Application of bulk segregant RNA-Seq (BSR-Seq) and allele-specific primers to study soybean powdery mildew resistance.

BACKGROUND: Powdery mildew (PM) is one of the important soybean diseases, and host resistance could practically contribute to soybean PM management. To date, only the Rmd locus on chromosome (Chr) 16 was identified through traditional QTL mapping and GWAS, and it remains unclear if the bulk segregant RNA-Seq (BSR-Seq) methodology is feasible to explore additional PM resistance that might exist in other varieties. RESULTS: BSR-Seq was applied to contrast genotypes and gene expressions between the resistant bulk (R bulk) and the susceptible bulk (S bulk), as well as the parents. The ∆(SNP-index) and G' value identified several QTL and significant SNPs/Indels on Chr06, Chr15, and Chr16. Differentially expressed genes (DEGs) located within these QTL were identified using HISAT2 and Kallisto, and allele-specific primers (AS-primers) were designed to validate the accuracy of phenotypic prediction. While the AS-primers on Chr06 or Chr15 cannot distinguish the resistant and susceptible phenotypes, AS-primers on Chr16 exhibited 82% accuracy prediction with an additive effect, similar to the SSR marker Satt431. CONCLUSIONS: Evaluation of additional AS-primers in the linkage disequilibrium (LD) block on Chr16 further confirmed the resistant locus, derived from the resistant parental variety 'Kaohsiung 11' ('KS11'), not only overlaps with the Rmd locus with unique up-regulated LRR genes (Glyma.16G213700 and Glyma.16G215100), but also harbors a down-regulated MLO gene (Glyma.16G145600). Accordingly, this study exemplified the feasibility of BSR-Seq in studying biotrophic disease resistance in soybean, and showed the genetic makeup of soybean variety 'KS11' comprising the Rmd locus and one MLO gene.

First Report of False Rust caused by Synchytrium psophocarpi on Winged Bean (Psophocarpus tetragonolobus) in Taiwan.

From September 2020 to January 2021, an unknown disease of winged bean (Psophocarpus tetragonolobus) was reported by local growers in the Toucheng Town, Yilan County (N24.91, E121.85). The disease occurs in all age of winged bean, and the occurrence tended to be higher in humid environment, such as branches in lower canopy or branches in high density. The disease symptoms, which also appeared to be the sign of the pathogen, were spherical pustules in yellow to orange color on the stems, leaves, and pods of winged bean. Severely infected plants also exhibited growth reduction, malformation, and curling of the leaves and pods. According to the disease literature of winged bean, this unknown disease was likely to be the false rust caused by a chytrid pathogen, Synchytrium psophocarpi (UK, CAB International. 1993); and the uredinia-liked pustules could be the sori, which contain numerous ovoid to globose sporangia inside. In order to characterize the pathogen identity, the sori were manually ruptured to assess the size of individual sporangium, which had an average of 26.71 ± 4.25 µm x 26.61 ± 4.60 µm (n=42), similar to the size reported in literature (Drinkall and Price. 1979). To confirm the molecular identity, the full genomic sequences from the small subunit (SSU) to the internal transcribed spacer-1 (ITS-1), 5.8S unit, and ITS-2 were amplified using the primer sets NS3 and ITS4. The 2,263 bp amplicon was cloned and sequenced to reveal the identity (Smith et al. 2014). The BLASTN results matched the SSU of our isolate (MW649126.1) to the Synchytrium minutum (HQ324138.1) with 96% similarity (1,075 out of 1,121 bp in length), Synchytrium decipiens isolate DAOM_87618 (KF160868.1) with 92% similarity (1,215 out of 1,326 bp in length) and S. decipiens isolate AFTOL-ID 634 (DQ536475.1) with 92% similarity (1210 out of 1316 bp in length). Phylogenetic analysis using the SSU sequence revealed this unknown pathogen was the grouped within the clade of Synchytrium genus with 100% bootstrapping confidence (Smith et al. 2014). Accordingly, the pathogen was confirmed to be a Synchytrium chytrid fungus. To complete the Koch's postulates, the sori were collected from infected tissue. After vortexing washing in 1% bleach for surface sterilization, the sori were gently crashed by a plastic tube pestle to harvest sporangia. The sporangia were sprayed onto healthy winged beans cultivated in pots, and the inoculated plants were kept in a moisture bag in 25 °C. While leaf curling and malformation could be observed about 14 days post inoculation, the yellow to orange sori could be observed around 30 to 40 days post inoculation on the whole plants cultivated in pots. The sori were collected to confirm the sporangia and the sequences were identical to the original pathogen. Collectively, this study not only presents the first report for the false rust of winged bean in Taiwan, but also documents the first reference sequence of S. psophocarpi that will be useful for future molecular diagnosis. Since S. psophocarpi has been only reported in tropic regions including Indonesia, Malay Peninsula, Malaysia, Papua New Guinea, and Philippines, this report provides the first observation of S. psophocarpi moving in the subtropic region.

Superoxide Initiates the Hyphal Differentiation to Microsclerotia Formation of Macrophomina phaseolina.

The infection of Macrophomina phaseolina often results in a grayish appearance with numerous survival structures, microsclerotia, on the plant surface. Past works have studied the development of fungal survival structures, sclerotia and microsclerotia, in the Leotiomycetes and Sordariomycetes. However, M. phaseolina belongs to the Dothideomycetes, and it remains unclear whether the mechanism of microsclerotia formation remains conserved among these phylogenetic clades. This study applied RNA-sequencing (RNA-Seq) to profile gene expressions at four stages of microsclerotia formation, and the results suggested that reactive oxygen species (ROS)-related functions were significantly different between the microsclerotia stages and the hyphal stage. Microsclerotia formation was reduced in the plates amended with antioxidants such as ascorbic acid, dithiothreitol (DTT), and glutathione. Surprisingly, DTT drastically scavenged H2O2, but the microsclerotia amount remained similar to the treatment of ascorbic acid and glutathione that both did not completely eliminate H2O2. This observation suggested the importance of [Formula: see text] over H2O2 in initiating microsclerotia formation. To further validate this hypothesis, the superoxide dismutase 1 (SOD1) inhibitor diethyldithiocarbamate trihydrate (DETC) and H2O2 were tested. The addition of DETC resulted in the accumulation of endogenous [Formula: see text] and more microsclerotia formation, but the treatment of H2O2 did not. The expression of SOD1 genes were also found to be upregulated in the hyphae to the microsclerotia stage, which suggested a higher endogenous [Formula: see text] stress presented in these stages. In summary, this study not only showed that the ROS stimulation remained conserved for initiating microsclerotia formation of M. phaseolina but also highlighted the importance of [Formula: see text] in initiating the hyphal differentiation to microsclerotia formation. IMPORTANCE Reactive oxygen species (ROS) have been proposed as the key stimulus for sclerotia development by studying fungal systems such as Sclerotinia sclerotiorum, and the theory has been adapted for microsclerotia development in Verticillium dahliae and Nomuraea rileyi. While many studies agreed on the association between (micro)sclerotia development and the ROS pathway, which ROS type, superoxide ([Formula: see text]) or hydrogen peroxide (H2O2), plays a major role in initiating hyphal differentiation to the (micro)sclerotia formation remains controversial, and literature supporting either [Formula: see text] or H2O2 can be found. This study confirmed the association between ROS and microsclerotia formation for the charcoal rot fungus Macrophomina phaseolina. Moreover, the accumulation of [Formula: see text] but not H2O2 was found to induce higher density of microsclerotia. By integrating transcriptomic and phenotypic assays, this study presented the first conclusive case for M. phaseolina that [Formula: see text] is the main ROS stimulus in determining the amount of microsclerotia formation.

First Report of Leaf Spot Caused by Diaporthe tulliensis on Boston Ivy (Parthenocissus tricuspidata) in Taiwan.

Starting from the May to August 2020 (average humidity 76.6% and temperature 25.2°C in Taipei), Boston ivy (Parthenocissus tricuspidata) plants on the campus of National Taiwan University (25°01'05.4"N 121°32'36.6"E) exhibited leaf rusts caused by Phakopsora ampelopsidis (Tzean et al., 2019) and leaf spots caused by an unknown pathogen. The leaf spots appeared reddish to brown color and mostly irregular to round shape on the simple and trifoliate leaflets (Supplemental Figure 1A-C). The leaf spots were surface-disinfected with 1% NaOCl for 30 seconds, and the margin of healthy and infected tissues was cut and placed onto water agar, which were incubated at room temperature. Hyphae grown out from leaf spots were sub-cultured on potato dextrose agar (PDA), and the majority of isolates exhibited white colony with black pycnidial conidiomata embedded in PDA. The pycnidial conidiomata of two-week-old has an average diameter of 463±193 µm (n=30) and the sizes of α-conidia were 5.71±0.49 µm in length and 2.42±0.32 µm in width (n=50) similar to the previous records (Crous et al. 2015). The α-conidium was one-celled, hyaline, and ovoid with two droplets (Supplemental Figure 1D-G). This putative pathogen was re-inoculated to confirm its pathogenicity on the leaves of Boston ivy plants. A PDA block with actively growing fungal edge was placed on the tiny needle-wounded leaves of detached branches (Supplemental Figure H-I) and the whole plants in pots (Supplemental Figure 1J-M) in a moist chamber at 28°C in dark. Reddish to brown leaf spots were observed by 2 days post-inoculation (dpi) and the leaf spots expanded by 5 dpi. To complete the Koch's postulates, the pathogen was re-isolated from inoculated leaves and the re-isolated pathogen exhibited identical morphology to the original isolate. The internal transcribed spacer (ITS), translational elongation factor subunit 1-α gene (EF1α), ß-tubulin (BT), and calmodulin (CAL) was amplified using the primers ITS1/ITS4 (Martin and Rygiewicz. 2005), EF1-728F/EF1-986R, Bt2a/Bt2b, and CAL-228F/CAL-737R, respectively (Manawasinghe et al. 2019). Using BLAST in the NCBI database, the ITS (MT974186), EF1α (MT982963), and ß-tubulin (MT982962) sequences showed 98.57% (NR_147574.1, 553 out of 561 bp), 98.04% (KR936133.1, 350 out of 357 bp), and 99.23% (KR936132.1, 518 out of 522 bp) identity to the Diaporthe tulliensis ex-type BRIP 62248a, respectively (Dissanayake et al. 2017). Phylogenetic analysis using concatenated sequences of ITS, EF1α, and ß-tubulin grouped the D. tulliensis isolated from Boston ivy leaf spots with the D. tulliensis ex-type (Supplemental Figure 1N). In summary, the morphological and molecular characterizations supported the causal pathogen of Boston ivy leaf spot as D. tulliensis. While Diaporthe ampelopsidis was reported to infect Parthenocissus quinquefolia and P. tricuspidata (Anonymous, 1960; Wehmeyer, 1933), there is no record for D. tulliensis infecting Boston ivy according to the USDA National Fungus Collections (Farr and Rossman. 2020). Because pathogens of Boston ivy such as P. ampelopsidis may also infect close-related crops like grape (Vitis vinifera L.) and D. tulliensis has been known to infect kiwifruits (Actinidia chinensis) and cocoa (Theobroma cacao) (Bai et al. 2016; Yang et al. 2018), the emergence of D. tulliensis should be aware to avoid potential damage to economic crops.

In Situ Cleaning and Fluorination of Black Phosphorus for Enhanced Performance of Transistors with High Stability.

Most two-dimensional (2D) semiconductors suffer from intrinsic instability under ambient conditions, especially 2D black phosphorus (BP). Although much effort has been made to study the passivation of 2D materials against corrosion by oxygen and water molecules, facile and effective passivation with long-term stability is still challenging; in particular, selective passivation, which is critical for integration into nanoelectronics, is still lacking. Here, we develop a novel passivation route for BP using a fluorinated self-assembled thin film of PFSA (perfluorosulfonic acid, PFSA), where the surface modifier with high hydrophobicity on BP presents extremely stable characteristics over five months under ambient conditions. Moreover, we report for the first time in situ cleaning and selective fluorination of only BP flakes on a SiO2/Si substrate by a spin-coating process followed by ultrasonication, which was attributed to the formation of P-F covalent bonds on the BP surface. Selectively fluorinated BP shows not only enhanced stability in air but also electrical properties of the BP field-effect transistor (FET), with the on-current of the BP FET increasing and presenting enhanced carrier mobility (125 cm2 V-1 s-1) and on/off ratio (104). This significant finding sheds light on fabricating vertical 2D heterostructures to realize high performance and reliability with versatile 2D materials. This work demonstrates an emerging passivation approach for long-term stability together with superior electrical properties, which paves the way for integrating 2D semiconductors into critical channel materials in FETs that are favorable for next-generation digital logic circuits.

Pool Boiling Heat Transfer Enhanced by Fluorinated Graphene as Atomic Layered Modifiers.

Graphene has been applied to thermal technology including boiling and condensation heat transfer, from which the pool boiling enhancement relies on adjusting the surface morphology and wettability that is favorable to catalyze the vaporization on the fluid/graphene interface. However, previous works using graphene or reduced graphene oxide (RGO) flake coatings, where the morphology of graphene coating is nonuniform and most of the underlying structured cavities are sealed by graphene flakes. For a long time, this hampered the unraveling of the mechanism behind the enhanced boiling performance by graphene coatings. Moreover, the previous work relied on using water-based pool boiling, which limits the scope of its practical applications since the versatile nonpolar refrigerant has been widely used in boiling heat transfer. The pool boiling was carried out on a plain copper surface to study the effect of fluorinated graphene (F-graphene) coating using nonpolar refrigerant R-141b as the working fluid along with bubble dynamic visualization. It was found that the increase of contact angle leads to more active cavities and enhances heat transfer performance up to twice as much, by applying the F-graphene coating. Moreover, the mechanism of graphene-enhanced heat transfer performance was unraveled and mainly attributed to the hydrophobic surface and effective cavity structure. This research provides a practical and reliable route for enhancing the heat transfer through F-graphene-coatings, which paves the way for potential application in graphene-based thermal technologies.

Spectroscopic and Electrical Characterizations of Low-Damage Phosphorous-Doped Graphene via Ion Implantation.

Development of n-/p-type semiconducting graphenes is a critical route to implement in graphene-based nanoelectronics and optronics. Compared to the p-type graphene, the n-type graphene is more difficult to be prepared. Recently, phosphorous doping was reported to achieve air-stable and high mobility of n-typed graphene. The phosphorous-doped graphene (P-Gra) by ion implantation is considered as an ideal method for tailoring graphene due to its IC compatible process; however, for a conventional ion implanter, the acceleration energy is in the order of kiloelectron volts (keV), thus severely destroys the sp2 bonding of graphene owing to its high energy of accelerated ions. The introduced defects, therefore, degrade the electrical performance of graphene. Here, for the first time, we report a low-damage n-typed chemical vapor deposition (CVD) graphene by an industrial-compatible ion implanter with an energy of 20 keV where the designed protection layer (thin Au film) covered on as-grown CVD graphene is employed to efficiently reduce defect formation. The additional post-annealing is found to heal the crystal defects of graphene. Moreover, this method allows transferring ultraclean and residue-free P-Gra onto versatile target substrates directly. The doping configuration, crystallinity, and electrical properties on P-Gra were comprehensively studied. The results indicate that the low-damaged P-Gra with a controllable doping concentration of up to 4.22 at % was achieved, which is the highest concentration ever recorded. The doped graphenes with tunable work functions (4.85-4.15 eV) and stable n-type doping while keeping high-carrier mobility are realized. This work contributes to the proof-of-concept for tailoring graphene or 2D materials through doping with an exceptional low defect density by the low energy ion implantation, suggesting a great potential for unconventional doping technologies for next-generation 2D-based nanoelectronics.

Solution-processed black phosphorus nanoflakes for integrating nonvolatile resistive random access memory and the mechanism unveiled.

In this study, we demonstrated the integration of black phosphorus (BP) nanoflakes in a resistive random access memory (RRAM) with a facile and complementary metal-oxide-semiconductor-compatible process. The solution-processed BP nanoflakes embedded in polystyrene (PS) as an active layer were sandwiched between aluminum electrodes (Al/BP:PS/Al). The device shows a figure of merit with typical bipolar behavior and forming-free characteristics as well as excellent memory performances such as nonvolatile, low operation voltage (1.75 V) and high ON/OFF ratio (>102) as well as the long retention time (>1500 s). The improved device performances were attributed to the formation of effective trap sites from the hybrid structure of the active layer (BP:PS), especially the BP nanoflakes and the partly oxidized species (P x O y ). Moreover, the extrinsic aluminum oxide layer was observed after the device operation. The mechanism of switching behavior was further unveiled through the carrier transport models, which confirms the conductive mechanisms of space-charge-limited current and Ohmic conductance at high resistance state (HRS) and low resistance state, respectively. Additionally, in the high electric field at HRS, the transfer curve was well fitted with the Poole-Frenkel emission model, which could be attributed to the formation of the aluminum oxide layer. Accordingly, both the trapping/de-trapping of carriers and the formation/rupture of conductive filaments were introduced as transport mechanisms in our devices. Although the partial P x O y species on BP were inevitable during the liquid phase exfoliation process, which was regarded as the disadvantages for various applications, it turns to a key point for improving performances in memory devices. The proposed approach to integrating BP nanoflakes in the active layer of the RRAM device could pave the way for next-generation memory devices.

Revisiting graphene-polymer nanocomposite for enhancing anticorrosion performance: a new insight into interface chemistry and diffusion model.

Graphene is impermeable to all molecules and has high chemical stability, which makes it an excellent anticorrosion coating for metals. However, current studies have indicated that galvanic coupling between graphene and a metal actually accelerates corrosion at the interface. Due to the insulating nature of polymers, graphene-polymer composite coatings with a strong interaction between the filler and the polymer matrix are an alternative means of addressing this issue. Nevertheless, such coatings require well-dispersed graphene flakes to lengthen the diffusion paths of gases or liquids, while preventing the formation of a conducting network from graphene to the metal. The difficulty in preparing such coatings was mainly due to problems with the control of the assembled phase during interfacial reactions. Herein, the interactions between the filler and the polymer were found to be a key factor governing anticorrosion performance, which has scarcely been previously reported. The advantage of graphene as a filler in anticorrosion coatings lies in its dispersibility and miscibility with both the casting solvent and the polymer. Electrochemically exfoliated graphene (EC-graphene) with appropriate surface functionalities that allow high miscibility with waterborne polyurethane (PU) and hydrophobic epoxy has been found to be an ideal filler that outperforms other graphene materials such as graphene oxide (GO) and reduced graphene oxide (rGO). Furthermore, a bilayer coating with EC-graphene additives for PU over epoxy has been found to reduce the corrosion rate (CR) to 1.81 × 10-5 mm per year. With a graphene loading of less than 1%, this represents the lowest CR ever achieved for copper and steel substrates and a diffusion coefficient that is lower by a factor of nearly 2.2 than that of the pristine polymer. Furthermore, we have shown that by controlling the amount of graphene loaded in the polymer galvanic corrosion favored by the formation of an interconnected graphene percolation network can successfully be limited. The present study, together with a facile and eco-friendly method of nanocomposite synthesis, may pave the way toward practical applications in the development of graphene-based anticorrosion coatings.

Ultra-large suspended graphene as a highly elastic membrane for capacitive pressure sensors.

In this work, we fabricate ultra-large suspended graphene membranes, where stacks of a few layers of graphene could be suspended over a circular hole with a diameter of up to 1.5 mm, with a diameter to thickness aspect ratio of 3 × 10(5), which is the record for free-standing graphene membranes. The process is based on large crystalline graphene (∼55 µm) obtained using a chemical vapor deposition (CVD) method, followed by a gradual solvent replacement technique. Combining a hydrogen bubbling transfer approach with thermal annealing to reduce polymer residue results in an extremely clean surface, where the ultra-large suspended graphene retains the intrinsic features of graphene, including phonon response and an enhanced carrier mobility (200% higher than that of graphene on a substrate). The highly elastic mechanical properties of the graphene membrane are demonstrated, and the Q-factor under 2 MHz stimulation is measured to be 200-300. A graphene-based capacitive pressure sensor is fabricated, where it shows a linear response and a high sensitivity of 15.15 aF Pa(-1), which is 770% higher than that of frequently used silicon-based membranes. The reported approach is universal, which could be employed to fabricate other suspended 2D materials with macro-scale sizes on versatile support substrates, such as arrays of Si nano-pillars and deep trenches.

The biomechanical analysis of relative position between implant and alveolar bone: finite element method.

BACKGROUND: The purpose of this study is to analyze biomechanical interactions in the alveolar bone surrounding implants with smaller-diameter abutments by changing position of the fixture-abutment interface, loading direction, and thickness of cortical bone using the finite element method. METHODS: Twenty different finite element models including four types of cortical bone thickness (0.5, 1, 1.5, and 2 mm) and five implant positions relative to bone crest (subcrestal 1, implant shoulder 1 mm below bone crest; subcrestal 0.5, implant shoulder 0.5 mm below bone crest; at crestal implant shoulder even with bone crest; supracrestal 0.5, implant shoulder 0.5 mm above bone crest; and supracrestal 1, implant shoulder 1 mm above bone crest) were analyzed. All models were simulated under two different loading angles (0 and 45 degrees) relative to the long axis of the implant, respectively. The three factors of implant position, loading type, and thickness of cortical bone were computed for all models. RESULTS: The results revealed that loading type and implant position were the main factors affecting the stress distribution in bone. The stress values of implants in the supracrestal 1 position were higher than all other implant positions. Additionally, compared with models under axial load, the stress values of models under off-axis load increased significantly. CONCLUSIONS: Both loading type and implant position were crucial for stress distribution in bone. The supracrestal 1 implant position may not be ideal to avoid overloading the alveolar bone surrounding implants.