Abstract
Morphological and molecular studies confirmed Paracercospora egenula as the primary cercosporoid fungus causing leaf spot of eggplant (Solanum melongena) in Hawaii. This is the first confirmed record of P. egenula in Hawaii, although it was previously reported as C. solani-melongenae, based on an interception at Oahu, Hawaii, in 1946. Another cercosporoid species, Cercospora melongenae, reported to cause leaf spot on eggplant in Hawaii, was not detected.
Avoid common mistakes on your manuscript.
Leaf spot of eggplant (Solanum melongena) is prevalent in vegetable gardens in Honolulu, Hawaii, with disease incidence reaching almost 100 % in the Manoa Valley, Oahu. The disease causes necrotic, angular or circular to oblong lesions of up to 7 mm in diam., occasionally with shot-holes in the centers (Fig. 1). Lesions may coalesce under favourable conditions and cause severe defoliation. Multiple cercosporoid species have been reported to cause leaf spot on eggplant worldwide. In his monograph of the genus Cercospora, Chupp (1954) listed C. deightonii, C. melongenae and C. solani-melongenae from eggplant. The only cercosporoid species recorded in the Hawaiian and USDA databases on eggplant in Hawaii is Cercospora melongenae (Anonymous 1960; Raabe et al. 1981). Deighton (1979) listed C. solani-melongenae as a heterotypic synonym of Paracercospora egenula. One of the herbarium specimens (IMI 90164 = BPI 441400) that Deighton (1979) studied was from eggplant intercepted at Oahu, Hawaii, and originally identified as C. solani-melongenae by Flora G. Pollack in 1946. Subsequently, Crous and Braun (2003) listed Hawaii in the geographical distribution of P. egenula, probably because Deighton (1979) had listed Hawaii as the origin of the intercepted specimen (IMI 90164). This specimen, however, was not subsequently listed in the Hawaiian and USDA databases as present in Hawaii, most likely because it represented a quarantine interception from another country. Our objective was to confirm the identity of the cercosporoid fungus associated with leaf spot of eggplant in Hawaii through morphological and molecular characterisation.
In November 2015, 13 isolates of cercosporoid fungi were collected from leaves of several varieties of eggplant at five community gardens at four locations (Manoa Valley, Ala Wai, Diamond Head and Makiki) in Honolulu, Hawaii. Conidia from individual lesions were collected by pipetting 50 μl of T-water (0.06 % v/v Tween-20 + 0.02 % w/v filter-sterilized ampicillin) onto the surface of sporulating lesions and transferring conidial suspensions onto water agar amended with ampicillin (0.02 % w/v). After 24 h incubation at room temperature, germinated conidia were transferred to Petri plates containing clarified V8-media (10 % v/v clarified V8 juice, 0.5 % CaCO3, 1.5 % w/v agar). Four monosporic isolates representing each of the four locations (HI-022, HI-027, HI-028, and HI-033; Table 1) were grown on potato dextrose agar (PDA), malt extract agar (MEA) and oatmeal agar (OA) for cultural characterisation.
Genomic DNA was extracted from isolates HI-022, HI-027, HI-028, and HI-033 using a QIAGEN DNeasy Plant Mini Kit according to the manufacturer’s instructions, from lyophilized mycelia derived from cultures grown in clarified V8 broth (10 % v/v clarified V8 juice, 0.5 % w/v CaCO3) on a shaker at 100 rpm at room temperature for 7 days. Standard primers were used to amplify the sequences of the ITS (White et al. 1990), LSU (Rehner and Samuels 1994), partial actin (ACT; Carbone and Kohn 1999), and translation elongation factor (EF-1α; Carbone and Kohn 1999), and all the sequences were deposited in the NCBI GenBank nucleotide database (Table 1). Maximum likelihood (ML) analysis was conducted on the concatenated alignment of the four loci (LSU: 1265; ITS: 488; EF-1α: 281; ACT: 189; total 2223), in RAxML v. 7.2.6 (Stamatakis 2006) using the GTRGAMMA model applied to the individual partitions with 1000 bootstraps. The resulting ML tree was viewed in TreeView v. 1.6.6. (Page 1996).
Mean colony diam. of the isolates ranged from 22 to 26 mm on PDA and OA; and from 21 to 27 mm on MEA, after incubation in the dark at room temperature (23–25 °C) for 6 weeks. Leaf spots amphigenous, scattered, circular to angular, 1–7 mm diam, with pale to dark brown centres, surrounded by diffuse chlorotic haloes. Conidiomata sporodochia-like, epiphyllous, up to 80 μm diam, pale to olivaceous brown. Conidiophores in dense fascicles arising from the upper cells of the conidiomata, reduced to conidiogenous cells or 1-septate, subcylindric, narrowed towards the tip, 10–25 × 3–5 μm, pale brown, smooth, conidiogenous loci apical and flattened with a circular darkened-refractive rim. Conidia cylindrical to obclavate, 25–75 × 3.5–5.5 μm, straight or curved to flexuous, apex obtuse, base obconically truncate, 2–6 septate, smooth, subhyaline to pale brown (Fig. 1), which was similar to the description of Paracercospora egenula (Deighton 1979; Crous et al. 2013). A representative culture has been lodged with the International Collection of Microorganisms from Plants, Auckland, New Zealand (ICMP 21323) and a herbarium specimen with the Queensland Plant Pathology Herbarium, Brisbane, Australia (BRIP 64766).
Paracercospora is characterized by circular conidiogenous loci with slightly thickened, darkened-refractive rims and pale conidia (Braun et al. 2013). It was recovered as a monophyletic genus sister to Pseudocercospora (Crous et al. 2013). The LSU and ITS sequences of all Hawaiian isolates were identical to that of P. egenula MUCC 883 and CBS 485.81 but 2 and 1 bp different from the LSU and ITS of CBS 132030, respectively. The ACT sequences were identical to those of MUCC 883, CBS 485.81 and CBS 132030. The EF-1α sequences were identical to MUCC 883 and 2 bp different from CBS 485.81 and CBS 132030. Maximum likelihood analysis showed that Hawaiian isolates formed a highly supported clade with the three P. egenula reference cultures sequenced by Crous et al. (2013) (Fig. 2). Paracercospora egenula was confirmed as the primary cercosporoid fungus causing leaf spot of eggplant in Hawaii while C. melongenae, previously reported on eggplant in Hawaii, was not detected.
References
Anonymous (1960) Index of Plant Diseases in the United States. US Dep Agric Handb 165:1–531
Braun U, Nakashima C, Crous PW (2013) Cercosporoid fungi (Mycosphaerellaceae) 1. Species on other fungi, Pteridophyta and Gymnospermae. IMA Fungus 4:265–345
Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553–556
Chupp C (1954) A monograph of the fungus genus Cercospora. Ithaca, New York, USA
Crous PW, Braun U (2003) Mycosphaerella and its anamorphs. 1. Names published in Cercospora and Passalora. CBS Biodiversity Series, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
Crous PW, Braun U, Hunter GC, Wingfield MJ, Verkley GJM, Shin HD, Nakashima C, Groenewald JZ (2013) Phylogenetic lineages in Pseudocercospora. Stud Mycol 75:37–114
Deighton FC (1979) Studies on Cercospora and allied genera. VII. New species and redispositions. Mycol Pap 144:1–56
Page RDM (1996) Treeview: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358
Raabe RD, Conners IL, and Martinez AP (1981) Checklist of plant diseases in Hawaii. College of Tropical Agriculture and Human Resources, University of Hawaii. Information Text Series No. 22. Hawaii Inst. Trop. Agric. Human Resources, 313 pp
Rehner SA, Samuels GJ (1994) Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycol Res 98:625–634
Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols. A guide to methods and applications. Academic, San Diego, pp. 315–322
Acknowledgments
This research was supported by the United States Department of Agriculture, National Institute of Food and Agriculture Hatch project NYG-625424, managed by the New York Agricultural Experiment Station, Cornell University, Geneva, NY. Thanks to Mr. David Strickland for excellent technical support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Vaghefi, N., Pethybridge, S.J., Shivas, R.G. et al. Confirmation of Paracercospora egenula causing leaf spot of eggplant in Hawaii. Australasian Plant Dis. Notes 11, 35 (2016). https://doi.org/10.1007/s13314-016-0223-4
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13314-016-0223-4