Development of new primers based on gapdh gene for Cercospora species and new host and fungus records for Iran

INTRODUCTION

Fungi in the genus Cercospora (Mycosphaerellaceae, Capnodiales) are known as serious plant pathogens, causing major losses on a wide range of crop plants worldwide including sugar beet (Weiland & Koch 2004; Bakhshi et al. 2011; Vaghefi et al. 2018), beans (Chand et al. 2015; Duangsong et al. 2016), faba beans (Kimber & Paull 2011), corn (Crous et al. 2006), carrots (Kushalappa et al. 1989), sesame (Bakhshi & Zare 2020) and soybean (Soares et al. 2015; Bakhshi & Zare 2020) as well as many vegetable and ornamental species. Several taxa are also considered potential biocontrol agents of weeds (Tessmann et al. 2001; Praveena & Naseema 2004).

Correct identification of Cercospora species has a crucial role in order to understand the epidemiology of the diseases caused by these taxa and to develop effective control measures. Due to the lack of useful morphological characters and high levels of intraspecific variation, morphology does not provide sufficient and informative characters for an accurate identification of Cercospora species (Groenewald et al. 2013; Bakhshi et al. 2012b, 2015a, 2015b). Therefore, traditional identification systems in Cercospora relied heavily on host plant association (Crous & Braun 2003). Molecular studies of Cercospora spp. in recent years revealed that many taxa have broader host ranges (Groenewald et al. 2013; Bakhshi et al. 2015a, 2018), consequently reliance on host data in Cercospora taxonomy has proven problematic.

Phylogenetic analyses based on DNA sequences have led to momentous progress in the systematics of the genus. In this regard, phylogenetic performance of sequence data of eight genomic loci including ITS, actA, cmdA, gapdh, his3, rpb2, tef1 and tub2, were assessed for Cercospora species based on the inter-/intraspecific distance ratio and clade recovery (Groenewald et al. 2013; Bakhshi et al.2015a, 2018; Bakhshi 2019). According to these results, none of the genes analyzed provides an effective barcode on its own across the entire genus. However, Bakhshi et al. (2018) showed that, gapdh is a strong candidate for improved species delimitation in Cercospora and this gene provided better insight, especially into species complexes. The amplification of gapdh with available primers (Berbee et al. 1999; Myllys et al. 2002) was, however, not easy and indicated the need of new primer designation for gapdh in Cercospora.

Therefor our primary aim was to designate an additional primer set for amplification of gapdh in Cercospora. In addition, our secondary aim was to characterize Cercospora species gained from the infected leaves of several plant species collected from different provinces of Iran, based on morphology, cultural characteristics and phylogenetic analyses of the DNA sequence data.

MATERIALS AND METHODS

Samples and morphology

Plant samples with Cercospora leaf spot symptoms were collected from seven provinces of Iran including Ardabil, Golestan, Guilan, Hormozgan, Khuzestan, Mazandaran and North Khorasan during the growing seasons 2017–2019, taken to the laboratory, and examined under a Nikon SMZ 445 stereo-microscope to observe sporulation. Fungal strains were isolated in pure culture by direct transfer of spores from a single leaf spot onto plates containing 2% malt extract agar (MEA; Fluka, Hamburg, Germany) with a sterile fine pointed needle as explained in Bakhshi et al. (2011). Representative samples of diseased specimens were dried in a plant press and deposited in the Fungal Herbarium of the Iranian Research Institute of Plant Protection (IRAN F). Representative isolates of the fungi were deposited in the Culture Collection of the Iranian Research Institute of Plant Protection (IRAN C), Tehran, Iran.

Morphological descriptions are based on structures from dried material. Diseased leaf tissues were examined under a Nikon SMZ 445 stereo-microscope and taxonomically informative morphological structures (stromata, conidiophores and conidia) were picked up from lesions with a sterile dissecting needle and mounted on glass slides in clear lactic acid. Structures were examined under an Olympus-BX51 (Olympus, Tokyo, Japan) light microscope, and photographed using a mounted Olympus DP 25 high definition color camera. Thirty measurements were made at ×1000 for each microscopic structure, and 95% confidence intervals were derived for the measurements with extreme values given in brackets.

DNA isolation, PCR amplification and sequencing

Mycelium from actively growing fungal cultures was scraped from the surface of MEA using a sterile scalpel blade and DNA was isolated using the protocol of Möller et al. (1992). The DNA samples were subsequently diluted 50–100 times in preparation for further DNA amplification reactions. The primers V9G (de Hoog & Gerrits van den Ende 1998) and ITS4 (White et al. 1990) were used to amplify part of the nuclear rRNA operon (ITS) spanning the 3’ end of 18S rRNA gene, the first internal transcribed spacer, the 5.8S rRNA gene, the second ITS region and the 5’ end of the 28S rRNA gene. Part of the actin gene (actA) was amplified using the primer set ACT-512F (Carbone & Kohn 1999) and ACT2Rd (Groenewald et al. 2013), whereas the primer set EF1-728F (Carbone & Kohn 1999) and EF-2 was used to amplify part of the translation elongation factor 1-alpha (tef1) gene. Primers employed for the amplification of calmodulin gene (cmdA) included CAL-228F and CAL-737R (Carbone & Kohn 1999) or CAL-2Rd (Groenewald et al. 2013), while the primer set CylH3F and CylH3R (Crous et al. 2004) was used to amplify part of the histone H3 gene (his3). The PCR amplifications were performed in a total volume of 25 µL on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, California). The protocols, and conditions for standard PCR amplification of the loci followed Bakhshi & Arzanlou (2017) and subsequent sequencing was performed in both directions using the PCR primers by Microsynth company (Balgach, Switzerland).

To amplify part of the gapdh, new primer set was designated here. For this purpose, the available sequences of gapdh for Cercospora spp. were retrieved from National Center for Biotechnology Information (NCBI) GenBank sequence database and were aligned with the MEGA v.7 (Molecular Evolutionary Genetics Analysis) software (Kumar et al. 2016). The forward and reverse primers were designed in regions showing similarity between different sequences using the OligoCalc (Oligonucleotide Properties Calculator) online software (http://biotools.nubic.northwestern.edu/OligoCalc.html) (Kibbe 2007). Synthesis of primers was carried out by Microsynth company. The different PCR mixtures and conditions were tested using the new primers to set the best condition and PCR mixture for amplification of part of the gapdh. Finally, the resulting fragments were sequenced in both directions using the PCR primers.

Sequence alignment and phylogenetic inference

The raw trace files were inspected and edited with MEGA v.7 software (Kumar et al. 2016), and consensus sequences were manually generated from the forward and reverse sequences. The newly generated sequences were blasted against the NCBI’s GenBank sequence database using MegaBLAST to identify related taxa. The obtained sequences from GenBank together with the novel sequences generated during this study were initially aligned with the MAFFT v.7 online interface using default settings (http://mafft.cbrc.jp/alignment/server/) (Katoh & Standley 2013) for each gene and, whenever necessary, manually improved in MEGA v.7.

For phylogenetic comparison, Bayesian inference (BI) analyses on individual gapdh gene and concatenated ITS, actA, cmdA, gapdh, his3 and tef1 loci were performed with MrBayes 3.2.6 (Ronquist et al. 2012). The best evolutionary model for each data partition was obtained using the software MrModelTest v. 2.3 (Nylander 2004). The heating parameter was set at 0.15 and the Markov Chain Monte Carlo (MCMC) analysis of four chains was started in parallel from a random tree topology and lasted until the average standard deviation of split frequencies came below 0.01. Trees were saved each 1 000 generations and the first 25% of saved trees were discarded as the ‘burn-in’ phase and posterior probabilities (PP) determined from the remaining trees. The resulting phylogenetic tree was printed with Geneious v. 5.6.7 (Drummond et al.2012). Sequences derived from this study were lodged at NCBI’s GenBank nucleotide database (http://www. ncbi.nlm.nih.gov; Table 1).

RESULTS AND DISCUSSION

Field survey

During the field survey of this study, leaf spot symptoms of various species of Cercospora were associated with different plant species including important crops and vegetables such as sugar beet (Beta vulgaris), celery (Apium graveolens), alfalfa (Medicago sativa), kohlrabi (Brassica oleracea), radish (Raphanus sativus), basil (Ocimum basilicum) and mint (Mentha longifolia), ornamentals such as Gazania sp. and Boston ivy (Parthenocissus tricuspidata), medical plants and or weeds such as mallow (Malva sp.), camel thorn (Alhagi maurorum), hemp-agrimony (Eupatorium cannabinum), sticky nightshade (Solanum sisymbriifolium), creeping cinquefoil (Potentilla reptans) etc. (Fig. 1).

Fig. 1. Disease symptoms associated with Cercospora spp. in the field. a. C. althaeina on Malva sp.; b. C. beticola and C. gamsiana on Beta vulgaris; c. C. beticola on Raphanus sativus; d–i. C. cf. flagellaris on d. Brassica oleracea, e. Ocimum basilicum, f.Mentha longifolia, g.Parthenocissus tricuspidata, h. Lapsana sp., i. Solanum sisymbriifolium;j. C. rumicis on Rumex sp.;k, l. Cercospora sp. G on k. Eupatorium cannabinum, l. Gazania sp.; m. Cercospora sp. T on Potentilla reptans; n. C. mercurialis onMercurialis annua; o.C. violae onViola sp.; p.C. zebrina on Alhagi maurorum.

Table 1. Collection details and GenBank accession numbers of Cercospora isolates included in this study.

Table 1. Continue…

Table 1. Continue …

* new host species and family records. ** new locality (province) record

Cercospora mercurialis was confirmed for the first time in Iran (Asia) using multi-gene molecular data. In addition, several new host species and families were recognized for the previously known Cercospora species including C. apii, C. beticola, C. cf. flagellaris, C. gamsiana, C. iranica, Cercospora sp. G & T in the world, and some species were recorded for the first time in some provinces of Iran. The species are treated as follows.

Fig. 2. part 2. Phylogenetic tree inferred by Bayesian analysis of the gapdh sequence alignment using MrBayes v.3.2.6. The scale bar indicates 0.01 expected changes per site. Cercospora species could be identified based on the results of the gapdh phylogeny, are indicated in colored blocks.

Fig. 3. Part 3. Phylogenetic tree inferred by Bayesian analysis of the combined 6-gene (ITS, tef1, actA, cmdA, his3 and gapdh) sequencealignment using MrBayes v.3.2.6. The scale bar indicates 0.01 expected changes per site. Cercospora species could be identified based on the results of the 6-gene phylogeny, are indicated in colored blocks.

Fig. 4. Cercospora althaeina. a, b. Fasciculate conidiophores; c–f. Conidia. Scale bars = 10 µm.

Cercospora althaeina Sacc., Michelia 1: 269 (1878) (Fig. 4)

Description.Leaf spots distinct, angular to irregular, mostly vein-limited, olivaceous-brown, sometimes grey-brown with dark brown margin, center becoming pale grey with black dots (= stroma with conidiophores). Caespituli amphigenous, mostly epiphyllous. Mycelium internal. Stromata well-developed, emerging through stomatal openings or erumpent through the cuticle. Conidiophores in divergent fascicles (6–18), pale olivaceous-brown at the base, paler upwards, 2–8-septate, straight to mildly curved, (50–)130–170(–250) × 3.5–6 µm, conically narrowed at the apex; loci conspicuous, apical or on shoulders formed by geniculation, 1.5–2 µm. Conidia solitary, obclavate-cylindrical to filiform, not acicular, straight to mildly curved, hyaline, 4–12-septate, obtuse at the apex, subtruncate or obconically truncate at the base, (40–)70–95(–145) × 3–5 µm. 

Specimen examined. IRAN, Mazandaran province, Amol, 36°28'31.21'' N, 52°27'56.69''E, on leaves of Malva sp. (Malvaceae), 2 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3920C, IRAN 17716F). Notes: Based on available literature (Ershad 2009; Bakhshi et al. 2012a, 2015a, 2018, Ershad et al. 2018), C. althaeina is reported here for the first time from Mazandaran Province.

Cercospora apii Fresen., emend. Groenewald et al., Phytopathology 95: 954 (2005)

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, Guilan province, Paresar, Pilembra, 37°35'43.51''N, 49°04'51.62''E, on leaves of Apium graveolens (Apiaceae), 17 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3921C, IRAN 17717F); Golestan province, Galikesh, 37°16'28.9"N 55°25'33.2"E, on leaves of Ipomoea hederacea (Convolvulaceae), 3 Nov. 2017, M. Bakhshi & A. Bahramishad (IRAN 3922C, IRAN 17718F). Notes. In this investigation, Cercospora apii was found for the first time on Ipomoea hederacea in the world based on multi-gene phylogeny and morphological data.

Cercospora beticola Sacc., emend. Groenewald et al., Phytopathology 95: 954 (2005)

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, Ardabil province, Moghan, 39°30'08.27''N, 48°02'38.62''E, on leaves of Beta vulgaris, 14 May 2018, M. Bakhshi (IRAN 3923C, IRAN 17720F) (P 631 I2, IRAN 17719F); Mazandaran province, Kelardasht, Goharkela, 36°28'59.04''N, 51°14'58.68''E, on leaves of B. vulgaris, 12 Aug. 2018, M. Bakhshi & A. Bahramishad  (IRAN 3924C, IRAN 17721F); Mazandaran province, Marzanabad, Foshkour, 36°21'29.2"N 51°11'43.0"E, on leaves of B. vulgaris, 12 Aug. 2018, M. Bakhshi & A. Bahramishad (P 656 R2, IRAN 17722F); IRAN, Khuzestan province, Shush-Dezful, 32°15'14.5"N 48°22'46.9"E, on leaves of Raphanus sativus (Brassicaceae), 22 Feb. 2018, M. Bakhshi & F. Ghamghami (IRAN 3925C, IRAN 17723F).

Notes. In the present research, C. beticolais found for the first time on Raphanus sativus in the world, thus a further family, Brassicaceae was added to the host range of this species.

Cercospora bizzozeriana Sacc. & Berl., Malpighia 2: 248 (1888)

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, North Khorasan province, Bojnourd, 37°28'35.27''N, 57°19'01.47''E, on leaves of Cardaria draba (Brassicaceae), 6 Nov. 2017, M. Bakhshi & A. Bahramishad (IRAN 3926C, IRAN 17724F).

Cercospora conyzae-canadensisM. Bakhshi, Arzanlou, Babai-ahari, Crous & U. Braun, Persoonia 34: 77 (2015a)

Description and illustration: Bakhshi et al. (2015a).

Specimens examined. IRAN, Mazandaran province, Sangdeh, 36°08'05.72''N, 53°12'49.12''E, on leaves of Conyza canadensis (Asteraceae), 31 Oct. 2017, M. Bakhshi & A. Bahramishad (IRAN 3927C, IRAN 17725F); Mazandaran province, Amol, Baudeh, 36°34'52.46''N, 52°20'59.88''E, on leaves of Conyza canadensis, 3 May 2018, M. Bakhshi & A. Bahramishad (IRAN 3928C, IRAN 17726F).

Notes: Cercospora conyzae-canadensiswas described recently by Bakhshi et al. (2015a) from Guilan and Zanjan provinces as host-specific to Conyza canadensis. Here the species recorded on this host, for the first time from Mazandaran Province.

Cercospora cylindracea M. Bakhshi, Arzanlou, Babai-ahari, Crous & U. Braun, Persoonia 34: 78 (2015a)

Description and illustration: Bakhshi et al. (2015a).

Specimens examined. IRAN, Mazandaran province, Galugah-Sefidchah, 36°41'50.38''N, 53°47'58.84''E, on leaves of Cichorium intybus (Asteraceae), 8 Nov. 2017, M. Bakhshi & A. Bahramishad (IRAN 3929C, IRAN 17727F); North Khorasan province, Eshghabad, Raz, 37°41'47.6"N 56°55'08.7"E, on leaves of Cichorium intybus, 7 Nov. 2017, M. Bakhshi & A. Bahramishad (IRAN 3930C, IRAN 17728F).

Notes: Cercospora cylindracea was described by Bakhshi et al. (2015a) from Ardabil, West Azerbaijan and Zanjan provinces on the host plants,Cichorium intybus and Lactuca serriola (Asteraceae) based on multi-gene phylogeny and morphological data. The species recorded here for the first time from North Khorasan and Mazandaran Provinces.

Cercospora cf. flagellaris

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, Guilan province, Rasht, 37°11'04.66''N, 49°39'34.09''E, on leaves of Conyza canadensis, 14 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3931C, IRAN 17729F); Golestan province, Gorgan, 36°50'26.22''N, 54°27'24.98''E, on leaves of Ocimum basilicum (Lamiaceae), 5 July 2017, M. Bakhshi & F. Ghamghami (IRAN 3932C, IRAN 17730F); Mazandaran province, Tonekabon, Sehezar Road, 36°36'14.26''N, 50°50'20.64''E, on leaves of Plantago major (Plantaginaceae), 13 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3933C, IRAN 17731F); Mazandaran province, Babol, Tazehabad, 36°33'01.58''N, 52°47'39.56''E, on leaves of Abutilon theophrasti (Malvaceae), 11 Oct. 2017, M. Bakhshi & F. Ghamghami (IRAN 3934C, IRAN 17732F); Guilan province, Shaft, Siahmazgi, Livandan, 37°01'19.13''N, 49°16'25.45''E, on leaves of Brassica oleracea (Brassicaceae), 16 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3935C, IRAN 17733F) (IRAN 3936C, IRAN 17734F); Mazandaran province, Tonekabon, Sehezar Road, 36°36'14.26''N, 50°50'20.64''E, on leaves of Calendula sp. (Asteraceae), 13 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3937C, IRAN 17735F); Guilan province, Talesh, Jokandan, on leaves of Fallopia convolvulus (Polygonaceae), 25 Aug. 2019, M. Kermanian (IRAN 3938C, IRAN 17736F); Guilan province, Havigh, Eshikaghasi, on leaves of Fallopia convolvulus, 25 Aug. 2019, M. Kermanian (IRAN 3939C); Guilan province, Shaft, Siahmazgi, Doudvazan Waterfall, 37°01'02.61''N, 49°15'01.21''E, on leaves of Lapsana sp. (Asteraceae), 16 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3940C, IRAN 17737F); Mazandaran province, Tonekabon, Sehezar Road, 36°36'14.26''N, 50°50'20.64''E, on leaves of Mentha longifolia (Lamiaceae), 13 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3941C, P 682I2, IRAN 17738F); Guilan province, Paresar, Pilembra, 37°35'43.51''N, 49°04'51.62''E, on leaves of Parthenocissus tricuspidata (Vitaceae), 17 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3942C, IRAN 17739F); Guilan province, Rasht, Saravan, 37°10'34.52''N, 49°35'50.17''E, on leaves of Solanum sisymbriifolium (Solanaceae), 16 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3943C, IRAN 17740F); Mazandaran province, Babol, Tazehabad, 36°33'01.58''N, 52° 47'39.56''E, on leaves of Sonchus sp. (Asteraceae ), 11 Oct. 2017, M. Bakhshi & F. Ghamghami (IRAN 3944C, IRAN 17741F). Mazandaran province, Tonekabon, Dohezar Road, Barseh, 36°38'28.1"N, 50°43'48.5"E, Unknown, 13 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3945C, IRAN 17742F).

Notes: Recently based on the combination of morphological and multi-gene phylogenetic analysis, it has been demonstrated that, C. cf. flagellaris is a plurivorous species with multiple family-associations in different groups of plants viz. agricultural crops, ornamentals, forest trees and weeds including Acerceae, Amaranthaceae, Araceae, Asteraceae, Balsaminaceae, Brassicaceae, Buxaceae, Caesalpinaceae, Campanulaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae, Geraniaceae, Hydrangeaceae, Malvaceae, Oleaceae, Onagraceae, Phytolaccaceae, Poaceae, Pontederiaceae, Rutaceae, Salicaceae, Solanaceae and Urticaceae, and is geographically distributed worldwide (Groenewald et al. 2013; Bakhshi et al. 2015a, 2018; Farr & Rossman 2020). Similar to Bakhshi et al. (2015a, 2018), in the present study, C. cf. flagellaris was the most common species in the country. Additionally, C. cf. flagellaris is newly recorded here on 10 new hosts, Brassica oleracea, Conyza canadensis, Fallopia convolvulus, Lapsana sp., Mentha longifolia, Ocimum basilicum, Parthenocissus tricuspidata, Plantago major, Solanum sisymbriifolium and Sonchus sp. in the world. Thus, three more plant families including Lamiaceae, Polygonaceae and Vitaceae were added to the host range of this species. In addition, it is reported for the first time from Golestan province.

Cercospora gamsiana M. Bakhshi & Crous, IMA Fungus 9: 321 (2018)

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, Mazandaran province, Kelardasht, Goharkela, 36°28'59.04''N, 51°14'58.68''E, on leaves of B. vulgaris, 12 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3946C, IRAN 17743F); Hormozgan province, Minab, 27°06'29.9"N 57°05'28.4"E, on leaves of Malva sp. (Malvaceae), 9 March 2018, M. Bakhshi (IRAN 3947C, IRAN 17744F).

Notes: Cercospora gamsiana was described recently by Bakhshi et al. (2018) on Malva spp., Rumex crispus, Sesamum indicum and Sonchus sp. from north and north-west of Iran. The species reported here for the first time from the south of the Iran (Hormozgan province). Furthermore, the report of this species on Beta vulgaris is new for the world.

Cercospora iranica M. Bakhshi, Arzanlou, Babai-ahari, Crous & U. Braun, Persoonia 34: 79 (2015a)

Description and illustration: Bakhshi et al. (2015a).

Specimens examined. IRAN, Guilan province, Siahkal, 37°11'58.61''N, 49°55'20.78''E, on leaves of Bidens tripartite (Asteraceae), 14 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3948C, IRAN 17745F).

Notes: Cercospora iranica was described by Bakhshi et al. (2015a) on Vicia faba (Fabaceae)and Hydrangea sp. (Hydrangeaceae). The report of this species on Bidens tripartita is new for the world.

Cercospora mercurialis Pass., in Thüm., Mycoth. Univ., No. 783. (1877) (Fig. 5)

Description. Leaf spots amphigenous, circular to subcircular, 1–5 mm, grey-brown, with dark brown border. Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in moderately loose fascicles (3–15), arising from a moderately-developed, erumpent, brown stroma, up to 25 μm diam; conidiophores pale to medium brown, aseptate or sparingly septate, straight to geniculate-sinuous due to sympodial proliferation, simple, uniform in width, sometimes constricted at the proliferating point, (15–)30–40(–55) × 3.5–4(–5) μm. Conidiogenous cells intercalary and terminal, sometimes conidiophores reduced to conidiogenous cells, pale brown, proliferating sympodially, 15–30 × 3.5–5 μm, multi-local; loci distinctly thickened, darkened and somewhat refractive, apical, lateral or formed on shoulders caused by geniculation, 2–3 μm diam. Conidia solitary, cylindrical to acicular, straight to slightly curved, hyaline, (25–)55–80(–120) × 2.5–5 μm, (3–)6–9(–15)-septate, with subobtusely rounded apices and subtruncate or obconically truncate bases; hila thickened, darkened, refractive, 1–2 μm diam.

Specimens examined. IRAN, Golestan province, Gorgan, 36°50'26.22''N, 54°27'24.98''E, on leaves of Mercurialis annua (Euphorbiaceae), 1 Nov. 2017, M. Bakhshi (IRAN 3949C, IRAN 17746F); on leaves of M. annua, 5 May 2018, M. Bakhshi (IRAN 3950C, IRAN 17747F)

Notes: Cercospora mercurialis was reported from Iran based on morphological data (Pirnia et al. 2010). To our knowledge, this study is the first molecular confirmation of C. mercurialis in Asia. Furthermore, part of the gapdh is sequenced for the first time in this species.

Cercospora plantaginis Sacc., Michelia 1: 267 (1878).

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, North Khorasan province, Eshghabad, Raz, 37°41'47.58''N, 56°55'08.65''E, on leaves of Plantago lanceolata (Plantaginaceae), 7 Nov. 2017, M. Bakhshi & A. Bahramishad (IRAN 3951C, IRAN 17748F); Azerbaijan-Iran border, Ardabil province, Mil-Mughan Water Reservoir, 39°25'55.7"N, 47°22'16.8"E, on leaves of Plantago lanceolata, 15 may 2018, M. Bakhshi (IRAN 3952C, IRAN 17749F).

Notes: Recently Bakhshi et al. (2018) have designated an epitype for C. plantaginis based on the combination of morphological and molecular data, and have shown that the species ishost-specific toPlantago lanceolata. This is the first report of this species from North Khorasan Province.

Cercospora rumicis Pavgi & U.P. Singh, Mycopathol. Mycol. Appl. 23: 191 (1964) (Fig. 6)

Description. Leaf spots circular to subcircular, with grey center and purple-brown margin, 2–8 mm diam. Mycelium internal. Caespituli amphigenous, brown. Conidiophores in divergent fascicles, arising from the upper cells of a moderately to well-developed, intraepidermal and substomatal, brown stroma; conidiophores pale brown to brown, 1–6-septate, straight, sinuous to distinctly geniculate, (40–)58–70 × 4– 5 μm, irregular in width, constricted at the parts of proliferation or at the septa. Conidiogenous cells terminal or intercalary, unbranched, pale brown, smooth, proliferating sympodially, multi-local; loci thickened, darkened, refractive, apical or formed on the shoulders caused by geniculation. Conidia solitary, subcylindrical to filiform, straight to mildly curved, hyaline, distinctly 2–15-septate, subobtuse at the apex, truncate at the base, (37–)80–110(–160) × 2.5–5 μm; hila thickened, darkened, refractive, 1.5–2.5 μm diam.

Specimen examined. IRAN, Mazandaran province, Amol, Najarmahalleh, 36°26'39.88''N, 52°27'11.02''E, on leaves of Rumex sp. (Polygonaceae), 3 May 2018, M. Bakhshi & A. Bahramishad (IRAN 3953C, IRAN 17750F).

Notes: Cercospora rumicisrecorded here for the first time from Mazandaran Province.

Cercospora sp. G sensu Groenewald et al. (2013)

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, Guilan province, Shaft, Siahmazgi, Doudvazan Waterfall, 37°01'02.61''N, 49°15'01.21''E, on leaves of Eupatorium cannabinum (Asteraceae), 16 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3954C, IRAN 17751F); Guilan province, Rasht, 37°11'04.66''N, 49°39'34.09''E, on leaves of Gazania sp.(Asteraceae), 14 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3955C, IRAN 17752F); Mazandaran province, Tonekabon, Dohezar Road, Barseh, 36°38'28.1"N, 50°43'48.5"E, on leaves of Lapsana sp., 13 Aug. 2018, M. Bakhshi & A. Bahramishad (IRAN 3956C, IRAN 17753F) (IRAN 3957C, IRAN 17754F).

Notes: Cercospora sp. G occurs on a wide host range such as Amaranthaceae, Asteraceae, Cucurbitaceae, Lamiaceae, Malvaceae, Plantaginaceae, Poaceae (Groenewald et al. 2013, Bakhshi et al. 2015a). Cercospora sp. G is found in this research, on three new hosts, Eupatorium cannabinum, Gazania sp. and Lapsana sp. in the world, and additionally for the first time from Mazandaran province.

Cercospora sp. T sensu Bakhshi et al. (2015a)

Description and illustration: Bakhshi et al. (2015a).

Specimens examined. IRAN, Mazandaran province, Salmanshahr, 36°42'22.4"N, 51°12'44.6"E, on leaves of Helianthus tuberosus (Asteraceae), 13 Aug 2018, M. Bakhshi & A. Bahramishad (IRAN 3958C, IRAN 17755F); Guilan province, Sowme’eh Sara, Lifshagard, 37°19'49.0"N, 49°25'12.6"E, on leaves of Mentha longifolia, 17 Aug 2018, M. Bakhshi & A. Bahramishad (IRAN 3959C, IRAN 17756F); Mazandaran province, Tonekabon, Sehezar Road, 36°36'14.26''N, 50°50'20.64''E, on leaves of Plantago major, 13 Aug 2018, M. Bakhshi & A. Bahramishad (P 686 I1); Mazandaran province, Salmanshahr, 36°42'22.4"N, 51°12'44.6"E, on leaves of Potentilla reptans (Rosaceae), 13 Aug 2018, M. Bakhshi & A. Bahramishad (IRAN 3960C, IRAN 17757F).

Notes: Cercospora sp. T was reported on Coreopsis sp. (Asteraceae) (Bakhshi et al. 2015a). In this research, Cercospora sp. T, is found on four new hosts, Helianthus tuberosus, Mentha longifolia, Plantago major and Potentilla reptans, therefore three more plant families including Lamiaceae, Plantaginaceae and Rosaceae were added to the host range of this species in the world. Inaddition, it is reported for the first time from Mazandaran province.

Cercospora uwebrauniana M. Bakhshi & Crous, IMA Fungus 9: 317 (2018)

Description and illustration: Bakhshi et al. (2018).

Specimens examined. IRAN, Golestan province, Gorgan-Aghghala, 36°52'15.4"N, 54°25'49.4"E, on leaves of Heliotropium europaeum (Boraginaceae), 1 Nov. 2017, M. Bakhshi & A. Bahramishad (IRAN 3961C, IRAN 17758F); Mazandaran province, Amol, Ejbarkola, 36°28'31.2"N 52°27'56.7"E, on leaves of Heliotropium europaeum, 14 Oct. 2017, M. Bakhshi & A. Bahramishad (IRAN 3962C).

Notes: Cercospora uwebraunianawas described recently by Bakhshi et al. (2018) and appears to be host specific to Heliotropium europaeum. Here we report this species for the first time from Golestan and Mazandaran provinces.

Cercospora violae Sacc., Nuovo Giron. Bot. Ital. 8: 187 (1876) (Fig. 7)

Description.Leaf spots circular to irregular, mostly vein-limited, dark brown, with concentric rings (= stroma with conidiophores), 2–8 mm diam. Mycelium internal. Caespituli amphigenous. Stromata lacking to moderately developed, dark brown, intraepidermal, and substomatal. Conidiophores in moderately dense fascicles, irregular in width, slightly attenuated at the upper portion, straight or mildly sinuous-geniculate, straight, simple, rarely branched, pale brown to brown, short conically truncate at the apex, wider at the base, 45–70(–90) × 3.5–4.5 µm, 2–12-septate. Conidiogenous cells integrated, terminal, rarely intercalary, proliferating sympodially, multilocal; loci distinct, thickened, apical or formed on shoulders caused by geniculation, 2–3.5 µm diam. Conidia solitary, hyaline, subcylindrical to obclavate or acicular, straight to slightly curved, truncate at the base, subobtuse at the apex, 44–95(–132) × 2.5–3.5 µm, 3–14-septate, smooth.

Specimen examined. IRAN, Golestan province,

Gorgan, Shastkola, 36°46'59.0"N, 54°21'58.0"E, on leaves of Viola sp.(Violaceae), 6 July 2017, M. Bakhshi & F. Ghamghami (IRAN 3963C, IRAN 17759F) (IRAN 3964C, IRAN 17760F).

Cercospora zebrina Pass., Hedwigia 16: 124 (1877) (Fig. 8)

Description.Leaf spots distinct, circular to irregular, brown to dark grey, without definite borders. Caespituli amphigenous. Mycelium internal. Stromata well-developed, intraepidermal or substomatal. Conidiophores in moderately dense fascicles (4–18), brown at the base, paler upwards, 1–6-septate, straight to mildly curved, (30–)50–65(–98) × 3.5–5 µm. Conidiogenous cells mostly terminal, pale brown, proliferating sympodially, uni-local to multi-local; loci conspicuous, thickened, darkened, refractive, apical, 2–3 µm. Conidia solitary, rarely catenate, cylindrical to obclavate-subcylindrical, straight to mildly curved, hyaline, 3–14-septate, obtuse at the apex, subtruncate or obconically truncate at the base, (30–)50–85(–135) × 3–5 µm.

Specimens examined. IRAN, Golestan province, Gorgan, 36°50'26.2"N 54°27'25.0"E, on leaves of Medicago sativa (Fabaceae), 5 July 2017, M. Bakhshi & F. Ghamghami (IRAN 3965C, IRAN 17761F); North Khorasan province, Ashkhaneh, 37°35'13.2"N 56°52'13.7"E, on leaves of Medicago sativa, 7 Nov. 2017, M. Bakhshi & A. Bahramishad (IRAN 3966C, IRAN 17762F); Mazandaran province, Galugah-Sefidchah, 36°41'50.38''N, 53°47'58.84''E, on leaves of Medicago sativa, 4 May 2018, M. Bakhshi & A. Bahramishad (IRAN 3967C, IRAN 17763F); Golestan province, Gorgan, Ghorogh Forest Park, 36°52'58.5"N, 54°40'47.2"E, on leaves of Oxalis sp. (Fabaceae), 7 May 2018, M. Bakhshi & A. Bahramishad (IRAN 3968C, IRAN 17764F); Golestan province, Aghghala_Incheboroun, Agh Ghabr, 37°00'42.6"N 54°23'43.1"E, on leaves of Alhagi maurorum (Fabaceae), 11 Nov. 2019, M. Bakhshi & A. Torabi (IRAN 3969C, IRAN 17765F).

Notes: To our knowledge, here, we report C. zebrina for the first time from Golestan and North Khorasan Provinces.

Fig. 5. Cercospora mercurialis. a. Leaf spot; b–e. Fasciculate conidiophores; f–h. Conidia. Scale bars = 10 µm.

Fig. 6. Cercospora rumicis. a–c. Fasciculate conidiophores; d–f. Conidia. Scale bars = 10 µm.

Fig. 7. Cercospora violae. a. Fasciculate conidiophores; b–c. Conidia. Scale bars = 10 µm.

Fig. 8. Cercospora zebrina. a. Fasciculate conidiophores; b–c. Conidia. Scale bars = 10 µm.

ACKNOWLEDGEMENTS

We gratefully acknowledge the Iran National Science Foundation (INSF), Research Deputy of the Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO) for financial support.