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Inoculation of root microorganisms for sustainable wheatericeand wheateblack gram rotations in India

Paul Mäder a,*, Franziska Kaiser a, Alok Adholeya b, Reena Singh b, Harminder S. Uppal b,Anil K. Sharma c, Rashmi Srivastava c, Vikram Sahai d, Michel Aragno e,Andres Wiemken f, Bhavdish N. Johri g, Padruot M. Fried h

aResearch Institute of Organic Agriculture (FiBL), Ackerstrasse, 5070 Frick, SwitzerlandbBiotechnology and Bioresources Division, The Energy and Resources Institute (TERI), Darbari Seth Block, India Habitat Centre, Lodhi Road, 110003 New Delhi, IndiacDepartment of Biological Sciences, CBSH, G.B. Pant University of Agriculture and Technology, 263 145 Pantnagar, UK, IndiadDepartment of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, 110016 New Delhi, Indiae Institute of Biology, Laboratory of Microbial Ecology, University of Neuchâtel, Rue Emile-Argand 11, PO Box 198, 2009 Neuchâtel, Switzerlandf Institute of Botany, University of Basle, Hebelstrasse 1, 4056 Basle, SwitzerlandgDepartment of Biotechnology, Barkatullah University, 462026 Bhopal, MP, IndiahAgroscope Reckenholz-Tänikon Research Station (ART), Reckenholzstrasse 211, 8046 Zurich, Switzerland

a r t i c l e i n f o

Article history:Received 14 July 2010Received in revised form10 November 2010Accepted 26 November 2010Available online 16 December 2010

Keywords:MicroorganismsMycorrhizaPGPRPseudomonasInoculationWheatYieldMineral nutrient concentrationMicro-elementsSoil enzymes

a b s t r a c t

The scarcity of non-renewable resources such as soils and fertilizers and the consequences of climatechange can dramatically in!uence the food security of future generations. Mutualistic root microor-ganisms such as plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF)can improve plant "tness. We tested the growth response of wheat (Triticum aestivum [L.]), rice (Orizasativa [L.]) and black gram (Vigna mungo [L.], Hepper) to an inoculation of AMF and PGPR alone or incombination over two years at seven locations in a region extending from the Himalayan foothills to theIndo-Gangetic plain. The AMF applied consisted of a consortium of different strains, the PGPR of two!uorescent Pseudomonas strains (Pseudomonas jessenii, R62; Pseudomonas synxantha, R81), derived fromwheat rhizosphere from one test region. We found that dual inoculation of wheat with PGPR and AMFincreased grain yield by 41% as compared to un-inoculated controls. Yield responses to the inoculantswere highest at locations with previously low yields. AMF or PGPR alone augmented wheat grain yield by29% and 31%, respectively. The bio-inoculants were effective both at Zero and at farmers’ practicefertilization level (70 kg N ha!1, 11 kg P ha!1 in mineral form to wheat crop). Also raw protein(nitrogen " 5.7) and mineral nutrient concentration of wheat grains (phosphorus, potassium, copper,iron, zinc, manganese) were higher after inoculation (#6% to #53%). Phosphorus use ef"ciency of wheatgrains [kg P grain kg!1 P fertilizer] was increased by 95%. AMF and PGPR application also improved soilquality as indicated by increased soil enzyme activities of alkaline and acid phosphatase, urease anddehydrogenase. Effects on rice and black gram yields were far less pronounced over two croppingseasons, suggesting that AMF and PGPR isolated from the target crop were more ef"cient. We concludethat mutualistic root microorganisms have a high potential for contributing to food security and forimproving nutrition status in southern countries, while safeguarding natural resources such as P stocks.

! 2010 Elsevier Ltd. All rights reserved.

1. Introduction

In India, rice, wheat and black gram are staple food crops (Guptaand Seth, 2007), on which the lives of more than 1.1 billion peoplerely. Although these crops can serve as constituents of a nutritiousdiet, malnutrition is a widespread phenomenon in the rural Indianpopulation (Menon et al., 2008; Vijayaraghavan and Rao,1998), andcurrently 231 million people are undernourished (FAO, 2008). Themain problems are energy de"ciency, lack of proteins, and the

* Corresponding author. Tel.: #41 62 865 72 32; fax: #41 62 865 72 73.E-mail addresses: paul.maeder@"bl.org (P. Mäder), [email protected]

(F. Kaiser), [email protected] (A. Adholeya), [email protected] (R. Singh), [email protected] (H.S. Uppal), [email protected] (A.K. Sharma), [email protected] (R. Srivastava), [email protected] (V. Sahai), [email protected] (M. Aragno), [email protected] (A. Wiemken), [email protected] (B.N. Johri), [email protected] (P.M. Fried).

Contents lists available at ScienceDirect

Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

0038-0717/$ e see front matter ! 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.soilbio.2010.11.031

Soil Biology & Biochemistry 43 (2011) 609e619


shortage of micronutrient supply (Vijayaraghavan and Rao, 1998).Food insecurity in the 21st century will even increase due to heatand drought stress induced by climate change, particularly intropical and subtropical regions (Battisti and Naylor, 2009). Thisimplies an urgent need to "nd new agricultural technologies toobtain suf"cient yields of good quality crops, also with respect toglobally limited natural resources such as fertilizers (Cordell et al.,2009) and the enormous loss of fertile soils (Adesemoye et al.,2009; Pimentel et al., 1995; Tilman, 1999; Tilman et al., 2002). InIndia, soils are often low in plant-available phosphorus (P) due tochemical sorption, which renders the application of readily solubleP-fertilizers highly inef"cient (Hegde et al., 1999). Mutualistic rootmicroorganisms such as arbuscular mycorrhizal fungi (AMF) andplant growth-promoting rhizobacteria (PGPR) have a high potentialto improve plant nutrient uptake, especially when they are appliedin combination (Artursson et al., 2006; Smith and Read, 2008; Toroet al., 1997). Furthermore, they are known to improve plant growthmost ef"ciently under poor growth conditions (Al-Karaki et al.,2004; Jat and Ahlawat, 2006; Nandakumar et al., 2001; Seciliaand Bagyaraj, 1994). Signi"cantly enhanced yields of differentcrops have been reported (Artursson et al., 2006; Hamel, 1996),especially from semi-arid climates and in nutrient-poor andP-sorbing soils (Hegde et al., 1999). Under such conditions, theinoculants may supplement chemical fertilizers and organicmanure (Hegde et al., 1999) by increasing the plant nutrient useef"ciency. In particular the secretion of phosphatase by phosphate-solubilising bacteria and/or by AMF is a common mode of facili-tating the conversion of insoluble forms of P to plant-availableforms, and thus to enhance plant P-uptake and plant growth(Kim et al., 1997).

AMF support plant growth directly by enhancing nutrient (P, Znand others) and water uptake (Hegde et al., 1999), as the extendedextra-radical hyphal network is able to explore more soil volumethan plant roots alone. AMF also support plant growth indirectly byimproving soil structure and resistance to certain root pathogens(Smith and Read, 2008; Treseder and Turner, 2007). Despite theenormous progress in research of plantemicrobe interactions atthe cellular and molecular level (Marx, 2004), there is a consider-able knowledge gap regarding the use of mutualistic root micro-organisms in agriculture. Recent research indicates that the bene"tthat a plant may draw from arbuscular mycorrhizal (AM) symbiosisdepends greatly on its compatibility with the fungal partner (Beveret al., 2008; Klironomos, 2002; Munkvold et al., 2004). This hasusually not been taken into account in studies attempting to useAMF or PGPR under competitive conditions with the indigenousmicrobial community in the rhizosphere of a speci"c crop.

PGPR comprise different functional and taxonomic groups ofbacteria like Pseudomonas, Bacillus, Rhizobia, Azospirillum, Azoto-bacter, and others (Benizri et al., 2001; Ghosh et al., 2002). Theirability to mobilize either minerally or organically bound nutrientsfrom the pedosphere or to "x atmospheric di-nitrogen and make itavailable to the plants is a crucial feature in their application.Furthermore, some stimulate plant growth directly by synthesizingplant hormones like indole acetic acid, or indirectly by suppressingsoil-borne pathogens (Benizri et al., 2001; Kloepper et al., 1980), orby inducing plant resistance (Benizri et al., 2001). Mycorrhizahelper bacteria were shown to stimulate AMF spore germination,hyphal growth and AMF root colonization directly (Artursson et al.,2006; Frey-Klett et al., 2007). Conversely, AMF in!uence thechemical composition of root exudates, which in turn are a majornutrient source for the bacteria in the rhizosphere (Artursson et al.,2006; Hegde et al., 1999). There is little knowledge on the effects ofa combined application of such bene"cial microorganisms on cropproductivity under local farmers’ practices (Artursson et al., 2006;Harris, 2009).

In a system approach, we investigated the effects of the recur-rent application of a consortium of AMF and PGPR on crop yield,grain and soil quality and nutrient uptake of the staple food cropwheat (Triticum aestivum [L.]) in a rotation with either rice (Orizasativa [L.]) or black gram (Vigna mungo [L.], Hepper). The two yearexperiments were performed in different agro-climatic zones ofIndia at seven locations extending from the Himalayan foothills tothe Indo-Gangetic plain.

2. Materials and methods

2.1. Locations and experiments

The study sites were located in the Indian states of Uttar Pra-desh, Uttarakhand and Haryana (Supplementary Table S1). Inocu-lants such as AMF and PGPR were inoculated and tested for plantgrowth response at two fertilizer levels in wheaterice andwheateblack gram cropping systems at three locations each. Thesites were selected for contrasting agro-climatic and soil condi-tions. They were previously used for at least 10 years as wheatericeor wheateblack gram cropping systems, and were entirelymanaged by farmers before experimental start. The crops weregrown as per agricultural practice prevalent in the respectiveregions, referred to here as farmers’ practice (FP).

The experiments were performed in a strip-plot design withfour "eld replicates per treatment. Inoculation treatments wererandomized in each replicate. Individual plot size was 5 " 5 m asa standard. On terraces at the Salary 1 and 2 locations (Table S1),plot dimensions were adjusted according to the terrace shape, butplot sizewas also at least 25m2. Low bunds of soil were constructedbetween the plots to minimize cross-contamination of theinoculants.

2.2. Soil properties

The soil texture at these locations ranged from loamy sand toloam (Table S1). Soils were classi"ed according to the USDA clas-si"cation system. Before planting the "rst experimental crops, soilsamples were taken from 0 to 15 cm depth in each replicate block ofthe experiment in the standing wheat crop (T. aestivum [L.]) at thebeginning of April 2005. Soils at all locations were alkaline andranged from pH(H2O) 7.59 to 7.76, and theywere low in soil organicmatter (0.35e0.42%). Olsen’s phosphorus (P) was measured ina sodium bicarbonate extract and potassium (K) in an ammoniumacetate extract. The diethylene triamine pentaacetic acid (DTPA)extractable micro elements copper (Cu), iron (Fe), zinc (Zn) andmanganese (Mn) were also measured (Table 1).

2.3. Crops and varieties

Seeds of wheat, rice and black gram were provided by theDepartment of Agronomy, College of Agriculture, GB Pant Univer-sity of Agriculture and Technology, Pantnagar, Uttarakhand State,India. Wheat (T. aestivum [L.]), variety UP 2338, was sown at a rateof 120 kg seeds ha!1 in the Rabi (OctobereMarch) season in 2006and 2007. Rice (O. sativa [L.]), variety Pant Dhan 4, was planted ata sowing rate of 50 kg seeds ha!1 in the Kharif (JulyeOctober)season in 2005 and 2006, and subsequently transplanted to the"eld at the beginning of July at a planting density of 25 hills m!2.The nursery plots were fertilized with 60 kg N ha!1 in the form ofurea in 2006 only. Before transplanting rice, at the location Bha# S,Sesbania aculeata [L.] was planted as a green manure crop for 45days. The "eld plots were irrigated once before sowing at the rate of20 kg seeds ha!1. The above-ground biomass was cut manually ata shoot length of about 1.0 m and then chopped using a plant cutter.

P. Mäder et al. / Soil Biology & Biochemistry 43 (2011) 609e619610


The green manure material was then manually mixed into the pre-irrigated "eld plots 10 days before rice planting. Black gram(V. mungo [L.], Hepper), variety Pant Urd 35, was grown at a rate of12.5 kg seeds ha!1 in the Kharif season in 2005 and 2006.

2.4. Inoculants and inoculation procedure

A natural AMF consortium (Mnat) and a single spore AMF strain(Mss2) were applied. Mnat was composed of an indigenous naturalAMF consortium, isolated from a "eld in Bhawanipur (Budaun) (790250 E/280 220 N) in the Mid-Western Plain Zone of Uttar Pradesh,India (Roesti et al., 2006). These "elds were planted with wheat forat least 10 years without the application of any fertilizers. It wasobtained and propagated at the CMCC (Centre for MycorrhizalCulture Collection) (Accession Code: CMCC Bht LL2) of TERI (TheEnergy and Resources Institute, Delhi, India) and was sub-culturedin trap cultures according to Oehl et al. (2003). The AMF strainMss2(TERI commercial, Glomus intraradices) was also obtained from theCMCC and propagated in a root organ culture (Accession Code:CMCC ROC1) as described by Adholeya et al. (2005).

Two bacterial strains, Pseudomonas jessenii (R62) and Pseudo-monas synxantha (R81) (Gaur et al., 2004; Roesti, 2005; Roesti et al.,2006), were used in the PGPR consortium (Ps) in all "eld experi-ments. These strains were selected among 3000 strains isolatedfrom wheat roots of the variety UP 2338 cultivated in low-input"elds under wheaterice rotation (Bhawanipur, same location asabove), providing a relatively high yield in spite of a low fertiliza-tion level. The in vitro selection of these strains was based primarilyon their plant growth-promoting characteristics, their potential tosuppress root pathogens, and their capability to interact positivelywith AMF (Table S2). Twenty strains were then selected and testedin different consortia for plant growth promotion in greenhousepot tests using soil from Bhawanipur. The two most promisingstrains, i.e. R62 and R81, were then selected for "eld assays (Roestiet al., 2006). These strains were grown separately in half-strengthKing’s B broth (KP, Difco Laboratories, Sparks, USA) in shake !asksincubated at 28 $C at 120 rpm until the late exponential phase.

For AMF inoculation purposes, Mss2 spores were extracted bydeionization of the media (Doner and Bécard, 1991), and subse-quently mixed with terragreen using TERI’s patented methodology(Adholeya, 2000). AMF were applied at the rate of ca. 20 infectiouspropagules per seed for all three crops using the seed encapsulationtechnique (Adholeya et al., 2005; Srivastava et al., 2007), for whichthe number of infectious propagules was determined prior toapplication (Gaur et al., 1998; Sharma et al., 1996). Rice plants wereadditionally inoculated at the time of transplantation at the rate of20 infectious propagules per plant by bare root dip treatment(Adholeya et al., 2005). Gum acacia was used as a binder (10 g kg!1

of the inocula). For the PGPR inoculations, Ps R62 and R81 cultureswere mixed with a charcoal-based carrier. Carboxymethyl cellulose("nal concentration 0.1%) was added in order to enable the bacteriato stick to the seeds. Via seed encapsulation, 105e106 colony-forming units (cfu) per seed were applied. Rice was inoculatedagain at the time of transplanting by bare root dip treatment at105e106 cfu per plant. Seeds were air-dried for 20 min after inoc-ulation prior to sowing. The seeds that were used for un-inoculatedcontrol plots were dipped in water for 20 min.

2.5. Fertilization

Plants were grown at zero (Zero) and at farmers’ practice (FP)fertilization levels. Each crop in the farmers’ practice plots wasamended by 3 t ha!1 of farmyard manure. Additionally, 60 kg (splitinto 3 " 20 kg) mineral N ha!1 was applied in the wheat and riceplots as urea, and 15 kg N ha!1 in the black gram plots. All cropsTa

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P. Mäder et al. / Soil Biology & Biochemistry 43 (2011) 609e619 611


received 25 kg P2O5 ha!1 as diammonium phosphate. In addition,the rice was fertilized with 12 kg ZnSO4 ha!1. At one marginal site,Sal2, wheat fertilization was restricted to 20 kg N ha!1 and to12.5 kg P2O5 ha!1 respectively. Plots grown at Zero fertilizationlevel were also amended by 3 t ha!1 of farmyard manure, but nomineral fertilizers were applied.

2.6. Assessments

AMF root colonization was assessed at the milk stage for wheatand rice, and at the !owering stage for black gram (Biermann andLindermann, 1981; Phillips and Hayman, 1970). Crop constituentswere determined in "nely ground grains of wheat, rice and blackgram. Crude protein was measured according to the Bradfordmethod (Stoscheck, 1990). Total nitrogen (N) was measured bytitration after Kjeldahl digestion. For the elements P, K, Cu, Fe, Zn,Mn, the plant material was digested by acid oxidation after expo-sure to microwaves prior to analysis (Kalra et al., 1989). P wasmeasured colorimetrically by the molybdivanado-phosphoric acidmethod, K by !ame photometry and Cu, Fe, Zn and Mn by atomicabsorption spectrophotometry. The ash concentration wasmeasured after digestion in a muf!e furnace at 575 $C. Phosphorususe ef"ciency of wheat was expressed as grain P accumulationef"ciency, calculated as the ratio of kg P grain/kg P fertilizeranalogue to the de"nition of grain N accumulation ef"ciency byDawson et al. (2008).

The use of soil biological markers related to microbial activity,such as enzyme activities, has been proposed by Naseby and Lynch(1997). The cell bound activity of dehydrogenase indicates theoverall biological activity, the exo-enzymes acid and alkalinephosphatase, and urease, are indicative for the capacity of a soil tomineralise organic N and P compounds (Kohler et al., 2007; Mäderet al., 2002). Soil samples for analysing soil enzyme activities weretaken at the milk stage for rice and wheat and at !owering for blackgram. Enzyme activities were measured in incubation experimentsafter addition of a substrate solution to the soils (Schinner et al.,1991). Soil dehydrogenase activity was measured by the triphe-nyltetrazolium chloride reduction method. Phosphatase activitywas determined colorimetrically as p-nitrophenyl released byphosphor-monoesterase from a p-nitrophenyl phosphate solutionadded to the soil. Ureasewas determined bymeasuring ammoniumaccumulation from an aqueous urea solution added to the soilsprior to incubation.

2.7. Statistical data analysis and calculations

Statistical analysis of the datawas carried out using the statisticspackage ‘JMP’, Version 5.0.1 (SAS Institute Inc., Cary, NC, USA).Analysis of variance (ANOVA) was performed at a signi"cance levelof p ' 0.05, 0.01 and 0.001 prior to a TukeyeKramer multiplecomparison test (p & 0.05) using an experimentwise error rate(Tukey’s honestly signi"cant difference HSD). A four-way ANOVAwas performed for data deriving from several locations and years(factors Inoculation, Fertilizer level, Location and Season). For cropyield, a three-way ANOVA was thereafter calculated separately forthe Zero and farmer’s practice fertilizer level, including the factorsInoculation, Location and Season and the respective interactions.A two-way ANOVA was conducted for data obtained from indi-vidual locations and years for the factors Inoculation and Fertilizerlevel and Inoculation " Fertilization level interaction.

3. Results

Bio-inoculants (AMF and PGPR), fertilizers and locations hadpronounced effects on crop yields, food and soil quality.

3.1. Crop yields

Evaluation of the data from all seven locations over the twoyears of cultivation revealed distinct responses of the differentcrops to the bio-inoculations (Fig. 1). The most marked effects weremeasured inwheat, inwhich themean grain yield was increased by41% in the treatment with the combined Mnat # Ps inoculationcompared to the un-inoculated control and across both fertilizerlevels applied. This yield increase was even higher than the overallfertilizer effect (27% compared to the non-fertilized plots; Fig. 1).Our data show that the wheat grain yield increase after inoculationwas mainly due to higher numbers of effective tillers, and thateffects on harvest index (grain yield/total above-ground cropbiomass), and thousand seed weight were far less important(Tables S3 and S4).

In rice, no effect of bio-inoculant applications on grain yield wasobserved over the two years (Fig. 1). However, planting S. aculeatagreen manure prior to rice as another biological measure enhancedmean rice and wheat grain yield by 25% and 23%, respectively overtwo cropping seasons (Fig. 1; Tables S5 and S6).

While a combined inoculation of AMF and PGPR was the mostsuccessful treatment in wheat, black gram showed the strongestpositive response to inoculation with AMF alone, whereby MnatandMss2 increased grain yield by 24% and 21%, respectively (Fig. 1).But these differences were not signi"cant over two croppingseasons (ANOVA). Black gram yield increase was achieved mainlyby an increase of the number of pods per plant and the plantdensity after inoculation, and less by harvest index and thousandseed weight (Table S7 and S8).

Remarkably, wheat yield increase due to inoculation wasconsistent over both years (Tables S3 and S4). However, the inoc-ulation had no signi"cant effect on rice and black gram in the "rstyear of application. Only in the second year were yield increasesalso observed in rice (Tables S5 and S6) and black gram (Table S7and S8).

We evaluated bio-inoculants in different environments withrespect to fertilization, green manuring and locations. The datashow that the inoculants had positive effects on crop yields withZero and farmers’ practice application of fertilizers (Table 2).Without any application of chemical fertilizers (Zero), wheat grainyield increased by 51% after inoculation of Mnat # Ps, while itincreased by 42% under farmers’ pratice fertilization. There was noeffect of the inoculants on rice grain yields at both fertilizer levels.Black gram grain yield was increased up to 19% (Mss2) in the Zerotreatment, and up to 31% at farmers’ practice (Mnat).

Moreover, in the wheaterice rotation at Bhawanipur, the bio-inoculants Mnat # Ps increased wheat yield by 68% when Sesbaniagreen manure was included in the rotation, and by 83% in a treat-ment without Sesbania. Thus, an additive yield increase can beachieved in wheat by using bio-inoculants, green manure andfertilizers. Not only the fertilizer level but also the locations hada strong in!uence on grain yields (Fig. 1). Our data revealed that atthe locations with low inherent soil fertility (i.e. Salari 2), the cropsbene"ted the most from the inoculants; that is, their relativeincrease of wheat grain yield was highest (78%; Fig. 2). But even atlocations with relatively high inherent soil fertility (Shyampur),wheat grain yield was increased by 15%.

3.2. Response of crop quality to inoculants

Inoculation not only enhanced yields, but also in!uenced thenitrogen andmineral nutrient grain concentration (Fig. 3; Table S9).In wheat, N concentration (corresponding to crude protein) as wellas all measured macroelements (P and K) and micro elements(Fe, Cu, Zn and Mn) were signi"cantly increased by 6%e53%. Except



for Fe, the highest level was reached by the combined inoculationMnat # Ps. In rice, inoculation with Ps alone enhanced mineralconcentration equal to or even more than Mnat # Ps, but only Mnand Zn were increased substantially. In black gram, inoculation didnot signi"cantly enhance the concentration of any of the macro-and microelements compared to the control.

The macro- and micronutrient uptake of wheat, as calculated bygrain dry matter yield " nutrient concentration of wheat grains,increased signi"cantly upon inoculation (Fig. 4). The uptake of P, K,Cu, and Zn was more than 100% higher in plots inoculated byMnat # Ps, whereas N, Fe and Mn increased by 30e70%. We foundthat P use ef"ciency, as calculated by the ratio P in grains ha!1/P infertilizers supplied ha!1, was 0.356 for the control plots and 0.695for the plots inoculated with Mnat # Ps (Table 3), which corre-sponds to a 95% increase of P-use ef"ciency upon inoculation withMnat # Ps. This means that P was much more ef"ciently taken upfrom the fertilized soils and translocated into the grains in theinoculated wheat plants.

3.3. Mycorrhizal root colonization and soil quality indicators

There is strong evidence that the positive in!uence of theinoculants onwheat yield and grain quality was due to a synergisticeffect of PGPR and AMF as indicated by the highest yields in dually

inoculated treatments. In all three crops, the percentage of AMFroot colonization was signi"cantly higher in plots inoculated withAMF than in the control plots (Table 4), showing that the inoculatedAMF could compete with the indigenous micro!ora. In rice andblack gram, inoculation with Ps slightly depressed AMF root colo-nization compared to the control, but not signi"cantly. All of theinoculants signi"cantly increased the activities of urease, alkalineand acid phosphatase and dehydrogenase in the soils of all threecrops (Fig. 5, Table S10). The increase of enzyme activities was mostpronounced in wheat, where they increased by 51%e98% with themixed inoculum Mnat # Ps as compared to the control.

4. Discussion

Microbial inoculants are promising components for integratedsolutions to agro-environmental problems because inoculantspossess the capacity to promote plant growth, enhance nutrientavailability and uptake, and support the health of plants(Adesemoye et al., 2009). However in many studies, effects of AMFon crop yields were detected in pot experiments, but not whencrops were inoculated in the "eld (Kaschuk et al., 2010). Ourinvestigation allowed the quanti"cation of the effects of microbialinoculants such as AMF and PGPR on the yield and nutrientconcentration of wheat in rotation with rice and black gram in

B

C

A

Fig. 1. Mean grain yield of wheat, rice and black gram from two cropping seasons. Grain yield of wheat grown in 2006 and 2007 and of rice and black gram grown in 2005 and 2006after four inoculation treatments at two fertilization levels at three to seven locations. Calculated means of 14 harvests of wheat and six harvests each of rice and black gram.Statistically signi"cant differences resulting from a four-way ANOVA including the factors Inoculation, Fertilization level, Location, Season are indicated by: *p < 0.05; **p < 0.01;***p < 0.001 (ns & not signi"cant). A Tukey-test was calculated post hoc after the ANOVA. Values not connected by the same letter differ signi"cantly. Value of grain yield is missingat some locations because the crop in question was not present in the rotation (zz). Values of site Shyampur for rice harvests 2005 and 2006 are missing because of a crop failure dueto drought (z). Values of site Mandori for wheat harvest 2007 are missing because of a severe hailstorm (y). Note the different scales of y-axes. For standard errors of means seeTables S3eS8. Cont & un-inoculated control, Mnat & natural AMF consortium, Ps & mix of two PGPR strains, Mnat # Ps & mix of a natural AMF consortium and two PGPR strains,Mss2 & single commercial AMF strain; Zero &without mineral fertilizers, FP & Farmers’ practice; Ujh & Ujhani, Bha - S & Bhawanipur without Sesbania, Bha # S & Bhawanipur withSesbania, Sal1 & Salari 1, Sal2 & Salari 2, Shy & Shyampur, Man & Mandori.



replicated, multi-location "eld experiments over two croppingseasons at two fertilizer levels. For practical reasons bio-inoculantshave to be effective at various agro-climatic and soil conditions. Inour study, the consistently higher wheat grain yields obtainedthrough inoculation at all locations (Table S1) with contrastinginherent soil fertility (Table 1) demonstrate that this biotechnologyis probably a valuable tool not only for farmers in remote areas, butis also useful on a broader geographic and edaphic range.

4.1. Performance of wheat

In our study, the higher wheat grain yield response obtainedthrough dual inoculation of AMF (Mnat) and PGPR (Ps) (#41%) ascompared to single inoculation of either AMF or PGPR alone(approx. #30%) supports the hypothesis that these bio-inoculantsact synergistically by mechanisms outlined in detail in the intro-duction section. Accordingly, Al-Karaki et al. (2004) reporteda wheat grain yield increase of 20%e41% after inoculation witheither a Glomus mossae or Glomus etunicatum AMF strain on a "nesandy loam in Texas, whereby this increase was dependent on thecultivar and strain combinations. Rosas et al. (2009) found that thebacterial inoculant Pseudomonas aurantiaca augmentedwheat grainyield on a "ne sandy loam inArgentinia by 36%, compared to the un-inoculated control. A study conducted in Turkey by Khan and Zaidi(2007) revealed that a combined inoculation of wheat withBacillus sp. (PGPR) and Glomus fasciculatum (AMF) even increasedwheat yield by 133%, whilst single application of either Bacillus sp.orG. fasciculatum resulted in 22% and 55% higherwheat grain yields,respectively. These effects are remarkable considering that the un-inoculated control plots were fertilized with 60 kg ha!1 N and40 kg ha!1 P, while the inoculated plots remained unfertilized.

For practical agricultural application in rural regions with low-input agricultural systems, it is of crucial importance to knowwhether the inoculants also develop their full potential under theconditions of fertilization associated with typical farmers’ practice.In our experiments, the inoculation treatments were conductedboth at Zero and farmers’ practice fertilization level (69.6 kg N ha!1

and 25 kg P2O5 ha!1 corresponding to 11.1 kg P ha!1). This low Pdose in the farmers’ practice fertilization level explains why growtheffects of dual inoculation onwheat yield was still 42% as comparedto 51% at Zero fertilization. However, these positive responses ofwheat to inoculants are confounded by other studies. For instance,Germida andWalley (1996) reported inconsistent growth responseof wheat to inoculationwith the pseudomonads P. cepacia R55, R85,

Table 2Grain yield response at Zero and Farmers’ Practice (FP) fertilization levels, 2005e2007.

Crop Inoculation Zero FP

LSQ mean [t ha!1] [%] Tukey s.e.m. n LSQ mean [t ha!1] [%] Tukey s.e.m. n

Wheat Cont 1.88 100 c %0.117 48 2.54 100 c %0.101 48Mnat 2.58 137 b %0.145 48 3.27 129 b %0.135 48Ps 2.58 137 b %0.154 48 3.30 130 b %0.132 48Mnat # Ps 2.84 151 a %0.172 48 3.60 142 a %0.120 48Mss2 2.56 136 b %0.143 48 3.18 126 b %0.121 48ANOVA *** ***

Rice Cont 2.72 100 a %0.261 24 3.08 100 a %0.165 24Mnat 2.98 110 a %0.396 24 3.11 101 a %0.258 24Ps 2.85 105 a %0.264 24 3.45 112 a %0.259 24Mnat # Ps 2.91 107 a %0.275 24 3.38 110 a %0.302 24Mss2 2.89 106 a %0.281 24 3.09 100 a %0.219 24ANOVA ns ns

Black gram Cont 0.281 100 b %0.0578 24 0.333 100 b %0.0693 24Mnat 0.327 116 a %0.0676 24 0.437 131 a %0.0938 24Ps 0.310 110 ab %0.0620 24 0.403 121 a %0.0831 24Mnat # Ps 0.296 105 ab %0.0552 24 0.394 118 a %0.0797 24Mss2 0.336 119 a %0.0628 24 0.409 123 a %0.0822 24ANOVA ** ***

n & number of observations.ANOVA: Three-way analysis of variance including the factors Inoculation, Location, Season. ns & not signi"cant, *p ' 0.05, **p ' 0.01, ***p ' 0.001.Tukey-test: Values not connected with the same letter differ signi"cantly.Inoculation: Cont & un-inoculated control, Mnat & natural mycorrhiza consortium, Ps & !uorescent Pseudomonas strains R62 # R81, Mss2 & single commercial AMF strain.Wheat: Locations Ujh, Bha - S, Bha # S, Sal1, Sal2, Shy (Mandori was excluded because of harvest failure in 2007).Rice: Locations Bha-S, Bha # S and Sal1.Black gram: Locations Ujh, Sal2, Man.

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Fig. 2. Wheat grain yield increase varies between locations with different inherent soilfertility. Average wheat grain yield (bars) of 2006 and 2007 in the control plots and inplots inoculated by arbuscular mycorrhizal fungi (“Mnat”) and !uorescent Pseudo-monas strains R62 and R81 (“Ps”). Error bars show standard error of means (n & 8). Theline depicts the percent yield increase of the inoculated plots compared to theun-inoculated control. Locations are ranked according to their productivity inthe un-inoculated control. See Fig. 1 for location names. For this calculation, values ofall plots with farmers’ practice fertilization were included.



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Crude protein***

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A Wheat B Rice

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Mss2Fig. 3. Food quality as indicated by crude protein, macro- and microelement concentration of wheat, rice and black gram grains. A) Wheat grown in 2006 and 2007, B) rice and C)black gram grown in 2005 and 2006 after four inoculation treatments across two fertilizer levels. Calculated means in percent (control & 100%) of four harvests of wheat and twoharvests of rice and black gram at the locations Bhawanipur and Mandori. Absolute values for the control (100%) are as follows: A) crude protein, 6.31% DM; P, 2671 ppm; K,3174 ppm; Cu, 13.9 ppm; Fe, 31.3 ppm; Zn, 31.1 ppm; Mn, 18.6 ppm; ash, 1.29% DM; B) crude protein, 7.33% DM; P, 3330 ppm; K, 4030 ppm; Cu, 2.85 ppm; Fe, 15.2 ppm; Zn,23.2 ppm; Mn, 35.1 ppm; ash, 1.09% DM; C) crude protein, 19.8% DM; P, 4600 ppm; K, 11600 ppm; Cu, 7.5 ppm; Fe, 45.6 ppm; Zn, 39.7 ppm; Mn, 9.86 ppm; ash, 3.34% DM.Statistically signi"cant differences between inoculation treatments are indicated by *p ' 0.05, **p ' 0.01 and ***p ' 0.001 (ANOVA). Constituents without asterisks did not differsigni"cantly between treatments. For standard errors of means see Table S9.

c c c c a c c

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** ** ** ** ** ** **

Macronutrients Micronutrients

Fig. 4. Nutrient uptake of wheat grains grown in 2006 and 2007 in four inoculation treatments across two fertilizer levels. Calculated means expressed as percent (control & 100%)of two harvests at the locations Bhawanipur and Mandori each are depicted. Statistically signi"cant differences between inoculation treatments are indicated by *p ' 0.05,**p ' 0.01 and ***p ' 0.001 (ANOVA). For macronutrient uptake per hectare by wheat grains, absolute values for the control (100%) are as follows: N, 31.8 kg ha!1; P, 7.56 kg ha!1;K, 8.20 kg ha!1. For micronutrient uptake per hectare by wheat grains, absolute values for the control (100%) are as follows: Cu, 30.3 g ha!1; Fe, 85.8 g ha!1; Zn, 76.1 g ha!1; Mn,51.0 g ha!1. Values of site Mandori in 2007 are missing because of a hailstorm.



P. aeruginosa R80, P. !uorescens R92 and P. putida R104, combinedwith AMF, and suggested that some PGPR inoculants may adverselyaffect mutualistic associations between plants and indigenous soilmicroorganisms. Having conducted a one year "eld study with thesame wheat variety (UP 2338) and the same AMF and PGPR inoc-ulants as used in the present study, Roesti et al. (2006) claimed thatthe positive response of wheat to AMF and PGPR might beexplained by the fact that these soil microorganisms have beenselected in the wheat rhizosphere from the variety UP 2338.

In rural areas of Central Asian and Middle Eastern countrieswheat can provide more than 70% of the daily energy intake of thepopulation (Cakmak et al., 2010). The per hectare energy produc-tion increases in parallel with wheat grain yields. This is in partic-ular interesting for the Northern States of India, where our studywas conducted. Their daily average wheat consumption is esti-mated at 254 g per capita (Ray, 2007). Assuming a wheat grainenergy content of 13.4 kJ g!1 (Souci et al., 1981) and a daily percapita calorie consumption norm of 10 MJ (Menon et al., 2008), this

Table 3Nutrient use ef"ciency for phosphorus [kg P grain kg!1 P fertilizer] in plots fertilized according to Farmers’ Practice (FP), calculated over two wheat harvests at Bhawanipur(without Sesbania) in 2006 and 2007 and one wheat harvest at Mandori in 2006.

Inoculation Input P Grain yield[t ha!1]

P concentrationgrain [kg P t!1 grain]

P grain[kg P grain ha!1]

Nutrient use ef"ciency[kg P grain kg!1 P fertilizer]

In FYM[kg ha!1]

In mineral fertilizer[kg ha!1]

Total[kg ha!1]

Cont 2.03 11.3 13.3 2.48 2.78 6.90 0.356Mnat 2.03 11.3 13.3 3.35 4.23 14.17 0.731Ps 2.03 11.3 13.3 3.45 4.04 13.93 0.719Mnat # Ps 2.03 11.3 13.3 3.33 4.04 13.48 0.695Mss2 2.03 11.3 13.3 3.28 3.84 12.59 0.649

Inoculation: Cont & un-inoculated control, Mnat & natural mycorrhiza consortium, Ps & !uorescent Pseudomonas strains R62 # R81, Mss2 & single commercial AMF strain.FYM & Farmyard manure.

Table 4AMF root colonization [% root length colonized] of wheat grown in 2006 and 2007 and of rice and black gram in 2005 and 2006 after four inoculation treatments.

Mycorrhizal root colonization

Wheat Rice Black gram

LSQ mean [%] Tukey n LSQ mean [%] Tukey n LSQ mean [%] Tukey n

InoculationCont 20.9 c 112 20.9 c 64 38.3 c 48Mnat 33.4 ab 112 40.0 a 64 58.2 ab 48Ps 22.0 c 112 17.2 c 64 34.6 c 48Mnat#Ps 28.5 b 112 27.6 b 64 52.2 b 48Mss2 34.1 a 112 39.3 a 64 61.5 a 48ANOVA *** *** ***

Fertilizer levelZero 28.1 a 280 30.5 a 160 50.0 a 120Farmers practice 27.5 a 280 27.5 b 160 47.9 a 120ANOVA ns * ns

LocationUjh 34.3 a 80 e e e 57.3 a 80Bha-S 29.3 abc 80 32.1 b 80 e e e

Bha#S 27.5 bc 80 31.3 b 80 e e e

Sal1 31.6 ab 80 42.0 a 80 e e e

Sal2 30.0 abc 80 e e e 55.9 a 80Shy 17.4 d 80 10.5 c 80 e e e

Man 24.5 c 80 e e e 33.6 b 80ANOVA *** *** ***

SeasonKharif 2005 e 33.0 a 160 50.3 a 120Rabi 2006 36.6 a 280 e e e e e

Kharif 2006 e 25.0 b 160 47.6 a 120Rabi 2007 19.0 b 280 e e e e e

ANOVA *** *** ns

Means of 14 harvests of wheat and six harvests each of rice and black gram.n & number of observations.ANOVA: Four-way analysis of variance including the factors Inoculation, Fertilizer level, Location, Season. ns & not signi"cant, *p ' 0.05, **p ' 0.01, ***p ' 0.001.Tukey-test: Values not connected with the same letter differ signi"cantly.Inoculation: Cont & un-inoculated control, Mnat & natural mycorrhiza consortia, Ps & !uorescent Pseudomonas strains R62 # R81, Mss2 & single commercial AMF strain.Locations: Ujh & Ujhani, Bha - S & Bhawanipur without Sesbania green manure in rotation, Bha # S & with Sesbania, Sal1 & Salari 1, Sal2 & Salari 2, Shy & Shyampur,Man & Mandori.e No crop planted.In Shyampur, there was a crop failure due to drought in rice harvests 2005 and 2006.In Mandori, the wheat harvest was destroyed by a severe hailstorm in 2007, but AMF root colonization was assessed.



corresponds to approximately one third of a persons’ daily calorieintake. In our experiments, in the control treatments withoutinoculation, 31.7 GJ ha!1 were produced, being suf"cient to supply25.5 people with one third of their daily energy requirement. Incontrast, from 1 ha inoculated with AMF and PGPR, 10.6 personsmore, i.e. 36.1 persons, could cover a third of their daily calorierequirement. As the experimental wheat yields in the control plots(2.54 t ha!1) are similar to the yields currently achieved in India(2.7e2.8 t ha!1, USDA (2008)), the data were extrapolated to thetotal area covered by wheat in Northern India (18.6 Mha, Trivedi(2008)). According to this extrapolation, approximately 197million more people could be supplied with a decisive amount oftheir daily energy requirement by wheat.

The higher wheat grain yields can be explained by the increasednutrient concentration obtained in the present study. Thereby, themixed inoculum Mnat # Ps showed the highest effect on theconcentration of crude protein, macro- and micronutrients, withincreases ranging from 6% to 53%, except for Fe concentrationwhich was highest when inoculated with AMF alone. Accordingly,increased nutrient concentration in wheat due to inoculation werereported by Al-Karaki et al. (2004), Roesti et al. (2006) and Khanand Zaidi (2007). The increased nutrient concentration and theextraordinary increase in nutrient uptake of wheat grains (approx.14e125%) found in this study have large implications in terms ofmalnutrition of the rural Indian population, where energy, protein,macro- and microelement shortage is widespread (Ray, 2007;Vijayaraghavan and Rao, 1998). On the other hand, these numbersare also important with respect to an ef"cient use of resources such

as P and N, as illustrated by a 95% increased P use ef"ciency ofwheat grains.

4.2. Performance of rice

None of the inoculation treatments revealed any signi"canteffect on rice grain yield over the two cropping seasons reported inthis study. On the contrary, Secilia and Bagyaraj (1994) obtainedincreased rice yields of 14% and 11% after inoculation withG. intraradices and phosphate fertilization with half of the fullrecommendation, respectively. An even more pronounced yieldincrease of 23%, obtained by Jha et al. (2009) upon rice inoculationwith a diazotrophic (N2-"xing) Pseudomonas sp. strain, was theresult of an inoculum directly derived from rice roots. Thus, there issome evidence that our results might be due to inappropriateselection of bio-inoculants for rice. It is worth noting that Jha et al.(2009) investigated plants which were "rst inoculated in an axenicinoculation system, then transplanted to pots, and thereafterre-transplanted to "eld. Remarkably, a positive effect of bio-inoc-ulants on rice grain yield was assessed in the second year of thepresent study. The reason for the different behaviour of the AMFand PGPR in the two experimental years remains unknown. Thissuggests that AMF and PGPR from preceding inoculations mighthave survived and still have affected yields.

In rice grains, only Mn and Zn concentration were substantiallyincreased by Mnat # Ps as well as by Ps alone. These "ndings are inline with Tariq et al. (2007), who in "eld microplots demonstratedthe ef"ciency of a commercial mixed PGPR consortium (containing

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Urease***

Alk. P.***

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Fig. 5. Soil quality as indicated by soil enzymes. Soil samples taken from A) wheat grown in 2006 and 2007, B) rice and C) black gram grown in 2005 and 2006 in four inoculationtreatments across two fertilizer levels. Calculated means in percentage (control & 100%) of four wheat and two rice and black gram cultivation periods at the locations Bhawanipurand Mandori. Absolute values for the control (100%) are as follows: A) DH, 46.7 mg TPF g!1 DM 16 h!1; Alk. P., 480 mg NP g!1DM 1 h!1; Acid P., 183 mg NP g!1 DM 1 h!1; Urease,192 mg N g!1 DM 2 h!1; B) DH, 25.3 mg TPF g!1 DM 16 h!1; Alk. P., 330 mg NP g!1 DM 1 h!1; Acid P., 194 mg NP g!1 DM 1 h!1; Urease, 122 mg N g!1 DM 2 h!1; C) DH, 54.5 mg TPF g!1

DM 16 h!1; Alk. P., 336 mg NP g!1 DM 1 h!1; Acid P., 204 mg NP g!1 DM 1 h!1; Urease, 199 mg N g!1 DM 2 h!1. Statistically signi"cant differences between inoculation treatments areindicated by *p ' 0.05, **p ' 0.01 and ***p ' 0.001 (ANOVA). Parameters without asterisks did not differ signi"cantly between treatments. For standard errors of means see TableS10. DH & Dehydrogenase activity, Alk. P. & alkaline Phosphatase activity, Acid. P & acid Phosphatase activity, Urease & Urease activity.



Pseudomonas sp. and other strains of PGPR) acting as Zn solubilizerand increasing Zn up to 157%. The improvement of Zn concentra-tion in rice is very relevant considering that Zn de"ciency wasranked as the "fth most important risk factor responsible for illnessand death in the developing world (WHO, 2002).

4.3. Performance of black gram

Despite the fact that the inoculation treatments did not signif-icantly increase the yield of black gram (ANOVA), there was a cleartendency in our study that AMF inoculation leads to a yield increase(24%). This tendency is in accordance with Tarafdar and Rao (1997),who obtained 23% yield increase after inoculation of mung bean(Vigna radiata [L.], Wilczek) crops with G. mosseae and G. fas-ciculatum in the Central Arid Zone of India. Conducting a "eldexperiment with lentils (Lens culinaris [L.]), Kumar and Chandra(2008) found a yield increase of 20% after inoculation with thePGPR Pseudomonas diminuta isolated from the lentil rhizosphere.Inoculation of Rhizobium leguminosarum, the phosphorus solubil-izer Bacillus megaterium and the P. diminuta even led to 39% highergrain yields than the control. Co-inoculation of the P-solubilisingbacterium P. jessenii and the N-"xing Mesorhizobium ciceri evenincreased grain yield of chickpea (Cicer arietinum) by about 50%compared to the un-inoculated crop (Valverde et al., 2006). Thisclearly shows that an appropriate selection of bio-inoculants for thetarget crops is also applicable for leguminous crops grown under"eld conditions. While none of the inoculation treatments had anyeffect on the quality of black gram grains in our experiments,Kumar and Chandra (2008) obtained enhanced N and P uptake of60% and 38%, respectively, in lentil crops inoculated withP. diminuta. This may once again be the result of a bacterial inoc-ulum directly isolated from the target crop lentil. Tarafdar and Rao(1997) and Valverde et al. (2006) obtained increased macro- and/ormicroelemental concentration in shoots of mung beans andchickpea, respectively, but not in grains. As grain yields of bothcrops were improved, it is possible that nutrients were diluted inthe harvested crop parts.

5. Conclusions

We conclude that the application of a consortium of microbialinoculants such as mycorrhiza and plant growth-promoting rhi-zobacteria can substantially improve wheat yield and quality onlow-input sites of India, in terms of mineral nutrient concentrationand uptake via an improved soil quality and a higher nutrientuptake. Soils clearly do not contain suf"cient densities of autocht-onous mutualistic microorganisms. The fact that inoculants origi-nating from the wheat rhizosphere had a stronger impact onwheatcrop yield and quality, compared to rice and black gram, points tothe need for crop speci"c inocula based on hostesymbiont speci-"city. The astonishing increase of P use ef"ciency by a factor oftwo upon inoculation demonstrates the enormous potential ofmicrobial inoculants for safeguarding natural resources such asphosphate deposits, which are expected to be depleted within thenext 50e100 years (Cordell et al., 2009). Microbial inoculants havebeen shown to be a valid option for sustainable high quality wheatproduction in low-input areas of India, promising to improve thenutritional status and health of the rural Indian population.

Acknowledgement

This research project was funded by the Swiss Agency forDevelopment and Cooperation, Government of Switzerland and theDepartment of Biotechnology, Government of India under the Indo-Swiss Collaboration in Biotechnology (ISCB). The publication does

not constitute an endorsement by the Governments of Switzerlandand India. We thank Estelle Berset for technical assistance inmanuscript preparation. We thank David Roesti and Thomas Bollerfor helpful discussions on former versions of the manuscript. Wethank all farmers, advisers, technicians and researchers whocontributed to this project.

Author contributions: A.W., A.A., M.A. and B.J. designed theproject. A.A. and Reena S. isolated, characterized and mass-multi-plied the mycorrhizal fungal strains. B.J., M.A., A.S., Rashmi S. andV.S. isolated, characterized and mass-multiplied the bacteriastrains. A.S., Rashmi S., A.A., Reena S. and H.U. conducted the "eldstudies and did the corresponding soil and plant analyses. P.F. andP.M. assisted in "eld experimental design. P.M. and F.K evaluatedthe "eld data and prepared the manuscript. P.F. co-ordinated andmanaged the second phase of the project. All authors contributed torevising the manuscript.

Appendix. Supplementary material

Supplementary material related to this article can be found atdoi:10.1016/j.soilbio.2010.11.031.

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