L. rotundifolia (Queen Palm) is one of the major ornamental plants belongs
to the family Arecaceae. L. rotundifolia is a round-leaf fountain palm
found in Southeast Asia. It is a common landscaping plant in the region. It
is a member of the genus Livistona. It is also called Footstool. It can
grow in sub-tropical climates and humid, tropical areas. Green Farms Ltd., Marawila,
Sri Lanka is a major exporter of queen palm mainly to Holland and substantial
amount to Japan, China and Switzerland as potted plant and cut leaves. L.
rotundifolia is cultivated as a potted plant with close spacing in the net
house. It succumb to outbreak of several pests viz., spider mites, grasshopper
and scales. Added to this, since 2006, an outbreak of a root-knot nematode
M. incognita becomes a main problem in this palm. M. incognita juvenile
stages caused severe damage in the active fresh roots of the queen palms thereby
affecting its export quality (personal observation).
The root knot nematodes M.incognita is sedentary endoparasites and is
among the most damaging agricultural pests, attacking a wide range of crops
(Katooli et al., 2010). The infections start
with root penetration of second stage juveniles, hatched in soil from eggs stored
in egg masses that have been laid by the female on the infected roots. Nematodes
pass through an embryonic stage, four juvenile stages (J1 - J4) and an adult
stage. Juvenile Meloidogynes hatch from eggs as vermiform second stage
juveniles (J2), the first moult having occurred within the egg. Newly-hatched
juveniles have a short free-living stage in the soil, in the rhizosphere of
the host plant. They may reinvade the host plant of their parent or migrate
through the soil to find a new host root. J2 larvae do not feed during the free
living stage, but use lipids stored in the gut (Jonathan and
Hedwig, 1991). These nematodes burrow into the soft tissues of root tips
and young root and cause the nearby root cells to divide and enlarge. Root knot
galls damage the vascular tissues of roots and thus interfere with the normal
movement of water and nutrient through the plant.
Chemical nematicide is one of the primary means of control for plant-parasitic
nematodes. However, the potential negative impact on the environment and ineffectiveness
after prolonged use have led to a total ban or restricted use of most nematicides
and an urgent need for safe and effective options. Application of microorganisms
antagonistic to Meloidogyne spp, or compound produced by these microbs,
could provide additional opportunity for managing the damage caused by root-
knot nematode (Khan et al., 2005).
Only a limited work has been done on this nematode pest infecting L. rotundifolia
in Sri Lanka. Indiscriminate use of pesticides causes a great harm to human
being, animals, vegetation and to environment as a whole due to their non-target
effect and hazardous nature (Vyas and Patel, 2002).
Therefore, biological control agents are gaining importance in the field of
nematode management. Another importance of these agents is their role as plant
growth promoting microorganism (Sharma and Pandey, 2009).
Many fungal and bacterial agents have been examined over a period of time for
their potential as bio control agents (Sharma and Pandey,
2009). Direct pathogenicity of fungal biocontrol agents is one of the main
mechanisms responsible for plant parasitic nematode control; however, secondary
metabolites from fungi also contain compounds which are toxic to plant parasitic
nematodes (Dababat and Sikora, 2007).
Several attempts have been made to use Trichoderma species to control
plant parasitic nematodes. Windham et al. (1989)
has reported the reduction of egg production by the root knot nematodes M.incognita
following soil treatment with a Trichoderma conidial suspension. Trichoderma
sp have been used as a bio control agents against plant parasitic nematodes
and this fungus may also promote plant growth and have the ability to colonize
root surfaces and the cortex. (Sharon et al., 2001).
Trichoderma species led to inhibition of the nematode activity and movements
in vitro during one week exposure. Trichoderma viride in combination
with organic amendments was also known to produce growth hormones, which were
observed to have added response in boosting the plant vigour. It has been reported
that Trichoderma has not only been proved to parasitize nematodes and
inactive pathogen enzymes but also help in tolerence to stress condition by
enhanced root development. It participates in solubilization of inorganic nutrients
(Sharma and Pandey, 2009). Prasad
and Anes (2008) reported that ethyl acetate and methanol extracts of T.
viride and T. harzianum significantly reduced the total number of
galls and M. incognita population in Okra.
The aim of this investigation was to isolate indigenous antagonistic fungi Trichoderma sp from ornamental foliage nursery and to determine their efficacy against root-knot nematode M. incognita infecting L. rotundifolia and also, their potential to control M.incognita was compared with using the standard chemical control agent: Carbofuran.
MATERIALS AND METHODS
The antagonistic fungi Trichoderma species were isolated from soil samples
obtained from Green Farms Ltd, Marawila, Sri Lanka using the soil dilution technique
(Year, 2005, 2006). Trichoderma sp. were isolated from organic rich soil
within a 15 cm depth by plating soil suspension after necessary serial dilution
directly on PDA. Five milliliter of soil suspension was placed in 15 mL molten,
cooling PDA, swirled and allowed to solidify. The set up was incubated 5-7 days
at 28°C. The isolates were purified by the single spore method. The fungi
were identified on the basis of their morphological and reproductive characters
(Anonymous, 2006; Bissett, 1991;
Lieckfeldt et al., 1999; Samuels
et al., 1998; Watanabe, 2002) and the pure
cultures of Trichoderma were maintained on PDA medium and stored at -
4°C. And further isolated species were identified through DNA sequencing.
Identified species were T. viride strain NRRL 6418 and T. harzianum
(Hypocrea lixii TWC1). These two species of Trichoderma were used
to evaluate their efficacy against root-knot nematode M.incognita infecting
In vitro study: Root knot nematode infected fields were randomly
selected in each locality and 5 media samples were taken from each bed (4x42
m) and pooled into one sample. The upper 3-5 cm surface media was removed and
about 250 g media along with 10 g feeder roots samples were collected from queen
palm rhizosphere to a depth of 5-10 cm (Santhosh et al.,
2005). The samples were placed in polythene bags, sealed labeld and then
brought to the laboratory for nematode isolation. M. incognita female
bodies and egg masses were removed aseptically from the palm, L. rotundifolia
roots. Eggs separated individually from egg masses using NaOCl @ 0.5% were
hatched in water to produce second juveniles (Sharon et
al., 2001). Ten petri dishes were filled with very thin layer of distilled
water. Young embryonated eggs (before appearing of first juvenile stage), Second
stage juveniles emerged from the eggs, females and young egg masses (including
gall) were collected from L. rotundifolia root galls. One milliliter
drop consisting approximately either 50 eggs or 50 second stage juveniles or
3 females were placed near the corner of the chamber and two such drops were
placed at two opposites corners of the chamber. Each chamber contains eggs,
juveniles and adult female bodies of the M. incognita. T. viride NRRL
6418 and T. harzianum (Hypocrea lixii) grown on potato dextrose
agar were separately introduced at the centre of the chamber as a small (4 mmØ)
agar disk (Sharon et al., 2001). Each life stage
was examined for infection of Trichoderma and the experiment was replicated
10 times, repeated two times. Control experiments were kept with the nematode,
M. incognita life stages without the antagonistic fungus. These chambers
were incubated at the room temperature of 27±3°C and the interaction
between the fungus and the nematodes was qualitatively monitored for one week
under the microscope. Egg masses were crushed on a slide to examine possible
infection of eggs by the fungus.
Field evaluation: Three field experiments were conducted in different locations, periods and for different age of the plants at Green Farms Ltd, Marawila, Sri Lanka. Experiments were carried out in the growing seasons 2007, 2008 and 2009 in soil naturally infested with M. incognita in a Farm located in the low country intermediate zone. These experiments were carried out in two seasons of the year. Climatic conditions of this research site are, Average Annual Rain fall (in 30 years)-1620 mm, minimum and maximum relative humidity of 60-90%, minimum and maximum day temperature 25-32°C, minimum and maximum night temperature 20-27°C. Standard agronomic practices were followed throughout the research period. The data was subjected to Analysis of Variance (ANOVA).
Trichoderma biomass and formulation production
Preparation of solid media: Paddy soaked in water for 6 h was parboiled
in a pressure cooker (1.1 kg cm-2 pressure for 45 min). After parboiling
the closed container was kept in a cooler room (15±2°C) for 2 h and
5 kg of parboiled paddy was equally distributed among 50 polyethylene bags.
The mouth of each bag was passed through a polyvinyl pipe of 2 cm diameter and
0.6 cm width and the mouth was thereafter plugged with a piece of sterilized,
non-absorbent cotton. A piece of paper was wrapped over the cotton plug and
the paper was kept intact using a rubber band. Plugs of uniform size of (4 mmØ)
were obtained from a pure culture of a 7-day old Trichoderma isolate
on PDA and used to inoculate the above media.
Trichoderma liquid formulation: One kg of 7-day-old mass cultures was flooded with 2 L of tap water and was shaken well in a closed container. The resulting suspension was filtered through muslin cloth. The filtrate was diluted with tap water to obtain conidia concentration range of 1014 cfu mL-1 for field application. One liter of this conidia suspension was mixed with 1 mL surfactant (wetting agent -commercial product from Lankem Ltd.) before applications.
Preparation of potting media: Potting mixture was prepared by mixing together coir dust, cow dung and compost in 7:2:1 ratio, respectively. pH level was between 4.5-5.5 and this level of pH was maintained throughout the experimental period. EC was maintained at lower than 1.8 mS cm-1
Evaluation of Trichoderma spp against root knot nematode M. incognita
Trial 1: This study was conducted at Green farm Ltd., Marawila
from July 2007 to June 2008. Healthy with out root knot damage L. rotundifolia
seedlings (7 cm pot) were arranged as follows in an area previously infected
with root knot nematode. Beds measuring, 3x30.6 m length and 3.6 m width accommodating
9720 numbers of potted queen palms was divided into 36 plots each plot consisting
270 potted queen palms seedlings. A randomized complete block design was used
and each treatment was replicated 12 times. Plots were marked using replicate
Treatments used in this experiment were given in below:
||T1Trichoderma harzianum (Hypocrea lixii) at
1x1014-1x1015 cfu g-1
||T2Carbofuran at 02 g pot-1
||T3Control (water) at 75 mL pot-1
T. harzianum (Hypocrea lixii) was used from the stock cultures
maintained at Green Farms Ltd. T. harzianum conidia suspensions were
prepared according to their concentrations and drenched to the pots in two weeks
intervals as a liquid formulation. Carbofuran granules were used as a standard
check, applied as spot application directly in to the pots at four weeks interval
as a granular application. Control was treated with field water.
A total of 270 palms were confined as a replicate from which 15 palms were randomly drawn to assess the effect of the treatments. No of galls /palm seedling were counted by visual method. Nematode infections such as formation of root galls were recorded in two weeks interval. The data was analyzed using SPSS version 10.
Study of growth parameters with different biological and chemical treatments:
In monthly interval, 12 random samples were taken in different treatments of
every replicates, separately. Then growth parameters such as height of plant
(cm) and weight of roots and shoots (g) were measured following the methods
suggested by Spiegel and Chet (1998). Then taken average
measurement and also drawn the bar chart separately for weight and height of
plants with different treatments.
Trial 2: The L. rotundifolia palm field naturally infected with
M. incognita was selected to evaluate the efficacy of Trichoderma
sp. 18 months old L. rotundifolia (12 cm) pot (750 mL) plants naturally
infected with root rot nematode M. incognita were selected for this trial.
T. harzianum (Hypocrea lixii) and T. viride strain NRRL
6418 were used from the stock cultures maintained at Green Farms Ltd. The conidial
suspension consisting T. viride NRRL 6418 and T. harzianum (Hypocrea
lixii) @ 1014 cfu mL-1 was prepared by equally mixing
the two cultures. Both species of Trichoderma together at 1014
cfu mL-1 were drenched at fortnight intervals and the other treatment
consisting of Cabofuran @2.5 g/pot (750 mL) kept as standard check. Cabofuran
was applied at four weeks interval as granular application. Two treatments such
||[T1]- T. viride NRRL 6418 + T. harzianum (Hypocrea
lixii) mixture at 1x 1014 cfu mL-1- as a liquid
formulation and at two weeks intervals
||[T2]- 2.5 gram 3% cabofuran/pot at as a granular application four weeks
Field experiment was arranged in a randomized complete block design. Treatments
were replicated five times. The plot size of each replicate was 3x42 m. Each
plot accommodating 1500 potted plants. Nematodes surviving at pre and post treatments
were counted. Number of root knot galls/plant and presence of females and eggs
inside the galls were recorded. First reading was taken three weeks after first
application and then continued at three weeks interval. Root knot disease incidence
was measured by means of number of root gall per plant. The data was analyzed
using SPSS version 10.
Trial 3: The methodology conducted in Trial 2 was repeated. Instead
of chemical treatment, irrigation water was used to find the efficacy of Trichoderma
sp. on controlling root knot nematode. 12 months old L. rotundifolia (12
cm) pot (750 mL) plants naturally infected with root rot nematode M. incognita
were selected in the plantation. A Field experiment was arranged in a randomized
complete block design. Treatments were replicated 5 times. The plot size of
each replicate was 3 mx42 m. Each plot accommodating 1500 potted plants. Two
treatments such as:
||[T1]- T. viride strain NRRL 6418 + T.harzianum
(Hypocrea lixii) mixture at 1x 1014 cfu mL-1-
two weeks intervals as a liquid formulation and
||[T2]- Untreated control was followed
Number of root knot galls/plant was recorded. The first reading was taken three
weeks after first application and then continued at three weeks interval. Root
knot disease incidence was measured by means of number of root gall per plant.
The data was analyzed using SPSS version 10.
RESULTS AND DISCUSSION
In vitro: T. harzianum (Hypocrea lixii) and T.
viride strain NRRL 6418 was tested for their capacity to reduce the incidence
of the root-knot nematode M. incognita on L. rotundifolia. Comprehensive
studies at the laboratory revealed the nematicidal potential of selected T.viride
and T. harzianum for controlling M. incognita. Direct interaction
of T.viride and T. harzianum with nematodes was observed in
vitro under sterile condition using the method described by Sharon
et al. (2001). Direct parasitism of Trichoderma strands on
separated egg/egg masses/female body was observed under the microscope (Goswami
et al., 2006). Trichoderma incubated in vitro with
the nematode gave promising results (Spiegel et al.,
2004). The hyphae of Trichoderma were penetrated and coiled the female
body. Eggs were also colonized and egg masses were penetrated by fungal strands.
T. viride and T. harzianum were able to colonize M. incognita
eggs and second juveniles and female. In vitro studies demonstrated that
both tested isolates were effective in causing nematode mortality compared with
the control (Fig. 1, 2).
Field evaluation: Experiments were carried out to study the effect of
two antagonistic fungal bio agents along with cow dung and cabofuran against
root knot nematode M. incognita infecting L. rotundifolia under
net house condition. Bio-agents viz., T. harzianum (Hypocrea lixii)
and T. viride strain NRRL 6418 in combination with cow dung promoted
plant growth, reduced number of galls/plant, female body and egg masses/root
system. The Trichoderma sp. along with cow dung showed least nematodes
reproduction factor as compared to untreated infested plants (Table
||Infection rate of Trichoderma viride strain NRRL 6418
on different live stages of Meloidogyne incognita under in vitro
||Infection rate of Trichoderma harzianum (Hypocrea
lixii) on different live stages of Meloidogyne incognita under
in vitro condition
||Comparison of effect of Trichoderma harzianum and Cabofuran
applications on the number Meloidogyne incognita root knot galls
||Comparison of efficacy of Trichoderma treatment and
Cabofuran applications on the root knot galls disease incidence in L.
||Comparison of efficacy of Trichoderma applications
with Un-treated control on the root knot galls disease incidence in L.
||Comparison of effect of Trichoderma and Cabofuran applications
on the growth parameters of Livistona rotundifolia
These results showed that the antagonistic fungus, T. viride NRRL 6418 and T. harzianum (Hypocrea lixii) excelled in suppressing the galls formation in the roots of the palm as well as significantly reduced the number of nematodes (Fig. 3, Table 1, 2).
In vitro: Results showed that these Trichoderma species
with different mechanism such as lysis of cell wall, inhibited growths of the
juveniles of M. incognita and parasitized the eggs /female body of M.
incognita and thus showed its antagonistic effects against causal agent
of root knot seedlings. T. viride NRRL 6418 and T. harzianum (Hypocrea
lixii) after seven days destructed and lysis the eggs and females bodies.
Identified Trichoderma species caused parasitize (to hyphal contact method)
eggs and female bodies (Fig. 1, 2). Trichoderma
had proved to infect on the eggs and adult females of the root knot nematode,
M. incognita. Since the female adult was sedentary in its movement, the
antagonistic fungus readily infected the nematode. But infection of Trichoderma
on eggs was prudent it is best opted at the beginning of the live stage
of the nematode to get controlled. Sharma and Saxena (1992)
reported that culture filtrate of T. viride adversely influenced hatching
of M. incognita larvae with highest inhibition of hatching occuring in
the standard concentration of the filtrate. When culture filtrates of the two
fungi were mixed together, the relative toxicity of the separate filtrates was
unaffected. Sahebani and Hadavi (2008); reported that
direct parasitism of Meloidogyne eggs through increase in extracellur
chitinase activity, which would be indicator of egg infection capability and
inducing plant defense mechanisms leading to systemic resistance are two main
suppression mechanisms used by T. harzianum against nematodes. T.
harzianum must be able to produce extracellur chitinase and proteinase because
of the proteinaceous and chitinase nature of nematode egg shell. Other extracellur
protein nature has been induced by colloidal chitin which may be involved in
nematode egg penetration.
Rao et al. (2000) reported that Trichoderma
sp. alone or in combination with either neem or caster cake, was most effective
in parasitizing the egg masses of the M. incognita and significantly
reduced root galling, nemtode population and fecundity of the nematode. T.harzianum
parasities eggs and larvae of M. incognita. The hypae penetrates the
eggs and larval cuticle by dissolving the chitin layer through enzymatic activity.
They proliferate within the organism and produce toxic metabolites (Dos-Santos
et al., 1992).
Dos-Santos et al. (1992) reported that T.
harzianum was a good egg parasite of M. incognita killing 53% eggs
in in vitro conditions.
In vivo: The efficacy of the potential bio control agent of Trichoderma
sp. in the management root knot nematode was achieved as the reduction in root
gall formation and also the reduction in the number of female M. incognita
and egg masses per root system of L. rotundifolia. Trichoderma treatment
was more effective than chemical treatment in reducing the gall formation in
L. rotundifolia. Palms treated with Trichoderma sp. was significantly
reduced the number of galls/plant compared with standard chemical treatment
(Fig. 3 and Table 1). Further application
of Trichoderma was able to infect the eggs, adult female and reduced
the number of galls/plant. Since, Cabofuran was used very frequently to control
the root knots nematodes, their usage came to standstill due to the development
of resistance against these inorganic chemicals. The response of application
of Cabofuran was ineffective and their persistence may pose ecological problem.
Therefore, bio control is suggested to be safer solution (Sharma
and Pandey, 2009). Trichoderma has good scope due to its effective
mode of action. Trichoderma parasitize the nematodes and control them
easily. In addition, the eco-friendly method of control of nematodes is prioritized
among the management tactics to protect the environment from polluting with
chemicals. In addition the consumers of these palms prefer organic products
from any countries and as such the Green Farm Ltd. at Marawila, Sri Lanka, apply
organic method of controlling insects and diseases to satisfy the requirements
of their clients.
The Trichoderma treatment also increased the fresh weight and growth
rate of the L. rotundifolia (Fig. 4). The plant height
and root weight were significantly improved compare to Cabofuran applications.
These results are also supported by Spiegel and Chet (1998),
Sharma and Pandey (2009).
Combination of isolated T. viride strain NRRL 6418 and T. harzianum
(Hypocrea lixii) @ 1014 cfu mL-1 proved that they
are capable to control M.incognita and showed good bio control activity
at the field level (Zhang and Zhang 2009). Root galling
was reduced and the number of new infection had been reduced. T. harzianum
(Hypocrea lixii) alone also significantly reduced gall formation and
improved palm growth. 3% carbofuran treatment showed a smaller reduction in
galling formation compare with T. harzianum (Hypocrea lixii) (Fig.
3) (Sharon et al., 2001; Siddiqui
and Shaukat, 2004). Both T. viride strain NRRL 6418 and T. harzianum
(Hypocrea lixii when added together with compost + cow dung showed significant
suppression of the egg masses per root system as compared to the control (Goswami
et al., 2006). Composts, on the other hand, can serve as an ideal
food base for biocontrol agents and offer an opportunity to introduce and establish
specific biocontrol agents into soils, which in turn leads to sustained biological
control based on the activities of microbial communities (Hoitink
and Boehm, 1999). Sharma and Pandey (2009) reported
that the Trichoderma has not only been proved to parasitize nematodes
but also help in tolerence to stress conditions by enhanced root development.
It participates in solubilization of inorganic nutrients.
This study identified antagonistic fungal isolates of T. viride strain NRRL 6418 and T. harzianum (Hypocrea lixii TWC1) capable of producing compounds active against root knot nematode M. incognita infecting L. rotundifolia.
The authors are grateful to Mr.Arne Svinningen, Chairman, Managing Director, Green Farms Ltd., Marawila, Sri Lanka for his technical help, writing assistance and provided and cared for study through-out the research period. Equally the authors wish to thank Mr. Arne Svinningen, Chairman, Managing Director, Green Farms Ltd., Marawila, Sri Lanka for his Financial and material support in conducting this study. The research work was fully supported (Financial and material support) by Green Farms, Ltd., Marawila. The authors also appreciate Mr.M.D.S.D Karunaratne, Technical Manager and Staff members, Laboratory unit, Green Farms Ltd., for their support to collected data through-out the research period.