Geosmithia Species Associated With Bark Beetles From China, With the Description of Nine New Species.

Fungi of the genus Geosmithia are frequently associated with bark beetles that feed on phloem on various woody hosts. Most studies on Geosmithia were carried out in North and South America and Europe, with only two species being reported from Taiwan, China. This study aimed to investigate the diversity of Geosmithia species in China. Field surveys in Fujian, Guangdong, Guangxi, Hunan, Jiangsu, Jiangxi, Shandong, Shanghai, and Yunnan yielded a total of 178 Geosmithia isolates from 12 beetle species. The isolates were grouped based on morphology. The internal transcribed spacer, β-tubulin, and elongation factor 1-α gene regions of the representatives of each group were sequenced. Phylogenetic trees were constructed based on those sequences. In total, 12 species were identified, with three previously described species (Geosmithia xerotolerans, G. putterillii, and G. pallida) and nine new species which are described in this paper as G. luteobrunnea, G. radiata, G. brevistipitata, G. bombycina, G. granulata (Geosmithia sp. 20), G. subfulva, G. pulverea (G. sp. 3 and Geosmithia sp. 23), G. fusca, and G. pumila sp. nov. The dominant species obtained in this study were G. luteobrunnea and G. pulverea. This study systematically studied the Geosmithia species in China and made an important contribution to filling in the gaps in our understanding of global Geosmithia species diversity.

Introduction

Members of Geosmithia are widely distributed fungal associates of phloem- and xylem-feeding beetles (Pitt, 1979; Kolařík et al., 2007, 2017; Lin et al., 2016), such as species in Bostrichidae and Curculionidae-Scolytinae (Coleoptera) (Juzwik et al., 2015; Kolařík et al., 2017). Geosmithia species are predominantly isolated from phloem-feeding bark beetles on broadleaved and conifer trees although they have been documented from many other substrates including soil (Kolařík et al., 2004), seed-feeding beetles (Huang et al., 2017), animal skin (Crous et al., 2018), indoor environment (Crous et al., 2018), insect-free plant tissues (McPherson et al., 2013), and food materials (Pitt and Hocking, 2009). To date, almost 60 phylogenetic and 21 formally described Geosmithia species have been recognized (Strzałka et al., 2021).

Geosmithia is similar to Penicillium and Paecilomyces in morphology, but it can be distinguished by the combination of stipe with or without a curved basal cell, verrucose conidiophores (including phialide), cylindrical phialide shape with a very short and cylindrical neck (collula), and ellipsoidal or cylindrical conidia (except globose conidia in Geosmithia eupagioceri and G. microcorthyli). The colony color could be in shades of white, yellow, brown, or red but never bluish-green or green (Kolařík et al., 2004; Kolařík and Kirkendall, 2010).

The spores of Geosmithia may be transmitted by attaching to the surface of the beetle vector, but the ecological role of most Geosmithia species in symbiosis with bark beetles is still unclear. Some species serve as the main food source or supplementary nutrition for the beetles (Kolařík and Kirkendall, 2010; Machingambi et al., 2014), but most are probably commensals with minimal or no benefit to the beetle (Veselská et al., 2019) because the vector beetles show neither any apparent morphological adaptation nor nutrient dependence (Huang et al., 2017, 2019). Some Geosmithia species exhibit extracellular antimicrobial and antifungal metabolites, but their ecological implications are unknown (Stodůlková et al., 2009; Veselská et al., 2019).

Some Geosmithia species can cause serious tree diseases. One example is the thousand cankers disease (TCD) of walnuts caused by G. morbida (Kolařík et al., 2011). Following high-density colonization by its beetle vector, the walnut twig beetle (Pityophthorus juglandis), in the phloem of walnut (Juglans spp.) or wingnut (Pterocarya spp.) trees, G. morbida causes numerous small lesions which eventually girdle the vascular tissue (Tisserat et al., 2009; Kolařík et al., 2011; Utley et al., 2012; Seybold et al., 2013; Hishinuma et al., 2015). TCD has affected many walnut trees in North America, especially in the western United States (Tisserat et al., 2009, 2011), and has recently been detected in Europe (Montecchio et al., 2014). Another mildly pathogenic species Geosmithia sp. 41 causes mild pathogenicity in Quercus argifolia (Kolařík et al., 2017), originally reported as G. pallida (Lynch et al., 2014).

After the discovery of the Geosmithia–beetle association (Kirschner, 2001), there has been an accumulation of reports describing Geosmithia fungi from phloem-feeding bark beetles around the world (Kolařík et al., 2004, 2007, 2008; Kubátová et al., 2004; Kolarik et al., 2005; Kolařík and Jankowiak, 2013; McPherson et al., 2013; Jankowiak et al., 2014; Machingambi et al., 2014; Pepori et al., 2015; Huang et al., 2019; Strzałka et al., 2021). Fungal communities associated with phloem-infected bark beetles are formed by a variety of biological and abiotic factors. The tree host is one of the most important selection factors (Skelton et al., 2018). Like other beetle-vectored fungi such as the ophiostomatoid fungi (Seifert et al., 2013), Geosmithia species display variable degrees of specificity to their beetle vectors and tree hosts, ranging from generalists to single-species specialists (Kolařík et al., 2007, 2008; Kolařík and Jankowiak, 2013; Jankowiak et al., 2014; Veselská et al., 2019). Other factors affecting the fungal community structure include beetle ecology, surrounding host tree community, and climatic factors (Six and Bentz, 2007; Jankowiak et al., 2014). These factors also influence the communities of Geosmithia, most notably by the fact that different beetles infesting the same host tree have similar Geosmithia assemblages (Kolařík et al., 2008; Machingambi et al., 2014).

At present, most of the studies of Geosmithia were conducted in North and South America and Europe, but the mycoflora of Asian bark beetles remain understudied. This study investigated the Geosmithia species from China using phylogenetic analyses and morphological and physiological features, and nine Geosmithia new species are described to fill the gap in our understanding of the global Geosmithia diversity.

Materials and Methods

Sampling, Isolating, and Preserving Fungal Isolates

The beetle gallery samples were collected in Fujian, Guangdong, Guangxi, Hunan, Jiangsu, Jiangxi, Shandong, Shanghai, and Yunnan Province from plant hosts of Altingia gracilipes (Altingiaceae), Gnetum luofuense (Gnetaceae), Lauraceae sp., Liquidambar formosana (Altingiaceae), L. styraciflua (Altingiaceae), Eriobotrya japonica (Rosaceae), Acacia pennata (Mimosaceae), Rhus chinensis (Anacardiaceae), Cupressus funebris (Cupressaceae), and Ulmus spp. (Ulmaceae) and kept individually in sealable bags (Table 1). The adult beetles were individually placed in Eppendorf tubes. Both galleries and adult beetles were kept at 4°C for further isolation. The beetle vectors included three groups: (1) Curculionidae-Scolytinae: Acanthotomicus suncei, Scolytus jiulianshanensis (Curculionidae-Scolytinae), S. semenovi, Microperus sp. L589, Cryphalus eriobotryae, C. kyotoensis, and Phloeosinus sp. and P. cf. hopehi, (2) Curculionidae-Platypodinae: Crossotarsus emancipates, and (3) Bostrichidae: Dinoderus sp. L489, Sinoxylon cf. cucumellae and Xylocis tortilicornis (Table 1). The fungal isolates were obtained by scraping wood tissue from the beetle galleries and crushing the beetle coating. The isolates were inoculated on 2% malt extract agar [MEA: 20 g agar (Solarbio, China), 20 g malt extract (Hopebio, China), and 1 L deionized water] amended with 0.05% streptomycin. The cultures were purified by hyphal-tip subculturing and incubated at 25°C. All the cultures obtained in this study were deposited in the culture collection (SNM) of Shandong Normal University, Jinan, Shandong Province, China. Isolates linked to type specimens of the fungal species were deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China. The holotype specimens (dry cultures) were deposited in the Herbarium Mycologicum, Academiae Sinicae (HMAS), Beijing, China (Table 2).

TABLE 1

Distribution and number of species of Geosmithia among 178 isolated strains.

Geosmithia species	Location	Tree host	Beetle species	Beetle groups	Gallery/beetle	No.
G. bombycina (2)	Fujian	Eriobotrya japonica	Cryphalus eriobotryae	Curculionidae-Scolytinae	Gallery	2
G. brevistipitata (18)	Shandong	Cupressus funebris	Phloeosinus cf. hopehi	Curculionidae-Scolytinae	Gallery	18
G. fusca (26)	Yunnan	Acacia pennata	Sinoxylon cf. cucumella	Bostrichidae	Beetle	8
	Guangdong	Phyllanthus emblica	Xylocis tortilicornis	Bostrichidae	Gallery	10
		Hibiscus tiliaceus	Ernoporus japonicus	Curculionidae-Scolytinae	Gallery	8
G. granulata (30)	Yunnan	Acacia pennata	Sinoxylon cf. cucumella	Bostrichidae	Beetle	2
	Guangdong	Hibiscus tiliaceus	Ernoporus japonicus	Curculionidae-Scolytinae	Gallery	26
	Jiangsu	Ulmus sp.	Scolytus semenovi	Curculionidae-Scolytinae	Gallery	2
G. luteobrunnea (39)	Jiangxi	Liquidambar formosana	Acanthotomicus suncei	Curculionidae-Scolytinae	Gallery	25
					Beetle	1
		Ulmus sp.	Scolytus jiulianshanensis	Curculionidae-Scolytinae	Gallery	5
	Shanghai	Liquidambar styraciflua	Acanthotomicus suncei	Curculionidae-Scolytinae	Gallery	8
G. pallida (2)	Yunnan	Acacia pennata	Sinoxylon cf. cucumella	Bostrichidae	Gallery	2
G. pulverea (33)	Guangdong	Gnetum luofuense	Dinoderus sp.	Bostrichidae	Gallery	1
	Shanghai	Liquidambar styraciflua	Acanthotomicus suncei	Curculionidae-Scolytinae	Gallery	1
	Yunnan	Acacia pennata	Sinoxylon cf. cucumella	Bostrichidae	Beetles	8
	Guangxi	Unknown	Crossotarsus emancipatus	Curculionidae-Platypodinae	Gallery	2
	Hunan	Unknown	Microperus sp. L589	Curculionidae-Scolytinae	Gallery	1
	Fujian	Eriobotrya japonica	Cryphalus eriobotryae	Curculionidae-Scolytinae	Gallery	1
	Shandong	Rhus chinensis	Cryphalus kyotoensis	Curculionidae-Scolytinae	Gallery	1
					Beetle	2
	Jiangsu	Ulmus sp.	Scolytus semenovi	Curculionidae-Scolytinae	Gallery	4
	Jiangxi	Liquidambar formosana	Acanthotomicus suncei	Curculionidae-Scolytinae	Gallery	1
		Unknown	Phloeosinus sp.	Curculionidae-Scolytinae	Gallery	6
		Ulmus sp.	Scolytus jiulianshanensis	Curculionidae-Scolytinae	Beetle	1
		Altingia gracilipes	Acanthotomicus suncei	Curculionidae-Scolytinae	Gallery	4
G. pumila (2)	Jiangsu	Ulmus sp.	Scolytus semenovi	Curculionidae-Scolytinae	Gallery	2
G. putterillii (6)	Jiangxi	Lauraceae	Phloeosinus sp.		Gallery	6
G. radiata (14)	Jiangxi	Liquidambar formosana	Acanthotomicus suncei	Curculionidae-Scolytinae	Gallery	7
		Ulmus sp.	Scolytus jiulianshanensis	Curculionidae-Scolytinae	Gallery	1
		Altingia gracilipes	Acanthotomicus suncei	Curculionidae-Scolytinae	Gallery	6
G. subfulva (5)	Guangdong	Hibiscus tiliaceus	Ernoporus japonicus	Curculionidae-Scolytinae	Beetle	2
	Fujian	Rhus chinensis	Hypothenemus sp. L636	Curculionidae-Scolytinae	Beetle	2
	Shandong	Rhus chinensis	Cryphalus kyotoensis	Curculionidae-Scolytinae	Gallery	1
G. xerotolerans (1)	Shandong	Cupressus funebris	Phloeosinus cf. hopehi	Curculionidae-Scolytinae	Gallery	1

TABLE 2

Cultures examined in this study and their GenBank accession numbers.

				GenBank accession no.
Species	Isolation no.	Beetle vectors	Tree host	ITS	TEF1-α	TUB2	RPB2	References
G. bombycine	SNM934	Cryphalus eriobotryae	Eriobotrya japonica	MZ519396	MZ514871	MZ514862	OL825679	Present study
	SNM933T = CGMCC3.20578	C. eriobotryae	E. japonica	MZ519395	MZ514870	MZ514861	OL825678	Present study
G. brevistipitata	SNM1616T = CGMCC3.20627	Phloeosinus cf. hopehi	Cupressus funebris	OK584392	OK632357	OK632375	OL825675	Present study
	SNM1610	Phloeosinus cf. hopehi	C. funebris	OK584393	OK632356	OK632373	OL825677	Present study
	SNM1611	Phloeosinus cf. hopehi	Cupressus funebris	OK584394	OK632355	OK632374	OL825676	Present study
G. brunnea	CBS 142634	Xylosandrus compactus	Liquidambar styraciflua	KY872741	KY872746	KY872751	KY882266	Huang et al., 2017
	CBS 142635	X. compactus	L. styraciflua	KY872742	KY872747	KY872752	KY882268	Huang et al., 2017
	CBS 142633T	Hypothenemus dissimilis	Quercus sp.	KY872743	KY872748	KY872753	KY882268	Huang et al., 2017
G. cnesini	CCF 3753	Cnesinus lecontei	Croton draco	AM947670				Kolařík and Kirkendall, 2010
	MK 1820	C. lecontei	C. draco	AM947671				Kolařík and Kirkendall, 2010
G. eupagioceri	MKA1-b	Eupagiocerus dentipes	Paullinia renesii	AM947666				Kolařík and Kirkendall, 2010
	CCF 3754				LR535705		LR535704	Kolarík et al., 2019*
G. fagi	CCF 6235	Taphrorychus bicolor	Fagus sylvatica	LR812775	LR813193	LR813119		Strzałka et al., 2021
	21114TBb	T. bicolor	F. sylvatica	LR812776		LR813120		Strzałka et al., 2021
	CCF 6234T	T. bicolor	F. sylvatica	LR812785	LR813141	LR813129		Strzałka et al., 2021
G. fassatiae	AK 31/98	S. intricatus	Quercus sp.	AM421039	MH580557			Kolařík et al., 2008
	CCF 4331			HF546239		KF853894		Kolařík et al., 2012*
	CCF 4340			HF546247		KF853895		Kolařík et al., 2012*
	CCF 3334 T		Quercus pubescens		MH580530			Kolarik et al., 2005
G. flava	CCF 3333T	Xiphydria sp.	Castanea sativa	AJ578483	MH580541			Kolařík et al., 2004
	CCF4337	Cerambycidae sp.	Pseudotsuga menziesii	HF546244	MH580542	KF853897		Kolařík et al., 2004
	CCF3354						LR535685	Kolarík et al., 2019*
G. fusca	SNM1577	Phyllanthus emblica	Xylocis tortilicornis	OK584387	OK632359	OK632371	OL825662	Present study
	SNM1578T = CGMCC3.20626	Phy. Emblica	Xylocis tortilicornis	OK584388	OK632358	OK632370	OL825661	Present study
	SNM1012 = CGMCC3.20486	Sinoxylon cf. cucumella	Acacia pennata	MZ519390	MZ514866	MZ514857	OL825664	Present study
	SNM1167	Sin. cf. cucumella	Aca. pennata	MZ519392	MZ514865	MZ514856	OL825663	Present study
G. granulate	SNM1015T = CGMCC3.20450	Sin. cf. cucumella	Aca. pennata	MZ519398	MZ514873	MZ514864	OL825667	Present study
	SNM1013	Sin. cf. cucumella	Aca. pennata	MZ519397	MZ514872	MZ514863	OL825668	Present study
G. lavendulan	CCF 3051		Laboratory contamination	AF033385				Kolařík et al., 2004
	CCF 3394	Carphoborus vestitus	Pistacia terebinthus	AM421098				Kolařík et al., 2007
	Hulcr 17347				MH580484			Present study
	CCF 4336					KF853902		Hamelin et al., 2013
G. langdonii	CCF 3332T	Scolytus intricatus	Quercus robur	KF808297	HG799876	HG799887	HG799928	Kolarik et al., 2005; Kolařík et al., 2017
	CCF 4338	C. pubescens	Sequoia serpervirens	HF546245	HG799877	HG799881	HG799929	Kolařík et al., 2017
G. longistipitata	RJ278m	Pityophthorus pityographus	Picea abies	HE604124				Strzałka et al., 2021
	CCF 4210T	P. pityographus	P. abies	HE604154	LR813194	LR813140		Strzałka et al., 2021
G. luteobrunnea	SNM261T = CGMCC3.20252	S. jiulianshanensis	Ulmus sp.	MW222399	MW592410	MW592395	OL825669	Present study
	SNM226	A. suncei	L. styraciflua	MW222404	MW592426	MW592392	OL825670	Present study
	SNM287	A. suncei	L. styraciflua	MW222393	MW592406	MW592398	OL825671	Present study
	SNM256 = CGMCC3.20254	A. suncei	L. formosana	MW222401	MW592423	MW592403	OL825674	Present study
G. microcorthyli	CCF 3861 T	Microcorthylus sp.	Cassia grandis	FM986798	MH580560	FM986793	FM986794	Kolařík and Kirkendall, 2010
G. morbida	CBS 124664		Juglans nigra	FN434081		KF853912	LR535706	Kolařík et al., 2011
	CCF 3881	Pityophthorus juglandis	J. nigra	FN434082	MH580543	KF853911		Kolařík and Kirkendall, 2010
	CCF 4576	P. juglandis	J. nigra		MH580544			Kolařík et al., 2007
G. obscura	CCF 3422T	S. intricatus	Q. robur	AJ784999	MH580539			Kolarik et al., 2005
	CCF 3425	S. carpini	Carpinus betulus	AM181460	MH580540	KF853914		Kolarik et al., 2005
G. omnicola	MK 1707	Hylesinus orni	Fraxinus sp.	AM181452	MH580558			Kolařík et al., 2008
	CNR115		Ulmus minor			KP990607		Pepori et al., 2015
	CNR5		Ulmus glabra			KP990546		Pepori et al., 2015
	IMI 194089	H. orni	Fraxinus sp.	AM181450				Kolařík et al., 2008
	CCF 3553	H. orni	Fraxinus sp.	AM181433				Kolařík et al., 2008
G. pallida	CCF 3053T		Cotton yarn	AJ578486		HG799817	HG799908	Kolařík et al., 2004, 2017
	CCF 3324		Soil		HG799846	HG799809	HG799900	Kolařík et al., 2004, 2017
	SNM1165	Sin. cf. cucumella	Aca. pennata	MZ519393	MZ514868	MZ514859	OL825666	Present study
	SNM1166	Sin. cf. cucumella	Aca. pennata	MZ519394	MZ514869	MZ514860	OL825665	Present study
G. pazoutovae	22015aSI	S. intricatus	Q. robur	LR812786		LR813130		Strzałka et al., 2021
	24Wa14SI	S. intricatus	Q. robur	LR812787		LR813131		Strzałka et al., 2021
	CCF 6233T	S. intricatus	Q. robur	LR812796	LR813142	LR813138		Strzałka et al., 2021
G. proliferans	CBS 142636T	Phloeotribus frontalis	Acer negundo	KY872744	KY872749	KY872754	KY882269	Huang et al., 2017
	CBS 142637	P. frontalis	A. negundo	KY872745	KY872750	KY872755	KY882270	Huang et al., 2017
G. pulverea	SNM885T = CGMCC3.20255	Dinoderus sp.	Gnetum luofuense	MW222410	MW592415	MW592388	OL825656	Present study
	SNM270	A. suncei	L. formosana	MW222398	MW592421	MW592387	OL825659	Present study
	SNM248	A. suncei	L. styraciflua	MW222402	MW592424	MW592386	OL825657	Present study
	SNM886	Crossotarsus emancipatus		MW222411	MW592416	MW592385	OL825658	Present study
	SNM887	C. emancipatus		MW222412	MW592417	MW592384	OL825660	Present study
	SNM888	Microperus sp.	Choerospondias axillaris	MW222409	MW592414	MW592389	OL825655	Present study
G. pumila	SNM1653T = CGMCC3.20630		Ulmus pumila	OK584389	OK632361	OK632366	OL825653	Present study
	SNM1657		Ulmus pumila	OK584390	OK632360	OK632367	OL825654	Present study
G. putterillii	CCF 3052T		Beilschmiedia tawa	AF033384	HG799853	HG799816	HG799907	Kolařík et al., 2004, 2017
	U 307		B. tawa	HF546306	MH580529			Kolařík et al., 2004, 2017
	SNM402	Phloeosinus sp.		MW584874	MW592419	MW592405	-	Present study
	SNM436	Phloeosinus sp.		MW584873	MW592418	MW592404	-	Present study
G. radiata	SNM279T = CGMCC3.20253	A. suncei	L. formosana	MW222397	MW592420	MW592402	OL825672	Present study
	SNM884	A. suncei	L. formosana	MW222406	MW592411	MW592400	OL825673	Present study
G. rufescens	MK 1800	C. lecontei	C. draco	AM947667				Kolařík and Kirkendall, 2010
	MK 1803	C. lecontei	C. draco	AM947668			LR535708	Kolařík and Kirkendall, 2010
	MK 1821	C. lecontei	C. draco	AM947669		KF853927		Kolařík and Kirkendall, 2010
	CCF 3752				LR535709			Kolarík et al., 2019*
G. subfulva	SNM1304T = CGMCC3.20579	Hibiscus tiliaceus	Ernoporus japonicus	OK584385	OK632363	OK632368	OL825651	Present study
	SNM1298	H. tiliaceus	Ernoporus japonicus	OK584386	OK632362	OK632369	OL825652	Present study
G. ulmacea	CCF 3559	S. multistriatus	Ulmus sp.	AM181439	MH580535			Kolařík et al., 2008
	1226	S. schevyrewi	Ulmus sp.	KJ716463				Zerillo et al., 2014*
	CNR23		U. minor			KP990560		Pepori et al., 2015
	CNR24		U. minor			KP990561		Pepori et al., 2015
G. xerotolerans	CCF 5270	Scolytus oregoni	P. menziesii		MH580534			Kolařík et al., 2017
	FMR 17085T			NR_169923		LS998791
	CCF 4280	H. ficus	F. carica	AM421049	MH580533			Kolařík et al., 2007
	SNM1618	Phloeosinus cf. hopehi	Cupressus funebris	OK584391	OK632354	OK632372	-	Present study
	CCF4334					KF853939		Hamelin et al., 2013
Geosmithia sp. 2	U107	Scolytus rugulosus	Prunus sp.	HF546256	HG799855	HG799818	HG799910	Kolařík et al., 2017
	MK 642	H. orni	Fraxinus ornus		HG799852		HG799906	Kolařík et al., 2017
Geosmithia sp. 3	CCF 4298	S. intricatus	Quercus dalechampii	AM181436	HG799851	HG799814	HG799905	Kolařík et al., 2008, 2017
	CCF 3481	Scolytus carpini	C. betulus	AM181467	HG799842	HG799805	HG799896	Kolařík et al., 2017
Geosmithia sp. 4	CCF 4278	Pteleobius vittatus	Ulmus laevis	AM181466	HG799850	HG799813	HG799904	Kolařík et al., 2008, 2017
Geosmithia sp. 5	CCF 3341	S. intricatus	Quercus petraea	AJ578487	HG799837	HG799801	HG799891	Kolařík et al., 2004, 2017
	CCF 4215	P. pityographus	P. abies	HE604117			HG799909	Kolařík and Jankowiak, 2013
	AK192/98	S. intricatus	Q. robur		HG799835		HG799889	Kolařík et al., 2017
Geosmithia sp. 8	CCF 3358	S. intricatus	Q. petraea	AM181421	MH580559	FM986788		Kolařík and Kirkendall, 2010
Geosmithia sp. 9	CCF 3564			AM181428				Kolařík et al., 2008
	CCF 3702			AM746018				Kolařík and Jankowiak, 2010*
	RJ0266	Ips cembrae	Larix decidua		MH580551			Kolařík and Jankowiak, 2013
Geosmithia sp. 11	CCF 3555	S. intricatus	Q. pubescens	AM181419	MH580545	KF853931		Kolařík et al., 2008
	CCF 3556	S. intricatus	Q. pubescens	AM181418				Kolařík et al., 2008
Geosmithia sp. 12	CCF 4320	Hylesinus oregonus	Fraxinussp.	HF546229	MH580532	KF853932		Kolařík et al., 2017
	CCF 3557	Leperisinus orni	F. excelsior	AM181431	MH580531			Kolařík et al., 2008
Geosmithia sp. 16	CCF 4201	P. pityographus	P. abies	HE604146	HE604206	HE604181	HE604234	Kolařík and Jankowiak, 2013
	RJ34m	P. pityographus	P. abies			HE604182	HE604259	Kolařík and Jankowiak, 2013
Geosmithia sp. 19	CCF 3658	Hypoborus ficus	Ficus carica	AM421085	MH580546			Kolařík et al., 2007
	CCF 3655	H. ficus	F. carica	AM421075				Kolařík et al., 2007
Geosmithia sp. 20	CCF 4316	Phloesinus fulgens	Calocedrus decurrens	HF546226	MH580547			Kolařík et al., 2017
	U193	Scolytus schevyrewi	Ulmus pumila	HF546287	MH580548			Kolařík et al., 2017
Geosmithia sp. 22	CCF 3645	Phloetribus scarabeoides	Olea europaea	AM421061	MH580552	KF853941		Kolařík et al., 2007
	CCF 3652	P. scarabeoides	O. europaea	AM421062	MH580553			Kolařík et al., 2007
Geosmithia sp. 23	CCF 3318	Scolytid beetles	Persea gratissima	AJ578489		HG799808	HG799899	Kolařík et al., 2004, 2017
	CCF 3639	Scolytus rugulosus	Prunus armeniaca	AM421068	HG799838	HG799802	HG799892	Kolařík et al., 2004, 2017
	U160	Scolytus multistriatus	U. pumila	HF546284			HG799911	Kolařík et al., 2017
Geosmithia sp. 24	MB136	Orthotomicus erosus	Pinus halepensis	KP691926		KP691936		Dori-Bachash et al., 2015
	MB242	Pityogenes calcaratus	Pinus brutia	KP691927		KP691937		Dori-Bachash et al., 2015
	MB322	O. erosus	P. brutia	KP691928		KP691938		Dori-Bachash et al., 2015
	CCF 4294	Pityogenes quadridens	P. sylvestris		MH580555			Kolařík and Jankowiak, 2013
	MK1772	P. pityographus	P. sylvestris		MH580556			Kolařík and Jankowiak, 2013
Geosmithia sp. 25	MK1832	Cryphalus abietis	Abies alba	HE604128	HE604218	HE604186	HE604250	Kolařík and Jankowiak (2013)
	CCF 4205	Cryphalus piceae	A. alba	HE604127	HE604219	HE604187	HE604253	Kolařík and Jankowiak, 2013
Geosmithia sp. 26	CCF 4222	Pinus sylvestris		HE604158	LN907595			Kolařík et al., 2017
Geosmithia sp. 27	CCF 4206	Pityogenes bidentatus	P. sylvestris	HE794978	HG799839		HG799893	Kolařík et al., 2017
	CCF 4605	Pityophthorus sp.	Pinus ponderosae	HF546309		HG799827	HG799919	Kolařík and Jankowiak, 2013
Geosmithia sp. 29	CCF 4221	C. piceae	A. alba	HE604125	HE604233	HE604184	HE604248	Kolařík and Jankowiak, 2013
Geosmithia sp. 30	CCF 4288	I. cembrae	L. decidua	HE604132	HE604216	HE604193	HE604242	Kolařík and Jankowiak, 2013
Geosmithia sp. 31	CCF 4196	P. pityographus	P. sylvestris		HE604230	HE604176	HE604256	Kolařík and Jankowiak, 2013
Geosmithia sp. 32	CCF 3554	Phloeosinus thujae	Chamaecyparis pisifera	AM181426	HG799874	HG799885	HG799926	Kolařík et al., 2008, 2017
	CCF 5242	Phloeosinus sequiae	S. serpervirens	HF546265	HG799873	HG799886	HG799925	Kolařík et al., 2008, 2017
Geosmithia sp. 33	CCF 4598	Scolytus praeceps	Abies concolor	HF546331	HG799869	HG799831	HG799923	Kolařík et al., 2017
Geosmithia sp. 34	CCF 4604	Ips plastographus	C. decurrens	HF546295	HG799866	HG799826	HG799918	Kolařík et al., 2017
	U417	S. praeceps	A. concolor	HF546330	HG799868	HG799830	HG799922	Kolařík et al., 2017
Geosmithia sp. 35.	U196	Pityophthorus sp	P. menziesii	HF546231		HG799823		Kolařík et al., 2017
Geosmithia sp. 36	CCF 4328	Pityophthorus sp.	Pinus muricata	HF546236				Kolařík et al., 2017
	MK1814		Cedrus atlantica		MH580538			Present study
Geosmithia sp. 37	U197	Pityophthorus sp.	P. menziesii	HF546288	HG799862	HG799824	HG799915	Kolařík et al., 2017
Geosmithia sp. 38	U79	Pseudopityophthorus pubipennis	Notholithocarpus densiflorus	HF546346	MH580537			Kolařík et al., 2017
	CCF 5241	P. pubipennis	Quercus acrifolia	HF546251	MH580536			Kolařík et al., 2017
Geosmithia sp. 39	U323	P juglandis	Juglans hindsii	HF546314		KC222335		Kolařík et al., 2017
Geosmithia sp. 40	CCF 5250	Pityophthorus sp.	Pinus ponderosa	HF546273	MH580550			Kolařík et al., 2017
	CCF 5245	I. plastographus	Pinus radiata	HF546304	MH580549			Kolařík et al., 2017
Geosmithia sp. 41	U215	Cossoninae sp.	Artemisia arborea	HF546292	HG799865	HG799825	HG799917	Kolařík et al., 2017
	CCF 4342	Bostrichidae sp.	Toxicodendron diversilobum	HF546249	HG799871	HG799833	HG799924	Kolařík et al., 2017
	U64	Scobicia declivis	Umbellularia californica	HF546342	HG799870	HG799832	HG799930	Kolařík et al., 2017
Geosmithia sp. 42	U166	P. canadensis	Chamaecyparis sp.	HF546279	HG799860	HG799821	HG799912	Kolařík et al., 2017
	CCF 5251	S. rugulosus	Prunus sp.	HF546285	HG799861	HG799822	HG799913	Kolařík et al., 2017
Geosmithia sp. 43	CCF 4203	Pityogenes knechteli	P. ponderosae	HF546223	HG799864		HG799916	Kolařík et al., 2017
Geosmithia sp. 44	CCF 4333	Pityophthorus sp.	Pinus sabiniana		LN907598			Kolařík et al., 2017
	CCF 4332	Pityophthorus sp.	P. sabiniana		LN907599			Kolařík et al., 2017
Geosmithia sp. 45	Hulcr 17004	Pityophthorus annectens	Pinus taeda		MH580482			Huang et al., 2019
	Hulcr 17006	P. annectens	P. taeda		MH580487			Huang et al., 2019
	Hulcr 18823	Pityophthorus pulicarius	P. taeda		MH580505			Huang et al., 2019
Geosmithia sp. 46	Hulcr 11575	Pseudopityophthorus minutissimus	Quercus laurifolia	MH426748	MH580479			Huang et al., 2019
	Hulcr 18077	Hypothenemus eruditus	J. nigra	MH426766	MH580490			Huang et al., 2019
	Hulcr 18201	H. eruditus	J. nigra	MH426776	MH580501			Huang et al., 2019
Geosmithia sp. 47	Hulcr 11904	H. dissimilis	Q. laurifolia	MH426749	MH580480			Huang et al., 2019
	Hulcr 19182	H. dissimilis	Carya illinoinensis	MH426789	MH580510			Huang et al., 2019
Geosmithia sp. 48	Hulcr 19190	Phloeosinus dentatus	Juniperus virginiana	MH426796	MH580514			Huang et al., 2019
	Hulcr 19192	P. dentatus	J. virginiana	MH426797	MH580515			Huang et al., 2019
Emericellopsis pallida	CBS 490.71	Pityophthorus sp.		NR_145052	KC998998	KC987138	KC999034	Grum-Grzhimaylo et al., 2013

The isolates recovered in the present study are in bold. Emericellopsis pallida was selected as the outgroup of phylogenies. Strains in italics were screened for morphological studies. *The sequences are available on NCBI but have not been published.

DNA Extraction, Amplification, and Sequencing

DNA was extracted by scraping fresh fungal tissue from pure cultures and adding to 50 μl extraction solution of the PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, United States). The samples were vortexed for 1 min, incubated at 100°C for 10 min, and then centrifuged at 5,000 rpm (MiniSpin Plus Centrifuge, Eppendorf 5453, Germany) for 5 min. The supernatant was transferred to a new Eppendorf tube and used as the template for polymerase chain reaction (PCR) amplification.

The rDNA region of ITS1-5.8S-ITS2, internal transcribed spacer (ITS), was amplified using the primer pair of ITS1-F (Gardes and Bruns, 1993) and ITS4 (White et al., 1990). The translation elongation factor 1-α gene (TEF1-α) was amplified using the primer pair of EF1-983F and EF1-2218R (Rehner and Buckley, 2005). β-Tubulin (TUB2) was amplified by using T10 and Bt2b (Glass and Donaldson, 1995; O’Donnell and Cigelnik, 1997). The second-largest subunit of the RNA polymerase II gene (RPB2) was amplified using the primer pair of fRPB2-5F/fRPB2-7cR (Liu et al., 1999). The PCR amplifications were carried out in a final 25-μl PCR reaction mixture consisting of 50–100 ng template DNA, 1.25 U Taq polymerase (Vazyme Biotech Co., Ltd., Nanjing, China), 200 μM dNTP, 0.5 μM of each primer, and 5% (v/v) dimethyl sulfoxide. The PCR conditions were as follows: 95°C for 3 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The final extension step was 72°C for 10 min. The amplified products were sequenced in Sangon Biotech, Qingdao, Shandong Province, China.

DNA Sequence Analyses

The sequences obtained using the forward and reverse primers were aligned in Geneious version 10.2.2 (Biomatters, Auckland, New Zealand). The reference sequences of Geosmithia species were retrieved from GenBank (Table 2). Emericellopsis pallida CBS 490.71 was chosen as the phylogenetic outgroup. The sequences were aligned by using the online version of MAFFT v. 7 (Katoh and Standley, 2013) with the default settings. The best nucleotide substitution model for each partition was determined in jModelTest v. 2.1.1 (Darriba et al., 2012). Maximum parsimony (MP) analyses were performed using MEGA v.10.2.0 with 1,000 bootstrap replicates; gaps were treated as a fifth-state character. Maximum likelihood (ML) phylogenetic analyses were conducted in the CIPRES Science Gateway (Miller et al., 2010) using RAxML v. 8.2.2 (Stamatakis, 2014) with the recommended partition parameters to assess the tree topology and bootstrap values from 1,000 replicate searches. Bayesian inference (BI) was estimated in the CIPRES Science Gateway (Miller et al., 2010) using MrBayes 3.2.7a (Ronquist et al., 2012). The MCMC runs of four chains were executed simultaneously from a random starting tree for 20 million generations, and every 100 generations were sampled, resulting in 200,000 trees. Chain convergence was determined with Tracer 1.7[1], and the effective sample size values over 200 are considered adequate. A total of 50,000 trees were discarded during burn-in. Posterior probabilities were estimated from the retained 150,000 trees. Phylogenetic trees were visualized and edited in FigTree v. 1.4.3. The final alignments used in this study have been submitted to TreeBase[2] (nos.: 28242).

Morphological Study

Morphological characters were observed and recorded using Olympus BX61 microscope (Olympus Corporation, Tokyo, Japan). The images were analyzed using ImageJ[3]. At least 50 measurements for each of the structures were measured. The results of the calculation are expressed as (minimum -) mean minus standard deviation -- mean plus standard deviation -- (- maximum). One-way ANOVA in SPSS version 26.0[4] was used to evaluate the morphological differences of the different species, with a significance level of 0.05 (Supplementary Figure S5).

Growth Study

Three independently isolated strains of each novel taxon were randomly selected for the growth experiments. The actively growing edge mycelia were inoculated at the center of 90-mm Petri dishes containing 2% MEA and incubated in darkness at temperatures ranging from 5 to 35°C at 5°C intervals and 37°C for 8 days; each temperature had three duplicates. The colony diameters were measured every 2 days, and then the optimum temperature of growth for each species and the high- and low-temperature conditions of growth were calculated.

Results

In total, 125 samples (N) were collected (Table 1). A total of 178 strains in the genus Geosmithia were isolated from 12 beetle species and their galleries. One hundred fifty-eight strains were from the galleries and 20 strains were from the beetles. There were 63 strains from Jiangxi, 47 from Guangdong, 23 from Shandong, 20 from Yunnan, 9 from Shanghai, 8 from Jiangsu, 5 from Fujian, 2 from Guangxi, and 1 from Hunan (Table 1).

Phylogenetic Analysis

The preliminary classification was carried out by BLAST on NCBI GenBank using the ITS marker (Supplementary Table S1). Subsequently, 32 representative strains were selected for multi-gene phylogenetic analysis, and 24 strains were screened for morphological studies (Table 2). The aligned sequences, including gaps, yielded 555 characters for ITS where 124 were parsimony informative, 899 characters for TEF1-α where 209 were parsimony informative, 1,066 characters for RPB2 where 380 were parsimony informative, and 653 characters for TUB2 where 321 were parsimony informative. The concatenated dataset comprised 162 sequences covering 3,173 characters where 1,028 were parsimony informative. The final average standard deviation of split frequency of MCMC analysis was 0.009591 for the concatenated dataset, 0.004862 for ITS, 0.006573 for TEF1-α, 0.008026 for RPB2, and 0.007595 for TUB2. The best substitution model for ITS, TEF1-α, RPB2, TUB2, and combined alignment was GTR + I + G. For all datasets (ITS, TUB2, TEF1-α, and RPB2), ML, MP, and Bayesian inference produced nearly identical topologies, with slight variations in the statistical support for each of the individual sequence datasets (Figure 1 and Supplementary Figures S1–S4). Phylograms obtained by ML are presented for all the individual datasets.

FIGURE 1

Maximum likelihood (ML) tree of Geosmithia generated from the combined ITS, TEF1-α, TUB2, and RPB2 sequence data. The sequences generated from this study are printed in bold. The bold branches indicate posterior probability values ≥0.9. Bootstrap values of ML/maximum parsimony ≥75% are recorded at the nodes. T, ex-type isolates.

Morphological Statistical Analysis

The results of the morphological comparison of the different species are presented in Supplementary Figure S5. The values are mean of 50 measurements (±) SD, and significant differences according to Dunnett-t3′ multiple-range tests at p < 0.05 levels were indicated and followed by different letters.

Taxonomy

Among the 178 strains obtained in this study, 12 species were identified. Nine of these species are new to science and are described as follows:

Geosmithia luteobrunnea R. Chang and X. Zhang, sp. nov.

MycoBank MB839256

Etymology: luteobrunnea, referring to the yellowish-brown appearance of the colony on MEA.

Diagnosis: The stipe of G. luteobrunnea is slightly thicker and shorter than that in other species. Geosmithia luteobrunnea can grow at 5 and 35°C, even grows slowly at 37°C (Figure 2).

FIGURE 2

Morphological characteristics of Geosmithia luteobrunnea sp. nov. (SNM261 = CGMCC3.20252, SNM226, SNM287). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. The stipe (indicated with arrows) is thick and short. Scale bars: 10 μm (B–E).

Type: China, Jiangxi Province, Ganzhou City, Longnan County, Jiulianshan National Nature Reserve (24°34′1′′ N, 114°30′ E), from the gallery of Scolytus jiulianshanensis on Ulmus sp., 5 May 2020, S. Lai, Y. Xu, S. Liao, Y. Wen and T. Li (HMAS 249919 – holotype, SNM261 = CGMCC3.20252 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like and (19.0–) 29.6–61.5 (–85.0)-μm long. Conidiophores borne mostly from aerial fungal hyphae, erect, determinate, solitary, sometimes funiculose, with all parts verrucose; base often consisting of a curved and atypically branched cell, stipe (6.4–) 11.3–40.1 (–78.4)-μm long, (1.5–) 1.7–3.2 (–6.0)-μm wide; penicillus, monoverticillate to terverticillate (penicilli of conidiophores on aerial funiculose mycelia are monoverticillate or biverticillate), symmetric or asymmetric, often irregularly branched, rami (1st branch) in whorls of 1–3, (4.1–) 5.2–7.0 (–8.7) × (1.2–) 1.7–2.5 (–3.2) μm, metulae (last branch) in whorls of 1 to 2, (4.0–) 4.9–6.5 (–7.6) × (1.4–) 1.8–2.3 (–2.6) μm; phialides in whorls of 1–3, cylindrical, without or with short cylindrical neck and smooth to verrucose walls, (4.2–) 5.1–7.5 (–10.2) × (1.1–) 1.5–2.3 (–2.7) μm. Conidia hyaline to subhyaline, smooth, narrowly cylindrical to ellipsoidal, (2.3–) 2.9–4.0 (–4.7) × (0.9–) 1.2–1.7 (–2.2) μm, produced in non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 50–64 mm at 20°C, 58–78 mm at 25°C, and 44–70 mm at 30°C. The hyphae grow slowly at 5 and 35°C. After 8 days of culture, the colony diameter was 1.5–4 mm and 11–14 mm, respectively. The optimal temperature for growth was 25°C. Colonies at 25°C, 8 days, were oppressed, velutinous, or floccose with raised mycelial cords; colony margin smooth, filamentous, diffuse; aerial mycelium sparse; substrate mycelium sparse; conidiogenesis moderate; milky white to light yellow; reverse lighter brown; absence of exudate; no soluble pigment. When incubated at 35°C, colonies were raised, slightly depressed at the center, rugose, or irregularly furrowed; margin undulate somewhat erose; aerial mycelia sparse to moderate; substratum mycelia dense, forming a tough basal felt; the colony was darker and yellowish-brown; reverse brown; soluble pigment was brown. MEA, 37°C, 8 days, germinating only.

Host: Liquidambar formosana, Liquidambar styraciflua, Ulmus sp.

Beetle vectors: Acanthotomicus suncei, Scolytus jiulianshanensis.

Distribution: Currently only known from Jiangxi and Shanghai.

Notes: Geosmithia luteobrunnea and G. radiata are phylogenetically close to each other on ITS, TUB2, RPB2, TEF1-α trees, and combined alignment tree (Figure 1 and Supplementary Figures S1–S4). The colony morphology of G. luteobrunnea and G. radiata are also similar, but there are many differences among those two species. First of all, their sequences are different (Table 3). Then, under the microscope, the morphological differences between them are more obvious (Supplementary Figure S5). The spore of G. radiata is shorter than the other specie. The stipe of G. radiata is thicker than the other specie, and the stipe of G. luteobrunnea is slightly shorter than the other two species (Supplementary Figure S5). Moreover, their growths at different temperatures are also different (Table 4). G. luteobrunnea can grow at both temperatures, especially at 35°C, even grows slowly at 37°C. Geosmithia radiata only grows a little at 5°C and grows slowly at 35°C. The growth speed of G. luteobrunnea is faster than that of G. radiata (Table 4). Geosmithia luteobrunnea and G. radiata form a species group outstanding by cream to yellow or brown color of sporulation accompanied by the darker (brownish to rusty) shades of the substrate mycelium and colony reverse. This feature is shared also by the phylogenetically related Geosmithia sp. 11 (Kolařík et al., 2007) which is known from Europe and the Mediterranean (Kolařík et al., 2007, 2008) and seems to be diagnostic for the whole species group.

TABLE 3

Summary of the variability between species of the Geosmithia luteobrunnea species complex.

Species	ITS rDNA (555 bp)		TEF1-α (899 bp)		TUB2 (666 bp)		RPB2 (1066 bp)
	G. radiata	G. luteobrunnea	G. radiata	G. luteobrunnea	G. radiata	G. luteobrunnea	G. radiata	G. luteobrunnea
G. radiata		5 (0.90%)		8–9 (0.89–1.0%)		4 (0.60%)		6 (0.56%)

TABLE 4

The colony diameter of G. subfulva, G. bombycine, G. luteobrunnea, G. radiata, G. granulate, and G. pallida species complex, G. brevistipitata and G. pumila, at different temperatures after 8 days on malt extract agar medium (unit: millimeter).

Species/T	5°C	20°C	25°C	30°C	35°C	37°C
G. bombycina	1	20–23	24–31	22–30	5–8	0
G. brevistipitata	2 to 3	20–30	23–34	8–12	0	0
G. fusca	1–6	21–26	25–36	26–32	1–11	≈0
G. granulata	<1	27–32	30–34	8–12	2–4	0
G. luteobrunnea	1–4	50–64	58–78	44–70	11–14	≈0
G. pulverea	0	23–29	30–37	31–36	1.5–4	0
G. pumila	7–10	25–29	25–33	22–26	≈0	0
G. radiata	1	50–58	59–69	49–60	1–4	0
G. subfulva	4–6	17–26	24–36	20–29	35	0

Additional cultures examined: China, Shanghai, from the gallery of Acanthotomicus suncei on Liquidambar styraciflua, April 2019, L. Gao (SNM226, SNM287).

Geosmithia radiata R. Chang and X. Zhang, sp. nov.

MycoBank MB839257

Etymology: radiata, referring to the radial appearance of the colony on MEA.

Diagnosis: The spore and the stipe of G. radiata are thicker than closely related species. Geosmithia radiata only grows a little at 5 and 35°C (Figure 3).

FIGURE 3

Morphological characteristics of Geosmithia radiata sp. nov. (SNM279 = CGMCC3.20253, SNM884). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. The sporulation structure is coarse, and the phialides (indicated with arrows) are abundant and compact. Scale bars: 10 μm (B–D) and 20 μm (E).

Type: China, Jiangxi Province, Ganzhou City, Longnan County (24°5′2.4′′ N, 114°47′2.4′′ E), from the gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May 2020, S. Lai (HMAS 249920 – holotype, SNM279 = CGMCC3.20253 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like and (22.6–) 35.6–85.7 (–119.3)-μm long. Conidiophores borne from the substrate or aerial hyphae, sometimes arising laterally from another conidiophore, erect, determinate, solitary, with all parts verrucose; stipe commonly (7.3–) 18.4–63.6 (–115.8)-μm long, (1.6–) 2.1–3.8 (–5.9)-μm wide, penicillus, with walls thick, septate; penicillus terminal, monoverticillate, biverticillate, or terverticillate, mostly symmetrical, rami (1st branch) in whorls of 2 to 3, (4.2–) 5.2–7.8 (–10.6) × (1.3–) 2.1–3.5 (–4.8) μm; metulae (last branch) in whorls of 1 to 2, (2.6–) 3.9–5.8 (–7.3) × (1.3–) 1.7–2.6 (–3.3) μm. Phialides in whorls of 1–5, (3.9–) 4.6–6.2 (–7.7) × (1.5–) 1.9–2.8 (–3.9) μm, cylindrical, without or with short cylindrical neck and smooth to verrucose walls. Conidia cylindrical to ellipsoidal, smooth, hyaline to subhyaline, (2.2–) 2.5–3.2 (–4.0) × (0.9–) 1.1–1.5 (–1.8) μm, formed in non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 50–58 mm at 20°C, 59–69 mm at 25°C, and 49–60 mm at 30°C. The hyphae grow slowly at 5 and 35°C. After 8 days of culture, the colony diameter was only 1 and 1–4 mm, respectively. The optimal temperature for growth is 25°C. Colonies at 25°C, 8 days, plane, slightly raised centrally, velutinous, with a slight overgrowth of aerial mycelium, with floccose and funiculose areas; substrate mycelium darker, aerial mycelium hyaline; sporulation moderate to heavy, pale cream; vegetative mycelium hyaline; reverse lighter yellow; soluble pigment and exudate absent. When incubated at 35°C, colonies were rising, slightly sunken in the center, furrowed, or irregularly fringed; the substratum hyphae were dense and formed a tough basal felt. The colony is dark and yellowish-brown. MEA, 37°C, 8 days: no growth.

Host: Liquidambar formosana, Ulmus sp.

Beetle vectors: Acanthotomicus suncei, Scolytus jiulianshanensis.

Distribution: Jiangxi.

Notes: See comparisons between Geosmithia luteobrunnea, G. radiata below the description of G. luteobrunnea.

Additional cultures examined: China, Jiangxi Province, Ganzhou City, Xunwu County (24°57′ N, 115°38′2′′ E), from the gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May 2020 (SNM884).

Geosmithia brevistipitata R. Chang and X. Zhang, sp. nov.

MycoBank MB841503

Etymology: brevistipitata, referring to the short conidiophore stipe, a character distinguishing it from other members of the species complex.

Diagnosis: Isolates of G. brevistipitata formed a monophyletic clade on all the phylogenetic trees (Figure 4).

FIGURE 4

Morphological characteristics of Geosmithia brevistipitata sp. nov. (SNM1616 = CGMCC3.20627, SNM1610, SNM1611). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. The stipe (indicated with arrows) is short and sometimes not smooth. Scale bars: 10 μm (B–E).

Type: China, Shandong Province, Linyi City, Tianfo scenic spot (35°5′ N, 118°2′ E), from the gallery of Phloeosinus cf. hopehi on Cupressus funebris, 8 August 2021, Y. Cao (HMAS 351566 - holotype, SNM1616 = CGMCC3.20627 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like and (9.5–) 15.5–42.3 (–77.9)-μm long. Conidiophores borne from the substrate or aerial hyphae, sometimes arising laterally from another conidiophore, erect, determinate, solitary, with all parts verrucose; stipe commonly (2.9–) 7.5–30.0 (–56.0) × (1.3–) 1.9–3.0 (–4.1) μm, penicillus, with walls thick, septate; penicillus terminal, monoverticillate or biverticillate, mostly symmetrical, metulae in whorls of 2–3, (4.6–) 6.3–9.1 (–11.2) × (1.8–) 2.0–2.7 (–3.2) μm. Phialides in whorls of 2–5, (3.2–) 5.0–8.7 (–11.4) × (1.3–) 1.7–2.4 (–2.8) μm, cylindrical, without or with short cylindrical neck and smooth to verrucose walls. Conidia cylindrical to ellipsoidal, smooth, hyaline to subhyaline, (2.2–) 2.4–3.1 (–3.8) × (1.2–) 1.5–1.9 (–2.2) μm, formed in non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 24–30 mm at 20°C, 23–34 mm at 25°C, and 8–12 mm at 30°C. The hyphae grow slowly at 5°C. After 8 days of culture, the colony diameter was only 2–3 mm. No growth at 35°C. The optimal temperature for growth is 20–25°C. Colonies at 25°C, 8 days, plane, granular, with a slight growth of aerial mycelium; substrate mycelium white; reverse white; sporulation moderate white; soluble pigment and exudate absent. MEA, 37°C, 8 days: no growth.

Host: Cupressus funebris.

Beetle vectors: Phloeosinus cf. hopehi.

Distribution: Shandong.

Notes: Isolates of G. brevistipitata formed a monophyletic clade on both ITS, TUB2, TEF1-α, and RPB2 trees. Its closely related species differ on various trees, such as according to ITS tree, isolates of G. brevistipitata were closely related to G. cnesini, G. xerotolerans, G. omnicola, G. ulmacea, and Geosmithia sp. 12 (Supplementary Figure S5), but according to the TUB2 tree, isolates of G. brevistipitata were connected to other species, e.g., G. microcorthyli and G. obscura (Supplementary Figure S2). Among the other species described, it is outstanding by the combination of slow growth and white colony color and short stipe.

Additional cultures examined: China, Shandong Province, Linyi City, Tianfo scenic spot (118°2′ N, 35°5′ E), from the gallery of Phloeosinus cf. hopehi on Cupressus funebris, 8 August 2021, Y. Cao (SNM1610).

Geosmithia granulata R. Chang and X. Zhang, sp. nov.

MycoBank MB 840646

Etymology: granulata, referring to the granular appearance of the colony on MEA.

Diagnosis: The conidia of G. granulata are shorter than the closely related species (Figure 5).

FIGURE 5

Morphological characteristics of Geosmithia granulata sp. nov. (SNM1015 = CGMCC3.20450, SNM1013). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. Conidia hyaline, smooth, wide oval shape, like an egg. Scale bars: 10 μm (B–E).

Type: China, Yunnan Province, Xishuangbanna City, Xishuangbanna Botanical Garden (21°55′1′′ N, 101°16′1′′ E), from the gallery of Sinoxylon cf. cucumella on Acacia pennata, 1 May 2021, Y. Dong and Y. Li (HMAS 351568 - holotype, SNM1015 = CGMCC3.20450 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like, (9.6–) 11.6–26.0 (–50.6) μm in length. Conidiophores emerging from hyphae, smooth, septate; stipe (4.0–) 4.8–8.3 (–14.3) × (1.1–) 1.4–2.1 (–2.6) μm; penicilli typically longer than the stipe, terminal, monoverticillate, biverticillate, or terverticillate, symmetric or asymmetric, often irregularly branched, rarely more; metulae in whorls of 1–2, (5.2–) 5.7–8.1 (–11.3) × (1.0–) 1.3–1.7 (–2.0) μm; phialides in whorls of 1–4, smooth, (3.3–) 4.9–7.1 (–8.8) × (1.0–) 1.3–1.8 (–2.2) μm. Conidia hyaline, smooth, wide oval shape, like an egg, (1.5–) 1.8–2.2 (–2.5) × (0.8–) 1.0–1.4 (–1.8) μm. Conidia formed in long, non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 27–32 mm at 20°C, 30–34 mm at 25°C, and 8–12 mm at 30°C. At 5°C: germinating only. At 35°C, the mycelia grew slowly. After 8 days of culture, the diameter of the colony was 2–4 mm. The optimal growth temperature is 20–25°C. At 25°C, 8 days: Colonies were flat and radiated, surface texture powdery; central hyphae slightly raised and wrinkled, conidiogenesis heavy; marginal colonies were similar to annual rings, slightly flocculent, hyphae were sparse, milky white, reverse creamyrice white; without exudate and insoluble pigment. MEA, 37°C, 8 days, no growth.

Host: Acacia pennata, Hibiscus tiliaceus, Ulmus sp.

Beetle vectors: Sinoxylon cf. cucumella, Ernoporus japonicus, Scolytus semenovi.

Distribution: Guangdong, Jiangsu, Yunnan.

Notes: According to the tree made by ITS and TEF1-α sequence, SNM1015 and SNM1013 were clustered with Geosmithia sp. 20 (Supplementary Figures S1, S3). TUB2 and RPB2 sequences of Geosmithia sp. 20 were not available on GenBank; therefore, Geosmithia sp. 20 was not included in TUB2 and RPB2 trees. These results suggested that our isolates and Geosmithia sp. 20 belonged to the same species, described as G. granulata sp. nov. This extends the geographical distribution of this species to the Mediterranean Basin (Kolařík et al., 2007) and western part of the United States (Kolařík et al., 2017) where it was found in association with many bark beetle species feeding on plants from the families Asteraceae, Fabaceae, Moraceae, Oleaceae, Ulmaceae (Mediterranean Basin), or Cupressaceae, Ulmaceae (Western United States).

Additional cultures examined: China, Yunnan Province, Xishuangbanna City, Xishuangbanna Botanical Garden (21°55′1′′ N, 101°16′1′′ E), from the gallery of Sinoxylon cf. cucumella on Acacia pennata, 1 May 2021, Y. Dong and Y. Li (SNM1013).

Geosmithia subfulva R. Chang and X. Zhang, sp. nov.

MycoBank MB 841505

Etymology: subfulva, referring to the beige appearance of the colony on MEA.

Diagnosis: Isolates of G. subfulva formed a monophyletic clade on all the phylogenetic trees (Figure 6).

FIGURE 6

Morphological characteristics of Geosmithia subfulva sp. nov. (SNM1304 = CGMCC3.20579, SNM1298). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. The metulae (indicated with arrows) branches are few and sparse. Scale bars: 10 μm (B–E).

Type: China, Guangdong Province, Zhuhai City (22°16′48′′ N, 113°30′28′′ E), from the gallery of Ernoporus japonicus in the twig of Hibiscus tiliaceus, 21 June 2021, W. Lin (HMAS 351569 - holotype, SNM1304 = CGMCC3.20579 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like and (13.3–) 21.0–43.5 (–62.5)-μm long. Conidiophores arising from substrate or aerial mycelium with all parts verrucose; stipe (5.3–) 9.3–26.4 (–36.6) × (0.9–) 1.5–2.2 (–3.1) μm; penicillus, biverticillate to quaterverticillate, symmetric or asymmetric, often irregularly branched, rarely more, rami (1st branch) in whorls of 1–2, (4.8–) 5.6–7.4 (–8.4) × (1.0–) 1.3–1.8 (–2.0) μm, metulae (last branch) in whorls of 1–3, (4.0–) 4.6–5.9 (–6.9) × (0.9–) 1.2–1.6 (–1.8) μm; phialides 1–3, cylindrical or ellipsoidal, without or with short cylindrical neck and smooth to verrucose walls, (3.6–) 4.8–6.9 (–10.0) × (0.8–) 1.1–1.4 (–1.6) μm. Conidia hyaline, smooth, wide oval shape, (1.1–) 1.5–2.2 (–2.2) × (1.0–) 1.1–1.5 (–1.7) μm. Conidia formed in long, non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 17–26 mm at 20°C, 24–36 mm at 25°C, and 20–29 mm at 30°C. At 5 and 35°C, the mycelia grew slowly. After 8 days of culture, the colony diameter was 4–6 and 3–5 mm. The optimal growth temperature is 25°C. Colonies at 25°C, 8 days, plane with radial rows and slightly raised centrally, texture velutinous (powdery); beige to off-white; reverse milky white; soluble pigment and exudate absent. When incubated at 35°C, the colonies are the same as described above. MEA, 37°C, 8 days: no growth.

Host: Hibiscus tiliaceus, Rhus chinensis.

Beetle vectors: Cryphalus kyotoensis, Ernoporus japonicus, Hypothenemus sp. L636.

Distribution: Fujian, Guangdong, Shandong.

Notes: Isolates SNM1304 and SNM1298 formed a monophyletic clade on both ITS, TUB2, TEF1-α, and RPB2 trees (Supplementary Figures S1–S4). On the ITS tree, SNM1304 and SNM1298 were distinct from all other species. On the TUB2 tree, SNM1304 and SNM1298 are linked to G. pulverea but have no strong support. On TEF1-α and RPB2 trees, they are nested in a clade including not only G. pulverea but also several other species.

Additional cultures examined: China, Guangdong Province, Zhuhai City (22°16′48′′ N, 113°30′28′′ E), from the gallery of Ernoporus japonicus LW320 in the twig of Hibiscus tiliaceus, 21 June 2021, W. Lin (SNM1298).

Geosmithia pulverea R. Chang and X. Zhang, sp. nov.

MycoBank MB839259

Etymology: pulverea, powdery in Latin. On MEA medium, G. pulverea has powdery sporulation.

Diagnosis: Geosmithia pulverea produces long spore chains, while its closely related species does not (Figure 7).

FIGURE 7

Morphological characteristics of Geosmithia pulverea sp. nov. (SNM885 = CGMCC3.20255, SNM270, SNM888). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. The stipe (indicated with arrows) are slender and abundant with spores. Scale bars: 10 μm (B–E).

Type: China, Guangdong Province, Shenzhen City (21°55′12″ N, 101°16′12″ E), from the gallery of Dinoderus sp. L489 in the vine of Gnetum luofuense, 12 April 2018, Y. Li (HMAS 249922 – holotype, SNM885 = CGMCC3.20255 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like and (17.5-) 30.9–84.3 (-120.1)-μm long. Conidiophores arising from substrate or aerial mycelium with all parts verrucose; base often consisting of curved and atypically branched cell; stipe (16.2–) 32.7–85.7 (–153.9) × (1.9–) 2.5–3.7 (–4.7) μm; penicillus, biverticillate to quaterverticillate, symmetric or asymmetric, often irregularly branched, rarely more, rami (1st branch) in whorls of 2–4, (8.2–) 10.2–14.4 (–18.9) × (2.2–) 2.5–3.3 (–3.9) μm, metulae (last branch) in whorls of 2–3, (6.3–) 7.5–10.9 (–15.8) × (1.8–) 2.1–2.8 (–3.5) μm; phialides 1–3, cylindrical or ellipsoidal, without or with short cylindrical neck and smooth to verrucose walls, (5.3–) 7.0–9.6 (–12.3) × (1.5–) 1.8–2.5 (–3.0) μm. Conidia hyaline, smooth, narrowly cylindrical to ellipsoidal, (2.1–) 2.5–3.4 (–5.1) × (1.1–) 1.2–1.6 (–2.0) μm. Conidia formed in long, non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 23–29 mm at 20°C, 30–37 mm at 25°C, and 31–36 mm at 30°C. No growth at 5°C. At 35°C, the mycelia grew slowly. After 8 days of culture, the colony diameter was 1.5–4 mm, with a yellow soluble pigment. The optimal growth temperature is 25–30°C. Colonies at 25°C, 8 days, plane with radial rows and slightly raised centrally, texture velutinous (powdery); sporulation abundant, light brownish yellow to buff; reverse yellowish to slightly avellaneous brown; soluble pigment and exudate absent. When incubated at 35°C, the colonies are the same as described above. MEA, 37°C, 8 days: no growth.

Host: Acacia pennata, Gnetum luofuense, Liquidambar formosana, L. styraciflua, Choerospondias axillaris, Lauraceae, Eriobotrya japonica, Rhus chinensis, Ulmus spp.

Beetle vectors: Sinoxylon cf. cucumella, Acanthotomicus suncei, Crossotarsus emancipatus, Dinoderus sp. L489, Microperus sp. L589, Phloeosinus sp., Scolytus semenovi, Scolytus jiulianshanensis, Cryphalus kyotoensis, Cryphalus eriobotryae.

Distribution: Fujian, Guangdong, Guangxi, Hunan, Jiangxi, Yunnan, Shandong, Shanghai.

Notes: Geosmithia pulverea colony was powdery and brown-yellow. One of the most obvious features is the long spore chain. According to the tree made by ITS sequence, SNM888, SNM885, and SNM248 were clustered with Geosmithia sp. 3, and SNM886, SNM887, and SNM270 were clustered with Geosmithia sp. 23 (Supplementary Figure S1). However, in the trees with TUB2, TEF1-α, and RPB2, these strains did not have a clear subclassification (Supplementary Figures S2–S4). It was consequently recognized, using multigene phylogeny, together with Geosmithia sp. 23, as a well-defined phylogenetic species inside the G. pallida species complex (Huang et al., 2017; Kolařík et al., 2017). The colony of G. pulverea was very similar to Geosmithia sp. 3 on MEA, but Geosmithia sp. 3 was darker and wrinkled (Kolařík et al., 2004). Geosmithia pulverea seems to have a smaller stipe size, but other features fit the morphology of Geosmithia sp. 3 (Kolařík et al., 2004). In this study, we are providing a formal description for the Chinese strains related to Geosmithia sp. 3 and sp. 23 which are known to be distributed over various bark beetle hosts in Temperate Europe in the case of Geosmithia sp. 3 (Kolařík et al., 2004, 2008; Strzałka et al., 2021) or seems to have a global distribution and many bark beetle hosts across Temperate Europe (Strzałka et al., 2021), the Mediterranean Basin (Kolařík et al., 2007), Northern America (Huang et al., 2017, 2019; Kolařík et al., 2017), and Seychelles (Kolařík et al., 2017). Further study is needed to assess the taxonomic relationships between G. pulverea, Geosmithia sp. 3, and Geosmithia sp. 23.

Additional cultures examined: China, Guangxi Province, Shangsi City, Shiwandashan Mt. (21°54′12′′ N, 107°54′14′′ E), from the body surface of Crossotarsus emancipates, 27 March 2018, Y. Li (SNM887).

China, Hunan Province, Changsha City, Yuelushan Mt. (28°10′56′′ N, 112°55′41′′ E), from the gallery of Microperus sp. L589 on the trunk of Choerospondias axillaris, 15 July 2019, Y. Li (SNM888).

Geosmithia fusca R. Chang and X. Zhang, sp. nov.

MycoBank MB841506

Etymology: fusca, referring to the brown appearance of the colony on MEA.

Diagnosis: The difference with closely related species G. cucumellae is reflected in such a way that the conidia of G. fusca are smooth and do not produce long spore chains (Figure 8).

FIGURE 8

Morphological characteristics of Geosmithia fusca sp. nov. (SNM1578 = CGMCC3.20626, SNM1577). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. Conidia hyaline, smooth, wide oval shape. Scale bars: 10 μm (B–E).

Type: China, Guangdong Province, Zhuhai City, Agricultural Science Research Center (22°18′9′′ N, 113°31′40′′ E), from the gallery of Xylocis tortilicornis on Phyllanthus emblica, 6 July 2021, W. Lin (HMAS 351570 - holotype, SNM1578 = CGMCC3.20626 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like and (16.3–) 20.2–55.8 (–94.3)-μm long. Conidiophores variable in shape and size, emerging from a surface mycelium, with all segments smooth or minutely verrucose to distinctly verrucose, septate, stipe (8.6–) 10.1–38.5 (–70.1) × (1.2–) 1.5–2.1 (–2.6) μm; penicilli typically shorter than the stipe, terminal, monoverticillate or biverticillate, symmetric or asymmetric, irregularly branched; metulae in whorls of 2–3, (4.9–) 6.0–8.3 (–9.9) × (1.1–) 1.3–1.8 (–2.1) μm; phialides in whorls of 1–3, smooth, (4.6–) 5.8–8.1 (–9.4) × (1.0–) 1.3–1.7 (–1.9) μm. Conidia cylindrical to ellipsoid, (1.5–) 2.0–2.7 (–3.4) × (0.9–) 1.1–1.7 (–1.7) μm. Conidia formed in long, non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 21–26 mm at 20°C, 25–36 mm at 25°C, and 26–32 mm at 30°C. At 5 and 35°C, the mycelia grew slowly. After 8 days of culture, the colony diameter was 1–3 and 7–11 mm, respectively. The optimal growth temperature is 25–30°C. At 25°C, 8 days: Colonies flat with radial rows, surface texture powdery; sporulation abundant, brown; central hyphae were raised and white flocculent; reverse yellowish to brown; without exudate and insoluble pigment. MEA, 37°C, 8 days: germinating only.

Host: Hibiscus tiliaceus, Phyllanthus emblica, Acacia pennata.

Beetle vectors: Ernoporus japonicus, Xylocis tortilicornis, Sinoxylon cf. cucumella.

Distribution: Guangdong, Yannan.

Notes: In the phylogenetic tree, SNM1012, SNM1067 and SNM1577, SNM1578 formed very close separate branches (Figure 1 and Supplementary Figures S1–S4), but combined with morphological analysis, it was found that SNM1012, SNM1067 and SNM1577, SNM1578 had no significant difference except for a small difference in spore length (Supplementary Figure S5). So, they are described as the same species.

Additional cultures examined: China, Guangdong Province, Zhuhai City, Agricultural Science Research Center (22.3025 N, 113.5277 E), from the gallery of Xylocis tortilicornis LW319 on Phyllanthus emblica, 6 July 2021, W. Lin (SNM1577).

China, Yunnan Province, Xishuangbanna City, Xishuangbanna Botanical Garden (21°55′1′′ N, 101°16′1′′ E), from the gallery of Sinoxylon cf. cucumella on the trunk of Acacia pennata, 1 May, 2021, Y. Dong and Y. Li (SNM1012, SNM1167).

Geosmithia pumila R. Chang and X. Zhang, sp. nov.

MycoBank MB841507

Etymology: pumila, referring to the tree host of Ulmus pumila where this species has been isolated.

Diagnosis: Isolates of G. pumila formed a monophyletic clade on all the phylogenetic trees (Figure 9).

FIGURE 9

Morphological characteristics of Geosmithia pumila sp. nov. (SNM1653 = CGMCC3.20630, SNM1657). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. Most phialides (indicated with arrows) are not smooth. Scale bars: 10 μm (B–E).

Type: China, Jiangsu Province, Nanjing City, Nanjing Forestry University (32°3′36′′ N, 118°48′36′′ E), from the gallery of Scolytus semenovi in the branch of Ulmus sp., 25 August 2021, S. Lai (HMAS 351571 - holotype, SNM1653 = CGMCC3.20630 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like and (12.9–) 35.9–72.7 (–109.4)-μm long. Conidiophores arising from substrate or aerial mycelium with all parts verrucose; stipe (9.9–) 19.7–51.5 (–77.9) × (1.2–) 1.4–2.2 (-2.6) μm; penicillus, monoverticillate or biverticillate, mostly monoverticillate, symmetric or asymmetric, often irregularly branched, rarely more, metulae in whorls of 2 to 3, (5.1–) 6.3–8.9 (–10.5) × (1.1–) 1.4–2.0 (–2.3) μm; phialides 1–3, smooth to verrucose walls, (5.0–) 5.7–7.3 (–8.5) × (1.1–) 1.2–1.6 (–1.8) μm. Conidia hyaline, smooth, ellipsoidal, (1.5–) 1.9–2.5 (–2.9) × (0.9–) 1.1–1.5 (–1.9) μm. Conidia formed in long, non-persistent conidial chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 25–29 mm at 20°C, 25–33 mm at 25°C, and 22–26 mm at 30°C. At 35°C: germinating only. At 5°C, the mycelia grew slowly. After 8 days of culture, the colony diameter was 7–10 mm. The optimal growth temperature is 20–25°C. Colonies at 25°C, 8 days, plane with radial rows, texture velutinous (powdery), slightly funiculus centrally; sporulation medium, light yellow to rice white; reverse milk-white; soluble pigment and exudate absent. MEA, 37°C, 8 days: no growth.

Host: Ulmus sp.

Beetle vectors: Scolytus semenovi.

Distribution: Jiangsu.

Notes: Based on ITS, TUB2, TEF1-α, and RPB2 trees (Supplementary Figures S1–S4), SNM1653 and SNM1657 were grouped with Geosmithia sp. 2. Therefore, we considered that SNM1653, SNM1657, and Geosmithia sp. 2 were the same species. This extends the geographical range to Europe, the Mediterranean Basin (Kolařík et al., 2007, 2008), the whole United States (Huang et al., 2017, 2019; Kolařík et al., 2017), Peru (Kolařík et al., 2004), and South Africa (Machingambi et al., 2014), which is reported to be in association with a large number of insect and tree hosts.

Additional cultures examined: China, Jiangsu Province, Nanjing City, Nanjing Forestry University (32°3′36′′ N, 118°48′36′′ E), from the gallery of Scolytus semenovi in the branch of Ulmus sp., 25 August 2021, S. Lai (SNM1657).

Geosmithia bombycina R. Chang and X. Zhang, sp. nov.

MycoBank MB 840535

Etymology: bombycina, referring to the cotton appearance of the colony on MEA.

Diagnosis: Isolates of G. bombycina formed a monophyletic clade on all the phylogenetic trees (Figure 10).

FIGURE 10

Morphological characteristics of Geosmithia bombycina sp. nov. (SNM933 = CGMCC3.20578, SNM934). (A) Eight-day-old culture on 2% malt extract agar. (B–E) Conidiophores and conidia. The penicilli (indicated with arrows) are typically shorter than the stipe, terminal, monoverticillate, biverticillate or terverticillate, symmetric or asymmetric. Scale bars: 10 μm (B–E).

Type: China, Fujian Province, Fuqing City (25°71′ N, 119°15′ E), from the gallery of Cryphalus eriobotryae on Eriobotrya japonica, 8 April 2021, Y. Li (HMAS 350284 – holotype, SNM933 = CGMCC3.20578 – ex-holotype culture).

Description: Sexual state not observed. Asexual state penicillium-like, (14.0–) 20.2–41.0 (–62.6) μm in length. Conidiophores emerging from hyphae, smooth, septate; stipe (5.4–) 9.4–30.0 (–47.5) × (0.9–) 1.4–2.0 (–2.4) μm; penicilli typically shorter than the stipe, terminal, monoverticillate, biverticillate or terverticillate, symmetric or asymmetric, often irregularly branched, rarely more; metulae in whorls of 1–2, (5.1–) 5.9–8.3 (–10.5) × (1.0–) 1.2–1.7 (–2.1) μm; phialides in whorls of 2–4, smooth, (4.9–) 5.8–9.4 (–12.6) × (0.9–) 1.3–1.7 (–2.0) μm. Conidia hyaline, smooth, narrow, and oval, (2.1–) 2.4–3.3 (–4.1) × (0.8–) 0.9–1.3 (–1.5) μm, produced in non-persistent chains. Substrate conidia absent.

MEA, 8 days: Colony diameter 20–23 mm at 20°C, 24–31 mm at 25°C, and 22–30 mm at 30°C. The hyphae grow slowly at 5 and 35°C. After 8 days of culture, the colony diameter was less than 1 and 5–8 mm, respectively. The optimal temperature for growth was 25–30°C. At 25°C, 8 days: The colonies were flat, like annual rings; central hyphae were raised and white flocculent; filamentous, diffuse, basal mycelium sparse; conidiogenesis moderate, milk-white; reverse creamy white; no exudate and insoluble pigment. When incubated at 35°C, the colonies grew, and the mycelia were sparse and snowflake-shaped, with no soluble pigment. MEA, 37°C, 8 days, no growth.

Host: Eriobotrya japonica.

Beetle vectors: Cryphalus eriobotryae.

Distribution: Currently only known from Fujian.

Notes: According to ITS, TUB2, and TEF1-α trees (Supplementary Figures S1–S4), SNM933 and SNM934 formed a monophyletic clade and nested with Geosmithia sp. 22, Geosmithia sp. 24, G. longistipitata, G. pazoutovae, and G. fagi. The RPB2 sequences for those species were not available on GenBank. Therefore, SNM933 and SNM934 formed a distinct clade that was far away from all the known species on the RPB2 tree.

Additional cultures examined: China, Fujian Province, Fuqing City (25°71′ N, 119°15′ E), from the gallery of Cryphalus eriobotryae on Eriobotrya japonica, 8 April 2021, Y. Li (SNM934).

Discussion

This is the first relatively comprehensive study of Geosmithia species associated with bark beetle in China. The samples were collected from 9 provinces, 12 tree hosts, and 12 bark and ambrosia beetles. A total of 178 strains of Geosmithia were isolated in this study. The analyses of ITS, RBP2, TUB2, and TEF1-α showed that those isolates were separated into 12 taxa, with three strains previously described, G. xerotolerans, G. putterillii, and G. pallida, and the other nine were novel species, described as G. luteobrunnea, G. radiata, G. brevistipitata, G. bombycina, G. granulata (Geosmithia sp. 20), G. subfulva, G. pulverea (Geosmithia sp. 3 and Geosmithia sp. 23), G. fusca, and G. pumila in this study. Those species were isolated from larvae, frass, and wood dust in the beetle galleries of dying, stressed, or weakened broad-leaf and conifer tree hosts, such as Liquidambar spp., Ulmus sp., and Cupressus sp.

The dominant species obtained in this study were G. luteobrunnea and G. pulverea, with 39 and 33 strains, respectively (Table 1). The reason for their abundance in our dataset is the fact that our study focused on sampling from Altinginaceae. Two species, G. putterillii and G. radiata, have only been isolated in Jiangxi (Table 1). The samples collected from Guangxi and Hunan only yielded G. pulverea.

Geosmithia putterillii was isolated from bark beetles feeding on plants from the family of Rosaceae (Kolařík et al., 2008) and Lauraceae in Europe (Kolařík et al., 2004) and on various families of angiosperms and gymnosperms in the Western United States (Kolařík et al., 2017). The type strain was isolated from timber in New Zealand (Pitt, 1979). In this study, G. putterillii was isolated from the gallery of Phloeosinus sp. on Lauraceae log (Jiangxi). This study is the first report of G. putterillii in China. It is becoming clear that G. putterillii is widely distributed globally, across many beetle hosts.

Another known species collected in this study is G. pallida, originally isolated from cotton yarn and soil (Kolařík et al., 2004). Later, it was found to be associated with beetles, such as ambrosia beetle Xylosandrus compactus (Vannini et al., 2017), and plants such as Brucea mollis (Deka and Jha, 2018). G. pallida was previously reported to induce dieback poisoning on coast live oak (Quercus agrifolia) by Lynch et al. (2014). Later, it was proved that the identification was incorrect, and the causal agent of this disease was confirmed to be Geosmithia sp. 41 (Kolařík et al., 2017). Two isolates were obtained from the gallery of Sinoxylon cf. cucumella on Acacia pennata in this study, which is the first report of G. pallida in China.

Most of G. luteobrunnea were isolated from the galleries of A. suncei (Table 1). Acanthotomicus suncei was recorded on Liquidambar in Fujian, Jiangsu, Jiangxi, Zhejiang, and Shanghai, China (Li et al., 2021). The hosts of this beetle were limited to sweet gum trees, such as L. styraciflua and L. formosana. The beetle was recorded as an agent of great damage to the imported American sweetgum L. styraciflua in Shanghai and neighboring Jiangsu Province (Gao and Cognato, 2018). The role of the fungus in this outbreak and the tree pathology remain uninvestigated, although the authors of this paper noted small lesions around the beetle galleries. The other five isolates were isolated from the galleries of S. jiulianshanensis on Ulmus sp., which suggests that G. luteobrunnea might colonize a wide range of tree hosts.

Geosmithia radiata was only isolated in samples from Jiangxi Province, from two plant families: Altinginaceae and Ulmaceae (Table 1). The colony of G. radiata is similar to G. luteobrunnea in morphology, but the difference can be seen in the micromorphology (Supplementary Figure S5). In addition, G. luteobrunnea can grow faster at 35°C, while G. radiata grows slower, and G. luteobrunnea could grow at 35°C, but G. radiata could not (Table 4).

Geosmithia brevistipitata and G. xelotolerans were isolated from the gallery of Phloeosinus cf. hopehi. This is not the first time that Geosmithia species were isolated from the gallery of Phloeosinus species. According to previous reports, G. flava, G. longdonii, G. putterillii, G. Lavandula, etc., are all related to Phloeosinus (Kolařík et al., 2017). It is now more certain that Phloeosinus and Geosmithia are closely related. Geosmithia xelotolerans is cosmopolite, known from the Mediterranean on many bark beetle species infecting Fabaceae, Moraceae, Oleaceae (Kolařík et al., 2007), in Western US on Cupressaceae, Pinaceae Fagaceae, Rosaceae (Kolařík et al., 2017), and in Eastern US on Cupressaceae, Fagaceae (Huang et al., 2017, 2019), and wall of the wall (Spain, Crous et al., 2018). Our study expanded the distribution range of G. xelotolerans.

Geosmithia bombycina was isolated from the gallery of C. eriobotryae on E. japonica. Cryphalus eriobotryae is one of the beetle pests that infest loquat (Zheng et al., 2019). This is the first study about the fungal association of this beetle.

Geosmithia granulata was isolated from the gallery of Sinoxylon cf. cucumella on Acacia pennata, Ernoporus japonicus on Hibiscus tiliaceus, and Scolytus semenovi on Ulmus sp. in this study. It was reported that it could be vectored by different beetle species which infested several plant hosts (Kolařík et al., 2007). In this study, we expanded the range of its beetle vectors and tree host.

Geosmithia pulverea is a species closely related to Geosmithia sp. 3 and Geosmithia sp. 23, which are known from various bark beetle hosts in Europe, United States, and Seychelles (Kolařík et al., 2007, 2008, 2017; Huang et al., 2017, 2019). Further study is needed to clarify the evolutionary relationship among these three lineages. In this study, we isolated G. pulverea from Aca. gracilipes, Alt. gracilipes, E. japonica, Gne. luofuense, L. formosana, L. styraciflua, Rhus chinensis, and Ulmus sp. (Table 1), which suggested that this species could colonize a very wide variety of plant hosts. It is also the most widely distributed species, isolated from Guangdong, Guangxi, Hunan, Jiangsu, Jiangxi, Shandong, and Shanghai (Table 1) and vectored by several beetle species, such as S. jiulianshanensis, A. suncei, C. emancipatus, C. kyotoensis, Dinoderus sp., Microperus sp., and Phloeosinus sp. (Table 1). Moreover, the abundance of Geosmithia species associated with A. suncei in the current study was also consistent with the frequent occurrence in Shanghai and Jiangxi (Gao et al., 2021).

In addition to G. pallida, Geosmithia pulverea, and Geosmithia fusca are the species found in the G. pallida species complex in this study. Only eight isolates of G. fusca were obtained from the gallery of Sinoxylon cf. cucumella on Acacia pennata. Two isolates of G. pallida, eight isolates of G. pulverea, and two isolates of G. granulata were also obtained from this beetle. Information about this beetle was very limited. As far as we know, it was found on Wendlandia tinctoria and distributed in the Himalayan mountain area and Burma, Thailand, Laos, and Vietnam (Liu, 2010; Liu and Beaver, 2018; Borowski, 2021). This is the first report in China, and this is the first study on its fungal associations.

Conclusion

This study does not provide sufficient data to determine the structure of the Geosmithia community in China, as was inferred in Europe and United States after a significantly greater sampling effort (Kolařík et al., 2007, 2008, 2017; Kolařík and Jankowiak, 2013; Jankowiak et al., 2014; Huang et al., 2017, 2019). Fungal communities are regulated by several factors, including geographic location, host tree species, and bark beetle vectors. Further sampling is needed to understand the determinants (Veselská et al., 2019). It is clear, however, that the diversity of China’s subcortical fungi is substantial. Fungal communities associated with trees need to be further investigated because many currently unknown species may cause plant diseases.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Author Contributions

RC, MD, and YL designed the research. YL, HS, and GZ collected the samples. XZ, RC, and YL isolated and purified the fungal cultures. XZ, RC, and XJ completed the data acquisition, analyses, and interpretation. XZ and RC completed the writing of the manuscript. MK, JH, and YL revised the text, taxonomy, and phylogeny. All authors approved the manuscript.

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Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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