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Annals of Microbiology, 55 (3) 171-174 (2005)

Pseudomonas fluorescens mediated vigour in black pepper (Piper nigrum L.) under green house cultivation

Paul DIBY*, Yammanuru Ramalinga SARMA, Veeraraghavan SRINIVASAN, Muthuswamy ANANDARAJ Indian Institute of Spices Research, Calicut, Kerala-673 012, India

Abstract - Pseudomonas fluorescens strains identified earlier as efficient biocontrol agents in black pepper vines enhanced the production of fibrous roots in the plant and increased the absorptive surface area of the roots. The root proliferation and enhanced vigour of black pepper brought about by P. fluorescens strains was attributed to the nutrient mobilisation in the rhizosphere by these beneficial strains thereby higher uptake by the plants. Significant uptake of nitrogen (N) and phosphorus (P) was noticed in bacterised plants. The study revealed the ability of the P. fluorescens strains to enhance nutrient mobilisation in the rhizosphere of black pepper, which resulted in enhanced plant vigour. Key words: Pseudomonas fluorescens, black pepper, nutrient mobilisation, rhizosphere.

INTRODUCTION Microbial populations are key components of the soil-plant systems where they are immersed in a framework of interaction affecting plant developments. Plant Growth Promoting Bacteria (PGPB) can benefit plant growth by different mechanisms (Bashan and de-Bashan, 2005). Their use as natural bio-inoculants is advantageous, not only from the economical, but also from the ecological point of view. The beneficial activities of PGPBs have been established in many crops including black pepper (Sarma et al., 2003; Lucy et al., 2004; Vestberg et al., 2004). Five strains of Pseudomonas fluorescens were found to be effective biocontrol agents against the foot rot disease in black pepper (Diby et al., 2005a, 2005b). Any biocontrol agent, which can act as a nutrient mobiliser in the rhizosphere, would be an added advantage. The present study deals with the efficacy of these five strains of P. fluorescens for mobilising the essential nutrient uptake in black pepper and thereby enhancing the total plant vigour.

rot disease of black pepper caused by Phytophthora capsici (Sarma et al., 2003; Diby et al., 2005a, 2005b), were obtained from the repository of rhizobacteria maintained at the Indian Institute of Spices Research, Calicut. Efficacy of Pseudomonas fluorescens in root initiation and proliferation in black pepper. The five selected strains of P. fluorescens were evaluated in green house for root initiation and proliferation in black pepper using 2-noded stem cuttings (~ 10-15 cm), which are the vegetative propagative units. The bacterial strains were mass multiplied in nutrient broth by incubation at 28 °C for 48 h. The cells were pelleted at 6000 x g for 10 min. Cells were resuspended in 10 mM MgSO4 and diluted to 108 CFU/ml of the final suspension. Bacterisation was carried out by dipping the stemcuttings in the bacterial suspension for 30 min. The cuttings were planted in sterile coir compost taken in polythene bags (10 cm x 20 cm) and the bags were drenched with 25 ml of the bacterial suspension. The six treatments included 5 strains of bacteria and an untreated control in 5 replicates, wherein single plant was a replicate. The bags were arranged in the greenhouse (28 °C, 600 µM light intensity) in random block design. The plants were irrigated with 10 ml of distilled water on every 3rd day. These cuttings were uprooted after 60 days and thorough examination of the root system viz. root length, total number of roots, the total area of roots and the root biomass were estimated after scanning and analysis using the software, GS-Root® (PP Systems, Winter Street, USA). The experiment was repeated twice. All the data were statistically analysed using Duncan's Multiple Range Test (DMRT) using MSTAT-C software.

MATERIAL AND METHODS The microorganisms used. The rhizobacterial strains, Pseudomonas fluorescens IISR-6, IISR-8, IISR-11, IISR-13 and IISR-51 which are proven biocontrol agents against foot

* Corresponding Author. Present Address: M.S. Swaminathan Research Foundation, 3rd Cross Street, Taramani Institutional Area, Chennai-600 113, India. Phone: +91-44-55299024; Mobile: +91-9841212996; Fax: +91-44-22541319; E-mail: [email protected]

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Green house assay for Pseudomonas fluorescens mediated nutrient flux. Two to three leaf stage cuttings of black pepper, rooted in sterile coir pith was used for the study. Bacterisation and planting was carried out as described above, the cuttings were replanted in sterile potting mixture (soil: sand: dried cow dung at 2:1:1 v/v/v). Sampling. The planting medium was sampled before planting in order to quantify the total and plant-available nitrogen (N), phosphorus (P) and potassium (K). The soil sampling was also done on 30, 60 and 90 days after treatment by destructive sampling. Soil adhering to the close proximity of the black pepper roots were collected, sieved and dried under shade for two days before analysis. The total dry weight of the plants in each treatment was taken on the final day of sampling that is on the 90th day for estimation of total and available quantity of N, P and K. Single plants served as a replicate and 5 replicates were analysed for a treatment. Screening of Pseudomonas fluorescens strains for in vitro phosphate solubilisation. The phosphate-solubilising test was done in both solid as well as in liquid medium. Medium used was Pikovskaya's medium (Pikovskaya, 1948). Agar plates were prepared and the bacterial strains were spot inoculated at the centre of the plates and incubated for 5-6 days. The plates were observed for clearing zone around the colony and the diameter of the clearing zone was measured. Pikovskaya's broth (100 ml) taken in conical flask was sterilized by autoclaving. The bacterial strains were inoculated and incubated for 48 h at 28 ± 2 °C. Medium without inoculation served as control. The culture was centrifuged at 6000 x g for 20 min at 4 °C. The supernatant was collected and the pellet was discarded. The supernatant (1 ml) was taken in a test tube and diluted by adding 6 ml of distilled water. Then 2 ml of chloromolybdic acid was added followed by 1 ml of chlorostanous acid. The absorbance of this reaction mixture was read at 660 nm. The intensity of colour developed was directly proportional to the amount of phosphate released. From the standard graph, the quantity of phosphates released from tricalcium phosphate by the bacteria was obtained. Estimation of total and available N, P and K in soil and plant: The total N in soil was estimated by H2SO4 digestion and Kjeldahl distillation (Bremner, 1996). The total P and K were estimated in HClO4 digested samples using Atomic Absorption Spectrophotometer (A-20, Varian), as outlined by Jackson (1967). The plant-available quantity of N in soil was estimated by alkaline KMnO4 distillation (Subbaiah and Asija, 1956), available P by Bray I extraction (Bray and Kurtz, 1945) and available K by NH4OAc extraction (Helmke and Sparks, 1996). The plant samples were digested in H2SO4 and N was estimated by Kjeldahl distillation. The P and K were estimated in triacid (HNO3: H2SO4: HClO4) digestion following standard procedures outlined by Jackson (1967). The total uptake of N, P and K by black pepper was calculated by multiplying their respective concentration in plant with the total dry weight of the plant. All the data were statistically analysed using Duncan's Multiple Range Test using MSTAT-C software.

RESULTS Efficacy of Pseudomonas fluorescens in root initiation and proliferation in black pepper Table 1 shows the enhanced root proliferation. The bacterial strains significantly increased the root biomass of the plants (30-135%). The highest increase among the bacterial strains was noticed with IISR-51. Most strains increased the root length in the treated plants (12-127%), highest being with IISR-6, which was on par with IISR-11 and IISR51. Similar trend was observed with the total root area after bacterisation (43-200%). The bacteria-treated plants had higher number of feeder roots as evidenced by the increased number of root tips in the treated plants (82-137%). IISR6, IISR-11 and IISR-51 were equally efficient in enhancing the number of roots in black pepper. Phosphate solubilisation in vitro In the Pikovskaya's agar plates, P. fluorescens strains produced 1.8 to 3.3 cm of clearing zones indicating phosphate solubilisation (Table 2). IISR-8 showed lowest potential in plate (1.8 cm) while a maximum clearing zone was observed with IISR-6 (3.3 cm). The broth assay implicated the amount of phosphate released (0.4-0.6 mg/l) from tricalcium phosphate to the medium by the strains of P. fluorescens (Table 2). Total dry matter production There was significantly higher dry matter yields (23.6440.83%) in plants treated with each strain of P. fluorescens compared to untreated control (Table 3), highest being by IISR-51 followed by IISR13 and IISR 6. Uptake of nitrogen, phosphorus and potassium by the plants All the strains were found to ease the uptake of N, P, and K by the plant roots (Table 3). The total quantity of N, P, and K in the P. fluorescens treated plants was higher compared to that in the untreated control. Except IISR-8, all the strains significantly increased the total quantity of N in plants (11.1013.39 mg), highest being by IISR-13 followed by IISR-11 and IISR-6. Highest P accumulation was observed in plants treated with P. fluorescens, IISR-6 (1.95 mg) and the lowest by IISR-8, which was on par with the untreated plants (0.88 mg). Significant increase with respect to the control plants was noticed with IISR-6, IISR-13 and IISR-51. Even though higher, the PGPB strains except IISR-13 did not have significant levels of K in treated plants. The total and plant available quantity of N, P, and K in soil did not show any significant change over time (data not shown).

DISCUSSION The bacterial strains under the present investigation significantly increased the total root biomass of the black pepper plants. The number of roots have been significantly increased apart from increasing the root length and thereby root area (Table 1). These beneficiary attributes can be corroborated with the enhanced nutrient mobilisation effected by the rhizobacteria. All the strains used in the study were found to solubilise complex forms of P to the plant-available form in the in vitro studies conducted. The strains also mobilised higher uptake of N and K in the treated plants. Since the

Ann. Microbiol., 55 (23), 171-174 (2005)

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TABLE 1 ­ Enhanced root proliferation effected in black pepper as a results of treatment with PGPB P. fluorescens strains IISR-6 IISR-8 IISR-11 IISR-13 IISR-51 Control Root biomass (g) 46.95 ab 48.39 ab 47.40 ab 33.06 bc 59.63 a 25.35 c Total root length (cm) 1048.046 a 0754.308 bc 0962.685 ab 0518.401 cd 0917.521 ab 0461.294 d Total root area (mm2) 2410.088 a 1349.492 bcd 2004.929 ab 1155.100 cd 1668.761 bc 0803.061 d Total number of roots 506.7 a 415.9 ab 480.9 a 389.8 ab 490.2 a 213.9 b

Values are mean of 5 replicates. Treatments showing a letter in common in the superscripts, do not differ significantly according to DMRT at P = 0.05.

TABLE 2 ­ Phosphate solubilisation potential of the Pseudomonas fluorescens strains Pikovskaya's agar and broth P. fluorescens strains IISR-6 IISR-8 IISR-11 IISR-13 IISR-51 Diameter of clearing zone (cm) 3.3 a 1.8 d 2.5 c 2.6 c 3.1 b Phosphate released from TCP* (mg/l of broth) 0.6 a 0.4 c 0.5 b 0.6 a 0.6 a

*TCP: tricalcium phosphate.

Values are mean of 4 replicates. Treatments showing a letter in common in the super

scripts, do not differ significantly according to DMRT at P = 0.05.

TABLE 3 ­ The total dry matter yields and uptake of nitrogen, phosphorus and potassium by the plants upon 90 days after treatment P. fluorescens strains IISR-6 IISR-8 IISR-11 IISR-13 IISR-51 Control Total dry matter yield (g) 7.28

ab

N uptake (mg) 11.27 8.29 12.72 13.39 11.10 8.12

a b a a a b

P uptake (mg) 1.95 0.88 1.31 1.39 1.41 0.88

a c bc b b c

K uptake (mg) 8.62 9.17 9.63 8.13

b

6.55 bc 7.12 bc 7.44 8.30 5.89

ab a c

8.39 b

ab a

10.62

ab b

Values are mean of 5 replicates. Treatments showing a letter in common in the superscripts, do not differ significantly according to DMRT at P = 0.05.

strains used in the study are proven biocontrol and growth promoting agent of black pepper, their ability to mobilise the essential nutrients required for the plant in the rhizosphere becomes an added quality of these bioinoculants. Microorganisms are critical for the transfer of P from poorly available forms and are important for maintaining P in readily available pools. The present study proved the P. fluorescens mediated P solubilisation and thereby enhanced uptake by the plants (122% over control), which resulted in increased root proliferation. The microorganisms used in the study are found producing siderophores (Diby et al., 2005a), which may in turn release P from complex forms of P through ligand exchange reactions by chelating metal ions associated with the bound P.

There also found an increased uptake of N by the bacterised plants (65% over control). A biological alternative for the extensive use of nitrogen fertilizers is the interaction between PGPB and the plant roots. The ability of Pseudomonas species to fix nitrogen is still debated. Vermeiren et al. (1999) proved that a diazotrophic rice endophyte, Pseudomonas stutzeri fix nitrogen and provide, thus improving plant growth. Even though not significant, higher levels of K uptake was noticed in PGPB treated plants (3-25% over control). Potassium is one of the essential nutrient for plant growth. Good K nutrition favours the rapid turn over of inorganic nitrogen in to proteins (Koch and Mangel, 1974). Even though there is no direct influence of rhizosphere microbes in K mobilisa-

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tion, the enhanced uptake of the same in the bacterised plants would have effected from the higher root / root hair growth, increased root surface area and better root health supported by the PGPBs. The present study revealed the ability of the bacterial strains to enhance nutrient mobilisation in the rhizosphere of black pepper, which resulted in enhanced plant vigour. Acknowledgements The financial support from the Department of Biotechnology, Government of India is gratefully acknowledged.

the foot rot pathogen of black pepper (Piper nigrum L.) Ann. Microbiol., 55 (2): 45-49. Helmke P.A., Sparks D.L. (1996). Lithium, Sodium, Potassium, Rubidium and Cesium. In: Sparks D.L., Page A.L., Helmke P.A., Loeppert R.H., Soltanpour P.N., Tabatabai M.A., Jhonston C.T., Sumner M.E, Eds, Methods of Soil Analysis, Part 3, Chemical Methods. ASA-SSSA, Madison, WI, pp. 559-560. Jackson M.L. (1967). Soil Chemical Analysis. Prentice-Hall of India, New Delhi, pp. 110-338. Koch K., Mangel K. (1974). Potassium in plant nutrition. J. Sci. Food Agric., 25: 465-471. Lucy M., Reed E.R., Glick B. (2004) Applications of free living plant growth-promoting rhizobacteria. Antonie Van Leeuwenhoek, 86 (1):1-25. Pikovskaya R.E. (1948). Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Mikrobiologia, 17: 362-370. Sarma Y.R., Krishnakumar V., Anandaraj M. (2003) Scope and role of PGPR in cropping systems in plantation crops and spices in relation to their agronomy and pathology. 6th International PGPR Workshop, 5-10 October 2003, Calicut, India. Subbaiah B.V., Asija G.L. (1956). A rapid procedure for determination of available nitrogen in rice soils. Curr. Sci., 25: 259260. Vermeiren H., Willems A., Schoofs G., de Mot R., Keijers V., Hai W., Vanderleyden J. (1999). The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri. Syst. Appl. Microbiol., 22: 215-224. Vestberg M., Kukkonen S., Saari K., Parikka P., Huttunen J., Tainio L., Devos N., Weekers F., Kevers C., Thonart P., Lemoine M.C., Cordier C., Alabouvette C., Gianinazzi S. (2004). Microbial inoculation for improving the growth and health of micropropagated strawberry. Appl. Soil Ecol., 27 (3): 243-258.

REFERENCES Bashan Y., de-Bashan L.E. (2005). Bacteria / Plant growth-promotion. In: Hillel D., Ed., Encyclopedia of Soils in the Environment. Vol. 1, Elsevier, Oxford, U.K., pp. 103-115. Bray R.H., Kurtz L.T. (1945). Determination of total, organic and available phosphorus in soils. Soil Sci., 59: 39-45. Bremner M.J. (1996). Nitrogen-Total. In: Sparks D.L., Page A.L., Helmke P.A., Loeppert R.H., Soltanpour P.N., Tabatabai M.A., Jhonston C.T., Sumner M.E, Eds, Methods of Soil Analysis, Part 3, Chemical Methods. ASA-SSSA, Madison, WI, pp. 10851121. Diby P., Anandaraj M., Kumar A., Sarma Y.R. (2005a). Antagonistic mechanisms of fluorescent pseudomonads against Phytophthora capsici in black pepper (Piper nigrum Linn.). J. Spices and Aromatic Crops, 14 (2): 94-101. Diby P., Saju K.A., Jisha P.J., Sarma Y.R., Kumar A., Anandaraj M. (2005b) Mycolytic enzymes produced by Pseudomonas fluorescens and Trichoderma spp. against Phytophthora capsici,

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