Determination of the In vitro Effect of Trichoderma harzianum on Phytopathogenic Strains of Fusarium oxysporum

Fusarium oxysporum is a well-known soil-borne fungi and it is difficult to control their pathogenic strains by conventional strategies. The cultures of two strains of Trichoderma harzianum (T16 and T23) were examined in laboratory conditions and with pot experiments for the control of pathogenic strains of Fusarium oxysporum f. sp. melongenae (Fomg), Fusarium oxysporum f. sp. lycopersici (Fol), Fusarium oxysporum f. sp. niveum (Fon) and F. oxysporum f. sp. melonis (Fom). The T16 and T23 strains showed significant inhibition of mycelial growth in the pathogenic strains of F. oxysporum and the maximum inhibition were recorded when the T. harzianum strain T16 was used (72.69%). Both T. harzianum strains produced volatile and non-volatile metabolites that inhibited growth of F. oxysporum strains on PDA medium. In vitro colonization study demonstrated the root-colonizing ability of these antagonists. The interaction between T. harzianum isolates (T16 and T23) and pathogenic F. oxysporum hyphae showed no overgrowth, hyphal coiling, cell wall degradation or any hyphal penetration around any of the tested F. oxysporum hyphae. Pre-treatment of soil with T16 significantly reduced the severity of Fusarium wilt disease. The disease severity in control plants reached to 90-95% whereas those of the T16-Fomg and T16-Fol treated seedlings of eggplants were 37.74% and 47.12%, respectively, on the 21 day. In this study, while both T. harzianum isolates had a considerable antagonistic effect on the tested pathogens, T16 was found to be more successful than T23. The strong repressive effect of T. harzianum (T16) towards pathogenic Fusarium oxsporum can be applied in biological control of these pathogens.


Introduction
Soil-borne fungi have a wide host range and persist for longer periods in soil by means of resistant resting spores.The plant diseases caused by such fungi are among the most difficult to control (Nelson et al., 1983;Yücel et al., 2007).Within these species, Fusarium oxysporum Schlechtend.:Fr. is one of the most important soil-inhabiting fungi and consists of both pathogenic and nonpathogenic strains (Gordon and Martyn, 1997).Individual pathogenic strains are known to be phylogenetically diverse and have a high degree of host specificity within F. oxysporum; they are generally known as species complexes which are assigned to intraspecific groups, called formae speciales (f.sp.) (Gordon and Martyn, 1997;Kistler, 2001).The usage of chemical compounds is a widely applied method to control soilborne diseases, but these have adverse effects on the environments such as like affecting the beneficial functions of microorganisms living in the soil and root ecosystem (Harman et al., 2004).Biological control has been advanced as an eco-friendly alternative to synthetic fungicides, and remarkable success has been achieved by utilizing antagonistic microorganisms.For example, Trichoderma species are common saprophytic fungi found in the soil and many studies have focused on their ability to reduce the incidence of the disease caused by plant pathogenic fungi (Elad, 2000;Freeman et al., 2004;Dubey et al., 2007).The Trichoderma species are useful avirulent plant symbionts that play an important role especially in controlling soil-borne fungal pathogens.Commercial biological products based on the Trichoderma species are manufactured and marketed worldwide as biofungicide, plant biostimulant and soil fertilizers for use on a wide range of crops.The use of these products for the management of diseases is eco-friendly, economical and also practical for improving soil health.
There are many biological control strategies for the control of soil-borne diseases.Among the potential biocontrol agents in the rhizosphere, several strains of the genus Trichoderma are reported to be effective in controlling a variety of fungal plant diseases (Menziez, 1993;Chet and Inbar, 1994;Freeman et al., 2004).

Dual culture technique
Two strains of T. harzianum (T16, T23) with pathogenic F. oxysporum strains were studied in a dual culture assay on PDA medium in 90 mm Petri plates as described by Altinok (2009).The experiment was arranged as a completely randomized design with ten replicates in a factorial arrangement of 2 x 4 (two T. harzianum strains and four F. oxysporum strains).The inoculated plates were incubated at 25 ± 1 °C until the end of the incubation period.The radial growth of the pathogen F. oxysporum isolates was measured on the 3 rd , 5 th and 7 th days after inoculation (DAI).The inhibition percent in the mycelial development of the pathogen fungus was calculated by the formula: R 1 =(C-T)/C x 100; Where R I is the inhibition percentage of the radial mycelial growth, C is the radial growth of the pathogen in the control (mm), and T is the radial growth of the pathogen in dual culture (Hajieghrari et al., 2008).The results were subjected to one-way ANOVA.Then the means were separated by Duncan's multiple range test to see the individual differences between the Fusarium isolates.

Effects of volatile and non-volatile metabolites
The effect of the volatile metabolites produced by T. harzianum on the mycelial growth of pathogenic strains of F. oxysporum was determined with the method described by Dennis and Webster (1971a).The effect of the non-volatile metabolites of the two isolates of T. harzianum on the mycelial growth of F. oxysporum strains was studied as described by Dennis and Webster (1971b).The filtrate was mixed together with molten PDA medium (10% w/v).After solidification, the Petri plates were inoculated with discs of the pathogen F. oxysporum strains and then incubated at 25±1 °C.There were 10 replicates for each treatment.Percent inhibition of pathogen radial growth was calculated as described above.

In vitro root colonization
The colonization of tested plants by Trichoderma species were studied using a method reported by Montealegre et al. (2003).For each treatment, 20 surface sterilized seeds were transferred to a sterile moist chamber.A one milliliter aliquot of each inoculum was added to the seeds in the moist chamber and the plates were incubated at room temperature for 1 h to allow binding of the antagonist fungi to the seed coat.Control seeds were inoculated with sterile distilled water.T. harzianum treated seeds and the control were incubated at 30 °C for five days in the dark for root development.One centimeter of root from each treatment was aseptically excised, then transferred to MgSO4 (0.1 M) solution, and diluted serially.From each dilution, a 0.1 ml aliquot was plated on PDA media and the plates were incubated at 30 °C for colony counts.The amount of fungal colonization in the root tissues was calculated as colony forming units per cm root (cfu cm root -1 ).

Slide culture method
A clean slide was placed in a 9 cm diameter Petri dish and autoclaved.Then a small amount of molten water agar was dropped onto it this and quickly spread over the slide to make a thin agar film.The inoculum of T. harzianum (10 8 conidia ml -1 ) and the pathogen (10 6 conidia ml -1 ) were placed on the slide, one cm apart from each other.A few ml of sterile water was added to Trichoderma species are filamentous soil antagonist fungi known to be an efficient biocontrol agent against a range of diseases in many economically important crops (Alabouvette et al., 2009;Carvalho et al., 2014).A number of Trichoderma-based commercial biofungicides which are of great importance as sources of enzymes, antibiotics and plant growth promoters have been developed.Trichoderma strains inhibit the infections caused by plant pathogens using different biocontrol mechanisms like competition, antibiosis, mycoparasitism, hyphal interactions, and enzyme secretion (Papavizas and Lumsden, 1980;Elad, 2000;Dubey et al., 2007;Hajieghrari et al., 2008;Poovendran et al., 2011).Recent studies have indicated that certain strains of Trichoderma can enhance plant growth and crop productivity, and can also induce systemic and localized resistance to several plant pathogens (Harman et al., 2004;Tran, 2010).Root colonization with Trichoderma strains can increase levels of defense-related plant enzymes, including various peroxidases, chitinases, β-1-3 glucanases, formation of calloseenriched wall appositions at sites of fungal penetration and pathogenesis-related proteins (Yedidia et al., 1999;Harman et al., 2004).
In the present study, the biological potential of Trichoderma harzianum isolates (T16 and T23) were evaluated with in in vitro experiments against four different Fusarium wilt pathogens (F.oxysporum f. sp.melongenae, Fomg; F. oxysporum f. sp.lycopersici, Fol; F. oxysporum f. sp.niveum, Fon and F. oxysporum f. sp.melonis, Fom) and their role in the control of Fusarium wilt disease in eggplant, tomato, watermelon and melon were determined with pot experiments.The results obtained from this work provides clues also for further field biocontrol studies.

Isolates
T. harzianum isolates (T16 and T23) were kindly provided from the culture collection of the Institute for Phytomedicine at the University of Hohenheim, Germany and these were used in this study.The F. oxysporum f. sp.melongenae, F. oxysporum f. sp.niveum, F. oxysporum f. sp.melonis and F. oxysporum f. sp.lycopersici isolates selected for this study were obtained from the collection of Fusarium spp. in Erciyes University, Agriculture Faculty, Department of Plant Protection Mycology Laboratory, Kayseri-Turkey.These isolates, which were isolated from the host plants (eggplant, watermelon, melon and tomato) and highly virulent isolates were used in this study.The isolates were maintained on a potato dextrose agar (PDA) medium and stored at 4°C for further use.
the Petri dish to prevent drying and was incubated at 25 ± 1 °C for five days.After incubation, the area where the hyphae of T. harzianum met the hyphae of the pathogen was observed under a light microscope (Zeiss Axiostar Plus) at 400X, in bright field mode.Three different fields within the observed area were examined for each slide.

Pot experiment
In this experiment, 50 ml conidial suspension of Trichoderma isolates was drenched into the soil in each pot.Preparation of T. harzianum (T16 and T23) spore suspensions: one ml spore suspension (10 8 spore ml -1 ) of 7-day-old T. harzianum cultured on PDA was used as the inoculum (Yedidia et al., 1999).The cfu numbers were determined by plating serial dilutions of conidial preparations of T16 and T23 onto PDA.Viability percentages were determined by comparing cfu with total conidia.One week later, eggplant, tomato, watermelon and melon seedlings were artificially inoculated with F. oxysporum conidia (1×10 6 conidia ml -1 ) by the root-dip method.Noninoculated seedlings served as negative control.The positive control plants were inoculated with only the pathogen inoculum and were treated with sterile distilled water instead of T. harzianum suspension.The inoculated plants were grown in the growth chamber at conditions stated above.The experiment was conducted as a 2 x 4 factorial arrangement of treatments in a completely randomized design with three replicates.The symptoms of the disease were recorded on the 7 th , 14 th and 21 st days after the exhibition of the first symptoms in infected plants with a Fusarium yellow scale of 0 to 4 (Altinok and Can, 2010).
plants were evaluated individually and a mean percent of disease severity was calculated for each assessment day based on the scale values according to the Townsend-Heuberger formula.The data were subjected to two-way ANOVA to identify interactions between the two factors.The differences among Fusarium isolates were also tested with one-way ANOVA for each of the Trichoderma strains, then the means were separated by Duncan's multiple range test (P ≤ 0.05) contained in the SPSS v.16 software (SPSS Inc., Chicago, IL, USA).

Dual culture technique
In the dual culture experiments, T16 and T23 showed a significant inhibitory effect on the mycelial growth of pathogenic strains of F. oxysporum when compared to the control.T. harzianum (T16) grew much faster on PDA than the tested pathogens under the same culture conditions.The maximum inhibition was recorded when the T. harzianum strain T16 was used (72.69%).The study concluded that T16 was more efficient than strain T23 in inhibiting colony growth of the pathogen F. oxysporum isolates (Table 1).

Effects of volatile and non-volatile metabolites
The efficiency of the volatile metabolites on the mycelial growth of the pathogenic strains of Fusarium oxysporum varied from 45.03 to 62.71.While Fomg was found to be the most susceptible to volatile inhibitors produced by T. harzianum strain T16, the minimum inhibition percentage was in Fom, which was paired with T23 (Table 2).
The maximum inhibition of Fomg radial growth was observed with the non-volatile metabolites of T. harzianum strain T16.Fom showed the minimum inhibition percentage of 49.61% and 44.92% by the non-volatile inhibitors of T16 and T23, respectively (Table 2).

In vitro root colonization
The in vitro root colonization study demonstrated that antagonist isolates were effective as root colonizers.The maximum count of the viable conidia was obtained from the eggplant, tomato, watermelon and melon roots after five days of germination.T16 and T23 strains were detected as successful colonizers in eggplant seeds (11.83 -12.25 cfu/cm root x 10 5 ) and tomato seeds (10.54 -8.36 cfu cm root -1 x 10 5 , respectively (Fig. 1).

Slide culture method
Hyphal interaction between the antagonist (T.harzianum) and tested pathogenic strains of the F. oxysporum was examined five days after incubation.Hyphal penetration or overgrowth was not observed on pathogenic F. oxysporum hyphae by T. harzianum strains.Although close contact occurred between T. harzianum (T16) and pathogen hyphae, no massive coiling, cell wall degradation or disintegrating structures were observed around any of the pathogen hyphae in three different microscopic fields.

Pot experiment
Prior to pot experiments, the viability of the conidia of T. harzianum strains was tested.The viability was 88% and 85% for T16 and T23, respectively.Both strains of T. harzianum successfully suppressed Fusarium wilt on tested plants.Initial symptoms appeared as yellowing of the older leaves on Fomg, Fol, Fon, Fom-treated and (+) control plants (only pathogen inoculated) seven days after inoculation.The systemic progress of the disease in the (+) control plants rapidly increased in time and by 21 DAI showed severe wilting.Browning areas were observed in the xylem of all infected plants, although the progress of the disease was much slower than that of positive control plants.Non-inoculated seedlings (negative control plants) showed no symptoms and appeared healthy throughout the course of the experiment.The soil applications of T. harzianum resulted in up to 50% reduction in Fusarium wilt disease progression in all tested seedlings at 21 st day, compared to the control.The effects of T16 and T23 treatments on the 7 th , 14 th and 21 st days after inoculation with the pathogen are presented in Fig. 2 and 3.
At the end of the experiment, the mean disease severity in positive control plants reached to 90-95% whereas in the T16-Fomg and T23-Fomg treated plants it was 37.74% and 50.12%, respectively.The maximum level of disease control was obtained from T16 treatment and the statistical differences between the pathogenic F. oxysporum isolates were found to be significant, F(3,36) = 4.84, P = 0.006.
The main effects of Trichoderma and Fusarium isolates were both found to be significant (F=37.67,F=8.12) while no interaction was observed between these factors (F=0.62).In adittion T16 was more effective in suppressing Fomg, on all scoring days (Fig. 2).T23, on the other hand, showed no difference in the virulence of the Fusarium isolates until 21 st day after inoculation.On the same day, the effect of T23 was significantly higher on Fomg-treated plants than in other Fusarium treatments.As seen in Fig. 2 and 3, the results of T. harzianum strains were similar at the end of the experiment (21 DAI).Therefore, the effects of T16 and T23 strains were compared for each of Fusarium isolates.No significant difference was found between T16 and T23, on Fol; Fusarium oxysporum f. sp.lycopersici (F (1,18) = 3.82, P = 0.066).The disease suppression ability of T23 was significantly higher than that of T16 on Fom=F.oxysporum f. sp.melonis [F(1,18) = 3.37, P = 0.021], oxysporum f. sp.melongenae [F(1,18) = 24.27,P < 0.001] and Fon=Fusarium oxysporum f. sp.niveum [F(1,18) = 18.01,P < 0.001].
The dual culture method as described by many researchers has been widely used in antagonistic studies (Dennis and Webster, 1971a;Bell et al., 1982;Küçük and Kivanç, 2004).In this study, the results of the dual culture experiment revealed the rapid colonization of the growth medium by T16 and T23.Both isolates evaluated were effective in controlling colony growth of the tested pathogenic F. oxysporum.The volatile and non-volatile metabolites also showed an effective performance on inhibiting the mycelial growth of the pathogens.Similar findings have been reported by Küçük and Kivanç (2004), Idris et al. (2007), Hajieghrari et al. (2008), and Perveen and Bokhari (2012).The substantial functions associated with Trichoderma species are their ability to produce a number of antibiotics as well as some cell wall degrading enzymes like chitinase and glucanase hydrolytic enzymes which are closely related to mycoparasitism (Elad, 2000).The volatile metabolites produced by antagonists may diffuse easily and inhibit the growth of soil-borne pathogens in vitro and even in soil (Dennis and Webster, 1971b).The in vitro root colonizing ability of these antagonists was found to be successful.Similar results were reported by Al-Jedabi (2009), after four days of germination, in that the cell counts obtained from the roots increased and the maximum count was achieved by T. harzianum.Microscopic studies showed no overgrowth, the hyphal penetration, or hyphal coiling (hyperparasitism) of isolates T16 and T23 around hyphae of pathogenic F. oxysporum strains, suggesting that mycoparasitism was not a major mechanism for the observed inhibitory effects.The pathogen and T. harzianum hyphae did not interfere with each other in dual cultures and this was explained by the production of extracellular mycolytic enzymes by T. harzianum (Calistru et al., 1997;El-Katatny et al., 2006).El-Katatny et al. (2006) suggested that the extracellular mycolytic enzymes secreted by Trichoderma might play an important role in antibiosis against pathogenic F. oxysporum.On the other hand, fungitoxic metabolite secretion by Trichoderma might not be the primary mechanism in biocontrol, instead it could be through the competition or parasitism of pathogen hyphae (Mukherjee and Raghu, 1997).The production of yellow pigment in an overlapped area of two colonies and mycelial coiling were also reported by other researchers (Dennis and Webster, 1971c;Küçük and Kivanç, 2004).The inoculation of the eggplant, tomato, watermelon and melon seedlings with pathogenic F. oxysporum caused symptoms similar to natural infections.The results showed that strains T16 and T23 were effective in reducing disease severity of Fusarium wilts.When T. harzianum was applied as seed coating, crown and root rot incidence was reduced by up to 80% in greenhouse conditions (Sivan et al., 1987).T. harzianum reduced the incidence of Fusarium crown and root rot in tomatoes (Van Steekelenburg, 1991;Ozbay et al., 2004).These results clearly indicate that the two T. harzianum strains exhibited strong ability to suppress pathogenic Fusarium species of different crops in pot experiments.Fig. 2. The inhibitory effect of soil application of T. harzianum (T16) against Fusarium wilt diseases caused by Fomg=Fusarium oxysporum f. sp.melongenae, Fol=Fusarium oxysporum f. sp.lycopersici, Fon=Fusarium oxysporum f. sp.niveum and Fom=F.oxysporum f. sp.melonis.
The seedlings were scored on 7 th , 14 th and 21 st days after pathogen inoculation, using 0-4 scale.Inhibitory effect was calculated by Abbott's formula.The same letters above means for each scoring day are not significantly different according to Duncan's Multiple Range Test (P ≤ 0.05).
Error bars indicate ± 1 standard error of the mean.

Table 1 .
Mycelial growth inhibition of pathogenic Fusarium oxysporum by T. harzianum isolates in dual culture Means within columns followed by the same letter are not significantly different according to Duncan's Multiple Range Test (P ≤ 0.05).Means starting with the same letter within lines are not significantly different at the 0.05 level using Tukey's HSD test.Values represent mean ± SD of ten replicates.Measurements of radial growth were taken 7 days after inoculation.**Fomg=Fusarium oxysporum f. sp.melongenae, Fol=Fusarium oxysporum f. sp.lycopersici, Fon=Fusarium oxysporum f. sp.niveum and Fom=F.oxysporum f. sp.melonis. *

Table 2 .
The effect of volatile and non-volatile metabolites by T. harzianum on pathogenic strains Fusarium oxysporum mycelial growth (mm) Means within columns followed by the same letter are not significantly different according to Duncan's Multiple Range Test (P ≤ 0.05).Means starting with the same letter within lines are not significantly different at the 0.05 level using Tukey's HSD test.Values represent mean ± SD of ten replicates.Measurements of radial growth were taken 7 days after inoculation.**Fomg=Fusarium oxysporum f. sp.melongenae, Fol=Fusarium oxysporum f. sp.lycopersici, Fon=Fusarium oxysporum f. sp.niveum and Fom=F.oxysporum f. sp.melonis. *