Endophytic Microorganisms Associated with Reversion of Silverleaf Disease Symptoms in Apple
- D. Grinbergs1 2 †
- J. Chilian1 †
- N. Padilla2
- M. Reyes1
- A. France1
- E. Moya-Elizondo2
- M. Gerding2
- 1Instituto de Investigaciones Agropecuarias, INIA, Chillán, Chile
- 2Universidad de Concepción, Chillán, Chile
Abstract
Silverleaf is caused by the fungus Chondrostereum purpureum, which produces wood necrosis and foliar silvering in woody plants. Field observations and studies in apple have shown the reversion of foliar symptoms. Because plants were clones and received identical agronomical management, it was hypothesized that reversion is driven by endophytic microbiota. Thus, the objectives of this study were to compare healthy, diseased, and reverted plants with respect to their physiology, endophytic microbial communities, antagonistic ability of their endophytes against C. purpureum, and defense genes expression. Water potential, stomatal conductance, chlorophyll content, and fluorescence were measured. Endophytic bacterial and fungal DNA were analyzed by denaturing gradient gel electrophoresis, and community richness and similarity were calculated. Wood cores were collected and bacterial and fungal endophytes were isolated and confronted with C. purpureum-virulent strains in dual-culture assays. Defense genes expression was measured by quantitative PCR. Results indicated that there were no differences in physiological parameters between healthy and reverted plants, except for fluorescence, and both type of plants differed from diseased ones. Bacterial and fungal community richness was similar in healthy and reverted plants and higher than in diseased ones. Endophytes from reverted and healthy plants showed high antagonism to C. purpureum. Furthermore, nonexpressor of pathogenesis-related gene 1 expression was upregulated in reverted plants, whereas phenylalanine ammonia lyase and polygalacturonase-inhibiting protein genes showed higher values in diseased plants. Overall, physiological, molecular, and microbial characteristics were similar between healthy and reverted plants, and both differed from diseased ones. Therefore, reversion of symptoms is associated with changes in the endophytic microbiota, which seems to be a promising source of biological control agents against C. purpureum.
LITERATURE CITED
- 2002. Population biology of Aureobasidium pullulans on apple leaf surfaces. Can. J. Microbiol. 48:500-513. https://doi.org/10.1139/w02-044 Crossref, Medline, ISI, Google Scholar
- 2003. Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia 95:388-398. https://doi.org/10.1080/15572536.2004.11833083 Crossref, Medline, ISI, Google Scholar
- 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology 88:541-549. https://doi.org/10.1890/05-1459 Crossref, Medline, ISI, Google Scholar
- 2019. The nonexpressor of pathogenesis-related genes 1 (NPR1) and related family: Mechanistic insights in plant disease resistance. Plant Sci. 10:102. https://doi.org/10.3389/fpls.2019.00102 ISI, Google Scholar
- 2008. Antibiosis, mycoparasitism, and colonization success for endophytic Trichoderma isolates with biological control potential in Theobroma cacao. Biol. Control 46:24-35. https://doi.org/10.1016/j.biocontrol.2008.01.003 Crossref, ISI, Google Scholar
- 1999. PCR based genetic markers for detection and infection frequency analysis of the biocontrol fungus Chondrostereum purpureum on Stika alder and trembling aspen. Biol. Control 15:71-80. https://doi.org/10.1006/bcon.1999.0693 Crossref, ISI, Google Scholar
- 2015. Bacteria in a wood fungal disease: characterization of bacterial communities in wood tissues of esca-foliar symptomatic and asymptomatic grapevines. Front. Microbiol. 6:1137. https://doi.org/10.3389/fmicb.2015.01137 Crossref, Medline, ISI, Google Scholar
- 2014. Analyses of the temporal dynamics of fungal communities colonizing the healthy wood tissues of esca leaf-symptomatic and asymptomatic vines. PLoS One 9:e95928. https://doi.org/10.1371/journal.pone.0095928 Crossref, Medline, ISI, Google Scholar
- 2006.
Understanding the roles of multifunctional mycorrhizal and endophytic fungi . Pages 281-298 in: Microbial Root Endophytes. B. J. E. Schulz, C. J. C. Boyle, and T. N. Sieber, eds. Springer-Verlag, Berlin, Germany. https://doi.org/10.1007/3-540-33526-9_16 Crossref, Google Scholar - 2012. Endophytic bacterial community living in roots of healthy and ‘Candidatus Phytoplasma mali’-infected apple (Malus domestica, Borkh.) trees. Antonie van Leeuwenhoek 102:677-687. https://doi.org/10.1007/s10482-012-9766-3 Crossref, Medline, ISI, Google Scholar
- 2009. Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR. J. Microbiol. 47:393-401. https://doi.org/10.1007/s12275-009-0082-1 Crossref, Medline, ISI, Google Scholar
- 1996. Preliminary screening of apple germplasm for resistance to silverleaf infection. N. Z. J. Crop Hortic. Sci. 24:1-6. https://doi.org/10.1080/01140671.1996.9513928 Crossref, ISI, Google Scholar
- 2015. Fungal endophytes: Modifiers of plant disease. Plant Mol. Biol. 90:645-655. https://doi.org/10.1007/s11103-015-0412-0 Crossref, Medline, ISI, Google Scholar
- 2016. Biological control of tree and woody plant diseases: an impossible task? BioControl 61:233-242. https://doi.org/10.1007/s10526-016-9737-0 Crossref, ISI, Google Scholar
- 2005. Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71:4951-4959. https://doi.org/10.1128/AEM.71.9.4951-4959.2005 Crossref, Medline, ISI, Google Scholar
- 2005. Genome wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139:5-17. https://doi.org/10.1104/pp.105.063743 Crossref, Medline, ISI, Google Scholar
- 1994. Conidiogenesis, nutritional physiology and taxonomy of Aureobasidium and Hormonema. Antonie van Leeuwenhoek 65:41-54. https://doi.org/10.1007/BF00878278 Crossref, Medline, ISI, Google Scholar
- 2018. Effect of Aureobasidium pullulans strains against Botrytis cinerea on kiwifruit during storage and on fruit nutritional composition. Food Microbiol. 72:67-72. https://doi.org/10.1016/j.fm.2017.11.010 Crossref, Medline, ISI, Google Scholar
- 2017. Isolation of a potential biocontrol agent Paenibacillus polymyxa NSY50 from vinegar waste compost and its induction of host defense responses against Fusarium wilt of cucumber. Microbiol. Res. 202:1-10. https://doi.org/10.1016/j.micres.2017.04.013 Crossref, Medline, ISI, Google Scholar
- 2016. Grapevine (Vitis vinifera) crown galls host distinct microbiota. Appl. Environ. Microbiol. 82:5542-5552. https://doi.org/10.1128/AEM.01131-16 Crossref, Medline, ISI, Google Scholar
- 2013. Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development. Front. Plant Sci. 4:1-9. https://doi.org/10.3389/fpls.2013.00049 Crossref, Medline, ISI, Google Scholar
- 2015. The effects of grapevine trunk diseases (GTDs) on vine physiology. Eur. J. Plant Pathol. 144:707-721. https://doi.org/10.1007/s10658-015-0770-0 Crossref, ISI, Google Scholar
- 2017. First detection of silverleaf (Chondrostereum purpureum) on rabbiteye blueberry (Vaccinium ashei) and disease damages. Acta Hortic. 1180:277-282. https://doi.org/10.17660/ActaHortic.2017.1180.37 Crossref, Google Scholar
- 2018. Incidence, identification, and mycoparasitic activity of Clonostachys epichloe, a hyperparasite of the fungal endophyte Epichloe typhyna. Plant Dis. 102:1973-1980. https://doi.org/10.1094/PDIS-02-18-0320-RE Link, ISI, Google Scholar
- 2020. A PCR-based method for the rapid detection of Chondrostereum purpureum in apple. Plant Dis. 104:702-707. https://doi.org/10.1094/PDIS-10-19-2086-RE Link, ISI, Google Scholar
- 2019. First report of silverleaf disease caused by Chondrostereum purpureum on murta (Ugni molinae Turcz.) in Chile. Plant Dis. 103:2140. https://doi.org/10.1094/PDIS-12-18-2285-PDN Link, ISI, Google Scholar
- 2016. The efficacy of six elite isolates of the fungus Chondrostereum purpureum against the sprouting of European aspen. J. Environ. Manage. 171:217-224. https://doi.org/10.1016/j.jenvman.2016.02.016 Crossref, Medline, ISI, Google Scholar
- 2015. The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol. Mol. Biol. Rev. 79:293-320. https://doi.org/10.1128/MMBR.00050-14 Crossref, Medline, ISI, Google Scholar
- 1987. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J. Ferment. Technol. 65:501-509. https://doi.org/10.1016/0385-6380(87)90108-7 Crossref, Google Scholar
- 2010. Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18s rDNA for denaturing-gradient gel electrophoresis (DGGE). Microbes Environ. 25:281-287. https://doi.org/10.1264/jsme2.ME10136 Crossref, Medline, ISI, Google Scholar
- 1985. Extinction coefficients of chlorophyll a and B in n,n-dimethylformamide and 80% acetone. Plant Physiol. 77:483-485. https://doi.org/10.1104/pp.77.2.483 Crossref, Medline, ISI, Google Scholar
- 2008. Diversity of predominant endophytic bacteria in European deciduous and coniferous trees. Can. J. Microbiol. 54:173-179. https://doi.org/10.1139/W07-134 Crossref, Medline, ISI, Google Scholar
- 2018. Multifaceted interactions between endophytes and plant: Developments and prospects. Front Microbiol. 9:2732. https://doi.org/10.3389/fmicb.2018.02732 Crossref, Medline, ISI, Google Scholar
- 2014. An important role of the pepper phenylalanine ammonia-lyase gene (PAL1) in salicylic acid-dependent signalling of the defence response to microbial pathogens. J. Exp. Bot. 65:2295-2306. https://doi.org/10.1093/jxb/eru109 Crossref, Medline, ISI, Google Scholar
- 2007. Some fungal endophytes from vegetable crops and their anti-oomycete activities against tomato late blight. Lett. Appl. Microbiol. 44:332-337. https://doi.org/10.1111/j.1472-765X.2006.02093.x Crossref, Medline, ISI, Google Scholar
- 2019. Evaluation of a novel endophytic Pseudomonas lactis strain for control of boxwood blight. Environ. Hortic. 37:39-43. https://doi.org/10.24266/0738-2898-37.2.39 Crossref, Google Scholar
- 1991.
16S/23S rRNA sequencing . Pages 115-175 in: Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt and M. Goodfellow, eds. John Wiley and Sons, New York. Google Scholar - 2010. Isolating endophytic fungi from evergreen plants and determining their antifungal activities. Afr. J. Microbiol. Res. 4:2243-2248. Google Scholar
- 2018. Apple endophytic microbiota of different rootstock/scion combinations suggests a genotype-specific influence. Microbiome 6:18. https://doi.org/10.1186/s40168-018-0403-x Crossref, Medline, ISI, Google Scholar
- 2015. Arabidopsis thaliana as a tool to identify traits involved in Verticillium dahliae biocontrol by the olive root endophyte Pseudomonas fluorescens PICF7. Front. Microbiol. 6:266. https://doi.org/10.3389/fmicb.2015.00266 Crossref, Medline, ISI, Google Scholar
- 2012. Postharvest biocontrol of Monilinia laxa, Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains. Biol. Control 60:132-140. https://doi.org/10.1016/j.biocontrol.2011.10.013 Crossref, ISI, Google Scholar
- 2009. DNA-dependent detection of the grapevine fungal endophytes Aureobasidium pullulans and Epicoccum nigrum. Plant Dis. 93:993-998. https://doi.org/10.1094/PDIS-93-10-0993 Link, ISI, Google Scholar
- 2008. How to get more out of molecular fingerprints: practical tools for microbial ecology. Environ. Microbiol. 10:1571-1581. https://doi.org/10.1111/j.1462-2920.2008.01572.x Crossref, Medline, ISI, Google Scholar
- 2008. Bacterial endophytes: Bacillus spp. from annual crops as potential biological control agents of black pod rot of cacao. Biol. Control 46:46-56. https://doi.org/10.1016/j.biocontrol.2008.01.022 Crossref, ISI, Google Scholar
- 1998. Denaturing gradient gel electrophoresis (DGGE) in microbial ecology. Molecular Microbiol. Ecol. Manual 3.4.4:1-27. Google Scholar
- 1993. Profiling of complex microbiol population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700. https://doi.org/10.1128/AEM.59.3.695-700.1993 Crossref, Medline, ISI, Google Scholar
- 2008. Fungal endophytes in xylem of healthy Chilean trees and their possible role in early wood decay. Fungal Divers. 33:77-86. ISI, Google Scholar
- 2006. Alteration of photosynthesis in grapevines affected by esca. Phytopathology 96:1060-1066. https://doi.org/10.1094/PHYTO-96-1060 Link, ISI, Google Scholar
- 2014. Induced systemic resistance by beneficial microbes. Annu. Rev. Phytopathol. 52:347-375. https://doi.org/10.1146/annurev-phyto-082712-102340 Crossref, Medline, ISI, Google Scholar
- 2019. Endophytes from wild rubber trees as antagonists of the pathogen Corynespora cassiicola. Phytopathology 109:1888-1899. https://doi.org/10.1094/PHYTO-03-19-0093-R Link, ISI, Google Scholar
- 2013. Biocontrol potential of an endophytic Bacillus pumilus JK-SX001 against canker. Biol. Control 67:421-430. https://doi.org/10.1016/j.biocontrol.2013.09.012 Crossref, ISI, Google Scholar
- 2009. Fungal endophytes: diversity and functional roles. New Phytol. 182:314-330. https://doi.org/10.1111/j.1469-8137.2009.02773.x Crossref, Medline, ISI, Google Scholar
- 2017. Greenhouse evaluation of grapevine microbial endophytes and fungal natural products for control of Pierce’s disease. Final Report for California Department of Food and Agriculture Agreement Number 16-0512-SA. Google Scholar
- 1994. Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17:915-919. ISI, Google Scholar
- 1965. Sap pressure in vascular plants: negative hydrostatic pressure can be measured in plants. Science 148:339-346. https://doi.org/10.1126/science.148.3668.339 Crossref, Medline, ISI, Google Scholar
- 2001. Characterization of an endopolygalacturonase gene cppg1 from phytopathogenic fungus Chondrostereum purpureum. J. Gen. Plant Pathol. 67:41-44. https://doi.org/10.1007/PL00012985 Crossref, Google Scholar
- 2006. Biocontrol of wood-rotting fungi with Streptomyces violaceusniger XL-2. Can. J. Microbiol. 52:805-808. https://doi.org/10.1139/w06-035 Crossref, Medline, ISI, Google Scholar
- 1998. Seasonal variation in susceptibility of xylem tissue of Malus, Pyrus, Prunus, and Salix species to Chondrostereum purpureum in New Zealand. Mycol. Res. 102:881-890. https://doi.org/10.1017/S0953756297005923 Crossref, Google Scholar
- 1987. Effects of silverleaf infection on ultrastructure of foliage of Prunus, Rosa, and Populus. N. Z. J. Bot. 25:411-423. https://doi.org/10.1080/0028825X.1987.10413358 Crossref, ISI, Google Scholar
- 2009. Susceptibility of silver birch pruning wounds to infection by white-rot fungus (Chondrostereum purpureum), a potential bioherbicide. Silva Fenn. 43:537-547. https://doi.org/10.14214/sf.179 Crossref, ISI, Google Scholar
- 2007. Fungal endophytes of native Gossypium species in Australia. Mycol. Res. 111:347-354. https://doi.org/10.1016/j.mycres.2006.11.004 Crossref, Medline, Google Scholar
- 2010. The characterization and diversity of bacterial endophytes of grapevine. Can. J. Microbiol. 56:209-216. https://doi.org/10.1139/W10-004 Crossref, Medline, ISI, Google Scholar
- 1990.
Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics . Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds. Academic Press, San Diego, CA. Crossref, Google Scholar - 2015. Synthetic herbicides were more effective than a bioherbicide based on Chondrostereum purpureum in reducing resprouting of Rhododendron ponticum, a host of Phytophthora ramorum in the UK. Forestry 88:336-344. https://doi.org/10.1093/forestry/cpv004 Crossref, ISI, Google Scholar
- 2019. Structure and function of the fruit microbiome in healthy and diseased kiwifruit. Pak. J. Agric. Sci. 56:577-585. Google Scholar
- . 2006. Statistical analysis of real-time PCR data. BMC Bioinformatics 7:85. https://doi.org/10.1186/1471-2105-7-85 Crossref, Medline, ISI, Google Scholar

