Isolation and Identification of Bacteriophage Effective on Xanthomonas Campestris Strains for Their Biocontrol

The genus Xanthomonas belongs to the Xanthomonadaceae family and the Gammaproteobacteria class, which includes short rod-shaped gram-negative bacteria that cause disease in more than 400 different plant hosts and are the most important pathogen in a wide range of plants, which results in a decrease in productivity in the agricultural industry.Pathogenic bacteria in plants including Xanthomonas campestris pv. campestris are one of the most important factors that reduce the productivity of agricultural products and are responsible for major economic losses in the agricultural industry. This bacterial strain is pathogenic in a wide range of plants such as rice, wheat, citrus fruits, tomatoes, peppers, cabbage, melons, bananas, and seeds such as beans. The methods of dealing with the disease that has been used against them so far involve environmental damage, the accumulation of toxins in the soil, and bacterial resistance. Therefore, it seems that more effective methods are needed. Bacteriophages, Viruses infect bacteria, which are exclusive to their host attack, and as new, safe, and effective inhibitory agents against plant pathogenic bacteria, they have been much considered in recent studies.In the studies conducted on isolated phages effective against Xanthomonas bacteria, promising results have been obtained in the field of plant disease management in laboratory and field conditions. In this regard, the identification and complete knowledge of the biological characteristics of phages effective against this bacterium is very crucial for the development of effective biological control products.

In the present study, isolation and identification of lytic bacteriophage effective on strains of Xanthomonas campestris bacteria were investigated to inhibit two forms of planktonic and biofilm bacteria so that it can be used in the future to fight the contamination of agricultural products with this pathogenic bacterium. Under sterile conditions and with a sterile loop, a single plaque was completely removed from the selective plate containing phage plaques and autoclaved in 5 ml of YMB medium and 10 μl of fresh suspension of the bacterial strain X. campestris DSM 1706 was added and incubated for 24 hours in the incubator was incubated at a temperature of 28 degrees Celsius and aeration at 150 rpm. Then the solution was centrifuged for 10 minutes at a speed of 6000 rpm and the supernatant was filtered with a 0.45 micron needle filter.Lytic phage is effective on the strain Xanthomonas campestris pv. campestris DSM1706. It was separated from the water of the Karun River by the two-layer agar method. The phage host range to the staining method was checked. Then, phage morphology through transmission electron microscopy (TEM) imaging, phage stability against pH and different temperatures, one-step proliferation curve, and the effect of phage on removing and inhibiting bacterial biofilm were investigated.

In this study, clear phage plaques, caused by the proliferation of lysing phages of the desired strain, were observed. Phage isolated on the morphological characteristic was based on the tectonic family. In addition to the strain Xanthomonas campestris pv. campestris DSM 1706, it had a lytic effect on three other pathogenic isolates obtained from infected cabbage fields including SAM 4209, SAM 4211, and SAM 4212. This phage had high stability in the temperature range of 20 °C to 50 °C and was inactivated at -70 °C. It also had good lytic activity in the range of pH 5 to 10 and became inactive at pH 3. According to the one-step proliferation curve, the phage proliferation cycle lasts about 70 minutes which includes a 25–30-minute incubation phase and a 40-minute phage release phase. Phage showed an 86% inhibitory effect and 93% elimination effect on the biofilm of this bacterium.In the present study, phage stability against different temperatures and acidity was studied as two main factors. The results showed that the phage has good lytic activity in acidity between 5 and 9, its performance decreases in alkaline acidity more than 9 and it becomes inactive in acidity 3 and does not show any lytic activity against the target bacteria. The best performance and the highest lytic activity were observed at an acidity of about 7.

Since the first discovery of phages in this study, it has been proven that phages as a biological control strategy are promising alternatives with many advantages for agricultural chemicals and antibiotics. Based on the results of this research, the obtained lytic phage has stability in the spectrum in laboratory conditions (a wide range of pH and temperatures) and was able to lyse the host bacteria in planktonic cell form and inhibit and remove the bacterial biofilm with a high percentage. As a result, it can be a biological agent with high potential in controlling plant diseases and replacing combat methods as a common and suitable candidate for phage therapy.In several studies in this field, it has been shown that the use of different compounds mixed with solutions containing phage increases the persistence of phage on leaf surfaces. For example, in one study, the use of skim milk and this combination led to improved control of tomato bacterial spot disease compared to standard treatment with chemical bactericides.In line with this study, it is suggested to investigate the effect of phage on other pathogenic strains of Xanthomonas bacteria and also on their biofilm in different ways. Among other things, specific lytic bacteriophages of Xanthomonas bacteria can be used individually or in combination with other isolated bacteriophages along with antibiotics or other control agents such as copper compounds in preventive and therapeutic measures as phage therapy against bacterial infections.