First Report of the Rust Fungus Melampsora ferrinii on Salix babylonica in China and a New Spermogonial and Aecial Host, Corydalis bungeana.

The occurrence of rust fungi on Corydalis bungeana Turcz. and Salix babylonica L. were found in same area of Hebei Province, China from 2022 to 2023. The life cycle connection of these rust fungi was suspected because Peng et al. (2022) reported the life cycle of Melampsora ferrinii Toome & Aime by inoculations, producing spermogonia and aecia on Corydalis species, and uredinia on S. babylonica. The morphology of the uredinial and telial stages on S. babylonica collected in the field was identical with the description of M. ferrinii by Toome and Aime (2015), and its identity was confirmed by phylogenetic analyses using the method of Ji et al. (2020) (LSU-PP087777, ITS-PP091274; Similarity with M. ferrinii: LSU-100%, ITS-99.85%). To confirm the life cycle of this rust fungus, inoculations were conducted on C. bungeana with basidiospores obtained from the teliospores on fallen leaves of Salix babylonica. The fallen leaves producing basidiospores were cut into small pieces (ca. 5 mm2) and placed on healthy leaves of C. bungeana. The inoculated plants were kept in a moist plastic box in darkness at 15-20℃ for 2 days and then transferred to the floor near windows at about 15-20℃ for observations. Ten days after inoculations small yellow spots of spermogonia appeared on the upper surface of the leaves of C. bungeana. About 7 days later, pale yellow aecia with aeciospores were produced mainly on the under surface of the leaves and petioles. The morphology of rust fungus on C. bungeana collected from the fields and obtained by inoculations was identical with the description by Peng et al. (2022). Phylogenetic analyses also showed that a specimen on C. bungeana collected from the field (LSU-OR607838, ITS-OR612063) were included into the same clade of M. ferrinii (Similarity: LSU-100 %, ITS-99.85). Based on morphology, inoculations and DNA sequence analyses, the rust fungi on C. bungeana and S. babylonica are identified as different stages of life cycle of M. ferrinii. This rust fungus has been reported to produce spermogonia and aecia on C. acuminata Franch., C. edulis Maxim. and C. racemosa (Thunb.) Pers. in China (Peng et al. 2022), and uredinia and telia on S. babylonica in USA, Argentina and Iran (Toome and Aime 2015, Abbasi et al. 2024), and on Salix sp. in Chile (Zapata 2016). Therefore, C. bungeana is a new host for M. ferrinii, and its field occurrence on S. babylonica is reported for the first time in China although Peng et al. (2022) reported successful results in its inoculations to S. babylonica in China. This report contributes to the control of rust diseases caused by this species. Specimens used in this experiment were deposited in the Fungal Herbarium of the Jilin Agricultural University, Changchun, China (HMJAU) and sequences newly analyzed were deposited in GenBank.

Phylogeny and taxonomy of three species of Blastospora (Pucciniales) from East Asia.

Three species of the rust fungus genus Blastospora, Bl. betulae, Bl. itoana, and Bl. smilacis, have been reported in East Asia. Although their morphological characteristics and life cycles have been investigated, their phylogenetic positions have not been clarified sufficiently. Phylogenetic analysis showed that these three species were included into Zaghouaniaceae of Pucciniales. However, Bl. betulae was phylogenetically distinct from Bl. itoana and Bl. smilacis and different from other genera. Based on this result, and applying recent International Code of Nomenclature decisions/recommendations/requirements, Botryosorus, gen. nov., and Bo. deformans,, comb. nov., were applied for Bl. betulae. Two new combinations, Bl. radiata for Bl. itoana and Bl. makinoi for Bl. smilacis, were also applied. Their host plants and distribution were described based on literature records. Zaghouania yunnanensis, comb. nov., was proposed for Cystopsora yunnanensis as a result of this analysis.

StPBS2, a MAPK kinase gene, is involved in determining hyphal morphology, cell wall development, hypertonic stress reaction as well as the production of secondary metabolites in Northern Corn Leaf Blight pathogen Setosphaeria turcica.

Mitogen activated protein kinase kinase (MAPKK) is a crucial component in the MAPK signaling pathway. However, the functions of MAPKKs in foliar pathogens remain poorly understood. In the current study, a MAPKK gene designated as StPBS2 was cloned from Setosphaeria turcica and the functions of this gene were investigated by RNAi technology. Four independent StPBS2 gene silence transformants with different efficiencies were confirmed by real time PCR. Compared to the wild type strain (WT), these transformants showed decreased colony growth, shortened hyphae cell length, broadened cell width and an obvious reduction in conidium yield. Moreover, the cell wall of the transformants was thicker and they were also more sensitive to substances that interfere with cell wall biosynthesis than WT. Additionally, the transformants displayed higher sensitivity to hypertonic stress than WT and the sensitivity was associated with the level of silencing of StPBS2. They were also resistant to the fungicides iprodione, procymidone and fludioxonil, to which WT almost completely sensitive. The transformants produced more red secondary metabolites than WT and the production was enhanced with increasing silencing level and increased glucose content in PDA medium. Our results suggest that StPBS2 is involved in morphogenesis, condiogenesis, cell wall development, hypertonic stress reaction and resistance to fungicides, as well as in the biosynthesis of secondary metabolites in S. turcica.

StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration.

In filamentous fungi, the pathogenic mitogen-activated protein kinase (PMK) pathway performs an important function in plant infection. STE12-like genes found in higher eukaryotes encode transcription factors and are regulated by the PMK pathway. However, the functions of STE12-like genes in foliar pathogens remain poorly understood. In this study, we cloned StSTE12 from Setosphaeria turcica and investigated its functions by RNA interference. Transformants ste12-3, ste12-2 and, ste12-1, in which the StSTE12 silencing efficiency increased in order, were confirmed by real time PCR. Compared with the wild-type (WT) strain, the transformants showed reduced growth rate, lighter colony color, and obviously decreased conidium production. More importantly, different to WT strain and ste12-3 with lower StSTE12silencing efficiency, ste12-1 and ste12-2 with higher StSTE12 silencing efficiency were nonpathogenic on intact leaves, but pathogenic on wounded leaves. However, the biological activity of HT-toxin from all transformants showed no difference on corn leaves. Furthermore, ste12-1 and ste12-2 did not penetrate artificial cellophane membrane and showed abnormal and delayed development appressoria. Although it could penetrate the cellophane membranes, ste12-3 formed appressoria after 48 h of inoculation more than WT. Therefore, StSTE12 was involved in vegetative growth, conidiation, appressorial development, penetration as well as the pathogenicity, but it was not related to HT-toxin biosynthesis. More interestingly, all the results suggested that StSTE12 was crucial for pathogenicity due to involvement in regulating appressoria development and penetration.

Chemiluminescent detection of DNA hybridization using gold nanoparticles as labels.

A chemiluminescent (CL) detection method has been developed for DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which gold nanoparticles modified with alkylthiol-capped oligonucleotide strands are used as probes to monitor the presence of the specific target DNA. The AuCl(4)(-), which is the dissolving product of the gold nanoparticles anchored on the DNA hybrids, serves as an analyte in the H(2)O(2)-luminol- AuCl(4)(-) CL reaction for the indirect measurement of the target DNA. The combination of the remarkable sensitivity of the CL analysis with the large number of AuCl(4)(-) released from each DNA hybrid allows a detection limit at levels as low as 0.1 pM of the target DNA. Moreover, with a further silver amplification step, the detection limit will be pushed down to the femtomolar domain.

A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels.

A sensitive chemiluminescent (CL) immunoassay of human immunoglobulin (IgG) which combined the inherent high sensitivity of CL analysis with the dramatic signal amplification of silver precipitation on colloidal gold tags was developed. First, the sandwich-type complex was formed in this protocol by the primary antibody immobilized on the polystyrene wells, the analyte in the sample, and the secondary antibody labeled with colloidal gold. Second, the colloidal gold was treated by an Ag(+) reduction solution, which resulted in the catalytic precipitation of silver on the surface of colloidal gold. Third, a large number of Ag(+) were oxidatively released in HNO(3) solution from the silver metal anchored on the sandwich-type complexes and then the human IgG was indirectly determined by a sensitive combined CL reaction of Ag(+)-K(2)S(2)O(8)-Mn(2+)- H(3)PO(4)-luminol. The chemiluminescence intensity depends linearly on the logarithm of the concentration of human IgG over the range of 0.02-50ngml(-1) and detection limit (3sigma) is 0.005ngml(-1) (i.e., approximately 3x10(-14)M, 3amol in 100-mul sample). This assay has been successfully applied to the determination of human IgG in human serum samples and showed great potential for numerous applications in immunoassay.