Establishment of an Artificial Inoculation Method of Ustilaginoidea virens without Damaging the Rice Panicle Sheaths

Rice false smut (RFS) is a destructive disease of rice worldwide caused by Ustilaginoidea virens. There is a lack of efficient and stable artificial inoculation method to simulate the natural infection of U. virens, which is an important factor limiting further research on the disease. The purpose of this study was to establish an artificial inoculation method, which can simulate the natural infection process of U. virens without destroying the panicle sheath structure of rice. In this research, rice plants were inoculated by soaking roots at the seedling stage, spraying at the tillering stage, injecting at the booting stage, and again spraying at the flowering stage to determine the appropriate artificial inoculation time. The panicle sheath instillation method and injection inoculation method were compared. The results show that stages 6 to 8 of young panicle differentiation are an important period for U. virens infection. There were no significant differences in the mean rates of infected panicles, mean rates of infected grains, and maximum infected grains per panicle between the two inoculation methods. However, the frequency of RFS ball occurrence at the upper part of the panicles was significantly higher on the spikelets inoculated by the injection method than that of spikelets inoculated by natural infection and panicle sheath instillation. Therefore, panicle sheath instillation method was more similar to the natural infection of U. virens in the field. This research exhibited an innovative artificial inoculation method for identification of U. virens pathogenicity and evaluation of rice resistance against RFS.

OsSPL9 Regulates Grain Number and Grain Yield in Rice

Rice grain yield consists of several key components, including tiller number, grain number per panicle (GNP), and grain weight. Among them, GNP is mainly determined by panicle branches and spikelet formation. In this study, we identified a gene affecting GNP and grain yield, OsSPL9, which encodes SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) family proteins. The mutation of OsSPL9 significantly reduced secondary branches and GNP. OsSPL9 was highly expressed in the early developing young panicles, consistent with its function of regulating panicle development. By combining expression analysis and dual-luciferase assays, we further confirmed that OsSPL9 directly activates the expression of RCN1 (rice TERMINAL FLOWER 1/CENTRORADIALIS homolog) in the early developing young panicle to regulate the panicle branches and GNP. Haplotype analysis showed that Hap3 and Hap4 of OsSPL9 might be favorable haplotypes contributing to high GNP in rice. These results provide new insights on high grain number breeding in rice.

First report of Fusarium proliferatum causing Sheath Rot Disease of Rice in Eastern India

Sheath rot is one of the most devastating diseases of rice because of its ability to reduce the yield significantly in all rice cultivating areas of the world (Bigirimana et al., 2015). Sheath rot disease is associated with various pathogens such as Sarocladium oryza, Fusarium fujikuroi complex and Pseudomonas fuscovaginae (Bigirimana et al., 2015). Hence, this disease has become more complex in nature and added more seriousness. From September to December 2018, plants were observed with typical sheath rot symptoms in research farm of ICAR-National Rice Research Institute and ten farmer’s fields of Cuttack district, Odisha, Eastern India. About 25 to 37% of sheath rot disease severity was recorded in the infected field. Diseased plants were observed with symptoms such as brownish or reddish brown irregular lesions, which were later, got enlarged with grayish centers. Further, rotting of the topmost leaf sheaths that surround the young panicle was observed. At the severe stages, the young panicle was partially emerged from sheath or completely rotted within the sheath. The white to pinkish powdery growth observed inside the infected sheath leading to chaffy and discolored grains. The sheath rot symptomatic plants were collected from the infected fields. To isolate the causal pathogen, infected sheath tissues were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed three times in sterile distilled water, and placed on potato dextrose agar medium (PDA) (HiMedia). Plates were incubated at 27 ± 1° C for 3 d. Further, fungal pathogen colonies were sub-cultured and purified to perform the pathogenicity test. On PDA, the colonies produced abundant white aerial mycelium with violet to pink pigmentation and hyphae were hyaline with septation. Abundant single celled, oval shaped microcondia (5.5-9 × 1.5-2 μm) were produced, whereas macrocondia were not produced and the fungal pathogen was tentatively identified as Fusarium sp. In order to characterize the pathogen at molecular level, ITS, alpha elongation factor gene (EF1-α), RNA polymerase II largest-subunit gene (RPB2), calmodulin gene (cld) were amplified using the primer pair of ITS1/ITS4, EF1/EF2, 5F/7CR and CLPRO1/CLPRO2 respectively and PCR amplicons were subjected to sequencing (White et al. 1990; O’Donnell et al. 1998; Chang et al. 2015). Furthermore, a species-specific primer Fp3-F/Fp4-R was used to identify the pathogen (Jurado et al., 2006). The resulting sequences were confirmed by BLAST analysis and the FUSARIUM-ID database (http://isolate.fusariumdb.org). BLASTn search showed 100% similarity between the query sequence and ITS, EF1-α, RPB2, Calmodulin gene sequences of F. proliferatum available in the Genbank. The following GenBank accession numbers were obtained; MT394055 for ITS; MT439867 for EF1-α; MT790774 for calmodulin; MT940224 for RPB2 and MT801050 for species-specific to F. proliferatum. To confirm the pathogenicity under glass house conditions, fungus grown on sterilized chaffy grains were placed in between boot leaf sheath and panicle and covered with moist cotton (Saravanakumar et al., 2009). After 15 days post inoculation (dpi), rotting symptoms were observed and these were similar to that of field symptoms. Pathogen was constantly re-isolated from symptomatic tissue, satisfying Koch’s postulates. Disease symptoms were not observed on un-inoculated plants. Morphological characters, pathogenicity test and molecular characterization have identified the pathogen as F. proliferatum. To the best of our knowledge, this is the first confirmed report of F. proliferatum causing sheath rot disease on rice from Eastern India.