First Report of Alternaria alternata Causing Leaf Spot Disease on Daylily in China.

Daylilies (Hemerocallis spp.; Xanthorrhoeaceae) originated from Eastern Asia and are widely cultivated as perennial ornamentals from the tropics to their native high latitudes. In June 2021, daylily cultivar 'Tao Hua Zhai' with leaf spot symptoms were found at the Shanghai Institute of Technology, Shanghai, China. The disease prevalence was about 14.5 % in a 33,000 m2 planting area indicated by survey statistics. Symptoms of the disease initially appeared as small, circular, brown spots on the leaves. As disease progressed, spots increased gradually until they were distributed uniformly over the lamina, the leaf tip became withered and the rest of the leaf became chlorotic. Symptomatic leaf tissue pieces (5 × 5 mm) from lesion margins were sterilized with 75 % ethanol for 1 min, rinsed three times with sterile distilled water, then incubated on potato dextrose agar (PDA) plates at 28 °C in the dark. A pure culture (ATHF-1) was obtained. Its upper surface on PDA was olive green with loose aerial hyphae, and its lower surface was brown.Conidiophores were brown, single or branched, producing numerous short chains conidia. Conidia were obclavate to obpyriform or ellipsoid, pale brown to dark brown, with a short cylindrical beak at the tip, contained 2-6 transverse septa and 0-4 longitudinal septa. The size of conidia were 15.9-47.3 µm × 7.6-16.6 µm (n=50), and length/width ratios were 1.51 to 4.92. Based on the morphological characteristics, the fungus was identified as Alternaria spp. (Simmons, 2007). For molecular characterization, three genes (the internal transcribed spacers [ITS], plasma membrane ATPase [ATPase] and major allergen Alt a 1) of ATHF-1 were amplified with primer pairs ITS1/ITS4 (White et al. 1990), ATPDF1/ATPDR1 (Lawrence et al. 2013) and Alt-for/Alt-rev (Hong et al. 2005), respectively. The sequences were deposited in GenBank (ITS, MZ983611; ATPase, MZ962978; Alt a 1, OK021654). Blastn searches showed the nucleotide sequences of ATHF-1 were highly similar to the reference sequences of Alternaria tenuissima (ITS, 99 % to KU982591; ATPase, 98 % to MT833928; Alt a 1, 100 % to MT109294). A phylogenetic tree based on the ITS, ATPase and Alt a 1 sequences was constructed by MEGA7.0, which showed that ATHF-1 was closely related to A. tenuissima and A. alternata. But according to Woudenberg et al. (2015), they were synonymized under the species name A. alternata. So, based on morphological and molecular characteristics, the fungus was identified as A. alternata. For pathogenicity tests, ten healthy two-month-old potted seedlings from tissue culture daylilies were sprayed with 20 ml of suspension (approximately 2×105 spores/ml), ten daylilies were used as controls and sprayed with sterile water. After covering with transparent plastic bags for 48 h to maintain humidity, the plants were placed in the greenhouse at 25 ℃ with 12 h photoperiod. The pathogenicity tests were repeated twice. Seven days after inoculation, lesions appeared on the plants inoculated with the pathogen, which were consistent with the symptoms observed in the field, while the controls remained symptomless. The morphological characteristics and gene sequences of the re-isolated strain from the diseased leaves were consistent with those of the inoculated strain. To our knowledge, this is the first report of A. alternata affecting leaf spot disease on daylily in China. Identification of the causal agent of the disease is important for developing effective disease management strategies. References: Hong, S.G., et al. 2005. Fungal Genet Biol. 42(2):119-129. https://doi.org/10.1016/j.fgb.2004.10.009 Lawrence, D.P., et al. 2013. Mycologia. 105(3):530-546. https://doi.org/10.3852/12-249 Simmons, E.G. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands. White, T. J., et al. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. PCR protocols: a guide to methods and applications, 18(1), 315-322. Woudenberg J.H.C., et al. 2015. Studies in Mycology. 82(82):1-21. https://doi.org/10.1016/j.simyco.2015.07.001.

Screening for Down's syndrome in early and late first and second trimester using six maternal serum markers.

The efficiency of six maternal serum markers for Down's syndrome (DS), alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), free beta-hCG, pregnancy-associated plasma protein-A (PAPP-A), the proform of eosinophil major basic protein (ProMBP), pregnancy-specific-beta-1-glycoprotein (SP(1)), and combinations thereof, was examined. Discriminant analysis in 156 DS pregnancies and 546 controls defined three effective combinations of serum marker logMoMs (multiples of the median in control samples) in three gestational age windows, i.e. Index I (weeks 7-9) = 0.52 logMoM ProMBP + 0.28 logMoM PAPP-A - logMoM SP(1); Index II (weeks 10-12) = 1.94 logMoM free beta-hCG - logMoM SP(1), and Index III (weeks 15-19) = 0.78 logMoM free beta-hCG + 1.12 logMoM ProMBP - logMoM AFP. The estimated detection rates of indices and age for a false-positive rate (FPR) of 5% were 73% for Index I, 69% for Index II, and 60% for Index III. Including the ultrasound marker nuchal translucency, using a DS at term risk of 1 : 400 as cut-off, the detection rates of the indices increased to 86, 83, and 82% for FPRs of 4.3, 4.1, and 5.8%, respectively. The indices are promising markers for screening for DS.

Development of highly fluorescent detection reagents for the construction of ultrasensitive immunoassays.

We developed two kinds of highly fluorescent streptavidin-based conjugates for use as universal detection reagents in ultrasensitive immunoassays. The direct conjugate was produced by covalently linking streptavidin to poly(Glu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to the conjugate of BSA-poly(Glu:Lys)-Eu chelate. Both direct and indirect conjugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA). Of two monoclonal antibodies used in the assay, one was coated on the well surface of the microtitration strips, and the other was biotinylated. When 10 microL of sample volume was used, we found that the assay using the indirect conjugate had a detection limit of 0.006 microg/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin. However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate. When 45 microL of sample volume was used, a detection limit of 0.001 microg/L was achieved by using the direct conjugate. This improvement in sensitivity should be equally obtainable for the analytes other than PSA. We further demonstrated that the final immunoassay performance was affected not only by the quality of the streptavidin-based conjugate used but also by the quality of the biotinylated antibody reagent. The universal detection reagents described here are believed to be particularly useful for the construction of ultrasensitive time-resolved fluorometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection.

The proform of eosinophil major basic protein: a new maternal serum marker for Down syndrome.

The proform of eosinophil major basic protein (proMBP), the most abundant protein in the eosinophil specific granule, is synthesized by the placenta and secreted into the maternal circulation, where it is found complex-bound to pregnancy-associated plasma protein-A (PAPP-A) and other proteins. We examined the potential of proMBP as a maternal serum marker for fetal Down syndrome (DS) by determining its maternal serum concentration (MSpMBP) in 25 Down syndrome (DS) pregnancies and 152 control pregnancies in the first trimester, and in 105 DS pregnancies and 156 control pregnancies in the second trimester. The median (95 per cent confidence interval) MSpMBP MoM in DS pregnancies (n=15) was 0.66 (0.49-0.79) in gestational weeks 5-9; 1.06 (0.71-1.97) in weeks 10-12 (n=10) and 1.62 (1.18-1.98) in weeks 14-20 (n=105). Using parameterized receiver operator characteristics analysis for proMBP as a single marker for DS, detection rates (DRs) of 22 per cent and 38 per cent, for false-positive rates (FPRs) of 5 per cent, were found in weeks 5-9 (using MSpMBP/=cut-off), respectively. When age and MSpMBP were used as markers in combination, a DR of 36.8 per cent for an FPR of 5.5 per cent was obtained in weeks 5-9 using a risk cut-off of 1:250. In weeks 14-20 the DR was 48.4 per cent for an FPR of 5.3 per cent using the same risk cut-off. This makes proMBP a marker comparable in diagnostic efficiency to human chorionic gonadotrophin (hCG), and exceeding that of alpha-fetoprotein (AFP) and unconjugated oestriol (uE3), in the second trimester.

Schwangerschaftsprotein 1 (SP1) as a maternal serum marker for Down syndrome in the first and second trimesters.

The potential of the maternal serum concentration of schwangerschaftsprotein 1 (MSSP1) as a marker for Down syndrome (DS) pregnancies was evaluated in the fifth to the 20th gestational week using 156 DS pregnancies and 546 unaffected control pregnancies. In DS pregnancies, the median of the multiple of the median (MOM) of MSSP1 was 0.27 [95 per cent confidence interval (CI) 0.11-0.59] in weeks 5-9 (n = 25) and 1.28 (CI 1.11-1.49) in weeks 14-20 (n = 117), significantly different from controls (P < 10(-6). In weeks 10-12, the median MSSP1 MOM was 0.89 (CI 0.20-2.09) (n = 14), not different from controls (P = 0.42). Using MSSP1 alone as a marker for DS gave--in empirical receiver-operator-characteristics (ROC) analysis--a detection rate of about 44 percent for a false-positive rate of about 5 per cent in weeks 5-9 (using MSSP1 MOM < or = cut-off), whereas a sensitivity of about 20 percent was found for a false-positive rate of 5 percent in weeks 14-20 (using MSSP1 MOM > or = cut-off). In parameterized ROC analysis, the detection rates were 38 and 18 percent for a false-positive rate of 5 per cent in weeks 5-9 and 14-20, respectively.

Double-monoclonal immunofluorometric assays for pregnancy-associated plasma protein A/proeosinophil major basic protein (PAPP-A/proMBP) complex in first-trimester maternal serum screening for Down syndrome.

Four double-monoclonal time-resolved immunofluorometric assays (TrIFMAs) have been developed for the specific determination of pregnancy-associated plasma protein A/proeosinophil major basic protein (PAPP-A/ proMBP) complex in first-trimester maternal serum samples. The assays have a functional sensitivity of < 4 mIU/L and a working range from 4 to 1000 mIU/L. These 4 assays, together with a polyclonal sandwich TrIFMA, were compared for their ability to discriminate between normal pregnancies (n = 149) and pregnancies carrying a Down syndrome fetus (n = 36) in maternal serum screening samples from gestational weeks 4-13. In 26 Down syndrome pregnancies from gestational weeks 7-12, the median PAPP-A multiples of the median concentration in controls (MoMs) determined by monoclonal antibody combinations 234-3/234-2*, 234-4/234-2*, 234-4/234-5*, and 234-5/234-6* were 0.35, 0.37, 0.42, and 0.44, respectively, whereas the median MoM determined by the polyclonal assay was 0.56. ROC curve analysis also showed that better overall diagnostic accuracy and detection rates were achieved by the monoclonal TrIFMAs than by the polyclonal TrIFMA. This report is the first to describe assays that specifically measure PAPP-A/proMBP complex without possible interference from other proMBP-containing complexes.

Time-resolved immunofluorometric assay of pregnancy-associated plasma protein A in maternal serum screening for Down's syndrome in first trimester of pregnancy.

A low maternal serum concentration of pregnancy associated plasma protein-A (MS-PAPP-A) in the first trimester has been suggested as a marker for the presence of a Down's syndrome (DS) fetus. We developed a time-resolved immunofluorometric assay (TrIFMA) for PAPP-A with a sensitivity < 3.9 mIU/l. In the 7-12 gestational weeks interval the median multiples of the median (MoM) was 0.57 (95%-confidence interval; 0.47-0.99) in DS pregnancies (n = 29) and lower than in controls (n = 223) (P < 0.005). The efficiency of MS-PAPP-A alone was evaluated using empirical receiver-operator-characteristics (ROC) and a sensitivity of about 25% was found for a false-positive rate of about 10% in the 7-12 gestational weeks interval. In parameterized ROC analysis a sensitivity of 9% was found for a false-positive rate of 5%. The TrIFMA PAPP-A assay seems to fulfil the quality criteria for an assay to be used in large-scale serum screening for Down's syndrome.