Environment Health Techniques 898
Sang-Mi Yu and Yong Hoon Lee
Research Article Genes involved in nutrient competition by Pseudomonas putida JBC17 to suppress green mold in postharvest satsuma mandarin Sang-Mi Yu1 and Yong Hoon Lee1,2 1
2
Division of Biotechnology, Chonbuk National University, 79 Gobong-ro, Iksan-si, Jeollabuk-do 570-752, Republic of Korea Advanced Institute of Environment and Bioscience, and Plant Medical Research Center, Chonbuk National University, Jeollabuk-do, Republic of Korea
Understanding the mechanisms underlying biocontrol activity in biocontrol agents is indispensable to implement biological control. However, the understanding of specific mechanisms for nutrient competition in nutrient limited environments is still limited. This study was performed to control green mold of postharvest satsuma mandarin (mandarin) using Pseudomonas putida JBC17 (JBC17), and identify the genes involved in nutrient competition. Treatment with JBC17 on wounded mandarin fruits at a concentration of 106 and 107 cfu ml1 suppressed the incidence of green mold with efficacy of 74.1 and 91.4%, respectively, compared to the untreated control. In spite of there being no antifungal activity in a dual culture test, JBC17 significantly inhibited the conidial germination of Penicillium digitatum. The results from the nutrient competition assay revealed that the inhibition of conidial germination was exerted by nutrient starvation. From the constructed transposon (Tn) library of JBC17, exopolyphosphatase (ppx) and Xaa-Pro aminopeptidase (pepP) were recognized as potential factors responsible for the inhibition of conidial germination. In conclusion, the understanding of nutrient depletion specific to the inhibition of conidial germination by JBC17 may ultimately lead to a deeper understanding of the bacterial metabolism and conidial metabolism for germination as well as biocontrol activity.
: Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: Aminopeptidase / Bicontrol mechanism / Conidia / Exopolyphosphatase / Postharvest spoilage Received: October 14, 2014; accepted: January 22, 2015 DOI 10.1002/jobm.201400792
Introduction Green mold of mandarins (Citrus unshiu Marc.) caused by Penicillium digitatum is one of the major factors limiting the storage of fruits, and causes severe economic losses worldwide in citrus [1, 2]. To control the postharvest spoilage, farmers mainly rely on the use of synthetic fungicides. However, the use of fungicides is becoming restricted due to the development of fungicide-resistant
Correspondence: Yong Hoon Lee, Division of Biotechnology, Chonbuk National University, 79 Gobong-ro, Iksan-si, Jeollabuk-do 570-752, Republic of Korea E-mail:
[email protected] Phone: þ82-63-850-0841 Fax: þ82-63-850-0834 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
pathogens and public concerns regarding human health and environment [2–4]. Biological control using microbial antagonists has received a great deal of attention as a promising alternative method to fungicides for controlling postharvest diseases in citrus. The use of microorganisms or their secondary metabolites to prevent postharvest disease, is environmental-friendly, safe, and may provide long-term protection [5, 6]. Hence, many bacteria, such as Bacillus amyloliquefaciens [7, 8], Bacillus subtilis [9], Pantoea agglomerans [10, 11], Pseudomonas cepacia [12], Pseudomonas fluorescence [13], and Pseudomonas syringae [14] have been reported to be effective biocontrol agents against postharvest diseases including citrus. Pseudomonas putida has also been explored as a potentially excellent biocontrol agent against plant
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J. Basic Microbiol. 2015, 55, 898–906
Genes for nutrient competition to inhibit conidial germination
pathogens [15, 16]. Preplant treatments of potato seed pieces and postharvest treatments of potato tubers with P. putida reduced soft rot caused by Erwinia spp. to 50 and 25%, respectively [17]. P. putida Cha 94 inhibited conidial germination of Fusarium oxysporum, Aspergillus sp., and Botrytis allii, which cause basal and neck rot during onion storage [18]. P. putida MGP1 also reduced postharvest blight caused by Phytophthora nicotianae in papaya [19]. Several mechanisms have been suggested to operate on biocontrol, including antibiosis, direct parasitism, competition for niche and limited resources, and induced resistance [1]. Recently, many studies have reported the role of competition for nutrients as an essential mechanism in postharvest biocontrol [11, 20]. However, the specificity of nutrients and genes, and the metabolism responsible for the competition between biocontrol agents and fungal pathogens in nutrient limited environments are still yet to be investigated. In this study, P. putida JBC17, a potential biocontrol agent, was tested for the suppression of green mold in wounded mandarin fruits, and its putative mode of action was determined. Furthermore, the genes responsible for the inhibition of conidial germination under nutrient limited conditions were identified using Tn mutants of JBC17. We believe that the results of this study will increase our understanding on nutrient competition in biocontrol activity.
Materials and methods Isolation of JBC17 and culturing of the bacteria The JBC17 was isolated from a soil sample collected from a strawberry greenhouse and identified as P. putida according to the results of the carbon source utilization pattern (Biolog, Inc., USA) and 16 S rDNA analysis. The bacterial strain was stored in 15% glycerol at 70 °C and revived as necessary. The bacterial cells which were grown in trypic soy broth at 28 °C and 200 rpm were harvested at the beginning of the stationary phase by centrifugation at 4000 g for 15 min and resuspended in 0.05 M phosphate buffer (pH 6.5) or distilled water (DW) to the desired concentration for the biocontrol assay. Culturing of green and blue mold pathogens P. digitatum KACC42258 isolated from citrus fruit was obtained from the Korean Agricultural Culture Collection (KACC) and maintained on potato dextrose agar (PDA) with periodic transfers to mandarin fruit to maintain pathogenicity. A conidial suspension for the inoculation of mandarin fruit was obtained by adding ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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10 ml of sterile DW with 0.01% of Silwet L-77 (Lehle Seeds, TX, USA) over the surface of 7–10 day-old PDA cultures and rubbing the surface with a loop. The conidial suspension was vortexed and number of conidia was counted in a hemocytometer, and diluted to the optimal concentration as needed. Biocontrol assay of the antagonists P. digitatum was inoculated on mandarin fruits which were surface-disinfested with 70% ethanol as described by Yu and Lee [8]. Briefly, the fruits were wounded with a toothpick by making an injury 1 mm deep, and a 10 ml suspension of P. digitatum at 105 conidia ml1 was applied to each wound, followed by inoculation with 20-ml suspension of JBC17 after 1 h. The treated fruits were incubated at 20 °C and 90% RH in closed plastic containers. The effect of the bacterial concentration on the incidence of green mold was assessed at 106, 107, 108, and 109 colony forming units (cfu) ml1. Ten fruits with four wounds per fruit were used for each treatment with three replicates. Data were recorded as the number of infected wounds 7 days after inoculation (DAI). Assay of antifungal activity The antagonistic activity of JBC17 on the mycelial growth of fungal pathogens infecting mandarin, such as Penicillium italicum (blue mold), Botrytis cinerea (gray mold), Altenaria alternata (black rot), Phytophthora citrophthora (Phytophthora rot), and Geotrichum candidum (sour rot) as well as P. digitatum was tested as described by Yu et al. [11]. An agar plug (8mm diameter) from the margins of a 7-10 day-old culture of the fungal pathogens was placed at the center of a PDA plate. And 10 ml of cell suspension of JBC17 was inoculated on paper disks (8 mm) which were laid 2.5 cm away from the center of the plate. The inhibition zones were recorded 7 DAI at 25 °C. Inhibition of conidial germination of P. digitatum and P. italicum by direct contact The inhibitory activity of JBC17 on the germination of conidia of P. digitatum was performed as described by Calvo et al. [21] with small modifications. Aliquots of 100 ml of JBC17 suspensions (1 106, 107, 108, and 109 cfu ml1) in 10% potato dextrose broth (PDB) were put in 24-well plates, and 100 ml of conidial suspension (5 105 conidia ml1) was immediately added. Controls were made with 100 ml of 10% PDB without cells of JBC17 and 100 ml of conidial suspension (5 105 conidia ml1). The well plates were incubated in a wet chamber at 25 °C for 24 h. The germination of conidia was stopped with lactophenol cotton blue, and the germination rate and
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J. Basic Microbiol. 2015, 55, 898–906
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Sang-Mi Yu and Yong Hoon Lee
germ tube length were measured with a light microscope. Five microscopic fields were selected and 20 conidia were observed per field. The number of germinated conidia (the conidium was considered to be germinated when the length of the germ tube was equal to length of the conidium) and the length of the germ tubes were measured with a micrometer. The percentages of inhibition were calculated by comparison to the control. Each experiment was repeated three times. Competition for nutrients The effect of nutrient depletion by JBC17 on the germination and growth of P. digitatum conidia was tested following Janisiewicz et al. [19] with minor modifications. Briefly, mandarin peel juice (0.5 and 5% w/v) which was prepared as needed by macerating fresh fruit peels was dispensed into the wells (0.9 ml per well) of 24-well tissue culture plates containing cylinder inserts with a hydrophilic polytetrafluoroethylene (PTFE) membrane (pore size 0.45 mm) attached at the bottom, with or without JBC17 (5 107 cfu ml1). The P. digitatum conidial suspension in DW (5 106 conidia ml1) was dispensed inside the cylinder inserts (0.1 ml per cylinder). The cylinders were placed in the wells and the plates were incubated at 25 °C. After 24 h of incubation, cylinders were removed from the wells and the membrane was blotted with tissue paper until all of the liquid from the inside of the cylinders was absorbed. The membrane was cut out with a scalpel, transferred to a glass slide and conidial germination was observed under a microscope. The germination rate was evaluated by comparing the size of the germ tube with the length of each conidium. The same set of culture plates with the inserts were prepared to determine viability of the conidia after 24 h of interaction with JBC17. After 24 h of incubation with the suspension, the membranes of the inserts were blotted and inserted into new wells containing only mandarin peel juice. After an additional 24 h of incubation, germination of conidia was observed as described above. Generation and screening of Tn insertion mutants Tn mutants were generated by tri-parental mating using plasposon TnMod-KmO and helper plasmid pRK2013 following Dennis and Zylstra [22]. Transposants were selected by plating on Luria-Bertani (LB) agar containing kanamycin (50 mg ml1) and vancomycin (50 mg ml1) for counter selection against the donor strain. Mutants were picked and transferred individually to 96-well microtiter plates containing LB medium with kanamycin and 20% (v/v) glycerol. After growing for 2 days at 28 °C, the plates ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
were stored at 80 °C. The Tn insertion mutants were individually multiplied on LB agar plates with kanamycin at 28 °C for 48 h and then screened for decreased ability to inhibit germination and growth of P. digitatum conidia as described above. The wild type JBC17 was used as a positive control. Two independent experiments were performed with two replicates of each mutant. Determination of the Tn insertion site The genes of interest were identified using inverse polymerase chain reaction (PCR) as described previously [23] with minor modifications. The chromosomal DNA was isolated and digested with restriction enzyme EcoRI, NcoI, or PstI (Promega Co. Madison, USA) as recommended by the manufacturer. The enzyme was inactivated by heating at 65 °C for 20 min. The DNA fragments were self-ligated using T4 DNA ligase and transformed into Escherichia coli DH5a. The colonies containing pTnMod-Okm plasmids in a portion of genomic DNA of JBC17 was selected with kanamycin. The DNA fragments containing Tn were isolated after culturing the strains and used for sequencing. In addition, the T4 DNA ligation mixture was used as a template for an inverse PCR with Tn-specific primers, pTnMod-OKm S1 (50 -CCCGTTGATTTGAGACA0 CAACGTGGC-3 ) and pTnMod-OKm AS1 (50 0 GTTCTTTCCTGGTACCGTCGACATGCA-3 ). The PCR products were separated by agarose gel electrophoresis, purified using the QIAGEN gel extraction kit, and then used as the templates for DNA sequencing which was performed at the DNA sequencing laboratory of SolGent Ltd. (Daejon, Korea). Two independent sequencing reactions were performed, each using one of the oligonucleotides pTnMod-OKm S1 or pTnMod-OKm AS1, and the genome regions flanking the transposon were sequenced. The resulting nucleic acid sequences were used to identify the mutated gene by comparison to genome sequences available at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm. nih.gov) and the Pseudomonas genome database (www. pseudomonas.com). All DNA techniques were performed as described by Sambrook and Russell [24]. Complementation of Tn mutants Each mutant in the exopolyphosphatase (mutant ID PPM90) or Xaa-Pro aminopeptidase gene (ID PPM1657) was complemented by amplifying the gene including a >100 bp upstream region from JBC17 genomic DNA by PCR. The primers, PPM90-F (50 -CTGCAGCGAATGACCGGGCAAC-30 , the PstI site is underlined) and PPM90-R (50 -GGATCCCTGGCTATCAGCTGCC-30 , the BamHI site is underlined), and PPM1657-F (50 -
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J. Basic Microbiol. 2015, 55, 898–906
Genes for nutrient competition to inhibit conidial germination
CTGCAGTGGAGTACCTGCGTG-30 , the PstI site is underlined), and PPM1657-R (50 -GGATCCCTGTAATGC0 CATCGCC-3 , the BamHI site is underlined) were used for PPM90 and PPM1657, respectively. Each PCR product was subcloned into the pUCP18 vector [25] using the PstI and BamHI restriction sites. The recombinant pUCP18 was transformed into each Tn mutant to create a complemented strain. The complementation of inhibition ability of each mutant for conidial germination was compared with the wild type JBC17.
Results Biocontrol efficacy of spoilage by green and blue mold We assayed various cell concentrations (106, 107, 108, and 109 cfu ml1) of JBC17 on wounded mandarin fruits to control green mold. In general, the higher the cell concentration of JBC17, the lower the disease incidence. The incidence of green mold at a concentration of 106 and 107 cfu ml1 of JBC17 decreased to 50 and 25%, respectively, in comparison to 95–100% incidence in nontreated fruits (Fig. 1). Furthermore, the biocontrol efficacy increased significantly (p < 0.05) to 95% upon raising the concentration of JBC17 to 108 cfu ml1. Antagonism on fungal pathogens The antagonism of JBC17 on fungal pathogens causing spoilage during postharvest storage of mandarin was
Figure 1. Incidence of green mold on wounded mandarin fruits treated with different concentrations of Pseudomonas putida JBC17. Wounded mandarin fruits inoculated with 105 conidia ml1 of Penicillium digitatum, followed by treatment with different concentrations of P. putida JBC17. Disease incidence was recorded after 7 days incubation at 20 °C and 90% RH. The same letters are not significantly different at p < 0.05 according to the Tukey’s test. Vertical bars indicate mean standard deviation (SD) of the replications. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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assayed to figure out the mode of action of the biocontrol activity. The JBC17 strain did not inhibit the mycelial growth of P. digitatum in a dual culture test on agar plates. In addition, the mycelial growth of other fungal pathogens such as P. italicum, B. cinerea, A. alternata, P. citrophthora, and G. candidum was not inhibited either (Supporting Information Fig. S1). Inhibition of conidial germination of P. digitatum by direct contact To determine the influence of JBC17 on the germination of conidia which are important for the dissemination and infection of green mold, we assayed the inhibition of conidial germination according to various concentrations of JBC17. The conidial germination of P. digitatum was approximately 86.7% in 10% PDB medium without JBC17 cells. However, the conidial germination of P. digitatum was significantly inhibited when cells of JBC17 were added (Fig. 2). The germination rate of P. digitatum significantly decreased to 26.0 and 4.0% in the presence of JBC17 at 106 and 107 cfu ml1, respectively. In addition, the average germ tube length of P. digitatum at 107 cfu ml1 of JBC17 was 1.0% compared to the control (Fig. 2). Competition for nutrients by JBC17 In spite of the effective biocontrol efficacy on green mold, the JBC17 strain did not antagonize the mycelial growth of many fungal pathogens including P. digitatum. However, the conidial germination of P. digitatum was significantly inhibited by direct contact with JBC17 as described above. To determine the mechanism under-
Figure 2. Percent germination and germ tube length of Penicillium digitatum conidia in the presence of Pseudomonas putida JBC17. The percentage of conidial germination (&) and germ tube length (&) of P. digitatum were observed with a light microscope at various concentration of JBC17. Both were recorded after 24 h incubation in 10% PDB at 25 °C. The same letters are not significantly different at p < 0.05 according to the Tukey’s test. Vertical bars indicate mean SD of the replications.
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J. Basic Microbiol. 2015, 55, 898–906
902
Sang-Mi Yu and Yong Hoon Lee
lying the inhibition of conidial germination, we assayed nutrient depletion using mandarin peel extract. The germination rate and scale of P. digitatum conidia increased as the concentration of mandarin peel extract increased from 0.5 to 5% (Fig. 3). However, the presence of JBC17 in the extract significantly inhibited the conidial germination rate and scale at both concentrations of peel extract during the first 24 h interaction. The increase of JBC17 cell numbers was negligible in affecting the inhibition efficacy of conidial germination during the interaction. In addition, reinserting the cylinders containing nongerminated conidia from wells containing JBC17 cells to wells without the bacterial cells resulted in the germination of most of the conidia. Screening of Tn mutants and identification of genes essential for inhibition of conidial germination Tn mutants of JBC17 were generated to identify factors responsible for inhibition of conidial germination. A total of 2804 mutants were individually assayed for their inhibition ability by direct contact and the mutants identified as having altered inhibition ability for conidial germination were reanalyzed using the nutrient competition assay described above. A final 15 mutants showing reduced inhibition ability were selected and the insertion sites of the Tn in the genomes of the mutants were determined by inverse PCR and sequencing. Excluding hypothetical proteins and unidentified sequences, mutations in nine of the selected mutants were successfully identified. The mutated genes identified have functions of exopolyphosphatase (ppx), acyl-
Figure 3. Percent germination of Penicillium digitatum conidia on PTFE membranes in cylinders. Conidia were incubated with Pseudomonas putida JBC17 (&) and without the antagonist (&) for 24 h. The interacted membranes were reinserted into new wells containing corresponding water or mandarin peel extract without JBC17 for an additional 24 h ( ). The same letters are not significantly different at p < 0.05 according to the Tukey’s test. Vertical bars indicate mean SD of the replications. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
CoA synthetase (fadD), acyl-CoA dehydrogenase (acdA), peptidase, Xaa-Pro aminopeptidase (pepP), auxin efflux carrier, methylthioribose-1-phosphate isomerase (mtnA), ATP-dependent DNA helicase (recG), and oligopeptidase B (prlC) (Table 1). Inhibition of conidial germination by PPM90 and PPM1657 The presence of JBC17 significantly inhibited conidial germination of P. digitatum. However, inhibition activity was significantly reduced in the PPM90 and PPM1657 mutant in which ppx and pepP was disrupted, respectively. The nongerminated conidia in 0.5% peel extract was reduced to 47 and 43%, respectively, in the presence of the PPM90 and PPM1657 cells compared to 87% in the presence of wild type JBC17 (Fig. 4). The inhibition activity for conidial germination was recovered in the complemented strains (PPM90
