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A new species of endophytic Balansia from Veracruz, Mexico

     Department of Plant Biology and Pathology, Cook College-Rutgers University, New Brunswick, New Jersey 08901

Gerald F. Bills

     Centro de Investigación Básica, Merck Sharp & Dohme de España, S.A.-Josefa Valcárcel 38, Madrid, E-28027, Spain

Gabriella Heredia
Manuela Reyes
Rosa Maria Arias

     Instituto de Ecología, A.C., Apartado Postal 63, 91000, Xalapa, Veracruz, Mexico

James F. White, Jr.

     Department of Plant Biology and Pathology, Cook College-Rutgers University, New Brunswick, New Jersey 08901

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 

A new graminicolous species of Clavicipitaceae, Balansia brunnans sp. nov., has been found to infect Panicum xalapénse. Staining of living host tissues indicates the presence of intercellular endophytic mycelium. Stromata develop just below the nodes on the culms. Balansia brunnans is comparable to Balansia aristidae, B. discoidea, B. gaduae, B. nigricans, and B. strangulans in development of stromata on culms and possession of an endophytic mycelial stage. Among the differences between Balansia brunnans and other comparable species is that it possesses a brown perithecial stroma, whereas comparable species have black perithecial stromata. A key is provided to distinguish B. brunnans from similar species.

Key words: Clavicipitaceae, endophyte, grass, stromata, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Members of the tribe Balansieae cause systemic infections of grasses and sedges. Species of warm-season grass symbionts in the tribe Balansieae, including genera Balansia Speg., Atkinsonella Diehl, and Myriogenospora Atk., are found in Asia, North, Central, and South America, Africa, and Australia (Diehl 1950, White and Glenn 1994). These genera possess ascomatal stromata that are brown to black and form stromata on inflorescences or tiller primordia, culms at nodes, or leaves. Those with epibiotic habits have mycelium that grows on the surfaces of inflorescences and leaf tissues, while endophytic species grow intercellularly in culm or leaf sheath tissues. Diehl (1950) distinguished the genus Balansia based on presence of macroconidia or ephelidial conidia (Ephelis Fr.).

Recently, we encountered an undescribed species of Balansia infecting Panicum xalapénse Kunth in the state of Veracruz, Mexico. In this report, we describe the new species, document its structure and biology, and compare it to other species of Balansia.

	MATERIALS AND METHODS

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Collections of the new species of Balansia infecting Panicum xalapénse Kunth were gathered from two populations in the state of Veracruz, Mexico. One population was located along a stream in the Reserva Ecológica Las Cañadas (19°11'N; 96°59'W) on 15 Jun 1999. A second population was located along a stream in the Botanical Garden ‘Javier Clavijero’ (19°30'N; 96°56'W) of the Instituto de Ecología in Xalapa, on 16 Jun 1999. Each population consisted of hundreds of infected plants, each bearing several stromata. Perithecia were observed on only a few collections from the Xalapa site. Collections were deposited in the Rutgers University Plant Pathology Herbarium. Isolations were made by surface-sterilizing culm pieces 3–5 mm long for 2 min in 50% Clorox®, rinsing in sterile water, and plating onto 2% potato dextrose agar (Difco). Once isolated, cultures were grown on PDA overlaid with cellulose acetate sheets. Cultures were maintained at approximately 21 C, 23 C, and 30 C in a light (10 h fluorescent room lighting)/dark (14 h) cycle. A culture was submitted to the American Type Culture Collection (MYA-2105).

Microscopy – To study mycelium in culm tissue and stroma anatomy, infected culms and stromata were fixed in FAA (five parts 40% formaldahyde : five parts glacial acetic acid : 90 parts 95% ethyl alcohol) for approximately 5 d. The tissue was then dehydrated and embedded in L. R. White® acrylic embedding medium (Polysciences, Inc., Warrington, Pennsylvania), and 1 µm sections were cut using glass knives. Sections were stained on a slide warmer for 30 s in toluidine blue stain (0.1% aqueous) and examined with a light microscope. Size of ascospores and conidia were an average of 20 individual measurements.

	TAXONOMY

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Balansia brunnans E.A. Lewis et J.F. White, sp. nov. Figs. 1–14.

View larger version (131K): [in this window] [in a new window]    FIGS. 1–10. Balansia brunnans from the type. 1. Cross-section of culm showing intercellular mycelium (arrows) in vascular tissues. 2. Cross-section of stroma and culm showing endophytic mycelium (arrows) growing around epidermal cells. 3. Stroma on culm. 4. Conidiomatal sporodochium (arrow). 5. Ephelis conidia. 6. Perithecial stroma (arrow) showing ostiola as minute dots. 7. Perithecial stroma. 8. Cross-section showing pseudoparenchyma in the perithecial stroma. 9. Immature asci. 10. Ascospores. Bars: 1 = 10 µm, 2 = 10 µm, 3 = 10 mm, 4 = 7 mm 5 = 18 µm, 6 = 10 mm, 7 = 7 mm, 8 = 10 µm, 9 = 40 µm, 10 = 10 µm

Anamorph: Ephelis sp.

Balansia nigricans (Speg.) Seaver similis sed subiculo in stromate pallide griseo atque stromate peritheciali brunneo differt. Mycelium intercellulari, in culmis foliisque endophyticum; stromata sub nodis culmi disposita, pallide grisea, subcoacta, 7–10 x 1.5–2 mm, sporodochia singula vel numerosa ferentia; sporodochia sub hypothallo erumpentia, in ambitu isodiametra vel elongata, subimmersa, hymenio e cellulis hyalinis conidiogenis atque conidiis composito praedita; conidia holoblastica unicellularia, hyalina, tenuitunicata, filamentosa, 17–23 x 0.7–0.8 µm, in verticillis disposita, in apicibus cellularum conidiogenarum prodientia; cellulae conidiogenae hyalinae, 15–25 x 0.8–1 µm, ad apicem usque 0.5 µm decrescentes, ad apicem in loco conidiogeno sinuatae, in termino hypharum subtendum singulatim vel in verticillis, prodientes; stroma perithecialia brunnea, stromata partim vel omnino obtegentia, elevata, per ostiola perithecialia punctata, e pseudoparenchymate tenuitunicato 5–12 µm diam composita; perithecia obpyriformia, in stromate immersa, 320–370 x 80–136 µm, collo ostiolari usqe 120 µm longo, eius pariete interiore periphysibus vestito, praedita; asci cylindrici in apice incrassati, 100–160 x 3.3–4 µm, 8-ascospori; ascosporae hyalinae, filamentosae, 8-septatae, in septis fasciculos guttularum continentes, post expulsionem in quattuor partes 28–43 x 0.6–0.7 µm disarticulantes. Status anamorphosis Ephelis, conidia 17–23 x 0.7–0.8 µm.

HOLOTYPUS. MEXICO. VERACRUZ: Xalapa, Reserva Ecológica Las Cañadas (19°11'N; 96°59'W), on Panicum xalapénse, 15 Jun 1999, J. White et G. Bills (RUTPP 1513, culture ATCC MYA-2105).

Balansia brunnans mycelium (Figs. 1–2), endophytic in culms and leaves, intercellular; stromata on culms below nodes (Fig. 3), subiculum pale gray, felty, 7–10 x 1.5–2 mm, bearing one to several sporodochia; sporodochia (Fig. 4) erumpent from beneath hypothallus, isodiametric to elongate in outline, subimmersed, with hymenium of hyaline conidiogenous cells and conidia; conidia (Fig. 5) holoblastic, unicellular, acicular, straight, hyaline, thin-walled, filamentous, 17–23 x 0.7–0.8 µm, produced in whorls at the tips of conidiogenous cells; conidiogenous cells hyaline, 15–25 x 0.8–1 µm, narrowing to 0.5 µm at tip, sinuate at tip where conidia produced, borne singly or in whorls at end of a supporting hypha; perithecial stroma (Figs. 6–8) brown, covering part to all of subiculum, raised, pulvinate, punctate due to perithecial ostioles, composed of thin-walled pseudoparenchyma 5–12 µm diam; perithecia obpyriform (Fig. 11), immersed in stroma, 320–370 x 80–136 µm, with ostiolar neck up to 120 µm long and bordered along its inner wall by periphyses (Figs. 12, 13); asci cylindrical, with a thickened tip, 100–160 x 3.3–4.0 µm, containing 8 ascospores; ascospores hyaline, filamentous, each with 8 septa, containing clusters of guttules at septa, disarticulating after ejection into four 1-septate units 28–43 x 0.6–0.7 µm. Colonies on PDA white; mycelium wooly in the center, becoming sparse and tan toward the margin. Reverse brown in the center, becoming lighter toward the margin (Fig. 14). Optimal temperature for growth on PDA was 21 C. Colony at 21 C PDA after 21 d 22–26 mm diam, at 23 C, 17–20 mm diam, at 30 C no growth was observed. Ephelis conidia produced after 10 d at 21 C.

View larger version (209K): [in this window] [in a new window]    FIGS. 11–14. Balansia brunnans. 11. Median longitudinal section through perithecium. 12. Section through ostiolar region. 13. Section of showing periphyses bordering ostiolar (arrow). 14. Reverse of colony (after 40 d in PDA at 23 C). Bars: 11 = 100 µm, 12 = 130 µm, 13 = 2 µm, 14 = 7 mm

Balansia brunnans is similar to several other species of Balansia in formation of stromata at nodes. However, B. brunnans has several features that distinguish it from these species. The following key distinguishes these similar species.

KEY TO BALANSIA SPP. FORMING STROMATA AT NODES OF GRASSES

1. Subiculum white-pale gray . . . . . 2

1. Subiculum tan, green, or purple . . . . . 3

     2. Perithecial stroma black, flat, sessile, forming discrete disks on surface of thin, white subiculum . . . . . B. discoidea

     2. Perithecial stroma brown, raised, covering surface of subiculum . . . . . B. brunnans

3. Masses of ephelidial conidia white; formed in linear sporodochia . . . . . B. strangulans

3. Masses of ephelidial conidia yellow-orange to tan, formed in an irregular shape on stroma surface . . . . . 4

     4. Stroma only partially surrounding grass culm . . . . . B. gaduae

     4. Stroma completely surrounding, or almost completely surrounding culm . . . . . 5

5. Perithecia completely embedded in stroma; stroma surface smooth . . . . . B. nigricans

5. Perithecia partially embedded in stroma, stroma surface tuberculate . . . . . B. aristidae

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Similar species – Balansia brunnans is comparable to Balansia aristidae (Atk.) Diehl, B. discoidea Henn, B. gaduae (Rehm) White, B. nigricans (Speg.) Seaver, and B. strangulans (Diehl) Diehl in development of stromata on culms and possession of an endophytic mycelial stage. However, aside from B. discoidea, none of the other species has a white to pale-gray subiculum, and all of them have a black perithecial stroma. Balansia brunnans possesses a white to pale-gray subiculum and is characterized by its brown perithecial stroma.

Balansia brunnans is most similar to Balansia nigricans as both have smooth punctate mature perithecial stromata. The two species differ in color of the conidial masses and perithecial stromata. In B. nigricans the conidial masses are yellow to orange and the stroma is black; while in B. brunnans conidial masses are white and the perithecial stroma is brown.

Endophytism – Although it is commonly believed that all of the graminicolous Clavicipitaceae are endophytes, the majority of species on a worldwide basis are epibiotic (Reddy et al 1998, Clay and Frentz 1993). Epibiotic species do not show intercellular mycelium in host tissues; instead all mycelium is evident outside the host epidermis. An analysis of stroma location among 32 endophytic and epibiotic species of Balansia and its allies, including Atkinsonella and Myriogenospora, suggests that only endophytic species are capable of stroma development at nodes of grasses (Diehl 1950, Reddy et al 1998). However, both epibionts and endophytes are capable of stroma development on grass leaves, inflorescences and tiller primordia. Balansia brunnans possesses mycelium that (Fig. 1, arrows) grows intercellularly in both xylem and phloem in the region of the stroma. This endophytic mycelium egresses from plant tissues by growing around epidermal cells and forming a subiculum on the surface of the culm (Fig. 2, arrow). The direct connection of endophytic mycelium with vascular tissues may provide the nutritional resources needed for stroma development at nodes of plants. Perhaps epibiotic species do not form stromata at nodes because they do not establish this direct connection to host vascular tissues. The epiphytic habit has been proposed to be more primitive than endophytism (Phelps et al 1993). Perhaps evolution of endophytism provided the ability to obtain enough nutrients from culms at nodes to fuel development of stromata.

Mating system – In B. brunnans the ephelidial conidiogenous cells develop in sporodochia (Fig. 4, arrow). The remaining surface of the stroma is composed of undifferentiated, possibly receptive hyphae. It is in this hyphal mat that the ascomata develop. Balansia brunnans may be heterothallic, in which case ephelidial conidia from a compatible mating type must be transferred to a receptive hypha in the subiculum to initiate ascomatal development. Heterothallic mating has been previously demonstrated for B. epichloë; and B. obtecta (White and Owens 1992, White et al 1995). It is not known whether insects are involved in the heterothallic mating of species of Balansia as has been demonstrated in genus Epichloë; (White and Bultman 1987, White 1994, Bultman et al 1995). A heterothallic mating system in B. brunnans could explain why most stromata in the two populations examined did not bear perithecia.

	FOOTNOTES

 
¹ Corresponding author, zabe{at}eden.rutgers.edu

Accepted for publication May 18, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Bultman TL, White JFJr,, Bowdish TI, Welch AM, Johnston J., 1995 Mutualistic transfer of Epichloë; spermatia by Phorbia flies. Mycologia 87:182-189

Clay K, Frentz IC., 1993 Balansia pilulaeformis, an epiphytic species. Mycologia 85:527-534

Diehl WW., 1950 Balansia and the Balansiae in America. USDA Agriculture Monograph No 4:1-82

Phelps RA, Morgan-Jones G, Owsley MR., 1993 Systematic and biological studies in the Balansieae and related anamorphs. IV. Host-pathogen relationship of Aristida purpurascens and Balansia aristidae. Mycotaxon 48:165-178

Reddy PV, Bergen MS, Patel R, White JFJr., 1998 An examination of molecular phylogeny and morphology of the grass endophyte Balansia claviceps and similar species. Mycologia 90:108-117

White JFJr., 1994 Endophyte-host associations in grasses. XX. Structural and reproductive studies of Epichloë amarillans sp. nov. and comparisons to E. typhina. Mycologia 86:571-580

———, Bultman TL., 1987 Endophyte-host associations in forage grasses. VIII. Heterothallism in Epichloë typhina. Amer J Bot 74:1716-1721

———, Glenn AE., 1994 A study of two fungal epibionts of grasses: structural features, host relationships, and classification in genus Myriogenospora Atk. (Clavicipitales). Amer J Bot 81:216-223

———, Owens JR., 1992 Stromal development and mating system of Balansia epichloë;, a leaf colonizing endophyte of warm-season grasses. Appl Environ Microbiol 58:513-519[Abstract/Free Full Text]

———, Sharp LT, Martin TI, Glenn AE., 1995 Endophyte-host associations in grasses. XXI. Studies on the structure and development of Balansia obtecta. Mycologia 87:172-181

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