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Rhizobacteria promoted yield of cucumber plants grown in perlite under Fusarium wilt stress
Scientia Horticulturae 153 (2013) 22–25
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Rhizobacteria promoted yield of cucumber plants grown in perlite under Fusarium wilt stress Ays¸e Gül a,∗ , Hatice Özaktan b , Funda Kıdo˘glu c , Yüksel Tüzel a a b c
Ege University, Faculty of Agriculture, Department of Horticulture, 35100 Bornova, Izmir, Turkey Ege University, Faculty of Agriculture, Department of Plant Protection, 35100 Bornova, Izmir, Turkey Ministry of Food Agriculture and Livestock, International Agricultural Research and Training Center, Menemen, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 24 February 2012 Received in revised form 8 January 2013 Accepted 9 January 2013 Keywords: Plant-growth-promoting rhizobacteria Bacillus spp. Bacillus amyloliquefaciens Pseudomonas putida Pseudomonas fluorescens Serratia marcescens Fusarium oxysporum f.sp. cucumerinum
a b s t r a c t This study was carried out to determine the effects of plant-growth-promoting rhizobacteria (PGPR) on cucumber (Cucumis sativus L. cv. Sardes F1 ) production in perlite under unheated greenhouse conditions. Four native bacterial strains (18/1K: Pseudomonas putida, 62: Serratia marcescens, 66/3: Bacillus spp., 70: Pseudomonas fluorescens) and one commercial product (FZB24: Bacillus amyloliquefaciens) were tested. Rhizobacteria inoculation took place before sowing and after transplanting. Plants were affected by Fusarium oxysporum f.sp. cucumerinum occurred naturally and effects of PGPR on cucumber yield were found statistically significant. Plants inoculated with Pseudomonas putida strain 18/1K and Serratia marcescens strain 62 gave significantly higher yield compared to the control plants. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Soilless culture has been increasingly popular in protected culture among commercial growers since it eliminates the problems originated from soil. Although it provides disease-free start to cultivation, but root diseases still can be major problem due to insufficient sterilization and/or contamination of the growing system. The strategy is to keep the growing systems as clean as possible; however, the sterility concept commonly used in soilless system has been broken with increased knowledge on the beneficial microflora. The new trend is introduction or stimulation of microflora in soilless system to induce resistance of plants against biotic and abiotic stress factors, and increase plant growth and yield (Alsanius et al., 2004; Koohakan et al., 2004; Deniel et al., 2006; Gravel et al., 2006; Sopher and Sutton, 2011). Among beneficial microorganisms for a sustainable agriculture, plant growth-promoting rhizobacteria (PGPR) are of great importance (Lucy et al., 2004). These bacteria colonize roots and cause either plant growth promotion or biological control of plant disease (Lee et al., 2010). Many PGPR have both of these effects on plants. Plant growth promotion is based on increasing nutrient cycling and/or producing biologically active substances such as auxins and
∗ Corresponding author. Tel.: +90 232 3111400; fax: +90 232 3881865. E-mail address: [email protected] (A. Gül). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.01.004
other plant hormones (Zhang et al., 2004; Khalid et al., 2004). PGPR mediated biological control is caused by several mechanisms such as competition, producing antibiotic substances and siderophores, and induced resistance (Pieterse et al., 1996; Zhang et al., 2004; Bakker et al., 2007). It is reported that studies on using microorganisms have been generally realized in growth chambers, laboratories or small field plots; and it is not possible in the farm scale to reach the same success obtained under controlled conditions (Vannacci and Gullino, 2000). More studies for specific crops in various geographical areas are needed to increase adoption of microbial inoculants (Kloepper et al., 2004). This study was carried out to determine the effects of PGPR on cucumber production in perlite under unheated greenhouse conditions. 2. Materials and methods 2.1. Bacterial strains Four bacterial strains from the collection of the Department of Plant Protection, Agricultural Faculty of Ege University (18/1K: Pseudomonas putida, 62: Serratia marcescens, 66/3: Bacillus spp., 70: Pseudomonas fluorescens) and one commercial product (FZB24: Bacillus amyloliquefaciens) were tested in this study. Bacterial strains were selected according to the results of our previous study on their effect on seedling growth (Kidoglu et al., 2008).
A. Gül et al. / Scientia Horticulturae 153 (2013) 22–25 7
2.2. Plant material and growing conditions
2.3. Bacterial inoculation Bacterial inoculation was carried out before sowing and after transplanting (Cummings et al., 2009; Singh et al., 2003). PGPR strains were grown on King’s medium B for 24 h at 24 ◦ C. Bacterial inoculants were suspended with 5 mL carboxyl methyl cellulose (CMC, 1.5%). The concentration of bacterial cells in the suspension was adjusted by diluting with sterile deionised water, thus a final concentration of 109 CFU/mL was obtained (Callan et al., 1990). Seeds were sterilized in 1% hypochlorite acid for 2 min and rinsed in distilled water. Seeds were then treated with the bacterial suspensions at the concentrations of 109 CFU/mL in Erlenmeyer flasks by shaking for 30 min at 140 rpm. In the control treatment (no-PGPR), seeds were shaken with CMC. After shaking, seeds were left on blotting papers for 24 h under sterile cabinet before sowing. Seedling production were realised in a commercial nursery. Seeds were sown manually in a commercial medium (a mixture of peat and perlite) in plastic viols. After germination, seedling trays were transferred to greenhouse used for organic seedling production and no pesticides were applied. The second bacterial inoculations were made 1 week after planting as root drenches. Each treatment was applied with 50 mL/plant of bacterial suspensions at the concentrations of 109 CFU/mL. Water was applied in the same way as a non-treated control.
2.4. Variables measured - Root colonization and population dynamics of native PGPR strains on cucumber roots: root samples were taken monthly in order to study colonization by introduced rhizobacteria during the growing period. Root samples were collected and placed into sterile flasks, 100 mL of 0.1 M phosphate buffer (pH 7.1) was added to each flask. Flasks were placed on a rotary shaker at 150 rpm for 10 min. Samples were diluted, spread on two replicate Petri dishes containing Kings medium B supplemented with rifampicin (100 g/mL) and bacterial colonies, which were rifampicin resistant mutants of tested PGPR strains were enumerated (Stockwell et al., 1998). - Production of IAA by PGPR: bacterial isolates from the collection of Ege University were assayed for their ability to produce indole-3-acetic acid (IAA) in vitro which was determined quantitatively according to Bric et al. (1991) and Asghar et al. (2002), and expressed as mg/L. Bacterial isolates were grown in liquid nutrient broth medium supplemented with l-tryptophan for 24 h.
6
Log 10 cfu/g root
The research was carried out in a polyethylene covered, nonheated greenhouse located at the Department of Horticulture, Ege University (38◦ 27 16.2 N, 27◦ 13 17.8 E). Cucumber (Cucumis sativus L. cv. Sardes F1 ) was used as plant material. Seedlings were produced in a commercial nursery, transferred to the experimental greenhouse at the five true-leaf stage and transplanted in plastic pots filled with perlite (8 L/plant). Pots were arranged in order to provide plant numbers of 3.48 per m2 equivalent to 34,800 plants per hectare. Experiment including 6 treatments (5 PGPR strains and control) was set up according to randomized blocks with four replicates and each plot had 9 plants. Complete nutrient solution was used to cover water and nutrient requirements of the plants (Papadopoulos, 1994) and applied via drip irrigation system with 2 L/h flow rate. The amount of nutrient solution was adjusted according to the drainage volume kept around 20%, and surplus solution was allowed to run to waste (open system).
23
5 4
18/1K
3
62
2
70
66/3
1 0 Sep.
Oct.
Nov.
Dec.
Fig. 1. Colonization of native rhizobacteria strains on cucumber roots and timedependent population dynamics.
Then, the culture filtrates of the bacteria were obtained and measured by spectrophotometer (535 nm). - Yield: plants were grown for 14 weeks and yield was recorded as fruit weight and number of fruits per plant. - In vitro antagonistic activity of the PGPR strains against Fusarium oxysporum f.sp. cucumerinum (FOC): plants were affected by FOC which occurred naturally. Therefore, in vitro experiments were setup to assay the effects of PGPR. We tested the inhibition of hyphae development of FOC, isolated from infected plants, by dual plate assay. In vitro antifungal activity was evaluated by measuring the diameter of fungal colony (mm) after the plates were incubated at 24 ◦ C for 7 days. Antagonistic activity of the PGPR strains was estimated by the inhibition of the fungal growth in comparison to a solely cultivated fungal agar disk. 2.5. Statistical analysis The yield data were subjected to ANOVA. Means were compared using Fisher’s protected least significant difference (LSD). Significance was set at P ≤ 0.05. Regression analyses were performed between in vitro production of IAA by native PGPR strains and yield. 3. Results 3.1. Population dynamics of native PGPR strains on cucumber roots and their ability to produce IAA Colonization dynamics of PGPR strains from Ege University on cucumber roots are shown in Fig. 1. Populations of PGPR on roots of cucumber seedlings at planting changed between 103 and 106 CFU/g root. It was determined that PGPR strains tested in this study could survive on cucumber plants through the vegetation period lasting 3 months. The best colonization level was obtained by 70. It was determined that all PGPR strains from Ege University had the ability to produce IAA. The highest IAA production were realized by 62 (0.700 mg/L) followed by P. putida strain 18/1K (0.610 mg/L) and 70 (0.160 mg/L), while 66/3 produced the lowest amount (0.065 mg/L). 3.2. Yield There were significant differences between treatments through the harvesting season. Yield changes were presented as 2 weekly cumulative yields (Table 1). Plants inoculated with 18/1K, 62, 70 and FZB24 gave higher yield compared to the control plants by the first 2 weeks. Increases varied from 78.5% (70) to 121.1% (18/1K) in native PGPR strains and commercial product of FZB24 was associated with a 66.1% higher yield compared to the control treatment. 18/1K and 62 ranked at the top also in further weeks. Yield increases
24
A. Gül et al. / Scientia Horticulturae 153 (2013) 22–25
Table 1 The effect of rhizobacteria on 2 weekly cumulative yields and total fruit number of cucumber plants grown in perlite. Values are the mean of 4 replicates and each replicate included 9 plants. Means in columns followed by different letters are significantly different at P ≤ 0.05 according to Fisher’s protected LSD test. PGPR strains
18/1K 62 66/3 70 FZB24 Control LSD0.05
Fruit yield (g/plant)
Total fruit no./plant
II
IV
VI
VIII
X
Total
535a 517a 221b 432a 402a 242b 152
1408a 1367ab 758d 1131abc 1068bcd 814cd 322
2291a 2291a 1323c 1883ab 1730bc 1390bc 507
2571a 2631a 1599c 2165ab 1988bc 1633bc 528
2966a 2999a 1981b 2486ab 2284b 1971b 584
3225a 3240a 2232b 2729ab 2522b 2274b 598
28a 28a 21bc 25ab 22bc 20c 4
Fig. 2. Growth of cucumber plants 12 days after planting in the treatments of Pseudomonas putida strain 18/1K (left) and control (right).
in the plants inoculated with 18/1K compared to control varied from 73.0% to 41.8% by the first 4 weeks and by the end of season, respectively. 62 which ranked at the same statistical group with 18/1K gave rise to increase in total yield 42.5% compared to control. 70 followed these rhizobacteria and resulted in 20% higher total yield compared to control treatment, however there was no statistically significant difference between 70 and control. Total yield of the plants inoculated with 66/3 and commercial PGPR product of FZB24 were not differed from that of the control plants. Native PGPR strains of 18/1K, 62 and 70 increased fruit number compared to the control. Control had the lowest fruit number value, 66/3 and FZB24 gave intermediate values between control and 70 (Table 1). It was determined that highly significant relationships between in vitro production of IAA by native PGPR strains and total fruit weight (r2 : 0.9241) and fruit number (r2 : 0.8759) of cucumber plants were found. 3.3. In vitro antagonistic activity of the PGPR strains against FOC Growth promotion of cucumber plants inoculated with PGPR differed from the control treatment after planting (Fig. 2). At the beginning of harvesting season, some plants exhibited symptoms of wilt and afterwards died. Isolated fungi from the affected plants were identified as FOC. Therefore, the effects of PGPR strains tested in this study on the control of fungal development of FOC were evaluated in vitro. As shown in Table 2, it was determined that tested PGPR strains inhibited the colony development of FOC in vitro by the rate of 11.0–40.5%, compared to the only pathogen grown plate. It was observed that the most effective rhizobacteria strain against FOC in vitro was B. amyloliquefaciens, the active ingredient of the commercial product of FZB24, and this followed Table 2 In vitro antagonistic activity of tested PGPR strains against Fusarium oxysporum f.sp. cucumerinum (FOC), the causal agent of Fusarium wilt of cucumber. Values are the mean of 4 replicates and each replicate included 2 Petri dishes (9 cm ∅). PGPR strains
Fungal colony diameter (mm)
Inhibition of FOC (%)
18/1K 62 66/3 70 FZB24 Pathogen alone
46.5 49.5 47.6 51.2 34.2 57.5
19.0 14.0 15.2 11.0 40.5 –
by 18/1K. Especially, 18/1K showed a strong antibiosis effect causing lysis of the hyphaes of FOC (Fig. 3). 4. Discussion It was determined that the initial populations of native PGPR strains tested in this study on roots of cucumber seedlings were around 103 and 106 CFU/g root. They could survive on roots of cucumber plants grown in perlite and generally maintain the initial population densities throughout the vegetation period lasting 3 or 4 months. Usual colonization level of PGPR was reported as 105–6 CFU/g root (Yan et al., 2003), therefore it can be said that tested PGPR strains could colonize on the cucumber roots under unheated greenhouse conditions during autumn or spring growing seasons. Effects of PGPR on cucumber yield were found statistically significant. Plants inoculated with 18/1K and 62 resulted in significantly higher fruit number and consequently higher yield compared to non treated control. This effect was determined at the beginning of harvesting season and lasted up to the end of vegetation period. It is thought that yield changes might be due to the different responses of plants against to Fusarium wilt infested naturally. However, there was no relationship between the effects of PGPR on the control of FOC in vitro and yield of cucumber plants. B. amyloliquefaciens (active ingredient of commercial product of FZB24) determined as the most effective rhizobacteria strain against FOC in vitro was not effective for increasing yield. It was determined that significant relationships between yield and in vitro IAA production by native PGPR strains tested in this study. This result might be resulted from stronger growth and decrease in disease severity of plants inoculated with PGPR having ability of IAA, although other mechanisms of action might have also contributed. Asghar et al. (2002) reported that PGPR influenced the growth and yield of inoculated plants by production of auxins. It has been reported that PGPR gave rise to yield increase in different plant species (Utkhede et al., 1999; Zhang et al., 2004; Mena-Violante and Olalde-Portugal, 2007; Gül et al., 2007, 2008; Kidoglu et al., 2009). In field trials, PGPR gave rise to generally 10–15% higher yield (Zhang et al., 2004). Yield of tomato plants grown under greenhouse conditions and inoculated with Bacillus subtilis strain BS13 was 21 and 25% higher compared with control plants (Mena-Violante and Olalde-Portugal, 2007). Studies related to the effects of PGPR on cucumber production showed that
A. Gül et al. / Scientia Horticulturae 153 (2013) 22–25
25
Fig. 3. The antagonistic effects of rhizobacteria strains, Bacillus amyloliquefaciens (active ingredient of commercial product of FZB24) and Pseudomonas putida strain 18/1K on mycelial development of FOC.
some pseudomonads PGPR increased fresh fruit weight up to 18% (McCullagh et al., 1996) and one strain of B. subtilis increased fruit yield by 14% compared with the Pythium aphanidermatum inoculated control (Utkhede et al., 1999). Yield increase detected in our study is quite high compared with these studies and might be related with an increased plant tolerance to Fusarium wilt infested naturally. The results obtained proved that rhizobacteria inoculation had positive effects on yield of cucumber plants grown in soilless media. Similar to our previous findings (Kidoglu et al., 2008, 2009), native PGPR strains were more effective for increasing the plant growth and yield compared to the commercial product. This effect may be explained by their high adaptability to the local climatic conditions. Among the tested PGPR, 18/1K and 62 were found to be the most effective PGPR strains for increasing the yield followed by 70. Acknowledgments This work was supported by Turkish Scientific Research Council – TUBITAK (Contract No: 105 O 571) and Ege University Scientific Research Fund (Contract No: 2005 ZRF 004). References Alsanius, B.W., Lundqvist, S., Persson, E., Gustafsson, K.A., Olsson, M., Khalil, S., 2004. Yield and fruit quality of tomato grown in a closed hydroponic greenhouse system as affected by Pythium ultimum attack and biological control agents. Acta Hortic. 644, 575–582. Asghar, H., Zahir, Z., Arshad, M., Khaliq, A., 2002. Relationship between in vitro production of auxins by rhizobacteria and their growth-promoting activities in Brassica juncea L. Biol. Fertil. Soils 35, 231–237. Bakker, P.A.H.M., Pieterse, C.M.J., Van Loon, L.C., 2007. Induced systemic resistance by fluorescent Pseudomonas spp. Phytopathology 97 (2), 239–243. Bric, M.J., Bostock, R.M., Silverstone, S.E., 1991. Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. App. Environ. Microbiol. 57 (2), 535–538. Callan, N.W., Mathre, D.E., Miller, J.B., 1990. Bio-priming seed treatment for biological control of Pythium ultimum pre-emergence damping-off in sh2 sweet corn. Plant Dis. 74, 368–372. Cummings, J.A., Miles, C.A., du Toit, L.J., 2009. Greenhouse evaluation of seed and drench treatments for organic management of soilborne pathogens of spinach. Plant Dis. 93, 1281–1292. Deniel, F., Renault, D., Tirilly, Y., Barbier, G., Rey, P.A., 2006. A dynamic biofilter to remove pathogens during tomato soilless culture. Agron. Sustainable Dev. 26, 185–193. Gravel, V., Martinez, C., Antoun, H., Tweddell, R.J., 2006. Control of greenhouse tomato root rot (Pythium ultimum) in hydroponic systems, using plant-growthpromoting microrganisms. Can. J. Plant Pathol. 28, 475–483. Gül, A., Kidoglu, F., Tuzel, Y., Tuzel, I.H., 2007. Different treatments for increasing sustainability in soilless culture. Acta Hortic. 747, 595–602.
Gül, A., Kidoglu, F., Tuzel, Y., Tüzel, I.H., 2008. Effects of nutrition and Bacillus amyloliquefaciens on tomato (Solanum lycopersicum L.) growing in perlite. Span. J. Agric. Res. 6 (3), 422–429. Khalid, A., Arshad, M., Zahir, Z.A., 2004. Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat. J. Appl. Microbiol. 96, 473–480. Kidoglu, F., Gul, A., Ozaktan, H., Tuzel, Y., 2008. Effect of rhizobacteria on plant growth of different vegetables. Acta Hortic. 801, 1471–1477. Kidoglu, F., Gul, A., Tuzel, Y., Ozaktan, H., 2009. Yield enhancement of hydroponically grown tomato plants. Acta Hortic. 807, 475–480. Kloepper, J.W., Reddy, M.S., Rodriguez-Kabana, R., Kenney, D.S., Kokalis-Burelle, N., Martinez-Ochoa, N., Vavrina, C.S., 2004. Application for rhizobacteria in transplant production and yield enhancement. Acta Hortic. 631, 217–229. Koohakan, P., Ikeda, H., Jeanaksorn, T., Tojo, M., Kusukari, S., Okada, K., Sato, S., 2004. Evaluation of the indigenous microorganisms in soilless culture: occurrence and quantitative characteristics in different growing systems. Sci. Hortic. 101, 179–188. Lee, S.-W., Ahn, L.-P., Sim, S.-Y., Lee, S.-Y., Seo, M.-W., Kim, S., Park, S.-Y., Lee, Y.H., Kang, S., 2010. Pseudomonas sp. LSW25R, antagonistic to plant pathogens, promoted plant growth, and reduced blossom-end rot of tomato fruits in a hydroponic system. Eur. J. Plant Pathol. 126, 1–11. Lucy, M., Reed, E., Glick, B.R., 2004. Applications of free living plant growthpromoting rhizobacteria. Antonie Van Leeuwenhoek 86, 1–25. McCullagh, M., Utkhede, R., Menzies, J.G., Punja, Z.K., Paulitz, T.C., 1996. Evaluation of plant growth-promoting rhizobacteria for biological control of Pythium root rot of cucumbers grown in rockwool and effects on yield. Eur. J. Plant Pathol. 102, 747–755. Mena-Violante, H.G., Olalde-Portugal, V., 2007. Alteration of tomato fruit quality by root inoculation with plant growth-promoting rhizobacteria (PGPR): Bacillus subtilis BEB-13bs. Sci. Hortic. 113, 103–106. Papadopoulos, A.P., 1994. Growing Greenhouse Seedless Cucumbers in Soil and Soilless Media. Agriculture and Agri-Food Canada Publication, 1920/E. Pieterse, C.M.J., van Wees, S.C.M., Hoffland, E., van Pelt, J.A., 1996. Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression. The Plant Cell 8, 1225–1237. Singh, U.P., Sarma, K.B., Singh, D.P., 2003. Effect of plant-growth promoting rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cicer arietinum). Curr. Microbiol. 46, 131–140. Sopher, C.R., Sutton, J.C., 2011. Quantitative relationships of Pseudomonas chlororaphis 63-28 to Pythium root rot and growth in hydroponic peppers. Trop. Plant Pathol. 36 (4), 214–224. Stockwell, V.O., Johnson, K.B., Loper, J.E., 1998. Establishment of bacterial antagonists of E. amylovora on pear and apple blossoms as influenced by inoculum preparation. Phytopathology 88, 506–513. Utkhede, R.S., Koch, C.A., Menzies, J.G., 1999. Rhizobacterial growth and yield promotion of cucumber plants inoculated with Pythium aphanidermatum. Can. J. Plant Pathol. 21 (3), 265–271. Vannacci, G., Gullino, M.L., 2000. Use of biocontrol agents against soil-borne pathogens: results and limitations. Acta Hortic. 532, 79–88. Yan, Z., Reddy, M.S., Kloepper, J.W., 2003. Survival and colonization of rhizobacteria in a tomato transplant system. Can. J. Microbiol. 49, 383–389. Zhang, S., Reddy, M.S., Kloepper, J.W., 2004. Tobacco growth enhancement and blue mold disease protection by rhizobacteria: relationship between plant growth promotion and systemic disease protection by PGPR strain 90-166. Plant and Soil 262, 277–288.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Rhizobacteria promoted yield of cucumber plants grown in perlite under Fusarium wilt stress Ays¸e Gül a,∗ , Hatice Özaktan b , Funda Kıdo˘glu c , Yüksel Tüzel a a b c
Ege University, Faculty of Agriculture, Department of Horticulture, 35100 Bornova, Izmir, Turkey Ege University, Faculty of Agriculture, Department of Plant Protection, 35100 Bornova, Izmir, Turkey Ministry of Food Agriculture and Livestock, International Agricultural Research and Training Center, Menemen, Izmir, Turkey
a r t i c l e
i n f o
Article history: Received 24 February 2012 Received in revised form 8 January 2013 Accepted 9 January 2013 Keywords: Plant-growth-promoting rhizobacteria Bacillus spp. Bacillus amyloliquefaciens Pseudomonas putida Pseudomonas fluorescens Serratia marcescens Fusarium oxysporum f.sp. cucumerinum
a b s t r a c t This study was carried out to determine the effects of plant-growth-promoting rhizobacteria (PGPR) on cucumber (Cucumis sativus L. cv. Sardes F1 ) production in perlite under unheated greenhouse conditions. Four native bacterial strains (18/1K: Pseudomonas putida, 62: Serratia marcescens, 66/3: Bacillus spp., 70: Pseudomonas fluorescens) and one commercial product (FZB24: Bacillus amyloliquefaciens) were tested. Rhizobacteria inoculation took place before sowing and after transplanting. Plants were affected by Fusarium oxysporum f.sp. cucumerinum occurred naturally and effects of PGPR on cucumber yield were found statistically significant. Plants inoculated with Pseudomonas putida strain 18/1K and Serratia marcescens strain 62 gave significantly higher yield compared to the control plants. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Soilless culture has been increasingly popular in protected culture among commercial growers since it eliminates the problems originated from soil. Although it provides disease-free start to cultivation, but root diseases still can be major problem due to insufficient sterilization and/or contamination of the growing system. The strategy is to keep the growing systems as clean as possible; however, the sterility concept commonly used in soilless system has been broken with increased knowledge on the beneficial microflora. The new trend is introduction or stimulation of microflora in soilless system to induce resistance of plants against biotic and abiotic stress factors, and increase plant growth and yield (Alsanius et al., 2004; Koohakan et al., 2004; Deniel et al., 2006; Gravel et al., 2006; Sopher and Sutton, 2011). Among beneficial microorganisms for a sustainable agriculture, plant growth-promoting rhizobacteria (PGPR) are of great importance (Lucy et al., 2004). These bacteria colonize roots and cause either plant growth promotion or biological control of plant disease (Lee et al., 2010). Many PGPR have both of these effects on plants. Plant growth promotion is based on increasing nutrient cycling and/or producing biologically active substances such as auxins and
∗ Corresponding author. Tel.: +90 232 3111400; fax: +90 232 3881865. E-mail address: [email protected] (A. Gül). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.01.004
other plant hormones (Zhang et al., 2004; Khalid et al., 2004). PGPR mediated biological control is caused by several mechanisms such as competition, producing antibiotic substances and siderophores, and induced resistance (Pieterse et al., 1996; Zhang et al., 2004; Bakker et al., 2007). It is reported that studies on using microorganisms have been generally realized in growth chambers, laboratories or small field plots; and it is not possible in the farm scale to reach the same success obtained under controlled conditions (Vannacci and Gullino, 2000). More studies for specific crops in various geographical areas are needed to increase adoption of microbial inoculants (Kloepper et al., 2004). This study was carried out to determine the effects of PGPR on cucumber production in perlite under unheated greenhouse conditions. 2. Materials and methods 2.1. Bacterial strains Four bacterial strains from the collection of the Department of Plant Protection, Agricultural Faculty of Ege University (18/1K: Pseudomonas putida, 62: Serratia marcescens, 66/3: Bacillus spp., 70: Pseudomonas fluorescens) and one commercial product (FZB24: Bacillus amyloliquefaciens) were tested in this study. Bacterial strains were selected according to the results of our previous study on their effect on seedling growth (Kidoglu et al., 2008).
A. Gül et al. / Scientia Horticulturae 153 (2013) 22–25 7
2.2. Plant material and growing conditions
2.3. Bacterial inoculation Bacterial inoculation was carried out before sowing and after transplanting (Cummings et al., 2009; Singh et al., 2003). PGPR strains were grown on King’s medium B for 24 h at 24 ◦ C. Bacterial inoculants were suspended with 5 mL carboxyl methyl cellulose (CMC, 1.5%). The concentration of bacterial cells in the suspension was adjusted by diluting with sterile deionised water, thus a final concentration of 109 CFU/mL was obtained (Callan et al., 1990). Seeds were sterilized in 1% hypochlorite acid for 2 min and rinsed in distilled water. Seeds were then treated with the bacterial suspensions at the concentrations of 109 CFU/mL in Erlenmeyer flasks by shaking for 30 min at 140 rpm. In the control treatment (no-PGPR), seeds were shaken with CMC. After shaking, seeds were left on blotting papers for 24 h under sterile cabinet before sowing. Seedling production were realised in a commercial nursery. Seeds were sown manually in a commercial medium (a mixture of peat and perlite) in plastic viols. After germination, seedling trays were transferred to greenhouse used for organic seedling production and no pesticides were applied. The second bacterial inoculations were made 1 week after planting as root drenches. Each treatment was applied with 50 mL/plant of bacterial suspensions at the concentrations of 109 CFU/mL. Water was applied in the same way as a non-treated control.
2.4. Variables measured - Root colonization and population dynamics of native PGPR strains on cucumber roots: root samples were taken monthly in order to study colonization by introduced rhizobacteria during the growing period. Root samples were collected and placed into sterile flasks, 100 mL of 0.1 M phosphate buffer (pH 7.1) was added to each flask. Flasks were placed on a rotary shaker at 150 rpm for 10 min. Samples were diluted, spread on two replicate Petri dishes containing Kings medium B supplemented with rifampicin (100 g/mL) and bacterial colonies, which were rifampicin resistant mutants of tested PGPR strains were enumerated (Stockwell et al., 1998). - Production of IAA by PGPR: bacterial isolates from the collection of Ege University were assayed for their ability to produce indole-3-acetic acid (IAA) in vitro which was determined quantitatively according to Bric et al. (1991) and Asghar et al. (2002), and expressed as mg/L. Bacterial isolates were grown in liquid nutrient broth medium supplemented with l-tryptophan for 24 h.
6
Log 10 cfu/g root
The research was carried out in a polyethylene covered, nonheated greenhouse located at the Department of Horticulture, Ege University (38◦ 27 16.2 N, 27◦ 13 17.8 E). Cucumber (Cucumis sativus L. cv. Sardes F1 ) was used as plant material. Seedlings were produced in a commercial nursery, transferred to the experimental greenhouse at the five true-leaf stage and transplanted in plastic pots filled with perlite (8 L/plant). Pots were arranged in order to provide plant numbers of 3.48 per m2 equivalent to 34,800 plants per hectare. Experiment including 6 treatments (5 PGPR strains and control) was set up according to randomized blocks with four replicates and each plot had 9 plants. Complete nutrient solution was used to cover water and nutrient requirements of the plants (Papadopoulos, 1994) and applied via drip irrigation system with 2 L/h flow rate. The amount of nutrient solution was adjusted according to the drainage volume kept around 20%, and surplus solution was allowed to run to waste (open system).
23
5 4
18/1K
3
62
2
70
66/3
1 0 Sep.
Oct.
Nov.
Dec.
Fig. 1. Colonization of native rhizobacteria strains on cucumber roots and timedependent population dynamics.
Then, the culture filtrates of the bacteria were obtained and measured by spectrophotometer (535 nm). - Yield: plants were grown for 14 weeks and yield was recorded as fruit weight and number of fruits per plant. - In vitro antagonistic activity of the PGPR strains against Fusarium oxysporum f.sp. cucumerinum (FOC): plants were affected by FOC which occurred naturally. Therefore, in vitro experiments were setup to assay the effects of PGPR. We tested the inhibition of hyphae development of FOC, isolated from infected plants, by dual plate assay. In vitro antifungal activity was evaluated by measuring the diameter of fungal colony (mm) after the plates were incubated at 24 ◦ C for 7 days. Antagonistic activity of the PGPR strains was estimated by the inhibition of the fungal growth in comparison to a solely cultivated fungal agar disk. 2.5. Statistical analysis The yield data were subjected to ANOVA. Means were compared using Fisher’s protected least significant difference (LSD). Significance was set at P ≤ 0.05. Regression analyses were performed between in vitro production of IAA by native PGPR strains and yield. 3. Results 3.1. Population dynamics of native PGPR strains on cucumber roots and their ability to produce IAA Colonization dynamics of PGPR strains from Ege University on cucumber roots are shown in Fig. 1. Populations of PGPR on roots of cucumber seedlings at planting changed between 103 and 106 CFU/g root. It was determined that PGPR strains tested in this study could survive on cucumber plants through the vegetation period lasting 3 months. The best colonization level was obtained by 70. It was determined that all PGPR strains from Ege University had the ability to produce IAA. The highest IAA production were realized by 62 (0.700 mg/L) followed by P. putida strain 18/1K (0.610 mg/L) and 70 (0.160 mg/L), while 66/3 produced the lowest amount (0.065 mg/L). 3.2. Yield There were significant differences between treatments through the harvesting season. Yield changes were presented as 2 weekly cumulative yields (Table 1). Plants inoculated with 18/1K, 62, 70 and FZB24 gave higher yield compared to the control plants by the first 2 weeks. Increases varied from 78.5% (70) to 121.1% (18/1K) in native PGPR strains and commercial product of FZB24 was associated with a 66.1% higher yield compared to the control treatment. 18/1K and 62 ranked at the top also in further weeks. Yield increases
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A. Gül et al. / Scientia Horticulturae 153 (2013) 22–25
Table 1 The effect of rhizobacteria on 2 weekly cumulative yields and total fruit number of cucumber plants grown in perlite. Values are the mean of 4 replicates and each replicate included 9 plants. Means in columns followed by different letters are significantly different at P ≤ 0.05 according to Fisher’s protected LSD test. PGPR strains
18/1K 62 66/3 70 FZB24 Control LSD0.05
Fruit yield (g/plant)
Total fruit no./plant
II
IV
VI
VIII
X
Total
535a 517a 221b 432a 402a 242b 152
1408a 1367ab 758d 1131abc 1068bcd 814cd 322
2291a 2291a 1323c 1883ab 1730bc 1390bc 507
2571a 2631a 1599c 2165ab 1988bc 1633bc 528
2966a 2999a 1981b 2486ab 2284b 1971b 584
3225a 3240a 2232b 2729ab 2522b 2274b 598
28a 28a 21bc 25ab 22bc 20c 4
Fig. 2. Growth of cucumber plants 12 days after planting in the treatments of Pseudomonas putida strain 18/1K (left) and control (right).
in the plants inoculated with 18/1K compared to control varied from 73.0% to 41.8% by the first 4 weeks and by the end of season, respectively. 62 which ranked at the same statistical group with 18/1K gave rise to increase in total yield 42.5% compared to control. 70 followed these rhizobacteria and resulted in 20% higher total yield compared to control treatment, however there was no statistically significant difference between 70 and control. Total yield of the plants inoculated with 66/3 and commercial PGPR product of FZB24 were not differed from that of the control plants. Native PGPR strains of 18/1K, 62 and 70 increased fruit number compared to the control. Control had the lowest fruit number value, 66/3 and FZB24 gave intermediate values between control and 70 (Table 1). It was determined that highly significant relationships between in vitro production of IAA by native PGPR strains and total fruit weight (r2 : 0.9241) and fruit number (r2 : 0.8759) of cucumber plants were found. 3.3. In vitro antagonistic activity of the PGPR strains against FOC Growth promotion of cucumber plants inoculated with PGPR differed from the control treatment after planting (Fig. 2). At the beginning of harvesting season, some plants exhibited symptoms of wilt and afterwards died. Isolated fungi from the affected plants were identified as FOC. Therefore, the effects of PGPR strains tested in this study on the control of fungal development of FOC were evaluated in vitro. As shown in Table 2, it was determined that tested PGPR strains inhibited the colony development of FOC in vitro by the rate of 11.0–40.5%, compared to the only pathogen grown plate. It was observed that the most effective rhizobacteria strain against FOC in vitro was B. amyloliquefaciens, the active ingredient of the commercial product of FZB24, and this followed Table 2 In vitro antagonistic activity of tested PGPR strains against Fusarium oxysporum f.sp. cucumerinum (FOC), the causal agent of Fusarium wilt of cucumber. Values are the mean of 4 replicates and each replicate included 2 Petri dishes (9 cm ∅). PGPR strains
Fungal colony diameter (mm)
Inhibition of FOC (%)
18/1K 62 66/3 70 FZB24 Pathogen alone
46.5 49.5 47.6 51.2 34.2 57.5
19.0 14.0 15.2 11.0 40.5 –
by 18/1K. Especially, 18/1K showed a strong antibiosis effect causing lysis of the hyphaes of FOC (Fig. 3). 4. Discussion It was determined that the initial populations of native PGPR strains tested in this study on roots of cucumber seedlings were around 103 and 106 CFU/g root. They could survive on roots of cucumber plants grown in perlite and generally maintain the initial population densities throughout the vegetation period lasting 3 or 4 months. Usual colonization level of PGPR was reported as 105–6 CFU/g root (Yan et al., 2003), therefore it can be said that tested PGPR strains could colonize on the cucumber roots under unheated greenhouse conditions during autumn or spring growing seasons. Effects of PGPR on cucumber yield were found statistically significant. Plants inoculated with 18/1K and 62 resulted in significantly higher fruit number and consequently higher yield compared to non treated control. This effect was determined at the beginning of harvesting season and lasted up to the end of vegetation period. It is thought that yield changes might be due to the different responses of plants against to Fusarium wilt infested naturally. However, there was no relationship between the effects of PGPR on the control of FOC in vitro and yield of cucumber plants. B. amyloliquefaciens (active ingredient of commercial product of FZB24) determined as the most effective rhizobacteria strain against FOC in vitro was not effective for increasing yield. It was determined that significant relationships between yield and in vitro IAA production by native PGPR strains tested in this study. This result might be resulted from stronger growth and decrease in disease severity of plants inoculated with PGPR having ability of IAA, although other mechanisms of action might have also contributed. Asghar et al. (2002) reported that PGPR influenced the growth and yield of inoculated plants by production of auxins. It has been reported that PGPR gave rise to yield increase in different plant species (Utkhede et al., 1999; Zhang et al., 2004; Mena-Violante and Olalde-Portugal, 2007; Gül et al., 2007, 2008; Kidoglu et al., 2009). In field trials, PGPR gave rise to generally 10–15% higher yield (Zhang et al., 2004). Yield of tomato plants grown under greenhouse conditions and inoculated with Bacillus subtilis strain BS13 was 21 and 25% higher compared with control plants (Mena-Violante and Olalde-Portugal, 2007). Studies related to the effects of PGPR on cucumber production showed that
A. Gül et al. / Scientia Horticulturae 153 (2013) 22–25
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Fig. 3. The antagonistic effects of rhizobacteria strains, Bacillus amyloliquefaciens (active ingredient of commercial product of FZB24) and Pseudomonas putida strain 18/1K on mycelial development of FOC.
some pseudomonads PGPR increased fresh fruit weight up to 18% (McCullagh et al., 1996) and one strain of B. subtilis increased fruit yield by 14% compared with the Pythium aphanidermatum inoculated control (Utkhede et al., 1999). Yield increase detected in our study is quite high compared with these studies and might be related with an increased plant tolerance to Fusarium wilt infested naturally. The results obtained proved that rhizobacteria inoculation had positive effects on yield of cucumber plants grown in soilless media. Similar to our previous findings (Kidoglu et al., 2008, 2009), native PGPR strains were more effective for increasing the plant growth and yield compared to the commercial product. This effect may be explained by their high adaptability to the local climatic conditions. Among the tested PGPR, 18/1K and 62 were found to be the most effective PGPR strains for increasing the yield followed by 70. Acknowledgments This work was supported by Turkish Scientific Research Council – TUBITAK (Contract No: 105 O 571) and Ege University Scientific Research Fund (Contract No: 2005 ZRF 004). References Alsanius, B.W., Lundqvist, S., Persson, E., Gustafsson, K.A., Olsson, M., Khalil, S., 2004. Yield and fruit quality of tomato grown in a closed hydroponic greenhouse system as affected by Pythium ultimum attack and biological control agents. Acta Hortic. 644, 575–582. Asghar, H., Zahir, Z., Arshad, M., Khaliq, A., 2002. Relationship between in vitro production of auxins by rhizobacteria and their growth-promoting activities in Brassica juncea L. Biol. Fertil. Soils 35, 231–237. Bakker, P.A.H.M., Pieterse, C.M.J., Van Loon, L.C., 2007. Induced systemic resistance by fluorescent Pseudomonas spp. Phytopathology 97 (2), 239–243. Bric, M.J., Bostock, R.M., Silverstone, S.E., 1991. Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. App. Environ. Microbiol. 57 (2), 535–538. Callan, N.W., Mathre, D.E., Miller, J.B., 1990. Bio-priming seed treatment for biological control of Pythium ultimum pre-emergence damping-off in sh2 sweet corn. Plant Dis. 74, 368–372. Cummings, J.A., Miles, C.A., du Toit, L.J., 2009. Greenhouse evaluation of seed and drench treatments for organic management of soilborne pathogens of spinach. Plant Dis. 93, 1281–1292. Deniel, F., Renault, D., Tirilly, Y., Barbier, G., Rey, P.A., 2006. A dynamic biofilter to remove pathogens during tomato soilless culture. Agron. Sustainable Dev. 26, 185–193. Gravel, V., Martinez, C., Antoun, H., Tweddell, R.J., 2006. Control of greenhouse tomato root rot (Pythium ultimum) in hydroponic systems, using plant-growthpromoting microrganisms. Can. J. Plant Pathol. 28, 475–483. Gül, A., Kidoglu, F., Tuzel, Y., Tuzel, I.H., 2007. Different treatments for increasing sustainability in soilless culture. Acta Hortic. 747, 595–602.
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