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Article

A New Species of Biscogniauxia Associated with Pine Needle Blight on Pinus thunbergii in China

1
Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2
The Connecticut Agricultural Experiment Station Valley Laboratory, Windsor, CT 06095, USA
*
Author to whom correspondence should be addressed.
Forests 2024, 15(6), 956; https://doi.org/10.3390/f15060956
Submission received: 27 April 2024 / Revised: 23 May 2024 / Accepted: 28 May 2024 / Published: 30 May 2024
(This article belongs to the Section Forest Health)

Abstract

:
In June 2020, needle blight symptoms on Pinus thunbergii were discovered in Bazhong City, Sichuan Province, China. Fungal isolates were obtained from the pine needles of P. thunbergii. After examining morphological characteristics and conducting multi-locus (ITS, ACT, TUB2 and RPB2) phylogenetic analyses, the isolates SC1–SC5 were determined to be a new species, Biscogniauxia sinensis. Genealogical Concordance Phylogenetic Species Recognition with a pairwise homoplasy index test was used to further verify the results of the phylogenetic analyses. The morphology and phylogenetic relationships between this new species and other related Biscogniauxia species were discussed. To our knowledge, this is also the first report of Biscogniauxia sinensis associated with pine needle blight on P. thunbergii in China. The needle damage of P. thunbergii associated with Biscogniauxia sinensis will detrimentally affect the carbon absorption and photosynthetic efficiency of P. thunbergii, further reduce the absorption of nutrients by Japanese black pine and may lead to the imbalance of pine forest conditions, which will have a negative impact on the forest ecological system.

1. Introduction

Pinus thunbergii (Japanese black pine) is native to Japan and Korea and is currently widely distributed in China due to its evergreen attributes, fast growth, and salinity tolerance [1]. It plays essential roles in ecological restoration, such as sand fixation and afforestation [2,3,4]. Despite the ecological effects, P. thunbergii in China were infected by many pathogens, which caused the wilt and mortality of pine trees. For instance, pine wilt disease caused by the pine wood nematode (Bursaphelenchus xylophilus) is the most devastating pine disease in China [5]. Needle cast disease caused by Lophodermium pinastri was extremely infectious, with a high incidence in young and mature forests. At the early stage, infected needles exhibited yellow spots, then turned to light brown, leading to the abscission of needles. In the spring of the subsequent year, the fallen needles became thin; black horizontal lines emerged and divided the needles into smaller portions. An oval, black fruiting body was then formed between these lines. Upon maturation, these fruiting bodies absorbed moisture, expanded, and developed a central longitudinal fissure, through which ascospores were released [6,7,8]. Brown-spot needle blight on P. thunbergii caused by Lecanosticta acicola initially produced small, discolored spots on infected needles, turned brown and died off quickly. The disease started at the base of the crown and gradually spread upward [9]. Such diseases are common with pine needles and also have posed severe threats to the growing conditions of P. thunbergii. However, the symptoms of pine needle blight with sporadic black spots are different from these aforementioned diseases.
In June 2020, pine needle blight symptoms were found on P. thunbergii at Nanyang Forest Farm, Bazhong city, Sichuan Province, China. Initial observations of discoloration and wilting on black pine needles were made in May, with a significant increase in yellowing and wilting symptoms occurring in June. The incidence peaked during July and August, coinciding with rising summer temperatures and the onset of the rainy season, sometimes extending into September. This disease progressed rapidly, infecting black pines of various ages and ultimately caused the needles to turn brown, even causing the complete death of the trees. Surveys indicated a disease incidence rate of 30%, signifying a high severity of this disease.
The objectives of this study are to determine a fungus isolated from Pinus thunbergii in Sichuan Province with subsequent morphological and phylogenetic analyses. GCPSR with a PHI test was conducted to further justify the placement of this species. Its morphology and molecular differences were compared with known species in the genus.

2. Materials and Methods

2.1. Sampling and Isolation

In total, 20 needles with typical symptoms were collected from Japanese black pines in a pine forest in Bazhong, Sichuan (31°85′88.09″ N, 106°75′36.69″ E (DMS)). All 20 needles were soaked in 1.5% sodium hypochlorite solution for 90 s for surface disinfection and rinsed in sterile water 3 times. The disinfected material was removed using a sterilized scalpel, and disease/health junction parts were cut into 2–3 × 2–3 mm blocks. These tissue blocks were cultivated on 2% potato dextrose agar medium (PDA) and incubated at 25 °C without light [10]. Conidia were removed from colonies with a needle, and pure cultures were obtained by single spore isolation using serial dilution techniques [11]. These pure isolates were stored in the Forest Pathology Laboratory of Nanjing Forestry University. The holotype of the new fungal taxon from this study was deposited at the China Forestry Culture Collection Center (CFCC).

2.2. Colony Observations

Morphological descriptions of the colonies and colony growth rates of isolates were evaluated with PDA and OA. The mycelium block (6 mm in diameter) was placed at the plate center and incubated at 25 °C. Each treatment had five replicates. Colony diameters were measured in perpendicular directions on 5th and 7th days for calculating the growth rate. Color, shape, and texture of the colonies on both sides were recorded.

2.3. Genomic DNA Extraction, PCR, and Sequencing

DNA was extracted from all isolates using the CTAB method [12], and DNA quality and quantity were assessed using Nanodrop, Qubit (Thermo Fisher, Waltham, MA, USA). According to the study on phylogenetic relationship of Obolarina/Biscogniauxia by Mirabolfathy et al. [13], four loci of ITS, ACT, TUB2 and RPB2 were amplified and sequenced with primer sets of ITS-5/ITS-4 [14], ACT-512F/ACT-783R [15], Bt2a/Bt2b [16], and fPB2-5F/fPB2-7cR [14], respectively. The polymerase chain reaction (PCR) was performed in a total volume of 20 µL, which contained 1 µL of DNA sample, 1 µL sense primer, 1 µL antisense primer, 7 µL ddH2O and 10 µL of 2× Green Taq Mix (Table 1). Sequencing of the PCR amplicons was performed by Shanghai Sangon Biotechnology Company.

2.4. Morphological Identification

Conidia, conidiogenous cells, and conidiophores were visualized and measured with a Zeiss Axio Imager A2m microscope equipped with Differential Interference Contrast optics (DIC) (Carl Zeiss, Oberkochen, Germany). Microphotographs were taken with ZEN 2 (blue edition, Axio Cam HR R3). The morphological characteristics were described based on the morphology of conidia, conidiogenous cells, and conidiophores [13].

2.5. Phylogenetic Analyses

All nucleotide sequences were searched in NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 15 June 2023) using BLAST toolkit. The BLASTn search based on ITS sequences indicated Biscogniauxia sp. SC3 had the highest sequence identity (100%) with Biscogniauxia maritima (MN844502). Based on the TUB2 and RPB2 sequences, Biscogniauxia sp. SC3 showed highest sequence identity with Biscogniauxia atropunctata (AY951673, 93.65%) and Biscogniauxia atropunctata (JX507778, 96.03%), respectively. While BLASTing with ACT sequence, Biscogniauxia sp. SC3 had the highest sequence identity (94.98%) with Obolarina persica (JX507798). The 72 available ITS sequences of all Biscogniauxia taxa were retrieved from the GenBank and a preliminary phylogenetic analysis was conducted using ITS sequences only. Based on the phylogenetic analysis of ITS, 21 closely related taxa with all multi-locus sequences (ITS + ACT + TUB2 + RPB2) were determined and retrieved from GenBank to construct a more reliable phylogenetic tree. The most closely related species outside the study group like Annulohypoxylon cohaerens [17] and Hypoxylon rubiginosum [18] were chosen as the outgroup [13,19]. The members of the genus Biscogniauxia included in phylogenetic analyses were listed in Table 2. BioEdit 7.0.5.3 [20] was used for multiple sequence alignment, clipping and merging to concatenated ITS + ACT + TUB2 + RPB2 sequence dataset [21]. The concatenated dataset was analyzed in PhyloSuite v1.2.2 [22,23]. ModelFinder was used to choose the most suitable model based on AIC and BIC criteria [24]. Maximum likelihood (ML) analysis was implemented with sequences of multi-loci using IQtree ver. 1.6.8. Bootstrap with 1000 replicates and the GTR + F+I + G4 model were used to improve the assessment of evolutionary branch stability and confidence level in the phylogenetic trees [25,26]. GTR + F + I + G4 model adjusted branch length distribution and the model parameters, until the likelihood value reached maximum. Bayesian Inference (BI) phylogenies were inferred using MrBayes 3.2.6 [27] with SYM + I + G model, 2 parallel runs and 2,000,000 generations. The initial 25% of sampled data were discarded as burn-in. The remaining trees were pooled, and the posterior probabilities (PP) of each branch system as a monophyletic system were calculated. Phylogenetic trees were visualized in FigTree v. 1.4.0 [28].

2.6. Genealogical Concordance Phylogenetic Species Recognition Analysis

The Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model and paired homology index (PHI or Φw) tests were used to make comparisons between new species and closely related taxa [47]. The PHI tests were applied in SplitsTree4 [48] to identify the level of recombination among closely related species using a four-locus dataset. A PHI index lower than 0.05 threshold (Φw < 0.05) indicated remarkable recombination in the dataset. The association between this new taxon and closely related species was visualized with splits graphs using the LogDet transformation and splits decomposition options [26].

3. Results

3.1. Sampling and Isolation

In June 2020, pine needle blight symptoms were found on P. thunbergii at Nanyang Forest Farm, Bazhong city, Sichuan Province, China. The incidence of the disease was ca. 30%, but severe. All infected pine needles showed brownish-yellow necrotic lesions at the tips, then the symptoms gradually spread to the entire pine needles. The pine needles became light brown, chlorotic, dry, and necrotic. In the late stage, infected pine needles defoliated.
Five fungal isolates (SC1, SC2, SC3, SC4 and SC5) with similar colony morphology were obtained from the infected pine needle tissues. The above isolates were used for subsequent phylogenetic analyses, and isolate SC3 was employed for morphology study.

3.2. Phylogenetic Analyses

The phylogenetic analysis using ITS sequences only showed that five fungal isolates (SC1, SC2, SC3, SC4 and SC5) were clustered in an independent clade, and they were closely related with B. atropunctata (G411 and SGSGf17), B. mediterranea var. microspora and B. nummularia var. merrillii (Figure 1).
The phylogenetic analyses using a four-locus (ITS, ACT, TUB2, RPB2) dataset of Biscogniauxia were conducted to verify the above classification result. This data set consisted of 84 sequences, including 21 ITS sequences, 21 ACT sequences, 21 TUB2 sequences and 21 RPB2 sequences from 17 taxa. Meanwhile, 20 novel sequences from 5 isolates (SC1-SC5) of Biscogniauxia sp. were generated from our collections and analyzed. Consequently, phylogenetic analyses were performed with a total of 1911 characters, including gaps that were ACT: 1–320, ITS: 321–654, RPB2: 655–1507, TUB2: 1508–1911. In this phylogenetic tree, five isolates (SC1-SC5) were clustered in a distinct clade with well-supported values (BI/ML = 1/94) (Figure 2), and these isolates differed from all other known species (Figure 2). Notably, this clade had a close relationship with B. atropunctata YMJ128. Based on the two aforementioned phylogenetic analyses, the five fungal isolates represented a new species, Biscogniauxia sinensis sp. nov.

3.3. Genealogical Concordance Phylogenetic Species Recognition

The GCPSR principle was applied to evaluate the boundary of different species. The PHI test (Figure 3) on Biscogniauxia sinensis showed that there was no significant recombination (Φw = 1.0) between Biscogniauxia sinensis and its closely related species, B. atropunctata (SGSGf17, B5A, YMJ128).

3.4. Taxonomy

Biscogniauxia sinensis Xiao-Lei Ding, Chang-Xia Qiao and D.W. Li, sp. nov. Figure 3.
Index Fungorum Number: IF 900185.
Etymology: The epithet refers to China where the holotype was collected.
Diagnosis: Differs from B. atropunctata in its shorter conidiogenous cells, is light brown and has smaller conidia. Differs from B. mediterranea in its smaller conidiogenous cells and shorter conidia. Differs from B. rosacearum in its shorter and wider conidia.
Description: The sexual state was not observed in vitro. Conidiophores, which are hyaline to yellowish, often occur on the aerial hyphae, composing of a principal axis and one or more branches, (7.5-) 8.2–11.8 (-13.7) × (1.9-) 2.2–3.0 (-3.6) µm, (mean ± SD = 10.0 ± 1.8 × 2.6 ± 0.4 µm, n = 40), with conidiogenous cells developing terminally, generally two to three. Conidiogenous cells are hyaline, oval, (3.8-) 4.5–6.5 (-7.9) × (2.9-) 3.3–4.1 (-4.5) µm, (mean ± SD = 5.5 ± 1.0 × 3.7 ± 0.4 µm, n = 40), and the apical area has remnants of conidial secession, with the apical end distorted due to producing a large number of conidia. The conidia are hyaline to light brown, globose or subglobose, smooth, and aggregated, (3.3-) 3.5–4.3 (-5.0) × (2.9-) 3.3–3.9 (-4.4) µm, (mean ± SD = 3.9 ± 0.4 × 3.6 ± 0.3 µm, n = 40).
Culture characteristics: Colonies on PDA grow fast, reaching the whole Petri dish in 5 d at 25 °C; they are initially white, cottony, and radial with abundant aerial mycelia and diffuse margins, and become floccose with the reverse side being reddish-brown with brown pigmentation after about 14 d (Figure 4G), even showing no sporulation. Colonies of Biscogniauxia sinensis that cover the entire OA Petri dish in 9 d at 25 °C, are regular and round. The mycelia changed from translucent at first to white and turned pale gray with aging. After 15 d, the reverse side became translucent to yellowish (Figure 4H). The mycelium was tight, and the aerial hyphae were raised, white and flocculent. Sporulation formed more frequently on aerial hyphae.
Holotype: China, Sichuan Province, Bazhong city, Nanyang Forest Farm, 31°85′88.09″ N, 106°75′36.69″ E (DMS), isolated from pine needles of Pinus thunbergii, June 2020, Xiao-Lei Ding, holotype CFCC59650. The holotype is a living specimen being maintained via lyophilization at the China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing, China, and the ex-type SC3 is preserved at the Forest Pathology Laboratory, Nanjing Forestry University.
Habitat and host: On the pine needles of Pinus thunbergii with blight.
Known distribution: Bazhong, Sichuan Province, China.
Additional specimens examined: China, Sichuan Province, Bazhong city, Nanyang Forest Farm, 31°85′88.09″ N, 106°75′36.69″ E (DMS), isolated from a pine needle of Pinus thunbergii, June 2020, Xiao-Lei Ding, CFCC 59,648 (=SC1), CFCC 59,649 (=SC2), CFCC 59,651 (=SC4), CFCC 59,652 (=SC5).
Note: In the multi-locus phylogenetic tree of the Biscogniauxia species, five strains of Biscogniauxia sinensis formed a single clade. Biscogniauxia sinensis can be distinguished from the closely related species B. atropunctata, based on the base-pair differences in ITS (36 out of 469), ACT (17 out of 278), TUB2 (45 out of 520), RPB2 (47 out of 1133). Morphologically, Biscogniauxia sinensis has shorter conidiogenous cells than those of B. atropunctata (4.5–6.5 × 3.3–4.1 vs. 5–10 × 3.5–4.5) µm [35]. Conidia are colorless to light brown, which are distinguished from the colorless conidia of B. atropunctata [49]. Moreover, Biscogniauxia sinensis has smaller conidia (3.5–4.3 × 3.3–3.9 µm vs. 4–5.5 × 3–4.5 µm) than those of B. atropunctata [49].

4. Discussion

Based on the morphological features, multi-locus phylogeny, and GCPSR analyses, the five isolates obtained from wilting needles of P. thumbergii were identified as a new species. According to the available information, this is also the first report of pine needle blight on P. thunbergii associated with Biscogniauxia sinensis in China.
It is worth noting that the new species associated with pine needle blight on P. thunbergii was closely related with B. atropunctata in the multi-locus and ITS-only phylogenetic analyses. By comparing the sequences, there are obvious differences between B. atropunctata and Biscogniauxia sinensis. To further compare the morphological characteristics of these two species [49], we found that the conidia size of isolates SC1 to SC5 are different to those of B. atropunctata; they also differed remarkably in their colony morphology and growth rates [49]. The radial growth rate of B. atropunctata on OA was 12.8 mm/d, and it was white felty, azonate, had diffuse and ropy margins and became floccose, with greenish-olivaceous or dull green patches. The reverse of B. atropunctata was dull green or greenish-olivaceous [49]. Nevertheless, the isolates SC1–SC5 had a growth rate of 9.8 mm/d, which is much slower than that of B. atropunctata. The colony of SC3 on OA (Figure 3) was different from that of B. atropunctata on morphology and color. The identification results showed that the fungus on P. thunbergii differed from B. atropunctata and a new species, Biscogniauxia sinensis.
Ju et al. (1998) wrote a monograph on the genus Biscogniauxia, in which 49 taxa were covered worldwide [50]. In their study, morphological differences among similar genera were discussed, and the critical identifying features among Biscogniauxia species were provided. This genus presents a synteny relationship with other related but morphologically different genera, such as Graphostroma, Camillea and Obolarina, in Xylariaceae [39,51,52], while, Wendt et al. (2018) reinstate and amended the family Graphostromataceae [44].
Graphostromataceae comprised Biscogniauxia, Graphostroma, Camillea, Obolarina, Theissenia, and Vivantia [53]. Biscogniauxia has been reported from various woody hosts around the world; more than 10 new hosts have been found in relevant studies recorded in the fungi database of the USDA [54]. Biscogniauxia was found in Malus, Mangifera, Prunus, Pyrus, and Vitis fruit trees. Most of these hosts are angiosperms, but B. atropunctata has been isolated from Juniperus virginiana in the United States [55]. Meanwhile, B. mediterranea usually occurs on Quercus suber in the Mediterranean basin [56,57]. But B. mediterranea has been reported as a pathogen of Pinus sylvestris in Spain [58]. In 2020, it was also isolated from a twig of Abies alba with needle browning symptoms in Poland [59]. This fungus has long been considered as an endophyte in all aerial organs (rarely in leaves) of oak trees and has an incubation period without symptoms [60]. However, when the host is weakened or stressed by other pathogens, B. mediterranea can become a facultative pathogen; it can accelerate the decline of the tree and eventually lead to the death of the hosts [61,62,63]. Similar study also indicates that, as an endophyte, it lies dormant within Scots pine (Pinus sylvestris) and can influence host growth, leading to slower growth rates in the pine trees [64]. Biscogniauxia nummularia is recognized as an endophyte in dicotyledonous angiosperms and some grass species [49]. In 2019, B. nummularia was first recorded in southern Poland as an endophyte of white fir (Abies alba) and Pinus × rhaetica Brugger [65,66]. Research has illuminated the endophytic characteristics of B. nummularia, enabling it to swiftly transition from a benign endophyte to a primary pathogen [67,68,69]. Similarly, Biscogniauxia, typically found in woody plants, has also been isolated from pines such as Pinus koraiensis, firs such as Abies nephrolepis, and Douglas fir, as well as from Taxus cuspidata as an endophyte [70].
Endophytes usually reside within the internal tissues of plants without causing apparent harm to the host [71]. Due to imbalances in nutrient exchange, environmental changes, and change in host, endophytes can transit into pathogens under such circumstances [72]. Biscogniauxia is globally distributed, usually acting as an ubiquitous wood decomposer and a common endophyte [69]. There is substantial evidence that Biscogniauxia species exist as endophytes within healthy trees and later become pathogenic to plants when adverse stress occurs [70,73]. Although Biscogniauxia has been characterized as an endophytic fungus, evidence from several studies indicate that these endophytes, akin to pathogenic bacteria, possess intrinsic virulence factors and exhibit prolonged latent periods. This virulence potential facilitates the establishment of infections in host plants, particularly when they are located in senescent branches or are subjected to environmental stressors [74]. One of the most significant influence factors on the occurrence of Biscogniauxia is climate change. It has been proved that warm temperatures and a prolonged summer drought favor the growth of Biscogniauxia species [75]. Most species of Biscogniauxia are particularly inclined to affect trees under water stress [76]. Biscogniauxia sinensis might act as an endophyte in many plants, potentially existing symbiotically within them. However, it could also function as a plant pathogen, particularly under stressful conditions such as drought. Thus, the application of sanitation and scarification practices along with appropriate fungicides can effectively prevent and reduce the occurrence of the Biscogniauxia on oak trees [77]. However, the control of fungi of this genus on Japanese black pine remains to be studied. Next, biocontrol agents and other control measures will be studied for effectively reducing the impact of Biscogniauxia sinensis on forests and ecological system.The climatic conditions during summer in Bazhong City, Sichuan Province, are high temperatures and drought, which enhance plant transpiration and trigger stress conditions. These conditions may induce the expression of pathogenic traits of endophytic fungi. Although the isolation of endophytic fungi was not the focus of our study during the initial field survey, it is necessary to conduct further comprehensive research on this phenomenon.
Generally, Biscogniauxia is not frequently reported to cause plant diseases in China. In 2008, B. mandshurica was found on dead branches of Malus sp. in Changbai Mountain, Jilin Province, China [78]. In March 2014, a new species, B. glaucae, was found on the dead bark of an unknown plant in Guizhou Province, China [36]. In 2014, B. petrensis was found on rock from two karst caves in Guizhou Province, China [79]. In November 2015, B. dendrobii was found in Dendrobium aphyllum stems from a nursery in Guizhou Province, China [35]. This is the first report that the new species identified here is associated with discolored and withered needles on P. thunbergii in forest. The symptoms have affected the potential value of pine trees, influenced the balance of the forest environment, and caused consequential economic and ecological losses [10].
The discovery and classification of the new species Biscogniauxia sinensis not only enhances the diversity of the genus but also provides new perspectives on its genetic diversity and evolutionary relationships with other species [80]. It will help us better understand its ecological role and interactions with host plants. Additionally, it aids in research on how these fungi adapt to various environments and hosts, which is crucial for maintaining the health and stability of forest ecosystems.

5. Conclusions

The morphological features, multi-locus phylogeny, and GCPSR analysis were employed to determine Biscogniauxia sinensis as a novel species associated with pine needle blight on P. thunbergii. The discovery and classification of Biscogniauxia sinensis will expand the current knowledge of this genus. It is necessary to conduct further studies on this disease from multiple perspectives (mycology, epidemiology, ecology and evolution, as well as economy). From the practical aspect, future studies should also focus on reducing its potential damage to P. thunbergii and forest ecosystems.

Author Contributions

C.Q.: conceptualization, writing—original draft and review and editing, data curation, investigation, methodology and visualization. R.Z.: writing—review and editing, investigation. X.D.: writing—review and editing, project administration, resources and supervision. D.L.: writing—review and editing, supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This project is supported by the National Natural Science Foundation of China 31800543.

Institutional Review Board Statement

Not applicable for studies not involving humans or animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors are very grateful to Mao-Jiao Zhang for her assistance in morphological and phylogenetic analyses.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic relationship of SC1, SC2, SC3, SC4 and SC5 with related taxa in Biscogniauxia derived from ITS. The tree is rooted with Annulohypoxylon cohaerens (YMJ 310). The values on the branches are Bayesian posterior probability (BI-PP) ≥ 0.90 and the ML bootstrap value (BS) ≥ 70, respectively. Isolates in this study are highlighted in red and holotype isolates are in bold.
Figure 1. Phylogenetic relationship of SC1, SC2, SC3, SC4 and SC5 with related taxa in Biscogniauxia derived from ITS. The tree is rooted with Annulohypoxylon cohaerens (YMJ 310). The values on the branches are Bayesian posterior probability (BI-PP) ≥ 0.90 and the ML bootstrap value (BS) ≥ 70, respectively. Isolates in this study are highlighted in red and holotype isolates are in bold.
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Figure 2. Phylogenetic relationship of five isolates (SC1, SC2, SC3, SC4 and SC5) with related taxa in Biscogniauxia derived from completed squences of ITS, ACT, TUB and RPB2. The tree is rooted with Annulohypoxylon cohaerens (YMJ 310) and Hypoxylon rubiginosum (YMJ 24). The values on the branches are Bayesian posterior probability (BI-PP) ≥ 0.90 and the ML bootstrap value (BS) ≥ 70, respectively. Isolates in this study are highlighted in red and holotype isolates are in bold.
Figure 2. Phylogenetic relationship of five isolates (SC1, SC2, SC3, SC4 and SC5) with related taxa in Biscogniauxia derived from completed squences of ITS, ACT, TUB and RPB2. The tree is rooted with Annulohypoxylon cohaerens (YMJ 310) and Hypoxylon rubiginosum (YMJ 24). The values on the branches are Bayesian posterior probability (BI-PP) ≥ 0.90 and the ML bootstrap value (BS) ≥ 70, respectively. Isolates in this study are highlighted in red and holotype isolates are in bold.
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Figure 3. Pairwise homoplasy index test among Biscogniauxia sinensis and their closely related taxa.
Figure 3. Pairwise homoplasy index test among Biscogniauxia sinensis and their closely related taxa.
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Figure 4. Morphological characteristics of Biscogniauxia sinensis from Pinus thunbergii. (A) Diseased needles in the field. (B) Conidiomata on needles. (CE) Conidiophores, conidiogenous cells and conidia. (F) Conidia. (G) Cultures on PDA from above (right) and reverse (left). (H) Cultures on OA from above (left) and reverse (right). Scale bars: (B) = 500 µm; (CG) = 10 µm.
Figure 4. Morphological characteristics of Biscogniauxia sinensis from Pinus thunbergii. (A) Diseased needles in the field. (B) Conidiomata on needles. (CE) Conidiophores, conidiogenous cells and conidia. (F) Conidia. (G) Cultures on PDA from above (right) and reverse (left). (H) Cultures on OA from above (left) and reverse (right). Scale bars: (B) = 500 µm; (CG) = 10 µm.
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Table 1. Primers and parameters used for PCR amplification.
Table 1. Primers and parameters used for PCR amplification.
LocusPCR Primers
(Forward/Reverse)
PCR: Thermal Cycles:
(Annealing Temperature in Bold)
ITSITS5/ITS495 °C: 5 min, (95 °C: 30 s, 55 °C: 30 s, 72 °C: 90 s) ×33 cycles, 72 °C: 10 min
ACTACT-512F/ACT-783R95 °C: 2 min, (95 °C: 30 s, 61 °C: 30 s, 72 °C: 60 s) ×35 cycles, 72 °C: 5 min
TUB2Bt-2a/Bt-2b95 °C: 2 min, (95 °C: 30 s, 55 °C: 30 s, 72 °C: 60 s) ×35 cycles, 72 °C: 5 min
RPB2fRPB2-5F/fRPB2-7cR95 °C: 2 min, (95 °C: 30 s, 54 °C: 30 s, 72 °C: 60 s) ×35 cycles, 72 °C: 5 min
Table 2. List of Biscogniauxia strains used in phylogenetic analysis.
Table 2. List of Biscogniauxia strains used in phylogenetic analysis.
SpeciesLocalityHost Strain Number 1GenBank AccessionsReference
ITSACTTUB2RPB2
Annulohypoxylon cohaerensFranceFagusYMJ 310 = BCRC34013 abEF026140AY951766AY951655GQ844766[29]
Biscogniauxia ancepsFranceFagusYMJ 123 = BCRC34029 abEF026132AY95178AY951671JX507777[29]
B. ancepsSpainEucalyptus sp.CBS 149687 aOQ990096NANANA[30]
B. ancepsEnglandunknownKM 201862 aMZ159582NANANAsubmitted directly
B. arimaMexicowoodYMJ 122 * = BCRC34030 abEF026150AY951784AY951672GQ304736[29]
B. atropunctataEcuadorPinusNY8660c aHQ108028NANANAsubmitted directly
B. atropunctataunknownSwitchgrassIUB:B.Whitaker:OTU41 aMH178710NANANAsubmitted directly
B. atropunctataCanadaAcerKC2031E5 aKX589181NANANAsubmitted directly
B. atropunctataunknownunknownKoLRI_053373 aMZ855376NANANAsubmitted directly
B. atropunctataunknownmilk thistleG411 aKM215648NANANAsubmitted directly
B. atropunctataunknownunknownK135 aMK304352NANANAsubmitted directly
B. atropunctataunknownunknownC226 aMK304016NANANAsubmitted directly
B. atropunctataunknownunknownW498 aMK247673NANANAsubmitted directly
B.atropunctataIranCeltis australisBIAT72 aMF497753NANANAsubmitted directly
B. atropunctataMexicoTaxus globosaSGSGf17 aEU715651NANANA[31]
B. atropunctataUSAwoodYMJ 128 abJX507799AY951785AY951673JX507778[13]
B. atropunctata var. intermedia Costa RicaQuercus sp.B70M = GB 4796 aAJ390412NANANA[31]
B. bartholomaeiUSAbranchesATCC 38992 aAF201719NANANA[32]
B. bartholomaeiUSAbranches108898191 aON692782NANANA[32]
B. capnodesChinawoodYMJ 138 = BCRC34032 bEF026131AY951787AY951675JX507779[29]
B. capnodesunknownunknownS74 aOR237607NANANAsubmitted directly
B. capnodesunknownunknownFS39 aMF770835NANANAsubmitted directly
B. capnodesGabonunknownGAB038 aKY250379NANANAsubmitted directly
B. capnodes var. capnodesNew ZealandLophozonia menziesii11682 aMH410019NANANAsubmitted directly
B. cinereolilacinaNorwayunknownCBS 10496 aMH862567NANANA[33]
B. citriformisUSACasuarina equisetifoliaYMJ 129 = BCRC34034 bJX507801AY951789AY951678JX507781[13]
B. citriformisChinawoodYMJ 88113012 abJX507800AY951790AY951677JX507780[13]
B. cylindrisporaChinaCinnamomumYMJ 89092701 = BCRC33717 abEF026133AY951791AY951679JX507782[34]
B. dendrobiiChinaDendrobium aphyllumMFLUCC 17 2607 * aNR172733NANANA[35]
B. formosanaChinaBark YMJ 89032201 * = BCRC33718 abJX507802AY951792AY951680JX507783[34]
B. formosanaunknownunknownGjav1ITS2OTU03662 aKY588559NANANAsubmitted directly
B. granmoAustriaPrunus padusYMJ 135 = BCRC34035 abJX507803AY951793AY951681JX507784[13]
B. glaucaeChinaQuercus glaucaGMBC 0007 abMT624046MT622656MT622654MT622652[36]
B. latirimaChinaBark YMJ 89101101 = BCRC33729 abJX507804AY951794AY951682JX507785[13]
B. latirimaChinaBark YMJ 90080703 = BCRC34036 abEF026135AY951795AY951683JX507786[29]
B. magnaThailandUnidentifiedMFLU 18-0850 aMW240616NANANA[37]
B. mangiferaeThailandMangifera indicaMFLU 18-0827 *aMN337232NANANA[38]
B. marginataSwitzerlandSorbus aucupariaZT-Myc-64233 aMW489534NANANAsubmitted directly
B. maritimaRussiaunknownLE 262845 aJQ247198NANANAsubmitted directly
B. maritimaRepublic of KoreaunknownKoLRI_EL004708 aMN844502NANANAsubmitted directly
B. maritimaRepublic of KoreaParmotremaKoRLI046050 aMN341558NANANAsubmitted directly
B. maritimaRepublic of KoreaunknownKACC 410582 aOR886237NANANAsubmitted directly
B. marginataGermanyMalus sp.CBS 124505 abKU684016KU684036KU684124KU684310[36]
B. mediterraneaItalyQ. pubescensB × 70 aKT253502NANANA[39]
B. mediterraneaItalyQ. pubescensB × 85 aKT253503NANANA[39]
B. mediterraneaFranceFagus sp.YMJ 147 = BCRC34037 bEF026134AY951796AY951684GQ844765[39]
B. mediterraneaJapan Malus sp.F8012 aAB693903NANANAsubmitted directly
B. mediterraneaUSAAcerVH04 aOR778793NANANAsubmitted directly
B. mediterraneaUSAwoodKern805 aOP038087NANANA[40]
B. nothofagiNew ZealandLophozonia menziesiiNZFS 3015 a MN007010NANANAHood, I.A submitted directly.
B. mediterranea var. microsporaThailandunknownBCC 1198 aAB376712NANANA[41]
B. nummulariaEngland Salix albaH86 = CCF 3919 bGQ428318GQ428312GQ428324FR715504[39]
B. nummulariaFranceFagus sp.MUCL 51395 * aJX658444NANANA[39]
B. nummulariaChinaunknownF8422 aON332160NANANAsubmitted directly
B. nummulariaPolandViscum album122H aOP699770NANANA[42]
B. nummulariaPolandPinusBn5L-19Pu aMN588203NANANAsubmitted directly
B. nummulariaIranZelkovaBINU72 aMF358880NANANAsubmitted directly
B. cf. nummularia unknownLewinskya2010_26_NT2 aMW907969NANANAsubmitted directly
B. nummularia var. merrilliiThailandbarkBCC 1213 aAB376714NANANA[41]
B. petrensisThailandDendrobium harveyanumMFLUCC 14-0151 * aMK951680NANANA[38]
B. cf. petrensis EcuadorwoodRLC_1445.1_iNat_7002890 aOQ878456NANANA[43]
B. petrensisJapanTeaST253 aLC685807NANANAsubmitted directly
B. petrensisChinaOsmanthus sp.HKAS 102388 aMW240615NANANA[37]
B. petrensisunknownunknownX2P3HRa aON921656NANANAsubmitted directly
B. philippinensis var. microspora ChinaBark YMJ 89041101 * = BCRC33720 abEF026136AY951797AY951685JX507787[39]
B. repandaUSAFagus sp.ATCC 62606 aKY610383NANANA[44]
B. repandaunknownunknownB75A aAJ390418NANANA[45]
B. rosacearumItaly P. domesticaBx26 = CBS141046 * aKT253493NANANA[39]
B. rosacearumItaly P. domesticaBx4KT253491NANANA[39]
B. rosacearumKhorramabadQuercus. brantiiIRAN 4288C aMZ359664NANANA[45]
B. rosacearumIranalmond treeIRNBS68 aMW452324NANANA[45]
B. rosacearumunknownunknownCSN1052 aMT813910NANANA[46]
Biscogniauxia sinensisChina Pinus thunbergiiSC1 = CFCC59648 abOR803753OR832182OR832177OR832187This study
Biscogniauxia sinensisChina P. thunbergiiSC2 = CFCC59649 abOR803754OR832183OR832178OR832188This study
Biscogniauxia sinensisChina P. thunbergiiSC3 = CFCC59650 * abOR803755OR832184OR832179OR832189This study
Biscogniauxia sinensisChina P. thunbergiiSC4 = CFCC59651 abOR803756OR832185OR832180OR832190This study
Biscogniauxia sinensisChina P. thunbergiiSC5 = CFCC59652 abOR803757OR832186OR832181OR832191This study
B. simpliciorFrance Rhamnus catharticaYMJ 136 = BCRC34038 abEF026130AY951798AY951686JX507788[39]
B. simpliciorunknownunknownB73C aAJ3904916NANANA[31]
B. uniapiculataChina BarkYMJ 90080608 = BCRC34039 abJX507805AY951799AY951687JX507789[39]
B. aff. uniapiculataGabonunknownGAB220 aKY250378NANANAsubmitted directly
B. whalleyiThailandwoodSWUF 13-85 abMW403821MZ466385MZ466386MZ466387Jantaharn, P.
submitted directly.
Hypoxylon rubiginosumUKFraxinusYMJ 24 = BCRC34116 bEF026143AY951862AY951751JX507791[39]
Note: NA, not applicable. Strains in this study are marked in bold. * = ex-type. a = ITS phylogenetic analyses. b = complete sequences of ITS + ACT + TUB2 + RPB2. 1 MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; MFLU: Mae Fah Luang University Herbarium, Thailand; CBS: Culture Collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CFCC: China Forestry Culture Collection Center, China; ATCC: American Type Culture Collection; MUCL: Agro–Food and Environmental Fungal Collection, Belgium; BCRC: Bioresource Collection and Research Center, China; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; CCF: Culture Collection of Fungi, Department of Botany, Faculty of Sciences, Charles University, Prague, Czech Republic; YMJ: Yu-Ming Ju’s private collection.
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MDPI and ACS Style

Qiao, C.; Zhao, R.; Li, D.; Ding, X. A New Species of Biscogniauxia Associated with Pine Needle Blight on Pinus thunbergii in China. Forests 2024, 15, 956. https://doi.org/10.3390/f15060956

AMA Style

Qiao C, Zhao R, Li D, Ding X. A New Species of Biscogniauxia Associated with Pine Needle Blight on Pinus thunbergii in China. Forests. 2024; 15(6):956. https://doi.org/10.3390/f15060956

Chicago/Turabian Style

Qiao, Changxia, Ruiwen Zhao, Dewei Li, and Xiaolei Ding. 2024. "A New Species of Biscogniauxia Associated with Pine Needle Blight on Pinus thunbergii in China" Forests 15, no. 6: 956. https://doi.org/10.3390/f15060956

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