Pythium oligandrum (PO) is a mycoparasite on a wide range of fungi and suppresses diseases caused by fungal pathogens when colonizing the rhizosphere. PO and its cell wall proteins (CWPs) have elicitor activity that induces defence responses in plants. The potential of a mycelial homogenate of PO to suppress bacterial diseases was studied in roots of tomato (Lycopersicon esculentum cv. Micro-Tom) inoculated with Ralstonia solanacearum. PO-treated plants showed enhanced resistance to R. solanacearum and reduction in severity of wilt symptoms. As ethylene often acts as one of the signal molecules for induced resistance, its production following treatment of tomato roots with the mycelial homogenate or CWP of PO was measured. The level of ethylene in PO- and CWP-treated plants was transiently elevated six- to 11-fold at 4–8 h after treatment, followed by high expression of three basic ethylene-inducible defence-related genes (PR-2b, PR-3b and PR-5b). Analysis of PR-5b gene expression in the leaves of PO- and CWP-treated plants suggested that PR gene expression was induced systemically. The expression of LeERF2 and LeETR4, which confer an ethylene-dependent signalling pathway, was also significantly accelerated by such treatments. These results indicate that PO has the potential to control bacterial wilt disease and that CWP may play an important role in the induction of resistance to R. solanacearum accompanying the activation of the ethylene-dependent signalling pathway.
Pythium oligandrum (PO), a soil-inhabiting nonpathogenic oomycete, colonizes the rhizosphere of many crop species (Martin & Hancock, 1987; Al-Rawahi & Hancock, 1997). PO has an ability to reduce soilborne plant diseases caused by a number of fungal pathogens by activating plant defence systems, and by microbial antagonism caused by nutrient competition, parasitism and the production of antimicrobial substances (Martin & Hancock, 1987; Lewis et al., 1989; Benhamou et al., 1997, 1999). The disease resistance induced against different fungal pathogens by PO is nonspecific in affording enhanced protection in some plant species such as tomato (Benhamou et al., 1997), sugar beet and wheat (Takenaka et al., 2003). However, it remains to be determined if PO has the potential to suppress bacterial diseases.
As the interaction between microorganisms and plant tissues probably requires cell surface molecules, the analysis of the elicitor activity of cell surface molecules is important for understanding the molecular basis of induced disease resistance. A family of small extracellular elicitor proteins, elicitins, produced by Phytophthora spp. have been studied extensively as to their biochemical and molecular characteristics and their biological activities (Ponchet et al., 1999). Some elicitin-like proteins have also been identified in Pythium spp. (Huet et al., 1995; Panabieres et al., 1997). Oligandrin, a ∼10 kDa elicitin-like protein, was isolated from culture filtrate of PO, and it has been suggested that oligandrin triggers cytological and biochemical modification in tomato cells, thereby inducing resistance to Phytophthora parasitica (Picard et al., 2000). Recently, Takenaka et al. (2003) also reported that two types of cell wall protein fractions (CWPs), d-type containing two major proteins with molecular mass of ∼28 and ∼24 kDa, and S-type containing one major protein with ∼27 kDa, seemed to act as an elicitor for inducing the defence reactions to Rhizoctonia solani AG2-2 in sugar beet plants. In addition, in sugar beet roots treated with CWP, the activities of phenylalanine ammonia lyase (PAL) and chitinase and the amount of cell wall-bound phenolic compounds increased (Takenaka et al., 2003). However, molecular mechanisms such as signalling pathway(s) of PO-mediated induced resistance are still unknown.
Ethylene is well known as an important compound for developing necrotic local lesions and inducing systemic acquired resistance against a broad range of pathogens in tobacco (de Laat & van Loon, 1983; Ohtsubo et al., 1999). Ethylene also regulates the susceptible response to pathogen infection in tomato (Lund et al., 1998). Induced systemic resistance (ISR) in several plant species colonized by Pseudomonas fluorescens strain WCS417r, a plant growth-promoting rhizobacterium, requires an ethylene-mediated signalling pathway in combination with a jasmonic acid (JA)-mediated signalling pathway (Pieterse et al., 2002). In Arabidopsis thaliana, ISR-expressing plants are primed to induce ethylene production and ethylene-responsive gene expression after elicitation by pathogen infection, suggesting that the increased capacity for ethylene production might contribute to an enhanced defensive capacity against pathogens (Hase et al., 2003). Exogenous application of ethylene to A. thaliana induces activity of defence genes, such as PR-1b and PDF1.2 (Xu et al., 1994; Penninckx et al., 1998). Furthermore, overexpression of ethylene response factor 1 (ERF1) and 5 (ERF5) in Arabidopsis and tobacco confers enhanced resistance to Botrytis cinerea, Plectosphaerella cucumerina, Fusarium oxysporum and Tobacco mosaic virus (Berrocal-Lobo et al., 2002; Berrocal-Lobo & Molina, 2004; Fischer & Droge-Laser, 2004). Therefore, the role of ethylene and ethylene-mediated signal transduction for the activation of defence responses is well established.
Although neither the parasitism between PO and phytopathogenic bacteria nor the suppression of bacterial diseases by PO has been reported, PO-mediated induced resistance may have the potential to control bacterial disease by elicitor-induced resistance. To extend our knowledge about the potential of PO to control bacterial diseases, the control of bacterial wilt disease caused by Ralstonia solanacearum was examined. Bacterial wilt disease results in severe loss of tomato production in Japan and thus effective control of this disease is desired.
The objective of this study was to test whether treatment of tomato roots with a mycelial homogenate of PO could reduce the severity of the bacterial wilt disease. Furthermore, ethylene production and the expression of ethylene-related genes encoding defence-related proteins, ethylene receptor and transcription factor were examined in tomato plants treated with the mycelial homogenate or CWP of PO.
Materials and methods
Treatment of tomato with CWP and mycelial homogenate of P. oligandrum
Lycopersicon esculentum cv. Micro-Tom, which is a laboratory-grown miniature tomato used in functional genomics studies (Shibata, 2005), was grown in quartz sand at 24°C in a growth chamber under continuous fluorescent light (70 µmol m−2 s−1) and fertilized with 1000-fold-diluted Hyponex solution (Hyponex Japan, Japan) at 3-day intervals (Takahashi et al., 2005). Pythium oligandrum (isolate MMR2) was cultured in 10% V-8 broth containing 0·015 m CaCO3 and 0·1% of wheat germ oil at 25°C for 3 weeks. The mycelial mats were collected by filtration and washed with distilled water.
The pots containing 14-day-old tomato plants grown in quartz sand were dipped into water and then the plants were carefully removed so as to minimize injury to the root tissue. After the roots had been rinsed with distilled water three times, the roots of one plant were dipped into 1 mL of mycelial homogenate which also contained ∼5 × 105 of oospores or 350 µL of solution containing 750 µg of d-type of CWP of PO in a 50 mL gas-tight serum flask and incubated at 24°C under continuous fluorescent light (70 µmol m−2 s−1). To prepare the mycelial homogenate, 5 g fresh weight of the washed mycelial mats were homogenized with 10 mL of distilled water (DW). CWP was extracted by a previously described method (Takenaka & Kawasaki, 1994).
Inoculation of tomato with R. solanacearum
The roots of plants were treated with mycelial homogenate or DW in a plastic multidish 12-well plate (Nunc) for 24 h as described above and transferred to 50 mL Falcon tubes containing 1000-fold-diluted Hyponex solution and incubated for an additional 4 days at 24°C. Ralstonia solanacearum (isolate 8242; race 1, biovar 4) was prepared as described previously (Nakaho et al., 2004). The plants were cut so that each retained 4 cm roots and were then transferred to 50 mL Falcon tubes containing 1 × 108 cfu mL−1 bacterial suspension. The inoculated plants were incubated in a growth chamber with 16 h of fluorescent light (70 µmol m−2 s−1) at 29°C. Plants were coded and inspected daily for wilting symptoms for 4–5 days after inoculation. Disease was rated (0, no wilting; 1, 1–25% wilting; 2, 26–50% wilting; 3, 51–75% wilting; 4, 76–100% wilting; or dead) and a disease index calculated (Roberts et al., 1988). Each assay was repeated in five successive trials and within each experiment, eight plants were inoculated.
Measurement of ethylene production
Each plant treated with mycelial homogenate and CWP was grown in a gas-tight serum flask. At the time of ethylene measurement, 1 mL gas samples were withdrawn through the rubber seal. The concentration of ethylene was determined by gas chromatography as described by de Laat & van Loon (1983). As a control, the plants were treated with DW. The mean (± SD) of ethylene concentration in three plants was calculated in each experiment. Each set of experiments was repeated twice.
Northern hybridization analysis
Expression of pathogenesis-related (PR) genes encoding basic β-1,3-glucanase (PR-2b), basic chitinase (PR-3b) and basic thaumatin-like protein (PR-5b) and two genes encoding ethylene receptor (LeETR4) and ethylene-related transcription factor (LeERF2) were analysed. Total RNA was isolated from the roots of tomato using an RNeasy Plant-Mini Kit (Qiagen) according to the manufacturer's instructions. A quantity of 15 µg of total RNA was loaded onto each line of a 1·2% denaturing agarose gel. Northern hybridization was performed according to a published protocol (Sambrook & Russell, 2001). To detect the expression of PR-2b, PR-3b and PR-5 genes, ∼1000 bp fragments of each gene were amplified by reverse PCR with the primers 5′-GTGCTTCTAGGATTACTTGTCGCCACC-3′ and 5′-CTCACTAGTGAGTGAAGAAGCAGTGC-3′ for PR-2b (van Kan et al., 1992), 5′-TTCTGTGCTTTTGCTGTCTGCCTCTG-3′ and 5′-TCCAAAAGACCTCTGATTGCCACAA-3′ for PR-3b (Danhash et al., 1993), and 5′-TCCATACACCGTCTGGGCGGCGTCG-3′ and 5′-TTCATCACTTGAGGGCATCTCCAAG-3′ for PR-5b (Rodrigo et al., 1993). The DNA fragments for detecting transcripts from LeETR4 and LeERF2 by Northern hybridization were also amplified by reverse PCR with primers 5′-CGGAAGTTTGGTAGCCACAACTGGC-3′ and 5′-GTGCTCCAGAACACTTAGAAATGGAGC-3′ for LeETR4 (Tieman & Klee, 1999) and 5′-GAGAAGCTCGTAAAATCAGGGGTAAG-3′ and 5′-CTCCAAAGCTCCATCGAGCCACCGC-3′ for LeERF2 (Tournier et al., 2003). First strand cDNA as a template for reverse PCR was reverse-transcribed from total RNA of tomato roots using a ThermoScript RT-PCR Kit (Life Technologies). One microgram of first strand cDNA was added to 50 µL of 10 mm Tris-HCl (pH 8·3) containing 50 mm KCl, 2 mm MgCl2, 0·2 mm each of dATP, dCTP, dGTP and dTTP, 0·2 µm of each primer, and 5 units of Taq DNA polymerase (Promega) for PCR. The reaction ran with the programme as follows: 30 cycles at 94°C for 1 min, 55°C for 2 min, and 73°C for 1 min. The PCR product was purified and cloned into the EcoRV site of pBluescript SK+ (Stratagene). To confirm that the expected DNA was cloned, the nucleotide sequence of each insert was determined by the Sanger method using an automated DNA sequencer ABI model 310 A (Applied Biosystems). The amplified PR-2b, PR-3b, PR-5b, LeETR4 and LeERF2 DNA fragments were labelled with [α-32P]dCTP using the Megaprime™ DNA labelling systems (Amersham-Pharmacia).
Induced resistance in tomato to R. solanacearum using PO mycelial homogenate
Tomato plants inoculated with R. solanacearum 5 days after 24 h treatment of roots with mycelial homogenate of PO were examined after 10 days. Wilt symptoms and mean disease index were significantly reduced in PO-treated tomato plants (DI = 0·6) compared with DW-treated control plants (DI = 2·4) (Fig. 1).
Elevated concentration of ethylene in mycelial homogenate or CWP-treated tomato
Compared with DW-treated control plants, a transiently elevated concentration of ethylene was observed in both mycelial homogenate-treated (PO) and CWP-treated tomato plants (Fig. 2a and b). In the PO treatment, the maximum relative increase of ethylene was about 11-fold at 4–8 h after treatment before decreasing (Fig. 2a) whilst CWP-treated plants produced a sixfold increase of ethylene before decreasing (Fig. 2b). In the latter case, ethylene concentration increased until 4–6 h after CWP treatment and then decreased gradually.
Pathogenesis-related protein genes in mycelial homogenate or CWP-treated tomatoes
To analyse the expression of three pathogenesis-related (PR) protein genes, PR-2b encoding basic β-1,3-glucanases, PR-3b encoding basic chitinase and PR-5b encoding basic thaumatin-like protein, their transcripts in mycelial homogenate or CWP-treated tomato roots were detected by Northern hybridization. PR-2b, PR-3b and PR-5b transcripts in tomato roots increased at 4, 8, 12 and 24 h after treatment with mycelial homogenate and CWP. Expression of these three PR genes was also induced by exogenous application with 1-aminocyclopropane-1-carboxylate (ACC), a precursor of ethylene, but not induced at 8 h after treatment with salicylic acid (SA), a well known inducer of acidic PR gene expression (Fig. 3).
To confirm the systemic induction of PR gene expression, PR-5b transcript in leaves was analysed at 6 and 12 h after treatment of tomato roots with mycelial homogenate or CWP. Figure 4 shows elevated amounts of PR-5b transcript detected at both 6 and 12 h following PO treatment and at 6 h after CWP treatment.
Activation of ethylene-mediated signalling pathway in PO- or CWP-treated tomatoes
Increased production of ethylene and basic PR transcripts resulting from PO and CWP treatments of tomato roots suggested that an ethylene-dependent signalling pathway might have to be activated in tomato plants. In general, the activation of the ethylene-dependent signalling pathway is accompanied by the expression of genes encoding ethylene receptors and/or ethylene-responsive transcription factors (Gutterson & Reuber, 2004; Klee, 2004). Transcripts of ethylene receptor homologue LeETR4 and ethylene-inducible transcription factor LeERF2 in the roots were analysed and the expression of both LeETR4 and LeERF2 genes was transiently induced in the roots at 4 and 8 h in CWP-treated plants and up to 12 h in PO-treated plants (see Fig. 5). These results indicate that the transient increase of ethylene production is likely to have been associated with the activation of the ethylene-mediated signalling pathway and induction of basic PR gene expression in mycelial homogenate and CWP-treated tomato plants, which resulted in the reduction of bacterial wilt disease caused by R. solanacearum.
The mycoparasite P. oligandrum (PO) reduced the development of bacterial wilt disease caused by R. solanacearum in tomatoes. Although the mechanism(s) for the control of the diseases in plants by colonization with PO are incompletely understood, the data presented here suggest the suppression of bacterial wilt disease in PO-treated tomatoes is likely to be caused by induced resistance (Butt & Copping, 2000). The expression of both basic defence-related genes and the activation of the ethylene-dependent signalling pathway were significantly induced in tomatoes treated separately with the mycelial homogenate of PO and CWP. This would suggest that the reduction of bacterial wilt by treatment with the mycelial homogenate is due to the elicitor activity of CWP because cell wall proteins were part of the mycelial homogenate.
Several elicitin-like proteins have been identified in Phytophthora and Pythium spp. (Huet et al., 1995; Panabieres et al., 1997). Two major d-type proteins of CWP seemed to be elicitin-like proteins according to the amino acid sequence deduced from the nucleotide sequence of cDNAs encoding them in CWP: POD1 and POD2 (DDBJ accessions AB217820 and AB217821) (S. Takenaka, unpublished data). In tomatoes treated with CWP, increased ethylene production and induction of basic PR gene expression accompanied induced resistance to R. solanacearum. Previously, the induction of ethylene production in cell suspension cultures of tomato and tobacco treated with elicitors of Phytophthora species has been reported (Toppan & Esquerré-Tugayé, 1984; Rickauer et al., 1989; Basse & Boller, 1992). Although the role of ethylene production in host–pathogen interactions has been partly elucidated by the study of symptom expression in compatible interactions and the hypersensitive response in gene-for-gene resistance (Reinhardt et al., 1991; Hoffman et al., 1999; Glazebrook, 2001; Arshad & Frankenberger, 2002), the relationship between ethylene production and elicitor-induced resistance to a broad range of pathogens has not been established. Therefore, the detection of increased ethylene production in CWP-treated tomatoes, coinciding with a significant reduction of the bacterial wilt disease, is interesting.
Advances in the analysis of the ethylene-dependent signal transduction pathway have occurred in the past decade. The upregulation of ethylene receptor genes and ethylene-responsive transcription factor genes, which apparently monitors activation of the ethylene-dependent signalling pathway, has been observed when the defence reactions to pathogen attack are induced (Gutterson & Reuber, 2004; Klee, 2004). In tomato, five ethylene receptor genes have been identified: LeETR1, LeETR2, NR, LeETR4 and LeETR5 (Klee, 2004). Infection of tomato plants with avirulent Xanthomonas campestris pv. vesicatoria results in rapid induction of LeETR4 and NR gene expressions (Ciardi et al., 2000). The ethylene responsive factors (ERFs), which contain a highly conserved DNA-binding domain known as the ERF domain, modulate the expression of many PR genes through interaction with the GCC box present in their promoters (reviewed in Wang et al., 2002). Tomato ERF (LeERF) genes are transcriptionally regulated by ethylene, abiotic stress and avirulent pathogen infection (Gu et al., 2000; Tournier et al., 2003). LeERF2 belongs to class IV ERF (Tournier et al., 2003). In Arabidopsis, a class IV ERF gene (AtEBP) is thought to play an important role in regulating ethylene-inducible gene expression during the plant defence response (Buttner & Singh, 1997). Recently Zhang et al. (2004) reported a transgenic tobacco overexpressing a tomato ERF (TSRF1) has enhanced resistance to R. solanacearum, suggesting that activation of ethylene signalling confers pathogen resistance. Therefore, transient induction of ethylene production by mycelial homogenate and CWP treatment, as shown in the data reported here, may activate the ethylene-mediated signalling pathway, thereby inducing basic PR gene expression in tomato plants and thereby enhancing resistance to R. solanacearum. Because ethylene has been demonstrated to stimulate, as well as to counteract disease development, ethylene-insensitive host mutants (Ciardi et al., 2000) should be tested for resistance to R. solanacearum using tomato mutants such as cv. Never ripe.
This work was supported by a grant of the Research and Development Programme for New Bio-industry Initiatives from the Bio-oriented Technology Research Advancement Institute, Japan. We wish to thank Dr D Shibata, Kazusa DNA Research Institute, Japan, for kindly supplying tomato seeds.