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Sugarcane smut is a serious disease caused by Sporisorium scitamineum, which causes significant losses to the sugar industry. It is critical to reveal the molecular pathogenic mechanism of S. scitamineum to explore a new control strategy for sugarcane smut. On the basis of transcriptome sequencing data of two S. scitamineum strains with different pathogenicity, we identified the gene, SsCI51640, which was predicted to encode kynurenine 3-monooxygenase. In this study, we obtained knockout mutants and complementary mutants of this gene and identified gene function. The results showed that the sporidial growth rate and acid production ability of knockout mutants were significantly higher and stronger than those of the wild-type and complementary mutants. The growth of knockout mutants under abiotic stress (osmotic stress and cell wall stress) was significantly inhibited. In addition, the sexual mating ability and pathogenicity of knockout mutants were significantly reduced, while this phenomenon could be restored by adding exogenous cyclic adenosine monophosphate (cAMP). It is thus speculated that the SsCI51640 gene may regulate sexual mating and pathogenicity of S. scitamineum by the cAMP signaling pathway. Moreover, the SsCI51640 gene enhanced the sporidial environmental adaptability, which promoted sexual mating and development of pathogenicity. This study provides a theoretical basis for the molecular pathogenesis of S. scitamineum.

Original pictures of agarose gel electrophoresis

Sugarcane is an important sugar crop and energy crop worldwide. Sugarcane smut caused by

Sugarcane mosaic disease (SMD) is one of the most destructive sugarcane diseases that occur around the world. Sugarcane streak mosaic virus (SCSMV) belongs to the genus Poacevirus of the family Potyviridae, and is a novel pathogen that causes SMD. The pathogen spreads primarily through infected sugarcane setts, and hence the use of disease-free planting materials is essential to prevent disease development in the field. In the present study, a species-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid and accurate detection of SCSMV. Based on the conserved polyprotein gene nucleotide sequences of SCSMV isolates that differ from Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV), four SCSMV specific primers, namely, P2-F3, P2-B3, P2-FIP, and P2-BIP, were designed and screened, using a panel of sugarcane viruses as controls. After optimization of the reaction conditions, the detection sensitivity of the RT-LAMP assay was 10-fold more sensitive than the conventional RT-PCR. The assay showed a high specificity in discriminating SCSMV from common viral pathogens of sugarcane such as SCMV, SrMV, Sugarcane yellow leaf virus (SCYLV), and sugarcane bacilliform virus (SCBV). The RT-LAMP assay also detected SCSMV in young sugarcane leaves with no visible sugarcane mosaic disease symptoms. The findings of this study provide a visual, simple, highly sensitive, rapid, and reliable technique for the detection of SCSMV, a tool that is useful for sugarcane quarantine and production of SCSMV-free seedcane. This is the first report of a RT-LAMP-based assay for the detection of SCSMV in sugarcane.

The life cycle of the sugarcane smut fungus Sporisorium scitamineum is a multistep process. Haploid sporidia of compatible (MAT-1 versus MAT-2) mating types fuse to generate pathogenic dikaryotic hyphae to infect the host. Within the host tissues, diploid teliospores are formed and induce a characteristic sorus that looks like a black whip. The diploid teliospores germinate to form haploid sporidia by meiosis. In order to monitor fungal development throughout the whole life cycle, we expressed the green fluorescent protein (GFP) and red fluorescent protein (RFP) in S. scitamineum MAT-1 and MAT-2 sporidia, respectively. Observation by epifluorescence microscope showed that conjugation tube formation and sporidia fusion occurred at 4 to 8 h, and formation of dikaryotic filaments was detected at 12 h after mating. The resultant teliospores, with diffused GFP and RFP, underwent meiosis as demonstrated by septated hypha with single fluorescent signal. We demonstrated that GFP- and RFP-tagged strains can be used to study the life cycle development of the fungal pathogen S. scitamineum, including the sexual mating and meiosis events. This dual-color imaging system would be a valuable tool for investigation of biotic and abiotic factors that might affect the fungal life cycle development and pathogenesis.

Downy blight caused by Peronophythora litchii is one of the most destructive diseases of lychee (Litchi chinensis Sonn.). Early diagnosis of the P. litchii pathogen should be the top priority for addressing disease epidemics and management. In this study, loop-mediated isothermal amplification (LAMP) assays targeting the M90 gene sequences were developed for specific detection of P. litchii. The detection limit of LAMP assay was 10 pg genomic DNA of P. litchii in 25 μL reaction mixtures. LAMP assay and nested polymerase chain reaction (PCR) assay showed higher sensitivity than conventional PCR. The LAMP assay could also detect the presence of P. litchii in infected leaves, fruits and soils. This study provides a simple, highly sensitive, rapid and reliable protocol for the early diagnosis of P. litchii on lychee.

The genetic relationships among 90 mating-type isolates of Sporisorium scitamineum collected from mainland China were evaluated based on the start codon-targeted (SCoT) marker technique. Fourteen selected SCoT primers amplified 135 DNA fragments, of which 98 (72.6 %) were polymorphic. The polymorphic information content values ranged from 0.649 to 0.910 with an average of 0.820, indicating that a high degree of genetic diversity exists among these Chinese S. scitamineum isolates. Cluster groups identified using the unweighted pair-group method with arithmetic mean (UPGMA) and principal component analysis (PCA) were similar and divided the 90 S. scitamineum isolates into three main groups (G1, G2, and G3) based on a Jaccard’s similarity coefficient of 0.74. The S. scitamineum isolates collected from the same province tended to cluster in the same main groups. This study showed that the genetic diversity of the 90 S. scitamineum isolates was associated with their geographic origin, with no evidence of coevolution between the host and S. scitamineum. The knowledge of genetic diversity of S. scitamineum is useful in breeding programs for development of smut-resistant sugarcane genotypes. The results of this study demonstrate that SCoT markers are informative and can be used to evaluate the genetic diversity of S. scitamineum.

Smut disease caused by Sporisorium scitamineum is one of the most destructive sugarcane diseases worldwide. The pathogen spreads primarily through infected sugarcane setts, and hence, the use of disease-free planting materials is essential for preventing disease development in the field. In this study, a species-specific loop-mediated isothermal amplification (LAMP) assay was developed for rapid and accurate detection of S. scitamineum. Based on the differences in internal transcribed spacer (ITS) sequences of S. scitamineum, a set of four species-specific primers, F3, B3, FIP and BIP, were designed by using a panel of fungal and bacterial species as controls. After optimization of the reaction conditions, the detection limit of LAMP assay was about 2 fg of the S. scitamineum genomic DNA in 25 µL reaction solution, 100-fold lower than that of conventional polymerase chain reaction. The assay showed high specificity to discriminate all S. scitamineum isolates from nine other fungal and bacterial pathogens. The LAMP assay also detected smut infection from young sugarcane leaves with no visible smut-disease symptoms. The findings from this study provide a simple, highly sensitive, rapid and reliable technique for early detection of S. scitamineum, which may be useful for sugarcane quarantine and production of smut-free seedcanes. This is the first report of LAMP-based assay for the detection of S. scitamineum in sugarcane.

A species-specific polymerase chain reaction (PCR) assay was developed for rapid and accurate detection of Ustilago scitaminea, the causal agent of sugarcane smut disease. Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of U. scitaminea, a pair of species-specific primers, SL1 (5`-CAGTGCACGAAAGTACCTGTGG-3`) and SR2 (5`-CTAGGGCGGTGTTCAGAAGCAC-3`) was designed by using a panel of fungal and bacterial species as controls. The primers SL1/SR2 specifically amplified a unique PCR product about 530 bp in length from U. scitaminea strains with a detecting sensitivity at 200 fg of the fungal genomic DNA in a 25 μl reaction solution. To increase sensitivity, a nested-PCR protocol was further established, which used ITS4/ITS5 as the first-round primers followed by the primer pair SL1/SR2. This protocol increased the detection sensitivity by 10,000-fold compared to the PCR method and could detect the fungal DNA as low as 20 ag. The nested-PCR detected U. scitaminea from young sugarcane leaves with no visible smut disease symptoms. The findings from this study provide a sensitive and reliable technique for the early detection of U. scitaminea, which would be useful for sugarcane quarantine and production of germ-free seedcanes.Keywords: Sugarcane, Ustilago scitaminea, nested-polymerase chain reaction (PCR), molecular detection

Sporisorium scitamineum is the causal agent of sugarcane smut, which is one of the most serious constraints to global sugarcane production. S. scitamineum and Ustilago maydis are two closely related smut fungi, that are predicted to harbor similar sexual mating processes/system. To elucidate the molecular basis of sexual mating in S. scitamineum, we identified and deleted the ortholog of mating-specific U. maydis locus b, in S. scitamineum. The resultant b-deletion mutant was defective in mating and pathogenicity in S. scitamineum. Furthermore, a functional b locus heterodimer could trigger filamentous growth without mating in S. scitamineum, and functionally replace the b locus in U. maydis in terms of triggering aerial filament production and forming solopathogenic strains, which do not require sexual mating prior to pathogenicity on the host plants.

Sugarcane smut disease caused by the fungus Sporisorium scitamineum is one of the important fungal diseases affecting sugarcane yield and sucrose content around the world. Cultivar resistance is the most appropriate control method for this disease. In this study, 37 BC1 lines chosen from the crossing YC96-40 (F1 of Erianthus arundinaceus) × CP84-1198 (commercial sugarcane cultivar) and 42 BC2 lines chosen from the crossing YCE01-116 (BC1 of E. arundinaceus) × Neijiang57-416 (commercial sugarcane cultivar) were evaluated for smut resistance using artificial inoculation. The results showed that of 79 tested BC1 and BC2 lines of E. arundinaceus, 10 (12.7%) were highly to moderately resistant to smut. BC1 of E. arundinaceus had more resistant lines than BC2 of E. arundinaceus. Of the 37 tested BC1 lines of E. arundinaceus, seven (18.9%) were highly to moderately resistant, while three (7.1%) of the 42 tested BC2 lines of E. arundinaceus were highly to moderately resistant to smut. The resistant lines identified in this study could be used as sources of smut resistance in sugarcane breeding programs.

The polymorphism and similarity relationships among 35 mating-type isolates of Ustilago scitaminea collected from Southern China were determined with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses. These fungal isolates were collected from 16 sugarcane cultivars including F134 that is resistant to the physiological race 1 but susceptible to the race 2 of U. scitaminea, and N: Co376 that is immune to both races 1 and 2. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis revealed that the U. scitaminea isolates could be divided into 2 groups with a coefficient of 0.74. The first group comprises two isolates collected from the sugarcane cultivar F134, while the remaining 33 isolates were clustered into the second group. The second group was further divided into two subgroups with most of the isolates from Guangdong Province which clustered in the same subgroup, and all the isolates from Guangxi and Yunnan Provinces were clustered in another subgroup. Given that the member of the second group could infect the cultivar N:Co376, which is immune to the races 1 and 2, our results suggest that majority of U. scitaminea in sugarcane-producing regions of Southern China may belong to or genetically similar to race 3. Key words: Ustilago scitaminea , sugarcane, inter-simple sequence repeat (ISSR), random amplified polymorphic deoxyribonucleic acid (DNA) (RAPD), genetic diversity.

Sugarcane improvement has traditionally been conducted by nationally focussed breeding programs, with some exchange of elite cultivars among some programs. It has been generally assumed by breeders that genotype × country interactions are large, and therefore selection trial data in one country may be of limited relevance to another. However, no studies quantifying clone × country interactions have been reported to our knowledge. In research reported here, a common set of mostly unselected sugarcane genotypes was evaluated in commercial production environments in Australia and China. Consistent with past studies, genotype × site interaction was an important source of variation within each country. Overall a moderate to high genetic correlation existed between production environments in China and Australia for cane yield and sugar content (0.77 for both traits). This suggests that despite difference in environmental conditions and crop management in production environments between countries, that selection trials in China have some relevance for selecting clones for Australian environments and vice versa. It also supports the hypothesis that regular exchange of selected germplasm from effective breeding programs between countries will have mutual benefits.