Mycologia, 104(5), 2012, pp. 1187–1199. DOI: 10.3852/11-403 # 2012 by The Mycological Society of America, Lawrence, KS 66044-8897
Epichloe¨ canadensis, a new interspecific epichloid hybrid symbiotic with Canada wildrye (Elymus canadensis) Nikki D. Charlton
INTRODUCTION
Forage Improvement Division, the Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401
Epichloe¨ endophytes, including asexual morphs formerly classified in the form genus Neotyphodium, are fungal symbionts of cool-season grasses that grow in the intercellular spaces of host tissues, often without causing disease. The sexually reproducing Epichloe¨ species are capable of forming stromata that engulf developing inflorescences and suppress seed production (‘‘choke’’ diseases), resulting in horizontal transmission, although some can colonize hosts asymptomatically and are transmitted vertically. In contrast, the asexually reproducing forms formerly classified as Neotyphodium spp. remain benign and grow into the developing ovule where they are transmitted within the seed (Freeman 1904). To date, 23 Neotyphodium species have been described based on morphology, host specificity and phylogenetic analyses (Schardl and Leuchtmann 2005, Moon et al. 2007, Chen et al. 2009, Iannone et al. 2009, Ji et al. 2009, Yan et al. 2009, Ghimire et al. 2011). Of particular utility, phylogenetic analyses of intron-rich sequences from the translation elongation factor 1-a gene (tefA) and b-tubulin gene (tubB) have been used to demonstrate the evolutionary relationships between Neotyphodium and Epichloe¨ spp. (Craven et al. 2001). The majority of Neotyphodium species have been identified from such analyses as interspecific hybrids between two or even three Epichloe¨ species (Schardl 2010). Some Epichloe¨ endophytes are known to provide fitness benefits to their hosts including drought tolerance, increased competitive ability and improved survival (Arachevaleta et al. 1989, West et al. 1993, Malinowski and Belesky 2000) and protection against insect and mammalian herbivory through the production of alkaloid compounds (Clay et al. 1985, Bacon et al. 1986). The four described classes of alkaloids include ergot alkaloids (such as ergovaline), indole-diterpenes (such as lolitrem B), a pyrrolopyrazine (peramine) and saturated aminopyrrolizidines (lolines). Ergot alkaloids and indole-diterpenes are documented most commonly for activity against vertebrates, causing fescue toxicosis (Bacon et al. 1977) and ryegrass staggers (Fletcher and Harvey 1981) respectively. Peramine is an insect feeding deterrent (Johnson et al. 1985, Rowan and Latch 1994), whereas lolines have potent insecticidal activity (Bush et al. 1997). The genes required for the biosynthesis of the four classes of alkaloids have been
Kelly D. Craven Plant Biology Division, the Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401
Shipra Mittal Andrew A. Hopkins1 Forage Improvement Division, the Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401
Carolyn A. Young2 Forage Improvement Division, the Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401
Abstract: Many Epichloe¨ endophytes found in coolseason grasses are interspecific hybrids possessing much or all of the genomes of two or three progenitors. Here we characterize Epichloe¨ canadensis sp. nov., a hybrid species inhabiting the grass species Elymus canadensis native to North America. Three distinct morphotypes were identified that were separated into two groups by molecular phylogenetic analysis. Sequence analysis of the translation elongation factor 1-a (tefA) and b-tubulin (tubB) genes revealed two copies in all isolates examined. Phylogenetic analyses indicated that allele 1 of each gene was derived from Epichloe¨ amarillans and allele 2 from Epichloe¨ elymi. This is the first documentation of an interspecific hybrid endophyte derived from parents of strictly North American origins. Alkaloid gene profiling using primers specific to genes in the peramine, loline, indole-diterpene and ergot alkaloid pathways may indicate chemotypic variation in the ergot alkaloid and loline pathways between the assigned morphotypes. All isolates have the gene enabling the production of peramine but lack genes in the indole-diterpene biosynthesis pathway. Morphology and phylogenetic evidence support the designation of isolates from El. canadensis as a new interspecific hybrid species. Key words: alkaloid diversity, endophyte, Epichloe¨ amarillans, E. elymi, lolines, Neotyphodium
Submitted 2 Dec 2011; accepted for publication 27 Feb 2012. 1 Current address: Dow AgroSciences, Inc., York, NE 68467. 2 Corresponding author. E-mail:
[email protected]
1187
1188
MYCOLOGIA
cloned and characterized (Panaccione et al. 2001, Wang et al. 2004, Spiering et al. 2005, Tanaka et al. 2005, Young et al. 2006, Fleetwood et al. 2007). The single perA gene is required for peramine (PER) biosynthesis (Tanaka et al. 2005). However, the loci required for loline (LOL), indole-diterpene (LTM) and ergot alkaloid (EAS) production each are found as complex gene clusters containing large stretches of associated repetitive elements (Young et al. 2005, 2006, 2009; Fleetwood et al. 2007; Kutil et al. 2007). Elymus canadensis (Canada wildrye) is a native coolseason bunch grass distributed throughout much of Canada, the northern and western United States and into Mexico. Canada wildrye provides forage and nesting habitat for wildlife and is an exceptional species for erosion control. Canada wildrye is known to harbor Epichloe¨ endophytes (White and Bultman 1987, Schardl and Leuchtmann 1999, Vinton et al. 2001) and many Elymus species, including El. canadensis, El. hystrix, El. villosus and El. virginicus (Virginia wildrye), have been reported to harbor Epichloe¨ elymi (Schardl and Leuchtmann 1999). Sequence analysis of tefA and tubB has been used more recently to identify a hybrid asexual endophyte in several populations of Canada wildrye with E. elymi and E. amarillans ancestral genomes (Saha et al. 2009). E. amarillans typically has been reported in Agrostis perennans, Calamagrostis canadensis, Sphenopholis nitida, S. obtusata and S. pallens (White 1994), whereas EviTG-1, an asexual non-hybrid with E. amarillans ancestry, was identified in El. virginicus (Moon et al. 2004). In this study we used morphological and molecular methods to characterize the asexual hybrid endophytes of Canada wildrye. Further, we describe alkaloid production potential based on PCR profiling of genes required for these biosynthetic pathways. Based on the new one-fungus, one-name rule (Miller et al. 2011), we formally describe the first interspecific hybridization of two endophyte parents of North American origins (E. elymi 3 E. amarillans) and propose a formal description of the new Epichloe¨ species, Epichloe¨ canadensis. MATERIALS AND
METHODS
Biological materials.— Original seed lines of Canada wildrye were collected from Texas and Mexico (TABLE I). Seeds from each line were germinated in Metro-Mix 830 (Sun Gro Horticulture, Bellevue, Washington), and plants were maintained in the greenhouse. Greenhouse conditions were 15 h day and 25/23 C day/night in summer and 22/ 20 C day/night in spring, fall and winter. Greenhouse plants (38 individuals representing three locations) initially were screened via PCR to determine endophyte infection and diversity based on alkaloid gene profiling. Initial
screening determined which stock plants would be used for endophyte isolation. Herbarium specimens were prepared (Pollack 1967) for each morphotype (CWR 5, CWR 15, CWR 34 represented by NRRL isolates 50005, 50003, 50004 respectively) and deposited with the Cornell University Plant Pathology Herbarium (CUP). Isolation of endophytes from plant tissues.—Pseudostems of endophyte-infected tillers were used to isolate the fungal endophyte. Tillers were cut into 3.0–5.0 cm pieces and surface-sterilized (95% ethanol 30 s, 70% ethanol 5 min, 0.6% NaOCl 25 min) followed by three washes with sterile distilled water. Plant material was blot dried, cut into 4– 5 mm pieces and placed on potato dextrose agar (PDA) plates amended with 100 mg/mL ampicillin, sodium salt (Agri-Bio, Bay Harbor, Florida), 50 mg/mL streptomycin sulfate (PhytoTechnology Laboratories, Shawnee Mission, Kansas) and 50 mg/mL chloramphenicol (PhytoTechnology Laboratories). Plates were sealed and incubated in the dark at 23 C and examined regularly for endophyte growth for up to 2 wk. Fungal isolates were subjected to hyphal tipping to obtain a pure culture (Whitney and Parmeter 1963). Morphological examination.—Colony morphology was examined from cultures grown on PDA. Plates (three/isolate) were inoculated with a 10 mL suspension of ground mycelium taken from the leading edge of an actively growing culture (3 mm plug in 200 mL sterile water). Cultures were incubated at 23 C in the dark, and measurements were taken from three colonies for each isolate every 7 d for 4 wk. Radial growth rate was recorded at 4 wk and averaged. Cultures were photographed at 3 wk. Microscopic observations of conidia and conidiogenous cells were performed after inoculating 1.5% water agar with a 10 mL suspension of ground mycelium as described above. Cultures were incubated at 23 C in the dark for 14 d. Agar blocks of cultures with conidiation were placed on a slide and cover slips were lightly pressed on the surface. Conidia and conidiophores were examined with an Olympus BX41TF light microscope (Olympus Corp., Tokyo, Japan) at 10003 magnification with oil immersion optics. Images were captured with an Olympus DP71 camera supported by DP-BSW application software (Olympus Corp.). Images were used to measure the length and width of conidia and the length and width of conidiogenous cells. Measurements of 20 fungal structures were recorded per isolate. The morphology of isolates was compared to E. amarillans (ATCC 200743, ATCC 200744), E. elymi (ATCC 201551, ATCC 200850) and descriptions from the literature (White 1994, Schardl and Leuchtmann 1999). DNA isolation and PCR.—Isolates were grown in potato dextrose broth (PDB) at 23 C with gentle shaking. After incubation 7–10 d, the mycelium was harvested and washed three times with sterile distilled water and lyophilized. DNA was isolated from approximately 20 mg ground mycelium using QIAGEN DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, California) according to manufacturer’s instructions. Total DNA from individual seeds (48/line) and tillers of stock plants were isolated with QIAGEN MagAttract 96 DNA
CHARLTON ET AL.: EPICHLOE¨ CANADENSIS TABLE I.
Plant collection locations, GPS coordinates, elevation and dates of collection
Elymus canadensis Plant lines 04CWR2 Mx NFCWR1a 04CWR6 a
1189
Collection location
Latitude
Longitude
Elevation (m)
Date
Nuevo Leo´n State, Mexico Throckmorton County, Texas Dawson County, Texas
25u209320N
100u169400W
1865
21 Oct 2004
33u239340N
99u079120W
407
16 Jul 1998
32u449470N
101u429200W
1000
23 Jul 2004
Formerly 98CWR8 (Saha et al. 2009).
Plant Core Kit (QIAGEN Inc.). Primers specific for tefA (SUPPLEMENTARY TABLE I) were used to detect the presence of endophyte. PCR was performed in a total volume of 25 mL containing 5 ng DNA, 1.0 U GoTaqTM DNA Polymerase (Promega Corp., Madison, Wisconsin), 13 Green GoTaqTM Reaction Buffer containing 1.5 mM MgCl2, 0.2 mM of each dNTP (Promega Corp.) and 1 mM target-specific primers. The cycling parameters were an initial denaturation step for 1 min at 94 C, 30 cycles of denaturation at 94 C for 15 s, annealing at 56 C for 30 s, extension at 72 C for 45 s, followed by a final synthesis step at 72 C for 10 min. Amplicons were analyzed by gel electrophoresis with a 1.5% agarose gel in 13 Tris-Boric-EDTA (TBE) buffer. DNA fragments were stained with ethidium bromide and viewed by UV transillumination. Sequencing and phylogenetic analysis of tefA and tubB genes.—DNA fragments of ITS, tefA and tubB were amplified from the Canada wildrye isolates by PCR. Primers ITS 4, ITS 5, tef1-exon1d, tef1-exon6u-1, T1.1 and T1.2 were used to amplify ITS, tefA and tubB respectively (SUPPLEMENTARY TABLE I). PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN Inc.) according to manufacturer’s instructions. Purified products were sequenced with Big Dye Terminator Chemistry 3.1 (Applied Biosystems, Foster City, California). Polymorphic peaks in the sequence traces indicated the presence of multiple alleles for both tefA and tubB of the Canada wildrye isolates, whereas identical sequences of the known haploid sexual species (E. elymi, E. amarillans) indicated single alleles. Therefore, the individual alleles from the Canada wildrye isolates were isolated by cloning the PCR product with pGEMH-T Easy Vector System I (Promega Corp.) and 2 mL of the ligation was used in the transformation of TOP10 competent cells (Invitrogen, Carlsbad, California). Clones were selected (12 clones/ isolate/gene) and grown in 96-well plates in Terrific Broth (Sambrook et al. 1989) 20 h at 470 rpm in the HiGroH (Genomic Solutions Inc., Ann Arbor, Michigan) microwell plate growth system. Plasmid DNA isolation was performed with Biomek FXP (Beckman Coulter Inc., Brea, California). DNA sequencing was performed as described above with primers SP6 and T7. Chromatograms were aligned and adjusted by visual examination with SequencherTM 5.0 (Gene Codes Corp., Ann Arbor, Michigan). Unique tefA and tubB sequences were submitted to GenBank (accession numbers JN886775–JN886778). The sequences from El. canadensis endophytes were aligned with sequences from representative Epichloe¨ species
(SUPPLEMENTARY TABLE II) including E. amarillans, E. baconii, E. brachyelytri, E. bromicola, E. clarkii, E. elymi, E. festucae, E. glyceriae, E. sylvatica, E. typhina and E. yangzii with Clustal W (Thompson et al. 1994). Alignments were checked manually for ambiguity and adjusted if needed with MacClade 4.0 (Maddison and Maddison 2000). Alignments for tefA and tubB were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S12041). Maximum parsimony (MP) analysis employed the branch and bound option in PAUP* 4.0 (Swofford 2003). For parsimony analysis, gaps were treated as missing data and character changes were weighted equally and unordered. Confidence of inferred phylogenies was estimated by 1000 bootstrap replications, and branches with values $ 70% were considered well supported. Maximum likelihood analysis (ML) parameters included proportion of variable sites, nucleotide frequencies and substitution rates, and gamma shape parameter. These parameters were estimated from the dataset with ML implemented in ModelTest 3.06 (Posada 2006). The parameters (GTR + G + I selected model) were used in the subsequent analysis. The ML tree was generated by random sequence additions, followed by tree-bisection reconnection. Starting branch lengths were obtained with the Rogers-Swofford approximation method implemented in PAUP*. Gene profiling.—Genes required for production of peramine, lolines, indole-diterpenes and ergot alkaloids (SUPPLEMENTARY TABLE I) were detected by PCR as described above. In addition, markers to detect mating-type genes mtAC and mtBA (SUPPLEMENTARY TABLE I) were used to determine genetic diversity within the mating type idiomorph.
RESULTS Characteristics of Elymus canadensis endophytes.— Endophyte infection frequencies of 48 individual seeds from the original lines of NFCWR1, 04CWR6 and 04CWR2 Mx were determined by PCR as 100, 98 and 38% respectively. Endophytes were isolated from each population from surface-sterilized tillers of El. canadensis plants grown in the greenhouse. A total of five isolates were maintained from the three lines, one isolate from NFCWR1 and two isolates each from 04CWR6 and 04CWR2 Mx.
1190 TABLE II.
MYCOLOGIA Morphological characteristics of Epichloe¨ elymi, E. amarillans and E. canadensis
Endophyte
Host
Conidiogenous cell (mm)
Growth on PDA (mm/week)
Conidial shape
Conidia size (mm)
Length
Width
4.0 6 0.4 3 2.2 6 0.2
17 6 3
2.0
4.5 6 0.7 3 1.9 6 0.2 23.4 6 6.5 1.7 6 0.2
E. elymia
Elymus sp.
13.7–20 at 24 C
E. amarillansb
Agrostis hiemalis
Ndc
Navicular to ellipsoidal Lunate to reniform
2–9 at 23 C
Obovate
6.3 6 0.7 3 3.1 6 0.2 24.4 6 4.5 2.2 6 0.4
5–14 at 23 C
Reniform
7.0 6 0.5 3 2.9 6 0.3 21.3 6 7.5 2.1 6 0.4
1–8 at 23 C
Obovate to reniform 6.6 6 0.5 3 3.2 6 0.3 23.6 6 6.4 1.7 6 0.2
E. canadensis El. canadensis (morphotype I)d E. canadensis El. canadensis (morphotype II)e E. canadensis El. canadensis (morphotype III)f a
Schardl and Leuchtmann 1999. White 1994. c Nd 5 no data. d Morphotype I 5 from 04CWR2 Mx isolates CWR 34 and 36 equivalent to NRRL 50004. e Morphotype II 5 from NFCWR1 isolate CWR 5 equivalent to NRRL 50005. f Morphotype III 5 from 04CWR6 isolates CWR 15 and 17 equivalent to NRRL 50003. b
The morphological characteristics of E. amarillans (ATCC 200743, ATCC 200744), E. elymi (ATCC 201551, ATCC 200850) and the isolates from Canada wildrye are summarized (TABLE II). Three morphotypes were identified from Canada wildrye based on differences in growth rate, colony convolution, colony felting and conidiogenous cells (FIG. 1). Morphotypes I (isolates CWR 34, 36; NRRL 50004), II (isolate CWR 5, NRRL 50005) and III (isolates CWR 15, 17, NRRL 50003) corresponded to plant lines 04CWR2 Mx, NFCWR1 and 04CWR6 respectively. Morphotype II (FIG. 1D, E) had the fastest growth, whereas morphotypes I (FIG. 1A, B) and III (FIG. 1G, H) both grew slightly slower (FIG. 1, TABLE II). Colony morphology distinguishes the three morphotypes where morphotype I has more distinct colony convolution than either morphotypes II and III. Morphotype III exhibits a more cottony phenotype, while morphotypes I and II appear more felt-like with less aerial growth than morphotype III. The conidia for the three morphotypes were significantly (P , 0.05) larger than E. amarillans and E. elymi, as determined by use of the unpaired t-test. Phylogenetic relationships.—Analysis of the ITS region from the three morphotypes revealed they were more similar to E. amarillans than E. elymi. However, because only one ITS copy is typically maintained after interspecific hybridization (Ganley and Scott 2002), phylogenetic analysis on tefA and tubB intron sequences also was conducted. Regardless of differences in morphology, all five isolates from Canada wildrye contained two tefA alleles of which allele 1 grouped with E. amarillans and allele 2
grouped with E. elymi (FIG. 2). Additional sequence variation was identified as a single nucleotide polymorphism (SNP) where morphotypes II and III have allele 1a and 2a while morphotype I has allele 1b and 2b. Phylogenetic analysis of these sequences with representative isolates from Epichloe¨ species (SUPPLEMENTARY TABLE II) resulted in 72 MP trees (one in FIG. 2). Relationships of tefA alleles from Canada wildrye endophytes had bootstrap support, $ 99, and all MP trees supported these relationships. Consistent with the tefA results, all five isolates also had two alleles of tubB of which allele 1 grouped with E. amarillans and allele 2 grouped with E. elymi (FIG. 3). The alleles that grouped with E. elymi were identical in sequence. However, the alleles that grouped with E. amarillans were distinguishable by a SNP, whereby three isolates (morphotypes II, III) shared identical sequence (allele 1a) and the two isolates representing morphotype I shared identical sequence (allele 1b). Phylogenetic analysis of these sequences with representative Epichloe¨ species resulted in nine MP trees (one in FIG. 3). Bootstrap values of $ 84 supported the inferred relationship of tubB copies from Canada wildrye endophytes, and all MP trees were identical in the placement of these genes. All relationships regarding placement of gene copies from the isolates from Canada wildrye were identical between MP and ML analyses, therefore ML trees are not shown. Alkaloid gene profiling.— All five Canada wildrye isolates were tested by PCR for alkaloid production genes, and representative isolates of E. elymi and E. amarillans were included for comparison (TABLE III).
CHARLTON ET AL.: EPICHLOE¨ CANADENSIS
1191
FIG. 1. Colony morphology, hyphae, conidiogenous cells and conidia of Epichloe¨ canadensis isolates from Elymus canadensis. Colony is from cultures grown 3 wk on PDA. From 04CWR2 Mx: (A) Surface, (B) reverse, (C) conidiogenous cells and conidia of morphotype I (isolate CWR 34). From NFCWR1: (D) surface, (E) reverse, (F) conidiogenous cells and conidia of morphotype II (isolate CWR 5). From 04CWR6: (G) surface, (H) reverse, (I) conidiogenous cells and conidia of morphotype III (isolate CWR 15).
All isolates apart from one, E. elymi ATCC 200850, tested positive for the peramine markers and are expected to be peramine producers. None of the genes required for the biosynthesis of indole-diterpenes were detected in any of the isolates (TABLE III). However, E. amarillans ATCC 200743 does produce an ltmG fragment as also shown by Young et al. (2009). Variation was detected among the ergot
alkaloid and loline biosynthesis genes. Of the nine genes tested from the EAS locus, all isolates contained fragments of dmaW, easE, easF and easG. Morphotype I isolates were segregated from morphotypes II and III due to the absence of cloA, easA, easH, lpsA and lpsB genes from the ergot alkaloid gene cluster (TABLE III). Morphotype I isolates showed the same EAS gene pattern as E. elymi ATCC 201551.
1192
MYCOLOGIA
FIG. 2. Phylogeny derived from maximum parsimony (MP) analysis of introns 1–4 of the tefA gene from representative haploid Epichloe¨ species and copies obtained from E. canadensis isolated from Elymus canadensis. Four additional isolates were examined and sequences obtained from two were identical to E. canadensis allele 1a and 2a representing morphotypes II and III, and the other two were identical to E. canadensis allele 1b and 2b representing morphotype I. MP tree is one of 72 obtained by branch and bound search. Number of parsimony informative characters 5 92, uninformative characters 5 52, tree length 5 186 steps, consistency index 5 0.8387, retention index 5 0.9198, rescaled consistency index 5 0.7714, midpoint root is at the left edge. Numbers at branches are the percentage of trees containing the corresponding clade in 1000 bootstrap replications. Letters after each endophyte refer to host designations as follows: Ah (Agrostis hyemalis), Ao (Anthoxanthum odoratum), Ap (Agrostis perennans), As (Agrostis stolonifera), Be (Bromus erectus), Bee (Brachyelytrum erectum), Bp (Brachypodium pinnatum), Bs (Brachypodium sylvaticum), Cv (Calamagrostis villosa), Ec (Elymus canadensis), Eh (Elymus hystrix), Em (Elymus macgregorii), Evl (Elymus villosus), Ev (Elymus virginicus), Fl (Festuca longifolia), Frr (Festuca rubra subsp. rubra), Gs (Glyceria striata), Hl (Holcus lanatus), Pn (Poa nemoralis), Pp (Poa pratensis), Rk (Roegneria kamoji), So (Sphenopholis obtusata).
CHARLTON ET AL.: EPICHLOE¨ CANADENSIS
1193
FIG. 3. Phylogeny derived from maximum parsimony (MP) analysis of introns 1–3 of the tubB gene from representative haploid Epichloe¨ species and two copies obtained from an E. canadensis isolated from Elymus canadensis. Four additional isolates were examined and sequences obtained from two were identical to E. canadensis allele 1a representing morphotypes II and III, and the other two were identical to E. canadensis allele 1b representing morphotype I. The sequences of the second copy for all isolates were identical to E. canadensis allele 2. MP tree is one of nine obtained by branch and bound search. Number of parsimony informative characters 5 46, uninformative characters 5 40, tree length 5 109 steps, consistency index 5 0.8807, retention index 5 0.9274, rescaled consistency index 5 0.8168, midpoint root is at the left edge. Numbers at branches are the percentage of trees containing the corresponding clade in 1000 bootstrap replications. Letters after each endophyte refer to host designations as listed in FIG. 2 legend.
+
2 2 2 2 2 2 2 2 2
+ + + + + + 2 + +
2 2 2 2 + 2 2 2
Ergot alkaloids (EAS) dmaW easE easF easG cloA easA easH lpsB lpsA
Lolines (LOL) lolA lolC lolD lolE lolF lolO lolP lolT lolU
Indole-diterpenes (LTM) ltmB ltmC ltmE ltmF ltmG ltmJ ltmK ltmM
721
Peramine (PER) perA
Plant line:
Strain (morphotype): ATCC 200743
2 2 2 2 2 2 2 2
+ + + + + + 2 + +
2 2 2 2 2 2 2 2 +Da
+
57
ATCC 200744
E. amarillans
2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
+ + + + 2 2 2 2 2
+
56
ATCC 201551
2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
+Da
184
ATCC 200850
E. elymi
Alkaloid gene profiling to determine alkaloid chemotype class
Species:
TABLE III.
2 2 2 2 2 2 2 2
+ + + + + +Da 2 + +
+ + + + 2 2 2 2 2
+
04CWR2 Mx
CWR 34 (I)
2 2 2 2 2 2 2 2
+ + + + + +Da 2 + +
+ + + + 2 2 2 2 2
+
04CWR2 Mx
CWR 36 (I)
2 2 2 2 2 2 2 2
+ + + + + + 2 + +
+ + + + + + + + +
+
NFCWR1
CWR 5 (II)
E. canadensis
2 2 2 2 2 2 2 2
+ + + + + + 2 + +
+ + + + + + + + +
+
04CWR6
CWR 15 (III)
2 2 2 2 2 2 2 2
+ + + + + + 2 + +
+ + + + + + + + +
+
04CWR6
CWR 17 (III)
1194 MYCOLOGIA
+D indicates the presence of a gene but associated with a deletion that may signify a non–functional gene. The predicted chemotype also may reflect the production of an intermediate in the pathway rather than end product. Boldface indicates confirmation of a compound in that class, as indicated by Schardl et al. 2012. b
a
per/eas/lol per/eas/lol per/eas/lol per/lol per/lol
per/eas
null
per/eas/lol
per/eas/lol
2 2 2 2 2 2 2 2
ltmP ltmQ Chemotype predictionb
2 2
2 2
2 2
2 2
2 2
04CWR6 04CWR6 NFCWR1 04CWR2 Mx 57 Plant line:
721
56
184
04CWR2 Mx
CWR 5 (II) CWR 36 (I) CWR 34 (I) ATCC 200850 ATCC 201551 ATCC 200744 Strain (morphotype): ATCC 200743
Species:
TABLE III.
Continued
E. amarillans
E. elymi
E. canadensis
CWR 15 (III)
CWR 17 (III)
CHARLTON ET AL.: EPICHLOE¨ CANADENSIS
1195
Of the nine genes tested from the LOL locus, all isolates appeared to lack a functional lolP gene (TABLE III), which has been shown to be required for conversion of N-methylloline to N-formylloline (Spiering et al. 2008). Morphotype II and III isolates have the same LOL gene profiles as the E. amarillans isolates ATCC 200743 and ATCC 200744. Morphotype I isolates have a 64 bp deletion in the second exon within the lolO gene (TABLE III). Primers to detect the mating type revealed that the morphotype I isolates have mtAC, indicating they are mating type 1 (MTA) and the remaining three isolates (morphotypes II and III) contained genes for both mating types (MTA and MTB). Individual seeds from the original collection were tested for mating type and the lolO deletion. Consistent with the separation of the morphotypes and lolO analysis, each endophyte-infected seed from 04CWR2 Mx was MTA and contained the deletion in lolO. While individual seed samples from NFCWR1 and 04CWR6 contained both MTA and MTB and had the complete lolO fragment. TAXONOMY Epichloe¨ canadensis N.D. Charlton et C.A. Young, sp. nov. FIG. 1 MycoBank MB 563748 Colonies on PDA white, cottony, moderate growing, attaining 27–48 mm diam in 28 d at 23 C. Colonies raised from agar surface, smooth to slightly convoluted; hyphae at periphery usually above surface; reverse of colony cream to light tan. Vegetative hyphae hyaline, septate, 1.5–2 mm wide. Conidiogenous cells hyaline, arising solitarily from hyphae, produced abundantly, 12.5–41.5 mm long, 1.5–3.0 mm wide at the base and tapering to approximately 0.5 mm at the tip. Conidia obovate to reniform, aseptate, hyaline, smooth, 5.75–8 3 2.5–4 mm. Genetic relationships to Epichloe¨ amarillans and E. elymi. Etymology. Referring to the host species Elymus canadensis. Holotype. CWR 5 USA, Texas, infecting Elymus canadensis, deposited in the Cornell University Plant Pathology Herbarium as CUP-067946. Known host range. Elymus canadensis L. Known distribution. As endophytic fungi inhabiting aerial tissues and seeds of Elymus canadensis from Texas, USA, and Nuevo Leo´n state, Mexico. Specimens examined. CWR 5 from El. canadensis, Throckmorton County, Texas, Jul 1998, A.A. Hopkins (HOLOTYPE, CUP-067946). CWR 15 and CWR 17 from El. canadensis, Dawson County, Texas, Jul 2004, A.A. Hopkins (CUP067947); CWR 34 and CWR 36 from El. canadensis, Nuevo Leo´n State, Mexico, Oct 2004, A.A. Hopkins (CUP-067945).
1196
MYCOLOGIA
Additional specimens examined. NRRL 50003 (also known as NFE1002) representing CWR 15 and 17 isolated from El. canadensis plant line 04CWR6, Dawson County, Texas, Jul 2004, A.A. Hopkins; NRRL 50005 (also known as NFE1000) representing CWR 5 isolated from El. canadensis plant line NFCWR1, Throckmorton County, Texas, Jul 1998, A.A. Hopkins; NRRL 50004 (also known as NFE1001) representing CWR 34 and CWR 36 isolated from El. canadensis plant line 04CWR2 Mx, Nuevo Leo´n State, Mexico, Oct 2004, A.A. Hopkins (Young and Hopkins 2008).
DISCUSSION Here we describe the first asexual endophyte to be characterized from Canada wildrye, and we propose the name Epichloe¨ canadensis. Elymus canadensis is native to North America, where its distribution has been reported to range from southern Alaska through Canada and most of the lower 48 states and into Mexico (Hitchcock 1971). It is most abundant in the Central and Great Plains and a common component in both tall and mixed grass prairies. The frequency of endophyte infection in Canada wildrye is relatively high, with 60–100% infected (White 1987, Vinton et al. 2001). However E. elymi, which reproduces sexually, causing choke on a portion of the plant’s infloresences, has been the most prominent species associated with Canada wildrye (White and Bultman 1987, Schardl and Leuchtmann 1999). This interspecific hybridization of E. elymi and E. amarillans represents the only known hybridization between two North American Epichloe¨ spp. Phylogenetic analysis confirmed that the endophyte from Canada wildrye possesses two alleles each of tefA and tubB genes, which were inherited from E. elymi and E. amarillans progenitors. The tefA and tubB alleles inherited from E. elymi were grouped consistently with those of E. elymi isolates from El. canadensis, El. hystrix, El. macgregorii, El. virginicus and El. villosus. However, the tefA and tubB alleles inherited from E. amarillans grouped differently, with the former aligning most closely with E. amarillans from Agrostis perennans and the latter with E. amarillans from Sphenopholis obtusata, both grass species (and their resident endophytes) native to North America. In keeping with their hybrid origin, both mating type idiomorphs, MTA (mating type 1) and MTB (mating type 2), are present in morphotypes II and III, which suggests that hybridization occurred between parents of opposite mating types and both idiomorphs are maintained. Epichloe¨ spp. lack a vegetative incompatibility system, therefore mating type does not influence heterokaryon formation (Chung and Schardl 1997). Morphotype I contains only the MTA idiomorph, suggesting either both parents were mating
type 1 with the potential for two copies of the MTA idiomorph present in the genome or that only one mating type was inherited after hybridization because we cannot discount the possibility of loss of one mating type through gene or chromosome loss. Exactly how and where such hybridizations have occurred is unknown, but our current understanding suggests that the process likely is mediated by somatic vegetative fusion (anastomosis) (Schardl and Craven 2003). Such events may happen after a horizontal ascospore dispersal of Epichloe¨ to a plant with a resident endophyte, resulting in a super infection. Because E. elymi is a common inhabitant of many Elymus spp., it is tempting to speculate that this progenitor was the initial inhabitant that subsequently fused with an opportunistic E. amarillans, but we cannot discount the possibility of the opposite order of events. Virginia wildrye (El. virginicus) has been shown to harbor E. amarillans, but the teleomorph is not expressed in this particular association (Moon et al. 2004), and it is yet to be determined whether that association is widespread. Interfertility between these two host species (Canada and Virginia wildrye) is well documented (Brown and Pratt 1960, Nelson and Tyrl 1978), and such events have been predicted to influence endophyte hybridization (Selosse and Schardl 2007). Retention of both progenitor genomes during hybrid formation results in an increased genome size. The observation of larger spore size in the hybrid endophytes as compared to the sexual species is consistent with studies that compared conidium sizes of E. festucae, E. typhina, N. coenophialum and N. sp. LpTG-2 with the genome sizes predicted by chromosome karyotyping and quantitative Southern blot hybridization (Kuldau et al. 1999). The sizes of conidia between the three morphotypes were not significantly different, and although the Canada wildrye isolates separated into three morphotypes this likely represents phenotypic variation among the species. Alkaloid diversity across the Epichloe¨ endophytes in many cases is due simply to the presence or absence of genes that are required for key pathway steps. Markers have been used to effectively genotype epichloid isolates, thus allowing a prediction regarding the alkaloid chemotype of the fungus (Young et al. 2009, Schardl et al. 2012). Alkaloid gene profiling revealed that all isolates contained the peramine gene but no indole-diterpene genes. E. elymi is known to produce the anti-feeding deterrent peramine (Siegel et al. 1990, Schardl and Leuchtmann 1999, Clay and Schardl 2002), whereas Siegel et al. (1990) has documented one E. amarillans isolate symbiotic with Agrostis hyemalis that produced peramine and low
CHARLTON ET AL.: EPICHLOE¨ CANADENSIS ergovaline. All isolates examined within this study tested positive for the two peramine gene markers, indicating the potential to produce peramine in planta (TABLE III). Of interest, one strain of E. elymi (ATCC 200850) lacks the 59 end of the perA gene likely rendering this strain a non-producer. The genes contained at the EAS locus are required for the production of the complex ergot alkaloid, ergovaline, which causes toxicosis in grazing livestock (Bacon et al. 1977). Genetic variation was detected among the morphotypes for the EAS genes. Morphotypes II and III contained all ergot alkaloid genes tested—dmaW, easA, easE, easF, easG, easH, cloA, lpsA and lpsB—suggesting that these isolates would have the ability to produce complex ergot alkaloids such as ergovaline (TABLE III) (Panaccione et al. 2001, Wang et al. 2004, Fleetwood et al. 2007, Schardl et al. 2012). Morphotype I isolates have EAS gene profiles similar to E. elymi ATCC 201551 because they do not have the full complement of ergot alkaloid biosynthesis genes and only tested positive for dmaW, easE, easF and easG. Some E. elymi isolates are known to contain EAS genes and at least one, El. canadensis-E. elymi symbiotum, produces chanoclavine (Schardl et al. 2012). Therefore, this suggests the ergot alkaloid genes likely originated from the E. elymi ancestor. However, the presence of a partial lpsA gene in E. amarillans ATCC 200744 indicates that EAS genes may still exist in other E. amarillans. The genes contained at the LOL locus are required for the production of lolines known for potent insecticidal activity (Bush et al. 1997). The LOL gene profiles of morphotypes II and III are similar to the two E. amarillans isolates that were tested. However, a 64 bp deletion in lolO was detected in morphotype I isolates that would result in a nonfunctional enzyme. Of note, while the two E. amarillans isolates have identical LOL gene profiles, they produce different intermediate compounds of the loline pathway (Spiering et al. 2008, Schardl et al. 2012). The presence of LOL genes in E. amarillans might indicate that the Canada wildrye hybrid endophyte most likely acquired the loline genes from the E. amarillans ancestor. However, the presence of lolC in E. elymi from El. hystrix was documented in Schardl et al. (2012) and studies have demonstrated that hybrid endophytes often retain genes involved in alkaloid biosynthesis despite the fact they are sometimes not present in existing relatives (Kutil et al. 2007, Schardl et al. 2012). Asexual hybrid epichloid endophytes seem more likely to produce a greater array of alkaloids as compared to their sexual counterparts, perhaps as a consequence of hybridization because alkaloid genes
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can be acquired from multiple parents. Examples of this phenomenon are seen in Neotyphodium uncinatum that has retained two copies of the LOL locus (Spiering et al. 2002, Spiering et al. 2005) while N. coenophialum e19 has a single LOL locus originating from the E. typhina parent (Spiering et al. 2002, Kutil et al. 2007) and two EAS loci of which one is from the E. festucae parent, and the origin of the other is yet to be determined (Wang et al. 2004, Florea et al. 2009, Liu et al. 2009). The pyramiding of alkaloid genes through hybridization in addition to variation within alkaloid gene clusters has the potential to confer greater fitness to hosts harboring these hybrid species. Based on gene profiling of the available alkaloid genes we predict that El. canadensis-E. canadensis symbiotum has the potential to produce peramine, ergot alkaloids or early pathway intermediates, and intermediates within the loline pathway (TABLE III). No indole-diterpenes would be expected. We have described E. canadensis, an interspecific hybrid of E. amarillans and E. elymi ancestry, from the wildrye El. canadensis using both morphological and phylogenetic species concepts. Further analysis using molecular markers against the alkaloid genes provided a comprehensive analysis of this new species indicating chemotypic diversity among the five isolates examined. We currently are evaluating whether this alkaloid diversity translates into differences in fitness and persistence of the host and exploring the distribution of chemotypic diversity within populations of Canada wildrye. ACKNOWLEDGMENTS We thank Christopher L. Schardl (University of Kentucky) and Adrian Leuchtmann for helpful discussion on new nomenclature rules, Christopher L. Schardl and Daniel G. Panaccione (West Virginia University) for valuable discussions regarding alkaloid biosynthesis, and Johanna E. Takach (Noble Foundation) and Christopher L. Schardl for critical review of this manuscript. We also thank Kirsti Burr, Cindy Crane, Ginger Swoboda, Lark Trammel (Forage Analysis Facility), the Genomic Core Facility and the Greenhouse Facility at the Samuel Roberts Noble Foundation for technical support and the Samuel Roberts Noble Foundation for financial support.
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