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Nitrate reduces the detrimental effect of potassium chlorate on longan (Dimocarpus Longan Lour.) trees
Scientia Horticulturae 108 (2006) 151–156 www.elsevier.com/locate/scihorti
Nitrate reduces the detrimental effect of potassium chlorate on longan (Dimocarpus Longan Lour.) trees Xu-Ming Huang a,*, Jie-Mei Lu b, Hui-Cong Wang a, Cheng-Lin Zhang c, Liang Xie b, Rui-Tao Yang a, Jian-Guo Li a, Hui-Bai Huang a a
Physiological Laboratory for South China Fruits, College of Horticulture, South China Agricultural University, Guangzhou 510642, China b Guangzhou Agricultural Technology Extension Center, Guangzhou 510520, China c College of Resources and Environment, South China Agricultural University, Guangzhou 510642, China Received 17 November 2005; received in revised form 22 January 2006; accepted 25 January 2006
Abstract A recent finding that potassium chlorate induces longan (Dimocarpus Longan Lour.) flowering at any seasons has led to wide use of this chemical in longan industry for off-season harvest, despite the risk of injury to the tree. In this paper, we examined the influences of pretreatment of nitrate by foliar spray with 3% potassium nitrate on the responses of potted longan (Dimocarpus longan Lour.) cv. Shixia (a subtropical ecotype) trees to soil applications of potassium chlorate at 10 and 20 g plant 1. Chlorate treatments caused severe leaf drop and chlorophyll breakdown and suppressed budbreak, flowering and shoot growth. Pretreatment of potassium nitrate reduced the severity of leaf drop and chlorophyll breakdown caused by chlorate but the extend was less significant in high dosage chlorate (20 g plant 1) treatment than low dosage (10 g plant 1) chlorate treatment. Potassium nitrate did not alleviate the suppression effect of chlorate on budbreak, flowering and shoot growth, although it promoted these processes per se. Chlorate treatment resulted in only a transient accumulation of chlorate in the leaves, which peaked around 14 days after chlorate treatment (DACT), while chloride accumulated constantly within 28 DACT. Pretreatment of potassium nitrate tended to promote chlorate accumulation but did not cause a significant change in chloride accumulation. Severity of leaf drop was not significantly correlated to chlorate accumulation but significantly to chloride accumulation, suggesting that toxicity of chlorate to longan tree was realized during the chlorideproducing process of chlorate reduction. Nitrate influenced the correlations between severity of leaf drop and chloride accumulation by reducing the slopes of the regressed lines of leaf drop versus chloride, indicating that nitrate reduced the sensitivity of longan trees to the toxic intermediates produced during chlorate reduction. The results indicate that the application rates were excessive for potted trees. The potential benefit of potassium nitrate in a longan production system based on application of chlorate needs to be evaluated in field trees. # 2006 Elsevier B.V. All rights reserved. Keywords: Chlorate; Dimocarpus longan Lour.; Flowering; Nitrate; Leaf drop
1. Introduction Longan (Dimocarpus Longan Lour.) is an evergreen woody species, which is generally classified into two ecotypes based on cool temperature requirement for flowering: the tropical type and the subtropical type (Menzel et al., 2005). The tropical type has much lower chill requirement and may produce several crops in a year, while the subtropical type requires a period of exposure to cool temperatures to induce flowering. Most
* Corresponding author. Present address: Physiological Laboratory for South China Fruits, College of Horticulture, South China Agricultural University, Guangzhou 510642, China. Fax: +86 20 85282107. E-mail address: [email protected] (X.-M. Huang). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.01.015
of commercial longan cultivars are subtropical and flower in spring and crop in early autumn. Recently, Yen et al. (2001) accidentally found that potassium chlorate may completely replace the cool temperature requirement and induce off-season flowering in subtropical longan trees. The discovery led to the technique of inducing off-season longan production with potassium chlorate. This technique has been successfully applied in Thailand, where fresh longan is nowadays marketed year round (Subhadrabandhu and Yapwattanaphun, 2001). Soil drenching (8–20 g m 2 subcanopy area) is most effective method of potassium chlorate application (Manochai et al., 2005). The potential for higher profit of off-season longan brought about an upsurge of studies and trials of application of potassium chlorate in other longan producing regions. In China,
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such trails have yielded only limited success due to the problems such as asynchronous flowering and poor fruit set, which rarely occur in untreated trees that flower normally. The effective dosage so far reported in China is 5–10 times higher than that used in Thailand (Huang et al., 2004). Chlorate applications to longan may also produce detrimental effects such as severe leaf drop, shoot dry-out, panicles dieback and even tree death (Zhu and Peng, 2002; Li et al., 2003; Hua et al., 2004). The toxicity of chlorate to plants is closely associated with nitrate reduction system. Being analogous to nitrate, chlorate can be readily reduced by nitrate reductase into more toxic chlorite or hypochlorite before it is further reduced into nontoxic chloride (Solomonsson and Vennesland, 1972; Hofstra, 1977; Van Wuk and Hutchinson, 1995; Stauber, 1998; Borges et al., 2004). Toxicity of chlorate depends upon the rate of its uptake by plant. Chlorate and nitrate share the same uptake mechanism, through nitrate transporters (Solomonsson and Vennesland, 1972). Nonnitrate utilizing organisms or plant mutants defective in either nitrate transport or nitrate reduction are immune to chlorate toxicity (Singh et al., 1977; Doddema et al., 1978; Nelson et al., 1983; Wilkinson and Crawford, 1993). Chlorate and nitrate compete for nitrate transporters as well as nitrate reductase. The affinity of nitrate reductase and nitrate transporters to nitrate is by far higher than to chlorate (Hofstra, 1977; Balch, 1987). Nitrate reductase and a type of high-capacity and high-affinity nitrate carrier can be induced by nitrate (Glass and Siddiqi, 1995). Hence, nitrate may exert a strong effect on the response to chlorate. Longan is the only known plant to flower in response to chlorate applications (Manochai et al., 2005), and the responsible mechanism is unknown (Huang et al., 2004; Manochai et al., 2005). Commercial use of this chemical is still based on trials and errors in longan production and same chlorate treatment may produce quite different results depending upon cultivar, soil type, tree age and irrigation and fertilization practices (Huang et al., 2004; Manochai et al., 2005). Potted trees with more concentrated root systems are more sensitive to the chlorate applications than filed specimens (He, 2002; Lu, 2005). The present study examines the response of potted trees of longan cv. ‘Shixia’, a subtropical ecotype, to potassium chlorate and the possibility of reducing the risk of its detrimental effects by pretreating plants with nitrate.
Fig. 1. Changes of air temperature (black spot) and humidity (open spot) in the greenhouse during the experiment.
The growth medium was five parts (in volume) of loamy red soil mixed with one part of chicken manure compost. The latest flush of all the trees had fully matured when they were given potassium chlorate and/or potassium nitrate treatment. 2.2. Treatment The trees were divided into six groups each with five trees (n = 5): control with no application of potassium nitrate or potassium chlorate (1); soil application of 10 or 20 g potassium chlorate (in 2 l) per pot (2 and 3); 3% potassium nitrate spray (4); 3% potassium nitrate, plus of 10 or 20 g potassium chlorate (5 and 6). Potassium nitrate was sprayed 2 days before application of potassium chlorate. 2.3. Shoot growth, leaf drop, bud break and flowering bud Three shoots from each tree were tagged and their new growth measured with a ruler. Dropped leaflets from each tree were collected and counted every two days. Numbers of broken buds and flowering buds were counted respectively 106 and 136 days after chlorate treatment (DACT) and the percentages of bud break and flowering buds were calculated: bud break (%) = (number of broken buds/number of total buds in a tree) 100%; flowering bud (%) = (number of buds with panicle emergency/number of broken buds in a tree) 100%.
2. Materials and methods
2.4. Changes in chlorophyll contents
2.1. Materials
Five mature leaflets from latest mature flushes from each tree were selected and tagged, and relative chlorophyll content in these leaves was represented by chlorophyll index, which was measured nondestructively with a SPAD-502 chlorophyll meter on the tagged leaflets (n = 5).
The experiment was conducted in South China Agricultural University, Guangzhou, China, from November 2004 to March 2005 with thirty 3-year-old longan cv. Shixia (subtropical ecotype) trees with even canopy size (around 1 m in diameter) planted individually in 50-l pots in a sunlight greenhouse without temperature and humidity control or artificial illumination. Changes in temperature and humidity in the greenhouse during the experiment, which were recorded with an autonomous data-logger every 30 min, are shown in Fig. 1.
2.5. Contents of chlorate and chloride in leaves Six leaflets from the latest mature flushes were sampled from each tree at each time for measurements of chlorate and chloride contents with a Dionex-120 ion chromatographer
X.-M. Huang et al. / Scientia Horticulturae 108 (2006) 151–156
based on the method described by Shen et al. (2001). 0.5 g sample was grounded into homogenate in a mortar with 5 ml deionized water and centrifuged at 12,000 g for 20 min. One millilitre of the supernatant was forced through a MilliporeTM filter (0.2 mm), and 25 ml of this filtrate was injected into the ion chromatographer equipped with an Ionpac AS11-HC separation column (at 25 8C), an Ionpac AG11-HC protection column and an electricity conductivity detector. Nitrogen was used as the carrier gas at a pressure of 0.3 kPa, and mobile phase was 6 mmol/l NaOH with a flowing rate of 1.08 ml/s. Using solutions of known concentrations of potassium chlorate and potassium chloride as standards, we obtained perfect linear correlation between peak areas and chloride or chlorate concentrations (Y = 1.6283X, P = 0.998 for chloride and Y = 6.5865X, P = 0.999 for chlorate). The analysis was done with five replicates (n = 5). 2.6. Statistics Multiple comparisons of leaf drop, budbreak, shoot growth and chlorophyll content among treatments with LSD tests, oneway ANOVA (n = 5), and correlation analyses between chlorate or chloride concentration and severity of leaf drop were conducted with SPSS 10.0. 3. Results 3.1. Shoot growth
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Table 1 The effects of potassium chlorate and potassium nitrate treatments on budbreak and flowering of potted longan of cv. ‘Shixia’ trees Treatment
Percentage of shoots with broken bud(s) (%)
Percentage of shoots with flowers (%)
Nitrate Nitrate + 10 g chlorate Nitrate + 20 g chlorate Control 10 g chlorate 20 g chlorate
56.2 4.8 a 17.22 9.40 c 5.4 3.4 c 3 7.2 5.9 b 22.5 5.0b c 12.8 6.4 c
30.3 9.9 a 0.0 0.0 26.1 10.5 a 0.0 0.0
Different letters (a–c) behind the values indicate significant difference at p = 0.05 among treatments based on LSD test, one-way AN0VA (n = 5).
3.2. Effects on bud break and flowering Chlorate treatments, especially at high dosage (20 g plant 1), significantly suppressed bud break of longan (Table 1). Potassium nitrate spray stimulated bud break compared to the control. However, spray of nitrate did not alleviate the suppression effect of chlorate on bud break. Instead, it enhanced this effect to some degrees. Percentage of shoots with flowers was 30.3 and 26.1%, respectively, in nitrate-treated trees and the control trees. Chlorate treatments strongly inhibited flowering in potted longan, and none of the treated trees with or without pretreatment of nitrate produced any flowers.
There was almost no new shoot growth in all trees within 74 DACT (Fig. 2). Foliar spray of nitrate stimulated growth of new flush. At 99 DACT, shoot growth was most prominent in the trees treated with just nitrate. After 123 DACT, shoot growth was not significantly different between the control trees and trees treated with only potassium nitrate, whereas potassium chlorate (with or without pretreatment of potassium nitrate) suppressed shoot growth, especially at 20 g plant 1.
3.3. Leaf drop
Fig. 2. The effect of potassium chlorate and/or potassium nitrate applications on shoot growth of potted ‘Shixia’ longan trees. Different letters above the columns indicate significant differences at p = 0.05 among treatments within the same day based on the LSD test, one-way ANOVA (n = 5). Vertical bars are standard errors.
Fig. 3. Cumulative changes in leaf drop from individual trees treated with potassium chlorate and/or potassium nitrate. Different letters behind the curves indicate significant difference at p = 0.05 among treatments at 49 days after chlorate treatment based on LSD test, one-way ANOVA (n = 5). Vertical bars are standard errors.
Potassium chlorate at 20 or 10 g plant 1 caused abrupt leaf drop, which continued until about 28 DACT when leaf drop tended to cease (Fig. 3). There was no significant difference in leaf drop among the two dosages. Potassium nitrate counteracted this response. Nitrate alone had no effect on leaf drop.
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Fig. 4. Effect of treatment with potassium chlorate and/or potassium nitrate on leaf chlorophyll content. Different letters behind the curves indicate significant difference at p = 0.05 among treatments at 56 days after chlorate treatment based on LSD test, one-way AN0VA (n = 5). Vertical bars are standard errors.
3.4. Chlorophyll While causing severe leaf drop, potassium chlorate treatments also resulted in significant decrease of chlorophyll content in the leaves, indicating that the treatment caused breakdown of the pigment (Fig. 4). There was no significant difference among the two application rates. Pretreatment of nitrate significantly reduced the decline of chlorophyll content caused by chlorate treatment in the trees applied at the lower rate (10 g plant 1) of chlorate. Such effect was not significant in trees treated with chlorate at a higher rate (20 g plant 1). 3.5. Changes in chlorate and chloride contents in leaves Application of potassium chlorate induced a short-term increase in chlorate concentration in the leaves, which peaked around 14 DACT and declined rapidly thereafter (Fig. 5A). Such changes were not seen in trees without chlorate application, where concentration of chlorate remained constantly low. Although not significant, pretreatment of potassium nitrate tended to accumulate more chlorate within 14 DACT, resulting in higher peaks as compared with applying chlorate alone at corresponding dosages (result of multiple comparisons not shown in the figure). Concentration of chloride increased within 28 DACT in all chlorate treatments followed by a gradual decline at different scales, while trees received no chlorate treatment had a constant low chloride concentration (Fig. 5B). Although not significant, higher dosage of chlorate tended to result in accumulation of more chloride (result of multiple comparisons not shown in the figure). Pretreatment of nitrate made no significant difference in the changes of chloride concentration. 3.6. Correlations among chlorate/chloride accumulation and detrimental effect (leaf drop) There was no significant correlation among leaf chlorate concentrations and leaf drop (r2 = 0.2835, P = 0.075). How-
Fig. 5. Changes in leaf chlorate and chloride concentrations of potted ‘Shixia’ longan trees after treatment with potassium chlorate and/or potassium nitrate (n = 5).
ever, chloride accumulated in the leaves was positively and significantly correlated to the severity of leaf drop (Y = 73.2X + 42.05, r2 = 0.68, P = 0.001**) (Fig. 6). Pretreatment of potassium nitrate reduced the slope of leaf drop versus chloride correlation (Y = 38.7X 36.2; r2 = 0.615, P = 0.012*, Fig. 6),
Fig. 6. Effects of pretreatment with potassium nitrate on the correlation between the leaf drop severity and the accumulation of chloride in the leaves as a result of chlorate application. Potassium nitrate solution (3%, w/v) was foliarly applied two days before potassium chlorate treatment. Chloride accumulation was calculated as the difference between the chloride concentration in leaves of chlorate treated trees and those of trees without chlorate treatment at 14 DACT when the peak chlorate accumulation occurred in chlorate treated trees (see Fig. 5). In this case, accumulation of chloride was arbitrarily regarded as zero in trees without chlorate treatment.
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indicating less impact at a given amount of chloride accumulation caused by chlorate treatment in trees pretreated with nitrate. 4. Discussions Longan is the only known plant in which flower may be induced by chlorate (Manochai et al., 2005). Since 2000, potassium chlorate has been widely used in the longan industry for off-season production in many countries (Huang et al., 2004), despite the risk of the detrimental effects of the chemical. Potassium chlorate at 10 or 20 g plant 1 failed to induce flowering in potted longan cv. ‘Shixia’, and instead inhibited flowering and budbreak and caused severe leaf drop and breakdown of chlorophyll. Similar detrimental effects of chlorate have been found in other plants (Van Wuk and Hutchinson, 1995; Mackown et al., 1996; Stauber, 1998; Borges et al., 2004). Application of potassium chlorate to longan trees caused only a transient accumulation of chlorate, which was rapidly converted into chloride (Fig. 5). Two evidences from our experiment suggest that the detrimental effects of potassium chlorate was not directly induced by chlorate per se: (1) the severity of leaf drop was not significantly correlated to chlorate accumulation (r2 = 0.2835, P = 0.075); (2) there was more chlorate accumulated but less leaf drop in trees pretreated with potassium nitrate. Leaf drop was instead significantly correlated to chloride accumulation (r2 = 0.6849, P = 0.001**) (Fig. 6). However, chloride is not toxic if not excessively accumulated (Kadman, 1963). A possible explanation is that the toxic effect of chlorate occurred during the process of its metabolism, i.e., stepwise reduction that finally converted chlorate into chloride. In other words, the actions of the intermediate products during chlorate reduction caused the detrimental effect on longan. Studies in other organisms have shown that, as an analog of nitrate, chlorate can be readily reduced by nitrate reductase into toxic chlorite or hypochlorite that embodies the toxicity of chlorate before it is further reduced into nontoxic chloride (Solomonsson and Vennesland, 1972; Hofstra, 1977; Van Wuk and Hutchinson, 1995; Stauber, 1998; Borges et al., 2004). Hence, our findings suggest that longan trees share the same toxicological responses to chlorate as other plants. Since the toxicity of chlorate depends upon the nitrate reduction system in plants, nitrate exerts a strong influence on the effect of chlorate (Hofstra, 1977; Balch, 1987). In this study, foliar spray of potassium nitrate overcame some of leaf drop and chlorophyll breakdown caused by chlorate. However, potassium nitrate treatment could not alleviate the inhibiting effect of chlorate on budbreak, flowering and shoot growth, despite that nitrate treatment per se promoted budreak and shoot growth. Therefore, the suppression of budbreak, flowering and shoot growth by chlorate must be due to a different mechanism. The effect of potassium nitrate in reducing leaf drop is considered a result of competition between nitrate and chlorate for nitrate reductase, and the affinity of nitrate reductase to nitrate is by far higher than to chlorate (Hofstra, 1977). Theoretically, application of nitrate suppresses the reduction of
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chlorate and reduces the production of chlorite, hypochlorite or chloride. However, in our study, pretreatment of nitrate resulted in a more prominent accumulation followed by a faster drop of chlorate in longan leaves, but the treatment did not influence the accumulation of chloride (Fig. 5), suggesting that conversion of chlorate to chloride was not suppressed by nitrate. A possible explanation is that nitrate application increased the activity of nitrate reductase, which in turn increased the rate of chlorate reduction and counter balanced the competative effect of nitrate against chlorate. Such a balance may be broken by changes in the dosages of chlorate and nitrate. Hence pretreatment of potassium nitrate overcome the leaf drop induced by chlorate at the low dose but not at the high dose. Such situation has been observed in algae, where toxicity of chlorate is dependent upon the ratio of nitrate to chlorate (Stauber, 1998; Solomonsson and Vennesland, 1972). Since nitrate did not suppress the chloride-producing chlorate reduction process, the effect of nitrate in reducing chlorate damage seems unlikely a simple result of blockage of chlorate reduction. Nitrate reduced the slope of the leaf drop versus chloride relationship, which indicates that application of nitrate reduced the sensitivity of longan trees to the toxic intermediates produced during the conversion of chlorate into chloride. We assume that nitrate and chlorate are reduced ‘‘side by side’’ by nitrate reduction system in the longan leaves, and the intermediates of nitrate reduction such as nitrite might block the action of the toxic intermediates of chlorate reduction, i.e., chlorite or hypochlorite before they are further reduced into nontoxic chloride. This assumption awaits further evidences. In conclusion, the chlorate used in our experiment were excessive for the potted longan trees and inhibited flowering while causing damages such as leaf drop and chlorophyll breakdown. Nitrate reduced the detrimental effects of chlorate on potted longan trees and the potential benefit of nitrate in a longan production system based on application of chlorate needs to be evaluated in field trees. Acknowledgments The study has been supported by Research Fund for Returned Scholars and Guangdong Natural Science Foundation. References Balch, W.M., 1987. Studies of nitrate transport by marine phytoplankton using 36 Cl–ClO3 as a transport analogue. I: physiological findings. J. Physiol. 23, 107–118. Borges, R., Miguel, E.C., Dias, J.M.R., da Cunha, M., Bressan-Smith, R.E., de Oliveira, J.G., de Souza Filho, G.A., 2004. Ultrastructural, physiological and biochemical analyses of chlorate toxicity on rice seedlings. Plant Sci. 166, 1057–1062. Doddema, H., Hofstra, J.J., Feenstra, W.J., 1978. Uptake of nitrate by mutants of Arabidopsis thaliana, disturbed in uptake or reduction of nitrate. I: effect of nitrogen source during growth on uptake of nitrate and chlorate. Physiol. Plant. 443, 343–350. Glass, A.D.M., Siddiqi, Y., 1995. Nitrogen absorption by plant roots. In: Srivastava, H.S., Singh, R.P. (Eds.), Nitrogen Nutrition in Higher Plants. Associated, New Delhi, pp. 21–56.
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He, H. Y. 2002. A physiological study on the flower inducing effect of potassium chlorate on longan. MS Thesis, South China Agricultural University, Guangzhou. Hofstra, J.J., 1977. Chlorate toxicity and nitrate reduction activity in tomato plants. Physiol. Plant. 41, 65–69. Hua, M., He, F., Wang, X.H., Chen, Y.G., Han, J., Li, X.H., 2004. Comparison of the flowering responses to potassium chlorate in ‘Chuliang’ and ‘Shixia’ longans. China South. Fruits 33, 35–42 (in Chinese). Huang, X.M., Lu, J.M., Wang, H.C., 2004. Current situation in research and application of chlorate in longan production. China South. Fruits 33, 29–30 (in Chinese). Kadman, A., 1963. The uptake and accumulation of chloride in avocado leaves and the tolerance of avocado seedlings under saline conditions. Proceedings of the American Society for Horticultural Science 83, 280–286. Li, J.G., Pan, X.W., Li, R., Dai, H.K., 2003. Advances in research of inducing longan flowering by potassium chlorate application. J. Fruit Sci. 20, 410– 414 (in Chinese with an English abstract). Lu, J. M, 2005. A physiological study on the biological effects of chlorate on longan. MS Thesis, South China Agricultural University, Guangzhou. Mackown, C.T., Vansanford, D.A., Rothwell, C.G., 1996. Nitrate uptake and assimilation and chlorate tolerance of wheat. Crop Sci. 36, 313–319. Manochai, P., Sruamsiri, P., Wiriyaalongkorn, W., Naphrom, D., Hegele, M., Bangerth, K., 2005. Year around off season flower induction in longan (Dimocarpus longan, Lour.) trees by KClO3 applications: potentials and problems. Sci. Horticult. 104, 379–390. Menzel, C.M., Huang, X.M., Liu, C.M., 2005. Cultivars and plant improvement. In: Menzel, C.M., Waite, G.K. (Eds.), Litchi and Longan, Botany, Production and Uses. CABI Publishing, Oxfordshire, UK, pp. 59–86.
Nelson, R.S., Ryan, S.A., Harper, J.E., 1983. Soybean mutants lacking constitutive nitrate reductase activity. II Nitrogen assimilation, chlorate resistance and inheritance. Plant Physiol. 61, 384–390. Shen, H., Yan, X.L., Zheng, S.L., Wang, X.R., 2001. Simultaneous analysis of organic acid and inorganicanions in common bean by ion chlormatography. J. South China Agric. Univ. 22, 8–10 (in Chinese with an English abstract). Singh, H.N., Sonie, K.C., Singh, H.R., 1977. Nitrate regulation of heterocyst differentiation and nitrogen fixation in a chlorate-resistant mutant of the blue-green alga Nostoc muscorum. Mutat. Res. 42, 447–452. Solomonsson, L.P., Vennesland, B., 1972. Nitrate reductase and chlorate toxicity in Chlorella vulgaris. Plant Physiol. 50, 421–424. Stauber, I.L., 1998. Toxicity of chlorate to marine microalgae. Aquat. Toxicol. 41, 213–227. Subhadrabandhu, S., Yapwattanaphun, C., 2001. Regulation off-season flowering of longan in Thailand. Acta Horticult. 558, 193–198. Van Wuk, D.J., Hutchinson, T.H., 1995. The ecotoxicity of chlorate to aquatic organisms: a critical review. Ecotoxicol. Environ. Safety 32, 244–253. Wilkinson, J.Q., Crawford, N.M., 1993. Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both NIA1 and NIA2 nitrate reductase structural genes. Mol. Gen. Genet. 239, 289–297. Yen, C.R., Chau, C.N., Chang, J.W., Tzeng, J.C., 2001. Effects of chemicals on flowering in Longan. J. Chin. Soc. Horticult. Sci. 47, 195–200 (in Chinese with an English abstract). Zhu, J.H., Peng, H.X., 2002. Technique of inducing longan flowering with chemicals. Guangxi Trop. Agricult. 3, 12–14 (in Chinese).
Nitrate reduces the detrimental effect of potassium chlorate on longan (Dimocarpus Longan Lour.) trees Xu-Ming Huang a,*, Jie-Mei Lu b, Hui-Cong Wang a, Cheng-Lin Zhang c, Liang Xie b, Rui-Tao Yang a, Jian-Guo Li a, Hui-Bai Huang a a
Physiological Laboratory for South China Fruits, College of Horticulture, South China Agricultural University, Guangzhou 510642, China b Guangzhou Agricultural Technology Extension Center, Guangzhou 510520, China c College of Resources and Environment, South China Agricultural University, Guangzhou 510642, China Received 17 November 2005; received in revised form 22 January 2006; accepted 25 January 2006
Abstract A recent finding that potassium chlorate induces longan (Dimocarpus Longan Lour.) flowering at any seasons has led to wide use of this chemical in longan industry for off-season harvest, despite the risk of injury to the tree. In this paper, we examined the influences of pretreatment of nitrate by foliar spray with 3% potassium nitrate on the responses of potted longan (Dimocarpus longan Lour.) cv. Shixia (a subtropical ecotype) trees to soil applications of potassium chlorate at 10 and 20 g plant 1. Chlorate treatments caused severe leaf drop and chlorophyll breakdown and suppressed budbreak, flowering and shoot growth. Pretreatment of potassium nitrate reduced the severity of leaf drop and chlorophyll breakdown caused by chlorate but the extend was less significant in high dosage chlorate (20 g plant 1) treatment than low dosage (10 g plant 1) chlorate treatment. Potassium nitrate did not alleviate the suppression effect of chlorate on budbreak, flowering and shoot growth, although it promoted these processes per se. Chlorate treatment resulted in only a transient accumulation of chlorate in the leaves, which peaked around 14 days after chlorate treatment (DACT), while chloride accumulated constantly within 28 DACT. Pretreatment of potassium nitrate tended to promote chlorate accumulation but did not cause a significant change in chloride accumulation. Severity of leaf drop was not significantly correlated to chlorate accumulation but significantly to chloride accumulation, suggesting that toxicity of chlorate to longan tree was realized during the chlorideproducing process of chlorate reduction. Nitrate influenced the correlations between severity of leaf drop and chloride accumulation by reducing the slopes of the regressed lines of leaf drop versus chloride, indicating that nitrate reduced the sensitivity of longan trees to the toxic intermediates produced during chlorate reduction. The results indicate that the application rates were excessive for potted trees. The potential benefit of potassium nitrate in a longan production system based on application of chlorate needs to be evaluated in field trees. # 2006 Elsevier B.V. All rights reserved. Keywords: Chlorate; Dimocarpus longan Lour.; Flowering; Nitrate; Leaf drop
1. Introduction Longan (Dimocarpus Longan Lour.) is an evergreen woody species, which is generally classified into two ecotypes based on cool temperature requirement for flowering: the tropical type and the subtropical type (Menzel et al., 2005). The tropical type has much lower chill requirement and may produce several crops in a year, while the subtropical type requires a period of exposure to cool temperatures to induce flowering. Most
* Corresponding author. Present address: Physiological Laboratory for South China Fruits, College of Horticulture, South China Agricultural University, Guangzhou 510642, China. Fax: +86 20 85282107. E-mail address: [email protected] (X.-M. Huang). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.01.015
of commercial longan cultivars are subtropical and flower in spring and crop in early autumn. Recently, Yen et al. (2001) accidentally found that potassium chlorate may completely replace the cool temperature requirement and induce off-season flowering in subtropical longan trees. The discovery led to the technique of inducing off-season longan production with potassium chlorate. This technique has been successfully applied in Thailand, where fresh longan is nowadays marketed year round (Subhadrabandhu and Yapwattanaphun, 2001). Soil drenching (8–20 g m 2 subcanopy area) is most effective method of potassium chlorate application (Manochai et al., 2005). The potential for higher profit of off-season longan brought about an upsurge of studies and trials of application of potassium chlorate in other longan producing regions. In China,
152
X.-M. Huang et al. / Scientia Horticulturae 108 (2006) 151–156
such trails have yielded only limited success due to the problems such as asynchronous flowering and poor fruit set, which rarely occur in untreated trees that flower normally. The effective dosage so far reported in China is 5–10 times higher than that used in Thailand (Huang et al., 2004). Chlorate applications to longan may also produce detrimental effects such as severe leaf drop, shoot dry-out, panicles dieback and even tree death (Zhu and Peng, 2002; Li et al., 2003; Hua et al., 2004). The toxicity of chlorate to plants is closely associated with nitrate reduction system. Being analogous to nitrate, chlorate can be readily reduced by nitrate reductase into more toxic chlorite or hypochlorite before it is further reduced into nontoxic chloride (Solomonsson and Vennesland, 1972; Hofstra, 1977; Van Wuk and Hutchinson, 1995; Stauber, 1998; Borges et al., 2004). Toxicity of chlorate depends upon the rate of its uptake by plant. Chlorate and nitrate share the same uptake mechanism, through nitrate transporters (Solomonsson and Vennesland, 1972). Nonnitrate utilizing organisms or plant mutants defective in either nitrate transport or nitrate reduction are immune to chlorate toxicity (Singh et al., 1977; Doddema et al., 1978; Nelson et al., 1983; Wilkinson and Crawford, 1993). Chlorate and nitrate compete for nitrate transporters as well as nitrate reductase. The affinity of nitrate reductase and nitrate transporters to nitrate is by far higher than to chlorate (Hofstra, 1977; Balch, 1987). Nitrate reductase and a type of high-capacity and high-affinity nitrate carrier can be induced by nitrate (Glass and Siddiqi, 1995). Hence, nitrate may exert a strong effect on the response to chlorate. Longan is the only known plant to flower in response to chlorate applications (Manochai et al., 2005), and the responsible mechanism is unknown (Huang et al., 2004; Manochai et al., 2005). Commercial use of this chemical is still based on trials and errors in longan production and same chlorate treatment may produce quite different results depending upon cultivar, soil type, tree age and irrigation and fertilization practices (Huang et al., 2004; Manochai et al., 2005). Potted trees with more concentrated root systems are more sensitive to the chlorate applications than filed specimens (He, 2002; Lu, 2005). The present study examines the response of potted trees of longan cv. ‘Shixia’, a subtropical ecotype, to potassium chlorate and the possibility of reducing the risk of its detrimental effects by pretreating plants with nitrate.
Fig. 1. Changes of air temperature (black spot) and humidity (open spot) in the greenhouse during the experiment.
The growth medium was five parts (in volume) of loamy red soil mixed with one part of chicken manure compost. The latest flush of all the trees had fully matured when they were given potassium chlorate and/or potassium nitrate treatment. 2.2. Treatment The trees were divided into six groups each with five trees (n = 5): control with no application of potassium nitrate or potassium chlorate (1); soil application of 10 or 20 g potassium chlorate (in 2 l) per pot (2 and 3); 3% potassium nitrate spray (4); 3% potassium nitrate, plus of 10 or 20 g potassium chlorate (5 and 6). Potassium nitrate was sprayed 2 days before application of potassium chlorate. 2.3. Shoot growth, leaf drop, bud break and flowering bud Three shoots from each tree were tagged and their new growth measured with a ruler. Dropped leaflets from each tree were collected and counted every two days. Numbers of broken buds and flowering buds were counted respectively 106 and 136 days after chlorate treatment (DACT) and the percentages of bud break and flowering buds were calculated: bud break (%) = (number of broken buds/number of total buds in a tree) 100%; flowering bud (%) = (number of buds with panicle emergency/number of broken buds in a tree) 100%.
2. Materials and methods
2.4. Changes in chlorophyll contents
2.1. Materials
Five mature leaflets from latest mature flushes from each tree were selected and tagged, and relative chlorophyll content in these leaves was represented by chlorophyll index, which was measured nondestructively with a SPAD-502 chlorophyll meter on the tagged leaflets (n = 5).
The experiment was conducted in South China Agricultural University, Guangzhou, China, from November 2004 to March 2005 with thirty 3-year-old longan cv. Shixia (subtropical ecotype) trees with even canopy size (around 1 m in diameter) planted individually in 50-l pots in a sunlight greenhouse without temperature and humidity control or artificial illumination. Changes in temperature and humidity in the greenhouse during the experiment, which were recorded with an autonomous data-logger every 30 min, are shown in Fig. 1.
2.5. Contents of chlorate and chloride in leaves Six leaflets from the latest mature flushes were sampled from each tree at each time for measurements of chlorate and chloride contents with a Dionex-120 ion chromatographer
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based on the method described by Shen et al. (2001). 0.5 g sample was grounded into homogenate in a mortar with 5 ml deionized water and centrifuged at 12,000 g for 20 min. One millilitre of the supernatant was forced through a MilliporeTM filter (0.2 mm), and 25 ml of this filtrate was injected into the ion chromatographer equipped with an Ionpac AS11-HC separation column (at 25 8C), an Ionpac AG11-HC protection column and an electricity conductivity detector. Nitrogen was used as the carrier gas at a pressure of 0.3 kPa, and mobile phase was 6 mmol/l NaOH with a flowing rate of 1.08 ml/s. Using solutions of known concentrations of potassium chlorate and potassium chloride as standards, we obtained perfect linear correlation between peak areas and chloride or chlorate concentrations (Y = 1.6283X, P = 0.998 for chloride and Y = 6.5865X, P = 0.999 for chlorate). The analysis was done with five replicates (n = 5). 2.6. Statistics Multiple comparisons of leaf drop, budbreak, shoot growth and chlorophyll content among treatments with LSD tests, oneway ANOVA (n = 5), and correlation analyses between chlorate or chloride concentration and severity of leaf drop were conducted with SPSS 10.0. 3. Results 3.1. Shoot growth
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Table 1 The effects of potassium chlorate and potassium nitrate treatments on budbreak and flowering of potted longan of cv. ‘Shixia’ trees Treatment
Percentage of shoots with broken bud(s) (%)
Percentage of shoots with flowers (%)
Nitrate Nitrate + 10 g chlorate Nitrate + 20 g chlorate Control 10 g chlorate 20 g chlorate
56.2 4.8 a 17.22 9.40 c 5.4 3.4 c 3 7.2 5.9 b 22.5 5.0b c 12.8 6.4 c
30.3 9.9 a 0.0 0.0 26.1 10.5 a 0.0 0.0
Different letters (a–c) behind the values indicate significant difference at p = 0.05 among treatments based on LSD test, one-way AN0VA (n = 5).
3.2. Effects on bud break and flowering Chlorate treatments, especially at high dosage (20 g plant 1), significantly suppressed bud break of longan (Table 1). Potassium nitrate spray stimulated bud break compared to the control. However, spray of nitrate did not alleviate the suppression effect of chlorate on bud break. Instead, it enhanced this effect to some degrees. Percentage of shoots with flowers was 30.3 and 26.1%, respectively, in nitrate-treated trees and the control trees. Chlorate treatments strongly inhibited flowering in potted longan, and none of the treated trees with or without pretreatment of nitrate produced any flowers.
There was almost no new shoot growth in all trees within 74 DACT (Fig. 2). Foliar spray of nitrate stimulated growth of new flush. At 99 DACT, shoot growth was most prominent in the trees treated with just nitrate. After 123 DACT, shoot growth was not significantly different between the control trees and trees treated with only potassium nitrate, whereas potassium chlorate (with or without pretreatment of potassium nitrate) suppressed shoot growth, especially at 20 g plant 1.
3.3. Leaf drop
Fig. 2. The effect of potassium chlorate and/or potassium nitrate applications on shoot growth of potted ‘Shixia’ longan trees. Different letters above the columns indicate significant differences at p = 0.05 among treatments within the same day based on the LSD test, one-way ANOVA (n = 5). Vertical bars are standard errors.
Fig. 3. Cumulative changes in leaf drop from individual trees treated with potassium chlorate and/or potassium nitrate. Different letters behind the curves indicate significant difference at p = 0.05 among treatments at 49 days after chlorate treatment based on LSD test, one-way ANOVA (n = 5). Vertical bars are standard errors.
Potassium chlorate at 20 or 10 g plant 1 caused abrupt leaf drop, which continued until about 28 DACT when leaf drop tended to cease (Fig. 3). There was no significant difference in leaf drop among the two dosages. Potassium nitrate counteracted this response. Nitrate alone had no effect on leaf drop.
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Fig. 4. Effect of treatment with potassium chlorate and/or potassium nitrate on leaf chlorophyll content. Different letters behind the curves indicate significant difference at p = 0.05 among treatments at 56 days after chlorate treatment based on LSD test, one-way AN0VA (n = 5). Vertical bars are standard errors.
3.4. Chlorophyll While causing severe leaf drop, potassium chlorate treatments also resulted in significant decrease of chlorophyll content in the leaves, indicating that the treatment caused breakdown of the pigment (Fig. 4). There was no significant difference among the two application rates. Pretreatment of nitrate significantly reduced the decline of chlorophyll content caused by chlorate treatment in the trees applied at the lower rate (10 g plant 1) of chlorate. Such effect was not significant in trees treated with chlorate at a higher rate (20 g plant 1). 3.5. Changes in chlorate and chloride contents in leaves Application of potassium chlorate induced a short-term increase in chlorate concentration in the leaves, which peaked around 14 DACT and declined rapidly thereafter (Fig. 5A). Such changes were not seen in trees without chlorate application, where concentration of chlorate remained constantly low. Although not significant, pretreatment of potassium nitrate tended to accumulate more chlorate within 14 DACT, resulting in higher peaks as compared with applying chlorate alone at corresponding dosages (result of multiple comparisons not shown in the figure). Concentration of chloride increased within 28 DACT in all chlorate treatments followed by a gradual decline at different scales, while trees received no chlorate treatment had a constant low chloride concentration (Fig. 5B). Although not significant, higher dosage of chlorate tended to result in accumulation of more chloride (result of multiple comparisons not shown in the figure). Pretreatment of nitrate made no significant difference in the changes of chloride concentration. 3.6. Correlations among chlorate/chloride accumulation and detrimental effect (leaf drop) There was no significant correlation among leaf chlorate concentrations and leaf drop (r2 = 0.2835, P = 0.075). How-
Fig. 5. Changes in leaf chlorate and chloride concentrations of potted ‘Shixia’ longan trees after treatment with potassium chlorate and/or potassium nitrate (n = 5).
ever, chloride accumulated in the leaves was positively and significantly correlated to the severity of leaf drop (Y = 73.2X + 42.05, r2 = 0.68, P = 0.001**) (Fig. 6). Pretreatment of potassium nitrate reduced the slope of leaf drop versus chloride correlation (Y = 38.7X 36.2; r2 = 0.615, P = 0.012*, Fig. 6),
Fig. 6. Effects of pretreatment with potassium nitrate on the correlation between the leaf drop severity and the accumulation of chloride in the leaves as a result of chlorate application. Potassium nitrate solution (3%, w/v) was foliarly applied two days before potassium chlorate treatment. Chloride accumulation was calculated as the difference between the chloride concentration in leaves of chlorate treated trees and those of trees without chlorate treatment at 14 DACT when the peak chlorate accumulation occurred in chlorate treated trees (see Fig. 5). In this case, accumulation of chloride was arbitrarily regarded as zero in trees without chlorate treatment.
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indicating less impact at a given amount of chloride accumulation caused by chlorate treatment in trees pretreated with nitrate. 4. Discussions Longan is the only known plant in which flower may be induced by chlorate (Manochai et al., 2005). Since 2000, potassium chlorate has been widely used in the longan industry for off-season production in many countries (Huang et al., 2004), despite the risk of the detrimental effects of the chemical. Potassium chlorate at 10 or 20 g plant 1 failed to induce flowering in potted longan cv. ‘Shixia’, and instead inhibited flowering and budbreak and caused severe leaf drop and breakdown of chlorophyll. Similar detrimental effects of chlorate have been found in other plants (Van Wuk and Hutchinson, 1995; Mackown et al., 1996; Stauber, 1998; Borges et al., 2004). Application of potassium chlorate to longan trees caused only a transient accumulation of chlorate, which was rapidly converted into chloride (Fig. 5). Two evidences from our experiment suggest that the detrimental effects of potassium chlorate was not directly induced by chlorate per se: (1) the severity of leaf drop was not significantly correlated to chlorate accumulation (r2 = 0.2835, P = 0.075); (2) there was more chlorate accumulated but less leaf drop in trees pretreated with potassium nitrate. Leaf drop was instead significantly correlated to chloride accumulation (r2 = 0.6849, P = 0.001**) (Fig. 6). However, chloride is not toxic if not excessively accumulated (Kadman, 1963). A possible explanation is that the toxic effect of chlorate occurred during the process of its metabolism, i.e., stepwise reduction that finally converted chlorate into chloride. In other words, the actions of the intermediate products during chlorate reduction caused the detrimental effect on longan. Studies in other organisms have shown that, as an analog of nitrate, chlorate can be readily reduced by nitrate reductase into toxic chlorite or hypochlorite that embodies the toxicity of chlorate before it is further reduced into nontoxic chloride (Solomonsson and Vennesland, 1972; Hofstra, 1977; Van Wuk and Hutchinson, 1995; Stauber, 1998; Borges et al., 2004). Hence, our findings suggest that longan trees share the same toxicological responses to chlorate as other plants. Since the toxicity of chlorate depends upon the nitrate reduction system in plants, nitrate exerts a strong influence on the effect of chlorate (Hofstra, 1977; Balch, 1987). In this study, foliar spray of potassium nitrate overcame some of leaf drop and chlorophyll breakdown caused by chlorate. However, potassium nitrate treatment could not alleviate the inhibiting effect of chlorate on budbreak, flowering and shoot growth, despite that nitrate treatment per se promoted budreak and shoot growth. Therefore, the suppression of budbreak, flowering and shoot growth by chlorate must be due to a different mechanism. The effect of potassium nitrate in reducing leaf drop is considered a result of competition between nitrate and chlorate for nitrate reductase, and the affinity of nitrate reductase to nitrate is by far higher than to chlorate (Hofstra, 1977). Theoretically, application of nitrate suppresses the reduction of
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chlorate and reduces the production of chlorite, hypochlorite or chloride. However, in our study, pretreatment of nitrate resulted in a more prominent accumulation followed by a faster drop of chlorate in longan leaves, but the treatment did not influence the accumulation of chloride (Fig. 5), suggesting that conversion of chlorate to chloride was not suppressed by nitrate. A possible explanation is that nitrate application increased the activity of nitrate reductase, which in turn increased the rate of chlorate reduction and counter balanced the competative effect of nitrate against chlorate. Such a balance may be broken by changes in the dosages of chlorate and nitrate. Hence pretreatment of potassium nitrate overcome the leaf drop induced by chlorate at the low dose but not at the high dose. Such situation has been observed in algae, where toxicity of chlorate is dependent upon the ratio of nitrate to chlorate (Stauber, 1998; Solomonsson and Vennesland, 1972). Since nitrate did not suppress the chloride-producing chlorate reduction process, the effect of nitrate in reducing chlorate damage seems unlikely a simple result of blockage of chlorate reduction. Nitrate reduced the slope of the leaf drop versus chloride relationship, which indicates that application of nitrate reduced the sensitivity of longan trees to the toxic intermediates produced during the conversion of chlorate into chloride. We assume that nitrate and chlorate are reduced ‘‘side by side’’ by nitrate reduction system in the longan leaves, and the intermediates of nitrate reduction such as nitrite might block the action of the toxic intermediates of chlorate reduction, i.e., chlorite or hypochlorite before they are further reduced into nontoxic chloride. This assumption awaits further evidences. In conclusion, the chlorate used in our experiment were excessive for the potted longan trees and inhibited flowering while causing damages such as leaf drop and chlorophyll breakdown. Nitrate reduced the detrimental effects of chlorate on potted longan trees and the potential benefit of nitrate in a longan production system based on application of chlorate needs to be evaluated in field trees. Acknowledgments The study has been supported by Research Fund for Returned Scholars and Guangdong Natural Science Foundation. References Balch, W.M., 1987. Studies of nitrate transport by marine phytoplankton using 36 Cl–ClO3 as a transport analogue. I: physiological findings. J. Physiol. 23, 107–118. Borges, R., Miguel, E.C., Dias, J.M.R., da Cunha, M., Bressan-Smith, R.E., de Oliveira, J.G., de Souza Filho, G.A., 2004. Ultrastructural, physiological and biochemical analyses of chlorate toxicity on rice seedlings. Plant Sci. 166, 1057–1062. Doddema, H., Hofstra, J.J., Feenstra, W.J., 1978. Uptake of nitrate by mutants of Arabidopsis thaliana, disturbed in uptake or reduction of nitrate. I: effect of nitrogen source during growth on uptake of nitrate and chlorate. Physiol. Plant. 443, 343–350. Glass, A.D.M., Siddiqi, Y., 1995. Nitrogen absorption by plant roots. In: Srivastava, H.S., Singh, R.P. (Eds.), Nitrogen Nutrition in Higher Plants. Associated, New Delhi, pp. 21–56.
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He, H. Y. 2002. A physiological study on the flower inducing effect of potassium chlorate on longan. MS Thesis, South China Agricultural University, Guangzhou. Hofstra, J.J., 1977. Chlorate toxicity and nitrate reduction activity in tomato plants. Physiol. Plant. 41, 65–69. Hua, M., He, F., Wang, X.H., Chen, Y.G., Han, J., Li, X.H., 2004. Comparison of the flowering responses to potassium chlorate in ‘Chuliang’ and ‘Shixia’ longans. China South. Fruits 33, 35–42 (in Chinese). Huang, X.M., Lu, J.M., Wang, H.C., 2004. Current situation in research and application of chlorate in longan production. China South. Fruits 33, 29–30 (in Chinese). Kadman, A., 1963. The uptake and accumulation of chloride in avocado leaves and the tolerance of avocado seedlings under saline conditions. Proceedings of the American Society for Horticultural Science 83, 280–286. Li, J.G., Pan, X.W., Li, R., Dai, H.K., 2003. Advances in research of inducing longan flowering by potassium chlorate application. J. Fruit Sci. 20, 410– 414 (in Chinese with an English abstract). Lu, J. M, 2005. A physiological study on the biological effects of chlorate on longan. MS Thesis, South China Agricultural University, Guangzhou. Mackown, C.T., Vansanford, D.A., Rothwell, C.G., 1996. Nitrate uptake and assimilation and chlorate tolerance of wheat. Crop Sci. 36, 313–319. Manochai, P., Sruamsiri, P., Wiriyaalongkorn, W., Naphrom, D., Hegele, M., Bangerth, K., 2005. Year around off season flower induction in longan (Dimocarpus longan, Lour.) trees by KClO3 applications: potentials and problems. Sci. Horticult. 104, 379–390. Menzel, C.M., Huang, X.M., Liu, C.M., 2005. Cultivars and plant improvement. In: Menzel, C.M., Waite, G.K. (Eds.), Litchi and Longan, Botany, Production and Uses. CABI Publishing, Oxfordshire, UK, pp. 59–86.
Nelson, R.S., Ryan, S.A., Harper, J.E., 1983. Soybean mutants lacking constitutive nitrate reductase activity. II Nitrogen assimilation, chlorate resistance and inheritance. Plant Physiol. 61, 384–390. Shen, H., Yan, X.L., Zheng, S.L., Wang, X.R., 2001. Simultaneous analysis of organic acid and inorganicanions in common bean by ion chlormatography. J. South China Agric. Univ. 22, 8–10 (in Chinese with an English abstract). Singh, H.N., Sonie, K.C., Singh, H.R., 1977. Nitrate regulation of heterocyst differentiation and nitrogen fixation in a chlorate-resistant mutant of the blue-green alga Nostoc muscorum. Mutat. Res. 42, 447–452. Solomonsson, L.P., Vennesland, B., 1972. Nitrate reductase and chlorate toxicity in Chlorella vulgaris. Plant Physiol. 50, 421–424. Stauber, I.L., 1998. Toxicity of chlorate to marine microalgae. Aquat. Toxicol. 41, 213–227. Subhadrabandhu, S., Yapwattanaphun, C., 2001. Regulation off-season flowering of longan in Thailand. Acta Horticult. 558, 193–198. Van Wuk, D.J., Hutchinson, T.H., 1995. The ecotoxicity of chlorate to aquatic organisms: a critical review. Ecotoxicol. Environ. Safety 32, 244–253. Wilkinson, J.Q., Crawford, N.M., 1993. Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both NIA1 and NIA2 nitrate reductase structural genes. Mol. Gen. Genet. 239, 289–297. Yen, C.R., Chau, C.N., Chang, J.W., Tzeng, J.C., 2001. Effects of chemicals on flowering in Longan. J. Chin. Soc. Horticult. Sci. 47, 195–200 (in Chinese with an English abstract). Zhu, J.H., Peng, H.X., 2002. Technique of inducing longan flowering with chemicals. Guangxi Trop. Agricult. 3, 12–14 (in Chinese).