Effect of plant growth promoting rhizobacteria on seed germination and seedling development of Zea mays

Transcription

1 National Conference ACGT 2015, 1-1 February 2015 Effect of plant growth promoting rhizobacteria on seed germination and seedling development of Zea mays Chandra Sengupta 1, Amita Bhosale 2, Swati Malusare 1, 2, Department of Microbiology, Sinhgad College of Science, Ambegaon (Bk.), Pune: 1 csengupta.msu@gmail.com 1 Abstract- Zea mays is an important cereal crop serving a major population world wide. Zea mays is traditionally used as a medicinal plant which has enormous uses as a cure in diabetes, renal diseases and cardiac diseases. Plant Growth promoting rhizobacteria (PGPR) are widely used as biofertilizer in combination with chemical fertilizers for improving fertility and sustainable agriculture. In the present study plant growth promoting bacteria were isolated from rhizosphere. Two isolates- Bacillus and were isolated from a sample collected from a field in Bhor, Pune, Maharashrta. Pseudomonas fluorescens (NCIM no. 287) and Pseudomonas putida (NCIM no. 5101) were used as reference strains. All these isolates were screened for their multiple plant growth promoting traits, such as production of Indole acetic acid (IAA), Ammonia production, Siderophore production, and Phosphate solubilization. Germination trials were carried out in which gave maximum vigour index. Evaluation of Zea mays seedling growth promotion with these isolates was also checked. showed best result as a plant growth promoter among all the isolates studied, followed by Pseudomonas fluorescens. Index Terms- Zea mays1;pgpr2; 1. INTRODUCTION Pathogenic microorganisms affecting crop production is the major and chronic threat to food production and ecosystem stability in global level. However, increasing use of chemical inputs causes several negative effects, i.e. development of pathogen resistance to the applied agents and their non-target environmental impacts. Plant growth promoting rhizobacteria (PGPR) are a heterogeneous group of bacteria that can be found in the rhizosphere, at root surfaces and in association with roots, which can improve the extent or quality of plant growth directly and or indirectly. In last few decades a large array of bacteria including species of Pseudomonas, Azospirillum,, Klebsiella, Enterobacter, Alcaligens, Arthobacter, Burkholderia, Bacillus and Serratia have reported to enhance plant growth (Kloepper et al, 1989; Glick, 1995). The direct promotion by PGPR includes either providing the plant with a plant growth promoting substances. The indirect promotion of plant growth occurs when PGPR lessen or prevent the deleterious effect of one or more phytopathogenic micro-organisms. Significant increases in growth and yield of agronomical important crops in response to inoculation with PGPR have been reported. (Asghar et al, 2002; Bashan et al, 200 and J.C. Biswas et al, 200) Bacterial inoculants are able to increase plant growth and germination rate, improve seedling emergence, responses to external stress factors and protect plants from disease (Lugtenberg et al, 2002) The effects of plant growth promoting rhizobacteria on growth and yield of some crop plants studied in previous works. But the effects of PGPR on growth parameter from germination to yield were not evaluated simultaneously. The main objective of this research was to determine if PGPR strains could affects on seed germination, growth parameters of Zea mays seedling in greenhouse and also grain yield of field grown Zea mays. Plant growth promoting activities like production of indoleacetic acid (IAA), ammonia (NH ), siderophore, and phosphate solubilisation. Mixtures of PGPR can elicit induced systemic resistance to fungal, bacterial, and viral diseases in the 2

2 National Conference ACGT 2015, 1-1 February 2015 four hosts tested. (Jetiyanon K and Kloepper JW, 2002) In the present study, Plant growth promoting rhizobateria were isolated, identified and characterized from rhizosphere sample. The PGPR activity Seed germination and Seedling development studies of the isolated bacteria compared to reference strains was done. 2. MATERIALS AND METHODS: Sample collection: Soil sample was collected from a field in Bhor, Pune, Maharashtra. Pseudomonas fluorescens (NCIM no. 287) and Pseudomonas putida (NCIM no. 5101) were obtained from NCIM. 2.1 Isolation and enrichment: For isolation and enrichment of rhizobacteria different media were used-- Nutrient agar medium, and Ashby s agar medium and King s B agar. 0.5 gm of Soil sample was inoculated into 50 ml Ashby s broth. The medium was incubated at 0 o C for 2-5 days. A film of surface growth was observed. A loopful of growth was taken and streaked on the Ashby s agar plate. The plate was kept for incubation at 0 o C for 2 days. Colony characteristics were observed after incubation. (Norris and Chapman, 1968) Similarly, a small 0.5 gm of sample was inoculated into 50 ml nutrient broth. The medium was kept for incubation at 0 o C for 2-5 days. After sufficient growth was observed, a loopful of growth was taken and streaked on the nutrient agar plate. The plate was incubated at 0 o C for 2-5 days. (Ahmad F et al, 2008).Pseudomonas fluorescens and Pseudomonas putida were grown on King s B agar plates. 2.2 Characterization and identification of rhizobacteria: The bacterial isolates were characterized by their cultural conditions, morphological and biochemical characteristics (Gram s reaction,hydrolysis of starch,nitrate reduction, utilization of glucose, maltose, mannitol and catalase reactions) using standard methods (Cappuccino and Sherman,1992). 2. Characterization of rhizobacteria for PGP traits: Production of Indole acetic acid: Indole acetic acid (IAA) production was detected as described by (Brick et al,1991). Bacterial cultures were grown for 72 h () and 8 h (Pseudomonas and Bacillus) on their respective media at 7 C. Fully grown cultures were centrifuged at 000 rpm for 0 min. The supernatant (2 ml) was mixed with two drops of orthophosphoric acid and ml of the Salkowski reagent (50 ml, 5% of perchloric acid,1 ml 0.5 M FeCl solution). Development of pink colour indicates IAA production. Production of ammonia: Bacterial isolates were tested for the production of ammonia in peptone water. Freshlygrown cultures were inoculated in 10 ml peptone water in each tube and incubated for 8 72 h at 7 C. Nessler s reagent (0.5 ml) was added in each tube. Development of brown to yellow colour was a positive test for ammonia production (Cappuccino and Sherman,1992). Siderophore production: Siderophore production was detected by the universal method of (Schwyn and Neilands,1987) using blue agar plates containing the dye chrom azurol S (CAS). Orange halos around the colonies on blue were indicative for siderophore production. Phosphate solubilisation: All isolates were first screened on Pikovskaya s agar plates for phosphate solubilization as described by (Gaur 1990). Zone of clearance around the colonies were indicative for phosphate solubilisation. 2. PGPR activity: Germination trial: Bacterial isolates- P.fluorescens, P. putida Bacillus, were used for Zea mays seed treatments. Seeds of Zea mays were surface sterilized with 0.02% Sodium hypochlorite for 2 min. and rinsed thoroughly in sterile distilled water. For inoculation, seeds were coated with coco peat as an

3 National Conference ACGT 2015, 1-1 February 2015 adhesive and rolled into the suspension of bacteria (10⁸ cfu ml ¹). Seeds treated with sterile distilled water, amended with coco peat served as non treated control. (Nezarat S. and Gholami A., 2009). 5 seeds for each treatment in triplicates were taken and germination tests were carried out in a tray. Incubated for 7 days at 28 o C. After 7 days the number of germinated seeds was counted. Root and shoot length of individual seedling was measured to determine the vigour index. A vigour is the sum total of those properties of the seed which determine the level of activity and performance of the seed or seed lot during germination and seedling emergence Vigour Index was calculated by using following formula: 2.5 Vigour index= (mean root length + mean shoot length) % germination. Zea mays Seedling development studies: For the evaluation of Zea mays seedling growth promotion with Plant growth promoting rhizobacteria, the Zea mays seedlings were transferred to the pots containing 1.5 kg. Two types of s were used, non-sterile and sterile. The seedlings were watered daily. After every 8 days shoot length, leaf length, leaf diameter, no. of leaves of each seedling were measured. (Nezarat S and Gholami A, 2009). RESULTS:.1 Characterization and Identification of the isolates: On the basis of cultural, morphological and biochemical characteristics (Endospore staining,sugar utilisation: Glucose, Maltose, Mannitol, Starch hydrolysis, Catalase, Nitrate reduction) isolates were partially identified as Bacillus (Isolate no. 1) and (Isolate no. 2).2 Characterization of bacteria for PGP (Plant the isolates studied. Siderophore production exhibited by P.fluorescens and. Phosphate solubilisation was shown by Bacillus. P.fluorescens and. Germination trial: Germination trial was carried out to see the in vivo effects of the isolates on the seeds of Zea mays. Results were recorded after 7 days, 16days, 2 days and 0 days. Root and shoot length of individual seedling was measured to determine the vigour index.. Zea mays Seedling development studies: For the evaluation of Zea mays seedling growth promotion with Plant growth promoting rhizobacteria, the Zea mays seedlings were transferred to the pots containing 1.5 kg. Two types of s were used, non-sterile and sterile. The seedlings were watered daily.after every 8 days shoot length, leaf length, leaf diameter, no. of leaves of each seedling were measured. (Nezarat S and Gholami A, 2009) Table 1: Characterization of bacteria for PGP traits: Bacteria IAA production Germination trials: Ammonia production Sidero phore production P.fluores cens P. putida + + Bacillus - + _ + Azotobac ter Table 2 : % germination of seeds and vigour index Isolate % seed Vigour index germination Control growth promoting traits): Isolates were screened for PGP traits. Screening results are depicted in table 2. P.fluorescens IAA production was shown by all the isolates except Table : Mean shoot length, mean leaf length, mean leaf diameter and no. o Bacillus. Ammonia production was observed for all Bacillus Phosphate Solubiliza tion

4 National Conference ACGT 2015, 1-1 February 2015 Mean shoot length length diameter No. of leaves Nonsterile Non- Non- Isolate sterile sterile Control P.fluorescens Bacillus Table (b): Mean shoot length, mean leaf length, mean leaf diameter and no. of leaves of the seedlings on 16th day Isolate Mean shoot length length diameter No. of leaves Control P.fluorescens Bacillus

5 International Journal of Research in Advent Technology (E-ISSN: (E ) 967) Special Issue National Conference ACGT 2015, February 2015 Table (c): Mean shoot length, mean leaf length, mean leaf diameter and no. of leaves of the seedlings on 2th 2 day and 0th day Mean shoot length Isolate Control P.fluorescens Bacillus length Control No. of leaves 21. Mean shoot length Isolate diameter sterile P.fluorescens 16.8 length Non Nonsterile sterile diameter Non sterile No. of leaves Nonsterile Bacillus Bacillus Fig. 1: Seedlings treated with isolates (after 8 days) P.fluorescens Control 6

6 National Conference ACGT 2015, 1-1 February 2015 control control control Bacillus Fig. 2: Seedlings treated with isolates (after 0 days). DISCUSSION: In the present study, production of IAA by Pseudomonas and is a general characteristic of our test isolates. The ability of bacteria to produce IAA in the rhizosphere depends on the availability of precursors and uptake of microbial IAA by plant. Growth promotion may be attributed to other mechanisms such as production of plant growth promoting hormones in the rhizosphere and other PGP activities (Arshad and Frankenberger, 199; Glick, 1995). P.fluorescens Another important trait of PGPR, that may indirectly influence the plant growth, is the production of 7

7 National Conference ACGT 2015, 1-1 February 2015 ammonia. All the isolates were able to produce ammonia. Pseudomonas fluorescens and isolates were siderophore producers. Siderophores chelates iron and other metals contribute to disease suppression by conferring a competitive advantage to biocontrol agents for the limited supply of essential trace minerals in natural habitats (Hofte et al, 1992; Ahemad and Kibret, 201). Siderophores may directly stimulate the biosynthesis of other antimicrobial compounds by increasing the availability of these minerals to the bacteria. Antibiotic and siderophores may further function as stress factors or singles including local and systematic host resistance. Phosphate solubilization was shown by P.fluorescens and Bacillus. In this way, some of the above-tested isolates could exhibit more than two or three PGP traits, which may promote plant growth directly or indirectly or synergistically. Further evaluation of the isolates exhibiting multiple plant growth promoting (PGP) traits on plant system is needed to uncover their efficacy as effective PGPR. This present work revealed that under in vitro conditions, seed treatment with PGPR strains improved seed germination, seedling vigour, seedling emergence and seedling stand over the control. Similar improvement of seed germination parameters by rhizobacteria has been reported in other cereals such as sorghum ( Raju NS et al, 1999) and pearl millet (Niranjan SR et al, 200) These findings may be due to the increased synthesis of hormones like gibberellins, which would have triggered the activity of specific enzymes that promoted early germination. Beside, significant increase in seedling vigour would have occurred by better synthesis of auxins. (Bharathi R et al, 200) In pot experiment, it was observed that inoculation with PGPR strains significantly promoted growth of seedling Zea mays under different conditions. In general, inoculation resulted in early seedling growth and development. Soil condition influenced growth promotion by bacterial strains. had more effect on growth parameters in sterile compared with non sterile. In contrast, inoculation with other bacterial treatments had a more stimulating effect on growth of plants in non-sterile than sterile condition. This may imply rhizobacteria had more competitive ability to survive and affect the growth of inoculated plants in the presence of indigenous micro flora. The present experiment revealed that seed inoculation with all bacteria resulted in an increased shoot length, leaf length, and leaf diameter. showed best result as a plant growth promoter among all the isolates studied, followed by Pseudomonas fluorescens. Both of these could be used as a potential biofertilizers. (Burd et al, 2000) reported that plant growth promoting rhizobacteria might enhance plant height and productivity by synthesizing phytohormones, increasing the local availability of nutrients, facilitating the uptake of nutrients by the plants decreasing heavy metal toxicity in the plants antagonizing plant pathogens. The effects of this plant growth promoting rhizobacteria on growth and yield of some crop plants studied in previous works. But the effects of PGPR on growth parameter from germination to yield were not evaluated simultaneously. In conclusion the results of this study suggest that simultaneous screening of rhizobacteria for their PGP traits, growth and yield promotion is a good tool to select effective PGPR for biofertilizer development biotechnology. 5.ACKNOWLEDGMENTS I would like to thank department of Microbiology, Sinhgad College of Science and principal to provide the facilities. 8

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