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Article

Characterization of Colletotrichum Isolates from Strawberry and Other Hosts with Reference to Cross-Inoculation Potential

by
Gunjan Sharma
†,
Marcel Maymon
,
Vineet Meshram
and
Stanley Freeman
*
Department of Plant Pathology and Weed Research, Institute of Plant Protection, Agricultural Research Organization-Volcani Institute, Rishon LeZion 7505101, Israel
*
Author to whom correspondence should be addressed.
†
Current address: Department of Plant Biotechnology, Gujarat Biotechnology University, Gandhinagar 382355, India.
Plants 2022, 11(18), 2373; https://doi.org/10.3390/plants11182373
Submission received: 1 July 2022 / Revised: 30 August 2022 / Accepted: 1 September 2022 / Published: 11 September 2022
(This article belongs to the Special Issue Interactions between Colletotrichum Species and Plants Ⅱ)
Browse Figures
Versions Notes

Abstract

:
Colletotrichum is an important phytopathogenic fungus that causes anthracnose disease in diverse agronomically important tropical food crops. Accurate pathogen identification is critical for early diagnosis and efficient management of anthracnose. ITS is not a reliable marker for this fungal genus due to its failure to phylogenetically resolve cryptic species. In this study, 36 Colletotrichum isolates belonging to the Acutatum, Boninense and Gloeosporioides species complexes were characterized using multigene phylogenetic analyses, morphology and pathogenicity assays. Additionally, the cross-inoculation potential of a representative subset of isolates was evaluated revealing that cross-infection potential is possible among the isolates belonging to the same species complex.

1. Introduction

Colletotrichum (Glomerellaceae, Glomerellales, Sordariomycetes) is a ubiquitous and important plant pathogenic filamentous fungal genus [1]. Colletotrichum species have high genetic variability and diverse survival lifestyles according to their hosts and the environment [2,3,4]. Members of the species can exhibit endophytic, hemibiotrophic or necrotrophic lifestyles on their host plants [2,5]. Hemibiotrophy is a common mode of survival for this species, allowing an initial interaction with the host plant before infection takes place [6,7]. As a hemibiotroph, a quiescent infection is initiated via melanized appressoria that penetrate the host tissues to form infection hyphae, eventually leading to necrotrophy. Pathogenic Colletotrichum species usually exhibit hemibiotrophic and necrotrophic modes of survival and infection.
This genus is considered a “catalogue of confusion” [8], comprising more than 248 accepted species names [9]. Previously, the Colletotrichum species were named based on host-association, which created a lot of nomenclatural ambiguities. Due to the presence of morphologically similar cryptic species within each species complex, genealogical concordance for phylogenetic species recognition [10] is an important approach applied to designate and characterize a Colletotrichum species. Colletotrichum taxonomy has undergone multiple amendments based on the use of multi-locus molecular phylogenetic analyses [11,12,13,14,15,16,17,18]. Along with the genetic diversity of a fungal population, it is also fundamental to assess their pathogenic potential and epidemiology to understand completely the pathogenic nature of the fungi associated with a certain crop. Thus, the use of DNA-sequence data is vital in plant pathology, as it assists in monitoring plant health, pathogen risk assessment and devising pathogen management practices [19].
The Colletotrichum isolates used in this study were originally characterized based on the banding patterns obtained from RFLPs (restriction fragment length polymorphisms) and AP-PCRs (arbitrarily primed polymerase chain reactions) [20,21,22]. However, considering the recent nomenclatural updates in Colletotrichum taxonomy, in this study, we utilized current molecular phylogenetic analyses incorporating multi-gene sequence data, morphology and pathogenicity assay to reveal the species level of identification.

2. Results

2.1. Morphological Studies

Distinct morphological characteristics (colony color, growth rate, conidial measurements) were observed and recorded for representative isolates (Colletotrichum godetiae—ALM-KSH10, ALM-BZR82; C. nymphaeae –ANE-NL4, ANE-HV83C, STR-101, TUT-5954; C. tamarilloi—TOM-10; C. acutatum—ANE-NL12; C. fioriniae– APL2, ALM-US4; C. karsti—MAN76, C. colombiense—TOM6, PASS33; C. brassicicola– PASS65, C. sydowii—PASS35, C. gloeosporioides s. l.—APL7, Cg-Sc-A1) from 7-day-old cultures grown on PDA at 25 °C. Details regarding the morphological characterization of the isolates used in this study are presented in Table 1.

2.2. Phylogenetic Analyses and Assignment of Species

The 36 representative Colletotrichum isolates used in this study were distributed to their respective species complex on the basis of the ITS sequence data and corresponding NCBI-BLAST result. Twenty-four Colletotrichum isolates were assigned to the C. acutatum species complex, eight Colletotrichum isolates were assigned to the C. boninense species complex, and three Colletotrichum isolates were assigned to the C. gloeosporioides species complex. One isolate originally isolated from passiflora in Colombia, was found to be related to C. sydowii, which is a singleton species with limited reference sequence data. Thus, the identity of PASS35 according to the percent sequence identity of the ITS, act, chs1, gapdh, his3 and tub2 sequences corresponds to the type strain sequences of C. sydowii. The phylogenies of the three isolates belonging to the C. gloeosporioides species complex have been described in the supplementary information (Supplementary Figure S1).

2.3. Phylogenetic Analyses of the Isolates Belonging to the C. acutatum Species Complex

A concatenated dataset of six genes (ITS, act, chs1, gapdh, his3 and tub2) was used for the phylogenetic analyses of 24 C. acutatum species complex members used in this study. Monilochaetes infuscans (CBS 869.96) was used as the outgroup, and 65 species in the Acutatum complex were used as a reference in the analysis. The multigene sequence alignment contained 2173 characters [ITS: 1–554; act: 555–817; chs1: 818–1094; gapdh: 1095–1368; his3: 1369–1755; tub2: 1756–2173], including gaps. Sixty ambiguous characters were excluded from the alignment, and, of the remaining 2113 included characters, 1431 characters were constant; 283 variable characters were parsimony-uninformative; and 399 characters were parsimony-informative. The parsimony analysis yielded 147 trees that were equally the most parsimonious; the topology of one is shown in Figure 1 [tree length (TL) = 1285, consistency index (CI) = 0.661, retention index (RI) = 0.905, rescaled consistency index (RC) = 0.598, homoplasy index (HI) = 0.339]. The Bayesian analysis of the combined alignment lasted 5,000,000 generations, and the resulting trees were used to calculate the posterior probabilities. The bootstrap support values of the MP analysis (MP > 50%), ML analysis (ML > 50%) and the BI posterior probabilities (PP > 0.90) are depicted at the branch nodes (Figure 1).
Based on the MP and ML phylogenetic analyses (Figure 1), the ANE-NL12 (anemone) isolate clustered with the ex-type sequences of C. acutatum sensu stricto CBS112996 (MP 100%, ML 100%, PP 1.0); PCN5 (pecan), PCH8 (peach), APL2 (apple), ALM-US4 (almond) clustered with the ex-type sequences of C. fioriniae CBS128517 (MP 100%, ML 100%, PP 1.0); and TOM10 (tamarilla) clustered with the ex-type sequences of C. tamarilloi CBS129814 (MP 100%, ML 100%, PP 1.0).
However, the isolates from almond, anemone and strawberry were placed within or next to the clades of C. godetiae, C. fioriniae or C. nymphaeae; that are already reported to possess high genetic diversity [12]. Thus, an additional phylogenetic analysis was performed incorporating more sequences from non-type strains of C. godetiae, C. fioriniae and C. nymphaeae. The multigene sequence alignment contained 2159 characters [ITS: 1–551; act: 552–822; chs1: 823–1096; gapdh: 1097–1361; his3: 1362–1748; tub2: 1749–2159], including gaps. Fifty ambiguous characters were excluded from the alignment and of the remaining 2109 included characters: 1564 characters were constant; 300 variable characters were parsimony-uninformative and 245 characters were parsimony-informative. The parsimony analysis yielded 205 equally most parsimonious trees, the topology of one of which is shown in Figure 2 [tree length (TL) = 703, consistency index (CI) = 0.869, retention index (RI) = 0.984, rescaled consistency index (RC) = 0.855, homoplasy index (HI) = 0.131]. Based on the Bayesian, MP and ML phylogenetic analyses (Figure 2), the isolates from almond were identified as C. godetiae and the isolates from anemone and strawberry were identified as C. nymphaeae. The NCBI-BLAST percent sequence identity of C. godetiae and C. nymphaeae with their closely related taxa is mentioned in the Supplementary Table S1. The number of parsimony-informative characters for each gene dataset is described in Supplementary Table S2.

2.4. Phylogenetic Analyses of Isolates Belonging to the C. boninense Species Complex

A concatenated dataset of six genes (ITS, act, chs1, gapdh, his3 and tub2) was used for phylogenetic analyses of eight C. boninense species complex members in this study. Monilochaetes infuscans (CBS 869.96) was used as the outgroup, and 50 species in the Boninense complex were used as references in the analyses. The multigene sequence alignment contained 2246 characters [ITS: 1–569; act: 570–849; chs1: 850–1126; gapdh: 1127–1427; his3: 1428–1821; tub2: 1822–2246], including gaps. Eighty-one ambiguous characters were excluded from the alignment and of the remaining 2165 included characters: 1383 characters were constant; 216 variable characters were parsimony-uninformative and 566 characters were parsimony-informative. The parsimony analysis yielded 63 equally most parsimonious trees, the topology of one of which is shown in Figure 3 [tree length (TL) = 1545, consistency index (CI) = 0.674, retention index (RI) = 0.870, rescaled consistency index (RC) = 0.586, homoplasy index (HI) = 0.326]. The Bayesian analysis of the combined alignment, lasted 5,000,000 generations, and resulting trees were used to calculate the posterior probabilities. Bootstrap support values of the MP analysis (MP > 50%), ML analysis (ML > 50%) and the BI posterior probabilities (PP > 0.90) are depicted at the branch nodes (Figure 3).
The overall branch support for the observed tree was high. Based on the MP and ML phylogenetic analyses, PASS33, PASS55, PASS62, PASS67 (passiflora) and TOM6 (tamarilla) isolates clustered with the ex-type sequences of C. colombiense CBS129818 (MP 100%, ML 100%, PP 1.0); PASS65 (passiflora) clustered with the ex-type sequences of C. brassicicola CBS101059 (MP 100%, ML 100%, PP 1.0); MAN 76 (mango) and PASS52 (passiflora) clustered with the sequences of C. karsti (MP 100%, ML 100%, PP 1.0). These isolates were also recovered as monophyletic with strong bootstrap support in four out of six individual gene trees (act, chs1, his3 and tub2) (data not shown). The phylogenetic analyses of the Boninense complex identified isolates of C. brassicicola, C. colombiense and C. karsti as pathogenic to non-host strawberry fruits.

2.5. Pathogenicity Testing

The pathogenicity of all the Colletotrichum isolates used in this study from their original host of isolation were already studied and published elsewhere [20,21,23]. In this study, we assessed the cross-inoculation potential of the Colletotrichum isolates to non-hosts. Strawberry was used as an experimental non-host, and symptoms were verified for the different representative Colletotrichum isolates (C. godetiae—ALM-KSH10, ALM-BZR82; C. nymphaeae—TUT-5954, STR-101, ANE-NL4, ANE-HV83C; C. tamarilloi—TOM-10, C. acutatum—ANE-NL12, C. fioriniae—APL2, ALM-US4; C. karsti—MAN76, PASS33; C. colombiense—TOM6, C. brassicicola—PASS65, C. sydowii—PASS35, C. gloeosporioides s. l.—Cg-Sc-A1, APL7). The C. gloeosporioides s. l. isolate Litchi-Cg2 could not be assessed for pathogenicity due to inadequate production of conidia.
The Colletotrichum species belonging to the Acutatum and Gloeosporioides species complexes exhibited anthracnose symptoms on strawberry fruits after 7 days of inoculation, whereas isolates belonging to the Boninense species complex and C. sydowii did not exhibit any noticeable anthracnose disease symptoms on inoculated fruit. Particularly, C. nymphaeae is an aggressive pathogen of strawberry in Israel, exhibiting 100% disease incidence and 75–100% disease severity in wounded inoculations. In unwounded inoculations, the disease incidence remained low. However, C. nymphaeae caused increased disease incidence and severity in unwounded inoculations as well. Interestingly, the C. acutatum sensu stricto and C. nymphaeae isolates from anemone were able to cross-infect strawberry. Similarly, C. godetiae and C. fioriniae isolates from almond, C. fioriniae isolate from apple and C. tamarilloi isolates from tamarillo were capable of infecting strawberry under wounded, as well as unwounded, conditions. This implies that the members of the Acutatum and Gloeosporioides species complexes used in this study are key pathogens of strawberry fruits and possess the potential for cross-infection. Detailed results of the pathogenicity tests are presented in Figure 4 and Figure 5 and in Supplementary Tables S3 and S4.

3. Discussion

3.1. Molecular Phylogeny of Isolates of Colletotrichum

Over the past decade, the phylogeny of the genus Colletotrichum has been in flux [24]. Prior to that, ITS sequencing was used to delineate isolates to the major “species”, e.g., C. gloeosporioides, C. acutatum, C. coccodes, etc. [24]. Thus, the single-gene phylogeny based on ITS sequencing did not result in reliable species diagnostics and identification [25]. However, since 2012, the main species clusters have been redefined according to multi-locus sequencing. For example, the Acutatum species complex has been defined according to phylogenetic analyses of the ITS, act, tub2, chs1, gapdh and his3 genes [12]; the Gloeosporioides species complex has been defined according to analyses of up to eight genes [18]; and the Boninense species complex has been defined according to phylogenetic analyses of the ITS, act, tub2, chs1, gapdh, his3 and cal genes [13]. Thus, the multi-locus sequencing of many populations of Colletotrichum has resulted in the renaming of undescribed cryptic species within the systematics of the genus. To date, a comprehensive systematics of the genus Colletotrichum includes a list of 16 species complexes, while an additional 15 novel single-isolate representatives were also described [26].

3.2. Previous Characterization of Strains

Past studies have characterized the current Colletotrichum isolate collection from Israel and elsewhere to species based on various molecular techniques. For example, isolates of C. gloeosporioides from avocado and other hosts were characterized according to AP-PCR, A+T-rich mitochondrial DNA, nuclear DNA and rDNA analyses; however, none of these methods were suitable in differentiating between the species from this complex [20]. Similarly, in additional studies, the C. acutatum isolates from various hosts, including anemone, olive and strawberry, were characterized to the specific complex using ITS sequencing, however, under limited conditions that did not coincide with pathogenicity tests [22]. Likewise, isolates within the species complex, Boninense, were originally identified based on ITS and rDNA sequencing [27]. In all of these past studies, the ITS sequence alone was not informative enough and did not delineate isolates to the species level within each Colletotrichum complex [20,21,22].

3.3. Colletotrichum of Almond

Under the circumstances, Colletotrichum affecting almond appears to be diverse; however, among the studied cases, the different populations were clustered within the Acutatum species complex. In Israel, the population of Colletotrichum is specific to almond and appears to be clonal and unique; however, ITS sequencing alone was not able to differentiate between the population from the US and those affecting almond from Australia [20]. The Australian populations were distinct from those found in Israel, however, and were more closely related to those affecting almond from the US [28].
As far as pathogenicity is concerned, all of the above populations of Colletotrichum affecting almond were pathogenic to the crop. In Israel, the specificity was demonstrated as follows: isolates that originated from almond were unique, and none of these isolates were detected within the populations affecting avocado, characterized within the Gloeosporioides complex; the anthracnose-affected almond orchards were cultivated adjacent, at a 5–10 m distance, to the affected avocado orchards [21]. Therefore, there is no need to be concerned that cross-inoculation from almond to avocado and vice versa takes place in the field. Thus, management protocols for disease reduction in each crop can be developed individually without any threat of cross-infection taking place between both of these crops.

3.4. Colletotrichum Affecting Anemone and Strawberry

Another case study in Israel, concerns the molecular identity of populations of Colletotrichum that affect anemone and strawberry. Anthracnose disease of anemone and strawberry is common, and both crops can be affected annually in Israel [21]. In certain instances, the same farmer can cultivate both crops in close proximity, when the infection takes place simultaneously. In the past, populations of the Acutatum complex were reported as causal agents of both crops, and it was shown that cross-inoculation of certain isolates/populations takes place under controlled conditions [21] and under field conditions (Freeman, pers. comm.).

3.5. Pathogenicity

The pathogenicity of Colletotrichum producing anthracnose symptoms can be evaluated on detached or attached plant structures. It has been shown, on the one hand, that detached fruit are not ideal “substrates” for pathogenicity testing; however, on the other hand, post-harvest evaluations are conducted primarily on detached fruit, e.g., pathogenicity and virulence on avocado [29]. In this study, the artificial inoculation of Colletotrichum isolates delineated to specific species within each complex were artificially inoculated on strawberry fruit. Reliable results were achieved that differentiated between specific species, indicating that this method is accurate in evaluating the pathogenic and virulence specificity for the isolates tested in this study.
Since the members of the Acutatum and Gloeosporioides species complexes are known pathogens of strawberry, all of the tested representative isolates belonging to these two species complexes were able to cross-infect strawberry fruits in the wounded as well as unwounded inoculations. However, slight differences in disease symptoms and severity were observed among the different isolates, such as those from anemone (ANE-NL4) and strawberry (TUT-5954) that may be attributed to the high genetic diversity within C. nymphaeae. Similar to the present study, C. acutatum and C. fioriniae have been previously reported to be associated with strawberry anthracnose symptoms. This is the first report of C. tamarilloi causing strawberry anthracnose.
The Colletotrichum isolates belonging to the Boninense species complex did not cause anthracnose symptoms in wounded or unwounded inoculations. Thus far, there is one report of the association of C. boninense and C. karsti with strawberry in China [30,31]. However, C. brassicicola, C. colombiense and C. sydowii have not been previously associated with strawberry [32], and, as in this study, C. sydowii was found to be non-pathogenic on detached strawberry fruits. Thus, according to the pathogenicity assays reported in this study, Colletotrichum isolates possess cross-infection potential within their respective species complex.

3.6. Colletotrichum Species Affecting Anemone, Strawberry and Almond

Within the Acutatum species complex, C. acutatum, C. fioriniae, C. godetiae, C. nymphaeae and C. simmondsii have been reported as pathogens of almond and strawberry fruit, while C. acutatum, C. fioriniae and C. nymphaeae have been reported on anemone [32]. In this study, a polyphasic approach of multigene phylogenetic analysis, morphological characterization and pathogenicity assays was used to characterize 36 Colletotrichum isolates that were previously identified based on AP-PCR and ITS sequence data alone. Similar to previous studies, C. godetiae was associated with almonds, and C. nymphaeae was associated with anemone and strawberry in Israel. The genealogical concordance was also evaluated using individual gene trees; however, there is a need to improve the phylogenetically informative barcode markers for the Acutatum species complex. The percent parsimony-informative characters for the various genes used for the phylogenetic analysis of the Acutatum species complex is quite low (Supplementary Table S2). In the case of the Gloeosporioides species complex, intergenic regions of the apn2 and MAT1-2-1 gene (ApMat) markers, along with the glutamine synthase (gs) gene, provide an efficient species-level resolution [33,34].

4. Materials and Methods

4.1. Fungal Isolates, Growth Conditions and Morphological Characterization

The monoconidial Colletotrichum isolates used in this study are listed in Table 2. The Colletotrichum isolates associated with almond, strawberry and anemone were isolated between 1993 and 1995, in Israel [20,21,22]. The isolates associated with passiflora, tamarillo and mango were isolated between 1998 to 2000, in Colombia [27]. The isolates associated with apple, pecan and peach originated in the US [22]. The Colletotrichum isolates were retrieved from the −80 °C facility of the Freeman laboratory at the ARO, revived on potato dextrose agar (PDA, Difco, USA) plates and grown at 25 °C for morphological characterization (colony morphology and growth rate). To increase sporulation, the isolates were grown on modified Mathur’s MS semi-selective (M3S) agar medium (per liter composition: 2.5 gm of MgSO4·7H2O, 2.7 gm of KH2PO4, 1 gm of peptone, 1 gm of yeast extract, 10 gm of sucrose and 20 gm of agar) [29] and synthetic nutrient agar (SNA) medium (per liter composition: 1 gm of KH2PO4, 1 gm of KNO3, 0.5 gm of MgSO4·7H2O, 0.5 gm of KCl, 0.2 gm of glucose, 0.2 gm of sucrose and 20 gm of agar) [35]. Microscopic slides were prepared in 1% lactic acid or water. For each representative isolate, the shape and size of the conidia and conidiogenous cells were measured using bright field microscopy (Nikon, Japan) and measured via NIS-Elements image analysis software (Nikon). At least 50 measurements were made for the length and width of the conidia. The growth rate was determined by measuring the colony diameter after 7 days (mm/day). Morphological characteristics are listed in Table 1.

4.2. DNA Extraction, PCR Amplification and Sequencing

The 36 representative Colletotrichum isolates used in this study were grown at 25 °C in 20 mL broth of glucose minimal media [29], without shaking. The fungal mycelia were harvested after 7 days by vacuum filtration. DNA was extracted according to Freeman et al. [36]. The partial sequence of actin (act), chitin synthase (chs1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), histone (his3), β-tubulin (tub2) and the internal transcribed spacer (ITS) region were amplified and sequenced using the primer pairs ACT-512F+ACT-783R [37], Bt-2A + Bt-2B [38], CHS-79F+CHS-354R [37], GDF1+GDR1 [39], CYLH3F+CYLH3R [40] and ITS-5+ITS-4 [41], respectively. The PCR reactions were carried out in a 20 µL volume. Each reaction tube contained 1.5 µL of total genomic DNA (100 ng/µL concentration), 10 µL of 2× PCRBIO HS Taq Mix Red (PCR Biosystems, www.pcrbio.com, accessed on 27 June 2022), 0.5 µL each of 10 µM forward and reverse primer and 7.5 µL of sterile water in a thermocycler (Biometra TAdvanced, Analytik-Jena, Jena, Germany). The cycling parameters were: initial denaturation at 95 °C for 4 min, followed by 34 cycles of denaturation at 95 °C for 30 s, annealing for 30 s (at 58 °C for act, 56 °C for chs1, 60 °C for gapdh, 52 °C for his3, 55 °C for tub2 and 54 °C for ITS), extension at 72 °C for 45 s and a final extension at 72 °C for 7 min. The PCR products were separated by 1.2% agarose gel electrophoresis, purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Dueren, Germany) and quantified using a Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, Wilmington, NC, USA). Both strands of the purified PCR products were sequenced at Macrogen Europe (http://www.macrogen.com, accessed on 27 June 2022). The sequences generated in this study were submitted to NCBI for GenBank accession numbers (Table 2).

4.3. Phylogenetic Analyses

The forward and reverse sequences for each gene were checked for quality and assembled using MEGA-X v. 10.1.17 [42]. The different gene regions were concatenated using SequenceMatrix v. 1.7.8 [43]. Ambiguous regions from the multiple sequence alignment were not included in the analyses, and the gaps were considered missing data (N). The six-gene dataset (act, cal, gapdh, his3, ITS and tub2) was used for the phylogenetic analyses of the Colletotrichum isolates belonging to the C. acutatum and C. boninense species complex. For the remaining three isolates belonging to the C. gloeosporioides species complex, the phylogenetic analyses comprised a five-gene dataset (act, chs1, gapdh, ITS and tub2). The reference type–strain sequences for the analyses were retrieved from GenBank [22] and are listed in Supplementary Tables S5–S8.
Phylogenetic analyses were conducted using a maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods. The maximum parsimony analysis was conducted using PAUP v. 4.0b10 [44], as detailed earlier in Sharma et al. [29]. The maximum likelihood phylogeny was inferred using RAxML-HPC2 under the GTR-GAMMA model in the CIPRES portal [45,46], and the branch support was evaluated by bootstrap analysis of 100 replicates (-m GTRGAMMA -p 12345 -f a -N 100 -x 12345 --asc-corr lewis). The Bayesian inference of the phylogeny was estimated using MrBayes version 3.2.7a [47] in the CIPRES portal with four MCMC chains of 5,000,000 generations. The sample trees were recorded every 103 generations, and 25% of the initial trees were discarded as burn-in. For each gene dataset, a suitable model for the estimation of phylogeny was evaluated, and ML analysis was carried out using MEGA-X and the “one click mode” tree analysis method, available at www.phylogeny.fr, accessed on 27 June 2022 [48] (data not shown). The resulting trees from each analysis were viewed in FigTree v. 1.4.4 [49] and edited in Microsoft PowerPoint 2016.

4.4. Pathogenicity Assay

The pathogenic potential of the representative isolates was evaluated by artificial inoculations of unripe strawberry fruit (light green to light red in color), cv. Peles, which is susceptible to anthracnose, that originated from a disease-free organic strawberry farm in the Sharon area of Israel. Prior to inoculation, the calyx and peduncle were trimmed, and the fruits were washed under running water. The conidia were harvested as described [29] and adjusted to 2 × 107 conidia/mL. After air drying, the fruits were inoculated with 10 µL of conidial solution at wounded (pin-pricked) and unwounded sites. In the control fruits, sterile saline solution was used for inoculation. The inoculated fruits were maintained in a moist chamber at room temperature (25–27 °C) and observed daily for the appearance of anthracnose symptoms. The disease severity and disease incidence were calculated as described in Sharma et al. [50] and detailed in Supplementary Table S3. The disease score was recorded and indicated in Supplementary Table S4. The statistical significance for the disease severity was calculated according to Tukey’s post-hoc test (p < 0.05) (Figure 5b).

5. Conclusions

ITS sequence data alone is not useful for identification and differentiation of species from the genus Colletotrichum. The derived classification is inaccurate and groups the isolates to a species complex but does not delineate them to species accurately. Currently, multi-locus sequence data has been adopted and is routinely used for delineating and accurately describing members of the Colletotrichum genus to new species within each complex. Additionally, the cross-infection potential of different Colletotrichum species was assessed, which provided insights into their diverse host-range and pathogenic potential on non-hosts. Plant breeders should consider the broad host range and high genetic diversity of the Colletotrichum species when developing disease resistant cultivars.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11182373/s1.

Author Contributions

Conceptualization and design of experiments, S.F., M.M. and G.S.; experimental work, G.S. and M.M.; statistical analysis and microscopy, V.M. and G.S.; data analysis and curation, G.S.; funding, S.F.; writing, G.S. and S.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Institute of Plant Protection, ARO, Volcani Institute, Israel, and the ARO postdoctoral fellowship award to G.S. and V.M.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequences generated in this study are deposited in the NCBI-GenBank.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. First of the 147 equally most parsimonious trees obtained from a heuristic search of the combined ITS, act, chs1, gapdh, his3 and tub2 sequence alignment of the Colletotrichum isolates in the Acutatum complex. The MP and ML bootstrap support values (>50%) and Bayesian posterior probabilities (>0.90) are displayed at the nodes (ML/MP/PP). The tree was rooted to Monilochaetes infuscans (CBS 869.96). The bar indicates 50 changes. The isolates from this study are highlighted in blue. (* = Type strain).
Figure 1. First of the 147 equally most parsimonious trees obtained from a heuristic search of the combined ITS, act, chs1, gapdh, his3 and tub2 sequence alignment of the Colletotrichum isolates in the Acutatum complex. The MP and ML bootstrap support values (>50%) and Bayesian posterior probabilities (>0.90) are displayed at the nodes (ML/MP/PP). The tree was rooted to Monilochaetes infuscans (CBS 869.96). The bar indicates 50 changes. The isolates from this study are highlighted in blue. (* = Type strain).
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Plants 11 02373 g002 550
Figure 2. First of 205 equally most parsimonious trees obtained from a heuristic search of the combined ITS, act, chs1, gapdh, his3 and tub2 sequence alignment of the representative Colletotrichum isolates belonging to the C. godetiae, C. nymphaeae, C. tamarilloi, C. fioriniae and C. acutatum clades. The MP and ML bootstrap support values (>50%) and Bayesian posterior probabilities (>0.90) are displayed at the nodes (ML/MP/PP). The tree was rooted to Monilochaetes infuscans (CBS 869.96). The bar indicates 50 changes. The isolates from this study are highlighted in blue. (* = Type strain).
Figure 2. First of 205 equally most parsimonious trees obtained from a heuristic search of the combined ITS, act, chs1, gapdh, his3 and tub2 sequence alignment of the representative Colletotrichum isolates belonging to the C. godetiae, C. nymphaeae, C. tamarilloi, C. fioriniae and C. acutatum clades. The MP and ML bootstrap support values (>50%) and Bayesian posterior probabilities (>0.90) are displayed at the nodes (ML/MP/PP). The tree was rooted to Monilochaetes infuscans (CBS 869.96). The bar indicates 50 changes. The isolates from this study are highlighted in blue. (* = Type strain).
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Plants 11 02373 g003 550
Figure 3. First of 63 equally most parsimonious trees obtained from a heuristic search of the combined ITS, act, chs1, gapdh, his3 and tub2 sequence alignment of the Colletotrichum isolates in the Boninense complex. The MP and ML bootstrap support values (>50%) and Bayesian posterior probabilities (>0.90) are displayed at the nodes (MP/ML/BYPP). The tree was rooted to Monilochaetes infuscans (CBS 869.96). The bar indicates 100 changes. The isolates from this study are highlighted in blue. (* = Type strain).
Figure 3. First of 63 equally most parsimonious trees obtained from a heuristic search of the combined ITS, act, chs1, gapdh, his3 and tub2 sequence alignment of the Colletotrichum isolates in the Boninense complex. The MP and ML bootstrap support values (>50%) and Bayesian posterior probabilities (>0.90) are displayed at the nodes (MP/ML/BYPP). The tree was rooted to Monilochaetes infuscans (CBS 869.96). The bar indicates 100 changes. The isolates from this study are highlighted in blue. (* = Type strain).
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Plants 11 02373 g004 550
Figure 4. Anthracnose lesions on detached unwounded (UW) and wounded (W) strawberry fruits 7 days after inoculation for each representative isolate and species, within each Colletotrichum species complex used in this study.
Figure 4. Anthracnose lesions on detached unwounded (UW) and wounded (W) strawberry fruits 7 days after inoculation for each representative isolate and species, within each Colletotrichum species complex used in this study.
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Plants 11 02373 g005 550
Figure 5. Bar diagram showing the (a) percent disease incidence and (b) percent disease severity of the representative Colletotrichum isolates used in this study. White bars = wounded fruits; gray bars = unwounded fruits. Data presented are the mean ± standard error. Means with different superscript letters are significant according to Tukey’s post-hoc test (p < 0.05).
Figure 5. Bar diagram showing the (a) percent disease incidence and (b) percent disease severity of the representative Colletotrichum isolates used in this study. White bars = wounded fruits; gray bars = unwounded fruits. Data presented are the mean ± standard error. Means with different superscript letters are significant according to Tukey’s post-hoc test (p < 0.05).
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Table
Table 1. Colony morphology and growth rate on PDA and conidial characteristics of the representative Colletotrichum species from this study.
Table 1. Colony morphology and growth rate on PDA and conidial characteristics of the representative Colletotrichum species from this study.
IsolateComplexColony MorphologyConidia Length (μm)Conidia Width (μm)Growth Rate (mm/Day)
C.godetiae ALM-KSH10Acutatumcottony, grey aerial mycelium; reverse dark grey to pale orange in center(9.6–18.9) mean = 13.81 ± 0.24(3.5–5.7) Mean = 4.68 ± 0.065.4
C.godetiae ALM-BZR82Acutatumcottony, grey aerial mycelium; reverse dark grey to pale orange in center(8.9–19.3) mean = 13.52 ± 0.46(2.4–5.6) Mean = 4.26 ± 0.155.3
C.nymphaeae ANE-NL4Acutatumcottony, white aerial mycelium; reverse pale yellow to pale orange in center, with rings(7.9–16.7) mean = 13.84 ± 0.25(2.7–4.7) Mean = 3.88 ± 0.077.0
C.nymphaeae ANE-HV83CAcutatumwhite colony with orange center, reverse pale white to orange(11.5–21.2) mean = 14.17 ± 0.20(2.6–5.5) Mean = 4.30 ± 0.086.9
C.nymphaeae STR-101Acutatumcottony, white aerial mycelium; reverse pale yellow to pale orange in center, with rings(9.3–16.5) mean = 12.82 ± 0.18(2.8–4.7) Mean = 3.64 ± 0.056.8
C.nymphaeae TUT-5954Acutatumcottony, white aerial mycelium, reverse pale white with orange center(7.6–15.7) mean = 12.07 ± 0.21(2.7–4.7) Mean = 3.74 ± 0.067.2
C. acutatum ANE-NL12Acutatumcottony, white aerial mycelium, reverse pale white with dark red center(9.5–16.4) mean = 13.49 ± 0.20(3.4–5.8) Mean = 4.62 ± 0.067.3
C. fioriniae ALM-US4Acutatumwhite to light pink with greenish gray center with conidial mass; reverse whitish to light pink in the center(7.0–13.2) mean = 10.69 ± 0.20(3.1–5.9) Mean = 4.45 ± 0.077.7
C. fioriniae APL2Acutatumpinkish red colony with greenish gray center, reverse light pink with dark pink rings(7.1–14.3) mean = 10.27 ± 0.22(2.5–4.9) Mean = 3.74 ± 0.077.7
C. tamarilloi TOM10Boninensewhite colony with orange center, reverse pale white to orange(8.1–16.3) mean = 11.44 ± 0.25(2.9–5.2) Mean = 3.79 ± 0.076.1
C. brassicicola PASS65Boninensewhite with orange and black conidiomata in the center and pale orange center(12.6–18.5) mean = 15.58 ± 0.15(4.3–7.8) Mean = 5.74 ± 0.083.1
C. columbiense TOM6Boninensegreyish black mycelia with white margins, reverse grey(13.1–17.5) mean = 14.98 ± 0.14(4.8–8.3) Mean = 6.56 ± 0.107.3
C. columbiense PASS33Boninensecottony, grey aerial mycelium with rings; reverse white with grey-orange center(11.5–17.2) mean = 14.66 ± 0.16(4.8–7.4) Mean = 5.90 ± 0.098.3
C. karsti MAN76Boninensecottony, white mycelium with grey rings of conidiomata, reverse pale white(10.5–18.9) mean = 15.20 ± 0.29(3.6–8.5) Mean = 6.02 ± 0.157.1
C. sydowii PASS35Sydowiigreyish black mycelia with white margins, reverse dark grey(14.8–22.5) mean = 18.74 ± 0.38(4.7–8.5) Mean = 6.36 ± 0.176.8
C. gloesporioides s. l. Cg-Sc-A1Gloeosporioidescottony, white aerial mycelium with grey center, reverse pale yellow with dark grey center(9.6–19.0) mean = 16.5 ± 0.22(4.0–6.6) Mean = 5.17 ± 0.0711.4
C. gloesporioides s. l. APL7Gloeosporioidescottony, white aerial mycelium with grey center, reverse pale yellow(9.6–15.9) mean = 13.19 ± 0.16(3.8–7.0) Mean = 5.30 ± 0.099.4
Table
Table 2. Details of host, country of origin and species complex of Colletotrichum isolates used in the phylogenetic analyses along with the GenBank accession numbers of the sequences (N.S. = not sequenced).
Table 2. Details of host, country of origin and species complex of Colletotrichum isolates used in the phylogenetic analyses along with the GenBank accession numbers of the sequences (N.S. = not sequenced).
IsolateComplexTaxonCountryHostITSactchs1gapdhhis3tub2
ANE-NL-12AcutatumColletotrichum acutatumNetherlandsAnemoneON631980ON707522ON707486ON707635ON707568ON707601
ALM-US-4AcutatumColletotrichum fioriniaeIsraelAlmondON631977ON707519ON707483ON707632ON707565ON707598
APL-2AcutatumColletotrichum fioriniaeUSAAppleON631983ON707525ON707489ON707638ON707571ON707604
PCH-8AcutatumColletotrichum fioriniaeUSAPeachON631995ON707537ON707501ON707650ON707580ON707614
PCN-5AcutatumColletotrichum fioriniaeUSAPecanON631996ON707538ON707502ON707651ON707581ON707615
ALM-BZR-82 = CBS 149276AcutatumColletotrichum godetiaeIsraelAlmondON631968ON707510ON707474ON707623ON707556ON707589
ALM-GOZ-36EAcutatumColletotrichum godetiaeIsraelAlmondON631969ON707511ON707475ON707624ON707557ON707590
ALM-GOZ-42BAcutatumColletotrichum godetiaeIsraelAlmondON631970ON707512ON707476ON707625ON707558ON707591
ALM-GVA-6AAcutatumColletotrichum godetiaeIsraelAlmondON631971ON707513ON707477ON707626ON707559ON707592
ALM-GZT-1FAcutatumColletotrichum godetiaeIsraelAlmondON631972ON707514ON707478ON707627ON707560ON707593
ALM-KN-17AcutatumColletotrichum godetiaeIsraelAlmondON631973ON707515ON707479ON707628ON707561ON707594
ALM-KSH-10 = CBS 149275AcutatumColletotrichum godetiaeIsraelAlmondON631974ON707516ON707480ON707629ON707562ON707595
ALM-KYZ-6WAcutatumColletotrichum godetiaeIsraelAlmondON631975ON707517ON707481ON707630ON707563ON707596
ALM-NRB-30KAcutatumColletotrichum godetiaeIsraelAlmondON631976ON707518ON707482ON707631ON707564ON707597
STR-101 = CBS 149278AcutatumColletotrichum nymphaeaeIsraelStrawberryON631997ON707539ON707503ON707652ON707582ON707616
STR-3AcutatumColletotrichum nymphaeaeIsraelStrawberryON631998ON707540ON707504ON707653ON707583ON707617
TUT-137AcutatumColletotrichum nymphaeaeIsraelStrawberryON632001ON707543ON707507ON707656ON707586ON707620
TUT-149AcutatumColletotrichum nymphaeaeIsraelStrawberryON632002ON707544ON707508ON707657ON707587ON707621
TUT-5954 = CBS 149277AcutatumColletotrichum nymphaeaeIsraelStrawberryON632003ON707545ON707509ON707658ON707588ON707622
ANE-3A = CBS 149280AcutatumColletotrichum nymphaeaeIsraelAnemoneON631978ON707520ON707484ON707633ON707566ON707599
ANE-HV-83C = CBS 149279AcutatumColletotrichum nymphaeaeIsraelAnemoneON631979ON707521ON707485ON707634ON707567ON707600
ANE-NL4AcutatumColletotrichum nymphaeaeNetherlandsAnemoneON631981ON707523ON707487ON707636ON707569ON707602
ANE-UK-31AcutatumColletotrichum nymphaeaeUnited KingdomAnemoneON631982ON707524ON707488ON707637ON707570ON707603
TOM-10AcutatumColletotrichum tamarilloiColombiaTamarilloON631999ON707541ON707505ON707654ON707584ON707618
PASS-65BoninenseColletotrichum brassicicolaColombiaPassifloraON631993ON707535ON707499ON707648ON707578ON707612
PASS-33BoninenseColletotrichum colombienseColombiaPassifloraON631988ON707530ON707494ON707643ON707573ON707608
PASS-55BoninenseColletotrichum colombienseColombiaPassifloraON631991ON707533ON707497ON707646ON707576ON707610
PASS-62BoninenseColletotrichum colombienseColombiaPassifloraON631992ON707534ON707498ON707647ON707577ON707611
PASS-67BoninenseColletotrichum colombienseColombiaPassifloraON631994ON707536ON707500ON707649ON707579ON707613
TOM-6BoninenseColletotrichum colombienseColombiaTamarilloON632000ON707542ON707506ON707655ON707585ON707619
MAN-76BoninenseColletotrichum karstiColombiaMangoON631987ON707529ON707493ON707642ON707572ON707607
PASS-52BoninenseColletotrichum karstiColombiaPassifloraON631990ON707532ON707496ON707645ON707575ON707609
PASS-35SydowiiColletotrichum sydowiiColombiaPassifloraON631989ON707531ON707495ON707644ON707574N.S.
Litchi-Cg2GloeosporioidesColletotrichum aenigmaColombiaLitchiON631986ON707528ON707492ON707641 N.S.
APL-7GloeosporioidesColletotrichum sp.USAAplleON631984ON707526ON707490ON707639N.S.ON707605
CG-SC-A1GloeosporioidesColletotrichum sp.IsraelSalicorniaON631985ON707527ON707491ON707640N.S.ON707606
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Sharma, G.; Maymon, M.; Meshram, V.; Freeman, S. Characterization of Colletotrichum Isolates from Strawberry and Other Hosts with Reference to Cross-Inoculation Potential. Plants 2022, 11, 2373. https://doi.org/10.3390/plants11182373

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Sharma G, Maymon M, Meshram V, Freeman S. Characterization of Colletotrichum Isolates from Strawberry and Other Hosts with Reference to Cross-Inoculation Potential. Plants. 2022; 11(18):2373. https://doi.org/10.3390/plants11182373

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Sharma, Gunjan, Marcel Maymon, Vineet Meshram, and Stanley Freeman. 2022. "Characterization of Colletotrichum Isolates from Strawberry and Other Hosts with Reference to Cross-Inoculation Potential" Plants 11, no. 18: 2373. https://doi.org/10.3390/plants11182373

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