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. 2015 Jun 18;10(6):e0129140. doi: 10.1371/journal.pone.0129140

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

Riccardo Baroncelli 1,2,¤,*, Antonio Zapparata 2, Sabrina Sarrocco 2, Serenella A Sukno 3, Charles R Lane 4, Michael R Thon 3, Giovanni Vannacci 2, Eric Holub 1, Surapareddy Sreenivasaprasad 5
Editor: Mark Gijzen6
PMCID: PMC4472692  PMID: 26086351

Abstract

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.

Fig 1

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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
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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.

Fig 2

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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).

Fig 3

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(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
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* 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
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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.

Supporting Information

S1 Table. Variability in aggressiveness of Colletotrichum species isolates on strawberry fruits and plants.

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)

Click here for additional data file. (50KB, xlsx)

Acknowledgments

The authors would like to dedicate this work to Maurizio Forti (University of Pisa), who passed away in December 2013 and to Dez Barbara (University of Warwick) who passed away in July 2012. The authors would like to thank Fera and the University of Warwick for funding this research and providing the strains set. They are especially thankful to: Ulrike Damm (CBS-KNAW Fungal Biodiversity Centre–The Netherlands), Paul Cannon and Alan Buddie (CABI—UK), Gunn Mari Strømeng (Norwegian University of Life Sciences—Norway), Katherine LoBuglio (Harvard University Herbaria, USA), Peter R. Johnston (Manaaki Whenua Landcare Research–New Zealand), James Cunnington (Institute for Horticultural Development—Australia), Amy Rossman (USDA-ARS–USA), Stanley Freeman (ARO Volcani Center—Israel), Daniel Buchvaldt Amby (University of Copenhagen–Denmark), Natalia Peres (University of Florida–USA) and Sheu Zong-ming (AVRDC–The World Vegetable Center–Taiwan) for kindly providing reference isolates.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors would like to thank University of Warwick for funding this research. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Variability in aggressiveness of Colletotrichum species isolates on strawberry fruits and plants.

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)

Click here for additional data file. (50KB, xlsx)

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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