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Gibberellin-producing Serratia nematodiphila PEJ1011 ameliorates low temperature stress in Capsicum annuum L.
European Journal of Soil Biology 68 (2015) 85e93
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Original article
Gibberellin-producing Serratia nematodiphila PEJ1011 ameliorates low temperature stress in Capsicum annuum L. Sang-Mo Kang a, Abdul Latif Khan a, b, Muhammad Waqas a, c, Young-Hyun You d, Muhammad Hamayun e, Gil-Jae Joo f, Raheem Shahzad a, Kyung-Sook Choi g, In-Jung Lee a, * a
School of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea UoN Chair of Oman's Medicinal Plants & Marine Natural Products, University of Nizwa, 616 Nizwa, Oman Department of Agriculture Extension, Buner 19290, Khyber Pakhtunkhwa, Pakistan d School of Life Sciences, Kyungpook National University, Daegu, Republic of Korea e Department of Botany, Abdul Wali Khan University Mardan, Pakistan f Institute of Agricultural Science and Technology, Kyungpook National University, Republic of Korea g Dept. of Agricultural Civil Engineering, Kyungpook National University, Daegu 702-701, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2014 Received in revised form 9 December 2014 Accepted 26 February 2015 Available online 27 February 2015
We studied the effects of plant growth-promoting rhizobacteria (PGPR) on the physio-hormonal attributes of pepper (Capsicum annuum L.) plants grown under low-temperature stress. After initial screening for growth promoting effect on gibberellin (GA) mutant Waito-C rice seeds, the PGPRs were analysed for gibberellins (GA) production through advanced chromatographic and spectroscopic techniques. Among 17 bacterial isolates, a novel isolate PEJ1011 produced bioactive GA4 (8.65 ng ml1) and physiologically inactive GA20 (6.21 ng ml1) and GA9 (1.64 ng ml1). The isolate PEJ1011 was identified as Serratia nematodiphila PEJ1011 using molecular techniques. To further assess it growth promoting effects, S. nematodiphila PEJ1011 was inoculated to pepper plant, where it significantly improved the growth attributes of pepper plants, while mitigated the deleterious effects of low temperature on pepper exposed to low temperature stress of 5 C. It was observed that the inoculated plants grown under normal and low temperature stress contained higher endogenous GA4 contents. To modulate cold stress, the beneficial association of PGPR up-regulated the endogenous ABA levels in pepper plants, while reduced the endogenous jasmonic acid and salicylic acid contents. This up and down regulation of stress hormones contribute to the immediate adaptation of plants exposed to low temperature stress. Current study showed the significance of S. nematodiphila PEJ1011 association to crops grown under adverse climatic conditions, and also reports the GA producing capacity of genus Serratia for the first time. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: PGPR Plant growth promotion Gibberellins Cold stress Hormonal modulation
1. Introduction Low temperatures greatly affect the physio-hormonal attributes of crops and reduce the crop productivity. The adverse effects of cold exposure include the hindrance of normal crop production, and in the worst situation, complete crop failure [20]. Crops may face stresses of varied origin, once or multiple times in their life cycle. However, sometimes a single, mild and brief stress is enough to eliminate the grower's profit. In the current global * Corresponding author. Crop Physiology Laboratory, School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea. E-mail address: [email protected] (I.-J. Lee). http://dx.doi.org/10.1016/j.ejsobi.2015.02.005 1164-5563/© 2015 Elsevier Masson SAS. All rights reserved.
environmental scenario, it is predicted that the various stresses will increase in frequency and intensity [31]. Among the environmental stresses, low temperature stress is known to affect the vegetative and reproductive phases of the plant life cycle. Cold stress can cause flower abortion during reproductive phase due to abscission, sterility of both male and female organs, and eventually reduce yields due to unsuccessful fruit set, which affect all of humanity [75] [85]. Therefore, it is very important to adopt eco-friendly strategies, which can protect crop plants and enhance their tolerance to low temperature stress. Phytohormones are key plant growth regulators and tend to facilitate physiological processes under normal and stressed conditions. The regulation of major phytohormones such as
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gibberellins (GAs), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) bear positive or negative effects on plant growth during cold stress [56]. GAs are considered to be plant growth promoters, while ABA, JA, and SA are grouped as stress-related hormones [48]. Phytohormones are known to interact with each other either in synergistic or antagonistic way, to modulate a particular physiological process under specific conditions and specific time [62]. During cold stress, each phytohormone has a distinguishable regulatory mechanism. ABA and SA are upregulated, as the endogenous levels of both increases in response to low temperature stress. Treatment with ABA and SA increases the cold stress tolerance or acclimation to low temperatures in various crops such as winter wheat, spinach, potato tubers, maize, and rice [35,56]. During cold temperatures, GAs act antagonistically and their endogenous levels, along with that of auxin are down-regulated, causing dwarfism [1,56]. JA decreases the negative effect of chilling stress and improves the postharvest storage of fruits [68,80]. Plant growth-promoting rhizobacteria (PGPRs) are best known for their beneficial effects and amelioration of abiotic stresses in crops. The PGPRs actively colonize the rhizosphere and enhance the yield and resistance of host plants [2,77]. Plant growth promotion by PGPR includes solubilisation, mobilisation, and enhanced uptake of unavailable major plant nutrients, commonly known as biofertilisation and the modulation of phytohormones, or phytostimulation. The suppression of plant pathogens with the help of PGPRs is called biocontrol, and this mechanism involve indirect plant growth promotion [2,52,77]. The phytohormones production capacity of putative PGPRs have been reported for several bacterial spp. like Bacillus cereus, B. macroides, B. pumilus, Azospirillum brasilense, Acinetobacter calcoaceticus and Burkholderia sp. and these PGPRs were extensively studied for their role in plant growth promotion [21,41,42,45]. The phytostimulatory mechanisms include production of GAs, cytokinin, ABA, indole-3-acetic acid (IAA), ethylene, and JA by the PGPRs. The PGPR hormones supplement those produced by the plants and as a result the biomass and yield of the host plant increases [60,77], due to an increase in size, branches and large surface areas of the roots and leaves. An increased in surface area of the leaves allow maximum sunlight absorption and the long and highly branched roots facilitate higher nutrients uptake. GAs are plant growth promoting hormones, which manipulate plant morphology and physiology by enhancing germination, promotes plant height, increase the number of flowers, promote floral organ development, quicken the onset of flowering and increase the leaf surface area [23,36,73,77]. However, there are few reports on PGPRs capable of GA production. Current study hypothesized that PGPR producing bioactive GAs might rescue plant growth under low temperatures by regulating endogenous GAs and related phytohormones, as reduced GAs biosynthesis is one of the major cause of dwarfism and malfunctioning of female sexual organs in crops during periods of low temperatures [69]. Therefore, we analysed the effects of a GA-producing PGPR (S. nematodiphila PEJ1011) on pepper under low temperature regime.
2. Materials and methods 2.1. Isolation of PGPR from pepper The rhizospheric soil samples were obtained from randomly collected pepper plants roots grown in horticulture fields at Kyungpook National University, Republic of Korea as described in Refs. [8] and [34]. In order to remove the bulk soil, pepper plants were vigorously shaken by hand for 7 min, paying attention to the roots integrity. The actual limit for shaking was considered as reached when roots loose or non-adhering soil particles were
completely removed [8]. The tightly adhered (rhizosphere) soil was then separated by using glass beads and then sieved with a 4 mm mesh [34].The rhizospheric soil samples were pooled together and about 10 g of rhizospheric soil was transferred to 250 ml flasks containing 100 ml of sterile Amies solution. The resulting suspensions were serially diluted (104) and 0.1 ml aliquots were grown on tryptic soy/agar (TSA; Merck Co., Germany) for the isolation of bacteria. The isolated bacterial strains were repeatedly inoculated on new petri plates till purification was confirmed and then incubated for 48 h at 30 C. Pure bacterial cultures were inoculated in nutrient broth with conditions as mentioned below and then screened for their plant growth promoting capacity by performing bioassay on dwarf gibberellins inefficient biosynthesis mutant Waito-C rice. The culture filtrate was harvested by centrifugation of culture broth at 5000 g at 4 C for 15 min. Supernatants (50 ml) were lyophilized in freeze dryer for 4 days, and then diluted in 1 ml of autoclaved distilled water. Waito-C seeds were surface sterilized with 2.5% sodium hypochlorite for 30 min, rinsed with autoclaved DDW, and then incubated for 24 h with 6.9 mM-uniconazol [38] to obtain equally germinated seeds. The equal size germinating rice seedlings were then transplanted in autoclaved pots containing 0.8% watereagar medium and kept in a growth chamber (day/night cycle: 14 h; 28 C/10 h; 18 C; relative humidity 60e70%; light intensity 1000 mmm2 s1 using natrium lamps). After attaining the two leaves stage, 10-ml from diluted supernatants of respective bacterial isolates was applied at the seedling apex. After a week, the shoot length, chlorophyll content, shoots’ fresh and dry weights were recorded and compared with ive and þive controls. The Burkholderia sp. KCTC 11096BP strain [42] was used as the positive control while autoclaved DDW was used as a negative control. A novel bacterial isolate PEJ1011, which induced maximum plant growth in rice (data not shown), was selected for further study and re-streaked on fresh TSA medium. For long-term preservation, PEJ1011 was stored in 50% glycerol at 80 C. PEJ1011 was checked for GAs production following standard procedure given in the section below. For GAs production, the PEJ1011 was grown in nutrient broth (NB) media for 3 days at 30 C and 200 rpm. 2.2. Identification of GAs-producing PEJ1011 The isolate PEJ1011 was identified on the basis of partial 16S ribosomal RNA (rRNA) gene sequence. The total DNA was isolated following standard procedures [70]. The 16S rRNA gene was PCR amplified using the 27F primer (50 -AGAGTTTGATC(AC)TGGCTCAG30 ) and 1492R primer (50 -CGG (CT) TACCTTGTTACGACTT-30 ), which were complementary to the 50 end and 30 end of the prokaryotic 16S rRNA, respectively [49]. The BLAST search program was used for nucleotide sequence homology of this bacterial isolate. The closely related sequences with the highest homology, query coverage and the lowest E values were selected and aligned by ClustalW using MEGA version 6.0software. Bacillus thioparans was used as outgroup during neighbour-joining tree generation using the same software. PEJ1011 showed maximum similarity for the subclade of S. nematodiphila (KC122708) and gave 72% bootstrap support when bootstrap 1K replications was used for the statistical support of the nodes in the phylogenetic tree. The 16S rRNA gene sequence of isolate PEJ1011 was submitted to NCBI GenBank under accession number KC819803. 2.3. PEJ1011 mediated plant growth promotion and cold stress resistance We analysed S. nematodiphila PEJ1011 for its plant growth-
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promoting activity and induction of cold stress resistance. A growth chamber experiment was conducted on pepper plants, which comprised both control and PEJ1011 treated pepper plants, with and/or without low temperature stress. Each treatment comprised 27 pepper plants and was replicated thrice. The glassware was properly sterilized before starting the experiment, in order to eliminate chances of contamination, which could affect our results. Pepper seeds were purchased from Seminis Korea Co. (Korea), surface sterilized with NaOCl (5%) for 10 min, rinsed 5 times with autoclaved double-distilled water (DDW), and treated with 20 ppm uniconazole. Seeds were initially sown in petri-plates containing filter paper, moistened with 5 ml DDW and kept for five days in an incubator. Pepper seedlings of almost identical lengths were transferred into plastic pots and moved to growth chamber (day/night cycle: 14/10 h; 28 C/10 h; 25 C; relative humidity 60e70%; light intensity 1000 mE m2 s1 from natrium lamps). The soil substrate in the plastic pots comprised of peat moss (13e18%), perlite (7e11%), coco-peat (63e68%) and 1 zeolite (6e8%) with the macro-nutrients NHþ 4 (e0.09 mg g ); NO3 (0.205 mg g1); P2O5 (0.35 mg g1), and K2O (e0.1 mg g1) [42]. Seedlings selected for inoculation were treated twice with 40 ml of bacterial suspension (grown in NB, 3 days of incubation at 30 C and 200 rpm) in split application, initially at the time of sowing in pots and then at 15 days after sowing. After the second application of the bacterial culture suspension, the pepper plants were left to grow for 1 week. In the low temperature (5 C) treatment, S. nematodiphila PEJ1011-inoculated and control plants were shifted to another growth chamber provided with controlled conditions (5 C; day/night cycle 14/10 h; light intensity 1000 mE m2 s1 from natrium lamps; and relative humidity 60%) for 4 h). Plant growth attributes i.e. shoot length, root length and fresh biomass were measured at the time of harvest, while the chlorophyll content of fully expanded leaves was analysed with the help of a chlorophyll meter (SPAD-502 Minolta, Japan). The plants were harvested, immediately frozen in liquid N and stored at 80 C.
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2.5. Analysis of plant endogenous ABA, JA and SA contents The endogenous ABA contents were extracted from the lyophilized pepper tissues following the method of [66]. The plant extracts were dried and methylated by adding diazomethane for GC-MS SIM (6890N network GC system, and 5973 network mass selective detector; Agilent Technologies, Palo Alto, CA, USA) analysis. For quantification, the Lab-Base (ThermoQuest, Manchester, UK) data system software was used to monitor responses to ions of m/z 162 and 190 for Me-ABA and 166 and 194 for Me-[2H6]-ABA. The endogenous JA contents were extracted following the protocol of [55]. The plant extracts were analysed with GC-MS SIM (6890N network GC system, and 5973 network mass selective detector; Agilent Technologies, Palo Alto, CA, USA). To enhance the sensitivity of the method, spectra were recorded in the selected ion mode, i.e. in case of JA determination, monitoring the fragment ion at m/z ¼ 83 corresponding to the base peaks of JA and [9, 10-2H2]-9, 10-dihydro-JA. The amount of endogenous JA was calculated from the peak areas of endogenous JA in comparison with the corresponding standards. Three replicates per treatment were used for the determination of JA. The SA contents were extracted and quantified, as described previously by Ref. [71]. The freeze-dried leaf samples were grounded to powder form, and 0.1 g was sequentially extracted with 90 and 100% methanol by centrifuging at 10,000 g. The combined methanol extracts were vacuum dried. Dry pellets were resuspended in 2.5 ml of 5% trichloroacetic acid and the supernatant was partitioned with ethyl acetate:cyclopentane:isopropanol (100:99:1, v/v). The top organic layer containing free SA was transferred to a 4 ml vial and dried with nitrogen gas. The dry SA was again suspended in 1 ml of 70% methanol. High performance liquid chromatography (HPLC) analyses were carried out on Shimadzu having a fluorescence detector (Shimdzu RF-10AXL, excitation and emission, 305 and 365 nm, respectively) fitted with a C18 reverse-phase HPLC column (HP hypersil ODS, particle size 5 mm, pore size 120 Å Waters). The flow rate was 1.0 ml min1.
2.4. Determination of pepper and S. nematodiphila PEJ1011 GAs 2.6. Statistical analysis The lyophilized plant samples were used for extraction and quantification of endogenous GAs. For GA analysis, 0.5 g lyophilized sample of pepper plant was used each time. For bacterial GA analysis, S. nematodiphila PEJ1011 inoculated NB media was partitioned (5000 g at 4 C for 15 min), and 50 ml of supernatant was used for analysing bacterial GAs. An established protocol for extraction and quantification of GAs was adopted, following [51] and [79]. For GA analysis, a gas chromatograph (Hewlett-Packard 6890, 5973N mass selective detector) with HA-1 capillary column (30 m 0.25 mm i.d., 0.25 mm film thickness) was programmed for a 1 min hold at 60 C, then a rise of 15 C min1 to 200 C, followed by 5 C min1 to 285 C. Helium carrier gas was kept at a head pressure of 30 kPa. The GC was directly connected to a mass selective detector with an interface and source temperature of 280 C and ionizing voltage of 70 eV, with dwell time of 100 ms. Full scan mode (the first trial) and three major ions of supplemented [2H2]GAs internal standards (the second trial) and the endogenous GAs were monitored simultaneously (standard GAs were purchased from Prof. Lewis N. Mander, Australian National University, Canberra, Australia). The endogenous GA contents of GA4, GA9, and GA20 were calculated from the peak area ratios of 284/286, 298/300, and 300/360, respectively. The data was calculated in nanograms per gram dry weight of plant samples and per ml of bacterial culture broth and each analysis was repeated two times.
To know the ameliorative effect of PEJ1011 symbiotic association with host plants under low temperature stress, the experiments was conducted in completely randomized design. For identification of the treatment and effects significances, analysis of variance (2Way ANOVA; Fisher's LSD and Bonferroni post-hoc test; P 0.05) was carried out on the data using Graph Pad Prism software (version 5.0, San Diego, California USA) and graphs were sketched with sigmaplot (version 12). The experiment was repeated three times. 3. Results and discussion 3.1. PGPR isolation, GAs analysis and growth conditions In current study, we collected 17 bacterial isolates from the rhizosphere of randomly selected pepper plants and subsequently screened for plant growth promoting capacity on GA mutant WaitoC rice (for detail see materials and methods). On the basis of screening results a novel bacterial isolate PEJ1011 was analysed for GAs production ability and found to produce three different types of GAs (GA4, GA20 and GA9). It was observed that PEJ1011 produced much higher contents of bioactive GA4 (8.65 ± 0.95 ng ml1), as compared to inactive GA20 (6.21 ± 1.54 ng ml1) and GA9 (1.64 ± 1.11 ng ml1). The GAs production potential of PGPRs have
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analysed IAA, ABA, and GA productions in Variovorax paradoxus previously isolated by Ref. [12]. The same bacterial isolate was reported to produce IAA, indole-3-carboxylic acid, and indole-3-lactic acid, but ABA or GA was not detected. In current study, PEJ1011 was grown in three different low temperature (2 C, 5 C, 10 C) and optimal temperature (30 C) regimes for 48 h to understand its growth rate during host-plant symbiotic association under control climate chamber experiment. The optical density of the bacterial mass growth was periodically analysed at 600 nm (OD 600), and almost log-linear growth was found from 4 h to 36 h of incubation, after which the mass was constant (stationary phase) for up to 48 h (Fig. 1) at 5 C, 10 C and 30 C. As shown in (Fig. 1) PEJ1011 was unable to grow at 2 C, however attained significantly higher growth rate at 30 C as compared to 10 C and 5 C. 3.2. Identification of bacterial isolate PEJ1011 Fig. 1. Growth rates of PEJ1011 at four different temperature regimes. Values in the form of different symbolic structures (i.e. to represent four temperature regimes) at regular intervals are means of (n ¼ 3). (*) mark indicates the significances of treatment effects in that particular growth conditions (temperature).
been reported previously [4,16], though bacterial strains were mostly studied for their IAA production [77], and rarely for GA production [15]. The GA biosynthesis pathway in fungi is well studied [3,25,46,78], but little is known about GA biosynthesis mechanism of GAs producing PGPRs [15,58]. Various bacterial spp. such as Rhizobium phaseoli, Azospirillum lipoferum, A. brasilense, Acetobacter diazotrophicus, Herbaspirillum seropedicae, Bacillus licheniformis, B. macroides, B. cereus, B. pumilus, B. amyloliquefaciens and Bradyrhizobium japonicum have been reported to produce bioactive and inactive GAs [4,10,16,36,39,41,53]. In most of the studied GA producing bacteria, the bioactive GAs include GA1, GA3, GA4, or GA7 [10,36,41,53]. PEJ1011 produced bioactive GA4 (the precursor for GA1 and GA3), and the same has also been reported in B. licheniformis, B. cereus, B. macroides, and B. pumilus [36,41]. The inactive GA9 and GA20 have been detected in B. cereus, B. licheniformis, B. macroides, and B. pumilus [36,41]. Occurrence of GA9 and GA20 in the PEJ1011 confirm pertinent previous reports. Current results are also in agreement with those of [40]; who
After confirming the GA producing potential of bacterial isolate PEJ1011, the isolate was phylogenetically assigned by sequencing of the 16S rRNA gene. BLAST search of the 16S rDNA sequence obtained from isolate PEJ1011 showed a high similarity (99%) to different S. nematodiphila strains. Four of these strains together with 9 reference strains of related species were used to generate a phylogenetic tree (Fig. 2). Based on BLAST search results and phylogenetic analysis the PEJ1011 was identified as a new strain of S. nematodiphila. Previous researches revealed that S. nematodiphila and other members of the genus Serratia have been isolated from diverse sources, including animals [84], forest soil [24], limonitic crust [63], and as plant endophytes [19]. S. nematodiphila isolated from soil and plants has been reported to produce various plant growth-promoting substances (PGPS). For instance, S. nematodiphila isolated from the forest soil was found to produce IAA, H2S, siderophores, and solubilised phosphate [24]. The same strain is capable to utilize citrate; reduce nitrate, oxidase and catalase; and hydrolyse esculin and casein. Endophytic S. nematodiphila isolated from Solanum nigrum L. growing in metal-polluted soil also possessed PGPSs, including 1-aminocyclopropane-1-carboxylic deaminase, IAA, siderophores and solubilised phosphate [19]. All the PGPSs produced by bacteria play a variable role in plant growth promotion and thus
Fig. 2. Identification of the bacterial isolate PEJ1011, by phylogenetic analysis. The neighbour-joining tree was constructed from 16S rDNA region sequences of the strains most similar to PEJ1011 using MEGA6 software. Accession numbers of all strains are written to their next.
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make a very complex phenomenon [24]. In earlier studies, authors linked plant growth promotion to a much better phosphorous and iron nutrition, possibly triggered by phosphate solubilization and siderophore production capacity of PGPR [24,26,50]. The plants were protected through biological means due to HCN [24], although IAA fabrication capacity of various bacteria was thought to be the main reason for plant growth and development [30]. 3.3. PEJ1011 and plant hormonal modulation during low temperature stress Plant inoculation with effective bacterial strains in a proper manner has gained considerable attention for sustainable and increased agriculture production [9]. The PGPRs enhance soil nutrients uptake in three different ways, i.e. solubilisation, mobilization and transformation by cycling organic and inorganic nutrients [24]. The interaction of PGPRs with a host plant is thus considered beneficial for plant health and soil quality [6,24]. However, another recognized important aspect of PGPRs is their phytohormone production or degradation capacity [65]. The widespread occurrence of phytohormones has been reported in many PGPRs belonging to several classes [28,30]. Most often, PGPR may produce one but rarely multiple types of phytohormones and their analogues [28]. In this study, GA-producing S. nematodiphila PEJ1011 were evaluated for pepper plant growth promotion and low temperature resistance (Table 1; Fig. 3a and b). The in vivo application of S. nematodiphila PEJ1011 under normal temperature in pepper markedly increased plant growth attributes as compared to control (Table 1). Shoot and root lengths were significantly increased by 55.8% and 12.9% respectively, than the control. Important plant growth parameters i.e. chlorophyll content in leaves and its effect in terms of increased fresh biomass, were 1.45 and 1.08 fold higher than control, under normal temperature. Randomly selected plants from both inoculated and control treatments grown in plastic pots, were subjected to low temperature 5 C stress for 4 h. The exposure to low temperature stress did
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Table 1 Growth promoting effects of PEJ1011 on shoot length (SL), root length (RL), fresh weight (FW), and chlorophyll contents (CC) of peppers grown under normal condition and cold shock stress. Treatment
SL (cm)
NT PEJ1011 CT CT þ PEJ1011
15.6 24.3 15.2 23.8
± ± ± ±
1.21b 0.57a 1.37b 0.56a
RL (cm) 11.6 13.1 11.3 13.2
± ± ± ±
1.06b 0.54a 1.01b 0.77a
FW (g) 2.25 3.27 2.21 3.19
± ± ± ±
CC (SPAD) 0.13b 0.06a 0.09b 0.09a
31.5 34.0 29.12 30.1
± ± ± ±
1.0b 1.2a 1.2b 1.1b
NT ¼ Normal temperature, CT ¼ Cold temperature/cold shock stress for 4 h at 5 C, SPAD ¼ soileplant analysis development unit for measuring leaf chlorophyll content. Each value in the columns represent mean ± standard deviation (n ¼ 3). Mean values in the same column denoted by different letter are significantly different at (P 0.05).
not affect growth characteristics of plants inoculated with S. nematodiphila PEJ1011 and maintain vigour and no visible sign of wilting or stunting was found. The shoot length, root length, and fresh weight was significantly increased by 1.57, 1.17 and 1.44 fold respectively, as compared to control, except chlorophyll contents under low temperature stress (Table 1). The plant growthpromoting traits of S. nematodiphila PEJ1011, similar to those of the other PGPRs in the current study, are in agreement with those of previous studies [19,24,59,74,82]. Previous studies have concluded that plants can achieve better growth under normal and stressful conditions in the presence of PGPRs, because of their ability to produce different phytohormones including GAs, IAA, cytokinin, ABA, and JA; nutrient solubilization; softening of the root membrane; biological nitrogen fixation; production of enzymes needed for the modulation of plant hormones and protection against microbial pathogens. Phytohormone-producing PGPRs modulate plant endogenous hormonal levels, by utilizing either their own biosynthetic pathway or further modifying the precursors released through root exudates [28]. The interaction of phytohormones for mediating plant physiological or phenotypic characteristics is a very complex process involving a network of signalling, cross-talking, and feedback
Fig. 3. Plant growth-promoting effect of Serratia nematodiphila PEJ1011. Gibberellic acid (GA4)-producing S. nematodiphila PEJ1011 grown in liquid culture was twice applied to the root zone of pepper plants. GA4 has a long distance effect and promotes the growth of other organs [14]. The pepper plants without inoculation (control, a) and with inoculation of S. nematodiphila PEJ1011 (b) were grown for 21 days. Twenty-seven randomly selected plants replicated three times from both treatments were exposed to cold shock stress for 4 h at 5 C. (a) A representative leaf sample from the control plant after exposure to shock stress. (b) A representative leaf sample from S. nematodiphila PEJ1011-inoculated plants is also shown for comparison.
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Fig. 4. In planta hormonal modulation ability of Serratia nematodiphila PEJ1011 in pepper plants under normal condition and cold shock stress. Gibberellic acid (GA4; a), abscisic acid (ABA; b), jasmonic acid (JA; c), and salicylic acid (SA; d). NT ¼ Pepper plants grown at normal temperatures without inoculation of S. nematodiphila PEJ1011. CT ¼ Pepper plants grown without inoculation of S. nematodiphila PEJ1011 and then subjected to cold temperatures/cold shock stress for 4 h at 5 C. PEJ1011 ¼ Application of S. nematodiphila PEJ1011 to pepper plants grown under normal temperatures. CT þ PEJ1011 ¼ Application of S. nematodiphila PEJ1011 to pepper plants and then subjected to cold temperatures/cold shock stress for 4 h at 5 C. Column bars sketched with error bars represents mean values ± standard deviation (n ¼ 3). In all figures, different letter(s) and (*) mark above column bars shows significant differences at (LSD; Bonferroni; P 0.05). In pairwise/group-wise comparison of normal and cold stress, (*) mark indicates the significances of treatment effects in that particular growth conditions.
mechanisms [7,76]. Most of the rhizobacteria modulate more than one endogenous plant hormone initially by affecting their levels in the roots and then establishing a long distance-signalling mechanism up to the shoot [11,27,28]. In view of this important aspect, the modulation of plant growth-regulating GAs and the defence hormones ABA, JA and SA was investigated in response to GAproducing S. nematodiphila PEJ1011 under low temperature stress. Since S. nematodiphila PEJ1011 was found to produce bioactive gibberellins GA4 (which is further converted into GA3 and GA1 [78], the response of pepper endogenous GA4 under normal condition and low temperature stress was analysed. S. nematodiphila PEJ1011 significantly increased the pepper endogenous GA4 content under normal condition and low temperature stress (Fig. 4a). The endogenous GA4 content of S. nematodiphila PEJ1011-treated pepper plants under normal condition and low temperature stress was increased by 78.40% and 85.58%, respectively. However, no significant differences were recorded for S. nematodiphila PEJ1011treated pepper plants grown under low temperature stress and control in normal growth condition. Different growth substances producing PGPRs have been frequently reported to have stimulatory effects on the endogenous phytohormone contents of plants [28]. The effect is usually reported to be more pronounced and synergistic for the corresponding phytohormone produced by the PGPRs [5,37,42e45]. Refs. [43,44] applied three different PGPR strains, Burkholderia cepacia SE4, Promicromonospora sp. SE188, and Acinetobacter calcoaceticus SE370, and found they significantly increased the endogenous GA4 content of cucumbers. The same effect was also reported for IAA- and cytokinin-producing PGPRs of the genera Pseudomonas, Bacillus, and Azospirillum for the
phytostimulation of the corresponding hormones [37]. However, during in same experiment, the higher IAA to cytokinin ratio in PGPRs played an antagonistic role in increasing the cytokinin contents of wheat plants. Ref. [13] treated lodgepole pine trees with IAA-producing PGPRs and found the same effect of increased IAA concentration in roots. It is a well established fact that PGPRs producing particular hormone(s) may affect other phytohormones either in a synergistic or antagonistic manner. The effect may be due to the modulation of the plant hormone corresponding to the PGPR hormone, which further regulates other hormones accordingly [28,43,44]. Otherwise, irrespective of the mechanism, PGPR may affect all or particular phytohormones even if it does not produce any hormones [18,67]. Therefore, we also studied major endogenous phytohormones of pepper under control and low temperature stress. The endogenous contents of drought-mediating hormone ABA was significantly enhanced (111.39%) in the control plants than in the S. nematodiphila PEJ1011-treated pepper plants (Fig. 4b). The decrease in ABA seems to be reasonable under normal condition, and it may be attributed to the high level of GA4 [81]. Both hormones act antagonistically under normal growth conditions when GAs are present in high amounts [32,81]. However, an opposite trend was observed for ABA regulation in the bacterium inoculated plants under low temperature stress, as the endogenous contents of ABA increased by 181% in S. nematodiphila PEJ1011-treated pepper plants compared to the control. An increased in ABA levels during drought, salinity, and low temperatures helped plants to adapt to these situations [83]. As a consequence, the expression of several genes that are known to respond to drought and cold stress was
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induced [72,83]. Nevertheless, the mechanism of ABA is not very clear, as some genes are expressed during cold or drought stress but never respond to exogenous ABA application [83]. Here the role of S. nematodiphila PEJ1011 application to pepper plants was appreciable under low temperature stress when the GA and ABA levels were compared to the control. The application of GA-producing PGPRs also increased the endogenous GA4 level to counteract the growth inhibitory effect of low temperature stress and might be tried synergistically with ABA content to render normal growth [56]. In comparison, the proportion of GA4 to ABA is very low in control plants under low temperature stress. Our results also confirm the previous observations that PGPRs have been reported to increase the ABA level during various kinds of abiotic stresses in different crops, including sunflowers, Arabidopsis, and maize [18,21,22,33]. JA plays an important role during biotic stress imposed by necrotrophic pathogens and herbivorous insects and abiotic cold stress [17,29,47,80]. The application of S. nematodiphila PEJ1011 caused significantly higher reductions in the level of JA in pepper plants than that in the controls under normal condition (1.49 ± 0.09b vs. 2.39 ± 0.12a) and low temperature stress (0.21 ± 0.13c vs. 1.35 ± 0.10b) (Fig. 4c). The level of JA was insignificant in pepper plants treated with S. nematodiphila PEJ1011 under normal growth conditions and control low temperature stress. Similar result was reported by Ref. [47]; where cold stress (both chilling and frost stress) decreased the JA level in short stature Arabis alpina. The short stature A. alpina was considered to be a model plant that adapted to extreme low temperatures and snow coverage, and hence, was added to the experimental group with two other ecotypes of the same species. In addition [48], showed the same trend on exposure to cold stress in winter and spring wheat cultivars, where the JA levels in the leaves and crowns reduced during the initial alarming phase of the cold response. Another plant defence hormone, SA, regulates various plant physiological functions and provides protection from microbial pathogenic infections [57]. The abiotic stress-mediating role of SA in salinity, drought, and low temperatures has been documented. The application of SA in low quantities reverts the cold stress effect in maize, rice, potatoes, and wheat; however, treatment with high amounts of SA has shown to induce the opposite effect [48,56]. In several experiments, a high amount of SA accumulation has been reported in different plant species in response to cold stress. Such a response of SA for a short time has been correlated with its positive role in cold stress resistance, and vice versa. In this study, pepper plants inoculated with S. nematodiphila PEJ1011 had significantly lesser SA during normal condition (9.50 ± 0.21c) and low temperature stress (10.63 ± 0.22b) than that in the controls (11.60 ± 0.22b and 13.32 ± 0.36a, respectively) (Fig. 4d). No significant difference was noted in the SA level between the control pepper plants grown under normal condition and S. nematodiphila PEJ1011-treated plants under low temperature stress. Overall, the current findings are very much in agreement with those of [48,64] and [61]. They concluded that plants with better cold stress resistance increased their ABA level and downregulated SA and JA levels in the initial hours of cold stress. This study, along with the aforementioned reports, suggests that ABA is antagonistic towards SA and JA during the initial cold period. This unique adaptation of pepper plants during low temperature stress is attributed to the presence of S. nematodiphila PEJ1011. 4. Conclusions The present results concluded that the application of novel PGPR strain like S. nematodiphila PEJ1011 might be a good alternative to synthetic fertilizer. The growth promoting potentials of
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this strain was also supported by the enhanced plant growth characteristics under normal and cold stress conditions. It was revealed that this increase in growth of inoculated plants was attained through relative increase in growth hormone like GA4 under normal and cold stress condition, and vice versa in case of stress hormones such as JA and SA. The S. nematodiphila PEJ1011 inoculation extended stress mitigation as shown by the increased level of stress responsive ABA in cold and decreased level in normal condition as compared to non-inoculated control plants. Further studies of S. nematodiphila PEJ1011 symbiotic association with host and other crops in open field conditions and exploration of its potentials at molecular level is recommended. Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A1004918). References [1] P. Achard, F. Gong, S. Cheminant, M. Alioua, P. Hedden, P. Genschik, The coldinducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism, Plant Cell 20 (2008) 2117e2129. [2] M. Ahemad, M. Kibret, Mechanisms and applications of plant growth promoting rhizobacteria: current perspective, J. King Saud Univ. Sci. 26 (2014) 1e20. [3] S. Albermann, T. Elter, A. Teubner, W. Krischke, T. Hirth, B. Tudzynski, Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi, Appl. Microbiol. Biotechnol. 97 (2013) 7779e7790. [4] R. Atzhorn, A. Crozier, C.T. Wheeler, G. Sandberg, Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots, Planta 175 (1988) 532e538. [5] O.Z. Barazani, J. Friedman, Is IAA the major root growth factor secreted from plant-growth-mediating bacteria? J. Chem. Ecol. 25 (1999) 2397e2406. n, C. Azco n-Aguilar, Microbial co-operation in the [6] J.M. Barea, M.J. Pozo, R. Azco rhizosphere, J. Exp. Bot. 56 (2005) 1761e1778. [7] R. Bari, J.D.G. Jones, Role of plant hormones in plant defence responses, Plant Mol. Biol. 69 (2009) 473e488. [8] C.D.C. Barillot, C.O. Sarde, V. Bert, E. Tarnaud, N. Cochet, A standardized method for the sampling of rhizosphere and rhizoplan soil bacteria associated to a herbaceous root system, Ann. Microbiol. 63 (2013) 471e476. [9] Y. Bashan, L.E. de-Bashan, S.R. Prabhu, J.P. Hernandez, Advances in plant growth-promoting bacterial inoculants technology: formulations and practical perspectives (1998e2013), Plant Soil (2014), http://dx.doi.org/10.1007/ s11104-013-1956-x. [10] F. Bastian, A. Cohen, P. Piccoli, V. Luna, R. Baraldi, R. Bottini, Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically defined media, Plant Growth Regul. 24 (1998) 7e11. [11] A.A. Belimov, I.C. Dodd, N. Hontzeas, J.C. Theobald, V.I. Safronova, W.J. Davies, Rhizosphere bacteria containing ACC deaminase increase yield of plants grown in drying soil via both local and systemic hormone signalling, New Phytol. 181 (2009) 413e423. [12] A.A. Belimov, N. Hontzeas, V.I. Safronova, S.V. Demchinskaya, G. Piluzza, S. Bullitta, B.R. Glick, Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.), Soil Biol. Biochem. 37 (2005) 241e250. [13] E. Bent, S. Tuzun, C.P. Chanway, S. Enebak, Alterations in plant growth and in root hormone levels of lodgepole pines inoculated with rhizobacteria, Can. J. Microbiol. (2001) 47793e47800. [14] H. Bidadi, K. Matsuoka, K. Sage-Ono, J. Fukushima, W. Pitaksaringkarn, M. Asahina, S. Yamaguchi, S. Sawa, H. Fukuda, Y. Matsubayashi, M. Ono, S. Satoh, CLE6 expression recovers gibberellin deficiency to promote shoot growth in Arabidopsis, Plant J. (2014), http://dx.doi.org/10.1111/tpj.12475. [15] R. Bottini, F. Cass an, P. Piccoli, Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase, Appl. Microbiol. Biotechnol. 65 (2004) 497e503. [16] R. Bottini, M. Fulchieri, D. Pearce, R.P. Pharis, Identification of gibberellins A1, A3, and iso-A3 in cultures of Azospirillum lipoferum, Plant Physiol. 90 (1989) 45e47. [17] L.C. Carvalhais, P.G. Dennis, D.V. Badri, G.W. Tyson, J.M. Vivanco, P.M. Schenk, Activation of the jasmonic acid plant defence pathway alters the composition of rhizosphere bacterial communities, PLoS One 8 (2013) e56457. [18] P. Castillo, M. Escalante, M. Gallardo, S. Alemano, G. Abdala, Effects of bacterial single inoculation and co-inoculation on growth and phytohormone production of sunflower seedlings under water stress, Acta Physiol. Plant. 35 (2013) 2299e2309.
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Original article
Gibberellin-producing Serratia nematodiphila PEJ1011 ameliorates low temperature stress in Capsicum annuum L. Sang-Mo Kang a, Abdul Latif Khan a, b, Muhammad Waqas a, c, Young-Hyun You d, Muhammad Hamayun e, Gil-Jae Joo f, Raheem Shahzad a, Kyung-Sook Choi g, In-Jung Lee a, * a
School of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea UoN Chair of Oman's Medicinal Plants & Marine Natural Products, University of Nizwa, 616 Nizwa, Oman Department of Agriculture Extension, Buner 19290, Khyber Pakhtunkhwa, Pakistan d School of Life Sciences, Kyungpook National University, Daegu, Republic of Korea e Department of Botany, Abdul Wali Khan University Mardan, Pakistan f Institute of Agricultural Science and Technology, Kyungpook National University, Republic of Korea g Dept. of Agricultural Civil Engineering, Kyungpook National University, Daegu 702-701, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2014 Received in revised form 9 December 2014 Accepted 26 February 2015 Available online 27 February 2015
We studied the effects of plant growth-promoting rhizobacteria (PGPR) on the physio-hormonal attributes of pepper (Capsicum annuum L.) plants grown under low-temperature stress. After initial screening for growth promoting effect on gibberellin (GA) mutant Waito-C rice seeds, the PGPRs were analysed for gibberellins (GA) production through advanced chromatographic and spectroscopic techniques. Among 17 bacterial isolates, a novel isolate PEJ1011 produced bioactive GA4 (8.65 ng ml1) and physiologically inactive GA20 (6.21 ng ml1) and GA9 (1.64 ng ml1). The isolate PEJ1011 was identified as Serratia nematodiphila PEJ1011 using molecular techniques. To further assess it growth promoting effects, S. nematodiphila PEJ1011 was inoculated to pepper plant, where it significantly improved the growth attributes of pepper plants, while mitigated the deleterious effects of low temperature on pepper exposed to low temperature stress of 5 C. It was observed that the inoculated plants grown under normal and low temperature stress contained higher endogenous GA4 contents. To modulate cold stress, the beneficial association of PGPR up-regulated the endogenous ABA levels in pepper plants, while reduced the endogenous jasmonic acid and salicylic acid contents. This up and down regulation of stress hormones contribute to the immediate adaptation of plants exposed to low temperature stress. Current study showed the significance of S. nematodiphila PEJ1011 association to crops grown under adverse climatic conditions, and also reports the GA producing capacity of genus Serratia for the first time. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: PGPR Plant growth promotion Gibberellins Cold stress Hormonal modulation
1. Introduction Low temperatures greatly affect the physio-hormonal attributes of crops and reduce the crop productivity. The adverse effects of cold exposure include the hindrance of normal crop production, and in the worst situation, complete crop failure [20]. Crops may face stresses of varied origin, once or multiple times in their life cycle. However, sometimes a single, mild and brief stress is enough to eliminate the grower's profit. In the current global * Corresponding author. Crop Physiology Laboratory, School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea. E-mail address: [email protected] (I.-J. Lee). http://dx.doi.org/10.1016/j.ejsobi.2015.02.005 1164-5563/© 2015 Elsevier Masson SAS. All rights reserved.
environmental scenario, it is predicted that the various stresses will increase in frequency and intensity [31]. Among the environmental stresses, low temperature stress is known to affect the vegetative and reproductive phases of the plant life cycle. Cold stress can cause flower abortion during reproductive phase due to abscission, sterility of both male and female organs, and eventually reduce yields due to unsuccessful fruit set, which affect all of humanity [75] [85]. Therefore, it is very important to adopt eco-friendly strategies, which can protect crop plants and enhance their tolerance to low temperature stress. Phytohormones are key plant growth regulators and tend to facilitate physiological processes under normal and stressed conditions. The regulation of major phytohormones such as
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gibberellins (GAs), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) bear positive or negative effects on plant growth during cold stress [56]. GAs are considered to be plant growth promoters, while ABA, JA, and SA are grouped as stress-related hormones [48]. Phytohormones are known to interact with each other either in synergistic or antagonistic way, to modulate a particular physiological process under specific conditions and specific time [62]. During cold stress, each phytohormone has a distinguishable regulatory mechanism. ABA and SA are upregulated, as the endogenous levels of both increases in response to low temperature stress. Treatment with ABA and SA increases the cold stress tolerance or acclimation to low temperatures in various crops such as winter wheat, spinach, potato tubers, maize, and rice [35,56]. During cold temperatures, GAs act antagonistically and their endogenous levels, along with that of auxin are down-regulated, causing dwarfism [1,56]. JA decreases the negative effect of chilling stress and improves the postharvest storage of fruits [68,80]. Plant growth-promoting rhizobacteria (PGPRs) are best known for their beneficial effects and amelioration of abiotic stresses in crops. The PGPRs actively colonize the rhizosphere and enhance the yield and resistance of host plants [2,77]. Plant growth promotion by PGPR includes solubilisation, mobilisation, and enhanced uptake of unavailable major plant nutrients, commonly known as biofertilisation and the modulation of phytohormones, or phytostimulation. The suppression of plant pathogens with the help of PGPRs is called biocontrol, and this mechanism involve indirect plant growth promotion [2,52,77]. The phytohormones production capacity of putative PGPRs have been reported for several bacterial spp. like Bacillus cereus, B. macroides, B. pumilus, Azospirillum brasilense, Acinetobacter calcoaceticus and Burkholderia sp. and these PGPRs were extensively studied for their role in plant growth promotion [21,41,42,45]. The phytostimulatory mechanisms include production of GAs, cytokinin, ABA, indole-3-acetic acid (IAA), ethylene, and JA by the PGPRs. The PGPR hormones supplement those produced by the plants and as a result the biomass and yield of the host plant increases [60,77], due to an increase in size, branches and large surface areas of the roots and leaves. An increased in surface area of the leaves allow maximum sunlight absorption and the long and highly branched roots facilitate higher nutrients uptake. GAs are plant growth promoting hormones, which manipulate plant morphology and physiology by enhancing germination, promotes plant height, increase the number of flowers, promote floral organ development, quicken the onset of flowering and increase the leaf surface area [23,36,73,77]. However, there are few reports on PGPRs capable of GA production. Current study hypothesized that PGPR producing bioactive GAs might rescue plant growth under low temperatures by regulating endogenous GAs and related phytohormones, as reduced GAs biosynthesis is one of the major cause of dwarfism and malfunctioning of female sexual organs in crops during periods of low temperatures [69]. Therefore, we analysed the effects of a GA-producing PGPR (S. nematodiphila PEJ1011) on pepper under low temperature regime.
2. Materials and methods 2.1. Isolation of PGPR from pepper The rhizospheric soil samples were obtained from randomly collected pepper plants roots grown in horticulture fields at Kyungpook National University, Republic of Korea as described in Refs. [8] and [34]. In order to remove the bulk soil, pepper plants were vigorously shaken by hand for 7 min, paying attention to the roots integrity. The actual limit for shaking was considered as reached when roots loose or non-adhering soil particles were
completely removed [8]. The tightly adhered (rhizosphere) soil was then separated by using glass beads and then sieved with a 4 mm mesh [34].The rhizospheric soil samples were pooled together and about 10 g of rhizospheric soil was transferred to 250 ml flasks containing 100 ml of sterile Amies solution. The resulting suspensions were serially diluted (104) and 0.1 ml aliquots were grown on tryptic soy/agar (TSA; Merck Co., Germany) for the isolation of bacteria. The isolated bacterial strains were repeatedly inoculated on new petri plates till purification was confirmed and then incubated for 48 h at 30 C. Pure bacterial cultures were inoculated in nutrient broth with conditions as mentioned below and then screened for their plant growth promoting capacity by performing bioassay on dwarf gibberellins inefficient biosynthesis mutant Waito-C rice. The culture filtrate was harvested by centrifugation of culture broth at 5000 g at 4 C for 15 min. Supernatants (50 ml) were lyophilized in freeze dryer for 4 days, and then diluted in 1 ml of autoclaved distilled water. Waito-C seeds were surface sterilized with 2.5% sodium hypochlorite for 30 min, rinsed with autoclaved DDW, and then incubated for 24 h with 6.9 mM-uniconazol [38] to obtain equally germinated seeds. The equal size germinating rice seedlings were then transplanted in autoclaved pots containing 0.8% watereagar medium and kept in a growth chamber (day/night cycle: 14 h; 28 C/10 h; 18 C; relative humidity 60e70%; light intensity 1000 mmm2 s1 using natrium lamps). After attaining the two leaves stage, 10-ml from diluted supernatants of respective bacterial isolates was applied at the seedling apex. After a week, the shoot length, chlorophyll content, shoots’ fresh and dry weights were recorded and compared with ive and þive controls. The Burkholderia sp. KCTC 11096BP strain [42] was used as the positive control while autoclaved DDW was used as a negative control. A novel bacterial isolate PEJ1011, which induced maximum plant growth in rice (data not shown), was selected for further study and re-streaked on fresh TSA medium. For long-term preservation, PEJ1011 was stored in 50% glycerol at 80 C. PEJ1011 was checked for GAs production following standard procedure given in the section below. For GAs production, the PEJ1011 was grown in nutrient broth (NB) media for 3 days at 30 C and 200 rpm. 2.2. Identification of GAs-producing PEJ1011 The isolate PEJ1011 was identified on the basis of partial 16S ribosomal RNA (rRNA) gene sequence. The total DNA was isolated following standard procedures [70]. The 16S rRNA gene was PCR amplified using the 27F primer (50 -AGAGTTTGATC(AC)TGGCTCAG30 ) and 1492R primer (50 -CGG (CT) TACCTTGTTACGACTT-30 ), which were complementary to the 50 end and 30 end of the prokaryotic 16S rRNA, respectively [49]. The BLAST search program was used for nucleotide sequence homology of this bacterial isolate. The closely related sequences with the highest homology, query coverage and the lowest E values were selected and aligned by ClustalW using MEGA version 6.0software. Bacillus thioparans was used as outgroup during neighbour-joining tree generation using the same software. PEJ1011 showed maximum similarity for the subclade of S. nematodiphila (KC122708) and gave 72% bootstrap support when bootstrap 1K replications was used for the statistical support of the nodes in the phylogenetic tree. The 16S rRNA gene sequence of isolate PEJ1011 was submitted to NCBI GenBank under accession number KC819803. 2.3. PEJ1011 mediated plant growth promotion and cold stress resistance We analysed S. nematodiphila PEJ1011 for its plant growth-
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promoting activity and induction of cold stress resistance. A growth chamber experiment was conducted on pepper plants, which comprised both control and PEJ1011 treated pepper plants, with and/or without low temperature stress. Each treatment comprised 27 pepper plants and was replicated thrice. The glassware was properly sterilized before starting the experiment, in order to eliminate chances of contamination, which could affect our results. Pepper seeds were purchased from Seminis Korea Co. (Korea), surface sterilized with NaOCl (5%) for 10 min, rinsed 5 times with autoclaved double-distilled water (DDW), and treated with 20 ppm uniconazole. Seeds were initially sown in petri-plates containing filter paper, moistened with 5 ml DDW and kept for five days in an incubator. Pepper seedlings of almost identical lengths were transferred into plastic pots and moved to growth chamber (day/night cycle: 14/10 h; 28 C/10 h; 25 C; relative humidity 60e70%; light intensity 1000 mE m2 s1 from natrium lamps). The soil substrate in the plastic pots comprised of peat moss (13e18%), perlite (7e11%), coco-peat (63e68%) and 1 zeolite (6e8%) with the macro-nutrients NHþ 4 (e0.09 mg g ); NO3 (0.205 mg g1); P2O5 (0.35 mg g1), and K2O (e0.1 mg g1) [42]. Seedlings selected for inoculation were treated twice with 40 ml of bacterial suspension (grown in NB, 3 days of incubation at 30 C and 200 rpm) in split application, initially at the time of sowing in pots and then at 15 days after sowing. After the second application of the bacterial culture suspension, the pepper plants were left to grow for 1 week. In the low temperature (5 C) treatment, S. nematodiphila PEJ1011-inoculated and control plants were shifted to another growth chamber provided with controlled conditions (5 C; day/night cycle 14/10 h; light intensity 1000 mE m2 s1 from natrium lamps; and relative humidity 60%) for 4 h). Plant growth attributes i.e. shoot length, root length and fresh biomass were measured at the time of harvest, while the chlorophyll content of fully expanded leaves was analysed with the help of a chlorophyll meter (SPAD-502 Minolta, Japan). The plants were harvested, immediately frozen in liquid N and stored at 80 C.
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2.5. Analysis of plant endogenous ABA, JA and SA contents The endogenous ABA contents were extracted from the lyophilized pepper tissues following the method of [66]. The plant extracts were dried and methylated by adding diazomethane for GC-MS SIM (6890N network GC system, and 5973 network mass selective detector; Agilent Technologies, Palo Alto, CA, USA) analysis. For quantification, the Lab-Base (ThermoQuest, Manchester, UK) data system software was used to monitor responses to ions of m/z 162 and 190 for Me-ABA and 166 and 194 for Me-[2H6]-ABA. The endogenous JA contents were extracted following the protocol of [55]. The plant extracts were analysed with GC-MS SIM (6890N network GC system, and 5973 network mass selective detector; Agilent Technologies, Palo Alto, CA, USA). To enhance the sensitivity of the method, spectra were recorded in the selected ion mode, i.e. in case of JA determination, monitoring the fragment ion at m/z ¼ 83 corresponding to the base peaks of JA and [9, 10-2H2]-9, 10-dihydro-JA. The amount of endogenous JA was calculated from the peak areas of endogenous JA in comparison with the corresponding standards. Three replicates per treatment were used for the determination of JA. The SA contents were extracted and quantified, as described previously by Ref. [71]. The freeze-dried leaf samples were grounded to powder form, and 0.1 g was sequentially extracted with 90 and 100% methanol by centrifuging at 10,000 g. The combined methanol extracts were vacuum dried. Dry pellets were resuspended in 2.5 ml of 5% trichloroacetic acid and the supernatant was partitioned with ethyl acetate:cyclopentane:isopropanol (100:99:1, v/v). The top organic layer containing free SA was transferred to a 4 ml vial and dried with nitrogen gas. The dry SA was again suspended in 1 ml of 70% methanol. High performance liquid chromatography (HPLC) analyses were carried out on Shimadzu having a fluorescence detector (Shimdzu RF-10AXL, excitation and emission, 305 and 365 nm, respectively) fitted with a C18 reverse-phase HPLC column (HP hypersil ODS, particle size 5 mm, pore size 120 Å Waters). The flow rate was 1.0 ml min1.
2.4. Determination of pepper and S. nematodiphila PEJ1011 GAs 2.6. Statistical analysis The lyophilized plant samples were used for extraction and quantification of endogenous GAs. For GA analysis, 0.5 g lyophilized sample of pepper plant was used each time. For bacterial GA analysis, S. nematodiphila PEJ1011 inoculated NB media was partitioned (5000 g at 4 C for 15 min), and 50 ml of supernatant was used for analysing bacterial GAs. An established protocol for extraction and quantification of GAs was adopted, following [51] and [79]. For GA analysis, a gas chromatograph (Hewlett-Packard 6890, 5973N mass selective detector) with HA-1 capillary column (30 m 0.25 mm i.d., 0.25 mm film thickness) was programmed for a 1 min hold at 60 C, then a rise of 15 C min1 to 200 C, followed by 5 C min1 to 285 C. Helium carrier gas was kept at a head pressure of 30 kPa. The GC was directly connected to a mass selective detector with an interface and source temperature of 280 C and ionizing voltage of 70 eV, with dwell time of 100 ms. Full scan mode (the first trial) and three major ions of supplemented [2H2]GAs internal standards (the second trial) and the endogenous GAs were monitored simultaneously (standard GAs were purchased from Prof. Lewis N. Mander, Australian National University, Canberra, Australia). The endogenous GA contents of GA4, GA9, and GA20 were calculated from the peak area ratios of 284/286, 298/300, and 300/360, respectively. The data was calculated in nanograms per gram dry weight of plant samples and per ml of bacterial culture broth and each analysis was repeated two times.
To know the ameliorative effect of PEJ1011 symbiotic association with host plants under low temperature stress, the experiments was conducted in completely randomized design. For identification of the treatment and effects significances, analysis of variance (2Way ANOVA; Fisher's LSD and Bonferroni post-hoc test; P 0.05) was carried out on the data using Graph Pad Prism software (version 5.0, San Diego, California USA) and graphs were sketched with sigmaplot (version 12). The experiment was repeated three times. 3. Results and discussion 3.1. PGPR isolation, GAs analysis and growth conditions In current study, we collected 17 bacterial isolates from the rhizosphere of randomly selected pepper plants and subsequently screened for plant growth promoting capacity on GA mutant WaitoC rice (for detail see materials and methods). On the basis of screening results a novel bacterial isolate PEJ1011 was analysed for GAs production ability and found to produce three different types of GAs (GA4, GA20 and GA9). It was observed that PEJ1011 produced much higher contents of bioactive GA4 (8.65 ± 0.95 ng ml1), as compared to inactive GA20 (6.21 ± 1.54 ng ml1) and GA9 (1.64 ± 1.11 ng ml1). The GAs production potential of PGPRs have
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analysed IAA, ABA, and GA productions in Variovorax paradoxus previously isolated by Ref. [12]. The same bacterial isolate was reported to produce IAA, indole-3-carboxylic acid, and indole-3-lactic acid, but ABA or GA was not detected. In current study, PEJ1011 was grown in three different low temperature (2 C, 5 C, 10 C) and optimal temperature (30 C) regimes for 48 h to understand its growth rate during host-plant symbiotic association under control climate chamber experiment. The optical density of the bacterial mass growth was periodically analysed at 600 nm (OD 600), and almost log-linear growth was found from 4 h to 36 h of incubation, after which the mass was constant (stationary phase) for up to 48 h (Fig. 1) at 5 C, 10 C and 30 C. As shown in (Fig. 1) PEJ1011 was unable to grow at 2 C, however attained significantly higher growth rate at 30 C as compared to 10 C and 5 C. 3.2. Identification of bacterial isolate PEJ1011 Fig. 1. Growth rates of PEJ1011 at four different temperature regimes. Values in the form of different symbolic structures (i.e. to represent four temperature regimes) at regular intervals are means of (n ¼ 3). (*) mark indicates the significances of treatment effects in that particular growth conditions (temperature).
been reported previously [4,16], though bacterial strains were mostly studied for their IAA production [77], and rarely for GA production [15]. The GA biosynthesis pathway in fungi is well studied [3,25,46,78], but little is known about GA biosynthesis mechanism of GAs producing PGPRs [15,58]. Various bacterial spp. such as Rhizobium phaseoli, Azospirillum lipoferum, A. brasilense, Acetobacter diazotrophicus, Herbaspirillum seropedicae, Bacillus licheniformis, B. macroides, B. cereus, B. pumilus, B. amyloliquefaciens and Bradyrhizobium japonicum have been reported to produce bioactive and inactive GAs [4,10,16,36,39,41,53]. In most of the studied GA producing bacteria, the bioactive GAs include GA1, GA3, GA4, or GA7 [10,36,41,53]. PEJ1011 produced bioactive GA4 (the precursor for GA1 and GA3), and the same has also been reported in B. licheniformis, B. cereus, B. macroides, and B. pumilus [36,41]. The inactive GA9 and GA20 have been detected in B. cereus, B. licheniformis, B. macroides, and B. pumilus [36,41]. Occurrence of GA9 and GA20 in the PEJ1011 confirm pertinent previous reports. Current results are also in agreement with those of [40]; who
After confirming the GA producing potential of bacterial isolate PEJ1011, the isolate was phylogenetically assigned by sequencing of the 16S rRNA gene. BLAST search of the 16S rDNA sequence obtained from isolate PEJ1011 showed a high similarity (99%) to different S. nematodiphila strains. Four of these strains together with 9 reference strains of related species were used to generate a phylogenetic tree (Fig. 2). Based on BLAST search results and phylogenetic analysis the PEJ1011 was identified as a new strain of S. nematodiphila. Previous researches revealed that S. nematodiphila and other members of the genus Serratia have been isolated from diverse sources, including animals [84], forest soil [24], limonitic crust [63], and as plant endophytes [19]. S. nematodiphila isolated from soil and plants has been reported to produce various plant growth-promoting substances (PGPS). For instance, S. nematodiphila isolated from the forest soil was found to produce IAA, H2S, siderophores, and solubilised phosphate [24]. The same strain is capable to utilize citrate; reduce nitrate, oxidase and catalase; and hydrolyse esculin and casein. Endophytic S. nematodiphila isolated from Solanum nigrum L. growing in metal-polluted soil also possessed PGPSs, including 1-aminocyclopropane-1-carboxylic deaminase, IAA, siderophores and solubilised phosphate [19]. All the PGPSs produced by bacteria play a variable role in plant growth promotion and thus
Fig. 2. Identification of the bacterial isolate PEJ1011, by phylogenetic analysis. The neighbour-joining tree was constructed from 16S rDNA region sequences of the strains most similar to PEJ1011 using MEGA6 software. Accession numbers of all strains are written to their next.
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make a very complex phenomenon [24]. In earlier studies, authors linked plant growth promotion to a much better phosphorous and iron nutrition, possibly triggered by phosphate solubilization and siderophore production capacity of PGPR [24,26,50]. The plants were protected through biological means due to HCN [24], although IAA fabrication capacity of various bacteria was thought to be the main reason for plant growth and development [30]. 3.3. PEJ1011 and plant hormonal modulation during low temperature stress Plant inoculation with effective bacterial strains in a proper manner has gained considerable attention for sustainable and increased agriculture production [9]. The PGPRs enhance soil nutrients uptake in three different ways, i.e. solubilisation, mobilization and transformation by cycling organic and inorganic nutrients [24]. The interaction of PGPRs with a host plant is thus considered beneficial for plant health and soil quality [6,24]. However, another recognized important aspect of PGPRs is their phytohormone production or degradation capacity [65]. The widespread occurrence of phytohormones has been reported in many PGPRs belonging to several classes [28,30]. Most often, PGPR may produce one but rarely multiple types of phytohormones and their analogues [28]. In this study, GA-producing S. nematodiphila PEJ1011 were evaluated for pepper plant growth promotion and low temperature resistance (Table 1; Fig. 3a and b). The in vivo application of S. nematodiphila PEJ1011 under normal temperature in pepper markedly increased plant growth attributes as compared to control (Table 1). Shoot and root lengths were significantly increased by 55.8% and 12.9% respectively, than the control. Important plant growth parameters i.e. chlorophyll content in leaves and its effect in terms of increased fresh biomass, were 1.45 and 1.08 fold higher than control, under normal temperature. Randomly selected plants from both inoculated and control treatments grown in plastic pots, were subjected to low temperature 5 C stress for 4 h. The exposure to low temperature stress did
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Table 1 Growth promoting effects of PEJ1011 on shoot length (SL), root length (RL), fresh weight (FW), and chlorophyll contents (CC) of peppers grown under normal condition and cold shock stress. Treatment
SL (cm)
NT PEJ1011 CT CT þ PEJ1011
15.6 24.3 15.2 23.8
± ± ± ±
1.21b 0.57a 1.37b 0.56a
RL (cm) 11.6 13.1 11.3 13.2
± ± ± ±
1.06b 0.54a 1.01b 0.77a
FW (g) 2.25 3.27 2.21 3.19
± ± ± ±
CC (SPAD) 0.13b 0.06a 0.09b 0.09a
31.5 34.0 29.12 30.1
± ± ± ±
1.0b 1.2a 1.2b 1.1b
NT ¼ Normal temperature, CT ¼ Cold temperature/cold shock stress for 4 h at 5 C, SPAD ¼ soileplant analysis development unit for measuring leaf chlorophyll content. Each value in the columns represent mean ± standard deviation (n ¼ 3). Mean values in the same column denoted by different letter are significantly different at (P 0.05).
not affect growth characteristics of plants inoculated with S. nematodiphila PEJ1011 and maintain vigour and no visible sign of wilting or stunting was found. The shoot length, root length, and fresh weight was significantly increased by 1.57, 1.17 and 1.44 fold respectively, as compared to control, except chlorophyll contents under low temperature stress (Table 1). The plant growthpromoting traits of S. nematodiphila PEJ1011, similar to those of the other PGPRs in the current study, are in agreement with those of previous studies [19,24,59,74,82]. Previous studies have concluded that plants can achieve better growth under normal and stressful conditions in the presence of PGPRs, because of their ability to produce different phytohormones including GAs, IAA, cytokinin, ABA, and JA; nutrient solubilization; softening of the root membrane; biological nitrogen fixation; production of enzymes needed for the modulation of plant hormones and protection against microbial pathogens. Phytohormone-producing PGPRs modulate plant endogenous hormonal levels, by utilizing either their own biosynthetic pathway or further modifying the precursors released through root exudates [28]. The interaction of phytohormones for mediating plant physiological or phenotypic characteristics is a very complex process involving a network of signalling, cross-talking, and feedback
Fig. 3. Plant growth-promoting effect of Serratia nematodiphila PEJ1011. Gibberellic acid (GA4)-producing S. nematodiphila PEJ1011 grown in liquid culture was twice applied to the root zone of pepper plants. GA4 has a long distance effect and promotes the growth of other organs [14]. The pepper plants without inoculation (control, a) and with inoculation of S. nematodiphila PEJ1011 (b) were grown for 21 days. Twenty-seven randomly selected plants replicated three times from both treatments were exposed to cold shock stress for 4 h at 5 C. (a) A representative leaf sample from the control plant after exposure to shock stress. (b) A representative leaf sample from S. nematodiphila PEJ1011-inoculated plants is also shown for comparison.
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Fig. 4. In planta hormonal modulation ability of Serratia nematodiphila PEJ1011 in pepper plants under normal condition and cold shock stress. Gibberellic acid (GA4; a), abscisic acid (ABA; b), jasmonic acid (JA; c), and salicylic acid (SA; d). NT ¼ Pepper plants grown at normal temperatures without inoculation of S. nematodiphila PEJ1011. CT ¼ Pepper plants grown without inoculation of S. nematodiphila PEJ1011 and then subjected to cold temperatures/cold shock stress for 4 h at 5 C. PEJ1011 ¼ Application of S. nematodiphila PEJ1011 to pepper plants grown under normal temperatures. CT þ PEJ1011 ¼ Application of S. nematodiphila PEJ1011 to pepper plants and then subjected to cold temperatures/cold shock stress for 4 h at 5 C. Column bars sketched with error bars represents mean values ± standard deviation (n ¼ 3). In all figures, different letter(s) and (*) mark above column bars shows significant differences at (LSD; Bonferroni; P 0.05). In pairwise/group-wise comparison of normal and cold stress, (*) mark indicates the significances of treatment effects in that particular growth conditions.
mechanisms [7,76]. Most of the rhizobacteria modulate more than one endogenous plant hormone initially by affecting their levels in the roots and then establishing a long distance-signalling mechanism up to the shoot [11,27,28]. In view of this important aspect, the modulation of plant growth-regulating GAs and the defence hormones ABA, JA and SA was investigated in response to GAproducing S. nematodiphila PEJ1011 under low temperature stress. Since S. nematodiphila PEJ1011 was found to produce bioactive gibberellins GA4 (which is further converted into GA3 and GA1 [78], the response of pepper endogenous GA4 under normal condition and low temperature stress was analysed. S. nematodiphila PEJ1011 significantly increased the pepper endogenous GA4 content under normal condition and low temperature stress (Fig. 4a). The endogenous GA4 content of S. nematodiphila PEJ1011-treated pepper plants under normal condition and low temperature stress was increased by 78.40% and 85.58%, respectively. However, no significant differences were recorded for S. nematodiphila PEJ1011treated pepper plants grown under low temperature stress and control in normal growth condition. Different growth substances producing PGPRs have been frequently reported to have stimulatory effects on the endogenous phytohormone contents of plants [28]. The effect is usually reported to be more pronounced and synergistic for the corresponding phytohormone produced by the PGPRs [5,37,42e45]. Refs. [43,44] applied three different PGPR strains, Burkholderia cepacia SE4, Promicromonospora sp. SE188, and Acinetobacter calcoaceticus SE370, and found they significantly increased the endogenous GA4 content of cucumbers. The same effect was also reported for IAA- and cytokinin-producing PGPRs of the genera Pseudomonas, Bacillus, and Azospirillum for the
phytostimulation of the corresponding hormones [37]. However, during in same experiment, the higher IAA to cytokinin ratio in PGPRs played an antagonistic role in increasing the cytokinin contents of wheat plants. Ref. [13] treated lodgepole pine trees with IAA-producing PGPRs and found the same effect of increased IAA concentration in roots. It is a well established fact that PGPRs producing particular hormone(s) may affect other phytohormones either in a synergistic or antagonistic manner. The effect may be due to the modulation of the plant hormone corresponding to the PGPR hormone, which further regulates other hormones accordingly [28,43,44]. Otherwise, irrespective of the mechanism, PGPR may affect all or particular phytohormones even if it does not produce any hormones [18,67]. Therefore, we also studied major endogenous phytohormones of pepper under control and low temperature stress. The endogenous contents of drought-mediating hormone ABA was significantly enhanced (111.39%) in the control plants than in the S. nematodiphila PEJ1011-treated pepper plants (Fig. 4b). The decrease in ABA seems to be reasonable under normal condition, and it may be attributed to the high level of GA4 [81]. Both hormones act antagonistically under normal growth conditions when GAs are present in high amounts [32,81]. However, an opposite trend was observed for ABA regulation in the bacterium inoculated plants under low temperature stress, as the endogenous contents of ABA increased by 181% in S. nematodiphila PEJ1011-treated pepper plants compared to the control. An increased in ABA levels during drought, salinity, and low temperatures helped plants to adapt to these situations [83]. As a consequence, the expression of several genes that are known to respond to drought and cold stress was
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induced [72,83]. Nevertheless, the mechanism of ABA is not very clear, as some genes are expressed during cold or drought stress but never respond to exogenous ABA application [83]. Here the role of S. nematodiphila PEJ1011 application to pepper plants was appreciable under low temperature stress when the GA and ABA levels were compared to the control. The application of GA-producing PGPRs also increased the endogenous GA4 level to counteract the growth inhibitory effect of low temperature stress and might be tried synergistically with ABA content to render normal growth [56]. In comparison, the proportion of GA4 to ABA is very low in control plants under low temperature stress. Our results also confirm the previous observations that PGPRs have been reported to increase the ABA level during various kinds of abiotic stresses in different crops, including sunflowers, Arabidopsis, and maize [18,21,22,33]. JA plays an important role during biotic stress imposed by necrotrophic pathogens and herbivorous insects and abiotic cold stress [17,29,47,80]. The application of S. nematodiphila PEJ1011 caused significantly higher reductions in the level of JA in pepper plants than that in the controls under normal condition (1.49 ± 0.09b vs. 2.39 ± 0.12a) and low temperature stress (0.21 ± 0.13c vs. 1.35 ± 0.10b) (Fig. 4c). The level of JA was insignificant in pepper plants treated with S. nematodiphila PEJ1011 under normal growth conditions and control low temperature stress. Similar result was reported by Ref. [47]; where cold stress (both chilling and frost stress) decreased the JA level in short stature Arabis alpina. The short stature A. alpina was considered to be a model plant that adapted to extreme low temperatures and snow coverage, and hence, was added to the experimental group with two other ecotypes of the same species. In addition [48], showed the same trend on exposure to cold stress in winter and spring wheat cultivars, where the JA levels in the leaves and crowns reduced during the initial alarming phase of the cold response. Another plant defence hormone, SA, regulates various plant physiological functions and provides protection from microbial pathogenic infections [57]. The abiotic stress-mediating role of SA in salinity, drought, and low temperatures has been documented. The application of SA in low quantities reverts the cold stress effect in maize, rice, potatoes, and wheat; however, treatment with high amounts of SA has shown to induce the opposite effect [48,56]. In several experiments, a high amount of SA accumulation has been reported in different plant species in response to cold stress. Such a response of SA for a short time has been correlated with its positive role in cold stress resistance, and vice versa. In this study, pepper plants inoculated with S. nematodiphila PEJ1011 had significantly lesser SA during normal condition (9.50 ± 0.21c) and low temperature stress (10.63 ± 0.22b) than that in the controls (11.60 ± 0.22b and 13.32 ± 0.36a, respectively) (Fig. 4d). No significant difference was noted in the SA level between the control pepper plants grown under normal condition and S. nematodiphila PEJ1011-treated plants under low temperature stress. Overall, the current findings are very much in agreement with those of [48,64] and [61]. They concluded that plants with better cold stress resistance increased their ABA level and downregulated SA and JA levels in the initial hours of cold stress. This study, along with the aforementioned reports, suggests that ABA is antagonistic towards SA and JA during the initial cold period. This unique adaptation of pepper plants during low temperature stress is attributed to the presence of S. nematodiphila PEJ1011. 4. Conclusions The present results concluded that the application of novel PGPR strain like S. nematodiphila PEJ1011 might be a good alternative to synthetic fertilizer. The growth promoting potentials of
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this strain was also supported by the enhanced plant growth characteristics under normal and cold stress conditions. It was revealed that this increase in growth of inoculated plants was attained through relative increase in growth hormone like GA4 under normal and cold stress condition, and vice versa in case of stress hormones such as JA and SA. The S. nematodiphila PEJ1011 inoculation extended stress mitigation as shown by the increased level of stress responsive ABA in cold and decreased level in normal condition as compared to non-inoculated control plants. Further studies of S. nematodiphila PEJ1011 symbiotic association with host and other crops in open field conditions and exploration of its potentials at molecular level is recommended. Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A1004918). References [1] P. Achard, F. Gong, S. Cheminant, M. Alioua, P. Hedden, P. Genschik, The coldinducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism, Plant Cell 20 (2008) 2117e2129. [2] M. Ahemad, M. Kibret, Mechanisms and applications of plant growth promoting rhizobacteria: current perspective, J. King Saud Univ. Sci. 26 (2014) 1e20. [3] S. Albermann, T. Elter, A. Teubner, W. Krischke, T. Hirth, B. Tudzynski, Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi, Appl. Microbiol. Biotechnol. 97 (2013) 7779e7790. [4] R. Atzhorn, A. Crozier, C.T. Wheeler, G. Sandberg, Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots, Planta 175 (1988) 532e538. [5] O.Z. Barazani, J. Friedman, Is IAA the major root growth factor secreted from plant-growth-mediating bacteria? J. Chem. Ecol. 25 (1999) 2397e2406. n, C. Azco n-Aguilar, Microbial co-operation in the [6] J.M. Barea, M.J. Pozo, R. Azco rhizosphere, J. Exp. Bot. 56 (2005) 1761e1778. [7] R. Bari, J.D.G. Jones, Role of plant hormones in plant defence responses, Plant Mol. Biol. 69 (2009) 473e488. [8] C.D.C. Barillot, C.O. Sarde, V. Bert, E. Tarnaud, N. Cochet, A standardized method for the sampling of rhizosphere and rhizoplan soil bacteria associated to a herbaceous root system, Ann. Microbiol. 63 (2013) 471e476. [9] Y. Bashan, L.E. de-Bashan, S.R. Prabhu, J.P. Hernandez, Advances in plant growth-promoting bacterial inoculants technology: formulations and practical perspectives (1998e2013), Plant Soil (2014), http://dx.doi.org/10.1007/ s11104-013-1956-x. [10] F. Bastian, A. Cohen, P. Piccoli, V. Luna, R. Baraldi, R. Bottini, Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically defined media, Plant Growth Regul. 24 (1998) 7e11. [11] A.A. Belimov, I.C. Dodd, N. Hontzeas, J.C. Theobald, V.I. Safronova, W.J. Davies, Rhizosphere bacteria containing ACC deaminase increase yield of plants grown in drying soil via both local and systemic hormone signalling, New Phytol. 181 (2009) 413e423. [12] A.A. Belimov, N. Hontzeas, V.I. Safronova, S.V. Demchinskaya, G. Piluzza, S. Bullitta, B.R. Glick, Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.), Soil Biol. Biochem. 37 (2005) 241e250. [13] E. Bent, S. Tuzun, C.P. Chanway, S. Enebak, Alterations in plant growth and in root hormone levels of lodgepole pines inoculated with rhizobacteria, Can. J. Microbiol. (2001) 47793e47800. [14] H. Bidadi, K. Matsuoka, K. Sage-Ono, J. Fukushima, W. Pitaksaringkarn, M. Asahina, S. Yamaguchi, S. Sawa, H. Fukuda, Y. Matsubayashi, M. Ono, S. Satoh, CLE6 expression recovers gibberellin deficiency to promote shoot growth in Arabidopsis, Plant J. (2014), http://dx.doi.org/10.1111/tpj.12475. [15] R. Bottini, F. Cass an, P. Piccoli, Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase, Appl. Microbiol. Biotechnol. 65 (2004) 497e503. [16] R. Bottini, M. Fulchieri, D. Pearce, R.P. Pharis, Identification of gibberellins A1, A3, and iso-A3 in cultures of Azospirillum lipoferum, Plant Physiol. 90 (1989) 45e47. [17] L.C. Carvalhais, P.G. Dennis, D.V. Badri, G.W. Tyson, J.M. Vivanco, P.M. Schenk, Activation of the jasmonic acid plant defence pathway alters the composition of rhizosphere bacterial communities, PLoS One 8 (2013) e56457. [18] P. Castillo, M. Escalante, M. Gallardo, S. Alemano, G. Abdala, Effects of bacterial single inoculation and co-inoculation on growth and phytohormone production of sunflower seedlings under water stress, Acta Physiol. Plant. 35 (2013) 2299e2309.
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