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

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derived from the published and unpublished work of others has been duly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

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 twoway 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

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

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.

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

Copyright owned by IPB, 2015

All rights reserved
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prior written permission from Bogor Agricultural University (IPB)

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

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

10

Thesis Title

Name
:
Student Number :
Major
:

Establishment of Vegetatively Propagated Khaya anthoteca

Pre-Inoculated with Arbuscular Mycorrhizae Fungi (AMF)
On an Ex-Coal Mined Site.
Philip Worlanyo Dugbley
E451138231
Tropical Silviculture (SVK)

Approved by,

Supervisory Committee:

Dr. Ir. Irdika Mansur, M.For.Sc

Head-supervisor

Dr. Ir. Basuki Wasis, MS

Co-supervisor

Endorsed by,

Head of Tropical Silviculture

Study Program

Dean of Graduate School

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

11

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

17

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

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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.

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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.

20

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 highclass 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.

21

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
PR of China
2 895.3
3 140.2
United States
987.6
996.1
India
566.1
570.4
Australia
411.6
424.1
Indonesia
291.2
325.0
Russian Federation
276.0
321.7
South Africa
249.5
254.5
Germany
183.6
182.3
Poland
135.2
133.2
Kazakhstan
100.9
110.9
Colombia
72.8
74.4
Turkey
79.5
73.4
Canada
62.9
67.9
Greece
64.9
56.5
Czech Republic
56.4
55.2
Other
346.4
359.9
World
6 835.6
7 201.1
a
b
Production includes recovered slurries. Estimated amount
Source: International Energy Agency, 2012.

2011b
3 471.1
1 004.1
585.9
414.3
376.2
333.8
253.1
188.6
139.2
116.7
83.8
78.1
67.1
58.8
54.4
391.4
7 678.4

22

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

23

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 biomagnification; 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

24

to undesirable pH levels, low fertility and poor soil physical properties but with
low contaminant
taminant concentrations (US EPA 2007).
2.1.4
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
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
Mining is viewed
positively
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
plans to increase the contribution of mining to the
national GDP over the coming years (Environmental
Environmental Leadership and Training
Initiative 2013)
While the coal mining industry has multiple benefits for the Indonesian
economy, the impact of mining on the biophysical
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).

Figure 2.1 Land under rehabilitation in Indonesia (Source: ELTI Asia Training
Program Workshop Report 2013)

25

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.

26

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.

27

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 (57%) 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).

28

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

29

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

30

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

32

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

33

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

34

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 macronutrients 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)

35

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).

36

Figure 2.5 Rhizosphere and mycorrhizosphere interactions

ons with heavy metals in soils
(Adopted from Giasson et al. 2008)

Mycorrhizal extraradical hyphae release organic acids that weather rocks

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 mycorrhizal arbuscules inside the root cell (Fig 2.5). In the
mycorrhizosphere,
rhizosphere, 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 515
5 15 cm from the infected root,
reaching farther and into smaller pores than could the plants own root hairs
(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 and Plant Water Relations
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
overing hundreds of studies, highlights a number of trends in AM
compared to non-mycorrhizal
mycorrhizal (NM) plants growing under water restrictions in pot
experiments. These studies include increased drought tolerance, greater depletion
of soil water, higher stomatal
stomatal conductance and transpiration, better supply of
diffusion-limited
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 dry
dry conditions than NM controls
(Duan et al. 1996), but the mechanisms underlying the effects remain elusive and
need further investigations. Some studies have also indicated that, improved
nutrition of AM compared with NM plants may be the main basis for im
improved
drought tolerance. About 80% of the studies assessed by Aug (2001) found that
AM plants grew better under drought stress than NM plants. In some cases the
drought-stressed
stressed AM plants were shown to absorb more P even when the NM
plants had adequate P nutrition. Neumann and George (2004) demonstrated

37

clearly that AM roots were able to absorb P more effectively from dry soil than
NM roots, regardless of plant water status. This effect could be explained by the
increased contribution of the AM uptake pathway as soil dries. Under these
conditions, continuity between water-filled pores decreases, the exposure of the
soil to root path increases (Tinker and Nye 2000), and soil hydraulic conductivity
is reduced. As a result, the rate of diffusion and mass flow of nutrients decreases
with decreasing soil moisture, and nutrients effectively become less available in
NM plants (Viets 1972).
2.5.4 AMF Host Specificity
Recent studies have indicated the importance of arbuscular mycorrhizal
fungi (AMF) in mediating plant coexistence (Hart et al. 2003). Traditionally,
AMF have been considered to be generalists with regard to the hosts that they
infect. They have also been considered to be functionally equivalent in their
effects on a host. These beliefs are based largely on the fact that most AMF can
successfully infect a wide range of plant species when grown experimentally in
monocultures. However, when different plants and fungi are grown together,
AMF growth and species composition is host specific (Bever 1996; Douds and
Millner 1999; Eom 2000; Kires 2000) Consequently, some AMF species are more
beneficial to a host plant than are others, because of genetic and/or physiological
incompatibilities between an AMF and its host (Bever 1999; Klironomos 2000).
In addition, AMF species differ in the services that they provide for host plants
such as nutrient uptake, protection against pathogens and water uptake
(Klironomos 2000). The consequence of AMFhost plant specificity for plant
coexistence is now being explored, both theoretically and experimentally. Bever
(1999) has hypothesized that AMF might have differential feedback effects on
plants. Experimental tests of his model confirm that both negative and positive
feedback occurs between plant and AMF communities, and that this feedback
could potentially contribute to plant coexistence. Although the roles of host
specificity and feedback are important for host plant coexistence, evidence for
both of these mechanisms remains indirect.
Giri et al. (2004) conducted a study on the effect of inoculation of two
arbuscular mycorrhizal fungi, Glomus fasciculatum, and G. macrocarpum, alone
and in combination, on establishment and growth of Acacia auriculiformis in a
wasteland soil studied under nursery and field conditions. According to the study,
Acacia auriculiformis exhibited a maximal mycorrhizal dependency of 79.6% on
dual inoculation. Mycorrhizal dependency differed with AM fungal isolates and
age of the plant. Under field conditions, AM colonization of A. auriculiformis
enhanced tree survival rates (85%) after transplantation. Arbuscular mycorrhizacolonized plants showed significant increase in height, biomass production, and
girth as compared to non-mycorrhizal plants. In general, all growth parameters
were higher on dual inoculation of G. fasciculatum and G. macrocarpum as
compared to uninoculated plants under both nursery and field conditions. The
study concluded that, mycorrhizal symbiosis play vital role in helping A.
auriculiformis to establish and thrive in alkaline wasteland soils. Hence, the
nursery inoculation programs with selected AM fungal species or combination of
synergistically interacting species may be helpful to produce vigorous seedlings to
survive under wasteland stress soil.

38

2.5.5 Mycorrhizae-Induced Growth Depressions

The obligatory symbiotic AM fungi are completely dependent on plants for
organic carbon (Grace et al. 2009). Explanations of the outcomes of symbiosis for
plants are often given simply in terms of the balance between net costs (C loss to
the fungus) and net benefits (additional P supply via the fungus). Where net costs
exceed net benefits then plant growth depressions follow. It is conventionally
assumed that the fungus is a parasite that cheats or exploits its host by
obtaining C but providing little or no soil P. This conventional explanation is
tenable when the fungi colonize the roots extensively (Li et al. 2008). However,
recent data and review of previous results have indicated that, large growth
depressions are not necessarily associated with high AM fungal colonization, but
also occur when there is very low internal root colonization and in some cases
also low external mycelium in soil (Smith et al. 2009).It is also possible that
growth depressions in the absence of high fungal biomass are the result of P
deficiency, induced by reduced activity of the direct P uptake pathway and
inadequate contribution of the AM pathway because of the low root colonization
or hyphal development in the soil (Li et al. 2008; Smith et al. 2009).
The regulatory interplay between AM phosphorus uptake and direct uptake
through epidermis and root hairs has yet to be fully revealed. The evolutionary
persistence of AM symbioses in non-responsive plants suggest that, benefits of
AM symbioses are not directly related to plant growth, such as tolerance to
pathogens or drought and effects on soil structure. These are certainly important
in some circumstances (Miller and Jastrow 2002). Nonetheless, it has recently
been revealed that the AM P uptake pathway operates in virtually all AM plants,
whether responsive or not in terms of P uptake and growth, additional
explanations based on AM P uptake become feasible, including effects on plant
competition. Furthermore, the effects that the external AM fungal mycelium has
on soil structure cannot be ignored, given that the latter plays a critical role in
plant water relations and maintenance of soil fertility.
2.5.6 Application of Mycorrhiza on Forest Tree Seedlings
Deficiency of plant nutrients especially P and prevalence adverse effects from
soils are serious problems in the tropics (Sanchez and Logan, 1992). The
problems can be alleviated through the application of large quantities of Pcontaining fertilizers, but this practice is not cost effective. An alternative of
increasing P supply to plants in these soils is to inoculate them with arbuscular
mycorrhizal fungi (AMF) (Marschner 1995). Some soil microorganisms can
solubilize insoluble forms of P (Azcon and Barea 1996; Di-Simine et al. 1998). A
study conducted by Osorio and Habte (2000) to assess the role that P solubilizing
microorganisms play in the P nutrition of mycorrhizal and mycorrhiza-free
Leucaena leucocephala showed that, soil microorganisms are able to solubilize
rock phosphate isolated from the rhizosphere of L. leucocephala naturally
growing on three different soils of Hawaii. The isolates were screened for their
ability to solubilize rock phosphate in culture medium. The highest activity was
observed with one of the fungal isolates. Phosphorus status of L. leucocephala
pinnules monitored as a function of time revealed that plants colonized by both
microorganisms had the highest P content followed by plants inoculated with the
mycorrhizal fungus alone. Their results suggest that, there is existence of

39

synergistic interaction between P solubilizing microorganisms and mycorrhizal

fungi, although the degree of synergism was more pronounced in terms of P
uptake than in terms of growth.
Giri et al. (2004) also conducted a study on the effect of inoculation of two
arbuscular rmycorrhizal fungi, Glomus fasciculatum, and G. macrocarpum, alone
and in combination, on establishment and growth of Acacia auriculiformis in a
wasteland soil, studied under nursery and field conditions. According to the study,
A. auriculiformis exhibited a maximal mycorrhizal dependency of 79.6% on dual
inoculation. Mycorrhizal dependency differed with AM fungal isolates and age of
the plant. Under field conditions, AM colonization of A. auriculiformis enhanced
tree survival rates (85%) after transplantation. Arbuscular mycorrhiza-colonized
plants showed significant increase in height, biomass production and girth as
compared to non-mycorrhizal plants. In general, all growth parameters were
higher on dual inoculation of G. fasciculatum and G. macrocarpum as compared
to uninoculated plants under both nursery and field conditions. The study
concluded that, mycorrhizal symbiosis play vital role in helping A. auriculiformis
to establish and thrive in alkaline wasteland soils. Hence, the nursery inoculation
programs with selected AM fungal species or combination of synergistically
interacting species may be helpful to produce vigorous seedlings to survive under
wasteland stress soil.
2.6 Compost
Composting is the control decomposition and the natural breakdown process
of organic residues. Raw organic waste materials are biologically transforms into
stable, humic substances that make excellent soil amendments (U.S. EPA 1998).
According to the US EPA (2007), compost is the stable soil conditioning product
that results from aerobically decomposing raw organic materials, such as yard
trimmings, food residuals or animal byproducts. The composting process involves
a proper carbon-to-nitrogen ratio, a favorable temperature regime, water, and air
to yield the compost end-product that is less in volume than the original material
and free from offensive odors. Composting is used frequently to significantly
reduce pathogens in organic waste streams since the process generates
temperature hot enough to achieve this reduction. Composts generally have a
lower N content than biosolids or manures. Compost is easier to handle than
manure and other raw organic materials, stores well and is odor-free.
2.6.1 Compost as Soil Amendment
Compost is an organic matter source with a unique ability to improve the
chemical, physical, and biological characteristics of soils. It improves water
retention in sandy soils and promotes soil structure in clayey soils by increasing
the stability of soil aggregates. Adding compost to soil increases soil fertility and
cation exchange capacity and can as well reduce fertilizer requirements up to
about 50%.Compost can greatly enhance the physical structure of soil. In finetextured (clay, clay loam) soils, the addition of compost will reduce bulk density,
improve friability (workability), porosity, and increase its gas and water
permeability, thus reducing erosion. When used in sufficient quantities, the
addition of compost has both an immediate and long-term positive impact on soil

40

structure. It resists compaction in fine textured soils and increases water holding
capacity and improves soil aggregation in coarse-textured (sandy) soils. The
frequency and intensity of irrigation may be reduced. Compost is a stabilized form
of organic matter, thus, it has become humified and is more slowly degradable.
Compared with uncomposted materials this has the advantage of reducing gaseous
and leaching losses of nutrients off-site and, hence, with less environmental effect
after compost application (Nason et al. 2007). Recent research also suggests that
the addition of compost in sandy soils can facilitate moisture dispersion by
allowing water to more readily move laterally from its point of application (The
United States Composting Council 2008).
Again, compost also improves the cation exchange capacity of soils,
enabling them to retain nutrients longer. It allows crops to more effectively utilize
nutrients, while reducing nutrient loss by leaching. For this reason, the fertility of
soils is often tied to their organic matter content. Improving the cation exchange
capacity of sandy soils by adding compost can greatly improve the retention of
plant nutrients in the root zone. The soil-binding properties of compost are due to
its humus content. Humus is a stable residue resulting from a high degree of
organic matter decomposition. The constituents of the humus act as soil glue,
holding soil particles together, making them more resistant to erosion and
improving the soils ability to hold moisture. Thus, it may provide greater drought
resistance and more efficient water utilization.
Furthermore, the addition of compost to soil may modify the pH of the final
mix. Depending on the pH of the compost and of the native soil, compost addition
may raise or lower the soil/compost blends pH. Therefore, the addition of a
neutral to slightly alkaline compost to an acidic soil will increase soil pH if added
in appropriate quantities. In specific conditions, compost has been found to affect
soil pH even when applied at quantities as low as 10-20 tons per acre. The
incorporation of compost also has the ability to buffer or stabilize soil pH,
whereby it will more effectively resist pH change (The United States Composting
Council 2008).Compost amendments reduce the bioavailability of heavy metals;
an important quality in the remediation of contaminated soils. A study to evaluate
the effect of the rhizosphere of Oleaeuropaea subsp. sylvestris and Rhamnus
lycioides, revealed that the addition of composted residue is an effective treatment
for increasing rhizosphere aggregate stability (Caravaca et al. 2002). Again,
compost also act as an amendment which improves soil morphological and
chemical properties.
2.6.2 Compost and Soil Microbes
Soil becomes microbially active and more suppressive to soil-borne and
foliar pathogens. Enhanced microbial activity also accelerates the breakdown of
pesticides and other synthetic organic compounds. The activity of soil organisms
is essential in productive soils and for healthy plants. Their activity is largely
based on the presence of organic matter. Soil microorganisms include bacteria,
protozoa, actinomycetes, and fungi. Microorganisms play an important role in
organic matter decomposition which, in turn, leads to humus formation and
nutrient availability. Microorganisms can also promote root activity as specific
fungi work symbiotically with plant roots, assisting them in the extraction of
nutrients from soils. Sufficient levels of organic matter also encourage the growth

41

of earthworms, which through tunneling; increase of water infiltration and

aeration (The United States Composting Council 2008). Micro-organisms
improve soil-structure because they help soil to aggregate. Some help to reduce
plant diseases while others establish the mycorrhizal fungi that allow plant roots
to access nutrients far below the reach of their roots. Mycorrhizal fungi make up
80-90% of plant systems, forming a close symbiotic relationship with roots.
There are two main types of mycorrhizal fungi, external ectomycorrhizae,
which form a dense web around tree roots, and internal endomycorrhizae, which
actually penetrate the roots of most other plants. All extend or enhance the plants
ability to reach distant nutrients. In return, plants provide the mycorrhizae with
carbohydrates for energy. Its a classic symbiotic system, benefiting both partners.
One of the most important mycorrhizae for the purposes of this study are the
arbuscular mycorrhizal fungi (AM), which infect the roots of many plants,
forming long, slender, branching systems of threads stretching from plant roots
into the soil below. These systems, which resemble roots, function like a second
set of roots, transporting nutrients from several meters below the plant. AM fungi
can penetrate much smaller spaces than can even the smallest root threads,
accessing nutrients that roots cannot. This is particularly important in allowing
plants to reach phosphorus and other immobile nutrients which are available only
in the extremely small volume of soil immediately surrounding the roots or in the
fungi that extend those roots (Smith and Smith 1990).
2.6.3 Compost and Plant Growth
Compost application significantly, also enhances increase in crop production and
mitigates the negative effect of a delay in sowing. The use of compost, therefore,
is a sound technology for combating soil degradation (Oudraogo et al. 2001).
Compost application leads to humus accumulation in the soil. Consequently, to
correctly assess the effect of the nutrients from compost, it is necessary to
differentiate between a short-term effect in the year of application, and a longterm effect over the following years. Generally, the nutrient effect of organic
fertilizers depends largely on the transformation processes that take place in the
soil after their application. This applies especially to nitrogen (N), for which its
plant availability is tightly related to microbial transformations. Primarily the
degradability and C/N ratio of the organic matter determines, whether it comes to
a net mineralization or immobilization of N after the application of an organic
fertilizer (European Commission, 2001). Other studies have also indicated that,
poultry manure as organic compost is also a valuable resource that enhances the
growth of plants although the effect is quiet significant on the primary growth of
most plants (Dugbley and Agbenyega 2013). Similar observations were made by
(Ghorbani et al. 2008) who studied the effects of organic amendments, synthetic
fertilizers and compost extracts on crop health, productivity and storability. Their
study concluded that, compost made of poultry manure appears to be a promising
ecological alternative to classical fertilizers.
2.6.4 Compost and AMF Interaction for Mined Land Reclamation
Arbuscular mycorrhizal (AM) fungi are obligate symbiotic soil fungi that
colonize the roots of most crop plants. The resulting mutualistic symbiosis (the
"mycorrhiza") is more efficient in the uptake of immobile soil nutrients such as P,

42

Zn, and Cu. The extraradical phase of the fungus functions, in effect, as an
extension of the root, exploring a greater volume of soil for nutrients. The
symbiosis can result in enhanced plant growth and yield, especially under nutrient
poor conditions. Even when no growth enhancement occurs, the majority of P
uptake by the plant can be attributed to the functioning of the mycorrhiza (Smith
et al. 2004; Li et al. 2006; Schnepf and Roose 2006). Application of compost
with AMF inoculation to enhance the growth of plants has been reported in many
studies. According to Douds et al. (2008), arbuscular mycorrhizal (AM) fungi are
potentially important tools due to their roles in tree crop nutrient uptake, disease
resistance, and water relations and in stabilizing soil aggregates. Inoculants of
these fungi can be effectively produced on-farm in mixtures of compost and
vermiculite with a suitable plant host. Success of this method, however, depends
upon utilizing the optimal compost and vermiculite mixture ratio. Experiments
conducted over two years utilizing a complete factorial design with three
composts, four mixture ratios and three AM fungi showed growth in yard
clippings and leaf composts; which were high in N, low in P, with moderate K
levels; produced more spores of AM fungi at mixture ratios.
Again, Caravaca et al. (2002) has also reported that soil aggregate stability
is one of the most important properties controlling plant growth in semiarid
Mediterranean environments. A field study carried out to evaluate the effect of the
rhizosphere of Olea europaea subsp. sylvestris and Rhamnus lycioides, the
addition of a composted residue and mycorrhizal inoculation with Glomus
intraradices on rhizosphere aggregate stability and on the viability of both plant
species in a semiarid structureless soil revealed that, for both plant species, water
soluble carbon (WSC) content and enzyme activities (urease, acid phosphatase
and h-glucosidase) measured in the rhizosphere aggregate were higher than in the
non-rhizosphere soil. Rhizosphere aggregate stability of both plant species was on
average 1.8-fold higher than that of non-rhizosphere aggregate. According to their
report, the addition of composted residue was the most effective treatment for
increasing rhizosphere aggregate stability. The mycorrhizal component was
increasingly important for improving the growth of both seedlings following the
addition of composted residue to soil under the severe climatic conditions of the
area.

43

3 RESEARCH METHODOLOGY
3.1 Time and Area of Study
This study was conducted was in two areas namely; glass house in the
Department of Silviculture, Faculty of Forestry, Bogor Agricultural University
(IPB) and field study at PT. Bukit Asam (Persero): a coal mining site located in
South Sumatera of Indonesia.
Indonesia The area falls within coordinates245
245S 10350E of
Indonesia. This area is characterized by a bimodal rainfall pattern with the major
wet season between May and July and experiences a short dry season in August
and a long dry season between December and March. The annual rainfall ranges
between 2000 mm and 3000 mm. The entire research was conducted from
February to December, 2014.
2014

Figure 3.1 Field research site,

s
PT. Bukit Asam-South Sumatra
(Source: Google map)

3.2 Research Materials and Tools

Materials and tools
t
used for the study included; record log sheets, clip
board, pencil, Rootone F hormone, composts, soil sample under K. anthoteca
plantation forest located
locate at Carita in the Banten province of Indonesia, Pueraria
javanica,, Sorghum bicolor,
bicolor culture pots, plastic rubber, sieves,
eves, zeolit, lab coat,
hand gloves, mounting glasses, crucible and spatula, 10% potassium hydroxide
(KOH), 3% Hydrogen peroxide,
peroxide, 1% Hydrochloric acid (HCl), heating plate, wash
bottle, trypan blue dye,
dye tape measure, electronic caliper, meter rule, analog
weighing scale, non-poisonous
non
maker, Microsoft excel, Minitab Version 16
16,
density gradient centrifuge machine, fine and rough forceps, dissecting and
compound microscopes.
microscopes

44

3.3 Research Designs

3.3.1 Vegetative Propagation of K. anthoteca
Taking stem tip cuttings is perhaps the most common way to vegetatively
propagate trees and shrubs. The process is relatively simple requiring only a
limited area for reproduction, whilst a single mother or stock plant can yield many
cuttings. Many houseplants, annuals, perennials, and woody plants can be
propagated by stem cuttings when they are in active growth stage and the stems
are soft. In this study, stem-tip cutting was used in propagating six (6) months old
K. anthoteca seedlings in the greenhouse obtained from Carita, Banten province
of Indonesia. Materials required the operation included; Shears (cutter), Cello
tape, plastic bag, tray container and media (sandy soil). The media was sterilized
by filling a metal pan (about 10 cm) deep with soil for at least 2 hours 30 minutes,
well-drained but able to retain enough moisture.

Figure 3.2 Setup for cutting experiment a) Materials for stem cutting b) Staked
cuttings in media c) Covered cuttings in greenhouse
Cutting of six-month old K. anthoteca seedlings was carried out at
glasshouse with natural light and day/night temperature between 25 and 32C and
relative humidity of 65-80%. A piece of stem, about 5 inches long was removed
from each seedling with about three sets of leaves on the cutting. The cuttings
were then trimmed in the following way:
a.
A cut at the bottom of the stem just below a node (where th eleaf and/or
the bud joins the stem) was made
b.
About 1/2 to 2/3 of the leaves removed, starting from the bottom of the
cutting. Large leaves were cut into half (Fig. 3.2b).
The lower inch of the cutting was dipped in rooting hormone, Root tone F and
placed in open space for about 3 minutes for hormone absorption. Holes for each
cutting was made using a cleaned rod and cuttings placed in the holes and rooting
mix around it gently firmed. Treatments administered were T1 (No Wounding, no
Hormone; W-H-), T2 (No wounding, with Hormone; W-H+), T3 (Wounding
without hormone; W+H-), and T4 (Hormone with Wounding; W+H+). The
wounding was done by removing the outer skin at the base to about 3 cm of the
cutting exposing the cambium layer. The cuttings were covered with plastic
rubbers and placed in an enclosure ensuring high temperature and humidity for
stem rooting. The rooting mix was checked every five days to make sure it was
moist and watered as and when necessary. Once roots have formed, the humidity

45

was slowly decreased around the plant by untying the plastic rubber gradually
each day.
3.3.2 Mycorrhizae Trapping Experiment
Soil samples (0-20 cm in depth) were collected from the rhizosphere of K.
anthoteca plantation site at Carita, Banten province for trapping and further
analysis of root segment colonization of AMF. Ten (10) trees were randomly
sampled for soil collection. P. javanica, K. anthoteca and S. bicolour were grown
in 600 ml culture trays in a planting media consisting of a 2:1 mixture of zeolit
and sample soil (50g) in the glasshouse. Each soil sample was used in growing all
the species giving a total of 30 sub-samples. P. javanica seeds were cleansed
using parazone for about 10 minutes and further subjected to warm water for 30
minutes and then germinated on a growing medium (zeolit) for a week. The plant
species for the trapping were watered each day over the course of the trapping
period of three months (Fig. 3.3)

Zeolit

Zeolit

Soil

Soil
Zeolit

Zeolit

Spore

Initial trapping time, T0

Final trapping time, Tf

Figure 3.3 Set-up for AMF trapping experiment

The 50 g soil samples from the pot cultures were passed through a 2-mm
sieve then transferred to a beaker. Soil aggregates were then crushed with a
spatula. Distilled or de-ionized water was added to obtain a 1-L suspension and
the suspension agitated manually for about 15 minutes. The purpose of these steps
was to disperse soil aggregates and release AMF spores. The soil suspension was
then passed through a stack of sieves of 425 m, 125 m, and 45 m with the
finest sieve being at the bottom of the stack. A stream of tap water was added to
facilitate the release of spores from the soil particles. The residue that remained in
the 45 and 125m aperture sieves were suspended in water and transferred to
centrifuge tubes with 60% w/v sucrose and centrifuged for 3 minutes at 2500rpm
separating spore sediments at the bottom of the tube, while organic materials
remain in suspension. Spores were isolated and collected under a dissecting
microscope, and number of spores associated with each plant species counted.
Roots were washed and cleared using 10% KOH heated to a temperature of 60oC
and stained using trypan blue in 1% HCl to bind the fungi structures. Roots were
prepared on glass slides and segment colonization was determined by the visual

46

estimation method. Ten (10) roots were prepared on each glass slide representing
a sample with each root observed at 40x magnification and 10 Fields
Fields-of-View
(FoV) according to Brundrett et al. (1996) with a compound microscope (Fig 3.4).

Roots in 10%
KOH at 60oC

Water

Rinsing of roots on
fine sieve after

Stain in trypan blue with

1% HCl

Figure 3.4 Clearing and staining of roots (A modification from Brundrett et al. 1996)

47

Soil samples under

K. anthoteca
plantation

Trapping of AMF using P.

javanica, S. bicolor, and K.
anthoteca

In the glasshouse

Wet sieving
and
centrifugation
with 60% w/v
sucrose

Isolation and counting of

arbuscular mycorrhizae
fungi (AMF) spores

Inoculation of K. anthoteca

Figure 3.5 A Schematic set up diagram for the trapping and isolation of AMF
(Picture adopted from Brundrett et al. 1996)

3.3.3

Field Experimental Design and Layout Procedures

48

The design for this field research was the Factorial experiment in
Completely Randomized Design (CRD). Two factors; mycorrhizae and compost
with four levels of each factor were used giving a total of sixteen (16) treatment
combinations and each treatment combination replicated four (4) times. The
treatments were randomly allocated to the various plots with each plot having one
of the treatment combinations. K. anthoteca species were inoculated with
commercial inoculums (mycofer) from the genera Glomus and Gigaspora with the
assumption that they are dominant species normally associated with most
terrestrial plants. In total, sixty-four (64) experimental units were used for this
study. The specified treatments (T) are as follows;
M0
M1
M2

M3

=
=
=
=

control (without mycorrhiza inoculation)

50 spores of Gigaspora margarita mycorrhizae

50 spores of Glomus manihotis mycorrhizae
25 spores each of G. margarita and G. manihotis mycorrhizae

Factor B (Compost treatment)

C0 = control (no compost)
C1 = 5Kg of Salvinia natans compost
C2 = 5Kg of Community compost
C3 = 2.5Kg each of Community and S. natans composts
Table 3.1 Factorial CRD treatment combinations
Planting space of 4 x 4m (625 plants/ha) was used for the study and the
Mycorrhizae
(M)
M0

C0
M0C0 =T1

Compost (C)
C1
C2
M0C1 =T2
M0C2 =T3

C3
M0C3 =T4

M1

M1C0 =T5

M1C1 =T6

M1C2=T7

M1C3 =T8

M2

M2C0 =T9

M2C1 =T10

M2C2 =T11

M2C3 =T12

M3

M3C0 =T13

M3C1 =T14

M3C2 =T15

M3C3 =T16

various treatment combinations randomly allocated to the plots (Fig 3.6).

Reference points of 5cm above the soil surface of the seedlings were marked with
non-poisonous indelible ink marker to provide consistency at the point of the
height and diameter measurement. The plant parameters measured were the height
(cm), stem diameter (mm), leaf counts and percentage survival. According to
Mohr and Schopfer (1995), height and stem diameter are some of the frequently
used methods of measuring the growth of multi-cellular living systems and also
often advantageous to use several characteristics for the same system.

49

28 m

28 m

Plot 1

Plot 2

Plot 3

Plot 4

Plot 5

Plot 6

Plot 7

Plot 8

T 10
Plot 9

T3
Plot 10

T5
Plot 11

T 12
Plot 12

T8
Plot 13

T4
Plot 14

T7
Plot 15

T 11
Plot 16

T9
Plot 17

T 16
Plot 18

T 13
Plot 19

T2
Plot 20

T 15
Plot 21

T 14
Plot 22

T6
Plot 23

T1
Plot 24

T 12
Plot 25

T9
Plot 26

T 11
Plot 27

T7
Plot 28

T 13
Plot 29

T8
Plot 30

T 15
Plot 31

T6
Plot 32

T5
Plot 33

T 16
Plot 34

T4
Plot 35

T1
Plot 36

T 10
Plot 37

T 14
Plot 38

T3
Plot 39

T2
Plot 40

T 15
Plot 41

T 11
Plot 42

T 16
Plot 43

T1
Plot 44

T 14
Plot 45

T 10
Plot 46

T3
Plot 47

T6
Plot 48

T4
Plot 49

T5
Plot 50

T 13
Plot 51

T8
Plot 52

T 12
Plot 53

T2
Plot 54

T7
Plot 55

T9
Plot 56

T6
Plot 57

T 15
Plot 58

T 13
Plot 59

T8
Plot 60

T 12
Plot 61

T 16
Plot 62

T1
Plot 63

T 10
Plot 64

T9
T2
T 14
T 11
T4
T7
T5
Figure 3.6 Random allocations of treatments on experimental plots.

T3

3.4 Cultural Practices on Experimental Plots

Vegetatively propagated propagules of K. anthoteca were inoculated and
transplanted with the transplanting exercise undertaken early morning after the
plots had been watered thoroughly. According to Hilary (2009), the best time of
day to transplant is in the late afternoon when the sun is not so hot. By taking
advantage of this time of day, the plants are able to acclimatize overnight. Strong
sun and wind have a potentially adverse effect on new transplants and unless
watered carefully, and in some cases sheltered from the wind and sun, they can
severely wilt. The plants can be stressed at the very beginning of their growing
cycle. Watering of the transplanted propagules was done immediately after
transplanting and once daily with each plot receiving about the same volume of
water. Plots were not watered after heavy rains (Swanson, 2000).

50

3.5 Data Collection

Data on the number of roots, length of longest roots and percentage survival
were collected for each treatment for the cutting potential of K. anthoteca in the
glasshouse. Isolation and counting of arbuscular mycorrhizae fungi (AMF) per 50
grams of soil sample and the percentage of root segment colonization were
conducted under dissecting and compound microscopes. For the field experiment,
an indelible ink was used to mark each plant 5 cm above the soil, where the
diameter and height readings were taken since irregularity of the soil around the
seedlings can affect the recording. Initial measurements of plant parameters such
as height (cm), diameter (mm) and number of leaves were recorded two days after
transplanting and fortnightly thereafter over sixteen weeks (4 months) of the field
research. Data on percentage survival was collected after the field experiment.

3.6 Data Management and Analysis

Data collected on number of spores and percentage root segment
colonization were correlated and percentage root segment colonization determined
according to Brundrett et al. (1996) and descriptive statistics and graphs used for
more clarifications where necessary. Root segment colonization and number of
spores counted were conducted in the laboratory of the research center for bioresources and biotechnology, Bogor Agricultural University (IPB), Indonesia.
Data on plant parameters for both cutting and field experiments were subjected to
a one-way and two-way analysis of variance (ANOVA) respectively at a
significance level of 5% (alpha 0.05). Microsoft excel was used in data entry and
computation for the increment of the various plant parameters measured (data
management). All statistical analyses were performed using the Minitab statistical
analysis package V.16. (Minitab, Inc.). Tukeys HSD test was used for multiple
comparison tests where treatment means differed significantly. The mathematical
models for the glasshouse and field research designs respectively are as follows;
1) % rootcolonization =

Number of fields - of - view containing mycorrhizae

Total observed fields - of - view

x 100%

2) Yij = + i + ij
Where:
Yij = the (ij)th observation for i=1, 2, 3 and 4; j = 1, 2, 3, 4 and 5
=parameter common to all treatments (the mean)
i= the ith treatmenteffect
ij= random error component with normal distribution
3) Yijk = + i + j + ()ij + ijk
Where:
Yijk = the (ijk)th observation for i=1, 2, 3 and 4; j = 1, 2, 3 and 4; and k =1,
2, 3 and 4

= parameter common to all treatments (the mean)

i
= the ith treatmenteffect of factor A (mycorrhizae)

51

j
= the jth treatment effect of factor B (compost)
()ij = the (ij)th interaction effect of factor A and B
ijk
= random error component with normal distribution

Stem cutting of K.
anthoteca (Experiment 1)

Trapping of AMF
(Experiment 2)

Soil inoculums
sample

Wet sieving of trapping media

(soil)

Clearing and staining

of root to estimate

AMF status of K.
anthoteca

Isolation and counting

of AMF spores

Establishment of inoculated K.
anthoteca on the field (Experiment 3)

Figure 3.7 Summary of experimental processes

p

52

4 RESULTS AND DISCUSSION

4.1 Vegetative Propagation of K. anthoteca
There was significant effect of the treatments administered on the number of
roots from cuttings of six months old seedlings of K. anthoteca. However, the
length of the longest root was not significantly affected although there were
differences among the treatment means. Again, all the cuttings recorded 100%
survival for the duration of the experiment. Analysis of variance (ANOVA) tested
at 5% significance level showed a significant effect of both hormone and
wounding effect on the rooting number of K. anthoteca cuttings over the control
treatment (Table 4.1). Aside mineral nutrition, hormone and wounding are some
of the treatments usually administered to cuttings to hasten root development.
Table 4.1 One-way analysis of variance for the cutting experiment
Source

DF

SS

MS

Treatments
Error
Totals

3
16
19

35.35
41.20
76.55

11.78
2.58

4.58*

0.017

*Significant at 5%
According to Hartmann et al. (1997), treating cuttings with auxins is
purposely to increase the percentage of cuttings that form roots, hasten root
initiation as well as increase uniformity of rooting. It was however emphasized
that, plant whose cuttings root easily may not justify the additional expense and
efforts of using these materials. Treatment means of wounding without hormone
was not significantly different from wounding with hormone. Other previous
studies have indicated that cuttings derived from juvenile stock plants are easier to
root (Browne et al. 1997; Berhe and Negash 1998; Bhardwaj and Mishra 2005;
Amri et al. 2010). This is a contributing factor to the observed percentage survival
the cuttings for all treatment. This suggests that wounding and growing stage of
plant play crucial roles in the rooting of cuttings especially from K. anthoteca
seedlings as the treatments means were not significantly different (Table 4.2).
The superior rooting ability of cuttings from seedlings over that of matured
trees has been attributed to the effect of changes in the woody plant
developmental process that occur with increasing age; these are known as
maturation or ontogenetic aging. Ontogenetic aging is often found to be most
advanced in the upper parts of a tree and least advanced near the base of the trunk,
with intermediate conditions between. As tree species possess meristems that are
normally perennially dormant and mature more slowly than active ones, these
meristems often produce vigorous sprouts (for example, stump sprouts) after the
release of dormancy (Bonga and von Aderkas 1993; Greenwood and Hutchison
1993; Hartmann et al. 2002). The rooting ability of juvenile cuttings has been
ascribed to optimum levels of sugars and the total carbohydrate content and low
nitrogen (Bhardwaj and Mishra 2005).

53

Figure 4.1 K. anthoteca cuttings under the various treatments a) Control treatment
b) No hormone with wounding c) Hormone without wounding d)
Hormone with wounding
Table 4.2 Grouping of means using Fisher method at 95% confidence
Treatments

Mean SE Mean
Number of
Length of Root
Roots
(cm)

W-, HW-, H+
W+, HW+, H+

5
5
5
5

2.20 0.49b
3.20 0.66b
4.20 0.66ab
5.80 0.97a

8.30 0.93a
8.74 2.30a
8.70 2.46a
13.72 3.47a

Rooting
Percentage
(%)
100%
100%
100%
100%

Means that do not share a letter in the same column are significantly different according
to Fisher at a confidence of 95% ( 5%)

The length of the longest root was not significantly affected although there
were differences among the treatment means. Auxin transport has naturally been
studied almost exclusively in young seedlings, where synthesis takes place in the
actively proliferating tissues. Some studies have also reported that wounding a
cutting initiates a chemical signal that induces changes in the metabolism of
affected cells (Wilson and van Staden 1990). Wounding the base of stem cuttings
can enhance root production and is one of the factors affecting the regeneration of
plants from cuttings. In this process, cell division and production of roots
primordia are stimulated through wounded tissues. This has been attributed
mainly to the natural accumulation of auxins and carbohydrates in the wounded

54

area and in the increase of respiration rate in the creation of a new s

sink area.
Injured tissues from wounding produce ethylene which may indirectly promote
adventitious root formations (Hartmann et al. 1997). The use of stem cuttings,
according to Leakey (1990), has become a common propagation method in
forestry and agroforestry.
estry. Root
oot initiation in stem cuttings requires that there is an
appropriate environment that would reduce post-severance
post severance and physiological
stress in the cutting. In this study, it is clear that wounding the base of stem
cuttings can enhance the number of root production as reported by Hartmann et al
(1997) and as such where hormone is unavailable wounding is adequate for the
vegetative propagation of K. anthoteca seedlings.
4.2 Arbuscular Mycorrhizae Fungi (AMF) Root Colonization

Percentage colonization (%)

Differences in percentage root segment colonization and mean number of

spores between the plant species were analyzed using descriptive statistics and
Analysis
nalysis of variance (ANOVA). The ANOVA test revealed no significant
differences at 5%
5% (F=2.05,
(F=2.0 p= 0.148 and F=2.93, p=0.071)) in both parameters
respectively for the species used for all the trapping experiment. A
After three
months of trapping of arbuscular mycorrhizae fungi (AMF) from soils under K.
anthoteca plantation,
lantation, the roots of Sorghum bicolor were more
re susceptible to AMF
infection compared to P. javanica and K. anthoteca recording the highest
percentage of root segment colonization and average number of spores per 50g of
soil of 43% and 5.1 respectively although these differences were not significant
(Figures 4.2 and 4.4).
).
Traditionally, AMF have been considered to be generalists with regard to
the hosts that they infect. They have also been considered to be functionally
equivalent in their effects on a host. These have been largely attributed to the fact
that most AMF can successfully infect a wide range of plant species when grown
experimentally in monocultures (Bever 1996; Douds and Millner 1999; Eom
2000; Kiers 2000).

50
45
40
35
30
25
20
15
10
5
0

43
40

P. javanica
32

S. bicolor

K. anthoteca

Figure 4.22 Average percentage of root segment

segmen colonization
olonization

55

Figure 4.3 a) Colonization of K. anthoteca root segments at a magnification of

40x b) Isolated single spore of AMF in soil sample
Again, most studies
tudies have also suggested that some plant species are baits for
AMF and therefore more susceptible. Root segment colonization by AM fungi
was generally moderate across all species, with few samples below 30
30% for P.
javanica and K. anthoteca and a substantial number above 40% of root
colonizationn (Appendix 5). Several hundred fungal species under natural
conditions are associated with and colonize plant roots. The preference or
specificity for partners in these symbiotic relationships is therefore a key to
understanding how the community structures of root-associated
root associated fungi and their
host plants influence each other in diverse ways (Toju et al.. 2013). According to
Smith and Read (2008), mycorrhizal fungi facilitate the soil nutrient acquisition of
plants and thereby enhance the
the competitive ability of their specific hosts in local
communities (Nara 2006).

Percentage Root Colonization

100

% Colonization = 10.011 number of Spore - 6.213

90

R2 = 0.797, r = 0.892, p= 0.000

80
70
60
50
40
30
20
10
1

10

Number of Spores

Figure 4.4 Correlation of percentage root colonization and number of spores

56

Brundrett et al. (1996) also reported that growth substrates, plant and fungal
materials, watering, nutrient supply and environmental conditions are some of the
major factors that significantly affect the performance of mycorrhizal plants in the
glass house. The chemical properties such as the pH of the soil are of vital
importance. Plants grown in glasshouse are typically grown from seeds. This
ensures uniformity since it aids in avoiding interference with treatment effects. K.
anthoteca used in this study were vegetatively propagated and may be a cause for
the observed differences in root colonization compared with the other species
(Fig. 4.2). Pearsons correlation analysis between the number of spores isolated in
the soil samples and the percentage of root segment colonization was very strong
with a correlation value (r) of 0.892 (Fig. 4.4). Again, no significant differences
were observed among the species with respect to the number of spores per 50g of
soil (Table 4.3)
Table 4.3 Average Number of Single Spores per 50 g of Soil Sample

Sample No.
Species Name
P. javanica
S. bicolor
K. anthoteca

Number of spores per sample soil (50g)

2
3
4
5
6
7
8
9 10

5
7
4

7
8
5

5
6
5

5
6
6

2
4
3

1
4
2

4
3
2

3
5
2

3
3
3

3
5
2

Avg.
number
3.8
5.1
3.4

4.3 Plant Growth

Growth is the irreversible increase in the size of a plant and has also been
considered as the product of its physiological processes. Plants have indeterminate
growth, thus, 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.
4.3.1 Growth of K. anthoteca on Ex-Coal Mined Site
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 (Russell 1998). Compost application to K.
anthoteca had a significant effect on the height growth of the species. Analysis of
variance (ANOVA) tested at 5% significance level showed a significant effect
(P<0.001) of compost on the mean height increment (Fig 4.6). There was no
significant effect (P>0.05) of mycorrhizae treatment as well as the interaction
between both factors on height growth (Table 4.5). Compost composition from a
mixture of S. natans and that prepared from the community of PT. Bukit Asam
(C3) recorded the highest increment in height of 9.31 cm while compost from S.
natans only (C1), community compost (C2) and control (C0) had increments of

57

9.00 cm, 5.78 cm and 4.47 cm respectively. However, there were no differences
at 95% confidence between increments of C3 and C1 (Table 4.9). An increment of
between 29.31-100.22% of the compost treatment over the control was recorded.
The use of compost to enhance the growth of plants has been reported and figure
4.5 shows the final height of the plants with the various compost treatments.

25.50cm

23.18cm

32.50cm

c
Figure 4.5 Growth of K. anthoteca on the field: a) Community compost b)
Without compost c) S. natans compost d) Mixed compost

33.81cm

58

No
1
2
3
4
5
6
7
8
9
10
11

Table 4.4 Analysis of composts

Salvinia natans
Parameter
Compost
Moisture Content (%) 78.27
pH
6.96
Ash content (%)
49.61
Organic material (%) 19.32
Organic carbon (%)a
15.94
C/N
13.86
a
N (%)
1.15
P (mg/100g)
19.34
K (mg/100g)
835.84
a
Mn (ppm)
914
a
Fe (ppm)
12349

Sample analyzed in the laboratory of SEAMEO Biotrop.

Bukit Asam (Persero).

Community
Compostb
40.28
6.55
72.35
5.34
5.23
4.98
1.05
11.84
158.67
50.32
b

Sample analyzed by PT.

Compost application significantly enhances increase in crop production and

mitigates the negative effect of a delay in sowing. The use of compost, therefore,
is a sound technology for combating soil degradation and often leads to humus
accumulation in the soil and thereby supplying nutrients in several binding forms
to the soil matrix. Compost also has dynamic impact on exchange processes
between root system and sorption complex. This is mainly caused by the humified
OM and the colloid properties of the humic substances (Oudraogo et al. 2001).
Generally, the nutrient effect of organic composts depends largely on the
transformation processes that take place in the soil after their application. This
applies especially to nitrogen (N), for which its plant availability is tightly related
to microbial transformations. According to the European Commission, (2001), the
degradability and C/N ratio of the organic matter determines whether it comes to a
net mineralization or immobilization of N after the application of an organic
fertilizer (European Commission, 2001). When used in sufficient quantities, the
addition of compost has both an immediate and long-term positive impact on soil
structure. It resists compaction in fine textured soils and increases water holding
capacity and improves soil aggregation in coarse-textured (sandy) soils.
Table 4.5 Two-way analysis of variance for height increment (cm)
Source
Mycorrhizae
Compost
Interaction
Error
Total

DF
3
3
9
48
63

SS
3.66
272.71
22.09
130.26
428.72

* Significant at alpha () 5%

MS
1.22
90.90
2.45
2.71
ns

Not significant

F-Value
ns

0.45
33.50*
ns
0.90

P-Value
0.72
0.00
0.52

59

Table 4.6 Analysis of soil sample from the research site

No.
1

Test Parameter
*pH
H2O (1:1)
CaCl2
(1:1)
*OrganicCarbon

*TotalNitrogen

4
5

C/N ratio
*Available P2O5

Method

Unit

Values

SNI 03-4787-2002

SNI 13-4720-1998
(Walkeyand Black)
SNI 13-4721-1998
(Kjeldahl)

SL-MU-TT-05
(Bray I/II)
6
Potential P2O5
SL-MU-TT-06 (HCl
25%)
7
Potential K2O
SL-MU-TT-08 (HCl
25%)
ExchangeableCations
8
*Ca
9
*Mg
SL-MU-TT-07 c
10
*K
(NH4O Ac 1.0N
11
*Na
extract. pH 7.0)
12
Total
13
CEC
14
BaseSaturation
Al-Hdd
3+
15
Al
SL-MU-TT-09
(KCl, 1N extract)
16
H+
Source: PT. Bukit Asam Tbk. 2014 Report

6.2
6.2
%

0.39

0.07

ppm

5.57
8.6

mg/100g

35

mg/100g

186

cmol/kg
cmol/kg
cmol/kg
cmol/kg
cmol/kg
cmol/kg
%

8.72
2.18
0.17
1.96
13.03
22.56
57.76

me/100g
me/100g

0.11
0.21

The results of this study are in conformity to earlier reports that, compost
application significantly enhances the growth of plants. Effect of compost
prepared from organic material, thus, S. natans (5 Kg/plant) on the height
increment of K. anthoteca was significantly different from that observed both in
the control and compost from a less organic material such as paddy husks (rice
hull) composts. Community compost made from rice hull and research site soil
analysis showed low levels of organic carbon and nitrogen of 5.23%; 1.05% and
0.39%; 0.07% respectively. On the other hand, compost prepared from S. natans
has 15.94% of organic carbon and 1.15% of total nitrogen.
Phosphorus (P), another important and macro nutrient for plant growth
responsible for photosynthesis and energy transfer was relatively high (19.34
mg/100 g) in compost from S. natans as compared to that of community compost
from paddy husk of 11.84mg/100 g (Table 4.4). This may account for the
observed differences in the height growth of K. anthoteca. Also, according to the
United States Composting Council (2008) compost products contain a

60

considerable variety of macro and micronutrients. Although often seen as a good

source of nitrogen, phosphorous, and potassium, compost also contains
micronutrients essential for plant growth. Since compost contains relatively stable
sources of organic matter, these nutrients are supplied in a slow-release form. It
also improves the cation exchange capacity of soils, enabling them to retain
nutrients longer. It allows tree crops to more effectively utilize nutrients, while
reducing nutrient loss through leaching.

Height Increment (cm)

25.0

20.0

15.0
10.0

5.0

0.0
Initial

Wk 2

Wk 4

Wk 6

Wk 8

Wk 10

Wk 12

Wk 14

Wk 16

Weeks After Transplanting

No Compost

Salvinia natans

Comminity compost

Mixed Compost

Figure 4.6 Effect of compost treatment on height increment

Microorganisms play an important role in organic matter decomposition
which, in turn, leads to humus formation and nutrient availability.
Microorganisms can also promote root activity as specific fungi work
symbiotically with plant roots, assisting them in the extraction of nutrients from
soils (United States Composting Council 2008). In a symbiotic association, fungus
colonizes plant 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. It has however been reported that, compost
applications as soil amendment is capable of enhancing plant growth. Effect of
AMF inoculation to K. anthoteca for height increment was not significantly
different from the control treatment although the means of the inoculated
propagules were relatively higher compared to the control. G. manihotis
inoculation recorded a height increment of 7.510.62 cm whiles G. margarita,
mixed culture and the control treatment recorded increments of 7.180.69 cm,
7.060.646 cm and 6.840.70 cm respectively. According to Douds and Millner

61

(1999), the AMF genera Gigaspora and Scutellospora produce only arbuscules
with extensive intraradical and extraradical hyphal networks whereas Glomus,
Entrophospora, Acaulospora, and Sclerocystis in addition, also produce vesicles.
Arbuscules can be dense and compact. Oval vesicles, which usually form between
root cortex cells, are present in many cases. These vesicles persist in roots and
often develop thickened and/or multi-layered walls. Although the arbuscular trunk
hyphae of Gigaspora normally are much longer and thicker and appear wispy due
to relatively long curving branches than those of Glomus, internal vesicles are not
present. Arbuscules are usually short-lived and begin to collapse after a few days
while vesicles are storage structures and can remain in roots for months or even
years (Brundrett et al. 1996).
4.3.2 Diameter Increment
Increase in plant diameter or girth has often been considered as a secondary
growth. However, with the addition of organic materials this process can be
hastened. Analysis of Variance (ANOVA) tested at 5% significance level
showed a significant effect (P0.000) of compost on the mean height increment
on plant diameter with steady increment for the study period (Fig 4.4). There was
no significant effect (P=0.148; 0.188) of mycorrhizae treatment as well as the
interaction between both factors (Table 4.7). In terms of diameter increment
compost from S. natans recorded the highest mean increment of3.610.238 mm
although there was no significant difference between the means of S. natans (C1)
and mixture compost (C3) according to Tukeys honest significant difference
(HSD) method at 95.0% confidence (Table 4.9). There was however a percentage
increases of between 9 and 48% of compost treatment over the control
experiment.
Table 4.7 Two-way analysis of variance for diameter increment (mm)
Source
Mycorrhizae
Compost
Interaction
Error
Total

DF
3
3
9
48
63

SS
3.11
17.44
7.34
26.72
54.61

* Significant at alpha () 5%

MS
1.04
5.81
0.82
0.56
ns

F-Value
1.86 ns
10.44 *
1.46 ns

P-Value
0.15
0.00
0.19

Not significant

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).
A laboratory test of both compost types used for this study had varied amount of
nutrients (Table 4.4). 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)

62

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 (Halley 1982).
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). The fungi of
vesicular-arbuscular mycorrhizae colonize considerable portions of the root
system and in spite of the carbon drain they impose on the host plant, their
presence within the root tissues can positively influence several aspects of the host
plant's physiology. In the majority of cases, improved phosphate uptake is the
primary cause of growth and yield enhancements in the mycorrhizal plants.
Mycorrhizae produced by Glomus Spp often result in simultaneous growth in 2
directions.
Mycorrhizal roots have different phosphate absorption kinetics and lower
threshold values than non-mycorrhizal roots. The external hyphae developing
around mycorrhizae explore a large volume of soil and absorb available phosphate
beyond the depletion zone at the root surface. Phosphate accumulating in the
external fungal hyphae is translocated to the internal mycelium by a welldeveloped transport system and transferred to the host tissues mainly across the
intracellular arbuscules (Gianinazzi-Pearson and Gianinazzi 1983). Certain
specialized enzyme activities are specifically associated with this alternative
pathway of phosphate nutrition in mycorrhizal plants. Available P2O5 in the soils
of the research site of 11.23 mg/100 g was relatively high in comparison with that
of S. natans 19.34 mg/100 g (193.4 ppm) and community compost 11.84 mg/100
g (118.4 ppm). According to Brundrett et al. (1996), the primary aim of
mycorrhizal inoculation is to increase the yield of plants grown for plantation
forestry. These growth responses depend on the mycorrhizal dependency of the
host plant species, soil properties; especially the availability of nutrients such as P
and the capacity of the fungi to provide benefits to the host plant. Since there is
relatively no difference between the amount of phosphorus in the soil and applied
composts the effect of mycorrhizae on the diameter increment of K. anthoteca
may not be remarkable. Although there was no significant different among the
means of the mycorrhizae treatment on diameter increment of K. anthoteca,
Glomus Spp had the highest mean increment of 3.250.17 mm compared to
Gigaspora Spp, mixed culture and non-mycorrhizal plants recording increment of
2.650.12 mm. It has been reported that, improved phosphate nutrition is not
always sufficient to explain the observed effects of vesicular-arbuscular
mycorrhizae on the host plant's physiology (Gianinazzi-Pearson and Gianinazzi
1983).

63

11.0

Diameter Increment (mm)

10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Initial Wk 2 Wk 4 Wk 6 Wk 8 Wk 10 Wk 12 Wk 14 Wk 16
Weeks After Transplanting
No Compost
Salvinia natans
Community Compost
Mixed Compost

Figure 4.7 Effect of compost treatment on diameter Increment

Again, aside the factors mentioned above, soil temperature and moisture
contents are other factor limiting the full potential of AMF (Brundrett et al. 1996).
The study site is quite critical with some extreme environmental conditions such
as high temperatures leading to low soil moisture due to high evaporation rates
even after watering. According to Nwoboshie (1982), 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.
Brundrett (1991) has again reported that there is considerable variation between
and within species of fungi in their response to those factors. The capacity for
fungi to produce soil hyphae is thought to be a major determinant of mycorrhizae
fungus benefits to their host and would result from inherent properties of the fungi
and interactions with soil conditions (Thomson et al. 1994).
4.3.3 Leaf Count
Unlike animals, plants only have 3 organs namely; the roots, the stems and
the leaves with the stems and leaves together forming the shoot of the plant.
Leaves differ from stems in that, they do not have an apical meristem, thus, leaves
are determinate (limited in their growth), while stems are indeterminate
(theoretically capable of growing forever).In this study, analysis of variance of
compost application showed a significance effect at a confidence level of 95%

64

(Table 4.8). The same was observed for both height and diameter increments with
significant differences among the various compost types (Tukeys HSD, p<0.05).
Leaves are produced in a very organized manner at the shoot apex. This
results in a predictable arrangement of the mature leaves on the stem. This
arrangement is normally referred to as phyllotaxis of the leaf. They are the main
site of photosynthesis, where sugars are made from water and carbon dioxide,
using sunlight energy and are therefore plant's food factory (Bell and Bryan
1993).The growth (roots, stem and leaves) of plants depends on the availability of
nutrients from the soil.

Source

Table 4.8 Two-way analysis of variance for leaf increment

DF
SS
MS
F-Value
P-Value

Mycorrhizae

19.81

6.60

2.60 ns

0.06

Compost

135.06

45.02

17.71*

0.00

Interaction

13.06

1.45

0.57 ns

0.81

Error

48

122.00

2.54

Total

63

289.94

* Significant at alpha () 5%

ns

Not significant

Thus, it is important that the soil should potentially provide nutrients for the
growth and development of plants. Prolonged uptake of nutrients by either
growing plants or excavation of the land for any purposes depletes soil of vital
nutrients, adversely affecting the growth of plants (Russell 1998). In this study,
plant biomass was substantially increased when green waste compost (S. natans)
was used as the organic matter component instead of rice husk. Green waste
compost has more nutrients available to plants due to its preconditioned state
(composted) and greater diversity of source material. It has been shown
previously that increasing the organic fraction of a substrate increases plant
growth (Nagase and Dunnett 2011). Composts incorporated into the soil help
increase plant growth and yield by providing one or more of the elements
essential for growth and development while 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). Compost application in this research showed a significant
increment in the number of leaves throughout the study period (Fig. 4.8).
However, compost from green waste such as S. natans was significantly different
from the other compost although a combination of the compost treatment had the
optimum effect on leaf count of 8.250.51 (Table 4.9)
As indicated in other plant parameters earlier, mycorrhizae effect in this
study did not significantly affect the growth of K. anthoteca on the field. In recent
times, there have been 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

65

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)

14

Number of Leaves

12
10
8
6
4
2
0
Initial

Wk 2

Wk 4

Wk 6

Wk 8 Wk 10 Wk 12 Wk 14 Wk 16

Weeks After Transplant

No Compost
Community Compost

Salvinia natans
Mixed Compost

Figure 4.8 Effect of compost treatment on leaf count

Table 4.9 Grouping information for compost treatment means for measured plant
parameters using Tukeys HSD method at a confidence of 95%
Mean SE Mean
Compost Treatments N
Height (cm)
Diameter (mm) Leaf Count
Without Compost

16

4.470.22b

2.440.15b

5.000.29

S. natans compost

16

9.00 0.45a

3.610.24a

7.690.45

Community compost 16

5.780.42b

2.530.19b

5.190.32

16

9.310.47a

3.420.20a

8.250.51

Mixed Compost

b
a

Means that do not share a letter in the same column are significantly different at a
confident of 95% ( 5%)

66

4.3.4 Survival of K. anthoteca on the Field

After four months of transplanting vegetatively propagated K. anthoteca to
the field, non-mycorrhizal (NM) treatment recorded the highest mortality of
56.25% with only 43.75% plants surviving while K. anthoteca propagules preinoculated with Glomus manihotis had the highest percentage survival of
81.25%.There were significant differences between NM treated plants and AMF
inoculated plants as well as between the various AM treated plants on the
percentage survival of K. anthoteca according to a t-test analysis for independent
samples (Fig 4.9). However, there were no significant differences between
compost and mycorrhizal applications on the percentage survival of K. anthoteca
P(Tt, 0.90) although the mean percentage survival of mycorrhizal treatment was
higher (64.06%) than the application of compost treatment (62.5%).

c
95

Percentage Survival

85

ab

75
65

55
45
35
25
15
5
Gi. margarita

Gl. manihotis
Mixed AMF
AMF Species

Non-AMF

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

anthoteca with one standard error bar from the mean
Association of fungi with plant roots has long been documented and they
function as plants root hair and acting 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 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 phytopathogenic fungi (Selvaraj and Chellappan 2006). The study area for this research
is characterized by high temperatures with low soil moistures hence propagules
with AMF inoculation were more tolerant as compared to NM plants. According

67

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. 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 stomatal conductance and transpiration, better supply of diffusion-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 dry conditions than NM controls (Duan et al. 1996). Some
studies have also indicated that, improved nutrition of AM compared with NM
plants may be the main basis for improved drought tolerance. About 80% of the
studies assessed by Aug (2001) also revealed AM plants grow better under
drought stress than NM plants.
In this study, G. manohotis inoculation recorded a higher percentage
survival compared to G. margarita and inoculums mix (G. smanihotis and G.
margarita) inoculations. According to Douds and Millner (1999), the AMF genera
Gigaspora and Scutellospora produce only arbuscules with extensive intraradical
and extraradical hyphal networks whereas Glomus and Entrophospora, in
addition, also produce vesicles. Arbuscules are usually short-lived and begin to
collapse after a few days while vesicles are storage structures and can remain in
roots for months or even years (Brundrett et al. 1996). However, vesicles persist
in roots and often develop thickened and/or multi-layered walls. There also seem
to be antagonism between the species (Fig 4.10). Some studies have however
suggested otherwise. Giri et al. (2004) conducted a study on the effect of
inoculation of two arbuscular rmycorrhizal fungi, Glomus fasciculatum, and G.
macrocarpum, alone and in combination, on establishment and growth of Acacia
auriculiformis in a wasteland soil, studied under nursery and field conditions.
According to the study, A. auriculiformis exhibited a maximal mycorrhizal
dependency of 79.6% on dual inoculation. Mycorrhizal dependency differed with
AM fungal isolates and age of the plant. Under field conditions, AM colonization
of A. auriculiformis enhanced tree survival rates (85%) after transplantation.
Arbuscular mycorrhiza-colonized plants showed significant increase in height,
biomass production and girth as compared to non-mycorrhizal plants. In general,
all growth parameters were higher on dual inoculation of G. fasciculatum and G.
macrocarpum as compared to uninoculated plants under both nursery and field
conditions. The study concluded that, mycorrhizal symbiosis play vital role in
helping A. auriculiformis to establish and thrive in alkaline wasteland soils.
Hence, the nursery inoculation programs with selected AM fungal species or
combination of synergistically interacting species may be helpful to produce
vigorous seedlings to survive under wasteland stress soil.

68

5 CONCLUSIONS AND RECOMMENDATION

5.1 Conclusions
Vegetative propagation using leafy stem cutting has been successful in K.
anthoteca. In this study, stem tip cutting of K. anthoteca seedlings for vegetative
propagation was successful without hormone (auxin) application. Within the
context of this study therefore, K. anthoteca seedlings can be propagated without
hormone application. Nonetheless, wounding will play crucial role in terms of the
number of roots. However, on the bases of survival or rooting ability, the
application of both hormone and wounding is unnecessary.
K. anthoteca species have been found to form symbiotic association with the
arbuscular mycorrhizae fungi. Again, correlation analysis indicated that there is
significant positive relationship between the number of available spores in the soil
and the colonization of root segments. Root segments colonization of K.
anthoteca cuttings was not significantly different from that of S. bicolor and P.
javanica.
K. anthoteca propagules can be established on an ex-coal mined site with
compost and AMF inoculation. Compost made from organic materials such as S.
natans is preferable for the growth and development of the plants. It must
however be emphasized that, vegetatively propagated K. anthoteca can as well be
established on the field (ex-coal mined site) without mycorrhizae inoculations.
However, inoculation of the plants with arbuscular mycorrhizae fungi (AMF) will
aid in the reintroduction of such organisms into the ecosystem.

5.2 Recommendations and Future Perspectives

Based on the outcome of this research, the following are hereby recommended;
1. K. anthoteca is an important tropical tree species with high economic
value and this study has shown that the species can be established on excoal mined sites. It is hereby recommended for revegetation of ex-coal
mined sites with compost application rate of 5 Kg/plant and the
propagules at heights between 30 cm and 40 cm.
2. Also, the establishment of K. anthoteca should be tested in other
resources mining areas such as gold, tin and manganese mining sites to
widen its application for revegetation.
3. For future studies, propagules from both seeds and vegetative
propagation should be tried comparing the growth regimes on the field
with AMF soil inoculums and compost, preferably from S. natans.
Other aquatic weeds should as well be experimented for composting and
subsequent application for revegetation of ex-coal mined sites.

69

REFERENCES
Al-Karaki GN. 2006. Nursery inoculation of tomato with arbuscular mycorrhizal
fungi and subsequent performance under irrigation with saline water. Scientia
Horticulture 17:109
Amri E, Lyaruu HVM, Nyomora AS, Kanyeka Z L. 2010. Vegetative propagation
of African Blackwood (Dalbergia melanoxylon Guill. & Perr.): effects of
age of donor plant, IBA treatment and cutting position on rooting ability of
stem cuttings, New Forests. 39:183194
Aswathanarayana U. 2003. Natural Resources and Environment. Geological
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APPENDICES
Appendix 1 Test result of soil sample from the research site
Request Number
: 526/TT/IV/2014
Sample number
: 1648-1650
Page
: 2 / Of 4
No.
1

Test Parameter
*pH
H2O (1:1)
CaCl2
(1:1)
*Organic
Carbon
*Total Nitrogen

4
5

C/N ratio
*Available P2O5

Method

Unit

Values

SNI 03-4787-2002

SNI 13-4720-1998
(Walkey and Black)
SNI 13-4721-1998
(Kjeldahl)

6.2
6.2
%

0.39

0.07

SL-MU-TT-05
ppm
(Bray I/II)
6
Potential P2O5
SL-MU-TT-06 (HCl
mg/100g
25%)
7
Potential K2O
SL-MU-TT-08 (HCl
mg/100g
25%)
Exchangeable Cations
8
*Ca
cmol/kg
9
*Mg
cmol/kg
SL-MU-TT-07 c
10
*K
cmol/kg
(NH4O Ac 1.0N
11
*Na
cmol/kg
extract.
pH
7.0)
12
Total
cmol/kg
13
CEC
cmol/kg
14
Base Saturation
%
Al-Hdd
15
Al3+
SL-MU-TT-09
me/100g
(KCl, 1N extract)
16
H+
me/100g
Source: PT. Bukit Asam Tbk. 2014 Report
Note:
- Sample test results was conducted with samples at 105C
- cmol/kg me/100g

5.57
8.6
35
186

8.72
2.18
0.17
1.96
13.03
22.56
57.76
0.11
0.21

Bogor, 20 June 2014

Soil and Plants Lab
Arif Nuryadin, BSc
Quality Control Manager
NIP. 196711172007011001

78

Appendix 2 Test result of Salvinia natans compost

Request Number
Sample number
Page

No.
1
2

:
:
:

Measured
Parameter
Organic carbon
Total Nitrogen

464/TT/IV/2014
1293-1294
2 / Of 2

Method

Unit

Compost
Sample code

Walkey &
Black

15.94

Kjeldahl

1.15

Total metals
(HNO3
3
Total Mn
ppm
914
HClO4) - AAS
(HNO3
4
Total Fe
ppm
12349
HClO4) - AAS
Note: Sample test results was conducted with samples at 105oC
Sample type: Compost
Bogor, 13 Mei 2014
Soil and plants Lab.
Arif Nuryadin, BSc
NIP. 196711172007011001

Appendix 3 Cultivation and preparation of Salvinia natans (Kiambang) composts

at PT. Bukit Asam, South Sumatra of Indonesia

S. natans growing in ponds

S. natans being composted

79

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

Appendix 5 Average root colonization of species by AMF

Sample No.
Species
P. javanica
S. bicolor
K. anthoteca

Average root colonization per Sample

3
4
5
6
7
8
9

50
60
36

58
51
42

57
41
35

58
45
40

28
41
38

20
37
31

36
31
25

31
45
24

31
35
27

10

Avg.
Col. (%)

31
42
26

40
43
32

Appendix 6 Laboratory analysis of root segment colonization by AMF

Philip Worlanyo D. at PAU laboratory, IPB

Sample roots on slides

80

Appendix 7 Soil sampling and set up for AMF trapping experiment

Soil sampling at Carita

Setting up of AMF trapping experiment

Appendix 8 Site selection, preparation and planting of K. anthoteca propagules

with AMF on the field

Planting site selection

Planting of K. anthoteca

Preparation of planting site

Planting gang

81

With field supervisor: Mr. Dedy Rosa

Field driver: Mr. Sahilun

Appendix 9 t-Test: Independent samples for survival of K. anthoteca

Mycorrhizae
Compost
Mean
64.0625
62.5
Variance
244.1406
338.5416667
Observations
4
4
Pooled Variance
291.3411
Hypothesized Mean Diff.
0
df
6
t Stat
0.12946
P(T<=t) one-tail
0.450612
t Critical one-tail
1.94318
P(T<=t) two-tail
0.901225*
t Critical two-tail
2.446912
*No significant difference among the factors with respect to percentage survival

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ABOUT THE AUTHOR

Philip Worlanyo Dugbley was born in Kumasi, Ashanti Region of Ghana on
11 April, 1987 to Mr. Anthony Dugbley and Mrs. Comfort Agyeibea. He is the
first child of his parents with three siblings and single. The author had his
elementary, junior and senior high education at public schools at Asato, Nkwanta
and Kpando respectively, all in Ghana.
In 2007, he started his university education in the Faculty of Renewable
Natural Resources of the Kwame Nkrumah University of Science and Technology
(KNUST) Kumasi, Ghana. During his undergraduate study, he served as the
chairman of the Renewable Natural Resources Students Association
(RENARSA) Council and again, as the chairman of the Electoral Commission for
the 2010/2011 academic year simultaneously. He later graduated with a
bachelors degree in Natural Resources Management in 2011 majoring
Silviculture and Forest Management.
After his first degree, he worked as a research and teaching assistant at
KNUST for about a year, thus, October, 2011 to August, 2012 and later became a
junior member of the Bogor Agricultural University (IPB) graduate school,
Indonesia, 2013 through the award of masters degree scholarship by the
government of Indonesia. While studying for his masters degree, the author
attended and presented posters in both national and international seminars and
conferences organized by Bogor Agricultural University, SEAMEO Biotrop, and
iYouLead.
The author has three publications with the titles; 1. The Establishment of
Vegetatively Propagated K. anthoteca Pre-Inoculated With Mycorrhizae (AMF)
with Compost Application on an Ex-Coal Mined Site published in the American
Journal of Agriculture and Forestry (March, 2015). 2. Susceptibility of
Vegetatively Propagated K. anthoteca to Arbuscular Mycorrhizae Fungi (AMF)
Soil Inoculum Infection published in Science Research (February, 2015). 3. The
Effect of Poultry Manure on the Early Growth Performance of Milicia excelsa
Seedlings published in the Journal of Tropical Forest Science and Technology
(December, 2013). He later completed his graduate studies with a Master of
Science degree in Tropical Silviculture in April 2015.
The author has interest in forest research specifically plant physiology,
mycorrhizae, tropical silviculture and ecological restoration. Aside academics, he
also likes peer counseling, watching movies playing and watching football as
hobbies. Currently, he is a peer reviewer for the Journal of Plant Sciences and
Journal of Science Research. This work is the authors master thesis report
presented to the postgraduate school of Bogor Agricultural University as a
requirement for the partial fulfillment of the award of Master of Science degree in
Tropical Silviculture.
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