PGPR in managing root rot disease and enhancing growth in mandarin (Citrus reticulata Blanco.) seedlings B.N. Chakraborty*, S. Allay, A.P. Chakraborty and U. Chakraborty Immuno-Phytopathology Laboratory Department of Botany, University of North Bengal Siliguri, Darjeeling, West Bengal - 734 013, India *E-mail: bncnbu@gmail.com ABSTRACT Decline in general plant-health and fruit production in mandarin influenced by abiotic and biotic factors is a major threat to cultivars grown in Darjeeling and Sikkim hills. Fusarium root rot, caused by F. oxysporum, is one of the most serious diseases afflicted during early plant growth stage in Citrus. To address this, seven PGPR isolates - Pseudomonas poae (RMK03), Bac illus str atos phe ric us (RHS/CL-01), Oc hrobactr um an thr opi, Pae nibacillus len tim orbus , Bac illus pum ilus, Bac illus m e gate rium and Bac illus amyloliquefaciens were isolated from the rhizosphere of Citrus reticulata, C. limonia and Camellia sinensis, and used for evaluating their effect on growth of mandarin seedlings. Pseudomonas poae showed in vitro antagonism to Fusarium oxysporum. Better growth enhancement was noticed with P. poae, B. stratosphericus, O. anthropi and B. pumilus. Enhanced activity of chlorophyll, total protein, phenol, four major defense enzymes- c as observed upon application of PGPR. P. poae also suppressed root rot caused by Fusarium oxysporum. Use of PGPR, which promote growth besides reducing disease severity to some extent, may lead to use of eco-friendly approaches for controlling plant diseases. Key words: PGPR, mandarin, root rot, Citrus, bacterial isolates INTRODUCTION Mandarin (Citrus reticulata Blanco.) is one of the most important fruit crops of Darjeeling and Sikkim hills. Economy of the farmers in this region is fully dependent on good, disease-free fruit production. However, several abiotic and biotic factors pose a threat to the plants, causing rapid decline in plant health resulting in decreased fruit production. Several diseases in citrus are attributed to soil-borne fungi. Fusarium root rot, caused by F. oxysporum, is one of the serious diseases in this crop. Soil application of Bacillus subtilis and Streptomyces griseoviridis has been reported to control root rot disease (Nemec et al., 1996; Ziedan and Eman, 2002). This has led to delivery of PGPR strains to the soil to improve population dynamics of the augmented bacterial antagonists and to suppress establishment of pathogenic microbes in horticultural crops (Esitken, 2011). Inte nsive inter ac tions ta ke pla c e in the rhizos pher e a mong soil, mic roorga nis ms and microfauna which may affect plant growth and yield positively or negatively (Antoun and Prevost, 2006). It has been proved that root inoculation and/or spraying with PGPR results in increased germination, seedling emergence, and, modified growth and yield in tomato (Mayak et al, 2004; Woitke et al, 2004), lettuce (Han and Lee, 2004; Barassi et al, 2006), radish (Yildirim et al, 2008a, b) and strawberry (Karlidag et al, 2011). Growth stimulation in plants by PGPR application may be a direct mechanism set-in by the production of seconda ry me tabolite s such as phytohormones, riboflavin and vitamins (Dakora, 2003), or, through improved nutrient availability (Glick, 1995; Chabot et al, 1996a; Yanni et al, 1997). Three commercial bio- fertilizers: phosphorein (phosphate dissolving bacteria - Bac illus me gate r ium var. Phos phatirc um), microbien (Azotobacter spp.) and Cerealien (nitrogen- fixing Cyanobacteria) controlled dry root-rot disease J. Hortl. Sci. Vol. 11(2): 104-115, 2017 105 of citrus caused by Fusarium solani (Mart.) Snyd & Hans, and improved the yield quality in mandarin (Mohamedy and Ahmed, 2009). Enhanced plant nutrition effected by PGPR is mainly through increased phosphorus uptake, by solubilization of inorganic phosphate or by mineralization of organic phosphate. Further, indirectly, PGPR act as biocontrol agents to reduce disease severity, or these stimulate other beneficial symbioses, or, protect the plant by degrading xenobiotics in contaminated soils (Jacobsen, 1997). More importa ntly, such PGPR may also induce systemic resistance in the host (ISR), thereby protecting plants against pathogen attack. This study aimed to understand the effect of PGPR in controlling root rot disease of mandarin caused by F. oxysporum using biochemical analysis during disease suppression. MATERIAL AND METHODS Test organisms Plant growth promoting rhizobacteria (PGPR): Pseudomonas poae (RMK03), isolated from the rhizos phere of Citr us re tic ulate ; Bac illus stratosphericus (RHS/CL-01), isolated from the rhizosphere of Citrus limonia; and, Ochrobactrum anthropi, P ae nibacillus le ntimor bus, Bacillus pumilus , Bac illus me gate r ium and Bac illus amyloliquefaciens, isolated from the rhizosphere of Camellia sinensis, were identified on the basis of morphological, microscopic and biochemical tests, and 16S rDNA sequencing. Fungal pathogen: F. oxysporum (RHS/M534), causing root rot of mandarin, was obtained from the rhizosphere of C. reticulata. BLAST query of the 18S rDNA sequence of F. oxysporum against GenBank database confirmed its identity. Morphologic a l and s ca nning e le ctron microscopic (SEM) studies: Pure cultures of the three isolates were streaked on Nutrient Agar (NA) plates for colony development. Individual colonies were examined for shape, size, structure and pigmentation. Gram-specific reactions of these isola tes were recorded as per Buchanan and Gibbson (1974). Gram positive/negative reaction and the shape of cells was recorded. SEM view of all the seven isolates was also recorded. Genomic DNA isolation and 16S rDNA amplification by PCR Extraction of genomic DNA was done from 24h old culture a s per Sta fford et al (2005) with modification. This was followed by spectrophotometric quantic a tion using a Colepa rme r UV–VIS Spectrophotometer and, quality was analyzed on 0.8% agarose gel. For PCR amplication, DNA was amplied by mixing the template DNA (50ng) with polymerase re ac tion buff er, dNT P mix, prime rs a nd Taq polymerase. Polymerase Chain Reaction (PCR) was performed in a total volume of 100 l, containing 78 l deionized water, 10 l 10x Taq polymerase buffer, 1 l 1U Taq polymerase enzyme, 6 l of 2 mM dNTPs, 1.5 l of 100 mM forwa rd (50-AGAGT RT GATCMTYGCT WAC-30) a nd re ve rse (50- CGYTAMC TTWTTACG RCT-30) primers, and 3.5 l of 50ng template DNA. PCR programming was as follows: initial denaturing at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 60s, annealing at 59°C for 60s and extension at 70°C for 2 min, and final extension at 72°C for 7 min, in a Primus 96 advanced gradient Thermocycler. The PCR product (20 l) was mixed with a loading buffer (8 l) containing 0.25% bromophenol blue, 40% w/v sucrose in water, and then loaded onto 2% Agarose gel with 0.1% ethidium bromide for examination under horizontal electrophoresis. T he PCR product was sent for sequencing to Chromous Biotech, Bengaluru, India. 16S rDNA sequence and phylogenetic analysis The 16S rDNA sequences obtained from PCR products were analyzed by NCBI-BLAST and aligned with ex-type isolate sequences from NCBI GenBank for identication. Evaluation of PGPR activity with bacterial isolates in vitro Phosphate solubilization: Primary phosphate solubilizing of all eight isolates was carried out by allowing the bacteria to grow on a selective medium, i.e ., Pikovs kaya’s a gar ( Pikovs ka ya, 1948). Appearance of a transparent halo around the bacterial colony indicated phosphate solubilizing activity of the bacterium. Side rophore produc tion: Produc tion of siderophore was detected by the standard method of PGPR in disease management in mandarin J. Hortl. Sci. Vol. 11(2): 104-115, 2017 106 Schwyn and Neiland (1987), using the blue indicator chrome azurol S (CAS). IAA production: For detection and quantification of IAA, selected bacterial cells were grown for 24h to 48h on high C/N ratio medium. Tryptophan (0.1mM) was added to enhance indole acetic acid (IAA) production. IAA in the culture supernatant was assayed by Pillet-Chollet method (Dobbelaere et al, 1999). Plant growth promotion One-year-old mandarin seedlings were selected and earthenware pots under open condition. Field-grown plants were watered regularly for maintenance. Growth promotion was studied in terms of increase in plant height and number of leaves. Observations were recorded at 4 and 8 months from application of the seven different bacterial strains. In vitro antagonism F. oxysporum was paired with P. poae on solid medium as per Chakraborty and Chakraborty (1989). Inoculum preparation and application Plant Growth Promoting Rhizobacteria: Initially, bacteria were cultured on Nutrient Broth medium (Himedia, M002-100G, ingredients: peptic digest of animal tissue 5.00g/litre, sodium chloride 5.00g/l, beef extract- 1.50g/l, yeast extract- 1.50g/l, final pH at 25°C 7.4±0.2), and allowed to grow with shaking at 37°C at 120rpm for 48h. At the end of the log phase, bacterial cultures were centrifuged at 10,000rpm for 15 min and the bacterial pellet collected. Bacterial aqueous suspension was prepared using distilled water as per requirement, to maintain a bacterial concentration of 108 c.f.u./ml. The aqueous suspension was then applied as foliar spray and soil-drench @ 100 ml/plant to the rhizosphe re of one -yea r-old manda rin pla nts. Application of suspension was done every month, repeated in three replications. Fungal pathogen: The pathogen (F. oxysporum) was grown in sand-maize meal medium (maize meal:sand:water 1:9:1 w:w:v) in autoclaved plastic bags (sterilized at 20lb pressure for 20 min) for a period of three weeks at 28ºC, until the mycelium completely covered the substrate. Mandarin seedlings were inoculated by adding 100g of previously prepared inoculum of F. oxysporum to the rhizosphere soil. Disease assessment Dise a se a ss es sme nt was done a s pe r Chakraborty et al (2006) at 15, 30 and 45d after inoculation. Disease intensity was assessed as root- rot index on a scale of 0-6, depending on underground as well as above-ground symptoms, as follows: 0=no symptom; 1=small roots turn brownish and start rotting; 2=leaves start withering and 20-40% of roots turn brown; 3=leaves withered and 50% of roots affected; 4=shoot tips also start withering and 60-70% roots affected; 5=shoots withered, with defoliation of lower withered leaves, 80% roots affected; 6=whole plants die, with upper withered leaves still attached to the shoot; roots fully rotted. Biochemical analysis All the biochemical analyses were performed with treated as well as Control mandarin leaves and roots. Enzyme assay Peroxidase (POX, EC1.11.1.7): Extraction and assay of peroxidase was done as per Chakraborty et al (1993). Specific activity was expressed as an Chitinase (CHT, EC 3.2.1.14): Chitinase was extracted from mandarin leaves and assayed following Boller and Mauch (1988). Phenylalanine Ammonia Lyase (PAL, EC 4.3.1.5): The enzyme was extracted and assayed as per Bhattacharya and Ward (1987). Enzyme activity was determined as the amount of cinnamic acid produced from L-phenyl alanine from 1g of tissue/min. 1,3- glucanase ( : glucanase was e xtracted and assayed from leaf samples as per Pan et al (1991). Enzyme activity was Phenolics: Phenols were extracted and estimated from leaf samples as per Mahadevan and Sridhar (1982). Quantification of total phenol and o-dihydroxy phenol was done using a standard of caffeic acid. Chlorophyll Total chlorophyll content was estimated by the method of Harborne (1973). Chakraborty et al J. Hortl. Sci. Vol. 11(2): 104-115, 2017 107 Protein Soluble proteins were extracted and estimated by the method of Lowry et al (1951). RESULTS AND DISCUSSION Microscopic observations and identification of PGPR Morphological observations of all the seven PGPR isolates showed that these were rod shaped and Gram +(ve) with the exception of RMK03 isolated from the rhizosphere of Citrus reticulata, which was rod shaped but Gram -(ve). All the bacilli of the group als o produc ed e ndos pore s. Sc a nning ele ctron micrographs also confirmed the structure of bacteria: B. amyloliquefaciens- larger, rod shaped (size ), B. pumilus –rod shaped (size O. anthropi- rod shaped (size 2µm), P. lentimorbus- rod shaped (size 2µm) and B. megaterium larger rod, shaped (size 2µm). Of the seven PGPR isolates, sequences of two isolates, RMK03 and RHS/CL-01, have been deposited with NCBI GenBank database, under accession Nos. KJ 917553.1 and KM 066950.1, for Pseudomonas poae and Bacillus stratosphericus, respectively. These two isola tes , Ps eudomonas poae and Bac illus stratosphericus, isolated from the rhizosphere of Citrus reticulata and C. limoni, respectively, were selected for the present study on the basis of their better response of phosphate solubilization and in vitro antagonism against F. oxysporum, causal agent of the root-rot disease in mandarin. BLAST query of the 18S rDNA sequence of F. oxysporum (RHS/M534) against GenBank database confirmed its identity. The sequence has been deposited with NCBI GenBa nk database as accession no. KF952602. In vitro PGPR activities The seven bacterial strains were tested for different PGPR activity, as described already under Materials and Methods. All these seven isolates produced a clear halo zone of approx. 3cm dia in Pikovskaya’s medium indicating, that, these could solubilize phosphate. Similarly, production of siderophore was confirmed by a change in colour of CAS from blue to yellow around the bacterial inoculum in the petri plate (Table 1). Antagonistic effects of P. poae against F. oxysporum PGPR in disease management in mandarin J. Hortl. Sci. Vol. 11(2): 104-115, 2017 Rhizosphere PGPR isolate Gen Bank Acc. No. Phosphate Characteristic IAA solubilization siderophore production production Citrus reticulata Pseudomonas poae KJ917553 + + + (RM-K-03) Citrus limoni Bacillus stratosphericus KM066950 + + + (CL-RH-01) Camellia sinensis Bacillus amyloliquefaciens JN983127 + + + (TRS 6) Ochrobactrum anthropi + + + (TRS 4) Paenibacillus lentimorbus + + + (TRS 5) Bacillus pumilus JQ765579 + + + (BRHS/T382) Bacillus megaterium JX312687 + + + (TRS 7) Table 1. In vitro PGPR characteristics of bacterial isolates + = Activity present 108 P. poae was tested for its antagonistic effect on F. oxysporum. In paired culture, P. poae inhibited growth of F. oxysporum (Fig. 1). Growth of mandarin seedlings P. poae reduced significant root-rot (Table 2). Disease index value of root-rot in one-year-old mandarin saplings following the treatment with F.oxysporum at 15 days after inoculation was 2.3, whereas, this value fell to 0.6 in plants treated with P. poae and challenge-inoculated with F.oxysporum. Even at 45 days after inoculation, root-rot index value was low in P. poae+F. oxysporum treatment, in comparison to the plants inoculated with F. oxysporum alone. Chlorophyll and phenol content Chakraborty et al J. Hortl. Sci. Vol. 11(2): 104-115, 2017 Fig 1. In vitro antagonistic effect of P. poae against F. oxysporum (A), P. poae+F. oxysporum (B) Growth promotion was studied in terms of increase in plant height and number of leaves, in comparison to the Control. Marked by enhanced growth of one-year-old mandarin seedlings was observed with all the isolates, but, superior growth enhancement was noticed on application of P. poae, B. stratosphericus, O. anthropi and B. pumilus. Per cent increase in plant height and number of leaves in mandarin plants were recorded at 4th and 8th month after application of the seven isolates as soil drench and foliar spray (Fig. 2). Effect of P. poae on root-rot of mandarin Fig 2. Effect of PGPR application on growth of mandarin seedlings at 4 and 8 months. T1- P. poae, T2- B. statosphericus, T3- B. amyloliquefaciens, T4- O. anthropi, T5- P. lentimorbus, T6- B. pumilus and T7- B. megaterium treated. Differences in height determined through t-test between Control and treatments significant at p=0.05.Different letters (a,b) above bars indicate significant difference in t-test at p=0.05 between control and treated. Table 2. Disease index for root rot incidence in one-year old mandarin saplings following tre atment with Pseudomonas poae and pathogen challenge Disease index Da ys of Root inoculated wi th Plant tr eated in ocul at i on F. oxysporum alone with P. poae and pathogen - F. oxysporum 15 2.3±0.145a 0.6±0.145b 30 3.4±0.115a 1.2±0.115b 45 5.9±0.057a 2.8±0.115b Rot index: 0- No symptoms; 1- Small roots turn brownish and start rotting; 2- Leaves start withering and 20-40% of roots turn brown; 3- Leaves withered and 50% of roots affected; 4- Shoot tips also start withering; 60-70% roots affected; 5- Shoots withered with defoliation of lower (withered) leaves, 80% roots affected; 6- Whole plant dies, with upper withered leaves still attached to shoot, roots fully rotted Average of thre e re plicates; Differences between F. oxys porum trea tment a nd P. poae + F. oxy sporum tr e a t m e nt s i gnif i c a nt a t p = 0. 05 ( St ude nt ’s t - test)Difference between F. oxysporum and P. poae + F. oxysporum teatments significant at p = 0.05 (students t- test) whe re superscript (a, b) are differe nt ; where seperate same, difference insignificant Bioc h em ic a l t e st s we re p e rf orme d to evaluate changes brought about by application of the PGPR isola te s. Enhanced a cc umulation of chlorophyll and total phenol was observed in the tre ate d se edling, as compa re d to the Control (Fig. 3). 109 Defense enzymes and protein The activity of defense enzymes and protein on seedling are presented in Table 3. There was considerable increase in the activity of the defense enzymes, viz, PAL, POX (Fig. 4 A-B), CHT and 1,3-GLU (Fig. 5 A-B), with all seven PGPR isolates. Significant increase in the activity of defense enzymes was observed in P. poae + F. oxysporum treatments in comparison to P. poae or F. oxysporum alone, or in Control plants (Fig. 6 A-B; Fig. 7 A-B). Chitinase content increased with increase in period of inoculation in both leaves and roots, whereas, peroxidase activity decreased with increase in the number of hours. Leaves showed a significant drop in peroxidase activity within 96 hours of inoculation. Protein content also increased significantly in plants following bacterial application, compared to that in untreated Control plants. Se ven PGPR isolates, Pseudomonas poae (RMK03), Bacillus stratosphericus (RHS/CL-01), Ochrobactrum anthropi, Paenibacillus lentimorbus, Bacillus pumilus, Bacillus megaterium and Bacillus amyloliquefaciens, were tested on mandarin seedlings in pot culture experiment, for plant growth promoting activity when applied as an aqueous suspension to non- sterile soil with the natural rhizospheric microflora. Of the seven PGPR isolates, sequence of two isolates (RMK03 and RHS/CL-01) has been deposited with NCBI GenBank database under the accession Nos. KJ 917553.1 and KM 066950.1 for Pseudomonas poae and Bacillus stratosphericus, respectively. The sequence of F. oxysporum (RHS/M534) has been PGPR in disease management in mandarin J. Hortl. Sci. Vol. 11(2): 104-115, 2017 Table 3. Protein content in leaf and root upon application of various PGPR isolates Average of three replicates; Differences between Control and treated significant at p=0.05 (Student’s t-test)Difference between control and treated significant at p = 0.05 (student’s t-test) where superscript (a, b) are different ; where superscipt same, difference insignificant Treatment Protein content Leaf Root Untreated (Control) 63.0 ± 0.57a 2.68 ± 0.397a Plant treated with: Pseudomonas poae 136.8 ± 1.18b 8.58 ± 0.881b Bacillus stratosphericus 106.4 ± 1.16b 6.37 ± 0.867b Bacillus amyloliquefaciens 107.7 ± 1.74b 4.04 ± 1.15b Ochrobactrum anthropi 104.2 ± 1.15b 6.29 ± 1.15b Paenibacillus lentimorbus 104.2 ± 0.58b 5.77 ± 1.73b Bacillus pumilus 76.4 ± 1.16b 5.81 ± 0.57b Bacillus megaterium 86.8 ± 1.19b 2.88 ± 0.57b gt = gram of tissue Fig 3. Effect of application of rhizobacterial strains on total chlorophyll content (A) and total phenol content (B) in man- darin leaves. T1- P. poae, T2- B. statosphericus, T3- B. amyloliquefaciens, T4- O. anthropi, T5- P. lentimorbus, T6- B. pumilus and T7- B. megaterium treated; mg /gt =milli- gram/gm tissue; Differences in chlorophyll and phenol con- tent determined through t-test between Control and treat- ment significant at p=0.05.Different letters (a,b) above bars indicate significant difference in t-test at p=0.05 between control and treated. 110 Chakraborty et al J. Hortl. Sci. Vol. 11(2): 104-115, 2017 Fig 4. Activity of phenylalanine ammonia lyase (A) and peroxidase (B) in leaf and root of mandarin following application of PGPR. T1- P. poae, T2- B. statosphericus, T3- B. amyloliquefaciens, T4- O. anthropi, T5- P. lentimorbus, T6- B. pumilus and T7- B. megaterium treated;µgcinnamic acid/gt/min=µgcinnamic acid/gm tissue/min; “O.D/gt/min=”O.D/gm tisuue/ min;Differences in PAL and POX activities determined through t-test between Control and treated leaf / root, significant at p=0.05.Different letters (a,b) above bars indicate significant difference in t-test at p=0.05 between control and treated. Fig 5. Activity of -1,3 glucanase (A) and chitinase (B) in leaf and root of mandarin following application of PGPR. T1- P. poae, T2- B. statosphericus, T3- B. amyloliquefaciens, T4- O. anthropi, T5- P. lentimorbus, T6- B. pumilus and T7- B. megaterium treated; µgglu/gt/min=µgglu/gm tissue/min; µgNacGlu/gt/hour=µgNacglu/gmtissue/hour; Differences in GLU and CHT activity determined through t-test between Control and treated leaf / root, significant at p=0.05.Different letters (a,b) above bars indicate significant difference in t-test at p=0.05 between control and treated. 111 Fig 6. Activities of phenylalanine ammonia lyase (A) and peroxidase (B) in leaf and root of mandarin see d- lings following application of P. poae and F. oxy sporum at 24 and 96 hours after inoculation;µgcinnamic a cid/ gt/min=µgcinnamic a cid/gm tissue/min; “O.D/gt/min=”O.D/gm tisuue /min; Differences in PAL and POX act iv- ity determine d through t-test be tween Control and tre ate d lea f / root, significant at p=0.05.Differ ent letters (a,b) above bars indicate signific ant difference in t-test at p=0. 05 between control and treated. Fig 7. Activity of -1, 3 glucanase (A ) and chitinase (B) in le af and root of mandar in se edlings following application of P. poae a nd F. oxysporum a t 24 and 96 hours a fte r inoculation;µgglu/gt/min=µgglu/gm tissue / min; µgNacGlu/gt/hour=µgNacglu/gm tissue/hour;Difference s in GLU and CHT activity determined through t- test betwe en Control a nd treate d le af / root, signific ant at p=0.05.Diffe rent le tte rs (a, b) above bars indicate significant differe nce in t-te st at p=0.05 betwe en control and tre ated. J. Hortl. Sci. Vol. 11(2): 104-115, 2017 PGPR in disease management in mandarin 112 deposited in NCBI GenBank database under the accession no. KF952602. Markedly enhanced growth of mandarin seedlings was observed with application of PGPR; however, superior growth enhancement was noticed with P. poae, B. stratosphericus, O. anthropi and B. pumilus application. All these isolates solubilized phospha te and produc ed siderophore s in vitro. Application of B. pumilus along with G. mosseae in the rhizosphere of Citrus plants led to an increase in growth of seedlings in terms of increase in plant height and number of leaves (Chakraborty et al, 2011). Acharya et al (2013) reported soil application and foliar spray of PGPR to be effective in promoting overall growth of Sualu plants, as well as a increase in the level of defense-related enzymes, phenols and protein in the leaves of treated plants. Similar results were rec orde d in tea see dlings upon a pplication of Ochrobactrum anthropi (Chakraborty et al, 2009). Nelson (2004) reported significant control of plant pathogens, or direct enhancement of plant development, by PGPR. The author also pointed out recent progress in understanding diversity, colonizing ability, mechanism of action, formulation and application of PGPR in management of sustainable agricultural systems. Lavania et al (2006) reported the ability of Serratia marcescens NBR11213 to promote growth and control foot and root rot disease of betel vine caused by Phytophthora nicotianeae, while, Chakraborty et al (2010) reported positive influence of S. marcescens (TRS-1) on growth of tea seedlings. The ability of B. megaterium to produce IAA (used for lateral-root induction) and to promote growth in Lactuca sativa, alone, or in combination with arbuscular mycorrhiza, was reported by Marulanda-Aguirre et al (2008). Orhan et al (2006) reported plant growth promoting effects of two Bacillus strains, OSU-142 (N2-fixing) and M3 (N2-fixing and phosphate solubilizing). Bacillus M3 strain stimulated plant growth and resulted in significant yield increase in raspberry (cv. Heritage) plants in terms of yield, growth and nutrient composition of leaf. Chakraborty et al (2013) reported that Bacillus amyloliquefaciens, Serratia marcescens and B. pumilus enhanced seedling growth in tea varieties in nursery as well as in the field. Effect of bacteria on metabolism in mandarin seedlings was also determined. To determine if the bacteria could induce systemic resistance in mandarin plants, accumulation of defense- related enzymes and phenolics was studied. Results revealed that the seven PGPR isolates also enhanced the activity of defense-related enzymes peroxidase, chitinase, 1,3-glucanase, phenylalanine ammonia lyase, protein content as well as total phenol. Greater increase in the activity of defense enzymes was observed in P. poae + F. oxysporum treatments, in comparison to P. poae or F. oxysporum-treated or Control plants. Enhanced activity of chlorophyll was also observed. Antibiotic-producing Pseudomonas chlororaphis strains DF190 and PA23, Bacillus cereus strain DFE4, and Bacillus amyloliquefaciens strain DFE16 were tested for elicitating induced systemic resistance (ISR) and direct antibiosis for control of black-leg in canola, caused by the fungal pathogen Leptosphaeria maculans. Bacteria were shown to control the black-leg disease in canola (Ramarathnam et al, 2011). Twenty-one isolates of Pseudomonas fluorescens were isolated and their identity confirmed through various biochemical tests, of which five tested positive for 2,4-DAPG production, with specific primers. The biocontrol potential of these isolates on groundnut stem-rot pathogen (Sclerotium rolfsii) was determined through in vitro dual culture assays. Eight isolates were found effective against S. rolfsii (causing up to 75% inhibition) in the dual culture method. All the five 2,4-DAPG-producing plant growth-promoting rhizobacteria isolates were highly antagonistic to S. rolfsii (Asadhi et al, 2013). Biological control re presents a promising approach for protection of plants against soil-borne pathogens. Fusarium wilt of cucumber, caused by F. oxysporum f. sp. Cucumerinum, has been successfully controlled by B. subtilis SQR 9, both in vitro and in vivo. Wilt incidence reduced significantly by 49%–61% (Cao et al, 2011). Yuan et al (2012) established that volatile compounds produced by B. amyloliquefaciens NJN-6 reduced mycelial growth and germination of spores in F. oxysporum f. sp. cubense in vivo by about 30-40%, and strongly antagonized F. oxysporum in the soil as well. Akhtar et al (2010) studied the effect of Bacillus pumilus, Pseudomonas alcaligenes and Rhizobium sp. on wilt of lentil caused by F. oxysporum f. sp. lentis. Combined application resulted in the greatest increase in plant growth, number of pods, nodulation, root colonization by rhizobacteria, and, reduced wilting. CONCLUSION Application of diffe re nt PGPR to Citr us reticulata resulted in improved growth of the crop, with simultaneous enhancement in activity of defense Chakraborty et al J. Hortl. Sci. Vol. 11(2): 104-115, 2017 113 PGPR in disease management in mandarin enzymes, and higher proteins, phenolics and chlorophyll. Root rot disease was successfully managed using one of the PGPR strains, Pseudomonas poae. These PGPR can be potentially developed as plant growth promoters having disease suppressing ability. ACKNOWLEDGEMENT We wish to thank University Grants Commission (UGC), New Delhi, for providing funds for this research. REFERENCES Akhtar, M.S., Shakeel, U. and Siddiqui, Z.A. 2010. Biocontrol of Fusarium wilt by Bacillus pumilus, Pseudomonas alcaligenes and Rhizobium sp. on lentil. Tur. J. 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