1

ESTABLISHMENT OF VEGETATIVELY PROPAGATED Khaya

anthoteca PRE-INOCULATED WITH ARBUSCULAR MYCORRHIZAE

FUNGI (AMF) ON AN EX-COAL MINED SITE

PHILIP WORLANYO DUGBLEY

GRADUATE SCHOOL BOGOR AGRICULTURAL UNIVERSITY

BOGOR 2015

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DECLARATION OF ORIGINALITY

I hereby declare that this thesis titled ESTABLISHMENT OF VEGETATIVELY

PROPAGATED Khaya anthoteca PRE-INOCULATED WITH ARBUSCULAR

MYCORRHIZAE FUNGI (AMF) ON AN EX-COAL MINED SITE and the

work reported herein were composed by and originated entirely from me under

the supervision of my supervisory committee. I therefore declare that, this is a true

copy of my thesis as approved by my supervisory committee and has not been

submitted for a higher degree to any other University or Institution. Information

derived from the published and unpublished work of others has been duly

acknowledged in the text as well as references given in the list of sources.

Bogor, April 2015

Philip Worlanyo Dugbley

Registration No.: E451138231

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SUMMARY

PHILIP WORLANYO DUGBLEY. Establishment of Vegetatively Propagated

Khaya anthoteca Pre-Inoculated with Arbuscular Mycorrhizae Fungi (AMF) On

an Ex-Coal Mined Site. Supervised by IRDIKA MANSUR and BASUKI

WASIS

Coal mining provides a means for creating wealth and significantly contributes to

export earnings, economic activity and employment whilst supporting regional

development. For example, the mining sector of Indonesia contributes to the

nations economy for about 11.54% of total GDP. However, coal mining is one of

the most severe disturbances in terrestrial ecosystems. It causes large-scale

deforestation and land degradation with complete loss of topsoil. Thus, the

removal of the natural vegetation and upper soil horizons for mining exploration

hinders the establishment and survival of plant and soil microbial communities.

Revegetation of coal mined lands is therefore required to enable the re-use of such

resources for other purposes. The establishment of tree species capable of

protecting the underlying soil and its micro-fauna and flora is one way of

achieving this aim. This study aims to; (i) determine the potential application of

stem cutting to propagate K. anthoteca seedlings. (ii) determine the status of

arbuscular mycorrhiza fungi (AMF) symbiosis of vegetatively propagated K.

anthoteca (iii) evaluate on the field, the effect of compost and pre-inoculation on

vegetatively propagated K. anthoteca for re-vegetation of ex-coal mined site in

South Sumatra of Indonesia. The design for the field study was the completely

randomized design (CRD) in factorial experiment. Four (4) levels of each factor

namely; C0 (control), C1 (5 kg of Salvinia natans compost), C2 (5 kg of

community/commercial compost) and C3 (2.5 kg each of Community and Salvinia

natans composts); M0 (control), M1 (50 spores of Gigaspora margarita

mycorrhizae), M2 (50 spores of Glomus manihotis mycorrhizae) and M3 (25

spores each of G. margarita and G. manihotis) mycorrhizae with four replicates.

Data collected on plant height, diameter and leave count were subjected to a two-

way analysis of variance (ANOVA) at a significance level of 5% ( 0.05) using

the Minitab statistical analysis package (Minitab Inc.). Tukeys HSD test was

used for multiple comparison tests for treatments that differed significantly.

Results of the cutting experiment demonstrated that, the species can be

vegetatively propagated through cutting without hormone application. Analysis of

variance tested at 0.05 revealed no significant difference between the

treatment means of hormone and wounding on the number of roots. Again, the

length of the longest root was not significantly different over the control

treatment. The trapping experiment also showed no significant difference between

K. anthoteca cuttings and other trapping plant species. However, this observation

was frequent in the young and meristematic segments of the roots for K.

anthoteca. An average root colonization of 32% was observed for K. anthoteca

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and the highest recorded by S. bicolor of about 43%. There was also a significant

positive correlation (r = 0.892; p 0.000) between percentage root segment

colonization and the number of counted spores. The results for the field

experiment showed that compost has significant effect (P < 0.001) on height,

diameter and leaf increments with steady increment for the study period. There

was no significant effect (P > 0.05) of mycorrhizae treatment as well as the

interaction between both factors (AMF and compost) on the growth of K.

anthoteca on the field. However, compost composition from a mixture of S.

natans and that prepared from the community of PT. Bukit Asam (C3) recorded

higher increment in height of 9.31 cm while compost from S. natans only (C1),

community compost (C2) and control (C0) had increments of 9.00 cm, 5.78 cm

and 4.47 cm respectively. The arbuscular mycorrhizae fungi played major role in

the survival of the species on the field. There was significant percentage

difference of between 18.5-37.5% over the control treatment. AMF from G.

manihotis had the highest plant percentage survival of 81.25% whiles the control

had the lowest percentage of 43.75%. The study concludes that wounding

treatment play more critical role in the vegetative propagation of K. anthoteca

seedlings as compared to hormone (auxin) application. Again, AMF soil

inoculums under K. anthoteca can be a good source of inoculants for the

establishment of the species in areas of degraded lands. Furthermore, AMF and

compost applications are feasible and sound technologies for establishing K.

anthoteca on an ex-coal mined site. Plants are also able to withstand harsh

environmental conditions through fungi-plant symbiosis enhancing the chances of

survival on the field, aiding plant establishment. Thus, K. anthoteca propagules

can be established on an ex-coal mined site with compost and AMF inoculation.

However compost made from organic materials such as S. natans is preferable for

the growth and development of the plants.

Keywords: Coal mining, K. anthoteca, AMF, Compost, Plant Hormone

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RINGKASAN

PHILIP WORLANYO DUGBLEY. Pertumbuhan Khaya Anthoteca Hasil Propagasi Vegetatif Diinokulasi Dengan Fungi Mikoriza Arbuskular (FMA) Pada Lahan Pasca Tambang Batu Bara. Dibimbing IRDIKA MANSUR dan BASUKI WASIS

Pertambangan batubara merupakan nilai kekayaan yang berarti dan

berkontribusi nyata dalam pendapatan ekspor, aktivitas ekonomi, dan penyerapan

lapangan pekerjaan sehingga dapat mendorong perkembangan daerah. Sebagai

contoh, sektor pertambangan Indonesia berkontribusi terhadap ekonomi negara

sebesar 11.54% dari GDP. Akan tetapi, pertambangan batubara merupakan

industri yang sangat merusak ekosistem. Hal tersebut dikarenakan deforestasi dan

degradasi lahan dalam skala besar dengan hilangnya seluruh top soil. Oleh karena

itu, hilangnya vegetasi alami dan horizon tanah di lapisan atasnya untuk

eksplorasi tambang menghambat berkembangnya tanaman dan bertahan hidupnya

tanaman serta komunitas mikroba tanah. Revegetasi lahan pasca tambang

diperlukan untuk memungkinkan penggunaan kembali sumberdaya untuk tujuan

lainnya. Penanaman pohon dapat melindungi lapisan tanah di bawahnya dan serta

mikro fauna dan flora yang ada di dalamnya. Penelitian ini bertujuan untuk: i)

menentukan pengaruh hormon dan perlakuan pelukaan pada perbanyakan

vegetatif bibit K. anthoteca ii) kelemahan stek pada infeksi inokulum FMA tanah

dibandingkan dengan species inang lainnya seperti Sorghum bicolor dan Puereria

javanica iii) untuk menduga di lapangan, pengaruh pre-inokulasi Fungi Mikoriza

Arbuskula (FMA) dan aplikasi kompos terhadap performa pertumbuhan dari

mahoni merah Afrika, K. anthoteca pada area pasca tambang. Rancangan yang

digunakan dalam studi ini adalah Rancangan Acak Lengkap (RAL) dalam

percobaan faktorial. Empat taraf dari masing-masing faktor di antaranya: C0

(kontrol), C1 (5 kg kompos Salvinia natans) C2 (5 kg kompos komersial), C3 (2.5

kg kompos komersil dan kompos Salvinia natans); M0 (kontrol) M1 (50 spora

Gigaspora margarita), M2 ( 50 spora Glomus manihotis), M3 (masing-masing 25

spora G. margarita dan G. manihotis) dengan empat ulangan. Data yang

dikumpulkan di antaranya tinggi tanaman, diameter batang, dan jumlah daun

dianalisis dengan ANOVA dengan tingkat kepercayaan 5% ( 0.05)

menggunakan analisis statistik Minitab. Uji Tukeys HSD digunakan untuk uji

perbandingan berganda untuk perlakuan yang berbeda nyata. Hasil eksperimen

stek menunjukkan bahwa jenis kaya ini dapat diperbanyak secara vegetatif

melalui strek tanpa aplikasi hormon. Uji ANOVA dengan tingkat kepercayaan

5% menunjukkan tidak ada perbedaan di antara perlakuan hormon dan pelukaan

pada jumlah akar. Selain itu, panjang dari akar terpanjang tidak berbeda secara

nyata dengan kontrol. Eksperimen trapping juga menunjukkan tidak ada

perbedaan nyata antara stek K. anthoteca dengan tanaman inang trapping lainnya.

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Akan tetapi, pengamatan ini secara berulang-ulang dilakukan pada segmen akar

muda dan maristematik dari K. anthoteca. Rata-rata kolonisasi akar K. anthoteca

adalah 32% dan tertinggi tercatat pada S. bicolor sebesar 43%. Selain itu juga

terdapat korelasi positif yang nyata (r = 0.892; p 0.000) antara persentasi

kolonisasi akar dan jumlah spora yang dihitung. Hasil eksperimen lapangan

menunjukkan bahwa kompos memiliki pengaruh yang signifikan (P < 0.001) pada

tinggi, diameter, dan riap daun dengan riap yang tetap untuk periode studi. Tidak

ada pengaruh yang nyata (P > 0.05) pada perlakuan mikoriza seperti halnya

intreaksi antara kedua faktor (FMA dan kompos) pada pertumbuhan K. anthoteca.

Akan tetapi, komposisi kompos dari gabungan S. natans dan pupuk yang

disediakan oleh PT. Bukit Asam (C3) tercatat memiliki riap teringgi pada tinggi

yaitu 9.31 cm sedangkan kompos S. natans (C1), kompos komersial (C2), dan

kontrol (CO) memiliki riap masing-masing sebesar 9.00 cm, 5,78 cm, dan 4.47

cm. Fungi mikoriza arbuskula memainkan peran yang penting dalam

keberlangsungan hidup tanaman di lapangan. Persentase perbedaan secara nyata

di antara 18.5-37.5% dibandingkan dengan kontrol. FMA dari G. manihotis

memiliki persentase hidup tanaman tertinggi sebesar 81.25% sedangkan kontrol

memiliki persentasi terkecil sebesar 43.75%. studi menyimpulkan bahwa

perlakuan pelukaan memainkan peran kritis pada perbanyakan vegetatif bibit K.

anthoteca dibandingkan dengan aplikasi hormon (auksin). Selain itu, inokulum

tanah FMA yang ada pada tegakan K. anthoteca dapat menjadi sumber yang

bagus pada inokulan untuk penanaman K. anthoteca di area terdegradasi.

Aplikasi FMA dan kompos mudah dikerjakan merupakan teknologi dalam

penanamannya pada lahan pasca tambang batubara. Tanaman juga dapat bertahan

pada kondisi lingkungan yang keras melalui simbiosis tanaman dan fungi

meningkatkan kesempatan bertahan hidup di lapangan. Oleh karena itu, propagul

K. anthoteca dapat ditanam di area pasca tambang batubara dengan kompos dan

inokulasi FMA. Akan tetapi, kompos yang terbuat dari bahan organik seperti S.

natans lebih baik untuk pertumbuhan dan perkembangan tanaman.

Kata kunci: Tambang batubara, K. anthoteca, FMA, Kompos, Hormon Tanaman

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Copyright owned by IPB, 2015

All rights reserved

No part of this document may be reproduced or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without

prior written permission from Bogor Agricultural University (IPB)

8

ESTABLISHMENT OF VEGETATIVELY PROPAGATED Khaya

anthoteca PRE-INOCULATED WITH ARBUSCULAR MYCORRHIZAE

FUNGI (AMF) ON AN EX-COAL MINED SITE

PHILIP WORLANYO DUGBLEY

A Thesis Submitted in partial fulfillment of the requirements for the degree of

Master of Science In

Tropical Silviculture

GRADUATE SCHOOL BOGOR AGRICULTURAL UNIVERSITY

BOGOR 2015

9

External Examiner: Prof. Dr. Ir. Sri Wilarso Budi R, MS

10

Thesis Title : Establishment of Vegetatively Propagated Khaya anthoteca

Pre-Inoculated with Arbuscular Mycorrhizae Fungi (AMF)

On an Ex-Coal Mined Site.

Name : Philip Worlanyo Dugbley

Student Number : E451138231

Major : Tropical Silviculture (SVK)

Approved by,

Supervisory Committee:

Dr. Ir. Irdika Mansur, M.For.Sc Dr. Ir. Basuki Wasis, MS Head-supervisor Co-supervisor

Endorsed by,

Head of Tropical Silviculture Dean of Graduate School

Study Program Prof. Dr. Ir. Sri Wilarso Budi R, MS Dr. Ir. Dahrul Syah, M.Sc. Agr. Final Exams: 13th March, 2015 Graduated on: 27th March, 2015

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ACKNOWLEDGEMENT

I am forever grateful to God Almighty for His everlasting love and

protection throughout my study. Much gratitude to my project supervisors Dr.

Irdika Mansur and Dr. Bakusi Wasis as well as Prof. Dr. Sri Wilarso Budi as an

external examiner whose invaluable supervisions, guidance, support, enthusiasm

and more importantly, constructive criticisms have perfected and facilitated the

completion of this work.

I also wish to acknowledge the government of Indonesia through the

Ministry of Education and Culture (DIKTI) for the grant of scholarship to pursue

my masters degree. Assistance from the laboratory experts of the research center

for bio-resources and biotechnology, Bogor Agricultural University (IPB) are

much appreciated. I again wish to thank the staff of PT. Bukit Asam (Persero),

Tbk. especially Mr. Muhamad Bagir and Dedy Saptaria Rosa for the provision of

boarding and lodging during the field work. I further wish to thank all staff and

colleagues in the Department of Silviculture, Forestry Faculty in the Bogor

Agricultural University (IPB) for their contributions and help in diverse ways

especially Dr. Noor Farikhah Haneda and Mr. Ismail during my masters degree

program in Indonesia.

Furthermore, I would like to express my profound love and special thanks to

my loving parents; Mr. Anthony Dugbley and Mrs. Comfort Agyeibea for their

constant pieces of advice, guidance and prayers. Supports and persistent

encouragements from my siblings; Clara Beatrice Mensah, Emelia Korantemaa

and all friends during this study are as well deeply appreciated.

Finally, I wish to acknowledge each and every person that directly and/or

indirectly contributed to the completion of this work piece. Indeed, you have all

played significant roles and may God richly bless you all. I hereby dedicate this

work to God Almighty and to my humble family whose love and constant pieces

of advice has brought me this far.

Bogor, April 2015

Philip Worlanyo Dugbley

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TABLE OF CONTENTS

Page

LIST OF TABLES ................................................................................................ xiv

LIST OF FIGURES ............................................................................................... xv

LIST OF APPENDICES ....................................................................................... xvi

1. INTRODUCTION ............................................................................................... 1

1.1 Background .................................................................................................... 1

1.2 Specific objectives ......................................................................................... 2

1.3 Research hypotheses ....................................................................................... 3

1.4 Problem statement ........................................................................... ...............3

1.5 Justification of study ...................................................................................... 4

2. LITERATURE REVIEW ................................................................................... 5

2.1 Coal mining ................................................................................................... 5

2.1.1 Coal mining in Indonesia ................................................................... 5

2.1.2 Some characteristics of coal mined soils ........................................... 6

2.1.3 Impact of coal mining ........................................................................ 6

2.1.4 Mined land rehabilitation in Indonesia .............................................. 8

2.2 Origin of the Khaya species .......................................................................... 9

2.2.1 Botanical description of K. anthoteca ................................................ 9

2.2.2 Distribution, habitat and ecology ..................................................... 10

2.2.3 Storage and viability of K. anthoteca seeds ..................................... 11

2.2.4 Planting and propagation ................................................................. 12

2.2.5 Growth and development of K. anthoteca ........................................ 12

2.2.6 Uses of K. anthoteca ......................................................................... 12

2.3 Environmental factors affecting plant growth .............................................. 13

2.3.1 Water ................................................................................................. 13

2.3.2 Sunlight ............................................................................................. 13

2.3.3 Temperature ...................................................................................... 14

2.3.4 Plant nutrient and fertilization........................................................... 14

2.4 Vegetative propagation ................................................................................ 15 2.4.1 Stem cutting in vegetative propagation ............................................. 16

2.5 The Arbuscular mycorrhizal fungi (AMF) ................................................. 17

2.5.1 General functions of arbuscular mycorrhizae (AM) ........................ 17

2.5.2 Arbuscular mycorrhizae and plant nutrient uptake ........................... 19

2.5.3 Arbuscular mycorrhizae and plant water relations .......................... 20

2.5.4 AMF host specificity ........................................................................ 21

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2.5.5 Mycorrhizae-induced growth depressions ........................................ 22

2.5.6 Application of mycorrhiza on forest tree seedlings ......................... 22

2.6 Compost ....................................................................................................... 23

2.6.1 Compost as soil amendment ............................................................. 23

2.6.2 Compost and soil microbes ............................................................... 24

2.6.3 Compost and plant growth ............................................................... 25

2.6.4 Compost and AMF interaction for mined land reclamation ............. 25

3. RESEARCH METHODOLOGY ........................................................................... 27

3.1 Time and area of study ................................................................................ 27

3.2 Research materials and tools ....................................................................... 27

3.3 Research designs ......................................................................................... 28

3.3.1 Vegetative propagation of K. anthoteca .......................................... 28

3.3.2 Mycorrhizae trapping experiment ..................................................... 29

3.3.3 Field experimental design and layout procedures ............................ 32

3.4 Cultural practices on experimental plots ..................................................... 33

3.5 Data collection ............................................................................................. 34

3.6 Data management and analysis ................................................................... 34

4. RESULTS AND DISCUSSION ........................................................................ 36

4.1 Vegetative propagation of K. anthoteca ...................................................... 36

4.2 Arbuscular mycorrhizae fungi (AMF) root colonization ............................ 38

4.3 Plant growth................................................................................................. 40

4.3.1 Growth of K. anthoteca on ex-coal mined site ................................ 40

4.3.2 Diameter increment ........................................................................... 45

4.3.3 Leaf count ......................................................................................... 47

4.3.4 Survival of K. anthoteca on the field ................................................ 50

5. CONCLUSIONS AND RECOMMENDATION .............................................. 52

5.1 Conclusions ................................................................................................. 52

5.2 Recommendations and future perspectives ................................................. 52

REFERENCES ...................................................................................................... 53

APPENDICES ....................................................................................................... 61

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LIST OF TABLES

Table Page

Table 2.1 Major coal producers ............................................................................... 5

Table 3.1 Factorial CRD treatment combinations ................................................. 32

Table 4.1 One-way analysis of variance for the cutting experiment .................... 36

Table 4.2 Grouping of means using Fisher method at confidence of 95% ............ 37

Table 4.3 Average number of single spores per 50 g of soil sample ..................... 40 Table 4.4 Analysis of composts ............................................................................ 42

Table 4.5 Two-way analysis of variance for height increment (cm) .................... 42

Table 4.6 Analysis of soil sample from the research site ...................................... 43

Table 4.7 Two-way analysis of variance for diameter increment (mm) ............... 45

Table 4.8 Two-way analysis of variance for leaf increment .................................. 48

Table 4.9 Grouping information for compost treatment means for measured plant

parameters using Tukeys HSD method at a confidence of 95% ......... 49

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LIST OF FIGURES

Figures Page

Figure 2.1 Land under rehabilitation in Indonesia .................................................. 8

Figure 2.2 Parts of K. anthoteca ............................................................................ 10

Figure 2.3 Stages in the germination of K. anthoteca seed ................................... 11

Figure 2.4 An overview of the functional diversity of arbuscular mycorrhizal

(AM) symbiosis in terrestrial ecosystems .......................................... 18

Figure 2.5 Rhizosphere and mycorrhizosphere interactions with heavy metals in

soils ..................................................................................................... 20

Figure 3.1 Field research site, PT. Bukit Asam-South Sumatra ........................... 27

Figure 3.2 Setup for cutting experiment ............................................................... 28

Figure 3.3 Set-up for AMF trapping experiment ................................................... 29

Figure 3.4 Clearing and staining of root segments ............................................... 30

Figure 3.5 A Schematic set up diagram for the trapping and isolation of AMF .... 31

Figure 3.6 Random allocations of treatments on experimental plots ..................... 33

Figure 3.7 Summary of experimental processes ................................................... 35

Figure 4.1 K. anthoteca cuttings under the various treatments .............................. 37

Figure 4.2 Average percentage of root segment colonization ............................... 38

Figure 4.3 Colonization of root segments of K. anthoteca and isolated single spore

of AMF ................................................................................................ 39

Figure 4.4 Correlation of percentage root colonization and number of spores...... 39

Figure 4.5 Growth of K. anthoteca on the field .................................................... 41

Figure 4.6 Effect of compost treatment on height increment ................................ 44

Figure 4.7 Effect of compost treatment on diameter increment ............................ 47

Figure 4.8 Effect of compost treatment on leaf counts .......................................... 49

Figure 4.9 Effect of mycorrhizae treatment on percentage survival of K.

anthoteca with one standard error bar from the mean ........................ 50

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LIST OF APPENDICES

Appendix Page

Appendix 1 Test result of soil sample from the research site ............................... 61

Appendix 2 Test result of S. natans compost ....................................................... 62

Appendix 3 Cultivation and preparation of S. natans compost at Bukit Asam ..... 62

Appendix 4 Philip Worlanyo .D at S. natans (Kiambang) stock pile site.............. 63

Appendix 5 Average root colonization of species by AMF................................... 63

Appendix 6 Laboratory analysis of AMF root colonization ................................. 63

Appendix 7 Soil sampling and set up for AMF trapping experiment .................... 64

Appendix 8 Site preparation and planting of K. anthoteca propagules with AMF

on the field ........................................................................................ 64

Appendix 9 Independent sample t-Test for survival of K. anthoteca .................... 65

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1 INTRODUCTION

1.1 Background

Coal mining provides a means for creating wealth. The mining sector is therefore, an important sector to Indonesia, as it is a significant provider of export earnings, economic activity and employment whilst supporting regional development. For example, the mining sector of Indonesia contributes to the nations economy currently, for about 11.54% of the total GDP. The outburst of the mining industry has contributed to the increase of over a hundred working mines in the country in the past ten years (Pamerindo Indonesia 2015). However, coal mining is one of the most severe disturbances in terrestrial ecosystems. It causes large-scale deforestation and land degradation with complete loss of topsoil. Thus, the removal of the natural vegetation and upper soil horizons for mining exploration hinders the establishment and survival of plant and soil microbial communities (Cunha et al. 2003). Mining also results in the formation of artificial habitats that are microbiologically poor, requiring human intervention for their proper restoration (Singh et al. 2000). Rehabilitation of coal-mined lands is required to enable the re-use of such lands for other purposes. The establishment of tree species capable of protecting the underlying soil and its micro-fauna and flora is one way of achieving this aim.

Khaya anthoteca commonly referred to as the African red mahogany belong to the family Meliaceae. This species is heavily exploited, particularly in the East and West of Africa. In places where parent trees are scarce, regeneration is poor and serious genetic erosion is believed to have occurred due to selective felling, habitat loss and degradation. The species is used in high-class cabinetwork and for production of veneers and any application where good quality, medium weight hardwood is needed (Hawthorne 1998). It also weathers well and is resistant to borers and termites. The dense crown makes it suitable as a shade tree and also popular for windbreaks and as an ornamental plant. It has been successfully introduced in South Africa, Cuba and Puerto Rico and on a limited scale in Indonesia and the Peninsular of Malaysia where it has been used in plantations and taungya systems. Although seeds are the most common way of natural plant regeneration, vegetative propagation methods/techniques however, offer several advantages. Furthermore, individuals may be recognized within populations that produce a higher quality of the desired product(s) or services. Reports also indicate that vegetative propagation using leafy stem cuttings has been successful in African mahoganies. Establishment of K. anthoteca on degraded lands such as an ex-coal mined site may aid in protecting the underlying soil as well as its micro-fauna and flora.

According to Prasetyo et al. (2010), the disturbance of soil changes the abundance and diversity of the mycorrhizal fungal population; even diminish the population of some soil microbe. On the other hand, the occurrence of soil microorganism is one of the success keys for restoration projects. It has been widely known that mycorrhizal fungi are capable of improving soil properties, and increasing plant access to relatively immobile mineral nutrients (Gaur and Adholeya 2004). Other studies have also suggested that, mycorrhizal fungi are the

18

main pathway through which most plants obtain mineral nutrients and as such, are critical in terrestrial ecosystem functioning (Smith and Read 1996). Mycorrhizal fungi have over the years played critical roles in nutrient cycling and ecosystem function. In this mutualistic symbiosis, plants exchange photosynthates, not only for mineral nutrients, but also for increased resistance to disease, drought and extreme temperatures. Thus, plants are able to withstand harsh environmental conditions through fungi-plant symbiosis. Mycorrhizal fungi are removed entirely in newly graded lands and always requires inoculation if the objective is a functional terrestrial ecosystem. Eroded land is also in nearly the same condition. The arbuscular mycorrhizal fungi (AMF) can therefore, be integrated in soil management (Hooker and Black 1995). These are structures resulting from the symbiosis between these fungi and plant roots, occurring in most soils and colonize roots of many plant species and directly involved in plant mineral nutrition. The symbiotic root-fungal association increases the uptake of less mobile nutrients (Ortas 2006), essentially phosphorus (P) but also of micronutrients like zinc (Zn) and copper (Cu), the symbiosis has also been reported as influencing water uptake. AMF can also benefit plants by stimulating the production of growth regulating substances, increasing photosynthesis, improving osmotic adjustment under drought and salinity stresses as well as increasing resistance to pests and soil borne diseases. These benefits have been mainly attributed to improved phosphorous nutrition (Al-Karaki 2006).

Furthermore, the addition of composted residue is an effective treatment for increasing rhizosphere aggregate stability. Caravaca et al. (2002) reported that, mycorrhiza is increasingly important for improving the growth of seedlings following the addition of composted residue to soil under severe climatic conditions. The study concluded that high proportion of stable aggregates of soil is mainly attributable to a higher microbial activity of root biomass and particularly to the presence of AMF in the rhizosphere aggregates. At the same time, reforestation techniques based on the addition of composted residue and mycorrhizal inoculation in the nursery could be used as a tool for improving soil structure, and subsequently improve plant growth. In this study, vegetatively propagated K. anthoteca seedlings pre-inoculated with AMF and compost were investigated on the field (ex-coal mined site).

1.2 Specific Objectives

It has been reported that the planting of seedlings pre-inoculated with AMF and grown in areas impacted by mining activities favors the development of plants. Again, plants with mycorrhizal roots may survive and grow better than non-inoculated plants after they are planted out on a project site. Studies also show that establishing a partnership with mycorrhizal fungi while the plants are in the nursery results in improved field growth (Baker et al. 2009). The objectives of this study therefore are;

1. To determine the potential application of stem cutting to propagate K. anthoteca

seedlings.

2. To determine the status of arbuscular mycorrhiza fungi (AMF) symbiosis of

vegetatively propagated K. anthoteca.

19

3. To evaluate on the field, the effect of compost and pre-inoculation on vegetatively

propagated K. anthoteca propagule for re-vegetation of ex-coal mined site in

South Sumatra of Indonesia.

1.3 Research Hypotheses

This study employs three (3) hypotheses; 1. K. anthoteca could be propagated vegetatively using stem cutting with the help of

growth hormone and wounding. 2. Vegetatively propagated propagules of K. anthoteca form symbiotic relationship

with the arbuscular mycorrhiza fungi. 3. K. anthoteca could survive and grow on ex-coal mined sites with the application

of mycorrhizae and compost.

1.4 Problem Statement

According to Singh et al. (2005), coal mining is the second large source of heavy metal contamination in soil after sewage sludge. Mining activities are well known for their deleterious effects on the environment, due to the deposition of large volumes of wastes on the soil. The formation of acid drainage generated at mine sites when metal sulfide minerals are oxidized under sufficient presence of water to mobilize the sulfur ion are common constituents in the host soil associated with metal mining activity. Acid generation can occur rapidly, or it may take years or decades to appear and reach its full potential. For that reason, even a long-abandoned site can intensify in regard to its environmental impacts. Dissolved metals in acid drainage may include lead, copper, silver, manganese, cadmium, iron, and zinc, among other metals. Elevated concentrations of these metals in the soil can preclude their use for plant growth, establishment and microorganism habitat.

Various amendment materials such as biosolids, digestates, lime, wood ash, cement kiln dust, red mud are among some of the soil amendments for land rehabilitation. However, the volume of soil amendments needed, their availability, transportation, and onsite storage issues are among the most important factors in determining per-acre costs of using soil amendments to remediate and revitalize a site. These costs can vary widely. A project in which amendments suitable for revitalization are already available on site may cost up to $1,000 per acre treated; a project requiring organic material alone to be delivered may cost up to about $10,000 per acre treated (Environmental Protection Agency of USA 2007). The costs involved in rehabilitating ex-coal lands are therefore huge. Simple but efficient technologies need to be tested through experimentation to supplement and reduce these costs. Composts are cheap and readily source of soil amendment coupled with mycorrhizae inoculation and considering the need for recovering an area disturbed by coal mining activities, inoculated vegetatively propagated K. anthoteca plants growing in soils with lower soil microorganisms and fertility were investigated.

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1.5 Justification of Study/Research Contribution

Considering the need to recover areas disturbed by coal mining activities, the effect of AMF inoculation on K. anthoteca plants growing in soils with increasing proportions of coal mining waste was investigated in this study. K. anthoteca is one of the most important timber woods in Africa. It is used in high-class cabinetwork and for production of veneers and any application where good quality, medium weight hardwood is needed. It has been successfully introduced in South Africa, Cuba and Puerto Rico and on a limited scale in Indonesia and Peninsular Malaysia where it has been used in Taungya systems. However, the species is heavily exploited, particularly in East and West Africa. K. anthoteca occurs in semi-deciduous forest, in both wetter and drier types, and in the transitional zone between dry semi-deciduous forest and savanna. Thus, it occurs in lowland rain-forests and riverine fringe forest at low to medium altitudes, up to 1500 m above sea level. In moist semi-deciduous forest it may occur together with K. ivorensis. In East and Southern Africa it is found in rainforest and riparian forest up to 1500 m altitude. Within the area of natural distribution it is widely grown in plantations and used in enrichment planting. Various countries have now imposed felling limits and bans on the export of its logs. K. anthoteca has been listed as a vulnerable species in 2002 IUCN species red list (Hawthorne 1998). The rehabilitation of ex-mined site through the establishment of mycorrhizal K. anthoteca will therefore help improve both soil conditions and the volume of the tree species for economic gains.

The use of mycorrhizal inoculums tolerant to heavy metals has been indicated for growth of tree seedlings planted in contaminated tropical areas allowing phyto-remediation of heavy metal polluted soils. Usually after mining the productivity of the land reduces drastically requiring restoration. Various amendment techniques are available, however the volume of soil amendments needed, their availability, transportation, and onsite storage issues are among the most important factors in determining the costs of using soil amendments to remediate and revitalize sites.

Again, the addition of composted residue is an effective treatment for increasing rhizosphere aggregate stability. Caravaca et al. (2002) have reported also that, mycorrhiza is increasingly important for improving the growth of seedlings following the addition of composted residue to soil under severe climatic conditions. Their study concluded that high proportion of stable aggregates of soil is mainly attributable to a higher microbial activity of root biomass and particularly to the presence of AMF in the rhizosphere aggregates. At the same time, reforestation techniques based on the addition of composted residue and mycorrhizal inoculation in the nursery could be used as a tool for improving soil structure, and subsequently improve plant growth under field conditions.

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2 LITERATURE REVIEW

2.1 Coal Mining

Mining is regarded as humankinds second earliest endeavors, granted that agriculture was the first. The two industries ranked together as the basic industries of early civilization. Little has changed in the importance of these industries since the beginning of civilization. If fishing and lumbering are considered as part of agriculture and oil and gas production as part of mining, then agriculture and mining continue to supply all the basic resources used by modern civilization. Resources extracted through this process can be marketed on the open market, enabling the countries that possess them to obtain valuable income. Coal is a family name for a variety of solid organic fuels and refers to a whole range of combustible sedimentary rock materials spanning a continuous quality scale. This has broadly been divided into two main categories, which are themselves divided into two subcategories; Hard coal and Brown coal (International Energy Agency 2012)

2.1.1 Coal Mining In Indonesia

Indonesias thermal coal mining industry is growing at a momentous pace. The countrys efforts to quench the demand of its rapidly growing continental peers have caused it to emerge as the leading exporter of the fossil fuel worldwide. Coal in Indonesia is mainly mined in the regions of East Kalimantan, Central Kalimantan and South Sumatra. Fueled by demand from both China and India, total output reached an estimated 390 million mt/year by the end of 2012, an 8% increase from 2011 production (Global business report 2012).

Table 2.1 Major Coal Producersa [Mt]

Country 2009 2010 2011b PR of China 2 895.3 3 140.2 3 471.1 United States 987.6 996.1 1 004.1 India 566.1 570.4 585.9 Australia 411.6 424.1 414.3 Indonesia 291.2 325.0 376.2 Russian Federation 276.0 321.7 333.8 South Africa 249.5 254.5 253.1 Germany 183.6 182.3 188.6 Poland 135.2 133.2 139.2 Kazakhstan 100.9 110.9 116.7 Colombia 72.8 74.4 83.8 Turkey 79.5 73.4 78.1 Canada 62.9 67.9 67.1 Greece 64.9 56.5 58.8 Czech Republic 56.4 55.2 54.4 Other 346.4 359.9 391.4 World 6 835.6 7 201.1 7 678.4 aProduction includes recovered slurries. bEstimated amount Source: International Energy Agency, 2012.

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Indonesia increased its exports of steam coal by 13 percent per year over the localization period from 58.30 mt in 2000 to 176.4 mt in 2009. Coal exports over the period 20002009 went mostly to other Asian countries with limited quantities being exported to Europe and the United States. Japan was Indonesias largest export customer until 2009 when China imported 34.3 mt of Indonesian coal, replacing Japan by a wide margin as the largest importer of Indonesian steam coal (Energy Publishing, 2010).

Over the past decade, coal producers located on Kalimantan have accounted for more than 90 percent of Indonesias coal production and exports. This industry concentration on Kalimantan is not surprising, given that the island accounts for more than 65 percent of economically recoverable reserves. The concentration of coal production capacity on Kalimantan is due to its proximity to the large power markets of Japan, Korea, Taiwan, and China, which have been the fastest-growing coal markets in Asia for the past 30 years. Again, Kalimantans coal reserves have been associated with higher typical calorific values (CVs) and also, are located closer to either the coast or navigable rivers such as the Barito and Mahakham. Between 2000 and 2009, Indonesias coal industry increased its output by 12 percent per year from 76.86 mt in 2000 to 214.60 mt in 2009 (Lucarelli 2010) 2.1.2 Some Characteristics of Coal Mined Soils

Soil pH is a measure of the level of active soil acidity, and is the most commonly used indicator of coal mined soil quality and has significant effect on the chemical properties as well as nutrient availability of the soil. Coal mined soils are essentially devoid of nitrogen (N) and so, the total amount of N required to sustain plant growth over time must come from initial fertilizations and subsequent symbiotic N-fixation by legumes. However, most coal mined soils contain sufficient calcium (ca), magnesium (Mg), and potassium (K) to supply plant growth over extended periods of time. This has been attributed to the abundance of these elements in readily available forms as they weather. On the other hand, old mined soils that have been leached and weathered for an extended period of time may become deficient in these essential cations. The bulk density of productive natural soils generally ranges from 1.1-1.5 g/cm3. Many coal mined soils are highly compacted (bulk density > 1.6 g/cm3) within several feet of the surface primarily due heavy machinery traffic. These compacted zones results from the repeated traffic of rubber tired loaders and haulers, and bulldozers to a lesser extent. There is also the presence of rock outcrops or extreme stoniness.

Again, some of the properties of ex-coal mined soils are disordered rock fragments, color variegations not associated with horizon formation or redoximorphic processes (often described as lithochromic mottling), splintered or sharp edges on rock fragments, bridging voids, and carbolithic rock fragments (Sencindiver and Ammons 2000)

2.1.3 Impact of Coal Mining

Coal mining is one of the most severe disturbances in terrestrial ecosystems. It causes large-scale deforestation and land degradation with complete loss of top soil (Maiti 2013). Coal mining may also be a possible cause of soils contaminated by heavy metals (Horvat et al. 2003). Many changes occur in the chemical, microbiological and physical properties of soils as result of storage. Soil is

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polluted due to disposal of industrial/mining and domestic solid wastes, wet and dry deposition from the atmosphere, infiltration of contaminated water and acid mined drainage (Aswathanarayana 2003; Singh and Singh 2004). Dumping of solid wastes on land can adversely introduce a wide range of pollutants to the soil.

However, on the other hand, there are compounds that do not occur naturally and may be introduces entirely through anthropogenic activities (Jung and Thornton 1997). In the process of open cast mining, the area is normally stripped completely of vegetation to remove the overburden covering the coal seam (Kundu and Ghose 1998). Some air pollution related impacts from coal mining includes; fugitive dust from blasting, drilling, materials handling (overburden, waste rock, coal, discard), vehicle entrainment, wind erosion, tipping, crushing and screening, sulphur dioxide, nitrogen oxide and carbon monoxide emissions from blasting operations as well as other volatile organic emissions from the spontaneous combustion of discard dumps. The removal of the natural vegetation and of the upper soil horizons for mining exploration hinders the establishment and survival of plant and soil microbial communities (Cunha et al. 2003). Thus, coal mining can results in the formation of artificial habitats that are microbiologically poor, requiring human intervention for their proper restoration (Singh et al. 2000).

Underground and surface coal mining leads to the contamination of ground and surface waters. Open cast coal mine effluents also usually contain high concentrations of suspended solids, total dissolved solids, heavy metals, oil, grease, sulphate, nitrates and a high value of hardness. Among the heavy metals: copper, cadmium, chromium, nickel and zinc, the mean concentration of zinc may range up to 7.10.4 mg/dm3 (Mishra et al. 2008). Heavy metals may have an influence on algae due to a disturbance in their metabolism and biological function, the inhibition of photosynthesis, and a reduction of cytochrome. This type of water pollution accumulates in algae and in such a way enters the food chain and may pose a serious threat to animals and to human health through bio-magnification; increase concentration of pollutant through food chain (Zhou et al. 2008). In rivers and streams subjected to Acid Mine Drainage stress through coal mining, typical physical and chemical parameters are observed: a lower value of pH, an increase of ion concentrations, mainly sulphates andiron concentration as a result of the pyrite oxidation through bacteria, and an elevated concentration of heavy metals including aluminum. Both a low value of pH and toxic concentrations of heavy metals eliminate macrophytes and animals, while the productivity, density and biomass of others are reduced. The loss of species richness of macro-invertebrates in streams affected by pollution from hard coal mines has been observed by many authors (Winterbourn et al. 2000; Cherry et al. 2001; Battaglia et al. 2005; Tripole et al. 2006). The macro-invertebrate taxa that are more sensitive to this type pollution are replaced by ones more tolerant.

In general, mining operations routinely modify the surrounding landscape by exposing previously undisturbed earthen materials. Erosion of exposed soils and extracted fine material in waste rock piles can also result in substantial sediment loading to surface waters and drainage ways. In addition, spills and leaks of hazardous materials and the deposition of contaminated windblown dust can lead to soil contamination. Normally, obstacles to ecosystem revitalization are related

to undesirable pH levels, low fertility and poor soil physical properties but with low contaminant concentrations (US EPA

2.1.4 Coal Mined Land Rehabilitation in Indonesia

Coal mining companies in Indonesia are obliged to perform reclamation effort to achieve sustainability restore after mined land to the initial condition. Theaffects the reclamation success is the loss of soil fertility due to soil erosion. Soil erosion causes the loss of top soil layer which has a very important role in plant growth because this layer contains the highest concentratiand microorganisms. Soil erosion is influenced by climate properties, soil properties, topographic properties, cropping management factor and human intervention in conservation practice factorpositively by both the national and regional governments because of its potential to contribute to the development of remote areas, where mining companies establish basic infrastructure and may be the only source of formal employment. The Indonesian government planational GDP over the coming yearsInitiative 2013)

While the coal economy, the impact of mining on the biophysworrisome. Mining areas are stripped of vegetation and soil, which impacts their ability to provide environmental services like the provision of forest products for local communities, soil stabilization, hydrological cycling,and habitat for biodiversity. Downstream communities, including riverine and marine areas, can also be significantly impacted through landslides, sedimentation, and the discharge of toxic materials

Figure 2.1 Land under rehabilitation

Program Workshop

to undesirable pH levels, low fertility and poor soil physical properties but with taminant concentrations (US EPA 2007).

1.4 Coal Mined Land Rehabilitation in Indonesia

Coal mining companies in Indonesia are obliged to perform reclamation effort to achieve sustainability of post mining land use and as much as possible to restore after mined land to the initial condition. The main factor that normally affects the reclamation success is the loss of soil fertility due to soil erosion. Soil erosion causes the loss of top soil layer which has a very important role in plant growth because this layer contains the highest concentration of organic matters and microorganisms. Soil erosion is influenced by climate properties, soil properties, topographic properties, cropping management factor and human intervention in conservation practice factor (Maryati, 2012).Mining is viewed

ly by both the national and regional governments because of its potential to contribute to the development of remote areas, where mining companies establish basic infrastructure and may be the only source of formal employment. The Indonesian government plans to increase the contribution of mining to the national GDP over the coming years (Environmental Leadership and Training

coal mining industry has multiple benefits for the Indonesian economy, the impact of mining on the biophysical environment is severe and worrisome. Mining areas are stripped of vegetation and soil, which impacts their ability to provide environmental services like the provision of forest products for local communities, soil stabilization, hydrological cycling, carbon sequestration, and habitat for biodiversity. Downstream communities, including riverine and marine areas, can also be significantly impacted through landslides, sedimentation, and the discharge of toxic materials (ELTI 2013).

under rehabilitation in Indonesia (Source: ELTI Asia Training Workshop Report 2013)

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to undesirable pH levels, low fertility and poor soil physical properties but with

Coal mining companies in Indonesia are obliged to perform reclamation post mining land use and as much as possible to

main factor that normally affects the reclamation success is the loss of soil fertility due to soil erosion. Soil erosion causes the loss of top soil layer which has a very important role in plant

on of organic matters and microorganisms. Soil erosion is influenced by climate properties, soil properties, topographic properties, cropping management factor and human

Mining is viewed ly by both the national and regional governments because of its potential

to contribute to the development of remote areas, where mining companies establish basic infrastructure and may be the only source of formal employment.

ns to increase the contribution of mining to the Environmental Leadership and Training

mining industry has multiple benefits for the Indonesian ical environment is severe and

worrisome. Mining areas are stripped of vegetation and soil, which impacts their ability to provide environmental services like the provision of forest products for

carbon sequestration, and habitat for biodiversity. Downstream communities, including riverine and marine areas, can also be significantly impacted through landslides,

ELTI Asia Training

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2.2 Origin of the Khaya species

The genera Khaya and Entandrophragma are the main source of Africa mahogany and are closely related to the South American genus Swietenia, the original source of mahogany wood (Tchimene et al. 2005). The African Khaya is a small genus with five species, three in tropical Africa and two in Madagascar. Species of Khaya includes; K. anthoteca (syn. K. nyasica), K. grandifoliola, K. ivorensis, K. madagascariensis and K. senegalensis. Mahoganies (Meliaceae) are among the most commercially important tropical timber tree species (Chalmers et al. 1994). Khaya species are found in all timber producing areas of West Africa, Central Africa and some parts of East Africa. The Khaya spp. occurs over a wide range of climatic, altitudinal, ecological, and edaphic conditions in Ghana as well. In fact, the geographic range of Khaya spp. extends from the wet evergreen high forest zone in the South to the Guinea savannah zone in the North. In Ghana, two species K. senegalensis and K. ivorensis display mutually exclusive distributions, the former confined to savannah vegetation while the latter to forest (Swaine 1996). The reason for this strikingly eco-geographic variation in complementary distribution is that K. senegalensisis better able to control water loss than Khaya ivorensis. Species distribution can thus be associated with a specific ecological zone, apparently defined by rainfall regime (Swaine 1996). Khaya anthoteca is another species of the Khaya family that occurs in lower rainfall regions of Africa. These regions can be found from Sierra Leone to Southeastern Nigeria, as well as in Uganda and the Democratic Republic of the Congo. Its distribution in Ghana is more or less within the dry semi-deciduous zones (Hawthorne and Gyakari 2006). The species name anthoteca is derived from the Greek words anthos (flower) and theke for the case/container of the flower.

2.2.1 Botanical Description of K. anthoteca

Khaya anthoteca, commonly referred to as the African red mahogany belongs to the family Meliaceae. It is a large or very large tree (up to 65 m tall) with a straight trunk that occurs in rainforest, riparian forests, and in savannah transitional zones. Leaves are alternate, evenly compound with 3-7 pairs of leaflets, 150-300 mm long and dark glossy green, base broadly tapering to round and slightly asymmetric, smooth and glossy, veins distinct on the lower surface, margin smooth. It is an evergreen or deciduous, monoecious, usually straight and cylindrical, up to 120(500) cm in diameter, with large buttresses up to 46 m high, sometimes extending into prominent surface roots; bark surface grey and smooth but exfoliating in small circular scales leaving a pock-marked, mottled grey and yellowish brown surface, inner bark pink to reddish; crown massive, rounded; twigs glabrous. It is a large tree, up to 60 m tall, with a straight bole that reaches a considerable height before branching. The bole is markedly buttressed, on large trees to a height of 6 m. Above the buttresses the trunk has a diameter of up to 4 m. Leaves are up to 40 cm long, compound, with 6-10 leathery leaflets, each up to 17 cm long. Flowers are unisexual, sweet-scented and white, in up to 40 cm long inflorescences. Male and female flowers are on the same tree (monoecious), they closely resemble each other as both have well developed but sterile, organs of the opposite sex.

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a) Trees of 10 years old b) Leaves; c) A fruiting twig; d) A dehiscent fruit; e)

Seeds of K. anthoteca (Hyde et al. 2015)

Figure 2.2 Parts of K. anthoteca a) Base bole of K. anthoteca b) Bark of K.

anthoteca c) K. anthoteca wood in transverse section (Hyde et al.

2015)

2.2.2 Distribution, Habitat and Ecology

Khaya anthoteca occurs in semi-deciduous forest, in both wetter and drier types, and in the transitional zone between dry semi-deciduous forest and savanna, in areas with 12001800 mm annual rainfall and a dry season of 24 months. Thus, it occurs in lowland rain-forests and riverine fringe forest at low to medium altitudes, up to 1500 m above sea level, in areas with 600-1600 mm rain/year. Native to tropical Africa between 20S and 10N, from Sierra Leone eastwards to Uganda and Tanzania and southwards to Angola, Zambia, Malawi, Mozambique and Zimbabwe but absent from the wettest forests of Upper and Lower Guinea forest ecosystems where it is replaced by K. ivoriensis. In moist semi-deciduous forest it may occur together with K. ivorensis. K. anthoteca species are frequently scattered on slopes along watercourses. In East and Southern Africa it is found in rainforest and riparian forest up to 1500 m altitude.

a b c

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Within the area of natural distribution it is widely grown in plantations and used in enrichment planting. In plantations it requires fertile deep soils and plenty of water. It is susceptible to fire. Seeds can germinate in full sun as well as in the shade, but natural regeneration may be very sparse in the forest.

In DR Congo it has been found that seedling survival and height growth are higher in gaps than in the forest understory, with 59% and 37% survival, respectively, and most seedlings in the forest understory being stunted. It has also been observed that secondary forest resulting on abandoned shifting-agricultural land offers favourable conditions for the regeneration of K. anthoteca. The species is heavily exploited, particularly in East and West Africa and in places where parent trees are scarce, regeneration is poor and serious genetic erosion is believed to have occurred. Various countries have now imposed felling limits and bans on the export of logs. The species is heavily exploited, particularly in East and West Africa and listed as vulnerable on the 2002 IUCN Red List of Threatened Species (Msanga 1998). 2.2.3 Storage and Viability of K. anthoteca Seeds

Seeds can be stored in gunny bags at 16 C or at room temperature for one year without significant loss of viability (Tanzania Seed Agency 1995). The seeds are tolerant to desiccation and should be dried down to low moisture content (5-7%) and stored in airtight containers. However, it is recommended to sow the seeds immediately upon receipt. Studies on optimal storage temperature indicate that the seeds may be chilling sensitive and that storage at 15C is better than 5 or -18C, but results are unclear (IPGRI/DFSC 1998). Even if the seeds are properly dried they retain high viability for no more than 6 months and after one year viability will normally have dropped to about half of the initial viability. One kilogram contains 4,000 seeds and may produce 3,600 seedlings under ideal conditions. In many cases only 3,000 seedlings per kg of seed will be obtained. The germination of fresh seeds is up to 90% in 2 - 3 weeks after sowing. The general recommendation is to sow seeds within one year after collection (Tanzania Tree Seed Agency 1995).

Figure 2.3 Stages in the Germination of K. anthoteca seed (Picture adopted from

Tanzania Tree Seed Agency, 1995).

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2.2.4 Planting and Propagation

Khaya anthoteca is propagated by seed. The 1000-seed weight is usually between 180 and 280g. The seeds are often already attacked by insects while they are still on the tree, and undamaged seeds should therefore be selected before sowing or storage. The seeds can be stored for up to 1 year in a cool and dry place. However, ash must be added to reduce insect damage. Fungi can cause serious losses of stored seed, with seeds stored at 18C and 5C showing higher occurrence of fungi and lower germination rates than seeds stored at 15C. The seeds are best sown in seed beds in the nursery and should they should be covered with only a thin layer of soil, or left partially uncovered. Germination takes 835 days. When seedlings are grown in small containers, they can be planted out when they reach 30 cm and have fully developed compound leaves. Seedlings can also be left in the nursery until they are 12 m tall, after which the root system is slightly pruned and leaves stripped off before planting into the field as striplings. In Cte dIvoire K. anthoteca has been planted in degraded or secondary forests at a distance of 725 m between lines and 37 m within the line. Pure plantations have also been established with trees planted at 3 m 3 m. 2.2.5 Growth and Development of K. anthoteca

Young trees of K. anthoteca have a slender stem and a small crown. Extensive lateral growth starts when the upper canopy of the forest has been reached. In Ghana the average height of seedlings was 2.5 m and average stem diameter 44.5 cm after 2.5 years. In Cte dIvoire K. anthoteca trees planted in the open in the semi-deciduous forest zone reached an average height of 12 m and an average bole diameter of 18 cm after 10 years. However, trees planted in the evergreen forest zone were only 6 m tall and 9 cm in diameter after 8 years. In Malawi planted K. anthoteca trees reached a height of 8 m and a diameter of 9 cm after 7 years. Trees may already develop fruits when they have a bole diameter of 18 cm, but abundant fruiting usually starts at diameters above 70 cm. This means that the removal of trees of diameter classes below 70 cm from the forest may result in lack of natural regeneration. Fruits are usually produced in the dry season, from January to March in Cte dIvoire and Guinea. Dispersal of the seeds is by wind. The dispersal distance can be over 50 m, but about 75% of all seeds are dispersed within 30 m of the parent tree.

2.2.6 Uses of K. anthoteca

The wood (trade names: African mahogany) is highly valued for furniture, cabinet work, decorative boxes and cases and veneer, and is also commonly used for window frames, paneling, doors and staircases. The species is suitable for light flooring, ship building, vehicle bodies, sporting goods, musical instruments, toys, novelties, carving, plywood and pulpwood. Traditionally, the wood is used for dugout canoes. It is also used as fuel wood and for charcoal production. The bitter-tasting bark is widely used in traditional medicine. It is taken to treat cough, whereas bark decoctions or infusions are taken to treat fever, colds, pneumonia, abdominal pain, vomiting and gonorrhea, and applied externally to wounds, sores and ulcers. Pulverized bark is taken as aphrodisiac and to treat male impotence. In Tanzania root decoctions are drunk to treat anaemia, dysentery and rectal

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prolapsed. The bark has been used by the Shambaa people for reddish brown dyeing. In DR Congo the leaves are said to be used for making an arrow-poison. K. anthoteca is fairly commonly planted as an ornamental shade tree and roadside tree. It is occasionally planted as a shade tree in agroforestry systems and The dense crown makes it suitable as a shade tree and it is also popular as an ornamental and in windbreaks.

2.3 Environmental Factors Affecting Plant Growth

Growth is the irreversible increase in the size of a plant. Plants have indeterminate growth which means they have the capacity to grow from the apical meristem indefinitely. Growth generally occurs in cycles such as seasonally or daily. Factors affecting the growth of plants are broadly categorized into genetic and environmental factors as well as the interaction between these factors. Genetic factors are quite species specific while environmental factors vary widely depending on the surroundings of the growing plant. Some of these factors that greatly affect plants growth include; light, water, nutrients and temperature.

2.3.1 Water

Of all the factors controlling seedling growth, water is the most critical. Water is the vehicle for all physiological and biochemical processes through which life is maintained. In the plant, opposing effect of transpiration and water absorption controls water. Whenever transpiration is greater than absorption, the plant becomes dehydrated. A decrease in hydration of protoplasm of cells in the meristematic tissues usually results in cessation or checking of cell division or cell enlargement or both. If there are no limiting growth factors, an increase in hydration of the protoplasm of a meristem usually results in an increase in the rate of cell division and the cell enlargement phase of tree growth. However, all phases of tree growth are not equally affected by the attenuation in the volume of water within the seedling (Nwoboshie 1982). 2.3.2 Sunlight

Light is the principal limiting factor for growth in all forests (Swaine et al. 1997). Light affects growth through its effects on photosynthesis. It affects photosynthesis in terms of its quality or wavelength composition, intensity or irradiance and duration. Light is important for many physiological processes such as stomatal action permeability, absorption of electrolytes as well as athocyanin and chlorophyll synthesis (Nwoboshie 1982). Spatial variation in light availability leads to variation in other physical and biotic environmental factors such as temperatures, herbivore abundance and activities of pathogenic fungi and bacteria. Thus, successful seedling establishment in the under story or light gaps hinges upon species-specific responses to these multiple factors confounded with light environment, not merely upon light intensity, spectral quality or sun flecks (Kitajima 1996).

Light regulates the elongation process in stems to develop the necessary strength to support itself above the ground. A stem kept in darkness will not have this control and will elongate rapidly and become very spindly in the dark. Light striking one side of a stem causes less growth hormone on that side and a

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concentration on the darker side. This light filtered through the leaves of other plants, for example in a dense forest, is of lower intensity than that above the tree canopy and this darkness appears to cause elongation of stems which has the effect of raising them nearer the light. However, plants native to a specific area and climate are able to time their activities (flowering, seed formation and dormancy) in relation to the seasons. The seasonal change in day length is a reliable cue and plants have evolved systems for measuring the relative lengths of night and day. 2.3.3 Temperature

Of all the planets, the thermal environment on earth is particularly fit to give rise to and sustain life. This is because life functions in an aqueous medium and the range of temperatures encountered over most of the earths surface generally ensures that sufficient water is maintained in the liquid state. The temperature at which biological processes can occur is generally limited by the freezing point of water on the low side and the irreversible denaturation of proteins on the high side. Plants are chemical machines and one universal characteristic of chemical machines is their sensitivity to temperature. Temperature, along with light and water, is one of the most critical factors in the physical environment of plants. This is especially so because, unlike homeothermic animals, plants are not able to maintain their tissues at a constant temperature (Hopkins and Hner 2008). Temperature affects plant growth through its effects on biochemical processes (Fitter and Hay 1987). Environmental temperature therefore exerts a profound influence on cellular metabolism and, as a result, plant growth and their geographic distribution. Plants in nature are subjected to a complex mosaic of fluctuating air and soil temperature regimes such that it is very difficult to study the effects of temperature in a natural setting. Air temperature, for example, fluctuates widely, and often rapidly, depending on the time of day, cloud cover, season, and other factors. Soil is a major heat sink as it absorbs and stores solar energy during the day. At night, some of this heat is radiated back into the atmosphere, which both cools the soil and warms the surface. Soil temperature also varies with the soil structure, organic content, and other physical characteristics as well as slope and aspect; the direction it faces with respect to the sun (Hopkins and Hner 2008) 2.3.4 Plant Nutrient and Fertilization

The growth of plants depends on the availability of nutrients from the soil. Thus, it is important that the soil should potentially provide nutrients for the growth and development of plants. Prolonged uptake of nutrients by growing plants depletes soil of vital nutrients, adversely affecting the growth of plants (Russell 1998).

Fertilizer/composts are substances which are incorporated into the soil to increase plant growth and yield by providing one or more of the elements essential for growth and development. Fertilizers are applied to promote healthy growth, assist plants to overcome adverse effects of diseases or insects or to correct mineral deficiencies, increase growth rate and maintain satisfactory vigor (Evans 1992). Fertilizer can be applied anytime during the growing season if a seedlings leaves turn yellowish, experience extreme slow growth or some other

31

signs. Where fertilizers are to be applied under hot, dry conditions, it is important to water the seedlings soon after the fertilizer application so that the salt from the fertilizer does not damage the seedling root system (Swanson 2000). Plants need a number of essential elements to enable them to grow and reproduce. These elemental nutrients may be classified as micronutrients and macronutrients. The macronutrients (primary nutrients) include Nitrogen (N), Phosphorus (P) and Potassium (K). Plants need these elements in relatively large quantities for their metabolism processes. The most important of the macronutrients is Nitrogen (N), which is the most limiting nutrient in the soil. It is generally known that nitrogen determines the yield of most crops more than any other nutrient element provided there is adequate rainfall or water supply. The micronutrients such as Copper (Cu), Zinc (Zn), Manganese (Mn), Boron (B), Molybdenum (Mo), Iron (Fe), Chlorine (Cl), Calcium (Ca), Magnesium (Mg) and Sulphur (S) are needed in relatively small amounts and are generally found in sufficient quantities in normal pH balanced soils. However, a deficiency in any of these nutrients can affect the health of seedlings. The macronutrients have a role in building the structure of plants, whereas micronutrients are important in enzyme systems and contribute to the plants function rather than its structure (Halley 1982).

Although soil may vary considerably in structure and in physical, chemical and biotic properties, the rate of growth of a seedling is influenced by those properties of the soil. From the soil, the plant derives its nutrients and it is a storehouse for water and oxygen, all of which are necessary for the physiological processes associated with growth. Hence the relative abundance of these factors in a particular soil, determine the rate at which the seedling will grow (Brady 1990).

2.4 Vegetative Propagation

Asexual propagation is the best way to maintain some species, particularly an individual that best represents that species (Relf and Ball 2009). Vegetative propagation is the reproduction of plant material so that the progeny will contain the exact characteristics of the parent material with regard to genotype and health status (Macdonald 1993). A major importance of asexual propagation is primarily due to its ability to produce uniform planting stock and also to capture major genetic gains in a single step (Hartmann et al. 1997). Establishment of plantations of most West African timber species has been difficult and largely unsuccessful due to pest and disease attacks as well as seed availability and viability. Important hardwoods like Milicia excelsa and the African mahogany species are known to have severe insect pest attacks by the Phytolyma lata, mahogany shoot borer and Hypsipyla robusta respectively. Diseases such as the die-back in Terminalia ivorensis and Ceiba pentandra also hinder the establishment of these species in plantations. Some seeds suffer serious predation by small animals, while others have short viability periods (Grogan and Galvao 2006). Reduced economic value of the timbers due to pest and disease attacks, coupled with the slow returns realized from capital invested in plantation establishment, makes indigenous tree cultivation very unattractive to growing farmers. There has, however, been growing interest in producing some of these species commercially, especially by means of vegetative propagation (Leakey et al. 1982b). Vegetative reproduction of superior individuals of such species will enhance rapid method for

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their multiplication. In forestry, vegetative propagation can be used to reproduce fast growing trees that can produce high quality timber (Opuni-Frimpong et al. 2008). A vegetative reproduction approach to plantation improvement, through the use of stem cuttings of elite trees, plays crucial role in enhancing uniformity, productivity and quality in forest plantations (Hartmann et al. 1997). Several methods have been applied in the vegetative propagation of these plants including grafting (budding), layering, cuttings and tissue culture (Macdonald 1993). According to Sukendro et al. (2010), vegetative propagation by grafting is an alternative method for propagating merbau (Instia bijuga Colebr.). In their study, one of the advantages of grafting is it application in seed productions that are planted in seed orchard and also useful for saving the genetic pool of merbau. According to the result of the research, it was revealed that the average survival percentage of merbau (I. bijuga) by grafting is about 21.67%.

The use of stem cuttings, according to Leakey (1990), has become a common propagation method in both forestry and agroforestry. Root initiation in stem cuttings requires that there is an appropriate environment that would reduce post-severance and physiological stress in the cutting (Hartmann et al. 1997). Again, the potting soil, or medium in which the plant grows must be of good quality. It should be porous for root aeration and drainage, but also capable of water and nutrient retention in order for the plants to form new root system, as well as able to supply moisture at the cut surface since oxygen, of course, is required for all living cells (Relf and Ball 2009). Propagation systems used in cultivation of stem cuttings are based on spraying mist, fogging or enclosing cuttings in polythene. 2.4.1 Stem Cutting in Vegetative Propagation

Many types of plants, both woody and herbaceous, are frequently propagated by cuttings. A cutting is a vegetative plant part which is severed from the parent plant in order to regenerate itself, thereby forming a whole new plant (Relf and Ball 2009). Propagation by stem cutting makes use of a portion of stem, root or leaf from a parent plant and then inducing the portion to develop roots and shoots by chemical, mechanical, and/or environmental manipulation (Hartmann et al. 1997). Stem cuttings can be categorized mainly into leafy softwood cuttings from young shoots and leafless hardwood cuttings from older shoots (Hartmann et al. 1997; Leakey 2004). Leafy stem cuttings have the ability to photosynthesize in the propagation bed once the favourable factors are met (Leakey 2004). Propagation by cuttings requires only that a new adventitious root system or both an adventitious root and shoot systems develop (Hartmann et al. 1997).

Regeneration through leafy stem cuttings has also been experimented. Natural regeneration of the mahoganies has been found to be far less than the rate of exploitation hence strong efforts are being made to develop other methods to regenerate mahogany seedlings to augment the low natural replacement and to ensure sustainability. A study conducted by Owusu et al. (2014) also provides useful information for vegetative propagation of leafy stem cutting of some other African mahogany species, which could contribute to regeneration and conservation of these important timber species in the tropics. According to Istomo et al. (2012), cutting is a plant propagation system which is relatively easy and produces seeds with good quality with similar characteristics to its parent plant

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and takes less time. A research on the effect of hormone IBA 100 ppm, NAA 100 ppm and combination of IBA 50 ppm and NAA 50 ppm on the growth of Combretocarpus rotundatus shoots cuttings has shown that the addition of plant growth regulators (IBA, NAA, and IBA + NAA) has no significant effect on the growth of C. rotundatus shoot cuttings (Istomo et al. 2012).

2.5 The Arbuscular Mycorrhizal Fungi (AMF)

Arbuscular mycorrhizae, originally referred to as vesiculararbuscular mycorrhizae, a name still used by some authors (Smith and Read 1990; 1997), are mutualistic symbiotic associations between the roots of most vascular plants and a small group of fungi in the new phylum Glomeromycota (Schler et al. 2001). Although some structural variation exists in this category, most arbuscular mycorrhizae are characterized by the presence of intraradical hyphae, arbuscules (finely branched hyphae involved in nutrient exchange), extraradical mycelium (hyphae that connect the root to the soil), and spores formed in the extraradical mycelium. Arbuscular mycorrhizae are normal part of the root system in most natural and agroecosystems, including polluted soils (Schler et al. 2001). They are considered as obligate symbiotic biotrophs, in that they cannot grow without a host plant supplying them with carbohydrates; glucose and sucrose (Martin et al. 2007).

In this symbiotic association, the fungus colonizes the plants root hairs through the cortex cells and acts as an extension of the root system. This type of association is characterized by the formation of arbuscles (finely branched hyphal structures) in the region of the root cortex that may function as nutrient organs or nutrient exchange sites between the symbionts as well as fungal multiplication. According to Douds and Millner (1999), the arbuscular mycorrhizal fungi (AMF) genera Gigasporaand Scutellospora produce only arbuscules with extensive intraradical and extraradical hyphal networks whereas Glomus, Entrophospora, Acaulospora, and Sclerocystis also produce vesicles. The formation of mycorrhizae induces great changes in the physiology of the roots, in the internal morphology of the plant, and in the mycorrhizosphere, thus, the soil surrounding the roots (Martin et al. 2007). The symbiotic association of AMF and plant roots has been considered to be the oldest symbiosis of plants and is suspected to ecologically be the most important symbiotic relationship between microorganisms and higher plants (Paszkowski 2006). Arbuscular mycorrhizal associations are reported to occur in about 80% of terrestrial plants including trees, shrubs, forbs and grasses. Many plants are able to establish symbiotic relationships with AMF (Helgason and Fitter 2009) and are therefore referred as mycorrhizal crops. 2.5.1 General Functions of Arbuscular Mycorrhizae (AM)

In this association the fungus takes over the role of the plants root hair and acts as an extension of the root system (Muchovej 2004). The beneficial effects of AM fungi result from one or several mechanisms. With mycorrhizal colonization in the roots, there is increased absorption surface area, greater soil area exposed greater longevity of absorbing roots, better utilization of low-availability nutrients and better retention of soluble nutrients, thus reducing reaction with soil colloids

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or leaching losses (Muchovej 2004; Selvaraj and Chellappan 2006). AM increase establishment, nodulation and atmospheric nitrogen fixation capacity in legumes (Turk et al. 2008).Mycorrhizae influence the colonization of roots by other microorganisms, and reduce the susceptibility of roots to soil-borne pathogens such as nematodes or phyto-pathogenic fungi (Selvaraj and Chellappan 2006). According to Muchovej (2004), AM also modify soil-plant-water relations, thus promoting better adaptation of plants to adverse conditions, such as drought, salinity or heat stress. At elevated heavy metal concentrations in soils, mycorrhizal fungi have been shown to detoxify the environment for plant growth. The real significance of mycorrhizal fungi is that they connect the primary producers of ecosystems, plants, to the heterogeneously distributed nutrients required for their growth, enabling the flow of energy-rich compounds required for nutrient mobilization whilst simultaneously providing conduits for the translocation of mobilized products back to their hosts. Therefore, proper understanding of the ecology and functioning of the AM symbiosis in the natural or agricultural ecosystem is essential for the improvement of plant growth and productivity.

Obligately depending on plant photosynthates as energy sources, the extensive mycelial systems (the vegetative parts of the fungus) effectively explore soil substrates and acquire soil inorganic nutrients including the major macro-nutrients N, P and K and some micro-nutrients, Cu, Fe and Zn, with some capacity for acquiring organic nitrogen and phosphorus. These soil-derived nutrients are not only essential for AM development but are also partly transferred to the host plant (Leake et al. 2004).

Figure 2.4 An overview of the functional diversity of arbuscular mycorrhizal

(AM) symbiosis in terrestrial ecosystems (Adopted from Garg and Chandel 2010)

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2.5.2 Arbuscular Mycorrhizae and Plant Nutrient Uptake

It has been well established that arbuscular mycorrhizae (AM) plants have two major pathways by which nutrients (particularly Phosphorus) can be absorbed namely; the direct uptake pathway the through epidermis and root hairs, and an AM pathway in which P absorbed by external fungal mycelium is translocated to structures inside the root across the symbiotic interface and finally to the plant cortical cells (Salley et al. 2010). Recent works, both molecular and physiological, has provided new insights into the integration of these two uptake pathways and how they influence plant nutrient acquisition (Bucher 2006; Javot et al. 2007; Smith and Read 2008).A large number of field and glasshouse investigations have proven that the outcome of the establishment of AM symbioses in low-P soil has a marked increase in plant growth and P uptake, compared with Non Mycorrhiza control plants of the same species, which of course do not usually exist in nature. Traditionally, this has been attributed to the fact that direct uptake of P (and probably other nutrients such as Zn and N) is supplemented by uptake through the AM pathway and the relief of P stress in the plant is considered to be the basis for increased growth, thus, the two pathways act additively. This simple view is now however being questioned (Smith et al. 2009).

In recent times, there have a number of cases which indicate that AM colonization does not result in any increases in growth or in total plant P, and sometimes the AM plants are smaller than the non mycorrhizal controls (Johnson et al. 1997; Smith et al. 2009). There is therefore a continuum of responses from strongly positive to negative, indicating considerable functional diversity in AM symbioses (Jakobsen et al. 2002).The AM-responsiveness in terms of plant growth is determined by properties of the plant genome such as development of extensive root systems and long root-hairs that enhance P uptake by the plant when it is non-mycorrhizal, the AM fungal genome, thus, inherent extensiveness of external hyphae and other features and plant-fungus genomic interactions (Smith and Read 2008; Smith et al. 2009). It is however crucial to recognize that for single plant species, responsiveness varies considerably with the identity of the fungal symbiont. This has important consequences for discussions of whether AM fungi which do not promote a positive growth response and can be viewed as parasitic, cheating their plant partners by receiving C, but delivering little or no P (Johnson et al. 1997; Jones and Smith 2004; Smith et al. 2009).

Figure 2.5 Rhizosphere

(Adopted from

Mycorrhizal extraradical hyphae release organic acids

and minerals in soils. Heavy metals are sequestered and extracted by AMF colonized roots. Nutrients and metals can be exchanged between the fungus and the host plant via mycorrhizalmycorrhizosphere, microscopic fungi naturally occur in soil to form a symbiosis with plant roots and produce a highly elaborated mycelium network. These fungal associations could grow into the soil some 5reaching farther and into (Brady and Weil 2008). AMF also have the capability of penetrating extremely small pores in soil and of accessing contaminants contained within. 2.5.3 Arbuscular Mycorrhizae

Many studies have indicated that AM symbioses can significantly alter plant water relations, but the reported effects have not been consistent between different investigations, and mechanisms are not clear. Nonetheless, an extensive review by Aug (2001) covering hundreds of studies, highlights a number of trends in AM compared to non-mycorrhizal (NM) plants growing under water restrictions in pot experiments. These studies include increased drought tolerance, greater depletion of soil water, higher stomatadiffusion-limited nutrients in dry soil and lower drought stress (assessed as reduced concentrations of xylem abscisic acid in AM plants). Such differences suggest that AM plants are under less stress in dr(Duan et al. 1996), but the mechanisms underlying the effects remain elusive and need further investigations.nutrition of AM