Abstract:

【Objective】 The objective of this study is to determine the antimicrobial activity of volatile organic compounds (VOCs), produced from tobacco rhizosphere soil Bacillus pumilus AR03 strain, and to analyze its main components.【Method】 The antifungal effect of VOCs on the colony, mycelium growth and spore germination of Phytophthora parasitica var. nicotianae and Alternaria alternata was determined by a double Petri dish assay and cavity slide method. The control effect of VOCs on tobacco black shank and brown spot by leaf inoculation was determined in vitro. VOCs were collected by head-space solid phase microextraction (HS-SPME) and identified by gas chromatography-mass spectrometry (GC-MS). Retention index (RI) and internal standard (IS) 1-pentadecence were used for qualitative and quantitative analysis.【Result】 VOCs released from B. pumilus AR03 strain had certain inhibitory effect on the two target pathogens, which showed that the mycelium of P. parasitica grew slowly and thinned, branches increased and twisted, and broke. The mycelium of A. alternata was deformed and no conidial pedicle was produced on the mycelium. Most of the inclusions gathered together and caused the mycelium to dry and constricted. Growth of exposed fungus colonies was inhibited by VOCs, the inhibition rates of VOCs were 56.21% and 59.23%, 64.75% and 59.86%, 66.13% and 61.10%, 67.04% and 70.00%, respectively, against P. parasitica and A. alternata cultured in sealed plates for 2, 4, 6 and 8 d. When the zoospores of P. parasitica and ascospores of A. alternata exposed to these volatile components for 6 h, the germination was delayed and the growth was slow. The number of sporocyst produced by P. parasitica obviously reduced. Most conidiophores of A. alternata expanded abnormally as cystic structure, indicating the fungicidal nature of the volatiles. Moreover, VOCs could significantly inhibit the disease severities of tobacco black shank and brown spot on leaves tests. Direct fumigation for 40 h and 80 h, black shank disease incidence was 92.50% on control and 70.83% on leaves treated by VOCs, the inhibitory of spot expansion was 62.35%. Brown spot disease incidence was 88.33% on control and 60.80% on leaves treated by VOCs, lesions expanded slowly and inhibitory rate was 65.75%. SPME GC-MS analysis showed that seven components of the volatiles were identified, all of which are sesquiterpenes with C₁₅H₂₄ structure. They are dihydrocurcumene (CAS NO. 1461-02-5), (E)-β-famesene (CAS NO. 18794-84-8), γ-curcumene (CAS NO. 451-55-8), α-zingiberene (CAS NO. 495-60-3), π-bisabolene (CAS NO. 495-61-4), β-sesquiphellandrene (CAS NO. 20307-83-9) and γ-E-bisabolene (CAS NO. 53585-13-0). When AR03 was cultured for 1 d, the relative content of β-sesquiphellandrene was the highest (80.64%), followed by (E)-β-famesene and α-zingiberene, the relative content was 7.20% and 6.67%, respectively. With the extension of culturing time, the species of each component were the same, but the relative content was different. Except for dihydrocurcumene, the content of other components showed a decreasing trend, when cultured for 6 d, other ingredients decreased more than 50%, besides dihydrocurcumene keeping relatively stable. 【Conclusion】 VOCs produced by B. pumilus AR03 could develop an additive antifungal effect against fungal pathogens on tobacco. B. pumilus AR03 has potential as an important microbial resource for developing antifungal metabolites and new drugs.

Key words: Bacillus pumilus AR03, volatile organic compounds, Phytophthora parasitica var. nicotianae, Alternaria alternata, SPME GC-MS