Turkish Journal of Agriculture and Forestry http://journals.tubitak.gov.tr/agriculture/
Research Article
Turk J Agric For (2015) 39: 300-309 © TÜBİTAK doi:10.3906/tar-1403-39
Effects of arbuscular mycorrhizal fungus, humic acid, and whey on wilt disease caused by Verticillium dahliae Kleb. in three solanaceous crops 1,
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3
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Semra DEMİR *, Suat ŞENSOY , Elvan OCAK , Şefik TÜFENKÇİ , Emre DEMİRER DURAK , 5 4 Çeknas ERDİNÇ , Hüsamettin ÜNSAL 1 Department of Plant Protection, Faculty of Agriculture, Yüzüncü Yıl University, Van, Turkey 2 Department of Horticulture, Faculty of Agriculture, Yüzüncü Yıl University, Van, Turkey 3 Department of Food Engineering, Faculty of Engineering and Architecture, Yüzüncü Yıl University, Van, Turkey 4 Department of Soil Science and Plant Nutrition, Faculty of Agriculture, Yüzüncü Yıl University, Van, Turkey 5 Department of Agricultural Biotechnology, Faculty of Agriculture, Yüzüncü Yıl University, Van, Turkey Received: 07.03.2014
Accepted: 01.12.2014
Published Online: 06.04.2015
Printed: 30.04.2015
Abstract: This study examined the effects of arbuscular mycorrhizal fungi (AMF), humic acid (HA), and whey (W) application on wilt disease caused by Verticillium dahliae Kleb. in tomato, pepper, and eggplant. Single, dual, and triple applications of AMF (2.5 g inocula of either Glomus mosseae or G. intraradices), HA (500 ppm), and W (50 mL kg–1) were found to improve the morphological growth and nutritional status of all three host species. Moreover, dual and triple applications reduced the severity of wilt disease caused by V. dahliae by between 40% and 70.5%. Triple application of AMF, HA, and W decreased the number of V. dahliae microsclerotia by 50%. Furthermore, W and HA application promoted AMF growth, with HA application resulting in significantly higher levels of AMF colonization and spore density when compared to untreated controls. Key words: Arbuscular mycorrhizal fungi, eggplant, humic acid, pepper, tomato, whey
1. Introduction Tomato, pepper, and eggplant are all members of the family Solanaceae and are among the most important vegetables grown in open fields as well as under protected cultivation. All three vegetable species are affected by plant protection problems that result in limited yields and other production-related problems, including root rot and wilt disease. Wilt disease can cause significant damage to all 3 vegetable species and represents a serious problem because of the difficulties involved in combating the source of the disease, the common soil-borne pathogen Verticillium dahliae Kleb. (V.d.) (Tjamos and Beckman, 1989). Chemical methods of controlling soil-borne root pathogens are inherently dangerous. Given the technical, environmental, and economic issues associated with chemical controls, it is no surprise that biological control methods are attracting more and more attention for their ability to provide effective, long-term protection with no adverse impact on the environment or on human health. Among these biological control agents are arbuscular mycorrhizal fungi (AMF), beneficial symbiotic microorganisms found in the rhizosphere whose presence can provide significant advantages to plants in terms of both resistance and * Correspondence:
[email protected]
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development (Azcón-Aguilar and Barea, 1996; Smith and Read, 2008). Recent studies have also suggested that organic materials that have been found to promote plant growth, such as humic acid (HA) and whey (W), could form part of an alternative agricultural practice (Cacco and Dell Agnolla, 1984; Wang et al., 1995; Valdrighi et al., 1996; Sonnleitner et al., 2003). Although their use is fairly uncommon, there is some evidence that these organic materials can have beneficial effects on soil microbial activity (Özrenk et al., 2003; Litterick et al., 2004; Türkmen at al., 2005; Loffredo et al., 2008; Demir and Ozrenk, 2009; Erman et al., 2011). Within this framework, the present study examined the effects of AMF, HA, and W on wilt disease caused by V.d. in tomato, pepper, and eggplant. 2. Materials and methods 2.1. Materials Study material included F1 hybrid tomato (Riva), pepper (Ergenekon), and eggplant (Pera) cultivars sensitive to V.d.; AMF isolates of Glomus intraradices and G. mosseae, which are known to have high relative mycorrhizal dependency with these cultivars; and the pathogen V.d., which was isolated from eggplant with a high level of virulence.
DEMİR et al. / Turk J Agric For 2.2. Methods 2.2.1. Seedling production Tomato, pepper, and eggplant cultivars were grown in combination with the most appropriate AMF (cv. Riva × G. mosseae, cv. Ergenekon × G. mosseae, cv. Pera × G. intraradices). Seeds were germinated in vials 5 cm in diameter and 6 cm deep containing a 1:1 growth medium of peat and perlite. HA [500 ppm, polymeric polyhydroxy acid 85% w/w Agrolig (commercial formulation)] and AMF inocula (2.5 g) were added to the AMF applications. Seeds were surface disinfected and covered with vermiculite (1 cm) after sowing. The study was conducted with a randomized block design of 16 different treatments per crop, with 15 seedlings in 3 replications for a total of 45 seedlings per treatment. Seedlings were cultivated in a growth chamber at 22 ± 2 °C, 60%–70% relative humidity, and 12 h of fluorescent illumination. Seedlings were irrigated with distilled water and fertilized 3 times with 5 mL of diluted nutrient solution per seedling. W was obtained from cheese produced using bovine milk and subjected to content analysis, which yielded the following information for W composition: fat, 0.55%; pH, 6.34; lactic acid, 0.18%; protein, 1.09%; N, 0.17%; dry matter, 7.19%; ash, 0.56%; lactose, 4.88%; P, 50.00 ppm; K, 1261.31 ppm; Ca, 385.73 ppm; Mg, 105.69 ppm; Mn, 0.01 ppm. At 1 week, seedlings were treated with a 50 mL kg–1 dose of W, which has been shown to cause no adverse effect on plant growth (Özrenk et al., 2003). 2.2.2. Pathogen inoculation and assessment of disease severity The V.d. pathogen was cultivated in a 1:1:4 medium of cornmeal, perlite, and water. At 6 weeks, seedlings were transferred to a growth medium containing equal amounts of peat and perlite, and the medium was inoculated with the pathogen at a ratio of 1:10. A control group of plants was cultivated in a similar peat–perlite medium without the addition of the pathogen. Disease symptoms were observed at 4 weeks following pathogen inoculation. Disease severity was evaluated according to degree of wilt [0 = healthy shoots; 1 = wilt in less than 25% of leaves; 2 = 25%–50% wilt (30% leaf loss); 3 = 50%–75% wilt (60% leaf loss); 4 = 75%–100% wilt (90% leaf loss); 5 = plant death] (Hwang et al., 1992) and vascular health (0 = healthy stem; 1 = 1%–33% brownish vascular systems; 2 = 34%–67% brownish vascular systems; 3 = 68%–100% brownish vascular systems) (Erwin et al., 1976). For both scales, the degree of disease severity was calculated using the following formula: Disease severity =
(0)(a) + (1)(b) + (2)(c) + (3)(d) × 100 , n = (a + b + c + d)
where a, b, c, and d represent the number of plants per group receiving a particular score, and n represents the total number of plants in that group. In addition to disease severity, V.d. microsclerotia density in growth medium was also evaluated. Soil samples were air-dried under ambient temperatures for 4 weeks and then passed through 2-mm and 250-µm sieves, respectively, after removal of plant residues. The number of microsclerotia per gram of soil was estimated by the method of Kabir et al. (2004). 2.2.3. AMF root colonization and spore density assessment Plant roots were fixed and stained (Phillips and Hayman, 1970), and AMF root colonization was assessed under a stereoscope microscope (4 × 10 and 10 × 10) using the grid-line intersect method (Giovanetti and Mosse, 1980). AMF spore density in growth media rhizosphere was evaluated using the wet sieving method (Gerdemann and Nicholson, 1963). 2.2.4. Plant growth parameter assessment Seedling total fresh weights were measured using a digital scale. Seedlings were dried for 48 h at 70 °C until a constant weight was obtained, and the dry weight was recorded. Morphologic developmental parameters [number of leaves, shoot length (cm), shoot diameter (cm), root length (cm)] were also measured. 2.2.5. Plant macro- and micronutrient content assessment Dried seedling samples (0.5 g) were ground, burned, and prepared for Ca, Mg, K, Fe, Mn, Zn, and Cu content analysis (ppm) using an atomic absorption spectrophotometer (Kacar and İnal, 2008). Total P in seedlings was determined by the vanadomolybdophosphoric yellow method in a spectrophotometer, whereas total N content was determined according to the Kjeldahl method (Kacar and İnal, 2008). 2.2.6. Statistical analysis Data were analyzed using the statistical software package SAS (1997). Means were grouped using the Duncan multiple comparison test. 3. Results and discussion 3.1. Effects of AMF, HA, W, and V.d. application on tomato, pepper, and eggplant seedling morphology Morphological parameters (number of leaves, shoot length, shoot diameter, root length, and shoot and root dry and fresh weights) of tomato, pepper, and eggplant seedlings by treatment are given in Tables 1–3. As the tables show, different combinations of AMF, HA, W, and V.d. had different effects on the in vitro growth of all 3 vegetables studied. Moreover, with the exceptions of shoot and root length in tomato and eggplant and stem diameter in eggplant, all differences were statistically significant
301
DEMİR et al. / Turk J Agric For Table 1. Growth parameters of tomato seedlings treated with different combinations of AMF (G. mosseae), HA, W, and V.d. Treatments Control AMF
SL * (cm)
RL (cm)
SD (cm)
LN
SFW (g)
RFW (g)
SDW (g)
RDW (g)
9.0 a**
34.2 a
4.51 ab
5.6 abc
4.5 abcd
3.9 ab
0.79 abc
0.28 abc
9.8 a
33.0 a
4.17 abc
5.5 abc
4.1 cde
3.7 ab
0.68 abcde
0.27 abc
W
10.4 a
34.6 a
4.40 ab
5.8 ab
5.4 a
4.2 ab
0.84 ab
0.31 ab
HA
8.9 a
36.0 a
4.36 ab
5.0 bcd
4.2 cde
3.8 ab
0.68 abcde
0.27 abc
W+AMF
9.7 a
36.4 a
4.57 ab
6.0 a
5.2 ab
4.1 ab
0.84 ab
0.30 ab
AMF+HA
9.0 a
35.1 a
4.18 abc
5.0 bcd
4.3 bcd
3.8 ab
0.75 abcd
0.28 abc
10.5 a
40.8 a
4.67 ab
5.6 abc
5.4 a
4.3 a
0.89 a
0.34 a
AMF+W+HA
9.8 a
34.0 a
4.46 ab
5.5 abc
4.6 abc
4.0 ab
0.80 abc
0.31 ab
V.d.
8.0 a
34.7 a
4.76 a
5.4 abcd
3.8 cde
3.3 ab
0.56 de
0.20 c
V.d .+AMF
9.0 a
38.1 a
4.09 abc
4.6 d
3.3 e
3.2 b
0.48 e
0.23 bc
V.d.+W
9.5 a
38.1 a
4.49 ab
5.5 abc
4.4 bcd
3.5 ab
0.62 bcde
0.25 abc
V.d.+HA
9.4 a
37.6 a
3.96 bc
4.6 d
3.3 e
3.3 ab
0.50 e
0.23 bc
V.d.+W+AMF
9.2 a
32.4 a
3.54 c
5.3 abcd
4.2 cde
3.3 ab
0.68 abcde
0.25 bc
V.d.+HA+AMF
8.9 a
37.0 a
4.16 abc
4.8 cd
3.5 de
3.7 ab
0.49 e
0.24 bc
8.7 a
32.4 a
4.19 abc
5.0 bcd
4.6 abc
3.8 ab
0.59 cde
0.25 abc
10. 1 a
35.1 a
4.21 abc
4.6 d
3.8 cde
3.8 ab
0.52 e
0.25 abc
W+HA
V.d.+W+HA V.d.+W+HA+AMF
*SL: Shoot length; RL: root length; SD: shoot diameter; LN: leaf number; SFW: shoot fresh weight; RFW: root fresh weight; SDW: shoot dry weight; RDW: root dry weight. **The same letters in the same column indicate insignificant differences (P < 0.05) according to Duncan’s test findings.
Table 2. Growth parameters of pepper seedlings treated with different combinations of AMF (G. mosseae), HA, W, and V.d. Treatments
SL * (cm)
RL (cm)
SD (cm)
LN
SFW (g)
RFW (g)
SDW (g)
RDW (g)
Control
9.4 ab **
25.6 ab
3.3 abc
7.7 a
2.0 abcd
2.8 abc
0.30 abcde
0.23 abcde
AMF
8.9 ab
25.7 ab
3.4 ab
7.1 ab
1.9 abcd
2.8 abc
0.34 ab
0.26 abc
W
9.5 ab
27.6 a
3.1 abc
8.2 a
2.4 ab
3.2 a
0.37 a
0.28 ab
HA
7.9 b
26.7 a
2.7 c
7.2 ab
1.7 bcd
2.9 ab
0.27 bcde
0.24 abcd
W+AMF
9.0 ab
26.5 a
3.2 abc
7.6 ab
2.4 ab
2.6 abc
0.34 abc
0.26 abc
AMF+HA
8.4 ab
24.0 ab
3.2 abc
7.2 ab
1.9 abcd
2.7 abc
0.26 cde
0.22 abcde
W+HA
9.7 ab
25.0 ab
3.7 a
8.2 a
2.5 a
3.1 a
0.32 abcde
0.28 ab
AMF+W+HA
9.9. ab
24.7 ab
3.6 a
7.9 ab
2.3 abc
3.2 a
0.36 ab
0.29 a
V.d.
8.6 ab
22.8 ab
3.2 abc
7.7 ab
1.6 cd
2.2 bc
0.25 de
0.17 de
V.d.+AMF
8.2 ab
21. 7 ab
3.3 abc
6.8 c
1.6 d
2.1 bc
0.24 e
0.15 e
V.d.+W
10.6 a
23.6 ab
3.4 ab
8.3 a
2.4 ab
3.0 ab
0.33 abcd
0.20 bcde
V.d.+HA
9.3 ab
24.0 ab
3.1 abc
7.4 ab
1.8 bcd
2.5 abc
0.29 abcde
0.21 bcde
V.d.+W+AMF
9.6 ab
23.7 ab
3.6 a
8.2 a
2.1 abcd
2.4 abc
0.30 abcde
0.17 de
V.d.+HA+AMF
9.1 ab
9.7 b
3.2 abc
7.5 ab
1.7 bcd
2.0 c
0.24 e
0.17 de
V.d.+W+HA
9.5 ab
24.9 ab
3.2 abc
8.4 a
2.2 abc
2.7 abc
0.30 abcde
0.22 abcde
V.d.+W+HA+AMF
9.1 ab
24.2 ab
2.9 bc
8.3 a
2.2 abc
2.6 abc
0.30 abcde
0.20 cde
*SL: Shoot length; RL: root length; SD: shoot diameter; LN: leaf number; SFW: shoot fresh weight; RFW: root fresh weight; SDW: shoot dry weight; RDW: root dry weight. **The same letters in the same column indicate insignificant differences (P < 0.05) according to Duncan’s test findings.
302
DEMİR et al. / Turk J Agric For Table 3. Growth parameters for eggplant seedlings treated with different combinations of AMF (G. intraradices), HA, W, and V.d. Treatments
SL (cm)
RL (cm)
SD (cm)
LN
SFW (g)
RFW (g)
SDW (g)
RDW (g)
Control
8.8 a
20.9 a
3.5 a
5.1 ab
3.4 ab
2.61 ab
0.66 abc
0.268 abc
AMF
8.3 a
23.4 a
3.7 a
4.6 ab
2.7 b
2.71 ab
0.40 d
0.231 abc
W
9.2 a
20.0 a
3.7 a
5.1 ab
3.6 ab
2.50 ab
0.69 abc
0.275 ab
HA
8.1 a
21.6 a
3.5 a
4.4 b
2.8 b
3.14 a
0.58 abcd
0.327 a
W+AMF
8.5 a
23.6 a
3.7 a
5.1 ab
3.3 ab
2.8 ab
0.66 abc
0.282 ab
AMF+HA
8.5 a
23.0 a
3.6 a
4.7 ab
2.8 b
2.8 ab
0.54 abcd
0.269 abc
W+HA
9.1 a
20.8 a
3.6 a
5.2 a
3.3 ab
2.6 ab
0.68 abc
0.278 ab
AMF+W+HA
8.8 a
22.9 a
3.8 a
4.6 ab
2.8 b
2.9 ab
0.60 abcd
0.287 ab
V.d.
8.6 a
21.0 a
3.7 a
4.6 ab
2.9 b
2.05 b
0.49 cd
0.208 bc
V.d.+AMF
8.7 a
20.2 a
3.6 a
4.8 ab
2.8 b
2.5 ab
0.51 cd
0.160 c
V.d.+W
8.7 a
20.2 a
3.7 a
4.9 ab
3.3 ab
2.47 ab
0.49 cd
0.230 abc
V.d.+HA
8.3 a
21.1 a
3.4 a
4.8 ab
3.0 ab
2.77 ab
0.53 bcd
0.211 bc
V.d.+W+AMF
9.2 a
21.5 a
3.5 a
5.2 a
3.6 ab
2.64 ab
0.76 ab
0.234 abc
V.d.+HA+AMF
9.3 a
20.5 a
3.3 a
4.9 ab
3.1 ab
2.77 ab
0.61 abcd
0.215 abc
V.d.+W+HA
9.2 a
20.1 a
3.5 a
5.3 a
3.8 a
2.77 ab
0.77 a
0.240 abc
V.d.+W+HA+AMF
9.5 a
22.6 a
3.5 a
4.6 ab
3.4 ab
2.78 ab
0.63 abc
0.244 abc
SL: Shoot length; RL: root length; SD: shoot diameter; LN: leaf number; SFW: shoot fresh weight; RFW: root fresh weight; SDW: shoot dry weight; RDW: root dry weight. The same letters in the same column indicate insignificant differences (P < 0.05) according to Duncan’s test findings.
when compared to controls. In general, growth parameters declined with the application of V.d. and improved with either single or multiple applications of HA, W, and AMF. For example, shoot diameter (4.76 cm) was highest with the V.d. application in tomato, shoot length (10.6 cm) and number of leaves (8.4) was highest with the V.d.+W+HA application in pepper, and number of leaves (5.3) and fresh and dry shoot weight (3.8 g, and 0.77 g, respectively) were highest with the V.d.+W+HA application in eggplant. Different studies have reported the stimulating effects of HA (Adani et al., 1998; Tüfenkçi et al., 2006; Aslanpay and Demir, 2012), W (Ocak and Demir, 2012), and AMF (Smith and Read, 2008) in terms of improving plant growth parameters and nutrient contents. However, few studies have examined how AMF, a key group of microbial symbionts in the rhizosphere, interact with HA and W. Some studies investigating potential sustainable agriculture strategies have reported that the combination of W and AMF promotes plant growth as well as AMF growth (Özrenk et al., 2003; Demir and Ocak, 2009). In view of these findings, it may be suggested that the high nutrient content of W improves the nutritional status of the plant, thereby positively affecting the development of AMF. Triple combinations of AMF, HA, and W have
also been shown to promote seedling development in watermelon (Halifeoğlu, 2011) and melon (Biçer, 2011). 3.2. Effects of AMF, HA, W, and V.d. application on tomato, pepper, and eggplant nutrient content Macro- and micronutrient contents of tomato, pepper, and eggplant seedlings are provided in Tables 4–6. As the tables show, in general, no statistically significant differences were found in plant nutrient contents; however, N and Cu contents in tomato (Table 4), P content in pepper (Table 5), and Mg content in eggplant (Table 6) were significantly higher with single or combined application of AMF, HA, and W when compared to controls. Many studies have suggested that AMF species play a role in macro- and micronutrient uptake, especially in soil with significant P limitations, which facilitates the supply of P to plants by AMF (Smith and Read, 2008). W has attracted attention as a plant fertilizer both in Turkey and abroad and has been shown to improve crop development and plant nutritional status (Sienkiewicz and Riedel, 1990; Özrenk et al., 2003; Demir and Ozrenk, 2009; Erman et al., 2011; Ocak and Demir, 2012). HA has been shown to facilitate plant growth by altering the chemical, physical, and biological characteristics of soil and improving plant nutritional status, mainly by playing an important role in
303
DEMİR et al. / Turk J Agric For Table 4. Macro- and micronutrient contents of tomato seedlings treated with different combinations of AMF (G. mosseae), HA, W, and V.d. Treatments
Macronutrients (%)
Micronutrients (ppm)
N
P
K
Ca
Mg
Fe
Cu
Control
0.83 cde*
0.48 ns
1.58 ns
1.84 ns
1.73 ns
91.65 ns
25.36 ab * 73.02 ns
AMF (G.m.)
0.72 e
0.54
1.55
2.16
1.77
75.20
W
0.82 cde
0.65
1.81
2.18
1.85
95.01
26.11 a
HA
0.96 abcd
0.62
1.76
2.03
1.68
W+AMF
0.95 abcd
0.67
1.12
2.19
1.77
AMF+HA
0.84 cde
0.60
1.08
2.27
W+HA
0.87 bcde
0.60
1.64
1.86
AMF+W+HA
0.85 cde
0.66
1.93
V.d. control
1.04 ab
0.67
1.63
V.d.+AMF
1.04 a
0.59
V.d.+W
0.96 abcd
0.55
V.d.+HA
0.95 abcd
V.d.+W+AMF V.d.+HA+AMF
9.26 cd
Zn
Mn 66.39 ns
45.07
62.88
51.87
60.44
79.18
13.05 abcd 73.19
62.88
96.11
22.15 abc
48.20
63.82
1.84
92.24
20.28 abcd 47.68
68.78
1.61
73.29
7.23 d
70.17
59.84
2.23
1.89
107.03
23.39 abc
53.79
70.75
1.99
1.72
93.56
21.45 abc
60.63
66.77
1.69
2.16
1.88
81.08
12.03 abcd 42.94
68.47
1.61
2.06
1.76
81.92
21.36 abc
52.36
62.66
0.58
1.61
2.06
1.82
89.69
14.33 abcd 66.04
62.60
0.99 abc
0.66
1.47
2.29
1.75
82.31
18.08 abcd 48.01
75.94
0.86 cde
0.57
1.64
1.86
1.63
78.67
15.42 abcd 49.98
65.97
V.d.+W+HA
0.80 de
0.53
1.69
2.03
1.88
77.14
22.82 abc
65.43
65.38
V.d .+W+HA+AMF
0.86 cde
0.64
1.64
2.13
1.83
100.20
11.40 bcd
88.34
57.83
*P < 0.05 (significant), ns: nonsignificant. Table 5. Macro- and micronutrient contents of pepper seedlings treated with different combinations of AMF (G. mosseae), HA, W, and V.d. Treatments
Macronutrients (%) N
Micronutrients (ppm)
P
K
Ca
Mg
Fe
Zn
Mn
Control
1.15 ns
0.53 c*
2.54 ns
1.83 ns
1.43 ns
77.55 ns
9.12 ns
31.73 ns
49.56 ns
AMF
1.19
0.59 bc
2.57
1.37
1.50
65.93
6.33
39.09
52.48
W
1.12
0.58 bc
3.34
1.54
1.55
71.29
10.50
38.38
49.55
HA
1.11
0.54 c
2.71
1.39
1.58
64.26
7.85
39.03
48.66
W+AMF
1.16
0.60 bc
3.16
1.46
1.61
62.71
6.08
39.79
51.45
AMF+HA
1.17
0.65 abc
2.71
1.60
1.67
68.92
7.25
40.47
51.28
W+HA
1.17
0.58 bc
2.61
1.30
1.49
70.24
10.50
33.59
48.47
AMF+W+HA
1.24
0.54 c
2.68
1.33
1.55
62.97
6.33
38.74
46.09
V.d. control
1.16
0.59 bc
3.18
1.38
1.52
95.05
7.71
31.55
46.84
V.d.+AMF
1.16
0.72 ab
2.84
1.37
1.52
97.88
12.72
42.35
55.64
V.d.+W
1.21
0.62 bc
3.63
1.41
1.58
75.59
10.62
33.48
51.41
V.d.+HA
1.16
0.68 abc
2.91
1.34
1.56
76.28
5.49
37.77
54.41
V.d.+W+AMF
1.23
0.59 bc
2.79
1.42
1.63
78.57
7.30
35.08
55.43
V.d +HA+AMF
1.31
0.71 ab
3.11
1.42
1.58
83.04
10.92
39.50
60.19
V.d.+W+HA
1.13
0.53 c
2.47
1.21
1.45
98.67
14.60
34.18
47.96
V.d.+W+HA+AMF
1.33
0.79 a
2.94
1.35
1.55
77.68
8.82
52.85
56.85
*P < 0.05 (significant), ns: nonsignificant.
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Cu
DEMİR et al. / Turk J Agric For Table 6. Macro- and micronutrient contents of eggplant seedlings treated with different combinations of AMF (G. intraradices), HA, W, and V.d. Treatments Control
Macronutrients (%)
Micronutrients (ppm)
N
P
K
Ca
Mg
Fe
1.14 ns
0.36 ns
1.13 ns
1.32 ns
0.97 b*
43.2 ns
Cu 4.06 ns
Zn
Mn
29.04 ns
51.44 ns
AMF
1.02
0.36
1.72
1.81
1.32 a
150.3
7.31
36.36
70.98
W
1.04
0.39
1.67
1.95
1.35 a
187.1
7.91
47.76
85.20
HA
0.99
0.42
1.50
1.83
1.36 a
147.4
8.07
33.76
79.95
W+AMF
1.00
0.38
1.67
1.68
1.34 a
220.8
7.78
41.32
82.36
AMF+HA
0.86
0.34
1.45
1.80
1.44 a
272.4
8.15
42.78
83.36
W+HA
1.03
0.37
2.19
2.02
1.27 a
217.3
5.62
38.50
74.91
AMF+W+HA
0.97
0.39
1.58
1.53
1.44 a
165.2
6.84
36.31
81.21
V.d. control
0.98
0.42
1.55
1.50
1.43 a
194.8
11.45
41.03
88.69
V.d.+AMF
0.97
0.42
1.25
1.22
1.25 a
171.9
5.93
33.85
63.19
V.d.+W
0.92
0.39
1.75
1.61
1.41 a
168.6
5.52
34.37
70.63
V.d.+HA
0.99
0.39
1.44
1.43
1.31 a
119.4
10.87
31.61
68.96
V.d.+W+AMF
0.92
0.40
1.18
1.24
1.26 a
142.4
5.02
35.72
59.12
V.d.+HA+AMF
0.98
0.42
1.45
1.67
1.47 a
274.0
9.98
33.39
81.08
V.d.+W+HA
1.02
0.37
1.66
1.62
1.44 a
193.1
4.73
31.25
75.04
V.d.+W+HA+AMF
1.96
0.45
1.61
1.56
1.40 a
194.1
15.50
30.15
92.91
*P < 0.05 (significant), ns: nonsignificant.
the uptake and transport of micronutrients (Wang, 1995; Adani et al., 1998). Özrenk et al. (2003), Demir and Ozrenk (2009), and Erman et al. (2011) found that a combined application of W+AMF significantly increased macro- and micronutrient contents of chickpeas and lentils grown in pots and in field conditions when compared to controls. Aslanpay and Demir (2012) reported that single and dual combinations of G. mosseae (AMF) and HA improved plant nutrient status in pepper. Biçer (2011) and Halifeoğlu (2011) demonstrated that a combination of W, AMF, and HA encouraged macro- and micronutrient uptake in melon and watermelon seedlings. 3.3. Effects of AMF, HA, and W application on wilt disease caused by V.d. in tomato, pepper, and eggplant Shoot wilt and stem vascular browning scores for tomato, pepper, and eggplant are given in Table 7. The highest disease incidence values for wilt and vascular browning were recorded in the tomato, pepper, and eggplant control groups (45.19% and 55.0%, 44.4% and 56.0%, and 40.74% and 52.0%, respectively). In tomato, the V.d.+HA, V.d.+W+HA, and V.d.+W+HA+AMF applications resulted in the lowest incidence of wilt (14.81%, 13.33%, and 13.33%) as well as the lowest incidence of vascular browning (33.0%, 34.0%, and 34.0%, respectively). In
pepper, the V.d.+W+HA application resulted in the lowest incidence of wilt (20.0%), whereas the lowest incidence of vascular browning occurred with the V.d.+AMF, V.d.+W, V.d.+W+AMF, and V.d.+HA+AMF applications (34.0%, 35.0%, 33.0%, and 33.0%, respectively). In eggplant, the lowest disease incidence values for both wilt (18.52%) and vascular browning (27.7%) occurred with the V.d.+W+AMF application. Overall, these findings reflect a reduction of V.d. rates of between 40% and 70.5%. V.d. causes tracheomycosis in plants by penetrating directly into young roots or wounds, establishing itself in the plant vascular system, and then systematically spreading throughout the plant. Given that the seedlings in the present study were not wounded, it was assumed that the V.d. pathogen was able to penetrate the young plants’ roots, whose inoculation with AMF had prompted physiological and morphological cell mediation. Both single and combined applications of AMF were found to significantly reduce the incidence of disease caused by V.d. in tomato, pepper, and eggplant (Table 7). After penetrating a host plant, AMF maintain a symbiotic relationship that leads to significant physiological changes and affects how the plant responds to disease. Considering that the roots are the main site of interaction between AMF and the host plant, research into the effects of
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DEMİR et al. / Turk J Agric For Table 7. Effects of AMF, HA, and W application on V.d. in tomato, pepper, and eggplant. Tomato Treatments
Wilt (%)**
Pepper Vasc. health Ms (%)***
Wilt (%)**
Eggplant Vasc. health Ms (%)***
Wilt (%)**
Vasc. health Ms (%)***
V.d. control
45.19 a*
55.0 a*
166 a *
44.44 a
56.0 a
144 a
40.74 a
52.0 a
155 a
V.d.+AMF
29.63 b
40.0 bc
112 b
28.15 b
34.0 c
100 c
19.26 c
38.0 c
100 b
V.d.+W
23.70 bc
45.0 b
100 b
15.56 d
35.0 c
89 d
29.63 b
40.7 bc
111 b
V.d.+HA
14.81 d
33.0 c
90 b
25.93 bc
45.0 b
111 bc
19.26 c
27.7 d
100 b
V.d.+W+AMF
22.22 bc
34.0 c
99 b
26.92 b
33.0 c
98 c
18.52 d
33.3 cd
90 bc
V.d.+HA+AMF
18.52 c
45.0 b
111 b
26.67 bc
33.0 c
122 b
21.48 c
39.0 c
88 bc
V.d.+W+HA
13.33 d
34.0 c
86 bc
20.00 c
44.0 b
100 c
22.96 bc
44.4 ab
88 bc
V.d.+W+HA+AMF
13.33 d
34.0 c
85 c
30.00 b
43.0 b
100 c
28.15 b
44.0 b
77 c
*P < 0.05 (significant). **Wilt symptoms in shoots were evaluated on a scale from 0 to 5. ***Vascular health was rated on a scale from 0 to 3. Ms: Number of V. dahliae microsclerotia in growth media.
AMF on plant disease has focused mainly on soil-borne pathogens. Dehne (1982) stated that AMF are capable of retarding the development of pathogens after they have been established on the root system. In addition, an earlier study by Dehne and Schönbeck (1978) reported that mycorrhizal fungi have excellent mechanical strength and are able to increase plant nutrient uptake because of their powerful transmission systems (xylem), which helps to reduce the damaging effects of plant pathogens. Demir (1998) reported that AMF (G. intraradices) reduced the disease severity of V.d. by 41% and restricted its ability to create inoculum. Karagiannidis et al. (2002) also showed that AMF (G. mosseae) reduced the incidence of disease caused by V.d., and Bars Orak and Demir (2011) reported that the application of AMF together with P (40 kg ha–1) significantly reduced the disease severity of V.d. The present study demonstrated that the application of HA to tomatoes, peppers, and eggplant significantly reduced disease severity (Table 7). Numerous studies have reported that HA promotes plant growth either directly or indirectly by impacting soil microorganisms such as Pythium ultimum, Phytophthora capsici, and Fusarium oxysporum (Pascual et al., 2002; Litterick et al., 2004; Türkmen et al., 2005; Loffredo et al., 2008; Biçer, 2011; Halifeoğlu, 2011; Aslanpay and Demir, 2012; Gülser et al., 2014). According to these studies, the functional and chemical properties of humic substances (total acidity, COOH groupings, elemental composition) allow HA to control soil-borne fungi and inhibit the formation of micelles. It has also been suggested that HA increases salt content in plants, thereby inhibiting disease formation (Gülser et al., 2014). The present study found that W combined with AMF and HA significantly suppressed the severity of V.d. disease
306
in both stems and shoots of solanaceous crops (Table 7). W is a substance of high biological value that possesses antimicrobial proteins and has been demonstrated to be effective in controlling powdery mildew and viral diseases (Crips et al., 2006; Pan et al., 2006); however, the number of studies examining the effects of W on soil-borne pathogens is limited. Biçer (2011) reported that W reduced the severity of wilt disease caused by Fusarium oxysporum f.sp. melonis by 46.7%; however, the mechanism by which W protects against soil-borne pathogens is unclear, although it was suggested that W promotes plant growth indirectly by improving nutrient uptake. The various applications of AMF, HA, and W used in the present study tended to reduce the microsclerotia density of V.d. in growth medium in all 3 solanaceous crops (Table 7). The triple application of AMF+HA+W was found to be especially effective in eggplant, resulting in close to a 50% reduction in microsclerotia density (Table 7). Reductions in V.d. development in plants lead to fewer propagules in growth medium. The findings of the present study are in line with those of Hwang et al. (1992), Demir (1998), Akköprü and Demir (2005), and Bars Orak and Demir (2011), which demonstrated that the reduction in the population of the pathogen was due to its interaction with AMF, which caused antibiotics and other inhibitive compounds to be secreted in plant roots as well as other changes in the microorganisms’ population in the rhizosphere. Despite variations in AMF colonization rates in seedling roots and soil spore density in response to the different applications, in general, the present study found that AMF development was negatively affected by applications that included the V.d. pathogen (Table 8). In
DEMİR et al. / Turk J Agric For Table 8. Means of % AMF colonized roots of tomato, pepper, and eggplant seedlings and spore density in soil applied with different combinations of HA, W, and V.d. Treatments
Tomato
Pepper
Eggplant
RC*
SD**
RC*
SD**
RC*
SD**
AMF
35.90 b
23 a
24.15 ab
20 a
26.40 a
30 b
AMF+W
38.67 b
25 a
26.59 ab
23 a
29.45 a
35 b
AMF+HA
44.10 a
21 a
31.64 a
19 a
30.74 a
47 a
AMF+W+HA
30.90 bc
12 b
21.82 b
15 ab
28.40 a
20 c
AMF+V.d.
28.10 c
15 b
13.17 c
5b
19.19 b
11 d
AMF+V.d.+W
28.07 c
15 b
17.18 b
12 ab
18.31 b
13 cd
AMF+V.d.+HA
23.75 cd
10 b
19.91 b
15 ab
19.79 b
13 cd
AMF+V.d.+HA+W
21.19 d
9c
16.97 bc
11 ab
17.64 b
10 d
* RC: AMF root colonization (%). ** SD: AMF spore density (cm3 soil).
contrast, applications that included HA increased AMF root colonization in all 3 plant species and increased soil spore density. The literature is still unable to present a clear picture as to how AMF colonization is affected by soil-borne pathogens. Whereas Zambolim and Schenck (1983), Hassan Dar et al. (1997), Akköprü and Demir (2005), and Aysan and Demir (2009) stated that various pathogens reduce AMF colonization of various hosts, Caron et al., (1985), Özgönen et al., (2001), and Aslanpay and Demir (2012) concluded that pathogen inoculation has no effect on AMF root colonization. There is also a limited number of studies examining how HA and W affect AMF colonization and sporulation. In some studies where W and AMF were used together, it was determined that both AMF and plant growth was ameliorated (Özrenk et al., 2003; Demir and Ozrenk, 2009). However, Biçer (2011) and Halifeoğlu (2011) determined that HA application promoted AMF colonization in melon and watermelon, while W inhibited colonization. In light of these studies, it may be deduced that the high nutrient content of W increases plant nutrient status and thus positively affects the growth of AMF, which are obligate microorganisms. In their study examining the effects of the interaction
between W and AMF on plant and soil nutrients (especially phosphorus) as well as on soil pH, salt, and CaCO3 content, Demir and Ozrenk (2009) found that plant P levels played an important role in AMF colonization and sporulation. The present study demonstrated that a combination of organic material (HA and W) and AMF, the most important symbiotic microorganisms found in the rhizosphere, improved the growth characteristics and disease resistance of three solanaceous vegetable species of high economic value (tomato, pepper, and eggplant). Having considered the positive effects of these agents in plant production, their helpful effect on seedling production cannot be avoided. Moreover, the use of W, a byproduct of the dairy industry, is thought to have both environmental and agricultural benefits, which makes this source an economically important input. The obtained encouraging results against Verticillium dahliae Kleb., an important soil-borne pathogen, could help plant protection efforts. Acknowledgment This project was supported by funds from the Scientific and Technological Research Council of Turkey (TÜBİTAK Project No. TOVAG-108O862).
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