Response of Free-Living Nematodes to Treatments Targeting Plant Parasitic Nematodes in Carnation
This study was carried out with the aim of evaluating
the effect of ecologically sound approaches for nematode management on
non-target organisms, free-living nematodes. The materials tested were
sugarcane bagasse, molasses, tea and flower composts, neem (Achook), a
biological agent (Paecilomyces lilacinus) and fenamiphos (Nemacur).
The treatments were administered before planting carnation var.
White Natila in flower beds that were naturally infested with nematodes.
Application of bagasse, molasses, tea and flower composts resulted in
increased abundance of free-living nematodes compared to the control where
nothing was applied. Bacterial feeders, fungal feeders, and predators
comprised 73, 14 and 13%, respectively of the free-living nematodes recovered.
Members of the genus Rhabditis were the most abundant (10%) among
the bacteriovores while Mononchus (10%) and Aphelenchoides
(14%) dominated among the predators and fungivorous trophic groups, respectively.
The highest numbers of free-living nematodes were recorded at 90 Days
after Planting (DAP) in plots treated with bagasse and molasses but the
numbers declined at 180 DAP. A steady increase in numbers of free-living
nematodes was observed in plots treated with tea and flower composts up
to 180 DAP. Significant reductions in abundance of free-living nematodes
were recorded in plots treated with fenamiphos and neem. This study has
established that application of organic substrates serve as a stimulus
to processes leading to build-up of free-living nematodes. The organic
substrates can strongly be recommended for use in sustainable carnation
The horticulture sub-sector has registered a phenomenal growth of 15-20%
in the past decade to rank third as a foreign exchange earner in Kenya.
It is currently the fastest growing agricultural sub-sector, with annual
earnings of US$ 300 million (Waceke et al., 2005). Fruits, vegetables
and cut-flower production are the main sectors of horticultural production
in Kenya. The cut flower industry dominates the horticultural exports,
earning US$ 500 million in 2003 and provides direct employment to over
500,000 people (Manda and Sen, 2005).
Carnation (Dianthus caryophyllus L.) is grown on more than
500 ha mainly under greenhouse conditions. Production of the crop has
been increasing steadily in response to the expanding market demand (Waceke
et al., 2001). Ultimately, Kenya has become the European Union`s
biggest source of the carnations, having overtaken Israel as market leader.
Nematodes have been rated among the principal constraints to carnation
production since they damage roots and reduce the ability of plants to
take up water or nutrients from soil (Masse et al., 2002). In ornamentals,
the problem is compounded by foliar nematodes (Aphelenchus spp.,
Aphelenchoides spp. and Ditylenchus spp.) which cause qualitative
losses. Losses due to nematodes in cut flower production are estimated
to be 10-20% worldwide (Agudelo, et al., 2006). Chemical nematicides
have dominated in the management of the nematodes but environmental concerns
have provided an added impetus to the search for alternative strategies
(Wang, 2004). Biological control, combined with other feasible strategies
is an area that is fast gaining popularity as a sustainable strategy in
nematode management (Mostafa, 2001; Kiewnick and Sikora, 2004). This approach
optimizes ecological synergies between biological components of the ecosystem,
enhancing biological efficiency of soil processes in order to maintain
soil fertility, productivity and crop protection in a new approach referred
to as ecologically based pest management, which aims at minimizing adverse
effects on non-target species and the environment (Steinberger et al.,
2001). While plant parasitic nematodes have a negative impact on the plant,
free living nematodes are known to play a role in nutrient cycling among
other benefits. According to Sánchez-Moreno and Navas (2007), abundance
and diversity of free-living nematodes is a good indicator of soil health,
given their role in decomposition and regulation of bacterial and fungal
microbes (Al-Sayed et al., 2007). It is, therefore, imperative
to conserve biodiversity of these free-living nematodes. Whilst large
data sets are available on the effect of organic substrates on plant parasitic
nematodes, few investigations have examined their effects on the free-living
nematode fauna (Ekschmitt et al., 2001). This study was undertaken
with the aim of determining the effects of treatments targeting plant
parasitic nematodes on free-living nematodes.
MATERIALS AND METHODS
The study was carried out between June 2006 and July 2007 at Kericho,
Kenya. The experiments were conducted under greenhouse conditions in an
area situated at an altitude of 1950 m a.s.l and receives mean annual
rainfall of 2000 mm with mean annual day and night temperatures of 25
and 11°C, respectively. The soil type is sandy loam humic nitisols
with an inherent pH of 4-4.5 but it is usually amended using agricultural
lime to pH 6 to make it suitable for growing of carnations.
Parallel flower beds, measuring 100 cm in width and raised to a height
of 25 cm, were made in a greenhouse, leaving a path of 50 cm between them.
Experimental plots, measuring 100x400 cm, were marked out in the flower
beds. The treatments tested were bagasse, tea and flower composts, molasses
(a by-product of sugarcane processing), neem, Paecilomyces lilacinus
(PL plus) and fenamiphos (Nemacur) while untreated plots were included
as a control. Bagasse, tea and flower composts were sun dried to a constant
weight. The composts and bagasse were applied at the rate of 300 t ha-1
as recommended for greenhouse usage (McSorley and Gallagher, 1995). Molasses
was applied at the rate of 667 mL m-3 (Schenck, 2001) while
neem was applied following the manufacturer`s recommendation at 1.5 mL
m-2 dissolved in 1000 L of water.
Paecilomyces lilacinus (PL-plus) was applied at the rate of 2
kg ha-1 while fenamiphos was applied 1 week before planting
at the rate of 30 g m-2. Spray carnations cv. White
Natila seedlings were transplanted into the plots. Treatments were arranged
in a completely randomized design with 6 replications.
Characterization of nematodes in the research site: Five soil
sub-samples were randomly collected to a depth 30 cm from the middle rows
of each plot. The nematodes were extracted from 200 cm3 soil
using the procedure described by Hooper et al. (2005). The sub-samples
were thoroughly mixed to form a composite sample before being placed in
plastic sampling bags, transported to the laboratory and stored at 4°C.
Soil sampling was done at planting, 90 and 180 days after planting. Nematodes
from each sample were fixed using rapid Seinhorst technique and thereafter
mounted on Cobb-type aluminium double cover glass slides that allow examination
from either side (Siddiqi, 2000). Identification of the nematodes was
based on morphological characteristics and pictorial keys using a high
power microscope (Hunt et al., 2005). After identification, the
nematodes were assigned to trophic groups as described by Yeates et
al. (1993). Nematode abundance was transformed to Log (x+1) and different
treatments were compared by two-way Analysis of Variance (ANOVA). Comparison
of the means was done with the Least Significant Difference (LSD) test.
Effect of organic substrates and bio-control agents on non-parasitic
nematodes in carnations: Application of bagasse, molasses, tea and
flower composts as organic amendments led to significant (p≤0.05) increase
in numbers of free-living nematodes in the soil (Table 1).
A decline in nematode numbers was recorded in plots treated with fenamiphos
and neem, compared to the control. The differences in mean numbers of
free-living nematodes were not significant in plots that were treated
with P. lilacinus, compared to the control.
Nematodes from 14 genera were recovered from carnation plots treated
with organic substrates and the bio-control agent, P. lilacinus
(Table 2). Among the free-living nematodes, bacterial
feeders accounted for 73% while fungal feeders and predators accounted
for 14 and 13%, respectively. The bacterivorous nematodes were members
of the genera Acrobeles, Rhabditis, Cephalobus,
Prodorylaimus, Bunonema, Eucephalobus, Heterocephalobus,
Plectus, Nygolaimus and Chromadora. The predators were
members of the genera Mononchus and Labronema while the
fungivores were assigned to the genera Aphelenchoides and Aphelenchus.
Members of the genus Rhabditis were predominant among the bacterial
feeding nematodes, representing 10% of the nematodes while Mononchus
(10%) and Aphelenchoides (14%) dominated the predacious and fungivorous
trophic groups, respectively. The treatments had a significant effect
on the free-living nematodes, with the exception of those in the genera
Cephalobus and Chromadora.
||Effect of different treatments on the free-living nematodes
in carnation production systems
|*Mean followed by different letter(s) are significantly
different along the row (p≤0.05)
||Occurrence and abundance of free living nematodes following
application of the various organic substrates in carnations
|ANS: Not Significant, **Significant differences
||Effect of different treatments on free-living nematodes
after varying durations from planting to 180 days after planting
|*Means followed by different letter(s) are significantly
different along the rows
||Effect of different treatments, applied to control parasitic
nematodes, on free living nematodes in the soil
|*Means within the same column followed by
different letter(s) are significantly different (p≤0.05)
The treatments had variable effects on numbers of free-living nematodes
over time, from application during planting throughout to 180 days after
planting (Table 3). With the exception of fenamiphos
and neem, all the other treatments led to a sharp increase in numbers
of free-living nematodes within the first 90 DAP. The nematode numbers
continued to increase in plots treated with tea and flower composts, fenamiphos
as well as in the control up to 180 days.
Free-living nematodes from different genera had varying responses to
the various treatments aimed at controlling plant-parasitic nematodes
(Table 4). The organic amendments namely bagasse, molasses,
tea and flower composts induced an increase in numbers of nematodes from
most of the genera of free-living nematodes. Bagasse led to an increase
in all the nematodes, except Nygolaimus and Eucephalobus
spp. While most organic substrates had a positive effect on the populations
of bacterivorous nematodes, P. lilacinus (biocontrol agent) greatly
increased the populations of fungivorous nematodes in the genera Aphelenchus
spp. and Aphelenchoides spp. Plots treated with fenamiphos and
neem had significantly lower numbers of free living nematodes compared
to the other treatments.
This study has demonstrated that amending soils with organic substrates
as well as with biological agents contribute to a change in nematode community
structure by increasing the abundance of free-living nematode populations.
Populations of free-living nematodes have been shown to increase rapidly
following the addition of organic substrates (Akhtar and Malik, 2000;
Agyarko and Asante, 2005). The mode of action of organic substrates leading
to stimulation of free-living nematodes is complex and dependent on the
nature of the substrate (El-Sherif et al., 2007). These mechanisms
stem from the decomposition process that leads to changes in the physical
and chemical properties of the soil. According to Sanchez and Navas (2007),
the nematode community structure is strongly impacted by changes in soil
systems since nematodes are highly dependent on soil properties. When
incorporated into the soil, organic substrates undergo a series of processes
that release NH4+, formaldehyde, phenols and volatile
fatty acids, among other compounds (Wang et al., 2004). The compounds
may act individually or collectively to stimulate build-up of beneficial
microbes including free-living nematodes (Desaeger and Rao, 2002). According
to Akhtar and Malik (2000), there could be a correlation between increase
in NH4+ and an increase in numbers of free-living
nematodes following addition of organic substrates. In addition, free-living
nematodes may accelerate the decomposition of soil organic matter and
increase mineralization of nitrogen and phosphorous thus triggering a
chain reaction that favours increase of the nematodes (Kimenju et al.,
From this study, the diversity of free-living nematodes recovered from
the carnation production system was lower compared to previous studies
in other production systems (Yeates et al., 1999; Zolda, 2006).
This can be attributed to the fact that cut-flower production is characterized
by usage of enormous amounts of agrochemicals, mainly in the form of fertilizers
and pesticides (Tenenbaum, 2002). Loss of biodiversity has indeed been
attributed to the adverse effects that are associated with agrochemicals,
especially the use of nematicides (Yeates et al., 1999). Fumigation
of soil to control soilborne pathogens and nematodes is recognized as
one of the most serious threats to the beneficial organisms such as free-living
nematodes (Pinkerton et al., 2000). Bacterial feeders dominated
the trophic groups isolated in this study and this finding is consistent
with reports from other workers (Wasilewska, 1997; Ekschmitt et al.,
2001). According to Zolda (2006), the elevated numbers of bacterial feeding
nematodes can be attributed to increased food resources for the microorganisms
in the soil. Indeed, bacterivorous nematodes responded quickly to increased
food supply (Yeates et al., 1999). In addition, decomposition pathways
in agricultural systems are mostly driven by bacteria which serve as a
stimulus to increased numbers of bacterial feeding nematodes (McSorley
and Frederick, 2000). Rapid changes in numbers of bacterivorous nematodes
can be anticipated, given their short generation time of 3-4 days (Ruess,
The dominance of fungal feeding genera (Aphelenchoides spp. and
Aphelenchus spp.) in soils amended with P. lilacinus (PL
Plus) is noteworthy because fungivorous genera are normally found at lower
densities than bacteriovores and predators (Knudsen and Bae, 2001). Paecilomyces
lilacinus is an ubiquitious soil hyphomycete which parasitizes
eggs of root-knot nematodes thus regulating populations of the nematodes
in field soil (Schenck, 2004). Numerous species of fungivores have been
found in soil with the most common genera being Aphelenchus and
Aphelenchoides (Yeates et al., 1999). A fungal-dominated
decomposition pathway is however likely, considering that species of one
of the most prevalent genus in this study, namely Acrobeles as
well as plant parasitic Filenchus and Tylenchus may also
feed on fungi (Zolda, 2006). The relative increase in fungal biomass occasioned
by application of P. lilacinus, could have resulted in the relative
increase of fungal feeding nematodes. Kiewnick and Sikora (1989) reported
that numbers of the mycophagous nematodes, Aphelenchoides sp. and
Aphelenchus avenae, increased several-fold within a few days after
adding flax to roots that had been precolonized by Rhizoctonia
solani. Moreover, abundance of bacterivorous nematodes has been
shown to reduce bacterial biomass, occasioning a relative increase of
fungal biomass (Soylu et al., 2005). Bae et al. (2001) observed
many nematodes associated with Trichoderma hyphae presumably feeding
and numerous nematode eggs adhering to hyphae. This led to the conclusion
that populations of fungivorous nematodes may increase rapidly following
addition of fungi as biocontrol agents.
The study revealed that neem and fenamiphos caused a reduction in numbers
of free-living nematodes. Pesticidal properties of azadirachtin, the active
ingredient in neem, have been clearly documented (Akhtar and Malik, 2000;
Agyarko and Asante, 2005). According to Agyarko and Asante (2005), neem
based products reduced egg hatch and the mobility of nematode juveniles.
Apart from azadirachtin, several other compounds namely salannin, nimbidin,
thionemone ammonia, phenol, formaldehyde and fatty acids are released
during decomposition of neem-based products (Yasmin et al., 2003).
It is possible that these compounds are individually or collectively detrimental
to free-living nematodes, thus accounting for the decline in their numbers
in treated plots. In conclusion, organic substrates are increasingly gaining
popularity as components of integrated pest management, developed with
the goal of reducing chemical usage in the control of plant parasitic
nematodes. The fact that addition of the organic substrates results in
build-up of beneficial organisms such as free-living nematodes is an added
An increase in numbers of free-living nematodes was recorded in all plots
where different organic substrates were applied with exception of neem.
A decline in numbers of the nematodes was observed in plots treated with
chemical nematicides (fenamiphos). It is recommended, the organic amendments
should be incorporated in carnation production given the range of other
benefits which include disease suppression, increased soil water holding
capacity, improvement of soil fertility and structure.
The authors are grateful to James Finlay (K) Ltd. for providing financial
and material support towards this study and the University of Nairobi
for providing laboratory space and equipment.
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