Abstract: Crossandra infundibuliformis var. Danica is a very valuable ornamental flowering potted plant that has been introduced to the international floriculture market. A wilt caused by Fusarium oxysporum is a major problem in the production of C. infundibuliformis plants. Control of F. oxysporum causing wilt disease of C. infundibuliformis var. Danica was investigated using Trichoderma isolates. The isolates effected the growth of Fusarium in laboratory experiments. The results suggest that the effect on fungicidal. The Trichoderma isolates also effectively reduced the wilt incidence in field experiments. Further, the isolation promoted the growth of the plants. The study strongly suggests that Trichoderma isolates, especially T. viride Tv1 can be exploited for the biological control of wilt disease at field level.
INTRODUCTION
Crossandra infundibuliformis (Fire cracker), which is widely used as a potted flowering, landscape plant in Sri Lanka and also has a great demand from many European and Asian countries. Sri Lanka, exports more than 600000 C. infundibuliformis rooted cuttings per annum. One of the major limiting factors of C. infundibuliformis monoculture is wilt caused by F. oxysporum. This is one of the most difficult pathogen to control and it causes destruction of the entire plant. The disease can affect the crop at any stage of growth. Characteristic symptoms are sudden drooping of leaves and petioles, no external rotting of roots, black internal discoloration involving xylem and pith. The control of F. oxysporum on C. infundibuliformis is difficult to achieve because the long survival of the pathogen in the soil as chlamydospores or as a mycelium in infected plant debris. The soil borne antagonistic fungi, T. viride and T. harzianum have been identified as naturally existing potential biological agent against F. oxysporum (Dubey et al., 2007; Shanmugam et al., 2008; Garcia et al., 1997). It has been suggested that Trichoderma sp. isolated from the root or rhizosphere of a specific crop may be better adapted to that crop and be more effective in controlling Fusarium sp. (Rubio-Perez et al., 2008). The pathogen is soil borne and hence chemical control is uneconomical and cause environment and groundwater pollution. In the recent years due to the development of resistance in the pathogen, the usage of chemical fungicides proved to be less effective in the control of Fusarium sp. (Reid et al., 2002). Further it is also reported that treatments with fungicides have been ineffective in controlling Fusarium sp. under conducive condition for disease development (Federico et al., 2007). Hence, the need has arisen to develop alternative methods to control the disease and one such promising method is the use of the antagonistic fungus Trichoderma (Gilardi et al., 2008). Trichoderma sp. are well documented as effective biological control agents of plant disease caused by both soil-borne fungi and leaf-and fruit-infecting plant pathogenic fungi. These mycoparasites not only have direct effect such as protection against attack by different plant pathogens in the rhizosphere, but also have a stimulating influence on the plant growth and induction of defense responses (Ilan et al., 2009). Root penetration is achieved by secretion of cellulolytic and proteolytic enzymes (Ilan et al., 2009). Trichoderma species are plant symbiont opportunistic virulent organisms, able to colonize plant root by mechanisms similar to those of mycorrhizal fungi. Root colonization by Trichoderma species frequently enhances root growth and development, crop productivity, resistance to abiotic stress and uptake and use of nutrients (Mukhopadhyay, 2009). Root-fungus association stimulates plant defense mechanism (Mukhopadhyay, 2009).
In this investigation, four local isolates belonging to two species of Trichoderma (T. viride and T. harzianum) were evaluated against F. oxysporum.
MATERIALS AND METHODS
This experiment was carried out in Green Farms Limited, Marawila, situated in low county intermediate zones of Sri Lanka. Climatic conditions of this research site are, average annual rain fall (in 30 years)-1620 mm, minimum and maximum relative humidity 60- 90%, minimum and maximum day temperature 25 -34°C, minimum and maximum night temperature 20- 27°C. In this study, potential Trichoderma sp. isolated from Marawila, Sri Lanka were tested for in vitro study and in field experiment antagonistic activities on the development of vascular wilt disease on C. infundibuliformis. Five strains of Trichoderma, originally isolated and identified from ornamental foliage cropped soil of Green Farms Ltd., Marawila, Sri Lanka and were identified as T. harzianum and T. viride. A strain of F. oxysporum originally isolated from naturally infected C. infundibuliformis, was used in in vitro study. These isolates were kept in 15% glycerol and frozen at -4°C. During the growing seasons of the years 2007/2008 (January 2007 to August 2008) the potential of different strains of T. harzianum and T. viride were evaluated to control F. oxysporum and the effect on plant growth promoter on C. infundibuliformis were determined.
In vitro Evaluation of Trichoderma Against F. oxysporum
Isolation of Microorganisms
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. Trichoderma species was isolated from organic rich soil within
a 15 cm depth by plating soil suspension after necessary serial dilution directly
on PDA. Five millliliter 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; Bisset, 1991; Lieckfeldt
et al., 1999; Samuels et al., 1998;
Watanabe, 2002a) and the pure cultures of Trichoderma
were maintained on PDA medium and stored at 4°C.
The following isolates were obtained, T. viride1 Tv1, T. viride2 Tv2, T. viride3 Tv3, T. harzianum1 Th1 and T. harzianum2 Th2.
Isolation of Fusarium oxysporum
Isolation from Plant Samples
Fusarium oxysporum was isolated from naturally infected C. infundibuliformis
var. Danica plants in the field plantation/Green Farms Ltd., Marawila, Sri Lanka.
Infected C. infundibuliformis plants were uprooted and collected from
a plantation. The samples were put in poly bags and transferred to the laboratory
and kept in moist polythene bags to enhance fungal growth. The plant material
(stem and root) were cut into 5 mm pieces and surface sterilized with 0.5% sodium
hypochlorite solution for 5 min and rinsed trice with sterilized water. The
pieces were dried with sterile filter paper and plated (3 pieces per plate)
on fresh Potato Dextrose Agar (PDA) medium impregnated with streptomycin (0.5
mg mL-1) and incubated for 7-10 days at 28 to 30°C. The resulting
F. oxysporum colonies were sub cultured by transferring small mycelia
plugs from the colony margins. Pure culture was obtained by sub-culturing three
times.
Isolation from Soil Sample
One gram of soil from vicinity of the roots of infected plants was collected
and the dilution plate technique was used to prepare soil suspensions of different
dilutions. One milliliter of each soil suspension was uniformly spread over
PDA. The resulting F. oxysporum colonies were sub cultured from PDA plates
by transferring small mycelia plugs from the colony margins. Pure culture was
obtained by sub-culturing three times. The fungus was identified based on morphology
and colony characteristics (Watanabe, 2002b). The pathogenicity
of the isolates was established by following the Koch’s postulates (Riley
et al., 2002).
Preparation of Conidia Suspensions
Conidia suspension of the isolates of Trichoderma and F.oxysporum
were prepared from 7 days old cultures grown 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 spore’s mL-1 using sterilized distilled water.
Antagonistic Effect of Trichoderma sp.
Dual Culture Technique
The isolates of T. harzianum and T. viride were screened
individually against F. oxysporum through the dual culture technique
(Singh et al., 2004). Complete randomized design
was used with five replicate and the same trial was repeated three times. Each
replicates has three plates. The test Trichoderma isolates and the F.
oxysporum isolate was inoculated at the center of two parallel radial lines
on 9 cm diameter PDA plate. The fungi for inoculations were obtained from the
margins of actively growing 7 day old cultures on PDA. The dual cultures were
incubated at 28-30°C for 10 days and measurement of radial mycelia growth
of the F. oxysporum were taken 3 and 7 days after inoculation. Fusarium
oxysporum alone was maintained as the control (Singh
et al., 2004). The percentage growth inhibition (I) was calculated
using the formula given below (Datta et al., 2004):
[I% = (C–T)/C] x100
Where:
| I | = | Percentage inhibition of pathogen by antagonists |
| C | = | Radial growth in control |
| T | = | Radial growth in the treatment |
Interaction Between Conidia of Trichoderma Isolates and F. oxysporum
One milliliter (1x104 conidia mL-1) of the conidia
suspension of the test Trichoderma isolate and F. oxysporum was
introduced in to sterile Petri dishes and thereafter 15 mL molten agar (30°C)
was poured. The plates were swirled round in a clockwise direction to ensure
mixing of the two suspensions and the plates were incubated at 28-30°C.
After 10 days inoculation the plates were observed for the presence of any pathogen
colonies.
Field Experiments
An experiment was carried out under field condition in the growing seasons
2007 and 2008 in soil naturally infested with F. oxysporum in a farm
located in the low country intermediate zone of Sri Lanka. Five treatments involving
soil and foliar applications of Trichoderma isolates were used together
with an untreated control. C. infundibuliformis was in field plots having
36 plants m-2. The size of a replicate plot was 3x10 m. Treatments
involving applications of Tv1, Tv2, Tv3 and Th1 isolates and the untreated control.
Treatments were replicated three times. They were in randomized completely block
designs. Standard agronomic practices were followed throughout the research
period. This experiments were carried out in two growing season. 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. 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. Plugs of uniform size of (4 mm) 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 kilogram 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 a conidia
concentration range of 1011 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.
Evaluation of Trichoderma sp. Against Wilt
To perform the experiment, a field was selected where C. infundibuliformis
var. Danica crop has been regularly grown and the soil was already infested
with. F. oxysporum, 6 week old C. infundibuliformis rooted cuttings
were raised on compost with dried cow dung mixed coir media. Three replication
of each treatment were laid out in a randomized complete block design and 15
plots each 3x10 m2 in size were used. Four Trichoderma isolates
which were found to be potent against the test pathogen during in vitro
tests were used in the field experiments and the liquid formulation as the conidial
suspension (1x1011 cfu mL-1 mixed with 0.1% surfactant)
prepared as described above was used to inoculate the plants and adjacent soil.
One square meter area was inoculated with 5 L of the Trichoderma liquid
formulation and the plots were left for 14 days with sufficient soil moisture.
The untreated control was drenched with water. Applications of liquid formulation
were done at 2-4 weeks intervals. Infected/wilted plants were removed from the
plots regularly and disease incidence/severity, percentage of disease control
was assessed as below.
Measurement of Disease Incidence/Severity
Wilt incidence was recorded at periodical (2 weeks) intervals up to end
of the experiment. Total wilted plants per plot were recorded. In each treatment
disease development was measured by means of percent infected plants per each
replicate. Rain fall, relative humidity and soil PH/EC were also measured through
out the study period. The level of disease severity is expressed as the mean
value of each treatment. Percentage Disease Control (PDC) was calculated by
using the following equation described by Engelhard (1997)
and Singh et al. (2002).
PDC = (DIck–DItr))/DIckx100
Where:
| DIck | = | Disease incidence in check plot |
| DItr | = | 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 0 treated plot had the same level of infection as the check plot.
Effect of Trichoderma Treatments on Growth of Plants
The effect of the treatments on the growth of the C. infundibuliformis
var. Danica was determined by measuring the growth parameters plant height,
root length and fresh weight of the plant at the end of the experimental periods.
Fifteen samples were randomly taken from different treatments of each replicates
separately.
Experimental Design and Data Analysis
In vitro experiments were arranged as a complete randomized design
with five replicates. Each replicate has three plates. Field experiments were
established as a randomized complete block design with three replicates. All
data were analyzed by one-way ANOVA, differences among the means were evaluated
for significant according to Turkey’s pair wise comparisons test (p<0.05)
(SPSS scientific software and mini-tab software were used for processing the
data) (Federico et al., 2007; Larry,
1997).
RESULTS
When both the species of Trichoderma sp. were evaluated against F. oxysporum, T. viride and T. harzianum were suppressed the growth of the mycelia of F. oxysporum.In vitro experiments, the best four isolates each in two above species were selected based on percentage of growth inhibition and have been tested for confirmation.
In dual culture technique three days after inoculation, the mycelium of both the cultures came in contact with each other. Three days after inoculation the hyphal growth of F. oxysporum was found to be inhibited by the hyphae of Trichoderma sp. Further, Trichoderma almost inhibited the mycelia growth of the F. oxysporum at 10 days after inoculation. The advancing hyphae of Trichoderma covered the entire Petri plates, suppressing the growth of F. oxysporum. Microscopic observations in dual culture revealed that coiling of antagonistic hyphae around F. oxysporum hyphae that ultimately resulted in a mycelial rope like appearance. Significant differences (p<0.05) have been found in each treatment. Ten days after inoculation growth of F. oxysporum was completely restricted whereas antagonist had proliferated growth with abundant sporulation. Its mycelia strands coiled around the hyphae of F. oxysporum forming a rope like structure and finally disintegrated. This study clearly demonstrated that the potential of Trichoderma sp. as a bio control agent against F. oxysporum causing wilt disease in C. infundibuliformis.In vitro examination of Trichoderma on F. oxysporum of C. infundibuliformis revealed that the F. oxysporum covered completely (4.5 cm) in the absence of antagonist fungus. T. viride 1, 2 and 3 reduced the growth of F.oxysporum by 93, 90.3 and 66.76%, respectively whereas T. harzianum 1 and 2 reduced it by 88.9 and 76.9%, respectively 7 days after inoculation.
In vitro (Hyphal Interaction)
The in vitro testes in dual cultures showed that all isolates of Trichoderma
had the ability to inhibit the growth of the F. oxysporum. The highest
inhibition was by Tv1, Tv2; Th1. Inhibition by Tv3 was lowest. The analysis
of variance showed that there were significant differences between treatments
and the control (Fig. 1). Macroscopic observation of fungal
growth in dual culture revealed that growth inhibition occurred soon after contact
between Trichoderma and F. oxysporum.
Interaction Between Conidia of Trichoderma Isolates and F. oxysporum
The entire plates were covered exclusively by the Trichoderma
isolate (Tv1, Tv2, Tv3, Th1 and Th2) 10 days after inoculation. It was not possible
to isolate F. oxysporum from the inoculated plates.
| Fig. 1: | Growth inhibition (%) of F. oxysporum by Trichoderma isolates. Th1: T. harzianum1, Th2: T. harzianum 2, Tv1: T. viride1, Tv2: T. viride 2, Tv3: T. viride3, FO-Fusarium oxysporum |
| Fig. 2: | Mean disease severity of F. oxysporum wilt disease with Tv1, Tv2, Tv3, Th1 and control treatments in year 2007/2008. Disease severity represents the percentage of the total number of plants that contained diseased plants per replicates at 2 weeks interval. Data are means of 5 replicates at 2 weeks intervals |
| Table 1: | The effect of Trichoderma treatments on wilt disease severity, wilt disease reduction and growth of Crossandra infundibuliformis var. Danica plants |
| Means in a column for each treatment followed by the same letter(s) are not significantly different according to Tukey's pair wise comparisons (p≤0.05) test. Disease incidence presented above is the average values of occurrence of the disease at two week intervals for one year obtained from all record combined. Data are average of three replicates in two growing seasons of the experiment | |
Field Experiments
Biological control has the potential role in the management of diseases. Trichoderma species has been identified as naturally existing potential biological agent against diseases caused by fungi. Isolation of different species of Trichoderma sp. from the soils of foliage nurseries had been an evident to its potential role against fungal pathogens.
In field experiment 4 isolates Tv1, Tv2, Tv3 and Th1 were used. Trichoderma treatments were most effective in increasing the percentage of disease control (PDC) and the frequency of healthy plants (Singh et al., 2002). All the Trichoderma treatments decreased disease incidence, with TV1 having the highest disease reduction (90.51%) followed by Th1 (89.16%), Tv2 (82.30%) and Th2 (77.71%) (Table 1 and Fig. 2, 3). The Trichoderma treatment was enhanced plant growth leading to a significant increase in plant height, weight as related to untreated control (Table 1).
| Fig. 3: | Percent F. oxysporum wilt disease control in C. infundibuliformis with Trichoderma treatments. PDC = (DIck -DItr)) / DIck x100. DIck mean disease incidence in control plot DItr mean disease incidence in treated plot |
DISCUSSION
In vitro (Hyphal Interaction)
This observation indicates that parasitization of Fusarium by Trichoderma
play a role in the inhibition of Fusarium. Such parasitation through
formation of mycelial coil has been reported by several workers (Dubey
et al., 2007; Almeida et al., 2007;
Irfan and Khalid, 2007; Ozbay and
Newman, 2004). Production of chitinases may have direct significance in
the parasitism of Trichoderma on F. oxysporum as these enzymes
function by breaking down the polysaccharides, chitin and β-glucan that
are responsible for the rigidity of fungal cell walls thereby destroying cell
wall integrity (Howell, 2003).
Interaction Between Conidia of Trichoderma Isolates and F. oxysporum
This suggests that the effect of Trichoderma isolates on Fusarium
is most likely fungicidal. Bashar and Rai (1994) reported
that T. viride amended in soil suppressed the growth of F. oxysporum
and exhibited strong fungistatic activity against germination of conidia
of test pathogen.
Field Trial
During 2007/2008, Assessment of F. oxysporum vascular wilt development
rate was done year around and the effect of Trichoderma species alone
and with the control application. Results of the field experiment conducted
in the year 2007/2008 were shown in Fig. 2 and 3.
But Trichoderma alone was excelled in suppressing the wilt development
and the rate was kept checked (Fig. 3). Application of spore
suspensions responded positively and had ever high populations of Trichoderma
sp. (9.2x106 cfu g-1) in the growing medium.
During 2007/2008, the vascular wilt disease development rate of F.oxysporum was kept checked after the application of Trichoderma alone however the control treatment were found continuously increasing the disease incidence (Fig. 2). There was no rigid correlation established between the Trichoderma isolates and the wilt disease development of F. oxysporum.
The vascular wilt development was faster in control treated plants than Trichoderma sp. treated plants. Since the wilt pathogen, F. oxysporum is capable to spread readily in C. infundibuliformis crop, controlling the disease requires both suppression of initial plant infections and reduction of infection rates. Applications of Trichoderma sp. were significantly inhibited disease severity during the initial stages of the disease development, most likely by reducing the inoculums level of F. oxysporum into the soil. To be successful in its performances, the bio control agent must be multiplied to sustain its effectiveness. Such effective antagonists must be established in C. infundibuliformis planting medium and remain active against target pathogens during periods favorable for F. oxysporum infections. Trichoderma applications enhanced the overall quality of C. infundibuliformis plants related to with control.
Thus, it could be concluded that use of Trichoderma sp. as bio-agent not only reduced the incidence of F. oxysporum fungi but also increased the growth and vigor of the C. infundibulifomis.
Repeated field investigations confirmed that Trichoderma sp. was a potential candidate to control wilt disease in C. infundibuliformis. Trichoderma sp. can, therefore, be used as a vital bio-controlling agent in developing effective disease management practices to manage F. oxysporum infections in C. infundibuliformis plantation (Gilardi et al., 2008). Trichoderma sp. provide plants with useful molecules such as glucose oxidase, that can increase their resistance to pathogens such as induce systemic resistance in plants (Brunner et al., 2005). Moreover, these fungi produce antibiotics such as gliotoxin, viridin and cell wall degrading enzymes and biologically active heat-stable metabolites such as ethyl acetate (Mujeebur et al., 2004). These substances may be involved in disease suppression. Trichoderma treatment were known to produce chitinase and β (1-3, glucocinase) enzymes which could degrade the cell wall leading to the lysis of hyphae of the pathogen as noted by Wu et al. (1986). Trichoderma sp. attacks to the host hyphae via coiling hooks and appressorium like bodies and penetrate the host cell wall by secreting lytic enzymes (Kubicek et al., 2001). β-1, 3-Glucanases and chitinases have been found to be directly involved in the mycoparasitism interaction between Trichoderma sp. and its hosts (Kubicek et al., 2001). Benitez et al. (2004) reported that the Trichoderma attacks to the pathogen via cell wall carbohydrates. Once it is attached, it coils around the pathogen and forms the appresoria. The next step consists of the production of all cell wall-degrading enzymes and peptaibols. The Trichoderma treatment effect was found significant (p = 0.05) in respect of wilt incidence and plant growth during the experimentation (Dubey et al., 2007; Federico et al., 2007; Mousseaux et al., 1998).
CONCLUSION
Reduction of F. oxysporum infection in C. infundibuliformis var. Danica was achieved by the use of Trichoderma isolates. Trichoderma treatment also increased growth of plants. The results of this study clearly revealed the potential of Trichoderma as a biological agent to control F. oxysporum wilt on C. infundibuliformis.
ACKNOWLEDGMENTS
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 was fully supported (Financial and material support) 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 to collected data through-out the research period.