Application of antimycoplasma properties of Bacillus subtilis surfactin in control of lime witches broom diseases

Acidic lime is one of the most important tropical and subtropical horticultural plants in south provinces of Iran, including Hormozgan, Sistan and Balouchestan, Fars and Kerman. Phytoplasma Candidatus "Phytoplasma aurantifolia" agent of lime witches broom disease (WBDL) causes a considerable loss in lime and other sensitive citruses production in Iran and other countries in Persian Gulf region. This agent is parasite of plant phloem, and therefore its control is very difficult. Recently, different methodologies, including quarantine programs, lime breeding for resistance, weed and vectors control, are suggested for control of WBDL. Surfactin is an antibiotic produced by the gram-positive bacterium Bacillus subtilis. This bacterial lipopeptid has detergent properties and has been known as an effective compound with antifungal, antibacterial, antiviral and antimycoplasma properties. In the present study, the effect of surfactin derived from Iranian native Bacillus subtilis isolates has been investigated against phytoplasma Candidatus "Phytoplasma aurantifolia". Two Handred ninety B. subtilis isolates were isolated from soil samples of fields and gardens of 5 provinces of Iran, including Golestan, Mazandaran, Gilan, Isfahan and Hormozgan. Screening of surfactant producing isolates was performed using biochemical methods, including Blood agar and Drop callopse. Isolates containing sfp gene, coding surfactin, were detected using PCR. Blood agar assays showed that 185 isolates produced different surfactants. PCR showed that 14 isolates contained the sfp gene. Drop callopse assays which detects isolates with high production of surfactin showed that four isolates produced high amont of surfactin. Production and concentration of surfactin in 14 isolates were evaluated useing HPLC. Isolates BS119M, BS116L and A28b (more than 1 gram/liter) have high surfactin production compared to other isolates and were identified as the best native isolates produced surfactin and were selected for surfactin extraction. After optimization of surfactin extraction, it was injected into witches broom phytoplasma-infected seedlings of lime by using syringe injection. To compare tetracycline effect with surfactin effect against phytoplasma activity and also to investigate probable synergistic effect of them, we planned to also treat plants with both mentioned antibiotics at the same time. Two Specific primers pairs were designed for qualitative and quantitative detection of phytoplasma in infected plants. Real Time PCR conjugated with Fluorescent SYBR® Green I dye and absolute quantification has been developed for rapid, sensitive and quantitative analysis to assess surfactin bioactivity against phytoplasma, and also to determine concentration of phytoplasma in infected seedlings before and after treatment. The results showed that the surfactin treatment had caused significant reduction of phytoplasma population in infected plants, and also the combination of surfactin and tetracycline had the highest toxicity against phytoplasma which shows their synrgic effects. Our results shopwed that combination of surfactin and tetracycline can be used as an efficient control method for WBDL agent.