Development of a sensitive nested-polymerase chain reaction (PCR) assay for the detection of Ustilago scitaminea

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 detectio

Genetic diversity of Ustilago scitaminea Syd. in Southern China revealed by combined ISSR and RAPD analysis

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

Digital Twin Driven Green Performance Evaluation Methodology of Intelligent Manufacturing: Hybrid Model Based on Fuzzy Rough-Sets AHP, Multistage Weight Synthesis, and PROMETHEE II

The design, planning, and implementation of intelligent manufacturing are mainly carried out from the perspectives of meeting the needs of mass customization, improving manufacturing capacity, and innovating business pattern currently. Environmental and social factors should be systematically integrated into the life cycle of intelligent manufacturing. In view of this, a green performance evaluation methodology of intelligent manufacturing driven by digital twin is proposed in this paper. Digital twin framework, which constructs the bidirectional mapping and real-time data interaction between physical entity and digital model, provides the green performance evaluation with a total factor virtual image of the whole life cycle to meet the monitoring and simulation requirements of the evaluation information source and demand. Driven by the digital twin framework, a novel hybrid MCDM model based on fuzzy rough-sets AHP, multistage weight synthesis, and PROMETHEE II is proposed as the methodology for the green performance evaluation of intelligent manufacturing. The model is tested and validated on a study of the green performance evaluation of remote operation and maintenance service project evaluation for an air conditioning enterprise. Testing demonstrates that the proposed hybrid model driven by digital twin can enable a stable and reasonable evaluation result. A sensitivity analysis was carried out by means of 27 scenarios, the results of which showed a high degree of stability

Interfacial Microstructure and Mechanical Properties of Titanium/Sapphire Joints Brazed with AuSn20 Filler Metal

In this study, C-plane (0001) sapphire was successfully brazed to titanium using AuSn20 filler metal, following metallization on the surface of the sapphire with Sn-3Ti (wt.%). At 1000 °C, Sn-3Ti had good wettability on the surface of the sapphire, with the lowest equilibrium contact angle of 57°. The reaction phases in the joints were identified, and the typical interfacial microstructure of the brazed joint brazed at 550 °C for 30 min was titanium substrate/Au-Sn-Ti layer/Ti6Sn5 + AuSn2 + AuSn4 + massive Au-Sn-Ti/TiO phase/sapphire. The shear test was utilized to evaluate the bonding strength of the titanium/sapphire joints. The highest shear strength reached 18.7 MPa when brazed at 550 °C for 35 min. The crack was initiated at the sapphire/brazing seam interface and propagated into the Au-Sn-Ti reaction layer

Interfacial Microstructure and Mechanical Properties of Titanium/Sapphire Joints Brazed with AuSn20 Filler Metal

In this study, C-plane (0001) sapphire was successfully brazed to titanium using AuSn20 filler metal, following metallization on the surface of the sapphire with Sn-3Ti (wt.%). At 1000 °C, Sn-3Ti had good wettability on the surface of the sapphire, with the lowest equilibrium contact angle of 57°. The reaction phases in the joints were identified, and the typical interfacial microstructure of the brazed joint brazed at 550 °C for 30 min was titanium substrate/Au-Sn-Ti layer/Ti6Sn5 + AuSn2 + AuSn4 + massive Au-Sn-Ti/TiO phase/sapphire. The shear test was utilized to evaluate the bonding strength of the titanium/sapphire joints. The highest shear strength reached 18.7 MPa when brazed at 550 °C for 35 min. The crack was initiated at the sapphire/brazing seam interface and propagated into the Au-Sn-Ti reaction layer

Hypha morphology of WT, mutants Δ<i>FoSlt2</i>, Δ<i>FoMkk2</i> and Δ<i>FoBck1</i>, and complemented strains Δ<i>FoSlt2</i>-c and Δ<i>FoMkk2</i>-c.

<p>Strains were incubated at 28°C for 4 days on PDA plates. A: Δ<i>FoSlt2</i>, B: Δ<i>FoMkk2</i>, C: Δ<i>FoBck1</i>, D: WT, E: Δ<i>FoSlt2</i>-c, F: Δ<i>FoMkk2</i>-c. Bars: 10μm.</p