Abstract: Aspergillus sp., Trichoderma viride strain 2 and T. viride strain 1 isolated from an animal manure compost are tested for their in vitro and in vivo antagonistic activity against Fusarium oxysporum f. sp. radicis-lycopersici, the causal agent of the Fusarium Crown and Root Rot of tomato. Dual culture experiments, observed after incubation at 25°C on PDA during 5 days, showed that all tested fungi significantly inhibited the mycelial growth of F. oxysporum f. sp. radicis-lycopersici comparatively to the untreated control. Inhibition varied from 25% for Trichoderma viride (strain 1) to 100% for Aspergillus sp. Competition for media was the predominant mechanism of action noted on PDA. In vivo, tomato plants (cv. Riogrande), simultaneously inoculated and treated individually by the compost fungi conidial suspensions (107 spores mL-1), showed reduced severity of the Fusarium Crown and Root Rot, when observed 30 days after transplantation, comparatively to the untreated control. The compost fungi T. viride (strain 1) was the most effective, it decreased severity of the disease by 48%.
INTRODUCTION
Fusarium Crown and Root Rot of tomato induced by Fusarium oxysporum f. sp. radicis-lycopersici is one of the most damaging soil-borne diseases of tomato causing heavy economic losses (Rekah et al., 1999). Complete suppression of this fungus from soil is difficult by the use of fungicides (Hibar et al., 2006). The difficulties in controlling this pathogen promoted scientists to search for other alternatives (Sivan and Chet, 1993). Composts prepared from heterogeneous organic wastes are some applicable means for the biological control for several plant diseases especially those caused by soilborne pathogens (Hoitink et al., 1991, 1997). Phytopathogenic fungi such as Phytophthora sp. (Aryantha et al., 2000), Rhizoctonia solani (Nelson and Hoitink, 1983) and Fusarium sp. (Chef et al., 1983; Cotxarrera et al., 2002) were successfully suppressed by composts. Several researches concluded that the microflora of composts plays the major role in suppression of plant pathogens. Indeed, Pasteurization of compost destroys their active microflora and nullifies their efficacy (Hoitink et al., 1991, 1997; Zhang et al., 1998; Bess, 2000; Quarles, 2001; Ingham, 2002; Camozzi, 2003). Beneficial microorganisms present in composts are implicated in various suppressive activities (Hoitink et al., 1991) and strains of Trichoderma sp. (Cotxarrera et al., 2002) and Bacillus subtilis (Phae et al., 1990), isolated from composts, were shown to be effective antagonists against several pathogens. Strains of T. hamatum suppressed F. oxysporum (Trialls-Gay et al., 1986). Isolates of Penicillium sp. and of Aspergillus sp. were suppressive to F. solani, F. graminearum, F. sambucinum and F. oxysporum f. sp. tuberosi (Daami-Remadi et al., 2006). Antagonistic interactions with other fungi and mechanisms involved in the biocontrol process are based on antibiosis, parasitism, induced resistance and competition (Hoitink et al., 1997).
Preliminary dual cultures of some compost extracts with F. oxysporum f. sp. radicis-lycopersici, showed inhibited mycelial growth of this pathogen (Kerkeni et al., 2007a). The aim of this study is to isolate fungi from the most suppressive previously tested compost extracts, to evaluate in vitro and in vivo their individually effects, on F. oxysporum f. sp. radicis-lycopersici and to assess their ability to decrease the Fusarium Crown and Root Rot of tomato severity.
MATERIALS AND METHODS
Pathogen
F. oxysporum f. sp. radicis-lycopersici used in this study
was isolated from tomato plants showing typical symptoms of crown and root rot.
It was cultured on PDA at 25°C for one week and stored at 4°C for long
preservation.
Compost Fungi
A mature compost (>12 months), composed of 40% cattle manure, 40% sheep
manure and 20% vegetable wastes and produced on 2006 at the composting-unit
of the Technical Centre of Organic Agriculture of Chott Mariem-Tunisia, was
used for antagonistic fungi isolations.
Potato Dextrose Agar (PDA; Sigma) supplemented with 5 mg L-1 Penicillium-G was used for fungal isolation. A sample of 10 g of solid compost was suspended in 90 mL of sterilized distilled water in 250 mL bottle. The sample was stirred for 1 h at 200 rpm. A serial dilution up to 10-3 was carried out and then 100 μL aliquots of this dilution were spread onto PDA medium plates. After incubation at 25°C for 5 days, fungal colonies obtained were individually transferred on PDA. The same procedure was repeated until having a purified fungal culture. Selected compost fungi were identified on the basis of their macroscopic and microscopic characteristics (El-Masry et al., 2002). They were cultured on PDA at 25°C for one week before use.
In vitro Bioassay of the Antagonistic Activity of the Compost Fungi
The study was conducted in the Laboratory of Phytopathology of the Regional
Centre of Research in Horticulture and Organic Agriculture of Chott Mariem (Tunisia).
The antifungal activity of each tested compost fungi against Fusarium oxysporum
f. sp. radicis-lycopersici was studied via the dual culture technique.
The method consists of placing an active mycelial disc (6 mm in diameter) of
the pathogen, 1 cm from the edge of a 9 cm Petri plate containing freshly prepared
PDA medium. Another disc (6 mm) of the antagonist fungi was deposited in a diametrically
opposed position 1 cm away from the other set of the plate. For untreated plates,
an agar disc of F. oxysporum f. sp. radicis-lycopersici was placed
at the center of the petri dish. All plates were then incubated at 25°C
and evaluated for pathogen growth inhibition after 4 days of incubation. Three
replicates were used per elementary treatment.
To determine the inhibition rate of this pathogen by each of the tested compost fungi, the fungal growth of F. oxysporum f. sp. radicis-lycopersici was recorded by measuring the F. oxysporum f. sp. radicis-lycopersici colony diameters (average of the two perpendicular diameters). These diameters (control and treated) served for the calculation of the inhibition rate of the fungal growth. This rate is calculated according to the following formula used by Hibar et al. (2005):
Inhibition rate (%) = (1–(Average diameter of
the treated/Average diameter of the control) x 100 |
In vivo Bioassay of the Antagonistic Activity of the Compost Fungi
Plant Material
Disease suppressiveness of substrates individually amended with the different
isolated fungi was tested in a bioassay using tomato (Lycopersicon esculentum
Mill. Priscas), cv. Riogrande plants. This later was chosen for its sensibility
to Fusarium oxysporum f. sp. radicis-lycopersici (Hibar, 2002).
Bioassay
One-month-old tomato plants, cv. Riogrande, were transferred from alveolar
flats into 10 cm diameter plastic pots containing an autoclaved peat (15 min
at 120°C).
For plant inoculation, mycelium taken from the edge colony of F. oxysporum f. sp. radicis-lycopersici was transferred to 150 mL of Potato Dextrose Broth (PDL) and incubated at 25°C for 5 days in a rotary incubator (120 rpm). The liquid culture was filtered and the conidial suspension was adjusted to 107 spores mL-1 by means of Malassez cystometer (Hibar et al., 2006). The same procedure was used for the preparation of the inoculum of the tested fungi.
Tomato plants already planted, were inoculated simultaneously by irrigation with 10 mL of conidial suspension (107 spores mL-1) of F. oxysporum f. sp. radicis-lycopersici and 10 mL of conidial suspension (107 spores mL-1) of compost fungi (inoculated separately). Plants inoculated with the pathogen and without compost fungi were used as control.
Bioassay was conducted under greenhouse conditions at 25°C and under 12 h photoperiod (Pharand et al., 2002). The plants were watered as needed. Ten replicate pots of each treatment were randomly placed. No fertilizer was added to plants. The experiment was conducted twice. Disease severity was determined 30 days after plantating (Woo et al., 1996), based on a symptom severity scale, where: 0 = asymptomatic plants; 1= weakly infected plants (<50% of leaves chlorotic or wilted); 2 = high infected plants (>50% of leaves wilted but plants not dead) and 3 = dead plants. At the end of the bioassay, the height, the mean shoot and root fresh and dry weights of plants per elementary treatment were determined.
Experimental Design and Statistical Analysis
Data were arranged as a completely randomized design. Ten replicate pots
of each elementary were used and the whole bioassay was repeated twice. Data
were analyzed using SPSS statistical program version 11.0. and subjected to
Analysis of Variance (ANOVA). Means were compared according to the Duncan test.
RESULTS
In vitro Inhibition of the F. oxysporum f. sp. radicis-lycopersici
Growth by the Tested Compost Fungi
The results shown in Table 1 showed that compost fungi,
significantly reduced the mycelial growth of Fusarium oxysporum f. sp.
radicis-lycopersici, after incubation at 25°C for 5 days. All fungi
tested were effective in reducing the mycelial growth more than 20% compared
to the untreated control.
| Table 1: | Inhibition rate of Fusarium oxysporum f. sp. radicis-lycopersici colonies in presence of the compost fungi (PDA, after five days of incubation at 25°C) |
| Different letter(s) within columns represent values that are significantly different at p = 0.05 based on ANOVA and Duncan test. Each value represents the mean of 3 values | |
| Fig. 1: | Total overlapping and inhibition of Fusarium oxysporum f. sp. radicis-lycopersici by the compost fungus Aspergillus sp. |
| Table 2: | Fusarium crown and root rot severity on tomato plants observed after 30 days, in sterilized peat treated with compost fungi in comparison to the untreated control (means of ten plants). Disease severity ranked from 0 (asymptomatic plants) to 3 (dead plants) |
| Different letter(s) represent values that are significantly different at p = 0.05 based on ANOVA and Duncan test | |
Inhibition varied from 25 to 100%. The most effective fungi were Aspergillus sp. and Trichoderma viride (strain 2), where pathogen growth was limited by 100 (Fig. 1) and 72%, respectively. The fungus T. viride (strain 1) showed lesser efficiency (25%).
In vivo Inhibition of the F. oxysporum f. sp. radicis-lycopersici
Growth by the Tested Compost Fungi
Disease Severity
The ability of the tested compost fungi to suppress F. oxysporum
f. sp. radicis-lycopersici on tomato plants, in sterilized peat, was
assessed one month post inoculation. Symptoms developed by F. oxysporum
f. sp. radicis-lycopersici were lesser in plants grown in substrates
treated with T. viride (strain 1) (Table 2). This later
showed a remarkable efficiency in reducing Fusarium crown and root rot
disease severity in comparison to the in vitro essay. In vivo,
it decreased disease severity by nearly 50%, compared to the untreated control.
Results showed also that Aspergillus sp., which was the most effective
in vitro, was comparable to the control. T. viride (strain 2)
reduced the disease development but its efficiency was lesser than T. viride
(strain 1).
Plant Growth Parameters
Plant Height
Results in the Table 3 showed that the amendment of substrates
with compost fungi had a significant effect on the tomato plant height. The
presence of Trichoderma viride (strain 2) in the substrate with F.
oxysporum f. sp. radicis-lycopersici enhanced significantly the plant
height comparatively to the control. Tomato plants growing in this substrate
had 23.75 cm. Whereas, those growing in the control substrates had only 13.4
cm. Statistically, plants inoculated with Aspergillus sp. and T. viride
(strain 1) were not different to the control.
| Fig. 2: | Effect of the treatment of substrates with compost fungi on the plant growth parameters of one month old tomato; Control: substrates with Fusarium oxysporum f. sp. radicis-lycopersici. (25°C, under 12 h photoperiod) |
| Table 3: | Effect of the treatment of substrates with compost fungi on plant height, shoot and root fresh weights and shoot and root dry weights, of one month old tomato. Control: substrates with Fusarium oxysporum f. sp. radicis-lycopersici |
| Different letter(s) represent values that are significantly different at p = 0.05 based on ANOVA and Duncan test; Each value represents the mean of 10 values | |
Plant Shoot and Root Weights
Table 3 showed that only the presence of T. viride
strain 2 in the substrates resulted in a pronounced increase in the tomato shoot
fresh weight, compared to the control and to substrates amended with the other
compost fungi. The improvement in shoot fresh weight of plants growing in the
presence of T. viride (strain 2) amounted to more than 50%, in comparison
to the untreated control (Table 3 and Fig. 2).
Root fresh weight increase for this treatment, was not different to the substrates
inoculated with T. viride (strain 1).
Table 3 showed also that the same substrate, inoculated with T. viride (strain 2) improved significantly tomato shoot dry weight, in comparison to all treatments. Whereas, no difference was obtained between all plants concerning root dry weights.
DISCUSSION
These results showed for the first time in Tunisia that fungi isolated from an animal manure compost are suppressive against the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici.
The current study showed that fungi isolated from compost were able to inhibit the growth of F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato. This results joined that obtained by Phae et al. (1990), Zhang et al. (1998) and Bess (2000), showing that compost contain microorganisms suppressive to plant pathogens. in vitro, Aspergillus sp., Trichoderma viride (strain 2) and T. viride (strain 1) showed an inhibitory effect towards F. oxysporum f. sp. radicis-lycopersici. Aspergillus sp. was the best in reducing the mycelial growth of F. oxysporum f. sp. radicis-lycopersici by 100%.
Earlier study conducted in vitro by Kerkeni et al. (2007a) showed that the whole compost extract used for these fungi isolation, inhibited the growth of this same isolate of F. oxysporum f. sp. radicis-lycopersici by only 42.6%. This suggests that composts and compost extracts contain biocontrol agents that are more efficient when used alone, probably this is the case with Aspergillus sp. and T. viride strain 2.
In the contrast, bioassay conducted in vivo, showed that this fungus was the least in reducing the disease severity on tomato plants. T. viride (strain 1) was the most effective in vivo but the least suppressive antagonist in vitro (25%). The fungus T. viride (strain 2) was always intermediate in suppression but the best in enhancing plant growth comparatively to the control and the other compost fungi. The variable efficiency of the tested fungi may be attributed to a variable mode of action and/or a variable type of antifungal metabolites produced by the antagonists (Williams and Asher, 1996). Tests based on in vitro mycelial inhibition do not always correlate with biocontrol efficacy under natural conditions. This finding proved that in vitro and in vivo results may be divergent as may be due to the variable physical and chemical properties within niches occupied by the biocontrol agents which may affect both root colonization and expression of biocontrol mechanisms.
The effectiveness of compost fungi against plant diseases as biocontrol agents was previously reported by Daami-Remadi et al. (2006), where an antagonistic effect of some filamentous fungi, isolated from compost, was noted against the Fusarium sp. complex, causing dry rot of potato. El Masry et al. (2002) and Muhammad and Amusa (2003) also isolated from compost, several fungal microorganisms such as Aspergillus niger, Rhizopus sp., Drechslera sp. and Trichoderma harzianum, which had an inhibitory effect against pathogens such as Pythium aphanidermatum, Fusarium oxysporum and Rhizoctonia solani. Cotxarrera et al. (2002), found that Trichoderma asperellum isolated from compost decrease disease severity of the Fusarium wilt of tomato. In a previous work, we also showed that compost fungi had an antagonistic effect against Pythium ultimum (Kerkeni et al., 2007b).
Competition for nutrients present in the media, traduced by an overlapping of the pathogen colonies by that of the tested fungi, was the main mechanism, employed by compost fungi in antagonism of F. oxysporum f. sp. radicis-lycopersici in the dual culture. The F. oxysporum f. sp. radicis-lycopersici colonies overlapping by antagonists is probably due to a physical contact between pathogen and the compost fungi leading to the pathogen parasitism and its mycelium destruction. The same mechanism was observed in the dual culture of some compost fungi and Pythium ultimum (Kerkeni et al., 2007b). In fact, multiple mechanisms of action including mycoparasitism, lysis, induction of mycelial cords and early chlamydospores formation were observed in dual cultures of some compost fungi with four Fusarium species (Ayed et al., 2006; Daami-Remadi et al., 2006).
Howell (2003), reported that biocontrol agents produce enzymes such as chitinase, protease and cellulase. These enzymes have been proved to be involved in the antagonistic activity. They act by breaking down and dissolving the polysaccharides, responsible for the rigidity of fungal cells walls. Chérif and Benhamou (1990) and Hibar et al. (2005) found that Trichoderma strains could produce enzymes that can diffuse in culture media. These substances induced reduction in F. oxysporum f. sp. radicis-lycopersici growth. However, in in vivo bioassay, Kerkeni et al. (2007b) did not show any correlation between the suppression of Pythium ultimum and production of cellulase in the growing substrate.
The compost fungus T. viride (strain 1) was the most effective in inhibiting the growth of F. oxysporum in vivo. Protective effect of this least fungus may be attributed to the induction of systemic resistance on tomato plants. This mechanism by which biocontrol agents acted, is not frequently detected in vitro (Schisler et al., 1997).
These results showed that isolated compost fungi are suitable products to suppress plant pathogenic fungi. All tested fungi showed an antagonistic activity in vitro and in vivo against Fusarium oxysporum f. sp. radicis-lycopersici, causal agent of Fusarium crown and root rot of tomato. This finding is interesting and showed the importance of antagonist potential employed and also the ability of compost isolated fungi to support environmental conditions. They could be a promising way for the biological control of plant diseases and so could reduce the need of fungicides use.