APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2011, p. 7063–7067 Vol. 77, No. 190099-2240/11/$12.00 doi:10.1128/AEM.05225-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Enhancement of Switchgrass (Panicum virgatum L.) BiomassProduction under Drought Conditions by the Ectomycorrhizal

Fungus Sebacina vermifera�†Sita R. Ghimire and Kelly D. Craven*

Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401

Received 20 April 2011/Accepted 2 August 2011

Experiments were conducted to examine the effects of cocultivating the important bioenergy crop switchgrasswith the ectomycorrhizal fungus Sebacina vermifera under severe drought conditions. Plants cocultivated withthe fungus produced significantly higher biomass and had a higher macronutrient content than uninoculatedcontrol plants under both adequately watered and drought conditions.

Drought is a predominant factor limiting plant growth andyield in both dry land and irrigated agriculture (6, 27). Lack ofsoil water has a wide range of effects on morphological andbiochemical processes in plants, including nutrient uptakefrom the soil, negatively impacting crop productivity (2). Un-fortunately, the influence of drought on agriculture is expectedto worsen in the future due to climate change (8) and increas-ing demands for water for municipal and residential consump-tion (9). Thus, various strategies are being developed to max-imize water use efficiency and minimize the effects of droughton agriculture (3, 12, 14, 18, 23, 26, 29). However, utilization ofnaturally occurring symbiotic microbes to enhance droughttolerance of agricultural crops has remained largely unex-plored.

Most plant species in natural ecosystems are in symbioticrelationships with mycorrhizal and/or endophytic fungi (21).Members of the newly defined basidiomycete order Sebacina-les naturally form a wide spectrum of mycorrhizal types ofrelationships (31) with the roots of various mono- and dicoty-ledonous plants (4, 11, 15, 28, 30). Two species in particular,Sebacina vermifera [Serendipita vermifera (Oberw.) P. Roberts,comb. nov] and its close relative Piriformospora indica, havestimulated considerable attention over the past several years,because they form endophytic and mycorrhiza-like associationswith most plant species studied to date (30, 32). This is of greatinterest, because both species are axenically cultivable, possessplant growth-promoting characteristics, and contribute severalother benefits to their host plants (4, 11, 15, 28, 30). Twoprevious studies have shown that colonization of roots by P.indica confers drought tolerance in Arabidopsis thaliana andChinese cabbage (24, 25). However, no similar studies havebeen performed to evaluate the potential of S. vermifera toimpart drought tolerance to host plants. Our objective herewas to investigate the effect of S. vermifera in mitigating bio-

mass losses in switchgrass due to drought, with the ultimategoal of maximizing the utility of this important bioenergy cropand the range of lands upon which it can be grown.

An in vitro study was performed using 175-ml plant contain-ers (65 mm in diameter by 65 mm in height) with lids. Thecontainers were filled with 25 ml of modified PNM culturemedium (24) and overlaid with a nylon disk (mesh pore size, 50�m). Two strains of S. vermifera, MAFF-305828 and MAFF-305830, were used in the study. One 5-mm-diameter plug offungal hyphae from a colony actively growing on malt extractagar (MEA) was placed at the center of the nylon disk andallowed to grow for 2 weeks, and a plug of similar size from anuninoculated MEA plate was used as a control. Subsequently,four germinated Alamo seeds with no visible contamination byfungi or bacteria were placed on each nylon disk at a distanceof 15 mm from the plug. Containers were incubated at 24°Cwith a light-dark cycle of 16 h and 8 h (light illumination of 165� mol m�2 s�1) for 6 weeks. The lids were then replaced withair-pore filters to allow evaporative and transpiration waterloss at ambient temperature. On the 7th day of drought expo-sure, leaf color, plant stature, and total fresh weight wererecorded. The fibrous roots were stained (16) and examinedunder a microscope for fungal colonization. Each treatmentconsisted of 10 to 13 experimental units (containers), and theexperiment was performed twice.

In a subsequent greenhouse experiment, 6-week-old rootedexplants of switchgrass genotype VS16 were grown in 3.8-literpots filled with a 4:1 (vol/vol) mixture of sterile Metromix-350medium (Scotts-Sierra Horticultural Products, Marysville,OH) and S. vermifera (strain MAFF-305828)-colonized sor-ghum grains (Fig. 1). Control seedlings were grown in a mix-ture prepared with sterile sorghum grains. Plants were main-tained at 24°C and 18°C in a light-dark cycle of 16 h and 8 h(light illumination of 165 � mol m�2 s�1) for 5 weeks. Indi-vidual seedlings from both treatments were subsequentlyplanted in 1-liter pots filled with a 3:1 (vol/vol) mixture ofMetromix-350 medium and sand and grown in the greenhousewith an average temperature and average relative humidity of20.2°C and 39.3%, respectively.

All plants were watered to saturation at planting. Thereaf-ter, half of the plants from the cocultivation and control treat-ments were subjected to drought whereas the remaining plants

* Corresponding author. Mailing address: Plant Biology Division,The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway,Ardmore, OK 73401. Phone: (580) 224-6960. Fax: (580) 224-6692.E-mail: kdcraven@noble.org.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print 12 August 2011.

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were watered normally. Experimental pots were checked indi-vidually for soil volumetric water content (VWC) on alternatedays using a Field Scout TDR 100 soil moisture probe (Spec-trum Technologies, Plainfield, IL). Plants receiving droughttreatments were allowed to grow until the VWC dropped to�1%, at which point subsets of the plants subjected to droughtand of the adequately watered plants were harvested. Plantswith normal watering were maintained at a constant 10 to 20%VWC but were harvested at the same time points as the plantssubjected to drought. These water levels were derived empir-ically in a previous experiment in which switchgrass seedlingsat 10 to 20% VWC grew normally, seedlings at 5% VWCshowed initial wilt symptoms, and plants at �1% VWC wiltedpermanently when grown for 2 days.

Experimental plants were arranged in a factorial random-ized-block design. Each treatment was applied to 48 individu-ally potted plants. One third of the experimental plants wereharvested after the first drought stress (i.e., when the averageVWC in the corresponding drought treatment pots reached1%). The remaining plants were subjected to a second droughtstress treatment, and half of those were subjected to a subse-quent third drought stress treatment. All experimental plantswere rewatered to saturation prior to subsequent droughtstress treatments. Data on tiller number, shoot length, rootlength, shoot dry matter (DM), and root DM were recorded ineach harvest. Leaf chlorophyll content was measured using aSPAD-502 Plus chlorophyll meter (Konica Minolta SensingAmericas Inc., Ramsey, NJ). The shoot and root tissues were

analyzed for nitrogen (N), phosphorous (P), potassium (K)calcium (Ca), magnesium (Mg), and sulfur (S) content at WardLaboratories, Inc. (Kearney, NE). Data on biomass and bio-mass-related parameters were analyzed using PROC GLM inSAS statistical software package version 9.1.3 (22), and least-significant-difference (LSD) tests were performed to comparetreatments at �95% confidence levels.

Roots of cocultivated plants from the in vitro study wereeffectively colonized by S. vermifera (Fig. 2). After a weeklongexposure to ambient temperature, the appearance of coculti-vated plants was barely affected whereas control seedlings werepale green and withered (Fig. 3). Plants cocultivated withMAFF-305828 and MAFF-305830 produced 71% and 53%higher levels of fresh biomass, respectively, than control plants(Table 1; P � 0.01). The absence of fungus-mediated waterand nutrient uptake might be the reason for the poor perfor-mance of control seedlings. Similar effects on the droughttolerance of Arabidopsis thaliana seedlings inoculated with P.indica were observed previously, with up to a 300% increase infresh biomass production (24).

In the greenhouse study, treatment results differed signifi-cantly with respect to shoot length, shoot DM, root DM, andshoot-to-root DM ratio (Table 2). Shoot lengths increased by109%, 59%, and 95% and shoot biomass increased by 337%,215%, and 267%, respectively, at the first, second, and thirdharvests in response to inoculation under conditions of ade-

FIG. 1. (a) Sorghum grains colonized by Sebacina vermifera. (b)Uninoculated control. FIG. 2. Switchgrass roots. (a) Uninoculated control. (b) Roots of

cocultivated switchgrass seedling. Aniline blue-stained mycelia on theroot surface are indicated with black arrows. Sebacina vermifera chla-mydospores in the epidermal cells are indicated with red arrows.

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quate watering. Intriguingly, cocultivated plants exposed todrought produced significantly taller plants with a higher shootDM level than well-watered control plants. Under conditionsof adequate watering, cocultivated plants consistently pro-duced higher levels (290%, 270%, and 166%, respectively, atfirst, second, and third harvest) of root biomass than the con-trols. Further, when subjected to one, two, or three droughtcycles, cocultivated plants produced 303%, 127%, and 112%higher root biomass levels, respectively, than the controls. Fur-

ther, as was evident for shoot tissues, cocultivated plants ex-posed to one, two, or three drought stress treatments pro-duced, respectively, 353%, 131%, and 148% higher rootbiomass levels than corresponding controls that had been ad-equately watered (Table 2; P � 0.01). Thus, plants colonized byS. vermifera exhibited simultaneous increases in both shoot androot biomass levels, indicating that above-ground biomassgains were not simply a consequence of reallocated carbohy-drate. Indeed, cocultivated plants consistently produced higher

FIG. 3. Switchgrass seedlings after exposure to mild drought stress. Cocultivated seedlings with Sebacina vermifera strain MAFF 305830 areshown on the left, with S. vermifera strain MAFF 305828 shown in the middle and controls shown on the right.

TABLE 1. Effect of Sebacina vermifera and switchgrass cocultivation on switchgrass biomass production underdrought stress in vitro conditionsa

Treatment

No. of seedlings per container after:Fresh wt (mg/container)

(mean � LSD)Planting Drought exposure(mean � LSD)

Expt 1Cocultivation

Strain MAFF 305828 4 3.46 � 0.26 A 148.62 � 13.45 AStrain MAFF 305830 4 3.69 � 0.26 A 143.08 � 13.45 A

Control 4 3.54 � 0.26 A 97.46 � 13.45 BSignificance for treatment results n.s. ��

Expt 2Cocultivation

Strain MAFF 305828 4 3.80 � 0.20 A 122.59 � 12.20 AStrain MAFF 305830 4 3.60 � 0.20 A 96.48 � 12.20 B

Control 4 3.10 � 0.20 B 58.11 � 12.20 CSignificance for treatment results �� ��

Expt 1 and 2Cocultivation

Strain MAFF 305828 4 3.61 � 0.17 A 137.30 � 10.65 AStrain MAFF 305830 4 3.65 � 0.17 A 122.82 � 10.65 A

Control 4 3.35 � 0.17 A 80.35 � 10.65 BSignificance for treatment results n.s. ��

a ��, P � 0.01; n.s., not significant. Values with different letters within the drought exposure and fresh weight columns for a given experiment represent significantlydifferent results at a �99% confidence level.

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root biomass levels than control plants, suggesting a greaterpotential to sequester carbon and hold soils, both highly de-sired properties in a crop grown under low-input conditions.

Except for the second harvest, cocultivated plants had sig-nificantly higher shoot-to-root DM ratios than the correspond-ing control plants (Table 2). In all treatments, the shoot-to-root DM ratio was highest at the first harvest and declined inthe subsequent harvests, falling between 66 and 77% from thefirst to the third harvest. These results suggest that nutrientavailability determines how plants allocate their resources toroot or shoot tissues. As experimental plants were grown inpots without supplemental nutrition, plants at the first harvestmay have had access to relatively higher nutrient conditionsthan those of second and third harvests. Accordingly, develop-ment prior to subsequent harvests may have shifted in favor ofroot growth (thereby reducing shoot-to-root DM levels) tomaximize nutrient acquisition potential. These observationsare consistent with other studies that reported increased bio-mass allocation to roots under low-nutrient conditions (7, 17).

Cocultivated plants had lower concentrations of several ma-

cronutrients in shoot and root tissues compared to controlplants (P � 0.05) (see Table S1 and Table S2 in the supple-mental material). However, the total acquisition of all macro-nutrients (except Ca in roots) was significantly higher in co-cultivated plants than in the controls, likely reflective of theirtaller stature. As the plants were grown on the same amount ofsoil substrate, larger plants would have depleted this resourcefaster than smaller plants. Moreover, most plant species, espe-cially perennials, effectively allocate resources to transport,growth, defense, and reproduction (10), with the remainderbeing committed to storage. The lower concentrations of thesenutrients in Sebacina-infected plants observed in this studymay reflect a depletion of cellular stores to fuel the demandsfor growth and maintenance in those substantially larger plants(19). Some of these macronutrients (e.g., N and Mg) are crit-ical constituents of chlorophyll, and their lower concentrationsin cocultivated plants might have affected the observed declinein leaf chlorophyll content (13).

This report confirms that cocultivation imparts extraordi-nary biomass gains to switchgrass such that the yield of such

TABLE 2. Effect of Sebacina vermifera colonization on switchgrass biomass production under conditions ofadequate watering and drought in greenhousea

Treatment Shoot length (cm)(mean � SE)

Root length (cm)(mean � SE)

Shoot DM(mg/plant)

(mean � SE)

Root DM(mg/plant)

(mean � SE)

Shoot-to-rootratio

(mean � SE)

No. of tillers(mean � SE)

Chlorophyllcontent (%)

(mean � SE)

First harvest (one droughtcycle)

CocultivationWatered 70.5 � 1.1 24.1 � 0.4 752 � 29 316 � 16 2.48 � 0.06 2.38 � 0.06 31.0 � 0.3Dry 56.9 � 1.0 30.5 � 0.9 500 � 23 367 � 16 1.40 � 0.03 2.06 � 0.03 30.9 � 0.3

ControlWatered 33.8 � 0.6 20.7 � 0.5 172 � 9 81 � 4 2.16 � 0.07 2.38 � 0.06 29.7 � 0.3Dry 28.5 � 0.7 20.3 � 0.4 105 � 6 91 � 5 1.23 � 0.07 1.88 � 0.10 31.7 � 0.3

Significance testsCocultivation �� �� �� �� � n.s. n.s.Drought �� � �� n.s. �� �� n.s.Interaction � �� � n.s. n.s. n.s. n.s.

Second harvest (two droughtcycles)

CocultivationWatered 73.6 � 1.0 36.2 � 0.6 1,600 � 53 1,236 � 80 1.46 � 0.05 2.56 � 0.06 28.6 � 0.3Dry 61.8 � 1.0 36.7 � 0.8 900 � 20 771 � 22 1.20 � 0.03 2.69 � 0.06 32.2 � 0.3

ControlWatered 46.2 � 0.8 37.9 � 0.7 508 � 17 334 � 11 1.56 � 0.05 2.63 � 0.06 31.1 � 0.3Dry 36.5 � 0.7 41.8 � 0.6 388 � 14 339 � 11 1.18 � 0.04 2.50 � 0.07 34.2 � 0.5

Significance testsCocultivation �� � �� �� n.s. n.s. ��Drought �� n.s. �� �� �� n.s. ��Interaction n.s. n.s. �� �� n.s. n.s. n.s.

Third harvest (three droughtcycles)

CocultivationWatered 72.9 � 0.7 43.3 � 0.9 1,598 � 35 2,342 � 41 0.68 � 0.01 2.69 � 0.08 16.7 � 0.9Dry 61.8 � 1.0 41.1 � 0.8 1,047 � 25 2,189 � 40 0.48 � 0.01 2.38 � 0.08 16.5 � 1.2

ControlWatered 37.4 � 1.1 43.8 � 0.9 435 � 20 882 � 36 0.49 � 0.01 2.25 � 0.07 24.7 � 0.3Dry 34.0 � 0.5 47.6 � 1.5 406 � 16 1,032 � 43 0.41 � 0.01 2.31 � 0.06 28.4 � 0.4

Significance testsCocultivation �� n.s. �� �� �� n.s. ��Drought �� n.s. �� n.s. �� n.s. n.s.Interaction � n.s. �� n.s. �� n.s. n.s.

a �, P � 0.05; ��, P � 0.01; n.s., not significant.

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plants grown under our defined drought stress conditions sig-nificantly exceeded that of control plants grown under normalor restricted-water conditions. The cocultivated plants consis-tently produced higher levels of root biomass than controlplants, suggesting a greater potential to sequester carbon andhold soils, both highly desired properties in a crop grown underlow-input conditions. As with many warm-season perennialgrasses, switchgrass can be difficult or slow to establish (1, 20),and this is a major impediment to its implementation as aprimary bioenergy crop (5). Therefore, both the shoot- androot-growth-promoting effects of S. vermifera, particularly atearly points in plant development, are likely to improve com-petitiveness of switchgrass seedlings, especially during the es-tablishment process.

The BioEnergy Science Center is a U.S. Department of EnergyBioenergy Research Center supported by the Office of Biological andEnvironmental Research in the DOE Office of Science.

The fungal strains used in these studies were obtained from theNational Institute of Agro-biological Sciences, Tsukuba, Ibaraki, Ja-pan. We are thankful to Nikki Charlton, Jeremey Bell, and MyoungChi for their help in greenhouse study and to Michael Udvardi andTwain Butler for reviewing the first draft of the manuscript.

REFERENCES

1. Aiken, G. E., and T. L. Springer. 1995. Seed size distribution, germinationand emergence of six switchgrass cultivars. J. Range Manage. 48:455–458.

2. Alam, S. M. 1999. Nutrient uptake by plants under stress conditions, p.285–313. In M. Pessarakli (ed.), Handbook of plant and crop stress, 2nd ed.Marcel Dekker Inc., New York, NY.

3. Anjum, S. A., et al. 2010. Desertification in Pakistan: causes, impacts andmanagement. J. Food Agric. Environ. 8:1203–1208.

4. Barazani, O., M. Benderoth, K. Groten, C. Kuhlemeier, and I. T. Baldwin.2005. Piriformospora indica and Sebacina vermifera increase growth perfor-mance at the expense of herbivore resistance in Nicotiana attenuata. Oeco-logia 146:234–243.

5. Beckman, J. J., L. E. Moser, K. Kubik, and S. S. Waller. 1993. Big bluestemand switchgrass establishment as influenced by seed priming. Agron. J. 85:199–202.

6. Begg, J. E., and N. C. Turner. 1976. Crop water deficits. Adv. Agron.28:161–217.

7. Boot, R. G. A., and M. Mensink. 1990. Size and morphology of root systemsof perennial grasses from contrasting habitats as affected by nitrogen supply.Plant Soil 129:291–299.

8. Burke, E. J., S. J. Brown, and N. Christidis. 2006. Modeling the recentevolution of global drought and projections for the twenty-first century withthe Hadley Centre climate model. J. Hydrometeorol. 7:1113–1125.

9. CAST. 2009. Water, people, and future: water availability for agriculturein the United States. Council for Agricultural Science and Technology,Ames, IA.

10. Chapin, F. S., E. D. Schulze, and H. A. Mooney. 1990. The ecology andeconomics of storage in plants. Annu. Rev. Ecol. Syst. 21:423–447.

11. Deshmukh, S., et al. 2006. The root endophytic fungus Piriformospora indicarequires host cell death for proliferation during mutualistic symbiosis withbarley. Proc. Natl. Acad. Sci. U. S. A. 103:18450–18457.

12. Endale, D. M., H. H. Schomberg, M. B. Jenkins, D. H. Franklin, and D. S.Fisher. 2010. Management implications of conservation tillage and poultrylitter use for Southern Piedmont U. S. A. cropping systems. Nutr. Cycl.Agroecosys. 88:299–313.

13. Ericsson, T. 1995. Growth and shoot: root ratio of seedlings in relation tonutrient availability. Plant Soil 168–169:205–214.

14. Fukai, S., P. Sittisuang, and M. Chanphengsay. 1998. Increasing productionof rainfed lowland rice in drought prone environments: a case study inThailand and Laos. Plant Prod. Sci. 1:75–82.

15. Ghimire, S. R., N. D. Charlton, and K. D. Craven. 2009. The mycorrhizalfungus, Sebacina vermifera, enhances seed germination and biomass pro-duction in switchgrass (Panicum virgatum L.). Bioenerg. Res. 2:51–58.

16. Grace, C., and D. P. Stribley. 1991. A safer procedure for routine staining ofvesicular arbuscular mycorrhizal fungi. Mycol. Res. 95:1160–1162.

17. Levang-Brilz, N., and M. E. Biondini. 2003. Growth rate, root developmentand nutrient uptake of 55 plant species from the Great Plains grasslands, U.S. A. Plant Ecol. 165:117–144.

18. Major, D. J., R. J. Morrison, R. E. Blackshaw, and B. T. Roth. 1991.Agronomy of dry land corn production at the northern fringe of the GreatPlains. J. Prod. Agric. 4:606–613.

19. Millard, P. 1988. The accumulation and storage of nitrogen by herbaceousplants. Plant Cell Environ. 11:1–8.

20. Moser, L. E., and K. P. Vogel. 1995. Switchgrass, big blue stem, andIndian grass, p. 409–420. In R. F. Barnes, D. A. Miller, and C. J. B.Nelson (ed.), Forage, an introduction to grassland agriculture, vol. 1.Iowa State University, Ames, IA.

21. Petrini, O. 1986. Taxonomy of endophytic fungi of aerial plant tissues, p.175–187. In N. J. Fokkema and J. van den Huevel (ed.), Microbiology of thephyllosphere. Cambridge University Press, Cambridge, United Kingdom.

22. SAS. 2004. Proc GLM, 9.1.3 ed. SAS Institute Inc., Cary, NC.23. Schittenhelm, S. 2010. Effect of drought stress on yield and quality of maize/

sunflower and maize/sorghum intercrops for biogas production. J. Agron.Crop Sci. 196:253–261.

24. Sherameti, I., S. Tripathi, A. Varma, and R. Oelmuller. 2008. The root-colonizing endophyte Pirifomospora indica confers drought tolerance inArabidopsis by stimulating the expression of drought stress-related genes inleaves. Mol. Plant-Microbe Inter. 21:799–807.

25. Sun, C. A., et al. 2010. Piriformospora indica confers drought tolerance inChinese cabbage leaves by stimulating antioxidant enzymes, the expressionof drought-related genes and the plastid-localized CAS protein. J. PlantPhysiol. 167:1009–1017.

26. Tabatabaee, J., and M. Y. Han. 2010. Rainwater harvesting potentials fordrought mitigation in Iran. Water Sci. Technol. 62:816–821.

27. Tazaki, T., K. Ishihara, and T. Ushijima. 1980. Influence of water stress onthe photosynthesis and productivity of plants in humid stress, p. 309–322. InN. C. Turner and P. J. Kratner (ed.), Adaptation of plants for water and hightemperature stress. Wiley, New York, NY.

28. Varma, A., S. Verma, Sudha, N. Sahay, B. Butehorn, and P. Franken. 1999.Piriformospora indica, a cultivable plant-growth-promoting root endophyte.Appl. Environ. Microbiol. 65:2741–2744.

29. Voloudakis, A. E., et al. 2002. Expression of selected drought-related genesand physiological response of Greek cotton varieties. Funct. Plant Biol.29:1237–1245.

30. Waller, F., et al. 2008. Systemic and local modulation of plant responses byPiriformospora indica and related Sebacinales species. J. Plant Physiol. 165:60–70.

31. Weiss, M., M. A. Selosse, K. H. Rexer, A. Urban, and F. Oberwinkler. 2004.Sebacinales: a hitherto overlooked cosm of heterobasidiomycetes with abroad mycorrhizal potential. Mycol. Res. 108:1003–1010.

32. Weiss, M., et al. 2011. Sebacinales everywhere: previously over-looked ubiquitous fungal endophytes. PLoS One 6:e16793. doi:10.1371/journal.pone.0016793.

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