Current Research Journal of Biological Sciences 3(3): 195-208, 2011
ISSN: 2041-0778
© Maxwell Scientific Organization, 2011
Received: February 04, 2011
Accepted: March 08, 2011
Published: May 05, 2011
In Search of Target Gene(s) to Quantify Pea Pathogenic Nectria haematococca
in Agricultural Soils
1,2
Ebimieowei Etebu and 3A. Mark Osborn
Department of Biological Sciences, Niger Delta University,
Wilberforce Island, Bayelsa State, Nigeria
3
Department of Biological Sciences, University of Hull, Cottingham Road,
Hull, HU6 7RX, United Kingdom
1
Abstract: Footrot disease due to N. haematococca (anamorph Fusarium solani f. sp. pisi) is a globally,
economically important disease of peas. The disease has been linked to the presence of six pea pathogenicity
(PEP) genes (PDA1, PEP1, PEP2, PEP3, PEP4 and PEP5) inherent in pathogenic forms of the causal fungus
N. haematococca MPIV. The disease is prevented only through avoidance of fields with high disease potential.
Identifying agricultural fields with a high disease potential prior to pea cultivation has been paramount in the
implementation of preventive measures. Although molecular techniques have been successfully used to quantify
pathogenic strains of N. haematococca in agricultural soils, targeting all six pathogenicity genes in these assays
would not be cost effective. This study therefore attempts to review the functions and roles of the different
genes linked with pea pathogenicity with the aim of identifying gene(s) that would serve as a logical target in
a quantitative molecular assay. Findings suggest that, whilst the PDA gene may be targeted in a preliminary
diagnostic measure, a conclusive assay, targeting the PEP3 gene may be required to affirm pea footrot disease
potential of agricultural fields. Agricultural fields with the PEP3 gene copy numbers of up to 100 per g soil
prior to cultivation may be deemed unsafe for peas.
Key words: Footrot disease, Fusarium solani f. sp. pisi, Nectria haematococca, pathogenicity genes, pea,
Pisum sativum
INTRODUCTION
N. haematococca (anamorph F. solani f. sp. pisi) is the
most important fungus (Hwang and Chang, 1989;
Sanssené and Didelot, 1995) being pathogenic on all
commercial processing pea cultivars (Hagedorn, 1991;
Grünwald et al., 2003), and contributing to pea yield
losses of up to 57% (Kraft, 1984; Oyarzun, 1993).
Footrot of peas due to Nectria haematococca
(anamorph Fusarium solani f. sp. pisi) is characterized by
reddish brown streaks at the primary and secondary roots
which later coalesce to form a dark reddish-brown lesion
on the primary root up to the soil line. Externally,
symptoms are characterised by stunted growth, yellowing,
and necrosis at the base of the stem (Kraft and
Kaiser, 1993; Kraft, 2001).
The disease has been linked to the presence of six pea
pathogenicity (PEP) genes (PDA1, PEP1, PEP2,
PEP3, PEP4 and PEP5) inherent in pathogenic forms
of the causal fungus N. haematococca MPIV
(Funnell et al., 2002; Funnell and VanEtten 2002;
Han et al., 2001; Temporini and VanEtten, 2002). These
PEP genes are clustered together on a conditionally
dispensable (CD) 1.6 Mb chromosome (Han et al., 2001;
Temporini and VanEtten, 2002; VanEtten et al., 1994).
Peas are grown in over 87 countries all over the
world (McPhee, 2003), providing food for humans and
feed for domestic animals (Hargrove, 1986; Hulse, 1994;
Patriarca et al., 2002). Total world production has
increased tremendously over the years amounting to about
10.5 million tonnes dry pea and 7 million tonnes fresh
peas (Duke, 1981; FAO, 1994, 2001).
Although, peas have enormous nutritional qualities
and have been considered to be the predominant export
crop in world trade, representing about 40% of the total
trade in pulses (Oram and Agcaoili, 1994), root (footrot)
rot disease has been identified as a major production
constraint (Biddle, 1984; Graham and Vance, 2003). Root
(foot) rots are known to occur wherever peas are grown
in the world (Fenwick, 1969; Hagedorn, 1976;
Persson et al., 1997).
Root rot of peas is caused by a variety of pathogenic
fungi, most notably Phoma medicaginis var. pinodella and
Nectria haematococca (anamorph Fusarium solani f. sp.
pisi) that are responsible for pea footrot disease in
particular (Hagedorn, 1991). Among the combination of
fungi responsible for root rot disease complex in peas,
Corresponding Author: Ebimieowei Etebu, Department of Biological Sciences, Niger Delta University, Wilberforce Island,
Bayelsa State, Nigeria
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Healthy pea root
Footrot symptom of infected pea root
Fig. 1: Root symptom of pea footrot disease, Disease symptoms were due to infection with N. haematococca (anamorph F. solani
f. sp. pisi) (Etebu, 2008)
pathogenic forms of N. haematococca in soils (Etebu and
Osborn, 2009; Funnell and VanEtten, 2002; Kistler and
VanEtten, 1988; Oyarzun, 1993; Oyarzun et al., 1994). In
contrast molecular (nucleic acid-based) approaches
utilising the Polymerase Chain Reaction (PCR) have been
used to selectively detect and quantify pathogenic forms
of the pathogen in agricultural soils without recourse to
culture (Etebu and Osborn, 2009, 2010) by targeting
pathogenicity genes inherent in pathogenic forms of the
fungus.
Although molecular techniques have been
successfully used to identify, and demonstrate the
presence of these genes in DNA extracted from pure
strains of N. haematococca, and agricultural soils with
prior footrot history. Targeting all six pathogenicity genes
in soil-DNA responsible for pea footrot disease in
molecular assays would not be cost effective. There is
therefore need to identify an indicator gene that would
serve as a reliable measure of inoculum density of the
pathogen. This study attempts to review the functions and
roles of the different genes linked with pea pathogenicity
with the aim of identifying gene(s) that would serve as a
logical target in a quantitative molecular assay.
So far, footrot disease in peas is prevented only
through avoidance of fields with high disease potential, as
neither genetic resistance nor chemical control is effective
in its management. Identifying agricultural fields with a
high disease potential has therefore been paramount in the
implementation of preventive measures (Oyarzun, 1993).
Soils with high disease potential are generally
characterized by high initial inoculum density of plant
pathogen(s) as the latter is correlated to disease incidence,
onset, and severity and yields of various crops (Bhatti and
Kraft, 1992; Navas-Cortés et al., 2000; Rush and
Kraft, 1986).
To this end, method(s) that would accurately
determine the population of pathogenic forms of
N. haematococca in soil prior to pea cultivation is/are
required to assess the likelihood of pea footrot disease
incidence and severity. In the past, several techniques
relying on culture-based analysis have been developed
over the years to detect and quantify various fungal
populations (including N. haematococca) in soil
(Duncan et al., 1987; Menzies, 1963; Stanghellini and
Kronland, 1985; Tivoli et al., 1987; Yarwood, 1946;
Oyarzun et al., 1994). However these approaches have
been shown to be unsuitable in the detection of
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Fig. 2: Early field symptoms of pea footrot disease (Processors and Growers Research Organisation, 1997)
PEA FOOTROT DISEASE
favoured by poor crop rotations, high soil temperatures
(22º - 30ºC), moist, acidic (pH 5.1-6.2) and low fertility
(Kraft, 1984; Tu, 1994). Also, soil compaction has been
shown to favour incidence and severity of the disease
(Tu, 1994)
Scoring or assessment of disease severity is very vital
in plant pathological experiments aimed at evaluating
disease resistance or susceptibility among different
varieties of a given species of plants, or of a given variety
of crop under different agricultural practices. Disease
severity scales often 0-5 or 1-9 corresponding to the
degree of damage in the infected plant have been
developed for many pathosystems (Infantino et al., 2006).
Research on the assessment of footrot disease on peas
has focused on laboratory and greenhouse experiments
(Han et al., 2001; Delserone et al., 1999; Ciuffetti and
VanEtten, 1996). Studies conducted to assess the response
of peas to footrot disease have often centred on a
disease index (DI) scale ranging from 0-5 to show
the differential symptomatic effect on peas infected
with Nectria haematococca (anamorph Fusarium solani
f. sp. pisi). 0 = no discolouration; 5 = totally discoloured
roots (Biddle, 1984; Grünwald et al., 2003;
Footrot of peas due to Nectria haematococca
(anamorph Fusarium solani f. sp. pisi), often termed
Fusarium root rot by some workers (Hagedorn, 1991) was
first reported in USA and Europe at about 1918
(Kraft, 2001). Spores of N. haematococca (anamorph F.
solani f. sp. pisi) germinate with 24 h in a 7 mm radius of
a germinating pea seed (Short and Lacy, 1974); triggered
by root exudates produced by the pea plant during growth
(Ayers and Thornton, 1968; Short and Lacy, 1976). The
fungus gains entry into the plant through the tap root just
above the point of cotyledon attachment (Bywater, 1959;
Integrated Pest Management, 2002; Kraft et al., 1981).
Disease symptoms are produced on infected peas as early
as 3 days of contact with pathogenic forms of the fungus
(Funnell et al., 2001); early symptoms appear as reddish
brown streaks at the primary and secondary roots and later
coalesce to form a dark reddish-brown lesion on the
primary root up to the soil line (Fig. 1). Externally,
symptoms are characterised by stunted growth, yellowing,
and necrosis at the base of the stem (Kraft and
Kaiser, 1993; Kraft, 2001) (Fig. 2). The disease is
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Cruickshank’s work formed the foundation which
established the concept that tolerance to a phytoalexin
might be important in pathogenesis (VanEtten et al.,
2001). Subsequent studies on the sensitivity of fungi to
phytoalexins, including pisatin, however, have revealed
additional exceptions to the correlation between
tolerance and fungal host range (Smith, 1982;
VanEtten et al., 1982).
Most plants resist infection caused by
microorganisms by producing antimicrobial secondary
metabolites. These are produced either during their
normal course of development or in response to pathogen
attack or stress (Kotchoni and Gachomo, 2006;
Links et al., 1929). Antimicrobial metabolites formed
constitutively within plants in course of their normal
development and growth are generally termed
phytoanticipin; known to potentially protect plants,
wherein they are formed, from attack against a wide range
of pathogens (Ingham, 1973; Mansfield, 1983;
Osbourn, 1996). Conversely, phytoalexins are produced
in response to pathogenic attack or stress. As such, they
are usually restricted to the infection locus and the
surrounding cells (Paxton, 1980, 1981; Grayer and
Harborne, 1994; Smith, 1996; Smith et al., 1982).
Interestingly, numerous studies have reportedly
demonstrated that pathogenic attack on plants elicits
phytoalexinic response in both disease resistant and
susceptible plants (Morrissey and Osbourn, 1999;
VanEtten et al., 2001). This notwithstanding,
phytoalexinic response represents one of the arrays of
induced defence mechanisms associated with plant
disease resistance (Dixon and Lamb, 1990; Lamb et al.,
1989), since the rate of production and amount produced
are significantly higher in resistant plants than in
susceptible ones (Morrissey and Osbourn, 1999).
Apparently, the mechanism of toxicity of phytoalexins
has been reported to involve disruption of membranes,
physiological and biochemical processes (Smith, 1996).
Molecular and genetic studies reveal that resistance
genes play a vital role in conferring resistance on plants
when induced by pathogenic invasion (Dangl and
Jones, 2001). They do so primarily through signal
transduction which leads to the activation of defence
genes (Dangl and Jones, 2001; Kotchoni and
Gachomo, 2006). Numerous defence genes have been
identified, most of which occur within plants as multigene
families (Corbin et al., 1987; Douglas et al., 1987;
Harrison et al., 1995). Genes associated with inducible
defence responses encode, among others, hydrolytic
enzymes such as chitinases and glucanases
(Bowels, 1990) and a number of other “Pathogenesis
Related (PR) proteins” whose functions are yet unknown
(van Loon et al., 1994). Also, in addition, these genes also
encode enzymes involved in the synthesis of antimicrobial
phytoalexins (Dixon and Paiva, 1995).
Oyarzun et al., 1997). Using the same scale
Grünwald et al. (2003) described the various levels of
footrot disease severity of the scale as follows: 0 = no
symptoms; 1 = slight hypocotyl lesions; 2 = lesions
coalescing around epicotyls and hypocotyls; 3 = lesions
starting to spread into the root system with the root tip
starting to be infected; 4 = epicotyl, hypocotyl and root
system almost completely infected and only slight amount
of white, uninfected tissue left; 5 = completely infected
root. A screening technique developed by the Perry
Foundation (2005) to evaluate the tolerance range of
commercial varieties of peas expressed footrot disease
severity as percentage discolouration of pea roots,
following infection. Thereafter, a scale of 0-5 was used to
differentiate the various level of disease severity as
follows: 0 = no discolouration; 1 = up to 20%
discolouration; 2 = 20-40% discolouration; 3 = 40-60%
discolouration; 4 = 60-80% discolouration; 5 = totally
discoloured. Etebu and Osborn (2009) in a more recent
work adopted the following scale to assess pea footrot
disease (0 = no root discolouration; 1#20%; 2 = 2140%; 3 = 41-60%; 4 = 61-80%; 5 = $81%).
Kraft and Boge (2001) in their study used differences
in root length, root surface area, and diameter as
measurable indices to compare root characteristic of peas
as affected by soil compaction and Fusarium root rot
caused by Fusarium solani f. sp. pisi. In all of these,
Infantino et al. (2006) prefers the use of percentage
scales, accommodating the full range of expression of
disease symptoms. Additionally, they opined that scoring
of root diseases of legume crops may be done on the
above ground organs, on principal roots or other plant
tissues.
As mentioned earlier, pathogens are able to cause
disease in plants after they get established within the
tissues. To get established within plant tissues plant
pathogens do overcome various barriers posed by the
plant, one of which is a group of secondary metabolites
collectively referred to as phytoalexins (Agrios, 1997).
Phytoalexinic response and pea pathogenicity: The
concept of phytoalexinic response was first reported by
Müller (1958). After him, very few studies on the subject
were reported until the early 1960s. Cruickshank (1962)
pioneered the study of pisatin detoxification by
pathogenic fungi and its role in disease resistance and/or
susceptibility. He exposed over 40 fungal pathogens to
pisatin, the major phytoalexin produced by the garden pea
(Cruickshank, 1963; VanEtten et al., 2001). His results
showed that five species were tolerant (<50% inhibition
by 100:g/mL) while the rest proved sensitive to pisatin.
Interestingly, all five fungal species, tolerant to pisatin
were pathogenic on pea while only one of the species,
sensitive to pisatin, was a pea pathogen. Thus,
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For pathogens to invade plants and get established
within the tissues to cause disease, they must have a way
to overcome or circumvent phytoalexins. Many fungi,
pathogenic on plants, have shown to do so by
metabolizing these ‘defence personnel’, neutralizing the
plant’s inducible defences rather than preventing their
expression (Gabriel and Rolfe, 1990; Keen, 1992;
Lamb et al., 1989). A number of workers have succinctly
demonstrated the existence of clear correlations between
tolerance of host plant phytoalexins in vitro and
pathogenicity with many phytopathogenic fungi
(VanEtten et al., 1982; 1989). The ability of a pathogen to
detoxify or metabolize phytoalexin is therefore an
apparent measure of its pathogenicity/virulence on a
specific plant.
Pisatin tolerance in fungi has been reportedly studied
most extensively in the garden pea pathogen Nectria
haematococca mating population (MP) VI (anamorph
Fusarium solani f. sp. pisi). So far, two mechanisms have
been proposed by various workers, which confer pisatin
tolerance to some isolates of Nectria haematococca.
These include enzymatic detoxification (VanEtten et al.,
1980; Tegtmeier and VanEtten, 1982) and/or a non
degradative tolerance mechanism (VanEtten et al., 2001).
Experimental studies on enzymatic detoxification have
been based on a substrate-inducible one step
demethylation of pisatin to a non-toxic product (Fig. 3)
(Matthews and VanEtten, 1983). Observations from these
studies have shown that all N. haematococca isolates
which possess pisatin demethylase activity (PDA+) are
more tolerant to pisatin than those which cannot
demethylate pisatin (PDA-) (VanEtten et al., 1980).
Pisatin demethylation is catalysed by a microsomal
cytochrome P450 monooxygenase, called Pisatin
demethylase (pdm) (Ciuffetti and VanEtten, 1996;
Matthews and VanEtten, 1983). Natural isolates of
N. haematococca vary quantitatively in pisatin
demethylating ability, and as a result, three whole cell
phenotypic groups have been classified (Matthews and
VanEtten, 1983), which include PDA-, PDAL and PDAH.
The first group lack the ability to detoxify pisatin (PDA-).
The second group produce low levels of pisatin after long
exposure to pisatin (PDAL) while the third group rapidly
produces moderate to high levels of PDA on exposure to
Pisatin (PDAH). In some earlier publications, PDAL
phenotype has been referred to as PDAn or PDALL and the
PDAH phenotype as PDAi, PDASH, or PDASM
(Mackintosh et al., 1989).
The existence of two alternative non degradative
tolerance mechanisms conferring phytoalexinic
tolerance has been advanced by some workers
(VanEtten et al., 2001; Khan and Straney, 1999). One of
these mechanisms has long been known among drug
resistant prokaryotes and eukaryotes. In this regard,
accumulation of toxic chemicals in the cell is prevented
O
CH 3O
OH
O
H
O
O
(+) Pisation
NADPH+H +
O2
NADP+
H2O
O
HO
OH
O
H
O
DMDP
+
Formaldehyde
O
Fig. 3: Detoxification of pisatin through demethylation pisatin
demethylated
to
3,6a-dihydroxy-8, 9
methylenedioxypterocarpan (DMDP) (Source: Matthews
and VanEtten, 1983)
through activation of membrane transporters
(Bolhuis et al., 1997). Denny et al. (1987) demonstrated
this while working with an isolate of N. haematococca
lacking pisatin metabolizing ability. Strains of
N. haematococca pathogenic on peas have been reported
to possess one of these genes that code for such
membrane-associated
toxin
efflux
systems
(Han et al., 2001). The second non degradative tolerance
mechanism that confers phytoalexinic tolerance among
pathogenic fungi was proposed by VanEtten et al. (2001)
suggesting this to involve the alteration of the cellular
component targeted by the toxin (phytoalexin).
Although most pathogens of pea have been
demonstrated to have pisatin demethylating ability, some
are able to infect peas without demethylating pisatin; in
vitro studies have also shown that this group of fungi are
sensitive to pisatin. Among these are Aphanomyces
euteiches
and Pythium species (Puekkpe and
VanEtten, 1976; VanEtten et al., 1982). Sweigard and
VanEtten (1987) assume that this group of plant
pathogens either escapes physical contact, by avoidance,
during infection or the in vitro bioassays do not reflect
their sensitivity in vivo.
Furthermore, fungi pathogenic to other plants
have also been shown to demethylate pisatin
(Delserone et al., 1999; Puekkpe and VanEtten, 1976).
For example, highly virulent isolates of Ascochyta pisi,
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Fusarium oxysporum f. sp. pisi, Mycosphaerella pinodes
and Phoma pinodella have all been shown to have a
pattern of pisatin demethylation activity similar to
virulent strains of N. haematococca MP VI (Delserone
et al., 1999), suggesting the presence of PDA genes in
these organisms. Suffice to mention that these workers
also observed that some isolates of Colletotrichum
graminicola, that is non pathogenic on peas, exhibited
PDA activity in a pattern similar to many of the pea
pathogens studied, confirming research by earlier workers
(Kistler and VanEtten, 1984a) which suggested that
pathogenicity on pea requires more than an ability to
rapidly detoxify pisatin.
Molecular detection of Nectria haematococca MPVI
pathogenic on garden peas: Pathogenicity on specific
hosts is dependent on the presence/expression of specific
genes inherent in pathogens. Such genes, called
‘Pathogenicity genes’ are usually clustered together on
chromosome(s) (Hacker et al., 1997), and their presence
or absence is being exploited in distinguishing between
pathogenic and non-pathogenic strains of the same species
of organism. In bacteria, clusters of pathogenicity genes
have been described as ‘Pathogenicity islands’
(Gabriel, 1999; Groisman and Ochman, 1996;
Hacker et al., 1997; Kjemtrup et al., 2000; Mecsas and
Strauss, 1996).
Although, in contrast to bacterial pathogens,
pathogenicity determinants among eukaryotes are
relatively poorly understood; studies on eukaryotic
pathogenic traits have been done most extensively with
members of N. haematococca Mating population V1
(MPVI) (Temporini and VanEtten, 2002; VanEtten and
Kistler, 1988).
The application of DNA-based techniques in the
detection of pea pathogenic N. haematococca started with
the sequencing of the first cloned PDA genes, PDAT9 and
PDA6-1, which confers the PDASH and PDALL phenotypes
respectively. Sequence results showed that these genes are
90% identical at the deduced amino acid level and 88%
identical at the nucleic acid level (Maloney and
VanEtten, 1994; Reimann and VanEtten, 1994). The
sequence and restriction site information of these
sequences formed a basis for comparison of the other
known and unknown PDA genes using Restriction
fragment length polymorphism (Maloney and
VanEtten, 1994; Miao et al., 1991a). Analyses of other
strains, as well as field isolates, using the strains from
which sequences were obtained, delineated PDA genes
into two broad groups. PDASH and PDASM genes were
reportedly placed into one group, leaving PDALL genes in
the second group (VanEtten et al., 1994). Earlier work
had pointed out that only phenotypes with PDASH and
PDASM genes were virulent on peas (Kistler and
VanEtten, 1984b; Mackintosh et al., 1989; Tegtmeier and
VanEtten, 1982).
Maloney and VanEtten (1994) demonstrated that a
1.3kb SacI fragment (Probe SacB) from a PDASH
phenotype of N. haematococca could hybridize to unique
DNA fragments in isolates known to possess PDA genes.
However, the probe also hybridized to DNA, not
attributed to pisatin demethylase genes. Using this same
probe, Delserone et al. (1999) were able to differentially
detect pea pathogenic isolates of N. haematococca from
some other fungi that possess pisatin demethylating
activity but were not pathogenic on peas. Also, they were
able to demonstrate, by Southern analysis that the
cytochrome P450 encoded for by the PDA genes of pea
The pathogen: Nectria haematococca:
Morphology and cultural characteristics: N.
haematococca (anamorph F. solani f. sp. pisi) is a
heterogeneous group of ascomycetous fungi composed of
both homothallic and heterothallic groups (Booth, 1971).
Members of mating population VI infect and cause
disease on nine plant species and one animal species;
occurs in 14 species of plants as secondary/tertiary
pathogens, and can exist as saprophytes in soil (Funnell
and VanEtten, 2002; VanEtten and Kistler, 1988). The
natural occurrence of perithecia from MPVI has been
reported only in Japan on mulberry branches (Matuo and
Snyder, 1972). They are, therefore, best known and
studied as pathogens of the Garden pea (Pisum sativum),
where they are often referred to as Fusarium solani
f. sp. pisi (Funnell et al., 2001).
The fungus Fusarium solani has been reported on a
wide variety of crops, soils, and plant debris (Booth,
1971; Domsch et al., 1980). The reported symptoms were
wilting, rotting of seeds, seedlings (damping off), roots,
lower stems, crowns, corms, bulbs and tubers (Farr et al.,
1989). Usually, asexual spores are produced, but under
certain conditions a perithecial stage identified as Nectria
haematococca has been found (Booth, 1971).
Colonies of Fusarium solani f. sp. pisi grown on
freshly prepared Potato dextrose Agar are characterized
by typical blue green to buff coloured sporodochia.
Macroconidia are hyaline, measuring between 27-40 :m
long, curved, and are usually three septate (Etebu and
Osborn, 2009; Kraft, 2001). Microconidia are generally
few except in liquid culture. Barreto et al. (2003) reported
that the macroconidia of Fusarium solani isolated from
infected roots of olive plants on potato dextrose agar
measured as much as 29.4×3.9 mm.
Morphological traits are not sufficient to delineate
between strains of N. haematococca, pathogenic and non
pathogenic on Garden peas, both of which are known to
exist in agricultural soils (Oyarzun, 1993). Hence
pathogenicity studies on strains of this organism in peas
have recently been hinged on DNA-based molecular
techniques, targeting pea pathogenicity genes.
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pathogenic and pisatin demethylating isolates of
Ascochyta pisi, Fusarium oxysporum f. sp. pisi,
Mycosphaerella pinodes and Phoma pinodella are very
divergent from those of N. haematococca. The only
exception was observed to be F. oxysporum f. sp. pisi.
Southern hybridization analysis with the PDA specific
probe SacB detected putative PDA homologues in
F. oxysporum f. sp. pisi. Furthermore, whilst, SacB
probing of XhoI/BamHI digested genomic DNA detected
a single band with N. haematococca f. sp. pisi strains,
known to possess PDA genes, the DNA of F. oxysporum
f. sp. pisi resulted in multiple bands, suggesting that
isolates of this species may possess more than one PDA
homologue.
One critical step in detection and investigation of
plant pathogens in the environment or in planta, using
DNA-based molecular approach (PCR), is designing or
selection of primer(s) that would selectively amplify
targeted sequences, unique to the pathogen(s) and/or
gene(s) of interest within the environment or plant. Primer
design or selection, therefore, is usually directed towards
specific genetic traits within the genome of the organisms
of interest. For phytopathogenic fungi, the target genes of
interest are those that confer pathogenicity, enabling them
to overcome the barriers and defence mechanisms of
plants. Prominent among them are genes that confer on
the pathogen(s) an ability to overcome phytoalexinic
response in plants. Molecular studies on the pathogenicity
of N. haematococca (anamorph F. solani f. sp. pisi) on
Pisum sativum (Garden peas) have also relied heavily on
this phenomenon (Ciuffetti and VanEtten, 1996; Hirschi
and VanEtten, 1996; Temporini and VanEtten, 2002).
unaffected compared to wild type. As a result they
concluded that the cutinase gene of N. haematococca is
not essential for pea infection. However, Schäfer (1994)
in his review, was uncertain as to the conclusion of Stahl
and Schäfer (1992). According to him, the unchanged
pathogenicity observed in cutinase-deficient mutants did
not rule out the possibility of other undetected cutinases.
Genes encoding for cutinase enzyme in N. haematococca
may not be ruled out being a determinant for
pathogenicity on peas.
Whilst the potential role of genes encoding cutinase
and pectate lyase enzymes in pea pathogenicity are
appreciated, they would not be logical target genes for
molecular quantification of the pea footrot disease
pathogen because the interaction between pea (Pisum
sativum) and the soil-borne fungal pathogen, Nectria
haematococca (anamorph Fusarium solani f. sp. pisi),
which leads to footrot disease of the former is in many
ways similar to what has been reviewed so far
(Funnell et al., 2002; ; Funnell and VanEtten, 2002;
George et al., 1998; George and VanEtten, 2001;
Han et al., 2001; Temporini and VanEtten, 2002). The
garden pea (Pisum sativum) produces an isoflavonoid
phytoalexin (+) Pisatin and many of the fungi that are
pathogenic on the pea plant are able to detoxify pisatin
via demethylation (VanEtten et al., 1989; Delserone
et al., 1999).
Although knowledge of the modulating influence of
biotic and abiotic soil-properties on this fungal-plant
relationship is yet inconclusive (Janvier et al., 2007),
phytopathogenic fungi are able to infect plants due
to the activity of some genes which they possess
(Gold et al., 2001; Idnurm and Howlett, 2001; Oliver and
Osbourn, 1995). The role of pathogenicity genes among
different fungal-plant relationship has received enormous
attention in recent times (Hamer and Holden, 1997;
Idnurm and Howlett, 2001; Kahmann and Basse, 1999;
Knogge, 1998; Maier and Schäfer, 1999; Oliver and
Osbourn, 1995; Schäfer, 1994). Apparently, this is borne
out of a desire to have a better and deeper insight into
disease processes, so that such genes could be targeted for
disease control. As a result, the identification and study of
pathogenicity genes have increased in recent years. As at
2001, at least 79 genes had been described, sequences of
most of them submitted to databases (Idnurm and
Howlett, 2001).
High virulence on peas due to N. haematococca
MPVI population has been linked to the ability to detoxify
the pea phytoalexin, pisatin (Kistler and VanEtten, 1984a,
b; Mackintosh et al., 1989; Tegtmeier and VanEtten,
1982; VanEtten et al., 1980). All field isolates that are
pathogenic on pea are known to produce a substrateinducible enzyme, pisatin demethylase. Pisatin
demethylation is catalysed by a microsomal cytochrome
P450 monooxygenase, called pisatin demethylase (pdm)
Pea pathogenicity genes of Nectria haematococca:
Identifying an indicator gene: A number of genes have
been reported to be involved in pea pathogenicity. One of
such genes is a pathogenicity gene NhL1 encoding for an
extracellular lipase enzyme in N. haematococca
(Nasser et al., 2001). Other genes that seem to confer
pathogenicity in N. haematococca against plants are pelA,
pelD and CutA (Dickman et al., 1989; Dong et al., 1992;
Gonzalez-Candelas and Kolattukudy, 1992; Ikonen et al.,
1992; Lin and Kolattukudy, 1978; Rogers et al., 1994;
Rogers et al., 2000). The pelA and pelD genes code for
pectate lyase and are involved in pectin degradation while
CutA encodes the enzyme cutinase and is responsible for
degrading the cuticle. Gene disruption experiments have
shown that disruption of the gene encoding for the
cutinase enzyme among N. haematococca (Fusarium
solani f. sp. pisi), pathogenic on peas, consistently
resulted in generation of non- pathogenic mutants
(Rogers et al., 1994). Earlier work by Stahl and
Schäfer (1992) had shown that although the absence of
cutinase-deficient mutants resulted in the loss of
detectable cutinase activity, pathogenicity remain
201
Curr. Res. J. Biol. Sci., 3(3): 195-208, 2011
PEP1
PEP2
PEP3
PDA
PEP5
PEP4
Fig. 4: Schematic representation of the PEP gene cluster, The cluster represents the PEP gene cluster found in strain 77-13-7 of N.
haematococca MPVI , and contains six genes that are expressed during infection of pea (black rectangles) and four ORFs
with homology to different class II fungal transposable elements (orange rectangles) (Han et al., 2001; Temporini and
VanEtten, 2002)
disrupted strains completely non-pathogenic (Wasmann
and VanEtten, 1996). Strains with their PDA gene
disrupted were expected to become non pathogenic on
peas, which was not the case. This observation of
reduction in pathogenicity in PDA site-disrupted strains
on peas rather than becoming non-pathogenic raised
questions on previous conventional genetic studies
(Kistler and VanEtten, 1984a, b) which had indicated that
PDA1 was inherited as a single gene and was an absolute
requirement for any pathogenicity on pea. Two findings
from subsequent studies helped diffuse this apparent
confusion. Firstly, it was found that the PDA1 gene is
located on a 1.6 million base pair (Mb) conditional
dispensable chromosome (Miao et al., 1991b; Wasmann
and VanEtten, 1996), and secondly, it was also observed
that additional gene(s) located on the same dispensable
chromosome is/are required for high virulence on peas
(Wasmann and VanEtten, 1996). This dispensable
chromosome is reported to possess six genes: PDA1,
PEP1, PEP2, PEP3, PEP4 and PEP5 (Fig. 4), all
clustered together (Liu et al., 2003; Temporini and
VanEtten, 2002). These genes are said to be expressed in
pathogenic N. haematococca during infection.
PDA1 is responsible for detoxification of pisatin
while the PEP5 gene is suggested to be involved in the
efflux of pisatin (Han et al., 2001). Previous work with
isolates of N. haematococca from diverse sources
consistently revealed a strong correlation between
pathogenicity and an ability to detoxify/tolerate pisatin
(VanEtten et al., 1980, 1989). VanEtten et al. (2001) in
their review, having observed that N. haematococca
tolerates pisatin through degradative (PDA1) and nondegradative (PEP5) means, suggested that both types of
tolerance mechanisms may operate in synergism during
infection, and that elimination of both mechanisms may
be required to make it non-pathogenic. Thus, by
inference, PDA1 and PEP5 genes may well serve as
adequate indicators of pathogenic strains among members
of N. haematococca in agricultural soils, since tolerance
to pisatin has long been known as a factor for
pathogenicity. However, to cause severe disease (highly
virulent), the presence and expression of other genes of
the PEP cluster appears indispensable.
All highly virulent isolates have been shown to
possess at least one homologue of each of the six genes
as stated earlier, and is encoded for by Pisatin
Demethylase Activity (PDA) genes (VanEtten
et al., 1995, 2001; Funnell et al., 2001, 2002;
Liu et al., 2003). Naturally occurring isolates of Nectria
haematococca that lack the ability to demethylate
pisatin (PDA-) normally lack PDA genes and are not
pathogenic on peas (Ciuffetti and VanEtten, 1996; Wu
and VanEtten, 2004).
Several PDA genes have progressively been
identified by various workers over the years. So far,
Genetic studies have identified nine highly similar PDA
genes: PDA 1-1, PDA1-2, PDA2, PDA3, PDA4, PDA5,
PDA6-1, PDA6-2, and PDA7 (Funnell et al., 2002; Kistler
and VanEtten, 1984b; Mackintosh et al., 1989; Miao and
VanEtten, 1992). PDA 1-1 and PDA1-2 are thought to be
allelic forms (Funnell et al., 2002; Funnell and
VanEtten, 2002); as are PDA 6-1 and PDA6-2 (Funnell
and VanEtten, 2002). PDA1 is associated with virulence
on garden peas, whereas PDA6 is not (Wasmann and
VanEtten, 1996). PDA6-1 is reported to be found only in
isolates of N. haematococca collected from hosts other
than pea (Hirschi, 1994). This suggests that this gene
encodes for a product involved in the detoxification of
other phytoalexins. N. haematococca strains possessing
PDA2, PDA3 and/or PDA6-1 genes are reported to be
non-pathogenic on pea (Delserone et al., 1999). The
chromosome, on which PDA6-1 gene is located, is
reported to also possess MAK1, essential for maximal
virulence on chickpea (Covert et al., 1996;
Enkerli et al., 1998).
PDA1, PDA4, PDA5 and PDA7 are known to confer
a rapidly induced PDA activity on isolates that possess
them and are linked to virulence on peas (George et al.,
1998; Maloney and VanEtten, 1994; Miao et al., 1991a).
PDA1 and PDA4 genes occur on small chromosomes of
1.6 Megabases (Mb), while the latter two genes, PDA5
and PDA7 unlike the other genes have been shown to be
linked on a relatively large chromosome of 4.9 Mb, and
have also been partly implicated with normal
growth among some strains on N. haematococca
(Funnell et al., 2002).
Site-directed disruption experiments of PDA genes in
strains led to increased sensitivity of the fungus to pisatin
and reduced the ability of the pathogen to cause necrotic
lesions on pea, as expected, but did not render the gene202
Curr. Res. J. Biol. Sci., 3(3): 195-208, 2011
except PEP4 (Temporini and VanEtten, 2002), and they
all appear to be clustered. Of the six genes located on the
dispensable chromosome of N. haematococca, PDA1,
PEP1, PEP2 and PEP5, generally termed the ‘Pea
Pathogenicity’ (PEP) cluster is noted as being
responsible for pathogenicity of N. haematococca on peas
(Han et al., 2001; Liu et al., 2003), essentially because
each of these genes is able to independently confer
pathogenic properties to non-pathogenic isolates of
N. haematococca that lack the conditional dispensable
chromosome
(Ciuffetti and VanEtten, 1996;
Han et al., 2001). Etebu and Osborn (2009) have recently
developed molecular assays and have successfully used
the same to amplify these genes, except PEP2, in
agricultural fields with a prior pea footrot disease without
recourse to culture. Recent studies show that N.
haematococca isolates that possessed only the PEP1
(Etebu and Osborn, 2008) or a combination of the PEP1
and PEP4 genes alone were either not pathogenic or
caused a low degree of disease (Etebu and Osborn, in
press). This further positions the PDA and PEP5 genes as
potential indicator genes of pathogenic strains among
members of N. haematococca in agricultural soils.
Interestingly, molecular assays have not only been
developed to detect these genes in soils without recourse
to culture, but the assays have been extended to generate
quantitative data (Etebu, 2008).
However, homologues of the PDA gene also occur in
other fungi that are not pathogenic on peas
(Delserone et al., 1999). Also, N. haematococca isolates
that possess only the PDA gene without other PEP gene
are not pathogenic on peas (Etebu and Osborn, 2009).
These findings about the PDA genes make it an unsuitable
choice of gene to target in the molecular risk assessment
of footrot disease prior to pea cultivation. The most
important finding that would probably render the PDA
gene an unsuitable logical target for molecular
quantification is that soil wherein the PDA gene alone was
detected did not cause disease on peas after 8 weeks of
planting (Etebu, 2008). Assays directed at the PDA gene
could therefore serve only as a preliminary diagnostic
measure. Whilst an agricultural field may be deemed safe
from pea footrot disease if the PDA gene is not detected,
presence of the gene may not also pose a threat.
Conversely, molecular assays, targeting the PDA gene
alongside the PEP5 or PEP3 gene has been suggested to
offer us the opportunity for quantitative prediction of pea
footrot infections in agricultural soils prior to cultivation
(Etebu, 2008).
Whilst the PEP5 gene have been shown to be a
reliable measure of pathogenic forms of N. haematococca
in soil it may still be unsuitable for prediction of pea
footrot infections in agricultural soils prior to cultivation.
This is because, like majority of the pea pathogenicity
genes, homologues of the PEP5 gene also occur in
isolates with low virulence. Enumeration of this gene in
soil may therefore not be a true representation of the
population of pathogenic forms of N. haematococca in
soil (Temporini and VanEtten, 2002; Han et al., 2001).
The PEP3 gene is usually not included among the
pea pathogenicity genes (Han et al., 2001; Liu et al.,
2003), apparently because of its inability to independently
confer pathogenic attributes when used to transform nonpathogenic strains. Also as with PEP1 and PEP2 the role
of PEP3 in pathogenicity is yet unknown (Idnurm and
Howlett, 2001; Temporini and VanEtten, 2002). This
notwithstanding, whereas homologues of all other pea
pathogenicity genes occur in isolates with low virulence,
the PEP3 homologue is the only gene that is present
exclusively in highly virulent isolates, pathogenic on peas
(Temporini and VanEtten, 2002; Han et al., 2001). It is
apparent that the PEP3 gene plays an important role in
pathogenicity toward pea. Recent studies have
consistently established the fact that the PEP3 gene is
present in all highly virulent isolates of N. haematococca
(Etebu and Osborn, 2009).The primers developed by
Etebu and Osborn (2009) was successfully used in
molecular assays to both detect, quantify and validate the
population of pathogenic forms of the pathogen in soil
(Etebu and Osborn, 2010). A density of the PEP3 gene
copy numbers of up to 100 per g soil has been sown to
constitute a threshold number for infection; potentially
capable of causing pea footrot disease of economic
proportion. The importance of the PEP3 gene in pea
pathogenicity was demonstrated by Temporini and
VanEtten (2002). They showed that the PDA and PEP3
genes are physically located between PEP2 and PEP5
(Fig. 4) and that isolates lacking PDA and/or PEP3 were
not highly virulent even when other pea pathogenicity
genes are present in the cluster of genes.
From the ongoing, the PDA gene may be targeted in
a preliminary diagnostic measure. An agricultural field
may be deemed safe from pea footrot disease if the PDA
gene is not detected, and further test may not be
necessary. However, if the PDA gene is detected, a follow
up and conclusive assay, targeting the PEP3 gene may be
required. Agricultural fields with the PEP3 gene copy
numbers of up to 100 per g soil prior to cultivation may
be deemed unsafe for peas. This review work has
identified the PDA gene as the potential gene of choice to
carry out preliminary assay while the PEP3 gene would
be the preferred choice of gene to confirm the likelihood
of footrot disease in agricultural soil prior to pea
cultivation.
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