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

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. Primers and parameters used for PCR amplification.

Locus	PCR Primers (Forward/Reverse)	PCR: Thermal Cycles: (Annealing Temperature in Bold)
ITS	ITS5/ITS4	95 °C: 5 min, (95 °C: 30 s, 55 °C: 30 s, 72 °C: 90 s) ×33 cycles, 72 °C: 10 min
ACT	ACT-512F/ACT-783R	95 °C: 2 min, (95 °C: 30 s, 61 °C: 30 s, 72 °C: 60 s) ×35 cycles, 72 °C: 5 min
TUB2	Bt-2a/Bt-2b	95 °C: 2 min, (95 °C: 30 s, 55 °C: 30 s, 72 °C: 60 s) ×35 cycles, 72 °C: 5 min
RPB2	fRPB2-5F/fRPB2-7cR	95 °C: 2 min, (95 °C: 30 s, 54 °C: 30 s, 72 °C: 60 s) ×35 cycles, 72 °C: 5 min

). 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. List of Biscogniauxia strains used in phylogenetic analysis.

Species	Locality	Host	Strain Number 1	GenBank Accessions				Reference
				ITS	ACT	TUB2	RPB2
Annulohypoxylon cohaerens	France	Fagus	YMJ 310 = BCRC34013 ab	EF026140	AY951766	AY951655	GQ844766	[29]
Biscogniauxia anceps	France	Fagus	YMJ 123 = BCRC34029 ab	EF026132	AY95178	AY951671	JX507777	[29]
B. anceps	Spain	Eucalyptus sp.	CBS 149687 a	OQ990096	NA	NA	NA	[30]
B. anceps	England	unknown	KM 201862 a	MZ159582	NA	NA	NA	submitted directly
B. arima	Mexico	wood	YMJ 122 * = BCRC34030 ab	EF026150	AY951784	AY951672	GQ304736	[29]
B. atropunctata	Ecuador	Pinus	NY8660c a	HQ108028	NA	NA	NA	submitted directly
B. atropunctata	unknown	Switchgrass	IUB:B.Whitaker:OTU41 a	MH178710	NA	NA	NA	submitted directly
B. atropunctata	Canada	Acer	KC2031E5 a	KX589181	NA	NA	NA	submitted directly
B. atropunctata	unknown	unknown	KoLRI_053373 a	MZ855376	NA	NA	NA	submitted directly
B. atropunctata	unknown	milk thistle	G411 a	KM215648	NA	NA	NA	submitted directly
B. atropunctata	unknown	unknown	K135 a	MK304352	NA	NA	NA	submitted directly
B. atropunctata	unknown	unknown	C226 a	MK304016	NA	NA	NA	submitted directly
B. atropunctata	unknown	unknown	W498 a	MK247673	NA	NA	NA	submitted directly
B.atropunctata	Iran	Celtis australis	BIAT72 a	MF497753	NA	NA	NA	submitted directly
B. atropunctata	Mexico	Taxus globosa	SGSGf17 a	EU715651	NA	NA	NA	[31]
B. atropunctata	USA	wood	YMJ 128 ab	JX507799	AY951785	AY951673	JX507778	[13]
B. atropunctata var. intermedia	Costa Rica	Quercus sp.	B70M = GB 4796 a	AJ390412	NA	NA	NA	[31]
B. bartholomaei	USA	branches	ATCC 38992 a	AF201719	NA	NA	NA	[32]
B. bartholomaei	USA	branches	108898191 a	ON692782	NA	NA	NA	[32]
B. capnodes	China	wood	YMJ 138 = BCRC34032 b	EF026131	AY951787	AY951675	JX507779	[29]
B. capnodes	unknown	unknown	S74 a	OR237607	NA	NA	NA	submitted directly
B. capnodes	unknown	unknown	FS39 a	MF770835	NA	NA	NA	submitted directly
B. capnodes	Gabon	unknown	GAB038 a	KY250379	NA	NA	NA	submitted directly
B. capnodes var. capnodes	New Zealand	Lophozonia menziesii	11682 a	MH410019	NA	NA	NA	submitted directly
B. cinereolilacina	Norway	unknown	CBS 10496 a	MH862567	NA	NA	NA	[33]
B. citriformis	USA	Casuarina equisetifolia	YMJ 129 = BCRC34034 b	JX507801	AY951789	AY951678	JX507781	[13]
B. citriformis	China	wood	YMJ 88113012 ab	JX507800	AY951790	AY951677	JX507780	[13]
B. cylindrispora	China	Cinnamomum	YMJ 89092701 = BCRC33717 ab	EF026133	AY951791	AY951679	JX507782	[34]
B. dendrobii	China	Dendrobium aphyllum	MFLUCC 17 2607 * a	NR172733	NA	NA	NA	[35]
B. formosana	China	Bark	YMJ 89032201 * = BCRC33718 ab	JX507802	AY951792	AY951680	JX507783	[34]
B. formosana	unknown	unknown	Gjav1ITS2OTU03662 a	KY588559	NA	NA	NA	submitted directly
B. granmo	Austria	Prunus padus	YMJ 135 = BCRC34035 ab	JX507803	AY951793	AY951681	JX507784	[13]
B. glaucae	China	Quercus glauca	GMBC 0007 ab	MT624046	MT622656	MT622654	MT622652	[36]
B. latirima	China	Bark	YMJ 89101101 = BCRC33729 ab	JX507804	AY951794	AY951682	JX507785	[13]
B. latirima	China	Bark	YMJ 90080703 = BCRC34036 ab	EF026135	AY951795	AY951683	JX507786	[29]
B. magna	Thailand	Unidentified	MFLU 18-0850 a	MW240616	NA	NA	NA	[37]
B. mangiferae	Thailand	Mangifera indica	MFLU 18-0827 *a	MN337232	NA	NA	NA	[38]
B. marginata	Switzerland	Sorbus aucuparia	ZT-Myc-64233 a	MW489534	NA	NA	NA	submitted directly
B. maritima	Russia	unknown	LE 262845 a	JQ247198	NA	NA	NA	submitted directly
B. maritima	Republic of Korea	unknown	KoLRI_EL004708 a	MN844502	NA	NA	NA	submitted directly
B. maritima	Republic of Korea	Parmotrema	KoRLI046050 a	MN341558	NA	NA	NA	submitted directly
B. maritima	Republic of Korea	unknown	KACC 410582 a	OR886237	NA	NA	NA	submitted directly
B. marginata	Germany	Malus sp.	CBS 124505 ab	KU684016	KU684036	KU684124	KU684310	[36]
B. mediterranea	Italy	Q. pubescens	B × 70 a	KT253502	NA	NA	NA	[39]
B. mediterranea	Italy	Q. pubescens	B × 85 a	KT253503	NA	NA	NA	[39]
B. mediterranea	France	Fagus sp.	YMJ 147 = BCRC34037 b	EF026134	AY951796	AY951684	GQ844765	[39]
B. mediterranea	Japan	Malus sp.	F8012 a	AB693903	NA	NA	NA	submitted directly
B. mediterranea	USA	Acer	VH04 a	OR778793	NA	NA	NA	submitted directly
B. mediterranea	USA	wood	Kern805 a	OP038087	NA	NA	NA	[40]
B. nothofagi	New Zealand	Lophozonia menziesii	NZFS 3015 a	MN007010	NA	NA	NA	Hood, I.A submitted directly.
B. mediterranea var. microspora	Thailand	unknown	BCC 1198 a	AB376712	NA	NA	NA	[41]
B. nummularia	England	Salix alba	H86 = CCF 3919 b	GQ428318	GQ428312	GQ428324	FR715504	[39]
B. nummularia	France	Fagus sp.	MUCL 51395 * a	JX658444	NA	NA	NA	[39]
B. nummularia	China	unknown	F8422 a	ON332160	NA	NA	NA	submitted directly
B. nummularia	Poland	Viscum album	122H a	OP699770	NA	NA	NA	[42]
B. nummularia	Poland	Pinus	Bn5L-19Pu a	MN588203	NA	NA	NA	submitted directly
B. nummularia	Iran	Zelkova	BINU72 a	MF358880	NA	NA	NA	submitted directly
B. cf. nummularia	unknown	Lewinskya	2010_26_NT2 a	MW907969	NA	NA	NA	submitted directly
B. nummularia var. merrillii	Thailand	bark	BCC 1213 a	AB376714	NA	NA	NA	[41]
B. petrensis	Thailand	Dendrobium harveyanum	MFLUCC 14-0151 * a	MK951680	NA	NA	NA	[38]
B. cf. petrensis	Ecuador	wood	RLC_1445.1_iNat_7002890 a	OQ878456	NA	NA	NA	[43]
B. petrensis	Japan	Tea	ST253 a	LC685807	NA	NA	NA	submitted directly
B. petrensis	China	Osmanthus sp.	HKAS 102388 a	MW240615	NA	NA	NA	[37]
B. petrensis	unknown	unknown	X2P3HRa a	ON921656	NA	NA	NA	submitted directly
B. philippinensis var. microspora	China	Bark	YMJ 89041101 * = BCRC33720 ab	EF026136	AY951797	AY951685	JX507787	[39]
B. repanda	USA	Fagus sp.	ATCC 62606 a	KY610383	NA	NA	NA	[44]
B. repanda	unknown	unknown	B75A a	AJ390418	NA	NA	NA	[45]
B. rosacearum	Italy	P. domestica	Bx26 = CBS141046 * a	KT253493	NA	NA	NA	[39]
B. rosacearum	Italy	P. domestica	Bx4	KT253491	NA	NA	NA	[39]
B. rosacearum	Khorramabad	Quercus. brantii	IRAN 4288C a	MZ359664	NA	NA	NA	[45]
B. rosacearum	Iran	almond tree	IRNBS68 a	MW452324	NA	NA	NA	[45]
B. rosacearum	unknown	unknown	CSN1052 a	MT813910	NA	NA	NA	[46]
Biscogniauxia sinensis	China	Pinus thunbergii	SC1 = CFCC59648 ab	OR803753	OR832182	OR832177	OR832187	This study
Biscogniauxia sinensis	China	P. thunbergii	SC2 = CFCC59649 ab	OR803754	OR832183	OR832178	OR832188	This study
Biscogniauxia sinensis	China	P. thunbergii	SC3 = CFCC59650 * ab	OR803755	OR832184	OR832179	OR832189	This study
Biscogniauxia sinensis	China	P. thunbergii	SC4 = CFCC59651 ab	OR803756	OR832185	OR832180	OR832190	This study
Biscogniauxia sinensis	China	P. thunbergii	SC5 = CFCC59652 ab	OR803757	OR832186	OR832181	OR832191	This study
B. simplicior	France	Rhamnus cathartica	YMJ 136 = BCRC34038 ab	EF026130	AY951798	AY951686	JX507788	[39]
B. simplicior	unknown	unknown	B73C a	AJ3904916	NA	NA	NA	[31]
B. uniapiculata	China	Bark	YMJ 90080608 = BCRC34039 ab	JX507805	AY951799	AY951687	JX507789	[39]
B. aff. uniapiculata	Gabon	unknown	GAB220 a	KY250378	NA	NA	NA	submitted directly
B. whalleyi	Thailand	wood	SWUF 13-85 ab	MW403821	MZ466385	MZ466386	MZ466387	Jantaharn, P. submitted directly.
Hypoxylon rubiginosum	UK	Fraxinus	YMJ 24 = BCRC34116 b	EF026143	AY951862	AY951751	JX507791	[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.

. 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.