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Comparative responses of two water hyacinth (Eichhornia crassipes) cultivars to different planting densities
Aquatic Botany 121 (2015) 1–8
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Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Comparative responses of two water hyacinth (Eichhornia crassipes) cultivars to different planting densities Xiao Shu, Qi Deng, QuanFa Zhang, WeiBo Wang ∗ Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 9 October 2014 Accepted 23 October 2014 Available online 1 November 2014 Keywords: Physiology Photosynthesis Radial oxygen loss Dissolved oxygen Microbial diversity Water hyacinth
a b s t r a c t Two water hyacinth cultivars, i.e., common water hyacinth (CWH) and purple root water hyacinth (PRWH), were used to investigate the effect of planting densities (i.e., 8, 16 and 24 plants per bucket with a volume of 1.1 m × 1 m (diameter × depth)) on root traits, physiological characteristics, and microbial diversity. The results indicated that the planting density significantly influenced root traits, photosynthesis, radial oxygen loss (ROL), dissolved oxygen (DO), and microbial diversity of water hyacinths. The root porosity, root diameter, and root chlorophyll of PRWH were higher than those of CWH, and CWH had higher chlorophyll and Pn in leaves. The microbial diversity decreased significantly with increasing plant density for CWH, while it increased and then decreased in PRWH and peaked at 16 plants bucket−1 . The results suggested that the aerenchyma of PRWH was more developed than those of CWH, and CWH had higher leaf photosynthesis. However, higher root chlorophyll a in PRWH indicated that its capacity for photon capture was higher than in CWH. The result of ROL suggests that larger root length and root porosity could help improve the dissolved oxygen of water column. The photosynthesis of CWH and PRWH can release oxygen into water column, and the capacity of PRWH was better than those of CWH. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Plant density depends on the genotype, environmental factors, cultural practices, etc. It should be noted that a thinner stand promotes the expression of an individual plant’s potential, whereas denser stands are conducive to a greater expression of the plants’ collective potential (Moravˇcevic´ et al., 2011). As planting density increases, plants with leaves on or above the water surface are able to capture a large proportion of incoming sunlight and hence shade out the root; both overwater and underwater intraspecific competition increases, potentially changing partitioning priorities (Steven et al., 2005; Dale and Gillespie, 1976). Higher planting density may increase biomass partitioning to fine roots to better compete for water and nutrients, or may increase partitioning to foliage to better compete for light (Steven et al., 2005). Planting density increases often results in a deficiency of oxygen (O2 ) and essential nutrients. To adapt to a low O2 environment, free-floating aquatic plants have developed aerenchyma tissues, which can be expressed quantitatively as porosity.
∗ Corresponding author. E-mail address: [email protected] (W. Wang). http://dx.doi.org/10.1016/j.aquabot.2014.10.007 0304-3770/© 2014 Elsevier B.V. All rights reserved.
Porosity in plant tissues results from the intercellular gas-filled spaces formed as a constitutive part of development (Raven, 1996), and it can be further enhanced by formation of aerenchyma. The phenomenon of aquatic plant roots releasing oxygen through the aerenchyma to the rhizosphere is termed radial oxygen loss (ROL) (Armstrong, 1979). ROL is an important characteristic of aquatic plants, which may relate to their adaptability to the water environment (Stottmeister et al., 2003) and nutrient removal (Sasikala et al., 2009). Rates of ROL have been reported to be markedly different between aquatic plant species (Li et al., 2011) and also between different genotypes of the same species. Previous studies have shown that the tolerance of plants to salinity and zinc exposure (Yang et al., 2014) are positively related to ROL. ROL would alert mobility and bioavailability of heavy metals, both on root surface and in rhizosphere, by process of oxidation and by altering pH, redox potential, microbial populations, which could eventually affect metal uptake and tolerance by plants (Jacob and Otte, 2003). ROL from roots is important for aerobic microbial activity and can cause the oxidation and/or immobilization of potential phytotoxins in rhizosphere to avoid the toxicity to roots (Taggart et al., 2009). The development of rhizosphere microbial communities is influenced by the plant and environment, but in turn, microorganisms exert profound effects on plant growth. Rhizosphere microbial
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communities carry out fundamental processes that contribute to nutrient cycling, plant growth, and root health. The extent to which these communities vary in relation to various environmental factors is thus of considerable interest to plant–microbial ecologists (Rachel et al., 2009). These include the physical and chemical characteristics of the environmental factors such as climate and vegetation, but little is known about the relative importance of these factors. In order to be able to manipulate microbial populations in the rhizosphere to the benefit of the plant, a better understanding of the relative importance of the environmental and plant factors for microbial rhizosphere communities is clearly needed. Water hyacinth (Eichhornia crassipes) is a perennial free-floating aquatic plant, it is native to South America, primarily the Amazon Basin. During the last century, it was introduced into other areas of the world as an ornamental garden pond plant. It has since spread into tropical and subtropical regions and leads to worldwide distribution (Zhang et al., 2010). As a free-floating hydrophyte, the growth rate of water hyacinth is higher than any known plant. Most of the problems associated with water hyacinth are due to its rapid growth rate, its ability to successfully compete with other aquatic plants, and its ease of propagation. Dense mats of water hyacinth can also lower dissolved oxygen levels in water bodies leading to reduction of aquatic fish production (Dandelot et al., 2008). Its enormous biomass production rate, its high tolerance to pollution, and its heavy-metal and nutrient absorption capacities (Ghabbour et al., 2004; Jayaweera and Kasturiarachchi, 2004; Soltan and Rashed, 2003; Mishra and Tripathi, 2009) qualify it for use in wastewater treatment. The purple root water hyacinth, an improved strain of water hyacinth, has recently succeeded in the pilot study of water purification in Dian Lake, southwest China’ Yunnan Province. Increasing the density of aquatic plant on a site generally increases the acquisition and use of resources such as light, nutrients and DO. To control free-floating water hyacinth, insight in the growth dynamics is necessary. We used two water hyacinth cultivars (common water hyacinth and purple root water hyacinth (CWH and PRWH)) to investigate the effects of planting densities on the root traits, physiological characteristics, and microbial diversity. In the current study, we addressed the following three questions: (1) how does planting density of water hyacinth impact on its root traits and physiology; (2) how does microbial diversity respond to water hyacinth grown under different planting densities; (3) do the responses of root traits, physiological characteristics, and microbial diversity to planting density differently between common water hyacinth (CWH) and purple root water hyacinth (PRWH)?
2.2. Determination of root traits
2. Materials and methods
2.5. Determination of microbial diversity in root surface
2.1. Plant material and growth conditions
Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) are a widely used combination of techniques for describing microbial community structure and diversity based on extracted DNA. In October 2011, samples from root surface were collected in separate sterile Petri dishes. The homogenate was centrifuged at 3000 × g for 10 min and the pellet was resuspended in 2 ml of PBS and used for community DNA extraction. The samples were used for determining microbial communities by the method of Ibekwe and Grieve (2004).
Young common water hyacinth (CWH) and purple root water hyacinth (PRWH) plants with a similar shape and size were collected from Wuhan (29◦ 53.112 N, 114◦ 14.116 E) and Kunming (25◦ 43.142 N, 102◦ 12.112 E) city, China, respectively. They were grown vegetative in pond in Wuhan city, Hubei, China. Wuhan belongs to subtropics with the average annual temperate of about 16.8 ◦ C and the average annual rainfall of about 1862.2 mm. On 5th June 2011, the plants were rinsed and transferred to outdoor buckets with a volume of 1.1 m × 1 m (diameter × depth). Each bucket started with 280 l of water (8.9 mg N L−1 , 0.8 mg P L−1 , pH = 6.7 ± 0.2), and the bottom of the bucket had 10 cm sludge. A series of different densities was used (8, 16 and 24 plants bucket−1 ). For each treatment, four replicates were applied. The initial root length was determined.
On 5th October 2011, the root lengths (mean of three longest roots in a plant) were measured. Increment of roots lengths was calculated according to roots = RLat – RLbt , where RLat and RLbt represented the mean length of longest roots after and before treatments, respectively. The roots diameters (50 mm behind the tip) were measured for every treatment. Porosity (% gas spaces per unit tissue volume) was measured on samples of roots by determining root buoyancy before and after vacuum infiltration of the gas spaces in the roots with water (Raskin, 1983), using the equations as modified by Thomson et al. (1990). 2.3. Determination of photosynthetic pigments and photosynthesis Plant material (0.1 g) was ground in chilled 80% acetone in the dark on 5th October 2011. After centrifugation at 10,000 × g for 10 min at 4 ◦ C, absorbance of the supernatant was taken at 470, 646 and 663 nm. The content of chlorophylls was estimated by the method of Lichtenthaler (1987) and that of carotenoid content by using the formula given by Duxbury and Yentsch (1956). Different parameters of photosynthesis including net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were determined in July, October and December 2011. In each treatment, four plants were selected randomly, and the leaf of each plant was used for determining photosynthesis by an infrared analyzer (LI-6400 System, LI-COR Company, USA). Average values of four plants were considered as one treatment. 2.4. Determination of radial oxygen loss (ROL) and dissolved oxygen (DO) In July, October and December 2011, ROL was measured colorimetrically with titanium citrate buffer (Kludze et al., 1994; Youssef and Saenger, 1996). Plants were put in an artificial climate incubator with the light intensity at 300 mol m−2 s−1 , temperature at 30 ± 2 ◦ C, and relative humidity of 60%. Their roots were entirely dipped in the deoxygenated titanium citrate covered with 2 cm liquid paraffin to avoid the oxygenation by air during the experiment. After 24 h, the buffer was measured at 527 nm by Shimadzu spectrometer in a deoxygenated box filled with nitrogen gas. Measure of ROL was positioned at 5, 10, 20 or 40 mm behind the root apex. Dissolved oxygen levels were measured at noon in depth of 0.20 m from the water surface throughout the experiment with an oxygen meter (YSIPro30).
2.6. Statistical analyses DNA fingerprints obtained from the 16S rRNA banding patterns on the DGGE gels were photographed and digitized using ImageMaster Labscan. The lanes were normalized to contain the same amount of total signal after background subtraction. An approach
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a
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Planting density(plant buckets ) Fig. 1. Effect of planting density on root length in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
was used to determine community structure based on peak height from the excel files for the different bacterial groups (16S rRNA bands) and was analyzed to generate diversity index (H). The peak height values generated from the sampling points were integrated and analyzed using the excel program. Data obtained were used to integrate the area under each peak for each lane in every treatment. For this analysis, each band was presumed to represent the ability of that bacterial species to be amplified. The Shannon index of diversity (H), Simpson (J) index and McIntosh (DMC) index were used to compare changes in diversity of microbial communities at each treatment (Ibekwe et al., 2001; Zhao et al., 2005). All data were processed by statistical package SPSS (version 11.5). Values reported were means of four replicates. Graphical work was carried out using Origin software 8.0. 3. Results 3.1. Effect of planting density on root traits There were significant differences between the two cultivars in root length, root diameter and root porosity. The root length of CWH and PRWH increased significantly with increasing planting density. The root length was relatively larger in PRWH than in CWH (Fig. 1). The root diameter increased and then decreased in PRWH and CWH. The root diameter was relatively larger in PRWH than in CWH (Fig. 2). With increasing planting density, root porosity increased in the PRWH, whereas it did not change in the CWH. The root porosity of PRWH was higher than that of CWH (Fig. 2). 3.2. Effect of planting density on photosynthetic pigments and photosynthesis There were significant differences between the cultivars in leaf chlorophyll (a and b), carotenoid and root chlorophyll (a and b). The leaf chlorophyll (a and b) and root chlorophyll b were relatively more in CWH than in PRWH across all planting density treatments (Fig. 3A–F). The leaf chlorophyll (a and b) of CWH and PRWH did not change significantly with increasing planting density. The root chlorophyll a of PRWH decreased significantly with increasing planting density, whereas it remained relatively constant in the CWH. There was higher content of root chlorophyll a in PRWH than in CWH. With increasing planting density, root chlorophyll b increased in the CWH, whereas it did not change in the PRWH. The chlorophyll a of PRWH and CWH were markedly higher relative to chlorophyll b. The chlorophyll a and chlorophyll b were found to be relatively more in leaf than in root. There was no
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Planting density (Plants bucket ) Fig. 2. Effect of planting density on root diameter (A) and root porosity (B) in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
difference in leaf chlorophyll a/b ratio between PRWH and CWH. The leaf chlorophyll a/b ratio of CWH and PRWH not changed significantly with increasing planting density. Root chlorophyll a/b ratio was significantly higher in PRWH than in CWH. The root chlorophyll a/b ratio of PRWH decreased significantly with increasing planting density, whereas it decreased and then increased in CWH. The Pn of water hyacinth were significantly affected by cultivar, time (different month), planting density, and significant interactions between cultivar and time or planting density and time were found (Table 1). Data regarding the Cond showed significant changes resulting from treatments of different cultivar or time (Table 1). The Tr of water hyacinth differed between different cultivars, among different time, cultivar × time and time × planting density interaction (Table 1). In July, the Pn of CWH and PRWH increased significantly with increasing planting density, and they decreased with increasing planting density in October and December. In CWH, Pn showed a similar trend as noted for Cond and Tr. The Pn and Cond of CWH were higher in leaf than those of PRWH (Fig. 4).
3.3. Effect of planting density on ROL and DO The results showed that ROL of water hyacinth was significantly affected by cultivar, time, planting density and position (distance from root tip) (Table 1), and interactions were detected between cultivar and time and between time and planting density. The ROL of PRWH was higher than those of CWH. From July to December, the ROL increased and then decreased in PRWH and CWH, the ROL peaked at October. The ROL of CWH and PRWH increased significantly with increasing planting density. The ROL of CWH and PRWH decreased significantly with increasing distance behind the root tip (Fig. 5). The DO of water hyacinth were significantly affected by cultivar, time, planting density, and significant interactions between cultivar and planting density or planting density and time were found (Table 1). The DO increased and then decreased in PRWH and CWH from July to December, the DO peaked in September. In July and October, the DO was found to be relatively more in treatment than in control. The DO of CWH and PRWH decreased significantly with increasing planting density except for PRWH in October. Moreover, in October and November, the DO of PRWH in water was higher than those of CWH (Fig. 6).
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Fig. 3. Effect of planting density on leaf chlorophyll, carotenoid, and root chlorophyll in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
3.4. Effect of planting density and cultivar on microbial diversity Fig. 7 showed a DGGE analysis of the 16S rRNA fragment amplified from the six treatments (cultivar × planting density July October December
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interaction). Differences were noted in band position, intensity, and number of bands present in the six different treatments (Fig. 7). Numerical analysis of the DGGE patterns with the Shannon (H) index, Simpson (J) index, and McIntosh (DMC) index showed
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Planting densit y(plants buc ket ) Fig. 4. Effects of planting density on photosynthetic rate, stomatal conductance (Cond), intercellular CO2 concentration (Ci), and transpiration rate (Tr) at difference months in PRWH (left) and CWH (right) cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
ROL
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Fig. 5. Radial oxygen loss (ROL) from roots in different cultivars, time, planting density and position (distance behind the root tip). The left panel is the CWH, the right panel is the PRWH. Data points and error bars represent mean ± SD of four replicates.
that the microbial communities of the six samples were different (Table 2). The diversity index of CWH decreased significantly with increasing planting density, whereas it increased and then decreased in PRWH. 4. Discussion 4.1. Effect of planting density on root traits Plant roots play a critical role in water and nutrient uptake and extensive data exist on functioning of seedling roots (Zobel et al., 2007). Optimum planting density is a key to achieve maximum root growth especially when nutrient is a limiting factor. Our results showed that maximum root length of the water hyacinth was
DO/ mg L
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12 8
obtained in the highest planting density. Induction in root length was possible due to competition for nutrition. When nutrition is limited, it may be advantageous for aquatic plants to allocate more resources to root growth (Larson, 2007). The longer root length in PRWH indicates that it has larger root growth capacity than CWH. The root diameter was also found to be relatively larger in PRWH than CWH. Porosity resulting from constitutive intercellular gas spaces can differ markedly among different species and genotypes, e.g. ranging from 2 to 22% in selected non-wetland species and from 15 to 52% in selected wetland species under O2 -deficient conditions (Colmer, 2003; Tanaka et al., 2005). Species with higher root porosity tend to form longer roots. High porosities are characteristic of plants adapted to grow in low-oxygen zones, as this enhances the
CWH
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Planting density (plants bucket ) Fig. 6. Effects of planting density on DO at difference months in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates.
Fig. 7. DGGE profile of amplified 16S rRNA fragments from different samples.
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Table 1 Statistically significant differences of net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), transpiration rate (Tr), ROL (radial oxygen loss), and DO (dissolved oxygen) based on ANOVA. Item
Source
df
% SS
P
Pn
Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Position (distance from root tip) Cultivar × Time Time × Planting density Cultivar Time Planting density Cultivar × Planting density Time × Planting density
1 2 2 2 2 4 1 2 2 2 2 4 1 2 2 2 2 4 1 2 2 2 2 4 1 2 2 3 2 4 1 5 3 3 15
15.84 66.35 2.16 7.38 0.05 5.15 25.27 22.62 2.75 0.12 1.21 15.88 4.22 57.85 10.04 0.75 0.66 9.82 2.34 92.72 0.11 1.27 0.02 1.08 9.35 78.24 1.29 0.93 5.24 0.31 0.40 52.02 11.90 0.75 26.70
0.0001** 0.0001** 0.0001** 0.0001** 0.7339 0.0001** 0.0001** 0.0001** 0.1311 0.9103 0.3966 0.0007** 0.0015** 0.0001** 0.0001** 0.3574 0.4019 0.0003** 0.0001** 0.0001** 0.3981 0.0002** 0.8416 0.0048** 0.0001** 0.0001** 0.0001** 0.0001** 0.0001** 0.0088** 0.018* 0.0001** 0.0001** 0.0162* 0.0001**
Cond
Ci
Tr
ROL
DO
* **
P < 0.05. P < 0.01.
internal movement of gases. Roots of plant contain large volumes of aerenchyma, providing a low-resistance pathway for O2 diffusion within the roots. The higher root porosity and root diameter in PRWH indicates that its aerenchyma was more developed than those in CWH. 4.2. Effect of planting density on photosynthetic pigments and photosynthesis There were no significant changes in leaf chlorophyll (a and b) of CWH and PRWH with increasing planting density. However, the root chlorophyll a of PRWH decreased significantly with increasing planting density, the reduction of chlorophyll a content could be explained partially by the effects of shading of the lower canopy, causing poor canopy interception of the photosynthetically active Table 2 Structure diversity of root surface microbe community under different planting densities. Sample
Simpson (J)
Shannon (H)
McIntosh (Dmc)
A B C D E F
0.5013 0.6677 0.5015 0.6679 0.5014 0.5014
0.9998 1.5843 1.0000 1.5849 1.0000 0.9999
0.3096 0.4418 0.3096 0.4417 0.3093 0.3095
A: PRWH 8 plants bucket−1 , B: PRWH 16 plants bucket−1 , C: PRWH 24 plants bucket−1 , D: CWH 8 plants bucket−1 , E: CWH 16 plants bucket−1 , F: CWH 24 plants bucket−1 .
radiation (Brahim et al., 1998). It has been widely accepted that photosynthetic pigments, mostly chlorophyll (a and b) tend to increase with decreasing irradiance to facilitate increased light harvesting in shade tolerant species (Givnish, 1988). In the present study, the CWH increased its root chlorophyll b content in high planting density, i.e., redirected resources toward more efficient photon capture. The higher leaf chlorophyll (a and b) in CWH indicates that its capacity for photon capture was higher than in PRWH in leaves tissue, and the higher root chlorophyll a in PRWH indicates that its capacity for photon capture was higher than this in CWH in roots tissue. The chlorophyll a/b ratio has been a key parameter to judge the shade tolerance of a particular species (Givnish, 1988), and it is a measurement of the proportion of light harvesting complex to other chlorophyll components. The root of CWH and PRWH display a lower ratio in high planting density compared to their grown in low planting density. It has been shown that CWH and PRWH species produce a higher proportion of chlorophyll b relative to chlorophyll a, which leads to a lower chlorophyll a/b ratio, to enhance the efficiency of blue light absorption in high planting density. Previous studies have shown that the chlorophyll a/b ratio as an indicator of shade tolerance of species in general (Beneragama and Goto, 2010). From an ecological point of view, shade tolerance refers to the capacity of a given photosynthetic organism to tolerate low light levels (Valladares and Niinemets, 2008). The higher root chlorophyll a/b ratio in PRWH indicates that its capacity for shade tolerance was higher than CWH. The photosynthetic apparatus performs photosynthesis, which involves the production of organic matter (carbohydrates, protein and fats). The leaves are the most important part of the plant for photosynthetic activity. Photosynthesis depends on a large number of factors (Moravˇcevic´ et al., 2011). Our study indicated that the Pn response to time (different month) depended on planting density and cultivar. The different cultivars explained the variation in Cond better than time, this result implies that the cultivar is most important factors that affect Cond of water hyacinths. Density-induced reduction in Pn of plants was probably caused by stomatal closure since density-treated plants were accompanied by a lower stomatal conductance as well as transpiration rate. The higher Pn and Cond in CWH also indicates that its capacity for photosynthetic was higher than in PRWH, this result is possible to be connected with leaf chlorophyll content (Makoi et al., 2010). The Pn closely mirrored leaf Chl levels in this study, with plants in low density exhibiting higher photosynthetic rates relative to those in high density. In CWH, Pn showed a similar trend as noted for Cond and Tr. The Cond or Tr of CWH and PRWH were higher in July than in October and December. 4.3. Effect of planting density on ROL and DO ROL is determined by anatomical, morphological, and physiological characteristics and demand for oxygen by the rhizosphere, and is highly related to plant species and cultivars (Peter et al., 2005). It was reported that ROL or rates of ROL from root zones differ markedly among aquatic plants and between different genotypes within a species (Jensen et al., 2005; Yang et al., 2010). The present results also showed significant differences between water hyacinth cultivars in ROL. Peter et al. (2005) suggested that the rates of ROL in different species and cultivars were mainly affected by root respiration, the presence of root aerenchyma and a barrier in the basal root zones. The ROL of CWH and PRWH decreased significantly with increasing distance behind the root tip, which are in agreement with the findings of Colmer et al. (2006). These findings suggest that roots of CWH and PRWH contain a barrier to ROL. Kotula et al. (2009) found that the levels of suberin and lignin increased along the roots toward the base, and concluded that ROL can be effectively restricted by the formation of a suberized
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exodermis and/or lignified sclerenchyma in the outer part of rice roots. These all may contribute to the formation of barriers in basal zones in stagnant conditions. In the present study, we observed a positive relationship between ROL and root length or root porosity of water hyacinth. The result of ROL suggests that larger root length and root porosity could help improve the dissolved oxygen of water column. DO in water is essential for the biochemical processes and submerged vegetation growth. Moreover, submerged vegetation can release oxygen into the water column (Sewwandi et al., 2010). Waters with high dissolved oxygen levels are considered healthier than waters with depressed levels. Our results showed that the DO response to planting density depended on time and cultivar. The DO of CWH and PRWH was highest in September, possibly due to intensive photosynthesis of water hyacinth. The aerenchyma and barrier to ROL enhance longitudinal diffusion of O2 toward the root tip. Due to aerenchyma is extensive in water hyacinth plants, O2 evolved during photosynthesis in leaves and roots, moves to the roots; this results are in agreement with the findings of Zhang et al. (2009). Moreover, in October and November, the capacity of PRWH release oxygen into the water column was better than those of CWH. The DO of CWH and PRWH decreased significantly with increasing planting density, this result is possible due to a large amount of water hyacinth’s biomass is on the water’s surface and form a closed-canopy, the lower layer leaves of water hyacinth are unable to produce oxygen by photosynthesis. 4.4. Effect of planting density and cultivar on microbial diversity The rhizosphere is believed to influence the survival, growth, and activity of microorganisms, depending on the plant species or cultivar and the developmental stage of the plant (Ibekwe and Grieve, 2004). Diversity index can be used as a parameter to reflect the structural diversity of the microbial community (Eichner et al., 1999). In our study, microbial diversity was significantly affected by planting density. In CWH, the microbial diversity decreased significantly with increasing plant density. However, the microbial diversity increased and then decreased in PRWH, with the largest value at 16 plants bucket−1 . The effect of different densities on microbial community structure in the rhizosphere may be indirect, via differences in plant growth and DO. Roots are known to excrete several forms of organic materials. The amounts and composition of these organic materials differ with plant species and planting density. The microbial species in root surface of plants are known to use different organic materials as a substrate with different densities. In addition, due to excessive plant materials acting as both a detrital source and a diffusion barrier, the high planting density can cause DO reduction in root surface of water hyacinth resulting in fewer species. In the present study, we observed a positive relationship between microbial diversity and DO in rhizosphere. By using the diversity index, we were able to monitor a whole range of community responses from the root surface. 5. Conclusions The planting density had significant effects on root traits, physiological characteristics, and microbial diversity of water hyacinths for both water hyacinth (Eichhornia crassipes) cultivars, common water hyacinth (CWH) and purple root water hyacinth (PRWH). PRWH has faster root growth capacity than CWH. The larger root porosity and root diameter in PRWH indicates that its aerenchyma is more developed than CWH. The higher contents of leaf chlorophyll and Pn in CWH indicate that its capacity for photosynthetic is higher than in PRWH in leaves tissue. However, PRWH has higher photon capture capacity than CWH by the higher chlorophyll a in
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roots tissue. The photosynthesis of CWH and PRWH can release oxygen into water column, but in October and November, PRWH release more oxygen into the water column than CWH. Acknowledgments This research was funded by the National High Technology Research and Development Program of China (863 Program) (No. 2010AA06Z301) and the National Key Technology Research and Development Program of China (Nos.: 2011BAD31B02 and 2012BAC06B03). References Armstrong, W., 1979. Aeration in higher plants. In: Woolhouse, H.W. (Ed.), Advances in Botanical Research, vol. 7. Academic Press, London, pp. 225–332. Beneragama, C., Goto, K.K., 2010. Chlorophyll a:b ratio increases under low-light in ‘shade-tolerant’ Euglena gracilis. Trop. Agric. Res. 22, 12–25. Brahim, K., Ray, D.T., Dierig, D.A., 1998. Growth and yield characteristics of Lesquerella fendleri as a function of plant density. Ind. Crops Prod. 9, 63–71. Colmer, T.D., 2003. 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Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Comparative responses of two water hyacinth (Eichhornia crassipes) cultivars to different planting densities Xiao Shu, Qi Deng, QuanFa Zhang, WeiBo Wang ∗ Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 9 October 2014 Accepted 23 October 2014 Available online 1 November 2014 Keywords: Physiology Photosynthesis Radial oxygen loss Dissolved oxygen Microbial diversity Water hyacinth
a b s t r a c t Two water hyacinth cultivars, i.e., common water hyacinth (CWH) and purple root water hyacinth (PRWH), were used to investigate the effect of planting densities (i.e., 8, 16 and 24 plants per bucket with a volume of 1.1 m × 1 m (diameter × depth)) on root traits, physiological characteristics, and microbial diversity. The results indicated that the planting density significantly influenced root traits, photosynthesis, radial oxygen loss (ROL), dissolved oxygen (DO), and microbial diversity of water hyacinths. The root porosity, root diameter, and root chlorophyll of PRWH were higher than those of CWH, and CWH had higher chlorophyll and Pn in leaves. The microbial diversity decreased significantly with increasing plant density for CWH, while it increased and then decreased in PRWH and peaked at 16 plants bucket−1 . The results suggested that the aerenchyma of PRWH was more developed than those of CWH, and CWH had higher leaf photosynthesis. However, higher root chlorophyll a in PRWH indicated that its capacity for photon capture was higher than in CWH. The result of ROL suggests that larger root length and root porosity could help improve the dissolved oxygen of water column. The photosynthesis of CWH and PRWH can release oxygen into water column, and the capacity of PRWH was better than those of CWH. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Plant density depends on the genotype, environmental factors, cultural practices, etc. It should be noted that a thinner stand promotes the expression of an individual plant’s potential, whereas denser stands are conducive to a greater expression of the plants’ collective potential (Moravˇcevic´ et al., 2011). As planting density increases, plants with leaves on or above the water surface are able to capture a large proportion of incoming sunlight and hence shade out the root; both overwater and underwater intraspecific competition increases, potentially changing partitioning priorities (Steven et al., 2005; Dale and Gillespie, 1976). Higher planting density may increase biomass partitioning to fine roots to better compete for water and nutrients, or may increase partitioning to foliage to better compete for light (Steven et al., 2005). Planting density increases often results in a deficiency of oxygen (O2 ) and essential nutrients. To adapt to a low O2 environment, free-floating aquatic plants have developed aerenchyma tissues, which can be expressed quantitatively as porosity.
∗ Corresponding author. E-mail address: [email protected] (W. Wang). http://dx.doi.org/10.1016/j.aquabot.2014.10.007 0304-3770/© 2014 Elsevier B.V. All rights reserved.
Porosity in plant tissues results from the intercellular gas-filled spaces formed as a constitutive part of development (Raven, 1996), and it can be further enhanced by formation of aerenchyma. The phenomenon of aquatic plant roots releasing oxygen through the aerenchyma to the rhizosphere is termed radial oxygen loss (ROL) (Armstrong, 1979). ROL is an important characteristic of aquatic plants, which may relate to their adaptability to the water environment (Stottmeister et al., 2003) and nutrient removal (Sasikala et al., 2009). Rates of ROL have been reported to be markedly different between aquatic plant species (Li et al., 2011) and also between different genotypes of the same species. Previous studies have shown that the tolerance of plants to salinity and zinc exposure (Yang et al., 2014) are positively related to ROL. ROL would alert mobility and bioavailability of heavy metals, both on root surface and in rhizosphere, by process of oxidation and by altering pH, redox potential, microbial populations, which could eventually affect metal uptake and tolerance by plants (Jacob and Otte, 2003). ROL from roots is important for aerobic microbial activity and can cause the oxidation and/or immobilization of potential phytotoxins in rhizosphere to avoid the toxicity to roots (Taggart et al., 2009). The development of rhizosphere microbial communities is influenced by the plant and environment, but in turn, microorganisms exert profound effects on plant growth. Rhizosphere microbial
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communities carry out fundamental processes that contribute to nutrient cycling, plant growth, and root health. The extent to which these communities vary in relation to various environmental factors is thus of considerable interest to plant–microbial ecologists (Rachel et al., 2009). These include the physical and chemical characteristics of the environmental factors such as climate and vegetation, but little is known about the relative importance of these factors. In order to be able to manipulate microbial populations in the rhizosphere to the benefit of the plant, a better understanding of the relative importance of the environmental and plant factors for microbial rhizosphere communities is clearly needed. Water hyacinth (Eichhornia crassipes) is a perennial free-floating aquatic plant, it is native to South America, primarily the Amazon Basin. During the last century, it was introduced into other areas of the world as an ornamental garden pond plant. It has since spread into tropical and subtropical regions and leads to worldwide distribution (Zhang et al., 2010). As a free-floating hydrophyte, the growth rate of water hyacinth is higher than any known plant. Most of the problems associated with water hyacinth are due to its rapid growth rate, its ability to successfully compete with other aquatic plants, and its ease of propagation. Dense mats of water hyacinth can also lower dissolved oxygen levels in water bodies leading to reduction of aquatic fish production (Dandelot et al., 2008). Its enormous biomass production rate, its high tolerance to pollution, and its heavy-metal and nutrient absorption capacities (Ghabbour et al., 2004; Jayaweera and Kasturiarachchi, 2004; Soltan and Rashed, 2003; Mishra and Tripathi, 2009) qualify it for use in wastewater treatment. The purple root water hyacinth, an improved strain of water hyacinth, has recently succeeded in the pilot study of water purification in Dian Lake, southwest China’ Yunnan Province. Increasing the density of aquatic plant on a site generally increases the acquisition and use of resources such as light, nutrients and DO. To control free-floating water hyacinth, insight in the growth dynamics is necessary. We used two water hyacinth cultivars (common water hyacinth and purple root water hyacinth (CWH and PRWH)) to investigate the effects of planting densities on the root traits, physiological characteristics, and microbial diversity. In the current study, we addressed the following three questions: (1) how does planting density of water hyacinth impact on its root traits and physiology; (2) how does microbial diversity respond to water hyacinth grown under different planting densities; (3) do the responses of root traits, physiological characteristics, and microbial diversity to planting density differently between common water hyacinth (CWH) and purple root water hyacinth (PRWH)?
2.2. Determination of root traits
2. Materials and methods
2.5. Determination of microbial diversity in root surface
2.1. Plant material and growth conditions
Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) are a widely used combination of techniques for describing microbial community structure and diversity based on extracted DNA. In October 2011, samples from root surface were collected in separate sterile Petri dishes. The homogenate was centrifuged at 3000 × g for 10 min and the pellet was resuspended in 2 ml of PBS and used for community DNA extraction. The samples were used for determining microbial communities by the method of Ibekwe and Grieve (2004).
Young common water hyacinth (CWH) and purple root water hyacinth (PRWH) plants with a similar shape and size were collected from Wuhan (29◦ 53.112 N, 114◦ 14.116 E) and Kunming (25◦ 43.142 N, 102◦ 12.112 E) city, China, respectively. They were grown vegetative in pond in Wuhan city, Hubei, China. Wuhan belongs to subtropics with the average annual temperate of about 16.8 ◦ C and the average annual rainfall of about 1862.2 mm. On 5th June 2011, the plants were rinsed and transferred to outdoor buckets with a volume of 1.1 m × 1 m (diameter × depth). Each bucket started with 280 l of water (8.9 mg N L−1 , 0.8 mg P L−1 , pH = 6.7 ± 0.2), and the bottom of the bucket had 10 cm sludge. A series of different densities was used (8, 16 and 24 plants bucket−1 ). For each treatment, four replicates were applied. The initial root length was determined.
On 5th October 2011, the root lengths (mean of three longest roots in a plant) were measured. Increment of roots lengths was calculated according to roots = RLat – RLbt , where RLat and RLbt represented the mean length of longest roots after and before treatments, respectively. The roots diameters (50 mm behind the tip) were measured for every treatment. Porosity (% gas spaces per unit tissue volume) was measured on samples of roots by determining root buoyancy before and after vacuum infiltration of the gas spaces in the roots with water (Raskin, 1983), using the equations as modified by Thomson et al. (1990). 2.3. Determination of photosynthetic pigments and photosynthesis Plant material (0.1 g) was ground in chilled 80% acetone in the dark on 5th October 2011. After centrifugation at 10,000 × g for 10 min at 4 ◦ C, absorbance of the supernatant was taken at 470, 646 and 663 nm. The content of chlorophylls was estimated by the method of Lichtenthaler (1987) and that of carotenoid content by using the formula given by Duxbury and Yentsch (1956). Different parameters of photosynthesis including net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were determined in July, October and December 2011. In each treatment, four plants were selected randomly, and the leaf of each plant was used for determining photosynthesis by an infrared analyzer (LI-6400 System, LI-COR Company, USA). Average values of four plants were considered as one treatment. 2.4. Determination of radial oxygen loss (ROL) and dissolved oxygen (DO) In July, October and December 2011, ROL was measured colorimetrically with titanium citrate buffer (Kludze et al., 1994; Youssef and Saenger, 1996). Plants were put in an artificial climate incubator with the light intensity at 300 mol m−2 s−1 , temperature at 30 ± 2 ◦ C, and relative humidity of 60%. Their roots were entirely dipped in the deoxygenated titanium citrate covered with 2 cm liquid paraffin to avoid the oxygenation by air during the experiment. After 24 h, the buffer was measured at 527 nm by Shimadzu spectrometer in a deoxygenated box filled with nitrogen gas. Measure of ROL was positioned at 5, 10, 20 or 40 mm behind the root apex. Dissolved oxygen levels were measured at noon in depth of 0.20 m from the water surface throughout the experiment with an oxygen meter (YSIPro30).
2.6. Statistical analyses DNA fingerprints obtained from the 16S rRNA banding patterns on the DGGE gels were photographed and digitized using ImageMaster Labscan. The lanes were normalized to contain the same amount of total signal after background subtraction. An approach
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Planting density(plant buckets ) Fig. 1. Effect of planting density on root length in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
was used to determine community structure based on peak height from the excel files for the different bacterial groups (16S rRNA bands) and was analyzed to generate diversity index (H). The peak height values generated from the sampling points were integrated and analyzed using the excel program. Data obtained were used to integrate the area under each peak for each lane in every treatment. For this analysis, each band was presumed to represent the ability of that bacterial species to be amplified. The Shannon index of diversity (H), Simpson (J) index and McIntosh (DMC) index were used to compare changes in diversity of microbial communities at each treatment (Ibekwe et al., 2001; Zhao et al., 2005). All data were processed by statistical package SPSS (version 11.5). Values reported were means of four replicates. Graphical work was carried out using Origin software 8.0. 3. Results 3.1. Effect of planting density on root traits There were significant differences between the two cultivars in root length, root diameter and root porosity. The root length of CWH and PRWH increased significantly with increasing planting density. The root length was relatively larger in PRWH than in CWH (Fig. 1). The root diameter increased and then decreased in PRWH and CWH. The root diameter was relatively larger in PRWH than in CWH (Fig. 2). With increasing planting density, root porosity increased in the PRWH, whereas it did not change in the CWH. The root porosity of PRWH was higher than that of CWH (Fig. 2). 3.2. Effect of planting density on photosynthetic pigments and photosynthesis There were significant differences between the cultivars in leaf chlorophyll (a and b), carotenoid and root chlorophyll (a and b). The leaf chlorophyll (a and b) and root chlorophyll b were relatively more in CWH than in PRWH across all planting density treatments (Fig. 3A–F). The leaf chlorophyll (a and b) of CWH and PRWH did not change significantly with increasing planting density. The root chlorophyll a of PRWH decreased significantly with increasing planting density, whereas it remained relatively constant in the CWH. There was higher content of root chlorophyll a in PRWH than in CWH. With increasing planting density, root chlorophyll b increased in the CWH, whereas it did not change in the PRWH. The chlorophyll a of PRWH and CWH were markedly higher relative to chlorophyll b. The chlorophyll a and chlorophyll b were found to be relatively more in leaf than in root. There was no
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Planting density (Plants bucket ) Fig. 2. Effect of planting density on root diameter (A) and root porosity (B) in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
difference in leaf chlorophyll a/b ratio between PRWH and CWH. The leaf chlorophyll a/b ratio of CWH and PRWH not changed significantly with increasing planting density. Root chlorophyll a/b ratio was significantly higher in PRWH than in CWH. The root chlorophyll a/b ratio of PRWH decreased significantly with increasing planting density, whereas it decreased and then increased in CWH. The Pn of water hyacinth were significantly affected by cultivar, time (different month), planting density, and significant interactions between cultivar and time or planting density and time were found (Table 1). Data regarding the Cond showed significant changes resulting from treatments of different cultivar or time (Table 1). The Tr of water hyacinth differed between different cultivars, among different time, cultivar × time and time × planting density interaction (Table 1). In July, the Pn of CWH and PRWH increased significantly with increasing planting density, and they decreased with increasing planting density in October and December. In CWH, Pn showed a similar trend as noted for Cond and Tr. The Pn and Cond of CWH were higher in leaf than those of PRWH (Fig. 4).
3.3. Effect of planting density on ROL and DO The results showed that ROL of water hyacinth was significantly affected by cultivar, time, planting density and position (distance from root tip) (Table 1), and interactions were detected between cultivar and time and between time and planting density. The ROL of PRWH was higher than those of CWH. From July to December, the ROL increased and then decreased in PRWH and CWH, the ROL peaked at October. The ROL of CWH and PRWH increased significantly with increasing planting density. The ROL of CWH and PRWH decreased significantly with increasing distance behind the root tip (Fig. 5). The DO of water hyacinth were significantly affected by cultivar, time, planting density, and significant interactions between cultivar and planting density or planting density and time were found (Table 1). The DO increased and then decreased in PRWH and CWH from July to December, the DO peaked in September. In July and October, the DO was found to be relatively more in treatment than in control. The DO of CWH and PRWH decreased significantly with increasing planting density except for PRWH in October. Moreover, in October and November, the DO of PRWH in water was higher than those of CWH (Fig. 6).
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X. Shu et al. / Aquatic Botany 121 (2015) 1–8
A
a
ab
1.5
ab
bc 1.0
c
c
0.5
Root chlorophyll a -1 (mg g FW)
-1
(mg g FW)
Leaf chlorophyll a
2.0
0.0
Roor chlorophyll b -1 (mg g FW)
bc
c
abc c
0.15 0.00 0.4
E
-1 (mg g FW)
Leaf carotenoid
a 0.3
b
a
a
ab
b
0.1
Leaf chlorophyll (a+b) -1 (mg g FW)
FW) (mg g
-1
Leaf chlorophyll b
C
a
ab
0.30
0.2
PRWH CWH
B
a
0.06
ab 0.03
bc
bc
bc c
0.00 0.10
0.60 0.45
0.09
ab
0.06 0.04
bc
c
8
bc
c
0.02 0.00 2.8
F
2.1
a
ab
ab
bc 1.4
c
c
0.7 0.0
0.0
D
a
0.08
16 Planting density -1 (Plants bucket )
8
16 Planting density -1 (Plants bucket )
24
Fig. 3. Effect of planting density on leaf chlorophyll, carotenoid, and root chlorophyll in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
3.4. Effect of planting density and cultivar on microbial diversity Fig. 7 showed a DGGE analysis of the 16S rRNA fragment amplified from the six treatments (cultivar × planting density July October December
24 16
cd
a
abc ab
abc
bc
e
d
a ab
0.5
a
d
f abc
ab
bc
a
cd de bc
cd f b
bc cd
a
a
b b
cd
bc bc
c
e a
ab bcd
a
bc
b b
b
c
0 1.5 1.0
a
a
de
8
interaction). Differences were noted in band position, intensity, and number of bands present in the six different treatments (Fig. 7). Numerical analysis of the DGGE patterns with the Shannon (H) index, Simpson (J) index, and McIntosh (DMC) index showed
cd d
d
0.0 390
bc e
260
a bc d
b
bc cd
b
130 0 12
a b
4
c
a bc
d
c
c 16
24
16
8
e
de
0 8
c
b
b
8
a
24
-1
Planting densit y(plants buc ket ) Fig. 4. Effects of planting density on photosynthetic rate, stomatal conductance (Cond), intercellular CO2 concentration (Ci), and transpiration rate (Tr) at difference months in PRWH (left) and CWH (right) cultivars. Data points and error bars represent mean ± SD of four replicates. Different letters indicate significant differences (P < 0.05) among the treatments.
ROL
ROL
ROL
ROL
μmolO2/(h g root) μmolO /(h g root) μmolO /(h g root) μmolO /(h g root) 2 2 2
X. Shu et al. / Aquatic Botany 121 (2015) 1–8
12 9
July October December
5 mm distance behind root tip
5
5 mm distance behind root tip
A
B
6 3 0 12
10 mm distance behind root tip
C
10 mm distance behind root tip
D
20 mm distance behind root tip
E
20 mm distance behind root tip
F
40 mm distance behind root tip
G
40 mm distance behind root tip
H
9 6 3 0 12 9 6 3 0 12 9 6 3 0
8
24
16
8
16
24
Planting density -1 (plants bucket )
Planting density -1 (plants bucket )
Fig. 5. Radial oxygen loss (ROL) from roots in different cultivars, time, planting density and position (distance behind the root tip). The left panel is the CWH, the right panel is the PRWH. Data points and error bars represent mean ± SD of four replicates.
that the microbial communities of the six samples were different (Table 2). The diversity index of CWH decreased significantly with increasing planting density, whereas it increased and then decreased in PRWH. 4. Discussion 4.1. Effect of planting density on root traits Plant roots play a critical role in water and nutrient uptake and extensive data exist on functioning of seedling roots (Zobel et al., 2007). Optimum planting density is a key to achieve maximum root growth especially when nutrient is a limiting factor. Our results showed that maximum root length of the water hyacinth was
DO/ mg L
-1
12 8
obtained in the highest planting density. Induction in root length was possible due to competition for nutrition. When nutrition is limited, it may be advantageous for aquatic plants to allocate more resources to root growth (Larson, 2007). The longer root length in PRWH indicates that it has larger root growth capacity than CWH. The root diameter was also found to be relatively larger in PRWH than CWH. Porosity resulting from constitutive intercellular gas spaces can differ markedly among different species and genotypes, e.g. ranging from 2 to 22% in selected non-wetland species and from 15 to 52% in selected wetland species under O2 -deficient conditions (Colmer, 2003; Tanaka et al., 2005). Species with higher root porosity tend to form longer roots. High porosities are characteristic of plants adapted to grow in low-oxygen zones, as this enhances the
CWH
July August September October November December
4 0
DO/ mg L
-1
PRWH 12 8 4 0 CK
8
24
16 -1
Planting density (plants bucket ) Fig. 6. Effects of planting density on DO at difference months in PRWH and CWH cultivars. Data points and error bars represent mean ± SD of four replicates.
Fig. 7. DGGE profile of amplified 16S rRNA fragments from different samples.
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Table 1 Statistically significant differences of net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), transpiration rate (Tr), ROL (radial oxygen loss), and DO (dissolved oxygen) based on ANOVA. Item
Source
df
% SS
P
Pn
Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Cultivar × Time Cultivar × Planting density Time × Planting density Cultivar Time Planting density Position (distance from root tip) Cultivar × Time Time × Planting density Cultivar Time Planting density Cultivar × Planting density Time × Planting density
1 2 2 2 2 4 1 2 2 2 2 4 1 2 2 2 2 4 1 2 2 2 2 4 1 2 2 3 2 4 1 5 3 3 15
15.84 66.35 2.16 7.38 0.05 5.15 25.27 22.62 2.75 0.12 1.21 15.88 4.22 57.85 10.04 0.75 0.66 9.82 2.34 92.72 0.11 1.27 0.02 1.08 9.35 78.24 1.29 0.93 5.24 0.31 0.40 52.02 11.90 0.75 26.70
0.0001** 0.0001** 0.0001** 0.0001** 0.7339 0.0001** 0.0001** 0.0001** 0.1311 0.9103 0.3966 0.0007** 0.0015** 0.0001** 0.0001** 0.3574 0.4019 0.0003** 0.0001** 0.0001** 0.3981 0.0002** 0.8416 0.0048** 0.0001** 0.0001** 0.0001** 0.0001** 0.0001** 0.0088** 0.018* 0.0001** 0.0001** 0.0162* 0.0001**
Cond
Ci
Tr
ROL
DO
* **
P < 0.05. P < 0.01.
internal movement of gases. Roots of plant contain large volumes of aerenchyma, providing a low-resistance pathway for O2 diffusion within the roots. The higher root porosity and root diameter in PRWH indicates that its aerenchyma was more developed than those in CWH. 4.2. Effect of planting density on photosynthetic pigments and photosynthesis There were no significant changes in leaf chlorophyll (a and b) of CWH and PRWH with increasing planting density. However, the root chlorophyll a of PRWH decreased significantly with increasing planting density, the reduction of chlorophyll a content could be explained partially by the effects of shading of the lower canopy, causing poor canopy interception of the photosynthetically active Table 2 Structure diversity of root surface microbe community under different planting densities. Sample
Simpson (J)
Shannon (H)
McIntosh (Dmc)
A B C D E F
0.5013 0.6677 0.5015 0.6679 0.5014 0.5014
0.9998 1.5843 1.0000 1.5849 1.0000 0.9999
0.3096 0.4418 0.3096 0.4417 0.3093 0.3095
A: PRWH 8 plants bucket−1 , B: PRWH 16 plants bucket−1 , C: PRWH 24 plants bucket−1 , D: CWH 8 plants bucket−1 , E: CWH 16 plants bucket−1 , F: CWH 24 plants bucket−1 .
radiation (Brahim et al., 1998). It has been widely accepted that photosynthetic pigments, mostly chlorophyll (a and b) tend to increase with decreasing irradiance to facilitate increased light harvesting in shade tolerant species (Givnish, 1988). In the present study, the CWH increased its root chlorophyll b content in high planting density, i.e., redirected resources toward more efficient photon capture. The higher leaf chlorophyll (a and b) in CWH indicates that its capacity for photon capture was higher than in PRWH in leaves tissue, and the higher root chlorophyll a in PRWH indicates that its capacity for photon capture was higher than this in CWH in roots tissue. The chlorophyll a/b ratio has been a key parameter to judge the shade tolerance of a particular species (Givnish, 1988), and it is a measurement of the proportion of light harvesting complex to other chlorophyll components. The root of CWH and PRWH display a lower ratio in high planting density compared to their grown in low planting density. It has been shown that CWH and PRWH species produce a higher proportion of chlorophyll b relative to chlorophyll a, which leads to a lower chlorophyll a/b ratio, to enhance the efficiency of blue light absorption in high planting density. Previous studies have shown that the chlorophyll a/b ratio as an indicator of shade tolerance of species in general (Beneragama and Goto, 2010). From an ecological point of view, shade tolerance refers to the capacity of a given photosynthetic organism to tolerate low light levels (Valladares and Niinemets, 2008). The higher root chlorophyll a/b ratio in PRWH indicates that its capacity for shade tolerance was higher than CWH. The photosynthetic apparatus performs photosynthesis, which involves the production of organic matter (carbohydrates, protein and fats). The leaves are the most important part of the plant for photosynthetic activity. Photosynthesis depends on a large number of factors (Moravˇcevic´ et al., 2011). Our study indicated that the Pn response to time (different month) depended on planting density and cultivar. The different cultivars explained the variation in Cond better than time, this result implies that the cultivar is most important factors that affect Cond of water hyacinths. Density-induced reduction in Pn of plants was probably caused by stomatal closure since density-treated plants were accompanied by a lower stomatal conductance as well as transpiration rate. The higher Pn and Cond in CWH also indicates that its capacity for photosynthetic was higher than in PRWH, this result is possible to be connected with leaf chlorophyll content (Makoi et al., 2010). The Pn closely mirrored leaf Chl levels in this study, with plants in low density exhibiting higher photosynthetic rates relative to those in high density. In CWH, Pn showed a similar trend as noted for Cond and Tr. The Cond or Tr of CWH and PRWH were higher in July than in October and December. 4.3. Effect of planting density on ROL and DO ROL is determined by anatomical, morphological, and physiological characteristics and demand for oxygen by the rhizosphere, and is highly related to plant species and cultivars (Peter et al., 2005). It was reported that ROL or rates of ROL from root zones differ markedly among aquatic plants and between different genotypes within a species (Jensen et al., 2005; Yang et al., 2010). The present results also showed significant differences between water hyacinth cultivars in ROL. Peter et al. (2005) suggested that the rates of ROL in different species and cultivars were mainly affected by root respiration, the presence of root aerenchyma and a barrier in the basal root zones. The ROL of CWH and PRWH decreased significantly with increasing distance behind the root tip, which are in agreement with the findings of Colmer et al. (2006). These findings suggest that roots of CWH and PRWH contain a barrier to ROL. Kotula et al. (2009) found that the levels of suberin and lignin increased along the roots toward the base, and concluded that ROL can be effectively restricted by the formation of a suberized
X. Shu et al. / Aquatic Botany 121 (2015) 1–8
exodermis and/or lignified sclerenchyma in the outer part of rice roots. These all may contribute to the formation of barriers in basal zones in stagnant conditions. In the present study, we observed a positive relationship between ROL and root length or root porosity of water hyacinth. The result of ROL suggests that larger root length and root porosity could help improve the dissolved oxygen of water column. DO in water is essential for the biochemical processes and submerged vegetation growth. Moreover, submerged vegetation can release oxygen into the water column (Sewwandi et al., 2010). Waters with high dissolved oxygen levels are considered healthier than waters with depressed levels. Our results showed that the DO response to planting density depended on time and cultivar. The DO of CWH and PRWH was highest in September, possibly due to intensive photosynthesis of water hyacinth. The aerenchyma and barrier to ROL enhance longitudinal diffusion of O2 toward the root tip. Due to aerenchyma is extensive in water hyacinth plants, O2 evolved during photosynthesis in leaves and roots, moves to the roots; this results are in agreement with the findings of Zhang et al. (2009). Moreover, in October and November, the capacity of PRWH release oxygen into the water column was better than those of CWH. The DO of CWH and PRWH decreased significantly with increasing planting density, this result is possible due to a large amount of water hyacinth’s biomass is on the water’s surface and form a closed-canopy, the lower layer leaves of water hyacinth are unable to produce oxygen by photosynthesis. 4.4. Effect of planting density and cultivar on microbial diversity The rhizosphere is believed to influence the survival, growth, and activity of microorganisms, depending on the plant species or cultivar and the developmental stage of the plant (Ibekwe and Grieve, 2004). Diversity index can be used as a parameter to reflect the structural diversity of the microbial community (Eichner et al., 1999). In our study, microbial diversity was significantly affected by planting density. In CWH, the microbial diversity decreased significantly with increasing plant density. However, the microbial diversity increased and then decreased in PRWH, with the largest value at 16 plants bucket−1 . The effect of different densities on microbial community structure in the rhizosphere may be indirect, via differences in plant growth and DO. Roots are known to excrete several forms of organic materials. The amounts and composition of these organic materials differ with plant species and planting density. The microbial species in root surface of plants are known to use different organic materials as a substrate with different densities. In addition, due to excessive plant materials acting as both a detrital source and a diffusion barrier, the high planting density can cause DO reduction in root surface of water hyacinth resulting in fewer species. In the present study, we observed a positive relationship between microbial diversity and DO in rhizosphere. By using the diversity index, we were able to monitor a whole range of community responses from the root surface. 5. Conclusions The planting density had significant effects on root traits, physiological characteristics, and microbial diversity of water hyacinths for both water hyacinth (Eichhornia crassipes) cultivars, common water hyacinth (CWH) and purple root water hyacinth (PRWH). PRWH has faster root growth capacity than CWH. The larger root porosity and root diameter in PRWH indicates that its aerenchyma is more developed than CWH. The higher contents of leaf chlorophyll and Pn in CWH indicate that its capacity for photosynthetic is higher than in PRWH in leaves tissue. However, PRWH has higher photon capture capacity than CWH by the higher chlorophyll a in
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roots tissue. The photosynthesis of CWH and PRWH can release oxygen into water column, but in October and November, PRWH release more oxygen into the water column than CWH. Acknowledgments This research was funded by the National High Technology Research and Development Program of China (863 Program) (No. 2010AA06Z301) and the National Key Technology Research and Development Program of China (Nos.: 2011BAD31B02 and 2012BAC06B03). References Armstrong, W., 1979. Aeration in higher plants. In: Woolhouse, H.W. (Ed.), Advances in Botanical Research, vol. 7. Academic Press, London, pp. 225–332. Beneragama, C., Goto, K.K., 2010. Chlorophyll a:b ratio increases under low-light in ‘shade-tolerant’ Euglena gracilis. Trop. Agric. Res. 22, 12–25. Brahim, K., Ray, D.T., Dierig, D.A., 1998. Growth and yield characteristics of Lesquerella fendleri as a function of plant density. Ind. Crops Prod. 9, 63–71. Colmer, T.D., 2003. 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