Introduction
A wide range of microorganisms, varying from pathogenic to beneficial, interact continuously with higher plants in the soil ecosystem, influencing the growth, development and functions of plants (Taghavi et al., 2009). Bacteria that are able to colonize plant root systems and promote plant growth are referred to as plant growth promoting rhizobacteria (PGPR). PGPR can affect plant growth either indirectly or directly. The indirect promotion of plant growth occurs when PGPR lessen or prevent the deleterious effects of one or more phytopathogenic organisms. The direct promotion of plant growth by PGPR involves either providing the plants with certain bacterial-synthesized compounds or facilitating the uptake of certain nutrients from the environment (Glick, 1995; Lugtenberg & Kamilova, 2009). On the other hand, deleterious rhizosphere bacteria (DRB) are defined as rhizobacteria that inhibit plant growth without causing disease symptoms (Brimecombe et al., 2007). Several mechanisms for growth inhibition by this undesirable group of rhizobacteria have been proposed, the most likely being the production of phytotoxins such as cyanide and other volatile and non-volatile compounds, as yet unidentified. An alternative mechanism by which DRB may inhibit plant growth is through the production of phytohormones (Brimecombe et al., 2007). Indole-3-acetic acid (IAA) produced by DRB has been shown to inhibit root growth in sugar beet and blackcurrant (Brimecombe et al., 2007). DRB may also compete with the plant and beneficial rhizobacteria for nutrients, contributing to the decreased plant growth and, therefore, lowered yields (Brimecombe et al., 2007). Further, DRB may indirectly reduce growth by counteracting the effects of nitrogen-fixing rhizobacteria (Brimecombe et al., 2007).
Auxins are a group of plant growth regulators that stimulate cell division and elongation. IAA is the principal auxin of higher plants, to which the amino acid L-tryptophan (L-Trp) plays a precursory role. Microbial synthesis of IAA has been known for a long time. This property is best documented for bacteria that interact with plants. IAA production by PGPR is one of the most studied and, perhaps, the most effective mechanism of plant growth promotion by these bacteria (Patten & Glick, 1996; Arshad et al., 2010). Plant roots secrete signalling chemicals (L-Trp, a precursor for IAA, as well as other amino acids and small molecules) into the rhizosphere soil, promoting the binding of bacteria to the root surface (Simons et al., 1997). The root-bound bacteria may use the L-Trp in the soil to produce IAA (Patten & Glick, 1996; Mirza et al., 2001; Gravel et al., 2007). The IAA is subsequently secreted from the PGPR and absorbed by the plant and used primarily to increase cell growth or proliferation (Glick et al., 1998). Therefore, it seems that IAA has a concentration-dependent dual role (Arshad & Frankenberger, 1992; Chauhan et al., 2009) with optimal IAA levels in plant roots being several orders of magnitude lower than its optimal levels in the shoots.
The genus Pseudomonas comprises a group of ubiquitous bacteria that have frequently been reported to play a dual role in plant health and growth (Meyer et al., 2008). For example, some of the bacteria in this group are known to be pathogenic (Hirano & Upper, 2000), while others are known to be involved in disease suppression (Halgren et al., 2011). In addition, some members of this group of bacteria appear to increase seed germination (Selvakumar et al., 2009), while others exert inhibitory effects on seed germination (McPhail et al., 2010; Lee et al., 2013). Germination arrest factors (GAFs) have been the subject of increased research in recent years. GAFs are defined as microbial-derived compounds that lead to irreversible arrest of seed germination in a wide range of graminaceous species (Banowetz et al., 2008). For example, the compounds 4-formylaminooxyvinylglycine (FVG) (McPhail et al., 2010) and L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) (Lee et al., 2013) are two GAFs produced by Pseudomonas species. However, there is a gap in knowledge regarding the impact of GAFs on wheat seed germination, in general, and on durum wheat (Triticum turgidum L.) seed, in particular. Durum wheat represents 10% of the wheat grown globally, occupying about 11 million hectares in the countries around the Mediterranean Basin. Durum wheat in Iran is cultivated across diverse environments, ranging from warm lowlands to cold highlands. The success of durum wheat in Iran, as a food security crop, is largely due to its good ability and capacity to produce well under drought-prone environments and marginal and poor management conditions where other crops would fail (Mohammadi et al., 2010).
Therefore, the aim of the present experiments was to evaluate the effect of some IAA–producing Pseudomonas isolates on seed germination, α-amylase activity, and seedling growth of durum wheat. In addition, because of concentration-dependent effects of IAA on plant growth, potential role of the exogenous IAA in germination behaviour of durum wheat seeds was also examined.
Material and methods
Bacteria strains and growth conditions
The Pseudomonas isolates UW3 (Pseudomonas sp.), UW4 (Pseudomonas putida) (Glick, 1995; Duan et al., 2013) have been described previously and isolates 550 (Pseudomonas fluorescens) and 57 (Pseudomonas sp.) were obtained from the Department of Soil Sciences, University of Tehran, Iran. The isolates were grown on nutrient agar (NA) (2 g yeast extract, 1 g meat extract, 5 g peptone, 5 g sodium chloride, 20 g agar-agar, 1000 mL distilled water) or nutrient broth (NB) for routine use and maintained in NB with 20% glycerol at – 80 °C for long-term storage. For preparing the bacterial cultures, single colony of each bacterial isolate was grown in 250-mL flasks containing 100 mL NB medium and incubated for 24 h at 28 ± 2 °C on a rotary shaker (KS 130 basic, IKA, Germany) at 120 rpm. After incubation, the cell suspension was centrifuged at 5,000 × g for 5 min at 4 °C and washed twice with sterile distilled water. The final pellet was resuspended in sterilized distilled water and the bacterial cultures were standardized to 108 colony-forming units (CFU)/mL and used, immediately, for seed germination experiments.
IAA production assay
The production of IAA by the isolates was determined as described by Glickman & Dessaux (1995). The isolates (108 cells/mL) were grown in 100 mL flasks containing 50 mL NB supplemented with L-Trp (100 µg/mL) for 48 h on a rotary shaker at 120 rpm. Then, cultures were centrifuged at 8,000 g for 10 min and the supernatants collected. Two mL of Salkowsky reagent (1 mL of 0.5 M FeCl3 in 50 mL of 35% HClO4) with 1 mL of the supernatant was allowed to react at 28 ± 2°C for 20 min at room temperature. Pink color developed indicating the presence of IAA was determined by measuring the absorbance in a spectrophotometer (HITACHI U1800) at 535 nm at the end of the incubation (Patten & Glick, 2002). A standard curve was plotted with IAA and Salkowsky reagent dissolved in NB medium to quantify the IAA (μg /mL) present in the culture supernatant. Concentration of IAA produced was estimated against standard curve of IAA in the range of 0–100 µg/mL.
Effect of bacterial strains on durum wheat seed germination traits
To evaluate the effect of Pseudomonas isolates on germination and emergence traits of durum wheat seeds (Triticum turgidum L. var durum), a laboratory experiment was conducted as a completely randomized design (CRD) with four replicates in Department of Agronomy and Plant Breeding, Isfahan University of Technology, Iran. Five treatments were made as follows: 1) seeds treated with isolate UW3; 2) seeds treated with isolate UW4; 3) seeds treated with isolate 550; 4) seeds treated with isolate 57; 5) uninoculated control. Seeds of durum wheat were obtained from the seed bank of Isfahan University of Technology. Four replicates of 25 seeds per treatment were used. Seeds were surface disinfected in a 2% (v/v) solution of sodium hypochlorite for 15 min and rinsed four times with sterile distilled water, and air-dried before being used in the germination experiments. All further manipulations were carried out under sterile conditions. The surface-sterilized seeds were immersed into individual bacterial suspensions for 30 min, shaking at 120 rpm. Twenty-five seeds were sown into sterile plastic 9-cm-diameter Petri dishes containing filter paper (Whatman No. 1) and watered with 7 mL of sterile distilled water. The Petri dishes were transferred into a dark growth chamber at 25 ± 2°C for 10 days, and the germinated seeds were counted on a two-day-interval basis in a certain time. A seed was considered as germinated when its radicle emerged by about 2 mm in length. Final germination percentage and rate were measured at day 10 after incubation. The germination rate was estimated according to the Eq. [1], modification of Timson’s index (Khan & Ungar, 1984). The root and shoot lengths were measured and seedling vigour index was determined according to the Eq. [2] (Abdul-Baki & Anderson, 1973).
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where, G is the percentage of seeds germinated at two-day-intervals, and t is the total germination interval period.
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Assay of α-amylase activity
For determination of α-amylase activity, durum wheat seeds were harvested at two, four, six, and 8 days after incubation with bacterial isolates as described above. To prepare the enzyme extract, five germinating seeds per Petri dish were weighed, frozen, ground to a fine powder in a pre-chilled mortar in liquid N2 using a pestle, homogenized with 5 mL of a 0.1 M sodium acetate buffer (pH 4.8), and filtered through Whatman filter paper to remove large particles. The extract was centrifuged at 12,000 × g for 20 min (5810R, Eppendorf refrigerated centrifuge, Germany). All of the preparations were carried out at 4°C. The supernatant was served as the crude enzyme extract for the α-amylase assay.
For the enzyme assay, the reaction medium (3 mL) contained 1 mL of the 0.1 M sodium acetate buffer, pH 4.8, 0.5 mL of enzyme extract diluted to 1 mL using acetate buffer, and 1 mL of 0.1% soluble starch (Merck #31231373) solution. The enzyme extract was diluted to obtain an absorbance change of less than one during the enzyme assay. The reaction medium was incubated for 10 min at room temperature (22 ± 2 °C), then the reaction was terminated by adding 1 mL of a 0.1% iodine reagent (6 g potassium iodide, 0.6 g iodine in 1 L of 0.05 N HCl) and 3 mL of 0.05 N HCl. The absorbance was measured at 620 nm and the decrease in the absorbance relative to the blank was considered as α-amylase activity (Beri & Gupta, 2007).
Effect of exogenous IAA on seed germination and seedling growth
To evaluate the effect of exogenous IAA on germination and emergence traits of durum wheat seeds, an in vitro experiment was conducted as a CRD with four replicates as well. Five treatments were made as follows: 1) seeds treated with 5 µg/mL IAA; 2) seeds treated with 10 µg/mL IAA; 3) seeds treated with 15 µg/mL IAA; 4) seeds treated with 20 µg/mL IAA; 5) untreated control. Briefly, seeds were placed in 9-cm diameter Petri dishes and, after subjecting to 7 mL of IAA (0, 5, 10, 15 , 20 µg/mL), incubated at 25 ± 2°C for 10 days, and finally the effect of the exogenous IAA levels on germination percent, germination rate, α-amylase activity, and seedling vigour index were measured and recorded as described above.
Statistical analysis
Analysis of variances (ANOVA) was conducted on the data and, when F values were significant (p ≤ 0.05), mean comparisons were conducted using least significant differences (LSD, 0.05) procedure. Data were reported as means ± the standard error of the mean (SEM).
Discussion
This study provided an initial assessment of the potential of some IAA-producing Pseudomonas isolates on durum wheat seed germination traits and α-amylase activity under in vitro conditions. The results of this experiment indicate that the germination process is slowed down due to the presence of bacteria in the in vitro conditions and this suppressing role is likely in consequence of a bacteria-induced increase in IAA level of the medium. There are some contradictory reports on the effect of PGPRs, in general, and Pseudomonas species, in particular, on seed germination. In some reports, inoculation with bacteria has promoted seed germination and rate (Ashrafuzzaman et al., 2009; Selvakumar et al., 2009; Zarrin et al., 2009; Noumavo et al., 2013), while in some other reports it has been found to decrease seed germination (Banowetz et al., 2008; McPhail et al., 2010). For example, Selvakuma et al. (2009) reported that P. fragi CS11RH1, an IAA-producing strain, significantly increased the germination percentage and rate, plant biomass and nutrient uptake of wheat seedlings. However, according to Banowetz et al. (2008), P. fluorescens WH6 suppressed germination of Poa annua seeds and some other graminaceous species due, presumably, to the production of 4-formylaminooxyvinylglycine.
The inhibitory action of bacteria on seed germination could be due to the elevated levels of IAA or some unknown metabolites produced by the bacteria or the stress induced by PGPR. In order to address these possibilities, the effects of different concentrations of exogenous IAA on the percentage and rate of seed germination were also examined. During germination, plant seeds accelerate their respiratory metabolism to produce metabolic energy and biosynthetic precursors (Perata et al., 1997). To maintain respiratory metabolism crucial to germination, readily available respiratory carbohydrates and soluble sugars must be supplied constantly. However, the amount of readily utilizable soluble sugars in plant seeds is usually very limited, with starch being the main reserve carbohydrate (Guglielminetti et al., 2000). The hydrolytic enzyme α-amylase is known to play a major role in degradation of reserve carbohydrates (i.e. starch) to soluble sugars during germination (Perata et al., 1997). Thus, the induction of α-amylase is essential to maintain an active respiratory metabolism and, therefore, seed germination in durum wheat seeds.
During seed germination in wheat, barley and other graminaceous species, gibberellic acid (GA) is formed in the embryo and transferred to the aleuronic layer, where it induces the synthesis of α-amylase (Beri & Gupta, 2007). Measurement of α-amylase activity in germinating seeds is a classical bioassay for determining the GA level (Beri & Gupta, 2007). In the present study, low concentrations of IAA (i.e., 5 and 10 µg/mL) promoted the induction of α-amylase in germinating durum wheat seeds, perhaps, because the exogenous IAA stimulated GA biosynthesis. This possibility is consistent with previous reports (Anitha, 2010; Li et al., 2012). Paulsen & Auld (2004) concluded that IAA inhibited the germination of wheat seeds and acted in concert with GA and cytokinins to regulate the germination process. The observation that L-Trp, a precursor of IAA, suppressed the sprouting of resistant wheat cultivars supports a role for IAA in controlling seed germination (Morris et al., 1988). Evidence has been accumulated (Chauhan et al., 2009; Roychowdhury et al., 2012) in support of a concentration-dependent role for IAA in seed germination, that is, low concentrations of exogenous IAA can promote, whereas high concentrations can inhibit seed germination. The regulatory role of bacterium-produced IAA in seed germination is supported by the evident biphasic response of germination of durum wheat seeds to IAA concentration (Fig. 2).
From the present results, it is concluded that IAA impaired the induction of α-amylase activity and, thus, germination in durum wheat seeds. There was a tendency for seed germination to increase with increasing the activity of α-amylase in the seeds. The bacterial strains used in the present study inhibited seed germination of durum wheat seeds due, presumably, to biosynthesis and accumulation of IAA in the inoculated seeds (Fig. 2).
The finding that high levels of IAA produced by bacteria-inoculated plants was probably the main cause of the inhibition of germination in durum wheat seeds contradicts some previous reports (Ashrafuzzaman et al., 2009; Zarrin et al., 2009). Zarrin et al. (2009) observed that coating of wheat seeds with IAA-producing PGPR strains (19.4-30.2 µg/mL) positively influenced their germination percentage and rate. Ashrafuzzaman et al. (2009) also reported that rice seed germination increased when seeds were pre-treated with IAA-secreting PGPR isolates and that the high-IAA producing isolates, PGB4 and PGG2, were proven to enhance the germination of the rice seeds. Our findings, however, agree with argument made by Glick et al. (1998). They have argued that plant roots may produce either optimal or suboptimal IAA levels endogenously. Then, the bacterial-produced IAA may enhance or inhibit plant growth, depending on whether the total IAA (that endogenous to the plant plus that produced by bacteria) is at optimal or supra-optimal levels, respectively.
The α-amylase enzyme activity in inoculated and non-inoculated seeds appeared to be low at the beginning, whereas it increased as germination progressed, the extent of the increase was much greater in non-inoculated, relative to the inoculated durum wheat seeds. The latter difference showed that the starch was metabolized faster in control, relative to the inoculated seeds. Thus, the bacterial strains used in the present study exerted inhibitory effects on the induction of α-amylase activity and, consequently, the germination of durum wheat seeds.
Despite the promoting effects exerted by the low concentrations of IAA (5-10 µg/mL) on the seedling vigour index, it seems that, in agreement with observations made by Chauhan et al. (2009), high concentrations (15-20 µg/mL) of this phytohormone negatively affect the seedling vigour index of durum wheat. The latter researchers reported a concentration-dependent response to IAA of seedling growth in black gram and horse gram plants. It is well known that auxins induce vascular differentiation in germinating embryos (Lovisolo et al., 2002; Pereyra et al., 2012). During seed germination and the early stages of seedling growth, the developing roots release some exudates, including L-Trp. Some rhizobacteria use the L-Trp in the soil to produce IAA. This positive interaction between plant root and bacterial IAA can, in turn, stimulate root development (Lambrecht et al., 2000).
Auxin, particularly at high concentrations, tends to exert inhibitory impacts on certain biological systems (e.g., root growth). This inhibitory effect has, almost, invariably been shown to be associated with the auxin-induced biosynthesis of ethylene (Davies, 2010). Although IAA is known as a prominent plant growth promoter, it can also stimulate transcription of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. The latter enzyme facilitates a key step in ACC oxidase-mediated ethylene biosynthesis (Mayak et al., 1999). The IAA-derived ethylene is believed to participate in the disruption of the normal growth (i.e. germination and seedling growth) of the host plant.
It is known that microbial functions such as nitrogen assimilation, siderophore secretion, and phosphorous mobilization occur only when deficiencies are present in the soil (Okon & Labandera-Gonzalez, 1994). According to Davies (2010), stem elongation (i.e. seedling height) is governed both by IAA and GA1. O’Neill & Ross (2002) reported that IAA may promote the biosynthesis of the active GA1 in shoots of pea seedlings. Therefore, even moderate changes in IAA supply can lead to physiologically significant changes in GA1 content and growth attributes of seedlings. IAA-producing isolates used in our study may have enhanced plant growth due to the increased production of fine roots (Saharan & Nehra, 2011). The latter findings are in concert with other reports (Sheng et al., 2008; Sinha & Mukherjee, 2008). It is seemed the performance of bacterial strains in terms of enhancing the seedling vigour index is greater in nutrient-deficient conditions, than in nutrient-sufficient conditions. In our study, no attempt was made to measure the bacterial-induced IAA production of inoculated seeds under nutrient-sufficient and deficient conditions. Instead, we measured the Pseudomonas potential for IAA production before inoculating the seeds. Therefore, the assumption that bacteria are able to produce and secret higher amounts of IAA in nutrient-deficient conditions than in sufficient conditions needs to be validated in future studies.
Based on the results obtained, Pseudomonas strains have, evidently, the capacity to suppress durum wheat seed germination. IAA-induced suppression of α-amylase activity seems to be a major contributing factor to the lowered germination percent. Despite the negative effect of moderate IAA levels on seed germination, production of comparable amounts of IAA by rhizobacteria appeared to exert promoting effects on the seedling growth. Since isolate 57 behaved as a high-IAA-producing bacterium and, therefore, induced certain adverse influences on both germination and seedling growth of durum wheat, its mechanism of action needs to be further investigated in the future.