Abstract
Severity of plant diseases is often influenced by the availability of nutrients, particularly N; however, its effect on the phyllosphere microbiome in foliar pathogen challenged plants is less investigated in rice. The tripartite interaction among the fungal pathogen (Magnaporthe oryzae), rice cultivars (basmati and non-basmati, blast resistant or susceptible) and nitrogen (N) fertilization (0, 120 and 180 N) was investigated. Plant growth, elicitation of defense responses and abundance of microbial members in the rice phyllosphere were monitored using biochemical and molecular methods. In general, photosynthetic pigments were distinct for each cultivar, and optimal N doses led to higher values. The susceptible var. CO-39 and resistant CO-39I exhibited higher contents of photosynthetic pigments and micronutrients such as zinc in leaves in response to N doses. Elicitation of defense and hydrolytic enzymes was significantly influenced by pathogen inoculation and modulated by N doses, but varietal effects were distinct. Scoring indices emphasized the pathogen susceptibility of var. CO-39 and PB-1, which showed almost 40–60% higher values than the resistant cultivars; the interactions of cultivars and N doses was also significant. Characteristic changes were recorded in the abundances of the gene copies, particularly, with an overall increase in the number of cyanobacterial 16S rRNA, and bacterial amoA in pathogen-challenged treatments, while nifH gene copies exhibited a reducing trend with increasing N doses, in the presence or absence of pathogen. The varietal differences in the cause and effect relationships can be valuable in crop protection for more effective foliar application of pesticides or biocontrol agents.
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Abanda-Nkpwatt D, Krimm U, Coiner HA, Schreiber L, Schwab W (2006) Plant volatiles can minimize the growth suppression of epiphytic bacteria by the phytopathogenic fungus Botrytis cinerea in co-culture experiments. Environ Exp Bot 56:108–119
Abril AB, Torres PA, Bucher EH (2015) The importance of phyllosphere microbial populations in nitrogen cycling in the Chaco semi-arid woodland. J Trop Ecol 21:1–5
Bates ST, Berg-Lyons D, Caporaso JG, Walters WA, Knight R, Fierer N (2011) Examining the global distribution of dominant archaeal populations in soil. ISME J 5:908–917
Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287
Bergmeyer N (1970) Methoden der enzymatischenanalyse, 1. Akademie Verlag, Berlin, pp 636–647
Breidenbach B, Conrad R (2014) Seasonal dynamics of bacterial and archaeal methanogenic communities in flooded rice fields and effect of drainage. Front Microbiol 5:752
Elbeltagy A, Nishioka K, Sato T, Suzuki H, Bin Y, Hamada T, Isawa T, Mitsui H, Minamisawa K (2001) Endophytic colonization and in planta nitrogen fixation by Herbaspirillum sp. isolated from wild rice. Appl Environ Microbiol 67(11):5285–5293
Ellur RK, Khanna A, Yadav A, Pathania S, Rajashekara H, Singh VK, Gopalakrishnan S, Bhowmick PK, Nagarajan M, Vinod KK, Prakash G, Mondal KK, Singh NK, Prabhu KV, Singh AK (2016) Improvement of Basmati rice cultivars for resistance to blast and bacterial blight diseases using marker assisted backcross breeding. Plant Sci 242:330–341
Ferrando L, Mañay JF, Scavino AF (2012) Molecular and culture-dependent analyses revealed similarities in the endophytic bacterial community composition of leaves from three rice (Oryza sativa) cultivars. FEMS Microbiol Ecol 80:696–708
Fierer N, Jackson JA, Vilgalys R, Jackson RB (2005) Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol 71:4117–4120
Forlani G (2010) Differential in vitro responses of rice cultivars to Italian lineages of the blast pathogen Pyricularia grisea. 2. Aromatic biosynthesis. J Plant Physiol 167:928–932
Fu MH, Xu XC, Tabatabai MA (1987) Effect of pH on nitrogen mineralization in crop-residue-treated soils. Biol Fertil Soils 5:115–119
Furnkranz M, Wanek W, Richter A, Abell G, Rasche F, Sessitsch A (2008) Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica. ISME J 2:561–570
Gechev TS, Van Breusegem F, Stone JM, Denev I, Laloi C (2006) Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays 28:1091–1101
Ghosh TK, Bailey HJ, Bisaria VS, Enari TM (1983) Measurement of cellulase activities. Final recommendations, Commission of Biotechnology. Pure Appl Chem 59:1–13
Giri S, Pati BR (2004) A comparative study on phyllosphere nitrogen fixation by newly isolated Corynebacterium sp. and Flavobacterium sp. and their potentialities as biofertilizer. Acta Microbiol Immunol Hung 51:47–56
Good AG, Shrawat AK, Muench DG (2004) Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production? Trends Plant Sci 9:597–605
Gothoskar SS, Scheffer RP, Walker JC, Stahmann MA (1995) The role of enzymes in the development of Fusarium Wilt of tomato. Ind Phytopathol 3:381–387
Gupta KJ, Brotman Y, Segu S, Zeier T, Zeier J, Presijn ST, Cristescu SM, Harren FJ, Bauwe H, Fernie AR, Kaiser WM (2013) The form of nitrogen nutrition affects resistance against Pseudomonas syringae pv. phaseolicola in tobacco. J Exp Bot 64:553–568
Ikeda S, Sasaki K, Okubo T, Yamashita A, Terasawa K, Bao Z, Liu D, Watanabe T, Murase J, Asakawa S, Eda S, Mitsui H, Sato T, Minamisawa K (2014) Low nitrogen fertilization adapts rice root microbiome to low nutrient environment by changing biogeochemical functions. Microbes Environ 29:50–59
Ikeda S, Tokida T, Nakamura H, Sakai H, Usui Y, Okubo T, Minamisawa K (2015) Characterization of leaf blade- and leaf sheath-associated bacterial communities and assessment of their responses to environmental changes in CO2, temperature and nitrogen levels under field conditions. Microbes Environ 30:51–62
Jackson ML (1967) Soil chemical analysis. Prentice Hall of India Pvt. Ltd., New Delhi
Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophyll a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanz 167:191–194
Jensen B, Munk L (1997) Nitrogen-induced changes in colony density and spore production of Erysiphe graminis f. sp. hordei on seedlings of six spring barley cultivars. Plant Pathol 46:191–202
Kanchan A, Simranjit K, Ranjan K, Prasanna R, Ramakrishnan B, Singh MC, Hasan M, Shivay YS (2018) Microbial biofilm inoculants benefit growth and yield of chrysanthemum varieties under protected cultivation through enhanced nutrient availability. Plant Biosyst. https://doi.org/10.1080/11263504.2018.1478904
Kant S, Bi YM, Rothstein SJ (2011) Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. J Exp Bot 62:1499–1509
Ke X, Angel R, Lu Y, Conrad R (2013) Niche differentiation of ammonia oxidizers and nitrite oxidizers in rice paddy soil. Environ Microbiol 15:2275–2292
Knief C, Delmotte N, Chaffron S, Stark M, Innerebner G, Wassmann R, Mering C, Vorholt JA (2012) Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice. ISME J 6:1378–1390
Koide Y, Kawasaki A, Yanoria MJ, Hairmansis T, Nguyet A, Bigirimana J, Fujita D, Kobayashi N, Fukuta Y (2010) Development of pyramided lines with two resistance genes, Pish and Pib for blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.). Plant Breed 129:670–675
Kong L, Xie Y, Hu L, Si J, Wang Z (2017) Excessive nitrogen application dampens antioxidant capacity and grain filling in wheat as revealed by metabolic and physiological analyses. Sci Rep 7:43363
Kumar A, Sheoran N, Prakash G, Ghosh A, Chikara SK, Rajashekara H, Singh UD, Aggarwal R, Jain RK (2017) Genome sequence of a unique Magnaporthe oryzae RMg-DI isolate from India that causes blast disease in diverse cereal crops, obtained using PacBio single-molecule and illumina HiSeq2500 sequencing. Genome Announc 5:e01570–e01516
Lindsay WL, Norvell WA (1978) Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci Soc Am J 42:421–428
Long DH, Lee FN, TeBeest DO (2000) Effect of nitrogen fertilization on disease progress of rice blast on susceptible and resistant cultivars. Plant Dis 84:403–409
Loper JE, Lindow SE (1994) A biological sensor for iron available to bacteria in their habitats on plant surfaces. Appl Environ Microbiol 60:1934–1941
Lopez-Berges MS, Rispail N, Prados-Rosales RC, Di Pietro A (2010) A nitrogen response pathway regulates virulence functions in Fusarium oxysporum via the protein kinase TOR and the bZIP protein MeaB. Plant Cell 22:2459–2475
Lv Q, Xu X, Shang J, Jiang G, Pang Z, Zhou Z, Wang J, Liu Y, Li T, Li X, Xu J (2013) Functional analysis of Pid3-A4, an ortholog of rice blast resistance gene Pid3 revealed by allele mining in common wild rice. Phytopathol 103:594–599
Mackill DJ, Bonman JM (1992) Inheritance of blast resistance in near isogenic lines of rice. Phytopathology 82:746–749
Mitchell CE, Reich PB, Tilman D, Groth JV (2003) Effects of elevated CO2, nitrogen deposition, and decreased species diversity on foliar disease interaction. Am Potato J 74:379–398
Müller H, Berg C, Landa BB, Auerbach A, Moissl-Eichinger C, Berg G (2015) Plant genotype-specific archaeal and bacterial endophytes but similar Bacillus antagonists colonize Mediterranean olive trees. Front Microbiol 6:138. https://doi.org/10.3389/fmicb.2015.00138
Mur LAJ, Simpson C, Kumari A, Gupta AK, Gupta KJ (2016) Moving nitrogen to the centre of plant defense against pathogens. Ann Bot 119:703–709
Murty MG (1983) Nitrogen fixation (acetylene reduction) in the phyllosphere of some economically important plants. Plant Soil 73:151–153
Neumann S, Paveley ND, Beed FD, Sylvester-Bradley R (2004) Nitrogen per unit leaf area affects the upper asymptote of Puccinia striiformis f. sp tritici epidemics in winter wheat. Plant Pathol 53:725–732
Nübel U, Garcia-Pichel F, Muyzer G (1997) PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl Environ Microbiol 63:3327–3332
Ohtakara A (1988) Chitosanase and β-N-acetyl hexosamine from Pycnosporus cinnabarinus. Methods Enzymol 168:464–468
Onaga G, Asea G (2016) Occurrence of rice blast (Magnaporthe oryzae) and identification of potential resistance sources in Uganda. Crop Protect 80:65–72
Ou SH (1980) Pathogen variability and host resistance in rice blast disease. Annu Rev Phytopathol 18:167–187
Pageau K, Reisdorf-Cren M, Morot-Gaudry JF, Masclaux-Daubresse C (2006) The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves. J Exp Bot 57:547–557
Panique E, Kelling KA, Schulte EE, Hero DE, Stevenson WR, James RV (1997) Potassium rate and source effects on potato yield quality and fungal plant disease. Glob Change Biol 9:438–451
Patti BR, Chandra AK (1981) Effect of spraying nitrogen-fixing phyllospheric bacterial isolates on wheat plants. Plant Soil 61:419–427
Pavlo A, Leonid O, Lryna Z, Natalia K, Maria PA (2011) Endophytic bacteria enhancing growth and disease resistance of potato (Solanum tuberosum L.). Biol Control 56:43–49
Poly F, Monrozier LJ, Bally R (2001) Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Res Microbiol 152:95–103
Prasanna R, Chaudhary V, Gupta V, Babu S, Kumar A, Singh R, Shivay YS, Nain L (2013) Cyanobacteria mediated plant growth promotion and bioprotection against Fusarium wilt in tomato. Eur J Plant Pathol 136:337–353
Prasanna R, Ramakrishnan B, Ranjan K, Venkatachalam S, Kanchan A, Solanki P, Monga D, Shivay YS, Kranthi S (2016) Microbial inoculants with multifaceted traits suppress Rhizoctonia populations and promote plant growth in cotton. J Phytopathol 164:1030–1042
Ribot C, Hirsch J, Balzergue S, Tharreau D, Nottéghem JL, Lebrun MH, Morel JB (2008) Susceptibility of rice to the blast fungus, Magnaporthe grisea. J Plant Physiol 165:114–124
Rigonato J, Alvarenga DO, Andreote FD, Dias ACF, Melo IS, Kent A, Fiore MF (2012) Cyanobacterial diversity in the phyllosphere of a mangrove forest. FEMS Microbiol Ecol 80:312–322
Rigonato J, Gonçalves N, Andreote APD, Lambais MR, Fiore MF (2016) Estimating genetic structure and diversity of cyanobacterial communities in Atlantic forest phyllosphere. Can J Microbiol 62:953–960
Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microbiol 63:4704–4712
Ruckstuhl M (1998) Population structure and epidemic of Bipolaris sorokiniana in rice–wheat cropping pattern in Nepal. In: Duveiller E, Dubin HJ, Reeves J, Mchab A (eds) Helminthosporium blights of wheat spot blotch and tan spot. CIMMYT, Veracruz, p 1328
Ruinen J (1971) The grass sheath as a site for nitrogen fixation. In: Preece TF, Dickinson CH (eds) Ecology of leaf surface microorganisms. Academic Press, London, pp 567–581
Sasaki K, Ikeda S, Ohkubo T, Kisara C, Sato T, Minamisawa K (2013) Effects of plant genotype and nitrogen level on bacterial communities in rice shoots and roots. Microbes Environ 28:391–395
Schlatter DC, Bakker MG, Bradeen JM, Kinkel LL (2015) Plant community richness and microbial interactions structure bacterial communities in soil. Ecology 96:134–142
Sebastian J, Chandra AK, Kolattukudy PE (1987) Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere. J Bacteriol 169:131–136
Sharma P (2007) Vegetables diseases, diagnosis and biomanagement. Aavishkar Publishers, Distributors, p 218
Smith KA, Thomson PE, Clayton H, McTaggart IP, Conen F (1998) Effects of temperature, water content and nitrogen fertilization on emissions of nitrous oxide by soils. Atmos Environ 32:3301–3309
Solomon PS, Tan KC, Oliver RP (2003) The nutrient supply of pathogenic fungi; a fertile field for study. Mol Plant Pathol 4:203–210
Suzuki MT, Taylor LT, DeLong EF (2000) Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays. Appl Environ Microbiol 66:4605–4614
Taffner J, Erlacher A, Bragina A, Berg C, Moissl-Eichinger C, Berg G (2018) What is the role of Archaea in plants? New insights from the vegetation of alpine bogs. mSphere 3:e00122–e00118. https://doi.org/10.1128/mSphere.00122-18
Takehisa H, Sato Y, Antonio BA, Nagamura Y (2013) Global transcriptome profile of rice root in response to essential macronutrient deficiency. Plant Signal Behav 8:e24409
Talbot NJ (2003) On the trail of a cereal killer, exploring the biology of Magnaporthe grisea. Annu Rev Microbiol 57:177–202
Tavernier V, Cadiou S, Pageau K, Laugé R, Reisdorf-Cren M, Langin T, Masclaux-Daubresse C (2007) The plant nitrogen mobilization promoted by Colletotrichum lindemuthianum in Phaseolus leaves depends on fungus pathogenicity. J Exp Bot 58:3351–3360
Thapa S, Prasanna R, Ranjan K, Velmourougane K, Ramakrishnan B (2017) Nutrients and host attributes modulate the abundance and functional traits of phyllosphere microbiome in rice. Microbiol Res 204:55–64
Thapa S, Ranjan K, Ramakrishnan B, Velmourougane K, Prasanna R (2018) Influence of fertilizers and rice cultivation methods on the abundance and diversity of phyllosphere microbiome. J Basic Microbiol 58:172–186
Trotel-Aziz P, Couderchet M, Biagianti S, Aziz A (2008) Characterization of new biocontrol agents Acinetobacter, Bacillus, Pantoea, and Pseudomonas sp. mediating grapevine resistance against Botrytis cinerea. Environ Expl Bot 64:21–32
Variar M, Vera Cruz CM, Carrillo MG, Bhatt JC, Sangar RBS (2009) Rice blast in India and strategies to develop durably resistant cultivars. In: Xiaofan W, Valent B (eds) Advances in genetics, genomics and control of rice blast disease. Springer, New York, pp 359–374
Walters DR, Bingham IJ (2007) Influence of nutrition on disease development caused by fungal pathogens: implications for plant disease control. Ann Appl Biol 151:307–324
Wang Y, Ke X, Wu L, Lu Y (2009) Community composition of ammonia-oxidizing bacteria and archaea in rice field soil as affected by nitrogen fertilization. Syst Appl Microbiol 32:27–36
Ward JL, Forcat S, Beckmann M, Miller SJ, Baker JM, Hawkins ND, Vermeer CP, Lu C, Lin W, Truman WM (2010) The metabolic transition during disease following infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato. Plant J 63:443–457
Wu LQ, Ma K, Li Q, Ke XB, Lu YH (2009) Composition of archaeal community in a paddy field as affected by rice cultivar and N fertilizer. Microb Ecol 58:819–826
Yadav RKP, Halley JM, Karamanoli K, Constantinidou HI, Vokou D (2004) Bacterial population on the leaves of Mediterranean plants: quantitative features and testing of distributional models. Environ Expl Bot 52:63–77
Yu ZH, Mackill DJ, Bonman JM (1987) Inheritance of resistance to blast in some traditional and improved rice cultivars. Phytopathology 77:323–326
Acknowledgements
The present study was partially supported by the ICAR-Network Project “Application of Microorganisms in Agricultural and Allied Sectors” (AMAAS), granted by Indian Council of Agricultural Research (ICAR), New Delhi to RP. We are thankful to the Post Graduate School, ICAR-IARI, New Delhi for the fellowship to the first author. We gratefully acknowledge the support and facilities provided by the Division of Microbiology and Division of Agronomy, ICAR-IARI, New Delhi during this study. We thank Dr. Gopalakrishnan, Division of Genetics, ICAR-IARI, New Delhi for providing the seeds of the four rice cultivars used in this study. We are thankful to Shri Gulab Singh, Dr. Y.S. Shivay and Dr. Dinesh Kumar for facilitating the analyses of macro- and micro-nutrients in the Division of Agronomy, ICAR-IARI, New Delhi.
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Concept and design of the study: ST, RP, BRK; acquisition and statistical analysis of data: ST, KV, NS, RP; drafting the manuscript and critical interpretation of the data: ST, RP, BRK, AK; supervision and critical inputs for experimental set-up and its maintenance: RP, AK. All the authors contributed to the preliminary data analyses and provided edits to the drafts of manuscript submitted.
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Communicated by Jorge Membrillo-Hernández.
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Supplementary Fig. 1 Comparison of the disease severity among the cultivars, after pathogen challenge by inoculation with Magnaporthe oryzae and application of different doses of N. Arrows in the first illustration indicate the characteristic symptoms of blast pathogen - the appearance of typical water soaked lesions on the leaf surface, leading to necrosis (PPTX 1148 KB)
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Thapa, S., Prasanna, R., Ramakrishnan, B. et al. Interactive effects of Magnaporthe inoculation and nitrogen doses on the plant enzyme machinery and phyllosphere microbiome of resistant and susceptible rice cultivars. Arch Microbiol 200, 1287–1305 (2018). https://doi.org/10.1007/s00203-018-1540-0
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DOI: https://doi.org/10.1007/s00203-018-1540-0