Collection of samples and isolation of fungi
A total of 76 strains in the genus Geosmithia were isolated from 6 beetle species and their galleries. The 73 strains were from the galleries and three strains (SNM887, SNM886, SNM885) from the beetles. Sixty-three strains were from Jiangxi, nine from Shanghai, two from Guangxi, one from Guangdong and one from Hunan (Table 1).
Phylogenetic analysis
The preliminary classification was carried out by BLAST on NCBI GenBank using the ITS marker. Subsequently, 20 representative strains were selected for multi-gene phylogenetic analysis and 10 strains were screened for morphological studies (Table 2). Aligned sequences including gaps yielded 562 characters for ITS, 907 characters for TEF1-α, and 632 characters for TUB2. The best substitution model for ITS, TEF1-α and TUB2 was GTR + I + G. For all datasets (ITS, TUB2, TEF1-α), ML, MP and Bayesian inference produced nearly identical topologies, with slight variations in the statistical support for each of the individual sequence datasets. Phylograms obtained by ML are presented for all the individual datasets.
Taxonomy
Among the 76 strains obtained in this study, five species were identified. Four of these species are new to science, and are described as follows:
Geosmithia jiulianshanensis R. Chang & X. Zhang, sp. nov. (Fig. 4)
MycoBank MB839256
Etymology: jiulianshanensis, referring to the predominant beetle vector Scolytus jiulianshanensis.
Diagnosis
The stipe of G. jiulianshanensis is slightly thicker and shorter than that in other species. Geosmithia jiulianshanensis can grow at 5 and 35°C, even grow slowly at 37°C.
Type
CHINA, Jiangxi Province, Ganzhou City, Longnan county, Jiulianshan National Nature Reserve (24°34′1″N, 114°30′E), from gallery of Scolytus jiulianshanensis on Ulmus sp., 5 May, 2020, S. C. Lai, Y. Xu, S. Liao, Y. Wen & T. Li (HMAS 249919 - holotype, SNM261 = CGMCC3.20252 - ex-holotype culture).
Description
Sexual state not observed. Asexual state penicillium-like. Conidiophores borne mostly from aerial fungal hyphae, erect, determinate, solitary, sometimes funiculose, with all parts verrucose; base often consisting of 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 (19.0-) 29.6–61.5 (-85.0) µm long, biverticillate to quaterverticillate (penicilli of conidiophores on aerial funiculose mycelia are monoverticillate or biverticillate), symmetric or asymmetric, often irregularly branched, rami (1st branch) in whorls of X-Y, (4.1-) 5.2-7.0 (-8.7) × (1.2-) 1.7–2.5 (-3.2) µm, metulae (last branch) in whorls of X-Y, (4.0-)4.9–6.5 (-7.6) × (1.4-) 1.8–2.3 (-2.6) µm; phialides in whorls of X-Y,, cylindrical, without or with short cylindrical neck and smooth to verruculose 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 d: Colony diam 59–64 mm at 20°C, 65–78 mm at 25°C, and 66–70 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 d were appressed, 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; absence of exudate; no soluble pigment. When incubated at 35 ℃, colonies raised, slightly depressed at center, rugose or irregularly furrowed; margin undulate somewhat erose; aerial mycelia sparse to moderate; substratum mycelia dense, forming a tough basal felt; the colony is darker and yellowish brown; soluble pigment is brown. MEA, 37°C, 8 d, 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 formosana, G. jiulianshanensis and G. jiangxiensis are phylogenetically close to each other on ITS, TUB2 and TEF1-α trees. The colony morphology of G. formosana, G. jiulianshanensis and G. jiangxiensis are also similar, but there are many differences among those three species. First of all, their sequences are quite different (Table 3). And then, under the microscope, the morphological differences between them are more obvious. The spore of G. jiangxiensis is thicker than the other two species. The stipe of G. formosana is thinner and longer than other two species, the stipe of G. jiangxiensis is obviously thicker than the other two species, and the stipe of G. jiulianshanensis is slightly thicker and shorter than that of G. formosana. Moreover, their growths at different temperatures are also different (Table 4). Geosmithia formosana cannot grow at 5 and 35°C while G. jiulianshanensis can grow at both temperatures, especially at 35°C, even grow slowly at 37°C. Geosmithia jiangxiensis only grows a little at 5°C, and grows slowly at 35°C. The growth speed of G. jiulianshanensis is faster than other two species (Table 4).
Additional cultures examined
CHINA, Jiangxi Province, Ganzhou City, Longnan county (24°5′2.4″N, 114°47′2.4″E), from gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May, 2020, S. C. Lai (SNM260, SNM246).
CHINA, Jiangxi Province, Ganzhou City, Xunwu county (24°57′N, 115°38′2″E), from gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May, 2020 (SNM882).
CHINA, Shanghai, from gallery of Acanthotomicus suncei on Liquidambar styraciflua, April 2019, L. Gao (SNM210, SNM226, SNM285, SNM286, SNM287).
Geosmithia jiangxiensis R. Chang & X. Zhang, sp. nov. (Fig. 5)
MycoBank MB839257
Etymology: jiangxiensis, referring to the place where this species was isolated, Jiangxi Province.
Diagnosis
The spore and the stipe of G. jiangxiensis is thicker than close related species. Geosmithia jianxiensis only grows a little at 5 and 35°C.
Type
CHINA, Jiangxi Province, Ganzhou City, Longnan county (24°5′2.4″N, 114°47′2.4″E), from gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May, 2020, S. C. Lai (HMAS 249920 - holotype, SNM279 = CGMCC3.20253 - ex-holotype culture).
Description
Sexual state not observed. Asexual state penicillium-like. Conidiophores borne from 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 (22.6-) 35.6–85.7 (-119.3) µm long, with walls thick, septate; penicillus terminal, mostly biverticillate, rerely terverticillate, mostly symmetrical, rami (1st branch) in whorls of X-Y, (4.2-) 5.2–7.8 (-10.6) × (1.3-) 2.1–3.5 (-4.8) µm,; metulae (last branch) in whorls of X-Y, (2.6-) 3.9–5.8 (-7.3) × (1.3-) 1.7–2.6 (-3.3) µm. Phialides in whorls of X-Y, (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 verruculose 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 d: Colony diam 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 mm and 1–4 mm respectively. The optimal temperature for growth is 25°C. Colonies at 25°C, 8 d, plane, slightly raised centrally, velutinous, with slight overgrowth of aerial mycelium, with floccose and funiculose areas; substrate mycelium white, aerial mycelium hyaline; sporulation moderate to heavy, pale cream; vegetative mycelium hyaline; reverse lighter; soluble pigment and exudate absent. When incubated at 35 ℃, colonies rising, slightly sunken in the center, furrowed or irregularly fringed; the substratum hyphae was dense and formed a tough basal felt. The colony is dark and yellowish-brown. MEA, 37°C, 8 d: no growth.
Host: Liquidambar formosana, Ulmus sp.
Beetle vectors: Acanthotomicus suncei, Scolytus jiulianshanensis.
Distribution
Jiangxi
Notes
See comparisons between Geosmithia jiulianshanensis, G. jiangxiensis and G. formosana below the description of G. jiulianshanensis.
Additional cultures examined
CHINA, Jiangxi Province, Ganzhou City, Longnan county, Jiulianshan National Nature Reserve (24°34′1″N, 114°30′E), from gallery of Scolytus jiulianshanensis on Ulmus sp., 5 May, 2020, S. C. Lai, Y. Xu, S. Liao, Y. Wen & T. Li (SNM280).
CHINA, Jiangxi Province, Ganzhou City, Xunwu county (24°57′N, 115°38′2″E), from gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May, 2020 (SNM883, SNM884).
Geosmithia formosana R. Chang & X. Zhang, sp. nov. (Fig. 6)
MycoBank MB839258
Etymology: formosana, referring to the tree host of Liquidambar formosana where this species has been isolated.
Diagnosis
The stipe of G. formosana is thinner and longer than close related species. Geosmithia formosana cannot grow at 5 and 35°C.
Type
CHINA, Jiangxi Province, Ganzhou City, Longnan county (24°5′2.4″N, 114°47′2.4″E), from gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May, 2020, S. C. Lai (HMAS 249921 - holotype, SNM256 = CGMCC3.20254 - ex-holotype culture).
Description
Sexual state not observed. Asexual state penicillium-like. Conidiophores borne from substrate or aerial mycelium, erect, determinate, solitary, with all parts verrucose; base often consisting of curved and atypically branched cell, stipe (9.2-) 16.7–62.6 (-108.0) × (1.0-) 1.7-3.0 (-3.5) µm; penicillus (21.2-) 41.0-88.8 (-113.9) µm long, penicillate conidiophores (penicilli) terminal, biverticillate to quaterverticillate (penicilli of conidiophores on aerial funiculose mycelia are monoverticillate or biverticillate), symmetric or asymmetric, often irregularly branched, rami (1st branch) in whorls of X-Y, (5.1-) 5.7–7.8 (-9.6) × (1.3-) 1.6–2.5 (-3.9) µm, metulae (last branch) in whorls of X-Y, (4.4-)5.1–6.5 (-7.3) × (1.1-) 1.6–2.4 (-2.9) µm; phialides in whorls of X-Y, cylindrical, without or with short cylindrical neck and smooth to verruculose walls, (3.0-)4.7–6.9 (-8.1) × (1.1-) 1.5–2.4 (-3.2) µm. Conidia hyaline to subhyaline, smooth, narrowly cylindrical to ellipsoidal, (2.3-)2.7–3.7 (-4.4) × (0.8-) 1.2–1.8(-2.2) µm, produced in non-persistent chains. Substrate conidia absent.
MEA, 8 d: Colony diam 50–54 mm at 20°C, 58–64 mm at 25°C, and 44–52 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 mm and close to 0 mm, respectively. At 35℃, there was little or no growth. The optimal growth temperature is 25°C. Colonies at 25°C, 8 d, appressed, white velutinous or floccose with raised mycelial cords; colony margin smooth, filamentous, diffuse, pale yellow; aerial mycelium hyaline, sparse; substrate mycelium hyaline, sparse; conidiogenesis moderate; light yellow to brown; absence of exudate; no soluble pigment. MEA, 37°C, 8 d: no growth.
Host: Liquidambar formosana.
Beetle vectors: Acanthotomicus suncei
Distribution
Jiangxi
Notes
See comparisons between G. jiulianshanensis, G. jiangxiensis and G. formosana below the description of G. jiulianshanensis.
Geosmithia pulverea R. Chang & X. Zhang, sp. nov. (Fig. 7)
MycoBank MB839259
Etymology: pulverea, powdery in Latin. On MEA medium, G. pulverea has powdery sporulation.
Diagnosis: Geosmithia pulverea produces long spore chain while its close related species does not.
Type
CHINA, Guangdong Province, Shenzhen City (22°37′54″N, 114°27′16″E), from gallery 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. Conidiophores arising from substrate or aerial mycelium with all parts verrucose, 40–250 µm tall; 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 (17.5-) 30.9–84.3 (-120.1) µm long, biverticillate to quaterverticillate, symmetric or asymmetric, often irregularly branched, 2–3×, rarely more, rami (1st branch) in whorls of X-Y, (8.2-) 10.2–14.4 (-18.9) × (2.2-) 2.5–3.3 (-3.9) µm, metulae (last branch) in whorls of X-Y, (6.3-) 7.5–10.9 (-15.8) × (1.8-) 2.1–2.8 (-3.5) µm; phialides X-Y, cylindrical or ellipsoidal, without or with short cylindrical neck and smooth to verruculose 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 d: Colony diam 23–29 mm at 20°C, 30–37 mm at 25°C, and 31–36 mm at 30°C. No grow at 5℃. At 35℃, mycelia grew slowly. After 8 days of culture, the colony diameter was 1.5-4 mm, with yellow soluble pigment. The optimal growth temperature is 25–30℃. Colonies at 25°C, 8 d, plane with radial rows and slightly raised centrally, texture velutinous (powdery); sporulation abundant, spore mass Light brownish yellow to buff; reverse yellowish to slightly avellaneous brown; soluble pigment and exudate absent. When incubated at 35 ℃, the colonies are the same as above. MEA, 37°C, 8 d: no growth.
Host: Gnetum luofuense, Liquidambar formosana, L. styraciflua.
Beetle vectors: Acanthotomicus suncei, Crossotarsus emancipatus, Dinoderus sp., Microperus sp.
Distribution
Gungdong, Guangxi, Hunan, Jiangxi, 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 was clustered with Geosmithia sp. 3, and SNM886, SNM887 and SNM270 were clustered with Geosmithia sp. 23 (Fig. 1). However, in the trees with TUB2 and TEF1-α, these strains did not have clear subclassification (Fig. 2 and Fig. 3). It was consequently recognized, using multigene phylogeny, together with Geosmithia sp. 23, as a well-defined phylogenetic species inside the G. pallida species complex (Kolařík et al. 2017; Huang 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 smaller stipe size, but other features fit to 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 the Temperate Europe in case of Geosmithia sp. 3 (Kolařík et al. 2004, 2008; Strzałka et al. 2021) or seems to have global distribution and many bark beetle hosts across Temperate Europe (Strzalka et al. 2021), Mediterranean basin (Kolařík et al. 2007), Northern America (Kolařík et al. 2017; Huang et al. 2017, 2019) and Seychelles (Kolařík et al. 2017). The 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 (21°54′12″N, 107°54′14″E), from body surface of Crossotarsus emancipates, 27 March, 2018, Y. Li (SNM887, SNM886).
CHINA, Hunan Province, Changsha City (28°10′56″N, 112°55′41″E), from gallery of Microperus sp. L589, 15 July, 2019, Y. Li (SNM888).
CHINA, Jiangxi Province, Ganzhou City, Longnan county (24°5′2.4″N, 114°47′2.4″E), from gallery of Acanthotomicus suncei on Liquidambar formosana, 5 May, 2020, S. C. Lai (SNM270).
CHINA, Shanghai, from gallery of Acanthotomicus suncei on Liquidambar styraciflua, April 2019, L. Gao (SNM248).