Biological control of Verticillium wilt on cotton by the use of fluorescent
Pseudomonas spp. under field conditions
Oktay Erdogan
a, Kemal Benlioglu
b,* aCotton Research Institute, Nazilli, Aydin, Turkey
b
Adnan Menderes University, Faculty of Agriculture, Plant Protection Dept., 09100 Aydin, Turkey
a r t i c l e
i n f o
Article history:
Received 17 January 2009 Accepted 23 November 2009 Available online 3 December 2009 Keywords: Verticillium Cotton Fluorescent Pseudomonas Serratia plymuthica Weed Field trial
a b s t r a c t
Four out of 59 fluorescent Pseudomonas spp. strains collected from cotton and weed rhizosphere were selected based on the following criteria: (1) inhibition of Verticillium dahliae in vitro, (2) disease suppres-sion on two cotton cultivars grown from bacterized seeds using stem-injection with the conidia of V. dah-liae, and (3) seedling vigor test (dry weight) under greenhouse conditions. Four selected Pseudomonas strains isolated from Xanthium strumarium (FP22), Portulaca sp. (FP23), Gossypium hirsitum (FP30), and Convolvulus arvensis (FP35), as well as the known biocontrol agent Serratia plymuthica (HRO-C48), were further tested for the impact on Verticillium wilt, growth parameters of cotton, and yield in a naturally infested field. The reduction of AUDPC by the seed bacterization with FP22, FP23, FP30, FP35, and HRO-C48 compared to non-bacterized control ranged from 39.2% to 50.9% and 22.1% to 36.8% in trials done in 2005 and 2006, respectively. The growth parameters (plant height, number of nodes on main stem, and NAWF-nodes above white flower) were significantly higher in seed bacterized plants compared to the untreated control. In the 2005 field trial, the increase of seed cotton yield by the treatment with four Pseudomonas strains and HRO-C48 ranged from 13.1% to 22.3% in Sayar 314 and 4.2% to 12.8% in Acala Maxxa. Seed cotton yield was not significantly influenced by the 2006 treatments. Our results indicate that seed treatment of cotton plants with our Pseudomonas spp. strains and the known strain Serratia ply-muthica can help in the biocontrol of V. dahliae and improve growth parameters in cotton fields.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
Cotton is a high value crop grown in western and southern Tur-key. In recent years, there has been a substantial increase in organ-ic cotton production worldwide, and Turkey has become the biggest producer of organic cotton in the world.1With 12,507 met-ric tons of organic cotton produced in 2007, Aydin province is the second-largest organic cotton producer in the country.2Verticillium wilt, caused by the soil inhabiting fungus Verticillium dahliae (Kleb.) is one of the most important diseases responsible for great economic losses in many crops (Tjamos et al., 2000). The disease was first dis-covered in the Manisa province of Turkey in 1941 (Iyriboz, 1941) and has since become widespread in the main cotton-growing areas of Turkey (Karaca et al., 1971; Esentepe, 1974; Dervis and Bicici, 2005). Verticillium wilt disease is difficult to control due to the broad host range of the pathogen, long viability of the resting structures
(microsclerotia), and the inability of fungicides to affect the patho-gen once it enters the xylem (Fradin and Thomma, 2006). Therefore, the most effective control of Verticillium wilt of cotton is achieved by an integrated management approach covering both growing adapted resistant cultivars and using cultural and management prac-tices known to reduce disease severity (El-Zik, 1985). However, the unavailability of commercially acceptable resistant cotton cultivars and the high cost and difficulty in large-scale application of soil solarization and fumigation to reduce microsclerotia in soil make it necessary to consider other control methods. The use of biological control agents has been increasing worldwide and is a promising alternative for controlling soil-borne diseases in sustainable and or-ganic agriculture. One of the recent approaches used to control Ver-ticillium wilt disease is rhizobacteria-mediated biological control. Previous studies have shown that antagonistic bacteria can be used successfully to control V. dahliae in plants (Leben et al., 1987; Saf-iyazov et al., 1995; Zhengjun et al., 1996; Berg et al., 2001; Tjamos et al., 2004; Cubukçu and Benlioglu, 2007). A registered biopesticide (RhizoStarÒ) strain, Serratia plymuthica (HRO-C48) isolated from the
rhizospheres of oilseed rape, was successfully used to control Verti-cillium wilt of strawberry and also increase the yield as shown in greenhouse and field trials (Kurze et al., 2001).
1049-9644/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2009.11.011
* Corresponding author. Fax: +90 256 7727233. E-mail address:kbenlioglu@adu.edu.tr(K. Benlioglu).
1 Organic Cotton Farm and Fiber Report, 2007, Organic Exchange publications,
www.organicexchange.org.
2 Organic Agriculture Statistics 2007, Ministry of Agriculture and Rural Affairs,
www.tarim.gov.tr.
Contents lists available atScienceDirect
Biological Control
Among biocontrol agents, root-associated fluorescent Pseudo-monas spp. has received special attention because of its excellent root colonizing ability (Gamliel and Katan, 1992a,b, 1993; Berg et al., 2006), potential to produce a wide variety of anti-microbial metabolites (O’Sullivan and O’Gara, 1992), and its induction of sys-temic resistance (Haas and Defago, 2005). Recent attempts to pro-duce biological control of V. dahliae have indicated that fluorescent Pseudomonas strains could effectively reduce the incidence and severity of wilt on olive planting stocks (Mercado-Blanco et al., 2004), eggplants (Malandraki et al., 2008), and potatoes (Uppal
et al., 2008) under greenhouse and field conditions.
The objective of this research was to determine whether fluo-rescent Pseudomonas isolated from cotton and weed rhizospheres could be effective in controlling Verticillium wilt in the field.
2. Materials and methods
2.1. Isolation and origin of microorganisms
Plant rhizosphere samples were collected from random cotton fields in five main cotton-growing regions (Aydın Merkez, Çine, Koçarlı, Nazilli and Söke) of Aydin province from July through Sep-tember in 2004. Bacteria were isolated from the rhizospheres of symptomless cotton plants and weeds (Chenopodium album, Sola-num nigrum, Xanthium strumarium, Datura stramonium, Portulaca sp., Sinapis sp., Convolvulus arvensis, and Malva sylvestris) known to be hosts of V. dahliae (Thanassoulopoulus et al., 1981). The plants were uprooted with rhizosphere soil and transported to the laboratory in polyethylene bags, where they were thoroughly washed with tap water. Approximately 5 g of root tissue was ex-cised from each plant samples and surface sterilized for 3 min in NaClO (0.5% available chlorine), then rinsed thoroughly in sterile distilled water. Root samples were shaken in 95 ml of sterile 0.05 M phosphate buffer (pH 7.2) for 1 h at room temperature. Samples were serially diluted with phosphate buffer and plated onto King’s medium B agar (KB) (King et al., 1954) supplemented with cycloheximide (100 ppm) and incubated at 24 °C for 48 h. Sin-gle fluorescent colonies were purified and stored in 30% glycerol at 76 °C until needed. Bacterial identification was performed based solely on the fluorescence in KB medium without any further at-tempt to identify the species of the most promising biocontrol strains.
One highly virulent V. dahliae strain (VD-11) selected among 32 V. dahliae strains collected from the same cotton fields as the bio-control strains was used for all in vitro and greenhouse tests (
Erdo-gan, 2007). In addition, the known biocontrol agent Serratia
plymuthica (strain HRO-C48), kindly provided by Gabriel Berg (Graz University of Technology – Austria), was used for comparison in field trials.
2.2. Preliminary screen
Fifty-nine bacterial strains were tested for their ability to pro-duce antifungal substances against V. dahliae using a dual-culture in vitro assay on PDA plates. Each plate was inoculated with four droplets of 10
l
l bacterial suspension (at a 108cfu/mlconcentra-tion) symmetrically placed on four sites at equal distances (2 cm) from the center of plate. After 24 h incubation at 24 °C, a single 5-mm-diameter mycelial disc was placed in the center. As a con-trol, a disc of V. dahliae was grown on a PDA plate. The radius of each fungal colony was measured after a 10-day incubation at 24 °C in darkness, and the relative growth inhibition was expressed as a percentage [(treatment–control)/control 100]. This experi-ment was conducted twice in triplicate.
2.3. Greenhouse experiments 2.3.1. Seed bacterization
Acid delinted seeds of two cotton cultivars, V. dahliae-sensitive local variety cv Sayar 314 (Göre et al., 2008) and V. dahliae-tolerant Upland cultivar Acala Maxxa (Bölek et al., 2005), were bacterized for pot and field trials. Cottonseeds were treated with antagonistic bacteria after surface disinfestations with 1% NaOCl for 1 min and followed by three washes in sterile 0.05 M phosphate buffer (PB, pH 7.4). The antagonistic bacteria were grown for 24 h at 25 °C on Tryptic soy broth (Difco) and pelleted by centrifugation (5000g, 5 min, 4 °C). After washing with PB, the pellets were resus-pended in 1.5% carboxy-methyl-cellulose (CMC) solution in sterile PB. Seeds were soaked for 30 min in a bacterial suspension (100 mg bacteria and 1 ml of 1.5% CMC solution per 20 seeds) or in the same volume of CMC without bacteria (the control treatment) and then dried under a laminar flow hood for 12 h and stored at 4 °C (
Quadt-Hallmann et al., 1997). Before sowing, the number of
colony-form-ing units of bacteria per seed was estimated by dilution platcolony-form-ing suspensions of 10 seeds in 10 ml PB on KB. The seed bacterization procedure resulted in a mean bacterial concentrations of 7.1 107–1.0 108cfu/seed in the greenhouse trials. The mean
number of bacteria ranged from 5 106 to 1.0 107cfu/seed in
2005, and from 1.1 107to 1.4 107cfu/seed in 2006 field trials.
2.3.2. Biological control of Verticillium wilt in the greenhouse The effects of fifteen fluorescent Pseudomonas strains against Verticillium wilt were tested in a growth room on two cultivars of cotton plants (cv Sayar 314 and Acala Maxxa). The bacterized and control seeds were each planted into 10-cm diameter plastic pots (400 ml) containing an autoclaved soil–sand–peat (1:1:1) mixture. Conidia from 10-day-old PDA plates inoculated with the V. dahliae strain (VD-11) were washed once with sterile water and diluted to a concentration of 3 107conidia/ml. Plants at the
six-true-leaf stage were wound-inoculated by puncturing the stem at the first internode above the soil line with a 22-gauge needle through a 5-
l
l drop of the conidial suspension (Hanson, 2000). Plants were incubated at 25 ± 2 °C with a 14-h photoperiod and fer-tilized once a week with liquid fertilizer (Sheffer 16-8-24N-P-K plus micronutrients). Control plants (bacterized and non-bacterized) were inoculated with sterile water. Fourteen days after inoculation, disease severity was assessed for each plant on a 0-to-4 rating scale according to the percentage of foliage affected by acropetal chloro-sis, necrochloro-sis, wilt, and/or defoliation (0 = healthy plant, 1 = 1–33%, 2 = 34–66%, 3 = 67–97%, 4 = dead plant) (Bejarano-Alcazar et al., 1995). Each combination of bacterial treatments, controls, and cul-tivars was replicated five times in a randomized complete design, which included three plants per replicate for each cultivar and the experiment was repeated twice.2.3.3. Plant dry weight in pods
A growth chamber experiment was conducted to compare the growth-promoting effects of fifteen Pseudomonas strains. Bacte-rized and non-bacteBacte-rized cottonseeds (Sayar 314 and Acala Maxxa) were planted in sterile 15 15 10 cm PVC containers filled with sterile sand. The water content of the sterile sand was adjusted to field capacity (13% moisture v/v) with sterile 10% liquid fertilizer (Sheffer) solution. Each treatment was replicated five times and in-cluded three plants per replicate for each cultivar. Treatments were randomized and the treated plants placed in a growth chamber at 25 ± 2 °C and a 14-h photoperiod. Plants were watered twice a week with sterile tap water and once a week with the fertilizer solution. After 24 days, plants were removed from the containers and washed. Whole plants were oven-dried at 105 °C for 48 h and then weighed. Plant dry weights were compared among treat-ments. The experiment was independently repeated.
2.4. Field trials
2.4.1. Field conditions and experimental design
Experiments were conducted to determine the ability of Pseudo-monas spp. strains (FP22, FP23, FP30 and FP35) and S. plymuthica (strain HRO-C48) to suppress wilt symptoms and to evaluate cotton yield potentials under field conditions. During 2005 and 2006, the experiments were carried out in a field that was naturally infested with a non-defoliant pathotype of V. dahliae, which has been repeat-edly used for cotton breeding field trials at Nazilli Cotton Research Institute since 1972. The natural population of microsclerotia of V. dahliae was determined from soil samples according to a standard-ized methodology from soil depths of 20–25 cm in an experimental field in May 2005 and 2006. Soil samples were air-dried under ambient temperatures for three weeks and then passed through a 0.8-mm sieve after the removal of stones and plant residues. The number of microsclerotia per g of soil was estimated by the wet sieving technique (Huisman and Ashworth, 1974) in five sub-sam-ples (ca. 25 g each) of sieved soil. The inoculum density was 14 ± 2.6 microsclerotia per g of soil in 2005 and 10 ± 3.2 in 2006.
The soil texture in the experimental field was a sandy clay with pH 7.85, 1.94% organic matter, 18.41% lime, and 0.11% salt, 603.7 ppm K2O and 6.9 ppm P2O5. Each year, 60 kg of N and
60 kg of P2O5per hectare were incorporated with a harrow before
cotton planting. The same amount of nitrogen was also applied be-tween rows just before flowering, and two sprays were made to control cotton pests like aphids and red spider mites. Maximum and minimum air temperatures were obtained from a data logger (Hobo, Onset Computer, USA) located in the experimental field during the cotton-growing season.
Treatments were applied in plots that were 0.7 m wide (in two rows of approximately 60 plants per row) by 12 m long in a ran-domized complete block design (at a 2-m interval between blocks) with two factors and four replicates.
2.4.2. Disease assessment and effect on yield
Verticillium wilt in each sampled plot was assessed three times each year during the period beginning early September and ending mid-October, in the 5–10%, 50–60%, and 75% of the bolls open stage. At each recording date, each individual plant was examined for the foliar symptoms of wilt and the disease severity was esti-mated for each plant using the same scale as previously described and the incidence (%) of infected plants was then determined for each plot. A disease severity index (DSI) was calculated for each plot by: (mean severity incidence%)/maximum severity rating. For DSI, the area under the DSI progress curve (AUDPC) was calcu-lated by trapezoidal integration (between day 0 and final disease assessment day) for each plot (Campbell and Madden, 1990).
Growths parameters, such as number of main stem nodes, NAWF (nodes above white flower), and plant height were also re-corded for ten randomly selected plants from each experimental plot. The number of nodes and NAFW were determined 2 or 3 days before the first disease rating at the 5–10% bolls open stage (8 Sep-tember 2005 and 9 SepSep-tember 2006). When counting the number of nodes, the cotyledonal node was counted as ‘‘0”. To determine NAWF, the number of nodes down from the terminal (terminal was considered 0) to the first white flower was counted. Plant height was measured one week after the last disease rating at the 75% of bolls open stage. At the end of the growing season (7 November 2005 and 21 November 2006), cotton seed yield was determined by hand harvesting from each plot.
2.5. Statistical analysis
Data were subjected to analysis of variance with the general lin-ear models procedure of JMP statistical software (Mac version
7.0.2, SAS Institute, Cary, NC, for Macintosh computer). When tests for inhibition in broth culture were carried out, differences in inhi-bition of V. dahliae by fluorescent Pseudomonas strains were tested by a one-way analysis of variance (ANOVA). To test differences in seedling vigor, disease suppression under greenhouse and field conditions, and plant growth parameters including yield among seed bacterization for two cultivars, two-way analysis of variance (ANOVA) was done. Mean separations were determined by Fisher’s protected least-significant-difference (FLSD) test. All data were checked for normality using a Shapiro–Wilk test (normality was assumed if P > 0.05) and data transformation was applied when necessary.
3. Results
3.1. Preliminary screen
Fifty-nine fluorescent Pseudomonas strains showing substantial inhibition zones against V. dahliae (VD-11) on PDA were isolated from roots. Eighteen were from cotton and five from each of fol-lowing weeds: C. album, S. nigrum, X. strumarium, D. stramonium, Portulaca sp., Sinapis sp., C. arvensis, and M. sylvestris, and 1 from Echinochloa colonum. Fifteen out of 59 Pseudomonas spp. strains showing high in vitro antifungal activity against V. dahliae were se-lected and used in subsequent in vitro inhibition tests and green-house experiments. Pseudomonas spp. with high antagonistic activity against V. dahliae were found in the rhizosphere of cotton and weed hosts of V. dahliae (Table 1). Strains FP5, FP15, FP21, FP29, FP35, and FP39 had the highest antagonistic activity in vitro. 3.2. Greenhouse experiments
Wilt symptoms developed on Verticillium-inoculated plants of both cultivars but were not observed on any of the water-inocu-lated plants. About 8 days after inoculation, the first symptoms ap-peared as blotchy, light yellow areas between leaf veins on the first and second mature leaves of the control plants of Sayar 314. Le-sions developed on mature leaves of all Verticillium-inoculated plants of both cultivars after 11 days. After 14 days, all inoculated control plants of Sayar 314 were dead. The in vivo effect of 15 fluo-rescent Pseudomonas strains on V. dahliae (VD-11) varied depend-ing on the cotton cultivar used (Table 1). The treatment of seeds with strains FP22, FP23, FP30, and FP35 significantly reduced symptom expression (by 33.3%) in artificially inoculated V. dah-liae-tolerant cotton plants (cv Acala Maxxa), while wilt symptoms were significantly reduced by 32.5% and 50% in plants of Sayar 314 treated with strains FP30 and FP35, respectively (Table 1).
Cotton seedling vigor, as measured by plant dry weight, was significantly increased (14.8–22%) by bacterization of cottonseed with seven strains of fluorescent Pseudomonas (FP1, FP22, FP23, FP25, FP29, FP30, and FP35) compared to that of non-bacterized control (P < 0.001) (Table 1). Significant interactions between culti-var and bacterial treatments were also observed for growth promo-tion of cotton seedlings in terms of plant dry weight.
3.3. Field trials
Four fluorescent Pseudomonas strains (FP22, FP23, FP30, and FP35) were selected for field trials after evaluating their ability for in vitro growth inhibition, disease suppression, and growth pro-motion under greenhouse conditions. Evidence of Verticillium wilt was apparent in all experimental plots in both 2005 and 2006. First symptoms, consisting of chlorotic areas between the main veins of the lower leaves, appeared in the first week of August (about three weeks after the first bloom) in the 2005 and 2006 growing seasons.
Seed bacterization with four fluorescent Pseudomonas strains (FP22, FP23, FP30, and FP35) and S. plymuthica (HRO-C48) resulted in significant reduction in AUDPC in both susceptible (Sayar 314) and tolerant (Acala Maxxa) cultivars compared to the non-bacte-rized control in 2005 (Table 2). In the 2006 trial, only two biocon-trol strains, FP35 and HRO-C48, caused a significant reduction in AUDPC in Acala Maxxa, while all strains significantly reduced AUDPC in Sayar 314. There was a significant interaction (P < 0.0001) between treatments and cultivar, and the tolerant cul-tivar had significantly lower AUDPC values then the susceptible one in both experimental years. The reduction of AUDPC by the seed bacterization with FP22, FP23, FP30, FP35, and HRO-C48 com-pared to non-bacterized control ranged from 39.2% to 50.9% and 22.1% to 36.8% in the 2005 and 2006 trials, respectively (Table 2).
However, ANOVA showed that seed bacterization with FP22, FP23, and FP30 had significantly a higher effect on Sayar 314 then on Acala Maxxa for disease suppression in both years.
In both years, growth parameters (plant height, number of nodes, and NAWF) and yield (except in 2006) in the two cotton cul-tivars were significantly affected by the seed bacterization with four fluorescent Pseudomonas strains (FP22, FP23, FP30, and FP35) and S. plymuthica (HRO-C48) under disease pressure (
Ta-ble 3). No significant interactions between cultivar and treatments
were observed for plant height, number of nodes, NAWF, and yield for each trial year. When recording was performed for approxi-mately 33 days after the first wilting symptoms appeared on leaves, the number of nodes on main stem was significantly higher for the treatments (FP22, FP23, and FP35 in 2005 and FP30, FP22,
Table 1
Effects of fluorescent Pseudomonas on in vitro inhibition of V. dahliae hyphal growth and development of Verticillium wilt on susceptible (cv Sayar 314) and tolerant (cv Acala Maxxa) cotton cultivars, and growth promotion under growth room conditions.
Strains Origin In vitro inhibition (%)a
Disease indexb
Plant dry weightc
Sayar 314 Acala Maxxa Sayar 314 Acala Maxxa FP1 Chenopodium album 48.3 bc 3.0 bc 3.0 bc 6.77 ab 6.87 a FP5 Portulaca sp. 56.0 a 3.0 bc 3.0 bc 5.07 h 5.47 fg FP11 Portulaca sp. 50.0 abc 3.0 bc 3.0 bc 5.23 h 5.63 f FP12 Gossypium hirsitum (cv BA 119) 43.9 cd 3.3 cd 3.0 bc 5.26 gh 5.86 e FP15 Solanum nigrum 53.1 ab 3.0 bc 3.0 bc 5.23 h 5.53 f FP18 Gossypium hirsitum (cv Carmen) 40.8 de 3.7 de 3.0 bc 5.27 gh 5.47 fg FP21 Solanum nigrum 53.0 ab 3.3 cd 3.0 bc 5.20 h 5.10 h FP22 Xanthium strumarium 48.6 bc 3.0 bc 2.0 a 6.53 cd 6.67 abc FP23 Portulaca sp. 43.9 cd 3.0 bc 2.0 a 6.67 abc 6.70 abc FP25 Chenopodium album 44.0 cd 3.0 bc 3.0 bc 6.43 d 6.63 bcd FP29 Portulaca sp. 53.2 ab 3.0 bc 3.0 bc 6.53 cd 6.63 bcd FP30 Gossypium hirsitum (cv Carmen) 44.0 cd 2.7 b 2.0 a 6.63 bcd 6.80 ab FP35 Convolvulus arvensis 56.0 a 2.0 a 2.0 a 6.73 abc 6.87 a FP39 Sinapis sp. 50.2 ab 3.7 de 3.0 bc 5.07 h 5.23 h FP53 Gossypium hirsitum (cv Giza 45) 37.7 e 3.7 de 3.0 bc 5.20 h 5.23 h
Control 4.0 e 3.0 bc 5.60 f 5.63 f
Interaction cultivar treatment 0.0001 0.0082
a,b,c
In each column, mean values followed by the same letter are not significantly different according to Fisher’s protected least significant differences at P = 0.05.
a
Data represent percentage inhibition of growth of Verticillium dahliae by Fluorescent Pseudomonas on PDA (relative to control).
b
Plants originated from bacterized and control seeds were stem-inoculated at the six-true-leaf stage (after 24 days from sowing) and the disease index was assessed 14 days after inoculation using a 0-to-4 rating scale.
c
Dry weight of plants from bacterized and control seeds in sterile sand culture 24 days after sowing.
Table 2
Effects of four fluorescent Pseudomonas strains and Serratia plymuthica (HRO-C48) on Verticillium wilt on susceptible (cv Sayar 314) and tolerant (cv Acala Maxxa) cotton cultivars in a naturally infested field with V. dahliae at the Nazilli Cotton Research Station during the 2005 and 2006 growing seasons.
Cultivar Treatments 2005 2006 AUDPCa Percent controlb AUDPCa Percent controlb Sayar 314 FP22 697.7 c 50.9 a 641.2 d 36.8 a FP23 702.8 c 50.5 a 646.0 cd 36.4 a FP30 789.8 bc 44.2 ab 726.3 bc 28.4 ab FP35 863.2 b 39.2 b 793.6 b 22.1 bc HRO-C48 846.6 b 40.5 b 778.5 b 23.5 bc Control 1423.2 a 1021.8 a Acala Maxxa FP22 358.1 e 40.4 b 391.5 ef 17.6 cd FP23 358.5 e 40.5 b 416.9 ef 16.8 cd FP30 355.3 e 40.8 b 395.9 ef 12.3 d FP35 427.8 e 28.8 c 390.0 f 17.9 cd HRO-C48 362.9 e 39.6 b 386.2 f 18.8 bcd Control 602.1 d 475.8 e
Interaction cultivar treatments 0.0001 NS 0.0001 0.0214
a,bIn each column, mean values followed by the same letter are not significantly different according to Fisher’s protected least significant difference at P = 0.05 level; NS, not
significant.
a
Area under disease severity index (DSI) progress curve, DSI was calculated for each plot by: (mean severity incidence%)/maximum severity rating after assessing at the 5–10% (12/9/2005 and 2006), 50–60% (28/9/2005 and 26/9/2006) and 75% (7/10/2005 and 5/10/2006) of the bolls open stage. Relative AUDPC were obtained by dividing AUDPC values by total observation days in 2005 and 2006.
b
and HRO-C48 in 2006) compared to the untreated control. Simi-larly, the plants in the HRO-C48-, FP22-, and FP30-treated plots in 2005, and in the FP22-, FP23-, FP35-, and HRO-C48-treated plots had significantly greater heights compared to untreated controls when measured 26 days before harvest. NAWF data, recorded five weeks after the appearance of the first symptoms, indicated that the mean values were slightly higher than NAWF = 5, which was generally accepted as a physiological cut-off (Bourland et al., 2001). However, the mean NAWF values for all treatments were significantly higher than those of untreated controls, while there was no significant difference between the two cultivars in 2005 and 2006. Plants were hand-harvested at 187 and 196 days after sowing in 2005 and 2006, respectively.
The control plants emerging from non-bacterized seeds pro-duced an average seed cotton yield of 3225 kg/ha and 3098 kg/ha from Sayar, 3496 kg/ha and 3161 kg/ha from Acala Maxxa, in 2005 and 2006 trials (Table 3). Two-way ANOVA showed that seed cotton yield significantly increased after seed bacterization with four fluorescent Pseudomonas strains and S. plymuthica (HRO-C48) compared to untreated control in 2005, while there was no statistical difference among any of the treatments (including un-treated controls) in 2006. In field trial 2005, the increase of seed cotton yield by treatment with FP22, FP23, FP30, FP35, and HRO-C48 compared with the non-treated control ranged from 13.1% to 22.3% in Sayar 314 and 4.2% to 12.8% in Acala Maxxa (Fig. 1).
Over-all, strains FP22, FP23, and HRO-C48 showed the highest level of yield increase in both cultivars.
4. Discussion
Pseudomonads are the most diverse and ecologically significant group of bacteria on the planet (Spiers et al., 2000). These bacteria have considerable potential for element cycling and the degrada-tion of biogenic and xenobiotic pollutants (Timmis, 2002), biore-mediation (Dejonghe et al., 2001), biocatalysis (Schmid et al., 2001), and as biocontrol agents in plant protection (Walsh et al.,
2001; Haas and Defago, 2005) and plant growth promotion (
Kloep-per, 1993). Our potential biocontrol strains were selected from
among 59 Pseudomonas strains isolated from the rhizosphere of V. dahliae host plants (cotton and weeds) in different locations of cotton-growing areas on the basis of their ability to inhibit V. dah-liae in vitro and on the basis of disease suppression and growth pro-motion ability in planta. In culture-dependent studies ofBerg and
colleagues (2002, 2006), it was found that Pseudomonas spp. was
the most abundant group of rhizobacteria showing in vitro antago-nistic activity towards V. dahliae, and was both site-specific and plant-specific.
As mentioned above, four (FP22, FP23, FP30, and FP35) out of 15 fluorescent Pseudomonas strains significantly reduced symptom
Table 3
Effects of four Fluorescent Pseudomonas strains and Serratia plymuthica (HRO-C48) on cotton growth parameters and seed cotton yield in a naturally infested field with V. dahliae at the Nazilli Cotton Research Station during the 2005 and 2006 growing seasons.
Cultivars Treatments 2005 2006 Plant heighta (cm) Number of nodesb NAWFc Yield kg/had Plant height (cm) Number of nodes NAWF Yield (kg/ha) Sayar 314 FP22 81.1 ae 11.0 a 5.85 a 3987 a 93.1 ab 10.7 ab 5.85 bc 3100 FP23 71.6 bcd 10.6 abcd 5.98 a 3778 bc 90.6 ab 10.3 bc 5.88 bc 3105 FP30 79.5 ab 10.8 abc 5.98 a 3901 ab 90.6 ab 10.6 ab 5.98 ab 3144 FP35 77.3 abc 10.7 abc 5.95 a 3684 cd 94.1 a 10.2 bcd 5.95 b 3173 HRO-C48 81.5 a 10.6 abcd 5.78 a 3855 ab 88.9 bc 10.4 bc 5.85 bc 3263 Control 72.9 bcd 09.9 d 5.18 b 3255 e 85.6 cd 09.7 d 5.38 d 3098 Acala Maxxa FP22 71.2 cde 11.0 a 5.90 a 3940 a 80.8 e 10.5 ab 5.90 b 3291 FP23 69.5 de 10.9 ab 6.05 a 3928 ab 81.8 de 10.5 ab 5.88 bc 3339 FP30 72.1 cde 10.0 cd 5.78 a 3756 bc 78.9 e 10.9 a 5.78 bc 3308 FP35 67.4 de 11.1 a 5.83 a 3641 cd 81.4 de 10.5 ab 5.93 b 3218 HRO-C48 72.7 cd 10.5 abcd 5.83 a 3938 a 81.9 de 10.5 ab 6.25 a 3272 Control 65.4 e 10.1 bcd 5.23 b 3496 de 77.9 e 09.9 cd 5.60 cd 3161 Interaction cultivar treatments NS NS NS NS NS NS NS NS
a
Plant height corresponds mean stem length of randomly selected ten plants in each plot at the 75% of bolls open stage.
b
Number of nodes indicates mean number of nodes of randomly selected ten plants in each plot before at before the first disease rating at the 5–10% bolls open stage.
c
NAWF (nodes above white flower) mean number of nodes down from the terminal (terminal is 0) to the first white flower at the 5–10% bolls open stage.
d
Mean total weight of seed cotton in each plot.
e
Values with the same letter within the same column do not differ significantly according to Fisher’s protected least significant difference (P = 0.05); NS, not significant.
Fig. 1. Yield increase (relative to control) in two cotton cultivars (Sayar 314 and Acala Maxxa) comparing seed bacterization of four fluorescent Pseudomonas strains and Serratia plymuthica (HRO-C48) in a naturally infested field with V. dahliae (14 ± 2.6 microsclerotia/g) at the Nazilli Cotton Research Station during the 2005 growing season. Error bars represent mean ± standard deviation. Mean values followed by the same letter are not significantly different according to Fisher’s protected least significant difference at the P = 0.05 level.
expression under greenhouse conditions (Table 1). This reduction occurred even when the pathogen was inoculated directly into the stems of cotton plants grown from bacterized seeds. During natural infection, infectious hyphae emerging from microsclerotia directly penetrate through the roots (El-zik, 1985); thus, the bacte-ria would be expected to influence the infection of V. dahliae pene-trating the roots. Therefore, many researchers conducted in vivo trials with rhizobacteria by using artificially inoculated soil with microsclerotia or conidia (Leben et al., 1987; Berg et al., 2001; Mer-cado-Blanco et al., 2004; Tjamos et al., 2004; Malandraki et al., 2008; Uppal et al., 2008) on several crop plants against V. dahliae. In other biocontrol studies in which the pathogen and antagonist were spatially separated,Chen et al. (1995)reported that six endo-phytic bacteria (including two Pseudomonas putida strains) reduced the disease severity on cotton bacterized at the 7-day seedling stage when inoculated by stem-injection with microconidia of Fusarium oxysporum f. sp. vasinfectum. Likewise, van Peer et al.
(1991)showed that when roots of carnations were bacterized with
Pseudomonas sp. strain (WCS417r) one week prior to stem inocula-tion with Fusarium oxysporum f. sp. dianthi, there was a signifi-cantly reduced number of diseased plants because of induced resistance in susceptible and moderately resistant carnation culti-vars. Multiple mechanisms have been implicated in the suppres-sion of fungal root diseases by biocontrol pseudomonads, including production of antibiotics, toxins, bio-surfactants, or lytic enzymes, competition for colonization sites, nutrients and miner-als, and induction of systemic resistance (Raaijmakers et al., 2002; Mavrodi et al., 2006; O’Sullivan and O’Gara, 1992; Whipps,
2001; van Loon et al., 1998; Haas and Defago, 2005). Further
stud-ies could expand our knowledge on how our pseudomonads strains control wilt of cotton, and whether they could establish a limited endophytic phase or penetrate the endodermis, crossing from the root cortex to the vascular system (Gamliel and Katan, 1993.
Kloepper et al., 1999).
During the 2005 and 2006 trials, seed bacterization with four fluorescent Pseudomonas strains and HRO-C48 significantly re-duced Verticillium wilt symptoms of both wilt-susceptible and wilt-tolerant cotton cultivars under field conditions. HRO-C48 (S. plymuthica), a rhizosphere-associated bacterium originally isolated from oilseed rape, was applied to protect strawberry roots against V. dahliae and become a registered product called RhizoStarÒ(
Mül-ler and Berg, 2008). The authors also stated that strain HRO-C48
was selected as a biocontrol agent according to the following crite-ria: (i) high antagonistic activity against several fungal pathogens in vitro (Kalbe et al., 1996; Frankowski et al., 2001); (ii) production of the plant growth hormone indole-3-acetic acid (Kalbe et al., 1996); (iii) harmless to human health and the environment; (iv) low level of antibiotic resistance (Berg, 2000), and, most impor-tantly, (v) because of its biocontrol and plant growth promotion ef-fect under field conditions (Kurze et al., 2001).
Although AUDPC (DSI) values were significantly influenced by seed bacterization, cultivars, and their interaction in both study years, this effect was slightly reflected in the seed cotton yield re-sponses. Only in the 2005 trials was the seed cotton yield signifi-cantly higher than the untreated control. The overall data indicated that seed cotton yield was not significantly influenced by the cultivar and its interaction with seed treatments, although a statistically significant interaction was found between cultivar and seed treatments for seedling vigor under greenhouse condi-tions. The major effect of Verticillium wilt on cotton plants was the inhibition of plant growth and development. However, the time of first foliar symptom expression was also negatively corre-lated with cotton yield. For example,Pulman and DeVay (1982) re-ported that cotton lint reductions due to the Verticillium wilt were small when foliar symptoms were seen after mid-August. Simi-larly, Bejerano-Alcazar et al. (1997) indicated that the greatest
yield reduction was observed in plants showing symptoms before opening of first flowers and the effect of wilt epidemics on yield was small or non-existent for plants that developed symptoms after opening of the first balls. Lower yield reduction in our field trials could be attributed to the first symptom appearance more than three weeks after the first bloom in both years. However, growth parameters such as plant height, number of nodes on main stem, and NAWF were significantly higher in seed bacterized plants compared to untreated control.Pulman and DeVay (1982)
found similar reductions in plant height during 1976–1980 trials and concluded that the earlier foliar symptoms appeared, the greater the reduction in plant height.
Our study demonstrates the potential of some fluorescent Pseu-domonas from weed and cotton rhizosphere and a known strain of S. plymuthica (HRO-C48) as effective biocontrol agents against non-defoliating V. dahliae in cotton field. To our best knowledge, this is the first report showing disease suppression and yield increase ef-fect of strain HRO-C48 (S. plymuthica) on cotton plants under field conditions. The study also provides a practical and promising alter-native for controlling Verticillium wilt of cotton under field condi-tions. Organic cotton production has been expanding in order to meet the growing market demand and become a viable and con-vincing alternative to conventional cotton production in cotton producing countries as well as in Turkey. However, field trials in combination with different cotton cultivars to biologically control defoliating pathotype of V. dahlia, which has been recently re-ported in cotton-growing areas of Aegean region in Turkey (Göre, 2007), efficient formulation procedure on the effect of our bacteria as in S. plymuthica (Müller and Berg, 2008), as well as studies on the mechanisms underlying disease suppression are the main goals of future research.
Acknowledgments
This work was carried out as part of a Ph.D study supported by the Ministry of Agriculture and Rural Affairs of Turkey. The authors thank to Dr. Gabriele Berg (Graz University of Technology – Aus-tria) for kindly providing Serratia plymuthica strain (HRO-C48).
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