Seed priming with boron and

Context: The production of chickpea (Cicer arietinum L.) is negatively affected by boron (B) deficiency. In Pakistan, the crop grown under B deficiency produces grains with low B concentration. Application of B-tolerant bacteria (BTB) is a promising option to improve B supply to plants grown under B deficiency. Aims: This study was focused on determining the appropriate concentration of B for seed priming, and its effects with BTB inoculation on growth, productivity and grain B concentration of chickpea. Methods: Chickpea seeds were primed in aerated solutions of B concentrations in the range 0.01–0.5% (w/v), with hydroprimed and dry seeds as controls. Concentrations >0.1% proved toxic and seeds failed to germinate. Hence, B was further diluted to concentrations in the range 0.0001–0.1%. Pots containing chickpea seeds were divided into two sets having all B treatments. One set was inoculated with BTB (5 mL per pot of pure Bacillus sp. MN54 culture at 10 9 cfu mL⁻¹); the other set was not inoculated. Key results: Seed priming with B along with BTB inoculation improved stand establishment, growth, nodulation, yield and grain B concentration of chickpea. Seed priming treatments with B at 0.001% and 0.0001% along with BTB inoculation were most effective for improving stand establishment, seedling growth and grain yield, whereas 0.1% B was more effective for improving grain B concentration. Conclusions: Seed priming with 0.001% B along with inoculation of Bacillus sp. MN54 improved seed germination, nodulation, yield and grain B concentration of chickpea under B-deficiency conditions. Implications: Seed inoculation with BTB i.e., Bacillus sp. MN54 coupled with seed priming in 0.001% B solution is a viable option to improve yield and grain B concentration of chickpea grown on B-deficient soils.

Boron (B) deficiency is the leading constraint to chickpea productivity in Pakistan. Results of this study elucidated that B seed priming at lower rates (0.001% and 0.0001%) along with seed inoculation with B-tolerant bacteria (Bacillus sp. MN54) improved the productivity and grain-B biofortification of chickpea. Higher levels of B seed priming (>0.1% B solution) proved toxic, with no germination recorded.

Keywords: Cicer arietinum; grain biofortification; growth; nodulation; seedling establishment; seed priming; yield; 100-grain weight.

More than half of the global population is affected by micronutrient malnutrition. Pregnant mothers and children below the age of 5 years are the most vulnerable to micronutrient malnutrition, particularly in the developing world ([5]). Boron (B) deficiency is regarded as a leading risk factor causing numerous diseases in all age groups ([68]). Sufficient B intake is necessary for human physiology, including metabolic activities regarding bones, lipids and minerals. In plants, B is responsible for cell wall construction and maintenance of the plasma membrane ([69]), photosynthetic rate ([66]) and sugar transport ([20]). Boron is an essential constituent of cell membranes, helping in lignification and respiration. Moreover, B initiates metabolism of RNA, indole-3-acetic acid (IAA), carbohydrate and phenols ([3]). For humans, the recommended daily intake of B is 1–13 mg, and the primary sources are fruits, vegetables, nuts and legumes ([40]). The average concentration of B in chickpea (Cicer arietinum L.) grains is 0.71 mg 100 g−1 ([35]).

Chickpea is ranked second among legume crops after soybean (Glycine max (L.) Merr.) in terms of global production, and it is a good source of edible proteins ([23]). Edible proteins from legumes represent a high proportion of the daily diet in South Asian countries ([46]), and in this region, the Thal Desert in Pakistan is the home of chickpea production ([46]). Deficiency of B is more prevalent than deficiency of other micronutrients in 132 crops from 80 countries, including Pakistan ([31]; [4]). It negatively affects grain setting in wheat (Triticum aestivum L.), eventually leading to reduced number of grains per spike, increased number of open spikelets and ultimately poor yield ([62]). Likewise, B deficiency has been reported to decrease lentil yield ([59]).

Biofortification is the application of fertilisers/nutrients directly to seed, soil and foliage, which improves nutrient status of plants without altering their genetic structure ([61]). Seed priming is a controlled hydration technique whereby seeds are soaked in water to allow metabolic progression without radicle protrusion and subsequently dried to their original weight ([12]). Good crop establishment and seedling emergence assure high crop productivity ([44]), and seed priming is a useful technique for improving early stand establishment, growth and yield of different crops ([27], [29]). Seed priming with micronutrients improves crop productivity and quality. For instance, seed priming with zinc improved grain yield of chickpea by 19% ([33]) and 36% ([6]).

Exogenous application of microbes can improve crop performance, nutrient dynamics and yield under stressed and benign environments ([65]; [18]). Collective use of microbes and fertilisers rapidly supplies nutrients, promotes growth and improves crop yield ([1]; [9]). Bacteria in rhizosphere are more versatile for solubilising, mobilising and transforming essential nutrients than those in bulk soils ([34]). Boron tolerant bacteria (BTB) are of biological interest owing to their ability to work under adverse environments. Hence, BTB can be used as plant growth-promoting rhizobacteria along with B application in B-deficient soils ([56]; [38]).

Seed priming is not new to farmers; most of them are familiar with this hydration technique and effectively use it for gap filling in late-sown crops. Seed priming is recommended for obtaining an ideal stand establishment; however, it has not been adequately adopted by farmers. Although performance of different crops with B seed priming has been improved, information about the effects of B seed priming on chickpea productivity is limited. Likewise, studies regarding the impact of interactive effects of B seed priming and BTB inoculation on chickpea productivity are rare. Thus, the present study was conducted to optimise B level for seed priming with BTB inoculation, to improve stand establishment, growth, grain B content and productivity of chickpea. We hypothesised that different levels of B seed priming will significantly differ in their impact on crop performance, and that higher B levels will prove toxic for seed germination and seedling emergence. The results will help to improve productivity and grain B concentration of chickpea.

Seeds of chickpea variety NIAB-2016 were collected from Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan. The bacterial strain Bacillus sp. MN54 (Accession no. KT375574) was collected from Soil and Environmental Microbiology Laboratory, Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan. The 16S rRNA genes were amplified by using the 8F (50-AGAGTTTGATCCTGGCTCAG-30) ([67]) and 1520R (50-AAGGAGGTGATCCAGCCGCA-30) ([21]) primers for identification of the bacterium. The DNA was extracted by using FastPrep kit (MP Biomedicals, Solon, OH, USA) following the manufacturer's instructions. Amplification was done by using PCR and Thermo Cycler (Thermo Fisher Scientific, Waltham, MA, USA) with 1520R primer (by LGC-Genomics, Berlin, Germany) sequence, and finally, the bacterium was identified by using BLAST analysis on the NCBI database (https://www.ncbi.nlm.nih.gov/). The bacterium strain has been reported to improve the growth of several crops such as maize (Zea mays L.), canola (Brassica napus L.) and chickpea ([49]; [56]; [38]). Boron tolerance of Bacillus sp. MN4 was tested by using different concentrations: 0, 60, 120 and 180 mM. The highest concentration (180 mM) caused minimum reduction in growth compared with the control. Finally, bacterial strain inoculum was prepared in a 500-mL Erlenmeyer flask containing 200 mL tryptic soy broth of 10% concentration. The broth was inoculated and shaken in a shaking incubator at 180 rpm for 24 h at 28 ± 2°C ([49]). The optical density of the strain was adjusted to 0.5 (bacterial population 109 cfu mL−1) before inoculation. Bacillus sp. MN 54 had been isolated from the rhizosphere of maize. The strain was then characterised for various growth-promoting traits (i.e. IAA production, phosphorus (P) solubilisation, ACC deaminase activity, siderophore production) and identified through molecular 16S rRNA gene sequencing. The accession number provided in the methodology was obtained after depositing the sequence to NCBI, Bethesda, MD, USA.

The pot experiment was conducted at the wire house of Department of Agronomy, Bahauddin Zakariya University, Multan, Pakistan, during the rabi season (winter) 2018–19. Before sowing, soil samples were collected and analysed to determine physico-chemical properties. The samples were air-dried and passed through a 2-mm sieve. The Walkley–Black method ([51]) was followed to determine organic matter content. Particle size distribution was determined by the hydrometer method in a sedimentation cylinder, using sodium hexametaphosphate as dispersing agent ([30]). The pH and electrical conductivity (EC) were measured in a saturated soil paste ([53]). Total P and potassium (K) were analysed by following [52] and [15], respectively. Total nitrogen (N) in the soil was determined by the Kjeldahl method ([13]). Boron was analysed according to [43]. The analysis revealed that the soil was a sandy-loam with pH 8.50, EC 2.66 dS m−1, 0.63% organic matter and 0.021% N. The B, P and K contents were 0.49, 7.80 and 240 mg kg−1, respectively.

Seeds were soaked in 0.01%, 0.1%, 0.2% and 0.5% (w/v) B solutions (osmo-priming) or in distilled water (hydro-priming) for 8 h under well-aerated conditions at a 1:5 (w/v) ratio. Dry and hydro-primed seeds were considered as control treatments. After priming, seeds were washed with distilled water three times and dried to their original weight under shade at 27 ± 3°C. These seeds were then used in the experiment. Pure culture of Bacillus sp. MN54 was applied at 5 mL per pot (bacterial population of 109 cfu mL−1). Higher B levels (0.1%, 0.2% and 0.5%) retarded seed germination. Therefore, treatments were revised to 0.0001%, 0.001%, 0.01% and 0.1% B by dilution. Thereafter, seeds were primed according to the revised treatments. Boric acid (H3BO3; Merck, Darmstadt, Germany) was used as B source for the preparation of solutions. There was no pH adjustment for the B solutions because of the low B concentration used. Two sets of pots with the B treatments were prepared for the experiment. One set was inoculated with BTB (Bacillus sp. MN54) and the other was left untreated (without BTB). The experiment was laid out as a completely randomised design with factorial arrangements and all treatments had three replications. Boron seed-priming levels were main plots, whereas BTB inoculation was randomised in subplots. Five seeds were sown in each pot on 27 October 2018; two plants were maintained until maturity from which to record data for yield-related traits. Earthen pots having 50 cm height and 22 cm internal diameter were filled with 20 kg soil and used for planting. Nitrogen and P were applied at sowing at 20 and 40 mg kg−1 soil, using urea and triple superphosphate fertilisers, respectively. Weeds were controlled manually. Pots were irrigated every 3–4 days to exclude the impacts of moisture stress on plant growth. The plants were harvested on 3 April 2019 to obtain data for yield and related traits.

The experiment was visited daily to record stand establishment data until seedling emergence became constant. The day at which the first seedling emerged after seed sowing was regarded as days to start emergence for the respective treatment. Time to complete 50% emergence (E50) and mean emergence time (MET) were calculated following the formulae of [24] and [22], respectively:

Graph: CP21377_UE1.gif

where N is total number of seedlings emerged, ni is number of seedlings emerged at time ti, and nj is number of seedlings emerged at time tj; and:

Graph: CP21377_UE2.gif

where D is number of days noted after emergence and n is total number of seedlings emerged on day D.

Final emergence percentage was calculated as a ratio of number of emerged seeds to total seeds sown. To count number of nodules per plant, roots were separated from each plants and washed it carefully with tap water. The nodules were counted manually from each root and mentioned as number of nodules per plant.

Total number of branches from each plant was counted at maturity, and data were averaged. The pods present on each plant were separated, counted and averaged to record number of pods per plant. Similarly, all pods present on each plant were opened and number of grains inside the pods was counted and the data averaged to record number of grains per pod. All grains present in the pods harvested from each plant were weighed to obtain grain weight per plant, which was divided by the total number of pods present on the plant to obtain grain weight per pod. From each treatment, 25 seeds were taken and weighed, and the weight was converted to 100-grain weight. The mature plants from each pot were harvested, dried and weighed to record biological yield per plant. Harvest index was calculated as:

Graph: CP21377_UE3.gif

Boron concentration in grains was calculated by placing a 1-g grain sample from each treatment in a porcelain crucible and ashing in muffle furnace for 6 h at 550°C ([17]). Ashed samples were extracted for 1 h by adding 10 mL 0.36 N H2SO4. After extraction, grain B concentration was determined by using spectrophotometer following [38].

Yield attribute and grain B concentration data were tested for normality by Shapiro–Wilk normality test ([57]), which indicated that the data were normally distributed. Therefore, original data were used in the statistical analysis. Two-way analysis of variance (ANOVA) was performed ([60]). Least significant difference (l.s.d. at P = 0.01) was used as post hoc test to separate means where ANOVA indicated significant differences. The interactions are presented and interpreted where significant, and main effects are discussed where the interaction was non-significant.

Seed priming with B significantly affected stand establishment traits time to start emergence, MET, E50 and final emergence percentage, whereas BTB inoculation had significant impact only on MET (Table 1). Seed priming × BTB inoculation interaction was non-significant for all stand establishment traits (Table 1). Seeds primed with 0.0001% and 0.001% B solution showed minimum time to start emergence, MET and E50, with higher FEP (Table 2).

Graph

Table 1.  Statistical summary indicating the impact of seed priming, BTB inoculation and their interaction on studied parameters.

*P < 0.05; **P < 0.01; n.s., not significant.

Graph

Table 2.  Impact of boron seed priming and BTB inoculation on early stand establishment of chickpea crop.

Values are means ± s.e. Within a column and main effect, means followed by the same letter are not significantly different at P = 0.01; n.s., not significant.

Boron seed priming combined with BTB inoculation significantly improved chickpea yield by increasing total number of nodules, number of branches and number of pods per plant. Seed priming also affected the number of grains per pod, whereas inoculation with BTB had no significant effect on number of grains per pod (Table 1). The interaction seed priming × BTB inoculation was significant only on number of nodules and number of branches per plant (Table 1). Seeds primed with 0.001% B solution produced the highest numbers of pods per plant and grains per pod (Table 3). Likewise, BTB inoculation significantly improved number of pods compared with no BTB inoculation (Table 3). Regarding the seed priming × BTB inoculation interaction, seed priming with 0.001% B combined with BTB inoculation recorded higher numbers of nodules and branches per plant than other treatments (Table 4).

Graph

Table 3.  Impact of boron seed priming and BTB inoculation on number of pods per plant, number of grains per pod and 100-grain weight of chickpea.

Values are means ± s.e. Within a column and main effect, means followed by the same letter are not significantly different at P = 0.01; n.s., not significant.

Graph

Table 4.  Interactive effect of boron seed priming and BTB inoculation on number of nodules and number of branches per plant of chickpea.

Values are means ± s.e. For each trait, means followed by the same letter are not significantly different at P = 0.01.

Boron seed priming combined with BTB inoculation had significant effects on 100-grain weight, grain and biological yields, and grain B content, but not on harvest index (Table 1). The interaction seed priming × BTB inoculation had significant effect on grain and biological yields, and grain B concentration (Table 1).

Seed priming with 0.001% B solution coupled with BTB inoculation significantly improved 100-grain weight (Table 3). Regarding the seed priming × BTB inoculation, priming with 0.001% B in combination with BTB inoculation recorded higher grain and biological yields per plant than dry seeds sown without BTB inoculation (Table 5). Moreover, priming with 0.1% B solution along with BTB inoculation recorded higher grain B concentration, whereas dry and hydroprimed seeds without BTB inoculation recorded the lowest B concentration in grain (Table 5).

Graph

Table 5.  Interactive effect of boron seed priming and BTB inoculation on grain and biological yields, and grains B concentration of chickpea.

Values are means ± s.e. For each trait, means followed by the same letter are not significantly different at P = 0.01.

Numbers of nodules, pods and seeds per plant had strong positive correlation with grain yield under both non-inoculated and BTB-inoculated treatments. However, number of seeds per pod had positive correlation with grain yield only under non-BTB inoculated treatments. However, 100-grain weight had no significant correlation with grain yield under either inoculation treatment (Table 6). Number of nodules, pods and seeds per plant, number of seeds per pod, and 100-grain weight had no significant correlation with grain B concentration under either inoculation treatment (Table 6).

Graph

Table 6.  Correlation of different traits of chickpea with grain yield and grain B concentrations.

*P < 0.05; **P < 0.01; n.s., not significant.

The study indicated that seed priming with B improved stand establishment, growth, yield and grain B concentration of chickpea compared with non-primed seeds. Moreover, B seed priming combined with BTB Bacillus sp. MN54 inoculation further improved the performance and grain B concentration of chickpea. Boron seed priming (0.001%) along with BTB inoculation resulted in early emergence and seedling establishment of chickpea, as indicated by lower values of time to start germination, E50, MET and final emergence percentage (Table 2). Seed priming results in faster and rapid germination, and improves crop uniformity ([47]). The reason for faster germination is the activation of hydrolytic enzymes, which enhances seed physiology ([7]). Furthermore, B is responsible for remobilisation of essential nutrients in seed during germination, which induce rapid germination ([10]). Seed priming reduces the time of the lateral germination stage, which promotes rapid emergence ([24]). Seed priming with B under no tillage resulted in quick and synchronised emergence, ending with higher yield and quality in wheat ([45]). However, non-primed seeds had irregular crop stand, which resulted in poor yield ([50]). Boron application improved rice (Oryza sativa L.) growth, yield and economic returns ([36]). Abiotic factors reduce crop yield, but seed priming imparts resistance so that the crop can thrive under unfavourable conditions ([25]). Boron deficiency is mostly observed in sandy, highly leached, calcareous and high pH soils ([11]; [5]). Seed priming reduces imbibition time, which increases germination ([14]), metabolite production and enzyme activation ([37]) and repair of damaged DNA ([26]), and regulates osmosis. [19] found that osmopriming with salts improved shoot and root length relative to control or non-primed seed.

The present study indicated that seed priming with 0.001% B solution along with BTB inoculation produced more nodules, branches and pods per plant and grains per pod, and finally improved grain yield, grain B content and biological yield (Tables 4 and 5). The study further revealed that increases in yield-related traits might be linked to better nodulation (Table 6). Grain yield directly depends on number of pods per plant and 100-grain weight; thus, higher number of pods would produce more grains and higher 100-seed weight ([55]). Seed priming boosts metabolic activities, resulting in early emergence. Seed germination can be enhanced by using physiological treatments, which are simple and easy approaches to improving seed quality ([8]). Boron application through seed priming increased grain B content of lentil (Lens culinaris Medik.), chickpea and wheat ([39]). [32] revealed that on-farm priming in different regions improved crop yields by 0–200% with an average increase of 30% ([32]). In our study, seed priming with low to medium B levels along with BTB inoculation improved chickpea grain yield by 43% (Table 5), whereas higher B levels proved toxic. According to [38], B application along with BTB inoculation improved 100-grain weight and grain and biological yield over no B application. Moreover, improved nodulation and number of branches due to seed priming directly correspond to higher water and nutrient absorption because of the extensively developed root system. The extent of correlations observed for grain B content, growth and yield-related traits are in line with the results reported by [16].

Higher grain B concentration was recorded with 0.1% B priming combined with BTB inoculation (Table 5). This might be due to sufficient B availability in the root-zone from the higher B level, and the BTB solubilising B and making it available for plant roots and finally translocation toward grains. Co-application of bacteria with synthetic fertilisers improves crop growth through increased uptake of essential micro- and macronutrients ([9]; [37]), and the same was recorded in our study. Bacteria capable of adhering to plant roots are more versatile in mobilising, solubilising and transforming nutrients ([34]). Inoculation with BTB (Bacillus sp. MN-54) improved yield and grain B contents of chickpea, which might be attributed to accelerated nutrient supply. Bacillus sp. MN-54, previously isolated from maize rhizosphere ([48]) and having the ability to improve growth, yield and physiology of various crops ([2]; [41]), was used along with B seed priming to improve growth and yield attributes of chickpea. Recently, [56] reported better canola growth due to auxin production after Bacillus sp. MN-54 inoculation. Seed priming improves seed germination, seedling emergence, stand establishment, crop growth, nodulation and productivity in pulses ([54]; [28]; [42]). Boron is an obligatory nutrient to crop plants and humans; however, the toxicity and deficiency range of B is very narrow. Our results confirmed that B seed priming at higher levels negatively affected seed germination of chickpea. Higher B levels (0.1%, 0.2% and 0.5%) initially used for priming resulted in no germination (data not shown). Boron toxicity at higher levels has been reported for chickpea ([63]). Principally, very little B is needed because its toxicity and deficiency are both damaging for crop plants. Boron toxicity radically affects plant physiology and biochemistry, leading to stunted growth, which hampers crop yield ([64]). A lower B level (0.001%) was more effective in improving growth, nodulation and productivity of chickpea.

Seed priming with boron (0.001% concentration) in combination with BTB (Bacillus sp. MN54) inoculation improved nodulation, yield and grain B contents of chickpea. Moreover, higher grain B concentration was recorded under higher B levels along with BTB inoculation. Higher B levels were found toxic for chickpea emergence. Therefore, seed priming with 0.001% solution along with BTB (Bacillus sp. MN54) inoculation is a suitable choice for improving yield and grain B concentration of chickpea.

All relevant data are within the paper. However, the raw replicated data that support this study will be shared upon reasonable request to the corresponding author.

The authors declare that they have no conflicts of interest.

The corresponding author (Dr Mubshar Hussain) is highly obliged to the Higher Education Commission of Pakistan for financial assistance to accomplish this study under NRPU grant no. 6998/Punjab/NRPU/R&D/HEC/2017.