Presented by: Ummu M. S. (080610091) Shelly F. N. (080610317)

1. Variation in Growth and Ion Uptake of Maize due to inoculation with Plant Growth Promoting Rhizobacteria Under Salt Stress Presented by: Ummu M. S. (080610091) Shelly F. N. (080610317)

2. HOME • INTRODUCTION • KEY WORDS • PROBLEM STATEMENT • PURPOSE • MATERIAL AND METHOD • RESULT • DISCUSSION • CONCLUSION

3. INTRODUCTION Among various environmental stresses soil salinity limits plant growth and production in many part of the world, particularly in arid and semi arid area (Shannon, 1984). Under salinity, growth depression results from increased level of ethylene (Nukui et al., 2001), decreased photosynthetic capacity (Drew et al., 1990), water deficit and ion imbalance (Wyn Jones, 1981). Many attemps have been made to reduce the drastic effect mostly focusing on chemical amelioration and saline agriculture. Recently, a biological approach using microorganism was attempt. HOME

4. Key words: • PGPR • GROWTH • IONS UPTAKE • MAIZE • SALINITY HOME

5. PGPR Plant Growth Promoting Rhizobacteria are free living microorganism having beneficial effects on plants by colonizing their roots (Nadeem et. al, 2006) and via direct or indirect mechanisms (Nelson, 2004) Kloepper and Schroth (1978) describe as soil bacteria that colonize the roots of plants following inoculation onto seed and that enhance plant growth. HOME

6. 1.1 Beneficial effect of PGPR • Production of phytohormones(auxin, cytokinins, and giberelins) (Garcia de Salamoneet al., 2001) • Enhancing release of the nutrients (Nautiyalet al., 2000; Idrisset al., 2002) • Preventing the deleterious effects of environmental stress (Kloepper and Schroth, 1978). • Produce antibiotics that protects roots from deasease, example: 2,4 –DAPG (2,4 – diacetylphloroglucinol) (Aragnoet al.,2004) • Also produce exopolysaccharides (EPSs) to bind cations including sodium (Geddie and Sutherland, 1993), thus help alleviating salt stress in plants grown under saline environment (Ashrafet al., 2004). HOME

7. THE KINDS OF PGPR • Bacillus vallismortis for mediated systemic resistance against Potato Virus • Bacillus subtilis • Pseudomonas flourescens • Bacillus polymixa • Serratia marcescens

9. Scanning electron micrograph of wheat root-colonizing Pseudomonas fluorescens OE 28.3 the role of Pseudomonas fluorescens cell envelope proteins (in particular the major outer membrane protein OprF) in adhesion to roots. Biocontrol of phytopathogens appears to be a major mechanism of plant growth promotion by these bacteria

10. Product of PGPR

13. 1.2 Environmental stress Nilsen and Orcutt in they book “Physiology of Plant Under Stress” explain several kinds of stress: • salinity • Temperature • Water (Flooding and drought) • pH • Light • nutrient HOME

14. SOIL SALINITY The dynamic nature of soil salinity in the rootzone affects performance of plants. Profile distribution of salts is affected by leaching fraction and changes with changing water content from irrigation and rootwater extraction. Soluble salts in soils are highly mobile and tranported by water through mass flow and dispersion. Irrigation water management is one of the keys in maintaing salt balance in the rootzone (Salinity by Andre Lauchli and Ulrich Luttge). HOME

15. GROWTH • Definition An increase in the size of an organism or part of an organism, usually as a result of an increase in the number of cells. Growth of an organism may stop at maturity, as in the case of humans and other mammals, or it may continue throughout life, as in many plants. • Growth is one of the best indices for evaluating plant responses to environmental or biotic stress (Nilsen and Orcutt, 1996) HOME

16. CLASSIFICATION OF MAIZE • Kingdom : Plantae • Plants Subkingdom : Tracheobionta • Vascular plants Superdivision : Spermatophyta • Seed plants Division : Magnoliophyta -Flowering plants • Class : Liliopsida -Monocotyledons • Subclass Commelinidae • Order Cyperales • Family Poaceae - Grass family • Genus Zea L. – corn • Species Zeamays L. – corn HOME

17. MORE HOME

18. PROBLEM STATEMENT • How does the effect of the strains of PGPR on growth and ion uptake of maize? • How does the effect of salt concentration on growth and ion uptake of maize? • How does the interaction between strain of PGPR and salt concentration on growth and ion uptake of maize? HOME

19. PURPOSE • To know the effect of the strains of PGPR on growth and ion uptake of maize • To know the effect of salt concentration on growth and ion uptake of maize • To know the interaction between strain of PGPR and salt concentration on growth and ion uptake of maize HOME

20. Rhizosphere soil were collected from salt-affected field of maize. Several bacterial strains were isolated by dilution plate technique using DF minimal medium (Dworkin and Foster, 1958). Further streaking on fresh plates purified the collected rhizobacterial strains Material and Methods 1 Prepared the bacteria HOME

21. S5 SHAKER NEXT S20 S5 S15 Incubated for 72 hours under shaking (100 rpm) condition 28 ± 1°C S15 Inoculum @ 60 mL broth S20 An optical density of 0.5 measured at a wavelength of 535 nm was achieved by dilution to maintain uniform cell density (107-108 cfu/mL) HOME

22. The selected strain were characterized by measuring ACC-deaminase activity (Honma and Shimomura, 1978) by monitoring the amount of α-ketobutyrate produced when the enzyme ACC-deaminase cleaves ACC (Aminocyclopropane-1-Carboylic Acid ) • In vitro auxin production was determined as IAA equivalent in the presence and absence of L-tryptophan by using protocol describe by Khalid et al. (2004) • Chitinase activity and phosphorus solubilizing activity was determined qualitatively as describe by Chernin et al. (1998) and Mehta and Nautiyal (2001). • Colonization ability of these strains was studied under axenic condition as describe by Simon et al. (1996) HOME

23. soil - surface disinfected - Inoculated with peat mixed with 10% sugar solution 3 2 Physicochemical characteristic was analyzed Maize seed Uninoculated seed was coated with sterilized peat in the sterilized broth • - sandy clay loam • pH • Ece • Organic matter • Total nitrogen • Olsen phosphorus • Exchangable potassium HOME

24. Inoculated seed POT Containing 12 kg soil S5 S15 A A B B C C D D S20 UNINOCULATED • Pot arranged in wire house under ambient light and temperature according to CRD • Recommended doses NPK fertilizer were applied in each pot as Urea, DAP, and SOP respectively • Phosporus and Potassium was applied as basal dose while Nitrogen was applied in two split A A B B C C D D A, B, C, D are four ECe level 0, 4, 8, and 12 dS/m respectively HOME

25. Thawed and crushed • For N and P analysis: • Dried leaf sample were digested with sulphuric acid and H2O2 • N was determined by Kjeldhal method • P content were determined by spectrophotometer after mixing with Barton reagents. The sap was collected in polypropylene by gilson pipette Centrifuge at 6500 rpm for 10 minutes • Chlorophyll pigment analysis: • 0.5 g of leaf samples of each treatment were homogenized with 80% acetone • The homogenate was filtered through filter paper • Absorbance of the resulting solution was read at 663, 645, and 480 nm for chlorophyll a, b, and carotenoids. Collected after 75 days The supernatant sap was collected and Na and K were determined using sherwood 410 Flame Photometer. Chloride content was determined by chloride analyzer Data were collected and analyzed statistically using a completely randomized design (Steel and Torrie, 1980) FLAME FLAME HOME

26. Variation in growth Shoot fresh weight Shoot dry weight Root fresh weight Root dry weight • Variation ion uptake Photosintetic pigments (Clorophyll a, b, and carotenoid contain), Ion composition (N, P, K and NaCl concentration) Result and discussion

27. Variation in growth • SFWS20,12 dS m-164% over c. • SDW S20,12 dS m-1 102% over c. • RFWS20,12 dS m-1 114% over c. • RDW S20,12 dS m-1102% over c.

28. Pot experiment of maize was observed that inoculation with these rhizobacterial strains significantly improved shoot/root fresh weight and shoot/root dry weight at all salinity levels. In general, growth was reduced with increase in salinity. However, inoculation was effective even in the presence of higher salinity levels (12 dS m-1).

29. Variation ion uptake • C aS20,0 and 8 dS m-113 and 76% over c. • C b S20,12 dS m-1 102% over c. • CarS20 et all EC level up to 58% over c. • N S20,12 dS m-155% over c.

30. Results of pot study showed that on overall basis the increase in salinity level decreased the photosynthetic pigments (chlorophyll a, b and carotenoid contents) of the maize seedlings. However, inoculation with PGPR containing ACCdeaminase activity significantly increased the pigments under salinity stress compared to control.

31. Variation ion uptake • PS20,12 dS m-156% over c. • K S20,12 dS m-1119% over c. • Na+S20,12 dS m-116% lower than c. • Cl- S20,12 dS m-140% lower than c.

32. Data in Table 2 and 3 described that N, P and K concentration significantly increased due to inoculation with PGPR. Na+ and Cl- concentration significantly increased with the increasing salinity particularly in control treatment.

33. Under salinity stress, ethylene is produced at higher concentration and it’s caused inhibitory to plant growth. Removing or blocking the effect of stress ethylene results can decrease of the stress effect.

34. Mechanism of PGPR Lowering the endogenous inhibitory levels of ethylene in roots because of their ACC metabolizing ability and also be due to the expolysaccharides (EPS) activity of bacteria. The PGPR strains can produce bacterial exopoloysaccahrides which bind cations including Na+ (Geddie and Sutherland, 1993) and decrease the content of Na available for plant uptake, thus helping decrease + salt stress in plants (Ashrafet al., 2004).

35. Gift of PGPR inoculum were statistically non significant on growth and ion uptake of maize. Gift of salt concentration were increased significantly on growth and ion uptake of maize. The interaction between strain of PGPR and salt concentration were increased significantly on growth and ion uptake of maize. CONCLUSION HOME

36. THANK YOU FOR THE ATTENTION THANK YOU FOR THE ATTENTION HOME HOME

37. DILUTION PLATE METHOD • DEFINITON OF DILUTION • the mixing of a small accurately measured sample with a large volume of sterile water or normal saline called (diluents or dilution blank) HOME

38. It has been known that if we use a larger volume we obtain a more accurate dilution. So for better results, we use 1:1000 dilution. And that is by adding 1ml of sample to 999 ml of diluent. But practically we cannot use 999 ml of diluent. So we do what is called a serial dilution. Serial Dilution: is a dilution made of a series of smaller dilution, and the total dilution is the product of each dilution in the series. DILUTION HOME

39. ACC deaminase • Hasnain and Sabri (1996) reported that inoculation with Pseudomonas sp. stimulated plant growth by reduction of toxic ion up take and production of stress- specific proteins in plant under stress. Certain microorganisms contain an enzyme 1-aminocyclopropane-1-carboylic acid (ACC) deaminase that hydrolyses ACC, the immediate precursor of ethylene thereby lowering the plant ethylene level (Glick et al., 1998) which eliminates the potential inhibitory effect of high ethylene concentration (Glicket al., 1999). ACC deaminase HOME

41. DUNCAN’S NEW MULTIPLE RANGE TEST • In statistics, Duncan's new multiple range test (MRT) is a multiple comparison procedure developed by David B. Duncan in 1955. Duncan's MRT belongs to the general class of multiple comparison procedures that use the studentizedrangestatisticqr to compare sets of means. • David B. Duncan developed this test as a modification of the Student-Newman-Keuls method that would have greater power. Duncan's MRT is especially protective against false negative (Type II) error at the expense of having a greater risk of making false positive (Type I) errors. Duncan's test is commonly used in agronomy and other agricultural research.