Insight into the Systematics of Novel Entomopathogenic Fungi Associated with Armored Scale Insect, Kuwanaspis howardi (Hemiptera: Diaspididae) in China

This study led to the discovery of three entomopathogenic fungi associated with Kuwanaspis howardi, a scale insect on Phyllostachys heteroclada (fishscale bamboo) and Pleioblastus amarus (bitter bamboo) in China. Two of these species belong to Podonectria: P. kuwanaspidis X.L. Xu & C.L. Yang sp. nov. and P. novae-zelandiae Dingley. The new species P. kuwanaspidis has wider and thicker setae, longer and wider asci, longer ascospores, and more septa as compared with similar Podonectria species. The morphs of extant species P. novae-zelandiae is confirmed based on sexual and asexual morphologies. Maximum likelihood and Bayesian inference analyses of ITS, LSU, SSU, tef1-α, and rpb2 sequence data provide further evidence for the validity of the two species and their placement in Podonectriaceae (Pleosporales). The second new species, Microcera kuwanaspidis X.L. Xu & C.L. Yang sp. nov., is established based on DNA sequence data from ITS, LSU, SSU, tef1-α, rpb1, rpb2, acl1, act, cmdA, and his3 gene regions, and it is characterized by morphological differences in septum numbers and single conidial mass.


Specimen Collection and Morphological Study
During spring to autumn from 2018 to 2021, the specimens were collected from the bamboo forests located in Ya'an City and a neighboring county (Sichuan Province, China), where the environment is characterized by river valley terraces and intermountain basins and a subtropical monsoon humid climate with abundant natural resources, and it is the transition zone from Qinghai-Tibet Plateau to Chengdu Plain. Specimens documented with host, locality, time, and distribution of taxa were returned to the laboratory in suitable containers separately with the collection detail tag, and the substrate with fruiting bodies was checked following the methods described in Senanayake et al. [24]. The fungi were isolated into pure culture using single conidium obtained from sporodochia and single ascospore from ascomata parasitic on Kuwanaspis howardi following the isolation via spore suspension detailed in Chomnunti et al. [25]. The spore suspension was sucked into a Pasteur pipette, small drops were placed on isolation media (potato dextrose agar, PDA) in an incubator (20 • C). Then the plates were examined for single germinated spores under a dissecting microscope, and germinating spores were transferred separately to at least three new PDA plates. After incubation on PDA plates at 20 • C for 20 to 40 days depending on the growth rate, colonies were examined for their diameter, shape, and appearance. Ascomata and sporodochia were observed and photographed using a dissecting microscope NVT-GG (Shanghai Advanced Photoelectric Technology Co. Ltd., Shanghai, China) fitted with a VS-800C micro-digital camera (Shenzhen Weishen Times Technology Co. Ltd., Shenzhen, China). Dimensions of asci, ascospores, pseudoparaphyses, hairs, ascomata wall, conidia, conidiophores, and numbers of septa were based on field samples and were photographed using an Olympus BX43 compound microscope fitted with an Olympus DP22 digital camera in association with ACDSee v3.1 software. Measurements were made using Tarosoft ® Image Frame Work v.0.9.7 (Tarosoft (R), Nontha Buri, Thailand). Lactophenol cotton blue reagent was used to observe the number of septa. The gelatinous appendage was observed in Black Indian ink. The type specimens were deposited at the Herbarium of Sichuan Agricultural University, Chengdu, China (SICAU). The extype cultures were deposited at the Culture Collection in Sichuan Agricultural University (SICAUCC), and MycoBank numbers are registered (http://www.MycoBank.org, accessed on 10 January 2021).

Phylogenetic Analyses
Phylogenetic analyses of a combined five-gene dataset (ITS, LSU, SSU, tef1-α, rpb2) Phylogenetic trees generated from ML and BI analyses were similar in overall topologies. Phylogeny from the combined sequence data analysis indicates that all the Pleosporalean families are monophyletic with strong bootstrap support values (Figure 1). Three species grouped with taxa in Podonectria with 100% ML and 1.00 BYPP support. A species (SICAUCC 21-0004, SICAUCC 21-0005) clustered with P. novae-zelandiae in a clade with 99% ML and 1.00 BYPP statistical support. Our novel species P. kuwanaspidis constitutes a moderately supported independent lineage (82% ML/-BYPP statistical support) between P. novae-zelandiae and P. coccicola.
DNA sequences of four known species of Microcera and our new taxon, M. kuwanaspidis, were used in the analyses. The combined dataset comprised 24 taxa within Nectriaceae and two outgroup taxa in Tilachlidiaceae ( Table 2) Parasitic fungus on scale insects, other fungi, or substrates previously colonized by other fungi. Sexual morph: Stromata byssoid, well-developed or scant, white to brown or dark-brown. Ascomata solitary or aggregated, superficial on or immersed in the stroma, globose to subglobose, obpyriform or ovoid, cream white to light yellow, or brown to dark brown, covered with hairs or absent. The hamathecium comprises numerous reticulate, filiform, septate, branched, pseudoparaphyses. Asci 8-spored, bitunicate, long clavate to cylindric. Ascospores long clavate to long cylindric, or vermiform, multiseptate. Asexual morph: Tetracrium-like. Sporodochia formed directly on cushion-shaped, white, orange, or brown, and hard stroma. Conidiophores moniliform. Conidia usually 1-4 "arms", narrowed toward the apex, joined at the basal cell, multiseptate.
Notes: The family Podonectriaceae was introduced to accommodate Podonectria by Dao et al. [5], in which descriptions of conidia, ascomata, asci, and ascospores were lacking. Here we emend those descriptions and the habitats of Podonectriaceae with the inclusion of fungi or substrates previously colonized by other fungi and not only scale insects [4,5,8,12]. This broadens the taxonomic concept of Podonectria, which is further supported by molecular analyses in this study.

Figure 2.
Phylogram generated from RAxML analysis based on combined ITS, LSU, tef1-α, rpb1, rpb2, acl1, act, tub2, cmdA, and his3 sequence data of Microcera isolates. Bootstrap support values for maximum likelihood (ML, left) higher than 70% and Bayesian posterior probabilities (BYPP, right) equal to or greater than 0.95 are indicated at the nodes respectively. The sequences from ex-type strains are in bold. The newly generated sequence is in red bold.
Notes: Here, we follow the recommendation of Rossman et al. [62] by adopting Podonectria over Tetracrium. The asexual morph of P. novae-zelandiae was reported by Dao et al. [5], and was supported with morphology and molecular data. Our observations agree with the descriptions provided by Rossman [4] and Dao et al. [5]. Nucleotide comparison of ITS and LSU (SICAUCC 21-0005) reveals high similarity to P. novae-zelandiae (isolate PUcS13, similarities = 473/476 (99%), 0 gaps (0%); similarities = 517/518 (99%), 0 gaps (0%), respectively) in Dao et al. [5]; however, the latter lack SSU, tef1-α, and rpb2 sequences for further comparisons. The conidia produced here in culture were similar to those on scale insects in the field.  Culture characters: Ascospores germinating on PDA within 12 h, and the cultures grow slowly on PDA. Colonies reach 2 cm in diameter after 25 days. Colonies from single ascospores are cottony, cling to the medium, with regular margin; the mycelium is creamy white to pale yellow but gradually becomes pale brown after 30 days.
Nectriaceae  Culture characters: Colonies from a single macroconidium on PDA grow slowly and reach approximately 2.2 cm in diameter after 12 days at 25 • C, circular, flat, whitish to bright orange with white mycelium on the surface forming concentric circles, and the back of colonies is bright orange.

Discussion
Mycologists have questioned the exact familial placement of Podonectria since the beginning of its establishment. Dingley [3] placed the genus in Clavicipitaceae (Hypocreales). Rossman transferred it into the Pleosporaceae (Pleosporales) due to its bitunicate asci rather than the unitunicate asci found in Hypocreales [4,7]. Barr transferred Podonectria to Tubeufiaceae [65], which was erected [66] to accommodate pleosporaceous taxa that are typically hyper saprobic on other fungi or substrates previously colonized by other fungi, hyperparasitic on foliicolous fungi, parasitic on scale insects, or occasionally parasitic on living leaves. This treatment was followed by subsequent authors [9,[67][68][69]. However, Tubeufiaceae, which was comprehensively reviewed by Boonmee et al. [70], was accommodated in a new order, Tubeufiales [58]. This placement was followed by Wijayawardene et al. [71,72] and Hongsanan et al. [73]. However, Dao et al. [5] proposed Podonectriaceae, a new family in Pleosporales, to accommodate this genus, which was confirmed by ITS and LSU data. This placement was supported by Yang et al. [12], in which Podonectria sichuanensis was identified based on morphological characteristics and phylogenetic analyses. Based on the phylogenetic results of combined ITS, LSU, SSU, tef1-α, and rpb2 data in this current study, we confirm Podonectriaceae as an accepted family in the suborder Pleosporineae [49]. Podonectriaceae is phylogenetically closely related to Pseudopyrenochaetaceae that has been established to accommodate two species, viz. Pseudopyrenochaeta lycopersici and P. terrestris [57]. However, the two families are morphologically distinct. Pseudopyrenochaetaceae has pycnidial conidiomata, filiform conidiophores, and aseptate, cylindrical to allantoid conidia, whereas Podonectriaceae comprises sporodochial conidiomata, moniliform or inconspicuous conidiophores, and 1-4 armed, multiseptated conidia. In addition, the coelomycete genera Tetranacrium that has septate tetraradiate conidia [74,75] was documented as the anamorph associated with Podonectria gahnia according to substrate observation [4]. However, the association is somewhat confused, as it lacks further phylogenetic investigations and taxonomic studies. Identical molecular sequences of Podonectria novae-zelandiae in our study confirmed the link between the sexual morphs and asexual morphs in Tetracrium. Podonectria was reported to be associated with scale insects on various hosts in previous studies [1,4,5,58]. In this paper, we isolated Podonectria sichuanensis (SICAUCC 21-0001) on the ascomata of Neostagonosporella sichuanensis in our sampling site and confirm that the Podonectria species are not only parasitic on scale insects but also on other fungi or substrates previously colonized by other fungi [12]. According to published studies, most species of Podonectria are associated with armored scale insects, in addition to being associated with the mostly reported hosts Citrus aurantium L. and C. nobilis Gräfenhan et al. [18] reported an association of Microcera to Fusarium, Cladosterigma Pat., Mycogloea L.S. Olive, Tetracrium Henn., and accepted four species in Microcera. Nowadays, taxonomic concepts based on multi-gene phylogenetic inference have provided a deeper understanding of phylogenetic relationships than those based on individual gene regions [76][77][78][79]. Recently, combined ITS-LSU-tef1-α-acl1-act-cmdA-his3-rpb1-rpb2-tub2 datasets were used to clarify intraspecific and intergeneric relationships within Nectriaceae [19], and combined ITS-LSU-tef1-α-cmdA-rpb2-tub2 datasets were similarly used for Hypocreales [80]. In this paper, Microcera kuwanaspidis can be distinguished from M. coccophila and is established as new species on account of base-pair differences, especially in the tef1-α (13%), acl1 (4%), act (3%), cmdA (5%), and his3 (9%). The Microcera species have been mostly reported associated with armored scale insects on citrus (Rutaceae), viz. Aonidiella aurantii, A. citrina, Lepidosaphes beckii, Unaspis citri, and Quadraspidiotus perniciosus on Pyrus communis, Prunus domestica and P. cerasus (Rosaceae), as well associated with nut scale Eulecanium tiliae (Hemiptera: Coccidae) on Salix sp. (Salicaceae) and Fraxinus excelsior (Oleaceae), and an unknown scale insect on Broussonetia kazinoki × B. papyrifera (Moraceae), Laurus nobilis (Lauraceae), Citrus maxima (Rutaceae), and apple trees [18,60,64].
In China, the entomopathogenic fungi associated with scale insects was mainly focused on commercial Citrus plants in the 1990s. Verticillium lecanii (Zimm) Viegas is the most common fungus that is parasitic on scale insects on Citrus since its discovery from Guizhou Province in 1982 [81]. Subsequently, Aschersonia duplex Berk., Beauveria bassiana (Bals.-Criv.) Vuill., Fusarium juruanum Henn., F. moniliforme Sheld., Microcera coccophila, Nigrospora sphaerica (Sacc) Mason, and Podonectria coccicola have also been reported to be associated with the scale insects on citrus [82][83][84]. Microcera and Podonectria were commonly encountered on scale insects within tree canopies and occurred throughout the year but were more noticeable under wet and humid conditions [5,64,85,86], consistent with the observations in this study. Presently, Microcera coccophila and Podonectria coccicola have been the most commonly and worldwide recorded species on scale insects, especially on orange trees [1, 4,7,[85][86][87][88][89]. This paper provides new records for three entomopathogenic fungi, Podonectria kuwanaspidis, P. novae-zelandiae, and Microcera kuwanaspidis on armored insect scale from bamboo in China. According to the field observation from 2015 to 2020, the three species are commonly associated with Kuwanaspis howardi on native bamboo, especially on Phyllostachys heteroclada, and they effectively cause the scale insect hosts to be infected, which ultimately results in death. As documented by Rossman [4] and Dao et al. [64], the role of entomopathogens in the biological control of destructive scale insects on citrus trees was usually controlled by chemical sprays. These entomopathogenic fungi should be further screened to assess their potential for commercial development as biological control agents.