Molecular Diversity of Anthracnose Pathogen Populations Associated with UK Strawberry Production Suggests Multiple Introductions of Three Different Colletotrichum Species.

Fragaria × ananassa (common name: strawberry) is a globally cultivated hybrid species belonging to Rosaceae family. Colletotrichum acutatum sensu lato (s.l.) is considered to be the second most economically important pathogen worldwide affecting strawberries. A collection of 148 Colletotrichum spp. isolates including 67 C. acutatum s.l. isolates associated with the phytosanitary history of UK strawberry production were used to characterize multi-locus genetic variation of this pathogen in the UK, relative to additional reference isolates that represent a worldwide sampling of the diversity of the fungus. The evidence indicates that three different species C. nymphaeae, C. godetiae and C. fioriniae are associated with strawberry production in the UK, which correspond to previously designated genetic groups A2, A4 and A3, respectively. Among these species, 12 distinct haplotypes were identified suggesting multiple introductions into the country. A subset of isolates was also used to compare aggressiveness in causing disease on strawberry plants and fruits. Isolates belonging to C. nymphaeae, C. godetiae and C. fioriniae representative of the UK anthracnose pathogen populations showed variation in their aggressiveness. Among the three species, C. nymphaeae and C. fioriniae appeared to be more aggressive compared to C. godetiae. This study highlights the genetic and pathogenic heterogeneity of the C. acutatum s.l. populations introduced into the UK linked to strawberry production.

Introduction

Fragaria × ananassa (common name: strawberry) is a hybrid species cultivated worldwide belonging to the Rosaceae family. Since the 1980s, the UK strawberry industry has expanded rapidly representing a significant component of fruit production in the country [1]. Anthracnose is a major disease of cultivated strawberry, caused by two species complexes of the fungus referred to as C. acutatum and C. gloeosporioides. C. acutatum is considered to be the dominant cause of strawberry anthracnose, and the second most important pathogen of strawberry after Botrytis cinerea [2-7]. The C. gloeosporioides complex includes C. fragariae, which is now considered synonymous with a new species C. theobromicola [8]. However, researchers have often continued to use the name C. fragariae when referring to a pathogen that was associated with strawberry anthracnose [9-12]. C. gloeosporioides is found only occasionally on strawberry in Europe [3,7].

C. acutatum s.l. was described for the first time as a strawberry pathogen in California in 1983 [13], and has since appeared to have spread worldwide, including the UK, through runners and propagating material [2,6,14-16]. A first extensive genetic characterization of C. acutatum s.l. representing the global diversity of the pathogen led to its sub-division into genetic groups named from A1 to A9 [6, 17]. More recently, the C. acutatum s.l. has been sub-divided into more than 30 species based on multi-locus phylogeny [18].

The first record of C. acutatum s.l. in the UK was in 1978, on Anemone sp. grown in Jersey [19]. In 1982, the first incidence of anthracnose disease in strawberries caused by C. acutatum s.l. was recorded in the UK, and was attributed to the importation of infected strawberry runners from the USA [20]. DNA sequences in public databases suggest two UK isolates (CBS198.35 and CBS199.35) that were collected in 1935 from the host Phormium spp. (common name “New Zealand flax”) belong to C. acutatum s.l. [18, 21]. CABI database records during 1978 to 1983 shows the incidence of the pathogens various hosts and in different locations in the UK (http://www.herbimi.info). However, it seems highly improbable that the first outbreak on strawberry led to the wide dispersal of the pathogen. In 1993, Lovelidge proposed that the continued introduction of infected strawberry material from abroad was so common that the disease was destined to become endemic in the UK [14]. In subsequent years, further outbreaks have been reported on strawberry linked to the importation of infected propagation material mainly from mainland Europe and on other important crop hosts [20,22,23].

Strawberry anthracnose symptoms produced by the two Colletotrichum species complexes are similar and can be found on all parts of the plant [12]. Flower blight and fruit rot are common symptoms in the field [24], whereas lesions on stolons, petioles and leaves are mainly found in plant nurseries [15]. Crown symptomatology is characterized by reddish-brown necrotic areas [25] and in some cases stunting and chlorosis have been associated with root necrosis [15].

Research has been carried out to characterize C. acutatum s.l. populations related to strawberry in specific geographic areas including Israel, France, Bulgaria, Spain, Belgium and other European countries [2-5,7,26] and from specific regions of the USA [25]. Other research has attempted to characterize C. acutatum s.l. related to strawberry using isolates collected worldwide [3], both by genomic fingerprinting (such as RFLP, apPCR, etc.) and sequence analysis based on the ITS region. Results have highlighted the presence of at least one representative “clonal” population suggesting a single source of origin and, consequentially, that the disease is spread through infected propagation material. However, ITS sequences alone or genomic fingerprinting are not suitable to discriminate among the newly assigned species designations.

In a recent study based on the analysis of more than two decades of anthracnose incidence data sets gathered by authorities responsible for plant health, trade was identified as the main route of entry and establishment of C. acutatum in the UK strawberry production. Over this period, various nurseries were importing planting material into the UK, and at least 55 cases of infested material that was planted in the field through imports that were not intercepted by the border inspection posts, were identified [20].

The focus of the present study was to assess the extent of the genetic and pathogenic diversity of these introduced pathogen populations mainly utilising a unique collection of C. acutatum s. l. isolates established through the plant health inspection surveys from the early 1980s onwards. We focused on C. acutatum s.l. because previous reports from France, Israel, UK, Bulgaria and Spain had described this taxa as a major widely distributed pathogen, compared with other species such as C. gloeosporioides s.l. that occur less frequently in Europe [2-5,12]. A range of historic and contemporary C. acutatum s. l. isolates including those from worldwide strawberry crops, other plant hosts in the UK, as well as worldwide representatives from different hosts building on our previous work were accessed as reference sources for determining the genetic and species identities of isolates associated with UK strawberry anthracnose phytosanitary control work. Based on multi-locus phylogenetic analysis, we have identified 12 different haplotypes that belong to three different species C. nymphaeae, C. godetiae and C. fioriniae suggesting multiple introductions of the strawberry anthracnose pathogen. Pathogenic and growth characteristics of these haplotype representatives further highlight the heterogeneity of the introduced pathogen populations.

Materials and Methods

Fungal isolates and culture conditions

A diverse collection of C. acutatum s.l. was assembled for this study including: 67 isolates associated with strawberry production in the UK (obtained from the UK Food and Environment Research Agency, or FERA responsible for plant health within the Department for Environment, Food and Rural Affairs), 27 C. acutatum s.l. isolates collected from strawberry in other countries, and 13 isolates collected from other host species in the UK. For further comparison, 33 isolates were added to represent other genetic groups, and novel species from previous studies [6,17,18]. This included two isolates of C. fruticola, two isolates of C. aenigma (belonging to C. gloeosporioides species complex [8]) associated with strawberry, two UK isolates of C. spinaciae and one isolate each of C. graminicola, C. higginsianum [27] and C. fioriniae [28]. Sequence data of the markers was retrieved from the reference genome sequences available from Genbank for C. graminicola and C. higginsianum (accession numbers: ACOD01000000 and CACQ02000000, respectively) used among out-groups in the phylogenetic analysis (Fig 1). Details of the isolate collection used in the present study are provided in Table 1.

Fig 1

Multilocus phylogenetic analysis of the Colletotrichum isolates used in this study.

Bayesian MCMC analysis tree constructed from the alignment based on the concatenation of rRNA, TUB, MAT1-2 and GPDH partial sequences of 140 Colletotrichum acutatum sensu lato isolates used in this study. The tree was rooted with sequences from C. graminicola and C. higginsianum retrieved from whole genome sequences and sequences of four C. gloeosporioides sensu lato and two C. spinaciae obtained experimentally. Isolates used to investigate variation in aggressiveness are highlighted in bold.

Table 1

Colletotrichum sp. strains used in this study with isolation details and GenBank accessions.

Strain Code	Genus	Species	Genetic group [6]	Country	Host	Accession numbers
						ITS	TUB	MAT1-2	GAPDH
Isolates from strawberry in UK
B88	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246514	KM251867	KM251969	KM252115
NI90	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	AF411766	AJ409294	KM251970	KM252116
CSL 1079	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246515	KM251868	KM251981	KM252118
CSL 2546	Colletotrichum	fioriniae	A3	United Kingdom	Fragaria x ananassa	KM246516	KM251870	KM251983	KM252120*
CSL 899	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246518	KM251872	KM251985	KM252122*
CSL 310	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246519	KM251873	KM251986	KM252123
CSL 915	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246520	KM251874	KM251987	KM252124*
CSL 886	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246521	KM251875	KM251988	KM252125
CSL 919	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246522	KM251876	KM251989	KM252126*
CSL 916	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246523	KM251877	KM251990	KM252127*
CSL 918	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246524	KM251878	KM251991	KM252128*
CSL 917	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246525	KM251879	KM251992	KM252129
CSL 223	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246526	KM251880	KM251993	KM252130
CSL 224	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246527	KM251881	KM251994	KM252131
CSL 225	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246528	KM251882	KM251995	KM252132
CSL 255	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246529	KM251883	KM251996	KM252133
CSL 256	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246530	KM251884	KM251997	KM252134*
CSL 258	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246531	KM251885	KM251998	KM252135
CSL 456	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria vesca	KM246532	KM251886	KM251999	KM252136
CSL 493	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246533	KM251887	KM252000	KM252137
CSL 494	Colletotrichum	godetiae	A4	United Kingdom	Fragaria vesca	KM246534	KM251888	KM252001	KM252138
CSL 604	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246535	KM251890	KM252003	KM252140
CSL 607	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246538	KM251893	KM252006	KM252143
CSL 608	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246539	KM251894	KM252007	KM252144
CSL 872	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246541	KM251896	KM252009	KM252146
CSL 903	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246542	KM251897	KM252010	KM252147
CSL 1001	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246543	KM251898	KM252011	KM252148
CSL 1258	Colletotrichum	fioriniae	A3	United Kingdom	Fragaria x ananassa	KM246544	KM251899	KM252012	KM252149
CSL 1259	Colletotrichum	fioriniae	A3	United Kingdom	Fragaria x ananassa	KM246545	KM251900	KM252013	KM252150*
CSL 1260	Colletotrichum	fioriniae	A3	United Kingdom	Fragaria x ananassa	KM246546	KM251901	KM252014	KM252151
CSL 1261	Colletotrichum	fioriniae	A3	United Kingdom	Fragaria x ananassa	KM246547	KM251902	KM252015	KM252152
CSL 1262	Colletotrichum	fioriniae	A3	United Kingdom	Fragaria x ananassa	KM246548	KM251903	KM252016	KM252153*
CSL 1305	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246549	KM251904	KM252017	KM252154
CSL 1376	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246550	KM251905	KM252018	KM252155
CSL 1377	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246551	KM251906	KM252019	KM252156
CSL 1378	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246552	KM251907	KM252020	KM252157
CSL 1379	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246553	KM251908	KM252021	KM252158
CSL 1380	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246554	KM251909	KM252022	KM252159
CSL 1381	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246555	KM251910	KM252023	KM252160
CSL 1382	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246556	KM251911	KM252024	KM252161
CSL 1383	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246557	KM251912	KM252025	KM252162
CSL 1384	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246558	KM251913	KM252026	KM252163
CSL 1385	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246559	KM251914	KM252027	KM252164
CSL 1386	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246560	KM251915	KM252028	KM252165
CSL 1387	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246561	KM251916	KM252029	KM252166
CSL 1388	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246562	KM251917	KM252030	KM252167
CSL 1389	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246563	KM251918	KM252031	KM252168
CSL 1390	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246564	KM251919	KM252032	KM252169
CSL 1391	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246565	KM251920	KM252033	KM252170
CSL 1392	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246566	KM251921	KM252034	KM252171
CSL 1393	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246567	KM251922	KM252035	KM252172
CSL 1394	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246568	KM251923	KM252036	KM252173
CSL 1395	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246569	KM251924	KM252037	KM252174
CSL 1396	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246570	KM251925	KM252038	KM252175
CSL 1397	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246571	KM251926	KM252039	KM252176
CSL 1398	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246572	KM251927	KM252040	KM252177
CSL 1429	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246573	KM251928	KM252041	KM252178
CSL 1441	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246574	KM251929	KM252042	KM252179
CSL 1442	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246575	KM251930	KM252043	KM252180
CSL 1443	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246576	KM251931	KM252044	KM252181
CSL 1444	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246577	KM251932	KM252045	KM252182
CSL 1449	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246578	KM251933	KM252046	KM252183
CSL 2064	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246579	KM251934	KM252047	KM252184
CSL 1002	Colletotrichum	godetiae	A4	United Kingdom	Fragaria x ananassa	KM246580	KM251935	KM252048	KM252185
CSL 892	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	KM246584	KM251938	KM252053	KM252188
IMI 299103 [18]	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria vesca	JQ948231	JQ949882	KM252069	JQ948561
PD88-857, CBS 125973 [18]	Colletotrichum	nymphaeae	A2	United Kingdom	Fragaria x ananassa	JQ948232	JQ949883	KM252100	JQ948562
C. acutatum sensu lato from strawberry worldwide
C2897	Colletotrichum	nymphaeae	A2	Australia	Fragaria x ananassa	AJ300558	AJ314718	KM251967	KM252113
CSL 397	Colletotrichum	nymphaeae	A2	USA	Fragaria x ananassa	AF411765	AJ409296	KM251968	KM252114
CSL 1053	Colletotrichum	godetiae	A4	Netherlands	Fragaria x ananassa	AJ536210	KM251869	KM251982	KM252119
CSL 891	Colletotrichum	nymphaeae	A2	Portugal	Fragaria sp.	EF622184	KM251889	KM252002	KM252139
CSL 511	Colletotrichum	nymphaeae	A2	France	Fragaria x ananassa	KM246536	KM251891	KM252004	KM252141
CSL 729	Colletotrichum	nymphaeae	A2	Switzerland	Fragaria x ananassa	KM246537	KM251892	KM252005	KM252142
CSL 1430	Colletotrichum	godetiae	A4	Norway	Fragaria vesca	KM246585	KM251939	KM252054	KM252189
CSL 1432	Colletotrichum	godetiae	A4	Norway	Fragaria x ananassa	KM246586	KM251940	KM252055	KM252190
PJ7 [28]	Colletotrichum	fioriniae	A3	New Zealand	Fragaria x ananassa	genome: JARH00000000
CSL 1020, IMI 301119 [18]	Colletotrichum	nymphaeae	A2	Kenya	Fragaria vesca	JQ948266	JQ949917	KM252070	JQ948596
IMI 311743 [18]	Colletotrichum	nymphaeae	A2	USA	Fragaria x ananassa	JQ948258	JQ949909	KM252071	JQ948588
IMI 335544	Colletotrichum	nymphaeae	A2	Italy	Fragaria x ananassa	KJ018636	KJ018648	KM252072	KJ018660
IMI 345026 [18]	Colletotrichum	godetiae	A4	Spain	Fragaria x ananassa	JQ948424	JQ950075	KM252073	JQ948755
CSL 1005, IMI 345027	Colletotrichum	nymphaeae	A2	France	Fragaria x ananassa	AJ536199	KM251946	KM252074	KM252198
IMI 345028	Colletotrichum	nymphaeae	A2	Colombia	Fragaria x ananassa	AF090853	KM251947	KM252075	KM252199
IMI 345029	Colletotrichum	nymphaeae	A2	Costa Rica	Fragaria x ananassa	KM246591	KM251948	KM252076	KM252200
CSL 1034, IMI345030	Colletotrichum	nymphaeae	A2	Costa Rica	Fragaria x ananassa	AJ536203	KM251949	KM252077	KM252201
IMI 345031	Colletotrichum	nymphaeae	A2	Italy	Fragaria x ananassa	KM246592	KM251950	KM252078	KM252202
IMI 345578 [18]	Colletotrichum	fioriniae	A3	New Zealand	Fragaria ananassa	JQ948334	JQ949985	KM252080	JQ948664
CSL 1046, IMI 346326	Colletotrichum	simmondsii	A2	Australia	Fragaria x ananassa	AJ536208	KM251952	KM252081	KM252204
IMI 345585 [18]	Colletotrichum	salicis	A7	New Zealand	Fragaria x ananassa	JQ948476	JQ950127	KM252084	JQ948807
CSL 1090, IMI 348160	Colletotrichum	nymphaeae	A2	USA	Fragaria x ananassa	AJ536200	KM251953	KM252086	KM252205
IMI 348177 [18]	Colletotrichum	nymphaeae	A2	USA	Fragaria x ananassa	KM246593	KM251954	KM252087	KM252206
IMI 348490	Colletotrichum	nymphaeae	A2	France	Fragaria x ananassa	KM246594	KM251955	KM252088	KM252207
CSL 1086, IMI 348498	Colletotrichum	nymphaeae	A2	France	Fragaria x ananassa	KM246595	KM251956	KM252089	KM252208
CSL 1049, IMI 348499	Colletotrichum	fioriniae	A3	France	Fragaria x ananassa	AJ536220	KM251957	KM252090	KM252209
IMI 360928 [18]	Colletotrichum	nymphaeae	A2	Switzerland	Fragaria x ananassa	JQ948243	JQ949894	KM252091	JQ948573
Strains isolated from different hosts in UK
RB-MAL-03 [23]	Colletotrichum	godetiae	A4	United Kingdom	Malus domestica	KF834206	KF834207	KM252049	KF834208
RB-MAL-04	Colletotrichum	godetiae	A4	United Kingdom	Malus domestica	KM246582	KM251936	KM252050	KM252186
CSL 1294	Colletotrichum	lupini	A1	United Kingdom	Lupinus polyphyllus	AJ300561	KM251944	KM252059	KM252194
CSL 287 [18]	Colletotrichum	acutatum	A5	United Kingdom	Statice sp.	JQ948389	JQ950040	KM252060	JQ948720
RB-VIT-01,CBS 129951 [22]	Colletotrichum	godetiae	A4	United Kingdom	Vitis vinifera	KF834203	KF834204	KM252061	KF834205
CSL 455 [18]	Colletotrichum	nymphaeae	A2	United Kingdom	Photinia sp.	JQ948217	JQ949868	KM252063	JQ948547
JC51, CBS 129948 [18]	Colletotrichum	fioriniae	A3	United Kingdom	Tulipa sp.	AJ749680	KM251945	KM252064	KM252195
CSL 302a	Colletotrichum	fioriniae	A3	United Kingdom	Nandina domestica	AJ749670	AJ748626	KM252065	KM252196
CSL 473 [18]	Colletotrichum	fioriniae	A3	United Kingdom	Liriodendron tulipifera	JQ948345	JQ949996	KM252066	JQ948675
CSL 318 [18]	Colletotrichum	fioriniae	A3	United Kingdom	Magnolia sp.	JQ948346	JQ949997	KM252067	JQ948676
IMI 350308	Colletotrichum	lupini	A1	United Kingdom	Lupinus sp.	AJ300561	KM251951	KM252079	KM252203
CBS 198.35 [18]	Colletotrichum	kinghornii	A7	United Kingdom	Phormium sp.	JQ948454	JQ950105	KM252083	JQ948785
PD93-1748, CBS 126527 [18]	Colletotrichum	godetiae	A4	United Kingdom	Prunus avium	JQ948408	JQ950059	KM252101	JQ948739
Isolates from different host worldwide and used as references for genetics groups / species
PT250, CBS 129953 [18]	Colletotrichum	rhombiforme	A6	Portugal	Olea europaea	JQ948457	JQ950108	KM251971	JQ948788*
PT135, CBS 129945 [18]	Colletotrichum	nymphaeae	A2	Portugal	Olea europaea	JQ948201	JQ949852	KM251972	JQ948531
PD85-694, CBS 126519 [18]	Colletotrichum	chrysanthemi	A2	Netherlands	Chrysanthemum sp.	JQ948272	JQ949923	KM251973	JQ948602
PD89-582, CBS 126524 [18]	Colletotrichum	simmondsii	A2	Netherlands	Cyclamen sp.	JQ948281	JQ949932	KM251974	JQ948611*
PT227, CBS 129952 [18]	Colletotrichum	acutatum	A5	Portugal	Olea europaea	JQ948364	JQ950015	KM251975	JQ948695*
Tom-21, CBS 129954 [18]	Colletotrichum	tamarilloi	A8	Colombia	Cyphomandra betacea	JQ948188	JQ949839	KM251976	JQ948518
Tom-12, CBS 129955 [18]	Colletotrichum	tamarilloi	A8	Colombia	Cyphomandra betacea	JQ948189	JQ949840	KM251977	JQ948519
CBS 193.32 [18]	Colletotrichum	godetiae	A4	Greece	Olea europaea	JQ948415	JQ950066	KM251978	JQ948746*
PT30	Colletotrichum	lupini	A1	Portugal	Lupinus albus	AJ300561	AJ292250	KM251979	KM252117*
CR46, CBS 129947 [18]	Colletotrichum	fioriniae	A3	Portugal	Vitis vinifera	JQ948343	JQ949994	KM251980	JQ948673*
9178	Colletotrichum	salicis	A7	Norway	Vaccinium corymbosum	KM246583	KM251937	KM252051	KM252187*
MP1, CBS 129972 [18]	Colletotrichum	salicis	A7	USA	Acer platanoides	JQ948466	JQ950117	KM252052	JQ948797*
PJ8	Colletotrichum	acutatum	A5	New Zealand	Pyrus pyrifolia	KM246587	KM251941	KM252056	KM252191*
ATCC MYA-663	Colletotrichum	fioriniae	A3	USA	Malus domestica	KM246589	KM251943	KM252058	KM252193*
HY09	Colletotrichum	lupini	A1	Canada	Lupinus albus	KJ018635	KJ018647	KM252062	KJ018659*
JL198	Colletotrichum	godetiae	A4	Serbia	Olea europaea	AJ749689	AJ748613	KM252068	KM252197*
AR3787, CBS 118191 [18]	Colletotrichum	phormii	A7	South Africa	Phormium sp.	JQ948453	JQ950104	KM252082	JQ948784*
CBS 607.94 [18]	Colletotrichum	salicis	A7	Netherlands	Salix sp.	JQ948460	JQ950111	KM252085	JQ948791*
ALM-NRB-30K	Colletotrichum	godetiae	A4	Israel	Prunus dulcis	DQ003129	KM251960	KM252094	KM252212*
CBS 101611 [18]	Colletotrichum	sp. 1	-	Costa Rica	Fern	JQ948196	JQ949847	KM252095	JQ948526*
BBA 70884, CBS 109225 [18]	Colletotrichum	lupini	A1	Ukraine	Lupinus albus	JQ948155	JQ949806	KM252096	JQ948485*
STE-U 164, CBS 112980 [18]	Colletotrichum	acutatum	A5	South Africa	Pinus radiata	JQ948356	JQ950007	KM252097	JQ948687*
STE-U 5303, CBS 112989 [18]	Colletotrichum	laticiphilum	A2	India	Hevea brasiliensis	JQ948289	JQ949940	KM252098	JQ948619
CBS 122122 [18]	Colletotrichum	simmondsii	A2	Australia	Carica papaya	JQ948276	JQ949927	KM252099	JQ948606*
CBS 211.78 [18]	Colletotrichum	costaricense	-	Costa Rica	Coffea sp.	JQ948181	JQ949832	KM252102	JQ948511
DPI 11711, CBS 292.67 [18]	Colletotrichum	brisbanense	A2	Australia	Capsicum annuum	JQ948291	JQ949942	KM252103	JQ948621
DPI 13483, CBS 294.67 [18]	Colletotrichum	simmondsii	A2	Australia	Carica papaya	JQ948277	JQ949928	KM252104	JQ948607*
ATCC 38896, CBS 526.77 [18]	Colletotrichum	nymphaeae	A2	Netherlands	Nymphaeae alba	JQ948199	JQ949850	KM252105	JQ948529
CBS 797.72	Colletotrichum	fioriniae	A3	New Zealand	Pinus radiata	KM246598	KM251961	KM252106	KM252213*
OCO-ARC-4	Colletotrichum	sp. 2	-	USA	Citrus x sinensis	EU647305	KM251962	KM252107	EU647318*
STF-FTP-10	Colletotrichum	sp. 2	-	USA	Citrus x sinensis	EU647306	KM251963	KM252108	EU647319
Coll-25	Colletotrichum	scovillei	A2	Taiwan	Capsicum annum	KJ018637	KJ018649	KM252109	KJ018661
Coll-154	Colletotrichum	scovillei	A2	Taiwan	Capsicum annum	DQ410028	KM251964	KM252110	KM252214
Isolates as out-group
CSL 311	Colletotrichum	fruticola	OG	USA	Fragaria x ananassa	KM246512	KM251865	KM251965	KM252111*
CSL 386	Colletotrichum	fruticola	OG	USA	Fragaria x ananassa	KM246513	KM251866	KM251966	KM252112*
CSL 780	Colletotrichum	aenigma	OG	UK	Fragaria x ananassa	KM246517	KM251871	KM251984	KM252121*
CSL 869	Colletotrichum	aenigma	OG	UK	Fragaria x ananassa	KM246540	KM251895	KM252008	KM252145*
CSL 593	Colletotrichum	spinaciae	OG	UK	Spinacia oleracea	KM246596	KM251958	KM252092	KM252210
CSL 739	Colletotrichum	spinaciae	OG	UK	Spinacia oleracea	KM246597	KM251959	KM252093	KM252211
M1.001 [27]	Colletotrichum	graminicola	OG	USA	Zea mais	genome: ACOD0100000000
IMI 349063 [27]	Colletotrichum	higginsianum	OG	Trinidad and Tobago	Brassica chinensis	genome: CACQ0200000000

Abbreviation

CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands IMI: Culture collection of CABI Europe UK Centre, Egham, UK CSL: Culture collection of The Food and Eviroment Research Agency, DEFRA, York, UK OG: out-group* strains used for pathogenicity tests

Cultures were maintained at 25°C on potato dextrose agar medium (PDA, Difco Laboratories, USA) for up to ten days under a 12 h light/ 12 h dark cycle. Long–term storage at 4°C involved cutting mycelial plugs from the edge of actively growing cultures on PDA and suspending them in sterile water.

Characterization of genetic variation

Genomic DNA was extracted according to the Chelex 100 protocol [29], with some modifications [30]. DNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, DE, USA).

Various target regions were used to characterise genetic diversity amongst the fungal isolates including: ITS region, partial sequence of the beta-tubulin 2 gene (TUB) (exons 3 through 6, including introns 2 through 4), partial sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and partial sequence of the mating type gene (MAT1-2) (the intron included in the conserved HMGbox region). Target regions were amplified using PCR reaction mixes (20 μl) that contained 1 μl of DNA, 1 μl each of primer (20 μM), 7 μl of H20 and 10 μl of ReadyMix RedTaq (Sigma).

PCR amplification of the target regions for sequencing was carried out as described below using previously published primers under conditions standardised for routine work. For ITS, primers ITS1Ext and ITS4Ext [31] were used. The amplification program consisted of 2 min of initial denaturation (95°C), 30 cycles of amplification (1 min at 94°C, 1 min at 55°C, and 1 min at 72°C) and a final extension at 72°C for 5 min. For TUB, primers TB5 and TB6 [31] were used. The amplification program consisted of 2 min initial denaturation (95°C), 30 cycles of amplification (1 min at 94°C, 1 min at 65°C and 1 min at 72°C) and a final extension at 72°C for 5 min. For GAPDH, primers GDF1 and GDR1 [32] were used. The amplification program consisted of 2 min initial denaturation at 95°C, 35 cycles of amplification (1 min at 94°C, 1 min at 60°C and 30 sec at 72°C) and a final extension at 72°C for 3 min. For MAT1-2, primers HMGacuF2 and HMGacuR [21] for C. acutatum s.l. and primers HMGgloeF1 and HMGgloeR1 for C. gloeosporioides s. l. [33] were used. The amplification program consisted of 5 min initial denaturation at 95°C, 40 cycles of amplification (1 min at 95°C, 1 min between 48°C and 55°C and 30s at 72°C) and a final extension of 20 min at 72°C. PCR products were separated using gel electrophoresis and purified using the QIAquick PCR purification kit (Qiagen, USA).

Sequencing of PCR products was carried out at the University of Warwick Genomics Centre, using an ABI Prism 7900HT or ABI3100 sequence detection system (Applied Biosystems, UK). PCR products were cleaned up and then quantified with reference to a ladder (Bioline EasyLadder I) containing DNA fragments of known concentration. One to five microliters of each sample (depending on DNA concentration) were used in sequencing reactions with the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, UK). ABI trace files were analyzed and consensus sequences were generated using Geneious 7.1.6 [34]. All the sequences were aligned using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) and were manually edited to optimise the alignment, as required. Multiple alignments were end trimmed in order to have comparable nucleotides.

Multiple sequence alignments were exported to MEGA5 [35] where best-fit substitution models were calculated for each separate sequence dataset. In order to evaluate whether the four sequenced loci were congruent and suitable for concatenation, tree topologies of 50% Neighbour-Joining bootstrap and maximum parsimony analysis (100,000 replicates) were separately performed for each gene and visually compared [36]. The multilocus concatenated alignment (ITS, TUB2, MAT1-2 and GAPDH) was performed with Geneious 7.1.6 [34]. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes 3.2.1 [37] for combined sequence datasets. Models of nucleotide substitution for each gene determined by MEGA5 were included for each locus. The analysis in MrBayes ran for 5000000 of generations to reach a P value lower than 0.01 with two parallel searches using three heated and one cold Markov chain sampled every 100 generations; 25% of generations were discarded as burn-in. Further phylogenetic analysis was performed by the neighbour-joining method with 1,000 bootstrap replicates under Kimura’s two-parameter correction using Geneious 7.1.6 [34] and the results are presented in Figs 1 and 2.

Fig 2

Percentage occurrence of Colletotrichum acutatum sensu lato species and relative numbers of haplotypes identified among 67 strains isolated from strawberry in UK.

Comparison of fungal growth in culture

The 67 fungal isolates collected from strawberry in the UK were compared with a subset of other isolates (chosen based on genetic, host and geographic diversity) including 49 isolates of C. acutatum s.l. and four isolates of C. gloeosporioides s.l. for in vitro growth studies on PDA (Potato Dextrose Agar, BD Difco). For experiments, a 7 mm diameter mycelial plug excised from the edge of an actively growing PDA culture was placed at the centre of a fresh PDA plate. In the growth experiment, two perpendicular colony diameters were measured daily and colony radius was calculated from cultures incubated at four different temperatures (15°C, 20°C, 25°C and 30°C) in darkness. Data corresponding to the linear growth phase were subjected to analysis of variance of regression in order to create growth curves for each isolate at each temperature. In both tests three plates were used as replicates. Statistical analysis was performed by SIGMAPLOT 10 program (Sigmaplot Software, USA). Colony characters were recorded after 15 days of incubation at 25°C under 12 h light/ 12 h dark cycle.

Pathogenicity tests

Representative isolates (highlighted with asterisks in Table 1) of each C. acutatum s.l. group isolated from strawberry in UK, together with reference isolates from other hosts, were used for pathogenicity tests on the generally susceptible strawberry cultivar Elsanta [38]. A conidial suspension was prepared for each isolates by flooding 10-day-old PDA culture plates with sterile deionised water. Spore concentration was adjusted to 105 spores ml-1 and 106 spores ml-1 for fruit and crown inoculation, respectively [7,38]. Unripe fruits (white fruit beginning to turn pink, as shown in Fig 3A) [39] were inoculated with a 5μl drop of conidial suspension. Before inoculation, fruit surfaces were disinfected for 5 min using NaClO (1% active chlorine) in 50% EtOH, washed three times in sterilized water, blotted dry and placed in a tray with moist sand on the bottom to prevent movement of the fruits during further procedures. After inoculation, fruits were incubated at 25°C under 12h light/ 12h in dark cycle.

Fig 3

Strawberry fruits and plants used for pathogenicity tests (A and C) and symptoms (B and D).

(A) Unripe fruits (phenological stage turning white-pink) used for artificial inoculations of Colletotrichum spp. (B) Strawberry fruits 7 days after inoculation with Colletotrichum sp. spores suspension showing typical black spot symptoms (bottom left) and with sterile water used as control (top right) (C) Three-month-old strawberry plants used to pathogenicity assays (D) Strawberry plant crown sectioned showing presence of red-brownish lesions characteristic of anthracnose caused by Colletotrichum spp.

Disease symptoms were evaluated 7 days after inoculation (d.a.i.) (Fig 3B) by recording the incidence of disease (% of infected fruits), and the aggressiveness of lesion development using the following severity scale: 0, no visible lesions; 1, lesions on less than 33% of fruit surface; 2, lesions covering 33–66% of fruit surface; and 3, lesions covering more than 66% of fruit surface. Three fruits inoculated with sterile distilled water (SDW) as well as fresh fruits served as non-inoculated controls. Four independent replicates were tested for each fungal isolate, consisting of three inoculated fruits for each replicate. At the end of the experiment, Colletotrichum isolates were re-isolated from infected fruits and cultured on PDA to confirm colony characteristics.

The capability of the isolates to produce crown rot symptoms was evaluated by injecting the crowns of three-months-old strawberry plants (Fig 3C) with 0.2 mL conidial suspension using a syringe [4,7]. Plants were placed in glasshouse at 23°C with 16h light / 8h darkness. After 24 days (d.a.i.), plants were evaluated for the presence of crown tissues with red-brownish discoloration, wilting and collapse of the plant, typical symptoms of Colletotrichum crown rot, according to the following severity scale: 0, no lesions; 1, crown tissues discoloration but no wilting or collapse; 2, wilting or collapse of part of the plant; and 3, plant death. Crowns of all plants were sectioned and examined for the presence of red-brownish lesions (Fig 3D). Crown infection was confirmed by re-isolation of the pathogen. Three plant crowns injected with SDW as well as untouched plants served as negative controls for each replicates. The experiment was independently replicated three times, with six plants for each replicate.

Values of disease severity were used to calculate a Disease Index (DI, average severity) according to the following formula: Σvn/N, where v represents the numeric value of the class, n is the number of plants or fruits assigned to the class, N is the total number of the plants or fruits assessed. Data for pathogenicity tests on both fruits and plants were subjected to analysis of variance ANOVA and means compared using Tukey’s multiple range test by Systat11 (Systat Software, USA).

Results

Characterization of genetic variation, and species identification

Phylogenetic trees were constructed using combined ITS, TUB2, GADPH and MAT1-2 sequence data set consisting of 148 Colletotrichum isolates (Table 1). As shown in Fig 1, most of the C. acutatum s.l. isolates (49/67) were identified as belonging to C. nymphaeae (= A2 genetic group), based on clustering with high bootstrap value with the reference isolates CBS 797.72, PT135, IMI345028 and other genetically similar isolates (identical sites = 1422/1438 or 98.9%; pairwise identity = 99.9%). A smaller proportion of isolates in the diversity collection (12/67) were identified as belonging to C. godetiae (= A4 genetic group) based on genetic clustering with reference isolates ALMNRB-30K, CBS 193.32 and JL198 (identical sites = 1411/1438 or 94.6%; pairwise identity = 99.4%). And finally, six isolates were identified as belonging to C. fioriniae (= A3 genetic group) based on clustering with the reference isolate ATCC 56813 (identical sites = 1.436 /1443 or 99.5%; pairwise identity = 99.9%).

Molecular characterisation of 67 Colletotrichum isolates collected from strawberry in the UK along with the reference isolates representing the host and geographic diversity (Figs 1 and 2) suggests that there have been multiple introductions of the anthracnose pathogen belonging to different Colletotrichum species into the country. Three different species C. nymphaeae, C. godetiae and C. fioriniae were identified based on sequence from four loci [6,17,18]. Incidence of these species is shown in Fig 2, where C. nymphaeae corresponds to 73%, followed by C. godetiae (18%) and C. fioriniae (9%). GAPDH is the locus that shows the highest variability across the nucleotide dataset, with 24.1% identical sites for the entire set of data (out-group included) and 59.3% within C. acutatum s.l. The MAT1-2 gene also shows a high variability with 34.4% identical sites of which 78.6% in C. acutatum s.l. TUB and ITS loci show lower percentage of variable sites. In detail, TUB has 58.1% of identical sites in the final alignment and 80.7% only considering C. acutatum s.l. While ITS has 77.8% and 92.4% of conserved nucleotides, respectively with and without out-groups. Based on the nucleotide variability referred to above, four haplotypes of C. nymphaeae, three haplotypes of C. fioriniae, and five haplotypes of C. godetiae were identified further highlighting the multiple introductions of the pathogens belonging to these species into the UK.

Fungal growth in plate culture

Radial growth data of C. acutatum s.l. and C. gloeosporioides s.l. isolates were subjected to analysis of variance of regression in order to obtain growth curves that were all statistically significant (R2≥0.9447 and P<0.0001), with the only exception of one isolate showing a R2 = 0.770 (C. nymphaeae CSL224 at 30°C). The slope for each isolate (three replicates for each isolate) belonging to the same species were averaged, in order to detect the hypothetical optimal growth temperature, and results are shown in Table 2. Almost all species, particularly those containing isolates from strawberry in the UK namely C. nymphaeae, C. fioriniae, and C. godetiae had highest growth rates at 25°C that was considered as optimum temperature. It is pertinent to mention that higher levels of strawberry anthracnose incidence in the UK have been reported in the southwest and southeast regions, where relatively high temperatures are most often reached [20]. However, C. phormii, C. kinghormii and C. rhombiforme showed the highest growth rate at the temperature of 20°C and they were not able to grow at 30°C. Interestingly, these three species are evolutionarily closely related, suggesting a specific adaptation to different environmental conditions compared to other members of the same complex. With respect to C. gloeosporioides s.l. isolates (C. aenigma CSL780 and CSL 869; C. fruticola CSL 311 and CSL386), used as out-groups, all the four isolates showed the highest growth rate at all the tested temperatures when compared with all the other isolates.

Table 2

Radial growth rate (mm h-1) of each Colletotrichum species at different temperatures.

	Species	15°C*	20°C*	25°C*	30°C*
out-group	C. aenigma	0.112 ± 0.001	0.199 ± 0.002	0.261 ± 0.008	0.124 ± 0.011
	C. fruticola	0.118 ± 0.004	0.209 ± 0.005	0.238 ± 0.019	0.150 ± 0.008
Colletotrichum acutatum species complex	C. rhombiforme	0.091 ± 0.001	0.135 ± 0.001	0.111 ± 0.002	0.000 ± 0.000
	C. kinghornii	0.073 ± 0.001	0.108 ± 0.001	0.077 ± 0.002	0.000 ± 0.000
	C. phormii	0.106 ± 0.001	0.166 ± 0.001	0.139 ± 0.002	0.000 ± 0.000
	C. salicis	0.094 ± 0.001	0.147 ± 0.002	0.179 ± 0.004	0.035 ± 0.005
	C. godetiae	0.094 ± 0.002	0.142 ± 0.003	0.163 ± 0.005	0.004 ± 0.000
	C. acutatum	0.054 ± 0.004	0.087 ± 0.006	0.148 ± 0.004	0.058 ± 0.005
	C. fioriniae	0.081 ± 0.003	0.136 ± 0.005	0.185 ± 0.004	0.083 ± 0.006
	Colletotrichum sp. 2	0.085 ± 0.002	0.140 ± 0.001	0.178 ± 0.002	0.075 ± 0.002
	C. lupini	0.086 ± 0.001	0.130 ± 0.003	0.152 ± 0.009	0.058 ± 0.002
	Colletotrichum sp. 1	0.083 ± 0.001	0.132 ± 0.001	0.138 ± 0.001	0.043 ± 0.001
	C. tamarilloi	0.069 ± 0.001	0.123 ± 0.002	0.148 ± 0.003	0.007 ± 0.000
	C. simmondsii	0.040 ± 0.003	0.092 ± 0.008	0.112 ± 0.014	0.089 ± 0.010
	C. laticiphilum	0.058 ± 0.002	0.113 ± 0.001	0.161 ± 0.001	0.121 ± 0.002
	C. nymphaeae	0.077 ± 0.001	0.135 ± 0.002	0.159 ± 0.004	0.063 ± 0.005
	C. chrysanthemi	0.050 ± 0.001	0.083 ± 0.001	0.111 ± 0.001	0.087 ± 0.002
	C. scovillei	0.036 ± 0.001	0.105 ± 0.001	0.115 ± 0.001	0.062 ± 0.002

* Values represent the average + SD of slopes (growth rates expressed as mm h-1) of all isolates belonging to the same species, three replicates for each isolate. The optimal temperature for each species is indicated in bold.

C. nymphaeae isolates developed white cottony aerial mycelium, light brownish conidial masses with peculiar colony colour from dark grey to dark brown. Twelve isolates belonging to C. godetiae were characterized by white aerial mycelium, and yellow pigmentation to white colour on the reverse side of the culture. C. fioriniae isolates were dark red on the reverse side of the cultures with orange conidial masses in large drops on the colony surface, and conidiomata formed directly on the hyphae. However, these characters are often difficult to describe reliably, and can change following sub-culturing or based on the length and type of storage. Thus, there is a need for further development of molecular methods for reliable and rapid diagnosis and monitoring of the pathogen populations belonging to different species associated with strawberry production in a specific geographic location.

Characterisation of variation in pathogenicity

Thirty-four C. acutatum s.l. isolates were chosen for pathogenicity tests on fruits and plants, including six representative isolates from each of the three species described above related to strawberry production in the UK (highlighted with * in Table 1 and in bold in Fig 1), and one or more isolates representative of all the major species of the C. acutatum complex. Four C. gloeosporioides s.l. isolates that were isolated from strawberry infected tissues from UK (CSL 780 and CSL 869, C. aenigma) and USA (CSL 311 and CSL 386, C. fruticola) were included in the experiments as an out-group.

C. acutatum s.l. isolates varied in aggressiveness on both host tissues. In the fruit assays, among the three species identified from the strawberry production systems in the UK, C. nymphaeae and C. fioriniae were more aggressive compared to C. godetiae. This was particularly noticeable for isolates originating from strawberry as reflected by the fruit disease index range for C. nymphaeae (2.08–3.00), C. fioriniae (1.92–2.75) and C. godetiae (0.75–2.08). Interestingly, with isolates originating from other hosts, C. nymphaeae isolates were less aggressive (0.67–1.67), and one or more isolates belonging to C. fioriniae (2.00–2.17) as well as C. godetiae (2.17) showed fruit disease index in the range of the strawberry isolates. Among the other species tested within the C. acutatum complex, C. acutatum s.s., C. simmondsii and Colletotrichum sp.2 included one or more isolates originating from non-strawberry hosts that showed medium level of aggressiveness with fruit disease index ranging from 1.17 to 2.08. Whereas, C. lupini (0.08–075), C. phormii (0.58), C. salicis (0.17–0.67), and C. rhombiforme (0.67) along with Colletotrichum sp.1 (0.33) isolates originating from various hosts other than strawberry were much less aggressive as reflected by the fruit disease index. The C. gloeosporioides s.l. isolates tested showed a fruit disease index ranging from 1.50 to 2.50 (Table 3).

Table 3

Variability in aggressiveness of Colletotrichum species isolates on strawberry fruits and plants.

	Isolate	Species	Isolation source	Origin	Fruit Disease index * , +		Plant Disease Index * , #
Colletotrichum acutatum species complex	CSL 256	C. nymphaeae	Fragraria	UK	2.50	abcd	0.50	bc
	CSL 899	C. nymphaeae	Fragraria	UK	3.00	a	0.83	abc
	CSL 915	C. nymphaeae	Fragraria	UK	2.08	abcdef	0.61	bc
	ATCC 38896	C. nymphaeae	Nymphaeae	Netherlands	0.67	defg	0.28	bc
	CSL 455	C. nymphaeae	Photinia	UK	1.08	bcdefg	0.56	bc
	PT135	C. nymphaeae	Olea	Portugal	1.67	abcdefg	0.89	abc
	CSL 916	C. godetiae	Fragraria	UK	1.92	abcdefg	0.39	bc
	CSL 918	C. godetiae	Fragraria	UK	0.75	cdefg	0.39	bc
	CSL 919	C. godetiae	Fragraria	UK	2.08	abcdef	0.67	bc
	ALM-NRB-30K	C. godetiae	Prunus	Israel	0.25	fg	0.11	c
	CBS 193.32	C. godetiae	Olea	Greece	0.75	cdefg	0.28	bc
	JL198	C. godetiae	Olea	Serbia	2.17	abcde	0.39	bc
	CSL 1259	C. fiorinae	Fragraria	UK	2.75	ab	0.72	bc
	CSL 1262	C. fiorinae	Fragraria	UK	1.92	abcdefg	1.00	ab
	CSL 2546	C. fiorinae	Fragraria	UK	2.67	abc	0.72	bc
	CBS 797.72	C. fiorinae	Pinus	New Zealand	1.08	bcdefg	0.39	bc
	ATCC MYA-663	C. fiorinae	Malus	USA	2.00	abcdef	0.83	abc
	CR46	C. fiorinae	Vitis	Portugal	2.17	abcde	0.33	bc
	PJ8	C. acutatum	Pyrus	New Zealand	2.08	abcdef	0.72	bc
	PT227	C. acutatum	Olea	Portugal	1.42	abcdefg	0.78	abc
	STE-U-164	C. acutatum	Pinus	South Africa	0.83	cdefg	0.28	bc
	CBS 122122	C. simmondsii	Carica	Australia	0.25	efg	0.22	bc
	CBS 294.67	C. simmondsii	Carica	Australia	1.17	abcdefg	0.61	bc
	PD89-582	C. simmondsii	Cyclamen	Netherland	1.83	abcdefg	0.44	bc
	BBA 70884	C. lupini	Lupinus	Ukraine	0.58	efg	0.33	bc
	HY09	C. lupini	Lupinus	Canada	0.08	g	0.17	bc
	PT30	C. lupini	Lupinus	Portugal	0.75	cdefg	0.56	bc
	9178	C. salicis	Vaccinium	Norway	0.50	efg	0.28	bc
	CBS 607.94	C. salicis	Salix	Netherlands	0.67	defg	0.17	bc
	MP1	C. salicis	Acer	USA	0.17	fg	0.22	bc
	CBS 101611	Colletotrichum sp. 1	Fern	Costa Rica	0.33	efg	0.06	c
	OCO-ARC-4	Colletotrichum sp. 2	Citrus	USA	1.42	abcdefg	0.11	c
	AR3787	C. phormii	Phormium	South Africa	0.58	efg	0.22	bc
	PT250	C. rhombiforme	Olea	Portugal	0.67	defg	0.33	bc
out-group	CSL 780	C. aenigma	Fragraria	UK	2.50	abcd	0.50	bc
	CSL 869	C. aenigma	Fragraria	UK	1.92	abcdefg	0.72	bc
	CSL 311	C. fruticola	Fragraria	USA	2.50	abcd	1.56	a
	CSL 386	C. fruticola	Fragraria	USA	1.50	abcdefg	0.22	bc

Disease Index data related to aggressiveness on strawberry fruits and crowns of representative Colletotrichum isolates.

*: Different letters within the same column correspond to significantly different values (ANOVA; P < 0.05). The values are the averages ± SD of four independent replicates, three fruits for each replicate and of three independent replicates, six plants for each replicate. Disease Index was calculated according to the following formula: Σvn/N, where v represents the numeric value of the class, n is the number of fruits or plants assigned to the class, N is the total number of the plants assessed.

+: 0, no visible lesions; 1, lesions on less than 33% of fruit surface; 2, lesions covering 33–66% of fruit surface; and 3, lesions covering more than 66% of fruit surface.

#: 0, no lesions; 1, crown tissues discoloration but no wilting or collapse; 2, wilting or collapse of part of the plant; and 3, plant death.

In the in vitro assays, anthracnose fruit rot symptoms were observed (e.g. Fig 3B) for various isolates tested with different levels of aggressiveness, as shown by the disease index ranging from 0.08 to 3.0 (Table 3). The variation in aggressiveness among different isolates was clearly reflected by the differences in incidence which ranged from 8.33 to 100% with only 4 out of 38 isolates showing 91.7 to 100% as well as the lesion type which ranged from 0.1 to 3.0 (S1 Table). When lesion morphology was evaluated, different kinds of lesions could be distinguished on fruits, ranging from brown ones containing orange drops of conidia to those entirely covered with aerial mycelium, with different lesion size. C. nymphaeae CSL899 was the most aggressive on strawberry fruits with the highest disease index (3.0, corresponding to symptoms covering more than 66% of fruit surface).

In the plant assays, varying degrees of crown rot symptoms were recorded 24 d.a.i, as reflected by the disease index range shown in Table 3. Symptom severity was generally low, with no isolate scoring higher than 2 (wilting and collapse of plant). Among the three species identified from UK strawberry production systems, C. fioriniae isolates originating from strawberry showed a higher range of disease index (0.72–1.00) compared to C. nymphaeae (0.5–0.83) and C. godetiae (0.39–0.67). The C. gloeosporioides s.l. isolate CSL 311 (C. fruticola from strawberry in USA) showed the highest disease index (1.6), this isolate was also amongst the most aggressive on fruit (Table 3). Colletotrichum isolates were recovered from all crowns showing symptoms.

Discussion

The UK strawberry industry has expanded rapidly in recent years, and this appears to correlate with increasing losses attributed to anthracnose caused by Colletotrichum spp. [6]. This study provides the first molecular characterization of C. acutatum sensu lato diversity related to strawberry production in the UK, combined with pathogenic characterization. A collection of 148 isolates representative of UK and global diversity of C. acutatum s.l. populations has been assembled. The isolates were chosen based on host association, geographic distribution, phylogenetic relationships and biological diversity.

On the basis of four sequence loci (ITS, TUB, GAPDH, and MAT1-2), the C. acutatum sensu lato isolates were assigned to three newly designated species C. nymphaeae, C. godetiae and C. fioriniae following a recent taxonomic re-assessment [18]. According to available literature, C. nymphaeae is the most common and C. godetiae is also often reported in European and American strawberry fields [6]. These two species were also the most representative in our dataset of isolates related to strawberry in the UK. C. fioriniae has a worldwide distribution and is common on strawberry but only a few isolates were identified in our collection, and this group was not commonly present in the fields in the UK. C. simmondsii, C. acutatum sensu stricto, C. salicis and C. miyabeana are common on strawberry in Oceania and have only been found sporadically in Europe. Isolates belonging to these species have not been detected on strawberry in the UK. The variability observed within the UK C. acutatum sensu lato species fits in part with previous reports of C. acutatum on strawberry within specific geographic regions. For example, in France, Israel, Bulgaria and Spain, the majority of strawberry anthracnose pathogen isolates clustered in the same species C. nymphaeae, and almost no intra-specific diversity was observed within each country [2-5]. A different situation has been observed on Belgian isolates, where the population represented: 33% isolates belonging to C. nymphaeae, 5% C. fioriniae, 50% C. godetiae, 3% C. acutatum s.s. and 6% C. salicis. A possible explanation to C. acutatum s.l. status in the UK might be recent introduction (late 70s) from a limited number of sources. The reason for the differences in the occurrence of various Colletotrichum species associated with strawberry production in different geographic locations still remains unclear, but the source of importation of the planting material and local trade have been heavily implicated [4,7].

The pathogenicity assays used in this work are based on a study in Belgium [7] in view of the similar molecular diversity of the anthracnose pathogen populations associated with strawberry production. These assays with the isolates representing the molecular diversity not only revealed variability in aggressiveness in different species described within C. acutatum s.l., but also complex patterns both between and within the species. For example, based on isolates originating from strawberry, C. fioriniae and C. nymphaeae appear equally aggressive on fruits with C. nymphaeae isolates indicating a degree of host-preference. Both C. fioriniae and C. godetiae included isolates originating from other hosts that showed comparable levels of aggressiveness to isolates from strawberry. Similar situation was observed with at least some non-strawberry isolates belonging to species such as C. acutatum s.s. and C. simmondsii. Furthermore, at least one C. godetiae isolate from strawberry was much less aggressive compared to others. These patterns suggest that some Colletotrichum species such as C. fioriniae and C. godetiae include populations that are capable of infecting a wider range of hosts, also influenced by environmental conditions. Further studies using a wider set of isolates of these three species and appropriate pathological and biological assays are required to gain additional insights into the evolution of pathogenicity in relation to field symptoms as well as any differential responses to host varieties and fungicides locally used in the UK strawberry production systems.

The study has highlighted the genetic and pathogenic heterogeneity of the introduced anthracnose pathogen populations belonging to three different Colletotrichum species emphasising the need for effective phytosanitary procedures linked to pathogen monitoring and characterisation to generally limit the entry of non-native pathogens. This also underlines the requirement of reliable and rapid diagnostic tools for further research and application in strawberry anthracnose management. The recent release of a whole genome sequence of C. fioriniae isolated from strawberry [28] along with the newly characterised isolates, based on multi-locus sequence and aggressiveness information reported here, represents a useful platform for further research into the genetic basis of C. acutatum s.l.—strawberry interactions.

a 0, no visible lesions; 1, lesions on less than 33% of fruit surface; 2, lesions covering 33–66% of fruit surface; and 3, lesions covering more than 66% of fruit surface. b no lesions; 1, crown tissues discoloration but no wilting or collapse; 2, wilting or collapse of part of the plant; and 3, plant death.

(XLSX)

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