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

ABSTRACT:

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.

Keywords:
Biological control; Colletotrichum (Sordariomycetes: Glomerellaceae); entomopathogen

Introduction

Colletotrichum Corda (Ascomycota: Sordiariomycetes: Glomerallaceae) is a cosmopolitan and speciose genus of fungi comprised largely of plant symbionts (Damm et al., 2012; 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 (Damm et al., 2012; 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.

Materials and Methods

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). Isolate ESALQ 1393 was collected in Limeira, state of Sao Paulo, 8 Feb 2007, Sylvio Baggio s.n., and isolate ESALQ 1368 from Cordeirópolis, state of Sao Paulo, 13 July 2005, Luiz Fernando Padulla s.n. Both isolates were preserved in a lyophilized state and in sterile 10 % glycerol at −80 °C as metabolically inactive strains.

Morphological study

Morphological study and descriptions were made from observations of fungal structures mounted in water 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.

Three loci were amplified: the entire nrDNA ITS region (ITS1-5.8S-ITS2), actin (ACT) and chitin synthase (CHS-1). The entire nrDNA ITS region (ITS1-5.8S-ITS2) was amplified using primer pairs ITS1F (Gardes and Bruns, 1993) + ITS4 (White et al., 1990), ACT and CHS-1 were amplified and sequenced using the primer pairs ACT-512F + ACT-783R and CHS-354R + CHS-79F (Carbone and Kohn, 1999), respectively. Primers and primer sequences are listed in Table 1. PCR was performed under the following conditions for ITS: step 1) 1 min at 95 °C, 2) 45 s at 95 °C, 3) 40 s, 50.8 °C, 4) 90 s at 72 °C, 5) return to step 2 34 times, 6) a final step of 10 min at 72 °C; for both ACT and CHS-1: step 1) 3 min at 95 °C, 2) 15 s at 95 °C, 3) 20 s at 58 °C, 4) 1 min at 72 °C, 5) return to step 2 34 times, 6) final step of 5 min at 72 °C. Samples were kept at 10 °C until electrophoresis was performed on a 1 % agarose TAE gel and visualized under UV light. PCR products were cleaned using a PCR purification kit and sent for sequencing. Sequences were assembled with Sequencher v. 5.3 and aligned manually in MEGA5 (Tamura et al., 2011).

Table 1
Primer sequences and references for loci amplified in this study: ACT = actin, CHS-1= chitin synthase 1 and ITS = Internal transcribed spacer (ITS) situated between the small-subunit ribosomal RNA.

Locus	Primers	Sequence (5'– 3')	Reference
ACT	ACT-512F	ATG TGC AAG GCC GGT TTC GC	Carbone and Kohn, 1999
	ACT-783R	TAC GAG TCC TTC TGG CCC AT	Carbone and Kohn, 1999
CHS-1	CHS-354R	TGG AAG AAC CAT CTG TGA GAG TTG	Carbone and Kohn, 1999
	CHS-79F	TGG GGC AAG GAT GCT TGG AAG AAG	Carbone and Kohn, 1999
ITS	ITS1F	CTT GGT CAT TTA GAG GAA GTA A	Gardes and Brunes, 1993
	ITS4	TCC TCC GCT TAT TGA TAT GC	White et al., 1990

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, ESALQ 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.

Table 2
Host information, geographical origin and ITS, ACT, CHS-1, GADPH and TUB2 GenBank accession numbers of the taxa/strains included in the molecular study. Types are designated by an asterisk. Insect hosts are underlined.

Species	Isolate/Strain	Host/substrate	Geographic origin	ITS	ACT	CHS-1	GADPH	TUB2
C. acerbum	CBS 128530*	Malus domestica	New Zealand	JQ948459	JQ949780	JQ949120	JQ948790	JQ950110
C. acutatum	CBS 144.29	Capsicum annuum	Sri Lanka	JQ948401	JQ949722	JQ949062	JQ948732	JQ950052
	CBS 112996*	Carica papaya	Australia	JQ005776	JQ005839	JQ005797	JQ948677	JQ005860
	CBS 979.69	Coffea arabica	Kenya	JQ948400	JQ949721	JQ949061	JQ948731	JQ950051
	CBS 370.73	Pinus radiata	New Zealand	JQ948351	JQ949672	JQ949012	JQ948682	JQ950002
	IMI 336479	Pistacia vera	Australia	JQ948367	JQ949688	JQ949028	JQ948698	JQ950018
	CBS 113006	Protea cynaroides	South Africa	JQ948390	JQ949711	JQ949051	JQ948721	JQ950041
C. australe	CBS 116478*	Trachycarpus fortunei	South Africa	JQ948455	JQ949776	JQ949116	JQ948786	JQ950106
	CBS 131325	Hakea sp.	Australia	JQ948456	JQ949777	JQ949117	JQ948787	JQ950107
C. brisbanense	CBS 292.67*	Capsicum annuum	Australia	JQ948291	JQ949612	JQ948952	JQ948621	JQ949942
C. cosmi	CBS 853.73*	Cosmos sp.	Netherlands	JQ948274	JQ949595	JQ948935	JQ948604	JQ949925
C. costaricense	CBS 330.75*	Coffea arabica	Costa Rica	JQ948180	JQ949501	JQ948841	JQ948510	JQ949831
	CBS 211.78	Coffea sp.	Costa Rica	JQ948181	JQ949502	JQ948842	JQ948511	JQ949832
C. cuscutae	IMI 304802*	Cuscuta sp.	Dominica	JQ948195	JQ949516	JQ948856	JQ948525	JQ949846
C. fioriniae	CBS 125396	Malus domestica	USA	JQ948299	JQ949620	JQ948960	JQ948629	JQ949950
	IMI 345578	Fragaria × ananassa	New Zealand	JQ948330	JQ949655	JQ948995	JQ948660	JQ949981
	CBS 126523	Berberis sp.	Netherlands	JQ948322	JQ949643	JQ948983	JQ948652	JQ949973
	CBS 786.86	Malus sylvestris	Italy	JQ948303	JQ949624	JQ948964	JQ948633	JQ949954
	CBS 112995	Malus domestica	USA	JQ948298	JQ949619	JQ949289	JQ948628	JQ949949
	CBS 981.69	Coffea arabica	Angola	JQ948327	JQ949648	JQ948988	JQ948657	JQ949978
	CBS 200.35	Rubus sp.	USA	JQ948293	JQ949614	JQ948954	JQ948623	JQ949944
	CBS 490.92	Solanum lycopersicum	New Zealand	JQ948326	JQ949647	JQ948987	JQ948656	JQ949977
	CBS 129948	Tulipa sp.	UK	JQ948344	JQ949665	JQ949005	JQ948674	JQ949995
	CBS 119292	Vaccinium sp.	New Zealand	JQ948313	JQ949634	JQ948974	JQ948643	JQ949964
C. godetiae	CBS 133.44*	Clarkia hybrida	Denmark	JQ948402	JQ949723	JQ949063	JQ948733	JQ950053
	CBS 129934	Prunus dulcis	Israel	JQ948431	JQ949752	JQ949092	JQ948762	JQ950082
	CBS 862.70	Sambucus nigra	Netherlands	JQ948437	JQ949758	JQ949098	JQ948768	JQ950088
	CBS 127561	Ugni molinae	Chile	JQ948442	JQ949763	JQ949103	JQ948773	JQ950093
C. guajavae	IMI 350839*	Psidium guajava	India	JQ948270	JQ949591	JQ948931	JQ948600	JQ949921
C. indonesiense	CBS 127551*	Eucalyptus sp.	Indonesia	JQ948288	JQ949609	JQ948949	JQ948618	JQ949939
C. johnstonii	IMI 357027	Citrus sp.	New Zealand	JQ948443	JQ949764	JQ949104	JQ948774	JQ950094
	CBS 128532*	Solanum lycopersicum	New Zealand	JQ948444	JQ949765	JQ949105	JQ948775	JQ950095
C. kinghornii	CBS 198.35*	Phormium sp.	UK	JQ948454	JQ949775	JQ949115	JQ948785	JQ950105
C. laticiphilum	CBS 112989*	Hevea brasiliensis	India	JQ948289	JQ949610	JQ948950	JQ948619	JQ949940
	CBS 129827	Hevea brasiliensis	Colombia	JQ948290	JQ949611	JQ948951	JQ948620	JQ949941
C. limetticola	CBS 114.14*	Citrus aurantifolia	USA, Florida	JQ948193	JQ949514	JQ948854	JQ948523	JQ949844
C. lupini	CBS 129944	Cinnamomum verum	Portugal	JQ948178	JQ949499	JQ948839	JQ948508	JQ949829
	CBS 513.97	Lupinus polyphyllus	Costa Rica	JQ948157	JQ949478	JQ948818	JQ948487	JQ949808
	CBS 466.76	Manihot utilissima	Rwanda	JQ948160	JQ949481	JQ948821	JQ948490	JQ949811
C. melonis	CBS 159.84*	Cucumis melo	Brazil	JQ948194	JQ949515	JQ948855	JQ948524	JQ949845
C. nymphaeae	CBS 130.80	Anemone sp.	Italy	JQ948226	JQ949547	JQ948887	JQ948556	JQ949877
	IMI 379162	Capsicum annuum	Zimbabwe	JQ948218	JQ949539	JQ948879	JQ948548	JQ949869
	CBS 129945	Olea europaea	Portugal	JQ948201	JQ949548	JQ948888	JQ948531	JQ949852
	IMI 360386	Pelargonium graveolens	India	JQ948206	JQ949527	JQ948867	JQ948536	JQ949857
	ESALQ 1368	Praelongorthezia praelonga	Brazil	—	—	—	—	KJ509200
	ESALQ 1393*	P. praelonga	Brazil	MG547976	MG572798	MG572799	—	KJ509199
	ARSEF 4360	P. praelonga	Brazil	EF593371	MG572800	MG572801	EF593346	EF593327
	CBS 361.79	Anemone coronaria	Netherlands	JQ948248	JQ949569	JQ948909	JQ948578	JQ949899
	CBS 126377	Fragaria × ananassa	Netherlands	JQ948233	JQ949554	JQ948894	JQ948563	JQ949884
	CBS 112202	Fragaria sp.	Spain	JQ948234	JQ949555	JQ948895	JQ948564	JQ949885
C. nymphaeae	CBS 129926	Litter	Thailand	JQ948216	JQ949537	JQ948877	JQ948546	JQ949867
	CBS 516.78	Nuphar luteum	Netherlands	JQ948198	JQ949519	JQ948859	JQ948528	JQ949849
	CBS 515.78*	Nymphaea alba	Netherlands	JQ948197	JQ949518	JQ948858	JQ948527	JQ949848
	CSL 455	Photinia sp.	UK	JQ948217	JQ949538	JQ948878	JQ948547	JQ949868
	EMA26	Praelongorthezia praelonga	Brazil	EF593372	—	—	EF593347	EF593328
	CBS 112992	Protea magnifica	South Africa	JQ948207	JQ949528	JQ948868	JQ948537	JQ949858
C. orchidophilum	CBS 631.80	Ascocenda sp.	USA	JQ948152	JQ949473	JQ948813	JQ948482	JQ949803
	CBS 632.80*	Dendrobium sp.	USA	JQ948151	JQ949472	JQ948812	JQ948481	JQ949802
C. paxtonii	CBS 502.97	Musa nana	West Indies	JQ948286	JQ949607	JQ948947	JQ948616	JQ949937
	IMI 165753*	Musa sp.	Saint Lucia	JQ948285	JQ949606	JQ948946	JQ948615	JQ949936
C. phormii	CBS 118191	Phormium sp.	South Africa	JQ948453	JQ949774	JQ949114	JQ948784	JQ950104
	CBS 118197	Phormium sp.	New Zealand	JQ948450	JQ949771	JQ949111	JQ948781	JQ950101
	CBS 483.82	Phormium tenax	New Zealand	JQ948451	JQ949772	JQ949112	JQ948782	JQ950102
C. pyricola	CBS 128531*	Pyrus communis	New Zealand	JQ948445	JQ949766	JQ949106	JQ948776	JQ950096
C. rhombiforme	CBS 129953 *	Olea europaea	Portugal	JQ948457	JQ949778	JQ949118	JQ948788	JQ950108
	CBS 131322	Vaccinium macrocarpum	USA	JQ948458	JQ949779	JQ949119	JQ948789	JQ950109
C. salicis	CBS 465.83	Araucaria excelsa	USA	JQ948468	JQ949789	JQ949129	JQ948799	JQ950119
	CBS 607.94*	Salix sp.	Netherlands	JQ948460	JQ949781	JQ949121	JQ948791	JQ950111
C. scovillei	CBS 126529*	Capsicum sp.	Indonesia	JQ948267	JQ949588	JQ948928	JQ948597	JQ949918
	CBS 120708	Capsicum annuum	Thailand	JQ948269	JQ949590	JQ948930	JQ948599	JQ949920
C. simmondsii	CBS 294.67	Carica papaya	Australia	JQ948277	JQ949598	JQ948938	JQ948607	JQ949928
	CBS 126524	Cyclamen sp.	Netherlands	JQ948281	JQ949602	JQ948942	JQ948611	JQ949932
C. sloanei	IMI 364297*	Protea cynaroides	Malaysia	JQ948287	JQ949608	JQ948948	JQ948617	JQ949938
C. tamarilloi	CBS 129814*	Theobroma cacao	Colombia	JQ948184	JQ949505	JQ948845	JQ948514	JQ949835
	CBS 129956	Solanum betaceum	Colombia	JQ948190	JQ949511	JQ948851	JQ948520	JQ949841
C. walleri	CBS 125472*	Coffea sp.	Vietnam	JQ948275	JQ949596	JQ948936	JQ948605	JQ949926
C. sp.	CBS 129810	Solanum betaceum	Colombia	JQ948179	JQ949500	JQ948840	JQ948509	JQ949830

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

Results

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.

Figure 1
One of 9,139 equally most-parsimonious phylograms from a maximum parsimony analysis of ITS + GADPH + CHS-1 + ACT + TUB2 combined sequence data from 73 isolates within the Colletotrichum acutatum species complex. Thickened branches lead to nodes with bootstrap support values ≥ 70 % (2000 replicates).

Table 3
Beta-tubulin gene sequence segment showing single nucleotide mutation present in all four isolates of C. nymphaeae var. entomophilum and shared by only one other taxon (C. australe) within the C. acutatum complex.

Taxon		Isolate	Beta-tubulin sequence
C. nymphaeae
	var. entomophilum	ARSEF 4360	TCAC-TCGTTCTCCAGTG
		EMA 26	TCAC-TCGTTCTCCAGTG
		ESALQ 1368	TCAC-TCGTTCTCCAGTG
		ESALQ 1393	TCAC-TCGTTCTCCAGTG
	var. nymphaeae	IMI 360386	TCAC-TCGTCCTCCAGTG
		CBS 515.78	TCAC-TCGTCCTCCAGTG
C. australe
		CBS 131325	TCTC-TTGTTCTCCAGTG
		CBS 116478	TCTC-TTGTTCTCCAGTG

Table 4
CHS-1 gene nucleotide differences between C. nymphaeae var. nymphaeae and C. numphaeae var. entomophilum.

Taxon	Isolate	Mutations and their position in the CHS-1 gene sequence
C. nymphaeae		217	247
var. entomophilum	ARSEF 4360	C	T
	ESALQ 1393	C	T
var. nymphaeae	CBS 515.78	T	C
	IMI 360386	T	C
	CBS 130.80	T	C

Taxonomy

Collectrichum nymphaeae var. entomophilum A.A. Wynns & I. Delalibera, var. nov.

MycoBank 823559 Figure 2A-H

Figure 2
A - H) Colletotrichum nymphaeae var. entomophilum (ESALQ 1393). A) culture on OA after eight days. B) culture on SNA after seven days. C) conidia. D - E) setae. F) appressorium. G - H) infected, sporulating, Praelongortheziidae praelonga cadavers. I - L) Plant pathogenic C. nymphaeae var. nymphaeae (CBS 515.78). I) culture on OA after seven days. J) culture on SNA after seven days. K) conidia. L) appressoria. Scale bars: A-J = 20 μm; C-F, K-L = 10 μm.

= C. gloeosporioides f. sp. ortheziidae (Marcelino et al., 2008)

Etymology: The varietal epithet, which means insect loving, indicates its predilection for insects.

Type: Limeira, state of São Paulo, isolated from a Praelongorthezia praelonga (Hemiptera: Ortheziidae) cadaver, 8 Feb 2007, Silvio Baggio s.n. Holotype, ESA 142963 permanently preserved in a metabolically inactive state as a dried culture in microscope slides of ESALQ 1393-H deposited in the herbarium of Escola Superior de Agricultura “Luiz de Queiroz” from the Universidade de São Paulo (USP/ESALQ/ESA - http://splink.cria.org.br/manager/detail?setlang=pt&resource=ESA).

Ex-holotype cultures are stored in lyophilized inactive state as ESALQ 1393-H in the Collection of Entomopathogenic Fungi, Laboratório de Patologia e Controle Microbiano, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo (USP/ESALQ), Piracicaba, state of São Paulo, and as CG1414 in the Invertebrate-Associated Fungal Collection of Embrapa (CG), Embrapa Genetic Resources and Biotechnology, DF-70770-970, Brazil (http://www.wfcc.info/ccinfo/collection/by_id/712).

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 SNA 9 cm at seven days, mycelium uniform, low, white, opaque (Figure 2B); colorless in reverse; acervuli few, colorless. Vegetative hyphae 1.1-8.0 μm, average 3.2 μm. Conidiomata acervular. Conidiogenous cells hyaline, 4.3 – 18.2 × 2.2 – 4.1 μm, ave. 11.3 × 3.2 μm. Conidia hyaline, pale orange in mass, 1-celled, 6.4 – 16.0 × 3.2 – 5.4 μm, ave. 10.9 × 4.2 μm (n = 41), L/W ratio 2.6, cylindrical to ellipsoidal with ends broadly or acutely rounded (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 μm, ave. 10.9 × 4.2 μm and the presence of setae (Table 5). The average length and width of conidia reported for 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 (Damm et al., 2012). 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 beta-tubulin gene (Table 3), two nucleotide mutations in the CHS-1 gene (Table 4) and by its insect, rather than plant host preference.

Table 5
Colony characteristics on oatmeal agar (OA) and synthetic nutrient-poor agar medium (SNA) at seven days and morphological measurements of SNA cultures for C. nymphaeae var. entomophilum and C. nymphaeae var. nymphaeae. Information not provided is indicated by a dash (-).

Colletotrichum nymphaeae	Colony diameter (cm) 7 days		Conidia		Setae
	SNA	OA	Length [ave] (μm)	Width [ave] (μm)
var. entomophilum	9.0	10.5	6.4-14.9 [10.9]	3.5-4.9 [4.2]	present
var. nymphaeae (CBS 515.78)	3.6	5.0	12.3-32.7 [17.2]	3.7-6.9 [5.0]	absent
nymphaeae (Damm et al., 2012)	1.65-2.6	1.45-2.9	10-19.5 [16.1]	3-6 [4.9]	absent
nymphaeae (Hemmi and Kawasi, 1954)	–	–	9-17 [-]	3-6 [-]	present

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 sensuDamm 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.

Additional specimens were examined at Cordeirópolis, São Paulo, 13 July 2005, L.F. Padulla s.n., ESALQ 1368, and Rio de Janeiro, state of Rio de Janeiro, Feb 1994, C.F. Robbs ARSEF 4360.

Colletotrichum nymphaeae var. nymphaeae

(Pass.) Aa, Netherlands Journal of Plant Pathology, Supplement 1 84 (3): 110, Fig. 20. 1978; ≡Ascochyta nymphaeae Pass., Hedwigia 16:120, 1877. Lectotype, Italy, Parma, from the leaf of Nymphaea alba (Nymphaeaceae), summer 1875, G. Passerini, 176820 (K, non vidi); Epitype, Netherlands, Ubbergen, Oude Waal near Nijmegen, from the leaf spots of Nymphaeae alba, 7 Aug 1978, G. van der Velde (CBS H-20787), ex-epitype culture CBS 515.78! = van der Aa No. 657. Figure 2I-L

Colonies on OA 3.6 cm in seven days, a low felty mycelium with white, floccose center (Figure 2I-J); acervuli salmon colored; culture in reverse dark brownish-grey. On SNA 5.0 mm in seven days, flat with a diffuse but well-defined irregular margin, hyaline to whitish; acervuli scattered, colorless. Vegetative hyphae 1.1 – 3.2 μm, septate, branched. Conidiogenous cells hyaline, 13.3 – 20.9 × 3.2 – 3.8 μm, ave. 17.1 × 3.6 μm. Conidia hyaline, pale orange in mass, 1-celled, 12.3 – 24 (-32.7) × 3.7 – 6.9 μm, ave. 17.2 × 5.0 μm (n = 31), L/W ratio 3.4, cylindrical, frequently narrowly obovate (Figure 2K). Appressoria reniform to elongate with crenulate margins, brown, 7.6 – 10.5 × 4.8 – 9.8 μm (Figure 2L). Setae not observed.

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 sensuDamm 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 (Damm et al., 2012). 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 (Damm et al., 2012; 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 (Damm et al., 2012). 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, 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.

Acknowledgments

This study is a part of the IMBICONT research project (Improved Biological Control for IPM in Fruits and Berries) (project number 1024151001) funded by the Innovation fund, Denmark.

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Edited by

Edited by: Richard V. Glatz

Publication Dates

• Publication in this collection
  20 Dec 2019
• Date of issue
  2020

History

• Received
  20 Aug 2018
• Accepted
  30 Apr 2019