Abstract: Dual culture of some bacterial isolates obtained from animal manure compost extracts with F. oxysporum f. sp. radicis-lycopersici, the causal agent of the Fusarium Crown and Root Rot of tomato, significantly inhibited the in vitro development of the pathogen comparatively to the untreated control. Among 14 isolates tested, 8 inhibited by 38 to 47% the mycelial growth of F. oxysporum f. sp. radicis-lycopersici. The transplantation of tomato seedlings (cv. Riogrande) in peat, previously treated by bacterial suspensions and inoculated with a conidial suspension of the pathogen (107 spores mL-1), significantly reduced the Fusarium Crown and Root Rot severity compared to the untreated control. The most effective isolates were identified by means of the API system, as Chryseomonas luteola, Serratia liquifaciens and Aeromonas hydrophila.
INTRODUCTION
Fusarium oxysporum f. sp. radicis-lycopersici causes the Fusarium Crown and Root Rot in tomato and induces serious yield losses (Rekah et al., 1999). Due to its soil borne origin, complete suppression of this pathogen from soil is difficult and fungicides use is limited by the risk of development of fungicide-resistant strains (Hibar et al., 2006). Thus, more efficient control alternatives are required (Sivan and Chet, 1993).
Under Tunisian conditions and for Fusarium wilts biocontrol, Hibar et al. (2005) and Ayed et al. (2006) showed the antagonistic activity of fungi isolated from suppressive soils, against Fusarim oxysporum f. sp. radicis-lycopersici and F. oxysporum f. sp. tuberosi. Daami-Remadi et al. (2006) reported the inhibitory effect of Bacillus sp. against Fusarium solani, F. graminearum, F. oxysporum f. sp. tuberosi and F. sambucinum, the causal agents of potato dry rot in Tunisia. Elsewhere, Idris et al. (2007) showed that some rhizobacteria isolated from the rhizosphere were efficient in controlling F. oxysporum Schlectend of sorghum.
For the biological control of Fusarium sp., composts and their extracts are also shown to be of potential value (Weltzien, 1992; McQuilken et al., 1994; Hoitink et al., 1997; Cotxarrera et al., 2002; Pharand et al., 2002) as compost microflora plays a major role in the suppression of plant pathogens. The pasteurization of compost destroyed its active microorganisms and consequently nullified their antagonistic effect (Hoitink et al., 1991, 1997; Zhang et al., 1998; Bess, 2000; Quarles, 2001; Ingham, 2002; Camozzi, 2003). Cotxarrera et al. (2002) showed that bacterial populations were frequent in compost and were implicated in the suppression of the Fusarium wilt of tomato. Some strains of Bacillus subtilis (Phae et al., 1990), isolated from compost, showed suppressive effect against several phytopathogenic fungi such as Verticillium dahliae and Fusarium oxysporum f. sp. lycopersici. Stindt (1990) found that 33 bacterial antagonists, belonging to the genera Pseudomonas, Bacillus and Enterobacter isolated from cattle manure compost extracts, inhibited the conidial germination of Botrytis cinerea. Similarly, Ketterer (1990) isolated from extracts of composted horse manure, two bacterial strains with antagonistic properties on detached potato leaves against Phytophthora infestans. Antagonistic interactions with phytopathogenic fungi and mechanism of biological control were based on antibiosis, parasitism, induced resistance and competition for space and limited resources (Hoitink et al., 1997).
Preliminary dual culture of some animal manure compost extracts with Fusarium oxysporum f. sp. radicis-lycopersici, showed inhibition of this pathogen (Kerkeni et al., 2007). The aim of this study was to evaluate the individually effect of some bacterial isolates, obtained from the most suppressive compost extracts, against F. oxysporum f. sp. radicis-lycopersici and their ability to decrease the severity of the Fusarium Crown and Root Rot of tomato.
MATERIALS AND METHODS
Pathogen
F. oxysporum f. sp. radicis-lycopersici used in this study
was isolated on 2006 from tomato plants showing typical symptoms of Fusarium
Crown and Root Rot. The pathogen was cultured on PDA at 25°C for one
week and stored at 4°C for long term preservation.
Isolation of Compost Bacteria
A mature compost (>12 months), composed by 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 compost extract preparation and bacteria isolation.
Compost bacteria were separately isolated on Glutamate-Mannitol (MG) medium based on yeast agar (Oxoid) (0.5 g L-1), Glutamic acid (2 g L-1), Mannitol (5 g L-1), KH2PO4.3H2O (0.5 g L-1), NaCl (0.2 g L-1), MgSO4.7H2O (0.2 g L-1) and agar (Oxoid No. 3) (20 g L-1). A serial dilution of compost extract up to 10-3 was carried out and then 10 μL aliquots of this dilution were spread onto MG medium plates. After 48 h of incubation at 27°C, bacterial colonies formed in the seeded media were individually resuspended into MG medium. The same procedure was repeated until having a purified bacterial culture. A total of fourteen bacterial isolates with different morphological characteristics were selected (CB1B1, CB1C, CB2A, CB2B, CB3A1, CB3B, CB3A2, CB3C, CB3D, CB4C, CB4D, CB5D, CB5B2 and CB7A1). They were sustained on King`s B medium (King et al., 1954) at 27°C. Their identification was realized by means of the API system (Idris et al., 2007).
In vitro Bioassay of the Antagonistic Activity of the Compost
Fungi
The antifungal activity of each bacterial isolate against Fusarium
oxysporum f. sp. radicis-lycopersici was tested via the dual
culture technique, as described by Fuchs (1993). The method consists of
placing an active mycelial disc (6 mm in diameter) of the pathogen at
the center of a 9 cm Petri plate containing freshly prepared King`s B
medium (King et al., 1954). Compost bacteria were applied on the
King`s B plate as shown in Fig. 1. For untreated plates,
an agar disc of F. oxysporum f. sp. radicis-lycopersici
was placed at the center of the Petri dish but sterile distilled water
was used instead of bacterial suspension. All plates were then incubated
at 25°C and evaluated for pathogen growth inhibition after 5 days
of incubation. Three replicates were used per elementary treatment.
To determine the inhibition rate of this pathogen by each of the tested compost bacteria, the radial fungal growth of F. oxysporum f. sp. radicis-lycopersici was noted by measuring the colony diameters for the control and treated plates (average of the two perpendicular diameters). The inhibition rate was calculated according to the formula used by Hibar et al. (2005) where:
| 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
Bacteria
Plant Material
Lycopersicon esculentum Mill. «Priscas», cv. Riogrande
was chosen for its susceptibility to Fusarium oxysporum f. sp.
radicis-lycopersici (Hibar, 2002).
Bacterial Inoculum Preparation
Colonies of 48 h old bacterial cultures on King`s B medium were used
for inoculation of 25 mL of a liquid M1 (i.e., (L-1) 0.98 g
K2HPO4.3H2O, 0.4 g MgSO4.7H2O,
0.4 g CaCO3, 3 g yeast agar and 10 g sucrose). The suspensions
were incubated at 27°C for 24 h under continuous agitation at 120
rpm. After incubation period, an aliquot of 200 μL of each suspension
was spread onto Petri plates containing King`s B medium and incubated
again for 24 h at 27°C. Cells from over-night cultures (27°C)
were washed three times in sterile distilled water. Twenty five milliliter
of bacterial suspensions were used for the treatment of tomato roots (Fuchs,
1993). Control plants were similarly treated but the bacterial suspension
was replaced by sterile distilled water.
Preparation of the Pathogen
F. oxysporum f. sp. radicis-lycopersici mycelium taken from
the colony edge was transferred to 150 mL of Potato Dextrose Broth (PDB)
and incubated at 25°C for 5 days under continuous agitation at 120
rpm. After incubation period, the liquid culture was filtered and the
conidial suspension was adjusted to 107 spores mL-1
by means of a Malassez cystometer (Hibar et al., 2006).
Bioassay
Compost bacteria inhibiting by more than 40% the F. oxysporum
f. sp. radicis-lycopersici mycelial growth in the in vitro
assay were tested under greenhouse conditions. One month old tomato plants,
cv. Riogrande, were transferred from alveolar flats; their roots washed
and soaked for 15 min in each of the bacterial suspension already prepared.
Plants were then transplanted into 10 cm diameter plastic pots containing
an autoclaved peat (15 min at 120°C).
Tomato plants already treated individually with compost bacteria were directly inoculated with F. oxysporum f. sp. radicis-lycopersici by irrigation with 10 mL of conidial suspension (107 spores mL-1). Plants inoculated with the pathogen and irrigated by sterile distilled water 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 transplantation (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), |
| 3 | = | Dead plants. |
At the end of the bioassay, the height, the mean shoot and root fresh weights of plants per elementary treatment were determined.
Identification of Bacterial Isolates
Based on the in vitro results, the most promising bacterial
isolates were selected and identified to the species level by means of
the API identification system assisted by Analytic Profile Index (API)
plus computer software (bioMétrieux® SA, Marcy-l`Etoile/France).
Gram negative rod isolates with fermentative reaction were identified
by The API® 20 E test strip while those with oxidative
reaction were identified by the API® 20 NE test strip.
Experimental Design and Statistical Analysis
Data were arranged as a completely randomized design. Ten replicate pots
per elementary treatment 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`s test.
RESULTS
In vitro Inhibition of F. oxysporum f. sp. radicis-lycopersici
Growth by Compost Bacteria
The results in Table 1 showed that all tested bacterial
isolates, significantly reduced the mycelial growth of Fusarium oxysporum
f. sp. radicis-lycopersici, after incubation at 25°C for
5 days. All tested isolates were effective in reducing the mycelial growth
by 3 to 47% compared to the untreated control; the most effective ones
were CB3B, CB2A, CB7A1 (Fig. 1), CB4D, CB3A2, CB3C,
CB3D and CB5B2, where pathogen growth was limited by more than 38%. The
isolates CB5D, CB1C, CB3A1 and CB1B1 showed lower efficiency (<10%).
In vivo Inhibition of F. oxysporum f. sp. radicis-lycopersici
Growth by Compost Bacteria
Disease Severity
The ability of four selected compost bacteria to reduce the F.
oxysporum f. sp. radicis-lycopersici development on tomato
was assessed one month post inoculation. Symptoms induced by F. oxysporum
f. sp. radicis-lycopersici were less severe in plants grown in
substrates treated with tested bacteria, in comparison to the untreated
control. The isolates CB3D and CB5B2 significantly inhibited by about
50% the tomato crown and root rot development, the disease severity was
reduced to 1.1 and 1.4%, respectively. Control plants not treated with
bacteria but inoculated only with the pathogen rendered 2.6% (Fig.
2).
| Table 1: | Inhibition rate of Fusarium oxysporum f. sp. radicis-lycopersici radial growth in presence of the compost bacteria (King`s B, after six 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`s test. Each value represents the mean of 3 values | |
| Table 2: | Fusarium crown and root rot severity on tomato plants observed after 30 days, in sterilized peat treated with compost bacteria 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`s test | |
| Fig. 1: | Inhibition of growth of Fusarium oxysporum f. sp. radicis-lycopersici by some bacteria isolated from compost extracts in comparison to the untreated control |
| Fig. 2: | Improvement of plant growth of one month old tomato plants (cv. Riogrande) with compost bacteria. Control: substrates with Fusarium oxysporum f. sp. radicis-lycopersici |
Plant Height
Results in Table 2 showed that the amendment of substrates
with compost bacteria significantly enhanced the plant height comparatively
to the untreated control when isolates such as CB3D, CB3B and CB5B2 were
used. In fact, tomato plants growing in those substrates were higher than
18 cm comparatively to 10.6 cm for the untreated control plants (Fig.
2).
| Table 3: | Effect of the treatment of substrates with compost bacteria on plant height, shoot and root fresh weights of one month old tomato plants (cv. Riogrande) |
| Control: substrates with Fusarium oxysporum f. sp. radicis-lycopersici. Each value represents the mean of 10 values. Different letter(s) represent values that are significantly different at p = 0.05 based on ANOVA and Duncan`s test | |
Shoot and Root Fresh Weights
The inoculation of plants by F. oxysporum f. sp. radicis-lycopersici
only significantly decreased the fresh weights of plants, compared to
plants treated by the bacterial isolates and inoculated (Table
3, Fig. 2). In fact, the treatment by CB3D, CB3B
and CB5B2 isolates enhanced the shoot fresh weight of plants by more than
60%, in comparison to the inoculated but untreated control.
Root fresh weights increased for all treatments with antagonistic bacteria, compared to the untreated control. No significant improvement in root weight was noted on plants treated by CB7A1 and CB3B isolates, which were shown to be less effective in reducing disease severity (Table 2). The increase in the root fresh weights by these both isolates was about 89% compared to the control.
Identification of Bacterial Isolates
Based on the results of the API tests, the four most effective bacterial
isolates tested, were identified as Chryseomonas luteola (CB7A1),
Serratia liquifaciens (CB3B) and Aeromonas hydrophila for
CB5B2 and CB3D.
DISCUSSION
The present study showed that some bacterial isolates obtained from an animal manure compost extract showed an inhibitory effect against the causal agent of the Fusarium Crown and Root Rot of tomato. Eight bacterial isolates, among the 14 tested, inhibited the pathogen growth by more then 38%. These results supported the findings of Phae et al. (1990), Stindt (1990), Ketterer (1990) and Weltzien (1992), who reported that compost extracts contain microorganisms, including bacteria with antagonistic potential against several pathogens.
In a previous work conducted in vitro, Kerkeni et al. (2007) showed that compost extracts used for isolation of tested bacterial isolates, also inhibited the growth of this same isolate of F. oxysporum f. sp. radicis-lycopersici by 42.6%. This suggests that compost extracts contain biocontrol agents that are more efficientwhen used alone as is the case of isolates CB3B (47%) and CB7A1 (44%).
In the bioassay, some tested isolates such as CB3D and CB5B2 were shown to be the most effective in reducing the Fusarium Crown and Root Rot severity and also in enhancing the plant growth parameters (height and shoot fresh weights). This suggests that these bacterial isolates are also able to promote the plant growth by induced plant resistance to inoculation and probably by affecting directly the pathogen by several modes of action and/or variable types of antifungal metabolites produced (Williams and Asher, 1996). In fact, Daami-Remadi et al. (2006) and Chérif et al. (2002) showed that antagonistic bacteria act by producing enzymes that cause hyphal alterations such as generalized cytoplasm disorganisation, complete protoplasm loss and fungal cell wall disintegration. Singh et al. (1999) reported that chitinolytic enzymes were important in the biological control of soil borne pathogens because of their ability to degrade fungal cell walls, of which a major component is chitin. These enzymes were proved to be involved in the antagonistic activity; they act by breaking down and dissolving the polysaccharides, responsible for the rigidity of fungal cell walls (Howell, 2003).
The effectiveness of bacteria isolated from compost, as biocontrol agents, against plant diseases was previously reported by Weltzien (1992), Ketterer (1990) and Stindt (1990). Phae et al. (1990), showed an antagonistic effect of some bacteria originating from compost, against several pathogens including F. oxysporum f. sp. lycopersici. Kwok et al. (1987) identified different species of bacteria such as Bacillus cereus, Pseudomonas sp. and Flavobacterium balustinum suppressive of Rhizoctonia solani. El-Masry et al. (2002) also isolated from compost several bacterial microorganisms such as Bacillus sp. with inhibitory effect against pathogens such as Pythium debaryanum, Fusarium oxysporum f. sp. lycopersici and Sclerotinia bataticola.
The four isolates CB3D, CB3B, CB7A1 and CB5B2, inhibiting by more than 40% the in vitro pathogen growth and shown to be effective in reducing disease severity in vivo, were identified as Chryseomonas luteola for CB7A1, Serratia liquifaciens for CB3B and Aeromonas hydrophila for CB5B2 and CB3D. The genus Serratia was found to possess antifungal properties (Kurze and Bahl, 2001). In the same way, Sneh et al. (1985) demonstrated that Serratia liquefaciens inhibited the Fusarium wilt of carnation. Inbar and Chet (1991) reported that strains of Aeromonas were effective biological control agents against Sclerotium rolfsii and Rhizoctonia solani. Further studies are required for elucidating the several modes of action of these bacterial isolates, quantifying their effect on disease incidence rather than on disease severity for their eventual test under field conditions and in naturally infected soils.