Root responses to cadmium in the rhizosphere

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采用流式细胞仪鉴定紫花苜蓿花药愈伤组织的变异

＊通讯作者。Ｅｍａ：ｈｎ — ｅ＠ｙｈｏｃｒ．ｎ — ｉｚｅｗｕｗｉａｏ．ｏｃｌｎ
第２０卷第３期
草业学报２１年０１
１２苜蓿花药愈伤组织的诱导培养．在苜蓿初花期到盛花期期间，集３个基因型紫花苜蓿单株上花药发育处于单核中、采晚期的花蕾（用醋酸使洋红染色压片法，观察、定发育时期。从外观上选择花冠最外面的旗瓣紧包，冠饱满，出花萼１确花伸～２ｍｍ，呈刀尖状的花蕾）。将采集的花蕾放置在４的冰箱内低温处理１～２； ℃ ２４ｈ然后将花蕾灭菌，无菌条件下剥出花在粉，接种到愈伤组织诱导培养基中（＋０５ｇＬ２４Ｄ＋０２ｇＬＮＡＡ＋０５ｇＬ６ＢＮＢ．／，一．／．／－Ａ＋２ｇＬＫ／Ｔ＋９蔗糖＋０７琼脂）在温度为２。光强１００ｌ光照时间１／、．，５Ｃ、０ｘ、６ｈｄ湿度４的环境下培养。诱导出愈伤组织后转０入继代培养基（ＮＢ＋３／糖＋０７琼脂）２６９蔗．，０ｄ为１个继代周期。培养条件同上。
色３～６，成细胞核染色，胞核悬浮液制备完成。ＯＯＳ完细２以已知的四倍体苜蓿叶片（ｎｚ２为对照。调整分析仪的放大倍数，对照的Ｄ）２一４一３）使ＮＡ峰值处在２００道附近。峰值处在１０道的样本为二倍体，０而处在３０和４０道的分别为三倍体和八倍体，００其余的为嵌合体。嵌合体又根据峰值位置确定其构成细胞的倍性。３将１所得细胞核悬浮液用调整好的流式细胞仪进行检测，ＮＡ含量的分布曲线图由仪器自动生成。））Ｄ

氯化锂对地塞米松诱导的大鼠胰岛β细胞凋亡的影响及其机制

University of Science andTechnology, Tangshan 063000, China）
ABSTRACT Objective： To construct the apoptosis model of islet INS-1 cells of the rats induced by dexamethasone （Dex） , and to investigate the effect of lithium chloride （LiCl） on the apoptosis of the islet 4 cclls induced by Dex and its possible mechanism. Methods： The INS-1 cells were divided into control group, 0. 1 gmol • L_lDex group and LiCl+0. 1 gmol • L_l Dex group. TUNEL staining and Annexin V /PI staining methods were used to detect the apoptotic rates of the INS-1 cells in various groups ； Real-time PCR method was used to detect the expression levels of superoxide dismutase （SOD）, inducible-nitric oxidesynthase （iNOS） , NADPH oxidase 4 （Nox4）, NADPH oxidase （p47phox）, and glycogen-synthase tinase-30 （GSK30） mRNA in theINS-1 cclls in various groups； Western blotting method was used to detect the expression levels of GSK-34， p-GSK-30, SOD, iNOS and Nox4

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Plant and Soil 260: 69–77, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.69Root-induced acidiﬁcation and excess cation uptake by N2 -ﬁxing Lupinus albus grown in phosphorus-deﬁcient soilJ. Shen1,2,4 , C. Tang3 , Z. Rengel2 & F. Zhang11 Department of Plant Nutrition, China Agricultural University,Key Laboratory of Plant-Soil Interactions, Ministry of Education, Beijing 100094, P. R. China. 2 Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley WA6009, Australia. 3 Department of Agricultural Sciences, La Trobe University, Bundoora, Vic. 3086, Australia. 4 Corresponding author∗Received 7 October 2002. Accepted in revised form 26 July 2003Key words: proton release, balance of cation-anion uptake, P deﬁciency, Lupinus albusAbstract White lupin plants (Lupinus albus L. cv. Kiev) were grown in soil columns under controlled conditions at 20/12 ◦ C (12/12 h) for 76 d to investigate the effect of phosphorus (P) deﬁciency on root-induced acidiﬁcation and excess cation uptake by N2 -ﬁxing plants. Phosphorus was added in each column as FePO4 at a level of 10 (limited P) or 200 µg P g−1 (adequate P). Supply of 10 µg P g−1 restricted plant growth from 58 d after sowing (DAS) and decreased P concentrations signiﬁcantly in shoots from 49 DAS and in roots from 40 DAS compared with plants supplied with 200 µg P g−1 . Phosphorus concentrations in shoots of plants receiving 10 µg P g−1 decreased steadily from 2.1 to 1.1 mg P g−1 dry weight from 40 to 76 DAS, but P concentrations in roots were constant with time. Total P uptake increased with time irrespective of P supply, and the P uptake by plants at 10 µg P g−1 was only 35–75% of that at 200 µg P g−1 . Plants fed with 10 µg P g−1 had higher Ca and Mg concentrations but lower S concentration in shoots than the plants fed with 200 µg P g−1 . The concentrations of excess cations in plants were higher at 10 µg P g−1 than 200 µg P g−1 after 49 DAS. Phosphorus deﬁciency decreased the pH of root exudate solution due to the enhanced release of protons (H+ ) from roots. The pH of root exudate solution decreased rapidly with time and dropped to the lowest (4.28) at 58 DAS in the 10 µg P g−1 treatment. The decreased pH of root exudate solution was correlated with the increased concentrations of excess cations in plants. The pH of root exudate solution showed a different pattern of change with time compared with citrate exudation, suggesting that exudation of citrate anions contributes only a part of total acidiﬁcation, but excess cation uptake dominantly contributes net proton release from roots of plants grown in P-deﬁcient soil. Plant tissue had a signiﬁcant accumulation of citrate in the treatment of 10 µg P g−1 compared with 200 µg P g−1 after 67 DAS. The results suggest that P deﬁciency enhances the excess cation uptake and concomitant proton release, and non-synchronous processes are involved in tissue accumulation and root exudation of organic anions under P deﬁciency.Introduction Phosphorus deﬁciency is one of the major yieldlimiting nutrition problems for plants in both acidic and calcareous soils due to low bio-availability of P (Barber, 1984; Marschner, 1995). Some plant species∗ FAX No: +86 10 62891016. E-mail: jbshen@have developed various morphological and physiological strategies to acquire sparingly soluble phosphorus from soil. The mechanisms based on morphological responses include enhanced root elongation (Barber, 1984; Raghothama, 1999), root-to-shoot ratio, root hair formation (Ma et al., 2001; Marschner, 1995), mycorrhizal associations (Smith et al., 1992),70 and decreased root radius (Föhse et al., 1991). In addition, physiological strategies are characterized by enhanced rhizosphere acidiﬁcation (Hedley et al., 1982; Hinsinger et al., 2003; Marschner, 1995; Tang et al., 2001), and increased root exudation of organic chelates (Dinkelaker et al., 1989; Gardner et al., 1983; Jones, 1998; Ryan and Delhaize, 2001), reducing agents and phosphatase (Dracup et al., 1984; Neumann et al., 1999, 2000). White lupin (Lupinus albus L.) is an important grain and forage crop widely grown in infertile soils. Under P deﬁciency, white lupin increases the formation of cluster roots, release of H+ and exudation of citrate (Dinkelaker et al., 1989; Gardner et al., 1983; Lamont, 2003; Neumann et al., 1999). For example, the enhanced net release of H+ from cluster roots acidiﬁed rhizosphere from pH 7.5 to 4.8 (Dinkelaker et al., 1989, 1995) and even as low as pH 3.6 in some cases (Li et al., 1997). Citrate concentration in root cluster rhizosphere can reach as high as 100 µmol g−1 soil, and the amount of citrate exudation from roots represented up to 23% of the total plant dry weight (Dinkelaker et al., 1989, 1995; Gerke et al., 1994). Large amounts of proton release and citrate exudation may facilitate P acquisition by white lupin from sparingly soluble P in soils (Gerke et al., 1994; Hinsinger, 1998; Marschner, 1995; Raghothama, 1999). Rhizosphere acidiﬁcation and citrate exudation induced by P deﬁciency have been studied mostly in white lupin grown in nitrate-containing nutrition solutions (Gardner et al., 1983; Johnson et al., 1994, 1996a, b; Keerthisinghe et al., 1998; Neumann et al., 1999; Neumann and Römheld, 1999; Shane et al., 2003; Watt and Evans, 1999a, b), or in soil culture with nitrogen supply as nitrate (Dinkelaker et al., 1989; Kamh et al., 1999) or ammonium nitrate (Li et al., 1997). Nitrogen forms have a marked inﬂuence on rhizosphere acidiﬁcation due to inﬂuencing the balance of anion and cation uptake by plants (Hinsinger, 2001; Hinsinger et al., 2003; Marschner, 1995). Only a few studies used N2 -ﬁxing P-deﬁcient white lupin plants to elucidate the pH changes caused by H+ release and root exudation (Sas et al., 2001; Shen et al., 2003; Tang et al., 1997, 2001). The legume plants dependent on N2 -ﬁxation could take up excess cations over anions, resulting in enhanced H+ release with an increasing amount of N2 ﬁxed (Tang et al., 1997, 2001). Moreover, localized rhizosphere acidiﬁcation and citrate exudation was also reported previously (Dinkelaker et al., 1989; Keerthisinghe et al., 1998; Neumann et al., 1999, 2000). However, littleTable 1. Basic nutrients applied to soil as solution form prior to planting. The nutrients added were thoroughly mixed with soil and then the soil mixture was carefully packed into plastic soil-column pots (diameter×height = 86×380 mm) for planting. There was 3-kg dry soil per pot Chemicals K2 SO4 CaCl2 .2H2 O MgSO4 .7H2 O MnSO4 .H2 O ZnSO4 .7H2 O CuSO4 .5H2 O H3 BO3 Na2 MoO4 .5H2 O CaCl2 .2H2 O MgSO4 .7H2 O Co2 SO4 .7H2 O Application rates (mg pot−1 ) 400 500 130 20 30 6 2 0.5 500 130 1information was available about patterns of pH change caused by H+ release by the whole root system and excess cation uptake of N2 -ﬁxing Lupinus albus grown in P-deﬁcient soil during various growth stages. The objective of the present study was to investigate the effect of P deﬁciency on root-induced acidiﬁcation and excess cation uptake by N2 -ﬁxing white lupin at different growth stages, and assess the relationship between balance of anion-cation uptake and H+ release, and organic acid anion exudation under P deﬁciency.Materials and methods Plant cultivation Seeds of white lupin (Lupinus albus L. cv. Kiev) were germinated and grown in column pots containing 3 kg of air-dried soil. Virgin brown sand (Uc4.22, Northcote, 1971) was collected from a bushland site 15 km south-east of Lancelin, WA (31.56 S, 115.20 E). The soil fertility was characterized as follows: organic carbon of 8.4 g kg−1, available P (Colwell P) of 5 µg g−1 , NO3 − -N of 2 µg g−1 and NH4 + -N of 1 µg g−1 . The soil conductivity was 0.0022 S m−1 , and the soil pH was 5 (CaCl2 ) or 5.7 (H2 O) (Shen et al., 2003). The soil texture is sand with 96% sand, 2% slit and 2% clay. The contents of oxy-hydroxides are very low with 0.54% Fe2 O3 and 0.11% Al2 O3 (Brennan et al., 1980). Phosphorus was added into the soil as FePO4 at71 a rate of 10 (limited P) or 200 µg P g−1 (adequate P). Nitrogen was supplied only via biological N2 ﬁxation through inoculating the seeds with Bradyrhizobium sp. (Lupinus) WU 425 by adding suspension solution (Sas et al., 2001; Tang et al., 2001). Other basic nutrients were added according to Table 1. The experiment was conducted in a controlled glass-house with temperature of 20/12 ◦ C (12-h day/12-h night) and relative humidity of 75–85%. The soil was watered up to 90% of ﬁeld capacity by weighing pots with plants once or twice daily. Three replicate pots of each treatment were used for root exudate collection, plant harvest, and nutrient uptake and carboxylate analysis at each of ﬁve harvests. The pots were completely randomized and repositioned weekly to minimize any effect of uneven environmental factors. for determination of mineral composition including total K, Na, Ca, Mg, P, S and Cl. Measurements The pH of the root exudate solution A 30-mL aliquot of collected solution was subsampled from the collected solution, and the pH was determined using an Orion 940E pH meter with a combined glass electrode. Organic anions Retention times and absorption spectra of 12 organic acid standards (oxalic, tartaric, formic, malic, malonic, lactic, acetic, maleic, citric, succinic, fumaric and aconitic acids) were used to identify the composition of organic anions in tissue extracts or root exudates. The 10-mL samples of plant extracts or root exudates were ﬁltered through sterile Millex GS Millipore 0.22-µm ﬁlters and directly analyzed for organic anions on a reversed phase column (Alltima C18 5 Micron, length 250 mm, i.d. 4.6 mm) using Waters HPLC (Shen et al., 2003). Mineral composition of plant tissues Concentrations of total K, Na, Ca, Mg, P and S were determined in roots and shoots after digesting plant materials in a mixture of concentrated nitric and perchloric acids (Johnson and Ulrich, 1959). Concentrations of total K, Na, Ca, Mg and S were determined using inductively coupled plasma emission spectrometry. Phosphorus was assayed using the vanadomolybdate method (Westerman, 1990). Chloride was analysed colourimetrically in the water extract. The concentrations of excess cations [cmol (+) kg−1 ] in plants were calculated as the sum of charge concentration of K+ , Ca2+ , mg2+ and Na+ minus the sum of H2 PO4 − , SO4 2− and Cl− according to the method described by Tang et al. (1997). Statistics Analysis of variance for comparisons among means was conducted using the SPSS statistical software (SPSS, 1998).Sampling of root exudates and plant materials Root exudates were collected by percolating the soil column containing intact plants with deionized water for 2 h (from 10:00 to 12:00 in the morning) at 40, 49, 58, 67 and 76 DAS according to the method described elsewhere (Shen et al., 2003). An aliquot of 600-mL deionized water was percolated through the soil column three times. The gravimetric analysis showed that 300 mL of solution was retained within the soil matrix, and thus the total volume for calculating carboxylate concentrations was 900 mL. The leachate contained water-soluble root exudates and was referred to as the ‘root exudate’. The pH of the leachate was determined immediately after collecting root exudate solution. Afterwards, Micropur (Sicheres Trinkwasser, Germany) at 0.01 g L−1 and three drops of concentrated H3 PO4 were added to the collected root exudates to inhibit the activity of microorganisms. A sub-sample of 10 mL from the collected solution was kept at −20 ◦ C for later analysis of carboxylates. After each root exudate collection, the plants were harvested, and roots and shoots were separated. Extraction of organic acids from plant materials was done according to the method reported by Neumann et al. (1999). Plant tissue samples were homogenized using ceramic mortar with 5% (v/v) H3 PO4 at the ratio of 1 mL H3 PO4 solution to 0.1 g fresh weight of plants. After centrifugation for 10 min at 10 000 g, the supernatant was diluted 10-fold with HPLC-elution buffer and analyzed by reversed-phase HPLC. Shoot and root weights were recorded after drying in an oven at 70 ◦ C for 3 d. After grinding, the plant materials were used72Figure 1. Fresh weights of shoots and roots (g plant−1 ) of white lupin with 10 (limited P) and 200 (adequate P) µg P g−1 soil at various growth stages. Vertical bars on symbols represent means±SE (n = 3 replicate pots). The separate bar represents LSD (P = 0.05) for any two means according to an analysis of variance for different treatments.Results Plant growth A signiﬁcant difference was observed in fresh weight between 10 and 200 µg P g−1 at 58 DAS for shoots, which occurred earlier than shoot dry weight changes (Shen et al., 2003), and at 67 DAS for roots (Figure 1). From day 58, plants receiving 10 µg P g−1 showed reduced growth as compared with those at 200 µg P g−1 . Fresh weights of shoots of white lupin at 10 µg P g−1 were 83%, 72% and 53%, respectively, of those at 200 µg P g−1 at 58, 67 and 76 DAS. The plants in two P treatments started to ﬂower at 55 DAS, and the number of ﬂowers in plants fed with 200 µg P g−1 was much greater than that at 10 µg P g−1 at 58 DAS. From day 58, the plants at 200 µg P g−1 also grew taller than those at 10 µg P g−1 (data not shown).Figure 2. P concentrations and contents in plants of white lupin grown at 10 or 200 µg P g−1 soil for 76 d. Vertical bars on symbols represent means±SE (n = 3 replicate pots). The separate bar represents LSD (P = 0.05) for any two means according to an analysis of variance for different treatments.Chemical composition of plant tissues The concentrations of P in shoots of plants fed with 10 µg P g−1 were signiﬁcantly lower than those at 200 µg P g−1 from 49 DAS, and there were the higher concentrations of P in roots of plants receiving 20073 than 10 µg P g−1 from 40 to 76 DAS (Figure 2). Phosphorus concentrations in shoots of plants receiving 10 µg P g−1 decreased steadily from 2.1 to 1.1 mg P g−1 shoot dry weight from 40 to 76 DAS. In contrast, P concentrations in roots supplied with 10 µg P g−1 had no signiﬁcant changes with time. At 200 µg P g−1 , there was a trend for decreasing P concentrations in both shoots and roots with time from 40 to 76 DAS. The P concentrations in shoots and roots of plants receiving 200 µg P g−1 were signiﬁcant lower at 76 than at 40 DAS. The higher P concentrations in shoots were observed at 58 compared with those at 49 and 76 DAS. The total P uptake increased with time irrespective of P supply, but the P uptake by the plants at 10 µg P g−1 was only 35–75% of that at 200 µg P g−1 (Figure 2). Plants fed with 10 µg P g−1 soil had higher Ca concentrations in shoots at 49 and 67 DAS, and had higher Mg concentrations in shoots at 49, 67 and 76 DAS (Figure 3). The concentrations of Ca in roots were higher at 67 DAS in the treatment of 10 than 200 µg P g−1 . Plants supplied with 10 µg P g−1 showed higher K concentrations in shoots at 49 DAS, and in roots at 67 and 76 DAS in comparison to plants receiving 200 µg P g−1 . However, Na concentrations in shoots were lower at 40 DAS in the treatment of 10 than 200 µg P g−1 . There was a lower S concentration at 58 DAS and higher Cl concentration at 67 DAS in the treatment of 10 than 200 µg P g−1 . However, there was no signiﬁcant difference in the concentrations of Na, Mg, S and Cl in roots between P treatments. Ca and Cl concentrations were higher, and Na, Mg and S concentrations were lower in shoots than in roots.Figure 3. Concentrations of K, Na, Ca, Mg, S and Cl in shoots and roots of white lupin grown at 10 or 200 µg P g−1 soil for 76 d. Vertical bars on symbols represent means ±SE (n = 3 replicate pots). The separate bar represents LSD (P = 0.05) for any two means according to an analysis of variance for different treatments.Root-induced acidiﬁcation Excess cation uptake Growing plants decreased the pH of soil leachate (root exudates) compared with the control without any plants irrespective of P treatments (Figure 4). The pH of root exudate solution in the treatment of 10 µg P g−1 was signiﬁcantly lower at 58 and 67 than at 40 or 49 DAS. In contrast, for the treatment of 200 µg P g−1 , the pH of root exudate solution remained constant with time. At 58 and 67 DAS, the pH of collected solutions from the plants fed with 10 µg P g−1 was signiﬁcantly lower than that from plants receiving 200 µg P g−1 . There was no signiﬁcant difference in the pH of root exudate solution between the treatments of 10 and 200 µg P g−1 at 40, 49 and 76 DAS. The concentrations of excess cations in plants were signiﬁcantly higher in the treatments of 10 than 200 µg P g−1 at 49 and 76 DAS in shoots, and only at 76 DAS in roots (Figure 5). The concentrations of excess cations in plants tended to be lower at 10 than 200 µg P g−1 soil at 40 DAS despite no signiﬁcant difference. However, after 49 DAS, the 10 µg P g−1 treatment surpassed the adequate P treatment in the concentrations of excess cations in plants. The highest concentrations of excess cations for the plants grown at 10 µg P g−1 were observed at 49 DAS for shoots and at 58 DAS for roots, and then the concentrations decreased with time.74Figure 4. The pH in root exudate solution of white lupin grown at 10 or 200 µg P g−1 soil for 76 d. Vertical bars on symbols represent means±SE (n = 3 replicate pots). The separate bar represents LSD (P = 0.05) for any two means according to an analysis of variance for different treatments. There was one replicate for the treatments without plants.The plants grown at 200 µg P g−1 showed a continuous decrease in excess cation concentrations in shoots with time, while in roots an increased concentration from 40 to 58 DAS and then a decrease till 76 DAS were observed. There were higher concentrations of excess cations in the whole plants grown at 10 µg P g−1 compared with the plants receiving 200 µg P g−1 after 49 DAS, and the difference in concentrations of excess cations between P treatments was signiﬁcant at 49 and 76 DAS. Concentrations of carboxylates in roots and shoots of plants and citrate exudation The concentrations of citrate in shoots or roots of the plants fed with 10 µg P g−1 signiﬁcantly increased with time from 40 to 76 DAS, but remained unchanged for the plants fed with 200 µg P g−1 over the experimental period (Figure 6). The citrate concentrations in shoots and roots were signiﬁcantly higher in the plants receiving 10 than 200 µg P g−1 after 67 DAS. The increased exudation of citrate from whole root system of white lupin in the limited P treatment was observed compared with the adequate P treatment over the period monitored (Figure 6, calculated from Shen et al., 2003). There was a higher exudation of citrate at 49 and 67 DAS in the treatment of 10 µg P g−1 , but the citrate exudation rates remained unchanged with time for the plants fed with 200 µg P g−1 from 40 to 76 DAS.Figure 5. Concentrations of excess cations in shoots and roots of white lupin grown at 10 or 200 µg P g−1 soil during 76 d. Vertical bars on symbols represent means ±SE (n = 3 replicate pots). The separate bar represents LSD (P = 0.05) for any two means according to an analysis of variance for different treatments.Discussion Effect of P limitation on plant growth and P uptake In the present study, P deﬁciency led to a signiﬁcant decrease in biomass of both shoots and roots, but the shoot growth appeared to be more sensitive to P deﬁciency than root growth. The decrease in plant growth due to P deﬁciency was also observed in the reduced75 laas et al., 2003). From the ﬁrst to ﬁnal harvests, the ratios between P uptake and P adding are 25–50% for 10 µg P g−1 , and 1.7–7.2% for 200 µg P g−1 . However, because P has a low diffusion in soil, particularly FePO4 , only the P close to root surface has a chance to be absorbed. According to the ratio of 10% [generally, 1–5% under ﬁeld condition (Marschner, 1995)] between root volume and total soil volume, only 3 mg and 60 mg FePO4 can be readily accessed by plants, and then the ratios between P uptake and the accessed FePO4 become 250–500% and 17–72%, suggesting that much P could be mobilized from soil. The signiﬁcant difference in P concentrations in plant tissues between P treatments occurred prior to plant fresh weights. The reduced growth of white lupin receiving 10 µg P g−1 was correlated with the decrease in P concentrations in shoots, but had no signiﬁcant correlation with P concentrations in roots. The results showed that the P concentrations in shoots were not completely dependent on the P concentrations in roots, being consistent with the previous report (Marschner, 1995). Root-induced acidiﬁcation and excess cation uptake The decreased pH of root exudate solution showed that the low P supply increased H+ release from roots. In symbiotically-grown legumes, rhizosphere acidiﬁcation may be caused by cation-anion balance, the excretion of organic anions and symbiotic nitrogen ﬁxation (Marschner, 1995; Tang et al., 1997). Under the speciﬁc experimental condition, it is not possible to separate the quantitative contribution of each of these processes to total net-release of protons. Moreover, in the present study, all process occurring in soil could have inﬂuenced the pH of the leachate and in fact we can not separate the net H+ release from other soil processes. However, the difference in pH between P treatments showed a clear effect of P limitation on H+ release from roots because P limitation caused excess uptake of cations, resulting in much H+ release from roots. Plant roots extrude a net amount of H+ to maintain charge balance when cation uptake exceeds anion uptake (Dinkelaker et al., 1989; Hedley et al., 1982; Marschner, 1995; Tang et al., 2001). In particular, excess cation uptake by plants reﬂected H+ release when N was supplied through N2 -ﬁxation (Tang et al., 1997). In the present experiment with N2 -ﬁxing Lupinus albus grown in P-deﬁcient soil, net H+ release was most likely caused by excess cation uptake. Phosphorus deﬁciency increased Ca concen-Figure 6. The concentrations of citrate [µmol (g FW)−−1 ] in shoot and root tissues and citrate exudation [µmol h−1 (g FW)−−1 ] from roots of white lupin grown with 10 and 200 µg P g−1 soil at different growth stages. Vertical bars on symbols represent means ±SE (n = 3 replicate pots). The separate bar represents LSD (P = 0.05) for any two means according to an analysis of variance for different treatments.plant height and number of ﬂowers per plant after 58 DAS. The reduced fresh weight and plant height were the ﬁrst visible symptoms of P deﬁciency. The increased fresh weights and P contents in the adequate P plants in comparison to the limited P plants showed that white lupin plants could take up large amounts of P from FePO4 added in soil, and that the amount of P taken up increased with the amounts of FePO4 added in soil despite FePO4 being a form of sparingly soluble P. The results showed a support for the viewpoint that white lupin plants have a great ability of mobilizing the sparingly soluble P through changing rhizosphere processes, particularly, citrate exudation (Hinsinger, 1998; Marschner, 1995; Venek-76 trations in shoots and roots, and Mg concentrations in shoots, but decreased S accumulation in shoots, showing that concentrations of excess cations was higher in the P-deﬁcient than P-sufﬁcient N2 -ﬁxing plants. Furthermore, a clear negative correlation was observed between the number of cluster roots (Shen et al., 2003) and the pH of root exudate solution, suggesting that cluster roots of white lupin are the main site for release of H+ under P deﬁciency, which is consistent with other studies (Dinkelaker et al., 1989, 1995; Marschner, 1995; Neumann and Römheld, 1999; Watt and Evans, 1999a; Yan et al., 2002). Relationship between proton release, accumulation and exudation of carboxylates Accumulation of citrate was found in shoot and root tissues of P-deﬁcient plants, which is in agreement with the observation of citrate accumulation in mature cluster roots under P deﬁciency (Neumann et al., 1999). However, in the present study, concentrations of citrate in shoots and roots of the plants fed with 10 µg P g−1 soil increased continuously with time (Figure 6), while a higher exudation of citrate was observed at 49 and 67 DAS in the treatment of 10 µg P g−1 (Figure 6 calculated from Shen et al., 2003). These results showed that the high concentrations of citrate in plants could be a prerequisite for citrate exudation, but citrate exudation did not show the consistent change pattern with time in comparison to citrate accumulation in plant tissue, being consistent with previous reports (Keerthisinghe et al., 1998; Neumann et al., 1999). Moreover, exudation of citrate showed a clearly different pattern of change with time compared with the pH of root exudate solution, showing a lack of dependence of proton release on exudation of citrate anions. The results suggest that exudation of citrate contributes only a part of total acidiﬁcation and excess cation uptake is the dominant factor affecting net proton release from roots of white lupin plants grown in P-deﬁcient soil. In conclusion, white lupin plants can take up large amounts of P from FePO4 as a sparingly soluble P form through changing rhizosphere processes. Phosphorus deﬁciency of N2 -ﬁxing plants grown in soil resulted in excess cation uptake and enhanced concomitant proton release, causing a decreased pH of root exudate solution. Citrate exudation did not show the consistent change pattern with time in comparison to citrate accumulation in plant tissue. Exudation of citrate anions contributes only a part of total acidiﬁcation, but excess cation uptake dominantly contributes net proton release from roots of white lupin plants grown in P-deﬁcient soil. Acknowledgements This study was supported by grants from the MSBRDP (Project No: G1999011709), by the National Natural Science Foundation of China (No. 30000102) and by AUSAid. ReferencesBarber S A 1984 Soil Nutrient Bioavailability: A mechanistic approach. John Wiley & Sons, Inc. New York. 398 p. Brennan R F, Gartrell J W and Robson A D 1980 Reactions of copper with soil affecting its availability to plants. I. Effect of soil type and time. Aust. J. Soil Res. 18, 447–459. Dinkelaker B, Romheld V and Marschner H 1989 Citric acid excretion and precipitation of calcium in the rhizosphere of white lupin (Lupinus albus L.). Plant Cell Environ. 12, 285–292. Dinkelaker B, Hengeler C and Marschner H 1995 Distribution and function of proteoid roots and other root clusters. Bot. Acta. 108, 183–200. Dracup M N H, Barrett-Lennard E G, Greenway H and Robson A D 1984 Effect of phosphorus deﬁciency on phosphatase activity of cell walls from roots of subterranean clover. J. Exp. Bot. 35, 466–480. Föhse D, Classen N and Jungk A 1991 Phosphorus efﬁciency of plants. II. Signiﬁcance of root radius, root hairs and cation-anion balance for phosphorus inﬂux in seven plant species. Plant Soil 132, 261–272. Gardner W K, Barber D A and Parbery K G 1983 The acquisition of phosphorus by Lupinus albus L. III. The probable mechanism by which phosphorus movement in the soil/root interface is enhanced. Plant Soil 70, 107–124. Gerke J, Roemer W and Jungk A 1994 The excretion of citric and malic acid by proteoid roots of Lupinus albus L. Effect on soil solution concentrations of phosphate, iron and aluminium in the proteoid rhizosphere in samples of an oxisol and luvisol. Z. Pﬂanzenernähr. Bodenkd. 157, 289–294. Hedley M J, White R E and Nye P H 1982 Plant-induced changes in the rhizosphere of rape (Brassica napus var. Emerald) seedlings. III. Changes in L value, soil phosphate fractions and phosphatase activity. New Phytol. 91, 45–56. Hinsinger P 1998 How do plant roots acquire mineral nutrients? Chemical properties involved in the rhizosphere. Adv. Agron. 64, 225–265. Hinsinger P 2001 Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: A review. Plant Soil 237, 173–195. Hinsinger P, Plassard C, Tang C and Jaillard B 2003 Origins of rootmediated pH changes in the rhizosphere and their responses to environmental constraints: A review. Plant Soil 248, 43–59. Johnson C M and Ulrich A 1959 Analytical methods for use in plant analysis. Cali. Agric. Exp. Stat. Bull. No.766. Johnson J F, Allan D L and Vance C P 1994 Phosphorus stressinduced proteiod roots show altered metabolism in Lupinus albus. Plant Physiol. 104, 657–665.。

GRE阅读3大类题型出题考点和解题思路分析指点

17. The primary purpose of the passage is to
(A) contrast the theories of Jean Wagner with those of other contemporary critics
(B) document the influence of Jean Wagner on the development of Afro-American poetry
GRE阅读题型思路分析：列举题
列举，顾名思义，就是符合某种条件的几个例子，例子可以是某几个事件，动作，情况或者人物。列举题有两种形式，一种是列举非，它问你四个选项中哪一个原文中没有提及。应对的办法仍然是定位原文，然后采用排除法，直至找到正确答案。第二种列举题要你在四个选项种找出哪一个选项在原文中提及，应对的措施仍然是根据关键词定位。其实，列举题并不难，出现的概率也比较大，每篇阅读大概出现一题。
It is Wagner who first demonstrated the essential fusion of racial and religious feeling in Afro-American poetry. The two, he argued, form a symbiotic union in which religious feelings are often applied to racial issues and racial problems are often projected onto a metaphysical plane. Wagner found this most eloquently illustrated in the Black spiritual, where the desire for freedom in this world and the hope for salvation in the next are inextricably intertwined.

Phytoremediation

Case study
- A liquid fertilizer was supplied weekly to the plants - grow time 4 weeks - Adding selected soil amendments - Plants were harvested 1 week after adding amendments - Shoots were washed - Were dried in an oven at 70°C - Analyzed for U by ICP-AES
Case study
Desorption to soil solution
200 fold increase from 1.2 to 240 mg pH was reduced by 0.5 to 1 unit
Case study
Case study
Case study: Results
- B. juncea is the best plant to accumulate U -Citric acid is the best acid to increase the U uptake of plants -U accumulation is associated with a stronger Pb accumulation -Even in one species there are differences in U uptake -For remediation, the soil has to be washed first with acid to desorp from soil particles - Hyperaccumulation is rapid

Interaction between arbuscular mycorrhizal fungi and Trichoderma harzianum

Applied Soil Ecology 47 (2011) 98–105Contents lists available at ScienceDirectApplied SoilEcologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /a p s o ilInteraction between arbuscular mycorrhizal fungi and Trichoderma harzianum under conventional and low input fertilization ﬁeld condition in melon crops:Growth response and Fusarium wilt biocontrolAinhoa Martínez-Medina ∗,Antonio Roldán,Jose A.PascualDepartment of Soil and Water Conservation and Organic Waste Management,CSIC-Centro de Edafología y Biología Aplicada del Segura.Campus Universitario de Espinardo,E-30100Espinardo,Murcia,Spaina r t i c l e i n f o Article history:Received 16April 2010Received in revised form 12November 2010Accepted 17November 2010Keywords:Biocontrol FertilizerFusarium oxysporum Glomus sp.MelonTrichoderma harzianuma b s t r a c tThe objective of this work was to evaluate the interactions between four arbuscular mycorrhizal fungi (AMF)(Glomus intraradices ,Glomus mosseae ,Glomus claroideum ,and Glomus constrictum )and the ben-eﬁcial fungus Trichoderma harzianum ,inoculated in a greenhouse nursery,with regard to their effects on melon crop growth under conventional and integrated-system ﬁeld conditions,and the biocontrol effect against Fusarium wilt.A synergistic effect on AM root colonization due to the interaction between T.harzianum and G.constrictum or G.intraradices ,was observed under a reduced fertilizer dosage,while no signiﬁcant effect was observed for G.claroideum or G.mosseae.With the reduced fertilizer input,AMF-inoculated plants and T.harzianum -inoculated plants had improved shoot weight and nutritional status,but the combined inoculation of AMF and T.harzianum did not result in an additive effect.Under the con-ventional fertilizer dosage,plant growth was not inﬂuenced by AM formation;however,it was increased signiﬁcantly in T.harzianum -inoculated plants.The AMF-inoculated plants were effective in controlling Fusarium wilt,G.mosseae -inoculated plants showing the greatest capacity for reduction of disease inci-dence.The T.harzianum -inoculated plants were more effective than AMF-inoculated plants with regard to suppressing disease incidence.Co-inoculation of plants with the AMF and T.harzianum produced a more effective control of Fusarium wilt than each AMF inoculated alone,but with an effectiveness similar to that of T.harzianum -inoculated plants.© 2010 Elsevier B.V. All rights reserved.1.IntroductionIn recent years,low-input agricultural systems have gained increasing importance in many industrialized countries,for reduc-tion of environmental degradation (Mäder et al.,2002).Integrated farming systems with reduced inputs of fertilizers and pesticides have been developed.It is under these conditions that plants are expected to be particularly dependent on beneﬁcial rhizosphere microorganisms (Smith et al.,1997).Arbuscular mycorrhizal fungi (AMF)are key components of soil microbiota and form symbiotic relationships with the roots of most terrestrial plants,improving the nutritional status of their host and protecting it against several soil-borne plant pathogens (Smith et al.,1997;Harrison,1999;Bi et al.,2007).The incidence and the effect of root colonization vary depending on the plant species and the AMF (Jeffries and Barea,2001);they are inﬂuenced by soil microorganisms and environmental factors (Azcón-Aguilar∗Corresponding author.Tel.:+34968396339;fax:+34968396213.E-mail address:ammedina@cebas.csic.es (A.Martínez-Medina).and Barea,1992;Bowen and Rovira,1999).Trichoderma sp.is a common component of rhizosphere soil and has been reported to suppress a great number of plant diseases (Chet,1987;Harman and Lumsden,1990;De Meyer et al.,1998;Elad,2000;Howell,2003).Some strains,also,have been reported to colonize the root sur-face,enhancing root growth and development,crop productivity,resistance to abiotic stresses,and the uptake and use of nutrients (Ousley et al.,1994;Björkman et al.,1998;Harman and Björkman,1998;Rabeendran et al.,2000;Harman et al.,2004).Several reports have demonstrated that the interaction of these two groups of microorganisms may be beneﬁcial for both plant growth and plant disease control (Linderman,1992;Barea et al.,1997;Saldajeno et al.,2008;Martínez-Medina et al.,2009a ).A syn-ergistic effect of some saprophytic fungi on AMF spore germination and colonization has been conﬁrmed (Calvet et al.,1993;McAllister et al.,1996;Fracchia et al.,1998).For example,it has been reported that some Trichoderma strains may inﬂuence AMF activity (Calvet et al.,1992,1993;Brimner and Boland,2003;Martinez et al.,2004;Martínez-Medina et al.,2009a ).Volatile and soluble exudates pro-duced by saprophytic fungi are involved in these effects (McAllister et al.,1994,1995;Fracchia et al.,1998).Nevertheless,the results0929-1393/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.apsoil.2010.11.010A.Martínez-Medina et al./Applied Soil Ecology47 (2011) 98–10599of research on the interactions between soil saprophytic and AM fungi differ widely,even when the same species of saprophytic fungi are involved.For example,Trichoderma harzianum has been found to have antagonistic,neutral,and stimulating effects on AMF (Rousseau et al.,1996;Siddiqui and Mahmood,1996;Fracchia et al., 1998;Godeas et al.,1999;Green et al.,1999;Martínez-Medina et al., 2009a).Little is known about the interactions between AMF and beneﬁcial saprophytic fungi,and the few studies published on this topic do not provide any conclusiveﬁndings(Green et al.,1999; Vázquez et al.,2000).Even more,the beneﬁcial effect attributable to these interactions under controlled experimental conditions may not be reﬂected inﬁeld experiments(Calvet et al.,1992;McAllister et al.,1997;Fracchia et al.,1998;Vázquez et al.,2000;Martinez et al.,2004).The aim of this work was to evaluate the effect of T.harzianum and four AMF,previously inoculated in a greenhouse nursery, with regard to melon plant growth and their potential biocontrol of Fusarium wilt,under different soil fertilization conditions.To achieve this aim,dual inoculation in a greenhouse nursery with four mycorrhizal fungi from the genus Glomus(G.constrictum,G. mosseae,G.claroideum and Glomus intraradices)and the fungus T. harzianum was evaluated in twoﬁeld experiments(under conven-tional conditions and with reduced fertilizer dosage,and under F. oxysporum pressure)for its effect on melon crops,with regard to(1)plant growth and(2)biocontrol of Fusarium wilt.2.Material and methods2.1.Host plant and fungal inoculaMelon plants(Cucumis melo L.,cv.“Giotto”)were used as the host plants.Plants were inoculated with T.harzianum and four dif-ferent AMF from the genus Glomus(G.constrictum,G.mosseae,G. claroideum,and G.intraradices)in a greenhouse nursery(Martínez-Medina et al.,2009a).Here,the AM inocula were mixed at a rate of20g kg−1of peat,while T.harzianum was added to reach a population density of1×106conidia g−1of peat,according to Martínez-Medina et al.(2009a).The AM fungal inoculum density was found to be35infective propagules per gram of inoculum. The isolate of T.harzianum,deposited in the Spanish Type Culture Collection(isolate CECT20714)by Centro de Edafología y Biologia Aplicada del Segura-CSIC(Spain),was chosen for this study owed to its high biocontrol capacity against F.oxysporum(Martínez-Medina et al.,2009a).T.harzianum inoculum was produced using a speciﬁc solid medium,prepared by mixing commercial oats,bentonite and vermiculite(1:2.5:5,w:v:v)according to Martínez-Medina et al. (2009b).The plants were grown in a peat-vermiculite mixture under nat-ural conditions,forﬁve weeks.They were irrigated manually,as necessary,during this period.Five weeks after planting,the melon plants were transplanted to theﬁeld,at the Estación Experimen-tal Cuatro Caminos(Spain)(38◦11 N;1◦03 W),where they were arranged in a randomized design.Monoconidial Fusarium oxysporum f.sp.melonis was isolated from infected melon plants from a greenhouse nursery.For the production of inocula,the pathogen was cultivated for5days on potato dextrose broth(Scharlau Chemie,Barcelona,Spain),at28◦C in darkness,on a shaker at120rpm.After the incubation period,the fungal culture was centrifuged at193×g,10min,re-suspended in sterilized water,and re-centrifuged.The fungal suspension con-tained1×108conidia mL−1.2.2.Experimental design and growth conditionsTwo experiments,using a completely randomized design,were conducted separately.Theﬁrst experiment had three factors,theﬁrst factor withﬁve levels:non-inoculation and inoculation with four AMF(G.constrictum,G.mosseae,G.claroideum,or G. intraradices)in the greenhouse nursery.The second factor had two levels:non-inoculation and inoculation with T.harzianum,in the greenhouse nursery.The third factor had two levels:conventional and reduced fertilization dosage.To assess the effect of the fertilization on the interactions between the AMF and T.harzianum,half of the experiment was fertirrigated with a conventional fertilization dose for melon plants in the Mediterranean area:0.51g L−1NH4NO3and0.51g L−1 NH4H2PO4.The other half of the experiment was fertirrigated with 1/3of this dose.Eight replicates were established for each of the20 treatments.The second experiment had three factors,theﬁrst factor with ﬁve levels:non-inoculation and inoculation in the greenhouse nursery with four AMF(G.constrictum,G.mosseae,G.claroideum,or G.intraradices).The second factor had two levels:non-inoculation and inoculation in the greenhouse nursery with T.harzianum.The third factor had two levels:non-inoculation and inoculation with F.oxysporum.To assess the effect of the interactions between the AMF and T.harzianum on the potential biocontrol of Fusarium wilt, four weeks after planting,half of the melon plants were infected by F.oxysporum to reach aﬁnal concentration of1×104conidia g−1 in the rhizosphere,while the other half was maintained as a control.Eight replicates were established for each of the20treat-ments.For both experiments,plants were planted1m apart,at a depth of10cm,in rows.The soil had a pH of8.04(1:1soil:water ratio), the NaHCO3-extractable P was26␮g g−1,total N was1mg g−1, and extractable K was289␮g g−1.The soil texture was39g kg−1 coarse sand,502g kg−1ﬁne sand,301g kg−1silt,and158g kg−1 clay.Plants were grown for eleven weeks in theﬁeld under natural conditions(the climate is semi-arid Mediterranean with an aver-age annual rainfall of300mm and a mean annual temperature of 19.2◦C;the potential evapo-transpiration reaches1000mm/year). Plants were fertirrigated automatically for10min every12h with 2.5L h−1water drippers.The fertilizer was added in fertigation at the following doses:0.13g L−1NH4NO3(total nitrogen:35.5%, nitric nitrogen:16.9%,ammoniacal nitrogen:17.6%)and0.13g L−1 NH4H2PO4(ammoniacal nitrogen:12%,soluble P2O5in neutral ammonium citrate:60%).Eleven weeks after planting,plants were harvested and rhi-zosphere samples were taken and stored at4◦C for biological and biochemical analyses.In theﬁrst experiment,the shoot fresh weight and the nitrogen,phosphorus,and potassium contents were recorded as well as the fruit production.In the second experiment, further F.oxysporum-infected plants were determined.2.3.Plant analysesPlant samples for nutrient content analysis were digested by a microwave technique,using a Milestone Ethos I microwave diges-tion instrument.A standard aliquot(0.1g)of dry,ﬁnely ground plant material was digested with concentrated nitric acid(HNO3) (8mL)and hydrogen peroxide(H2O2)(2mL).Subsequently,the phosphorus and potassium contents were analyzed using ICP(Iris intrepid II XD2Thermo).Plant nitrogen content was determined using a Flash1112series EA carbon/nitrogen analyzer.Roots were softened with10%KOH in water bath and stained with0.05%trypan blue(Phillips and Hayman,1970).The per-centage root length colonized by AMF was calculated by the line intersect method(Giovannetti and Mosse,1980).Positive counts for AM colonization included the presence of vesicles,arbuscules, or typical mycelium within the roots.To determine the F.oxysporum colonization of inoculated plants, stem segments(∼1.5cm)from inoculated plants were cut imme-100 A.Martínez-Medina et al./Applied Soil Ecology47 (2011) 98–105 diately above crowns,surface-sterilized by soaking in1%sodiumhypochlorite for5min,and rinsed with sterilized water.Thesegments were incubated on PDA at28◦C for6days,and theappearance of F.oxysporum colonies was considered to be indica-tive of infected plants.The percentage of infected plants was usedto determine the disease incidence.2.4.Soil biological analysesSerial dilutions of1g of rhizosphere soil from the top0.3m,insterile,quarter-strength Ringer solution,were used for quantify-ing the T.harzianum colony forming units(CFU)by a plate counttechnique using PDA(Scharlau Chemie,Barcelona,Spain)amendedwith50mg L−1rose bengale and100mg L−1streptomycin sulfate.The plates were incubated at28◦C for5days.After the incubationperiod,CFUs were counted.Komada medium(Komada,1975)wasused for quantiﬁcation of F.oxysporum.2.5.Statistical analysisThe data were subjected to analysis of variance(ANOVA)usingSPSS software(SPSS system for Windows,version15.0,SPSS Inc,Chicago,II).The statistical signiﬁcance of the results was deter-mined by performing Duncan’s multiple-range test(P<0.05).3.Results3.1.Experiment I3.1.1.Plant shoot fresh weightIn treatments involving reduced fertilization,plants inoculatedwith T.harzianum alone had signiﬁcantly increased shoot freshweight relative to the non-inoculated plants(Table1).The AMF-inoculated plants also showed increases in fresh weight,with nodifferences among the AMF.Plants co-inoculated with T.harzianumand AMF showed fresh weights similar to those of AMF-inoculatedplants.Conventionally fertilized plants had a higher fresh weightthan plants receiving a reduced dosage of fertilizer,the factor fer-tilization being highly signiﬁcant(P<0.001)(Tables1and4).T.harzianum-inoculated plants receiving the conventional fertilizerdosage had an increased fresh weight compared with the non-inoculated plants,while the fresh weight of AMF-inoculated plantsdid not differ from that of the non-inoculated plants.Co-inoculated(T.harzianum-AMF)plants which were fertilized conventionallyexhibited fresh weights similar to those of AMF-inoculated plants(Table1).Table1Fresh shoot weight(g)of plants inoculated or not with Trichoderma harzianumand/or Glomus constrictum,Glomus mosseae,Glomus claroideum,or Glomusintraradices,under conventional and reduced fertilization dosages.Treatment Reduced fertilizerdose Conventional fertilizer doseNon-inoculated772±28c1156±27cG.constrictum890±64b1160±22cG.mosseae854±69b1291±56abc G.claroideum881±22b1154±45cG.intraradices884±47b1257±19abc T.harzianum1057±35a1378±80abG.constrictum×T.harzianum885±9b1166±77cG.mosseae×T.harzianum978±42ab1307±75abc G.claroideum×T.harzianum818±19b1233±121bc G.intraradices×T.harzianum911±14ab1288±55abcData are means±standard error of eight replicates.Values in the same column with the same letters,represent no signiﬁcant difference between treatments according to Duncan’s multiple range test(P≤0.05),n=8.3.1.2.Nutrient contentWith the low fertilizer dosage,total plant nitrogen was increased(P<0.001)in AMF-inoculated plants(Tables2and4). Inoculation of plants with T.harzianum increased the nitrogen content signiﬁcantly.No additive effect for nitrogen content was observed in plants co-inoculated with T.harzianum and AMF;even negative effect could be observed in the case of plants co-inoculated with G.constrictum and T.harzianum.The plants co-inoculated with T.harzianum and G.mosseae showed higher nitrogen contents than plants co-inoculated with any other AMF.The conventional fer-tilization dose increased the plant nitrogen concentration in all treatments(P<0.001)(Tables3and4).The nitrogen content was increased in T.harzianum-inoculated plants,while no differences in nitrogen content were found in AMF-inoculated plants,compared to the non-inoculated plants.Co-inoculation with T.harzianum and AMF gave nitrogen values similar to those of plants inoculated with the AMF alone.Under reduced fertilizer dosage,the phosphorus concentration was increased(P<0.001)in plants which had been inoculated in the greenhouse nursery with G.mosseae,G.claroideum,or G.intraradices alone,but it was unaffected in G.constrictum-inoculated plants(Tables2and4).The phosphorus content of T.harzianum-inoculated plants at the reduced fertilizer dosage was increased with respect to the non-inoculated plants.The plants co-inoculated with T.harzianum and AMF showed phos-phorus levels similar to those of plants inoculated with the AMF alone.The conventional fertilizer application increased(P<0.001) the shoot phosphorus level in all the treatments compared with the lower dose(Tables3and4).The phosphorus content of T. harzianum-inoculated plants was not altered with respect to non-inoculated plants.A decreased phosphorus level was observed in AMF-inoculated plants,and in plants co-inoculated with AMF and T. harzianum,relative to non-inoculated plants.Co-inoculated plants showed lower phosphorus contents than T.harzianum-inoculated plants.With the low fertilizer dosage,the plant potassium con-tent was increased in plants which had been inoculated in the greenhouse nursery with the AMF(Table2).The potas-sium content of T.harzianum-inoculated plants was increased with respect to the non-inoculated plants,at the reduced fertil-izer dosage.The potassium contents of plants co-inoculated with T.harzianum and AMF were similar to those of plants inocu-lated with each AMF alone,with no differences among them or with respect to T.harzianum-inoculated plants.The factor fer-tilization was not signiﬁcant for the plant potassium content (Table4).At the conventional fertilizer dosage,no differences in plant potassium content were found among the treatments (Table3).3.1.3.AM root colonizationInoculation in the greenhouse nursery with the different AMF produced a signiﬁcant increase(P<0.001)in the AM root coloniza-tion underﬁeld conditions(Fig.1).Under low fertilizer dosage,G. constrictum-and G.intraradices-inoculated plants showed higher percentages of AM root colonization than any other AMF tested. The lowest percentage of AM colonization was observed in G. claroideum-inoculated plants.Under the reduced fertilizer dosage, AM root colonization by G.constrictum or G.intraradices was increased(P<0.001)in plants which were also co-inoculated with T.harzianum,with respect to plants inoculated with AMF alone,but it was unaffected in plants co-inoculated with G. mosseae or G.claroideum and T.harzianum.The conventional fer-tilizer dosage produced,in general,a decreased percentage of AM root colonization(P<0.001)compared with the reduced fertilizer dose.A.Martínez-Medina et al./Applied Soil Ecology47 (2011) 98–105101Table2Shoot nitrogen,phosphorus,and potassium contents(g per plant)of melon plants inoculated or not with Trichoderma harzianum and/or Glomus constrictum,Glomus mosseae, Glomus claroideum,or Glomus intraradices,under reduced fertilization dosage.Treatment Nitrogen Phosphorous PotassiumNon-inoculated 1.90±0.01c0.37±0.02c 1.18±0.09cG.constrictum 2.11±0.01b0.36±0.02c 1.56±0.16abG.mosseae 2.49±0.01ab0.62±0.17ab 1.73±0.40aG.claroideum 2.08±0.16b0.47±0.05b 1.51±0.16abG.intraradices 2.05±0.05b0.45±0.02b 1.34±0.21bT.harzianum 2.42±0.06ab0.88±0.09a 1.51±0.13abG.constrictum×T.harzianum 1.82±0.29c0.39±0.09bc 1.52±0.05abG.mosseae×T.harzianum 2.63±0.07a0.48±0.05b 1.66±0.44abG.claroideum×T.harzianum 1.94±0.11bc0.49±0.19b 1.29±0.59bG.intraradices×T.harzianum 2.07±0.23b0.41±0.01bc 1.33±0.46bData are means±standard error of eight replicates.Values in the same column with the same letters,represent no signiﬁcant difference between treatments according to Duncan’s multiple range test(P≤0.05),n=8.Table3Shoot nitrogen,phosphorus,and potassium contents(g per plant)of melon plants inoculated or not with Trichoderma harzianum and/or Glomus constrictum,Glomus mosseae, Glomus claroideum,or Glomus intraradices,under conventional fertilization dosage.Treatment Nitrogen Phosphorous PotassiumNon-inoculated 4.74±0.01bcd 1.49±0.15a 1.20±0.14abG.constrictum 4.39±0.05bcde 1.11±0.40bc 1.56±0.49abG.mosseae 4.80±0.49bcd 1.16±0.21bc 1.55±0.46abG.claroideum 4.19±0.34cde0.95±0.06c 1.19±0.28abG.intraradices 5.46±0.27ab 1.21±0.09bc 1.70±0.62aT.harzianum 5.72±0.81a 1.59±0.57a 1.78±0.84aG.constrictum×T.harzianum 3.73±0.32de0.98±0.42c 1.00±0.21abG.mosseae×T.harzianum 5.02±0.64abc 1.10±0.36bc 1.47±0.35abG.claroideum×T.harzianum 3.56±0.04e0.99±0.14c0.99±0.03bG.intraradices×T.harzianum 4.98±0.17abc0.93±0.02c 1.77±0.03aData are means±standard error of eight replicates.Values in the same column with the same letters,represent no signiﬁcant difference between treatments according to Duncan’s multiple range test(P≤0.05),n=8.3.1.4.T.harzianum populationT.harzianum was detected in the rhizosphere,reaching values around1×104CFU g−1and showing similar CFU values in all the treatments which included inoculation with T.harzianum;in non-inoculated treatments,its density was below1×102CFU g−1(data not shown).3.1.5.Number of fruitsWith the reduced fertilizer dosage,AMF-inoculated plants had an increased number of fruits(P<0.01)compared with non-inoculated plants(Fig.2).T.harzianum-inoculated plants did not differ in their fruit number relative to non-inoculated plants. At the reduced fertilizer dose,and compared with the AMF-inoculated plants,the number of fruits was decreased signiﬁcantly by T.harzianum–G.constrictum or T.harzianum–G.intraradices co-inoculation,whereas T.harzianum–G.mosseae co-inoculation signiﬁcantly increased the number of fruits.The conventional fertilization dose reduced signiﬁcantly the number of fruits(P<0.001),compared with the lower dose(Fig.2). No.signiﬁcant differences in fruit number were produced by AMF or T.harzianum inoculation,alone or in combination,at the con-ventional fertilizer dosage,compared to non-inoculated plants. 3.2.Experiment II3.2.1.T.harzianum populationT.harzianum was detected in the rhizosphere,reaching values around1×104CFU g−1and showing similar CFU values in all the treatments which included inoculation with T.harzianum;in non-inoculated treatments,its density was below1×102CFU g−1(data not shown).3.2.2.Disease incidenceThe disease incidence in AMF-inoculated plants was reduced by up25–50%,G.mosseae-inoculated plants showing the low-est percentage of infection(Fig.3).The disease incidence in T. harzianum-inoculated plants was reduced by60%with respect to non-inoculated plants.Plants co-inoculated with T.harzianum and AMF showed a lower percentage infection than AMF-inoculated plants.Table4The three-factor ANOVA(arbuscular mycorrhizal fungi(AMF)inoculation,Trichoderma harzianum inoculation,and fertilization(F)level)for all parameters studied.P signiﬁcant values.Parameters studied AMFinoculation AM T.harzianuminoculation ThFertilization F InteractionAM×ThInteractionAM×FInteractionTh×FInteractionAM×Th×FShoot fresh weight0.0290.008<0.0010.0200.005NS NS Nitrogen content<0.0010.05<0.001<0.001<0.001NS NS Phosphorus content<0.0010.045<0.001NS NS NS NS Potassium content0.030.05NS0.045NS NS NS AM root colonization<0.001NS<0.001<0.001<0.001<0.001NS Fruit number0.0060.027<0.0010.017NS NS NS T.harzianum population NS<0.001NS<0.001NS<0.001NSNS:non-signiﬁcant.102 A.Martínez-Medina et al./Applied Soil Ecology47 (2011) 98–105Fig.1.Percentage of root length colonized by Glomus constrictum ,Glomus mosseae ,Glomus claroideum ,and Glomus intraradices in melon plants receiving conventional or reduced fertilization and co-inoculated or not with Trichoderma harzianum .Bars indicate standard error of eight replicates.Values with the same letter do not differ signiﬁcantly according to Duncan’s multiple range test (P ≤0.05),n =8.4.DiscussionThe results show a synergistic effect on AM root colonization due to the interaction between T.harzianum and G.constrictum or G.intraradices ,while no signiﬁcant effect was observed for G.claroideum and G.mosseae .Although saprophytic fungi have been reported to inﬂuence AM colonization and host plant response (Fracchia et al.,2000),the effects of the saprophytic fungi on AM formation differ depending on the inherent characteristic of both agents (Martinez et al.,2004;Saldajeno et al.,2008;Martínez-Medina et al.,2009a ).A synergistic interaction between T.aureoviride and G.mosseae has been reported for AM root col-onization (Calvet et al.,1993).Fracchia et al.(1998)found that T.harzianum did not affect the percentage of soybean root length col-onized by G.mosseae ,whereas T.pseudokoningii increased it.Calvet et al.(1992)reported a stimulation of G.mosseae spore germination by T.harzianum and T.aureoviride .The synergistic effect produced by the interaction between T.harzianum and G.constrictum or G.intraradices in our experiment could have been caused by a direct beneﬁcial action of soluble exudates and volatile compounds pro-duced by the saprophytic fungus (Calvet et al.,1992).No negative interaction was observed in our results,in contrast to previous results (McAllister et al.,1996;Green et al.,1999;Martinez et al.,2004).Our results further demonstrate that,under reduced fertilizer dosage,AMF and T.harzianum inoculation resulted in an improve-ment in shoot weight and nutritional status.Soil microorganisms and their activities play important roles in thetransformationFig.2.Number of fruits produced by plants inoculated with Trichoderma harzianum and/or Glomus constrictum ,Glomus mosseae ,Glomus claroideum ,or Glomus intraradices ,under conventional and reduced fertilization doses.Bars indicate standard error of eight replicates.Values with the same letter do not differ signiﬁcantly according to Duncan’s multiple range test (P ≤0.05),n =8.A.Martínez-Medina et al./Applied Soil Ecology47 (2011) 98–105103Fig.3.Disease incidence(%)in plants inoculated with Trichoderma harzianum and/or Glomus constrictum,Glomus mosseae,Glomus claroideum,or Glomus intraradices, seven weeks after pathogen inoculation.Bars indicate standard error of eight repli-cates.Values with the same letter do not differ signiﬁcantly according to Duncan’s multiple range test(P≤0.05),n=8.of plant nutrients from unavailable to available forms and the improvement of soil fertility(Adesemoye and Kloepper,2009).The capacity of AMF to promote plant growth and enhance phospho-rous availability and uptake has been widely reported over the years(Ames et al.,1983;Smith et al.,1997;Barea et al.,2002; Tawaraya et al.,2006).Several investigations indicated as well,that plant interaction with Trichoderma sp.correlates with improved phosphorous availability and plant growth(Harman and Björkman, 1998;Altomare et al.,1999).However,the combined inoculation of AMF and T.harzianum did not result in an additive effect.In general, for co-inoculated plants,both growth and nutrient uptake were maintained at values similar to those of plants inoculated with the AMF alone.In contrast to our results,Haggag and Abd-El latif(2001) found that the combined inoculation of G.mosseae and T.harzianum enhanced growth of geranium plants.Similarly,combined inocu-lation of T.aureoviride and G.mosseae had a synergistic effect on the growth of marigold plants(Calvet et al.,1993).However,root and shoot weights of soybean were decreased by co-inoculation with T.pseudokoningii and Gigaspora rosea(Martinez et al.,2004). The interaction between AMF and T.harzianum and its effect on plant growth may vary depending on the inherent characteristics of the AMF and the T.harzianum strain(Saldajeno et al.,2008).In our experiment,this interaction was in fact negative in the case of plants co-inoculated with G.constrictum and T.harzianum,which showed a decrease in nitrogen content relative to plants inocu-lated with the AMF or T.harzianum alone.However,an increase in the plant nitrogen content was observed in plants which had been co-inoculated with T.harzianum and G.mosseae in the greenhouse nursery,relative to plants inoculated with the saprophyte alone. Co-inoculation with T.harzianum and G.mosseae was more effec-tive than any other combination tested with regard to increases in the uptake of nitrogen.Under the conventional fertilizer dose,plant growth was not inﬂuenced by AM formation,but it was signiﬁcantly increased when T.harzianum was inoculated alone.However,the growth pro-motion mediated by T.harzianum was decreased at this fertilizer rate.Rabeendran et al.(2000)hypothesized that when plants are grown under optimal conditions growth promotion by Trichoderma is unlikely,whereas under suboptimal conditions enhanced growth can be achieved.Our results show that differences in growth pro-motion by T.harzianum and AMF are related to differences in growing conditions,being more pronounced in soils relatively poor in nutrients.It is noteworthy that plants co-inoculated with the AMF and T.harzianum had growth which was similar to that of non-inoculated plants under these conditions.The negative impact of high N and P levels on mycorrhizal root colonization has been reported(Rubio et al.,2002;Kohler et al.,2006).In our experiment,under the higher fertilization dose, the beneﬁcial effect of the AMF disappeared and the effect was even negative in the case of phosphorus uptake.The suppression of extraradical mycelium development,which occurs in soil fol-lowing a high fertilizer application(Azcón et al.,2003),could not explain ourﬁndings,since under this condition no effect on plant growth should be expected.This negative effect may be explained by an alteration in the rhizosphere microbial population due to the nutrient supply(Liu et al.,2000;Rengel and Marschner,2005). Stimulation of the rhizospheric population may increase competi-tion between plant roots and the microbial population,which has particular nutrient requirements(Germida et al.,1998;Grifﬁths et al.,1999),microorganisms being,in many cases,superior com-petitors(Kaye and Hart,1997;Hodge et al.,2000).Similar results have been reported by Azcón et al.(2003),who observed that a higher application of nitrogen and phosphorus to the soil reduced the nutrient uptake in AM-compared with non-AM-lettuce plants. Ourﬁndings may indicate not only the lack of mycorrhizal ben-eﬁt at these high fertilizer doses,but also a negative inﬂuence of AMF on mechanisms associated with the mineral nutrition of plants when grown in a highly fertilized soil.These results suggest that the beneﬁcial mycorrhizal effect on plant nutrition is only evident under lower fertility levels and that fertilizer application can reduce or even eliminate it.Interestingly,a decrease in fruit number was observed due to an increase in the fertilizer dose.Imbalanced fer-tilizer use in soil has been reported to cause yield decline(Manna et al.,2005).In our experiment,T.harzianum was able to increase plant nitrogen uptake even at the higher fertilizer level.Ourﬁnd-ings indicate an improvement in plant active-uptake mechanisms, and an increase in the effectiveness of nitrogen-containing fertil-izer.These results contrast markedly with the absence of effects observed in tomato plants under fertilized conditions:no effects on plant nutritional status occurred following inoculation with Tri-choderma(Inbar et al.,1994).The AMF-inoculated plants showed a signiﬁcant decrease in Fusarium wilt incidence,G.mosseae-inoculated plants showing the greatest reduction.AM symbiosis has been shown to reduce the damage caused by soil-borne plant pathogens(Azcón-Aguilar and Barea,1996;Bi et al.,2007).Com-petition for host photosynthates or sites,microbial changes in the mycorrhizosphere due to AM,and induction of local and systemic defense responses have been proposed(Azcón-Aguilar and Bago, 1994;Caron,1989;Liu et al.,2007).With regard to suppress-ing disease incidence,T.harzianum was more effective than the AMF.Several studies report the biocontrol capacity of Trichoderma sp.(Chet,1987;Chet et al.,1997;De Meyer et al.,1998;Yedidia et al.,1999;Harman,2000;Howell et al.,2000;Yedidia et al., 2003;Shoresh et al.,2005;Martínez-Medina et al.,2009b).Various mechanisms of biocontrol have been reported,such as mycopara-sitism,antibiotic production,competition,or induction of local and systemic defense responses(Howell,2003;Yedidia et al.,2003; Harman et al.,2004).Co-inoculated plants showed disease sup-pression similar to that of T.harzianum-inoculated plants.Datnoff et al.(1995)reported a higher suppressive effect against Fusarium crown and root rot of tomato with the combination of T.harzianum and G.intraradices than with each biological agent applied alone. However,there are several examples of combinations of different biocontrol agents providing no better or,in some cases,worse bio-control than the isolates used singly(Larkin and Fravel,1998;de Boer et al.,1999).。

Critical Reviews in Oral Biology & Medicine

/Critical Reviews in Oral Biology & Medicine/content/14/2/128The online version of this article can be found at:DOI: 10.1177/1544111303014002062003 14: 128CROBM T.M.T. Waltimo, B.H. Sen, J.H. Meurman, D. Ørstavik and M.P.P. HaapasaloYeasts in Apical PeriodontitisPublished by: On behalf of:International and American Associations for Dental Research can be found at:Critical Reviews in Oral Biology & Medicine Additional services and information for/cgi/alerts Email Alerts:/subscriptions Subscriptions: /journalsReprints.nav Reprints:/journalsPermissions.nav Permissions:What is This?- Mar 1, 2003Version of Record >>IntroductionA pical periodontitis is an inflammatory process of the peri-apical area caused by an infection of the dental root canal system. In most cases, chemo-mechanical preparation of the root canal and local medication with calcium hydroxide fol-lowed by filling of the root canal with gutta-percha and sealer result in elimination of the infection and healing of the lesion. Occasionally, apical periodontitis does not respond favorably to root canal therapy, and periapical inflammation caused by the root canal infection may persist months or even years despite treatment. Several factors may contribute to the failure of the treatment of persistent cases. Most commonly, these fac-tors are related to difficulties in chemo-mechanical prepara-tion of the root canals. Occasionally, micro-organisms resistant to conservative therapy may also be involved (Byström, 1986; Sirén et al., 1997). Literature on the microbiological findings in persistent apical periodontitis not responding favorably to conservative therapy is limited (Bender and Selzer, 1952; Grahnen and K rasse, 1963; Engström, 1964; Goldman and Pearson, 1969; Haapasalo et al., 1983; Ranta et al., 1988; Sirén et al., 1997; Molander et al., 1998; Hancock et al., 2001; Kalfas et al., 2001; Love, 2001). However, it is known that a few species are more frequently isolated from persistent cases compared with primary cases. These include the Enterococcus faecalis/faecium group, enteric Gram-negative facultative rods, i.e., coliforms, and Pseudomonas species (Engström, 1964; Haapasalo et al., 1983; Ranta et al., 1988; Sirén et al., 1997; Hancock et al., 2001;Love, 2001). Recently, there has also been increasing interest in the presence and role of yeasts in infections resistant to con-servative root canal therapy (Nair et al., 1990; Sen et al., 1995, 1997a,b). This review presents the contemporary knowledge of the occurrence and biotypes of yeasts in endodontic infections, and susceptibility, in vitro, of yeasts to endodontic irrigants, local disinfectants, and antifungal agents.Taxonomy and General Characteristics of Yeasts Yeasts belong to a separate kingdom of living organisms, fungi. Contrary to bacteria, fungi are eukaryotic organisms, i.e., their genome is organized in a nucleus which is surrounded by a membrane. This membrane is continuous with the endoplas-mic reticulum, and organelles, such as mitochondria, ribo-somes, and different storage inclusions, are present. Fungal cell walls are rigid structures composed mainly of glucan, mannan, and chitin. For nutrition, fungal organisms are dependent on nitrogen and carbon compounds which are taken up through the cell wall (De Hoog and Guarro, 1995).Yeasts are present in various sites in the human body as members of the normal flora. They occur, e.g., in the gastro-intestinal tract, vagina, and perineal area (Jarvis, 1996). The oral cavity has suitable environmental conditions for yeast colo-nization. Oral yeasts belong to the division Ascomycota and class Endomycetes, which is divided further into four families: Saccharomycetaceae, Endomycetaceae, Dipodascaceae, and Lipomycetaceae. Clinically, the most important oral yeastsY EASTS IN A PICAL P ERIODONTITIST.M.T. Waltimo1*B.H. Sen2J.H. Meurman3D. Ørstavik4M.P.P. Haapasalo51Institute of Dentistry, University of Turku, Lemminkäisenkatu 2, 20520 Turku, Finland; 2Department of Restorative Dentistry and Endodontics, Ege University, Izmir, Turkey; 3Institute of Dentistry, University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Finland; 4NIOM, Scandinavian Institute of Dental Materials, Haslum, Norway; and 5Department of Endodontics, Dental Faculty, University of Oslo, Norway; *corresponding author, tuomas.waltimo@utu.fiABSTRACT: Microbiological reports of apical periodontitis have revealed that yeasts can be isolated from approximately 5-20% of infected root canals. They occur either in pure cultures or together with bacteria. Almost all isolated yeasts belong to the genus Candida, and the predominant species is C. albicans. Pheno- and genotypic profiles of C. albicans isolates show hetero-geneity comparable with those of isolates from other oral sites. C. albicans expresses several virulence factors that are capable of infecting the dentin-pulp complex, including dentinal tubules. This causes, consequentially, an inflammatory response around the root apex, which suggests a pathogenic role for this organism in apical periodontitis. Yeasts are particularly associated with persistent root canal infections that do not respond favorably to conservative root canal therapy. This may be due to the resis-tance of all oral Candida species against a commonly used topical medicament, calcium hydroxide. However, other antimicro-bial agents may offer alternative therapeutic approaches and improve the treatment of these persistent cases of apical perio-dontitis.Key words.Apical periodontitis, Candida, endodontics, yeast infection.128Crit Rev Oral Biol Med14(2):128-137(2003)belong to the family Saccharomycetaceae and to the genus Candida . Reproduction of Candida is based on multilateral bud-ding, which may take place anywhere on the mother cell (de Hoog and Guarro, 1995).Oral Yeast SpeciesCandida albicans is the most dominant oral yeast species, fol-lowed by C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C.kefyr, and C. parapsilosis (Odds, 1988). Recent findings also sug-gest the occasional occurrence of C. dubliniensis, which is a species closely related to C. albicans (Hannula et al ., 1997). Other yeast genera have also been isolated from the oral normal flora,e.g., Saccharomyces spp. and Geotrichum spp. (Tawfik et al ., 1989;Stenderup, 1990; Heinic et al ., 1992). Isolation of other fungi from the oral cavity has also been reported, but they are usual-ly seen in association with systemic disease—for example, pul-monary cryptococcosis caused by Cryptococcus neoformans (Stenderup, 1990).Virulence Factors of CandidaThe transition of C. albicans from a harmless commensal to apathogenic organism appears to be dependent on minorchanges in predisposing conditions which cause the expres-sion of a variety of virulence factors (Shepherd, 1992; Sweet,1997). These factors include adherence, hyphal formation,thigmotropism, protease secretion, and phenotypic switch-ing phenomenon.Adherence of micro-organisms is a complex, multifactori-al process involving several types of cell-surface adhesinswhich are essential for colonization and infection of the host.The main adhesin molecules of C. albicans responsible foradhesion to host cells seem to be cell wall mannoproteins(Sweet, 1997). However, several other factors also contribute tothe adherence of yeasts, e.g., cell-surface hydrophobicity, envi-ronmental pH, and concentrations of iron, calcium, zinc, andcarbon dioxide (Ener and Douglas, 1992; K lotz, 1994;Samaranayake et al ., 1995; Sohnle et al ., 2001). Furthermore,environmental proteins from saliva and gingival crevicularfluid as well as extracellular matrix components affect thecomplex adherence of Candida to host cells and tissues(Calderone et al ., 2000; Holmes et al ., 2002). C. albicans is a pleo-morphic micro-organism demonstrating different growthforms such as germ tubes, yeasts (blastospores), pseudo- andtrue hyphae, and chlamydospores (Odds, 1988; de Hoog andGuarro, 1995). All growth patterns except chlamydosporesmay show conversion to each form of growth, depending onthe environmental conditions. Therefore, the term 'dimorphic',often used in the literature, is semantically inaccurate toexplain C. albicans morphogenesis. Although hyphal formation is not a prerequisite for pathogenicity of C.albicans , biopsies of candidal infections often revealhyphal adherence to and penetration through epithe-lial tissues, indicating increased pathogenicity in com-parison with ovoid yeast forms (Sweet, 1997). It seemsthat the hyphal penetration into tissues is enhanced by thigmotropism, i.e., contact sensing by hyphae to find intracellular junctions or microscopic breaks on mucosal surfaces (Sherwood et al ., 1992; Gow et al .,1994; Sweet, 1997). One of the key virulence determi-nants of Candida species is their ability to produce and secrete aspartyl proteases which digest a variety of host proteins. The virulence of these proteases has beendemonstrated with animal experiments showing that the amount of protease is directly comparable with the patho-genicity of the strain (MacDonald and Odds, 1983; K wong-Chung et al ., 1985; Okamoto et al ., 1993; Togni et al ., 1994).Therefore, the higher rate of protease activity of C. albicans in comparison with other Candida species also suggests higher virulence. In addition to these major virulence factors, C. albi-cans has a tendency to phenotypic alteration, which con-tributes to environmental adaptation. Phenotypic alterations include change of colony morphology and protease activity (Slutsky et al ., 1985; White and Agabian, 1995). This genetical-ly controlled phenomenon is known as phenotypic switching,and it may occur relatively frequently, especially under stress (Soll, 1988). Phenotypic switching may assist in survival of and colonization by the yeasts, and it may also lead to genetic selection of adaptive strains (Sweet, 1997). Virulence factors and their possible contributions to apical periodontitis are list-ed in Table 1.Oral Yeast Infections A characteristic feature of yeast infections is that they develop when the host provides the environmental conditions and nutrients essential for attachment, growth, and reproduction of fungi. In other words, yeasts are opportunistic pathogens.Thus, local or general predisposing factors are required for yeast infection to develop (Shepherd, 1992). These factors can be classified into four categories: (i) host factors, such as nor-mal and pathological changes in physiological status of the host; (ii) dietary factors, such as carbohydrate-rich diets and vitamin deficiencies; (iii) mechanical factors, such as denture-wearing; and (iv) iatrogenic factors, such as administration of broad-spectrum antibiotics and corticosteroids (Odds, 1988).The clinical form of candidosis is often related to a predispo-sing factor, e.g., acute pseudomembraneous candidosis (thrush) is often associated with natural factors such as immunological and microbiological instability at birth, angu-lar cheilitis with dietary factors, chronic atrophic candidosis (denture-associated stomatitis) with mechanical factors, and acute atrophic candidosis with iatrogenic factors (Lynch,1994). In dental practice, chronic atrophic candidosis (denture stomatitis) is perhaps one of the most frequently encountered oral Candida infections (Wilson, 1998). There has also been an increasing interest in the presence of yeasts in infected perio-dontal pockets and their possible role in the pathology of dif-ferent forms of periodontitis (Slots et al ., 1988; Zambon et al .,1990; Rams and Slots, 1991; Dahlén and Wickström, 1995;Hannula et al ., 1997, 2001).14(2):128-137 (2003)Crit Rev Oral Biol Med129TABLE 1Virulence Factors of C. albicans and Their Possible Contributions to Apical Periodontitis Virulence Factor Possible Contribution to Apical Periodontitis Adherence Colonization of dental hard tissues Hyphal formation Penetration into dentinal tubules Thigmotropism Penetration into dentinal tubulesProtease secretion Survival in conditions with limited nutrient supply Phenotypic alteration Adaptation in ecologically harsh conditionsMicrobiology of Apical PeriodontitisApical periodontitis is a host defense response to infection of necrotic pulp (Miller, 1894;Kronfeld, 1939; Kakehashi et al .,1965). The host has an array of defense mechanisms consisting of several types of inflammato-ry cells, such as polymorphonu-clear leukocytes and lympho-cytes, intercellular messengers,such as cytokines, and chemical weapons such as proteolytic enzymes (Nair, 1997). Despite these defenses, the body cannot eliminate the micro-organisms residing in the necrotic root canal, and therefore the inflam-matory process does not result in healing. The interaction between root canal infection and the host defense mecha-nisms eventually cause destruc-tion of periapical tissues and formation of apical periodonti-tis (Nair, 1997).More than 300 species of micro-organisms colonize the human oral cavity, but only a limited number of these have been isolated from infected root canals with apical periodontitis (Moore, 1987). Several factors contribute to the selection of micro-organisms. Primarily , the selection takes place among those micro-organisms entering the root canal, which depends on the pathway to the pulp. For example, a deep caries lesion may serve as a pathway and limit the number of possible microbial species. In addition,the host defense mechanisms in the infected but still vital pulp reduce the number of surviving species. Furthermore, environ-mental factors of the necrotic root canal, e.g., redox-potential and source of nutrients, give an advantage to species with pro-teolytic activity and ability to survive in anaerobic conditions.Finally, microbial interactions—either negative (such as compe-tition for nutrients, secreted toxic metabolites, and specific bacteriocins) or positive (i.e.,symbiosis of different spe-cies)—regulate the microflora of130Crit Rev Oral Biol Med14(2):128-137(2003)TABLE 2Micro-organisms Commonly Associated with Chronic Periodontitis, Apical Periodontitis, and Persistent Root Canal InfectionsDisease Group Micro-organismsReferencesChronic Gram-negative Fusobacterium nucleatum Moore and Moore, 1994periodontitisanaerobic rodsPorphyromonas gingivalis,Haffajee and Socransky, 1994Prevotella intermedia,Slots, 1999Campylobacter rectus,Hannula et al ., 2001Selenomonas spp.Treponema denticola Bacteroides forsythus Gram-negative Actinobacillusanaerobic rods actinomycetemcomitans Eikenella corrodens Gram-positive Peptostreptococcus micros anaerobic cocci Gram-positive Eubacterium spp.anaerobic rods Gram-positive Streptococcus intermedius facultative cocci YeastCandida albicans ApicalGram-negative Prevotella spp.Sundqvist, 1994periodontitisanaerobic rodsFusobacterium nucleatum,Sundqvist et al ., 1998Porphyromonas spp.Haapasalo, 1989Campylobacter rectus Selenomonas spp.Gram-positive Peptostreptococcus micros anaerobic cocci Gram-positive Eubacterium spp.anaerobic and Propionibacterium acnes facultative rods Actinomyces ctobacillus spp.Gram-positive Streptococcus spp.facultative cocciPersistent root Gram-positive Enterococcus faecalis Haapasalo et al ., 1983canal infectionfacultative cocci Streptococcus spp.Sirén et al ., 1997Waltimo et al ., 1997Gram-positive Peptostreptococcus spp.Sundqvist et al ., 1998anaerobic cocci Molander et al ., 1998Hancock et al ., 2001Gram-positive Actinomyces spp.anaerobic and facultative rods Gram-negative Bacteroides spp.anaerobic and facultative rods YeastCandida albicansthe infected root canal (Sundqvist, 1994).Apical periodontitis is a polymicrobial infection domina-ted by obligate anaerobes (Bergenholtz, 1974; Kanz and Henry, 1974; Sundqvist, 1976; Byström et al., 1985; Haapasalo, 1986; Sundqvist et al., 1989; Baumgartner and Falkler, 1991). Usually, the number of isolated species is between two and eight, and monoinfections are rare (K anz and Henry, 1974; Sundqvist, 1976, 1994; Haapasalo, 1986). Before root canal therapy, the most frequently isolated micro-organisms are: Gram-negative anaerobic rods, such as Prevotella spp., Porphyromonas spp., Fusobacterium nucleatum, Campylobacter rectus, and Selenomonas spp.; Gram-positive anaerobic cocci, such as Peptostreptococcus spp.; Gram-positive anaerobic and facultative rods, such as Eubacterium spp., Propionibacterium acnes, Actinomyces spp., and Lactobacillus spp.; and Gram-positive facultative Streptococcus species (Sundqvist, 1976; Haapasalo, 1986). The microbiology of root canal infections is still not clear in many regards, e.g., the data concerning the occurrence of uncultivable species such as spirochetes are scarce (Dahle et al., 1993).The literature on microbiological findings in persistent root canal infections is also relatively limited (Bender and Selzer, 1952; Grahnen and Krasse, 1963; Engström, 1964; Goldman and Pearson, 1969; Haapasalo et al., 1983; Ranta et al., 1988; Sirén et al., 1997; Molander et al., 1998). However, it is known that a few species are frequently isolated from persistent cases. These include the Enterococcus faecalis/faecium group, enteric Gram-negative facultative rods (i.e., coliforms), and Pseudomonas species (Engström, 1964; Haapasalo et al., 1983; Ranta et al., 1988; Molander et al., 1998; Sundqvist et al., 1998; Hancock et al., 2001; Love, 2001). Micro-organisms commonly associated with chronic periodontitis, apical periodontitis, and persistent root canal infections are listed in Table 2.Yeasts in Apical Periodontitis Microbiological investigations of apical periodontitis during the past 50 years have revealed that yeasts can be isolated from infected root canals (Grossman, 1952; Slack, 1953, 1957; Macdonald et al., 1957; Hobson, 1959; Goldman and Pearson, 1969; Matusow, 1981; Nair et al., 1990; Najzar-Fleger et al., 1992; Sen et al., 1995, 1997a,b; Waltimo et al., 1997; Molander et al., 1998). Slack (1953, 1957) reported that yeasts exist in about 5% of cases of apical periodontitis. According to Grossman (1952), as many as 17% of infected root canals may contain Candida species. Hobson (1959) reported that Candida albicans was often isolated from root canal infections, although their pathogenici-ty in the root canal was unclear. However, according to a case report, a pure culture of Candida albicans caused acute pulpal-alveolar cellulitis (Matusow, 1981).In another case report, C. albicans was found in root canals and in periapical granulomas of a patient suffering from chron-ic urticaria (Eidelmann et al., 1978). The complete cure of the patient was achieved only after the extraction of the infected teeth. Histological examination revealed that the granuloma exhibited an invasive Candida infection composed of acute and chronic granulation tissue along with hyphae and yeast cells. The root canal surfaces were covered by dense masses of yeast cells, and dentinal tubules were totally filled with hyphae. In addition to these cases, Damm et al. (1988) described two cases of cancer patients having dentinal candidosis. In the first case, carious dentin of the patient's deciduous teeth contained numerous oval to filamentous Candida cells. The teeth exhibit-ed either acute irreversible pulpitis or acute apical plete healing was accomplished after extraction of all deciduous teeth. The second case demonstrated exposed coro-nal dentin with heavy colonization by C. albicans. Pseudohyphae and yeast cells were present not only in pulp tissue but also in cervical and apical soft tissues. As seen in these cases, extensive invasion by fungi seems to be mostly associated with the immunocompromised state of the patients. However, Kinirons (1983) described a similar clinical case with no systemic illness.Nair et al. (1990) studied therapy-resistant root canal infec-tions and found micro-organisms in 6 of 9 specimens. Bacteria were shown in 4 of the 6 cases, while yeast-like organisms were found in 2 cases as judged by electron microscopy. The pres-ence of intraradicular fungi in the endodontically treated human teeth was associated with periapical lesions that per-sisted after treatment. Sen et al. (1995) observed bacteria and fungi with scanning electron microscopy in infected root canals and dentinal tubules associated with periapical lesions. They found that 4 out of 10 root canals were heavily infected with yeasts, confirming the association between yeasts and root canal infections. They formed dense but separate colonies, and, in one specimen, hyphal elements were also present. Since the patients in this study did not have any systemic disease, the presence of yeasts in root canals may be attributed to poor oral hygiene.In a report by Waltimo et al. (1997), the occurrence of yeasts was studied in 967 microbiological samples taken from cases of apical periodontitis not responding favorably to conventional treatment. Micro-organisms were found in 692 (72%) samples, whereas 275 (28%) showed no growth. Forty-eight fungal strains were isolated from 47 samples, which represented 7% of the culture-positive samples. The fungi were endomyceteous yeasts, and they were isolated either in pure culture (6 cases, 13%) or together with bacteria (41 cases, 87%). The identifica-tion of yeasts was carried out with conventional clinical labo-ratory procedures, showing results comparable with those of earlier studies. Almost all isolates belonged to the genus Candida, and C. albicans was the most common species. C.g labrata, C. g uilliermondii, C. inconspicua, and Geotrichum can-didum were also isolated.In studies of the initial microbial flora of root canal infec-tions, yeasts have usually not been found (Haapasalo, 1989; Sundqvist et al., 1989). However, according to a recent study of randomly selected patients with periapical radiolucencies, C. albicans was detected in 5 out of 24 samples (21%) taken from infected root canals by means of the polymerase chain-reaction-based (PCR) molecular detection technique (Baumgartner et al., 2000). The PCR was carried out conventionally with a detection limit of 10-4 ng of DNA. Although the material was limited, the high percentage may be due to the higher sensitivity of the method in comparison with detection of micro-organisms by conventional culture procedures. However, the finding indi-cates that yeasts may be present in low numbers at the start of root canal treatment, and that they may reach higher propor-tions during conventional treatment procedures. In another recent study, intact root canals with pulp necrosis were exam-ined microbiologically (Lana et al., 2001). C. tropicalis and S. cerevisiae were recovered from two canals (7.4%) before root canal therapy. C. guilliermondii and C. parapsilosis were cultivat-ed in the second and third collections, respectively, of root canal contents. According to these findings, it is also possible that yeasts which are common opportunistic pathogens of the oral14(2):128-137 (2003)Crit Rev Oral Biol Med131cavity gain access to the root canal as contaminants duringendodontic therapy (Sirén et al ., 1997). This emphasizes the importance of aseptic treatment procedures in the prevention of persistent infections. Regardless of the source and means of entry for yeasts into the root canal, their presence in cultivable numbers may have clinical importance in persistent cases.Influence of Necrotic Root Canalon Strain SelectionNecrotic root canals provide harsh ecological conditions for micro-organisms in comparison with other oral sites. The influ-ence of these conditions for yeast strain selection was examined in a recent study that compared the phenotypes and genotypes of C. albicans isolates from root canals and periodontal crevices (Waltimo et al ., 2001). Briefly , the phenotyping was based on the presence of 5 enzymes (valine arylamidase, phosphoamidase,alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase), ability of the strains to assimilate 11carbohydrates (glycerol, L-arabinose, xylose, adonitol,xylitol, sorbitol, methyl-D-glucoside, N-acetyl-D-glu-cosamine, sucrose, trehalose, and melezitose), and the resistance to boric acid (Williamson et al ., 1987).Genotyping was based on randomly amplified polymor-phic DNA (RAPD) profiles obtained with the use of two different primers.A total of 14 different phenotypes was found among the 37 root canal isolates of C. albicans . The majority of the isolates (26) were classifiable into three major phenotypes, described by Williamson et al . (1987):16 isolates (43.2%) belonged to phenotype A1R, 6(16.2%) to A1S, and 4 (10.8%) to B1S. Interestingly, C.albicans phenotype A1R, which was predominant, has been associated mainly with patients with symptomatic C. albicans infections but not with asymptomatic carri-ers. This may indicate a higher virulence of this pheno-type in comparison with other phenotypes (Xu and Samaranayake, 1995). The genotypic characterization with use of the combination of the two different primers yielded 32 different profiles for the 37 C. albicans strains,demonstrating high genotypic divergence of the iso-lates.Analysis of the current data implies genotypic het-erogeneity of C. albicans isolates from root canals in humans. However, frequently encountered phenotypes were similar to the ones reported from other oral and non-oral sources (Bostock et al ., 1993; Tsang et al ., 1995;Xu and Samaranayake, 1995; Matee et al ., 1996). This implies that phenotypically unusual strains of C. albicans are not frequently involved in root canal infections.Therefore, it seems that the root canal, an ecologically harsh niche with regard to redox-potential and nutrients supply, may not have an impact on strain selection that differs from those of other oral sites. Thus, it seems that a general characteristic of C. albicans is its ability to tolerate a wide variety of different environmental conditions.Accompanying Bacteria in Yeast InfectionsRecent studies have shown that yeasts can survive as a monoinfection of the root canal (Matusow, 1981; Waltimo et al ., 1997). However, they are usually found in mixed cultures together with bacteria. Yeasts may often be iso-lated together with facultative Gram-positive bacteria such as a- and non-hemolytic Streptococcus species, whereas Gram-negative isolates are rare (Waltimo et al ., 1997). The dom-inance of the facultative Gram-positive accompanying bacteria may be due to the special ecological conditions of the root canal during prolonged treatment, which could favor yeasts and streptococci. There may also be synergism between these micro-organisms. However, no or only negative association of Streptococcus spp. with other bacteria has been reported in root canals (Sundqvist, 1992). However, it has been reported that C.albicans may prolong the viability of ␤-hemolytic streptococci (Burnet and Sherp, 1968). Furthermore, C. albicans co-aggre-gates with a variety of streptococci such as S. g ordonii, S.mutans, and S. sanguis (Holmes et al ., 1995; Nikawa et al ., 2001).This may promote their colonization and thus explain the con-comitant occurrence of these microbial species. Further investi-gations concerning microbial interactions are needed for a bet-132Crit Rev Oral Biol Med14(2):128-137(2003)Figure 1. Scanning electron micrograph of C. albicans blastospores on root canal surface in vitro . The bar indicates 10 ␮m.Figure 2.Scanning electron micrograph of C. albicans hyphae penetrating a dentinal tubule. The bar indicates 2 ␮m.。

不同程度镉污染对棉花生长和镉富集特征的影响

新疆农业科学 2021,53(5) ：922 -923Xinjiang Agricultural Sciexcosdel ： 14.0443/j ；算x. 1447 -4334. 9207.25.216不同程度镉污染对棉花生长和镉富集特征的影响陈丽丽，李俊华，鲁伟丹，罗彤，田爽(石河子大学农学D/新疆生产建设6789生态农业；点实验？，新疆石河子334OO7)摘要：【目的】开究不同程度镉污染土壤下棉花生长和镉富集的特征。

【方法】p 用盆栽模拟方法，添加外源镉,分析棉花种植后土壤pH 和镉含量的变化，以及镉胁迫对棉花生长和镉积累量的影响。

【结果】帛花根系具有酸化作用，使其根际土壤pH 下降，随镉胁迫浓度的增加，酸化受到抑制,土壤有效态镉含量随之显著降低。

棉花株高和地上部生物量随镉浓度增加逐渐降低，根系则相反。

棉花各器官镉含量、转移系数与积累量随镉浓度的增加显著升高。

在不同镉胁迫下，棉花根系镉富集系数均＞7。

在O mg/kg 镉胁迫下茎、叶和蕾富集系数分别达到4. 98、4. 33和4. 63；镉富集量分别为酮.47、39. 5和93. 608 平盆,表现出较强的积累能力。

【结论】在镉胁迫下棉花根系生物量增加，地上部生物量降低，随镉浓度的增加，棉花镉积累量增大，在5和O mOkg 镉胁迫下棉花地上部镉积累量显著高于地下部。

关键词棉花生长;土壤镉污染;富集特征;镉积累量中图分类号:S562 文献标识码:A 文章编号：047 -4334(2421245 -0922 -094引言【研究意义】农业土壤中的镉污染及其对作物吸收［］。

镉既容易积聚在植物体内对生理过程有很强的抑用［2］。

对棉花研究不同程度镉污染土棉花生长和镉的。

【前人研究进展】植复技术复治理污染的有效之一,其中植物提取是利用或超植物吸收和转运，并累积在植物地，随后收获地分中处理的技术7］,该技术应用广泛，修复成本低、环境友好、土壤破坏小，适用复大面积、中污染的土壤⑷。

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Journal of Experimental Botany,Vol.62,No.1,pp.21–37,2011 doi:10.1093/jxb/erq281Advance Access publication20September,2010REVIEW PAPERRoot responses to cadmium in the rhizosphere:a reviewAlexander Lux1,*,Michal Martinka1,Marek Vaculı´k1and Philip J.White21Department of Plant Physiology,Faculty of Natural Sciences,Comenius University in Bratislava,Mlynska´dolina B2,84215Bratislava, Slovakia2Scottish Crop Research Institute,Invergowrie,Dundee DD25DA,UK*To whom correspondence should be addressed:E-mail:lux@fns.uniba.skReceived17June2010;Revised17August2010;Accepted18August2010AbstractThis article reviews the responses of plant roots to elevated rhizosphere cadmium(Cd)concentrations.Cadmium enters plants from the soil solution.It traverses the root through symplasmic or apoplasmic pathways before entering the xylem and being translocated to the shoot.Leaf Cd concentrations in excess of5–10m g g21dry matter are toxic to most plants,and plants have evolved mechanisms to limit Cd translocation to the shoot.Cadmium movement through the root symplasm is thought to be restricted by the production of phytochelatins and the sequestration of Cd-chelates in vacuoles.Apoplasmic movement of Cd to the xylem can be restricted by the development of the exodermis,endodermis,and other extracellular barriers.Increasing rhizosphere Cd concen-trations increase Cd accumulation in the plant,especially in the root.The presence of Cd in the rhizosphere inhibits root elongation and inﬂuences root anatomy.Cadmium concentrations are greater in the root apoplasm than in the root symplasm,and tissue Cd concentrations decrease from peripheral to inner root tissues.This article reviews current knowledge of the proteins involved in the transport of Cd across root cell membranes and its detoxiﬁcation through sequestration in root vacuoles.It describes the development of apoplastic barriers to Cd movement to the xylem and highlights recent experiments indicating that their maturation is accelerated by high Cd concentrations in their immediate locality.It concludes that accelerated maturation of the endodermis in response to local Cd availability is of functional signiﬁcance in protecting the shoot from excessive Cd loads.Key words:Accumulation,apoplasm,cadmium,endodermis,maize,root,suberin lamellae,symplasm,tissue asymmetry, transport.IntroductionCadmium(Cd)is toxic to plant cells,even at low concen-trations.Leaf concentrations greater than5–10l g Cd gÀ1 DM are toxic to most plants(White and Brown,2010), although some ecotypes of a few plant species have adapted to grow on soils with high Cd concentrations and can tolerate leaf concentrations in excess of100l g Cd gÀ1DM(Reeves and Baker,2000;Broadley et al.,2001;Verbruggen et al., 2009).These Cd-hyperaccumulator plants include ecotypes of Noccaea(formerly Thlaspi)caerulescens(J&C Presl.)FK Mey (Reeves et al.,2001),Arabidopsis(formerly Cardaminopsis) halleri(L.)O’Kane and Al-Shehbaz(Bert et al.,2002),Sedum alfredii Hance(Yang et al.,2004),Viola baoshanensis Shu, Liu et Lan.(Wei et al.,2004),Thlaspi praecox Wulf.(Vogel-Mikusˇet al.,2005),Picris divaricata Vant.(Tang et al.,2009),and Phytolacca americana L.(Liu et al.,2010b).The roots of some of these exceptional plant species proliferate in Cd-enriched patches of soil,which contrasts with the behaviour of roots of most plant species that generally avoid such patches(Whiting et al.,2000;Liu et al.,2010a).To prevent Cd accumulation in shoot tissues,plants have evolved various mechanisms to restrict the entry of Cd to the xylem.This article reviews our current knowledge of these mechanisms, which include(i)the production of Cd-chelates in the cytoplasm of root cells and the sequestration of Cd-chelates in the vacuole to restrict Cd delivery to the xylem from the symplast,and(ii)the development of physical barriers to the extracellular movement of Cd to the xylem to restrict Cd delivery to the xylem from the apoplasm.ªThe Author[2010].Published by Oxford University Press[on behalf of the Society for Experimental Biology].All rights reserved. For Permissions,please e-mail:journals.permissions@ at South China Agricultural University on December 5, Downloaded fromCadmium in the soilTo limit Cd concentrations in edible produce,Cd concen-trations lower than3l g gÀ1dry soil have been recommended for agriculture and horticulture(Mengel et al.,2001). Cadmium concentrations in non-polluted soils increase with clay concentration,but are generally lower than1l g gÀ1dry soil(Mengel et al.,2001).Cadmium is concentrated in the topsoil,where it is associated with organic matter.Solutions extracted from non-polluted soils generally have Cd concen-trations less than40–300nM(Wagner,1993).Cadmium availability to plants is greater in acid soils(Mengel et al., 2001;Tudoreanu and Phillips,2004;Kirkham,2006),and its solubility is increased by root exudates(Zhu et al.,1999). Cadmium occurs in the soil solution predominantly as Cd2+, but also as Cd-chelates(Tudoreanu and Phillips,2004).Low Cd2+concentrations in the soil solution,in combination with low diffusion coefﬁcients for Cd2+in aqueous solutions, suggest that transpiration-driven mass-ﬂow of the soil solution will dominate the delivery of Cd2+to plant roots (Sterckeman et al.,2004).This is consistent with reports that Cd accumulation by plants grown in soil is directly related to transpiration(Ingwersen and Streck,2005).Higher soil Cd concentrations can occur either naturally or through anthro-pogenic activities(Mengel et al.,2001;He et al.,2005; Kirkby and Johnson,2008).Natural mineral outcrops can be enriched in Cd through the weathering of Cd-rich rocks. Cadmium pollution of the environment has occurred through the mining and reﬁning of metal ores,and through the appli-cation of Cd-containing phosphate fertilizers,sewage sludge, and municipal composts to agricultural soils.The ability to grow on soils with high Cd concentrations is generally related to the ability of roots to exclude Cd from the plant and/or of plant tissues to chelate Cd as a non-toxic com-pound or sequester it in a non-vital cellular compartment. Plants tolerating high Cd concentrations,and especially plants accumulating this metal in their above-ground parts, have potential utility for the phytostabilization and phytor-emediation of contaminated soils(Raskin and Ensley,2000; Schwitzgue´bel et al.,2009).Cadmium uptake by plant rootsIn nature,shoot Cd concentrations vary greatly.Although much of this variation can be attributed to environmental factors,there is appreciable phylogenetic variation in shoot Cd concentrations(Broadley et al.,2001;Watanabe et al., 2007).When grown in the same environment,shoot Cd concentrations are generally highest in species from the Caryophyllales and Lamiales,and lowest in monocot species (Broadley et al.,2001).Shoot Cd concentrations are de-termined largely by Cd entry to the root,sequestration within root vacuoles,translocation in the xylem and phloem, and dilution within the shoot through growth.Cadmium concentrations are often(but not always)greater in roots than in shoots,suggesting that Cd transport to the xylem is restricted in most plants,and lowest in seeds,fruits,and tubers,suggesting that Cd is not readily translocated in the phloem(Seregin and Kozhevnikova,2008;Conn and Gilliham,2010).Grafting experiments have suggested that shoot Cd concentrations in Nicotiana(N.tabacum L.and N.rustica L.;Wagner et al.,1988),Solanum(S.integrifolium, S.melongena,and S.torvum;Arao et al.,2008),and Noccaea/ Thlaspi(Noccaeae caerulescens and Thlaspi perfoliatum; Guimara˜es et al.,2009)are controlled by root properties. Shoot Cd concentration often increases to a maximum value with increasing Cd concentration in the soil(Logan et al.,1997;Zhao et al.,2003;Sterckeman et al.,2004;Vogel-Mikusˇet al.,2005;Chen et al.,2008;Peng et al.,2009;Liu et al.,2010b),bulk soil solution,or nutrient solution(Yang et al.,1995;Perriguey et al.,2008;Street et al.,2009),al-though non-saturating relationships have also been reported (Salt et al.,1995;Ingwersen and Streck,2005).The con-centration dependence of Cd uptake from hydroponic solu-tions measured over short periods into either excised roots or intact plants generally follows the sum of a single Michaelis–Menten component plus a linear component(Table1).The linear component is often attributed to tight Cd binding to cell walls,but it could also represent an apoplasmic Cdﬂux to the xylem(cf.White,2001;White et al.,2002;Broadley et al.,2007).Estimates of the K m value for‘high-afﬁnity’Cd uptake commonly fall between20–1000nM(Table1). Cadmium uptake and accumulation by plant roots is generally inhibited by La3+,Ca2+,Cu2+,Fe2+,Zn2+or Mn2+ in the rhizosphere solution(Cataldo et al.,1983;Costa and Morel,1993,1994;Cohen et al.,1998;Lombi et al.,2001; Hart et al.,2002;Zhao et al.,2002;Berkelaar and Hale, 2003a;Han et al.,2006;Zhao et al.,2006),but Cd uptake by the Cd-hyperaccumulating Ganges ecotype of N.caerulescens was not inhibited by divalent cations nor by La3+(Lombi et al.,2001;Zhao et al.,2002).The presence of organic acid anions in hydroponic solutions increases the capacity of both the Michaelis–Menten component and the linear component of Cd uptake,but the reasons for this are unclear(Berkelaar and Hale,2003a;Han et al.,2006).Cadmium can enter root cells as Cd2+through ZIP(Zinc-regulated transporter/Iron-regulated transporter-like Protein) transporters,such as orthologues of AtIRT1and TcZNT1/ TcZIP4,through orthologues of the wheat TaLCT1transpor-ter,or via cation channels,such as depolarization-activated calcium channels(DACC),hyperpolarization activated cal-cium channels(HACC),and voltage-insensitive cation chan-nels(VICC),all of which are relatively non-selective between cations(Fig.1;Clemens et al.,1998;Cohen et al.,1998; White and Broadley,2003;White,2005;Plaza et al.,2007; DalCorso et al.,2008;Pedas et al.,2008;Verbruggen et al., 2009).In addition,Cd might enter root cells as Cd-chelates through YSL(Yellow-Stripe1-Like)proteins(Curie et al., 2009).Cadmium can then reach the stele through a symplas-mic pathway formed by the cytoplasms of individual root cells connected by plasmodesmata.The Cd species trans-ported through the symplasm are unknown,but could include Cd2+or Cd-chelates(Verbruggen et al.,2009). Cadmium is loaded from the symplasm into the xylem by heavy metal P1B-ATPases,such as orthologues of AtHMA2and AtHMA4,and possibly also by YSL proteins22|Lux et al.at South China Agricultural University on December 5, Downloaded from(Mills et al.,2005;DalCorso et al.,2008;Wong and Cobbett,2008;Verbruggen et al.,2009).The Arabidopsis ABC trans-porter AtPDR8has been implicated in Cd efﬂux across the plasma membrane of root hairs and epidermal cells (Kim et al.,2007).In many plant species,Cd tolerance is related to Cd accumulation in the vacuole (Chardonnes et al.,1998;Cosio et al.,2005;Korenkov et al.,2007;Seregin and Kozhevnikova,2008).Cadmium can be translocated across the tonoplast by H +/Cd 2+-antiporters,such as orthologues of AtCAX2and AtCAX4(Korenkov et al.,2007,2009),by heavy metal P 1B -ATPases,such as orthologues of AtHMA3(Morel et al.,2009),and as Cd-chelates by ABC transporters,such as orthologues of AtMRP3(Tommasini et al.,1998;Table 1.Published relationships between cadmium (Cd)uptake (V )by excised roots or intact plants and Cd in the external solution ([Cd]ext ),described by Michaelis–Menten functions using the terms V max (V ,when [Cd]ext ¼N )and K m ([Cd]ext ,when V ¼0.5V max ),and a linear term k (V /[Cd]ext )*Original data expressed on the basis of root surface area,V max ¼400pmol m À2s À1.**Values converted from DM to FW values using the conversion factor DM/FW ¼0.1.[Cd]ext (m M)K m (nM)V max(nmol g À1FW h À1)K m (m M)V max(nmol g À1FW h À1)k(nmol g À1FW h À1m M À1)ReferenceSoybean (Glycine max (L.)Merr.)Intact plants 0.000002.5–0.57622.9 1.2232ND Cataldo et al.,1983Maize (Zea mays L.)Intact plants 0.0063–0.16430–100*NDNDND Mullins andSommers,1986Maize (Zea mays L.)Intact plants 0.25–5.020020.31ND ND 16Han et al.,2006Maize (Zea mays L.)Intact plants 0.00001–100ND ND ND ND Yes Perriguey et al.,2008Maize (Zea mays L.)Excised roots 0.05–5026023.6**ND ND 3.6**Redjala et al.,2009Lupin (Lupinus albus L.)Intact plants 0.00005–5.04211.6NDNDND Costa and Morel,1993Lettuce (Lactuca sativa L.)Intact plants 0.00005–5.08–3032–43**0.011–0.0542420–7740**ND Costa and Morel,1994Pea (Pisum sativum L.)[Fe-replete]Excised roots 1–10060034ND ND 5.3Cohen et al.,1998Bread wheat(Triticum astivum L.)Intact plants 0.05–1.252026ND ND 28Hart et al.,1998Bread wheat(Triticum astivum L.)Intact plants 0.05–1.55933ND ND Yes Hart et al.,2002Durum wheat (Triticum turgidum L.var.durum )Intact plants0.05–1.254029NDND22Hart et al.,1998Durum wheat (Triticum turgidum L.var.durum )Intact plants 0.05–1.56738ND ND Yes Hart et al.,2002Durum wheat (Triticum turgidum L.var.durum )Intact plants 0.005–1.81660.87ND ND 1.69Harris and Taylor,2004Noccaeacaerulescens (Prayon)Intact plants 0.2–5026033ND ND 6.0Lombi et al.,2001Noccaeacaerulescens (Prayon)Intact plants 0.2–5093021.8ND ND 4.2Lombi et al.,2002Noccaeacaerulescens (Ganges)Intact plants 0.2–50180160ND ND 11.2Lombi et al.,2001Noccaeacaerulescens (Ganges)Intact plants 0.2–501000187.6ND ND 3.6Lombi et al.,2002Noccaeacaerulescens (Ganges)Intact plants <5450143ND ND ND Zhao et al.,2002Noccaeacaerulescens (Nc-H)Excised roots 0.05–50390130**ND ND 1.38**Redjala et al.,2009Noccaeacaerulescens (Nc-L)Excised roots 0.05–507023.4**ND ND 2.22**Redjala et al.,2009Arabidopsis halleri Intact plants 0.2–1035039.7ND ND 15Zhao et al.,2006Rice (Oryza sativa L.)Intact plants 0.25–50254044ND ND 0.8He et al.,2007Eggplant(Solanum melongena )Intact plants 0.04–1.2380270**ND ND ND Mori et al.,2009Solanum torvumIntact plants0.04–1.2353**NDNDNDMori et al.,2009Root responses to cadmium |23at South China Agricultural University on December 5, 2010 Downloaded fromCobbett,2000;DalCorso et al.,2008;Verbruggen et al.,2009).Within plant cells,Cd is bound to S-containing ligands,such as those present in glutathione,metallothio-neins,and phytochelatins (DalCorso et al.,2008).In many plant species,the synthesis of phytochelatins is induced by Cd exposure (see Salt et al.,1995;Schat et al.,2002;Clemens,2006;Ernst et al.,2008;Seregin and Kozhevnikova,2008;Persson et al.,2006)and Cd is often sequestered in the vacuole as Cd-phytochelatin complexes (Cobbett,2000;Clemens,2006;Ernst et al.,2008).The involvement of phytochelatins in Cd detoxiﬁcation is consistent with observations that mutants deﬁcient in phytochelatin synthesis show lower Cd tolerance than wild-type plants and mutants with enhanced phytochelatin synthesis can show higher Cd tolerance than wild-type plants (Cobbett,2000;Clemens,2006).However,differ-ences in Cd tolerance among natural ecotypes of several plant species appear to be unrelated to differences in phytochelatin synthesis,suggesting additional mechanisms of Cd-detoxiﬁcation (Schat et al.,2002).In some plant species,metallothioneins play an important role in Cd detoxiﬁcation (Ernst et al.,2008;Shim et al.,2009).The production of metallothioneins is also induced by exposure to Cd,and plants genetically engineered to produce metal-lotheioneins in greater concentrations show increased Cd tolerance (see DalCorso et al.,2008;Korenkov et al.,2009).Cadmium is thought to be exported from the vacuole by NRAMP (Natural Resistance-Associated Macrophage Pro-tein)transporters,such as orthologues of AtNRAMP3and AtNRAMP4(Thomine et al.,2003;Verbruggen et al.,2009).It is also possible for Cd 2+and Cd-chelates to reach the xylem solely via an extracellular,apoplasmic pathway in regions of the root lacking a Casparian band (Berkelaar and Hale,2003b ;Lux et al.,2004).The uptake of cationic ele-ments reaching the xylem via an apoplasmic pathway is generally restricted to the extreme root tip and to regions in which lateral roots are being initiated (White,2001;Moore et al.,2002).Although the relative contributions of the sym-plastic and apoplasmic pathways to the delivery of Cd to the xylem are unknown,it is likely that the relative contri-bution of the apoplasmic pathway will increase as the Cd concentration in the rhizosphere solution increases,as has been proposed for both Zn and Na (White et al.,2002;Plett and Møller,2010).Supporting the presence of an apoplasmic pathway,are observations that the root tip is the most active region of the root for Cd 2+inﬂux (Pin ˜eros et al.,1998),and that Cd accumulation by wheat cultivars is positively correlated with their number of root apices (Berkelaar and Hale,2000).A linear component to the concentration dependence of Cd uptake on Cd concentration in the nutrient solution is almost universally observed in short duration studies on hydroponically-grown plants (Table 1).This phenomenon can be interpreted as the result of an apoplasmic Cd ﬂux to the xylem.In challenging this view,Xing et al.(2008)showed that,among accessions of N.caerulescens with contrasting abilities to take up Cd and Zn,the amount of Cd taken up by roots over a 24h period and translocated to the shoot was inversely related to apoplasmic water ﬂows assayed using a membrane-impermeable ﬂuorescentdye.Fig.1.Proteins thought to be responsible for Cd inﬂux to the symplasm,sequestration in the vacuoles of root cells,and efﬂux to the rhizosphere and to the xylem (see text for details).24|Lux et al.at South China Agricultural University on December 5, 2010 Downloaded fromHowever,this is inconsistent with studies of the effects of transpiration on Cd accumulation by other plant species (Ingwersen and Streck,2005).Reducing transpiration by the addition of ABA to the nutrient solution has also been shown to reduce both xylem sap Cd concentration and shoot Cd accumulation of hydroponically grown plants (Salt et al.,1995;Hsu and Kao,2003;Zhao et al.,2006;Liu et al.,2010b),but this has been interpreted as an effect of ABA on symplastic loading of Cd into the xylem since Cd uptake by roots themselves was unaffected by the presence of ABA(Salt et al.,1995;Zhao et al.,2006).More persua-sive arguments for symplasmic delivery of Cd to the xylem are(i)that there is competition between Cd2+and other cations for uptake by roots(Hart et al.,2002)and(ii)the contrasting abilities of N.caerulescens accessions to accu-mulate Cd and Zn independently in the shoot provides evidence of transport selectivity(Xing et al.,2008),both of which can be taken as evidence for protein-mediated symplasmic transport(White,2001).Barriers to apoplasmic movement of solutes to the xylemThe isolation of the stele from the peripheral cell layers of the root is critical for the control of solute transport to the shoot.It is effected by cell wall impregnations and the most important impregnating substance in this context is suberin (Franke and Schreiber,2007).Cadmium uptake by plant roots is generally restricted to young subapical regions of actively growing roots(Pin˜eros et al.,1998).The peripheral cell layers in these regions are specialized for solute uptake.The epidermal layer,which constitutes the outermost cell layer and differs from the epidermal layer of above-ground plant parts both in lacking cutin and through the development of root hairs,is termed the rhizodermis(von Guttenberg,1968).The rest of the peripheral tissues are termed the cortex.The endodermis separates the cortex from the stele and extracellular move-ment of solutes to the xylem is restricted by suberin deposi-ted in endodermal cell walls.Suberin,together with lignins, form the impregnation material of Casparian bands de-veloped in radial and transverse endodermal cell walls (Schreiber et al.,1999;White,2001).This impregnation,the lack of intercellular spaces between endodermal cells,and a tight junction between cell walls and the plasma mem-brane form the apoplasmic barrier of the root(White, 2001).The endodermis with its Casparian bands represents a considerable,but not impenetrable,barrier to solute movement through the apoplasm(Steudle et al.,1993; White,2001;Ranathunge et al.,2005).Casparian bands represent theﬁrst endodermal ontogenic stage(Stage I),which is only rarely(e.g.in some aquatic species;Seago,2002)theﬁnal stage.The deposition of lamellar suberin,in this case on the whole inner cell wall surface,is the commonly occurring second endodermal ontogenic stage(White,2001).Stage II endodermis presents a more complete apoplasmic barrier to the radialﬂow of water and solutes to the xylem in more mature parts of theroot(Melchior and Steudle,1993;Peterson et al.,1993; Steudle and Peterson,1998;White,2001).Nevertheless,the absolute amount of suberin deposited in root cell walls affects the radial transport of water and ions,as demon-strated by the enhanced suberin1(esb1)mutant of Arabidop-sis thaliana Heynh.(Baxter et al.,2009).This mutant has elevated amounts of suberin in the root,most likely in the endodermis,reduced waterﬂow to the xylem,and a decreasein shoot Ca,Mn,and Zn accumulation(Baxter et al.,2009). However,quantitative differences in root suberin concen-trations may not be the only factor inﬂuencing the apo-plasmic movement of water and solutes to the xylem,andboth the chemical nature of the suberins,together with the microstructure of deposits,must also be taken into consid-eration(Schreiber et al.,2005).Differences in suberin composition along the root axis during the development ofthe endodermis are indicated by an increase in fatty acidx-hydroxylation(Thomas et al.,2007;Ho¨fer et al.,2008). However,association of these changes with functional properties of suberized barriers in the endodermis remainsto be demonstrated.In some plant species,more distant from the root apex,ineven older parts of the root,the endodermis may pass to thethird stage(von Guttenberg,1968;White,2001).Stage III endodermis is characterized by thick cellulose secondarywalls(sometimes classiﬁed as tertiary walls)deposited overthe suberin lamellae.This cell wall layer,together with the original primary wall,is often ligniﬁed,and in some species impregnation of walls with silicon may occur(Sangster and Parry,1976;Lux et al.,1999).In Stage III,the function ofthe endodermis is already mostly mechanical and radial transport of water and solutes is limited(Melchior and Steudle,1993;White,2001).The gradual changes in cell wall composition of the endodermis can extend for a considerable distance along the root.Theﬁrst stage,characterized by Casparian bands,often starts very close to the root apex.The zone of gradual development of the second stage,the deposition of suberin lamellae,is usually very long.It may vary from several millimetres to several hundred millimetres from the root apex.Throughout this distance the number of endodermalcells without suberin lamellae decreases,and the cells not covered by suberin lamellae are called passage cells.Passagecells can be present even when the majority of endodermalcells have entered the third stage of development.Indeed, passage cells can remain in theﬁrst state permanently, although in the majority of the plant species this does not occur.Hypodermal layers,characterized by a gradual thickeningof cell walls,are often present in older roots,especially in monocotyledons.A suberized periderm is developed in olderroots of dicotyledons and gymnosperms.Periderm has been shown to act as a barrier preventing the movement of waterand ions(Vogt et al.,1983),gases(De Simone et al.,2003),and pathogen incursion(Lulai and Corsini,1998).From a functional viewpoint,these older regions of the root con-tribute little to water uptake(Melchior and Steudle,1993)Root responses to cadmium|25at South China Agricultural University on December 5, Downloaded fromand are mostly engaged in the long-distance transport of water and solutes.This function is concentrated in inner root tissues,within the vascular cylinder,or in the secondary vascular tissues.In the majority of angiosperms another apoplasmic barrier,the exodermis,can develop in parallel with the endodermis(Perumala et al.,1990;Peterson and Perumala, 1990;Hose et al.,2001;Ma and Peterson,2003).The exodermis develops in the same three stages as the endo-dermis.The exodermis can be uniseriate or multiseriate,in contrast to the uniseriate endodermis.The exodermis usually develops at a greater distance from the root apex than the endodermis(Ma and Peterson,2003).However, environmental conditions can modify the rate of develop-ment of the exodermis(Zimmermann and Steudle,1998) and accelerated development of the exodermis has been associated with reduced Cd uptake by roots(T Redjala and I Zelko,personal communication).In some plant species, and under speciﬁc environmental conditions,the exodermis may differentiate earlier than the endodermis.This has been observed in some wetland plants(Seago et al.,1999;Soukup et al.,2002),in tea(Homma et al.,2000;Tanimoto et al., 2004),and in maize(Zea mays L.)grown in soil(T Redjala and I Zelko,personal communication).The importance of the exodermis as an environmentally variable barrier to the uptake of water and ions was recognized by Peterson et al. (1993),and several subsequent studies have conﬁrmed this conclusion(Peterson,1997;Meyer et al.,2009).Under some conditions,such as in hydroponics,the exodermis can be absent,even in species in which it is normally present (Zimmerman and Steudle,1998).The rhizodermis,exodermis,and endodermis have all been shown to act as barriers to the apoplasmic movement of toxic elements,including Cd(Gierth et al.,1999;White, 2001;Enstone et al.,2003;Seregin et al.,2004;Seregin and Kozhevnikova,2008).The additional peri-endodermal layer of cells with ligniﬁed cell walls present in N.caerulescens may function similarly(Zelko et al.,2008).These apoplas-mic barriers develop closer to the root apex when roots are exposed to high concentrations of potentially toxic ele-ments.Accelerated development of both the endodermis and exodermis have been observed in various plant species in response to salinity(Reinhardt and Rost,1995;Karahara et al.,2004),and the multiple environmental stresses caused by cultivation in municipal solid waste slag with high salt and heavy metal content have been shown to induce extensive thickening of the inner tangential walls of maize endodermal cells(Degenhardt and Gimmler,2000).Exposure to Cd has been found to result in the formation of Casparian bands and suberin lamellae closer to the root apex in several plant species,including A.thaliana (Schreiber et al.,1999),Silene dioica(Martinka and Lux, 2004),woody shrub species such as Karwinskia humboldti-ana(Zelko and Lux,2004),and maize(Fig.2;Vaculı´k et al., 2009).Maturation of the endodermis closer to the root apex can be attributed partly to a reduction in the rate ofrootFig.2.The development of endodermal suberin lamellae in the roots of maize plants after10d growth in Hoagland’s solution containingno cadmium(Cd0)or5l M Cd(NO3)2.4H2O(Cd5).Three regions of the root can be distinguished:A,a region in which endodermalsuberin lamellae are fully developed(solid green lines),B,a region in which the suberin lamellae are partially developed(broken green lines),and C,a region lacking endodermal suberin lamellae.Because the length of roots grown in the absence and presence of Cddiffered,the distance from the root tip is expressed as percentage of the total root length.Endodermal suberin lamellae developed closerto the root apex in roots exposed to Cd,when expressed in either absolute or percentage terms.White arrows indicate suberin lamellaein the endodermis.26|Lux et al.at South China Agricultural University on December 5, Downloaded from。