Neofusicoccum cryptomeriae sp. nov. and N. parvum Cause Stem Basal Canker of Cryptomeria japonica in China

Cryptomeria japonica D. Don is a coniferous tree species widely grown in southern China for its high ornamental value. Recently, during disease surveys in China, a symptom of dieback occurred on C. japonica in Nanjing, Jiangsu Province, China. A total of 130 trees were surveyed and more than 90% showed the same symptom. The crowns of affected trees were brown when viewing from a distance, and the bark showed no difference from the healthy ones. In this study, 157 isolates were isolated from the 3 affected plants of C. japonica, and based on the living culture on PDA, the fungal isolates were preliminarily divided into 6 groups. Thirteen representative isolates were selected for the pathogenicity test, and seven of them showed obvious pathogenicity on C. japonica, causing stem basal canker. These isolates were identified based on comparisons of the DNA sequences of the internal transcribed spacer regions (ITS), partial translation elongation factor 1-alpha (tef1), β-tubulin (tub2), and DNA-directed RNA polymerase II subunit (rpb2) and combined with their morphological characteristics. Results showed that these seven isolates belong to two taxa in Neofusicoccum, including a species new to science. The new species, Neofusicoccum cryptomeriae, was hereby described and illustrated. The other species was N. parvum. Both species were pathogens of stem basal canker of Cryptomeria japonica.


Introduction
Cryptomeria is a monotypic genus of conifer in Cupressaceae, and its only species, Cryptomeria japonica (Linn. f.) D. Don, Japanese cedar, is a monoecious coniferous tree species native to Japan and has been introduced to Jiangsu, Guangxi, Shandong, Zhejiang, and other provinces in China as an ornamental tree species and for lumber production [1,2]. Cryptomeria japonica has been introduced and cultivated in China for a millennium or more, and some trees on Tianmu Mountain are estimated to be nearly 1000 years old, and the oldest one, ca. 1500 years old [3,4]. It has been introduced to other countries: Azores (Portugal), former Czechoslovakia, Føroyar (Denmark), Korea, Mauritius, New Zealand, Réunion (France), Sweden, Turkey, and the UK [2].
Cryptomeria japonica has extensive application values. Its cones and unusual needles have high aesthetic appeal. As an environmental-friendly tree, C. japonica showed a good effect on the absorption of cesium [5,6]. Many studies have shown that the bark, core material, and needles of C. japonica contain a variety of monoterpenoids, sesquiterpenes, and diterpenoids [7]. These substances show a wide range of biological activities, such were incubated at 25 • C in the dark for five days, and hyphae tips at the edge of the colony were transferred to the new PDA plates.

Pathogenicity Tests
To determine the pathogenicity of the isolates on Cryptomeria japonica, the trunk of two-year-old seedlings was wounded with a sterile scalpel to expose the cambium. The wounds were located approximately 2 cm above the soil level. For inoculation, 5 mm plugs were cut out from the growing edges of 5-day-old cultures placed into the wounds with the mycelia facing the cambium, and then the inoculating site was wrapped with Parafilm. Thirteen representative fungal isolates (G1, G2, G4, G11, G15, G16, G18, G23, G24, G74, G91, G92, and C7) were used for inoculation, and 3 to 5 seedlings were inoculated with each isolate. Five control seedlings were treated with sterile PDA agar using the same aforementioned method [31]. All inoculated plants were kept in a greenhouse (relative humidity > 80%, 25 ± 2 • C). The inoculated fungi were re-isolated as described above and confirmed by both morphological characteristics and ITS sequence analysis.

Morphological Identification
After the pathogenicity experiment, seven pathogenic isolates were cultured on PDA for seven days, and the colony color, texture, and pigment production of the isolates were observed and recorded. To induce sporulation, the fungi were cultured on Petri dishes containing synthetic nutrient-poor agar medium (SNA) or 2% tap water agar (WA) supplemented with double autoclaved pine needles on their surface [32][33][34]. All colonies were placed under near-ultraviolet light at 25 • C for 1-2 months. The morphology and size of 30 pycnidia of each isolate were observed and recorded using a Zeiss stereomicroscope (SteRo Discovery v20). Relevant morphological characteristics were observed and recorded using a Zeiss Axio Imager A2m microscope. The lengths, widths, and shapes of 20 conidiophores, 20 conidiogenous cells, and 50 conidia of the 7 isolates were measured [32].

Phylogenetic Analyses
Initial identities of the isolates were determined using BLASTn of the NCBI GenBank with sequences generated in this study. ITS, tef1, tub2, and rpb2 sequences of phylogenetically related Neofusicoccum species and Botryosphaeria dothidea (CBS 115476) as an outgroup were obtained from GenBank ( Table 1). The sequences of Neofusicoccum isolates obtained during this study were aligned based on loci with reference sequences, respectively, in PhyloSuite V1.2.2 using the 'FFT-NS-2 (default)' strategy and normal alignment mode of MAFFT V7.313, and then edited manually where necessary [52,53]. Two phylogenetic analyses were conducted using IQtree ver. 1.6.8 for the maximum likelihood (ML) analysis and MrBayes 3.2.6 for Bayesian Inference (BI) analysis [54,55]. ModelFinder was used to select the best-fit model for the multi-locus phylogenetic analyses [56]. For ML analysis, with 1000 bootstrap replicates, we utilized the best-fit model: GTR + F + I + G4. For BI analysis, we used the GTR + I + G + F model (2 parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in. All phylogenetic trees were viewed using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 12 February 2023)).

Genealogical Concordance Phylogenetic Species Recognition Analysis
The concatenated dataset (ITS, tef1, tub2, and rpb2) was used to analyze the new species, their species limits, and their most closely related taxa, as described by Quaedvlieg et al. [57], through the GCPSR concept with a PHI test performed in SplitsTree v.4.14.6. A PHI index below 0.05 (Φ w < 0.05) indicates the presence of significant recombination in the dataset. The relationships between this new taxon and closely related species were visualized in splits graphs with both the LogDet transformation and splits decomposition options.

Symptoms in the Field and Fungal Isolation
The results of drone aerial photography and field investigation showed that 90 percent of C. japonica showed symptoms of dieback (130 trees in total) ( Figure 1A,B). Affected trees have normal bark and reddish-brown canopies ( Figure 1C,D). After stripping the bark, lesions can be observed in the phloem, which spread from the root color to the breast height of the trunk ( Figure 1E-H). The lesions were dry and brownish, and the healthy xylem was yellowish and moist. A total of 157 fungal isolates were isolated and were divided into 6 groups according to the colony morphology. Thirteen representative isolates (G1, G2, G4, G11, G15, G16, G18, G23, G24, G74, G91, G92, and C7) were selected for pathogenicity experiments.

Pathogenicity Assays
Three days after the inoculation, brown spots began to appear at the inoculation points of isolates G1, G2, G15, G16, G24, G91, and G92. The lesions gradually expanded in about a week, and after 20 days, half of the whole plant withered from the bottom to the top, and the whole plant withered in 1 month ( Figure 2B-H). These symptoms were consistent with those observed in the field. At the same time, the control, G4, G11, G18, G23, G74, and C7 did not develop symptoms (Figure 2A). The inoculated fungal isolates were re-isolated from the lesions on the inoculated seedlings, and no fungi were isolated from the control. Therefore, the seven isolates (G1, G2, G15, G16, G24, G91, and G92) were identified as the causal agents of stem basal canker on C. japonica.

Molecular Identification of the Fungal Isolates
Phylogenetic analyses showed that four isolates (G15, G16, G91, and G92) were in the same cluster with N. parvum (ex-type: ATCC 58191). Three isolates (G1, G2, and G24) were clustered in a distinct clade, which was distinct from all other known species and a sister clade to the clade of N. sinense (ex-type: CGMCC 3.18315) (Figure 3). Based on the phylogenetic analyses using the concatenated sequences of the ITS, tef1, tub2, and rpb2 sequences, four isolates (G15, G16, G91, and G92) were N. parvum, and three isolates (G1, G2, and G24) were a new species of Neofusicoccum. The tree topologies of ML and BI phylogenetic trees were consistent, where maximum likelihood bootstrap support values (ML ≥ 50) and Bayesian posterior probability (PP ≥ 0.90) are shown at the nodes (ML/PP). Furthermore, the PHI test on N. cryptomeriae revealed that there was no significant recombination (Φ w = 0.163) among their closely related taxa: N. sinense, N. brasiliense, and N. kwambonambiense (Figure 4). Thus, the isolates G1, G2, and G24 were confirmed to be new species.

Pathogenicity Assays
Three days after the inoculation, brown spots began to appear at the inoculation points of isolates G1, G2, G15, G16, G24, G91, and G92. The lesions gradually expanded in about a week, and after 20 days, half of the whole plant withered from the bottom to x FOR PEER REVIEW 8 of 17 from the control. Therefore, the seven isolates (G1, G2, G15, G16, G24, G91, and G92) were identified as the causal agents of stem basal canker on C. japonica.

Morphology and Taxonomy
For isolates G1, G2, and G24, morphological differences were observed compared to the most closely related species (Neofusicoccum sinense CGMCC 3.18315) based on phylogenetic analyses. Therefore, the results of the phylogenetic analyses and morphological studies support the conclusion that three isolates (G1, G2, and G24) were a Neofusicoccum species new to science. The new species is described as follows: Neofusicoccum Culture characteristics: Colonies on PDA were initially white with fluffy mycelia. After five days, the aerial mycelia were dense at the edge of the colony and sparse in the middle, and hyphae at the center of the front and back sides are gray.
The measured data of isolates G1 and G2 are listed in Table 2

Morphology and Taxonomy
For isolates G1, G2, and G24, morphological differences were observed compared to the most closely related species (Neofusicoccum sinense CGMCC 3.18315) based on phylogenetic analyses. Therefore, the results of the phylogenetic analyses and morphological studies support the conclusion that three isolates (G1, G2, and G24) were a Neofusicoccum species new to science. The new species is described as follows: Neofusicoccum Culture characteristics: Colonies on PDA were initially white with fluffy mycelia. After five days, the aerial mycelia were dense at the edge of the colony and sparse in the middle, and hyphae at the center of the front and back sides are gray.
The measured data of isolates G1 and G2 are listed in Table 2. Host/distribution: from Cryptomeria japonica in Nanjing, Jiangsu, China.
J. Fungi 2023, 9, x FOR PEER REVIEW 12 of 17 Notes: Phylogenetically, N. cryptomeriae is closely related to N. sinense. They were distinguished based on 14 nucleotides in the concatenated alignment, of which 9 were distinct in ITS, 4 in tef1, and 1 in tub2. N. sinense has no sequence data of rpb2 for comparison. Morphologically, N. cryptomeriae differs from N. sinense by its longer conidia (23-26.1 × 7-7.8 µm vs. 17.6-20.4 × 7.4-8 µm) ( Table 2). Fusicoccum cryptomeriae was a pathogen causing leaf spots on C. japonica, and it was differentiated from N. cryptomeriae by its much smaller conidia: 6.5-8 × 2.5 µm vs. 23.0-26.1 × 7.0-7.8 µm [14].    Based on analyses of DNA sequence data, four isolates (G15, G16, G91, and G92) were in the same cluster with N. parvum. Comparing these four isolates with the morphological characteristics of N. parvum, including colony, conidiomata, conidiophores, conidiogenous cells, and conidia, showed that the morphologies of the four isolates were the same as N. parvum. Therefore, these four isolates were N. parvum. The morphological characteristics of the representative isolate G15 are as follows ( Figure 6). On PDA, G15 was initially white, and after 5 days, it developed an abundant greyish-white aerial mycelium. Conidiomata pycnidial, produced on pine needles on WA within 30 days, solitary or in groups, covered by mycelium, dark-brown to black, up to 172-247 µm-wide, and 144-440-µm high. Conidiogenous cells were hyaline and short subcylindrical:  Based on analyses of DNA sequence data, four isolates (G15, G16, G91, and G92) were in the same cluster with N. parvum. Comparing these four isolates with the morphological characteristics of N. parvum, including colony, conidiomata, conidiophores, conidiogenous cells, and conidia, showed that the morphologies of the four isolates were the same as N. parvum. Therefore, these four isolates were N. parvum. The morphological characteristics of the representative isolate G15 are as follows ( Figure 6). On PDA, G15 was initially white, and after 5 days, it developed an abundant greyish-white aerial mycelium. Conidiomata pycnidial, produced on pine needles on WA within 30 days, solitary or in groups, covered by mycelium, dark-brown to black, up to 172-247 µm-wide, and 144-440-µm high. Conidiogenous cells were hyaline and short subcylindrical:

Discussion
In this study, the pathogen causing stem basal canker of C. japonica in Nanjing, China, was determined by the pathogenicity tests using Japanese cedar seedlings. Based on morphological, GCPSR principle, and phylogenetic studies, the pathogens were identified as two species of Neofusicoccum, N. parvum and a new species, Neofusicoccum cryptomeriae.
As early as 2013, studies have shown that N. parvum is a widely distributed and common pathogen to plants, occurring on 90 host species across 6 continents [58]. At present, this species has been reported in 181 plants [59]. In subsequent studies, N. parvum was

Discussion
In this study, the pathogen causing stem basal canker of C. japonica in Nanjing, China, was determined by the pathogenicity tests using Japanese cedar seedlings. Based on morphological, GCPSR principle, and phylogenetic studies, the pathogens were identified as two species of Neofusicoccum, N. parvum and a new species, Neofusicoccum cryptomeriae.
As early as 2013, studies have shown that N. parvum is a widely distributed and common pathogen to plants, occurring on 90 host species across 6 continents [58]. At present, this species has been reported in 181 plants [59]. In subsequent studies, N. parvum was found to be one of the most virulent species based on the extent of necroses it causes [60][61][62][63]. Neofusicoccum spp. have not been reported to infect Japanese cedar, but many species of Neofusicoccum have been reported on other conifers, including many endangered species. For example, N. nonquaesitum was reported to cause branch cankers on Sequoiadendron giganteum in North America [64]. N. parvum was reported to cause canker and dieback of S. giganteum in the Geneva Lake area, Switzerland [65]. N. nonquaesitum has also been reported to cause branch dieback and decline in Araucaria araucana, and this tree species has been listed as an endangered species on the Red Data List of the International Union for Conservation of Nature [66].
The asexual state developed under natural conditions is very important for the morphological identification of fungi [67]. Many genera of Botryosphaericeae, including Neofusicoccum, Botryosphaeria, Pseudofusicoccum, and Neoscytalidium, share similar morphological characteristics of their asexual states, and most of their conidia are narrow ellipsoids [16]. Similar morphological characteristics make it difficult to differentiate the genera. Botryosphaericeae can grow well on culture media, but it is difficult to produce conidia [16]. These conditions have led to some challenges in the morphological identification of fungi in this family. Many species of Neofusicoccum are similar in morphology, and the molecular methods provide supplementary methods of fungal identification and classification. In recent years, phylogenetic and phytopathological studies on Neofusicoccum have used concatenated sequences of ITS, tef1, tub2, and rpb2 [24,28,68]. Phylogenetic analyses using sequences of multiple loci can better distinguish and identify some closely related species in the genus.
The research on Neofusicoccum fungi was mainly focused on the identification of plant pathogens in China, where there is a lack of systematic taxonomic research. New species of this genus have been continuously discovered, indicating that the fungal resources of the genus Neofusicoccum are relatively abundant in China. It is necessary to collect a large number of specimens to establish a complete and reasonable classification system for this genus to provide mycological and molecular information and a scientific basis for disease prevention and control. Data Availability Statement: All sequences generated in this study were submitted to GenBank.

Conflicts of Interest:
"Jiangsu PIESAT Information Technology Co., Ltd." is Yuan-Zhi Si and Yi-Na Chen's current work unit, and there is no conflict of interest.