Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE Characterisation of 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; 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 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 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 -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 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 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 of 20 mL, activities were evaluated using methods described by per pot, each day. The wheat was harvested 25 days post Sietsma & Wessels (1979). Cellulase production was planting, and the disease index of sharp eyespot disease detected as described by Teather & Wood (1982). Each was determined according to the methods of Lipps and assay was performed in four replicates. The production Herr (1982). The disease inhibition activity was calculated of indole acetic acid (IAA) was determined using the as follows: method of Patten & Glick (2002) in nutrient broth −1 Sharp eyespot disease inhibition (%) = [(InC − InT ) /InC] with and without tryptophan (0.5 g L ). Phosphate solubilisation was detected using National Botanical × 100 Research Institute’s Phosphate growth medium (NBRIP) −1 −1 −1 where InT is sharp eyespot disease index of the wheat containing 10 g L glucose, 5 g L Ca3(PO4)2,5gL −1 −1 treated by a bacterial culture or validamycin A, and InC is MgCl2 6H2O, 0.25 g L MgSO4 7H2O, 0.2 g L KCl, −1 −1 disease index of the wheat treated with sterilised water. 0.1 g L (NH4)2SO4 and 18.0 g L agar (Nautiyal, 1999). Four replicates were performed for each assay. Production of HCN was observed by HCN-sensitive paper according to Identification of Pa40 strain the method of Castric & Castric (1983) and quantitative Phenotypic characterisation analysis of HCN production was assessed in LB broth with and without glycine (4.5 g L−1) according to the The ability of strain Pa40 to use different carbon method of Lambert et al. (1975). Siderophore production sources for growth was tested using the Biolog GN2 was determined by chrome azurol S (CAS) assay Microplate method (Biolog, Hayward, CA, USA). Other (Schwyn & Neilands, 1987). Biosurfactant production morphological, cultural, biochemical and physiological was analysed using a drop-collapse method (Bodour & characteristics of Pa40 were examined according to Miller-Maier, 1998). Each test was conducted using four methods previously reported by Peix et al. (2007). All replicates. other biochemical characteristics were determined in accordance with methods described in Bergey’s Manual of Systematic Bacteriology, 2nd Edn (Palleroni, 2005). The Detection of genes by PCR cell morphology, including flagella, was observed by transmission electron microscopy (TEM) according to To analyse the distribution of genes involved in antibiotic previously described methods (Guo et al., 2007). production by this pseudomonad, gene-specific primers were used in PCR. Specific primers, PCA2a and PCA3b for phenazine (phzCD), Phl2a and Phl2b for 2,4-DAPG Molecular characterisation and phylogenetic analysis (phlD), PrnCf and PrnCr for pyrrolnitrin (prnC), and PltBf Genomic DNA of Pa40 was extracted and purified accord- and PltBr for pyoluteorin (pltB) were employed according ing to the methods described by Sambrook & Russell to methods described by Zhang et al. (2006). Primers (2001). 16S rRNA genes were amplified using the primers 30-84XBA and PHZO10 as described by Delaney et al. 8F 5CGGGATCCAGAGTTTGATCCTGGCTCAGAACGAA (2001) were used to target 2-hydroxylated phenazine CGCT3 and 1506R 5CGGGATCCTACGGCTACCTTGTT (phzO). The PCR programs and conditions were processed ACGACTTCACCCC3 (Xin et al., 2004). PCR products of following the references mentioned above. P. fluorescens atpD, carAandrecA genes were amplified according to the Pf-5 was served as a control in all PCR reactions. Pa40 methods of Hilario et al. (2004). DNA sequence similar- PCR products were sequenced to confirm similarity ity searches were performed with the online Basic Local to 2,4-DAPG, pyoluteorin, phenazine, 2-hydroxylated Alignment Search Tool (BLAST) search engine in the phenazine and pyrrolnitrin biosynthesis gene sequences. National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST). Similarity anal- Induced hypersensitive response (IHR) assay ysis of the sequences was performed with DNAMAN DNA analysis software package (DNAMAN version 5.22; Eight-week-old tobacco (Nicotiana tabacum cv. Xanthi) Lynnon Biosoft, Montreal, Canada). Phylogenetic analy- leaves (n = 4) were inoculated with strain Pa40 using a ses were conducted using the neighbour-joining method leaf infiltration method (Liu et al., 2009). Symptoms of with Molecular Evolutionary Genetics Analysis version plant-hypersensitive response were inspected every 24 h, 4.0 (Tamura et al., 2007). for 10 days.

446 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

Statistical analysis and nucleotide sequences accession number

Analysis of variance (ANOVA) was performed using GLM procedure in SPSS (version 13.0, SPSS Inc, Chicago, IL, USA) and data were subjected to compared using Fisher’s least significant difference (LSD) test (P < 0.01). The 16S rDNA, atpD, carAandrecA gene sequences of Pa40 strain have been deposited in GenBank under accession numbers FJ515772-FJ515775 and accession numbers of the 16S rRNA gene sequences of antagonistic bacteria isolated here are listed as JX122095-JX122119. The accession number of genes involved in biosynthesis of 2-hydroxylated phenazine, phenazine and pyrrolnitrin Figure 1 In planta wheat sharp eyespot disease inhibition by antagonis- tic bacterial strains. Columns with the same letters are not significantly in Pa40 are KC020730-KC020732. different (P < 0.01).

Results Isolation, screening and characterisation of antagonistic bacteria

A total of 582 bacterial strains were isolated from rhizospheres soil associated with wheat plants that were infected by R. cerealis. Of these, 26 isolates showed highly antagonistic activity towards R. cerealis in vitro and were identified from their 16S rRNA (a) (b) (c) (d) gene sequences. The antagonistic community was comprised of eight genera; Pseudomonas (50.0%), Bacillus (19.2%), Flavobacterium (7.7%), Arthrobacter (7.7%), Figure 2 Representative greenhouse results depicting the onset of wheat sharp eyespot disease post seed treatment. Treatments and Pedobacter (3.8 %), Stenotrophomonas (3.8 %), Alcaligenes control. (a) Seed treated with sterile water and planted in sterile soil. (3.8 %), and Agrobacterium (3.8 %) (Table 1). A (b) Seed treated with sterile water and planted in sterile soil mixed with phylogenetic, neighbour-joining tree, which represents Rhizoctonia cerealis. (c) Seed treated with validamycin A and planted in four phyla, , Firmicutes, Bacteroidetes and sterile soil mixed with R. cerealis. (d) Seed treated with Pa40 strain and Actinobacteria, is available in Fig. S1. Eleven of the planted in sterile soil mixed with R. cerealis. 26 strains inhibited R. cerealis growth in vitro by more than 50%. Ten of the strains, displaying the greatest (Table 2). On day 6 inhibitions ranged from 47% to 85%, zones of inhibition on plates, were selected for further depending on the pathogen, and in each case inhibition investigation in greenhouse experiments and the results increased between day 4 and day 6. Six of the eight of this assay are displayed in Fig. 1. All strains tested bacterial pathogens assessed were also inhibited by Pa40 inhibited sharp eyespot in planta by 10% or greater and which represent four genera (Table 2). treatment with three of the strains reduced symptoms The results of PCR using specific primers showed by more than 50%. In this assay, Pa40 exemplified the that Pa40 contained genes encoding enzymes involved best inhibition of disease, averaging 65%. Fig. 2 depicts in biosynthesis of phenazine, 2-hydroxylated phenazine the presence or absence of lesions and browning of stems and pyrrolnitrin and showed no amplification for a gene associated with wheat sharp eyespot disease onset. related 2,4-diacetylphloroglucinol production (Fig. 3e). Pa40 yielded a product of larger size than P. fluorescens Pf-5 when PltBf and PltBr were used to target a gene involved Characteristics of Pa40 biocontrol in pyrrolnitrin production (Fig. 3e) and the sequence Disease inhibition by Pa40 treatment was similar to that of this product showed that it was not a pyrrolnitrin of chemical treatment with validamycin A and both biosynthesis gene. The sequences of the PCR products treatments show clear suppression when compared to from Pa40 showed 99%, 98% and 99% similarities to the an infected, non-inoculated control (Fig. 2). The results genes involved in phenazine, 2-hydroxylated phenazine of in vitro assays showed inhibition of 11 phytopathogenic and pyrrolnitrin biosynthesis in P. chlororaphis strains fungal strains (representing four classes) after 4 and 6 days GP72, 30–84 and Pa23. Pa40 tested positive for HCN,

Ann Appl Biol 163 (2013) 444–453 © 2013 China Agricultural University 447 Annals of Applied Biology © 2013 Association of Applied Biologists P. chlororaphis biocontrol of wheat sharp eyespot Z. Jiao et al.

Table 1 Identification and Rhizoctonia cerealis-antagonistic activity of bacteria strains isolated from soil

Strains Inhibition of growth (%) Identification Strains Inhibition of growth (%) Identification

MOA1-16 73 ± 2 aAb Pedobacter sp. MOC27 48 ± 1EFG Pseudomonas sp. MOB1-17 70 ± 1AB Alcaligenes sp. CDA1-5 48 ± 1EFG Agrobacterium sp. MOC1-22 69 ± 1AB Pseudomonas sp. CDB11 46 ± 1FG Pseudomonas sp. CDA1-15 69 ± 1AB Pseudomonas sp. COB10 45 ± 2HFG Bacillus sp. MOB13 68 ± 1B Pseudomonas sp. MOC1-4 41 ± 3HI Pseudomonas sp. CKC35 67 ± 1BC Pseudomonas sp. MOC14 37 ± 1JI Pseudomonas sp. MOC16 65 ± 2BC Pseudomonas sp. CKC9 36 ± 2J Stenotrophomonas sp. Pa40 55 ± 1D Pseudomonas sp. MOA1-4 22 ± 3K Flavobacterium sp. MOC3 56 ± 2D Bacillus sp. CKC31 22 ± 3K Arthrobacter sp. MOD23 55 ± 3D Bacillus sp. CDA-19 20 ± 1LK Pseudomonas sp. CCB27 53 ± 1ED Bacillus sp. MOA1-25 18 ± 3LK Arthrobacter sp. MOB29 50 ± 2EF Pseudomonas sp. MOC20 18 ± 1LK Flavobacterium sp. MOA1-19 49 ± 4EF Pseudomonas sp. MOC28 16 ± 1L Bacillus sp. aEach data represents the mean of four independent replicates and corresponding standard error. bDifferent letters indicate statistical significance (P < 0.01).

Table 2 Antagonistic activity of Pseudomonas chlororaphis subsp. glycine, respectively. Pa40 did not show β-glucanase or aurantiaca Pa40 to the indicator phytopathogenic fungi and phy- chitinase activities and produced no biosurfactant. It was topathogenic bacteria not determined to be an IAA producer and tested negative Inhibition of growth (%) for phosphorus solubilisation. Indicator 4 Days 6 Days

Phytopathogenic fungi Ceratocystis fimbriata 44 ± 1a 51 ± 4 Identification of strain Pa40 ± ± Monilinia laxa 50 2594 Gram staining of strain Pa40 determined it was Gram- Magnaporthe grisea 55 ± 166± 3 Phytophthora capsici Leonian 56 ± 266± 4 negative and TEM imagery depicted strain Pa40 as a polar- Alternaria solani 53 ± 261± 2 flagellate, rod-shaped bacterium. Metabolic fingerprinting Botrytis cinerea 77 ± 285± 2 by the Biolog system identified Pa40 as P. chlororaphis ± ± Cladosporium fulvum 51 1633 (Flour. Biotype D) with a 0.51 similarity index and Septoria apiicola 49 ± 157± 3 f.sp. vasinfectum 61 ± 370± 3 distance indices of 7.6 to P. chlororaphis and8.90tothe Fusarium moniliforme 38 ± 247± 4 next likely species, P. aurantiaca. Enzyme and substrate Fusarium oxysporum f.sp. Lili 46 ± 256± 4 usage analysed in the Biolog assay and also reported b Fusarium.oxysporum f.sp. niveum – – for P. chlororaphis subsp. aurantiaca ATCC type strain are Botrytis cinerea Pers. – – comparatively displayed in Table S1. Of the 34 parameters Inhibition zone tested in each strain, only three showed different results Phytopathogenic bacteria diameter (mm) such as urease reaction and using raffinose or D-arbitol. Xanthomonas malvacearum (E. F. Smith) Dowson 18.4 ± 6.8 Factors used to determine the subspecies of Pa40 are Ralstonia solanacearum 24.7 ± 4.0 included in Table 3. Analysis of 16S rDNA sequence ± Pseudomonas syringae 26.7 0.5 information from Pa40 displayed greater than 98% Pseudomonas corrugata ICMP5819 35.4 ± 0.3 identity to those of three P. chlororaphis subspecies. Agrobacterium vitis K308 20.7 ± 0.4 Agrobacterium rhizogenes K27 6.9 ± 0.9 The sequences of recA, atpDandcarA showed strong Agrobacterium tumefaciens C58 – identities, 96.8% and higher, to all of the P. chlororaphis Burkholderia cepacia ICMP5796 – subspecies (Table 3). Phylogenetic analyses of 16S rRNA, aStandard error of fifteen independent experiments. recA, atpDandcarA genes all revealed close relatedness b –: No growth inhibitory activity. of Pa40 to P. chlororaphis subspecies aurantiaca (Fig. S2a–d). Pa40 carbon source usage included L-arabinose, but not 5-ketogluconate and orange pigmentation was protease activity and siderophore production and also visually confirmed; all characteristics which show high stimulated the hypersensitive response in tobacco after similarity to P. chlororaphis subspecies aurantiaca (Table 7 days (Fig. 3a to Fig. 3d). Pa40 produced 15.3 μgmL−1 3). After comparative analysis to type strain, NCIMB and 6.9 μgmL−1 HCN in LB broth with and without 10068T (=ATCC 33663T = CIP 106718T)asdescribedby

448 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

(a) (b) R. cerealis biocontrol capabilities. In this work, the R. cerealis-antagonists were predominantly Pseudomonas spp. and Bacillus spp., which are often ubiquitous in (Table 1 and Fig. S1). These genera have consistently been reported with respect to biocontrol and have shown antagonistic behaviour towards Verticillium dahliae and Phytophthora nicotianae in strawberry, potato, oilseed rape and tobacco rhizospheres (Berg et al., 2002; Jin et al., 2011). A culture-independent method also pointed (c) (d) to γ -proteobacteria as a predominant group when a microbiome of a Rhizoctonia solani-suppressive soil was completed (Mendes et al., 2011). All of the above mentioned reports show some degree of overlap with our findings and antagonistic Pseduomonas spp. and Bacillus spp. were present in all studies. The Pseudomonas genus, in particular, was consistently represented by multiple species, isolated from a variety of host rhizospheres, which showed antagonistic behaviour towards an array of plant- pathogens. This genus has been the focus of biological soil (e) treatments for decades and considering this versatility, a strain of Pseduomonas sp. could hold great potential in the development of a broad-spectrum biocontrol strategy. The most promising biocontrol strain found in this study was selected for complete identification. All morphological characteristics exemplified by Pa40 are typical of the genus Pseudomonas, as described by Palleroni (2005). Metabolic profiling using the Biolog system suggests proper genus identification with high confidence and also indicated the P. chlororaphis species classification. Figure 3 Assays for characteristics related to biocontrol. (a) Siderophore Classification based on 16S rRNA, recA, atpDandcarA production by Pseudomonas chlororaphis subsp. aurantiaca Pa40. (b) gene sequences has led to greater resolution of related Protease production by P. chlororaphis subsp. aurantiaca Pa40 on species in the Pseudomonas genus (Hilario et al., 2004). The skimmed milk agar plates. (c) HCN production by Escherichia coli DH5α sequences of these genes denote the P. chlororaphis species (left) and P. chlororaphis subsp. aurantiaca Pa40 (right) detected with classification, as all show high identities to members HCN-sensitivepaper.(d)HypersensitiveresponseofP.chlororaphissubsp. aurantiaca Pa40 (left sterile buffered saline inoculated as the negative of this species (Table S1; Fig. S2). In accordance with control, right bacteria suspension of Pa40). (e) Detection of antibiotic procedures described by Peix et al. (2007) to accurately genes by PCR, lane M DNA marker, Lane 1is blank control, lane 2 is P. differentiate P. chlororaphis subspecies, Pa40 is additionally fluorescens Pf-5, and lane 3 is P. chlororaphis subsp. aurantiaca Pa40. resolved to be P. chlororaphis subsp. aurantiaca,by comparing carbon substrate usage and pigmentation to (Peix et al., 2007), Pa40 was further labelled as P. that of three type strains representing the subspecies of chlororaphis subspecies aurantiaca. P. chlororaphis (Table 3). Unlike the P. chlororaphis subsp. aurantiaca type strain, Pa04 produces urease, does not use raffinose or D-arbitol, and does not produce a green Discussion pigmentation. These differences do not indicate a high Some soils are known to be naturally disease-suppressive; uncertainty associated with this classification, but suggest a property most closely associated with high population that strain Pa40 has some unique biochemical properties densities of diverse bacteria that are antagonistic towards apart from its subspecies. The proposed identifications, a phytopathogen. Approximately 5% of the bacteria based on both molecular sequencing and metabolic isolated from the rhizosphere of wheat infected by R. profiling, coincide and together provide strong evidence cerealis were determined to be antagonistic. Chen et al. for the accuracy of this classification. (2010) determined a large portion of wheat rhizobacterial Pa40 displayed the ability to reduce the onset of isolates showing antagonism to R. cerealis.Bothresults wheat sharp eyespot in planta by 65% (Fig. 1) as well indicate the presence of many indigenous bacteria with as growth inhibition of various, economically important

Ann Appl Biol 163 (2013) 444–453 © 2013 China Agricultural University 449 Annals of Applied Biology © 2013 Association of Applied Biologists P. chlororaphis biocontrol of wheat sharp eyespot Z. Jiao et al.

Table 3 Phenotypes used to distinguish subspecies and % similarity of 16S rRNA, atpD, recAandcarA gene sequences between Pseudomonas chlororaphis subsp. type strains and Pa40

Pseudomonas chlororaphis Pseudomonas chororaphis Pseudomonas chororaphis subsp. aurantiaca subsp. aureofaciens subsp. chlororaphis Characteristic ATCC33663/Pa40 ATCC13985/Pa40 ATCC9446/Pa40

Non-fluorescent pigment Green + /−−/−+/− Orange +/++/+−/+ Nitrate reduction −/−−/– +/− 5-Ketogluconate −/−+/−+/− L-Arabinose +/++/+−/+ 16S rRNA (%) 98.4 98.8 98.8 atpD (%) 99.0 99.5 98.8 recA (%) 98.8 98.6 97.6 carA (%) 99.2 96.8 97.1

+: positive reaction; −: negative reaction; v; variable. phytopathogens (Table 2). In a survey of R. cerealis- treatment of wheat seedlings with Pa40 showed greater infected wheat fields by Clarkson and Cook (1983) disease inhibition than other strains tested (Fig. 1). This slight infections, defined as those in which lesions were could reflect enhanced rhizosphere competence of Pa40 present on less than half of a stem’s circumference, over other strains, a biochemical change which occurs corresponded to negligible yield loss. The lesions observed in the rhizosphere environment, or Pa40’s ability to post Pa40 treatment (Fig. 2) fall within the category trigger hypersensitive response (Fig. 3d), enhancing plant of a slight infection, as defined by Clarkson & Cook resistance to the pathogen. (1983). Hence, both the inoculation method described Biochemical analyses of the mechanisms by which here and Pa40 strain appears to be effective for R. Pa40 interacts with R. cerealis revealed that there was not cerealis disease-suppression in greenhouse conditions. The a single antifungal agent employed by these bacteria, but percent inhibition, resulting from Pa40 treatment was that several compounds may be involved in controlling similar to that for plants produced from seeds treated the infection and spread of this fungal pathogen. Pa40 with validamycin A, with neither displaying enhanced produced volatile HCN, which reduces the growth of efficacy in disease control over the other (Figs 1 and fungi (Haas & Keel, 2003); enzymatic proteases, which 2). Validamycin A is actively being used to control act to disrupt bacterial and fungal cell membranes (Muleta wheat sharp eyespot in China and Japan (Hamada et al., 2007); and siderophores, which sequester essential et al., 2011). However, treatment with this fungicide iron away from phytopathogens (Weller, 2007) (Fig. 3a will likely threaten non-target fungi, causing a decline to Fig. 3c). Multiple antimicrobial metabolites will play in populations of non-pathogenic R. cerealis-competitors an important role in a pathogenesis (Haas & Defago, or plant-beneficial fungi, such as mycorrhizae. Several 2005). The quantity of HCN released by Pa40 doubled studies have tested the use of beneficial soil organisms with glycine addition, indicating its role as a metabolic for the control of R. cerealis (Hamada et al., 2011). For precursor in the HCN production pathway of this strain example, Innocenti et al. (2003) showed Trichoderma (Castric, 1977). Compared to previous research, HCN atroviride LF 312 treatment to reduce the incidence of production quantified in this study was higher than those wheat sharp eyespot. However, there are additional determined for two other Pseudomonas spp. (Gardner benefits to using P. chlororaphis. Previous research on et al., 1984; Bano & Musarrat, 2003) and lower than P. chlororaphis demonstrated its plant growth promoting that of Pseudomonas corrugata P94 (Guo et al., 2007). properties, which were linked to improved wheat growth Spencer et al. (2003) demonstrated that the biocontrol and yield, as well as its ability to serve as an excellent capabilities of P. chlororaphis 06 are related to both biocontrol agent against several, cereal, seed-borne phenanzine production and induction of resistance in pathogens (Johnsson et al., 1998; Carlier et al., 2008). tobacco. Orange pigmentation of Pseudomonas spp., as But this is the first report on P. chlororaphis biocontrol observed in Pa40 colonies (Fig. 3a and 3b), often of wheat sharp eyespot. In vitro plate assays displayed reflects phenanzine production (Spencer et al., 2003; the robustness of Pa40 for biological disease control Peix et al., 2007) and Pa40 was shown to contain (Table 2). Pa40 did not show the highest degree of R. genes related to those involved in the production of cerealis inhibition on laboratory plates (Table 1). However, pyrrolnitrin, phenazine and 2-hydroxylated phenazine.

450 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

These compounds have well-studied antibiotic properties diversity of antagonistic rhizobacteria isolated from and also serve to inhibit a broad range of phytopathogens different verticillium host plants. Applied and Environmental as each offers a unique antifungal spectrum (Park Microbiology, 68, 3328–3338. et al., 2011). Additionally, many phenazines have been Bodour A.A., Miller-Maier R.M. (1998) Application of a indicated to elicit ISR in plants (Pierson & Pierson, 2010) modified drop-collapse technique for surfactant quantita- and here induction of the hypersensitive response was tion and screening of biosurfactant-producing microorgan- observed in tobacco leaves injected with Pa40 (Fig. 3d). isms. Journal of Microbiological Methods, 32, 273–280. This can improve crop immunity by stimulating systemic Carlier E., Rovera M., Rossi Jaume A., Rosas S. (2008) resistance and is also noted to boost plant resistance Improvement of growth, under field conditions, of wheat against an array of phytopathogens (Van Wees et al., inoculated with Pseudomonas chlororaphis subsp. aurantiaca 2008). Interestingly, two strains of Botrytis cinerea and SR1. World Journal of Microbiology and Biotechnology, 24, two strains of Fusarium oxysporum fungi were affected 2653–2658. Castric P.A. (1977) Glycine metabolism by Pseudomonas differently by Pa40 (Table 2). The ability of Pa40 to trigger aeruginosa: hydrogen cyanide biosynthesis. Journal of plant hypersensitive response may broaden the range Bacteriology, 130, 826–831. of Pa40 disease inhibition if tested in planta. Additional Castric K.F., Castric P.A. (1983) Method for rapid detection of research into the magnitude and mechanisms involved cyanogenic bacteria. Applied and Environmental Microbiology, in Pa40 positive regulation of plant defence genes in 45, 701–702. wheat, when applied to seedlings, would confirm the Chen F., Guo Y.B., Wang J.H., Li J.Y., Wang H.M. (2007) capability of Pa40 to enhance plant resistance. Pa40 biological control of grape crown gall by Rahnella aquatilis tested negative for biosurfactant production and IAA HX2. Plant Disease, 91, 957–963. production while strains of the same species are known Chen H.-G., Cao Q.-G., Xiong G.-L., Li W., Zhang A.- to produce rhamnolipids and IAA (Gunther et al., 2005; X., Yu H.-S., Wang J.-S. (2010) Composition of wheat Kang et al., 2006). Variable characteristics within this rhizosphere antagonistic bacteria and wheat sharp eyespot species suggest that mechanisms of biocontrol used by as affected by rice straw mulching. Pedosphere, 20, 505–514. Pa40 may be unique to this strain. After the examination Clarkson J.D.S., Cook R.J. (1983) Effect of sharp eyespot of a community of R. cerealis antagonists, this novel (Rhizoctonia cerealis) on yield loss in winter wheat. Plant research recommends the use of P. chlororaphis subsp. Pathology, 32, 421–428. aurantiaca strain Pa40 as a biocontrol agent for the Daayf F., Adam L., Fernando W.G.D. (2003) Comparative suppression of wheat sharp eyespot with potential for screening of bacteria for biological control of potato late additional control of other plant diseases. blight (strain US-8), using in-vitro, detached-leaves, and whole-plant testing systems. Canadian Journal of Plant Acknowledgements Pathology, 25, 276–284. Delaney S.M., Mavrodi D.V., Bonsall R.F., Thomashow L.S. We thank Dr. David E. Crowley (Department of Envi- (2001) phzO, a Gene for Biosynthesis of 2-Hydroxylated ronmental Sciences, University of California, Riverside) Phenazine Compounds in Pseudomonas aureofaciens for his valuable advice. We are grateful to Dr. Li Jinyun 30–84. Journal of Bacteriology, 183, 318–327. (Department of Plant Pathology, China Agricultural Uni- Gardner J., Chandler J., Feldman A. (1984) Growth pro- versity) for kindly providing phytopathogenic bacterial motion and inhibition by antibiotic-producing fluorescent strains and Dr. Lu Chaige (Plant and Environment Pro- pseudomonads on citrus roots. Plant and Soil, 77, 103–113. tection Institute, The Beijing Academy of Agriculture and Gunther N.W., Nunez˜ A., Fett W., Solaiman D.K.Y. (2005) Forestry Sciences, Beijing, China) for kindly providing Production of Rhamnolipids by Pseudomonas chlororaphis, phytopathogenic fungal strains. This work was supported a nonpathogenic bacterium. Applied and Environmental by The National Key Technology R&D Program in Rural Microbiology, 71, 2288–2293. Areas (2012BAD14B07), National Natural Science Foun- Guo Y., Zheng H., Yang Y., Wang H. (2007) Characterization dation of China (31200386&31170489), and Chinese of Pseudomonas corrugata strain p94 isolated from soil in Universities Scientific Fund (2011JS167&2012QJ159). Beijing as a potential biocontrol agent. Current Microbiology, 55, 247–253. Haas D., Defago G. (2005) Biological control of soil-borne References pathogens by fluorescent pseudomonads. Nature Reviews Bano N., Musarrat J. (2003) Characterization of a new Microbiology, 3, 307–319. Pseudomonas aeruginosa strain NJ-15 as a potential Haas D., Keel C. (2003) Regulation of antibiotic production biocontrol agent. Current Microbiology, 46, 324–328. in root-colonizing Pseudomonas spp. and relevance for Berg G., Roskot N., Steidle A., Eberl L., Zock A., Smalla biological control of plant disease. Annual Review of K. (2002) Plant-dependent genotypic and phenotypic Phytopathology, 41, 117–153.

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Hamada M.S., Yin Y., Chen H., Ma Z. (2011) The escalating the ability to induce a hypersensitive reaction in tobacco. threat of Rhizoctonia cerealis, the causal agent of sharp Journal of General Plant Pathology, 75, 369–380. eyespot in wheat. Pest Management Science, 67, 1411–1419. Mendes R., Kruijt M., de Bruijn I., Dekkers E., van der Hilario E., Buckley T.R., Young J.M. (2004) Improved Voort M., Schneider J.H.M., Piceno Y.M., DeSantis T.Z., resolution on the phylogenetic relationships among Andersen G.L., Bakker P.A.H.M., Raaijmakers J.M. (2011) Pseudomonas by the combined analysis of atp D, car Deciphering the rhizosphere microbiome for disease- A, rec A and 16S rDNA. Antonie van Leeuwenhoek, 86, suppressive bacteria. Science, 332, 1097–1100. 51–64. Mew T., Ho W.C., Chu L. (1976) Infectivity and survival Innocenti G., Roberti R., Montanari M., Zakrisson E. (2003) of soft-rot bacteria in Chinese cabbage. Phytopathology, 66, Efficacy of microorganisms antagonistic to Rhizoctonia 1325–1327. cerealis and their cell wall degrading enzymatic activities. Muleta D., Assefa F., Granhall U. (2007) In vitro antagonism Mycological Research, 107, 421–427. of rhizobacteria isolated from Coffea arabica L. against Jin F., Ding Y., Ding W., Reddy M.S., Fernando W.G.D., Du emerging fungal coffee pathogens. Engineering in Life B. (2011) Genetic diversity and phylogeny of antagonistic Sciences, 7, 577–586. bacteria against Phytophthora nicotianae isolated from Nautiyal C.S. (1999) An efficient microbiological growth tobacco rhizosphere. International Journal of Molecular medium for screening phosphate solubilizing microorgan- Sciences, 12, 3055–3071. isms. FEMS Microbiology Letters, 170, 265–270. Johnsson L., Hokeberg¨ M., Gerhardson B. (1998) Perfor- Palleroni N.J. (2005) Genus I. Pseudomonas. In Bergey’s Manual mance of the Pseudomonas chlororaphis biocontrol agent MA of Systematic Bacteriology. 2nd edn, Volume 2, pp.323–379. 342 against cereal seed-borne diseases in field experiments. Eds D.J. Brenner, N.R.J. Krieg, T. Staley, and G.M. Garrity. European Journal of Plant Pathology, 104, 701–711. East Lansing: Springer. Kang B., Yang K., Cho B., Han T., Kim I., Lee M., Anderson Park J.Y., Oh S.A., Anderson A.J., Neiswender J., Kim J.-C., A., Kim Y. (2006) Production of indole-3-acetic acid in Kim Y.C. (2011) Production of the antifungal compounds the plant-beneficial strain Pseudomonas chlororaphis O6 is phenazine and pyrrolnitrin from Pseudomonas chlororaphis negatively regulated by the global sensor kinase GacS. 06 is differentially regulated by glucose. Letters in Applied Current Microbiology, 52, 473–476. Microbiology, 52, 532–537. Keel C., Schnider U., Maurhofer M., Voisard C., Laville Patten C.L., Glick B.R. (2002) Role of Pseudomonas putida J., Burger U., Wirthner P., Haas D., Defago G. (1992) indoleacetic acid in development of the host plant Suppression of root diseases by Pseudomonas fluorescens root system. Applied and Environmental Microbiology, 68, CHAO: importance of bacterial secondary metabolite, 2,4- 3795–3801. diacetylphoroglucinol. Molecular Plant-Microbe Interaction, 5, Paulsen I.T., Press C.M., Ravel J., Kobayashi D.Y., Myers 4–13. G.S.A., Mavrodi D.V., DeBoy R.T., Seshadri R., Ren Q., Kim Y.C., Miller C.D., Anderson A.J. (2000) Superoxide Madupu R., Dodson R.J., Durkin A.S., Brinkac L.M., dismutase activity in Pseudomonas putida affects utilization Daugherty S.C., Sullivan S.A., Rosovitz M.J., Gwinn M.L., of sugars and growth on root surfaces. Applied and Environmental Microbiology, 66, 1460–1467. Zhou L., Schneider D.J., Cartinhour S.W., Nelson W.C., King E.O., Ward M.K., Raney D.E. (1954) Two simple media Weidman J., Watkins K., Tran K., Khouri H., Pierson E.A., for the demonstration of pyocyanin and fluorescein. The Pierson L.S., Thomashow L.S., Loper J.E. (2005) Complete Journal of Laboratory and Clinical Medicine, 44, 301–307. genome sequence of the plant commensal Pseudomonas Kumar R.S., Ayyadurai N., Pandiaraja P., Reddy A.V., fluorescens Pf-5. Nature Biotechnology, 23, 873–878. Venkateswarlu Y., Prakash O., Sakthivel N. (2005) Peix A., Valverde A., Rivas R., Igual J.M., Ramırez-Bahena´ Characterization of antifungal metabolite produced by a M.-H., Mateos P.F., Santa-Regina I., Rodrıguez-Barrueco´ new strain Pseudomonas aeruginosa PUPa3 that exhibits C., Martınez-Molina´ E., Velazquez´ E. (2007) Reclassifica- broad-spectrum antifungal activity and biofertilizing traits. tion of Pseudomonas aurantiaca as a synonym of Pseudomonas Journal of Applied Microbiology, 98, 145–154. chlororaphis and proposal of three subspecies, P. chlororaphis Lambert J.L., Ramasamy J., Paukstelis J.V. (1975) Stable subsp. chlororaphis subsp. nov., P. chlororaphis subsp. aure- reagents for the colorimetric determination of cyanide ofaciens subsp. nov., comb. nov. and P. chlororaphis subsp. by modified Koenig reactions. Analytical Chemistry, 47, aurantiaca subsp. nov., comb. nov. International Journal of 916–918. Systematic and Evolutionary Microbiology, 57, 1286–1290. Lipps P.E., Herr L.J. (1982) Etiology of Rhizoctonia cerealis Pierson L., Pierson E. (2010) Metabolism and function of sharp eyespot of wheat. Phytopathology, 72, 1574–1577. phenazines in bacteria: impacts on the behavior of bacteria Liu Y., Kanda A., Yano K., Kiba A., Hikichi Y., Aino in the environment and biotechnological processes. Applied M., Kawaguchi A., Mizoguchi S., Nakaho K., Shiomi Microbiology and Biotechnology, 86, 1659–1670. H., Takikawa Y., Ohnishi K. (2009) Molecular typing of Raaijmakers J., Paulitz T., Steinberg C., Alabouvette C., Japanese strains of Ralstonia solanacearum in relation to Moenne-Loccoz¨ Y. (2009) The rhizosphere: a playground

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and battlefield for soilborne pathogens and beneficial Weller D.M. (2007) Pseudomonas biocontrol agents of soil- microorganisms. Plant and Soil, 321, 341–361. borne pathogens: looking back over 30 years. Phytopathol- Sambrook J., Russell D.W. (2001) Molecular cloning: a ogy, 97, 250–256. laboratory manual, 3rd edn. New York, USA: Cold Spring Xin Y.H., Zhou Y.G., Zhou H.L., Chen W.X. (2004) Harbor Laboratory Press. Ancylobacter rudongensis sp. nov., isolated from roots of Schwyn B., Neilands J.B. (1987) Universal chemical assay for Spartina anglica. International Journal of Systematic and the detection and determination of siderophores. Analytical Evolutionary Microbiology, 54, 385–388. Biochemistry, 160, 47–56. Yuan C.-L., Mou C.-X., Wu W.-L., Guo Y.-B. (2011) Effect Sietsma J.H., Wessels J.G.H. (1979) Evidence for covalent of different fertilization treatments on indole-3-acetic acid linkages between chitin and β-glucaninafungalwall. producing bacteria in soil. Journal of Soils and Sediments, 11, Journal of General Microbiology, 114, 99–108. 322–329. Spencer M., Ryu C.-M., Yang K.-Y., Kim Y.C., Kloepper Zhang, Y., Fernando W.G.D., de Kievit T.R., Berry C., Daayf J.W., Anderson A.J. (2003) Induced defence in tobacco F., Paulitz T.C. (2006) Detection of antibiotic-related genes by Pseudomonas chlororaphis strain O6 involves at least from bacterial biocontrol agents with polymerase chain the ethylene pathway. Physiological and Molecular Plant reaction. Canadian Journal of Microbiology, 52, 476–481. Pathology, 63, 27–34. Tamura K., Dudley J., Nei M., Kumar S. (2007) MEGA4: Supporting Information molecular evolutionary genetics analysis (MEGA) soft- ware version 4.0. Molecular Biology and Evolution, 24, Additional Supporting Information may be found in the 1596–1599. online version of this article: Teather R.M., Wood P.J. (1982) Use of Congo red- Fig. S1. Phylogenetic relationships among 16S rDNA polysaccharide interactions in enumeration and charac- sequences of Antagonistic bacteria isolated from soil. terization of cellulolytic bacteria from the bovine rumen. Fig. S2. Phylogenetic relationships analysis of (A) 16S Applied and Environmental Microbiology, 43, 777–780. rDNA, (B) atpD, (C) carAand(D)recA of Pa40. Van Wees S.C.M., Van der Ent S., Pieterse C.M.J. (2008) Table S1. Biochemical and physiological characteristics Plant immune responses triggered by beneficial microbes. of P. chlororaphis subsp. aurantiaca Pa40 and the strain Current Opinion in Plant Biology, 11, 443–448. ATCC33663 reported by Peix et al. (2007).

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