Morphological and phylogenetic resolution of <i>Conoideocrella luteorostrata</i> (Hypocreales: Clavicipitaceae), a potential biocontrol fungus for <i>Fiorinia externa</i> in United States Christmas tree production areas

The entomopathogenic fungus <i>Conoideocrella luteorostrata</i> has recently been implicated in natural epizootics among exotic elongate hemlock scale (EHS) insects in Christmas tree farms in the eastern United States. Since 1913, <i>C. luteorostrata</i> has been reported from various plant feeding Hemiptera in the southeastern United States, but comprehensive morphological and phylogenetic studies of U.S. populations are lacking. The recovery of multiple strains of <i>C. luteorostrata</i> from mycosed EHS in North Carolina provided an opportunity to conduct pathogenicity assays and morphological and phylogenetic studies to investigate genus- and species-level boundaries among the Clavicipitaceae. Pathogenicity assays confirmed that <i>C. luteorostrata</i> causes mortality of EHS crawlers, an essential first step in developing this fungus as a biocontrol. Morphological studies revealed that conidia aligned with previous measurements of the <i>Paecilomyces</i>-like asexual state of <i>C. luteorostrata</i>, with conidiophore morphology consistent with historical observations. Additionally, a <i>Hirsutella</i>-like synanamorph was observed in select <i>C. luteorostrata</i> strains. In both a four-locus, 54-taxon Clavicipitaceae-wide phylogenetic analysis including D1–D2 domains of the nuclear 28S rRNA region (28S), elongation factor 1 alpha (<i>EF1-α</i>), DNA-directed RNA polymerase II subunit 1 (<i>RPB1</i>), and DNA-directed RNA polymerase II subunit 2 (<i>RPB2</i>) and a two-locus, 38-taxon (28S and <i>EF1-α</i>) phylogenetic analysis, all three <i>Conoideocrella</i> species were resolved as strongly supported monophyletic lineages across all loci and both methods (maximum likelihood and Bayesian inference) of phylogenetic inference except for 28S for <i>C. tenuis</i>. Despite the strong support for individual <i>Conoideocrella</i> species, none of the analyses supported the monophyly of <i>Conoideocrella</i> with the inclusion of <i>Dussiella</i>. Due to the paucity of <i>RPB1</i> and <i>RPB2</i> sequence data, <i>EF1-α</i> provided superior delimitation of intraspecies groupings for <i>Conoideocrella</i> and should be used in future studies. Further development of <i>C. luteorostrata</i> as a biocontrol against EHS will require additional surveys across diverse Hemiptera and expanded pathogenicity testing to clarify host range and efficacy of this fungus.