Endophytic Bacterium Lysobacter firmicutimachus Strain 5-7 Is a Promising Biocontrol Agent Against Rice Seedling Disease Caused by Pythium arrhenomanes in Nursery Trays.

Rice root rot disease caused by Pythium spp. is a highly destructive disease in rice nurseries. Biocontrol with endophytic bacteria was developed in this study to control rice seedling diseases. An in planta screening assay revealed that two bacterial endophytes, strains 5-7 and 6-4, displayed strong protection of rice seedlings from attack by Pythium arrhenomanes. Phylogenetic analysis indicated that strain 5-7 is Lysobacter firmicutimachus, while strain 6-4 belongs to the Kitasatospora genus. To quickly evaluate the disease severity of the root system damaged by Pythium spp. in nursery trays, a root surface area measurement assay was developed. By using this measurement, the control efficacy in nursery trays was evaluated, and L. firmicutimachus 5-7 showed promising biocontrol activity against Pythium disease. In a field trial, the two endophytes exhibited significant disease control efficacy on rice brown spot disease caused by Bipolaris oryzae naturally occurring in a commercial nursery field. The two endophytes exhibited multiple enzymatic activities and broad-spectrum antagonistic activities against multiple rice pathogens. The two endophytes colonized the root surface and inside of the root. L. firmicutimachus 5-7 primarily colonized the intercellular space and aerenchyma. Antibiosis is the major mechanism used by strain 5-7 to cause Bipolaris hyphal swelling and inhibit Pythium zoospore germination and sporangium formation, while a hyperparasitism-like phenomenon was found in the interaction of strain 6-4 with Pythium and Bipolaris hyphae. In conclusion, we report the promising biocontrol agent L. firmicutimachus 5-7 and the potential biocontrol agent Kitasatospora sp. 6-4 for disease control of rice seedlings in commercial nursery trays and their possible mechanisms of action.

A Large-Scale Hydroponic Evaluation of Rice Mutants for Pythium Resistance.

Rice is a major staple crop worldwide. However, the occurrence of rice diseases during cultivation poses a significant challenge to achieving optimal yields. Among the major pathogens, Pythium species, which cause seedling blight, are of particular concern. Pythium infects rice roots through zoospores, mycelia, or oospores, leading to root rot, stunting, yellowing, and ultimately seedling damping-off. While many disease resistance-related genes have been reported in rice, only very limited research has been associated with resistance to Pythium infection. In this study, we aimed to establish a rapid screening system to identify rice lines that are resistant or susceptible to Pythium pathogen in rice nurseries. We conducted evaluations on important factors, including virulence, inoculation method, seed soaking period, and the measurement of disease severity. As a result, we successfully developed a screening system that allows for high-throughput and rapid screening of the Taiwan Rice Insertional Mutant (TRIM) library for mutant lines exhibiting resistance to P. arrhenomanes. Furthermore, we identified a slightly resistant TRIM line and explored potential genes encoding endglucanase-1 precursor and malonyl-CoA decarboxylase that may be involved in conferring resistance to P. arrhenomanes.

First report of Pythium aristosporum causing root rot on rice seedling in Taiwan.

Rice (Oryza sativa L.) is the principle staple crops in the World and its production can be severely damaged by Pythium species. Several Pythium species including P. afertile, P. arrhenomanes, P. dissotocum, P. elongatum, P. spinosum, have been recorded to cause rice seedling root rot in Taiwan (List of Plant Diseases in Taiwan edited by Tzean et al., 2019). During the survey of rice seedling diseases, we identified a new species of Pythium that causes seedling root rot on rice in commercial nursery trays in two nursery fields in 2019 in Taichung, Taiwan. Stunting and root rot symptom were found on the affected plants and up to 20% seedlings in a nursery tray showed similar symptoms. To isolate the pathogen, symptomatic roots were surface sterilized with 75% ethanol for 1 min and rinsed in sterile water. The margin of lesion was cut off, placed on 1.5% water agar and incubated at 28 ℃. After 24 h, the hyphal tips of a white colony growing from the diseased region were transferred to potato dextrose agar (PDA) medium. Koch's postulates were fulfilled by inoculating the germinated rice seeds with mycelia. Rice seeds of O. sativa var. Tainan11 (TN11) were treated with 75% ethanol and then 1.2% NaOCl for 15 min. The sterilized seeds were soaked in sterile water under dark condition for 3 days and the water was replaced every day. Five of the pre-germinated seeds with 2~5 mm embryonic shoot were placed in a sterile petri-dish and inoculated with 3-ml mycelial suspension (OD600 = 0.045) prepared by blending the mycelia of a 3-days PDA culture using an Oster 10 speed blender 6640 (Oster, USA). The seeds-mycelia were then covered with sterilized soil mixture of Akadama soil and rice husk (1:1, volume to volume) and incubated in a growth chamber at 28 ℃. Seven days post-inoculation, the inoculated seedlings showed stunting with short and necrotic roots (Fig. S1). The pathogen was reisolated from the diseased seedlings and identified with morphology and molecular methods. For morphological characterization, the pathogen was cultured on V8 agar to produce oogonia and zoospore (Chamswarng and Cook 1985). Globose oogonia with multiple antheridia (1-5 per oogonium), inflated filamentous sporangia, vesicle with abundant zoospores, main hypha with up to 6.57 µm wide and mature aplerotic oospores with diameter 24.35-30.81 µm (average= 27.22 µm; n=20) were observed (Fig. S1) that are similar to the descriptions for P. aristosporum (van der Plaats-Niterink 1981). Genomic DNA was extracted with CTAB method (Wang and White 1997) and the sequences of the internal transcribed spacer (ITS) region and gene region of ß-tubulin (tub) and cytochrome c oxidase subunit II (cox II) were amplified with published primers (Villa et al., 2006). The obtained sequences were submitted to GenBank (accession nos: OL701302 (ITS), OL763269 (tub), and OL763270 (cox II); Fig. S2). Phylogenetic relationships between this Pythium pathogen and other 55 Pythium isolates, including the type species of P. aristosporum (ATCC11101), were conducted with the concatenated sequences of tub and cox II and analyzed by Bayesian interference (Fig. S3). Based on the tree built with tub and cox II sequences, this pathogen was identified as P. aristosporum that has not been reported in rice and other plants in Taiwan. It was observed in laboratory assays that this pathogen caused significant root-rot symptoms on several major rice varieties grown in Taiwan, including TN11, Tainung67 and Kaoshiung139. It may potentially cause severe crop loss in rice production, especially in nurseries. This identification provides important information on rice disease management.