Integrated genetic analysis of leaf blast resistance in upland rice: QTL mapping, bulked segregant analysis and transcriptome sequencing.

Elite upland rice cultivars have the advantages of less water requirement along with high yield but are usually susceptible to various diseases. Rice blast caused by Magnaporthe oryzae is the most devastating disease in rice. Identification of new sources of resistance and the introgression of major resistance genes into elite cultivars are required for sustainable rice production. In this study, an upland rice genotype UR0803 was considered an emerging source of blast resistance. An F2 mapping population was developed from a cross between UR0803 and a local susceptible cultivar Lijiang Xintuan Heigu. The individuals from the F2 population were evaluated for leaf blast resistance in three trials 7 days after inoculation. Bulked segregant analysis (BSA) by high-throughput sequencing and SNP-index algorithm was performed to map the candidate region related to disease resistance trait. A major quantitative trait locus (QTL) for leaf blast resistance was identified on chromosome 11 in an interval of 1.61-Mb genomic region. The candidate region was further shortened to a 108.9-kb genomic region by genotyping the 955 individuals with 14 SNP markers. Transcriptome analysis was further performed between the resistant and susceptible parents, yielding a total of 5044 differentially expressed genes (DEGs). There were four DEGs in the candidate QTL region, of which, two (Os11g0700900 and Os11g0704000) were upregulated and the remaining (Os11g0702400 and Os11g0703600) were downregulated in the susceptible parent after inoculation. These novel candidate genes were functionally annotated to catalytic response against disease stimulus in cellular membranes. The results were further validated by a quantitative real-time PCR analysis. The fine-mapping of a novel QTL for blast resistance by integrative BSA mapping and transcriptome sequencing enhanced the genetic understanding of the mechanism of blast resistance in upland rice. The most suitable genotypes with resistance alleles would be useful genetic resources in rice blast resistance breeding.

Carbamoyl phosphate synthetase subunit Cpa1 interacting with Dut1, controls development, arginine biosynthesis, and pathogenicity of Colletotrichum gloeosporioides.

Carbamoyl phosphate synthetase is involved in arginine biosynthesis in many organisms. In this study, we investigate the biological function of Cpa1, a small subunit of carbamoyl phosphate synthetase of Colletotrichum gloeosporioides. The deletion of the CPA1 gene affected vegetative growth, arginine biosynthesis, and fungal pathogenicity. Genetic complementation with native CPA1 fully recovered all these defective phenotypes. We observed that Cpa1-RFP fusion protein is localized at the mitochondria, which is consistent with Cpa2, a large subunit of carbamoyl phosphate synthetase. We identified the proteins that interact with Cpa1 by using the two-hybrid screen approach, and we showed that Dut1 interacts with Cpa1 but without Cpa2 in vivo. Dut1 is dispensable for hyphal growth, appressorial formation, and fungal pathogenicity. Interestingly, the Dut1-Cpa1 complex is localized at the mitochondria. Further studies showed that Dut1 regulates Cpa1-Cpa2 interaction in response to arginine. In summary, our studies provide new insights into how Cpa1 interacts with its partner proteins to mediate arginine synthesis.

First Report of Stem Rot of Huangjing (Polygonatum sibiricum) Caused by Sclerotium rolfsii.

Huangjing (Polygonatum sibiricum) is a medicinal plant widely distributed in China, Japan, and Korea. The dried rhizome of Huangjing has been reported to have many pharmacological applications and biological activities, such as antioxidants, immunity enhancement, anti-fatigue, anti-osteoporosis, and anti-aging activity (Cui et al., 2018). In June 2018, we observed some wilted Huangjing plants in commercial plantings in Shuicheng, Guizhou, China (26.22 N, 104.76 E). Symptoms began as moderate to severe wilting of stems and necrosis of leaves, followed by the death of plants. The collar rot appeared on the stem near to the soil. When incubated at 28°C and 100% relative humidity (RH) for 8 to 10 days, the infected stem produced brown sclerotia. We picked the sclerotia and cultured them on potato dextrose agar (PDA) supplemented with 50 µg/ml of streptomycin. The hyphal tips generated by the sclerotia was isolated under microscopic field and transferred to the fresh PDA. Three isolates (HJ-1, HJ-6 and HJ-10) came from the hyphal tips formed the typical clamp connection structure at 6-7 days post-incubation and the sclerotia of them were white and the late ones turned dark brown. The matured sclerotia were globular, 1.5 to 3.3 mm (avg. 2.2) in diameter. The morphologic observation revealed that three isolates were consistent with Athelia rolfsii (Paul et al., 2017). To further confirm the fungal species, the ribosomal internal transcribed spacer (ITS) sequences were amplified and sequenced. Primers and PCR amplification were referenced as previously described (Paul et al., 2017). The sequences were compared to type sequences in GenBank. The ITS sequences (GenBank accession MT478452, MT949696 and MT949697) of the isolates (HJ-10, HJ-1 and HJ-6) were 99% identical with strain 13M-0091 (GenBank accession KT222898) of A. rolfsii, respectively (Paul et al., 2017). A maximum likelihood tree was constructed using MEGA-X version 10.1.6 (Kumar et al., 2018) based on the ITS sequences of the three strains (HJ-10, HJ-1 and HJ-6) and that of Athelia spp. previously deposited in GenBank (Paul et al., 2017). Phylogenetic analysis showed that the isolates (HJ-10, HJ-1 and HJ-6) belong to the A. rolfsii clade. Based on morphology and DNA sequencing, the isolates (HJ-10, HJ-1 and HJ-6) were identified as A. rolfsii. To verify pathogenicity, Huangjing seedlings were inoculated with colonized agar discs of the isolates. Additional Huangjing plants inoculated with uncolonized agar discs were used as the control. After inoculation, Huangjing seedlings were moved to the inoculation chamber under high humidity and 28°C for 3 days and then transferred to a greenhouse. The typical wilting symptoms appeared 8 days after inoculation and were similar to those observed in the field, while control plants remained symptomless. The causing agents were isolated from the lesions and the ITS sequences of them were sequenced again. The alignment analysis of the ITS sequences showed the causing agents are consistent with the original isolates. These studies fulfilled Koch's postulates. To our knowledge, this is the first report of A. rolfsii causing stem rot on Huangjing.