Application of Trichoderma Hz36 and Hk37 as Biocontrol Agents against Clubroot Caused by Plasmodiophora brassicae
Abstract
:1. Introduction
2. Materials and Methods
2.1. Isolation of Fungal Strains inside the Rapeseed Root
2.2. Morphological Characterization of Strains Hz36 and Hk37
2.3. Fungus DNA Extraction, PCR and ITS Sequencing
2.4. Phylogenetic Analyses of Hz36 and Hk37 Strains
2.5. Fungal Spore Preparation
2.6. Plant Materials, P. brassicae Inoculation and Growth Conditions
2.7. Germination Assay of P. brassicae Resting Spores
2.8. Plant RNA Isolation, Plant DNA Isolation and qPCR Analysis
2.9. Microscopic Analysis
2.10. Seed Treatment
2.11. Statistical Analysis
3. Results
3.1. Identification of Strains Hz36 and Hk37
3.2. Biocontrol Effects of Hz36 and Hk37 on A. thaliana Clubroot Disease
3.3. Biocontrol Effects of Hz36 and Hk37 on Rapeseed Clubroot Disease
3.4. Inhibitory Effects of Hz36 and Hk37 on the Development of P. brassicae
3.5. Inhibitory Effects of Hz36 and Hk37 on Resting Spore Germination
3.6. Effects of Hz36 and Hk37 Strains on Rapeseed Seed Germination and Early Root Development
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Dixon, G.R. The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease. J. Plant Growth Regul. 2009, 28, 194–202. [Google Scholar] [CrossRef]
- Zheng, X.; Koopmann, B.; Ulber, B.; Von Tiedemann, A. A global survey on diseases and pests in oilseed rape—Current challenges and innovative strategies of control. Front. Agron. 2020, 2, 590908. [Google Scholar] [CrossRef]
- Botero-Ramirez, A.; Hwang, S.F.; Strelkov, S.E. Plasmodiophora brassicae inoculum density and spatial patterns at the field level and relation to soil characteristics. Pathogens 2021, 10, 499. [Google Scholar] [CrossRef]
- Struck, C.; Rusch, S.; Strehlow, B. Control strategies of clubroot disease caused by Plasmodiophora brassicae. Microorganisms 2022, 10, 620. [Google Scholar] [CrossRef] [PubMed]
- Hirai, M.; Harada, T.; Kubo, N.; Tsukada, M.; Suwabe, K.; Matsumoto, S. A novel locus for clubroot resistance in Brassica rapa and its linkage markers. Theor. Appl. Genet. 2004, 108, 639–643. [Google Scholar] [CrossRef] [PubMed]
- Rocherieux, J.; Glory, P.; Giboulot, A.; Boury, S.; Barbeyron, G.; Thomas, G.; Manzanares-Dauleux, M.J. Isolate-specific and broad-spectrum QTLs are involved in the control of clubroot in Brassica oleracea. Theor. Appl. Genet. 2004, 108, 1555–1563. [Google Scholar] [CrossRef]
- Chu, M.G.; Song, T.; Falk, K.C.; Zhang, X.G.; Liu, X.J.; Chang, A.; Lahlali, R.; McGregor, L.; Gossen, B.D.; Peng, G.; et al. Fine mapping of Rcr1 and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae. BMC Genom. 2014, 15, 1166. [Google Scholar] [CrossRef] [Green Version]
- Fähling, M.; Graf, H.; Siemens, J. Pathotype separation of Plasmodiophora brassicae by the host plant. J. Phytopathol. 2003, 151, 425–430. [Google Scholar] [CrossRef]
- Abbasi, P.A.; Lazarovits, G. Effect of soil application of AG3 phosphonate on the severity of clubroot of bok choy and cabbage caused by Plasmodiophora brassicae. Plant Dis. 2006, 90, 1517–1522. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liu, C.; Yang, Z.; He, P.; Munir, S.; Wu, Y.; Ho, H.; He, Y. Deciphering the bacterial and fungal communities in clubroot-affected cabbage rhizosphere treated with Bacillus subtilis XF-1. Agric. Ecosyst. Environ. 2018, 256, 12–22. [Google Scholar] [CrossRef]
- Mendis, H.C.; Thomas, V.P.; Schwientek, P.; Salamzade, R.; Chien, J.T.; Waidyarathne, P.; Kloepper, J.; de La Fuente, L. Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions. PLoS ONE 2018, 13, e0193119. [Google Scholar]
- Peng, G.; McGregor, L.; Lahlali, R.; Gossen, B.D.; Hwang, S.F.; Adhikari, K.K.; Strelkov, S.E.; McDonald, M.R. Potential biological control of clubroot on canola and crucifer vegetable crops. Plant Pathol. 2011, 60, 566–574. [Google Scholar] [CrossRef]
- Zhu, M.L.; He, Y.W.; Li, Y.; Ren, T.R.; Liu, H.; Huang, J.B.; Jiang, D.H.; Hsiang, T.; Zheng, L. Two new biocontrol agents against clubroot caused by Plasmodiophora brassicae. Front. Microbiol. 2020, 10, 3099. [Google Scholar] [CrossRef] [PubMed]
- Sood, M.; Kapoor, D.; Kumar, V.; Sheteiwy, M.S.; Ramakrishnan, M.; Landi, M.; Araniti, F.; Sharma, A. Trichoderma: The “Secrets” of a multitalented biocontrol agent. Plants 2020, 9, 762. [Google Scholar] [CrossRef]
- Alfiky, A.; Weisskopf, L. Deciphering Trichoderma-plant-pathogen interactions for better development of biocontrol applications. J. Fungi 2021, 7, 61. [Google Scholar] [CrossRef]
- Yu, X.X.; Zhao, Y.T.; Cheng, J.; Wang, W. Biocontrol effect of Trichoderma harzianum T4 on brassica clubroot and analysis of rhizosphere microbial communities based on T-RFLP. Biocontrol Sci. Technol. 2015, 25, 1493–1505. [Google Scholar] [CrossRef]
- Li, J.; Philp, J.; Li, J.; Wei, Y.; Li, H.; Yang, K.; Ryder, M.; Toh, R.; Zhou, Y.; Denton, M.D.; et al. Trichoderma harzianum inoculation reduces the incidence of clubroot disease in Chinese cabbage by regulating the rhizosphere microbial community. Microorganisms 2020, 8, 1325. [Google Scholar] [CrossRef]
- Turner, D.; Kovacs, W.; Kuhls, K.; Lieckfeldtc, E.; Peter, B.; Arisan-Atac, I.; Strauss, J.; Samuels, G.J.; Börner, T.; Kubicek, C.P. Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum and associated Hypocrea species. Mycol. Res. 1997, 101, 449–459. [Google Scholar]
- Carbone, I.; Kohn, L.M. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 1999, 91, 553–556. [Google Scholar] [CrossRef]
- Samuels, G.J.; Ismaiel, A. Trichoderma evansii and T. lieckfeldtiae: Two new T. hamatum-like species. Mycologia 2009, 101, 142–156. [Google Scholar] [CrossRef]
- White, T.J.; Bruns, T.; Lee, S.; Taylor, J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press: New York, NY, USA, 1990; pp. 315–322. [Google Scholar]
- Rehner, S.A. Primers for Elongation Factor 1-Alpha (EF1-Alpha). 2001. Available online: https://www.docin.com/p-1613748809.html (accessed on 15 July 2022).
- Chaverri, P.; Gazis, R.; Samuels, G.J. Trichoderma amazonicum, a new endophytic species on Hevea brasiliensis and H. guianensis from the Amazon basin. Mycologia 2011, 103, 139–151. [Google Scholar] [CrossRef] [Green Version]
- Chaverri, P.; Samuels, G.J. Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): Species with green ascospores. Stud. Mycol. 2003, 48, 1–116. [Google Scholar]
- Chaverri, P.; Samuels, G.J. Evolution of habitat preference and nutrition mode in a cosmopolitan fungal genus with evidence of interkingdom host jumps and major shifts in ecology. Evolution 2013, 7, 2823–2837. [Google Scholar] [CrossRef] [PubMed]
- Katoh, K.; Kuma, K.-I.; Toh, H.; Miyata, T. MAFFT 5: Improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005, 33, 511–518. [Google Scholar] [CrossRef] [PubMed]
- Stamatakis, A. RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 2006, 22, 2688–2690. [Google Scholar] [CrossRef]
- Asano, T.; Kageyama, K.; Hyakumachi, M. Surface disinfestation of resting spores of Plasmodiophora brassicae used to infect hairy roots of Brassica spp. Phytopathology 1999, 89, 314–319. [Google Scholar] [CrossRef] [Green Version]
- Siemens, J.; Nagel, M.; Ludwig-Muller, J.; Sacristan, M.D. The interaction of Plasmodiophora brassicae and Arabidopsis thaliana: Parameters for disease quantification and screening of mutant lines. J. Phytopathol. 2002, 150, 592–605. [Google Scholar] [CrossRef]
- Zhang, Z.L.; Li, Y.H.; Li, J.C.; Zhao, Z.G. Disease-resistance of Shaanxi Brassica napus to various Plasmodiophora brassicae. Fujian J. Agric. Sci. 2019, 34, 581–586. [Google Scholar]
- Jäschke, D.; Dugassagobena, D.; Karlovsky, P.; Vidal, S.; Ludwigmüller, J. Suppression of clubroot (Plasmodiophora brassicae) development in Arabidopsis thaliana by the endophytic fungus Acremonium alternatum. Plant Pathol. 2010, 59, 100–111. [Google Scholar] [CrossRef]
- Niwa, R.; Nomura, Y.; Osaki, M.; Ezawa, T. Suppression of clubroot disease under neutral pH caused by inhibition of spore germination of Plasmodiophora brassicae in the rhizosphere. Plant Pathol. 2008, 57, 445–452. [Google Scholar] [CrossRef]
- Chen, T.; Bi, K.; Zhao, Y.L.; Lyu, X.L.; Gao, Z.X.; Zhao, Y.; Fu, Y.P.; Cheng, J.S.; Xie, J.T.; Jiang, D.H. MAPKK inhibitor U0126 inhibits Plasmodiophora brassicae development. Phytopathology 2018, 108, 711–720. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Allen, G.C.; Flores-Vergara, M.A.; Krasnyanski, S.; Kumar, S.; Thompson, W.F. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat. Protoc. 2006, 1, 2320–2325. [Google Scholar] [CrossRef]
- Chen, T.; Bi, K.; He, Z.C.; Gao, Z.X.; Zhao, Y.; Fu, Y.P.; Cheng, J.S.; Xie, J.T.; Jiang, D.H. Arabidopsis mutant bik1 exhibits strong resistance to Plasmodiophora Brassicae. Front. Physiol. 2016, 7, 402. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Card, S.; Johnson, L.; Teasdale, S.; Caradus, J. Deciphering endophyte behaviour: The link between endophyte biology and efficacious biological control agents. Fems Microbiol. Ecol. 2016, 92, fiw114. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Van Wees, S.C.M.; Van der Ent, S.; Pieterse, C.M.J. Plant immune responses triggered by beneficial microbes. Curr. Opin. Plant Biol. 2008, 11, 443–448. [Google Scholar] [CrossRef] [Green Version]
- Lapsansky, E.R.; Milroy, A.M.; Andales, M.J.; Vivanco, J.M. Soil memory as a potential mechanism for encouraging sustainable plant health and productivity. Curr. Opin. Biotechnol. 2016, 38, 137–142. [Google Scholar] [CrossRef] [PubMed]
- Mayo, S.; Gutiérrez, S.; Gutierrez, S.; Malmierca, M.G.; Lorenzanal, A.; Campelol, M.P.; Hermosa, R.; Casquero, P.A. Influence of Rhizoctonia solani and Trichoderma spp. In growth of bean (Phaseolus vulgaris L.) and in the induction of plant defense related genes. Front. Plant Sci. 2015, 6, 685. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vitti, A.; Monaca, E.L.; Sofo, A.; Scopa, A.; Cuypers, A.; Nuzzaci, M. Beneficial effects of Trichoderma harzianum T-22 in tomato seedlings infected by cucumber mosaic virus (CMV). Biocontrol 2015, 60, 135–147. [Google Scholar] [CrossRef]
- Bae, H.; Roberts, D.P.; Lim, H.S.; Strem, M.D.; Park, S.C.; Ryu, C.M.; Melnick, R.L.; Bailey, B.A. Endophytic Trichoderma isolates from tropical environments delay disease onset and induce resistance against Phytophthora apsica in hot pepper using multiple mechanisms. Mol. Plant-Microbe Interact. 2011, 24, 336–351. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Harman, G.E.; Howell, C.R.; Viterbo, A.; Chet, I.; Lorito, M. Trichoderma species-opportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2004, 2, 43–56. [Google Scholar] [CrossRef]
- Atanasova, L.; Crom, S.L.; Gruber, S.; Coulpier, F.; Seidl-Seiboth, V.; Kubicek, C.P.; Druzhinina, I.S. Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism. BMC Genom. 2013, 14, 121–135. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Adnan, M.; Islam, W.; Shabbir, A.; Khan, K.A.; Ghramh, H.A.; Huang, Z.; Chen, H.Y.H.; Lu, G. Plant defense against fungal pathogens by antagonistic fungi with Trichoderma in focus. Microb. Pathog. 2019, 129, 7–18. [Google Scholar] [CrossRef] [PubMed]
- Gruber, S.; Seidl-Seiboth, V. Self-versus non-self: Fungal cell wall degradation in Trichoderma. Microbiology 2012, 158, 26–34. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Plaszkó, T.; Szűcs, Z.; Vasas, G.; Gonda, S. Interactions of fungi with non-isothiocyanate products of the plant glucosinolate pathway: A review on product formation, antifungal activity, mode of action and biotransformation. Phytochemistry 2022, 200, 113245. [Google Scholar] [CrossRef]
- Iula, G.; Miras-Moreno, B.; Lucini, L.; Trevisan, M. The mycorrhiza-and Trichoderma-mediated elicitation of secondary metabolism and modulation of phytohormone profile in tomato plants. Horticulturae 2021, 7, 394. [Google Scholar] [CrossRef]
- Kamble, M.V.; Joshi, S.M.; Hadimani, S.; Jogaiah, S. Biopriming with rhizosphere Trichoderma harzianum elicit protection against grapevine downy mildew disease by triggering histopathological and biochemical defense responses. Rhizosphere 2021, 19, 100398. [Google Scholar] [CrossRef]
- Maddu, S.; Ravuri, J.M. Elicitation of induced systemic resistance in groundnut (Arachis Hypogaea L.) plants by Trichoderma Spp. against Sclerotium Rolfsii. Lect. Notes Netw. Syst. 2021, 215, 343–353. [Google Scholar]
- Vinale, F.; Ghisalberti, E.L.; Sivasithamparam, K.; Marra, R.; Ritieni, A.; Ferracane, R.; Woo, S.; Lorito, M. Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens. Lett. Appl. Microbiol. 2009, 48, 705–711. [Google Scholar]
- Sessitsch, A.; Mitter, B. 21st century agriculture: Integration of plant microbiomes for improved crop production and food security. Microb. Biotechnol. 2015, 8, 32–33. [Google Scholar] [CrossRef]
- Zhao, Y.; Gao, Z.X.; Tian, B.N.; Bi, K.; Chen, T.; Liu, H.Q.; Xie, J.T.; Cheng, J.S.; Fu, Y.P.; Jiang, D.H. Endosphere microbiome comparison between symptomatic and asymptomatic roots of Brassica napus infected with Plasmodiophora brassicae. PLoS ONE 2017, 12, e0185907. [Google Scholar] [CrossRef] [Green Version]
- Zhang, J.H.; Ahmed, W.; Dai, Z.L.; Zhou, X.H.; He, Z.L.; Wei, L.F.; Ji, G.H. Microbial consortia: An engineering tool to suppress clubroot of Chinese cabbage by changing the rhizosphere bacterial community composition. Biology 2022, 11, 918. [Google Scholar] [CrossRef] [PubMed]
- Mueller, U.G.; Sachs, J.L. Engineering microbiomes to improve plant and animal health. Trends Microbiol. 2015, 23, 606–617. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mazzola, M.; Freilich, S. Prospects for biological soilborne disease control: Application of indigenous versus synthetic microbiomes. Phytopathology 2017, 107, 256–263. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mendes, R.; Garbeva, P.; Raaijmakers, J.M. The rhizosphere microbiome: Significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. Fems Microbiol. Rev. 2013, 37, 634–663. [Google Scholar] [CrossRef]
- Lebreton, L.; Guillerm-Erckelboudt, A.-Y.; Gazengel, K.; Linglin, J.; Ourry, M.; Glory, P.; Sarniguet, A.; Daval, S.; Manzanares Dauleux, M.J.; Mougel, C. Temporal dynamics of bacterial and fungal communities during the infection of Brassica rapa roots by the protist Plasmodiophora brassicae. PLoS ONE 2019, 14, e0204195. [Google Scholar] [CrossRef] [Green Version]
- Niu, B.; Wang, W.; Yuan, Z.; Sederoff, R.R.; Sederoff, H.; Chiang, V.L.; Borriss, R. Microbial interactions within multiple-strain biological control agents impact soil-borne plant disease. Front. Microbiol. 2020, 11, 585404. [Google Scholar] [CrossRef]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Zhao, Y.; Chen, X.; Cheng, J.; Xie, J.; Lin, Y.; Jiang, D.; Fu, Y.; Chen, T. Application of Trichoderma Hz36 and Hk37 as Biocontrol Agents against Clubroot Caused by Plasmodiophora brassicae. J. Fungi 2022, 8, 777. https://doi.org/10.3390/jof8080777
Zhao Y, Chen X, Cheng J, Xie J, Lin Y, Jiang D, Fu Y, Chen T. Application of Trichoderma Hz36 and Hk37 as Biocontrol Agents against Clubroot Caused by Plasmodiophora brassicae. Journal of Fungi. 2022; 8(8):777. https://doi.org/10.3390/jof8080777
Chicago/Turabian StyleZhao, Yanli, Xingfu Chen, Jiasen Cheng, Jiatao Xie, Yang Lin, Daohong Jiang, Yanping Fu, and Tao Chen. 2022. "Application of Trichoderma Hz36 and Hk37 as Biocontrol Agents against Clubroot Caused by Plasmodiophora brassicae" Journal of Fungi 8, no. 8: 777. https://doi.org/10.3390/jof8080777