Abstract
Citrus cultivation has socioeconomic relevance, and gummosis Phytophthora diseases considerably reduce its yield. This infection has motivated the development of diagnostic procedures in the Citrus-Phytophthora pathosystem to assist breeding programs in selecting resistant rootstocks. The objective of this study was to standardize a method for detecting nuclei Phytophthora sp. for study the colonization process and the life cycle of the pathogen in infected stems of Rangpur lime rootstocks using flow cytometry. The stems were inoculated with a Phytophthora sp. isolated from a citrus population of Bom Jesus da Lapa, Bahia, Brazil. Firstly, the DNA ploidy level and nuclear genome size were determined from the mycelium cells of Phytophthora sp. and leaf cells of Rangpur lime, using the saline extraction buffer, procedure of preparation of suspensions in two stages (isolation and staining), with centrifugation of the nuclei suspensions and discard of the supernatant. The mean number of 1C (gametangia) and 2C (other tissues) Phytophthora sp. nuclei in Rangpur lime was estimated based on the reference values relative to the position of G0/G1 peaks of the 2C nuclei of Phytophthora sp. and of the Rangpur lime rootstock and based on the standardization of the amount of infected tissue, and the volume of nuclear suspension analyzed. In infected Rangpur lime, there is a higher mean number of 2C nuclei of Phytophthora sp., which correspond to hyphae in the vegetative growth stage, dispersion spores, and infection structures of the pathogen’s life cycle. On the other hand, 1C nuclei, in smaller quantities, correspond to gametangia. Therefore, the established flow cytometry procedure was reproducible and relatively fast for study the colonization process and the life cycle of the Phytophthora sp. on diseased plants of Rangpur lime.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abbate L, Panno S, Mercati F, Davino S, Del Bosco SF (2019) Citrus rootstock breeding: response of four allotetraploid somatic hybrids to Citrus tristeza virus induced infections. Eur J Plant Pathol 153:837–847. https://doi.org/10.1007/s10658-018-1599-0
Adaskaveg JE, Förster H. (2014) Integrated Postharvest Strategies for Management of Phytophthora Brown Rot of Citrus nos Estados Unidos. Em: Prusky D., Gullino M. (eds) Post-harvest Pathology. Plant Pathology in the 21st Century (Contributions to the 9th International Congress), vol 7. Springer, Cham. https://doi.org/10.1007/978-3-319-07701-7_11
Afek U, Sztejnberg A (1990) A Rapid Method for Evaluating Citrus Seedlings for Resistance to Foot Rot Caused by Phytophthora citrophthora. Plant Dis 74(1):66–68. https://doi.org/10.1094/PD-74-0066
Ah-Fong AMV, Kim KS, Judelson HS (2017) RNA-seq of life stages of the oomycete Phytophthora infestans reveals dynamic changes in metabolic, signal transduction, and pathogenesis genes and a major role for calcium signaling in development. BMC Genomics 18(1):198. https://doi.org/10.1186/s12864-017-3585-x
Bennett MD, Leitch IJ, Price HJ, Johnston JS (2003) Comparisons with Caenorhabditis (∼100 Mb) and Drosophila (∼175 Mb) using flow cytometry show genome size in Arabidopsis to be ∼157 Mb and thus ∼25% larger than the Arabidopsis genome initiative estimate of ∼125 Mb. Ann Bot 91:547–557. https://dx.doi.org/10.1093%2Faob%2Fmcg057
Bertier L, Leus L, D’Hondt L, Awam DC, Höfte M (2013) Host adaptation and speciation through hybridization and polyploidy in Phytophthora. PLoS ONE 8(12):e85385. https://doi.org/10.1371/journal.pone.0085385
Bleichrodt RJ, Read ND (2019) Flow cytometry and FACS applied to filamentous fungi. Fungal Biol Rev 33(1):1–15. https://doi.org/10.1016/j.fbr.2018.06.001
Carvalho CR, Fernandes RC, Carvalho GMA, Barreto RW, Evans HC (2011) Cryptosexuality and the Genetic Diversity Paradox in Coffee Rust. Hemileia Vastatrix. PLoS ONE 6(11)
Carvalho GMA, Carvalho CR, Barreto RW, Evans HC (2014) Coffee rust genome measured using flow cytometry: Does size matter? Plant Pathol 63:1022–1026. https://doi.org/10.1111/ppa.12175
Catal M, King L, Tumbalam P, Wiriyajitsomboon P, Kirk WW, Adams GC (2010) Heterokaryotic nuclear conditions and a heterogeneous nuclear population are observed by flow cytometry in Phytophthora infestans. Cytometry A 77(8):769–775. https://doi.org/10.1002/cyto.a.20888
Clarindo WR, Carvalho CR (2011) Flow cytometric analysis using SYBR Green I for genome size estimation in coffee. Acta Histochem 113:221–225. https://doi.org/10.1016/j.acthis.2009.10.005
D'Hondt L, Höfte M, Van Bockstaele E, Leus L (2011) Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status. Mol Plant Pathol 12(8):815-28. https://doi.org/10.1111/j.1364-3703.2011.00711.x
Doležel J, Bartoš J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry 51(2):127–129. https://doi.org/10.1002/cyto.a.10013
Doležel J, Bartoš J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95(1):99–110. https://doi.org/10.1093/aob/mci005
Doležel J, Sgorbati S, Lucretti S (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plant 85:625–631. https://doi.org/10.1111/j.13993054.1992.tb04764.x
Drenth A, Guest DI (2004) Diversity and management of Phytophthora in Southeast Asia. Australian Centre for International Agricultural Research, Canberra, pp 227–231
Ehgartner D, Herwig C, Fricke J (2017) Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry—the fast alternative to microscopic image analysis. Appl Microbiol Biotechnol 101:7675–7688. https://doi.org/10.1007/s00253-017-8475-2
Fabritius AL, Cvitanich C, Judelson HS (2002) Stage-specific gene expression during sexual development in Phytophthora infestans. Mol Microbiol 45(4):1057–1066. https://doi.org/10.1046/j.1365-2958.2002.03073.x
Ferreira EA, Pio LAS, Batista LA, Nogueira VHB, Silveira FA (2016) Morphological responses of Navelate orange tree grafted on different rootstocks under water deficit. Citrus Res Technol 37(2):132–137. https://doi.org/10.4322/crt.ICC062
Fry WE (2016) Phytophthora infestans: New tools (and old ones) lead to new understanding and precision management. Annu Rev Phytopathol 54(1):529–547. https://doi.org/10.1146/annurev-phyto-080615-095951
Fry WE, Patev SP, Myers KL, Bao K, Fei Z (2019) Phytophthora infestans sporangia produced in culture and on tomato leaflet lesions show marked differences in indirect germination rates, aggressiveness, and global transcription profiles. Mol Plant Microbe Interact 32(5):515–526. https://doi.org/10.1094/MPMI-09-18-0255-TA
Garavello M, Cuenca J, Dreissig S, Fuchs J, Houben A, Aleza P (2019) Assessing Ploidy Level Analysis and Single Pollen Genotyping of Diploid and Euploid Citrus Genotypes by Fluorescence-Activated Cell Sorting and Whole-Genome Amplification. Front Plant Sci 10:1174. https://doi.org/10.3389/fpls.2019.01174
Hamed BH, Gisi U (2013) Generation of pathogenic F1 progeny from crosses of Phytophthora infestans isolates differing in ploidy. Plant Pathol 62(3):708–718. https://doi.org/10.1111/j.1365-3059.2012.02655.x
Hao W, Gray MA, Förster H, Adaskaveg JE (2019) Evaluation of new oomycota fungicides for management of Phytophthora root rot of Citrus in California. Plant Dis 103(4):619–628. https://doi.org/10.1094/pdis-07-18-1152-re
Husson C, Aguayo J, Revellin C, Frey P, Ioos R, Marçais B (2015) Evidence for homoploid speciation in Phytophthora alni supports taxonomic reclassification in this species complex. Fungal Genet Biol 77:12–21. https://doi.org/10.1016/j.fgb.2015.02.013
Judelson HS, Blanco FA (2005) The spores of Phytophthora: Weapons of the plant destroyer. Nat Rev Microbiol 3(1):47–58. https://doi.org/10.1038/nrmicro1064
Jung T, Pérez-Sierra A, Durán A, Horta Jung, M, Balci Y, Scanu B (2018) Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands. Persoonia 40:182–220. https://doi.org/10.3767/persoonia.2018.40.08
Kamiri M, Stift M, Costantino G, Dambier D, Kabbage T, Ollitrault P, Froelicher Y (2018) Preferential Homologous Chromosome Pairing in a Tetraploid Intergeneric Somatic Hybrid (Citrus reticulata + Poncirus trifoliata) Revealed by Molecular Marker Inheritance. Front Plant Sci 9:1557. https://doi.org/10.3389/fpls.2018.01557
Khankahdani HH, Rastegar S, Golein B, Golmohammadi M, Jahromi AA (2018) Genetic Variation of Lime (Citrus sp.) Accessions using flow cytometry technique, morphological characteristics and molecular markers. Int J Hortic Sci 5(2):199–208. https://doi.org/10.22059/ijhst.2018.258553.241
Lima RPM, Máximo HJ, Merfa MV, Dalio RJD, Cristofani-Yaly M, Machado MA (2018) Genetic tools and strategies for citrus breeding aiming at resistant rootstocks to gummosis disease. Tropical Plant Pathology 43(4):279–288. https://doi.org/10.1007/s40858-018-0229-x
Mahmoudi K, Handaji N, Ibriz M, Benyahya H (2020) Research of citrus triploid hybrids by embryo rescue and flow research of citrus triploid hybrids by embryo rescue and flow cytometry from two oranges varieties Pineapple and parson brown. Plant Cell Biotechnol Mol Biol 21(1–2):19–27. https://www.ikprress.org/index.php/PCBMB/article/view/4914
Otto F (1990) DAPI Staining of Fixed Cells for High-Resolution Flow Cytometly of Nuclear DNA. Methods Cell Biol 33:105–110. https://doi.org/10.1016/s0091-679x(08)60516-6
Praça-Fontes MM, Carvalho CR, Clarindo WR (2011) C-value reassessment of plant standards: An image cytometry approach. Plant Cell Rep 30(12):2303–2312. https://doi.org/10.1007/s00299-011-1135-6
Puglisi I, De Patrizio A, Schena L, Jung T, Evoli M, Pane A, Van Hoa N, Van Tri M, Wright S, Ramstedt M, Olsson C, Faedda R, Lio S, GMdi, Cacciola SO, (2017) Two previously unknown Phytophthora species associated with brown rot of Pomelo (Citrus grandis) fruits in Vietnam. PLoS ONE 12(2):e0172085. https://doi.org/10.1371/journal.pone.0172085
Ramallo AC, Cerioni L, Olmedo GM, Volentini SI, Ramallo J, Rapisarda VA (2019) Control of Phytophthora brown rot of lemons by pre- and postharvest applications of potassium phosphite. Eur J Plant Pathol 154:975–982. https://doi.org/10.1007/s10658-019-01717-y
Ramos AP, Tavares S, Tavares D, MdoC S, Loureiro J, Talhinhas P (2015) Flow cytometry reveals that the rust fungus, Uromyces bidentis (Pucciniales), possesses the largest fungal genome reported-2489Mbp. Mol Plant Pathol 16(9):1006–1010. https://doi.org/10.1111/mpp.12255
Rocha LO, Tralamazza SM, Reis GM, Rabinovitch L, Barbosa CB, Corrêa B (2014) Correction (2019): Multi-Method Approach for Characterizing the Interaction between Fusarium verticillioides and Bacillus thuringiensis Subsp. Kurstaki PLOS ONE 14(5):e0216693. https://doi.org/10.1371/journal.pone.0216693
Silva AR, S Pinto KN, Maserti BE, Santos-Filho HP, Gesteira AS (2021) Systematic review of defense responses against Phytophthora and strategies to manage Phytophthora diseases in citrus. Funct Plant Biol. Jun 15. https://doi.org/10.1071/FP20349
Siviero A, Furtado EL, Boava LP, Barbasso DV, Machado MA (2002) Avaliação de métodos de inoculação de Phytophthora parasitica em plântulas e plantas jovens de citros. Fitopatol Bras 27(6):574–580. https://doi.org/10.1590/S0100-41582002000600003
Vercauteren A, Boutet X, D’Hondt L, Van Bockstaele E, Maes M, Leus L, Chandelier A, Heungens K (2011) Aberrant genome size and instability of Phytophthora ramorum oospore progenies. Fungal Genet Biol 48(5):537–543. https://doi.org/10.1016/j.fgb.2011.01.008
Wang J, Presser JW, Goss EM (2016) Nuclear DNA content of the hybrid plant pathogen Phytophthora andina determined by flow cytometry. Mycologia 108(5):899–904. https://doi.org/10.3852/15-107
Wen G, Cao R, Wan Q, Tan L, Xu X, Wang J, Huang T (2020) Desenvolvimento de métodos de coloração de esporos de fungos para quantificação por citometria de fluxo e sua aplicação na desinfecção à base de cloro. Chemosphere 243: 125453. https://doi.org/10.1016/j.chemosphere.2019.125453
Author information
Authors and Affiliations
Contributions
All authors contributed to this study, revised the manuscript, carried out the experiments and analyzed the data, searched the literatures, drafted the manuscript and have approved the final manuscript.
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Silva, A.R., Pinto, K.N., Silva, J.C. et al. Measurement of Phytophthora nuclear DNA amount by flow cytometry enables study the colonization process and the life cycle of the pathogen on citrus. J Plant Pathol 103, 1265–1274 (2021). https://doi.org/10.1007/s42161-021-00911-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s42161-021-00911-4