Diaporthe taoicola and D. siamensis, Two New Records on Citrus sinensis in China.

Two Diaporthe species isolated from fruit of Citrus sinensis in China were characterized based on morphology and multilocus phylogeny of ITS, tef1, and tub2 gene sequences. The phylogeny indicated that the two species match Diaporthe taoicola and D. siamensis. A critical examination of phenotypic characteristics confirmed the phylogenetic results. Diaporthe taoicola was morphologically characterized by producing Alpha conidia with tapering toward both ends. Meanwhile, D. siamensis produced cylindrical or ellipsoidal Alpha conidia with two oil drops. Pathogenicity tests revealed that both species were pathogenic to fruit of C. sinensis. To our knowledge, the two species were firstly reported on Citrus sinensis in China.

Citrus (Rutaceae) is worldwide cultivated because of its nutritional values and the medicinal benefits (e.g., anti-hypertensive) [1]. In 2018, citrus production in Zigui county, Hubei province, the larger Citrus sinensis production area in China, has reached 23.3 thousand ha. As citrus has diversified as a commercial crop, it became a host for various pathogens from nursery to the storage stage.

Diaporthe species are present as pathogens, endophytes, or saprophytes on a wide range of cultivated or wild trees and ornamentals [2-5]. Species in this genus have been reported as the pathogens of blight, canker, decay, dieback, wilt, leaf spot, fruit rot and root rot across a diverse range of plant species [6-9]. Members of Diaporthe are also frequently associated with citrus diseases worldwide [10,11]. Diaporthe citri (anamorph=Phomopsis citri) caused melanose and stem-end rot of fruit, which are important in most citrus-growing areas with high humidity. Besides, D. foeniculina has also been found from New Zealand, Spain and USA associated with stem end rot on fruit [11]. In China, Diaporthe citri, D. eres and D. unshiuensis have been reported on fruit of Citrus spp. [12].

Preliminarily, species in Diaporthe were identified mainly based on morphological characters and host associations. However, morphology has been conferred to be inconsistent for identification due to inter- and intra-species variability [13]. Molecular analyses inferred that Diaporthe species are not highly host-specific [14]. More than one species is often present on one host, or one species may occur on more than one host [15]. Studies converging on the diversity of Diaporthe have been progressed in recent years in China. Huang et al. (2015) studied Diaporthe on Citrus in China found eight known species and seven novel species based on morphological comparison and multi-gene analyses. Moreover, Diaporthe associated with peach trees [16], pear shoot canker [5], and dieback diseases involving 16 host genera [17] were reported in China. These references provided bounteous information for the study of Diaporthe in China.

During the investigation of fungal pathogens associated with Citrus sinensis in Zigui county, Diaporthe isolates were encountered based on morphology. Three Diaporthe-like isolates, YZU 181047, YZU 181403, and YZU 181223 were found pathogenic to fruit of C. sinenesis. The main objectives of this study were to identify them based on morphological observations and sequence analyses of multiple gene regions.

In 2018, diseased citrus fruit was collected from commercial orchards in Zigui county. Tissues from the margin of infected lesions were cut into segments, which contained both diseased and healthy parts. All segments were surface sterilized in 2% sodium hypochlorite for 2 min, followed by 75% ethanol for 30 s and rinsed in sterile distilled water for three times. All samples were dried with sterile filter paper and plated onto potato dextrose agar (PDA, Difco, USA). Plates were incubated at 25 °C in darkness until mycelia grow. Then, mycelia from colony margin were taken and transferred on fresh PDA plates. Pure cultures were stored in the Fungi Herbarium of Yangtze University (YZU) in Jingzhou, China.

Genomic DNA was extracted from mycelium developed on PDA medium according to Cenis [18]. The primers ITS4 and ITS5 [19] was used to amplify the ITS region of the nuclear ribosomal RNA operon, including the 3′ end of the 18S rRNA, the first internal transcribed spacer region, the 5.8S rRNA gene; the second internal transcribed spacer region and the 5′ end of the 28S rRNA gene. The primers EF1-728F and EF1-986R [20] were used to amplify part of the translation elongation factor 1-α (tef1) gene, and the primers Bt2a and Bt2b [21] were used to amplify the partial beta-tublin (tub2) gene. The PCRs were performed in a 25 μL reaction mixture consisted of 12.5 μL of 2 × Taq PCR StarMix (Genstar, Beijing, China), 2 μL genomic DNA, 1.25 μL of each primer, and 8 μL distilled water (ddH2O). The thermal cycling program was completed on a thermal cycler using the following conditions: initial denaturation at 94 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at (52 °C for ITS, and 56 °C for tef1, and 60 °C for tub2) for 30 s, extension at 72 °C for 60 s, with a final extension step at 72 °C for 5 min. Successful PCR amplification products were purified and sequenced at BGI (Beijing Genomics Institute).

All the obtained sequences were analyzed in the basic-local-alignment search tool (BLASTn) (http://blast.ncbi.nlm.nih.gov/) to retrieve the most similar taxa sequences. Relevant sequences were selected from the studies of Gomes et al. [3], Dissanayake et al. [16], Yang et al. [17], and Tibpromma et al. [22]. All sequences were aligned and combined in the MEGA 7.0 program [23]. Maximum Parsimony (MP) analysis was performed in PAUP version 4.0 b10 [24], generating a heuristic search option of 1000 random-addition replicates and a tree bisection-reconnection (TBR) as a branch-swapping algorithm. MaxTrees were set to 1000, branches of zero length collapsed, and all equally parsimonious trees were saved. Other scores in parsimony were calculated as tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC). The maximum likelihood (ML) [25] phylogeny of the combined dataset was constructed with 1000 bootstrap replicates using GTRGAMMAI model. Additionally, Bayesian (BI) analysis was conducted in MrBayes v. 3.2.6 with 1,000,000 Markov chain Monte Carlo (MCMC) generations and a sampling frequency of every 100th generations. The best-fit evolutionary model was determined via MrModelTest v. 2.3 [26]. At the end of the analysis, the first 25% of the samples were excluded as burn-in, and consensus trees were generated using the 50% majority-rule consensus tree criteria. The tree was viewed in Figtree v.1.3.1 [27]. Posterior probability (PP) values of BI analysis and bootstrap (BP) values of ML and MP analyses were shown at the nodes of branches. The out-group of the phylogeny was Diaporthella corylina CBS 121124.

Three isolates were characterized for their colonial and conidial morphology. Agar plugs (6 mm diam.) of each isolate were taken from the edge of actively growing cultures and transferred onto the center of petri dishes (9 cm diam), containing potato dextrose agar (PDA) and oatmeal agar (OA) for cultural feature. Plates containing 2% water agar (WA) with autoclaved Citrus sinensis leave tissues were incubated at 25 °C under a 12-h near-ultraviolet light/12-h dark cycle to induce sporulation. The culture was checked periodically for the development of ascomata and conidiomata. Morphology was recorded including colony color, texture, microconidia, and sporocarps formation. Conidia were mounted in sterile water for microscopic observation using a light microscope (Nikon DS-Ri2, Tokyo, Japan), equipped with a Nikon DS-Ri2 digital camera. Conidia (n = 50) were measured for each species.

Pathogenicity tests were performed on fruit of Citrus sinensis. Mature and healthy fruit were surface-sterilized in 2% sodium hypochlorite for 2 min and washed three times with sterile distilled water. Fruit was wounded with a sterile scalpel around 6 mm × 6 mm in size. The mycelial plugs from 3 days old cultures grown on PDA were transferred onto the wounds. Controls were treated with sterile PDA. The inoculated fruits were maintained at 25 °C and 80 to 100% relative humidity (RH). The development of disease symptoms was checked daily for one week. The pathogen was re-isolated from the inoculated fruit and identified based on morphology to satisfy Koch's postulates. The pathogenicity tests were conducted with three replicates for each isolate and repeated three times.

A total of nine new sequences were generated and deposited in GenBank (Table 1). The combined multi-gene phylogeny (ITS, tef1, and tub2) contained 30 strains, of which 27 were obtained from NCBI (https://www.ncbi.nlm.nih.gov/) (Table 1, Figure 1). A total of 1276 characters (ITS 494, tef1 377, tub2 405) were included after alignment. Among them, 804 were constant, 190 were variable, and 282 were parsimony uninformative. The heuristic search generated 5 parsimonious trees (TL = 908, CI = 0.664, HI = 0.336, RI = 0.819, RC = 0.544). For the BI analysis, the HKY + I model was recommended by MrModeltest. The topology of ML phylogeny was identical to the results of BI and MP analyses, and it was used as a basal tree.

Table 1.

Isolates and GenBank accession numbers used in the phylogenetic analyses of Diaporthe.

Species	Strain	Host/Locality	GenBank accession number
			ITS	tef1	tub2
Diaporthe arecae	CBS 161.64	Areca catechu/India	KC343032	KC343758	KC344000
Diaporthe arengae	CBS 114979	Arenga engleri/China, HongKong	KC343034	KC343760	KC344002
Diaporthe batatas	CBS 122.21	Ipomoea batatas/USA	KC343040	KC343766	KC344008
Diaporthe citri	CFCC 53079	Citrus sp./China	MK573940	MK574615	MK574635
	CFCC 53080	Citrus sp./Brazil	MK573941	MK574616	MK574636
Diaporthe endophytica	CBS 133811	Schinus terebinthifolius	KC343065	KC343791	KC344033
Diaporthe eugeniae	CBS 444.82	Eugenia aromatica/Indonesia	KC343098	KC343824	KC344066
Diaporthe oxe	CBS 133186	Maytenus ilicifolia/Brazil	KC343164	KC343890	KC344132
Diaporthe perseae	CBS 151.73	Persea americana/Netherlands	KC343173	KC343899	KC344141
Diaporthe pseudomangiferae	CBS 101339	Mangifera indica/Dominican Republic	KC343181	KC343907	KC344149
Diaporthe pseudophoenicicola	CBS 462.69	Phoenix dactylifera/Spain	KC343184	KC343910	KC344152
Diaporthe siamensis	MFLUCC 10-0573a	Dasymaschalon sp./Thailand	JQ619879	JX275393	JX275429
	MFLUCC 10-0573 b	Dasymaschalon sp./Thailand	JQ619880	JX275395	JX275430
	MFLUCC 10-0573c	Dasymaschalon sp./Thailand	JQ619881	JX275396	JX275431
	MFLUCC 17-0591	Pandanaceae/Thailand	MT908796	MG646989	MG646925
	YZU 181403	Citrus sinensis/China	MW160357	MW160363	MW160360
Diaporthe sojae	FAU 635	Glycine max/USA	KJ590719	KJ590762	KJ610875
Diaporthe taoicola	PSCG 292	Pyrus pyrifolia/China	MK626871	MK654800	MK691232
	PSCG 386	Pyrus pyrifolia/China	MK626868	MK654797	MK691222
	PSCG 413	Pyrus pyrifolia/China	MK626890	MK654814	MK691238
	PSCG 485	Pyrus pyrifolia/China	MK626869	MK654812	MK691238
	MFLUCC 16-0117	Prunus persica/China	KU557567	KU557635	KU557591
	MFLUCC 16-0118	Prunus persica/China	KU557568	KU557636	KU557592
	MFLUCC 16-0119	Prunus persica/China	KU557569	KU557637	KU557593
	MFLUCC 16-0120	Prunus persica/China	KU557570	KU557638	KU557594
	YZU 181047	Citrus sinensis/China	MW160355	MW160361	MW160358
	YZU 181223	Ctrus sinensis/China	MW160356	MW160362	MW160359
Diaporthe yunnanensis	LC 6168	Coffea sp.	KX986796	KX999188	-
	SAUCC 0254	Unknown/China	MT376663	MT376663	MT376634
Diaporthella corylina	CBS 121124	Corylus sp./China	KC343004	KC343730	KC343972

The present strains are shown in bold.

Figure 1.

Phylogram of Diaporthe strains based on combined gene sequences of ITS, tef1 and tub2. Values at the branch nodes indicated maximum parsimony bootstrap (MP BP ≥ 60%), Bayesian posterior (BI PP ≥ 0.6) and maximum likelihood bootstrap (ML BP ≥ 60%), respectively. The tree is rooted with Diaporthella corylina. Strains in the current study are in bold.

The phylogenetic tree showed that isolates YZU 181047 and YZU 181223 fell into a clade containing reference strains of Diaporthe taoicola supported with PP values of 0.93 and clustered together with D. taoicola PSCG 485 [5] with the BP or PP values of 94/1.0/95 (MP/BI/ML). The result indicated that both isolates were D. taoicola. However, the isolate YZU 181403 clustered with reference strains of D. siamensis with high BP or PP values of 87/1.0/95 (MP/BI/ML). The result showed that it was D. siamensis.

Morphological examination confirmed the phylogenetic results, the isolates YZU 181047 and YZU 181223 were identified as Diaporthe taoicola [5,16], and the isolate YZU 181403 was D. siamensis [22,28] based on the colony and conidia characteristics.

Sexual morph: Not observed. Asexual morph: Conidiomata 160–230 μm in size, pycnidial, subcuticular, scattered to confluent, dark brown to black, uniloculate, broadly spherical to flattened, cream conidial droplets exuding from central ostioles (Figure 2(d–f)). Conidiophores 16–28 × 2–3 μm, hyaline, smooth, densely aggregated, cylindrical, straight, or slightly curved, tapering toward the apex (Figure 2(g)). Alpha conidia 6–9 × 2–3 μm (av. 8 × 2.7 μm) hyaline, smooth, fusiform to ellipsoid, tapering toward both ends, straight (Figure 2(h)).

Figure 2.

Diaporthe taoicola (YZU 181047). (a, b). Front and back view, respectively of colonies on PDA (a) and OA (b); (c). Pathogenicity test on Citrus sinensis fruit for 7 d; (d, e). Conidiomata; (f): Section view of conidiomata; (g). Conidiophores; (h): Alpha conidia. Scale bars: d, e, f = 100μm; g, h: 10 μm.

Colony morphology: Colonies on PDA covering the entire Petri dishes after 7 days, ropey with abundant tufted white aerial mycelium, reverse buff with zonate and irregular lines (Figure 2(a)), 79–81 mm in diam., with aerial mycelium dense in the center and sparse at the marginal area. Colonies on OA flat with white felty aerial mycelium, turning white to dark brown aerial mycelium, conidiomata irregularly distributed on the medium surface after 15-day incubation (Figure 2(b)).

Materials examined. China, Hubei province, Zigui county, on fruit of Citrus sinensis, August, 2018, M. J. Cui (cultures YZU 181047 and YZU 181223).

Notes: The size and shape of Alpha conidia from the present isolates were identical to that firstly reported by Dissanayake et al. (7–9 × 2–3 μm) [16]. However, Beta conidia were not observed during the present study, which was same as the strains found on pear shoot canker by Guo et al. [5].

Sexual morph: Not observed. Asexual morph: Conidiomata 130–240 μm wide, 94–200 μm high, solitary, single conical neck erumpent through leave tissues, 80–160 × 54–85 μm in size (Figure 3(d–f)). Conidiophores 11–23 × 1–2.5 μm, cylindrical, hyaline, straight, or curved, tapering toward the apex (Figure 3(g)). Alpha conidia 6–8 × 3–3.5 μm (av. 7 × 3.2 μm) hyaline, aseptate, ellipsoidal to oval, biguttulate, rounded at both ends (Figure 3(h)).

Figure 3.

Diaporthe siamensis (YZU 181403). (a, b). Front and back view, respectively of colonies on PDA (a) and OA (b); (c). Pathogenicity test on Citrus sinensis fruit for 7 d; (d, e). Conidiomata; (f): Section view of conidiomata; (g). Conidiophores; (h): Alpha conidia. Scale bars: d, e, f = 100μm; g, h: 10 μm.

Colony morphology: Colonies on PDA mycelia growing full of Petri-dishes after 7 d with zones of the dirty white and umber, reverse umber patches (Figure 3(a)). Colonies on OA flat with white felty aerial mycelium, turning white to reddish-brown, with irregular black zones (Figure 3(b)).

Materials examined. China, Hubei province, Zigui county, on fruit of Citrus sinensis, August, 2018, M. J. Cui (culture YZU 181403).

Note: Alpha conidia of the present isolate were identical to that firstly reported by Udayanga et al. with (3.5–)4–5(–6) × (2–)2.5(–3) μm in size, collected from diseased leaves of Dasymaschalon sp. (Annonaceae) [28]. Besides, its cultural characteristics on PDA were identical to D. siamensis reported by Tibpromma et al., as an endophytic fungus from a Pandanaceae host (Pandanus sp.) [22]. Unfortunately, Beta conidia and Gamma conidia were not observed in the present study.

In the pathogenicity tests, all isolates caused brown fruit rot (Figures 2(c), 3(c)) on Citrus sinensis, exposing mycelia on surface and severe rotting inside. The initial symptoms appeared as tiny, watery lesions, which gradually expanded eventually led to fruit rot on 7th day. However, the diameters of the lesions varied among different species; D. siamensis caused larger lesions (33–37 mm, av. 34 mm) than D. taoicola (26–30 mm, av. 28 mm) during the tests. In parallel, no lesions developed on the fruit that were inoculated with PDA disks as control. These results showed that all the present isolates were responsible agents for fruit rot of Citrus sinensis.

Presently, the identification of Diaporthe is mainly based on morphological characters and phylogenetic analysis [3,28]. In recent reported studies, nearly 65 Diaporthe species were associated with Chinese hosts, from which 15 were founded on Citrus spp. (Table. 2). According to Huang et al. [15] and Li et al. [36], phylogeny inferred from combined gene loci of ITS, tef1, and tub2 could be used for further identification of Diaporthe species.

Table 2.

Diaporthe species isolated from various hosts in China.

Species	Authority	Host	Locality (Province)	Reference
Diaporthe acerigena	C.M. Tian & Q. Yang,	Acer tataricum	Shaanxi	Yang et al. [17]
D. acuta	Y.S. Guo & G.P. Wang	Pyrus pyrifolia	Hubei	Guo et al. [5]
D. acutispora	Y.H. Gao & L. Cai	Coffea sp.	Yunnan	Gao et al. [4]
D. alangii	C.M. Tian & Q. Yang	Alangium kurzii	Zhejiang	Yang et al. [17]
D. amygdali	Udayanga, Crous & K.D. Hyde	Pyrus pyrifolia	Jiangxi, Yunnan	Bai et al. [30]
		Camellia sp.	Sichuan	Gao et al. [4]
D. apiculata	Y.H. Gao & L. Cai	Camellia sp.	Jiangxi, Guangxi	Gao et al. [4]
D. aquatica	D.M. Hu, L. Cai & K.D. Hyde	aquatic habitats	Guizhou	Hu et al. [31]
D. betulae	C.M. Tian & X.L. Fan,	Betula platyphylla	Sichuan	Du et al. [8]
D. betulicola	C.M. Tian & Z. Du,	Betula albo-sinensis	Shaanxi	Du et al. [8]
D. betulina	C.M. Tian & Q. Yang	Betula sp.	Heilongjiang	Yang et al. [17]
D. biconispora	F. Huang, K.D. Hyde & H.Y. Li	Citrus sinensis	Jiangxi, Guangxi, Fujian	Huang et al. [12]
D. biguttulata	F. Huang, K.D. Hyde & H.Y. Li	Citrus limon	Yunnan	Huang et al. [12]
		Juglans regia	Zhejiang	Yang et al. [17]
D. biguttusis	Y.H. Gao & L. Cai	Lithocarpus glabra	Zhejiang	Gao et al. [4]
D. camptothecicola	C.M. Tian & Qin Yang	Camptotheca acuminata	Jiangsu	Yang et al. [32]
D. caryae	C.M. Tian & Q. Yang	Carya illinoensis	Jiangsu	Yang et al. [32]
		Pyrus pyrifolia	Jiangsu	Guo et al. [5]
D. cercidis	C.M. Tian & Q. Yang	Carya illinoensis	Jiangsu	Yang et al. [17]
		Pyrus pyrifolia	Shandong, Zhejiang, et al.	Guo et al. [5]
D. chensiensis	C.M. Tian & Q. Yang	Abies chensiensis	Shaanxi	Yang et al. [17]
D. chongqingensis	Y.S. Guo & G.P. Wang	Pyrus pyrifolia	Chongqing	Guo et al. [5]
D. cinnamomi	C.M. Tian & Q. Yang	Cinnamomum sp.	Zhejiang	Yang et al. [17]
D. citri	F.A. Wolf	Citrus sp.	Zhejiang, Huangyan, Jiangxi	Huang et al. [15]
D. citriasiana	F. Huang, K.D. Hyde & H.Y. Li	Citrus sp.	Shaanxi, Jiangxi, Zhejiang	Huang et al. [15]
D. citrichinensis	F. Huang, K.D. Hyde & H.Y. Li	Citrus sp.	Shaanxi, Guangxi, Fujian	Huang et al. et al. [15]
D. compacta	Y.H. Gao & L. Cai	Camellia sp.	Jiangxi	Gao et al. [4]
D. conica	C.M. Tian & Q. Yang	Alangium chinense	Zhejiang	Yang et al. [17]
D. discoidispora	F. Huang, K.D. Hyde & H.Y. Li	Citrus sp.	Jiangxi	Huang et al. [12]
D. elaeagni-glabrae	Y.H. Gao & L. Cai	Elaeagnus glabra	Jiangxi	Gao et al. [29]
D. ellipicola	Y. H. Gao & L. Cai	Lithocarpus glabra	Zhejiang	Gao et al. [4]
D. endophytica	R.R. Gomes, C. Glienke & Crous	Citrus sp.	Fujian	Huang et al. [12]
D. eres	Nitschke	Aralia elata	northeastern China	Bai et al. [30]
		Citrus sp.	Guangxi, Jiangxi, Zhejiang	Huang et al. [12]
		Vitis vinifera	Beijing, Zhejiang	Dissanayake et al. [16]
		Juglans regia	Zhejiang	Yang et al. [17]
		Camellia sp.	Sichuan	Gao et al. [29]
D. fraxinicola	C.M. Tian & Q. Yang,	Fraxinus chinensis	Shaanxi	Yang et al. [17]
D. fulvicolor	Y.S. Guo & G.P. Wang	Pyrus pyrifolia	Hubei	Guo et al. [5]
D. fusicola	Y.H. Gao & L. Cai	Pyrus pyrifolia	Jiangxi, Fujian, Zhejiang	Guo et al. [5]
D. ganjae	R.R. Gomes	Pyrus pyrifolia	Guizhou	Guo et al. [5]
D. hongkongensis	R.R. Gomes, C. Glienke & Crous	Citrus sp.	Zhejiang, Guangxi	Huang et al. [12]
		Vitis vinifera	Beijing	Dissanayake et al. [16]
		Camellia sp.	Guangxi	Gao et al. [4]
D. incompleta	Y.H. Gao & L. Cai	Camellia sinensis	Yunnan	Gao et al. [4]
D. juglandicola	C.M. Tian & Q. Yang	Juglans mandshurica	Beijing	Yang et al. [32]
D. kadsurae	C.M. Tian & Q. Yang	Kadsura longipedunculata	Jiangxi	Yang et al. [17]
D. lithocarpus	Y.H. Gao, W. Sun & L. Cai	Lithocarpus sp.	Zhejiang	Gao et al. [4]
D. longicolla	(Hobbs) J.M. Santos, Vrandečić & A.J.L. Phillips	Pyrus pyrifolia	Jiangxi, Fujian, Hubei	Bai et al. [30]
D. mahothocarpus	Y.H. Gao, W. Sun & L. Cai	Lithocarpus sp.	Zhejiang	Gao et al. [4]
D. multigutullata	F. Huang, K.D. Hyde & H.Y. Li	Citrus sp.	Fujian	Huang et al. [12]
D. neotheicola	A.J.L. Phillips & J.M. Santos	Pyrus bretschneideri	Yunnan, Jiangxi, Fujian	Bai et al. [30]
D. oraccinii	Y.H. Gao & L. Cai	Camellia sp.	Jiangxi	Gao et al. [29]
D. ovalispora	F. Huang, K.D. Hyde & H.Y. Li	Citrus sp.	Yunnan	Huang et al. [12]
D. ovoicicola	Y. H. Gao & L. Cai	Citrus sp.	Zhejiang	Gao et al. [4]
D. padina	C.M. Tian & Q. Yang	Padus racemosa	Jiangxi	Yang et al. [17]
D. parvae	Y.S. Guo & G.P. Wang	Pyrus pyrifolia	Yunnan	Guo et al. [5]
D. pentriteum	Y.H. Gao & L. Cai	Camellia sp.	Jiangxi	Gao et al. [29]
D. pescicola	Dissanayake et al.	Pyrus bretschneideri	Yunnan	Guo et al. [5]
D. phaseolorum	(Cooke & Ellis) Sacc.	Vitis vinifera	Beijing	Huang et al. [12]
D. phragmitis	Crous	Phragmitis australis	Beijing	Crous et al. [33]
D. podocarpi-macrophylli	Y.H. Gao & L. Cai	Podocarpus macrophyllus	Zhejiang	Gao et al. [29]
D. rostrata	C.M. Tian, X.L. Fan & K.D. Hyde	Juglans mandshurica	Gansu	Fan et al. [34]
D. sambucusii	C.M. Tian & Q. Yang	Sambucus williamsii	Heilongjiang	Yang et al. [35]
D. schisandrae	C.M. Tian & Q. Yang	Schisandra chinensis	Heilongjiang	Yang et al. [35]
D. sojae	Lehman	Vitis vinifera	Beijing	Huang et al. [12]
		Citrus sp.	Shaanxi	Huang et al. [12]
		Pyrus pyrifolia	Guizhou, Jiangsu	Guo et al. [5]
D. spinosa	Y.S. Guo & G.P. Wang	Pyrus pyrifolia	Zhejiang, Jiangsu, Guizhou	Guo et al. [5]
D. subclavata	F. Huang, K.D. Hyde & H.Y. Li	Citrus sp.	Fujian, Guangdong	Huang et al. [12]
D. taoicola	Dissanayake, X.H. Li & K. D	Prunus persica	Hubei	Dissanayake et al. [16]
		Pyrus pyrifolia	Zhejiang, Jiangsu, Guizhou	Guo et al. [5]
D. ternstroemia	Y.H. Gao, W. Sun & L. Cai	Ternstroemia sp.	Zhejiang	Gao et al. [4]
D. unshiuensis	F. Huang, K.D. Hyde & H.Y. Li	Citrus sp.	Guangxi	Huang et al. [12]
		Carya illinoensis	Jiangsu	Yang et al. [17]
D. velutina	Y.H. Gao & L. Cai	Pyrus pyrifolia	Fujian, Guizhou	Guo et al. [5]
D. xishuangbanica	Y.H. Gao & L. Cai	Camellia sinensis	Yunnan	Gao et al. [29]
D. yunnanensis	Y.H. Gao & L. Cai	Coffea sp.	Yunnan	Gao et al. [29]
D. zaobaisu	Y.S. Guo & G.P. Wang	Pyrus bretschneideri	Yunnan	Guo et al. [5]

Diaporthe taoicola was firstly isolated from diseased shoots of Prunus persica in Hubei province, China, 2017, proved being able to cause necrotic lesions on detached peach shoots [16]. From then on, it had been only reported on Pyrus pyrifolia causing shoot canker symptoms in China with high phylogenetic diversity [5]. Guo et al. [5] inoculated D. taoicola from pear shoots on wounded twigs of different fruit crops to evaluate its host range, which could induce symptoms on citrus, apple, peach, and kiwifruit. It is worth noting that D. taoicola might pose threats to fruit trees in China. The present study firstly confirmed that it also pathogenic to Citrus sinensis fruit.

Diaporthe siamensis had been reported on diseased leaves of Dasymaschalon sp. in the family of Annonaceae in Thailand [28], but without pathogenicity test on the host plant. Then, it was also found as an endophytic fungus from Pandanus sp. (Pandanaceae) in Thailand [22], also probably as endophyte on Garcinia parvifolia from Malaysia [28]. Regretfully, the pathogenicity evaluation of the species remained a lack in previous studies. Except a detailed description of D. siamensis given in this study, the pathogenicity tests revealed that it could induce fruit rot on Citrus sinensis, stronger than D. taoicola. To the best of our knowledge, this is the first report of Diaporthe taoicola and D. siamensis from Citrus sinensis in China, which could induce fruit rot on the host.