Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2015, V. 50, № 6, pp. 832-838.

UDC 636.085:579.62/.63:577.21:579.083.1

doi: 10.15389/agrobiology.2015.6.832eng

Acknowledgements:
Supported by grant of Russian Science Foundation, project № 14-16-00114

THE INVESTIGATION OF ENDOPHYTIC MICROORGANISMS
AS A SOURCE FOR SILAGE MICROBIOCENOSIS FORMATION
USING NGS-SEQUENCING

E.A. Yildirim1, G.Yu. Laptev1, L.A. Il’ina1, I.N. Nikonov1, V.A. Filippovav1, V.V. Soldatova1, E.A. Brazhnik1, N.I. Novikova1, T.Yu. Gagkaeva2

1Biotrof+ Ltd, 7-N,8, lit. A,
Malinovskaya ul., St. Petersburg, 196602 Russia,
е-mail deniz@biotrof.ru, laptev@biotrof.ru, ilina@biotrof.ru, nikonov@biotrof.ru, dumova@biotrof.ru, biotrof@biotrof.ru, bea@biotrof.ru;
2All-Russian Research Institute of Plant Protection, Federal Agency of Scientific Organizations,
3, sh. Podbel’skogo, St. Petersburg, 196608 Russia,
e-mail t.gagkaeva@mail.ru

Received June 30, 2015

The composition of plant and silage microflora affects the fermentation processes in the silage and its final quality. To date, reports about studying fodder plants and silage microbiota by means of molecular genetic methods are few and limited to descriptions of composition and function of some groups of microorganisms. Moreover, the NGS (next generation sequencing) data on diversity of epiphytic microflora and silage microbiocoenosis are not still reported. We first used this approach in studying phyllosphere and silage microbiom, and reported it to be rather rich in composition and abundance that is in contrast with conventional understanding. At that, the pathogenic and non-culturable microbes were detected in the microbiota, including specific inhabitants of mammalian gastrointestinal tract. So using NGS we examined the structure and diversity of bacterial community of Dactylis glomerata L. harvested plants and the biomass ensilaged with chemical preservative AIV 2000 Plus («KEMIRA OYJ, Inc.», Finland) composed of mixture of formic, propionic and benzoic acids. Assays were carried out on days 3, 7, 14 and 30 of ensilaging. The results showed that the bacterial community of silage from D. glomerata sharply differed from the composition of foliage microorganisms and varied greatly in the course of successive changes which occurred during maturation of silage preserved by mixture of organic acids. The composition of plant microorganisms and silage were found to be very various in contrast with a traditional view. Among foliar microorganisms of D. glomerata there were mostly the bacteria of phylum Proteobacteria (94.1 %), and in the silage the bacteria of phylums Bacteroidetes and Firmicutes were main representatives (up to 59.5 % and 74.9 %, respectively). In taxonomic diversity of the order Lactobacillales, mainly involved in ensilaging, there were genera Lactobacillus (up to 39.6%), Enterococcus (up to 36.36 %), Lactococcus (up to 14.4 %), Pediococcus (to 1.45 %) and the family Leuconostocaceae (to 3.52 %). Interestingly, in the silage there were the bacteria of phylum Bacteroidetes, families Ruminococcaceae, Lachnospiraceae and Selenomonadales considered the common inhabitants of the mammals’ gastrointestinal tract, and also the uncultured and pathogenic microorganisms. Particularly, these were 15 genera of family Enterobacteriaceae,including genera Klebsiella, Salmonella, Yersinia, etc., among which the dangerous mammalian pathogens are frequent. On days 3 and 7, the phylum Bacteroidetes prevailed (59.53 and 48.91 %, respectively). On days 14 and 30, the phylum Firmicutes was dominant (up to 74.85 %) with the facultative aerobic bacteria of order Lactobacillales mostly found (up to 74.76 %). Using NGS, a total of 70 genera were attributed in the plant phyloshere, and in the silage there were 84 genera on day 3, 96 genera on day 7, 51 genera on day 14, and 69 genera on day 30. Classical microbiology methods are not enough to detect these bacteria among silage microbiota.

Keywords: silage microorganisms, epiphytic microflora, NGS-sequencing, organic acids.

 

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REFERENCES

  1. Kosolapov V.M., Trofimov I.A. Zernobobovye i krupyanye kul'tury, 2013, 6(2): 59-63.
  2. Mak-Donal'd P. Biokhimiya silosa [Silage biochemistry]. Moscow, 1985.
  3. Lin C., Bolsen K.K., Brent B.E., Hart R.A., Dickerson A.M., Feyerherm A.M., Aimutis W.R. Epiphytic microflora on alfalfa and whole-plant corn. J. Dairy Sci., 1992, 75: 2484-2493 CrossRef
  4. Corsetti A., Gobbetti M., Rossi J., Damiani P. Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. J. Appl. Microbiol. Biotechnol., 1998, 50: 253-256 CrossRef
  5. Mansfield M.A., Kuldau G.A. Microbiological and molecular determination of mycobiota in fresh and ensiled maize silage. Mycologia, 2007, 99: 269-278 CrossRef
  6. Driehuis F. Silage and the safety and quality of dairy foods: a review. Agricult. Food Sci., 2013, 22: 16-34.
  7. Amann R.I., Ludwig W., Schleifer K.H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microb. Rev., 1995, 59: 143-169.
  8. Said E., Yimin C., Yasuhito F. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl. Environ. Microbiol., 2003, 69(1): 444-451 CrossRef
  9. Eikmeyer F.G., Köfinger P., Poschenel A., Jünemann S., Zakrzewski M., Heinl S., Mayrhuber E., Grabherr R., Pühler A., Schwab H., Schlüter A. Metagenome analyses reveal the influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass ensiling. J. Biotechnol., 2013, 167(3): 334-343 CrossRef
  10. Muck E. Recent advances in silage microbiology. Agricult. Food Sci., 2013, 22: 3-15.
  11. Paola D., Ernesto T., Luca C., Giorgio B. Microbial dynamics during aerobic exposure of corn silage stored under oxygen barrier or polyethylene films. Appl. Environ. Microbiol., 2011, 77(21): 7499-7507 CrossRef
  12. Lakin G.F. Biometriya [Biometry]. Moscow, 1990.
  13. Voznyakovskaya Yu.M. V sbornike: Ispol'zovanie mikroorganizmov v sel'skom khozyaistve [In: Use of microorganisms in agriculture]. Moscow-Leningrad, 1962: 100-112.
  14. Voznyakovskaya Yu.M. Mikroflora rastenii i urozhai [Plant microflora and yield]. Moscow, 1969.
  15. Tarakanov B.V. Metody issledovaniya mikroflory pishchevaritel'nogo trakta sel'skokhozyaistvennykh zhivotnykh i ptitsy [Study of gastrointestinal microflora in farm animals and poultry]. Moscow, 2006.
  16. Ling J.R., Robert E. Hungate’s The rumen and its microbes after 25 years. Lett. Appl. Microbiol., 1991, 13(4): 179-181 CrossRef
  17. Ushakova N.A., Nekrasov R.V., Meleshko N.A., Laptev G.Yu., Il'i-
    na L.A., Kozlova A.A., Nifatov A.V. Mikrobiologiya, 2013, 82(4): 456-563 CrossRef
  18. Kvasnikov E.I. Biologiya molochnokislykh bakterii [Biology of lactic acid bacteria]. Tashkent, 1960.
  19. Orla-Jensen S. The lactic acid bacteria. Kopenhagen, 1919. 
  20. Lin C., Bolsen K.K., Brent B.E., Fung D.Y.C. Epiphytic lactic acid bacteria succession during the pre-ensiling and ensiling periods of alfalfa and maize. J. Appl. Bacteriol., 1992, 73: 375-387 CrossRef
  21. Yang J., Cao Y., Cai Y., Terada F. Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation. J. Dairy Sci., 2010, 93(7): 3136-3145 CrossRef
  22. Pang H., Tan Z., Qin G., Wang Y., Li Z., Cai Y. Phenotypic and phylogenetic analysis of lactic acid bacteria isolated from forage crops and grasses in the Tibetan Plateau. J. Microbiol., 2012, 50: 63-71 CrossRef
  23. Heron S.J.E., Wilkinson J.F., Carol M. D. Enterobacteria associated with grass and silages. J. Appl. Bacteriol., 1993, 75: 13-17 CrossRef
  24. Lindgren S., Petterson K.L., Jonsson A., Lingvall P., Kaspersson A. Silage inoculation: Selected strains, temperature, wilting and practical application. J. Agric. Res., 1985, 15: 9-18.

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