Australasian Plant Disease Notes (2020) 15: 38 https://doi.org/10.1007/s13314-020-00407-7 Alternaria dauci causes leaf spots and leaf blight of coriander (Coriandrum sativum) in Brazil Paloma S. Mansur 1 & André L. Silva 1 & Sara S. Salcedo 1 & Robert W. Barreto 1 & Adans A. Colmán 1,2 Received: 18 November 2019 / Accepted: 8 October 2020 / Published online: 19 October 2020 # Australasian Plant Pathology Society Inc. 2020 Abstract Alternaria dauci is fully confirmed to cause leaf spots and leaf blight of coriander (Coriandrum sativum) in Brazil based on observations made of blighted plants at a demonstration plot (Infectarium) at Viçosa (state of Minas Gerais) Brazil. Identification was supported by a combination of morphological, cultural and molecular features. A representative herbarium sample and an isolate were deposited in public collections as well as DNA sequences. Pathogenicity tests were conducted and Koch’s postulates were fulfilled. Keywords Etiology . Phylogeny . Section Porri . Pathogenicity . Pleosporaceae Coriander (Coriandrum sativum), also known as cilantro (coentro in Brazil) is an annual herb of the Apiaceae. It is widely cultivated and used as a condiment herb worldwide (Simpson and Connor-Ogorzaly 2001) and also in Brazil. Several fungal pathogens have been reported as causing leaf spots on coriander in Brazil (Farr and Rossman 2019) and Alternaria leaf spot is considered an important disease of coriander in Brazil (Reis and Lopes 2016). In July 2018, all plants growing in a demonstration plot in the Infectarium – the plant disease garden - of the Departamento de Fitopatologia of the Universidade * Adans A. Colmán adan-colman@hotmail.com Paloma S. Mansur paloma.mansur@ufv.br André L. Silva als9528@gmail.com Sara S. Salcedo taphrina10@gmail.com Robert W. Barreto rbarreto@ufv.br 1 Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais 36570-900, Brazil 2 Present address: Facultad de Ciencias Agrarias, Universidad Nacional de Asunción, San Lorenzo, Paraguay Federal de Viçosa (municipality of Viçosa, state of Minas Gerais, Brazil) were found to be attacked by a disease leading to severe leaf blight symptoms. Firstly small round to oval, 3–5 mm diam., leaf spot appeared which quickly coalesced and turned into necrotic blight of entire leaves. Samples were collected and examined under a dissecting microscope and a dematiaceous fungus was found to be regularly associated with the necrotic tissues. Under a dissecting microscope (Olympus SZX7) conidia were taken from infected leaves with a sterile fine pointed needle and transferred onto previously marked positions of V8 juice-agar plates. Each position of those plates was then examined, under the high power of the dissecting microscope, to select positions where single conidia were deposited. Those points of the plates were further marked, at the underside of the plates, indicating that colonies from these positions represented single-spore isolates. After 1 week of growth in the incubator, at 25 °C, a fragment of the margin of one selected single spore colony was taken and transferred to a tube containing potato carrot-agar. This representative culture was deposited in the culture collection of the Universidade Federal de Viçosa (Acc No COAD 2594) and used in this study. Additionally, a representative specimen of diseased coriander was dried in a plant press and deposited in the local herbarium (Acc. No VIC 47142). Slides were prepared by scraping fungal structures from sporulating colonies on leaves and mounting these on a drop 38 Page 2 of 5 of lactophenol. Observations were made under a microscope (Olympus BX 53) equipped with differential interference contrast (DIC) illumination and connected to an Olympus Q-color 5™ camera. The fungus on coriander had the following morphology: conidiophores forming loose groups, subcylindrical, straight to somewhat sinuose, 27.5–75 × 5–10 μm, 1–4 septate, pale brown to brown, smooth; conidiogenous cells subcylindrical, geniculate, (7.5) 15–27.5 (37.5) × 5–10 μm, conidiogenous loci terminal to intercalary 2.5–7.5 μm diam.; conidia solitary, (125) 200–300 (340) × 17.5– 22 μm, 1–8 longitudinally and 8–15 transversally septae, beak up up 280 μm long, pale brown, walls somewhat verruculose (Fig. 1). DNA was extracted from mycelium of COAD 2594 grown in 20 ml of potato dextrose broth at 23 ± 2 °C for 4 days. Extraction was performed with a Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s recommendations. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) region was amplified with the primers gpd1 and gpd2 (Berbee et al. 1999), the RPB2 region with RPB25F2 (Sung et al. 2007) and fRPB2–7cR (Liu et al. 1999) and the Alt a1 region with the primers Alt-for and Alt-rev (Hong et al. 2005). PCR was performed following the procedure described in Colmán et al. (2018) and phylogenetic analyses were performed as described in Woudenberg et al. (2013). Bayesian inference analyses were performed with the CIPRES web portal using MrBayes v. 3.2.6. (Miller et al. 2015). The jmodeltest (Darriba et al. 2012) was used for estimation of sequence evolution. Based on the phylogenetic analyses results combined GAPDH, Alt 1 and RPB2 gene regions, our isolate COAD 2594 was identified as belonging to Alternaria dauci (Fig. 2). Sequences obtained in this work were deposited in GenBank (Table 1). To confirm the pathogenicity of COAD 2594, 4 month old healthy coriander plants were inoculated. Additionally, groups of four pots (one healthy plant per pot) containing either carrot, celery or parsley were also inoculated with COAD 2594. A conidial suspension was prepared by flooding sporulating 7-day-old cultures formed on V8 juice-agar plates with sterile distilled water (SDA) and scraping their surface with a rubber spatula. A haemocytometer was used to calculate the concentration of spores/ml which was then adjusted to 106 conidia/ml. Plants were sprayed until runoff and covered with a plastic bag for 48 h to keep humidity levels high. One healthy plant of each species treated similarly but sprayed with SDA only served as controls. Typical symptoms of disease were observed 2–4 days after inoculation. Control plants remained healthy. Alternaria Australasian Plant Dis. Notes (2020) 15: 38 Fig. 1 Alternaria dauci on coriander (Coriandrum sativum). a Leaf spot and some blight symptoms on coriander; b Group of conidiophores of A. dauci; c Conidia with long septate beaks, d Colony of A. dauci on V8 juice-agar; e-f Pathogenicity test on coriander plants, control (e) and inoculated plants (f) 4 days after inoculation. Scale bars 20 μm dauci sporulated abundantly on necrotic tissues and was reisolated on V8 juice-agar plates producing sporulating cultures. The morphology of the fungus reisolated from necrotic tissues of inoculated plants was examined under the microscope and found to have a morphology identical to COAD 2594 fulfilling Koch’s postulates. Several species of Alternaria have been reported on C. sativum (Farr and Rossman 2019). Of these (A. poonensis) was originally described by Raghunath Australasian Plant Dis. Notes (2020) 15: 38 Table 1 Page 3 of 5 38 GenBank accession number of sequences obtained in this study and other Alternaria spp. used for phylogenetic analysis Species Name Host Strain number Country GenBank accession number Alt 1 GPDH RPB2 Alternaria anagallidis Alternaria anagallidis Alternaria argyroxiphii Alternaria argyroxiphii Alternaria argyroxiphii Alternaria bataticola Alternaria bataticola Alternaria bataticola Alternaria bataticola Alternaria dauci Alternaria dauci A. dauci (syn. A. poonensis) Alternaria dauci Alternaria dauci Anagallis arvensis Anagallis arvensis Argyroxiphium sp Ipomoea batatas Ipomoea batatas Ipomoea batatas Ipomoea batatas Ipomoea batatas Ipomoea batatas Daucus carota Daucus carota Coriadrum sativum Coriadrum sativum Coriadrum sativum CBS 117128 CBS 117129 CBS 117122 PPRI 11848 PPRI 11971 CBS 531.63 CBS 532.63 CBS 117095 CBS 117096 CBS 111.38 CBS 345.79 CBS 117.100 USFL-3 NB625 New Zealand New Zealand USA South Africa South Africa Japan Japan Australia Australia Italy New Zealand Puerto Rico USA Algeria KJ718628 KJ718629 KJ71863 KJ718633 KJ718634 JQ646433 KJ718637 KJ718638 KJ718639 KJ718673 KJ718675 KJ718680 MF595071 – KJ717961 KJ717962 JQ646350 KJ717965 KJ717966 JQ646349 KJ717969 KJ717970 KJ717971 KJ718005 KJ718007 JQ646348 MF595072 MK513412 KJ718282 KJ718283 KJ718286 KJ718287 KJ718288 KJ718291 KJ718292 KJ718293 KJ718294 KJ718331 KJ718333 KJ718338 – MK513423 Alternaria dauci Alternaria dauci Alternaria echinaceae Alternaria echinaceae Alternaria euphorbiicola Alternaria euphorbiicola Alternaria ipomeae Alternaria ipomeae Alternaria neoipomeae Alternaria neoipomeae Alternaria tagetica Alternaria tagetica Alternaria tagetica Alternaria tropica Alternaria tropica Coriadrum sativum Coriadrum sativum Echinaceae sp. Echinaceae sp. Euphorbia hyssopifolia Euphorbia pulcherrima Ipomoea batatas Ipomoea batatas Ipomoea batatas Ipomoea batatas Tagetes sp. Tagetes sp. Tagetes erecta Passiflora edulis Passiflora edulis NB622 COAD 2594 CBS 116117 CBS116118 CBS 133874 CBS 19886 CBS 219.79 PPRI 8988 PPRI 8990 PPRI 13903 CBS 297.79 CBS 298.79 CBS 479.81 CBS 631.93 CBS 117216 Algeria Brazil New Zealand New Zealand USA USA Ethiopia South Africa South Africa South Africa UK UK UK USA USA – MN433697 KJ718684 KJ718685 KJ718019 KJ718017 KJ718689 KJ718690 KJ718706 KJ718709 KJ718759 KJ718760 KJ718761 KJ718768 KJ718769 MK513409 MT050451 KJ718015 KJ718016 KJ718687 KJ718686 KJ718020 KJ718021 KJ718035 KJ718038 KJ718080 KJ718081 KC584143 KJ718088 KJ718089 MK513420 MN433698 KJ718343 KJ718344 KJ718347 KJ718345 KJ718348 KJ718349 KJ718370 KJ718373 KJ718428 KJ718429 KC584434 KJ718436 KJ718437 (1963) based on a sample of diseased coriander from India. This was based on morphological features and supposed host-specificity. Morphology of COAD 2594 was found to be rather similar to that described by Raghunath (1963) for A. poonensis, including for the absence of bifurcated beaks, thought to be typical for A. dauci but not seen in COAD 2594 (either from in planta or in vitro materials). Nevertheless, based on their molecular phylogenetic analysis Woudenberg et al. (2014), placed A. poonensis as a synonym of A. dauci. Results of our phylogenetic analysis of COAD 2594 were in agreement with Woudenberg et al. (2014) (Fig. 2). COAD 2594 grouped with the type strain of A. dauci (CBS 111.38) and other A. dauci strains isolated from coriander in the United States US-FL-1 (Poudel and Zhang 2018) and Algeria NB622 (Bessadat et al. 2019). Alternaria dauci is now recognized to be a polyphagous pathogen infecting several plants in the Apiaceae. COAD 2594 was also proven to be capable of infecting carrots, parsley and celery in our inoculation studies, further confirming the polyphagous status (within the Apiaceae) of A. dauci. In Brazil this species has already been reported in association with coriander seeds and plants (Reis et al. 2006). Nevertheless these reports were never supported by molecular data and no voucher specimens were deposited in herbaria or culture collections. Here the identity of Alternaria dauci as the ethiological agent of leaf spots and blight of coriander in Brazil is fully confirmed for the first time. 38 Page 4 of 5 Australasian Plant Dis. Notes (2020) 15: 38 Fig. 2 Multilocus phylogenetic tree of Alternaria species inferred from Bayesian analysis based on combined Alt a 1, GAPDH and RPB2 sequences. Bayesian posterior probabilities above 0.7 are indicated above the nodes. Isolate from Brazil are highlighted in bold. 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