Characterization of rhizosphere fungi that mediate resistance in tomato against bacterial wilt...

Journal of Experimental Botany, Vol. 64, No. 12, pp. 3829–3842, 2013doi:10.1093/jxb/ert212

© The Author [2013]. Published by Oxford University Press on behalf of the Society for Experimental Biology. For permissions, please email: [email protected]

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Characterization of rhizosphere fungi that mediate resistance in tomato against bacterial wilt disease

Sudisha Jogaiah1,2, Mostafa Abdelrahman3, Lam-Son Phan Tran4 and Ito Shin-ichi1,*1 Laboratory of Molecular Plant Pathology, Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan2 Downy Mildew Research Laboratory, Department of Studies in Biotechnology, University of Mysore, Mysore-570 006, Karnataka, India3 Laboratory of Vegetable Crop Science, Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan4 Signaling Pathway Research Unit, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan

* To whom correspondence should be addressed. E-mail: [email protected]

Received 8 April 2013; Revised 7 June 2013; Accepted 13 June 2013

Abstract

Plant immunization for resistance against a wide variety of phytopathogens is an effective strategy for plant dis-ease management. Seventy-nine plant growth-promoting fungi (PGPFs) were isolated from rhizosphere soil of India. Among them, nine revealed saprophytic ability, root colonization, phosphate solubilization, IAA production, and plant growth promotion. Seed priming with four PGPFs exhibited early seedling emergence and enhanced vigour of a tomato cultivar susceptible to the bacterial wilt pathogen compared to untreated controls. Under greenhouse condi-tions, TriH_JSB27 and PenC_JSB41 treatments remarkably enhanced the vegetative and reproductive growth param-eters. Maximum NPK uptake was noticed in TriH_JSB27-treated plants. A significant disease reduction of 57.3% against Ralstonia solanacearum was observed in tomato plants pretreated with TriH_JSB27. Furthermore, induction of defence-related enzymes and genes was observed in plants pretreated with PGPFs or inoculated with pathogen. The maximum phenylalanine ammonia lyase (PAL) activity (111 U) was observed at 24 h in seedlings treated with TriH_JSB27 and this activity was slightly reduced (99 U) after pathogen inoculation. Activities of peroxidase (POX, 54 U) and β-1,3-glucanase (GLU, 15 U) were significantly higher in control plants inoculated with pathogen after 24 h and remained constant at all time points. A similar trend in gene induction for PAL was evident in PGPFs-treated tomato seedlings with or without pathogen inoculation, whereas POX and GLU were upregulated in control plus pathogen-inoculated tomato seedlings. These results determine that the susceptible tomato cultivar is triggered after percep-tion of potent PGPFs to synthesize PAL, POX, and GLU, which activate defence resistance against bacterial wilt disease, thereby contributing to plant health improvement.

Key words: Bacterial wilt, defence-related enzymes and genes, growth promotion, induced systemic resistance, plant growth-promoting fungi, phosphate solubilization, tomato.

Introduction

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop in the world. It is estimated that 4.4 million ha of tomatoes are grown annually worldwide

producing more than 151.7 million tonnes. In Japan, it is grown in an area of 0.0123 million ha producing about 0.7 million tonnes annually (FAOSTAT, 2010) with 5860 kg/10

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acre yield per annum. The crop is infected by a large number of diseases, among which bacterial wilt by Ralstonia solan-acearum is the most destructive one and poses a great threat to tomato production. It is a soil-borne pathogen, occurs throughout most tomato growing areas. and causes severe crop damage to both greenhouse and field depending upon the varieties of tomato cultivars used and the environmen-tal conditions (Vanitha et al., 2009a). Most infected seedlings die, and those that survive are stunted and do not flower. The pathogen also infects a wide range of economically impor-tant crops such as potato, brinjal, chilli, and non-solanaceous groundnut and banana (Vanitha et al., 2009a).

Although the use of wilt-resistant cultivars can provide greater degree of disease resistance, the emergence of new races of the pathogen that overcome resistance is a continu-ing problem. In Japan, the most effective method applied to control tomato bacterial wilt is the use of chemicals such as methyl bromide and chloropicrin (Ogawa, 1998). However, application of methyl bromide has been prohibited by interna-tional treaty because of its property to damage the ozone layer (Tateya, 1996). The use of chloropicrin is not recommended either as it adversely affects useful soil micro- organisms, human health, and the environment. In this context, novel eco-friendly and safe strategies have been explored for the management of plant diseases. Thus, induced systemic resist-ance (ISR) is one of the important methods and is gaining worldwide importance and acceptance (Pieterse et al., 1998; Van Wees et al., 2008).

There are a number of plant growth-promoting fungi (PGPFs) in rhizosphere soils, such as species of the genera Trichoderma, Penicillium, Fusarium, and Phoma, which have the ability to stimulate the plant immune response upon enemy attack and are considered as one of the safest mode for ISR and growth promotion in crop plants (Harman et al., 2004; Van Wees et al., 2008; Shoresh et al., 2010). A growing body of evidence indicates that, in addition to promotion of plant growth (Fontenelle et al., 2011; Nagaraju et al., 2012), PGPFs are beneficial to plants in reducing the impacts of various fungi (Fontenelle et al., 2011; Nagaraju et al., 2012; Murali et al., 2013), bacteria (Hossain et al., 2008; Sultana et al., 2009; Yoshioka et al., 2012), viruses (Elsharkawya et al., 2012), and nematodes (Gotlieb et al., 2003) by elicit-ing ISR. Several PGPF isolates are known for solubilization of phosphates, minerals, and micronutrients, which contrib-ute an important role in plant growth promotion, includ-ing resistance (Altomare et al., 1999; Whitelaw, 2000; Alam et al., 2011; Singh et al., 2011). The main source of resistance induced by PGPF-treated plants is associated with the biosyn-thesis of defence enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (POX), and β-1,3-glucanase (GLU), which determine the degree of host resistance (Harman et al., 2004; Shoresh et al., 2005, 2010; Lamba et al., 2008; Sultana et al., 2009; Yoshioka et al., 2012; Murali et al., 2013).

PAL is a key enzyme of the phenylpropanoid pathway that catalyses the deamination of phenylalanine to trans-cinnamic acid, a precursor for both phytoalexins and lignin, which pre-vents cell-wall penetration by the pathogen (Dixon, 2001). Recently, the involvement of phenylpropanoid pathway in

Trichoderma hamatum-induced ISR regulated by jasmonates and ethylene has been reported (Mathys et al., 2012). POX and GLU are well-known pathogen-related proteins (PR), which activate the salicylic acid pathway for plant defence. POX (PR-9) catalyses the formation of lignin and is produced to limit cellular spreading of infection through the establish-ment of structural barriers by producing reactive oxygen and nitrogen species (Marjamaa et al., 2009). GLU (PR-2) degrades the fungal cell wall and exerts an antimicrobial effect against various phytopathogenic fungi (Fritig et al., 1998; Van Wees et al., 2008). In spite of many studies having been undertaken on these enzymes involved in plant resist-ance mechanisms against phytopathogenic fungi, the under-standing of the mechanisms in the tripartite interaction of PGPF, tomato, and Ralstonia solanacearum remains limited.

The aim of the present study is to develop environmentally acceptable and durable protection methods to minimize yield losses caused by the bacterial wilt pathogen Ralstonia solan-acearum as well as to minimize the excessive use of hazard-ous chemicals. This study isolated and characterized PGPF isolates from rhizosphere soils and tested them for elicitation of resistance against tomato bacterial wilt disease. The plant growth promotion and disease reduction ability of the four PGPF isolates were tested using a susceptible tomato cultivar (Oogata-Fukuju). The induction of defence-related genes, namely jasmonate-responsive PAL and SA-responsive POX and GLU, were also assessed.

Materials and methods

Host and pathogenSeeds of tomato cv. Oogata-Fukuju, a common Japanese cultivar that is highly susceptible to bacterial wilt, were procured from Takii Seed Co, Kyoto, Japan. The pathogen Ralstonia solanacearum YA12-1, a causal agent of bacterial wilt, was obtained from Dr H Kajihara (Yamaguchi Prefectural Technology Center for Agricultural and Forestry, Yamaguchi, Japan). A pure culture of strain YA12-1 was maintained on nutrient agar and Kelman’s TZC media plates by streaking. The disease response of the susceptible cultivar was con-firmed by repeated screening experiments. Inocula were prepared by streaking loop-fulls of cells on Kelman’s TZC media plates and incubation at 30 °C for 2 days. Cells were harvested in sterile distilled water by centrifugation (Tomy LC-220, Tokyo, Japan) at 10 000 rpm for 10 min. The pellet was resuspended in sterile distilled water and the optical density at 610 nm of bacterial suspension was adjusted to 0.45 using a UV-visible spectrophotometer (Hitachi U-2001, Tokyo, Japan) to obtain the concentration of 1 × 108 cfu ml−1 for use as inoculum.

Isolation of PGPFs from rhizosphere soil and rhizoplane samplesRhizosphere soil samples along with root segments were collected from healthy vegetables, sunflower, legumes, and cereal crops culti-vated in farmer’s and experimental fields of Karnataka, Tamil nadu, Andrapradesh, Kerala, Goa, Rajasthan, Gujarat, Maharashtra and West Bengal states, India during 2009–2010. Rhizosphere soils from grass plants located in a hilly region from these states were also col-lected. The collected soil samples were stored in sterilized polythene bags at 4 °C until further use. Soil samples (1 g) from each collection site were subjected to the serial dilution technique. Root bits in the soil samples were also evaluated by placing on potato dextrose agar. The plates were incubated under a 12/12 h near ultraviolet light/dark



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at 23 ± 2 °C for 7 days. Observations for growth and identification of the fungi were made based on morphological microscopic examina-tion and molecular characterization of internal transcribed spacer (ITS) region markers.

Screening for potential PGPFsPathogenicity of PGPF isolates was determined according to Koch’s postulates (http://phytopath.ca/education/kochspostulates.html). The saprophytic nature of PGPFs was detected using sor-ghum as a substrate and performed following the method of Meera et al. (1994). Root colonization and rhizosphere competence were measured according to Murali et al. (2013). An agar disc diffusion method was performed to determine the ability of each PGPF iso-late to inhibit growth of Ralstonia solanacearum YA12-1.

Biochemical characterization of the PGPF isolatesScreening of phosphate solubilizersIn order to detect phosphate-solubilizing fungi, PGPF isolates (5 mm disc) were placed on Pikovskaya’s agar (PVK, contain-ing per litre: 0.5 g yeast extract, 10 g dextrose, 5 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.2 g KCl, 0.1 g MgSO4.7H2O, 0.0001 g MnSO4.H2O, 0.0001 g FeSO4.7H2O and 15 g agar, pH 7.2). The plates were incu-bated at 25 ± 2 °C for 10 days and isolates that induced a clear zone around the growth were considered as positive. The phosphate solu-bilization index was calculated as (fungal growth diameter ± halo-zone diameter)/fungal growth diameter.

pH changeOne ml of spore/mycelial suspension (1 × 107 spores ml−1) of each PGPF isolate was inoculated in 250 ml conical flask containing 100 ml sterile PVK broth (pH 7.0) and incubated at 25 ± 2 °C for 21 days. The change in pH was recorded after 1 week by a digital pH meter (Horiba D-51, Okayama, Japan).

Quantitative estimation of phosphate solubilization in broth mediumPGPF cultures were grown in PVK broth for 6 days with continuous shaking (120 rpm) at 25 ± 2°C. Ten ml were taken from each sam-ple culture and centrifuged at 9000 rpm for 10 min. Phosphorus (P) in solution was extracted with ammonium bicarbonate diethylene triamine penta acetic acid and the P content was determined by the ascorbic acid method (Watanabe and Olsen, 1965) The opti-cal density of the blue colour developed after 15 min was measured at 880 nm by spectrophotometry. The concentration of available P (µg ml−1) was calculated against the standard KH2PO4 curve.

Determination of indole-3-acetic acidThe amount of indole-3-acetic acid (IAA) produced by each PGPF isolate was determined using potato dextrose broth supplemented with 1 mg l-tryptophan ml−1 and incubated at 26°C for 4 days. After incubation, the inoculated medium was centrifuged and the super-natant was used for the estimation of IAA by the ferric chloride-perchloric acid assay (Gordon and Weber, 1951).

Molecular characterization of the PGPF isolatesFungal genomic DNA was amplified using ITS1 and ITS4 primers (1 μl): (F: 5′-CCGTAGGTGAACCTGCGG-3′ and R: 5′-TCCTCC GCTTATTGATATGC-3′). PCR was performed using a Thermal cycler TP600 (Takara Bio, Shiga, Japan) with a 20-μl reaction according to the method described by Hermosa et al. (2000). The amplification products were sequenced using a ABI PRISM 3100 genetic analyser (Applied Biosystems, USA). Sequence assembly was performed with Finch TV version 1.4.0 (www.geospiza.com) and sequence alignment was done using multiple-sequence align-ment in clustal w. Sequence similarity searches were performed using the advanced blastn program. Sequence data were submitted to the National Center for Biotechnology Information (accession numbers JQ665253–JQ665267).

Seed priming with PGPF isolatesThe susceptible tomato cultivar was treated with pure culture of each prescreened PGPF isolate at the rate of 1 × 107 spores ml−1 by mixing 400 seeds with 15 ml conidial suspension. Treated seeds were kept at 23 ± 2 °C in a rotary shaker (70 rpm) for 5 h to facilitate the penetration of the PGPF isolate inside the seeds. These treated seeds were then used for all the greenhouse and laboratory experiments. The treatments consisted of (i) seed priming with PGPF alone, (ii) seed priming with PGPF plus pathogen inoculation, (iii) seeds or seedlings soaked with sterile distilled water (untreated control), and (iv) untreated control plus pathogen inoculation.

Assays for seed germination and seedling vigourPrescreened seven bioactive PGPFs treated to Oogata-Fukuju tomato seeds (four replicates of 100 seeds) were plated equidistantly on moistened blotter discs placed in glass Petri plates to evalu-ate seed germination. For evaluation of seedling vigour, another set of treated seeds and sterile distilled water (SDW) control were subjected to the ‘between-paper method’ to record seedling vigour (Abdul Baki and Anderson, 1973).

Measurement of nitrogen (N) P, and potassium (K) uptakeSeeds treated with four PGPFs, namely Trichoderma harzianum TriH_JSB27, Phoma multirostrata PhoM_JSB17, Trichoderma har-zianum TriH_JSB36, and Penicillium chrysogenum PenC_JSB41 and controls were raised in plastic pots containing peat-based substrate (Napura Yoda, Yanmar Agricultural Equipment, Tokyo, Japan). Seedlings were cultivated in the growth chamber (75 mol m–2 s−1 photosynthetically active radiation, 12/12 h light/dark photoperiod, 25 °C) for 15 d. In these conditions, plants reached the two-leaf stage. These plants were transferred into earthen pots filled with pot-ting medium mixture of peat moss and vermiculite (1:1, v/v) con-taining 50 mg N kg−1, 500 mg P kg−1, and 100 mg K kg−1 (pH 6–6.5, water-holding capacity ~70%, NapuraYodo, Yanmar Agricultural Equipment, Tokyo, Japan) as a root-supporting medium that was thoroughly mixed with individual PGPF (100 ml of 1 × 107 spores ml−1) as soil treatment. The pots were arranged in a randomized complete block design and grown in greenhouse conditions. One plant from each pot was randomly selected and its NPK content was analysed to determine the NPK uptake. The selected plants were cut into small pieces (1 cm size) and dried at 65 °C for 2 days. The dried samples were ground using stainless steel hand mill. One g of pow-dered samples was taken and wet digested in concentrated H2SO4 for determination of total N, and in di-acidic mixture HNO3 and HCIO4 (4:1 ratio) for determination of total P and K. The total P and N contents in digesting samples were determined by the vanadomolyb-date phosphoric acid method and the Kjeldahl method, respectively (Jackson, 1973). Total K content was determined by a flame pho-tometer method (Jackson, 1962).

Evaluation of the effect of PGPFs priming on tomato growth and developmentThe number of days required to form the first flower was recorded. Treatment effects on plant height, stem girth, shoot and root lengths, and fruit weight were measured at maturity. The experiment was conducted in four replications with 10 plants/replication and repeated twice.

Determination of the time required for PGPFs-treated tomato plants to induce resistance against bacterial wilt diseaseTreated and untreated Oogata-Fukuju seeds raised in the growth chamber were sown in earthen pots under greenhouse conditions. Three-week-old tomato plants raised from seeds primed with PGPFs were uprooted and were challenged to infection with 50 ml suspension of Ralstonia solanacearum YA12-1 (1 × 108 cfu ml−1)



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by root-dip inoculation for 30 min at 30 °C. Similarly, inocula-tion was given to plants 1, 2, 3, and 4 days later (22, 23, 24, and 25-day-old plants). The pots were then arranged in a randomized complete block design and maintained under greenhouse condi-tions. The plants were watered at regular intervals and 0.1% NPK (Hyponex 6:10:5, Osaka, Japan) dissolved in water was provided twice. The SDW seed treatment served as controls. Each tomato treatment consisted of 16 plants in four replications and repeated twice. Plants were observed daily and the progression of the dis-ease was recorded. Plants were rated diseased when they showed at least one of the typical symptoms of bacterial wilt disease, such as wilting, brown discoloration in the vascular system of leaves, and milky white bacterial ooze was observed when plant material was placed in water. The disease incidence was recorded up to 30 days after pathogen inoculation and percentage disease protection was calculated as (no. of diseased plants in control – no. of diseased plants in the treatment) × 100/total no. of plants observed for each treatment.

Determination of the levels of defence enzymes after PGPF primingInoculation of seedlings and samplingThree-week old seedlings raised in pots pretreated with Trichoderma harzianum TriH_JSB27 and Penicillium chrysogenum PenC_JSB41 were uprooted, washed, and dipped separately in 25 ml of aqueous PGPF suspensions (1 × 107 spores ml−1), respec-tively, or in SDW for 15 min. Subsequently, treated seedlings were root-dip inoculated with 25 ml suspension of Ralstonia solan-acearum (1 × 108 cfu ml−1) for 15 min and incubated at 30°C in the dark humid chamber. From each treatment, 1 g of seedlings was harvested at 0, 4, 12, 24, 48, and 96 h post pathogen infec-tion (hpi), immediately wrapped in aluminium foil, and stored at –70°C until enzyme assay and RNA extraction. Seedlings mock inoculated with SDW and harvested at the same time intervals served as untreated controls.

One g of harvested samples was ground to a fine powder in liq-uid nitrogen and used for extraction of individual enzymes by homogenization in different buffers (3 ml g−1 seedlings) at 4 °C. The homogenates were centrifuged at 10 000 g for 15 min at 4 °C and the supernatants were used for enzyme assays. The protein content in the extract was estimated using the protein dye binding method of Bradford (1976) with bovine serum albumin (Sigma, USA) used as a standard.

PAL assayFresh seedlings (1 g) were extracted in 25 mM sodium borate buffer (pH 8.8) containing 32 mM β-mercaptoethanol. The homogenate was centrifuged at 20 000 g for 20 min at 4 °C in a refrigerated high-speed centrifuge. PAL was assayed as described by Nagarathna et al. (1993) using trans-cinnamic acid as a standard. The enzyme activity was expressed as mol trans-cinnamic acid (mg protein)−1 h−1.

POX assayFresh seedlings (1 g) were homogenized in 3 ml of 0.1 M sodium phosphate buffer (pH 7.0) and the activity was measured described by Hammerschmidt et al. (1982) using guaiacol as the hydrogen donor. The enzyme activity was expressed as units at 470 nm (mg protein)−1 min−1.

GLU assayFresh seedlings (1 g) were homogenized in 0.05 M sodium acetate buffer (pH 5.2) and assayed according to the method of Kini et al. (2000) with 0.1% laminarin (Sigma, USA) used as the substrate. Enzyme activity was measured at 540 nm and expressed in mole (mg protein)−1 min−1.

Expression analysis of defence genes in PGPF-primed tomatoTotal RNA was isolated from TriH_JSB27- and PenC_JSB41-treated and control sets harvested at 0, 4, 12, 24, and 48 hpi. Frozen samples (1 g) were ground into fine powder in liquid nitrogen and total RNA was extracted using Sepasol (Nacalai tesque, Kyoto, Japan) accord-ing to manufacturer’s protocol. Reverse transcription was per-formed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) as per the manufacturer’s instructions. The specific primers for POX (F: 5′-GCTTTGTCAGGGGTTGTGAT-3′ and R: 5′-TGCATCT CTAGCAACCAACG-3′) and reference gene β-actin (F: 5′-TTGCCG CATGCCATTCT-3′ and R: 5′-TCGGTGAGGATATTCATCAG GTT-3′) were designed using Primer3 software (http://simgene.com/Primer3), the PAL primer (F: 5′-TTCAAGGCTACTCTGGC-3′ and R: CAAGCCATTGTGGAGAT-3′) and GLU primer (F: 5′-GGAACAG GAACACAAGAAACAGTGA-3′ and R: 5′-CCCAATCCATTA GTGTCCAATCG-3′) was obtained from NCBI (accession num-ber X58548). Real-time quantitative PCR was performed by add-ing 10 μl Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan) to each well containing 1 μl template cDNA and 0.5 μl 12 pmol forward and reverse primers, 50× ROX reference dye (0.4 μl) in a 20-μl reaction. PCR cycling consisted of an initial denaturation step of 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min (7300 Real Time PCR system, Applied Biosystems). The experiment was conducted in quadruplicate and repeated twice. The fold-increase of expression levels of each gene was calculated as (quantity of target gene in treated sample/quantity of reference gene in treated sample)/(quantity of target gene in SDW treatment at 0 hpi/quantity of reference gene at 0 hpi).

Statistical analysisAll statistical analyses of the data were performed using SPSS version 18.0 software with advanced models (SPSS Japan, Tokyo, Japan). Differences between means were located using Tukey’s mul-tiple-comparison test (P < 0.05). The in vitro experiments were based on Scheffe’s multiple post hoc test. Dunnett’s test was employed to compare the mean data between growth parameters, N, P, and K, and controls. The data of disease resistance, defence enzymes and genes in the control and treated plants were subjected to independ-ent Student’s t-test.

Results

Isolation and screening of PGPFs for characteristic traits

A total of 79 PGPFs belonging to six genera, namely Trichoderma, Fusarium, Phoma, Penicillium, Aspergillus, and Pythium were isolated from rhizosphere soils of healthy food crops and grass plants from different agro climatic zones of India. Of these, 43 were found to be non-pathogenic upon artificial inoculation in tested host crop plants; 37 of which showed saprophytic ability with fair to good growth (maximum 75–81%) after 3 weeks of incubation. Among these 37 isolates, only 15 solubilized phosphate on Pikovskaya’s agar, produc-ing clear zones (Table 1). Soluble phosphate was ranged from 117 μg ml−1 (Fusarium oxysporum FusO_JSB63) to 352 μg ml−1 (Penicillium chrysogenum PenC_JSB9) on day 9 of inoculation. With Aspergillus flavus AspF_JSB23, a significant reduction in the pH of PVK broth, from pH 7.0 to 3.3, was observed after 9 days of incubation. Of the 15 phosphate-solubilizing strains, nine showed positive IAA production (Table 1).

Seven isolates were selected for further study based on their ability to colonize tomato root and promote plant



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growth under laboratory conditions (Table 1). PCR ampli-fication with specific primers for the ITS region generated bands ranging from 540 to 610 bp. These fragments were sequenced separately, and the sequence data were deposited in the GenBank of the National Center for Biotechnology Information (NCBI) under the accession number JQ665253–JQ665267. The closest relative were firstly determined based on the similarity degrees of their ITS sequences obtained by a direct blast search in NCBI GenBank. The results revealed that this study’s seven PGPF sequences showed clos-est matches with those of the rhizosphere soil microbes or plant endophytes, with 85–99% homology to those of species that are classified as the plant biocontrol agents and/or plant growth promoters. The ITS sequence of TriH_JSB27 (474 bp) revealed high similarity (99% sequence identity, E-value = 0) to that of the most similar PGPF T. harzianum isolates from rhizosphere soil, saprophytes on decaying woods, and endo-phytes across the world, while that of PenC_JSB41 (470 bp) showed 99% nucleotide sequence identity (E-value = 0) to those of 54 Penicillium chrysogenum strains isolated from soil or plant endophytes from India and China. Moreover, most

of the T. harzianum and Penicillium chrysogenum sequences that showed sequence similarity with TriH_JSB27 and PenC_JSB41 were reported for the production of bioactive second-ary metabolites and their role in plant protection.

Test for antagonism

Two of the seven isolates, TriA_JSB1 and TriAt_JSB2 revealed a completely antagonistic nature against Ralstonia solanacearum after 5 days of incubation. The remaining five isolates showed lower, from fair to weak, inhibition levels against Ralstonia solanacearum. Among them, the highest inhibition was noticed for PhoP_JSB5 (22.5% inhibition), while the lowest inhibition was recorded for PenC_JSB41 (3.4% inhibition) (Table 1).

Seed germination and seedling vigour after PGPFs priming

Among seven root colonizing isolates, seed treatment with four isolates revealed significantly (P < 0.05) enhanced early

Table 1. In vitro screening of non-pathogenic rhizosphere fungi for beneficial characteristic traits

Values are mean ± SE (n = 4). Treatment means followed by the same letter(s) within the column are not significantly different according to Scheffe’s multiple post-hoc test (P < 0.05). The data presented are from representative experiments that were repeated at least twice with similar results.

Species (accession no.)

Strain P solubilization index (9th day)

pH in broth P solubilization (μg ml−1)

IAA (μg ml−1) Inhibition of R. solancearum (%)

Root colonization

Penicillium chrysogenum (JQ665253)

PenC_JSB9 1.28 ± 0.85a 4.6 ± 0.22cd 352 ± 5.0a 35 ± 3.16bc 9.6 ± 0.58hij w

Phoma multirostrata (JQ665254)

PhoM_JSB17 0.84 ± 0.27e 4.2 ± 0.14de 273 ± 6.69cde 44.2 ± 3.09f 7.1 ± 0.29efghij +

Phoma putaminum (JQ665255)

PhoP_JSB5 0.46 ± 0.14cd 5 ± 0.09c 265 ± 2.65d 0 ± 3.0de 22.5 ± 1.37c –

Trichoderma asperellum (JQ665256)

TriA_JSB1 0.55 ± 0.31de 3.5 ± 0.15ef 304 ± 4.81bcd 0 ± 0f 93.4 ± 1.14b +

Trichoderma atroviride (JQ665257)

TriAt_JSB2 1.06 ± 0.4abc 5.1 ± 0.16c 193 ± 4.74ghi 42.7 ± 2.39bcd 98.1 ± 0.62a +


PenC_JSB41 0.81 ± 0.2cd 6.2 ± 0.06a 310 ± 7.6cde 81.2 ± 1.84a 3.4 ± 0.27j +

Trichoderma harzianum (JQ665259)

TriH_JSB27 0.97 ± 0.42c 5.1 ± 0.05c 335 ± 7.5ab 68 ± 2.27ab 9.5 ± 0.34fg +

Aspergillus flavus (JQ665260)

AspF_JSB23 1.27 ± 0.33ab 3.3 ± 0.05f 309 ± 6.2bc 63 ± 2.54b 13.8 ± 0.17d –


TriH_JSB40 1.08 ± 0.42abc 6.1 ± 0.04a 139 ± 8.3bcd 0 ± 0f 39.1 ± 3.25fghij w


PenC_JSB31 0.99 ± 0.32bc 5 ± 0.01c 254 ± 4.3ef 61 ± 4.41e 23.2 ± 1.54efghi w


TriH_JSB36 0.95 ± 0.31c 6.6 ± 0.03a 304 ± 5.1ij 52.2 ± 3.44de 11.5 ± 0.84def +


TriH_JSB72 1.32 ± 0.41a 5.1 ± 0.01c 207 ± 3.0g 0 ± 0f 4.3 ± 0.4ij w

Penicillium funiculosum (JQ665265)

PenF_JSB59 0.60 ± 0.22de 6.3 ± 0.01a 214 ± 4.9fg 0 ± 0f 8.3 ± 0.25efgh –

Fusarium oxysporum (JQ665266)

FusO_JSB63 0.49 ± 0.21e 5.3 ± 0.02bc 117 ± 5.6j 37 ± 2.12d 5.7 ± 0.26ghij w

Fusarium oxysporum (JQ665267)

FusO_JSB47 1.07 ± 0.58abc 6.0 ± 0.01ab 160 ± 5.0hi 0 ± 0f 11 ± 0.18de +

+, Positive; w, weakly positive; –, negative.



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emergence, germination, and seedling vigour: PenC_JSB9, PenC_JSB41, TriH_JSB27, and TriH_JSB36 (Fig. 1). The highest germination (95%) and seedling vigour score (1924) with early emergence (4 days after sowing) were recorded for TriH_JSB27 (accession number JQ665259), followed by PenC_JSB41, whose treatment showed 93% germination, vig-our score 1645, and seedling emergence on day 5, whereas in the control treatment, germination and vigour index were found to be 87% and 1002, respectively (Fig. 1).

Uptake of macronutrients and growth of tomato after PGPFs priming

All four PGPF treatments showed moderate to significant dif-ferences (P < 0.05) in the amount of increased NPK uptake compared to the untreated control plants (Table 2). Among the treatments, higher nutrient uptake of 1.9, 0.5, and 1.6 mg (g shoot)−1 of N, P, and K was noticed with TriH_JSB27, whereas control plants showed 0.7, 0.2, and 0.8 mg (g shoot)−1 for the uptake of N, P, and K, respectively.

The tested isolates exhibited significant (P < 0.05) enhance-ment of tomato growth parameters. However, the degree of growth promotion varied with the isolates (Table 2). When compared to the untreated control plant height (105 cm), the maximum plant height was increased by 41 and 36% in TriH_JSB27- and TriH_JSB36-treated plants, respectively. Early flowering ranged between 63–65 days and was noticable in all treatments compared to untreated controls (72 days).

A significant enhancement of stem girth of about 1.12 cm in untreated controls was increased by 44.6% with TriH_JSB36, followed by 41.07% with PenC_JSB41, 38.3% TriH_JSB27, and 37.5% PhoM_JSB17 treatments. Similarly, shoot and root weight significantly increased in the treated plants. Fruit weight and days to maturity were advanced in all the four treatments over the untreated controls. Maximum fruit weight of 223 g was recorded for TriH_JSB27, followed by 207 g in TriH_JSB36 treatment, whereas the untreated control plants offered only 162 g of fruit weight. Furthermore, days for plant maturity in the untreated control plants (117 days) was signif-icantly (P< 0.05) reduced to 102, 106, 110, and 111 days for TriH_JSB36, TriH_JSB27, PenC_JSB41, and PhoM_JSB17 treatments, respectively (Table 2).

PGPFs priming elicited resistance against bacterial wilt disease

In a separate experiment, nature of resistance offered by the inducers was studied by inoculating 3-week-old tomato plants with Ralstonia solanacearum at intervals of 1–5 days between plant and pathogen inoculation. Plants treated with TriH_JSB27 and PenC_JSB41 offered significant disease suppression after 51 days of sowing (Fig. 2). TriH_JSB27 showed significantly (P < 0.05) increased disease protection (27.6 to 56.3%) with pathogen inoculation 2 days after PGPF priming, followed by PenC_JSB41, in plants inoculated with pathogen 3 days after priming (27–48% protection), when

Fig. 1. Seed priming effects of PGPF isolates on seed emergence, germination, and seedling vigour. Values are means of four independent replications. Bars represent standard error. Treatment means annotated above by the same letter are not significantly different according to Scheffe’s multiple post hoc multiple test (P < 0.05). The data presented are from representative experiments that were repeated at least twice with similar results (this figure is available in colour at JXB online).

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500

1000

1500

2000

2500

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compared to the untreated control plants. This trend in dis-ease protection was consistently maintained throughout the experiment, suggesting that TriH_JSB27- and PenC_JSB41-treated tomato plants require a minimum of 2 and 3 days, respectively, for maximum expression of disease resistance. The other two isolates, TriH_JSB36 and PhoM_JSB17, offered low disease protection (13 and 11%, respectively). However, both treatments still remain significant when com-pared to the untreated control plants.

Induction of PAL, POX, and GLU enzyme activities after PGPFs priming

This study observed varied temporal changes in tested enzyme activities in seedlings pretreated with PGPF or not treated before and after pathogen inoculation. PAL activity in both TriH_JSB27- and PenC_JSB41-pretreated seedlings challenged with or without Ralstonia solanacearum was sig-nificantly higher when compared to SDW control seedlings (Fig. 3A). Maximum PAL activity was noticed with TriH_JSB27 and pathogen inoculation, which was initiated early (0 hpi), with 34.3 U reaching a maximum of 117 U (3.4-fold increase) at 12 hpi, which then remained stable. PenC_JSB41 and pathogen inoculated recorded lower PAL activity: 14.3 U initiated at 4 hpi. This activity further increased with time and reached maximum activity of 63.9 U (4.5-fold increase) at 48 hpi. In treated seedlings alone, TriH_JSB27 recorded higher PAL activity (111 U) at 24 hpi, which was significantly better compared to that of PenC_JSB41 (72.4 U) at 48 hpi. Interestingly, during the later stage, this activity was not much altered. SDW control seedlings inoculated with the pathogen showed very low PAL activity (16.9 U, 12h) and were on par with the uninoculated control at all time intervals.

POX activity in control seedlings inoculated with Ralstonia solanacearum was 39.2 U at 4 hpi and reached a maximum of 54 U at 24 hpi, and this activity remained unchanged at further time periods (Fig. 3B). In TriH_JSB27- and PenC_JSB41-treated samples, no primed increase in POX activity was recorded. However, upon inoculation with the pathogen, the activity was significantly increased, reaching a peak of 18.9 U (48 hpi) and 40.1 U (12 hpi), respectively. Although, the decline in POX activity was observed in TriH_JSB27, POX activity was still significantly higher relative to uninocu-lated seedlings, which recorded the highest activity of 2.2 U at 96 hpi.

GLU in control seedlings inoculated with Ralstonia sola-nacearum showed 3- and 5-fold increases in activity com-pared to the seedlings treated with PGPFs and challenged with the pathogen. In SDW control plus pathogen-inoculated seedlings, significant GLU activity was noticed at early time intervals of 4 hpi with 4.7 U, and later this activity was rap-idly enhanced to 3.5-fold reaching a higher peak by 15 U at 24 hpi and thereafter it remained stable. Similarly, seedlings pretreated with TriH_JSB27 or PenC_JSB41 plus patho-gen recorded increased GLU activity beyond 12 hpi with maximum activity of 3.1 (24 hpi) and 5.9 U (48 hpi), respec-tively; however, both treatments showed reduced activities during the initial time intervals (Fig. 3C), In uninoculated Ta

ble

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PGPFs-treated seedlings, GLU activity was on par with the control seedlings raised from SDW without pathogen.

Expression of PAL, POX, and GLU after PGPFs priming

A differential expression of defence response genes was clearly evident among all treatments in this study. In TriH_JSB27 treatment alone, a 3.7-fold increase in PAL transcripts was noticed at 12 h, while upon pathogen inoculation, a minor reduction in gene expression (0.1-fold) was noticed at the same time intervals. Induction of PAL in TriH_JSB27-treated plus pathogen-inoculated plants was observed at 4 hpi (3.1-fold) and this significant upregulation was evident at all time points, while in PenC_JSB41 treatments, the signal of PAL expression was low compared to TriH_JSB27, with maximum induction of 2.1- and 1.8-fold noticed at 48 and 24 hpi in PenC_JSB41 alone and inoculated samples, respec-tively (Fig. 4A). The expression levels in SDW control with or without the pathogen showed minor induction of PAL transcripts.

On the other hand, the expression levels of POX and GLU were significantly higher in SDW control plus pathogen-inoc-ulated samples which recorded an early induction (4 h) and reached maximum at 12 hpi with 5.8- and 4.7-fold increase for POX and GLU, respectively. This expression level was predominantly consistent throughout the experimental time

points (Fig. 4B, C). In PenC_JSB41-inoculated plants, a maxi-mum enhancement of just over 3.7-fold (4 hpi) of POX and 1.8-fold increase (12 hpi) in GLU expression were observed at all time intervals, which were significantly higher when com-pared to TriH_JSB27 treatments. However, the expression level trend of these genes was drastically decreased after 12 and 24 hpi and later in both the treatments. On the contrary, PGPF treatments without pathogen inoculation showed no or weak POX and GLU induction and the expression levels were par with the uninoculated control samples. Among the genes examined, PAL was differentially expressed in TriH_JSB27 treatment alone at 4 h (just after treatment), and its expression was significantly increased at 12 h (just after pathogen inocula-tion). Although the expression levels of POX and GLU were found in TriH_JSB27 treatment, these were less superior when compared to those in PenC_JSB41 treatment, Interestingly, in SDW control plus pathogen-inoculated samples, both POX and GLU were significantly induced when compared to those of TriH_JSB27 and PenC_JSB41 treatments (Fig. 4B, C).

Discussion

Beneficial traits of the PGPF isolates

In this study, 79 rhizosphere soil fungi were isolated from various food crops and grass and screened for potent

Fig. 2. PGPF priming to tomato seeds and the spatiotemporal effect in induction of resistance against bacterial wilt disease under greenhouse conditions. Values are means of four independent replications. Bars represent standard error. *, ***, and **** on bars indicate significant difference between control and inoculated samples (P < 0.05, 0.005, 0.001, respectively; independent Student’s t-test). The data presented are from representative experiments that were repeated at least twice with similar results (this figure is available in colour at JXB online).

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Pathogen inoculation time gap (days) in primed plants

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Fig. 3. Time course induction of defence-related enzymes PAL (A), POX (B), and GLU (C) in 3-week-old tomato seedlings cv. Oogata-Fukuju pretreated with TriH_JSB27 or PenC_JSB41 or untreated, and challenged with or without the pathogen Ralstonia solanacearum at 0, 4, 12, 24, 48, and 96 hours post inoculation. Values are means of four independent replicates. Bars represent standard error. *, **, ***, and **** on bars indicate significant difference between control and inoculated samples (P < 0.05, 0.01, 0.005, 0.001, respectively; independent Student’s t-test). The data presented are from representative experiments that were repeated at least twice with similar results (this figure is available in colour at JXB online).

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Fig. 4. Expression of defence-related genes in 3-week-old tomato seedlings cv. Oogata-Fukuju raised after pretreatment with TriH_JSB27 or PenC_JSB41 or untreated. Expression of PAL (A), POX (B), and GLU (C) was analysed at 0, 4, 12, 24 and 48 h after root-dip inoculation with Ralstonia solanacearum. Values are means of four independent replicates. Bars represent standard error. *, **, ***, and **** on bars indicate significant difference between control and inoculated samples (P < 0.05, 0.01, 0.005, 0.001, respectively; independent Student’s t-test). Expression levels for each gene are reported as the fold-increase (normalized with reference gene) relative to those of the untreated controls. The data presented are from representative experiments that were repeated at least twice with similar results (this figure is available in colour at JXB online).

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characteristic traits of PGPFs. Of these, nine isolates were found positive for phosphate solubilization and IAA produc-tion in various pH conditions. Seven isolates were positive for both root colonization and in vitro plant growth-promoting abilities (Table 1). It is suggested that phosphate solubiliza-tion, production of IAA, and other related compounds by the fungus will interact with plants as part of its colonization, leading to growth promotion, induced resistance, and modi-fication of basal plant defence mechanisms (Altomare et al., 1999; Harman et al., 2004; Contreras-Cornejo et al., 2009; Yadav et al., 2011). Additionally, the comparative analysis of the ITS sequences of the identified PGPFs indicated that the TriH_JSB27 and PenC_JSB41 have high identity with known beneficial microbes that have several potential characteristic traits, such phosphate solubilization, IAA production, plant growth promoter, and biocontrol properties, which can be utilized for agriculture crop production.

PGPFs priming improved seed emergence and seedling vigour

In comparison with the SDW control, a significant enhance-ment of early seedling emergence, germination, vigour, and plant growth was noticed in PGPFs-treated plants, with maximum germination of 97% and seedling vigour index score of 2119 with early emergence (4 days after sowing) by TriH_JSB27 (Fig. 1). Recently, this study group has dem-onstrated that seeds treated with T. harzianum in sunflower (Nagaraju et al., 2012) and Penicillium chrysogenum in pearl millet (Murali et al., 2013) showed enhanced seed germina-tion and seedling vigour over the control. Delgado-Sanchez et al. (2011) reported that Opuntia streptacantha seeds treated with Penicillium chrysogenum, Phoma sp., and Trichoderma koningii also showed higher seed germination compared to the controls. Similar observations of increase in seed germina-tion and vigour were reported in chilli (Islam et al., 2011) and palm (Jegathambigai et al., 2009) using various Trichoderma isolates as seed-treatment fungi.

PGPFs priming enhanced nutrient uptake and growth responses of tomato

This study also reported a similar trend of increase in plant growth parameters in TriH_JSB27-pretreated plants (Table 2). The findings are in line with a previous study (Azarmi et al., 2011) in which the authors demonstrated that T. harzianum-treated tomato seedlings showed significant increases in shoot height, shoot diameter, and fresh and dried shoot and root weights as well as in NPK uptake. The results of the present study support this previous investigation with the observation of increased uptake of NPK nutrients and yield enhance-ment in TriH_JSB27-pretreated plants (Table 2). The increase in NPK uptake might be due to the increased biomass of the tomato crop treated with PGPFs, which can be directly linked to increase in tomato yield. This is true for T. harzi-anum, which has strong capacity to mobilize and take up soil nutrients and promote yield (Benitez et al., 2004). Similar correlations were made by Singh et al. (2011), who found

high uptake of nutrients in ratoon sugarcane plants grown on soil supplemented with biocontrol fungus. The authors sug-gested that Trichoderma spp. can facilitate increased availabil-ity and efficient uptake of soil nutrients, thereby improving yield of a ratoon crop. In the present study, the improvement of plant nutrition and crop was directly associated with the beneficial growth effect of inoculation with T. harzianum on the root surface. Recently, treatment of tomato plants with Penicillium sp. EU0013, which is known to induce resistance against Fusarium wilt disease in tomato and cabbage, was also shown to significantly mobilize nitrogen from soil to plants and enhance the growth-yield parameters (Alam et al., 2011).

Elicitation of induced resistance in tomato due to PGPFs priming

The current findings revealed that, under greenhouse condi-tions, a significant disease protection of 57.3 and 49% was observed in tomato plants pretreated with TriH_JSB27 and PenC_JSB41, respectively, and challenged with the patho-gen, compared to the SDW control (Fig. 2). Various reports on applications of PGPF isolates in agricultural practices as biocontrol agents, biofertilizers, and soil amendments for the management of phytopathogens and crop improve-ment in many crop plants have been documented (Saldajeno and Hyakumachi, 2011; Elsharkawya et al., 2012). Recently, the current study group also demonstrated the application of T. harzianum and Penicillium chrysogenum induced sys-temic resistance against downy mildews of sunflower and pearl millet (Nagaraju et al., 2012; Murali et al., 2013). In another independent study, Yoshioka et al. (2012) reported that Arabidopsis thaliana plants treated with a barley grain spore inoculum and culture filtrate of Trichoderma asperellum (SKT-1) possessed ISR against bacterial leaf speck disease Pseudomonas syringae pv. tomato. Additionally, the induction of systemic resistance by Trichoderma isolates in tomato upon subsequent inoculation with Xanthomonas euvesicatoria and Alternaria solani pathogens was shown to confer plant protec-tion at all time intervals by a spatiotemporal study (Fontenelle et al., 2011). Root colonization of PGPF Penicillium sp. GP16-2 and its culture filtrate induces systemic resistance against Pseudomonas syringae pv. tomato in Arabidopsis thali-ana (Hossain et al., 2008). Dong et al. (2003) demonstrated that soil amendment with 5% penicillin induced resistance to Verticillium wilt in cotton. The application of dry mycelium of Penicillium chrysogenum to cucumber and tomato plants in the field induced resistance against root-knot nematode (Gotlieb et al., 2003). Also, the present study noticed that the other two tested PGPFs displayed fair to moderate disease protection in addition to their growth promotion. These results are interest-ing because they show that the growth-promoting action of PGPF isolates is dependent on tripartite interactions.

Upregulation of defence-related enzymes and genes in tomato mediated by PGPF treatments

Plants are able to defend themselves upon numerous phy-topathogen attacks by producing a wide spectrum of defence



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enzymes that enhance both cellular protection and disease resistance. In this study, maximum PAL activity was observed in seedlings treated with TriH_JSB27 alone, and this activ-ity was reduced after 12 h of pathogen infection (Fig. 3A). Similar correlations have been established with increase in PAL enzyme activity after sunflower plants primed with T. harzianum (Lamba et al., 2008). PenC_JSB41-treated plants inoculated with pathogen showed comparatively lower activity than those treated with TriH_JSB27. The SDW con-trol seedlings with or without pathogen infection recorded the lowest PAL activities without much variation (Fig 3A). The activities of POX and GLU were significantly higher in SDW control plus pathogen-inoculated plants with a relevant increase from 4 hpi, and this increase trend was maintained throughout the time intervals. Although the activities of POX and GLU were noticed in PenC_JSB41-treated plus patho-gen-inoculated seedlings, they were significantly lower when compared to those detected in SDW control plus pathogen-inoculated samples (Fig 3B, C). These results are in agree-ment with a previous study (Van Wees et al., 2008) in which the authors illustrated that defence responses in primed plants are not activated directly but are accelerated upon attack by pathogens or insects, resulting in faster and stronger resist-ance to the attacker encountered.

This study also noticed that priming tomato seedlings with TriH_JSB27 and PenC_JSB41 induced PAL gene expression significantly by 3.7- and 2.1-fold, respectively, and that these expression levels were slightly reduced (0.1–0.3 fold) upon pathogen inoculation (Fig. 4A). However, neither TriH_JSB27 or PenC_JSB41 treatment did not directly induce systemic expression of POX and GLU. Upon subsequent stimulation by Ralstonia solanacearum attack, POX and GLU were more highly upregulated in PenC_JSB41-primed plants than in TriH_JSB27-primed ones. Additionally, the expres-sion levels of these two genes were significantly higher (2- to 4-fold increase) in the SDW control plus pathogen-inoculated plants, while no induction was observed in uninoculated SDW control seedlings (Fig 4B, C). These data indicate that both pathogen and PGPFs are needed to induce expression of POX and GLU, whereas priming of PGPFs alone is suf-ficient for PAL gene induction. This finding is well supported by the study of Shoresh et al. (2005), who reported high induction of PAL in cucumber plants treated with T. asperel-lum. The authors also observed a much higher upregulation of POX, GLU, and other disease-related genes in treated plants challenged to Pseudomonas syringae pv. lachrymans. Hossain et al. (2007) reported that multiple defence mech-anisms are involved in PGPF Penicillium simplicissimum-mediated ISR in Arabidopsis plants against Pseudomonas syringae pv. tomato. Pearl millet susceptible plants primed with PGPF Penicillium chrysogenum showed higher basal levels of defence gene expression after pathogen challenge as compared to non-primed plants (Murali et al., 2013).

Earlier reports support the finding of the current study. The accumulation of PAL, POX, and GLU mediated by the beneficial microbe Pseudomonas fluorescens was reported for the tomato-Fusarium oxysporum f. sp. lycopersici interaction (Ramamoorthy et al. 2002). Similarly, systemic resistance

was enhanced in response to Ralstonia solanacearum chal-lenge in tomato due to high accumulation of defence enzymes (Vanitha et al., 2009b). Further, Vanitha and Umesha (2009c) demonstrated that rapid and high induction of PAL and POX was noticed in Pseudomonas fluorescens-pretreated tomato seedlings, which were inoculated with Ralstonia solanacearum. The interaction between tomato and Verticillium dahliae trig-gered enhanced activities of POX and PAL, phenylpropanoid metabolism, and synthesis of lignins (Gayoso et al., 2010). Recently, the involvement of POX imparting basal resistance of tomato to Rhizoctonia solani was demonstrated by Nikraftar et al. (2013). Several lines of evidence report that increased induction in the activity profile of GLU in tomato plants was able to overcome the damage caused by plant pathogens and that the elicitation of GLU enzyme or its gene was mediated by plant growth-promoting rhizobacteria or PGPF in tomato plants (Ramamoorthy et al., 2002; Tucci et al., 2011).

In conclusion, the current investigation has lead to the iden-tification of phosphate-solubilizing and IAA-producing PGPF isolates, T. harzianum (TriH_JSB27) and Penicillium chrysoge-num (PenC_JSB41) which were able to trigger the susceptible tomato cultivar into developing resistance against bacterial wilt pathogen. Further, these results also provide evidence of interconnecting ISR and defence responses. The induction of SA-responsive genes (POX and GLU) was upregulated when tomato plants were in contact with the pathogen Ralstonia solanacearum, while TriH_JSB27 and PenC_JSB41 treatments stimulated the immune response of tomato before and after challenging with Ralstonia solanacearum pathogen by activat-ing PAL, a defence gene which confers ISR. The primed plants had improved plant health by enhanced nutrient uptake from the soil. These two PGPFs are useful in the development of pro-tection against bacterial wilt to bestow high-yielding tomatoes.

Acknowledgements

JS was financially supported by the Japan Society for Promotion of Science (JSPS) through a Foreign Postdoctoral Researcher Scholarship (2011–2013), Government of Japan. JS kindly acknowledges Mr Sasaki Kazunori and Mr Imada Kiyoshi for their kind help. Also, the authors thank the Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, Japan for pro-viding laboratory and greenhouse facilities.

References

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Altomare C, Norvell WA, Bjorkman T, Harman GE. 1999. Solubilization of phosphates and micronutrients by the plant growth promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Applied and Environmental Microbiology 65, 2926–2933.



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Characterization of rhizosphere fungi that mediate resistance in tomato against bacterial wilt...

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