Bacillus tequilensis strain 'UPMRB9' improves biochemical attributes and nutrient accumulation in different rice varieties under salinity stress.

Soil salinity exert negative impacts on agricultural production and regarded as a crucial issue in global wetland rice production (Oryza sativa L.). Indigenous salt-tolerant plant growth-promoting rhizobacteria (Bacillus sp.) could be used for improving rice productivity under salinity stress. This study screened potential salt-tolerant plant growth-promoting rhizobacteria (PGPR) collected from coastal salt-affected rice cultivation areas under laboratory and glasshouse conditions. Furthermore, the impacts of these PGPRs were tested on biochemical attributes and nutrient contents in various rice varieties under salt stress. The two most promising PGPR strains, i.e., 'UPMRB9' (Bacillus tequilensis 10b) and 'UPMRE6' (Bacillus aryabhattai B8W22) were selected for glasshouse trial. Results indicated that 'UPMRB9' improved osmoprotectant properties, i.e., proline and total soluble sugar (TSS), antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Moreover, 'UPMRB9' inoculated rice plants accumulated higher amount of nitrogen and calcium in tissues. Therefore, the indigenous salt-tolerant PGPR strain 'UPMRB9' could be used as a potential bio-augmentor for improving biochemical attributes and nutrient uptake in rice plants under salinity stress. This study could serve as a preliminary basis for future large-scale trials under glasshouse and field conditions.

Spermine mediated improvements on stomatal features, growth, grain filling and yield of rice under differing water availability.

Rice which belongs to the grass family is vulnerable to water stress. As water resources get limited, the productivity of rice is affected especially in granaries located at drought prone areas. It would be even worse in granaries located in drought prone areas such as KADA that receives the lowest rainfall in Malaysia. Spermine (SPM), a polyamine compound that is found ubiquitiosly in plants is involved in adaptation of biotic and abiotic stresses. The effect of SPM on growth,grain filling and yield of rice at three main granaries namely, IADA BLS, MADA and KADA representing unlimited water, limited water and water stress conditions respectively, were tested during the main season. Additinally, the growth enhancer was also tested during off season at KADA. Spermine increased plant height, number of tillers per hill and chlorophyll content in all three granaries. Application of SPM improved yield by 38, 29 and 20% in MADA, KADA and IADA BLS, respectively. Harvest index showed 2.6, 6 and 16% increases at IADA BLS, KADA and MADA, respectively in SPM treated plants as compared to untreated. Except for KADA which showed a reduction in yield at 2.54 tha-1, SPM improved yield at MADA, 7.21 tha-1 and IADA BLS, 9.13 tha-1 as compared to the average yield at these respective granaries. In the second trial, SPM increased the yield to 7.0 and 6.4 tha-1 during main and off seasons, respectively, indicating that it was significantly higher than control and the average yield reported by KADA. The yield of SPM treatments improved by 25 and 33% with an increment of farmer's income at main and off seasons, respectively. Stomatal width was significantly higher than control at 11.89 µm. In conclusion, irrespective of the tested granaries and rice variety, spermine mediated plots displayed increment in grain yield.

The Dynamic Responses of Oil Palm Leaf and Root Metabolome to Phosphorus Deficiency.

Inorganic phosphate (Pi) starvation is an important abiotic constraint that affects plant cellular homeostasis, especially in tropical regions with high acidic soil and less solubilizable Pi. In the current work, oil palm seedlings were hydroponically maintained under optimal Pi-supply and no Pi-supply conditions for 14 days, and metabolites were measured by gas chromatography-mass spectrometry (GC-MS), from leaves and roots, after seven and 14 days of treatment, to investigate biochemical pathways in relation to P-utilizing strategy. After seven days of limited Pi, plant leaves showed increased levels of most soluble sugars, and after 14 days, the sugars' level decrease, except for erythritol, mannose, fructose, and glucose, which showed the highest levels. Rather in root samples, there were different but overlapping alterations, mainly on sugars, amino acids, and organic acids. The leaf sample was shown to have the highest response of sugars with myo-inositol playing a vital role in the redistribution of sugars, while maltose levels increased, indicating active degradation of starch in the root. High levels of glycerol and stearate in both roots and leaves suggest the metabolism of storage lipids for cellular energy during Pi-deficient conditions.

Characterization of salt-tolerant plant growth-promoting rhizobacteria and the effect on growth and yield of saline-affected rice.

In this study, we characterized, identified, and determined the effect of salt-tolerant PGPR isolated from coastal saline areas on rice growth and yield. A total of 44 bacterial strains were isolated, and 5 were found to be tolerant at high salt concentration. These isolates were further characterized for salinity tolerance and beneficial traits through a series of quantitative tests. Biochemical characterization showed that bacterial survivability decreases gradually with the increase of salt concentration. One of the strains, UPMRB9, produced the highest amount of exopolysaccharides when exposed to 1.5M of NaCl. Moreover, UPMRB9 absorbed the highest amount of sodium from the 1.5M of NaCl-amended media. The highest floc yield and biofilm were produced by UPMRE6 and UPMRB9 respectively, at 1M of NaCl concentration. The SEM observation confirmed the EPS production of UPMRB9 and UPMRE6 at 1.5M of NaCl concentration. These two isolates were identified as Bacillus tequilensis and Bacillus aryabhattai based on the 16S rRNA gene sequence. The functional group characterization of EPS showed the presence of hydroxyl, carboxyl, and amino groups. This corresponded to the presence of carbohydrates and proteins in the EPS and glucose was identified as the major type of carbohydrate. The functional groups of EPS can help to bind and chelate Na+ in the soil and thereby reduces the plant's exposure to the ion under saline conditions. The plant inoculation study revealed significant beneficial effects of bacterial inoculation on photosynthesis, transpiration, and stomatal conductance of the plant which leads to a higher yield. The Bacillus tequilensis and Bacillus aryabhattai strains showed good potential as PGPR for salinity mitigation practice for coastal rice cultivation.

GCTTCA as a novel motif for regulating mesocarp-specific expression of the oil palm (Elaeis guineensis Jacq.) stearoyl-ACP desaturase gene.

KEY MESSAGE: TAAAAT and a novel motif, GCTTCA found in the oil palm stearoyl-ACP desaturase (SAD1) promoter are involved in regulating mesocarp-specific expression. Two key fatty acid biosynthetic genes, stearoyl-ACP desaturase (SAD1), and acyl-carrier protein (ACP3) in Elaeis guineensis (oil palm) showed high level of expression during the period of oil synthesis in the mesocarp [12-19 weeks after anthesis (w.a.a.)] and kernel (12-15 w.a.a.). Both genes are expressed in spear leaves at much lower levels and the expression increased by 1.5-fold to 2.5-fold following treatments with ethylene and abscisic acid (ABA). Both SAD1 and ACP3 promoters contain phytohormone-responsive, light-responsive, abiotic factors/wounding-responsive, endosperm specificity and fruit maturation/ripening regulatory motifs. The activities of the full length and six 5' deletion fragments of the SAD1 promoter were analyzed in transiently transformed oil palm tissues by quantitative ß-glucuronidase (GUS) fluorometric assay. The highest SAD1 promoter activity was observed in the mesocarp followed by kernel and the least in the leaves. GUS activity in the D3 deletion construct (- 486 to + 108) was the highest, while the D2 (- 535 to + 108) gave the lowest suggesting the presence of negative cis-acting regulatory element(s) in the deleted - 535 to - 486 (49 bp). It was found that the 49-bp region binds to the nuclear protein extract from mesocarp but not from leaves in electrophoretic mobility shift assay (EMSA). Further fine-tuned analysis of this 49-bp region using truncated DNA led to the identification of GCTTCA as a novel motif in the SAD1 promoter. Interestingly, another known fruit ripening-related motif, LECPLEACS2 (TAAAAT) was found to be required for effective binding of the novel motif to the mesocarp nuclear protein extract.

In vitro evaluation of Pseudomonas bacterial isolates from rice phylloplane for biocontrol of Rhizoctonia solani and plant growth promoting traits.

The ability for biocontrol and plant growth promotion of three Pseudomonas bacterial isolates namely Pseudomonas fluorescens (UMB20), Pseudomonas aeruginosa (KMB25) and Pseudomonas asplenii (BMB42) obtained from rice plants was investigated. Fungal growth inhibition by the isolates ranged from 86.85 to 93.15% in volatile and 100% in diffusible metabolites test. Among the isolates, BMB42 showed fungal growth inhibition significantly in the volatile metabolite test. Isolates UMB20 and BMB42 were able to synthesis chitinase with chitinolytic indices of 13.66 and 13.50, respectively. In case of -1,3-glucanase, all the isolates showed activity to produce this enzyme at varied levels and isolate KMB25 showed significantly highest activity (53.53 ppm). Among the three isolates, KMB25 showed positive response to protease production and all of them were negative to pectinase and lipase and positive to the production of siderophore, and HCN, and were able to solubilize tricalcium phosphate. All the three bacterial isolates were capable of forming biofilm at different levels. Above results suggest that phylloplane Pseudomonas bacterial isolates have potential for antifungal activities and plant growth promotion.

Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes.

Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and examined their effect on salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme activities (SOD, APX, and CAT) and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR) were observed in PGPR inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under salinity stress.

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

Phylogenetic relationship of phosphate solubilizing bacteria according to 16S rRNA genes.

Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field.

Isolation and identification of antagonistic bacteria from phylloplane of rice as biocontrol agents for sheath blight.

A total of 325 bacteria were isolated from both healthy and sheath blight infected leaf samples of rice plants, collected from different places of Malaysia, following dilution technique. Sheath blight pathogen was isolated from infected samples by tissue plating method. Out of 325, 14 isolates were found to be antagonist against the pathogen in pre evaluation test. All the 14 isolates were morphologically characterized. Antagonistic activity of these isolates was further confirmed by adopting the standard dual culture and extracellular metabolite tests. The best isolates were selected, based on the results. In dual culture test, the selected bacterial isolates KMB25, TMB33, PMB38, UMB20 and BMB42 showed 68.44%, 60.89%, 60.22%, 50.00% and 48.22% fungal growth inhibition, respectively and in extracellular metabolite test these bacterial isolates exhibited 93.33%, 84.26%, 69.82%, 67.96% and 39.26% of the same, respectively. Biochemical tests of selected isolates were performed following standard procedure. These bacterial isolates were tentatively identified as fluorescent pseudomonas by morphological and biochemical characterization. The identities were further confirmed by Biolog microstation system as P. fluorescens (UMB20), P. aeruginosa (KMB25, TMB33 and PMB38) and P. asplenii (BMB42) with similarity index ranging from 0.517 to 0.697. The effective bacterial isolates obtained from the present study can be used in the management of soil borne fungal pathogen Rhizoctonia solani, causing sheath blight of rice.

Specific genomic fingerprints of phosphate solubilizing Pseudomonas strains generated by BOX elements.

Primers corresponding to conserved bacterial repetitive of BOX elements were used to show that BOX-DNA sequences are widely distributed in phosphate solubilizing Pseudomonas strains. Phosphate solubilizing Pseudomonas was isolated from oil palm fields (tropical soil) in Malaysia. BOX elements were used to generate genomic fingerprints of a variety of Pseudomonas isolates to identify strains that were not distinguishable by other classification methods. BOX-PCR, that derived genomic fingerprints, was generated from whole purified genomic DNA by liquid culture of phosphate solubilizing Pseudomonas. BOX-PCR generated the phosphate solubilizing Pseudomonas specific fingerprints to identify the relationship between these strains. This suggests that distribution of BOX elements' sequences in phosphate solubilizing Pseudomonas strains is the mirror image of their genomic structure. Therefore, this method appears to be a rapid, simple, and reproducible method to identify and classify phosphate solubilizing Pseudomonas strains and it may be useful tool for fast identification of potential biofertilizer strains.