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Transformation of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol of rhizoctonia root rot and in situ antibiotic production
FEMS Microbiology Ecology 49 (2004) 243–251 www.fems-microbiology.org
Transformation of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol of rhizoctonia root rot and in situ antibiotic production Zhengyu Huang a
a,1
, Robert F. Bonsall b, Dmitri V. Mavrodi Linda S. Thomashow a,b
b,*
, David M. Weller
a,b
,
USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Washington State University, Pullman, WA 99164-6430, USA b Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA Received 9 December 2003; received in revised form 4 February 2004; accepted 14 March 2004 First published online 7 April 2004
Abstract A seven-gene operon for the synthesis of phenazine-1-carboxylic acid was introduced into Pseudomonas fluorescens Q8r1-96, an aggressive root colonizer that produces 2,4-diacetylphloroglucinol and consistently suppresses take-all of wheat. The recombinant strains produced both antifungal metabolites and maintained population sizes comparable to those of Q8r1-96 over a seven-week period in the rhizosphere of wheat. The strains were no more suppressive of take-all or Pythium root rot than was Q8r1-96, but suppressed Rhizoctonia root rot at a dose of only 102 CFU per seed, one to two orders of magnitude lower than the dose of Q8r1-96 required for comparable disease control. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Genetically modified microorganisms; Rhizosphere; Pseudomonas; Biological control
1. Introduction The term plant growth-promoting rhizobacteria (PGPR) applies to diverse genera of root-associated bacteria with the ability to improve the growth of plants when applied to seeds, soil, or subterranean plant parts [1]. Growth promotion occurs as a result of direct stimulation of plant growth, induction of host plant systemic resistance, or suppression of plant pathogens [2–4]. Antifungal metabolites produced in situ by PGPR have key roles in the suppression of soilborne pathogens that cause damping-off, root rots, and wilt diseases [1,4].
*
Corresponding author. Tel.: +1-509-335-3269; fax: +1-509-3357674. E-mail address: [email protected] (D.V. Mavrodi). 1 Present address: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268-1054, USA.
The polyketide metabolite 2,4-DAPG is a key determinant in the control of black root rot of tobacco, crown and root rot of tomato, Pythium damping-off of cucumber, and take-all of wheat by P. fluorescens CHA0 [5,6]; Pythium damping-off of sugar beet and cyst nematode and soft rot of potato by Pseudomonas sp. F113 [7,8]; and take-all of wheat by P. fluorescens strains Q2-87 [9] and Q8r1-96 [10]. In some soils, 2,4-DAPGproducing fluorescent Pseudomonas spp. are largely responsible for the natural suppression of take-all known at take-all decline [11]. The 2,4-DAPG biosynthetic locus includes six genes that function in regulation, synthesis, and export [12]. Other antifungal metabolites, the phenazines, are produced via the shikimic acid pathway by the biocontrol strains P. fluorescens 2-79 and P. chlororaphis 30-84. These compounds have a major role in the suppression of take-all by both strains [13,14] and contribute to the long-term survival of the bacteria on roots [15]. The
0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.03.010
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Z. Huang et al. / FEMS Microbiology Ecology 49 (2004) 243–251
biosynthetic locus for phenazine-1-carboxylic acid (PCA), the precursor of the various phenazine compounds produced by biocontrol strains, contains a sevengene operon that is highly conserved among fluorescent pseudomonads [16] while additional, linked genes are required for the conversion of PCA to other phenazine derivatives [17–19] that differ from PCA in their activity against target pathogens. In the Pacific Northwest of the United States, wheat is damaged by one or more root diseases including takeall, caused by Gaeumannomyces graminis var. tritici, Rhizoctonia root rot caused by R. solani AG-8 and R. oryzae, and Pythium root rot caused by various Pythium spp., most notably P. irregulare and P. ultimum [20]. The three diseases often occur as a complex in the same field and are exacerbated by direct seeding and intensive cereal production [21]. Commercial wheat varieties have no resistance to these diseases, and chemical treatments perform inconsistently. Biological control with strains of fluorescent Pseudomonas and Bacillus spp. applied as seed treatments has provided significant suppression of take-all [22,23], Pythium root rot [22], and Rhizoctonia root rot [22]. However, inconsistent performance has impeded the commercialization of PGPR for control of root diseases of wheat and other crops. A treatment may provide significant control in one field or season but not the next. Consequently, only a handful of PGPR have been developed for commercial use [24]. Approaches to improve biocontrol performance have included the application of larger or multiple doses of bacterial inoculum, the development of new formulations, the use of strain combinations, and strain improvement by genetic engineering or the transfer of ‘‘biocontrol genes’’ to recipient strains with other desirable attributes [1]. Here we report on recombinant biocontrol PGPR constructed by introducing the PCA biosynthetic locus
from P. fluorescens 2-79 into the 2,4-DAPG-producing strain Q8r1-96, an exceptionally aggressive colonizer of the roots of wheat [10]. PCA and 2,4-DAPG are not known to occur naturally within the same strain, but both are active against root pathogens. The recombinant strains produced both antibiotics in situ and suppressed Rhizoctonia root rot at a lower dose than did either parental strain.
2. Materials and methods 2.1. Bacterial strains, plasmids and wheat root pathogens Strains and plasmids used in this study are listed in Table 1. Escherichia coli and spontaneous rifampin-resistant derivatives of P. fluorescens strains [10,25,26] were grown in LB or M9 media [27] at 37 and 28 °C, respectively. Antibiotics were added to media at the following concentrations: ampicillin, 80 lg ml1 ; rifampicin, 75 lg ml1 ; kanamycin, 100 lg ml1 ; and tetracycline, 12.5 lg ml1 . G. graminis var. tritici isolate R3-111a-1 [26], R. solani AG-8 isolate C1 [28], and an isolate of P. irregulare from the roots of wheat [28] were used in all laboratory and greenhouse assays. 2.2. Construction of recombinant phenazine-producing derivatives of P. fluorescens Q8r1-96 Standard methods were used for DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation [27]. The phenazine biosynthetic operon phzABCDEFG from P. fluorescens 279, cloned previously as pT7-6A-G [16], was excised as a 6.7-kb BglII–XbaI DNA fragment and introduced into pALTER-Ex1 (Promega, Madison, WI) distal to the tac
Table 1 Bacterial strains and plasmids used in this study Strain or plasmid
Relevant characteristicsa
Source or reference
Strains P. fluorescens 2-79 RN10 Q2-87 Q8r1-96
Phzþ Rifr Nalr Phzþ Rifr Phlþ Rifr
[25] [26] [10]
E. coli JM109 S17-1(kpir)
F’ traD36 proAþ proBþ lacIq lacZDM15/recA1 gyrA96 thi hsdR17 supE44 relA1 D(lac-proAB) mcrA thi pro hsdR hsdM recA rpsL RP4-2(Tcr ::Mu) (Kmr ::Tn7) kpir
Promega [18]
Promega [29] [30] [16]
pUTKm-phz
T7, SP6 and tac promoters ColE1 f1ori Tetr ColE1 bla; identical to pUC18 but with polylinker flanked by SfiI sites Tn5-based delivery plasmid with Kanr ; bla oriR6K mobRP4 tnp* gene of Tn5-IS50R pT7-6 containing 6.9 kb BglII–XbaI fragment from pPHZ108A with phzABCDEFG genes from strain 2-79 pUTKm containing phzABCDEFG genes under control of the tac promoter
a
r
Plasmids pALTER-Ex1 pUC18Sfi pUTKm1 pT7-6A-G
This study
bla, b-lactamase; Kan , kanamycin resistance; Tet , tetracycline resistance; Rif , rifampin resistance; Nal , nalidixic acid resistance; Phzþ , production of phenazine-1-carboxylic acid; Phlþ , production of 2,4-diacetylphloroglucinol. r
r
r
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promoter. The 6.7-kb PtacphzABCDEFG fragment was then subcloned into pUC18Sfi [29], and finally, the 6.7kb phz fragment, now flanked with SfiI sites, was moved into the SfiI site of mini-Tn5Km1 [30], yielding pUTKm-phz. This plasmid was transformed into E. coli S17-1(kpir) and mobilized into P. fluorescens Q8r1-96 by using a biparental filter mating technique [18]. Transformants were selected on M9 minimal medium supplemented with kanamycin, and isolates containing presumptive transposed phz genes in genomic DNA were transferred ten times on King’s medium B agar [31] to assure that kanamycin resistance and colony morphology, color, and fluorescence remained stable. The Q8r1-96 genetic background of each recombinant clone was confirmed by fingerprinting with a RAPD technique [10], and the presence of the PCA and 2,4-DAPG biosynthetic genes was confirmed by PCR with phenazineand 2,4-DAPG-gene-specific primers [18,32]. Finally, growth kinetics in vitro of wild-type Q8r1-96 and its PCA-producing derivatives were compared in M9 broth amended with 0.05% yeast extract and in KMB broth. Turbidity (OD 600nm ) was monitored at 4, 8, 12, 20, and 24 h with a Dynatech MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA). 2.3. Fungal inhibition in vitro Growth inhibition of the three root pathogens by Q8r1-96 and its PCA-producing derivatives was tested on potato dextrose agar (PDA) (Becton, Dickinson and Company, Franklin Lakes, NJ). In general, two 3-ll drops from an overnight culture of bacteria in LB broth were spotted about 1 cm from opposite edges of a PDA plate and a plug 0.5 cm in diameter, cut from the leading edge of a fungal colony grown on PDA, was placed at the center. The plates were incubated at 28 °C and the distance between the edges of the bacterial spots and the fungal mycelium was measured. For G. graminis var. tritici, the bacteria were spotted 24 h after introduction of the fungus and the zone of inhibition was measured 5 d later. For R. solani AG-8, the bacteria and the fungus were introduced at the same time and the inhibition zone was measured after 5 d. For P. irregulare, the bacteria were spotted 48 h prior to fungal inoculation and the zone of inhibition was measured after two additional days. 2.4. Seed treatment Spring wheat (cv. Penawawa) was coated with bacteria at approximately 102 , 103 , or 104 per seed by using the methods described by Pierson and Weller [26]. The bacterial inoculum was grown on KMB agar plates for 48 h at 27 °C. Bacterial cells were then scraped from the plates, washed twice by centrifugation in 40 ml of sterile distilled water to remove residual 2,4-DAPG and PCA, suspended in 0.5% methylcellulose (Sigma Chemical Co.) to a volume
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of 15 ml, and serially diluted. For each seed treatment, 25 g of wheat seed was thoroughly mixed with 9 ml of an appropriately diluted cell suspension in 0.5% methylcellulose and air-dried overnight in a laminar flow hood. The actual number of CFU per seed was determined by dilution plating on KMB immediately before planting. 2.5. Disease suppression assays Tube assays were used to test the biocontrol activity of the parental and recombinant derivative strains against take-all, Rhizoctonia root rot, and Pythium root rot. Inocula were prepared by growing each fungal pathogen on autoclaved whole oat grains [26,28]. The colonized grains were stored at 4 °C until used. Plastic tapered tubes (2.5 cm 20 cm) with holes at the bottom were plugged with cotton balls to a height of about 3 cm and half-filled with fine vermiculite followed by 13 g of Quincy virgin soil, a Shano sandy soil [10], with or without inoculum of a pathogen. For assays of take-all and Rhizoctonia root rot, the soil was amended with 1% (w/w) of freshly ground oat kernel inoculum (sieved to collect particle sizes of 0.25–0.50 mm), and each tube received 10 ml of metalaxyl (2.5 mg of active ingredient ml1 ; Syngenta, Greensboro, NC) to minimize interference by Pythium spp. indigenous to soils of the Pacific Northwest. For Pythium root rot assays, one whole oat kernel colonized by P. irregulare was introduced into the top layer of soil in each tube and the tube was watered with 10 ml of tap water. The tubes were seeded immediately for assays of take-all suppression, or after incubation at room temperature for 48 h for assays of Pythium and Rhizoctonia root rot control. Two wheat seeds were sown per tube, covered with about 5 ml of fine vermiculite, and watered with 3 ml of water for Pythium assays or water plus metalaxyl (2.5 mg ml1 ) for take-all and Rhizoctonia assays. The tubes were arranged in a randomized complete block design and incubated in a growth chamber for 4–5 weeks (12-h photoperiod, 16 °C). After seedling emergence (3–4 d), each tube was watered twice weekly with 5 ml of onethird-strength Hoagland’s solution [26]. All experiments were repeated 3 times with 5 or 6 replicates per treatment and 5 tubes per replicate. Rhizoctonia root rot, Pythium root rot, and take-all were evaluated by determining the percentage of infected roots [22], plant height [22], and disease severity on a scale of 0–8 [33], respectively. All experiments were conducted three times with similar results. 2.6. Colonization of the wheat rhizosphere by fluorescent Pseudomonas spp. Wheat seed was treated with bacterial strains at 103 CFU/seed, planted, and grown as described above. Population sizes of the introduced strains in the
Z. Huang et al. / FEMS Microbiology Ecology 49 (2004) 243–251
2.7. Antibiotic isolation and detection PCA and 2,4-DAPG produced in situ were extracted from the roots of wheat as described by Bonsall et al. [34]. Briefly, 30 g of roots with adhering rhizosphere soil were extracted with 40 ml of 80% acetone acidified to pH 2.0 with trifluoroacetic acid (TFA). The extracts were filtered through Whatman No. 1 paper. Soil particles were removed by centrifugation and the filtrate was evaporated to a volume of approximately 8 ml. Samples were acidified again with TFA, extracted twice with 10 ml of ethyl acetate, and the pooled ethyl acetate extracts were dried. Prior to analysis by high performance liquid chromatography (HPLC), samples were suspended in 1 ml of 35% acetonitrile and 0.1% TFA. Antibiotic concentrations were determined relative to phenazine and 2,4-DAPG standards and related to bacterial population size. PCA and 2,4-DAPG synthesized in vitro were isolated and identified as described previously [34]. 2.8. Statistical analyses Plant heights and percentages of diseased roots were compared with STATISTIX (version 7, Analytical Software, St. Paul, MN). Differences among treatments were determined at each bacterial dilution by standard one-way analysis of variance, and mean comparisons among treatments were performed by using Fisher’s protected least significant difference test at P ¼ 0:05.
3. Results 3.1. Selection and characterization in vitro of recombinant strains Mating of P. fluorescens Q8r1-96 with E. coli S171(kpir)/(pUTKm-phz) yielded thirty-five recombinant colonies, five of which successfully survived selection on M9 agar containing kanamycin. These clones, designated Z30-97, Z31-97, Z32-97, Z33-97, and Z34-97, remained kanamycin-resistant after ten successive transfers on KMB agar and were stable with regard to colony size, morphology, color, and fluorescence. The
presence of the introduced phenazine biosynthetic genes was confirmed by PCR with the phenazine-specific primers PHZ1 and PHZ2 [18], and the RAPD banding patterns generated from these recombinant strains with the M13 primer were indistinguishable from those of the parental strain Q8r1-96 (data not shown). Cultures of Q8r1-96 attained maximal turbidity in M9 broth amended with 0.05% yeast extract by 12 h, whereas the turbidity of cultures of the recombinant strains continued to increase for up to 24 h. At 12 h, cell densities of Z31-97 and the other recombinant strains were about 50% and 30% lower, respectively, than that of Q8r1-96 (data not shown). Similar results were obtained when the experiment was performed in LB broth except that strain Q8r1-96 reached its maximal density by 8 h. Strain Z31-97 also grew more slowly and produced smaller colonies than Q8r1-96 and the other recombinant strains on KMB agar, and was not included in further tests. Cultures of the recombinant strains produced both PCA and 2,4-DAPG in vitro. At 40 h, PCA produced per 108 CFU in cultures of the recombinant strains ranged from about 1.5 to up to 27 times greater than in cultures of wild type 2-79 (Fig. 1). Similarly, cultures of the recombinant strains accumulated up to 2.7 times more 2,4-DAPG by 24 h than did those of strain Q8r1-
Integrated Area (CFU 108)-1
rhizosphere were determined after 66 h and 1, 3, 5, and 7 weeks as described by Raaijmakers and Weller [10]. Briefly, four seedlings were selected at random from each replicate and shaken to remove the loosely-adhering soil. One gram of roots with associated soil was suspended in 5 ml of sterile water, agitated vigorously by Vortex mixing and sonication, and serial dilutions of the wash suspension were spread onto KMB agar amended with rifampicin and cycloheximide at 100 lg ml1 . Colonies were enumerated after 48 h.
0.3 (A)
0.25 0.2 0.15 0.1 0.05 0
Integrated Area (CFU 108)-1
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1.0 (B)
0.8 0.6 0.4 0.2 0 0
8
16
24 Time (h)
32
40
48
Fig. 1. Production in vitro of phenazine-1-carboxylic acid (A) and 2,4diacetylphloroglucinol (B) by wild-type and recombinant strains. Cultures were grown with constant shaking at 27 °C in modified KMB broth. Antibiotics were extracted and viable counts were determined at 8, 16, 24, 32, 40, and 48 h as described in Section 2. The strains are Q8r1-96 ðjÞ; Z30-97 ðMÞ; Z32-97 (X); Z33-97 ðÞ; Z34-97 ðsÞ; and 279 ð}Þ.
Z. Huang et al. / FEMS Microbiology Ecology 49 (2004) 243–251 Table 2 Inhibition in vitro of wheat root pathogens by Q8r1-96 and recombinant derivative strains Strain
Z34-97 Z33-97 Z32-97 Z30-97 Q8r1-96
Zone of inhibition (cm)A Gaeumannomyces graminis var. tritici
Rhizoctonia solani AG-8
Pythium irregulare
1.7aB 1.7ab 1.1c 1.6b 0.5d
0.6a 0.6a 0.5a 0.6a 0.2b
0.7a 0.7a 0.7a 0.8a 0.5b
A
Width of the zone of inhibition was measured from the edge of the fungal colony to the edge of the bacterial colony. B Means in the same column followed by the same letter are not significantly different at P ¼ 0:05 according to the protected LSD.
96. Strain Z34-97 produced maximal amounts of 2,4-DAPG earlier than did the other strains. These increased levels of antibiotic production were correlated with enhanced antifungal activity in vitro. All of the recombinant strains produced larger zones of inhibition against the three fungal pathogens than did Q8r1-96 (Table 2). Inhibition of P. irregulare by the recombinant strains was 43–70% greater than that by Q8r1-96, and inhibition of G. graminis var. tritici and R. solani AG-8 was 2–3 times, and over 3 times greater, respectively, than inhibition by the parental strain. 3.2. Root disease suppression in soil Preliminary studies indicated that recombinant strains applied at doses of 105 per seed or greater sometimes were toxic to wheat, causing reduced emergence and stunting of the roots and shoots. To avoid these symptoms of phytotoxicity, the bacteria in this study were applied at doses of 102 , 103 , and 104 CFU per seed. P. fluorecens Q8r1-96 suppressed take-all significantly (P ¼ 0:05) as compared to the nontreated and
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methylcellulose controls at all three doses tested. For example, the root disease ratings for Q8r1-96 applied at 102 , 103 , and 104 CFU per seed and for the two controls were 3.8, 2.9, 2.6, 4.2, and 4.2, respectively, on a scale of 0–8. However, none of the recombinant strains showed increased suppression of take-all as compared to Q8r196 (data not shown). Strain Q8r1-96 applied at 104 per seed, but not at lower doses, significantly (P ¼ 0:05) increased the height of wheat planted in soil infested with Pythium irregulare as compared to the methylcellulose and nontreated controls (Table 3). In contrast, the four recombinant strains significantly increased plant height at all three doses compared to the controls. Except for Z34-97 at 103 and 104 CFU per seed, however, the recombinant strains did not differ significantly from Q8r1-96 at each of the three doses, which were compared individually to the controls because of strain by dose interactions. Results were similar in subsequent experiments. Strain Q8r1-96 at a dose of 104 CFU per seed and all four recombinant strains at all three doses significantly ðP ¼ 0:05Þ reduced the percentage of roots with symptoms of Rhizoctonia root rot as compared to the methylcellulose and nontreated controls (Fig. 2, Table 3). Treatments were compared within doses because of strain by dose interactions. At a dose of 103 CFU per seed, all four recombinant strains reduced disease significantly more ðP ¼ 0:05Þ that did Q8r1-96. At doses of 102 , recombinant strains Z32-97, Z33-97 and Z34-97 reduced disease significantly ðP ¼ 0:05Þ more than Q8r1-96; with Z30-97 the disease reduction was not significant compared to the parental strain. In subsequent experiments at a dose of 102 , Z30-97 was significantly more suppressive than Q8r1-96 (data not shown). At a dose of 104 , all recombinant strains except Z34-97 reduced disease significantly more than Q8r1-96; Z34-97 reduced disease more than the parental strain but the difference was not significant. Similar results
Table 3 Suppression of rhizoctonia and pythium root rot by wild type and recombinant strains of Pseudomonas fluorescens Treatment
% Roots with rhizoctoniaA 102 C
Q8r1-96 Z30-97 Z32-97 Z33-97 Z34-97 2-79 MCD Nontreated A
E
50.3ab 37.3bc 28.6c 30.9c 31.7c . . .F 53.1a 54.9a
Height (cm) of Pythium-infected plantsB
103
104
102
103
104
46.1a 19.1b 21.5b 29.2b 28.7b ... 53.1a 54.9a
42.4bc 30.9d 24.2d 30.7d 34.0cd 47.3ab 53.1a 54.9a
30.7ab 32.0a 31.4a 32.2a 32.5a ... 28.8b 28.7b
30.9bc 31.7ab 32.9ab 31.1b 33.9a ... 28.7c 28.6c
31.4b 32.2b 32.7b 31.9b 34.8a ... 28.7c 28.6c
Percentage of seedling roots with typical symptoms of Rhizoctonia root rot 31 days after planting. Shoot length of seedlings with roots infected with Pythium 31 days after planting. C Approximate dose of each strain (CFU per seed) as determined by dilution plating. D Seed treated with 0.5% methylcellulose only. E Means in the same column followed by the same letter are not significantly different at P ¼ 0:05 according to Fisher’s protected LSD. F Not tested at this dose. B
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Fig. 2. Suppression of Rhizoctonia root rot by wild-type and recombinant strains of Pseudomonas fluorescens. Wheat seed treated with 0.5% methylcellulose alone (control) or with bacterial suspensions of Z34-97, Q8r1-96 (each at 103 per seed) or 2-79 (104 per seed) was sown in soil infested with Rhizoctonia solani AG-8. Root rot symptoms included darkened lesions and reduced root mass, and were most severe on the control roots, least severe on roots colonized by Z34-97, and intermediate in severity on roots colonized by strains Q8r1-96 and 279.
were obtained when the experiments were repeated except that no significant differences occurred between Q8r1-96 and the recombinant strains at a dose of 104 . At a dose of 104 , Q8r1-96 but not 2-79, had significantly less disease than the methylcellulose and non-treated controls. Although PCA is more inhibitory of R. solani than 2,4-DAPG, the greater control by Q8r1-96 reflects its superior root colonization ability. 3.3. Colonization and antibiotic production in the wheat rhizosphere Strains Q8r1-96, Z30-97, Z32-97, Z33-97, Z34-97, and Q2-87 applied at 103 CFU per seed multiplied rapidly on the seeds and in the wheat rhizosphere. The population sizes of all of the strains had increased about 1000-fold on the seed coat and the radical by 66 h (0.4 week), and peaked after one week (Fig. 3). Populations of the recombinant strains and Q8r1-96 did not differ in size throughout the 7-week study, and were significantly higher than those of Q2-87 at weeks 1, 3, 5, and 7. The
Population, CFU (g root fresh weight)
-1
109 108 107 106 105 104 103 102 0
1
2 3 Weeks after planting
4
5
Fig. 3. Population dynamics of wild-type and recombinant strains of Pseudomonas fluorescens in the rhizosphere. Wheat seed (cv. Penawawa) was coated with bacterial strains at a density of 103 CFU per seed (approximately 2 104 CFU g1 of seed), sown in Quincy virgin soil, and seedlings were harvested after 5 weeks. Error bars represent the standard error of four replicates. The strains are Q8r1-96 ðjÞ; Z30-97 ðMÞ; Z32-97 (X); Z33-97 ðÞ; Z34-97 ðsÞ; and Q2-87 ð}Þ, an ‘‘average’’ root colonizer.
recombinant strains produced both PCA and 2,4-DAPG in the rhizosphere and, consistent with results obtained in vitro, production of both antibiotics was greater for the recombinant than for the wild-type strains. Strains Z33-96 and Z34-96 produced 4–5 times more PCA per 108 CFU than did strain 2-79 (P ¼ 0:008; Kruskal– Wallis one-way analysis of variance), and Z34-97 about 11-fold more 2,4-DAPG than did Q8r1-96 (P ¼ 0:003; one-way analysis of variance) (Table 4).
4. Discussion Wheat growing worldwide can be damaged by one or more of the root diseases take-all, Pythium root rot, and Rhizoctonia root rot, which often occur in the Pacific Northwest as a complex in the same field. These diseases
Table 4 Production in the rhizosphere of 2,4-diacetylphloroglucinol (2,4-DAPG) and phenazine-1-carboxylic acid (PCA) by P. fluorescens Q8r1-96, P. fluorescens 2-79, and recombinant strains Strains
Populationb (CFU g1 root)
2,4-DAPGc (ng g1 of root)
PCAc (ng g1 of root)
2,4-DAPGc (ng (105 )1 CFU)
PCAc (ng (105 )1 CFU)
Q8r1-96 (103 )a 2-79 (104 ) 2-79 (106 ) Z34-97 (103 ) Z33-97 (103 )
2.30 108 0.18 108 0.41 108 0.87 108 0.64 108
291.4 134.8 N/A N/A 1248.9 322.1 344.3 61.3
N/A 15.9 1.3 16.8 10.6 313.6 80.6 269.6 80.8
0.13 0.06 N/A N/A 1.45 0.35 0.44 0.1
N/A 0.08 0.01 0.04 0.03 0.35 0.08 0.42 0.13
a
Values in brackets indicate the approximate dose at which strains were applied to wheat seeds. CFU of introduced bacteria per gram of root as determined by dilution plating. c Mean standard deviation ðn ¼ 3Þ; N/A, not applicable. b
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are exacerbated when wheat is direct-seeded (no-till) [21], a practice gaining in popularity because it reduces soil erosion and rebuilds soil organic matter. Certain strains of fluorescent Pseudomonas spp. can suppress these diseases, but most strains fail to perform consistently and no single strain is effective against all three phytopathogens. The goal of this study was to enhance the performance of biocontrol agents or to extend their activity to a wider range of wheat root diseases. We attempted to improve biocontrol activity by inserting foreign genes that confer the ability to produce a biologically active metabolite into the biocontrol strain P. fluorescens Q8r1-96. A key characteristic that makes Q8r1-96 an ideal host for such manipulations is its unusual aggressiveness in colonizing the roots of wheat and pea and its ability to suppress take-all when applied to seeds or soil at doses of 104 or fewer CFU per seed or gram of soil [10,35]. Other Pseudomonas strains with biocontrol activity require doses 100- to 10,000-fold greater in order to achieve take-all suppression [25]. We modified Q8r1-96 through a stable insertion of genes for biosynthesis of PCA, which has broad-spectrum activity against a variety of soilborne pathogens [3]. Unlike the polyketide 2,4-DAPG, PCA is synthesized via the shikimic acid pathway [16], reducing the probability that the two pathways would compete directly for the same biosynthetic precursors. To our knowledge, recombinant derivatives of Q8r196 used in the study represent the first example of fluorescent Pseudomonas spp. that have the capacity to produce both phenazines and the polyketide metabolite 2,4-DAPG. Bacteria that produce both antibiotics are not known in nature. Introduction of the constitutivelyexpressed PCA biosynthetic genes, even in single copy, clearly placed a metabolic burden on Q8r1-96 because all of the recombinant strains grew more slowly in vitro than did Q8r1-96. Nevertheless, Q8r1-96 and its recombinant derivatives had similar population dynamics in the short-term colonization studies reported here, indicating that the metabolic cost of PCA synthesis in pure culture is at least partially offset in the more nutritionally and microbiologically heterogeneous rhizosphere environment. The recombinant strains produced PCA and 2,4-DAPG in quantities equal to or greater than those produced by the parental strains, both in vitro and in the rhizosphere (Fig. 1, Table 4). Increased accumulation of PCA likely was due to constitutive expression of the phz genes from the tac promoter, but the observed overproduction of 2,4-DAPG is less easily explained. In wild-type P. fluorescens strains CHA0 and F113, 2,4-DAPG induces its own synthesis, and synthesis is repressed by salicylate and certain other aromatic compounds [36]. We speculate that the constitutively expressed PCA pathway drains the chorismate pool enough to limit the synthesis of salicylate or another potential repressor of 2,4-DAPG syn-
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thesis. The differences observed between individual recombinant strains in amounts of PCA and 2,4-DAPG produced and in biocontrol activity suggest that positional effects are associated with sites of insertion of the phz operon. Despite their enhanced antibiotic production, the recombinant biocontrol agents were not necessarily more effective than their parental strains against the three diseases tested. For example, no recombinant strain provided greater suppression of take-all than did Q8r196. G. graminis var. tritici is highly sensitive to 2,4-DAPG (complete inhibition at 3 lg/ml), Q8r1-96 is highly effective against take-all, and isolates genotypically identical to Q8r1-96 are responsible for the natural biological control of take-all known as take-all decline in Washington State soils [11]. Thus, Q8r1-96 probably already is ‘‘finely tuned’’ to suppress take-all, and not much more can be done to improve its performance even though the pathogen is also highly sensitive to PCA. It is likely that there is a threshold dose of both antibiotics in the rhizosphere above which no further inhibition is derived. The recombinant strains were only slightly more effective than Q8r1-96 against Pythium root rot as indicated by the fact that plants treated with Q8r1-96 were no different in height than control plants or those treated with recombinant strains, but plants treated with recombinant strains were significantly taller than control plants. Pythium irregulare is less sensitive to both 2,4DAPG (complete inhibition at 10 lg ml1 ) and PCA than is the take-all pathogen, but the elevated levels of one or both of the antibiotics present on roots colonized by the recombinant strains was correlated with measurably better shoot growth than seen with seedlings colonized by Q8r1-96. While further work will be required to determine whether 2,4-DAPG, PCA, or a combination of the two was responsible for this effect, it is noteworthy that overproduction of 2,4-DAPG alone by P. fluorescens CHA0 did not improve protection of sweet corn and cress against Pythium damping-off [37], nor was an overproducing strain of P. fluorescens F113 more effective than the wild-type strain against P. ultimum on sugarbeet [38]. In contrast, PCA-producing derivatives of P. fluorescens SBW25 containing the same phenazine gene construct described here were significantly more suppressive of damping-off disease of pea seedlings caused by P. ultimum than was the parental strain, and the effectiveness of the transformed strains was directly correlated with the level of PCA biosynthesis [39]. Finally, the recombinant strains were substantially more suppressive of Rhizoctonia root rot than was Q8r1-96. This difference was most apparent at lower doses, where wheat treated with recombinant strains had about 40–50% less diseased roots compared to control plants treated with Q8r1-96. The PCA-producing recombinant derivatives retained the unique colonization
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properties of strain Q8r1-96 and were highly effective against Rhizoctonia when applied at only 102 CFU per seed. In theory, a one-liter culture of Z34-97 at 1010 CFU per ml, easily attainable in standard growth media after overnight incubation, should be sufficient to treat the seed (at 102 CFU per seed) needed to plant 10,000 acres of wheat. Obtaining consistent biocontrol with such low doses has long been a goal of PGPR research and would remove barriers related to inoculum production, formulation and cost that now limit more widespread use of Pseudomonas biocontrol agents. Finally, the use of transgenic strains may offer certain benefits over the application of strain consortia in which components may compete for the same niche, leading especially at low doses to rapid displacement of the less rhizosphere-competent strain by its more aggressive counterpart. Rhizoctonia solani has a very broad host range and the recombinant strains have applicability to other crops commonly grown in cereal-based production systems. However, we fully realize that the effects of such genetically-modified strains on the environment must be carefully considered when proposing their release for control of soilborne pathogens. Studies of the long-term fate of these recombinant strains, their impact on indigenous rhizosphere microflora, and their competitiveness with the parental strain are underway.
Acknowledgements Dmitri V. Mavrodi and Robert F. Bonsall contributed equally to this study. This work was supported by grant 96-35303-3242 from the U.S. Department of Agriculture, Office of Grants and Program Systems, National Research Initiative, Competitive Grants Program.
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Z. Huang et al. / FEMS Microbiology Ecology 49 (2004) 243–251 [22] Kim, D.-S., Cook, R.J. and Weller, D.M. (1997) Bacillus sp. L32492 for biological control of three root diseases of wheat grown with reduced tillage. Phytopathology 87, 551–558. [23] Weller, D.M. and Cook, R.J. (1983) Suppression of take-all of wheat by seed treatments with fluorescent pseudomonads. Phytopathology 73, 463–469. [24] Cook, R.J. (1993) Making greater use of introduced microorganisms for biological-control of plant-pathogens. Annu. Rev. Phytopathol. 31, 53–80. [25] Weller, D.M. (1983) Colonization of wheat roots by a fluorescent pseudomonad supressive to take-all. Phytopathology 73, 1548– 1553. [26] Pierson, E.A. and Weller, D.M. (1994) Use of mixtures of fluorescent pseudomonads to suppress take-all and improve the growth of wheat. Phytopathology 84, 940–947. [27] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1995) Short protocols in molecular biology. J. Wiley and Sons, NY. [28] Mazzola, M. and Cook, R.J. (1991) Effects of fungal root pathogens on the population dynamics of biocontrol strains of fluorescent pseudomonads in the wheat rhizosphere. Appl. Environ. Microbiol. 57, 2171–2178. [29] de Lorenzo, V. and Timmis, K.N. (1994) Analysis and construction of stable phenotypes in Gram-negative bacteria with Tn5-derived and Tn10-derived minitransposons. Meth. Enzymol. 235, 386–405. [30] deLorenzo, V., Herrero, M., Jakubzik, U. and Timmis, K.N. (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J. Bacteriol. 172, 6568–6572. [31] King, E.O., Ward, M.K. and Raney, D. (1954) Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44, 301–307. [32] Raaijmakers, J.M., Weller, D.M. and Thomashow, L.S. (1997) Frequency of antibiotic-producing Pseudomonas spp. in natural environments. Appl. Environ. Microbiol. 63, 881–887.
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Transformation of Pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol of rhizoctonia root rot and in situ antibiotic production Zhengyu Huang a
a,1
, Robert F. Bonsall b, Dmitri V. Mavrodi Linda S. Thomashow a,b
b,*
, David M. Weller
a,b
,
USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Washington State University, Pullman, WA 99164-6430, USA b Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA Received 9 December 2003; received in revised form 4 February 2004; accepted 14 March 2004 First published online 7 April 2004
Abstract A seven-gene operon for the synthesis of phenazine-1-carboxylic acid was introduced into Pseudomonas fluorescens Q8r1-96, an aggressive root colonizer that produces 2,4-diacetylphloroglucinol and consistently suppresses take-all of wheat. The recombinant strains produced both antifungal metabolites and maintained population sizes comparable to those of Q8r1-96 over a seven-week period in the rhizosphere of wheat. The strains were no more suppressive of take-all or Pythium root rot than was Q8r1-96, but suppressed Rhizoctonia root rot at a dose of only 102 CFU per seed, one to two orders of magnitude lower than the dose of Q8r1-96 required for comparable disease control. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Genetically modified microorganisms; Rhizosphere; Pseudomonas; Biological control
1. Introduction The term plant growth-promoting rhizobacteria (PGPR) applies to diverse genera of root-associated bacteria with the ability to improve the growth of plants when applied to seeds, soil, or subterranean plant parts [1]. Growth promotion occurs as a result of direct stimulation of plant growth, induction of host plant systemic resistance, or suppression of plant pathogens [2–4]. Antifungal metabolites produced in situ by PGPR have key roles in the suppression of soilborne pathogens that cause damping-off, root rots, and wilt diseases [1,4].
*
Corresponding author. Tel.: +1-509-335-3269; fax: +1-509-3357674. E-mail address: [email protected] (D.V. Mavrodi). 1 Present address: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268-1054, USA.
The polyketide metabolite 2,4-DAPG is a key determinant in the control of black root rot of tobacco, crown and root rot of tomato, Pythium damping-off of cucumber, and take-all of wheat by P. fluorescens CHA0 [5,6]; Pythium damping-off of sugar beet and cyst nematode and soft rot of potato by Pseudomonas sp. F113 [7,8]; and take-all of wheat by P. fluorescens strains Q2-87 [9] and Q8r1-96 [10]. In some soils, 2,4-DAPGproducing fluorescent Pseudomonas spp. are largely responsible for the natural suppression of take-all known at take-all decline [11]. The 2,4-DAPG biosynthetic locus includes six genes that function in regulation, synthesis, and export [12]. Other antifungal metabolites, the phenazines, are produced via the shikimic acid pathway by the biocontrol strains P. fluorescens 2-79 and P. chlororaphis 30-84. These compounds have a major role in the suppression of take-all by both strains [13,14] and contribute to the long-term survival of the bacteria on roots [15]. The
0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.03.010
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biosynthetic locus for phenazine-1-carboxylic acid (PCA), the precursor of the various phenazine compounds produced by biocontrol strains, contains a sevengene operon that is highly conserved among fluorescent pseudomonads [16] while additional, linked genes are required for the conversion of PCA to other phenazine derivatives [17–19] that differ from PCA in their activity against target pathogens. In the Pacific Northwest of the United States, wheat is damaged by one or more root diseases including takeall, caused by Gaeumannomyces graminis var. tritici, Rhizoctonia root rot caused by R. solani AG-8 and R. oryzae, and Pythium root rot caused by various Pythium spp., most notably P. irregulare and P. ultimum [20]. The three diseases often occur as a complex in the same field and are exacerbated by direct seeding and intensive cereal production [21]. Commercial wheat varieties have no resistance to these diseases, and chemical treatments perform inconsistently. Biological control with strains of fluorescent Pseudomonas and Bacillus spp. applied as seed treatments has provided significant suppression of take-all [22,23], Pythium root rot [22], and Rhizoctonia root rot [22]. However, inconsistent performance has impeded the commercialization of PGPR for control of root diseases of wheat and other crops. A treatment may provide significant control in one field or season but not the next. Consequently, only a handful of PGPR have been developed for commercial use [24]. Approaches to improve biocontrol performance have included the application of larger or multiple doses of bacterial inoculum, the development of new formulations, the use of strain combinations, and strain improvement by genetic engineering or the transfer of ‘‘biocontrol genes’’ to recipient strains with other desirable attributes [1]. Here we report on recombinant biocontrol PGPR constructed by introducing the PCA biosynthetic locus
from P. fluorescens 2-79 into the 2,4-DAPG-producing strain Q8r1-96, an exceptionally aggressive colonizer of the roots of wheat [10]. PCA and 2,4-DAPG are not known to occur naturally within the same strain, but both are active against root pathogens. The recombinant strains produced both antibiotics in situ and suppressed Rhizoctonia root rot at a lower dose than did either parental strain.
2. Materials and methods 2.1. Bacterial strains, plasmids and wheat root pathogens Strains and plasmids used in this study are listed in Table 1. Escherichia coli and spontaneous rifampin-resistant derivatives of P. fluorescens strains [10,25,26] were grown in LB or M9 media [27] at 37 and 28 °C, respectively. Antibiotics were added to media at the following concentrations: ampicillin, 80 lg ml1 ; rifampicin, 75 lg ml1 ; kanamycin, 100 lg ml1 ; and tetracycline, 12.5 lg ml1 . G. graminis var. tritici isolate R3-111a-1 [26], R. solani AG-8 isolate C1 [28], and an isolate of P. irregulare from the roots of wheat [28] were used in all laboratory and greenhouse assays. 2.2. Construction of recombinant phenazine-producing derivatives of P. fluorescens Q8r1-96 Standard methods were used for DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation [27]. The phenazine biosynthetic operon phzABCDEFG from P. fluorescens 279, cloned previously as pT7-6A-G [16], was excised as a 6.7-kb BglII–XbaI DNA fragment and introduced into pALTER-Ex1 (Promega, Madison, WI) distal to the tac
Table 1 Bacterial strains and plasmids used in this study Strain or plasmid
Relevant characteristicsa
Source or reference
Strains P. fluorescens 2-79 RN10 Q2-87 Q8r1-96
Phzþ Rifr Nalr Phzþ Rifr Phlþ Rifr
[25] [26] [10]
E. coli JM109 S17-1(kpir)
F’ traD36 proAþ proBþ lacIq lacZDM15/recA1 gyrA96 thi hsdR17 supE44 relA1 D(lac-proAB) mcrA thi pro hsdR hsdM recA rpsL RP4-2(Tcr ::Mu) (Kmr ::Tn7) kpir
Promega [18]
Promega [29] [30] [16]
pUTKm-phz
T7, SP6 and tac promoters ColE1 f1ori Tetr ColE1 bla; identical to pUC18 but with polylinker flanked by SfiI sites Tn5-based delivery plasmid with Kanr ; bla oriR6K mobRP4 tnp* gene of Tn5-IS50R pT7-6 containing 6.9 kb BglII–XbaI fragment from pPHZ108A with phzABCDEFG genes from strain 2-79 pUTKm containing phzABCDEFG genes under control of the tac promoter
a
r
Plasmids pALTER-Ex1 pUC18Sfi pUTKm1 pT7-6A-G
This study
bla, b-lactamase; Kan , kanamycin resistance; Tet , tetracycline resistance; Rif , rifampin resistance; Nal , nalidixic acid resistance; Phzþ , production of phenazine-1-carboxylic acid; Phlþ , production of 2,4-diacetylphloroglucinol. r
r
r
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promoter. The 6.7-kb PtacphzABCDEFG fragment was then subcloned into pUC18Sfi [29], and finally, the 6.7kb phz fragment, now flanked with SfiI sites, was moved into the SfiI site of mini-Tn5Km1 [30], yielding pUTKm-phz. This plasmid was transformed into E. coli S17-1(kpir) and mobilized into P. fluorescens Q8r1-96 by using a biparental filter mating technique [18]. Transformants were selected on M9 minimal medium supplemented with kanamycin, and isolates containing presumptive transposed phz genes in genomic DNA were transferred ten times on King’s medium B agar [31] to assure that kanamycin resistance and colony morphology, color, and fluorescence remained stable. The Q8r1-96 genetic background of each recombinant clone was confirmed by fingerprinting with a RAPD technique [10], and the presence of the PCA and 2,4-DAPG biosynthetic genes was confirmed by PCR with phenazineand 2,4-DAPG-gene-specific primers [18,32]. Finally, growth kinetics in vitro of wild-type Q8r1-96 and its PCA-producing derivatives were compared in M9 broth amended with 0.05% yeast extract and in KMB broth. Turbidity (OD 600nm ) was monitored at 4, 8, 12, 20, and 24 h with a Dynatech MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA). 2.3. Fungal inhibition in vitro Growth inhibition of the three root pathogens by Q8r1-96 and its PCA-producing derivatives was tested on potato dextrose agar (PDA) (Becton, Dickinson and Company, Franklin Lakes, NJ). In general, two 3-ll drops from an overnight culture of bacteria in LB broth were spotted about 1 cm from opposite edges of a PDA plate and a plug 0.5 cm in diameter, cut from the leading edge of a fungal colony grown on PDA, was placed at the center. The plates were incubated at 28 °C and the distance between the edges of the bacterial spots and the fungal mycelium was measured. For G. graminis var. tritici, the bacteria were spotted 24 h after introduction of the fungus and the zone of inhibition was measured 5 d later. For R. solani AG-8, the bacteria and the fungus were introduced at the same time and the inhibition zone was measured after 5 d. For P. irregulare, the bacteria were spotted 48 h prior to fungal inoculation and the zone of inhibition was measured after two additional days. 2.4. Seed treatment Spring wheat (cv. Penawawa) was coated with bacteria at approximately 102 , 103 , or 104 per seed by using the methods described by Pierson and Weller [26]. The bacterial inoculum was grown on KMB agar plates for 48 h at 27 °C. Bacterial cells were then scraped from the plates, washed twice by centrifugation in 40 ml of sterile distilled water to remove residual 2,4-DAPG and PCA, suspended in 0.5% methylcellulose (Sigma Chemical Co.) to a volume
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of 15 ml, and serially diluted. For each seed treatment, 25 g of wheat seed was thoroughly mixed with 9 ml of an appropriately diluted cell suspension in 0.5% methylcellulose and air-dried overnight in a laminar flow hood. The actual number of CFU per seed was determined by dilution plating on KMB immediately before planting. 2.5. Disease suppression assays Tube assays were used to test the biocontrol activity of the parental and recombinant derivative strains against take-all, Rhizoctonia root rot, and Pythium root rot. Inocula were prepared by growing each fungal pathogen on autoclaved whole oat grains [26,28]. The colonized grains were stored at 4 °C until used. Plastic tapered tubes (2.5 cm 20 cm) with holes at the bottom were plugged with cotton balls to a height of about 3 cm and half-filled with fine vermiculite followed by 13 g of Quincy virgin soil, a Shano sandy soil [10], with or without inoculum of a pathogen. For assays of take-all and Rhizoctonia root rot, the soil was amended with 1% (w/w) of freshly ground oat kernel inoculum (sieved to collect particle sizes of 0.25–0.50 mm), and each tube received 10 ml of metalaxyl (2.5 mg of active ingredient ml1 ; Syngenta, Greensboro, NC) to minimize interference by Pythium spp. indigenous to soils of the Pacific Northwest. For Pythium root rot assays, one whole oat kernel colonized by P. irregulare was introduced into the top layer of soil in each tube and the tube was watered with 10 ml of tap water. The tubes were seeded immediately for assays of take-all suppression, or after incubation at room temperature for 48 h for assays of Pythium and Rhizoctonia root rot control. Two wheat seeds were sown per tube, covered with about 5 ml of fine vermiculite, and watered with 3 ml of water for Pythium assays or water plus metalaxyl (2.5 mg ml1 ) for take-all and Rhizoctonia assays. The tubes were arranged in a randomized complete block design and incubated in a growth chamber for 4–5 weeks (12-h photoperiod, 16 °C). After seedling emergence (3–4 d), each tube was watered twice weekly with 5 ml of onethird-strength Hoagland’s solution [26]. All experiments were repeated 3 times with 5 or 6 replicates per treatment and 5 tubes per replicate. Rhizoctonia root rot, Pythium root rot, and take-all were evaluated by determining the percentage of infected roots [22], plant height [22], and disease severity on a scale of 0–8 [33], respectively. All experiments were conducted three times with similar results. 2.6. Colonization of the wheat rhizosphere by fluorescent Pseudomonas spp. Wheat seed was treated with bacterial strains at 103 CFU/seed, planted, and grown as described above. Population sizes of the introduced strains in the
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2.7. Antibiotic isolation and detection PCA and 2,4-DAPG produced in situ were extracted from the roots of wheat as described by Bonsall et al. [34]. Briefly, 30 g of roots with adhering rhizosphere soil were extracted with 40 ml of 80% acetone acidified to pH 2.0 with trifluoroacetic acid (TFA). The extracts were filtered through Whatman No. 1 paper. Soil particles were removed by centrifugation and the filtrate was evaporated to a volume of approximately 8 ml. Samples were acidified again with TFA, extracted twice with 10 ml of ethyl acetate, and the pooled ethyl acetate extracts were dried. Prior to analysis by high performance liquid chromatography (HPLC), samples were suspended in 1 ml of 35% acetonitrile and 0.1% TFA. Antibiotic concentrations were determined relative to phenazine and 2,4-DAPG standards and related to bacterial population size. PCA and 2,4-DAPG synthesized in vitro were isolated and identified as described previously [34]. 2.8. Statistical analyses Plant heights and percentages of diseased roots were compared with STATISTIX (version 7, Analytical Software, St. Paul, MN). Differences among treatments were determined at each bacterial dilution by standard one-way analysis of variance, and mean comparisons among treatments were performed by using Fisher’s protected least significant difference test at P ¼ 0:05.
3. Results 3.1. Selection and characterization in vitro of recombinant strains Mating of P. fluorescens Q8r1-96 with E. coli S171(kpir)/(pUTKm-phz) yielded thirty-five recombinant colonies, five of which successfully survived selection on M9 agar containing kanamycin. These clones, designated Z30-97, Z31-97, Z32-97, Z33-97, and Z34-97, remained kanamycin-resistant after ten successive transfers on KMB agar and were stable with regard to colony size, morphology, color, and fluorescence. The
presence of the introduced phenazine biosynthetic genes was confirmed by PCR with the phenazine-specific primers PHZ1 and PHZ2 [18], and the RAPD banding patterns generated from these recombinant strains with the M13 primer were indistinguishable from those of the parental strain Q8r1-96 (data not shown). Cultures of Q8r1-96 attained maximal turbidity in M9 broth amended with 0.05% yeast extract by 12 h, whereas the turbidity of cultures of the recombinant strains continued to increase for up to 24 h. At 12 h, cell densities of Z31-97 and the other recombinant strains were about 50% and 30% lower, respectively, than that of Q8r1-96 (data not shown). Similar results were obtained when the experiment was performed in LB broth except that strain Q8r1-96 reached its maximal density by 8 h. Strain Z31-97 also grew more slowly and produced smaller colonies than Q8r1-96 and the other recombinant strains on KMB agar, and was not included in further tests. Cultures of the recombinant strains produced both PCA and 2,4-DAPG in vitro. At 40 h, PCA produced per 108 CFU in cultures of the recombinant strains ranged from about 1.5 to up to 27 times greater than in cultures of wild type 2-79 (Fig. 1). Similarly, cultures of the recombinant strains accumulated up to 2.7 times more 2,4-DAPG by 24 h than did those of strain Q8r1-
Integrated Area (CFU 108)-1
rhizosphere were determined after 66 h and 1, 3, 5, and 7 weeks as described by Raaijmakers and Weller [10]. Briefly, four seedlings were selected at random from each replicate and shaken to remove the loosely-adhering soil. One gram of roots with associated soil was suspended in 5 ml of sterile water, agitated vigorously by Vortex mixing and sonication, and serial dilutions of the wash suspension were spread onto KMB agar amended with rifampicin and cycloheximide at 100 lg ml1 . Colonies were enumerated after 48 h.
0.3 (A)
0.25 0.2 0.15 0.1 0.05 0
Integrated Area (CFU 108)-1
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1.0 (B)
0.8 0.6 0.4 0.2 0 0
8
16
24 Time (h)
32
40
48
Fig. 1. Production in vitro of phenazine-1-carboxylic acid (A) and 2,4diacetylphloroglucinol (B) by wild-type and recombinant strains. Cultures were grown with constant shaking at 27 °C in modified KMB broth. Antibiotics were extracted and viable counts were determined at 8, 16, 24, 32, 40, and 48 h as described in Section 2. The strains are Q8r1-96 ðjÞ; Z30-97 ðMÞ; Z32-97 (X); Z33-97 ðÞ; Z34-97 ðsÞ; and 279 ð}Þ.
Z. Huang et al. / FEMS Microbiology Ecology 49 (2004) 243–251 Table 2 Inhibition in vitro of wheat root pathogens by Q8r1-96 and recombinant derivative strains Strain
Z34-97 Z33-97 Z32-97 Z30-97 Q8r1-96
Zone of inhibition (cm)A Gaeumannomyces graminis var. tritici
Rhizoctonia solani AG-8
Pythium irregulare
1.7aB 1.7ab 1.1c 1.6b 0.5d
0.6a 0.6a 0.5a 0.6a 0.2b
0.7a 0.7a 0.7a 0.8a 0.5b
A
Width of the zone of inhibition was measured from the edge of the fungal colony to the edge of the bacterial colony. B Means in the same column followed by the same letter are not significantly different at P ¼ 0:05 according to the protected LSD.
96. Strain Z34-97 produced maximal amounts of 2,4-DAPG earlier than did the other strains. These increased levels of antibiotic production were correlated with enhanced antifungal activity in vitro. All of the recombinant strains produced larger zones of inhibition against the three fungal pathogens than did Q8r1-96 (Table 2). Inhibition of P. irregulare by the recombinant strains was 43–70% greater than that by Q8r1-96, and inhibition of G. graminis var. tritici and R. solani AG-8 was 2–3 times, and over 3 times greater, respectively, than inhibition by the parental strain. 3.2. Root disease suppression in soil Preliminary studies indicated that recombinant strains applied at doses of 105 per seed or greater sometimes were toxic to wheat, causing reduced emergence and stunting of the roots and shoots. To avoid these symptoms of phytotoxicity, the bacteria in this study were applied at doses of 102 , 103 , and 104 CFU per seed. P. fluorecens Q8r1-96 suppressed take-all significantly (P ¼ 0:05) as compared to the nontreated and
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methylcellulose controls at all three doses tested. For example, the root disease ratings for Q8r1-96 applied at 102 , 103 , and 104 CFU per seed and for the two controls were 3.8, 2.9, 2.6, 4.2, and 4.2, respectively, on a scale of 0–8. However, none of the recombinant strains showed increased suppression of take-all as compared to Q8r196 (data not shown). Strain Q8r1-96 applied at 104 per seed, but not at lower doses, significantly (P ¼ 0:05) increased the height of wheat planted in soil infested with Pythium irregulare as compared to the methylcellulose and nontreated controls (Table 3). In contrast, the four recombinant strains significantly increased plant height at all three doses compared to the controls. Except for Z34-97 at 103 and 104 CFU per seed, however, the recombinant strains did not differ significantly from Q8r1-96 at each of the three doses, which were compared individually to the controls because of strain by dose interactions. Results were similar in subsequent experiments. Strain Q8r1-96 at a dose of 104 CFU per seed and all four recombinant strains at all three doses significantly ðP ¼ 0:05Þ reduced the percentage of roots with symptoms of Rhizoctonia root rot as compared to the methylcellulose and nontreated controls (Fig. 2, Table 3). Treatments were compared within doses because of strain by dose interactions. At a dose of 103 CFU per seed, all four recombinant strains reduced disease significantly more ðP ¼ 0:05Þ that did Q8r1-96. At doses of 102 , recombinant strains Z32-97, Z33-97 and Z34-97 reduced disease significantly ðP ¼ 0:05Þ more than Q8r1-96; with Z30-97 the disease reduction was not significant compared to the parental strain. In subsequent experiments at a dose of 102 , Z30-97 was significantly more suppressive than Q8r1-96 (data not shown). At a dose of 104 , all recombinant strains except Z34-97 reduced disease significantly more than Q8r1-96; Z34-97 reduced disease more than the parental strain but the difference was not significant. Similar results
Table 3 Suppression of rhizoctonia and pythium root rot by wild type and recombinant strains of Pseudomonas fluorescens Treatment
% Roots with rhizoctoniaA 102 C
Q8r1-96 Z30-97 Z32-97 Z33-97 Z34-97 2-79 MCD Nontreated A
E
50.3ab 37.3bc 28.6c 30.9c 31.7c . . .F 53.1a 54.9a
Height (cm) of Pythium-infected plantsB
103
104
102
103
104
46.1a 19.1b 21.5b 29.2b 28.7b ... 53.1a 54.9a
42.4bc 30.9d 24.2d 30.7d 34.0cd 47.3ab 53.1a 54.9a
30.7ab 32.0a 31.4a 32.2a 32.5a ... 28.8b 28.7b
30.9bc 31.7ab 32.9ab 31.1b 33.9a ... 28.7c 28.6c
31.4b 32.2b 32.7b 31.9b 34.8a ... 28.7c 28.6c
Percentage of seedling roots with typical symptoms of Rhizoctonia root rot 31 days after planting. Shoot length of seedlings with roots infected with Pythium 31 days after planting. C Approximate dose of each strain (CFU per seed) as determined by dilution plating. D Seed treated with 0.5% methylcellulose only. E Means in the same column followed by the same letter are not significantly different at P ¼ 0:05 according to Fisher’s protected LSD. F Not tested at this dose. B
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Fig. 2. Suppression of Rhizoctonia root rot by wild-type and recombinant strains of Pseudomonas fluorescens. Wheat seed treated with 0.5% methylcellulose alone (control) or with bacterial suspensions of Z34-97, Q8r1-96 (each at 103 per seed) or 2-79 (104 per seed) was sown in soil infested with Rhizoctonia solani AG-8. Root rot symptoms included darkened lesions and reduced root mass, and were most severe on the control roots, least severe on roots colonized by Z34-97, and intermediate in severity on roots colonized by strains Q8r1-96 and 279.
were obtained when the experiments were repeated except that no significant differences occurred between Q8r1-96 and the recombinant strains at a dose of 104 . At a dose of 104 , Q8r1-96 but not 2-79, had significantly less disease than the methylcellulose and non-treated controls. Although PCA is more inhibitory of R. solani than 2,4-DAPG, the greater control by Q8r1-96 reflects its superior root colonization ability. 3.3. Colonization and antibiotic production in the wheat rhizosphere Strains Q8r1-96, Z30-97, Z32-97, Z33-97, Z34-97, and Q2-87 applied at 103 CFU per seed multiplied rapidly on the seeds and in the wheat rhizosphere. The population sizes of all of the strains had increased about 1000-fold on the seed coat and the radical by 66 h (0.4 week), and peaked after one week (Fig. 3). Populations of the recombinant strains and Q8r1-96 did not differ in size throughout the 7-week study, and were significantly higher than those of Q2-87 at weeks 1, 3, 5, and 7. The
Population, CFU (g root fresh weight)
-1
109 108 107 106 105 104 103 102 0
1
2 3 Weeks after planting
4
5
Fig. 3. Population dynamics of wild-type and recombinant strains of Pseudomonas fluorescens in the rhizosphere. Wheat seed (cv. Penawawa) was coated with bacterial strains at a density of 103 CFU per seed (approximately 2 104 CFU g1 of seed), sown in Quincy virgin soil, and seedlings were harvested after 5 weeks. Error bars represent the standard error of four replicates. The strains are Q8r1-96 ðjÞ; Z30-97 ðMÞ; Z32-97 (X); Z33-97 ðÞ; Z34-97 ðsÞ; and Q2-87 ð}Þ, an ‘‘average’’ root colonizer.
recombinant strains produced both PCA and 2,4-DAPG in the rhizosphere and, consistent with results obtained in vitro, production of both antibiotics was greater for the recombinant than for the wild-type strains. Strains Z33-96 and Z34-96 produced 4–5 times more PCA per 108 CFU than did strain 2-79 (P ¼ 0:008; Kruskal– Wallis one-way analysis of variance), and Z34-97 about 11-fold more 2,4-DAPG than did Q8r1-96 (P ¼ 0:003; one-way analysis of variance) (Table 4).
4. Discussion Wheat growing worldwide can be damaged by one or more of the root diseases take-all, Pythium root rot, and Rhizoctonia root rot, which often occur in the Pacific Northwest as a complex in the same field. These diseases
Table 4 Production in the rhizosphere of 2,4-diacetylphloroglucinol (2,4-DAPG) and phenazine-1-carboxylic acid (PCA) by P. fluorescens Q8r1-96, P. fluorescens 2-79, and recombinant strains Strains
Populationb (CFU g1 root)
2,4-DAPGc (ng g1 of root)
PCAc (ng g1 of root)
2,4-DAPGc (ng (105 )1 CFU)
PCAc (ng (105 )1 CFU)
Q8r1-96 (103 )a 2-79 (104 ) 2-79 (106 ) Z34-97 (103 ) Z33-97 (103 )
2.30 108 0.18 108 0.41 108 0.87 108 0.64 108
291.4 134.8 N/A N/A 1248.9 322.1 344.3 61.3
N/A 15.9 1.3 16.8 10.6 313.6 80.6 269.6 80.8
0.13 0.06 N/A N/A 1.45 0.35 0.44 0.1
N/A 0.08 0.01 0.04 0.03 0.35 0.08 0.42 0.13
a
Values in brackets indicate the approximate dose at which strains were applied to wheat seeds. CFU of introduced bacteria per gram of root as determined by dilution plating. c Mean standard deviation ðn ¼ 3Þ; N/A, not applicable. b
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are exacerbated when wheat is direct-seeded (no-till) [21], a practice gaining in popularity because it reduces soil erosion and rebuilds soil organic matter. Certain strains of fluorescent Pseudomonas spp. can suppress these diseases, but most strains fail to perform consistently and no single strain is effective against all three phytopathogens. The goal of this study was to enhance the performance of biocontrol agents or to extend their activity to a wider range of wheat root diseases. We attempted to improve biocontrol activity by inserting foreign genes that confer the ability to produce a biologically active metabolite into the biocontrol strain P. fluorescens Q8r1-96. A key characteristic that makes Q8r1-96 an ideal host for such manipulations is its unusual aggressiveness in colonizing the roots of wheat and pea and its ability to suppress take-all when applied to seeds or soil at doses of 104 or fewer CFU per seed or gram of soil [10,35]. Other Pseudomonas strains with biocontrol activity require doses 100- to 10,000-fold greater in order to achieve take-all suppression [25]. We modified Q8r1-96 through a stable insertion of genes for biosynthesis of PCA, which has broad-spectrum activity against a variety of soilborne pathogens [3]. Unlike the polyketide 2,4-DAPG, PCA is synthesized via the shikimic acid pathway [16], reducing the probability that the two pathways would compete directly for the same biosynthetic precursors. To our knowledge, recombinant derivatives of Q8r196 used in the study represent the first example of fluorescent Pseudomonas spp. that have the capacity to produce both phenazines and the polyketide metabolite 2,4-DAPG. Bacteria that produce both antibiotics are not known in nature. Introduction of the constitutivelyexpressed PCA biosynthetic genes, even in single copy, clearly placed a metabolic burden on Q8r1-96 because all of the recombinant strains grew more slowly in vitro than did Q8r1-96. Nevertheless, Q8r1-96 and its recombinant derivatives had similar population dynamics in the short-term colonization studies reported here, indicating that the metabolic cost of PCA synthesis in pure culture is at least partially offset in the more nutritionally and microbiologically heterogeneous rhizosphere environment. The recombinant strains produced PCA and 2,4-DAPG in quantities equal to or greater than those produced by the parental strains, both in vitro and in the rhizosphere (Fig. 1, Table 4). Increased accumulation of PCA likely was due to constitutive expression of the phz genes from the tac promoter, but the observed overproduction of 2,4-DAPG is less easily explained. In wild-type P. fluorescens strains CHA0 and F113, 2,4-DAPG induces its own synthesis, and synthesis is repressed by salicylate and certain other aromatic compounds [36]. We speculate that the constitutively expressed PCA pathway drains the chorismate pool enough to limit the synthesis of salicylate or another potential repressor of 2,4-DAPG syn-
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thesis. The differences observed between individual recombinant strains in amounts of PCA and 2,4-DAPG produced and in biocontrol activity suggest that positional effects are associated with sites of insertion of the phz operon. Despite their enhanced antibiotic production, the recombinant biocontrol agents were not necessarily more effective than their parental strains against the three diseases tested. For example, no recombinant strain provided greater suppression of take-all than did Q8r196. G. graminis var. tritici is highly sensitive to 2,4-DAPG (complete inhibition at 3 lg/ml), Q8r1-96 is highly effective against take-all, and isolates genotypically identical to Q8r1-96 are responsible for the natural biological control of take-all known as take-all decline in Washington State soils [11]. Thus, Q8r1-96 probably already is ‘‘finely tuned’’ to suppress take-all, and not much more can be done to improve its performance even though the pathogen is also highly sensitive to PCA. It is likely that there is a threshold dose of both antibiotics in the rhizosphere above which no further inhibition is derived. The recombinant strains were only slightly more effective than Q8r1-96 against Pythium root rot as indicated by the fact that plants treated with Q8r1-96 were no different in height than control plants or those treated with recombinant strains, but plants treated with recombinant strains were significantly taller than control plants. Pythium irregulare is less sensitive to both 2,4DAPG (complete inhibition at 10 lg ml1 ) and PCA than is the take-all pathogen, but the elevated levels of one or both of the antibiotics present on roots colonized by the recombinant strains was correlated with measurably better shoot growth than seen with seedlings colonized by Q8r1-96. While further work will be required to determine whether 2,4-DAPG, PCA, or a combination of the two was responsible for this effect, it is noteworthy that overproduction of 2,4-DAPG alone by P. fluorescens CHA0 did not improve protection of sweet corn and cress against Pythium damping-off [37], nor was an overproducing strain of P. fluorescens F113 more effective than the wild-type strain against P. ultimum on sugarbeet [38]. In contrast, PCA-producing derivatives of P. fluorescens SBW25 containing the same phenazine gene construct described here were significantly more suppressive of damping-off disease of pea seedlings caused by P. ultimum than was the parental strain, and the effectiveness of the transformed strains was directly correlated with the level of PCA biosynthesis [39]. Finally, the recombinant strains were substantially more suppressive of Rhizoctonia root rot than was Q8r1-96. This difference was most apparent at lower doses, where wheat treated with recombinant strains had about 40–50% less diseased roots compared to control plants treated with Q8r1-96. The PCA-producing recombinant derivatives retained the unique colonization
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properties of strain Q8r1-96 and were highly effective against Rhizoctonia when applied at only 102 CFU per seed. In theory, a one-liter culture of Z34-97 at 1010 CFU per ml, easily attainable in standard growth media after overnight incubation, should be sufficient to treat the seed (at 102 CFU per seed) needed to plant 10,000 acres of wheat. Obtaining consistent biocontrol with such low doses has long been a goal of PGPR research and would remove barriers related to inoculum production, formulation and cost that now limit more widespread use of Pseudomonas biocontrol agents. Finally, the use of transgenic strains may offer certain benefits over the application of strain consortia in which components may compete for the same niche, leading especially at low doses to rapid displacement of the less rhizosphere-competent strain by its more aggressive counterpart. Rhizoctonia solani has a very broad host range and the recombinant strains have applicability to other crops commonly grown in cereal-based production systems. However, we fully realize that the effects of such genetically-modified strains on the environment must be carefully considered when proposing their release for control of soilborne pathogens. Studies of the long-term fate of these recombinant strains, their impact on indigenous rhizosphere microflora, and their competitiveness with the parental strain are underway.
Acknowledgements Dmitri V. Mavrodi and Robert F. Bonsall contributed equally to this study. This work was supported by grant 96-35303-3242 from the U.S. Department of Agriculture, Office of Grants and Program Systems, National Research Initiative, Competitive Grants Program.
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