Diversity of Colletotrichum species on strawberry ( Fragaria × ananassa ) in Germany

caused by Colletotrichum species is an important disease of strawberries ( Fragaria × ananassa ), but the species causing this disease in Germany have not been investigated based on modern systematics. By using multi-locus phylogenetic analyses (ITS, act , gapdh , chs-1 , his3 , tub2 ), 58 Colletotrichum isolates from previous and recent collections, obtained mainly from fruit anthracnose of cultivated strawberries in Germany, were identified or re-identified as C. fioriniae , C. godetiae and C. nymphaeae ( C. acutatum species complex) as well as C. anthrisci and C. line-ola ( C. dematium complex). Colletotrichum nymphaeae was dominant; most of the iso-lates belonged to one clonal lineage that occurs on strawberries throughout Europe, the United States of America, and some African and Asian countries. One of the other two haplotypes was distantly related and only represented by recently collected material. All other species, each of one haplotype, had only been isolated once or twice from German strawberries. This is the first report of C. anthrisci in Germany and for the genus Fragaria worldwide; all the other isolated fungi are newly reported for this genus in Germany. Comparisons of morphological characteristics of the species identified demonstrate that these features are of limited use for identification, even to species complex level. In pathogenicity tests, all five species caused anthracnose symptoms on ripe fruit of Fragaria × ananassa ‘Asia’.


INTRODUCTION
Strawberries (Fragaria × ananassa, Rosaceae) are the most common fruit crops after apples in Germany, with a harvested volume of 133,135 t in 2022 (Lehari, 2002;Statistisches Bundesamt, 2023).Intensive strawberry breeding, which has been primarily aimed at high yields, influenced the gene structure and thus immunity to pathogens in strawberries (Hardigan et al., 2018), so strawberry cultivation is associated with high production risks.In the presence of warm temperatures and high humidity, rapid outbreaks of anthracnose caused by Colletotrichum species can be expected (Howard et al., 1992).Trading of young plantlets with latent infections can rapidly spread the disease over long distances (Howard et al., 1992).Anthracnose of strawberry fruit is characterised as brown, sunken spots, in which orange conidial mucilage develops.Main symptoms of green tissues (runners, leaves and stalks) include dark, sunken, necrotic areas (Howard et al., 1992).Brooks (1931) reported a new strawberry disease in Florida, United States of America (USA), called anthracnose, and described the causative pathogen as C. fragariae.In the 1980s, C. acutatum sensu lato was first reported from strawberries in the USA (Smith and Black, 1986).Anthracnose symptoms in strawberry fields in Germany (Baden, Palatinate) and other European countries including France, Bulgaria, Sweden, Denmark, Spain and Belgium have been reported at least since the 1990s and attributed to C. acutatum and C. gloeosporioides, while C. fragariae was only found within material from the USA (Denoyes and Baudry, 1995;Laun and Fried, 1996;Bobev et al., 2002;Nilsson et al., 2005;Sundelin et al., 2005;Garrido et al., 2008;Debode et al., 2009).The first molecular study of Colletotrichum in Germany by Nirenberg et al. (2002) focusing on C. lupini, included a strain from strawberry in Germany that had also been identified as C. acutatum based on ITS sequence data.Within this investigation, several Colletotrichum strains from different hosts and countries, including from strawberries in Germany, had been collected and are maintained at the culture collection of the Biologische Bundesanstalt für Land-und Forstwirtschaft (BBA) Berlin, now Julius Kühn-Institut (JKI), Bundesforschungsinstitut für Kulturpflanzen (Federal Research Centre for Cultivated Plants), Institut für Epidemiologie und Pathogendiagnostik, Braunschweig, Germany.
The systematics of Colletotrichum has changed within the last 20 years, due to application of molecular data, especially in multi-locus DNA sequence analyses.Colletotrichum acutatum and C. gloeosporioides that had been previously regarded as causal agents of strawberry anthracnose were shown to be large species complexes (Damm et al., 2012a;Weir et al., 2012).The number of accepted Colletotrichum species, those that have been revised or newly described based on multi-locus DNA sequences, is constantly increasing; more than 300 Colletotrichum species in at least 15 species complexes and more than ten further species are currently accepted (Liu et al., 2022;Talhinhas and Baroncelli, 2023).To date, at least 22 species are known from strawberry, belonging to the C. acutatum, C. boninense, C. coccodes, C. dematium, C. gloeosporioides and C. truncatum species complexes (Farr and Rossman, 2024).Colletotrichum acutatum sensu stricto is only confirmed from strawberry in Australia (Damm et al., 2012a), while most reports of C. acutatum on strawberry in the USDA Fungal Databases (Farr and Rossman, 2024), including that from Germany (Nirenberg et al., 2002), date back to the pre-molecular era, or were from before C. acutatum was shown to be a species complex (Damm et al., 2012a).Therefore, these fungi should be considered as C. acutatum sensu lato and could include other species within this complex.
To date, only Colletotrichum strains infecting a few random, often exotic host plants collected in Germany have been identified or described based on modern systematics (e.g.Damm et al., 2012aDamm et al., , b, 2014Damm et al., , 2019;;Weir et al., 2012); while there are no records of Colletotrichum species from Fragaria hosts in Germany identified on the same basis.
The aims of the present study were to investigate the diversity of Colletotrichum on strawberries from all over Germany based on multi-locus sequence data, to characterise the species morphologically and to test their pathogenicity to fruit of cultivated strawberry (Fragaria × ananassa) 'Asia'.

Isolates
Symptomatic material of cultivated strawberry plants (Fragaria × ananassa) was collected in different regions of Germany, including Saxony (Dresden, Görlitz, Ore Mountains), Brandenburg (Spreewald), North Rhine-Westphalia (Münsterland), Lower Saxony (Lüdersfeld) and Mecklenburg-Western Pomerania (Mecklenburg Lake District).The collected material also included fruit bought at markets in Germany (some of Polish origin) and one sample from wild strawberry (Fragaria vesca).
Most of the newly collected material consisted of infected fruit of F. × ananassa, which had symptoms of brown, sunken spots of necrotic tissue that spread radially.Orange conidial masses sometimes developed in the centres of these spots.Flat, pale to dark mouse-grey mycelium often formed at the edges and sometimes over the entire infection sites.Elongated dark brown necroses initially formed on host stems and petioles, which spread and developed into stem-encompassing necroses and constrictions, on which conidia were sometimes observed.On affected leaves of F. × ananassa, roundish to oval necrotic areas were observed, each with dark brown irregular edges and a light brown centre, which partially merged.Symptoms of the F. vesca leaf sample were similar; grey-brown spots of various sizes and shapes with paler centres and darker irregular margins formed especially at the edges of affected leaves.
To obtain single-conidium isolates, conidia formed on the necrotic host tissues were spread on the surface of petri dishes with synthetic nutrient-poor agar (SNA; Nirenberg, 1976) using a drop of sterile water.On the following day, single germinating conidia were transferred to oatmeal agar (OA; Crous et al., 2019).Plant parts, on which no conidia were present, were surface sterilised (1 min in 3.5% NaClO, 30 s in 70% ethanol), washed in sterile water and placed in petri dishes on sterile filter paper with sterile water until conidia were formed, from which single-conidium isolates were produced.
A further 22 isolates from the culture collection of the former BBA had been collected more than 20 years ago in the federal states of Mecklenburg-Western Pomerania, Saxony, Baden-Württemberg, Hesse, Rhineland-Palatinate and Brandenburg and included one strain bought in a supermarket that originated from the Netherlands.
Although it was not possible to complete and confirm all collection data, it is assumed that all the BBA isolates originated from anthracnose symptoms, especially from fruit rot, because the material had been deposited by plant protection offices in different German federal states in the years when strawberry anthracnose spread across Europe.For one of the isolates from Saxony there was information provided that a 2-year-old stock of 'Selva' (imported plants) was very heavily infested, and a 1-year-old stock was over 50% infected.Most of the strawberry runners from which isolates had been obtained were characterised as necrotic, while one isolate had a noticeable reddish discolouration at the infection site.Two isolates originated from brown roots of F. vesca var.semperflorens.There was no information on the leaf symptom and no further information on fruit symptoms than fruit rot.
The isolates were stored in the culture collection of the Senckenberg Museum of Natural History Görlitz, Germany (GLMC; Table 1).Selected isolates were deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany.

Morphological analyses
The Colletotrichum strains were cultivated on SNA with autoclaved filter paper and double-autoclaved stems of Anthriscus sylvestris and on OA.Cultures were incubated at 20°C under near UV light (12 h daily photoperiod) for 10 d.Microscopic preparations were made in clear lactic acid.Measurements of microscopic structures of the fungi were carried out according to Damm et al. (2007), with 30 measurements per structure and strain, using a Nikon SMZ1000 dissecting microscope (DM) and a Nikon Eclipse 80i compound microscope with differential interference contrast (DIC).Appressoria were observed on the reverse sides of the SNA cultures.Colony features on SNA and OA were observed after the incubation period.To calculate colony growth rates, the diameters of the colonies were determined after 7 and 10 d.Colony colour was characterised according to Rayner (1970).
For preliminary identification and selection of reference strains, blastn searches were carried out with ITS sequences in NCBI GenBank.Maximum parsimony (MP) analyses were carried out on the multi-locus alignments (ITS,gapdh,his3,act,tub2) with Phy-logenetic Analysis Using Parsimony (PAUP) v. 4.0b10 (Swofford, 2003), using the heuristic search option with 100 random sequence additions and tree bisection and reconstruction as the branch-swapping algorithm.Alignment gaps were treated as missing, and all characters were unordered and of equal weight.No more than ten trees of score (length) greater than or equal to ten were saved in each replicate.Tree length, consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for the resulting trees.The robustness of the obtained trees was evaluated by 10,000 bootstrap replications, using the fast-stepwise addition algorithm (Hillis and Bull, 1993).Maximum likelihood (ML) analyses were calculated online using IQ-TREE 1.6 (http://iqtree.cibiv.univie.ac.at;Nguyen et al., 2015;Trifinopoulos et al., 2016) and model testing under the Bayesian information criterion (BIC) (Kalyaanamoorthy et al., 2017).Branch support was obtained by 5000 replicates of ultrafast bootstrap (ufb; Hoang et al., 2018;Minh et al., 2013).Support values ≥ 95% were considered relevant, following Guindon et al. (2010).

Pathogenicity tests
Selected Colletotrichum isolates were incubated on OA at 20°C under near UV light for 10 d.To harvest the conidia, 10 mL of sterile distilled water was added to an OA culture of each isolate and swirled thoroughly.The resulting conidium suspensions were adjusted to a final concentration of 2 × 10 4 conidia mL -1 .Ripe fruit of F. × ananassa 'Asia' (Obsthof Rüdiger, Dresden, Germany) of the quality standard EU No. 543/2011 class Extra (Anonymous, 2011) were surface sterilised with 1% NaClO for 6 min, washed three times with sterile distilled water and placed under sterile conditions on moist filter paper in covered glasses (Gourmet glass, round edge, 300 mL, Weck).Each fruit was inoculated with a 5 μL droplet of conidium suspension (without wounding) or treated with 5 μL of sterile distilled water (experimental control).Five repetitions (fruit) were made for each Colletotrichum strain and the control.The covered glasses with inoculated strawberries were placed randomly in a climate cabinet (20°C, 14 h fluorescent light/10 h dark daily cycle, humidity up to 100%).The experiment was carried out twice.Seven d post inoculation (dpi), resulting lesion sizes were determined by counting infected unit squares on a grid placed over scaled photographs.Symptoms were evaluated visually and photographed 7 dpi or, in the case of delayed infection development, 10 dpi.Re-isolations were made from lesion edges and resulting fungi were identified.

Phylogenetic analyses
Based on preliminary morphological examinations and blastn searches of the ITS sequences on NCBI Gen-Bank, 56 of the 58 isolates were identified as belonging to the C. acutatum species complex.The other two isolates were identified as belonging to the C. dematium complex.
In the multi-locus phylogenetic analyses of the C. acutatum species complex (gene boundaries of ITS: 1-546, gapdh: 557-829, chs-1: 840-1121, his3: 1132-1519, act: 1530-1777, tub2: 1788-2285) 181 isolates, including a selection of 32 (of the 56) newly sequenced isolates from strawberry, 147 reference strains of the C. acutatum species complex, and the outgroup C. orchidophilum CBS 632.80 and IMI 309357 (Table 1, Supplementary table 1) and 2285 characters including the alignment gaps were processed.Of these, 429 characters were parsimony-informative, 154 parsimony-uninformative and 1702 constant.After a heuristic search using PAUP, the maximum of 590 equally most parsimonious trees were retained (length = 1203 steps, CI = 0.615, RI = 0.947, RC = 0.582, HI = 0.385).One of these trees is shown in Figure 1.The topology of these trees was similar, which was verified for a large section of trees.They differed in the position of taxa within subclades.The consensus tree of the ML analysis confirmed the tree topology obtained with parsimony.The bootstrap values of the two analyses generally agreed with each other.The phylogeny consists of five main clades representing clades 1 to 5 in Damm et al. (2012a).Most of the isolates clustered within C. nymphaeae (clade 2), where they formed three haplotype groups; most of the strains were of one haplotype.This was the same as several other isolates from strawberry in Europe, Northern America and Africa.According to a preliminary analysis, 46 isolates from the present study belonged to this haplotype, and 24 of these isolates were included in the final analyses.Isolates GLMC 2599, GLMC 2600 and GLMC 2606 represented a second haplotype with other strains from strawberry in Europe and the USA and a strain from Anemone in the Netherlands.The third haplotype included isolates GLMC 2653, GLMC 2654 and a strain from Fragaria in the USA, forming a distinct subclade (bootstrap support MP/ML: -/100).Two isolates (GLMC 2660, GLMC 2661) clustered in C. fioriniae (clade 3); the haplotype was the same as that of a strain from apple in Italy and from Grevillea in Germany.Three further strains, two from Germany (GLMC 2589, GLMC 2590) and one from the Netherlands (GLMC 2583), grouped with C. godetiae (clade 5).This haplotype was the same as that of strains from strawberry and other hosts from other European countries, including an isolate from Mahonia in Germany.

Colletotrichum anthrisci
Cultural characteristics.Colonies on SNA flat with entire margins, medium hyaline, pale cinnamon to pale peach, partly covered by whitish aerial mycelium, Anthriscus stems partly covered by saffron spore masses, colony reverse sides same colours; 21-26 mm in 7 d (30-38 mm in 10 d).Colonies on OA flat with undulate margins, surface coral to luteous, almost entirely covered by felty, flesh to rosy vinaceous aerial mycelium and saffron spore masses, colony reverse sides red, coral, brick to apricot; 9-18 mm in 7 d (14-30 mm in 10 d).Conidia in mass saffron.
Notes: Sequence data and morphology of C. godetiae are very variable, including the shape and size of the conidia that are studied and discussed in Damm et al. (2012a).While the ex-type strain and a strain from Fragaria in the Netherlands (CBS 125972) formed comparatively fusiform conidia, conidia of other strains, including those from Fragaria in Germany studied here, were rather cylindrical, and those of further strains were even clavate (Damm et al., 2012a; this study, Figure 5L).
Cultural characteristics.Colonies on SNA flat with entire margins, medium hyaline, pale cinnamon to pale ochreous, medium, filter papers and Anthriscus stems partly covered by tiny olivaceous black spots, aerial mycelium lacking, colony reverse sides same colours; 29-36 mm in 7 d (38->40 mm in 10 d).Colonies on OA flat with entire margins, medium buff to honey, with tiny olivaceous grey to olivaceous black spots, aerial mycelium lacking, colony reverse sides honey to vinaceous buff; 21-26 mm in 7 d (36->40 mm in 10 d).Conidia in mass whitish.
Notes: The morphology of isolate GLMC 2587 agreed with the description in Damm et al. (2009) that based on material from Apiaceae hosts, except for the slightly longer conidia of the isolate from strawberry formed on Anthriscus stems.

Pathogenicity
Seven dpi, all tested isolates of the five Colletotrichum species caused symptoms on strawberry fruit F. × ananassa 'Asia'.However, for almost all isolates and species in both experiments, some fruit did not develop symptoms or showed symptoms that were very slight 7 dpi.
Due to infection by other fungi or collapsing of individual strawberries, some were excluded from both experiments, and resulted in failures, especially after inoculations with C. nymphaeae isolates GLMC 2445, GLMC 2588, GLMC 2600, GLMC 2610, GLMC 2658, C. lineola isolate GLMC 2587 and C. fioriniae isolate 2661.However, all inoculated fruit in the experiments showed symptoms 10 dpi.
The species that were inoculated were respectively re-isolated from all symptoms developing on inoculated strawberries.No symptoms developed after control treatments with sterile, distilled water.

DISCUSSION
This study has demonstrated that Colletotrichum is widespread on strawberries in Germany, although neither the BBA collection nor recent sampling described here covered all regions of this country.The Colletotrichum collection of the BBA shows occurrence of Colletotrichum on strawberries around the year 2000 in up to seven federal states of Germany, including Brandenburg, Lower Saxony, Saxony, Mecklenburg-Western Pomerania, Baden-Württemberg, Hesse and (probably) Rhineland-Palatinate.The new collections (2019-2021) confirm this genus in Brandenburg, Lower Saxony, Saxony, Mecklenburg-Western Pomerania, and also include collections from North Rhine-Westphalia.
Most of the samples in these collections were of C. nymphaeae, which is the most common Colletotrichum species on strawberry in Germany.This species belongs to the C. acutatum species complex, occurs on several hosts and is common on strawberry in Europe, Iran and North America (Damm et al., 2012a;Baroncelli et al., 2015;Karimi et al., 2017;Grammen et al., 2019;Wang et al., 2019;Tsvetkova and Kuznetsova, 2022).Based on the present study, C. nymphaeae is present in all federal states of Germany for which data are available and occurs on F. × ananassa and F. vesca var.semperflorens, a cultivated variety of wild strawberry.Colletotrichum nymphaeae was mostly isolated from fruit of F. × ananassa in the field and from the market.Fruit bought from the marked in Görlitz originated from Poland, confirming a recent report of C. nymphaeae on strawberries in that country (Tsvetkova and Kuznetsova, 2022).This species was also isolated from symptoms on different green plant parts of F. × ananassa, both from established plants in the field and from purchased young plants from German propagation culture.
In a survey of the C. acutatum species complex on strawberry in the USA, C. nymphaeae dominated (97.7% of the isolates), and almost all isolates were of one clonal lineage regardless of the isolation source (Wang et al., 2019).One representative of these strains (isolate 16-320) was included in the phylogeny of the present study, and this isolate grouped with most of the isolates from strawberry and with the majority of those examined by Damm et al. (2012a) and Baroncelli et al. (2015).Thus, this one clonal lineage is almost entirely restricted to strawberry, is distributed throughout Europe, also occurs in Israel and some African countries, and is demonstrated here to occur in Germany and Poland and to be the dominating lineage in Germany, both in historical and recent collections (Damm et al., 2012a; the present study).Wang et al. (2019) suggested that this clonal lineage had been distributed throughout the USA and Canada via quiescently infected strawberry transplants, which could also explain its widespread occurrence in European countries.In contrast, the dominating species on strawberry in China, Korea, Japan and Taiwan belong to the C. gloeosporioides complex.The species detected in Korea, Japan and Taiwan were completely different from those found in Europe, while in China C. nymphaeae is also present, but to a lesser extent (Nam et al., 2013;Han et al., 2016;Jayawardena et al., 2016;Gan et al., 2017;Chung et al., 2020).
The other two haplotypes of C. nymphaeae from strawberry in Germany detected in the present study were also identical to haplotypes from the USA, as described by Wang et al. (2019).This indicates three possibly independent introductions of this pathogen to German strawberries.One of these two haplotypes represented a clone that is closely related to the most common haplotype, which also includes some strains from strawberry from other European countries.The German isolates were part of the old collection.In contrast, the other haplotype has several nucleotide differences to the other haplotypes occurring on strawberry and included only three isolates, one from strawberry in the USA and two from a recent collection on strawberry in Brandenburg, Germany.This could represent a new lineage of C. nymphaeae which has restricted distribution and unknown impacts.Despite its dominating incidence on strawberries, there are few records of C. nymphaeae in Germany prior to the present study, including a collection from Freyburg, Saxony-Anhalt by H. Jage and one from a dried-up pond close to Flemsdorf, Brandenburg by J. Kruse (Dämmrich et al., 2023).Both collections are from Nymphaea, and both specimens are kept in the fungarium of the Senckenberg Museum of Natural History Görlitz (GLM-F127296, GLM-F129560).However, these identifications were not confirmed by DNA sequence data.
Colletotrichum godetiae also belongs to the C. acutatum species complex and occurs on several hosts including many woody plants as well as strawberry (Damm et al., 2012a).Although this fungus is probably common on strawberry in Europe (Damm et al., 2012a;Baroncelli et al., 2015;Tsvetkova and Kuznetsova, 2022), it was found only twice on strawberries from Germany in the present study and was not detected in the new collections.Despite proven infectivity of C. godetiae on F. × ananassa 'Asia' under laboratory conditions, there has been no invasive spread in German strawberry stocks within the last 20 years.In contrast to C. nymphaeae and C. fioriniae, C. godetiae is not known from strawberry in the USA and Canada (Baroncelli et al., 2015;Wang et al., 2019).The only North American strain of this species included in Damm et al. (2012a) was from a different host, and the haplotype represented by the strains from strawberry in the present study occurs both on strawberry and on other hosts.This suggests spread of this species on strawberries within Europe, rather than an introduction from the USA or occasional transmissions to strawberries by other hosts.This species was previously found in Germany, on leaf spots of Mahonia aquifolium and in necrotic wood of sour cherry (Prunus cerasus) (Damm et al., 2012a;Bien and Damm, 2020).However, the present report is the first from strawberry in Germany.A clade that was previously regarded as a subclade of C. godetiae and comprised exclusively strains from South America was recently described as new species, C. americanum (Zapata et al., 2024).
Colletotrichum fioriniae is another species belonging to the C. acutatum species complex.This pathogen occurs on several hosts, mainly crops, and is common on fruit of apple and strawberry as well as Vaccinium species in Europe, the USA and New Zealand and on Persea in Australia.In Germany, C. fioriniae has previously only been reported from an indoor collection of Grevillea sp.(Proteaceae) (Damm et al., 2012a), an exotic genus from Australia.However, the present report is the first of C. fioriniae from strawberry in Germany, and is also the first report from an outdoor cultivated crop in Germany.In contrast to C. nymphaeae, the haplotype of C. fioriniae detected in the present study, is not specialised to strawberry.There are several haplotypes of this species that occur both on strawberry and other hosts (Damm et al., 2012a;this study).This suggests a different transmission route of this pathogen to strawberries, possibly by host-jumps rather than in planting material of the same host.The high variability between isolates of C. fioriniae from the same hosts could also be related to recombination, as the formation of sexual morphs and of hybrids with C. acutatum (sensu stricto) are known (Marcelino et al., 2008;Damm et al., 2012a).In the USA, C. fioriniae was determined to be of low incidence on strawberries (five of 217 Colletotrichum isolates; Wang et al., 2019).In the present study, C. fioriniae was isolated only twice from recently collected material from one strawberry field.However, since this fungus was able to infect F. × ananassa 'Asia' under laboratory conditions with development of large necrotic areas and abundant sporulation, there is a potential risk of the pathogen spreading to other strawberry stocks.
As the two subclades of C. fioriniae reported in the phylogeny of Damm et al. (2012a) were not supported and had similar hosts and distributions, they were treated as one species.The phylogeny of Chen et al. (2022) that included new strains from Malus domestica in China supported the two subclades, and the second subclade was described as C. orientale [actually as "orientalis"], but invalidly (Art.40.8 Shenzhen), while Zhang et al. (2023a) described two further related strains from China as C. radermacherae.Since new strains from ornamental plants in China were intermediate between all three species, Zhang et al. (2023b) reduced both new species to synonymy with C. fioriniae.In the phylogeny of the present study that included one "C.radermacherae" strain (GZCC 21-0814) but not the intermediate strains of Zhang et al. (2023b), the two subclades were supported by one analysis (ML) that was not applied in Damm et al. (2012a).The strains from strawberry in Germany belong to the subclade containing the ex-type strain of C. fioriniae.
Colletotrichum lineola belongs to the C. dematium species complex and had been isolated from dead plant parts and diseases of several plants, mainly in Central Europe and North America.These hosts included Fragaria (petiole) in Canada (Damm et al., 2009).Tsvetkova and Kuznetsova (2022) also isolated C. lineola from strawberries in Russia.In the present study, this fungus was found on strawberry fruit in Germany for the first time.In the pathogenicity tests, this species also caused very small symptoms on fruit, and it was not detected in the new collections.Therefore, C. lineola can be regarded as of minor importance for strawberry cultivation.
Another species of the C. dematium complex, C. anthrisci, was described from dead stems of A. sylvestris in the Netherlands (Damm et al., 2009) and was recently classified as highly endangered (Talhinhas and Baroncelli, 2021).In contrast, this fungus was found to be ubiquitous in a forest in Japan, where it was isolated from seedlings of several trees, including Prunus grayana, Fraxinus lanuginosa, Cornus controversa and Magnolia obovata that had been killed by damping-off (Konno et al., 2011).Colletotrichum anthrisci was also recorded on avocado fruit with anthracnose symptoms in Chile and was confirmed to cause this disease (Bustamente et al., 2022).In the present study, C. anthrisci was isolated from leaf spots of F. vesca in a forest in Germany and was shown to cause fruit anthracnose of cultivated strawberry.
Thus, this species is neither host-specific nor rare, and is known from three continents where it is at least locally very common.This is the first report of C. anthrisci both from Germany and from Fragaria worldwide and the first evidence of this species causing anthracnose on cultivated strawberry fruit under laboratory conditions.However, the symptoms caused on F. × ananassa 'Asia' were mild and C. anthrisci has to date not been collected from cultivated strawberry in the field.
Prior to the present study, most of the Colletotrichum strains from strawberry in Europe had been identified as C. acutatum.This also applies to the isolates from the BBA collection examined, one of which was the basis of the report by Nirenberg et al. (2002).In the present study, isolates previously identified as C. acutatum were re-identified as C. nymphaeae, except for the isolate originating from the Netherlands that was shown to be C. godetiae.None of the isolates were C. acutatum (sensu stricto).Colletotrichum acutatum (sensu stricto) has been found predominantly in the southern hemisphere (Damm et al., 2012a) and is known to be associated with fruit rot of strawberry, but only in Australia (Sreenivasaprasad and Talhinhas, 2005;Damm et al., 2012a).To date, there is no confirmed occurrence of C. acutatum (sensu stricto) on strawberries elsewhere in the world.Nearly all reports from strawberry prior to the treatment of the C. acutatum complex by Damm et al. (2012a) and some later reports, refer to other species within this complex, which was confirmed here for the pathogens causing strawberry anthracnose in Germany.
Other previously unreported Colletotrichum strains from the BBA collection had been identified as C. truncatum, C. fragariae and C. gloeosporioides, based on the original strain list.In the present study, C. truncatum was re-identified as C. lineola, and C. fragariae and C. gloeosporioides were re-identified as C. godetiae.Colletotrichum truncatum has not been reported from Germany (Farr and Rossman, 2024).This fungus forms curved conidia, but with different shape than C. lineola and belongs to the C. truncatum species complex, while C. lineola belongs to the C. dematium complex (Damm et al., 2009).In contrast, C. fragariae and C. gloeosporioides form cylindrical conidia and belong to the C. gloeosporioides complex.However, no species of the C. gloeosporioides complex were found among the isolates from Germany that were examined here.However, the conidia of the C. godetiae isolates from strawberry studied here were more cylindrical than fusiform (Figure 5L).The cylindrical conidium shape of isolates of C. godetiae, like those from strawberries studied here, as well as of some other species of the C. acutatum species complex, is reminiscent of species of the C. gloeosporioides complex (Damm et al., 2012a;Weir et al., 2012).This can cause confusion because identifications of isolates by morphology only are not reliable, even to species complex level.This also applies to species with curved conidia, as for the C. lineola isolate examined in this study.In contrast, C. theobromicola (syn.C. fragariae) has only been reported from strawberries from a few countries, of which only isolates from the USA have been confirmed by sequence data, while sequences of C. fragariae reported from the United Kingdom and Japan suggest also C. godetiae and a different species in the C. gloeosporioides complex, respectively (Nirenberg et al., 2002;Moriwaki et al., 2003;Weir et al., 2012;Farr and Rossman, 2024).The only reliable reports of C. gloeosporioides (sensu stricto) from strawberries are from China (e.g.Han et al., 2016).As the present study was the first molecular identification of Colletotrichum strains from strawberries in Germany after the revisions of the respective Colletotrichum species complexes based on multi-locus sequence data (Damm et al., 2009(Damm et al., , 2012a;;Weir et al., 2012), all species detected in this study are new reports on strawberries in Germany.
While symptoms caused by C. eriobotryae and C. nymphaeae on loquat fruit were indistinguishable (Damm et al., 2020), those caused by the species tested on strawberry fruit in the present study were very different from each other.This could be attributed to the high genetic distance of the species studied here that belong to different species complexes or at least different main clades within the C. acutatum complex.The two species from loquat both belonged to clade 2 of the C. acutatum complex.Although the symptoms caused by the five different species were typical for the individual species, it is unlikely that species can be identified based on host symptoms alone.
Colletotrichum nymphaeae and C. fioriniae were more aggressive than C. godetiae in strawberry fruit assays reported by Baroncelli et al. (2015).This can be tentatively confirmed by the present study, although our data could not be statistically analysed due to large variations and nil results.In pathogenicity tests, MacKenzie et al. (2009) showed that Colletotrichum isolates from strawberry and blueberry from Florida (USA) that were later identified as C. nymphaeae and C. fioriniae, respectively, based on ITS and gapdh sequences (Damm et al., 2012a), caused anthracnose on strawberry fruits; the lesions caused by C. nymphaeae isolates were larger than those caused by C. fioriniae isolates.This cannot be confirmed by the present study, because lesion sizes caused by one of the C. fioriniae isolates belonged to the largest in the tests.Because of variability in lesion sizes caused by these two species, no conclusions can be drawn about relative virulence of these species.Based on the number of isolates and virulence, C. nymphaeae is likely to be the most important strawberry anthracnose pathogen in Europe, representing the highest economic risk for commercial strawberry production.This was previously confirmed for the United Kingdom and Russia and in the present study also for Germany.
The present study has identified the pathogens causing anthracnose of cultivated strawberry in Germany, which provides the basis for application and development of targeted control measures for management of these pathogens in commercial strawberry cultivation.This will require assessments of fungicide effectiveness and pathogen resistance.Targeted strawberry breeding should be aimed at resistance to specific Colletotrichum pathogens defined at species and/or haplotype level.Monitoring of pathogens in the field and testing of acquired, especially imported planting material using molecular methods would help to detect possible new pathogen species and haplotypes, as the pathogen spectrum could change due to changes in prevailing climates.strains, and Dr Alexandra Wichura, Landwirtschaftskammer Niedersachsen, Pflanzenschutzamt, Sachgebiet Obstund Gemüsebau, Hannover, Germany, for providing diseased plant specimens.The authors also thank the other supporters who contributed material and data for this research.The research was financially supported by the Deutsche Bundesstiftung Umwelt, Osnabrück, Germany.

Figure 1 .
Figure 1.The first of 590 most parsimonious trees obtained from a heuristic search of the combined ITS, act, gapdh, chs-1, his3, tub2 sequence alignment of Colletotrichum isolates from Fragaria and representative strains of the C. acutatum species complex.Bootstrap support values of the MP analysis >70% and of the ML analysis >95% are shown at the nodes.Colletotrichum orchidophilum CBS 632.80 and IMI 309357 were used as outgroup.Numbers of ex-type strains are indicated by an asterisk.Isolates obtained or sequenced in this study are shown in bold font.Strain numbers are followed by substrate (host genus) and country of origin (NL = Netherlands, NZ = New Zealand, ZA = South Africa).(Continued)

Figure 2 .
Figure 2. The first of 440 most parsimonious trees obtained from a heuristic search of the combined ITS, act, gapdh, chs-1, his3, tub2 sequence alignment of Colletotrichum isolates from Fragaria and representative strains of the C. dematium species complex.Bootstrap support values of the MP analysis >70% and of the ML analysis >95% are shown at the nodes.Colletotrichum chlorophyti IMI 103806 was used as outgroup.Numbers of ex-type strains are indicated by an asterisk.Strains obtained or sequenced in this study are emphasised in bold font.Strain numbers are followed by substrate (host genus) and country of origin (NL = Netherlands, CR = Czech Republic).

Table 1 .
, 36 isolates, includ-ing the two newly sequenced isolates from strawberry, 33 reference strains and the outgroup C. chlorophyti IMI 103806 (Table1, Supplementary table 1), along with 2298 characters including the alignment gaps were processed.Of these, 415 characters were parsimony-informative, 205 parsimony-uninformative and 1678 constant.After a heuristic search using PAUP, the maximum of 440 equal-Strains of Colletotrichum spp.used in this study, with collection details and GenBank accession numbers.

Table 1 .
(Continued).Culture collection of Senckenberg Museum of Natural History Görlitz, Görlitz, Germany; BBA: Culture collection of the Biologische Bundesanstalt für Land und Forstwirtschaft, Berlin, Germany; DSM: German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.
(Continued) Diversity of Colletotrichum species on strawberry (Fragaria × ananassa) in Germany a GLMC: b ITS, 5.8S nuclear ribosomal RNA gene with the two flanking internal transcribed spacers; gapdh, 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase gene; chs-1, partial sequences of the chitin synthase 1 gene; his3, partial sequences of the histone H3 gene; act, partial sequences of the actin gene; tub2, partial sequences of the β-tubulin gene.Table 1. (Continued).