﻿Seven new species of Alternaria (Pleosporales, Pleosporaceae) associated with Chinese fir, based on morphological and molecular evidence

﻿Abstract Chinese fir (Cunninghamialanceolata) is a special fast-growing commercial tree species in China and has significant ecological and economic value. However, it experienced damage from leaf blight caused by pathogenic fungi of the genus Alternaria. To determine the diversity of Alternaria species associated with leaf blight of Chinese fir in China, infected leaves were collected from five major cultivation provinces (Fujian, Henan, Hunan, Jiangsu and Shandong provinces). A total of 48 fungal strains of Alternaria were obtained. Comparison of morphology and phylogenetic analyses, based on nine loci (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2) of the representative isolates as well as the pairwise homoplasy index tests, revealed that the fungal strains belonged to seven undescribed taxa of Alternaria, which are described here and named as Alternariacunninghamiicolasp. nov., A.dongshanqiaoensissp. nov., A.hunanensissp. nov., A.kunyuensissp. nov., А. longqiaoensissp. nov., A.shandongensissp. nov. and A.xinyangensissp. nov. In order to prove Koch’s postulates, pathogenicity tests on detached Chinese fir leaves revealed significant pathogenicity amongst these species, of which A.hunanensis is the most pathogenic to Chinese fir. This study represents the first report of A.cunninghamiicola, A.dongshanqiaoensis, A.hunanensis, A.kunyuensis, A.longqiaoensis, A.shandongensis and A.xinyangensis causing leaf blight on Chinese fir. Knowledge obtained in this study enhanced our understanding of Alternaria species causing leaf blight on Chinese fir and was crucial for the disease management and the further studies in the future.

For example, Alternaria species have been recorded as endophytes in grasses, angiosperms, rice and other herbaceous plants and shrubs (Fisher and Petrini 1992;Schulz et al. 1993;Rosa et al. 2009;Polizzotto et al. 2012) and have been also isolated from soil (Hong and Pryor 2004).Many Alternaria species are saprobes on a variety of plant tissues in different habitats (Thomma 2003;Liu et al. 2015b;Wanasinghe et al. 2018).Some Alternaria species, such as A. alternata, produce host-specific toxins (Hyde et al. 2018).Several taxa are also important postharvest pathogens, for example, A. alternata and A. solani (El-Goorani and Sommer 1981;Reddy et al. 2000), or airborne fungal allergens/pathogens-causing upper respiratory tract infections and asthma in humans (Mitakakis et al. 2001;Woudenberg et al. 2015;Hyde et al. 2018).Due to the significant negative health effects of Alternaria on humans and their surroundings, a correct and rapid identification of Alternaria species would be of great significance to researchers, plant pathologists, medical mycologists, other biological professionals and the public alike (Woudenberg et al. 2013).
The taxonomy of Alternaria species especially small-spored species within the alternata species group are particularly challenging because few morphological characters are able to clearly differentiate taxa and these characters are strongly influenced by the environment.Morphological characteristics, such as colour, size, shape of conidia and sporulation patterns have been used for the identification and classification of Alternaria species (Simmons 1992).Wiltshire (1945) divided Alternaria into three major sections, Brevicatenatae, Longicatenatae and Noncatenatae, based on conidial catenation.However, this division is unreliable as some of these characters overlap amongst species and vary depending on the cultural conditions, such as temperature and substrate (Simmons and Roberts 1993).Simmons (1992Simmons ( , 1995) ) arranged several species groups within Alternaria based on the morphological similarity amongst species.Some other genera, such as Stemphylium (Wallroth, 1833) and Ulocladium (Preuss, 1852) also produce phaeodictyospores and are morphologically similar to Alternaria, and this has further led to taxonomic complications (Bigelow 2003).Simmons (2007) revised Alternaria taxonomy, based on morphology and 275 species were recognised.At the same time, Simmons (2007) proposed three new genera Alternariaster, Chalastospora and Teretispora for some species that were previously described in Alternaria.
However, molecular phylogeny has revealed polyphyletic taxa within Alternaria and Alternaria species clades, which do not always correlate with morphological species-groups (Inderbitzin et al. 2006;Runa et al. 2009;Lawrence et al. 2012).Pryor and Gilbertson (2000) elucidated relationships amongst Alternaria, Stemphylium and Ulocladium based on ITS and SSU sequence data and revealed that Stemphylium species were phylogenetically distinct from Alternaria and Ulocladium species.Most Alternaria and Ulocladium clustered together in a large Alternaria/Ulocladium clade (Pryor and Gilbertson 2000).Chou and Wu (2002) confirmed that filament-beaked Alternaria species constitute a monophyletic group distinct from the other members in this genus and hypothesised that this group is evolutionarily distinct, based on phylogenies of ITS sequence.Two new species groups, A. panax and A. gypsophilae were introduced by Lawrence et al. (2013) with phylogenetic evidence and they accepted eight well supported asexual species-sections within Alternaria, while the taxa with known sexual morphs, the A. infectoria species-groups, were not given the similar rank.Woudenberg et al. (2013) delineated taxa within Alternaria and allied genera, based on SSU, LSU, ITS, GAPDH, RPB2 and TEF1 sequence data.The generic circumscription of Alternaria was emended and 24 internal clades in the Alternaria complex were treated as sections, together with six monotypic lineages (Woudenberg et al. 2013;Gannibal et al. 2022).Woudenberg et al. (2013) also demoted the genera Allewia, Brachycladium, Chalastospora, Chmelia, Crivellia, Embellisia, Lewia, Nimbya, Sinomyces, Teretispora, Ulocladium, Undifilum and Ybotromyces to synonymy with Alternaria.Therefore, the use of DNA sequence data is very important in resolving Alternaria taxonomy.
Chinese fir (Cunninghamia lanceolata (Lamb.)Hook.) is an important fast-growing timber species in China and its afforestation area and timber volume rank first amongst forest plantations; it plays an important role in forest carbon sequestration, increasing farmers' income and rural revitalisation (Yan 2020).Average timber volume is estimated at 500-800 m 3 /ha and in China, Chinese fir contributes 40% of the total commercial timber production (Zheng et al. 2016).However, Chinese fir is often damaged by many diseases and insects (Lan et al. 2015) (Anonymous 1976;Kobayashi and Zhao 1987;Wang et al. 1995;Chen 2002;Lan et al. 2015;Liu et al. 2015a;Xu and Liu 2017;Huang et al. 2018;Tian et al. 2019;Zhou and Hou 2019;Cui et al. 2020a, b;He et al. 2022).However, there is a lack of comprehensive study on Alternaria causing leaf blight disease on Chinese fir including diversity, occurrence and pathogenicity of the pathogens.
Surveys of fungal diseases on foliage of Chinese fir in its main cultivation regions in China were conducted from 2016 to 2020, 48 isolates of Alternaria spp.were collected and examined.The main aims of the present study were to determine the Alternaria spp.associated with leaf blight disease on Chinese fir using a polyphasic approach of fungal morphology and phylogenetic analyses, based on multi-locus sequences of ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2.

Isolation of the potential fungal pathogen
A total of 48 isolates of Alternaria spp.were isolated from leaf blight samples of Chinese fir, which were collected in five provinces (Fujian, Henan, Hunan, Jiangsu and Shandong) in China (Suppl.material: table S1).Small pieces (2 × 3 mm) were cut from the margins of infected tissues and surface sterilised in 75% alcohol for 30 s, then in 1% sodium hypochlorite (NaOCl) for 90 s, followed by three rinses with sterile water (Huang et al. 2016), then blotted dry with sterilised filter paper, placed on 2% potato dextrose agar (PDA) Petri plates with 100 mg/l ampicillin and then cultured for 3 days at 25 °C in the dark.Fungal isolates were purified with the monosporic isolation method described by Li et al. (2007).Single-spore isolates were maintained on PDA plates.The obtained isolates were stored in the Forest Pathology Laboratory of Nanjing Forestry University.Holotype specimens of new species from this study were deposited at the China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing, China.

DNA extraction, PCR amplification and sequencing
Genomic DNA of 48 isolates was extracted using a modified CTAB method (Damm et al. 2008).The fungal plugs of each isolate were grown on the PDA plates for 5 days and then collected in a 2 ml tube.Then, 500 µl of chloroform and 500 µl of hexadecyltrimethyl ammonium bromide (CTAB) extraction buffer (0.2 M Tris, 1.4 M NaCl, 20 mM EDTA, 0.2 g/l CTAB) were added into the tubes, which were placed in a shaker at 25 °C at 200 rpm for 2 h.The mixture was centrifuged at 15,800 × g for 5 min.Three hundred µL of the supernatant was transferred into a new tube and 600 µl of 100% ethanol was added.The suspension was centrifuged at 15,800 × g for 5 min.Then, 600 µl of 70% ethanol was added into the precipitate.The suspension was centrifuged at 15,800 × g for 5 min and the supernatant was discarded.The DNA pellet was dried and resuspended in 30 µl ddH 2 O.
The polymerase chain reaction (PCR) amplification was conducted as described by Woudenberg et al. (2015).PCR was performed in a 30 µl reaction volume containing 2 µl of genomic DNA (ca.200 ng/µl), 15 µl of 2× Taq Plus Master Mix (Dye Plus) (Vazyme P212-01), 1 µl of 10 μM forward primer, 1 µl of 10 μM reverse primer and 11 µl of ddH 2 O.The PCR conditions consisted of an initial denaturation step of 4 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C for ITS, GAPDH and endoPG, 35 cycles of 30 s at 94 °C, 30 s at 62 °C and 45 s at 72 °C for OPA10-2 and Alt a1, and 35 cycles of 30 s at 94 °C, 30 s at 59 °C and 60 s at 72 °C for RPB2, TEF1, LSU and SSU, and a final elongation step of 10 min at 72 °C.All DNA sequencing was performed at Shanghai Sangon Biotechnology Company (Nanjing, China).Sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic analyses
The sequences generated in this study were compared against nucleotide sequences in GenBank using BLAST to determine closely-related taxa.Alignments of different loci, including the sequences obtained from this study and the ones downloaded from GenBank, were initially performed with the MAFFT v.7 online server (https://mafft.cbrc.jp/alignment/server/)(Katoh and Standley 2013) and then manually adjusted in MEGA v. 10 ( Kumar et al. 2018).The post-alignment sequences of multiple loci were concatenated in PhyloSuite software (Zhang et al. 2020).Maximum-Likelihood (ML) and Bayesian Inference (BI) were run in PhyloSuite software using IQ-TREE ver.1.6.8 (Nguyen et al. 2015) and MrBayes v. 3.2.6 (Ronquist et al. 2012), respectively.ModelFinder was used to carry out statistical selection of best-fit models of nucleotide substitution using the corrected Akaike information criterion (AIC) (Kalyaanamoorthy et al. 2017).For ML analyses, the default parameters were used, and bootstrap support (BS) was carried out using the rapid bootstrapping algorithm with the automatic halt option.Bayesian analyses included two parallel runs of 2,000,000 generations, with the stop rule option and a sampling frequency set to each 1,000 generations.The 50% majority rule consensus trees and posterior probability (PP) values were calculated after discarding the first 25% of the samples as burn-in.Phylogenetic trees were visualised in FigTree v. 1.4.2(http://tree.bio.ed.ac.uk/ software/figtree/) (Rambaut 2014).Phylogenetically-related, but ambiguous species were analysed using the genealogical concordance phylogenetic species recognition (GCPSR) model by performing a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. (2014).The PHI test was performed in SplitsTree4 (Huson 1998;Huson and Bryant 2006) in order to determine the recombination level within phylogenetically closely-related species using a concatenated multi-locus dataset (ITS, SSU, LSU, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2).If the pairwise-homoplasy index results were below a 0.05 threshold (Ф w < 0.05), it indicates significant recombination present in the dataset.The relationship amongst the closely-related species was visualised by constructing splits graphs.

Morphological study
One representative isolate was randomly selected from each Alternaria species for morphological research according to the method of Simmons (2007).Mycelial plugs (5 mm) of purified cultures were transferred from the growing edge of 5-d-old cultures to the centre of 7-mm-diameter potato carrot agar (PCA) plates (Crous et al. 2009b) in triplicate at 25 °C.Colony diameters were measured from 3 to 6 days to calculate mycelial growth rates (mm/d).Colony colour, size and density were also recorded.The morphology and size of conidial chains were studied and recorded using a Zeiss stereo microscope (SteRo Discovery v.20).The shape, colour and size of conidiophores and conidia were observed using a ZEISS Axio Imager A2m microscope (ZEISS, Germany) with differential interference contrast (DIC) optics.At least 30 measurements per structure were performed using Carl Zeiss Axio Vision software to determine their sizes, unless no or fewer individual structures were produced.
For in-vitro inoculation, detached leaves were surface-sterilised with 75% ethanol, washed three times with sterile water and air-dried on sterile filter paper.A 10 µl aliquot of conidial suspension (1.0 × 10 6 conidia/ml) was transferred to a sterile plastic tube (20 × 6 mm), in which a leaf was placed so that the base of the leaf was immersed in the conidial suspension.The control was treated with the same amount of double-distilled water.Leaves in the tubes were then placed in plastic trays (40 × 25 cm), covered with a piece of plastic wrap to maintain relative humidity at 99% and incubated at 25 °C in the dark for 5 days.Each treatment had twelves replicates and the experiment was conducted three times.Symptom development on each detached leaf was evaluated by determining the means of lesion lengths at 5 days post-inoculation (dpi).The data were analysed by analysis of variance (ANOVA) using SPSS v. 18 software.LSD's range test was used to determine significant differences amongst or between different treatments.Origin v. 8.0 software was used to draw histograms (Li et al. 2020).Pathogens were re-isolated from the resulting lesions and identified as described above.

Taxonomy
Based on morphology and multi-locus sequence data, a total of 48 obtained isolates from Chinese fir were assigned to seven species of Alternaria, which represented seven undescribed taxa and were described below.
Host/distribution.From C. lanceolata in Dongshanqiao Forest Farm, Nanjing City, Jiangsu Province, China.
Culture characteristics.Colonies on PCA incubated at 25 °C in the dark growing at 9.3 ± 0.1 mm/d; aerial hypha cottony, white to pale grey; reverse centre dark green to black; sporulation sparse; diffusible pigment absent.
Etymology.Epithet is after Dongshanqiao Forest Farm, Nanjing City, Jiangsu Province where the type specimen was collected.
Host/distribution. from C. lanceolata in Dongshanqiao Forest Farm, Nanjing City, Jiangsu Province, China.
Culture characteristics.Colonies on PCA incubated at 25 °C in the dark growing at 7.8 ± 0.2 mm/d; aerial hyphae cottony, greyish-green, with grey margins; reverse centre black, with white margins.
Etymology.Epithet is after Longqiao Town, Yiyang City, Hunan Province where the type specimen was collected.
Culture characteristics.Colonies on PCA incubated at 25 °C in the dark growing at 7.8 ± 0.1 mm/d; aerial hypha cottony, pale gray to greyish-green, with white to pale grey margins; reverse centre brownish to dark green with pale grey margins; sporulation sparse; diffusible pigment absent.
Etymology.Epithet is after Kunyu Mountain, Yantai City, Shandong Province where the type specimen was collected.
Culture characteristics.Colonies on PCA incubated at 25 °C in the dark growing at 7.5 ± 0.2 mm/d; aerial hypha sparse, olive green to dark green; reverse centre grey; sporulation abundant; diffusible pigment absent.
Etymology.Epithet is after Longqiao Town, Yiyang City, Hunan Province where the type specimen was collected.
Culture characteristics.Colonies on PCA incubated at 25 °C in the dark growing at 8.3 ± 0.4 mm/d; aerial hypha cottony, dark green to black, with pale green margins; reverse centre black with pale grey margins; sporulation abundant; diffusible pigment absent.
Etymology.Epithet is after Shandong Province where the type specimen was collected.

Alternaria xinyangensis
Culture characteristics.Colonies on PCA incubated at 25 °C in the dark growing at 7.2 mm/d; aerial hyphae cottony, olive green, with white margins; reverse centre black to greyish; sporulation abundant; diffusible pigment absent.
Additional materials examined.China, Henan Province, Xinyang City, Zhenlei Mountain, 32°04'51"N, 114°07'23"E, isolated from leaf spots of Cunninghamia lanceolata, May 2017, Wen-Li Cui, ZLS1-1, ZLS1-2, ZLS1-3, ZLS1-4; China, Henan Province, Xinyang City, Xinyang University, 32°08'20"N, 114°02'06"E, isolated from leaf spots of C. lanceolata, May 2017, Wen-Li Cui, XYXY06, XYXY8-2, XYXY15, XYXY15-1, XYXY15-2, XYXY15-3, XYXY15-4, XYXY16.A previous multi-locus phylogenetic study Woudenberg et al. (2013) established the taxonomic conclusions of morpho-species known under A. alternata based on the multi-locus phylogenetic analysis.Subsequently, Woudenberg et al. (2015) used the same analysis to determine the discrete lineages of Alternaria spp. in section Alternaria, which showed a 97-98% genomic similarity, concluding that species, such as A. angustiovoide, A. citri,A. lini,A. mali (CBS 106.24),A. malvae and A. tenuissima (CBS 918.96) did not make discrete groupings, but all are synonymous with A. alternata sensu stricto.Although Woudenberg et al. (2015) assigned 35 morpho-species as synonyms of Alternaria alternata, their affinities are still unclear due to inconsistencies, lack of morphological details and a comparison of single nucleotide polymorphisms.However, further studies, based on combined multi-locus phylogeny, showed that recent A. alternata species may not constitute a monophyletic group in DNA sequence-based phylogenies (Li et al. 2023).Morphological characters and phylogenetic analyses of the nine loci showed all 48 Alternaria isolates clustered in the Sect.Alternata in the phylogenetic tree and divide into seven distinct clusters in the current study.We compared these strains, based on morphology and phylogeny.Interestingly, our phylogenetic analyses show that the morpho-species of A. alternata can be separated into different clades and our novel taxa from Chinese fir are both morphologically and phylogenetically distinct from the A. alternata complex and other species in Alternaria sect.Alternaria.Herein, based on these most recent classifications, these isolates from Chinese fir in this study are, thus, identified as the A. alternata complex including A. cunninghamiicola, A. dongshanqiaoensis, A. hunanensis, A. kunyuensis, A. longqiaoensis, A. shandongensis and A. xinyangensis.The results of pathogenicity tests indicate that the seven new Alternaria species were pathogenic to Chinese fir.Alternaria hunanensis exhibited the strongest virulence in the Alternaria species from the present study, and A. xinyangensis, A. kunyuensis and A. cunninghamiicola with weaker virulence especially in shoots of Chinese fir.Nevertheless, compared with our previous study, Alternaria species showing weaker virulence than those of Colletotrichum spp.(He et al. 2022) and Fusarium spp.(unpublished) and the results may explain why most of Alternaria species are facultative parasites and their pathogenicities are not too strong.Alternaria spp.may prefer to be saprobes or secondary pathogens growing in senescent, near-dead or dead plant tissues.The diseases caused by these pathogens often attack senescent and diseased leaves before crop maturity or when the growth of the hosts is poor.In addition, according to previous studies, some Alternaria taxa carry out facultative parasitism life cycles mainly depending on the following three aspects: damaging the cell walls of their hosts by mechanical penetration and the degrading enzymes, producing mycotoxins that target the cytoplasmic membrane, mitochondria, chloroplast and influencing the activity of enzymes related metabolisms, and mediating pathogenicity through signal transduction (Thomma 2003;Kang et al. 2013).At present, there are few studies on the pathogenic mechanism of Alternaria species, without revealing the specific process of host infection.Therefore, the thorough study of its pathogenic mechanism is the basis and key to solving the damage from Alternaria.
Until now, over 360 species of Alternaria are reported as plant pathogens and saprobes, resulting in the decline of forest quality and fruit decay during

Figure 1 .
Figure 1.Phylogenetic relationships of 116 isolates of the Alternaria species complex with related taxa with concatenated sequences of the SSU, LSU, ITS, GAPDH, RPB2, TEF1, Alt a1, endoPG and OPA10-2 loci using Bayesian inference (BI) and Maximum-likelihood (ML) methods.Bootstrap support values from ML ≥ 70% and BI posterior values ≥ 0.9 are shown at nodes (ML/BI).Alternaria alternantherae CBS 124392 was the outgroup.* and red font indicates strains of this study.T indicates the ex-type strains, ET indicates the ex-epitype strains, HT indicates the ex-holotype strains.

Figure 2 .
Figure 2. Splitgraphs showing the results of the pairwise homoplasy index (PHI) test of newly described taxa and closely-related species using both LogDet transformation and splits decomposition A the PHI of Alternaria xinyangensis sp.nov.and A. dongshanqiaoensis sp.nov.with their phylogenetically related isolates or species B the PHI of A. shandongensis sp.nov., A. kunyuensis sp.nov., A. hunanensis sp.nov.and A. longqiaoensis sp.nov.with their phylogenetically related isolates or species C the PHI of A. cunninghamiicola sp.nov.with their phylogenetically-related isolates or species.PHI test value (Φ w ) < 0.05 indicate significant recombination within a dataset.* indicates strains of this study.T indicates the ex-type strains, ET indicates the ex-epitype strains, HT indicates the ex-holotype strains.

Table 1 .
Isolates used in this study and their GenBank accession numbers.

Table 2 .
Distinguishing characteristics of the new species and similar known species of Alternaria spp.under growth conditions a