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Nutrition and nitrogen-fixation in Malaysian
Pterocarpus indicus Willd.
By
LOK ENG HAI
Dip. (Forestry), BSc. (Forestry),
MSc. (Plant Science)
Thesis submitted in fulfillment of the requirements for the
degree of the Doctor of Philosophy
School of Biological Sciences & Biotechnology
Faculty of Sustainability, Environmental and Life Sciences
Murdoch University,
Perth, Western Australia
June 2011
II
DECLARATION
I declare that this thesis is my own account of my research and contains as its main
content work which has not previously been submitted for a degree at any tertiary
education institution.
……………… ……………. Lok Eng Hai
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Abstract
Pterocarpus indicus is a promising tropical woody legume for the
establishment of forest plantations in Malaysia. Woody legumes that form
symbiotic relationships with nitrogen-fixing bacteria also play an important
role for forest restoration on degraded land. Although P. indicus has been
widely planted as an amenity tree in SE Asia, its silvicultural requirements
have not been determined. There are no recommendations for fertilizer or for
inoculation with nitrogen-fixing bacteria. This thesis explores the phosphorus
(P) and nitrogen (N) requirements of seedlings and identifies a range of
nitrogen-fixing bacteria capable of forming root nodules under glasshouse
conditions.
Four glasshouse experiments were undertaken on two soil types: Yalanbee
sandy gravel (YB) and yellow sand (YS) to determine the P and N
concentration ranges in the foliage of deficient and healthy plants and to define
critical nutrient concentrations for the diagnosis of deficiency. There was a
narrow range in rates of P fertilizer, supplied as aerophos, Ca(H2PO4)2.H2O,
between deficiency and toxicity in both soil types. The relationship between
yield and P concentration in the youngest fully expanded leaf (YFEL) enabled
critical P concentrations for the diagnosis of deficiency (0.17%) and toxicity
(0.41%) to be determined at 90% maximum yield from linear regressions fitted
to the data. The foliar P concentration ranges for deficiency and toxicity were
similar to other nitrogen-fixing trees. Only plants in YS responded to inorganic
N fertilizer, and soil analysis suggested that seedlings may take up ammonium-
N in preference to nitrate-N at luxury supply. A critical concentration for the
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diagnosis of N deficiency was not able to be determined due to the lack of data
points. Plants with adequate N fertilizer had YFEL N concentrations of 2-3.5%
dry weight.
To determine whether P. indicus is more sensitive to luxury soil P supply than
other fast-growing legume trees, the P response of P. indicus in YB was
compared with three other woody legumes, Pterocarpus macrocarpus, Acacia
mangium and Sesbania formosa. The sensitivity of P. indicus to high P was
confirmed and the response shown to be similar to P. macrocarpus. Both
species showed severe symptoms of P toxicity, namely leaf necrosis and
stunted growth. In contrast, shoot and root yields of A. mangium and S. formosa
were not reduced at luxury P supply and yield x fertilizer relationships were
able to be fitted to the Mitscherlich model. Critical P concentrations for the
diagnosis of P deficiency in A. mangium and S. formosa, derived using the
Mitscherlich model, were estimated to be 0.2-0.3% dry weight for the YFELs.
Two glasshouse inoculation trials were carried out using diverse strains of root
nodule bacteria in order to identify strains suitable for inoculation in the
nursery. There were eight strains from Bradyrhizobium, five from Rhizobium,
three from Sinorhizobium and two Mesorhizobium strains. P. indicus formed
nodules with strains from Bradyrhizobium, Rhizobium, Sinorhizobium and
Mesorhizobium, which suggests it is a promiscuous host. Nodules formed
were globose, single and of aeschynomenoid type. In the first trial,
Bradyrhizobium strain WSM 2096, promoted shoot growth while in the second
trial, Bradyrhizobium strain WSM 3712 promoted shoot growth. Growth
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stimulation was similar to the uninoculated control supplied with inorganic N
as KNO3 but was inferior to plants given (NH4)2SO4.
The response of P. indicus to low soil P in inoculated and uninoculated plants
was studied in a pot trial comprising two P treatments (nil, just adequate).
Plants were grown for 3 and 6 weeks. At nil fertilizer P, uninoculated P.
indicus seedlings had higher total root length and root dry weight than those
with adequate P. Inoculation with WSM 3712 suppressed root growth relative
to uninoculated plants.
Information gathered in this thesis has application for the production of
planting stock in forest nurseries. Firstly, care needs to be taken to ensure that
rates of P supplied as hard or liquid fertilizer are not in the range likely to cause
toxicity. Secondly, if any starter inorganic N fertilizer is to be used then it
should be supplied either as ammonium-N or as urea. Thirdly, more research is
required in order to identify effective strains of rhizobia for widespread
commercial application. Fourthly, the critical foliar P concentrations identified
for the diagnosis of P deficiency and P toxicity can be used to help interpret
foliar analysis of seedlings in the future.
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Publications arising from this research
Lok E.H., O’Hara, G.W. and Dell, B. 2006. Nodulation of the legume Pterocarpus
indicus Willd. by diverse strains of rhizobia. Journal of Tropical Forest Science
18(3): 188-194.
It is anticipated that further publications will arise from this work.
VII
List of abbreviations
ANOVA Analysis of variance
CRS Centre for Rhizobium Studies at Murdoch University
DBH Diameter at breast height (1.3 m)
DMRT Duncan’s New Multiple Range test
FRIM Forest Research Institute Malaysia
INRA Institute national de la Recherche Agronomique, France
N Nitrogen
n Number of samples
P Phosphorus
PCR Polymerase chain reaction
R Rhizobium
R2
Correlated regression coefficients
RCBD Randomized complete block design
S.E. Standard error
Sp. Species (singular; spp., plural)
T Temperature ( oC)
WA Western Australia
WSM Western Australia Soil Microbiology
YB Yalanbee soil
YFEL Youngest fully expanded leaf
YMA Yeast mannitol agar medium
YS Yellow sand
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Acknowledgement
Foremost, I would like to express my sincere gratitude to Prof. Dr. Bernie Dell, for
his supervision, guidance, directing my thoughts to develop this thesis, endless
patience, constant encouragement, support and many critical comments throughout
the course of this study.
My immense gratitude and thanks also to Associate Professor Dr. Graham O’Hara
for his helpful suggestions, valuable guidance, generous assistance and critical
comments. These studies would not have been possible without his consistent advice,
guidance, kind encouragement and interest in the progress of the study. I am also
thankful and grateful to all my supervisors for their undivided support and assistance
extended to me and my family during our memorable stay in Australia.
This postgraduate study was made possible through the Malaysian Government
scholarship generously offered by the Ministry of Science, Technology and
Innovation (MOSTI) and Public Service Department (PSD) for which it is greatly
acknowledged. My special thanks to Dato’ Dr. Abdul Latif Mohmod, Director
General of Forest Research Institute Malaysia (FRIM) and Mrs. Rahmah Bt. Wan
Raof, Human Resources and Development (FRIM) for granting a study leave and
their support to pursue this study. Similarly, I am thankful to the Murdoch University
for the financial support in granting me a Murdoch University Studentship during the
final stage of my study.
Special thanks are due to Professor John Howieson and his team from the Centre for
Rhizobium Studies (CRS), Murdoch University for his permission to use the
Centre’s facilities, technical help and useful advice at the Centre’s laboratory. I
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would also like to thank Mr. Gordon Thomson for his superb histology works and for
sharing his microscopic laboratory facilities. My sincere thanks are also due to Mr.
Kim Tan, Max Dawson and Kose’ Minetto for their companionship and assistance
during my study at the glasshouse. A big thanks to Associate Professor Dr. Mike
Calver for his invaluable advice on statistical analysis during the process of the
study.
I am greatly appreciative to Ben Bradshaw, Bill Dunstan, Paul Baba and Alan Chen
YingLong for their kind assistance and companionship throughout the course of my
study. To my close friend, Carlos Raphael, thanks for ever willing helps, always
reminding and inviting me to eat regularly and the coffee break hour together
especially during the final stage of my writing.
I would like to extend my deepest gratitude and appreciation to my wife, Doris Loh
Lee Fah and my children, Vince Lok Xia Ren and Jessica Lok Xia Shin for their
love, prayers and sacrifices. Thanks also to my sisters and brother who never missed
calling to ask on my well-being. Last but not least, I would also like to dedicate this
work to my late father and mother for their love and sacrifices, who never lost faith
on me.
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Table of Contents
Declaration II
Abstract III
Publications arising from this thesis VI
List of Abbreviations VII
Acknowledgements VIII
Chapter 1 Introduction, literature review and thesis aims
1.1 Introduction 1
1.2 History of planted forests in Peninsular Malaysia 2
1.3 Pterocarpus and its background 3
1.4 Nutrition and symbiotic requirements of tropical woody legumes 14
1.5 Principles of plant analysis 18
1.6 Alternative approaches for determining nutrient disorders 21
1.7 N-fixing trees and root nodule bacteria 23
1.7 Conclusions 24
1.8 Aims of the thesis 25
Chapter 2 Diagnosis of phosphorus and nitrogen disorders in Pterocarpus
indicus seedlings
2.1 Introduction 28
2.2 Materials and methods 29
2.3 Results 35
2.4 Discussion 43
Chapter 3 The phosphorus response of Pterocarpus indicus seedlings in a
lateritic soil compared to three tropical tree legumes
3.1 Introduction 54
3.2 Materials and methods 56
3.3 Results 59
3.4 Discussion 74
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Chapter 4 Nodulation of Pterocarpus indicus by diverse strains of root nodule
bacteria
4.1 Introduction 77
4.2 Materials and methods 78
4.3 Results 83
4.4 Discussion 88
Chapter 5 Effective rhizobia for nodulation and nitrogen fixation on Pterocarpus
indicus seedlings
5.1 Introduction 92
5.2 Materials and methods 93
5.3 Results 94
5.4 Discussion 99
Chapter 6 Effects of phosphorus and root nodule bacteria on root growth in
Pterocarpus indicus seedlings
6.1 Introduction 104
6.2 Materials and methods 105
6.3 Results 107
6.4 Discussion 114
Chapter 7 General discussion 119
Appendices 127
References 133
1
Chapter 1
Introduction, literature review and thesis aims
1.1 Introduction
Plantations of timber and multiple-purpose tree species play an important role in
restoring soil productivity and ecosystem stability. They may also enhance biological
diversity for degraded tropical lands. The rapid population growth in developing
countries, especially in the tropics and sub-tropics, has resulted in many social and
economic problems that cause unabated need for food, fodder, energy and wood. It is
estimated that about 13 million ha/year of the tropical forests and woodlands are
being destroyed or seriously degraded through agricultural expansion, logging, land
clearing for development and fuel wood collection (Landly, 1982; Parrotta, 1996;
Wan Razali, 2003; FAO, 2005).
Tropical deforestation is often associated with the conversion of forest lands to
agricultural uses mostly to replace land that has lost productivity due to unsuitable
farming practices, shifting cultivation and illegal timber harvesting, among others
(Whitmore 1984; Harwood et al., 1993; Lamb, 1994). This is also true in Malaysia
where the major causes of deforestation and forest degradation are over-logging and
the conversion of forests to agricultural plantations (Appanah and Ismail, 1996;
Nambiar and Brown, 1997; Appanah and Razak, 1998). Currently, most of the
remaining forests are confined to the fragile hilly parts of the country and the vital
watershed areas (Baharuddin and Chin, 1986; Bordeleau and Prevost, 1994).
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Plantations have now been recognized as integral parts of sustainable forest
management and complementary to natural forests (Darus and Radhuan, 1990). In
order to overcome the anticipated shortage of raw materials for the wood, pulp and
paper industries in Southeast Asia, plantations of both indigenous and fast-growing
exotic species are being widely exploited and much basic research is needed to
underpin these developments. Among the popular species used in the region,
including Malaysia, are Acacia mangium, Azadirachta excelsa, Dyera costulata,
Endospermum malaccensis, Eucalyptus spp., Gmelina arborea, Hevea brasiliensis,
Khaya ivorensis, Neolamarkia cadamba, Octomeles sumatrana, Paraserianthes
falcataria, Swietenia macrophylla and Tectona grandis. Other promising indigenous
species, such as dipterocarps and Pterocarpus indicus are also being considered
(FRIM in Focus, 2002; Krishnapillay and Appanah, 2002; MTIB, 2007). The later
species P. indicus is the subject of this thesis.
1.2 History of planted forests in Peninsular Malaysia
Planting of timber trees in Peninsular Malaysia started with the commercial planting
of Hevea brasiliensis (rubber) for latex in the late 1800s. A brief history of artificial
regeneration of rubber and forest plantation in Peninsular Malaysia has been
described by Appanah and Weinland (1993). By the end of 1999, a total of 1,700 ha.
model rubber plantations had been established at several locations in Peninsular
Malaysia. These include some of the promising clones tested like RRIM623,
RRIM900s series, PM10, PB235, PB260 and the newly launched RRIM2000s
clones. Some of these clones are expected to yield between 1,500-3,000 kg of latex
annually and with wood volume of between 0.75 to 1.3 m3 per tree after 15 years.
These figures are about 300% higher as compared to the older clones that are
currently being used.
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Meanwhile, the search for other fast-growing hardwood species continues. Several
potential exotics and indigenous species have been identified for short-rotation tree
crops to produce general utility timber (Darus et al., 1990). In 1982, the
Compensatory Forest Plantation Project (CFPP) was launched in the states of
Pahang, Johor, Selangor and Negri Sembilan with a projected target of 188,000 ha.
However, due to the widespread occurrence of heart rot disease in Acacia mangium,
only about 50% of the targeted area was achieved by the end of 1999 (Hashim et al.,
1996). Heart rot has led to increased interest in other forest species (Forest
Department Annual Report, 2001; MTIB, 2007). Furthermore, data obtained in a
survey throughout Peninsular Malaysia indicate that there has recently been a
considerable increase in the area of forest plantations of 75,672 ha resulting from
some of the recommendations made by FRIM in 1996 and 1997 (Krishnapillay and
Ong, 2003).
1.3 Pterocarpus and its background
The genus Pterocarpus, a close relative of Dalbergia, belongs to the family
Leguminosae-subfamily Papilionoideae (Fagaceae) and is the largest family of
flowering plants (Polhill, 1981). It produces many beautiful, highly decorative
timbers that are rank among the finest luxury timbers. Among them is Pterocarpus
indicus Willd. which is locally known as narra in the Philippines, sonokembang or
angsana in Indonesia, pradoo or padauk in Thailand, rosewood in Papua New
Guinea, nanara in Vanuatu, liki in Solomon Islands and sena or angsana in Malaysia,
Brunei and Singapore (Corner, 1988). It has the status of National Tree in the
Philippines and is a favourite timber for the manufacture of fine, high quality
furniture. Due to its scarcity and very limited timber supply from planted trees, many
native stands are now fast disappearing. In Singapore, it was part of the country‟s
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garden city planting program with many avenues graced by this attractive species,
and in Malaysia it has been used as a shade tree for over 200 years (Wong, 1982;
Corner, 1988; Ng, 1992). More recently, this species has been planted along roads in
West Kalimantan, Indonesia (Setiadi, 1995). The first extensive planting of the
species was recorded in 1802 by the British in Penang and earlier in Malacca in 1778
(Wee and Corlett, 1986). Since then, systematic planting has been carried out
especially in Singapore, and in some of the states of Malacca and Penang. The
National Academy of Sciences (NAS, 1979) concluded that despite their commercial
importance, Pterocarpus species had been almost entirely overlooked by science and
that research on all Pterocarpus species, including P. indicus, was important (Ang,
1988; Munyanziza and Oldeman, 1994; Valencia and Umali-Garcia, 1994; Desmet et
al., 1996; Effendi et al., 1996).
1.3.1 Biology and silviculture of Pterocarpus indicus
a) Natural distribution and habitat of Pterocarpus
The genus Pterocarpus is pantropical and comprises of 20 species with 11 in tropical
western Africa, 5 in the Indo-Pacific region and 4 in tropical South America (Rojo,
1977). Most of these species are large trees with the African and Asian species
producing high-quality wood. Two main species of Pterocarpus dominate in
Southeast Asia and some Pacific islands, namely a) Pterocarpus indicus that is native
to the Philippines, Malaysia, Indonesia, Vanuatu, Solomon Islands and Papua New
Guinea and b) Pterocarpus macrocarpus which is native to Myanmar, Thailand,
Laos, Cambodia and Vietnam. There are nine species which are commercially
valuable while six other species are listed as threatened for being over-exploited.
These include P. indicus and P. santalinus. P. santalinus is sometimes called red
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sandalwood of India and its international trade is listed under International Union for
Conservation of Nature and Natural Resources (IUCN, 2006) as Red List of
Threatened Species except for P. santalinus which is classified in Appendix II under
the Convention of International Trade in Endangered Species (CITES) (Rojo, 1977;
Oldfield et al., 1998) (Table 1.1). The species showed that it may only be traded
internationally if accompanied by appropriate permits.
Due to its wide geographical distribution, there is considerable morphological and
ecological variation in P. indicus. It is possible that trees planted at any given locality
tend to be uniform because of extensive local clonal propagation (Rojo, 1977; Ng,
1992). However, due to over-exploitation and selected felling in Peninsular
Malaysia, P. indicus is no longer naturally available except for some relic trees that
are confined to a few states of Penang, Melaka, Port Dickson (Negri Sembilan),
Rompin (Pahang) and Taiping (Perak) (Ridley, 1922; NAS, 1979; Wong, 1982;
Wong, 1983; Corner, 1988; Soerianegara and Kartawinata, 1985).
In general, P. indicus is found at low to medium altitudes (up to 750 m) but seems to
thrive up to 1300 m in primary and secondary forests. It grows on a variety of soil
types from fertile agricultural to rocky soils and along inundated river banks, swamps
and lagoons (Allen and Allen, 1981; Corner, 1988; Ng, 1992). The actual habitat, the
associated tree species, occurrence, and soil conditions required in which it can grow
well have not been studied in Malaysia in contrast to Pterocarpus macrocarpus in
Thailand (Dhanmanonda, 1992; Liengsiri et al., 1998).
b) Botanical description
Pterocarpus indicus is a large, deciduous tree growing up to 33 m tall with a
maximum diameter of 7.5 m at breast height. Leaves are shed during the monsoon or
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hot dry season (February to April). The bole is usually short with small thick
buttresses and a spreading crown of drooping twigs and foliage. Large buttresses,
however, sometimes occur and have a well developed near-surface lateral rooting.
Crowns are large and bear many long branches that are at first ascending but
eventually arch over and sometimes droop at the ends. Trees with long, willowy,
drooping branches are particularly conspicuous and attractive for playgrounds and
roadside planting (Figure 1.1). Open-grown trees tend to be buttressed and branching
with poor bole form. However, the bole form may improve if trees are planted with
close spacing and with other silvicultural treatments such as pruning and fertilization
(Ng Lian Teck, pers. comm.).
Table 1.1 Commercial Pterocarpus species, distribution, common name and uses
Species/Region Distribution Local name Use
Asia: 5 species
dalbergiodes South Asia Andaman
padauk
Highly sought hardwood
indicus South East Asia Narra, sena,
angsana
Classic furniture
macrocarpus Myanmar and
Thailand
Burma padauk Luxury timber, furniture
marsupium India and Sri Lanka - Important after teak
santalinus India Red
sandalwood
Fragrant wood, substitute
for Santalum album
Africa: 4 species
angolensis South Africa Muninga Furniture and decorative
erinaceus Senegal - Furniture and decorative
osun and soyauxii Nigeria to Zaire African padauk Furniture and decorative
Source: Modified from Rojo (1977)
The bark is smooth, pale grey on young trees, later becoming rough, dark brownish
grey and longitudinally cracked about 1 cm thick. The inner bark is yellowish, tinged
with pink and when cut, the bark exudes a bright, dark red resin which is believed to
have medicinal values (Burkill, 1935). The leaves are compound-pinnate with 6-12
alternate leaflets. The leaflets range from 7 x 3.5 to 11 x 5.5 cm, ovate to elliptic in
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Figure 1.1 Mature trees of Pterocarpus indicus that are commonly planted at (a) an
urban playground and (b, c) along roadsides for recreational and amenity purposes
in Peninsular Malaysia.
(a)
(b)
(c)
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shape with pronounced acuminate tips. In areas with seasonal climate, P. indicus is
noted for its gregarious flowering as this is stimulated by a sudden drop in
temperature caused by a heavy downpour on a hot day (Holttum, 1954; Wong, 1982;
Corner, 1988). This synchronous flowering ensures good cross-pollination. The
flowers are yellow, fragrant and borne in large axillary panicles. The fruit is a
compressed, indehiscent samara, disk-like or sometimes falcate, broadly winged with
a thickened central, usually woody or corky, seed-bearing central portion containing
1-2 seeds (Figure 1.2 a, b). The seed is kidney-shaped, with a smooth to undulating,
brown to blackish seed coat (Figure 1.2c). It takes about 3-4 months to mature.
Unlike many legumes, the fruit is indehiscent and is dispersed both by wind and
water. A good description of the species is given by Claveria (1952), Assidao and
Nastor (1961), Corner (1988), Ng (1992), Appanah and Weinland (1993).
c) Wood properties and utilization
The sapwood is light yellowish brown to grey and generally quite narrow. The
heartwood is bright yellowish red to golden brown. The heartwood is lustrous when
first exposed but ages to a dull yellowish hue. The wood is classified as moderately
heavy, hard, strong and durable (550-900 kg/m3 at 15% moisture). Texture is
moderately fine to moderately coarse with grain sometimes wavy and interlocked.
The wood is ranked as the finest for furniture, paneling, cabinet work, carvings and
tool handles while other known uses include decorative sliced veneer, interior wall
paneling, feature flooring (including strip and parquet), musical instruments, boat
building and specialized joinery (Eddowes, 1977).
In the Philippines, the export of narra wood has declined substantially from 3 million
kg in 1985 to 2.3 million kg (57% processed) and 430,000 kg (processed) in 1986
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and 1987, respectively. However, a total ban on the felling of P. indicus has been
imposed in the Philippines due to decline in the resource. The species has now been
classified as vulnerable in CITES, Appendix 2. Following this ban, countries like
Papua New Guinea, the Solomon Islands and Vanuatu have also banned round-wood
for export of this species. However, processed wood is allowed for export. The price
for sawn boards was estimated to be about US $600-800/m3 FOB (Evans, 2000;
Grace, 2000; FAO, 2001).
1.3.2 Silvicultural characteristics
a) Seed germination
Germination is epigeous with one fruit producing one to three seeds. There are about
2450 seeds available in every 100 g of seeds. Seed is preferred over dewinged,
scarified fruit for sowing because of the shorter germination time (1-2 weeks versus
>2 months) and a more uniform and higher germination rate (>85%). An optimum
temperature range of 25 to 35 oC is recommended for both P. indicus and P.
macrocarpus (Hundley, 1956; Liengsiri and Hellum, 1988; Coles and Boyle, 1999)
but in the Forest Research Institute of Malaysia (FRIM), seeds of P. indicus sowed at
this temperature range can give a germination rate of between 75-85% without any
pre-treatment (Nor Asma, pers. comm.).
b) Natural regeneration
It has been suggested that P. indicus behaves like a pioneer species and grows best
in the open although some shade is required in the early stage of establishment (Ng,
1992). This also applies to other species of Pterocarpus such as P. macrocarpus in
Thailand (Piewluang et al., 1997). For instance, an initial study by Piewluang (1996)
showed that germination, survival and height growth of P. macrocarpus seemed to
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improve as conditions become more open and less competitive. As a rule, Troup
(1921) indicates that natural regeneration is reported to be good only in rather dry,
open forest but is scant or absent in more moist forest where the vegetation cover is
dense. However, this appears to be anecdotal as no inventory data are available. In
Myanmar, natural regeneration of P. macrocarpus was reported to be poor and this
may be due to the tough fibrous outer covering of the fruit and the hard testa as
germination was only 40-50 % (U Saw Yan, 1993).
c) Artificial regeneration
Early attempts at establishing plantations of Pterocarpus had mixed success. For
example, Troup (1921) reported that attempts to establish a P. macrocarpus
plantation by direct sowing pods or planting nursery-raised seedlings have generally
been unsuccessful. The establishment of a 60 years old P. indicus trial in FRIM also
failed to perform well due to high mortality (Ang, 1988). This failure may be due to
a lack of understanding of the silvicultural requirements of the species including site
preference. While many of the early trials were poorly managed, it became evident
that young seedlings were not competitive with Imperata or other tall grasses
suggesting that regular weeding activities are necessary.
d) Vegetative propagation
Vegetative propagation by cuttings is generally a preferred way for ornamental and
roadside plantings because growth of seedlings is slow and there is considerable
variation in vigour (Wong, 1982). Clonal material through vegetative cuttings also
provides a mean of perpetuating desirable characters such as clear, straight boles
suitable for timber production. P. indicus is also unique among the big timber trees
as it has the capacity to root easily in different soil types and rooting capacity is not
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lost with age (Claveria, 1952; Assidao and Nastor, 1961; Allen and Allen, 1981;
Wong, 1982). In urban and some reforestation activities, cuttings are used to produce
„instant shade‟ and fast-growing trees when transplanted with a high survival rate of
90% in the Philippines (Claveria, 1952; Assidao and Cerna, 1960; Sardina, 1961;
Wong, 1982). Hence, to ensure that cuttings propagate easily, stem size of more than
3 m long and 5 to 10 cm diameter is normally used. Cuttings should be procured
from a straight, healthy branch that comprises several nodes with preference for
young woody branches (Browne, 1955; Wong, 1982).
e) Growth and yield
There is very little published data on the growth and yield of P. indicus and these are
summarized in Table 1.2. It has been suggested (Evans, 1982) that early maintenance
of P. indicus is necessary to improve plant growth in the field and that initial
inorganic fertilizer application using 50 gm of 15:15:15 (NPK) is recommended one
month after planting, followed by 100 g after every six months. Pruning also plays an
important role in the silvicultural treatment of P. indicus as undesirable branches
should be removed to prevent knot formation which may affect growth and wood
quality (Evans, 1982).
f) Pests and diseases
P. indicus is a fairly disease and pest resistant tree species. A problem occurred in the
period 1875 to 1925 which was attributed to leafhopper transmission of a fungal
pathogen (Corner, 1988). Other related pests include the leaf miner, Neolithocollettis
pentadesma which in 1990 caused severe leaf defoliation to trees planted in the open
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Figure 1.2 Fruits and seeds of Pterocarpus indicus: a) green and b) dried winged
fruits and c) extracted mature seeds (scale bar shows size of seed).
(a)
(b)
(c)
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and along roadsides throughout Peninsular Malaysia (Ahmad et al., 1996) and
Crematopsyche pendulla that caused severe damage to leaves resulting in necrosis
and holes (Ahmad, 1993).
Table 1.2 Growth data of Pterocarpus indicus recorded for isolated individuals or
in plantations.
Note: GBHOB = Girth at breast height over bark and DBHOB = Diameter at breast
height over bark . * unknown age. Source: (Lok, 1996).
1.3.3 Gaps in knowledge
There is an urgent need for studies on the nutrition requirements of P. indicus
primarily on phosphorus (P) supply, given that the species will be introduced to
either poor or marginal soils in Peninsular Malaysia. There is also a need to explore
the symbiotic relationship with nitrogen (N)-fixing bacteria given that the conversion
of secondary forests into plantations is likely to result in a loss of soil microbial
diversity. Constraints due to P and N are also likely to be exacerbated on severely
compacted sites such as skid trails and log landings where all vegetation and top soil
Age
(yr)
Total height (m) GHOB (m) DBHOB (cm) Author
5 8 0.3 - NAS (1979)
60 7.1 0.5 - Ang (1988)
100 27 4.6 - Wong (1982)
* 36.4 3.6 12.6 Browne (1955)
* - 1.0-1.6 2.4-4.4 Nwoboshi (1982)
* 15.2 - - Troup (1921)
* 30.3 - - Corner (1988)
* - > 4 - Foxworthy (1927)
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has been removed. If P. indicus plantations are to be set up, good nursery and
silvicultural practices will have to be developed for the planting stock and nutrition
could be a key factor in their success. Again, there is little information on nursery
and silvicultural practices for P. indicus.
1.4 Nutrition and symbiotic requirements of tropical woody legumes
a) Role of phosphorus and nitrogen on tree growth
Most tropical forest plantations are likely to be established on either poor or marginal
sites where essential nutrients in soils for tree growth are low or poorly available
(Holford, 1997; Evans and Turnbull, 2004). For example, P has been identified as
one of the major limitations to tree growth on reforestation sites but little information
is available on the response of tropical plantation timber species to P fertilizers
(Gobbina et al., 1989; Goncalves et al., 1997; Tiessen, 1998). In the tropics,
deficiencies of many macro and micronutrients can result from soil leaching
(Stoorvogel et al., 1993; Goncalves et al., 1997; Sanchez et al., 1997). Phosphorus
and nitrogen have been identified as the main limiting macronutrients that affect
plant growth, particularly biomass accumulation (Begon et al., 1990; Shepherd et al.,
1996). Deficiencies in P and N may also lead to poor bole form, reduced microbial
activity and increased susceptibility to pests and disease during the nursery stage and
in young plants after out-planting (Kadeba, 1978; Kanowski et al., 1991;
McLaughlin, 1996; Norton and Firestone, 1996; Otsamo, 1996; Goncalves et al.,
1997; Kaye and Hart, 1997; Dell et al., 2001; Xu et al., 2001 ). Hence, a critically
short supply in these nutrients can lead to the failure to sustain tree growth. Not
surprisingly, fertilization is a consideration in many industrial tree estates in SE Asia.
An example of the responses that can be achieved with the addition of fertilizer is a
15
study carried out in Papua New Guinea on some indigenous tree species in a
degraded grassland using NPK (17:5:22) at 500 kg/ha (Table 1.3).
Historically, the use of fertilizer in forestry has been less emphasized than in
agriculture due to the slow growth of trees, long gestation period, uncertain
economic returns and use of species that are adapted well to low P sites (Evans,
1992). Applications of P are sometimes necessary to ensure early growth rates on
some soils such as with conifers (Ballard, 1974) (see also Table 1.3), or to shorten
harvest rotations (Evans, 1992). In south China, P fertilization has been regarded as
essential to ensure growth for fast-growing eucalypts in plantations (Wang and Zhou,
1996; Xu, 2001).
Most P fertilizer fixed by soil components is converted into forms which are not
readily available to plants (Grove and Keraitis, 1976; Subba Rao, 1982). Several
biological methods of enhancing nutrient uptake may reduce the amount of inorganic
fertilizer required for optimum growth. Legumes are known to have a high P
requirement for optimum growth, nodule formation and nitrogen fixation (Mosse et
al., 1976). Besides direct uptake of these nutrients, beneficial soil symbionts such as
arbuscular mycorrhizal fungi (AMF) and rhizobia (root nodule bacteria) can play an
important role in P and N cycling (Craswell et al., 1986; Brundrett, 1991). The
contributions derived from these symbioses by plants can also contribute to minimize
the use of fertilizers (Barea and Azcon-Aguilar, 1983). Dual symbioses are also
known to significantly increase the uptake of nutrients and improve the production of
seeds compared to plants that are singly treated with either one of the symbionts
(Barea and Azcon-Aguilar, 1983; Ikram et al., 1987; Tian et al., 2003).
16
For N, the largest contribution of biological N2-fixation in land plants is derived from
the symbiosis between legumes and root nodule bacteria (Burris and Roberts, 1993).
A significant proportion of these are tropical tree species whose relationship with
root nodule bacteria continue to be widely studied (e.g. Allen and Allen, 1981;
Moreira et al., 1992; Oyaizu et al., 1993; Odee et al., 1995; Sprent, 1995; Sprent and
Parsons, 2004; Sprent et al., 2010). Nitrogen-fixing bacteria make N directly
available to plants as NH3 in symbiotic associations using the nitrogenase enzyme in
bacteroids of root nodules (Bergersen et al., 1988; Giller and Wilson, 1991).
Table 1.3 Comparisons between height increment for P in unfertilized and fertilized
four-years old stands in Kunjingini, Papua New Guinea (Extracted from Lamb,
1975).
Species
Height increment without
fertilizer (m)
Year 1 Year 4
Height increment with
fertilizer (m)
Year 1 Year 4
Acacia auriculiformis 1.77 3.99 2.68 9.66
Eucalyptus deglupta 0.79 1.49 3.67 7.65
Eucalyptus tereticornis 0.94 2.04 2.77 4.78
Intsia bujuga 0.58 0.36 0.49 0.97
Pterocarpus indicus 0.67 0.73 1.31 1.61
Tectona grandis 0.21 dead 2.60 3.04
Terminalia brassii 0.52 1.13 2.50 4.69
Terminalia complanata 0.40 dead 0.76 1.34
b) Nutrient functions
Phosphorus
Phosphorus is essential for plant growth as it is involved in most metabolic
processes. It is a constituent of nucleic acids, phospholipids, phosphoproteins,
phosphate esters, dinucleotides and adenosine triphoshate. It is required for the
storage and transfer of energy, photosynthesis, electron transport processes,
17
regulation of some enzymes such as the synthesis of sugars and starch and the
transport of carbohydrates. Since P is phloem mobile, purple symptoms of P
deficiency are normally first observed in old leaves (Dell et al., 2001).
Nitrogen
Nitrogen is a constituent of amino acids, amides, amines, N bases, nucleic acids,
alkaloids, chlorophyll and many coenzymes. It is also a structural component of cell
walls in plants. The main symptom of N deficiency is leaf chlorosis which is due to
reduced chlorophyll formation or enhanced senescence. Symptoms typically first
appear in mature leaves and then spread to the whole canopy. In young eucalypt
seedlings, the symptoms usually occur in young leaves (Dell et al., 2001). Since
amino acids are also precursors of the polypeptide chains of proteins, N also
influences many enzyme reactions. Inorganic N is absorbed by roots as nitrate or
ammonium forms depending on the preference for some species and iron availability
(Marschner, 1995). For example, E. globulus seedlings utilize ammonium more
effectively than nitrate and are susceptible to nitrate toxicity when moderate to high
levels of nitrate-containing fertilizer are used (Shedley et al., 1995).
c) Diagnosis by symptoms
Where symptoms are specific, diagnosis by visual symptoms is a fast and simple
indicator for nutrient status in plants. Often, diagnosis of nutrient disorders by visual
assessment is applied together with soil analysis and plant tissue analysis due to
insufficient specific symptoms being expressed and other limitations. Details of
commonly encountered limitations have been described by Ulrich and Hills (1967),
Dell and Robinson (1993), Dell and Malajczuk (1994), Grundon et al. (1997),
Uchida (2000) and Wong (2005).
18
1.5 Principles of plant analysis
Limitation in using visual symptoms to diagnose nutrient deficiencies has resulted in
plant analysis being the preferred means of delineating the deficiency and sufficiency
levels of plants. This is because concentrations of nutrients in plant tissue are
determined by the combined effects of all the processes affecting the supply of
nutrients in soil and uptake by roots.
Principles were originally defined by Ulrich (1952) and Ulrich and Hills (1967)
using field crops but they were later applied to woody plants (Chapman, 1967;
Brown, 1970; Ballard, 1980; Dow and Roberts, 1982; Lambert, 1984; Mead, 1984;
Weeman and Wells, 1990; Paudyal and Mjid, 1992; Shedley et al., 1995; Timmer,
1997; Orlander, 1998; Dell et al., 2001; Landis et al., 2004). Using the relationship
between nutrient concentration and plant yield, commonly shoot biomass (Smith,
1986), critical concentrations have been established for many economic field crop
species, but is less certain for timber species. The generalized relationship between
plant yield and nutrient concentration is shown in Figure 1.3 which shows four
zones:
1. Deficiency zone where with small changes in plant nutrient concentration,
plant growth increases sharply. The slight increase in nutrient concentration
may be attributed to the fact that for each additional increment of nutrient
absorbed by the plant, there is a corresponding increment of additional
growth;
2. Marginally deficiency zone where the increase in yield slows as the nutrient
concentration increases;
19
3. Adequate-luxury supply zone where an increase in nutrient markedly
increases the nutrient concentration in the plant but has no effect on its
growth, this suggests that the added nutrient is no longer the factor that limits
growth; and
4. Toxicity zone where an increase in nutrient supply increases nutrient
concentration in the plant to a toxic level causing a decline in plant growth.
One variation in the general calibration curve is known as the “Piper-Steenbjerg”
effect or “C-shaped” curve (Piper, 1942; Steenbjerg, 1951) which results when the
addition of a limiting nutrient to a severely deficient plant increases yield but
decreases nutrient concentration (Figure 1.3, lower). Plant analysis therefore, has
very limited value for diagnostic purposes unless the “C-shaped curve” can be
avoided. Ulrich and Hills (1967) suggested that this effect can be minimized by
sampling when early visible symptoms first appear. Alternatively, the “Piper-
Steenbjerg” effect may sometimes be avoided by sampling specific parts for nutrient
analysis. For example, by contrast with the whole shoot, young leaves of Cu-
deficient wheat, subterranean clover and peanut showed no “Piper-Steenbjerg” effect
(Robson, 1977; Loneragan et al., 1980; Reuter et al., 1981).
The diagnosis of nutrient deficiencies from plant analysis is based on the concept of
“critical nutrient concentration”. Ulrich (1952) has suggested that the critical
concentration of nutrient deficiency should be defined as the nutrient concentration
that is just deficient for maximum growth, adequate for maximum growth or the
concentration separating the zone of deficiency from the zone of adequacy and this
concentration is often taken at 5 or 10% below the maximum yield. He also
concluded that plant parts should be chosen in the narrow transition zone between
deficiency and luxury concentrations. Others have suggested that the critical
20
concentration should be in a range rather than a single value (Follet, 1965; Dow and
Roberts, 1982).
The relationships shown in Figure 1.3 are usually determined by growing plants with
a range of nutrient levels of the element being examined with all other essential
elements being adequate. For annual species, experiments may be undertaken in the
field or in pot culture. For long-lived tree species, it is much more difficult to obtain
field data to plot the relationship in Figure 1.3, and for this reason effort has
generally been placed on seedlings or cuttings. Whether plants can grown in the field
or in a greenhouse, harvest and chemical analyses are carried out, when there are
deficiency symptoms at the lower treatment or visible growth response is evident.
Figure 1.3 Relationships between nutrient concentration in plant tissues and growth
(Upper: Smith and Loneragan 1997; Lower: Ulrich and Hills, 1967).
21
The most common procedures for deriving the critical concentration of a given
nutrient have been the hand-fitted curve, the Mitscherlich equation (Ware et al.,
1982), the graphical and the statistical Cate Nelson procedures (Cate and Nelson,
1971), and the Smith and Dolby two phase linear model (Smith and Dolby, 1977).
However, the selection of a defined point on a hand-fitted curve has been the most
common method for deriving a critical nutrient concentration in early studies (Smith,
1986). The two main criticisms of the latter method are the subjective assessment
involved in the location of the curve through the points especially in the marginal
zone and the arbitrary use of 90% maximum yield to determine the corresponding
critical concentration.
1.6 Alternative approaches for determining nutrient disorders
Although plant analysis is the common method for nutrient deficiency diagnosis in
plant tissues, other methods have been applied with varying success. These are
briefly described below.
a) Comparison of healthy and unhealthy trees
Comparing nutrient concentrations in healthy and unhealthy plant tissues is a quick
and common way for preliminary assessment of nutrient disorders. Difficulties may
arise in the following situations: i) changes in severity of nutrient deficiencies with
plant age, ii) presence of symptoms caused by non nutritional factors, iii) similarity
of some symptoms associated with different nutrients, and iv) multiple effects from
multiple nutrient disorders. The disadvantage of using unhealthy tissue is that
symptoms may only appear when the nutrient supply to roots has markedly reduced
growth or caused stem malformation (Lambert, 1984; Mead, 1984; Dell, 1996;
22
Uchida, 2000; Dell et al., 2001). Detail on the sampling of plant tissues has been
described by Dell et al. (2001) for the diagnosis of nutrient disorders in eucalypts.
b) Nutrient ratios
This concept called Diagnostic and Recommendation Integrated System (DRIS) was
first developed by Beaufils (1957; 1971; 1973) and is described by Walworth and
Sumner (1987). The method involves measurement of the ratio of two or more
nutrients within a plant tissue and comparison with a predetermined range where
plant growth will not be limited. For example, using this method Schaffer (1988)
found that a combination of both manganese (Mn) and iron (Fe) are responsible for
the decline in mango yield.
Unlike critical nutrient concentrations, DRIS is a predictive rather than diagnostic
technique and thus may not be universally useful for plant analysis in forestry
practices (Smith and Loneragan, 1997). DRIS has been applied to identify yield-
limiting nutrients, sensitivity and long-term variations of different doses of
fertilization practices in fruit and other cash crops such as corn, mango, maize,
pineapple, sugarcane and sweet potato (Beaufils, 1971; Elwali and Gascho, 1983).
c) Biochemical tests
Biochemical techniques are more commonly applied in agricultural and horticultural
crops for nutrient-status of plants but have little application so far in forestry.
However, biochemical tests for P and Cu in eucalypts have been suggested (Dell,
1996). Biochemical tests are mostly based on the activity of enzymes which require
essential elements for normal functioning. For example, phosphatase activity in plant
tissues of E. diversicolor seedlings was shown to be a more useful indicator of P
status than foliar P concentrations in a glasshouse study (O‟Connell and Grove,
23
1987) and a lignin stain has been used to distinguish between Cu-deficient and Cu-
adequate E. maculata trees (Dell and Bywaters, 1989; Dell, 1994).
Unfortunately, none of these tests are presently being used commercially in forestry.
Biochemical assays may be better than nutrient analysis for predicting the nutrient
status of a plant where significant amounts of nutrients are held in physiologically
inactive forms (Leece, 1976) but with the lack of specificity and complexity in their
measurements they are seen as disadvantages.
1.7 N-fixing trees and root nodule bacteria
Quite a number of commercial timber species are N-fixing trees and these include
genera like Acacia, Casuarina, Dalbergia, Paraserianthes and Pterocarpus.
However, despite their abundance, diversity and economic importance as prized
timber species, few have been examined for their ability to nodulate and fix
atmospheric N symbiotically with root nodule bacteria (Allen and Allen, 1981; de
Faria et al. 1989; Wester and Hogberg, 1989; Sprent, 2006). Not only do some of
these species have potential for hardwood plantations but they could play an
important role as pioneer species in the rehabilitation of degraded and over-exploited
forests (Sprent, 2001). Generally, plantations of N-fixing trees are known to have
higher rates of nutrient cycling than plantations of non-fixing trees but little is known
of the long-term influence on soil properties (Binkley et al., 2000).
The N-fixing symbionts normally gain access to carbon substrates, nutrients and
protection from desiccation in return for fixing atmospheric N which is transferred to
the host plant for further processing into amino acids and proteins (Long, 1996;
Waters et al., 1998). This symbiotic relationship is at an advantage where N is a
limiting nutrient for plant growth, such as in many tropical ecosystems. Tropical
24
woody legumes, especially in the sub-family Papilionceae, are capable of nodulating
with local soil root nodule bacteria but little is known about the specificity, symbiotic
relationships, soil type preference or the capacity to fix N, especially in P. indicus
and Dalbergia spp. (Allen and Allen, 1981; de Faria, 1989; Moreira et al., 1998;
Roggy and Prevost, 1999; Sprent and Parsons, 2000; Sprent, 2001; Diabate et al.,
2005). According to de Faria et al. (1989) and Sprent (1995), only 57% and 20% of
the genera and species respectively in the tropical legumes have been examined but
poorly studied.
Planting of tropical woody legumes is normally integrated with agro-forestry
practices (Brewbaker, 1983). They are used as nurse or shade trees and in land
reclamation (Hegazi et al., 1994). Favoured species include Acacia mangium,
Leucaena leucocephala, Paraserianthes falcataria, Parkia javanica and Tamarindus
indica (Hashim et al.,1996). However, there is still a lack of knowledge regarding
tropical woody legumes. Attention is being focused on their role in nutrient cycling,
in their usefulness in reforestation projects, potential uses in wood and non-wood
products, and capacity to become invaders of new habitats such as Australian acacias
in South Africa and in some Asian regions (Allen and Allen, 1981; Brewbaker et al.,
1982; de Faria et al., 1987; Wilson and Tilman, 1991; Witkowski, 1991a, 1991b;
Berger, 1993; Streeter, 1994; Sprent, 1995; Unkovich et al., 1997; Dart, 1998;
Chang and Handley, 2000).
1.8 Conclusions
There is an urgent need for studies on the nutrition requirements of P. indicus
primarily on phosphorus supply, given that most forest plantation trees will be
introduced on rather poor and marginal soils in Peninsular Malaysia. There is also a
25
need to explore the symbiotic relationship between N-fixing bacteria given that the
conversion of secondary forests into plantations is likely to result in a loss of soil
microbial diversity (Hau and Corlett, 2003). More severe impacts are likely on most
high impacted sites such as skid trails and log landings where all vegetation and most
of the top soil have been depleted. If P. indicus plantations are to be set up, good
nursery practices will have to be developed for the planting stock and nutrition could
be a key factor. Again, there is little information on nursery practices for P. indicus.
1.9 Aims of the thesis
Pterocarpus indicus is a promising tree legume species with fast growth rates,
desirable characteristics for plantation forestry and an important tree for high quality
timber. However, plantations of this species are in their infancy although serious
attention has been given to the species as a result of over-exploitation in its natural
habitat. The silviculture requirements of P. indicus, including fertilizer requirements
and foliar nutrient standards of seedlings at the nursery stage or at field
establishment, are poorly understood. The capacity to form an effective symbiotic
relationship with root nodule bacteria and mycorrhizal fungi are also highly
recommended given the likelihood that most forest plantations in the tropics are
likely to be established on sites with silvicultural constraints. Hence, the overall aim
of this study is to provide a scientific basis for diagnosing nutrient deficiency in
order to recommend on fertilizer use and application of effective N-fixing symbionts
so as to optimize nursery operations and productivity of Pterocarpus indicus after
out-planting.
26
The specific objectives are:
a) Examine the sensitivity of P. indicus to changes in external P supply and identify
foliar nutrient concentrations that define the P status of seedlings under nursery
conditions;
b) Identify effective strains of root nodule bacteria for nodulation and N-fixing
capabilities;
c) Document responses in P. indicus roots at low external P supply; and
d) Provide recommendations on management of P and inoculation of P. indicus in
tropical nurseries.
Figure 1.4 shows the structure of the thesis in relation to these objectives.
27
Figure 1.4 shows the structure of the thesis in relation to these objectives.
Identifying
limiting
nutrients
Changes in root
morphology
Major nutrient constraints on growth,
diagnosis and symbiosis effects with root
nodule bacteria in nitrogen-fixing Pterocarpus
indicus (Chapter 1)
Identifying
limiting nutrients
Lack of information
on effective root
nodule bacteria
Identify effective root nodule
bacteria for nodulation and
nitrogen-fixation
(Chapter 5)
General discussion and recommendations (Chapter 7)
Diagnosis of P and N
deficiencies using
plant analysis
(Chapter 2)
Root nodule
bacteria for
nodulation
(Chapter 4)
Changes in root
morphology due to P
supply and root
nodule bacteria
(Chapter 6)
Comparison of P response to
3 other leguminous tree
species
(Chapter 3)
28
Chapter 2
Diagnosis of phosphorus and nitrogen disorders in
Pterocarpus indicus seedlings
2.1 Introduction
Pterocarpus indicus, is a potential tree species for forest plantation establishment in
Peninsular Malaysia (Soerianegara and Lemmens, 1993; FRIM 2002). It is expected
that constraints on macronutrient soil fertility may limit productivity (Holford, 1997;
Evans and Turnbull, 2004). Deficiency of phosphorus (P) or nitrogen (N) can
severely restrict tree growth rates, leading to poor bole form and may increase
susceptibility of trees to pests and diseases (Kadeba, 1978; Kanowski et al., 1991;
Otsamo, 1996; Goncacalves et al., 1997). Hence, in order to achieve satisfactory tree
growth these nutrients must be adequately supplied. In addition, appropriate
silvicultural operations and good forest management practices are necessary to
maintain soil fertility, reduce soil erosion and conserve water through subsequent
rotations so that the productive potential of the site can be maintained. Appropriate
forest plantation management techniques also favour the maintenance of biological
diversity (Troup, 1921; Bunyavejchewin, 1983; Sprent et al., 1989; Moreira et al.,
1992; Marschner, 1995; Odee et al., 1995; Oldfield et al., 1998; Sylla et al., 1998).
Analysis of foliar nutrient concentrations could provide information on the
nutritional status and requirements for long term survival and optimal growth of P.
indicus in plantations. Although the relationship between foliar nutrients and growth
has been broadly studied and successfully applied to pines and eucalypts (Lambert,
1984; Turner and Lambert, 1986; Drechsel and Zech, 1991; Dell et al., 2001), it has
29
not been widely applied to many tropical hardwood species. These relationships are
generally derived from fertilizer rates trials which are tedious to undertake. So far,
such trials are lacking for P. indicus.
Hence, glasshouse trials were undertaken with P. indicus with the following
objectives:
a) to determine the nutrient concentration ranges in the foliage of deficient
and healthy plants;
b) where possible to define critical concentrations for diagnosis of N and P
deficiencies; and
c) to define fertilizer responses in order to establish suitable rates of N and P
for experiments on root nodulation.
The data obtained could provide useful information for nursery managers and serve
as a guide for identifying some macronutrient constraints in seedlings following out-
planting in the field.
2.2 Materials and methods
2.2.1 Experimental design
Separate P and N pot trials were carried out under glasshouse conditions on two soil
types: Yalanbee sandy loam (YB) and a yellow sand (YS). A randomized complete
block design (RCBD) was used for each pot trial, consisting of ten treatments and
four replicate pots. Four germinated seedlings of P. indicus were planted per pot and
later thinned to three per pot to prevent self shading. The P treatments for the YB
trial were 0, 5, 10, 20, 40, 80, 160, 320, 640 and 1280 mg P kg-1
soil, hereafter
designated as P0, P5, …and P1280 and for YS were 0, 2, 4, 8, 16, 32, 64, 128, 256
and 512 mg P kg-1
sand, hereafter designated as P0, P2, …and P512. Phosphorus was
30
supplied as aerophos, Ca(H2PO4)2.H2O (from Albright and Wilson Australian Ltd).
The N treatments for the YB trial were 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 mg N kg-1
soil, and hereafter designated as N0, N5, …and N45 and for YS were 0, 5, 10, 20, 30,
40, 50, 60, 70 and 80 mg N kg-1
sand, hereafter designated as N0, N5, …and N80.
Nitrogen was supplied as NH4NO3. The P and N trials on YB were carried out from
October, 2002 to January, 2003 (Spring-Summer) and the P and N trials on YS were
undertaken from July-October, 2003 (Winter-Spring), in an evaporatively cooled
glasshouse at Murdoch University, Western Australia. The annual mean temperatures
for both trials are given in Appendix I.
2.2.2 Seed and plant material
Seeds were obtained from large trees in the campus ground of the Forest Research
Institute of Malaysia (FRIM), Kuala Lumpur, Malaysia. Seeds were surface sterilized
in 70% (v/v) ethanol for 60 seconds, followed by 45 seconds in freshly prepared 4%
(v/v) sodium hypochlorite, and then rinsed in sterile distilled water before sowing.
The sterilized seeds were sown in plastic trays 29 cm wide x 35 cm length,
containing yellow sand which had been sterilized by autoclaving for 20 minutes at
121oC. Four germinated seedlings of P. indicus at the two-leaf stage were then
transferred into each treatment pot after about ten days.
2.2.3 Soil information
The YB soil is a typical laterite soil occurring on the Darling Range in Western
Australia. Generally, these laterite soils are infertile, have poor water holding
capacity, and strongly adsorb phosphate (Seddon, 1972). Crops here require inputs of
N and P fertilizers to produce commercial returns and in some places these can
induce secondary deficiencies of micronutrients such as Zn. The native vegetation is
31
a dry sclerophyll, open eucalypt forest. Soils in the region are deep sandy gravels
with the top soil classified as sand to sandy loam and the subsoil as sand to clayey
sand. Soil which had never been fertilized was collected at 0-20 cm depth from the
Allandale Research Farm Station in Wundowie which is about 63 km east of Perth
(31o47’S, 116
o22’E) and immediately north of the Great Eastern Highway. The soil
was sieved through a 4 x 4 mm stainless steel field mesh and mixed thoroughly
(Figure 2.1). The yellow sand was obtained from under virgin banksia woodland on
the Swan Coastal Plain near Gnangara north of Perth (31o46 ’S, 115
o51’E). Chemical
properties of the two virgin soil types are given in Table 2.1. These soils have
previously been used in studies on eucalypt nutrition and mycorrhizal responses
(Brundrett et al., 1994; McLaughlin, 1996; Karopoulos, 1999; Asher et al., 2002).
2.2.4 Background on climate for Perth, Western Australia
Perth is subjected to a Mediterranean climate, distinctively characterised by cool, wet
winters and very hot, dry summers. Heavy rainfall with gusty wind is typical in
winter. Approximately 80-95% of the annual average rainfall of 869 mm falls
between the months of May to October (Gerritse and Schofield, 1989).
2.2.5 Soil preparation and nutrient treatments
All trial soil was steam pasteurized twice at 90 oC for one hour to kill any pathogenic
or symbiotic organisms (Landis et al., 1989), allowed to cool for 24 hours, and re-
steamed. Similar pot culture trials have also been described by Brundrett et al.
(1996). Many soils in south-western Australia are infested with Phytophthora. The
pasteurized soil was then spread thinly over clean plastic and air-dried for five days.
Three kilograms dry soil was transferred into each non-draining plastic pot (150 cm
diameter x 17.5 mm height) lined with a clear plastic bag.
32
Figure 2.1 Yalanbee soil collection at the Allandale Research Station, Wundowie,
Western Australia. a1-a2) Sites where soil was collected to 20 cm depth, b1-b2)
Digging and collecting into gunny sacks, c) Soil that has been sieved and air-dried
before being potted.
(a1) (a2)
(b1) (b2)
(c)
33
Basal nutrients were applied to the surface of soil in solution (mg/kg soil): YB-51
Ca, 20 P, 20 N, 52 K, 7.4 Mg, 3 Mn, 2.3 Cu, 3 Zn, 0.1 Co, 0.6 Mo, 0.6 Na, and YS-
31 Ca, 48 P, 20 N, 90 K, 25 Mg, 15 Mn, 12.3 Cu, 13.8 Zn, 1 Co, 1.2 Mo, 3.3 Na.
When dry, the basal nutrients and treatments were mixed throughout by shaking the
soil in a plastic container for two minutes by hand. Additional N was supplied
fortnightly in an aqueous solution over 11 weeks to give a total of 99 mg N/pot. The
plants were not inoculated with N-fixing bacteria. The pots were watered by weight
to 10% field capacity and left to incubate for two days before planting (Figure 2.2).
After planting, pots were daily watered to field capacity.
2.2.6 Harvesting and plant analysis
The stem heights of the plants were taken before they were harvested at 10 weeks
old. Shoots were separated from the roots at one cm above the soil level and then
partitioned into the youngest fully expanded leaf (YFEL) and the rest of the shoot.
Roots were recovered by gently washing the soil over a 2 mm sieve and care was
taken to minimize loss of fine roots and root tips. Total root length was measured
using ASSESS software (Lamari, 2002). The plant parts were oven-dried at 70 oC for
at least 48 hours to constant weight. The dried YFELs were ground by hand in a
porcelain mortar and pestle. For P, the plant material was digested in concentrated
nitric acid (70% w/w) and H2O2 (30% w/w) using an open-vessel microwave oven
(CEM Mars 5) as described by Huang et al. (2004). All chemicals used were of
analytical grade. Total Kjeldahl N was determined following digestion of material in
concentrated sulphuric acid (98% w/w) as described by Zarcinas et al. (1987). Blank
solutions of sulphuric acid at 0, 0.5, 1, 2 and 4 mL together with a standard reference
material, Eucalypt 2000, were used in each digest batch to check for contamination.
34
Figure 2.2 Glasshouse trials using a) Yalanbee soil and b) yellow sand for the P-
growth response of Pterocarpus indicus seedlings. The surface of the yellow sand
was covered with white sterile polyvinyl chloride beads to reduce evaporation.
(a)
(b)
35
Total P and N were measured by induction coupled plasma spectroscopy. Combined
core soil samples of 200 g from each treatment were collected and sent to a
commercial laboratory (Westfarmers CSBP Limited) for analysis (Table 2.1).
Table 2.1 Mean soil chemical properties for the virgin Yalanbee soil and yellow sand
2.2.7 Statistical analysis
Data at harvest were analyzed using SPSS version 11.5. The effects of fertilizer
treatments on shoot dry weight, root biomass, root-whole plant biomass and root-
length ratio were subjected to analysis of variance (ANOVA). Means were compared
using Duncan’s New Multiple Range Test (DMRT) where ANOVA showed that
there was a significant difference between treatment means (P≤0.05). All data were
checked for normality before being analyzed and square root transformation was
carried out where appropriate to satisfy the homogeneity test by using Bartlett test at
F≤ 0.01.
A hand-fitted curve method was used for data analysis.
2.3 Results
2.3.1 Growth
a) Phosphorus
Property
Yalanbee soil Yellow sand
P (Colwell) (mg kg-1
) 20 4
Nitrate-N (mg kg-1
) 1 <1
Ammonium-N (mg kg-1
) 5 5
K (Colwell) (mg kg-1
) 60 20
S (mg kg-1
) 8.8 11.8
Organic carbon (%) 2.26 0.06
pH (H2O) 5.7 5.6
pH (CaCl2) 6.3 6.2
36
There was a significant shoot growth response of P. indicus to P fertilizer (P≤0.05).
Generally, growth of seedlings also increased with increasing P fertilizer treatments
in all soil types (Figures 2.3a and 2.4a) up to a maximum. There was a steep increase
at low P rates, a narrow optimum range and shoot growth at high P rates was
depressed. Maximum seedling growth occurred at lower rates of P in YS [(P128
(0.56 ± 0.04 g)] than in YB [P320 (3.60 ± 0.13 g)]. The stem height in YB plants was
about 50% higher than that in YS plants (Appendix II). Growth measured as shoot
dry weight, root biomass and total root length was severely depressed at low P rates
(P0-P32) in YS plants and at P0-P40 for YB plants. Withholding P reduced plant
yield by up to 390% and 330% in YS and YB, respectively.
Interestingly, both soil types shared a similar Colwell P concentration of 118 mg/kg
soil at maximum shoot dry weight (Figures 2.3b and 2.4b) even though the YB had a
greater buffering capacity at higher fertilizer rates and plant toxicity symptoms were
less severe (Figure not shown). Soil analysis showed that Colwell P was lower in YS
than YB at low fertilizer rates (Table 2.1).
b) Nitrogen
There was a significant growth response (P≤0.05) in YS to N fertilizer. As there was
no response in growth of P. indicus to N fertilizer in YB, this experiment is not
discussed further here. In YS, shoot dry weight increased linearly with N rates in the
deficiency range (N0-N5) then remained constant (N10-N80) (Figure 2.5). Soil
analysis suggests that P. indicus may have had a preference for ammonium-N over
nitrate-N at luxury N supply (Table 2.2). Concentrations of NH4+
remained low at 1
mg/kg across all N treatments whereas soil NO3-
concentrations increased at high
(50-80) N rates.
37
0 200 400 600 800 1000 1200 1400
P treatment (mg P/kg soil)
0
1
2
3
4
5
Sh
oo
t d
ry w
eig
ht
(g/p
ot)
(a)
0 100 200 300 400 500
Colwell phosphorus (mg/kg soil)
0
1
2
3
4
5
Sh
oo
t d
ry w
eig
ht
(g/p
ot)
(b)
Figure 2.3 Effects of P fertilizer (a) on shoot dry weight of Pterocarpus indicus and
(b) soil available P (Colwell) in Yalanbee soil. Data are means of 4 replicates with
standard error bars.
38
0 100 200 300 400 500 600
P treatment (mg P/kg soil)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Sh
oot
dry
weig
ht
(g/p
ot)
(c)
0 100 200 300 400 500
Colwell phosphorus (mg/kg soil)
0.0
0.2
0.4
0.6
0.8
Sh
oot
dry
weig
ht
(g/p
ot)
(d)
Figure 2.4 Effects of P fertilizer (a) on shoot dry weight of Pterocarpus indicus and
(b) soil available P (Colwell) in yellow sand. Data are means of 4 replicates with
standard error bars.
(a)
(b)
39
2.3.2 Foliar nutrient concentrations and plant symptoms
a) Phosphorus
The P concentration in the YFEL increased linearly with increase in P fertilizer from
0 to 19.2 mg/g (at P0-P512) in the YS plants and 0 to 5.5 mg/g (at P0-P1280) in YB
plants (Figure 2.6). Symptoms of P deficiency and toxicity are described in Table 2.3
along with P concentration ranges in the corresponding YFELs. The foliar contents
on different treatments are as in Appendix III.
Table 2.2 Mean properties of the soils at harvest for the P trial in Yalanbee (YB) and
yellow sand (YS) and the N trial in yellow sand.
1. YB-Phosphorus 0 5 10 20 40 80 160 320 640 1280
Colwell P (mg kg-1
) 6 9 12 14 24 45 85 118 298 458
Nitrate-N (mg kg-1
) 51 59 60 51 40 42 54 52 67 66
Ammonium-N (mg kg-1
) 71 79 82 61 46 49 51 51 57 80
pH (H2O) 5.8 5.9 5.8 5.8 5.9 5.8 5.7 5.7 5.8 5.9
pH (CaCl2) 5.3 5.4 5.3 5.2 5.3 5.2 5.2 5.3 5.3 5.4
2. YS-Phosphorus 0 2 4 8 16 32 64 128 256 512
Colwell P (mg kg-1
) 1 1 2 1 5 13 29 70 137 305
Nitrate-N (mg kg-1
) 48 51 47 41 56 43 35 30 32 27
Ammonium-N (mg kg-1
) 19 13 10 11 9 10 8 7 15 22
pH (H2O) 5.9 5.8 5.8 5.6 5.5 5.5 5.8 6.1 6.0 6.5
pH (CaCl2) 5.1 5.3 5.3 5.0 5.1 5.3 5.3 5.6 5.4 6.1
3. YS-Nitrogen 0 5 10 20 30 40 50 60 70 80
Colwell P (mg kg-1
) 20 23 20 20 23 25 23 23 21 22
Nitrate-N (mg kg-1
) 1 1 1 1 3 7 14 17 19 37
Ammonium-N (mg kg-1
) 1 1 1 3 1 1 1 1 1 1
pH (H2O) 5.6 5.7 5.6 5.6 5.5 5.6 5.6 5.6 5.5 5.5
pH (CaCl2) 5.9 6.1 6.4 5.1 5.0 5.1 5.0 5.0 5.0 5.0
40
0 10 20 30 40 50 60 70 80 90
N treatment (mg N/kg soil)
0.0
0.1
0.2
0.3
0.4
0.5
Sh
oot
dry
weig
ht
(g/p
ot)
Figure 2.5 Effect of N fertilizer on shoot dry weight of Pterocarpus indicus in
yellow sand. Data are means of 4 replicates with standard error bars.
y = 0.0375x
R2 = 0.9498
y = 0.0043x
R2 = 0.8821
0
5
10
15
20
25
0 200 400 600 800 1000 1200 1400
Fertiliser P rates (mg/kg soil)
YF
EL
co
ncen
trati
on
(m
g/g
)
YS Yellow sand YB Yalanbee soil
Figure 2.6 Change in total P concentration in the youngest fully expanded leaf
(YFEL) of Pterocarpus indicus seedlings in response to P fertilizer. Points represent
means of four replicates. Functions with R2
values are fitted separately to individual
replicate data for the YS and YB experiments.
41
Generally, P. indicus seedlings grown in YS produced smaller leaves than those
grown in YB. This is likely to have been due to the lower glasshouse temperatures
and shorter days in winter when YS trial was setup as compared to summer in the
YB trial. However, YS plants showed early deficiency symptoms (Figure 2.7a) at
lower P fertilizer treatments (P0-P32) than in YB plants (P0-P80). These seedlings
had depressed shoot dry weight, reduced size of the older leaves, light green or
chlorotic foliage and short stems that were highly branched and usually rosette-like
in appearance (Table 2.3). There was also reduced root development (Figure 2.8).
The YS plants with high YFEL concentration (at P256-512) had depressed (P≤0.05)
growth (root biomass and total root length) (Appendix IV). Similarly, YB plants
grown at P640-1280 had high foliar P concentration and depressed (P≤0.05) shoot
dry weight (Figure 2.11). Symptoms are given in Figure 2.7c and Figure 2.9.
b) Nitrogen
Unlike for P in the P rates trials, there was no linear relationship between foliar N
concentration and fertilizer rate for plants grown in YS (Figure 2.10). From N0 to
N40, foliar N concentrations were low but increased four-fold at N50 and above.
These two groups correspond to deficient and adequate plants respectively. Low or
deficient seedlings showed stunted growth (Table 2.3) with smaller leaves that were
yellowish green (Figure 2.7d) and had reduced root length (Appendix 2.3). At the
highest N rate, leaves were small, flaccid and the tips were necrotic, suggesting N
toxicity.
2.3.3 Critical phosphorus concentrations
Critical P concentrations in the YFEL of P. indicus seedlings corresponding to 90%
of maximum shoot dry weight were derived from linear relationships fitted to the
42
Table 2.3 Related symptoms and nutrient concentrations in the youngest fully
expanded leaves of Pterocarpus indicus.
shoot dry weight and the foliar P data (Figure 2.11). The fitted lines were divided
into three zones, namely: 1) zone of deficiency, 2) zone of adequacy and 3) zone of
toxicity. Data points for seedlings with severe toxicity symptoms were omitted for
the line fitting exercise. The regression coefficients (R2) for deficiency, adequacy and
Element Description Symptom Concentration
(mg g-1
dry
weight)
P Deficient
Mildly
deficient
Adequate
Mildly
toxic
Toxic
Malformed, stunted growth with soft
small leaves, early leaf shedding,
stunted stems, short internodes and
malformed apical buds
Leaves light green
Dark green healthy leaves and taller
seedlings
Mature leaves with marginal and
interveinal chlorosis
Shorter, stunted stems and coppicing,
branches with rosette-like small
leaves, shoot dying back from apex,
old leaves pale yellow with necrotic
spots, poor root development
<0.48 - 0.90
0.97 - 1.49
2.42 - 3.56
3.57-4.64
>4.65
N Deficient
Stunted growth with leaves pale,
uniform yellow
<4.0
Mildly
Deficient
Adequate
Mature leaves with marginal and
interveinal chlorosis, with young
pale green leaves
All leaves were dark green
4.0-7.0
20-35
43
toxicity zones were 0.39, 0.70 and 0.99, respectively. The estimated P concentration
in the YFEL for the diagnosis of P deficiency at 90% maximum shoot yield, was
0.17% and for diagnosis of P toxicity was 0.41%. These critical P concentrations
were estimated from bivariate fits using JMP.SAS (Version 5).
2.3.4 Critical nitrogen concentration and yield
In the nitrogen trial in YS, due to the wide gap in N concentrations in the YFEL of
deficient and adequate plants, it was not possible to define a critical leaf
concentration for the diagnosis of deficiency or toxicity. No linear relationship
between foliar N concentration and fertilizer rate for plants growth in YS was
obtained and this was previously explained under subheading 2.3.2 b.
2.4 Discussion
There was a strong growth response of Pterocarpus indicus to fertilizer P in the two
soils. The soils were selected because they had low concentrations of available P and
differ in P adsorption capacity (Schoknecht, 1997). Historically in the region, acute P
deficiency occurs when the land is first cleared for agriculture or plantation forestry.
Usually, when P fertilizers are applied the water-soluble P component reacts rapidly
with the surfaces of soil constituents (by adsorption to iron and aluminium oxides)
and with cations in soil solution (e.g. by precipitation with ions of calcium, iron and
aluminium) (Barrow, 1980) restricting P available for absorption by plant roots or
mycorrhizal fungi. As fertilizer P increased, there was an increase in Colwell P
(Colwell, 1963) and plant growth. This sodium bicarbonate soil test is widely used in
Western Australia and has been applied to prescribe fertilizer P requirements
44
Figure 2.7 Leaf symptoms observed in Pterocarpus indicus seedlings in response to
soil P in Yalanbee soil and soil N in yellow sand. Upper left to right: (a) changes in
leaf size with yellowish small malformed leaf for P10, healthy green P-adequate for
P320 leaves and chlorosis reduced size leaves for P1280 and (b) Lower left to right:
N-deficient leaves showing stunted growth and uniform pale yellow leaves in N5
treatment, mildly N-deficient N30 pale yellow leaves in N30 and healthy uniform
dark green leaves in N70.
(a)
(b)
P10 P320 P1280
N5
N30
N70
45
Figure 2.8 P deficient symptoms in Pterocarpus indicus seedlings at a) P0 treatment
showing stunted stems, b) adequate P160 treatment with tall green seedlings, c) (i) P-
deficient plant, (ii-iii) just adequate-P plants and iv) plant with P-toxicity – note the
stunted root growth.
(a)
(b)
(c)
(i) (ii)
(iii)
(iv)
46
Figure 2.9 P toxicity symptoms in Pterocarpus indicus at P640 and P1280
treatments. (a) mature leaves with interveinal chlorosis, (b) leaves with necrotic spots
and (c) stunted plants showing coppicing and rosette-like small leaves.
(b)
(a)
(c)
47
0
5
10
15
20
25
30
35
40
0 20 40 60 80 100
Fertiliser, N rates (mg/kg soil)
N C
on
cen
trati
on
(m
g/g
)
Figure 2.10 Relationship between N fertilizer rates and N concentration in the
youngest fully expanded leaf for Pterocarpus indicus grown in yellow sand.
y = 63.622x - 13.231
R2 = 0.3969
y = -0.4131x + 100.8
R2 = 0.7007
y = -5.7174x + 114
R2 = 0.9965
0
20
40
60
80
100
120
0 5 10 15 20Total P Concentration (mg/g)
Maxim
um
shoot
dry
weig
ht
(%)
Deficiency range Adequate range Toxicity range
Figure 2.11 Relationship between foliar P concentration (mg/g) and total shoot dry
weight (%) for Pterocarpus indicus seedlings using the combined data from the two
P experiments. The fitted lines were obtained using JMP.SAS linear fit estimates.
The replicates with the highest yield = 100%.
48
(Bolland and Wilson, 1994). However, soil tests only provide crude estimates for
predicting yields of annual crops and pasture (Bolland et al., 1989). This is the main
reason why this thesis focuses on plant, rather than soil P status, for diagnosis of P
deficiency.
Compared to the growth response of other woody plants and field crops in these
soils, there was surprisingly a narrow response range between P deficiency and P
toxicity for P. indicus. Although P toxicity was anticipated at the high fertilizer rate
in the yellow sand due to its low buffering capacity (Schoknecht, 1997), it was not
expected in the Yalanbee soil where the P adsorption capacity is much higher
(Barrow, 1977). This raises the possibility that P. indicus is an unusual tree that may
be sensitive to levels of fertilizer P supply that would normally not adversely affect
the growth of other legume tree species with fast growth potential. This will be
explored further in Chapter 3.
There was no evidence that P. indicus can function better at low soil P than other
species. The foliar P concentration range in the most deficient plants was 0.05-0.09%
which was similar to juvenile leaves in Acacia auriculiformis (Zech, 1990).
Deficiency ranges for Acacia mangium and Casuarina equisetifolia seedlings
obtained in pot trials (Table 2.4) are somewhat higher but these values merely reflect
the extent of P deficiency obtained in different experiments with soils differing in P
status. However, the adequate P foliar concentration range in P. indicus (0.24-0.36%)
was higher than in A. auriculiformis (0.12-0.25%) (Zech, 1990) but was similar to
other nitrogen-fixing trees such as Acacia mangium (0.35-0.5%) and Casuarina
equisetifolia (>0.25%) (Walker et al., 1993). Overall, the adequate P concentration
range in most legume tree species is comparatively higher than in non nitrogen-
49
fixing tree species such as Eucalyptus urophylla (0.1-0.3%) (Dell and Robinson,
1993), Swietenia macrophylla (>0.07-0.09%) (Yao, 1981) and Hevea brasiliensis
(0.14-0.17%) (Yew and Pushparajah, 1984). The foliar deficiency concentration
range was lower than reported in fruit trees such as cherry (Prunus avium) (<0.09%)
(Leece, 1976), cashew (Anacardium occidentale) (<0.14%) (Marchal, 1987) and
hazel-nut (Corylus avellana) (<0.1%) (Righetti et al., 1988). It was also lower than
oil palm (Elaeis guineensis) (<0.19%) (Coulter, 1958). Other broad–leaved tropical
tree species reported to have low P concentrations in deficient plants include Cedrela
odorata (<0.10%), Eucalyptus camaldulensis (0.05-0.08%), Dalbergia sissoo
(<0.08%) and Gmelina arborea (0.05%) (Drechsel and Zech, 1991). Differences in
the spread of the deficiency and adequate concentration ranges measured may arise
due to differences in soil P buffering capacity, the plant part sampled, the age of the
plant when samples were taken, the developmental stage of growth (e.g. vegetative
reproductive), and differences between species and provenances as reported by Dell
et al. (1987), Grove (1990), Dreshsel and Zech (1991), Netera et al. (1992),
Palomarki and Holopainen (1994), Sanginga et al. (1994) and Robinson et al. (1997).
The P toxicity range for P. indicus was 0.47-1.68% and this range has been reported
in six years old Acacia mangium (>0.5%) (Amir et al., 1993), and in the young
mature leaves of five years old Hevea brasiliensis (>3.0%) in Malaysia. The toxicity
range in other examples of broad–leaved tropical and fruit trees species are Pinus
radiata (>0.8% in the young mature foliage in the field) (Humphreys and Truman,
1964), Prunus persica (>0.40%) (Leece, 1976); Malus domestica (>3.0%)
(Goldspink, 1983), Prunus armeniaca (>0.4%) (Robinson and Nicholas, 1983) and
citrus (>0.25%) (Lee and Mayer, 1993).
50
Table 2.4 Comparisons of P concentrations for nitrogen-fixing and non-nitrogen
fixing tree species in the tropical and subtropical regions (Boardman et al., 1997).
A) Nitrogen-fixing
trees:
P concentration range (%)
Deficient Adequate Toxic
*Growth
stage
#Plant
part
1. Acacia
auriculiformis
<0.034-0.064 >0.12-0.25 Juv YMP
2. Acacia mangium <0.20-0.35 >0.35-0.50 >0.35-0.50 Juv YMP
3. Casuarina
equisetifolia
<0.15 >0.25 Seedl YS
4. Pterocarpus indicus
(present study)
0.05-0.09 0.24-0.36 0.47-1.68 Seedl YFEL
B) Non nitrogen-fixing
trees:
5. Eucalyptus urophylla <0.05-0.09 >0.1-0.3 Juv YMF
6. Hevea brasiliensis <0.127-0.131 >0.137-0.170 Seedl YB
7. Swietenia
macrophylla
<0.08-0.12 >0.07-0.09 Seedl WS
See note in Table 2.5 below.
Table 2.5 Comparisons of N concentration for N-fixing and other non-leguminous
tree species in the tropical and subtropical regions (Boardman et al., 1997).
Note: *Growth stage: Juv = Juvenile; Seedl = seedlings.
#Plant parts: YMP =
youngest mature petiolar organ; YFEL = youngest fully expanded leaf; YS = young
shoot; YB/YS = youngest leaflet/young leaf blade; and WS = whole shoot.
A) Nitrogen-fixing trees: N concentration range (%)
Deficient Adequate Toxic
*Growth
stage
#Plant
part
1. Acacia auriculiformis 0.81-0.84 >1.68-2.81 Juv YMP
2. Acacia mangium 2.04-3.0 Juv YMP
3. Casuarina equisetifolia <1.4 <2.53 Seedl YS
4. Pterocarpus indicus
(present study)
2.0 >2.5 5.6 Seedl YFEL
B) Non nitrogen-fixing
trees:
5. Eucalyptus urophylla <0.86 >1.1-3.0 Juv YMF
6. Hevea brasiliensis <1.49 >1.61-2.26 Seedl YB
7. Swietenia macrophylla <1.09-1.67 Seedl WS
51
Whilst foliar concentration ranges are widely used to assist in making decisions on
fertilizer management in plantations, such ranges need to be calibrated in the field.
Although the lower end of the deficiency range and the upper end of the adequate
range can be expected to vary between sites differing in soil fertility, critical
concentrations for the diagnosis of deficiency should be independent of site and
fertilizer type for a particular plant part and plant age. Furthermore, plant tissue tests
are independent of fertilizer placement.
Linear fits were used to determine critical P concentrations for the diagnosis of
deficiency and toxicity of P. indicus using the relationships between foliar P
concentration and maximum shoot dry weight (Figure 2.7). The linear regression
lines were based and determined on the highest correlated regression coefficients
(R2). Examination of Figure 2.7 shows that the fit of the lines in the regions where
growth is just affected by deficiency or toxicity is imperfect, and may underestimate
true critical concentrations.
No N response was obtained in the YB soil even though the two soil types used had
similar levels of nitrate and ammonium-N at the start of the trial (Table 2.1).
However, the YB soil had 2.26% organic C compared to 0.06% in YS. It seems that
micro-organisms may have made organic N available for plant growth in the YB soil
and this is why there was no N response. It was decided to focus research more on N-
fixation and P fertilizer and not investigate the lack of N response.
There was an early response of P. indicus to N fertilizer in the yellow sand. Growth
was depressed further in the deficiency range, at 2% foliar N in six N treatments
(from N0 to N40). There was no possibility of defining the concentration ranges in
the toxicity zone (Table 2.5). P. indicus seedlings appeared to have some preference
52
for ammonium-N over nitrate-N. Ammonium-N was depleted more than nitrate-N in
the N trial at luxury rates of N supply. There was greater depletion of ammonium-N
in the P fertilizer trial in YS but not in YB (Table 2.2). The conclusion that P. indicus
may prefer ammonium-N over nitrate-N assumes that soil microbes did not
substantially affect the balance of soil inorganic N pools. Soil pH effects (Moore and
Keraitis, 1971) observed in some eucalypt seedling studies (Bougher et al., 1990;
Dell et al., 1991; Shedley et al., 1995) are unlikely to have influenced N uptake as
the pH of the YS was similar across all fertilizer treatments. Shedley et al. (1995)
undertook a trial on Eucalyptus globulus using similar N-free yellow sand (pH 6.0).
They showed that foliar N concentrations in the YFEL ranged from 1% for N-
deficient plants to 3.3% for N-adequate plants. These values are lower than those
obtained for N-deficient plants in P. indicus (2%). Dell et al. (1991) reported that
preference for ammonium in E. diversicolor seedlings diminished when the seedlings
become mycorrhizal but this was not studied for P. indicus.
Some symptoms of nutrient disorders have been described previously for P. indicus.
Srivastava (1979), grew P. indicus using river sand and reported an increase in the
number of branches under P deficiency resulting in rosette-like plants. This symptom
was only observed for high P treatments (Figure 2.7) in this study. He also observed
that buds did not develop in plants that were severely N deficient. However, no P and
N analyses were carried out and no detail was provided of the trial duration.
Where symptoms are severe, this provides an early indication on the P status of a
crop. However, symptoms in the mild deficiency/toxicity ranges are often difficult to
detect and can easily be confused with some other limiting nutrients or stress agents.
53
This is particularly the case for loss of chlorophyll which can also be a consequence
of S deficiency. Hence, the emphasis on foliar analysis is this thesis.
In conclusion, the P concentration ranges and the critical P concentration may be
useful for monitoring the P status of nursery stock and in young seedlings after out-
planting. Whether benefit can be obtained by inoculation with strains of root nodule
bacteria will be discussed in Chapter 4.
54
Chapter 3
The phosphorus response of Pterocarpus indicus seedlings in a
lateritic soil compared to three tropical tree legumes
3.1 Introduction
In the previous chapter, there was surprisingly a narrow range between P deficiency
and toxicity responses for Pterocarpus indicus in two soils that are naturally low in
P. Whilst P toxicity was anticipated at the high fertilizer rate in the yellow sand due
to the low buffering capacity of this soil, it was not expected in the Yalanbee soil due
to its high P adsorption capacity (Barrow, 1977). Soils with similar structural and
chemical properties from the Darling Plateau have been routinely used in Western
Australia to study the nutrient requirements of field crops and some tree species
(Barrow, 1977; Wallace et al., 1986; Burgess et al., 1990; Dell and Robinson, 1993;
Dell and Xu, 1995; Shedley et al., 1995; Lu et al., 1998; Cheng et al., 2002). For
example, Barrow (1977) grew seedlings of Eucalyptus diversicolor (karri) and
Trifolium subterraneum (subterranean clover) in a lateritic soil and obtained response
curves with broad plateaus to fertilizer P in the range of 250 to 1000 mg P/pot
(Figure 3.1a). Each pot contained 0.86 kg soil and P was added in solution as
potassium dihydrogen phosphate before planting. Similar responses were obtained
for E. marginata (jarrah), Corymbia calophylla (marri), Acacia pulchella (prickly
acacia) and Pinus radiata (radiata pine) in a second experiment (Barrow, 1977).
These response curves have also been reported by other workers, including E.
marginata (Dell et al., 1987); and subterranean clover and P. pinaster (Robson and
Gilkes, 1980). Clearly, these published accounts contrast sharply with the response
shown for Pterocarpus indicus in Figure 3.1B. Even in the yellow sand used in
55
Chapter 2, published shoot responses (e.g. Bougher et al., 1990) to fertilizer P reveal
a broad plateau where moderate increases in soil P do not alter shoot biomass.
This raises the possibility that P. indicus is more sensitive to luxury soil P supply
than other legume tree species with fast growth potential. Hence, in order to
investigate this hypothesis, this chapter compares the P response of P. indicus with
three other woody leguminous species, viz. Acacia mangium Willd., Sesbania
formosa F. Muell. and Pterocarpus macrocarpus Kurz. Acacia mangium was chosen
because it is widely grown in plantations and for rehabilitation of degraded soils in
tropical humid zones (Sim, 1987; Udarbe and Hepburn, 1987; Turnbull et al., 1998).
Pterocarpus macrocarpus is valued for its high quality timber (Troup, 1921;
Soerianegara and Lemmens, 1994) and S. formosa is used as a host species for the
Indian sandalwood (Santalum album) in plantations in northern Australia
(Radomiljac, 1998). The experiment was undertaken using Yalanbee soil because of
the broad plateau shown in Figure 3.1A over which the response of P. indicus to
fertilizer P could be compared. The soil was collected as close as possible to where
Yalanbee soil was sourced in Chapter 2.
0 200 400 600 800 1000 1200 1400
P treatment (mg P/kg soil)
0
1
2
3
4
5
To
tal sh
oo
t d
ry w
eig
ht
(g/p
ot)
Figure 3.1 Comparison of effects of P fertilizer applied to lateritic soils from
southwestern Australia on shoot dry weight of (a) Eucalyptus diversicolor (solid line)
and Trifolium subterraneum (dashed line) taken from Barrow (1977); and (b)
Pterocarpus indicus (Chapter 2).
(a) (b)
56
3.2 Materials and methods
3.2.1 Experimental design
A randomized complete block design (RCBD) consisting of four tropical tree
legumes (Acacia mangium, Sesbania formosa, Pterocarpus macrocarpus and
Pterocarpus indicus), seven P treatments and four replicate pots was used. Initially,
four seedlings per pot were planted but these were progressively thinned to two by
the end of the trial. The treatments used were 0, 40, 80, 160, 320, 640 and 1280 mg P
kg-1
soil, hereafter designated as P0, P40, P80, P160, P320, P640 and P1280. The
trial was carried out from June to September, 2005 in a heated glasshouse at
Murdoch University, Western Australia (Figure 3.2) with mean minimum and
maximum temperature (± S.E.) of 15.22 ± 0.19 and 27.88 ± 0.26 oC, respectively.
Seed was sourced as follows: A. mangium from a commercial nursery, Perth,
Western Australia; S. formosa from trees at Kununurra, Western Australia; P. indicus
from Forest Research Institute Malaysia and P. macrocarpus from Faculty of
Forestry, Kasetsart University, Thailand.
3.2.2 Seed treatment and germination
Prior to sowing, the seeds were treated as follows. Seeds of A. mangium and S.
formosa were immersed in rapidly boiling water and removed after about 60 seconds.
They were then surface dried and put onto moistened sterile filter paper in 20 cm
diameter plastic Petri plates. The plates were covered with aluminum foil and placed
in a controlled temperature room at 25 oC, in the dark for 4 days and 2 days in the
light at the same temperature for germination.
Seeds of P. indicus and P. macrocarpus were surface sterilized in 70% (v/v) ethanol
for 60 seconds, followed by 45 seconds in freshly prepared 4% (v/v) sodium
57
hypochlorite, and then rinsed in sterile distilled water (x6) before sowing in sterilized
plastic trays, dimensions of 315 (W) x 425 (L) x 120 (H) cm, containing autoclaved
yellow sand. The seeds for all four species were sowed at a depth of about 1 cm from
the soil surface. They were transplanted into the pots when two cotyledons had
emerged from the soil (Chapter 2). Watering was carried out twice daily to field
capacity (10% by weight).
Figure 3.2 Phosphorus pot trial of four leguminous tree species in the glasshouse.
3.2.3 Soil information and preparation
Soil at 0-20 cm depth was collected from the same site used in Chapter 2. Soil
preparation was subjected to the same procedure described in Chapter 2 except that it
was not pasteurized. The soil had been shown to be free of Phytophthora by other
workers (Brundrett et al., 1994). The chemical properties of the batch of Yalanbee
soil used are given in Table 3.1. Low levels of many nutrients in the base rock
granite combined with prolonged deep-weathering and crystallization of kaolinite
and sesquioxides had resulted in a soil of poor nutrient status. The physical and
58
chemical properties of the soil series have been described by McArthur and Bettenay
(1960), Gilkes et al. (1973), Barrow (1977) and Robson and Gilkes (1980).
Table 3.1 Soil chemical properties for the virgin Yalanbee soil.
The same basal nutrients were applied and the plants were managed according to the
procedures described in Chapter 2 except for the following variations (mg/pot):
K2SO4 468; MgSO4.7H2O 73 and CaSO4.2H2O 400. Nitrogen was supplied weekly in
aqueous solution for 11 weeks as (NH4)2SO4 to give a total of 99 mg N/pot. The
plants were not inoculated with N-fixing bacteria.
3.2.4 Harvesting and plant analysis
Harvesting was staggered due to the differences in growth rates between species. The
harvesting sequence was S. formosa: (15 weeks), A. mangium (17 weeks), P.
macrocarpus (19 weeks) and P. indicus (21 weeks) after planting. The objective was
to obtain growth curves in relation to P supply, not to compare absolute growth rates.
Prior to harvest, stem height and collar diameter were taken using a ruler and scale
caliper, respectively. The plants were separated into the youngest fully expanded leaf
(YFEL), rest of the shoot, stem and roots. Roots were recovered by washing the root
ball over a 2 mm sieve and care was taken to minimize loss of fine roots. Shoots and
roots were dried in an oven at 70 oC for 48 hours to a constant weight. The dried
Property
Value
P (Colwell) (mg kg-1
) 4
Nitrate-N (mg kg-1
) 2
Ammonium-N (mg kg-1
) 1
K (Colwell) (mg kg-1
) 61
S (mg kg-1
) 4.0
Organic carbon (%) 2.6
pH (H2O) 5.6
pH (CaCl2) 6.3
59
samples were ground in a stainless steel home electric grinder and digested in
concentrated nitric acid using an open-vessel microwave oven (CEM Mars 5,
manufactured by CEM Corp., USA) followed by elemental quantification using an
ICP-AES as described in Chapter 2 and by Huang et al. (2004). Standard eucalypt
reference material was used for each digestion batch.
3.2.5 Statistical analysis
Multivariate analysis of variance (P≤0.01 and P≤0.05) was used depending on the
homogeneity of variances to compare growth rate of the species. The phosphorus
fertilizer treatments, however, were also analyzed separately for each species using
ANOVA (SPSS version 17.0) and the means were examined using Duncan’s New
Multiple Range Test.
The effects of phosphorus fertilizer treatments on growth were fitted where possible
to a Mitscherlich model equation Y = a-be–cx
; where Y = is the plant yield at the
nutrient concentration X, a, is the estimated maximum yield, and b and c are
parameters to be estimated (Ware et al., 1982). A hand-fitted curve method was also
tested for data analysis.
3.3 Results
3.3.1 Effect of phosphorus treatment on growth
There was a significant species x P interaction for all dependent variables measured
(Table 3.2). For convenience, the measured and observed parameters are considered
under five headings below.
a) Height and diameter
Generally, growth was poor in all P0 plant for the four tree species. P. indicus grew
the slowest, reaching maximum height and diameter of 12.9 ± 1.5 cm and 3.6 ± 0.3
60
mm at P60, respectively, at age 21 weeks (Figure 3.3). A similar height growth of
12.7 ± 1.3 cm was also obtained for P. indicus in the previous trial (Chapter 2). The
P. macrocarpus was also slow growing, obtaining maximum height and diameter
growth (data not presented) of 16.1 ± 0.8 cm and 3.8 ± 0.4 mm at P80, respectively,
at age 19 weeks.
By contrast, both A. mangium and S. formosa were fast growing with S. formosa
reaching height and diameter growth of 27.2 ± 0.3 cm and 4.9 ± 0.1 mm at P320 after
15 weeks and A. mangium with 16.7 ± 0.3 cm, 4.0 ± 0.3 mm at P1280 after 17
weeks, respectively.
0
5
10
15
20
25
30
0 40 80 160 320 640 1280
P Treatment (g/kg)
Heig
ht
(cm
)
Acacia mangiumSesbaniaP. indicusP.macrocarpus
Figure 3.3 Height growth of four leguminous tree species at harvest. Each data point
is the mean of four replicates with standard error bars.
b) Shoot and root dry weight growth
The response of shoot and root dry weights to P fertilizer differed markedly between
species. There were two groups of responses, with A. mangium and S. formosa
forming one group, where shoot and root dry weight responses could be fitted using
the Mitscherlich model (Figures 3.4, 3.5). P. indicus and P. macrocarpus formed a
P treatment (mg/kg soil)
61
second group where there were steep declines in growth at higher P treatments and
these response surfaces could not be fitted to the Mitscherlich model (Figures 3.6,
3.7). Maximum yields for shoot and root dry weight of 0.70 ± 0.15 and 0.35 ± 0.11
g/seedling at P160 for P. indicus were obtained, respectively. These yields were
more than three times higher than those at P0. Growth was further depressed by more
than 45% of maximum yield when high P fertilizer treatments (>P640) were applied
(Figures 3.6a, 3.7a). The response of P. macrocarpus was quite similar to P. indicus
with maximum shoot and root dry weight occurring at P160 and there was a
maximum yield depression of more than 55% at high P treatments (Figures 3.7a, b).
Table 3.2 MANOVA of main factors and their interaction and dependent variables.
a) Factor Wilks’
Lambda
F df1 df2 Sig.
Species (1) 0.011 103.714 24 224 <0.001
Phosphorus (2) 0.081 5.309 48 383 <0.001
(1) x (2) 0.009 3.566 144 580 <0.001
b) Source Dependent variable df1 df2 F Sig.
Species
Height
Diameter
Shoot dry weight
Root dry weight
Root/shoot ratio
2266
128
36
16
0.99
3
3
3
3
3
214.020
216.653
210.184
138.736
22.779
<0.001
<0.001
<0.001
<0.001
<0.001
Phosphorus Height
Diameter
Shoot dry weight
Root dry weight
Root/shoot ratio
1139
27
17
5
0.26
6
6
6
6
6
53.774
22.819
47.500
23.615
3.040
<0.001
<0.001
<0.001
<0.001
<0.001
Species x
Phosphorus
Height
Diameter
Shoot dry weight
Root dry weight
Root/shoot ratio
545
12
9
4
0.57
18
18
18
18
18
8.570
3.501
9.085
5.173
2.165
<0.001
<0.001
<0.001
<0.001
<0.001
62
Sh
oo
t d
ry w
eig
ht
(g/s
ee
dlin
g)
0 200 400 600 800 1,000 1,200 1,400
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
P treatment (mg/kg soil)
Root
dry
weig
ht
(g/
seedlin
g)
0 200 400 600 800 1,000 1,200 1,400
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Figure 3.4 The relationship between P supply and a) shoot dry weight and b) root dry
weight of Acacia mangium. The fitted curve was obtained using the Mitscherlich
model.
Y = 1.97–1.70 exp (-0.03X)
R2 = 0.84
Y = 1.28-1.05 exp (-0.02X)
R2 = 0.82
(a)
(b)
63
Sh
oo
t d
ry w
eig
ht
(g/s
ee
dlin
g)
0 200 400 600 800 1,000 1,200 1,400
0.0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3.0
P treatment (mg/kg soil)
Ro
ot
dry
we
igh
t (g
/se
ed
ling
)
0 200 400 600 800 1,000 1,200 1,400
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Figure 3.5 The relationship between P supply and a) shoot dry weight and b) root dry
weight of Sesbania formosa. The fitted curve was obtained using the Mitscherlich
model.
Y = 2.24-1.98 exp (-2.63X)
R2 = 0.83
Y = 1.32-1.09 exp (-1.68X)
R2 = 0.65
(a)
(b)
64
a
ab
ab
ab
ab
ab
c(a)
0 200 400 600 800 1000 1200 1400
P treatments (mg/kg soil)
0.0
0.2
0.4
0.6
0.8
Sh
oo
t d
ry w
eig
ht
(g/s
ee
dlin
g)
a
a
ab
b
abab
ab
(b)
0 200 400 600 800 1000 1200 1400
P treatment (mg/kg soil)
0.0
0.1
0.2
0.3
0.4
Roo
t d
ry w
eig
ht
(g/s
ee
dlin
g)
Figure 3.6 Effects of P supply on a) shoot dry weight and b) root dry weight of
Pterocarpus indicus. Data points with the same letter are not significantly different at
P≤0.05 using DMRT.
65
a
abc
bc
c
abc
abc
ab
(a)
0.2
0.4
0.6
0.8
1.0
1.2
Sh
oo
t d
ry w
eig
ht
(g/s
ee
dlin
g)
a
c
cc
c c
b
(b)
0 200 400 600 800 1000 1200 1400
P treatment (mg/kg soil)
0.1
0.2
0.3
0.4
0.5
0.6
Roo
t d
ry w
eig
ht
(g/s
ee
dlin
g)
Figure 3.7 Effects of P supply on a) shoot dry weight and b) root dry weight of
Pterocarpus macrocarpus. Data points with the same letter are not significantly
different at p≤0.05 using DMRT.
66
In contrast to P. indicus and P. macrocarpus, there was no reduction in either shoot
or root yields for A. mangium and S. formosa at high P rates (P640 and P1280). The
fitted Mitscherlich curves (Figures 3.4, 3.5) showed that the maximum shoot and root
yields occurred between P160 and P320 for both species. Although the R2
values
were reasonable, ranging from 0.65 to 0.84, the fits of the curves to data points in the
region between deficiency and adequacy were not perfect (Figures 3.4, 3.5).
c) Root-shoot ratio
Root-shoot dry weight ratios were higher in P0 plants of A. mangium and S. formosa
than those in plants supplied with adequate P due to adaptive response of the species.
By contrast, root-shoot ratios for P. indicus and P. macrocarpus did not alter with
change in external P supply. The change and adaptive response are explained in
subheading 3.4.2. Root-shoot ratios in P0 for A. mangium and S. formosa were twice
those of the two Pterocarpus species (Table 3.3).
Table 3.3 Effect of P treatment on the relative root/shoot ratio of four
Leguminous tree species.
*Columns with the same letter are not significantly different using Duncan’s
New Multiple Range Test at P≤0.01.
P mg/kg A. mangium
S. formosa
P. indicus
P. macrocarpus
0 0.94c* 0.80b 0.48a 0.46a
40 0.61ab 0.50a 0.44a 0.58a
80 0.69ab 0.60ab 0.43a 0.59a
160 0.67ab 0.46a 0.46a 0.50a
320 0.61ab 0.54a 0.48a 0.48a
640 0.78bc 0.64ab 0.35a 0.56a
1280 0.57a 0.66ab 0.42a 0.48a
67
d) Symptoms
Both Pterocarpus species shared similar visible symptoms (Figures 3.8, 3.9). At low
P rates (P0 and P40), P-deficient plants were short, stunted and leaves were pale,
light green. At high P treatments (P640 and P1280), most of the plants suffered from
P toxicity and showed tip burn, spot necrosis in the lamina of older leaves, shorter
internodes and there were rosette-like leaves starting to develop as coppice.
Symptoms of P deficiency also occurred in A. mangium and S. formosa at P0. The P
deficient plants were short, stunted and leaves were pale, light green. These P
deficiency symptoms were similar to both Pterocarpus species. However, no
symptoms of P toxicity were expressed at high P rates in P640 and P1280, although
these plants had dark green leaves (Figure 3.11).
e) Relationship between shoot yield and foliar P concentration
In P. indicus and P. macrocarpus, shoot dry weight increased with increase in P
concentrations in the YFEL and then declined (Figure 3.12). Due to the small
number of data points obtained for P-adequate plants, no function was able to be
fitted to the data. Hence, critical values for the diagnosis of either efficiency or
toxicity could not be obtained.
Although there were large gaps in the deficiency range data for the relationship
between yield and P concentration in the YFEL for A. mangium and S. formosa
plants, it was possible to fit the Mitscherlich model to the data (Figure 3.13).
Tentative critical values for diagnosis of P deficiencies, at 10% below the plateaus,
were estimated to be 2-3 mg P/g dry weight for the two species.
68
Figure 3.8 Symptoms of P supply for Pterocarpus indicus seedlings. Left to right: a)
height growth variations from low to high P, and b) symptoms of leaves at low, toxic
and c) adequate P supply.
(a)
(b)
Low
Toxic
Adequate
69
Figure 3.9 Height and foliar symptoms in Pterocarpus indicus seedlings. a) Small
plant size at deficient P (P40), b) Smaller and shorter seedlings at increased P (P320)
supply, and c) stunted stem, yellowish and smaller leaves at high P (P640).
(b)
(c)
(a)
70
Figure 3.10 Symptoms of P supply for Pterocarpus macrocarpus seedlings. Left to
right: a) Seedlings at low (P0) and high P (P1280) supply, b) Leaves at P0 and P40,
c) All leaves at high P (P1280) showing necrotic tissue.
(a)
(b)
(c)
71
Figure 3.11 Symptoms associated with P supply for Acacia mangium (a,b) and
Sesbania formosa (c) seedlings. a) Left to right: P0, P80, P160, P320 and P1280, b)
Leaves from P0, P40, P80, P320 and P1280, c) P0, P40 and P320. Generally, leaves
were smaller in low P and larger in high P supply. No P toxicity symptom was
observed for both species.
(a)
(b)
(c)
72
0
20
40
60
80
100
0 2 4 6 8 10
Shoot dry
weig
ht (%
)
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
P concentration (mg/g)
Shoot dry
weig
ht (%
)
Figure 3.12 The relationship between P concentration in the youngest fully expanded
leaves at maximum yield of (a) Pterocarpus indicus and (b) Pterocarpus
macrocarpus and shoot dry weight (% maximum yield). Each data point is the mean
of 4 replicates.
(a)
P concentration (mg/g)
P concentration (mg/g)
(b)
73
Y = 75.67-223.90 exp (-1.11x)
R2 = 0.82
Shoot
dry
weig
ht
(%)
0 2 4 6 8 10 12 14 16
0
10
20
30
40
50
60
70
80
90
100
P concentration (mg/g)
Sho
ot
dry
we
igh
t (%
)
0 2 4 6 8 10 12 14 16
0
10
20
30
40
50
60
70
80
90
100
Figure 3.13 The relationship between P concentration in the youngest fully expanded
leaves of (a) Acacia mangium and (b) Sesbania formosa and shoot dry weight (%
maximum yield). The fitted lines were obtained using the Mitscherlich model. Each
data point is the mean of the four replicates.
(a)
(b)
Y = 80.11-125.72 exp (-1.04x)
R2 = 0.80
74
3.4 Discussion
3.4.1 Growth response to application of phosphorus
The main finding from this study is that the response of Pterocarpus indicus to P
differs markedly from those of Acacia mangium and Sesbania formosa. The similar
response of P. macrocarpus to P. indicus further suggests that Pterocarpus may be
very sensitive to P fertilizer, even in soil that has a high capacity to adsorb
phosphate. The repeatability of the results for P. indicus with Chapter 2 confirmed
that the toxicity response is real for this species when external P is high and the plant
started to exhibit foliar symptoms of P toxicity.
In A. mangium and S. formosa, the response to fertilizer was not greatly different
from those obtained previously by other workers for subterranean clover and
eucalypts (Figure 3.1) where no reduction in either shoot or root yields were obtained
at luxury supply and these relationships were able to be fitted with the Mitscherlich
model (Lamb, 1976; Barrow, 1977). Similar Mitscherlich-type responses have also
been reported in crops such as wheat (Triticum aestivum), lupin (Lupinus
angustifolius), Sesbania spp., Calliandra and Cedrela odorata (Bolland, 1992; Elliot
et al., 1997; Webb et al., 2000).
3.4.2 Root-shoot ratio
The increased allocation of dry matter to roots under P-deficiency stress in A.
mangium and S. formosa is a typical and important adaptive response that has been
observed in many woody plants and field crops including Hakea, Triticum, Medicago
and Lupinus (Maschner, 1995; Raghotama, 1999; Lamont, 2003; Shane et al., 2004;
Lambers et al., 2006). Adaptive responses to P deficiency can involve accumulation
of P in roots and stems that can then be used when shoot growth predominates (Dell,
1987; McCully, 1994; Raaimakers and Lambers, 1996). For instance, roots usually
75
behave as the preferential site for P storage in H. sericea and the capacity for storage
also can exceed the P concentration in leaves when the latter increases to values
where toxicity symptoms start to appear (Konowski et al., 1991; Sousa et al., 2007).
There may be other root adaptive responses to P deficiency and these include an
increase in mass ratio, root hair length, root hair density and specialized responses
such as formation of ‘proteoid’ or ‘cluster roots’ (Purnell, 1960; Dinkelaker et al.,
1995; Kirk and Du, 1997). Many plants also respond to low P with increased
exudation of carboxylates that play an important role in nutrient mobilization from
inorganic sources in the soil e.g., addition of carboxylates to soil such as citrate has
been shown to mobilize nutrients such as P and Fe (Jones and Darrah, 1994; Lambers
et al., 2002).
By contrast, the lack of root biomass response in the two Pterocarpus species is
unusual and this suggests that these plants may rely on other mechanisms for
facilitating P fertilizer uptake at low external supply since they appear not to depend
on adaptive responses such as increase in root length to access soil P (Jecke and Pate,
1995). However, root systems in P. indicus have not being studied in any detail even
though they contribute significantly to the ecophysiological function of the plant
system for water and nutrient uptake in the field (Van de Driessche, 1984;
Keerthisinghe et al., 1998; Gazal and Kubiske, 2004; Gazal et al., 2004). This will be
discussed further in Chapter 6.
3.4.3 Critical phosphorus concentration for the diagnosis of phosphorus deficiency
Tentative critical values for the diagnosis of P deficiency and P toxicity in Chapter 2
for P. indicus were not able to be confirmed in the present experiment due to the
limited data set for plants grown with adequate P. Furthermore, the tentative critical
76
values obtained from the fit of the Mitscherlich model to the data for A. mangium
and S. formosa are likely to have overestimated the true values, even though the R2
values were 0.8. There are two reasons for this conclusion. The first is the gap for
plants in the range from minor to serious deficiency. The second is that the fitted
maximum yields from the Mitscherlich model were less than 80% of the observed
maximum yield.
The critical P concentration for diagnosis of deficiency in Acacia mangium and
Sesbania formosa obtained in this study was 0.2-0.3%. This range is higher than
some critical P concentrations for diagnosis of deficiency reported for acacias and
some other woody plants. Examples are A. mangium (0.2-0.35%) in the youngest
mature phyllode in a sand pot trial (Sun et al. (1992), A. auriculiformis (0.03-0.06%)
in the youngest mature phyllode in the field (Zech, 1990), Swietenia macrophylla
(0.08-0.12%) in the whole shoot in the field (Yao, 1981), Eucalyptus camaldulensis
(0.05-0.08%) in the mature leaves in the field (Zech, 1990), Anacardium occidentale
(<0.14%) in the mature leaves in the field (Richards, 1992), Actinidia chinensis
(<0.13%) in the first leaf above the fruit in the field (Cresswell, 1989) and Prunus
persica (<0.09%) obtained from the field (Nicholas and Robinson, 1977; Cripps and
Goldspink, 1983).
In conclusion, this chapter shows that the response of P. indicus to P fertilizer is
similar to that in P. macrocarpus seedlings and differs from A. mangium and S.
formosa. It also shows that, by comparison to A. mangium and S. formosa, both
Pterocarpus species are sensitive to luxury levels of P fertilizer and readily exhibit
symptoms. Chapter 6 will investigate further the root adaptive response of P. indicus
seedlings to soil P supply.
77
Chapter 4
Nodulation of Pterocarpus indicus by diverse
strains of root nodule bacteria
4.1 Introduction
Pterocarpus indicus is a tropical legume with potential for commercial forest
plantations (Chapter 1). The ability of some legume tree species to nodulate
effectively has generated interest in providing a new avenue for forestry application
practices. These include maintaining forest ecosystem fertility, land reclamation, soil
stabilization and revegetation programs (Dreyfus and Dommergues, 1981; Faria et
al., 1987; Wilson and Tilman, 1991; Pate et al., 1993; Chang and Handley, 2000;
Sprent and Parsons, 2000; Perez-Fernandez and Lamont, 2003; Fougnies, 2007).
Legumes are often used for managing agricultural ecosystems in order to improve
their organic fertility, N economy or farming system flexibility (Andrew and
Kamprath, 1978; Reeves and Ewing, 1993; Brockwell et al., 1995; Sprent, 2001;
Howieson et al., 2004). Often, optimum performance of the N-fixing symbiosis
depends upon pre-selection of both symbiotic partners for adaptation to the target
environment which in turn present a challenge to root nodule bacteria nodulation and
survival (Chatel and Parker, 1973; Howieson and Ballard, 2004). In order to
investigate symbiotic partners for P. indicus, this chapter was undertaken as a
prerequisite to determine the effective strains for nodulation in P. indicus.
Studies on nodulation in legume crops are reported to be closely associated with
Rhizobium and Bradyrhizobium (Dreyfus et al., 1979) but this information is still
lacking in woody legumes where host-strain relations are unknown. Trees that
78
nodulate effectively with fast-growing root nodule bacteria are recognized as being
specific for nodulation (Gibson et al. 1982; Duhoux and Dommergues, 1985)
whereas species that nodulate with Bradyrhizobium are less specific but tend to be
promiscuous in nature. P. indicus is of no exception and this has been reported to be
a promiscuous host tree. For example, the African P. indicus introduced in Brazil
were found to nodulate with Bradyrhizobium in Brazilian soils (Moreira et al. 1992;
de Faria and de Lima, 1998) in the field. Similar observations have been recorded in
Laguna, Philippines (Oyaizu, 1993) with B. japonicum and another Bradyrhizobium
sp. and in Thailand (Manassila et al., 2007). No further studies have been carried out
reported and there is still a lack of understanding on the specificity, symbiotic
relationships and capacity to fix nitrogen in P. indicus (Lim, 1976; Allen and Allen,
1981; Oyaizu et al., 1993; de Faria and de Lima, 1998; Sprent, 2001).
Inoculation of tree legumes, using effective strains of root nodule bacteria (or
“rhizobia”), can be useful in commercial forest nurseries where increased N2-fixation
capability promotes sustained growth after out-planting (Turk and Keyser, 1992;
Martin-Laurent et al., 1999; Perez-Fernandez and Lamont, 2003). Since N deficiency
is a major fertility constraint in agriculture and forestry in the tropics (Dakora and
Keya, 1997; Kass et al., 1997; Leblanc et al., 2005), there is an urgent need to
exploit N2-fixing tree legumes for sustained productivity. The aim of this study
therefore, was to investigate the ability of Malaysian P. indicus to form nodules with
diverse strains of root nodule bacteria and to identify effective strains with potential
for application as inocula in the nursery and field (Chapter 5).
4.2 Materials and methods
4.2.1 Experimental design
79
A complete randomized block design was used with 18 strains of rhizobia (Table
4.1) and two control treatments (C1-uninoculated without added inorganic N and C2-
uninoculated with plus inorganic N, supplied as KNO3). There were four replicates
per treatment. Each replicate was a single pot (15 cm diameter x 17.5 cm depth)
containing three P. indicus seedlings. The trial was carried out between September
2003 and December 2003 in an evaporative-cooled glasshouse at Murdoch
University, Western Australia. The mean minimum and maximum temperatures (±
S.E.) were 18 ± 2 oC and 30 ± 3
oC, respectively.
4.2.2 Plant and soil material
Seeds were obtained from large trees in the campus ground of the Forest Research
Institute of Malaysia (FRIM), Kuala Lumpur, Malaysia as in Chapters 2 and 3.
Before sowing, the seeds were surface sterilized in 70% (v/v) ethanol for 60 seconds,
followed by 45 seconds in freshly prepared 4% (w/v) sodium hypochlorite, followed
by six rinses in sterile distilled water. Seeds were sown in plastic trays (365 cm
length x 315 cm width x 120 cm height) at a depth of about 1 cm from the surface of
the sterilized yellow sand. The sand was autoclaved for 20 minutes at 121oC and left
to dry for about 48 hours prior to sowing. After one week, seedlings with two open
cotyledons and 5 cm radicles were transplanted into the pots. The pots had been
soaked overnight in 4% (w/v) sodium hypochlorite, rinsed with sterile deionised
water, lined with a sterile paper towel and filled in a 3 kg pot N-free sterile yellow
sand at pH 6.0 (Chapter 2). The physical and chemical properties of the soil are as
described by Bettenay et al. (1960); Bougher et al. (1990) and Brundrett et al.
(1996).
80
To remove traces of inorganic nitrogen, the sand-filled pots were flushed twice with
two litres of boiling water and allowed to drain to field capacity. These pots were
then covered with sterile cling plastic sheeting until ready for use. After transplanting
and inoculation, all the pots were given a pulse of 20 ml aqueous solution containing
KNO3 (5 g/L) and the soil surface was covered with white sterile polyvinyl chloride
beads to reduce evaporation and to prevent contamination. Pots were watered with
deionised water as required, and weekly with sterile complete nutrient solution
(minus N) (Howieson et al., 1995). The nutrient solution contained (g/L): 0.31
MgSO4.7H2O, 0.21 KH2PO4, 0.44 K2SO4, 0.06 FeEDTA, 0.05 CaSO4 and trace
elements (mg/L) 0.116 H3BO4, 0.0045 Na2MoO4.2H2O, 0.134 ZnSO4.7H2O, 0.01
MnSO4.H2O, 0.03 CoSO4.H2O and 0.03 CuSO4.5H2O. The nitrogen-fed control was
supplied weekly with 5 ml of KNO3 (5 g/L). Water and nutrients were supplied
through a sterile white PVC tube inserted in the centre of each pot and covered with
a plastic cap.
4.2.3 Root nodule bacteria strains
The strains of root nodule bacteria used in this study are listed in Table 4.1. Strains
WSM 3712 were isolated from nodules of P. indicus collected in 2002. These
nodules were collected from a potted seedling in FRIM’s nursery and were stored
over silica gel. No description on the nodules was provided. Five nodules were taken
randomly for isolation irrespective of the size and colour, left to soak in distilled
water for four hours, surfaced sterilized by immersion in 70% (v/v) ethanol for 60
seconds, followed by 45 seconds in freshly prepared 4% (v/v) sodium hypochlorite
and by six rinses of sterile distilled water (Vincent, 1970; Beck et al., 1993). The
nodule was crushed in a drop of sterile water and the contents transferred using a
81
sterile wire loop onto yeast mannitol agar medium (YMA) as described by Howieson
et al. (1988) (Appendix VI).
Sixteen strains of WSM and TAL, obtained from the Centre for Rhizobium Studies
(CRS) at Murdoch University, Western Australia were recovered from vacuum-dried
ampoules using the CRS method (Howieson et al., 1988). The Rhizobium strain,
R602 (R. gallicum), was provided by the Institute National de la Recherche
Agronomique, INRA (France).
Table 4.1 Details of root nodule bacteria used in the study
Strain No. Species Host plant **
Source
TAL 643 Bradyrhizobium sp. Canavalia
gladiata
RRIM
TAL 648 Bradyrhizobium sp. Psophocarpus
tetragonolobus
RRIM
TAL 651 Bradyrhizobium sp. Calopogonium
mucunoides
RRIM
TAL 656 Bradyrhizobium sp. Pachyrhizus
erosus
RRIM
*R 602 Rhizobium gallicum Phaseolus
vulgaris
INRA
WSM 2096 Bradyrhizobium elkanii Glycine max CRS
WSM 2097 Bradyrhizobium japonicum Glycine max CRS WSM 2098 Bradyrhizobium liaoningense Glycine max CRS WSM 2100 Mesorhizobium ciceri Cicer arietinum CRS WSM 2105 Rhizobium galegae Galega sp. CRS WSM 2106 Rhizobium hainanense Desmodium sp. CRS WSM 2108 Rhizobium leguminosarum Pisum sativum CRS WSM 2110 Rhizobium tropici Phaeseolus
vulgaris
CRS
WSM 2114 Sinorhizobium meliloti Medicago sativa CRS WSM 2115 Sinorhizobium saheli Acacia senegal CRS WSM 2116 Sinorhizobium terangae Acacia senegal CRS WSM 2117 Mesorhizobium loti Lotus edulis CRS WSM 3712
Bradyrhizobium sp. Pterocarpus
indicus
FRIM
Source: *
Strain obtained from INRA, **
CRS = Centre for Rhizobium Studies, INRA =
Institute National de la Recherche Agronomique (France), FRIM = Forest Research
Institute of Malaysia and RRIM = Rubber Research Institute of Malaysia.
82
4.2.4 Inoculation
The bacteria were initially grown at 28 oC on YMA plates to produce sufficient
culture material for inoculation. The fast-growing strains of Rhizobium and
Sinorhizobium were incubated for 3-4 days, the medium-growing strains of
Mesorhizobium for 4-5 days and the slow-growing strains of Bradyrhizobium for 7-
10 days (Cowan, 1974; Howieson et al., 1988; O’hara et al., 2000). These colonies
were then washed from the Petri plates using sterile sucrose solution 1% (w/v) and a
suspension of 10 ml was used to inoculate each pot. Seedlings were inoculated two
days after transplanting using a sterile syringe to distribute the suspension evenly
onto the soil surface around each seedling.
4.2.5 Harvest
Plants were harvested after three months (15 weeks) when there was a visible
difference and stability in plant shoot and height growth between treatments. Roots
were extracted from the sand with running water and nodulation was assessed based
on the number of nodules, size, color and position on the roots (Howieson et al.,
1995). The shoots were separated and oven-dried at 70 oC for 48 hours or till
constant weight. Nodules used for microscopic studies were fixed overnight at 4 oC
in gluteraldhyde (3% v/v) and in phosphate (0.025) buffer before being dehydrated
and embedded in Spurr’s resin (Spurr, 1969). Thin sections, 1 µm in thickness, were
obtained using glass knives on a Reichert-Jung 2050 microtome. They were stained
in 1% methylene blue plus 1% Azur II in 1% sodium tetraborate (Richardson et al.,
1960). Observations on nodules were made using stereo (Olympus SZX 12) and
compound (Olympus BX 51) microscopes.
83
4.2.6 Identification of root nodule bacteria strain with primer, RPO1
The polymerase chain reaction (PCR) method was used to differentiate five
inoculants and nodule isolates strains comprising the four genera in WSM 3712,
WSM 2096, R602, WSM 2114 and WSM 2100. This method has been previously
used for differentiating and identifying rhizobia (Welsh and McClelland, 1990;
Williams et al., 1990; Richardson et al., 1995; Hebb et al., 1998; Nandasena, 2004).
The technique was used to confirm the nodules had developed with the inoculants
organisms and there had not been contamination in the glasshouse.
4.2.7 Statistical analysis
The data were subjected to analysis of variance (ANOVA) using the program SPSS
version 17.0. Data transformation was carried out where necessary before Duncan’s
New Multiple Range Test was used as a post hoc test to identify differences between
means at P ≤ 0.05.
4.3 Results
4.3.1 Nodulation in Pterocarpus indicus
The uninoculated controls remained nodule free indicating that soil sterilization was
successful and there was no contamination problem in the glasshouse. Nodules were
present on roots of P. indicus inoculated with strains from all four genera,
Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium after three months.
Ten strains (TAL 643, TAL 651, R602, WSM 2096, WSM 2098, WSM 2100, WSM
2106, WSM 2110, WSM 2114 and WSM 3712) formed nodules (Table 4.1) whereas
eight strains (TAL 648, TAL 656, WSM 2097, WSM 2105, WSM 2108, WSM 2115,
WSM 2116 and WSM 2117) did not nodulate. P. indicus inoculated with four slow-
84
growing Bradyrhizobium strains (WSM 3712, TAL 643, WSM 2096 and TAL 651)
formed greater mean number of nodules per seedling whereas nodulated plants using
medium-growing strains (Mesorhizobium) and fast-growing (Rhizobium and
Sinorhizobium) formed low number of nodules (Table 4.2). Strain WSM 3712, which
was collected from the host plant, was identified as a Bradyrhizobium sp. based on
its growth rate on YMA.
4.3.2 Nodule location and size
Plants inoculated with Bradyrhizobium (WSM 3712, WSM 2096, TAL 643) formed
nodules on both the tap and main lateral roots near the root collar (Table 4.2, Figure
4.1). Nodules formed with WSM 3712 and WSM 2096 were larger in size and pink
internally, whereas nodules derived from other strains were smaller in size and white
internally (not illustrated in figures). There were also larger number and bigger
nodule sizes observed near the root collar and tap root than those of lateral roots
(Figure 4.1). P. indicus nodules were mostly globose, smooth, internally
aeschynomenoid in shape, having determinate growth in oblate form and containing
central infected tissues with few or no uninfected cells (astragaloid in the
terminology of Corby 1971, 1988; Brown and Walsh, 1994) (Figure 4.2).
4.3.3 Shoot dry weight
Plants nodulated with Bradyrhizobium elkanii (WSM 2096) and Bradyrhizobium sp.
(WSM 3712) were observed to have dark green shoots compared to the uninoculated
control (C1) plants without added inorganic N. However, only WSM 2096 had
significantly (P≤ 0.05) increased shoot dry weight at the time of harvest (Figure 4.3).
This increased in shoot dry weight was similar to the plants in the uninoculated
85
control (C2) that were supplied with inorganic N. The shoot dry weight obtained was
about three times greater than the control (C1) without inorganic N (Figure 4.3).
Table 4.2. Mean nodule number, nodulated seedlings, nodule distribution, nodule
size and nodule colour in Pterocarpus indicus seedlings for ten treatments.
** Nodule distribution (%) at: A = 0-3 cm; B = 3-5 cm and C = >5 cm from the root
collar for all four replicates, #Size (%):
L = large: > 5mm, M = medium: 3-5 mm and
S = small: < 3mm in diameter for all four replicates (Howieson et al., 1995).
Strain
Mean number of
nodules per
nodulated
seedling (± S.E.)
Nodulated
seedlings
(%)
**Nodule
distribution1
(%)
A B C
#Nodule size2
(%)
L M S
Nodule
colour
WSM 3712 25.3±6.3 83 93 7 - - 18 82 pink
TAL 643 17.3±5.0 83 57 28 15 - 19 81 white
WSM 2096 19.0±9.0 42 38 46 16 16 42 42 pink
TAL 651 16 8 100 - - - - 100 white
WSM 2106 3 8 100 - - - 100 - white
WSM 2114 2 8 100 - - - 100 - white
WSM 2100 1 8 100 - - - - 100 white
R 602 1 8 100 - - - - 100 white
WSM 2098 1.0±0.0 8 100 - - - - 100 white
WSM 2110 1 8 100 - - - - 100 white
86
Figure 4.1 Pterocarpus indicus seedlings nodulated with WSM 2096 at 15 weeks.
Left to right: a) control (C2) treatment, nodulated with WSM 2096 and control (C1)
seedlings, b) the location of nodules on 1) the tap root and 2) the lateral roots, and (c)
nodules at the root collar.
(a) (b)
(c)
1
1
2
87
Figure 4.2 Morphology and anatomy of Pterocarpus indicus nodules formed with
Bradyrhizobium elkanii (WSM 2096). (a) typical morphology of aeschynomenoid
nodules (Scale bar = 1 mm), (b) transverse section of 3 month old nodule. The darker
central stained region (X) shows infected tissues with determinate growth.
b
b
a
a a
aa a
a
a a a
C1 (
-N)
TA
L 6
51
WS
M 2
110
R 6
02
WS
M 2
100
TA
L 6
43
WS
M 2
098
WS
M 2
106
WS
M 2
114
WS
M 3
712
WS
M 2
096
C2 (
+N
)
Strain
0.0
0.1
0.2
0.3
0.4
Sh
oo
t D
ry W
eig
ht
(g)
a
b
b
Figure 4.3 Mean shoot dry weight of Pterocarpus indicus seedlings. Bars with the
same letter are not significantly different at P ≤ 0.05.
Shoot
dry
wei
ght
(g/
seed
ling
X
(a) (b)
88
4.3.4 Identification of strains by PCR
The five inoculant strains comprising species from Bradyrhizobium (WSM 3712,
WSM 2096), Rhizobium (R602), Sinorhizobium (WSM 2114) and Mesorhizobium
(WSM 2100) were differentiated with amplified fragment length from each other
using primer RPO1 (Figure 4.4). The strains isolated from the root nodule bacteria
had the same gel band patterns as the inoculant organisms (Figure 4.4).
bp L 1 2 3 4 5 6 L 1 2 3 4 5 6
Figure 4.4 PCR amplification profiles for detecting strains obtained from the a)
inoculants and b) nodule isolates (corresponding in lanes 1 to 6) for P. Indicus.
Strains left to right (1) WSM 3712, (2) WSM 2096, (3) R 602, (4) WSM 2114, (5)
WSM 2100 and 6) TAL 643. The standard ladder size in lane L is 1 kb.
4.4 Discussion
Knowledge of root nodule bacteria associated with P. indicus is limited. A
significant finding from this study was that P. indicus can be nodulated by diverse
strains of rhizobia from all four genera, namely Bradyrhizobium, Rhizobium,
Sinorhizobium and Mesorhizobium. Nodulation of Pterocapus indicus from slow-
(a) (b) 12,216
4072
3054
2036
89
growing Bradyrhizobium has been previously reported (Lim, 1976; Allen and Allen,
1981; de Faria et al., 1989; Oyaizu et al., 1993; Parker, 2004; Rasolomampianina et
al., 2005) but this is the first report of nodulation for P. indicus by Mesorhizobium,
Rhizobium and Sinorhizobium. This study also showed that P. indicus was able to
form root nodules with seven species of root nodule bacteria. This result provides
strong evidence to support the suggestion by Sprent and Parsons (2000) that
nodulating species of Pterocarpus, such as P. indicus, are promiscuous. It also
suggests that symbiotic promiscuity may be a benefit for legumes growing in natural
ecosystems but could also present problems for inoculation with improved strains in
the nursery or field. When there is competition for nodulation, more than one root
nodule bacteria genotype resident within the soil may be capable of nodulating the
legume (Amarger, 1981; Thies et al., 1991). This may then manifest over the year of
establishment of the legume in the field despite seed inoculation (Barran and
Bromfield, 1997; Denton et al., 2002) or may develop progressively as inoculant
strains become displaced by naturalised root nodule bacteria in seasons subsequent to
inoculation (Streeter, 1994; Evans and Turnbull, 2004). However, competition for
nodulation is often intra-specific, that is between strains of a single species of root
nodule bacteria, but there are examples of different species being able to co-nodulate
a specific legume genotype. For example, Pheseolus vulgaris may be nodulated by at
least five species of root nodule bacteria and in this case, inter-specific competition is
possible (Amarger et al., 1997).
Previous observations on nodulation in P. indicus were associated with the ability of
P. indicus to nodulate but little has been reported concerning the effectiveness of
these symbiotic relationships for increasing productivity of the host (Ssali and Keya,
1980; de Faria, 1989; Sprent, 1995; Moreira et al., 1998 and Sprent and Parsons
90
2000). Only one of the 10 strains used, Bradyrhizobium elkanii (WSM 2096) was
assumed to be partially effective for N2-fixation in P. indicus due to the visible pink
nodules and significantly increased shoot dry weight under soil limiting conditions.
Nodule effectiveness is discussed further in Chapter 5.
The nodules of P. indicus were found to be globose, single and of aeschynomenoid
type and this was similar to earlier reports of Higashi et al. (1987), Corby (1988),
Lim (1976), Allen and Allen (1981) and Sprent (2001). Similar nodule types are also
reported in other woody legumes such as Albizia paraserianthes and Acacia
auriculiformis (Corby, 1988, Lim, 1976; Allen and Allen, 1981 and Sprent, 2001).
Further inoculation studies with a wide range of strains of local root nodule bacteria,
including those isolated from taxonomically related genera such as Dalbergia, are
required so that these effective strains can be developed for potential use as
commercial inocula for Pterocarpus. Whether woody legumes require inoculation in
the nursery depends on the availability of compatible and effective root nodule
bacteria in the soils where they are to be out planted. This should be investigated
with any new plantation tree legume. The current study indicates that P. indicus is a
promiscuous host with the ability to form symbioses with both slow and fast growing
root nodule bacteria. However, the results show that this tree legume may have a
narrow range of effective root bacteria for forming a highly effective N2-fixing
symbiosis. Further work is required to quantify the role of these strains and their
effectiveness in N2-fixation. This also includes identifying more effective strains for
nursery use and to establish whether there are any indigeneous strains in silvicultural
sites in Malaysia that could promote efficient N2-fixation in this tree species.
91
In conclusion, this study showed that P. indicus can be nodulated by a diverse range
of root nodule bacteria and that it is a promiscuous host species. The ability to
nodulate however, does not necessary indicate these strains are effective for N2-
fixation as only one strain, isolated from P. indicus as WSM 2096, promoted growth
and that further studies are required.
92
Chapter 5
Growth response to nodulation and nitrogen-fixation in
Pterocarpus indicus seedlings
5.1 Introduction
In the previous chapter, a diverse range of root nodule bacteria were able to nodulate P.
indicus but only one promoted growth. The fact that nodulation can be variable and is
affected by many edaphic and climatic factors, raises the need for local nodulation
studies. These factors include soil pH (Cheng et al., 2002), water availability (Streeter,
2003) and availability of soil nutrients especially phosphorus (Manhart and Wong, 1980;
Van Kessel and Roskoski, 1981; Sprent, 2001) which are environmental factors known
to affect nodulation and nitrogen fixation in legumes. The possible preference of
ammonium-N over nitrate-N for P. indicus, reported in Chapter 2, prompted this repeat
study to use two inorganic N sources, namely, KNO3 and (NH4)2SO4. The failure to find
nodules in the field does not conclusively indicate the nodulation status of a species and
nodulation should be confirmed by inoculation tests (Chapter 4). Though nodule
inspections are relatively simple, they are technically important for exploitation and
sustainable management of nitrogen-fixing legumes.
The overall nodulation pattern in P. indicus showed a promiscuous response to
inoculation in Chapter 4. Comments on the effectiveness of nodules were based on the
internal colouration of the nodules at harvest (also indicated by Odee, 1997), shoot dry
weight and number of nodules produced (Bergersen, 1980). The number of nodules
93
produced in the plants does not necessarily indicate that they are effective. The aim of
this chapter is to confirm and investigate the nodulation pattern and ability of P. indicus
to fix nitrogen.
5.2 Materials and methods
5.2.1 Experimental design
A complete randomized block design consisting of ten strains of root nodule bacteria,
three control treatments and four replicate pots were used. The strains used were R602,
TAL 643, TAL 651, WSM 2096, WSM 2098, WSM 2100, WSM 2106, WSM 2110,
WSM 2114, WSM 3712 (Table 4.1, Chapter 4). The control treatments were: C1 (non-
inoculated and nil inorganic nitrogen), C2 (non-inoculated with inorganic N, KNO3) and
C3 (non-inoculated and with inorganic N, (NH4)2SO4. The seedlings were grown for 13
weeks from 28th March, 2005 to 30th June, 2005 in an evaporative-cooled glasshouse at
Murdoch University, Western Australia. The mean minimum and maximum
temperatures (± S.E.) were 18.6 ± 0.3 oC and 27.3 ± 0.2
oC, respectively. The procedures
used were as described in Chapter 4 except where stated below.
5.2.2 Soil preparation, inoculation and enumeration of root nodule bacteria
A sand mixture consisting of 1:1 mix of yellow sand: washed white river sand was used.
The basal nutrients were supplied as in Chapter 4 except for a reduced concentration
(mg/pot) of: K2SO4 450; MgSO4.7H2O 12.5and CaSO4.2H2O 250. Nodulation in terms
of mean number of nodules per seedling, total nodule number, % plants nodulated and
nodule location, size and colour were recorded at harvest. Other measurements taken
94
were plant height, shoot dry weight, root dry weight and total nitrogen concentrations in
the shoot and root without nodules.
5.2.3 Statistical analysis
Data were analyzed using the SPSS Statistical Analysis (version 14.0) to perform a one-
way ANOVA between the strains, shoot and root dry weight. Means were compared
using Duncan‟s New Multiple Range Test (DMRT) where the ANOVA showed there
were significant differences between means at P≤0.05.
5.3 Results
5.3.1 Nodulation
There was a significant effect (P≤0.05) of inoculation on nodulation. WSM 3712
(Bradyrhizobium sp.) formed the most nodules with a mean total of 48 per seedling
(Figure 5.1). TAL 643 was intermediate with 23 nodules per seedling. WSM 2110 and
WSM 2096 had elevated number of nodules but were not significantly different from the
remaining six inoculant treatments which had fewer than 10 nodules per seedling
(Figure 5.1). Nodulation was inconsistent among some replicates of some strains.
Bradyrhizobium strain, WSM 3712, TAL 643 and WSM 2096 were the only inoculated
treatments where all plants in all replicate pots formed nodules (Table 5.2). In WSM
2106, only 17% of the plants became nodulated. There was no nodule formed in the
control treatments.
5.3.2 Nodule location
Root nodules were mostly located on the tap and main lateral roots of the plants in the
collar region (Figure 5.2). The location of nodules has been described and used for
95
studies of agricultural crops (Howieson et al., 1995). Generally, nodules formed by the P.
indicus plants were abundant in the collar region and sparse in the lateral roots (Table
WS
M 3
71
2
TA
L 6
43
WS
M 2
11
0
WS
M 2
09
6
R 6
02
WS
M 2
09
8
WS
M 2
11
4
WS
M 2
10
6
TA
L 6
51
WS
M 2
10
0
Strains
0
10
20
30
40
50
No.
of
no
du
le p
er
se
ed
ling
c
b
abab
aa a
a a a
Figure 5.1 Mean numbers of nodules produced by Pterocarpus indicus using ten strains
of root nodule bacteria. Data are means of 4 replicates. Data with the same letters are not
significantly different at P≤0.05 using DMRT.
5.2). Large nodules formed in plants inoculated with WSM 2106, WSM 2114 and WSM
2098. Smaller nodules occurred in plants inoculated with WSM 2106, TAL 651 and
WSM 2100, and these were confined to the root collar region. The latter plants were
considered to be ineffectively nodulated.
5.3.3 Plant growth
Height
Strain
No.
of
nodule
s per
see
dli
ng
96
There was a significant increase (P≤0.05) in plant height for WSM 3712, TAL 643 and
R 602 (Table 5.3). Height increments in these plants were 20% higher than the
uninoculated control plants that were not supplied with inorganic N (C1) and were
Table 5.2 Total nodule number, % nodulated plants, nodule location, size and nodule
cross-section colour in Pterocarpus indicus.
*For all four replicates,
** Nodule distribution (%) at: A = 0-3 cm; B = 3-5 cm and C
= >5 cm from the root collar for all four replicates, #Size (%):
L = large: > 5mm, M =
medium: 3-5 mm and S = small: < 3mm in diameter
similar to plants supplied with nitrate-N (C2). However, height growth for all other
strains was inferior to the uninoculated plants supplied with nitrate and ammonium-N
(C2 and C3), respectively.
Shoot dry weight
There was a significant (P≤0.05) increase in shoot dry weight for WSM 3712 and TAL
643 (Table 5.3), being 200% and 140% higher, respectively, than the uninoculated
plants without inorganic N (C1).
Strain *Total
number of
nodules
% nodulated
plants (n=12)
**Nodule
distribution (%)
A B C
#Nodule
size (%)
L M S
Colour in
section
WSM 3712 378 100 75 14 11 4 21 75 pink
TAL 643 190 100 76 22 2 3 11 86 pink
WSM 2096 103 100 66 10 24 1 16 83 light pink
WSM 2110 92 42 53 32 15 - 11 89 white
R 602 28 50 86 7 7 4 42 54 white
WSM 2098 27 58 63 7 30 11 63 26 white
WSM 2114 19 50 100 - - 11 42 47 white
WSM 2106 8 17 63 37 - 12 12 76 white
TAL 651 10 33 60 20 20 - 60 40 white
WSM 2100 8 25 100 - - - 62 38 white
97
Figures 5.2 Nodule distribution for Pterocarpus indicus in strain WSM 3712 along the
tap root (a, b) 0-3 cm from the root collar; and (c,d) on the main lateral roots in strain
WSM 2096.
(a) (b)
(c) (d)
98
Table 5.3 Mean stem height, shoot and root dry weight of Pterocarpus indicus
inoculated with 10 strains of rhizobium or uninoculated and supplied with or without
inorganic N.
*1 = uninoculated and without N, *
2 = uninoculated with KNO3, *
3 = uninoculated with
(NH4)2 SO4, #Weight for root dry weight not included with root nodules. Data are means
of 4 replicates. Data with the same letters are not significantly different at P≤0.05 using
DMRT.
Shoot dry weight of the above two inoculated treatments was similar to the C2 plants.
Plants inoculated with WSM 3712 and TAL 643 had uniformly dark green leaves
whereas the non-inoculated controls in C2 and C3 had medium green leaves (Figure 5.3).
Leaves in all other inoculated plants remained light green and were similar to the
uninoculated control without inorganic N (C1).
Strains Stem height
(cm)/seedling
Shoot dry
weight (g)/pot
#Root dry weight
(g)/seedling
WSM 3712 7.39 ± 1.40b 0.45 ± 0.10c 0.23 ± 0.05cd
TAL 643 7.61 ± 0.68b 0.36 ± 0.05bc 0.18 ± 0.04bcd
WSM 2096 6.88 ± 0.31ab 0.28 ± 0.03ab 0.19 ± 0.02bcd
WSM 2110 6.58 ± 0.38ab 0.26 ± 0.01ab 0.18 ± 0.03bcd
R 602 7.57 ± 0.43b 0.27 ± 0.01ab 0.18 ± 0.03bcd
WSM 2098 6.57 ± 0.34ab 0.23 ± 0.01ab 0.15 ± 0.03abc
WSM 2114 6.57 ± 0.10ab 0.25 ± 0.03ab 0.20 ± 0.01cd
WSM 2106 5.98 ± 0.34a 0.17 ± 0.02a 0.16 ± 0.02abc
TAL 651 7.16 ± 0.38ab 0.25 ± 0.02ab 0.15 ± 0.01abc
WSM 2100 6.15 ± 0.35a 0.24 ± 0.02ab 0.10 ± 0.02ab
*1C1 6.18 ± 0.27a 0.15 ± 0.01a 0.08 ± 0.01a
*2C2 7.60 ± 0.29b 0.37 ± 0.09bc 0.15 ± 0.04abc
*3C3 13.11 ± 0.45c 0.78 ± 0.07d 0.27 ± 0.04d
99
Root dry weight
There was a significant (P≤0.05) increase in root dry weight for WSM 3712, TAL 643,
WSM 2096, WSM 2110, R602 and WSM 2114 (Table 5.3), being about 16-20% higher
than the uninoculated plants in C1. The root dry weight for all other strains was similar
to the controls in C1 and C2 but was inferior to the C3 which produced more root dry
weight (Table 5.3).
5.3.4 Total nitrogen content in shoot, root and whole plants
There was a significant increase (P≤0.05) in nitrogen content in the shoot of seedlings
inoculated with WSM 3712 (Table 5.4). This content was twice that of the C1 control,
and in other inoculated seedlings, and not different from the C2 control. However, the
shoot N content for WSM 3712 seedlings was four times lower than the C3 control
(Table 5.4). There was no significant difference in N content in the other uninoculated
seedlings (Table 5.4). The C3 control plants had the highest shoot and root N contents of
all treatments. Related N concentrations in the shoot and root are given in Appendix IV.
5.4 Discussion
The absence of nodules in non-inoculated control treatments (C1, C2 and C3) showed
that soil sterilization was successful and that contamination was not present. The study
confirmed the nodulating ability of the ten strains used earlier (Chapter 4) on P. indicus
and that P. indicus is a „promiscuous‟ host.
Interestingly, earlier the most effective strains for nodulation were slow-growing
Bradyrhizobium WSM 3712 and WSM 2096. Due to the fact that many “rhizobia” were
100
Figures 5.3 Effects of inorganic nitrogen and inoculation treatments on height and shoot
growth of Pterocarpus indicus plants at 13 weeks. From left to right: (a) Controls (C1,
C2, C3); b) Controls (C1, C2, C3) and WSM 3712; and (c) Front row: left to right
Controls (C2, C1) with strains TAL 651, WSM 2106 Rear row: left to right and Control
C3 with strains WSM 3712, TAL 643 and R 602.
(a)
(b)
(c)
101
Table 5.4 Total N content (mg/seedling) present in the shoot, root and total root+shoot
of Pterocarpus indicus, inoculated with 10 strains of root nodule bacteria and control
treatments that were or supplied with or without inorganic N.
C2*- N supplied as KNO3; C3
*- N supplied as (NH4)2SO4. Data are means of four
replicates. Data with the same letters are not significantly different at P≤0.05 using
DMRT.
able to nodulate P. indicus (Chapter 4 and 5) but only a few were effective, much more
research is required in commercial nurseries. None of the inoculated plants achieved the
same growth as plants supplied with adequate levels of inorganic N supplied as
(NH4)2SO4. The greater response to (NH4)2SO4 than KNO3 confirms the observation in
Chapter 3 that P. indicus is better able to utilize NH4-N for growth than NO3-N.
Treatment Shoot Root Total root+shoot
C1
(control minus N)
2.87±0.213a 1.24±0.09a 0.87±0.06a
WSM 3712 7.78±1.67c 2.19±0.30a 4.98±1.95a
TAL 643 5.50±1.04ab 2.06±0.16a 5.26±1.81a
WSM2096 5.06±0.40ab 2.03±0.15a 3.56±0.51a
R 602 4.53±0.10a 1.93±0.48a 2.82±0.82a
WSM2100 3.98±0.39a 1.88±0.30a 2.64±0.19a
TAL 651 3.52±0.33a 1.63±0.26a 2.44±0.26a
WSM 2110 4.05±0.27a 1.23±0.41a 2.22±0.19a
WSM2114 3.83±0.33a 1.47±0.11a 2.09±0.11a
WSM 2098 4.14±0.18a 1.81±0.38a 1.75±0.33a
WSM 2106 3.25±0.46a 1.17±0.29a 1.49±0.28a
C2*
(control plus N)
16.60±3.15c 3.43±0.72b 5.98±2.20a
C3*
(control plus N)
30.08±1.30d 5.69±0.58c 35.33±4.54b
102
Similar nodulation trends for slow-growing Bradyrhizobium spp. have been observed in
pot trials with related genera such as in Dalbergia sisso (De Faria, 1987; Bala et al.,
1990). Bradyrhizobium is a slow-growing genus that belongs to the non-specialized
cowpea group which is prevalent in many tropical soils (Dreyfus and Dommergues,
1981). There are some incompleted studies for Bradyrhizobium on P.indicus in the field
in Singapore and Brazil (Lim, 1976; Allen and Allen, 1981; Oyaizu et al., 1993; Sprent
and Parsons, 2000; Rasolomampianina et al., 2005) but nothing for Sinorhizobium and
Mesorhizobium. Further studies are required to understand the symbiotic relationships
under local conditions. Nodulation ability has been reported to vary among and within
plant species under different locations and environmental conditions. An example is
nodulation by P. indicus introduced into Brazil (Moreira et al., 1992; de Faria, 1998;
Perret et al., 2000). Furthermore, others include Acacia mangium in Australia (Lawrie,
1981) and Leucaena species in Fujian Province of China (Zou et al., 1998).
Unfavourable conditions such as soil pH, water, nitrogen and phosphorus levels (Chee et
al., 1989; Zahran, 1999) may alter symbiosis in the field.
Although Pterocarpus erinaceus and P. lucens was reported to be nodulated by
Bradyrhizobium elkanii (WSM 2096) and Bradyrhizobium japonicum (WSM 2097)
(Sylla et al., 1998), in this thesis however, they were ineffective in growth promotion
under N-limiting conditions. Longer trials are required to establish whether these
bacteria would be effective in the field. Because Bradyrhizobium (WSM 3712) was
originally isolated from P. indicus, it was anticipated that it would be a strong performer.
However, in pot trials it was only partially effective. In this and the previous study
(Chapter 4), it was not possible to quantify N-fixation. This was not done due to the
103
variations in nodules size even though the plants were supplied with adequate levels of
inorganic N, the fact that the majority of plants had ineffective nodules and the short
duration in which the pot trials were carried out. The presence of pink nodules indicates
functional nodules but not the rate of N fixation. The high N content for WSM 3712
plants suggest effective N fixation.
Many methods have been applied to quantify N in woody legumes. For example,
nitrogen-fixing abilities have been assessed using the acetylene-reduction assay, as
described by Hardy et al. (1968) and used by Ng and Hau (2009). Other approaches that
can be used to quantify N in woody legumes are the N balance method that estimates the
net increase in total N of a plant-soil system, 15
N natural abundance, 15
N isotope dilution
and ureide methods which aims to separate plant N into the fraction taken up from the
soil (Unkovich et al., 2008). These methods are also being used to quantify N2 fixation
in crop and pasture plants (e.g Chalk, 1985; Peoples and Herridge, 1990).
In conclusion, further trials are recommended using a broad collection of Bradyrizobium
from under forest plantations in Malaysia. These should be evaluated for N-fixation
capacity under both nursery and field conditions and compared to the best two
performers identified in this and an earlier chapter.
104
Chapter 6
Effects of phosphorus and root nodule bacteria on
root growth in Pterocarpus indicus seedlings
6.1 Introduction
The sensitivity of Pterocarpus to P fertilizer (Chapters 2 and 3) raises the question as to how
well this species is adapted to soils of low P status or with high P fixation. Phosphate
presence in the soil is regarded as one of the primary constraints for tree legume
development due to its immobilization in all but sandy soil types (Kadiata et al., 1996;
Franco and de Faria, 1997; Palmer and Young, 2000). However, no detailed research has
been undertaken on P. indicus or other taxa in this genus regarding root development and P
acquisition. In general, the root system of plants contributes significantly to
ecophysiological function for anchorage, uptake of water and nutrients, storage of
carbohydrates and synthesis of growth regulators (Gazal et al., 2004). Many plants adapt to
low soil P availability by increasing their ability to acquire P (Vadex et al., 1995;
Raghothama, 1999) and/or developing root architectures which maximize nutrient
acquisition (Ma et al., 2001; Waisel et al., 2002). These responses in P are normally
associated and enhanced through P uptake kinetics, production of cluster roots (such as in
Banksia and Lupinus species), differential root hair growth, mycorrhiza symbioses and/or
increase in root phosphatase activity (Reddell, 1993; Marschner, 1995; Gahoonia and
Nielsen, 1997; Bates and Lynch, 2000).
105
A common response in field and tree crop species is for increased allocation of biomass
below-ground in soils of low available P. This is one of the earliest responses observed in
Chapter 3 for Sesbania and Acacia but not for Pterocarpus. The objective of this chapter
was to quantify the morphological effect of early root and shoot development in response to
low soil P. It was hypothesised that the root system would show no adaptive changes to low
external P.
6.2 Materials and methods
6.2.1 Experimental design
A randomized complete factorial block design comprising two P treatments (0, 60 mg P/kg
soil, hereafter designated as P0 and P60, respectively), two rhizobia treatments (no
inoculum, inoculated with WSM 3712, hereafter designated as –R and +R, respectively) and
two harvests, hereafter designated as H1 and H2 for 3 and 6 weeks, respectively, and three
replicate pots was used. The P60 treatment was chosen because it was nearly optimum for
growth of plants in Chapter 2. The experiment was carried out during summer, from 4th
January-15th
February, 2006 in an evaporatively-cooled glasshouse. The mean minimum and
maximum (± S.E.) temperatures were 26.9 oC (± 0.3) and 29.2
oC (± 0.4), respectively.
6.2.2 Plant material, soil and basal nutrients
Seeds of P. indicus were treated and germinated in sterilized yellow sand as described in
Chapter 3. When two weeks of age, five seedlings were transplanted into yellow sand
(described in Chapter 2), that had been steam pasteurized to prevent contamination from
root nodule bacteria. Each pot contained 3 kg pot air-dried sand. The seedlings were thinned
after two weeks to three seedlings per pot to prevent self shading. Basal nutrients were
106
applied to all pots at half the dosage used in Chapter 3 due to the shorter growing period.
The total amount of nitrogen supplied was 9 mg N/pot for the duration of the trial. This rate
was chosen based on the results in Chapter 5.
6.2.3 Data collection
Shoot height was taken from the root collar to the shoot tip and tap root length from the root
collar to the root tip using a measuring ruler. The number of lateral roots and root orders
were designated on their presence from the tap root (Figure 6.1). Total root length was
determined using ASSESS: Image Analysis Software for Plant Disease Quantification, 2002
(Lamari, 2002).
The shoot was cut near the root collar of the stem and the root system washed over a 10 mm
sieve. Plant parts were dried in an oven at 70 oC for 48 hours to a constant weight. The
relative root hair density (%) was visually rated on the 1st lateral root along the tap root and
scores were given as: a) none, b) scarce and c) dense. Nodule number was recorded based on
the mean nodule number and % nodulated seedlings as described in Chapter 4.
6.2.4 Statistical analysis
Data were analyzed using the SPSS Statistical Analysis version 16 and SAS Program
version 6.12 to perform a one-way ANOVA. Means were compared using Duncan’s New
Multiple Range Test (DMRT) where the ANOVA showed there were significant differences
between means at P≤0.05.
107
Figure 6.1 Root system diversity: Lateral root order (1 and 2) from the root collar (A) of the
tap root for Pterocarpus indicus plants
6.3 Results
6.3.1 Shoot height, tap root length and relative root hair abundance
Generally, there was no difference in shoot height or tap root length across treatments at 3
and 6 weeks. The taproot obtained was almost three times longer than the shoot. Root hairs
were dense and comprising of about 89% along the taproot and lateral roots of the seedlings
throughout the duration of the study (Table 6.1).
6.3.2 Shoot and root dry weight
Shoot and root dry weights did not differ between treatments at 3 weeks (Figure 6.1a, c,
Table 6.2). By week 6, the plants inoculated with Bradyrhizobium had significantly (p<0.05)
less shoot dry weight than the uninoculated plants (Figure 6.2d). There was however, no
difference due to P treatments. The root response in dry weight was similar to that shown for
the shoot except that the P60-R plants had less biomass than the P0-R plants (Figure 6.2b).
1 2 2
1
1 2
2
A
108
6.3.3 Total root length
Total root length was significantly (P≤0.05) affected by P and R and there were 2-way and
3-way interactions (Table 6.2). Root length of P0-R plants was maximum at both H1 and H2
(Figure 6.3). Furthermore, inoculation with Bradyrhizobium (+R) appeared to suppress total
root length in P0 plants during early growth at week 3 three but had little effect in P60
plants.
Table 6.1 Shoot height, taproot length and relative root hair abundance of six weeks old
Pterocarpus indicus seedlings grown at two levels of phosphorus (P) fertiliser and without (-
R) or with Bradyrhizobium (+R). Symbols with the same letter are not significantly different
using Duncan’s New Multiple Range Test at P≤0.05. Values are means of three (3)
replicates with standard errors (S.E.) for two harvests.
* Means for two harvest **n = 72.
6.3.4 Nodulation
Nodule formation was observed at week 3. However, these nodulation were evaluated and
harvested at week 6 on all treated +R plants. There was a significant interaction between P x
R x H (Table 6.2). The plants that were inoculated with Bradyrhizobium and have adequate
P supply were also found to be necessary for high nodulation capability as these can
significantly increased nodule production by more than 60% (Figure 6.4).
Treatment
Shoot height (cm) ± S.E.
H1 H2
Tap Root length (cm) ± S.E.
H1 H2
Relative root hair
abundance (%)
Nil Few Dense
P0 - R 4.07±0.28 * 5.32±0.21 13.97±0.14b
* 14.40±0.35 - 11
** 89
P60 - R 4.07±0.20 5.47±0.22 13.57±0.14ab 14.63±0.22 - 22 78
P0 + R 3.83±0.20 5.05±0.26 13.13±0.18a 14.43±0.20 - 11 89
P60 + R 3.80±0.32 5.15±0.13 13.50±0.11ab 14.50±0.15 - 11 89
109
6.3.5 Lateral root number - orders 1 and 2
Phosphorus application significantly (P≤0.05) increased the total number of lateral roots in
order 1 at H1 and H2 and order 2 at H2 (Figure 6.5 a-c). There is also a significant effect of
lateral root (P≤0.05) on Bradyrhizobium (+R) treated plants on Order 1 and 2 (Table 6.3)
(Appendix 6.1). However, the lateral root orders 1 and 2 were significantly reduced in P
deficient plants where P was not added (P0) (Table 6.3).
Table 6.2 Analysis of variance (ANOVA) for effects of phosphorus (P), root nodule bacteria
(R) and harvest (H) on shoot and root length, shoot and root dry weight, lateral root orders 1
and 2, total root length and nodule number interactions on both harvests.
*Significant at P≤0.05, otherwise unless stated, ns = not significant
1Lateral root order 2 and nodule numbers were only obtained at H2.
Variable/
Contrast
Shoot
height
Root
length
Shoot
dry
weight
Root
dry
weight
Lateral
root
order 1
1Lateral
root
order 2
Total
root
length
Nodule
number
P 0.75ns 0.03* 0.54ns 0.22ns 0.00* 0.02* 0.00* 0.00*
R 0.13ns 0.00* 0.00* 0.00* 0.01* 0.00* 0.00* 0.00*
H 0.00* 0.00* 0.00* 0.00* 0.00* - 1.00 -
Rep 0.59ns 0.20ns 0.13ns 0.89ns 0.48ns 0.85ns 0.15ns 0.51ns
P x H 0.69ns 0.03* 0.12ns 0.16ns 0.42ns - 1.00 -
P x R 0.90ns 0.01 0.27ns 0.00 0.31ns 0.02 0.00* 0.00
R x H 0.91ns 0.71ns 0.00* 0.00* 0.24ns - 1.00 -
P x R x H 0.99ns 0.90ns 0.16ns 0.00* 0.68ns - 1.00 -
110
Figure 6.2 Effects of P treatment and inoculation with Bradyrhizobium on the root (a,b) and
shoot (c,d) dry weight in three (a,c) and six (b,d) weeks old Pterocarpus indicus seedlings.
Each data point is the mean of three replicates with standard error bars. Symbols with the
same letter are not significantly different using Duncan’s New Multiple Range Test at
P≤0.05.
a
a a
a
a
a a
(a)
(c)
(d) (b)
a
c
b
a ab
b
b
a a
111
Figure 6.3 Effects of P treatment and inoculation with Bradyrhizobium on total root length
of (a) three and (b) six week old Pterocarpus indicus seedlings. Each bar is the mean of
three replicates with standard error bars. Symbols with the same letter are not significantly
different using Duncan’s New Multiple Range Test at P≤0.05.
(a)
(b)
c
b
a ab
b
a a
a
112
Figure 6.4 Effects of P and inoculation with Bradyrhizobium on nodulation in the six week
old Pterocarpus indicus seedlings. Each data point is the mean of three replicates with
standard error bars. Symbols with the same letter are not significantly different using
Duncan’s New Multiple Range Test at P≤0.05.
Table 6.3 Effects on number of lateral root orders 1 and 2 in three (H1) and six (H2) weeks
old Pterocarpus indicus seedlings grown at two levels of phosphorus (P) fertiliser and with
(+R) and without Bradyrhizobium (-R). Symbols with the same letter are not significantly
different using Duncan’s New Multiple Range Test at P≤0.05. Values are means with
standard errors (S.E.) for two harvests.
Treatment Lateral root order 1 (L1) ± S.E.
H1 H2
Lateral root order 2 (L2) ± S.E.
H2
P0 - R 34.67±3.48ab 55.67±5.04a 46.00±6.11a
P60 - R 41.00±5.13a 69.33±4.10a 62.67±6.89a
P0 + R 25.67±1.86b 42.00±1.15b 25.00±4.04b
P60 + R 40.67±4.37a 59.33±5.36a 45.00±2.08a
a
a
b
a
b
(a)
b
113
Figure 6.5 Effects of P treatment and Bradyrhizobium on the first order lateral roots at three
(a) and six (b) weeks and second order lateral roots at six weeks (c) old Pterocarpus indicus
seedlings. Each data point is the mean of three replicates with standard error bars. Symbols
with the same letter are not significantly different using Duncan’s New Multiple Range Test
at P≤0.05.
a
b
a
b
a
b
a
b
(b)
(c)
a
b
a
b
(a)
114
6.4 Discussion
In P. indicus, root development was strongly affected by P supply. In the absence of
phosphatic fertilizer, total root length and total root dry weight were enhanced in
uninoculated plants. However, surprisingly P deficiency reduced the number of first order
lateral roots and did not seem to enhance root hairs in any visual way. Many plant species
like Arabidopsis thaliana are known to exhibit increased extension of root hairs per unit root
length under P starvation (Foehse and Jungk, 1983; Bates and Lynch, 1996; Terence and
Lynch, 2000; Zhang et al., 2003; Hermans et al., 2007; Sousa et al., 2007). This response
may be an important strategy that can enhance P acquisition by growing denser and longer
root hairs. Other components of root architecture, such as root elongation, were influenced
by the low P status of the soil. In A. thaliana, leaves responded to P deficiency by
accumulating sugars and starch in the leaves but this was not explored in P. indicus
(Cakmak et al., 1994; de Groot et al., 2003; Sanchez-Calderon et al., 2005). The other main
finding was that inoculation with Bradyrhizobium altered the plant’s response to fertilizer P.
For instance, neither root dry weight nor total root length were enhanced at low P in
nodulated plants. In addition, inoculated plants had lower biomass and less root length than
uninoculated plants. It is well known that early adaptive response for most plant species to
overcome P deficiency include enhanced internal remobilization of P through early leaf
senescence (Marschner, 1995), and an increased root to shoot ratio (Asher and Loneragon,
1967; Lambers et al. 1998) due to a greater proportion of the photosynthate being allocated
to root growth. Root morphology is important for the acquisition of P, a nutrient for which
supply to the plant depends on root exploration of soil and on diffusion to the root surface.
Many plants also respond to low P with increased exudation of carboxylates which play an
115
important role in nutrient mobilisation from inorganic sources in the soil (Gardner et al.
1983; Hinsinger, 1998; Jones, 1998). The addition of carboxylates, such as citrate, to soil
has been shown to mobilise nutrients such as P and Fe (Jones and Darrah, 1994; Lambers,
2002). Similarly, root-exuded citrate has been reported to enhance P uptake in Oryza sativa
(Kirk et al. 1999).
The mean root-shoot ratio at P0 , uninoculated plants was 0.45 at H1 and 0.48 at H2 (Table
6.4) and this was similar to uninoculated plants grown without P fertilizer in Chapters 2 and
3 (Table 6.4). However, at just adequate P (P60), the root-shoot ratio was lower in Chapter 2
than Chapter 6. The root-shoot ratio at H2 was the same as that observed in P80 plants in
Chapter 3. Differences in plants age may account for some of these differences but the
results obtained at 6 weeks in this chapter was different from older plants in Chapter 2. All
plants were grown in yellow sand with Colwell P of 4 mg/kg. Hence, other unexplained
factors must have contributed to the unrepeatability of the results between experiments. In
the Philippines, initial study on the root growth capacity or root regeneration at day 14,
showed that P. indicus transplants can response quickly to different levels of moisture
regimes within 7 -14 days, resulting in height, shoot, length of taproot, lateral root and root
total biomass increased but no further data were given (Russell, 1973; Gazal et al., 2004).
This immediate response to P. indicus was attributed to the ability of the tree species to
adapt to different environmental stress conditions. However, the nutrient status of the soil
was not studied.
There is an assumption that species with fine, extensive root systems have low external P
requirements for maximum growth and those with thick, small root systems generally have
116
Table 6.4 Comparison of mean root-shoot ratio for uninoculated Pterocarpus indicus
plants grown at low or just adequate P supply in yellow sand.
na = mean value not available
high external P requirements. It has been suggested that these intrinsic root characteristics
can be better determinants of P requirement than changes in root morphology under P
deficiency (Hill et al. 2006). Relative root hair presence in P. indicus did not increase with P
deficiency and this is contrast with plants such as Arabidopsis where root hairs tend to grow
longer and denser in response to low P availability (Foehse and Jungk, 1983; Bates and
Lynch, 1996; Ma et al. 2001). Root hairs are reported to be an important site for perception
of chemical signals with root nodule bacteria that may lead to nodule formation in legumes
(Marschner, 1995; Ridge and Emons, 2000; Jebara et al., 2005). These changes in root hairs,
such as increased extension, also enhance P uptake (Bates and Lynch, 2000; Svistoonoff et
al., 2007) by overcoming constraints imposed by the P-depletion zones around roots and
therefore P is taken up from a greater soil volume (Anghinoni and Barber, 1980; Misra et
al., 1988). Changes in root hairs probably involve auxins (Bates and Lynch, 1996; Nibau,
2008) as indole-3-acetic acid (IAA) promoted the formation of long root hairs in
Arabidopsis thaliana plants of adequate P status whereas the auxin inhibitor 2-(p-
chlorophenoxyl)-2-methylpropionic acid (CMPA) caused a reduction in root hair length in
plants grown under low P conditions (Peoples et al., 1995; Skene and James, 2000; Cahalan,
Treatment Chapter 2 Chapter 3 Chapter 6 H2
Age (weeks) 15 21 6
P0 0.402 0.440 0.476
P60
0.286 na 0.433
P80 na 0.432 na
117
2001; Sousa et al., 2007). The responses to P deficiency at week six showed that P. indicus
has an ability to enhance P uptake. This behaviour is similar to many crop plants. The
hypothesis that the root system of P. indicus would show no adaptive changes to low
external P is therefore not supported.
In this study, nodule formation was formed as early as week three but was only well
developed (pink nodules) in week six on plants that were inoculated with Bradyrhizobium in
P-sufficient plants. In earlier chapters, the period of early nodule formation was not studied.
Nodule number was significantly reduced due to P deficiency. This phenomenon was
observed in earlier trials on P. indicus where nodulation increased with sufficient P supply
(Chapters 4 and 5). Phosphorus had been reported to have an atypical pattern of effects on
nodule biomass and on host plant growth (Tang et al., 2001; Serraj, 2004). The deficiency of
P may limit nitrogen fixation through its effects on growth and survival of root nodule
bacteria, nodule formation, nodule function and host plant growth. For example, Robson et
al. (1981), Sessitsch et al. (2002), Howieson and Ballard (2004) observed that mineral
nitrogen (NH4)2NO3) increased the growth response of nodulated plants of subterranean
clover to P supply and suggested that P increased the symbiotic nitrogen-fixation through its
effect on host plant growth. In constrast, this relationship in soybean required high P supply
for optimal functioning than either the host plant growth or N assimilation in the plant (Gan
et al., 2004; Matiru and Dakora, 2004; Walch-Liu et al., 2006). For this, it has been
suggested that P deficiency affects subsequent nodule formation by restricting metabolite
supply from the host plant. In other studies, P deficiency has been demonstrated to decrease
nodule mass more than shoot growth, as well as nodule number and specific nitrogenase
activity of nodules in crops such as soybean (Singleton et al., 1985; Drevon and Hartwig,
118
1997); pea (Jakobsen, 1985), white clover (Hart, 1989) and Leucaena leucocephala
(Brandon et al., 1997). There are conflicting results that indicate P deficiency may or may
not affect nodule number per unit shoot mass (Ribet and Drevon, 1995; Vadez et al., 1996;
Drevon et al., 1997; Tang et al., 2001). However, such occurrences in nodulation capability
towards P deficiency may be associated with the legume species, genotype and experimental
conditions and as well as the inoculant strains. Interestingly, inoculated seedlings were
found to have lower biomass and less root length than the uninoculated plants. In some
plants, nodules which appeared to be a strong sink for P, with functions reliant on high P
requirements may have stored and act as a reservoir (Robson et al., 1981; Jakobson, 1985;
Hart, 1989; Vadex et al., 1995; Drevon and Hartwig, 1997).
In conclusion, this study showed that P deficiency affected root morphology resulting in
higher total root length and root dry weight in uninoculated plants but there was no marked
change in root hair morphology.
119
Chapter 7
General discussion
This is the first study to investigate nutritional requirements of Pterocarpus indicus.
Key findings were: i) seedling growth was depressed strongly in P-deficient soils
similar to most other plants but moderate rates of P fertilizer led to expression of P
toxicity symptoms; ii) foliar P concentration ranges for deficiency and toxicity were
similar to other nitrogen-fixing trees; iii) the relationship between yield and P
concentration in the youngest fully expanded leaf (YFEL) enabled critical P
concentrations to be determined for the diagnosis of deficiency (0.17% dry wt) and
toxicity (0.41% dry wt). Critical P concentrations exist for very few woody plant
species. These values can be used to help interpret foliar analysis of seedlings, and
young out-planted stock, in the future; and iv) uninoculated P. indicus seedlings
responded to P deficiency by increasing allocation of biomass to roots (increased
root length and root dry weight). Furthermore, glasshouse inoculation trials showed
that seedlings were able to form nodules with strains from Bradyrhizobium,
Rhizobium, Sinorhizobium and Mesorhizobium, suggesting P. indicus is a
promiscuous host. However, only two strains of Bradyrhizobium promoted shoot
growth, one of these was isolated from P. indicus in Malaysia and the other from
Glycine max in Africa. Another finding was that uninoculated seedlings grew better
when the N was supplied as ammonium-N than when supplied as nitrate-N.
Phosphorus, one of the main essential elements for plant growth, is often a limiting
nutrient in plantation soils. Hence addition of P is usually necessary to achieve good
120
establishment, survival and growth under adverse conditions (Ballard, 1974). The
application of fertilizer containing P can shorten forest plantation rotations especially
in areas where nutrients have been depleted following clearing of vegetation (Evans,
1992; Ndufa et al., 1999). Given that the growth of P. indicus seedlings was
markedly reduced in soils of low P status in this study, it is likely that they will
respond to fertilizer P when plantations become established on marginal soils in
Malaysia and elsewhere. This response is likely to be similar to P responses obtained
in other studies, such as for Acacia mangium (Nik Muhamad and Paudyal, 1999).
Many plants respond to a low availability of soil P by increasing exudation of
carboxylates which play an important role in nutrient mobilisation from inorganic
sources in soil (Gardner et al., 1983; Hinsinger, 1998). The addition of carboxylates
to soil, such as citrate, has been shown to mobilise P and Fe (Lambers et al., 2003;
Shane et al., 2004). Plant growth and P uptake of Lotus pedunculatus increased due
to P mobilisation from the action of exuded carboxylates on calcium phosphate
(Bolan et al., 1997). Similarly, P uptake by Oryza sativa was enhanced due to its
mobilisation by root-exuded citrate (Kirk et al., 1999).Very fast rates of carboxylate
exudation are exhibited by plants that produce cluster roots (Keerthisinghe et al.,
1998). The carboxylate composition of cluster root exudates from Banksia grandis
depends on soil type and the form of P in the soil (Lambers et al., 2002). When P
was supplied as aluminium phosphate, the major carboxylates were di- and
tricarboxylates. When P was supplied as iron phosphate, a mixture of mono-, di- and
tricaroxylates were exuded (Lambers et al., 2003).
121
Species differ in their ability to modify their root systems or in allocation of dry
matter below ground in response to low nutrient supply, but this may not be related
to their internal nutrient requirements or ability to take up nutrients from soil. For
example, Austrodanthonia richardsonii and Hordeum leporinum had a similar ability
to adjust to low P but had significantly different critical external P requirements and
growth rates under low P (Hill et al., 2006). In this study, Pterocarpus species were
sensitive to high P supply in contrast to some other leguminous tree species.
However, examination of the root morphology of P. indicus at low P supply did not
reveal any special adaptive features that might explain the response to adequate-
luxury soil P. For example, P. indicus did not develop cluster roots as occurs in some
lupins.
Symbiotic partnerships may alter the response of P. indicus to soil nutrients. For
example, it is possible that P. indicus may be able to adapt to infertile soils by
forming effective associations with mycorrhizal fungi but these were not examined in
this thesis. Furthermore, N-fixing rhizobia may alter uptake kinetics of P as, in white
clover (Hart, 1989) and soybean (Israel, 1987), nodules appeared to have a strategy
to regulate P influx which minimized P deficiency when the supply was low but
avoided excess when the supply was high. More studies are required to extend these
findings to tree legumes.
In field crops, the probability of developing symptoms of P toxicity increases as leaf
P concentrations exceed 1% dry wt (Marschner, 1995). What distinguish the
development of P toxicity in some members of the Proteaceae is not symptoms but
its occurrence at relatively low external P concentrations compared with those that
122
induce P toxicity in other species (Lambers et al., 2003; Shane et al., 2004). A
similar conclusion can be drawn for Pterocarpus. Some of the possible explanations
for the development of P toxicity in P. indicus, which require investigations are:
Interference with leaf water relations at high cellular P concentrations (Bhatti
and Loneragan, 1970). Inability to function with very low concentrations of
leaf P and capacity to translocate a large fraction of P from senescing leaves
before leaf shedding (Lambers et al., 1998).
Inability to reduce P-uptake rates at elevated, but still relatively low, external
P concentrations (Shane et al., 2003). These also resulted in the failure to
down regulate the uptake of high P in between the compartments of the roots
(Harrison and Helliwell, 1979; Raghothama, 1999; Hermans, 2006). In
addition to tolerating high P concentrations by transporting P between
compartments, roots may also down-regulate the uptake of P (Harrison and
Heliwell, 1989).
Failure to decrease the expression of genes encoding P transporters (Smith et
al., 2003). The possible existence of multiple Pi transporters genes with
different functional characteristics in plant cell membranes can result in
variation of internal P status or external concentration (Smith et al., 2003).
Plants have adapted a number of morphological and biochemical strategies to
reduce the depletion zone around roots and increase the concentration of Pi in
solution immediately adjacent to the sites of uptake. Some species also
develop highly branched root systems with more root apices that are efficient
in acquiring P. For instance, studies on Phaseolus have shown that P-
acquisition efficient genotypes have an altered geotropic response which
enables their lateral roots to grow out at angles that better explore the upper
layers of the soil (Lynch and Beebe, 1995).
Inability to store P in the roots and stems during early age of the plants and to
maintain low leaf P concentrations so as to prevent P-toxicity symptoms from
123
developing (Jeschke and Pate, 1995; Parks et al., 2000). A low P-absorption
capacity might be expected in slow-growing plants adapted to infertile soils
because: a) diffusion to the root surface is the major rate-limiting step in low-
P soils and this cannot be overcome by increased absorption capacity
(Nye,1977) and b) P demand to support growth is the major determinant of P-
absorption rate (Clarkson, 1985). In order, to temporarily avoid the toxic
effects of excessive P accumulation in the cytoplasm, P can be stored in
vacuoles, mostly as inorganic phosphate in higher plants (Raghothama,
1999).
Thus, in the future, much remains to be done in order to understand the P nutrition of
P. indicus including basic studies on the behaviour of P in the plant and identification
of storage pools of P as done previously with some tree species (Dell, 1987; Chen,
2005).
The ability of P. indicus to nodulate with diverse genera of root nodule bacteria
confirmed the promiscuous behaviour of the species. Two strains of Bradyrhizobium
were able to form functioning nodules but the work was not able to be repeated
between chapters 4 and 5. In the field, variability in nodulation in some situations
may be caused by the effect of nitrate on nodulation. Since legume symbioses do not
react uniformly to nitrate, some symbioses can delay nodulation and nodulation may
proceed when the soil reserves of nitrate-N become diminished (Howieson and
Ballard, 2004). However, this is unlikely to be the case in the pot trials as no starter
N fertilizer was used and the soil had very low soluble N levels. The duration of the
trials (3 months) should have allowed adequate time for nodulation to occur and
functionality to become established. Interestingly, the two most effective
Bradyrhizobium strains promoted growth in a commercial nursery (Figure 7.1).
From the research findings in this thesis, the following recommendations can be
made:
Firstly, care needs to be taken that rates of P supplied as hard or liquid
fertiliser are not in the range likely to cause toxicity in forest nurseries
124
producing P. indicus or in the field after outplanting. It is also essential to
identify fertiliser management practises that suit local soil conditions and
cultivation practices. Whether organic or inorganic fertilizers are used, P-
release characteristics of these should be checked and their appropriateness
for P. indicus determined. In the field, soft rock phosphate may be a safer
option particularly where fertilizer is applied close to the planting stock, such
as in the planting hole or heeled in at the time of planting. Most tropical
phosphate rock deposits are unreactive and require processing before use.
However, there are some lower-cost options to be considered include partial
acidulation, blending with soluble P sources or microbial solubilisation.
Figure 7.1 Four month old seedlings of Pterocarpus indicus in a commercial nursery
in tropical Western Australia. The two plants on the left are uninoculated, those in
the centre were inoculated with Bradyrhizobium WSM 2096 and those on the right
with WSM 3712.
125
The foliar nutrient standards, defined for P in chapter 2, can be used to inform
management decisions both for nursery stock and for seedlings after
outplanting in the first season of growth (Specht and Turner, 2006). The use
of foliar as opposed to soil analysis as indicators of site suitability has the
advantage that they are species-specific, and the concentrations present in the
leaf are the consequence of several factors, in particular, actual availability of
the element to that species given its root architecture, rate of growth and its
competitive environment (Specht and Turner, 2006). Leaves are considered
the most reliable of all plant parts and this is why foliar analysis was used in
this study on P. indicus. Foliar phosphorus has been shown in Eucalyptus and
Pinus to be positively correlated with growth (Lamb, 1977; Xu et al., 2002;
Palmer et al., 2005). Over time, critical concentrations for the diagnosis of P
deficiency can be defined in field rates trials once sites with P-limiting soils
have been identified (Simpson et al., 1998). These can then be used to
calibrate tree growth with suitable measures of soil P availability. At the same
time, foliar sampling guidelines can be established for trees of different age in
the field (Nik Muhamad et al., 1998)
The observation that P. indicus seedlings were possibly more able to use
ammonium-N than nitrate-N has ramifications for N fertilizer use. If any
starter inorganic N fertilizer is to be used in the nursery, then it should be
supplied either as ammonium-N or as urea. Also, the level of N needs to be
low so nodulation is not repressed. High levels of nitrate can be a potent
inhibitor to the plants for N2 fixation (People et al., 1995). If effective
nodulation occurs in the forest nursery, there should be no need to add
inorganic N fertilizer in the field.
Whilst the two Bradyrhizobium strains that promoted growth in pot trials
could be tried in a commercial nursery in Malaysia, more research is required
in order to identify effective strains of rhizobia for widespread commercial
application. It is important that inoculation be considered to improve the N
126
economy of commercial plantings (Howieson et al., 2000). Desirable strain
characters have been enunciated by Brockwell et al. (2005) as Table 12 on
page 73.
In the longer term, research should include development of more site specific
nutrient management practices for improving fertilizer use, considering factors such
as indigenous nutrient supply at each site. Preferably these should be done using
clonal lines of improved P. indicus.
127
Appendix I
Table 1. Glasshouse temperature for P and N trials
Year Minimum (oC) Maximum (
oC) Mean (
oC)
Mean S.E. Mean S.E. Mean S.E.
2002
Jan 21.69 0.199 32.10 0.235 26.90 0.169
Feb 21.43 0.149 30.57 0.327 26.00 0.206
Mar 21.24 0.169 29.93 0.424 25.59 0.234
Apr 19.71 0.205 27.96 0.416 23.84 0.191
May 18.55 0.196 26.34 0.901 22.45 0.509
Jun 17.31 0.198 22.50 0.518 19.90 0.301
Jul 17.63 0.316 22.04 0.304 19.83 0.236
Aug 18.14 0.280 23.48 0.214 20.81 0.205
Sep 17.93 0.460 28.34 0.502 23.14 0.354
Oct 19.10 0.487 28.31 0.427 23.71 0.362
Nov 19.62 0.333 32.54 0.601 26.08 0.344
Dec 20.88 0.501 33.88 0.581 27.38 0.496
2003
Jan 20.10 0.407 34.17 0.412 27.14 0.324
Feb 20.50 0.526 33.07 0.485 26.79 0.406
Mar 19.21 0.497 30.90 0.625 25.05 0.526
Apr 16.59 0.64 28.21 0.48 22.40 0.515
May 16.00 0.449 25.97 0.408 20.98 0.316
Jun 16.03 0.615 24.59 0.833 20.31 0.627
Jul 17.06 0.347 29.10 0.26 23.08 0.239
Aug 16.93 0.325 29.63 0.357 23.28 0.232
Sep 18.39 0.226 30.86 0.67 24.63 0.349
Oct 17.96 0.580 30.37 0.772 24.27 0.494
Nov 18.86 0.363 30.36 0.622 24.61 0.395
Dec 20.10 0.336 31.55 0.363 25.82 0.307
Note: Letters in bold/italic are the trials Chapter 2 and 4. Values are means with
standard errors (S.E.)
128
Appendix II
Table A1: Height measurement for 10 weeks old Pterocarpus indicus seedlings in
YB and YS Trials
YB trial
Treatments
Height (cm)±S.E YS trial
Treatments
Height (cm)±S.E
N0 11.625±1.143 N0 4.100±0.549
N5 12.625±0.987 N5 5.075±0.103
N10 12.500±1.486 N10 6.050±0.456
N15 11.700±1.798 N20 6.525±0.287
N20 10.125±0.427 N30 5.600±0.245
N25 10.500±1.041 N40 5.400±0.245
N30 11.050±2.438 N50 5.475±0.335
N35 11.750±0.924 N60 5.525±0.377
N40 12.125±0.554 N70 6.375±0.461
N45 12.625±1.179 N80 4.675±0.290
YB trial
Treatments
Height (cm)±S.E YS trial
Treatments
Height (cm)±S.E
P0 9.250±0.323 P0 5.425±0.397
P5 8.750±0.144 P2 5.525±0.206
P10 8.375±0.625 P4 5.500±0.453
P20 8.250±0.250 P8 5.700±0.123
P40 9.275±0.401 P16 5.600±0.212
P80 10.250±0.595 P32 5.325±0.197
P160 12.425±0.855 P64 7.800±0.337
P320 12.725±1.301 P128 6.175±0.527
P640 10.675±0.568 P256 5.200±0.498
P1280 12.200±0.363 P512 4.500±0.141
129
Appendix III
Table A2. Foliar analysis results for phosphorus and other micronutrients present
using Yalanbee soil (YB) and yellow sand (YS) trials
1(a)YB
P
mg/g
K
mg/g
Mg
mg/g
S
mg/g
Ca
mg/g
Na
mg/g
Fe
µg/g
Mn
µg /g
Zn
µg /g
Cu
µg /g
B
µg /g
P0 3.8 30.18 2.49 2.30 8.24 16.26 103.38 342.5 79.25 9.73 77.43
P5 4.2 33.47 2.98 2.01 7.49 20.40 124.33 281.90 95.90 11.33 111.3
P10 4.7 35.44 3.28 2.16 8.26 29.25 136.8 333.48 126.98 7.95 67.6
P20 5.9 37.60 2.12 2.11 6.49 37.91 102.83 255.55 99.85 7.93 64.88
P40 7.9 31.67 2.70 2.63 7.56 15.34 98.85 261.55 131.00 9.10 60.00
P80 8.6 28.49 2.42 2.93 8.28 10.12 108.33 291.63 108.33 9.23 60.48
P160 13.5 24.58 1.96 3.12 7.18 4.39 106.15 235.40 43.43 8.13 59.55
P320 34.0 24.93 1.96 3.09 9.75 5.98 101.10 306.53 53.43 9.50 70.58
P640 41.7 28.67 1.74 5.18 12.13 9.77 131.1 360.8 49.03 11.48 64.73
P1280 116.7 27.30 1.53 5.20 12.45 7.42 83.85 412.75 32.23 9.05 63.33
1(b)
YS
P0 0.70
P2 0.70
P4 0.79 35.21 2.28 3.75 7.73 0.71 148.95 398.40 34.50 15.25 23.55
P8 0.49
P16 0.80 32.81 1.97 3.19 7.49 1.38 115.25 459.10 37.80 14.10 22.90
P32 0.88
P64 1.34 28.01 2.69 3.11 7.94 0.60 68.20 288.30 39.45 13.70 20.00
P128 3.29 27.96 2.54 4.11 7.46 0.54 60.80 209.65 39.80 16.55 19.20
P256 10.15 39.72 1.67 5.17 11.73 1.04 104.90 282.35 28.00 21.75 19.30
P512 14.14
130
Appendix IV
Table A3. Effect of P supply on mean total shoot dry weight, root biomass,
root/whole plant ratio and total root length of Pterocarpus indicus seedlings.
Means followed by the same letter within a column are not significantly different at
P ≤ 0.05
YB
(mg P/kg
soil)
Shoot dry weight
(g/pot)
Root biomass
(g/pot)
Root/whole plant
(g/pot)
0 0.363 ±0.031f 0.401 ±0.042c 0.523 ±0.045ab
5 0.406 ±0.094f 0.431 ±0.036c 0.572 ±0.062a
10 0.339 ±0.039f 0.356 ±0.024c 0.516 ±0.034ab
20 0.278 ±0.043f 0.430 ±0.034c 0.542 ±0.068a
40 0.479 ±0.061f 0.482 ±0.049c 0.445 ±0.025abc
80 0.847 ±0.044e 0.623 ±0.018bc 0.425 ±0.010abc
160 2.241 ±0.027c 0.953 ±0.053a 0.306 ±0.009bcd
320 3.598 ±0.127a 0.860 ±0.208ab 0.140 ±0.038d
640 2.592 ±0.049b 0.558 ±0.065bc 0.184 ±0.024d
1280 1.818 ±0.023d 0.700 ±0.058abc 0.261 ±0.000cd
YS
(mg P/kg
soil)
Shoot dry weight
(g/pot)
Root biomass
(g/pot)
Root/whole
plant
Total root length
(cm/pot)
0 0.215 ±0.144bc 0.154 ±0.011bcd 0.402 ±0.018a 243.444 ±21.617abc
2 0.289 ±0.018bc 0.165 ±0.007bcd 0.363
±0.018ab
301.427 ±24.907abc
4 0.228 ±0.012bc 0.176 ±0.010abc 0.417 ±0.018a 250.567 ±14.859abc
8 0.240 ±0.018bc 0.179 ±0.017abc 0.428 ±0.018a 268.615 ±21.757abc
16 0.208 ±0.009bc 0.112 ±0.012cd 0.386 ±0.020a 187.960 ±16.635bc
32 0.261 ±0.021bc 0.163 ±0.016bcd 0.422 ±0.018a 236.048 ±33.519abc
64 0.508 ±0.035a 0.202 ±0.015ab 0.286 ±0.018b 363.826 ±29.478a
128 0.564 ±0.044a 0.251 ±0.014a 0.358
±0.018ab
329.108 ±44.454ab
256 0.310 ±0.039b 0.181 ±0.021ab 0.382 ±0.020a 258.898 ±34.346abc
512 0.172 ±0.016c 0.082 ±0.013d 0.430 ±0.021a 163.335 ±39.434c
131
Appendix V
Table A4. Growth media for preparing yeast mannitol agar (YMA) for
rhizobia (Based on recipe published by Howieson et al., 1988.
Inorganic nutrients Amount (g/L)
D-glucose 3g/l
Mannitol 2g/l
Yeast extract 1g/l
K2HPO4 0.5g/l
MgSO4.7H2O 0.2g/l
NaCl 0.1g/l
CaSO4. 2H2O 0.05g/l
NH4Cl 0.1g/l
Agar 15g/l
Note: 500mls of the agar solutions can prepare 20 Petri dish
132
Appendix VI
bc
c
bcabc
abcabc
abcabcab aba
d
d
C1(m
inu
s in
org
anic
N)
WS
M 3
712
WS
M 2
096
TA
L 6
43
R 6
02
WS
M 2
106
WS
M 2
100
TA
L 6
51
WS
M 2
110
WS
M 2
098
WS
M 2
114
C2 (
plu
s in
org
anic
N)
C3 (
plu
s in
org
anic
N)
Strain
0.0
1.0
2.0
3.0
4.0
5.0
% N
in
sho
ot
abc
d
cdbc
ab
abab
ab aba a
e
e
C1 (
min
us
ino
rgan
ic N
)
WS
M 3
712
TA
L 6
43
WS
M 2
100
WS
M 2
096
WS
M 2
098
R 6
02
WS
M 2
110
TA
L 6
51
WS
M 2
106
WS
M 2
114
C2 (
plu
s in
org
anc N
)
C3 (
plu
s in
org
anic
N)0.0
0.5
1.0
1.5
2.0
2.5
3.0
% N
in r
oot
c
c
a
b
ab
abab
abab
abab
ab ab
C1 (
min
us
inorg
an
ic N
)
WS
M 3
71
2
WS
M 2
10
0
TA
L 6
43
WS
M 2
09
6
WS
M 2
09
8
WS
M 2
10
6
R 6
02
WS
M 2
11
0
WS
M 2
11
4
TA
L 6
51
C2 (
plu
s in
org
anic
N)
C3 (
Plu
s in
org
an
ic N
)
Strain
0.0
0.9
1.8
2.7
3.6
4.5
% N
in to
tal pla
nt
Figure 1: Total N concentration (%) in a) shoot, b) root, c) total root+shoot of
Pterocarpus indicus seedlings. Data are means of 4 replicates. Data with the same
letter are not significantly different at P≤0.05 using DMRT.
(a) (b)
(c)
132
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