Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in
Inoculation with Selected Microbial Consortia Enhanced the Growth and Yield of Withania somnifera under Polyhouse Conditions Anuroopa, N1&2 & Bagyaraj, D. J.3 1
2
PRIST University, Vallam, Trichy-613403 Department of Microbiology, Government Science College, Nrupathunga Road, Bangalore-560001 3 Centre for Natural Biological Resources and Community Development(CNBRCD), #41, RBI Colony, Anand Nagar, Bangalore – 560024.
Abstract: Withania somnifera is an important medicinal plant. The roots are rich in withanolides, which is the active constituent of this medicinal plant. An earlier pot culture study brought out that Bacillus licheniformis (Bl) to be the best PGPR and Aculospora laevis (Al) followed by Claroideoglomus etunicatum (Ce) to be the best AM fungi for inoculating W. somnifera. In the present investigation, a pot culture experiment was conducted in a poly house, to study the effect of individual as well as microbial consortia of Bl, Al and Ce on the growth of W. somnifera. Plant growth, dry biomass, plant nitrogen and phosphorus, spore count and root colonization were found to be maximum in plants inoculated with microbial consortia, maximum being found in plants inoculated with Bl+Al when compared with individual treatments and uninoculated plants. It was concluded that the inoculated microorganisms interact synergistically and the microbial consortia containing Bl+Al are best for inoculating W. somnifera. Keywords: AM Withanaloide
fungi,
PGPR,
Biofertlizer,
1. Introduction Withania somnifera is an important medicinal plant widely cultivated for its roots that contain withanolides which are of medicinal value. In India W. somnifera is cultivated around 17,780 ha with the production of 8429 tonnes/year. The annual demand of this herb increased from 7028 tonnes (2001-2001) to 9127 tonnes (2004-2005). This 29.8% increase in demand has led to an increase in area under its cultivation for higher production with good quality [1]. Medicinal plants cultivation demands fine tuning between the applications of inorganic, organic and biofertilizers [2]. Nowadays emphasis is given on growing plants using microbial inoculants that serve as an alternative to chemical fertilizers. Research on the use of plant growth promoting rhizobacteria (PGPR) and Arbuscular mycorrhiza (AM) to
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promote plant growth has increased dramatically over the last few years due to potential benefits observed under field conditions. The mechanisms by which these organisms enhance plant growth are multitudinous and include production of plant growth-regulating substances or phytohormones, suppression of plant pathogens through antibiosis, bacteriocinogenic action or siderophore production, nitrogen fixation, and mineralization of organic phosphorus and enhancement of mineral uptake, increasing tolerance towards abiotic stress and also by their interaction with AM fungi [3]. AM fungi and bacteria interact synergistically through a range of mechanisms that include improved nutrient acquition, inhibition of fungal plant pathogens and promote plant growth and yield, and also improve soil quality [4]. This beneficial interaction has lead to increased use of these organisms in promoting plant growth. Their interactions can have important implications in agriculture [5]. The interactions between rhizotrophic microorganisms can influence their activities, soil conditions and hence plant growth [6]. Synergistic interactions between PGPR and AM fungi have been reported to increase the yield and biomass in several medicinal plants under nursery and field conditions [7]. There are reports showing a strong stimulatory impact of PGPR on the growth of AM fungi [8]. Garbaye [9] proposed the term ‘ Mycorrhization helper bacteria’ for rhizobacteria that increase the ability of the root to establish symbiotic interactions with mycorrhizal fungi due to helper effect such as stimulation of root development, enhanced recognition and susceptibility to root colonization. Several reports have demonstrated that the presence of PGPR in the rhizosphere has enhanced AM fungal colonization [10]. Studies have also suggested that Gram-positive bacteria and γ- proteobacteria are more commonly associated with AM fungi than Gram-negative bacteria [11]. Several PGPR have been shown to be excellent root colonizers and a number of surface components have been demonstrated to play a role in the physical interactions between such bacteria and plant roots Page 127
Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in [12]. Bianciotto et al. [13] reported that interactions between rhizobacteria such as Rhizobium and Pseudomonas species with AM fungi were mediated by soluble factors or physical contact. The biological features of attachment of five different gfp-tagged bacteria to AM fungus Glomus claroideum were studied by Toljander et al. [14] which indicated that bacterial strains differ in their ability to attach to different physiological states of hyphae. One possible way that attachment could benefit both partners, would be through facilitation of certain metabolic interactions, such as nutrient and carbon exchange and this would rely on close cell contact between the bacterial and fungal components [11]. The beneficial effect of the interaction on plants by some bacteria is by directly affecting the AM fungal germination and growth rate [15]. AM fungi together with specific bacteria support plant growth by nutrient acquition, inhibition of plant pathogenic fungi and enhancement of root branching [16]. AM fungi are not host specific but are known to have host preference [17]. Earlier workers have emphasized that it is better to screen and select the best symbiotic AM fungus for a particular host so that it can be used for inoculation [18]. Studies have shown that some bacterial species respond to the presence of certain AM fungi [3], suggesting a high degree of specificity between bacteria associated with AM fungi. Stimulation of certain bacterial species by specific AM fungi may be that those bacteria are activated by species-specific fungal exudates. Interaction studies between AM fungi and PGPR has been proved to form a consortium benefitting the growth of a few plant species. Though it is a beneficial interaction, there is specificity seen in this interaction, therefore it is necessary to test for the most suitable combination of plant, PGPR and AM fungi to get maximum growth benefits. Earlier studies have shown that B. licheniformis is the best PGPR among the different PGPRs screened for inoculating W. somnifera [19]. A. laevis is the best AM Fungus followed by C. etunicatum for inoculating W. somnifera [20]. In order to develop microbial consortia for sustainable cultivation of W. somnifera, interaction between B. licheniformis, A. laevis and C. etunicatum was studied under pot culture conditions.
2. Material and methods 2. 1. Polyethylene house study The investigation was carried out to study the effect of microbial consortia (selected from the pot culture studies) on growth, plant nutrition, biomass, total withanolides, spore count and root colonization under poly ethylene house conditions. The interaction between PGPR, B. licheniformis and AM fungi, A. laevis and C. etunicatum on growth and
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yield of W. somnifera was studied. B. licheniformis was obtained from Hyderabad University and A. laevis and C. etunicatum isolated and maintained at CNBRCD, Bangalore, India was used. The following six different treatments: T1: Uninoculated; T2: Inoculated with Bacillus licheniformis (Bl) T3: Inoculated with Acaulospora laevis (Al) T4: Inoculated with Claroideoglomus etunicatum (Ce) T5: Inoculated with Bl+Al T6: Inoculated with Bl+Ce The substrate used was soil:sand:vermicompost mix (1:1:0.25) (v/v/v). The soil used in the study was a finekaolinitic, isohyperthermic, typical kanhaplustalf. The soil pH was 7.0, EC 0.65, organic carbon 0.89%, available N 276 kg/ha, available P 18.33 kg/ha and available K 300 kg/ha. The potting mixture was filled in polyethylene bags of size 22 × 14 cm holding 2 kg substrate. One month old Seedlings of W. somnifera obtained from Central Institute of Medicinal and Aromatic Plants, Bengaluru, were used for transplantation. A planting hole was made in each polyethylene bag. 10 g of the inoculum A. laevis (containing 2.8 × 103IP/g) and C. etunicatum (containing 2.3 × 103 IP/g) was added to the planting hole depending on the treatment. Ten milliliters of B. licheniformis inoculum (with 108cfu/mL) was added to the planting hole based on the treatment. Total number of treatments was six and each treatment was replicated ten times. The plants were maintained in a polyhouse 10 m long and 5 m wide and watered whenever necessary.
2.2. Microbial inoculums The microbial inoculum consisting of the AM fungus A. laevis and C. etunicatum the PGPR B. licheniformis (based on our earlier pot culture studies) was used in this investigation. B. licheniformis was grown in one liter flask containing 500mL of Luria–Bertani (LB) broth and placed on a rotary shaker for 24 h. The broth after incubation harbored 1x108 cfu/mL. A. laevis and C. etunicatum was multiplied in plastic tubs using soilrite, perlite and vermiculite (1:1:1 v/v/v basis) as the substrate and Rhodes grass (Chloris gayana) as the host. The mixture consisting of the root system plus substrate was finely chopped and air dried. The hyphae, spores and root segments present in the air dried substrate served as inoculum. The infective propagules of A. laevis and C. etunicatum were determined by MPN method [21] and it was found to be 2.8×103IP/g and 2.3×103IP/g. Ten grams/ten ml of microbial inoculum was added to each planting hole as per the treatment.
2.3. Plant parameters studied Each treatment had 10 plants. Plants from each plot were harvested and plant growth parameters
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in were recorded. Plant height was measured from soil surface to the growing tip of the plant and stem girth was measured one cm above the soil surface using digital Vernier calipers during harvesting. Biovolume index (BI) (a measure of total volume of a plant) was determined using the formula ‘Biovolume index = Plant height (cm) × Stem girth (mm)’ given by Hatchell [22]. The plants were uprooted on 140 DAT. For this, shoot of the plants were severed at harvest and kept in an oven at 60 ◦C for 48 h. Dry weight of shoot was recorded. The concentration of N, P and K in the shoot was determined by methods outlined by Jackson [23]. Total withanolide concentration in roots was determined by HPLC [24].
to control. The plants inoculated with Bl+Ce had highest value in height and was statistically significant when compared to other treatments. Stem girth and biovolume index was statistically significant in plants treated with Bl+Al and differed significantly from all other treatments, the least being in uninoculated plants (Table 1). The highest shoot dry weight was observed in the treatments Bl+Al, followed by Bl+Ce, both differing significantly from the uninoculated control. The root dry weight and also total plant dry weight was statistically significant in plants treated with the microbial consortia Bl+Al differing significantly from all other treatments, the least was recorded in uninoculated plants (Table 2).
2.4. Mycorrhizal parameters studied
3.2. Nutrient parameters studied
Roots of the plants were extracted from soil to measure the percent mycorrhizal colonization as described by Philips and Hayman [25]. The roots were cut into 1 cm bits, washed, cleared with KOH, acidified with HCl and stained with trypan blue in lacto glycerol. The stained roots were observed under microscope for determining per cent mycorrhizal colonization. The AMF spore numbers in the root zone soil were determined by wet sieving and decantation method [26].
Increase in nutrient uptake was also observed in inoculated plants compared to uninoculated plants. The shoot N concentration was more over control in Bl+Al treatment and was on par with all the treatments excepting plants inoculated with Bl and uninoculated plants. Shoot phosphorus concentration did not vary significantly among treatments. The shoot potassium concentration was significantly high in plants inoculated with Al alone and also in microbial consortia Bl+Al (Table 3). Similarly the root N content also did not vary significantly among treatments. The root P concentration was significantly high in microbial consortia inoculated plants and was on par with Bl inoculation but differed significantly from single AM inoculation as well as control plants. The root K concentration was also significantly high in microbial consortia inoculated plants and also in Al inoculated plants differing significantly from uninoculated control. The root withanolide concentration was statistically significant in plants inoculated with microbial consortia when compared to single inoculation. Single inoculation with either AM fungi or PGPR significantly enhanced the root withanolide concentration compared to uninoculated plants. The least root withanolide concentration as recorded in uninoculated control plants (Table 4).
2.5.Enumeration population
of
Bacillus
species
The rhizosphere population of Bacillus species was enumerated by the standard dilution plating technique [27] after subjecting the diluents to 80◦C in a water bath for 10 min and plating on LB medium. The colony characteristics and the number of colonies were noted and the population was expressed as cfu g−1 of soil. The total bacterial population in the rhizosphere soil was also enumerated using nutrient medium
2.6. Statistical analysis The data collected in field study was subjected to statistical analysis suitable to CRBD using the ASSISTAT (7.7 beta) software. Data were subjected to the analysis of variance at significant level (<0.05) and means were compared by Duncan’s multiple range test.
3. Results 3.1. Plant growth parameters studied The results of the poly-house experiment showed that the combination of the AM fungus A. laevis with PGPR B. licheniformis enhanced the growth of W. sominifera. The plant height was more in plants treated with microbial inoculants, compared
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3.3. Mycorrhizal parameters The Plants inoculated with only PGPR had significantly lower mycorrhizal colonization compared to AM inoculated treatments. Highest colonization was observed in the treatment Bl+Al, which differed significantly from other treatments. It is clear that B. licheniformis used in the present study not only enhanced significant mycorrhizal colonization by AM fungi but showed preference towards Al. This result indicates that, there is specific interaction between the selected PGPR and AM fungus in improving the growth and nutrition of
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in W. somnifera. Maximum number of mycorrhizal spores in root zone soil was observed in Bl+Al treatment therefore supporting the colonization in the root zone soil of Bl+Al. The least spore count was observed in the uninoculated control (Table 5).
3.4. Soil bacterial population as influenced by different treatments The results of the total bacterial population in rhizosphere soil indicated that they were significantly higher in MC treatments followed by Bl inoculation alone. All the three treatments were on par with each other. The least number of bacteria were encountered in control. The Bacillus species population was significantly high in plants inoculated with Bl which was on par with Bl+Al, and differed significantly from other treatments indicating that the inoculated Bacillus species persists well in the rhizosphere soil (Table 5).
4. Discussion Biofertilizers are integral part of organic cultivation of medicinal plants. Boosting yield, reducing production cost and improving soil health are interlinked components of sustainable agriculture. The results of the present study showed that combination of the AM fungus A. laevis with PGPR B. licheniformis enhanced the growth of W. sominifera. The plants inoculated with Bl+Al gave significantly higher results in all the plant parameters like the stem girth, biovolume index, shoot and root dry weight and also total plant dry weight. This upholds the views expressed by earlier workers that inoculation with microbial consortia improves efficiency and performance compared to inoculation with individual microbial inoculants [28]. Arpana and Bagyaraj [29] reported that inoculation with AM fungus + PGPR significantly increased the plant height compared to uninoculated control or single inoculation in the medicinal plant kalmegh. Thilagar et al. [30] reported that dual inoculation with F. mosseae and B. licheniformis consortia resulted in better growth and yield of chilly compared to inoculation with either one of them. Coinoculation of AM fungi with one or more than one PGPR has been reported to enhance growth and productivity in different crops [30, 31]. In the present study Bl+Al gave better results in growth and yield parameters of W. somnifera. Shoot N and P concentration did not vary significantly among treatments however there was significant variation seen in the shoot K concentration. Root nitrogen concentration also did not vary significantly, but variation was seen in the root P and K concentration. Singh et al [32] reported that Coleus forskohlii plants treated with Pseudomonas monteilii and Glomus fasciculatus had
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higher N, P and K uptake. Highest root P was recorded in plants inoculated with Bl+Al which differed significantly from other treatments. Similarly highest root K was reported in plants inoculated with Al alone followed by consortial treatments Bl+Ce and Bl+Al. Higher N, P and K accumulation in shoots of Stevia rebaudiana plants due to dual or combined inoculation was reported by Das et al [33] under glasshouse conditions. Bagyaraj et al [34] reported that P uptake is enhanced by AM fungi from labile pools of soil P. Arthurson et al [35] reported that AM fungi together with an efficient PGPR changed the P uptake pattern which upholds the results of the present study. Enhanced secondary metabolite accumulation in medicinal plants is of great importance to the industries interested in medinical plant cultivation. Research has revealed the molecular basis and principles of the plant microbe interactive mechanisms [36] which indicates that plant secondary products usually act as signal molecules or respond to symbiont colonization. The root withanolide concentration was statistically significant in plants inoculated with microbial consortia when compared to single inoculation and varied significantly from the control. Similar results of microbial consortium enhancing withanolide concentration in W. somnifera have been reported by Rajashekar and Elango [37]. Singh et al [32] reported that forskolin content of tubers was significantly increased by the inoculation of P. monteilii + G. fasciculatum in Coleus forskohlii. Thilagar et al [30] reported that B. licheniformis acts as mycorrhization helper bacterium and helps mycorrhiza to colonize plant roots better which is also supported the present study that dual inoculation with an AM fungi supported root colonization and sporulation in the root zone. Similar results were obtained with co-inoculation of P. monteilii with R. fasciculatus which significantly improved the percent AM root colonization and spore numbers retrieved from Coleus forskohlii soil [32]. It is clear that B. licheniformis used in the present study not only enhanced significant mycorrhizal colonization by AM fungi but had a positive interaction in improving the growth and nutrition of W. somnifera. The total bacterial population and Bacillus species were found to be high in inoculated plants when compared to control indicating that the inoculated organisms persist well in the rhizosphere soil. Similar results were obtained by Kuan et al [38] where inoculation with Klebsiella sp. Br1 and Acinetobacter sp. S3r2 gave significantly higher bacterial populations in their respective soils, with around 250â&#x20AC;&#x201C;300% over the uninoculated control (1.98 x 107 cfu gâ&#x2C6;&#x2019;1) in maize under green house conditions.
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Imperial Journal of Interdisciplinary Research (IJIR) Vol-3, Issue-2, 2017 ISSN: 2454-1362, http://www.onlinejournal.in The present study revealed that inoculation with B. licheniformis + A. laevis resulted in increase in plant growth parameters like stem girth and Biovolume index, and shoot, root and total plants dry weight. The nutrient uptake and total withanolide concentration was also significantly more in plants inoculated with Bl+Al. The Bacillus count and mycorrhizal parameters were also high in this treatment. Therefore it can be concluded that B. licheniformis + A. laevis is the best microbial consortium for inoculating W. somnifera.
5.Conclusions Polyethylene house study suggested that the best microbial consortia for inoculating W. somnifera were the combination of PGPR B. licheniformis and AM fungus A. laevis. The results of the interaction study between B. licheniformis and A. laevis and G. etunicatum brought that the interaction is synergistic. Thus, dual inoculation with B. licheniformis and A. laevis is very effective than single inoculation or inoculation with B. licheniformis + G. etunicatum in enhancing the growth, nutrition and yield of W. somnifera.
Acknowledgement This study was supported by UGC (MRP(S) 1314/KABA016/UGC-SWRO). Table 1: Influence of microbial inoculum on plant height, stem girth and biovolume index of W. somnifera under poly house conditions. Height Girth Biovolume Treatments (cm/plant) (mm/plant) Index Control 32.74f 7.42f 245.1f e b Bl 36.61 9.30 335.5c d e Al 37.83 8.30 305.8e Ce 38.00c 8.57d 332.0d Bl+Al Bl+Ce
40.94b 41.56a
9.61a 9.01c
396.5a 370.7b
Table 3: Influence of microbial inoculum on shoot NPK and total withanolide concentration of W. somnifera under polyhouse conditions. Treatments Shoot nutrient concentration N% P% K% Control 1.62c 0.52 0.7c Bl 1.89b 0.53 0.92b a Al 2.21 0.59 1.18a a Ge 2.17 0.52 0.85bc a Bl+Al 2.22 0.53 1.13a Bl+Ce 2.17a 0.61 0.88b NS Legend as in table 1, NS=Non significant Table 4: Influence of microbial inoculum on shoot NPK and total withanolide concentration of W. somnifera under polyhouse conditions. Root nutrient Total concentration Treatments withanolide (%) N% P% K% Control 2.17 0.58c 0.96c 0.12d Bl 2.32 0.81a 1.10bc 0.14c a Al 2.22 0.66ab 1.3 0.17b c Ge 2.32 0.64bc 1.07 0.15c Bl+Al 2.37 0.86a 1.27ab 0.21a a Bl+Ce 2.18 0.84a 1.29 0.20a NS Legend as in table 1, NS=Non significant
Table 5: Influence of microbial inoculum on mycorrhizal root colonization, mycorrhizal spore count total bacterial and Bacillus count of W. somnifera under polyhouse conditions.
Control
82.2e
Mycorrhizal spore count/ 50g soil 101.33e
Bl
84.4d
131.00de
58a
17a
c
dc
b
Treatments
Mycorrhizal root colonization (%)
Bacterial population 108 cfu/ g of soil 18b
Bacillus 105 cfu/g of soil 5c
Note: means with same letter do not differ significantly at P = 0.05, Bl = Bacillus licheniformis, Al = Acaulospora laevis and Ce = Claroideoglomus etunicatum.
Al Ge
90.0 92.4bc
191.66 153.33cd
25 27b
8bc 8bc
Bl+Al
94.4a
279.33a
48a
12ab
Table 2: Influence of microbial inoculum on shoot, root and total dry weight of W. somnifera under poly house conditions. Shoot dry Root dry Total dry Treatments weight weight weight (g/plant) (g/plant) (g/plant) Control 3.91e 2.07f 5.98f Bl 3.35c 4.32c 7.67c d e Al 4.22 3.06 7.28e Ce 4.39b 3.18d 7.57d a a Bl+Al 5.55 4.42 9.97a a b Bl+Ce 5.56 3.53 9.09b
Bl+Ce 93.3b Legend as in table 1
227.33b
62a
11.33b
Legend as in table 1
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