Acta Biologica Hungarica 65(4), pp. 451–468 (2014) DOI: 10.1556/ABiol.65.2014.4.9
THE POTENTIALITY OF TRICHODERMA HARZIANUM IN ALLEVIATION THE ADVERSE EFFECTS OF SALINITY IN FABA BEAN PLANTS G. K. Abd El-Baki1 * and Doaa Mostafa2 1 Botany
and 2 Microbiology Department, Faculty of Science, Minia University, El-Minia, 61519, Egypt (Received: January 17, 2014; accepted: March 24, 2014)
The interaction between sodium chloride and Trichoderma harzianum (T24) on growth parameters, ion contents, MDA content, proline, soluble proteins as well as SDS page protein profile were studied in Vicia faba Giza 429. A sharp reduction was found in fresh and dry mass of shoots and roots with increasing salinity. Trichoderma treatments promoted the growth criteria as compared with corresponding salinized plants. The water content and leaf area exhibited a marked decrease with increasing salinity. Trichoderma treatments induced a progressive increase in both parameters. Both proline and MDA contents were increased progressively as the salinity rose in the soil. Trichoderma treatments considerably retarded the accumulation of both parameters in shoots and roots. Both Na+ and K+ concentration increased in both organs by enhancing salinity levels. The treatment with Trichoderma harzianum enhanced the accumulation of both ions. Exposure of plants to different concentrations of salinity, or others treated with Trichoderma harzianum produced marked changes in their protein pattern. Three types of alterations were observed: the synthesis of certain proteins declined significantly, specific synthesis of certain other proteins were markedly observed and synthesis of a set specific protein was induced de novo in plant treated with Trichoderma harzianum. Keywords: Lipid peroxidation – proline – proteins – potassium – salinity – sodium – Trichoderma
Introduction High concentrations of salts in soils account for large decreases in yield of a wide variety of crops all over the world. Globally, more than 770,000 km2 of land is saltaffected by secondary salinization: 20% of irrigated land, and about 2% of dry agricultural land [7]. Salt stress affects many aspects of plant metabolism and, as a result, growth and yields are reduced. Excess salt in the soil solution may adversely affect plant growth either through osmotic inhibition of water uptake by roots or specific ion effects, specific ion effects may cause direct toxicity or, alternatively, the insolubility or competitive absorption of ions may affect the plant’s nutritional balance. Salinity was shown to increase the uptake of Na+ or decrease the uptake of Ca2+ and K+, the * Corresponding author; e-mail address: [email protected] 0236-5383/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
452
G. K. Abd El-Baki and D. Mostafa
growth of plants is ultimately reduced by salinity stress although plant species differ in their tolerance to salinity [24]. Plant growth-promoting rhizobacteria (PGPR) and fungi can facilitate plant growth indirectly by reducing plant pathogens, or directly by facilitating the uptake of nutrients from the environment, by influencing phytohormone production (e.g. auxin, cytokinin, or gibberellin), and/or by enzymatic lowering of plant ethylene levels [12]. In addition to facilitating the growth of plant, plant growth-promoting microorganisms can protect plants from the deleterious effects of some stresses including phytopathogens [15]. It is thought that the depressive effect of salinity on germination could be related to a decline in endogenous levels of hormones, however, incorporation of Trichoderma during seed biopriming treatments in many cereal and vegetable crops has resulted in increased levels of plant growth hormones and improved seed performance [18]. Biocontrol agent, Trichoderma, releases a variety of compounds that induce resistance responses to biotic and abiotic stresses, several studies have shown also that root colonization by Trichoderma harzianum results in increased level of plant enzymes, including various peroxidases, chitinases, β-1,3-glucanases, lipoxygenase-pathwayhydroperoxidelyase and compounds like phytoalexins and phenols to provide durable resistance against stress [9]. Accumulation of some compatible solutes has been observed under salt stress conditions and has been suggested as part of mechanism(s) that controls salt tolerance in plants. Proline is one of the best known solutes, however, its relative importance for tolerance and precise protective function during stress require further investigations. The objective of this study was to investigate the effects of seed inoculation of faba bean with Trichoderma strains upon growth, physiological and biochemical parameters under normal and saline conditions. In this study we have examined and compared the proline content of treated and untreated bean plants with different degrees of salt stress, in order to exploit Trichoderma strains in relation to salt stress tolerance in beans. This research is important as it may reveal the role of Trichoderma in imparting stress resistance and provides insight into the potential of bean plants to adapt to saline conditions.
Materials and Methods Faba bean seeds (Vicia faba Giza 429) were germinated in pots using sand clay soil (2:1 w/w). The plants were divided in two groups, one of them was treated with NaCl alone and the other with NaCl + Trichoderma harzianum (T24) spore suspension (108 CFU ml–1). Five seeds per pot, three replications for each, were cultivated and inoculated with 200 µl spore suspension for each or left without inoculation (NaCl alone). The seeds were left to germinate and grow for about three weeks and then treated with different salt concentrations (0.0, 50, 100, 150 and 200 mM NaCl) by top irrigation. The plants left to further growth with different treatments for about 40 days from Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
453
germination, 20-days from salt treatment under day night temperature of (20/25 °C and relative humidity 85%) plants were harvested. Subsequently roots were rapidly rinsed in cold water or with cold glycinebetaine solutions equimolar to the salted nutrient solution in order to prevent osmotic shock. After rinsing, roots were carefully blotted with tissue paper. To determine the dry matter, the freshly harvested organs (shoots and roots) were dried in an aerated oven at 105 °C for 24 hours. Water content in both organs were determined. Leaf area were carried out by Wiersma et al. [31]. The level of lipid peroxidation was measured in terms of malondialdehyde (MDA) content. MDA a product of lipid peroxidation following the method of [16]. Polyacrylamide gel electrophoresis was carried out according to Laemmli [20] with 12% acrylamide + 1% SDS. The soluble proteins were determined according to the method adopted by Lowery et al. [21]. Free proline was determined according to Bates et al. [3]. Soluble minerals were determined in boiled water extract by flame photometer method [32] using a Carl Zeiss flame photometer. The data of all experiments were subjected to one-way analysis variance and means were compared using the least significant difference test (L.S.D.) using statistical program (Sta. Base. Exe.) on computer.
Results From the data in Table 1 and Fig. 2 it can be seen that, there is a sharp reduction in fresh and dry mass of shoots and roots of faba bean (Vicia faba Giza 429) with increasing salinity in the soil. Trichoderma treatments promoted the growth criteria of bean plants as compared with corresponding salinized plants. This promotion in growth criteria was more pronounced at lower and moderate salinity levels used. The relative water content of both shoots and roots was exhibited a slight reduction (99.4% and 99.2%), however, the treatment of bean plants with Trichoderma harzianum (T24) resulted in higher relative water content especially in shoots (100.1% and 99.3%) of absolute control at 200 mM NaCl. It is worth to mention that Trichoderma treatments enhanced the relative water content of both shoots and roots especially at lower and moderate salinity levels used. The results concerning the leaf area (cm2/plant) of the various treatments are given in Table 1. The leaf area of faba bean Giza 429 exhibited a marked and progressive decrease with increasing salinity in the soil. The data also denoted that NaCl salinity exerted a great inhibitory effect on leaf area at all salinization levels used. The plants salinized with NaCl exhibited a reduction in shoot length, however, the root length was more dense and longer, compared with control especially at moderate salinity levels. The treatment of bean plants with Trichoderma resulted in the promotion of both shoot and root lengths up to 150 mM NaCl compared with untreated plants. Trichoderma treatments induced a marked and progressive increase in leaf area compared with the corresponding salinized plants. Moreover, the inhibitory effect of salinity was completely eliminated at all salinization level applied.
Acta Biologica Hungarica 65, 2014
454
G. K. Abd El-Baki and D. Mostafa
12
11
1
6
2
7
Fig. 1a. Electrophoretic banding profile of protein extracted from the leaves of faba bean (Vicia faba Giza 429) treated with NaCl alone or inoculated with Trichoderma harizianum (T24) + NaCl. Lane 1: control, lane 6: Control + T, lane 2: 50 mM NaCl, lane 7: 50 mM NaCl + T and lane 11: protein marker, lane 12: protein profile
Acta Biologica Hungarica 65, 2014
455
Interaction between NaCl and Trichoderma in Vicia faba
3
8
4
9
5
10
Fig. 1b. Electrophoretic banding profile of protein extracted from the leaves of faba bean (Vicia faba Giza 429) treatded with NaCl alone or inoculated with Trichoderma harizianum (T24) + NaCl. Lane 3: 100 mM NaCl, lane 8: 100 mM NaCl + T, lane 4: 150 mM NaCl, lane 9: 150 mM + T, lane 5: 200 mM NaCl and lane 10: 200 mM + T
Acta Biologica Hungarica 65, 2014
Acta Biologica Hungarica 65, 2014 81.1 3.7±1.5 22.6
%
200
% 4.19
13.3±1.41
150
5%
69.5
%
0.23
25.3
0.19±0.09
89.3
0.67±0.04
73.3
0.55±0.1
74.7
75 11.4±2.4
%
50.7 0.56±0.2
56.1 12.3±2.1
%
50
100
0.38±0.1
100
0.75±0.0
74.6
0.56±0.03
85.3
0.64±0.01
9.25±1.7
0
85.9
%
76.9
13.6±0.21
150 82.9
69.5
%
14.1±0.98
11.4±1.3
100
%
95.7
%
200
0.71±0.09
15.7±0.28
50
100
100
%
0.75±0.16
DW
16.4±3.8
FW
Root
0
NaCl (mM)
5.59
91.4
16±1.4
102.9
18.0±0.0
100
17.5±2.1
108.6
19±4.2
105.7
18.5±2.1
100
17.5±2.1
111.4
19.5±2.1
108.5
19±2.8
100
17.5±3.5
100
17.5±2.1
L
2.16
99.3
94.7
99.5
94.9
99.7
95.1
99.9
95.4
100.2
95.6
99.2
94.68
99.2
94.7
99.6
95.1
100.5
95.9
100
95.4
RWC
Data calculated as % of absolute control and means of 3 replications ±SD.
LSD
NaCl+ Trichoderma
NaCl
Treatment
3.49
25.8
6.1±1.6
78.03
16.7±2.1
83.6
17.9±1.1
102.3
21.9±0.5
90.6
19.4±1.7
81.3
17.4±1.4
75.7
16.2±1.3
68.2
14.6±1.6
101.8
21.8±1.1
100
21.4±2.1
FW
0.39
27.9
0.51±.014
79.2
1.45±0.3
85.8
1.57±0.1
99.5
1.82±0.1
94.5
1.73±0.16
92.3
1.69±0.16
80.3
1.47±0.03
67.2
1.23±0.07
100.5
1.84±0.08
100
1.83±0.37
DW
Shoot
5.43
48.1
12.5±3.5
78.8
20.5±2.1
84.6
22±1.4
109.6
28.5±2.1
100
26±0.0
67.3
17.5±3.5
73
19±1.4
75.7
19.7±1.9
96.1
25±0.0
100
26±4.2
L
2.15
100.1
91.5
99.9
91.3
99.8
91.2
100.2
91.6
99.8
91.2
99.4
90.9
99.4
90.9
100.1
91.5
100.1
91.5
100
91.4
RWC
1.26
64.8
19.5±0.01
81.7
24.5±0.04
85.3
25.6±0.9
96.4
29.01±0.0
93.4
28.1±0.1
44.5
13.4±0.6
33.2
16.9±0..1
45.5
13.7±0.1
54.4
16.4±0.6
100
30.1±0.0
Leaf area
Table 1 Fresh and dry weight (g), leaf area (cm2), length (cm) and relative water content of Vicia faba Giza 429 grown for 40 days from sowing on NaCl alone or NaCl+ Trichoderma
456 G. K. Abd El-Baki and D. Mostafa
Acta Biologica Hungarica 65, 2014 9.1±3.9 211.6 14.2±1.3 330.2
150 % 200 % 3.84
120.9
5%
5.2±0.14
%
325.6
%
100
14±2.4
200
144.1
125.6
%
%
5.4±1.3
150
6.2±1.5
243.9
%
50
10.1±0.52
100
295.3
162.7
%
12.7±0.6
7.0 ±1.0
50
0
100
%
%
4.3±1.2
Soluble protein
0
NaCl (mM)
0.55
84.6
0.44±0.05
307.6
1.6±1.4
173.1
0.9±0.02
38.5
0.2±0.03
96.2
0.5±0.03
461.5
2.4±0.13
175
0.91±0.4
211.5
1.1±0.26
140.4
0.73±0.14
100
0.52±0.1
Proline
Root
Data calculated as % of absolute control and means of 3 replications ±SD.
LSD
NaCl+ Trichoderma
NaCl
Treatment MDA
1.02
87.3
112.4±9.4
58.8
74.4±0.0
52.7
67.8±0.0
73.2
94.2±0.05
84.5
108.8±0.01
89.8
115.6±0.04
54.9
70.7±0.02
98.2
126.5±0.01
76.3
98.3±0.02
100
128.7±0.03
8.45
73.6
25.1±1.1
108.8
37.1±2.3
75.9
25.9±0.7
93.5
31.9±0.6
120.5
41.1±1.1
99.4
33.9±0.97
155.1
52.9±5.3
98.2
33.5±2.4
138.4
47.2±2.7
100
34.1±9.6
Soluble protein
1.54
118.8
1.2±0.60
207.9
2.1±0.12
178.2
1.8±0.3
192.1
1.94±1.4
178.2
1.8±0.1
277.2
2.8±0.41
277.2
2.8±0.9
425.7
4.3±1.1
200.9
2.03±0.21
100
1.01±0.12
Proline
Shoot
1.02
67.1
34.1±0.14
94.6
48.1±0.01
92.7
47.1±0.16
52.6
26.7±0.0
78.1
39.7±0.4
153.7
78.1±0.2
148.8
75.6±1.2
130.5
66.3±0.6
112.4
57.1±0.0
100
50.8 ±0.05
MDA
Table 2 Proline, soluble proteins (mg/gDW) and MDA content (n mol g–1 FW) in faba bean (Vicia faba Giza 429) grown for 40 days from sowing on NaCl alone or NaCl+ Trichoderma
Interaction between NaCl and Trichoderma in Vicia faba
457
458
G. K. Abd El-Baki and D. Mostafa
Fig. 2. Enhanced shoot development of plants developed from seeds treated with NaCl alone, or inoculated with Trichoderma harizanum (T24) + NaCl. Plants grown for 40 days from sowing
Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
459
Fig. 2. (cont.)
Acta Biologica Hungarica 65, 2014
460
G. K. Abd El-Baki and D. Mostafa
Fig. 2. (cont.)
The proline content increased progressively in shoots and roots of faba bean Giza 429 as the salinity rose in the soil (Table 2). Trichoderma treatments considerably retarded the accumulation of proline in shoots and roots as compared with corresponding salinized plants. Moreover, the amount of proline in shoots was markedly lower than in the absolute control samples, even at higher salinization level. The soluble protein contents in both shoots and roots of untreated plants were accumulated with increasing salinity especially at lower and moderate salinity levels applied, however, the treatments of plants with Trichoderma harzianum resulted in reduction of proteins especially in roots as compared with untreated plants (Table 2). The data demonstrated also that, all salinity levels induced irregular stimulation in soluble protein in both shoots and roots. Therefore, the highest values in roots were recorded at 200 mM NaCl, while, in shoots were recorded with 150 mM NaCl compared with absolute control. In roots, Trichoderma treatment resulted in a marked induction and irregular activation in the biosynthesis of the soluble proteins. This stimulatory effect in soluble protein biosynthesis accompanied with Trichoderma treatment was highly significant at higher salinity levels used, on the other hand, the shoot soluble fraction decreased at all salinity levels compared with corresponding salinized plants.
Acta Biologica Hungarica 65, 2014
461
Interaction between NaCl and Trichoderma in Vicia faba Table 3 Mineral contents of shoot and root (mg/g DW) of faba bean (Vicia faba Giza 429). Plants grown for 40 days from sowing under NaCl alone or (NaCl+ Trichoderma) Treatment
NaCl
NaCl + Trichoderma
LSD
NaCl (mM)
0
Root
Shoot
Na+
K+
K+/Na+
Na+
K+
K+/Na+
18.1±0.12
13.5±1.2
0.74
12.4±3.3
20.9±2.1
1.68
%
100
100
100
100
100
100
50
19.7±0.74
19.5±0.8
0.98
19.5±2.4
21.4±5.2
1.1
%
108.8
144.4
132.4
157.2
102.3
65.5
100
11.7±1.5
14.7±1.2
1.3
20.8±6.7
24.1±7.1
1.2
%
64.6
108.8
175.6
167.7
115.3
71.4
150
16.6±1.4
20±1.0
1.2
21.3±2.9
24.1±4.7
1.1
%
91.7
148.1
162.1
171.7
115.3
65.5
200
24.8±3.4
19.3±0.4
0.77
20.3±1.3
20.8±0.5
1.02
%
137.01
142.9
104.1
163.7
99.5
60.7
0
19.5±0.14
17.1±0.1
0.87
14.1±0.41
19.6±2.4
1.39
%
107.8
126.7
117.6
113.7
93.8
82.7
50
15±0.56
12.5±0.21
0.83
15.6±0.56
19.4±1.6
1.24
%
83.3
92.6
112.2
125.8
92.8
73.8
100
21.8±5.1
16.5±4.1
0.75
19.3±3.2
20±4.1
1.03
%
120.4
122.2
101.3
155.6
95.6
61.3
150
17.1±4.3
16±0.28
0.93
18.7±7.2
19.9±1.9
1.06
%
94.4
118.5
125.7
150.8
95.2
63.1
200
19.4±0.84
12.8±1.1
0.65
29.3±5.0
23.2±4.8
0.79
%
107.2
94.8
87.8
236.2
111
47.02
5%
5.56
3.38
0.61
8.98
8.74
0.97
Data calculated as % of absolute control and means of three replications ±SD.
The data in Table 2 revealed also that MDA content increased progressively in both shoot and root with increasing salinity in the soil. This increasing trend was highly significant at all salinization levels used. Trichoderma treatment progressively inhibited the accumulation of MDA as compared with the corresponding salinized plants, regardless the salinity level used. It is worth to mention also that, MDA content in shoots were much lower than roots whatever the salinity level applied. The mineral composition of bean plants assimilated in both Na+ and K+ as well as K+/Na+ ratios in both shoots and roots were presented in Table 3. Both Na+ and K+ ions were increased by increasing salinity except at (100 mM NaCl in roots) which
Acta Biologica Hungarica 65, 2014
Acta Biologica Hungarica 65, 2014 16
Band1 Band2 Band3 Band4 Band5 Band6 Band7 Band8 Band9 Band10 Band11 Band12 Band13 Band14 Band15 Band16 Band17 Band18 Band19 Band20 Band21 Band22
Total bands
50
14
270.845 223.823 201.588 165.749 152.080 143.600 134.000 120.282 113.192 104.209 98.564 79.282 67.539 64.422 – – – – – – – –
Plants grown for 40 days from sowing.
0.0
271.761 223.823 203.296 165.749 145.552 134.000 130.651 120.282 112.621 102.465 87.287 83.119 79.015 74.358 68.111 64.639 – – – – – –
MW-bp
18
268.569 222.693 199.894 165.190 152.080 143.600 134.000 125.042 117.276 113.192 105.625 98.564 95.939 91.975 79.954 74.988 66.073 64.096 – – – –
100
15
267.213 221.568 189.706 161.606 151.312 143.116 140.721 133.549 123.992 119.271 114.151 107.061 102.812 98.564 95.939 – – – – – – –
150
18
268.569 221.568 214.215 187.162 160.248 153.369 143.116 138.133 134.000 123.992 119.271 114.151 110.363 106.520 102.465 98.564 93.068 75.623 – – – –
200
14
265.864 226.482 199.894 184.964 175.537 169.425 160.248 154.147 141.914 131.093 121.917 113.192 105.625 101.948 – – – – – – – –
C+T
19
261.415 250.193 226.482 199.894 184.964 175.537 170.285 159.439 154.147 144.329 136.511 128.899 122.329 113.767 105.625 100.242 96.426 91.975 88.325 – – –
50+T
20
263.630 251.463 226.482 199.894 185.590 176.130 170.285 159.439 152.594 145.061 133.549 126.102 115.703 111.298 102.465 95.939 92.286 88.325 85.393 69.622 – –
100+T
19
267.213 255.742 226.482 199.894 185.590 177.024 167.153 159.439 152.080 145.061 133.549 125.677 117.276 111.298 104.209 95.939 92.755 88.325 85.682 – – –
150+T
Table 4 SDS electrophoresis analysis of soluble protein bands produced by the faba bean (Vicia faba Giza 429) cultivated under NaCl alone or NaCl+ Trichoderma 200+T
22
262.742 248.091 226.482 199.894 185.590 177.024 167.153 152.080 145.061 137.668 125.677 117.672 112.241 102.465 96.426 92.755 88.325 85.682 71.527 69.037 66.633 54.143
462 G. K. Abd El-Baki and D. Mostafa
Interaction between NaCl and Trichoderma in Vicia faba
463
exhibited slight reduction in both ions. The treatment of bean plants with Trichoderma harzianum enhanced the accumulation of both ions regardless the tissue tested or organ analyzed. The K+/Na+ ratios exhibited different responses with different treatments. In salinized bean plants the ratio decreased in shoots and increased in roots except at 200 mM NaCl. The treatment of plants with Trichoderma harzianum decreased the K+/Na+ ratios with increasing salinity regardless the organ tested. It is worth to mention that the ratios of K+/Na+ in plants treated with Tricoderma were less compared with corresponding salinized plants. Exposure of plants to different concentrations of NaCl salinity and others treated with Trichoderma harzianum produced marked changes in their protein pattern, three types of alterations were observed as follows (see also Fig. 2a,b). (i) The Synthesis of certain proteins declined significantly, however, (14:18 KDa) protein bands were detected only under salinity. (ii) Specific synthesis of certain other proteins were markedly observed in both treatments, however the number of new protein bands in plants treated with Trichoderma were much higher than those of salted plants. (iii) Synthesis of a set specific protein was induced de novo in plant treated with Tricho derma harzianum. The results revealed also that (14–18 protein bands) were detected only under salinity while, plants treated with Trichoderma harzianum displayed (14–22 protein bands) Table 4. The 143 KDa protein bands were specific markers for faba bean Giza 429 under salinity and 134 KDa protein band was specific for high salinity dose only. However, five protein bands of molecular weight (226.482; 199.894; 185.590; 152.08 and 88.325 KDa) were specific markers for interaction between Trichoderma and salinity. It is worth to mention that, some low molecular weight of (71–54 KDa) were specific for 200 mM NaCl + Trichoderma. Salinity specifically enhanced the synthesis of the set proteins and also induced de novo synthesis of another set of proteins. On the other side, interaction between salinity and Trichoderma not only improved growth but also enhanced protein synthesis. A number of protein bands also appeared in plants treated with Trichoderma harzianum as compared with control (untreated plants).
Discussion The growth response of bean plants (Vicia faba Giza 429) to salt stress was different from those treated with Trichoderma harzianum (T24). The fresh weight was decreased by (81.5%) of control plants, however, the reduction in dry weight was less compared with the reduction in fresh weight (92.3%) under 200 mM NaCl. The treatment with Trichoderma harzianum resulted in reduction (91.5% and 29.4%) of both fresh and dry weight under 200 mM NaCl. Certain Trichoderma spp. have beneficial effects on plant growth and enhance resistance to both biotic and abiotic stresses, it can colonize plant root and its rhizosphere, second it improves plant health through increasing plant growth, ultimately, root growth [30]. Early work revealed that, Trichoderma promotes growth response in radish, pepper, cucumber and tomato [5]. Acta Biologica Hungarica 65, 2014
464
G. K. Abd El-Baki and D. Mostafa
Further studies demonstrated also that Trichoderma increases root development and crop yield, the proliferation of secondary roots, seedlings fresh weight and foliar area [14]. Recently, Tucci et al. [29] have demonstrated the effects of the plant genetic background on the outcome of the interaction between different tomato lines and two bio-control strains of Trichoderma atroviride, Trichoderma harzianum and in at least one tomato cultivar the Trichoderma treatment did not exert any plant growth promotion effect and was even seen to be detrimental. Studies also showed that, Trichoderma treatment in tobacco increased fresh weight of root and leaf area index (300%), lateral roots (300%) and the leaf (140%) [1]. This is in accordance with our results of bean plants treated with Trichoderma harzianum (T24) that improved widely the leaf area (155.7%) of control. The increase in relative water content of bean plants under different treatments revealed that, there is no detrimental effect in water uptake especially when the plants treated with Trichoderma harzianum which exert water content (100.3%) of control plants. Recently, Mastouri et al. [22] reported that the treatment of tomato seeds with Trichoderma harzianum ameliorates water, osmotic, salinity, chilling and heat stresses by inducing physiological protection in plants against oxidative damage. Trichoderma spp. produce auxins that are able to stimulate plant growth and root development. The proline content of salted plants as well as inoculated with Trichoderma harzianum was enhanced with increasing salinity, however, soluble proteins increased with salinity and decreased with Trichoderma treatments. The proline accumulation was used as a suitable marker for salt tolerance in plants treated with Trichoderma harzianum which enhanced proline synthesis under salt stress conditions. The accumulation of proline under stress protects the cell by balancing the osmotic level of cytosol with that of vacuole and external environment, additionally, these solutes interact with cellular macromolecules such as enzymes and stabilize their structure and functions [11]. It has been reported that higher osmolyte concentration helps in the maintenance of comparatively higher relative water content and antioxidant enzyme activity which in accordance with our results in water content (Table 1) and MDA contents in Table 3. In cucumber [26] similar trends of increase in proline with increasing salinity concentration were reported. However, proline accumulation cannot be used as a sole criterion for salt tolerance as it also accumulates under other stresses such as higher temperature and drought [17]. Numerous proteins induced in response to Trichderma were involved in stress and defense responses. One of common mechanisms could be the control of damage caused by the reactive oxygen species. These molecules that play a crucial signaling role during abiotic stresses at higher concentrations cause cellular and molecular damage, most ROS are unstable and are quickly converted to H2O2 which is either reduced to water during the ascorbate GSH cycle, converted to water and oxygen by catalase enzyme, or used as a substrate of peroxidase enzyme [8]. However, in the presence of transition metal ions, H2O2 is converted to hydroxyle radicals, which start chain reactions leading to the peroxidation of membrane lipids [2] that resulted in the loss of membrane integrity and also damage to other macromolecules. Thus measurement of MDA contents Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
465
served as a reliable indication of oxidative damage. During a biotic stress we found an increase of (MDA) contents in faba bean Giza 429 under salt stress, but plants treated with Trichoderma harzianum had lower (MDA) content which follow the results of Harman and Mattick [13]. This evidence supports that Trichoderma harzianum stimulate plant growth as well as leaf area and ameliorates stress by inducing plant physiological protection against oxidative damage. The mechanisms by which Trichoderma spp. induce such changes are not known, however, enhanced ROS level could act as a signal to regulate expression of the related genes. An increase in ROS has been detected 5 to 10 minutes after treating soybean cell culture with culture filtrate of Trichoderma otroviride [25]. Such signals along with Ca+2 signaling can induce plant ROS scavenging mechanisms resulting in elevated protection against the oxidative damage. MDA, which is indicative of oxidative stress, increased in plants. Lipid peroxidation is the main index of the increase in active free radicals, and MDA is the main by-product of the lipid peroxidation process. Our finding showed that the degree of accumulation of MDA content has been reported to be indicative of the rate of lipid peroxidation due to salt stress, as lipid peroxidation is the symptom mostly ascribed to oxidative damage it is often used as an indicator of increased damage [19]. Lowest MDA content was recorded under inoculation of Trichoderma harzianum (Table 3), which might be due to an increase in expression of stress related proteins. Similar effect was observed by the inoculation of Tricoderma harzianum in maize plants. Under environmental stress, when ROS are produced, these detoxifying proteins triggered by Trichoderma inoculation act as scavenging enzyme and play a central role in protecting the cell from oxidative damage. Salinity dominated by Na+ and Cl– not only reduces K+ availability but also reduces K+ transport and mobility to growing regions of the plant that affects the quality of both vegetative and reproductive organs [10]. Moreover, a number of studies have shown that high concentrations of Na+ and Cl– in the soil solutions may depress nutrient–ion activities and produce extreme ratios of Na+/K+ in the plants, making the plants to be susceptible to osmotic and specific ion injury as well as to nutritional disorders that result in reduced yield and quality [27]. The results of this study showed that salinity caused an increase in Na+ as well as K+ concentrations which means there is no antagonistic effect between Na+ and K+ in this cultivar which may be used as a marker for salt tolerance criteria. However, Na+ concentration decreased under treatments of seeds with Trichoderma harzianum especially under lower and moderate salinity levels (till 150 mM NaCl). However some biological treatments reduced the Na+ uptake of plants and/or increased the K+ uptakes compared to control treatment under salt stress, thus increasing the ratio of K+/Na+. A positive correlation was found between fresh weight and K+ especially at lower salinity levels, however, a negative correlation between fresh weight and Na+ content was observed. Some studies indicate that an increase in concentration of K+ and Ca+2 in plants under salt stress could ameliorate the deleterious effects of salinity on growth and yield [27]. The response of crops to salinity is a complex phenomenon involves changes in certain metabolites, alteration in the behavior of many enzymes and synthesis of new Acta Biologica Hungarica 65, 2014
466
G. K. Abd El-Baki and D. Mostafa
sets of proteins [23]. Studying the pattern of protein synthesis under salt stress may help to identify a protein(s) associated with stress. In the present study, salinity induced the synthesis of newly proteins and simultaneously reduced other protein sets as compared with control plants. In that manner some authors working with chickpea (Cicer arietinum) reported that the total number of protein bands do not change under low 20 mM NaCl but was dramatically reduced at high salt used 40 mM NaCl. Tammam [28] found also that, salt treatment of broad bean seedling resulted in the disappearance of five polypeptides, while the peptides with low molecular mass increased in their intensity on the gel. The disappearance of certain proteins under salt stress may be due to either an inhibition of its mRNA transcription and/or translation, and increase in RNA-ase or through dissociation of polyribosomes under stress [4]. The treatments of faba bean Giza 429 with Trichoderma harzianum resulted in marked changes in protein patterns, several polypeptides were apparently suppressed whereas others were induced. The results revealed also that four bands at molecular weight 54.91, 46.80, 34.34 and 18.06 kDa were induced under Trichoderma treatment as compared with untreated plants, the induction of these bands differed with differing dose of salinity. In this respect [6] working with soybean and chickpea treated with Trichoderma demonstrated that the ability of Trichoderma to reverse the lowering of protein level caused by salt stress might be due to enhancing of nitrogen fixation and nitrogen uptake by plants. They also revealed that Trichoderma harzianum could produce nitrogen oxide (NO) which is the coding for enzyme involved in L-arginine which is important for protein and arginine biosynthesis. In addition, numerous proteins induced in response to Trichoderma harzianum could involved in stress and defense responses. These extra bands of protein which induced in faba bean Giza 429 under NaCl alone or inoculated with Trichoderma suggested that these protein bands may play an important role in the mechanism of salinity tolerance. The newly synthesized proteins together with proline could act as components of a salt tolerance mechanism in our bean plants. They may also function as compatible cytoplasmic solutes in osmotic adjustment to equalize the osmotic potential of the cytoplasm with the vacuoles under adverse conditions of salinity. Acknowledgement The authors would like to thank Prof. Dr. M. A. A. Shaddad, Assuit University for his valuable advices and revising the manuscript. References 1. Altintas, S., Bal, U. (2008) Effects of the commercial product based on Trichoderma harzianum on plant, bulb and yield characteristics of onion. Sci. Hortic. 116, 219–222. 2. Aust, S. D., Morehouse, L. A., Thomas, C. (1985) Role of metals in oxygen radical reactions. J. Free Radic. Biol. Med. 1, 3–26. 3. Bates, L. S., Waldern, R. P., Teare, I. D. (1973) Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
467
4. Bewlfey, J. D., Oliver, M. J. (1983) Responses to a changing environment at the molecular level: Does desiccation, modulate protein synthesis at the transcriptional or translational level in a tolerant plant? Current Topics Plant Biochem. Biophys. 2, 145–146. 5. Chang, Y. C., Baker, R., Kleifeld, O., Chet, I. (1986) Increased growth of plants in the presence of the biological control agent Trichoderma harzianum, Plant Disease 70, 145–148. 6. Egberongbe, H. O., Akintokun, A. K., Babalola, O. O., Bankole, M. O. (2010) The effect of Glomus mosseae and Tricoderma harizianum on proximate analysis of soybean (Glycine max. (L.) Merrill) Seed grown in sterilized and unsterilized soil. J. Agric. Extension Rural Development 2, 54–58. 7. FAO (2000) Global network on integrated soil management for sustainable use of salt effected soils, Available in: http://www.fao.org/ag/AGL/agll/spush/intro.htm. 8. Foyer, C. H., Kiddle, G., Antoniw, J., Bernard, S., Verrier, P. J., Pastori, G. M., Noctor, G. (2003) The role of antioxidant-mediated signal transduction during stress. Mol. Cell. Proteomics 2, 682. 9. Gachomo, E. W., Kotchoni, S. O. (2008) The use of T. harzianum and T. viride as potential biocontrol agents against peanut microflora and their effectiveness in reducing aflatoxin contamination of infected kernels. Biotechnol. 7, 439–447. 10. Grattan, S. R., Grieve, C. M. (1999) Salinity–mineral nutrient relations in horticultural crops. Sci. Hortic. 78, 127–157. 11. Greenway, H., Munns, H. (1980) Mechanisms of salt tolerance in non halophytes. Annu. Rev. Plant Physiol. 31, 149–190. 12. Grichko, V. P., Glick, B. R. (2001) Amelioration of flooding stress by ACC deaminase containing plant growth-promoting bacteria. Plant Physiol. Biochem. 39, 11–17. 13. Harman, G. E., Mattick, L. R. (1976) Association of lipid oxidation with seed aging and death. Nature 260, 323–324. 14. Harman, G. E. (2000) Myths and dogmas of biocontrol – changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Disease 84, 377–393. 15. Harman, G. E., Björkman, T. (1998) Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement. In: Harman, G. E., Kubicek, C. P. (eds), Trichoderma and Gliocladium. 2, Taylor & Francis, London, United Kingdom, pp. 229–265. 16. Heath, R. L., Packer, L. (1968) Photoperoxidation in isolated chloroplast. 1. Kinetics and stiochiometry of fatty acid peroxidation. Arch. Bioch. Biophys. 125, 189–198. 17. Hong, Z. L., Lakkineni, K., Zhang, Z. M., Verma, D. P. S. (2000) Removal of feedback inhibition of delta (1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol. 122, 1129–1136. 18. Howell, C. R. (2003) Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, 4–10. 19. Khan, M. H., Panda, S. K. (2008) Alterations in root lipid peroxidation and antioxidative responses in two rice cultivars under NaCl salinity stress. Acta Physiol. Plant. 30, 91–98. 20. Laemmli, U. K. (1970) Cleavage of structure proteins during assembly of the head of bacteriophage T4. Nature 277, 680–685. 21. Lowery, O. H., Rosebrough, N. H., Farr, A. L., Randall, R. J. (1951) Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 291–297. 22. Mastouri, F., Björkman, T., Harman, G. E. (2010) Seed treatment with Trichoderma harzianum alleviates biotic, abiotic, and physiological stresses in germinating seeds and seedlings. Phytopathol. 100, 1213–1221. 23. Michal, S. G., Harman, E. (2008) The molecular basis of shoot responses of maize seedling to Trichoderma harizianum T22 inoculation of the root. Plant Physiol. 147, 2147–2163. 24. Munns, R., Termaat, A. (1986) Whole plant responses to salinity. Aust. J. Plant Physiol. 13, 143–160. 25. Navazio, L., Baldan, B., Moscatiello, R., Zuppini, A., Woo, S. L., Mariani, P., Lorito, M. (2007) Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride. BMC Plant Biol. 7, 41–49. 26. Shiqing, S., Shirong, G., Qingmao, S., Zhigang, Z. (2006) Physiological effects of exogenous salicylic acid on cucumber seedling under salt stress. Acta Hortic. Sin. 33, 68–72. Acta Biologica Hungarica 65, 2014
468
G. K. Abd El-Baki and D. Mostafa
27. Sivritepe, N., Sivritepe, H. O., Eris, A. (2003) The effects of NaCl priming on salt tolerance in melon seedlings grown under saline conditions. Sci. Hortic. 97, 229–237. 28. Tammam, A. A. (2003) Response of Vicia faba plants to the interactive effect of sodium chloride salinity and salicylic acid treatment. Acta Agron. Hungarica 51, 239–248. 29. Tucci, M., Ruocco, M., De Masi, L., De Palma, M., Lorito, M. (2011) The beneficial effect of Trichoderma spp. on tomato is modulated by the plant genotype. Mol. Plant Pathol. 12, 341–354. 30. Vinale, F., Sivasithamparam, K., Ghisalberti, E. L., Marra, R., Barbetti, M. J., Li, H., Woo, S. L., Lorito, M. (2008) A novel role for R. Hermosa and others Trichoderma secondary metabolites in the interactions with plants. Physiol. Mol. Plant Pathol. 72, 80–86. 31. Wiersma, T. V., Bailey, T. B. (1975) Estimation of leaflet, trifoliate and total leaf area of soybean. Agron. J. 176, 26–30. 32. Williams, S., Twine, M. (1960) Flame photometric method for sodium, potassium and calcium. In: Peach, K., Tracey, M. V. (eds) Modern Methods of Plant Analysis. Vol. 5. Springer Verlag, Berlin, pp. 3–5.
Acta Biologica Hungarica 65, 2014
THE POTENTIALITY OF TRICHODERMA HARZIANUM IN ALLEVIATION THE ADVERSE EFFECTS OF SALINITY IN FABA BEAN PLANTS G. K. Abd El-Baki1 * and Doaa Mostafa2 1 Botany
and 2 Microbiology Department, Faculty of Science, Minia University, El-Minia, 61519, Egypt (Received: January 17, 2014; accepted: March 24, 2014)
The interaction between sodium chloride and Trichoderma harzianum (T24) on growth parameters, ion contents, MDA content, proline, soluble proteins as well as SDS page protein profile were studied in Vicia faba Giza 429. A sharp reduction was found in fresh and dry mass of shoots and roots with increasing salinity. Trichoderma treatments promoted the growth criteria as compared with corresponding salinized plants. The water content and leaf area exhibited a marked decrease with increasing salinity. Trichoderma treatments induced a progressive increase in both parameters. Both proline and MDA contents were increased progressively as the salinity rose in the soil. Trichoderma treatments considerably retarded the accumulation of both parameters in shoots and roots. Both Na+ and K+ concentration increased in both organs by enhancing salinity levels. The treatment with Trichoderma harzianum enhanced the accumulation of both ions. Exposure of plants to different concentrations of salinity, or others treated with Trichoderma harzianum produced marked changes in their protein pattern. Three types of alterations were observed: the synthesis of certain proteins declined significantly, specific synthesis of certain other proteins were markedly observed and synthesis of a set specific protein was induced de novo in plant treated with Trichoderma harzianum. Keywords: Lipid peroxidation – proline – proteins – potassium – salinity – sodium – Trichoderma
Introduction High concentrations of salts in soils account for large decreases in yield of a wide variety of crops all over the world. Globally, more than 770,000 km2 of land is saltaffected by secondary salinization: 20% of irrigated land, and about 2% of dry agricultural land [7]. Salt stress affects many aspects of plant metabolism and, as a result, growth and yields are reduced. Excess salt in the soil solution may adversely affect plant growth either through osmotic inhibition of water uptake by roots or specific ion effects, specific ion effects may cause direct toxicity or, alternatively, the insolubility or competitive absorption of ions may affect the plant’s nutritional balance. Salinity was shown to increase the uptake of Na+ or decrease the uptake of Ca2+ and K+, the * Corresponding author; e-mail address: [email protected] 0236-5383/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
452
G. K. Abd El-Baki and D. Mostafa
growth of plants is ultimately reduced by salinity stress although plant species differ in their tolerance to salinity [24]. Plant growth-promoting rhizobacteria (PGPR) and fungi can facilitate plant growth indirectly by reducing plant pathogens, or directly by facilitating the uptake of nutrients from the environment, by influencing phytohormone production (e.g. auxin, cytokinin, or gibberellin), and/or by enzymatic lowering of plant ethylene levels [12]. In addition to facilitating the growth of plant, plant growth-promoting microorganisms can protect plants from the deleterious effects of some stresses including phytopathogens [15]. It is thought that the depressive effect of salinity on germination could be related to a decline in endogenous levels of hormones, however, incorporation of Trichoderma during seed biopriming treatments in many cereal and vegetable crops has resulted in increased levels of plant growth hormones and improved seed performance [18]. Biocontrol agent, Trichoderma, releases a variety of compounds that induce resistance responses to biotic and abiotic stresses, several studies have shown also that root colonization by Trichoderma harzianum results in increased level of plant enzymes, including various peroxidases, chitinases, β-1,3-glucanases, lipoxygenase-pathwayhydroperoxidelyase and compounds like phytoalexins and phenols to provide durable resistance against stress [9]. Accumulation of some compatible solutes has been observed under salt stress conditions and has been suggested as part of mechanism(s) that controls salt tolerance in plants. Proline is one of the best known solutes, however, its relative importance for tolerance and precise protective function during stress require further investigations. The objective of this study was to investigate the effects of seed inoculation of faba bean with Trichoderma strains upon growth, physiological and biochemical parameters under normal and saline conditions. In this study we have examined and compared the proline content of treated and untreated bean plants with different degrees of salt stress, in order to exploit Trichoderma strains in relation to salt stress tolerance in beans. This research is important as it may reveal the role of Trichoderma in imparting stress resistance and provides insight into the potential of bean plants to adapt to saline conditions.
Materials and Methods Faba bean seeds (Vicia faba Giza 429) were germinated in pots using sand clay soil (2:1 w/w). The plants were divided in two groups, one of them was treated with NaCl alone and the other with NaCl + Trichoderma harzianum (T24) spore suspension (108 CFU ml–1). Five seeds per pot, three replications for each, were cultivated and inoculated with 200 µl spore suspension for each or left without inoculation (NaCl alone). The seeds were left to germinate and grow for about three weeks and then treated with different salt concentrations (0.0, 50, 100, 150 and 200 mM NaCl) by top irrigation. The plants left to further growth with different treatments for about 40 days from Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
453
germination, 20-days from salt treatment under day night temperature of (20/25 °C and relative humidity 85%) plants were harvested. Subsequently roots were rapidly rinsed in cold water or with cold glycinebetaine solutions equimolar to the salted nutrient solution in order to prevent osmotic shock. After rinsing, roots were carefully blotted with tissue paper. To determine the dry matter, the freshly harvested organs (shoots and roots) were dried in an aerated oven at 105 °C for 24 hours. Water content in both organs were determined. Leaf area were carried out by Wiersma et al. [31]. The level of lipid peroxidation was measured in terms of malondialdehyde (MDA) content. MDA a product of lipid peroxidation following the method of [16]. Polyacrylamide gel electrophoresis was carried out according to Laemmli [20] with 12% acrylamide + 1% SDS. The soluble proteins were determined according to the method adopted by Lowery et al. [21]. Free proline was determined according to Bates et al. [3]. Soluble minerals were determined in boiled water extract by flame photometer method [32] using a Carl Zeiss flame photometer. The data of all experiments were subjected to one-way analysis variance and means were compared using the least significant difference test (L.S.D.) using statistical program (Sta. Base. Exe.) on computer.
Results From the data in Table 1 and Fig. 2 it can be seen that, there is a sharp reduction in fresh and dry mass of shoots and roots of faba bean (Vicia faba Giza 429) with increasing salinity in the soil. Trichoderma treatments promoted the growth criteria of bean plants as compared with corresponding salinized plants. This promotion in growth criteria was more pronounced at lower and moderate salinity levels used. The relative water content of both shoots and roots was exhibited a slight reduction (99.4% and 99.2%), however, the treatment of bean plants with Trichoderma harzianum (T24) resulted in higher relative water content especially in shoots (100.1% and 99.3%) of absolute control at 200 mM NaCl. It is worth to mention that Trichoderma treatments enhanced the relative water content of both shoots and roots especially at lower and moderate salinity levels used. The results concerning the leaf area (cm2/plant) of the various treatments are given in Table 1. The leaf area of faba bean Giza 429 exhibited a marked and progressive decrease with increasing salinity in the soil. The data also denoted that NaCl salinity exerted a great inhibitory effect on leaf area at all salinization levels used. The plants salinized with NaCl exhibited a reduction in shoot length, however, the root length was more dense and longer, compared with control especially at moderate salinity levels. The treatment of bean plants with Trichoderma resulted in the promotion of both shoot and root lengths up to 150 mM NaCl compared with untreated plants. Trichoderma treatments induced a marked and progressive increase in leaf area compared with the corresponding salinized plants. Moreover, the inhibitory effect of salinity was completely eliminated at all salinization level applied.
Acta Biologica Hungarica 65, 2014
454
G. K. Abd El-Baki and D. Mostafa
12
11
1
6
2
7
Fig. 1a. Electrophoretic banding profile of protein extracted from the leaves of faba bean (Vicia faba Giza 429) treated with NaCl alone or inoculated with Trichoderma harizianum (T24) + NaCl. Lane 1: control, lane 6: Control + T, lane 2: 50 mM NaCl, lane 7: 50 mM NaCl + T and lane 11: protein marker, lane 12: protein profile
Acta Biologica Hungarica 65, 2014
455
Interaction between NaCl and Trichoderma in Vicia faba
3
8
4
9
5
10
Fig. 1b. Electrophoretic banding profile of protein extracted from the leaves of faba bean (Vicia faba Giza 429) treatded with NaCl alone or inoculated with Trichoderma harizianum (T24) + NaCl. Lane 3: 100 mM NaCl, lane 8: 100 mM NaCl + T, lane 4: 150 mM NaCl, lane 9: 150 mM + T, lane 5: 200 mM NaCl and lane 10: 200 mM + T
Acta Biologica Hungarica 65, 2014
Acta Biologica Hungarica 65, 2014 81.1 3.7±1.5 22.6
%
200
% 4.19
13.3±1.41
150
5%
69.5
%
0.23
25.3
0.19±0.09
89.3
0.67±0.04
73.3
0.55±0.1
74.7
75 11.4±2.4
%
50.7 0.56±0.2
56.1 12.3±2.1
%
50
100
0.38±0.1
100
0.75±0.0
74.6
0.56±0.03
85.3
0.64±0.01
9.25±1.7
0
85.9
%
76.9
13.6±0.21
150 82.9
69.5
%
14.1±0.98
11.4±1.3
100
%
95.7
%
200
0.71±0.09
15.7±0.28
50
100
100
%
0.75±0.16
DW
16.4±3.8
FW
Root
0
NaCl (mM)
5.59
91.4
16±1.4
102.9
18.0±0.0
100
17.5±2.1
108.6
19±4.2
105.7
18.5±2.1
100
17.5±2.1
111.4
19.5±2.1
108.5
19±2.8
100
17.5±3.5
100
17.5±2.1
L
2.16
99.3
94.7
99.5
94.9
99.7
95.1
99.9
95.4
100.2
95.6
99.2
94.68
99.2
94.7
99.6
95.1
100.5
95.9
100
95.4
RWC
Data calculated as % of absolute control and means of 3 replications ±SD.
LSD
NaCl+ Trichoderma
NaCl
Treatment
3.49
25.8
6.1±1.6
78.03
16.7±2.1
83.6
17.9±1.1
102.3
21.9±0.5
90.6
19.4±1.7
81.3
17.4±1.4
75.7
16.2±1.3
68.2
14.6±1.6
101.8
21.8±1.1
100
21.4±2.1
FW
0.39
27.9
0.51±.014
79.2
1.45±0.3
85.8
1.57±0.1
99.5
1.82±0.1
94.5
1.73±0.16
92.3
1.69±0.16
80.3
1.47±0.03
67.2
1.23±0.07
100.5
1.84±0.08
100
1.83±0.37
DW
Shoot
5.43
48.1
12.5±3.5
78.8
20.5±2.1
84.6
22±1.4
109.6
28.5±2.1
100
26±0.0
67.3
17.5±3.5
73
19±1.4
75.7
19.7±1.9
96.1
25±0.0
100
26±4.2
L
2.15
100.1
91.5
99.9
91.3
99.8
91.2
100.2
91.6
99.8
91.2
99.4
90.9
99.4
90.9
100.1
91.5
100.1
91.5
100
91.4
RWC
1.26
64.8
19.5±0.01
81.7
24.5±0.04
85.3
25.6±0.9
96.4
29.01±0.0
93.4
28.1±0.1
44.5
13.4±0.6
33.2
16.9±0..1
45.5
13.7±0.1
54.4
16.4±0.6
100
30.1±0.0
Leaf area
Table 1 Fresh and dry weight (g), leaf area (cm2), length (cm) and relative water content of Vicia faba Giza 429 grown for 40 days from sowing on NaCl alone or NaCl+ Trichoderma
456 G. K. Abd El-Baki and D. Mostafa
Acta Biologica Hungarica 65, 2014 9.1±3.9 211.6 14.2±1.3 330.2
150 % 200 % 3.84
120.9
5%
5.2±0.14
%
325.6
%
100
14±2.4
200
144.1
125.6
%
%
5.4±1.3
150
6.2±1.5
243.9
%
50
10.1±0.52
100
295.3
162.7
%
12.7±0.6
7.0 ±1.0
50
0
100
%
%
4.3±1.2
Soluble protein
0
NaCl (mM)
0.55
84.6
0.44±0.05
307.6
1.6±1.4
173.1
0.9±0.02
38.5
0.2±0.03
96.2
0.5±0.03
461.5
2.4±0.13
175
0.91±0.4
211.5
1.1±0.26
140.4
0.73±0.14
100
0.52±0.1
Proline
Root
Data calculated as % of absolute control and means of 3 replications ±SD.
LSD
NaCl+ Trichoderma
NaCl
Treatment MDA
1.02
87.3
112.4±9.4
58.8
74.4±0.0
52.7
67.8±0.0
73.2
94.2±0.05
84.5
108.8±0.01
89.8
115.6±0.04
54.9
70.7±0.02
98.2
126.5±0.01
76.3
98.3±0.02
100
128.7±0.03
8.45
73.6
25.1±1.1
108.8
37.1±2.3
75.9
25.9±0.7
93.5
31.9±0.6
120.5
41.1±1.1
99.4
33.9±0.97
155.1
52.9±5.3
98.2
33.5±2.4
138.4
47.2±2.7
100
34.1±9.6
Soluble protein
1.54
118.8
1.2±0.60
207.9
2.1±0.12
178.2
1.8±0.3
192.1
1.94±1.4
178.2
1.8±0.1
277.2
2.8±0.41
277.2
2.8±0.9
425.7
4.3±1.1
200.9
2.03±0.21
100
1.01±0.12
Proline
Shoot
1.02
67.1
34.1±0.14
94.6
48.1±0.01
92.7
47.1±0.16
52.6
26.7±0.0
78.1
39.7±0.4
153.7
78.1±0.2
148.8
75.6±1.2
130.5
66.3±0.6
112.4
57.1±0.0
100
50.8 ±0.05
MDA
Table 2 Proline, soluble proteins (mg/gDW) and MDA content (n mol g–1 FW) in faba bean (Vicia faba Giza 429) grown for 40 days from sowing on NaCl alone or NaCl+ Trichoderma
Interaction between NaCl and Trichoderma in Vicia faba
457
458
G. K. Abd El-Baki and D. Mostafa
Fig. 2. Enhanced shoot development of plants developed from seeds treated with NaCl alone, or inoculated with Trichoderma harizanum (T24) + NaCl. Plants grown for 40 days from sowing
Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
459
Fig. 2. (cont.)
Acta Biologica Hungarica 65, 2014
460
G. K. Abd El-Baki and D. Mostafa
Fig. 2. (cont.)
The proline content increased progressively in shoots and roots of faba bean Giza 429 as the salinity rose in the soil (Table 2). Trichoderma treatments considerably retarded the accumulation of proline in shoots and roots as compared with corresponding salinized plants. Moreover, the amount of proline in shoots was markedly lower than in the absolute control samples, even at higher salinization level. The soluble protein contents in both shoots and roots of untreated plants were accumulated with increasing salinity especially at lower and moderate salinity levels applied, however, the treatments of plants with Trichoderma harzianum resulted in reduction of proteins especially in roots as compared with untreated plants (Table 2). The data demonstrated also that, all salinity levels induced irregular stimulation in soluble protein in both shoots and roots. Therefore, the highest values in roots were recorded at 200 mM NaCl, while, in shoots were recorded with 150 mM NaCl compared with absolute control. In roots, Trichoderma treatment resulted in a marked induction and irregular activation in the biosynthesis of the soluble proteins. This stimulatory effect in soluble protein biosynthesis accompanied with Trichoderma treatment was highly significant at higher salinity levels used, on the other hand, the shoot soluble fraction decreased at all salinity levels compared with corresponding salinized plants.
Acta Biologica Hungarica 65, 2014
461
Interaction between NaCl and Trichoderma in Vicia faba Table 3 Mineral contents of shoot and root (mg/g DW) of faba bean (Vicia faba Giza 429). Plants grown for 40 days from sowing under NaCl alone or (NaCl+ Trichoderma) Treatment
NaCl
NaCl + Trichoderma
LSD
NaCl (mM)
0
Root
Shoot
Na+
K+
K+/Na+
Na+
K+
K+/Na+
18.1±0.12
13.5±1.2
0.74
12.4±3.3
20.9±2.1
1.68
%
100
100
100
100
100
100
50
19.7±0.74
19.5±0.8
0.98
19.5±2.4
21.4±5.2
1.1
%
108.8
144.4
132.4
157.2
102.3
65.5
100
11.7±1.5
14.7±1.2
1.3
20.8±6.7
24.1±7.1
1.2
%
64.6
108.8
175.6
167.7
115.3
71.4
150
16.6±1.4
20±1.0
1.2
21.3±2.9
24.1±4.7
1.1
%
91.7
148.1
162.1
171.7
115.3
65.5
200
24.8±3.4
19.3±0.4
0.77
20.3±1.3
20.8±0.5
1.02
%
137.01
142.9
104.1
163.7
99.5
60.7
0
19.5±0.14
17.1±0.1
0.87
14.1±0.41
19.6±2.4
1.39
%
107.8
126.7
117.6
113.7
93.8
82.7
50
15±0.56
12.5±0.21
0.83
15.6±0.56
19.4±1.6
1.24
%
83.3
92.6
112.2
125.8
92.8
73.8
100
21.8±5.1
16.5±4.1
0.75
19.3±3.2
20±4.1
1.03
%
120.4
122.2
101.3
155.6
95.6
61.3
150
17.1±4.3
16±0.28
0.93
18.7±7.2
19.9±1.9
1.06
%
94.4
118.5
125.7
150.8
95.2
63.1
200
19.4±0.84
12.8±1.1
0.65
29.3±5.0
23.2±4.8
0.79
%
107.2
94.8
87.8
236.2
111
47.02
5%
5.56
3.38
0.61
8.98
8.74
0.97
Data calculated as % of absolute control and means of three replications ±SD.
The data in Table 2 revealed also that MDA content increased progressively in both shoot and root with increasing salinity in the soil. This increasing trend was highly significant at all salinization levels used. Trichoderma treatment progressively inhibited the accumulation of MDA as compared with the corresponding salinized plants, regardless the salinity level used. It is worth to mention also that, MDA content in shoots were much lower than roots whatever the salinity level applied. The mineral composition of bean plants assimilated in both Na+ and K+ as well as K+/Na+ ratios in both shoots and roots were presented in Table 3. Both Na+ and K+ ions were increased by increasing salinity except at (100 mM NaCl in roots) which
Acta Biologica Hungarica 65, 2014
Acta Biologica Hungarica 65, 2014 16
Band1 Band2 Band3 Band4 Band5 Band6 Band7 Band8 Band9 Band10 Band11 Band12 Band13 Band14 Band15 Band16 Band17 Band18 Band19 Band20 Band21 Band22
Total bands
50
14
270.845 223.823 201.588 165.749 152.080 143.600 134.000 120.282 113.192 104.209 98.564 79.282 67.539 64.422 – – – – – – – –
Plants grown for 40 days from sowing.
0.0
271.761 223.823 203.296 165.749 145.552 134.000 130.651 120.282 112.621 102.465 87.287 83.119 79.015 74.358 68.111 64.639 – – – – – –
MW-bp
18
268.569 222.693 199.894 165.190 152.080 143.600 134.000 125.042 117.276 113.192 105.625 98.564 95.939 91.975 79.954 74.988 66.073 64.096 – – – –
100
15
267.213 221.568 189.706 161.606 151.312 143.116 140.721 133.549 123.992 119.271 114.151 107.061 102.812 98.564 95.939 – – – – – – –
150
18
268.569 221.568 214.215 187.162 160.248 153.369 143.116 138.133 134.000 123.992 119.271 114.151 110.363 106.520 102.465 98.564 93.068 75.623 – – – –
200
14
265.864 226.482 199.894 184.964 175.537 169.425 160.248 154.147 141.914 131.093 121.917 113.192 105.625 101.948 – – – – – – – –
C+T
19
261.415 250.193 226.482 199.894 184.964 175.537 170.285 159.439 154.147 144.329 136.511 128.899 122.329 113.767 105.625 100.242 96.426 91.975 88.325 – – –
50+T
20
263.630 251.463 226.482 199.894 185.590 176.130 170.285 159.439 152.594 145.061 133.549 126.102 115.703 111.298 102.465 95.939 92.286 88.325 85.393 69.622 – –
100+T
19
267.213 255.742 226.482 199.894 185.590 177.024 167.153 159.439 152.080 145.061 133.549 125.677 117.276 111.298 104.209 95.939 92.755 88.325 85.682 – – –
150+T
Table 4 SDS electrophoresis analysis of soluble protein bands produced by the faba bean (Vicia faba Giza 429) cultivated under NaCl alone or NaCl+ Trichoderma 200+T
22
262.742 248.091 226.482 199.894 185.590 177.024 167.153 152.080 145.061 137.668 125.677 117.672 112.241 102.465 96.426 92.755 88.325 85.682 71.527 69.037 66.633 54.143
462 G. K. Abd El-Baki and D. Mostafa
Interaction between NaCl and Trichoderma in Vicia faba
463
exhibited slight reduction in both ions. The treatment of bean plants with Trichoderma harzianum enhanced the accumulation of both ions regardless the tissue tested or organ analyzed. The K+/Na+ ratios exhibited different responses with different treatments. In salinized bean plants the ratio decreased in shoots and increased in roots except at 200 mM NaCl. The treatment of plants with Trichoderma harzianum decreased the K+/Na+ ratios with increasing salinity regardless the organ tested. It is worth to mention that the ratios of K+/Na+ in plants treated with Tricoderma were less compared with corresponding salinized plants. Exposure of plants to different concentrations of NaCl salinity and others treated with Trichoderma harzianum produced marked changes in their protein pattern, three types of alterations were observed as follows (see also Fig. 2a,b). (i) The Synthesis of certain proteins declined significantly, however, (14:18 KDa) protein bands were detected only under salinity. (ii) Specific synthesis of certain other proteins were markedly observed in both treatments, however the number of new protein bands in plants treated with Trichoderma were much higher than those of salted plants. (iii) Synthesis of a set specific protein was induced de novo in plant treated with Tricho derma harzianum. The results revealed also that (14–18 protein bands) were detected only under salinity while, plants treated with Trichoderma harzianum displayed (14–22 protein bands) Table 4. The 143 KDa protein bands were specific markers for faba bean Giza 429 under salinity and 134 KDa protein band was specific for high salinity dose only. However, five protein bands of molecular weight (226.482; 199.894; 185.590; 152.08 and 88.325 KDa) were specific markers for interaction between Trichoderma and salinity. It is worth to mention that, some low molecular weight of (71–54 KDa) were specific for 200 mM NaCl + Trichoderma. Salinity specifically enhanced the synthesis of the set proteins and also induced de novo synthesis of another set of proteins. On the other side, interaction between salinity and Trichoderma not only improved growth but also enhanced protein synthesis. A number of protein bands also appeared in plants treated with Trichoderma harzianum as compared with control (untreated plants).
Discussion The growth response of bean plants (Vicia faba Giza 429) to salt stress was different from those treated with Trichoderma harzianum (T24). The fresh weight was decreased by (81.5%) of control plants, however, the reduction in dry weight was less compared with the reduction in fresh weight (92.3%) under 200 mM NaCl. The treatment with Trichoderma harzianum resulted in reduction (91.5% and 29.4%) of both fresh and dry weight under 200 mM NaCl. Certain Trichoderma spp. have beneficial effects on plant growth and enhance resistance to both biotic and abiotic stresses, it can colonize plant root and its rhizosphere, second it improves plant health through increasing plant growth, ultimately, root growth [30]. Early work revealed that, Trichoderma promotes growth response in radish, pepper, cucumber and tomato [5]. Acta Biologica Hungarica 65, 2014
464
G. K. Abd El-Baki and D. Mostafa
Further studies demonstrated also that Trichoderma increases root development and crop yield, the proliferation of secondary roots, seedlings fresh weight and foliar area [14]. Recently, Tucci et al. [29] have demonstrated the effects of the plant genetic background on the outcome of the interaction between different tomato lines and two bio-control strains of Trichoderma atroviride, Trichoderma harzianum and in at least one tomato cultivar the Trichoderma treatment did not exert any plant growth promotion effect and was even seen to be detrimental. Studies also showed that, Trichoderma treatment in tobacco increased fresh weight of root and leaf area index (300%), lateral roots (300%) and the leaf (140%) [1]. This is in accordance with our results of bean plants treated with Trichoderma harzianum (T24) that improved widely the leaf area (155.7%) of control. The increase in relative water content of bean plants under different treatments revealed that, there is no detrimental effect in water uptake especially when the plants treated with Trichoderma harzianum which exert water content (100.3%) of control plants. Recently, Mastouri et al. [22] reported that the treatment of tomato seeds with Trichoderma harzianum ameliorates water, osmotic, salinity, chilling and heat stresses by inducing physiological protection in plants against oxidative damage. Trichoderma spp. produce auxins that are able to stimulate plant growth and root development. The proline content of salted plants as well as inoculated with Trichoderma harzianum was enhanced with increasing salinity, however, soluble proteins increased with salinity and decreased with Trichoderma treatments. The proline accumulation was used as a suitable marker for salt tolerance in plants treated with Trichoderma harzianum which enhanced proline synthesis under salt stress conditions. The accumulation of proline under stress protects the cell by balancing the osmotic level of cytosol with that of vacuole and external environment, additionally, these solutes interact with cellular macromolecules such as enzymes and stabilize their structure and functions [11]. It has been reported that higher osmolyte concentration helps in the maintenance of comparatively higher relative water content and antioxidant enzyme activity which in accordance with our results in water content (Table 1) and MDA contents in Table 3. In cucumber [26] similar trends of increase in proline with increasing salinity concentration were reported. However, proline accumulation cannot be used as a sole criterion for salt tolerance as it also accumulates under other stresses such as higher temperature and drought [17]. Numerous proteins induced in response to Trichderma were involved in stress and defense responses. One of common mechanisms could be the control of damage caused by the reactive oxygen species. These molecules that play a crucial signaling role during abiotic stresses at higher concentrations cause cellular and molecular damage, most ROS are unstable and are quickly converted to H2O2 which is either reduced to water during the ascorbate GSH cycle, converted to water and oxygen by catalase enzyme, or used as a substrate of peroxidase enzyme [8]. However, in the presence of transition metal ions, H2O2 is converted to hydroxyle radicals, which start chain reactions leading to the peroxidation of membrane lipids [2] that resulted in the loss of membrane integrity and also damage to other macromolecules. Thus measurement of MDA contents Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
465
served as a reliable indication of oxidative damage. During a biotic stress we found an increase of (MDA) contents in faba bean Giza 429 under salt stress, but plants treated with Trichoderma harzianum had lower (MDA) content which follow the results of Harman and Mattick [13]. This evidence supports that Trichoderma harzianum stimulate plant growth as well as leaf area and ameliorates stress by inducing plant physiological protection against oxidative damage. The mechanisms by which Trichoderma spp. induce such changes are not known, however, enhanced ROS level could act as a signal to regulate expression of the related genes. An increase in ROS has been detected 5 to 10 minutes after treating soybean cell culture with culture filtrate of Trichoderma otroviride [25]. Such signals along with Ca+2 signaling can induce plant ROS scavenging mechanisms resulting in elevated protection against the oxidative damage. MDA, which is indicative of oxidative stress, increased in plants. Lipid peroxidation is the main index of the increase in active free radicals, and MDA is the main by-product of the lipid peroxidation process. Our finding showed that the degree of accumulation of MDA content has been reported to be indicative of the rate of lipid peroxidation due to salt stress, as lipid peroxidation is the symptom mostly ascribed to oxidative damage it is often used as an indicator of increased damage [19]. Lowest MDA content was recorded under inoculation of Trichoderma harzianum (Table 3), which might be due to an increase in expression of stress related proteins. Similar effect was observed by the inoculation of Tricoderma harzianum in maize plants. Under environmental stress, when ROS are produced, these detoxifying proteins triggered by Trichoderma inoculation act as scavenging enzyme and play a central role in protecting the cell from oxidative damage. Salinity dominated by Na+ and Cl– not only reduces K+ availability but also reduces K+ transport and mobility to growing regions of the plant that affects the quality of both vegetative and reproductive organs [10]. Moreover, a number of studies have shown that high concentrations of Na+ and Cl– in the soil solutions may depress nutrient–ion activities and produce extreme ratios of Na+/K+ in the plants, making the plants to be susceptible to osmotic and specific ion injury as well as to nutritional disorders that result in reduced yield and quality [27]. The results of this study showed that salinity caused an increase in Na+ as well as K+ concentrations which means there is no antagonistic effect between Na+ and K+ in this cultivar which may be used as a marker for salt tolerance criteria. However, Na+ concentration decreased under treatments of seeds with Trichoderma harzianum especially under lower and moderate salinity levels (till 150 mM NaCl). However some biological treatments reduced the Na+ uptake of plants and/or increased the K+ uptakes compared to control treatment under salt stress, thus increasing the ratio of K+/Na+. A positive correlation was found between fresh weight and K+ especially at lower salinity levels, however, a negative correlation between fresh weight and Na+ content was observed. Some studies indicate that an increase in concentration of K+ and Ca+2 in plants under salt stress could ameliorate the deleterious effects of salinity on growth and yield [27]. The response of crops to salinity is a complex phenomenon involves changes in certain metabolites, alteration in the behavior of many enzymes and synthesis of new Acta Biologica Hungarica 65, 2014
466
G. K. Abd El-Baki and D. Mostafa
sets of proteins [23]. Studying the pattern of protein synthesis under salt stress may help to identify a protein(s) associated with stress. In the present study, salinity induced the synthesis of newly proteins and simultaneously reduced other protein sets as compared with control plants. In that manner some authors working with chickpea (Cicer arietinum) reported that the total number of protein bands do not change under low 20 mM NaCl but was dramatically reduced at high salt used 40 mM NaCl. Tammam [28] found also that, salt treatment of broad bean seedling resulted in the disappearance of five polypeptides, while the peptides with low molecular mass increased in their intensity on the gel. The disappearance of certain proteins under salt stress may be due to either an inhibition of its mRNA transcription and/or translation, and increase in RNA-ase or through dissociation of polyribosomes under stress [4]. The treatments of faba bean Giza 429 with Trichoderma harzianum resulted in marked changes in protein patterns, several polypeptides were apparently suppressed whereas others were induced. The results revealed also that four bands at molecular weight 54.91, 46.80, 34.34 and 18.06 kDa were induced under Trichoderma treatment as compared with untreated plants, the induction of these bands differed with differing dose of salinity. In this respect [6] working with soybean and chickpea treated with Trichoderma demonstrated that the ability of Trichoderma to reverse the lowering of protein level caused by salt stress might be due to enhancing of nitrogen fixation and nitrogen uptake by plants. They also revealed that Trichoderma harzianum could produce nitrogen oxide (NO) which is the coding for enzyme involved in L-arginine which is important for protein and arginine biosynthesis. In addition, numerous proteins induced in response to Trichoderma harzianum could involved in stress and defense responses. These extra bands of protein which induced in faba bean Giza 429 under NaCl alone or inoculated with Trichoderma suggested that these protein bands may play an important role in the mechanism of salinity tolerance. The newly synthesized proteins together with proline could act as components of a salt tolerance mechanism in our bean plants. They may also function as compatible cytoplasmic solutes in osmotic adjustment to equalize the osmotic potential of the cytoplasm with the vacuoles under adverse conditions of salinity. Acknowledgement The authors would like to thank Prof. Dr. M. A. A. Shaddad, Assuit University for his valuable advices and revising the manuscript. References 1. Altintas, S., Bal, U. (2008) Effects of the commercial product based on Trichoderma harzianum on plant, bulb and yield characteristics of onion. Sci. Hortic. 116, 219–222. 2. Aust, S. D., Morehouse, L. A., Thomas, C. (1985) Role of metals in oxygen radical reactions. J. Free Radic. Biol. Med. 1, 3–26. 3. Bates, L. S., Waldern, R. P., Teare, I. D. (1973) Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Acta Biologica Hungarica 65, 2014
Interaction between NaCl and Trichoderma in Vicia faba
467
4. Bewlfey, J. D., Oliver, M. J. (1983) Responses to a changing environment at the molecular level: Does desiccation, modulate protein synthesis at the transcriptional or translational level in a tolerant plant? Current Topics Plant Biochem. Biophys. 2, 145–146. 5. Chang, Y. C., Baker, R., Kleifeld, O., Chet, I. (1986) Increased growth of plants in the presence of the biological control agent Trichoderma harzianum, Plant Disease 70, 145–148. 6. Egberongbe, H. O., Akintokun, A. K., Babalola, O. O., Bankole, M. O. (2010) The effect of Glomus mosseae and Tricoderma harizianum on proximate analysis of soybean (Glycine max. (L.) Merrill) Seed grown in sterilized and unsterilized soil. J. Agric. Extension Rural Development 2, 54–58. 7. FAO (2000) Global network on integrated soil management for sustainable use of salt effected soils, Available in: http://www.fao.org/ag/AGL/agll/spush/intro.htm. 8. Foyer, C. H., Kiddle, G., Antoniw, J., Bernard, S., Verrier, P. J., Pastori, G. M., Noctor, G. (2003) The role of antioxidant-mediated signal transduction during stress. Mol. Cell. Proteomics 2, 682. 9. Gachomo, E. W., Kotchoni, S. O. (2008) The use of T. harzianum and T. viride as potential biocontrol agents against peanut microflora and their effectiveness in reducing aflatoxin contamination of infected kernels. Biotechnol. 7, 439–447. 10. Grattan, S. R., Grieve, C. M. (1999) Salinity–mineral nutrient relations in horticultural crops. Sci. Hortic. 78, 127–157. 11. Greenway, H., Munns, H. (1980) Mechanisms of salt tolerance in non halophytes. Annu. Rev. Plant Physiol. 31, 149–190. 12. Grichko, V. P., Glick, B. R. (2001) Amelioration of flooding stress by ACC deaminase containing plant growth-promoting bacteria. Plant Physiol. Biochem. 39, 11–17. 13. Harman, G. E., Mattick, L. R. (1976) Association of lipid oxidation with seed aging and death. Nature 260, 323–324. 14. Harman, G. E. (2000) Myths and dogmas of biocontrol – changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Disease 84, 377–393. 15. Harman, G. E., Björkman, T. (1998) Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement. In: Harman, G. E., Kubicek, C. P. (eds), Trichoderma and Gliocladium. 2, Taylor & Francis, London, United Kingdom, pp. 229–265. 16. Heath, R. L., Packer, L. (1968) Photoperoxidation in isolated chloroplast. 1. Kinetics and stiochiometry of fatty acid peroxidation. Arch. Bioch. Biophys. 125, 189–198. 17. Hong, Z. L., Lakkineni, K., Zhang, Z. M., Verma, D. P. S. (2000) Removal of feedback inhibition of delta (1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol. 122, 1129–1136. 18. Howell, C. R. (2003) Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis. 87, 4–10. 19. Khan, M. H., Panda, S. K. (2008) Alterations in root lipid peroxidation and antioxidative responses in two rice cultivars under NaCl salinity stress. Acta Physiol. Plant. 30, 91–98. 20. Laemmli, U. K. (1970) Cleavage of structure proteins during assembly of the head of bacteriophage T4. Nature 277, 680–685. 21. Lowery, O. H., Rosebrough, N. H., Farr, A. L., Randall, R. J. (1951) Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 291–297. 22. Mastouri, F., Björkman, T., Harman, G. E. (2010) Seed treatment with Trichoderma harzianum alleviates biotic, abiotic, and physiological stresses in germinating seeds and seedlings. Phytopathol. 100, 1213–1221. 23. Michal, S. G., Harman, E. (2008) The molecular basis of shoot responses of maize seedling to Trichoderma harizianum T22 inoculation of the root. Plant Physiol. 147, 2147–2163. 24. Munns, R., Termaat, A. (1986) Whole plant responses to salinity. Aust. J. Plant Physiol. 13, 143–160. 25. Navazio, L., Baldan, B., Moscatiello, R., Zuppini, A., Woo, S. L., Mariani, P., Lorito, M. (2007) Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride. BMC Plant Biol. 7, 41–49. 26. Shiqing, S., Shirong, G., Qingmao, S., Zhigang, Z. (2006) Physiological effects of exogenous salicylic acid on cucumber seedling under salt stress. Acta Hortic. Sin. 33, 68–72. Acta Biologica Hungarica 65, 2014
468
G. K. Abd El-Baki and D. Mostafa
27. Sivritepe, N., Sivritepe, H. O., Eris, A. (2003) The effects of NaCl priming on salt tolerance in melon seedlings grown under saline conditions. Sci. Hortic. 97, 229–237. 28. Tammam, A. A. (2003) Response of Vicia faba plants to the interactive effect of sodium chloride salinity and salicylic acid treatment. Acta Agron. Hungarica 51, 239–248. 29. Tucci, M., Ruocco, M., De Masi, L., De Palma, M., Lorito, M. (2011) The beneficial effect of Trichoderma spp. on tomato is modulated by the plant genotype. Mol. Plant Pathol. 12, 341–354. 30. Vinale, F., Sivasithamparam, K., Ghisalberti, E. L., Marra, R., Barbetti, M. J., Li, H., Woo, S. L., Lorito, M. (2008) A novel role for R. Hermosa and others Trichoderma secondary metabolites in the interactions with plants. Physiol. Mol. Plant Pathol. 72, 80–86. 31. Wiersma, T. V., Bailey, T. B. (1975) Estimation of leaflet, trifoliate and total leaf area of soybean. Agron. J. 176, 26–30. 32. Williams, S., Twine, M. (1960) Flame photometric method for sodium, potassium and calcium. In: Peach, K., Tracey, M. V. (eds) Modern Methods of Plant Analysis. Vol. 5. Springer Verlag, Berlin, pp. 3–5.
Acta Biologica Hungarica 65, 2014
