Biological control of poplar anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.

Poplar anthracnose is one of the most serious diseases caused by the fungal pathogen Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. Biocontrol is an efficient green way for the disease control, and numerous researches have focused on exploring the potential biocontrol bacteria strains against C. gloeosporioides. In this study, antifungal activities against C. gloeosporioides of 108 rhizosphere soil isolates from healthy polar plants were investigated in vitro by the dual culture assay. The results suggested that strain ZSH-1 showed the highest level of antifungal activity, as it inhibited C. gloeosporioides at a distance of 10.00 mm. Based on the morphological, physiological-biochemical characteristics, and phylogeny analysis, strain ZSH-1 was identified as Bacillus subtilis. The sterile culture filtrate, crude protein, and crude lipopeptide extracts from the culture filtrate, and volatile compound(s) of ZSH-1 displayed a strong antagonism towards 7 fungal phytopathogens (C. gloeosporioides, Fusarium oxysporum, Alternaria tenuissima, Cytospora chrysosperma, Botryosphaeria dothidea, Mucor sp., and Absidia sp.), with inhibition rates ranging from 44.0 to 89.1%, 26.7 to 85.4%, 11.6 to 89.7%, and 7.8 to 63.2%, respectively. Moreover, ZSH-1 exhibited cell wall-degrading traits by producing 3 lytic enzymes (cellulose, β-1,3-glucanase, and protease). Finally, the greenhouse studies also revealed that strain ZSH-1 had a 47.6% (12 days) efficacy in controlling poplar anthracnose when compared with the control. In concluding, obtained results demonstrate the potential biocontrol effect of B. subtilis ZSH-1, and it can be used as a promising biocontrol agent against poplar anthracnose and other fungal phytopathogens.


Background
Poplar is one of the most important trees in China where it has a major role in farmland shelterbelts, soil conservation, and environmental protection. Poplar anthracnose, as a serious branch and leaf disease of poplar, is mainly caused by Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. The disease generally occurs in various poplar cultivated areas in the north and south of the country (Li et al. 2012). Currently, applications of chemical fungicides are still the effective means to prevent the infection and dispersal of poplar anthracnose (Song et al. 2016). However, the excessive and long-term use of chemical fungicides has led to the evolution of drug resistance of pathogenic fungi. Even more, chemical fungicides result in serious environmental pollution because of the difficult degradation and non-target toxicity (Furuya et al. 2011). Therefore, more and more researches have focused on developing and applying alternative methods that are less reliant on fungicides and more environmentally friendly. Biological control is high efficiency and safer for the environment to be used in disease management (Shafi et al. 2017).
Many microorganisms have been used for the control of fungal phytopathogens. Among them, the Bacillus genus is recognized as a promising group with a wide distribution in soils. Due to their strong antagonistic activity, broad inhibitory spectrum, and high viability, members of the Bacillus species have been effectively used in controlling soil-borne, air-borne, and postharvest diseases (Pérez-García et al. 2011;Shafi et al. 2017). Antagonistic strains control fungal phytopathogens through several mechanisms, such as competition, mycoparasitism, the production of antibiotics, and the induction of defense responses (Guo et al. 2019;Jinal and Amaresan 2020). Production of a variety of metabolites is an important mechanism by which the Bacillus genus exerts biocontrol. These inhibitory metabolites are mainly including lipopeptides, proteins, and volatile compound(s) (Baysal et al. 2013;Shafi et al. 2017). In addition, lysates of antagonistic bacteria have also been reported to possess antifungal activity (Yu et al. 2013).
The objectives of this study were to explore the potential biocontrol antagonistic bacteria from rhizosphere soil against the poplar anthracnose pathogen C. gloeosporioides and to identify the strain, characterize the main ingredients for antagonistic, and clarify the cell walldegrading traits.

Isolation of rhizobacteria
Rhizobacteria were isolated from the rhizosphere soil of healthy poplar plants in Haidian and Changping districts of Beijing City, China. Five grams of soil sample was transferred to 45 ml of sterilized water and shaken at 200 rpm for 20 min prior to use (Kurabachew and Wydra 2013). Gradient dilutions of the soil suspension were spread onto Luria-Bertani (LB) agar plates, and the plates were incubated at 30°C in darkness. A single bacterial colony that is well separated from the other colonies were selected and re-streaked onto a new LB agar plate with an inoculating loop. Then, the plate was incubated at 30°C for 24 h, and then stored at − 80°C in LB with 30% glycerol (v/v).
In vitro screening of rhizobacteria against C. gloeosporioides The rhizobacteria isolates were screened for antifungal activity against C. gloeosporioides using the dual culture assay described by Huang et al. (2014). The inhibition distance was defined as the width of the inhibition zone between the bacterial and fungal strains. The strain showed the highest antifungal activity was selected for further study.
Morphology, culture conditions, and physiologicalbiochemical characteristics of selected strain Morphology, culture conditions, and physiologicalbiochemical characteristics were performed according to previously published methods of Dong et al. (2001) and Furuya et al. (2011).

Phylogenetic analysis of selected strain
The 16S rRNA gene was amplified and sequenced, using a single intact colony of selected strain according to a previously described method (Huang et al. 2014). The primers 63f (5′-CAGGCCTAACACATGCAAGTC-3′) and 1387r (5′-GGGCGGWGTGTACAAGGC-3′) were used for amplification. Polymerase chain reaction (PCR) conditions were as follows: 94°C for 4 min, 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, and a final extension at 72°C for 10 min. The PCR products were sequenced by Invitrogen Corporation (Beijing, China). The sequences were blasted in the GenBank database, using a blastn search and aligned using Clustal W (ver. 1.82). Phylogenetic analysis was performed using MEGA (ver. 5.0) and a neighbor-joining phylogenetic tree was constructed by bootstrap analysis with 1000 replicates.

Evaluation of the effects of sterile culture filtrate
The strain selected from the in vitro test was grown in 100 ml modified medium no. 3 (g L −1 ; glucose 10.0, peptone 5.0, soybean meal 5.0, KH 2 PO 4 1.0, MgSO 4 ·7H 2 O 0.5, NH 4 Cl 3.0, Na 2 HPO 4 1.0, and yeast extract 0.5; pH 7.0-7.2) in a 500-ml Erlenmeyer flask at 28°C with 200 rpm. Culture broth was incubated for 3 days and centrifuged with 10,000 rpm at 4°C for 20 min. Sterile culture filtrate (SCF) was obtained by passing the supernatant through a sterile membrane filter (0.22 μm; Pall, Ann Arbor, USA). The antifungal effects of SCF against 7 fungal phytopathogens were assayed by measuring the mycelial growth inhibition rates (Song et al. 2012). Briefly, 2 ml of SCF was mixed with 20 ml of PDA at 40-45°C in a culture plate; no SCF was added for the control. After the medium was solidified, freshly growing fungal mycelial plugs (6 mm) were collected and inoculated onto the center of the plate using a sterile steel borer. The plates were incubated at 28°C. The diameters of the fungal colonies were measured when those of the control colonies covered more than three quarters of the diameter of the dish according to the crisscross method. All experiments were repeated 3 times. The percentage Huang et al. Egyptian Journal of Biological Pest Control (2020)  of growth inhibition was calculated using the following formula (Yang et al. 2011): where P is the inhibitory rate, C is the diameter of a control colony, T is the diameter of a treated colony, and d is the diameter of the mycelial plug.
Evaluation of the effects of crude protein and crude lipopeptide extracts Crude protein and crude lipopeptide extracts were obtained respectively from SCF according to a previously described method (Huang et al. 2014). Then, the extracts were sterilized by passing through a sterile membrane filter (0.22 μm; Pall). The antifungal effects of crude protein and crude lipopeptide extracts against 7 fungal phytopathogens were assayed by measuring the mycelial growth inhibition rates using the procedure described above.

Evaluation of the effects of volatile compound(s)
The antifungal effects of the volatile compound(s) (VOCs) against the 7 fungal phytopathogens were tested by co-cultivating the fungi and bacteria on twocompartment plates (Yuan et al. 2012). One half of each plate was filled with PDA, while the other half contained TYB medium. A 6-mm diameter fungal plug was placed on the PDA side, and the candidate antagonistic strain was inoculated on the other side 24 h later. Plates not inoculated with the test strain served as the controls. Then, the plates were sealed with parafilm and incubated at 28°C. The diameters of fungal colonies and percentages of growth inhibition were measured as described above. All experiments were repeated three times.

Characterization of cell wall-degrading traits
The cellulase activity and β-1, 3-glucanase activity of the test strain was determined, using a method described previously (Essghaier et al. 2009). The activities of β-1,3glucanase, chitinase, protease, and lipase were determined using aniline blue pachyman agar plate, 0.2% colloidal chitin plate, skim milk agar plate, and 1% Tween 80 agar plate, respectively (Dong et al. 2001;Gao et al. 2009;Ren et al. 2013). Briefly, a single colony of test strain collected from an overnight culture was seeded onto the center of the plates; then, the plates were monitored for the appearance of a clear zone around the developing bacterial colonies at 28°C.

Evaluation of biocontrol activity of the candidate strain under greenhouse conditions
To evaluate the biocontrol activity of the candidate strain towards poplar anthracnose caused by C. gloeosporioides, a pot assay was performed using 1-year-old poplar seedlings (Populus ×euramericana cv. '74/76') with 7-10 expanded leaves (Huang et al. 2014). Each treatment consisted of 15 plants with 3 replicates (5 plants per replicate). Three treatments were set up as follows: SZ, the candidate strain and C. gloeosporioides sprayed on leaves at the same time; CK1, only sterile distilled water sprayed on leaves; and CK2, leaves only sprayed with C. gloeosporioides. For the phytopathogen inoculation, a spore suspension (1.0 × 10 5 spores ml −1 ) of C. gloeosporioides was sprayed on both sides of the leaves (2 ml per leaf) using an artist's airbrush (YD12-F111, Yudi, Zhejiang, China). Sterile distilled water and the suspension (1.0 × 10 9 cfu ml −1 ) of the candidate strain were sprayed on the leaves using the same method as for C. gloeosporioides. The diseased leaves rate (DLR), disease severity (DS), disease index (DI), and greenhouse control efficacy (GCE) were investigated 12 days after C. gloeosporioides inoculation, respectively. The DS was expressed as a percentage of lesion area over the total surface area per leaf and divided into 6 ratings: 0, no lesion; 1, lesion area ≤ 5%; 2, lesion area 5-25%; 3, lesion area 25-45%; 4, lesion area 45-65%; and 5, lesion area > 65%. The DLR, DI, and GCE were calculated using the following formulas:

Statistical analysis
The mean values and standard deviations were calculated, and statistically compared using analysis of variance (ANOVA) and Duncan's multiple range tests (p ≤ 0.05) using SPSS software version 20.0 (SPSS Inc., Chicago, Illinois).

Isolation and screening of antifungal bacteria in vitro
A total of 108 bacterial isolates were obtained from 5 rhizosphere soil samples, and 15 of them showed antifungal activity in vitro. Of the 15 bacterial strains, ZSH-1 showed the highest level of antifungal activity, as it inhibited C. gloeosporioides at a distance of 10.00 mm ( Morphology, culture conditions, and physiologicalbiochemical characteristics of strain ZSH-1 The colonies of strain ZSH-1 grown on LB agar medium were off-white to gray-yellow, oblong, smooth, moist, and slightly convex and had an entire margin. The cells were rod-shaped, 0.5-0.8 μm wide, and 1.2-2.5 μm long. Strain ZSH-1 can grow at temperatures ranging from 10 to 50°C, in pH values ranging from 5 to 10, and in NaCl concentrations ranging from 0.2 to 10% (w/v) ( Table 2). The morphological and physiological-biochemical characteristics of strain ZSH-1 were found to be in complete accordance with B. subtilis, using interpreting software cluster analysis (Todorova and Kozhuharova 2010).

Phylogenetic analysis of strain ZSH-1
Based on the morphological and physiologicalbiochemical characteristics, strain ZSH-1 was identified as B. subtilis. To precisely determine the species of strain ZSH-1, 1281 bp (GenBank accession KM114000.1) of the 16S rDNA sequence was aligned with those of the most closely related B. subtilis by the Clustalx program. The 16S rDNA sequence of Escherichia coli U5/41 (GenBank accession NR_024570.1) was used as an out group. Bootstrap analysis showed strain ZSH-1 was grouped with B. subtilis (GenBank accession HE582781.1) with a sequence similarity of 89% (Fig. 1). The phylogenetic analysis of 16S rDNA sequence revealed that strain ZSH-1 belongs to B. subtilis.

Antifungal activities of sterile culture filtrate
The SCF of ZSH-1 exhibited strong antifungal activities against 7 fungal phytopathogens (C. gloeosporioides, F.  oxysporum, A. tenuissima, C. chrysosperma, B. dothidea, Mucor sp., and Absidia sp.) belonging to various taxonomic groups. The inhibition rates ranged from 44.0 to 89.1% (Fig. 2a). Moreover, when the colonies of C. gloeosporioides were measured every day, the diameters were significantly reduced by treatment with SCF of ZSH-1 than the control. The inhibition rate was gradually increased over time from day 1 to day 6 (Fig. 2b). The antifungal activity of sterile culture filtrate was also stable after treatment at temperatures from 40°C to 100°C for 30 min, or autoclaved at 121°C for 20 min, as determined by inhibition of mycelial growth of C. gloeosporioides (Fig. S1). Numerous studies have reported that the culture filtrates of Bacillus genus have a broad spectrum of activities. The culture filtrate of B. safensis B21 showed antifungal activity against Magnaporthe oryzae, which causes rice blast disease (Rong et al. 2020). Dong et al. (2019) reported that culture filtrate of B. amyloliquefaciens Rdx5 strongly inhibited the growth of M. oryzae. These indicated the great potential of Bacillus genus in control plant disease.
Antifungal activities of crude protein and crude lipopeptide extracts from sterile culture filtrate The 7 fungal phytopathogens that were strongly inhibited by the SCF were used to determine the antifungal activities of the crude protein extracts and crude lipopeptide extracts from the SCF. The crude protein and crude lipopeptide extracts strongly inhibited the growth of all 7 fungal phytopathogens, with inhibition rates in the range of 26.7 to 85.4% and 11.6 to 89.7%, respectively (Fig. 2c)  . Rong et al. (2020) reported that B. safensis B21 produced iturin A2 and A6 that inhibited the growth of hyphae of M. oryzae by changing membrane permeability. Ren et al. (2019) reported that a novel protein obtained from the culture filtrate of B. subtilis XB-1 inhibited the mycelial growth of M. fructicola.

Antifungal activities of volatile compound(s)
Strain ZSH-1 produced antifungal VOCs that reduced the mycelial growth of the 7 fungal phytopathogens with inhibition rates in the range of 7.8 to 63.2% (Fig.  2a). The result consists with previous findings that VOCs produced by Bacillus genus had antifungal activities. Yuan et al. (2012) reported that the VOCs produced by B. amyloliquefaciens NJN-6 inhibited the growth and spore germination of F. oxysporum. Currently, the VOCs produced by Bacillus genus are generally grouped into amides, aldehydes, alcohols, ketones, and phenols. Zhang et al. (2013) reported that O-anisaldehyde is one of the most abundant VOCs produced by B. atrophaeus CAB-1 with the highest inhibitory effect on the mycelial growth of B. cinerea. Three VOCs (2nonanone, β-benzeneethanamine, and 2-decanone) produced by B. pumilus and B. thuringiensis had great inhibitory effects on C. gloeosporioides (Zheng et al. 2013). VOCs of ZSH-1 probably contain many kinds of antifungal components, and different fungi have different sensitivities to the antifungal action of a same component, so there is difference in the inhibitory effects of VOCs from ZSH-1 against different fungal phytopathogens. Moreover, when the colony diameters of C. gloeosporioides were measured every 12 h, the mycelial growth was significantly inhibited after 24 h by the VOCs produced by strain ZSH-1, and there was a gradual increase in the inhibition rate from 0 to 60 h (Fig.  2d). This result is in agreement with the study that the inhibitory effect of volatiles produced by B. amyloliquefaciens NJN-6 gradually increased with treatment time (Yuan et al. 2012). The increasing inhibition rate may be due to the creation of a hypoxic environment by the antifungal bacteria (Boukaew et al. 2013).

Cell wall-degrading traits
The cell wall-degrading traits of strain ZSH-1 was determined by the activities of 5 lytic enzymes (cellulose, β-1, 3-glucanase, chitinase, protease, and lipase). When the colony of ZSH-1 was cultured after 4 days, clear zones around the colonies on different culture plates were observed. This indicated that ZSH-1 have activities of cellulase (Fig. 3a), β-1,3-glucanase (Fig. 3b), and protease (Fig. 3c). No clear zone was found in the culture plates containing chitinase or lipase (not shown). Several cell wall-degrading enzymes such as chitinase, β-1,3-glucanase, cellulase, protease, and lipase are produced by Bacillus species which are involved in the antifungal activity against fungal phytopathogens. The enzymes mainly play an important role in the decomposition of fungal cell walls and subsequent cell death (Tseng et al. 2008). Ren et al. (2013) reported that B. pumilus JK-SX001 produced cellulase and protease. Dong et al. (2019) demonstrated that cellulase and protease were secreted by B. amyloliquefaciens Rdx5. In the present study, the cell walldegrading traits of strain ZSH-1 may be one reason that this bacterium is equally effective against different fungal phytopathogens with variable cell wall compositions. At present, the main disease control measure for poplar anthracnose is done by applying with fungicides (Song et al. 2016). Many Bacillus species have been used as biocontrol agents for C. gloeosporioides that causes plant anthracnose (Fira et al. 2018;Guerrero-Barajas et al. 2020). However, used Bacillus species for control poplar anthracnose that caused by C. gloeosporioides is rarely reported. In the previous study, B. atrophaeus XW2 was used as an effective biocontrol agent against C. gloeosporioides, it had a 49.1% efficacy in controlling poplar anthracnose under controlled greenhouse conditions (Huang et al. 2014). In the present study, strain ZSH-1 obviously inhibited the development of poplar anthracnose in greenhouse experiments (Table 3). After 12 days inoculation, the diseased leaves rate and the disease index of plants inoculated with ZSH-1 were significantly lower than the values of plants un-inoculated with ZSH-1. ZSH-1 had 47.6% control effect against poplar anthracnose that was caused by C. gloeosporioides. The leaves treated with sterile distilled water had no disease symptoms. The pot experiments in greenhouse indicated that strain ZSH-1 has a good biocontrol efficacy against C. gloeosporioides in vivo and can be used as a potential biocontrol agent for more study. However, biocontrol of the poplar anthracnose in the greenhouse may not effectively control the disease in the field. Additional studies about the biological characteristics of strain ZSH-1, such as colonization, survival ability, and product formulation, are needed to confirm the biocontrol effect of strain ZSH-1 in the field (Liu et al. 2019). Meanwhile, the abilities of strain ZSH-1 to promote plant growth and activate induced systemic resistance (ISR) of plant in the field should also be studied in the future (Lyu et al. 2019;Tiwari et al. 2019, andJinal andAmaresan 2020).

Conclusion
In the present study, an antagonistic bacteria B. subtilis ZSH-1 against C. gloeosporioides from the rhizosphere soil of poplar was evaluated. The sterile culture filtrate, crude protein, and crude lipopeptide extracts from the culture filtrate and volatile compound(s) of ZSH-1 strongly inhibited the fungal phytopathogens growth in vitro. Moreover, the greenhouse trial revealed that strain ZSH-1 had a good biocontrol efficacy in controlling C. gloeosporioides in vivo. Therefore, the results proved that B. subtilis ZSH-1 can be used as a promising biocontrol agent against C. gloeosporioides and other fungal phytopathogens.
Additional file 1: Fig. S1. Effect of temperature on antifungal activity of sterile culture filtrate of strain ZSH-1.   Values with the different letter within the same column are significantly different at P ≤ 0.05 according to Duncan's test. Numbers followed by the "±" are standard errors (SE). "\" indicates the data is incalculable revising the paper scientifically and checking analysis. All authors read and approved the final manuscript.