Geosmithia Species Associated With Bark Beetles From Southern China, With the Description of Four 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 were reported from Taiwan, China. The aim of this study was to investigate the diversity of Geosmithia species in southern China. Field surveys in Guangdong, Guangxi, Hunan, Jiangxi and Shanghai yielded a total of 76 fungal isolates from six beetle species. Isolates were grouped based on morphology. The ITS, β-tubulin and elongation factor 1-α gene regions of representatives of each group were sequenced. Phylogenetic trees were constructed based on those sequences. In total ve species were identied, with one previously described species G. putterillii and four new species which were described as G. jiulianshanensis, G. jiangxiensis, G. formosana, and G. pulverea (Geosmithia sp. 3 and Geosmithia sp. 23) sp. nov., in this paper.


Introduction
Members of Geosmithia are widely distributed fungal associates of phloem-and xylem-feeding beetles (Kolařík et al. 2007(Kolařík et al. , 2017; Lin et al. 2016;Pitt 1979), 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 (Kolarík et al. 2004), seed-feeding beetles (Huang et  Geosmithia is similar to Penicillium and Paecilomyces in morphology, but it can be distinguished by the combination of stipe with or without curved basal cell, verrucous conidiophores (incl. phialide), cylindrical phialide shape with very short and cylindrical neck (collula) and by ellipsoidal or cylindrical conidia (except of globose conidia in G. eupagioceri and G. microcorthyli). Colony color could be in shades of white, yellow, brown or red, but newer bluish green or green (Kolarík et al. 2004; Kolařík and Kirkendall 2010).
The spores of Geosmithia may be transmitted by attaching to the surface of beetle vector, but the ecological role of most Geosmithia species in symbiosis with bark beetles is still unclear. Some species serve as a main food source or supplementary nutrition for the beetles ( 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 (WTB, 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 ( ). TCD has affected many walnut trees in North America, especially in the western United province, China. Isolates linked to type specimens of the fungal species were deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China. Holotype specimens (dry cultures) were deposited in the Herbarium Mycologicum, Academiae Sinicae (HMAS), Beijing, China.
DNA extraction, ampli cation, 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). Samples were vortexed after incubated at 100 ℃ for 10 min and then centrifuged at 5000 rpm for 5 min. The supernatant was transferred to a new Eppendorf tube and used as template for polymerase chain reaction (PCR) ampli cation.

DNA sequence analyses
The sequences obtained using the forward and reverse primers were aligned in Geneious version 10.2.2 (Biomatters, Auckland, New Zealand). Reference sequences of Geosmithia species were retrieved from GenBank ( Morphological study Morphological characters were observed and recorded using the Olympus BX61 microscope (Olympus Corporation, Japan). The images were analyzed using ImageJ (https://imagej.net/). 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).

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

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 classi cation 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, ve species were identi ed. Four of these species are new to science, and are described as follows: Geosmithia jiulianshanensis R. Chang & X. Zhang, sp. nov. (Fig. 4) 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. occose with raised mycelial cords; colony margin smooth, lamentous, 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.

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). 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.

Discussion
A total of 76 strains of Geosmithia were isolated in this study. Analyses of ITS, TUB2 and TEF1-α showed those isolates were separated into ve taxa, with one of these strains has been named in previous, G. putterillii, and the other four were novel species, described as G. jiulianshanensis, G. jiangxiensis, G. formosana and G. pulverea in this study. Those species were isolated from larvae, frass and wood dust in beetle galleries of dying, stressed or weakened broad-leaf tree host, such as Liquidambar spp. and Ulmus sp.
The dominant species obtained in this study were G. jiulianshanensis and G. pulverea, with 38 and 18 strains respectively ( Table 1). The reason for their abundance in our dataset is the fact that our study focused on sampling from Altinginaceae; it does not mean that the fungus is dominant in other tree taxa.
Four species, G. putterillii, G. jiangxiensis and G. formosana have only been isolated in Jiangxi ( Table 1). The samples collected from Guangdong, Guangxi and Hunan only yielded G. pulverea.
Geosmithia putterillii was isolated from bark beetles feeding on plants from the family of Rossaceae (Kolařík et al. 2008) and Lauraceae in Europe (Kolařík et al. 2004) and on various families of Angiosperms and Gymnosperms in the Western U.S. (Kolařík et al. 2017). The type strain was isolated from the timber in the New Zealand (Pitt 1979). In this study, G. putterillii was isolated from gallery of Phloeosinus sp. on Lauraceae sp. log (Jiangxi). This study is the rst report of G. putterillii in China. It is becoming clear that G. putterillii is widely distributed globally, across many beetle hosts.
Most of G. jiulianshanensis were isolated from the galleries of A. suncei (Table 1). Acanthotomicus suncei was recorded on Liquidambar in Fujian, Jiangsu, Jiangxi, Zhejiang, and Shanghai, China ). The hosts of this beetle were limited to sweet gum trees, such as L. styraci ua and L. formosana. The beetle was recorded as an agent of great damage to the imported American sweetgum L. styraci ua in Shanghai and neighbouring Jiangsu province (Gao and Cognato 2018). The role of the fungus in this outbreak and in the tree pathology remains uninvestigated, though the authors of this paper noted small lesions around the beetle galleries. The other ve isolates were isolated from the galleries of Scolytus jiulianshanensis on Ulmus sp, which suggests that G. jiulianshanensis might colonize wide range tree hosts.
Geosmithia jiangxiensis was only isolated in samples from Jiangxi province, from two plant families: Altinginaceae and Ulmaceae ( Table 1). The colony of G. jiangxiensis is similar to G. jiulianshanensis in morphology, but the difference can be seen in the growth rate and micromorphology.
Geosmithia pulverea, is a species closely related to Geosmithia sp. 3 and Geosmithia sp. 23 which are know from various bark beetle hosts in Europa, USA and Seychelles (Kolařík et al. 2007(Kolařík et al. , 2008(Kolařík et al. , 2017Huang et al. 2017Huang et al. , 2019, and further study need to clarify among these three lineages. In this study, we isolated G. pulverea from A. gracilipes, Gne. luofuense, L. formosana 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, Jiangxi, and Shanghai (Table 1) and vectored by several beetle species, such as, S. jiulianshanensis, A. suncei, C. emancipatus, Dinoderus sp. Microperus sp. and Phloeosinus sp. (Table 1). Moreover, the abundant of Geosmithia species associated with Acanthotomicus suncei in the current study was also consistent with the frequent occurrence in Shanghai and Jiangxi ).

Conclusions
This study does not provide su cient data to determine the structure of the Geosmithia community in southern China, as was inferred in Europe and USA after a signi cantly greater sampling effort (Kolařík et al. 2007(Kolařík et al. , 2008(Kolařík et al. , 2013 Availability of data and materials The datasets generated for this study (