Zamioculcas zamiifolia is an important plant grown in Sri Lanka for
export. Zamioculcas is the genus of flowering plant in the family Araceae,
containing the single species Z. zamifolia. It is a tropical perennial,
herbaceous plant growing to 45-60 cm tall, from a stout underground, succulent
rhizome. It is normally evergreen, but becomes deciduous during drought, surviving
drought due to large potato-like rhizome that stores water until rainfall resumes.
The leaves are pinnate, 40-60 cm long, with 8-10 pairs of leaflets 6-10 cm long:
they are smooth shiny and dark green. The export quality of Z. zamiifolia
declined due to the repeated occurrence of a collar rot caused by Sclerotium
rolfisii. The collar rot disease cycle begins with the germination of sclerotia.
Mycelium fans out in all directions from the sclerotia, slowly growing across
the surface of the soil in warm, wet weather. Symptoms begin to appear on Z.
zamifolia after period of prolonged hot, humid weather. The lower leaves
turn yellow, then brown and finally wilt from the margins back toward the base.
The upper leaves soon collapse. Widespread occurrence of the collar rot affected
Z. zamiifolia production causing a significant setback to the floriculture
industry in Sri Lanka. Consumer concern has made it necessary to develop eco-friendly
methods to control this disease. Biological control of plant pathogens by microorganisms
has been considered a more natural and environmentally acceptable alternative
to the existing chemical treatment methods and also found that many isolates
of Trichoderma species produced non-volatile antibiotics, which were
active against a range of fungi (Eziashi et al.,
2007). One such method is the use of biological controlling agents such
as Trichoderma. The bioagent, Trichoderma species are known antagonists
of other fungi and have been shown to be very efficient biocontrol agents of
several soil borne plant pathogenic fungi (Barakat et
al., 2006; Karthikeyan et al., 2006).
Trichoderma sp. is known mycoparasites of a number of plant pathogens.
T. harzianum colonizes S. rolfsii hyphae, disrupts mycelial growth
and kills the organism. Trichoderma has the ability to suppress the growth
of many pathogenic fungi (Chet et al., 2009;
Mukhopadhyay, 2009). It has also been reported that Trichoderma species
with different mechanism such as lysis of sclerotia, inhibited mycelial growth
of S. rolfsii with volatile metabolites producing and parasitized the
hyphal trends of disease agent (Shaigan et al., 2008).
Developing appropriate formulation and delivery systems is the pre-requisite
for implementing biological control using microbial antagonists and the formulation
of biological control agents depends upon biomass production and maintaining
viability at the end of the process (Thangavelu et al.,
2004). The aim of this study was to evaluate the efficacy of Trichoderma
species to control collar rot in Z. zamiifolia plantation under field
conditions. In this investigation antagonistic effect of three isolates of Trichoderma
viride and one isolate of Trichoderma harzianum were tested against
MATERIALS AND METHODS
The experiment was carried out under field condition in the growing seasons of 2006, 2007 and 2008 in soil naturally infested with S. rolfsii in an Ornamental foliage nursery located in the low country intermediate zone of Sri Lanka.
Isolation of fungi Trichoderma sp.: The Trichoderma species
used in this study were isolated from soil samples obtained from Green Farms
Ltd, Marawila, Sri Lanka using the soil dilution technique (Subba,
2003). Pure cultures of the Trichoderma isolates were maintained
on PDA at 25±2°C and the isolates were identified using morphological
and reproductive characters (Anonymous, 2006; Bisset,
1991; Lieckfeldt et al., 1999; Samuels
et al., 1998; Watanabe, 2002a).
Sclerotium rolfsii: Infected Z. zamiifolia plants were
uprooted from the field at Green Farms Ltd and were immediately brought to the
laboratory. The plants were kept in moist polythene bags to enhance mycelial
growth prior to isolation of pathogen on Potato Dextrose Agar (PDA) medium.
The fungus was identified based on morphology and colony characteristics (Sarama
et al., 2002; Punja and Damini, 1996; Harlton
et al., 1995; Watanabe, 2002b) The pathogenicity
of the isolated S. rolfsii was established by following the Kochs
postulates (Riley et al., 2002) as described below.
Sclerotium rolfsii grown in pure culture was used to inoculate Z.
zamiifolia plants through topical application to the basal portion of the
stem. Six months old Z. zamiifolia were transplanted in polythene bags.
Spore suspension of the fungus was prepared in distilled water using 7- day-
old cultures grown on PDA. The culture was flooded with 10 mL sterilized distilled
water and then scraping the culture surface was scraped with a sterile glass
rod. (Hong and Hwang, 1998). After filtering the resulting
suspension through double layers of cheese cloth, the spore concentration was
adjusted using sterile distilled water to 104 spores mL-1
(Hong and Hwang, 1998). Base of the stem of the plants
at soil line was pricked by a blade and the S. rolfsii spore suspension
was smeared on the injured portion. Occurrence of symptom of collar rot was
Antagonistic effects of Trichoderma sp. Dual culture technique:
Sclerotium rolfsii and the test Trichoderma isolate was inoculated
at the center of two parallel radial lines on a 9 cm diameter PDA plate. The
fungi for inoculation were obtained from the margin of actively growing 7-day
old cultures on PDA. The radial mycelia growth of the Trichoderma sp.
and S. rolfsii were measured daily for 7 days. The treatments
were replicated five times. Control was with S. rolfsii alone (Singh
et al., 2004). This trial was repeated three times. The percentage
growth inhibition (I) was calculated using the formula given below (Datta
et al., 2004):
||Percentage inhibition of pathogen by antagonists
Radial growth in control
Radial growth in the treatment
Poison food technique: Poisoned food technique (Bhanumathi
and Ravishanker, 2007) was followed to determine the inhibitory effect of
Trichoderma isolate Tv1 on S. rolfsii. Conidia suspensions (1
mL) of Trichoderma isolates prepared as described below was poured into
a sterilized Petri dish followed by 15 mL of PDA. One milliliter of distilled
water was used instead of the conidia suspension in the control. Four mm diameter
discs were obtained from the actively growing region of a 7-day old S. rolfsii
culture on PDA and were transferred aseptically to the center of each Trichoderma
isolate amended PDA medium. The treatment were replicated five times in a completely
randomized design and repeated three times. The Petri dishes were incubated
at 28±2°C and 72±4% RH.
Growth of S. rolfsii was determined at 3 and 7 days after inoculation by measuring the mycelial growth diametrically.
Preparation of Trichoderma conidia suspensions: Conidia suspension
of the test isolates of Trichoderma was prepared from seven-day- old
cultures on PDA. A 9 cm diameter PDA plate was flooded with 10 mL sterilized
distilled water and shaken for a few minutes. The resulting suspension was filtered
through muslin cloth (Hong and Hwang, 1998) and the
conidia concentration of the filtrate was adjusted to 104 spores
mL-1 using sterilized distilled water.
Effect of T. viride on sclerotia of S. rolfsii: Among
the Trichoderma isolates, T. viride Tv1 which showed the highest
percentage inhibition (in-vitro) was selected for this trial. Mycelia
of S. rolfsii having sclerotia were collected from naturally infected
Z. zamiifolia plants and were transferred to the laboratory. The samples
were surface sterilized with 1% sodium hypochlorite solution for 2 min. Thereafter,
1 cm diameter discs were taken from the samples and were immersed in a conidia
suspension of T. viride Tv1 prepared as described above but having 1x1011
conidia mL-1 for 10 min and placed on a PDA plate (Henis
et al., 1983). In the control the pathogen samples were immersed
in sterilized distilled water for 10 min. The germination of sclerotia was determined.
Five replicates were used with three Petri plates per replicate.
Preparation of Trichoderma inocula for field tests Mass production of Trichoderma Solid media: Paddy soaked in water for 6 hours was parboiled in a pressure cooker (1.1 kg cm-2 pressure for 45 min) having sufficient amount of water. After parboiling, the closed container was kept in, a cooler room (15±2°C) for 2 h. Five kilogram of the parboiled paddy was equally distributed among 50 polyethylene bags. Mouth of the 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. The same procedure was followed with other grains - barley, maize, sorghum, white pericarp cowpea and brown pericarp cowpea.
Liquid media Paddy extract: The extract water resulting from parboiling described above was used as liquid medium to grow Trichoderma. This extract was dispensed in flat 250 mL bottles (each bottle having 50 mL of extract) and was sterilized.
Rice extract: Rice (250 g) was cooked by adding 2 L of water. Once the rice was cooked the excess water was drained off and 50 mL of this water was dispensed in flat bottles of 250 mL. The bottles were sterilized thereafter.
Semi-solid media: Potato (200 g) was cut into small pieces, boiled in 1 L of water and filtered through muslin cloth and 20 g of dextrose was added to the filtrate. The suspension was made up to 1 L and 50 mL was poured in to flat bottles of 250 mL and sterilized.
Inoculation of media: Plugs of 4 mm diameter obtained from pure cultures of 7-day old Trichoderma isolates on PDA were used to inoculate the above media.
Growth of the fungus: The growth of the fungus was determined by measuring the conidia yield in 1.0 mL or 1.0 g of the culture after appropriate serial dilutions at 7 and 14 days after inoculation. A double ruled Neubauers haemocytometer was used to count the conidia. Initiation and cover of medium with mycelium was also observed daily. Four replicates were used for each treatment.
Pot trials: Stem cuttings (30 cm) of Z. zamiifolia were surface sterilized by immersion in 0.1% aqueous sodium hypochlorite for 2 min and thoroughly rinsed in sterile distilled water prior to being rooted in 12 cm pots containing sterile, moist coir dust. Pots were kept in propagation beds for 3 weeks. After the cuttings rooted they were carefully taken from the pots and repotted in a mixture of compost + coir dust (1:1) at a density of two rooted cuttings per 19 cm pot. Plants were fertilized with the fertilizer mixture (N-P-K, 12-11-18). With the application of the fertilizer, the pH of the media was adjusted to 4.8-5.3 and the electrical conductivity was adjusted to 1.8 mS.
The resulting six -weeks old Z. zamiifolia seedlings were inoculated with a conidial suspension of Tv1 (1011 conidia mL-1), as close as possible to the root system (2-3 cm) with a sterile syringe. Control plants were treated similarly but with sterile water only. Four days later, plants were inoculated by introducing two plugs (5 mm diameter) of actively growing mycelium of S. rolfsii obtained from a 7-day old on PDA, as close as possible to the root system (3-5 cm). Controls were treated with fungus-free PDA disks. The experimental design included the following treatments: (1) Controls (2) Tv1 only, without S.rolfsii, (3) S. rolfsii only, without Tv1 and (4) Tv1 and S. rolfsii both. Twenty-five plants were used for each treatment and the experiment was repeated twice. Five replicates and five plants in each replicate. After 5 days the stem were pulled out of the substrate and examined for fungal infection (visible necrotic lesions). Samples from the stem (color) were collected 5 to 7 days after pathogen inoculation and either inoculated on PDA or processed for microscopy.
Field trials: The most promising antagonistic T. viride, Tv1 was also used for field trails. A six month old Z. zamiifolia plantation naturally infected with S rolfisii was selected as the experimental area. Z. zamifolia were in field plots having 12 bushes m-2. The size of a plot was 1x30 m2. Treatments involving soil and foliar applications of T. viride Tv1 isolates and the untreated control. Treatments were replicated five times. They were in randomized completely block designs. T. viride Tv1 was mass cultured on sterilized parboiled paddy as described above (section 126.96.36.199 and 2.3.2). T. viride Tv1 formulations were prepared as follows.
Liquid formulation: One kilogram of 7-day-old Trichoderma viride Tv1 cultures on parboiled paddy was flooded with 2 L of tap water and shaken well in a closed container. The resulting suspension was filtered through muslin cloth. The conidia concentration of the filtrate was adjusted to 1011 cfu mL-1 using tap water. The number of conidia was determined by counting in a double ruled Haemocytometer. One liter of this T. viride Tv1 conidia suspension was mixed with 1 mL surfactant (Lankem Ltd, Colombo, Sri Lanka) before being applied as a foliar spray.
Powder formulation: One kilogram of 7-day-old mass cultures was mixed
with 500 g of Talc powder (W.H. HENDRICK and SONS LTD. Colombo 11, Sri Lanka)
by using a mixing machine (Green Farms Ltd) for 1 h (22 rpm). Talc mixed paddy
was sieved through 2x2 mm GI wire mesh. The conidia concentration of the extract
was adjusted to 1011 CFU g-1 with Talc powder. This powder
formulation was mixed with cow dung (1:5) (Sangle and Bambawala,
2004) before application.
Method of applications: In foliar applications, each plant (i.e., plant
with 2-3 suckers) was sprayed with 200 mL of T. viride Tv1 conidia suspension
prepared as described above. The plants were sprayed with the above Tv1 conidial
suspension until runoff at 2 to 4 weeks intervals. The plants were sprayed with
irrigation water until runoff in the control plots. The cow dung mixed powder
formulation was also applied as a top dressing at 2 to 4 weeks intervals together
with foliar applications at the rate of 100 g m-2. This mixture was
distributed by hand as uniformly as possible over the bed area. Time of the
application of T. viride Tv1 was done based on the collar rot disease
development rate records. However, some of the applications were applied at
weekly intervals when disease severity became very high (Lo
et al., 1997). In both methods of applications cultural practices
such as field sanitation and manual removal of infected plants were strictly
followed throughout the experimental period. Further 40 g of fertilizer mixture
(N%- P2O5%- K2O% 11 -12-14) per one square meter was applied
at 2 weeks intervals.
Measurement of disease: Disease assessments were recorded at two weeks
intervals in both this treated plants and the untreated controls up to the end
of the trials. The assessments were carried out according to the Horsfall-Barratt
rating scale given below (Egel and Harmon, 2001; Horsfall
and Barratt, 1945).
Rating scale 1-5:
Rainfall, relative humidity and soil PH/EC were also measured through out the
study period. Percentage Disease Control (PDC) was calculated by using the following
equation described by Engelhard (1997)
Mean disease incidence in check plot
Mean disease incidence in treated plot
The effect of the transformation is to relate the efficacy of candidate material to that of control. When PDC is 100, infection is not present in treated plot. When PDC is zero treated plot had the same level of infection as the check plot.
Study of growth parameters with T. viride Tv1 and Un-treated control.
At the end of the experiment 50 samples were randomly taken in different treatments of each replicates separately. Then growth parameters (height of plant in cm and weight of roots/shoots in g and number of suckers/bush) were measured.
Experimental design and data analysis: In-vitro experiments were arranged as a complete randomized design with five replicates. All pot experiments and field experiments were established as a randomized complete block design with five replicates. All data were analyzed by one-way ANOVA, Differences among the means were evaluated for significant according to Turkeys pair wise comparisons test (p<0.05) (SPSS scientific software and mini-tab software were used for processing the data).
RESULTS AND DISCUSSION
Isolation of Trichoderma: Four different forms of Trichoderma were isolated from the soil samples obtained from Green Farms Ltd Marawila, Sri Lanka. They were T. viride Tv1, T. viride Tv2, T. viride Tv3 and T. harzianum Th1.
Effect of Trichoderma isolates on S. rolfsii In-vitro tests:
In Dual culture technique and poisoned food technique all isolates of Trichoderma
suppressed the growth of S. rolfsii. The isolates T. viride Tv1
showed the highest suppression (Fig. 1) (Johnson
et al., 2008).
Mass production of Trichoderma sp.: The highest mycelia growth was observed in the Barley and Paddy media (Table 1). The highest number of conidia was also produced in these two media. In the semi-solid potato dextrose medium though the growth was high sporulation was low. Hence Paddy medium was used to mass produce Trichoderma.
Pot trials: Treatment with T. viride Tv1 prior to S. rolfsii
inoculation resulted in a significant decrease in the occurrence of the disease.
In the absence of Tv1 treatment the seedlings exhibited typical symptoms as
early as 5-days after inoculation (Table 2).
||Growth inhibition (%) of S. rolfsii by Trichoderma
Further in all such infected plants stem damage was always associated with
the presence of S. rolfsii.
Field trials: The combined application of T. viride Tv1 conidia suspension having 0.1% surfactant and the powder formulation having cattle manure reduced the severity of the collar rot disease (Fig. 2a, b, Table 3).
In-vitro: The results revealed that the variation of antagonistic potential between isolates was due to the variation in mycelium-coiling rate, sporulation, fungitoxic metabolites, induced growth response and temperature effect (Barkat et al., 2006). The results showed that Tv1 was the most effective isolate inhibited S. rolfsii mycelial growth.
In dual culture technique all the isolates had the ability to parasitize the
mycelium of S. rolfsii. Microscopic examination revealed the formation
of coils around the hyphae of S. rolfsii by Trichoderma isolates,
causing lysis of the hyphal walls. Such observation has been reported by many
workers (Mukherjee and Raghu, 1997).
|| Growth and spore yield of T. viride Tv1 in different
||Effect of T. viride Tv1 on the growth of S. rolfsii
in the pot trials under field conditions
||S. rolfsii collar rot disease severity with T. viride
Tv1 treatment and un-treated control
Data are means of five replicates at two weeks intervals. Disease severity
represents the percentage of the total number of plants that contained diseased
plants per replicates at two weeks interval Since the T. viride Tv1 appears
to be the most efficient isolate, T. viride Tv1 was used for further
trials in this investigation.
||(a) Mean collar rot Disease severity with T. viride
Tv1 treatments (both soil and foliar application together). Year 2006/2007.
Disease severity represents the percentage of the total number of plants
that contained diseased plants per replicates at two weeks interval. Data
are means of five replicates at two weeks intervals and (b) Mean Disease
severity of Collar rot disease with T. viride Tv1 treatment (both
soil and foliar application together) Year 2007/2008. Disease severity represents
the percentage of the total number of plants that contained diseased plants
per replicates at two weeks interval. Data are means of five replicates
at two weeks intervals
In the poisoned food technique the conidial suspension of Tv1 completely suppressed
the growth of S. rolfsii. This was also due mainly to formation of coils
around the hyphae of S. rolfsii by Tv1. The sclerotia of S. rolfsii
failed to germinate when inoculated with Tv1. After 14 days, sporulation of
Tv1 on the sclerotia was observed. The sclerotia lost their rigidity, darkened
and finally degraded after about 4 weeks. In the control the sclerotia germinated
profusely. Some researchers have expressed doubts about the ability of Trichoderma
to parasitize healthy sclrotia (Henis et al., 1983).
The results of this investigation clearly indicate that Trichoderma has
the ability to parasitize healthy sclerotia
Mass production of Trichoderma isolates Growth of Trichoderma isolates in different substrates: A more complete cover of the medium by T. harzianum and T. viride was observed in Barley and Paddy within a short period than in other substrates. Initiation of mycelium occurred early in these solid substrates. Tv1 isolate of T. viride grew well in Barley within 3 days. In semisolid and liquid substrates complete cover of the medium by Trichoderma sp occurred in 5 and 6 days respectively, due to high nutrient content. But spore yield was very low in these substrates. High nutrient content in the substrate facilitated the growth of Trichoderma sp. As the fungi grow faster, the nutrients become depleted from the substrate indicating the reduction of spore yield in the subsequent days.
Spore yield in solid, semi-solid and liquid substrates: Among the solid substrates Barley seeds, Paddy and Brown/White peri-carp cowpea yielded a significantly higher spore count 7 days after inoculation, while a higher spore count in maize and sorghum medium produced on or at 14 days after inoculation. However maize yielded significantly greater amount of spores than sorghum. In general, liquid and semi-solid substrates were produced poor spore load compared to solid substrates. Both liquids, semi-solid substrates produced a higher number of spores at 14 days after inoculation and afterwards the spore number declined. Rice extract was proved as good as paddy extract to produce the spores of Trichoderma spp. T. harzianum was superior in producing spores in all the liquid substrates. Both the species of Trichoderma were produced at or above 1x1010 spores g-1 in barley as well as well as in paddy 7 days after inoculation. However, T. viride Tv1 spores were significantly higher in barley and paddy. White bran and red-bran-seed-coated cowpea stands the next best substrates for the production of spores of Trichoderma sp. Potato dextrose semi-solid medium did not support the spore yield 14 days after inoculation and were produced as low as 1x107 spores g-1. Paddy and rice extract were able to produce the spores of T. harzianum, T. viride at or above 1x109 spores g-1 at 14 days after inoculation.
The overall performance of T. viride Tv1 isolate is found to be superior
among the isolates of Trichoderma with respect to inhibition of S.
rolfsii and amenable to producing the highest spore load in solid as well
as liquid substrates.
The difference of spore yield could be considered for the selection of substrate as a whole for the production of Trichoderma. Among the isolates tested, T. viride Tv1 isolates proved to be the most potent isolates among all of the substrates. Rice and paddy extraction were able to produce significant spore counts at 14 days after inoculation; they did not support a steady production of spores afterwards. Since these are considered as waste materials they can be incorporated with paddy to produce Trichoderma at a lower cost.
Pot trials: The T. viride Tv1 treated plants also harbored a
vigorous root system. Though a few small brownish lesion were present on the
main stem. Their frequency and severity never reached the level observed in
the non-Tv1 treated plants. The ability of T. viride Tv1 to control
S. rolfsii infection has been attributed to the ability of Trichoderma
to parasitize the sclerotia (Mukherjee and Raghu, 1997).
Present earlier observation on the effect of Tv1 on sclerotia of S. rolfsii
support this attribution.
Field trial: The isolates T. viride Tv1 reduced the stem rot
significantly compared to control. However, penetration alone doesnt lead
to sclerotial degradation. And naturally produced sclerotia will have an attached
soil. The environmental factors and the properties of this antagonists that
lead to sclerotial attack and degradation remain to be elucidated (Henis
et al., 1983).
Since, the collar rot pathogen is capable of rapid spread, control of the disease
requires suppression of initial infection and reduction of infection rate. The
soil application of the powder formulation reduces the level of the pathogen
inoculum in the soil and thus the initial infections. The foliar application
reduces the spread of the pathogen through leaves or the rate of infection.
The T. viride Tv1 treatment also effectively increased the mean % of
disease control (PDC) and the frequency of healthy plants (Fig.
3, Table 4). The Effect of Trichoderma on Z.
zamiifolia growth was obvious; height, shoot and root weight were increased.
It was observed that the T. virirde Tv1 treated plants growth, shoot
weight and root weight were increased.
||Z. zamiifolia Plant growth parameters with T. vrirde
Tv1 treatment and Un- treated control at the end of the experiment
|Data are average of five replicates in two growing seasons
of the experiment. Means in a column for each treatment followed by the
same superscripted letters are not significantly different according to
Tukey's pair wise comparisons (p = 0.05) test
||Percent Collar rot Disease control in Z. zamifolia
with Trichoderma treatment. PDC = (DIck-DItr))/DIckx100.
DIck-mean disease incidence in check plot. DItr - -
mean disease incidence in treated plot
The increase was significantly different from the control (Table
4). Trichoderma sp. is also known to provide plants with useful molecules
such as glucose oxidase and growth stimulating compounds that can increase their
growth and vigor (Brunner et al., 2005; Gravel
et al., 2006).
Trichoderma isolates especially T. viride Tv1, has the potential to be used as a biological controlling agent against collar rot of Z. zamiifolia caused by S. rolfsii. Paddy or Barley based media can be used to mass produce the antagonistic fungus Trichoderma.
The authors are grateful to Mr. Arne Svinningen, Chairman, Managing Director, Green Farms Ltd, Marawila, Sri Lanka for his kind assistance throughout the research period. The research work was supported by Green Farms, Ltd, Marawila. The authors also appreciate Mr. M.D.S.D Karunaratne, Technical Manager, Ms. K.P.Rashani and Staff members, Laboratory unit, Green Farms Ltd, for their support throughout the research period.