Metarhiziumpuerense (Hypocreales, Clavicipitaceae): a new species from Yunnan, south-western China

Abstract Background As a genus within the Clavicipitaceae, Metarhizium exhibits rich morphological and ecological diversity, with a wide distribution and a variety of hosts. Currently, sixty-eight species of Metarhizium have been described. New information A new species of Metarhizium, M.puerense (Hong Yu bis), was described in Pu'er City, Yunnan Province, south-western China. Based on morphological characteristics and multilocus phylogenetic analyses, Metarhiziumpuerense was confirmed to be phylogenetically related to M.album, but was clearly separated and formed a distinct branch. In contrast, the host of Metarhiziumalbum was plants and leafhoppers and that lepidopteran larvae were the host of M.puerense. The diagnostic features of M.puerense were solitary to multiple stromata and smooth-walled, cylindrical with rounded apices conidia.


New information
A new species of Metarhizium, M. puerense (Hong Yu bis), was described in Pu'er City, Yunnan Province, south-western China.Based on morphological characteristics and multilocus phylogenetic analyses, Metarhizium puerense was confirmed to be phylogenetically related to M. album, but was clearly separated and formed a distinct branch.In contrast, the host of Metarhizium album was plants and leafhoppers and that lepidopteran larvae were the host of M. puerense.The diagnostic features of M. puerense were solitary to multiple stromata and smooth-walled, cylindrical with rounded apices conidia.‡ ‡ ‡ ‡ ‡ ‡, ‡

Introduction
Metarhizium, as a group with rich morphological and ecological diversity in Clavicipitaceae, is very rich in widely distributed and complex habitats (Bischoff et al. 2009).The type species Metarhizium anisopliae (Metschn 1879) was used by Sorokīn (1883) to establish the new asexual genus Metarhizium ( Tulloch 1976).Advances in molecular systematics have led to the emergence of multigene systematic analysis as a new technical method for the taxonomic identification of Metarhizium sp., combining functional protein genes with rDNA gene fragments (Sung et al. 2007and Kepler et al. 2012and Kepler et al. 2014).In their study of genetic diversity within Metarhizium species, Driver et al. (2000) were the first to utilise molecular biology techniques.They solved the problem of classification at the species and varietal levels by identifying four variants in the Metarhizium anisopliae complex groups, five variants in the M. flavoviride (Gams 1973) complex groups and delineating M. album (Petch 1931).In a multi-gene phylogenetic study of Metarhizium anisopliae and M. flavoviride lineages, Bischoff et al. 2006 andBischoff et al. 2009 elevated and accepted Metarhizium varieties to species rank by using additional protein-coding genes (EF-1α, RPB1, RPB2 and TUB).The reexamination of Metarhizium and related genera led to the establishment of six new genera: Keithomyces, Marquandomyces, Papiliomyces, Purpureomyces, Sungia and Yosiokobayasia ( Mongkolsamrit et al. 2020) Chen et al. 2018aand Chen et al. 2018band Chen et al. 2018cand Chen et al. 2023and Li et al. (2023).Currently, sixty-eight species of Metarhizium have been described.
According to the latest classification system, the Metarhizium genus belongs to the Fungi, Ascomycota, Sordariomycetes, Hypocreales and Clavicipitaceae.Its typical morphological characteristics are: Sexual form: Stromata single or multiple, unbranched or irregularly branched, mostly fleshy, with the main colours being pale yellow, green to greenish-brown or dark purple; fertile parts columnar or rod-shaped; perithecia partially or completely immersed; asci mainly columnar, ascospores linear, fusiform, breaking into secondary ascospores upon maturity or not.Asexual form: Growing rapidly on PDA medium, the colonies are flat and velvety, initially white and turn yellow-green or green after sporulation; phialides are morphologically diverse, single on the aerial hyphae or verticillate on the conidiophores; conidia are smooth, oval to columnar, spherical to subspherical, ovoid, aggregated in chains or clusters (Liang 2007).The typical characteristics of M. puerense were solitary to multiple stromata and smooth-walled, brownish in colour and producing a large number of green powdery conidia at the tip.
Metarhizium species that parasitise lepidopteran larvae were collected from Yunnan for this investigation.Phylogenetic location was elucidated, based on Bayesian Inference (BI) and Maximum Likelihood (ML) analyses, which involved concatenating sequences of the six loci.The results revealed that the species in question belong to the genus Metarhizium, specifically Metarhizium puerense.

Collection and isolation of strains
Specimens were collected from the broad-leaved evergreen forest of Pu'er, Yunnan Province, China, 2 August 2023, 22°71.33'N,100°95.57'E,alt.1358 m.The samples were preserved in sterile tubes and stored at 4°C.To obtain pure cultures, fresh specimens were cleaned and surface-sterilised by soaking in 30% hydrogen peroxide for approximately one minute.The samples were then washed with sterile water to remove residual hydrogen peroxide and the residual water was aspirated with a sterile filter paper.The worms were dissected on an ultra-clean bench, picked up with a sterilised scalpel with an appropriate amount of white tissue in the sclerotium centre, inoculated on potato dextrose agar medium (PDA: fresh potato 200 g/l, dextrose 20 g/l and agar 18 g/l) (Wang et al. 2020) and allowed to incubate at room temperature.The collected specimens were placed in the Yunnan Herbarium of the Yunnan University (YHH).The obtained strains were preserved at the Yunnan Fungal Culture Conservation Center (YFCC).

Morphological characterization
Fresh specimens, including the stromata and hosts, were photographed using a Canon 750D camera.For descriptions of colony appearance and microscopic features, the colonies on PDA plates were cultured for two weeks and the colony characteristics (size, texture and colour) were photographed with a Canon 700D camera to characterise the morphology of the colonies.Observations, measurements and photographs of the phialides and conidia were obtained using a light microscope (Olympus BX53).

DNA extraction, PCR and sequencing
DNA extraction was performed using a ZR Fungal DNA kit (Zymo, California, USA).DNA was preserved at -20˚C and used as a template for PCR amplification of the six loci.To amplify the largest and second-largest subunit sequences of RNA polymerase II (RPB1 and RPB2), the primer pair RPB1-5'F and RPB1-5'R, as well as the primer pair RPB2-5'F and RPB2-5'R, were applied (Bischoff et al. 2006).The nuclear ribosomal small and large subunits (SSU and LSU) were amplified using the primer pairs used by 18S-CoF and 18S-CoR (Wang et al. 2015), as well as LR5 andLR0R (Vilgalys andHester 1990 andRehner andSamuels 1994).The translation elongation factor 1α (EF-1α) gene was amplified using the primer pair EF1α-EF and EF1α-ER (Bischoff et al. 2006 andSung et al. 2007).PCR primers used to amplify the internal transcribed spacers were ITS4 and ITS5 (White et al. 1990).All PCR reactions were performed in a final volume of 50 μl and contained 25 μl of 2 × Taq PCR Master Mix (Tiangen, Beijing, China), 0.5 µl forward and reverse primers (10 μM), 1 μl template DNA (1 ng/μl) and 23 μl sterile distilled water.The polymerase chain reaction (PCR) was performed as described by Wang et al. (2015).

Phylogenetic analysis
The data matrix included 72 sequences from 48 species in Metarhizium and two outgroup taxa.Sequences of six loci (ITS, SSU, LSU, EF-1α, RPB1 and RPB2) were retrieved from GenBank.Sequences were aligned using MUSCLE software (Tamura et al. 2013).After alignment, the gene sequences were concatenated.Clonostachys rosea (GJS 90-227) and Hydropisphaera peziza (CBS 102038) were designated as the outgroup taxa.Phylogenetic analyses were conducted using BI and ML methods with MrBayes v.3.1.2and RaxML 7.0.3,respectively (Ronquist andHuelsenbeck 2003 andStamatakis et al. 2008).The GTR+G+I model was determined using jModelTest version 2.1.4(Darriba et al. 2012) with five million generations for the BI analysis.GTR+I was selected as the optimal model for the ML analysis and 1,000 rapid bootstrap replicates were performed on the dataset.

Description
Sexual morph: Sexual morphs were not found.
Asexual morph: Stroma arising from the larvae of Lepidoptera larva buried in soil, solitary or multiple, brownish in colour and producing a large number of green powdery conidia at the tip.Colonies on PDA grew at 25°C, reaching 25-28 mm diam. in 14 days, cottony with high mycelium density, white to light yellow and reverse yellow; 45-52 mm in diameter in 30 days at 25°C, first white turning to green, powdery while sporulating, white mycelium at the margin.Hyphae septate, smooth-walled.

Etymology
Named after Pu'er City, where the species were first collected.
Morphological comparisons of Metarhizium puerense with its related species.

Discussion
To date, multi-locus phylogenetics, based on the joint analysis of ribosomal DNA and functional protein-coding genes, have been widely used in the phylogenetic study of fungi and have achieved many results (Sung et al. 2007and Luangsa-ard et al. 2017and Mongkolsamrit et al. 2020).In this study, we conducted an investigation, searched for and retrieved the Metarhizium nuclear gene sequences from the NCBI database.Subsequently, the sequences were compared with the obtained data.Additionally, a phylogenetic tree was constructed, based on multilocus database analyses (ITS, SSU,  LSU, EF-1α, RPB1 and RPB2) to elucidate the phylogenetic position of M. puerense.Phylogenetically, Metarhizium puerense is closely related to M. album.However, in terms of morphological characteristics, M. puerense parasitises the larvae of Lepidoptera, either solitary or multiple and produces a large number of green conidia.There were also differences in the sizes of phialides and conidia.
In the forests of Pu'er City, Yunnan Province, China, which are characterised by a warm and humid climate, a diverse array of entomopathogenic fungi thrive.Amongst these, Metarhizium is a fungal insecticide with large-scale production capabilities.It offers significant value owing to its environment-friendly nature, extended efficacy period and low resistance potential.This makes it an important asset for pest control.Hence, it is crucial to accurately identify the Metarhizium species and determine their host range to facilitate the development and utilisation of this potent insecticidal agent.In the current study, a new species collected from Pu'er City, Metarhizium puerense, is described.The phylogenetic and morphological evidence presented in this study supports the classification of the species as a new taxon within the genus Metarhizium.This research contributes to the expansion of diversity within Metarhizium species, enhances our understanding of host interactions, morphology, distribution and pure culture characteristics and provides valuable taxonomic and phylogenetic information for further detailed investigations of the genus.Additionally, this opens up new possibilities for the development of fungal insecticides.
In the investigation of entomogenous fungi resources in Yunnan, a new species of Metarhizium was discovered and identified.This work not only increases the diversity of species in the genus Metarhizium, enriches the biological fungal species resource pool in Yunnan Province, but also lays a certain foundation for the distribution of Metarhizium species in Yunnan Province and other regions in China.Additionally, it deepened our understanding of the morphology, distribution and pure culture characteristics of the Metarhizium genus and provided taxonomic and phylogenetic information for a more detailed study of the genus's systematics.
The combined six-locus dataset contained 4862 base pairs (bp) of sequences after alignment: 607 bp for ITS, 914 bp for SSU, 802 bp for LSU, 902 bp for EF-1α, 688 bp for RPB1 and 1101 bp for RPB2.
Clonostachys rosea (GJS 90227) and Hydropisphaera peziza (CBS 102038) were designated as outgroup taxa in the phylogenetic tree.In phylogenetic trees based on both Bayesian Inference (BI) and Maximum Likelihood (ML) analyses, the samples collected in Yunnan formed a strongly-supported clade that was sister to M. album (BI posterior probability = 1.00,ML bootstrap =100%).This result indicates that it is a new species of Metarhizium, named M. puerense (Fig.2).

Table 1 .
GenBank accession numbers of materials used in this study.