Alternaria alternata, the Causal Agent of a New Needle Blight Disease on Pinus bungeana

Pinus bungeana, an endangered and native coniferous tree species in China, has considerable timber and horticulture value. However, little is known about needle diseases in P. bungeana. A needle blight of P. bungeana has been observed in Hebei Province, China. P. bungeana inoculated with mycelial plugs of fungal isolates presented symptoms similar to those observed under field conditions. Ten virulent fungal isolates were identified as a small-spored Alternaria species based on morphological observations. Maximum likelihood and Bayesian phylogenetic analyses carried out with multilocus sequence typing of eight regions (SSU, LSU, ITS, gapdh, tef1, Alt a 1, endoPG, OPA10-2) assigned the pathogen to Alternaria alternata. This is the first report of A. alternata causing needle blight on P. bungeana in China.


Introduction
Bunge's pine (Pinus bungeana Zucc. ex Endl.), a distinctive and evergreen coniferous tree species within the genus Pinus of the family Pinaceae, is mainly distributed across warm temperate areas and the north-subtropical and middle subtropical climatic zones [1]. It is known as an endemic and endangered coniferous tree species in China with high ornamental value, and is widely used in landscaping and afforestation owing to its ability to endure drought and cold climates [2]. Furthermore, the wood of Bunge's pine is commonly used for construction, furniture and stationery [3]. In addition, P. bungeana plays a key role in local forest ecosystems, with strong resistance to sulphur dioxide, ozone and soot pollution in nature [4]. Due to its ecological and economic value, this species has been the subject of many investigations, mainly on its phylogeny, morphology, genetic diversity and biological characteristics [5,6]. Few diseases of Bunge's pine have been reported.
The genus Alternaria Nees was described in 1816 [7]. Since then, more than 1100 names have been published, and 275 Alternaria species have been recognised [8,9]. Alternaria is a ubiquitous fungal genus that includes saprophytic, endophytic and pathogenic species [10]. Some Alternaria species are famous as pathogens of plants and animals [11]. In addition, those pathogenic species harm more than 4000 host plants and are distributed worldwide, with a broad host range, including agronomic plants, ornamentals, vegetables, fruit trees and animals [10,12,13]. Leaf blight, leaf spot, black point, stem cancer, fruit rot and mouldy cores are well-known symptoms of infection by Alternaria species [14][15][16].
In the past, Alternaria spp. have been classified based exclusively upon their morphological characteristics, which include cultural morphology, shape and size of conidia, septation, beak formation, branching patterns of conidial chains, and sporulation patterns [17]. This approach is effective when distinguishing large-spored Alternaria spp. from small-spored catenulate species due to conidia that are distinct and easy to recognise. Nevertheless, the identification of small-spored species based on morphological characteristics

Disease Investigation and Isolate Collection
In September 2020, leaf spot of Bunge's pine was found in Hebei Province, China. Thirty symptomatic tissues, the margin between the lesioned and healthy pine leaves, were cut into 3 to 5 mm long pieces. These tissues were surface sterilised for 45 s in ethanol (75%), washed thrice in sterilised distilled water and blotted dry with sterile paper. Pieces were transferred to 2% potato dextrose agar (PDA) in Petri plates, supplemented with ampicillin at 100 µg/mL and incubated at 25 • C (±1 • C) in the dark for 4 days. The single-spore isolation technique was used to obtain purified fungal isolates [34]. Single-spore isolates were cultured on PDA and stored in the Forest Pathology Laboratory of Nanjing Forestry University, Nanjing, China and the representative strain are being deposited to China Center for Type Culture Collection, Wuhan, China (CCTCC).

Pathogenicity Tests
All isolates were cultured on PDA and used for virulence tests on detached P. bungeana needles under controlled conditions. Asymptomatic needles of P. bungeana were surface disinfected and air-dried. Then, one piercing wound was made on the mid-upper region of each needle with a sterile needle (insect pin, 0.71 mm in diameter). The inoculation was performed by placing mycelial blocks (5 mm in length) from actively growing colony margins onto each stab wound. Needles inoculated with noncolonised PDA blocks were treated as negative controls. Each control and treatment, involving three needles per replicate, was placed into a Petri dish (9 cm) with moist sterile filter paper and sealed with plastic wrap to maintain a high relative humidity. Then, they were incubated at 25 • C in a growth chamber with a 12 h photoperiod. The whole experiment was carried out three times.
Ten isolates that were confirmed to be pathogenic on the detached needles were selected to determine pathogenicity on potted Bunge's pine. Bunge's pine needles were disinfected with 75% ethanol and air dried. Then, wound inoculation was conducted on 2-year-old potted, healthy Bunge's pine with a sterile needle. The blocks (3 mm in length) from colony margins with actively growing mycelia of 3-day-old isolates were placed on each wounded site. Blocks were removed 2 days post-inoculation. PDA discs with no mycelia were used as controls. Three potted plants were treated as one replicate, and three replicates were used. The inoculated plants were placed into a controlled-environment greenhouse. The size of the disease spot was recorded until representative symptoms appeared. The same procedure was carried out on 2-month-old seedlings of Korean pine (P. koraiensis Sieb. et Zucc.).
Re-isolations were performed from the margins of needles inoculated with ten isolates, and morphological and phylogenetic comparisons were conducted to meet Koch's postulates.

Morphological Study
Isolates were cultured on PDA for 7 days at 25 • C (±1 • C) to observe the colony morphology [35]. Micromorphological features were observed from those cultured on synthetic nutrient-poor agar plates (SNA) [36]. The characteristics of sporulation formation, including the length of conidial chains, branching patterns of conidial chains and presence of secondary conidiophores, were captured with a Zeiss stereo microscope (SteRo Discovery v20) [35]. A ZEISS Axio Imager A2m microscope (Carl Zeiss, Göttingen, Germany) equipped with differential interference contrast (DIC) optics was used to capture conidial chains and conidia. Fifty mature conidia mounted in sterile water were measured at random under a light microscope at ×100 magnification.

DNA Sequencing and Phylogenetic Analysis
The reference sequences of 43 Alternaria spp. described by Woudenberg et al. [20] selected for the phylogenetic analyses are also listed in Table 2 together with their corresponding GenBank accession numbers. Sequences in Table 2 were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/ (accessed on 10 February 2021)). A. alternantherae (CBS 124392) was used as the outgroup. The alignments of nucleotide sequences were obtained by using Clustal W in BioEdit software [45]. Treating gaps in the alignment as a fifth character, all of the characters had equal weight [46].
Phylogenetic trees of combined genes were constructed with two independent optimality search criteria, Bayesian inference (BI) phylogenetic analysis and maximum likelihood (ML) analysis. The ML analysis was performed using IQ-TREE [47], choosing the GTR + G + I model, and branch stability was estimated by 1000 bootstrap replicates.

Symptoms in Nature
Symptoms appeared on Bunge's pine needles and enlarged constantly. The colour of infected needles is off-white at the early stage and then turns to light brown gradually, with dark-brown spots appearing one by one ( Figure 1B,C). At the later stage of the disease, a large number of needles are infected, and the growth of the tree is inhibited ( Figure 1A). In total, 20 single-spore fungal isolates were collected. disease, a large number of needles are infected, and the growth of the tree is inhibited ( Figure 1A). In total, 20 single-spore fungal isolates were collected.

Pathogenicity Tests
Ten isolates were pathogenic, and healthy needles exhibited symptoms similar to those in nature, while mock-inoculated control needles showed no symptoms (Figure 2). Light-brown lesions were first observed at two days after inoculation and then expanded gradually, and dark-brown segments were noticed 14 days after mycelial plug inoculation ( Figure 2B). Ten lesions of each strain were counted and there was no significant difference in virulence among the three strains. Symptoms in nature appeared on Korean pine seedlings ( Figure 2C). The fungus was re-isolated from inoculated needles, and its colony morphology and molecular sequence were consistent with those of the original isolates.

Pathogenicity Tests
Ten isolates were pathogenic, and healthy needles exhibited symptoms similar to those in nature, while mock-inoculated control needles showed no symptoms (Figure 2). Light-brown lesions were first observed at two days after inoculation and then expanded gradually, and dark-brown segments were noticed 14 days after mycelial plug inoculation ( Figure 2B). Ten lesions of each strain were counted and there was no significant difference in virulence among the three strains. Symptoms in nature appeared on Korean pine seedlings ( Figure 2C). The fungus was re-isolated from inoculated needles, and its colony morphology and molecular sequence were consistent with those of the original isolates. disease, a large number of needles are infected, and the growth of the tree is inhibited ( Figure 1A). In total, 20 single-spore fungal isolates were collected.

Pathogenicity Tests
Ten isolates were pathogenic, and healthy needles exhibited symptoms similar to those in nature, while mock-inoculated control needles showed no symptoms (Figure 2). Light-brown lesions were first observed at two days after inoculation and then expanded gradually, and dark-brown segments were noticed 14 days after mycelial plug inoculation ( Figure 2B). Ten lesions of each strain were counted and there was no significant difference in virulence among the three strains. Symptoms in nature appeared on Korean pine seedlings ( Figure 2C). The fungus was re-isolated from inoculated needles, and its colony morphology and molecular sequence were consistent with those of the original isolates.

Morphology of Fungal Isolates
The virulent isolates shared similar colony morphologies. The colonies, with a regular prominent white margin, were olive green to black 10 d post-incubation. The bottom of the colonies was black surrounded with a light-brown circle. The aerial hyphae were thick and cottony and turned from colourless to pale brown ( Figure 3A). Conidiophores arose singly and were separated and pale brown. The conidia were solitary or in chains, the conidial body was 18.09-37.61 µm × 9.15-19.90 µm (average 24 × 14 µm, n = 50), typically obclavate, subglobose and ellipsoid, with 1-5 transverse septa and 1-3 longitudinal septa that slightly constricted near several septa. The conidia were yellow-brown and later turned black-brown ( Figure 3B-F). The morphological characterization of ten isolates revealed Alternaria-like morphology.

Morphology of Fungal Isolates
The virulent isolates shared similar colony morphologies. The colonies, with a regular prominent white margin, were olive green to black 10 d post-incubation. The bottom of the colonies was black surrounded with a light-brown circle. The aerial hyphae were thick and cottony and turned from colourless to pale brown ( Figure 3A). Conidiophores arose singly and were separated and pale brown. The conidia were solitary or in chains, the conidial body was 18.09-37.61 μm × 9.15-19.90 μm (average 24 × 14 μm, n = 50), typically obclavate, subglobose and ellipsoid, with 1-5 transverse septa and 1-3 longitudinal septa that slightly constricted near several septa. The conidia were yellow-brown and later turned black-brown ( Figure 3B-F). The morphological characterization of ten isolates revealed Alternaria-like morphology.

Phylogenetic Analysis
A multilocus phylogenetic analysis was conducted on ten pathogenic isolates based on the sequences from eight genes: SSU, LSU, ITS, gapdh, tef1, Alt a 1, endoPG and OPA10-2 (GenBank accession numbers MZ835355 to MZ835364, MZ835345 to MZ835354, MZ823461 to MZ823470, MZ835385 to MZ835394, MZ835395 to MZ835404, MZ802959 to MZ802968, MZ835375 to MZ835384, MZ835365 to MZ835374). For these ten isolates, the PCR amplification and sequencing of each gene generated product sizes were about 1072, 942, 733, 619, 259, 516, 491 and 753 or 777 bp, respectively. The alignments (including the gaps) for eight genes were 1021, 849, 522, 579, 241, 473, 448 and 634 bp in size, respectively. The ten sequences of isolates along with sequences from 33 Alternaria strains were concatenated for the construction of a phylogenetic tree. The alignment of the eight-locus concatenated dataset consisted of 4767 characters, with 4356 constant characters, 245 parsimony-uninformative characters, and 166 parsimony-informative characters.
ML and BI analyses generated basically the same tree topology, which demonstrated that the evolutionary relationships of the fungus isolates were statistically supported. A single tree with bootstrap proportions (BP) from ML and Bayesian posterior probabilities (BPP) from BI was generated (Figure 4). The phylogenetic analysis showed that all isolates herein clustered into two clades, with a highly supported clade (≥92% BP/0.91 BPP) with A. alternata CBS 121455 and CBS 121336. Two phylogenetic analyses revealed that all isolates with aggressiveness showed >95% similarity to the A. alternata isolates reported previously.

Phylogenetic Analysis
A multilocus phylogenetic analysis was conducted on ten pathogenic isolates based on the sequences from eight genes: SSU, LSU, ITS, gapdh, tef1, Alt a 1, endoPG and OPA10-2 (GenBank accession numbers MZ835355 to MZ835364, MZ835345 to MZ835354, MZ823461 to MZ823470, MZ835385 to MZ835394, MZ835395 to MZ835404, MZ802959 to MZ802968, MZ835375 to MZ835384, MZ835365 to MZ835374). For these ten isolates, the PCR amplification and sequencing of each gene generated product sizes were about 1072, 942, 733, 619, 259, 516, 491 and 753 or 777 bp, respectively. The alignments (including the gaps) for eight genes were 1021, 849, 522, 579, 241, 473, 448 and 634 bp in size, respectively. The ten sequences of isolates along with sequences from 33 Alternaria strains were concatenated for the construction of a phylogenetic tree. The alignment of the eightlocus concatenated dataset consisted of 4767 characters, with 4356 constant characters, 245 parsimony-uninformative characters, and 166 parsimony-informative characters.
ML and BI analyses generated basically the same tree topology, which demonstrated that the evolutionary relationships of the fungus isolates were statistically supported. A single tree with bootstrap proportions (BP) from ML and Bayesian posterior probabilities (BPP) from BI was generated (Figure 4). The phylogenetic analysis showed that all isolates herein clustered into two clades, with a highly supported clade (≥92% BP/0.91 BPP) with A. alternata CBS 121455 and CBS 121336. Two phylogenetic analyses revealed that all isolates with aggressiveness showed >95% similarity to the A. alternata isolates reported previously.

Discussion
Because of its ability to assimilate harmful material in the needles, graceful appearance and fine timber, Bunge's pine plays an essential role in ecology and the economy. Needle blight disease can not only worsen the pine appearance but also influence apical dominance. The loss of apical dominance reduces wood quality. Moreover, the death of trees can occur in severe cases. Generally, the diseases affecting Bunge's pine damage the economy and ecology. Based on morphological characteristics and molecular identification with phylogenetic analysis of multiple gene sequences, A. alternata was confirmed to be the causal agent of needle blight on Bunge's pine in China. This is the first report of A. alternata on P. bungeana.
Several small-spore Alternaria spp. are frequently misidentified due to morphological overlap with A. alternata [35]. The dimensions of conidia in this study were very different from those described by Moumni et al. [49], but were similar to those reported by Gao et al. [50]. This phenomenon could be attributed to the morphological plasticity exhibited by most Alternaria species. Conidial morphology is dependent on culture conditions and conidium age [35]. The number of conidia produced with conidial chains was related to the

Discussion
Because of its ability to assimilate harmful material in the needles, graceful appearance and fine timber, Bunge's pine plays an essential role in ecology and the economy. Needle blight disease can not only worsen the pine appearance but also influence apical dominance. The loss of apical dominance reduces wood quality. Moreover, the death of trees can occur in severe cases. Generally, the diseases affecting Bunge's pine damage the economy and ecology. Based on morphological characteristics and molecular identification with phylogenetic analysis of multiple gene sequences, A. alternata was confirmed to be the causal agent of needle blight on Bunge's pine in China. This is the first report of A. alternata on P. bungeana.
Several small-spore Alternaria spp. are frequently misidentified due to morphological overlap with A. alternata [35]. The dimensions of conidia in this study were very different from those described by Moumni et al. [49], but were similar to those reported by Gao et al. [50]. This phenomenon could be attributed to the morphological plasticity exhibited by most Alternaria species. Conidial morphology is dependent on culture conditions and conidium age [35]. The number of conidia produced with conidial chains was related to the nutrition that the fungi obtained. In addition, the numbers of longitudinal and transverse septa were variable. It is suggested that morphological characteristics are not stable.
Due to morphological variability and minimal molecular variation, the taxa of Alternaria spp. were reclassified by Woundenberg et al. [51]. Whole-genome sequencing and transcriptome sequencing were used to distinguish 168 Alternaria isolates, and nine gene regions (SSU, LSU, ITS, gapdh, tef1, Alt a 1, endoPG, OPA10-2 and rpb2) were selected to distinguish sect. Alternaria more effectively [20]. Phylogenetic analyses and species identification are challenging in small-spored Alternaria due to lineage sorting, recombination and horizontal transfer [52]. Multilocus species identification was confirmed to be necessary among Alternaria sections for low resolution of species delimitation in small-spored Alternaria [10]. The analysis with a concatenation of six gene regions (ITS, rpb2, endoPG, tef1, Alt a 1 and OPA10-2) was able to separate A. alternata from the A. arborescens species complex [10]. A slowly evolving gene (rpb2) was excluded, while additional molecular markers (gaphd, SSU and LSU) were included in this study as proposed by Woudenberg et al. [20]. The combined phylogenetic tree shows consistency with other studies [10,15,17,20].
Alternaria alternata was reported as a ubiquitous pathogen in the great majority of crops and some broad-leaved trees [17,26,30,31,[53][54][55][56]. In particular, A. alternata is the most important mycotoxin-producing genus as a result of the wide reports of TA, AME, AOH, ALT and ATX produced [57]. In addition, A. alternata can not only colonise the phylloplane but also penetrate into living leaves [58]. Nevertheless, A. alternata was reported to be the dominant endophytic fungal taxon in the bark and needles of Chinese oil pine (Pinus tabulaeformis Carr.) and isolated from various plants [59]. In addition, as an endophytic fungus, it showed strong antifungal activity against Raffaelea quercusmongolicae [60]. When examining the abundance and diversity of fungi on needles of Pinus sylvestris, A. alternata was found to be a common primary or secondary saprotroph [61]. It is difficult for A. alternata to colonise Bunge's pine needles without wounding, which may be related to plant resistance or pathogenic activity. The result of unwounded inoculation indicated that wounding may play a significant role in the pathogenicity of A. alternata. In nature, needles are prone to chafing, which can induce laceration as a result of the wind. This may provide an opportunity for A. alternata to be virulent. In addition, the virulence of A. alternata may have been obtained horizontally from a recent common saprophytic ancestor [52].
According to previous studies, A. alternata, as a pathogen of pine needles, has never been reported. Although the thicker epidermis and cuticle of needles make it more difficult for fungi to invade plants, it is noteworthy that wounds appearing on needles may lead to disease prevalence. Pathogenicity test results indicate that A. alternata has the ability to infect other Pinus species, and it is necessary to investigate the distribution and propagation of the disease caused by A. alternata. A. alternata may pose a great threat to ecology because the hosts that the pathogen can invade are increasing, especially in Pinus species. Studies on the pathogenicity mechanism of A. alternata and disease management should be conducted in the future.  Institutional Review Board Statement: Not applicable for studies not involving humans or animals.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this article.

Conflicts of Interest:
The authors declare no conflict of interest.