The aim of this research was to isolate and characterize bacteria from spores of arbuscular mycorrhizal fungi (AMF). We designated these bacteria ‘probable endobacteria’ (PE). Three bacterial strains were isolated from approximately 500 spores of Gigaspora margarita (Becker and Hall) using a hypodermic needle (diameter, 200 μm). The bacteria were identified by morphological methods and on the basis of ribosomal gene sequences as Bacillus sp. (KTCIGM01), Bacillus thuringiensis (KTCIGM02), and Paenibacillus rhizospherae (KTCIGM03). We evaluated the effect of these probable endobacteria on antagonistic activity to the soil‐borne plant pathogens (SBPPs) Fusarium oxysporum f. sp. lactucae MAFF 744088, Rosellinia necatrix, Rhizoctonia solani MAFF 237426, and Pythium ultimum NBRC 100123. We also tested whether these probable endobacteria affected phosphorus solubilization, ethylene production, nitrogenase activity (NA), and stimulation of AMF hyphal growth. In addition, fresh samples of spores and hyphae were photographed using an in situ scanning electron microscope (SEM) (Quanta 250FEG; FEI Co., Japan). Bacterial aggregates (BAs), structures similar to biofilms, could be detected on the surface of hyphae and spores. We demonstrate that using extraction with an ultrathin needle, it is possible to isolate AMF‐associated bacterial species that are likely derived from inside the fungal spores.

Aims: The aim of this work was to observe bacteria associated with the spores of Gigaspora margarita, an arbuscular mycorrhizal fungus (AMF). Methods and Results: First, a direct analysis of DNA from sterilized spores indicated the bacteria belonging to the genus Janthinobacterium. In the second assay, two bacterial strains were isolated by osmosis from protoplasts, which were derived from spores by using two particular enzymes: lysing enzymes and yatalase. After isolation, cultivation and identification by their DNA as performed in the first experiment, the species with the closest relation were Janthinobacterium lividum (KCIGM01) and Paenibacillus polymyxa (KCIGM04) isolated with lysing enzymes and yatalase respectively. Morphologically, J. lividum was Gram negative and oval, while P. polymyxa was also oval, but Gram positive. Both strains had antagonistic effects to the pathogenic fungi Rosellimia necatrix, Pythium ultimum, Fusarium oxysporum and Rhizoctonia solani. In particular, J. lividum was much stronger in this role. However, in phosphorus (P) solubilization P. polymyxa functioned better than J. lividum. Conclusions: This experiment had revealed two new bacteria species (P. polymyxa and J. lividum), associated with AMF spores, which functioned to suppress diseases and to solubilize P. Significance and Impact of the Study: AMF spores could be a useful source for bacterial antagonists to soil-borne diseases and P solubilization.

This research evaluated the monthly variation of plant mineral nutrition in six species of fruit trees over a year. Leaf samples were taken from the fruit trees and nutritional status (N, P, K, Ca, Mg, Fe, Zn, Cu, Mn, and Mo) was determined in the leaves in a month basis from April until November for apple, persimmon and peach. Mandarin mineral nutrition was monitored for one year, and grape and fig from May to November. Using this data, the Diagnosis Recommendation Integrated System (DRIS) was also calculated to evaluate the nutrient balance in the plants. The concentration of N and P had seasonal differences, especially in apple and peach, which reached the peak during the summer. Apple, fig, and grape trees had large ranging on their mineral contents over the year, especially with the P and K levels reaching the minimum during the harvesting season. However, the seasonal changes in leaf micronutrient concentrations were not uniform and not affected by phenological stage. The DRIS data demonstrated that mandarin had the best nutrient balance compared to others and that K was the most limiting element among the fruit trees. In summary, the current data suggest the occurrence of a significant seasonality in mineral nutrition in these six fruit trees, especially in temperate ones.