Post-Translational Modification ß-Hydroxybutyrylation Regulates Ustilaginoidea virens Virulence.

Lysine ß-hydroxybutyrylation (Kbhb) is an evolutionarily conserved and widespread post-translational modification that is associated with active gene transcription and cellular proliferation. However, its role in phytopathogenic fungi remains unknown. Here, we characterized Kbhb in the rice false smut fungus Ustilaginoidea virens. We identified 2204 Kbhb sites in 852 proteins, which are involved in diverse biological processes. The mitogen-activated protein kinase UvSlt2 is a Kbhb protein, and a strain harboring a point mutation at K72, the Kbhb site of this protein, had decreased UvSlt2 activity and reduced fungal virulence. Molecular dynamic simulations revealed that K72bhb increases the hydrophobic solvent-accessible surface area of UvSlt2, thereby affecting its binding to its substrates. The mutation of K298bhb in the septin UvCdc10 resulted in reduced virulence and altered the subcellular localization of this protein. Moreover, we confirmed that the NAD+-dependent histone deacetylases UvSirt2 and UvSirt5 are the major enzymes that remove Kbhb in U. virens. Collectively, our findings identify regulatory elements of the Kbhb pathway and reveal important roles for Kbhb in regulating protein localization and enzymatic activity. These findings provide insight into the regulation of virulence in phytopathogenic fungi via post-translational modifications.

Epicoccum layuense Causing Leaf Spot on Oxalis corymbosa in China.

Oxalis corymbosa DC. is a perennial herb used as the groundcover in the urban landscape and traditional Chinese medicine. In May 2019, a leaf spot-like disease was observed on about 20% of O. corymbosa plants in one park in Hefei, Anhui, China (N31°52´, E117°15´). Symptoms appeared as yellowish, irregular or circular areas that later turned pale brown, coalesced and produced large necrotic areas of various shapes. Ten leaves with typical symptoms were removed from 5 different O. corymbosa plants (2 leaves per plant) and disinfected with 75% ethanol for 30 s. The tissue pieces (4×4 mm) were cut from the margin of the leaf lesion, dipped in 2% NaClO for 1 minute, washed 3 times with sterile distilled water, placed on Potato Dextrose Agar (PDA) medium with 50 µg/ml each of ampicillin and kanamycin, and cultured at 25℃ under a 12-h light/dark cycle. The white mycelia grew out of each leaf tissue pieces 3 days later and the hyphal tips were sub-cultured into a new PDA medium for purification. After 5 days of incubation at 25℃, the produced colonies were yellow with irregular white margin from the top with floccose aerial mycelia, ranged in a diameter from 60 to 70 mm, and produced red orange pigments into PDA medium. Colonies on the reverse side were dark red. Conidia produced on PDA were ranged in size from 5.5 to 35.5 µm in diameter (n = 800), subglobose to pyriform, and yellowish-brown. The representative isolate YHF0519 was chosen for further analysis. Fragments of four genes including the internal transcribed spacer (ITS), beta-tubulin, 28S large subunit ribosomal RNA gene (LSU), and the second largest subunit of RNA polymerase II (RPB2) were amplified from the extracted genomic DNA of YHF0519 and sequenced. The corresponding primer sets were ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), LSU1Fd/LR5 (Crous et al. 2009; Vilgalys and Hester 1990), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007; Liu et al. 1999), respectively. All resulting sequences were deposited into GenBank and received assigned accession numbers: MN418006 for ITS; MN428043 for beta-tubulin; MN428044 for LSU; MZ437946 for RPB2. A phylogenetic tree generated by the maximum likelihood method with 1,000 bootstrapping replications based on the combined ITS, beta-tubulin, LSU, and RPB2 sequences revealed that YHF0519 was clustered closest to the isolate CGMCC 3.18362 of Epicoccum layuense Qian Chen, Crous & L. Cai (Chen et al. 2017; Jayasiri et al. 2017). The pathogenicity test was performed on leaves of healthy O. corymbosa plants in the field and repeated twice. The average air temperature was about 28°C during the pathogenicity test. A total of 126 and 127 leaves were used for treatment and control, individually. Leaves were inoculated by spraying 30 ml of spore suspension (1×106 spores/ml) of YHF0519 or sterilized distilled water. Eight day-post inoculation, about 92% of the leaves inoculated with spores developed pale brown spots, which are identical to those occurred naturally in the field. No symptoms were detected on control leaves. The fungus was re-isolated from the symptomatic leaf tissues and showed the same morphological and molecular characteristics as YHF0519. E. layuense has been reported on Perilla sp., oat, and tea plant (Chen et al. 2017; Chen et al. 2019; Chen et al. 2020). This is the first report of E. layuense causing leaf spot on O. corymbosa in China. Due to the potential threat on the ornamental and medicinal value of O. corymbosa by E. layuesne, it will be necessary to develop local management strategies against leaf spot on O. corymbosa.

Nigrospora oryzae Causing Leaf Spot on Asiatic Dayflower in Chongqing, China.

Asiatic dayflower (Commelina communis L.) is an annual herbaceous weed that is distributed throughout China. A foliar disease on Asiatic dayflowers was discovered in one farm field in Dianjiang County, Chongqing, China (N30°3´22″, E107°18´5″) in summer, 2019. The disease incidence was observed on about 10% (13/127) of the plants. Symptoms appeared as round-shaped tan lesions (2-5 mm) in diameter that occurred randomly and irregularly on the whole leaves. The centers of lesions become grayish white with reddish borders as the disease progressed. The leaves with typical symptoms were detached and wiped with 70% ethanol for surface disinfestation before isolating the causal agent. Subsequently, three pieces (3-4 mm2) of tissue were taken from the margin of the leaf lesion, disinfested in 1.5% NaClO for 1 min, rinsed 3 times in sterilized distilled water, and placed onto Potato Dextrose Agar (PDA) medium containing 50 µg/ml each of kanamycin and ampicillin. A fungus was exclusively and consistently isolated from the disinfested leaf lesion sections. The colonies on PDA grew rapidly and covered the entire petri dish within 5 days at 28℃. Colonies were at first grayish white, cotton wool-like, round, with abundant aerial mycelium, and later turned black as conidia produced. The abundant conidia formed on PDA were initially yellow brown and gradually became black, oblate to ellipsoidal, smooth, single-celled, and ranged in size from 4 to 10 × 3.5 to 9 µm. They were borne on a colorless, hyaline, and inverted flask-shaped cell at the tip of each conidiophore. The morphology characteristics were consistent with those of Nigrospora spp. (Wang et al. 2017). Genomic DNA was extracted from one representative isolate NDJ0819. The amplification and sequencing of the gene fragments including the internal transcribed space (ITS) region of ribosomal DNA and beta-tubulin were performed using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Fragments of 536 bp for ITS and 408 bp for beta-tubulin were obtained. A phylogram of the combined ITS and beta-tubulin sequences reconstructed using the maximum likelihood bootstrapping method implemented in the software MEGA version 7.0 (Kumar et al. 2016) indicated that isolate NDJ0819 clustered with Nigrospora oryzae. Both ITS and beta-tubulin sequences were deposited into GenBank (accession no. MT140353 and MT157509, respectively). Pathogenicity test was performed by rub-inoculating needle-wounded leaves of three 4-week-old Asiatic dayflowers with spore suspension (2.6 × 106 conidia/ml) of NDJ0819 prepared in water containing 0.05% Tween-20, and holding plants at 28℃ in the growth chamber. The pathogenicity test was repeated twice. Brown, round-shaped lesions developed on leaves inoculated with spores at 15 days post-inoculation. However, the centers of the lesion did not become grayish white, compared to those of lesions seen in naturally infected leaves. No symptoms developed on leaves inoculated with sterilized distilled water. N. oryzae was re-isolated from the lesions. All results described above indicated that N. oryzae was responsible for the leaf spot of Asiatic dayflower. To our knowledge, this is the first report of N. oryzae causing leaf spot on Asiatic dayflower in China. Research into the potential use of N. oryzae as a candidate biological agent against the weed is worth being initiated.

Study on Fabrication of ZnO Waveguide Layer for Love Wave Humidity Sensor Based on Magnetron Sputtering.

The ZnO waveguide layer for the Love wave humidity sensor was fabricated by radio frequency (RF) magnetron sputtering technique using ZnO as the target material. To investigate the effect of RF magnetron sputtering temperature on the ZnO waveguide layer and Love wave device, a series of Love wave devices with ZnO waveguide layer were fabricated at different sputtering temperatures. The crystal orientation and microstructure of ZnO waveguide was characterized and analyzed, and the response characteristics of the Love wave device were analyzed by network analyzer. Furthermore, a humidity measurement system is designed, and the performance of the Love wave humidity sensor was measured and analyzed. The research results illustrate that the performance of the ZnO waveguide layer is improved when the sputtering temperature changes from 25 °C to 150 °C. However, when the sputtering temperature increases from 150 °C to 200 °C, the performance of the ZnO waveguide layer is degraded. Compared with the other sputtering temperatures, the ZnO waveguide layer fabricated at 150 °C has the best c-axis orientation and the largest average grain size (53.36 nm). The Love wave device has the lowest insertion loss at 150 °C. In addition, when the temperature of the measurement chamber is 25 °C and the relative humidity is in the range of 10% to 80%, the fabricated Love wave humidity sensor with ZnO waveguide layer has good reproducibility and long-term stability. Moreover, the Love wave humidity sensor has high sensitivity of 6.43 kHz/RH and the largest hysteresis error of the sensor is 6%.