Culturable endophytic bacteria from stems and leaves of garden pea (Pisum sativum L.)

Cover Page
Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription or Fee Access

Abstract


Background. Endophytic microorganisms inhabit internal tissues of most plants. However, little is known about endophytic community of the garden pea (Pisum sativum L.), an agriculturally important crop.

Materials and methods. Culturable endophytic bacteria were isolated from sterilized stems and leaves of three pea genotypes: K-8274 (cv. Vendevil), K-3358 (unnamed cultivar), and cv. Triumph. The taxonomic position of isolates was determined by 16S rRNA gene sequencing. The plant growth-promoting capabilityof identified bacteria was tested on the roots of watercress (Lepidium sativum L.).

Results. In total, out of 118 morphotypes of culturable endophytic bacteria identified, for 80 the taxonomic position was determined. Proteobacteria and Firmicutes were dominant phyla, and Actinobacteria were present in minority. Eight bacterial isolates demonstrated the plant growth-promoting capability, and one of them – KV17 (Rahnella sp.) maintained this capability after several passages and prolonged storage.

Conclusion. The plant growth-promoting bacteria isolated from pea stems and leaves can become a component of microbiological preparations.


Full Text

Restricted Access

About the authors

Ekaterina N. Vasileva

All-Russia Research Institute for Agricultural Microbiology

Author for correspondence.
Email: evasilieva@arriam.ru
ORCID iD: 0000-0001-5599-0361

Russian Federation, Saint-Petersburg

Technician, Laboratory of Genetics of Plant-Microbe Interactions

Gulnar A. Akhtemova

All-Russia Research Institute for Agricultural Microbiology

Email: ahgulya@yandex.ru
ORCID iD: 0000-0001-7957-3693

Russian Federation, Saint-Petersburg

PhD, Senior Scientist, Laboratory of Genetics of Plant-Microbe Interactions.

Alexey M. Afonin

All-Russia Research Institute for Agricultural Microbiology

Email: afoninalexeym@gmail.com
ORCID iD: 0000-0002-8530-0226

Russian Federation, Saint-Petersburg

PhD student, Laboratory of Genetics of Plant-Microbe Interactions

Alexey Yu. Borisov

All-Russia Research Institute for Agricultural Microbiology

Email: ayborisov@yandex.ru
ORCID iD: 0000-0001-9834-7368

Russian Federation, Saint-Petersburg

DSc, Chief Researcher

Igor A. Tikhonovich

All-Russia Research Institute for Agricultural Microbiology; Saint Petersburg State University

Email: arriam2008@yandex.ru
ORCID iD: 0000-0001-8968-854X

Russian Federation, Saint-Petersburg

DSc, Scientific Director; Professor, Dean, Faculty of Biology

Vladimir A. Zhukov

All-Russia Research Institute for Agricultural Microbiology

Email: vzhukov@arriam.ru
ORCID iD: 0000-0002-2411-9191

Russian Federation, Saint-Petersburg

PhD, Head, Laboratory of Genetics of Plant-Microbe Interactions

References

  1. Partida-Martínez LP, Heil M. The microbe-free plant: fact or artifact? Front Plant Sci. 2011;2:100. https://doi.org/10.3389/fpls.2011.00100.
  2. Штарк О.Ю., Жуков В.А., Сулима А.С., и др. Перспективы использования многокомпонентных симбиотических систем бобовых // Экологическая генетика. – 2015. – Т. 13. – № 1. – С. 33–46. [Shtark OYu, Zhukov VA, Sulima AS, et al. Prospects for the use of multi-component symbiotic systems of the legumes. Ecological genetics. 2015;13(1):33-46. (In Russ.)]. https://doi.org/10.17816/ecogen13133-46.
  3. Bais HP, Weir TL, Perry LG, et al. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol. 2006;57:233-266. https://doi.org/10.1146/annurev.arplant.57.032905.105159.
  4. Цавкелова Е.Л., Климова С.Ю., Чердынцева Т.Л., Нетрусов Л.И. Микроорганизмы-продуценты стимуляторов роста растений и их практическое применение (обзор) // Прикладная биохимия и микробиология. – 2006. – T. 42. – № 2. – C. 133–143. [Tsavkelova EA, Klimova SYu, Cherdyntseva TL, Netrusov LI. Microbial producers of plant growth stimulators and their practical use: a review. Prikladnaya biokhimiya i mikrobiologiya. 2006; 42(2): 133-143. (In Russ.)]
  5. Wilson D. Endophyte: the evolution of a term, and clarification of its use and definition. Oikos. 1995;73(2):274-276. https://doi.org/10.2307/3545919.
  6. Hardoim PR, van Overbeek LS, Elsas JD. Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol. 2008;16(10):463-471. https://doi.org/ 10.1016/j.tim.2008.07.008.
  7. Compant S, Clément C, Sessitsch A. Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem. 2010;42(5): 669-678. https://doi.org/10.1016/j.soilbio.2009. 11.024.
  8. Santoyo G, Moreno-Hagelsieb G, Orozco-Mosqueda Mdel C, Glick BR. Plant growth-promoting bacterial endophytes. Microbiol Res. 2016;183:92-99. https://doi.org/10.1016/j.micres.2015.11.008.
  9. Чеботарь В.К., Макарова Н.М., Шапошников А.И., Кравченко Л.В. Антифунгальные и фитостимулирующие свойства ризосферного штамма Bacillus subtilis Ч-13 продуцента биопрепаратов // Прикладная биохимия и микробиология. – 2009. – T. 45. – № 4. – C. 465–469. [Chebotar VK, Makarova NM, Shaposhnikov AI, Kravchenko LV. Antifungal and phytostimulating characteristics of Bacillus subtilis Ch-13 rhizospheric strain, producer of bioprepations. Prikladnaya biokhimiya i mikrobiologiya. 2009;45(4):465-469. (In Russ.)]
  10. Чеботарь В.К., Заплаткин А.Н., Щербаков А.В. и др. Микробные препараты на основе эндофитных и ризобактерий, которые перспективны для повышения продуктивности и эффективности использования минеральных удобрений у ярового ячменя (Hordeum vulgare L.) и овощных культур // Сельскохозяйственная биология. – 2016. – Т. 51. – № 3. – С. 335–342. [Chebotar VK, Zaplatkin AN, Shcherbakov AV, et al. Microbial preparations on the basis of endophytic and rhizobacteria to increase the productivity in vegetable crops and spring barley (Hordeum vulgare L.), and the mineral fertilizer use efficiency. Sel’skokhoziaistvennaia biologiia. 2016;51(3):335-342. (In Russ.)]. https://doi.org/10.15389/agrobiology.2016.3.335rus.
  11. Васильева Е.Н., Ахтемова Г.А., Жуков В.А., Тихонович И.А. Эндофитные микроорганизмы в фундаментальных исследованиях и сельском хозяйстве // Экологическая генетика. – 2019. – Т. 17. – № 1. – С. 19–32. [Vasileva EN, Akhtemova GA, Zhukov VA, Tikhonovich IA. Endophytic microorganisms in fundamental research and agriculture. Ecological genetics. 2019;17(1):19-32. (In Russ.). https://doi.org/10.17816/ecogen17119-32.
  12. Гарипова С.Р., Гарифуллина Д.В., Маркова О.В., и др. Изучение бактериальных ассоциаций эндофитов клубеньков, способствующих увеличению продуктивности бобовых растений // Агрохимия. – 2010. – № 11. – С. 50–58. [Garipova SR, Garifullina DV, Markova OV, et al. Bacterial endophyte associations of nodules increasing the productivity of legumes. Agrochemistry. 2010;(11):50-58. (In Russ.)]
  13. Гарифуллина Д.В. Эндофитные бактерии растений гороха как активный компонент бобово-ризобиальной симбиотической системы: Автореф. дис. … канд. биол. Наук. – Уфа, 2012. [GarifullinaDV. Endofitnyye bakterii rasteniy gorokha kak aktivnyy komponent bobovo-rizobial’noy simbioticheskoy sistemy. [dissertation abstract] Ufa; 2012. (In Russ.)]. Доступно по: https://revolution.allbest.ru/agriculture/00982191_0.html. Ссылка активна на 02.02.2020.
  14. Гарипова С.Р., Гарифуллина Д.В., Маркова О.В., и др. Комплексная биологическая активность in vitro эндофитных бактерий, выделенных из клубеньков гороха и фасоли // Известия Уфимского научного центра Российской академии наук. – 2015. – № 4–1. – С. 25–28. [Garipova SR, Garifullina DV, Markova OV, et al. Complex biological activity in vitro of endophytic bacteria isolated from pea and bean nodules. Izvestiya Ufimskogo Nauchnogo Tsentra Rossiyskoy Akademii Nauk. 2015;(4-1):25-28. (In Russ.)]
  15. Гарипова С.Р., Гарифуллина Д.В., Баймиев А.Х., Хайруллин Р.М. Межмикробные взаимоотношения бактерий Serratia sp. Ent16 — симбионта клубенька гороха и колонизация ими эндоризосферы хозяина // Прикладная биохимия и микробиология. – 2017. – T. 53. – № 3. – C. 299–307. [Garipova SR, Garifullina DV, Baimiev AH, Khairullin RM. Intermicrobial relationships of the pea nodule symbiont Serratia sp. Ent16 and its colonization of the host endorhizosphere. Prikladnaya biokhimiya i mikrobiologiya. 2017;53(3):299-307. (In Russ.)]. https://doi.org/10.7868/S0555109917030060.
  16. Selvakumar G, Kundu S, Gupta AD, et al. Isolation and characterization of nonrhizobial plant growth promoting bacteria from nodules of Kudzu (Pueraria thunbergiana) and their effect on wheat seedling growth. Curr Microbiol. 2008;56(2):134-139. https://doi.org/10.1007/s00284-007-9062-z.
  17. Tariq M, Hameed S, Yasmeen T, Ali A. Non-rhizobial bacteria for improved nodulation and grain yield of mung bean [Vigna radiata (L.) Wilczek]. Afr J Biotechnol. 2012;11:15012-15019. https://doi.org/10.5897/AJB11.3438.
  18. Elvira-Recuenco M, van Vuurde JW. Natural incidence of endophytic bacteria in pea cultivars under field conditions. Can J Microbiol. 2000;46(11):1036-1041. https://doi.org/10.1139/ w00-098.
  19. Shtark OY, Borisov AY, Zhukov VA, Tikhonovich IA. Mutually beneficial legume symbioses with soil microbes and their potential for plant production. Symbiosis. 2012;58(1-3):51-62. https://doi.org/10.1007/s13199-013-0226-2.
  20. Штарк О.Ю., Данилова Т.Н., Наумкина Т.С., и др. Анализ исходного материала гороха посевного (Pisum sativum L.) для селекции сортов с высоким симбиотическим потенциалом и выбор параметров для его оценки // Экологическая генетика. – 2006. – Т. 4. – № 2. – С. 22–28. [Shtark OYu, Danilova TN, Naumkina TS et al. Analysis of pea (Pisum sativum L.) source material for breeding of cultivars with high symbiotic potential and choice of criteria for its evaluation. Ecological genetics. 2006;4(2): 22-28. (In Russ.)]. https://doi.org/10.17816/ecogen4222-28.
  21. Данилова Т.Н. Эффективность взаимодействия гороха (Pisum sativum L.) с комплексом полезной почвенной микрофлоры. Новый признак селекции зернобобовых культур: Автореф. дис. … канд. биол. наук. – СПб., 2011. – 18 с. [Danilova T.N. Effektivnost’ vzaimodeistviya gorokha (Pisum sativum L.) s kompleksom poleznoi pochvennoi microflory. Novyi priznak selektsii zarnobobovykh kul’tur. [dissertation abstract] Saint Petersburg; 2011. 18 p. (In Russ.)]. Доступно по: https://search.rsl.ru/ru/record/01004859442. Ссылка активна на 02.02.2020.
  22. Гарипова С.Р. Экологическая роль эндофитных бактерий в симбиозе с бобовыми растениями и их применение в растениеводстве // Успехи современной биологии. –2012. – T. 132. – № 5. – С. 493–505. [Garipova SR. The ecological role of endophytic bacteria in symbiosis with legumes and their use in plant breeding. Advances in modern biology. 2012;132(5): 493-505. (In Russ.)]
  23. Жуков В.А., Ахтемова Г.А., Жернаков А.И., и др. Симбиотическая эффективность генотипов гороха посевного (Pisum sativum L.) при моделировании в вегетационном эксперименте // Сельскохозяйственная биология. – 2017. – Т. 52. – № 3. – С. 607–614. [Zhukov VA, Akhtemova GA, Zhernakov AI, et al. Evaluation of the symbiotic effectiveness of pea (Pisum sativum L.) genotypes in pot experiment. Sel’skokhoziaistvennaia biologiia. 2017;52(3):607-614. (In Russ.)]. https://doi.org/10.15389/agrobiology.2017.3.607rus.
  24. Наумкина Т.С., Борисов А.Ю., Штарк О.Ю., и др. Использование симбиозов бобовых при создании высокоэффективных растительно-микробных систем для адаптивного растениеводства // Аграрная Россия. – 2011. – № 3. – С. 35–37. [Naumkina TS, Borisov AYu, Shtark OYu, et al. Use of symbioses of pod-bearing plants for building of highly effective plant-microbic systems for adaptive plant growing. Agrarian Russia. 2011;3:35-37. (In Russ.)]. https://doi.org/10.30906/1999-5636-2011-3-35-37.
  25. Hallmann J, Berg G. Spectrum and population dynamics of bacterial root endophytes. In: Schulz BJ, Boyle CJ, Sieber TN, eds. Microb. Root Endophytes. Part of the Soil Biology book series (SOILBIOL, vol. 9). Berlin, Heidelberg: Springer Berlin Heidelberg; 2006. Р. 15-31. https://doi.org/10.1007/3-540-33526-9_2.
  26. Wilson K. Preparation of genomic DNA from bacteria. Curr Protoc Mol Biol. 2001; Ch. 2: Unit 2.4. https://doi.org/10.1002/0471142727.mb0204s56.
  27. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1-2):203-214. https://doi.org/10.1089/10665270050081478.
  28. Берестецкий О.А. Фитотоксины почвенных микроорганизмов и их экологическая роль. Фитотоксические свойства почвенных микроорганизмов. – Л.: Изд-во ВНИИ сельскохозяйственной микробиологии, 1978. – С. 7–31. [Berestetskiy O.A. Fitotoksiny pochvennykh mikroorganizmov i ikh ekologicheskaya rol’. Fitotoksicheskiye svoystva pochvennykh mikroorganizmov. Leningrad: Izd-vo VNII sel’skokhozyaystvennoy mikrobiologii; 1978. P. 7-31. (In Russ.)]
  29. Mamontova T, Afonin AM, Ihling C, et al. Profiling of seed proteome in pea (Pisum sativum L.) lines characterized with high and low responsivity to combined inoculation with nodule bacteria and arbuscular mycorrhizal fungi. Molecules. 2019;24(8). pii: E1603. https://doi.org/10.3390/molecules24081603.
  30. López-López A, Rogel MA, Ormeño-Orrillo E, et al. Phaseolus vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. nov. Syst Appl Microbiol. 2010;33(6):322-327. https://doi.org/10.1016/j.syapm.2010.07.005.
  31. Dong Y, Iniguez AL, Triplett EW. Quantitative assessments of the host range and strain specificity of endophytic colonization by Klebsiella pneumoniae 342. Plant Soil 2003;257(1):49-59. https://doi.org/10.1023/A:1026242814060.
  32. Соловьева Е.А., Алещенкова З.М., Сафронова Г.В., и др. Микробный препарат Бактопин в технологии возделывания однолетних цветочных растений // Роль ботанических садов и дендрариев в сохранении, изучении и устойчивом использовании разнообразия растительного мира: Материалы Международной научной конференции, посвященной 85-летию Центрального ботанического сада Национальной академии наук Беларуси: в 2-х частях. – Минск, 2017. – С. 284–287. [Solovyova EA, Aleshenkova ZM, Safronova GV, et al. Application of microbial preparation Bactopin in technology of growing ornamental plants. (Conference proceedings) Rol’ botanicheskih sadov i dendrariev v sohranenii, izuchenii i ustojchivom ispol’zovanii raznoobraziya rastitel’nogo mira: Materialy Mezhdunarodnoy nauchnoy konferentsii, posvyashchennoy 85-letiyu Tsentral’nogo botanicheskogo sada Natsional’noy akademii nauk Belarusi: v 2-kh chastyakh. Minsk; 2017. P. 25-28. (In Russ.)]
  33. Malfanova N, Kamilova F, Validov S, et al. Characterization of Bacillus subtilis HC8, a novel plant-beneficial endophytic strain from giant hogweed. Microb Biotechnol. 2011;4(4):523-532. https://doi.org/10.1111/j.1751-7915.2011.00253.x.
  34. Kandel SL, Firrincieli A, Joubert PM, et al. An in vitro study of bio-control and plant growth promotion potential of salicaceae endophytes. Front Microbiol. 2017;8:386. https://doi.org/10.3389/fmicb.2017.00386.
  35. Тихонович И.А., Андронов Е.Е., Борисов А.Ю., и др. Принцип дополнительности геномов в расширении адаптационного потенциала растений // Генетика. – 2015. – Т. 51. – № 9. – С. 973. [Tikhonovich IA, Andronov EE, Borisov AYu, et al. The principle of genome complementarity in the enhancement of plant adaptive capacities. Genetika. 2015;51(9):973. (In Russ.)]. https://doi.org/10. 7868/S001667581509012X.

Supplementary files

Supplementary Files Action
1.
Fig. 1. Morphotypes of endophytic bacteria isolated from the internal tissues of the stems and leaves of peas

Download (58KB) Indexing metadata
2.
Fig. 2. Representation of phyla endophytic bacteria isolated from the stems and leaves of pea plants of various genotypes (in the case of plants of the leafless Triumph variety, stipules were studied instead of leaves)

Download (26KB) Indexing metadata
3.
Fig. 3. Representatives of the endophytic community of pea plants. The diagrams show the number of representatives of various genera of bacteria.

Download (151KB) Indexing metadata
4.
Fig. 4. The length of the root of watercress during inoculation with endophytic bacteria isolated from stems and leaves (stipules). Numbers 1–5 denote experiments, for each of which a separate control was set. The significance of differences in the length of plant roots from the control: * p <0.05; ** p <0.01; *** p <0.001, **** p <0.0001; ns - lack of significant differences

Download (103KB) Indexing metadata
5.
Fig. 5. Results of a repeated test of KV17 for growth-promoting activity. Reliability of differences in plant root length from control: ** p <0.01; **** p <0.0001; ns - lack of significant differences

Download (10KB) Indexing metadata

Statistics

Views

Abstract - 103

PDF (Russian) - 4

Cited-By


PlumX

Dimensions


Copyright (c) 2020 Vasileva E.N., Akhtemova G.A., Afonin A.M., Borisov A.Y., Tikhonovich I.A., Zhukov V.A.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies