Effects of Root Exudates on Specific Diazotroph-Rice Genotype Association

Diazotrophs are known to utilize root exudate carbon compounds, form natural associations with rice plants and subsequently fix nitrogen. The specific association can be influenced by the bacterial strains and rice genotypes. A series of experiments were conducted in laboratory and glasshouse conditions with the following objectives; (i) to isolate and characterize the indigenous diazotrophs, (ii) to determine the root exudate sugars and amino acids of different rice genotypes (iii) to determine the utilization of root exudates sugars by the diazotrophs (Rhizobium sp. and Corynebacterium sp.) during colonization and (iv) to determine the effects of specific sugars on plant-diazotrophs associations, biological nitrogen fixation and growth of different rice genotypes. The diazotrophs were isolated from Tanjong Karang rice irrigation project area using N-free semi-solid media and the nitrogenase enzyme activity was determined by Acetylene Reduction Assay (ARA) technique. The isolated diazotrophs were identified using Biolog Identification method. Root exudates sugars and amino acids of the three rice genotypes (Mahsuri, Mayang Segumpal and MR219) were determined using high performance liquid chromatography (HPLC). The root (rhizosphere and endophytic) colonization were visually observed by using Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM). The diazotroph–plant association and biological nitrogen fixation (BNF) was estimated using 15N dilution technique in glasshouse condition. Results showed that the indigenous diazotrophic populations were significantly (P<0.01) influenced by soil types, plant age and rice varieties. Bacterial populations were significantly higher in soil (1.8-2.2 x 106 cfu g-1soil) and rhizosphere (1.4-4.2 x 107 cfu g-1 root) of rice grown in Organic Clay & Muck, Bakau, Sedu and Serong soil series. Molecular analysis indicated a diverse group of diazotrophic strains were present in the different soil types. Ten of the strains were identified by the Biolog Identification method as Rhizobium, Burkholderia and Corynebacterium spp.. Biochemical tests of 19 isolates showed that these strains were positive for N2 fixation, capable of degrading cellulose and able to produc high amounts of indoleacetic acid (IAA) which ranged from 15 to 69 mg L-1. The diazotrophs exhibited differences in the specific growth rate, generation time, and utilized mono and disaccharide sugars as sole energy sources. A total of seven sugars and 16 amino acids were determined from rice root exudates. The concentration of root exudate sugars, amino acids and their release patterns differed significantly among rice genotypes. Mahsuri released the highest root exudate sugars (25.73%) followed by MR219 (23.14%) and Mayang Segumpal (20.85%). Inoculated plants produced different amounts of sugars and amino acids compared to non-inoculated plants. Mahsuri inoculated with Corynebacterium sp. (Sb26) released the highest amount of fructose and arabinose, while Mayang Segumpal inoculated with Rhizobium sp. (Sb16) produced the highest amount of sucrose in the root exudate. All rice genotypes produced significantly higher amounts of glycine and isoleucine in root exudates as compared to other amino acids. In general, plants inoculated with Sb16 produced higher amounts of total sugars and amino acids in their root exudates compared to those inoculated with Sb26. A significant relationship was observed between diazotrophic populations and utilization of root exudates sugars and amino acids in the rice genotypes. The Sb16 strain utilized higher amounts of sugars and stimulating higher rhizosphere population compared to Sb26 strain. The diazotrophs were able to colonize and proliferate endophytically in the rice roots. SEM micrographs showed the occurrence of bacterial colonization on surfaces of primary and lateral roots, root hair zone, lateral root junction, in crevices and root tips. TEM view of roots revealed the presence of diazotroph in the intercellular spaces of cortical parenchyma, within epidermis, inner cortex, and near vascular tissue. The results of invitro and glasshouse study using 15N dilution studies showed that Mayang Segumpal inoculated with Sb16 and applied with galactose significantly increased plant-N content and fixed 42 % of atmospheric N (Ndfa). This association increased 147-245 % plant biomass compared to non-inoculated control and 8-52 % over 60 kg ha-1 N-fertilizer application.The study proved that diazotroph inoculation enhanced root exudate sugar production and provides specific sugars for specific diazotroph-rice plant association. Application of galactose and arabinose as external carbon source enhanced the growth and N2 fixation activity of the Rhizobium sp. (Sb16) and Corynebacterium sp. (Sb26), respectively. The association of Mayang Segumpal with Sb16 and MR219 rice with Sb26 significantly improved nitrogen fixation and subsequently plant growth

Beneficial effects of mycorrhizal association for crop production in the tropics - a review.

Mycorrhiza plays a significant role in sustainable agriculture and has mutualistic symbiotic association with plant roots. There are several species of mycorrhiza and among the species studied Glomus mosseae is well known to colonize several vegetables, fruits, cereals and industrial crops. This paper highlighted the symbioses and beneficial effects of arbuscular mycorrhiza fungi (AMF) with tomato (Solanum lycopersicum), brinjal (S. melongena), potato (S. tuberosum), lady's finger (Abelmoschus esculentus), cucumber (Cucumis sativus), bean (Phaseolus vulgaris), pepper (Capsicum annuum), wheat (Triticum aestivum), aerobic rice (Oryza sativa), corn (Zea mays), durian (Durio zibethinus), rambutan (Nephelium lappaceum), pineapple (Ananas comosus), citrus (Citrus sinensis), banana (Musa acuminata), oil palm (Elaeis guineensis) and kenaf (Hibiscus cannabinus). Application of AMF increased nutrient uptake, water relations and perform as bio-protectants against pathogens and toxic stresses. In order to further improve their benefits, it is necessary to ensure the management practices comprising low tillage, abridged use of chemical fertilizers, especially the phosphatic fertilizers

Effect of urea-N on growth and indoleacetic acid production of Stenotrophomonas maltophilia (Sb16) isolated from rice growing soils in Malaysia

Growth and activity of N2-fixing bacteria can be affected by N fertilizer application. A study was conducted at Universiti Putra Malaysia, Malaysia, to determine the effect of urea-N on the growth and indoleacetic acid (IAA) production of diazotrophic Stenotrophomonas maltophilia strain Sb16 (accession number, JQ820255), previously isolated from rice (Oryza sativa L.) growing soils of Malaysia. Five rates of N from urea fertilizer were applied (0, 50, 100, 150, and 200 kg ha-1) to the flooded rice soil and incubated in the control environment for 6 wk. Results showed that the population growth increased with increased N rates and highest population (8.2 log10 cfu mL-1) recorded in soil-standing water at the second week of incubation. The population decreased with the reduction of total N. Soil and soil water pH increased from 7.1 to 8.4 at the first week of incubation. Production of indoleacetic acid was significantly reduced with N fertilizer application. The highest indoleacetic acid (2.6 mg mL-1) was produced in the control treatment. Application of high rates of N fertilizer increased the population of diazotrophic strain Sb16 but significantly reduced indoleacetic acid production

Application of boron and zinc in the tropical soils and its effect on maize (Zea mays) growth and soil microbial environment.

An experiment was conducted at glasshouse of Universiti Putra Malaysia with boron (B) and zinc (Zn) deficient soil (Serdang series) to evaluate the effect of B and Zn on maize crop and the behavior of soil microbial communities with various levels of boron and zinc. Among the six tested soil series, Malawi soil was found the most deficient in boron (0.06 ppm). Four levels of B from Borax as 0, 0.5, 1.0, and 1.5 kg ha -1 and two levels of Zn from Zinc Sulphate, 0 and 5 kg ha -1 were applied. The highest plant height (109 cm), root length (30.67 cm), leaf area index, chlorophyll content, shoot (5.38 g) and root dry weight (0.23 g) were obtained at B 0.5 + Zn 5.0 kg ha -1 treatment. The interaction effect between boron × zinc in the soil was significant. Antagonistic effect occurred for B uptake at higher rates of B with 5 kg ha -1 of Zn application. The addition of B at lower level alone or in combination with Zn significantly increased soil bacterial population. The highest rhizosphere bacterial population was found at B 0.5 + Zn 5 kg ha -1 treatment. The addition of B 1.0 kg ha -1 with zinc Zn 5 kg ha -1 showed a toxic effect on soil bacterial population. Although B produced substantial biomass yield increment with extended rates, the excess amount caused toxicity in the soil bacterial environment. It can be concluded that 0.5 kg ha -1 of B in combination with 5 kg ha -1 Zn can exhibit higher yield of maize and is friendly to this particular soil environment

Culturable total and beneficial microbial occurrences in long-term nutrient deficit wetland rice soil

A study was conducted to find the effect of long term (24 years) soil macro nutrient deficit condition on total soil microbial population and occurrences of free-living nitrogen fixing and phosphate solubilizing bacteria (PSB) population in wetland rice cultivation system. After the 48th crop cycle (two crops of rice grown per year) soil and plant samples were collected before transplanting and maximum tillering stages from plots with the following treatments: (i) Complete fertilizers (NPKSZn), (ii) NPSZn (-K), (iii) NKSZn (-P) (iv) PKSZn (-N), and (v) without fertilizer. The total bacteria, fungus, actinomycetes, free-living nitrogen fixing bacteria and phosphate solubilizing bacteria populations were determined from each treatment. Results showed that total bacteria, fungus and actinomycetes population were high in the "complete fertilizer" treatment and low in "without fertilizer" treatments, which proved that the absence of any single nutrient element (N or P or K) decreased total soil microbial populations. Significantly high free-living nitrogen fixing bacterial population and high biological nitrogen fixation was found in "without N" fertilizer treatment. The highest atmospheric nitrogen fixed in the nitrogen-free broth culture (3.7%) was by the endophytic strains isolated from "without N" treatment. The Population of PSB was high in the "complete fertilizer" treatment. The highest phosphate (P) solubilizing activity (80%) was observed with the isolated PSB in "complete fertilizer" treatments. The isolated strains produced indoleacetic acid at rates of 1-9 mg l-1. The results of soil chemical analysis showed that the soil N reserve was not reduced as compared to P and K, although the soil received the same treatments over the 24 years. In general, total microbial population was significantly affected by the absence of nutrient elements, whereas the absence of nitrogen increased the free-living nitrogen fixing bacteria

Assessment of phosphorus use efficiency and various phosphorus application methods on wheat in calcarious soil

Phosphorus has been recognized as an essential constituent for plants. A field experiment was conducted in calcareous soil to evaluate the phosphorus fertilizer use efficiency (PFUE), P uptake and plant growth yield through different P fertilizer application on wheat crop. The treatments were: T1 (control); T2, 80 kg ha-1 P2O5 broadcasting method; T3, 80 kg ha-1 P2O5 banding (applied with 1% H2SO4 at the 1st irrigation); T4, 60 kg ha-1 P2O5 banding; T5, 60 kg ha-1 P2O5 banding (applied with 1% H2SO4 at 1st irrigation); T6, 40 kg banding + 20 kg ha-1 P2O5 top dressing (applied with 1% H2SO4 at the 1st irrigation); and T7, 40 kg ha-1 P2O5 banding applied with 1% H2SO4 at the 1st irrigation. The result showed that the band plus acid treatment was more effective than broadcasting method of application. There were no significant differences found among the various banding applications. However, significant differences were observed among the various application methods, in terms of PUE, grain nutrient ratio (GNR), P uptake and VCR. Significantly high PFUE (straw 8.52% and grain 17.48%), GNR (13.5%), P uptake (straw 4.21, and grain 8.18 kg ha-1) and VCR were found using 40 kg banding + 20 kg ha-1 P2O5 top dressing method along with 1% H2SO4

Bio-Fertilizer, ground magnesium limestone and basalt applications may improve chemical properties of Malaysian acid sulfate soils and rice growth

Acid sulfate soils are normally not suitable for crop production unless they are appropriately ameliorated. An experiment was conducted in a glasshouse to enhance the growth of rice, variety MR219, planted on an acid sulfate soil using various soil amendments. The soil was collected from Semerak, Kelantan, Malaysia. Ground magnesium limestone (GML), bio-fertilizer, and basalt (each 4 t ha−1) were added either alone or in combinations into the soil in pots 15 d before transplanting. Nitrogen, P and potash were applied at 150, 30, and 60 kg ha−1, respectively. Three seven-day-old rice seedlings were transplanted into each pot. The soil had a pH of 3.8 and contained organic C of 21 g kg−1, N of 1.2 g kg−1, available P of 192 mg kg−1, exchangeable K of 0.05 cmolc kg−1, and exchangeable Al of 4.30 cmolc kg−1, with low amounts of exchangeable Ca and Mg (0.60 and 0.70 cmolc kg−1). Bio-fertilizer treatment in combination with GML resulted in the highest pH of 5.4. The presence of high Al or Fe concentrations in the control soil without amendment severely affected the growth of rice. At 60 d of growth, higher plant heights, tiller numbers and leaf chlorophyll contents were obtained when the bio-fertilizer was applied individually or in combination with GML compared to the control. The presence of beneficial bacteria in bio-fertilizer might produce phytohormones and organic acids that could enhance plant growth and subsequently increase nutrient uptake by rice. Hence, it can be concluded that addition of bio-fertilizer and GML improved rice growth by increasing soil pH which consequently eliminated Al and/or Fe toxicity prevalent in the acid sulfate soil

Specific growth rate and carbon sugar consumption of diazotrophs isolated from rice rhizosphere

A study was conducted in in vitro condition to determine the specific growth rate, sugar consumption and generation time of four diazotrophic bacterial species, Burkholderia, Rhizobium and two Corynebacterium sp. in four different carbon substrates. Growth of all bacterial strains was significantly affected by the sugars. Burkholderia sp. (Sb13) rapidly attained maximum population in glucose, fructose and sucrose and the specific growth rate best fitted with exponential model. The cell generation times of Burkholderia sp. (Sb13) in glucose, fructose and sucrose substrate were 0.4, 0.55 and 0.1 h, respectively. Rhizobium sp. (Sb16) followed an exponential growth model in fructose and its generation time was 0.62 h. The growth of Corynebacterium sp. (Sb26) was higher in arabinose and followed logarithmic growth model with generation time of 0.68 h. Corynebacterium sp. (Sb35) showed faster generation time (1 h) in sucrose substrates and the specific growth rate in all four carbon substrates followed logarithmic model of growth phase. Burkholderia sp. and Corynebacterium sp. (Sb26) consumed 100% of arabinose after 36 h of incubation period. The total sugar consumption by the diazotrophs were higher in logarithmic model of growth phase compared to exponential, power and polynomial model

Isolation of Diazotrophs from Different Soils of Tanjong Karang Rice Growing Area in Malaysia

Isolation and biochemical characterization of diazotrophs were carried out on seven soil series/types of Tanjong Karang Rice Irrigation Project area. The soil population ranged from 4 × 104 to 2.2 × 106 cfu g-1 soil. Diazotrophic populations were significantly (P<0.01) influenced by soil types, plant age and rice varieties. Higher soil and rhizosphere populations were recorded in Organic Clay and Muck, Bakau, Sedu and Serong soils. The highest root (6.3 × 107) and shoot (2.5 × 107) populations were found in MR219 rice planted in Organic Clay and Muck and Sedu soil series, which had higher C, N and P contents. The highest acetylene reduction assay (ARA) value (1.26 × 10-6 μmoL C2H4 cfu-1 hr1) was found in isolate Sb35. Several diazotrophic strains produced 32 to 69 mg L-1 of indoleacetic acid(IAA). The highest IAA was produced by the diazotrophic strain Sb41 (Corynebacterium sp). Eleven of the diazotrophic strains isolated from root and shoot of the rice varieties were capable of producing cellulose degrading enzyme. Tanjong Karang rice growing area harbor a diverse group of bacteria and most of the isolates belonged to the genera of Rhizobium, Burkholderia and Corynebacterium

Influence of root exudate carbon compounds of three rice genotypes on rhizosphere and endophytic diazotrophs

Root exudates play an important role in microbial colonization of the rhizosphere. An in vitro experiment was conducted to study the root exudate sugars and production of amino acids of three different rice (Oryza sativa) genotypes, as well as the influence of these compounds on Rhizobium sp. (Sb 16) and Corynebacterium sp. (Sb26) colonization. Using HPLC, a total of 7 carbohydrate sugars and 16 amino acids were identified from the Mahsuri, Mayang Segumpal and MR219 rice root exudates. A significant (p<0.05) relationship was observed between diazotrophic population growth and root exudates sugar and amino acid consumption of the three rice varieties. Higher bacterial population was found in the plant rhizosphere, as compared to the endosphere. Rhizobium sp. consumed more sugar and produced higher rhizosphere population as compared to Corynebacterium sp. The Rhizobium sp. consumed 100% of mannose, xylose, arabinose and sucrose in the root exudates of three rice genotypes. The differences in sugar consumption by Corynebacterium sp. were observed between the rice varieties. Corynebacterium sp. consumed 100% mannose, xylose and fructose in Mahsuri rice, 100% xylose and arabinose in Mayang Segumpal and 100% arabinose and sucrose in MR219 variety. The identification of the preferred carbon sources by the diazotrophs and the selection of genotypes which produce these compounds may increase the root colonization and subsequently N fixation in the rice plants