﻿Morphological and molecular analyses reveal two new species of Microcera (Nectriaceae, Hypocreales) associated with scale insects on walnut in China

﻿Abstract The fungal genus Microcera consists of species mostly occurring as parasites of scale insects, but are also commonly isolated from soil or lichens. In the present study, we surveyed the diversity and assess the taxonomy of entomopathogenic fungi in Sichuan Province, China. Two new species of Microcera, viz. M.chrysomphaludis and M.pseudaulacaspidis, were isolated from scale insects colonising walnut (Juglansregia). Maximum Likelihood and Bayesian Inference analyses of ITS, LSU, tef1-α, rpb1, rpb2, acl1, act, tub2, cmdA and his3 sequence data provide evidence for the validity of the two species and their placement in Nectriaceae (Hypocreales). Microcerapseudaulacaspidis primarily differs from similar species by having more septate and smaller cylindrical macroconidia, as well as DNA sequence data. Meanwhile, Microcerachrysomphaludis has elliptical, one-septate ascospores with acute ends and cylindrical, slightly curved with 4–6 septate macroconidia up to 78 µm long. Morphological descriptions with illustrations of the novel species and DNA-based phylogeny generated from analyses of multigene dataset are also provided to better understand species relationships.

Morphological and molecular analyses reveal two new species of Microcera (Nectriaceae, Hypocreales) associated with scale insects on walnut in China

Introduction
The genus Microcera Desm. (Nectriaceae, Hypocreales) was introduced in the 19 th century and was typified by M. coccophila Desm., commonly known as the "red-headed fungus". Microcera has been considered to be a synonym of the Fusarium Link in some major taxonomic revisions (Booth 1971;Nelson et al. 1983;Leslie and Summerell 2006). The genus is characterised by superficial, flame-like conidiomata, forming a fusarium-like asexual stage (Gräfenhan et al. 2011;Herrera et al. 2013). Microcera species exhibit diverse ecological characteristics and are typically regarded as entomogenous fungi that are associated with scale insects, although they can occasionally be isolated from other substrates, such as aphids, adelgids, lichens and soil (Gräfenhan et al. 2011;Crous et al. 2021aCrous et al. , b, 2022a.
Currently, there are eight accepted species within the genus Microcera (Bills et al. 2009;Gräfenhan et al. 2011;O'Donnell et al. 2012;Herrera et al. 2013;Dao et al. 2015Dao et al. , 2016Lombard et al. 2015;Crous et al. 2021bCrous et al. , 2022aXu et al. 2021). Based on DNA sequence data and ecological association, Gräfenhan et al. (2011) revised many anamorph-and teleomorph-typified genera of the Nectriaceae, resurrected Microcera and accepted four Microcera species, viz., M. coccophila, M. diploa (Berk. & M.A. Curtis) Gräfenhan & Seifert, M. rubra Gräfenhan & Seifert and M. larvarum (Fuckel) Gräfenhan, Seifert & Schroers. Lombard et al. (2015) supported Microcera as a monophyletic group distantly related to Fusarium, based on further phylogenetic inferences from DNA sequence data. Xu et al. (2021) (Crous et al. 2021b(Crous et al. , 2022a. During a survey of entomopathogenic fungi in Sichuan Province, China, two Microcera species, in association with the two scale insects Pseudaulacaspis pentagona and Chrysomphalus aonidum on walnut, were isolated. Microcera pseudaulacaspidis sp. nov. and M. chrysomphaludis sp. nov. are introduced here based on the morphological characteristics and multi-locus analyses (DNA based). They were compared morphologically with existing taxa. In this study, comprehensive descriptions, micrographs of macroscopic and microscopic morphological characteristics, as well as DNA sequence data, are provided to support the establishment of the new species.

Specimen collection and isolation
Three specimens of scale insects (SICAU 22-0161, SICAU 22-0162 and SICAU 22-0163) that were infected, were collected from Neijiang City (29°48′15″N, 105°06′44″E) and Liangshan Yi Autonomous Prefecture (26°56′43″N, 102°16′16″E), Sichuan Province, on 16 April and 8 October 2022. The specimens were placed in sterilised tubes or plastic boxes and returned to the laboratory as described by Senanayake et al (2020). The fungi were isolated, based on the single spore isolation technique described by Chomnunti et al. (2014). Cultures were grown on PDA for 20-40 days, at 25 °C, under 12 h light/12 h dark for recording growth rates, shape, texture and colour of the colonies. Ascomata and sporodochia were observed and photographed using a dissecting microscope NVT-GG (Shanghai Advanced Photoelectric Technology Co. Ltd., Shanghai, China). We observed microscopic characteristics, such as asci, ascospores, pseudoparaphyses, ascomata wall, conidia, conidiophores, number of septa, metulae and conidiophores using an Olympus BX43. No fewer than 20 measurements of the two species were made for each feature using the Image Frame Work (IFW 0.9.0.7). The type specimens were deposited at the Herbarium of Sichuan Agricultural University, Chengdu, China (SICAU). The ex-type cultures were deposited at the Culture Collection in Sichuan Agricultural University (SICAUCC).

DNA extraction, PCR amplification and nucleotide sequencing
The New Plant Genomic DNA Kit (Beijing Aidlab Biotechnologies Co., Ltd, Beijing, China) was used to extract genomic DNA from fresh fungal mycelium. The extracted DNA to be used was stored at -20 °C. Amplified gene markers and their corresponding primers are shown in Table 1. Polymerase chain reaction (PCR) was performed in 25 µl reaction mixture containing 22 µl Master Mix (Beijing LABLEAD Biotech Co., Ltd., Beijing, China), 1 µl DNA template and 1 µl each of forward and reverse (10 µM) primers. The amplification reactions were performed as described by Gräfenhan et al. (2011), Lombard et al. (2015, Dai et al. (2016) and Wanasinghe et al. (2021). PCR products were sequenced at Hangzhou Youkang Biotech Co., Ltd., Chengdu, China. The newly-generated sequences were deposited in Gen-Bank. New species are established as recommended by Jeewon and Hyde (2016).

Sequence alignment and phylogenetic analyses
Based on BLAST searches in GenBank and recent publications (Bills et al. 2009;Gräfenhan et al. 2011;O'Donnell et al. 2012;Herrera et al. 2013;Dao et al. 2015Dao et al. , 2016Lombard et al. 2015;Xu et al. 2021), using the large subunit of the ATP citrate lyase (acl1), actin (act) regions, calmodulin (cmdA), histone H3 (his3), the internal transcribed spacer (ITS), the partial large subunit nuclear rDNA (LSU), the RNA polymerase II largest subunit (rpb1), the RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1-α), β-tubulin (tub2) and sequence data, reference sequences were downloaded and separate phylogenetic analyses, based on single gene datasets were carried out to initially determine the placement of the two species. Information on the taxa used and GenBank AGTTGTCGG GACGGAAGAG Crous et al. (2006) accession numbers of our novel species are listed in Table 2. Alignments for the individual locus matrices were generated with the online version of MAFFT version 7.429 (Katoh and Standley 2013) and ambiguous regions were excluded using BioEdit version 7.0.5.3 (Hall 1999). Combined sequences of ITS, LSU, tef1-α, rpb1, rpb2, acl1, act, tub2, cmdA and his3 were performed by SequenceMatrix v.1.7.8 (Vaidya et al. 2011). Maximum Likelihood (ML) and Bayesian Inference (BI) were constructed as described in Xu et al (2020). The phylogenetic tree constructed was viewed and edited using FigTree version 1.4.2 and Adobe Illustrator CS6.

Genealogical concordance phylogenetic species recognition analysis
Phylogenetically closely-related species were analysed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model by performing a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. (2014). The PHI test was performed in SplitsTree v.4.17.1 (Huson 1998;Huson and Bryant 2006) in order to determine the recombination level within phylogenetically closely-related species using a 6-locus concatenated dataset (ITS, LSU, tef1-α, acl1, cmdA and his3). The results can be visualised by constructing a split graph using LogDet conversion and the Splits options. Pairwise homoplasy index below a 0.05 threshold (Ф w < 0.05) indicates significant recombination present in the dataset. The relationship between closely-related species was visualised by constructing a Splits graph.

Phylogenetic analyses
The  Fig. 1 where the isolates from this study formed two distinct, well-supported lineages (MLBS = 100%, BIPP = 1.00) and, thus, were considered to represent two previously-unknown species.
Asexual state. Stromata byssoid, pale yellow, formed directly on margin of host scales with 1-6 sporodochia. Sporodochia conical, erupted, yellowish, scattered or aggregated. Macroconidia 73-89 long, 6.9-10.6 µm wide (x -= 78.8 × 8.5 μm, n = 50), hyaline, cylindrical, slightly curved, slender towards each end, 2-7 septa, mostly 4-6 septa, slightly constricted at septum, difficult to distinguish apical cell and basal cell. Microconidia and chlamydospores were not observed. Culture characters. Ascospores germinate on PDA within 12 h and cultures grow slowly on PDA. Colonies reach 2.4 cm in diameter after 20 days. Colonies from single conidia flocculent, clinging to medium, with irregular margin, white to pink mycelium on surface and back of colonies dark orange. Mycelium creamy-white starting at centre, but gradually becoming pale pink after 20 days, forming sparsely distributed mycelial clumps near edge of colony. Conidia germinate on PDA within 12 h, cultures grow slowly on PDA. Colonies 2.5 cm in diameter after 20 days. Colonies from single ascospores cottony and hard, with regular margin; mycelium creamy-white to pale pink, with concentric rings; back of colonies pale yellow.

Discussion
In this study, two new species (Microcera chrysomphaludis and M. pseudaulacaspidis) associated with scale insects from walnut were introduced, based on phylogenetic inferences of a combined ITS, LSU,acl1,act,cmdA,his3,rpb1,rpb2 and tub2 DNA sequence dataset and morphological evidence. Ecologically, Microcera species are mainly distributed in tropical regions, but they have also been reported in the subtropical and temperate regions. Most of the Microcera species are pathogens of scale insects (Gräfenhan et al. 2011;O'Donnell et al. 2012;Dao et al. 2015Dao et al. , 2016Crous et al. 2021a;Xu et al. 2021), However, two new species have recently been described from lichens (Crous et al. 2021b(Crous et al. , 2022a. Most Microcera species infecting scale insects occur in the tree canopy and are more noticeable under moist conditions (Dao et al. 2015(Dao et al. , 2016Xu et al. 2021), consistent with the findings of this study. Morphologically, the sexual morph in this genus is characterised by orange to dark red perithecia with a blunt papilla producing cylindrical to narrowly clavate asci and 1(-3)-septate ascospores, while the asexual morph is predominantly fusarium-like, with verticillate to penicillate conidiophores producing small macroconidia (Gräfenhan et al. 2011;Lombard et al. 2015;Crous et al. 2021a, b). Similar morphs were observed and documented in this study to provide further evidence of a connection between our isolates and other Microcera species (e.g. Figs 4, 5). Gräfenhan et al. (2011) analysed an association of Microcera to Fusarium, Cladosterigma Pat., Mycogloea L.S. Olive and Tetracrium Henn. and accepted four species in Microcera. In recent years, numerous newly-discovered species have been described by employing extensive sampling coupled with multigene phylogenies (Sung et al. 2007;Lombard and Crous 2012;Wei et al. 2019;Lucking et al. 2021). Lombard et al. (2015) performed a multi-gene phylogenetic analysis, using combined datasets of ITS, LSU, tef1-α, acl1, act, cmdA, his3, rpb1, rpb2 and tub2 to clarify intraspecific and intergeneric relationships within Nectriaceae. In this paper, M. pseudaulacaspidis was distinguished from M. kuwanaspidis and established as a new species, based on base-pair differences, particularly in the LSU (17.67%), tef1-α (3.22%) and his3 (3.82%). Additionally, M. chrysomphaludis formed a distinct and well-supported subclade and was found to be morphologically distinct from M. coccophila in terms of the size of asci, ascospores and macroconidia (Gräfenhan et al. 2011;O'Donnell et al. 2012;Dao et al. 2015). Through multigene phylogenetic analysis, the connection between the sexual and asexual morphs of M. chrysomphaludis was also confirmed.
Entomopathogenic fungi are common on scale insects and have great potential in biological control (Zha et al. 2019;Sharma et al. 2020). Based on field trials, Microcera larvarum has been reported to have a significant biological control effect of Saissetia oleae, an economically important pest of olive and citrus plants (Cozzi et al. 2002). Microcera species have also been exploited for various biopharmaceuticals in recent years due to their secondary metabolites with medicinal properties. For instance, parnafungins, extracted from M. larvarum, have intrinsic antifungal activity (Parish et al. 2008). Isaka et al. (2015) isolated two new ascochlorin derivatives from cultures of Microcera sp. BCC 17074 and demonstrated their significant cytotoxic activities against various cancer cells. Furthermore, Cadelis et al. (2020) isolated four new secondary metabolites from M. larvarum isolates, which exhibited potent antimicrobial activity.
This paper presents novel findings of two new entomopathogenic fungi, Microcera chrysomphaludis and M. pseudaulacaspidis, which were isolated from scale insects found on walnut trees in China. We conducted surveys in numerous walnut orchards across Sichuan Province and observed significant infections of scale insects by these two species, resulting in high mortality rates, particularly in wet and humid conditions. Further screening and evaluation of these entomopathogenic fungi could facilitate their potential use as commercial biological control agents.