First Report of Root Rot Caused by Calonectria montana on Sugar Beet in Heilongjiang Province, China.

Sugar beet (Beta vulgaris L.) is an important crop that has significant economic value in northern regions of China, especially in Heilongjiang Province. In October 2019, root rot was discovered on the sugar beet cultivar HDW09 in Hulan (126°64' E, 46°00' N), Heilongjiang Province, China. Typical symptoms included lesions on root tissues, which were initially small and dark brownish, then gradually turned into irregular shapes and black in color. As the disease progressed, the extent of necrosis penetrated from external layers into inner tissue. Root tissues suffered from severe decay, which resembles symptoms of several previously reported root rot diseases of sugar beet(Harveson 2006). To identify the pathogen, pieces of the transition zones (3-5mm) between asymptomatic and symptomatic tissues were surface sterilized for 15 seconds in 1% NaClO, rinsed twice with sterilized distilled water， plated on corn meal agar supplemented with penicillin G (50 mg/L), and incubated at 25 ± 2°C in the dark. Isolates belonging to a Calonectria sp. were recovered and purified using the hyphal tipping technique. Four isolates including A1, A5, A6, and A7 were used for morphological characterization and identification by DNA sequencing. The seven-day-old colonies on malt extract agar produced buff and wooly aerial mycelia. They were sienna to umber in color. Chlamydospores and microsclerotia were produced abundantly throughout the medium. For further identification, the isolates were cultured on potato dextrose agar (PDA) at room temperature(25°C) under near-ultraviolet light irradiation. Macroconidiophores comprised of a stipe, a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle. Stipe extension were septate, straight to flexuous, 61-117 µm long, 2-4 µm wide at the apical septum, terminating in a sphaeropedunculate vesicle, 5-9 µm diam. Conidiogenous apparatus were 31-177 µm long, and 16-110 µm wide(n=30). Primary branches of conidiogenous apparatus were aseptate or 1-septate, 16-51 × 3-7 µm; secondary branches aseptate, 6-31 × 2-7 µm; tertiary branches aseptate, 8-19 × 2-6 µm, each terminal branch producing 1-6 phialides. Conidia cylindrical were rounded at both ends, straight, 32-53 × 3-5 µm, (mean = 47 × 4 µm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime(n=100). Partial sequences of calmodulin (Carbone et al. 1999), histone H3, the translation elongation factor 1-alpha, and beta-tubulin 2 (Crous et al. 2004) genes of the four isolates were obtained and deposited into GenBank under accession numbers MW118652 to MW118667. BLAST results showed that the calmodulin, histone H3, the translation elongation factor 1-alpha and beta-tubulin 2 sequences of A1, A5, A6 and A7 were highly identical to the sequences of Ca. montana strain CERC 8957 MF527082.1 (CAL) (99-100%), CERC 8930 MF527061.1 (HIS) ( 98-99%), HSP4 MN356465.1 (EF1-alpha) (100%) and HSP4 MN356460.1 (tub2) (98-99%), respectively. A phylogenetic tree using the maximum likelihood algorithm and sequences of the four concatenated genes was reconstructed in RAxML and revealed that the four isolates clustered in the clade of Ca. montana. The pathogen was identified as Ca. montana based upon these morphological and molecular traits(Liu et al. 2017; Stepniewska et al. 2020). Ten eight-week-old sugar beet plants without root rot symptoms were selected for pathogenicity test. The roots near the ground were carefully cleaned with hands. One mycelial plug (5 mm in diameter) from a seven-day-old colony of isolate A1 were used to inoculate each sugar beet root. Ten plants inoculated with plugs of noncolonized PDA served as the control plants. Pathogenicity tests were repeated three times. Plants were incubated under greenhouse conditions at 25 ± 2°C and watered when the surface soil appeared dry. All inoculated plants showed symptoms that resembled those in the field after 30 days, while the control plants remained healthy. The symptomatic tissues were plated in corn meal agar for 7 days at 25°C. Ca. montana was reisolated from 100% of the inoculated tissues, and identification was confirmed by molecular sequencing, validating Koch's postulates. This is the first report of Ca. montana in China causing root rot on sugar beet. The study suggests its broader host range and wider geographical distribution than ever known and lays a basis for further monitoring and managing this important pathogen.

Deep sequencing profiles MicroRNAs related to Aspergillus fumigatus infection in lung tissues of mice.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a severe opportunistic infection with high mortality in patients with compromised immunity. The full repertoire of microRNAs (miRNAs) involved in the regulation of IPA infection remains to be established. METHODS: We established a mouse IPA model and analyzed small RNA transcriptomes in lung tissues of immunodeficient IPA mice (IPA group) and matched immunodeficient control mice (control group) through next-generation sequencing. RESULTS: A total of 3759 known miRNAs were detected, in which 23 miRNAs were identified to be related to IPA. IPA-associated miRNAs include upregulated mmu-let-7b-3p, mmu-miR124-3p, mmu-miR21a-3p, mmu-miR29c-5p, mmu-miR3473b and mmu-miR3473e, and downregulated mmu-miR-150-3p and mmu-miR-503-5p. The expression levels of eight identified miRNAs were quantified in a validation cohort (n = 40) by qRT-PCR, and results revealed the same change patterns. MiRNA target prediction revealed that all IPA-related miRNAs possibly engage a cooperative regulation of key elements in the NF-kappa B signaling pathway. CONCLUSION: We conclude that deep-sequencing small RNAs can uncover miRNA pool-regulating IPA. Our results may lead to further understanding IPA pathogenesis and gain insight into the complexity and diversity of small RNA molecules that regulate immunodeficient IPA.

Activation of NF-κB and respiratory burst following Aspergillus fumigatus stimulation of macrophages.

Dectin-2, a C-type lectin receptor (CLR), plays an essential role in mediating nuclear factor-kappa B (NF-κB) activation and anti-fungal immunity in response to Candida albicans infection. However, the molecular mechanisms and function of Dectin-2 signaling in response to infection by the pathogenic fungus Aspergillus fumigatus have not been characterized. In order to characterize Dectin-2 signaling in response to A. fumigatus infection, activation of Dectin-2 was analyzed at both transcriptional and translational levels. Spleen tyrosine kinase (Syk) phosphorylation, NF-κB activation and cytokine production downstream of Dectin-2 activation were also investigated. In addition, Dectin-2-Syk function and its ability to mediate reactive oxygen species (ROS) production and elimination of A. fumigatus conidia was examined. We demonstrate that Syk is involved in Dectin-2-induced IκBα (inhibitor of kappa B protein) phosphorylation and NF-κB activation following A. fumigatus stimulation in a time dependent manner. Silencing of Dectin-2 and Syk as well as Syk inhibition blocks NF-κB activation and cytokine secretion. Furthermore, the killing of A. fumigatus conidia and ROS production are significantly affected by Dectin-2 or Syk silencing as well as Syk inhibition. Swelling and germination of the fungus followed by hyphae formation and not the resting and heat-inactivated form of A. fumigatus mediate the activation of Dectin-2 signaling. In conclusion, Syk plays an essential role in IκBα kinase phosphorylation, NF-κB activation, and ROS production mediated by Dectin-2 activation in response to A. fumigatus infection.

Dectin-2 is predominately macrophage restricted and exhibits conspicuous expression during Aspergillus fumigatus invasion in human lung.

We investigated the features of Dectin-2 expression both at transcriptional and translational levels during Aspergillus fumigatus infection in human lung. Simultaneously, the expression of CD206 was assayed as an activated marker of alveolar macrophages. The characteristic of Dectin-2 expression were then confirmed in Monocyte-derived macrophages (MDM) after A. fumigatus stimulation by Flow Cytometry. We found that the expression of Dectin-2 was low in normal lung, while it revealed a markedly up-regulation during A. fumigatus invasion. Dectin-2 expression was predominantly restricted to CD206 positive cells. There was salient positive correlation between Dectin-2 expression and CD206. We conclude that Dectin-2 expression is largely restricted to alveolar macrophages in human lung. The conspicuous expression of Dectin-2 during A. fumigatus invasion suggests its notable contribution to antifungal defenses in pulmonary aspergillosis.

[The expressions of nerve growth factor and its tyrosine kinase A receptor on alveolar macrophages in a rat model of chronic obstructive pulmonary disease].

OBJECTIVE: To observe the expressions of nerve growth factor (NGF) and its tyrosine kinase A (TrkA) receptor on alveolar macrophage in a rat model of chronic obstructive pulmonary disease (COPD). METHODS: Forty healthy male SD rats were randomly divided into a control group and a COPD group. The COPD model was established by exposing the rats to cigarette smoke for 6 months, and lung function changes were measured. Lung histopathological changes were detected by HE staining. The expression of NGF protein in the supernatant of alveolar macrophage (AM) culture medium was detected by ELISA. Confocal microscopy was used to identify the separation and purification of AM from bronchoalveolar lavage fluid, and to detect semi-quantitatively the expression of TrkA receptor on AM. NGF and its TrkA receptor at the mRNA level were evaluated by real-time PCR. The differences among groups were calculated by one way ANOVA, and comparison between groups was made by t test. RESULTS: Significant decrease of pulmonary compliance [(0.15 ± 0.03) ml/cm H(2)O (1 cm H2O = 0.098 kPa)] and minute ventilation [(0.045 ± 0.004) L], and significant increase of airway resistance [(0.038 ± 0.004) cm H2O×L(-1)×s(-1)] were found in the COPD group compared with the control group [(0.42 ± 0.05) ml/cm H2O and (0.102 ± 0.010) L and (0.016 ± 0.002) cm H2O×L(-1)×s(-1), t = 9.46 - 12.99, respectively, all P < 0.01]. Alveolar wall thinning, alveolar septa breakdown, alveolar enlargement and emphysema were significant in the COPD rats. The expression of NGF protein in the supernatant of AM culture medium was enhanced in the COPD group [(3.79 ± 1.52) ng/L] compared with the controls [(0.94 ± 0.27) ng/L, t = 4.13, P < 0.05]. Mean fluorescence intensity of TrkA protein on AM in the COPD group (19.5 ± 1.5) was higher than that in the control group (11.2 ± 1.9, t = 7.95, P < 0.05). The expressions of NGF and TrkA at mRNA level in the COPD group (24.8 ± 6.0 and 9.0 ± 3.3) were increased compared with the control group (1.0 ± 0.2 and 1.0 ± 0.4, t = 8.48 and 5.16, all P < 0.05). CONCLUSIONS: The expressions of NGF and its TrkA receptor on AM in COPD group were increased, indicating that NGF and its TrkA receptor might be involved in the pathogenesis of COPD mediated by AM.