1. Introduction
Welsh onion (
Allium fistulosum L.) is an important vegetable crop in Vietnam and other Asian countries, such as China and India, which are by far the largest onion producers in the world. This vegetable is mainly grown for its green leaves, which are widely used in Asian cuisine. Welsh onion, which is intensively cultivated throughout the year, is unfortunately also prone to bacterial infections. Bacterial leaf blight, caused by
Xanthomonas axonopodis pv.
allii (Xaa), is distributed worldwide and is one of the most important diseases for several onion species. Epidemic outbreaks have already been reported in several
Allium spp. [
1]. The bacterium infects all stages of the plant. Primary inoculum commonly comes from infected seeds, seedlings and plant debris [
2,
3]. The bacterium penetrates through the leaf stomata and disease rapidly progresses during periods of high humidity. Consequently, during wet and rainy seasons, the leaves rapidly collapse and the plant eventually dies [
4]. Therefore, crop yields are significantly reduced, which has been reported for many growing areas worldwide [
1,
5,
6,
7].
To manage this bacterial disease, farmers mainly rely on chemical bactericides such as copper compounds and antibiotics, which can have negative effects on beneficial microbial communities and promote the development of resistant strains. In addition, overuse of antibiotics in agriculture would promote the transmission of antibiotic resistance genes from plant bacterial pathogens to human pathogens [
8,
9,
10]. Moreover, chemical residues gradually build up in the environment and the food chain, which is undesirable [
11,
12].
Currently, biocontrol with bacteriophages is considered a promising alternative strategy for bacterial disease management [
13,
14]. The application of phages has been shown to be successful in controlling both soilborne [
15,
16] and airborne plant diseases [
17,
18,
19]. For instance, Lang et al. [
20] showed that biweekly phage applications could reduce disease severity symptoms of bacterial leaf blight on onion equal to or better than weekly applications of copper hydroxide plus mancozeb.
This study aims at controlling bacterial leaf blight on Welsh onion using phage biocontrol in Vietnam. We screened phages isolated from Xaa infected onion leaves and we selected three promising ones based on host range and plaque/halo size. These were then characterized by whole genome sequencing. The selected lytic phages were evaluated for their potential of controlling X. axonopodis pv. allii both in vitro and in greenhouse and field conditions.
4. Materials and Methods
4.1. Host Bacterium Isolation and Phage Manipulations
Infected leaf blight samples were collected from different provinces in the Mekong Delta and were used for isolation of Xaa. After surface sterilization, the infected leaves were inspected for bacterial oozing under a microscope. Next, one drop of suspension containing bacterial ooze was plated on King’s B agar medium and streaked for individual colonies and then incubated for 48 h at 25 °C. Single colonies were picked up, then tested for their pathogenicity by spraying bacterial suspensions (OD600nm = 0.3; corresponding to 3 × 108 CFU/mL) on Welsh onion and scoring symptom development and used as a representative host for further experiments.
For phage isolation, the infected onion leaves were chopped and crushed using mortar and pestle. The homogenized leaves were mixed with an equal volume of water and subsequently centrifuged at 6000 rpm. The supernatant was transferred and treated with chloroform at a concentration of 3–5% and incubated for 5 min before another round of centrifugation (6000 rpm, 5 min). Phages were visualized by mixing 100 µL of this supernatant with 10 mL of 0.8% King’s B soft agar containing a bacterial suspension (the bacterial strain isolated from the same leaf sample) and pouring it on an agar plate. After 24 h incubation, individual plaques were picked up with a sterile toothpick and streaked on a fresh bacterial lawn with a cotton swab. A single plaque was harvested in water as a phage suspension and stored at 4 °C.
Phages were routinely amplified by cotton swab streaking of this phage stock on fresh soft King’s B agar plates containing host bacterium. After 24 h incubation at 25 °C, water was added for harvesting the phages, after excluding the remaining bacterial cells by centrifugation and chloroform treatment.
The host range of the different phages was tested by spotting 5 µL of phage suspension on a bacterial lawn containing the test strain. In short, these bacterial lawns of each test strain were prepared by adding 100 µL of bacterial suspension at an OD600nm of 0.3 to 10 mL of King’s B soft agar (0.8%). Plates were incubated for 24 h at room temperature. The lysis zone was recorded for all strains to determine the hot range.
Promising phages with broad host ranges were selected for further experiments. Plaque formation for the different phages was compared by plating 5 × 102 pfu/mL in triplicate (one plate per replicate). The diameter of the halos around the plaques was recorded 24, 48 and 72 h after infection (incubation at room temperature in darkness).
4.2. Phage Characterization
The virion morphology was determined by TEM analysis. The phage suspension was first allowed to adsorb for 3 min on carbon and formvar-coated copper–palladium grids, which were then rinsed several times with water. Next, the grids were negatively stained with aqueous 0.5% uranyl acetate and the excess fluid was removed with filter paper. Observations and photographs were made with a Philips CM10 transmission electron microscope (TEM) (Eindhoven, The Netherlands), operating at 80 kV. Micrograph films were developed and digitally acquired at high resolution with a D800 Nikon camera. Finally, the images were trimmed and adjusted for brightness and contrast using the Fiji software [
36].
Next, to analyze the genome of these phages, 10
10 pfu/mL phage suspensions were used for DNA phenol-chloroform extraction [
37]. A sequencing library was then obtained using the Illumina Nextera flex kit and sequenced on an Illumina MiniSeq. The reads were assembled and annotated with RAST [
38] using the PATRIC platform [
39]. Phage sequences were compared to homologous phage sequences on NCBI using BLASTn [
40]. Protein sequences were manually verified using BLASTp and Artemis [
41] was used to polish the genbank files. Genome maps were drawn using EasyFig [
42]. The comparison between the phage genomes was performed by mapping the reads on the reference genome using Bowtie2 [
43] and variants were called using iVar [
44]. The data were visualized using an integrated genome viewer [
45].
4.3. Evaluation of the Efficacy of Phage Treatment in Greenhouse Conditions
The first greenhouse experiment was used to compare the efficacy of different phage treatments for controlling bacterial leaf blight on Welsh onion. The experiment was a completely randomized design with six treatments (three monophage treatments, one treatment with a cocktail of three phages (containing 1/3rd of each phage), a control treatment without phage application and a treatment with oxolinic acid). Each treatment included five replicates, each in a different pot.
Thirty-day-old Welsh onion plants were used for experiments. The phage suspension (108 PFU/mL) of each treatment was sprayed over the leaves (25 mL/pot). After 2 h, plants were inoculated with a phage-susceptible Xaa strain (XaaBL11, OD600nm of 0.3) on the leaf surface using a hand sprayer (again 25 mL/pot). The pots were covered with plastic bags for 24 h in darkness, at 25 °C and 100% humidity in a growth chamber. After 24 h, the plastic bags were removed, and the plants were grown in greenhouse conditions. The percentage of infected leaf area was recorded at several time points until the control treatment was almost fully infected. In addition, the bacteriophage density on the leaf surface of differently treated plants was determined at 0, 48 and 72 h after pathogen inoculation (three leaves per plant; three plants per treatment).
In the second greenhouse experiment, the optimal phage titer was determined using the same experimental setup. Pots were arranged completely randomly for five treatment conditions (four different phage titers, i.e., 105, 106, 107 and 108 pfu/mL and one control treatment without phage application).
4.4. Evaluation of the Efficacy of Phage Biocontrol in Field Conditions
A field trial was conducted in a 500 m2 Welsh onion field in the An Giang province. This experiment was set up as a completely randomized block design, in which four treatments were evaluated: (1) control treatment without the application of phage or chemicals, (2) phage Φ31, (3) a cocktail of three phages (Φ16, Φ17 and Φ31) and (4) bactericide (oxolinic acid) treatment. There were four replicates per treatment and the treatments were applied 30 days after planting the onion seedlings.
Phages were applied (phage Φ31 or the three-phage cocktail) by spraying a phage suspension (108 pfu/mL) at 1 L/25 m2 one hour before pathogen inoculation and at 3, 8 and 13 days after pathogen inoculation (dai). Bactericide treatment consisted of Starner 20 WP (20 g oxolinic acid/16 L water) at 1 L/25 m2, which was first applied when the percentage infection was around 5–10% (3 dai) and which was repeated at 8 and 13 dai. Pathogen inoculation was done by spraying XaaBL11 (OD600nm = 0.15, corresponding to 108 CFU/mL; 1 L/25 m2) on the leaf surface 30 days after planting. The disease index was recorded at 5, 9 and 15 dai. The actual yield (the whole plant) and commercial yield (without infected leaves) were recorded as well.
4.5. Statistical Analyses
All statistical analyses were carried out in MSTAT-C (Statistical software developed by the Crop and Soil Science Department of Michigan State University, USA). First, a Shapiro–Wilk test was used to test for normality of the experimental data with or without transformation by taking the square root of each datapoint. Next, Bartlett’s test was run to determine the equality of variances. Finally, means were separated pairwise using Duncan’s or Tukey’s range test, resulting in a significance level letter report.
5. Conclusions
During this research, ten bacteriophages were isolated from twelve bacterial blight-infected onion leaf samples and three promising phages Φ16, Φ17A and Φ31 were selected based on their host range and plaque/halo diameter. The three podoviruses are lytic phages based on whole-genome sequencing and form a new phage species. Phage Φ31 shows higher disease reduction compared to phage Φ16, Φ17A and phage cocktail in greenhouse conditions, and the optimal phage titer for disease control lies at 107 and 108 pfu/mL as these concentrations performed equally well. During field trials, phage Φ31 reduced disease symptoms equally compared to the bactericide Starner and provided a significant increase in crop yield.