Colletotrichum Species Causing Anthracnose of Citrus in Australia

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.

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].

Table 1.

Strains of Colletotrichum species used in the phylogenetic analyses with details of host and location, and GenBank accession numbers of the sequences.

Species	Accession Number	Host	Location	GenBank Accession Numbers
				ITS	GAPDH	ACT	TUB2	gs	ApMat	CHS-1	HIS3
Gloeosporioides complex
C. aenigma	ICMP 18608 *	Persea americana	Israel	JX010244	JX010044	JX009443	JX010389	JX010078	KM360143	JX009774	–
C. aeschynomenes	ICMP 17673; ATCC 201874 *	Aeschynomene virginica	USA	JX010176	JX009930	JX009483	JX010392	JX010081	KM360145	JX009799	–
C. alatae	ICMP 17919 *	Dioscorea alata	India	JX010190	JX009990	JX009471	JX010383	JX010065	KC888932	JX009837	–
C. alienum	ICMP 12071 *	Malus domestica	New Zealand	JX010251	JX010028	JX009572	JX010411	JX010101	KM360144	JX009882	–
C. asianum	ICMP 18580; CBS 130418 *	Coffea arabica	Thailand	FJ972612	JX010053	JX009584	JX010406	JX010096	FR718814	JX009867	–
C. aotearoa	ICMP 18537 *	Coprosma sp.	New Zealand	JX010205	JX010005	JX009564	JX010420	JX010113	KC888930	JX009853	–
C. artocarpicola	MFLUCC 18–1167 *	Artocarpus heterophyllus	Thailand	MN415991	MN435568	MN435570	MN435567	–	–	MN435569	–
C. australianum	VPRI 43074; UMC001	Citrus reticulata	Australia, Vic	MG572137	MG572126	MK473452	MG572148	MG572159	MG572170	MW091986	–
	VPRI 43075; UMC002 *	Citrus sinensis	Australia, Vic	MG572138	MG572127	MN442109	MG572149	MG572160	MG572171	MW091987	–
	BRIP 63695	Capsicum annuum	Australia	KU923677	MN442115	MN442105	KU923693	KU923737	KU923727	MW092000	–
	BRIP 63696	Capsicum annuum	Australia	KU923678	–	–	KU923694	KU923738	KU923728	–	–
	BRIP 63697	Capsicum annuum	Australia	KU923679	–	–	KU923695	KU923739	KU923729	–	–
	BRIP 63698	Capsicum annuum	Australia	KU923680	MN442116	MN442106	KU923696	KU923740	KU923730	MW092001	–
	BRIP 63699	Capsicum annuum	Australia	KU923681	MN442117	MN442107	KU923697	KU923741	KU923731	MW092002	–
	BRIP 63700	Capsicum annuum	Australia	KU923682	MN442118	MN442108	KU923698	KU923742	KU923732	MW092003	–
C. camelliae	CGMCC 3.14925 *	Camellia sinensis	China	KJ955081	KJ954782	KJ954363	KJ955230	KJ954932	KJ954497	–	–
Glomella cingulate f. sp. camelliae	ICMP 10643 *	Camellia × williamsii	UK	JX010224	JX009908	JX009540	JX010436	JX010119	KJ954625	JX009891	–
C. changpingense	MFLUCC 15-0022	Fragaria ×ananassa	China	KP683152	KP852469	KP683093	KP852490	–	–	KP852449	–
C. chrysophilum	CMM4268 *	Musa sp.	Brazil	KX094252	KX094183	KX093982	KX094285	KX094204	–	KX094083	–
C. conoides	CAUG17 *	Capsicum annuum	China	KP890168	KP890162	KP890144	KP890174	–	–	KP890156	–
C. cordylinicola	MFLUCC 090551; ICMP 18579 *	Cordyline fruticosa	Thailand	JX010226	JX009975	HM470235	JX010440	JX010122	JQ899274	JX009864	–
C. clidemiae	ICMP 18658 *	Clidemia hirta	USA, Hawaii	JX010265	JX009989	JX009537	JX010438	JX010129	KC888929	JX009877	–
C. endophytica	CAUG28	Capsicum annuum	China	KP145441	KP145413	KP145329	KP145469	–	–	KP145385	–
C. fructicola	ICMP 18581; CBS 130416 *	Coffea arabica	Thailand	JX010165	JX010033	FJ907426	JX010405	JX010095	JQ807838	JX009866	–
	LC2923; LF130	Camellia sinensis	China	KJ955083	KJ954784	KJ954365	KJ955232	KJ954934	KJ954499	–	–
	VPRI 43079; UMC006	Citrus reticulata	Australia, Qld	MG572142	MG572131	MK473454	MG572153	MG572164	MG572175	MW091991	–
	BRIP 65028a; VPRI 43034; B03-43034	Citrus latifolia	Australia, Qld	MK470007	MK470025	MK470097	MK470061	MK470043	MK470079	MW091983	–
C. fructicola (syn. C. ignotum)	ICMP 18646	Tetragastris panamensis	Panama	JX010173	JX010032	JX009581	JX010409	JX010099	JQ807839	JX009874	–
C. fructivorum	CBS 133125 *	Vaccinium macrocarpon	USA	JX145145	–	–	JX145196	–	–	–	–
C. gloeosporioides	IMI 356878; ICMP 17821; CBS 112999 *	Citrus sinensis	Italy	JX010152	JX010056	JX009531	JX010445	JX010085	JQ807843	JX009818	–
	LC3110; LF318	Camellia sinensis	China	KJ955127	KJ954828	KJ954407	KJ955275	KJ954978	KJ954541	–	–
	LC3312; LF534	Camellia sinensis	China	KJ955158	KJ954859	KJ954434	KJ955305	KJ955009	KJ954569	–	–
	LC3382; LF604	Camellia sinensis	China	KJ955176	KJ954877	KJ954450	KJ955323	KJ955026	KJ954584	–	–
	LC3686; LF916	Camellia sinensis	China	KJ955226	KJ954927	KJ954493	KJ955371	KJ955076	KJ954629	–	–
	VPRI 43076; UMC003	Citrus sinensis	Australia, Vic	MG572139	MG572128	MN442110	MG572150	MG572161	MG572172	MW091988	–
	VPRI 43078; UMC005	Citrus aurantifolia	Australia, Qld	MG572141	MG572130	MN442111	MG572152	MG572163	MG572174	MW091990	–
	VPRI 43080; UMC007	Citrus reticulata	Australia, Qld	MG572143	MG572132	MK473455	MG572154	MG572165	MG572176	MW091992	–
	VPRI 43081; UMC008	Citrus reticulata	Australia, Qld	MG572144	MG572133	MN442112	MG572155	MG572166	MG572177	MW091993	–
	VPRI 43082; UMC009	Citrus reticulata	Australia, Qld	MG572145	MG572134	MN442113	MG572156	MG572167	MG572178	MW091994	–
	VPRI 43084; UMC011	Citrus japonica	Australia, Vic	MG572147	MG572136	MN442114	MG572158	MG572169	MG572180	MW091996	–
	VPRI 43648; UMC012	Citrus sinensis	Australia, Vic	MW081160	MW081163	MW081166	MW081169	MW081175	MW081172	MW091997	–
	VPRI 43649; UMC013	Citrus limon	Australia, Vic	MW081161	MW081164	MW081167	MW081170	MW081176	MW081173	MW091998	–
	VPRI 43650; UMC014	Citrus japonica	Australia, Vic	MW081162	MW081165	MW081168	MW081171	MW081177	MW081174	MW091999	–
	VPRI 10312; A01-10312	Citrus sinensis	Australia, Vic	MK469996	MK470014	MK470086	MK470050	MK470032	MK470068	MW091972	–
	VPRI 10347; A02-10347; BRIP 54771	Citrus limon	Australia, Vic	MK469997	MK470015	MK470087	MK470051; KU221374	MK470033	MK470069	MW091973	–
	WAC 12803; BRIP 63680a; VPRI 43024; A05-43024	Citrus sinensis	Australia, WA	MK469998	MK470016	MK470088	MK470052	MK470034	MK470070	MW091974	–
	BRIP 66210a; VPRI 43026; A07-43026	Citrus reticulata	Australia, SA	MK470000	MK470018	MK470090	MK470054	MK470036	MK470072	MW091976	–
	BRIP 66210b; VPRI 43027; A08-43027	Citrus reticulata	Australia, SA	MK470001	MK470019	MK470091	MK470055	MK470037	MK470073	MW091977	–
	BRIP 28546a; VPRI 43028; A09-43028	Citrus sinensis Navel	Australia, Qld	MK470002	MK470020	MK470092	MK470056	MK470038	MK470074	MW091978	–
	BRIP 28754a; VPRI 43030; A11-43030	Citrus reticulata	Australia, Qld	MK470003	MK470021	MK470093	MK470057	MK470039	MK470075	MW091979	–
	BRIP 53157d; VPRI 43031; A12-43031	Citrus aurantifolia Tahiti	Australia, Qld	MK470004	MK470022	MK470094	MK470058	MK470040	MK470076	MW091980	–
	BRIP 66135a; VPRI 43032; B01-43032	Citrus reticulata Imperial Blanco	Australia, Qld	MK470005	MK470023	MK470095	MK470059	MK470041	MK470077	MW091981	–
	BRIP 28831a; VPRI 43033; B02-43033	Citrus sinensis	Australia, Qld	MK470006	MK470024	MK470096	MK470060	MK470042	MK470078	MW091982	–
	VPRI 42955; G01-42955	Citrus limon	Australia, NSW	MK470008	MK470026	MK470098	MK470062	MK470044	MK470080	MW091984	–
	VPRI 42956; H01-42956	Citrus sinensis	Australia, NSW	MK470009	MK470027	MK470099	MK470063	MK470045	MK470081	MW091985	–
C. grevilleae	CBS 132879 *	Grevillea sp.	Italy	KC297078	KC297010	KC296941	KC297102	KC297033	–	KC296987	–
C. grossum	CGMCC3.17614T; CAUG7 *	Capsicum sp.	China	KP890165	KP890159	KP890141	KP890171	–	–	KP890153	–
	CAU31	Capsicum sp.	China	KP890166	KP890160	KP890142	KP890172	–	–	KP890154	–
	CAUG32	Capsicum sp.	China	KP890167	KP890161	KP890143	KP890173	–	–	KP890155	–
C. hebeiense	MFLUCC13-0726 *	Vitis vinifera cv. Cabernet Sauvignon	China	KF156863	KF377495	KF377532	KF288975	–	–	KF289008	–
C. helleniense	CPC 26844; CBS 142418 *	Poncirus trifoliata	Greece	KY856446	KY856270	KY856019	KY856528	–	–	KY856186	–
C. henanense	LC3030; CGMCC 3.17354; LF238 *	Camellia sinensis	China	KJ955109	KJ954810	KM023257	KJ955257	KJ954960	KJ954524	–	–
C. horii	ICMP 10492 *	Diospyros kaki	Japan	GQ329690	GQ329681	JX009438	JX010450	JX010137	JQ807840	JX009752	–
C. hystricis	CPC 28153; CBS 142411 *	Citrus hystrix	Italy	KY856450	KY856274	KY856023	KY856532	–	–	KY856190	–
C. jiangxiense	LF687 *	Camellia sinensis	China	KJ955201	KJ954902	KJ954471	KJ955348	KJ955051	KJ954607	–	–
C. cigarro	ICMP 18534	Kunzea ericoides	New Zealand	JX010227	JX009904	JX009473	JX010427	JX010116	HE655657	JX009765	–
C. kahawae	IMI 319418; ICMP 17816 *	Coffea arabica	Kenya	JX010231	JX010012	JX009452	JX010444	JX010130	JQ894579	JX009813	–
C. musae	ICMP 19119; CBS 116870 *	Musa sp.	USA	JX010146	JX010050	JX009433	HQ596280	JX010103	KC888926	JX009896	–
	ICMP 17817	Musa sapientum	Kenya	JX010142	JX010015	JX009432	JX010395	JX010084	–	JX009815	–
C. nupharicola	ICMP 18187 *	Nuphar lutea subsp. polysepala	USA	JX010187	JX009972	JX009437	JX010398	JX010088	JX145319	JX009835	–
C. pandanicola	MFLUCC 17-0571	Pandanaceae	Thailand	MG646967	MG646934	MG646938	MG646926	–	–	MG646931	–
C. proteae	CBS 132882 *	Protea sp.	South Africa	KC297079	KC297009	KC296940	KC297101	KC297032	–	KC296986	–
C. psidii	ICMP 19120 *	Psidium sp.	Italy	JX010219	JX009967	JX009515	JX010443	JX010133	KC888931	JX009901	–
C. queenslandicum	ICMP 1778 *	Carica papaya	Australia	JX010276	JX009934	JX009447	JX010414	JX010104	KC888928	JX009899	–
	CPC 17123	Syzygium australa	Australia	KP703357	KP703282	–	KP703439	KP703693	KP703778	–	–
	ICMP 18705	Coffea sp.	Fiji	JX010185	JX010036	JX009490	JX010412	JX010102	–	JX009890	–
	CMM3233	Anacardium occidentale	Brazil, Pernambuco state	–	MF110849	–	MF111058	MF110996	MF110639	–	–
	CMM3241	Anacardium occidentale	Brazil, Pernambuco state	–	MF110848	–	MF111059	MF111000	MF110642	–	–
	CMM3236	Anacardium occidentale	Brazil, Pernambuco state	–	MF110850	–	MF111060	MF110997	MF110640	–	–
	CMM3240	Anacardium occidentale	Brazil, Pernambuco state	–	MF110852	–	MF111061	MF110999	MF110644	–	–
	CMM3237	Anacardium occidentale	Brazil, Pernambuco state	–	MF110853	–	MF111062	MF110998	MF110641	–	–
	CMM3242	Anacardium occidentale	Brazil, Pernambuco state	–	–	–	MF111063	MF111001	MF110643	–	–
C. rhexiae	CBS 133134 *	Rhexia virginica	USA	JX145128	–	–	JX145179	–	–	–	–
C. salsolae	ICMP 19051 *	Salsola tragus	Hungary	JX010242	JX009916	JX009562	JX010403	JX010093	KC888925	JX009863	–
C. siamense	ICMP 18578 CBS 130417 *	Citrus arabica	Thailand	JX010171	JX009924	FJ907423	JX010404	JX010094	JQ899289	JX009865	–
	VPRI 43077; UMC004	Citrus limon	Australia, NSW	MG572140	MG572129	MK473453	MG572151	MG572162	MG572173	MW091989	–
	BRIP 54270b; VPRI 43029; A10-43029	Citrus australasica	Australia, Qld	MK469995	MK470013	MK470085	MK470049	MK470031	MK470067	MW091971	–
C. siamense (syn. C. jasmini-sambac)	CBS 130420; ICMP 19118	Jasminum sambac	Vietnam	HM131511	HM131497	HM131507	JX010415	JX010105	JQ807841	JX009895	–
C. siamense (syn. C. hymenocallidis)	CBS 125378; ICMP 18642; LC0043	Hymenocallis americana	China	JX010278	JX010019	GQ856775	JX010410	JX010100	JQ899283	GQ856730	–
C. siamense (syn. C. murrayae)	GZAAS 5.09506	Murraya sp.	China	JQ247633	JQ247609	JQ247657	JQ247644	JQ247621	–	–	–
C. syzygicola	DNCL021; MFLUCC 10-0624 *	Syzygium samarangense	Thailand	KF242094	KF242156	KF157801	KF254880	KF242125	–	–	–
C. temperatum	CBS 133122 *	Vaccinium macrocarpon	USA	JX145159	–	–	JX145211	–	–	–	–
C. theobromicola	ICMP 18649; CBS 124945 *	Theobroma cacao	Panama	JX010294	JX010006	JX009444	JX010447	JX010139	KC790726	JX009869	–
C. theobromicola (syn. C. fragariae)	ICMP 17927; CBS 142.31; MTCC 10325T	Fragaria × ananassa	USA	JX010286	JX010024	JX009516	JX010373	JX010064	JQ807844	JX009830	–
	VPRI 43083; UMC010	Citrus aurantifolia	Australia, Qld	MG572146	MG572135	MK473456	MG572157	MG572168	MG572179	MW091995	–
C. ti	ICMP 4832 *	Cordyline sp.	New Zealand	JX010269	JX009952	JX009520	JX010442	JX010123	KM360146	JX009898	–
C. tropicale	ICMP 18653; CBS 124949 *	Theobroma cacao	Panama	JX010264	JX010007	JX009489	JX010407	JX010097	KC790728	JX009870	–
C. viniferum	GZAAS 5.08601 *	Vitis vinifera, cv. ‘Shuijing’	China	JN412804	JN412798	JN412795	JN412813	JN412787	–	–	–
C. wuxiense	CGMCC 3.17894 *	Camellia sinensis	China	KU251591	KU252045	KU251672	KU252200	KU252101	KU251722	KU251939	–
C. xanthorrhoeae	BRIP 45094; ICMP 17903; CBS 127831 *	Xanthorrhoea preissii	Australia	JX010261	JX009927	JX009478	JX010448	JX010138	KC790689	JX009823	–
Colletotrichum sp.	BRIP 58074a; VPRI 43025; A06-43025	Citrus australasica	Australia, Qld	MK469999	MK470017	MK470089	MK470053	MK470035	MK470071	MW091975	–
Boninense complex
C. annellatum	CBS 129826 *	Hevea brasiliensis	Colombia	JQ005222	–	JQ005570	JQ005656	–	–	JQ005396	JQ005483
C. beeveri	CBS 128527 *	Brachyglottis repanda	New Zealand	JQ005171	–	JQ005519	JQ005605	–	–	JQ005345	JQ005432
C. boninense	ICMP 17904; CBS 123755 *	Crinum asiaticum ‘Sinicum’	Japan	JQ005153	–	JQ005501	JQ005588	–	–	JQ005327	JQ005414
C. brassicicola	CBS 101059	Brassica oleracea var. gemmifera	New Zealand	JQ005172	–	JQ005520	JQ005606	–	–	JQ005346	JQ005433
C. brasiliense	CBS 128501*	Passiflora edulis	Brazil	JQ005235	–	JQ005583	JQ005669	–	–	JQ005409	JQ005496
C. catinaense	CBS 142417; CPC 27978 *	Citrus reticulata	Italy, Catania	KY856400	–	KY855971	KY856482	–	–	KY856136	KY856307
C. citricola	CBS 134228 *	Citrus unchiu	China	KC293576	–	KC293616	KC293656	–	–	KY856140	KY856311
C. constrictum	CBS 128504	Citrus limon	New Zealand	JQ005238	–	JQ005586	JQ005672	–	–	JQ005412	KY856313
C. colombiense	CBS 129818 *	Passiflora edulis	Colombia	JQ005174	–	JQ005522	JQ005608	–	–	JQ005348	JQ005435
C. cymbidiicola	IMI 347923 *	Cymbidium sp.	Australia	JQ005166	–	JQ005514	JQ005600	–	–	JQ005340	JQ005427
C. dacrycarpi	CBS 130241 *	Dacrycarpus dacrydioides	New Zealand	JQ005236	–	JQ005584	JQ005670	–	–	JQ005410	JQ005497
C. hippeastri	CBS 125376 *	Hippeastrum vittatum	China	JQ005231	–	JQ005579	JQ005665	–	–	JQ005405	JQ005492
C. karstii	CBS 126532	Citrus sp.	South Africa	JQ005209	–	JQ005557	JQ005643	–	–	JQ005383	JQ005470
	CBS 128551	Citrus sp.	New Zealand	JQ005208	–	JQ005556	JQ005642	–	–	JQ005382	JQ005469
	CBS 129829	Gossypium hirsutum	Germany	JQ005189	–	JQ005537	JQ005623	–	–	JQ005363	JQ005450
	CPC 27853	Citrus sinensis	Italy, Catania	KY856461	–	KY856034	KY856543	–	–	KY856202	KY856377
	CPC 31139	Citrus sinensis	Italy, Catania	KY856467	–	KY856040	KY856549	–	–	KY856208	KY856383
	CBS 129833	Musa sp.	Mexico	JQ005175	–	JQ005523	JQ005609	–	–	JQ005349	JQ005436
	CBS 861.72	Bombax aquaticum	Brazil	JQ005184	–	JQ005532	JQ005618	–	–	JQ005358	JQ005445
	DAR 25017a; VPRI 42941; D02-42941	Citrus sinensis Valencia	Australia, NSW	MK470103	–	MK470109	MK470106	–	–	MK470115	MK470112
	DAR 29821a; VPRI 42943; F02-42943	Citrus sinensis Valencia	Australia, NSW	MK470104	–	MK470110	MK470107	–	–	MK470116	MK470113
	DAR 29826a; VPRI 42944; G02-42944	Citrus sinensis Valencia	Australia, NSW	MK470105	–	MK470111	MK470108	–	–	MK470117	MK470114
	VPRI 43651; UMC015	Citrus limon	Australia, Vic	MW081178	–	MW081186	MW081182	–	–	MW081190	MW081194
	VPRI 43652; UMC016	Citrus sinensis	Australia, Vic	MW081179	–	MW081187	MW081183	–	–	MW081191	MW081195
	VPRI 43653; UMC017	Citrus sinensis	Australia, Vic	MW081180	–	MW081188	MW081184	–	–	MW081192	MW081196
	VPRI 43654; UMC018	Citrus sinensis	Australia, Vic	MW081181	–	MW081189	MW081185	–	–	MW081193	MW081197
C. limonicola	CBS 142410; CPC 31141 *	Citrus limon	Malta, Gozo	KY856472	–	KY856045	KY856554	–	–	KY856213	KY856388
C. novae-zelandiae	CBS 128505 *	Capsicum annuum	New Zealand	JQ005228	–	JQ005576	JQ005662	–	–	JQ005402	JQ005489
C. oncidii	CBS 129828 *	Oncidium sp.	Germany	JQ005169	–	JQ005517	JQ005603	–	–	JQ005343	JQ005430
C. parsonsiae	CBS 128525 *	Parsonsia capsularis	New Zealand	JQ005233	–	JQ005581	JQ005667	–	–	JQ005407	JQ005494
C. petchii	CBS 378.94 *	Dracaena marginata	Italy	JQ005223	–	JQ005571	JQ005657	–	–	JQ005397	JQ005484
C. phyllanthi	CBS 175.67 *	Phyllanthus acidus	India	JQ005221	–	JQ005569	JQ005655	–	–	JQ005395	JQ005482
C. torulosum	CBS 128544 *	Solanum melongena	New Zealand	JQ005164	–	JQ005512	JQ005598	–	–	JQ005338	JQ005425
Truncatum complex
C. truncatum	CBS 151.35 *	Phaseolus lunatus	USA	GU227862	–	GU227960	GU228156	–	–	GU228352	GU228058

Vic: Victoria, NSW: New South Wales, Qld: Queensland, WA: Western Australia, SA: South Australia. * Ex-holotype or ex-epitype cultures.

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.

Table 2.

Best-fit model for each gene locus selected by MrModeltest.

Dataset	Substitution Models
	ITS	tub2	act	chs-1	his3
boninense complex	SYM + I+G	HKY + I	HKY + G	GTR + G	HKY + I
	ITS	gapdh	tub2	act	ApMat	gs	chs-1
gloeosporioides complex	SYM + I	HKY + I	SYM + I	HKY + I	HKY + G	GTR + G	K80 + G

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 ($\mathrm{percentage} (\%)=\frac{infected points}{inoculated points}\times 100\%$).

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 ($\mathrm{percentage} (\%)=\frac{infected points}{inoculated points}\times 100\%$).

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 ($\mathrm{percentage} (\%)=\frac{infected points}{inoculated points}\times 100\%$).

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).

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.

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).

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.

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).

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.

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.

Table 3.

Morphological characters of Colletotrichum species.

Taxon	Conidial Length (μm)	Conidial Width (μm)	Conidial Shape	Growth Rate (mm/day) 1
C. gloeosporioides	(10.2–) 13.8–14.3 (–16.1)	(4.2–) 5.3–5.5 (–7.3)	Subcylindrical	10.4–10.8
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)	Cylindrical	10.5–10.7
Colletotrichum sp.	(13.1–) 15.6–16 (–18.0)	(4.6–) 6.1–6.3 (–7.7)	Cylindrical	8.9–9.7
C. karstii	(11.3–) 13.2–13.6 (–14.8)	(6.4–) 7.1–7.3 (–8.3)	Cylindrical	9.4–9.6
New species	(12.7–) 14.1–14.5 (–17.2)	(3.9–) 4.5–4.7 (–5.5)	Cylindrical with one end acute	9.7–10.3
C. queenslandicum 2	(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].

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).

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.

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).

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 (I–K). Scale bars: D, 500 µm; F, G, H, I, J, K, 20 µm.

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.

Table 4.

Incidence of infection on Washington Navel orange fruit and leaves and Meyer lemon leaves by Colletotrichum species.

Culture	Fungus Species	Infection Incidence %
		Fruit Bioassay (Wound)	Leaf Bioassay		Petal Bioassay
			Orange Leaf	Lemon Leaf
VPRI 43075	C. australianum sp. nov.	95.8	0	0	100
VPRI 43076	C. gloeosporioides	0	0	0	100
VPRI 43079	C. fructicola	75	0	0	100
VPRI 43083	C. theobromicola	95.8	0	83.3	100
BRIP 58074a	Colletotrichum sp.	95.8	0	0	100
VPRI 43654	C. karstii	100	100	100	100

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.