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. 2016 Aug 26;119(5):815–827. doi: 10.1093/aob/mcw169

Sugarcane smut: shedding light on the development of the whip-shaped sorus

João Paulo R Marques 1,2, Beatriz Appezzato-da-Glória 2, Meike Piepenbring 3, Nelson S Massola Jr 4, Claudia B Monteiro-Vitorello 1, Maria Lucia Carneiro Vieira 1,*
PMCID: PMC5378185  PMID: 27568298

Abstract

Background and Aims Sugarcane smut is caused by the fungus Sporisorium scitamineum (Ustilaginales/Ustilaginomycotina/Basidiomycota), which is responsible for losses in sugarcane production worldwide. Infected plants show a profound metabolic modification resulting in the development of a whip-shaped structure (sorus) composed of a mixture of plant tissues and fungal hyphae. Within this structure, ustilospores develop and disseminate the disease. Despite the importance of this disease, a detailed histopathological analysis of the plant–pathogen interaction is lacking.

Methods The whip-shaped sorus was investigated using light microscopy, scanning and transmission electron microscopy, histochemical tests and epifluorescence microscopy coupled with deconvolution.

Key Results Sorus growth is mediated by intercalary meristem activity at the base of the sorus, where the fungus causes partial host cell wall degradation and formation of intercellular spaces. Sporogenesis in S. scitamineum is thallic, with ustilospore initials in intercalary or terminal positions, and mostly restricted to the base of the sorus. Ustilospore maturation is centrifugal in relation to the ground parenchyma and occurs throughout the sorus median region. At the apex of the sorus, the fungus produces sterile cells and promotes host cell detachment. Hyphae are present throughout the central axis of the sorus (columella). The plant cell produces callose around the intracellular hyphae as well as inside the papillae at the infection site.

Conclusions The ontogeny of the whip-shaped sorus suggests that the fungus can cause the acropetal growth in the intercalary meristem. The sporogenesis of S. scitamineum was described in detail, demonstrating that the spores are formed exclusively at the base of the whip. Light was also shed on the nature of the sterile cells. The presence of the fungus alters the host cell wall composition, promotes its degradation and causes the release of some peripheral cells of the sorus. Finally, callose was observed around fungal hyphae in infected cells, suggesting that deposition of callose by the host may act as a structural response to fungal infection.

Keywords: Callose, histopathological analysis, plant anatomy, Saccharum spp., smut disease, Sporisorium scitamineum

INTRODUCTION

Sugarcane (Saccharum spp.) is a crop used to produce sugar in many tropical and sub-tropical countries, characterized by a long growing season and adaptation to large-scale farming. Currently, there are >100 countries producing sugarcane in diverse climates and growing conditions worldwide. Advanced research is opening up new paths for sugarcane, demonstrating that it can be the source of many renewable energy products, besides the commonly known ethanol and bioelectricity. Brazil is the world’s largest producer, responsible for 35 % of global production (http://sugarcane.org). Cane fields cover 9 Mha (2·8 % of the country’s arable land), and the 2016/2017 sugarcane harvest is expected to yield 655 Mt (Conab, 2016).

Cane juice is also used to produce the biofuel ethanol, through microbial fermentation and distilling processes. Second-generation bioethanol production from the crop is also possible through the use of cell wall-degrading enzymes for cellulose depolymerization of sugarcane bagasse, which offer a more sustainable approach than traditional chemical methods (Waclawovsky et al., 2010; Mamo et al., 2013). The wide-scale use of ethanol, combined with the expanding use of bioelectricity produced from the leftover biomass or bagasse, explains why sugarcane is already the second largest energy source in Brazil, and is considered the cleanest in the world (http://sugarcane.org).

Sugarcane production is affected by important diseases, with sugarcane smut, caused by Sporisorium scitamineum, occurring across all growing areas globally (Sundar et al., 2012). Infected plants show profound metabolic modification, resulting in the development of a whip-shaped structure (sorus) at the stalk apex, which contains a mixture of plant tissues and fungal hyphae. Within these structures, large numbers of dark ustilospores develop and disseminate the disease. Sugarcane plants are prone to pathogen attack in the early stages of plant growth.

Sorus development, which occurs during later stages of the infection, arises through changes in the shoot apical meristem and is the main diagnostic feature of this disease (Waller, 1970). Fungal infection also causes a reduction in the diameter and length of basal internodes and the number of tillers for industrialization, as well as increased fibre and lower sucrose content, which means that less sugar is extracted, impairing sugarcane productivity (Huang, 2004). The most economical and environmentally sustainable method for preventing the disease involves selecting for host resistance in existing breeding programmes.

The smut whip is an expanded apical internode (columella) composed of both fungal structures and plant tissues, covered with a silver peridium formed by the detached host epidermis and some sub-epidermal cells (Piepenbring, 1996). The whip varies in size from a few centimetres to > 1 m long, appearing as a dark mass of ustilospores beneath the peridium. As the peridium breaks up, the ustilospores are exposed and disseminated by wind and water. It is estimated that each sorus can produce up to 1011 spores of S. scitamineum (Waller, 1970).

Smut fungi are facultative biotrophs, since they grow saprotrophically as yeast-like cells on culture media but require biotrophic infection of host cells to complete their life cycle (Piepenbring, 2015). In the host tissue, these fungi grow both inter- and intracellularly, developing haustoria, and colonize the shoot meristem, affecting the reproductive development of infected plants (Langdon and Fullerton, 1975; Maytac, 1985; Martinez et al., 1999; Ghareeb et al., 2011). Once induced, ustilospores develop mostly embedded in the host tissue covered by a peridium that can be formed by plant and/or fungal cells.

While the genome sequence for the fungal pathogen has been described (Que et al., 2014b; Taniguti et al., 2015) together with transcriptome (Wu et al., 2013; Que et al., 2014a) and proteome analyses (Song et al., 2013), relevant for identification of potential effectors implicated in the infection process (Que et al., 2014b; Taniguti et al., 2015), our knowledge of the mechanisms involved in infection, colonization, sorus ontogeny and sporogenesis remain insufficient. In this study, we employed a combination of light and electron microscopy methods in order to elucidate in detail the infection, colonization of S. scitamineum hyphae in injured plant tissues, sporogenesis in S. scitamineum, sorus formation and host tissue structural modifications in response to the pathogen.

MATERIALS AND METHODS

Plant material

Whip-shaped sori at different developmental stages, as well as internodes occurring below the sorus and young inflorescences, were obtained from the ‘RB925345’ sugarcane cultivar. This plant material belongs to the Federal University of São Carlos, Brazil, a member of the Interuniversity Network Sugarcane Genetic Breeding Program (RIDESA) (http://pmgca.dbv.cca.ufscar.br). ‘RB925345’ exhibits an intermediate reaction to smut infection on the basis of the percentage of infected stools (12·6–15·5 %), adopting the disease scale reported by Lemma et al. (2015).

To obtain the sori, 250 buds from miniature stalks were collected from the middle third of 7-month-old plants. Heat treatment was used for disinfection (52 °C in water, 30 min), followed by washing in a 0·01 % sodium hypochlorite solution (10 min) and three washes in autoclaved distilled water. Before inoculation, buds were pre-sprouted by incubation in plastic boxes (24 h) lined with sterile filter paper moistened with sterile water. The mini-stalks were arranged with buds facing upward and covered with a sheet of moistened filter paper. After inoculation, the mini-stalks were planted in pots (0·5 L) containing a commercial substrate (Tropstrato®) and NPK fertilizer (10:10:10), and kept in a greenhouse. To analyse the different stages of sorus development, evaluations commenced 100 days after inoculation (DAI) and samples were collected every 15 d. Mature sori were analysed using 15 sori collected 220 DAI.

Inoculation procedure

Sporisorium scitamineum (Syd.) M. Piepenbr., M. Stoll & Oberw., ustilospores were collected from sori that developed on susceptible sugarcane genotype IACSP98-2053 at the Instituto Agronômico de Campinas, Brazil. Prior to inoculation, a viability test was carried out by incubating a 100 μL ustilospore suspension (1 × 104 ustilospores mL–1) on 0·1 % agar–water medium (8 h, 28 °C). The percentage germination was estimated by observing 100 ustilospores on a slide, with inoculum selected with a germination rate >80 %. Inoculum consisted of 0·5 g of ustilospores and 1 mL of a sterile aqueous solution containing 0·05 % NaCl and 0·01 % Tween-20. Inoculation was performed by wounding 200 buds with a sterile needle previously immersed in the inoculum (Wu et al., 2013). Fifty additional buds were inoculated with sterile water as controls.

Light microscopy and histochemical tests

For examining the sori by light microscopy, samples were fixed (24 h) in Karnovsky solution (Karnovsky, 1965), dehydrated in an ethanol series and embedded in hydroxyethyl-methacrylate (Leica Microsystems, Wetzlar, Germany). Transverse and longitudinal sections (5 μm) were cut with a rotary microtome (Leica RM 2045), and subsequently stained in 0·05 % toluidine blue, pH 4·3 (O’Brien et al., 1964). Images were digitally captured using a Leica DMLB microscope with a video camera coupled to a computer running IM50 software (Leica Microsystems).

The following histochemical tests were then performed: an aqueous solution of ruthenium red to detect the presence of acidic polysaccharides (Chamberlain, 1932); Sudan black B to detect lipids (Pearse, 1968); and Xylidine Ponceau to identify proteinaceous compounds (Cortelazzo and Vidal, 1991).

Epifluorescence microscopy and image deconvolution

To detect callose, 7 μm sections were stained with 1 % aniline blue (diluted in 0·1 m PO4 buffer, pH 9) for 20 min (Ruzin, 1999), and examined under the A4 filter set at 340–380 nm excitation and 450–490 nm emission wavelengths. Microscopic analysis was performed using an epifluorescence microscope (Leica DM 5500), and deconvolution of image stacks was performed with LAS AF software (Leica Microsystems) using a calculated point-spread function.

Electron microscopy

Samples to be examined under transmission electron microscopy (Tecnai Spirit; FEI Company, Hillsboro, OR, USA) at 80 kV were fixed in 3 % glutaraldehyde and 0·2 m sodium cacodylate buffer (pH 7·25, 24 h), post-fixed in 1 % osmium tetroxide in 0·1 m phosphate buffer (pH 7·2), and processed using standard methods (Roland, 1978). Ultra-thin sections were treated with 5 % uranyl acetate and lead citrate 2 % (15 min each) (Reynolds, 1963). To examine samples under scanning electron microscopy, the different regions of the whip-shaped sorus were fixed (48 h) in Karnovsky’s solution (Karnovsky, 1965), dehydrated in an ethanol series and dried to their critical point (Horridge and Tamm, 1969). These dried samples were glued on aluminium stubs, gold coated, and examined with a scanning electron microscope (FEI Quanta 200; FEI Company) at 20 kV.

RESULTS

General observations

The first whip-shaped sori were observed in plants 150 DAI, with a total of 63 plants producing sori. The emergence of smut whips continued until 270 DAI. Plants from which the smut whips were not observed had all died within 10 months after inoculation, with 15 % of inoculated plants dying within 20 DAI. Control plants exhibited vegetative growth throughout the experimental period.

Development of whip-shaped sori

Over the first 3 months, fungal cells were present in meristematic tissues without causing any visible impact on the development of plant tissues. At the early stages of soral development, the sorus was contorted and threadlike, with no structures related to floral organs (Fig. 1A, B). The young sorus was covered by the leaf sheath and did not show any morphological alteration up to 90 DAI, as observed based on a fortnightly harvesting schedule.

Fig. 1.

Fig. 1.

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Stages of soral development in the ‘RB925345’ sugarcane cultivar. (A) At 120 days after inoculation (DAI), the first recognizable stage consists of a filiform apical structure. (B) Detail (red rectangle) of (A). Note the absence of flower-like structures. (C) At 150 DAI, the whip-shaped sorus exhibits a white and a black zone. Internodes (IN) are shorter near the sorus base (arrows). (D) Detail (red rectangle in C) of the sorus. (E) At 230 DAI, mature sorus divided into white (I) and black (H) basal zones, and median (F) and apical (G) regions.

After this phase, the whip-shaped sori remained hidden by leaf sheaths, with two distinct regions recognized: a white basal region and a black median to apical region (Fig. 1C). Internodes directly below the sorus were closer to each other than the more basal nodes. The absence of spikelets or lateral branches was also observed (Fig. 1D). The mature sorus was formed by a filiform columella consisting of a mixture of plant tissues and fungal hyphae, and in some cases it was >1 m long. In this phase, it was possible to divide the sorus into three distinct regions (Fig. 1E–I). The basal part was hidden by leaf sheaths, and could be split into a white and black zone. The median region was partially hidden, whereas the apical region was thinner and completely exposed. Each of these regions had specific histopathological features, as described below.

The white base of the sorus exhibited an intercalary meristem with high mitotic activity and continuous acropetal growth of plant cells (Fig. 2A). Fungal hyphae were localized between or inside meristematic cells (Fig. 2B), and the cells continuously formed by the intercalary meristem were axially elongated and had sinuous walls (Fig. 2C). Continuous intercalary meristem activity enlarged the sorus (Fig. 2A). Therefore, even if the apical region of the sorus was removed, it did not stop growing. The fungal hyphae initiated sporogenesis primarily in the white base of the sorus and then extended acropetally, mediated by axial growth promoted by intercalary meristem activity. Initially, hyphal growth was intercellular, causing the middle lamellae to dissolve, with plant cell walls changing to a smooth fibrillar texture. This schizogenous process was particularly noticeable below the surface of the white base, culminating in the formation of intercellular spaces that accommodated the sporogenous hyphae. These spaces coalesced forming larger cavities (Fig. 2D–I). In the white base of the sorus, the sporogenous hyphae began to grow and form ustilospores. This region was pushed from the base towards the apex (acropetally) by the high mitotic activity of the intercalary meristem, with the ustilospores maturing in the black regions, where thallic chains of ustilospores were evident.

Fig. 2.

Fig. 2.

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Anatomy of the basal (white) zone of the sorus in the ‘RB925345’ sugarcane cultivar. (A–C, F) Longitudinal sections of the base under light microscopy and (D, E, G–I) under transmission electron microscopy. (A) Sporogenous hyphae (upper arrows), intercalary meristem (red rectangle) and internodes below the base of the sorus. (B) Detail of the intercalary meristem with intra- and intercellular hyphae (arrows). (C) Elongation of axial cells in the region above the intercalary meristem. Note the abundance of hyphae (arrows). (D–I) Details of the development of sporogenous hyphae. (D, E) The onset of the cell wall alterations, (D) hyphae among the host cells (arrows) and (E) fibrillar aspect of the host cell walls (*). (F, G) Schizogenous process of space formation (* in F) in G, sporogenous hyphae (arrows) in a mucilaginous matrix (*). (H) The space (*) increases without cell lyses. (I) Sporogenous hyphae occupying the intercellular space.

Apical thallic sporogenesis occurred in both black and white regions (Fig. 3A–J), while thallic sporogenesis occurred exclusively in the white region (Fig. 1I;Supplementary Data Fig. S1a–c). In apical thallic sporogenesis, the ustilospore began to swell in the apical region of the sporogenic hyphae. After this stage, a septum was formed centripetally, separating the sporogenous hypha from the ustilospore initial. The last stage consisted of detachment of the ustilospore initial. Detachment of the ustilospore produced scars in the apical region of sporogenous hyphae. In contrast, thallic sporogenesis usually occurred within pockets formed by intercellular spaces (Fig. S1a–c). This type of sporogenesis was concomitant with the fragmentation of the dikaryotic hyphae. The latter stages were followed by maturation in a mucilaginous matrix (Fig. 3k;Fig. S1k, l). Ustilospore maturation occurred centrifugally in relation to the ground parenchyma (Fig. 3k).

Fig. 3.

Fig. 3.

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Sporogenesis in Sporisorium scitamineum on the ‘RB925345’ sugarcane cultivar: aspects of thallic sporogenesis in white and black zones of the sorus. (A, K) Light micrographs. (B, G–J) Scanning electron micrographs. (C–F) Transmission electron micrographs. (A–C) Sporogenous hyphae (SH) (* in A). (D–F) Thallic sporogenesis at SH apex. Note the centripetal septum formation (arrows) and the fibrillar appearance of the mucilaginous matrix. (G–I) Ustilospore initials (TI, indicated by arrows in G and H) start in the SH apex. (I) Septum formation (arrows). (J) TI release promotes scar formation (arrows). (K) Ustilospore formation and maturation in distinct sectors of the black zone at the base.

After ustilospore detachment, the ustilospore initials developed a gelatinous thick wall and did not exhibit any vacuoles (Supplementary Data Fig. S1d). Vacuoles and lipid droplets then began to appear in the cytoplasm, and electron-dense echinulations developed starting at the plasma membrane (Fig. S1e, f, h, i). The mature ustilospore exhibited numerous lipid droplets (Fig. S1g–j) and electron-opaque inner and electron-dense outer walls with superficial echinulations (Fig. S1d–g). Sporogenesis led to the rupture of both epidermal and sub-epidermal layers, releasing the ustilospores (Fig. S1k).

Ultrastructural aspects of fungal hyphae interacting with plant cells

The hyphae of S. scitamineum can grow intercellularly or enter the protoplast. Intracellular hyphae can be ramified and colonize more than one cell. When the fungal cell penetrates a plant cell, the host plasma membrane invaginates, often close to the host cell nucleus (Fig. 4A–D, I), which frequently appears hypertrophied and lobate (Fig. 4I), exhibiting conspicuous nuclear pores. Mitochondria appeared in large numbers in the infected host cells, with abundant cristae and dictyosomes yielding several vesicles toward the intracellular hyphae (Fig. 4E–G). The endoplasmic reticulum of the host cell had a circular arrangement and in some cases was located close to the plasma membrane (Fig. 4H).

Fig. 4.

Fig. 4.

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Biotrophic interface between Sporisorium scitamineum hyphae and sugarcane cells. (A) Light micrograph. (B, C) Scanning electron micrographs. (D–I) Transmission electron micrographs. (A) Intercellular (*) and intracellular fungal hyphae (arrows). (B) Ramified intracellular hyphae (arrows). (C) Fungal hypha that apparently grew from one host cell into the neighbouring cell (arrow). (D) Fungal intracellular hypha (FU) close to the host nucleus (NU). (E, F) The host cell exhibits numerous dictyosomes (DI) and some vesicles fused with the plasma membrane (PM) close to the intracellular hyphae–host interface (arrows in F). Note the electron-dense material between the fungus and the host cell (*). (G) Several mitochondria close to the intracelullar hyphae (arrows). (H) Circular endoplasmatic reticulum (RE) that can be close to the plasma membrane (arrow). (I) Host cell exhibiting a lobed nucleus. CW, cell wall; FCW, fungal cell wall.

The presence of the fungus in vascular tissues changed both the phloem and xylem arrangements (Fig. 5). At the sorus periphery, tracheary elements of vascular bundles were separated, changing the organization into collateral vascular bundles (Fig. 5A, B). The fungus penetrated tracheary elements between the secondary lignified walls. Once inside, the fungus usually grew so as to avoid the secondary lignified walls (Fig. 5C, D). Throughout sorus development, the phloem exhibited several structural alterations. In the longitudinal section, inclusions close to the sieve plates of the sieve tube elements (STEs) were observed (Fig. 5E). The proteinaceous nature of these inclusions was confirmed by histochemical staining with Xylidine Ponceau (Fig. 5F). Ultrastructural analysis showed that STEs had type-P plastids (Fig. 5G), and cuneiform protein inclusions were found close to the sieve plate pores (Fig. 5H) that were also occluded by callose deposition (Fig. 5I;Supplementary Data Video S1).

Fig. 5.

Fig. 5.

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Vascular tissue of sugarcane altered by Sporisorium scitamineum. (A–F) Light micrographs. (G, H) Transmission electron micrographs. (I) Epifluorescence micrograph. (A, B) Vascular bundle at the periphery of the sorus columella. (B) Some phloem (PH) cells colonized by hyphae (arrows). Sporogenous hyphae and ustilospore initials (*). (C) Fungal hyphae in tracheary elements (TR). (D) Fungal colonization of a tracheary element. Arrows indicate how hyphae avoid lignified secondary walls. (E) Sieve tube elements (STE) exhibiting protein inclusions close to the sieve plate (arrow) (stained with Toluidine blue). (F) Evidence of the proteinaceous nature of the inclusions by staining with Xylidine Ponceau. (G) Plastid-P (PP) in sieve tube elements with cuneiform protein inclusions. (H) Cuneiform protein (PR) inclusion near the sieve plate with callose deposition in sieve pores (*). Note the complete pore obstruction in the sieve plate. (I) Phase contrast merge image under an A4 filter after aniline blue staining, showing callose deposition on the sieve plate (arrows).

When fungal hyphae came into contact with host cells, electron-dense material possibly produced by the host cell was present close to the surface of fungal cells, both inter- and intracellularly (Fig. 6A–C). Plant cell wall material was observed around intracellular hyphae, mainly in young sori. Using transmission electron microscopy and histochemical tests, we observed that many hyphae were completely encased by sheaths consisting mainly of callose (Fig. 6D–F;Supplementary Data Video S2). In addition, fungal penetration was restricted by the formation of papillae (Fig. 6G). In median and apical regions of the sorus, callose deposition was present on the host cell walls of the columella, mainly at the periphery (Fig. 6H–J).

Fig. 6.

Fig. 6.

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Sugarcane defence response against infection by Sporisorium scitamineum. (A–C, F–G) Transmission electron micrographs. (D) Light micrograph. (E, H–J) Epifluorescence micrographs under an A4 filter after aniline blue staining. (A, B) Intercellular growth. Note the deposition of electron-dense material around the hyphae (arrows in B). (C) This hypha apparently could not penetrate the plant cell wall (arrows). (D–F) Callose encapsulation of intracellular hyphae (arrows). (D) Callose encasement around intracellular hyphae (arrows). (E) Image showing the callose encasement (arrowheads and arrows). (F) Callose deposition around intracellular hyphae. (G) Longitudinal section of an intracellular hypha showing the callose sheath (*) completely surrounding it. (H, I) Periphery of the sorus observed under fluorescence and bright field optics. (J) Merged images of (H) and (I). (H–J) Cells in contact with ustilospores (TE) have thick walls with callose deposition. CW, cell wall; HC, host cell; FU, fungal hyphae; NU, nucleus; PH, phloem; XL, xylem.

The median and the apical regions of the sorus did not exhibit any sporogenous hyphae, the peridium was lost and the sorus surface was covered by mature ustilospores (Fig. 7A–C). Under polarized light, the birefringence of the plant cell wall was lost in the cells of the whip's periphery (Fig. 7D), indicating that the crystalline structure of the cellulose was modified. In their classic study, Bateman and Basham (1976) suggested that the loss in birefringence of plant cell walls in tissues invaded by pathogens is an indication of the enzymatic destruction of crystalline cellulose by the pathogen.

Fig. 7.

Fig. 7.

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Anatomical features of the apical and median regions of the sorus. (A–C, J, L–N) Light micrographs. (D, F–I) Polarized light. (E, K) Transmission electron micrographs. (A) Overview of a transverse section of the columella in its apical part. Note the absence of the peridium. (B, C) The sorus surface is depressed (arrows in B) by the constant release of host cells (HC) (arrows in C). (D) The loss in birefringence of plant cell walls (arrowed) in the periphery of the sorus (white arrowheads). (E, F) The release process of host cells starts with hyphae installation between two host cells. (G-I) Note the birrefringence of the cell walls of the detached host cells (arrows). (J–L) Early developmental stage sterile fungal cells (SC) (arrow). (K, L) Divided sterile fungal cells retaining the wall (arrows). (M) Mature sterile fungal cells showing thickened cell walls. (N) Sterile fungal cells among the ustilospores (TE) containing lipid substances stained with Sudan Black B (I). FU, fungal hyphae; VB, vascular bundle.

The fungus grew intercellularly within the ground parenchyma, promoting host cell detachment (Fig. 7E, F). Some fungal cells apparently divided while attached to the host cells and were subsequently released (Figs. 7G–I). The formation and maturation of sterile fungal cells were concurrently observed. These cells are formed among the ustilospores by asymmetrical divisions (Fig. 7J) and retain the mother cell wall (Fig. 7K). Young sterile cells divide several times and have a thickened cell wall when mature (Fig. 7L, M). They were shown to contain compounds of a lipophilic nature (Fig. 7 N).

DISCUSSION

Whip-shaped sorus development

Smut fungi on Poaceae can lead to floral induction, colonizing the reproductive structures by a mechanism called floral reversion or phyllody (Matheussen et al., 1991; Ghareeb et al., 2011). In the 1970s, the whip-shaped sorus of the sugarcane smut was described as a modified inflorescence (Waller, 1970; Leu et al., 1976). More recently, Piepenbring et al. (2002) recognized the structure to be a whip, rather than an inflorescence. However, our anatomical analyses indicate that this filiform structure is an elongated internode produced by continuous intercalary meristem activity. No flower-like structures were observed.

Once formed, the sorus shows indeterminate growth mediated by high mitotic activity in the intercalary meristem at its basal region, previously referred to as the soral meristem (Langdon and Fullerton, 1975). The occurrence of intra- and intercellular hyphae in the intercalary meristem suggests that the pathogen secretes substances that act as a trigger to induce continuous mitosis, favouring the growth of an indeterminate acropetal sorus.

Establishment and sporogenesis of S. scitamineum

The establishment of sporogenous hyphae occurs only at the sorus base. Similar findings have been reported for other pathosystems such as Ustilago maydis–Zea mays (Snetselaar and Mims, 1994), Sporisorium provinciale–Andropogon gerardii (Snetselaar and Tiffany, 1990), Ustilago esculenta–Zizania latifolia (Yang and Leu, 1978) and Sporisorium caledonicum–Heteropogon contortus (Langdon and Fulleton, 1975). The development of sporogenous hyphae was associated with the dissolution of the middle lamellae. Both this dissolution and the smooth fibrillar appearance of the cell wall suggest that S. scitamineum secretes cell wall hydrolytic enzymes promoting tissue disintegration and resulting in large cavities (Evert, 2006). However, further investigation is needed in order to histolocalize and characterize such enzymes related to the development of the sporogenous hyphae.

Thallic sporogenesis occurs in S. scitamineum, confirming its placement in the ‘Ustilago group’ (Piepenbring et al., 1998). We observed that dikaryotic hyphae grow intra- or intercellularly in the host tissue or on its surface and disintegrate into hyphal segments, or ustilospore initials, embedded in a hyaline matrix, as shown by Piepenbring et al. (1998). In S. scitamineum, spore initials are formed at the apex of sporogenous hyphae and are not directly connected to vegetative hyphae. They dissociate in clumps of sporogenous hyphae, where there is a separation of ustilospore initials from sporogenic hyphae, as also demonstrated by Trione (1980). Sporisorium scitamineum was shown herein to exhibit sporogenesis in the black zone at the sorus base. This process of spore formation is similar to that reported for S. consanguineum (Fernandez and Hess, 1978).

Ustilospore maturation occurs immersed in a matrix, similar to that observed in the sori of U. esculenta (Yang and Leu, 1978). The matrix is mucilaginous, providing a hydrophilic environment for S. scitamineum sporogenesis. Ustilospore wall ornamentation and accumulation of lipid droplets observed herein for S. scitamineum was similar to that of S. sorghi (Mims and Snetselaar, 1991). In Ustilago nuda, lipid and glycogen are the main reserve compounds of ustilospores (Laere and Franzen, 1989). In S. scitamineum, ustilospores contain lipid droplets that possibly act as a source of energy during germination.

Biotrophic interface

Our results show that the plant–pathogen interface in sugarcane smut is comparable with that of other Ustilaginaceae fungi (Langdon and Fullerton, 1975; Mims and Snetselaar, 1991). Sporisorium scitamineum does not develop haustoria. Instead it develops intracellular hyphae with indeterminate growth. Intracellular hyphae of S. scitamineum are similar to those of S. reilianum (Martinez et al., 1999) and S. provinciale (Snetselaar and Tiffany, 1994). According to Perfect and Green (2001), intracellular hyphae tend to be less specialized than haustoria and, due to their indeterminate growth, they can spread from cell to cell remaining in the biotrophic phase.

Here we have shown that S. scitamineum hyphae are distributed both inter- and intracellularly throughout the sorus, as previously reported by Sundar et al. (2012). The intercellular growth involves partial degradation of the sugarcane cell wall. According to Souza et al. (2012), the sugarcane cell wall contains cellulose closely associated with xyloglucan and arabinoxylan, and cellulose strongly bound to pectins. A set of cell wall-degrading enzymes expressed during early and late stages following S. scitamineum infection was reported in sugarcane (Taniguti et al., 2015). This arsenal involves cellulose-, pectin- and hemicellulose-degrading enzymes, allowing S. scitamineum to colonize the sugarcane tissues, including lignified tissues such as tracheary elements.

The vascular bundles in sugarcane are collateral (van Dillewijn, 1952). However, along with the sorus, the separation of tracheary elements causes the formation of abnormal bundles. Vascular bundle alteration has also been reported in arabidopsis leaves infected by Verticillium longisporum (Reusche et al., 2012). For the first time, we describe the presence of S. scitamineum within tracheary cells, similar to S. reilianum in maize (Maytac, 1985), representing a strategy for fungal locomotion, since these cells do not have protoplasts. Fungi that colonize only vessel elements, such as Fusarium oxysporum f. sp. lycopersici, can secrete small proteins within the xylem (SIX, secreted in xylem), associated with virulence (Rep et al., 2005). We were not able to determine the role that the colonization of tracheary elements plays in S. scitamineum infection.

The median and apical regions of the sorus continuously release parenchyma cells, and this releasing process could be related to wall-degrading enzymes produced by S. scitamineum, as mentioned above. The process causes a reduction in sorus diameter in the exposed portions, endowing it with a characteristic morphology. The participation of phytohormones in this rapidly induced cell division should be considered in future studies. In addition, these cells start to accumulate lipophilic compounds and remain interspersed with the spores. It is difficult at this point to be sure about the function, if any, of these released cells.

Sugarcane structural responses to S. scitamineum infection

For the first time we observed that a callose sheath might encapsulate infective intracellular hyphae, and be accumulated inside the papillae. Callose and papillae are known to be effective physical barriers, formed and deposited at infection sites in response to pathogen invasion (Luna et al., 2011; Giraldo and Valent, 2013; Chowdhury et al., 2014; Voigt, 2014). Callose deposition is usually triggered by pathogen-associated molecular patterns (PAMPs) (Luna et al., 2011; Giraldo and Valent, 2013). Regarding its pivotal role in cell wall-associated defence response triggered by PAMPs, callose deposition is considered to happen late after PAMP-triggered immunity elicitation (Li et al., 2016). Additionally, in arabidopsis, elevated early amounts of callose deposition resulted in complete resistance to fungal penetration (Ellinger et al., 2013). While our observations of callose deposition may indicate a role in the formation of structural barriers during S. scitamineum colonization, ‘RB925345’ exhibits an intermediate reaction to smut infection, and the fungus efficaciously colonizes the entire sorus, allowing many intracellular hyphae to establish themselves.

Conclusions

We have described the ontogeny of the whip-shaped sorus and suggest that the fungus can incite acropetal growth via the intercalary meristem. Sporogenesis in S. scitamineum is thallic, with ustilospore initials in intercalary or terminal positions, and mostly restricted to the base of the sorus. The presence of the fungus promotes host cell wall degradation, induces the release of some peripheral cells and also alters their composition. Additionally, callose was observed around fungal hyphae in infected cells, suggesting that deposition of callose by the host may act as a structural response to fungal infection. Our novel findings related to pathogen infection and colonization, together with sorus development, clearly advanced our understanding of this pathosystem.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Figure S1: aspects of the development of ustilospores in sori of Sporisorium scitamineum on the ‘RB925345’ sugarcane cultivar. Video S1: animation of callose deposition close to the sieve plate magnified ×63. Video S2: animation of callose encapsulation around the intracellular hyphae magnified ×63.

Supplementary Material

Supplementary Data
Click here for additional data file. (19.3MB, zip)

ACKNOWLEDGEMENTS

The authors acknowledge the following Brazilian institutions: Fundação de Amparo à Pesquisa do Estado de São Paulo for the fellowship given to J.P.R.M. (2009/25315-1); Centro de Microscopia Eletrônica, Universidade Estadual Paulista, Campus de Botucatu; Universidade Federal de São Carlos (L. F. D. Pereira) for supplying the plant material; Laboratório de Microscopia Eletrônica e Microanálise, Universidade Estadual de Londrina (M. H. P. Fungaro and C. G. T. de Jesus), Centro de Microscopia e Imagem, Faculdade de Odontologia, Universidade Estadual de Campinas (E. A.O. Narvaes and F. S. M. Rodrigues) for microscopy facilities. We also thank Mr Steve Simmons for proofreading the manuscript.

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