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Opportunities for New-Generation Ganoderma boninense Biotechnology
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DOI: 10.1007/978-3-030-29541-7_17
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Chapter 17
Opportunities for New-Generation
Ganoderma boninense Biotechnology
Nisha Govender, Wong Mui-Yun, and Robert Russell Monteith Paterson
1 Introduction
Malaysia and Indonesia are the highest oil palm growing countries and exporters of
palm oil and products. Straightforward establishment, high output and low cost
make the crop lucrative (Dislich et al. 2017). The industry is a crucial contributor
to the economies of various other tropical countries, such as Thailand, Nigeria and
Colombia (Liew et al. 2015).
Commercial production of palm oil was established in Malaysia in 1917 (Basiron
2007), and plantations cover 5.74 and 11.9 million hectares of land in Malaysia and
Indonesia, respectively (MPOB 2017; Indonesian Statistics Agency, BPS 2017),
85% of which is produced by Indonesia and Malaysia alone (Khairil and Hasmadi
2010; Ommelna et al. 2012). Oil palm produces 3–8 times more oil than any other oil
crop (Barcelos et al. 2015), and palm oil is a large global industry, worth over
USD50 billion annually (Murphy 2007). World palm oil production yields in
Malaysia and Indonesia increased between 1998 and 2008 by 4% annually but
N. Govender
Institute of Plantation Studies (IKP), Universiti Putra Malaysia, Serdang, Malaysia
Centre for Research in Biotechnology for Agriculture (CEBAR), University Malaya,
Kuala Lumpur, Malaysia
W. Mui-Yun (*)
Institute of Plantation Studies (IKP), Universiti Putra Malaysia, Serdang, Malaysia
Faculty of Agriculture, Department of Plant Protection, Universiti Putra Malaysia, Serdang,
Malaysia
e-mail: muiyun@upm.edu.my
R. R. M. Paterson
Faculty of Agriculture, Department of Plant Protection, Universiti Putra Malaysia, Serdang,
Malaysia
Centre of Biological Engineering, University of Minho, Braga, Portugal
© Springer Nature Switzerland AG 2020
H. Nevalainen (ed.), Grand Challenges in Fungal Biotechnology, Grand Challenges
in Biology and Biotechnology, https://doi.org/10.1007/978-3-030-29541-7_17
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with a persistent reduction in yield recently. Reasons for this decline include adverse
weather (Kamil and Omar 2016; Harun et al. 2010), restrictive government labour
and immigration policies, ageing trees (USDA 2017) and plant diseases (Ariffin
et al. 2000; Flood et al. 2011; Pilotti 2005; Susanto et al. 2005).
Oil palm cultivation causes significant environmental damage in terms of deforestation and haze production (Dislich et al. 2017). If the existing plantations can be
managed as healthily as possible, then pressure to develop new plantations may be
reduced in addition to reducing losses caused by Ganoderma disease from existing
fields (Paterson and Lima 2018). Hence, it is very important to understand how
diseases occur and how they can be controlled.
Oil palm is prone to attack by basal stem rot (BSR) caused by the fungus
Ganoderma boninense—the most serious disease of oil palm in Malaysia and
Indonesia. However, strategies for its early detection and control are ineffective.
The growing of oil palm under unsuitable climate from climate change (Paterson
et al. 2004, 2015, 2017) may result in lowered resistance to BSR (Paterson et al.
2013; Paterson 2019). A global expansion of 43% in oil palm cultivation of
15 million has been reported (Koh and Wilcove 2008). The yields of existing
plantations would be improved by controlling BSR, decreasing the pressure to
develop new plantations where it is incumbent upon the industry to maximize
production from existing fields (Paterson et al. 2004). This current review summarizes the biology and pathogenic nature of G. boninense and highlights current and
future advancements available for BSR disease management using biotechnology.
1.1
Ganoderma
The name Ganoderma was introduced by a Finnish mycologist Petter Adolf Karsten
in 1881. The identification of Ganoderma species was performed mainly on host
specificity, geographical distribution and macromorphological features of the
fruiting body. For instance, traits such as colour and the shape of the margin of
pileus and whether the fruit body was stipitate or sessile were observed. The colour
of the pileus surface and hymenophore varies from deep red, non-laccate, laccate and
light yellow to white. The morphology has been reported to differ between isolates
(Shin and Seo 1988) and is highly influenced by environmental conditions during the
basidiocarp development. The size and colour of the basidiocarp show significant
differences between specimens. However, the pore sizes are similar (Gilbertson and
Ryvarden 1986).
Generally, the Ganodermataceae show a trimitic hyphal system; however, occasional dimitic system may also be observed. The generative hyphae are hyaline, thin
walled, branched, septate or not and clamped. The youngest parts of the hymenium
and fresh specimens have shown clamp connections. Skeletal hyphae are pigmented,
thick walled and arboriform or aciculiform. Binding hyphae are usually colourless
with terminal branching. Hyphal characters are highly influenced by environmental
factors but are useful for species identification (Zhao 1989). Basidia and basidiospores are considered as the most important characters for species identification in
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basidiomycetes. The Ganodermataceae basidia attain a large size and range from
typically clavate to pyriform. Intermediate forms are often seen in the same specimen. Basidiospores show several dependable characters for identification (Seo and
Kirk 2000). Ganodermataceae have a unique double-walled basidiospore. Basidiospores of Ganoderma are ovoid or ellipsoid-ovoid, occasionally cylindric-ovoid and
always truncate at the apex. The wall is not uniformly thickened, with the apex
always thicker than the base. It is very distinctly double-walled, with the outer wall
hyaline and thinner, and the inner one usually coloured and thicker and echinulate or
not. Microscopic observations, such as the size and morphology of basidiospores,
have been adopted as the characters for the taxonomy of Ganoderma. The basidiospores commonly have double walls and are ellipsoid and brownish and vary in size
(Zhao and Zhang 1994). For G. boninense, the artificial production of mycelium,
protoplast and fruiting body has been described by Govender et al. (2016).
1.2
The Ganoderma boninense-Oil Palm Pathosystem
A plant is defined as diseased when any of the plant organ functions are disturbed or
deranged. In the Ganoderma boninense-oil palm pathosystem, the pathogen establishes within the oil palm root tissues either by root contact or sporulation and
utilizes two distinct nutrition modes during pathogenesis: biotroph and
hemibiotroph. The pathogen attaches to and penetrates the host surface primarily
via root contact. At the initial stage of infection, the biotroph pathogen shows no
visible symptoms on the host. As disease progresses, the pathogen switches to
hemibiotroph mode, evident by visible necrotic symptoms on host tissues. Penetration aided by micro-hyphae is thereafter followed by colonization whereby the
pathogen absorbs nutrients, reproduces within the host and incites the host to behave
abnormally, leading to the expression of BSR (Govender et al. 2017; Rees et al.
2009).
In old palms, the onset of the BSR is characterized by the collapse of the lower
leaves, which hang vertically downwards from their petioles. BSR manifests with
the drooping of younger leaves, which turn pale, olive green and die back from the
tip. The leaflets roll back around the rachis, and the desiccated leaves heavily cloak
the top of the stem. Next, the base of the stem blackens, gum exudation may be
observed, and fruitifications of G. boninense appear. As disease progresses, the
whole head of the palm may fall, or the trunk collapses. Infected roots show
brown colouration of the cortex and blacken stele (Rees et al. 2009). Large numbers
of fruiting bodies or sporophores are observed during disease, initially small in size
and rounded which changes into a typical bracket. The upper surface of the sporophore is reddish brown, uneven and shiny. There is a clear band at the outer edge.
The underside is pale yellow-brown, and it is covered with minute pores. The
symptoms of young palm infection are variable: the internal symptoms are similar
to those of infected old palms. The leaf openings fail, and sporophores appear at an
early stage of disease development. In addition, unopened spears are seen in the
centre of the crown (Turner 1981).
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The relationship between pathogenicity, disease and virulence during BSR development is poorly understood and differs highly amongst different infections under
various environmental factors. The sequences of events of the expression of disease
are highly complex and are interconnected. Therefore, acquiring essential understanding on each key event is critical to understanding the biology of the
G. boninense-oil palm pathosystem.
Generally, a pathogenic fungus is capable of producing three types of specialized
cellular structures, namely, the appressoria, haustoria and micro-hyphae (Nicole
et al. 1994) for host surface attachment and penetration. Apart from these specialized
structures, a pathogen also recruits several enzymes (e.g. cutinase, pectinase and
cellulase) for mechanical alteration of the host cell wall. The G. boninense produces
lignin-degrading enzymes in abundance making it different from other fungal
pathogens (Paterson 2007). Appressoria are spore-like organs derived from germ
tubes and provide anchorage for infection pegs or germ tubes with substantial ability
to overcome the host’s physical barriers such as cuticles (Hoch and Staples 1987).
Haustoria vary morphologically from short and lobed to highly branched. During
attachment and penetration, the haustorium elongates to form a continuous membrane, which adheres to the host plasmalemma (Mims 1991). Micro-hyphae, also
known as fine hyphae, are produced from the active differentiation of fungi. They
can extend for appreciable distances into host walls and cause the breakdown of host
tissues. Micro-hyphae have been reported in Ascomytina and Basidiomycotina
(Hale and Eaton 1985). For BSR, micro-hyphae with needle-shaped structures
have been reported to penetrate the root tissues of artificially infected oil palm
plantlets at early-stage infection (Govender and Wong 2017).
1.3
Lignin-Degrading Ganoderma boninense
The wood component of palm trees is made of predominantly cellulose, hemicellulose and lignin. Oil palm trunk fibre had the following chemical composition (% dry
wt, w/w): cellulose 41.2%, hemicelluloses 34.4%, lignin 17.1%, ash 3.4%, extractives 0.5% and ethanol soluble 2.3% (Paterson et al. 2009). Lignin, together with
cellulose and hemicellulose, supports the vascular system for transportation of water
and minerals. Lignification establishes the first-line defence response during pathogen invasion in plants (Maeda et al. 2011). Strong covalent bonds are formed
between lignin and cellulose which protects cellulose from degradation, and the
whole structure is referred to as lignocellulose. Cellulose is the structural polysaccharide that allows plants to remain upright. G. boninense degrades polysaccharides
and the lignin of host tissue during BSR development (Govender et al. 2017;
Paterson 2007).
Immediate lignifications have been reported to occur as a function of plant
defence mechanism. However, G. boninense can degrade the lignin produced de
novo making it different from conventional pathogens. Many studies demonstrated
the accumulation of lignin and lignin-like material during elicitor-receptor
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interaction (Nicholson and Hammerschmidt 1992; Bhuiyan et al. 2009). Elicitor
treatments (artificial infection of phytopathogens) on plants have shown to elevate
the levels of flux in the general phenylpropanoid pathway via the quantification of
enzymatic activity and relative defensive gene expression in banana (De Ascensao and
Dubery 2000) and flax (Kostyn et al. 2012). Inducible lignin differs in composition and
content in comparison to constitutive lignin synthesized for growth and development of
a plant. Elicitor-receptor interactions activate nuclear genes to induce the stress-related
enzymes and the release of other associated defensive compounds. The initial recognition between plant and pathogen commences a cascade of biochemical events, in
which each accumulating effect contributes to the expression of defence
(Narayanasamy 2011). In G. boninense-oil palm interactions, an immediate lignification has been observed at the early stage of infection (Govender et al. 2017) which may
explain the defence response against G. boninense. However, how effective this will be
is debatable as the fungus can readily degrade lignin, unlike most conventional fungi.
White rot fungi such as G. boninense were named after their ability to selectively
degrade lignin in the wood allowing exposure of the “white” cellulose (Paterson
2007). They can often degrade cellulose and hemicelluloses, whilst the lignin is
degraded. Biodegradation of lignin is an energy demanding procedure (Paterson
2007). White rot fungi produce extracellular oxidative enzymes which oxidize
unspecific one-electrons found on the benzene rings of the different lignin substructures (Chen et al. 2010) during degradation. The initial step of lignin degradation involves oxidative lignin breakdown by a series of lignolytic enzymes. The
catabolic lignin degradation involves cleavage of ether bonds that exist between the
monomers, followed by oxidative cleave of the propane side chains. Next, the
methyl group (demethylation) is removed, and finally the benzene ring is cleaved
into ketoadipic acids. Generally, it is members of the basidiomycetes that can fully
undertake these procedures; however, small numbers of ascomycetes are also characterized with the similar but lesser ability.
Amongst the wood-rotting fungi such as brown rot and ascomycetes, the white rot
fungi are the only group that is able to degrade lignin completely into water and
carbon dioxide (Seiboth et al. 2011). The fungi employ two distinct patterns for
delignification: selective and non-selective. The first degrades cellulose, hemicelluloses and lignin simultaneously, whilst the latter performs sequential decay beginning with lignin and hemicellulose and finally attacking cellulose. The patterns of
lignifications have been reported to vary amongst the different species of white rot
fungi and also amongst the strains of the same species (Eriksson et al. 1990).
However, the sequential decay is more strictly a white rot. The initial attack to
degrade lignin is reported to be oxidative, non-specific and non-hydrolytic (Higuchi
1990; Hatakka 1994). Lignolytic enzymes or the lignin-modifying enzymes (LMEs),
for example, laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase
(LiP), mineralize lignin completely into carbon dioxide and water (Lundell et al.
2010). Both MnP and LiP belong to the fungal class II. Production of Lac, a coppercontaining protein, and also a phenol-oxidizing enzyme has been reported in numerous microorganisms such as Rhizoctonia solani, Pseudomonas fluorescens (Crowe
and Olsson 2001), Trametes versicolor and Trichoderma harzianum (Baldrian 2004)
and in plants.
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In G. boninense, the following lignin-degrading and hydrolytic enzymes have
been characterized: (1) lignin-degrading enzymes (Lac, LiP and MnP) (Surendran
et al. 2018a) and (2) hydrolytic enzymes (cellulase and amylase) (Surendran et al.
2018b). Inhibition of lignin-degrading enzymes is highly recommendable for BSR
management (Paterson et al. 2008). Lignin cements the wood components of host
tissues, and therefore an immediate rupture of the lignin polymer would directly
affect the adhering and structural components such as cellulose and hemicellulose. In
a study which investigated effect of lignolytic enzymes produced in G. boninense in
axenic culture, Lac showed the highest concentration amongst the other enzymes
investigated; it was fivefold higher than LiP and MnP (Surendran et al. 2018a).
Laccase plays dual role to assist G. boninense pathogenesis; first it degrades lignin,
and second it overcomes the host defence response which includes oxidative burst of
phenolic compounds. A relatively high concentration of Lac may explain on how
this enzyme smoothly oxidizes plant defence metabolites, clearing the route to
colonization. The interaction of oil palm laccase with G. boninense laccase may be
a fruitful area of research.
1.3.1
Lignin and Other Degrading Enzymes: Strategies Towards BSR
Control
Ganoderma boninense rot of oil palm is a potentially devastating disease which has
been problematic for decades (Flood and Bridge 2000). There has been little
progress in disease control despite considerable effort (Flood and Bridge 2000;
Mohd As’wad et al. 2011; Muniroh et al. 2014; Ho and Tan 2015), related to an
incomplete understanding of the disease process as enzymatic degradation. Indeed,
cell wall degrading enzymes are the only pathogenicity factors definitely implicated
in the disease (Mohd As’wad 2015) eventually leading to a collapse of the OP
(Fig. 17.1). The biomass of the fungus will increase within the OP which acts as a
measure of disease (Mohd As’wad et al. 2011; Muniroh et al. 2014) making the
malady unlike others of OP.
The wilt caused by Fusarium oxysporum f. sp. elaeidis is better understood: the
fungus penetrates via the petioles and secretes pectinases which degrade pectin. A
gel is formed blocking the vascular transmitting cells resulting in disease symptoms
(Flood 2006; Ploetz 2006). Fatal yellowing or lethal bud rot of OP is caused by
Thielaviopsis paradoxa which attacks non-lignified tissue, providing a useful contrast to G. boninense in terms of understanding the diseases (Paterson et al. 2013),
especially as the collapse of the palm is not as frequent as in G. boninense disease,
because the structural cellulose remains intact.
G. boninense possesses overwhelming enzymatic weaponry to attack OP to
achieve growth as mentioned. The fungus produces (a) enzymes to metabolize
carbohydrates allowing it to colonize OP and (b) ligninases which degrade the
major defence of the plant, lignin. Previously, reports considered lignin as the key
factor in the disease (Paterson 2007; Paterson et al. 2008, 2009) and investigated
subsequently by others (Goh et al. 2014b; Govender 2016; Goh 2016; Tan et al. 2016).
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Fig. 17.1 Collapsed oil palm after weakening from degradation of particularly the structural
cellulose components. Red arrows indicate the infected site by the white rot, Ganodema boninense
However, it is also necessary to focus on carbohydrate metabolism which allows
establishment in the OP. In reality lignin is not present in plants as a separate
compound but is intimately attached to cellulose and other carbohydrates as lignocellulose. Hence, this chapter presents a novel and more comprehensive description,
allowing consideration of alternative control measures (Paterson 2007; Paterson et al.
2008, 2009; Mohd As’wad et al. 2011; Muniroh et al. 2014).
A similar phenomenon to BSR is the decay of dead plant material by white rot
fungi (Tuor et al. 1995; Leonowicz et al. 1999). Fungi obtain the name from the
appearance of dead, white tree trunks and from which these fungi were isolated:
whiteness comes from exposed cellulose after the lignin is degraded. White rot fungi
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can degrade uniquely the lignin in the wood to CO2 and H2O, although lignin
degradation per se will not support growth of the fungi as the process is too energy
intensive. Sufficient energy can be obtained from the cellulose after lignin is
removed (Azadpour et al. 1997).
Living OP may itself respond when it is attacked by G. boninense, although this
requires better proof. In theory it can involve OP genes being (a) upregulated to
strengthen host defences, including genes encoding pathogenesis-related proteins,
cofactors and enzymes, or (b) downregulated due to the suppression of the host
defence system by the pathogen (Goh 2016), such as the genes encoding antimicrobial peptides, and proteins (Ho and Tan 2015). Production of secondary metabolites
by the fungus (Lim et al. 2016) and phytohormones by the plant (Ho and Tan 2015)
may be employed.
The lignification response towards infection requires careful consideration
(Govender 2016; Goh 2016; Tan et al. 2016), an area of study growing rapidly.
These responses have been well-established for non-white rot fungi in other plants
(Wu et al. 1999; Paterson et al. 2009) where the increase in lignification by the plant
cannot be degraded by the non-ligninolytic pathogen. White rot fungi such as
G. boninense may produce, inter alia, laccase or phenylalanine ammonia lyase
enzymes, as does OP, where the fungal and plant enzymes will be involved in
depolymerization and polymerization, respectively, depending partly on the physiological environment of the enzymes. However, repolymerization of the lignin
degradation products is also possible by white rot fungi (Rahmanpour et al. 2016).
Determining the regulation of these enzymes upon infection of the OP could be
misinterpreted, as the plant and fungal enzymes may both be present. Comparisons
with other pathogens such as Fusarium may only give partial answers to the disease
because of this fundamental ability by Ganoderma to degrade lignin, which is
different from other pathogens. In summary, employing the hypersensitive reaction
HP response that involves lignification of cell walls by the oil palm may not be
effective because G. boninense is a very efficient lignin degrader.
1.4
Relevant Oil Palm Chemical Constituents
Oil palm (OP) contains the major polymers and carbohydrates required for a plant to
live: (a) structural carbohydrates, e.g. cellulose, hemicellulose and pectin
(Roongsattham et al. 2016), and (b) nonstructural carbohydrates, e.g. starch, glucose
and sucrose: the palm also contains lignin (Paterson 2007, 2006). The cell wall in
higher plants is composed primarily of three families of polysaccharides, cellulose,
hemicellulose and pectin, but also proteins, ions, water and lignin. The properties
and functions of these molecules and their interactions are complex which determine
the development of cells and organs and the mechanical behaviour of plant tissues
(Cosgrove 2005).
Cellulose is formed from repeating units of glucose connected by β1-4 glycosidic
bonds. It is at high concentration in most plants and is the most abundant natural
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polymer. Cellulose exists as a crystalline structure which is somewhat difficult to
degrade and in an amorphous form which is not. The two forms of the molecule are
readily degraded by organisms with efficient cellulolytic systems such as
Ganoderma (Paterson 2007; Paterson et al. 2009). Cellulose provides plants with
rigidity and strength to withstand gravity or high winds. The mechanical properties
of the plant cell wall are provided by cellulose microfibrils and xyloglucan links,
which are known to confer tensile resistance and mechanical anisotropy to the wall.
This structural network is embedded into a matrix of pectins which influences the
mechanical behaviour of the cellulose-xyloglucan network. Pectin is a
heteropolysaccharide and is one of the binding polysaccharides in plant cell walls
and is in low concentrations. There are various pectinases that are involved in its
efficient degradation (Miller et al. 1995). Starch is often an energy storage molecule
and is in OP at high concentrations. It is formed by repeating units of glucose
molecules connected by glycosidic bonds and is readily degraded by amylases.
Proteins are present in cell walls at low concentration but will provide a source of
nitrogen for invading pathogens and is degraded by proteinase (Paterson et al. 2000).
Lignin is quite different. The function is to protect cellulose from damage,
especially microbial, and it is highly recalcitrant. It can be composed of more than
one monomer, and each monomer can form various bonds to other monomers, as
such the chemical structure is highly complex. It is the second most abundant
polymer globally and is found bound to cellulose as lignocellulose (Paterson 2007,
2006). Importantly, it is degraded fully only by the white rot fungi.
1.4.1
Ganoderma boninense: Oil Palm Degrading Enzymes
The components present in OP allow G. boninense to infect the plant and form the
basis of the disease, if not representing the complete aetiology where secondary
metabolites and phytohormones may contribute (Ho and Tan 2015; Lim et al. 2016).
Surprisingly, it is only recently that ligninolytic (Goh et al. 2014b), cellulolytic and
amylolytic enzymes (Naidu et al. 2015) from G. boninense have been reported.
Naidu et al. (2015) investigated tannic acid degradation as an indicator of phenol
oxidases activity although this may not indicate an ability to utilize lignin as
Thormann et al. (2002) pointed out. Naidu et al. (2015) described xylanase, and
Miller et al. (1995) investigated pectinase isoenzymes from G. boninense, where it
was suggested that pectinases are involved in OP “tissue” degradation. The fungus
will degrade healthy OP with these enzymes in a manner akin to white rot of dead
plant material. Furthermore, G. boninense can utilize glucose as indicated in the
various media used to grow the fungus and probably sucrose. Hence, the pathogen
can produce the significant carbohydrate utilizing and ligninolytic enzymes required
to infect OP and sustain fungal growth.
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1.4.2
N. Govender et al.
Utilization of Oil Palm Constituents by Ganoderma boninense
Readily degraded, high-energy-yielding carbohydrates are required for G. boninense
establishment in OP. Nevertheless, previous authors emphasized lignin degradation
as being the most important factor in the disease, largely because G. boninense is a
white rot fungus and the protection extended to structural cellulose will cease to exist
if lignin is degraded (Paterson 2007; Paterson et al. 2008, 2009; Goh et al. 2014a).
G. boninense will degrade the digestible carbohydrates first, especially glucose, then
starch and finally free cellulose, as the fungus accesses the stem and roots of OP. The
fungus will grow into the OP producing large amounts of biomass and enzymes
leaving the indigestible lignocellulose. The Ganoderma may simultaneously produce ligninase and cellulase enzymes to degrade the lignocellulose. The final
symptom of the disease is collapse of OP from gravity or winds when the cellulose
has become incapable of supporting the tree (Fig. 17.1). The above description
provides a theoretical model of how G. boninense infects OP. Furthermore, the
steps will not necessarily be separated in time, e.g. some lignin degradation may
occur whilst starch or other carbohydrates are being degraded; G. boninense will
continue to grow on the OP after collapse and completely degrade the plant on the
plantation floor.
Furthermore, Paterson et al. (2009) suggested developing high lignin OP to resist
G. boninense attack, and Paterson et al. (2008) considered inhibitors of ligninolytic
enzymes as a potential method to control the fungus. Inhibitors of cellulase
(e.g. Hildebrand et al. 2016), amylase (e.g. Sun et al. 2016) and glycolysis
(e.g. Ugochukwu and Babady 2003) may be a more effective and immediate approach
to control the disease. Ergosterol analysis (Mohd As’wad et al. 2011; Muniroh et al.
2014) will be effective at determining the success of potential control methods because
it determines the amount of growth in OP which is otherwise problematic (Steudler and
Bley 2015).
1.5
Biotechnology of Lignocellulose in Understanding Basal
Stem Rot
Interestingly, the greatest understanding of lignocellulose degradation is obtained
from biotechnology. Ruminant animal feed or biofuel can be produced by increasing
enzymatic access to cellulose, in otherwise indigestible crop byproducts (e.g. straw)
because of high lignin content. Increasing the digestibility of these wastes by, for
example, treatment with white rot fungi can potentially lead to them being used
because the cellulose is available for further conversion. The production of ethanolic
biofuels is possible by increasing accessibility to cellulose by treatment with white
rot fungi. By degrading cellulose, glucose is produced, which can then be fermented
to produce ethanol (García-Torreiro et al. 2016).
By analogy, G. boninense degrades the lignin in OP and allows the fungus to
further metabolize cellulose and create biomass for further infection and ultimately
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collapse of the palm (Fig. 17.1). The optimal method for determining to what extent
plant material is suitable for fungal degradation is by sequential enzymatic treatment
in vitro and which is referred to as a digestibility assay (Aufrère and Guérin 1996;
VanSomeren et al. 2015).
G. boninense BSR of OP requires to be considered from a biochemical point of
view. We present a novel theory of how G. boninense will degrade OP based on a
general understanding of fungal physiology. The fungus will colonize OP using the
readily available carbon sources first (e.g. glucose), and then it will degrade significantly the more amenable polymers such as starch and unprotected cellulose
allowing the biomass to increase once established. The final stage will be when
the fungus degrades the lignocellulose allowing access to structural cellulose. It may
be beneficial in terms of control to inhibit glycolysis at the early stage rather than
inhibiting ligninolytic enzymes when the fungus is well-established (Paterson 2007;
Paterson et al. 2009). Finally, Ganoderma disease of OP requires consideration as
one of carbohydrates and not only lignin biodegradation, for suitable disease control
method establishment.
1.6
1.6.1
Biotechnological Approaches in Understanding
Ganoderma Basal Stem Rot and Novel Control Strategies
Ganoderma boninense Genome and Transcriptome Data
Presently, there are two G. boninense genome assemblies found under the NCBI
depository. Draft genome sequence of G. boninense strain G3 (accession number
GCA002900995.2) is 79.24 Mb with 55.9% GC content. It attained 495 scaffolds,
N50 scaffold length of 272.644 Kb and a maximum scaffold size of 1,452,774 bp.
There are 26,226 predicted coding sequences (CDS) with an average of 330 genes
per Mb sequences (Utomo et al. 2018). On the other hand, the G. boninense strain
NJ3 (accession number GCA001855635.1) has a total sequence length of 60,325,
844 and 18,903 number of contigs. Table 17.1 indicates publicly available genome
and transcriptome data associated to G. boninense and G. boninense-OP
pathosystem. High-throughput next generation technology coupled with vigorous
bioinformatics analyses creates an avenue for G. boninense pathogenesis and housekeeping candidate gene identification. Despite flourishing downstream application
of bioinformatics tools in other fungi organisms, G. boninense remains understudied
in regard to system-level gene function studies, followed by biotechnology manipulation. Gene co-expression network model is not available for G. boninense. Most
studies have focused on host transcriptome data, whilst similar studies on the
counterpart pathogen remain scarce.
There are very few reports found in relation to G. boninense transcripts expressed
during Ganoderma-oil palm interaction. Ho et al. (2016) utilizing mass RNA
sequencing and de novo assembly of RNA-seq were able to detect a high number
of Ganoderma transcripts involved in lignin metabolism such as manganese
muiyun@upm.edu.my
muiyun@upm.edu.my
Data sets presented are reported as of 19 April 2019
Leaf transcriptomes of oil palm (Elaeis
guineensis Jacq.) control (uninfected) and
infected by Ganoderma boninense
SRA
Oil palm transcriptome during early interaction
with necrotrophic Ganoderma boninense
SRA
Ganoderma boninense PER71 transcriptome or
gene expression
Genome sequencing and assembly of Ganoderma
boninense strain NJ3 (monoisolate)
SRA
SRA
Study
Genome sequencing and assembly of Ganoderma
boninense strain G3
Source
SRA
ERP019877
ERX 2940247
ERX 2940246
ERX 2940245
ERX 2940244
ERX 2940243
ERX 2940242
ERX 2940241
ERX 2940240
ERX 2940239
ERX 2940238
ERX 2940237
ERX 2940236
ERX 2940235
ERX 2940234
ERX 2940233
ERX 2940232
SRX809922
GCA_001855635.1
Gene bank
accession number
GCA_002900995.2
Illumina
HiSeq
2000
Illumina
HiSeq
2000
Illumina
HiSeq
2000
Illumina
HiSeq 454
Platform
PacBio
Paired
Paired
Layout
Assembly
level:
contig
Assembly
level:
contig
Single
Table 17.1 Ganoderma boninense-oil palm associated genome and transcriptome data publically available
Universiti
Kebangsaan
Malaysia
Universiti Putra
Malaysia
Universiti Putra
Malaysia
CIRAD
Institute
PT SMART Tbk
15
Apr
2015
8 Dec
2014
3Dec
2018
28 Oct
2016
Date
26 Jan
2018
PRJEB17971
PRJNA269646
PRJEB27915
PRJNA287769
Bio-project
PRJNA421251
488
N. Govender et al.
17
Opportunities for New-Generation Ganoderma boninense Biotechnology
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peroxidase and laccases, transcripts encoding cell wall degrading/modification
enzymes such as glycoside hydrolases and glycosyltransferases from various families, carbohydrate-binding modules, cellulases and polysaccharide lyase. This finding corroborates with reports on the detection of corresponding enzyme activities in
G. boninense as described in Sects. 1.4.1 and 1.4.2.
More recently, the transcriptome of G. boninense at three different stages
(monokaryon, mating junction and dikaryon) was reported (Isaac et al. 2018). The
data made publicly available will be useful for investigation of the mating process of
this fungus. However, annotation and functional studies of these differentially
expressed genes at different stages are yet to be carried out.
1.6.2
Genetic Transformation of Ganoderma boninense
Agrobacterium tumefaciens is a soil-borne, gram-negative, rod-shaped bacterium.
The motile bacterium causes crown gall disease in economically important crops
such as grapes and apples. The crown gall or tumour tissues are manifested in hosts
during infection. It transfers bacterial genes into plant genomes during infection. The
startling feature of this microbe is its ability to allow inter-kingdom gene transfer.
This unique feature has been adopted in the field of biotechnology to assist genetic
transformation of plants. A. tumefaciens infect plants at wound site resulting from the
chemotaxis response by damaged plant which releases sugars and phenolic compounds and subsequently causes disease symptoms. Crown gall formation is associated with the presence of Ti (tumour inducing) plasmid in A. tumefaciens. Part of
this plasmid is being transferred into plant genome, and the transferred region of
plasmid is known as the transfer-DNA (T-DNA). Next, T-DNA successfully integrates into the plant genome, and genes coding for tumour tissues are expressed, thus
resulting in the formation of crown gall. The T-DNA carries genes that encode
proteins involved in both hormone synthesis (auxin and cytokinin) and biosynthesis
of novel plant metabolites such as opines and agropines (Michielse et al. 2005).
Agrobacterium receives signals such as phenolic compounds and sugars which
are released by the wounded plant. These substances are the product of plant defence
mechanisms, being involved in phytoalexin and lignin synthesis. The substances
render plants cells’ competence for transformation via vir gene induction which
concurrently takes place in Agrobacterium. There are number of virulence genes
present in Agrobacterium. The Vir-A senses phenolics and auto-phosphorylates
which subsequently activates Vir-G, whereas an induced Vir-G would serve as the
precursor for the expression of all the vir genes. Sugar compounds such as glucose,
galactose and xylose are vir gene induction enhancers. Upon vir gene induction,
T-strand production takes place. The left and right borders are recognized by a
Vir-D1Vir-D2 complex, and vir-D2 produces single-stranded nicks in the DNA. The
T-DNA-Vir-D2 complex is exported from the bacterial cell by a T-pilus composed
of proteins encoded by the Vir-B operon and Vir-D4. Vir-E2 and Vir-F are also
exported from the bacterial cell (Michielse et al. 2005).
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Agrobacterium-mediated transformation of phytopathogenic fungi is widely used
to understand the molecular basis underpinning numerous plant diseases inflicting
economically important crops throughout the world. With advent technologies, a
descriptive molecular insight of host-pathogen interaction offers crucial information
on both the host tolerance/susceptibility and pathogen virulent factors. Factors that
influence host specificity and the role of bioactive compounds secreted by host in a
pathosystem can be determined (Nakamura et al. 2012). The Agrobacterium-mediated transformation method is a powerful research tool for gene identification and
functional analysis (Jones 1994). The knowledge provides significant potential for
direct elimination of varietal factors contributing to disease development. Nevertheless, optimization of an efficient Agrobacterium-mediated transformation protocol
for fungi is often considered challenging primarily for the following reasons:
(1) variable nutrient response, (2) variable selection response, (3) environmental
conditions and (4) evolving pathogenicity.
Disease development recruits compatible host-pathogen interaction, as proposed
by the gene-for-gene concept on flax rusts (Flor 1956). A fungal pathogen exerts a
high degree of variation to support their survival at adverse environments
(Narayanasamy 2011). The pathogenicity genes of a virulent G. boninense underpinning the expression of BSR remain unknown. Through next generation sequencing technology and bioinformatics, the gene sequences that are highly expressed
during disease could be determined (transcriptome data). To assign function on a
putative gene, the Agrobacterium-mediated transformation method is employed to
unravel gene function in respect to pathogenicity (Malonek and Meinhardt 2001). In
the case of G. boninense, an efficient and species-specific Agrobacterium-mediated
transformation protocol has been developed for tagging G. boninense with green
fluorescent protein (GFP) gene and tracking mycelia penetration in oil palm roots
(Govender and Wong 2017). Upon recognizing genes responsible for pathogenicity,
a number of selection pressures can be performed on the host to disturb the
compatible relationship between host pathogens. For example, it is possible to
apply chemicals to discriminate the protein products derived from pathogenicity
genes. In addition, introduction of host resistance genes, which counter-attacks the
virulent genes, creates an avenue to complete resistance. Therefore, molecular
studies to understand gene functions underlying G. boninense pathogenicity are
the foremost fundamental step towards BSR management.
1.6.3
RNA Silencing in Fungi
RNA silencing is a natural cellular process used by most eukaryotes in developmental gene regulation and defence responses and was first discovered in the nematode,
Caenorhabditis elegans (Fire et al. 1998). In fungi, the phenomenon is called
quelling, and there are two RNA silencing pathways operating at different stages
in the life cycle of a fungus: quelling in the vegetative phase and meiotic silencing by
unpaired DNA (MSUD) in the sexual phase (Siu et al. 2001), each with discrete set
of RNA silencing components. Quelling is equivalent to RNA interference (RNAi)
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in animals or post-transcriptional gene silencing (PTGS) in plants, as all these
pathways utilize typical RNA silencing components such as RNase-III-like enzyme
(DICER), Argonaute (Ago) proteins and RNA-dependent RNA polymerase (RdRP)
to target complementary mRNA for degradation and triggered by double-stranded
RNA (dsRNA) molecules (Fagard et al. 2000). MSUD pathway consists of enzymes
with similar functions as those in the RNAi pathway (Siu et al. 2001). RNAimediated gene silencing was demonstrated in fungi of various phyla—Ascomycota,
Basidiomycota and Zygomycota—and of the oomycetes such as Phytophthora spp.
(Nunes and Dean 2011). RNAi was also demonstrated to silence a complete gene
family (Salame et al. 2010).
1.6.4
Bidirectional Cross-Kingdom RNA Silencing
RNAi is a powerful genetic tool to study the function of genes in an organism. The
application of RNAi is not restricted to gene function studies. The discovery of
mobile small RNAs (sRNAs) that act as RNAi signals across kingdoms presents a
new innovative strategy for crop protection. RNAi signals are mobile small RNAs
(sRNAs) including short interfering RNAs (siRNAs), microRNAs (miRNAs),
tRNA-derived RNA fragments (tRFs) and piwi-interacting small RNAs (piRNAs),
where siRNAs and miRNAs are studied more extensively in plants and animals
(Nunes and Dean 2011). However, sRNAs in fungi are largely unknown.
MicroRNA-like RNAs (milRNAs), Dicer-independent small interfering RNAs
(disiRNAs) and tRFs were reported in Neurospora crassa, Magnaporthe oryzae
and Aspergillus fumigatus (Nunes and Dean 2011). Computational analysis
performed on the genome of Ganoderma lucidum, the medicinal mushroom,
revealed 89 potential miRNAs, and they were differentially expressed in different
developmental stages (Mu et al. 2015). Cross-kingdom RNAi has been observed in
animal and plant systems (Weiber and Jin 2015). RNAi signals originating from
plants were found in their interacting organisms such as fungi, oomycetes, nematodes, pests and parasitic plants (Weiberg et al. 2015). Conversely, fungal pathogens
were found to transfer sRNA effectors into their host plants to suppress host
immunity genes (Weiberg et al. 2013). Application of sRNAs or dsRNAs on host
surfaces infected with fungal pathogens confers plant protection in the case of
Fusarium graminearum and Botrytis cinerea (Koch et al. 2016; Wang et al. 2016).
1.6.5
RNAi as an Efficient Tool for Functional Genomics Study
in Basidiomycota and Ganoderma boninense
RNAi has proven to be a powerful genetic tool for gene function study in fungi due
to the ability to attenuate expression of genes at different levels whilst keeping the
organism viable though involving target genes important for cell survival
(Nakayashiki et al. 2005). For plant pathogenic fungi, the process of infection in
the host plant consists of several key steps such as spore production, penetration
structures, development of invasive hyphae in the host cells, dealing with host
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defence and colonization in the hosts. Within Basidiomycota plant pathogenic fungi,
such as Puccinia striiformis f. sp. tritici, RNAi was employed to study genes that are
involved in infection of host plants such as invertase, Ran (an important family of
small GTP-binding proteins) and MAPK cascades (Chang et al. 2017; Cheng et al.
2016; Zhu et al. 2017), in addition to hydrophobin, peroxiredoxin, effector proteins
and mating type gene in Moniliophthora perniciosa, causal agent of witches’ broom
disease of cacao (Caribe dos Santos et al. 2009; Lawrence et al. 2010; Laurie et al.
2008).
In G. lucidum, RNAi-mediated gene silencing was used to study the roles of 14-33 proteins responsible for multiple physiological processes and demonstrated that
two homologs were involved in mycelial growth, hyphal branching, ganoderic acid
biosynthesis and response to abiotic stress (Zhang et al. 2018). However, the
application of RNAi to study developmental or virulent genes in G. boninense is
lacking even though the genome has been sequenced (Utomo 2018). The role of
genes involved in ergosterol biosynthetic pathway in G. boninense utilizing RNAimediated gene silencing is currently being investigated in the current authors’
laboratory. Other potential developmental/virulent genes encoded in the genome
of G. boninense, such as genes related to lignolytic enzymes, laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP), and genes related to small RNA
effectors, DICER-LIKE (DCL) and Argonaute (AGO), should also be studied for
their gene functions. The identification and verification of such candidate genes are
crucial for the application of these target genes in RNAi-based crop protection such
as host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS)
which are described in the section below.
1.6.6
RNAi as a Tool for Potential Crop Protection Strategy Against
Diseases Caused by Basidiomycota and Ganoderma boninense
RNAi-based crop protection is a novel strategy that is simple and environmentalfriendly. The efficiency of RNAi to improve control of bacteria, viruses, fungi,
insects, nematodes and parasitic weeds was reported (Saurabh et al. 2014). There
are several types of RNAi-mediated gene silencing. Virus-induced gene silencing
(VIGS) employs engineered viruses by replacing some portion of the viral genome
with the target gene to be silenced. When plants were infected with recombinant
viruses containing homology sequence to the target gene and challenged with the
corresponding pathogen, the silencing mechanism in the plants will be activated and
thereby causing the silencing of the target gene in the pathogen (Cooper and
Campbell 2017). Host-induced gene silencing (HIGS) was developed using genetically modified plants expressing sRNAs with high homology to the target genes of a
pathogen, usually genes involved in infection.
HIGS has great potential to be utilized as an effective crop protection strategy in
the near future. However, the application of HIGS requires the transformation of
plants with efficient RNAi vector, and this causes public concern regarding genetically modified organisms (GMOs). Furthermore, established crop transformation
protocols are unavailable for most crop plants. These concerns gave rise to the
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development of spray-induced gene silencing (SIGS). Following the success in crop
protection of agricultural insects and nematodes utilizing exogenous RNA molecules
targeting their essential genes, Koch et al. (2016) and Wang et al. (2016) demonstrated that external application of sRNAs targeting cytochrome P450 lanosterol
C-α-demethylases of Fusarium graminearum and DCL genes of Botrytis cinerea
inhibited fungal growth and conferred plant protection in barley and fruits and
vegetables, respectively.
In basidiomycetes, studies on RNAi-mediated silencing for disease control are
lacking, where only a few studies had been reported, mainly on rust fungi and
Rhizoctonia solani employing VIGS and HIGS strategies (Cooper and Campbell
2017; Chang et al. 2017; Zhou et al. 2016; Zhu et al. 2017; Yang et al. 2018). For
Ganoderma species, there is a lack of information on studies related to application of
RNA silencing in crop protection. A study on the potential application of RNA
silencing targeting DCL genes of G. boninense to confer protection against basal
stem rot is in progress in the current authors’ laboratory. The availability of
G. boninense genome data in public database (NCBI) enables potential candidate
genes to be identified for testing and designing of efficient silencing constructs to
avoid off-target transcripts, whilst the availability of the oil palm genome data helps
to ensure the silencing constructs do not target and negatively affect the host. Due to
substantial economic losses caused by Ganoderma BSR disease to the oil palm
industry in Malaysia and Indonesia, there is an urgent need to develop RNA-based
crop protection strategy to control this devastating disease. In addition, due to the
nature of Ganoderma infection which is protected in the roots and basal stems of its
hosts, external application of RNA molecules as mobile signals to trigger host
immunity is expected to be a powerful and cost-effective strategy to combat this
disease. RNAi-based strategy will be the crop protection of the future.
1.6.7
CRISPR/Cas Systems
Clustered regularly interspaced short palindromic repeats (CRISPR) were found in
bacteria and archaea to provide adaptive immunity against bacteriophages (Barrangou
et al. 2007). CRISPR-CRISPR-associated protein 9 (Cas9) which is from the type II
CRISPR system is one of the most famous genome editing tools and was first described
in 2012 (Gasiunas et al. 2012). CRISPR-Cas9 system comprises of two crucial
components: (1) Cas9 nuclease and (2) single-guide RNA (sgRNA) comprised of
small, interfering CRISPR RNAs (crRNAs). Gasiunas et al. (2012) demonstrated that
the Cas9-crRNA complex functions as an RNA-guided endonuclease that uses RNA
for target site recognition and Cas9 for DNA cleavage. Subsequently, triggering the
host’s DNA repair process comprises of non-homologous end joining (NHEJ) and
high-fidelity homology-directed repair (HDR) pathways (Qin et al. 2017). CRISPRCas9 technology provides an extremely precise and powerful tool for modifying
genomes compared to other genome editing tools such as zinc-finger nucleases
(ZEN) and transcription activator-like effector nucleases (TALEN), with countless
potential applications particularly in crop improvements such as increased abiotic stress
or pests and disease tolerance and higher-yielding varieties.
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1.6.8
N. Govender et al.
CRISPR/Cas9 as an Efficient Tool for Functional Genomics Study
and Potential Sustainable Crop Protection Strategy Against Fungi
Particularly Ganoderma boninense
For plant pathogenic fungi and fungal-like pathogens, the application of CRISPRCas9 for functional genomics studies leading to application for crop protection is
lacking. There are some studies done in Sclerotinia sclerotiorum, a necrotrophic
fungal pathogen causing rotting and blighting diseases in a broad range of host plants
(Li et al. 2018); Trichoderma reesei, used as a biocontrol agent against plant
pathogens (Liu et al. 2015); Beauveria bassiana, a popular entomopathogenic
fungus used as a biocontrol agent against insect pests (Chen et al. 2017); and
human fungal pathogens such as Aspergillus fumigatus, Cryptococcus neoformans
var. neoformans, C. neoformans var. grubii, Candida albicans and C. glabrata
(Mitchell 2017) as well as in oomycete, Phytophthora sojae (Fang and Tyler 2016).
As for fungi from basidiomycota group, very few studies related to the employment
of CRISPR-Cas system for functional genomics studies were reported. Five genes of
the effector eff1 family of the smut fungus, Ustilago maydis, were simultaneously
disrupted using CRISPR-Cas9 technology (Schuster et al. 2016; Schuster et al. 2017)
as well as in G. lucidum and G. lingzhi (Qin et al. 2017). For pathogenic Ganoderma
species such as G. boninense, the potential of employing CRISPR-Cas9 system for
functional genomics studies and as crop protection strategy is great considering the
availability of G. boninense genome for genetic manipulation and unavailability of
effective control measure in the field. Such study targeting polygalacturonases and
URA3 genes of G. boninense is in progress in our laboratory.
As with RNAi technology, CRISPR-Cas9 technology makes it possible to study
multiple gene functions in an efficient manner for underexplored G. boninense, and
the technology is considered a non-transgenic strategy and, thus, may not be
regulated under the existing regulations governing genetically modified organisms
(GMOs) and will sidestep many of the consumer concerns over these GMOs.
Furthermore, the edited traits are inherited in the next generations, thus the sustainability of CRISPR-Cas9 technology for crop protection. Similarly, CRISPR-Cas9
technology could be applied in the host, oil palm, to produce genotypes that are
resistant against G. boninense.
2 Conclusion
In this chapter, we focused on Ganoderma boninense-oil palm pathosystem and how
fundamental pathology studies in line with modern genomic tools could be
employed to understand key pathogenicity genes involving lignin biodegradation
during basal stem rots of OP. Genetic modification of oil palm, though feasible, may
pose huge risk and cost as replanting new planting materials, replacing the existing
ones, requires ample of time and management. In addition, GM plants are
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homogenous, and with climate change predicted and currently taking place, GM
planting materials would not fit to withstand the adversely changing environmental
conditions. Ideally, basal stem rot of OP could be managed with compounds such as
small/large polymers or peptides or RNA molecules that would counter-attack or
inhibit pathogenesis factors of G. boninense. We have summarized the research
status of G. boninense and discussed future prospective fitted for a sustainable BSR
disease management.
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