Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2022.841604 Microbiology Original Research Species Diversity and Virulence Potential of the Beauveria bassiana Complex and Beauveria scarabaeidicola Complex Wang Yao 1 2 Fan Qi 1 2 Wang Dong 1 2 Zou Wei-Qiu 1 2 Tang De-Xiang 1 2 Hongthong Preeyanat 3 Yu Hong 1 2 * 1Yunnan Herbal Laboratory, School of Ecology and Environmental Science, Yunnan University, Kunming, China 2The International Joint Research Center for Sustainable Utilization of Cordyceps Bioresources in China and Southeast Asia, Yunnan University, Kunming, China 3Faculty of Agricultural Technology, Rajamangala University of Technology, Thanyaburi, Thailand

Edited by: Dimitrios Georgios Karpouzas, University of Thessaly, Greece

Reviewed by: Andreas Leclerque, Darmstadt University of Technology, Germany; Samantha Chandranath Karunarathna, Qujing Normal University, China

 *Correspondence: Hong Yu, hongyu@ynu.edu.cn

This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology

 04 03 2022 2022 13 841604 22 12 2021 14 02 2022 Copyright © 2022 Wang, Fan, Wang, Zou, Tang, Hongthong and Yu. 2022 Wang, Fan, Wang, Zou, Tang, Hongthong and Yu

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Beauveria is a very important fungal resource. Some Beauveria species have great economic and ecological value. Through surveying Beauveria in China and Thailand over the past 4 years, 15 Beauveria spp. were collected and identified. Three new species—namely, B. polyrhachicola, B. songmingensis, and B. subscarabaeidicola—were described and illustrated based on morphological characteristics and molecular data. The phylogenetic positions of the 15 species were evaluated according to phylogenetic inferences based on six loci (nrSSU, nrLSU, TEF, RPB1, RPB2, and Bloc). Nine species of Beauveria in our study were isolated from adult scarab beetles (Coleoptera: Scarabaeidae). The pathogenicity of the isolates from the B. bassiana complex and B. scarabaeidicola complex was determined with three bioassays using B. mori and T. molitor larvae, in addition to Protaetia brevitarsis adults. The results indicated that the B. bassiana complex isolates had great potential in the biocontrol of the three insects; by contrast, the B. scarabaeidicola complex isolates showed obvious host specificity and low virulence.

 Taxonomy Beauveria phylogenetic analyses new species biological control 31870017 31760011 32160005 2018FY001(-006) National Natural Science Foundation of China10.13039/501100001809 Yunnan Provincial Science and Technology Department10.13039/501100008871 Introduction

Beauveria is a very important fungal resource, with some species having great economic and ecological value (Zimmermann, 2007; Rehner et al., 2011; Wang Y. et al., 2020). Beauveria bassiana (Bals.-Criv.) Vuill. and B. brongniartii (Sacc.) Petch are well-known environmentally safe alternatives to using chemical pesticides to control agricultural pests (Zimmermann, 2007; Rehner et al., 2011). Beauveria pseudobassiana S.A. Rehner and Humber has also been shown to have great potential in the biocontrol of numerous insect pests (Wang Y. et al., 2020). The entomopathogenic fungi Beauveria spp. are a class of environmentally friendly fungal pathogens that play an important role in controlling insect populations in nature (Luo et al., 2018; McKinnon et al., 2018). Some Beauveria species, as endophytes or soil and rhizosphere inhabitants, have been considered for potential use as biocontrol agents against plant pathogens by concerned practitioners, such as agriculturalists and plant pathologists. These species can produce an array of bioactive metabolites that limit the growth of some fungal plant pathogens and induce plant systemic resistance against the pathogenic bacterium (Ownley et al., 2010).

Beauveria bassiana is the most widely used fungus available commercially for controlling agricultural and forestry pests (Li et al., 2011). Products based on this species have been developed in many countries around the world (Goettel et al., 2005; Faria and Wraight, 2007; Li et al., 2011). However, a growing body of molecular evidence has demonstrated that B. bassiana, originally known as a generalist with a global distribution, encompasses cryptic lineages adapted to specific hosts or ecologies (Li et al., 2011; Rehner et al., 2011). Many initially identified B. bassiana isolates may belong to any of the species in the B. bassiana complex, such as B. rudraprayagi Y. Agrawal, Mual and Shenoy, B. staphylinidicola (Kobayasi and Shimizu) B. Shrestha, Kepler and Spatafora, and B. peruviensis D.E. Bustamante, M.S. Calderon, M. Oliva, and S. Leiva (Rehner et al., 2011; Agrawal et al., 2014; Kepler et al., 2017; Bustamante et al., 2019). Therefore, the abovementioned mycoinsecticide formulations of B. bassiana are not likely all based on B. bassiana.

Beauveria scarabaeidicola (Kobayasi) S.A. Rehner and Kepler is widely distributed in Oceania and Asia and named after its host adult beetle (Coleoptera: Scarabaeidae). It was originally described as Cordyceps scarabaeicola occurring in its sexual morph on an adult scarab beetle in New Guinea (Kobayasi and Shimizu, 1976). Cordyceps scarabaeicola has also been reported occasionally from many Asian countries, including China, Japan, and Korea (Shrestha et al., 2014). In an important phylogenetic study of Beauveria, a new entomopathogenic species, B. sungii S.A. Rehner and R.A. Humber, was described as a scarab-killing pathogen (hosts of all B. sungii isolates were identified as scarabs) (Rehner et al., 2011). Later, however, Shrestha et al. (2014) demonstrated that the telemorphic stage of B. sungii was C. scarabaeicola based on morphological and phylogenetic evidence. Because C. scarabaeicola was described earlier than B. sungii, Kepler et al. (2017) recommended B. scarabaeicola as the name of this species. Recently, Chen et al. (2019) proposed a new species, B. yunnanensis, a Chinese species parasitic on Lepidoptera pupa buried in soil that was a sister lineage to B. scarabaeicola.

During surveys of entomopathogenic fungi from different regions in Yunnan Province, China, and Chiang Rai Province, Thailand, over the past 4 years, approximately 15 Beauveria spp. were found and identified (Table 1). In this study, we aimed to: (1) reveal the hidden species diversity of the B. bassiana complex and B. scarabaeidicola complex based on phylogenetic analyses and morphological observation and (2) assess the biocontrol potential of species in the B. bassiana complex and B. scarabaeidicola complex through pathological tests on the lepidopteran Bombyx mori and the coleopteran Tenebrio molitor larvae as well as Protaetia brevitarsis adults.

Specimen information and GenBank accession numbers for sequences used in this study.

Taxon Voucher information Host/substrate GenBank accession number References
TEF RPB1 RPB2 Bloc
Cordyceps cicadae RCEF HP090724-31 Hemiptera: Cicadidae MF416496 MF416653 MF416447 Kepler et al., 2017
Cordyceps tenuipes ARSEF 5135 Lepidopteran pupa JF416020 JN049896 JF416000 Kepler et al., 2012
Beauveria acridophila HUA 179219T Orthoptera: Acrididae JQ958613 JX003857 JX003841 Sanjuan et al., 2014
Beauveria acridophila HUA 179220 Orthoptera: Acrididae JQ958614 JX003852 JX003842 Sanjuan et al., 2014
Beauveria amorpha ARSEF 2641T Hymenoptera: Formicidae AY531917 HQ880880 HQ880952 HQ880739 Rehner et al., 2011
Beauveria araneola GZAC 150317T Araneae KT961699 KT961701 KT961698 Chen et al., 2017
Beauveria asiatica ARSEF 4850T Coleoptera: Cerambycidae AY531937 HQ880859 HQ880931 HQ880718 Rehner et al., 2011
Beauveria asiatica YFCC 5600 Coleoptera: Cerambycidae MN576996 MN576886 MN576940 MW168177 Wang Y. B. et al., 2020; This study
Beauveria australis ARSEF 4598T Soil HQ880995 HQ880861 HQ880933 HQ880720 Rehner et al., 2011
Beauveria baoshanensis CCTCC AF 2018011T Coleoptera: Chrysomelidae MG642897 MG642854 MG642867 Chen et al., 2019
Beauveria bassiana ARSEF 1564T Lepidoptera: Arctiidae HQ880974 HQ880833 HQ880905 HQ880692 Rehner et al., 2011
Beauveria bassiana ARSEF 7518 Hymenoptera: Pamphiliidae HQ880975 HQ880834 HQ880906 HQ880693 Rehner et al., 2011
Beauveria bassiana YFCC 3369 Coleoptera: Scarabaeidae MN576994 MN576884 MN576938 MW168176 Wang Y. B. et al., 2020; This study
Beauveria blattidicola MCA 1727T Blattodea: Blattidae MF416483 MF416640 Kepler et al., 2017
Beauveria blattidicola MCA 1814 Blattodea: Blattidae MF416484 MF416641 Kepler et al., 2017
Beauveria brongniartii ARSEF 617T Coleoptera: Scarabaeidae HQ880991 HQ880854 HQ880926 HQ880713 Rehner et al., 2011
Beauveria brongniartii YFCC 3240 Coleoptera: Scarabaeidae MN576995 MN576885 MN576939 MW168175 Wang Y. B. et al., 2020; This study
Beauveria caledonica ARSEF 2567T Soil EF469057 HQ880889 HQ880961 HQ880748 Rehner et al., 2011
Beauveria caledonica YFCC 7025 Coleoptera: Cerambycidae MN576997 MN576887 MN576941 MW168178 Wang Y. B. et al., 2020; This study
Beauveria diapheromeriphila QCNE 186272T Phasmatodea: Diapheromeridae JQ958610 JX003848 Sanjuan et al., 2014
Beauveria diapheromeriphila QCNE 186714 Phasmatodea: Diapheromeridae MF416491 MF416648 Kepler et al., 2017
Beauveria hoplocheli Bt116 Coleoptera: Melolonthidae KC339703 KM453957 KM453966 KM453967 Robène-Soustrade et al., 2015
Beauveria hoplocheli MNHN-RF-06107T Coleoptera: Melolonthidae KC339702 KM453954 KM453963 KM453971 Robène-Soustrade et al., 2015
Beauveria kipukae ARSEF 7032T Homoptera: Delphacidae HQ881005 HQ880875 HQ880947 HQ880734 Rehner et al., 2011
Beauveria lii ARSEF 11741T Coleoptera: Coccinellidae JN689371 JN689374 JN689370 JN689373 Zhang et al., 2012
Beauveria locustiphila TS881 Orthoptera: Romaleidae JQ958619 JX003847 JX003845 Sanjuan et al., 2014
Beauveria majiangensis GZAC GZU12141T Coleoptera: Scarabaeoidea MG052640 MG052644 MG052639 Chen et al., 2018
Beauveria majiangensis YFCC 852 Coleoptera: Scarabaeidae MW168229 MW168195 MW168212 MW168179 This study
Beauveria malawiensis ARSEF 7760T Coleoptera: Cerambycidae DQ376246 HQ880897 HQ880969 HQ880756 Rehner et al., 2011
Beauveria malawiensis YFCC 853 Coleoptera: Scarabaeidae MW168230 MW168196 MW168213 MW168180 This study
Beauveria medogensis 2898 Soil KU994833 KU994835 KU994834 KU994836 Imoulan et al., 2016
Beauveria medogensis YFCC 854 Coleopteran adult MW168231 MW168197 MW168214 MW168181 This study
Beauveria peruviensis UTRP19 = ARSEF 14196T Coleoptera: Curculionidae MN094781 MN100118 MN094757 Bustamante et al., 2019
Beauveria peruviensis UTRF35 Coleoptera: Curculionidae MN094771 MN100115 MN094755 Bustamante et al., 2019
Beauveria polyrhachicola YFCC 859T Hymenoptera: Formicidae MW168236 MW168202 MW168219 MW168184 This study
Beauveria polyrhachicola YFCC 867 Hymenoptera: Formicidae OM373098 OM373099 OM304364 OM373100 This study
Beauveria pseudobassiana ARSEF 3405T Lepidoptera: Tortricidae AY531931 HQ880864 HQ880936 HQ880723 Rehner et al., 2011
Beauveria pseudobassiana YFCC 1806007 Coleoptera: Scarabaeidae MN523553 MN523582 MN523611 MN868289 Wang Y. et al., 2020
Beauveria rudraprayagi MTCC 8017T Lepidoptera: Bombycidae JQ990914 JQ990892 JQ990870 JQ990848 Agrawal et al., 2014
Beauveria rudraprayagi YFCC 858 Lepidopteran larva MW168235 MW168201 MW168218 MW168183 This study
Beauveria scarabaeidicola ARSEF 1685 Coleoptera: Scarabaeidae AY531899 HQ880881 HQ880953 HQ880740 Rehner et al., 2011
Beauveria scarabaeidicola ARSEF 5689 Coleoptera: Scarabaeidae DQ522335 DQ522380 DQ522431 HQ880741 Rehner et al., 2011
Beauveria scarabaeidicola ARSEF 7043 Coleoptera: Scarabaeidae AY531948 HQ880883 HQ880955 HQ880742 Rehner et al., 2011
Beauveria scarabaeidicola ARSEF 7279 Coleoptera: Scarabaeidae HQ881009 HQ880885 HQ880957 HQ880744 Rehner et al., 2011
Beauveria scarabaeidicola ARSEF 7281 Coleoptera: Scarabaeidae HQ881011 HQ880887 HQ880959 HQ880746 Rehner et al., 2011
Beauveria scarabaeidicola YFCC 865 Coleoptera: Scarabaeidae MW168243 MW168209 MW168226 MW168191 This study
Beauveria sinensis BUB 504 Orthoptera: Grylloidea MG642895 MG642852 MG642865 Chen et al., 2019
Beauveria sinensis RCEF 3903T Lepidoptera: Geometridae HQ270151 JX524283 JX524284 Chen et al., 2013
Beauveria songmingensis YFCC 860T Coleoptera: Scarabaeidae MW168238 MW168204 MW168221 MW168186 This study
Beauveria songmingensis YFCC 861 Coleoptera: Scarabaeidae MW168239 MW168205 MW168222 MW168187 This study
Beauveria staphylinidicola ARSEF 5718 Coleoptera: Staphylinidae EF468776 EF468881 AY883807 Sung et al., 2007
Beauveria staphylinidicola YFCC 855 Coleoptera: Cerambycidae MW168232 MW168198 MW168215 MW168182 This study
Beauveria subscarabaeidicola YFCC 863T Coleoptera: Scarabaeidae MW168241 MW168207 MW168224 MW168189 This study
Beauveria subscarabaeidicola YFCC 864 Coleoptera: Scarabaeidae MW168242 MW168208 MW168225 MW168190 This study
Beauveria varroae ARSEF 8257T Coleoptera: Curculionidae HQ881002 HQ880872 HQ880944 HQ880731 Rehner et al., 2011
Beauveria vermiconia ARSEF 2922T Soil AY531920 HQ880894 HQ880966 HQ880753 Rehner et al., 2011
Beauveria yunnanensis CCTCC AF 2018010T Lepidopteran pupa MG642900 MG642857 MG642870 Chen et al., 2019
Beauveria yunnanensis YFCC 3105 Coleoptera: Scarabaeidae MN576999 MN576889 MN576943 Wang Y. B. et al., 2020
Beauveria yunnanensis YFCC 862 Coleoptera: Scarabaeidae MW168240 MW168206 MW168223 MW168188 This study

Boldface: data generated in this study. Tex-type material.

 Materials and Methods Soil and Specimen Collection

All the soil samples and the majority of Beauveria specimens were collected from Yunnan Province in China. Some specimens were collected from Chiang Rai Province in Thailand. Soil samples and specimens were noted and photographed in the fields, and then carefully placed in plastic containers at low temperature. Afterward, they were carried to the laboratory and stored at 4°C before examination and isolation.

 Fungal Isolation and Culture

Beauveria strains were isolated from the soil samples using the Tenebrio molitor baiting method (Keyser et al., 2015). Conidia developing on insect cadavers were transplanted onto plates of potato dextrose agar (PDA; potato 200 g/L, dextrose 20 g/L, agar 20 g/L) and cultured at 25°C. Teleomorph specimens were rinsed with tap water, washed with sterile distilled water, and then dried on sterile filter paper. To obtain axenic cultures, white tissue inside the sclerotia of the teleomorph specimens was removed and inoculated onto PDA plates using a sterilized dissecting knife. Colonies of the isolated filamentous fungi appearing in the culture were transferred onto fresh PDA media. The purified fungal strains were maintained in a culture room at 25°C or transferred to PDA slants and stored at 4°C. Specimens were deposited in the Yunnan Herbal Herbarium (YHH) at the Institute of Herb Biotic Resources of Yunnan University. Cultures were stored in the Yunnan Fungal Culture Collection (YFCC) at the Institute of Herb Biotic Resources of Yunnan University.

 Morphological Observations

Specimens were examined using an Olympus SZ61 stereomicroscope (Olympus Corporation, Tokyo, Japan). Cultures on PDA slants were transferred to PDA plates and then incubated at 25°C for 14 days. For morphological evaluation, microscope slides were prepared by placing mycelia from the cultures on PDA medium blocks (5 mm diameter) and then overlaid with a coverslip. Medan dye solution was used to observe asci and ascospores. Other structures were mounted in water. Micro-morphological observations and measurements were performed using a light microscope (CX40, Olympus Corporation, Tokyo, Japan) and a scanning electron microscope (Quanta 200 FEG, FEI Company, Hillsboro, United States). Length to width ratios are given as Q. Mean values for length, width, and Q are indicated by Lm, Wm, and Qm, respectively.

 DNA Extraction, PCR and Sequencing

Specimens and axenic living cultures were prepared for DNA extraction. Genomic DNA was extracted using the Genomic DNA Purification Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The primer pair nrSSU-CoF and nrSSU-CoR was used to amplify a fraction of the nuclear ribosomal small subunit (nrSSU) (Wang et al., 2015). Primer pair LR5 and LR0R (Vilgalys and Hester, 1990; Rehner and Samuels, 1994) was used to amplify a fraction of the nuclear ribosomal large subunit (nrLSU) and EF1α-EF and EF1α-ER (Bischoff et al., 2006; Sung et al., 2007) for the translation elongation factor 1α (TEF). For amplification of the largest and second largest subunits of the RNA polymerase II (RPB1 and RPB2), PCR primer pairs RPB1-5′F/RPB1-5′R and RPB2-5′F/RPB2-5′R (Bischoff et al., 2006; Sung et al., 2007) were employed. The Bloc fragment was amplified using primer pair B5.1F/B3.1R (Rehner et al., 2006). All the PCR reactions were performed in a final volume of 50 μL and contained 25 μL of 2 × Taq PCR Master Mix (Tiangen Biotech Co. Ltd, Beijing, China), 0.5 μL of each primer (10 μM), 1 μL of genomic DNA, and 23 μL of RNase-free water. Target gene amplification and sequencing were performed according to the methods described in our previous study (Wang Y. B. et al., 2020).

 Phylogenetic Analyses

Phylogenetic analyses were based on six gene (nrSSU, nrLSU, TEF, RPB1, RPB2, and Bloc) sequences. The sequences were retrieved from GenBank and combined with those generated in our study. Taxon information and GenBank accession numbers were provided in Supplementary Table 1 and Table 1. Sequences were aligned using MAFFT v.7.1 After alignment, the sequences of the genes were concatenated. Conflicts among the six genes were tested using PAUP* 4.0b10 (Swofford, 2002). The results revealed that the phylogenetic signals in the six genes were not in conflict. The data partitions were defined for the combined dataset using PartitionFinder V1.1.1 (Lanfear et al., 2012). Phylogenetic analyses were conducted using BI and ML methods employing MrBayes v3.1.2 and RaxML 7.0.3, respectively (Ronquist and Huelsenbeck, 2003; Stamatakis et al., 2008). The BI analysis was run on MrBayes v3.1.2 for five million generations using a GTR+G+I model determined by jModelTest version 2.1.4 (Darriba et al., 2012). GTR+I was selected as the optimal model for ML analysis, and 1,000 rapid bootstrap replicates were performed on the dataset.

The first analysis based on the combined five-gene (nrSSU+nrLSU+TEF+RPB1+RPB2) dataset was performed using the following taxa: Akanthomyces, Amphichorda, Ascopolyporus, Beauveria, Blackwellomyces, Cordyceps, Gibellula, Hevansia, Samsoniella, and Simplicillium. Two taxa of Trichoderma were designated as outgroups. The second analysis based on the combined four-gene (TEF+RPB1+RPB2+Bloc) sequences was performed using Beauveria taxa.

We applied a (phylo-) genetic distance matrix calculation for the combined four-gene (TEF+RPB1+RPB2+Bloc) sequences to assess species boundaries in the B. bassiana complex and B. scarabaeidicola complex (Table 2). The pairwise genetic distances of most Beauveria lineages (Supplementary Table 2) were measured based on the Kimura 2-parameter model using MEGA6 software (Tamura et al., 2013).

Genetic distance (p-distances) of species in the B. bassiana complex and B. scarabaeidicola complex.

Group Taxa Marker
TEF+RPB1+ RPB2+Bloc
The B. bassiana complex B. bassiana—B. peruviensis 0.015
B. bassiana—B. polyrhachicola 0.019
B. bassiana—B. rudraprayagi 0.042
B. bassiana—B. staphylinidicola 0.010
B. peruviensis—B. polyrhachicola 0.011
B. peruviensis—B. rudraprayagi 0.044
B. peruviensis—B. staphylinidicola 0.015
B. polyrhachicola—B. rudraprayagi 0.045
B. polyrhachicola—B. staphylinidicola 0.019
B. rudraprayagi—B. staphylinidicola 0.045
The B. scarabaeidicola complex B. scarabaeidicola—B. songmingensis 0.013
B. scarabaeidicola—B. subscarabaeidicola 0.017
B. scarabaeidicola—B. yunnanensis 0.014
B. songmingensis—B. subscarabaeidicola 0.012
B. songmingensis—B. yunnanensis 0.013
B. subscarabaeidicola—B. yunnanensis 0.013
Conidial Viability of <italic>Beauveria</italic> spp. Isolates

A total of 19 Beauveria spp. isolates (Table 3) were analyzed for their conidial viability using the method described by Imoulan et al. (2011). The conidial viability of each isolate was confirmed by inoculating three tubes of 3 ml PDB media (potato 200 g/L, dextrose 20 g/L) with 0.1 ml of conidia suspension (3 × 106 conidia/ml). Only isolates with conidial viability greater than 65% were tested for pathogenicity toward B. mori, T. molitor, and P. brevitarsis.

Conidial viability of Beauveria spp. isolates used in this study.

Group Species and isolate Host/substrate Location Conidial viability ± SE (%)#
The B. bassiana complex B. bassiana
YFCC 841 Lepidopteran larva Yunnan province, China 86.33 ± 3.48bc
YFCC 842 Hymenoptera: Vespidae Yunnan province, China 50.86 ± 0.41
YFCC 843 Lepidoptera: Geometridae Yunnan province, China 36.80 ± 4.72
YFCC 844 Soil Yunnan Province, China 95.00 ± 1.53a
YFCC 3369 Coleoptera: Scarabaeidae Yunnan province, China 84.67 ± 3.76bc
B. polyrhachicola
YFCC 859 Hymenoptera: Formicidae Chiang Rai Province, Thailand 89.33 ± 0.67ab
B. rudraprayagi
YFCC 858 Lepidopteran larva Chiang Rai province, Thailand 91.67 ± 1.20ab
B. staphylinidicola
YFCC 845 Coleoptera: Staphylinidae Yunnan province, China 25.31 ± 0.69
YFCC 855 Coleoptera: Cerambycidae Yunnan province, China 81.33 ± 2.33c
The B. scarabaeidicola complex B. scarabaeidicola
YFCC 846 Coleoptera: Scarabaeidae Yunnan province, China 0
YFCC 847 Coleoptera: Scarabaeidae Chiang Rai Province, Thailand 65.67 ± 1.20d
YFCC 865 Coleoptera: Scarabaeidae Yunnan province, China 69.00 ± 2.65d
B. songmingensis
YFCC 848 Coleoptera: Scarabaeidae Yunnan Province, China 42.33 ± 0.67
YFCC 860 Coleoptera: Scarabaeidae Yunnan province, China 68.00 ± 3.79d
YFCC 861 Coleoptera: Scarabaeidae Yunnan province, China 0
B. subscarabaeidicola
YFCC 863 Coleoptera: Scarabaeidae Yunnan province, China 18.69 ± 3.25
YFCC 864 Coleoptera: Scarabaeidae Yunnan province, China 14.33 ± 2.33
B. yunnanensis
YFCC 862 Coleoptera: Scarabaeidae Yunnan province, China 69.00 ± 3.46d
YFCC 3105 Coleoptera: Scarabaeidae Yunnan province, China 46.67 ± 1.20

#Only isolates with % of conidial viability ≥ 65% were significance tested. Different lowercase letters in the same column indicate significant differences at 5% level.

 Virulence Assay of <italic>Beauveria</italic> spp. Isolates

A total of 10 Beauveria spp. isolates from the B. bassiana complex and B. scarabaeidicola complex were tested for their pathogenicity to B. mori and T. molitor larvae in addition to P. brevitarsis adults. Conidia for each isolate were obtained from 4-week-old cultures grown on malt extract agar plates, suspended in a sterile aqueous solution of 0.01% Tween 80, and mixed vigorously until homogeneous conidial suspensions were produced. Quantification of the conidia was performed using a hemocytometer under a light microscope at 400 × magnification. All of the suspensions were adjusted to 1 × 108 conidia/ml. The tested insects were individually placed in sterilized rearing boxes and 10 μl of conidial suspension was applied to the surface of each insect. A diet was provided for each insect and renewed as needed. Control groups were treated with the same volume of a sterile aqueous solution of 0.01% Tween 80. The test was replicated three times with 50 insects per replicate. All of the test groups were kept at 25°C under a 12:12 h photoperiod cycle. The numbers of dead insects were recorded every 12 h for a 30 day period, which was used to determine the percentage of mortality. Cadavers were removed, immediately surface-disinfected, and individually placed and maintained in rearing box chambers. Mycelium samples from cadavers were aseptically removed and cultured on PDA for microscope examination, DNA extraction, and TEF sequencing to confirm that mortality was caused by the inoculated fungal strain.

 Results Sequencing and Phylogenetic Analyses

The combined five-gene dataset included sequences from 123 fungal taxa. The final dataset consisted of 5,001 bp of sequence data (nrSSU 1,138 bp, nrLSU 910 bp, TEF 1,047 bp, RPB1 781 bp, and RPB2 1,125 bp). Eleven well-supported clades were recognized based on both Bayesian inference (BI) and maximum likelihood (ML) analyses of the combined five-gene dataset of 123 taxa from Cordycipitaceae and Trichoderma, which accommodate species of the genera Akanthomyces, Amphichorda, Ascopolyporus, Beauveria, Blackwellomyces, Cordyceps, Gibellula, Hevansia, Samsoniella, Simplicillium, and Trichoderma (Supplementary Figure 1). The phylogenetic analyses also revealed the species diversity of the B. bassiana complex and B. scarabaeidicola complex in Beauveria clades. This suggested that the groups composed of the B. bassiana complex and B. scarabaeidicola complex should be genetically composed of at least four species (Supplementary Figure 1). Phylogenetic analyses based on combined partial TEF+RPB1+RPB2+Bloc sequences consisting of 59 fungal taxa resolved most Beauveria lineages in separate terminal branches (Figure 1). This revealed a similar tree and clustering topology, as shown in Supplementary Figure 1. It was proposed that the two strains YFCC 859 and YFCC 867, which formed a strongly supported clade, might be a new species in the B. bassiana complex, named B. polyrhachicola (Figure 1). Our analyses also revealed that two newly discovered species, B. songmingensis and B. subscarabaeidicola, were phylogenetically clustered with B. yunnanensis and B. scarabaeidicola, but they were clearly distinguished from the latter two by forming two separate clades in the B. scarabaeidicola complex (Figure 1 and Supplementary Figure 1). The genetic divergence comparisons showed that the minimum threshold (p-distance) to distinguish genetic species in Beauveria was 0.010 for the combined four-gene (TEF+RPB1+RPB2+Bloc) sequences, as occurred between B. araneola and B. medogensis, between B. asiatica and B. majiangensis, between B. australis and B. brongniartii, and between B. bassiana and B. staphylinidicola (Table 2 and Supplementary Table 2). These results also suggested that: (1) the B. bassiana complex, which is made up of five species, should include B. bassiana, B. peruviensis, B. polyrhachicola, B. rudraprayagi, and B. staphylinidicola and (2) the B. scarabaeidicola complex should be composed of four species, including B. scarabaeidicola, B. songmingensis, B. subscarabaeidicola, and B. yunnanensis.

Phylogenetic analysis of Beauveria species based on combined partial TEF+RPB1+RPB2+Bloc sequences. Statistical support values (≥ 0.5/50%) are shown at the nodes for Bayesian inference (BI) posterior probabilities/maximum likelihood (ML) bootstrap support. Isolates representing ex-type material are marked with “T”. Isolates in bold type are those analyzed in this study.

 Morphological Features

The morphological characteristics of the three new species as well as photomicrographs of morphological structures are shown in Figures 2–4. The detailed fungal morphological descriptions are provided in the Taxonomy section.

Morphology of Beauveria polyrhachicola. (A,B) The type specimen (YHH 859). (C) Bombyx mori larva infected by B. polyrhachicola. (D) Tenebrio molitor larva infected by B. polyrhachicola. (E) Protaetia brevitarsis adult infected by B. polyrhachicola. (F) Culture character on PDA medium. (G–J) Conidiogenous cells and conidia. Scale bars: (A) = 10 mm; (B) = 2 mm; (C–E) = 10 mm; (F) = 20 mm; (G) = 20 μm; (H–J) = 10 μm.

Morphology of Beauveria songmingensis. (A) The type specimen (YHH 860). (B,C) Protaetia brevitarsis adults infected by B. songmingensis. (D,E) Culture character on PDA medium. (F–I) Conidiogenous cells and conidia. Scale bars: (A–C) = 10 mm; (D,E) = 20 mm; (F–H) = 20 μm; (I) = 10 μm.

Morphology of Beauveria subscarabaeidicola. (A) Perithecial stroma as encountered in the field. (B) The type specimen (YHH 863). (C) The brown host of B. subscarabaeidicola. (D) Surface of fertile structure of perithecial stroma showing emerging apical parts of immersed perithecia. (E,F) Perithecia. (G,H) Asci. (I,J) Ascospores. (K,L) Culture character on PDA medium. (M,N) Conidiogenous cells and conidia. Scale bars: (A,B) = 10 mm; (C) = 5 mm; (D) = 2 mm; (E,F) = 500 μm; (G–J) = 40 μm; (K,L) = 20 mm; (M,N) = 10 μm.

 Conidial Viability of the <italic>Beauveria bassiana</italic> Complex and <italic>Beauveria scarabaeidicola</italic> Complex Isolates

Percentage of conidial germination was used to determine conidial viability. The conidial viability of the B. bassiana complex isolates was high, but the highest value (x¯ = 95%) was found on isolates of YFCC 844 from soil (see Table 3). The values of the conidial viability of the isolates in the B. scarabaeidicola complex were significantly lower than those in the B. bassiana complex. Only four B. scarabaeidicola complex isolates with conidial viability were greater than 65%, and their conidial viability values were not significantly different (P < 0.05).

 Virulence of the <italic>Beauveria bassiana</italic> Complex and <italic>Beauveria scarabaeidicola</italic> Complex Isolates

Ten isolates had conidial viability greater than 65%. These isolates were then selected for pathogenicity tests against B. mori and T. molitor larvae as well as P. brevitarsis adults (Table 4). The B. bassiana complex isolates were shown to have great potential for use in the management of various insect pests; by contrast, the B. scarabaeidicola complex isolates showed obvious host specificity and low virulence. All tested isolates in the B. bassiana complex inflicted mycoses on B. mori and T. molitor larvae and caused over 80% mortality, whereas those in the B. scarabaeidicola complex did not. It was determined that the 10 isolates were pathogenic to P. brevitarsis adults but demonstrated different levels of virulence. Like the conidial viability, the mortalities of P. brevitarsis adults caused by the B. scarabaeidicola complex isolates were significantly lower than those of isolates in the B. bassiana complex (P < 0.05), strengthening the hypothesis that the virulence of certain entomopathogenic fungi is related to their conidial viability (Butt et al., 1994; Fernandes et al., 2007). Additionally, B. bassiana YFCC 844, which was isolated from soil and exhibited the highest conidial viability, showed high virulence against B. mori and T. molitor larvae, as well as P. brevitarsis adults, causing (94.00 ± 1.15)% mortality against B. mori larva, (95.33 ± 1.45)% mortality against T. molitor larva, and (79.00 ± 1.53)% mortality against P. brevitarsis adults (Table 4). Mycelium samples from cadavers were aseptically removed and cultured on PDA. Microscopic examination recovered the same morphological characters of conidiophores and conidia as the inoculated fungal strain. Further, TEF sequenced from DNA extracted from recultures of the external mycelium of cadavers were found to match that of inoculated strain perfectly.

The lethal effect of Beauveria spp. isolates on B. mori and T. molitor larvae, and P. brevitarsis adult.

Group Species and isolate Mortality ± SE (%)#
B. mori larva T. molitor larva P. brevitarsis adult
The B. bassiana complex B. bassiana
YFCC 841 89.33 ± 0.88a 85.00 ± 1.73c 67.67 ± 1.76bc
YFCC 844 94.00 ± 1.15a 95.33 ± 1.45a 79.00 ± 1.53a
YFCC 3369 81.33 ± 2.03b 91.33 ± 2.03ab 74.67 ± 2.60ab
B. polyrhachicola
YFCC 859 82.00 ± 1.53b 84.00 ± 3.06c 67.00 ± 3.79bc
B. rudraprayagi
YFCC 858 91.33 ± 2.03a 87.00 ± 1.15bc 78.67 ± 2.33a
B. staphylinidicola
YFCC 855 80.67 ± 1.86b 81.67 ± 1.45c 62.33 ± 2.03c
The B. scarabaeidicola complex B. scarabaeidicola
YFCC 847 0 0 31.33 ± 3.18e
YFCC 865 0 0 48.00 ± 5.20d
B. songmingensis
YFCC 860 0 0 49.33 ± 4.70d
B. yunnanensis
YFCC 862 0 0 49.67 ± 5.90d

#Corrected mortality. Different lowercase letters in the same column indicate significant differences at 5% level.

 Taxonomy

Beauveria polyrhachicola H. Yu & Y. Wang, sp. nov. Figure 2

MycoBank number 841450.

Etymology: “polyrhachicola” refers to the host (Polyrhachis sp.).

Sexual morph: Undetermined.

Asexual morph: Colonies on PDA reached 20–38 mm in diameter after 14 days at 25°C, white, circular, velutinous, and closely appressed to the agar surface; reverse yellowish white. Odor indistinct. Vegetative hyphae septate, branched, hyaline, smooth-walled, 1.2–2.3 μm wide. Conidiogenous cells, long cylindrical to long flask shaped, solitary or occurring in dense lateral clusters, base cylindrical to ampulliform and 1.4–3.0 μm wide, sympodially branched neck tapering into a long, slender, denticulate rachis, produced laterally on aerial hyphae or from subtending cells, 11.8–40.9 × 1.4–3.0 μm. Conidia 2.0–3.8 × 1.7–2.6 μm, Q = 1.0–1.8 μm (Lm = 2.7 μm, Wm = 2.1 μm, Qm = 1.3), globose, subglobose, slightly ellipsoid, oblong, or cylindrical, hyaline, aseptate, walls smooth and thin.

Type: Thailand, Chiang Rai Province, Khun Tan District (19.9233°N, 100.3133°E, 396 m above sea level), on an adult worker of Polyrhachis sp. emerging from leaf litter on the forest floor, May 2019, collected by Preeyanat Hongthong (holotype: YHH 859; ex-type living culture: YFCC 859).

Distribution: Khun Tan District, Chiang Rai Province, Thailand; Simao District, Yunnan Province, China.

Other material examined: China, Yunnan Province, Puer City, Simao District (22.7113°N, 100.9579°E, 1,360 m above sea level), on an adult worker of Polyrhachis sp. emerging from leaf litter on the forest floor, August 26, 2021, Yao Wang (YHH 867, 868; living culture: YFCC 867).

Notes: Regarding phylogenetic relationships, B. polyrhachicola forms a distinct lineage in the B. bassiana complex, and it is closely related to B. peruviensis, B. staphylinidicola, B. bassiana, and B. rudraprayagi (Figure 1). Morphologically, B. polyrhachicola is similar to B. bassiana, B. kipukae, B. pseudobassiana, B. varroae, and B. peruviensis in terms of the shape and size of the conidia (Rehner et al., 2011; Bustamante et al., 2019). However, B. polyrhachicola can be distinguished from them by its long conidiogenous cells (11.8–40.9 × 1.4–3.0 μm).

Beauveria songmingensis H. Yu & Y. Wang, sp. nov. Figure 3

MycoBank number 841451.

Etymology: named after the location Songming County where this species was collected.

Sexual morph: Undetermined.

Asexual morph: Colonies on PDA reaching 20–35 mm in diameter after 14 days at 25°C, yellowish white, pale yellow, or light yellow, circular; reverse pale yellow, light yellow, or shades of orange to deep orange. Odor indistinct. Vegetative hyphae septate, branched, hyaline or translucent pale yellow, smooth-walled, 2.2–4.5 μm wide. Conidiogenous cells, cylindrical to long flask shaped, solitary but usually in dense clusters of five or more, base cylindrical to ampulliform and 2.7–5.6 μm wide, apex with an indeterminate 1 μm wide geniculate, denticulate rachis, produced laterally on aerial hyphae or from subtending cells, mostly 9.6–34.1 × 2.7–5.6 μm. Conidia 3.6–6.8 × 2.8–3.9 μm, Q = 1.0–2.0 μm (Lm = 5.6 μm, Wm = 3.4 μm, Qm = 1.6), subglobose, broadly ellipsoid, ellipsoid, or oblong, hyaline, aseptate, walls smooth and thin.

Type: China, Yunnan Province, Kunming City, Songming County, Dashao Village (25.3924°N, 102.5589°E, 2,700 m above sea level), on an adult of Pseudosymmachia flavescens (Coleoptera: Scarabaeidae), August 12, 2018, collected by Yao Wang, (holotype: YHH 860; ex-type living culture: YFCC 860).

Distribution: at present known only in Dashao Village, Songming County, Yunnan Province, China.

Other material examined: China, Yunnan Province, Kunming City, Songming County, Dashao Village (25.3924°N, 102.5589°E, 2,700 m above sea level), on an adult of Pseudosymmachia sp. emerging from leaf litter on the forest floor, August 12, 2018, Yao Wang (YHH 848, 861; living culture: YFCC 848, 861).

Notes: Morphologically, B. songmingensis resembles the phylogenetically sister species B. scarabaeidicola and B. subscarabaeidicola. They were found to be parasitic on adult beetles (Coleoptera: Scarabaeidae), and they could be easily recognized by their distinctly yellow colony pigmentation and ellipsoid or oblong conidia. However, our morphological observation revealed a significant difference of conidia sizes between B. songmingensis (3.6–6.8 × 2.8–3.9 μm) and B. scarabaeidicola (2.5–3.5 × 1.5–2.5 μm). B. songmingensis differs from B. subscarabaeidicola by its long conidiogenous cells (9.6–34.1 × 2.7–5.6 μm) and large conidia (3.6–6.8 × 2.8–3.9 μm). Both morphological study and phylogenetic analyses of combined TEF, RPB1, RPB2, and Bloc sequence data support that this fungus is a distinctive species in the genus Beauveria.

Beauveria subscarabaeidicola H. Yu, Y. Wang & Q. Fan, sp. nov. Figure 4

MycoBank number 841452.

Etymology: “subscarabaeidicola” refers to morphologically resembling Beauveria scarabaeidicola but phylogenetically distinct.

Sexual morph: Stromata solitary, fleshy, pale yellow to orange, arising on adult scarab beetles buried in soil or decayed leaves, 30–45 mm long. Stipes cylindrical to clavate, yellowish white to deep yellow, 1.1–2.0 mm wide. Fertile parts clavate, being slightly wider than and indistinct from the stipes, deep yellow to orange, 5.2–26.0 mm long, 1.4–3.3 mm wide. Perithecia semi-immersed and crowded at the apex of the stromata, ampuliform, pyriform, ovoid to oblong, 265–700 × 180–320 μm (n = 50). Asci hyaline, cylindrical, 124.5–257.4 × 3.7–5.2 μm (n = 50). Apical caps prominent, hemiglobose, 2.7–3.9 μm wide, 2.4–3.2 μm high (n = 50). Ascospores hyaline, filiform, multi-septate, finally breaking into secondary ascospores, 75.6–188.5 × 1.0–1.5 μm (n = 30). Secondary ascospores cylindrical, hyaline, 6.9–11.2 × 1.0–1.5 μm (n = 50).

Asexual morph: Colonies on PDA reaching 28–42 mm in diameter after 14 days at 25°C, yellowish white, pale yellow, or light yellow, circular; reverse pale yellow, light yellow, or shades of orange to deep orange. Odor indistinct. Vegetative hyphae septate, branched, hyaline or translucent pale yellow, smooth-walled, 1.2–2.5 μm wide. Conidiogenous cells, phialidic, solitary but usually in dense clusters of five or more, base subspherical to ampulliform and 2.8–5.0 μm wide, apex with an indeterminate 1 μm wide geniculate, denticulate rachis, produced laterally on aerial hyphae or from subtending cells mostly 4.8–6.9 × 2.0–4.6 μm. Conidia 2.6–4.2 × 1.9–3.5 μm, Q = 1.0–1.4 μm (Lm = 3.4 μm, Wm = 2.8 μm, Qm = 1.2), subglobose or broadly ellipsoid, hyaline, aseptate, walls smooth and thin.

Type: China, Yunnan Province, Kunming City, Songming County, Dashao Village (25.2398°N, 102.5617°E, 2,697 m above sea level), on an adult of Anomala exoleta (Coleoptera: Scarabaeidae), July 23rd, 2019, collected by Dexiang Tang, (holotype: YHH 863; ex-type living culture: YFCC 863).

Distribution: at present known only from Dashao Village, Songming County, Yunnan Province, China.

Other material examined: China, Yunnan Province, Kunming City, Songming County, Dashao Village (25.2398°N, 102.5617°E, 2,697 m above sea level), on an adult of Anomala exoleta, July 23, 2019, Dexiang Tang (YHH 864; living culture: YFCC 864).

Notes: Beauveria subscarabaeidicola is practically indistinguishable in morphology to B. scarabaeidicola. Our morphological observation revealed no significant differences in the morphological characteristics of teleomorph and anamorph between the two species (Kobayasi and Shimizu, 1976; Rehner et al., 2011; Shrestha et al., 2014). The lack of diagnostic morphological features to distinguish B. subscarabaeidicola and B. scarabaeidicola was overcome by delimiting the two species using DNA-based methodologies.

 Discussion

It is generally agreed that distinguishing individual Beauveria species can be difficult using only morphological characters, as several species in the genus are morphologically cryptic species. In this study, we conducted a comprehensive investigation of the cryptic species diversity of the B. bassiana complex and B. scarabaeidicola complex. The molecular phylogeny clearly suggested the existence of distinct species in the B. bassiana complex and B. scarabaeidicola complex that we accordingly propose as new species: B. polyrhachicola (Figure 2), B. songmingensis (Figure 3), and B. subscarabaeidicola (Figure 4). Beauveria polyrhachicola is practically indistinguishable in morphology from other members of the B. bassiana complex. The shape and size of the conidia and the colony color of B. polyrhachicola, among other morphological features, have been observed in B. bassiana, B. rudraprayagi, B. staphylinidicola, and B. peruviensis (Rehner et al., 2011; Agrawal et al., 2014; Kepler et al., 2017; Bustamante et al., 2019). In the B. scarabaeidicola complex, the macromorphology of B. scarabaeidicola, B. songmingensis, and B. subscarabaeidicola is very similar, and thus species cannot be distinguished visually. The macroscopic and microscopic observations performed during our investigation revealed the extensive overlap in morphological characters and the lack of distinctive phenotypic variation, supporting the notion of cryptic species in a species complex.

At present, multi-locus phylogenetic analyses have gained importance in delimiting the species within the entomopathogenic fungi Beauveria. Rehner et al. (2011) divided B. bassiana s. lat. and B. brongniartii s. lat. into several cryptic species and described six new species based on the Bloc nuclear intergenic region and three nuclear genes encoding elongation factor 1-a (TEF), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2). Subsequently, more than seven new species and new combinations were confirmed using combined analysis of the four-locus sequence data (Zhang et al., 2012; Chen et al., 2013, 2017, 2018; Agrawal et al., 2014; Robène-Soustrade et al., 2015; Imoulan et al., 2016). In more recent studies, six species were added to the genus based on multilocus (nrSSU, nrLSU, TEF, RPB1, and RPB2) sequence data: B. acridophila, B. blattidicola, B. diapheromeriphila, B. locustiphila, B. scarabaeidicola, and B. staphylinidicola (Kepler et al., 2017). In this study, we analyzed most species of the newly circumscribed genus Beauveria based on phylogenetic inferences of six nuclear molecular markers (nrSSU, nrLSU, TEF, RPB1, RPB2, and Bloc). Phylogenetic analyses based on the five-gene (nrSSU, nrLSU, TEF, RPB1, and RPB2) dataset and the combined four-gene (TEF+RPB1+RPB2+Bloc) sequences produced trees with similar topologies that resolved most Beauveria lineages in separate terminal branches (Figure 1 and Supplementary Figure 1). The results of the present work indicate that the first dataset was conducive to determining the phylogenomic relationships between Beauveria and its related genera, and the use of the latter was essential to establish robust Beauveria species boundaries, particularly the B. bassiana complex and B. scarabaeidicola complex.

Scarab beetles are leaf and root feeding pests of grasses, grains, sugarcane, strawberry, potato tubers, and young nursery plants (Crocker et al., 1996; Yokoyama et al., 1998). Based on the published literature, there are about six Beauveria spp. that parasitize adult scarab beetles: B. asiatica, B. bassiana, B. brongniartii, B. majiangensis, B. pseudobassiana, and B. scarabaeidicola (Rehner et al., 2011; Kepler et al., 2017; Chen et al., 2018; Khonsanit et al., 2020; Wang Y. et al., 2020). Here, we identified an extension of the members to also include B. malawiensis, B. songmingensis, B. subscarabaeidicola, and B. yunnanensis, as shown in Figure 1. Chen et al. (2019) emphasized that hosts of B. yunnanensis isolates were Lepidoptera pupae. However, our morphological observations of specimens from a type locality of B. yunnanensis indicated that their hosts were adult scarab beetles. Moreover, the host of B. yunnanensis was not shown in their publication (Chen et al., 2019). It seems that the host of Lepidoptera pupa is doubtful. There is reason to believe that members of the B. scarabaeidicola species complex are host-specific.

Not all scarab-killing pathogens are suitable for mycoinsecticide formulations that control scarab beetles. Our data suggested that the B. scarabaeidicola complex isolates showed low virulence. In addition, mortalities in P. brevitarsis adults caused by the B. scarabaeidicola complex isolates were significantly lower than those of isolates in the B. bassiana complex. Additional research is needed to determine the effectiveness of other species before future consideration of isolates for biological pest control.

 Conclusion

The B. bassiana complex and B. scarabaeidicola complex, as special groups in the genus Beauveria, are rich in species diversity and have a wide distribution in nature. The B. bassiana complex, which is made up of five species, is a cosmopolitan group of soilborne necrotrophic arthropod-pathogenic fungi that have been shown to have great potential for the management of various insect pests. The B. scarabaeidicola complex is composed of pathogens specific to scarab beetles, and it is found on leaf litter or buried in soil. Species in this complex are morphologically highly similar and can hardly be distinguished macroscopically. In this study, we reported the discovery and description of three new species: B. polyrhachicola, which was found in the B. bassiana complex, and B. songmingensis and B. subscarabaeidicola, which were found in the B. scarabaeidicola complex. In addition, 10 species of Beauveria were found to be parasitic on scarab beetles. However, not all members are suitable for mycoinsecticide formulations for controlling scarab beetles. Our data suggested that the B. scarabaeidicola complex isolates showed obvious low virulence. Additionally, the mortality of Protaetia brevitarsis adults caused by the B. scarabaeidicola complex isolates was significantly lower than that of isolates in the B. bassiana complex.

 Data Availability Statement

The datasets presented in this study can be found in GenBank. The accession numbers can be found in the article/Supplementary Material.

 Author Contributions

YW and HY: conceptualization. YW: methodology, writing—original draft preparation, and formal analysis. YW and QF: software. QF, W-QZ, and DW: validation. YW, DW, D-XT, PH, and HY: investigation. YW, D-XT, and PH: resources. HY: writing—review and editing and funding acquisition. All authors reviewed and approved the final manuscript.

 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.

 Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 Funding

This work was supported by the National Natural Science Foundation of China (No. 31870017, 31760011, and 32160005) and the Department of Science and Technology of Yunnan Province [No. 2018FY001(-006)].

We thank Dr. Yuan-Bing Wang for help in naming the new fungal species.

 Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.841604/full#supplementary-material

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