A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria.

AIMS: To develop a rapid and simple genus-specific polymerase chain reaction (PCR) method for detecting and identifying isolates of the genus Azospirillum which is well-recognized as plant growth-promoting rhizobacterium. METHODS AND RESULTS: Nine pairs of PCR primers were designed based on the Azospirillum 16S rRNA, ipdC, nifA and nifH genes to assess their genus specificity by testing against 12 Azospirillum (from seven species) and 15 non-Azospirillum reference strains, as compared with the fAZO/rAZO pair reported by Baudoin et al. (J Appl Microbiol, 108, 2010, 25). Among the primer pairs assessed, the Az16S-A pair designed on the 16S rRNA gene sequence showed the highest genus specificity: it successfully yielded a single amplicon of the expected size in all the 12 Azospirillum strains and for a close relative, Rhodocista centenaria. The PCR with the Az16S-A primers generated a detectable amount of the amplicon from ≥10³ CFU ml⁻¹ of Azospirillum cell suspensions even in the presence of contaminants and accurately discriminated Azospirillum and non-Azospirillum species in both 35 Azospirillum-like and 70 unknown isolates from plant roots and rhizosphere soils. CONCLUSIONS: We developed a rapid and simple PCR method for detecting and identifying Azospirillum isolates within populations of rhizosphere bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The method developed would serve as a useful tool for isolating a variety of indigenous Azospirillum bacteria from agricultural samples.

Proteomic analysis of secreted proteins from aseptically grown rice.

Plants are known to secrete a variety of compounds into the rhizosphere. These compounds are thought to play important roles in the regulation of soil chemical properties and soil microorganisms. To determine the composition of proteins secreted from rice roots, aseptic hydro culture was performed, and the collected proteins were analyzed. Over 100 proteins were identified; most were identified using the rice database (RAP-DB), and about 60% of the identified proteins were suspected to have a signal peptide. Functional categorization suggested that most were secondary metabolism- and defense-related proteins. Pathogenesis- and stress-related proteins were the major proteins found in the bathing solution under aseptic conditions. Thus, we propose that rice plants constitutively secrete a large variety of proteins to protect their roots against abiotic and/or biotic stresses in the environment.

Allocation of 14C-carbon in two species of larch seedlings infected with ectomycorrhizal fungi.

The flow of labeled carbon in ectomycorrhizal and non-ectomycorrhizal seedlings of Japanese larch (Larix kaempferi Sarg.) and its F1 hybrid (Larix gmelinii Rupr. x L. kaempferi) was studied. Larch seedlings were grown in a greenhouse for 110 days with larch forest soil (FM) or Suillus grevillei (SM) inoculum, or in the absence of ectomycorrhizal fungi (NM). Shoots of colonized and NM seedlings were exposed to a pulse of 14CO2 for 1 h under natural light. Seedlings were harvested following 0, 6 and 24 h of exposure to 14CO2. At the final harvest, SM seedlings of Japanese larch and hybrid larch allocated 2.6 and 2.5% more 14C, respectively, to roots than NM seedlings. In contrast, FM seedlings of Japanese larch and hybrid larch allocated 6.5 and 18.0% more 14C, respectively, to the stem than NM seedlings. Of the total 14C detected in needle, stem and root fractions, FM and SM seedlings allocated a greater proportion than NM seedlings, perhaps because FM and SM seedlings had significantly (P < 0.05) higher photosynthetic rates than NM seedlings. As a result, FM and SM seedlings had greater dry masses than NM seedlings. Concentrations of nitrogen and phosphorus in FM and SM seedlings were significantly (P < 0.05) higher than in NM seedlings, as was stomatal conductance.

Starch properties of the sago palm (Metroxylon sagu Rottb.) in different soils.

We investigated the relationships between starch concentrations and activities of starch synthetic enzymes in sago palms (Metroxylon sagu Rottb.) under acid sulfate and mineral soil conditions. Plants grown naturally that had reached their maturated stage were sampled. We found that the growth in acid sulfate soil is lower than that in mineral soil and that starch granules were larger and there was more amylase activity in acid sulfate soil than in mineral soil. Lower amylase activity in mineral soil could eliminate the degradation of starch, making the smaller granules suitable for storing large amounts of starch in a limited space inside cells.

Mineral characteristics of leaves of plants from different phylogeny grown in various soil types in the temperate region.

The objective of this research was to analyze selected minerals in leaves of plants, belonging to 166 species growing in alluvial, low pH, brown forest and serpentine soils. Mineral characteristics of the soils involved were also determined. For the macronutrients, in trees grown in alluvial soil, N, P, Ca, and Mg concentrations of leaves were higher in recently evolved plants than in plants with a longer period of evolution; K concentration remained constant regardless of evolution. In grasses grown in alluvial soil, it was difficult to detect the general tendency of mineral concentration. N, P, and K concentrations in alluvial soil were closely related to those in low pH and serpentine soils. Ca concentration in alluvial soil was lower than that in low pH and serpentine soils. Mg concentration in alluvial soils was higher than that in low pH soils, while lower than that in serpentine soil. Therefore, N, P, and K accumulated according to the plant characteristics for these elements, while Ca and Mg accumulation was strongly affected by the soil properties. For the micronutrients, in trees, Fe and Mn remained constant regardless of evolution; Zn concentration was lower in recently-evolved plants than in plants with a longer period of evolution. In grasses, Fe, Mn, and Zn concentrations in Caryophyllids were high. Except for Caryophyllids, Fe and Cu concentrations remained constant. Mn concentration decreased with evolution, Zn concentration was higher in recently-evolved plants than in plants with a longer period of evolution.

Mineral characteristics of the leaves of 166 plant species with different phylogeny in the temperate region.

The P-N regression coefficient was moderate in Magnoliales, Coniferopsidae, Pteridophyta, and Asterids, but very high in Caryophyllids, and very low in Rosids. The K-N regression coefficient in trees remained constant regardless of evolution, and that in grasses was high except for Rosids compared with that in trees. The coefficient was very high in Caryophyllids and was very low in Rosids. The N-Ca and N-Mg relationship was not estimated at all, suggesting that the mechanism of Ca and Mg accumulation was completely different from that of N related accumulation. The Zn and Cu concentrations were related to the N concentration. The Al concentration in leaves was negatively correlated with the N, P, K, Ca, Mg, Na, Cu, Zn, Mn, and Fe concentrations, while the N, P, K, and Mn concentrations in leaves increased slightly with the increase of Al concentration in the high Al concentration. The Na concentration in leaves related negatively to the P, Ca, Cu, Zn, Mn and Al concentration range. Thus, it was demonstrated that most of the minerals in leaves accumulate negatively Al and Na indicating that there are antagonistic mechanisms for mineral accumulation in leaves among Al or Na and other mineral elements.

Differences in nitrogen economy of temperate trees.

Twenty-four temperate tree species were classified into three groups based on cluster analysis of relative growth rate, nitrogen concentration, nitrogen-production efficiency, nitrogen-distribution ratio and nitrogen-use efficiency as follows: Group I (Asteridae and Rosidae), Group II (Dilleniidae and Hamamelidae) and Group III (Coniferopsidae). Relative growth rate (RGR) was high in Group II, moderate in Group I and low in Group III. The regression coefficient for the relationship between RGR and leaf nitrogen concentration was higher in Group II than in Group I, and no relationship was observed in Group III. Parameter analysis of RGR indicated that RGR per unit leaf nitrogen was important for all three groups, but that the allocation of nitrogen to leaves was particularly important in Groups I and II. The ratio of dark respiratory rate (R) to net photosynthetic rate (A) was higher in Group I than in Group II. Neither A nor R was measured in the Group III species. A linear relationship was observed between leaf nitrogen concentration and A in Group II, but this relationship was not evident in Group I.