Alternaria arborescens and A. italica Causing Leaf Blotch on Celtis julianae in China

Celtis julianae Schneid. is widely planted as a versatile tree species with ecological and economic significance. In September 2022, a leaf blotch disease of C. julianae was observed in Nanjing, Jiangsu, China, with an infection incidence of 63%. The disease led to severe early defoliation, significantly affecting the ornamental and ecological value of the host tree. The accurate identification of pathogens is imperative to conducting further research and advancing disease control. Koch’s postulates confirmed that the fungal isolates (B1–B9) were pathogenic to C. julianae. The morphology of the characteristics of the pathogen matched those of Alternaria spp. The internal transcribed spacer region (ITS), large subunit (LSU) and small subunit (SSU) regions of rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen gene (Alt a 1), RNA polymerase second largest subunit (RPB2), and portions of translation elongation factor 1-alpha (TEF1-α) genes were sequenced. Based on multi-locus phylogenetic analyses and morphology, the pathogenic fungi were identified as Alternaria arborescens and A. italica. The findings provided useful information for disease management and enhanced the understanding of Alternaria species diversity in China. This is the first report of A. arborescens and A. italica causing leaf blotch of C. julianae in China and worldwide.


Introduction
Celtis julianae Schneid.(Ulmaceae) is a large deciduous tree that can reach a height of over 25 m.It serves as a valuable landscaping and shelter tree species due to its ability to withstand soot and toxic gases [1].Additionally, its kernels are used as raw material for soap and lubricating oil production due to their high oil content.The numerous hairs on leaf surfaces enable it to trap airborne particulates, contributing to air purification [2].In September 2022, a leaf blotch disease was observed on C. julianae at Nanjing Forestry University (118 • 48 26 E, 32 • 4 52 N).The disease led to early defoliation in severe cases, resulting in a reduction of its ornamental and ecological value.
The traditional identification of plant pathogenic fungi has mainly relied solely on morphological characteristics and host association.However, when relying solely on morphological characteristics, such as conidia, conidiophores, conidiogenous cells, and fruiting body, the pathogenic fungi may not be accurately classified and determined.Phylogenetic analyses have been widely applied for decades, leading to the discovery of numerous new species [21][22][23].Initially, the internal transcribed spacer region (ITS) was commonly used for taxonomic purposes.However, studies have pointed out the limitations of using ITS sequence data [24][25][26].In many fungal genera, the ITS locus only resolves taxa to the genus level due to the intraspecific and even intragenomic polymorphisms commonly observed [27,28].Moreover, a substantial percentage of ITS sequences in GenBank are derived from misidentified specimens or cultures [29,30].As a result, secondary DNA barcodes have been proposed for various genera of plant pathogenic fungi [31][32][33].Nowadays, multi-locus phylogenetic analysis is considered more appliable and reliable in fungal taxonomy [34].
Similarly, Alternaria species were traditionally classified based on morphological characteristics of their reproductive structures and sporulation patterns under various conditions [35].Alternaria spp.are dematiaceous fungi, exhibiting grey-olive/brown colonies.The conidia of Alternaria are dictyospores.These can either be found alone or in chains [36].However, the subtle differences between Alternaria spp.and the morphological variation under different conditions can make identification based on solely on morphological characteristics challenging.With the advent of molecular analyses, researchers have examined phylogenetic relationships among Alternaria species.Lawrence et al. [12,18] first determined 27 sections in Alternaria through multi-locus phylogeny.Woudenberg et al. [6] used six loci to establish 24 sections, including 16 newly described sections.By combining seven loci, a consensus phylogeny was generated.Ghafri et al. [37] introduced Alternaria section Omanenses in 2019, and Gannibal et al. [38] introduced Alternaria section Helianthiinficiens in 2022, bring the total number of Alternaria species to 29 sections.Currently, a clear and stable phylogenetic classification has been established at the species level among Alternaria spp., providing a reliable basis for this study.
The objectives of this study were (1) to isolate and identify the pathogen of leaf blotch on C. julianae according to morphological characteristics and multilocus phylogenetic analyses, and (2) to confirm the pathogenicity of the isolates on C. julianae.

Disease Symptoms and Fungal Isolations
The leaf blotch disease was observed on C. julianae at the campus of Nanjing Forestry University, China, with an incidence of 63% (63/100 plants).On average, 80% of the leaves per tree showed the presence of this leaf blotch disease (Figure 1A,B).The symptoms initially appeared as small, light brown spots.Measuring 1-2 mm in diameter, they were surrounded by yellow halos (Figure 1C).Subsequently, the spots gradually expanded into irregular necrotic blotches with dark brown borders, and the halos developed around the dark brown borders, leading to an increase in the size of the blotches over time (Figure 1D).Eventually, the blotches coalesced into large necrotic areas, resulting in leaf shrinkage, wrinkling, and collapse (Figure 1E).Numerous brown conidial chains were observed on the lesions of infected leaves (Figure 1F,G).Diseased leaves were collected twice for isolating fungal pathogens in Septembe in October 2022, respectively.In the September experiment, three types of colonies found with a frequency of 63%, 24%, and 13%, respectively.Similarly, in the Octob periment, three types of colonies were found with a frequency of 54%, 38%, and 8% spectively.According to the ITS sequence alignment, the three types of colonies belo to the genera Nigrospora, Alternaria and Nothophoma, respectively (Table 1).

Pathogenicity Tests
Five days post-inoculation in vitro, all detached leaves inoculated with nin lates (B1-B9) of Alternaria spp.appeared to display brown spot symptoms at th oculation sites, whereas leaves inoculated with Nigrospora sp., Nothophoma sp Diseased leaves were collected twice for isolating fungal pathogens in September and in October 2022, respectively.In the September experiment, three types of colonies were found with a frequency of 63%, 24%, and 13%, respectively.Similarly, in the October experiment, three types of colonies were found with a frequency of 54%, 38%, and 8%, respectively.According to the ITS sequence alignment, the three types of colonies belonged to the genera Nigrospora, Alternaria and Nothophoma, respectively (Table 1).

Pathogenicity Tests
Five days post-inoculation in vitro, all detached leaves inoculated with nine isolates (B1-B9) of Alternaria spp.appeared to display brown spot symptoms at the inoculation sites, whereas leaves inoculated with Nigrospora sp., Nothophoma sp. and control leaves did not show any symptoms.Subsequently, in vivo experiments were conducted that used control leaves did not show any symptoms.Subsequently, in vivo experiments were conducted that used three representative fungal isolates (B1, B2 and B3) of the Alternaria species to inoculate healthy C. julianae seedlings.Seven days after inoculation, the inoculated leaves appeared to be brown spots with yellow halos, resembling the early symptoms observed on the leaves infected in the field.In contrast, the control leaves remained healthy (Figure 2).The same fungus was re-isolated from the lesions, and no other fungi were isolated from the control leaves.This fulfills Koch's postulates, confirming that isolates B1, B2 and B3 are the causal agents of leaf blotch on C. julianae.

Discussion
The genus Alternaria was first described by Nees von Esenbeck (1816), with A. tenuis as the type of species.Historically, the identification and classification of Alternaria species heavily relied on measurements and descriptions of morphological characteristics [41], complemented later by molecular phylogeny [42,43].However, recent studies have challenged the morphological basis for the identification of some species in Alternaria [11,13,44].Currently, the genus Alternaria contains 29 sections, and the main morphological characteristic of Alternaria sect.Alternaria is the production of short conidia in chains [6,18,45].Woudenberg et al. [6] established species in Alternaria sect.Alternaria based on ITS.Subsequent whole-genome sequencing, transcriptome comparisons, and multi-gene sequencing further rearranged Alternaria sect.Alternaria into 11 phylogenetic species and one species complex [13].Following a number of studies conducted by Li et al. [3,4], Cannibal et al. [45,46], Wanasinghe et al. [47], Jayawardena et al. [48] and Nishikawa et al. [49], currently over 89 species constitute the sect.Alternaria.Traditional morphological methods with molecular phylogeny are vital for identifying Alternaria species.In this study, a multi-locus phylogeny based on a concatenated ITS, LSU, SSU, GAPDH, Alt a 1, RPB2 and TEF1-α sequences, combined with morphological characteristics, revealed two known species, A. arborescens and A. italica, within the sect Alternaria.Additionally, our study also reported new host records for A. arborescens and A. italica.It is worth noting that many pathogenic fungal species are named after their hosts, and that the two Alternaria species identified in this study belong to the same section and is isolated from the same host.The similarities in their morphological features highlight the importance of taking cautious approaches in identifying and naming Alternaria species.
Alternaria is among the primary mycotoxigenic fungal genera found in cereals worldwide [50].It can produce a variety of mycotoxins derived from secondary metabolism, with about 70 toxic metabolites described so far [35].Some pathogenic Alternaria spp.utilize a diverse array of pathogenic toxins to infect plant tissues [51,52].Certain species of Alternaria produce host-specific toxins (HSTs) that influence their pathogenicity and virulence.For example, the Japanese pear pathotype associated with Alternaria produces AK toxins I and II, which only exhibit toxicity in susceptible pear cultivars [35].In addition, HSTs affect the taxonomy of the genus as fungal systematics include the analysis of secondary metabolites, providing information for use in species differentiation.At present, several researchers have employed chemotaxonomy and pathogenicity characteristics to assist with classification [53][54][55].However, the method cannot be used independently for classification, because members within the same group may not share a common metabolite profile [50].Therefore, the application of polyphasic approaches could provide strong support in fungal taxonomy, and further studies are warranted.
In our study, we observed that the isolation rates of Nigrospora spp.were consistently higher than those of Alternaria spp. in two independent fungal isolations.However, coexistence observation and pathogenicity testing showed that Alternaria spp. was the pathogen causing leaf blotching of C. julianae.Previous studies have indicated that species the of genus Nigrospora possess a strong saprobic nature and commonly occur as plant endophytes or saprobes on different hosts [56,57].Nigrospora spp.may have more advantages in competing for nutrients in substrates compared to Alternaria spp., which can slow the growth of Alternaria.This could be the reason for the higher isolating rates of Nigrospora than those of Alternaria spp.
Celtis julianae is an important ornamental species mainly found in the temperate biome, with its native range overlaying the central and southern parts of China [58].It has a high level of wind resistance due to its deep roots, making it an ideal species for embankment protection and water conservation [59].Additionally, C. julianae helps maintain air humidity, reduce heat, and create various shadows with its large and dense leaves [60].Economically, C. julianae has rich bark fiber, which can be used for making paper, and the core of its fruit can be used in oil and soap manufacture.In summary, C. julianae is a multi-functional species with ornamental, ecological and economic value.Currently, research on diseases affecting C. julianae is insufficient.Therefore, the investigation of leaf blotch on C. julianae and identification of pathogens have significance in providing the theoretical basis for reducing the economic and ecological losses caused by this disease.

Sampling and Isolation of Fungi
From September to October 2022, the isolation of fungi was conducted twice.Each time, 25 symptomatic leaves were collected from three infected plants on the campus of Nanjing Forestry University.The symptomatic leaves were first rinsed under running water and dried on sterilized filter paper.One-hundred small tissue (3 × 3 mm) samples were cut from lesion margins and surface-sterilized in 75% ethanol for 30 s, followed by the use of 1% NaClO for 90 s.The samples were then rinsed 3 times in sterile water, dried on sterilized filter paper, and finally plated onto potato dextrose agar (PDA) supplemented with ampicillin.The plates were incubated at 25 • C in an incubator, MIR-553 (Sanyo, Osaka Plants 2023, 12, 3113 9 of 20 Japan) [61].After 3 days, the growing edges of the mycelium from the plant materials were cut and transferred to fresh PDA media to obtain a pure culture [62].

Pathogenicity Tests
To determine the pathogenicity of the isolates, experiments were conducted on both detached leaves and attached leaves.Healthy leaves from the field were collected, rinsed under tap-water for 15 min, and then wounded with sterile needles after drying on sterilized filter paper.For each type of test, two isolates were selected.For inoculation, plugs (5 mm diam.) were cut out from the growing edges of 5-day-old cultures and placed onto the wounds.The PDA plugs were used as controls.Five wounded leaves were treated with isolate and control plug.After inoculation, the leaves were placed into Petri dishes to maintain their humidity and kept at 25 • C in an incubator.Subsequently, nine representative fungal isolates (B1-B9) were selected from pathogenic species for in vivo inoculation with mycelial plugs.To confirm Koch's postulates, further pathogenicity tests were performed by inoculating conidial suspensions on leaves of C. julianae seedlings that were wounded with a sterile needle.The leaves were inoculated with the three representative isolates (B1-B3) using 10 µL of conidial suspensions (10 6 conidia/mL).Five plants were used for each treatment, and three leaves of each plant were inoculated.The control was treated with 10 µL of sterile water.All inoculated seedlings were covered with plastic bags, and sterilized water was sprayed into the bags daily to maintain a moist microclimate.All seedlings were kept in a growing chamber at 20 ± 2 • C/10 ± 2 • C (day/night) and observed regularly.Leaves that showed typical symptoms after the inoculation were used for re-isolations.

DNA Extraction and PCR Amplification
Nine isolates were cultured on potato dextrose agar (PDA), V8 agar (V-8) and potatocarrot agar (PCA) media at 25 • C in a constant temperature incubator with a 12/12 h light/dark cycle.Morphological identification was based on colony morphology and characteristics of conidia, conidiophores, conidiogenous cells, such as the shape and color of the colony, as well as the shape, size, color, septation, and presence of a beak in the conidia.A Zeiss Axio Imager A2m microscope (Carl Zeiss AG, Oberkochen Germany) and Zeiss stereo microscope (SteRo Discovery v20) were used to observe, describe, and measure conidia and other structures (n = 30).

Figure 1 .
Figure 1.Symptoms of leaf blotch on Celtis julianae in the field.(A,B) Symptoms on diseased l (C-E) diseased leaves on early stage, middle stage, and later stage, respectively; (F,G) co chains on a lesion of a leaf, scale bars: F = 100 µm, G = 50 µm.

Figure 1 .
Figure 1.Symptoms of leaf blotch on Celtis julianae in the field.(A,B) Symptoms on diseased leaves; (C-E) diseased leaves on early stage, middle stage, and later stage, respectively; (F,G) conidial chains on a lesion of a leaf, scale bars: F = 100 µm, G = 50 µm.

Table 1 .
Fungi isolated from diseased leaves of Celtis julianae.

Table 1 .
Fungi isolated from diseased leaves of Celtis julianae.

Number of Tissues Number of Colonies Nigrospora sp. Alternaria sp. Nothophoma sp.
• C for ITS, 52.4 • C for LSU, 53 • C for SSU and RPB2, 59.5 • C for GAPDH, 60.5 • C for Alt a 1, extension at 72 • C for 30 s, and a final elongation at 72 • C for 10 min.The cycling parameter for TEF1-α was as follows: 94 • C for 5 min; 40 cycles at 94 • C for 30 s; 59 • C for 30 s; 72 • C for 45 s; and a final extension at 72 • C for 7 min.The PCR products were sequenced at Sangon Biotech Co., Ltd.

Table 2 .
Primers used for PCR amplification in molecular identification of nine isolates (B1-B9).

Table 3 .
Isolates of Alternaria spp.used in this study and corresponding GenBank accession numbers.