Research article

Characterization of the brown leaf spots pathosystem in Brazilian pecan orchards: pathogen morphology and molecular identification

Tales Poletto, Marlove Fátima Brião Muniz, Vinícius Spolaor Fantinel, Laís da Silva Martello, Lucas Graciolli Savian, Ricardo Harakava, Eduardo Guatimosim, Igor Poletto, Valdir Marcos Stefenon

Tales Poletto
Federal University of Santa Maria
Marlove Fátima Brião Muniz
Federal University of Santa Maria
Vinícius Spolaor Fantinel
Federal University of Santa Maria
Laís da Silva Martello
Federal university of Santa Maria
Lucas Graciolli Savian
Federal University of Santa Maria
Ricardo Harakava
Biological Institute of São Paulo
Eduardo Guatimosim
Federal University of Rio Grande
Igor Poletto
Federal University of Pampa
Valdir Marcos Stefenon
Universidade Federal de Santa Catarina. Email: valdir.stefenon@ufsc.br

Online First: June 28, 2021
Poletto, T., Muniz, M., Fantinel, V., Martello, L., Savian, L., Harakava, R., Guatimosim, E., Poletto, I., Stefenon, V. 2021. Characterization of the brown leaf spots pathosystem in Brazilian pecan orchards: pathogen morphology and molecular identification. Annals of Forest Research DOI:10.15287/afr.2020.1957


Due to the increase in pecan nuts demand, plantation areas are expected to expand around the world and more frequent epidemics caused by fungal pathogens may occur in orchards and nurseries. Ragnhildiana diffusa is a pathogenic fungus reported as causing brown leaf spots in pecan in Mexico, South Africa, and the U.S.A. The scarcity of comprehensive information in symptoms on the host and morphology of the fungus lead this disease to be initially incorrectly identified in Brazil. In this study, we employed different approaches to characterize the pathogen morphology and pathogenicity and to molecularly identify the organism causing brown leaf spots in 10 different orchards in southern Brazil. A phylogenetic analysis based on the ITS and the LSU gene sequences confirmed R. diffusa as the causal pathogen of the disease in all orchards. Inoculation tests on healthy leaflets confirmed that all sampled isolates were pathogenic, although some variation in their virulence was observed. Variation in the morphology of the asexual stage was observed among and within isolates. The accurate and prompt identification of the disease may assist controlling further spread of the pathogen into orchards and nurseries still free of the disease in South America.


Almeida A.M., Piuga F.F., Marin S.R., Binneck E., Sartori F., Costamilan L.M., Teixeira M.R.O., Lopes M., 2005. Pathogenicity, molecular characterization, and cercosporin content of Brazilian isolates of Cercospora kikuchii. Fitopatologia Brasileira, 30(6): 594-602. https://doi.org/10.1590/S0100-41582005000600005

Bach E.E., Kimati H., 1999. Purification and characterization of toxins from wheat isolates of Drechslera tritici-repentis, Bipolaris bicolor, and Bipolaris sorokiniana. Journal of Venomous Animals and Toxins 5(2): 184-199. http://dx.doi.org/10.1590/S0104-79301999000200006

Bach E.E., Limiro C., Rodrigues E., 2005. Extração e ação de toxinas de Exserohilum turcicum em plantas de milho. [Extraction and action of Exserohilum turcicum in mays plants]. ConScientiae Saúde 4: 105-113 https://doi.org/10.5585/conssaude.v4i0.423

Chupp C.C., 1953. Monograph on the fungus genus Cercospora. Ithaca, NY, 1886-1967.

Crous P.W., Braun U., 2003. Mycosphaerella and its anamorphs: 1. Names published in Cercospora and Passalora. Centraalbureau voor Schimmelcultures (CBS). 571p.

de Oliveira L.O., Beise D.C., Dos Santos D.D., Nagel J.C., Poletto T., Poletto I., Stefenon V.M., 2021. Molecular markers in Carya illinoinensis (Juglandaceae): from genetic characterization to molecular breeding. The Journal of Horticultural Science and Biotechnology, https://doi.org/10.1080/14620316.2021.1892534

Dhingra O.D., Sinclair J.B., 1995. Basic plant pathology methods 2.ed. Boca Raton FL. Lewis Publishers. 448p.

Doyle J.J., Doyle J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15.

Ellis M.B., 1976. More dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 507 p.

Fernandes M.R., 1993. Manual para laboratório de fitopatologia. [Manual of phytopathology laboratory]. EMBRAPA – CNPT (Passo Fundo, RS) CIDA, 128 p.

Hall T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98 / NT. Nucleic Acids Symposium Series 41: 95-98.

Jiménez M.M., Bahena S.M., Espinoza C., Trigos Á., 2010. Isolation, characterization and production of red pigment from Cercospora piaropi, the biocontrol agent for waterhyacinth. Mycopathology 169: 309-314. https://doi 10.1007/s11046-009-9257-x

Kluepfel M., Blake J.H., Reilly C.C., 2014. Pecan diseases. Factsheet HGIC 2211. Pecan diseases | Home & Garden information center. Clemson Cooperative Extension. Clemson University – USA. https://hgic.clemson.edu/factsheet/pecan-diseases/

Lynch F.J., Geoghegan M.J., 1979. The role of pigmentation in survival of the leaf spot fungus Cercospora beticola. Annals of Applied Biology 91(3): 313-318. https://doi.org/10.1111/j.1744-7348.1979.tb06506.x

Madero E.R., Trabichet F.C., Pepé F., Wright E.R., 2016. Manual de manejo del huerto de nogal pecán. Ediciones INTA; Estación Experimental Agropecuaria Delta del Paraná, 94 p.

Maguire J.D., 1962. Speed of germination-aid selection and evaluation for seedling emergence and vigor. Crop Science 2: 176-177.

Miller M.A., Pfeiffer W., Schwartz T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans. https://doi.org/10.1109/GCE.2010.5676129

Nagel J.C., de Oliveira Machado L., Lemos R.P.M., Matielo C.B.D.O., Poletto T., Poletto I., Stefenon V.M., 2020. Structural, evolutionary and phylogenomic features of the plastid genome of Carya illinoinensis cv. Imperial. Annals of Forest Research, 63(1): 3-18. https://doi.org/10.15287/afr.2019.1413

Nylander J., 2004. MrModeltest v2. Program distributed by the author. Evol Biol Cent Uppsala Univ 2:1–2.

Pascholati S.F., 2011. Physiology of parasitism: how pathogens attack plants. In: Amorim L., Rezende J.A.M., Bergamin Filho A. (eds) Manual: principles and control. São Paulo: Agronomic Publishing Ceres, 704 p.

PMR Analysis, 2020. Pecan Market: Global Industry Analysis 2015-2019 and Opportunity Assessment 2020-2030.

Poletto T., Muniz M.F.B, Fantinel V.S., Favaretto R.F., Poletto, I., Reiniger, L., Blume, E., 2018. Culture medium, light regime and temperature affect the development of Sirosporium diffusum. Journal of Agricultural Science 10(6): 310-318. https://doi.org/10.5539/jas.v10n6p310

Poletto T., Muniz M.F.B., Blume E., Mezzomo R., Braun U., Videira S.I.R., Harakava R., Poletto I., 2017. First Report of Sirosporium diffusum causing brown leaf spot on Carya illinoinensis in Brazil. Plant Disease 101(2): 381-381. https://doi.org/10.1094/PDIS-06-16-0820-PDN

Poletto T., Muniz M.F.B., Lucio A.D.C., Fantinel V.S., Heldwein A.B., Reiniger L.R.S., Blume E., 2020. Diagrammatic scale for quantifying severity of brown leaf spot on Carya illinoinensis. Annals of the Brazilian Academy of Sciences 92: e20180889. http://dx.doi.org/10.1590/0001-3765202020180889

Reino J.L., Hernández‐Galán R., Durán‐Patrón R., Collado I.G., 2004. Virulence–toxin production relationship in isolates of the plant pathogenic fungus Botrytis cinerea. Journal of Phytopathology 152(10): 563-566. https://doi.org/10.1111/j.1439-0434.2004.00896.x

Ronquist F., Teslenko M., Van der Mark P., Ayres D.L., Darling A., Höhna S., Larget B., Liu L., Suchard M.A., Huelsenbeck J.P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029

Schmitz A., Riesner D., 2006. Purification of nucleic acids by selective precipitation with polyethylene glycol 6000. Analytical Biochemistry 354(2): 311-313. https://doi: 10.1016/j.ab.2006.03.014

Shaner G., Stromberg E.L., Lacy G.H., Barker K.R., Pirone T.P., 1992. Nomenclature and concepts of pathogenicity and virulence. Annual Review of Phytopathology 30(1): 47-66. https://doi.org/10.1146/annurev.py.30.090192.000403

Stein L.A., Mceachern G.R., Nesbitt M.L., 2012. Texas Pecan Handbook. Texas A & M AgriLife Extension Service, College Station, 199 p.

Trabichet F.C., Madero E.R., Bruno N.R., Grassi A.L., Gadea T., 2016. Guía de buenas prácticas agrícolas para la producción de nuez pecán. Ediciones INTA, 47 p.

Upchurch R.G., Walker D.C., Rollins J.A., Ehrenshaft M., Daub M.E., 1991. Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity. Applied and Environmental Microbiology 57(10): 2940-2945.

Videira S.I.R., Groenewald J.Z., Nakashima C., Braun U., Barreto R.W., de Wit P.J.G.M., Crous P.W., 2017. Mycosphaerellaceae – chaos or clarity? Studies in Mycology 87: 257-421. https://doi.org/10.1016/j.simyco.2017.09.003

Vilgalys R., Hester M., 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172(8): 4238-4246. https://doi: 10.1128/jb.172.8.4238-4246.1990

Walker C., Muniz M., Martins R.D.O., Rabuske J., Santos A.F.D., 2018. Susceptibility of pecan cultivars to Cladosporium cladosporioides species complex. Floresta e Ambiente, 25(4): e20170267. http://dx.doi.org/10.1590/2179-8087.026717

White T.J., Bruns T., Lee S.J.W.T., Taylor J.W., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18(1): 315-322. https://doi:10.1016/B978-0-12-372180-8.50042-1



No Supplimentary Material available for this article.
No metrics available for this article.