A NOVEL EPIPHYTIC CYANOBACTERIAL SPECIES FROM THE GENUS BRASILONEMA CAUSING DAMAGE TO EUCALYPTUS LEAVES 1

J. Phycol. 44, 1322–1334 (2008)  2008 Phycological Society of America DOI: 10.1111/j.1529-8817.2008.00584.x

A NOVEL EPIPHYTIC CYANOBACTERIAL SPECIES FROM THE GENUS BRASILONEMA CAUSING DAMAGE TO EUCALYPTUS LEAVES 1 Rosane Aguiar2 Plant Biology Department, Federal University of Vic¸osa, Vic¸osa, Minas Gerais, Brazil

Marli Fatima Fiore Center for Nuclear Energy in Agriculture, University of Sa˜o Paulo, Piracicaba, Sa˜o Paulo, Brazil

Maione Wittig Franco, Marı´lia Contin Ventrella Plant Biology Department, Federal University of Vic¸osa, Vic¸osa, Minas Gerais, Brazil

Adriana Sturion Lorenzi Center for Nuclear Energy in Agriculture, University of Sa˜o Paulo, Piracicaba, Sa˜o Paulo, Brazil

Cla´udia A. Vanetti Microscopy & Microanalysis Center, Federal University of Vic¸osa, Vic¸osa, Minas Gerais, Brazil

and Acelino Couto Alfenas Plant Pathology Department, Federal University of Vic¸osa, Vic¸osa, Minas Gerais, Brazil

A cyanobacterial mat colonizing the leaves of Eucalyptus grandis was determined to be responsible for serious damage affecting the growth and development of whole plants under the clonal hybrid nursery conditions. The dominant cyanobacterial species was isolated in BG-11 medium lacking a source of combined nitrogen and identified by cell morphology characters and molecular phylogenetic analysis (16S rRNA gene and cpcBA-IGS sequences). The isolated strain represents a novel species of the genus Brasilonema and is designated Brasilonema octagenarum strain UFV-E1. Thin sections of E. grandis leaves analyzed by light and electron microscopy showed that the B. octagenarum UFV-E1 filaments penetrate into the leaf mesophyll. The depth of infection and the mechanism by which the cyanobacterium invades leaf tissue were not determined. A major consequence of colonization by this cyanobacterium is a reduction in photosynthesis in the host since the cyanobacterial mats decrease the amount of light incident on leaf surfaces. Moreover, the cyanobacteria also interfere with stomatal gas exchange, decreasing CO2 assimilation. To our knowledge, this is the first report of an epiphytic cyanobacterial species causing damage to E. grandis leaves.

Abbreviations: MP, neighbor joining

maximum

parsimony;

NJ,

Cyanobacteria are oxygenic photosynthetic bacteria that occur in a wide range of habitats. These microorganisms have a selective advantage in many ecological settings, particularly given their ability to adjust their photosynthetic apparatus to changes in light intensity and color, and capacity to adapt to other changes in environmental conditions (Stal 2000). The ability of cyanobacteria to form symbiotic associations with a broad range of hosts, including plants (mosses, hornworts, liverworts, cycads, Gunnera, Azolla), fungi (lichens and Geosiphon pyriforme), algae (diatoms and dinoflagellates), and animals (sponges, ascidians, echiuroid worms, and midge larvae), is well documented (see reviews by Adams 2000, Rai et al. 2002). Many cyanobacterial symbionts are filamentous and have the ability to fix atmospheric nitrogen. N2 fixation occurs in specialized cells termed heterocytes (heterocysts) that provide the necessary anaerobic conditions for nitrogenase function (Wolk et al. 1994). A few unicellular and nonheterocyte-forming filamentous cyanobacteria have particular physiological strategies that also allow them to fix atmospheric nitrogen (Stal 2000). In most cases, the cyanobiont provides fixed nitrogen and carbon to the host, which, in turn, protects the cyanobacteria from environmental extremes, such as intolerably high light intensities

Key index words: Brasilonema; cyanobacteria; Eucalyptus grandis; leaf anatomy; novel species; ultrastructure 1

Received 28 May 2007. Accepted 14 March 2008. Author for correspondence: e-mail [email protected].

2

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and desiccation, and from predation (Adams 2000). Many filamentous symbiotic cyanobacteria develop hormogonia, short motile filaments that serve as the infective agents in many of the symbioses, particularly those involving plants (Cohen and Meeks 1997). Cyanobacteria can also form looser associations with plants. Several cyanobacterial species grow epiphytically on mosses; on the bark of trees (Zhu 1987); on submerged roots and stems of rice plants in flooded fields (Whitton et al. 1988); on the lower epidermis and reproductive pockets of Lemna leaves (Duong and Tiedje 1985); on trunks, branches, and leaves in mangrove forests (Toledo et al. 1995); attached to the underwater matrix of stems and roots of floating macrophytes in the Amazon floodplain (Fiore et al. 2005); and on Hydrilla spp. leaves (Wilde et al. 2005). These associations are also mutually beneficial in that the cyanobacteria provide fixed nitrogen to the host, while the host serves as an attachment substratum for the cyanobacteria (Duong and Tiedje 1985, Rother et al. 1988, Toledo et al. 1995). Here we report the occurrence of cyanobacterial mats on the leaves of the angiosperm E. grandis and their effects on the leaf morphology and ultrastructure. Eucalyptus is of significant economic importance being widely cultivated in Australia, Asia, South America, and parts of southern Europe for use in the paper, cellulose, and wood pulp industries. The dominant unique epiphytic cyanobacterial species, characterized using a polyphasic approach of phenotypic and molecular analyses, is responsible for serious damage to the leaves that can affect the growth and development of the entire plant under nursery conditions. MATERIALS AND METHODS

Field surveys. Cyanobacterial mats were collected in June 2005 from E. grandis leaves cultivated in a clonal nursery garden at Timo´teo City, Minas Gerais State, Brazil (1934¢ S, 4238¢ W). Young plants 45 d old were cultivated in small pots (one plant per pot) in enriched soil. Plants were watered daily with water provided by a small reservoir covered with a dark net that permitted 50% of the daylight to penetrate to the water surface. In the nursery garden, the pots were placed on wooden desks 0.84 m above the ground. The plants were uncovered and open to the sky. Fresh samples of epiphytic cyanobacterial mats were collected from leaves and shoots and transported to the laboratory in plastic bags. Cyanobacterial strain isolation and morphological evaluation. Small pieces of leaf (2 · 2 cm) were cut off and placed in petri dishes. They were then immersed in BG-11 medium, some with and some without a source of combined nitrogen (Allen 1968). After 2 weeks, numerous cells of Brasilonema appeared in the enriched culture. Small filaments were isolated into monoculture using a micropipette to transfer the filaments followed by serial dilution (Kugrens et al. 2000). The filaments were grown in BG-11 without combined nitrogen under continuous illumination (30 lmol photons Æ lm)2 Æ s)1) provided by white fluorescent light at 21 ± 1C in a 16:8 light:dark (L:D) regime. The monoculture was maintained in the culture collection at the Plant Biology Department, Federal University of Vic¸osa, MG, Brazil, and in the culture collection of CENA ⁄ USP in Piracicaba, SP, Brazil, under the strain number UFV-E1.

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The systematic scheme of Koma´rek and Anagnostidis (1989) and recent literature describing the type species of Brasilonema (Fiore et al. 2007) were used for the morphological characterization. The morphology of the new species was analyzed using an Olympus AX 70-TRF microscope (Olympus Optical, Tokyo, Japan), fitted by a U-PHOTO system (Universal Photo System, Moldel U-PHOTO, Olympus Optical). Molecular and phylogenetic analyses. Total genomic DNA of B. octagenarum UFV-E1 and of B. octagenarum UFV-OR1, isolated from orchid leaves collected in Venda Nova do Imigrante, Espı´rito Santo, Brazil (2020¢ S, 4108¢ W), was extracted from cultured cells using the procedure previously described (Fiore et al. 2000). PCR-amplification and sequencing of the small subunit (16S) rRNA gene and the cpcBA-IGS gene sequences were also carried out as previously described (Fiore et al. 2007). The sequences obtained in this study and related ones retrieved from GenBank were aligned and used to infer a phylogenetic tree on the basis of the neighbor-joining (NJ) and maximum-parsimony (MP) methods included in the program package MEGA version 3.1 (Kumar et al. 2004). Statistical confidence of the inferred evolutionary relationships was assessed by bootstrapping (1,000 replicates). The accession numbers for the analyzed nucleotide sequences obtained from B. octagenarum UFV-E1 are EF150854 (16S rRNA gene) and EF153636 (cpcBA-IGS); and from B. octagenarum UFV-OR1, EF150855 (16S rRNA gene) and EF153637 (cpcBA-IGS). Micromorphology and anatomy surveys. For the LM analysis, leaves covered by cyanobacterial mats and healthy leaves were removed from the stems. Leaves were fixed in FAA50, for 48 h, followed by storage in 70% ethanol (Johansen 1940). Selected regions of the leaves were cut out in small squares of 0.5 cm2. Part of the samples were sectioned using a table microtome and stained with astra-blue ⁄ basic fucsin or sudan IV. The other samples were embedded in methacrylate medium (Historesin; Leica Microsystems Nussloch, Heidelberger, Germany), according to the methodology described by Carmello-Guerreiro (1995). Thin sections of 7 lm were obtained using a rotating microtome (model RM2155; Leica Microsystems Inc., Deerfield, IL, USA). Sections were stained with toluidine blue O pH 4.0 (O’Brien et al. 1965) and mounted in synthetic resin (Permount; Fisher Scientific, Fair Lawn, NJ, USA). The slides were used to investigate the interactions between cyanobacterial mats and the leaf tissues as well as to examine necrotic areas observed on the leaf surface covered by mats. All sections were examined using a light microscope (Olympus AX 70-TRF; Olympus Optical, Tokyo, Japan). EM. For the SEM analysis, leaf samples were fixed for 1 h in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer adjusted to pH 7.0. After fixation, leaf pieces were dehydrated in an ethanol series and dried in a critical-point drier (CPD 030; Bal-Tec, Balzers, Liechtenstein) followed by gold sputtering (20 nm thickness) in a FDU 010 apparatus (FDU 010; BalTec). All material was viewed with a scanning electron microscope (LEO 1430 VP, Zeiss, Jena, Germany) operated at 15 kV. For the TEM analysis, leaf samples were soaked in 0.05 M sodium phosphate buffer for 2 h and fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0) (Kugrens et al. 2000). After several buffer rinses, the cells were postfixed in a 2% osmium tetroxide solution, also buffered with 0.05 M sodium phosphate. After a graded series of dehydrations in ethanol, the material was embedded in Spurr’s Low Viscosity Resin (Spurr 1969). Thin sections were obtained with a Sorvall MT-2 ultramicrotome (Cambridge Scientific, Cambridge, MA, USA) and poststained with a 1% aqueous uranyl acetate solution followed by lead citrate. Stained thin sections were viewed with a transmission electron microscope (EM 109; Zeiss, Oberkochen, Germany), belonging to the Electron Microscopy Center (NMM) from the Federal University of Vic¸osa.

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Under field conditions, cyanobacterial mats were observed growing on all aerial parts of the eucalyptus plants, including apical shoots (meristematic regions), terminal and axillary buds, and upper and lower leaf surfaces (Fig. 1). Growing epiphytically on leaf blades and stems of E. grandis, the macroscopic thallus of B. octagenarum formed flat, woolly velvet mats that creeped on the substrates. The mat color varied from yellowish-brown to blackish-green. The major biomass in ‘‘mature’’ mats was composed of B. octagenarum but also contained some other microorganisms, including other cyanobacteria, such as Nostoc and Oscillatoria spp. B. octagenarum was found throughout the nursery garden, including inside and outside the greenhouses and on plant pots, wooden desks, pipes from the irrigation

system, and on the ground surface as several small spots. Filaments of this species were scarcely found in the partially covered reservoir used to store water for plant irrigation. Molecular characterization. Nearly complete nucleotide sequences of 1,450 base pairs (bp) of the 16S rRNA gene were determined for the two falsebranching B. octagenarum strains UFV-E1 and UFVOR1 and were found to be virtually identical (99.9% similarity). The BLAST analysis of the 16S rRNA gene sequences from these strains matched them to a high degree with the type strain B. bromeliae SPC951 (96% similarity) and B. sennae CENA114 (97% similarity) (Table 1), supporting their assignment to the genus Brasilonema. A comparison of the cpcBA-IGS between B. octagenarum strains UFV-E1 and UFV-OR1 also revealed high

Fig. 1. Field-collected specimens of epiphytic Brasilonema octagenarum UFV-E1, on young plants of Eucalyptus grandis. (A) General view of the cyanobacterial mats showing the woolly velvet mats on upper portion of a leaf blade. (B) Mats are also spread on lower portion of a leaf and on stems (arrows). (C) The mat is covering the whole blade (leaf on the right), and chlorotic areas appear when the mat is removed (leaf on the left). (D) Necrotic areas on the leaves. Note the details of the damaged areas (arrow).

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Table 1. 16S rRNA gene sequence similarity in the Brasilonema octagenarum strains UFV-E1 and UFV-OR1 with sequences of related cyanobacterial strains obtained from GenBank. Similarity (%). Strain

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Brasilonema octagenarum UFV-E1 Brasilonema octagenarum UFV-OR1 Brasilonema bromeliae SPC951 Brasilonema sennae CENA114 ‘‘Symphyonemopsis’’ VAPOR 1 Mastigocladopsis repens MORA Symphyonema sp.1517 Symphyonema sp.1269-1 Scytonema hyalinum F1-8A Scytonema hoffmannii PCC7110 Scytonema sp. U-3-3 Scytonema sp. IAM M-262

2

3

4

5

6

7

8

9

10

11

12

99.9

96.5 96.7

97.1 97.2 99.3

96.0 96.0 96.1 96.0

94.7 94.8 94.6 94.6 95.2

94.2 94.2 93.7 94.3 94.2 95.6

94.5 94.6 94.3 94.6 94.2 95.6 99.2

94.9 95.0 93.4 93.8 93.7 95.0 96.5 96.3

92.8 92.8 92.8 93.2 93.4 92.9 93.0 92.8 92.7

92.6 92.9 92.3 92.2 92.9 91.8 91.5 91.6 91.5 95.2

93.2 93.3 92.3 92.7 92.9 92.0 91.4 91.6 91.1 95.5 97.5

sequence similarity (99.7% similarity). However, both strains shared relatively low cpcBA-IGS sequence similarity (91%) with B. bromeliae SPC951 and B. sennae CENA114. Furthermore, the length of the IGS region on both B. octagenarum strains was 127 bp, which differed from B. bromeliae SPC951 (132 bp) and B. sennae CENA114 (131 bp). In the phylogenetic analysis of the 16S rRNA gene and of the cpcBA-IGS gene sequences, the B. octagenarum strains UFV-E1 and UFV-OR1 clustered together with the previously published Brasilonema strains with high bootstrap support (Figs. 2 and 3, respectively). However, B. octagenarum strains formed a distinct internal cluster within these larger clades for both of the analyzed sequence sets. The 16S rRNA gene sequences of Brasilonema strains (Nostocales) formed a separate but closely related branch to Symphyonemopsis VAPOR1 (Stigonematales). At this time, the cpcBA-IGS sequences for Symphyonemopsis, Symphyonema, and Mastigocladopsis strains are not available. Consequently, the closest branch to the Brasilonema strains in the phycocyanin tree was made up of members of the Nostocales. Morphological identification and characterization. The morphological analysis of the dominant cyanobacterium colonizing E. grandis leaves is consistent with its assignment the genus Brasilonema, a recently described scytonematoid, filamentous, heterocyteforming cyanobacterial genus (Fiore et al. 2007). This genus is morphologically similar to the genus Scytonema, but with sporadic false branching. The newly isolated strain differs at the species level from the two Brasilonema spp. described to date (B. bromeliae SPC951 and B. sennae CENA114) and is designated Brasilonema octagenarum strain UFV-E1. Brasilonema octagenarum sp. nov. Latin diagnosis: Filamenta in stratum macroscopicum, brunescens vel atro-viride plus minusve paralleliter in fasciculis consociata, irregulariter flexuosa, cylindrical, 9.8–18.5 lm lata, apice cylindrica et rotundata. Trichoma cylindrica, 9.5–18.4 lm lata, ad septa haud constricta, ad apices not attenuata. Vagi-

nae cylindricae, tenues, firmae, surperficie leves, sine colore, paucim lamellosae, adultae apice apertae. Cellulae cylindricae, in hormogoniis et partes apicalibus curtae, in trichomatis adultis ad isodiametricae vel paucim longiores quam latae, contentu granuloso, olivaceo-viridi vel brunescente-violaceo, saepe impletae cum structuras vacuolis similares. Dimensiones cellularum 8.4–13.9 · 6.9–2.6 lm. Heterocytae terminales vel intercalares, solitariae, plus minusve cylindricae. Ramificatio tolypotrichoideum vel scytonematoideum rarissime praesens. Cum akinetae solitariis 6.5–10.2 · 6.1–10.6 lm. Holotypus hic designatus: Exsiccatum no. BRNM HY 1416, isotypus hic designatus: figura nostra 4. Habitatio: Epiphytice, subaerophytice intra Eucalyptus grandis; locus classicus: nursery garden, Timo´teo, Minas Gerais, Brasilia (coll. Jun. 2005). Etymologia: Hoc species nominata est honorem memoriam ocotgesimum anuum Universitatis Federalis Vicosae. Formal description (diagnosis and typification). B. octagenarum, description of the new species (Fig. 4 and Fig. S1 in the supplementary material): Filaments 9.8–18.5 lm, trichomes 9.5–18.4 lm wide, cylindrical. Thallus joined to flat macroscopic, velvet, yellowish-brown to blackish-green mats, densely fasciculate, creeping on the substrate, slightly irregularly coiled, cylindrical, erect fascicles. Filaments are ensheathed, intensely fasciculated, parallel situated, long, 9.8–18.5 lm wide, cylindrical, of the same width along the whole length. Sheaths thin, firm, cylindrical, sometimes slightly lamellated, colorless. Cells 1.5–13.3 lm long, granulated, olive-green or brownish-violet content; cylindrical; without constrictions at cross walls; in liberated hormogonia with short cells slightly constricted at cross walls; vacuole-like structures sometimes in cells, rows of neighboring cells, both in apical (young) and differentiated cells. Fasciculation of filaments with very rare false branching. Hormogonia with short, more granulated cells, 4.8–12.6 lm wide. Heterocytes, solitary, terminal or intercalary, discoid or more or less cylindrical, 8.4–13.9 · 6.9–2.6 lm; hormocysts ± isodiametric, thick walls, 6.5–10.2 · 6.1–10.6 lm.

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Fig. 2. Phylogenetic relationships among Brasilonema octagenarum UFV-E1, B. octagenarum UFV-OR1, and related cyanobacteria based on 16S rRNA gene sequences (878 bp) with Gloeobacter violaceus PCC 7421 as the outgroup. Numbers near nodes indicate bootstrap values >50% for neighbor-joining (left) and maximum-parsimony (right) analyses.

Habitat: Epiphytic, covering leaves, stems, and apical buds of young E. grandis plants in a nursery garden in Timo´teo City, Minas Gerais, Brazil. Holotype here designated: Exsiccatum no. BRNM HY 1416. Isotype here designated: Figure 4, this publication. Type strain (reference strain): UFV-E1 (deposit in the Federal University of Vic¸osa [UFV] Culture Collection, Vic¸osa, Brazil, and CENA ⁄ USP, Piracicaba, Brazil). Etymology: This species is named in honor of UFV on the occasion of its 80th anniversary.

Morphology of B. octagenarum UFV-E1. Cyanobacterial mats of B. octagenarum were examined in the specimens from the nursery garden located in Timo´teo, Minas Gerais, Brazil. This species has an unusual ecology, growing epiphytically on living and dead leaves, stems, and apical buds of young plants of E. grandis. The thallus is macroscopic, flat, woolly velvet, creeping on substrates (Fig. 4, A–G). The mats form a dense layer, yellowish-brown to brackish-green in color, composed of densely arranged, erect fascicle filaments oriented mostly perpendicular to the substrate (Fig. 4, A and B; Fig. S1, A–L). They are

CYANOBACTERIAL DAMAGE ON LEAVES

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Fig. 3. Phylogenetic relationships among Brasilonema octagenarum UFV-E1, B. octagenarum UFV-OR1, and related cyanobacteria based on cpcBA-IGS sequences (544 bp) with Gloeobacter violaceus PCC 7421 as the outgroup. Numbers near nodes indicate bootstrap values >50% for neighbor-joining (left) and maximum-parsimony (right) analyses.

irregularly coiled, cylindrical along the entire length, long, roundly open at the end after hormogonia are released. Filaments are surrounded by a thin sheath (Fig. 4, C–E). Mucilaginous sheaths around the cells and filaments probably assist them in the colonization of subaerophytic habitats. These sheaths absorb and store water as well as associated nutrients from rain and moisture found in the substrate. They protect the cells from desiccation during periods of drought and retain dust that can provide nutrients for growth. If conditions are favorable, the filaments will grow, form new sheaths, and extend over larger and deeper areas (Sheridan 2001, Murphy 2002). Trichomes are cylindrical (9.5–18.4 lm wide), not constricted at cross walls, at least in the older filaments, and not attenuated toward the ends (Fig. 4, C and D; Fig. S1, A–D). The filaments are sparsely branched with tolypotrichoid or scytonematoid branching at free ends (Fig. 4, D and E; Fig. S1, K). Cells (1.5–13.3 lm long) are more granulated, blue-green, olive-green, or brownish-violet in color (Fig. 4, F–H). Vacuolelike structures are sometimes present in rows of neighboring cells in the apical and intercalary parts of the trichomes (Fig. 4F; Fig. S1, A, B, and J). Heterocytes are terminal or intercalary, solitary,

cylindrical (8.4–13.9 · 6.9–2.6 lm) (Fig. 4, B, C, and H; Fig. S1, B). Reproduction is mainly by hormogonia, often with two or more cells (Fig. 4G; Fig. S1, I and L). The similarity of B. octagenarum with B. bromeliae and B. sennae (Fiore et al. 2007) is in the fasciculation of creeping filaments on the substrate, development of vacuole-like structures, and sparse tolypotrichoid or scytonematoid branchings. Differences are in the ecology (type of substrate), cell dimensions (width of filaments, trichomes, and heterocytes), and sheath color (Table 2). Micromorphological and anatomical leaf alterations. Healthy leaves of E. grandis are made up of flat, tabular, uniseriate epidermal cells coated by a thick wax and cuticle on their outer walls. Stomata are mainly on the lower surface. The internal leaf tissue is dorsiventral, and the mesophyll is composed of a palisade parenchyma below the adaxial surface, formed by two to three layers of cells, and the spongy parenchyma, above the abaxial surface, also formed by two to three layers of cells (Fig. 5A). Cyanobacterial mats colonizing the upper or lower surfaces of leaves formed an extensive tuft that caused severe damage on leaf tissues (internal injuries and necrosis) (Fig. 5B). There is a significant

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Fig. 4. Light micrographs of Brasilonema octagenarum UFV-E1. (A) Microscopic shape of cyanobacterial mats. Note the growth pattern of erect, irregular, and creeping filaments. Scale bars, 200 lm. (B) Higher magnification of filaments showing the typical vegetative cells. Note numerous granules inside vegetative cells. Filaments with ‘‘vacuolized cells’’ and heterocytes can also be observed. Scale bar, 20 lm. (C) Higher magnification of filament end (arrow). Note the smooth sheaths. Scale bar, 20 lm. (D–E) False tolypotrichoid branching with mucilage and necridic cells (arrows). Scale bars, 20 lm. (F) Parts of trichomes showing rows of cells with vacuole-like structures (arrow). Scale bar, 100 lm. (G–H) Liberating and germinating of hormogonia (G) with two differentiated heterocyte cells (H). Note the thick wall surrounding hormocyst. Scale bars, 20 lm. h, heterocytes; hc, hormocyst; ho, hormogonia; w, vacuole-like.

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Table 2. Comparative morphological features of Brasilonema octagenarum with the type strain B. bromeliae SPC951 and B. sennae. Features

B. octagenarum

B. bromeliae

B. sennae

Width of filaments (lm) Width of trichomes (lm) Morphology of sheaths

9.8–16–18.5 9.5–14.9–18.4 Thin, firm, later lamellated Colorless to yellow-brown Brownish, olive-green, or rarely violet Mats to irregular fascicles, creeping fascicles Dirty-green, brownish, or blackish-green ±Cylindrical 5.4–15.6 · 10–17.6 Epiphytic on living and dead leaves, stem, and buds of Eucalyptus grandis plants (only in nursery garden) Brazil (MG), Timo´teo, (Clonal Nursery Garden) Jun 2005

10–14.8–21 8–13.2–18 Thin, firm

10–11.5–20 6–7.7–12.5 Thin, firm, later lamellated

Colorless Grayish blue or brownish, olive-green, or violet Free fascicles

Colorless to yellow-brown Blue-green or olive-green

Blackish-green to blackish violet ±Cylindrical 4–19 · 15–16.8 Subaerophytic, epiphytic on living and dead leaves of bromeliads (inside of leaf rosettes)

Dirty-green, brownish, or blackish-green Cylindrical 6.8–15.4 · 10.2–11.2 Subaerophytic, edge of springs on wet wooden stony and iron substrates

Brazil (SP), Sa˜o Paulo (Botanical Garden) 2002–2004

Brazil (SP), Paranapiacaba

Color of sheaths Color of cells Form of thallus Color of thallus Heterocytes (lm) Ecology

Distribution Year of sampling

interaction between the cyanobacterial mats and leaf tissues. The incident light on the leaf surfaces is drastically decreased. Consequently, a serious restriction to the photosynthetic process is expected. Plant growth and plant productivity are severely reduced over time. The onset of the symptoms occurred after the establishment of the mats on the adaxial or abaxial leaves of young plants with subsequent internal cell damage. Transverse sections of colonized leaves showed an intimate association of B. octagenarum filaments with internal leaf tissues (Fig. 5C). The structural analysis of the injuries caused by B. octagenarum filaments showed the occurrence of groups of epidermal cells ruptured and exposed. Near the adaxial surface, the injury development resulted in total collapse of epidermis and mesophyll cells (Fig. 5B). The palisade parenchyma cells near necrotic areas collapsed, displaying rounded chloroplasts with large starch grains in the cells. A cicatrization tissue (peridermis) in a transition region between the necrotic and healthy tissue could be observed. It was composed of long cells with suberized walls formed as a result of the mesophyll cell division in necrotic leaves (arrows in Fig. 5C). Adjacent cells of cicatrized tissue showed hypertrophy and contents blue-green in color as a result of phenolic compounds produced in response to the stress. The same symptoms were observed near the abaxial surface blade (Fig. 5, D–F). Figure 5, E and F, shows the injury process occurring on an abaxial leaf surface colonized by B. octagenarum mats. Morphological damage in this region was also identified not only in the mesophyll cells but also in the secretory cavities. Hyperplasia and hypertrophy were observed in cortical cells leading to expansion of the necrotic areas.

Regular erect fascicles

2002–2004

Ultrastructure. Scanning electron micrographs of B. octagenarum mats on E. grandis leaf surfaces are displayed in Figure 6, A–D. Figure 6A shows the surface view of healthy epidermis densely covered by fasciculated filaments creeping on the substrate (adaxial leaf surface). The transverse section of healthy leaf (Fig. 6B) shows some details of the palisade and spongy parenchyma cells. Note the close interaction between cyanobacterial filaments and the epidermis surface. Figure 6C (arrow) shows the presence of small cylindrical and segmented filaments. These filaments correspond to the hormogonia surrounded by a thin sheath. These motile hormogonia become detached from the parent filament and move away, developing a new filament (Lee 1999). Their presence is a major means of cyanobacterial mat formations on E. grandis leaf surfaces and is probably responsible for cyanobacterium proliferation and establishment over the substrates. It was observed that when the mats were mechanically removed from the leaf, deep scars on the epidermis tissues were revealed (Fig. 6D). Sporadic false branching is shown in Figure 6D, enclosed by a firm, thin sheath. Transmission electron micrographs show the matrix-building cyanobacterial mats colonizing E. grandis leaf blades in abundance (Fig. S2, A–B, in the supplementary material). These mats are embedded in thick mucilage as they generate hormogonia. Hormogonia in the process of detaching from the parental filaments followed by one- or two-celled hormocysts and vegetative cells surrounded by thick mucilage were the predominant cellular forms in the mats. Inclusions of unidentified function were observed in the protoplasts (Fiore et al. 2007). Other structures revealed by TEM surveys are the

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Fig. 5. Transverse thin section of Eucalyptus grandis leaves stained with toluidine blue. (A) Healthy leaf showing a typical dorsiventral mesophyll differentiated into palisade parenchyma and spongy parenchyma. Details of the midvein covered by Brasilonema octagenarum UFV-E1 mats. (B) The arrows show the damage on adaxial surface developing from epidermis to cortical regions. Note the presence of cells with phenolic compounds in cortical region (*), which is identified by the blue-green color (toluidine blue O dye). (C) Details of leaf necrosis. Note the periderm cicatrization tissue (arrows) followed by hypertrophied and phenolized cells. The epidermis ruptured by growth of cyanobacterial filaments and the collapse of mesophyll cells is exposed. (D) The matrix-building mats on the lower epidermis. (E, F) Details of leaf necrosis (abaxial surface). Necrosis in the secretory cavity and cortical cells (hypertrophied cells) and induced production of phenolic compounds. Advanced necrosis (arrows) developing from abaxial to adaxial leaf surface. Note that the neighboring cells (*) are hypertrophic and also show hyperplasia. Mesophyll cells are compacted. Scale bars, 100 lm. ab, abaxial surface; ad, adaxial surface; f, filament; pp, palisade parenchyma; sc, secretory cavity; sp, spongy parenchyma.

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Fig. 6. Ultrastructure of Brasilonema octagenarum UFV-E1. (A) SEM of filaments creeping on substrate (upper epidermis). (B) Part of healthy exposed mesophyll showing palisade (pp) and spongy parenchyma (sp). Note cyanobacterial mats on upper epidermis. Scale bars, 100 lm. (C) Filaments of B. octagenarum strongly adhered on the leaf surface. Note false tolypotrichoid branching (arrow) and hormogonia (ho). Scale bars, 30 lm. (D) Note the scars (arrow) left on leaf surface after filament removal. Scale bar, 20 lm. ep, epidermis; pp, palisade parenchyma; sp, spongy parenchyma.

bi- or multiconcave disks of intercellular substances toward the ends of the cells, which generally initiate the fragmentation of trichomes with formation of new hormogonia (Fig. S2, B). The disks are composed of an electron-dense material (Fiore et al. 2007). Ultrathin sections also showed that the constrictions at cross walls of the trichome cells are more evident than can be seen by LM. Carboxysomes in the cytoplasm of hormogonia were observed (Fig. S2, C). As was described by Fiore et al. (2007) in B. bromeliae, trichome and filament cells contain large vacuole-like areas in the protoplasts (Fig. S2, D and E). They usually occur in rows of cells, all with large, solitary, central vacuole-like formations. The ultrathin sections revealed that they are not true vacuoles, but free spaces within the protoplast, enveloped by enlarged thylakoids of different densities. The function, if any, of these structures remains unknown. TEM sections also con-

firmed the intimate interaction between the cyanobacterial mats and leaf epidermis (Fig. S3, A, in the supplementary material). Filaments of B. octagenarum firmly adhere to the epidermis cells. The cyanobacterial colonization of leaves initiated and propagated the disintegration of cell wall components as shown in Figure S3 (B and C, arrows). DISCUSSION

A survey of microorganisms associated with damage to E. grandis leaves has led to the discovery of a novel cyanobacterial species. The B. octagenarum occurring in mats on E. grandis leaves was associated with other microorganisms, including other cyanobacteria, such as Nostoc and Oscillatoria spp. However, B. octagenarum was the dominant component of the cyanobacterial mats. The morphological and ecological features together with molecular

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phylogenetic analysis support the inclusion of the epiphytic isolate into the recently described nostocalean genus Brasilonema (Fiore et al. 2007). The B. octagenarum strains share morphological characters with B. bromeliae (Fiore et al. 2007), such as typical fasciculation of creeping filaments on the substrate, rare false branching, solitary heterocytes, and large vacuole-like regions occurring in rows of cells. However, diagnostic features include differences in the dimensions of cells (width of filaments and trichomes), coloration of the cells and sheaths, morphology of sheaths, and ecology (type of substrate). The 16S rRNA gene sequence divergence of the B. octagenarum reference strain from the closest phylogenetically related species was >2.5%, consistent with the proposal that it represents a novel species (Wayne et al. 1987, Stackebrand and Goebel 1994). The cpcBA-IGS regions were similarly informative and support this conclusion. Results of morphological and molecular analyses demonstrate that a nearly identical strain colonizing orchid leaves can also be assigned to B. octagenarum. Identifying the precise mechanism leading to tissue damage of Eucalyptus leaves by this mat-forming cyanobacterium is complicated, and further studies are needed. However, there are several possibilities that could account for tissue injuries. Cyanobacterial mats were initially formed in an irregular, threedimensional surface and eventually expanded to cover whole leaves. The incidence of light was drastically decreased over the upper surface of the leaves, resulting in a serious restriction of photosynthesis. Furthermore, as the B. octagenarum filaments grow, the external mucilage becomes thicker while adhering to the substrate, causing increased structural stress to the leaf surface. Secondary metabolites produced by cyanobacterial cells may be involved in the disintegration of the epidermis cuticle of eucalyptus leaves. Cyanobacterial mats composed of an N2-fixing genus, as is the case of Brasilonema, have been considered corrosive due to the metabolic secretion of organic acid (Murphy 2002). In the presence of water (stored in the sheath), the ammonium resulting from the N2-fixation process is oxidized by a common nitrifying bacteria, forming highly corrosive nitric acid. The acid can also interact with ‘‘lime’’ (calcium oxide) to form highly soluble ammonium sulfates that attack the substrates via water migration (Sheridan 2001, Murphy 2002, Crispim et al. 2004). In stone monuments, the cyanobacterial mats allowed the growth of more complex microbial consortia formed by heterotrophic microorganisms, and these contributed significantly to the subsequent deterioration (Tomaselli et al. 2000). The establishment of cyanobacterial mats on eucalyptus leaves may also provide a suitable microhabitat for the development of other parasitic microorganisms, such as fungi, yeast, and other bacteria. It is therefore possible that cyanobacterial mats participate in

the decay process directly, causing aesthetic damage and subsequent structural damages, as well as indirectly, by facilitating the growth of other microorganisms. Deterioration of leaf tissue may also be a consequence of toxins produced by cyanobacterial cells. It is well known that cyanobacteria are able to produce a range of toxic compounds that are hazardous to human and animal populations (Chorus and Bartram 1999). Among the known toxins, a large number of oligopeptides of nonribosomal origin, with unknown function, have been isolated and characterized from cultured strains and field samples (Welker and von Do¨hren 2006). Several studies were conducted to investigate the toxicity of microcystins, the most commonly known cyanotoxin, for plants or seedlings, and they are summarized in Babica et al. (2006). In Phaseolus vulgaris, for example, the exposure of leaves to the microcystin-LR reduced their CO2-saturated rate of photosynthesis and carboxylation efficiency and caused necrosis (Abe et al. 1996). The toxicity of microcystins is due to the inhibition of eukaryotic protein phosphatases 1 and 2A (Honkanen et al. 1990, MacKintosh et al. 1990). Protein phosphatases are involved in important cellular processes of plants, such as ion channel activity, carbon and nitrogen metabolism, gene expression, growth, and developmental processes (Smith and Walker 1996, Luan 1998, 2000, 2003, Toroser and Huber 2000). Production of microcystins has been detected in all cyanobacterial orders, including terrestrial genera such as Nostoc and Hapalosiphon (Sivonen and Jones 1999). Toxins produced by the cyanobacterial mats may have been delivered to eucalyptus leaves through cell excretion or cell lysis. The economic impact of colonization of E. grandis by cyanobacterial mats is a particular concern, since growth of the plants in the clonal nursery garden can be dramatically reduced and colonized young eucalyptus plants usually are not selected for commercial sale. E. grandis is of major economic importance due to its many uses. This study presents the first comprehensive data of a member of the family Scytonemataceae forming dense mats on E. grandis leaves in tropical and subtropical regions of Brazil. The new cyanobacterial species is responsible for serious damage to the leaves, stems, and buds of E. grandis and may constitute a major threat to these plants in clonal hybrid nursery gardens. This study was conducted with the support of the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG CRA ⁄ 2004) and the Botany Graduated Program supported by Federal University of Vic¸osa (UFV) and Brazilian Sponsoring Agencies: CNPq and CAPES. Research held at CENA ⁄ USP was supported by a grant from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP 2005 ⁄ 56303-5). A. S. Lorenzi received a graduate scholarship from CNPq (National Council for Scientific and Technological

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Development – 140327 ⁄ 2004-5). The authors greatly acknowledge Dr. Jirˇi Koma´rek (Institute of Botany, University of ˇ eske´ Bud South Bohemia, Faculty of Biology, C ejovice, Czech Republic) and Dr. Sea´n Turner (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD, USA) for valuable comments and suggestions on the manuscript. Abe, T., Lawson, T., Weyers, J. D. B. & Cood, G. A. 1996. Microcystin-LR inhibits photosynthesis of Phaseolus vulgaris primary leaves: implications for current spray irrigation practice. New Phytol. 133:651–8. Adams, D. G. 2000. Symbiotic interactions. In Whitton, B. A. & Potts, M. [Eds.] The Ecology of Cyanobacteria. Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht, the Netherlands, pp. 523–61. Allen, M. B. 1968. Simple conditions for growth of unicellular bluegreen algae on plates. J. Phycol. 4:1–4. Babica, P., Blaha, L. & Marsalek, B. 2006. Exploring the natural role of microcystins – a review of effects on photoautotrophic organisms. J. Phycol. 42:9–20. Carmello-Guerreiro, S. M. 1995. Technique for plant material inclusion using historesin. In Regional Meeting of Anatomists from Sa˜o Paulo State 1, 1995. UNESP. Rio Claro, Sa˜o Paulo, pp. 1 – 7. Chorus, I. & Bartram, J. [Eds.] 1999. Toxic Cyanobacteria in Water. A Guide to Their Public Health Consequences, Monitoring and Management. World Health Organization, E & FN Spon, London, 416 pp. Cohen, M. F. & Meeks, J. C. 1997. A hormogonium regulating locus, hrmUA, of the cyanobacterium Nostoc punctiforme strain ATCC 29133 and its response to an extract of a symbiotic plant partner Anthoceros punctatus. Mol. Plant-Microbe Interact. 10:280– 9. Crispim, C. A., Gaylarde, C. C. & Gaylarde, P. M. 2004. Biofilms on church walls in Porto Alegre, RS, Brazil, with special attention to cyanobacteria. Int. Biodeterior. Biodegrad. 54:121–4. Duong, T. P. & Tiedje, J. M. 1985. Nitrogen fixation by naturally occurring duckweed-cyanobacterial associations. Can. J. Microbiol. 31:327–30. Fiore, M. F., Moon, D. H., Tsai, S. M., Lee, H. & Trevors, J. T. 2000. Miniprep DNA isolation from unicellular and filamentous cyanobacteria. J. Microbiol. Methods 39:159–69. Fiore, M. F., Neilan, B. A., Copp, J. N., Rodrigues, J. L. M., Tsai, S. M., Lee, H. & Trevors, J. T. 2005. Characterization of nitrogen-fixing cyanobacteria in the Brazilian Amazon floodplain. Water Res. 39:5017–26. Fiore, M. F., Sant’Anna, C. L., Azevedo, M. T. P., Koma´rek, J., Kasˇtovsky´, J., Sulek, J. & Lorenzi, A. S. 2007. The cyanobacterial genus Brasilonema, gen. nov., a molecular and phenotypic evaluation. J. Phycol. 43:789–98. Honkanen, R. E., Zwiller, J., Moore, R. E., Daily, S. L., Khatra, B. S., Dukelow, M. & Boynton, A. L. 1990. Characterization of microcystin-LR, a potent inhibitor of type 1 and type 2A protein phosphatases. J. Biol. Chem. 265:19401–4. Johansen, D. A. 1940. Plant Microtechnique. McGraw-Hill, New York, 423 pp. Koma´rek, J. & Anagnostidis, K. 1989. Modern approach to the classification system of cyanophytes. 4 – Nostocales. Arch. Hydrobiol. 56:247–345. Kugrens, P., Clay, B. & Aguiar, R. 2000. Ultrastructure of Lobocharacium coloradoensis, gen. et. sp. nov. (Chlorophyta, Characiosiphonaceae), an unusual coenocyte from Colorado. J. Phycol. 36:421–32. Kumar, S., Tamura, K. & Nei, M. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5:150–63. Lee, R. E. [Ed.] 1999. Phycology. 3rd ed. Cambridge University Press, Cambridge, UK, 614 pp. Luan, S. 1998. Protein phosphatases and signaling cascades in high plants. Trends Plant Sci. 3:271–5.

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Supplementary Material The following supplementary material is available for this article: Fig. S1. Drawing of Brasilonema octagenarum UFV-E1 filaments. Fig. S2. TEMs of cyanobacterial mats associated with Eucalyptus grandis leaves. Fig. S3. (A–C) TEM. The sequence of events shows the interaction of cyanobacterial mats with leaf and the subsequent damages occurring on cell walls. This material is available as part of the online article from: http://www.blackwell-synergy.com/ doi/abs/10.1111/j.1529-8817.2008.00584.x. (This link will take you to the article abstract.) Please note: Wiley-Blackwell are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.