Fusarium graminearum was provided by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. Colletotrichum gloeosporioides (an apple fruit pathogen), Rhizoctonia solani (a cotton pathogen), Sclerotium rolfsii (a rice pathogen), and Alternaria alternata (a pear fruit pathogen) were acquired from the lab of Pathogenic Fungi and Genomics in College of Agriculture, Yangtze University, Jingzhou, China.

The samples were collected by five-point sampling method in Jingzhou, Hubei Province from plants grown in a field with severe wheat scab outbreak (N30°21′32.53″ E112°08′22.05″). The roots, stems, leaves, and spikelets of wheat were cut into small segments of about 5 cm and washed with water for 30 min. The following operations were carried out on a clean bench: samples were washed 4 times with sterile water, soaked in 75% ethanol for 5 min, washed with sterile water two times, soaked in 1% mercuric chloride for 3 min, and finally rinsed three times with sterile water. The sterilized plant material was placed in a sterile mortar containing an appropriate amount of quartz sand. The sample was grinded with 10 ml of sterile water, 100 μL of the solution was plated on Potato Dextrose Agar (PDA: potato 20 g/L, glucose 20 g/L, agar 15 g/L) and Nutrient Agar (NA: peptone 10 g/L, beef extract 3 g/L, glucose 2.5 g/L, agar 18 g/L) kept at a constant temperature of 28°C After three days of culture, colonies were separated and cultured on PDA and NA media, respectively. Pure strains were obtained after three times’ sub-culturing.

Adopting dual culture assays tested the antagonistic effects of isolations on Fusarium graminearum (Su et al., 2010). In each repetition, a 5 mm mycelium pellet of F. graminearum was inoculated in the central portion of the PDA medium with a diameter of 90 mm, and the endophyte was attached to the periphery. Plates grown with F. graminearum alone served as controls, and three repetitions were set for each treatment. These were cultured at 28°C in the dark for 3 to 4 days until control dishes were covered with the F. graminearum. The inhibition zone was measured and statistically analyzed. The diameter of the zone of inhibition was the growth width of the antagonistic colonies, measured in a criss-cross method. Diameter of inhibition zone is calculated using the following formula: (A1+A2)/2, where A1 and A2 are respectively the diameter of inhibition zone measured by the criss-cross method. Two independent experimental replicates were performed for each strain. Dual culture assays were used to detect the inhibitory effect of antagonistic endophyte on the previous pathogenic fungi. Three replicates were performed for each treatment.

DNA of the antagonize endophytes were extracted by using CTAB method (Liu et al., 2019). The extracted DNA was used as a template for polymerase chain reaction (PCR) to amplify with the 16S rDNA universal primers (27F: 5’-CAGAGTTTGATCCTGGCT-3’, 1492R: 5’-AGGAGGTGATCCAGCCGCA-3’) (Redburn and Patel, 1993). Amplification process order for: 94°C 3 min, 94°C 40 s, 65°C 40 s, 72°C 1 min, a total of 30 cycles; then 72°C 8 min. The amplified product was sequenced by Sangon Biotech. The sequencing results were used to perform BLAST searching on the NCBI website (http://www.ncbi.nlm.nih.gov/) to determine the species level. Phylogenetic tree was completed by the phylogenetic software MEGA 7.0 (Yin et al., 2017).

Five common antibiotics, chloramphenicol (Ch1), ampicillin (Amp), tetracycline (Tet), streptomycin (Str) and kanamycin (Kan), were used to test the tolerance of strain XY-1. Bacterial suspensions from the colonies developed in NB were standardized to 108 CFU/mL and transferred (10 μL) to NA plates containing different concentrations of antibiotics (25 μg/mL CH1, 100 μg/mL Amp, 25 μg/mL Tet, 50 μg/mL Str, 10 μg/mL Kan).The plates were incubated at 28°C for 48 hand evaluated for the presence of bacterial growth. The colony diameter of 0-8 mm indicated that the strain had no drug resistance to antibiotics, the colony diameter of 9-17 mm had weak drug resistance, and the colony diameter larger than 17 mm had drug resistance (Xia et al., 2019).

The antagonistic bacteria was cultured in NA liquid medium and incubated in the dark at 28°C and pH 7.0 with shaking at 180 rpm. The OD₆₀₀ value was measured every 4 h until 36 h, and then measured every 12 h until 84 h, NA liquid medium alone was used as a control. At each time point, OD₆₀₀ was measured by a spectrophotometer at an absorbance wavelength of 600 nm (Zhang et al., 2019).

The pH was adjusted to 3.0, 5.0, 6.0, 7.0, 7.4, 8.0, 10.0, and 12.0 with NaOH and HCl, respectively, and 0.1 mL of bacterial suspensions was cultured in 100 mL of NA liquid medium with different pH values, shaken at 28°C, 180 r/min, and the OD₆₀₀ value was measured at 600 nm after 48 h. NA liquid medium alone was used as a control, and three independent biological replicates were set for each treatment.

The optimum growth temperature curve was drawn with 0.1 mL of bacterial suspensions in 100 mL of NA culture medium (pH 7.4). The endophyte bacterial suspensions was cultured at different temperatures (4°C,10°C, 16°C, 22°C, 28°C, 34°C, 40°C, 46°C, and 52°C). The OD₆₀₀ value was measured at 600 nm after 48 h. NA liquid medium alone was used as a control, and three independent biological replicates were set for each treatment.

A liquid medium with glucose (2.5 g/L) and peptone (10 g/L) as carbon source and nitrogen source, respectively, was used as the basic medium. Then, the carbon source of glucose was replaced by sucrose and lactose, and the nitrogen source of peptone was replaced by beef extract or ammonium nitrate to explore the best combination of carbon and nitrogen sources for endophyte fermentation.

0.5 mL of bacterial suspensions was inoculated into 50 mL of the optimized liquid medium and incubated for 2 days at 28°C with shaking at 180 r/min. The culture supernatant was collected from the culture broth by centrifugation at 8,000 rpm for 30 min at 4°C. The bacteria XY-1 in the culture supernatant was filtered out through a 0.22 µm microfiltration membrane. One group was poured 5 mL of the culture broth into a petri dish containing 10 mL of PDA medium, and inoculated the center of the dish with F. graminearum mycelia plug. The other group was inoculated with F. graminearum mycelia plug alone. Then they were cultured in a constant temperature incubator at 28°C for 3 to 5 days. The medium at the junction of the inhibition ring was observed under a microscope, and the morphological characteristics of the mycelium of the treatment group and the control group were compared. Each treatment contains 3 replicates.

Testing for the antagonistic effect of endophyte against F. graminearum was performed on detached spikelets from wheat cultivar “Emai 170”. Detached spikelets were soaked in a suspension of endophyte or control water for 2 min, before being placed on plates filled with water agar medium (3 g/L). Five days later, a conidia suspension of Fusarium graminearum (10⁵ conidia/mL) was sprayed on spikelets. Under conditions of 20/15°C day/night temperature, 16/8 h light/dark photoperiod, and 65% of relative humidity. Two weeks later, morbidity, disease index and 1000-grain weight was recorded. Each treatment consisted of two independent experimental replicates with at least 30 spikelets per replicate.

Data were subjected to analysis of variance using SPSS 19.0, where F tests were significant at p < 0.05, and the means were separated by Duncan’s Multiple Range test at p < 0.05.

According to the differences of colony morphology and color, a total of 45 bacterial strains were isolated from different tissues of wheat. Seven strains antagonistic to Fusarium graminearum were screened by dual culture assays. Among them, the stain XY-1 isolated from root, exhibited the best antagonistic effect. The average diameter of the inhibition zone was larger than other strains, which was 29.7 mm ( Figure 1 ). Therefore, XY-1 was selected for further study.

Strain XY-1 was tested for antagonism against several common crops and fruit tree pathogens by using dual culturing assays. The results showed that XY-1 displayed an inhibitory effect on the growth of four tested pathogens, namely Colletotrichum gloeosporioides, Rhizoctonia solani, Sclerotium rolfsii, and Alternaria alternate. The zone of inhibition ranged from 17.7 to 32 mm. In addition, XY-1 exhibited the best inhibitory effect on Alternaria alternata, followed by Sclerotium rolfsii ( Table 1 ).

In order to observe the growth of strain XY-1, the bacterial solution was inoculated into the NA liquid medium, incubated at 28°C with shaking (160 r/min), and its OD₆₀₀ value was measured at a wavelength of 600 nm. As shown in Figure 2A , XY-1 was in the exponential phase within 24 h after inoculation into the NA culture medium; it was in the stable phase within 24-60 h of inoculation; it entered the recession phase after 60 h ( Figure 2A ).

Figure 2B showed that XY-1 could grow at pH 4.0-10.0, but not at pH 3.0. The pH range for growth was 5.0 to 9.0, with an optimum pH of 7.4 ( Figure 2B ). Strain XY-1 was placed in NA broth at pH 7.4 and placed at different temperatures. The results showed that strain XY-1 could grow at 10–50°C with an optimum temperature of 28°C ( Figure 2C ).

In this experiment, sucrose, glucose and lactose were used as carbon sources, and peptone, beef extract and ammonium nitrate were used as nitrogen sources. The results showed that XY-1 could utilize all carbon and nitrogen sources tested in this study. Among them, glucose was the most suitable carbon source, and beef extract was the most suitable nitrogen source ( Figure 2D ).

In order to test the resistance of the strain XY-1 to common antibiotics, chloramphenicol (Ch1), streptomycin (Str), tetracycline (Tet), kanamycin (Kan), and ampicillin (Amp) was placed in NA liquid medium, respectively. The plaque diameters were counted after culturing in a constant temperature incubator at 28°C for 2 days. The results showed that the resistance of XY-1 to the five antibiotics was different, that XY-1 had better resistance to chloramphenicol and ampicillin ( Table 2 ).

The sequencing data showed that the length of the 16S rDNA sequence of the strain XY-1 was 1207 bp. The sequence was blast aligned in the NCBI database (http://www.ncbi.nlm.nih.gov/), and the phylogenetic tree was constructed using Mega7.0 software ( Figure 3 ). The analysis showed that XY-1 belonged to Bacillus, and the sequence similarity with Bacillus amyloliquefaciens (EU855184.1) was 95%. Based on its physiological and biochemical characteristics, the strain XY-1 was preliminarily classified as Bacillus amyloliquefaciens.

The medium at the junction of the inhibition ring was observed under a microscope, and the morphological characteristics of the mycelium of the treatment group and the control group were compared. The results showed that the hyphae of F. graminearum grew alone normally ( Figure 4A ), and the hyphae of the pathogenic fungi with culture supernatant of XY-1 were obviously inhibited ( Figure 4B ). As shown in Figure 4C , the mycelium morphology of F. graminearum grown alone was normal, slender and long ( Figure 4C ). By comparison, the mycelium morphology of F. graminearum with culture supernatant was malformation, thick and short ( Figure 4D ).

The antagonistic effect of XY-1 against F. graminearum was further tested in isolated wheat spikelets. Spikes inoculated with alone developed typical wheat head blight symptoms within two weeks, and inoculated with distilled water alone did not cause any visible disease symptom ( Figure 5A ). As shown in Figure 5B , CK were the isolated spikelets that were not inoculated with Fusarium graminearum, and they were symptomless. The incidence rate of isolated spikelets that were immersed in the filtered culture supernatant and then inoculated with PH-1 was 13.5%, and the incidence rate of the isolated spikelets that were inoculated with F. graminearum alone was 100%. The results showed that the culture supernatant of strain XY-1 reduced the incidence of F. graminearum by 86.5% ( Figure 5B ). The study showed that bacteria XY-1 reduced the disease index by 56.7% ( Figure 5C ). The 1000-grain weight measurement data for each group of wheat showed that the group inoculated with F. graminearum alone was 15 g, the treatment group with culture supernatant of XY-1 just reduced by 3g compared with CK ( Figure 5D ). The application of XY-1 greatly reduced the yield decline caused by FHB.