Colletotrichum nymphaeae var . entomophilum var . nov . a natural enemy of the citrus scale insect , Praelongorthezia praelonga ( Hemiptera : Ortheziidae )

The citrus scale insect Praelongorthezia praelonga (Douglas), a major pest of citrus and other economically important crops, has only two commonly documented natural enemies: an entomopathogenic strain of the fungus Colletotrichum nymphaeae (Pass.) Aa and several parasitoids. The entomopathogenic strain of C. nymphaeae, formerly recognized under the synonym C. gloeosporioides f. sp. ortheziidae, is under development for commercial application as a biological control agent in citrus in Brazil-the top exporter of citrus globally. The synonomy of C. gloeosporioides f. sp. ortheziidae with C. nymphaeae remains based on limited DNA sequence data and without morphological study. To qualify for future approval as a biological control agent by federal agencies in Brazil and the European Union, the circumscription of a microorganism must be explicit and without ambiguities. Herein, through morphological study and phylogenetic analysis of five DNA regions we clarify the circumscription and affinity of entomopathogenic C. nymphaeae and describe it as a new variety.


Introduction
Colletotrichum Corda (Ascomycota: Sordiariomycetes: Glomerallaceae) is a cosmopolitan and speciose genus of fungi comprised largely of plant symbionts Hanlin, 1998). The host range within the genus is broad with both pathogenic and endophytic species reported from all major lineages of land plants (MacKenzie et al., 2009;Manamgoda et al., 2013;Photita et al., 2005). Two Colletotrichum taxa are entomopathogenic: C. fiorinae and C. nymphaeae. Colletotrichum fiorinae was described from the hemlock scale insect Fiorna externa Ferris; however, C. nymphaeae is a widespread plant pathogen in which entomopathogencity is an exception and limited to strains isolated from the citrus scale insect Praelongorthezia praelonga (Douglas) (syn. Orthezia praelonga Douglas) (Hemiptera: Ortheziidae) (Marcelino et al., 2008).
Entomopathogenic C. nymphaeae is of particular interest because the citrus scale insect (P. praelonga), its primary host, is a serious pest of Citrus spp. and other major economic plants such as coffee, figs and ornamentals (Garcia-Roa, 1995). Citrus scale is considered a pest wherever it is found because of its elevated reproductive rate and highly polyphagous nature (Kondo et al., 2013). The regularly occurring natural enemies of citrus scale are currently limited to C. nymphaeae and several parasitoids (Kondo et al., 2013;Ramos et al., 2018). Studies on entomopathogenic C. nymphaeae have focused on its utility and development as a biological control agent of P. praelonga in citrus production (Teixeira et al., 2001;Teixeira et al., 2004). Natural outbreaks of the fungus with high mortality of citrus scale are observed in conventional citrus groves; outbreaks appear to be density dependent with increased prevalence during rainy and warm weather (Mascarin et al., 2016).
In Brazil, C. nymphaeae on citrus scale is colloquially known as the salmão (salmon) fungus-a reference to the characteristic salmon pink color of the conidial masses on infected insects. The salmão fungus, previously C. gloeosporioides f. sp. ortheziidae, was transferred to C. nymphaeae based on a single locus and without a comparative morphological study Marcelino et al., 2008). The designation formae speciales (f. sp.) was dropped by this transfer; therefore, entomopathogenic isolates of C. nymphaeae are no longer distinguished by name from its plant pathogenic conspecifics. Because of its potential for biological control, the taxonomy and affinity of the salmão fungus require further study. In this study, through morphological and molecular studies, we take a polyphasic approach to clarifying the taxonomy of this important natural enemy of the citrus scale insect.

Fungal isolation and preservation
Two isolates of Colletotrichum sp., collected from the citrus scale insect, were purified in vitro on potatodextrose-agar and subsequently deposited in the Laboratório de Patologia e Controle Microbiano, Departamento de Entomologia e Acarologia of the Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo (USP/ESALQ

Morphological study
Morphological study and descriptions were made from observations of fungal structures mounted in wa-

Agricultural Microbiology
Research Article scale insect, Praelongorthezia praelonga (Hemiptera: Ortheziidae) Insect pathogenic C. nymphaeae Sci. Agric. v.77, n.5, e20180269, 2020 ter on a glass slide. Measurements and light photomicrographs were taken using an Olympus AX70 Provis compound light microscope and Cell ˄A analysis image processing software as well as an Olympus SZX16 dissecting microscope. Herbarium acronyms followed those of the Index Herbariorum (Thiers, 2018). Morphological characters were recorded from cultures grown on sabouraud dextrose agar (SDA), oatmeal agar (OA) (Crous et al., 2009), and synthetic nutrient-poor agar medium (SNA) (Nirenberg, 1976) and maintained at 22 °C with a light regime of 18 h of darkness and 6 h of light. Figures were assembled with Adobe Photoshop CS4.

Molecular study
Genomic DNA was extracted from C. nymphaeae isolates from infected P. praelonga individuals collected from citrus orchards in Brazil. One isolate, ARSEF 4360, was obtained from the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) and ESALQ 1393 was obtained from the culture collection of the Laboratório de Patologia e Controle Microbiano, Universidade de São Paulo (USP/ESALQ). DNA from ESALQ 1393 and for ARSEF 4360 were extracted as described in Mascarin et al. (2016); genomic DNA was obtained by grinding the mycelium with a plastic pestle inside a 1.5 mL Eppendorf tube. DNA was then isolated using a commercial plant DNA extraction kit using the standard protocol and eluted with 100 µL sterile deionized water.
Using the newly obtained sequences as well as sequences from GenBank, including those generated by Damm et al. (2012), five datasets were produced: ITS and TUB2 datasets each having 77 sequences, and glyceraldehyde-3-phosphate dehydrogenase (GADPH), CHS-1 and ACT datasets each having 76 sequences. A total of three strains of C. nymphaeae isolated from P. praelonga were included in the molecular study: ESALQ 1368, ES-ALQ 1393, ARSEF 4360; however, only the last two, for which multiple loci were obtained, were included in the combined phylogenetic analysis. The taxa and isolates used in the study are shown in Table 2.
The individual ITS, GDPH, CHS-1, ACT, TUB2 matrices were exported in NEXUS format and converted to a combined data file in PAUP* v. 4.0.10b. The individual datasets were analyzed by maximum parsimony using a heuristic search with random addition sequence, TBR swapping and 1,000 heuristic replicates, saving no more than ten best trees per replicate, followed by a final search of saved trees. The phylograms of each dataset were visually inspected for topological congruence and then combined into a single dataset. The combined dataset was bootstrapped (2,000 replicates) using the same maximum parsimony search parameters. Following Damm et al. (2012), C. orchidophilum was used as the outgroup for the C. acutatum complex. A final phylogram with added bootstrap values was prepared using the Adobe Illustrator Professional. The combined dataset was deposited in TreeBase and can be accessed at http:// purl.org/phylo/treebase/phylows/study/TB2:S18116

Molecular study
The combined ITS+GDPH+CHS-1+ACT+TUB2 dataset included 1,661 characters of which 1,233 were constant, 50 were variable but not parsimony-informative, and 378 were parsimony-informative. A total of 600 equally most-parsimonious trees of 432 steps were recovered: a phylogram of one of these trees is shown in Figure 1. Isolates from the citrus scale insect are in a well-supported C. nymphaeae clade [97 % bootstrap support (bs)] ( Figure 1). The combined dataset recovered two major clades for C. nymphaeae with respective bootstrap support values of 86 % and 89 %. The citrus  scale insect isolates are in the same clade as isolate CBS 515.78, the type of C. nymphaeae designated as C. nymphaeae var. nymphaeae in Figure 1. In the beta-tubulin dataset, a single nucleotide mutation was present in all four entomopathogenic isolates of C. nymphaeae: this mutation is shared only by one other species, C. australe, which is also in the C. acutatum complex, but distantly related to C. nymphaeae (Table 3; Figure 1). Isolates of C. nymphaeae from the citrus scale insect differed from all other C. nymphaeae isolates by two nucleotide changes in the CHS-1 gene (Table 4). Insect pathogenic C. nymphaeae, colloquially referred to as the salmão fungus is described herein as a new variety of C. nymphaeae.
MycoBank 823559 Figure 2A-H = C. gloeosporioides f. sp. ortheziidae (Marcelino et al., 2008) Etymology: The varietal epithet, which means insect loving, indicates its predilection for insects.     (Figure 2C). Appressoria reniform, brown, smooth-walled, margin entire, 5.9 -9.8 × 3.9 -7.7 µm ( Figure 2F). Setae on OA rare, brown, thick-walled, irregular margin, tip acute, 68.5 -73.1 × 3.4 -3.7 µm ( Figure 2D and E). Host: Praelongorthezia praelonga. Figure 2G-H. Diagnosis: Collectrichum nymphaeae var. entomophilum is distinguished morphologically from C. nymphaeae var. nymphaeae by its smaller conidia 6.4 -16 × 3.2 -5.4 mm, ave. 10.9 × 4.2 mm and the presence of setae ( Colonies on OA 10.5 cm at seven days, a low cottony mycelium with concentric alternating white, salmon and grey rings ( Figure 2A); salmon color from numerous small acervuli oozing conidia; culture in reverse pale greyish-brown with a greyish-salmon center. On C. nymphaeae is 16.1 × 4.9 µm with the exception of one strain, CBS 526.77, which was reported as having conidia measuring 9 -13 × 3 -4.5 µm . In addition to morphological differences, C. nymphaeae var. entomophilum is distinct from all other isolates of C. nymphaeae by a single nucleotide mutation in the betatubulin gene (Table 3), two nucleotide mutations in the CHS-1 gene (Table 4) and by its insect, rather than plant host preference. Additional notes. Colletotrichum nymphaeae var. entomophilum more closely matches the description of Gloeosporium (Colletotrichum) nymphaeae Hemmi and Kawase, isolated from leaves of the Nymphaea in Japan (Hemmi and Kawase, 1954), than the description of C. nymphaeae sensu Damm et al. (2012). Hemmi and Kawase report dark brown thick-walled setae, 19 -76 µm long and conidia ranging from 9 -17 × 3 -6 µm. Unfortunately, the specimens cited by Hemmi and Kawasi (1954) cannot be found and no living cultures exist. According to Damm et al. (2012), C. nymphaeae as currently circumscribed is probably not the same organism described by Hemmi and Kawasi primarily because setae have never been observed in C. nymphaeae, neither by Damm et al. (2012) nor by van der Aa (1978); however, Batista and Bezerra (1966) described abundant setae in citrus orthezia insects infected by a "special strain" of C. gloeosporioides. Batista and Bezerra (1966) did not cite specimens in their study; however, the special strain they discovered and reported for the first time was likely C. nymphaeae var. entomophilum.

Discussion
Delineating taxa and resolving the relationships within Colletotrichum is a challenge because of high sequence homogeneity and morphological uniformity within the genus. For this reason, multiple genes are required not just for elucidating the relationships between taxa but also for species identification. Using DNA sequence data from five gene regions: GDPH+TUB2+ITS+ACT+CHS-1, we found strong support for including the insect pathogenic isolates of C. nymphaeae (formerly C. gloeosporioides f. sp. ortheziidae in the species C. nymphaeae (97 % bs) (Figure 1). On the basis of morphological and molecular characters we recognize the insect pathogenic isolates of C. nymphaeae as a new variety and designate the name C. nymphaeae var. entomophilum. A single nucleotide mutation in the GADPH gene, two mutations in CHS-1, and the presence of setae readily distinguishes this new variety from its closest relatives. Once additional informative markers are found for Colletotrichum, it may be possible to better resolve the relationships within C. nymphaeae and determine if C. nymphaeae var. entomophilum should continue to be recognized as a variety or as a distinct species. Until then, the circumscription of C. nymphaeae sensu Damm et al., 2012, should be modified to include setae. Setae are present in C. nymphaeae var. entomophilum but have so far not been observed in plant symbiont isolates of C. nymphaeae. In order to clarify their taxonomic utility, further studies should be conducted to confirm the presence of setae in situ and to elucidate any effects that sub-culturing or other environmental conditions might have on the production of setae in vitro.
Both entomopathogenic Colletotrichum taxa, C. nymphaeae var. entomophilum and C. fiorniae, display remarkably diverse lifestyle strategies with the ability to live as insect pathogens, plant pathogens and endophytes. At least 50 phytopathogenic isolates of C. fiorinae have been identified by DNA sequencing . Additionally, C. fiorinae has been isolated from fruit rot and detected as an endophyte in 28 species of plants in the region of epizootic outbreaks on the hemlock scale insect Marcelino et al., 2009). Colletotrichum nymphaeae var. entomophilum has also been recovered as an endophyte but only in plants inoculated with the fungus (Marcelino et al., 2009). The occurrence of C. nymphaeae var. entomophilum in nature, either as an endophyte or on additional insect hosts has not been studied. This is an area of research that merits prompt attention given that endophytic entomopathogens may serve as defensive mutualists when living in plant tissue (Bultman and Faeth, 2002;Crouch et al., 2014;Redman et al., 2002). For example, if C. nymphaeae var. entomophilum occurs endophytically in nature, it may potentially play a larger role in controlling citrus scale and other sap-sucking insects than previously realized.
The significance of the varied lifestyle strategies of C. fiorniae and C. nymphaeae var. entomophilum has previously been downplayed because the insect hosts of these fungi are plant sucking insects . A close relationship between insect pathogenic fungi and grass endosymbionts has previously been shown (Spatafora et al., 2007). Comparative genomic analyses showed that insect pathogenic Metarhizium spp. are more closely related to endophytes and plant pathogens compared to animal pathogens (Gao et al., 2011). The evolutionary transition from plant pathogen to endophyte in Colletotrichum is thought to occur relatively easily; for example, in at least one species of Colletotrichum, a single-locus mutation converts the fungus from a symptomatic plant pathogen to an endophyte. However, transitioning from plant pathogen or endophyte to a pathogen of plant sucking insects may represent a more significant and complex physiological shift than is assumed with genes co-opted, evolved or acquired by horizontal gene transfer from a plant-associated fungus (Barelli et al., 2016). Gene expression in Colletotrichum is highly dependent on plant signaling (Crouch et al., 2014;O'Connell et al., 2012). In vitro signaling has been shown to be markedly different from in planta signaling even from the point of spore germination to the ultimate necrotrophic phase characteristic of plant pathogens (O'Connell et al., 2012). Transcriptomic studies comparing gene expression of the entomopathogenic Colletotrichum taxa in planta, in insecta and in vitro may provide insight into the mechanisms behind inter-kingdom host shifts or the potential of a dual life style as insect pathogens and endophytes; in turn, these insights may help to determine how much or little weight should be given to host preferences for delimiting Colletotrichum taxa.
Much remains to be known about C. nymphaeae var. entomophilum. For example, how it is dispersed, its insect host-range, if it occurs endophytically both in nature and in citrus orchards and if endophytic growth affects the fitness of the citrus scale insect. Improved development of this important natural enemy should include studies on the below ground control of Ortheziidae scale insects since these insects also live in soil litter in humid habitats and feed on fungi, mosses and plant roots (Kondo et al., 2013). Entomopathogenic endophytes are well-known among the fungi, especially from the order Hypocreales e.g., Acremonium, Beauveria, Clonostachys, Isaria, Lecanicillium, Verticillium (Vega, Insect pathogenic C. nymphaeae Sci. Agric. v.77, n.5, e20180269, 2020 2008; Vega et al., 2008); however, few are aware that the common plant pathogenic genus Colletotrichum (Glomerellales) also contains endophytic entomopathogens. Clarifying the taxonomy of C. nymphaeae var. entomophilum may provide the impetus for further research on this overlooked entomopathogenic fungus. The assignment of a formal name along with morphological and molecular characterization provides a solid framework for facilitating the evaluation and approval of this important fungus for biological control.