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Article

Colletotrichum Species Causing Anthracnose of Citrus in Australia

1
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia
2
Agriculture Victoria, Department of Jobs, Precincts and Regions, AgriBio Centre, 5 Ring Road, La Trobe University, Bundoora, VIC 3083, Australia
3
School of Applied Systems Biology, La Trobe University, Bundoora, VIC 3083, Australia
4
Faculty of Science, The University of Melbourne, Parkville, VIC 3010, Australia
5
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
*
Author to whom correspondence should be addressed.
J. Fungi 2021, 7(1), 47; https://doi.org/10.3390/jof7010047
Received: 21 December 2020 / Revised: 4 January 2021 / Accepted: 6 January 2021 / Published: 12 January 2021
(This article belongs to the Special Issue Fungal Biodiversity and Ecology)

Abstract

Colletotrichum spp. are important pathogens of citrus that cause dieback of branches and postharvest disease. Globally, several species of Colletotrichum have been identified as causing anthracnose of citrus. One hundred and sixty-eight Colletotrichum isolates were collected from anthracnose symptoms on citrus stems, leaves, and fruit from Victoria, New South Wales, and Queensland, and from State herbaria in Australia. Colletotrichum australianum sp. nov., C. fructicola, C. gloeosporioides, C. karstii, C. siamense, and C. theobromicola were identified using multi-gene phylogenetic analyses based on seven genomic loci (ITS, gapdh, act, tub2, ApMat, gs, and chs-1) in the gloeosporioides complex and five genomic loci (ITS, tub2, act, chs-1, and his3) in the boninense complex, as well as morphological characters. Several isolates pathogenic to chili (Capsicum annuum), previously identified as C. queenslandicum, formed a clade with the citrus isolates described here as C. australianum sp. nov. The spore shape and culture characteristics of the chili and citrus isolates of C. australianum were similar and differed from those of C. queenslandicum. This is the first report of C. theobromicola isolated from citrus and the first detection of C. karstii and C. siamense associated with citrus anthracnose in Australia.
Keywords: anthracnose; citrus; Colletotrichum australianum; phylogenetic analysis; taxonomy anthracnose; citrus; Colletotrichum australianum; phylogenetic analysis; taxonomy

1. Introduction

Edible citrus (Citrus spp.) are important fruit crops globally, produced in temperate and tropical climates [1]. Cumquat (Citrus japonica), grapefruit (Citrus × paradisi), lemon (Citrus limon), lime (Citrus aurantifolia), mandarin (Citrus reticulata), and orange (Citrus × sinensis) are all commercially important citrus species [1,2]. Australia is a major citrus producer with citrus grown in every mainland state [3,4]. In 2019, there was approximately 25,500 ha of citrus production in Australia [5]. Citrus is one of the largest fresh fruit exports from Australia. Australia exported 251,594 tonnes of citrus in 2018, with a total value of $A452.9 million [6].
In citrus, anthracnose caused by Colletotrichum spp. is a serious disease limiting production globally. Preharvest anthracnose reduces yield, while postharvest anthracnose affects fruit quality, negatively impacting fruit export and marketability [7]. Colletotrichum species are difficult to identify based on morphological characters. Molecular phylogeny has reinvigorated Colletotrichum taxonomy [8], with over 220 Colletotrichum species in 14 species complexes now recognised [9,10].
Globally, multiple Colletotrichum species within several species complexes have been identified as causing citrus anthracnose. Colletotrichum gloeosporioides was reported to be associated with anthracnose in Australia [8], Vietnam [11], China [12], Italy [8,13], Morocco [14], Mexico [15,16], Pakistan [17], Ghana [18,19], Brazil [11,20], Algeria [21], Greece [8], Malta [8], New Zealand [8], Portugal [8,22], South Africa [11], Spain [8], Tunisia [23,24], United States [8] and Zimbabwe [11]. Colletotrichum karstii was reported in Southern Italy [13], China [25,26,27], Portugal [23], South Africa [11], Europe [8], United States [28], Tunisia [16], Turkey [29], and New Zealand [25]; C. fructicola was reported in China [26,27,30]; and C. siamense was reported in Vietnam [11], Bangladesh [11], Egypt [11], China [31], Mexico [22], and Pakistan [17,32]. Additionally, C. abscissum, C. acutatum, C. boninense, C. brevisporum, C. catinaense, C. citri, C. citricola, C. citri-maximae, C. constrictum, C. godetiae, C. helleniense, C. hystricis, C. johnstonii, C. cigarro, C. limetticola, C. limonicola, C. novae-zelandiae, C. queenslandicum, C. simmondsii, C. tropicicola, and C. truncatum have all been associated with citrus anthracnose [8,11,25,27,33,34,35,36].
Colletotrichum acutatum, C. fructicola, C. gloeosporioides, and C. nymphaeae have been reported as pathogens associated with citrus anthracnose in Australia. However, C. acutatum was identified based on morphology, and C. nymphaeae was verified by a single tub2 sequence [37,38]. Citrus fruits and plants with anthracnose symptoms are very common both in home gardens and in commercial orchards in Australia. Hence, it is necessary to accurately characterize the Colletotrichum species causing anthracnose diseases of citrus in Australia to help develop appropriate disease management strategies and provide a baseline for plant biosecurity, trade, and market access.
In this study, a representative collection of Colletotrichum isolates from eastern Australian citrus was established from symptomatic leaves, twigs, and fruit, and from culture collections. Colletotrichum species were determined by utilising a polyphasic approach, in which informative gene loci were sequenced. Multigene phylogenetic analyses, morphological characters, and pathogenicity bioassays were used to confirm the taxonomy and phylogenetic relationships of Colletotrichum spp. pathogens causing citrus anthracnose in Australia.

2. Materials and Methods

2.1. Sample Collection

A total of 147 Colletotrichum isolates were collected from anthracnose lesions on citrus stems and leaves of trees growing in Victoria and New South Wales and from citrus fruits with anthracnose disease symptoms from supermarkets in Melbourne, Victoria. In addition, 21 isolates originating from citrus plants were obtained from State fungaria (the Victorian Plant Pathology Herbarium (VPRI), the Queensland Plant Pathology Herbarium (BRIP), and the NSW Plant Pathology Collection (DAR)).

2.2. Isolate Preparation

Infected fruits, stems, and leaves were surface sterilized by dipping in 2.3% (active ingredient) sodium hypochlorite (NaOCl) for 2 min and rinsed five times with sterile distilled water (SDW). Tissue pieces (2 mm2) were excised from the margins of infected lesions and plated onto potato dextrose agar (PDA). The plates were incubated at 25 °C in continuous dark for 7 d as described by Guarnaccia et al. [8]. Subcultures of mycelia on PDA plates were maintained under the same growing conditions for a further 7 d. All isolates were established as single spore cultures, as described in De Silva et al. [39].

2.3. Morphological and Cultural Analyses

Plugs (2 mm2) of actively growing mycelia were taken from the edge of 7-d-old cultures and transferred onto PDA and synthetic nutrient-poor agar (SNA), as described by Guarnaccia et al. [8]. After 7 d of incubation at 25 °C under continuous near-ultraviolet light, colony growth was determined by measuring two diameters perpendicular to each other per plate and determining the average of six plates. At 10 d, colony colour was determined using colour charts [40]. Acervuli were induced by inoculating pieces of sterilized mandarin rind with mycelia and incubating on water agar (WA) and SNA, at 25 °C for 10 d.
Appressoria were induced using the slide culture technique described by Johnston and Jones [41]. The length and width of 30 appressoria/slide were measured using X1000 magnification with a Leica DM6000 LED compound microscope, Leica DMC2900 camera, and Leica LAS v. 4.5.0 software.
Slide preparations of morphological structures were prepared in lactic acid, and at least 30 observations were recorded for conidia, conidiophores, and conidiogenous cells per isolate, as well as presence or absence of setae. The range, mean, and standard error (SE) were calculated for each isolate.

2.4. Multigene Phylogenetic Analysis

2.4.1. DNA Extraction, PCR Amplification, and Sequencing

1. DNA extraction
Genomic DNA was extracted from pure (single-spored) mycelia of Colletotrichum isolates grown on PDA at 25 °C for 7 d using DNeasy Plant Mini kits (Qiagen, Australia), following the manufacturer’s instructions. DNA concentration was determined using NanoDrop, then diluted to 2 ng∙µL−1 and stored at −20 °C until further use [39].
2. PCR amplification and sequencing
Isolates were assigned to a species complex based on morphology and internal transcribed spacer and intervening 5.8S nrDNA gene (ITS) and β-tubulin (tub2) gene sequences data. Isolates in the gloeosporioides species complex were further characterised using seven gene loci: ITS, glyceraldehyde-3-phosphate dehydrogenase (gapdh), actin (act), tub2, the Apn2–Mat1–2 intergenic spacer and partial mating type (Mat1–2) (ApMat), glutamine synthetase (gs), and chitin synthase 1 (chs-1) genes. Isolates in the boninense species complex were further characterised using five gene loci: ITS, tub2, act, chs-1, and histone (his3). These gene sequences were amplified and sequenced by using primer pairs: ITS-1F (ITS; [42]) and ITS4 (ITS; [43]), GDF1 and GDR1 (gapdh; [44]), ACT-512F + ACT-783R (act; [45]), Btub2Fd and Btub4Rd (tub2; [46]), AMF1 and AMR1 (ApMat; [47]), GSF1 and GSR1 (gs; [48]), CHS-79F and CHS-354R (chs-1; [45]), and CYLH3F and CYLH3R (his3; [49]).
PCR was performed in a 2720 Thermal Cycler (Applied Biosystems, Australia). The total volume of PCR mixture was 25 µL. The PCR of the ITS, gapdh, act, tub2, gs, chs-1, and his3 genes followed the protocol described by De Silva et al. [39] and contained 1× PCR buffer, 2 mM MgCl2, 0.2 mM dNTP, 1 U Taq DNA polymerase (MangoTaq DNA polymerase; Bioline, Australia), 0.4 µM of each primer, and 6 ng template DNA. The PCR annealing temperatures were adjusted to 55 °C for ITS, gapdh, and his3; 58 °C for act, tub2, and gs; and 66 °C for chs-1.
For ApMat, in the 25 µL PCR mixture, the concentration of each primer was adjusted to 0.5 µM, and the template DNA was adjusted to 10 ng. The PCR amplification protocols were performed according to Silva et al. [47], except the annealing temperature of ApMat was adjusted to 62 °C.
All PCR products were purified using QIA-quick PCR Purification Kit (Qiagen, Australia) following the manufacturer’s instructions. Purified PCR products were sequenced in both the forward and reverse sense at the Australian Genome Research Facility (AGRF, Melbourne), then aligned to produce a consensus sequence for each isolate using ClustalW in MEGA 6.06 [50]. The consensus sequences were deposited in GenBank.

2.4.2. Phylogenetic Analyses

The sequences of reference isolates were retrieved from GenBank for use in phylogenetic analyses (Table 1). All the sequences were aligned by using ClustalW in MEGA 6.06 and manually edited when necessary. The ITS and tub2 sequences of morphologically different isolates were compared to determine which species complex each isolate belonged based on maximum likelihood analysis (ML) by using MEGA 6.06 [10]. For isolates from the gloeosporioides species complex, phylogenetic analyses of combined seven gene sequences (ITS, gapdh, act, tub2, ApMat, gs, and chs-1) and combined two gene sequences (ApMat and gs) were carried out with selected reference sequences [39,51]. For isolates from the boninense species complex, phylogenetic analysis of combined five gene sequences (ITS, tub2, act, chs-1, and his3) was constructed [8].
Further phylogenetic analyses were based on Bayesian Inference analyses (BI) by using MrBayes v. 3.1.2 and ML analysis by using MEGA 6.06 [39]. For BI analyses, MrModeltest2.3 was used to determine the best-fit model for each locus [52] (Table 2). MrBayes v. 3.2.6 was used to generate phylogenetic trees. Four chains were used in the Markov Chain Monte Carlo (MCMC) analysis and were run for 1,000,000,000 generations. The trees were sampled every 100 generations and the heating parameter was set to 0.2. Analyses stopped once the average standard deviation of split frequencies was below 0.01. For ML analysis, analyses were done by using MEGA 6.06. The phylogeny test was the Bootstrap method with 1000 replicates. The substitution model was the Tamura–Nei model based on nucleotide type. The tree inference option was Nearest-Neighbor-Interchange (NNI) ML heuristic method.

2.5. Pathogenicity Testing

One isolate of each Colletotrichum species (except for C. siamense, which did not sporulate in culture) was used in the pathogenicity tests to inoculate orange (Washington Navel) fruits, orange leaves, lemon (Myer) leaves, and orange flower petals according to the method of Guarnaccia et al. [8].

2.5.1. Fruit Bioassay

Conidial suspensions of each isolate were prepared by adding 10 mL of SDW to 7-d-old cultures, scraping the mycelia then filtering through muslin cloth. The concentration of spore suspension was adjusted to 106 conidia mL−1. Organically grown orange fruits (Citrus sinensis) purchased from a market (Queen Victoria Market in Melbourne) were washed with tap water and then submerged in 70% ethanol for 10 min, and finally rinsed in SDW twice. The orange fruits were marked in the middle to divide into two parts and inoculated with both wound (W) and non-wound (NW) methods. For the wound method, the orange skin was pricked with a sterilized pipette tip to about 1 mm depth. Six wound points were made, and each inoculated with 6 µL spore suspension. In the non-wound method, six drops of 6 µL spore suspension were placed directly on the orange skin. For the control group, 6 µL of SDW was used to treat orange fruit in both wound and non-wound methods. There were three replicates per treatment per isolate and the experiments replicated twice. The fruit was transferred to a plastic box and incubated at 25 °C with 100% humidity in dark. After 10 d, fruits were examined for symptom development, and the percentage of infection was calculated ( percentage   ( % ) = i n f e c t e d   p o i n t s i n o c u l a t e d   p o i n t s × 100 % ).

2.5.2. Leaf Bioassay

Young, healthy, fully expanded orange and lemon leaves were collected from trees growing in pots. The leaves were washed with tap water, then submerged in 70% ethanol for 2 min, and finally rinsed in SDW twice. The petioles of leaves were wrapped with damp cotton wool and the leaves were placed into petri dishes, three leaves per dish. Three drops of 6 µL spore suspension (106 conidia/mL) were individually placed directly onto the leaf upper surfaces. For the control group, 6 µL of SDW was used. Each set of three leaves per petri dish was inoculated with a different isolate. The petri dishes were placed inside a plastic box and the leaves incubated at 25 °C with 100% humidity and 12/12 h fluorescent light/dark cycle. After 10 d, the leaves were examined for symptom development, and the percentage of infection was calculated ( percentage   ( % ) = i n f e c t e d   p o i n t s i n o c u l a t e d   p o i n t s × 100 % ).

2.5.3. Petal Bioassay

Healthy orange flower petals were collected from the same trees. Petals were washed in tap water, then submerged in 70% ethanol for 30 s, and finally rinsed in SDW twice. One drop of 6 µL spore suspension (103 conidia/mL) was carefully placed on the middle of each petal without wounding. For the control group, 6 µL of SDW was used. Seven flower petals were used per isolate. The inoculated petals were put in a plastic box and incubated at 25 °C with 100% humidity and 12/12 h fluorescent light/dark cycle. After 3 d, the petals were examined for symptom development, and the percentage of infection was calculated ( percentage   ( % ) = i n f e c t e d   p o i n t s i n o c u l a t e d   p o i n t s × 100 % ).

3. Results

3.1. Phylogenetic Analyses

The 147 isolates were separated into 18 morphological groups based on culture characteristics. One isolate from each morphological group and 18 isolates from State fungaria from different hosts and location were selected for phylogenetic analyses. Among the 36 Colletotrichum isolates, 29 were identified to be in the gloeosporioides complex and seven were identified to be in the boninense complex based on analysis of combined ITS and tub2 gene sequences. All the isolates in the gloeosporioides complex were isolated from stems, leaves, or fruit, while six of the seven isolates in the boninense complex were isolated from infected orange leaf, while another one was from infected lemon leaf (Table S1).

3.1.1. Gloeosporioides Species Complex

1. Seven-gene tree of citrus isolates in gloeosporioides species complex
The seven-gene phylogenetic analysis consisted of 29 citrus isolates and 29 reference sequences from the gloeosporioides species complex. Colletotrichum boninense (ICMP 17904T) was used as the out-group. A total of 3703 characters (ITS: 504, gapdh: 271, act: 271, tub2: 510, ApMat: 898, gs: 914, chs-1: 275 and 10 N to separate each two sequences) were analysed. The Bayesian analysis lasted 825,000 generations, resulting in 11,995 total trees, of which 8997 trees were used to calculate the posterior probabilities. The BI posterior probabilities were plotted on the ML tree (Figure 1).
2. Two-gene tree of citrus isolates in gloeosporioides species complex
Analysis using the ApMat and gs sequence alignment consisted of 29 citrus isolates and 44 reference sequences from the gloeosporioides species complex. Colletotrichum horii (ICMP 10492T) was used as the out-group. A total of 1832 characters (ApMat: 903, gs: 919 and 10 N to separate two sequences) were analysed. The Bayesian analysis lasted 240,000 generations, resulting in 3601 total trees of which 2701 trees were used to calculate the posterior probabilities. The BI posterior probabilities were plotted on the ML tree (Figure 2).
Five species and one unknown Colletotrichum sp. were identified from the two trees (Figure 1 and Figure 2). Twenty-one (72%) of citrus isolates were identified as C. gloeosporioides, two isolates clustered with three reference isolates of C. siamense, two isolates clustered with three reference isolates of C. fructicola, and one isolate was identified to be C. theobromicola. Two isolates were identified and described as a new species, which was phylogenetically close but significantly different to C. queenslandicum with high support (100/1 in both trees). Isolate BRIP 58074a formed a significantly separate clade (96/1 in both trees) close to C. cordylinicola.

3.1.2. Boninense Species Complex

The five gene phylogenetic analysis consisted of seven citrus isolates and 26 reference sequences from the boninense complex. Colletotrichum truncatum (CBS 151.35T) was used as the out-group. A total of 2048 characters (ITS: 559, tub2: 503, act: 280, chs-1: 282, his3: 395) were analysed. The Bayesian analysis lasted 135,000 generations, resulting in 1994 total trees, of which 1496 trees were used to calculate the posterior probabilities. The BI posterior probabilities were plotted on the ML tree. The phylogenetic analysis of the boninense species complex identified the seven citrus isolates as C. karstii (Figure 3).

3.2. Morphological Analysis

Morphological characters including conidial size, conidial shape, and growth rate of seven Colletotrichum species were recorded (Table 3). Their conidial size, conidial shape, and growth rate overlapped.
Morphological characters of the type specimen of C. queenslandicum (ICMP 1778) were according to Weir et al. [36] (Table 3). The new species varied morphologically from the type specimen of C. queenslandicum (ICMP 1778) by having different spore shape. Although the range of spore size overlapped between the new species and C. queenslandicum, the average conidial length of the new species was smaller than that of C. queenslandicum [36].

3.3. New Colletotrichum Species

3.3.1. Two-Gene Tree of New Colletotrichum Species

The two gene phylogenetic analysis consisted of six chili (Capsicum annuum) and two citrus isolates of the new Colletotrichum species, 34 reference sequences from the C. gloeosporioides species complex, including eight isolates of C. queenslandicum. Colletotrichum theobromicola (ICMP 18649T) was used as the out-group. A total of 1820 characters (ApMat: 900, gs: 910 and 10 N to separate two sequences) were analysed. The Bayesian analysis lasted 115,000 generations, resulting in 1709 total trees, of which 1282 trees were used to calculate the posterior probabilities. The BI posterior probabilities were plotted on the ML tree (Figure 4).
The six isolates from chili [39] clustered with the two citrus isolates of the new Colletotrichum species in the two-gene tree, which were significantly different from C. queenslandicum (Figure 4).

Taxonomy

Morphological characters and phylogenetic analyses indicated that the Colletotrichum species isolated from infected mandarin and orange fruits collected from Melbourne and Dunkeld, Victoria, respectively, and isolated from infected chili fruit collected from Brisbane, Queensland, Australia, was a new species, for which the name Colletotrichum australianum is proposed.
Colletotrichum australianum W. Wang, D. D. De Silva, and P. W. J. Taylor, sp. nov. (Figure 5).
MycoBank Number: MB830323.
Etymology: Named after the country where the pathogen was first isolated, Australia.
Holotype: Australia, Victoria, Dunkeld, on fruit of Citrus sinensis, May 2016, J. Kennedy (VPRI 43075–holotype; UMC002–ex-type culture).
Asexual morph on SNA. Conidiomata on SNA inconspicuous or absent, 41–140 µm diam, formed from hyphae, lacking setae. Conidia hyaline, smooth, aseptate, straight, cylindrical with one end slightly acute, granular, and guttulate, (13.2–) 14.4–14.6 (–15.9) × (4.8–) 5.6–5.7 (–6.1) µm. Appressoria single, medium to dark brown, ovoid with an undulate margin, (6.1–) 8.5–8.9 (–12.2) × (4.6–) 6.7–7.1 (–9.3) µm.
Asexual morph on PDA. Conidiomata on PDA formed on hyphae or on a brown central stroma, lacking setae. Conidiophores hyaline, smooth-walled, septate, branched, 28–58 × 2–3 μm. Conidiogenous cells hyaline, smooth-walled, subcylindrical, straight to curved, phialidic with visible periclinal thickening at the apex, 14–30 × 2–3 µm. Conidia hyaline, smooth, aseptate, straight, cylindrical with one end acute, granular and guttulate, (12.7–) 14.1–14.5 (–17.2) × (3.9–) 4.5–4.7 (–5.5) µm. Appressoria single, medium to dark brown, ovoid with an undulate margin, (7.2–) 8.1–8.3 (–9.5) × (5.4–) 6.5–6.7 (–7.6) µm.
Mycelia on mandarin rind were colourless to white. Conidiomata salmon, smooth. Conidia hyaline, smooth-walled, aseptate, straight, cylindrical with one end acute, granular and guttulate, (12.9–) 14.7–15.1 (–16.1) × (4.3–) 4.8–5 (–5.4) µm.
Culture characteristics: Colonies on SNA flat, entire margin, hyaline, 45–55 mm diam in 7 d. Colonies on PDA 65–75 mm in 7 d; pale yellow to white aerial mycelia, changing to grey in the centre, reverse have a uniform concentric ring with pinkish outside and inside pale grey to grey in the centre. Colonies on MEA flat, entire margin, white aerial mycelia, 52–78 mm in 7 d.
Notes: Colletotrichum australianum is phylogenetically close to C. queenslandicum but are separable using ApMat and gs sequences. The closest match in a Blastn search with the gs sequence was GenBank KP703693, C. queenslandicum strain CPC 17123, with 98 % identity.

3.4. Pathogenicity Assay

For the fruit bioassay, C. australianum, C. fructicola, C. theobromicola, Colletotrichum sp., and C. karstii developed brown lesions on wounded orange fruits. Colletotrichum karstii had the highest infection incidence at 100%, while the C. gloeosporioides isolate did not cause obvious symptoms (Table 4). None of the Colletotrichum species were able to infect non-wounded orange fruit.
For the leaf bioassay, C. karstii developed lesions on both orange and lemon leaves, while C. theobromicola only developed lesions on lemon leaves (Table 4). Other Colletotrichum isolates did not cause obvious symptoms on both orange and lemon leaves.
In the petal bioassay, all isolates infected orange petals.

4. Discussion

Six Colletotrichum species were identified from citrus stems, leaves, and fruits with anthracnose symptoms in Australia. Colletotrichum australianum was isolated from orange and mandarin fruit in Victoria, Australia, and identified and described as a new species causing anthracnose of citrus in Australia. Isolates from chili (Capsicum annuum) from Queensland and previously identified as C. queenslandicum [39] were also reidentified as C. australianum. Phylogenetic analyses clearly showed C. australianum to be a new species closely related to C. queenslandicum. There were also differences in morphological characters between these two species. The ApMat and gs sequences clearly distinguished C. australianum. These genes are considered as informative markers to identify species within the C. gloeosporioides species complex [10,36,51,53].
Colletotrichum gloeosporioides sensu lato was the most frequently isolated in diseased citrus. There was no preference for a particular Citrus sp. or infected organ tissue. Colletotrichum gloeosporioides was isolated from various citrus species, including cumquat, finger lime, grapefruit, lemon, lime, mandarin, orange, Persian lime, and Tahitian lime. Colletotrichum gloeosporioides was previously cultured from lemon (Citrus limon) and orange (Citrus sinensis) in Australia [37]. The isolate VPRI 10347 from lemon from Victoria and previously identified as C. nymphaeae [37] was also reidentified as C. gloeosporioides. The prevalence of Colletotrichum species that cause anthracnose of citrus in Australia, is in accordance with recent global studies on the major cause of anthracnose of citrus [8,11,12,13,14,19,20,21,23,24,27].
This is the first report in Australia of Colletotrichum siamense being associated with citrus anthracnose. Colletotrichum siamense was isolated from lemon fruit and finger lime fruit and has been recorded as a pathogen of a broad range of plants in Australia [37,39]. Colletotrichum siamense was previously reported to be isolated from catmon (Citrus pennivesiculata) in Bangladesh and Egypt, mandarin (C. reticulata Blanco cv. Shiyue Ju) in China, and mandarin (C. reticulata cv. Kinnow) in Pakistan [11,31,32,54]. Colletotrichum siamense isolate BRIP 54270b was collected in 2011 in Queensland, suggesting C. siamense has been a citrus pathogen for at least 10 years in Australia. However, both C. siamense isolates were collected from citrus fruits, and no C. siamense isolate was found on citrus leaves or stems, suggesting C. siamense is more likely to be a postharvest pathogen of citrus in Australia.
Colletotrichum theobromicola is for the first time reported as a pathogen of citrus. Colletotrichum theobromicola was isolated from lime fruit from Queensland but was recently neotypified from cacao tree (Theobroma cacao) in Panama [36]. Colletotrichum theobromicola has been recorded as a pathogen of a broad range of plants in Australia including jointvetch (Aeschynomene falcata), arabica coffee (Coffea arabica), olive (Olea europaea), pomegranate (Punica granatum), stylo (Stylosanthes guianensis), and sticky stylo (Stylosanthes viscosa) [37].
Colletotrichum fructicola was reported for the first time, associated with anthracnose symptoms from mandarin fruit in Australia. Isolate BRIP 65028 from Tahitian lime growing in Queensland was previously identified as C. fructicola in 2018 [38]. Colletotrichum fructicola was also isolated from avocado (Persea americana) in Australia [37]. In China, C. fructicola was reported to be associated with bergamot orange (Citrus bergamia), pomelo (C. grandis), mandarin (C. reticulata cv. nanfengmiju), oranges (C. sinensis), and kumquat (Fortunella margarita) [26,27,30]. Colletotrichum fructicola was found to cause both preharvest and postharvest citrus disease in Australia.
Colletotrichum karstii was the second dominant pathogen and was isolated from infected orange and lemon leaves in both New South Wales and Victoria. Colletotrichum karstii is the only species in the boninense species complex found to be associated with citrus anthracnose in Australia. Three C. karstii isolates were collected from orange leaves in the 1970s and were maintained in State fungaria, suggesting C. karstii has been a citrus pathogen for over 50 years in Australia but was misidentified as C. gloeosporioides. Colletotrichum karstii was reported to infect citrus and to have a wide global distribution [8,11,13,16,23,25,26,27,28,29]. Previously, C. karstii was reported from other hosts such as black plum (Diospyros australis), strawberry (Fragaria x ananassa), and banana (Musa banksia) in Australia [37].
Six Colletotrichum isolates from chili (Capsicum annuum) that had been previously identified as causing anthracnose fruit rot of chili in Brisbane, Queensland, Australia [39], were also identified as C. australianum. These six Colletotrichum isolates were morphologically similar to C. australianum from citrus rather than the type specimen of C. queenslandicum (ICMP 1778), which was originally isolated from infected papaya. The identification of C. australianum from diverse hosts such as orange, mandarin, and chili, suggests that C. australianum may have a broad host range. Further studies are required on the host range of this pathogen, which may have biosecurity implication for the export of Australian fruit. The occurrence of C. australianum in both Victoria and Queensland indicates the wide geographic spread across different climatic zones in Australia.
The species identification of Colletotrichum isolates based on ApMat and gs gene sequences were as similar as the results from phylogenetic analysis of seven-gene combination, proving that the locus ApMat was effective in identifying Colletotrichum species within the gloeosporioides species complex. The phylogenetic analysis of combined ApMat and gs sequences can identify species within the gloeosporioides species complex [10,47,51,53]. The efficiency of the ApMat gene to identify species was also supported by Sharma et al. [55] and Sharma, Pinnaka, and Shenoy [56], who differentiated Colletotrichum isolates in India. The isolate VPRI 10347 was identified to be Colletotrichum nymphaeae in Shivas et al. [37] based on single tub2 sequence. However, in this study, ApMat and gs gene sequences identified isolate VPRI 10347 as C. gloeosporioides, same as the result from phylogenetic analysis of the seven-gene combination. However, the limitation of using the ApMat gene in constructing phylogenetic trees is that several reference Colletotrichum species in the gloeosporioides species complex in GenBank have not been sequenced for ApMat. For example, the isolate VPRI 43083 was phylogenetically close to C. grevilleae and C. grossum based on analysis of combined ITS and tub2 gene sequences (Supplementary Figure S1) but due to a lack of ApMat sequence of C. grevilleae and C. grossum, these species were not included in either the seven-gene nor the two-gene trees, whereas VPRI 43083 was identified as C. theobromicola based on seven gene combination and two gene combination analyses with high bootstrap value. Due to a lack of replicate isolates, as well as a lack of reference sequences, especially ApMat gene data of Colletotrichum species close to BRIP 58074a, the unknown Colletotrichum sp. (BRIP 58074a) isolate cannot be further described taxonomically or phylogenetically at this stage.
Colletotrichum acutatum has been reported from lemon (DAR 80516, from Tasmania in 2009, and DAR 72160, from NSW in 1998) previously [38]. However, C. acutatum was not found in this study. The two C. acutatum isolates were identified based on morphology but have not been confirmed by molecular analysis. Gene sequences of isolates DAR 80516 and DAR 72160 should be analysed to accurately identify these two isolates.
Pathogenicity tests of five Colletotrichum species from citrus showed that all species except for C. gloeosporioides were capable of infecting wounded fruit. In contrast, none of the five Colletotrichum species caused disease on the non-wounded fruit. These results are consistent with previous reports where wound inoculated citrus fruits were used in postharvest pathogenicity testing of Colletotrichum species [8,27]. Variable maturity of the fruit may also be a reason for lack of infection. Mature fruits are reported to be more sensitive to Colletotrichum species [57]. The fruit used for inoculation in this study may not have been fully mature, although they were selected based on the colour of mature fruit; thus, they were not conducive for Colletotrichum spores to attach to the cuticle, germinate, and form appressoria prior to infection.
Different Colletotrichum species had various degrees of aggressiveness on wounded orange fruit and non-wounded orange and lemon leaves. Colletotrichum karstii was the most aggressive species when infecting orange fruit and orange and lemon leaves. The variable aggressiveness of different Colletotrichum species has been reported by Guarnaccia et al. [8]. Colletotrichum gloeosporioides isolate VPRI 43076 was non-pathogenic on fruit and leaves but was pathogenic on orange petals. Conversely, Guarnaccia et al. [8] reported C. gloeosporioides to be the most aggressive species when infecting orange fruit. Pathogenic variation has been reported within populations of a Colletotrichum species [10,58,59]. Hence, VPRI 43076 was likely to have been an isolate of Colletotrichum gloeosporioides, which had weak aggressiveness on citrus fruit. Further assessment of pathogenicity of isolates from each species needs to be undertaken to determine the variability of aggressiveness.

5. Conclusions

Six Colletotrichum spp. were identified to cause anthracnose of citrus in Australia that included one novel species C. australianum, and one undetermined species. In addition, this was the first report of C. theobromicola as a pathogen of citrus globally, and the first report of C. karstii and C. siamense to be associated with citrus anthracnose in Australia.

Supplementary Materials

The following are available online at https://www.mdpi.com/2309-608X/7/1/47/s1: Figure S1: Phylogram generated from maximum likelihood analysis of all available Colletotrichum species in the gloeosporioides species complex and the boninense species complex based on combined ITS and tub2 sequences data, Table S1: Information of the 36 Colletotrichum isolates selected for phylogenetic analyses.

Author Contributions

Conceptualization: W.W., J.E., P.K.A. and P.W.J.T.; data curation: W.W. and J.E.; formal analysis: W.W., D.D.d.S., A.M., and J.E.; funding acquisition: P.W.J.T. and J.E.; methodology: W.W., D.D.d.S., P.K.A., and P.W.J.T.; project administration: P.W.J.T.; resources: W.W. and P.W.J.T.; supervision: P.K.A., P.W.C., and P.W.J.T.; writing—original draft: W.W.; writing—review and editing: W.W., D.D.d.S., A.M., J.E., P.K.A., P.W.C., and P.W.J.T. All authors have read and agreed to the published version of the manuscript.

Funding

W.W. was supported by a University of Melbourne postgraduate scholarship. Financial support was also received from the Innovation Seed Fund for Horticulture Development between Agriculture Victoria Research and The University of Melbourne.

Institutional Review Board Statement

Not applicable

Informed Consent Statement

Not applicable

Data Availability Statement

Alignments generated during the current study are available in TreeBASE (accession http://purl.org/phylo/treebase/phylows/study/TB2:S27542). All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis of the combined ITS, gapdh, act, tub2, ApMat, GS, and chs-1 sequence alignment of Colletotrichum isolates in the gloeosporioides complex. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
Figure 1. Phylogenetic analysis of the combined ITS, gapdh, act, tub2, ApMat, GS, and chs-1 sequence alignment of Colletotrichum isolates in the gloeosporioides complex. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
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Figure 2. Phylogenetic analysis of the combined ApMat and GS sequence alignment of Colletotrichum isolates in the gloeosporioides complex. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
Figure 2. Phylogenetic analysis of the combined ApMat and GS sequence alignment of Colletotrichum isolates in the gloeosporioides complex. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
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Figure 3. Phylogenetic analysis of the combined ITS, tub2, act, chs-1, and his3 sequence alignment of Colletotrichum isolates in the boninense complex. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
Figure 3. Phylogenetic analysis of the combined ITS, tub2, act, chs-1, and his3 sequence alignment of Colletotrichum isolates in the boninense complex. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
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Figure 4. Phylogenetic analysis of the combined ApMat and GS sequence alignment of Colletotrichum australianum sp. nov. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
Figure 4. Phylogenetic analysis of the combined ApMat and GS sequence alignment of Colletotrichum australianum sp. nov. The bootstrap support values (ML > 75%) of maximum likelihood analysis and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (ML/PP). Black circle denotes isolates from Citrus spp.
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Figure 5. Morphological characteristics of Colletotrichum australianum sp. nov.: One-week-old culture on PDA (A,B), conidiomata on mandarin rind (C), conidiomata on SNA (D), conidiomata on PDA (E), conidiophores (F,G), conidia (H) and appressoria (IK). Scale bars: D, 500 µm; F, G, H, I, J, K, 20 µm.
Figure 5. Morphological characteristics of Colletotrichum australianum sp. nov.: One-week-old culture on PDA (A,B), conidiomata on mandarin rind (C), conidiomata on SNA (D), conidiomata on PDA (E), conidiophores (F,G), conidia (H) and appressoria (IK). Scale bars: D, 500 µm; F, G, H, I, J, K, 20 µm.
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Table 1. Strains of Colletotrichum species used in the phylogenetic analyses with details of host and location, and GenBank accession numbers of the sequences.
Table 1. Strains of Colletotrichum species used in the phylogenetic analyses with details of host and location, and GenBank accession numbers of the sequences.
SpeciesAccession NumberHostLocationGenBank Accession Numbers
ITSGAPDHACTTUB2gsApMatCHS-1HIS3
Gloeosporioides complex
C. aenigmaICMP 18608 *Persea americanaIsraelJX010244JX010044JX009443JX010389JX010078KM360143JX009774
C. aeschynomenesICMP 17673; ATCC 201874 *Aeschynomene virginicaUSAJX010176 JX009930JX009483JX010392JX010081KM360145JX009799
C. alataeICMP 17919 *Dioscorea alataIndiaJX010190JX009990JX009471 JX010383JX010065KC888932JX009837
C. alienumICMP 12071 *Malus domesticaNew Zealand JX010251JX010028JX009572 JX010411JX010101KM360144JX009882
C. asianumICMP 18580; CBS 130418 *Coffea arabicaThailandFJ972612 JX010053JX009584 JX010406JX010096FR718814JX009867
C. aotearoaICMP 18537 *Coprosma sp.New ZealandJX010205JX010005JX009564JX010420JX010113KC888930JX009853
C. artocarpicolaMFLUCC 18–1167 *Artocarpus heterophyllusThailandMN415991MN435568MN435570MN435567MN435569
C. australianumVPRI 43074; UMC001Citrus reticulataAustralia, VicMG572137MG572126MK473452MG572148MG572159MG572170MW091986
VPRI 43075; UMC002 *Citrus sinensisAustralia, VicMG572138MG572127MN442109MG572149MG572160MG572171MW091987
BRIP 63695Capsicum annuumAustraliaKU923677MN442115MN442105KU923693KU923737KU923727MW092000
BRIP 63696Capsicum annuumAustraliaKU923678KU923694 KU923738KU923728
BRIP 63697Capsicum annuumAustraliaKU923679KU923695 KU923739KU923729
BRIP 63698Capsicum annuumAustraliaKU923680MN442116MN442106KU923696KU923740KU923730MW092001
BRIP 63699Capsicum annuumAustraliaKU923681MN442117MN442107KU923697KU923741KU923731MW092002
BRIP 63700Capsicum annuumAustraliaKU923682MN442118MN442108KU923698 KU923742KU923732MW092003
C. camelliaeCGMCC 3.14925 *Camellia sinensisChinaKJ955081KJ954782KJ954363KJ955230KJ954932KJ954497
Glomella cingulate f. sp. camelliaeICMP 10643 *Camellia × williamsiiUKJX010224JX009908JX009540JX010436JX010119KJ954625JX009891
C. changpingenseMFLUCC 15-0022Fragaria ×ananassaChinaKP683152KP852469KP683093KP852490KP852449
C. chrysophilumCMM4268 *Musa sp.BrazilKX094252KX094183KX093982KX094285KX094204KX094083
C. conoidesCAUG17 *Capsicum annuumChinaKP890168 KP890162KP890144 KP890174KP890156
C. cordylinicolaMFLUCC 090551; ICMP 18579 *Cordyline fruticosaThailandJX010226JX009975HM470235JX010440JX010122JQ899274JX009864
C. clidemiaeICMP 18658 *Clidemia hirtaUSA, HawaiiJX010265 JX009989JX009537JX010438JX010129KC888929JX009877
C. endophyticaCAUG28Capsicum annuumChina KP145441KP145413KP145329KP145469KP145385
C. fructicolaICMP 18581; CBS 130416 *Coffea arabicaThailandJX010165JX010033FJ907426JX010405JX010095JQ807838JX009866
LC2923; LF130Camellia sinensisChinaKJ955083KJ954784KJ954365KJ955232KJ954934 KJ954499
VPRI 43079; UMC006Citrus reticulataAustralia, QldMG572142MG572131MK473454MG572153MG572164MG572175MW091991
BRIP 65028a; VPRI 43034; B03-43034Citrus latifoliaAustralia, QldMK470007MK470025MK470097MK470061MK470043MK470079MW091983
C. fructicola (syn. C. ignotum)ICMP 18646Tetragastris panamensisPanamaJX010173JX010032JX009581JX010409JX010099JQ807839JX009874
C. fructivorumCBS 133125 *Vaccinium macrocarponUSAJX145145JX145196
C. gloeosporioidesIMI 356878; ICMP 17821; CBS 112999 *Citrus sinensisItalyJX010152JX010056JX009531JX010445JX010085JQ807843JX009818
LC3110; LF318Camellia sinensisChinaKJ955127KJ954828KJ954407KJ955275KJ954978KJ954541
LC3312; LF534Camellia sinensisChina KJ955158KJ954859KJ954434KJ955305KJ955009KJ954569
LC3382; LF604Camellia sinensisChinaKJ955176KJ954877KJ954450KJ955323KJ955026KJ954584
LC3686; LF916Camellia sinensisChina KJ955226KJ954927KJ954493 KJ955371 KJ955076KJ954629
VPRI 43076; UMC003Citrus sinensisAustralia, VicMG572139MG572128MN442110MG572150MG572161MG572172MW091988
VPRI 43078; UMC005Citrus aurantifoliaAustralia, QldMG572141MG572130MN442111MG572152MG572163MG572174MW091990
VPRI 43080; UMC007Citrus reticulataAustralia, QldMG572143MG572132MK473455MG572154MG572165MG572176MW091992
VPRI 43081; UMC008Citrus reticulataAustralia, QldMG572144MG572133MN442112MG572155MG572166MG572177MW091993
VPRI 43082; UMC009Citrus reticulataAustralia, QldMG572145MG572134MN442113MG572156MG572167MG572178MW091994
VPRI 43084; UMC011Citrus japonicaAustralia, VicMG572147MG572136MN442114MG572158MG572169MG572180MW091996
VPRI 43648; UMC012Citrus sinensisAustralia, VicMW081160MW081163MW081166MW081169MW081175MW081172MW091997
VPRI 43649; UMC013Citrus limonAustralia, VicMW081161MW081164MW081167MW081170MW081176MW081173MW091998
VPRI 43650; UMC014Citrus japonicaAustralia, VicMW081162MW081165MW081168MW081171MW081177MW081174MW091999
VPRI 10312; A01-10312Citrus sinensisAustralia, VicMK469996MK470014MK470086MK470050MK470032MK470068MW091972
VPRI 10347; A02-10347; BRIP 54771Citrus limonAustralia, VicMK469997MK470015MK470087MK470051; KU221374MK470033MK470069MW091973
WAC 12803; BRIP 63680a; VPRI 43024; A05-43024Citrus sinensisAustralia, WAMK469998MK470016MK470088MK470052MK470034MK470070MW091974
BRIP 66210a; VPRI 43026; A07-43026Citrus reticulataAustralia, SAMK470000MK470018MK470090MK470054MK470036MK470072MW091976
BRIP 66210b; VPRI 43027; A08-43027Citrus reticulataAustralia, SAMK470001MK470019MK470091MK470055MK470037MK470073MW091977
BRIP 28546a; VPRI 43028; A09-43028Citrus sinensis NavelAustralia, QldMK470002MK470020MK470092MK470056MK470038MK470074MW091978
BRIP 28754a; VPRI 43030; A11-43030Citrus reticulataAustralia, QldMK470003MK470021MK470093MK470057MK470039MK470075MW091979
BRIP 53157d; VPRI 43031; A12-43031Citrus aurantifolia Tahiti Australia, QldMK470004MK470022MK470094MK470058MK470040MK470076MW091980
BRIP 66135a; VPRI 43032; B01-43032Citrus reticulata Imperial Blanco Australia, QldMK470005MK470023MK470095MK470059MK470041MK470077MW091981
BRIP 28831a; VPRI 43033; B02-43033Citrus sinensisAustralia, QldMK470006MK470024MK470096MK470060MK470042MK470078MW091982
VPRI 42955; G01-42955Citrus limonAustralia, NSWMK470008MK470026MK470098MK470062MK470044MK470080MW091984
VPRI 42956; H01-42956Citrus sinensisAustralia, NSWMK470009MK470027MK470099MK470063MK470045MK470081MW091985
C. grevilleaeCBS 132879 *Grevillea sp.Italy KC297078KC297010KC296941KC297102KC297033KC296987
C. grossumCGMCC3.17614T; CAUG7 *Capsicum sp.ChinaKP890165 KP890159 KP890141 KP890171KP890153
CAU31Capsicum sp.ChinaKP890166KP890160 KP890142KP890172KP890154
CAUG32Capsicum sp. ChinaKP890167KP890161KP890143KP890173KP890155
C. hebeienseMFLUCC13-0726 *Vitis vinifera cv. Cabernet SauvignonChinaKF156863KF377495KF377532KF288975KF289008
C. hellenienseCPC 26844; CBS 142418 *Poncirus trifoliataGreeceKY856446KY856270KY856019KY856528KY856186
C. henanenseLC3030; CGMCC 3.17354; LF238 *Camellia sinensisChinaKJ955109KJ954810KM023257KJ955257KJ954960KJ954524
C. horiiICMP 10492 *Diospyros kakiJapanGQ329690GQ329681JX009438 JX010450JX010137JQ807840JX009752
C. hystricisCPC 28153; CBS 142411 *Citrus hystrixItalyKY856450KY856274KY856023KY856532KY856190
C. jiangxienseLF687 *Camellia sinensisChinaKJ955201KJ954902KJ954471KJ955348KJ955051KJ954607
C. cigarroICMP 18534Kunzea ericoidesNew ZealandJX010227JX009904JX009473JX010427JX010116HE655657JX009765
C. kahawaeIMI 319418; ICMP 17816 *Coffea arabicaKenyaJX010231JX010012JX009452JX010444JX010130JQ894579JX009813
C. musaeICMP 19119; CBS 116870 *Musa sp. USAJX010146 JX010050JX009433 HQ596280JX010103KC888926JX009896
ICMP 17817Musa sapientumKenyaJX010142JX010015JX009432JX010395JX010084JX009815
C. nupharicolaICMP 18187 *Nuphar lutea subsp. polysepalaUSAJX010187JX009972JX009437 JX010398JX010088JX145319JX009835
C. pandanicolaMFLUCC 17-0571PandanaceaeThailandMG646967MG646934MG646938MG646926MG646931
C. proteaeCBS 132882 *Protea sp.South AfricaKC297079KC297009KC296940KC297101KC297032KC296986
C. psidiiICMP 19120 *Psidium sp. ItalyJX010219JX009967JX009515JX010443JX010133 KC888931JX009901
C. queenslandicumICMP 1778 *Carica papayaAustraliaJX010276JX009934JX009447 JX010414JX010104KC888928JX009899
CPC 17123Syzygium australaAustraliaKP703357KP703282KP703439KP703693KP703778
ICMP 18705Coffea sp.FijiJX010185JX010036 JX009490JX010412JX010102JX009890
CMM3233Anacardium occidentaleBrazil, Pernambuco stateMF110849MF111058MF110996MF110639
CMM3241Anacardium occidentaleBrazil, Pernambuco stateMF110848MF111059MF111000MF110642
CMM3236Anacardium occidentaleBrazil, Pernambuco stateMF110850MF111060MF110997MF110640
CMM3240Anacardium occidentaleBrazil, Pernambuco stateMF110852MF111061MF110999MF110644
CMM3237Anacardium occidentaleBrazil, Pernambuco stateMF110853MF111062MF110998MF110641
CMM3242Anacardium occidentaleBrazil, Pernambuco stateMF111063MF111001MF110643
C. rhexiaeCBS 133134 *Rhexia virginicaUSAJX145128JX145179
C. salsolaeICMP 19051 *Salsola tragusHungaryJX010242JX009916JX009562JX010403JX010093KC888925JX009863
C. siamenseICMP 18578 CBS 130417 *Citrus arabicaThailand JX010171JX009924FJ907423JX010404JX010094JQ899289JX009865
VPRI 43077; UMC004Citrus limonAustralia, NSWMG572140MG572129MK473453MG572151MG572162MG572173MW091989
BRIP 54270b; VPRI 43029; A10-43029Citrus australasicaAustralia, QldMK469995MK470013MK470085MK470049MK470031MK470067MW091971
C. siamense (syn. C. jasmini-sambac)CBS 130420; ICMP 19118Jasminum sambacVietnam HM131511HM131497HM131507 JX010415JX010105JQ807841JX009895
C. siamense (syn. C. hymenocallidis)CBS 125378; ICMP 18642; LC0043Hymenocallis americanaChina JX010278JX010019GQ856775JX010410JX010100JQ899283GQ856730
C. siamense (syn. C. murrayae) GZAAS 5.09506Murraya sp.ChinaJQ247633JQ247609JQ247657 JQ247644 JQ247621
C. syzygicolaDNCL021; MFLUCC 10-0624 *Syzygium samarangenseThailandKF242094KF242156KF157801KF254880KF242125
C. temperatumCBS 133122 *Vaccinium macrocarponUSAJX145159JX145211
C. theobromicolaICMP 18649; CBS 124945 *Theobroma cacaoPanamaJX010294JX010006JX009444JX010447JX010139KC790726JX009869
C. theobromicola (syn. C. fragariae)ICMP 17927; CBS 142.31; MTCC 10325TFragaria × ananassaUSAJX010286 JX010024 JX009516JX010373JX010064JQ807844JX009830
VPRI 43083; UMC010Citrus aurantifoliaAustralia, QldMG572146MG572135MK473456MG572157MG572168MG572179MW091995
C. tiICMP 4832 *Cordyline sp.New ZealandJX010269JX009952JX009520JX010442JX010123KM360146JX009898
C. tropicaleICMP 18653; CBS 124949 *Theobroma cacaoPanamaJX010264JX010007JX009489 JX010407JX010097KC790728JX009870
C. viniferumGZAAS 5.08601 *Vitis vinifera, cv. ‘Shuijing’ China JN412804JN412798JN412795JN412813JN412787
C. wuxienseCGMCC 3.17894 *Camellia sinensisChina KU251591 KU252045KU251672KU252200KU252101KU251722KU251939
C. xanthorrhoeaeBRIP 45094; ICMP 17903; CBS 127831 *Xanthorrhoea preissiiAustraliaJX010261 JX009927JX009478JX010448JX010138KC790689JX009823
Colletotrichum sp.BRIP 58074a; VPRI 43025; A06-43025Citrus australasicaAustralia, QldMK469999MK470017MK470089MK470053MK470035MK470071MW091975
Boninense complex
C. annellatumCBS 129826 *Hevea brasiliensisColombia JQ005222JQ005570 JQ005656 JQ005396 JQ005483
C. beeveriCBS 128527 *Brachyglottis repandaNew Zealand JQ005171 JQ005519 JQ005605 JQ005345 JQ005432
C. boninenseICMP 17904; CBS 123755 *Crinum asiaticum ‘Sinicum’Japan JQ005153 JQ005501JQ005588 JQ005327 JQ005414
C. brassicicolaCBS 101059Brassica oleracea var. gemmiferaNew ZealandJQ005172JQ005520 JQ005606 JQ005346 JQ005433
C. brasilienseCBS 128501*Passiflora edulisBrazilJQ005235 JQ005583JQ005669 JQ005409 JQ005496
C. catinaenseCBS 142417; CPC 27978 *Citrus reticulataItaly, Catania KY856400 KY855971 KY856482 KY856136 KY856307
C. citricolaCBS 134228 *Citrus unchiuChina KC293576 KC293616KC293656 KY856140KY856311
C. constrictumCBS 128504Citrus limonNew ZealandJQ005238JQ005586JQ005672JQ005412KY856313
C. colombienseCBS 129818 *Passiflora edulisColombiaJQ005174 JQ005522 JQ005608 JQ005348 JQ005435
C. cymbidiicolaIMI 347923 *Cymbidium sp.AustraliaJQ005166 JQ005514JQ005600 JQ005340JQ005427
C. dacrycarpiCBS 130241 *Dacrycarpus dacrydioidesNew Zealand JQ005236 JQ005584 JQ005670 JQ005410 JQ005497
C. hippeastriCBS 125376 *Hippeastrum vittatumChinaJQ005231 JQ005579 JQ005665 JQ005405JQ005492
C. karstiiCBS 126532Citrus sp.South Africa JQ005209 JQ005557 JQ005643 JQ005383JQ005470
CBS 128551Citrus sp.New Zealand JQ005208JQ005556JQ005642JQ005382JQ005469
CBS 129829Gossypium hirsutumGermanyJQ005189JQ005537 JQ005623JQ005363JQ005450
CPC 27853Citrus sinensisItaly, CataniaKY856461 KY856034 KY856543KY856202KY856377
CPC 31139Citrus sinensisItaly, CataniaKY856467KY856040KY856549KY856208KY856383
CBS 129833Musa sp.MexicoJQ005175JQ005523JQ005609JQ005349JQ005436
CBS 861.72Bombax aquaticumBrazilJQ005184JQ005532JQ005618 JQ005358JQ005445
DAR 25017a; VPRI 42941; D02-42941Citrus sinensis ValenciaAustralia, NSWMK470103MK470109MK470106MK470115MK470112
DAR 29821a; VPRI 42943; F02-42943Citrus sinensis ValenciaAustralia, NSWMK470104MK470110MK470107MK470116MK470113
DAR 29826a; VPRI 42944; G02-42944Citrus sinensis ValenciaAustralia, NSWMK470105MK470111MK470108MK470117MK470114
VPRI 43651; UMC015Citrus limonAustralia, VicMW081178MW081186MW081182MW081190MW081194
VPRI 43652; UMC016Citrus sinensisAustralia, VicMW081179MW081187MW081183MW081191MW081195
VPRI 43653; UMC017Citrus sinensisAustralia, VicMW081180MW081188MW081184MW081192MW081196
VPRI 43654; UMC018Citrus sinensisAustralia, VicMW081181MW081189MW081185MW081193MW081197
C. limonicolaCBS 142410; CPC 31141 *Citrus limonMalta, Gozo KY856472KY856045 KY856554KY856213 KY856388
C. novae-zelandiaeCBS 128505 *Capsicum annuumNew ZealandJQ005228JQ005576 JQ005662 JQ005402JQ005489
C. oncidiiCBS 129828 *Oncidium sp.GermanyJQ005169 JQ005517 JQ005603 JQ005343 JQ005430
C. parsonsiaeCBS 128525 *Parsonsia capsularisNew Zealand JQ005233 JQ005581JQ005667 JQ005407 JQ005494
C. petchiiCBS 378.94 *Dracaena marginataItalyJQ005223 JQ005571 JQ005657 JQ005397 JQ005484
C. phyllanthiCBS 175.67 *Phyllanthus acidusIndiaJQ005221 JQ005569JQ005655 JQ005395 JQ005482
C. torulosumCBS 128544 *Solanum melongenaNew Zealand JQ005164 JQ005512 JQ005598JQ005338 JQ005425
Truncatum complex
C. truncatumCBS 151.35 *Phaseolus lunatusUSAGU227862GU227960 GU228156 GU228352 GU228058
Vic: Victoria, NSW: New South Wales, Qld: Queensland, WA: Western Australia, SA: South Australia. * Ex-holotype or ex-epitype cultures.
Table 2. Best-fit model for each gene locus selected by MrModeltest.
Table 2. Best-fit model for each gene locus selected by MrModeltest.
DatasetSubstitution Models
ITStub2actchs-1his3
boninense complexSYM + I+GHKY + IHKY + GGTR + GHKY + I
ITSgapdhtub2actApMatgschs-1
gloeosporioides complexSYM + IHKY + ISYM + IHKY + IHKY + GGTR + GK80 + G
Table 3. Morphological characters of Colletotrichum species.
Table 3. Morphological characters of Colletotrichum species.
TaxonConidial Length (μm)Conidial Width (μm) Conidial ShapeGrowth Rate (mm/day) 1
C. gloeosporioides(10.2–) 13.8–14.3 (–16.1)(4.2–) 5.3–5.5 (–7.3)Subcylindrical10.4–10.8
C. siamense(12.0–) 13.1–13.4 (–15.8)(4.8–) 5.4–5.5 (–5.9)Fusoid 10.9–11.5
C. fructicola(12.7–) 14.2–14.6 (–17.1)(4.6–) 5.1–5.2 (–5.7)Cylindrical 10.5–11.1
C. theobromicola(10.8–) 15.2–16 (–21.2)(4.0–) 4.8–5 (–5.8)Cylindrical10.5–10.7
Colletotrichum sp.(13.1–) 15.6–16 (–18.0)(4.6–) 6.1–6.3 (–7.7)Cylindrical8.9–9.7
C. karstii(11.3–) 13.2–13.6 (–14.8)(6.4–) 7.1–7.3 (–8.3)Cylindrical9.4–9.6
New species (12.7–) 14.1–14.5 (–17.2)(3.9–) 4.5–4.7 (–5.5)Cylindrical with one end acute9.7–10.3
C. queenslandicum2(12–) 14.5–16.5 (–21.5)(3.5–) 4.5–5 (–6)Cylindric, straight, sometimes slightly constricted near center, ends broadly rounded/
1 Seven Colletotrichum species incubated at 25 °C for 7 d. Colony growth was determined by measuring two diameters perpendicular to each other per plate and determining the average of six plates. 2 C. queenslandicum ICMP 1778, MycoBank MB563593 [36].
Table 4. Incidence of infection on Washington Navel orange fruit and leaves and Meyer lemon leaves by Colletotrichum species.
Table 4. Incidence of infection on Washington Navel orange fruit and leaves and Meyer lemon leaves by Colletotrichum species.
CultureFungus SpeciesInfection Incidence %
Fruit Bioassay (Wound)Leaf BioassayPetal Bioassay
Orange LeafLemon Leaf
VPRI 43075C. australianum sp. nov.95.800100
VPRI 43076C. gloeosporioides000100
VPRI 43079C. fructicola7500100
VPRI 43083C. theobromicola95.8083.3100
BRIP 58074aColletotrichum sp.95.800100
VPRI 43654C. karstii100100100100
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