CC BY-NC-ND
0July 2016. Horticultural Plant Journal, 2 (4): 229-239.
Horticultural Plant Journal
CrossMark
Available online at www.sciencedirect.com The journal's homepage: http://www.joumals.elsevier.com/horticultural-plant-joumal
Chrysanthemum Growth Gains from Beneficial Microbial Interactions and Fertility Improvements in Soil Under Protected Cultivation
Radha Prasannaa*, Amrita Kanchana, Simranjit Kaura, Balasubramanian Ramakrishnana, Kunal Ranjana, Mam Chand Singhb, Murtaza Hasanb, Anil Kumar Saxenaa, and Yashbir Singh Shivay c
a Division of Microbiology, ICAR — Indian Agricultural Research Institute, New Delhi 110012, India b Centre for Protected Cultivation Technology (CPCT), ICAR —Indian Agricultural Research Institute, New Delhi 110012, India c Division of Agronomy, ICAR — Indian Agricultural Research Institute, New Delhi 110012, India
Received 14 May 2016; Received in revised form 20 June 2016; Accepted 7 July 2016
Available online 29 November 2016
Abstract
An investigation was undertaken to analyse the influence of microbial inoculants on growth and enzyme activities elicited, and soil microbiome of two varieties of Chrysanthemum morifolium Ramat, which were grown under protected mode of cultivation. Rhizosphere soil sampling at 45 and 90 DAT (days after transplanting of cuttings) revealed up to four- to five-fold enhancement in the activity of defence-, and pathogenesis-related, and antioxidant enzymes, relative to the uninoculated control. Plant growth and soil microbial parameters, especially soil microbial biomass carbon and potential nitrification exhibited significant increases over control. Available soil nitrogen concentrations showed 40%-44% increment in inoculated treatments. Scanning electron microscopy of the root tissues revealed biofilm-like aggregates and individual short bits of cyanobacterial filaments. Analyses of DGGE profiles of archaeal and bacterial communities did not show temporal variations (between 45 and 90 DAT). However, distinct influences on the number and abundance of phylotypes due to microbial inoculants were recorded. The inoculants — Cyanobacterial consortium (BF1- 4) and Anabaena sp.-Trichoderma sp. biofilm (An-Tr) were particularly promising in terms of the plant and soil related parameters, and remained distinct in the DGGE profiles generated. The effect of Trichoderma viride-Azotobacter biofilm on soil bacterial and archaeal communities was unique and distinct as a separate cluster. This study highlights that microbial inoculants exert positive effects, which are specific even to the rhizosphere soil microbiome of chrysanthemum varieties tested. Such inoculants can serve as soil fertility enhancing options in protected floriculture.
Keywords: microbial interaction; biofilm; cyanobacteria; DGGE; floriculture; soil fertility
1. Introduction
Protected cultivation of crops has emerged as a promising option globally in the last decade. The major advantage of protected mode of cultivation is the significant reduction in losses, due to extremes of temperature, pests and disease incidence. However, due to intensive cultivation year round and a closed environment, a rapid decline in organic matter and nutrient levels is observed along with deterioration in physical properties of soil. Improved physical, chemical and biological properties of these soils demand the application of management practices such as the application of organic materials (Saha et al., 2008). The sustainability of soils, especially in relation to the quality and
time dependent changes of the soil, deserves immediate attention in the protected mode of cultivation (Karlen et al., 1997).
Biofertilizers are a low cost environment friendly sustainable agronomic option as they can contribute to mobilization, mineralization and recycling of nutrients in an effective manner (Chaudhary, 2010). Among biofertilizers, cyanobacteria are commonly deployed in rice and more recently in other crops including wheat, cotton, legumes and vegetables (Prasanna et al., 2014, 2015). They produce a wide range of bioactive molecules known to be necessary for plant growth (Obana et al., 2007; Maqubela et al., 2009). Cyanobacteria are also known to enhance the production of secondary metabolites in plants, including essential oils in Mentha piperita L. and antioxidants in Lilium alexandrae
* Corresponding author. Tel.: +91 11 25847649 E-mail address: radhapr@gmail.com
Peer review under responsibility of Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)
http://dx.doi.org/10.1016/j.hpj.2016.08.008
2468-0141/© 2016 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
(Shariatmadari et al., 2015). Several bacteria, including cyano-bacteria, as well as fungi mediate such processes which results in better growth and nutrient mobilization (Pieterse et al., 2014; Prasanna et al., 2014; Triveni et al., 2015). The deployed microbial consortia and biofilmed inoculants in this investigation have been used extensively in various crops and positive impacts on growth, yields an soil nutrient mobilization have been recorded (Prasanna et al., 2013, 2014, 2015; Triveni et al., 2015; Manjunath et al., 2016). Although a number of reports are published on biofertilizer-mediated improvement in flowering and quality attributes in gladiolus, tuberose and jasmine (Dalve et al., 2009; Srivastava et al., 2013), their role in improving the fertility of soil or elicitation of plant innate immunity, particularly in such a protected mode of cultivation is a less investigated area.
Chrysanthemum displays a wide range of variability among the economic floral traits and has excellent keeping quality among its cultivars due to a wide range of colours, size, and forms. It is an annual plant that requires a long day for vegetative growth and a short day for flowering, with availability throughout the year.
Soil microbial community is highly complex and dynamic with variations in the composition both spatially and temporally. Inoculation through enrichment of soil or seed bacterization can lead to changes in the structure of the indigenous microbial communities. Viewing the microbiota from an ecological perspective can provide new insights into how to promote soil health and plant productivity. The proposed research work is aimed towards understanding the following: (i) Can microbial inoculants improve the growth and improve the fertility of soil in the chrysanthemum crop under protected mode of cultivation, (ii) Does this inoculation influence the bacterial and archaeal microbial communities in the rhizosphere, and (iii) Are there any relationships between the PCR-DGGE (denaturing gradient gel electropho-resis) analyses and the performance of inoculants, in terms of changes brought on plant growth or soil nutrient parameters under protected cultivation of chrysanthemum.
2. Material and methods
2.1. Experimental site
The study was conducted at the Centre for Protected Cultivation and Technology (CPCT), ICAR-Indian Agricultural Research Institute (IARI), New Delhi-110012 (latitude 28°38' N, longitude 77°12'E and altitude 228.4 m) between November 2014 and March 2015. Two cultivated varieties of Chrysanthemum morifolium Ramat (thereafter referred to as chrysanthemum)-'Golden Ball'and 'White Star', were planted in November 2014 in soil beds (15 m long and 1.25 m wide) at a density of 4 plants • m-2 (with three rows) in a randomized block design with 3 replications, and each replication contained 20 plants in a naturally ventilated greenhouse. The 30 d old plug plants raised in soil less media composed of a mixture coco-peat, vermiculite and perlite (3:1:1, w/w), were transplanted. The plants were maintained as mother stock and grown under long days (>13 h day length, with the light given for fixed hours during the night); thus retained at the vegetative stage as a result of low temperature. After planting, these plug plants (cuttings) were drip-fertigated on the 10 cm raised beds in a naturally ventilated greenhouse.
The average monthly temperature and humidity of the greenhouse varied from 14 °C to 22 °C and 62% to 75%, respectively. The average minimum and maximum solar radiation was found to be between 225 and 285 Watt • m-2 inside the greenhouse. The number of irrigations for chrysanthemum varied from 4 to 8 during the study period. The irrigation quantity varied from 200 to 400 litres for the chrysanthemum grown in 50 m2 area. Fertigation for the major nutrients- N, P and K varied from 75-90, 40-60 and 60-80 ^g • L-1 during the study period, with an average of 21, 13 and 9 g • m-2, respectively. During the study period, the EC and pH were maintained at 1.3-1.5 dS • m-1 and 7.5 respectively for irrigation and 1.8-2.2 dS • m-1 and 7.0 for fertigation.
2.2. Details of microorganisms and inoculation treatments
All the strains used in this investigation are available in the germplasm of the Division of Microbiology, ICAR — Indian Agricultural Research Institute, New Delhi and their details are given in earlier investigations (Prasanna et al., 2008, 2011, 2014).
Formulations were prepared using optimized protocols with compost: vermiculite as carrier with bacterial or fungal partners maintained at 107-1010cfu • g-1 and chlorophyll a content of 100 ^g • g-1 carrier, as described in our earlier investigations (Prasanna et al., 2011, 2014; Triveni et al., 2012, 2015). Microbial inoculation at the rate of 1 g per cutting (plug plants) was done before placing the cutting in the holes. The treatments included: Control, no inoculation; Anabaena sp.-Trichoderma sp. biofilm (An-Tr); Anabaena-Azotobacter sp. biofilm (An-Az); Cyanobacterial consortium of BF1 Anabaena torulosa; BF2 Nostoc carneum; BF3 Nostocpiscinale; BF4 Anabaena doliolum (BF1-4); Trichoderma viride-Azotobacter biofilm (Tr-Az). The rhizosphere soil sampling was done after 45 days and 90 days after transplanting of cuttings (plug plants).
2.3. Assay of defence and hydrolytic enzyme activities in root and shoot tissues
The whole plant samples were collected after 45 and 90 days of transplanting of the cuttings. The leaf and root tissue samples were homogenized using 50 mmol • L-1 Tris-HCl buffer, Polyphenol oxidase (PPO) activity was measured using catechol, which served as the substrate (Jennings et al., 1969). The enzyme activity was determined spectrophotometrically at 546 nm and the changes in absorbance were recorded at 30 s intervals for 3 min. Phenylalanine ammonia-lyase (PAL) activity was assayed in the tissue extracts of leaves and roots by measuring the amount of trans-cinnamic acid formed from L-phenylalanine spectropho-tometrically at a wavelength of 290 nm against the blank (Beaudoin-Eagan and Thorpe, 1985).
Chitosanase (EC 3.2.1.99), endoglucanase (6-1,3-glucanase and ^-1,4-glucanase; EGases, EC 3.2.1.39 and EC 3.2.1.4 respectively) activities were assayed spectrophotometrically using glycol chitosan, laminarin and carboxy methyl cellulose respectively, as substrate, as given in Prasanna et al. (2013, 2015).
2.4. Analyses of soil microbial parameters and available nutrient concentrations
Soil samples from the rhizosphere of two varieties were collected at 90 DAT from 0 to 20 cm depth. By using the 6 g soil
samples, dehydrogenase activity was assayed, after incubation with 3% Triphenyl Tetrazolium Chloride (TTC), extracting with methanol and the absorbance was measured at 485 nm (Casida et al., 1964). The values were expressed as micrograms ofTriphenyl Formazon (TPF) per g of soil per day. For the potential nitrification activity, the soil samples incubated at the medium of Schmidt and Belser (1982) and the activity was expressed as Potential Nitrification Activity (PNA) as described by Olsson and Falkengren-Grerup (2000). Microbial carbon biomass was measured (Nunan et al., 1998) using aliquots of K2SO4 extracts through dichromate digestion, and calculated after back titration with ferrous ammonium sulphate using the equation: where EC biomass C = 2.64 x EC = (Organic C from fumigated soil) - (Organic C from unfumigated soil), expressed as mg • kg-1 soil; where EC is the amount of C extracted by fumigation.
Following the method of Lin (2005), the total polysaccha-rides were assayed in the soil samples and OD recorded at 490 nm. Based on comparison with known standard solutions of glucose, the polysaccharide content was expressed in mg • g-1 soil. Soil organic carbon was measured using the methodology of Hesse (1971) and the values were expressed as percent carbon. The concentrations of available nitrogen were estimated using the alkaline permanganate method (Subbiah and Asija, 1956). Available phosphorus (P) concentration in soil was estimated by the method of Olsen et al. (1954) and the blue colour intensity of reduced solution recorded at 600 nm.
2.5. Plant growth and biometric parameters in White Star/Golden Ball
Fresh weight of the plants was taken on 90 DAT. Dry weight of plants was estimated by keeping the plants at 60 °C in hot air oven, till constant weight was obtained. The plant biometric parameters such as plant height, number of leaves, stem diameter were measured by standard procedures. The photosynthetic pigments were measured using fresh leaves by extraction with DMSO (dimethyl sulphoxide), following the method of Hiscox and Israelstam (1979). The spectrophotometric measurements were taken at 480, 510, 645 and 663 nm using a UV VIS spectrophotometer (Model Evolution 300, Thermo Scientific) and values of chlorophyll a, b and carotenoids calculated using the equations given by Arnon (1949). All measurements were done with a minimum of three plants.
2.6. Scanning Electron Microscopy (SEM) of root samples
Root segments (1 mm diameter) of variety White Star and Golden Ball were taken from the plants at 45 DAT and the processed samples were observed using SEM (Zeiss, EVO MA10, Germany) using 20 kV/EHT between 1.0 KX to 7.0 KX after 24 nm palladium coating.
2.7. Soil DNA extraction and PCR DGGEprofiling of archaeal and bacterial communities
Plants (three individual plants for each treatment) of variety 'White Star' were randomly pulled out gently from the soils on 45 DAT and 90 DAT. After tapping the roots mildly, the soil remained on the roots was collected as the rhizosphere soil. The total DNA in these samples was extracted using the Power Soil DNA Isolation kit according to the instructions (MO BIO Laboratories, Inc., Carlsbad, California, USA).The total DNA extracted from the rhizosphere soils of three replicate samples were pooled together and then amplified using QB-96 Gradient Thermal Cycler [Biotron Healthcare (India) Pvt. Ltd, Mumbai]. The specific primers for the archaea (Heuer et al., 1997) and bacteria (Bano et al., 2004) were used in a total PCR reaction volume of (25 ^L) using RedTaq® ReadyMix (12.5 ^L, Sigma-Aldrich Biotechnology), Bovine serum albumin (1 ^L of 10 mg • L-1), and the rhizosphere soil DNA as the template (10-15 ng). The profiling ofpolymerase cycling was performed as: at 94 °C for 4 min, 30 cycles at 94 °C for 30 s, 57 °C for 30 s, 72 °C for 45 s, 72 °C for 10 min.
The PCR amplicons (250 ng) were used for the Denaturing Gradient Gel Electrophoretic profiling. The profiling was carried out at a constant voltage of 38 V and at 60 °C for 15 h.
2.8. Statistical analysis
The triple sets of data were analysed by analysis of variance (ANOVA). Standard deviation (SD) values were calculated using Microsoft Excel and depicted in graphs as error bars.
3. Results
3.1. Plant growth and biometric parameters
The concentration of chlorophyll a was highest in samples from treatment BF1-4 from 'Golden Ball'; all the microbial inoculants performed better than control (Fig. 1). The concentrations
Fig. 1 Photosynthetic pigments at 90 DAT in 'Golden Ball' and 'White Star' as influenced by microbial inoculation
Fig. 2 Fresh and dry weight of plants at 90 DAT in 'Golden Ball' and 'White Star' as influenced by microbial inoculation
of chlorophyll b revealed that BF1-4 treatment was superior to An-Tr, while the concentration of carotenoids was higher in BF1-4, followed by An-Tr treatments. The 'White Star' plants treated with the microbial inoculants performed better than that of control on 90 DAT, in terms of enhancement in photosynthetic pigments, however, the pigment content was considerably lower than those recorded in 'Golden Ball' (Fig. 1). The chlorophyll a concentration was highest in An-Tr, followed by Tr-Az treatments on 90 DAT, while in terms of chlorophyll b, with An-Tr treatment showing 101% enhancement over control. The carotenoid content showed significant increases due to the microbial inoculation treatments, with the treatment Tr-Az treatment recording an increase of 147%, followed by An-Tr treatment samples, which exhibited an enhancement of 133% over control.
The plant fresh weight values of 'Golden Ball' ranged from 267 to 722 g with the highest value in An-Tr, followed by An-Az; the dry weight of plants ranged from 81.3 to 268.3 g, with the highest value in An-Tr (Fig. 2). Microbial inoculants brought about significant enhancement in plant fresh and dry weight in 'White Star' samples, over control; however, no significant differences among the inoculants were observed for these parameters (Fig. 2).
The plant height as an index of plant growth was recorded, which ranged from 63 to 72 cm in samples from 'Golden Ball'. The highest plant height was recorded in the treatment An-Tr. The plant height of 'White Star' samples, recorded at 90 DAT, was about 67.8-69.9 cm. The highest plant height was recorded in BF1-4, followed by Tr-Az, and An-Tr treatments.
3.2. Activity of plant defence and hydrolytic enzymes
The activity of plant defence enzymes such as polyphenol oxidase and phenylalanine ammonia-lyase enzymes was analysed in the shoots and roots sampled of 'Golden Ball' on 45 DAT (Tables 1 and 2). Polyphenol oxidase activity in the shoot on 45 DAT was higher in treatment BF1-4, followed by Tr-Az. In the root tissues, highest PAL activity was recorded in treatment Tr-Az, followed by An-Az. Lowest activity was observed in the control treatment (Table 2). On 90 DAT, highest PAL activity in the roots was recorded in the treatment An-Tr, while highest percent increases in root and shoot tissues were observed in the treatments — An-Tr and BF1-4, respectively from 45 to 90 DAT. Highest polyphenol oxidase activity in root samples from 'White Star' was recorded in treatment BF1-4 on both 45 and 90 DAT. On 45 DAT, highest PAL activity in roots was recorded in treatment Tr-Az, followed by BF1-4. On 90 DAT, the highest PAL activity in roots was in the treatment BF1-4. Similarly, highest PAL activity in shoots on 45 and 90 DAT was recorded in the treatment BF1-4. In general, the activity of enzymes elicited was much higher in 'Golden Ball', although microbial inoculation exerted significant effects in both varieties.
The activity of ¡-1,3 endoglucanase at 45 and 90 DAT (Fig. 3), in root tissues of 'Golden Ball' showed a four-fold enhancement in the inoculated treatment Tr-Az as compared to control. The activity of ¡3-1, 3 endoglucanase in root tissues of 'White Star' on 45 and 90 DAT showed significant increases in the inoculated treatments, with BF1-4 treatment recording an almost 40% increase
Table 1 Polyphenol oxidase activity in chrysanthemum 'Golden Ball' and *White Star' as influenced by microbial formulations I U • g 1 FW
Treatment Root Shoot
Golden Ball White Star Golden Ball White Star
45 DAT 90 DAT 45 DAT 90 DAT 45 DAT 90 DAT 45 DAT 90 DAT
Control An-Tr An-Az BF1-4 Tr-Az 3.19 ± 0.510 7.56 ± 0.120 5.27 ± 0.040 9.82 ± 0.260 9.83 ± 0.300 0.30 ± 0.028 0.19 ± 0.028 0.19 ± 0.043 0.33 ± 0.059 0.61 ± 0.049 2.74 ± 0.150 3.20 ± 0.043 2.41 ± 0.043 4.56 ± 0.081 2.71 ± 0.086 0.21 ± 0.015 0.35 ± 0.019 0.65 ± 0.034 0.91 ± 0.013 0.62 ± 0.043 3.07 ± 0.17 7.20 ± 0.05 7.43 ± 0.19 9.46 ± 0.08 8.60 ± 0.03 0.39 ± 0.042 4.74 ± 0.230 4.25 ± 0.086 1.03 ± 0.139 0.69 ± 0.059 1.70 ± 0.110 1.70 ± 0.030 1.88 ± 0.050 4.23 ± 0.140 3.30 ± 0.110 0.23 ± 0.021 0.45 ± 0.037 0.42 ± 0.019 0.62 ± 0.021 0.58 ± 0.026
Note: DAT: Days after transplanting of cutting (plug plants).
Table 2 Phenylalanine ammonia lyase activity in chrysanthemum 'Golden Ball' and 'White Star' as influenced by microbial formulations,
grown in the greenhouse pmol • min-1 • g-1 FW
Treatment Root Shoot
Golden Ball White Star Golden Ball White Star
45 DAT 90 DAT 45 DAT 90 DAT 45 DAT 90 DAT 45 DAT 90 DAT
Control An-Tr An-Az BF1-4 Tr-Az 5.26 ± 0.13 6.23 ± 0.12 13.56 ± 0.24 11.22 ± 0.15 15.74 ± 0.33 15.83 ± 0.76 24.68 ± 0.71 21.30 ± 0.39 19.41 ± 0.42 21.02 ± 0.66 10.776 ± 0.12 16.536 ± 0.15 16.941 ± 0.20 17.796 ± 0.07 18.403 ± 0.12 6.187 ± 0.10 6.862 ± 0.17 7.289 ± 0.15 7.604 ± 0.15 6.884 ± 0.10 3.31 ± 0.04 6.65 ± 0.12 6.21 ± 0.13 2.71 ± 0.10 4.54 ± 0.33 14.35 ± 0.42 19.28 ± 0.23 21.75 ± 0.20 28.05 ± 0.23 21.12 ± 0.10 21.35 ± 0.20 25.58 ± 0.05 28.75 ± 0.20 39.12 ± 0.12 33.14 ± 0.18 6.41 ± 0.15 7.89 ± 0.05 8.37 ± 0.15 14.78 ± 0.48 14.47 ± 0.24
Note: DAT: Days after transplanting of cutting (plug plants).
over control. The values ranged from 0.477 to 1.601 IU • g-1 FW of shoot tissues of 'Golden Ball', while the values were lower, ranging from 0.125 to 0.656 IU • g-1 FW in roots at 45 DAT. A significant increase was observed in shoot tissues at 45 DAT, in the treatment BF1-4 in both varieties. At 90 DAT, a significant percent increase was observed in the shoot tissues in the treatment An-Az, followed by control and An-Tr treatments.
At 45 DAT, the CMCase (^-1,4 endoglucanase) activity in shoot tissues of 'Golden Ball' was higher as compared to that of root tissues; the highest values were recorded in the treatment BF1-4 (Table 3). The values ranged from 0.093 to 0.111 IU • g-1 FW of root tissues on 90 DAT, with highest values in the treatment Tricho-derma viride-Azotobacter. On 45 and 90 DAT, the activity of fi-1,4 endoglucanase in shoot tissues was highest in the treatment
Fig. 3 fi-1, 3 endoglucanase activity in the plant tissues of 'Golden Ball' and 'White Star', as influenced by microbial inoculation
Table 3 Evaluation of CMCase (^-1,4 endoglucanase) enzyme activities, as elicited by the application of microbial formulations
in 'Golden Ball' and 'White Star' crop, grown in CPCT IU • g-1 FW
Treatment Root Shoot
Golden Ball White Star Golden Ball White Star
45 DAT 90 DAT 45 DAT 90 DAT 45 DAT 90 DAT 45 DAT 90 DAT
Control An-Tr An-Az BF1-4 Tr-Az 0.051 ± 0.003 0.097 ± 0.003 0.090 ± 0.004 0.152 ± 0.002 0.067 ± 0.003 0.071 ± 0.006 0.093 ± 0.005 0.101 ± 0.002 0.080 ± 0.003 0.111 ± 0.002 0.125 ± 0.002 0.149 ± 0.001 0.196 ± 0.004 0.150 ± 0.005 0.148 ± 0.007 0.298 ± 0.009 0.403 ± 0.007 0.468 ± 0.007 0.339 ± 0.007 0.444 ± 0.009 0.086 ± 0.001 0.152 ± 0.002 0.153 ± 0.002 0.364 ± 0.003 0.161 ± 0.004 0.130 ± 0.006 0.211 ± 0.002 0.221 ± 0.005 0.374 ± 0.004 0.308 ± 0.009 0.237 ± 0.005 0.340 ± 0.001 0.356 ± 0.003 0.320 ± 0.003 0.335 ± 0.002 0.195 ± 0.002 0.358 ± 0.012 0.339 ± 0.007 0.419 ± 0.004 0.483 ± 0.011
Table 4 Evaluation of chitosanase enzyme activities, as elicited by the application of microbial formulation in 'Golden Ball' and
'White Star' crop, grown in CPCT IU •
Treatment
Golden Ball
White Star
Golden Ball
White Star
45 DAT
90 DAT
45 DAT
90 DAT
45 DAT
90 DAT
45 DAT
90 DAT
Control
0.148 ± 0.011 0.313 ± 0.014 0.327 ± 0.007 0.198 ± 0.005 0.373 ± 0.009
0.155 ± 0.027 0.285 ± 0.009 0.237 ± 0.004 0.211 ± 0.012 0.187 ± 0.003
0.602 ± 0.007 0.971 ± 0.005 0.976 ± 0.004 1.024 ± 0.001 0.932 ± 0.004
0.159 ± 0.007 0.296 ± 0.010 0.213 ± 0.004 0.374 ± 0.007 0.265 ± 0.009
0.206 ± 0.003 0.675 ± 0.010 0.070 ± 0.248 0.244 ± 0.009 0.382 ± 0.008
0.194 ± 0.012 0.289 ± 0.019 0.220 ± 0.019 0.238 ± 0.013 0.219 ± 0.015
0.174 ± 0.004 0.484 ± 0.002 0.194 ± 0.004 0.228 ± 0.009 0.202 ± 0.007
0.334 ± 0.010 0.495 ± 0.005 0.424 ± 0.005 0.491 ± 0.007 0.569 ± 0.010
BF1-4, followed by Trichodermaviride-Azotobacter and Anabaena-Azotobacter treatments. In 'White Star', the activity of CMCase (8-1,4 endoglucanase) in shoot tissues on 45 DAT was higher than that of root tissues. Highest values in root tissues were recorded in An-Az treatment on both 45 and 90 DAT. Similar trend in the activity in shoot tissues on 45 DAT was recorded with the treatment An-Az exhibiting highest values. However, on 90 DAT, the treatment, Tr-Az treatment recorded highest values. The samples from 'White Star' showed, in general, higher values.
The chitosanase activity in root tissues of 'Golden Ball', on 45 DAT was highest in the treatment Tr-Az, while at 90 DAT, the highest values were recorded in the sample from An-Tr treatment (Table 4). At 45 DAT, the shoot tissues of the samples from An-Tr and Tr-Az treatments showed the highest value of 0.675 IU • g-1 FW At 90 DAT, the shoot tissue samples from An-Tr and BF1-4 treatments showed the highest values. In 'White Star', chitosanase activity in root tissues on 45 and 90 DAT was highest in BF1-4 treatment. In the 45 DAT shoot tissue samples, An-Tr and BF1-4 treatments showed the highest values; on 90 DAT, the treatments Tr-Az and An-Tr showed the highest values, followed by T4 (BF1-4). In 'White Star', the plant height, recorded at 90 DAT, was about 67.869.9 cm (Table 4). The highest plant height was recorded in BF1-4 treatment and the number of leaves which ranged from 21 to 26, with highest values in the Tr-Az treatment.
3.3. Soil microbiological and nutrient parameters
The soil microbiological analyses were taken up using rhizo-sphere soil samples only at 90 DAT (Table 5). The dehydrogenase activity in samples from 'Golden Ball' ranged from 15.49 to 17.45 |g • g-1 • d-1 and was highest in BF1-4 treatment, followed by the samples from An-Tr treatment. On 90 DAT, the dehydrogenase activity in the rhizosphere of 'White Star was between 85.82 and 116.15 |g • g-1 • d-1, with the highest activity in An-Tr treatment. The potential nitrification activity in samples from 'Golden
Ball' ranged from 3.11 to 6.58 nmol • g-1 • h-1, with the highest value in BF1-4, followed by An-Tr treatments. The potential nitrification activity in this soil samples from 'White Star' ranged from 2.53 to 7.52 nmol • g-1 • h-1, with the highest values in An-Az, followed by Tr-Az treatment. The soil microbial biomass carbon concentrations in the rhizosphere of plants of 'Golden Ball' were between 42.60 and 78.96 |g • g-1, with the highest value in An-Az, followed by An-Tr and BF1-4 treatments. The concentration of microbial biomass carbon in the rhizosphere in samples from White Star' was between 288.15 and 769.93 |g • g-1 soil, with highest was in Tr-Az treatment, followed by control. The concentrations of polysaccharide from 'White Star' were about 2.87 to 4.53 mg • g-1. Highest value was recorded in An-Tr treatment, while they ranged between 3.87 and 5.18 mg • g-1 in 'White Star'; the samples from treatment Tr-Az had the highest concentration, followed by An-Tr. The samples from 'White Star' showed four-to five-fold higher dehydrogenase activity and microbial biomass carbon, as compared to Golden Ball.
The concentrations of soil organic carbon enhanced from 0.19% in the control to 0.3-0.4 in the microbial inoculant treatments, with highest in An-Az; in variety White Star, values ranged between 0.2 and 0.24% with highest in An-Tr, followed by Tr-Az treatments (Table 6). The concentrations of available nitrogen ranged from 135.23 in Control to the highest value of 236.97 mg • g-1 in BF1-4 treatment. All the inoculated treatments recorded 30% higher values in terms of available P and organic C in soil. The concentrations of available phosphorus ranged from 15-16 mg • kg-1 in the inoculated treatments (with highest in BF1-4, as against 11.33 mg • kg-1 in Control. In samples from 'White Star', available nitrogen concentration ranged from 213.3 to 276.0 mg • kg-1 soil while the available phosphorus concentration was between 43.04 and 46.29 mg • kg-1 (Table 6). The available phosphorus concentration did not vary significantly among treatments including that of control.
Table 5 Microbiological parameters as influenced by the application of microbial formulations in 'Golden Ball' and 'White Star'
under protected cultivation
Treatment Dehydrogenase activity/(|g • g 1 • d ') PNA /(nmol • g-1 • h-1) Microbial biomass carbon/(|g • g ') Polysaccharide content/(mg • g ')
Golden Ball White Star Golden Ball White Star Golden Ball White Star Golden Ball White Star
Control An-Tr An-Az BF1-4 Tr-Az 15.49 ± 0.62 16.82 ± 0.55 15.65 ± 0.16 17.45 ± 0.39 16.82 ± 0.08 85.82 ± 1.08 116.15 ± 2.97 105.47 ± 4.94 96.98 ± 2.24 114.66 ± 3.90 3.11 ± 0.09 6.07 ± 0.25 3.22 ± 0.13 6.58 ± 0.39 4.08 ± 0.16 2.53 ± 0.22 3.12 ± 0.09 7.52 ± 2.17 3.11 ± 0.09 3.69 ± 0.14 42.60 ± 2.16 66.53 ± 2.42 78.96 ± 2.57 66.53 ± 2.04 54.83 ± 0.95 288.15 ± 4.73 376.99 ± 3.09 328.87 ± 6.44 520.05 ± 10.95 769.93 ± 5.01 3.87 ± 0.49 5.18 ± 0.14 4.77 ± 0.32 4.69 ± 0.36 3.99 ± 0.23 2.87 ± 0.25 3.82 ± 0.97 3.12 ± 0.15 3.52 ± 0.45 4.53 ± 0.13
Table 6 Soil nutrient availabilities as influenced by the application of microbial formulations in 'Golden Ball' and 'White Star'
under protected cultivation
Treatment
Soil organic carbon/%
Available nitrogen/(kg • hm 2)
Golden Ball
White Star
Golden Ball
White Star
Available phosphorus/(kg • hm 2)
Golden Ball
White Star
Control
0.19 ± 0.002 0.42 ± 0.001 0.35 ± 0.003 0.31 ± 0.003 0.28 ± 0.001
0.19 ± 0.030 0.24 ± 0.015 0.21 ± 0.009 0.21 ± 0.034 0.20 ± 0.023
135.23 ± 23.02 179.50 ± 21.10 109.07 ± 15.14 236.97 ± 12.10 147.27 ± 13.12
163.2 ± 34.14
213.3 ± 78.90 219.5 ± 53.92 275.8 ± 64.54 276.0 ± 61.54
11.33 ± 0.72 15.67 ± 0.63 16.33 ± 0.51 16.67 ± 0.54 15.00 ± 0.61
40.32 ± 3.91 43.31 ± 3.21 44.80 ± 3.38 46.29 ± 3.91 43.04 ± 1.80
3.4. Scanning electron microscopic analysis
The SEM analysis of the root tissues of both varieties was undertaken at 45 DAT and similar degree of colonization of the inoculated organisms was observed. Fig. 4 illustrates the colonization of 'White Star' variety plant roots by filaments/biofilm-like aggregates (Fig. 4, A) and short filaments of cyanobacteria (Fig. 4, B), in the samples from treatments BF1-4 and An-Tr respectively.
3.5. Analyses of PCR-DGGEprofiles ofarchaeal and bacterial communities in the rhizosphere of'White Star'
The archaeal and bacterial communities were analysed by the PCR-DGGE profiling method using rhizosphere soil samples taken at 45 and 90 DAT in both varieties of Chrysanthemum; however, only the samples from 'White Star' variety showed the distinct influences of the microbial inoculants tested (Figs. 5 and 6). The maximum number of archaeal 'phylotypes' deduced from the prominent bands increased from 12 to 22 with the increase in days after inoculation from 45 to 90 d (Fig. 5, A,B). On 45 d, the archaeal community structures were influenced by the inoculants tested; the archaeal community structure of control plants clustered with that of An-Tr treatment (Fig. 6, A). But, the mi-crobial inoculant Tr-Az had a distinctive effect on the archaeal community structure which was also observed on 90 d (Fig. 6, B). Interestingly, both BF1-4 and Tr-Az treatments had similar effects as the archaeal community structures of the rhizo-sphere soils under these two treatments clustered together on 90 d. Other two inoculant treatments such as An-Az and An-Tr influenced the archaeal community like that of the control. The cluster analysis of the archaeal community structures showed the
stronger influences of the microbial inoculants tested on 90 d.
The predominant bacterial phylotypes in these soils were 1214 on both 45 (Fig. 7, A) and 90 d (Fig. 7, B). The cluster analysis of bacterial DGGE profiles suggested that the microbial inoculants tested had a stronger effect on 45 d (Fig. 8, A), relative to those of 90 d (Fig. 8, B). There were three different clusters as Control and An-Az and An-Tr and BF 1-4 which were different from that of Tr-Az. The bacterial community structure under Tr-Az was also different from that of other treatments on 90 d. The stronger effect of the microbial inoculants on the bacterial communities was evident as the clusters of the DGGE profiles were similar in both 45 and 90 DAT samples.
45 d 90 d
1 2 3 4 5
Fig. 5 Archaeal community structure in the rhizosphere of variety 'white Star', as influenced by microbial inoculation
1: Control; 2: An-Tr; 3: An-Az; 4: BF1-4; 5: Tr-Az. A, B: Data correspond to the community patterns of archaea obtained in DGGE on 45 and 90 DAT.
Fig. 7 Bacterial community structure in the rhizosphere of 'White Star', as influenced by microbial inoculation
1: Control, 2: An-Tr, 3: An-Az; 4: BF1-4 and 5: Tr-Az. A, B: Data correspond to the community patterns of bacteria obtained in DGGE on 45 and 90 DAT.
Fig. 6 Dendrograms from multivariate cluster analysis of archaeal community patterns using Dice's method
Community patterns are provided in DGGE on 45 and 90 DAT.
Fig. 8 Dendrograms from multivariate cluster analysis of bacterial community patterns using Dice's method
Community patterns are provided in DGGE on 45 and 90 DAT.
4. Discussion
Microbial interactions in the rhizosphere have great significance in ecological and sustainable resource management in agriculture, as they not only aid in nutrient exchange, but also elicit exudation and enzymes involved in systemic and induced resistance to abiotic/biotic stresses (Adesemoye et al., 2009). The application of microbial inoculants can facilitate plant growth directly or indirectly by enhancing nutrient availability to the host plant, influencing root growth and morphology by strengthening beneficial symbiotic and associative relationships (Ruzzia and Arocab, 2015).
Cyanobacteria play a key role in sustained nitrogen management and soil fertility which is known to be responsible for maintaining sustainable yields in rice (Singh, 1961; Roger and Watanabe, 1986). The ability of cyanobacteria to promote plant growth was reported by Karthikeyan et al. (2007) in wheat crop under glasshouse and controlled environmental conditions. This aspect has now been extended to several other crops including maize, vegetables, legumes and cotton (Prasanna et al., 2014, 2015; Manjunath et al., 2016). Evidence exists that nitrogen fixed by cyanobacteria is made available to rice as well as other plant or microbial life (Roger and Watanabe, 1986; Mandal et al., 1998)
using 15N balance studies. The plant growth promoting rhizobacteria are also known to increase the availability of nutrients in the rhizosphere, which may be through solubilization of unavailable forms of nutrients and/or siderophore production and facilitating transport of nutrients. In the present experiment, all the formulations showed significant growth enhancement of chrysanthemum varieties over the uninoculated control. In the varieties 'Golden Ball' and 'White Star', the inoculants BF1-4 and An-Tr were the top performers. The availability of N in the soils of White Star was greater in Tricho-derma viride-Azotobacter and BF1-4 treatments, than in the uninoculated control. A significant increase ranging from 30% to 50% in terms of nutrient availability (N, P and C) was also recorded in all the inoculated treatments in the variety of 'Golden Ball'. In an earlier report, the beneficial effect of biofertilizers in improving plant growth and yield has been reported in China aster, and an increase in bulb yield might be due to enhanced N availability to the plants (Srivastava et al., 2013).
Plant-mediated defence responses elicited by plant growth-promoting rhizobacteria (PGPR) can be exerted through diverse mechanisms, including root architecture modification, production of phytohormones and antibiotic compounds, and triggering of specific defence signalling pathways known as induced
systemic resistance (ISR) (van Loon, 2007). In the present study, a significant level of elicitation of plant defence/pathogenesis-related enzymes was recorded in both varieties of chrysanthemum on 45 DAT, over control. However, there was a differential response, recorded on 90 DAT, in both the varieties for different enzyme activities. The ¡5-1,3 endoglucanase activity showed almost 50%-80% increase in the inoculated treatments at 90 DAT in 'Golden Ball', however, up to 50% reduction was recorded in 'White Star'. The enhancement in the degree of elicitation, which primes the plants, leading to better growth and robustness, represents an important facet of plant-microbe interactions for their effective use as inoculants. Conversely, Pérez-Montaño et al. (2013) observed that eukaryotes, including plants, can interfere with bacterial QS (Quorum Sensing) systems by synthesizing molecules that interfere with bacterial QS systems. Their work illustrated that plants are able to enhance or to inhibit the bacterial QS systems depending on the bacterial strain, illustrating the significance of specificity in the interactions of plants with microbes. Further studies in this direction, deploying our inoculants would contribute to a better understanding of plant-bacteria relationships at the molecular level.
Soil microbial biomass is fundamental to maintaining soil functions, also controls the build-up and breakdown of organic matter, the decomposition of organic residues, and an indicator of changes in soil management, heavy metal contamination and fertilizer practices. This is reflected in terms of dehydrogenase activity, potential nitrification and accumulation of polysaccharides in soil. In the present study, Anabaena-Trichoderma viride in 'White Star' recorded highest in organic carbon percent and dehydro-genase activity. In 'Golden Ball', the dehydrogenase activity was higher in BF1-4 treatment as compared to uninoculated control. In 'White Star', microbial biomass carbon content was higher in Trichoderma viride-Azotobacter treatment and in 'Golden Ball', in Anabaena-Azotobacter treatment as compared to uninoculated control. Similar observations have been recorded with these biofilmed formulations in cotton (Triveni et al., 2015). Although polysaccharide content in soil was higher in 'Golden Ball', the microbial biomass carbon, and dehydrogenase activity were much lower compared to 'White Star'. This illustrates the importance of variety-specific root exudates and their role in influencing soil microbiological and plant growth parameters in the two varieties grown under similar soil conditions.
Biofilms represent a novel means of inoculation, as the polysaccharides in the bacteria/fungal/cyanobacterial matrix provide an ideal home for colonization by other microbes. Earlier studies on the development of such biofilms using fungi/cyanobacteria (Seneviratne, 2003; Prasanna et al., 2011, 2014, 2015) illustrated their promise, especially in cyanobacterial biofilmed biofertilizers (CBBs) which can supply both photosynthates and fixed nitrogen (Prasanna et al., 2014, 2015). Cyanobacteria can colonize wheat and rice roots, as shown in hydroponic, pot and field experiments (Karthikeyan et al., 2007; Prasanna et al., 2014, 2015; Priya et al., 2015). In the present investigation, SEM analyses revealed the presence of biofilm-like structures and short pieces of cyanobacterial trichomes in the root tissues. This validates the colonization potential of the applied inoculants, which repre-
sents an important determinant for effective plant growth promotion, facilitated by plant-microbe interactions.
Rodriguez Navarro et al. (2000) reported that incorporation of vermicompost 20% with or without chemical fertilizer increased the chlorophyll content in gerbera. In the present study, the plants of 'Golden Ball' generally possessed higher chlorophyll pigments, as compared to those of 'White Star'. In 'Golden Ball', BF1-4 treatment recorded highest values, while in 'White Star' chlorophyll a and b were higher in the plants under different cyanobacterial treatments, as compared to control. Interesting observations were recorded related to the significant enhancement of carotenoid content in all the microbial inoculants treatments. This is an important observation, which needs to be investigated in future studies as microbial inoculants can be targeted for modulating flower colour and intensity.
Cyanobacterial formulations maybe specific for crop or varieties, in terms of enhancing growth and may even vary from species to species, but the inoculants BF1-4 and An-Tr were found to be superior and most suited to these varieties for enhancement in biomass and metabolic activity. The consortium BF1-4 has shown immense promise as an inoculant for the rice-wheat cropping system (Prasanna et al., 2014, 2015). The balanced supply from biofertilizers promotes the translocation of phyto-hormones to the shoot, and lead to better growth, as observed in gladiolus (Srivastava et al., 2013). The combined application of Azospirillum and Phosphobacterium with a reduced level of NPK led to enhanced plant height, dry matter production, pedicel length, and in African marigold (Velmurugan, 1998). Inoculation of the cutting Jasmimum sambac with Azospirillum and phosphobacteria at the time of planting root cuttings, brought about a significant increase in plant height, leaf area, number of branches, etc, besides providing a savings of 25 percent of N and P fertilizers (Manonmani, 1992). The use of multiple inoculants, vis a vis single, showed synergistic effects across 11 plant species demonstrating that members of root microbiome complement one another in mobilizing nutrients and help in better ecosystem functions (van der Heijden et al., 2016). This supports the use of the microbial consortia and biofilmed inoculants in our investigation.
It is important to study the temporal changes in soil micro-bial communities because this type of analyses may help to comprehensively explain the intricacies of soil function and provide insight into the environmental niches that are inhabited by diverse soil microbes (Rasche et al., 2011; Lauber et al., 2013). DGGE analyses of the samples from all the treatments at both 45 and 90 DAT revealed significant changes in archaeal and bacterial communities. A temporal change in community profile was recorded with bacterial communities being modulated at 45 DAT, while archaeal communities were more resilient and distinct influence was recorded only at 90 DAT.
Roesti et al. (2006) analysed the effects of PGPR/AMF inoculations on bacterial community structure of wheat rhizosphere and illustrated that inoculants induced a significant modification in the bacterial community structure. However, in terms of impact on the bacterial community structure, the type of PGPR consortium, rather than the AMF contributed to the variance (28.3% and 10.6% respectively). In the present study, both the
top ranking inoculants — Consortium BF1-4 and An-Tr were always observed to remain as distinct clusters, away from control, especially in the bacterial DGGE profiles. The presence of Tr-Az treatment as a separate cluster in both the PCR-DGGE profiles of archaeal and bacterial communities and over time (45 and 90 DAT), highlights its uniqueness as an inoculant.
Felici et al. (2008) showed that a combination of the two rhizobacteria (Bacillus subtilis and Azospirillum brasilense) had no synergistic or comparable effects on plant biomass in tomato (Lycopersicon esculentum), with respect to their single applications. This suggested that plant-growth reduction and root-architectural alterations, elicited by the microbial combination, may involve independent signalling pathways of the two bacterial species, and it is important to select synergistic combinations.
5. Conclusions
Modification in the bacterial community structure caused by inoculation is often buffered by ecosystem resilience, which is driven by the level of diversity and interactions of the plant-soil-biota. Our study highlighted that the effect of microbial inoculant on the soil microbiome especially under the greenhouse conditions of protected cultivation is specific to the plant variety and the inoculant and shows a distinct spatial and temporal variation, despite the soil effect. The separation of the promising inoculants in the PCR-DGGE profiles reveals that it may be possible to relate their effects on microbial communities with their distinctness as a promising inoculant. However, more in-depth studies are needed to substantiate such observations in other varieties and soil conditions.
Acknowledgements
The study was partly funded by the AMAAS Network Project on Microorganisms (IARI Code :12/122), granted by ICAR to RP and the SERB project (SR/S0/PS/164/2010), DST, Government of India granted to BR. We are thankful to the Centre for Protected Cultivation Technology (CPCT), ICAR-IARI, New Delhi, for the facilities to conduct the experiment. We gratefully acknowledge the Division of Agronomy and Division of Microbiology, ICAR-IARI, New Delhi for providing necessary facilities for undertaking this study.
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