New Species of Large-Spored Alternaria in Section Porri Associated with Compositae Plants in China

Alternaria is a ubiquitous fungal genus including saprobic, endophytic, and pathogenic species associated with a wide variety of substrates. It has been separated into 29 sections and seven monotypic lineages based on molecular and morphological data. Alternaria sect. Porri is the largest section, containing the majority of large-spored Alternaria species, most of which are important plant pathogens. Since 2015, of the investigations for large-spored Alternaria species in China, 13 species were found associated with Compositae plants based on morphological comparisons and phylogenetic analyses. There were eight known species and five new species (A. anhuiensis sp. nov., A. coreopsidis sp. nov., A. nanningensis sp. nov., A. neimengguensis sp. nov., and A. sulphureus sp. nov.) distributed in the four sections of Helianthiinficientes, Porri, Sonchi, and Teretispora, and one monotypic lineage (A. argyranthemi). The multi-locus sequence analyses encompassing the internal transcribed spacer region of rDNA (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen gene (Alt a 1), translation elongation factor 1-alpha (TEF1), and RNA polymerase second largest subunit (RPB2), revealed that the new species fell into sect. Porri. Morphologically, the new species were illustrated and compared with other relevant large-spored Alternaria species in the study. Furthermore, A. calendulae, A. leucanthemi, and A. tagetica were firstly detected in Brachyactis ciliate, Carthamus tinctorius, and Calendula officinalis in China, respectively.


Introduction
Alternaria is a cosmopolitan and widely distributed fungal genus described originally by Nees (1816), which is characterized by the dark-coloured phaeodictyospores in chains and a beak of tapering apical cells [1]. It is also associated with nearly every environmental substrate including animal, plant, agricultural product, soil, and the atmosphere. Species of Alternaria are known as serious plant pathogens, causing enormous losses on many crops [1,2]. The taxonomy is mainly based on sporulation patterns and their conidial shape, size, and septation [2,3]. Around 280 species are summarised and recognised on the basis of morphology [2], comprising two groups, large-spored (60-100 µm long conidial body) and small-spored (below 60 µm conidial body) [4][5][6].
Due to the effects of Alternaria on humans and their surroundings, the identification is particularly important to agriculture, medicine, and science. The Compositae plants serve as food plant, oil seed, seed plant, ornamental, and sources of medicine and insecticide worldwide [22], of which nearly 3000 species almost 240 genera have been found in China [23]. Most Alternaria are commonly plant pathogens leading to substantial economic losses caused by Alternaria leaf spots and defoliation [18,[24][25][26]. Large-spored Alternaria species encompassing 148 species are almost phytopathogenic demonstrated [2].
During the investigation of large-spored Alternaria in China, five new species were encountered from diseased leave samples of composite plants. The objectives of this study were to identify them on the basis of the cultural and conidial morphology incorporate with multi-loci phylogeny (ITS, GAPDH, Alt a 1, TEF1, and RPB2). The present multilocus analysis supplemented with cultural and morphological data forms an example for Alternaria species recognition. The five new species described in this study add species diversity to large-spored Alternaria and provide theoretical and practical basis for the further identification and disease management.

Sample Collection and Fungal Isolation
Symptomatic samples of composite plants (14) have been randomly collected from different provinces in China since 2015. For fungal isolation, the samples were put into sterile plastic bags and taken to the laboratory. Small leaf segments (2 mm) with disease lesions were placed into petri dishes with moist filter papers and incubated at 25 • C in dark for conidial sporulation. Single spore of large-spored Alternaria was picked by a sterilized glass needle under the stereoscopic microscope and transferred to potato dextrose agar (PDA: Difco, Montreal, Canada). Over ten similar spores were randomly picked from a sample for sub-culturing to obtain the pure cultures, and two to three strains were selected for deposition when exhibiting similar cultural morphology on PDA. A total of 81 strains were kept in test-tube slants and deposited at 4 • C. Living ex-type strains were preserved in the Fungi Herbarium of Yangtze University (YZU), in Jingzhou, Hubei, China.

Morphological Observations
To determine cultural characteristics including growth rate, color and texture of colonies [27], mycelial plugs (6 mm in diameter) were taken from the edge of colonies grown on PDA. Then, the plugs were put on fresh PDA plates (90 mm) at 25 • C for 7 days in darkness. To observe the conidial morphology (conidial sporulation patterns, shape, size, etc.), mycelia were grown on potato carrot agar (PCA) and V8 juice agar (V8A) inoculated at 22 • C with a light period of 8 h light/16 h dark [2]. After 7 days, conidia and sporulation patterns were observed. Conidiophores and conidia were mounted with lactophenol picric acid solution and photographed with a Nikon ECLIPSE Ni-U microscope (Nikon, Japan). Randomly selected conidia (n = 50) were separately measured for each characterization.
Successfully amplified PCR products were purified and sequenced by TSINGKE company (Beijing, China).

Phylogenetic Analyses
The resulted sequences were examined by BioEdit v.7.0.9 [34] and assembled with PHYDIT 3.2 [35]. All newly generated sequences were deposited in GenBank (Table 1). Relevant sequences [4] were retrieved from NCBI database based on the results of BLAST searches ( Table 1). The concatenated sequence dataset of multiple loci was aligned using MEGA v.6.0 [36]. Phylogenetic analyses of each alignment were performed using maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was conducted using RAxML v.7.2.8 [37]. Bootstrapping with 1000 replicates was performed using the model of nucleotide substitution obtained by MrModeltest. For the BI analysis, it was performed using parameters including 1,000,000 Markov chain Monte Carlo (MCMC) algorithm with Bayesian posterior probabilities [38]. MrModel test v.2.3 used the best-fit model (GTR+I+G) according to the Akaike Information Criterion (AIC). Two MCMC chains were run from random trees for 10 6 generations, and the trees were sampled every 100th generation. After discarding the first 25% of the samples, the 50% majority rule consensus tree and posterior probability values were calculated. Finally, the resulting trees were edited in FigTree v.1.3.1 [39]. Branch support of the groupings (>60%/0.6 for ML bootstrap value-BS/posterior probability-PP) were indicated in the phylogram. Alternaria gypsophilae CBS 107.41 in sect. Gypsophilae was used as an outgroup.

Results
In the present study, large-spored Alternaria species associated with Compositae leaf spot in China since a survey from 2015 are summarized based on the phylogenetic analysis of GAPDH and RPB2 gene fragments ( Figure S1 and Table S1). A total of 13 species including the present five new taxa revealed in four sections of Helianthiinficientes (A. helianthiinficiens), Porri (A. calendulae, A. tagetica and A. zinniae), Sonchi (A. cinerariae and A. sonchi), and Teretispora (A. leucanthemi), and one monotypic lineage (A. argyranthemi) ( Figure S1). Meanwhile, a comprehensive description of the five new species in sect. Porri are described as A. anhuiensis sp. nov., A. coreopsidis sp. nov., A. nanningensis sp. nov., A. neimengguensis sp. nov., and A. sulphureus sp. nov..

Phylogenetic Analysis
The multi-gene phylogeny was constructed to determine the accurate positions of the new Alternaria based on five sequence loci (ITS + GAPDH + Alt a 1 + TEF1 + RPB2) ( Table 1). The analysis comprised sequences of the ITS (504 characters), GAPDH (526 characters), Alt a 1 (457 characters), TEF1 (342 characters), and RPB2 (672 characters) gene regions with a total length of 2501 characters. The tree topologies (Figure 1) computed from the ML and BI analyses, were similar to each other, resulting in identical species-clades and the ML topology was presented as basal tree. The present strains fell into five separate branches in sect. Porri of Alternaria. Strain YZU 171206 was sister to A. alternariacida supported with a PP value of 1.0, which close to A. silybi with low BS and PP values surpport. Strains YZU 161159 and YZU 161160 formed an independent clade (BS/PP = 100%/1.0). Strain YZU 171523 fell into an individual branch close to A. obtecta and A. tillandsiae well-supported by 97%/0.99 (BS/PP). Strain YZU 171784 was clustered with A. cirsinoxia, A. centaureae, A. cichorii, and A. cantannaches supported by values of 79%/1.0 (BS/PP). Strain YZU 191448 was out group of strain YZU 171206, A. silybi and A. alternariacida with BS and PP values below 60% and 0.6. The results indicated that the five branches represent five new species from three different hosts (Coreopsis basalis, Cosmos sulphureus, and Lactuca seriola). Notes: Phylogenetic analysis of the species based on a combined dataset of ITS, GAPDH, Alt a 1, TEF1, and RPB2 gene fragments falls in an individual clade close to A. alternariacida and A. silybi in sect. Porri (Figure 1). Morphologically, its primary conidiophores can generate geniculate conidiogenous loci at or near apex which differed from those two species (Figure 2, Table 2). It can be easily distinguished from A. alternariacida by producing more transverse septa and shorter beaks.       Notes: Phylogenetically, the species falls into an independent lineage outside of a clade comprising type species of A. porri of sect. Porri (Figure 1). It can be delimited based on either of GAPDH and RPB2 gene sequences ( Figure S1). The species is characterized by producing conidia with false beak swollen at the apex up to 8-13 (-16.5) × 4.5-6.5 µm (Figure 3; Table 2).
Notes: The species is phylogenetically recognized as a distinct species in sect. Porri based on ITS, GAPDH, Alt a 1, TEF1, and RPB2 which displays a close relationship with A. obtecta, A. tillandsiae, and A. steviae (Figure 1). Compared with them, it is quite different by producing smaller conidia with short beaks (Figure 4; Table 2). Furthermore, its conidia are smooth-walled while some conidia of A. obtecta and A. steviae are minutely punctulate. Alternaria nanningensis forms simple conidiophores (solitary). But many conidiophores of A. steviae produce geniculate extensions and additional conidia, yielding tiny distal clumps of sporulation.  Notes: In the phylogeny, the species is sister to A. cirsinoxia, A. centaureae, A. cichorii, and A. catananches (Figure 1). The conidiophores are distinct to A. cirsinoxia whose are 2-3 arm branches near a conidiophore tip and progressively geniculate, yielding tufts of several conidia. They are different from A. cichorii whose are frequently branch or proliferate in a geniculate manner near the apex, yielding terminal clumps of 4-5 conidia. In conidial morphology, it is obviously different from those four species by producing larger conidia (Table 2).
Alternaria sulphureus L. Zhao and J.X. Deng, sp. nov. (Figure 6). Notes: This species is phylogenetically related to A. silybi, A. alternariacida and A. anhuiensis sp. nov. in sect. Porri (Figure 1). It could be distinguished from A. silybi and A. alternariacida by forming larger conidia ( Figure 6; Table 2) and is quite different from A. alternariacida by producing multiple and shorter beaks.

Discussion
Thirteen large-spored Alternaria species associated with Compositae leaf spot in China were assigned to four sections and one monotypic lineage in this study. Among theses species, five new species (A. anhuiensis sp. nov., A. coreopsidis sp. nov., A. nanningensis sp. nov., A. neimengguensis sp. nov., and A. sulphureus sp. nov.) were clearly recognized in section Porri. The section is speciose assessing encompassing 117 large-spored Alternaria [5]. In 2014, the section is reduced 82 morphospecies in to 63 phylogenetic species [2]. They are commonly pathogenic and could induce typical black necrotic lesions surrounded by chlorotic areas. There are some important famous plant pathogens, such as A. porri on Allium plants (Liliaceae), A. solani for potato (Solanaceae), A. sesami for sesame (Pedaliaceae) and A. dauci for carrot (Umbelliferae) [2]. Twenty-one species are comprised in sect. Porri associated with the Compositae family [4]. This study provides new data supplements for the Alternaria taxonomy of sect. Porri.
Morphologically, large-spored Alternaria species in sect. Porri are characterised by broadly ovoid, obclavate, ellipsoid, subcylindrical or obovoid, medium to large conidia containing multiple transverse and longitudinal septa, solitary or in short chains with a simple or branched, long to filamentous beak [4]. Among these characteristics, sporulation patterns, conidial body, transverse septa, and beak type provide useful information for the preliminary separation into sections [2]. Morphology is quite important for new fungal species identification, which can be defined based on unique morphological characters when the molecular data is not well-supported [41]. Morphological comparisons of the present new species and their relevant species in sect. Porri were conducted (Table 2). For the sporulation patterns, the conidia of A. anhuiensis, A. nanningensis, A. neimengguensis, and A. sulphureus are solitary produced except A. coreopsidis, which similar to A. alternariacida, A. cichorii, A. cirsinoxia, and A. steviae forming chain of 2 (-3) units [2,4]. In conidial morphology, A. anhuiensis, A. nanningensis, A. neimengguensis, and A. sulphureus are distinguishable from their closely related species based on the size of conidial bodies (Table 2) and also the wall ornamentations [2,4]. On the other hand, A. anhuiensis, A. neimengguensis, and A. sulphureus are readily be distinguished by producing multiple beaks. By the way, there are no significant differences on conidial morphology of PCA and V8A medium for all species.
In addition, morphological variation and fundamental pleomorphism complicate the Alternaria species recognition, and host plants reflect some evidences for the identification [3]. With the discovery of Alternaria species, it has been found from various plants of Compositae [1,4,21,42,43]. Alternaria calendulae has been reported from Calendula officinalis in Czech Republic [2], Germany [4], Japan [4], and Korea [44]. It also is found on C. officinalis in China and firstly on Brachyactis ciliata in the study. Alternaria leucanthemi has previously been found on Chrysanthemum maximum from Netherlands [1] and Helianthus annuus from China [45]. It is firstly isolated from Carthamus tinctorius in this study. In addition, A. tagetica is commonly associated with Tagetes plants (Tagetes erecta and Tagetes patula) [3,4,[46][47][48], which firstly encountered from Calendula officinalis in this study. Interestedly, the five new species are isolated from three different composite hosts (Coreopsis basalis, Cosmos sulphureus, and Lactuca seriola) and A. cinerariae are found on five different composite plants in China ( Figure S1; Table S1). The results suggest that an Alternaria species may associated with several host plants.

Conclusions
The present data indeed revealed a diversity of large-spored Alternaria associated with Compositae plants in China. A total of 13 large-spored Alternaria species were obtained and circumscribed as eight known species and five new species belonging to the four sections of Helianthiinficientes, Porri, Sonchi, and Teretispora, and one monotypic lineage (A. argyranthemi) based on the morphological characteristics and molecular properties of multiple DNA sequences (ITS, GAPDH, Alt a 1, TEF1, and RPB2). Alternaria calendulae, A. leucanthemi, and A. tagetica were firstly isolated from Brachyactis ciliate, Carthamus tinctorius, and Calendula officinalis in China, respectively. Since large-spored Alternaria species are almost demonstrated phytopathogens, further study on the pathogenicity is needed to verify in the future.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8060607/s1, Figure S1: Phylogenetic tree of large-spored Alternaria from the Compositae family in China using a maximum likelihood (ML) analysis based on combined GAPDH and RPB2 gene sequences. The RAxML bootstrap support values > 60% (ML) and Bayesian posterior probabilities >0.6 (PP) are given at the nodes (ML/PP); Table S1: The other Altenraria species associated with the Compositae plants from China analyzed by phylogeny. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The sequences newly generated in this study have been submitted to the GenBank database.