
Thirty-four strains of bacteria were isolated from
At present, the main control method of plant diseases is the use of chemical pesticides. However, long-term use of chemical pesticides causes environmental pollution, and pesticide residues lead to an increase in disease resistance and reduced soil fertility (Passari et al., 2018). Moreover, chemical control causes the deterioration of natural resources, degradation of product quality, and destruction of ecological balance, with serious impacts on human health and ecological safety (Abbas et al., 2017). In contrast, biocontrol technology, which utilizes beneficial microorganisms to control the occurrence and development of disease, has been proposed as a solution (Iasur-Kruh et al., 2018). Indeed, the development of effective biological agents against pathogenic fungi is an important method to controlling plant diseases (Boukaew et al., 2017). Biocontrol bacteria are ubiquitous in the environment, with a wide range of prevention and control properties; furthermore, such bacteria exhibit good effects in controlling plant diseases, and there is no pathogen resistance. Thus, biocontrol bacteria are highly promising for plant disease control (Boukaew et al., 2017; Etesami and Alikhani, 2017; Kamal et al., 2016). Biocontrol microbes are abundant and include bacteria, fungi and actinomycetes (Baltz, 2016). The relevant bacteria are largely
To date, there have been few reports on canker rot in
One-yearold healthy
The pathogenic fungus
Culture medium: (1) PDA: potato 200 g, glucose 20 g, agar 15–20 g, distilled water 1000 ml, pH 7.0. Potato glucose agar was used for pathogen culture. (2) NA: peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 15–20 g, distilled water 1000 ml, pH 7.4–7.6. Beef peptone solid medium was used for the isolation and purification of bacteria.
To isolate endophytic bacteria, the bark of
According to the antagonistic bacterium screening method of Edwards and Mcbride (1975), antagonistic bacteria were preliminarily screened by the confrontation culture method. Strains with an obvious inhibition circle were selected, and their widths were measured. After purification, the strains were screened again. LB culture medium was selected for culture of antagonistic strains in a shaking bed incubator set at a constant temperature of 25°C and 135 r/min for 2 d. The fermentation culture filtrate was collected and filtered through filter paper and centrifuged at 4,000 r/min for 8 min, and the upper layer was filtered through a 0.22 μm membrane to prepare a sterile culture filtrate. The growth rate method of Bekierkunst and Szulga (1954) was used to determine strain antagonism. LB plates spread with sterile water and no fermentation culture filtrate were used as a control. Samples were cultured at 25°C constant temperature, and the process was repeated three times.
Morphologic observation: The antagonistic bacterium was inoculated onto NA medium and cultured in an incubator at 25°C. Morphological characteristics of colonies were assessed according to the Manual of Identification of the Common Bacterial System (Montanari et al., 2004; Zou et al., 2015).
Physiological and biochemical testing: The antagonistic bacterium was tested for Gram staining and spore staining as well as for the gelatine liquefaction test, nitrate reduction reaction, starch hydrolysis test, sugar oxidation fermentation test, contact enzyme reaction, acetyl methyl methanol test and hydrogen sulfide test.
Molecular biological identification: The strain was cultured in 20 ml of NA liquid medium for 24 h under constant temperature shock. The culture liquid was collected into 2 ml centrifuge tubes and centrifuged at 4°C and 12,000 rpm for 10 min. DNA was extracted according to the bacterial genome DNA kit (Tiangen Biochemistry Technology Company of Limited Liability). The extracted DNA was amplified by Polymerase Chain Reaction using the bacterial 16S rDNA universal primer (27F5’-AGAGTTTGATCCTGGCTCAG-3’) and (1492R5’-GGTTACCTTGTTACGAC -T T-3’).
Each 25 μl PCR mixture contained 0.5 μl DNA template, 12.5 μl 2 × Taq Master Mix, 1 μl each upstream and downstream primers, and 10 μl ddH2O. The reaction consisted of a 4-min pre-denaturation at 94°C, 35 cycles of denaturation 40 s at 94°C, annealing 30 s at 55°C, and extension for 1 min at 72°C, and a 10 min final elongation at 72°C. Five microlitres of the amplified product was assessed by 1% agarose gel electrophoresis and sequenced. The sequences were analysed by Blast homologous alignment, and the 16S rDNA sequences of highly homologous strains were downloaded from GenBank. Finally, a phylogenetic tree was constructed using the MEGA5.0 software adjacency method, and the phylogenetic status of the antagonistic strain was determined.
The antagonistic bacterium was incubated for 48 h in NA liquid medium in a shaking bed incubator set at 28°C and 180 r/min. The culture medium was prepared with different concentrations of 104, 106, and 108 cfu/ml. Five pieces of pathogenic fungus mycelium (Φ=8 mm) were obtained by the punch method and added to each bottle of PDA liquid medium; pathogen spores were at a concentration of 105/ml after 72 h. One hundred healthy annual
Field-control trials were conducted in a pure forest of
Inhibitory effect of the antagonistic bacterium on the pathogenic fungus: (1) Effect on the mycelial growth of the pathogenic fungus - an antagonism test was carried out using the perforating method; sterile water was used as the control, and samples were assessed at 24, 48, 72, 96 and 120 h after addition of the antagonist supernatant. This assay was used to determine the diameter of the fungal colony and the diameter of the control fungal colony; each treatment was repeated 3 times. A growth curve of the disease after confrontation was drawn. After the control mycelium was covered with a flat plate, the difference between the mycelia of the surrounding colony and the normal mycelium of the control colony was observed under a light microscope (Hoyt et al., 2015). (2) Effects on the germination and production of spores by the pathogenic fungus: 107 cfu/ml of spore suspension was prepared, and a sterile filtrate was obtained. A conidial suspension of 2 μl of the pathogenic fungus was placed onto aseptic slides, and aseptic filtrates with an equal volume of concentrations of 1%, 5%, 25% and 50% were added to the conidial suspension droplets. Sterile water was used as the control, and the treatment was repeated 3 times. Conidial germination was detected at 5 h, 12 h and 24 h under the condition of moisture retention at 25%. Germination of the spore bud tube was considered at more than 1/2 of the spore diameter. (Sørensen et al., 2003). For each treatment, 200 spores were observed under a microscope; the total number of spores and number of sprouts were recorded, and the spore germination rate was calculated (Li et al., 2011).
All data were subjected to one-way analysis of variance to determine the significance of individual differences at the
A total of 34 strains with different morphologies were isolated from healthy bark by tissue separation and named B1–B34. Five strains (B9, B18, B26, B30, and B34) with different inhibitory effects on the pathogenic fungus were screened by confrontation culture. The antagonistic effect of B18 was the best, with an inhibition rate of 85.4% (Table 1).
Morphological characteristics: Strain B18 was positive for Gram staining. The cell is a long rod in shape, with a size of 0.7–0.8 μm × 2.0–3.0 μm; endophytic spores formed are endophytic to secondary, elliptic or columnar, and slightly expanded (
The control effects after inoculation with the B18 strain for 30 days in pot experiments are shown in Table 3, presenting symptoms such as those depicted in
The control effects at 15 days after spraying treatment are shown in Table 4. The cure rate of the original was up to 96%. Better than 80% control was observed with concentrations of 108, 2 × 106, 5 × 105 and 2.5 × 105 cfu/ml, and the difference was not significant. The control effect of the concentration of 1.25 × 105 cfu/ml was significantly reduced to approximately 66%; while the disease was serious in the negative control group.
A growth curve of the pathogenic fungus (Fig. 2A) based on confrontation culture (Fig. 2B) was generated according to culture time, and the control group mycelium continued to increase in size. The colour of the mycelium and culture medium of the pathogenic fungus gradually deepened and darkened due to a control effect. Under an optical microscope, the morphology of the marginal mycelium of the inhibited colony was destroyed, mainly manifesting as swelling of the mycelium and deformity of the conidium. In contrast, the mycelia of the control group were vigorous and well developed (Fig. 2C). The results of the spore germination test are shown in Table 5. The B18 sterile culture filtrate demonstrated a distinct inhibitory effect on germination of the pathogen (Fig. 2D), whereas the spores in the control group were able to germinate normally. The germination rate was 77%.
The beneficial bacteria of healthy plant tissue play an important role in controlling plant diseases. In this study, 34 strains of bacteria were isolated from the bark of healthy
The five strains exhibited more than 50% inhibition of the growth of
Several studies have shown that
This research was supported financially by National Natural Science Foundation of China (31700568) and China Postdoctoral Science Foundation (2016M602705).