First telomere-to-telomere gapless assembly of the rice blast fungus Pyricularia oryzae.

Rice blast caused by Pyricularia oryzae (syn., Magnaporthe oryzae) was one of the most destructive diseases of rice throughout the world. Genome assembly was fundamental to genetic variation identification and critically impacted the understanding of its ability to overcome host resistance. Here, we report a gapless genome assembly of rice blast fungus P. oryzae strain P131 using PacBio, Illumina and high throughput chromatin conformation capture (Hi-C) sequencing data. This assembly contained seven complete chromosomes (43,237,743 bp) and a circular mitochondrial genome (34,866 bp). Approximately 14.31% of this assembly carried repeat sequences, significantly greater than its previous assembled version. This assembly had a 99.9% complement in BUSCO evaluation. A total of 14,982 genes protein-coding genes were predicted. In summary, we assembled the first telomere-to-telomere gapless genome of P. oryzae, which would be a valuable genome resource for future research on the genome evolution and host adaptation.

Mitochondrial characteristics of the powdery mildew genus Erysiphe revealed an extraordinary evolution in protein-coding genes.

The genus Erysiphe was an obligate parasite causing powdery mildew disease on a wide range of higher plants. However, the knowledge of their mitogenome architecture for lifestyle adaptability was scarce. Here, we assembled the first complete mitogenome (190,559 bp in size) for rubber tree powdery mildew pathogen Erysiphe quercicola. Comparable analysis of the Erysiphe mitogenomes exhibited conserved gene content, genome organization and codon usage bias, but extensive dynamic intron gain/loss events were presented between Erysiphe species. The phylogeny of the Ascomycota species constructed in the phylogenetic analysis showed genetic divergences of the Erysiphe species. Compared with other distant saprophytic and plant pathogenic fungi, Erysiphe had a flat distribution of evolutionary pressures on fungal standard protein-coding genes (PCGs). The Erysiphe PCGs had the highest mean selection pressure. In particular, Erysiphe's cox1, nad1, cob and rps3 genes had the most elevated selection pressures among corresponding PCGs across fungal genera. Altogether, the investigations provided a novel insight into the potential evolutionary pattern of the genus Erysiphe to adapt obligate biotrophic lifestyle and promoted the understanding of the high plasticity and population evolution of fungal mitogenomes.

Characterization of conidial autofluorescence in powdery mildew.

Autofluorescence is produced by endogenous fluorophores, such as NAD(P)H, lipofuscin, melanin, and riboflavin, indicating the accumulation of substances and the state of energy metabolism in organisms. As an obligate parasite, powdery mildew is wildly spread by air and parasitic crops. However, most identification studies have been based on morphology and molecular biology which were far too time- and labor-consuming, thus lacking characteristic, simple, and effective means. Using microscopy under the blue and cyan channels, we elaborated visible conidial autofluorescence in three powdery mildew species, Erysiphe quercicola, E. cichoracearum, and Podosphaera hibiscicola, with a sharp increase during the conidia senescence in E. quercicola. Additionally, the main spectral excitation detected by fluorescence spectrometery was 375 nm for these species, with a common emission peak at approximately 458-463 nm, and an additional trend at 487 nm for P. hibiscicola. Because NAD(P)H has a similar spectral feature, we further investigated the relation between NAD(P)H and conidial autofluorescence by fluorescence spectra. We observed that the reduced coenzymes prominently contributed to conidial autofluorescence; however, the conidial autofluorescence in P. hibiscicola displayed a different trend that may be affected by the oxidized coenzyme -NAD. Finally, the normalized average spectra of these three powdery mildew species and standard samples showed that the spectral trend of each species was similar but that the features in detail were specific and distinct based on principal component analysis. In conclusion, we showed and characterized conidial autofluorescence in three powdery mildew species for the first time. The specific conidial autofluorescence in these species provides a new idea for the development of field spore capture and identification devices for the discrimination of powdery mildew at the species level.

First Report of Stemphylium lycopersici Causing Leaf Spot on Hevea brasiliensis in China.

Hevea brasiliensis is widely planted in tropical and subtropical regions and is the main source of natural rubber production. The growth of rubber trees is plagued by various leaf diseases, resulting in decreased rubber production. From January to March in 2020, a severe leaf spots disease on Hevea brasiliensis found in Agricultural Science Base in Haidian campus of Hainan University (20° 03' 31″ N, 110° 19' 07″ E), Haikou, Hainan province, China. Spots were only observed on the mature green rather than young and bronze-colored leaves. This symptom has never been reported on the leaves of Hevea brasiliensis. During the early stages of the disease, gray leaf spots were concentrated to the leaf margins, but later expanded forming irregular gray lesions with chlorotic edges (Figure 1A). Eventually, lesions became necrotic shot holed, and leaves curled, wilted, and dropped. Five small pieces were cut from the margin of spots from different infected leaves, and were surface disinfected with 75% alcohol three times for five seconds each time and 1% sodium hypochlorite solution (NaClO) for 60 s. After washing twice with sterile water, leaf pieces were placed in the center of plates with Potato Dextrose Agar (PDA) medium and incubated for one week at 28 °C. After 7 days, mycelium developed and colonies were single-spore cultured for further study. One of the strains labeled HN01 developed a yellowish-brown to reddish-brown pigment on PDA, and the colonies were gray and cottony. The colony and pigment feature very consistent with Stemphylium sp. (Figure 2) (Li et al. 2017). Conidiophore were solitary, transparent to pale, mostly 102.1-228.8 µm × 4.0-5.8 µm, with 2-3 septa and apical vesicular swellings 6.5-7.9 µm. The dimensions of conidia were 28.3-45.1 × 11.5-17.5 µm and one septum (Figure 3). Conidia of S. lycopersici were solitary, oblong with a conical end at the apex, with 1-2 septa, and constricted at the transverse septum. The internal transcribed spacer region of rDNA was amplified with primers ITS1/ITS4 (5'-TCCGTAGGTGAACCTGCGG-3'/5'-TCCTCCGCTTATTGATATGC-3'), glyceraldehyde-3-phosphate dehydrogenase (gpd) was amplified with primers GPD-F/R (5'-GCACCGACCACAAAAATC-3'/ 5'-GGGCCGTCAACGACCTTC-3'), calmodulin region (cmdA) was amplified with the primers CALDF1/CALDR2 (5'-AGCAAGTCTCCGAGTTCAAGG-3'/5'-CTTCTGCATCATCAYCTGGACG3') from genomic DNA of strain HN01 (Xie et al. 2018), and PCR products were sequenced. The ITS sequence of strain HN01 (GenBank Accession No. MZ496930) had 99.64% identity with isolates sl001, sl110, sl111, and sl112 of Stemphylium lycopersici (GenBank Accession No. KX858848.1, MF480547.1, MF480548.1, MF480549.1). Similarly GPD sequences (GenBank Accession No. MZ505106) had 100% identity with strain xiqing, HZ2114 and HZ2115 of Stemphylium lycopersici (GenBank Accession No. KR911809.1, KR911810.1, KT957742.1 and KT957743.1), and CMDA sequences (GenBank Accession No. MZ505105) had 99.85% identity with Stemphylium lycopersici strain LJ1609270201 (GenBank Accession No. MG742412.1). A phylogenetic analysis constructed by MEGA6.0 based on concatenated sequences of the HN01 and another 17 strains from GenBank by using the maximum-likelihood (ML) method showed that the HN01 was clustered and matched with Stemphylium lycopersici LJ1609270201 (Figure 4). To satisfy Koch's postulates, we inoculated mature green leaves of Hevea brasiliensis with mycelial plugs (diameter = 5 mm) of pure cultured strain HN01. All leaves of Hevea brasiliensis were wrapped in a freezer bag to maintain relative humidity >85%, and the temperature of greenhouse is 28ºC. The disease developed on the inoculated leaves after 2-3 days, but not on control leaves (Figure 1B). We used the same method as before to re-isolate the pathogen, which had the same morphology and genotypes as the original isolate. S. lycopersici has been reported to infect the leaves of a variety of plants, including pepper, tomato, eggplant, watermelon, Physalis alkekengi. (Yang et al.2017; Ben et al. 2017; Yang et al. 2020). To our knowledge, this is the first record of S. lycopersici causing leaf spot of Hevea brasiliensis in China, and Hevea brasiliensis is the global new host of S. lycopersici. Hevea brasiliensis is the main source of natural rubber and is widely planted in southern China. Therefore, it is imperative to implement disease management measures to prevent potential threats.

Antibacterial Properties and Application of Nanosilver Material in the Tracheal Tube.

To investigate the antibacterial ability of a new type of antibacterial tracheal tube coated with nanosilver/polyurethane in rats. In January 2016, 48 male SD rats of SPF grade, provided by the medical center of Hong Kong University of science and technology, Peking University, Shenzhen, were selected as the study objects. Twenty-four healthy rats, who underwent endotracheal intubation and retained nanosilver/polyurethane-coated new antibacterial endotracheal tube in vivo, were randomly selected as the experimental group, while 24 healthy rats who underwent endotracheal intubation at the same time and retained common endotracheal tube in vivo were randomly selected as the control group. At 12, 24, 48, and 72 hours after the operation, the number of colonies in the alveolar lavage fluid of the two groups was measured using the plate count method, and the thickness of the biofilm formed by the built-in catheter of the two groups was observed by microscope. Twelve hours after operation, there was no significant difference between the two groups (P <0.05). The colony number in BALF in the experimental group was significantly lower than that in the control group (P < 0.05). At 12 and 24 hours after operation, there was no significant difference in the biofilm thickness between the two groups (P > 0.05). In the experimental group, the thickness of biofilm that had formed by catheterization 48 and 72 hours after operation was significantly lower than that in the control group (P < 0.05). The new type of antibacterial tracheal tube, coated with nanosilver/polyurethane, has stronger antibacterial and anti-biofilm proliferation performance than that of the common tracheal tube.

[TLR3c.1377, TLR9-1486, and TLR9 2848 gene polymorphisms and multiple sclerosis].

OBJECTIVE: To investigate the relationship between the gene polymorphism of TLR3c.1377,TLR9-1486,and TLR9 2848 and susceptibility to multiple sclerosis(MS)in Han people of south China. METHODS: A total of 123 unrelated MS patients from South China with a clinical or laboratory definition MS according to 2005 Revisions to the McDonald Criteria were studied. Another 126 controls were randomly selected from hospital staff of non-autoimmune diseases and healthy individuals. Toll like receptor (TLR) 3 and TLR 9 genotypes were determined by PCR and digested by specific restriction enzymes. RESULTS: There was significant difference in genotype and allele distribution of TLR3c.1377 polymorphism between the MS patients and the controls (P<0.05), and the MS patients with T allele had a lower risk (OR=0.532, P=0.014). There was no significant difference in genotypes and allele distribution of TLR9-1486 polymorphism between the MS patients and the controls. There was higher TLR9 2848 A allele frequency in the MS patients than in the controls (39.8% vs. 30.6%;P=0.037), and higher risk in MS patients with A allele than those without (OR=1.837, P=0.020). There was no significant interaction among the TLR3c.1377, TLR9-1486 and TLR9 2848 allele. Strong linkage disequilibrium was found between TLR9-1486 and TLR9 2848, but there was no significant interaction between the polymorphism of TLR9-1486 and TLR9 2848 in the MS patients. CONCLUSION: TLR3c.1377 and TLR9 2848 polymorphisms may be related to MS in Han people in south China. TLR3c.1377 and TLR9 2848 may be linked with susceptibility genes.

Study of Cbl-b dynamics in peripheral blood lymphocytes isolated from patients with multiple sclerosis.

E3 ubiquitin ligase Casitas B cell lymphoma-b (Cbl-b) has been recently highlighted as a negative regulator of T-cell activation and which dysfunction usually results in autoimmunity. To present, however, the possible involvement of Cbl-b in multiple sclerosis (MS), an autoimmune demyelinating disease mediated by T-helper 1 (Th1) cells is still unclear. To clarify this, using reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analyses, we thus investigated the levels of Cbl-b mRNA and protein in peripheral blood T-lymphocytes isolated from 11 MS patients in acute relapse phase and 20 cases in remission phase. 16 healthy subjects were used as normal control. Cbl-b mRNA and protein levels were found both significantly down-regulated in peripheral blood lymphocytes isolated from MS patients (P<0.0001). Interestingly, this decrease of Cbl-b protein but not mRNA levels was significantly more marked in samples of relapsed patients than that of remitted cases (P<0.0001). In addition, it was shown that Cbl-b mRNA levels being inversely correlated with the frequency of MS clinical relapses (P<0.0001). Altogether, the data show for the first time that Cbl-b dynamics in peripheral blood T-lymphocyte subset and which possible relationship with the clinical onsets during MS.