Antiviral activity of Pseudomonas putida A3 strain crude extract against TMV in Tobacco

Antiviral activity of Pseudomonas putida A3 strain ethyl acetate crude extracts against Tobacco mosaic virus in Tobacco
Jinguang Yang, Cong Guo, Xilun Zhai, Lili Shen, Yumei Qian, Fenglong Wang* (Open Project Program of Key Laboratory of Tobacco Pest Monitoring Controlling & Integrated Management，State Tobacco Monopoly Bureau, Qingdao,266101, China)
Abstract:
Keyword: Antiviral activity; Pseudomonas putida; Tobacco mosaic virus; crude extract
Introduction
Tobacco mosaic virus(TMV) is one of the most devastating virus that infecting many crops worldwide including vegetables, ornamentals, legumes, and other important crops, especially tobacco and other members of the family Solanaceae. Since its first description during the late 19th century, and the diseases of TMV infecting still caused heavy losses of agriculture world-widely. TMV is endemic in all tobacco planting areas of China where it causes important virus diseases of field-grown tobacco. Because of its epidemiological features, the only effective control of TMV is through planting resistance varieties, and the conventional chemical control often showed less effective in the TMV diseases control. In practice, over-appling of chemical anti-viral agents led to serious pesticide residue in crops and environmental problems. Thus, there is a pressing need for effective anti-viral natural products against TMV to address these problems and concerns.
Many rhizosphere microbe showed anti-viral activity, which secondary metabolites were possible anti-viral materials and biological agents. A Pseudomonas putida A3 strain was screened, which has been shown to be useful against TMV in tobacco. Therefore, the present investigation was carried out to assess the antiviral effects of P. putida A3 strain ethyl acetate crude extracts in plant and cell levels.
Material and Methods
Isolation of bacteria and preparation of extracts
The bacterial isolates were screened for anti-viral against TMV. Of 126 bacterial isolates, 12 bacterial strains were found to possess antiviral activity against TMV. And P. putida A3 was shown highest antiviral activity through half leaf methods (data bot shown) . P. putida A3 was grown in liquid Luria-Bertani broth (LB) with ampicillin (100 mg/L) . Cultures were grown with shaking (210 rpm) for 2 days at 37℃.
The P. putida A3 cultures were extracted with 3 volume ethyl acetate in the shade at room temperature (25-33℃）for 48 h. The extract was concentrated under reduced pressure of 22-26 mmHg at 45℃, and the residue obtained was dissolved in normal PBS and adjusted to stock concentration (1.0g/L) prior to application to the cells and seeding, and stored at 4℃.
Tobacco seeding and antiviral activity
Nicotiana tabacum cv. 三生烟plants were grown under standard greenhouse conditions at
24-26 ℃, with a 14 h light /10 h dark cycle. The ethyl acetate crude extracts were diluted to 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.4 g/L, 0.6 g/L, 0.8 g/L, 1.0 g/L, and mixed with equal volume TMV(_) and inoculated directly onto leaves of 6-week-old Nicotiana tabacum
Tobacco protoplasts, tobacco seeding and TMV virus
N. benthamiana protoplasts were prepared from callus cells by treatment with 0.5 g of cellulysin and 0.1 g of macerase (Calbiochem) in 50 ml of protoplast incubation media (Kong et al 1997; Nagy et al 1999 and Nagy et al 2001) for 4.5 h with gentle shaking at 25°C. After incubation, the protoplasts were filtrated through a sieve set and a sterile nylon cloth. The protoplasts were washed twice with 0.5 M mannitol and once with the electroporation buffer (10 mM HEPES, 10 mM NaCl, 120 mM KCl, 4 mM CaCl2, 200 mM mannitol); 5 × 105 protoplasts were electroporated with either 2 μg of in vitro transcribed gCNV RNA or the combination of 2 μg of gCNV RNA and 1 μg of DI-72 RNA. In the two-component studies (Fig. 6 and Fig. 7), we used 4 μg of p92Y and 2 μg of DI-p33 RNA transcripts. Electroporation was done using Gene pulser II (Bio–Rad, the settings were at 0.2 kV and 0.5 μF). After electroporation, samples were kept on ice for 30 min, followed by adding 1.8 ml of protoplast culture medium (Kong et al., 1997) to each sample. Protoplasts were incubated in 35 × 10 mm petri dishes in the dark for 24–48 h at 22°C.
Results
Discussion
Reference。