Materials
Seeds of wheat (Triticum spp. cv. Xinmai 18) were commercially from the Mianyang Seed Company, Sichuan, China. The purple soil (Eutric Regosol, FAO Soil Classification System) was collected at 5-10 cm depth from a field within the National Monitoring Station of Soil Fertility and Fertilizer Efficiency on Purple Soils, locating in the Southwest University campus (E 106°24′37″; N 29°48′32″) Beibei, Chongqing, China. The collected purple soils were thoroughly mixed after the removal of debris, dried at 30°C, then sieved to 2 mm and autoclaved before the pot experiments, and the basic chemical properties were shown in Table 1.
Table 1 Chemical properties of the experimental purple soil
Chemical properties
|
OM
g/kg
|
TN
g/kg
|
TP
g/kg
|
TK
g/kg
|
AN
mg/kg
|
AP
mg/kg
|
AK
mg/kg
|
pH
|
Purple soil
|
10.62
|
0.84
|
0.63
|
21.33
|
70.43
|
31.21
|
117.22
|
5.52
|
OM: Organic matter; TN: Total nitrogen; TP: Total phosphorus; TK: Total potassium; AN: Available nitrogen; AP: Available phosphorus; AK: Available potassium.
Two fungal strains were examined: B. adust M1 was isolated from the above-mentioned rhizosphere soil in April 2016, and F. graminearum, a pathogenic fungus resulting in the wheat scab, was from the Institute of Plant Ecology and Pathology, Southwest University.
A chemical fungicide: carbendazim [50% N-(benzimidazlyl-2) methyl carbamate] was from the Sichuan Guoguang Agrochemical Company, Jianyang, Sichuan, China.
The Potato dextrose agar (PDA) medium was used for cultivating the strains of B. adusta M1 and F. graminearum (Farias et al. 2010). The basic broth medium (20 g glucose, 10 g NH4Cl, 1 g MgSO4, 1 g CaCl2, 2 g KH2PO4, and 1,000 mL distilled water) was used for obtaining the fermentation broth of B. adusta M1 and the spore suspension of B. adusta M1 and F. graminearum.
Preparation of spore suspension of F. graminearum
F. graminearum was inoculated in the PDA plate and cultivated at 28°C for 5 d until the mycelia filled with the plate, and then added 5 mL sterilized basic broth medium into the plate, gently scraped off the conidia with the inoculating spear. The liquid was filtrated with 4 layers of gauzes. Meanwhile, the spore numbers of the filtrate were counted by the globulimeter under ordinary optical microscope microscope, and finally diluted to 4.5×105 pcs/mL basic broth medium.
Preparation of FB
B. adusta M1 was inoculated in 250 mL triangular bottle with 100 mL basic broth medium, which was shaken at 32°C and 150 r/min for 5 d, and then centrifuged at 3000 r/min for 1 min. The supernatant was filtrated with 0.22 μm sterile filter, and the filtrate was termed as FB.
The inhibitory effects of B. adusta M1 on F. graminearum
The inhibitory effect of B. adusta M1 on F. graminearum was measured by the antifungal test as follows. Both the B. adusta M1 and F. graminearum, with a size of 5 mm-diameters, were inoculated on the same PDA plate (9 cm × 9 cm), while the two strains were 3 cm apart to form a two-point confrontation. The sole F. graminearum that was inoculated on the PDA was the control. After cultivated at 32°C for 5 d, the inhibitory effect of B. adusta M1 on F. graminearum could be observed. The fungal colony sizes in the control and confronting plates were recorded and percent inhibition was calculated as
Where A1 and A2 are the areas in mm2 of the indicator pathogen in the control and treatment, respectively. The tests were performed in three replications.
The inhibitory effects of FB on F. graminearum
The antifungal effect of FB on F. graminearum was determined by the antifungal test (Campanile et al. 2007) and the relevant inhibition rate was calculated with the above formula 1. Briefly, F. graminearum was inoculated on PDA plate (9 cm × 9 cm) with 10%, 20% or 40% FB (the control was different concentration basic broth medium) and cultivated at 32°C for 5 d, and repeated for 3 times. The inhibition rate was calculated with the formula 1.
The competition and mycoparasitism effects of B. adusta M1 against F. graminearum
Two groups of experiments were conducted. B. adusta M1 and F. graminearum, both with 5 mm-diameters, was inoculated on the same PDA plate, and the two strains were 3 cm apart, forming a two-point confrontation. One group was used to observe the competition effect of B. adusta M1 on F. graminearum (only F. graminearum was inoculated on the PDA as control group). For another group, a 2 cm × 2 cm foil paper was laid on the surface of the PDA plate between two strains, and the fungal strains were cultivated at 32°C for about 3 d until they intermingled on the foil paper. Then the foil paper was removed to prepare microscope slides and the mycoparasitism effect of B. adusta M1 on F. graminearum was observed under a scanning electron microscope.
The control effects of FB on wheat scab
Firstly, wheat seeds were immersed in 75% alcohol for 5 min, and treated with 20% sodium hypochlorite for 3 min, and rinsed several times with sterile water, and then cultivated in a constant temperature incubator at 28°C for 3 d to promote their germination. The germinated seeds were sowed in a seedling tray (20 cm × 30 cm) that was filled with aseptic nutrient soil. Three wheat seedlings with two cotyledons were transplanted to the pot (18 cm × 24 cm) with 2 kg sterilized soil. After 30 d of transplanting, the wheat seedlings were irrigated with 10 mL F. graminearum spore suspension. After the wheat seedlings got sick, four treatments were examined: The wheat seedlings were irrigated with 10 mL basic broth medium (CK), 10 mL B. adust M1 fermentation broth (FB), or 10 mL 0.08 mg/mL chemical fungicide polymyxin carbendazim (CF). Each test was repeated 3 times. After 24 h of treatment, the disease index and control effects were respectively counted once every 5-day for a total of 3 times (this article showed the last statistical results) according to the formula 2 and 3. The disease classification criteria were as follows: Grade 0: no disease spots, or the stem leaves infected the disease are less 1%; Grade 1: there are small disease spots, the stem leaves infected the disease are 1% ~ 25%; Grade 2: there are large disease spots, the stem leaves infected the disease are 26% ~ 50%; Grade 3: there are a large number of disease spots, blight and curly, the stem leaves infected the disease are 51% ~ 75%; Grade 4: there are a large number of disease spots, the stem leaves infected the disease are 76% ~ 90%; Grade 5: the stem leaves infected the disease are more than 90%, or whole plant die of disease (Lian et al. 2017).
Wheat leaves were collected after the last count of plant disease, and the activities of leaf catalase (CAT), peroxidase (POD) and phenylalanine ammonia lyase (PAL) were measured by the (UV) spectrophotometry and guaiacol method (Pandey et al. 2018). The leaf cell membrane permeability and malondialdehyd was analyzed by the electrolyte infiltration and thiobarbital (TBA) colorimetry (Yuan et al. 2005).
Data analyses
The statistical software SPSS 21.0 (Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL) was used to analyze the data obtained from our experiments. The significant differences between treatments were compared with the Duncan’s multiple range tests at a significance level of P < 0.05. The figures and tables were generated with Excel 2017, Oringin 9.0.