Taxonomical Study of Noteworthy Species of Botryosphaeria in Japan

Figures & data

Table 1. List of Japanese Botryosphaeria isolates used in this study.

Fungal species	Isolate No.	Material No.	Host Family	Host species	Regions	Identified by previous study
B. dothidea	MUCC 157	MUMH 10467	Daphniphyllaceae	Daphniphyllum macropodum	Aichi	—
	MUCC 221	MUMH 10395	Ericaceae	Leucothoe fontanesiana	Aichi	—
	MUCC 245	MUMH 10425	Daphniphyllaceae	Daphniphyllum macropodum	Aichi	—
	MUCC 248	MUMH 10429	Lauraceae	Lindera obtusiloba	Aichi	—
	MUCC 2521 (MAFF 410826)	—	Rosaceae	Prunus sp.	Ibaraki	B. dothidea
	MUCC 254	MUMH 10437	Saxifragaceae	Saxifraga stolonifera	Aichi	—
	MUCC 2543 (FFPRI 411204)	—	Myrtaceae	Eucalyptus viminalis	Tokyo	Dothiorella sp.
	MUCC 2627	—	Rosaceae	Pyrus pyrifolia	Mie	—
	MUCC 2748	—	Fagaceae	Castanea crenata	Ibaraki	—
	MUCC 2749	—	Fagaceae	Castanea crenata	Kumamoto	—
	MUCC 2750	—	Fagaceae	Castanea crenata	Kumamoto	—
	MUCC 2751	—	Rosaceae	Prunus persica	Ibaraki	—
	MUCC 2755	—	Fagaceae	Castanea crenata	Kumamoto	—
B. qingyuanensis	MUCC 321	MUMH 10273	Araliaceae	Gamblea innovans	Aichi	—
B. sinensis	MUCC 2522 (MAFF 410827)	—	Rosaceae	Prunus sp.	Ibaraki	B. dothidea
	MUCC 2533 (FFPRI 411202)	—	Aucubaceae	Aucuba japonica	Tokyo	Dothiorella sp.
	MUCC 2537 (FFPRI 411203)	—	Paulowniaceae	Paulownia tomentosa	Niigata	Dothiorella sp.
B. tenuispora	MUCC 237	MUMH 10420	Ericaceae	Leucothoe fontanesiana	Aichi	—
	MUCC 2900	—	Aucubaceae	gall on Aucuba japonica	Ibaraki	—
Botryosphaeria sp.	MUCC 2754	—	Fagaceae	Castanea crenata	Kumamoto	—
	MUCC 2897	—	Aucubaceae	pupa of Asphondylia aucubae	Ibaraki	—
	MUCC 2898	—	Aucubaceae	gall on Aucuba japonica	Ibaraki	—
	MUCC 2899	—	Aucubaceae	gall on Aucuba japonica	Ibaraki	—
	MUCC 2901	—	Aucubaceae	gall on Aucuba japonica	Ibaraki	—

Table 2. PCR primer sets and annealing temperatures.

Region	Primer F	Primer R	Annealing temperature (°C)
ITS	ITS1 (White et al. [16])	ITS4 (White et al. [16])	48
TEF1-α	EF1-728F (Carbone and Kohn [17])	EF1-986R (Carbone and Kohn [17])	52
TUB2	BT2A (Glass and Donaldson [18])	BT2B (Glass and Donaldson [18])	55
RPB2	RPB2-5f2 (Liu et al. [19])	fRPB2-7cR (Liu et al. [19])	60→58→54

Table 3. List of Botryosphaeria species used for phylogenetic analysis.

Fungal species	Isolates No.	Host	Country	Accession numbers
				ITS	tef1	tub2	rpb2
B. agaves	CBS 133992	Agave sp.	Thailand	JX646791	JX646856	JX646841	—
B. auasmontanum	CBS 121769	Acacia mellifera	Namibia	EU101303	EU101348	—	—
B. corticis	CBS 119047	Vaccinium corymbosum	USA	DQ299245	EU017539	EU673107	—
B. dothidea	CBS 115476	Prunus sp.	Switzerland	AY236949	AY236898	AY236927	EU339577
B. dothidea	MUCC 157	Daphniphyllum macropodum	Japan	LC585280	LC585152	LC585176	LC585198
B. dothidea	MUCC 221	Leucothoe fontanesiana	Japan	LC585282	LC585154	LC585178	LC585200
B. dothidea	MUCC 245	Daphniphyllum macropodum	Japan	LC585273	LC585145	LC585169	LC585192
B. dothidea	MUCC 248	Lindera obtusiloba	Japan	LC585275	LC585147	LC585171	LC585194
B. dothidea	MUCC 2521 (MAFF 410826)	Prunus sp.	Japan	LC585270	LC585142	LC585166	LC585189
B. dothidea	MUCC 254	Saxifraga stolonifera	Japan	LC585274	LC585146	LC585170	LC585193
B. dothidea	MUCC 2543 (FFPRI 411204)	Eucalyptus viminalis	Japan	LC585271	LC585143	LC585167	LC585190
B. dothidea	MUCC 2627	Pyrus pyrifolia	Japan	LC585284	LC585156	LC585180	LC585202
B. dothidea	MUCC 2748	Castanea crenata	Japan	LC585283	LC585155	LC585179	LC585201
B. dothidea	MUCC 2749	Castanea crenata	Japan	LC585289	LC585161	LC585185	LC585207
B. dothidea	MUCC 2750	Castanea crenata	Japan	LC585269	LC585141	LC585165	—
B. dothidea	MUCC 2751	Prunus persica	Japan	LC585281	LC585153	LC585177	LC585199
B. dothidea	MUCC 2755	Castanea crenata	Japan	LC585272	LC585144	LC585168	LC585191
B. fabicerciana	CBS 127193	Eucalyptus sp.	China	HQ332197	HQ332213	KF779068	MF410137
B. fusispora	MFLUCC 10-0098	Entada sp.	Thailand	JX646789	JX646854	JX646839	—
B. guttulata	CGMCC 3.20094	Dead wood	China	MT327839	MT331606	—	—
B. kuwatsukai	CBS 135219	Malus domestica	China	KJ433388	KJ433410	—	—
B. minutispermatia	GZCC 16-0013	Dead wood	China	KX447675	KX447678	—	—
B. pseudoramosa	CGMCC 3.18739	Eucalyptus hybrid	China	KX277989	KX278094	KX278198	MF410140
B. qingyuanensis	CGMCC 3.18742	Eucalyptus hybrid	China	KX278000	KX278105	KX278209	MF410151
B. qingyuanensis	MUCC 321	Gamblea innovans	Japan	LC585291	LC585163	LC585187	—
B. ramosa	CBS 122069	Eucalyptus camaldulensis	Australia	EU144055	EU144070	KF766132	—
B. rosaceae	CGMCC 3.18007	Malus sp.	China	KX197074	KX197094	KX197101	—
B. scharifii	CBS 124703	Mangifera indica	Iran	JQ772020	JQ772057	—	—
B. sinensis	CGMCC 3.17722	Populus sp.	China	KT343255	—	—	—
B. sinensis	MUCC 2522 (MAFF 410827)	Prunus sp.	Japan	LC585277	LC585149	LC585173	LC585195
B. sinensis	MUCC 2533 (FFPRI 411202)	Aucuba japonica	Japan	LC585268	LC585140	LC585164	LC585188
B. sinensis	MUCC 2537 (FFPRI 411203)	Paulownia tomentosa	Japan	LC585279	LC585151	LC585175	LC585197
B. tenuispora	MUCC 237	Leucothoe fontanesiana	Japan	LC585278	LC585150	LC585174	LC585196
B. tenuispora	MUCC 2900	gall on Aucuba japonica	Japan	LC585276	LC585148	LC585172	–
B. wangensis	CGMCC 3.18744	Cedrus deodara	China	KX278002	KX278107	KX278211	MF410153
Botryosphaeria sp.	MUCC 2754	Castanea crenata	Japan	LC585288	LC585160	LC585184	LC585206
Botryosphaeria sp.	MUCC 2897	pupa of Asphondylia aucubae	Japan	LC585285	LC585157	LC585181	LC585203
Botryosphaeria sp.	MUCC 2898	gall on Aucuba japonica	Japan	LC585286	LC585158	LC585182	LC585204
Botryosphaeria sp.	MUCC 2899	gall on Aucuba japonica	Japan	LC585290	LC585162	LC585186	—
Botryosphaeria sp.	MUCC 2901	gall on Aucuba japonica	Japan	LC585287	LC585159	LC585183	LC585205
C. atrovirens	CBS 124934	Pterocarpus angolensis	South Africa	FJ888473	FJ888456	—	—

Ex-type strains are in boldface.

Figure 1. Phylogenetic tree of Botryosphaeria spp. constructed by ML using the combined ITS, RPB2, TEF1-α, and TUB2 gene sequence datasets. ML bootstrap values and Bayesian PPs are given near the branches (BS/PP). Ex-type strains are in boldface.

Table 4. Morphological characteristics of the genus Botryosphaeria.

Species	Conidia			Isolate	Literature
	Conidial bodies (μm)	Average	L/W
B. auasmontanum	(8.1–)8.8–11.3(–13) × (2.5–)2.9–3.9(–5)	10.1 × 3.4	3.0	—	Slippers et al. [25]
B. dothidea	(20–)23–27(–30) × 4–5(–6)	26.2 × 5.4	4.9	—	Slippers et al. [25]
B. dothidea	22–30 × 3.9–6.3	25.8 × 5.2	4.9	MUCC 157	This study
B. dothidea	21–29 × 3.8–6	25.0 × 5.1	4.8	MUCC 221	This study
B. dothidea	20–30 × 5.3–7	25.1 × 6.1	4.1	MUCC 245	This study
B. dothidea	18.5–30 × 3.8–8.8	25.2 × 5.2	4.8	MUCC 248	This study
B. dothidea	16–24 × 3.3–7.3	19.7 × 5.7	3.4	MUCC 2521 (MAFF 410826)	This study
B. dothidea	17–30 × 4.2–6.2	25.6 × 5.1	4.9	MUCC 254	This study
B. dothidea	19–26 × 4.8–6	22.9 × 5.4	4.2	MUCC 2543 (FFPRI 411204)	This study
B. dothidea	22.5–29.4 × 4.2–6.6	26.1 × 5.4	4.8	MUCC 2627	This study
B. dothidea	15–26 × 4–7.4	22.0 × 5.5	4.0	MUCC 2748	This study
B. dothidea	25–33 × 3.8–6.3	28.7 × 5.1	5.6	MUCC 2749	This study
B. dothidea	17–36 × 3.8–6.9	26.7 × 5.2	5.1	MUCC 2750	This study
B. dothidea	22.5–31 × 4.4–6.9	26.1 × 5.4	4.8	MUCC 2751	This study
B. dothidea	21–35 × 4–6.7	26.8 × 5.1	5.2	MUCC 2755	This study
B. guttulata	(17.1–)18.5–19.3(– 20.3) × (4.1–)4.4–4.9(– 5.2)	18.9 × 4.7	4.0	—	Chen et al. [13]
B. minutispermatia	8–14 × 3–4	13.0 × 3.5	3.7	—	Ariyawansa et al. [26]
B. qingyuanensis	(15–)19.5–24.5(–28.5) × (5–)6–6.5(–7.5)	22.0 × 6.2	3.5	—	Li et al. [27]
B. qingyuanensis	17–24 × 3.6–6.5	21.4 × 5.2	4.1	MUCC 321	This study
B. sinensis	(15–)19–29 × 5–7	24.3 × 5.9	4.1	—	Zhou et al. [28]
B. sinensis	16–23 × 4.8–6	20.4 × 5.4	3.7	MUCC 2522 (MAFF 410827)	This study
B. sinensis	14–27 × 3–5.8	24.0 × 4.6	5.2	MUCC 2537 (FFPRI 411203)	This study
B. tenuispora	23–32 × 4–6.7	27.3 × 5.1	5.4	MUCC 237	This study
B. wangensis	(20.5–)22–26(–29) × (4.5–)5.5–6.5(–7.5)	23.8 × 6.0	3.9	—	Li et al. [27]
Botryosphaeria sp.	14.6–29 × 3.2–6.2	22.8 × 5.2	4.4	MUCC 2899	This study

Ex-type strains are in boldface.

Figure 2. Morphological features of Botryosphaeria tenuispora [A–F: MUMH 10420 (MUCC 237) and G–I: MUCC 2900]. (A) Specimen MUMH 10420. (B) Symptoms with pycnidia forming on the leaf of Leucothoe fontanesiana. (C) Vertical section of pycnidium in the leaf tissue. (D) Conidia and conidiophores. (E, F) Conidia. (G) Conidiomata formation on BMA after 7 days. (H) Conidiomata. (I) Conidium and conidiophores. (J) Conidium. Scale bars, 200 μm (C and H) and 25 μm (D–F and I–J).

Figure 3. Morphological features of Botryoshaeria sp. (A and B: MUCC 2899). (A) fruit galls by Asphondylia aucabae on Aucuba japonica. (B) Conidiomata formation on the BMA after 7 d. (C) Vertical section of pycnidium in the leaf tissue. (D–E) Conidia and conidiophores. (F–G) Conidia. Scale bars, 200 μm (C) and 25 μm (D–G).

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