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Article

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

by
Yuan-Zhi Si
1,2,3,†,
Jian-Wei Sun
1,2,4,†,
Yu Wan
1,2,
Yi-Na Chen
3,
Jiao He
1,2,
Wei-Zheng Li
1,5,
De-Wei Li
6 and
Li-Hua Zhu
1,2,*
1
College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
3
Jiangsu PIESAT Information Technology Co., Ltd., Xuzhou 221116, China
4
Zhouning County Bureau of Forestry, Ningde 355400, China
5
Advanced Analysis and Testing Center, Nanjing Forestry University, Nanjing 210037, China
6
The Connecticut Agricultural Experiment Station Valley Laboratory, Windsor, CT 06095, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2023, 9(4), 404; https://doi.org/10.3390/jof9040404
Submission received: 23 February 2023 / Revised: 18 March 2023 / Accepted: 23 March 2023 / Published: 24 March 2023
(This article belongs to the Special Issue Phylogeny and Taxonomy of Ascomycete Fungi)
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Abstract

:
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.

1. 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 as antibacterial and insect resistance [8,9,10], and the hepatoprotective phytocompounds from C. japonica have a potential function in inflammatory mediators [11]. In addition, as a common timber species, C. japonica was widely used in the production of particleboard [12]. However, during the seedling stage and the afforestation process, C. japonica was often attacked by many kinds of pathogenic fungi, causing a number of diseases, including the trunk rot caused by Fomitiporia torreyae Y. C. Dai & B. K. Cui [13], leaf spots and new shoot canker caused by Fusicoccum cryptomeriae Sawada [14], and the shoot blight caused by Pestalotiopsis neglecta Thüm [15]. The occurrence of the disease has detrimentally affected the ecological functions and economic value of C. japonica and restricted the development of this species.
Neofusicoccum was proposed by Crous et al. in 2006 with the type species of Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips [16]. It belongs to Botryosphaericeae, and the fungi of this family include various non-host-specific pathogens, saprobes, endophytes, and potential pathogens [17]. In the past several years, diseases caused by Neofusicoccum species were frequently reported in many countries. N. parvum was reported as a pathogen of many plants, including the stem and branch blight disease of Zanthoxylum bungeanum in Sichuan, China [18], nut rot of chestnut (Castanea sativa) in Italy [19], and dieback and canker of hemp (Cannabis sativa L.) in the United States [20]. N. luteum (Pennycook & Samuels) Crous & al., N. batangarum Begoude & al., N. mangiferae Syd. & P. Syd, and some other species in Neofusicoccum were also reported to relate to some plant diseases [21,22,23]. Due to the similarity of morphological characteristics in Neofusicoccum, some species were erroneously classified in the past. With the development of phylogenetic analysis, many new species were found, and some known species were reclassified. Five novel species (N. dianense G.Q. Li & S.F. Chen, N. magniconidium G.Q. Li & S.F. Chen, N. ningerense G.Q. Li & S.F. Chen, N. parviconidium G.Q. Li & S.F. Chen) were described by Li et al. [24]. In a recent taxonomic study of Botryosphaericeae, 11 species in Neofusicoccum were reduced to synonymy, and 2 novel species, named N. podocarpi W. Zhang & Crous and N. rapaneae W. Zhang & Crous, were described [25]. Since then, several studies have been published and described a number of new taxa, such as Neofusicoccum caryigenum M.T. Brewer & C.J. Cameron [26], Neofusicoccum sichuanense X. L. Xu & C. L. Yang [27], N. hyperici Y. Hattori & C. Nakash, N. miyakoense Y. Hattori & C. Nakash, and N. okinawaense Y. Hattori & C. Nakash [28], Neofusicoccum moracearum Tennakoon, C.H. Kuo & K.D. Hyde [29], and Neofusicoccum mystacidii Crous [30]. At present, there are 52 species of Neofusicoccum [25,27,28].
Recently, during disease surveys in Nanjing, Jiangsu Province, China, a new disease of C. japonica was found. A total of 130 trees were surveyed and more than 90% showed the same symptom of dieback of the stem and branch. It is different from the other reported diseases of C. japonica. The infected plants showed an obvious distinction in the conjunct area of healthy and infected parts, both in color and texture in the xylem and the interior side of the bark. The occurrence of the disease seriously damaged the ornamental value of C. japonica. The aims of this study were to: (1) test the pathogenicity of representative isolates, and (2) determine the identities of the fungi causing stem basal canker of C. japonica based on morphological characteristics and phylogenetic analyses and describe a new fungal species, which is also pathogenic to C. japonica.

2. Materials and Methods

2.1. Sample Collection and Fungal Isolation

Disease surveys were conducted at Nanjing Forestry University from September to October 2020. Approximately 50 bark pieces from the edges of both healthy and infected trunks were collected from 3 symptomatic trees. The interior side of the bark was cut into small pieces (3–5 mm2) using a sterile scalpel. The pieces were submerged in 75% ethanol for 30 s and then in 1.5% NaClO for 90 s, washed 3 times in sterile water, blotted dry with sterilized filter paper, and placed onto potato dextrose agar (PDA) with 100 µg/mL of ampicillin (Nanjing Zebra Experimental Equipment Co., Ltd., Nanjing, China). Cultures 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.

2.2. 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.

2.3. 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].

2.4. DNA Extraction, PCR Amplification, and Sequencing

Total genomic DNA from isolates in this study was extracted from the fungal mycelia of 7-day-old cultures using the cetyltrimethylammonium bromide (CTAB) protocol. Four loci, including internal transcribed spacer (ITS), partial translation elongation factor 1-alpha (tef1), partial β-tubulin (tub2), and partial DNA-directed RNA polymerase II subunit (rpb2), were amplified with the primer pairs ITS1/ITS4 [35], EF1-728F/EF1-986R [36], BT-2a/BT-2b [37], and RPB2bot6F/RPB-2bot7R [38,39], respectively. The polymerase chain reaction consisted of 25 μL of Taq DNA polymerase mix, 2 μL of genomic DNA, 2 μL of each primer, and 19 μL of double-distilled water. The amplification conditions consisted of an initial denaturation step at 95 °C for 5 min, 34 cycles of 95 °C for 30 s, and annealing at a suitable temperature for 30 s for each locus: 56 °C (ITS), 52 °C (tef1), 60 °C (tub2), and 55 °C (rpb2), and then 72 °C for 30 s, followed by a final elongation step at 72 °C for 10 min and a cool-down step to 4 °C. Primers were synthesized and PCR products were sequenced by the Shanghai Jieli Biotechnology Co. Ltd., Nanjing, Jiangsu Province, China. All sequences of the isolates from this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov (accessed on 28 July 2022)) (Table 1).

2.5. 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)).

2.6. 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 (Φw < 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.

3. Results

3.1. 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.

3.2. 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.

3.3. 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 (Φw = 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.

3.4. 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 cryptomeriae Li-Hua Zhu, Yuan-Zhi Si, Jian-Wei Sun & D. W. Li, sp. nov. (Figure 5).
Index Fungorum number, IF 900283.
Etymology. Latin: cryptomeriae referring to the host genus Cryptomeria.
Sexual state: Undetermined. Asexual state: Conidiomata pycnidial, produced on pine needles on SNA within 30 days, solitary or aggregated, covered by mycelium, dark-brown to black, stylolitic, ellipsoidal or spherical, up to 183–381 μm-wide, and 463–1152 μm-high. Conidiophores hyaline, cylindrical, branched, and smooth: (20.6–)25–34.1(–39) × (3.1–)3.6–3.8(–5.2) μm (av. = 29.6 × 4.2 μm, n = 20). Conidiogenous cells holoblastic, hyaline, cylindrical, and phialidic, with periclinal thickening: (11.6–)13.8–22.4(–25.8) × (3–)3.4–4.2(–4.9) μm (av. = 18.1 × 3.8 μm, n = 20). Paraphyses not observed. Conidia 1-celled, hyaline, thin-walled, smooth with granular contents, fusiform, initially non-septate, and subsequently becoming 1–2 septate: (20.9–)23.0–26.1(–27.5) × (6.8–)7.0–7.8(–8.3) μm (av. = 24.6 × 7.4 μm, n = 50; L/W = 3.3).
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.
Holotype: China, Jiangsu, Nanjing, 32°04′53.11″ N, 118°49′10.27″ E, isolated from Cryptomeria japonica, 2 September 2020, Jian-Wei Sun, CFCC 55721 (=G24). The holotype specimen is a living specimen being maintained via lyophilization at the China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing, China.
Additional materials examined: China, Jiangsu, Nanjing, 32°04′53.11″ N, 118°49′10.27″ E, isolated from Cryptomeria japonica, 2 September 2020, Jian-Wei Sun, CFCC 55720 (=G1). China, Jiangsu, Nanjing, 32°04′53.11″ N, 118°49′10.27″ E, isolated from Cryptomeria japonica, 2 September 2020, Jian-Wei Sun, CFCC 55728 (=G2).
Host/distribution: from Cryptomeria japonica in Nanjing, Jiangsu, China.
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].
Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips.
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: (11.4–)13.4–18.4(–19.4) × (2.5–)2.6–3.8(–4.2) μm (av. = 15.9 × 3.2 μm, n = 20). Conidia 1-celled, hyaline, ellipsoidal to fusiform: (15.5–)17–18.7(–20.3) × (5.2–)5.8–6.3(–6.8) μm (av. = 18.3 × 6.2 μm, n = 50; L/W = 3), and when mature, became brown, septate. The measured data of isolates G16, G91, and G92 are listed in Table 2.

4. 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.

Author Contributions

Y.-Z.S., J.-W.S., L.-H.Z. and D.-W.L. designed the study and were involved in writing the paper; Y.-Z.S., J.-W.S. and L.-H.Z. were responsible for sample collections; W.-Z.L. provided the aerial view; J.-W.S. was responsible for pathogenicity tests; Y.-Z.S., J.-W.S. and Y.W. were involved in morphological identification; Y.-Z.S., J.-W.S. and J.H. were involved in phylogenetic analyses; L.-H.Z., D.-W.L., Y.-Z.S. and Y.-N.C. contributed to planning and editing of the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the National Key R&D Program of China (2022YFD1401005).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

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.

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Figure 1. Symptoms of stem basal canker disease on C. japonica in the field. (A,B) Aerial view. (C,D) Stem basal canker of C. japonica. (E–G) Lesions on the phloem of the tree trunk. (H) Lesions on the phloem of the tree root. Scale bars: (G,H) = 5 cm.
Figure 1. Symptoms of stem basal canker disease on C. japonica in the field. (A,B) Aerial view. (C,D) Stem basal canker of C. japonica. (E–G) Lesions on the phloem of the tree trunk. (H) Lesions on the phloem of the tree root. Scale bars: (G,H) = 5 cm.
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Figure 2. Symptoms caused by fungal isolates in this study 20 days after inoculation. (A) Control, G4, G11, G18, G23, G74, and C7, showing the absence of lesion development on C. japonica. G1, G2, G15, and G92, showing the lesion development on C. japonica. (B–H) Lesions produced on C. japonica by isolates (B) G1, (C) G2, (D) G15, (E) G16, (F) G24, (G) G91, and (H) G92.
Figure 2. Symptoms caused by fungal isolates in this study 20 days after inoculation. (A) Control, G4, G11, G18, G23, G74, and C7, showing the absence of lesion development on C. japonica. G1, G2, G15, and G92, showing the lesion development on C. japonica. (B–H) Lesions produced on C. japonica by isolates (B) G1, (C) G2, (D) G15, (E) G16, (F) G24, (G) G91, and (H) G92.
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Figure 3. Phylogenetic relationship of Neofusicoccum cryptomeriae and N. parvum with closely related taxa derived from a maximum likelihood (ML) analysis and Bayesian Inference using combined ITS, tef1, tub2, and rpb2 sequence alignment, with Botryosphaeria dothidea (CBS 115476) as the outgroup. Maximum likelihood bootstrap support values (ML ≥ 50) and Bayesian posterior probability (PP ≥ 0.90) are shown at the nodes (ML/PP). Ex-type strains are marked in bold, the species are delimited with colored blocks, and isolates in this study are marked in red.
Figure 3. Phylogenetic relationship of Neofusicoccum cryptomeriae and N. parvum with closely related taxa derived from a maximum likelihood (ML) analysis and Bayesian Inference using combined ITS, tef1, tub2, and rpb2 sequence alignment, with Botryosphaeria dothidea (CBS 115476) as the outgroup. Maximum likelihood bootstrap support values (ML ≥ 50) and Bayesian posterior probability (PP ≥ 0.90) are shown at the nodes (ML/PP). Ex-type strains are marked in bold, the species are delimited with colored blocks, and isolates in this study are marked in red.
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Figure 4. Pairwise homoplasy index (PHI) test of Neofusicoccum cryptomeriae and closely related N. sinense, N. brasiliense, and N. kwambonambiense using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset.
Figure 4. Pairwise homoplasy index (PHI) test of Neofusicoccum cryptomeriae and closely related N. sinense, N. brasiliense, and N. kwambonambiense using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset.
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Figure 5. Morphology of Neofusicoccum cryptomeriae (G24). (A) Five-day-old front and back view culture on PDA. (B,C) Conidioma formed on pine needle culture. (D) Conidiophores, conidiogenous cells, and developing conidia. (E) Conidiogenous cells. (F) Conidia with 1 septum (indicated by arrow). (G) Conidia. Scale bars: (B,C) = 500 μm; (D–G) = 10 μm.
Figure 5. Morphology of Neofusicoccum cryptomeriae (G24). (A) Five-day-old front and back view culture on PDA. (B,C) Conidioma formed on pine needle culture. (D) Conidiophores, conidiogenous cells, and developing conidia. (E) Conidiogenous cells. (F) Conidia with 1 septum (indicated by arrow). (G) Conidia. Scale bars: (B,C) = 500 μm; (D–G) = 10 μm.
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Figure 6. Morphology of Neofusicoccum parvum (G15). (A) Five-day-old front and back view culture on PDA. (B) Conidioma formed on pine needle culture. (C) Conidiogenous cells and developing conidia. (D) Hyaline conidia. (E) Brown, septate conidia (indicated by arrow). Scale bars: (B) = 200 μm; (C–E) = 10 μm.
Figure 6. Morphology of Neofusicoccum parvum (G15). (A) Five-day-old front and back view culture on PDA. (B) Conidioma formed on pine needle culture. (C) Conidiogenous cells and developing conidia. (D) Hyaline conidia. (E) Brown, septate conidia (indicated by arrow). Scale bars: (B) = 200 μm; (C–E) = 10 μm.
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Table
Table 1. Strains obtained in this study and downloaded from GenBank with accession numbers used for phylogenetic analyses.
Table 1. Strains obtained in this study and downloaded from GenBank with accession numbers used for phylogenetic analyses.
GenBank Accession Number
SpeciesIsolateITS ctef1 ctub2 crpb2 cReference
Neofusicoccum arbutiCBS 116131 aAY819720KF531792KF531793KX464003[32]
N. arbutiCBS 117090AY819724KF531791KF531794N/A[32]
N. australeCBS 110865AY343408KX464661KX464937KX464005[40]
N. australeCMW 6837 aAY339262AY339270AY339254EU339573[40]
N. batangarumCBS 124923FJ900608FJ900654FJ900635FJ900616[41]
N. batangarumCBS 124924 aFJ900607FJ900653FJ900634FJ900615[41]
N. brasilienseCMM 1285JX513628JX513608KC794030N/A[42]
N. brasilienseCMM 1338 aJX513630JX513610KC794031N/A[42]
N. buxiCBS 113714KX464164KX464677KX464954KX464009[40]
N. buxiCBS 116.75 aKX464165KX464678N/AKX464010[40]
N.caryigenumCBS 146964 aMW405114MW393657MW393679MW393668[26]
N. cordaticolaCBS 123634 aEU821898EU821868EU821838EU821928[38]
N. cordaticolaCBS 123635EU821903EU821873EU821843EU821933[38]
N. cryptoaustraleCMW 23785 aFJ752742FJ752713FJ752756KX464014[40]
N. cryptomeriaeG24 = CFCC 55721 a,bON209700OP056461OP056458OP056455this study
N. cryptomeriaeG1 = CFCC 55720 bON209698OP056459OP056456OP056453this study
N. cryptomeriaeG2 = CFCC 55728 bON209699OP056460OP056457OP056454this study
N. eucalypticolaCBS 115679 aAY615141AY615133AY615125N/A[43]
N. eucalypticolaCBS 115766AY615143AY615135AY615127N/A[43]
N. eucalyptorumCBS 115791 aAF283686AY236891AY236920N/A[44]
N. eucalyptorumCMW 10126AF283687AY236892AY236921N/A[44]
N. grevilleaeCBS 129518 aJF951137N/AN/AN/A[45]
N. hellenicumCERC1947 aKP217053KP217061KP217069N/A[46]
N. hellenicumCERC1948KP217054KP217062KP217070N/A[46]
N. hongkongenseCERC2968KX278051KX278156KX278260KX278282[17]
N. hongkongenseCERC2973 aKX278052KX278157KX278261KX278283[17]
N. hypericiMUCC 241 aLC589125LC589137LC589147LC589160[28]
N. hypericiMUCC 2509LC589126LC589138LC589148LC589161[28]
N. illiciiCGMCC 3.18310 aKY350149N/AKY350155N/A[34]
N. illiciiCGMCC 3.18311KY350150KY817756KY350156N/A[34]
N. kwambonambienseCBS 123639EU821900EU821870EU821840EU821930[38]
N. kwambonambienseCBS 123641EU821919EU821889EU821859EU821949[38]
N. lumnitzeraeCMW 41228KP860882KP860725KP860803KU587926[47]
N. lumnitzeraeCMW 41469 aKP860881KP860724KP860801KU587925[47]
N. luteumCBS 562.92 aKX464170KX464690KX464968KX464020[40]
N. macroclavatumCBS 118223 aDQ093196DQ093217DQ093206KX464022[48]
N. magniconidiumCSF5875MT028611MT028777MT028943MT029084[24]
N. magniconidiumCSF5876 aMT028612MT028778MT028944MT029085[24]
N. mangiferaeCBS 118531 aAY615185DQ093221AY615172N/A[48]
N. mangiferaeCBS 118532AY615186DQ093220AY615173KX464023[48]
N. mediterraneumCBS 121718 aGU251176GU251308GU251836KX464024[49]
N. microconidiumCERC3497 aKX278053KX278158KX278262MF410203[17]
N. microconidiumCERC3498KX278054KX278159KX278263MF410204[17]
N. miyakoenseMUCC 2585 aN/ALC589146LC589157LC589170[28]
N. miyakoenseMUCC 2586LC589133LC589144LC589155LC589168[28]
N. moraceariumMELU 19-2758 aNR174834MW183808N/AN/A[29]
N. moraceariumMFLU 19-0316MW063187MW183809N/AN/A[29]
N. mystacidiiCBS 147079 aNR173012MW890094MW890133MW890065[30]
N. ningerenseCSF6028MT028613MT028779MT028945MT029086[24]
N. ningerenseCSF6030MT028614MT028780MT028946MT029087[24]
N. nonquaesitumCBS 126655 aGU251163GU251295GU251823KX464025[49]
N. nonquaesitumPD 301GU251164GU251296GU251824N/A[49]
N. occulatumCBS 128008 aEU301030EU339509EU339472EU339558[39]
N. occulatumMUCC 286EU736947EU339511EU339474EU339560[39]
N. okinawaenseMUCC 789 aLC589134LC589145LC589156LC589169[28]
N. parviconidiumCSF5667 aMT028615MT028781MT028947MT029088[24]
N. parviconidiumCSF5677MT028619MT028785MT028951MT029092[24]
N. parvumATCC 58191 aAY236943AY236888AY236917EU821963[44]
N. parvumCMW 9080AY236942AY236887AY236916EU821962[44]
N. parvumG15 = CFCC 55724 bON209685OP095379OP095383OP095387this study
N. parvumG16 = CFCC 55718 bON209686OP095380OP095384OP095388this study
N. parvumG91 = CFCC 55719 bON209687OP095381OP095385OP095389this study
N. parvumG92 = CFCC 55723 bON209688OP095382OP095386OP095390this study
N. pennatisporumWAC 13153 aNR136987EF591976EF591959N/A[50]
N. pistaciaeCBS 595.76 aKX464163KX464676KX464953KX464008[40]
N. podocarpiCBS 115065MT587507MT592222MT592714MT592411[25]
N. podocarpiCBS 131677 aMT587508MT592223MT592715MT592412[25]
N. protearumCBS 114176 aAF452539KX464720KX465006KX464029[40]
N. rapaneaeCBS 145973 aMT587511MT592226MT592718MT592415[25]
N. rapaneaeCPC 32578MT587512MT592227MT592719MT592416[25]
N. ribisCBS 114306MT587514MT592229MT592721MT592418[25]
N. ribisCBS 115475 aAY236935AY236877AY236906EU821958[25]
N. sichuanenseSICAUCC22-0099 aOP058990OP066336OP066363OP066355[27]
N. sichuanenseSICAUCC22-0093OP058984OP066333OP066357OP066349[27]
N. sinenseCGMCC 3.18315 aKY350148KY817755KY350154N/A[34]
N. sinoeucalyptiCERC2005 aKX278061KX278166KX278270KX278290[17]
N. sinoeucalyptiCERC3416KX278064KX278169KX278273KX278293[17]
N. stellenboschianaCBS 110864 aAY343407AY343348KX465047KX464042[40]
N. terminaliaeCBS 125263 aGQ471802GQ471780KX465052KX464045[40]
N. terminaliaeCBS 125264GQ471804GQ471782KX465053KX464046[40]
N. umdonicolaCBS 123645 aEU821904EU821874EU821844EU821934[40]
N. umdonicolaCBS 123646EU821905EU821875EU821845EU821935[40]
N. ursorumCMW 23790FJ752745FJ752708KX465057N/A[40]
N. ursorumCMW 24480 aFJ752746FJ752709KX465056KX464047[40]
N. variabileCMW 37739 aMH558608N/AMH569153N/A[51]
N. variabileCMW 37742MH558609MH576585MH569154N/A[51]
N. viticlavatumCBS 112878 aAY343381AY343342KX465058KX464048[32]
N. viticlavatumCBS 112977AY343380AY343341KX465059N/A[32]
N. vitifusiformeCBS 110880AY343382AY343344KX465008N/A[40]
N. vitifusiformeCBS 110887AY343383AY343343 KX465061KX464049[40]
N. yunnanenseCSF6034MT028672MT028838MT029004MT029117[24]
N. yunnanenseCSF6142 aMT028667MT028833MT028999MT029112[24]
Botryosphaeria dothideaCBS 115476 aAY236949AY236898AY236927EU339577[44]
a Ex-type cultures are shown in bold. b Isolates used in this study. c ITS, internal transcribed spacer; rpb2, DNA-directed RNA polymerase II subunit; tef1, partial translation elongation factor 1-alpha gene; tub2, partial beta-tubulin gene.
Table
Table 2. Synoptic characters of two species of Neofusicoccum in this study and related Neofusicoccum spp.
Table 2. Synoptic characters of two species of Neofusicoccum in this study and related Neofusicoccum spp.
SpeciesIsolatesConidiogenous Cells (μm)Conidia (μm)Reference
Neofusicoccum cryptomeriaeG1 = CFCC 5572016.4–24.1 × 3–3.920.5–25.7 × 7.1–8.9this study
G2 = CFCC 5572815.8–24.2 × 3.2–420.1–22.6 × 6.8–8this study
G24 = CFCC 5572113.8–22.4 × 3.4–4.223–26.1 × 7–7.8this study
N. parvumG15 = CFCC 5572413.4–18.4 ×2.6–3.817–18.7 × 5.8–6.3this study
G16 = CFCC 5571811.4–19.5 × 2.1–3.616.8–18.1 × 5.7–6.3this study
G91 = CFCC 5571913.7–20.9 × 2.5–3.317.1–19.5 × 5.7–6.4this study
G92 = CFCC 5572312.2–21.4 × 2.6–3.417.1–18.8 × 5.6–6.3this study
N. sinenseCGMCC 3.18315not observed17.6–20.4 × 7.4–8[34]
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MDPI and ACS Style

Si, Y.-Z.; Sun, J.-W.; Wan, Y.; Chen, Y.-N.; He, J.; Li, W.-Z.; Li, D.-W.; Zhu, L.-H. Neofusicoccum cryptomeriae sp. nov. and N. parvum Cause Stem Basal Canker of Cryptomeria japonica in China. J. Fungi 2023, 9, 404. https://doi.org/10.3390/jof9040404

AMA Style

Si Y-Z, Sun J-W, Wan Y, Chen Y-N, He J, Li W-Z, Li D-W, Zhu L-H. Neofusicoccum cryptomeriae sp. nov. and N. parvum Cause Stem Basal Canker of Cryptomeria japonica in China. Journal of Fungi. 2023; 9(4):404. https://doi.org/10.3390/jof9040404

Chicago/Turabian Style

Si, Yuan-Zhi, Jian-Wei Sun, Yu Wan, Yi-Na Chen, Jiao He, Wei-Zheng Li, De-Wei Li, and Li-Hua Zhu. 2023. "Neofusicoccum cryptomeriae sp. nov. and N. parvum Cause Stem Basal Canker of Cryptomeria japonica in China" Journal of Fungi 9, no. 4: 404. https://doi.org/10.3390/jof9040404

APA Style

Si, Y. -Z., Sun, J. -W., Wan, Y., Chen, Y. -N., He, J., Li, W. -Z., Li, D. -W., & Zhu, L. -H. (2023). Neofusicoccum cryptomeriae sp. nov. and N. parvum Cause Stem Basal Canker of Cryptomeria japonica in China. Journal of Fungi, 9(4), 404. https://doi.org/10.3390/jof9040404

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