Ann Microbiol (2013) 63:1177–1185 DOI 10.1007/s13213-012-0576-7

ORIGINAL ARTICLE

Promotion of plant growth, biological control and induced systemic resistance in maize by aurantiaca JD37

Rui Fang & Jia Lin & Shanshan Yao & Yujing Wang & Jing Wang & Chenhao Zhou & Huijie Wang & Ming Xiao

Received: 18 May 2012 /Accepted: 20 November 2012 /Published online: 12 December 2012 # Springer-Verlag Berlin Heidelberg and the University of Milan 2012

Abstract Some Pseudomonas aurantiaca strains have been yield and disease control (Kloepper 1992; Dey et al. 2004; found to facilitate plant growth. A P. aurantiaca JD37 strain Wang et al. 2010b). The use of these in sustainable isolated from a suburb of Shanghai, China, was found to agriculture is steadily increasing and offers an attractive effectively colonize the and internal roots of alternative to the application of chemical fertilizers, maize (Zea mays L.) and promote maize growth. Agar pesticides and supplements. diffusion assays and biocontrol effect experiments showed Rhizobacteria and endophytic bacteria can affect plant that strain JD37 had significant antagonistic activity against growth either directly or indirectly. Factors that directly Bipolaris maydis, as well as a high biocontrol effect on promote plant growth include biological nitrogen fixation southern maize leaf blight caused by B. maydis. PCR detec- (Christiansen-Weneger 1992), solubilization of soil phos- tion, associated with reverse-phase high-performance liquid phorus and iron (De Freitas et al. 1997), synthesis of several chromatography assays, showed that strain JD37 might pro- different phytohormones (Bastian et al. 1998) and a number duce a number of important antibacterial substances, such as of enzymes, such as 1-aminocyclopropane-1-carboxylate phenazine-1-carboxylic acid, pyrrolnitrin and 2,4-diacetyl- (ACC) deaminase (Safronova et al. 2006). Indirect promo- phloroglucinol. The crude bacterial extracts and the cell-free tion of plant growth has shown that some bacteria can supernatant of strain JD37 were found to induce resistance produce to suppress diseases caused by plant in maize against B. maydis and reduce plant disease. Our pathogens (Mavrodi et al. 2001), degrade pollutants, such results indicate the potential of some bacteria for producing as phenol (Wang et al. 2007), compete for nutrients and bacterial compounds that serve as inducers of disease resis- suitable niches on the root surface (Glick 1995)and tance, which is an attractive alternative to the application of induce plant systemic resistance (Bakker et al. 2007;De chemical fertilizers, pesticides and supplement in agricultural Vleesschauwer et al. 2008). A specific plant growth- practices. promoting bacterium/rhizobacterium (PGRB/PGPR) may affect plant growth and development by using any one Keywords Pseudomonas aurantiaca JD37 . Plant growth or more of these mechanisms. In recent years, many of promotion . Biocontrol . Induced systemic resistance . the mechanisms of PGPB/PGPR have been identified Bipolaris maydis (Bashan and de-Bashan 2005; Lugtenberg and Kamilova 2009; Bashan and de-Bashan 2010) and studied in plants. It is likely that the mechanisms of PGPB affect environ- Introduction mental remediation and the inhibition of pathogen and plant growth. Rhizobacteria and endophytic bacteria have been applied to The antibiotics produced by the PGPB mainly contain various crops to enhance growth, seed emergence, crop 2,4-diacetylphloroglucinol (2,4-DAPG), phenazine-1- : : : : : : carboxylic acid (PCA) and pyrrolnitrin (Prn) (Van Pee et R. Fang J. Lin S. Yao Y. Wang J. Wang C. Zhou al. 1983; Mavrodi et al. 2001; Makarand et al. 2007), all of : * H. Wang M. Xiao ( ) which actively contribute to the inhibition of plant patho- College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, People’s Republic of China gens. One easy approach to determine whether a PGPB can e-mail: [email protected] inhibit a specific pathogen and thus enhance the plant’s 1178 Ann Microbiol (2013) 63:1177–1185 ability to fight off that pathogen is to challenge that available P 42.43 μg·g−1; available K 158.7 μg·g−1;pH pathogen with a PGPB on the plant’s leaves (Bashan 8.08) and then maintained in a greenhouse [daytime tem- and de-Bashan 2002). perature 30°C; night temperature 25°C; relative humidity The colonization of roots by selected strains of nonpatho- 50 %; light intensity 60,000–100,000 μmol m−2s−1 (sunny genic PGPR leads to a phenotypically similar form of in- day), 1,000–10,000 μmolm−2s−1 (rainy day) ]. Each set duced resistance commonly referred to as induced systemic contained ten pots, and each pot contained four seeds. The resistance (ISR) (Van Loon et al. 2008), which is a state of bacterial solutions of the above density were poured into enhanced defensive capacity of plants which enables the each pot again after 1 week. Vegetative growth parameters mobilization of appropriate cellular defense responses be- (seedling height, shoot dry weight and root dry weight) were fore or upon pathogen attack (Bakker et al. 2007). Success- determined after 28 days (five-leaf stage) from 30 seedlings ful establishment of ISR depends on the recognition of randomly chosen from each set. Control experiments bacterial determinants by the plant roots, such as lipopoly- were performed in parallel with non-inoculated seed- saccharides, bacterial flagellins and pseudobactin (Bakker et lings; other treatments were the same as described al. 2007; Tran et al. 2007). These defense responses com- above. Each treatment was replicated three times. prise the oxidative burst characterized by the generation of active oxygen species (AOS) which is one of the earliest Colonization of P. aurantiaca strain JD37 in rhizosphere events produced in cell suspension cultures in response to soil and within the root tissue of maize rhizobacterial elicitors that can be linked to the development of ISR in whole plants (Van Loon et al. 2008). The inoculated seedlings were planted in soil in which P. Pseudomonas aurantiaca strains have been shown to aurantiaca JD37 was not originally found. The population enhance plant growth (Rosas et al. 2009), but they have density of P. aurantiaca JD37 in the rhizosphere was not been found to have any biocontrol features. We recently determined at 4-day intervals throughout the 28-day study isolated a P. aurantiaca strain, named JD37, from a suburb period. Roots from plants grown in soil were thoroughly of Shanghai, China, and found that it could inhibit plant shaken to remove adhering soil particles. Then, serial dilutions pathogens (Wang et al. 2010a). In the study reported here, of these soil samples were inoculated (three repetitions) on we found that antagonistic activity of strain JD37 may have KB plates supplemented with ampicillin (50 μg·mL−1)and promoted maize growth and led to ISR. Based on these chloramphenicol (30 μg·mL−1). After 48 h, the orange- results, we suggest that this specific strain can be used as a pigmented colonies on the KB plates were counted biofertilizer or biocide to promote the growth of crops and and characterized by amplification and sequencing of a replace chemical fertilizers, pesticides and supplements. partial sequence (1,034 bp) of the 16S rDNA gene, as described by Wang et al. (2010a). Colonization of strain JD37 within the root tissue of Materials and methods maize was determined as described above except that the roots were excised and any attached soil particles washed Seed treatment and growth promotion of maize off the root surface; the roots were then surface sterilized by P. aurantiaca JD37 with 0.3 % NaClO for 15 min, rinsed and weighed, cut it into pieces and homogenized in sterile distilled water. Pseudomonas aurantiaca JD37 (accession no. GQ358919, deposited in China General Microbiological Culture Collec- Antagonistic activity assays tion Center), with resistance to ampicillin and chloramphen- icol, was grown in King B (KB) medium at 28 °C (King et Antagonistic activity of P. aurantiaca JD37 was evaluated al. 1954; Wang et al. 2010a). Healthy maize (Zea mays L.) in vitro by the agar diffusion method (Bonev et al. 2008). seeds were surface sterilized in a 0.3 % sodium hypochlorite The JD37 strain was cultured in KB medium at 28 °C for (NaClO) water solution for 30 min, then rinsed ten times 28 h. Three samples were prepared: (1) bacterial fermenta- with sterile water and incubated on wet sterile filter paper in tion broth (1.2×108CFU·mL−1), (2) cell-free suspension sterile plates for 4 days at 28 °C to induce germination. obtained by centrifuging the above-mentioned bacterial fer- Seedlings were placed with their roots in a solution of P. mentation broth at 7,100 g for 10 min at 4 °C and (3) a aurantiaca JD37 [1.2×108 colony forming units (CFU) bacterial suspension prepared by resuspending the pellet mL−1, log phase] for 10 min. This experiment was replicated obtained following centrifugation (as described) in distilled three times. water (1.2×108CFU·mL−1). Three samples were inoculated The inoculated seedlings were transferred into pots into a hole at the center of three plates, respectively, of (height 18 cm, diameter at bottom 12 cm) containing 4 kg dextrose agar (PDA) medium. The 200 μLofBipo- of soil (organic matter 2.12 %; available N 173.61 μg·g−1; laris maydis spore suspension (104 conidia·mL−1), provided Ann Microbiol (2013) 63:1177–1185 1179 by Shanghai Research and Development Center for Pesti- at 11,000 g (4 °C, 15 min) and the supernatant collected and cides, was evenly dispersed on the above PDA medium then extracted twice with the same volume (about 0.5 L) of (three repetitions). After incubation for 5–6 days at 28 °C, dichloromethane analytical reagents (AR). After rotary antifungal activity was determined by measuring the zone of evaporation, the sample was dissolved with the mixture of inhibition (mm) around the holes. The plates inoculated with acetidin (AR) and methanol (AR) and then filtered through a only fungus served as a control. 0.22-μm filtration membrane (strain JD37 that may exist The non-inoculated seedlings were planted in pots under after centrifugation was discarded). greenhouse conditions [30±4 °C, 16/8-h (light/dark) photo- The antagonistic activity of the filtered sample was ana- period]. The P. aurantiaca JD37 suspension (1.2×108CFU lyzed using the agar-diffusion assays described above, with mL−1) was spread onto maize leaves after 28, 31 and 35 reverse-phase (RP)-HPLC (Agilent Technologies, Santa days, respectively, and then the conidial suspension of Bipo- Clara, CA) with a diode array detector. The C18 analytical laris maydis (1×104 conidia·mL−1) was sprayed onto the column (AQ-C18; 150×4.6 mm, i.d. 5 μm) was washed same site on the leaves and moistened with a humidifier for with 10 mL of mobile phase (methanol:water:phosphonic 1 week. The disease index and disease reduction were acid, 65:35:0.1, v/v/v), and 1–2 μLofthesamplewas calculated according to the formulas below. added; the temperature of the C18 column was 25 °C and P the flow rate was 1.0 mL·min−1. UV spectra of the ðÞ ¼ di li Disease index LN 100 compounds were scanned on a UV spectrophotometer Disease reduction ¼ ðÞI0 Ii =I0 100% (Persee, Shanghai, China). Where, d 0represents for the grade of disease severity, I ISR and hydrogen peroxide production lI0the number of leaves at different grades of disease, L0the number of all investigated leaves, N0the highest grade of Induced resistance assays were performed basically as de- disease severity, I 0the disease index of control, and I 0the 0 I scribed previously (De Vleesschauwer et al. 2006), with the disease index of different treatment groups. As a control, results indicating that non-pathogenic plant rhizosphere micro- bacteria-free deionized water was spread on leaves. organisms or the chemical factors they produced induced plant disease resistance responses to pathogens. Briefly, the non- Detection of genes responsible for the biosynthesis inoculated seedlings were dipped in the bacterial suspensions of antibiotics (1.2×108CFU·mL−1) for 10 min, transplanted to perforated plastic trays (23×16×6 cm) containing the autoclaved soil A PCR was used to detect biosynthesis genes and then transferred to the greenhouse [30±4 °C, 16/8-h according to the standard method with the specified primers (light/dark) photoperiod]; the bacterial inoculum was applied and with the genome of P. aurantiaca JD37 as templates a second time as a soil drench 12 days later. The leaves were (Sambrook et al. 1989). The PCR primers used for the ampli- detached from the treated 4-week-old seedlings and challenged fication of gene fragments responsible for the biosynthesis of with B. maydis. The maize plants were subjected to the same ′ PCA, Prn and 2,4-DAPG were 5 -TTGCCAAGCCTCGCT experiment except that salicylic acid (SA, 0.1 mM) (Shimono et ′ ′ ′ CCAAC-3 and 5 -CCGCGTTGTTCCTCGTTCAT-3 al. 2007), instead of strain JD37, was used as a positive control. ′ (Raaijmakers et al. 1997), 5 -CCACAAGCCCGGCCA Sterilized distilled water, instead of strain JD37, was used as a ′ ′ GGAGC-3 and 5 -GAGAAGAGCGGGTCGA negative control. The disease index and disease reduction were ′ ′ TGAAGCC-3 ,and5-GAGGACGTCGAAGAC determined as described above 6 days after inoculation. CACCA-3′ and 5′-ACCGCAGCATCGTGTATGAG-3′ Quantitative analyses of hydrogen peroxide (H2O2) produced (Mavrodi et al. 2001), respectively. PCR products were by leaves excised from the plant challenged with the pathogen detected by agarose gel electrophoresis (1 %) and then were performed using the Ti (IV)-PAR colorimetric method sequenced. The sequencing results were detected by the (A508nm) (Chiyo et al. 1983;Liuetal.2000). The amount of BLAST program from GenBank and compared to the −1 H2O2·g leaves was calculated on the basis of the standard sequences of published strains, such as Pseudomonas curve using an UV-spectrophotometer (A508nm) at the indicated chlororaphis (EU188755) and Burkholderia cenocepacia time points. The concentration of H2O2, between 0 to strain K56-2 (EU874251). 30 μmol·L−1, is allowed for in the Lambert–Beer’slaw(absor-

bance value is proportional to the concentration of H2O2). Analysis of antifungal substance by high-performance liquid chromatography Protection assays on detached leaves

Strain JD37 was grown for 60 h at 28 °C and 200 rpm in KB Protection assays on detached leaves were conducted as broth medium. Bacterial fermentation broth was centrifuged described previously (Verhagen et al. 2010). Strain JD37 1180 Ann Microbiol (2013) 63:1177–1185 was cultured in KB broth media. The bacterial fermentation Antagonistic activity of P. aurantiaca strain JD37 broth (1.2×108CFU·mL−1) was centrifuged at 11,000 g at 4 °C for 15 min to obtain the supernatant. The bacterial To analyze the cause of plant growth promotion by strain cells,whichwereharvestedandresuspendedin10mM JD37, we prepared three samples (sample 1: the bacteria

MgSO4 solution at the above-mentioned concentration, fermentation broth; sample 2: the cell-free suspension; sam- served as the live bacterial solution. The live bacterial solu- ple 3: bacterial suspension) and subjected these to antago- tion was boiled at 95 °C for 15 min to prepare crude cell nistic experiments against B. maydis. The agar diffusion extracts, designated the bacterial extract. assays showed that all three samples produced an inhibition Leaves were excised from 4-week-old maize plants zone (sample 1: 3.96±2.30 cm; sample 2: 2.76±3.21 cm; and treated with the above-mentioned supernatant, the sample 3: 3.24±2.07 cm). Sample 1 produced the largest live bacterial solution and the bacterial extract, respectively. inhibition zone, implying that the combined action of the

The detached leaves treated with MgSO4 solution, subjected bacterial cells and metabolites might be the most effective, to the same experimental treatment, served as a control. After while the presence of antagonistic activity in sample 2 48 h, leaves were rinsed with deionized water and placed on suggested that the supernatant of strain JD37 might have wet absorbing paper in dishes and then challenged with 200 contained antifungal substances. μLofaB. maydis conidial suspension (1×104 conidia·mL−1). The assay for the biocontrol effect of strain JD37 on Disease development was quantified as the average diameter southern maize leaf blight (Tatum 1971) caused by B. may- of lesions formed at 5 days post-inoculation. Disease index dis was performed on maize leaves. The disease index of the and disease reduction were calculated as previously inoculated leaves was significantly inhibited compared with mentioned. the control (Fig. 2a), indicating that strain JD37 was able to control southern maize leaf blight. This result was supported Statistical analysis by data from experiments on disease reduction (Fig. 2b).

All experiments were performed at least three times per Antagonistic substance produced by P. aurantiaca strain treatment and the data analyzed by analysis of variance JD37 (ANOVA). When ANOVA showed treatment effects (P< 0.05), the least significant difference test (LSD) and To determine whether strain JD37 could produce antagonistic Duncan’s multiple range test (for maize) were applied substances, we amplified the genes responsible for the bio- to make comparisons among the means. The statistical synthesis of antibiotic using the PCR primers described in the package SPSS ver. 17.0 (SPSS, Chicago, IL) was used section Detection of genes responsible for the biosynthesis of for all analyses. antibiotics. A 693- and 1,086-nucleotide gene sequence asso- ciated to the biosynthesis of Prn and PCA, respectively, were obtained (Fig. 3a). However, no gene was found for the Results biosynthesis of 2,4-DAPG (data not shown). The results from NCBI BLAST programs showed that the 693-nucleotide gene Growth promotion of maize by P. aurantiaca JD37 shared 98 % identity with the same gene of (EU188755), while the 1,086-nucleotide gene Maize plants were inoculated with strain JD37 to examine shared 98 % identity with that of Burkholderia cenocepacia its effect on plant growth promotion. In terms of promoting strain K56-2 (EU874251). plant growth, the bacterial suspension significantly affected The purified supernatants of strainJD37showedantifungal seedling height, shoot dry weight and root dry weight, activity against B. maydis on PDA plates. RP-HPLC chromato- compared with the controls (Table 1). Inoculation with P. grams showed four different absorption peaks at different re- aurantiaca JD37 therefore impacted favorably on maize tention times, indicating at least four ingredients in the bacterial growth. supernatants (Fig. 3b). From the full-band scan of UV-visible The colonization experiments verified the presence of absorption spectra, the two ingredients of the purified com- strain JD37 in the rhizosphere soil and in the internal roots pounds showed maximum UV-absorption at 255 and 370 nm, of the plants (Fig. 1). A bacterial population of 107CFU was which were similar to the maximum absorption of Prn (Van Pee observed at 4 days post-inoculation, which was maintained et al. 1983) and PCA (Makarand et al. 2007), respectively, at at about 106CFU during the following period (8–28 days similar times (Fig. 3c). Thus, we preliminarily considered that post-inoculation) per gram of rhizosphere soil. In each gram the purified compounds from the culture supernatants of strain of root tissue, the population density increased up to 4.2× JD37 may contain the two kinds of antibiotics, Prn and PCA, 104CFU by 4 days post-inoculation and remained at 1×104 consistent with the results of the PCR analysis. The other two until 28 days post-inoculation. absorption peaks showed maximum UV adsorption only in the Ann Microbiol (2013) 63:1177–1185 1181

Table 1 Growth promotion of maize by Pseudomonas aurantiaca strain JD37

Treatmenta Average seedling height (cm) Average shoot dry weight (g) Average root dry weight (g)

Control 12.42±0.622 a 0.16±0.039 a 0.097±0.014 a JD37 14.72±0.395 b 0.25±0.032 b 0.142±0.016 b

Values represent the mean of three replicates ± standard deviation (SD). Values followed by different lowercase letters are statistically significant different at P<0.05 according to the LSD test a Bacterial suspensions of P. aurantiaca JD37: 1.2×108 CFU·mL−1 . Thirty seedlings were randomly chosen from each set for analysis. The experiment was replicated three times low band (data not shown), and we speculate that these were that bacterial colonization was confined to the root zone most likely impurities. (data not shown).

We next compared pathogenesis-related H2O2 generation, – Plant ISR caused by P. aurantiaca strain JD37 which is one of the most unique defense responses in plant pathogen interactions (Van Loon et al. 2008). Compared to the To verify if P. aurantiaca JD37 was capable of inducing control, the amount of H2O2 from the leaves treated with strain μ resistance in maize, which might be an indirect factor pro- JD37 increased more rapidly, reaching approximately 9 mol −1 moting maize growth, we grew maize plants in soil contain- g leaves by day 4 post-inoculation (Fig. 4b), indicating that ing P. aurantiaca JD37 and subsequently challenged the addition of JD37 to the rhizosphere soil of maize was detached leaves with the pathogen B. maydis. Six days after capable of inducing an oxidative burst of AOS. the challenge, the inoculated plants produced a resistance phenotype characterized by the appearance of many small Protection assays on detached maize leaves (diameter <6 mm) dark-brown necrotic spots on the leaves and exhibited a marked disease reduction (28.56 %) To further analyze the ISR of plants by strain JD37, we (Fig. 4a). The application of SA, one of the most extensively performed protection assays on detached leaves. Five days studied plant defense activators in plants (Shimono et al. 2007), induced an even higher level of protection, reducing the number of susceptible-type lesions by as much as 41.9 %. In contrast, the leaves of non-induced control plants developed large, spindle-shaped lesions with a yellow brown center (diameter >6 mm), often surrounded by chlorotic or necrotic tissue (Fig. 4a). To rule out the possibility that the observed disease protection was due to direct effects of JD37 on B. maydis, possible spreading of root-inoculated bacteria to foliar tis- sues was assessed by plating leaf extracts from induced plants onto selective KB agar plates. However, JD37 bacte- ria were not detected in the internal leaf tissues, indicating

Fig. 2 The biocontrol effect of P. aurantiaca strain JD37 on southern maize leaf blight caused by Bipolaris maydis. Deionized water (lane 1) Fig. 1 Bacterial colonization assays. The maize seeds inoculated with or strain JD37 suspension (1.2×108CFU·mL−1)(lane 2) was spread on P. aurantiaca strain JD37 (1.2×108CFU·grain−1) were sown in the maize leaves and the leaves subsequently challenged with the conidial soil. The bacterial population density in maize rhizosphere soil and in suspension of B. maydis (1×104 conidia·mL−1). After 1 week, the the internal roots of the plants were determined at indicated time average disease index (a) and disease reduction (b) were calculated. points. Values represent the mean of three replicates±standard Values represent the mean of three replicates. Different letters denote a deviation (SD) significant difference according to the LSD test (P<0.05) 1182 Ann Microbiol (2013) 63:1177–1185

Fig. 3 Detection of antagonistic substances produced by P. aurantiaca strain JD37. a Genes responsible for the biosynthesis of antagonistic substances were analyzed by PCR. Lanes: M DL2000 DNA marker, 2, 3 DNA fragments amplified with the specified primers for gene clusters responsible for the biosynthesis of phenazine-1- carboxylic acid (PCA)(1) and pyrrolnitrin (Prn)(2). b PCA- or Prn-like antagonistic sub- stances were obtained by reverse-phase high- performance liquid chromatog- raphy. 1, 2, 3, 4 Four peaks. c UV-visible absorption spectra of the purified compounds. The two substances of the purified compounds both showed maxi- mum UV adsorption at 255 and 370 nm, respectively

after challenge with pathogens, the control leaves treated diazotrophicus, Herbaspirillum seropedicae (Bastian et al. with a MgSO4 solution developed large necrotic lesions 1998), Burkholderia cepacia (Mendes et al. 2007), Pseudo- (diameter >20 mm). The detached leaves incubated with monas putida (Glick et al. 1997)andAzospirillum (Bashan et the live bacterial solution, the corresponding bacterial al. 2004; Bashan and de-Bashan 2010) The first case of P. extracts and the supernatant exhibited a significant reduction aurantiaca enhancing plant growth was reported by Rosas et in disease symptoms (Fig. 4c). The treatment with the al. (2009). In our study, another P. aurantiaca strain, JD37, extracts and the supernatant resulted in obvious disease was also found to have a plant growth-promoting property. reduction, suggesting that the two samples may contain the Pseudomonas spp. have been reported as being able to determinants of ISR; however, this remains to be verified in biologically control different fungi, including species of future experiments. Rhizoctonia, Fusarium, Sclerotium, Pythium, Erwinia and Macrophomina (Negi et al. 2005; Li et al. 2011). The broad- spectrum antagonistic activity of the pseudomonads are due Discussion to the secretion of a number of metabolites, including anti- biotics (Haas and Keel 2003), volatile hydrocyanic acid Plant growth promotion by rhizobacteria and endophytic bac- (Bhatia et al. 2003) and siderophores (Gupta et al. 2002). teria has been the focus of research for both academic and Our investigations of the antagonistic activity of strain practical reasons because beneficial interactions between JD37 in the PDA plates and our evaluation of the biocontrol these bacteria and plants have a tremendous potential for field effect of strain JD37 on maize plants revealed: (1) that strain applications. Examples of such bacteria include Acetobacter JD37 had an antagonistic activity against B. maydis and (2) Ann Microbiol (2013) 63:1177–1185 1183

Fig. 4 Induction of the systemic resistance of maize plants by strain P. was calculated at the indicated time points. c Detached leaves were aurantiaca JD37. a Plants were grown in soil drenched with salicylic incubated for 48 h with a 10 mM MgSO4 solution (1), live bacterial acid (SA, 0.1 mM; 1), a suspension of strain JD37 (1.2×108CFU solution (2), crude bacterial extract (3) or supernatant (4), and after- mL−1; 2) or deionized water (3), and then challenged with the conidial wardswashedwithdeionizedwaterandthenchallengedwithB. suspension of B. maydis (1×104 conidia·mL−1). Photographs depicting maydis. Disease reduction (%) was calculated 5 days after challenge representative symptoms were taken 6 days after inoculation (left); with B. maydis. Different letters indicate significant differences be- disease reduction was also calculated (right). b Induction of oxidative tween treatments according to Duncan’s multiple range test (α00.05). −1 burst in maize leaves by strain JD37. The amount of H2O2 g leaves All experiments were performed at least three times that strain JD37 was able to control the southern maize leaf against and Pseudomonas syringae blight caused by B. maydis. The data from RP-HPLC assays (Corné et al. 1996). showed that strain JD37 might produce two kinds of anti- Some bacteria can trigger plant ISR, based on multiple biotics, namely, PCA and Prn. PCA and Prn are important mechanisms, including the enhancement of the capacity of antibiotic substances found in many PGPB (Bashan and plants to mobilize cellular defense responses before or upon Holguin 1998). Previous reports indicated that the biosyn- pathogen challenge. ISR has been observed in some PGPB thesis of PCA and Prn was regulated by the responsible (Bakker et al. 2007; Van Loon et al. 2008). To determine genes (Van Pee et al. 1983; Makarand et al. 2007). Indeed, whether strain JD37 triggered plant ISR, we performed our PCR method using the specified primers for the gene different protection assays on maize leaves; in all of these clusters responsible for the biosynthetic antibiotic substan- assays, there was a reduction in plant disease. These findings ces showed the corresponding DNA sequence. The results suggest that the JD37-provoked disease suppression was not of these analyses indicated that the JD37 strain may produce due to microbial antagonism but rather resulted from the PCA and Prn and inhibit phytopathogens, and so be very activation of the plant’s own defensive repertoire. Moreover, useful in the biological control of plant diseases. These the production of AOS, one of the most unique defense antibiotic substances presumably endow strain JD37 with responses in plant–pathogen interactions (De Vleesschauwer antagonistic activity, which is a probable cause of the et al. 2008; Van Loon et al. 2008), was also detected in leaf biocontrol. cells of maize seedlings grown in soil inoculated with strain

Research on biological control mechanisms by PGPR JD37. H2O2 was extracted from leaves treated with strain and endophytic bacteria has revealed that some strains JD37, SA (positive control) or sterilized distilled water are able to protect plants against pathogen infection (negative control), and the H2O2 amount was measured through ISR, without provoking any symptoms them- by the Ti (IV)-PAR colorimetric method. The results show selves; for example, biocontrol strain Pseudomonas flu- that there was a positive correlation between oxidative bursts orescens WCS417r caused ISR in Arabidopsis thaliana and induced resistance. 1184 Ann Microbiol (2013) 63:1177–1185

Some factors, such as lipopolysaccharides in cellular that causes charcoal rot of ground nut. Indian J Exp Biol – membranes, bacterial flagellins, pseudobactin and even cer- 41:1441 1446 Bonev B, Hooper J, Parisot J (2008) Principles of assessing bacterial tain antibiotics act as the bacterial determinants of ISR susceptibility to antibiotics using the agar diffusion method. J (Meziane et al. 2005). The crude bacterial extracts and the Antimicrob Chemother 61:1295–1301 cell-free supernatant of strain JD37 also triggered ISR in Chiyo M, Yuji N, Yasushi Y, Kiyoko T (1983) A spectrophotometric maize leaves, implying that the above extracts and superna- method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase. Anal tant might contain bacterial determinants of ISR. These Biochem 130:128–133 results suggest that some compounds released after boiling Christiansen-Weneger C (1992) N2-fixation by ammonium- the bacteria were sufficient for maize ISR. Effects of bacte- excreting Azospirillum brasilense in auxin-induced tumours – rial extracts and supernatants, which contain cell-wall frag- of wheat (Triticum aestivum L.). Biol Fertil 12:85 100 Corné MJ, Pieterse, Saskia CM, Van Wees, Ellis Hoffland, Johan A, ments, have also suggested a possible role of membrane Van Pelt, Leendert C, Van Loon (1996) Systemic resistance in lipopolysaccharides in inducing disease resistance (Van Arabidopsis induced by biocontrol bacteria is independent of Loon et al. 2008). Therefore, the above components might salicylic acid accumulation and pat hogenesis-related gene – be present in JD37 and act as the elicitors of defense expression. Plant Cell 8:225 1237 De Freitas JR, Banerjee MR, Germida JJ (1997) Phosphate solubilizing responses in maize; however, this remains to be verified in rhizobacteria enhance the growth and yield but no phosphorus future studies. uptake of canola (Brassica napus L.). Biol Fertil Soils 24:358– In this study, we have performed a preliminary 364 exploration of the ability of P. aurantiaca strain JD37 De Vleesschauwer D, Cornelis P, Hofte M (2006) Redox-active pyo- cyanin secreted by Pseudomonas aeruginosa 7NSK2 triggers to promote the plant growth-promoting mechanism as systemic resistance to Magnaporthe grisea but enhances Rhizoc- well as the induction of plant systemic resistance. The tonia solani susceptibility in rice. Mol Plant Microbe Interact underlying mechanisms and the induction of systemic 19:1406–1419 resistance determinants require further study. De Vleesschauwer D, Djavaheri M, Bakker PAHM, Hofte M (2008) Pseudomonas fluorescens WCS374r-Induced systemic resistance in rice against Magnaporthe oryzae is based on pseudobactin- Acknowledgments This work was supported by the National mediated priming for a salicylic acid-repressible multifaceted Natural Science Foundation of China (30930019), the Shanghai defense response. Plant Physiol 148:1996–2012 Municipal Science and Technology Commission (11440502300), Dey R, Pal KK, Bhatt DM, Chauhan SM (2004) Growth promotion the Shanghai Municipal Education Commission (11YZ87) and and yield enhancement of peanut (Arachis hypogaea L.) by ap- Shanghai Normal University (SK201111). plication of plant growth-promoting rhizobacteria. Microbiol Res 159:371–394 Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41:107–120 References Glick BR, Liu C, Ghosh S, Dumbroff EB (1997) Early development of canola seedlings in the presence of the plant growth-promoting rhizobacterium Pseudomonas putida GR 12–2. Soil Biol Biochem – Bakker PAHM, Pieterse CMJ, Van Loon LC (2007) Induced systemic 29:233 1239 resistance by fluorescent Pseudomonas spp. Phytopathology Gupta CP, Dubey RC, Maheshwari DK (2002) Plant growth enhance- 97:239–243 ment and suppression of Macrophomina phaseolina causing char- Bashan Y, de-Bashan LE (2002) Protection of tomato seedlings against coal rot of peanut by fluorescent Pseudomonas. Biol Fertile Soils – infection by Pseudomonas syringae pv tomato by using the plant 35:399 405 growth-promoting bacterium Azospirillum brasilense. Appl Environ Haas D, Keel D (2003) Regulation of antibiotic production in root Microbiol 68:2637–2643 colonizating Pseudomonas spp. and relevance for biological – Bashan Y, de-Bashan LE (2005) Bacteria/Plant growth-promotion. In: control of plant diseases. Annu Rev Phytopathol 41:17 153 Hillel D (ed) Encyclopedia of soils in the environment, vol 1. King EO, Ward MK, Raney DE (1954) Two simple media for the Elsevier, Oxford, pp 103–115 demonstration of phycocyanin and fluorescin. J Lab Clin Med – Bashan Y, de-Bashan LE (2010) How the plant growth-promoting 44:301 307 bacterium Azospirillum promotes plant growth—a critical assessment. Kloepper JW (1992) Plant growth-promoting rhizobacteria as biological Adv Agron 108:77–136 control agents. In: Metting FB Jr (ed) Soil microbial ecology: Bashan Y, Holguin G (1998) Proposal for the division of plant growth- applications in agricultural and environmental management. Marcel – promoting rhizobacteria into two classifications: biocontrol- Dekker, New York, pp 255 274 PGPB (plant growth-promoting bacteria) and PGPB. Soil Biol Li H, Li H, Bai Y, Wang J, Nie M, Bo L, Xiao M (2011) The use of Biochem 30:1225–1228 Pseudomonas fluorescens P13 to control sclerotinia stem rot Bashan Y, Holguin G, de-Bashan LE (2004) Azospirillum–plant (Sclerotinia sclerotiorum) of oilseed rape. J Microbiol 49:884– relationships: physiological, molecular, agricultural, and environmental 889 advances (1997–2003). Can J Microbiol 50:521–577 Liu J, Bo LB, Xu LL (2000) An improved method for the determination Bastian F, Cohen A, Piccoli P, Luna V, Baraldi R, Bottini R (1998) of hydrogen peroxide in leaves. Prog Biochem Biophys 27:548– Production of indole-3-acetic acid and gibberellins A1 and A3 by 551 Acetobacter diazotrophicus and Herbaspirillum seropedicae in Lugtenberg B, Kamilova F (2009) Plant-growth-promoting rhizobacteria. chemically-defined culture media. Plant Growth Regul 24:7–11 Annu Rev Microbiol 63:541–556 Bhatia S, Dubey RC, Maheshwari DK (2003) Antagonistic effect of Makarand RR, Prashant DS, Bhushan LC (2007) Detection, isolation fluorescent pseudomonads against Macrophomina phaseolina and identification of phenazine-1-carboxylic acid produced by Ann Microbiol (2013) 63:1177–1185 1185

biocontrol strains of Pseudomonas aeruginosa. J Sci Ind Res Shimono M, Sugano S, Nakayama A, Jiang CJ, Ono K, Toki S, Takatsuji 66:627–631 H (2007) Rice WRKY45 plays a crucial role in benzothiadiazole- Mavrodi OV, Gardener BBM, Mavrodi DV (2001) Genetic diversity of inducible blast resistance. Plant Cell 19:2064–2076 phlD from 2,4-diacetylphloroglucinol-producing fluorescent Tatum LA (1971) The southern corn leaf blight epidemic. Science Pseudomonas spp. Phytopathology 91:35–43 171:1113–1116 Mendes R, Pizzirani-Kleiner AA, Araujo WL, Raaijmakers JM (2007) Tran H, Ficke A, Asiimwe T, Hofte M, Raaijmakers JM (2007) Role of Diversity of cultivated endophytic bacteria from sugarcane: the cyclic lipopeptide massetolide A in biological control of genetic and biochemical characterization of Burkholderia cepacia Phytophthora infestans and in colonization of tomato plants by complex isolates. Appl Environ Microbiol 73:7259–7267 Pseudomonas fluorescens. New Phytol 175:731–742 Meziane H, Sluis I, Van Loon LC, Höfte M, Bakker PAHM (2005) Van Loon LC, Bakker PAHM, Van Der Heijdt WHW, Wendehenne D, Determinants of Pseudomonas putida WCS358 involved in in- Pugin A (2008) Early responses of tobacco suspension cells to ducing systemic resistance in plants. Mol Plant Pathol 6:177–185 rhizobacterial elicitors of induced systemic resistance. Mol Plant Negi YK, Garg SK, Kumar J (2005) Cold tolerant fluorescent Microbe Interact 21:1609–1621 Pseudomonas isolates from Garhwal Himalayas as potential Van Pee KH, Salcher O, Fischer P (1983) The biosynthesis of brominated plant growth promoting and biocontrol agents in pea. Curr pyrrolnitrin derivatives by Pseudomonas aureofaciens. J Antibiot Sci 89:2151–2156 (Tokyo) 36:1735–1742 Raaijmakers J, Weller DM, Thomashow LS (1997) Frequency of Verhagen BWM, Trotel-Aziz P, Couderchet M, Hofte M, Aziz A antibiotic producing Pseudomonas spp. in natural environments. (2010) Pseudomonas spp.-induced systemic resistance to Botrytis Appl Environ Microbiol l66:881–887 cinerea is associated with induction and priming of defence Rosas SB, Avanzini G, Carlier E, Pasluosta C, Pastor N, Rovera M responses in grapevine. J Exp Bot 61:249–260 (2009) Root colonization and growth promotion of wheat and Wang Y, Xiao M, Geng X, Liu J, Chen J (2007) Horizontal transfer of maize by Pseudomonas aurantiaca SR1. Soil Biol Biochem 21 genetic determinants for degradation of phenol between the (9):1802–1806 bacteria living in plants and its rhizosphere. Appl Microbiol Safronova VI, Stepanok VV, Engqvist GL, Alekseyev YV, Belimov AA Biot 77:733–739 (2006) Root-associated bacteria containing 1-aminocyclopropane-1- Wang H, Song J, Zhu Y, Zhou Y, Xiao M (2010a) Screening of an carboxyate deaminase improve growth and nutrient uptake by pea antagonistic bacterial strain against plant pathogenic fungi and its genotypes cultivated in cadmium supplemented soil. Biol Fertil Soils antimicrobial mechanism. J Microbiol 30:7–13 42:267–272 Wang Y, Li H, Zhao W, He X, Chen J, Geng X, Xiao M (2010b) Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a Induction of toluene degradation and growth promotion in corn laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold and wheat by horizontal gene transfer within endophytic bacteria. Spring Harbor Soil Biol Biochem 42:1051–1057