Four New Species of Small-Spored Alternaria Isolated from Solanum tuberosum and S. lycopersicum in China

Small-spored Alternaria species have been frequently isolated from diseased leaves of Solanum plants. To clarify the diversity of small-spored Alternaria species, a total of 118 strains were obtained from leaf samples of S. tuberosum and S. lycopersicum in six provinces of China during 2022–2023. Based on morphological characterization and multi-locus phylogenetic analysis of the internal transcribed spacer of the rDNA region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase gene (EndoPG) and an anonymous gene region (OPA10-2), seven species were determined, including four novel species and three known species (A. alternata, A. gossypina and A. arborescens). The novel species were described and illustrated as A. longxiensis sp. nov., A. lijiangensis sp. nov., A. lycopersici sp. nov. and A. solanicola sp. nov.. In addition, the pathogenicity of the seven species was evaluated on potato leaves. The species exhibited various aggressiveness, which could help in disease management.


Introduction
The plants of the Solanaceae family are widely cultivated worldwide and have significant economic, medicinal and ornamental values.There are approximately 115 species belonging to 25 genera in China [1][2][3].For example, potatoes, peppers, tomatoes, eggplants, tobacco, etc. are important cash crops.Solanum is the largest genus, including more than 2000 plants including potato and tomato.(http://www.iplant.cn/)(accessed on 5 July 2023) As the fourth most important crop after rice, wheat and maize, potato (S. tuberosum) has become a staple food in many European countries because of its high nutritional value, rich in starch as well as protein, multivitamins and trace elements [4][5][6].China's potato acreage and its total production have consistently held a leading position in the world [7,8].Tomato (S. lycopersicum) is a popular fruit and vegetable containing abundant vitamin C, cultivated all over the world [9].Tomato fruit can be eaten raw, cooked, in processed ketchup, juice and so on, whose plant can also be used as potted ornamentals [10].Moreover, it has great medicinal properties, of which lycopene, a powerful antioxidant may protect against cardiovascular disease and some cancers [11].
The two Solanum crops are susceptible to fungal pathogen infection during their cultivation, which severely threatens their yield and quality [12,13].Especially for Alternaria spp., they are widely distributed with a great impact on economic returns of crops.When encountering favorable conditions, it can cause serious yield reduction or even no harvest [14,15].Alternaria foliar disease caused by large-spored Alternaria species has been frequently reported from section (sect.)Porri comprising A. solani, A. blumeae, A. linariae, A. grandis and A. protenta infecting both potatoes and tomatoes [14,16].Another type of Alternaria, Symptomatic samples of potato and tomato resembling Alternaria leaf spot or blight were randomly collected from 6 provinces in 2022-2023 (Table 1).The samples were preserved in sterile plastic bags and taken to laboratory for further isolation.The leaf fragments from edge of the lesions were cut and placed on moist filter papers in Petri dishes and incubated at 25 • C in dark forsporulation.Single spore was picked using a sterile glass needle under a stereomicroscope and inoculated onto potato dextrose agar (PDA: Difco, Montreal, Canada).Strains were kept into test-tube slants stored at 4 • C in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China.

Morphology and Culture Characteristics
Strains representing each species were selected and cultured on PDA at 25 • C in dark for 7 days to determine the cultural features.To confirm the conidial morphology (conidial size, shape, sporulation patterns, etc.), fresh fungal mycelia were transferred onto potato carrot agar (PCA) and V8 juice agar (V8A) media, and then incubated at 22 • C with 8 h light/16 h dark period [21].After 7 days, conidia and sporulation patterns were examined and photographed with a Nikon Eclipse Ni-U microscope system (Nikon, Tokyo, Japan).Fifty randomly selected conidia were measured for each strain.

Phylogenetic Analysis
The resulting sequences were checked by the BioEdit v. 7.2.3 [35] and primarily aligned using the program of PHYDIT v.3.2 [36].Phylogenetic analysis of RPB2 gene for all strains was performed for pre-test.Then, each of ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG and OPA10-2 gene sequences was analyzed by BLAST search in NCBI (https: //www.ncbi.nlm.nih.gov/)(accessed on 15 May 2023).Their relevant sequences were downloaded from the GenBank database.All gene sequences were spliced and edited through manual processing in MEGA v.7.0 [37].The multi-locus phylogenetic trees were constructed based on Bayesian inference (BI) and maximum likelihood (ML) analyses.
The ML analyses were performed using RAxML v.7.2.8 [38] using the GTRCAT model and bootstrapping with 1000 replicates.MrModeltest v.2.3 [39] used the Akaike Information Criterion (AIC) to determine the best-fit model (GTR + I + G) of nucleotide substitution, which was used for the Bayesian analyses performed with MrBayes v. 3.1.2[40].The analyses of two simultaneous Markov Chain Monte Carlo (MCMC) chains were run from random trees for 10, 000, 000 generations and sampled every 100th generations.The first 25% of the samples were discarded.Finally, the resulting trees were edited in FigTree v. 1.3.1. [41].Branch support of the analysis (>60%/0.6 for ML bootstrap value-BS/posterior probability-PP) was indicated in the phylogram.

Pathogenicity Tests
Twelve representative strains were selected to determine the pathogenicity of those present identified species, among which the four new species did not induce symptoms on tomato according to pre-tests except potato.Hence, the local grown potato cultivars were transplanted in pots and grown in greenhouse (25 • C, 12 h light period) for two weeks used for the experiment.The Alternaria strains were cultured on PDA at 25 • C for 3-5 days, and a 6 mm diameter disc was obtained from the colony edges and inoculated on healthy living leaves sterilized with 70% ethanol in the same greenhouse.The disease development was observed daily.After 7 days, the developed symptoms were recorded and the disease lesion size (LS) was measured.Control experiments were carried out simultaneously using clean PDA discs.For each strain, two potato plants were used and three sites were inoculated for each plant.To maintain the accuracy of the results, potato leaves of the same growing period with uniform size were selected for each test.The treatment was conducted four times.To complete Koch's postulates, the same inoculated Alternaria was successfully reisolated from their induced symptoms for morphology identification and RPB2 gene sequence analysis.The LS values were the mean value of four replicates ± standard deviation.The least significant difference test (p < 0.05) was conducted using IBM SPSS Statistics 23 for analysis.

Phylogenetic Analysis
A total of 118 small-spored Alternaria strains were obtained according to morphological traits.The preliminary RPB2 gene sequence analysis showed that all strains belonged to sect.Alternaria, of which 88.13% was A. alternata and the others appeared as six taxa with different culture characteristics.Two strains of A. alternata and strains of the six taxa were further determined using the other six gene loci.Phylogenetic analysis included 88 strains from sect.Alternaria (Table 2), which comprised both reference strains and the present strains.The analysis was based on a combined ITS, GAPDH, TEF1, RPB2, Alt a 1, OPA10-2 and EndoPG sequence dataset, which included 494, 501, 215, 596, 437, 627 and 442 characters after alignment, respectively.Alternaria alternantherae (CBS 124392) from sect.Alternantherae was chosen as the outgroup taxon.The BI and ML analyses exhibited similar topologies.The ML tree was used as the basal tree (Figure 1).The result was similar to the RPB2 gene phylogram.Four selected strains were well merged into the clades of three known Alternaria species, A. alternata, A. arborescens and A. gossypina, supported with PP/BS values of 0.97/79, 0.99/99 and 0.95/98, respectively.The other eight strains fell into four well independent clades supported with 0.99-1.00PP values and 99-100% BP values, close to A. orobanches and A. ovoidea [42,43] in a branch supported with 0.80/73 PP/BS values.The results indicate that the four clades represent four new species.
Notes: Based on the combined dataset of ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG and OPA10-2 gene fragments, the results reveal that the strains fall in an individual wellsupported clade representing a new species, which is closer to A. longxiensis sp.nov.and A. solanicola sp.nov., near to A. orobanches.After a nucleotide pairwise comparison, the present species can be readily differentiated from the other two related novel species based on Alt a 1, RPB2 and OPA10-2 gene regions, which has 21 bp differences in the Alt a 1 region, 17 bp in RPB2 and 19 bp in OPA10-2 when compared with A. longxiensis.
For A. solanicola sp.nov., there are 18 bp nucleotide differences in Alt a 1 region, 16 bp in RPB2 and 20 bp in the OPA10-2 region.Morphologically, its conidia are smallest, clavate and clearly smooth-walled with shorter apical beaks compared to the other two new species.(Figure 2, Table 3).
Notes: Based on the combined dataset of ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG and OPA10-2 gene fragments, the results reveal that the strains fall in an individual wellsupported clade representing a new species, which is closer to A. longxiensis sp.nov.and A. solanicola sp.nov., near to A. orobanches.After a nucleotide pairwise comparison, the present species can be readily differentiated from the other two related novel species based on Alt a 1, RPB2 and OPA10-2 gene regions, which has 21 bp differences in the Alt a 1 region, 17 bp in RPB2 and 19 bp in OPA10-2 when compared with A. longxiensis.
For A. solanicola sp.nov., there are 18 bp nucleotide differences in Alt a 1 region, 16 bp in RPB2 and 20 bp in the OPA10-2 region.Morphologically, its conidia are smallest, clavate and clearly smooth-walled with shorter apical beaks compared to the other two new species.(Figure 2, Table 3) Notes: The species is phylogenetically recognized as a distinct species that forms a subclade with A. longxiensis sp.nov. in a branch containing A. lycopersici sp.nov.and A. orobanches.On the bases of the RPB2, OPA10-2 and Alt a 1 gene sequences, this species, respectively, comprises 3 bp, 20 bp and 18 bp nucleotide differences from A. longxiensis.According to morphology, it can be differentiated from A. longxiensis by producing Notes: The species is phylogenetically recognized as a distinct species that forms a subclade with A. longxiensis sp.nov. in a branch containing A. lycopersici sp.nov.and A. orobanches.On the bases of the RPB2, OPA10-2 and Alt a 1 gene sequences, this species, respectively, comprises 3 bp, 20 bp and 18 bp nucleotide differences from A. longxiensis.
According to morphology, it can be differentiated from A. longxiensis by producing conidia with longer beaks and fewer septa and by comprising fewer spore units in a chain with 1-2 branches.(Figure 3, Table 3).
Notes: Phylogenetic analysis shows that the species falls into an independent lineage sister to the A. orobanches, A. ovoidea and three new species.Morphologically, it can be easily distinguished from the relevant species by producing conidia with the longest beak (up to 40 μm).(Figure 5, Table 3) Notes: Phylogenetic analysis shows that the species falls into an independent lineage sister to the A. orobanches, A. ovoidea and three new species.Morphologically, it can be easily distinguished from the relevant species by producing conidia with the longest beak (up to 40 µm).(Figure 5, Table 3).

Pathogenicity Assays
Pathogenicity tests indicated that the seven small-spored Alternaria species (A.alternata, A. arborescens, A. gossypina, A. lycopersici, A. solanicola, A. longxiensis and A. lijiangensis) of sect.Alternaria were all pathogenic to S. tuberosum and showed varying degrees of pathogenicity.No symptoms were observed in the controls.(Figure 6, Table 4) After three days, black spots began to appear on the leaves and gradually expand, accompanied by yellow halos.Strains of A. lijiangensis exhibited the most severe symptom with LS up to 30.5 mm (av.= 27.5)resulting in a whole dark brown leaf wilting.The disease severity was followed by A. gossypina (LS up to 24 mm) and A. arborescens with LS around 18 to 20 mm, and then A. longxiensis sp.nov., A. lycopersici sp.nov., and A. solanicola sp.nov..The weakest pathogenicity on potato was A. alternata among all the tested species.

Pathogenicity Assays
Pathogenicity tests indicated that the seven small-spored Alternaria species (A.alternata, A. arborescens, A. gossypina, A. lycopersici, A. solanicola, A. longxiensis and A. lijiangensis) of sect.Alternaria were all pathogenic to S. tuberosum and showed varying degrees of pathogenicity.No symptoms were observed in the controls.(Figure 6, Table 4) After three days, black spots began to appear on the leaves and gradually expand, accompanied by yellow halos.Strains of A. lijiangensis exhibited the most severe symptom with LS up to 30.5 mm (av.= 27.5)resulting in a whole dark brown leaf wilting.The disease severity was followed by A. gossypina (LS up to 24 mm) and A. arborescens with LS around 18 to 20 mm, and then A. longxiensis sp.nov., A. lycopersici sp.nov., and A. solanicola sp.nov..The weakest pathogenicity on potato was A. alternata among all the tested species.

Pathogenicity Assays
Pathogenicity tests indicated that the seven small-spored Alternaria species (A.alternata, A. arborescens, A. gossypina, A. lycopersici, A. solanicola, A. longxiensis and A. lijiangensis) of sect.Alternaria were all pathogenic to S. tuberosum and showed varying degrees of pathogenicity.No symptoms were observed in the controls.(Figure 6, Table 4) After three days, black spots began to appear on the leaves and gradually expand, accompanied by yellow halos.Strains of A. lijiangensis exhibited the most severe symptom with LS up to 30.5 mm (av.= 27.5)resulting in a whole dark brown leaf wilting.The disease severity was followed by A. gossypina (LS up to 24 mm) and A. arborescens with LS around 18 to 20 mm, and then A. longxiensis sp.nov., A. lycopersici sp.nov., and A. solanicola sp.nov..The weakest pathogenicity on potato was A. alternata among all the tested species.

Discussion
Simmons dedicates to the morphological taxonomy of Alternaria using a life time.A total of 276 species are comprehensively illustrated based on sporulation patterns and conidial morphology, comprising 128 small-spored Alternaria species [21].With the continuous research on the taxonomy of small-spored Alternaria aided with molecular approach, the sect.Alternaria is one of the largest sections containing 11 species and one species complex [20], of which A. alternata includes 35 morph-species described by Simmons [21].There are 27 new species latterly defined as new members of sect.Alternaria [27,42,43,45].In this study, four novel species (A.lycopersici sp.nov., A. solanicola sp.nov., A. longxiensis sp.nov.and A. lijiangensis sp.nov.) were as found in China and added in the section.
Phylogenetically, nine commonly used genetic regions (SSU, LSU, ITS, GADPH, RPB2, TEF1, Alt a1, EndoPG, and OPA10-2) have been used for the delamination of species within sect.Alternaria [46][47][48].Among them, the RPB2 gene is considered as a nuclear gene with the advantages of being single-copy and having a slow evolutionary rate.It is capable of effectively distinguishing species for both large-spored and small-spored type [49,50], and is also applicable for the identification of other pathogenic fungi [51].In this present study, seven gene loci without SSU and LSU were performed and clearly separated the four new taxa.In addition, the RPB2 gene was again confirmed for the ability of fungal classification, which resulted in similar consequences to distinguish the present seven Alternaria species (Figure 1).The present novel species fell into four independent clades together with A. orobanches and A. ovoidea, which were isolated from Orobanche sp. and Dactylis glomerata in Italy, respectively [42,43].
Morphologically, these four new species can be distinguished by their conidia and sporulation patterns.Among them, A. lijiangensis has conidia with the longest beaks among the four present novel species and its relevant species, A. orobanches and A. ovoidea.The other three new species had their own characteristics and could be differentiated with A. orobanches and A. ovoidea by sporulation pattern (more conidial units per chain).(Table 3) For example, conidia of A. lycopersici has a shorter apical cell secondary conidiophore (beak); conidia of A. longxiensis has a longer beak; A. solanicola has 1-2 branches near the main chain.The results indicated the correlation between morphology and molecular composition, and also stated the usefulness of morphological traits (conidial morphology and sporulation pattern) described by Simmons [20] for the taxonomy of small-spored Altenraria.
The large-spored A. solani and A. linariae, are considered to be the primary pathogen of foliar disease in the Solanaceae family [14,44,52], but small-spored Alternaria are also frequently isolated from symptomatic tissues worldwide.Nabahat et al. [17] found that species within sect.Alternaria were dominant populations in Solanaceae plants during

Figure 1 .
Figure 1.Phylogenetic tree based on the combined gene sequences of ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG and OPA10-2 generated from Alternaria spp. on potatoes and tomatoes.The Bayesian

Figure 1 .
Figure 1.Phylogenetic tree based on the combined gene sequences of ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG and OPA10-2 generated from Alternaria spp. on potatoes and tomatoes.The Bayesian posterior probabilities (PP > 0.60) and maximum likelihood bootstrap values (BS > 60%) are given at the nodes (PP/BS).Examined present strains are in bold.

Figure 6 .
Figure 6.Pathogenicity of the seven present small-spored Alternaria species on Solanum tuberosum.

Table 1 .
The numbers of Alternaria strains collected from potato and tomato in different provinces of China.

Table 4 .
Disease incidence and lesion size of seven present small-spored Alternaria species on Solanum tuberosum.Disease incidence (DI) was evaluated by counting the percentage of diseased leaves.Lesion size (LS) values are the mean value of three replicates ± standard deviation.Values followed by different lowercase letters within a column are significantly different according to the least significant difference test (p < 0.05) using IBM SPSS Statistics 23. Notes: