Morphological and histo-anatomical traits reflect die-back in Phragmites australis (Cav.) Steud.

Aquatic Botany 103 (2012) 122–128

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Morphological and histo-anatomical traits reflect die-back in Phragmites australis (Cav.) Steud. Lara Reale ∗ , Daniela Gigante, Flavia Landucci, Francesco Ferranti, Roberto Venanzoni Department of Applied Biology, University of Perugia, I-06121 Perugia, Italy

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Article history: Received 10 November 2011 Received in revised form 10 July 2012 Accepted 13 July 2012 Available online 23 July 2012 Keywords: Callus Lignification Phragmites australis Plant traits Reed decline Rhizome Root Starch Vascular occlusion

a b s t r a c t The decline of Phragmites australis (Cav.) Steud., a well-known phenomenon in Central and Western Europe, was detected recently also in the Mediterranean Basin. A range of parameters were quantified in healthy and affected stands as a screening for indicators of die-back. These were: stem dimension, density and pattern, starch storage, presence of occlusions in the vessel cells, abnormal lignification and calli in the root parenchyma. First, high stem density, occlusions, lignification and calli did not always coincide with die-back. Second, starch content appeared a reliable indicator. The higher starch amount detected in the healthy stands can be related to the greater ability to perform photosynthesis and accumulate reserves. Furthermore, the starch lack detected in the declining stands might result from the increased starch demand, due to the shoot regeneration connected to the clumped habit. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Phragmites australis (Cav.) Steud. is one of the most widespread angiosperm species in the world (Tucker, 1990). It grows in lowland temperate regions of both hemispheres. It is most abundant in North America and Eurasia, with the northern limit at 70◦ N in Norway, and is also widespread in non-tropical Africa and temperate parts of Australia (Engloner, 2009). Reed beds are considered valuable ecosystems and are often protected because of their important ecological functions (Brix, 1999; Berthold et al., 1993). In fact they provide habitats for different groups of fauna, especially birds and insects (Berthold et al., 1993; Brix, 1999; Tscharntke, 1999; Tewksbury et al., 2002). Reed beds are also used for land reclamation and erosion prevention (Coops et al., 1994), and for wastewater treatment (Cooper and Green, 1995). The decline of reed in parts of Europe has prompted much research (Van der Putten, 1997); its occurrence in the Mediterranean Basin was noticed in the last decade (Fogli et al., 2002; Gigante et al., 2008, 2010, 2011; Bresciani et al., 2009), disproving the general opinion that this phenomenon is absent in Mediterranean areas.

∗ Corresponding author at: Department of Applied Biology, Borgo XX Giugno 74, I-06121 Perugia, Italy. Tel.: +39 075 5856407; fax: +39 075 5856407. E-mail address: [email protected] (L. Reale). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2012.07.005

Reed die-back can be induced by several factors, including sudden changes in the water table (Weisner and Graneli, 1989; Gutteridge and Halliwell, 1990), mechanical damage (Dinka et al., 1995; Sukopp and Markstein, 1989) and subsequent flooding (Hellings and Gallagher, 1992), intensely reducing soil conditions and eutrophication (Cizkova-Koncalová et al., 1992), phytotoxin accumulation (Kovacs et al., 1989), raised temperatures and insect or fungal damage (Armstrong and Armstrong, 1995; Armstrong et al., 1996a). These factors interfere with the internal aeration and carbohydrate balance and possibly with the water and mineral uptake of reed, locally leading to stunted growth and an abnormally high mortality of plant parts. Accumulation of organic matter and production of phytotoxins may cause plant death and prevent recolonization in the short term, thus promoting the vicious circle known as the die-back syndrome (Armstrong et al., 1996b). Armstrong et al. (1996b) observed that die-back in P. australis was associated with callus occlusions in the intercellular space, blockages in the vascular systems, wall lignification and suberization in the normally absorptive regions of the root system. Other symptoms include altered ratios between various amino acids and between amino acids and sugars in culm bases (Kohl and Henning, 1987; Kohl et al., 1998) and lower levels of starch in rhizomes (Cizkova et al., 1996). These anatomical and physiological changes possibly are adaptive responses to minimize the negative impact of adverse environmental factors, mechanical and chemical injury or pathogenic attack.

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Table 1 Names, ID, geographical coordinates and main environmental features of sites and plots. Site

Plot ID

Geographical coordinates

Water depth, end Aug 2009 (cm)

Presence of litter

Oasi La Valle

OAS1 OAS3 OAS5 OAS6

N43◦ 05.699 N43◦ 05.694 N43◦ 05.681 N43◦ 05.686

E12◦ 11.034 E12◦ 11.044 E12◦ 10.946 E12◦ 10.983

35 70 80 85

Abundant Abundant Abundant Abundant

Porto di Panicarola

POR3

N43◦ 04.848 E12◦ 06.523

0

Absent

Rio Pescia

POM1 POM2

N43◦ 06.752 E12◦ 02.943 N43◦ 06.747 E12◦ 02.933

0 0

Absent Absent

Passignano

PAS1 PAS2

N43◦ 11.412 E12◦ 06.491 N43◦ 11.415 E12◦ 06.495

0 0

Absent Absent

Considering that several physiologic processes and a number of macroscopic and histologic traits are involved in the reed die-back syndrome, we tested a set of parameters generally considered as die-back indicators in order to point out (1) which of them actually co-occur in a manifest condition of reed decline and (2) which can be reliably used as valid diagnostic traits. 2. Materials and methods 2.1. Site details Lake Trasimeno has an average size of ca. 121.5 km2 and average depth of 4.2 m (considered period: 1963–1997; Dragoni and Evangelisti, 1999). It has a pluviseasonal-oceanic Mediterranean macrobioclimate, upper meso-Mediterranean bioclimatic belt with subhumid ombrotype (data recorded in Monte del Lago; Gigante and Venanzoni, 2007). On the ground of former analyses carried out in 2006–2007 in the same area (Gigante et al., 2010, 2011), 9 plots (1 m × 1 m) were fixed along the littoral reed belt. The plots were located both in sites without evident macroscopic signs of decay, identified as “healthy stands” (PAS1, PAS2, POM1, POM2, POR3), and in stands seemingly affected by decline and retreat, identified as “dying-back stands” (OAS1, OAS3, OAS5, OAS6), as indicated by Gigante et al. (2011). Names, codes, geographical coordinates and some environmental features of sites and plots are reported in Table 1. 2.2. Plant traits Four macroscopic traits were selected based on Van der Putten (1997) and former studies in the same area (Gigante et al., 2010, 2011): stem height (cm), diameter (mm), density (nm−2 ), clumping habit (nm−2 and %). Regarding stem height and diameter, 15 stems of reed were randomly chosen in each plot (for a total amount of 135 stems) and measured with a metric tape and a caliber, respectively. The height included the stem between the ground and the base of the inflorescence; the diameter was measured at about 120 cm above ground. Stem density was measured in each plot counting the total number of stems. The presence of clumping habit, caused by breaking of apical dominance and resulting in uncontrolled outgrowth of dormant buds (Van der Putten, 1997), was quantified by counting the number of clumped stems in each plot. This value was related to the total stem number in order to obtain the rate of clumped stems. Measurements were carried out in August 2009. 2.3. Cyto-histological observations Anatomical and cyto-histological analyses were carried out on material from the same plots where morphological investigations were performed. For each of the 9 plots, 6 rhizomes and 10 roots have been collected in spring and autumn 2009.

Histo-anatomical structure was investigated by observation of hand made and semi-thin sections of samples embedded in epoxy resin. To obtain semi-thin sections, portions of rhizomes (from the middle internodal region) and roots (both at 1–2 cm from the apex and near the insertion on the rhizomes) were fixed in 3% (w/v) glutaraldehyde in 0.075 M cacodylate buffer, pH 7.2, for 5 h. Then samples were washed four times for 15 min each in 0.075 M cacodylate buffer, pH 7.2 and post-fixed in 1% (w/v) OsO4 . At this stage, samples were dehydrated in increasing concentrations of ethanol and then included in resin (Epon, 2-dodecenylsuccinic anhydride, and methylnadic anhydride mixture). The semi-thin sections (1–2 ␮m) were cut with an ultramicrotome (OmU2, Reichert, Heidelberg) equipped with a glass blade, stained with toluidine blue and observed under a light microscope (DMLB, Leica, Wetzlar, Germany). 2.4. Evaluation of polysaccharide content To evaluate the starch deposition in rhizomes (at the start and at the end of the vegetative season) and adventitious roots (at the start of the vegetative season), fresh sections cut with a hand microtome were treated with iodide iodine solution (Johansen, 1940) and observed under a light microscope. Semi-thin sections of the same samples after deresination were rehydrated and treated with iodide iodine solution (Johansen, 1940) and observed under a light microscope. The presence of starch grains was indicated in both cases by a blue-dark colour. To quantify starch content, the image of each section, stained with iodide iodine solutions, was analysed by the image processing program “ImageJ” (Abramoff et al., 2004). The amount of starch was reported as relative units (pixel ␮m−2 ), calculated as the number of pixels, measured by the software, related to the observed surface (␮m2 ). In the adventitious roots the starch content was also checked by Periodic Acid Schiff’s reaction (O’Brien and McCully, 1981). Slides were observed under a light microscope; the presence of starch was indicated by a magenta colour, while proteins appeared blue. Presence and kind of gels inside vessels were analysed by staining with toluidine blue O. The fresh sections of roots cut with a hand microtome were treated for 15 min with toluidine blue 0.025% in 0.1 M citrate buffer pH 4, rinsed with demineralized water and observed under a light microscope; the presence of phenols was indicated by a green colour, while mucine/pectins appeared pink to purple. 2.5. Fluorescent staining procedure for lignin and suberin The presence of callus associated with abnormal lignification or suberization of cell walls was detected in the adventitious roots

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Fig. 1. (A) Transversal hand-cut section of rhizome stained with crystal violet, bar = 500 ␮m. (B and C) Transversal semi-thin sections of adventitious root, near to the rhizome (B) and near to the apex (C), stained with toluidine blue, bar = 200 ␮m. (D–F) Transversal hand-cut sections of adventitious roots, near to apex (D) and near to the rhizome (E and F), collected from OAS6 (D) and PAS2 (E and F), after staining with toluidine blue O, bars = 200 ␮m. (G) Transversal hand-cut sections of adventitious roots from a POM site, after staining with berberine, bar = 500 ␮m. (H and I) Transversal hand-cut sections of adventitious roots collected from PAS1 (H, bar = 500 ␮m) and OAS6 (I, bar = 250 ␮m) after staining with berberine. a = aerenchyma; e = endodermis; ex = exodermis; p = pith; v = vascular bundle; x = xylem.

by berberine staining. Freehand sections, taken approximately 10–20 mm from the apex of roots collected at different sites, were stained with 0.1% (w/v) berberine hemi-sulphate (Brundrett et al., 1988). Slides were observed with a UV epifluorescence microscope (Leica DMR HC) using excitation filter BP 340–380 and suppression filter LP 425. The presence of lignin and suberin was indicated by a white colour.

2.6. Statistical treatment of the data The normal distribution of stem height and diameter was tested by Shapiro–Wilk test and Skewness and Kurtosis coefficients. Their statistical significance was then tested by an independent-samples t-test. The stem density and the presence of starch were tested by non-parametric Mann–Whitney U-test, since their distribution did

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Table 2 Results of the morphological and histo-anatomical analyses (av. ± SE) for the two groups of plots. Variable name Stem height (cm) Stem diameter (mm) Stem density (nm−2 ) Starch amount in the rhizomes (pixel ␮m−2 ) Starch amount in the rhizomes (pixel ␮m−2 ) Starch amount in the adventitious roots (pixel ␮m−2 ) a

Healthy stands 259.40 7.80 65.40 95.80 1.309.20 6.89

± ± ± ± ± ±

9.19 0.27 8.97 28.59 227.11 2.30

Dying-back stands

p t-testa

± ± ± ± ± ±

<0.001 <0.001 <0.05 <0.05 <0.05 <0.05

188.04 5.47 170.67 0.15 397.87 0.17

11.13 0.27 34.21 0.03 258.38 0.05

For stem density and starch amount a Mann–Whitney rank test was used, because data distribution did not conform to normality.

not conform to normality. The correlations have been tested by applying Spearman’s rank correlation coefficient. All the statistical analyses have been performed by using the software Analyst Soft Stat Plus: mac v2009. 3. Results 3.1. Plant traits The results of the morphological analysis are reported in Table 2. In the whole population, the stem diameter ranged between 2.2 and 13.5 mm with an average of 6.9 mm; stem height varied between 53 and 399 cm with an average of 232.6 cm; stem density ranged between 45 and 237 stems m−2 . Both stem height and diameter showed remarkable differences among the 2 considered groups of plots (Table 2, t-test: p < 0.001); stem density was remarkably higher in the declining stands (Table 2, Mann–Whitney U-test: p < 0.05). The clumping habit occurred in all the plots belonging to the dying-back group, where it affected a remarkably high rate of stems, 93.7% on average (OAS1: 85%, OAS3: 100%, OAS5: 90%, OAS6: 100%), while it was completely absent in the healthy plots. When we confronted the stem density and the clumping occurrence, the highest density appeared strongly correlated to the presence of clumped growth (Spearman’s rank correlation coefficient: 0.817, p < 0.01). 3.2. Cyto-histological observations The histo-anatomical structure of rhizomes (Fig. 1A) and roots (Fig. 1B and C), observed in all stands, can be considered quite typical for P. australis (Soukup et al., 2000, 2002). No structural or histological differences were found between samples taken from different sites. Root samples collected at different distance from the apex (Fig. 1B and C) showed only differences related to different stage of development. The presence of occlusions in the vessel cells, mainly due to the presence of mucine and/or pectin (pink coloured), was detected in all sites (Fig. 1D–F) except OAS1, OAS5 and PAS2. 3.3. Callus and abnormal lignifications detection Calli (white coloured), wholly or partly blocking the cortical aerenchyma, were found in samples collected from all sites (Fig. 1G). Abnormal lignification, indicated by a white colour of the aerenchyma cell walls, normally pectocellulosic, was found in samples collected from healthy stands (Fig. 1H) and from only one of the dying-back plots (OAS5) (Fig. 1I). 3.4. Starch detection In the rhizome of the healthy stands, in autumn, starch (indicated by black dots except in Fig. 2K and L where it appears

magenta) was abundant and present in all the portions of the rhizome (Fig. 2A and B) while in spring it was scarce and present only in the central cylinder (Fig. 2D) or absent (Fig. 2E). In the dyingback stands, it appeared remarkably scarce and present only in the central cylinder in autumn (Fig. 2C); it was totally absent in spring (Fig. 2F). Starch was never detected in the roots collected from dyingback stands (Fig. 2G). The roots collected from healthy stands showed high amounts of starch in autumn (Fig. 2H and K) while it resulted poor (Fig. 2I) or absent in spring (Fig. 2J and L). The starch amount in reed rhizomes and adventitious roots, expressed by relative units (pixel ␮m−2 ), is reported in Table 2; the quantitative analyses confirmed the qualitative observations. The differences between the 2 groups of healthy and dying-back plots were evident and statistically significant (Mann–Whitney U-test, p < 0.05). 4. Discussion The morphologic analyses confirmed the detection of 2 groups of healthy and dying-back stands in the reed population, the latter displaying smaller stems and clumped habit, often indicated as peculiar die-back symptoms (Mochnacka-Lawacz, 1974; Dinka, 1986; Hofmann, 1986; Den Hartog et al., 1989; Van der Putten, 1994, 1997; Rolletschek et al., 1999; Hardej and Ozimek, 2002; Haslam, 2010). Similar signs of decay, together with a remarkable incidence of dead buds and higher rates of viable seeds compared to the healthy stands, had been formerly reported in the same area and plots (Gigante et al., 2010, 2011; Reale et al., 2011). The dying-back stands were characterized by permanent submersion and a pronounced presence of organic matter and standing litter (Table 1). The excessive production and deposition of litter, especially in aquatic stands, might result in growth reduction or decline also due to toxic effects (Granéli, 1989; Weisner and Graneli, 1989; Van der Putten, 1993; Weisner, 1996; Graveland and Coops, 1997; Van der Putten et al., 1997; Clevering, 1998; Armstrong et al., 1994, 1996a,b). Although chemical analyses of the sediment have not been carried out in the present study, bibliographic data show remarkably higher amounts of toxic compounds in a sector of the lake very close to the dying-back stands (De Bartolomeo et al., 2004). The highest rates of clumping co-occurred with the highest stem density, with a statistically significant correlation. A high shoot density has often been considered as an indication of reed health (Van der Putten, 1997) although, like in the present study, it can be due to the presence of multiple, thinner stems resulting from the apical dominance breaking down, another symptom of reed die-back (Armstrong et al., 1996a; Clevering, 1999). This indicates that the stem density alone might not be a robust trait to detect reed die-back.A correlation between die-back and lack of starch in roots and rhizomes, previously suggested by Cizkova et al. (1996), has been confirmed. Starch was largely detected in rhizomes and roots from healthy stands, being respectively very scarce or absent in rhizomes and roots from declining plots. As concerns the adventitious roots, in spring some differences were observed also among

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Fig. 2. (A–C) Transversal hand sections, stained with iodide iodine solution, of rhizomes collected in autumn from healthy stands PAS (A) and POM (B) and dying-back stands (C), bars = 500 ␮m. (D–F) Transversal hand sections, stained with iodide iodine solution, of rhizomes collected in spring from PAS (D), POM (E) and OAS (F), bars = 500 ␮m. (G) Transversal hand section, stained with iodide iodine solution, of adventitious root from an OAS site, bar = 200 ␮m. (H–J) Transversal hand sections, stained with iodide iodine solution, of adventitious roots collected from the healthy stands PAS in spring (I and J) and POR in autumn (H), bars = 200 ␮m. (K and L) Transversal semi-thin sections, stained with Schiff’s reagent and Amido Black solution, of adventitious roots collected from POM healthy sites in autumn (K, bar = 200 ␮m) and in spring (L, bar = 500 ␮m). a = aerenchyma; ex = exodermis; p = pith; v = vascular bundle; x = xylem.

the healthy stands. It should be considered that, differently from the rhizome, the adventitious root is not a storage organ and can accumulate starch only if this is very abundant in the plant. In this light, the starch amount in adventitious roots might be used as a more stringent and revealing parameter.The higher starch amount detected in the healthy stands is to be related to the greater ability to perform photosynthesis and accumulate reserves. The suffering plants, which had a lower photosynthetic activity in the previous year and could not store reserves, undergo a self-increasing process of storage-lack that may lead to death. Furthermore, the starch lack may be a result of the increased starch demand, due to the shoot

regeneration in the stands affected by clumped habit, as suggested by Dinka and Szeglet (1999). No differences in the anatomical structure of rhizomes and roots were observed among the analysed stands and in the rhizome nodal septa we did not detect any blockages. With regard to cyto-histological parameters, occlusions in the vessel cells and abnormal lignification or calli in the root parenchyma, generally considered symptoms of die-back (Armstrong et al., 1996b), were not selectively detected in the declining sites. Occlusions were sometimes present in stands where reed showed an optimal growth condition and abnormal

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lignification of aerenchyma cells was not detected in the dying-back stands. We hypothesize that these traits alone might not always be good indicators of die-back, considering that they may be also induced by mechanical damage or fungal attack. A rich endophytic community was indeed detected in the analysed stands by Angelini et al. (2012).

Acknowledgement This work was partially funded by the Umbria Region (POR FSE 2007-2013 Asse II Occupabilità, obiettivo specifico “E” – Asse IV “Capitale Umano”, Obiettivo specifico “L”, Risorse CIPE”).

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