.271 Std Err of Coef. 55.946 142.493 2.464 5.052 t = b/s(b) 1.603 -2.677 -0.394 0.450 t A 2 2.569 7.168 0.155 0.202 ns * * ns ns t(0.01) 2.760 (0.05) 4.180 122 Regression Output:Ah site minus P,N,Ca Constant 34.653 Std Err of Y Est 12.855 R Squared 0.487 No. of Observations 30.000 Degrees of Freedom 26.000 b1 b2 b4 X Coefficient(s) 104.499 -395.590 0.980 Std Err of Coef. 40.664 135.656 3.779 t = b/s(b) 2.570 -2.916 0.259 t A 2 6.604 8.504 0.067 * * * * ns 2.980 4.640 Regression Output:Ah site minus N,P,Ca,Mg Constant 37.546 Std Err of Y Est 12.631 R Squared 0.486 0.697 s(r) A 2 = (1-rA 2)/(n-M) No. of Observations 30.000 s(r)A 2 = 0.018 Degrees of Freedom 27.000 s(r) = 0.136 b1 b2 X Coefficient(s) 103.487 -382.787 t= 5.143 Std Err of Coef. 39.771 124.147 t A 2 = 26.448 F= 26.448 t 2.602 -3.083 t A 2 6.771 9.507 F0.05(2,27) 3.350 ** ** 0.01(2,27) 5.490 Y = 37.54628 +103.4874X1-382.787X2 (X1)(Y): (X1,X2)(Y): Regression Output: Regression Output: Constant 12.397 Constant 37.55 Std Err of Y Est 14.422 Std Err of Y Est 12.631 R Squared 0.305 R Squared 0.486 No. of Observations 30.000 No. of Obseivations 30.00 Degrees of Freedom 28.000 Degrees of Freedom 27.00 b1 b1 b2 X Coefficient(s) 148.143X Coefficient(s) 103.487 -382.787 Std Err of Coef. 42.294 Std Err of Coef. 39.771 124.147 R A 2 increment R A 2X1 R A 2X2 0.305 0.181 0.305 0.181 * * * Ftab(1,27) 0.050 4.210 0.010 7.680 R critical value 123 0.050 0.135 0.010 0.221 Regression OutputAm site general Constant 440.444 Std Err of Y Est 21.295 R Squared 0.354 R A 2 0.126 No. of Observations 30.000 t=R/s(r) 0.006 ns Degrees of Freedom 23.000 X Coefisient(s) bl b2 b3 b4 b5 b6 -484.673 -2345.048 5.644 -8.299 -55.345 -0.028 Std Err of Coef. 351.210 1699.903 12.426 62.359 203.419 3.299 t = b/s(b) -1.380 -1.380 0.454 -0.133 -0.272 -0.009 1.904 1.903 0.206 0.018 0.074 0.000 t A 2 ns ns ns ns ns ns 2.530 3.710 Regression OutputAm site minus N-tot Constant 439.170 Std Err of Y Est 20.846 R Squared 0.354 No. of Observations 30.000 Degrees of Freedom 24.000 X Coefficient(s) b1 b2 b3 b4 b5 -483.396 -2343.537 5.619 -8.305 -54.041 Std Err of Coef. 311.100 1655.075 11.830 61.042 131.630 t=b/s(b) -1.554 -1.416 0.475 -0.136 -0.411 t A 2 2.414 2.005 0.226 0.019 0.169 ns ns ns ns ns t(0.05) 2.620 (0.01) 3.900 Regression Output:Am site minus N,Ca Constant 403.282 Std Err of Y Est 20.433 R Squared 0.354 No. of Observations 30.000 25.000 b1 b2 b3 b5 X Coefficient(s) -444.902 -2138.408 4.050 -60.377 Std Err of Coef. 126.772 669.144 2.588 120.676 t = b/s(b) -3.509 -3.196 1.565 -0.500 t A 2 12.316 10.213 2.449 0.250 124 " ** ns ns t(0.05) 2.760 (0.01) 4.180 Regression Output:Am site minus N,Ca,P Constant 375.242 Std Err of Y Est 20.136 R Squared 0.347 No. of Observations 30.000 Degrees of Freedom 26.000 b1 b2 b3 X Coefficient(s) -430.736 -1968.323 3.135 Std Err of Coef. 121.775 567.987 1.804 t = b/s(b) -3.537 -3.465 1.737 t A 2 12.511 12.009 3.018 * * * * * t(0.05) 2.980 (0.01) 4.640 Regression Output:Am site with XI variable Constant 109.501 Std Err of Y Est 23.562 R Squared 0.038 No. of Observations 30.000 Degrees of Freedom 28.000 b1 X Coefficient(s) -89.498 Std Err of Coef. 85.331 Regression Output:Am with X1,X2 Constant 333.168 Std Err of Y Est 20.875 R Squared 0.272 No. of Observations 30.000 Degrees of Freedom 27.000 b1 b2 X Coefficient(s) -360.080 -1347.083 Std Err of Coef. 118.996 457.483 R A 2 increment Rx1 Rx2 Rx3 0.038 0.234 0.076 X1 X2 X3 R A 2 0.038 0.234 0.076 ns ** ns F(1,26) 4.230 (0.05) 7.720 (0.01) Crit value R A 2 125 (0.05) 0.140 (0.01) 0.229 Regression OutpufcSm site general Constant 255.842 Std Err of Y Est 10.294 R Squared 0.288 No. of Observations 28.000 Degrees of Freedom 21.000 X Coefficient(s) b1 b2 b3 b4 b5 b6 33.556 -53.351 -1.111 -0.885 -40.797 -8.853 Std Err of Coef. 596.196 957.785 11.544 24.747 87.043 66.232 t=b/s(b) 0.056 -0.056 -0.096 -0.036 -0.469 -0.134 0.003 0.003 0.009 0.001 0.220 0.018 ns ns ns ns ns ns Regression Output:Sm site minus Mg Constant 203.992 Std Err of Y Est 10.057 R Squared 0.288 No. of Observations 28.000 Degrees of Freedom 22.000 X Coefficient(s) b1 b2 b3 b5 b6 15.135 -21.497 -0.714 -43.236 -6.598 Std Err of Coef. 293.085 343.431 3.127 52.820 19.766 t 0.052 -0.063 -0.228 -0.819 -0.334 t A 2 0.003 0.004 0.052 0.670 0.111 Regression OutputSm site minus Mg,Na Constant 194.491 Std Err of Y Est 9.505 R Squared 0.294 No. of Observations 30.000 Degrees of Freedom 25.000 b2 b3 b5 b6 X Coefficient(s) -11.226 -0.632 -42.139 -6.035 Std Err of Coef. 300.627 2.675 23.448 16.910 t -0.037 -0.236 -1.797 -0.357 0.001 0.056 3.230 0.127 ns ns * ns Regression Output:Sm site minus Mg,Na,K Constant 179.770 Std Err of Y Est 9.321 R Squared 0.294 No. of Observations 30.000 126 Degrees of Freedom 26.000 b3 b5 b6 X Coefficient(s) -0.534 -42.715 -5.415 Std Err of Coef. 0.472 17.321 3.209 t -1.131 -2.466 -1.688 t A 2 1.279 6.081 2.848 Regression Output:Sm site minus Na,KCa,Mg Constant 103.183 Std Err of Y Est 9.369 R Squared 0.259 No. of Observations 30.000 Degrees of Freedom 27.000 b5 b6 X Coefficient(s) -49.562 -1.968 Std Err of Coef. 16.313 1.007 -3.038 -1.954 9.231 3.819 * * * Regression Output:X5 Constant 59.384 Std Err of Y Est 9.829 R Squared 0.154 No. of Observations 30.000 Degrees of Freedom 28.000 b5 X Coefficient(s) -25.890 Std Err of Coef. 11.462 R A 2 increment X5 X6 0.154 0.105 0.154 0.105 * ns Ftab(1,27) (0.05) 4.210 (0.01) 7.680 Crit value of R A 2 (0.05) 0.135 (0.01) 0.221 Regression Output:Tg site general Constant 68.094 Std Err of Y Est 11.857 R Squared 0.737 No. of Observations 30.000 Degrees of Freedom 23.000 X Coefficient(s) b1 b2 b3 b4 b5 b6 131.535 -340.444 -1.765 10.160 -57.128 0.515 Std Err of Coef. 127 186.617 249.639 t=b/s(b) 0.705 -1.364 0.497 1.860 ns ns 3.742 10.018 -0.472 1.014 0.222 1.029 ns ns 17.022 1.600 -3.356 0.322 11.263 0.104 Regression Qutput:Tg site minus Ntot Constant 72.475 Std Err of Y Est 11.634 R Squared 0.735 No. of Observations 30.000 Degrees of Freedom 24.000 X Coefficient(s) b1 b2 b3 b4 b5 124.807 -310.309 -1.731 11.425 -56.703 Std Err of Coef. 181.947 227.059 3.670 9.043 16.651 t 0.686 -1.367 -0.471 1.263 -3.405 t A 2 0.471 1.868 0.222 1.596 11.597 ns ns . ns ns ** Regression Output:Tg site minus Ntot,Ca Constant 67.180 Std Err of Y Est 11.451 R Squared 0.733 No. of Observations 30.000 Degrees of Freedom 25.000 b1 b2 b4 b5 X Coefficient(s) 46.660 -407.214 15.505 -57.580 Std Err of Coef. 73.888 95.001 2.583 16.287 t 0.631 -4.286 6.002 -3.535 t A 2 0.399 18.373 36.027 12.498 Regression OutputTg site minus Ntot,Ca,Na Constant 77.146 Std Err of Y Est 11.318 R Squared 0.729 No. of Observations 30.000 Degrees of Freedom 26.000 b2 b4 b5 X Coefficient(s) -419.971 16.211 -52.450 Std Err of Coef. 91.749 2.301 13.952 t=b/s(b) -4.577 7.044 -3.759 t A 2 20.953 49.615 14.132 * * * * * * Regression Output:X2 and X4 Constant 38.025 Std Err of Y Est 13.799 R Squared 0.581 No. of Observations 30.000 128 Degrees of Freedom 27.000 b2 b4 X Coefficient(s) -318.301 14.896 Std Err of Coef. 106.888 2.773 Regression Output:X2, X4 and X5 Constant 77.146 Std Err of Y Est 11.318 R Squared 0.729 No. of Observations 30.000 Degrees of Freedom 26.000 b2 b4 b5 X Coefficient(s) -419.971 16.211 -52.450 Std Err of Coef. 91.749 2.301 13.952 Regression Output: X2 Constant 73.987 R A 2 increment Std Err of Y Est 19.489 X2 X4 X5 R Squared 0.134 0.134 0.448 0.147 No. of Observations 30.000 0.133 0.447 0.147 Degrees of Freedom 28.000 ns ** * b2 X Coefficient(s) -313.660 F(1,26) 4.230 (0.05) Std Err of Coef. 150.956 7.720 (0.01) Crit value of R A 2 (0.05) 0.140 (0.01) 0.229 

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