1887

Abstract

Here we assessed the time course of rhizoplane colonization by the endophytic insect pathogenic fungus Metarhizium robertsii. We describe a method of quantifying root colonization of bean plants by M. robertsii using quantitative polymerase chain reaction (qPCR). Results of this method were compared to the standard plate count method using colony-forming units (c.f.u.). Both the c.f.u. and qPCR methods were used to monitor the time-course of haricot bean (Phaseolus vulgaris) colonization by a strain of M. robertsii that expresses the green fluorescent protein (ARSEF 2575-GFP) for colony verification. There was a strong correlation between the results of the c.f.u. and qPCR methods, indicating that both methods are well suited for the determination of colonization of P. vulgaris roots by M. robertsii. Primers for a catalase gene (cat) amplified DNA from M. robertsii, M. brunneum and M. guizhouense. Primers for a nitrogen response-regulator (nrr) additionally detected M. acridum and M. flavoviride, whereas Metarhizium perilipin-like protein (mpl) primers were specific to M. robertsii alone. However, cat was the only target that specifically amplified Metarhizium in experiments utilizing non-sterile soil. Endophytic colonization of P. vulgaris at 60 days post-inoculation with M. robertsii was detected from surface-sterilized roots with more sensitivity using our qPCR technique over the c.f.u. method. Our results suggest that there is a prolonged period of rhizoplane colonization by Metarhizium with transient, low-level endophytic colonization of the root system of P. vulgaris that persists for the entirety of the plant life cycle.

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2018-10-12
2019-12-08
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References

  1. Shah PA, Pell JK. Entomopathogenic fungi as biological control agents. Appl Microbiol Biotechnol 2003;61:413–423 [CrossRef][PubMed]
    [Google Scholar]
  2. Gao Q, Jin K, Ying SH, Zhang Y, Xiao G et al. Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet 2011;7:e1001264 [CrossRef][PubMed]
    [Google Scholar]
  3. Meyling NV, Eilenberg J. Ecology of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in temperate agroecosystems: Potential for conservation biological control. Biological Control 2007;43:145–155 [CrossRef]
    [Google Scholar]
  4. Vega FE, Goettel MS, Blackwell M, Chandler D, Jackson MA et al. Fungal entomopathogens: new insights on their ecology. Fungal Ecol 2009;2:149–159 [CrossRef]
    [Google Scholar]
  5. Behie SW, Padilla-Guerrero IE, Bidochka MJ. Nutrient transfer to plants by phylogenetically diverse fungi suggests convergent evolutionary strategies in rhizospheric symbionts. Commun Integr Biol 2013;6:e2232122329 [CrossRef][PubMed]
    [Google Scholar]
  6. Sasan RK, Bidochka MJ. The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development. Am J Bot 2012;99:101–107 [CrossRef][PubMed]
    [Google Scholar]
  7. Behie SW, Zelisko PM, Bidochka MJ. Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science 2012;336:1576–1577 [CrossRef][PubMed]
    [Google Scholar]
  8. Brundrett M. Understanding the roles of multifunctional mycorrhizal and endophytic fungi. Microb Root Endophytes 2006;9:387
    [Google Scholar]
  9. Liao X, O'Brien TR, Fang W, St Leger RJ. The plant beneficial effects of Metarhizium species correlate with their association with roots. Appl Microbiol Biotechnol 2014;98:7089–7096 [CrossRef][PubMed]
    [Google Scholar]
  10. Khan AL, Hamayun M, Khan SA, Kang SM, Shinwari ZK et al. Pure culture of Metarhizium anisopliae LHL07 reprograms soybean to higher growth and mitigates salt stress. World J Microbiol Biotechnol 2012;28:1483–1494 [CrossRef][PubMed]
    [Google Scholar]
  11. Sasan RK, Bidochka MJ. Antagonism of the endophytic insect pathogenic fungus Metarhizium robertsii against the bean plant pathogen Fusarium solani f. sp. phaseoli. Can J Plant Pathol 2013;35:288–293 [CrossRef]
    [Google Scholar]
  12. Behie SW, Bidochka MJ. Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect-pathogenic fungi: an additional branch of the soil nitrogen cycle. Appl Environ Microbiol 2014;80:1553–1560 [CrossRef][PubMed]
    [Google Scholar]
  13. Lovett B, St Leger RJ. Stress is the rule rather than the exception for Metarhizium. Curr Genet 2015;61:253–261 [CrossRef][PubMed]
    [Google Scholar]
  14. Behie SW, Jones SJ, Bidochka MJ. Plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and Beauveria. Fungal Ecol 2015;13:112–119 [CrossRef]
    [Google Scholar]
  15. Porras-Alfaro A, Bayman P. Hidden fungi, emergent properties: endophytes and microbiomes. Annu Rev Phytopathol 2011;49:291–315 [CrossRef][PubMed]
    [Google Scholar]
  16. Maciá-Vicente JG, Jansson HB, Talbot NJ, Lopez-Llorca LV. Real-time PCR quantification and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusarium equiseti and Pochonia chlamydosporia. New Phytol 2009;182:213–228 [CrossRef][PubMed]
    [Google Scholar]
  17. Tellenbach C, Grünig CR, Sieber TN. Suitability of quantitative real-time PCR to estimate the biomass of fungal root endophytes. Appl Environ Microbiol 2010;76:5764–5772 [CrossRef][PubMed]
    [Google Scholar]
  18. Landa BB, López-Díaz C, Jiménez-Fernández D, Montes-Borrego M, Muñoz-Ledesma FJ et al. In-planta detection and monitorization of endophytic colonization by a Beauveria bassiana strain using a new-developed nested and quantitative PCR-based assay and confocal laser scanning microscopy. J Invertebr Pathol 2013;114:128–138 [CrossRef][PubMed]
    [Google Scholar]
  19. Wen K, Seguin P, St-Arnaud M, Jabaji-Hare S. Real-Time Quantitative RT-PCR of Defense-Associated Gene Transcripts of Rhizoctonia solani-Infected Bean Seedlings in Response to Inoculation with a Nonpathogenic Binucleate Rhizoctonia Isolate. Phytopathology 2005;95:345–353 [CrossRef][PubMed]
    [Google Scholar]
  20. Huggett JF, Foy CA, Benes V, Emslie K, Garson JA et al. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem 2013;59:892–902 [CrossRef][PubMed]
    [Google Scholar]
  21. Greenfield M, Gómez-Jiménez MI, Ortiz V, Vega FE, Kramer M et al. Beauveria bassiana and Metarhizium anisopliae endophytically colonize cassava roots following soil drench inoculation. Biol Control 2016;95:40–48 [CrossRef][PubMed]
    [Google Scholar]
  22. Ékk F, Keyser CA, Rangel DEN, Foster RN, Roberts DW. CTC medium: a novel dodine-free selective medium for isolating entomopathogenic fungi, especially Metarhizium acridum, from soil. Biol Control 2010;54:197–205
    [Google Scholar]
  23. Healey A, Furtado A, Cooper T, Henry RJ. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods 2014;10:21–28 [CrossRef][PubMed]
    [Google Scholar]
  24. Mutune B, Ekesi S, Niassy S, Matiru V, Bii C et al. Fungal endophytes as promising tools for the management of bean stem maggot Ophiomyia phaseoli on beans Phaseolus vulgaris. J Pest Sci 2016;89:993–1001 [CrossRef]
    [Google Scholar]
  25. Dutta P, Kaushik H, Bhawmick P, Puzari KC, Hazarika GN. Metarhizium anisopliae as endophyte has the ability of plant growth enhancement. Int J Curr Res 2015;7:14300–14304
    [Google Scholar]
  26. Golo PS, Gardner DR, Grilley MM, Takemoto JY, Krasnoff SB et al. Production of destruxins from Metarhizium spp. fungi in artificial medium and in endophytically colonized cowpea plants. PLoS One 2014;9:e104946 [CrossRef][PubMed]
    [Google Scholar]
  27. Kaushik H, Dutta P. Establishment of Metarhizium anisopliae, an entomopathogen as endophyte for biological control in tea. Res Crop 2016;17:375–387 [CrossRef]
    [Google Scholar]
  28. Wyrebek M, Huber C, Sasan RK, Bidochka MJ. Three sympatrically occurring species of Metarhizium show plant rhizosphere specificity. Microbiology 2011;157:2904–2911 [CrossRef][PubMed]
    [Google Scholar]
  29. Pava-Ripoll M, Angelini C, Fang W, Wang S, Posada FJ et al. The rhizosphere-competent entomopathogen Metarhizium anisopliae expresses a specific subset of genes in plant root exudate. Microbiology 2011;157:47–55 [CrossRef][PubMed]
    [Google Scholar]
  30. Keyser CA, de Fine Licht HH, Steinwender BM, Meyling NV. Diversity within the entomopathogenic fungal species Metarhizium flavoviride associated with agricultural crops in Denmark. BMC Microbiol 2015;15:249 [CrossRef][PubMed]
    [Google Scholar]
  31. Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S et al. The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 2015;79:293–320 [CrossRef][PubMed]
    [Google Scholar]
  32. Hyde KD, Soytong K. The fungal endophyte dilemma. Fungal Divers 2008;33:e173
    [Google Scholar]
  33. McKinnon AC. Plant Tissue Preparation for the Detection of an Endophytic Fungus In Planta. Methods Mol Biol 2016;1477:167–173 [CrossRef][PubMed]
    [Google Scholar]
  34. Ment D, Churchill AC, Gindin G, Belausov E, Glazer I et al. Resistant ticks inhibit Metarhizium infection prior to haemocoel invasion by reducing fungal viability on the cuticle surface. Environ Microbiol 2012;14:1570–1583 [CrossRef][PubMed]
    [Google Scholar]
  35. Zamioudis C, Pieterse CM. Modulation of host immunity by beneficial microbes. Mol Plant Microbe Interact 2012;25:139–150 [CrossRef][PubMed]
    [Google Scholar]
  36. Mwajita MR, Murage H, Tani A, Kahangi EM. Evaluation of rhizosphere, rhizoplane and phyllosphere bacteria and fungi isolated from rice in Kenya for plant growth promoters. Springerplus 2013;2:606–609 [CrossRef][PubMed]
    [Google Scholar]
  37. Morgan JA, Bending GD, White PJ. Biological costs and benefits to plant-microbe interactions in the rhizosphere. J Exp Bot 2005;56:1729–1739 [CrossRef][PubMed]
    [Google Scholar]
  38. Smith SE, Read D. Mycorrhizal Symbiosis, 3rd ed. New York: Academic Press; 2008
    [Google Scholar]
  39. Lareen A, Burton F, Schäfer P. Plant root-microbe communication in shaping root microbiomes. Plant Mol Biol 2016;90:575–587 [CrossRef][PubMed]
    [Google Scholar]
  40. Kobae Y, Fujiwara T. Earliest colonization events of Rhizophagus irregularis in rice roots occur preferentially in previously uncolonized cells. Plant Cell Physiol 2014;55:1497–1510 [CrossRef][PubMed]
    [Google Scholar]
  41. Rodriguez RJ, White JF, Arnold AE, Redman RS. Fungal endophytes: diversity and functional roles. New Phytol 2009;182:314–330 [CrossRef][PubMed]
    [Google Scholar]
  42. Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM et al. Trichoderma: the genomics of opportunistic success. Nat Rev Microbiol 2011;9:749–759 [CrossRef][PubMed]
    [Google Scholar]
  43. Elena GJ, Beatriz PJ, Alejandro P, Lr E. Metarhizium anisopliae (Metschnikoff) Sorokin promotes growth and has endophytic activity in tomato plants. Adv Biol Res 2011;5:22–27
    [Google Scholar]
  44. Pasche JS, Mallik I, Anderson NR, Gudmestad NC. Development and validation of a real-time pcr assay for the quantification of Verticillium dahliae in potato. Plant Dis 2013;97:608–618 [CrossRef]
    [Google Scholar]
  45. Alkan N, Gadkar V, Coburn J, Yarden O, Kapulnik Y. Quantification of the arbuscular mycorrhizal fungus Glomus intraradices in host tissue using real-time polymerase chain reaction. New Phytol 2004;161:877–885 [CrossRef]
    [Google Scholar]
  46. Schneider S, Rehner SA, Widmer F, Enkerli J. A PCR-based tool for cultivation-independent detection and quantification of Metarhizium clade 1. J Invertebr Pathol 2011;108:106–114 [CrossRef][PubMed]
    [Google Scholar]
  47. Entz SC, Johnson DL, Kawchuk LM. Development of a PCR-based diagnostic assay for the specific detection of the entomopathogenic fungus Metarhizium anisopliae var. acridum. Mycol Res 2005;109:1302–1312 [CrossRef][PubMed]
    [Google Scholar]
  48. Bell AS, Blanford S, Jenkins N, Thomas MB, Read AF. Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: technique validation and first applications. J Invertebr Pathol 2009;100:160–168 [CrossRef][PubMed]
    [Google Scholar]
  49. Guo L-D. Molecular diversity and identification of endophytic fungi. Molecular Identification of Fungi Berlin, Heidelberg: Springer; 2010; pp.277–296
    [Google Scholar]
  50. Burgdorf RJ, Laing MD, Morris CD, Jamal-Ally SF. A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies. Braz J Microbiol 2014;45:977–983 [CrossRef][PubMed]
    [Google Scholar]
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