Annals of Applied Biology ISSN 0003-4746 RESEARCH ARTICLE Characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40 with the ability to control wheat sharp eyespot disease Z. Jiao1†,N.Wu1†,L.Hale2,W.Wu1,D.Wu1 & Y. Guo1 1 Department of Ecology and Ecological Engineering, College of Resources and Environmental Science, China Agricultural University, Beijing 100193, China 2 Department of Environmental Sciences, University of California, Riverside, CA 92521, USA Keywords Abstract Characterisation; Pseudomonas chlororaphis subsp. aurantiaca; Rhizoctonia cereal; wheat This study details the isolation and characterisation of Pseudomonas chlororaphis sharp eyespot. subsp. aurantiaca strain Pa40, and is the first to examine P. chlororaphis for use in suppression of wheat sharp eyespot on wheat. Pa40 was isolated during Correspondence an investigation aimed to identify biocontrol agents for Rhizoctonia cerealis. Dr Yanbin Guo, Department of Ecology and Over 500 bacterial strains were isolated from the rhizosphere of infected Ecological Engineering, College of Resources and Environmental Science, China Agricultural wheat and screened for in vitro antibiosis towards R. cerealis and ability to University, Beijing 100193, China. Email: provide biocontrol in planta. Twenty-six isolates showed highly antagonistic [email protected] activity towards R. cerealis,inwhichPseudomonas spp. and Bacillus spp. were predominant members of the antagonistic community. Strain Pa40 exhibited †These authors contributed equally to this clear and consistent suppression of wheat sharp eyespot disease in a greenhouse work. study and suppression was comparable to that of chemical treatment with validamycin A. Pa40 was identified as P. chlororaphis subsp. aurantiaca by Received: 31 October 2012; revised version accepted: 12 August 2013. the Biolog identification system combined with 16S rDNA, atpD, carAand recA sequence analysis and biochemical and physiological characteristics. To doi:10.1111/aab.12068 determine broad-spectrum applicability and the specific mechanisms involved in Pa40’s pathogen suppression this strain was tested for antibiosis towards various phytopathogens and assayed for many biocontrol activities and plant- beneficial traits. Strain Pa40 inhibited the growth of 10 of 13 phytopathogenic fungal strains and six of eight phytopathogenic bacteria tested. This original work characterises HCN, protease and siderophore production in P. chlororaphis. Each of these characteristics likely contributed to Pa40’s biocontrol capabilities as well as stimulation of the hypersensitive response in tobacco and the presence of genes involved in the biosynthesis of phenazine, 2-hydroxylated phenazine and pyrrolnitrin. Introduction death of the shoot spikes. The fungus is soil-borne and transmitted from plant debris, and as propagules in the Wheat sharp eyespot, a disease caused by the soil-borne, form of resistant sclerotia. To date, there has been no fungal pathogen, Rhizoctonia cerealis, is rapidly becoming study of interactions of this fungus with soil antagonist one of the most serious diseases of wheat (Triticum Pseudomonas chlororaphis.Thelatterisofparticularinterest aestivum L.), and has been recently detected in Europe, for the development of inoculants that might be used for North America, Africa, Oceania and Asia (Hamada et al., biocontrol of this disease. 2011). In infected wheat plants, R. cerealis may destroy Among the various microorganisms that inhabit the the stem and sheath tissues of the host plants, primarily plant rhizosphere, certain strains of Pseudomonas spp. have by blocking phloem transport which leads to lodging and the ability to control soil-borne, pathogenic fungi (Haas 444 Ann Appl Biol 163 (2013) 444–453 © 2013 China Agricultural University Annals of Applied Biology © 2013 Association of Applied Biologists Z. Jiao et al. P. chlororaphis biocontrol of wheat sharp eyespot & Defago, 2005; Weller, 2007). Root-colonising pseu- cerealis was estimated based on the percent inhibition of domonads produce a diversity of extracellular metabo- growth: lites with antimicrobial activity, and that have an = − × important role in disease suppression. These substances Inhibition of growth (%) [(C T) /C] 100. include 2,4-diacetylphloroglucinol (2,4-DAPG), pyolute- Highly antagonistic bacteria were defined as those which orin, phenazines, pyrrolnitrin, cyclic lipopeptides and inhibited growth by greater than 15%. Antagonistic hydrogen cyanide (HCN) (Weller, 2007). Effective bio- bacterial strains were identified based on their partial control pseudomonads, such as Pseudomonas fluorescens 16S rRNA gene sequences. The 16S rRNA partial genes CHA0 and Pf-5, produce multiple antimicrobial metabo- were amplified according to the method of Yuan et al. lites with overlapping or different degrees of activity (2011). against specific pathogens (Haas & Keel, 2003; Paulsen et al., 2005). Besides the antimicrobial metabolites, phyto- hormones (Keel et al., 1992), siderophores (Gardner et al., Bacterial and fungal strains, media and culture 1984; Bano & Musarrat, 2003) and the ability to con- conditions fer induced systemic resistance (ISR) (Raaijmakers et al., Pa40, which was isolated from this survey, was identified 2009) contribute to the efficacy of these bacteria. In this in this study as P. chlororaphis subsp. aurantiaca.Itis article, we surveyed a community of bacteria for their available through the China General Microbiological potential antagonism towards R. cerealis. Among these Culture Collection Center (CGMCC), collection No. 2764. bacteria, we focused in particular on one strain that was Pseudomonas sp. strains were cultured on King’s B medium especially effective in disease suppression. This bacterium, (King et al., 1954) and other bacterial strains were cultured P. chlororaphis subsp. aurantiaca strain Pa40, was further on Luria-Bertani (LB) broth or agar plates (Sambrook & characterised with respect to specific traits that were asso- Russell, 2001) at 28°C. All fungal strains were cultured ciated with biocontrol. In addition to R. cerealis, Pa40 was on potato dextrose agar (PDA) medium (100 g L−1 potato, also investigated for its antagonistic activity to several 10 g L−1 dextrose and 15 g L−1 agar) at 25°C. other phytopathogenic fungi and bacteria. Determination of antimicrobial activity in vitro Materials and methods The ability of strain Pa40 to inhibit growth of the Bacteria isolation and screening for R. cerealis phytopathogenic bacteria was tested on King’s B Medium antagonists according to the method of Chen et al. (2007). The Bacteria surveyed for this research were isolated from inhibitory effect of Pa40 against the various pathogenic an agricultural field located at the North China Inten- fungi was calculated against fungal plugs on PDA plates sive Agro-ecosystem Experimental Station (35°00N, (25°C) after 4 days and again after 6 days, as described 114°24E), Huantai County, Shandong Province, China. above. Soil samples were collected from the rhizospheres of wheat infected by R. cerealis. After removing approxi- Biocontrol assay against wheat sharp eyespot mately 3–5 cm of soil from the surface layer, the infected wheat roots with soil were placed in sterile plastic bags. The ability of Pa40 to control sharp eyespot disease The bacteria were isolated from soil according to meth- caused by R. cerealis on wheat was examined in a ods used by Mew et al. (1976). Each isolated colony was greenhouse experiment. R. cerealis was incubated in maize streaked 3–4 times to ensure isolation. The inhibition medium [maize powder, sand, and water (1:1:1, w/w/w) activity of each isolate against R. cerealis was tested using autoclaved at 121°Cfor1h]at25°C for 15 days. The the modified method of Daayf et al. (2003). A 5 mm fungal inocula prepared using this medium were then mycelial plug of R. cerealis was placed in the centre of a added to a mixture of sterile clay soil and vermiculite (1:1, PDA plate. Two 5 μL aliquots of isolate-cell-suspensions v/v) at ratio of 1:30 (v/v) in each pot (25 cm in diameter), were spotted on opposite sides of these plates, 2.5 cm which was used as a medium to culture wheat plants. from the centre of the fungal plug. A single plug of fun- Wheat seeds (Triticum aestivum Luyuan301, Shandong gus placed on a PDA plate served as a control. The plates Academy of Agricultural Sciences) were soaked with a were incubated at 25°C for 4 days. The mean distance solution of each treatment strain according to the methods (n = 4) of the treated fungal colony, measured from the described previously (Kim et al., 2000). Controls consisted plug centre to the edge nearest to the bacterial colony of, non-inoculated plants, prepared by soaking seeds in (T), was compared to the radius of the control plate fun- sterilised water, as well as seeds treated with a solution gal colony (C). The inhibitory effect of isolates against R. containing 20 mg L−1 validamycin A. Every treatment Ann Appl Biol 163 (2013) 444–453 © 2013 China Agricultural University 445 Annals of Applied Biology © 2013 Association of Applied Biologists P. chlororaphis biocontrol of wheat sharp eyespot Z. Jiao et al. was replicated using 10 pots per treatment (10 seeds Determination of physiological activities and traits per pot), and the experiment was repeated four times. Protease production was determined using skim milk agar The pots were maintained under a 14-h photoperiod at plates (Kumar et al., 2005). Chitinase and β-glucanase 22 ± 4°C. Sterile water was provided at rate