Alleviation of boron stress through plant derived smoke extracts in sorghum bicolor

Boron is an essential micronutrient required to maintain structural integrity of plant cell wall and metabolic pathways. The optimum B content for most of the plants is 20-100 ppm. Excess levels of Boron results in B toxicity as the B requirements and toxicity levels varies among plants. Boron availability in soil and in irrigation water is an important factor in agricultural production (Tanaka and Fujiwara, 2007). Boron occurs primarily as boric acid (H 3 BO 3 ) in soil solution, which can be leached under high rainfall situations (Shorrocks, 1997; Yan et al ., 2006). Due to this, B becomes deficient for plants that grow there and plant growth is affected as highly leached soils are not able to retain boron. On the other hand, low rainfall conditions can not sufficiently leach B and therefore may accumulate to levels that are toxic to plant growth (Reid, 2007b).

Plant-derived smoke has the ability to break dormancy and stimulate germination of Audouinia capitata, a fynbos species that grows in a fire-prone habitat (De Lange and Boucher, 1990). Since this discovery originated an idea that aqueous smoke extracts can be used to improve seed germination in a broad range of plants related to many families, irrespective of their fire sensitivity (Dixon et al., 1995; Roche et al., 1997; Brown and Botha, 2004). This phenomenon is potentially applicable in seed technology and has been comprehensively discussed in a number of reviews (Brown and Van Staden, 1997; Van Staden et al., 2000). Recently, a highly active and water soluble germination promoting compound, butenolide, 3-methyl-2H-furo [2, 3-c] pyran-2-one has been identified in smoke of burnt fynbos Passerina vulgaris and grass Themeda triandra L. (Van Staden et al., 2004) as well as from the combustion of cellulose (Flematti et al., 2004). This compound has the ability to stimulate germination at very low concentrations, i.e. 10 -9 M (Flematti et al., 2004; Van Staden et al., 2004). The present research work is the first systematic attempt to investigate the alleviating effects of plant derived smoke extracts against toxicity caused by boron.

MATERIALS AND METHODS

Seeds of Sorghum ( Sorghum bicolor ), DS-2003 were obtained from NARC (National Agriculture Research Centre) Islamabad, Pakistan.

Plant materials of M. azedarach, C. jwarancusa, P. harmala, D. alba and E. camaldulensis were collected from Kohat region. Shade dried plant parts of these plants were used for preparation of concentrated aqueous smoke extracts by following methods of De Lange and Boucher (1990) as well as Dixon et al . (1995) with slight modifications. The smoke extracts were diluted up to 1:100, 1:200, 1:300, 1:400 and 1:500 by using distilled water.

Initially the Sorghum seeds were treated with different boron concentrations, i.e. 5; 10; 15; 20 and 25 ppm respectively. Furthermore, smoke solutions with significant results (1:500) were mixed with boron solutions to prepare “B stress alleviating solutions” for assessment of alleviation potential of plant-derived smoke against B stress.

Ten seeds of Sorghum per petri plate were moistened and chilled in Petri dish covered with double layer filter papers and then kept at 25 ⁰C for two days to obtain uniform germination. After germination the seeds with equal radical size were placed on a filter paper soaked with respective B stress alleviating solutions. The filter paper was rolled like a cigar and was placed in a growth chamber under controlled conditions at 25 ⁰C for ten days. Seed germination percentage was recorded for ten days of incubation by following Chantachume et al . (1995) method. Root and shoot length as well as fresh and dry weight of seedlings were recorded after ten days of incubation.

For determination of B level, oven dried roots and shoots of Sorghum plant were digested in sulfuric acid and hydrogen peroxide in 2:1 ratio and analyzed by spectrophotometer following Azomethine – H method reviewed by Basson et al . (1969).

Statistics 9 software was used to analyze the data. One-way ANOVA and LSD- tests were applied to determine the correlation among different variables. P-value less than 0.05 considered as significant value. RESULTS

Present results revealed that smoke extracts collected from plant materials, improved seed germination (Fig. 1a). Significant increase in germination was observed by treating seeds with different smoke dilutions of C. jwarancusa (1:100 and 1:500), D. alba (1:300, 1:400 and 1:500), M azedarach at dilutions (1:100 and 1:500), while all dilutions E. camaldulensis and P. harmala (1:100, 1:200 and 1:500) (Fig. 1b). Significant decrease in final germination of Sorghum with (1:200) dilution of both C. jwarancusa and M. azedarach was resulted. Similarly concentrated smoke extracts of all the plants significantly reduced seed germination percentage.

After 24 hours of germination, smoke treatment (1:500) of E. camaldulensis showed most significant results and increased germination percentage was observed with smoke treatment (Fig 2a), while inhibition resulted with B treatments. With the application of combined solutions of smoke and B, smoke alleviated the inhibitory effects of B on seed germination percentage (Fig. 2b).

Root length of Sorghum was significantly increased with application of E. camaldulensis smoke at dilutions (1:200 and 1:500) and P. harmala at dilution of 1:100 (Fig. 3a) while decreased by the application of D. alba dilutions (1:300, 1:400 and 1:500) and M. azedarach dilutions (1:100, 1:300 and 1:400). Similarly shoot length was significantly increased with the somoke dilutions (1:200 and 1:500) of E. camaldulensis and P. harmala (1:100 and 1:500) while dilutions of Melia and Datura as well as concentrated smoke extracts of plants decreased shoot length (Fig. 3b).

Number of secondary roots of Sorghum were significantly increased when treated with smoke dilutions (1:200 and 1:500) of E. camaldulensis while 1:400 dilution of P. harmala increased the number of secondary roots as compared to control. Similarly, 1:100 and 1:500 of C. jwarancusa, all dilutions of D. alba and M. azedarach as well as concentrated smoke extracts of all the plants significantly decreased the number of secondary roots (Fig. 4).

With application of plant-derived smoke, fresh and dry weight of plant roots were increased. Dilutions of E. camaldulensis (1:100, 1:200 and 1:500) and P. harmala (1:100) significantly increased root fresh weight, while D. alba and M. azedarach decreased root fresh weight at all dilutions. Similarly concentrated smoke extract of all the plants reduced fresh weight of roots. Similarly root dry weight was increased with treatment of E. camaldulensis and P. harmala at the same dilutions as for fresh weight (Table 1). However, smoke solutions of D. alba and M. azedarach as well as concentrated smoke of all

Fresh and dry weight of Sorghum shoot was increased with the smoke solutions of E. camaldulensis and P. harmala . E. camaldulensis smoke dilutions (1:200 and 1:500) and P. harmala dilution (1:500) showed promotory effects while decrease in fresh and dry weight was observed with D. alba and M. azedarach (1:300, 1:400 and 1:500). Concentrated smoke extracts of all the plants also resulted inhibition and significantly decreased shoot fresh and dry weights (Table 2).

With increase in B concentration, significant reduction in root and shoot length was found (Fig. 5 -7). However, plant derived smoke significantly alleviated the stress of B on root length (Fig. 5a and 7). Though an improvement was found in shoot length with application of alleviated solutions as compared to B stress but it was not significant (Fig. 5b and 7).

the plants decreased root dry weight

Figure 1. Effect of C. jwarancusa (Cym), D. alba (Dat), E. camaldulensis (Euc), M. azedarach (Mel) and P. harmala (Peg) derived smoke on the germination (%) of Sorghum after 24 (a) and 48 (b) hours. (P < 0.05 S).

Figure 2. Germination (%) of Sorghum treated with boron and alleviating solutions ( E. camaldulesis + B) after 24 (a) and 48 (b) hours. Vertical bar shows LSD at P < 0.05 S.

Figure 3 . Effects of C. jwarancusa (Cym), D. alba (Dat), E. camaldulensis (Euc), M. azedarach (Mel), P. harmala (Peg) and their concentrated (Con) smoke on root (a) and shoot length (b) of Sorghum (P < 0.05 S).

□ DW □ 1:100 ■ 1:200 □ 1:300 □ 1:400 □ 1:500 □ Con

Figure 4. Effects of C. jwarancusa (Cym), D. alba (Dat), E. camaldulensis (Euc), M. azedarach (Mel), P. harmala (Peg) and their concentrated (Con) smoke on secondary root of Sorghum (P < 0.05 S).

Figure 5. Effect of B and alleviating solutions on root (a) and shoot length (b) of Sorghum. Vertical bar shows LSD at P < 0.05. Each data point shows mean of three replicates.

Figure 6 . Effect of B on root and shoot length of

Figure 7. Effect of alleviating solutions on root and shoot length of Sorghum.

Sorghum

Figure 8. B contents of Sorghum root (a) and shoot (b). Each data point represents the mean of three

replicates. The vertical bar represents LSD at p < 0.05.

Table 1. Effect of smoke dilution on root fresh and dry weight of sorghum

Root fresh weight                                    dry weight

Root

Smoke dilutions

	Cym	Euc	Peg	Dat	Mel
DW	0.164b	0.164d	0.164b	0.164a	0.164a
1:100	0.160b	0.288be	0.318a	0.101b	0.090b
1200	0218ab	0.345ab	0.184ab	0.061bc	0.092b
1:300	0.192ab	0.241cd	0.183ab	0.054bc	0.073b
1:400	0.161b	0.236cd	0.168b	0.081bc	0.066bc
1:500	0237ab	0.390a	0.165b	0.078b	0.088b
Cone.	0.011c	0.015e	0.017c	0.018c	0.016c
LSD	0.0993	0.0969	0.1454	0.0542	0.051

Cym	Euc	Peg	Dat	Mel
0.028bc	0.028c	0.028bc	0.028a	0.028a

0.053ab

0.082ab   0.103a    0.011b    0.01

S 8 g У

0.054ab    0.085ab   0.059b    0.011b0.01

0.049ab    0.056bc   0.038bc   0.010b0.01

0.041abc   0.054bc   О.ОЗОЬс   0.010b0.01

0.064ab    0.088a    0.034bc   0.011b    0.011b

0.007d     0.006c    0.004d    0.003c    0.002b

0.0395     0.0250    0.0364    0.0151    0.0154

All the data points shown are mean of three replicates with ten seeds in each Petri dish.

Table 2. Effect of smoke on shoot fresh weight and dry weight of Sorghum

Shoot						Shoot dry weight
	fresh weight
Smoke diutions	Cym	Euc	Peg	Dat	Mel	Cym	Euc	Peg	Dat	Mel
DW	0.372a	0.372bc	0.372bc	0.372a	372a	0.090a	0.090bc	0.090bc	0.090a	0.090a
1:100	0.473a	0.559ab	0.750ab	0271 abc	0.247abc	0.106a	0.192b	0244ab	0.025ab	0.025ab
1200	0.413a	0.837a	0.383bc	0.318ab	0.329ab	0109a	0.328a	0.099bc	0.035ab	0.022ab
1:300	0310a	0381bc	0.506ab	0.074c	0.086be	0.087a	0.181b	0.147bc	0.0167b	0.015b
1:400	0.350a	0.580ab	0.386bc	0.100bc	0.097bc	0.117a	0.190b	0.127bc	0.021b	0.011b
1:500	0.400a	0.928a	0.876a	0.105b	0.125bc	0.065a	0.415a	0.386a	0.017b	0.019b
Cone.	0.023b	0.012d	0.029d	0.041b	0.0361c	0.009b	0.009d	0.004d	0.004c	0.006b
LSD	02540	0.3988	0.4085	02372	0.2447	0.0690	0.1337	0.1637	0.0684	0.0713

All the data points shown in the table 1 are mean of three replicates with ten seeds in each Petri dish.

Table 3. Effect of boron on root and shoot fresh and dry weight of Sorghum

Treatments В (ppm)	Root fresh weight	Root dry weight	Shoot fresh weight	Shoot dry weight
DW	0.164a	0.028a	0.372a	0.090a
5	0.123ab	0.031a	0.355ab	0.034ab
10	0.062bc	0.013b	0.205abc	0.032ab
15	0.056bc	0.012b	0.262ab	0.025ab
20	0.058be	0.010b	0.101c	0.012b
25	0.052c	0.011b	0.108 be	0.008b
LSD	0.0679	0.0127	0.2535	0.0731

All the data points shows mean of three replicates and each replica contain ten seeds

Table 4. Effect of alleviating solution of boron and smoke on root and shoot fresh and dry weight of Sorghum

Treaments	Root	Root	Shoot	Shoot
В (ppm)	Fresh weight	dry weight	fresh weight	dry weight

D. W	0.164a	0.028a b	0.505a	0.053a
5+1:500	0.227a	0.043a	0.451 ab	0.037ab
10+1:50	0.192a	0.040a	0.383ab	0.032ab
15+1:500	0.014b	0.011c	0.285be	0.027b
20+1:500	0.013b	0.011c	0.276bc	0.022b
25+1:500	0.044b	0.010c	0.187c	0.017b
LSD	0.0674	0.0159	0.1776	0.0241

All the data point shows mean of three replicates and each replica contain ten seeds.

Effect of B stress and alleviating solutions on _{roots and shoots (Table 4).}

root and shoot fresh and dry weight

Analysis of B in Sorghum roots and shoots

In case of root fresh and dry weight, there was

Present investigation showed that smoke solution found no effect at lower concentrations of B but higher potentially alleviated boron stress probably by concentrations showed a significant reduction in both reducing the uptake of B. However B uptake was the parameters (Table 3). Present results also reduced significantly in roots as compared to shoot revealed that combined solutions of B and smoke

(Fig. 6a and b). Though smoke solution reduced the

(alleviating solutions) significantly alleviated the toxic uptake of B in the shoot, but the reduction was not so effects of B and improved fresh and dry weight of significant.

DISCUSSION

This study highlights the effect of C. jwarancusa , E. camaldulensisa , P. harmala , D. alba and M. azedarach extracted smoke on the germination and seedling vigor of Sorghum in order to alleviate boron stress. Smoke collected from these plants enhanced seed germination significantly (Fig.1a). In smoke extracts of the plants, concentrated solutions showed inhibition against all the parameters studied. Present results are in accordance with the investigations of Brown and Van Staden (1997) who reported that smoke extracts at lower concentrations improve seed germination. Improvement in germination at lower concentrations of smoke could be attributed to the removal of germination inhibitors such as ABA and phenolics (Da Cuhna and Casali, 1989; Hilhorst and Karssen, 1992). Similarly in case of root and shoot length, plant derived smoke significantly overcame the inhibitory effects caused by excessive amount of B and co-relates with the results of Sparge et al . (2006). Present investigations also indicated that, smoke solutions resulted an increase in seedling fresh and dry weights (Table 1 and 2). These results are in agreement with that of Kulkarni et al . (2006). Similarly number of secondary roots were significantly increased by eucalyptus at the dilution of 1:200 and 1:500 (Fig. 4).

Excess concentration of B in soil may interrupt growth and development of wheat and other higher plants. The most frequent symptoms of boron toxicity in cereals crops include chlorosis and necrosis (Cartwright et al ., 1984), delay in development, reduced shoot growth and plant height (Paull et al .,

1988) and root growth suppression (Huang and Graham, 1990). Significant yield losses due to excess boron have been observed in cereals (Cartwright et al ., 1984; Paull et al ., 1992). Present work Indicates that treatment of plants with B solutions inhibited both root and shoot growth (Fig. 5 and 6). On the other hand plant derived smoke extracts has the potential to overcome the inhibitory effects of B stress through alleviating solutions. Present investigations revealed that Sorghum has lower B contents in shoots as compared to root (Fig. 8). The selection of smoke dilution i.e. 1:500 of E. camaldulensis for alleviating solution was done on the basis of strong vigour among all the dilutions. The germination (%) and the root and shoot length were significantly increased by smoke as well as alleviating solutions overcome the toxicity of boron stress considerably (Fig. 2 and 5).

A significant relationship was found between diverse factors that could manage B tolerance in Sorghum while treating with alleviating solutions. There was a significant interaction between B stress, alleviating solutions and boron contents of seed and seedlings. In addition, Sorghum seeds treated with boron gave lower seed germination and low root and shoot length. Present results suggested that higher dose of boron retard the growth of Sorghum. Similarly, alleviating effects of smoke extracts against B toxicity is just because smoke has the potential to reduce the uptake of boron which ultimately leads to tolerance of Sorghum to B. However the mechanism by which plant-derived smoke solutions reduce Boron uptake is still a mystery to be unveiled.

The present study investigated that, smoke has the potential to alleviate phytotoxicity caused by B stress and improved seed germination and seedling vigour in Sorghum under B stress condition. Investigations also revealed that, B toxicity has direct relation to B contents of root and shoot, i.e. more B contents, resulted lower growth of root and shoot while, alleviating solutions of smoke and B, smoke reduced the uptake of B resulted an increase in root and shoot length. Hence, the tolerance to B toxicity could be attributed to lower B uptake. Present work also suggested that, smoke is involved in physiological and metabolic activities that improves plant growth under B toxic condition. It is predicted that this study will provide a basic strategy for reducing the risks of B toxicity and maintaining sustainable plant production.