Supplemental Figure S1. Effects of spore suspension concentration B. pumilus TUAT1 strain on the growth (fresh weight) of seedlings of O. sativa cv. Hitomebore at 3 weeks after sowing. Inocula consisted of 4 mL of water or bacterial suspensions and were added to the soil of each seedling once weekly from just after sowing. Values are provided as mean ± SE (n = 8). ** indicates significant differences at the 1% level, compared to the control-inoculated plants, as determined by t-test.

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Legumes establish symbiosis with nitrogen-fixing rhizobia through root nodules to acquire nitrogen. Legumes control nodule number through systemic (autoregulation of nodulation) as well as local regulation. Moreover, plants defend themselves against bacteria and other pathogens through the induction of localized (localized acquired resistance) and systemic (SAR, systemic acquired resistance; ISR, induced systemic resistance) responses. Herein, we show that the number of root nodules is suppressed by programmed cell death (PCD), and is simultaneously controlled by SAR and ISR in soybean (Glycine max [L.] Merr.). The wild-type soybean cultivar Williams 82 showed markedly fewer root nodule primordia and PCD symptoms, including accelerated DNA degradation, enhanced generation of reactive oxygen species (visualized by 3,3′-diaminobenzidine staining), and excessive cell death (detected on staining with trypan blue) compared to the hypernodulation mutant NOD1-3. These results suggest that PCD suppresses the formation of root nodules in wild-type soybean. In addition, microarray and gene ontology analyses showed that essential components of hypersensitive response (HR) or disease resistance, such as resistance (R) genes, mitogen-activated protein kinase cascade, SAR, salicylic acid, jasmonic acid, ethylene, etc., were activated in wild-type plants. These analyses corroborate the above findings, demonstrating that the suppression of root nodule formation by PCD is accompanied by HR, and is simultaneously controlled by SAR and ISR in soybean. These findings provide new insight into the control of nodulation to balance nutritional requirements and energy status in legumes.

Biofertilizers are agricultural materials capable of reducing the usage amounts of chemical fertilizers. Spore-forming microorganisms (SFM) could be used for plant growth promotion or to improve plant health. Until now, biofertilizers based on SFM have been applied for rice and other crops. In this study, we isolated and characterized SFM, which were colonized on the Oryza sativa L. roots. SFM were analyzed regarding the short-term effects of biofertilization on the nursery growths. Analysis was performed without nitrogen or any inorganic fertilizer and was divided into two groups, including bacteria and fungi. SF-bacteria were dominated by the Firmicutes group, including species from Viridibacillus, Lysinibacillus, Solibacillus, Paenibacillus, Priestia, and mainly Bacillus (50%). The fungi group was classified as Mucoromycota, Basidiomycota, and mainly Ascomycota (80%), with a predominance of Penicillium and Trichoderma species. In plant performance in comparison with B. pumilus TUAT1, some bacteria and fungus isolates significantly improved the early growth of rice, based on 48 h inoculum with 107 CFU mL−1. Furthermore, several SFM showed positive physiological responses under abiotic stress or with limited nutrients such as phosphorous (P). Moreover, the metabolic fingerprint was obtained. The biofertilizer based on SFM could significantly reduce the application of the inorganic fertilizer and improve the lodging resistances of rice, interactively enhancing better plant health and crop production.

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