The use of wastewater as an alternative water source for irrigation needs specific studies since this water contains organic matter, salt, heavy metals and high boron concentrations, which may be toxic to many crops. Grafting of vegetable plants became a common practice with the main goals to control soil-borne diseases and nematodes and improve tolerance to environmental stresses such as flooding, salin-ity and high boron concentrations. The aim of the present study was to explore how boron damage to melon plants under wastewater irrigation is reduced by grafting onto pumpkin rootstocks. Six melon/pumpkin combinations were examined. Boron decreased shoot and root dry weights of all plant types, but by much less in those with pumpkin root systems than in those on melon roots, indicating that pumpkins are more boron-tolerant than melons and can impart this tolerance onto plants grafted on them. Boron concentrations in roots were much lower than in shoots, and similar in all plant types, implying that boron was not retained within the pumpkin root system. Shoot dry weights were much higher in plants grafted on pumpkin than in those grafted on melon, therefore transpiration rates and consequently boron transport to the shoots were expected to be higher in the former; in fact, they were significantly lower, implying that boron exclusion by the pumpkin root system at least partly accounted for the lower boron damage to plants grafted onto pumpkin. The concentrations of boron in xylem sap exudates were significantly lower for plants with pumpkin root systems than for those on melon roots, and exudation rates were markedly higher for the former, regardless of boron concentrations. However, the total amounts of boron exuded from plants grafted on pumpkin were still lower than those from plants grafted on melon, indicating that pumpkin roots blocked boron uptake from the growth media, to some extent. The study demonstrates that pumpkin root systems are capable of partially excluding boron when its concentra-tions in the growing media is high, thus avoiding damage to boron-sensitive plants, such as melons grafted onto pumpkins. The grafting technique can thus be adopted for agricultural setups where effluents with high boron concentration are used for irrigation.
Although boron deficiency is known to occur to a higher extent than any other plant micronutrient deficiency (Gupta, 1979), excess of boron, leading to toxicity, is very common in arid and semiarid regions (Yau and
Ryan, 2008). Crops may be exposed to boron toxicity by two main factors: (a) growth in soils with high levels of naturally occurring boron, usually accompanied by
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salinity; (b) irrigation with water containing high levels of boron (Keren and Bingham, 1985). High boron con-tent in irrigation water may be associated with salinity, but it could also be a significant constituent in effluent water, even if the salt content is not very high, because sewage treatment does not reduce boron concentration (Ben-Hur, 2004).
The negative effects of excess boron concentrations on crop production are widely documented. Bingham et al. (1985) concluded that wheat, barley, and sorghum were sensitive, moderately tolerant, and very tolerant to boron, respectively. Francois (1984) found a threshold boron concentration for tomatoes of 5.7 mg L–1, and a productivity decrease of 3.7% for each additional 1.1 mg L–1 of boron in the soil solution. Lauter et al. (1989) studied the effects of high concentrations of boron on peanut (Arachishypogaea cv. ‘Shulamit’) and found that grain yield was reduced when the boron concentration in the nutrient solution exceeded a threshold of 3.1 mg L–1. Ben-Gal and Shani (2002) reported a negative re-sponse of tomatoes to boron, and Goldberg et al. (2003) showed that melons are sensitive to excess boron. The problem of boron toxicity was documented in many dry areas such as West Asia, North Africa, South Australia, and western USA (Yau and Ryan, 2008). Boron toxicity exerts various negative effects on vascular plants, the outcome of which is decreased shoot and root growth (Nable et al., 1997; Edelstein et al., 2005), and marked decreases in crop yield (Eaton, 1994; Goldberg et al., 2003). Although decreases in chlorophyll content and photosynthesis rates have been mentioned (Nable et al., 1997), the physiological background for the decrease in yield is not very clear. Reid et al. (2004) suggested three main explanations of the effect of excess boron on crops: (a) alterations in the structure of cell walls; (b) binding of boron to ribose of ATP, NADH, or NADPH, thus disrupting metabolic activity; and (c) binding of boron to ribose of RNA, thus interfering with protein synthe-sis and cell division. Toxicity symptoms in cereals and legumes such as alfalfa, faba beans, chickpeas, lentils, etc., were summarized as chlorosis or necrosis of leaf tips and margins, that is more intensive in older leaves (Yau and Ryan, 2008). In fruit trees, brown lesions were found along stems and petioles, as well as twig dieback or similar symptoms (Brown and Hu, 1998).
Plant sensitivity to boron differs widely among species and even among cultivars of the same species (Marschner, 1998). For instance, for wheat, barley and sorghum, respectively, boron concentration thresholds of 0.3, 3.4, and 7.4 mg L–1 in the nutrient solution were found, above which yield was decreased (Bingham et al., 1985). Choi et al. (2006) showed that a variety of crop and weed species showed symptoms of toxicity at an ex-
tractable boron concentration in subsoil of 12.2 mg/kg, and root growth and water uptake were inhibited at lower concentrations. Reid et al. (2004) claimed that internal plant boron concentrations in the 1–5 mM range (de-pending on species) inhibited growth. However, Torun et al. (2003) showed that the decrease in yield of various barley cultivars was not related to internal boron concen-trations and that, despite the differences in sensitivity, the internal boron concentrations were very similar.
Many efforts have aimed to prevent or minimize boron toxicity effects on crop plants. Agronomic means, such as leaching boron out of the root zone, are only suitable when the soil, and not the irrigation water, is contaminated with boron. Moreover, much more water (which may not be available in dry regions) is needed to leach boron than to leach salts (Bingham et al., 1985). The practical way to overcome boron toxicity has been, therefore, to select or breed species and cultivars with high boron tolerance (Yau and Ryan, 2008). A potential criterion for selection could be boron accumulation in the shoot, since boron-tolerant cultivars were suggested to be characterized by lower boron concentrations in their leaf tissues compared to sensitive ones, probably because of lower uptake rates (Nable et al., 1990). Other researchers showed that an efflux-type borate trans-porter accumulated in a transgenic boron-tolerant line of Arabidopsis thaliana (Miwa et al., 2007), and that this enhanced the efflux of boron and thereby reduced boron accumulation in the plant, and improved growth. Sutton et al. (2007) demonstrated disposal of boron from leaves of barely plants by guttation via hydathodes. The abil-ity of boron-tolerant cultivars to maintain a lower shoot boron concentration may thus be due to lower rates of entry into roots, and possibly also to boron extrusion. Published surveys of germplasm of several boron-toler-ant cultivars of crop species such as barley, durum and bread wheat, rice, lentils, and peas are being used to in-corporate boron tolerance into some of these crop plants (Nable et al., 1997; Yau and Ryan, 2008).
In addition to the difficulties presented and the time needed for breeding high-value crop plants, it should be noted that this approach was not utilized for numeri-ous other crops of high agronomic importance. In our earlier studies, the approach of grafting boron-sensitive plants onto more tolerant plants was shown to be an effective means to reduce boron accumulation in the shoot (Edelstein et al., 2005, 2007). When melon plants, which are boron-sensitive, were grafted onto Cucur-bita rootstocks, which are more boron-tolerant, they accumulated less boron in their shoots than ungrafted melons, and it was suggested that the root systems of the grafted plants had lower boron uptake rates than those of ungrafted ones. Moreover, fruit yield and dry weight
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production of the grafted melons were less sensitive to boron accumulation in their vegetative organs than those of ungrafted plants. It was suggested that boron sensitivity of the root system of ungrafted plants could have impaired their water and nutrient uptakes, thereby reducing fruit production (Edelstein et al., 2007). A comparison between plant combinations in which mel-ons and Cucurbita were grafted onto each other as well as onto themselves could lead to a better understanding of the scion–rootstock interactive effects on boron up-take and accumulation.
The extended use of effluent water for irrigation in arid regions, due to shortage in fresh water, may intensi-fy the problem of boron toxicity. Boron can be found at relatively high concentrations in raw sewage water; for instance, in Israeli industrial sewage water its average concentration was 0.6 mg L–1, and after radical changes it dropped to 0.3 mg L–1 (Weber and Juanico, 2004). It was shown that boron concentration in the effluent was in many cases not reduced significantly after treatments (Tsadilas, 1997; Ben-Hur, 2004), and its concentration is still in the damaging range for most crops. Optimal utilization and development of the grafting technique for cultivation under the toxic level of boron is depen-dent on increased understanding of the physiological processes involved. The objective of the present study was to understand the mechanism responsible for reduc-ing boron concentrations in melon grafted onto pump-kin, by comparing dry weight production, and boron uptake, accumulation, and compartmentalization in the various plant organs in ungrafted melons and pumpkins, melons grafted on melons and pumpkins, and pumpkins grafted on pumpkins and melons.
MATERIALS AND METHODS
The experiment was conducted in a heated (hot water), naturally illuminated greenhouse at the Newe Ya’ar Re-search Center in northern Israel. Day temperature was 25 ± 2 ºC and night temperature was 18 ± 2 ºC. Grafted and ungrafted seedlings were planted on January 30, 2006 in 10-L pots containing Perlite no. 2 (Agrical, Habonim, Israel), one seedling per pot. Melon (Cucumismelo L.) plants ‘Arava’ (Hazera Genetics Ltd. Berurim, Israel), and the commercial pumpkin rootstock Cucur-bitamaxima Duchesne × Cucurbitamoschata Duchesne ‘TZ-148’ plants (Tezier, Almeria, Spain) were used. The melons and pumpkins were grown as ungrafted, grafted on themselves, melons grafted on pumpkins (Edelstein et al., 1999), and pumpkins grafted on melons. Plants were subjected to three boron concentrations—0, 5, and 10 mg L–1—by adding the appropriate amounts of boric acid (H3BO3) to the irrigation solutions.
The plants were irrigated to excess five times a day, so that ~35% of the irrigation water drained, to enable leaching of excess salt out of the pot. The fertilizer Shefer (Fertilizers & Chemicals Ltd., Haifa, Israel), containing nitrogen, phosphorus, and potassium in the proportions of 7–3–7 (N:P2O5:K2O) was applied in the irrigation water. The concentration of the fertilizer in the irrigation water was 0.2% (by volume). The experiment was conducted under a completely randomized design, with four replicates (four pots) for each treatment.
The shoot (stems and leaves) and roots of each plant were harvested 30 days after planting, prior to formation of fruits. The vegetative organs were weighed, washed gently in deionized water, and dried at 70 ºC for 48 h, after which the shoots and the roots were stored pending chemical analysis. At the time of harvest and immedi-ately after the last irrigation event, the stem of each plant was cut ~5 cm above the surface of the pot, when this cut in the grafted plants was ~1 cm below the grafting site, and the xylem sap exudates from the cut stem were collected for 2 h. The quantities of sap were recorded, and the liquid of three replicates was collected into 20-ml plastic tubes and immediately frozen at –20 ºC pending chemical analysis.
Scion and rootstock stem diameters (four replicates) were measured one cm above or below the grafting site with a mechanical caliper.
Leaf stomatal conductance (three replicates) was measured between 10:00 to 12:00 PM, with an LI-1600 Steady State Porometer (LI-COR , Lincoln, NE, USA).
Chemical analysis
Boron was analyzed by ashing 0.25 g of each sample in a furnace at 600 ºC for 5 h. Five milliliters of 1 M HCl were added to the cooled ash, and the solution was filtered after 15 min and analyzed by ICPES (Spectro GmbH, Kleve, Germany).
RESULTS AND DISCUSSION
Growth rates of both shoots and roots of pumpkins were much higher than those of melons. Dry weights of shoots and roots of pumpkin control plants, i.e., cul-tivated without the addition of boron, were more than three times higher, compared to melons (Table 1). The addition of boron to the irrigation water reduced both shoot and root dry weights of all plant types tested, but the sensitivity of pumpkins was much less than that of melons. The decreases of pumpkin shoot and root dry weights were only about 10% with boron at 5 mg L–1, and 20–25% at 10 mg L–1, whereas the correspond-ing decreases of melon dry weights were 26–35 and 55–76%, respectively. A protective effect of the pump-
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Table 1Dry weight formation of ungrafted pumpkins (P), ungrafted melons (M), melons grafted on their own rootstock (M/M), pump-kins grafted on their own rootstock (P/P), melons grafted on pumpkin (M/P), and pumpkins grafted on melon (P/M), as affected
by various boron concentrations
Plant type Boron in irrigation water (mg L–1) 0 5 10
kin rootstock against dry weight reduction in boron-ex-posed plants can be seen in melons grafted on pumpkins (Table 1), whereas reciprocal grafting did not improve pumpkin tolerance, and the reduction of dry weight of pumpkin with increasing boron concentration was enhanced by grafting the pumpkins onto a melon root-stock. In some cases, there was no decrease of root dry weight with boron at 5 mg L–1, when pumpkins served as rootstocks. Because of the differences in weight of the different types of untreated plants, the boron sensi-tivity of their dry weight production is better illustrated by comparing relative weights (Fig. 1). The results demonstrate that the plants evaluated can be divided ac-cording to their responses into two groups: plants with a pumpkin root system exhibited lower boron sensitivity than those with a melon root system which exhibited high sensitivity. Thus, the melons grafted on pumpkins were the least sensitive plants to boron, and ungrafted melons were the most sensitive. It can thus be concluded that the root system and not the shoot is the source of boron sensitivity in the investigated plants. This agrees with our earlier hypothesis that vegetative dry weight production by the shoot, as well as the fruit yield, were strongly affected by boron sensitivity of the root system (Edelstein et al., 2007).
Since the responses of ungrafted melon and pumpkin plants and of plants grafted on themselves were similar, the concentrations of boron in shoots and roots at plant harvest are presented only for the grafted plants, i.e., M/M, P/P, M/P and P/M (Tables 2 and 3). The concen-
trations of boron in the shoots of the various plant types were increased by 5.4- to 8.4-fold with irrigation boron at 5 mg L–1, and by 9.2- to 12.6-fold at 10 mg L–1. Boron accumulation in shoots of melons grafted on pumpkin rootstocks was significantly lower than in those of mel-ons grafted on melon (Table 2A). On the other hand, its accumulation in shoots of pumpkins grafted on melon was much higher than that of pumpkins grafted on pumpkin. These results are in agreement with our earlier findings with the same cultivars (Edelstein et al., 2005, 2007), which showed that the concentration of boron in melons grafted on pumpkins was lower than that in ungrafted melons.
It was previously demonstrated that boron is trans-ported to plant shoots via the transpiration stream, and is confined within the leaves (Dannel et al., 2000; Ben-Gal and Shani, 2002). Provided that boron is transported only in the transpiration stream and that transpiration rates are directly proportional to shoot size, then the amount of boron in the shoot should be proportional to the shoot size, i.e., to its dry weight. We, therefore calcu-lated the expected amount of boron in the shoots of the various scions, according to the amounts of boron in the shoots of ungrafted plants, adjusted according to their dry weight ratios (Table 2B). We also calculated the to-tal amounts of boron in the shoots of the grafted plants, by multiplying the measured concentrations of boron (Table 2A) by the shoot dry weight (Table 1A). It can be seen that under 5 mg L–1 supply the ratios between the actual and expected amounts of boron in M/M and M/P
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Fig. 1. Relative shoot (A) and root (B) dry weights of ungrafted pumpkins (P), ungrafted melons (M), melons grafted on their own rootstock (M/M), pumpkins grafted on their own rootstock (P/P), melons grafted on pumpkin (M/P), and pumpkins grafted on melon (P/M), as affected by various boron concentrations.
were 1.25 and 0.73, respectively, and at 10 mg L–1 these ratios were 0.92 and 0.71, respectively. This indicates that the melon rootstock enhanced boron accumulation compared with the expected rate, or had no effect or the accumulation rate, whereas the pumpkin rootstock in-hibited the accumulation. The ratios between the actual and expected amounts of boron in P/M and P/P plants, under 5 mg L–1 supply, were 1.41 and 1.03, respectively,
and at 10 mg L–1 were 1.36 and 1.02, respectively. This suggests again that melon rootstocks enhanced boron accumulation, even when the scion was pumpkin, whereas pumpkin rootstocks had no effect.
Boron concentrations in the roots were much lower than those in the shoots, and were increased by only 1.8- to 3.6-fold and by 2.3- to 4.2-fold with irrigation boron, at 5 and 10 mg L–1, respectively (Table 3A). Moreover,
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Table 2Boron in shoots as affected by boron concentration in the irrigation water: A—Concentration; B—Expected total amount; and C—Measured total amount. In roots of melons grafted on their own rootstock (M/M), pumpkins grafted on their own rootstock
(P/P), melons grafted on pumpkin (M/P), and pumpkins grafted on melon (P/M)
Boron in irrigation water (mg L–1)Plant type 0 5 10
Table 3Boron in roots as affected by irrigation boron concentration: A—Concentration; B—Expected total amount; and C—Measured total amount. In roots of melons grafted on their own rootstock (M/M), pumpkins grafted on their own rootstock (P/P), melons
grafted on pumpkin (M/P), and pumpkins grafted on melon (P/M)
Boron in irrigation water (mg L–1)Plant type 0 5 10
the concentrations of boron in roots were quite similar in all plant types (Table 3A), which indicates that previous finding of low root boron concentrations in ungrafted melons and melons grafted onto pumpkins (Edelstein et al., 2007) is also valid for all other combinations of sci-ons and rootstocks. We may, therefore, conclude that the hypothesis that boron retention within the root system serves as the mechanism by which the pumpkin root-stock protects plant growth has to be rejected. It seems, rather, that the mechanism responsible for restriction of boron accumulation in shoots of plants grafted on pumpkin is different from that which restricted sodium accumulation in melons grafted onto pumpkin rootstock (Edelstein et al., 2011). In spite of the low boron con-tent in roots, we also calculated the expected and actual amounts of boron in roots, similarly to the calculation for shoots (Tables 3B, C). It can be seen that also in roots the actual amount of boron in melons grafted on pump-kin was significantly lower than the expected amount, and that in pumpkins grafted on melon it was higher. All these findings support the hypothesis that exclusion of boron by the pumpkin root system was responsible for its lower concentration in the grafted shoots. It may be concluded on the basis of the ratios between actual and expected boron contents in shoots that this exclusion could reduce boron content by 25–30%. The finding that dry weight production by plants grafted on pumpkin was more markedly enhanced than that by those grafted on melon might be due to a different response of dry weight production to boron.
The uptake of boron by the root systems, and its po-tential transfer to the shoots by the different rootstocks was also determined by analyzing the boron in stem exudates (Fig. 2). As may be expected, the higher the concentration of boron in the irrigation water the higher was its concentration in the exudates. The low but non-zero boron concentrations found in exudates of control plants when no boron was added to the irrigation water, indicate that the water and fertilizers contain very low amounts of boron. The concentrations of boron in the exudates of ungrafted melons, melons grafted on mel-ons, and pumpkin grafted on melons were significantly higher than those in the exudates of ungrafted pumpkins or plants grafted on pumpkins (Fig. 2A). This suggests that boron uptake and/or transport was markedly lower in plants with pumpkin roots.
Determination of the exudation rates of the different plants showed that plants with pumpkin roots, regard-less of the scion, exuded at a markedly higher rate than plants with melon roots (Fig. 2B). These higher rates could be due to one of the following reasons: (a) the root systems of the pumpkin were much larger and more proliferated than those of melon, as shown previously
Fig. 2. (A) Boron concentration in xylem sap exudate, (B) rate of exudation, and (C) quantity of exudated boron. Letters un-der horizontal axis indicate: P—ungrafted pumpkins, M—un-grafted melons, M/M—melons grafted on their own rootstock, P/P—pumpkins grafted on their own rootstock, M/P—melons grafted on pumpkin, and P/M—pumpkins grafted on melon, as affected by various boron concentrations. Vertical bars indicate ± SE (n = 4).
(Edelstein et al., 2007), so that many more of the boron molecules distributed in the growth media could be ex-tracted; (b) the larger canopies of plants on the pumpkin rootstock had larger transport capacities, in order to supply water and nutrients, and could, therefore, exude boron at higher rates than those on melon, even after be-ing excised. The higher exudation rates of plants grafted on pumpkins than of those grafted on melons lessened the effect of pumpkin rootstock on boron concentrations in the exudates (Fig. 2C). Nevertheless, however, the total amounts of exuded boron were higher in all plant types grafted on melons at both boron concentrations (Fig. 2C).
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Fig. 3. Leaf conductance of P—pumpkin, M—melon, M/P—melon grafted onto pumpkin, P/M—pumpkin grafted onto melon, as affected by various boron concentrations. Vertical bars indicate ± SE (n = 4).
Stomatal conductance was determined several times during the growing period for ungrafted melons and pumpkins, and also for pumpkins grafted on melon and melons grafted on pumpkin. Figure 3 shows that, in the absence of added boron, stomatal conductance was higher in pumpkin leaves than in melon leaves. The con-ductance in ungrafted melons and in pumpkins grafted on melon was reduced by the boron treatments. On the other hand, boron had no significant effect on conduc-tance of pumpkin leaves or of melon leaves grafted on pumpkin. The higher stomatal conductance could have led to higher photosynthetic CO2 uptake, leading, in turn, to higher rates of dry matter production.
It was shown earlier that the pumpkin rootstock had a distinct supportive effect on the activity and growth of the scion, and that the melon rootstock inhibited them. Further evidence of the effect of the rootstock on scion growth and vice versa can be obtained by measuring stem diameter above and below the graft, as shown in Fig. 4. The diameters of the P and P/P scions were significantly larger than those of the M and M/M scions (Fig. 4A). However, the diameter of the melon scion grafted on pumpkin was enhanced by the pumpkin root-stock and was significantly larger than those of M and M/M. On the other hand, the diameter of the pumpkin scion was decreased by the melon rootstock and was smaller than those of P and P/P. The diameters of the rootstock stems also responded as the scion, as can be seen in Fig. 4B. It is interesting that boron, even at the highest concentration in the irrigation water, in most cases had only negligible effects on stem diameters of both rootstock and scion.
Fig. 4. Diameter of scion (A) and rootstock (B) of ungrafted pumpkins (P), ungrafted melons (M), melons grafted on their own rootstock (M/M), pumpkins grafted on their own root-stock (P/P), melons grafted on pumpkin (M/P), and pumpkins grafted on melon (P/M), as affected by various boron concen-trations. Vertical bars indicate ± SE (n = 4).
The study clearly demonstrates that melon plants, which are sensitive to boron in the irrigation water, responded less negatively to boron accumulation when grafted on pumpkin, a tolerant plant. The mechanism of this phenomenon is mainly the capability of the root-stock to partly exclude boron when its concentration in the irrigation water is high. The grafting can thus be used as a tool to prevent boron damage when effluent with high boron concentration is used.
ACKNOWLEDGMENTS
The authors thank A. Porat and F. Baumkoler for technical assistance. This study was supported by the Chief Scientist’s Fund of the Ministry of Agriculture and Rural Development of the State of Israel, under
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research project 255-0663-04. This support is gratefully acknowledged.
Contribution No. 125/2009 from the Agricultural Research Organization, the Volcani Center, Bet Dagan 50250, Israel.
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