Autochthonous halotolerant plant growth-promoting rhizobacteria promote bacoside A yield of Bacopa monnieri(L.)Nash and phytoextraction of salt-affected soil

2020-10-30UmeshPANKAJDurgeshNarainSINGHPoojaMISHRAPoojaGAURVivekBABUKarunaSHANKERandRajeshKumarVERMA

Umesh PANKAJ,Durgesh Narain SINGH,Pooja MISHRA,Pooja GAUR,C.S.Vivek BABU,Karuna SHANKER and Rajesh Kumar VERMA

1Department of Soil Science,CSIR-Central Institute of Medicinal and Aromatic Plants,Lucknow 226015(India)

2Division of Biotechnology,CSIR-Central Institute of Medicinal and Aromatic Plants,Lucknow 226015(India)

3Division of Microbial Technology,CSIR-Central Institute of Medicinal and Aromatic Plants,Lucknow 226015(India)

4Analytical Chemistry Department,CSIR-Central Institute of Medicinal and Aromatic Plants,Lucknow 226015(India)

5Research Centre,CSIR-Central Institute of Medicinal and Aromatic Plants,Bangalore 560065(India)

ABSTRACT Phytoremediation is a promising approach for reclamation of salt-affected soil.Phytoextraction is the most commonly used process,which exploits plants to absorb,immobilize,and accumulate salt in their shoots.In this study,halotolerant plant growth-promoting rhizobacteria(PGPR)were isolated from the rhizosphere of wild grasses growing naturally in salt-affected areas of Lucknow,Uttar Pradesh(India)and were tested for their efficacies of salt-tolerance and plant growth-promoting(PGP)abilities.Based on 16S rRNA sequences,the most efficient halotolerant isolates possessing PGP traits were identified as Pseudomonas plecoglossicida(KM233646),Acinetobacter calcoaceticus(KM233647),Bacillus flexus(KM233648),and Bacillus safensis(KM233652).Application of these isolates as bio-inoculants significantly(P ＜0.05)increased the growth and bacoside A yield of a medicinal plant,Bacopa monnieri(L.)Nash,grown on natural salt-affected soil.The phytoremediation of salt-affected soil was evident by the substantial increase in shoot Na+:K+ ratio of bio-inoculant-treated plants.When compared to un-inoculated control plants,the soil physico-chemical properties of bio-inoculant-treated plants were improved.The shoot and root biomass(fresh and dry weights),soil enzymes,and soil nutrient parameters showed significant positive correlations with the shoot Na+:K+ ratio.Consequently,the halotolerant PGPR screened in this study could be useful for the reclamation of saline soils concomitant with improved plant growth and bacoside A yield.

Key Words: bio-inoculant,phytoremediation,plant productivity,soil physico-chemical property,soil salinity

INTRODUCTION

Soil salinity is a major limiting factor for plant growth and productivity. Detrimental effects of salinity are the results of the complex interactions between morphological,physiological, and biochemical processes affecting plant growth and productivity.Osmotic stress,nutrient imbalance,and membrane destabilization are major constraints for plant growth under salt-affected soils(Singh and Chatrath,2001;Zhu,2007;Akbarimoghaddamet al.,2011).However,most plants possess different mechanisms to reduce the deleterious effects of salinity,including synthesis of compatible solutes,induction of anti-oxidative enzymes and plant hormones,compartmentalization of ions, and altered photosynthetic pathways(Rojas-Tapiaset al.,2012;Bhartiet al.,2013).In addition to reduced crop productivity, salinity stress also leads to abandonment of agricultural areas. To decrease the negative effects of soil salinity,salt is removed by scraping of soil surface layer, soil irrigation with clean water, and soil treatment with organic matter(USEPA,2000;Alberta Environmental Sciences Division, 2001). Besides being costly,the other major drawback of these techniques includes the lack of suitable clearance of contaminants. Extensive research is required for the development of an economical method for the re-establishment of vegetation, as well productivity in salt-affected soils(USEPA,2000).

Phytoremediation is a cost-effective and eco-friendly process that uses plants for absorption(uptake),immobilization,and accumulation of soil contaminants(Komives and Gullner,2000;USEPA,2000).Phytoextraction is the most common type of phytoremediation method applied for the reclamation of saline soil.In the process of phytoextraction,plants accumulate salt in their shoots and afterward salt can be removed by harvesting of foliage (USEPA, 2000).However,the efficacy of phytoextraction depends on several factors,including soil characteristics,plant growth,and plant biomass.Thus,efficient phytoextraction requires sufficient above-ground plant biomass,and this is a matter of concern because salinity stress inhibits seed germination and plant growth.Different strategies have been developed by many researchers to reduce the toxic effects of salinity to plants,such as traditional breeding method,development of transgenic,and usage of plant growth promoting rhizobacteria(PGPR)(Flowers and Yeo,1995;Bimponget al.,2016).

The PGPR,often referred to as‘biofertilizers’,are known for their ability to promote growth and secondary metabolite content of plants directly or indirectly(Dimkpaet al.,2009;Singhet al.,2014).The direct mechanism involves biological nitrogen fixation,phosphate solubilization,siderophore production, and synthesis of plant hormones such as indole acetic acid,cytokinins,or gibberellins(Rojas-Tapiaset al.,2012).The indirect mechanism entails suppression of plant pathogens by the production of antagonistic molecules(Kloepperet al.,2004).In recent years,PGPR have drawn considerable attention towards the alleviation of salt stress and crop improvement under saline environments.Different studies had shown beneficial effects of halotolerant PGPR in phytoremediation concomitant with improved plant growth in salt-affected soils (Mayaket al., 2004; Chang, 2007;Nadeemet al.,2007;Greenberget al.,2010).Salt-affected soil is an extreme habitat where halophilic microorganisms grow amply at the extreme pH and salinity.For better survival and optimal performance of any PGPR in salt-affected soils,both plant growth promoting(PGP)and salt-tolerant abilities are desired (Bhartiet al., 2013). Therefore, isolation of indigenous halotolerant microorganisms from salt-affected soils and screening for their PGP traits is a promising way for the selection of efficient halotolerant strains that could be used as bio-inoculants for plant growth promotion and phytoremediation of salt-affected soil(Shrivastava and Kumar,2015).

MATERIALS AND METHODS

Soil sampling and physico-chemical properties

For isolation of halotolerant PGPR,salt-affected soil was collected in March 2015 from the rhizosphere ofSaccharum munja(family Poaceae),which is the most prominent naturally growing grass in salt-affected areas of Bachhra wan (26°29′N, 81°7′E), Khantari (27°2′N, 80°59′E),and Ranipur(26°37′N,80°48′E)regions of Uttar Pradesh,India.Soil samples were collected from private land after obtaining permission from the land owner.In these areas,salinity developed through sedimentary processes involved in the accumulation of salts in large quantities with parent materials in the semi-arid climate and alluvial plains,primarily by transportation and deposition along the major rivers,originating from the Himalayas(Bhargavaet al.,1981).Soil samples, which were closely adhered to the root surfaces ofS. munja, were collected. The collected samples were transported to the laboratory and processed immediately for isolation of salt-tolerant PGPR.Soil physico-chemical properties,such as pH,electrical conductivity(EC),cation exchange capacity(CEC),exchangeable sodium percentage(ESP),and sodium adsorption ratio(SAR),were analyzed using standard protocols as described elsewhere (Gupta,2007). The high values of soil EC (4.60–6.90 dS m－1),pH (9–11), ESP (50%–57%), and SAR (17–22) depicted the high salinity level of the sites selected for isolation of halophiles(Table SI,see Supplementary Material for Table SI).

Isolation and characterization of halotolerant PGPR

Halotolerant bacteria were isolated from rhizosphere soil by enrichment culture techniques(Singh and Jha,2016).In brief,5.0 g soil was mixed with 100 mL nutrient broth medium amended with 5%(weight:volume)NaCl,and incubated for 24 h under shaking condition at 170 r min－1and 30°C;and then 0.1 mL of serially diluted inoculums from the enrichment culture were spread on nutrient agar plates amended with varying concentrations(2%–12%)of NaCl(Upadhyayet al.,2009;Bhartiet al.,2013).Colony counts were recorded after 48 h of incubation at 30°C.In comparison to solid medium,microbial cells were exposed to salt to a greater extent in broth medium, and thereby,the minimum inhibitory concentration was reduced.Thus,strains which showed the maximum tolerance to NaCl on nutrient agar plates were further tested for their hyper-tolerant ability in broth medium.In broth medium,strains were not able to grow beyond 6%–8%NaCl,and only strains depicting tolerance to 6%–8%NaCl were selected for characterization of PGP attributes(Upadhyayet al.,2009).

Subsequently, the biochemical and enzymatic assays were performed in respective medium amended with 5%NaCl. The production of indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate(ACC)deaminase activity were determined in tryptophan-amended medium as described by Gordon and Weber(1951)and Piromyouet al.(2011),respectively.The selected isolates were screened for phosphate solubilization and their phosphate solubilizing index was calculated as a ratio of the sum of the colony diameter and halo zone diameter to colony diameter(Pikovskaya,1948;Edi-Premonoet al.,1996).Siderophore and hydrogen cyanide production were detected as described elsewhere(Schwyn and Neilands, 1987; Alström and Burns, 1989).Formation of a clear halo zone around colonies because of a change in colour from dark blue to yellow is indicative of the production of siderophores(Schwyn and Neilands,1987). The diameter of the halo zone was measured in millimeter. ThenifHgene genomic DNA of isolates was extracted using the Wizard genomic DNA purification kit(Promega,USA).Genomic DNA ofAzospirillum brasilensewas taken as the positive control.Approximately 30–50 ng of genomic DNA were used as a template for amplification of thenifHgene using universalnifHgene primers PolF(5′-TGCGAYCCSAARGCBGACTC-3′)and PolR(5′-ATSGCCATCATYTCRCCGGA-3′)(Coelhoet al.,2008).The gene was amplified in a 25 μL reaction mixture containing 200–250 μmol L－1of forward and reverse primers and 1.0 unit of Taq DNA polymerase(Genei,Inida).The amplification conditions were set as follows:an initial denaturation of 3 min at 95°C,followed by 30 cycles of 1 min at 94°C,1 min at 55°C,and 1 min at 72°C,with a final extension of 5 min at 72°C.The amplicons were visualized on 1.5%agarose gel at 5 V cm－1under an ultraviolet(UV)light using an Alpha imager(Alpha Innotech,UK).In subsequent steps,thenifHamplicon was sequenced at CSIR-Central Institute of Medicinal and Aromatic Plants(CIMAP),Lucknow,India using the forward primer PolF(5′-TGCGAYCCSAARGCBGACTC-3′)and a BigDye Terminator v3.1 cycle sequencing kit(Applied Biosystems,USA)in an ABI 3130 automated DNA sequencer (Applied Biosystems). Additionally, the growth of isolates was also tested in a nitrogen-free Jensen’s medium(M710,HiMedia,India).Growth pattern of halotolerant isolates was monitored in nutrient broth (control)and nutrient broth amended with varying concentrations of NaCl.The nutrient broth medium inoculated with overnight grown culture was incubated at 30°C and 170 r min－1in an incubator shaker.Growth pattern of isolates was determined by measuring the absorbance of culture at 600 nm(A600)with a spectrophotometer(Perkin Elmer,USA),at regular intervals of 3 h until the culture reached stationary phase.

Identification of halotolerant PGPR

Halotolerant PGP strains were identified based on the 16S rRNA gene sequence.Briefly,the 16S rRNA gene was amplified using universal bacterial primers 8f(5′-AGAGTTTGATYMTGGCTCAG-3′)and 1495r(5′-CTACGGCTACCTTGTTACG-3′).A 50 μL reaction mixture included 30–50 ng of genomic DNA,200–250 μmol L－1of primers, and 1.0 unit of Taq DNA polymerase.Amplification conditions were set as follows:an initial denaturation of 5 min at 95°C,followed by 35 cycles of 1 min at 94°C,1 min at 51°C,and 1 min at 72°C,with a final extension of 5 min at 72°C.The 16S rRNA gene amplicons were visualized in 0.8%agarose gel at 5 V cm－1under a UV light using an Alpha imager(Alpha Innotech).For sequencing,the 16S rRNA genes were purified using the Wizard SV gel PCR purification kit (Promega)and quantification was conducted using a NanoDrop-1000 spectrophotometer(Thermo Fisher Scientific,USA).Direct sequencing was performed at CSIR-CIMAP,using forward universal primer 8f (5′-AGAGTTTGATYMTGGCTCAG-3′)and a BigDye Terminator v3.1 Cycle sequencing kit(Applied Biosystems) in an ABI 3130 automated DNA sequencer (Applied Biosystems). Isolates were identified by comparison of their 16S rRNA gene sequences with nucleotide sequences present in the National Center for Biotechnology Information(NCBI)database using a standard nucleotide BLAST search.The 16S rRNA gene sequences of isolates were aligned with similar sequences retrieved from the NCBI database using the ClustalW programme(Mega 6.0),and a phylogenetic tree was constructed using the neighbour-joining method of MEGA 6.0(Singhet al.,2012).

Experimental design

A pot experiment was conducted in a completely randomized design with five treatments and four replications(n=4)under glass house conditions with an average temperature of 28±0.5°C at CSIR-CIMAP. For planting,healthy erect shoots ofB. monniericv. CIM-Jagriti were obtained from the gene bank of CSIR-CIMAP.Plant terminal part(5–8 cm)was excised and washed under running tap water to remove adhering soil particles. Plants were sterilized with 70% ethanol for 30 s followed by washing with sterile deionized water three times. The halotolerant PGPR(P.plecoglossicida,A.calcoaceticus,B.flexus,andB.safensis)possessing plant growth promotion traits,such as production of phyto-hormones(e.g.,IAA),siderophore,and ACC deaminase, phosphate solubilization, and biological nitrogen fixation, were used as bio-inoculants to promote the growth and bacoside A yield ofB.monniericoncomitant with phytoremediation of salt-affected soil. The bacterial inoculum was prepared by growing cultures to the exponential phase in 200 mL nutrient broth amended with 5%NaCl.The culture suspension was centrifuged at 7 000 r min－1for 10 min and the cell pellet was washed twice with sterile normal saline and re-suspended in 50 mL sterilized normal saline to adjust theA600value to 1.0(1×107cells mL－1).Surface sterilized plants were pre-treated with the respective bacterial culture by dipping in the culture suspension for 30 min.After treatment, plants were planted(2 plants pot－1)in plastic pots containing 1 kg sterile(autoclaved)salt-affected soil from Khantari region(Bhartiet al.,2013).After planting,10 mL of specific bacterial suspension(1×107cells mL－1)was poured into the respective treatment,whereas normal saline was used for the control.To allow the initial establishment of plants in salt-affected soil,one-third of the recommended dose of N and P(N:P =30:20)was added to the experimental soil before planting.Soil was not amended with potassium because it was already rich(360 kg ha－1)in the initial soil.Pots were irrigated with sterilized water whenever required to maintain the optimum moisture level. We also confirm that this study did not involve any endangered or protected species.

Measurement of plant growth parameters

Metabolic parameters of un-inoculated plants,as well as PGPR-inoculated plants,were analyzed to assess the stress amelioration by bio-inoculants. Total chlorophyll (Tchl)content was spectrophotometrically quantified at 645 and 663 nm(Harborne,1998).Superoxide dismutase(SOD,EC 1.15.1.1) activity was assayed as described by Becanaet al.(1998),and proline accumulation was quantified using the standard method as described by Bateset al. (1973).Plant height and shoot and root fresh and dry weights were recorded manually.

Quantification of total bacoside A content

For quantification of bacoside A content,aboveground biomass samples from each treatment were collected,dried,and powdered. One gram of dried plant material was extracted with ethanol using continuous acoustic exposure in a Microclean-109 ultrasonic bath(300 mm×250 mm×125 mm,34±3 kHz,PZT Sandwich type six transducer,250 W)(Oscar Ultrasonics,India),and the extract was vacuum concentrated at 40°C.The residue obtained was re-dissolved in 5 mL of high performance liquid chromatography(HPLC)-grade methanol and centrifuged at 10 000 r min－1. The supernatant was filtered with a 0.45-μm nylon syringe filter prior to HPLC analysis.HPLC analysis was conducted on Waters HPLC equipment(600E pump,717plus auto-injector,996 photodiode array detector)using a Merck Chromolith®RP18e column (100 mm×4.6 mm inner diameter); the mobile phase was HPLC-grade acetonitrile-water (30:70,volume:volume)(Merck,Germany)with a flow rate of 0.8 mL min－1.Detection was made at 205 nm(Bhandariet al.,2006). Data acquisition and computation were performed using Empower software.The standard of bacoside A(Sigma Aldrich,USA)was purchased from Life Technologies,India and was confirmed by co-injection of standard and sample.

Post-harvest soil analysis

Analysis of rhizosphere soil collected after harvesting of plants was performed to monitor the changes in nutrients status,as well as biological properties of the soil.Colony forming unit(CFU)of rhizosphere soil was measured by the standard serial dilution method.Serially diluted soil samples(0.1 mL)were spread on nutrient agar plates amended with 6%NaCl.Plates were incubated at 30°C for 48 h,and the number of colonies was recorded.Soil organic carbon(SOC)was determined by the rapid dichromate oxidation method(Walkley and Black,1934),total Kjeldahl nitrogen(TKN)by the micro Kjeldahl method,available phosphorus(P)by the Olsen’s method(Olsenet al.,1954),and available potassium by flame photometer using the neutral normal ammonium acetate method (Pageet al., 1982). Soil dehydrogenase(DHA),alkaline phosphatase(ALP),and acid phosphatase(ACP)activities were determined as described by Tabatabai(1982).

Data analysis

Data(n=4)were analyzed using the SPSS 17 software package for Windows and subjected to a one-way analysis of variance.The Tukey’s test was used forpost-hoccomparisons to determine differences between means.Differences were considered significant atP ≤0.05.The Pearson’s correlation coefficient(r)was also performed among various observed parameters using SPSS 17 software.

RESULTS

Screening of halotolerant PGPR

The number of colonies observed on nutrient agar plates decreased with increasing NaCl concentration.The maximum number of colonies appeared on the plates amended with 2%NaCl,whereas the minimum number of colonies was observed on the plates amended with 10%NaCl.No growth was observed on the plates amended with 12%NaCl(data not shown).On the plates amended with 10%NaCl,numbers of distinct colonies were 28, 17, and 11 from Khantari,Ranipur,and Bachhrawan samples,respectively.When these halotolerant isolates were grown in nutrient broth medium amended with different NaCl concentrations,isolates from Khantari and Ranipur samples were not able to grow beyond 6%NaCl,and Bachhrawan isolate was proficiently growing at 8%NaCl(Fig.1).Among these halotolerant strains,only two isolates(UP4 and UP5)from Ranipur,one isolate(UP6)from Bachhrawan,and one isolate(UP10)from Khantari soil exhibited PGP traits.Growth patterns of halotolerant PGP isolates in nutrient broth amended with 6%NaCl(Ranipur and Khantari isolates)and 8%NaCl(Bachhrawan isolate)exhibited an extended lag phase when compared to the control (without NaCl) (Fig. 1). Amplification of a 360-base pair fragment from the genome of Ranipur isolate (UP4)indicated the presence of thenifHgene,which was absent in all other isolates.The PGP traits of halotolerant isolates of this study are listed in Table SII(see Supplementary Material for Table SII).

Fig.1 Growth patterns of different halotolerant isolates UP4(identified as Pseudomonas plecoglossicida),UP5(identified as Acinetobacter calcoaceticus),UP6(identified as Bacillus flexus),and UP10(identified as Bacillus safensis)in nutrient broth medium amended with 0,2%,4%,6%,8%,and 10%NaCl.Vertical bars indicate standard errors of the means(n=4).

Identification of bacterial isolates

Halotolerant strains growing well in medium amended with 6%and 8%NaCl and possessing PGP traits were identified using 16S rRNA gene sequencing.Identification based on comparison of 16S rRNA gene sequences and phylogenetic analysis of isolates showed that isolates UP4 and UP5 from Ranipur soil were similar toPseudomonas plecoglossicidaandAcinetobacter calcoaceticus,respectively.Isolate UP6 from Bachhrawan soil was identified asBacillus flexus,and isolate UP10 from Khantari soil asBacillus safensis(Fig.2).The 16S rRNA gene sequences of halotolerant isolates described in this study have been deposited in GenBank under accession numbers KM233646 (UP4), KM233647(UP5),KM233648(UP6),and KM233652(UP10).

Performance of halotolerant bio-inoculants

Performance of bio-inoculants in this study was evaluated after their application to the rhizosphere region ofB.monnieriplants grown in natural saline soil.The bio-inoculant-treated plants showed better growth in terms of plant (shoot and root)fresh weight,plant dry weight,and plant height when compared to that of the un-inoculated control plants(Fig.3a,b).Increased plant height,plant dry weight,and root fresh and dry weight were observed in the bio-inoculant-treated plants. As compared to that of the un-inoculated control,the maximum increase in plant fresh weight was observed in the plants inoculated withP. plecoglossicida(88.2%),followed byA.calcoaceticus(64.2%),B.flexus(61.3%),andB. safensis(17.0%) (Fig. 3b). When compared to the uninoculated plants,higher chlorophyll content was observed in the bio-inoculant-treated plants(Fig.3c).Intriguingly,a reduction in proline content and SOD activity was observed in the bio-inoculant-treated plants when compared to the uninoculated control.In comparison to the un-inoculated plants,the maximum decrease in proline content was observed in the plants treated withP.plecoglossicida(61.8%),followed byB.flexus(59.7%),A.caloaceticus(30.5%),andB.safensis(22.9%)(Fig.3d).Likewise,the maximum reduction in SOD activity was observed in the plants treated withB. flexus(47.5%)andP.plecoglossicida(36.4%)(Fig.3d).Moreover,increased levels of Na+:K+ratio and P content(P uptake)in shoots of bio-inoculant-treated plants were observed(Fig.4).

Fig.2 16S rRNA gene-based phylogenetic relationships of halotolerant isolates UP4(identified as Pseudomonas plecoglossicida)(a),UP5(identified as Acinetobacter calcoaceticus)(b),and UP6(identified as Bacillus flexus)and UP10(identified as Bacillus safensis)(c)with other representatives of taxonomic groups in the National Center for Biotechnology Information(NCBI)database.The tree was constructed by a neighbour-joining method using MEGA 6.0 software.Bootstrap values are indicated at each branching point,and accession numbers of selected sequences are given in parentheses.

Qualitative evaluation of B.monnieri

Total bacoside A is a mixture of bacoside A3,bacopaside II,bacopaside X,and bacopasaponin C(Fig. S1, see Supplementary Material for Fig. S1), and therefore four well-separated peaks a–d were observed in the HPLC chromatogram(Fig.5a).All plants inoculated with PGPR showed a significant increase in total bacoside A content when compared to the un-inoculated control. In comparison to the un-inoculated control, the maximum increase in bacoside A content was observed in the plants inoculated withP.plecoglossicida(121.1%),followed byB.flexus(53.4%),A.caloaceticus(45.0%),andB.safensis(44.7%)(Fig.5b).

Fig.3 Effects of halotolerant bio-inoculants Pseudomonas plecoglossicida(Pp),Acinetobacter calcoaceticus(Ac),Bacillus flexus(Bf),and Bacillus safensis(Bs)on physiological traits of Bacopa monnieri grown on salt-affected soil.The un-inoculated plants were used as control(CK).Image was taken at the harvest stage(a),and growth parameters(b),chlorophyll contents(c),and proline content and superoxide dismutase(SOD)activity(d)of the plants inoculated with different bio-inoculants were recorded.Vertical bars show standard errors of means(n=4).Means with the same letter(s)are not significantly different at P ≤ 0.05 by Tukey’s test.FW=fresh weight;DW=dry weight;Chl a=chlorophyll a;Chl b=chlorophyll b;TChl=total chlorophyll.

Fig.4 Comparative analysis of Na+:K+ ratio and P uptake in the shoots of Bacopa monnieri plants, treated with bio-inoculants Pseudomonas plecoglossicida(Pp),Acinetobacter calcoaceticus(Ac),Bacillus flexus(Bf),and Bacillus safensis(Bs),grown on natural saline soil.The un-inoculated plants were used as control (CK). Vertical bars show standard errors of means(n=4).Means with the same letter(s)are not significantly different at P ≤ 0.05 by Tukey’s test.

Post-harvest soil evaluation

Post-harvest soil biological and physico-chemical properties were evaluated to monitor the establishment and proliferation of inoculated strains within the rhizosphere,as well as their performance under stressed conditions(Table I).Successful establishment and proliferation of bio-inoculants were measured by quantifying microbial load (CFU g－1soil) and soil enzyme activities in terms of DHA, ALP,and ACP.The population of rhizobacteria was substantially high(66.1×106to 92.4×106CFU g－1soil)in the bioinoculant-treated plants when compared to the un-inoculated control,which had negligible populations(Table I).Likewise,rhizosphere soil from the bio-inoculant-treated plants showed an elevation in ALP(7–8 folds),ACP(2–3 folds),and DHA(3–4 folds)activities when compared to that of the un-inoculated control(Table I).Moreover,decreases in Na+:K+ratio, EC,ESP,SAR,and pH of rhizosphere soil from the bio-inoculant-treated plants were observed (Table I).Changes in nutrient status of post-harvest rhizosphere soil were assessed by quantification of soil TKN,SOC,and available P levels.Increases in soil TKN,SOC,and available P levels were observed in the rhizosphere soil from the bio-inoculant-treated plants(Table I).

Correlation coefficients among different parameters

Fig.5 Analysis of total bacoside A yield:high performance liquid chromatography chromatogram depicting bacoside A peaks a–d(a)and quantitative measurement of bacoside A yield(b)in Bacopa monnieri plants,treated with bio-inoculants Pseudomonas plecoglossicida(Pp),Acinetobacter calcoaceticus(Ac),Bacillus flexus(Bf),and Bacillus safensis(Bs),grown on natural saline soil.The un-inoculated plants were used as control(CK).Vertical bars show standard errors of means(n=4).Means with the same letter(s)are not significantly different at P ≤ 0.05 by Tukey’s test.

TABLE I Biological and physico-chemical propertiesa) of rhizosphere soil from Bacopa monnieri plants,treated with bio-inoculants Pseudomonas plecoglossicida,Acinetobacter calcoaceticus,Bacillus flexus,and Bacillus safensis,grown on natural saline soil

Pearson’s correlations between various physiological traits ofB.monnieriand properties of post-harvest soil were evaluated(Table II).The data revealed that plant SOD and proline contents were negatively correlated with chlorophyll contents(Chla,Chlb,and TChl),plant height,shoot and root biomass(fresh and dry weights),and soil chemical and biological properties(r=-0.01 to-0.87**).High degrees of significant positive correlation were observed between Chlband TChl(r=0.99**),and between soil ALP activity and CFU(r=0.96**).Plant shoot and root biomass(fresh and dry weights)showed significant positive correlations with total bacoside A yield(r=0.55*to 0.91**).The growth and yield ofB.monnieriwere positively correlated with plant nutrient uptake.Shoot P uptake showed significantly positive correlations with PH,PDW,RFW,and TBA(r=0.54*to 0.66**).The population of rhizobacteria(i.e.,CFU)showed significant positive correlations with soil enzyme activities(r=0.80**,0.89**,and 0.96**for ACP,DHA,and ALP,respectively),SOC(r=0.84**),and TKN(r=0.85**).

DISCUSSION

P GPR and cultivation of B.monnieri on salt-affected soil

a/K SN Na/K root fresh;available P U P 0.14 1 W=RFP=SP 1 0.24 1 0.02 -eiicg hCt;;A TK 0.43-0.37 00.360.63 N.1**9 plant dry w OC=soil organ 0.28 AP 1 W=* 0.51* 1 6 0.40 -PD SO 0.22 -.8 hatase;S C.3* 10.276*eight;* 0** 0.80*P* 0 AL 0.36*** 0.69*0.45*plant fresh w** 0.84*0.50* -alkaline phosp 1 0.91 ACP** 1 0.21 0.57 0.66 0.53 W=PF 0.710.47* -AL plant height;A P=DH 0.38 0.89 1* 0.96** 0.95**0.58 phosphatase;0.35 0.63** -**il*****So CFU 0.59** 0.35 0.85**1 0.89 H=acid**0.680.44* -perties of post-harvest soil P=0.23 4*3*.5.5.5.6* 0.84** 0.80**BA 6*0*0.45 AC 6*W T 1** 0.74** 1 1* 0.57** 0.4 0.62** 0.80** 0.86**0.23 ase;RL=proline;P RD 3* 0.53* 0 0.42 -0.06 -2** 1 0.16 -drogen dehy A=** 0* 0 0.19 -0.47* 0 0.55* 0* 0 0.12 W RFW.7.9 4** 0.72** 0 2* 0.60** 0.5.6* 0.5.5.6 0.55* 1 e dismutase;P-0.32 -0.70 0.53* 0.42 DH 6** 0.30 PD* 0.54*onnieri and pro 0.07 -0.38 eroxid0.44.07 0 nit;a+SN 0.64* 0.45* 0.49*soil N.05 W 1 0.12 -0.61******0.63 0.66 0.06-0* 0.86** 1:K+ ratio.* 0.73** 0.74*0.57** 00.20**0.73** 0* 0.63** 0.68*acopa m 0.51 PF OD=sup:K+ ratio;9*2*130 f B.4.6.4.1.4.7.6.7.7.5 PH* 0.59** 0.64*8*5*8*9*8*1*1** 0.4** 0 a+** 0** 0** 0 hysiological traits o 0.58** 0.39** 0 0.58 0.74** 0* 0 0.75** 0** 0 FU=colony forming u 0.54* 0.41 Chl=e A0.69 L 1-0.32** 0* -0.46 0.66 0.87* -0.75** -0.69 0.56* --0.45* --0.26 0.16-0.69*ectively.* -Na/K=p* -0.61 0.81 tal bacosid D lant N 1 0.73;C 3** -SOPR 0.13 to hl a/K=* 0.37 0.43**0.43-0.77*0.39-0.32 -≤0.01,resp the p TC 0.23 0.60** -A=.6 0.42-0.85*0.76* 1 total chlorophyll;S* -cients amo 0.46*3** -0.06 -0.17-0.53* -0.16-0.50* -0.27-0.39 00..5586**** - -* 0 0.62** 0 0.49 0.82** --0.36 0.74** -.6ng=chlorophyll b;T TB 0.39-0.41 0.29-0.28 5 and P 0.14 0.22 Ch 0.15 l b 0.23-0.61*coeffiuptake;P 0.42 0.46.99*hl b PU=shoot P 0.43 0.48*0.27 -0.35 0.27.59*0.250.46.0≤0;C t Chl a.1.0*1 -7 -0.17 0.64 0.06 -ahl N.0* --0 0.21.53*0.48 0-.1099 -0.23 0.66 0.62 0.34** 0 0.06*0.52 Parametera) Plan** 00.53*0.24-0 0.27-0 0.47 0* -*icant at P 0.32 jeld DW=root d 1 C h l b E II if tal K Plant Chl a to PH eight;Pearson’s correlation DL 0.30.5 BL SO** 1 T C h l 0.7 4** 0 PR WWWWA SoilCF PF PD Sign RD RF TB UAPPC DH AP=chlorophyll ary w;S AC**AL SO N TK Ua/K l a PN TA SP a/K SNa)Chht;R*,wTKeigN=

Soil salinity affects plant growth and productivity by imposing osmotic as well ionic stress to plants(Shabala and Cuin,2008).The PGPR are well known for their abilities in alleviating detrimental effects of environmental stresses(Lugtenberg and Kamilova,2009).The role of PGP bacteria in phytoremediation of contaminated soil has been reported earlier.The role of arsenic-resistant endophytic PGP bacteria in phytoextraction of arsenic has been reported(Tiwariet al.,2016).Likewise,Maet al.(2015)reported the role of metal-resistant endophytic bacteria in phytoremediation of multi-metal contaminated soil.This study was conducted to explore naturally salt-affected soils for competent halotolerant PGPR,which can efficiently promote phytoextraction in salt-affected soils concomitant with improved growth of a medicinal plantB.monnieriin natural salt-affected soils.Earlier studies had reported improved growth and bacoside A content ofB.monnierigrown under normal soil or artificially created saline soil(Bhartiet al.,2013;Singhet al.,2014).However,only a few reports are available for exploitation of indigenous halotolerant PGPR for the phytoremediation of salt-affected soils concomitant with improved plant growth(Kishore and Singh,2005;Shrivastava and Kumar,2015).For isolation of efficient halotolerant PGPR, enrichment culture technique was followed to ensure the isolation of under-represented taxa,because with the use of the minimal medium the possibility of losing under-documented taxa would be always high (Bhartiet al., 2013). In this study,the most efficient halotolerant PGP isolates were used as bio-inoculants for improvement of growth and bacoside A content ofB. monnierion salt-affected soils. Application ofP.plecoglossicida,B.flexus,andA.calcoaceticussignificantly enhanced the growth and bacoside A content ofB.monnierion salt-affected soils. Congruently, halotolerant PGPPseudomonas,Acinetobacter,andBacillusstrains have been reported by other researchers(Hrenovic and Ivankovic,2009;Upadhyayet al.,2012;Huanget al.,2013;Nakbanpoteet al., 2014). In our findings, alleviation of salinity stress to plants and PGP activity of bio-inoculants can be attributed to their ability to produce ACC deaminase enzyme(P.plecoglossicida,A.calcoaceticus,andB.safensis),siderophores(P.plecoglossicidaandA.calcoaceticus),and phosphate solubilization(P.plecoglossicidaandB.safensis).In situphosphate solubilizing activity ofP.plecoglossicidawas confirmed by the significant increase(by 31.92%)of phosphorus level(P uptake)in the shoot ofP.plecoglossicida-treated plants(Fig.4).The maximum P uptake in the shoot ofP.plecoglossicida-treated plant could be ascribed to the highest phosphate solubilizing activity ofP.plecoglossicidain comparison to those of other bio-inoculants(Table SII).Maximum root biomass(fresh and dry weights)production was also reported inP.plecoglossicida-treated pots(Fig.3b).This suggests that because of better root system,P.plecoglossicida-treated plants can absorb more phosphorus from the rhizosphere soil, leading to the maximum P uptake in shoots(Fig.4)and minimum available P level in rhizosphere soil ofP.plecoglossicida-treated plants(Table I).

Under stressed environments, plants produce ‘stress ethylene’, which regulates plant homeostasis and results in reduced plant growth (Shrivastava and Kumar, 2015).Within plants, ACC is a precursor for the production of ethylene, and bio-inoculants of this study produced ACC deaminase enzyme,which degraded plant ACC for nitrogen and energy.Moreover,bacterial degradation of ACC reduced the deleterious effects of ‘stress ethylene’and alleviated salinity stress to plants(Glicket al.,2007).Singh and Jha(2016)reported that halotolerant PGPRSerratia marcescensalleviated salinity stress to wheat by production of ACC deaminase.Achromobacter piechaudii, ACC deaminaseproducing PGPR,significantly reduced ethylene stress and increased biomass of tomato plants cultivated in saline soil(Mayaket al., 2004). Higher root biomass (fresh and dry weights)of treated plants is ascribed to IAA production by bio-inoculants. Congruently, IAA produced by rhizobacteria increased chickpea root biomass(Fierro-Coronadoet al., 2014). The PCR amplification ofnifHgene from the genome ofP.plecoglossicida(UP4)does not reflect its activity,and thus the growth ofP.plecoglossicida(UP4)was tested in nitrogen-free Jensen’s medium.The growth ofP.plecoglossicidain Jensen’s medium illustrated the nitrogen fixation properties under deficient nitrogen condition.However,any considerable increase in nitrogen content was not detected in rhizosphere soil ofP. plecoglossicida(UP4)-treated plants,and this suggested inhibition of nitrogenase by salinity stress and available nitrogen.Earlier studies reported thatAzospirillum brasilensegrown in nitrogen-free medium was relatively more sensitive to the presence of NH+4than high NaCl(Tripathiet al.,2002;Chowdhuryet al.,2007).The increase in chlorophyll content of bio-inoculant-treated plants showed that bio-inoculants counter the negative effects of salinity stress on the photosynthetic pathways and plant growth(Ahireet al.,2013;Singh and Jha,2016).The concentration of reactive oxygen species(ROS)is elevated under saline conditions and leads to cellular toxicity in plants grown under salinity stress(Bowleret al.,1992;Hasegawaet al.,2000).Plants possess antioxidant system(e.g.,SOD,a well-known scavenger of ROS)to combat ROS.The SOD activity in the bio-inoculant-treated plants was significantly reduced as compared to that of the un-inoculated control.Similar findings were reported in lettuce(Han and Lee,2005)and wheat crops(Upadhyayet al.,2012)grown under salinity stress.Likewise,decreased proline content of bio-inoculanttreated plants reproduced the stress-ameliorating abilities of bio-inoculants.Hamdiaet al.(2004),Nadeemet al.(2007),and Rojas-Tapiaset al. (2012) reported that plant proline content increased with salinity level, but decreased in the bio-inoculant-treated plants.Contrary to these observations,previous evidence suggested a positive correlation between plant proline accumulation and adaptation to salt stress,but results are yet under debate(Chandler and Thorpe, 1987;Ashraf and Fooland 2007;Rojas-Tapiaset al.,2012).The results from this study revealed that under salt stress,bioinoculants decreased plant SOD level and proline content concomitantly with improved plant growth(plant height and biomass).Siliniet al.(2016)reported that two varieties of wheat(Waha and Bousselam)inoculated withAzotobacter chroococcumAZ6 had decreased proline content in shoots under salt stress condition;however,un-inoculated varieties strongly accumulated proline.Similarly,Kumariet al.(2015)reported that bacterial inoculated plants exhibited decreased SOD and catalase activities under salinity stress,indicating the stress amelioration by bio-inoculants.

PGPR and phytoremediation of saline soils

This study described the phytoremediation of saltaffected soils through the cultivation ofB.monnieriunder the influence of halotolerant PGPR. The positive role of bio-inoculants in this study in reclamation of saline soil was evident by the decreased levels of rhizosphere soil EC,SAR,Na+:K+ratio,and ESP from the bio-inoculant-treated plants(Table I).Moreover,the increased Na+:K+ratio in the shoots of bio-inoculant-treated plants indicated the phytoextraction of salts by their efficient root system.This was corroborated by the correlation study, where plant Na+:K+ratio was significantly correlated with shoot and root biomass(fresh and dry weights) (Table II). Thus, the increased Na+:K+ratio in the shoots of bio-inoculant-treated plants may be due to the greater plant growth.Na+absorbed by roots is rapidly translocated to shoots through xylem and accumulates there with the evaporation of water.The transport of Na+to shoots is unidirectional causing its accumulation in shoots and foliage(Tester and Davenport,2003).Excessive accumulation of Na+causes damage to leaves, leading to necrosis and eventually reducing plant growth and yield(Munns,1993).To alleviate the adverse effects of Na+in plants,Na+is stored in vacuoles within plant cells(Apseet al.,1999;Blumwaldet al., 2000). Moreover, Na+has been reported essential for few plant species(Brownell,1980),and is required for growth and productivity in several plants(Truoget al.,1953;Hyltonet al., 1967; Marschner, 1971; Rush and Epstein,1981).Substantial evidence exists showing plants growing under saline conditions accumulate a large amount of salt and Na+,in particular.In most of plants,Na+is not a necessary nutrient required for growth,but only when the K+supply is limited,Na+promotes the growth of some plants by contribution to turgor formation and mineral nutrition(Dhillionet al.,1995;Maathuis and Amtmann,1999;Xiong and Zhu,2002;Tester and Davenport,2003).In this study,improved plant growth (height and biomass) contributed to the increased bacoside A content ofB.monnieriplants.Likewise,increased bacoside A content ofB.monnierigrown under normal soil or artificially created saline soil was correlated with improved plant growth(Bhartiet al.,2013;Singhet al.,2014).Furthermore,few studies have demonstrated positive effects of salt stress on secondary metabolite accumulation.Ahireet al.(2013)reported that accumulation of bacoside A increased with the increase in NaCl concentration up to 100 mmol L－1and that a further increase in NaCl decreased the bacoside A content.Decreased bacoside A content with an increased NaCl concentration might be due to the inhibition of enzymes involved in the biosynthesis of bacoside(Ahireet al.,2013).In this study,deleterious effect of salinity toB.monnieriplants was reduced by bio-inoculants and thus,there was no inhibition of bacoside-synthesizing enzymes in the inoculated plants when compared to the un-inoculated control.

Reclamation of saline soil was also illustrated by improved biological properties and nutrient status of bioinoculant-treated rhizosphere soil.The considerable increases in microbial count and enzyme activities of rhizosphere soil are the indicators for the improved biological properties of soil.Dehydrogenase and other enzyme activities are the indicators of microbial respiration as well as the measure of microbial activity in soil(Dhillionet al.,1995).An increase in soil DHA,ALP,and ACP activities indicated establishment, proliferation, and larger biomass of bio-inoculants within the rhizosphere region.Rhizobacteria can induce the expression of flavonoid genes,as evidenced by an extended exudation of plant flavonoids by a secondary inoculation ofAzospirilluminPhaseolus-Rhizobiuminteractions(Dimkpaet al.,2009).Therefore,increases in TKN,SOC,and available P levels in rhizosphere soil of the inoculated plants are accredited to higher microbial biomass and extended root exudation.

CONCLUSIONS

Our study emphasized the reclamation of saline soil concomitant with improved growth of a high-value medicinal plant,Bacopa monnieri,on natural salt-affected soil.Through the PGP activity,bio-inoculants in this study improved growth and bacoside A content ofB.monnieriplants by protecting them against inhibitory effects of salinity.Improved morphological,physiological,and biochemical traits of the bio-inoculant-treated plants contributed to a reduced Na+:K+ratio in rhizosphere soil and increased bacoside A content ofB.monnieri. Isolates in this study were able to ameliorate salt stress toB. monnieriplants and therefore might be used as bio-inoculants for improving plant growth and reclamation of salt-affected areas as a sustainable approach.Moreover,further comprehensive research is required for elucidation of mechanisms involved in stress amelioration by autochthonous halotolerant PGPR.

ACKNOWLEDGEMENT

The authors are thankful to director CSIR-Central Institute of Medicinal and Aromatic Plants,Lucknow(India)for providing all the necessary facilities required for this work.

SUPPLEMENTARY MATERIAL

Supplementary material for this article can be found in the online version.

Autochthonous halotolerant plant growth-promoting rhizobacteria promote bacoside A yield of Bacopa monnieri(L.)Nash and phytoextraction of salt-affected soil_参考网 (2024)