﻿Acremoniumcapsici and A.guizhouense, two new members of Acremonium (Hypocreales, Sordariomycetes) isolated from the rhizosphere soil of Capsicumannuum

﻿Abstract Two new species, Acremoniumcapsici and A.guizhouense, isolated from the rhizosphere soil of Capsicumannuum, are described and illustrated. Two-locus DNA sequences based on phylogeny, in combination with the morphology of the asexual morph, were used to characterize these species. In the phylogenetic tree, both new species clustered into a monophyletic clade with strong support, distinct from other previously known species of Acremonium. The new species differed from their allied species in their morphology.


Introduction
Capsicum annuum L. is a globally grown and consumed spice crop that is rich in vitamins. C. annuum originated from the tropical and subtropical regions of Central and South America. It was introduced into China at the end of the Ming Dynasty, and has a long history of cultivation in China. According to the Food and Agriculture Organization of the United Nations, global C. annuum production reached approximately 36.1 million ton in 2020, with China producing the most in the world. Link (1809) erected the genus Acremonium, whose members are geographically widespread and involve many substrates (Yang et al. 2019). As described by Gams (1971), the main diagnostic criteria of the genus Acremonium are conidiophores simple or verticillate; phialides narrow, solitary, generally cylindrical and gradually tapered towards the tips; conidia unicellular, hyaline to light-pigmented, spherical to cylindrical, arranged in slimy heads or unconnected chains or both; chlamydospores and sclerotia present or absent. The genus Acremonium is similar to some genera -Sarocladium W. Gams &D. Hawksw., Brunneomyces Giraldo, Gené &Chordomyces Bilanenko, M.L. Georgieva &Grum-Grzhimaylo etc. (Giraldo et al. 2015, 2017), including some of the simplest morphologies of all filamentous anamorphic fungi (Summerbell et al. 2011), so the morphological delimitation between them is challenging (Yang et al. 2019). Recent phylogenetic studies have documented that the genus Acremonium is polyphyletic, including sexual and nomenclaturally complex asexual morphs (Summerbell et al. 2011;Giraldo et al. 2012). To date, Acremonium has 219 records in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, retrieval on Dec. 2022). However, many Acremonium taxa have been reported, but there are no trustworthy classification systems and little sequence data are available in GenBank for multigene analyses (Park et al. 2017). In the future, the classification of Acremonium will become clearer with the increase of molecular data.
In this study, seven strains of Acremonium were isolated in the process of investigating the rhizosphere fungal diversity of cultivated Capsicum annuum in Guizhou Province, southwest China, based on a culturable method. Identification of these strains in combination with morphological characteristics and phylogenetic analysis showed that these strains belong to two previously undescribed species of Acremonium. The new species differed from their allied species in their morphology.

Fungal isolation and morphology
Capsicum annuum plants were cultivated in farmlands located in Guiyang, Guizhou Province, China (26°45'75"N, 106°64'87"E). One composite rhizosphere soil sample was taken from five randomly selected C. annuum plants. The roots were shaken vigorously to separate soil that is not tightly attached to the roots, and the remaining soil attached to the region 2-3 mm from the plant root was collected as the rhizosphere soil sample (Smalla et al. 2001). Fungi were isolated and purified using a dilution plate method as follows: 2 g samples were weighed with glass beads in a conical flask containing 20 mL sterile water, mixed evenly using eddy shock for 10 min, diluted to 1:10,000, and cultured on Martin's medium supplemented with chloramphenicol and cycloheximide.
The purified isolates were transferred to potato dextrose agar (PDA), oatmeal agar (OA), malt extract agar (MEA), and corn meal agar (CMA) at 25 °C in darkness for 14 days to examine the macroscopic and morphological characteristics of the colonies. Photomicrographs of the diagnostic structures were obtained using an OLYMPUS BX53 microscope equipped with differential interference contrast optics, an OLYMPUS DP73 high-definition color camera, and cellSens software v.1.18. Both dry and living cultures were deposited at the Institute of Agro-bioengineering, Guizhou University.
The best-fit substitution model was selected using the corrected Akaike information criterion, in ModelFinder (Kalyaanamoorthy et al. 2017). The maximum likelihood (ML) and Bayesian inference (BI) methods were used in the analysis. The ML analysis was implemented in IQ-TREE v1.6.11 (Nguyen et al. 2015) with 10,000 bootstrap tests, using the ultrafast algorithm (Minh et al. 2013). For the BI, MrBayes v3.2 (Ronquist et al. 2012) was used and Markov chain Monte Carlo simulations were run for 5,000,000 generations with a sampling frequency of every 500 generations and a burnin of 25%. The above analyses were carried out in PhyloSuite v1.16 (Zhang et al. 2020).
Known distribution. Guiyang City, Guizhou Province, China. Notes. In a phylogenetic tree based on LSU + ITS sequences, Acremonium capsici forms a separate clade sister to A. variecolor in Acremonium sensu lato (Bionectriaceae). In a comparison of LSU and ITS nucleotides, A. capsici (Type strain SQT01) has 93% and 83% similarity, in LSU (459/492 bp, one gap) and ITS (388/468 bp, 16 gaps), which is different from A. variecolor (CBS 130360). They are distinguished by the appearance of colonies on OA, MEA, and PDA: colonies of A. capsici grow slowly (less than 25 mm), and are white, while colonies of A. variecolor grow faster (more than 40 mm), and are white to yellowish (Giraldo et al. 2012). In addition, A. capsici bear simple phialides, while conidiophores of A. variecolor are mostly branched, bearing whorls of two to five phialides (Giraldo et al. 2012). A. variecolor produces sessile conidia, which is not seen in A. capsici (Giraldo et al. 2012). Etymology. Referring to the country where this fungus was first isolated.

Discussion
Traditionally, a polyphasic approach based on morphology, physiology, biochemistry, or reactions to chemical tests, has been used to differentiate species (Senanayake et al. 2020). Currently, many new fungal taxa have been reported based on DNA sequences. Phylogenetic analysis is becoming increasingly important in reporting new taxa of fungi, and has gradually become a mandatory component. However, many previously published fungal taxa lack DNA molecular data, and even specimens have been lost . Thus, there are still many undetermined, questionable, or misidentified taxa that warrant taxonomic investigations (Summerbell et al. 2018). Since most species of the genus Acremonium have only LSU and ITS sequences Li et al. (2022), we used only ribosomal sequences (LSU + ITS) for phylogenetic analysis, while the sequencing of other loci was aimed at establishing a database for future studies.
Members of the genus Acremonium are geographically widespread and ecologically diverse, and seem to colonize all types of substrates, including endophytes, epiphytes, saprophytes, human and plant pathogens, lichens, insects, or arthropods taxa (Yang et al. 2019). In addition, Acremonium species have various functions, such as biological control (Shang et al. 2018), enhancing drought tolerance of grasses, and promoting nectar production of beans (Jaber and Vidal 2009), as well as improving plant resistance to plant pathogens (Kasselaki et al. 2006). In the present study, all the isolates were obtained from the rhizosphere soils of Capsicum annuum. Therefore, more studies are necessary to further confirm their relationship with their host plant Capsicum annuum.
In summary, seven isolates of Acremonium were obtained from the rhizosphere soils of Capsicum annuum. Morphological characteristics in combination with two-locus (LSU + ITS) phylogenetic analysis were used for delimitation. Therefore, two new species of Acremonium capsici (three isolates) and Acremonium guizhouense (four isolates) are introduced. This study contributes to our understanding of the rhizosphere microbial population of Capsicum annuum and also of Acremonium species.