Plant growth promoting rhizobacteria ( PGPR ) induces resistance against Fusarium wilt and improves lycopene content and texture in tomato

Plant growth promoting Bacillus subtilis (BS2) was found effective against tomato wilt caused by Fusarium oxysporum f sp. lycopersici under field conditions. Pretreatment of tomato plants with B. subtilis BS2 significantly induced the activities of defense related enzymes viz., peroxidase, polyphenol oxidase, chitinase and phenylalanine ammonialyase and phenolics when challenged with the pathogen. Apart from disease control, BS2 improved the fruit quality with high lycopene (76.30 mg/kg against control, 40.34 mg/kg) and texture (90.5 Fmax against control, 56.35 Fmax) during harvest and even 15 days after harvest, similar trend was maintained unequivocally indicating that plant growth promoting rhizobacteria (PGPR) can improve the nutritional quality as well as shelf life of the fruits.


INTRODUCTION
Tomato is an important worldwide cultivated solanaceous vegetable playing a major role in human nutrition.Wilt caused by Fusarium oxysporum f.sp.lycopersici is a serious concern in tomato cultivation (Loganathan et al., 2009).Management of the disease through an ecofriendly approach will nullify the ill effects such as environmental pollution, residual toxicity and fungicidal resistance in pathogens due to use of chemicals.Many bacterial communities living in the rhizosphere and found associated with plant growth promotion activity are called plant growth promoting rhizobacteria (PGPR) (Kloepper et al., 1980).The PGPR are known to act as biocontrol agents through competition for ecological niche/substrate and production of siderophores, antibiotics, hydrogen cyanide and fungal cell wall lysing enzymes (Glick and Bashan 1997;Wang et al., 2000;Saravanakumar et al., 2007).PGPR are also involved in induced systemic resistance (ISR) against many diseases in a wide range of crops (Kloepper and Beauchamp, 1992;Liu et al., 1995;Chen et al., 2000;Sangeetha et al., 2010).Although the application of PGPR and their effects on disease or pest control and yield have been well established for a wide range of crops, information on fruit quality in terms of lycopene content and texture is very limited.Lycopene, a carotenoid present in tomato is an important compound in human nutrition as it plays a key role in reducing cardiovascular as well as prostate cancer diseases (Giovannucci, 1999;Giovannucci et al., 2002).Apart from the medicinal value, the market quality of the tomato fruit is also affected by lycopene because fruit color is determined by carotenoids, and primarily lycopene (Fraser et al., 1994;Shi and Le Maguer, 2000).Shelf life of tomato fruits is dependent on fruit firmness.Main factor affecting firmness of the fruit during maturity is microbial spoilage (Kramer et al., 1992).In our earlier study we identified two potential PGPR: Bacillus amyloliquefaciens isolate BA1 (earlier reported as chilli 1) and Bacillus subtilis isolate BS2 (earlier reported as chilli 2), based on preliminary studies related to plant growth promotion and plant disease control (Loganathan et al., 2010).In that study, BA1 and BS2 manifested high level of in vitro efficacy in producing plant growth promoting compounds (indole acetic acid and siderophores) and solublizing phosphates apart from inhibiting mycelial growth of wilt fungus, F. oxysporum fsp.lycopersici.In the present study, field efficacy of PGPR bio-formulations (BA1 and BS2) on Fusarium wilt and quality of fruits (lycopene content and texture) during harvest and postharvest were determined.

Testing the talc bio-formulation efficacy under field conditions
Talc formulation of PGPR isolates BA1 and BS2 were prepared using a standard protocol (Vidhyasekaran and Muthamilan, 1995) with some minor modifications.The PGPR isolates were cultivated in nutrient broth (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 48 h at temperature of 28±2°C under shaking at 20 g.Four hundred milliliters of bacterial culture containing 8 x 10 8 cfu/ml was mixed with 1 kg talc (Talc India, Rajasthan, India) sterilized at 105°C for 12 h along with 10 g carboxy methyl cellulose (adhesive agent) and 15 g calcium carbonate (to neutralize the pH to 7.0).The bioformulation was shade dried for 12 h at ambient temperature (28±2°C) and packed in polythene packs.Bacterial population in the talc bio-formulation at the time of application was 6 x 10 8 cfu/g.Two field trials were conducted in years 2009 (trial I at Upland Block at Indian Institute of Vegetable Research, Varanasi, India) and 2010 (trial II at Lowland Block at Indian Institute of Vegetable Research, Varanasi, India).Both blocks have recorded 40-60% Fusarium wilt incidence in tomato (cv DVRT-1) in the previous year.The seeds of tomato (cv.DVRT-1 obtained from Seed Production Unit, Indian Institute of Vegetable Research, Varanasi, India) were treated with the talc formulation (10 g formulation/kg of seeds) and shade dried for 12 h.The treated seeds were sown in nursery beds applied with talc formulation (mixed thoroughly 50 g talc formulation with 5 kg well decomposed Farm Yard Manure (FYM) and applied to the 3m 2 bed).In chemical treatment, seeds treated with 1 g of carbendazim 50% WP (Dhanuka Agritech Ltd., Gurgaon, India) per kg of seeds were sown in nursery beds applied with well decomposed FYM (5 kg/3m 2 ).In control, nursery beds were prepared as described in chemical treatment but seeds were sown without any treatment.After 25 days, under bio-formulation treatment, main field was applied with 2.5 kg bioformulation in 50 kg of well decomposed FYM ha -1 .The seedlings under bio-formulation treatment were uprooted and the root portion was dipped in talc based bio-formulation solution (1.0%) for 30 min and transplanted in the main field.Similarly, for the chemical and untreated controls, the seedlings were dipped for 30 min in 0.1% carbendazim solution and water respectively and transplanted separately in the main field and FYM alone (50 kg/ha) was applied.The experiment was conducted in randomized block design with four replications of each treatment.Observations on disease control and measurement of plant height were recorded 45 days after transplanting.

Testing quality of fruits
In all the treatments the mature fruits were harvested from the field separately and analyzed for fruit texture and lycopene content.In tomato, third harvest normally gives maximum yield since it matches with exact maturity.Hence third harvest fruits were used in all fruit quality analyses.For each of these analyses, five fruits were randomly collected from each replication (in each treatment there were four replications hence total number of fruits were 20/treatment).

Analysis of fruit texture
Texture of the fruit was analyzed by a puncture test using a Texture Analyzer TA-XT (Stable Micro Systems), loading (2 mm s -1 ) at a distance of 15 mm on two opposite points along the equatorial plane (Mena-Violante and Olalde-Portugal, 2007).Finally an average maximum force (F max) per fruit was calculated.

Estimation of lycopene
Lycopene content was estimated by a spectrophotometric method as described by Anthon and Barrett (2001).The samples were extracted with a solution containing ethanol and hexane (4:3 v/v) and the phases were separated.Amount of light absorption were recorded from hexane phase at 503 nm and the results were interpreted using the value of 172/nM as the extinction coefficient for lycopene in hexane and expressed in mg/kg of fruits

Sample collection for analyses of induced defense proteins and phenolics
Pot mixture (consists of red soil : sand : well decomposed FYM at 1:1:1 ratio) was autoclaved for 1 h for two consecutive days at 24 h interval and filled in pots (20 cm diameter and 30 cm height).Virulent isolate of F. oxysporum f.sp.lycopersici (FOL) was mass multiplied in sterilized sand-maize (19:1 ratio) medium for 15 days and mixed in the pot soil (10 g sand maize fungal culture/kg of soil).Tomato seeds were treated with talc formulation as described in field experiments and sown on sterilized soil.After 24 days, the seedlings were uprooted and the root portions of the seedlings were dipped in 1.0% talc formulation solution for 30 min.The treated seedlings were transplanted into the pot containing sterilized pot mixture treated with PGPR talc formulation (10 g/kg of soil) with or without pathogen according to the treatment details mentioned in Table 5.For analysis of defence related enzymes and chemical, root portion of 45 days old seedlings (21 days after transplanting) from different treatments was used.Five seedlings per replication were uprooted and thoroughly washed with running tap water.Root portions were removed from the seedlings and cut into pieces and powdered using liquid nitrogen and the frozen powder was used for each analysis.The protein content of sample was estimated by Lowry et al. (1951) using bovine serum albumin as standard.

Assay of phenylalanine ammonia-lyase (PAL)
One gram of plant root sample was homogenized in 3 ml of ice cold 0.1 M sodium borate buffer (pH 7.0) containing 1.4 mM of 2mercaptoethanol and 50 mg of insoluble polyvinylpyrrolidone (PVP) by using pestle and mortar.The extract was filtered through cheese cloth to remove debris, and centrifuged at 20,000 g for 10 min at 4°C and the supernatant was used as the enzyme source.PAL activity was determined as the rate of conversion of Lphenylalanine to trans-cinnamic acid at 290 nm (Dickerson et al., 1984) and was expressed in nmol trans-cinnamic acid min -1 mg -1 of protein

Assay of chitinase
Plant root sample (1 g) was extracted with 5 ml of 0.1 M sodium citrate buffer (pH 5.0) and the content was centrifuged at 20,000 g at 4°C for 10 min and the supernatant was used as enzyme source.The colorimetric assay of chitinase was carried out as described by Boller and Mauch (1988) and the enzyme activity was expressed as nmoles GlcNAc equivalents min -1 mg -1 protein.

Assay of peroxidase (PO)
One gram of plant root sample was homogenized with 2 ml of 0.1 M phosphate buffer (pH 7.0) at 4°C and then centrifuged at 15,000 g and 4°C for 15 min.The supernatant (0.5 ml) was mixed with 0.5 ml of 0.0 5M pyrogallol and 0.5 ml of 1% H2O2 and incubated at room temperature (28 ± 2°C).The absorbance was recorded at 420 nm at 30 s intervals up to 3 min and the activity was expressed as changes in the absorbance min −1 mg protein −1 (Hammerschmidt et al., 1982).

Assay of phenol
Root samples (1 g) were homogenized in 80% methanol as described by Zieslin and Ben-Zaken (1993).The reaction mixture consisting of 1 ml methanolic extract, 5 ml distilled water and 250 μl Folin-Ciocalteau reagent (1 N) was prepared and kept at 25°C.The development of blue colour was measured at 725 nm and compared with standard catechol and the amount of phenolics was expressed as μg catechol mg protein −1 .

Native PAGE analysis of polyphenol oxidase (PPO)
The effect of PGPR treatment on induction of PPO isoforms was visualized in native PAGE electrophoresis (Laemmli 1970).Root tissue (1 g) was homogenized in 2 ml of 0.1 M sodium phosphate buffer (pH 6.5) at 4°C and centrifuged at 20,000 g for 10 min at 4°C and the supernatant was used as enzyme extract.Sample (60 µg) containing protein was loaded in polyacrylamide gel (8%).After electrophoresis, the gel was equilibrated in 0.1% p-phenylene diamine in 0.1 M potassium phosphate buffer (pH 7.0) for 30 min followed by 10 mM catechol in the same buffer.Finally, the PPO isoforms were visualized by adding catechol which resulted in appearance of dark brown discrete bands (Jayaraman et al., 1987).

Statistical analysis
Data were statistically analyzed (Gomez and Gomez,1984) 2).The yield parameter had a positive relationship with plant growth as it was highest in BS2 (57.7 cm) followed by BA1 (51.95 cm), chemical (48.15 cm) and control (44.80 cm).

PGPR treatment on quality of fruits
Fruits collected at different harvesting periods were analyzed for lycopene content and texture (Tables 3 and  4).Lycopene content was increasing invariably towards maturity of the plants irrespective of the treatments.However, under PGPR treatments the content was significantly higher than in the chemical and untreated control.Among the treatments, BS2 recorded the highest content in all the harvest (71.28 mg/kg) when compared with BA1 (46.21 mg/kg), chemical (39.12 mg/kg) and control (35.21 mg/kg).Apart from this, fruits were analyzed for texture and lycopene content at the time of harvest and after 15 days of harvest.Results indicate that in all cases, there was a decline in texture profile after 15 days of storage while lycopene content increased (Table 4).At harvest, the highest texture profile (F max ) was recorded in plants inoculated with BS2 (90.5) followed by BA1 (79.16), carbendazim (61.96) and control (56.35).After 15 days, there was a declining trend in F max value in all the treatments but in BS2 the content was high (11.10)as compared to control (2.14) and other    treatments.Post harvest lycopene analysis indicated that there was an increase in the content after 15 days of storage in all the treatments (Table 4) but the increase was greater in BS2 treatment (82.70 mg/kg) followed by carbendazim (48.65 mg/kg), BA1 (48.34 mg/kg) and control (44.70 mg/kg).

Induction of defense related enzymes
Induction of phenyl alanine ammonia lyase (PAL), peroxidase (PO) polyphenol oxidase (PPO), chitinase and phenol was observed in plants treated with PGPR upon challenged with FOL (Table 5).Induction of PAL was more in PGPR treated plants challenged with FOL and the effect was greater in BS2 (30.53 nmol min -1 mg -1 ) followed by BA1 (29.10 nmol min -1 mg -1 ), pathogen inoculated control (22.00 nmol min -1 mg -1 ) and untreated control (20.00 nmol min -1 mg -1 ).Similarly, all PGPR treated plants challenged with pathogen showed high level induction of PO, chitinase and phenol as compared to the control.However, in the case of PPO, the induction was significantly high in BS2 treated plants challenged with pathogen followed by BA1 and other treatments.
Native PAGE analysis of tomato plants treated with BS2 with or without pathogen inoculation showed four isoforms (PPO1, PPO2, PPO3 and PPO4) in BS2 treated plants challenged with FOL (Figure 1).Among the four isoforms, PPO3 and PPO4 were induced commonly in healthy control, pathogen inoculated control and BS2 with or without pathogen treated plants.On the other hand, PPO2 was absent in healthy and present in the other treatments and PPO1 was exclusively present in BS2 treated plants challenged with pathogen.Presence of strong signal of PPO3 and PPO4 isoforms was observed in the BS2 treated plants challenged with pathogens.

DISCUSSION
Majority of PGPR isolated for the management of plant diseases are Pseudomonas spp., (Ramamoorthy et al., 2002) or Bacillus spp., (Schisler et al. 2004).In our preliminary study, out of 142 rhizospheric PGPR tested, two Bacillus spp.: B. amyloliquefaciens and B. subtilis were identified as potential isolates for use against fungal pathogens such as F. oxysporum f.sp.lycopersici and Sclerotium rolfsii apart from plant growth promotion through production of indole acetic acid and phosphate solubilization (Loganathan et al., 2010).In the present study, among the two isolates (BS2 and BA1), BS2 proved to be more effective against wilt pathogen, F. oxysporum f.sp.lycopersici under field conditions.Similarly, PGPR were reported to control fungal diseases caused by soil borne and leaf spot pathogens in wide range of crops (Wei et al., 1996;Viswanathan and Samiyappan, 2001;Ramamoorthy et al., 2002;Vivekananthan et al., 2004).
In the present study, the lycopene content in DVRT1 was found to increase from first harvest onwards and reached maximum at the third harvest.However, the influence of PGPR treatment, especially BS2 on lycopene content was greater since in all three harvests, the content was significantly higher than other treatments indicating that BS2 has a pivotal role in enhancing the content.It has been reported that environment conditions and plant nutrient status played a major role on status of lycopene content (Abushita et al., 2000;Binoy et al., 2004).By keeping environmental conditions constant, there was a clear difference between PGPR treated and untreated plant fruits indicating the direct or indirect influence or both on lycopene content.Apart from lycopene, important quality of fruit is shelf life or post harvest keeping quality.Fruits from the BS2 treated plot exhibited considerably high level texture even at well ripening stage (15 days after harvest).Fruits with high texture showed extended keeping quality.The fruit texture is associated with low production ethylene production (Alexander and Grierson, 2002) and it has been demonstrated in plants treated with PGPR (Glick et al., 1998).In the present study, alternation of ethylene production is indirectly reflected in promoting growth of the tomato plants (Table 2).Though there is a relationship between PGPR treatment and improvement of fruit texture, further research in this line is required.This has been found to be of significant importance since less textured/firm fruits are more prone to spoilage by microbes (Mena-Violante and Olalde-Portugal, 2007).
Application of PGPR for plant disease management is gaining importance, due to induction of defense related enzymes (Viswanathan and Samiyappan, 2001) and chemicals (Chen et al., 2000) to suppress the pathogen thereby enabling a process called induced systemic resistance (ISR).In the present study, PAL, PO, PPO, chitinase and phenol were triggered in PGPR treated plant inoculated with FOL indicating activation of ISR mechanism by PGPR.The defense related enzymes play a major role in biosynthesis of lignin, oxidation of phenol and synthesis of antimicrobial phytoalexins (Daayf et al., 1997).The defense reaction was observed in several crops against a wide range of pathogens.Tomato plants treated with Pseudomonas fluorescens Pf1 recorded high level induction of defence enzymes in response to Pythium aphanidermatum attack (Ramamoorthy et al., 2002).Similarly, mango trees sprayed with P. fluorescens FP7 recorded a greater amount of the enzymes against Colletotrichum gloeosporioides infection ( Vivekanadan et al., 2004).Mixture of Pseudomonas (NFP6), P. fluorescens (Pf3a), and B. subtilis (BS1) was found to induce defence reaction in banana against crown rot caused by Lasiodiplodia theobromae and Colletotrichum musae under in vivo conditions (Sangeetha et al., 2010).Phenolics are known to have fungitoxic effect and a role in strengthening the host cell wall.PGPR treated plants showed high level accumulation of phenolic compounds when challenged with pathogens (M'Piga et al., 1997).Similarly, pre-treatment with PGPR had shown induction of phenolics against F. oxysporum f. sp.pisi and Pythium ultimum in pea and cucumber, respectively (Benhamou et al., 1996(Benhamou et al., , 2000)).In the present study, among the different defense related enzymes, PPO was found to be induced significantly in BS2 treated plants challenged with FOL.Similarly in native PAGE, induction of new or enhanced accumulation of PPO in tomato pretreated with BS2 against FOL indicated that PPO also played a crucial role in protecting the crop from the soil borne pathogen.Ramamoorthy et al. (2002) also noted unique induction of PPO in native PAGE analysis in PGPR treated tomato and chilli against P. aphanidermatum.
In conclusion, induction of defense related enzymes and phenolics by Bacillus PGPR, was found to have protective role in combating the wilt disease caused by F. oxysporum f.sp.lycopersici in tomato which in turn improved the fruit yield and quality.
means of three replicates; Means in a column followed by same superscript letters are not significantly different according to Duncan's multiple range test at P=0.05.

of PGPR isolates against Fusarium wilt disease of tomato under field conditions
and theLoganathan et al.  1107treatment means were compared by Duncan's multiple range test (DMRT).The package IRRISTAT version 92 developed by the International Rice Research Institute Biometrics unit, Philippines was used in all the analyses. RESULTSEfficacy

Table 1 .
Effect of PGPR treatment on fusarial wilt disease of tomato under field conditions.

Table 2 .
Effect of PGPR on plant growth and yield of tomato under field conditions.Means in a column followed by same superscript letters are not significantly different according to Duncan's multiple range test at P=0.05.
rd harvest; *Cumulative yield of three harvests;

Table 3 .
Effect of PGPR treatment on lycopene content at different stages of fruit harvest.
Means in a column followed by same superscript letters are not significantly different according to Duncan's multiple range test at P=0.05.

Table 4 .
Effect of preharvest application of PGPR on post harvest fruits quality.

Table 5 .
Induction of defense related enzymes and phenol in tomato plants challenged with FOL.