Promotion of Plant Growth, Biological Control and Induced Systemic Resistance in Maize by Pseudomonas Aurantiaca JD37
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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 Pseudomonas 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 bacteria in sustainable isolated from a suburb of Shanghai, China, was found to agriculture is steadily increasing and offers an attractive effectively colonize the rhizosphere soil 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 antibiotics 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 potato 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.