Species of Dendrostoma (Erythrogloeaceae, Diaporthales) associated with chestnut and oak canker diseases in China.

Dendrostoma was recently proposed in Erythrogloeaceae (Diaporthales, Sordariomycetes), with all known members recorded as being plant pathogenic on economically important tree hosts. During our collections of Dendrostoma species in China, mild to severe canker symptoms were observed on sweet chestnut (Castaneamollissima) and oak (Quercus spp.) trees. Dead and dying plant tissues exhibiting Dendrostoma canker symptoms were sampled for fungal isolation. A total of 37 Dendrostoma isolates were obtained and analysed using morphological characteristics and molecular data (ITS, LSU, RPB2, TEF1-α). Based on these data, 10 novel clades could be distinguished, which also proved to represent morphologically distinct species described here as Dendrostomaaurorae, D.castaneae, D.castaneicola, D.chinense, D.dispersum, D. parasiticum, D.qinlingense, D.quercus, D.shaanxiense and D.shandongense spp. nov. A key to species of the genus is also provided.

Introduction

The family was established to accommodate , , and , which exhibit epiphyllous acervuli along with subcylindrical to ampulliform conidiogenous cells and aseptate conidia (Senanayake et al. 2017). (Petrak 1953) is the type genus of and causes severe anthracnose on in South America (Ferreira et al. 1992). was first proposed in , being associated with leaf spots on spp. in Australia (Crous et al. 2012a), but was subsequently transferred to , based on DNA sequence data (Senanayake et al. 2017). was introduced with two initial species, and , discovered on diseased leaves in Australia (Crous et al. 2012b). Two additional species, and , were subsequently reported as foliar pathogens of (Crous et al. 2016, 2017).

(, ) was recently introduced as a phytopathogenic fungal genus causing canker diseases on several economic hardwoods such as , and (Fan et al. 2018). Subsequently, on trees was transferred from based on ITS and LSU sequences analysis (Senanayake et al. 2018). represents one of four genera in the family, but is the only one known to have a sexual morph. Hence, can be distinguished from the other diaporthalean families by multiguttulate and bicellular ascospores that are constricted at the septum and acervular conidiomata, with subcylindrical to ampulliform conidiogenous cells and hyaline to olivaceous, aseptate conidia (Rossman et al. 2007, Voglmayr and Jaklitsch 2014, Senanayake et al. 2017, Voglmayr et al. 2017, Fan et al. 2018).

The , including , , and , represent a family of fungal pathogens occurring on several commercially important tree genera such as , , , , and in Australia, Brazil, China and Costa Rica (Petrak 1953, Ferreira et al. 1992, Crous et al. 2012a, b, 2016, 2017, Fan et al. 2018). Considering the importance of these tree diseases and the lack of taxonomic information on , we conducted several surveys for members of the genus in China.

The aims of present study were (i) to describe the important spp. associated with canker diseases on chestnut and oak trees in China and (ii) to provide a multi-gene phylogeny for the genus based on a large set of freshly collected specimens in China. In agreement with previous taxonomic studies in , where different spp. were discovered on (Crous et al. 2012a, b, 2016, 2017), several spp. were found on ( and ), being associated with mild to severe canker diseases. The species were subsequently classified based on morphological characteristics and phylogenetic data.

Materials and methods

Sample collections and fungal isolates

Surveys for species were conducted in plantations, nurseries, parks, gardens, on mountains and natural reserves in Beijing, Hebei, Shaanxi, Shandong, Tianjin and Zhejiang Provinces in China from 2017 to 2018. Typical canker symptoms were observed on stems, branches and twigs of different hosts, including , , , and other species (Fig. 1). Diseased samples were collected and placed in paper bags, then transferred to the laboratory for further study.

Figure 1.

Chestnut plantations and canker symptoms. A A chestnut plantation on the mountain B A chestnut plantation on the plain C Collection of the dead trees killed by pathogens D–H canker symptoms on host branches.

A total of 37 isolates were established by removing a mucoid spore mass from sporulating ascomata and conidiomata produced on diseased bark, spreading the suspension on the surface of potato dextrose agar (PDA) plates and incubating the plates at 25 °C in the dark for up to 24 h. Single germinating spores were then transferred to clean plates under a dissecting microscope with a sterile needle. Specimens and isolates were deposited in the Museum of Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis

The identification of spp. was based on morphological features observed on the natural substrates. Cross-sections for ascomata and conidiomata from tree barks were prepared by hand using a double-edged blade under a dissecting microscope. At least 10 conidiomata/ascomata, 10 asci and 50 conidia/ascospores were measured to calculate the mean size and standard deviation. Measurements are reported as maxima and minima in parentheses and the range representing the mean plus and minus the standard deviation of the number of measurements is given in parentheses (Voglmayr et al. 2017). Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004). Cultural characteristics were recorded for isolates incubated on PDA in the dark at 25 °C.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from fungal colonies growing on PDA using a modified cetyl trimethyl ammonium bromide method (CTAB; Doyle and Doyle 1990, Zhang et al. 2010). The ITS region was amplified using the primers ITS1 and ITS4 (White et al. 1990), the LSU region with the primers LR0R and LR5 (Vilgalys and Hester 1990), the RPB2 region with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999) and the partial TEF1-α gene with the primers EF1-728F and EF1-986R (Carbone and Kohn 1999). The PCR mixture for all regions consisted of 1 μl genomic DNA, 3 mM MgCl2, 20 μM of each dNTP, 0.2 μM of each primer and 0.25 U rTAQ DNA polymerase (TaKaRa, Shiga). Amplification of LSU and ITS were accomplished by an initial step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 51 °C and 40 s at 72 °C, with a final extension of 10 min at 72 °C. For TEF1-α amplification, the 35 cycles consisted of initiation at 95 °C for 8 min, denaturation at 95 °C for 15 s, annealing at 55 °C for 20 s, elongation at 72 °C for 1 min and a final extension of 5 min at 72 °C. For RPB2, amplification of 35 cycles consisted of initiation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 52 °C for 1 min, elongation at 72 °C for 1 min and a final extension of 10 min at 72 °C. The DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with BigDye Terminater Kit v. 3.1 (Invitrogen, Carlsbad) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing).

Phylogenetic analyses

Sequences generated from the above primers of the different genomic regions (ITS, LSU, TEF1-α and RPB2) were analysed in comparison with those of (CFCC 52102), (CBS 187.37), (CFCC 52106, CFCC 52107, CFCC 52108 and CFCC 52109) and (CFCC 52103, CFCC 52104 and CFCC 52105) from Fan et al. (2018) and Senanayake et al. (2018). (CBS 132528), (CBS 132183) and (CBS 132184) were selected as the outgroup taxa (Crous et al. 2012a, b). All sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and edited manually using MEGA v. 6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v. 4.0b10 for maximum parsimony (MP) analysis (Swofford 2003) and PhyML v. 3.0 for Maximum Likelihood (ML) analysis (Guindon et al. 2010). The first analyses were performed on the combined multi-gene dataset (ITS, LSU, TEF1-α and RPB2) to compare isolates of species to ex-type sequence data from recent studies (Table 1).

Table 1.

Isolates and GenBank accession numbers used in the phylogenetic analyses.

Species	Culture	Location	Host	Host family	GenBank accession numbers
					ITS	LSU	TEF1−a	RPB2
Chrysocrypta corymbiae	CBS 132528*	Australia	Corymbia sp.	Myrtaceae	JX069867	JX069851	MH545457	MH545415
Dendrostoma aurorae	CFCC 52753*	China	Castanea mollissima	Fagaceae	MH542498	MH542646	MH545447	MH545405
	CFCC 52754	China	Castanea mollissima	Fagaceae	MH542499	MH542647	MH545448	MH545406
Dendrostoma castaneae	CFCC 52745*	China	Castanea mollissima	Fagaceae	MH542488	MH542644	MH545437	MH545395
	CFCC 52746	China	Castanea mollissima	Fagaceae	MH542489	NA	MH545438	MH545396
	CFCC 52747	China	Castanea mollissima	Fagaceae	MH542490	NA	MH545439	MH545397
	CFCC 52748	China	Castanea mollissima	Fagaceae	MH542491	NA	MH545440	MH545398
	CFCC 52749	China	Castanea mollissima	Fagaceae	MH542492	MH542645	MH545441	MH545399
	CFCC 52750	China	Castanea mollissima	Fagaceae	MH542493	NA	MH545442	MH545400
	CFCC 52751	China	Castanea mollissima	Fagaceae	MH542494	NA	MH545443	MH545401
	CFCC 52752	China	Castanea mollissima	Fagaceae	MH542495	NA	MH545444	MH545402
Dendrostoma castaneicola	CFCC 52743*	China	Castanea mollissima	Fagaceae	MH542496	NA	MH545445	MH545403
	CFCC 52744	China	Castanea mollissima	Fagaceae	MH542497	NA	MH545446	MH545404
Dendrostoma chinense	CFCC 52755*	China	Castanea mollissima	Fagaceae	MH542500	MH542648	MH545449	MH545407
	CFCC 52756	China	Castanea mollissima	Fagaceae	MH542501	MH542649	MH545450	MH545408
	CFCC 52757	China	Castanea mollissima	Fagaceae	MH542502	MH542650	MH545451	MH545409
	CFCC 52758	China	Castanea mollissima	Fagaceae	MH542503	MH542651	MH545452	MH545410
Dendrostoma dispersum	CFCC 52730*	China	Quercus sp.	Fagaceae	MH542467	MH542629	MH545416	MH545374
	CFCC 52731	China	Quercus sp.	Fagaceae	MH542468	MH542630	MH545417	MH545375
Dendrostoma leiphaemia	CBS 187.37	NA	Quercus sp.	Fagaceae	MH855882	MH867393	NA	NA
Dendrostoma mali	CFCC 52102*	China	Malus spectabilis	Rosaceae	MG682072	MG682012	MG682032	MG682052
Dendrostoma osmanthi	CFCC 52106*	China	Osmanthus fragrans	Oleaceae	MG682073	MG682013	MG682033	MG682053
	CFCC 52108	China	Osmanthus fragrans	Oleaceae	MG682074	MG682014	MG682034	MG682054
	CFCC 52107	China	Osmanthus fragrans	Oleaceae	MG682075	MG682015	MG682035	MG682055
	CFCC 52109	China	Osmanthus fragrans	Oleaceae	MG682076	MG682016	MG682036	MG682056
Dendrostoma parasiticum	CFCC 52761	China	Castanea mollissima	Fagaceae	MH542480	MH542636	MH545429	MH545387
	CFCC 52763	China	Castanea mollissima	Fagaceae	MH542481	MH542637	MH545430	MH545388
	CFCC 52762*	China	Quercus wutaishanica	Fagaceae	MH542482	MH542638	MH545431	MH545389
	CFCC 52764	China	Quercus aliena	Fagaceae	MH542483	MH542639	MH545432	MH545390
	CFCC 52765	China	Castanea mollissima	Fagaceae	MH542484	MH542640	MH545433	MH545391
	CFCC 52766	China	Quercus aliena var. acuteserrata	Fagaceae	MH542485	MH542641	MH545434	MH545392
Dendrostoma qinlingense	CFCC 52732*	China	Quercus wutaishanica	Fagaceae	MH542471	MH542633	MH545420	MH545378
	CFCC 52733	China	Quercus aliena var. acuteserrata	Fagaceae	MH542472	MH542634	MH545421	MH545379
Dendrostoma quercinum	CFCC 52103*	China	Quercus acutissima	Fagaceae	MG682077	MG682017	MG682037	MG682057
	CFCC 52104	China	Quercus acutissima	Fagaceae	MG682078	MG682018	MG682038	MG682058
	CFCC 52105	China	Quercus acutissima	Fagaceae	MG682079	MG682019	MG682039	MG682059
Dendrostoma quercus	CFCC 52734	China	Quercus sp.	Fagaceae	MH542473	NA	MH545422	MH545380
	CFCC 52735	China	Quercus sp.	Fagaceae	MH542474	NA	MH545423	MH545381
	CFCC 52737	China	Quercus sp.	Fagaceae	MH542475	NA	MH545424	MH545382
	CFCC 52739*	China	Quercus sp.	Fagaceae	MH542476	MH542635	MH545425	MH545383
	CFCC 52738	China	Quercus sp.	Fagaceae	MH542477	NA	MH545426	MH545384
	CFCC 52736	China	Quercus sp.	Fagaceae	MH542478	NA	MH545427	MH545385
	CFCC 52740	China	Quercus sp.	Fagaceae	MH542479	NA	MH545428	MH545386
Dendrostoma shaanxiense	CFCC 52741*	China	Castanea mollissima	Fagaceae	MH542486	MH542642	MH545435	MH545393
	CFCC 52742	China	Castanea mollissima	Fagaceae	MH542487	MH542643	MH545436	MH545394
Dendrostoma shandongense	CFCC 52759*	China	Castanea mollissima	Fagaceae	MH542504	MH542652	MH545453	MH545411
	CFCC 52760	China	Castanea mollissima	Fagaceae	MH542505	MH542653	MH545454	MH545412
Disculoides eucalypti	CBS 132183*	Australia	Eucalyptus sp.	Myrtaceae	JQ685517	JQ685523	MH545455	MH545413
Disculoides eucalyptorum	CBS 132184*	Australia	Eucalyptus viminalis	Myrtaceae	JQ685518	JQ685524	MH545456	MH545414

A partition homogeneity test with heuristic search and 1000 replicates was performed using PAUP v. 4.0b10 to assess the discrepancy amongst the ITS, LSU, TEF1-α and RPB2 sequence datasets in reconstructing phylogenetic trees. MP analysis was run using a heuristic search option of 1000 search replicates with random-additions of sequences with a tree bisection and reconnection algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). ML analysis was performed using a GTR site substitution model including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated using a bootstrapping method of 1000 replicates (Hillis and Bull 1993). Phylograms were shown using FigTree v. 1.3.1 (Rambaut and Drummond 2010). Novel sequences generated in the current study were deposited in GenBank (Table 1) and the aligned matrices used for phylogenetic analyses in TreeBASE (accession number: S22929).

Results

The alignment based on the combined sequence dataset (ITS, LSU, TEF1-α and RPB2) included 46 ingroup taxa and three outgroup taxa, comprising 3536 characters in the aligned matrix. Of these, 2612 characters were constant, 175 variable characters were parsimony-uninformative and 749 characters were parsimony informative (101 from ITS, 21 from LSU, 389 from TEF1-α and 238 from RPB2). The MP analysis resulted in 108 equally most parsimonious trees (TL = 1590, CI = 0.744, RI = 0.897, RC = 0.668); the first tree is shown in Fig. 2. The phylogram based on the four gene sequences indicated 10 new species in (Fig. 2), as described below.

Figure 2.

Phylogenetic tree based on an MP analysis of a combined DNA dataset of ITS, LSU, TEF1-α and RPB2 gene sequences for the species of . Bootstrap values ≥ 50% for MP and ML analyses are presented at the branches. Isolates representing ex-type material are marked with *.

Taxonomy

X.L. Fan & C.M. Tian, Persoonia 40: 126 (2018)

Type species.

X.L. Fan & C.M. Tian.

Description.

Sexual morph: Pseudostromata small to large, distinct, circular, erumpent, consisting of an inconspicuous ectostromatic disc, semi-immersed to superficial, causing a pustulate bark surface. Ectostromatic disc flat or concave, orange, surrounded by bark flaps. Central column beneath the disc more or less conical. Stromatic zones lacking. Ascomata perithecial, conspicuous, umber to fuscous black, embedded in orange to umber pseudostromatic tissue, regularly scattered, surrounding the ectostromatic disc, with small to long ostioles that emerge within the ectostromatic disc. Ostioles flat in the disc or sometimes slightly projecting, cylindrical, sometimes obscuring the disc, covered by an orange, umber to fuscous black crust. Paraphyses deliquescent. Asci fusoid, 8-spored, 2–3-seriate, with an apical ring, becoming detached from the perithecial wall. Ascospores hyaline, fusoid to cylindrical, symmetrical to asymmetrical, straight to curved, bicellular, with a median septum, constricted at the septum, smooth, multiguttulate. Asexual morph: Conidiomata pycnidial, spherical to conical to pulvinate, occurring separately, immersed to semi-immersed in bark; wall of several layers of yellow textura angularis. Central column beneath the disc conical or not. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of cavity, hyaline, smooth, subcylindrical to ampulliform. Conidia hyaline, aseptate, smooth, multiguttulate or not, thin-walled, ellipsoid to fusoid, straight to curved.

C.M. Tian & N. Jiang sp. nov.

MB826795

Figure 3

Figure 3.

Morphology of from (BJFC-S1561). A–C Habit of conidiomata on branches D Transverse section of conidioma E Longitudinal section through conidioma F, H Conidia G Conidiogenous cells. Scale bars: 1 mm (A); 0.5 mm (B, C, E); 0.2 mm (D); 5 μm (F, H); 10 μm (G).

Diagnosis.

differs from and by the existence of obvious central column.

Holotype.

CHINA. Shaanxi Province: Lan’gao County, chestnut plantation, , 1820 m a.s.l., on branches of , 3 Jul. 2017, N. Jiang (holotype: BJFC-S1561; ex-type culture: CFCC 52753).

Etymology.

Aurorae, referring to the orange conidiomata with exuding conidial tendrils.

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, conical to pulvinate, occurring separately, bright yellow to orange, semi-immersed in bark, 300–500 μm high, 800–1400 μm diam.; wall of several layers of bright yellow textura angularis; conidiomata exuding slimy orange masses of conidia; central column beneath the disc more or less conical, pale yellow. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, subcylindrical to ampulliform, 4–15 × 2.5–4 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, ellipsoid to fusoid, straight to curved, (7.2–)8.1–9.8(–10.3) × (2.1–)2.3–2.6(–2.8) μm, l/w = (2.7–)3.2–4.1(–4.2) (n = 50).

Culture characters.

On PDA, cultures are initially white, becoming isabelline after 2 weeks. The colonies are flat with irregular edge; texture uniform within 1 month at 25 °C in the dark.

Additional specimen examined.

CHINA. Shaanxi Province: Lan’gao County, chestnut plantation, , 1820 m a.s.l., on branches of , 3 Jul. 2017, N. Jiang, living culture CFCC 52754 (BJFC-S1562).

Notes.

was discovered on stems of dying chestnut trees and appears morphologically similar to the chestnut blight pathogen, . However, these two diaporthalean pathogens can be distinguished by the existence of a central column inside the conidiomata of . In the genus , differs from and by the existence of an obvious central column.

C.M. Tian & N. Jiang sp. nov.

MB826796

Figure 4

Figure 4.

Morphology of from (BJFC-S1553). A, B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section through conidioma E, G Conidia F Conidiogenous cells. Scale bars: 1 mm (A–D); 10 μm (E–G).

is distinguished from the phylogenetically closely related species by its narrower conidia.

CHINA. Hebei Province: Xinglong County, chestnut plantation, , 256 m a.s.l., on branches of , 27 Apr. 2018, N. Jiang & C.M. Tian (holotype: BJFC-S1553; ex-type culture: CFCC 52745).

Castaneae, referring to the host genus, .

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, pulvinate, occurring separately, bright yellow to orange, immersed in bark, 400–600 μm high, 900–2200 μm diam.; wall of several layers of brown textura angularis; central column beneath the disc irregular, pale yellow. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, subcylindrical to ampulliform, 3–10 × 2–3.5 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, ellipsoid, straight to curved, (9.3–)10.4–12.3(–13.3) × (2.1–)2.2–2.7(–2.9) μm, l/w = (3.4–)4.2–5.2(–5.9) (n = 50).

On PDA, cultures are initially white, exhibiting grey after 2 weeks. Colonies are flat with irregular edge; texture initially uniform, producing concentric circles with faint orange conidiomata distributed outside the rim within 1 month at 25 °C in the dark.

Additional specimens examined.

CHINA. Hebei Province: Chengde City, Xinglong County, chestnut plantation, , 256 m a.s.l., on branches of , 27 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52748 (BJFC-S1556); Hebei Province: Chengde City, Xinglong County, chestnut plantation, , 256 m a.s.l., on branches of , 27 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52751 (BJFC-S1557); Hebei Province: Chengde City, Xinglong County, chestnut plantation, 40°21'44"N, 117°51'29"E, 256 m a.s.l., on branches of , 27 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52747 (BJFC-S1559); Hebei Province: Chengde City, chestnut plantation, , 256 m a.s.l., on branches of , 28 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52750 (BJFC-S1558); Hebei Province: Chengde City, chestnut plantation, , 256 m a.s.l., on branches of , 28 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52752 (BJFC-S1560); Tianjin City: Jizhou District, chestnut plantation, , 185 m a.s.l., on branches of , 25 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52749 (BJFC-S1554); Tianjin City: Jizhou District, chestnut plantation, , 185 m a.s.l., on branches of , 25 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52746 (BJFC-S1555).

is the most common species in this genus occurring on the host in China and is associated with canker symptoms on stems and branches. As shown in Fig. 2, is the closest relative of ; however, they can be distinguished by conidial width (2.2–2.7 μm in vs. 3.2–3.8 μm in ).

C.M. Tian & N. Jiang sp. nov.

MB826797

Figure 5

Figure 5.

Morphology of from (BJFC-S1551). A, B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section through conidioma E, G Conidia F Conidiogenous cells. Scale bars: 1 mm (A); 0.5 mm (B–D); 5 μm (E, G); 10 μm (F).

differs from the two phylogenetically closely related species, and , by its white central column.

CHINA. Hebei Province: Chengde City, chestnut plantation, , 262 m a.s.l., on branches of , 28 Apr. 2018, N. Jiang & C.M. Tian (holotype: BJFC-S1551; ex-type culture: CFCC 52743).

Castaneicola, referring to the host genus, .

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, conical to pulvinate, occurring separately, reddish-orange, semi-immersed in bark, 300–550 μm high, 900–1600 μm diam.; wall of several layers of faint yellow textura angularis; central column beneath the disc more or less conical, white. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, subcylindrical to ampulliform, 5–14 × 2–3.5 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, ellipsoid to fusoid, straight, (9.3–)10.5–12.8(–13.8) × (3.1–)3.2–3.8(–4.1) μm, l/w = (2.3–)3–4(–4.4) (n = 50).

On PDA, cultures are initially white, becoming black after 2 weeks. The colonies are flat with irregular edge; texture uniform, producing a circle with faint orange conidiomata distributed along the edge of the circle within 1 month at 25 °C in the dark.

CHINA. Hebei Province: Chengde City, Xinglong County, chestnut plantation, , 256 m a.s.l., on branches of , 27 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52744 (BJFC-S1552).

, and comprise three closely related pathogen species causing chestnut canker diseases in China, all three species occurring on . They differ with regard to conidiomatal characteristics, including conidial dimensions (Table 2) and the central column colour (pale yellow central column in vs. white in vs. bright yellow in ). Additionally, was only discovered in the Shaanxi Province, whereas and were both distributed in Hebei Province.

Table 2.

Conidial size of species from natural host barks, species with * were measured from conidia produced in PDA.

Species	Conidial length (μm)	Conidial width (μm)	Length/width ratio
Dendrostoma aurorae	8.1–9.8	2.3–2.6	3.2–4.1
Dendrostoma castaneae	10.4–12.3	2.2–2.7	4.2–5.2
Dendrostoma castaneicola	10.5–12.8	3.2–3.8	3–4
Dendrostoma chinense	7.7–9.1	3.4–3.7	2.2–2.6
Dendrostoma dispersum	11.1–12.2	2–2.3	4.9–5.9
Dendrostomamali*	3.5–4.5	2–2.5	NA
Dendrostomaosmanthi*	7.5–10.5	2–2.5	NA
Dendrostoma parasiticum	9.3–11.7	2.8–3.3	3–3.9
Dendrostoma qinlingense	16–18	3.3–3.7	4.4–5.2
Dendrostomaquercinum*	10.5–14	2.5	NA
Dendrostoma quercus	13.3–16.1	3.5–4.2	3.3–4.4
Dendrostoma shaanxiense	9.5–11.1	2.5–3.1	3.3–4.2
Dendrostoma shandongense	8.1–8.8	3.8–4.3	1.9–2.3

C.M. Tian & N. Jiang sp. nov.

MB826798

Figure 6

Figure 6.

Morphology of from (BJFC-S1563). A, B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section through conidioma E, G Conidia F Conidiogenous cells. Scale bars: 1 mm (A); 0.5 mm (B–D); 10 μm (E–G).

differs from by the appearance of conidiomata and is again similar to in its conidial characteristics.

CHINA. Shandong Province: Rizhao City, Donggang District, chestnut plantation, , 452 m a.s.l., on branches of , 14 Apr. 2017, N. Jiang (holotype: BJFC-S1563; ex-type culture: CFCC 52755).

Chinense, referring to the country, China.

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, spherical, occurring separately, black, semi-immersed in bark, 250–450 μm high, 600–850 μm diam.; wall of several layers of white textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, ampulliform, 7–14 × 1–2.5 μm. Conidia hyaline, aseptate, smooth, multiguttulate or not, thin-walled, fusoid to ellipsoid, apex acutely rounded, base truncate, (6.9–)7.7–9.1(–9.7) × (3.3–)3.4–3.7(–3.9) μm, l/w = (1.9–)2.2–2.6(–2.7) (n = 50).

On PDA, cultures are initially white, becoming olive green in the outer zone after 2 weeks. Colonies are flat with a regular edge; texture uniform within 1 month at 25 °C in the dark.

CHINA. Shandong Province: Rizhao City, Donggang District, chestnut plantation, , 452 m a.s.l., on branches of , 14 Apr. 2017, N. Jiang, living culture CFCC 52756 (BJFC-S1564); Hebei Province: Chengde City, chestnut plantation, , 262 m a.s.l., on branches of , 29 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52757 (BJFC-S1565); Hebei Province: Chengde City, chestnut plantation, , 262 m a.s.l., on branches of , 29 Apr. 2018, N. Jiang & C.M. Tian, living culture CFCC 52757 (BJFC-S1566).

and have been occasionally discovered on the same branches and share similar conidial shape and dimensions. However, the conidiomatal appearance of these two species is quite different (black conidiomata in vs. orange conidiomata in ).

C.M. Tian & N. Jiang sp. nov.

MB826799

Figure 7

Figure 7.

Morphology of from sp. (BJFC-S1537). A, B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section through conidioma E, G Conidiogenous cells F Conidia. Scale bars: 1 mm (A); 0.5 mm (B–D); 10 μm (E, F), 5 μm (G).

can be distinguished from the phylogenetically closely related and based on its conidial dimensions.

CHINA. Shaanxi Province: Beijing City: Mentougou District, Xiaolongmen Forest Park, , 1670 m a.s.l., on branches of sp., 15 Aug. 2017, N. Jiang & X.L. Fan (holotype: BJFC-S1537; ex-type culture: CFCC 52730).

Dispersum, referring to the conidiomata scattered on the bark surface.

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, conical to spherical, occurring separately, bright yellow, semi-immersed in bark, 500–800 μm high, 900–1500 μm diam.; wall of several layers of bright yellow textura angularis; central column beneath the disc conical, bright yellow. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, subcylindrical to ampulliform, 6–15 × 2.5–5 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, ellipsoid to fusoid, straight to curved, (10.9–)11.1–12.2(–12.8) × (1.9–)2–2.3(–2.4) μm, l/w = (4.8–)4.9–5.9(–6.3) (n = 50).

On PDA, cultures are initially white, becoming faint yellow after 2 weeks. The colonies are flat with regular edge; texture uniform, producing concentric circles within 1 month at 25 °C in the dark.

CHINA. Beijing City: Yanqing District, Yudu Mountain, , 840 m a.s.l., on branches of sp., 12 Mar. 2018, N. Jiang, X.L. Fan, Y.M. Liang & C.M. Tian, living culture CFCC 52731 (BJFC-S1538).

is phylogenetically close to and (Fig. 2). Conidial dimensions of and were described from PDA plates (Fan et al. 2018) and can be differentiated from by having much longer conidia (11.1–12.2 μm in vs. 3–4.5 μm in ) and from by narrower conidia (2–2.3 μm in vs. 2.5–3 μm in ).

C.M. Tian & N. Jiang sp. nov.

MB826822

Figure 8

Figure 8.

Morphology of from (BJFC-S1570). A, B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section through conidioma E, G Conidia F Conidiogenous cells. Scale bars: 2 mm (A); 1 mm (B); 0.5 mm (C, D); 10 μm (E–G).

is distinguished from by its shorter and narrower conidia.

CHINA. Shaanxi Province: Shangluo City, Zhashui County, Longtougou Village, , 2504 m a.s.l., on branches of , 8 Jul. 2017, N. Jiang (holotype: BJFC-S1570; ex-type culture: CFCC 52762).

Parasiticum, referring to the fungus causing canker diseases on different hosts.

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, conical to spherical, occurring separately, yellow, semi-immersed in bark, 350–600 μm high, 1000–1800 μm diam.; wall of several layers of bright yellow textura angularis; central column beneath the disc conical, bright yellow. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, subcylindrical to ampulliform, 7–12 × 2–3.5 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, fusoid, straight, (9.2–)9.3–11.7(–13.6) × (2.7–)2.8–3.3(–3.6) μm, l/w = (2.7–)3–3.9(–4.2) (n = 50).

On PDA, cultures are initially white, becoming dark orange after 2 weeks. The colonies are flat with irregular edge; texture uniform, producing concentric circles within 1 month at 25 °C in the dark.

CHINA. Shaanxi Province: Shangluo City, Zhashui County, chestnut plantation, , 2504 m a.s.l., on branches of , 8 Jul. 2017, N. Jiang, living culture CFCC 52762 (BJFC-S1569); Shaanxi Province: Ankang City, Xiangxidong Park, , 2504 m a.s.l., on branches of , 29 Jun. 2017, N. Jiang, living culture CFCC 52763 (BJFC-S1571); Beijing City: Mentougou District, Xiaolongmen Forest Park, , 452 m a.s.l., on branches of , 17 Aug. 2017, N. Jiang & X.L. Fan, living culture CFCC 52764 (BJFC-S1572); Beijing City: Yanqing District, Yudu Mountain, 40°53'48"N, 115°54'48"E, 840 m a.s.l., on branches of , 12 Mar. 2017, N. Jiang, X.L. Fan, Y.M. Liang & C.M. Tian, living culture CFCC 52765 (BJFC-S1573); Hebei Province: Chengde City, chestnut plantation, , 262 m a.s.l., on branches of , 15 Oct. 2017, N. Jiang, living culture CFCC 52766 (BJFC-S1574).

constitutes a widely distributed species occurring on several tree species including , , and . appears to be associated with tree dieback, canker and even tree death, although its pathogenicity remains unproven. is close to in the phylogram (Fig. 2), but differs from with shorter (9.3–11.7 μm in vs. 13.3–16.1 μm in ) and narrower (2.8–3.3 μm in vs. 3.5–4.2 μm in ) conidia.

C.M. Tian & N. Jiang sp. nov.

MB826823

Figure 9

Figure 9.

Morphology of from (BJFC-S1539). A, B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section through conidioma E, G Conidiogenous cells F Conidia. Scale bars: 1 mm (A); 0.5 mm (B–D); 10 μm (E–G).

produces the largest conidia amongst known species of the genus.

CHINA. Baoji City, Mei County, Taibai Mountain, , 2752 m a.s.l., on branches of , 13 Jul. 2017, N. Jiang (holotype: BJFC-S1539; ex-type culture: CFCC 52732).

Qinlingense, referring to the Qinling Mountain.

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, conical to pulvinate, occurring separately, dark yellow, semi-immersed in bark, 400–700 μm high, 1100–1600 μm diam.; wall of several layers of bright yellow textura angularis; central column beneath the disc conical, dark orange. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, ampulliform, 6–22 × 2–3.5 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, fusoid, straight, (15.6–)16–18(–18.6) × (3.1–)3.3–3.7(–3.8) μm, l/w = (4.2–)4.4–5.2(–5.8) (n = 50).

On PDA, cultures are initially white, exhibiting light grey after 2 weeks. The colonies are flat with irregular edge; texture uniform, producing concentric circles with sparse conidiomata irregularly distributed on the centre of the plate within 1 month at 25 °C in the dark.

CHINA. Shaanxi Province: Baoji City, Mei County, Taibai Mountain, , 2752 m a.s.l., on branches of , 13 Jul. 2017, N. Jiang, living culture CFCC 52733 (BJFC-S1540).

was discovered on two species on the Qinling Mountain in northwest China. This species is phylogenetically related to on . However, differs from by much larger conidia (16–18 × 3.3–3.7 μm in vs. 7.5–10 × 2–2.5 μm in ).

C.M. Tian & N. Jiang sp. nov.

MB826824

Figure 10

Figure 10.

Morphology of from sp. (BJFC-S1547). A–C Habit of psedostromata on branches D Transverse section of pseudostroma E, H Habit of conidiomata on branches F Transverse section of conidioma G Longitudinal section through conidioma I Conidiogenous cells producing dimorphic conidia J Secondary conidia K Asci and ascospores L Ascospores M Primary conidia. Scale bars: 1 mm (A, H); 0.5 mm (B–G); 10 μm (I, K–M); 5 μm (J).

is recognised by the existence of dimorphic conidia, which is unique in the genus.

CHINA. Hebei Province: Qinhuangdao City, Zu Mountain, , 1125 m a.s.l., on branches of sp., 2 May 2018, N. Jiang & C.M. Tian (holotype: BJFC-S1547; ex-type culture: CFCC 52739).

Quercus, referring to the host genus, .

Sexual morph: Pseudostromata erumpent, consisting of an inconspicuous ectostromatic disc, semi-immersed to superficial, causing a pustulate bark surface, 1000–1500 µm diam. Ectostromatic disc flat or concave, pale brown to brown, sometimes concealed by ostioles, surrounded by bark flaps, 400–800 µm diam.; central column yellowish to brownish. Stromatic zones lacking. Perithecia conspicuous, umber to fuscous black, 350–500 µm diam. Ostioles 5–8 per disc, flat in the disc or sometimes slightly projecting, cylindrical, covered by an orange, umber to fuscous black crust, 60–80 µm diam. Paraphyses slightly deliquescent. Asci fusoid to slightly fusiform, 8-spored, ascospores regularly disposed, with an apical ring, 55–65 × 8–11 µm. Ascospores hyaline, fusoid to cylindrical, smooth, often containing one guttule per cell to multiguttulate, symmetrical to asymmetrical, straight curved, bicellular, (13.4–)13.8–15.6(–16.6) × (5.1–)5.3–5.8(–5.9) μm, l/w = (2.4–)2.5–2.8(–2.9) (n = 50). Asexual morph: Conidiomata pycnidial, conical, occurring separately, pale yellow, semi-immersed in bark, 700–1000 μm high, 700–950 μm diam.; wall of several layers of pale yellow textura angularis; central column beneath the disc conical, yellow. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, subcylindrical to ampulliform, 4.5–9 × 2–4 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, dimorphic, type one (> 99%) ellipsoid to fusoid, straight to curved, (11–)13.3–16.1(–16.9) × (3.4–)3.5–4.2(–4.5) μm, l/w = (2.6–)3.3–4.4(–4.9) (n = 50); type two (< 1%) fusoid, apex acutely rounded, 13–16 × 4–6 μm.

On PDA, cultures are initially white, becoming dark grey after 2 weeks. The colonies are flat with irregular edge; texture uniform, producing concentric circles with sparse conidiomata irregularly distributed within 1 month at 25 °C in the dark.

CHINA. Hebei Province: Qinhuangdao City, Zu Mountain, , 1125 m a.s.l., on branches of sp., 2 May 2018, N. Jiang & C.M. Tian, living culture CFCC 52734 (BJFC-S1548); Hebei Province: Qinhuangdao City, Zu Mountain, , 1125 m a.s.l., on branches of sp., 2 May 2018, N. Jiang & C.M. Tian, living culture CFCC 52735 (BJFC-S1541); Hebei Province: Qinhuangdao City, Zu Mountain, , 1125 m a.s.l., on branches of sp., 2 May 2018, N. Jiang & C.M. Tian, living culture CFCC 52736 (BJFC-S1542); Hebei Province: Qinhuangdao City, Zu Mountain, , 1125 m a.s.l., on branches of sp., 2 May 2018, N. Jiang & C.M. Tian, living culture CFCC 52737 (BJFC-S1543); Hebei Province: Qinhuangdao City, Zu Mountain, , 1125 m a.s.l., on branches of sp., 2 May 2018, N. Jiang & C.M. Tian, living culture CFCC 52738 (BJFC-S1544); Hebei Province: Qinhuangdao City, Zu Mountain, , 1125 m a.s.l., on branches of sp., 2 May 2018, N. Jiang & C.M. Tian, living culture CFCC 52740 (BJFC-S1545).

is associated with oak branch cankers and forms both sexual and asexual fruiting structures beneath cankered bark. Within the genus, produces the second largest conidia, smaller only than those of (Table 2). The presence of dimorphic conidia in , however, is a feature unique to .

C.M. Tian & N. Jiang sp. nov.

MB826825

Figure 11

Figure 11.

Morphology of from (BJFC-S1549). A, B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section through conidioma E, G Conidia F Conidiogenous cells. Scale bars: 1 mm (A); 0.5 mm (B–D); 10 μm (E–G).

is distinguished from the closely related species by smaller l/w ratio and from by its narrower conidia.

CHINA. Shaanxi Province: Ankang City, Xiangxidong Park, , 1079 m a.s.l., on branches of , 1 Jul. 2017, N. Jiang (holotype: BJFC-S1549; ex-type culture: CFCC 52741).

Shaanxiense, referring to the Shaanxi Province in China.

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, conical to pulvinate, occurring separately, dark orange, semi-immersed in bark, 350–650 μm high, 1050–1400 μm diam.; wall of several layers of bright yellow textura angularis; central column beneath the disc conical, bright yellow. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of the cavity, hyaline, smooth, subcylindrical to ampulliform, 5–11 × 2.5–3.5 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, ellipsoid to fusoid, straight to curved, (8.6–)9.5–11.1(–11.7) × (2.3–)2.5–3.1(–3.4) μm, l/w = (2.8–)3.3–4.2(–4.9) (n = 50).

On PDA, cultures are initially white, turning purple after 2 weeks on PDA. The colonies are flat with irregular edge; texture uniform, producing concentric circles within 1 month at 25 °C in the dark.

Shaanxi Province: Ankang City, Xiangxidong Park, , 1079 m a.s.l., on branches of , 1 Jul. 2017, N. Jiang, CFCC 52742 (BJFC-S1550).

, and are phylogenetically closely related species occurring on the same host, (Fig. 2). However, has conidia with a smaller l/w ratio than (3.3–4.2 in vs. 4.2–5.2 in ) and has narrower conidia than (2.5–3.1 μm diam. in vs. 3.2–3.8 μm diam. in ).

C.M. Tian & N. Jiang sp. nov.

MB826826

Figure 12

Figure 12.

Morphology of from (BJFC-S1567). A–C Habit of conidiomata on branches D Transverse section of conidioma E Longitudinal section through conidioma F Conidiogenous cells G Conidia. Scale bars: 1 mm (A); 0.3 mm (B–D); 5 μm (F); 5 μm (G).

is distinguished from its closest relative by the colour of conidiomata.

CHINA. Shandong Province: Rizhao City, Donggang District, chestnut plantation, , 452 m a.s.l., on branches of , 14 Apr. 2017, N. Jiang (holotype: BJFC-S1567; ex-type culture: CFCC 52759).

, referring to the Shandong Province in China.

Sexual morph not observed. Asexual morph: Conidiomata pycnidial, spherical, occurring separately, reddish-orange, semi-immersed in bark, 250–400 μm high, 450–650 μm diam.; wall of several layers of black textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner walls of cavity, hyaline, smooth, ampulliform, 6.5–13 × 1–2.5 μm. Conidia hyaline, aseptate, smooth, multiguttulate, thin-walled, fusoid to ellipsoid, apex acutely rounded, base truncate, (7.8–)8.1–8.8(–9) × (3.7–)3.8–4.3(–4.8) μm, l/w = (1.6–)1.9–2.3(–2.4) (n = 50).

On PDA, cultures are white. The colonies are flat with irregular edge; texture uniform, producing sparse conidiomata irregularly distributed near the centre of the plate within 1 month at 25 °C in the dark.

Shandong Province: Rizhao City, Donggang District, chestnut plantation, , 452 m a.s.l., on branches of , 14 Apr. 2017, N. Jiang, CFCC 52760 (BJFC-S1568).

and occasionally occur on the same branches. These species are best distinguished by the appearance of their conidiomata, which are black in and orange in .

Discussion

In this study, we reviewed the taxonomic circumscription of using molecular and morphological data. This is the first study that presents a robust phylogeny using a number of isolates from different geographic origins. The results revealed up to 14 species in based on the observation of type specimens and ex-type cultures ( was not observed), of which 10 species were shown to represent new species, namely , , , , , , , , and .

The 13 type specimens in (except ) were examined to establish robust morphological characteristics amongst specific ranks. Amongst these, 3 species, , and , were discovered to only have a sexual morph on natural hosts; 9 species, , , D. , , , , , and , were observed with only an asexual morph and only one species, , was represented by both asexual and sexual morphs. Hence, morphological differences amongst species were mainly established based on conidiomata produced on diseased host tissues, including colours of conidiomata, culture characteristics (Fig. 13), existence or non-existence of a central column, conidial shape and dimensions.

and are similar in conidial shape and size, but differ markedly from the other species. Additionally, and comprise the only two species in the genus with conidiomata lacking a central column structure, although they differ considerably with regard to in conidiomatal appearance (Figs 6, 13). The remaining eight species differ by the existence of a central column inside the conidiomata and can be further distinguished by their conidial characteristics, namely length, width and l/w ratio. Additionally, a key to the 14 species is provided below.

Figure 13.

cultures on PDA after 1 month at 25 °C, ABCD. DEF–GHIJ; KL.

Key to species

The genus was initially proposed to include three presumed plant pathogens causing canker diseases on hardwood trees, namely on , on and on (Fan et al. 2018). Consistent with the previous study, the newly described 10 species were all isolated from fruiting structures associated with typical canker symptoms on several hardwood tree species, namely and spp.

The tree genera and in contain numerous important and common tree species in China, including , , , , , , , and (Flora of China website: http://frps.eflora.cn/). constitutes one the most important crop tree species widely cultivated in 26 provinces in China. However, many plantations and nurseries planting Chinese chestnut suffer from fungal diseases that cause high production losses (Jiang et al. 2018). In particular, chestnut blight caused by represents the most serious fungal disease, reducing host vitality and potentially killing the host (Jiang et al. 2018, Rigling and Prospero 2018).

In the present study, seven species were observed on the host including , , , , D. , and , causing chestnut canker diseases, termed canker herein. canker constitutes a newly discovered disease that has been observed in chestnut plantations and nurseries. Species of usually infect host branches and stems, with occasional infection of twigs. Maturation of the fruiting structures from June to July resulted in death of the infected branches. Notably, no sexual fruiting structures were discovered during our investigations on chestnut trees.

Accurate recognition and identification of plant diseases are essential as fungal pathogens are constantly evolving and traditional control methods are frequently insufficient for disease control. In comparison, in the present study, canker is considered to be caused by up to eight different species of . Further studies are, however, required to confirm their pathogenicity and fully resolve their ecology.

1	Asexual morphs with or without sexual morphs known from natural substrates	2
–	Only sexual morph known from natural substrates	11
2	Central column absent, length/width ratio of conidia < 3	3
–	Central column present, length/width ratio of conidia > 3	4
3	Conidiomata orange	D. shandongense
–	Conidiomata black	D. chinense
4	Conidia dimorphic	D. quercus
–	Conidia monomorphic	5
5	Conidial length > 15 μm	D. qinlingense
–	Conidial length < 15 μm	6
6	Conidial length/width ratio > 4.2	7
–	Conidial length/width ratio < 4.2	8
7	Conidial length/width ratio 4.2–5.2, conidial width 2.2–2.7 μm	D. castaneae
–	Conidial length/width ratio 4.9–5.9, conidial width 2–2.3 μm	D. dispersum
8	Central column white	D. castaneicola
–	Central column bright yellow or pale yellow	9
9	Central column pale yellow	D. aurorae
–	Central column bright yellow	10
10	Conidial width 2.8–3.3 μm, length/width ratio 3–3.9	D. parasiticum
–	Conidial width 2.5–3.1 μm, length/width ratio 3.3–4.2	D. shaanxiense
11	Ascospores width > 5 μm	D. leiphaemia
–	Ascospores width < 5 μm	12
12	Ascospores length > 15 μm	D. quercinum
–	Ascospores length < 15 μm	13
13	On Osmanthus, Ascospores 11.5–14.5 × 3.5–4 μm	D. osmanthi
–	On Malus, Ascospores 12–14 × 3–4 μm	D. mali