Screening of antagonistic Trichoderma strains and their application for controlling stalk rot in maize

Journal of Integrative Agriculture 2020, 19(1): 145–152 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Screening of antagonistic Trichoderma strains and their application for controlling stalk rot in maize LU Zhi-xiang1, TU Guang-ping2, ZHANG Ting1, LI Ya-qian1 , WANG Xin-hua1, Zhang Quan-guo5, SONG Wei5, CHEN Jie1, 3, 4 1

School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, P.R.China

2

Plant Protection College, Shenyang Agricultural University, Shenyang 110866, P.R.China State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, P.R.China 4 Key Laboratory of Urban Agriculture, Ministry of Agriculture, Shanghai 200240, P.R.China 5 Institute of Cereal and oil Crops, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, P.R.China 3

Abstract Maize is one of the major crops in China, but maize stalk rot occurs nationwide and has become one of the major challenges in maize production in China. In order to find an environment-friendly and feasible technology to control this disease, a Trichoderma-based biocontrol agent was selected. Forty-eight strains with various inhibition activities to Fusarium graminearum, and Fusarium verticillioides were tested. A group of Trichoderma strains (DLY31, SG3403, DLY1303 and GDFS1009) were found to provide an inhibition rate to pathogen growth in vitro of over 70%. These strains also prevented pathogen infection over 65% and promoted the maize seedling growth for the main root in vivo by over 50%. Due to its advantage in antifungal activity against pathogens and promotion activity to maize, Trichoderma asperellum GDSF1009 was selected as the most promising strain of the biocontrol agent in the Trichoderma spectrum. Pot experiments showed that the Trichoderma agent at 2–3 g/pot could achieve the best control of seedling stalk rot and promotion of maize seedling growth. In the field experiments, 8–10 g/hole was able to achieve over 65% control to stalk rot, and yield increased by 2–11%. In the case of natural morbidity, the control efficiency ranged from 27.23 to 48.84%, and the rate of yield increase reached 11.70%, with a dosage of Trichoderma granules at 75 kg ha–1. Based on these results, we concluded that the Trichoderma agent is a promising biocontrol approach to stalk rot in maize. Keywords: stalk rot in maize, biocontrol, Trichoderma, Fusarium, granules

1. Introduction Received 25 July, 2018 Accepted 3 July, 2019 LU Zhi-xiang, E-mail: [email protected]; Correspondence CHEN Jie, Tel: +86-21-34206141, E-mail: [email protected] © 2020 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(19)62734-6

Maize is one of the major crops in China, however, maize growth suffers from a variety of pathogenic infections. In recent years, maize stalk rot has occurred nationwide and has become a major challenge for maize production in China (Duan et al. 2018). Maize stalk rot is caused by Pythium spp. and Fusarium spp. (Ma et al. 2017). The relevant Fusarium spp. mainly include F. graminearum, F. verticillioides,

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F. proliferatum and F. subglutinans, among which F. graminearum is the major species infecting the roots, the stalks, the internodes, and eventually resulting in lodging (Ghini et al. 2016). Fusarium spp. are soil inhabitants, which can survive in the diseased plant remains and soil for a long time. Therefore, Fusarium stalk rot is difficult to control by traditional chemical means. Furthermore, in our previous study (data not shown), stems infected by Fusarium have been detected with high amounts of toxin accumulation. As a result, another potential threat faces the livestock feed industry, where toxin contaminated feed would lead to livestock products with poor quality and safety. In order to deal with the stalk rot challenge, many chemical fungicides have been applied; however, so far specific chemical fungicides which are effective in controlling this disease have not been found. At the same time, the usage of chemical fungicides on such a large scale would bring more problems, such as the resistance of the pathogens and the resurgence of the disease. In these cases, the antagonistic microorganisms have been applied worldwide to prevent the further spread of these diseases. As a group of well-known biocontrol fungi, Trichoderma spp. are mainly isolated from soil, and are thus very applicable for use in controlling soil borne diseases such as stalk rot. There are various mechanisms by which Trichoderma can control plant diseases, such as competition for space and nutrients with the pathogens, mycoparasitism, antagonism to the pathogens, or induction of the plant’s natural resistance. It is widely believed that multiple mechanisms govern the control of stalk rot by Trichoderma. In particular, the induction of resistance and improvement of soil ecological diversity are believed to play vital roles in the sustainable control of this disease. Trichoderma produces and releases a variety of compounds that can induce localized or systemic resistance responses, and they can protect the plants from the seedling to adult stages. Fusarium infection usually occurs at the seedling stage and can last to the adult stage, so the infection period is longer than other soilborne diseases. Therefore, as a symbiont with the plant, Trichoderma can enhance root growth and development, crop productivity, and the use of nutrients, as well as tolerance to abiotic stresses, providing long-term protection for the plants (Harman et al. 2004). Trichoderma harzianum has been applied to the seeds to protect the maize against the F. verticillioides (Nayaka et al. 2010). Trichoderma asperellum has been used in the biocontrol of the disease caused by F. verticillioides (Amy et al. 2018). In China, before marketing to farmers seeds are usually coated by chemical pesticides, so it would be difficult to re-coat Trichoderma on the outside of the chemically-coated seed surface. Therefore, using Trichoderma to treat soil is more

practical. Previous work has demonstrated the possibility of using Trichoderma spp. to control the maize stalk rot (Wu et al. 2015). Therefore, finding an environment-friendly and feasible control technology is significant. In this study, we screened the Trichoderma spp. isolated from the soil in farmland and obtained some strains with antifungal activity against the pathogen of maize stalk rot. Some strains that are effective in controlling stalk rot in corn fields have been tested, and then extended into some fields in the countrywide.

2. Materials and methods 2.1. Materials Strains The pathogens of corn stalk rot, F. verticilliodes and F. graminearum, were collected from diseased plants in the field and kept in the Laboratory of Phytopathology in the Shanghai Jiao Tong University, China. Forty-eight strains of Trichoderma spp. were isolated from farming soils from eastern China. Medium Potato dextrose agar (PDA) medium was used for confront culture of the pathogens (F. graminearum and F. verticilliodes) and Trichoderma spp. Potato dextrose (PD) medium is the fermentation medium that is typically used for the reproduction of Trichoderma.

2.2. The screening of the antagonistic Trichoderma strains In vitro screening Trichoderma and pathogenic fungi were cultured on the PDA medium for 4 days. The fungi disks were obtained from the edge of the colony by a 5-mm puncher. The disks of Trichoderma and pathogens were placed on each side (at the distance of 5 cm) of the PDA dish, and cultured in a 28°C incubator for 6 days. Each treatment group had three replicates (three dishes). In the control group, the side opposite the pathogenic fungus was blank. After 6 days, the linear growth distance of pathogens in the treatment and control groups were measured. Then, the antifungal effect was calculated by the formula: Antifungal rate (%)=(Sc–St)/Sc×100, where Sc is the mean linear growth distance of pathogens in the control groups, and the St is the mean linear growth distance of pathogens in treatment groups. In vivo screening Seven strains (DLY31, G3302, SG3403, DLY1303, LYI1208, GDFS1009 and GZ2101) with highly significant antifungal activity in vitro were further evaluated for their plant growth promotion and disease control activities. Strains were grown in PD medium for 7 days, until the concentration of spores was over 107 cfu mL–1. The spore suspension was then diluted 50 folds, and was used

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to soak maize seeds for 6 h. Treated seeds were planted in potting soil containing a mixture of F. graminearum spore suspension (105 cfu mL–1), and each strain was evaluated in three replicates (six pots in each replicate and five plants/pot, for a total of 30 plants). All treatment groups were examined at the 3–5 leaf stage to confirm whether the Trichoderma strains showed significant activity in the promotion of seedling growth and the inhibition of pathogen infection in the seedling roots.

2.3. Preparation of Trichoderma granules and pathogen inoculum Individual spores of T. asperellum GDFS1009 were cultivated in PDA medium (28°C, for 3 days) as the primary culture. They were then transferred to five dishes of the culture (obtained by 9 mm puncher) into the PD medium, and cultivated in a shaker (28°C, 180 r min–1) for 5 days as the secondary cultures. Subsequently, the secondary culture was mixed with the fermentation broth (300 L), which contained maize flour (10 kg), zinc sulfate (0.4 g), ammonium sulfate (220 g), manganese sulfate (0.5 g), magnesium sulfate (100 g), potassium dihydrogen phosphate (766 g), sodium chloride (200 g), sodium nitrate (284 g) at weight ratio of 1:20, and cultivated at 28°C for 7 days. Finally, the fermentation product was mixed with diatomite, wheat bran, corn flour, humic acid, and zinc sulfate fertilizer in a blender, and the mixture was prepared into granules which were dried in a drying machine at 45–50°C, until the moisture level was below 10%. Each kilogram of granules contained 500 g of Trichoderma liquid fermentation broth, 687.5 g of diatomaceous earth, 156.25 g of wheat bran, 156.25 g of corn flour, 6.25 g of humic acid and 1.75 g of zinc sulfate (90%). The final preparation of Trichoderma granules was named TG1009. Fusarium graminearum was cultivated in PD medium for 7 days in the rocking incubator at 28°C, 180 r min–1. Then the solution with the pathogens was transferred to the sterilized maize kernels for another 7 days of incubation at 28°C until the surface of the maize kernels was covered with hyphae.

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inoculation, seedling growth indicators were measured, including plant height, root length, root fresh weight, disease index and incidence. In the field experiment, fields of uniform fertility were chosen. Seven groups were designed to compare different dosages of Trichoderma granules under the same usage levels of chemical fertilizer (45 kg/667 m2). The dosages of granules varied from 10 g/hole to 2 g/hole. The treatment with a single chemical fertilizer was used as a blank. Each group was planted with 100 corn seedlings (three replicates, for a total of 300 seedlings). All treatments were conducted at the sowing stage. Inoculum preparation was as described above (Section 2.3). The pathogen inoculation was conducted at the bellmouthed stage by placing the inoculum into the basal part of the stem. Plants were monitored until the maturity stage for plant growth indicators and disease index (150 plants were chosen for investigation from each group).

2.5. Demonstration of Trichoderma granule against stalk rot The biocontrol tests of Trichoderma granules against stalk rot were conducted in naturally infected fields. At the sowing period, Trichoderma granules (TG1009) and granules mixed with compound fertilizer (Trichoderma granule 5 kg/667 m2; compound fertilizer 45 kg/667 m2) were applied into farming soil. The blank control (CK) was only treated with the compound fertilizer at 45 kg/667 m2. At the same time, the seed coating agent or seed dressing agent that was widely used in this locality were used as the positive controls. The maize varieties used for this biocontrol demonstration were those that are widely planted in this locality. At the maturity stage (R6), three rows were chosen randomly for investigation of stalk rot in each experimental field. In each row, 100 plants were selected for determining the incidence of stalk rot. The morbidity levels were counted and calculated.

3. Results

2.4. Pot and field experiment

3.1. Screening of Trichoderma strains against stalk rot pathogen

To ensure that the optimal amounts of Trichoderma granules were used in the field, three dosages of Trichoderma granules (0.5–3 g/pot) were compared under the same amount of compound fertilizer (5 g/pot) applied in experimental pots. Each treatment was set up with three replicates (six pots in each replicate and five plants/pot). The maize seedlings at the 3-leaf stage were inoculated with F. graminearum spore suspension (105 cfu mL–1) in the roots that had been wounded. A total of 15 days after pathogen

In general, the majority of strains showed a good control efficiency in vitro against stalk rot pathogen (Fig. 1-A and B). In total, 48 strains were screened based on confront culture on PDA with the pathogens (F. graminearum and F. verticillioides). The results showed that 77.1% of the strains gave inhibitory ratios against F. graminearum over 60%. Strains DLY31, SG3302, GDFS1009, DLY1301, LYI1208, GZ2101, ZQ3206, YZ2203, FZ1205, SG3403 and SH2103 gave inhibitory ratios of 70–83%. In addition,

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56.25% of strains gave inhibitory ratios against F. vertillioides

were selected for further testing their activity for maize

over 60% and strains DLY1303, DLY31, LYI1208, SG3403,

seedling growth promotion and the control of root rot

GZ2101 and GDFS1009 had inhibitory ratios of 70–80%.

in seedlings. GDFS1009 showed the most significant

Furthermore, seven Trichoderma strains (DLY31,

promotion of root and stem growth, however, DLY31 showed

SG3302, SG3403, DLY1303, LYI1208, GDFS1009 and

the best control efficiency to root rot caused by the stalk rot

GZ2101) with high inhibitory ratios for the pathogen in vitro

pathogen (Table 1).

A

Fusarium graminearum

Fusarium verticillioides

90 The antifungal rate (%)

80 70 60 50 40 30 20 10

1.

ZQ 2. 120 3. DLY 2 S 3 4. G33 1 Z 0 5. Q3 2 SG 20 6. 3 6 D 4 7. LY1 03 D 10 L 8. Y1 2 LY 30 9. I1 3 N 2 10 C3 08 . G 20 11 Z2 8 . 1 12 FZ1 01 . 3 13 SZ1 01 . G 20 14 Z2 2 . 1 15 YZ2 05 . A 61 16 Q3 2 . 3 17 FZ1 03 . G 30 18 Z2 2 .S 1 19 H1 08 . 4 20 YZ2 04 . H 20 21 A2 3 . L 40 Y 4 2 I1 23 2. 101 . D HF 24 LY 31 . L 13 YI 01 21 03

0

Strain

100 90 The antifungal rate (%)

80 70 60 50 40 30 20 10

25

. 26 JA1 . 1 27 LY 03 . Z 11 0 28 Q3 3 . L 10 29 Y2 1 . 5 30 LC1 04 . N 20 31 B 2 . 11 32 QZ 07 . 31 33 SG 03 . S 33 34 G3 05 . 3 35 LC 06 . H 23 36 M 01 . S 24 37 H2 03 38 . FZ 103 . D 12 39 LY 05 . S 23 40 G3 12 . 1 41 SZ3 02 . 1 42 LC1 04 . L 20 43 C31 1 44 . DL 02 . H Y3 F 4 4 25 46 5. 03 . L JA 47 YI1 14 48 . 2 . G LY 03 D I11 FS 12 10 09

0

Strain

B

FG (CK) FG

GDFS1009

Fig. 1 The antifungal properties of Trichoderma against Fusarium spp. A, the antifungal rate of the different strains. Bars donate standard deviation (n=3). B, the antifungal assay of Trichoderma asperellum GDFS1009 against with Fusarium graminearum (FG).

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3.2. Development of application technology of Trichdoderma strains against stalk rot at the seedling stage Control efficiency of Trichoderma granules on stalk rot at the maize seedling stage In the pot experiment, with increasing amounts of granules applied in soil without chemical fertilizer, the maize seedling root length and plant height were improved, in which the 2 g/hole dosage showed the most significant promotion of stem and root growth. Also, 2 g/hole was the optimal dosage for the stalk rot control, since at that dosage the incidence of the stalk rot and the disease index was the lowest (Table 2). Control efficiency of the application of Trichoderma granules combined with chemical fertilizer on stalk rot and maize growth duration in the field In the field experiment, the dosage of Trichoderma granules at 10 g/hole

with 45 kg/667m2 chemical fertilizer showed the optimum control efficiency for stalk rot and maize growth duration. In this treatment, the control efficiency reached 72.05% and yield increased by 11.28% compared with the blank control (CK). The results showed that the disease incidence was very serious, as high as 58.86% in the control treatment without Trichoderma granules (Table 3), and suggested that the application of Trichoderma granules can reduce the incidence of maize stalk rot and significantly promote the yield. Therefore, the Trichoderma granule is promising and its use should be demonstrated nationwide. The demonstration of Trichoderma granule application in a farming field The Trichoderma granule application was demonstrated in some experimental stations in representative maize producing areas in China. In Sichuang, Yunnan and Liaoning provinces, the incidence of stalk rot and the yield of corn were investigated in

Table 1 Efficiency of different Trichoderma strains on promoting maize growth and control of root rot Trichoderma strain DLY31 SG3302 SG3403 DLY1303 LYI1208 GDFS1009 GZ2101 H6 CK

Main root (cm) 10±0.57 eD 7±0.70 deCD 10±0.57 bAB 9±0.60 bcBC 6±0.60 eD 12±1.15 aA 7±0.55 deCD 8±0.60 cdBCD 6±0.60 eD

Stem height (cm) 32±1.53 bB 24±2.00 cD 38±1.50 aA 26±0.58 cCD 26±1.50 cCD 40±2.08 aA 24±1.00 cD 31±0.60 bBC 23±1.60 cD

The length of rotted area in main root (cm) 0±0 cD 1.0±0.21 abABC 0.3±0.20 cCD 0±0 cD 1.2±0.15 aAB 0.5±0.11 bcBCD 1.0±0.17 abABC 1.5±0.45 aA 1.5±0.22 aA

The control efficiency (%) 100 33.33 80.00 100 20.00 66.67 33.33 0 –

The variance analysis was conducted by SPSS, the confidence interval: 95%; multiple comparison by SSR (one-way ANOVA), the lower-case letters indicate α=0.05, the capitals indicate α=0.01. Bars denote standard deviation (n=3).

Table 2 Efficiency of Trichoderma granules on stalk rot at maize seedling stage (pot experiment) TG1009 (g/pot) 3 2 1 0.5 0

Height (cm) 37.9±0.82 ab 38.2±1.04 a 36.5±1.36 abc 35.7±1.23 abcd 34.1±1.55 bcd

Root length (cm) 28.3±0.82 a 28.0±0.75 a 25.6±1.31 ab 21.2±1.74 cd 17.8±1.45 de

Fresh weight (g) 1.85±0.08 a 1.89±0.10 a 1.83±0.15 a 1.77±0.17 a 1.65±0.26 a

Incidence (%) 63.3±1.04 de 60.0±1.38 e 71.2±0.69 cd 80.0±1.66 b 96.7±2.83 a

Disease index 33.1 f 32.5 f 41.6 de 57.8 c 70.2 a

The variance analysis was conducted by SPSS, the confidence interval: 95%; multiple comparison by SSR (one-way ANOVA), the lower-case letters indicate α=0.05. Bars denote standard deviation (n=3).

Table 3 Effects of increased Trichoderma granules with a fixed amount of chemical fertilizer on the control of stalk rot in maize (field experiment) TG1009 (g/hole) 10 8 6 5 4 2 CK

Disease index 16.45 e 20.32 de 26.15 d 24.26 d 33.65 c 45.78 b 58.86 a

Incidence (%) 51.67±2.13 d 65.00±2.62 c 70.00±3.08 c 63.33±2.94 c 81.67±3.76 b 88.33±3.49 ab 98.33±2.98 a

Control efficiency (%)

Yield (kg)

72.05 a 65.48 ab 55.57 c 58.78 bc 42.83 d 22.22 e –

549.9±18.2 a 541.2±15.6 a 528.9±12.7 a 535.2±13.8 a 526.8±20.1 a 504.8±22.3 a 494.1±25.4 a

Yield increase rate (%) 11.28 a 9.53 b 7.16 c 8.32 bc 6.65 c 2.21 d –

The variance analysis was conducted by SPSS, the confidence interval: 95%; multiple comparison by SSR (one-way ANOVA), the lower-case letters indicate α=0.05. Bars denote standard deviation (n=3).

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experimental fields in 2016. The results suggested that the control efficiency varied in different areas. In the case of natural morbidity, the control efficiency ranged from 27.23 to 48.84%. The control efficiency varied due to unstable environmental factors (Table 4). In the demonstration site of Yunnan Province, the yield increased by 11.70%. The yields also increased over 10% in some sites in Liaoning Province (Table 5).

4. Discussion This study successfully screened a group of Trichoderma strains with significant inhibitory activities to stalk rot pathogens F. graminearum and F. verticilloides. According to the in vivo experiments, four strains (DLY31, SG3403, DLY1303 and GDFS1009) showed the best performance in

inhibiting pathogen infection and promoting maize growth. Those strains all revealed significant control efficiency for stalk rot at the seedling stage, because the pathogens start infecting the roots from the seedling stage and continue throughout the whole season. Therefore, demonstrating whether the Trichoderma agent can effectively inhibit pathogen attack in the whole growing season is vital for showing the value of the Trichoderma-based biocontrol approach for future outreach. Our results clearly show that the Trichoderma agent has advantages for controlling the soil-borne diseases like stalk rot featured throughout the whole growth period of infection, and this was probably achieved by the lasting root colonization effect generated by Trichoderma mycelia along the plant growth period (Harman et al. 2004). It has been suggested that Trichoderma can induce maize plant defense gene systemic expression

Table 4 Effects of Trichoderma granules on corn stalk rot at experimental stations Demonstration site Zhuanghe (Liaoning Province)

Varieties Jincheng 9

Haicheng (Liaoning Province)

Haydan 9

Fengcheng (Liaoning Province)

Danyu 405

Qujing (Yunnan Province)

Senhai 2

Mianyang (Sichuan Province)

Yudan 8

1)

Treatment1) TG1009 PC CK TG1009 PC CK TG1009 PC CK TG1009 PC CK TG1009 PC CK

Incidence (%) 15.50 6.70 21.30 2.20 3.30 4.30 0 0 0 0 0 0.75 4.00 4.25 6.25

Control efficiency (%) 27.23 68.54 – 48.84 23.26 – 100 100 – 100 100 – 32.00 36.00 –

TG1009, Trichoderma granules; PC, the positive control that was treated by the seed coating agent or seed dressing agent that is currently widely used at the location; CK, the blank control.

Table 5 Trichoderma granules effect on yield at experimental stations Demonstration county Zhuanghe, Liaoning Province

Varieties Jincheng 9

Haicheng, Liaoning Province

Haydan 9

Fengcheng, Liaoning Province

Danyu 405

Qujing, Yunnan Province

Senhai 2

Mianyang, Sichuang Province

Yudan 8

1)

Treatment1) TG1009 PC CK TG1009 PC CK TG1009 PC CK TG1009 PC CK TG1009 PC CK

Test production (kg/667 m2) 498.50 512.49 506.52 699.52 712.68 635.47 627.12 638.59 599.38 469.90 445.37 424.30 398.00 430.50 356.50

Yield increase rate (%) –1.60 1.18 – 10.08 12.15 – 4.60 6.50 – 10.75 4.97 – 11.70 20.90 –

TG1009, Trichoderma granules; CK, the blank control; PC, the positive control that was treated by the seed coating agent or seed dressing agent that is currently widely used at the location.

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throughout the whole growth period, as a rhizosphere fungus which closely interacts with maize root cortex tissue leading to long-term defense signal transduction (Nawrocka and Małolepsza 2013). Furthermore, Trichoderma strains have been found to colonize the rhizosphere of maize, which enrich soil microbiome diversity, and as a result, Fusarium spp. populations are significantly reduced (Saravanakuma et al. 2017). Our previous studies have also demonstrated that T. harzianum secretes cellulaseelicitor and hydrophobin Hyd1, which enable it to induce maize systemic resistance against foliar diseases through long distance transduction of JA/ET and BR signals. It has been implied that the Trichoderma agent systemic induction effect truly works (Saravanakumar et al. 2016). According to previous research, Trichoderma has demonstrated various positive effects on plants’ growth, resilience and yield. These effects have been attributed, in part, to symbiotic interactions between the plants and the microbes that live around, on and inside them (Doni F et al. 2017). Chemical compound fertilizers are widely applied in maize production, therefore finding the best ratio of fertilizer to combine with the Trichoderma agent for controlling stalk rot in the field is an indispensable measure at the sowing stage. From an economic point of view, the combined use of Trichoderma agent and chemical fertilizer would be very cost-effective. Based on previous tests, chemical fertilizers have a slight impact on Trichoderma growth and reproduction (data not shown) in the field. In the current study, we ensured that the dosage of Trichoderma granules at 10 g/hole was effective for controlling stalk rot, even in cases when the diseases occurred seriously. According to our last five years of field practice, Trichoderma granules at 2–5 kg/667 m2 combined with the use with fertilizer usually generates good control efficiency against stalk rot in the field. However, we also found that the amounts of chemical fertilizer applied, if beyond the optimal limit, would negatively impact the stalk rot control effects. This means that the synergistic effects of both agents were positively working only within a certain range of chemical fertilizer use, but currently the mechanism is still unclear. A previous field study has found that chemical fertilizer could be used at 30% less than the normal amounts applied if the Trichoderma agent was added at the dosage of 2–5 kg/667 m2, which could enhance the nutritional utility of the maize plant. The yield reached the same or higher levels when compared with the chemical fertilizer applied at the full level. A potential ecological benefit of this combined use of the Trichoderma agent and compound fertilizer is that it avoids leaching of the superfluous fertilizer into the soil, so it prevents the destruction of the soil ecological system. This destruction would lead to plant resilience decline due to stress conditions, such as pathogen infection and the salinization

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of soil. On the other hand, chemical seed coating agents are commonly used throughout the world, so before sowing or planting seeds are already coated by chemical agents. In this case, it is difficult for the seed surface to be recoated with Trichoderma. Thus, Trichoderma granules provide an alternative approach for use even though the seeds are already coated in advance with chemicals agents before sowing.

5. Conclusion The Trichoderma strains DLY31, SG3403, DLY1303 and GDFS1009 were showed the best performance for inhibiting pathogen infection and promoting maize growth. The results suggest that Trichoderma agents have advantages for controlling the soil-borne diseases like stalk rot during whole growth period infection.

Acknowledgements The research has been supported by the National Key Research and Development Program of China (2017YFD0200403), the Key International Intergove­rn­ mental Scientific and Technological Innovation Cooperation Project, China (2017YFE0104900), the National Natural Science Foundation of China (31750110455, 31672072), the Agriculture Research System of Shanghai, China (201710) and the earmarked fund for the China Agriculture Research System (CARS-02).

References Amy V, Suhail R M, Murray S L . 2018. Trichoderma asperellum isolated from African maize seed directly inhibits Fusarium verticillioides growth in vitro. European Journal of Plant Pathology, 153, 279–283. Doni F, Zain C R C M, Isahak A, Fathurrahman F, Sulaiman N, Uphoff N, Yusoff W M W. 2017. Relationships observed between Trichoderma inoculation and characteristics of rice grown under system of rice intensification (SRI) vs. conventional methods of cultivation. Symbiosis, 72, 1–15. Duan H M, Li Y U, Huang W D, Yu H B. 2018, Inhibitory activity of six fungicides to Fusarium graminearum maize stalk rot at different temperatures and mixture screening with antagonistic bacteria fermentation supernatant. Jiangsu Journal of Agricultural Sciences, 34, 41–49. (in Chinese) Ghini R, Perondi F N L, Navas-Cortés J A, Silva C A, Bettio W. 2016. Combined effects of soil biotic and abiotic factors, influenced by sewage sludge incorporation, on the incidence of corn stalk rot. PLoS ONE, 11, e0155536. Harman G E, Howell C R, Viterbo A, Chet I, Lorito M. 2004. Trichoderma species - opportunistic, avirulent plant symbionts. Nature Reviews Microbiology, 2, 43.

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LU Zhi-xiang et al. Journal of Integrative Agriculture 2020, 19(1): 145–152

Harman G E, Petzoldt R, Comis A, Chen J. 2004. Interactions between Trichoderma harzianum strain T22 and maize inbred line Mo17 and effects of these interactions on diseases caused by Pythium ultimum and Colletotrichum graminicola. Phytopathology, 94, 147–153. Ma H X, Zhang H J, Sun H, Shi J, Chen D, Guo N. 2017. Comparison of pathogen detection methods for corn stalk rot. Plant Protection, 43, 149–153. (in Chinese) Nayaka S C, Niranjana S R, Shankar A C U, Raj S N, Reddy M S, Prakash H S, Mortensen C N. 2010. Seed biopriming with novel strain of Trichoderma harzianum for the control of toxigenic Fusarium verticillioides and fumonisins in maize. Archives of Phytopathology and Plant Protection, 43, 264–282. Nawrocka J, Małolepsza U. 2013. Diversity in plant systemic

resistance induced by Trichoderma. Biological Control, 67, 149–156. Saravanakumar K, Fan L L, Fu K H, Yu C J, Wang M, Xia H, Sun J N, Li Y Q, Chen J. 2016. Cellulase from Trichoderma harzianum interacts with roots and triggers induced systemic resistance to foliar disease in maize. Scientific Reports, 6, 35543. Saravanakumar K, Li Y Q, Yu C J, Wang Q Q, Wang M, Sun J N, Gao J X, Chen J. 2017. Effect of Trichoderma harzianum on maize rhizosphere microbiome and biocontrol of Fusarium stalk rot. Scientific Reports, 7, 1771. Wu X R, Chen S W, Yang Y H, Wang Y H, Liu Y Z, Chen J. 2015. Application of Trichoderma granules in the control of corn stalk rot. Journal of Plant Protection, 42, 1030–1035. (in Chinese)

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