Volume 63, Issue 5 p. 1062-1069
Original Article
Free Access

Biological control agents (BCAs) of verticillium wilt: influence of application rates and delivery method on plant protection, triggering of host defence mechanisms and rhizosphere populations of BCAs

D. J. Angelopoulou

D. J. Angelopoulou

Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos street, 11855 Athens, Greece

These authors contributed equally to this work.Search for more papers by this author
E. J. Naska

E. J. Naska

Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos street, 11855 Athens, Greece

These authors contributed equally to this work.Search for more papers by this author
E. J. Paplomatas

E. J. Paplomatas

Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos street, 11855 Athens, Greece

Search for more papers by this author
S. E. Tjamos

Corresponding Author

S. E. Tjamos

Laboratory of Plant Pathology, Agricultural University of Athens, 75 Iera Odos street, 11855 Athens, Greece

E-mail: [email protected]Search for more papers by this author
First published: 17 January 2014
Citations: 67

Abstract

Verticillium dahliae causes severe yield reductions in a variety of important annual crops worldwide. Control of verticillium wilt has relied on soil fumigation; however, the use of the main soil fumigant, methyl bromide, has been banned in the European Union since 2010, creating a demand for novel crop protectants. As such, the use of biocontrol agents (BCAs) is an appealing management strategy. Prerequisites for the development of a successful BCA are an understanding of the modes of action of the antagonist, its ecological fitness and an efficient and economically feasible delivery system. Therefore, two BCAs (Paenibacillus alvei K165 or the nonpathogenic Fusarium oxysporum F2) and two release strategies (seed coating or amendment of the transplant soil plug) were assessed against verticillium wilt of aubergine (eggplant). Mixing the transplant soil plug with K165 or F2, at a rate of 10 and 20% (v/v), respectively, reduced verticillium wilt symptom development. Furthermore, a positive correlation was revealed between the release strategy and the BCA rhizosphere population. Correlation analysis also showed that disease severity was negatively correlated to the rhizosphere size of the BCA population. In addition, qPCR analysis showed that both BCAs induced the expression of the pathogenesis-related (PR) proteins PR1 and PR4 in the stem of aubergines before and after inoculation with V. dahliae in a manner that suggests a link with the rhizosphere size of the BCA population.

Introduction

Verticillium dahliae is a widely distributed soilborne pathogen causing vascular wilt on more than 160 plant species and resulting in billions of dollars in crop losses, annually, worldwide (Pegg & Brady, 2002). Verticillium dahliae survives in the soil in the form of microsclerotia, the primary infection inoculum in the field, for several years. Because of its long-term persistence in the field, inaccessibility during infection, broad host range and scarcity of resistance in host germplasm, control of verticillium wilt has relied heavily on soil fumigation, a protective measure that is contingent on the economic returns from the crop (Klosterman et al., 2009). In 2005, the use of methyl bromide, the main soil fumigant, was banned under the Montreal Protocol on Substances that Deplete the Ozone Layer. Therefore, the development and use of biocontrol agents (BCAs) against V. dahliae is an appealing management strategy for both the conventional and organic farming industry.

A prerequisite for the commercial use of BCAs is that inoculum retains high cell viability and can easily be transported and applied (Kloepper & Schroth, 1981). Furthermore, proper timing and placement of applications can greatly reduce the amount of BCA required (Fravel, 2005). It can be supposed that BCA application in greenhouses or nurseries is preferable to large-scale field treatments because it is easier in practice and less expensive and time-consuming. Therefore, a biocontrol strategy based on the application of BCAs to transplant soil plugs would be the most effective, in terms of economic returns and agricultural practice strategy, for crops that are initially grown in the greenhouse or nursery and then transplanted in the field for protection against soilborne pathogens such as V. dahliae.

A crucial factor affecting many aspects of biocontrol performance is the formulation of BCAs (Fravel, 2005). It has been postulated that a dry product is more favourable than other formulations, because it is less weight to ship and at lower risk of possible contamination (Fravel, 2005). Kloepper & Schroth (1981) tested a number of different powder formulations of a plant growth-promoting rhizobacterium (PGPR), and suggested that a formulation based on xanthan gum was the most efficacious preparation for releasing BCAs in the field.

Several bacteria and fungi have been reported as BCAs against V. dahliae (Tjamos et al., 2004; Malandraki et al., 2008; Zheng et al., 2011; Veloso & Díaz, 2012). These BCAs are capable of colonizing the root system and protecting plants against V. dahliae by employing several mechanisms of action, such as antibiosis, competition for nutrients or space on roots, and triggering of induced systemic resistance (ISR) (Tjamos et al., 2005; Malandraki et al., 2008; Veloso & Díaz, 2012; Li et al., 2013). ISR offers the prospect of broad spectrum disease control and can result in the direct activation of defence genes, but can also lead to priming, wherein plant defences are potentiated for enhanced expression upon pathogen attack. The development of ISR is associated with the coordinated expression of a specific set of genes encoding pathogenesis-related (PR) proteins in a salicylic acid (SA)- or ethylene/jasmonic acid (ET/JA)-dependent pathway. The PR proteins have been classified in 17 families that include chitinases (PR3, PR4, PR8), glucanases (PR2), proteases (PR7) and families with unknown properties (e.g. PR1) (van Loon et al., 2006).

The BCAs of V. dahliae include the bacterial isolate Paenibacillus alvei K165 and the fungal isolate Fusarium oxysporum F2 (Tjamos et al., 2004; Malandraki et al., 2008). The use of a transformed F2 strain with the enhanced green fluorescent protein (EGFP) reporter gene revealed that competition for space or nutrients on the root surface is the main mode of action of F2 against V. dahliae (Pantelides et al., 2009). The protective activity of K165 has been attributed to the triggering of plant defence mechanisms via a SA-dependent pathway and the reduction of V. dahliae microsclerotia germination (Tjamos et al., 2005; Antonopoulos et al., 2008). Therefore, knowing to a certain extent the mode of action of these two BCAs, the aims of this study were to: (i) assess the effectiveness of two different BCA release strategies, seed coating and amendment of the transplant soil plug, of the bacterial and the fungal BCAs, K165 and F2, respectively, against verticillium wilt of aubergine; and (ii) relate the observed plant protective activity to the rhizosphere BCA population and the induction of the plant defence-associated genes PR1 and PR4.

Materials and methods

Pathogen preparation

A V. dahliae isolate with known pathogenic activity against aubergine was used in the experiments (Malandraki et al., 2008). The isolate was cryopreserved by freezing a suspension of 107 conidia mL−1 in 25% aqueous glycerol at −80°C. Before being used, the fungus was transferred to potato dextrose agar (PDA; Merck) at 25°C for 5 days. Verticillium dahliae microsclerotia were prepared in sucrose sodium nitrate (SSN) liquid medium. The liquid cultures were shaken in an orbital incubator at 22°C for 3 weeks. Microsclerotia were centrifuged at 10 000 g at 20°C for 10 min to remove growth medium then air dried. Microsclerotia were resuspended in sterile distilled water and filtered through 70 μm mesh to select large (>70 μm) microsclerotia, which germinate easily and show high levels of pathogenicity (Hawke & Lazarovits, 1994).

Biocontrol agents preparation

A rifampicin-resistant strain of K165 and a hygromycin B-resistant strain of F2 (Tjamos et al., 2004; Pantelides et al., 2009) were used throughout the experiments.

K165 and F2 were grown in liquid cultures of nutrient broth plus glycerol (NG) and SSN, respectively, in an orbital incubator at 180 rpm at 30°C for 48 h. The K165 and F2 suspensions were centrifuged at 5865 g for 10 min and resuspended in 50 mm phosphate buffer, pH 7·02, providing a concentration of 108 cells mL−1. The BCAs were formulated as a dry powder inoculum with 10% xanthan gum according to Kloepper & Schroth (1981), resulting in a final concentration of 107 colony-forming units (CFU) g−1.

Verticillium dahliae–BCA bioassays

Two different release strategies of the powder formulations of K165 and F2 were evaluated under greenhouse conditions. For this purpose, seeds of aubergine cv. Black Beauty were either coated with the powder formulation of the BCA (107 CFU g−1 of seed) or planted in plastic pots (4 × 4 × 4·5 cm) containing soil (Potground; Klasmann) amended with the K165 or F2 powder formulation at a rate of 1, 5, 10 and 20% (v/v). A control of untreated seeds planted in unamended soil was also included. At the three-leaf stage, plants were transplanted to plastic pots (9 × 9 × 10 cm) containing soil infested or not (mock-inoculated plants) with 20 V. dahliae microsclerotia g−1. Aubergine plants were maintained at 25 ± 3°C with a 12 h light and dark cycle. Each treatment consisted of 10 plants and the experiment was conducted three times. Verticillium wilt symptoms were recorded every 2 days after the onset of symptom development for 14 days. Disease severity at each observation was calculated as the percentage of leaves that showed wilting. Subsequently, disease ratings were plotted over time to generate disease progress curves. The area under the disease progress curve (AUDPC) was calculated by the trapezoidal integration method (Campbell & Madden, 1990). Disease severity was expressed as a percentage of the maximum possible AUDPC for the whole period of the experiment and is referred to as relative AUDPC.

Determination of the K165 and F2 rhizosphere populations

The ability of K165 and F2 to colonize the rhizosphere of aubergine following the different treatments was evaluated at 10 and 1 day(s) before transplantation to V. dahliae-infested soil (20 and 30 days after sowing, respectively) as well as 10, 20 and 30 days post-inoculation (dpi), using the dilution plating technique. Five plants per treatment were sampled and the experiment was performed three times (a total of 15 plants per treatment). To estimate rhizosphere populations, 0·5 g rhizosphere soil was collected and shaken for 45 min in 50 mm phosphate buffer (pH 7·02) containing Tween 20 (0·02%). The suspension was plated onto PDA supplemented with rifampicin (100 μg mL−1) or hygromycin B (75 μg mL−1), in the case of K165- or F2-treated plants, respectively. After incubation at 30°C for 48 h, the number of K165 or F2 CFU g−1 of rhizosphere soil was determined. The rhizosphere microbial populations were plotted over time to generate microbial progression curves; subsequently the area under the microbial progression curve (AUMPC) was calculated by the trapezoidal integration method (Campbell & Madden, 1990).

RNA isolation and qPCR determination of PR1 and PR4 transcript levels

For Verticillium-caused wilt diseases, the resistance or susceptibility of the host plant is generally considered to be determined mainly by the cellular interactions between the plant and the fungus occurring in the stem (Robb et al., 2007), because in the roots only the reinforcement of structural barriers upon pathogen invasion has been observed (Daayf et al., 1997). In the present study, nine plants from the most effective BCA treatments (10 and 20% for K165 and F2, respectively) and less effective BCA treatments (seed coated with K165 or F2), along with control plants (pathogen- and mock-inoculated plants) were harvested at 0, 5, 10 and 20 dpi. For each sampled plant, a 5 cm long stem segment was cut at soil level, ground to a fine powder using an autoclaved mortar and pestle in the presence of liquid nitrogen and stored at −80°C. For each sample, total RNA was extracted from 100 mg of ground tissue using TRIzol (Invitrogen) according to the manufacturer's instructions. The RNA samples were treated with DNase I (Invitrogen) to eliminate traces of contaminating genomic DNA. The RNA concentration was measured in a spectrophotometer (ND-1000; NanoDrop). First-strand cDNA was synthesized using SuperScript II (Invitrogen) following the manufacturer's procedure.

The expression levels of the PR1 and PR4 aubergine genes were detected by using the following primer sequences: for PR1 (GenBank accession no. AB222697), forward PR1S 5′-GCCGTGAAGATGTGGGTCGA-3′ and reverse PR1A 5′-GCACATCCAAGTACGTACCGAGTT-3′; and for PR4 (GenBank accession no. AB222698), forward PR4S 5′-GGACCGCTTTCTGTGGCCCCG-3′ and reverse PR4A 5′-ATAAGGTGGCCTTGCTGGTAGCC-3′ (Kiba et al., 2006). PCR efficiency for each amplicon was calculated by employing the linear regression method on log (fluorescence) per cycle number data, using lin-regpcr software. Quantitative real-time PCRs (qPCR) were performed in duplicate. The absence of nonspecific products and primer dimers was confirmed by the analysis of melting curves. The expression level of the aubergine actin gene, detected using the primer pair ACTIN-F 5′- TTCCGTTGCCCAGAGGTCCT-3′ and ACTIN-R 5′-TTCCGTTGCCCAGAGGTCCT-3′ (Chen et al., 2007), was used as an internal standard to normalize small differences in cDNA template amounts. Average threshold cycle (Ct) values were calculated for each gene of interest on the basis of three independent biological samples.

Statistical analysis

Data on relative AUDPC and AUMPC were transformed with the √(+ 1) transformation before analysis of variance (anova) was performed. When a significant (P ≤ 0·05) F-test was obtained for treatments, data were subjected to means separation by Tukey's multiple range test. Overall relationships between the measured variables were analysed with correlation analysis.

Results

Verticillium dahliae–F2 and –K165 bioassays

The efficacy of K165 and F2 as powder formulations against V. dahliae was investigated by employing two different release strategies: seed coating and amendment of the transplant soil plug at different rates (1, 5, 10 and 20%, v/v). BCA applications significantly reduced verticillium wilt symptom development in aubergine compared to the control treatment (Fig. 1). However, the release of K165 and F2 as a soil amendment was more effective against V. dahliae than the seed coating treatment.

Details are in the caption following the image
Verticillium wilt disease severity on aubergine plants either seed-coated with a biocontrol agent (BCA) or grown in transplant soil plugs amended with BCA at a rate of 1, 5, 10 and 20% (v/v) then transplanted to Verticillium dahliae-infested soil containing 20 microsclerotia per gram of soil. The BCA was Paenibacillus alvei K165 (a) or Fusarium oxysporum F2 (b). Disease ratings were plotted over time to generate disease progression curves; subsequently the area under the disease progression curve (AUDPC) was calculated by the trapezoidal integration method (Campbell & Madden, 1990). (c) Results expressed as the relative AUDPC, i.e. the disease level as a percentage of the maximum possible area for the whole period of the experiment. Columns with different letters are significantly different according to Tukey's multiple range test at < 0·001.

In the case of K165, the most suppressive treatments were the ones in which K165 was incorporated in the transplant soil plug at a rate of 5, 10 and 20% (v/v). Verticillium wilt symptoms started 18 dpi, when wilt symptoms were observed in the control and the seed coating treatment. These two treatments showed the most prominent verticillium wilt symptoms, progressing rapidly until 28 dpi when a plateau was reached. On the other hand, the onset of wilt symptoms in the treatments where K165 was incorporated as a soil amendment was delayed compared to the control and the seed coating treatment. This delay was followed by a rather slow progress of symptom development that ranged from 8 to 28% between the different soil amendment treatments at 32 dpi, while the percentage of disease severity was 69 and 47% in the control and seed coating treatments, respectively (Fig. 1a). Statistical analysis of the relative AUDPC values revealed that amending the transplant soil plug with the K165 powder formulation at a rate of 5, 10 or 20% conferred the same level of resistance to the plants (Fig. 1c). The incorporation of K165 at a rate of 1% and seed coating resulted in substantially less protection compared to the other K165 treatments (Fig. 1c).

Likewise, the application of F2 resulted in significant protection against V. dahliae (Fig. 1c). The onset of symptom development was delayed in all F2 treatments by 2–8 days compared to the control plants (Fig. 1b). Also, the progress of the disease severity was two to seven times lower in the F2-treated plants compared to the control treatment at 32 dpi (Fig. 1b). In the case of F2, statistical analysis of the relative AUDPC values showed that there was no significant difference between the different F2 soil amendment treatments. Furthermore, the use of F2 as a seed coating preparation resulted in a significantly lower relative AUDPC value than the corresponding K165 seed treatment (Fig. 1c).

Determination of the BCA rhizosphere populations

The rhizosphere population of K165 depended on the release strategy, according to correlation analysis (r = 0·91, P = 0·001, d.f. = 13). Indeed, the treatments of soil amendment at a rate of 10 and 20% exhibited the highest K165 populations on the root system of aubergine, followed by the treatments of 5%, 1% and the seed coating treatment (Fig. 2a, c). However, the correlation analysis between the K165 population in the different treatments over time showed that the bacterial population declined with time in all treatments. Nevertheless, on the basis of correlation analysis, an inverse correlation occurred between the population size of K165 on the rhizosphere and the relative AUDPC values of the different treatments (= 0·55, = 0·05, d.f. = 16). Therefore, the release strategy and rate of application of K165 affected the level of plant protection against V. dahliae.

Details are in the caption following the image
The rhizosphere population of Paenibacillus alvei K165 (a) and Fusarium oxysporum F2 (b) following the use of two different release strategies (seed coating; amendment of the transplant soil plugs at four different rates, 1, 5, 10 and 20%, v/v). The microbial population was plotted over time to generate microbial progression curves; subsequently the area under the microbial progression curve (AUMPC) was calculated by the trapezoidal integration method (Campbell & Madden, 1990) for P. alvei K165 (c) and F. oxysporum F2 (d). Columns with different letters are significantly different according to Tukey's multiple range test at < 0·001.

Although statistical analysis of the AUMPC values revealed that there was no significant difference between the treatments that mixed F2 with the transplant soil plug at a rate of 1, 5 and 10%, the rhizosphere population of F2 was significantly correlated with release strategy (= 0·87, P = 0·001, d.f. = 13; Fig. 2b, d). In contrast to the K165 population that declined over time, correlation analysis between the F2 population in the different treatments and time revealed that in the 1, 5 and 20% soil amendment treatments the population did not decline with time, while in the 10% treatment it increased over time. The F2 population declined with time in the seed coated plants only. Thus, disease severity was negatively correlated to the population size of F2 in the rhizosphere (r = 0·63, P = 0·01, d.f. = 16), as was the case for K165.

qPCR determination of PR1 and PR4 transcript levels

The transcript levels of two previously studied aubergine PR genes (Kiba et al., 2006) were examined by qPCR analysis to test if they could be induced by the BCAs, and if there was a differential induction between the most and the least efficacious treatments. For this purpose, the induction of PR1 and PR4 was studied in the most effective treatments (where K165 and F2 were mixed in the transplant soil plug at a rate of 10 and 20%, respectively), the least effective K165 and F2 seed coating treatments, and the inoculated and mock-inoculated control treatments.

The qPCR analysis showed that PR1 was over-expressed in the plants that were grown in the 10% K165-treated soil at all time points, reaching a peak at 10 dpi and then declining (Fig. 3a). Interestingly, PR1 was under-expressed in the control plants compared to the mock-inoculated plants at all time points (Fig. 3a). PR1 was also under-expressed in all treatments except 10% K165 at 5 and 10 dpi (Fig. 3a). However, in both F2 treatments the under-expression of PR1 at 5 and 10 dpi was reversed at 20 dpi (Fig. 3a). Plants responded to the presence of the BCAs by over-expressing PR1 (except in the case of the F2 seed coating treatment) prior to the application of the pathogen at 0 dpi. A similar observation was made for PR4, which was over-expressed in all BCA treatments at 0 dpi. After the inoculation of the pathogen, in the F2 and the 10% K165 treatments the PR4 over-expression increased to reach the highest level at 10 dpi (Fig. 3b). On the other hand, PR4 expression in the control and the K165 seed coated plants was similar to PR1 expression, being under-expressed at all time points after inoculation with the pathogen.

Details are in the caption following the image
Changes in relative transcript abundance of PR1 (a) and PR4 (b) in aubergine plants seed coated with Paenibacillus alvei K165 or Fusarium oxysporum F2, or grown in transplant soil plugs amended with 10% (v/v) K165 or 20% (v/v) F2, then transplanted to Verticillium dahliae-infested soil with 20 microsclerotia per gram of soil. Total RNA was isolated from the above-ground parts of plants 0, 5, 10 and 20 days post-inoculation, converted to cDNA, and used as template in quantitative PCR assays. Transcript levels of the genes were normalized to the expression of actin measured in the same samples and expressed relative to the normalized transcript levels in mock-inoculated plants. Each column represents mean data with error bars from three independent biological samples.

Discussion

The delivery of fungal or bacterial BCAs capable of colonizing the rhizosphere and achieving their biocontrol potential is a key issue in the use of biocontrol inoculants for protection of crops against soilborne plant pathogens (El-Hassan & Gowen, 2006). The present study assessed whether a dry formulation of K165 and F2 applied as a seed coating preparation or soil amendment of the transplant soil plug at different rates could implement the biocontrol potential of K165 and F2, protecting aubergine plants against V. dahliae. Seed treatment is considered as an ideal method for introducing BCAs to control specific pathogens, because it allows the BCA to be placed in close proximity to the plant and the BCA's growth can be supported by the plant it protects through the organic compounds released in root exudates (El-Hassan & Gowen, 2006). The concept of introducing BCAs into the rhizosphere using the transplant soil plug is based on the hypothesis that BCA establishment in the relatively clean environment of the planting medium should provide the opportunity to develop stable populations in the seedling rhizosphere, and that these populations would then persist in the field (Kokalis-Burelle et al., 2006). It has been also hypothesized that early exposure to BCAs might precondition young plants to resist pathogen attack after transplantation in the field (Kokalis-Burelle et al., 2006).

Indeed, the data of the present study support the concept of introducing BCAs into the rhizosphere using the transplant plug, because this application was more effective than the seed coating treatment for both BCAs (Fig. 1). The incorporation of BCAs in the transplant amendments was introduced by Gagné et al. (1993) and has been followed by other researchers (Kloepper et al., 2004; Kokalis-Burelle et al., 2006). However, the objective of these studies was mainly to assess the applied microbial agents as PGPRs rather than BCAs of particular plant pathogens. In the present study, it was shown that incorporation of K165 and F2 in the transplant plug can effectively protect aubergine against V. dahliae, over a range of different application rates. Furthermore, the formulation of K165 and F2 as a dry powder preparation with 10% xanthan gum was shown to be a successful formulation strategy without having an adverse effect on the viability of the cells, as has been noted in other formulation techniques such as sodium alginate (Sabaratnam & Traquair, 2002; Schoina et al., 2011).

The monitoring of the BCA population in the rhizosphere revealed that the K165 population declined with time, while the F2 population remained stable (1, 5 and 20% F2-treated soil) or increased (10% F2-treated soil), except in seed-coated plants. It is accepted that plant protection depends on the population density of the BCA or, more precisely, on the ratio of pathogen to the BCA (Larkin & Fravel, 1999). Indeed, statistical analysis of the data in the current study revealed that disease severity was negatively correlated to the population size of F2 or K165 in the rhizosphere. Ecological fitness is a requirement for a successful BCA and in a number of studies the rhizosphere population of many different potential BCAs, mainly bacteria belonging to the Pseudomonas or Bacillus genera, has been recorded (Kloepper & Schroth, 1981; Tjamos et al., 2004; Kokalis-Burelle et al., 2006; Malandraki et al., 2008; Li et al., 2013). In most of these studies, a decline in the rhizosphere population was observed over time, as in the case of K165 during the present study (Kloepper & Schroth, 1981; Malandraki et al., 2008; Li et al., 2013). This decline has been attributed to a number of parameters, such as temperature, water content and pH of the soil (summarized by Weller, 1988).

The root colonization pattern of protective Fusarium isolates has been extensively studied using fluorescently tagged strains. These studies have revealed that protective Fusarium strains grow on the root surface and use the mechanism of competition for niches and nutrients as a biocontrol strategy against pathogenic F. oxysporum and V. dahliae (Olivain et al., 2006; Pantelides et al., 2009). On the other hand, the only published study that has dealt with the population levels of a Fusarium BCA is by Edel-Hermann et al. (2009), who demonstrated the ability of a benomyl-resistant mutant to establish in two different soils. One year after its introduction at two different concentrations in autoclaved soils, the BCA established at similarly high population densities, whereas in non-autoclaved soils it survived at lower densities which were related to the initial concentrations at which it was introduced (Edel-Hermann et al., 2009). These results, along with the present data, show the ability of non pathogenic Fusarium strains to establish in the soil and rhizosphere for extensive time periods in adequate numbers to confer resistance to the plants against vascular wilt pathogens.

Many studies have reported the ability of BCAs to trigger plant defences against plant pathogens. The aim of most of these studies was to find out whether the BCAs under study could trigger plant defence mechanisms, mainly the expression of PRs, and elucidate this interaction by using Arabidopsis mutants. In the present study, it was investigated whether different BCA delivery methods and rates in the rhizosphere affect the expression of the SA- and JA/ET-inducible genes PR1 and PR4, respectively. The ability of bacterial BCAs to induce the expression of PR1 and PR4 has been shown in many different studies in plants other than aubergine. In the present research effort, it was noted that, overall, the induction of both PRs was greater with larger BCA rhizosphere populations. Indeed, dose–response studies for BCAs have revealed that high populations are needed for effective control of diseases and triggering of ISR (Raaijmakers et al., 1995). In the case of K165 seed-treated plants, the PR1 and PR4 genes were under-expressed upon pathogen infection, as was also observed in the control treatment. It can be deduced that the pathogen suppresses the expression of PR1 and PR4, avoiding the deployment of certain plant defence mechanisms, as in the case of Pseudomonas syringae pv. tomato DC3000 that secretes the phytotoxin coronatine to repress PR gene expression and promote disease (Zhao et al., 2003).

While PR gene expression has been widely studied in biocontrol interactions, only a few studies have dealt with the plant response to inoculation with a protective strain of F. oxysporum. An increased activity of chitinases, β-1,3-glucanase, peroxidase and PR1 in tomato plants inoculated with the protective strain Fo47 compared to the non-inoculated control has been reported (Duijff et al., 1998; Cachinero et al., 2002). In a more recent study, Aimé et al. (2008) compared the accumulation of five PR protein transcripts in tomato cell cultures and in roots and leaves of tomato plants challenged with either the protective strain Fo47 or the pathogenic strain Fol8. Results showed a lower expression of PR genes in the plants or cells inoculated with the protective strain. In the present study, PR4 was over-expressed in the F2-treated plants compared to the control plants at 5 and 10 dpi. These results may suggest that the protective isolate F2 acts as an elicitor of plant defence reactions. A similar conclusion has also been drawn by Veloso & Díaz (2012) after observing increased accumulation of PR1, chitinase and sesquiterpene cyclase 1 transcripts in pepper plants inoculated with the protective strain Fo47 and challenge-inoculated with V. dahliae.

The success of biological control requires an understanding of the modes of action of the antagonist, its interactions with the plant and the pathogen, and on the mode and dose of application (Alabouvette et al., 2009). It also depends on the ecological fitness of the BCAs, especially when they target soilborne plant pathogens. Accordingly, the present study demonstrated that applying a bacterial or fungal BCA as a soil amendment of the transplant plug conferred an adequate BCA population size in the rhizosphere, reduced disease development and primed plant defence.