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Colletotrichum Species on Cultivated Solanaceae Crops in Russia

Colletotrichum Species on Cultivated Solanaceae Crops in Russia agriculture Article 1 1 2 2 1 , 2 Maria Yarmeeva , Irina Kutuzova , Michael Kurchaev , Elena Chudinova , Ludmila Kokaeva , 3 4 2 1 2 Arseniy Belosokhov , Grigory Belov , Alexander Elansky , Marina Pobedinskaya , Archil Tsindeliani , 1 , 5 1 , 2 , Yulia Tsvetkova and Sergey Elansky * Lomonosov Moscow State University, 119991 Moscow, Russia Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia Michurina St., 54, Michurinsk, 393760 Tambov, Russia Russian Potato Research Centre, 140051 Moscow, Russia All-Russian Plant Quarantine Center (VNIIKR), 140150 Moscow, Russia * Correspondence: elanskiy_sn@pfur.ru Abstract: Colletotrichum species are the causal agents of potato and tomato diseases, such as black dot and anthracnose. Several new species and species complexes were recently established. Thereby, a reassessment of the genus diversity is required. The study revealed two species, Colletotrichum coccodes and Colletotrichum nigrum, as Russia’s main disease agents of cultivated Solanaceae plants. Black dot and anthracnose in potato were caused exclusively by C. coccodes, whereas the same diseases in tomato, eggplant, and pepper were predominately caused by C. nigrum. However, one isolate of C. coccodes was also identified as an agent of the tomato disease. Five potentially hybrid isolates were discovered. Morphological examination and pathogenicity assessment revealed no significant differences between the two Colletotrichum species. All isolates were sensitive to the fungicides azoxystrobin, difenoconazole, and thiabendazole, which are currently used in agriculture. This is the first report of the occurrence of C. nigrum in Russia. Keywords: black dot; anthracnose; pathogen; potato; tomato; Colletotrichum; multi-gene phylogeny Citation: Yarmeeva, M.; Kutuzova, I.; Kurchaev, M.; Chudinova, E.; Kokaeva, L.; Belosokhov, A.; Belov, 1. Introduction G.; Elansky, A.; Pobedinskaya, M.; Colletotrichum is a well-known causal agent of potato and tomato diseases such as black Tsindeliani, A.; et al. Colletotrichum dot and anthracnose, dramatically damaging both underground and aboveground plant Species on Cultivated Solanaceae parts. Although several revisions of the genus Colletotrichum were recently published [1–4], Crops in Russia. Agriculture 2023, 13, it still remains taxonomically puzzling. Currently, 16 species complexes and 15 singleton 511. https://doi.org/10.3390/ species (e.g., C. coccodes and C. nigrum) are established within Colletotrichum [5]. Given the agriculture13030511 complicated systematics of the genus, the species identification is based on morphological Academic Editor: Yuan Li features, combined with molecular data. C. coccodes predominantly infects Solanaceae plants, including chilli fruit [6], potato Received: 30 December 2022 tubers [7,8], tomato [9], sweet pepper [10–12], black nightshade [13], and eggplant [14]. Revised: 12 February 2023 Nevertheless, its wide host range is not limited to Solanaceae, since the species was reported Accepted: 16 February 2023 to infect strawberry [15], pumpkin [16], or onion [17]. Published: 21 February 2023 There are very few reports concerning C. nigrum. The species was first described as an agent of pepper anthracnose from Gloucester County, New Jersey, USA [18], but it can be associated with tomato and eggplant diseases [19,20] as well. Liu and colleagues [17,21] Copyright: © 2023 by the authors. reviewed the species’ description, introduced a neotype to C. coccodes, selected an epitype Licensee MDPI, Basel, Switzerland. to C. nigrum, and stated the ability of both species to induce anthracnose. This article is an open access article In Russia, the diversity within the genus is poorly described, due to the predomi- distributed under the terms and nance of the morphological identification of causal agents. The review by Kotova and conditions of the Creative Commons Kungurtseva [22] specified that C. coccodes is the only cause of potato and tomato black Attribution (CC BY) license (https:// dot and anthracnose. Several studies in Russia investigated the diversity of Colletotrichum creativecommons.org/licenses/by/ species on potato and tomato leaves using genetic markers [9,23,24] directly from the plant 4.0/). Agriculture 2023, 13, 511. https://doi.org/10.3390/agriculture13030511 https://www.mdpi.com/journal/agriculture Agriculture 2023, 13, 511 2 of 20 material, without isolating the species in axenic cultures. Kazartsev and colleagues [25] recently scrutinized the diversity of Colletotrichum species on several wild and cultivated plants (no potato or tomato plants were included into the analysis), using molecular and morphological approaches to identify the species. C. coccodes strains were isolated from Ambrosia artemisiifolia, Beta vulgaris, Brassica napus, Cannabis sativa, Galinsoga parviflora, and Portulaca oleracea. Poluektova and colleagues [26] analysed the glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase genes of four C. coccodes strains from Russian potato. To the best of our knowledge, these are the only molecular investigations of the genus in Russia to date. This study focuses on the disease agents of several cultivated Solanaceae crops in Russia. Our research combines both morphological and molecular approaches to reveal the diversity within the genus Colletotrichum. To this end, four genetic markers were used: ITS1-5.8S-ITS2 region (ITS) as a well-established barcode, the glyceraldehyde-3-phosphate dehydrogenase gene intron (gaphd), considered the most reliable genetic marker for Col- letotrichum species, the actin intron (act), and the glutamine synthetase intron (gs). To estimate the agricultural risks of the disease spread, we assessed the sensitivity to some fungicides that are officially used for tuber treatment, and the pathogenicity range towards the tomato fruit and potato tuber slices. 2. Materials and Methods 2.1. Sampling and Isolation of Cultures Samples were collected from the fruits of tomato, eggplant, and pepper (Table 1 and Figure 1) as well as from potato tubers, leaves, and stems. Seed potato tubers from the Netherlands, Germany, Australia, Cyprus, and Uganda were taken for comparison. All isolation sources were surface-sterilized with sodium hypochlorite (2% solution) to remove possible contamination, sliced, and put in wet chambers at 24  1 C. For isolation, small black sclerotia from the tuber peel or diseased tissue were taken using a preparation needle under a binocular microscope (MBS10, Russia), and transferred to culture media (potato- dextrose agar, PDA) amended with antibiotic (benzylpenicillin sodium salt, 100 mg/L). Table 1. Details of isolates used in the study. GenBank Accession Numbers ** Origin * Isolation Year of Strain Identifier Host Plant (Figure 1) Source Isolation ITS gaphd act gs C13V(GH)PT1/1 1 Potato Tuber 2013 OP718477 OP743730 OP743793 OP743860 C13K(S)PT11 2 Potato Tuber 2013 OP718470 OP743723 OP743786 OP743853 C13K(S)PT14 2 Potato Tuber 2013 OP718471 OP743724 OP743787 OP743854 C13K(S)PT15 2 Potato Tuber 2013 OP718472 OP743725 OP743788 OP743855 C13K(S)PT17 2 Potato Tuber 2013 OP718473 OP743726 OP743789 OP743856 C13K(S)PT21 2 Potato Tuber 2013 OP718474 OP743727 OP743790 OP743857 C13K(S)PT34 2 Potato Tuber 2013 OP718475 OP743728 OP743791 OP743858 C13K(S)PT58b 2 Potato Tuber 2013 OP718476 OP743729 OP743792 OP743859 C13HPT29/2 3 Potato Tuber 2013 OP718469 OP743722 OP743836 OP743890 C13G(B)PTde8/2 4 Potato Tuber 2013 OP718463 OP743716 OP743830 OP743844 C13G(B)PTde9 4 Potato Tuber 2013 OP718464 OP743717 OP743831 OP743845 C13G(B)PTde12 4 Potato Tuber 2013 OP718461 OP743714 OP743828 OP743842 C13G(B)PTde23 4 Potato Tuber 2013 OP718462 OP743715 OP743829 OP743843 C13G(B)PTes6 4 Potato Tuber 2013 OP718466 OP743719 OP743833 OP743847 C13G(B)PTes19 4 Potato Tuber 2013 OP718465 OP743718 OP743832 OP743846 C13G(B)PTal15 4 Potato Tuber 2013 OP718456 OP743709 OP743823 OP743837 C13G(B)PTal19 4 Potato Tuber 2013 OP718457 OP743710 OP743824 OP743838 C13G(B)PTal20 4 Potato Tuber 2013 OP718458 OP743711 OP743825 OP743839 C13G(B)PTal23 4 Potato Tuber 2013 OP718459 OP743712 OP743826 OP743840 C13G(B)PTal24 4 Potato Tuber 2013 OP718460 OP743713 OP743827 OP743841 C13G(B- 5 Potato Tuber 2013 OP718468 OP743721 OP743835 OP743849 Sh)PTsa5 C13G(B- 5 Potato Tuber 2013 OP718467 OP743720 OP743834 OP743848 Sh)PTsa29 C14M(Ch)PT6 6 Potato Tuber 2014 OP718479 OP743732 OP743795 OP743862 Agriculture 2023, 13, 511 3 of 20 Table 1. Cont. GenBank Accession Numbers ** Origin * Isolation Year of Strain Identifier Host Plant (Figure 1) Source Isolation ITS gaphd act gs C14M(Ch)PT18/2 6 Potato Tuber 2014 OP718478 OP743731 OP743794 OP743861 C15M(L)PT1 7 Potato Tuber 2015 OP718480 OP743733 OP743796 OP743863 C15M(L)PT1/2 7 Potato Tuber 2015 OP718481 OP743734 OP743797 OP743864 C15M(L)PT4 7 Potato Tuber 2015 OP718482 OP743735 OP743798 OP743865 C15M(L)PT5 7 Potato Tuber 2015 OP718483 OP743736 OP743799 OP743866 C15M(L)PT6 7 Potato Tuber 2015 OP718484 OP743737 OP743800 OP743867 C15M(L)PT7 7 Potato Tuber 2015 OP718485 OP743738 OP743801 OP743868 C16ME(Y-O)PL7 8 Potato Leaf 2016 OP718490 OP743743 OP743806 OP743873 C16ME(Y- 8 Potato Leaf 2016 OP718489 OP743742 OP743802 OP743872 O)PL11 C16M(G)PS9 9 Potato Stem 2016 OP718488 OP743741 OP743805 OP743871 C16M(G)PS15 9 Potato Stem 2016 OP718486 OP743739 OP743803 OP743869 C16M(G)PS16b 9 Potato Stem 2016 OP718487 OP743740 OP743804 OP743870 C17K(K)TF5-2 10 Tomato Fruit 2017 OP718492 OP743745 OP743808 OP743875 C17K(K)TF5-14 10 Tomato Fruit 2017 OP718491 OP743744 OP743807 OP743874 C17K(S)PTrs9 11 Potato Tuber 2017 OP718494 OP743747 OP743810 OP743877 C17K(S)PTrs11/1 11 Potato Tuber 2017 OP718493 OP743746 OP743809 OP743876 C18M(L)TF1/1 7 Tomato Fruit 2018 OP718496 OP743749 OP743822 OP743889 C18K(S)TF1/2 12 Tomato Fruit 2018 OP718495 OP743748 OP743811 OP743878 C18U(G)TF1/1 13 Tomato Fruit 2018 OP716941 OP730520 OP743774 OP743898 C18U(G)PT4 13 Potato Tuber 2018 OP718500 OP743753 OP743815 OP743882 C18U(G)PT6 13 Potato Tuber 2018 OP718501 OP743754 OP743816 OP743883 C18U(G)PT7 13 Potato Tuber 2018 OP718502 OP743755 OP743817 OP743884 C18U(G)PT11 13 Potato Tuber 2018 OP718499 OP743752 OP743814 OP743881 C18TPS8 14 Potato Stem 2018 OP718497 OP743750 OP743812 OP743879 C18TPS9 14 Potato Stem 2018 OP718498 OP743751 OP743813 OP743880 C19CyPT1/2 15 Potato Tuber 2019 OP718503 OP743756 OP743783 OP743850 C19CyPT2/1 15 Potato Tuber 2019 OP718504 OP743757 OP743784 OP743851 C20AuPT5a 16 Potato Tuber 2020 OP718505 OP743758 OP743785 OP743852 C20UgLaPT1/1 17 Potato Tuber 2020 OL405711 OP743762 OP743821 OP743888 C20UgKgPT1 17 Potato Tuber 2020 OP718506 OP743759 OP743818 OP743885 C20UgKgPT2 17 Potato Tuber 2020 OP718508 OP743761 OP743820 OP743887 C20UgKgPT12 17 Potato Tuber 2020 OP718507 OP743760 OP743819 OP743886 C21KST1F1 12 Tomato Fruit 2021 OP716934 OP730512 OP743775 OP743891 C21KSTF9 12 Tomato Fruit 2021 OP716939 OP730517 OP743780 OP743896 C21KST3F1 12 Tomato Fruit 2021 OP716935 OP730513 OP743776 OP743892 C21KST3F2 12 Tomato Fruit 2021 OP716936 OP730514 OP743777 OP743893 C21KSTF88 12 Tomato Fruit 2021 OP716938 OP730516 OP743779 OP743895 C21KSTF77 12 Tomato Fruit 2021 OP716937 OP730515 OP743778 OP743894 C21KSTF97 12 Tomato Fruit 2021 OP716940 OP730518 OP743781 OP743897 C21KSTF98 12 Tomato Fruit 2021 OP716941 OP730519 OP743782 OP743899 C21KSPeF3 12 Pepper Fruit 2021 OP716931 OP743706 OP743771 OP743908 C21KSPeF4 12 Pepper Fruit 2021 OP716932 OP743707 OP743772 OP743909 C21KSPeF6 12 Pepper Fruit 2021 OP716933 OP743708 OP743773 OP743910 C21KSPeF20 12 Pepper Fruit 2021 OP716930 OP743705 OP743770 OP743907 C21KSPeF19 12 Pepper Fruit 2021 OP716929 OP743704 OP743769 OP743906 C21KSEgF1 12 Eggplant Fruit 2021 OP716923 OP743698 OP743763 OP743900 C21KSEgF3 12 Eggplant Fruit 2021 OP716924 OP743699 OP743764 OP743901 C21KSEgF4.1 12 Eggplant Fruit 2021 OP716925 OP743700 OP743765 OP743902 C21KSEgF5 12 Eggplant Fruit 2021 OP716926 OP743701 OP743766 OP743903 C21KSEgF6 12 Eggplant Fruit 2021 OP716927 OP743702 OP743767 OP743904 C21KSEgF7 12 Eggplant Fruit 2021 OP716928 OP743703 OP743768 OP743905 * Geographical origins of the isolates. Russia: 1—Vladimir Region; 2, 11—Kostroma Region; 6, 7, 9—Moscow Region; 8—the Mari El Republic; 10, 12—Krasnodar Krai; 13—Primorsky Krai; 14—the Republic of Tatarstan; 3—the Netherlands; 4, 5—Germany; 15—the Republic of Cyprus; 16—Australia; 17—Uganda. ** ITS—ITS1-5, 8S-ITS2 region, gaphd—glyceraldehyde-3-phosphate dehydrogenase gene intron, act—actin intron, gs—glutamine synthetase intron. Agriculture 2023, 13, 511 4 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 3 of 21 Figure 1. Location of collection sites (see also Table 1). Russia: 1—Vladimir Region; 2, 11—Kostroma Figure 1. Location of collection sites (see also Table 1). Russia: 1—Vladimir Region; 2, 11—Kostroma Region; 6, 7, 9—Moscow Region; 8—the Mari El Republic; 10, 12—Krasnodar Krai; 13—Primorsky Region; 6, 7, 9—Moscow Region; 8—the Mari El Republic; 10, 12—Krasnodar Krai; 13—Primorsky Krai; 14—the Republic of Tatarstan; 3—the Netherlands; 4, 5—Germany; 15—the Republic of Cy- Krai; 14—the Republic of Tatarstan; 3—the Netherlands; 4, 5—Germany; 15—the Republic of Cyprus; prus; 16—Australia; 17—Uganda. 16—Australia; 17—Uganda. Table 1. Details of isolates used in the study. 2.2. DNA Isolation, PCR, Sequencing, and Phylogenetic Analysis Origin * To Host extract DIsolation NA, the m ycY elear ium of o f fungi wa GenB s groank wn o Ac n cession Num a liquid pea m bers ** edium (180 g Strain Identifier of green pea boiled for 10 min in 1 L of water, then filtered and autoclaved for 30 min at (Figure 1) Plant Source Isolation ITS gaphd act gs 1 atm). DNA was extracted according to the standard CTAB protocol [27,28]. ITS, act, and C13V(GH)PT1/1 1 Potato Tuber 2013 OP718477 OP743730 OP743793 OP743860 gaphd amplifications were performed in a SSI microtube strips in a 25 L total volume re- C13K(S)PT11 2 Potato Tuber 2013 OP718470 OP743723 OP743786 OP743853 action containing 1 L of a DNA template (50 ng/L), 2.5 L of 10 PCR buffer (Applied C13K(S)PT14 2 Potato Tuber 2013 OP718471 OP743724 OP743787 OP743854 Biosystems, Waltham, MA, USA), 0.5 L of 10 mM each deoxyribonucleotide triphosphates C13K(S)PT15 2 Potato Tuber 2013 OP718472 OP743725 OP743788 OP743855 (dNTP), 0.4 L of 100M each primer (Evrogen Co, Moscow, Russia), 1.5 U of Taq polymerase C13K(S)PT17 2 Potato Tuber 2013 OP718473 OP743726 OP743789 OP743856 (5U/L, Promega, Madison, WI, USA), and Milli-Q water (MQ). For the amplification of gs C13K(S)PT21 2 Potato Tuber 2013 OP718474 OP743727 OP743790 OP743857 2.8 L of each dNTP was used; the concentrations of the other components remained the C13K(S)PT34 2 Potato Tuber 2013 OP718475 OP743728 OP743791 OP743858 same. The following primers were used: ITS1 5’-TCCGTAGGTGAACCTGCGG-’3 and ITS4 5’- C13K(S)PT58b 2 Potato Tuber 2013 OP718476 OP743729 OP743792 OP743859 TCCTCCGCTTATTGATATGC-3’ for the ITS region [29], GSF1 5’-ATGGCCGAGTACATCTGG- C13HPT29/2 3 Potato Tuber 2013 OP718469 OP743722 OP743836 OP743890 ’3 and GSR1 5’-GAACCGTCGAAGTTCCAC-’3 for the gs gene [30], GDF-1 5’-GCCGTCAACG C13G(B)PTde8/2 4 Potato Tuber 2013 OP718463 OP743716 OP743830 OP743844 ACCCCTTCATTGA-’3 and GDR-1 5’-GGGTGGAGTCGTACTTGAGCATGT-’3 for gaphd [31], C13G(B)PTde9 4 and ACPota T-51to 2F 5’-ATTuber GTGCAAGG2C013 CGGTTOP71 TCGC 846 -’34 andOP74 ACT-371 7837 OP74 R 5’-TAC383 GAG1 TCOP74 CTTC384 TG5 G CCCAT-’3 for act [32]. C13G(B)PTde12 4 Potato Tuber 2013 OP718461 OP743714 OP743828 OP743842 The PCR protocol included initial denaturation at 94 C for 3 min, 35 amplification C13G(B)PTde23 4 Potato Tuber 2013 OP718462 OP743715 OP743829 OP743843 cycles, and an additional extending step at 72 C for 3 min. For the primer pair ITS1/ITS4, C13G(B)PTes6 4 Potato Tuber 2013 OP718466 OP743719 OP743833 OP743847 Agriculture 2023, 13, 511 5 of 20 the amplification cycles were 94 C for 30 s, 55 C for 30 s, and 72 C for 45 s. For the primer pairs GDF-1/GDR-1 and ACT-512F/ACT783R, the amplification cycles were 94 C for 30 s, 60 C for 30 s, and 72 C for 30 s. For the primer pair GSF1/GSR1, the amplification cycles were 94 C for 30 s, 61 C for 30 s, and 72 C for 120 s. The amplification was performed on a T100 Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). PCR products were run in 0.7–1.2% agarose gel amended with ethidium bromide; the agarose concentration depended on the PCR fragment length. The gel extraction was performed with a Cleanup Mini Kit (Evrogen Co., Russia). The PCR frag- ments were sequenced using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and the Applied Biosystems 3730  l automated sequencer (Applied Biosystems, USA). Each fragment was sequenced in both directions. Consensus sequences for each locus were assembled using Geneious version 7.1 (created using Biomatters) and MEGA X [33], and aligned with available type species sequences (Table 2). Species identification was based primarily on the gaphd sequence. Table 2. Reference strains used in this study. GenBank Accession Numbers * Species Strain Species Host Complex Identifier ITS act gaphd gs C. nigrum singleton CBS 69.49 Capsicum sp. NR163523 JX546646 JX546742 - Solanum C. nigrum singleton CBS 132450 JX546845 JX546653 JX546749 - lycopersicum Cichorium C. nigrum singleton CBS 127562 JX546842 JX546650 JX546746 - intybus Alternanthera C. dianense singleton YMF 1.04943 OL842189 OL981258 OL981284 - philoxeroides Solanum C. coccodes singleton CBS 369.75 HM171679 HM171667 HM171673 HM171676 tuberosum Cyphomandra C. gigasporum Gigasporum CBS 101881 KF687736 KF687797 KF687841 KF687745 betacea * ITS—ITS1-5.8S-ITS2 region, gaphd—glyceraldehyde-3-phosphate dehydrogenase gene intron, act—actin intron, gs—glutamine synthetase intron. 2.3. Morphological Analysis The cultures were grown on synthetic nutrient-poor agar medium (SNA) [34] amended with Anthriscus sylvestris double autoclaved stems [16] for 10 days. Micro- scopic preparations were made in clear lactic acid. The width and length of conidia were measured, with 30 measurements per structure, using a Leica DM 2500 (Leica Microsystems, Wetzlar, Germany). 2.4. Pathogenicity Tests To compare the pathogenic activity, 10 isolates (two from potato, two from pepper, three from tomato, and three from eggplant) were chosen. Healthy cherry tomato fruits and potato tubers (cultivar “Gala”) were washed and surface-sterilised in 0.5% sodium hypochlorite solution for 5 min, rinsed in distilled water, and air-dried. The potato tubers were sliced to imitate wounding. Two types of tomato fruit were used: wounded with sterile tips and unwounded. The experiment was conducted with three repeats for each strain of each kind of inoculation. The wounded fruits were internally inoculated with 100 L of conidial suspension (concentration 10 spores/mL). The unwounded fruits and potato slices were surface-inoculated with mycelium and conidia, and placed in sterile wet chambers. The control fruits and tubers were surface or internally inoculated with distilled water. Each wet chamber was stored at 10 C for 21 or 35 days, and the radius of the lesion was measured. To fulfil Koch’s postulate, a small tissue sample was taken from the margin of the disease area with a sterile scalpel and placed in a Petri dish on PDA. Agriculture 2023, 13, 511 6 of 20 2.5. In Vitro Assessment of Fungicide Sensitivity Three chemical fungicides: azoxystrobin (Quadris , Syngenta, Basel, Switzerland), ® ® difenoconazole (Score , Syngenta), and thiabendazole (Tecto , Syngenta) were chosen to evaluate their efficiencies against Colletotrichum isolates. The fungicides were selected based on their current use in Russia for tuber or in-furrow treatment, and they were obtained from local suppliers. The sensitivity was evaluated in Petri plates with PDA. The fungicides were added at different concentrations to autoclaved PDA medium to produce a concentration series of 0, 0.1, 1, 10, and 100 mg/L for each fungicide (active ingredient). The mycelial plug (5 mm in diameter) of each isolate was punched from the margin of an actively growing colony of a 5-day-old culture and placed in the centre of a 90 mm PDA plate amended with fungicide, as well as on non-amended PDA plates (controls). Three replicates per treatment were produced, and the plates were incubated at 24  1 C for 4 days. The diameter of the fungal colony on each plate was measured at perpendicular angles. The average of the two measurements was used to calculate the fungicide concentration inhibiting linear colony growth of 50% over control (EC ) [35,36]. 3. Results In total, 74 isolates were analysed: 50 from potato (Solanum tuberosum L.), 11 from tomato (Solanum lycopersicum L.), 7 from pepper (Capsicum annuum L.), and 6 from eggplant (Solanum melongena L.). All strains isolated from potato, regardless of the plant organ, were identified as C. coccodes, while those from eggplant and pepper proved to be related to C. nigrum (Figures 2–6). Almost all tomato isolates except one strain (C18M(L)TF1/1) were identified as C. nigrum. The aligned concatenated sequence dataset of the original isolates was 1690 bp long (440, 207, 233, and 810 bp for the ITS, act, gaphd, and gs sequences, respectively). It contained 65 variable sites: 2 in the ITS region, 2 in the act intron, 12 in the gaphd intron, and 49 the in gs second intron (including three deletions); among them, 2 in act, 8 in gaphd, and 17 in the gs intron seemed to be specific to either C. coccodes or C. nigrum, and they may be used to differentiate between these species. Most C. coccodes, but also several C. nigrum (possible hybrids) isolates, contained a 25 bp insertion in the gs intron (Figure 7). All the nucleotide differences were found in the non-coding regions. All the phylogenetic trees, except one based on the ITS region (Figure 3), showed two well-delimited clades corresponding to two segregate species: C. coccodes and C. nigrum (Figures 2 and 4–6). The recently described C. dianense was in the same clade as C. nigrum (Figures 4 and 5). Among the tomato and pepper C. nigrum strains, five intriguing isolates (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20) were found (Figure 7). Presumably, they shared the features of both species—C. coccodes and C. nigrum. All of the isolates were identified as C. nigrum, based on the gaphd sequence (Table 3). The similarity to C. dianense is discussed further. In total, within 810 bp sequences of the gs gene, we revealed one 25 bp insertion (positions 225–249) and one 1 bp (position 470) insertion typical of C. coccodes CBS 369.75, and 17 single nucleotide polymorphisms (SNP) atypical of C. coccodes (positions 11, 51, 102, 122, 143, 144, 193, 320, 326, 357, 467, 471, 515, 588, 629, 675, and 724). Morphological differences were not found between the sizes of the C. coccodes and C. nigrum conidia (for C. coccodes, the conidial length was 18.23  6.13 m and the width was 4.49  1.04 m; for C. nigrum, the conidial length was 21.17  5.10 m and the width was 4.34  1.03 m; Figure 8). The obtained conidial measurements overlapped with those of the type strains of C. coccodes, C. nigrum, and C. dianense [16,21,37]. All of the isolates produced aseptate, smooth-walled, hyaline, oval to cylindrical conidia with acute, subacute or obtuse apices, typical of C. coccodes or C. nigrum (Figure 9). Agriculture 2023, 13, 511 7 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 8 of 21 Figure 2. Phylogenetic tree inferred from maximum-likelihood analysis of the concatenated align- Figure 2. Phylogenetic tree inferred from maximum-likelihood analysis of the concatenated align- ment, including the ITS region, and the partial act, gaphd, and gs gene regions. The confidence values ment, including the ITS region, and the partial act, gaphd, and gs gene regions. The confidence values are indicated at the branches. Green indicates C. coccodes clade, and blue indicates the C. nigrum are indicated at the branches. Green indicates C. coccodes clade, and blue indicates the C. nigrum clade. clade. Agriculture 2023, 13, 511 8 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 9 of 21 Figure 3. Phylogenetic tree inferred from maximum-likelihood analysis of the ITS1-5, 8S-ITS2 region Figure 3. Phylogenetic tree inferred from maximum-likelihood analysis of the ITS1-5, 8S-ITS2 region alignment. Boo alignment. Bootstrap tstrap 1000 rep 1000 replicates licates. The . Theco confidence nfidence value values s are areindicat indicated ed at the branche at the branches. s. Green Green indicates C. coccodes isolates; blue indicates C. nigrum isolates, and red marks C. dianense type spe- indicates C. coccodes isolates; blue indicates C. nigrum isolates, and red marks C. dianense type species. cies. Agriculture Agricultur 2023 e 2023 , 13, x FOR PEER , 13, 511 REVIEW 10 of 21 9 of 20 Figure 4. Phylogenetic tree inferred from maximum-likelihood analysis of the actin intron alignment. Figure 4. Phylogenetic tree inferred from maximum-likelihood analysis of the actin intron align- ment. Boot Bootstrap strap 1000 replicate 1000 replicates. sThe . The confidence confidence va values luesar are e indicated indicated at at the b the branches. ranches. Green Green indicate indicates s C. C. coccodes clade, blue indicates the C. nigrum clade, and red marks C. dianense type species. coccodes clade, blue indicates the C. nigrum clade, and red marks C. dianense type species. Agriculture Agricultur 2023 e 2023 , 13 ,, x FOR PEER 13, 511 REVIEW 11 of 10 21 of 20 Figure 5. Phylogenetic tree inferred from maximum-likelihood analysis of the glyceraldehyde-3- Figure 5. Phylogenetic tree inferred from maximum-likelihood analysis of the glyceraldehyde-3-phos- phate dehydrogenase phosphate dehydr intron alignment. Bootstr ogenase intron alignment. ap 1000 replica Bootstrap 1000 tes. rThe confidence va eplicates. The confidence lues are indica values tedar e at the indicated branches at . G therbranches. een indicat Gr es een C. cocc indicates odes clade, C. coccodes blue ind clade, icates blue the indicates C. nigrumthe clade C.,nigrum and red marks clade, and C. dianense type species. red marks C. dianense type species. Agriculture 2023, 13, 511 11 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 12 of 21 Figure 6. Phylogenetic tree inferred from maximum-likelihood analysis of the glutamine synthetase Figure 6. Phylogenetic tree inferred from maximum-likelihood analysis of the glutamine synthetase intron alignment. Bootstrap 1000 replicates. The confidence values are indicated at the branches. intron alignment. Bootstrap 1000 replicates. The confidence values are indicated at the branches. Green indicates C. coccodes clade; blue indicates C. nigrum clade. Green indicates C. coccodes clade; blue indicates C. nigrum clade. Agriculture 2023, 13, 511 12 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 13 of 21 Figure 7. Comparison of glutamine synthetase second intron sequences of intriguing isolates Figure 7. Comparison of glutamine synthetase second intron sequences of intriguing isolates (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20) to type strain C. coccodes (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20) to type strain C. coccodes CBS 369.75. SNPs, including A, T, C, and G, are marked with red, green, blue, and yellow, respec- CBS 369.75. SNPs, including A, T, C, and G, are marked with red, green, blue, and yellow, respectively. tively. Table 3. Comparison of intriguing isolates to type strains *. Morphological differences were not found between the sizes of the C. coccodes and C. Percentage of Similarity to Type Strains nigrum conidia (for C. coccodes, the conidial length was 18.23 ± 6.13 μm and the width was Isolate 4.49 ± 1.04 μm; for C. nigrum, the conidial length was 21.17 ± 5.10 μm and the width was ITS Sequence act Sequence gaphd Sequence gs Sequence ** 4.34 ± 1.03 μm; Figure 8). The obtained conidial measurements overlapped with those of 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% the type strains of C. coccodes, C. nigrum, and C. dianense [16,21,37]. All of the isolates pro- C18U(G)TF1/1 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes duced aseptate, smooth-walled, hyaline, oval to cylindrical conidia with acute, subacute 100% C. nigrum 97% C. coccodes 97% C. coccodes or obtuse apices, typical of C. coccodes or C. nigrum (Figure 9). 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KST1F1 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KST3F1 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KSTF77 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KSPeF20 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes * C. coccodes CBS:369.75, C. nigrum CBS:169.49, and C. dianense YMF 1.04943. ITS—ITS1-5.8S-ITS2 region, gaphd— glyceraldehyde-3-phosphate dehydrogenase gene intron, act—actin intron, gs—glutamine synthetase intron. ** No gs sequences of type C. nigrum or C. dianense isolates were found. Agricultur Agriculture e 2023 2023,, 13 13,, x FOR PEER 511 REVIEW 14 of 13 of 21 20 Agriculture 2023, 13, x FOR PEER REVIEW 14 of 21 Figure 8. Comparison of the conidial lengths (a) and widths (b) of Colletotrichum isolates and type Figure 8. Comparison of the conidial lengths (a) and widths (b) of Colletotrichum isolates and type Figure 8. Comparison of the conidial lengths (a) and widths (b) of Colletotrichum isolates and type strains. Boxes indicate quartiles (first and third), whiskers indicate the minimum and the maximum strains. Boxes indicate quartiles (first and third), whiskers indicate the minimum and the maximum strains. Boxes indicate quartiles (first and third), whiskers indicate the minimum and the maximum values, and points outside the boundary are outliers. values, and points outside the boundary are outliers. values, and points outside the boundary are outliers. Figure 9. Conidia of C. coccodes isolate C20UgKgPT2 (a) and C. nigrum isolate C21KSPeF6 (b). Figure 9. Conidia of C. coccodes isolate C20UgKgPT2 (a) and C. nigrum isolate C21KSPeF6 (b). Figure 9. Conidia of C. coccodes isolate C20UgKgPT2 (a) and C. nigrum isolate C21KSPeF6 (b). All the tested strains of both species were able to cause tomato fruit and potato tuber All the tested strains of both species were able to cause tomato fruit and potato tuber All the tested strains of both species were able to cause tomato fruit and potato tuber infection (Table 4). Regardless of the species used to infect the tomatoes, the infected fruit infection (Table 4). Regardless of the species used to infect the tomatoes, the infected fruit infection (Table 4). Regardless of the species used to infect the tomatoes, the infected fruit had typical dark lesions of anthracnose with sclerotia, milky-white swellings with conidia had typical dark lesions of anthracnose with sclerotia, milky-white swellings with conidia had typical dark lesions of anthracnose with sclerotia, milky-white swellings with conidia occasionally developed. In the case of tomato wound inoculation, the C. coccodes- and C. occasionally developed. In the case of tomato wound inoculation, the C. coccodes- and occasionally developed. In the case of tomato wound inoculation, the C. coccodes- and C. nigrum-caused disease radii were 5.2–6.2 mm and 3.2–8.3 mm, respectively, after 21 days C. nigrum-caused disease radii were 5.2–6.2 mm and 3.2–8.3 mm, respectively, after 21 days nigrum-caused disease radii were 5.2–6.2 mm and 3.2–8.3 mm, respectively, after 21 days of infection. On the intact fruit, the disease radius did not exceed 1.2 mm after 21 days for of infection. On the intact fruit, the disease radius did not exceed 1.2 mm after 21 days of infection. On the intact fruit, the disease radius did not exceed 1.2 mm after 21 days for all the isolates of both species, but after 35 days, one anthracnose-causing strain of C. all the isolates of both species, but after 35 days, one anthracnose-causing strain of C. Agriculture 2023, 13, 511 14 of 20 for all the isolates of both species, but after 35 days, one anthracnose-causing strain of C. nigrum reached a disease radius of 9.8 mm in width. All the tested strains of C. coccodes and C. nigrum were able to spread on potato slices. The disease radius was 2.5–5.2 mm for C. coccodes and 1.2–8.3 mm for C. nigrum after 21 days. No correlation between the disease severity and the original host was found: the isolates from tomatoes and potatoes could infect plants of both species. Nevertheless, the tomato fruit disease caused by both species was more rapid and extensive under the same temperature conditions compared with the potato tuber disease. Table 4. Pathogenicity tests. Average Disease Radius Average Disease Radius (mm) on Tomato after (mm) on Potato after Strain Species Host 21 days 35 days 21 days Identifier Wound Surface Surface Wound Inoculation Inoculation Inoculation Inoculation C21KSEgF7 C. nigrum Eggplant 4.8 0.0 0.9 2.3 C21KSEgF3 C. nigrum Eggplant 3.2 0.1 2.0 2.5 C21KSEgF4.1 C. nigrum Eggplant 3.2 0.1 2.3 4.3 C21KSPeF6 C. nigrum Pepper 3.7 1.0 9.8 2.0 C21KSPeF19 C. nigrum Pepper 5.2 0.3 2.5 8.3 C20AuPT5a C. coccodes Potato 6.2 0.2 2.0 5.2 C20UgKgPT2 C. coccodes Potato 5.2 1.2 2.2 2.5 C21KSTF88 C. nigrum Tomato 8.3 0.5 3.3 1.8 C21KSTF97 C. nigrum Tomato 7.5 0.5 1.2 1.2 C21KST3F2 C. nigrum Tomato 6.3 0.2 3.2 4.0 No isolate resistant to any examined fungicide was found (Table 5). Thiabendazole EC for C. coccodes was 0.65–58.38 mg/L, and that for C. nigrum was 0.58–20.29 mg/L. Six isolates (five C. coccodes from potato tubers and stem, and one C. nigrum from tomato fruit) were less sensitive to the chemical (EC > 10 mg/L). No resistance was found for azoxystrobin, EC for C. coccodes was 0.05 and 9.07 mg/L, EC for C. nigrum was 50 50 0.08–8.50 mg/L. Difenoconazole was the most effective chemical; EC for all the tested isolates was less than 0.12 mg/L. Table 5. Sensitivity to fungicides. EC , mg/L ** Isolation 50 Strain Identifier Species Source * Thiabendazole Azoxystrobin Difenoconazole C13V(GH)PT1/1 C. coccodes PT 4.24 0.08 0.06 C13K(S)PT11 C. coccodes PT 5.07 7.75 0.07 C13K(S)PT14 C. coccodes PT 7.75 0.08 0.06 C13K(S)PT15 C. coccodes PT 4.47 0.28 0.07 C13K(S)PT17 C. coccodes PT 10.20 5.82 0.12 C13K(S)PT21 C. coccodes PT 7.90 3.68 - C13K(S)PT34 C. coccodes PT 0.78 0.07 0.06 C13K(S)PT58b C. coccodes PT 3.47 - 0.07 C13HPT29/2 C. coccodes PT 0.91 0.05 0.05 C13G(B)PTde9 C. coccodes PT 50.30 0.07 0.06 C13G(B)PTde12 C. coccodes PT 0.85 0.06 0.05 C13G(B)PTde23 C. coccodes PT 0.93 0.09 0.06 C13G(B)PTes6 C. coccodes PT 33.38 0.10 0.06 C13G(B)PTes19 C. coccodes PT 8.78 0.10 0.06 C13G(B)PTal15 C. coccodes PT 0.96 0.09 0.09 Agriculture 2023, 13, 511 15 of 20 Table 5. Cont. EC , mg/L ** Isolation Strain Identifier Species Source * Thiabendazole Azoxystrobin Difenoconazole C13G(B)PTal19 C. coccodes PT 0.96 0.09 0.06 C13G(B)PTal20 C. coccodes PT 0.99 0.08 0.06 C13G(B)PTal23 C. coccodes PT 6.13 0.07 0.07 C13G(B)PTal24 C. coccodes PT 0.85 0.06 0.05 C13G(B- C. coccodes PT 1.00 0.08 0.06 Sh)PTsa29 C14M(Ch)PT6 C. coccodes PT - 0.09 0.06 C14M(Ch)PT18/2 C. coccodes PT - 0.08 0.06 C15M(L)PT1 C. coccodes PT 0.82 0.06 0.08 C15M(L)PT1/2 C. coccodes PT 0.94 - 0.09 C15M(L)PT4 C. coccodes PT 0.89 0.08 0.09 C15M(L)PT5 C. coccodes PT 0.85 0.08 0.09 C15M(L)PT6 C. coccodes PT - 0.08 0.06 C15M(L)PT7 C. coccodes PT 0.95 - 0.09 C16ME(Y-O)PL7 C. coccodes PL - - 0.09 C16ME(Y- C. coccodes PL - - 0.08 O)PL11 C16M(G)PS9 C. coccodes PS 0.85 0.08 0.09 C16M(G)PS15 C. coccodes PS 0.84 4.09 0.06 C16M(G)PS16b C. coccodes PS 58.38 0.07 0.08 C17K(K)TF5-2 C. nigrum TF 0.91 0.09 0.09 C17K(K)TF5-14 C. nigrum TF - 0.08 0.09 C17K(S)PTrs9 C. coccodes PT - 0.08 0.06 C17K(S)PTrs11/1 C. coccodes PT 0.87 0.08 0.07 C18M(L)TF1/1 C. coccodes TF 0.74 6.32 0.12 C18K(S)TF1/2 C. nigrum TF 20.29 8.50 - C18U(G)TF1/1 C. nigrum TF - - 0.07 C18U(G)PT4 C. coccodes PT 6.07 - - C18U(G)PT7 C. coccodes PT 0.65 7.75 0.10 C18U(G)PT11 C. coccodes PT 25.43 9.07 0.08 C18TPS8 C. coccodes PS - 7.75 0.09 C18TPS9 C. coccodes PS - 3.31 0.09 C19CyPT1/2 C. coccodes PT 0.95 0.07 0.09 C19CyPT2/1 C. coccodes PT 0.85 0.07 0.09 C20AuPT5a C. coccodes PT 0.75 0.08 0.07 C20UgLaPT1/1 C. coccodes PT 0.71 0.07 0.07 C20UgKgPT1 C. coccodes PT 0.82 0.08 0.07 C20UgKgPT2 C. coccodes PT 0.73 0.08 0.07 C20UgKgPT12 C. coccodes PT 0.83 0.07 0.07 C21KST3F2 C. nigrum TF 0.66 0.08 0.07 C21KSTF88 C. nigrum TF 0.67 0.08 0.07 C21KSTF97 C. nigrum TF 0.65 0.08 0.07 C21KSPeF6 C. nigrum PeF 0.68 0.08 0.07 C21KSPeF19 C. nigrum PeF 0.68 0.08 0.07 C21KSEgF3 C. nigrum EF 0.58 0.08 0.07 C21KSEgF4.1 C. nigrum EF 0.64 0.08 0.07 C21KSEgF6 C. nigrum EF 0.71 0.08 0.07 C21KSEgF7 C. nigrum EF 0.66 0.09 0.07 * PT—potato tuber, PS—potato stem, PL—potato leaf, TF—tomato fruit, PeF—pepper fruit, EF—eggplant fruit. ** EC —effective concentration. 4. Discussion The efficiencies of the known genetic markers in differentiating Colletotrichum species vary among different species complexes [4]. The ITS region is widely used in routine studies, although the result may be doubtful. For instance, in northern Italy, C. coccodes was reported as an agent of pepper root disease [10]. Undoubtedly, the species can cause root Agriculture 2023, 13, 511 16 of 20 disease; still, ITS-based identification remains insufficient. In Turkey, unusual symptoms of Colletotrichum disease leading to extremely high crop losses were discovered, and the pathogen was identified as C. coccodes [38]. However, the only molecular marker used in the study was the ITS region, so the identification seems uncertain. Dos Santos Vieira and colleagues [39] propose using gaphd and several other regions to distinguish between Colletotrichum species, while ITS and act are less effective. According to our study, both the act and gaphd genes are suitable, at least for C. coccodes and C. nigrum division (Figures 5 and 6). The Gs intron also proved useful for delineating Colletotrichum species. This gene sequence is mainly used to distinguish the species within C. gigasporum, C. orbiculare, and C. gloeosporoides species complexes. Thus, up to date, GenBank lacks the gs region sequences of the type material for many species. Several GenBank accession numbers marked as the C. coccodes gs gene (GU935816 and GU935817) presumably belong to C. nigrum, as they differ by approximately 2–3% from C. coccodes CBS164.49 or CBS369.75 (GenBank accession numbers HM171675 and HM171676, respectively) but they are similar to our strains that are identified as C. nigrum, based on the act or gaphd genes. We propose that at least 17 single nucleotide changes underlie the differences between the gs second intron of the two species. The C. nigrum currently presumed occurrence and host range seem to be lower than the real ranges. We assume that several reports of C. coccodes, for example [17], may display C. nigrum disease instead. According to the pertinent literature, the sexual process is unknown for C. coccodes or C. nigrum. The only way for strains of these species to exchange genetic material is via a parasexual process or through a vegetative compatibility reaction [40]. Based on the gs sequence of five isolates (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20), we suppose that they might represent hybrids between C. coccodes and C. nigrum. Whereas we detected SNPs in all the isolates identified as C. nigrum based on gaphd, we assume these SNPs to be specific to C. nigrum (Figure 2). At least one of the isolates (C18U(G)TF1/1) was collected from tomato fruit grown near potato plants; therefore, it might have had a possibility of interfering with C. coccodes strains. Notwithstanding these putative hybrid isolates, we assume that the second intron of the gs gene is useful for distinguishing between C. coccodes and C. nigrum, and we propose a more active use of the GSF1—GSR1 primers for identifying the Colletotrichum species. Both C. coccodes and C. nigrum are currently considered as singleton species. According to Liu et al. [16], all potato-associated isolates belong to C. coccodes. At the same time, both C. nigrum and C. coccodes were able to infect tomato and pepper. The statement is supported by other studies [3–5] and by our data. Until now, we found no information regarding C. nigrum in Russia. Based on ITS region sequencing, only one Colletotrichum species—C. coccodes—was previously reported from potato and tomato leaves in Russia [9,23,24,26]. Belov and colleagues [9] used a specific primer pair (Cc1F1 and Cc2R1) [41] to detect C. coccodes [41]. Both test systems [23,42] were developed based on the ITS region, considered the universal fungi barcode [43]. However, they were of limited use in distinguishing C. coccodes and C. nigrum. C. dianense, which is very similar to C. nigrum, was recently described [37]. The authors of the study stated that it could be distinguished from C. nigrum by its conidial shape and apex. The ITS region and the act gene of the C. dianense type isolate YMF 1.04943 is 100% identical, and the gaphd gene is 99.66% similar to the C. nigrum type strain CBS 169.49 (one nucleotide difference, position 185). We compared our isolates to both species. Although, in our opinion, the two species are slightly different, we named our isolates from tomato, pepper, and eggplant C. nigrum, as C. nigrum is a well-known and earlier described species. The cross-virulence of Colletotrichum species was reported a while ago [44]. Yet, there are no literature reports of C. nigrum potato infections, as the species was only found on tomato and pepper [3,16], while the GenBank database contains several C. nigrum isolates (e.g., KU821311) reported from potatoes. Colletotrichum disease is known as post-harvest, Agriculture 2023, 13, 511 17 of 20 and is particularly harmful to climacteric fruits (e.g., tomato) [45]. Even though our study demonstrated the possibility of C. nigrum potato infection, no C. nigrum strains were isolated from potato. Another Colletotrichum species, C. acutatum s. str., was reported as a tomato and pepper infectious agent [5,46]. The presence of C. acutatum s.l. in potato leaves and mini tubers was revealed in our previous studies (unpublished data) based on the reaction with species- specific primers CaInt2/ITS4 [47]. Although C. acutatum s.l. has not been proven to be a potato disease agent, the possibility of its presence on potato tubers and leaves should be kept in mind. Liu and colleagues [16,21] mentioned that C. coccodes strains from tomato or other hosts produce larger conidia than C. coccodes from potato, and C. nigrum forms longer conidia than C. coccodes. Our results do not support these statements and show no significant difference between the conidial length or width among the three studied species (Figure 8). Contrary to Zheng and colleagues [37], we suppose that the morphological differentiation within the C. coccodes—C. nigrum—C. dianense clade may not be significant. All the tested chemicals—azoxystrobin, thiabendazole, and difenoconazole—proved to be effective against Colletotrichum spread. The results were in line with previous stud- ies [48–50]: normally, EC is less than 1 mg/L for all of the tested chemicals. In addition, azoxystrobin reduced black dot on tubers in field conditions [50]. Resistance to azoxys- trobin or thiabendazole was reported in other Colletotrichum species complexes [51,52]. We discovered five strains (C13G(B)PTde9, C13G(B)PTes6, C16M(G)PS16b, C18K(S)TF1/2, and C18U(G)PT11), with thiabendazole EC ranging over 20–50 mg/L. Sanders and Ko- rsten [52] classified strains with 66–70% growth on 0.5–2.5 mg/L thiabendazole as resistant, but we named them as less sensitive after Leite [53]. In our previous study, we exam- ined the -tubulin gene, but no specific mutations in any of the Colletotrichum isolates was found [54], contrary to Colletotrichum musae [53], less sensitive strains, Colletotrichum siamense [55], or Helminthosporium solani highly resistant strains [56]. Because even the highest EC values for Colletotrichum spp. are much lower than the concentrations in the working liquid for treatment (e.g., 170–250 mg/L for azoxystrobin, 4800–5600 mg/L for thiabendazole, and 187–625 mg/L for difenoconazole), we conclude that in general, all of the studied chemicals could still be considered as an effective strategy for anthracnose control on Solanaceae in Russia. 5. Conclusions Here, we present the results of the first extensive molecular and morphological anal- ysis of Colletotrichum species affecting Solanaceae plants in Russia. Two morphologically indistinguishable species, C. coccodes and C. nigrum, were revealed. The act and gaphd gene introns are suggested as the most suitable molecular markers to differentiate between these species. The Gs intron sequences give rise to the hypothesis of a parasexual process between these two species; therefore, further research is required. Eggplant and pepper plants were found to be infected exclusively by C. nigrum; tomato plants were infected by both species. Potato infection was caused only by C. coccodes. However, in vitro, both species showed an ability to infect tomato fruit and potato tubers. Three studied chemicals, azoxystrobin, difenoconazole, and thiabendazole, were effective against the isolates of both species, although several isolates were less sensitive to thiabendazole. Author Contributions: Conceptualization, S.E.; methodology, E.C., S.E. and M.Y.; software, E.C., S.E. and L.K.; validation, I.K., M.Y., M.K., A.B., E.C. and L.K.; formal analysis, E.C., S.E., M.Y. and M.K.; investigation, I.K., M.K., M.Y., A.B., G.B., A.E., M.P., A.T. and Y.T.; resources, S.E., E.C., A.B., M.Y. and I.K.; data curation, E.C., M.Y. and S.E.; writing—original draft preparation, M.Y.; writing—review and editing, S.E., A.B. and L.K.; visualization, M.Y.; supervision, S.E.; project administration, S.E.; funding acquisition, S.E., L.K. and E.C. All authors have read and agreed to the published version of the manuscript. Agriculture 2023, 13, 511 18 of 20 Funding: This research was funded by the RUDN University Scientific Grant System (project № 202193-2-000), and by the Russian Foundation for Basic Research (grant № 20-016-00139). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Newly generated ITS, act, gaphd, and gs sequences are deposited in GenBank under the accession numbers specified in Table 1. Acknowledgments: The authors are grateful to Anastasia Sharapkova and Tatiana Gavrilova (Rosetta Stone MSU) for the English improvement of the article. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. Marin-Felix, Y.; Hernández-Restrepo, M.; Iturrieta-González, I.; García, D.; Gené, J.; Groenewald, J.Z.; Cai, L.; Chen, Q.; Quaedvlieg, W.; Schumacher, R.K.; et al. Genera of phytopathogenic fungi: GOPHY 1. Stud. Mycol. 2017, 86, 99–216. [CrossRef] [PubMed] 2. Jayawardena, R.S.; Bhunjun, C.S.; Hyde, K.D.; Gentekaki, E.; Itthayakorn, P. 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Colletotrichum Species on Cultivated Solanaceae Crops in Russia

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Abstract

agriculture Article 1 1 2 2 1 , 2 Maria Yarmeeva , Irina Kutuzova , Michael Kurchaev , Elena Chudinova , Ludmila Kokaeva , 3 4 2 1 2 Arseniy Belosokhov , Grigory Belov , Alexander Elansky , Marina Pobedinskaya , Archil Tsindeliani , 1 , 5 1 , 2 , Yulia Tsvetkova and Sergey Elansky * Lomonosov Moscow State University, 119991 Moscow, Russia Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia Michurina St., 54, Michurinsk, 393760 Tambov, Russia Russian Potato Research Centre, 140051 Moscow, Russia All-Russian Plant Quarantine Center (VNIIKR), 140150 Moscow, Russia * Correspondence: elanskiy_sn@pfur.ru Abstract: Colletotrichum species are the causal agents of potato and tomato diseases, such as black dot and anthracnose. Several new species and species complexes were recently established. Thereby, a reassessment of the genus diversity is required. The study revealed two species, Colletotrichum coccodes and Colletotrichum nigrum, as Russia’s main disease agents of cultivated Solanaceae plants. Black dot and anthracnose in potato were caused exclusively by C. coccodes, whereas the same diseases in tomato, eggplant, and pepper were predominately caused by C. nigrum. However, one isolate of C. coccodes was also identified as an agent of the tomato disease. Five potentially hybrid isolates were discovered. Morphological examination and pathogenicity assessment revealed no significant differences between the two Colletotrichum species. All isolates were sensitive to the fungicides azoxystrobin, difenoconazole, and thiabendazole, which are currently used in agriculture. This is the first report of the occurrence of C. nigrum in Russia. Keywords: black dot; anthracnose; pathogen; potato; tomato; Colletotrichum; multi-gene phylogeny Citation: Yarmeeva, M.; Kutuzova, I.; Kurchaev, M.; Chudinova, E.; Kokaeva, L.; Belosokhov, A.; Belov, 1. Introduction G.; Elansky, A.; Pobedinskaya, M.; Colletotrichum is a well-known causal agent of potato and tomato diseases such as black Tsindeliani, A.; et al. Colletotrichum dot and anthracnose, dramatically damaging both underground and aboveground plant Species on Cultivated Solanaceae parts. Although several revisions of the genus Colletotrichum were recently published [1–4], Crops in Russia. Agriculture 2023, 13, it still remains taxonomically puzzling. Currently, 16 species complexes and 15 singleton 511. https://doi.org/10.3390/ species (e.g., C. coccodes and C. nigrum) are established within Colletotrichum [5]. Given the agriculture13030511 complicated systematics of the genus, the species identification is based on morphological Academic Editor: Yuan Li features, combined with molecular data. C. coccodes predominantly infects Solanaceae plants, including chilli fruit [6], potato Received: 30 December 2022 tubers [7,8], tomato [9], sweet pepper [10–12], black nightshade [13], and eggplant [14]. Revised: 12 February 2023 Nevertheless, its wide host range is not limited to Solanaceae, since the species was reported Accepted: 16 February 2023 to infect strawberry [15], pumpkin [16], or onion [17]. Published: 21 February 2023 There are very few reports concerning C. nigrum. The species was first described as an agent of pepper anthracnose from Gloucester County, New Jersey, USA [18], but it can be associated with tomato and eggplant diseases [19,20] as well. Liu and colleagues [17,21] Copyright: © 2023 by the authors. reviewed the species’ description, introduced a neotype to C. coccodes, selected an epitype Licensee MDPI, Basel, Switzerland. to C. nigrum, and stated the ability of both species to induce anthracnose. This article is an open access article In Russia, the diversity within the genus is poorly described, due to the predomi- distributed under the terms and nance of the morphological identification of causal agents. The review by Kotova and conditions of the Creative Commons Kungurtseva [22] specified that C. coccodes is the only cause of potato and tomato black Attribution (CC BY) license (https:// dot and anthracnose. Several studies in Russia investigated the diversity of Colletotrichum creativecommons.org/licenses/by/ species on potato and tomato leaves using genetic markers [9,23,24] directly from the plant 4.0/). Agriculture 2023, 13, 511. https://doi.org/10.3390/agriculture13030511 https://www.mdpi.com/journal/agriculture Agriculture 2023, 13, 511 2 of 20 material, without isolating the species in axenic cultures. Kazartsev and colleagues [25] recently scrutinized the diversity of Colletotrichum species on several wild and cultivated plants (no potato or tomato plants were included into the analysis), using molecular and morphological approaches to identify the species. C. coccodes strains were isolated from Ambrosia artemisiifolia, Beta vulgaris, Brassica napus, Cannabis sativa, Galinsoga parviflora, and Portulaca oleracea. Poluektova and colleagues [26] analysed the glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase genes of four C. coccodes strains from Russian potato. To the best of our knowledge, these are the only molecular investigations of the genus in Russia to date. This study focuses on the disease agents of several cultivated Solanaceae crops in Russia. Our research combines both morphological and molecular approaches to reveal the diversity within the genus Colletotrichum. To this end, four genetic markers were used: ITS1-5.8S-ITS2 region (ITS) as a well-established barcode, the glyceraldehyde-3-phosphate dehydrogenase gene intron (gaphd), considered the most reliable genetic marker for Col- letotrichum species, the actin intron (act), and the glutamine synthetase intron (gs). To estimate the agricultural risks of the disease spread, we assessed the sensitivity to some fungicides that are officially used for tuber treatment, and the pathogenicity range towards the tomato fruit and potato tuber slices. 2. Materials and Methods 2.1. Sampling and Isolation of Cultures Samples were collected from the fruits of tomato, eggplant, and pepper (Table 1 and Figure 1) as well as from potato tubers, leaves, and stems. Seed potato tubers from the Netherlands, Germany, Australia, Cyprus, and Uganda were taken for comparison. All isolation sources were surface-sterilized with sodium hypochlorite (2% solution) to remove possible contamination, sliced, and put in wet chambers at 24  1 C. For isolation, small black sclerotia from the tuber peel or diseased tissue were taken using a preparation needle under a binocular microscope (MBS10, Russia), and transferred to culture media (potato- dextrose agar, PDA) amended with antibiotic (benzylpenicillin sodium salt, 100 mg/L). Table 1. Details of isolates used in the study. GenBank Accession Numbers ** Origin * Isolation Year of Strain Identifier Host Plant (Figure 1) Source Isolation ITS gaphd act gs C13V(GH)PT1/1 1 Potato Tuber 2013 OP718477 OP743730 OP743793 OP743860 C13K(S)PT11 2 Potato Tuber 2013 OP718470 OP743723 OP743786 OP743853 C13K(S)PT14 2 Potato Tuber 2013 OP718471 OP743724 OP743787 OP743854 C13K(S)PT15 2 Potato Tuber 2013 OP718472 OP743725 OP743788 OP743855 C13K(S)PT17 2 Potato Tuber 2013 OP718473 OP743726 OP743789 OP743856 C13K(S)PT21 2 Potato Tuber 2013 OP718474 OP743727 OP743790 OP743857 C13K(S)PT34 2 Potato Tuber 2013 OP718475 OP743728 OP743791 OP743858 C13K(S)PT58b 2 Potato Tuber 2013 OP718476 OP743729 OP743792 OP743859 C13HPT29/2 3 Potato Tuber 2013 OP718469 OP743722 OP743836 OP743890 C13G(B)PTde8/2 4 Potato Tuber 2013 OP718463 OP743716 OP743830 OP743844 C13G(B)PTde9 4 Potato Tuber 2013 OP718464 OP743717 OP743831 OP743845 C13G(B)PTde12 4 Potato Tuber 2013 OP718461 OP743714 OP743828 OP743842 C13G(B)PTde23 4 Potato Tuber 2013 OP718462 OP743715 OP743829 OP743843 C13G(B)PTes6 4 Potato Tuber 2013 OP718466 OP743719 OP743833 OP743847 C13G(B)PTes19 4 Potato Tuber 2013 OP718465 OP743718 OP743832 OP743846 C13G(B)PTal15 4 Potato Tuber 2013 OP718456 OP743709 OP743823 OP743837 C13G(B)PTal19 4 Potato Tuber 2013 OP718457 OP743710 OP743824 OP743838 C13G(B)PTal20 4 Potato Tuber 2013 OP718458 OP743711 OP743825 OP743839 C13G(B)PTal23 4 Potato Tuber 2013 OP718459 OP743712 OP743826 OP743840 C13G(B)PTal24 4 Potato Tuber 2013 OP718460 OP743713 OP743827 OP743841 C13G(B- 5 Potato Tuber 2013 OP718468 OP743721 OP743835 OP743849 Sh)PTsa5 C13G(B- 5 Potato Tuber 2013 OP718467 OP743720 OP743834 OP743848 Sh)PTsa29 C14M(Ch)PT6 6 Potato Tuber 2014 OP718479 OP743732 OP743795 OP743862 Agriculture 2023, 13, 511 3 of 20 Table 1. Cont. GenBank Accession Numbers ** Origin * Isolation Year of Strain Identifier Host Plant (Figure 1) Source Isolation ITS gaphd act gs C14M(Ch)PT18/2 6 Potato Tuber 2014 OP718478 OP743731 OP743794 OP743861 C15M(L)PT1 7 Potato Tuber 2015 OP718480 OP743733 OP743796 OP743863 C15M(L)PT1/2 7 Potato Tuber 2015 OP718481 OP743734 OP743797 OP743864 C15M(L)PT4 7 Potato Tuber 2015 OP718482 OP743735 OP743798 OP743865 C15M(L)PT5 7 Potato Tuber 2015 OP718483 OP743736 OP743799 OP743866 C15M(L)PT6 7 Potato Tuber 2015 OP718484 OP743737 OP743800 OP743867 C15M(L)PT7 7 Potato Tuber 2015 OP718485 OP743738 OP743801 OP743868 C16ME(Y-O)PL7 8 Potato Leaf 2016 OP718490 OP743743 OP743806 OP743873 C16ME(Y- 8 Potato Leaf 2016 OP718489 OP743742 OP743802 OP743872 O)PL11 C16M(G)PS9 9 Potato Stem 2016 OP718488 OP743741 OP743805 OP743871 C16M(G)PS15 9 Potato Stem 2016 OP718486 OP743739 OP743803 OP743869 C16M(G)PS16b 9 Potato Stem 2016 OP718487 OP743740 OP743804 OP743870 C17K(K)TF5-2 10 Tomato Fruit 2017 OP718492 OP743745 OP743808 OP743875 C17K(K)TF5-14 10 Tomato Fruit 2017 OP718491 OP743744 OP743807 OP743874 C17K(S)PTrs9 11 Potato Tuber 2017 OP718494 OP743747 OP743810 OP743877 C17K(S)PTrs11/1 11 Potato Tuber 2017 OP718493 OP743746 OP743809 OP743876 C18M(L)TF1/1 7 Tomato Fruit 2018 OP718496 OP743749 OP743822 OP743889 C18K(S)TF1/2 12 Tomato Fruit 2018 OP718495 OP743748 OP743811 OP743878 C18U(G)TF1/1 13 Tomato Fruit 2018 OP716941 OP730520 OP743774 OP743898 C18U(G)PT4 13 Potato Tuber 2018 OP718500 OP743753 OP743815 OP743882 C18U(G)PT6 13 Potato Tuber 2018 OP718501 OP743754 OP743816 OP743883 C18U(G)PT7 13 Potato Tuber 2018 OP718502 OP743755 OP743817 OP743884 C18U(G)PT11 13 Potato Tuber 2018 OP718499 OP743752 OP743814 OP743881 C18TPS8 14 Potato Stem 2018 OP718497 OP743750 OP743812 OP743879 C18TPS9 14 Potato Stem 2018 OP718498 OP743751 OP743813 OP743880 C19CyPT1/2 15 Potato Tuber 2019 OP718503 OP743756 OP743783 OP743850 C19CyPT2/1 15 Potato Tuber 2019 OP718504 OP743757 OP743784 OP743851 C20AuPT5a 16 Potato Tuber 2020 OP718505 OP743758 OP743785 OP743852 C20UgLaPT1/1 17 Potato Tuber 2020 OL405711 OP743762 OP743821 OP743888 C20UgKgPT1 17 Potato Tuber 2020 OP718506 OP743759 OP743818 OP743885 C20UgKgPT2 17 Potato Tuber 2020 OP718508 OP743761 OP743820 OP743887 C20UgKgPT12 17 Potato Tuber 2020 OP718507 OP743760 OP743819 OP743886 C21KST1F1 12 Tomato Fruit 2021 OP716934 OP730512 OP743775 OP743891 C21KSTF9 12 Tomato Fruit 2021 OP716939 OP730517 OP743780 OP743896 C21KST3F1 12 Tomato Fruit 2021 OP716935 OP730513 OP743776 OP743892 C21KST3F2 12 Tomato Fruit 2021 OP716936 OP730514 OP743777 OP743893 C21KSTF88 12 Tomato Fruit 2021 OP716938 OP730516 OP743779 OP743895 C21KSTF77 12 Tomato Fruit 2021 OP716937 OP730515 OP743778 OP743894 C21KSTF97 12 Tomato Fruit 2021 OP716940 OP730518 OP743781 OP743897 C21KSTF98 12 Tomato Fruit 2021 OP716941 OP730519 OP743782 OP743899 C21KSPeF3 12 Pepper Fruit 2021 OP716931 OP743706 OP743771 OP743908 C21KSPeF4 12 Pepper Fruit 2021 OP716932 OP743707 OP743772 OP743909 C21KSPeF6 12 Pepper Fruit 2021 OP716933 OP743708 OP743773 OP743910 C21KSPeF20 12 Pepper Fruit 2021 OP716930 OP743705 OP743770 OP743907 C21KSPeF19 12 Pepper Fruit 2021 OP716929 OP743704 OP743769 OP743906 C21KSEgF1 12 Eggplant Fruit 2021 OP716923 OP743698 OP743763 OP743900 C21KSEgF3 12 Eggplant Fruit 2021 OP716924 OP743699 OP743764 OP743901 C21KSEgF4.1 12 Eggplant Fruit 2021 OP716925 OP743700 OP743765 OP743902 C21KSEgF5 12 Eggplant Fruit 2021 OP716926 OP743701 OP743766 OP743903 C21KSEgF6 12 Eggplant Fruit 2021 OP716927 OP743702 OP743767 OP743904 C21KSEgF7 12 Eggplant Fruit 2021 OP716928 OP743703 OP743768 OP743905 * Geographical origins of the isolates. Russia: 1—Vladimir Region; 2, 11—Kostroma Region; 6, 7, 9—Moscow Region; 8—the Mari El Republic; 10, 12—Krasnodar Krai; 13—Primorsky Krai; 14—the Republic of Tatarstan; 3—the Netherlands; 4, 5—Germany; 15—the Republic of Cyprus; 16—Australia; 17—Uganda. ** ITS—ITS1-5, 8S-ITS2 region, gaphd—glyceraldehyde-3-phosphate dehydrogenase gene intron, act—actin intron, gs—glutamine synthetase intron. Agriculture 2023, 13, 511 4 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 3 of 21 Figure 1. Location of collection sites (see also Table 1). Russia: 1—Vladimir Region; 2, 11—Kostroma Figure 1. Location of collection sites (see also Table 1). Russia: 1—Vladimir Region; 2, 11—Kostroma Region; 6, 7, 9—Moscow Region; 8—the Mari El Republic; 10, 12—Krasnodar Krai; 13—Primorsky Region; 6, 7, 9—Moscow Region; 8—the Mari El Republic; 10, 12—Krasnodar Krai; 13—Primorsky Krai; 14—the Republic of Tatarstan; 3—the Netherlands; 4, 5—Germany; 15—the Republic of Cy- Krai; 14—the Republic of Tatarstan; 3—the Netherlands; 4, 5—Germany; 15—the Republic of Cyprus; prus; 16—Australia; 17—Uganda. 16—Australia; 17—Uganda. Table 1. Details of isolates used in the study. 2.2. DNA Isolation, PCR, Sequencing, and Phylogenetic Analysis Origin * To Host extract DIsolation NA, the m ycY elear ium of o f fungi wa GenB s groank wn o Ac n cession Num a liquid pea m bers ** edium (180 g Strain Identifier of green pea boiled for 10 min in 1 L of water, then filtered and autoclaved for 30 min at (Figure 1) Plant Source Isolation ITS gaphd act gs 1 atm). DNA was extracted according to the standard CTAB protocol [27,28]. ITS, act, and C13V(GH)PT1/1 1 Potato Tuber 2013 OP718477 OP743730 OP743793 OP743860 gaphd amplifications were performed in a SSI microtube strips in a 25 L total volume re- C13K(S)PT11 2 Potato Tuber 2013 OP718470 OP743723 OP743786 OP743853 action containing 1 L of a DNA template (50 ng/L), 2.5 L of 10 PCR buffer (Applied C13K(S)PT14 2 Potato Tuber 2013 OP718471 OP743724 OP743787 OP743854 Biosystems, Waltham, MA, USA), 0.5 L of 10 mM each deoxyribonucleotide triphosphates C13K(S)PT15 2 Potato Tuber 2013 OP718472 OP743725 OP743788 OP743855 (dNTP), 0.4 L of 100M each primer (Evrogen Co, Moscow, Russia), 1.5 U of Taq polymerase C13K(S)PT17 2 Potato Tuber 2013 OP718473 OP743726 OP743789 OP743856 (5U/L, Promega, Madison, WI, USA), and Milli-Q water (MQ). For the amplification of gs C13K(S)PT21 2 Potato Tuber 2013 OP718474 OP743727 OP743790 OP743857 2.8 L of each dNTP was used; the concentrations of the other components remained the C13K(S)PT34 2 Potato Tuber 2013 OP718475 OP743728 OP743791 OP743858 same. The following primers were used: ITS1 5’-TCCGTAGGTGAACCTGCGG-’3 and ITS4 5’- C13K(S)PT58b 2 Potato Tuber 2013 OP718476 OP743729 OP743792 OP743859 TCCTCCGCTTATTGATATGC-3’ for the ITS region [29], GSF1 5’-ATGGCCGAGTACATCTGG- C13HPT29/2 3 Potato Tuber 2013 OP718469 OP743722 OP743836 OP743890 ’3 and GSR1 5’-GAACCGTCGAAGTTCCAC-’3 for the gs gene [30], GDF-1 5’-GCCGTCAACG C13G(B)PTde8/2 4 Potato Tuber 2013 OP718463 OP743716 OP743830 OP743844 ACCCCTTCATTGA-’3 and GDR-1 5’-GGGTGGAGTCGTACTTGAGCATGT-’3 for gaphd [31], C13G(B)PTde9 4 and ACPota T-51to 2F 5’-ATTuber GTGCAAGG2C013 CGGTTOP71 TCGC 846 -’34 andOP74 ACT-371 7837 OP74 R 5’-TAC383 GAG1 TCOP74 CTTC384 TG5 G CCCAT-’3 for act [32]. C13G(B)PTde12 4 Potato Tuber 2013 OP718461 OP743714 OP743828 OP743842 The PCR protocol included initial denaturation at 94 C for 3 min, 35 amplification C13G(B)PTde23 4 Potato Tuber 2013 OP718462 OP743715 OP743829 OP743843 cycles, and an additional extending step at 72 C for 3 min. For the primer pair ITS1/ITS4, C13G(B)PTes6 4 Potato Tuber 2013 OP718466 OP743719 OP743833 OP743847 Agriculture 2023, 13, 511 5 of 20 the amplification cycles were 94 C for 30 s, 55 C for 30 s, and 72 C for 45 s. For the primer pairs GDF-1/GDR-1 and ACT-512F/ACT783R, the amplification cycles were 94 C for 30 s, 60 C for 30 s, and 72 C for 30 s. For the primer pair GSF1/GSR1, the amplification cycles were 94 C for 30 s, 61 C for 30 s, and 72 C for 120 s. The amplification was performed on a T100 Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). PCR products were run in 0.7–1.2% agarose gel amended with ethidium bromide; the agarose concentration depended on the PCR fragment length. The gel extraction was performed with a Cleanup Mini Kit (Evrogen Co., Russia). The PCR frag- ments were sequenced using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and the Applied Biosystems 3730  l automated sequencer (Applied Biosystems, USA). Each fragment was sequenced in both directions. Consensus sequences for each locus were assembled using Geneious version 7.1 (created using Biomatters) and MEGA X [33], and aligned with available type species sequences (Table 2). Species identification was based primarily on the gaphd sequence. Table 2. Reference strains used in this study. GenBank Accession Numbers * Species Strain Species Host Complex Identifier ITS act gaphd gs C. nigrum singleton CBS 69.49 Capsicum sp. NR163523 JX546646 JX546742 - Solanum C. nigrum singleton CBS 132450 JX546845 JX546653 JX546749 - lycopersicum Cichorium C. nigrum singleton CBS 127562 JX546842 JX546650 JX546746 - intybus Alternanthera C. dianense singleton YMF 1.04943 OL842189 OL981258 OL981284 - philoxeroides Solanum C. coccodes singleton CBS 369.75 HM171679 HM171667 HM171673 HM171676 tuberosum Cyphomandra C. gigasporum Gigasporum CBS 101881 KF687736 KF687797 KF687841 KF687745 betacea * ITS—ITS1-5.8S-ITS2 region, gaphd—glyceraldehyde-3-phosphate dehydrogenase gene intron, act—actin intron, gs—glutamine synthetase intron. 2.3. Morphological Analysis The cultures were grown on synthetic nutrient-poor agar medium (SNA) [34] amended with Anthriscus sylvestris double autoclaved stems [16] for 10 days. Micro- scopic preparations were made in clear lactic acid. The width and length of conidia were measured, with 30 measurements per structure, using a Leica DM 2500 (Leica Microsystems, Wetzlar, Germany). 2.4. Pathogenicity Tests To compare the pathogenic activity, 10 isolates (two from potato, two from pepper, three from tomato, and three from eggplant) were chosen. Healthy cherry tomato fruits and potato tubers (cultivar “Gala”) were washed and surface-sterilised in 0.5% sodium hypochlorite solution for 5 min, rinsed in distilled water, and air-dried. The potato tubers were sliced to imitate wounding. Two types of tomato fruit were used: wounded with sterile tips and unwounded. The experiment was conducted with three repeats for each strain of each kind of inoculation. The wounded fruits were internally inoculated with 100 L of conidial suspension (concentration 10 spores/mL). The unwounded fruits and potato slices were surface-inoculated with mycelium and conidia, and placed in sterile wet chambers. The control fruits and tubers were surface or internally inoculated with distilled water. Each wet chamber was stored at 10 C for 21 or 35 days, and the radius of the lesion was measured. To fulfil Koch’s postulate, a small tissue sample was taken from the margin of the disease area with a sterile scalpel and placed in a Petri dish on PDA. Agriculture 2023, 13, 511 6 of 20 2.5. In Vitro Assessment of Fungicide Sensitivity Three chemical fungicides: azoxystrobin (Quadris , Syngenta, Basel, Switzerland), ® ® difenoconazole (Score , Syngenta), and thiabendazole (Tecto , Syngenta) were chosen to evaluate their efficiencies against Colletotrichum isolates. The fungicides were selected based on their current use in Russia for tuber or in-furrow treatment, and they were obtained from local suppliers. The sensitivity was evaluated in Petri plates with PDA. The fungicides were added at different concentrations to autoclaved PDA medium to produce a concentration series of 0, 0.1, 1, 10, and 100 mg/L for each fungicide (active ingredient). The mycelial plug (5 mm in diameter) of each isolate was punched from the margin of an actively growing colony of a 5-day-old culture and placed in the centre of a 90 mm PDA plate amended with fungicide, as well as on non-amended PDA plates (controls). Three replicates per treatment were produced, and the plates were incubated at 24  1 C for 4 days. The diameter of the fungal colony on each plate was measured at perpendicular angles. The average of the two measurements was used to calculate the fungicide concentration inhibiting linear colony growth of 50% over control (EC ) [35,36]. 3. Results In total, 74 isolates were analysed: 50 from potato (Solanum tuberosum L.), 11 from tomato (Solanum lycopersicum L.), 7 from pepper (Capsicum annuum L.), and 6 from eggplant (Solanum melongena L.). All strains isolated from potato, regardless of the plant organ, were identified as C. coccodes, while those from eggplant and pepper proved to be related to C. nigrum (Figures 2–6). Almost all tomato isolates except one strain (C18M(L)TF1/1) were identified as C. nigrum. The aligned concatenated sequence dataset of the original isolates was 1690 bp long (440, 207, 233, and 810 bp for the ITS, act, gaphd, and gs sequences, respectively). It contained 65 variable sites: 2 in the ITS region, 2 in the act intron, 12 in the gaphd intron, and 49 the in gs second intron (including three deletions); among them, 2 in act, 8 in gaphd, and 17 in the gs intron seemed to be specific to either C. coccodes or C. nigrum, and they may be used to differentiate between these species. Most C. coccodes, but also several C. nigrum (possible hybrids) isolates, contained a 25 bp insertion in the gs intron (Figure 7). All the nucleotide differences were found in the non-coding regions. All the phylogenetic trees, except one based on the ITS region (Figure 3), showed two well-delimited clades corresponding to two segregate species: C. coccodes and C. nigrum (Figures 2 and 4–6). The recently described C. dianense was in the same clade as C. nigrum (Figures 4 and 5). Among the tomato and pepper C. nigrum strains, five intriguing isolates (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20) were found (Figure 7). Presumably, they shared the features of both species—C. coccodes and C. nigrum. All of the isolates were identified as C. nigrum, based on the gaphd sequence (Table 3). The similarity to C. dianense is discussed further. In total, within 810 bp sequences of the gs gene, we revealed one 25 bp insertion (positions 225–249) and one 1 bp (position 470) insertion typical of C. coccodes CBS 369.75, and 17 single nucleotide polymorphisms (SNP) atypical of C. coccodes (positions 11, 51, 102, 122, 143, 144, 193, 320, 326, 357, 467, 471, 515, 588, 629, 675, and 724). Morphological differences were not found between the sizes of the C. coccodes and C. nigrum conidia (for C. coccodes, the conidial length was 18.23  6.13 m and the width was 4.49  1.04 m; for C. nigrum, the conidial length was 21.17  5.10 m and the width was 4.34  1.03 m; Figure 8). The obtained conidial measurements overlapped with those of the type strains of C. coccodes, C. nigrum, and C. dianense [16,21,37]. All of the isolates produced aseptate, smooth-walled, hyaline, oval to cylindrical conidia with acute, subacute or obtuse apices, typical of C. coccodes or C. nigrum (Figure 9). Agriculture 2023, 13, 511 7 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 8 of 21 Figure 2. Phylogenetic tree inferred from maximum-likelihood analysis of the concatenated align- Figure 2. Phylogenetic tree inferred from maximum-likelihood analysis of the concatenated align- ment, including the ITS region, and the partial act, gaphd, and gs gene regions. The confidence values ment, including the ITS region, and the partial act, gaphd, and gs gene regions. The confidence values are indicated at the branches. Green indicates C. coccodes clade, and blue indicates the C. nigrum are indicated at the branches. Green indicates C. coccodes clade, and blue indicates the C. nigrum clade. clade. Agriculture 2023, 13, 511 8 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 9 of 21 Figure 3. Phylogenetic tree inferred from maximum-likelihood analysis of the ITS1-5, 8S-ITS2 region Figure 3. Phylogenetic tree inferred from maximum-likelihood analysis of the ITS1-5, 8S-ITS2 region alignment. Boo alignment. Bootstrap tstrap 1000 rep 1000 replicates licates. The . Theco confidence nfidence value values s are areindicat indicated ed at the branche at the branches. s. Green Green indicates C. coccodes isolates; blue indicates C. nigrum isolates, and red marks C. dianense type spe- indicates C. coccodes isolates; blue indicates C. nigrum isolates, and red marks C. dianense type species. cies. Agriculture Agricultur 2023 e 2023 , 13, x FOR PEER , 13, 511 REVIEW 10 of 21 9 of 20 Figure 4. Phylogenetic tree inferred from maximum-likelihood analysis of the actin intron alignment. Figure 4. Phylogenetic tree inferred from maximum-likelihood analysis of the actin intron align- ment. Boot Bootstrap strap 1000 replicate 1000 replicates. sThe . The confidence confidence va values luesar are e indicated indicated at at the b the branches. ranches. Green Green indicate indicates s C. C. coccodes clade, blue indicates the C. nigrum clade, and red marks C. dianense type species. coccodes clade, blue indicates the C. nigrum clade, and red marks C. dianense type species. Agriculture Agricultur 2023 e 2023 , 13 ,, x FOR PEER 13, 511 REVIEW 11 of 10 21 of 20 Figure 5. Phylogenetic tree inferred from maximum-likelihood analysis of the glyceraldehyde-3- Figure 5. Phylogenetic tree inferred from maximum-likelihood analysis of the glyceraldehyde-3-phos- phate dehydrogenase phosphate dehydr intron alignment. Bootstr ogenase intron alignment. ap 1000 replica Bootstrap 1000 tes. rThe confidence va eplicates. The confidence lues are indica values tedar e at the indicated branches at . G therbranches. een indicat Gr es een C. cocc indicates odes clade, C. coccodes blue ind clade, icates blue the indicates C. nigrumthe clade C.,nigrum and red marks clade, and C. dianense type species. red marks C. dianense type species. Agriculture 2023, 13, 511 11 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 12 of 21 Figure 6. Phylogenetic tree inferred from maximum-likelihood analysis of the glutamine synthetase Figure 6. Phylogenetic tree inferred from maximum-likelihood analysis of the glutamine synthetase intron alignment. Bootstrap 1000 replicates. The confidence values are indicated at the branches. intron alignment. Bootstrap 1000 replicates. The confidence values are indicated at the branches. Green indicates C. coccodes clade; blue indicates C. nigrum clade. Green indicates C. coccodes clade; blue indicates C. nigrum clade. Agriculture 2023, 13, 511 12 of 20 Agriculture 2023, 13, x FOR PEER REVIEW 13 of 21 Figure 7. Comparison of glutamine synthetase second intron sequences of intriguing isolates Figure 7. Comparison of glutamine synthetase second intron sequences of intriguing isolates (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20) to type strain C. coccodes (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20) to type strain C. coccodes CBS 369.75. SNPs, including A, T, C, and G, are marked with red, green, blue, and yellow, respec- CBS 369.75. SNPs, including A, T, C, and G, are marked with red, green, blue, and yellow, respectively. tively. Table 3. Comparison of intriguing isolates to type strains *. Morphological differences were not found between the sizes of the C. coccodes and C. Percentage of Similarity to Type Strains nigrum conidia (for C. coccodes, the conidial length was 18.23 ± 6.13 μm and the width was Isolate 4.49 ± 1.04 μm; for C. nigrum, the conidial length was 21.17 ± 5.10 μm and the width was ITS Sequence act Sequence gaphd Sequence gs Sequence ** 4.34 ± 1.03 μm; Figure 8). The obtained conidial measurements overlapped with those of 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% the type strains of C. coccodes, C. nigrum, and C. dianense [16,21,37]. All of the isolates pro- C18U(G)TF1/1 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes duced aseptate, smooth-walled, hyaline, oval to cylindrical conidia with acute, subacute 100% C. nigrum 97% C. coccodes 97% C. coccodes or obtuse apices, typical of C. coccodes or C. nigrum (Figure 9). 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KST1F1 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KST3F1 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KSTF77 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes 100% C. coccodes 100% C. nigrum 100% C. nigrum 97% C21KSPeF20 100% C. dianense 100% C. dianense 100% C. dianense C. coccodes 100% C. nigrum 97% C. coccodes 97% C. coccodes * C. coccodes CBS:369.75, C. nigrum CBS:169.49, and C. dianense YMF 1.04943. ITS—ITS1-5.8S-ITS2 region, gaphd— glyceraldehyde-3-phosphate dehydrogenase gene intron, act—actin intron, gs—glutamine synthetase intron. ** No gs sequences of type C. nigrum or C. dianense isolates were found. Agricultur Agriculture e 2023 2023,, 13 13,, x FOR PEER 511 REVIEW 14 of 13 of 21 20 Agriculture 2023, 13, x FOR PEER REVIEW 14 of 21 Figure 8. Comparison of the conidial lengths (a) and widths (b) of Colletotrichum isolates and type Figure 8. Comparison of the conidial lengths (a) and widths (b) of Colletotrichum isolates and type Figure 8. Comparison of the conidial lengths (a) and widths (b) of Colletotrichum isolates and type strains. Boxes indicate quartiles (first and third), whiskers indicate the minimum and the maximum strains. Boxes indicate quartiles (first and third), whiskers indicate the minimum and the maximum strains. Boxes indicate quartiles (first and third), whiskers indicate the minimum and the maximum values, and points outside the boundary are outliers. values, and points outside the boundary are outliers. values, and points outside the boundary are outliers. Figure 9. Conidia of C. coccodes isolate C20UgKgPT2 (a) and C. nigrum isolate C21KSPeF6 (b). Figure 9. Conidia of C. coccodes isolate C20UgKgPT2 (a) and C. nigrum isolate C21KSPeF6 (b). Figure 9. Conidia of C. coccodes isolate C20UgKgPT2 (a) and C. nigrum isolate C21KSPeF6 (b). All the tested strains of both species were able to cause tomato fruit and potato tuber All the tested strains of both species were able to cause tomato fruit and potato tuber All the tested strains of both species were able to cause tomato fruit and potato tuber infection (Table 4). Regardless of the species used to infect the tomatoes, the infected fruit infection (Table 4). Regardless of the species used to infect the tomatoes, the infected fruit infection (Table 4). Regardless of the species used to infect the tomatoes, the infected fruit had typical dark lesions of anthracnose with sclerotia, milky-white swellings with conidia had typical dark lesions of anthracnose with sclerotia, milky-white swellings with conidia had typical dark lesions of anthracnose with sclerotia, milky-white swellings with conidia occasionally developed. In the case of tomato wound inoculation, the C. coccodes- and C. occasionally developed. In the case of tomato wound inoculation, the C. coccodes- and occasionally developed. In the case of tomato wound inoculation, the C. coccodes- and C. nigrum-caused disease radii were 5.2–6.2 mm and 3.2–8.3 mm, respectively, after 21 days C. nigrum-caused disease radii were 5.2–6.2 mm and 3.2–8.3 mm, respectively, after 21 days nigrum-caused disease radii were 5.2–6.2 mm and 3.2–8.3 mm, respectively, after 21 days of infection. On the intact fruit, the disease radius did not exceed 1.2 mm after 21 days for of infection. On the intact fruit, the disease radius did not exceed 1.2 mm after 21 days of infection. On the intact fruit, the disease radius did not exceed 1.2 mm after 21 days for all the isolates of both species, but after 35 days, one anthracnose-causing strain of C. all the isolates of both species, but after 35 days, one anthracnose-causing strain of C. Agriculture 2023, 13, 511 14 of 20 for all the isolates of both species, but after 35 days, one anthracnose-causing strain of C. nigrum reached a disease radius of 9.8 mm in width. All the tested strains of C. coccodes and C. nigrum were able to spread on potato slices. The disease radius was 2.5–5.2 mm for C. coccodes and 1.2–8.3 mm for C. nigrum after 21 days. No correlation between the disease severity and the original host was found: the isolates from tomatoes and potatoes could infect plants of both species. Nevertheless, the tomato fruit disease caused by both species was more rapid and extensive under the same temperature conditions compared with the potato tuber disease. Table 4. Pathogenicity tests. Average Disease Radius Average Disease Radius (mm) on Tomato after (mm) on Potato after Strain Species Host 21 days 35 days 21 days Identifier Wound Surface Surface Wound Inoculation Inoculation Inoculation Inoculation C21KSEgF7 C. nigrum Eggplant 4.8 0.0 0.9 2.3 C21KSEgF3 C. nigrum Eggplant 3.2 0.1 2.0 2.5 C21KSEgF4.1 C. nigrum Eggplant 3.2 0.1 2.3 4.3 C21KSPeF6 C. nigrum Pepper 3.7 1.0 9.8 2.0 C21KSPeF19 C. nigrum Pepper 5.2 0.3 2.5 8.3 C20AuPT5a C. coccodes Potato 6.2 0.2 2.0 5.2 C20UgKgPT2 C. coccodes Potato 5.2 1.2 2.2 2.5 C21KSTF88 C. nigrum Tomato 8.3 0.5 3.3 1.8 C21KSTF97 C. nigrum Tomato 7.5 0.5 1.2 1.2 C21KST3F2 C. nigrum Tomato 6.3 0.2 3.2 4.0 No isolate resistant to any examined fungicide was found (Table 5). Thiabendazole EC for C. coccodes was 0.65–58.38 mg/L, and that for C. nigrum was 0.58–20.29 mg/L. Six isolates (five C. coccodes from potato tubers and stem, and one C. nigrum from tomato fruit) were less sensitive to the chemical (EC > 10 mg/L). No resistance was found for azoxystrobin, EC for C. coccodes was 0.05 and 9.07 mg/L, EC for C. nigrum was 50 50 0.08–8.50 mg/L. Difenoconazole was the most effective chemical; EC for all the tested isolates was less than 0.12 mg/L. Table 5. Sensitivity to fungicides. EC , mg/L ** Isolation 50 Strain Identifier Species Source * Thiabendazole Azoxystrobin Difenoconazole C13V(GH)PT1/1 C. coccodes PT 4.24 0.08 0.06 C13K(S)PT11 C. coccodes PT 5.07 7.75 0.07 C13K(S)PT14 C. coccodes PT 7.75 0.08 0.06 C13K(S)PT15 C. coccodes PT 4.47 0.28 0.07 C13K(S)PT17 C. coccodes PT 10.20 5.82 0.12 C13K(S)PT21 C. coccodes PT 7.90 3.68 - C13K(S)PT34 C. coccodes PT 0.78 0.07 0.06 C13K(S)PT58b C. coccodes PT 3.47 - 0.07 C13HPT29/2 C. coccodes PT 0.91 0.05 0.05 C13G(B)PTde9 C. coccodes PT 50.30 0.07 0.06 C13G(B)PTde12 C. coccodes PT 0.85 0.06 0.05 C13G(B)PTde23 C. coccodes PT 0.93 0.09 0.06 C13G(B)PTes6 C. coccodes PT 33.38 0.10 0.06 C13G(B)PTes19 C. coccodes PT 8.78 0.10 0.06 C13G(B)PTal15 C. coccodes PT 0.96 0.09 0.09 Agriculture 2023, 13, 511 15 of 20 Table 5. Cont. EC , mg/L ** Isolation Strain Identifier Species Source * Thiabendazole Azoxystrobin Difenoconazole C13G(B)PTal19 C. coccodes PT 0.96 0.09 0.06 C13G(B)PTal20 C. coccodes PT 0.99 0.08 0.06 C13G(B)PTal23 C. coccodes PT 6.13 0.07 0.07 C13G(B)PTal24 C. coccodes PT 0.85 0.06 0.05 C13G(B- C. coccodes PT 1.00 0.08 0.06 Sh)PTsa29 C14M(Ch)PT6 C. coccodes PT - 0.09 0.06 C14M(Ch)PT18/2 C. coccodes PT - 0.08 0.06 C15M(L)PT1 C. coccodes PT 0.82 0.06 0.08 C15M(L)PT1/2 C. coccodes PT 0.94 - 0.09 C15M(L)PT4 C. coccodes PT 0.89 0.08 0.09 C15M(L)PT5 C. coccodes PT 0.85 0.08 0.09 C15M(L)PT6 C. coccodes PT - 0.08 0.06 C15M(L)PT7 C. coccodes PT 0.95 - 0.09 C16ME(Y-O)PL7 C. coccodes PL - - 0.09 C16ME(Y- C. coccodes PL - - 0.08 O)PL11 C16M(G)PS9 C. coccodes PS 0.85 0.08 0.09 C16M(G)PS15 C. coccodes PS 0.84 4.09 0.06 C16M(G)PS16b C. coccodes PS 58.38 0.07 0.08 C17K(K)TF5-2 C. nigrum TF 0.91 0.09 0.09 C17K(K)TF5-14 C. nigrum TF - 0.08 0.09 C17K(S)PTrs9 C. coccodes PT - 0.08 0.06 C17K(S)PTrs11/1 C. coccodes PT 0.87 0.08 0.07 C18M(L)TF1/1 C. coccodes TF 0.74 6.32 0.12 C18K(S)TF1/2 C. nigrum TF 20.29 8.50 - C18U(G)TF1/1 C. nigrum TF - - 0.07 C18U(G)PT4 C. coccodes PT 6.07 - - C18U(G)PT7 C. coccodes PT 0.65 7.75 0.10 C18U(G)PT11 C. coccodes PT 25.43 9.07 0.08 C18TPS8 C. coccodes PS - 7.75 0.09 C18TPS9 C. coccodes PS - 3.31 0.09 C19CyPT1/2 C. coccodes PT 0.95 0.07 0.09 C19CyPT2/1 C. coccodes PT 0.85 0.07 0.09 C20AuPT5a C. coccodes PT 0.75 0.08 0.07 C20UgLaPT1/1 C. coccodes PT 0.71 0.07 0.07 C20UgKgPT1 C. coccodes PT 0.82 0.08 0.07 C20UgKgPT2 C. coccodes PT 0.73 0.08 0.07 C20UgKgPT12 C. coccodes PT 0.83 0.07 0.07 C21KST3F2 C. nigrum TF 0.66 0.08 0.07 C21KSTF88 C. nigrum TF 0.67 0.08 0.07 C21KSTF97 C. nigrum TF 0.65 0.08 0.07 C21KSPeF6 C. nigrum PeF 0.68 0.08 0.07 C21KSPeF19 C. nigrum PeF 0.68 0.08 0.07 C21KSEgF3 C. nigrum EF 0.58 0.08 0.07 C21KSEgF4.1 C. nigrum EF 0.64 0.08 0.07 C21KSEgF6 C. nigrum EF 0.71 0.08 0.07 C21KSEgF7 C. nigrum EF 0.66 0.09 0.07 * PT—potato tuber, PS—potato stem, PL—potato leaf, TF—tomato fruit, PeF—pepper fruit, EF—eggplant fruit. ** EC —effective concentration. 4. Discussion The efficiencies of the known genetic markers in differentiating Colletotrichum species vary among different species complexes [4]. The ITS region is widely used in routine studies, although the result may be doubtful. For instance, in northern Italy, C. coccodes was reported as an agent of pepper root disease [10]. Undoubtedly, the species can cause root Agriculture 2023, 13, 511 16 of 20 disease; still, ITS-based identification remains insufficient. In Turkey, unusual symptoms of Colletotrichum disease leading to extremely high crop losses were discovered, and the pathogen was identified as C. coccodes [38]. However, the only molecular marker used in the study was the ITS region, so the identification seems uncertain. Dos Santos Vieira and colleagues [39] propose using gaphd and several other regions to distinguish between Colletotrichum species, while ITS and act are less effective. According to our study, both the act and gaphd genes are suitable, at least for C. coccodes and C. nigrum division (Figures 5 and 6). The Gs intron also proved useful for delineating Colletotrichum species. This gene sequence is mainly used to distinguish the species within C. gigasporum, C. orbiculare, and C. gloeosporoides species complexes. Thus, up to date, GenBank lacks the gs region sequences of the type material for many species. Several GenBank accession numbers marked as the C. coccodes gs gene (GU935816 and GU935817) presumably belong to C. nigrum, as they differ by approximately 2–3% from C. coccodes CBS164.49 or CBS369.75 (GenBank accession numbers HM171675 and HM171676, respectively) but they are similar to our strains that are identified as C. nigrum, based on the act or gaphd genes. We propose that at least 17 single nucleotide changes underlie the differences between the gs second intron of the two species. The C. nigrum currently presumed occurrence and host range seem to be lower than the real ranges. We assume that several reports of C. coccodes, for example [17], may display C. nigrum disease instead. According to the pertinent literature, the sexual process is unknown for C. coccodes or C. nigrum. The only way for strains of these species to exchange genetic material is via a parasexual process or through a vegetative compatibility reaction [40]. Based on the gs sequence of five isolates (C18U(G)TF1/1, C21KST1F1, C21KST3F1, C21KSTF77, and C21KSPeF20), we suppose that they might represent hybrids between C. coccodes and C. nigrum. Whereas we detected SNPs in all the isolates identified as C. nigrum based on gaphd, we assume these SNPs to be specific to C. nigrum (Figure 2). At least one of the isolates (C18U(G)TF1/1) was collected from tomato fruit grown near potato plants; therefore, it might have had a possibility of interfering with C. coccodes strains. Notwithstanding these putative hybrid isolates, we assume that the second intron of the gs gene is useful for distinguishing between C. coccodes and C. nigrum, and we propose a more active use of the GSF1—GSR1 primers for identifying the Colletotrichum species. Both C. coccodes and C. nigrum are currently considered as singleton species. According to Liu et al. [16], all potato-associated isolates belong to C. coccodes. At the same time, both C. nigrum and C. coccodes were able to infect tomato and pepper. The statement is supported by other studies [3–5] and by our data. Until now, we found no information regarding C. nigrum in Russia. Based on ITS region sequencing, only one Colletotrichum species—C. coccodes—was previously reported from potato and tomato leaves in Russia [9,23,24,26]. Belov and colleagues [9] used a specific primer pair (Cc1F1 and Cc2R1) [41] to detect C. coccodes [41]. Both test systems [23,42] were developed based on the ITS region, considered the universal fungi barcode [43]. However, they were of limited use in distinguishing C. coccodes and C. nigrum. C. dianense, which is very similar to C. nigrum, was recently described [37]. The authors of the study stated that it could be distinguished from C. nigrum by its conidial shape and apex. The ITS region and the act gene of the C. dianense type isolate YMF 1.04943 is 100% identical, and the gaphd gene is 99.66% similar to the C. nigrum type strain CBS 169.49 (one nucleotide difference, position 185). We compared our isolates to both species. Although, in our opinion, the two species are slightly different, we named our isolates from tomato, pepper, and eggplant C. nigrum, as C. nigrum is a well-known and earlier described species. The cross-virulence of Colletotrichum species was reported a while ago [44]. Yet, there are no literature reports of C. nigrum potato infections, as the species was only found on tomato and pepper [3,16], while the GenBank database contains several C. nigrum isolates (e.g., KU821311) reported from potatoes. Colletotrichum disease is known as post-harvest, Agriculture 2023, 13, 511 17 of 20 and is particularly harmful to climacteric fruits (e.g., tomato) [45]. Even though our study demonstrated the possibility of C. nigrum potato infection, no C. nigrum strains were isolated from potato. Another Colletotrichum species, C. acutatum s. str., was reported as a tomato and pepper infectious agent [5,46]. The presence of C. acutatum s.l. in potato leaves and mini tubers was revealed in our previous studies (unpublished data) based on the reaction with species- specific primers CaInt2/ITS4 [47]. Although C. acutatum s.l. has not been proven to be a potato disease agent, the possibility of its presence on potato tubers and leaves should be kept in mind. Liu and colleagues [16,21] mentioned that C. coccodes strains from tomato or other hosts produce larger conidia than C. coccodes from potato, and C. nigrum forms longer conidia than C. coccodes. Our results do not support these statements and show no significant difference between the conidial length or width among the three studied species (Figure 8). Contrary to Zheng and colleagues [37], we suppose that the morphological differentiation within the C. coccodes—C. nigrum—C. dianense clade may not be significant. All the tested chemicals—azoxystrobin, thiabendazole, and difenoconazole—proved to be effective against Colletotrichum spread. The results were in line with previous stud- ies [48–50]: normally, EC is less than 1 mg/L for all of the tested chemicals. In addition, azoxystrobin reduced black dot on tubers in field conditions [50]. Resistance to azoxys- trobin or thiabendazole was reported in other Colletotrichum species complexes [51,52]. We discovered five strains (C13G(B)PTde9, C13G(B)PTes6, C16M(G)PS16b, C18K(S)TF1/2, and C18U(G)PT11), with thiabendazole EC ranging over 20–50 mg/L. Sanders and Ko- rsten [52] classified strains with 66–70% growth on 0.5–2.5 mg/L thiabendazole as resistant, but we named them as less sensitive after Leite [53]. In our previous study, we exam- ined the -tubulin gene, but no specific mutations in any of the Colletotrichum isolates was found [54], contrary to Colletotrichum musae [53], less sensitive strains, Colletotrichum siamense [55], or Helminthosporium solani highly resistant strains [56]. Because even the highest EC values for Colletotrichum spp. are much lower than the concentrations in the working liquid for treatment (e.g., 170–250 mg/L for azoxystrobin, 4800–5600 mg/L for thiabendazole, and 187–625 mg/L for difenoconazole), we conclude that in general, all of the studied chemicals could still be considered as an effective strategy for anthracnose control on Solanaceae in Russia. 5. Conclusions Here, we present the results of the first extensive molecular and morphological anal- ysis of Colletotrichum species affecting Solanaceae plants in Russia. Two morphologically indistinguishable species, C. coccodes and C. nigrum, were revealed. The act and gaphd gene introns are suggested as the most suitable molecular markers to differentiate between these species. The Gs intron sequences give rise to the hypothesis of a parasexual process between these two species; therefore, further research is required. Eggplant and pepper plants were found to be infected exclusively by C. nigrum; tomato plants were infected by both species. Potato infection was caused only by C. coccodes. However, in vitro, both species showed an ability to infect tomato fruit and potato tubers. Three studied chemicals, azoxystrobin, difenoconazole, and thiabendazole, were effective against the isolates of both species, although several isolates were less sensitive to thiabendazole. Author Contributions: Conceptualization, S.E.; methodology, E.C., S.E. and M.Y.; software, E.C., S.E. and L.K.; validation, I.K., M.Y., M.K., A.B., E.C. and L.K.; formal analysis, E.C., S.E., M.Y. and M.K.; investigation, I.K., M.K., M.Y., A.B., G.B., A.E., M.P., A.T. and Y.T.; resources, S.E., E.C., A.B., M.Y. and I.K.; data curation, E.C., M.Y. and S.E.; writing—original draft preparation, M.Y.; writing—review and editing, S.E., A.B. and L.K.; visualization, M.Y.; supervision, S.E.; project administration, S.E.; funding acquisition, S.E., L.K. and E.C. All authors have read and agreed to the published version of the manuscript. Agriculture 2023, 13, 511 18 of 20 Funding: This research was funded by the RUDN University Scientific Grant System (project № 202193-2-000), and by the Russian Foundation for Basic Research (grant № 20-016-00139). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Newly generated ITS, act, gaphd, and gs sequences are deposited in GenBank under the accession numbers specified in Table 1. Acknowledgments: The authors are grateful to Anastasia Sharapkova and Tatiana Gavrilova (Rosetta Stone MSU) for the English improvement of the article. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. Marin-Felix, Y.; Hernández-Restrepo, M.; Iturrieta-González, I.; García, D.; Gené, J.; Groenewald, J.Z.; Cai, L.; Chen, Q.; Quaedvlieg, W.; Schumacher, R.K.; et al. Genera of phytopathogenic fungi: GOPHY 1. Stud. Mycol. 2017, 86, 99–216. [CrossRef] [PubMed] 2. Jayawardena, R.S.; Bhunjun, C.S.; Hyde, K.D.; Gentekaki, E.; Itthayakorn, P. 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Journal

Agriculture – Multidisciplinary Digital Publishing Institute

Published: Feb 21, 2023

Keywords: black dot; anthracnose; pathogen; potato; tomato; Colletotrichum; multi-gene phylogeny

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