Penicillin Trunk Injection Affects Bacterial Community Structure in Citrus Trees


Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), an uncultured α-proteobacterium, is the most destructive disease of citrus trees worldwide. In previous studies, trunk injections of penicillin reduced CLas titers and HLB symptoms in citrus. However, antibiotic effects on the whole plant microbial community, which include effects on taxa that interact with CLas, have not yet been addressed. In this study, we investigated the effects of penicillin injection (0, 1000, and 6000 mg L−1) on rhizospheric and endophytic bacterial communities of grapefruit trees in field and greenhouse experiments through culture-independent high-throughput sequencing. DNA extractions from petioles and roots were subjected to 16S rRNA high-throughput sequencing, and reads were clustered by sequence similarity into operational taxonomic units (OTUs). Principal coordinates analysis based on weighted-UniFrac distances did not reveal differences in bacterial communities among treatments in any of the sample sources. However, pairwise linear discriminant analysis indicated significant differences in relative abundance of some taxa (including CLas) among treatments. Network analysis showed that penicillin produced major changes in root bacterial community structure by affecting interspecific microbial associations. This study provides new knowledge of the effect of antimicrobial treatments on interspecific relationships in citrus microbial communities.

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  1. 1.

    Bové JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 88(1):7–37

    Google Scholar 

  2. 2.

    Farnsworth D, Grogan KA, van Bruggen AH, Moss CB (2014) The potential economic cost and response to greening in Florida citrus. Choices 29:1–6

    Google Scholar 

  3. 3.

    da Graça JV, Douhan GW, Halbert SE, Keremane ML, Lee RF, Vidalakis G, Zhao H (2016) Huanglongbing: an overview of a complex pathosystem ravaging the world’s citrus. J. Integr. Plant Biol. 58:373–387.

    Article  PubMed  Google Scholar 

  4. 4.

    Hodges AW, Spreen TH (2012) Economic impacts of citrus greening (HLB) in Florida, 2006/7–2010/11. University of Florida Department of Food and Resource Economics

  5. 5.

    NASS (2016) United States Department of Agriculture. Agricultural Marketing Service. National Agricultural Statistics Service. 2015–2016. Florida Citrus Statistics

  6. 6.

    Shimwela MM, Narouei-Khandan HA, Halbert SE, Keremane ML, Minsavage GV, Timilsina S, Massawe DP, Jones JB, van Bruggen AHC (2016) First occurrence of Diaphorina citri in East Africa, characterization of the Ca. Liberibacter species causing huanglongbing (HLB) in Tanzania, and potential further spread of D. citri and HLB in Africa and Europe. Eur. J. Plant Pathol. 146:349–368.

    Article  Google Scholar 

  7. 7.

    Boina DR, Bloomquist JR (2015) Chemical control of the Asian citrus psyllid and of huanglongbing disease in citrus. Pest Manag. Sci. 71:808–823.

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Shen W, Halbert SE, Dickstein E et al (2013) Occurrence and in-grove distribution of citrus huanglongbing in north central Florida. J. Plant Pathol.:361–371

  9. 9.

    Shen W, Cevallos-Cevallos JM, Da Rocha UN et al (2013) Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with Huanglongbing. Eur. J. Plant Pathol. 137:727–742

    Article  CAS  Google Scholar 

  10. 10.

    McManus PS, Stockwell VO, Sundin GW, Jones AL (2002) Antibiotic use in plant agriculture. Annu. Rev. Phytopathol. 40:443–465.

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    EPA (2013) Streptomycin; pesticide tolerances for emergency exemptions. Environmental Protection Agency 40 CFR Part 180 [EPA–HQ–OPP–2011–0852; FRL–9385–3]. Federal Register 78 (96):29049

  12. 12.

    Graham JH, Dewdney MM, Myers ME (2010) Streptomycin and copper formulations for control of citrus canker on grapefruit. Proc Fla State Hortic Soc 123:92–99

    Google Scholar 

  13. 13.

    Zhang M, Guo Y, Powell CA, Doud MS, Yang C, Duan Y (2014) Effective antibiotics against ‘Candidatus Liberibacter asiaticus’ in HLB-affected citrus plants identified via the graft-based evaluation. PLoS One 9:e111032.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Zhang M, Powell CA, Guo Y, Benyon L, Duan Y (2013) Characterization of the microbial community structure in Candidatus Liberibacter asiaticus-infected citrus plants treated with antibiotics in the field. BMC Microbiol. 13:112.

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Zhang M, Powell CA, Benyon LS, Zhou H, Duan Y (2013) Deciphering the bacterial microbiome of citrus plants in response to Candidatus Liberibacter asiaticus-infection and antibiotic treatments. PLoS One 8:e76331.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Hu J, Jiang J, Wang N (2018) Control of citrus huanglongbing via trunk injection of plant defense activators and antibiotics. Phytopathology 108:186–195.

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Buchholz A, Baur P, Schönherr J (1998) Differences among plant species in cuticular permeabilities and solute mobilities are not caused by differential size selectivities. Planta 206:322–328

    Article  CAS  Google Scholar 

  18. 18.

    Buchholz A, Schönherr J (2000) Thermodynamic analysis of diffusion of non-electrolytes across plant cuticles in the presence and absence of the plasticiser tributyl phosphate. Planta 212:103–111

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Kumar K, C. Gupta S, Chander Y, Singh AK (2005) Antibiotic use in agriculture and its impact on the terrestrial environment. In: Advances in agronomy. Elsevier, pp 1–54

  20. 20.

    Stockwell VO, Duffy B (2012) Use of antibiotics in plant agriculture. Rev Sci Tech Int Off Epizoot 31:199–210

    Article  CAS  Google Scholar 

  21. 21.

    Chung K, Zhisheng W (1991) A study on the use of antibiotics to control citrus huanglongbing. Proceedings of the 6th international Asia Pacific Workshop on Integrated Health Management. FAO, Rome, pp 40–48

    Google Scholar 

  22. 22.

    Aćimović SG, Zeng Q, McGhee GC et al (2015) Control of fire blight (Erwinia amylovora) on apple trees with trunk-injected plant resistance inducers and antibiotics and assessment of induction of pathogenesis-related protein genes. Front. Plant Sci. 6.

  23. 23.

    Poudel R, Jumpponen A, Schlatter DC, Paulitz TC, Gardener BBMS, Kinkel LL, Garrett KA (2016) Microbiome networks: a systems framework for identifying candidate microbial assemblages for disease management. Phytopathology 106:1083–1096.

    Article  CAS  PubMed  Google Scholar 

  24. 24.

    Franklin AM, Aga DS, Cytryn E, Durso LM, McLain JE, Pruden A, Roberts MC, Rothrock MJ, Snow DD, Watson JE, Dungan RS (2016) Antibiotics in agroecosystems: introduction to the special section. J. Environ. Qual. 45:377.

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Shin K, Ascunce MS, Narouei-Khandan HA, Sun X, Jones D, Kolawole OO, Goss EM, van Bruggen AHC (2016) Effects and side effects of penicillin injection in huanglongbing affected grapefruit trees. Crop Prot. 90:106–116.

    Article  CAS  Google Scholar 

  26. 26.

    Grünwald NJ, Hu S, van Bruggen AHC (2000) Short-term cover crop decomposition in organic and conventional soils: characterization of soil C, N, microbial and plant pathogen dynamics. Eur J Plant Pathol 106:37–50.

  27. 27.

    Zelenev VV, van Bruggen AH, Semenov AM (2000) “BACWAVE,” a spatial-temporal model for traveling waves of bacterial populations in response to a moving carbon source in soil. Microb. Ecol. 40:260–272

    CAS  PubMed  Google Scholar 

  28. 28.

    Zelenev VV, van Bruggen AHC, Semenov AM (2005) Short-term wavelike dynamics of bacterial populations in response to nutrient input from fresh plant residues. Microb. Ecol. 49:83–93.

    Article  CAS  PubMed  Google Scholar 

  29. 29.

    Yocum RR, Rasmussen JR, Strominger JL (1980) The mechanism of action of penicillin: penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase. J. Biol. Chem. 255:3977–3986

    CAS  PubMed  Google Scholar 

  30. 30.

    Zhang Q, Dick WA (2014) Growth of soil bacteria, on penicillin and neomycin, not previously exposed to these antibiotics. Sci. Total Environ. 493:445–453.

    Article  CAS  PubMed  Google Scholar 

  31. 31.

    Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U. S. A. 108(Suppl 1):4516–4522.

    Article  PubMed  Google Scholar 

  32. 32.

    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7:335–336.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Rideout JR, He Y, Navas-Molina JA, Walters WA, Ursell LK, Gibbons SM, Chase J, McDonald D, Gonzalez A, Robbins-Pianka A, Clemente JC, Gilbert JA, Huse SM, Zhou HW, Knight R, Caporaso JG (2014) Subsampled open-reference clustering creates consistent, comprehensive OTU definitions and scales to billions of sequences. PeerJ 2:e545.

    Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Lozupone CA, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 71:8228–8235.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Lozupone CA, Hamady M, Kelley ST, Knight R (2007) Quantitative and qualitative diversity measures lead to different insights into factors that structure microbial communities. Appl. Environ. Microbiol. 73:1576–1585.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C (2011) Metagenomic biomarker discovery and explanation. Genome Biol. 12:R60.

    Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Friedman J, Alm EJ (2012) Inferring correlation networks from genomic survey data. PLoS Comput. Biol. 8:e1002687.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Csardi G, Nepusz T (2006) The igraph software package for complex network research. InterJournal Complex Syst 1695:1–9

    Google Scholar 

  39. 39.

    Core Team R (2016) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  40. 40.

    Nguyen NH, Smith D, Peay K, Kennedy P (2015) Parsing ecological signal from noise in next generation amplicon sequencing. New Phytol. 205:1389–1393.

    Article  CAS  PubMed  Google Scholar 

  41. 41.

    van der Heijden MGA, Hartmann M (2016) Networking in the plant microbiome. PLoS Biol. 14:e1002378.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Trivedi P, Duan Y, Wang N (2010) Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots. Appl. Environ. Microbiol. 76:3427–3436.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Gourion B, Rossignol M, Vorholt JA (2006) A proteomic study of Methylobacterium extorquens reveals a response regulator essential for epiphytic growth. Proc. Natl. Acad. Sci. 103:13186–13191.

    Article  CAS  PubMed  Google Scholar 

  44. 44.

    Meena KK, Sorty AM, Bitla UM, Choudhary K, Gupta P, Pareek A, Singh DP, Prabha R, Sahu PK, Gupta VK, Singh HB, Krishanani KK, Minhas PS (2017) Abiotic stress responses and microbe-mediated mitigation in plants: the omics strategies. Front. Plant Sci. 8.

  45. 45.

    Boutin S, Bernatchez L, Audet C, Derôme N (2013) Network analysis highlights complex interactions between pathogen, host and commensal microbiota. PLoS One 8:e84772.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Kyselková M, Kopecký J, Frapolli M, Défago G, Ságová-Marečková M, Grundmann GL, Moënne-Loccoz Y (2009) Comparison of rhizobacterial community composition in soil suppressive or conducive to tobacco black root rot disease. ISME J 3:1127–1138.

    Article  PubMed  Google Scholar 

  47. 47.

    Adam M, Westphal A, Hallmann J, Heuer H (2014) Specific microbial attachment to root knot nematodes in suppressive soil. Appl. Environ. Microbiol. 80:2679–2686.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Madhaiyan M, Poonguzhali S, Lee J-S, Lee KC, Saravanan VS, Santhanakrishnan P (2010) Microbacterium azadirachtae sp. nov., a plant-growth-promoting actinobacterium isolated from the rhizoplane of neem seedlings. Int. J. Syst. Evol. Microbiol. 60:1687–1692.

    Article  CAS  PubMed  Google Scholar 

  49. 49.

    Madigan MT, Martinko JM, Parker J (1997) Brock biology of microorganisms. Prentice Hall, Upper Saddle River

    Google Scholar 

  50. 50.

    Gartemann K-H, Abt B, Bekel T, Burger A, Engemann J, Flugel M, Gaigalat L, Goesmann A, Grafen I, Kalinowski J, Kaup O, Kirchner O, Krause L, Linke B, McHardy A, Meyer F, Pohle S, Ruckert C, Schneiker S, Zellermann EM, Puhler A, Eichenlaub R, Kaiser O, Bartels D (2008) The genome sequence of the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis ncppb382 reveals a large island involved in pathogenicity. J. Bacteriol. 190:2138–2149.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Zhang L, Xu Z (2008) Assessing bacterial diversity in soil. J. Soils Sediments 8:379–388

    Article  CAS  Google Scholar 

  52. 52.

    Bergmann G, Bates ST, Eilers KG et al (2011) The under-recognized dominance of Verrucomicrobia in soil bacterial communities. Soil Biol. Biochem. 43:1450–1455.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Dubourg G, Lagier J-C, Armougom F, Robert C, Audoly G, Papazian L, Raoult D (2013) High-level colonisation of the human gut by Verrucomicrobia following broad-spectrum antibiotic treatment. Int. J. Antimicrob. Agents 41:149–155.

    Article  CAS  PubMed  Google Scholar 

  54. 54.

    Choudhary DK, Johri BN (2009) Interactions of Bacillus spp. and plants—with special reference to induced systemic resistance (ISR). Microbiol. Res. 164:493–513.

    Article  CAS  PubMed  Google Scholar 

  55. 55.

    Yang C, Powell CA, Duan Y, Shatters R, Fang J, Zhang M (2016) Deciphering the bacterial microbiome in huanglongbing-affected citrus treated with thermotherapy and sulfonamide antibiotics. PLoS One 11:e0155472.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    He J, Sun J, Deem MW (2009) Spontaneous emergence of modularity in a model of evolving individuals and in real networks. Phys. Rev. E 79:031907.

    Article  CAS  Google Scholar 

  57. 57.

    Earl DJ, Deem MW (2004) Evolvability is a selectable trait. Proc. Natl. Acad. Sci. 101:11531–11536.

    Article  CAS  PubMed  Google Scholar 

  58. 58.

    Lorenz DM, Jeng A, Deem MW (2011) The emergence of modularity in biological systems. Phys Life Rev 8:129–160.

    Article  PubMed  PubMed Central  Google Scholar 

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This research was funded by the Florida Department of Agriculture and Consumer services; additional funding was provided by the University of Florida (UF) IFAS Research Early Career Seed Fund, the UF Emerging Pathogens Institute, the UF Department of Plant Pathology, and the Esther B. O’Keeffe Foundation. We are grateful to Robert H. Moore of the Pesticide Registration Review Section, Bureau of Scientific Evaluation and Technical Assistance, Division of Agricultural Environmental Services, Florida Department of Agriculture and Consumer Services, Tallahassee, FL, for providing the exemption to use penicillin in an experimental field, and in a BSL2 greenhouse and laboratory. Hossein A. Narouei-Khandan, Oluwaseun Olawale Kolawole, Cody Neff, Hannah Fahsbender, Ellen Dickstein, and Yorley Nikary Bustamante participated in collecting and processing citrus samples. Mark G. Kann and Jim A. Boyer took care of the young citrus trees in the greenhouse at Citra. We wish to thank the UF Plant Pathology Department for providing support for greenhouse experiments, especially Mike Stilwell and his team. We are also grateful to the High Performance Computing Center and the HiPerGator cluster support staff for their assistance. We want to thank the editor and the reviewers for comments on an earlier version of the manuscript.

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Correspondence to Marina S. Ascunce.

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Ascunce, M.S., Shin, K., Huguet-Tapia, J.C. et al. Penicillin Trunk Injection Affects Bacterial Community Structure in Citrus Trees. Microb Ecol 78, 457–469 (2019).

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  • Citrus
  • Huanglongbing
  • Microbiomes
  • 16S rRNA
  • Antibiotics
  • Networks