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. 2020 Mar 3;10(4):154. doi: 10.1007/s13205-020-2141-6

Priming maize seeds with cyanobacteria enhances seed vigour and plant growth in elite maize inbreds

Vikas Sharma 1, Radha Prasanna 1,✉, Firoz Hossain 2, Vignesh Muthusamy 2, Lata Nain 1, Shrila Das 3, Yashbir Singh Shivay 4, Arun Kumar 5
PMCID: PMC7054569  PMID: 32181116

Abstract

Biofilm formation of a nitrogen-fixing cyanobacterium Anabaena torulosa with a beneficial fungus Trichoderma viride (An-Tr) was examined under laboratory conditions. A gradual enhancement in growth over A. torulosa alone was recorded in the biofilm, with 15–20% higher values in nitrogen fixation, IAA and exopolysaccharide production illustrating the synergism among the partners in the biofilm. To investigate the role of such biofilms in priming seed attributes, mesocosm studies using primed seeds of two maize inbred lines (V6, V7) were undertaken. Beneficial effects of biofilm (An-Tr) were recorded, as compared to uninoculated treatment and cyanobacterial consortium (Anabaena–Nostoc; BF 1-4) at both stages (7 and 21 DAS, days after sowing) with a significant increase of more than 20% in seedling attributes, along with 5–15% increment in seed enzyme activities. More than three- to fivefold higher values in nitrogen fixation and C-N mobilizing enzyme activities, and significant increases in leaf chlorophyll, proteins and PEP carboxylase activity were observed with V7-An-Tr biofilm. Cyanobacterial inoculation brought about distinct changes in the soil phospholipid fatty acid profiles (PLFA); particularly, significant changes in those representing eukaryotes and anaerobic bacteria. Principal component analyses illustrated the significant role of dehydrogenase activity and microbial biomass carbon and distinct elicited effects on soil microbial communities, as evidenced by the PLFA. This investigation highlighted the promise of cyanobacteria as valuable priming options to improve mobilization of nutrients at seed stage, modulating the abundance and activities of various soil microbial communities, thereby, enhanced plant growth and vigour of maize plants.

Electronic supplementary material

The online version of this article (10.1007/s13205-020-2141-6) contains supplementary material, which is available to authorized users.

Keywords: Cyanobacteria, Biofilms, Maize, Nitrogen, Rhizosphere community, PLFA

Introduction

Cyanobacteria evolved 3.5 billion years back and are considered as one of the oldest life forms on Earth’s biosphere. They are known for generating biogenic oxygen in the ancient environment and represent a major source of fixed carbon and nitrogen (Berman-Frank et al. 2003). Widespread over a range of ecological locations including rice paddies, desert soils, swampy land, etc., these organisms exhibit diverse morphologies and are involved in several beneficial activities in agriculture and environmental sustainability (Venkataraman 1981; Gupta et al. 2013; Renuka et al. 2018). Microbial inoculation is practiced in several crops, as a means of enhancing yields and soil fertility, among which cyanobacterial inoculants play a significant role. They are the primary producers involved in C–N cycling, not only in flooded paddies, but also play beneficial roles in other cereals, legumes, vegetables and flower crops (Mandal et al. 1999; Pandey et al. 2019; Prasanna et al. 2015; Bidyarani et al. 2016; Manjunath et al. 2016; Kanchan et al. 2018).

It is now established that most microorganisms often exist in organized structures called biofilms, when colonizing soil or different plant surfaces, in contrast to their more well-studied planktonic states (Rudrappa et al. 2008). Biofilm mode of growth improves their colonization potential and facilitates proliferation, benefitting both the soil and plant (Velmourougane et al. 2017). Biofilms comprising cyanobacteria along with other plant growth promoting bacteria/fungi are known to facilitate increased nutrient cycling and biocontrol of plant pathogens, and their inoculation can bring about accretion of 40–50 kg N ha−1 (Prasanna et al. 2016; Thapa et al. 2017).

In India, maize occupies 9.21 million hectares, with a production of 28.72 million metric tons. The maize yield in India (3115 kg/ha) is about half as compared to the world (5754 kg/ha) average, which illustrates that there is a lot of scope for improvement in terms of yield and quality traits. Employing high doses of fertilizers to achieve more yields is indispensible under intensive cropping, but blanket application of chemical fertilizers is considered as one of the major constraints to sustain crop production (Chand and Pavithra 2015). The use of inorganic fertilizers is not an ecologically benefitting option, as it is fraught with many undesirable changes in soil health, including increased soil acidity, nutrient imbalance and erosion of microfaunal and microfloral diversity (Verma et al. 2011; Singh and Ryan 2015; Rahman and Zhang 2018). Inorganic fertilizers are fairly expensive for the small and marginal farmers, but the lack of widespread awareness and access to organic options has made farmers rely on chemical fertilizers. Schmidt and Gaudin (2018) undertook a meta-analysis to examine the agronomic potential of biofertilizers in maize and identified important pointers for developing a better framework for their use in future; however, their analyses focussed more on Azospirillum as an inoculant. To understand the benefits of inoculants, and their effects on the diversity and abundance of active microbes in the soil, phospholipid fatty acids (PLFAs) are gaining importance to serve as biomarkers to determine the shape and the structure of rhizosphere microbial communities (Frostegard and Baath 1996; Ramsey et al. 2006).

The intricate interactions between plant genotype and nutrition are known to bring about distinct changes in the microbial activities, besides the taxonomic composition of the microbiome (Baudoin et al. 2003; Berendsen et al. 2003). In our earlier studies, the use of cyanobacterial inoculants proved promising in improving plant growth, soil fertility and providing 25% N savings in elite hybrids of maize (Prasanna et al. 2015, 2016), which is significant as maize is a high-N demanding crop. In the present investigation, a set of elite inbreds were evaluated for their interactions with two promising cyanobacterial formulations, mainly focussing on plant (seed, leaves and roots) and microbiological attributes, besides analyses of microbial communities using PLFA. The hypothesis of this study was that cyanobacterial priming of seeds can lead to stimulation of plant growth, as a result of increased seed metabolic activity; this may result in modulation of soil biological activities and better availability of nutrients to the plant.

Materials and methods

Growth and maintenance of cultures

The organisms used in the study include the cyanobacterium Anabaena torulosa and the fungus Trichoderma viride as the biofilm partner; the members of the cyanobacterial consortium BF1-4 comprising BF1 Anabaena torulosa; BF2 Nostoc carneum; BF3 Nostoc piscinale; BF4 Anabaena doliolum (Prasanna et al. 2009). All these cyanobacterial strains belong to the Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi, while Trichoderma viride (ITCC 2211) was obtained from the Indian Type Culture Collection, Division of Pathology, IARI; New Delhi, India. Potato dextrose agar was used as growth media for Trichoderma and the broth was maintained as a static culture at 30 °C. The axenization of cyanobacterial cultures was done using the method given by Kaushik (1987) and the cultures were grown and maintained in nitrogen-free BG11 medium under a temperature of 27 ± 1 °C, using white light (50–55 µmol photons m−2 s−1) (16:8 h of light:dark cycles) as static cultures in Haffkine flasks, as given earlier (Prasanna et al. 2011, 2013).

Development and characterization of biofilms

Anabaena torulosa was grown in N-free BG11 medium, under light:dark cycles 16:8 under white light (50–55 µmol photons m−2 s−1) and 28 ± 2 °C. Trichoderma viride was grown in potato dextrose broth, incubated at 30 °C in stationary conditions for 1 week. A. torulosa culture was grown for seven days followed by inoculation with one-week-old culture of T. viride, as optimized earlier (Prasanna et al. 2011) and kept under white light (with light: dark cycles 16:8; 50–55 µmol photons m−2 s−1) at 28 ± 2 °C. After one, two, three and four weeks of coculturing, the biofilms were taken out and washed with distilled water and subjected to centrifugation followed by vortexing to generate a homogeneous suspension for analyses (Prasanna et al. 2013). The A. torulosa culture and A. torulosa–T. viride (An–Tr) biofilm were analyzed in terms of the chlorophyll content (for cyanobacterial growth) and colony forming unit (for fungal growth) (Prasanna et al. 2011). Samples were collected in triplicate for analyses using three biological replicate flasks.

To measure total chlorophyll, hot extraction method was employed (MacKinney 1941) and the methanolic extract was used for measurement of the optical density of the supernatant, at 645 and 665 nm, and total chlorophyll calculated in terms of mg/ml culture. Acetylene reduction activity (as an index of nitrogenase activity) was determined using a gas chromatograph (GC), following the method of Hardy et al. (1973). The ethylene produced, denoted as nmoles ethylene produced mg chlorophyll−1 h−1, was measured using gas chromatograph, containing a Porapak N column, attached to a flame ionization detector. IAA was estimated using 2 ml of culture extract, acidified with 1–2 drops of orthophosphoric acid followed by 30 min of incubation with 4 ml of Salkowski reagent (blend of 2% 0.5 M FeCl3 and 35% perchloric acid) in dark at normal room temperature. The absorbance was measured using spectrophotometer at a wavelength of 535 nm and calculated using the standard curve of IAA (Patten and Glick 2002).

PEP carboxylase activity was measured using modified method of Wu and Wedding (1985) by reading absorbance at 340 nm and expressed as µmoles of oxidised NADH mg−1 chlorophyll min−1. Glutamine synthetase activity was measured by recording optical density spectrophotometrically, at 540 nm and expressed as µmol γ-glutamyl hydroxamate produced mg−1 fresh wt min−1, following the method of Dharmawardene et al. (1973). Nitrate reductase activity was measured by diazocoupling method given by Lowe and Evans (1964) by measuring the absorbance at 540 nm and calculated using the standard curve of sodium nitrite. Values were expressed as units/mg fresh weight, where one unit can reduce 1.0 µmole of nitrate to nitrite per minute in a β-NADH system at pH 7.3 at 30 °C.

Glomalin (also referred to as Glomalin-like soil proteins/GRSPs) were estimated using Bradford dye method (Wright and Upadhyaya 1996). The amount of GRSPs was expressed as µg/g fresh weight of culture. To estimate the exopolysaccharide (EPS) production, the biofilm was harvested by filtering the biomass, and analysed following the method of Quesada et al. (1993) and expressed in terms of dry weight. EPS from the supernatant was obtained by centrifugation at 10,000 × g for 20 min at 4 °C and all the samples were stored at 4 °C.

Setting up of mesocosm experiment

Based on screening carried out in field experiments over the last 2 years (2017 and 2018) and their response to microbial inoculation, two inbred genotypes of maize viz., V6 (HKI323PV) and V7 (HKI161PV) were selected. The seeds were obtained from the Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi. Cyanobacteria based formulations viz., BF1-4 (BF1 Anabaena torulosa; BF2 Nostoc carneum; BF3 Nostoc piscinale; BF4 Anabaena doliolum) referred to as (B1), identified as promising, based on the results from earlier field experiments (Prasanna et al. 2015, 2016) and Anabaena torulosa–Trichoderma viride biofilm (An–Tr biofilm-B2), earlier characterized in this study, were used. The formulations were prepared by the addition of cyanobacterial dried biomass equivalent to 100 µg chlorophyll/g carrier, as optimized in our earlier published reports (Prasanna et al. 2015, 2016). The total chlorophyll content in the formulations was determined by extracting with 4 ml of DMSO–acetone mixture (1:1) per 1 g sample of the formulation. The samples were incubated for 48–96 h in the dark at room temperature with intermittent shaking after 24 h. After incubation, the residual filtrate after centrifugation was analysed spectrophotometrically at 663, 645 and 630 nm, and chlorophyll concentration was determined, following the method given by Nayak et al. (2004).

Mesocosm experiments were undertaken in plastic pots of 4″ diameter containing 500 g soil and conducted in the glasshouse located at the National Phytotron Facility, ICAR-Indian Agricultural Research Institute (IARI), New Delhi, during mid-December 2018–mid-January 2019. A total of three plants were maintained for three weeks in the pot under controlled temperature and relative humidity conditions (28 ± 2 °C, 65 ± 5%). Soil was taken from maize field and found to contain 105 kg/ha available N and 0.52% organic C; 60% moisture was maintained in the pots for this experiment. A completely randomized design (CRD) was used to assess the effects of different combinations on various plant and soil parameters.

Overnight presoaked seeds of the two maize inbreds (V6 and V7) were primed with the formulations (BF1-4 and An–Tr). Approximately, 10 g formulation was mixed with 100 seeds, and sticker (1% carboxymethyl cellulose) was added to facilitate a smooth coating on the seeds. Such coated seeds were air-dried in shade, prior to sowing. Uninoculated seeds were used as control. The seeds were not surface sterilized, so that the observations could be applicable to field experiments. All the treatments were applied in triplicate.

Seed germination and enzyme activities

Germination was recorded 96 h after sowing and root length, shoot length, fresh weight and dry weight of plants were measured 21 days after sowing. The activity of seed germination-related and nutrient-mobilising enzymes viz. α-Amylase and invertase were also examined in the seedlings, 96 h after sowing.

For the estimation of amylase activity, 500 mg of crushed seeds each was taken from the inoculated and uninoculated treatments and ground using 0.1 M sodium citrate buffer (pH 5.5) and centrifuged at 10,000 × g at 4 °C for 15 min. The amylase activity was measured from the supernatant (Miller 1959). The amount of enzyme that produced reducing sugars equivalent to 1 µmol of glucose from starch in one minute at 45 °C was defined as one unit of amylase activity.

Invertase activity was measured following the method given by Krishnan et al. (1985). Samples of 500 mg seeds were crushed with 0.02 M sodium phosphate buffer (pH 7.0). The resultant extract was centrifuged at 10,000 × g, 4 °C for 10 min and supernatant was used for enzyme estimation. In terms of liberated reducing sugars by DNSA, the assay mixture containing 100 µl supernatant, 500 µl of 100 mM sucrose and 400 µl of sodium acetate buffer (pH 5.0) was kept for incubation at 37 °C for 30 min. The reaction was stopped by adding 3 ml of 1% DNSA solution and boiled for 10 min. Reducing sugars were measured at 540 nm spectrophotometrically. The amount of enzyme that can hydrolyze sucrose to 1 µmol of glucose in one minute at 37 °C at pH 4.6 was defined as one unit of invertase activity.

Plant parameters

Plants samples were analysed for leaf chlorophyll, leaf PEPcase and leaf proteins. The total leaf chlorophyll was measured using DMSO (dimethyl sulphoxide) extraction method, as stated by Hiscox and Isrealstam (1979). Absorbance was recorded at two wavelengths viz. 645 and 663 nm using a UV–VIS spectrophotometer (Model Evolution 300, Thermo Scientific). Phosphoenol pyruvate carboxylase activity was estimated following the method given by Wu and Wedding (1985). As given earlier; the amount of protein was measured spectrophotometrically at 595 nm by the Bradford assay, using bovine serum albumin as standard.

Phosphate buffer (50 mM; pH 7.5) was used for extracting the contents of the root or shoot samples. IAA was estimated in root samples using Salkowski reagent (blend of 2% 0.5 M FeCl3 and 35% perchloric acid) in dark, at room temperature. The absorbance was measured using spectrophotometer at wavelength of 535 nm and calculated using the standard curve of IAA (Patten and Glick 2002). Lowry method (Lowry et al. 1951) was followed for the estimation of total proteins in the samples.

Root biofilm formation was measured as an index of the colonization potential of the inoculated cyanobacteria/biofilm. The washed roots of maize, devoid of soil particles were taken in a glass tube; 100 mg of root samples was used for analysis. The roots were dipped in 3 ml of 0.1% crystal violet for biofilm assay for 15 min (O'Toole 2011). Surplus stain was discarded and the roots were washed with tap water. Thereafter, 4 ml of 30% acetic acid was used for destaining with incubation period of 10 min. The destained solution was measured spectrophotometrically at 660 nm to calculate the extent of biofilm formation. Microscopic observations on biofilm formation were also undertaken.

Soil parameters

The soil samples were also examined for dehydrogenase activity and microbial biomass carbon estimation. The dehydrogenase assay was done using 6 g of soil, incubated with triphenyl tetrazolium chloride (3%) for 24 h in dark. The enzymatic reaction was terminated using methanol and the absorbance at 485 nm was measured using the supernatant (Casida et al. 1964). The dehydrogenase activity was expressed as µg triphenyl formazon (TPF) produced g−1 d−1. The microbial biomass carbon was estimated using fumigation extraction modified method of Nunan et al. (1998). The amount of MBC was calculated by determining the differences in absorbance at 280 nm in fumigated and non-fumigated soil samples and expressed as µg g−1 soil sample. Soil chlorophyll, as an index of photosynthetic biomass accretion, was measured as described earlier for the formulations spectrophotometrically at 663, 645 and 630 nm, following the protocol optimized by Nayak et al. (2004).

Rhizosphere microbial community analysis

Microbial community analysis was carried out by preparing PLFAs, using the method given by Buyer et al. (2010). Rhizosphere soil samples were collected by bringing the pots to the laboratory, removing the plants and collecting the soil adhering to the roots from all the treatments, at one week and three weeks after sowing. The modified Blight and Dyer extraction method was employed for the estimation of lipid fractions from the rhizosphere soil (5 g each) samples, using 60 m DB-23 column having ID 0.25 mm and 0.25 µm film thickness. Fatty acid methyl esters (FAMEs) were separated using 25 m column with 0.2 mm ID and 0.33 µm film thickness. The conditions used for analysis included: initial oven temperature at 190 °C initially, with ramping up to 285 °C at the rate of 10 °C min−1 rate of change, further to 310 °C for 60 °C min−1, subsequently holding at 310 °C for 2 min. The temperature of the flame ionization detector (FID) was 300 °C whereas injector temperature was set at 250 °C. An internal standard of methyl nonadecanoate was used to calculate the FAMEs concentrations. Various fatty acid types were defined as nmol g−1 soil. Diverse ‘microbial assemblages’ were notionally reckoned from these fatty acids as advocated by Frostegard and Baath (1996), Ringelberg et al. (1997) and Zelles (1999).

The concentrations of PLFAs representing different microbial groups were ordinated using principal component analysis based on the correlation to explore the variability among the treatments using the statistical analysis program R (version 3.5.1).

Statistical analysis

All the data were recorded using three biological samples. Statistical analysis program R (version 3.5.1) was used to undertake the Principal Component analyses based on correlation to explore the variability among the treatments. The interaction effects and means of different treatment combinations were compared using the Duncan multiple range test (DMRT), as given by Duncan (1955).

Results

Biofilm development and characterization

First, biofilm development was investigated by measuring the changes in metabolism and growth of both partners in the biofilms. In terms of growth, highest amount of chlorophyll (4.62 µg ml−1 culture) at 4 weeks after co-culturing (WAC) was observed in the An–Tr biofilm (Fig. 1a), while fungal counts of the partner Trichoderma sp. increased from 106 at the time of inoculation to 107 after 2 weeks, followed by 108 CFU ml−1 in the biofilm after 4 weeks. IAA production was significantly higher in the biofilms, as compared to BF1 at 2, 3 and 4 weeks (Fig. 1b), with almost 25% higher values at the 4 WAC. Biofilm formation also enhanced the concentration of glomalin-related soil proteins, ranging from 37 to 62% increase in the first and fourth week after culturing (WAC), respectively (Fig. 1c). Exopolysaccharide production was positively and significantly influenced by biofilm formation at all weeks of sampling (25–26% enhancement) (Fig. 1d).

Fig. 1.

Fig. 1

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Characterization of BF1 and An–Tr biofilm in terms of a total chlorophyll; b IAA production; c GRSPs and d EPS production. Error bars represent standard deviations in the graphs. Superscripts denote the highest values for the respective weeks based on Duncan’s multiple range test at p, 0.05. a and b in the graphs denote the highest values among the treatments

Nitrogen-fixing potential (nmol ethylene mg−1 chlorophyll h−1), as measured by the acetylene reduction assay (ARA) ranged from 11.84 to 18.59 in these cultures, with highest values in the An–Tr biofilm in the fourth week of coculturing (Fig. 2a); while lowest values were in uninoculated control. On the contrary, the activity of PEP carboxylase showed no significant differences in both BF1 and An–Tr at all stages of growth, although the values increased gradually over the period (Fig. 2b). The T. viride inoculation and biofilm formation led to 5–50% increase over BF1 only, in the glutamine synthatase (GS) activity from first to fourth week after co-culturing (WAC), with a sharp peak after 3 WAC. The nitrate reductase (NR) activity was found to increase by 6% in An–Tr, compared to BF1 alone at the fourth week after co-culturing (WAC), with almost twofold higher values at the 4 WAC stage (Fig. 2).

Fig. 2.

Fig. 2

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Activity of C and N mobilizing enzymes in BF1 and An–Tr biofilm. a Nitrogenase activity (measured as acetylene reducing activity—ARA); b PEP carboxylase activity; c glutamine synthetase activity and d nitrate reductase activity. Error bars represent standard deviations in the graphs. Superscripts denote the highest values for the respective weeks based on Duncan’s multiple range test at p 0.05. a and b in the graphs denote the highest values among the treatments

Maize seed and growth attributes, as influenced by inoculation with cyanobacterial consortium and biofilm

Seed priming of two maize inbreds (V6 and V7) using both the inoculants led to a significant enhancement in germination related enzyme activities and biometric characters, which was more distinct with the biofilm treatment (Table 1). Highest germination (93%) was recorded in the B2 (An–Tr biofilm) inoculated seeds, followed by the B1 (BF1-4) treatment in V6 genotype; while in V7 genotype, both the treatments showed a similar germination pattern (96.7%), but higher than uninoculated control. Plumule length was found significantly higher in treatments B1 (8.67) and B2 (10.67) compared to control (3.67 cm) in V6 as well as in V7 in which B1, B2 and control recorded 10.0, 8.0 and 3.33 cm, respectively (Table 1). Radicle length also increased significantly upon inoculation with these two formulations. In V6, highest length measured in B1 and B2 (24.67 and 24.33 cm, respectively) which was about 24% more than the control. An enhancement of 27.16% and 42.39% was recorded in B1 and B2 inoculated seeds over the control.

Table 1.

Seed germination parameters at 96 h after sowing, as influenced by the application of cyanobacteria-based inoculants in maize inbreds

Treatmenta Germination (out of 10) Plumule length (cm) Radicle length (cm) α-Amylase activity (IU g−1) Invertase activity (IU g−1)
V6B0 8.00b 3.67b 19.67e 723.43f 61.31e
V6B1 9.00ab 8.67a 24.67d 754.23e 64.50c
V6B2 9.33a 10.67a 24.33d 770.73d 69.34a
V7B0 9.00ab 3.33b 30.67c 818.77c 59.95f
V7B1 9.67a 10.00a 39.00b 840.03b 62.00d
V7B2 9.67a 8.00a 43.67a 898.33a 67.98b
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Treatments in column having different superscript are statistically different at 5% level of significance

aV6, V7 denote maize inbreds; B0, uninoculated control; B1, BF1-4; B2, Anabaena torulosa–Trichoderma viride (An–Tr) biofilm

The α-amylase activity ranged from 723.43 to 770.73 IU g−1 with the highest values (898.33 IU g−1) recorded in the genotype V7. Invertase enzyme activity was 5.2% and 13.1% higher (64.50, 69.34 and 61.31 IU g−1 in B1, B2 and uninoculated, respectively) compared to control in the genotype V6; while it was more by 3.4% and 13.4% higher (62.0, 67.98 and 59.95 IU g−1 in B1, B2 and un-inoculated, respectively) compared to control in the genotype V7 (Table 1).

The analyses on plant growth parameters were done on 7, 14 and 21 DAS. Inoculation with Anabaena–Nostoc consortium (BF1-4) and An–Tr biofilms, both in V6 and V7, was higher in terms of total plant length, relative to uninoculated plants (Supplementary Table S1). Root length was 16.44% and 13.72% higher in the inoculated treatments (BF1-4 B1, An–Tr B2) as compared to control (B0) in the genotype V6; in contrast to 2% and 6% higher (17.0, 17.67 and 16.67 cm in BF1-4, An–Tr and un-inoculated, respectively) compared to control in the genotype V7. Fresh weight in V6 showed increases of 12.28% and 21.3% due to inoculation of BF1-4 and An–Tr, respectively; on the other hand these two treatments showed 28.69% and 22.44% higher fresh weight over un-inoculated control in V7. Dry weight in genotype V6 treatments led to enhancement of 3.22% and 8.06% as a result of inoculation of BF1-4 and An-Tr, respectively; while both these treatments showed significantly higher (31.42% and 22.86%) dry weight, as compared to uninoculated control in V7.

Impact on leaf parameters

The PEP carboxylase activity in the leaves of maize inbreds tested with the two inoculants varied from 2.15 to 4.07 µmoles mg−1 fresh wt min−1 in V6 genotype and from 2.11 to 4.12 µmoles mg−1 fresh wt min−1 in V7 genotype (Table 2). The highest values were recorded in the B2 treatment (An–Tr biofilm formulation), with V7 genotype, on both days of sampling. The amount of total chlorophyll in the leaves ranged between 1.46 and 2.25 mg g−1 fresh wt in V6 while it ranged between 1.66 and 2.42 mg g−1 fresh wt in V7 genotype. The highest amounts were recorded in the B2 (An–Tr biofilm formulation) treatment tin V7 genotype, followed by B1 inoculation in the same genotype. Protein content in leaves showed a similar trend, as observed in terms of chlorophyll. In V6 genotype, the highest increase (11.4%) in the amount of proteins was measured with the treatment B1 (BF1-4) over B0 in 7DAS whereas in V7, the highest enrichment (8.63%) was found with B2 (An–Tr biofilm formulation) at 14DAS.

Table 2.

Plant growth attributes (measured in the leaves of maize inbreds) and soil parameters, as influenced by the application of cyanobacteria-based inoculants at 7, 14 and 21 DAS

Treatments* PEP Carboxylase (µmoles g−1 fw min−1) Chlorophyll (mg−1 g fw) Proteins (mg g−1) MBC (µg g−1 soil) Dehydrogenase activity (µg TPF released g−1 soil d−1) Soil chlorophyll (µg g−1 soil)
14 DAS 21 DAS 7 DAS 14 DAS 21 DAS 7 DAS 14 DAS 21 DAS 7 DAS 14 DAS 21 DAS 7 DAS 14 DAS 21 DAS 7 DAS 14 DAS 21 DAS
V6B0 2.15e 2.73e 1.46f 1.56f 1.62f 3.07e 3.20e 3.52f 104.28d 143.51e 244.69d 44.73d 52.75e 55.90f 0.11e 0.14e 0.11e
V6B1 2.92d 3.60c 1.57e 1.89e 1.95e 3.27c 3.37d 3.62d 193.07b 327.29c 591.60b 58.73c 65.96c 70.73d 0.13b 0.16c 0.13b
V6B2 3.05b 4.07b 1.85c 2.17c 2.25d 3.42b 3.52c 3.70c 240.56a 391.30a 658.71a 63.13b 67.69c 74.19c 0.12b 0.17b 0.12b
V7B0 2.11f 2.73e 1.66d 2.05d 2.37c 3.25d 3.36d 3.59e 98.08d 206.49d 304.58c 47.72d 59.67d 62.76e 0.11d 0.15d 0.11d
V7B1 2.96c 3.47d 1.88b 2.26b 2.60b 3.42b 3.55b 3.78b 156.93c 334.52c 626.71ab 62.66b 75.24b 76.44b 0.12c 0.16c 0.12c
V7B2 3.19a 4.12a 2.10a 2.45a 2.82a 3.51a 3.65a 3.85a 205.46b 357.23b 671.10a 69.42a 80.74a 85.61a 0.13a 0.18a 0.13a
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Treatments in column having different superscript are statistically different at 5% level of significance

*V 6, V7 denote maize inbreds; B0, uninoculated control; B1, BF1-4; B2, Anabaena torulosa–Trichoderma viride (An–Tr) biofilm

Impact of cyanobacterial inoculation on soil biological parameters

Microbial biomass carbon (MBC) showed a significant three- to fourfold enhancement, as a result of inoculation over the entire experimental duration in the rhizospheric soil. It ranged from 98.08 to 671.1 µg g−1 with significant increments from 7 to 21 DAS. Highest values were recorded in B2 treatment in both genotypes at 21 DAS (Table 2). Dehydrogenase enzyme activity showed a similar trend in terms of increase over the period of experimentation, and highest values were recorded on all the days of sampling in V7B2. Rhizospheric soils from both the genotypes had higher dehydrogenase activity in inoculated treatments (B1 and B2) over uninoculated treatments (B0). In V6 genotype, highest increase (41.13%) was recorded with the treatment B2 over B0 in 7DAS, whereas in V7, highest enhancement (45.47%) was also found with B2 at 7DAS.

Soil chlorophyll in the rhizosphere samples ranged from 0.11 to 0.17 (mg g−1soil) in V6 and from 0.11 to 0.18 (mg g−1 fresh wt) in V7 with highest values in the treatment B2 at 14 DAS in both the genotypes. The ethylene produced (nmol g−1 h−1) as a measure of nitrogenase activity was found to increase throughout the period of experiment, with B2 leading to a 5-fold and 7.5-fold increase over B0 at 21 DAS in V6 and V7 genotypes, respectively (Fig. 3). Root biofilm formation showed a significant increase in inoculated treatments compared to plants without inoculation in both the genotypes at 7, 14 and 21 DAS. The genotype V7 inoculated with An–Tr biofilm led to the highest relative fold change in root biofilm formation over uninoculated control (0.84, 0.62 and 0.53 relative fold increase over control at 7, 14 and 21 DAS) (Fig. 3).

Fig. 3.

Fig. 3

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Soil biological parameters in the mesocosm experiment with two maize inbreds and inoculation of BF1-4 (Anabaena–Nostoc consortium) and An–Tr biofilm, expressed as fold increase, over uninoculated control B0 in (a) nitrogenase activity (measured as acetylene reducing activity—ARA) and b root biofilm formation. V6: genotype HKI323PV; and V7: genotype HKI161PV; B1: inoculation with BF1-4; B2: inoculation with An–Tr biofilm. Error bars represent standard deviations in the graphs. Superscripts denote the highest values for the respective weeks based on Duncan’s multiple range test at p 0.05. a and b in the graphs denote the highest values among the treatments

Change in microbial community composition

Community profiling by PLFA method was undertaken on 7 and 21 DAS. In the present investigation, the total PLFAs varied from 158.33 to 1211.78 nmol g−1 soil, with the least in V7B0 (control) at 7DAS and highest in V6B2 (HKI323PV inoculated with An–Tr biofilm, B2) (Supplementary Table S2). On 7 and 21 DAS, the soils inoculated with BF1-4 and An–Tr showed higher quantity of total PLFAs in contrast to control without inoculation.. Straight chain and polyunsaturated fatty acids (PUFA) were the highest among all fatty acid types. Distinct variations among the treatments were observed. PUFA comprised 21–45%, while straight chain fatty acids (14–43%) were next in abundance (Supplementary Table S2). Cyanobacterial/biofilm inoculation in these genotypes led to higher concentrations of PLFAs representing eukaryotes and anaerobic bacteria, as compared to those in respective control treatment; however, the concentration of representative PLFA of actinobacteria declined at 7 and 21 DAS (Fig. 4a, b).

Fig. 4.

Fig. 4

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Concentrations of biomarker PLFAs, notionally identified as different groups (calculated as molar percentage of the total area of the chromatogram) and denoted as fold changes compared to the respective soils without inoculation in soil, as influenced by the inoculation with BF1-4 (Anabaena–Nostoc consortium) and An–Tr biofilms, in V6: inbred genotype HKI323PV and V7: inbred genotype HKI161PV of maize. B1: inoculation with BF1-4; B2: inoculation with An–Tr biofilm; a rhizosphere soil samples from 7 and b 21DAS. Soil was sterilized before inoculation

The correlation among various plant and soil attributes along with total PLFA revealed a significantly strong and positive correlation of amounts of total PLFA with leaf PEP carboxylase (0.93, p < 0.05), root biofilm formation (0.86, p < 0.05), soil ARA (0.85, p < 0.05), soil total chlorophyll (0.86, p < 0.05) and with microbial biomass carbon (0.9, p < 0.05). Similarly, soil ARA found to show a significantly very strong correlation with soil total chlorophyll (0.98, p < 0.05), microbial biomass carbon (0.96, p < 0.05), dehydrogenase activity (0.95, p < 0.05), leaf PEP carboxylase activity (0.97, p < 0.05), leaf protein content (0.89, p < 0.05), root biofilm formation (0.98, p < 0.05). Leaf chlorophyll was found to have moderately negative correlation with plant fresh weight (− 0.49, p < 0.05) while strong negative correlation with plant dry weight (0.67, p < 0.05), shoot length (0.94, p < 0.05) and root length (0.69, p < 0.05) (Supplementary Table S3).

Principal component analysis (PCA) illustrated that cyanobacterial/biofilm inoculation had a significant influence on plant, microbial and soil attributes (Fig. 5). On the basis of Eigen values, the first four principal components were determined as major contributors representing 96.76% of cumulative variance in the 7 DAS. Further, the first dimension was able to explain 42.24% (Fig. 5), while the second dimension explained 25.45% of the variance. As affirmed from the loading, leaf related attributes viz. chlorophyll and protein content influenced MBC positively in dimension 1. Further in dimension 2, methanotrophs were identified as dominant factors and their population affected negatively (loading value = − 0.52) by variables in this dimension. At 21 DAS, the uninoculated soils of both the genotypes formed a distinct group away from the PC1 axis of inoculated soils, while both the inoculations in the genotype V7 separated into one group (Fig. 6). It was found that out of six principal components obtained, first three were found as major determinants, depicting 90.27% of cumulative variance. Additionally, the first dimension explained 42.65% and the second dimension explained 38.12% of the variance. Soil microbial attributes such as MBC and dehydrogenase activity were identified as the principal determinants in dimension 1. Further in dimension 2, gram positive bacteria were identified as dominant factors and affected eukaryotes negatively (loading value = − 0.52) in this dimension. The genotype V7 inoculated with BF1-4 was always correlated with greater dehydrogenase activity, MBC, leaf chlorophyll and leaf proteins. In terms of organisms, Gram negative bacteria, fungi, actinobacteria and AM fungi clustered as one group. PCA also distinctly showed that both the uninoculated treatments were found to be away from the grouping of total PLFA, microbial activities and plant attributes (Fig. 6). A total of six dimensions were obtained, among which, based on the size of the Eigenvalues, the first two principal components were identified as the major contributors to 92.34% of cumulative variance. As per the loading, soil related attributes viz. soil chlorophyll, microbial biomass carbon, nitrogenase and dehydrogenase activity were identified as the principal determinants which affected leaf protein content and PEP carboxylase activity positively in dimension 1 (Fig. 7). In dimension 2, plant biometric attributes such as plant fresh weight, root length and shoot length were identified as the dominant factors which affected plant biomass accumulation positively.

Fig. 5.

Fig. 5

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Principal component analysis to illustrate the relationships among plant, soil parameters and abundances of biomarker PLFAs representing different microbial groups in the rhizosphere soils of maize inbred genotypes, as influenced by the inoculation with BF1-4 and An–Tr biofilm. V6: maize inbred genotype HKI323PV; V7: maize inbred genotype HKI161PV; B1: inoculation with Anabaena–Nostoc consortium BF1-4; B2: inoculation with An–Tr biofilm at 7 DAS. The soil for mesocosm study was sterilized before inoculation

Fig. 6.

Fig. 6

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Principal component analysis to illustrate the relationships among plant, soil parameters and abundances of biomarker PLFAs representing different microbial groups in the rhizosphere soils of maize inbred genotypes, as influenced by the inoculation with BF1-4 and An–Tr biofilm. V6: maize inbred genotype HKI323PV; V7: maize inbred genotype HKI161PV; B1: inoculation with Anabaena–Nostoc consortium BF1-4; B2: inoculation with An-Tr biofilm at 21DAS. The soil for mesocosm study was sterilized before inoculation

Fig. 7.

Fig. 7

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Principal component analysis to illustrate the relationships among plant, soil parameters and total amount of PLFAs in the rhizosphere soils of maize inbred genotypes, as influenced by the inoculation with BF1-4 and An-Tr biofilm. V6: maize inbred genotype HKI323PV; V7: maize inbred genotype HKI161PV; B1: inoculation with Anabaena–Nostoc consortium BF1-4; B2: inoculation with An–Tr biofilm at 21DAS

Discussion

Agriculturally important microorganisms employ quorum sensing to adopt biofilm mode during colonization and establishment (Seneviratne et al. 2010), which can help in enhancing the effects of plant–soil–microbe interactions through improved N2 fixation and micro- and macronutrient uptake. Cyanobacteria are important biofertilizers being used in different crops including rice, wheat, maize, tomato and other vegetables (Pedurand and Reynaud 1987; Misra and Kaushik 1989; Karthikeyan et al. 2007; Rana et al. 2012; Prasanna et al. 2016). In the investigation undertaken, firstly, the analyses of biofilm development was undertaken, using cyanobacterium Anabaena torulosa as the matrix with Trichoderma viride as partner, followed by its use as inoculant, using two genotypes of maize. Seed enzyme attributes and germination related parameters were utilized as suitable indices to understand the cyanobacterium-elicited effects on the physiology of the seed for generating a healthy crop.

Biofilm development was investigated by measuring the changes in metabolism and growth of both partners in the biofilms developed using the cyanobacterium Anabaena torulosa; the optimization of biofilm formation using this as the matrix and its significant role as biofertilizers in various crops is well documented (Prasanna et al. 2013, 2015). Results revealed a synergism among the partners leading to gradual but significant increases in the selected attributes, as compared to the individual culture of Anabaena torulosa. An enhancement in IAA in the biofilms compared to A. torulosa alone, indicates that these two organisms need an interaction period to express this activity synergistically. T. viride inoculation with A. torulosa appeared to be collaborative during biofilm formation, illustrated by the increased production of glomalin like proteins and exopolysaccharides which impart stability and ecological fitness in An–Tr biofilm state. Further, the amounts of glomalin and EPS increased over the course of experiment implying the progressive and effective attachment of T. viride mycelia/spores on A. torulosa filaments during biofilm formation, as has been reported earlier (Prasanna et al. 2013). This is the first time that the N-assimilation enzymes have been evaluated in biofilms, which is of significance to improving N-availability to crops in agriculture. Among the various times of sampling, highest growth was shown in the fifth week, although the rate of increase was higher upto the fourth week. Biofilm formation is known to involve an orchestrated series of events, when involving two beneficial and synergistically interacting organisms and results in increments in physiological activities and metabolite production (Rudrappa et al. 2008). Similar results were obtained in the present study, which also support our previously published work on biofilms using A. torulosa (Prasanna et al. 2013).

Further, in order to evaluate the promise of such biofilms, seed priming with cyanobacteria—as monoculture or as biofilm preparations was undertaken. Significant improvement in seed germination, vigour, plant biometric and soil microbial activity were recorded in the two elite maize inbreds. This supports the previously published work on the use of biofertilizers in maize (Prasanna et al. 2015, 2016; Dicko et al. 2018; Dineshkumar et al. 2019). Soil microbiological indicators such as dehydrogenase activity, microbial biomass carbon and soil chlorophyll content were measured; these are important parameters while estimating the contribution of microorganisms in the mobilization of nutrients in the soil (Filip 2002).

In the present investigation, dehydrogenase activity, microbial biomass carbon and total chlorophyll content exhibited a positive correlation with one another, in the inoculated treatments, in both the genotypes. A direct effect of microbial inoculation was recorded on increase in soil ARA values, compared to control. The difference in soil ARA values denotes that type of formulation, specifically, whether a consortium of cyanobacteria or in biofilm mode with T. viride, is decisive in N fixation in rhizosphere soils. In addition, both the genotypes by and large responded in a disparate manner Additionally, synergistic effects of microbial inoculation on plant growth and development could be due to the indirect effect of available N on the soil via increased dehydrogenase activity, microbial biomass carbon and total soil chlorophyll content. Biofilms are well-known to impart additional advantages to the participating organisms, including improved resistance to biotic and abiotic stresses, protection from predators, increased nutrient cycling (Jayasinghearachchi and Seneviratne 2004; Velmourougane et al. 2017), thereby, stimulating plant growth, both directly and indirectly. Dehydrogenase activity is regarded as indicator of microbial activity, as it measures the oxidation–reduction reactions in soil, while MBC is an index of total microbial origin biomass in the soil (exclusive of fresh or decomposed organic matter). The enhanced enzyme activities reflect the stimulation by cyanobacterial inoculation. Mandal et al. (1999) emphasized that cyanobacteria have important roles, besides nitrogen fixation, such as improving the cellular moieties, eliciting enzymatic machinery and plant health as observed in this investigation. Enhancement of most microbiological and plant parameters, illustrated that cyanobacteria/biofilm shows a positive interaction with the maize genotypes tested. Seed priming is one of the most effective methods of improving plant growth and productivity in an environment-friendly manner that employs beneficial microbes which can elicit the enzyme machinery of the seeds leading to greater vigour (Moshynets and Kosakivska 2010). The enhanced growth can be attributed to the enhanced IAA production by biofilms and improved N-availability in soil.

The qualitative and quantitative study of microbial community structure for the presence and abundance of key microbial groups by analysis of phospholipid fatty acid (PLFA) is suggested to be more useful among various polyphasic approaches (Ramsey et al. 2006). The concentrations of total PLFAs that represent active microorganisms increased by seed priming at both stages and highest concentration as observed in BF1 or An–Tr inoculated treatments in the present study. Diverse maize genotypes vary in generating root exudate composition (Corrales et al. 2007). Maize root exudates largely include 2/3 sugars, 1/3 organic acids and a small fraction of amino acids (Baudoin et al. 2003). Plant nutrition is known to affect the root exudate composition, which in turn, results in higher microbial biomass and altered soil community structure (Neumann and Römheld 2001). Microbial inoculation resulted in increased nitrogen availability by affecting N-cycle enzymes and production of growth supporting substances which can lead to variation in community composition. The availability of nutrients and growth stimulants can mutually alter plant exudation, resulting in changes in the dynamics of microbial communities in the rhizosphere. In the present investigation, cyanobacterial inoculation elicited greater relative changes in the composition of eukaryotes and anaerobes, as compared to uninoculated control. Distinct but divergent fatty acid profiles were generated which reflected the significant effects of cyanobacterial consortium /biofilms, as well as the uniqueness of each maize genotype. The overall concentrations of different microbial groups, as denoted by biomarker PLFAs, illustrated that cyanobacterial inoculation distinctly influenced the rhizosphere microbiome. PCA further emphasized that cyanobacterial inoculation influenced not only total PLFAs, but also the composition and abundance of microbial communities, thereby influencing metabolic activities in soil and plant growth and metabolic attributes.

A significant enhancement in these two important microbiological parameters reflects the positive effects on soil beneficial activities. Recently, Chen et al. (2019) emphasized that the root microbiomes of maize cultivars are more influenced by soil characteristics, rather than cultivar type; this brings to fore the importance of microbial activities in the rhizosphere. The rhizosphere microbial community composition and their activity are liable to change as a function of time and host genotype, since the changes occur in the root morphology, nutrient availability and exudation patterns of roots with plants age and genotype (Di Cello et al. 1997; Berendsen et al. 2012). By provisioning organic compounds/nutrient supply, the root exudates play a significant role as a key plant-derived factor which also elicits colonization of roots (Lugtenberg et al. 1999) biofilm attachment (Walker et al. 2004; Noirot-Gros et al. 2018). Additionally, water availability or hydration is one of the most important factors in successful establishment of the biofilms in the rhizosphere (Chang and Halverson 2003).

PCA further helped to substantiate the significant role of cyanobacterial inoculation in improving the microbial activities, which in turn improved photosynthetic biomass, as a result of improved C and N cycling in soil. Beauregard et al. (2013) emphasized on the role of biofilms and plant-associated polysaccharides in improving colonisation of inoculated microbes, such as Bacillus subtilis. Analyses of these fatty acid types as biomarkers to denote the notional groups of microorganisms, showed that the cyanobacterial inoculation led to a distinct influence on the rhizosphere microbiome, as observed earlier in rice (Ranjan et al. 2016). It is well established that biofilm formation is an integral part of plant–microbe interactions and several PGPR, especially Rhizobium, Bacillus species are known to use the biofilm mode for colonisation, however, cyanobacterial biofilms are a less investigated area of research.

Conclusions

An understanding of the mutual interaction between modulation of rhizosphere environment through microbial inoculation and genetic determinants of host plants appears to be a prerequisite for evaluating the impact produced by a microbial inoculant which could affect an array of plant growth promoting activities. Consequently, the influence exerted by bioinoculants on microbial community structure has practical importance; the results can be used to assess the intrinsic patterns of interactions for developing stronger linkages. Although there is a lot of published work on using chemical stimulants to prime the seeds, the use of microbes to bring about similar effects is a more promising option, as these represent multifunctional supplements, which can modulate the populations and activities of the microbial communities in the rhizosphere, towards a beneficial holobiome and higher crop productivity.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOC 73 kb) (73.5KB, doc)

Acknowledgements

The authors are thankful to the Division of Microbiology, Post Graduate School and Director, ICAR-IARI (New Delhi, India) for providing necessary facilities towards the Ph.D. program of the first author, who also is grateful to SKRAU, Bikaner, for deputation on study leave. This study was supported partially by the grants from the ICAR-Network Project on Microorganisms ‘Application of Microorganisms in Agriculture and Allied Sectors’ (AMAAS) granted by Indian Council of Agricultural Research (ICAR), New Delhi to RP. The authors are also thankful to Mr. Gulab Singh for his help in the soil analyses and Mr. Suresh Kumar, Division of Agricultural Economics, ICAR-IARI, New Delhi for assisting in the statistical analyses. Funding was provided by National Bureau of Agriculturally Important Microorganisms (T12/22).

Author contributions

VS conducted the experiments, recorded the scientific data. RP outlined the hypothesis, designed, assisted in the execution of experiments, and facilitated lab resources, including instrumentation and chemicals to VS. RP and VS interpreted the data and wrote the final manuscript. FH and VM provided plant material and guided in the establishment of experiments. LN and SD provided facilities and expertise for analyses. YSS facilitated the nutrient analyses and its interpretation. AK assisted in the arrangements for experimental setup and its maintenance.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest and no competing financial interests.

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