Species Diversity of Phaeobotryon Associated with Tree Canker and Dieback Diseases in Xinjiang, China

Abstract

Withered branch disease is a major threat to the health of forest trees, resulting in the death of susceptible branches and even the whole plant. Botryosphaeriaceae members (e.g., Phaeobotryon spp.) are common pathogenic fungi that cause withered branches, canker and dieback disease symptoms in woody plants. This study aimed to identify the Phaeobotryon species inhabiting tree species with canker and dieback symptoms in Xinjiang Uyghur Autonomous Region, China, based on both morphological and phylogenetic approaches. In the current study, branches and twigs showing typical symptoms of canker and dieback were collected from Elaeagnus angustifolia, Juglans regia, Malus pumila, Malus ‘Royalty’, Prunus armeniaca (wild apricot) and Rhus typhina growing on Tianshan Mountain in the Xinjiang Uygur Autonomous Region of China. Phaeobotryon species isolated from these samples were characterized based on methods of morphology and molecular phylogeny. As a result, two species were identified: P. rhoinum and a new species Phaeobotryon mali sp. nov. Here, we provide a description and illustrations of this new species for science.

1. Introduction

The genus Phaeobotryon Theiss. and Syd. of Botryosphaeriaceae Theiss. and Syd. was established to accommodate the species Dothidae cercidis Cooke as Phaeobotryon cercidis [1]. Phaeobotryon is a monophyletic genus in Botryosphaeriaceae phylogenetically and morphologically distinguished from the other genera in this family [2,3,4,5]. This genus is characterized by 2-septate brown ascospores with conical apiculate-elliptic to oblong or obovoid shapes at both ends and hyaline or brown conidia [3,6,7,8]. Given the many differences between Phaeobotryon and Botryosphaeria in terms of morphology and phylogeny, the Phaeobotryon genus was redefined based on the characteristics of 2-septate ascospore with bipolar conical apiculi. The type of species is P. cercidis, and an additional species was then proposed named P. mamane [4]. Subsequently, Abdollahzadeh et al. introduced an endophyte isolated from the stem of North American cypress, named P. cupressi [9,10]. The morphology of the anamorph and the size of conidia are the basis for the classification of different species of Phaeobotryon [5]. At present, 13 species of Phaeobotryon are listed in Species Fungorum (2023); however, only nine of these species (viz., P. aplosporum, P. caraganae, P. cupressi, P. mamane, P. negundinis, P. rhoinum, P. rhois, P. spiraeae and P. ulmi) have been verified and studied based on molecular phylogeny [2,3,5,7,8,11,12,13].

Species of Phaeobotryon have been widely reported to live parasitically on gymnosperms and angiosperms, sometimes as endophytes or potential pathogens. When the host is under environmental stress or weakening, Phaeobotryon species can cause dry rot, sticky shoot blight and other cankers [14]. The main symptoms of disease on the host are yellowish brown stripes in the cankered tissues of the branches. As the disease develops, the branches turn yellowish brown, and then the whole plant dies [14]. Phaeobotryon species can inhabit a wide range of host species, including Acer negundo, Calocedrus decurrens, Caragana arborescens, Cupressus sempervirens, Dioscorea nipponica, Forsythia × intermedia, Juniperus scopulorum, Ligustrum vulgare, Platycladus orientalis, Rhamnus dahuricus, Rhus typhina, Rubus crataegifolius, Sophora chrysophylla, Syzygium aromaticum and Ulmus pumila [3,4,5,8,9,10,11,12,13,14,15,16].

Four species of Phaeobotryon have been reported in China: P. rhois, P. rhoinum, P. aplosporum and P. caraganae [6,7,8,14]. P. rhois was first reported as a pathogen causing Rhus typhina canker in Ningxia Province [6] and has also been found in Beijing and Heilongjiang Provinces. P. rhoinum and P. aplosporum were reported from the branches of Rhus typhina in Dongling and Yudu Mountains in Beijing. P. caraganae has been observed on Caragana arborescens plants [14]. However, the species, hosts and distribution of Phaeobotryon in Xinjiang are not clear before the present study.

This study aimed to identify the Phaeobotryon isolates obtained from diseased samples of host plants growing in Xinjiang Uygur Autonomous Region based on phylogenetic analysis of partial sequence combinations of the ITS region, LSU and tef1 loci and morphology and culture characteristics.

2. Materials and Methods

2.1. Sampling and Isolation

Disease investigations were conducted in the north slope of Tianshan Mountain in Xinjiang, China. Canker and dieback diseases of Elaeagnus angustifolia, Juglans regia, Malus pumila, Malus ‘Royalty’, Prunus armeniaca and Rhus typhina were observed and collected. Then, sample surfaces were washed with sterile water and dried with sterile cotton cloth, and small pieces of conidial mass were removed from conidiomata and transferred onto the surface of Petri dishes containing standard potato dextrose agar (PDA) medium. The PDA plates were then incubated at 25 °C in the dark for up to 24 h. After incubation, single germinating conidia were transferred to the new PDA plates, which were kept in a refrigerator at 4 °C. Representative isolates from the present study were deposited in China Forestry Culture Collection Center (CFCC).

2.2. Morphological Observation

Morphological Observation was conducted mainly based on the conidiomata naturally formed on the host tissues, including size, shape and color. Macromorphological photographs were obtained using a Leica stereomicroscope (M205, Leica, Wetzlar, Germany). Micromorphological observations including size and shape of conidiophores and conidia were performed by a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). Twenty conidiomata were sectioned using a sterile scalpel, and then the lengths and widths of 50 randomly chosen conidia were measured. Colony morphology and growth rates were recorded.

2.3. DNA Extraction, PCR Amplification and Sequencing

The fungal genomic DNA was obtained from 10-day-old colonies from PDA plates based on the CTAB method. ITS, LSU and tef1 sequence data were generated as follows. The ITS region was amplified using ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) primers [17]. The LSU region was amplified using LROR (5′-GTA CCC GCT GAA CTT AAG C-3′) and LR5 (5′-ATC CTG AGG GAA ACT TC-3′) primers [18]. The TEF-1α region was amplified using TEF1-688F (5′-CGG TCA CTT GAT CTA CAA GTG C-3′) and TEF1-1251R (5′-CCT CGA ACT CAC CAG TAC CG-3′) primers [19]. The PCR conditions were set as follows: an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 50 s at 52 °C (ITS and LSU) or 54 °C (tef1) and 1 min at 72 °C and a final elongation step of 7 min at 72 °C [20,21]. PCR amplifications were performed using a DNA Engine (PTC-200) Peltier Thermal Cycler (Bio-Rad, California, USA), and amplification products were visually estimated by performing electrophoresis in 1% agarose gels. DNA sequencing was performed using an ABI PRISM^®3730XL DNA analyzer (ABI, California, USA) with a BigDye^®120 Terminator Kit v. 3.1 (Invitrogen, Beijing, China). The positive transformants were sequenced at Sangon Biological Engineering Co., Ltd. (Beijing, China).

2.4. Phylogenetic Analyses

The quality of the chromatograms of the present study was checked, and the nucleotide sequences were assembled using SeqMan v.7.1.0. Reference sequences and newly generated nucleotide sequences were listed in

Table 1. Isolates and GenBank accession numbers used in the phylogenetic analyses.

Species	Isolate No.	Host	Locality	GenBank Accession Numbers
				LSU	ITS	tef1
Phaeobotryon aplosporum	CFCC 53774	Syzygium aromaticum	China	MN215871	MN215836	MN205996
P. aplosporum	CFCC 53775	Rhus typhina	China	MN215872	MN215837	NA
P. aplosporum	CFCC 53776	Rhus typhina	China	MN215873	MN215838	MN205997
P. caraganae	NEFU817	Caragana arborescens	China	NA	MH014076	MF193891
P. caraganae	NEFU816	Caragana arborescens	China	NA	MH036714	MF509765
P. cupressi	IRAN 1455C	Cupressus sempervirens	Iran	KX464539	FJ919672	FJ919661
P. cupressi	IRAN 1454C	Cupressus sempervirens	Iran	KX464538	FJ919673	FJ919662
P. cupressi	IRAN 1445C	Cupressus sempervirens	Iran	NA	KF766208	KF766428
P. mali	XJAU 2930	Malus pumila	China	MW367101	MW326854	MW509519
P. mali	XJAU 2772	Juglans regia	China	MW367094	MW326853	MW509520
P. mali	XJAU 2782	Malus ‘Royalty’	China	MW367092	MW326852	MW509516
P. mali	XJAU 3094	Elaeagnus angustifolia	China	MW367100	MW326858	MW509517
P. mali	XJAU 3100	Rhus typhina	China	MW367093	MW326878	MW509518
P. mamane	CPC 12442	Sophora chrysophylla	USA	DQ377899	EU673333	EU673299
P. mamane	CPC 12440	Sophora chrysophylla	USA	EU673248	KF766209	EU673298
P. mamane	CPC 12443	Sophora chrysophylla	USA	EU673249	EU673334	EU673300
P. negundinis	CAA 797	Acer negundo	Russia	KU820971	KX061513	KX061507
P. negundinis	CAA 798	Ligustrum vulgare	Russia	NG069332	KX061514	KX061508
P. rhoinum	CFCC 52449	Rhus typhina	China	MH133940	MH133923	MH133957
P. rhoinum	CFCC 52450	Rhus typhina	China	MH133941	MH133924	MH133958
P. rhoinum	CFCC 52451	Rhus typhina	China	MH133942	MH133925	MH133959
P. rhoinum	XJAU 1468	Prunus armeniaca	China	MW367102	MW326857	MW509522
P. rhoinum	XJAU 2764	Prunus armeniaca	China	MW367095	MW326855	MW509524
P. rhoinum	XJAU 3049	Prunus armeniaca	China	MW367096	MW326856	MW509523
P. rhoinum	XJAU 3168	Prunus armeniaca	China	MW367097	MW326877	MW509521
P. rhois	CFCC 89662	Rhus typhina	China	KM030591	KM030584	KM030598
P. rhois	CFCC 89663	Rhus typhina	China	KM030592	KM030585	KM030599
P. spiraeae	CFCC 53925	Spiraea salicifolia	China	OM049432	OM049420	NA
P. spiraeae	CFCC 53926	Spiraea salicifolia	China	OM049433	OM049421	NA
P. spiraeae	CFCC 53927	Spiraea salicifolia	China	OM049434	OM049422	NA
Oblongocollomyces variabilis	CBS 121774	Acacia karroo	Namibia	KX464536	NR136994	EU101357

Note: NA, not applicable. Strains in this study are marked in bold.

. Then sequences were aligned using MAFFT v. 6 and manually corrected by MEGA 7.0.21 [22,23].

The phylogeny of combined gene loci ITS, LSU and tef1 was analyzed based on both Maximum Likelihood (ML) and Bayesian Inference (BI) methods. The ML analyses were performed using RAxML-HPC BlackBox 8.2.10 on the website of CIPRES Science Gateway portal (https://www.phylo.org; accessed on 5 February 2023) [24]. During the analyses, a GTRGAMMA substitution model was employed with 1000 bootstrap replicates. The Bayesian posterior probabilities were analyzed by MrBayes v. 3.2.6 using Markov Chain Monte Carlo sampling [25]. The resulted phylograms were visualized by FigTree v.1.3.1 and edited in Adobe illustrator. Newly sequenced gene data were uploaded to GenBank (Table 1).

3. Results

3.1. Phylogenetic Analysis

The gene loci of ITS, LSU and tef1 were combined and analyzed to infer the phylogenetic placement of our isolates in the genus Phaeobotryon. The dataset includes 30 sequences; of these Oblongocollomyces variabilis (CBS 121774) was set as the outgroup taxon. Of the dataset, 450 characters in ITS, 576 characters in LSU and 304 characters tef1 including gaps were included in the phylogenetic analysis. In the dataset, 1165 characters were constant; 67 characters were variable and parsimony-uninformative; and 98 were parsimony-informative. The best ML tree (lnL = −3042.15) is shown as Figure 1. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Figure 1). Isolates XJAU 3168, XJAU 2764, XJAU 3049 and XJAU 1468 obtained from the present study identified as Phaeobotryon rhonium and placed together with CFCC 52449 the ex-type strain. Isolates XJAU 2782, XJAU 2930, XJAU 2772, XJAU 3094 and XJAU 3100 clustered in a new clade close to P. caraganae and P. rhois and recognized as a new species in this genus.

3.2. Taxonomy

3.2.1. Description of the New Species Phaeobotryon mali

Phaeobotryon mali H.Y. Jia & R. Ma, sp. nov.

Figure 2.

MycoBank: MB838239

Etymology: Named after the host genus Malus.

Holotype: CHINA, Xinjiang Uygur Autonomous Region, Bole County, 44°56′10.66″ N, 81°56′39.36″ E, alt. 581 m, on branches of Malus pumila, H.Y. Jia and R. Ma, 14 August 2018, (Holotype XJAU 293001, ex-type culture XJAU 2930).

Description: Conidiomata pycnidial, stromatic, scattered to gregarious, multiloculate, immersed to erumpent from bark surface, (341–)468–503.5(–639) μm in diameter (av = 469 μm, n = 50). Locules multiple, irregular arrangement with common walls, ectostromatic disc and ostiole inconspicuous, (126–)142–175(–217) μm (av = 163.5 μm, n = 30). Paraphyses hyaline, cylindrical, aseptate, unbranched, arising amongst the conidiogenous cells, up to 55 × 3.5–7 μm. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, thin walled, cylindrical to doliiform, holoblastic, phialidic, formed from the cells lining the inner walls, 8.5–42.5 × 5–12 μm. Conidia initially hyaline, becoming dark brown, 1-septate, obtusely round at both ends, obviously constricted in the middle, and slightly wider in the transverse septum, 22.0–31.5 × 12–16.5 μm (av = 26.1 × 14.3 μm, n = 50), with a L/W ratio of 1.83.

Culture characteristics: Colonies on PDA flat, spreading, with flocculent aerial mycelium, white initially, becoming olive green to black after 8 days, reaching a 90 mm diameter after 10 days and forming abundant black conidiomata after 25 days at 25 °C.

Additional materials examined: CHINA, Xinjiang Uygur Autonomous Region, Xinyuan County, 43°23′05.46″ N, 83°35′52.55″ E, alt. 1273 m, on branches of Malus ‘Royalty’, H.Y. Jia and R. Ma, 29 July 2017 (XJAU 278201 and culture XJAU 2782). Xingyuan County, 43°23′05.46″ N, 83°35′52.55″ E, alt. 1273 m, on twigs of Juglans regia, H.Y. Jia and R. Ma, August 20, 2017 (XJAU 277201, culture XJAU 2772). Xinyuan County, 43°24′41.18″ N, 83°45′05.12″ E, alt. 1120 m, on twigs of Elaeagnus angustifolia, H.Y. Jia and R. Ma, 6 July 2020 (XJAU 309401, culture XJAU 2772). Xinyuan County, 43°24′41.18″ N, 83°45′05.12″ E, alt. 1120 m, on twigs of Rhus typhina, H.Y. Jia and R. Ma, 6 July 2020 (XJAU 310001, culture XJAU 3100).

Notes: Phylogenetic analysis of the sequence data of LSU, ITS and tef1 indicated that P. mali formed a distinct clade closely related to P. caraganae and P. rhois [14]. Morphologically, P. mali differs from P. caraganae and P. rhois by conidial size (22.0–31.5 × 12–16.5 μm in P. mali vs. 31–36.1 × 18–22.6 μm in P. caraganae and 19–25 × 10–12 μm in P. rhoi) [6,14].

3.2.2. Description of Phaeobotryon rhoinum

Phaeobotryon rhoinum X.L. Fan, Phytotaxa 348: 67 (2018)

Figure 3.

Description: Conidiomata pycnidial, stromatic, scattered to gregarious, multiloculate, immersed to erumpent from bark surface, (356.5–)402.5–506(–542.5) μm in diameter (av = 443.1 μm, n = 50). Locules multiple, irregular arrangement with common walls, ectostromatic disc and ostiole inconspicuous, (95–)146–184(–235) μm (av = 162.3 μm, n = 30). Paraphyses hyaline, cylindrical, aseptate, unbranched, arising amongst the conidiogenous cells, up to 45 × 3–6 μm. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, thin walled, cylindrical to doliiform, holoblastic, phialidic, formed from the cells lining the inner walls, 7–36.5 × 5.5–13 μm. Conidia initially hyaline, becoming dark brown, aseptate, obtusely round at both ends, 19–28 × 9.5–12 μm (av = 26.1 × 14.3 μm, n = 50), with a L/W ratio of 2.7.

Culture characteristics: Colonies on PDA flat, spreading, with flocculent aerial mycelium, white initially, becoming dark to black after 10 days, reaching a 90 mm diameter after 12 days and forming abundant black conidiomata after 25 days at 25 °C.

Materials examined: CHINA, Xinjiang Uygur Autonomous Region, Tekes County, 43°8′42.85″ N, 81°46′1.84″ E, alt. 1262 m, on branches of Prunus armeniaca, H.Y. Jia and R. Ma, August 6, 2016 (XJAU 146801, culture XJAU 1468). Xingyuan County, 43°32′33.69″ N, 83°26′11.81″ E, alt. 1127 m, on branches of Prunus armeniaca, R. Ma, 5 July 2020 (XJAU 304901, culture XJAU 3049). Xinyuan County, 43°24′00.44″ N, 83°36′41.32″ E, alt. 1306 m, on branches of Prunus armeniaca, H.Y. Jia and R. Ma, 20 August 2017 (XJAU 276401, culture XJAU 2764).

Notes: Phaeobotryon rhoinum was introduced as a new species from branches of Rhus typhina in Beijing of China [7]. In the present study, three specimens from Prunus armeniaca were collected from Xinjiang of China. Morphologically, conidia from P. armeniaca are aseptate, which are different from those observed from the host Rhus typhina. Phylogenetically, isolates from Prunus armeniaca and Rhus typhina clustered in a well-supported clade in this genus (Figure 1).

4. Discussion

In the present study, we discovered a novel fungal species named Phaeobotryon mali associated with canker and dieback diseases on woody hosts Elaeagnus angustifolia, Juglans regia, Malus pumila, Malus ‘Royalty’ and Rhus typhina. In addition, Phaeobotryon rhoinum was firstly discovered on the host Prunus armeniaca and in Xinjiang, China. The findings of this study suggest that withered branch disease caused by Phaeobotryon species could become a threat to the health of forest trees in Xinjiang Uygur Autonomous Region of China.

Phaeobotryon species are widely distributed and considered to be potential or opportunistic pathogens [26,27,28,29,30]. Some species are associated with various host plants, while the others are discovered on only one host [14,31]. In this study, two species of Phaeobotryon were identified, including a new species Phaeobotryon mali and a new host for P. rhoinum. Both species were isolated from various hosts, which confirmed the non-host specificity of the identified species.

An increase in the discovery of new species within a genus is greatly affected by sampling and the number of known species. The rate of discovery of new species has not slowed down in the past decade. Our understanding of fungal species diversity may be mainly promoted by the expansion of geographical sampling. In this study, we investigated nine different sites in Xinyuan, Gongliu, Bole and Tekes counties in the north slope of Tianshan Mountain in Xinjiang and revealed species diversity of Phaeobotryon in Xinjiang.

5. Conclusions

In the present study, we introduced a new species of Phaeobotryon, named P. mali, from five tree hosts Elaeagnus angustifolia, Juglans regia, Malus pumila, Malus ‘Royalty’ and Rhus typhina from Xinjiang, China. P. rhoinum was firstly discovered on the host Prunus armeniaca.