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The effects of Trichoderma on preventing cucumber fusarium wilt and regulating cucumber physiology
Journal of Integrative Agriculture 2019, 18(3): 607–617 Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
The effects of Trichoderma on preventing cucumber fusarium wilt and regulating cucumber physiology LI Mei1, MA Guang-shu2, LIAN Hua2, SU Xiao-lin2, TIAN Ying2, HUANG Wen-kun1, MEI Jie1, JIANG Xiliang1 1
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
2
College of Agronomy, Heilongjiang Bayi Agricultural University, Daqing 163319, P.R.China
Abstract In our previous studies, we identified 3 Trichoderma strains with anti-Fusarium oxysporum activity, including T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886. Here, we evaluated the effects of these 3 Trichoderma strains on preventing cucumber fusarium wilt through pot culture and greenhouse culture experiments. All 3 Trichoderma strains demonstrated higher control effects toward cucumber fusarium wilt than previous studies, with efficacies over 78%. Additionally, inoculation with the 3 Trichoderma strains significantly promoted the quality and yield of cucumbers. Among the 3 strains, Trichoderma 866 was the most effective, with disease control efficacy of 78.64% and a cucumber yield increase of 33%. Furthermore, seedlings inoculated with Trichoderma exhibited significantly increased measures of plant height, stem diameter, leaf area, aboveground fresh weight, underground fresh weight, chlorophyll content, and nitric nitrogen content, as well as the activities of several stress-resistance enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), and ascorbate oxidase (AAO). In addition, the plants inoculated with Trichoderma showed decreased cell membrane permeability and malondialdehyde (MDA) content in the leaves. Together, our results suggest that T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886 inoculations inhibit F. oxysporum infection, stimulate the metabolism in cucumbers, and enhance the activities of stress-resistance enzymes, which consequently promote the growth of cucumber plants, prevent cucumber fusarium wilt, and improve the yield and quality of cucumbers. T. harzianum is a commonly used biocontrol fungus, while few studies have focused on T. asperellum or T. koningense. In this study, strains of T. asperellum and T. pseudokoningii showed excellent plant disease prevention and growth promoting effects on cucumber, indicating that they also have great potential as biocontrol fungi. Keywords: Trichoderma, cucumber fusarium wilt, physicochemical features, control effect, Fusarium oxysporum f. sp. cucumerinum Owen
Received 12 June, 2018 Accepted 24 July, 2018 LI Mei, Tel: +86-10-82106381, E-mail: [email protected]; Correspondence JIANG Xi-liang, Tel/Fax: +86-10-82106381, E-mail: [email protected]; LIAN Hua, Tel: +86-459-6819184, Fax: +86-459-6819170, E-mail: [email protected] © 2019 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(18)62057-X
1. Introduction Cucumber fusarium wilt is a soil-borne fungal disease caused by Fusarium oxysporum f. sp. cucumerinum Owen. It can occur at any stage of cucumber growth and severely impairs the yield and quality of cucumbers (Lievens et al. 2007). Biological control approaches for disease management are
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typically safe and environmentally friendly, and thus, have been broadly applied in the control of vegetable soil-borne diseases (Naranjo et al. 2015).
2. Materials and methods 2.1. Materials
Trichoderma spp. includes important strains of fungi for biological control with key features including high adaptability, fast growth and broad antibiotic spectrum. In particular, they have demonstrated significant inhibitory effects on major soil-borne fungal pathogens, such as Fusarium spp., Pythium spp., Phytophthora spp., and Rhizoctonia solani (Mukherjee et al. 2012; Prabhakaran et al. 2015). The ability of Trichoderma spp. to prevent cucumber fusarium wilt has been established in several studies. For example, Cheng et al. (2010) found that T. viride T23 and T. harzianum T22 prevented cucumber fusarium wilt; Bi (2016) showed that T. longibrachiatum and T. viride prevented cucumber fusarium wilt; Chen (2011) demonstrated an inhibitory effect of two Trichoderma stains TG and TM leachate on fusarium pathogenic fungi; and Deng et al. (2013) reported that Trichoderma not only prevented cucumber fusarium wilt but also promoted cucumber growth and yield. All the studies above focused on the disease control effects of Trichoderma on cucumber F. oxysporum and the subsequent yield. However, the impact of Trichoderma on the physiological and biochemical features of cucumbers remains unclear. Additionally, little is known about the correlation between the Trichoderma-induced physiological changes and the efficacy of Trichoderma for disease prevention and cucumber yield improvement. In our previous screening studies, we obtained 3 strains of Trichoderma with anti-F. oxysporum activity, including T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886. In this study, we further evaluated the effects of these 3 strains on cucumber fusarium wilt incidence, in addition to cucumber seedling growth, biomass accumulation, physicochemical features, and product quality. We also explored the mechanism by which Trichoderma prevents cucumber fusarium wilt and promotes cucumber growth. Together, our results provide scientific evidence for the benefits of appying Trichoderma in the field and the technical basis for improving the safety, yield, and product quality in cucumber cultivation.
The cucumber cultivar used in this study was Changchun Mici, from Yuyuan Seed Co., Ltd., Xintai, Shandong, China. The strains of Trichoderma and pathogenic fungi used in this study are listed in Table 1.
2.2. Preparation of the spore suspensions of Tricho derma and pathogenic fungi Trichoderma were cultured on PDA culture medium (200 g of potato, 20 g of dextrose, 10 g of agar, 1 000 mL of distilled water, and neutral pH) for 7 d in the dark at 28°C, and the spores were collected by washing the agar surface with distilled water. For preparation of the spores of phytopathogenic fungi, 5 samples with 5 mm diameter were taken from the edge of each fungal colony and inoculated into 100 mL of the PD liquid culture medium described above in 500-mL conical flasks. The flasks were cultured in the dark for 3 d in a shaking incubator (250 r min–1) at 28°C to obtain the spore suspensions. The spore counts were determined using a hemocytometer. Each Trichoderma spore suspension was diluted to a concentration of 1.5×108 spores mL–1, and the pathogenic fungi spore suspensions were diluted to a concentration of 1×105 spores mL–1.
2.3. Evaluation of the effect of Trichoderma on the quality of cucumber seedlings and the prevention of cucumber fusarium wilt Cultivating seedlings of cucumber This experiment was performed in a modern greenhouse at the Teaching Base of College of Agronomy, Heilongjiang Bayi Agricultural University, China. The soil was mixed well and placed in 50-hole plastic seedling culture dishes (3.5 cm×24 cm× 11 cm). In each dish, 100 soaked and germinated seeds were planted. After germination, 50 seedlings with similar size were retained. Three replicates of 5 dishes were used in each treatment for the field tests and the yield/quality evaluations. The basic physicochemical properties of the potting soil were as follows: 198.56 mg kg–1 of total nitrogen,
Table 1 Fungal strains used in this study Codes of isolates 525 610 886 B
Scientific name Trichoderma asperellum Trichoderma harzianum Trichoderma pseudokoningii Fusarium oxysporum f. sp. cucumerinum Owen
Source This lab This lab This lab Isolated from cucumber plants showing fusarium wilt symptoms and stored in this lab
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164.78 mg kg–1 of alkali-hydrolyzed nitrogen, 15.76 mg kg–1 of total phosphorus, 210 mg kg–1 of fast-acting potassium, 59.60 g kg–1 of organic matter, and pH value of 8.04. Inoculation of Trichoderma and pathogenic fungi The cucumber seeds were disinfected using a 40% formalin solution that was diluted 100-fold. After soaking in the formalin solution for 30 min, the seeds were rinsed with water and soaked in water for 8–12 h at 25–30°C. The seeds were then cultured at 28–30°C, and the seeds started to germinate after 12 h. Germinated seeds were sown in pots, and when the true leaves started to unfold 5 d after sowing, cucumber seedlings of relatively consistent size were inoculated with Trichoderma and/or pathogenic fungi spore suspensions through root irrigation. Each seedling was inoculated with 3 mL of the corresponding suspension. Each of the 8 different treatments in this study included 5 dishes, and each dish included 50 seedlings. All seedlings were randomly selected, and the entire experiment was repeated 5 times. The treatment groups were as follows: 1) T. asperellum 525 only (T1); 2) T. harzianum 610 only (T2); 3) T. pseudokoningii 886 only (T3); 4) T. asperellum 525 and pathogenic fungus (T1B); 5) T. harzianum 610 and pathogenic fungus (T2B); 6) T. pseudokoningii 886 and pathogenic fungus (T3B); 7) Pathogenic fungus F. oxysporum f. sp. cucumerinum Owen only (B); 8) PD liquid medium control (P). Greenhouse transplanting and follow-up management In the soil at the greenhouse, 5 000 kg of organic fertilizer and 150 kg of ammonium dihydrogen phosphate per hectare were applied. The soil was fully plowed for beds. High beds were arranged with a lower bed width of 120 cm, an upper bed width of 100 cm, a bed height of 13–15 cm, and a bed length of 5 m. Two rows of plants were sown in the upper beds, with plant spacing of 25 cm and row spacing of 60 cm. Each bed was set as an experimental plot with a size of 12 m2. A randomized block design was used for the experimental plots, with 5 replicates. When the seedlings had grown to the stage of 4-leaf and 1-heart, healthy seedlings of consistent size were collected and transplanted to the greenhouse, with 42 plants randomly chosen and transplanted to each experimental plot. Upon recovery, each plant was irrigated with 100 mL of the indicated treatment solution (Trichoderma spore suspension or water). The T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886 strain treatments were named T-1, T-2, and T-3, respectively, while the water control was named CK. After the treatments, vine hanging and single vine pruning were performed in a timely manner, and all plants were watered every 3 d. Analysis of cucumber seedlings in pots A total of 7 d after the seedlings were inoculated with Trichoderma and/
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or pathogenic fungi, the growth morphological, physiological, biochemical, and disease resistance indexes were measured. The plant height was determined by the distance between the stem base and the growing point of each seedling. The stem diameter was measured at 1 cm below the cotyledon section (Zhang S P et al. 2016). The root volume was measured by the displacement approach (Zhang S P et al. 2016). The leaf area was measured by the weighing method (Cao et al. 2017). First, a leaf with certain area (A1) was collected and stacked with other leaves. Then, a 1-cm puncher pin was used to obtain round sheets; the weights of the round sheets and the remaining leaves were measured, from which the weight of the leaf with a certain area (W1) and the weights of the remaining leaves (W2) were calculated. From these measurements, the leaf total area (A) was calculated using the formula: A=[A1×(W1+W2)]/W1 The root weight, shoot weight and the seedling dry weight were measured according to Hao et al. (2007). From these values, the seedling vigor index was calculated using the formula: Seedling vigor index=(Stem diameter/Plant height+Root weight/Shoot weight)×Seedling dry weight (Li et al. 2017) For aboveground and underground fresh weights, the plants were repeatedly rinsed with water and dried with absorbent paper. A 1/1 000 electronic scale was used to measure the fresh weights (Chen et al. 2002). The physiological biochemical indexes included chlorophyll content, soluble carbohydrate content, nitric nitrogen content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, polyphenol oxidase (PPO) activity, ascorbate oxidase (AAO) activity, root activity, total root absorption area, and root specific surface area. The soluble carbohydrate content was measured by the anthrone colorimetric method, as shown in Huang et al. (2009). Nitric nitrogen was measured by the phenol disulfonic acid method (Ma et al. 2017). The activities of SOD were measured by the NBT method (Giannopolitis and Ries 1977). The activities of POD were measured by the guaiacol method (Vieria 1998). The activities of CAT and AAO were measured according to the method of Cakmak and Marschner (1992). The activities of PPO were measured by titration (Bao et al. 2015). The content of chlorophyll was measured by the acetone-ethanol method (Hao et al. 2007). The leaf cell membrane permeability was evaluated based on the relative conductivity (Tian et al. 2017). The malondialdehyde (MDA) content was measured by the thiobarbituric acid method (Sun et al. 2001). Root activity was determined by the alpha-naphthylamine oxidation method (Zhang 2002). The total root absorption area, root active absorption area, and root specific surface area were determined by the methylene blue absorption method (Rubín et al. 2010) using the following formulas:
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2.4. Statistical analysis Microsoft Excel 2007 was used for preparing figures and
tables. The average and standard deviation of either 3 or 5 independent replicates for each treatment were calculated. The significances of differnces between samples were calculated using the DPS7.05 data processing system.
3. Results 3.1. The preventive effect of Trichoderma on cucumber fusarium wilt A total of 7 d after inoculation of Trichoderma and the pathogenic fungi onto cucumber seedlings, we evaluated the survival rate, disease incidence, disease index, and control effects. As shown in Fig. 1, the disease indexes of the seedlings treated with both Trichoderma and pathogenic fungus (T1B, T2B, and T3B), or pathogenic fungus only (B), were 9.12, 10.69, 10.55, and 49.39, respectively. The control effects of T1B, T2B, and T3B were 81.53, 78.36, and 78.64%, respectively, with no significant differences among the three groups. These results indicated that all 3 strains of Trichoderma exhibited good control effects on cucumber fusarium wilt.
3.2. The effects of Trichoderma on the yield and quality of cucumbers Trichoderma significantly increased overall cucumber yield, average fruit weight, and the contents of soluble solids, soluble carbohydrates, soluble proteins, and vitamin C in cucumber fruits, as shown in Fig. 2 and Table 2. Compared with the Trichoderma-untreated group (CK), T-1, T-2, and T-3-treated plants exhibited 25.57, 23.54, and 33.85% increases in cucumber yield, respectively. T-3-treated plants exhibited a significantly higher cucumber yield, and the difference between the T-1- and T-2-treated plants was not Control efficiency 90 80 70 60 50 40 30 20 10 0
Disease index
a
a
a
b
b
b
a
b T1B
T2B
T3B
B
60 50 40 30 20 10 0
Disease index
Total root absorption area (m2)=[(C1−C1´)×V1]+[(C2− C2´)×V2]×1.1 Root active absorption area (m2)=[(C3−C3´)×V3]×1.1 Root specific surface area (m2 mL–1)=Total root absorption area/Total root volume, in which C indicates the original concentration (mg mL–1) of the solution, C´ indicates the concentration (mg mL–1) of the solution after extraction, and 1, 2, and 3, indicate the numbers of the subsequent rinses. The disease resistance indexes included plant survival rate, disease incidence rate, disease index, and control effect. The grading system of cucumber fusarium wilt severity was according to Zhang S P’s standard (2016), and the disease index was calculated according to the method of Zong and Kang (2002): Grade 0: no symptoms; Grade 1: The yellowing or wilting area of true leaves and cotyledon does not exceed 50% of the total area; Grade 2: The yellowing or wilting area of true leaves and cotyledon exceeds 50% of the total area; Grade 3: Leaves are wilted or dead with only growing points surviving; Grade 4: The entire plant is severely wilted or dead. Disease index=∑(Plant number in the grade×Grade number)/(Total plant number×The highest grade number)×100% Control effect (%)=(Disease index in the control group− Disease index in the treated group)/Disease index in the control group×100 Determination of cucumber yield and quality indexes After harvesting cucumbers 2–4 times, 5 cucumber fruits were randomly selected from each experimental plot. The quality of fruits was evaluated, and the average quality value was calculated (Ma et al. 2016). For the average single-fruit weight and yield, 10 plants from each experimental plot were tagged and separately managed. An electronic scale with 0.01 g precision was used to measure the weight of individual cucumbers. In addition, the yield of each plot was determined, and the average yield was calculated. From these, the yield per square meter was calculated according to the planting density and the plot area. The soluble solids content was determined using a handheld refractometer. The soluble carbohydrate content was determined using the anthrone colorimetric method (Huang et al. 2009). The vitamin C content was evaluated by ultraviolet spectrophotometry (Zhang et al. 2009). The soluble protein content was determined by the Coomassie brilliant blue G-250 staining method (Oshima et al. 1987). The nitric nitrogen content was determined by the phenol disulfonic acid method (Ma et al. 2017).
Control efficiency (%)
610
Fig. 1 The effects of Trichoderma on preventing cucumber Fusarium wilt. The disease resistance indexes of cucumber seedlings were detected 7 d after treatment. B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B. Data are mean±SD. Different lowercase letters indicate significant differences at the 0.05 probability level.
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T3
Fig. 2 Effects of Trichoderma treatment on yield of cucumber. One average sized cucumber fruit was selected and photographed from each experimental plot after harvesting 4 times. CK, water; T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only.
Soluble sugar Soluble sugar Increase (%) (%) 2.42±0.97 c – 3.51±0.37 a 45.04 2.94±0.63 b 21.49 3.78±0.84 a 56.20 Soluble solid Soluble solid Increase (%) (%) 4.52±0.18 c – 5.17±0.21 a 14.38 4.87±0.38 b 7.74 5.34±0.34 a 18.14 Weight per fruit Weight per fruit Increase (g) (%) 117.81±2.45 c – 139.99±2.65 a 18.83 128.48±4.26 b 9.06 145.93±2.87 a 23.87 Increase (%) – 25.57 23.54 33.85 Yield Yield (kg m–2) 5.67±0.04 c 7.12±0.10 b 7.00±0.10 b 7.58±0.07 a CK T-1 T-2 T-3
T2
Treatment1)
T1
Table 2 The effects of Trichoderma on the quality and yield of cucumber
CK
Vitamin C Vc Increase (mg 100 g–1 FW) (%) 15.21±0.93 c – 18.65±0.53 a 22.62 16.87±0.71 b 10.91 19.65±0.64 a 29.19
The 7 d after treatment with Trichoderma and/or the pathogenic fungus, the cucumber seedlings were examined for growth, morphology, and biomass accumulation. As shown in Table 3 and Fig. 3, the T1- and T3-treated cucumber seedlings exhibited greater plant height than T1B-, T2B-, and T3B-treated cucumber seedlings. T2-treated cucumber seedlings exhibited a plant height similar to T2B. T1-, T2-, and T3-treated cucumber seedlings exhibited greater leaf area, root volume, and biomass accumulation compared to the T1B-, T2B-, and T3B-treated cucumber seedlings. The PD medium (P)-treated cucumber seedlings exhibited similar levels for seedling vigor indexes, underground fresh weight, aboveground fresh weight and stem diameter, but showed higher levels of plant height, root volume, and leaf area than the B-treated seedlings. Moreover, the Trichoderma treatments promoted the growth and biomass accumulation of cucumber seedlings. The T1-, T2-, and T3-treated cucumber seedlings exhibited 24, 8.6, and 25% increases in plant height, respectively, compared with the control (P), and the growth-promoting effects of T1 and T3 were significantly higher than that of T2. The pathogen treatment (B) led to a decrease in growth and biomass accumulation, which was attenuated upon the treatment together with Trichoderma. The seedling vigor indexes in the T1-, T2-, and T3-treated seedlings were not significantly different. The underground
1)
3.3. The effects of Trichoderma on cucumber seedling growth
CK, water; T-1, T. asperellum 525; T-2, T. harzianum 610; T-3, T. pseudokoningii 886. Values are mean value±SE. Lowercase letters indicate the samples with significant differences in each column at 0.05 significance level.
Soluble protein Soluble protein Increase (mg 100 g–1） (%) 20.98±1.32 c – 24.51±2.27 a 14.40 23.29±1.76 b 9.92 25.21±3.65 a 16.78
significant. Compared with the T-2-treated plants, the T-1- and T-3-treated plants produced cucumbers with a higher quality. Among all the fruit quality indexes of soluble solids, soluble carbohydrates, soluble proteins, and vitamin C, the changes in soluble carbohydrates were the most significant in the Trichoderma-treated plants. Specifically, the T-1-, T-2-, and T-3treated cucumbers exhibited 45.04, 21.49, and 56.20% increases in soluble carbohydrate content compared to the control, respectively. These results suggest that the Trichoderma treatments improved the yield and quality of cucumbers to varying extents.
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Table 3 The effects of Trichoderma on cucumber seedling growth Treatment1)
Plant height (cm)
Root volume (mL)
Sound seedling index
Leaf area (cm2)
T1B T2B T3B B T1 T2 T3 P
8.04±0.05 b 7.97±0.02 bc 7.80±0.07 b 6.66±0.04 e 8.49±0.02 a 7.43±0.04 c 8.58±0.02 a 6.84±0.04 d
0.51±0.02 c 0.52±0.01 bc 0.53±0.01 b 0.51±0.01 c 0.60±0.05 a 0.61±0.02 a 0.60±0.00 a 0.55±0.00 b
0.11±0.01 ab 0.09±0.01 c 0.10±0.01 bc 0.09±0.01 c 0.12±0.01 a 0.11±0.01 ab 0.11±0.01 ab 0.10±0.01 bc
34.96±1.22 b 34.13±0.33 c 34.76±1.41 bc 31.50±0.91 e 36.39±0.83 a 34.96±0.37 b 35.19±2.45 b 32.19±00.82 d
1)
Underground fresh weight (g/plant) 0.36±0.03 b 0.29±0.02 c 0.33±0.03 a 0.29±0.04 c 0.35±0.02 ab 0.31±0.02 bc 0.36±0.02 a 0.30±0.03 c
Stem diameter (mm) 2.43±0.02 c 2.44±0.05 c 2.62±0.03 b 2.02±0.02 d 2.53±0.04 c 2.57±0.04 bc 2.73±0.02 a 2.13±0.02 d
Aboveground fresh weight (g/plant) 1.29±0.07 b 1.22±0.02 c 1.26±0.02 c 1.21±0.07 bc 1.30±0.07 a 1.23±0.05 ab 1.28±0.02 b 1.25±0.04 b
The growth morphological indexes of cucumber seedlings were measured 7 d after treatment. T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; P, PD liquid medium control. Values are mean value±SE. Different lowercase letters within the same column indicate significant differences at 0.05 probability level.
T1B
T2B
T3B
B
T1
T2
T3
P
Fig. 3 Effect of Trichoderma treatment on the growth of cucumber seedlings. The photo was taken on the 7 d after inoculation. T1B, T. asperellum 525 and B; T2B, T. harzianugm 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; P, PD liquid medium control.
fresh weight, aboveground fresh weight, and plant height of the T1-treated seedlings were equivalent to those of the T3-treated seedlings, but were higher than those of the T2treated seedlings. The leaf area of the T2-treated seedlings was similar to that of the T3-treated seedlings but was lower than that of T1-treated seedlings. Furthermore, among the T1-, T2-, and T3-treated seedlings, the T3-treated seedlings exhibited the largest stem diameters, and T1-treated seedlings exhibited the smallest stem diameters. Together, these data suggested that among the T1-, T2-, and T3-treatments, the T3 treatment exhibited the greatest effect on promoting seedling growth and biomass accumulation, while the T2 treatment exhibited the smallest effect on promoting seedling growth and biomass accumulation.
the different strains of Trichoderma were collected 7 d after
3.4. The effects of Trichoderma on cucumber seedling physiology
of the T1B-, T2B- and T3B-treated seedlings, which were
The leaves derived from cucumber seedlings treated with
promoting seedling physiology.
treatment, and analyzed for several physiological features. As shown in Figs. 4–5, compared with the leaves from the other groups, the leaves derived from the T3-treated cucumber seedling exhibited significantly higher levels of chlorophyll content, nitric nitrogen content, root activity, total root absorption area, root active absorption area, and root-specific surface area, while the B-treated seedlings exhibited significantly lower levels of all the corresponding physiological indexes. The Trichoderma treatment promoted the physiological activities of cucumber seedlings and enhanced the efficiency of photosynthesis and nitric nitrogen absorption. Specifically, the physiological activities of the T1-, T2- and T3-treated seedlings were higher than those higher than those of the B-treated seedlings. Among all 3 strains of Trichoderma, T3 exhibited the highest effect on
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B
1.6
a
1.4 1.2
c
d
de
b
c
d
e
1.0 0.8 0.6 0.4 0.2 0
cd
8
Nitrate nitrogen content (×1 000 μg mg–1)
Chlorophyll content (mg g–1)
A
a
e
c
b
d
e
7
f
6 5 4 3 2 1 0
T1
T2
T3
T1B T2B T3B
B
T1
P
T2
T3
Treatment
T1B T2B T3B
B
P
Treatment
Fig. 4 The effects of Trichoderma on chlorophyll and nitric nitrogen content in cucumber seedling leaves. The chlorophyll (A) and nitric nitrogen contents (B) in cucumber seedlings were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
1.2
Total absorption (m2)
a
ab
a
a
b
bc d
0.8
0.4
Root specific surface area (m2 mL–1)
Active absorption area (m2) c
1.0
0.6
Root activity (×500 μg g–1 FW h–1)
b
c bc
a
a
c b
a
a
T1
T2
T3
c
d
c
c c
c b
d
cd
c
e
a
b
c
c
T2B
T3B
B
P
0.2 0
T1B
Fig. 5 The effects of Trichoderma on the physiology of roots in cucumber seedlings. The physiological indexes in roots of cucumber seedlings were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
3.5. The effects of Trichoderma on the stress-resistance of cucumber seedlings The activities of stress-resistance enzymes in seedling leaves were analyzed following treatment with Trichoderma and pathogenic fungi. As shown in Fig. 6, the T3-treated seedlings exhibited the highest activities of SOD, POD, CAT, PPO, and AAO, compared with the other treatments. The PD liquid medium-treated seedlings exhibited the lowest enzymatic activities, and the B-treated seedlings only exhibited a slight increase in enzymatic activities. These results indicated that both the pathogenic fungus and Trichoderma treatments enhanced the activities of stressresistance enzymes, while the effects of Trichoderma were stronger than those of the pathogenic fungus.
The contents of MDA in cucumber seedlings are shown in Fig. 7-A. The contents of MDA in the T1-, T2-, and T3treated seedling leaves were equivalent to each other, but higher than the MDA contents in the T1B-, T2B-, T3B-treated seedling leaves. The T3B- and P-treated leaves showed the lowest contents of MDA. These data indicated that either Trichoderma or pathogenic fungus alone increased the content of MDA, but the treatment of pathogenic fungus in combination with Trichoderma decreased the content of MDA to a different extent. Furthermore, the effects of the treatments on leaf cell membrane permeability were as follows: B>T1>T2>T3 (Fig. 7-B). In addition, leaves treated with T1B, T2B, and T3B also showed different levels of decrease in leaf permeability. Together, our data indicated that compared to the P treatment, the Trichoderma
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SOD (×100 U g–1) PPO (mg g–1 min–1)
6 5 Enzyme activity
d
bc b b
4
bab
a b
a
b
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Plasma membrane permeability (%)
Fig. 6 The effects of Trichoderma on the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO) and ascorbate oxidase (AAO) in cucumber seedlings. The antioxidant enzyme activities were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
60 50
b
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a c
d
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30 20 10 0
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T2
T3 T1B T2B T3B
B
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Fig. 7 The effects of Trichoderma on the malondialdehyde content and cell membrane permeability in cucumber seedling leaves. The malondialdehyde (MDA) content (A) and plasma permeability (B) were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
and pathogenic fungus treatments increased the MDA
control have primarily used strains such as T. harzianum
content and cell membrane permeability, in which the
(Yedidia et al. 2001; Cheng et al. 2010; Chen et al. 2012),
pathogenic fungus treatment induced a stronger response.
T. viride (Zhuang et al. 2005; Cheng et al. 2010; Bi 2016),
Additionally, the combined Trichoderma and pathogenic
T. longibrachiatum (Li et al. 2010; Bi 2016; Zhang S W
fungus treatment reduced both the MDA content and cell
et al. 2016), T. reesei (Luo et al. 2016), and T. atroviride
membrane permeability.
(Han et al. 2013). Very few studies have focused on
4. Discussion
In this study, we isolated 3 strains of Trichoderma,
Recent studies on the role of Trichoderma in biological
T. pseudokoningii 886 (T3), which have been identified
T. asperellum and T. koningense (Qi and Zhao 2013). including T. asperellum (T1), T. harzianum 610 (T2), and
LI Mei et al. Journal of Integrative Agriculture 2019, 18(3): 607–617
as T. asperellum, T. harzianum, and T. pseudokoningii, respectively. Functional analysis showed that all 3 strains of Trichoderma prevented cucumber fusarium wilt and promoted the quality and yield of cucumber. Particularly, the efficacy of T1 and T3 was better than that of T2. Our results indicate that T. asperellum and T. koningense may have great potential for application to plant disease control. In our preliminary studies, using a dual culture assay, we screened some Trichoderma strains from our laboratory that were antagonistic against F. oxysporum. Among these strains, T. asperellum 525 (T1), T. harzianum 610 (T2), and T. pseudokoningii 886 (T3) exhibited 80.24, 76.86, and 74.17% growth inhibitory rates against F. oxysporum, respectively (data not shown). To further characterize the antagonistic strains, we evaluated the effect of these 3 strains on preventing cucumber fusarium wilt in both pot and greenhouse culture experiments. The results showed that the effects of T1, T2, and T3 on preventing cucumber fusarium wilt were 81.53, 78.36, and 78.64%, respectively. The results from greenhouse culture experiments were consistent with the results from the dual culture assay, indicating that dual culture assay could be a reliable method to screen Trichoderma strains for their ability to prevent cucumber fusarium wilt. Abo-Elyousr (2014) reported that the results from dual culture assay and greenhouse culture experiments were not positively correlated. We speculate that the lack of correlation between those two experiments might have been due to the variance in pathogenic fungi, Trichoderma strains, or culture conditions. Further validation is required to confirm the correlation between the dual culture assay data and the greenhouse culture experiment data when those variables are taken into account. In this study, 7 d after inoculation was chosen as the time-frame for evaluating the effects of different Trichoderma strains. The 7 d after the treatment with Trichoderma (or 12 d after the sowing of cucumber) was the period when the first true leaves of the cucumber seedlings were fully unfolded (the one leaf and one heart period). The growth of cucumber seedlings was vigorous at this time, so the effects of Trichoderma treatments on morphological, physiological and biochemical indexes of cucumber seedlings were dramatic, which could be used to easily evaluate the effects of the Trichoderma strains. Several studies have shown that Trichoderma prevented cucumber fusarium wilt. For example, Cheng et al. (2010) observed that T. viride T23 and T. harzianum T22 prevent cucumber fusarium wilt, with an efficacy of 66.04%; while Zhuang et al. (2005) demonstrated that upon treatment with T. viride T23 conidia and chlamydospores, cucumber seedlings exhibited a decrease in the disease index from 33.69 to 13.12 and 10.28, respectively. Bi et al. (2016) showed that T. longibrachiatum and T. viride prevent
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cucumber fusarium wilt, with efficacies of 75.74 and 70.76%, respectively. In our study, all three strains of Trichoderma exhibited control efficacy above 78%, and treatments with Trichoderma reduced the disease index from 49.93 in the control to 9.12–10.55. Our data showed higher control efficacy of Trichoderma on cucumber fusarium wilt compared to the published results, indicating the great application value of the 3 Trichoderma strains used here in the field. In addition to inhibiting several pathogenic fungi, Trichoderma also promoted the growth and yield of plants (Hyakumachi et al. 1994; Masunaka et al. 2011; Deng et al. 2013). For example, Yedidia et al. (2010) reported that T. harzianum T203 in the soil increased the cucumber root area by 95%, the plant dry weight by 80%, the root length by 75%, the plant height by 45%, and the leaf area by 80%. Zhang S W et al. (2016) demonstrated that T. longibrachiatum T6 spore suspension significantly promoted the growth of Meloidogyne incognita-inoculated cucumbers. In addition, several strains of Trichoderma also promoted the plant growth and biomass accumulation in other plants, such as cabbage (Xing et al. 2017) and Sedum plumbizincicola (Luo et al. 2016). The plant chlorophyll content and the leaf nitric nitrogen content are important indicators for evaluating photosynthesis and nitrogen assimilation and utilization. In this study, T. pseudokoningii 886 (T3) treatment significantly increased the chlorophyll content, nitric nitrogen content, root activity, total root absorption area, and root specific surface area, effects that were consistent with the published data. Our results indicated that the Trichoderma treatments enhanced the physiological and metabolic activities in cucumbers, and increased the cucumber yields. Notably, compared to all the other treatments, T3 induced higher physiological and metabolic activities, more biomass accumulation, and higher cucumber yield (33.85%), as well as more dramatic changes in all the other indexes. It is well known that Trichoderma may have a variety of action mechanisms such as competition, antagonism, mycoparasitism, and the stimulation of plant resistance. The results suggested that the antagonistic effect of T3 on pathogenic fungi was lower than those of T1 and T2, but T3 also had a strong effect on inducing plant resistance, which could promote physiological activities, increase yield and quality of cucumber. T3 was identified as T. pseudokoningii. Little is known about T. pseudokoningii as a potential biocontrol agent, and its biocontrol mechanisms need further research. It has been established that enhanced activities of POD, CAT, PPO, AAO, and SOD increase the stress-resistance capability of plants. Additionally, MDA is one of the final products of lipid peroxidation in cell membranes. It causes membrane lipid peroxidation, damages the structure and function of biomembranes, and changes the permeability
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of the membranes, which affect a series of physiological and biochemical reactions of cells. Therefore, the content of MDA reflects the level of cell membrane damage (Huang 2005). The higher the membrane permeability and MDA content, the greater the extent of cell membrane damage. Liu et al. (2013) reported that T. viride broth inhibited the growth of mango anthracnose pathogens and enhanced the activities of antioxidant enzymes in mango. Liu et al. (2014) demonstrated that T. harzianum T23 induced the generation of phytoalexin and lignin in eggplants and enhanced the activities of PAL, PPO, POD, and SOD, which enhanced the resistance of eggplants to fusarium wilt. Consistent with these published data, the current study showed that 3 Trichoderma strains induced different levels of increase in the activities of antioxidant enzymes, suggesting that all 3 Trichoderma strains enhanced the disease resistance in cucumber plants by elevating the activities of stress-resistance enzymes. Compared to the other controls, pathogenic fungi-treated cucumber seedlings exhibited significantly enhanced stress-resistance enzyme activities, higher leaf cell membrane permeability, and higher MDA content, indicating that the pathogenic fungi damaged cucumber seedlings. In addition, the cell membrane permeability and MDA content of the Trichoderma and pathogenic fungi-treated seedlings were higher than those of the control group (P), indicating that the Trichoderma and pathogenic fungi stimulated or damaged the seedlings at varying levels. However, the combined treatment of Trichoderma and the pathogenic fungi decreased the cell membrane permeability and MDA content to various extents, suggesting that the interaction between Trichoderma and the pathogenic fungi ameliorates pathogen damage to the seedlings. The level of efficacy was associated with the specific Trichoderma strains, and the exact mechanisms require further analysis.
5. Conclusion T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886 inhibit F. oxysporum infection, stimulate the metabolism of cucumber plants, and enhance the activities of stressresistance enzymes, which consequently promote the growth of cucumber plants, prevent cucumber fusarium wilt, and improve the yield and quality of cucumbers.
Acknowledgements The authors are grateful for the financial support from the National Key R&D Program of China (2018YFD0201202), the National Science and Technology Basic Work, China (2014FY120900) and the 948 Program of China (2011-G4).
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RESEARCH ARTICLE
The effects of Trichoderma on preventing cucumber fusarium wilt and regulating cucumber physiology LI Mei1, MA Guang-shu2, LIAN Hua2, SU Xiao-lin2, TIAN Ying2, HUANG Wen-kun1, MEI Jie1, JIANG Xiliang1 1
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
2
College of Agronomy, Heilongjiang Bayi Agricultural University, Daqing 163319, P.R.China
Abstract In our previous studies, we identified 3 Trichoderma strains with anti-Fusarium oxysporum activity, including T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886. Here, we evaluated the effects of these 3 Trichoderma strains on preventing cucumber fusarium wilt through pot culture and greenhouse culture experiments. All 3 Trichoderma strains demonstrated higher control effects toward cucumber fusarium wilt than previous studies, with efficacies over 78%. Additionally, inoculation with the 3 Trichoderma strains significantly promoted the quality and yield of cucumbers. Among the 3 strains, Trichoderma 866 was the most effective, with disease control efficacy of 78.64% and a cucumber yield increase of 33%. Furthermore, seedlings inoculated with Trichoderma exhibited significantly increased measures of plant height, stem diameter, leaf area, aboveground fresh weight, underground fresh weight, chlorophyll content, and nitric nitrogen content, as well as the activities of several stress-resistance enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), and ascorbate oxidase (AAO). In addition, the plants inoculated with Trichoderma showed decreased cell membrane permeability and malondialdehyde (MDA) content in the leaves. Together, our results suggest that T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886 inoculations inhibit F. oxysporum infection, stimulate the metabolism in cucumbers, and enhance the activities of stress-resistance enzymes, which consequently promote the growth of cucumber plants, prevent cucumber fusarium wilt, and improve the yield and quality of cucumbers. T. harzianum is a commonly used biocontrol fungus, while few studies have focused on T. asperellum or T. koningense. In this study, strains of T. asperellum and T. pseudokoningii showed excellent plant disease prevention and growth promoting effects on cucumber, indicating that they also have great potential as biocontrol fungi. Keywords: Trichoderma, cucumber fusarium wilt, physicochemical features, control effect, Fusarium oxysporum f. sp. cucumerinum Owen
Received 12 June, 2018 Accepted 24 July, 2018 LI Mei, Tel: +86-10-82106381, E-mail: [email protected]; Correspondence JIANG Xi-liang, Tel/Fax: +86-10-82106381, E-mail: [email protected]; LIAN Hua, Tel: +86-459-6819184, Fax: +86-459-6819170, E-mail: [email protected] © 2019 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(18)62057-X
1. Introduction Cucumber fusarium wilt is a soil-borne fungal disease caused by Fusarium oxysporum f. sp. cucumerinum Owen. It can occur at any stage of cucumber growth and severely impairs the yield and quality of cucumbers (Lievens et al. 2007). Biological control approaches for disease management are
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typically safe and environmentally friendly, and thus, have been broadly applied in the control of vegetable soil-borne diseases (Naranjo et al. 2015).
2. Materials and methods 2.1. Materials
Trichoderma spp. includes important strains of fungi for biological control with key features including high adaptability, fast growth and broad antibiotic spectrum. In particular, they have demonstrated significant inhibitory effects on major soil-borne fungal pathogens, such as Fusarium spp., Pythium spp., Phytophthora spp., and Rhizoctonia solani (Mukherjee et al. 2012; Prabhakaran et al. 2015). The ability of Trichoderma spp. to prevent cucumber fusarium wilt has been established in several studies. For example, Cheng et al. (2010) found that T. viride T23 and T. harzianum T22 prevented cucumber fusarium wilt; Bi (2016) showed that T. longibrachiatum and T. viride prevented cucumber fusarium wilt; Chen (2011) demonstrated an inhibitory effect of two Trichoderma stains TG and TM leachate on fusarium pathogenic fungi; and Deng et al. (2013) reported that Trichoderma not only prevented cucumber fusarium wilt but also promoted cucumber growth and yield. All the studies above focused on the disease control effects of Trichoderma on cucumber F. oxysporum and the subsequent yield. However, the impact of Trichoderma on the physiological and biochemical features of cucumbers remains unclear. Additionally, little is known about the correlation between the Trichoderma-induced physiological changes and the efficacy of Trichoderma for disease prevention and cucumber yield improvement. In our previous screening studies, we obtained 3 strains of Trichoderma with anti-F. oxysporum activity, including T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886. In this study, we further evaluated the effects of these 3 strains on cucumber fusarium wilt incidence, in addition to cucumber seedling growth, biomass accumulation, physicochemical features, and product quality. We also explored the mechanism by which Trichoderma prevents cucumber fusarium wilt and promotes cucumber growth. Together, our results provide scientific evidence for the benefits of appying Trichoderma in the field and the technical basis for improving the safety, yield, and product quality in cucumber cultivation.
The cucumber cultivar used in this study was Changchun Mici, from Yuyuan Seed Co., Ltd., Xintai, Shandong, China. The strains of Trichoderma and pathogenic fungi used in this study are listed in Table 1.
2.2. Preparation of the spore suspensions of Tricho derma and pathogenic fungi Trichoderma were cultured on PDA culture medium (200 g of potato, 20 g of dextrose, 10 g of agar, 1 000 mL of distilled water, and neutral pH) for 7 d in the dark at 28°C, and the spores were collected by washing the agar surface with distilled water. For preparation of the spores of phytopathogenic fungi, 5 samples with 5 mm diameter were taken from the edge of each fungal colony and inoculated into 100 mL of the PD liquid culture medium described above in 500-mL conical flasks. The flasks were cultured in the dark for 3 d in a shaking incubator (250 r min–1) at 28°C to obtain the spore suspensions. The spore counts were determined using a hemocytometer. Each Trichoderma spore suspension was diluted to a concentration of 1.5×108 spores mL–1, and the pathogenic fungi spore suspensions were diluted to a concentration of 1×105 spores mL–1.
2.3. Evaluation of the effect of Trichoderma on the quality of cucumber seedlings and the prevention of cucumber fusarium wilt Cultivating seedlings of cucumber This experiment was performed in a modern greenhouse at the Teaching Base of College of Agronomy, Heilongjiang Bayi Agricultural University, China. The soil was mixed well and placed in 50-hole plastic seedling culture dishes (3.5 cm×24 cm× 11 cm). In each dish, 100 soaked and germinated seeds were planted. After germination, 50 seedlings with similar size were retained. Three replicates of 5 dishes were used in each treatment for the field tests and the yield/quality evaluations. The basic physicochemical properties of the potting soil were as follows: 198.56 mg kg–1 of total nitrogen,
Table 1 Fungal strains used in this study Codes of isolates 525 610 886 B
Scientific name Trichoderma asperellum Trichoderma harzianum Trichoderma pseudokoningii Fusarium oxysporum f. sp. cucumerinum Owen
Source This lab This lab This lab Isolated from cucumber plants showing fusarium wilt symptoms and stored in this lab
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164.78 mg kg–1 of alkali-hydrolyzed nitrogen, 15.76 mg kg–1 of total phosphorus, 210 mg kg–1 of fast-acting potassium, 59.60 g kg–1 of organic matter, and pH value of 8.04. Inoculation of Trichoderma and pathogenic fungi The cucumber seeds were disinfected using a 40% formalin solution that was diluted 100-fold. After soaking in the formalin solution for 30 min, the seeds were rinsed with water and soaked in water for 8–12 h at 25–30°C. The seeds were then cultured at 28–30°C, and the seeds started to germinate after 12 h. Germinated seeds were sown in pots, and when the true leaves started to unfold 5 d after sowing, cucumber seedlings of relatively consistent size were inoculated with Trichoderma and/or pathogenic fungi spore suspensions through root irrigation. Each seedling was inoculated with 3 mL of the corresponding suspension. Each of the 8 different treatments in this study included 5 dishes, and each dish included 50 seedlings. All seedlings were randomly selected, and the entire experiment was repeated 5 times. The treatment groups were as follows: 1) T. asperellum 525 only (T1); 2) T. harzianum 610 only (T2); 3) T. pseudokoningii 886 only (T3); 4) T. asperellum 525 and pathogenic fungus (T1B); 5) T. harzianum 610 and pathogenic fungus (T2B); 6) T. pseudokoningii 886 and pathogenic fungus (T3B); 7) Pathogenic fungus F. oxysporum f. sp. cucumerinum Owen only (B); 8) PD liquid medium control (P). Greenhouse transplanting and follow-up management In the soil at the greenhouse, 5 000 kg of organic fertilizer and 150 kg of ammonium dihydrogen phosphate per hectare were applied. The soil was fully plowed for beds. High beds were arranged with a lower bed width of 120 cm, an upper bed width of 100 cm, a bed height of 13–15 cm, and a bed length of 5 m. Two rows of plants were sown in the upper beds, with plant spacing of 25 cm and row spacing of 60 cm. Each bed was set as an experimental plot with a size of 12 m2. A randomized block design was used for the experimental plots, with 5 replicates. When the seedlings had grown to the stage of 4-leaf and 1-heart, healthy seedlings of consistent size were collected and transplanted to the greenhouse, with 42 plants randomly chosen and transplanted to each experimental plot. Upon recovery, each plant was irrigated with 100 mL of the indicated treatment solution (Trichoderma spore suspension or water). The T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886 strain treatments were named T-1, T-2, and T-3, respectively, while the water control was named CK. After the treatments, vine hanging and single vine pruning were performed in a timely manner, and all plants were watered every 3 d. Analysis of cucumber seedlings in pots A total of 7 d after the seedlings were inoculated with Trichoderma and/
609
or pathogenic fungi, the growth morphological, physiological, biochemical, and disease resistance indexes were measured. The plant height was determined by the distance between the stem base and the growing point of each seedling. The stem diameter was measured at 1 cm below the cotyledon section (Zhang S P et al. 2016). The root volume was measured by the displacement approach (Zhang S P et al. 2016). The leaf area was measured by the weighing method (Cao et al. 2017). First, a leaf with certain area (A1) was collected and stacked with other leaves. Then, a 1-cm puncher pin was used to obtain round sheets; the weights of the round sheets and the remaining leaves were measured, from which the weight of the leaf with a certain area (W1) and the weights of the remaining leaves (W2) were calculated. From these measurements, the leaf total area (A) was calculated using the formula: A=[A1×(W1+W2)]/W1 The root weight, shoot weight and the seedling dry weight were measured according to Hao et al. (2007). From these values, the seedling vigor index was calculated using the formula: Seedling vigor index=(Stem diameter/Plant height+Root weight/Shoot weight)×Seedling dry weight (Li et al. 2017) For aboveground and underground fresh weights, the plants were repeatedly rinsed with water and dried with absorbent paper. A 1/1 000 electronic scale was used to measure the fresh weights (Chen et al. 2002). The physiological biochemical indexes included chlorophyll content, soluble carbohydrate content, nitric nitrogen content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, polyphenol oxidase (PPO) activity, ascorbate oxidase (AAO) activity, root activity, total root absorption area, and root specific surface area. The soluble carbohydrate content was measured by the anthrone colorimetric method, as shown in Huang et al. (2009). Nitric nitrogen was measured by the phenol disulfonic acid method (Ma et al. 2017). The activities of SOD were measured by the NBT method (Giannopolitis and Ries 1977). The activities of POD were measured by the guaiacol method (Vieria 1998). The activities of CAT and AAO were measured according to the method of Cakmak and Marschner (1992). The activities of PPO were measured by titration (Bao et al. 2015). The content of chlorophyll was measured by the acetone-ethanol method (Hao et al. 2007). The leaf cell membrane permeability was evaluated based on the relative conductivity (Tian et al. 2017). The malondialdehyde (MDA) content was measured by the thiobarbituric acid method (Sun et al. 2001). Root activity was determined by the alpha-naphthylamine oxidation method (Zhang 2002). The total root absorption area, root active absorption area, and root specific surface area were determined by the methylene blue absorption method (Rubín et al. 2010) using the following formulas:
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2.4. Statistical analysis Microsoft Excel 2007 was used for preparing figures and
tables. The average and standard deviation of either 3 or 5 independent replicates for each treatment were calculated. The significances of differnces between samples were calculated using the DPS7.05 data processing system.
3. Results 3.1. The preventive effect of Trichoderma on cucumber fusarium wilt A total of 7 d after inoculation of Trichoderma and the pathogenic fungi onto cucumber seedlings, we evaluated the survival rate, disease incidence, disease index, and control effects. As shown in Fig. 1, the disease indexes of the seedlings treated with both Trichoderma and pathogenic fungus (T1B, T2B, and T3B), or pathogenic fungus only (B), were 9.12, 10.69, 10.55, and 49.39, respectively. The control effects of T1B, T2B, and T3B were 81.53, 78.36, and 78.64%, respectively, with no significant differences among the three groups. These results indicated that all 3 strains of Trichoderma exhibited good control effects on cucumber fusarium wilt.
3.2. The effects of Trichoderma on the yield and quality of cucumbers Trichoderma significantly increased overall cucumber yield, average fruit weight, and the contents of soluble solids, soluble carbohydrates, soluble proteins, and vitamin C in cucumber fruits, as shown in Fig. 2 and Table 2. Compared with the Trichoderma-untreated group (CK), T-1, T-2, and T-3-treated plants exhibited 25.57, 23.54, and 33.85% increases in cucumber yield, respectively. T-3-treated plants exhibited a significantly higher cucumber yield, and the difference between the T-1- and T-2-treated plants was not Control efficiency 90 80 70 60 50 40 30 20 10 0
Disease index
a
a
a
b
b
b
a
b T1B
T2B
T3B
B
60 50 40 30 20 10 0
Disease index
Total root absorption area (m2)=[(C1−C1´)×V1]+[(C2− C2´)×V2]×1.1 Root active absorption area (m2)=[(C3−C3´)×V3]×1.1 Root specific surface area (m2 mL–1)=Total root absorption area/Total root volume, in which C indicates the original concentration (mg mL–1) of the solution, C´ indicates the concentration (mg mL–1) of the solution after extraction, and 1, 2, and 3, indicate the numbers of the subsequent rinses. The disease resistance indexes included plant survival rate, disease incidence rate, disease index, and control effect. The grading system of cucumber fusarium wilt severity was according to Zhang S P’s standard (2016), and the disease index was calculated according to the method of Zong and Kang (2002): Grade 0: no symptoms; Grade 1: The yellowing or wilting area of true leaves and cotyledon does not exceed 50% of the total area; Grade 2: The yellowing or wilting area of true leaves and cotyledon exceeds 50% of the total area; Grade 3: Leaves are wilted or dead with only growing points surviving; Grade 4: The entire plant is severely wilted or dead. Disease index=∑(Plant number in the grade×Grade number)/(Total plant number×The highest grade number)×100% Control effect (%)=(Disease index in the control group− Disease index in the treated group)/Disease index in the control group×100 Determination of cucumber yield and quality indexes After harvesting cucumbers 2–4 times, 5 cucumber fruits were randomly selected from each experimental plot. The quality of fruits was evaluated, and the average quality value was calculated (Ma et al. 2016). For the average single-fruit weight and yield, 10 plants from each experimental plot were tagged and separately managed. An electronic scale with 0.01 g precision was used to measure the weight of individual cucumbers. In addition, the yield of each plot was determined, and the average yield was calculated. From these, the yield per square meter was calculated according to the planting density and the plot area. The soluble solids content was determined using a handheld refractometer. The soluble carbohydrate content was determined using the anthrone colorimetric method (Huang et al. 2009). The vitamin C content was evaluated by ultraviolet spectrophotometry (Zhang et al. 2009). The soluble protein content was determined by the Coomassie brilliant blue G-250 staining method (Oshima et al. 1987). The nitric nitrogen content was determined by the phenol disulfonic acid method (Ma et al. 2017).
Control efficiency (%)
610
Fig. 1 The effects of Trichoderma on preventing cucumber Fusarium wilt. The disease resistance indexes of cucumber seedlings were detected 7 d after treatment. B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B. Data are mean±SD. Different lowercase letters indicate significant differences at the 0.05 probability level.
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T3
Fig. 2 Effects of Trichoderma treatment on yield of cucumber. One average sized cucumber fruit was selected and photographed from each experimental plot after harvesting 4 times. CK, water; T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only.
Soluble sugar Soluble sugar Increase (%) (%) 2.42±0.97 c – 3.51±0.37 a 45.04 2.94±0.63 b 21.49 3.78±0.84 a 56.20 Soluble solid Soluble solid Increase (%) (%) 4.52±0.18 c – 5.17±0.21 a 14.38 4.87±0.38 b 7.74 5.34±0.34 a 18.14 Weight per fruit Weight per fruit Increase (g) (%) 117.81±2.45 c – 139.99±2.65 a 18.83 128.48±4.26 b 9.06 145.93±2.87 a 23.87 Increase (%) – 25.57 23.54 33.85 Yield Yield (kg m–2) 5.67±0.04 c 7.12±0.10 b 7.00±0.10 b 7.58±0.07 a CK T-1 T-2 T-3
T2
Treatment1)
T1
Table 2 The effects of Trichoderma on the quality and yield of cucumber
CK
Vitamin C Vc Increase (mg 100 g–1 FW) (%) 15.21±0.93 c – 18.65±0.53 a 22.62 16.87±0.71 b 10.91 19.65±0.64 a 29.19
The 7 d after treatment with Trichoderma and/or the pathogenic fungus, the cucumber seedlings were examined for growth, morphology, and biomass accumulation. As shown in Table 3 and Fig. 3, the T1- and T3-treated cucumber seedlings exhibited greater plant height than T1B-, T2B-, and T3B-treated cucumber seedlings. T2-treated cucumber seedlings exhibited a plant height similar to T2B. T1-, T2-, and T3-treated cucumber seedlings exhibited greater leaf area, root volume, and biomass accumulation compared to the T1B-, T2B-, and T3B-treated cucumber seedlings. The PD medium (P)-treated cucumber seedlings exhibited similar levels for seedling vigor indexes, underground fresh weight, aboveground fresh weight and stem diameter, but showed higher levels of plant height, root volume, and leaf area than the B-treated seedlings. Moreover, the Trichoderma treatments promoted the growth and biomass accumulation of cucumber seedlings. The T1-, T2-, and T3-treated cucumber seedlings exhibited 24, 8.6, and 25% increases in plant height, respectively, compared with the control (P), and the growth-promoting effects of T1 and T3 were significantly higher than that of T2. The pathogen treatment (B) led to a decrease in growth and biomass accumulation, which was attenuated upon the treatment together with Trichoderma. The seedling vigor indexes in the T1-, T2-, and T3-treated seedlings were not significantly different. The underground
1)
3.3. The effects of Trichoderma on cucumber seedling growth
CK, water; T-1, T. asperellum 525; T-2, T. harzianum 610; T-3, T. pseudokoningii 886. Values are mean value±SE. Lowercase letters indicate the samples with significant differences in each column at 0.05 significance level.
Soluble protein Soluble protein Increase (mg 100 g–1） (%) 20.98±1.32 c – 24.51±2.27 a 14.40 23.29±1.76 b 9.92 25.21±3.65 a 16.78
significant. Compared with the T-2-treated plants, the T-1- and T-3-treated plants produced cucumbers with a higher quality. Among all the fruit quality indexes of soluble solids, soluble carbohydrates, soluble proteins, and vitamin C, the changes in soluble carbohydrates were the most significant in the Trichoderma-treated plants. Specifically, the T-1-, T-2-, and T-3treated cucumbers exhibited 45.04, 21.49, and 56.20% increases in soluble carbohydrate content compared to the control, respectively. These results suggest that the Trichoderma treatments improved the yield and quality of cucumbers to varying extents.
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Table 3 The effects of Trichoderma on cucumber seedling growth Treatment1)
Plant height (cm)
Root volume (mL)
Sound seedling index
Leaf area (cm2)
T1B T2B T3B B T1 T2 T3 P
8.04±0.05 b 7.97±0.02 bc 7.80±0.07 b 6.66±0.04 e 8.49±0.02 a 7.43±0.04 c 8.58±0.02 a 6.84±0.04 d
0.51±0.02 c 0.52±0.01 bc 0.53±0.01 b 0.51±0.01 c 0.60±0.05 a 0.61±0.02 a 0.60±0.00 a 0.55±0.00 b
0.11±0.01 ab 0.09±0.01 c 0.10±0.01 bc 0.09±0.01 c 0.12±0.01 a 0.11±0.01 ab 0.11±0.01 ab 0.10±0.01 bc
34.96±1.22 b 34.13±0.33 c 34.76±1.41 bc 31.50±0.91 e 36.39±0.83 a 34.96±0.37 b 35.19±2.45 b 32.19±00.82 d
1)
Underground fresh weight (g/plant) 0.36±0.03 b 0.29±0.02 c 0.33±0.03 a 0.29±0.04 c 0.35±0.02 ab 0.31±0.02 bc 0.36±0.02 a 0.30±0.03 c
Stem diameter (mm) 2.43±0.02 c 2.44±0.05 c 2.62±0.03 b 2.02±0.02 d 2.53±0.04 c 2.57±0.04 bc 2.73±0.02 a 2.13±0.02 d
Aboveground fresh weight (g/plant) 1.29±0.07 b 1.22±0.02 c 1.26±0.02 c 1.21±0.07 bc 1.30±0.07 a 1.23±0.05 ab 1.28±0.02 b 1.25±0.04 b
The growth morphological indexes of cucumber seedlings were measured 7 d after treatment. T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; P, PD liquid medium control. Values are mean value±SE. Different lowercase letters within the same column indicate significant differences at 0.05 probability level.
T1B
T2B
T3B
B
T1
T2
T3
P
Fig. 3 Effect of Trichoderma treatment on the growth of cucumber seedlings. The photo was taken on the 7 d after inoculation. T1B, T. asperellum 525 and B; T2B, T. harzianugm 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; P, PD liquid medium control.
fresh weight, aboveground fresh weight, and plant height of the T1-treated seedlings were equivalent to those of the T3-treated seedlings, but were higher than those of the T2treated seedlings. The leaf area of the T2-treated seedlings was similar to that of the T3-treated seedlings but was lower than that of T1-treated seedlings. Furthermore, among the T1-, T2-, and T3-treated seedlings, the T3-treated seedlings exhibited the largest stem diameters, and T1-treated seedlings exhibited the smallest stem diameters. Together, these data suggested that among the T1-, T2-, and T3-treatments, the T3 treatment exhibited the greatest effect on promoting seedling growth and biomass accumulation, while the T2 treatment exhibited the smallest effect on promoting seedling growth and biomass accumulation.
the different strains of Trichoderma were collected 7 d after
3.4. The effects of Trichoderma on cucumber seedling physiology
of the T1B-, T2B- and T3B-treated seedlings, which were
The leaves derived from cucumber seedlings treated with
promoting seedling physiology.
treatment, and analyzed for several physiological features. As shown in Figs. 4–5, compared with the leaves from the other groups, the leaves derived from the T3-treated cucumber seedling exhibited significantly higher levels of chlorophyll content, nitric nitrogen content, root activity, total root absorption area, root active absorption area, and root-specific surface area, while the B-treated seedlings exhibited significantly lower levels of all the corresponding physiological indexes. The Trichoderma treatment promoted the physiological activities of cucumber seedlings and enhanced the efficiency of photosynthesis and nitric nitrogen absorption. Specifically, the physiological activities of the T1-, T2- and T3-treated seedlings were higher than those higher than those of the B-treated seedlings. Among all 3 strains of Trichoderma, T3 exhibited the highest effect on
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B
1.6
a
1.4 1.2
c
d
de
b
c
d
e
1.0 0.8 0.6 0.4 0.2 0
cd
8
Nitrate nitrogen content (×1 000 μg mg–1)
Chlorophyll content (mg g–1)
A
a
e
c
b
d
e
7
f
6 5 4 3 2 1 0
T1
T2
T3
T1B T2B T3B
B
T1
P
T2
T3
Treatment
T1B T2B T3B
B
P
Treatment
Fig. 4 The effects of Trichoderma on chlorophyll and nitric nitrogen content in cucumber seedling leaves. The chlorophyll (A) and nitric nitrogen contents (B) in cucumber seedlings were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
1.2
Total absorption (m2)
a
ab
a
a
b
bc d
0.8
0.4
Root specific surface area (m2 mL–1)
Active absorption area (m2) c
1.0
0.6
Root activity (×500 μg g–1 FW h–1)
b
c bc
a
a
c b
a
a
T1
T2
T3
c
d
c
c c
c b
d
cd
c
e
a
b
c
c
T2B
T3B
B
P
0.2 0
T1B
Fig. 5 The effects of Trichoderma on the physiology of roots in cucumber seedlings. The physiological indexes in roots of cucumber seedlings were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
3.5. The effects of Trichoderma on the stress-resistance of cucumber seedlings The activities of stress-resistance enzymes in seedling leaves were analyzed following treatment with Trichoderma and pathogenic fungi. As shown in Fig. 6, the T3-treated seedlings exhibited the highest activities of SOD, POD, CAT, PPO, and AAO, compared with the other treatments. The PD liquid medium-treated seedlings exhibited the lowest enzymatic activities, and the B-treated seedlings only exhibited a slight increase in enzymatic activities. These results indicated that both the pathogenic fungus and Trichoderma treatments enhanced the activities of stressresistance enzymes, while the effects of Trichoderma were stronger than those of the pathogenic fungus.
The contents of MDA in cucumber seedlings are shown in Fig. 7-A. The contents of MDA in the T1-, T2-, and T3treated seedling leaves were equivalent to each other, but higher than the MDA contents in the T1B-, T2B-, T3B-treated seedling leaves. The T3B- and P-treated leaves showed the lowest contents of MDA. These data indicated that either Trichoderma or pathogenic fungus alone increased the content of MDA, but the treatment of pathogenic fungus in combination with Trichoderma decreased the content of MDA to a different extent. Furthermore, the effects of the treatments on leaf cell membrane permeability were as follows: B>T1>T2>T3 (Fig. 7-B). In addition, leaves treated with T1B, T2B, and T3B also showed different levels of decrease in leaf permeability. Together, our data indicated that compared to the P treatment, the Trichoderma
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SOD (×100 U g–1) PPO (mg g–1 min–1)
6 5 Enzyme activity
d
bc b b
4
bab
a b
a
b
POD (U g–1 min–1) AAO (×2 mg g–1 min–1) a
a
d
a
a
cc be
cc
cc
CAT (H2O2 mg g–1 min–1)
bb
e
d
d
c
cd
d
bc
3
e
f
f e e
e
f
B
P
2 1 0
T1
T2
T3
T1B T2B Treatment
T3B
MDA content (μmol g–1)
A 3.0 b
2.5
b
b
c
bc
B
a d
d
2.0 1.5 1.0 0.5 0
T1
T2
T3 T1B T2B T3B
B
P
Treatment
Plasma membrane permeability (%)
Fig. 6 The effects of Trichoderma on the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO) and ascorbate oxidase (AAO) in cucumber seedlings. The antioxidant enzyme activities were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
60 50
b
b
40
a c
d
c
d
e
30 20 10 0
T1
T2
T3 T1B T2B T3B
B
P
Treatment
Fig. 7 The effects of Trichoderma on the malondialdehyde content and cell membrane permeability in cucumber seedling leaves. The malondialdehyde (MDA) content (A) and plasma permeability (B) were measured 7 d after treatment. T1, T. asperellum 525 only; T2, T. harzianum 610 only; T3, T. pseudokoningii 886 only; T1B, T. asperellum 525 and B; T2B, T. harzianum 610 and B; T3B, T. pseudokoningii 886 and B; B, pathogenic fungus B (Fusarium oxysporum f. sp. cucumerinum Owen); P, PD liquid medium control. Data are mean±SD. Different lowercase letters indicate significant differences among different treatments at 0.05 probability level.
and pathogenic fungus treatments increased the MDA
control have primarily used strains such as T. harzianum
content and cell membrane permeability, in which the
(Yedidia et al. 2001; Cheng et al. 2010; Chen et al. 2012),
pathogenic fungus treatment induced a stronger response.
T. viride (Zhuang et al. 2005; Cheng et al. 2010; Bi 2016),
Additionally, the combined Trichoderma and pathogenic
T. longibrachiatum (Li et al. 2010; Bi 2016; Zhang S W
fungus treatment reduced both the MDA content and cell
et al. 2016), T. reesei (Luo et al. 2016), and T. atroviride
membrane permeability.
(Han et al. 2013). Very few studies have focused on
4. Discussion
In this study, we isolated 3 strains of Trichoderma,
Recent studies on the role of Trichoderma in biological
T. pseudokoningii 886 (T3), which have been identified
T. asperellum and T. koningense (Qi and Zhao 2013). including T. asperellum (T1), T. harzianum 610 (T2), and
LI Mei et al. Journal of Integrative Agriculture 2019, 18(3): 607–617
as T. asperellum, T. harzianum, and T. pseudokoningii, respectively. Functional analysis showed that all 3 strains of Trichoderma prevented cucumber fusarium wilt and promoted the quality and yield of cucumber. Particularly, the efficacy of T1 and T3 was better than that of T2. Our results indicate that T. asperellum and T. koningense may have great potential for application to plant disease control. In our preliminary studies, using a dual culture assay, we screened some Trichoderma strains from our laboratory that were antagonistic against F. oxysporum. Among these strains, T. asperellum 525 (T1), T. harzianum 610 (T2), and T. pseudokoningii 886 (T3) exhibited 80.24, 76.86, and 74.17% growth inhibitory rates against F. oxysporum, respectively (data not shown). To further characterize the antagonistic strains, we evaluated the effect of these 3 strains on preventing cucumber fusarium wilt in both pot and greenhouse culture experiments. The results showed that the effects of T1, T2, and T3 on preventing cucumber fusarium wilt were 81.53, 78.36, and 78.64%, respectively. The results from greenhouse culture experiments were consistent with the results from the dual culture assay, indicating that dual culture assay could be a reliable method to screen Trichoderma strains for their ability to prevent cucumber fusarium wilt. Abo-Elyousr (2014) reported that the results from dual culture assay and greenhouse culture experiments were not positively correlated. We speculate that the lack of correlation between those two experiments might have been due to the variance in pathogenic fungi, Trichoderma strains, or culture conditions. Further validation is required to confirm the correlation between the dual culture assay data and the greenhouse culture experiment data when those variables are taken into account. In this study, 7 d after inoculation was chosen as the time-frame for evaluating the effects of different Trichoderma strains. The 7 d after the treatment with Trichoderma (or 12 d after the sowing of cucumber) was the period when the first true leaves of the cucumber seedlings were fully unfolded (the one leaf and one heart period). The growth of cucumber seedlings was vigorous at this time, so the effects of Trichoderma treatments on morphological, physiological and biochemical indexes of cucumber seedlings were dramatic, which could be used to easily evaluate the effects of the Trichoderma strains. Several studies have shown that Trichoderma prevented cucumber fusarium wilt. For example, Cheng et al. (2010) observed that T. viride T23 and T. harzianum T22 prevent cucumber fusarium wilt, with an efficacy of 66.04%; while Zhuang et al. (2005) demonstrated that upon treatment with T. viride T23 conidia and chlamydospores, cucumber seedlings exhibited a decrease in the disease index from 33.69 to 13.12 and 10.28, respectively. Bi et al. (2016) showed that T. longibrachiatum and T. viride prevent
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cucumber fusarium wilt, with efficacies of 75.74 and 70.76%, respectively. In our study, all three strains of Trichoderma exhibited control efficacy above 78%, and treatments with Trichoderma reduced the disease index from 49.93 in the control to 9.12–10.55. Our data showed higher control efficacy of Trichoderma on cucumber fusarium wilt compared to the published results, indicating the great application value of the 3 Trichoderma strains used here in the field. In addition to inhibiting several pathogenic fungi, Trichoderma also promoted the growth and yield of plants (Hyakumachi et al. 1994; Masunaka et al. 2011; Deng et al. 2013). For example, Yedidia et al. (2010) reported that T. harzianum T203 in the soil increased the cucumber root area by 95%, the plant dry weight by 80%, the root length by 75%, the plant height by 45%, and the leaf area by 80%. Zhang S W et al. (2016) demonstrated that T. longibrachiatum T6 spore suspension significantly promoted the growth of Meloidogyne incognita-inoculated cucumbers. In addition, several strains of Trichoderma also promoted the plant growth and biomass accumulation in other plants, such as cabbage (Xing et al. 2017) and Sedum plumbizincicola (Luo et al. 2016). The plant chlorophyll content and the leaf nitric nitrogen content are important indicators for evaluating photosynthesis and nitrogen assimilation and utilization. In this study, T. pseudokoningii 886 (T3) treatment significantly increased the chlorophyll content, nitric nitrogen content, root activity, total root absorption area, and root specific surface area, effects that were consistent with the published data. Our results indicated that the Trichoderma treatments enhanced the physiological and metabolic activities in cucumbers, and increased the cucumber yields. Notably, compared to all the other treatments, T3 induced higher physiological and metabolic activities, more biomass accumulation, and higher cucumber yield (33.85%), as well as more dramatic changes in all the other indexes. It is well known that Trichoderma may have a variety of action mechanisms such as competition, antagonism, mycoparasitism, and the stimulation of plant resistance. The results suggested that the antagonistic effect of T3 on pathogenic fungi was lower than those of T1 and T2, but T3 also had a strong effect on inducing plant resistance, which could promote physiological activities, increase yield and quality of cucumber. T3 was identified as T. pseudokoningii. Little is known about T. pseudokoningii as a potential biocontrol agent, and its biocontrol mechanisms need further research. It has been established that enhanced activities of POD, CAT, PPO, AAO, and SOD increase the stress-resistance capability of plants. Additionally, MDA is one of the final products of lipid peroxidation in cell membranes. It causes membrane lipid peroxidation, damages the structure and function of biomembranes, and changes the permeability
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of the membranes, which affect a series of physiological and biochemical reactions of cells. Therefore, the content of MDA reflects the level of cell membrane damage (Huang 2005). The higher the membrane permeability and MDA content, the greater the extent of cell membrane damage. Liu et al. (2013) reported that T. viride broth inhibited the growth of mango anthracnose pathogens and enhanced the activities of antioxidant enzymes in mango. Liu et al. (2014) demonstrated that T. harzianum T23 induced the generation of phytoalexin and lignin in eggplants and enhanced the activities of PAL, PPO, POD, and SOD, which enhanced the resistance of eggplants to fusarium wilt. Consistent with these published data, the current study showed that 3 Trichoderma strains induced different levels of increase in the activities of antioxidant enzymes, suggesting that all 3 Trichoderma strains enhanced the disease resistance in cucumber plants by elevating the activities of stress-resistance enzymes. Compared to the other controls, pathogenic fungi-treated cucumber seedlings exhibited significantly enhanced stress-resistance enzyme activities, higher leaf cell membrane permeability, and higher MDA content, indicating that the pathogenic fungi damaged cucumber seedlings. In addition, the cell membrane permeability and MDA content of the Trichoderma and pathogenic fungi-treated seedlings were higher than those of the control group (P), indicating that the Trichoderma and pathogenic fungi stimulated or damaged the seedlings at varying levels. However, the combined treatment of Trichoderma and the pathogenic fungi decreased the cell membrane permeability and MDA content to various extents, suggesting that the interaction between Trichoderma and the pathogenic fungi ameliorates pathogen damage to the seedlings. The level of efficacy was associated with the specific Trichoderma strains, and the exact mechanisms require further analysis.
5. Conclusion T. asperellum 525, T. harzianum 610, and T. pseudokoningii 886 inhibit F. oxysporum infection, stimulate the metabolism of cucumber plants, and enhance the activities of stressresistance enzymes, which consequently promote the growth of cucumber plants, prevent cucumber fusarium wilt, and improve the yield and quality of cucumbers.
Acknowledgements The authors are grateful for the financial support from the National Key R&D Program of China (2018YFD0201202), the National Science and Technology Basic Work, China (2014FY120900) and the 948 Program of China (2011-G4).
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