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Transfusion of autologous blood is a timesaving, convenient, safe, and effective therapy from a clinical perspective, and often employed for the treatment of diabetic patients. Stabilization of HIF-1α has been widely reported to be a critical factor in the improvement of wound healing in diabetes. Therefore, our study reveals the roles of improved autologous blood in wound healing in diabetes, through autologous blood transfusion in a mouse model. Initially, BALB/c mice were subjected to streptozotocin for diabetic mouse model establishment. Diabetic mice were transfused with improved or standard autologous blood in perfusion culture system. Roles of improved autologous blood in mediating HIF-1α pathway were determined by measuring expression of VEGF, EGF, HIF-1α, and HSP-90. In order to assess the detailed regulatory mechanism of improved autologous blood in perspective of wound healing, cell proliferation, migration and cell cycle, fibroblasts isolated from diabetic mice were transfected with HIF-1α siRNA. Mice transfused with improved autologous blood exhibited increased levels of CD31 and α-SMA in skin tissues, and reduced TNF-α, IL-1β, and IL-6 levels, indicating that improved autologous blood promoted wound healing ability and reduced the release of inflammatory factors. Diabetic mice transfused with improved autologous blood presented activated HIF-1α pathway. The survival rate, proliferation, and migration of fibroblasts were elevated via activation of the HIF-1α pathway. Taken together, improved blood preservation solution could enhance the oxygen carrying capacity of red blood cells and wound healing in mice with diabetes, which is achieved through regulation of HIF-1α pathway.

The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24 Rhizoctonia solani isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS/ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than IGS/ITS-MP-PCR because of their higher redundancy in the fungal genome. The number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used. Each primer used could differentiate all of the fungal isolates examined in this study. The profiles generated were identical and reproducible between repeated PCR experiments. PRACTICAL APPLICATIONS Combining the intergenic spacer/internal transcribed spacer-microsatellite-primed polymerase chain reaction technique with microsatellite–detection assay allows the rapid and specific detection of Rhizoctonia solani anastomosis groups and different phytopathogenic fungi. The utility of this approach stems from its simplicity and reproducibility, the high number of polymorphisms revealed, the very small amounts of DNA needed, rapidity, and ease of performance. The improved technique will present valuable information on the role of some phytopathogenic fungi and R. solani in agriculturally important plant diseases.

A real-time PCR assay was applied to evaluate the effects of two fungicide treatments on Mycosphaerella graminicola leaf blotch in the field and compared with two usual visual methods. The three fungicides Fandango, Input and Prosaro used in the two treatments showed high inhibitions against germ tube growth and conidial germination in vitro, suggesting that they had high curative and protective activities. In the field, the fungicide effect was assessed for two times on 17 May and 14 June, 2004. M. graminicola DNA, disease index and pycnidial number per leaf reduced obviously on most leaf layers after both treatments. According to the real-time PCR data, significant effects were still observed on leaf 3, 4 and 5 even after 25 days of treatment (17 May). And on 14 June, treatment A significantly decreased the DNA amount by 99.42, 87.68, 24.12 and 68.87% on leaf 1, 2, 3 and 4 and treatment B by 98.83, 98.12, 70.79 and 64.15% respectively. In contrast, effects could neither be assessed by disease classification on leaf 5 of treatment B on 17 May and leaf 3 and 4 of treatment A on 14 June and nor by pycnidia-counting method on leaf 3 and 5 of treatment B on 17 May and leaf 4 of both treatments on 14 June. The band intensity of final real-time PCR products could be hardly distinguished on agarose gel amongst those samples with disease index >2.7, although their initial DNA amounts quantified by real-time PCR were different, indicating the superiority of the real-time PCR assay over those PCR assays estimating initial DNA input with endpoint measurement. All this shows that the real-time PCR assay may serve as an alternative method for accurate assessment of the fungicide effects on M. graminicola leaf blotch.

Mycosphaerella graminicola is a prevalent and economically important fungal pathogen in wheat. Presymptomatic and accurate detection of viable pathogen structures in infected host plants is desirable for determining latent periods of epidemics and for timely fungicide application. A reverse transcription polymerase chain reaction (RT–PCR) assay was used to detect and identify M. graminicola in wheat with the specific primer set E1/STSP2R. One single fragment was amplified only from total RNA of M. graminicola and infected leaves, but not from those of healthy leaves or five other common fungal wheat pathogens. The sensitivity of one-step RT–PCR was compared with that of two-step RT–PCR. The detection limit of two-step RT–PCR was markedly lower (100 pg total RNA) than that of one-step RT–PCR (5 ng total RNA). RT–PCR was used to monitor M. graminicola disease development in inoculated wheat plants and in naturally infected F1 leaves (the second leaf from top) during the epidemic period. RT–PCR could detect M. graminicola at least 4 days before lesions appeared in inoculated plants. After symptoms were visible on the leaves, the band intensities of the amplified RT–PCR products were in general agreement with the visual disease assessment.

An effective and rapid method for RNA extraction from conidia and mycelium of S. tritici was developed, in which guanidinium isothiocyanate, CTAB and insoluble PVP were combined in one step, simplifying the extraction process greatly. The procedure can be completed in less than 6 h. Yield and purity of RNA could be estimated by electrophoresis on 1% formaldhyde-denatured agarose gel and by measuring the absorbance at 280, 260, 230 and 210 nm by UV spectroscopy. The result of electrophoresis showed two clear bands of 18S and 28S rRNA and no DNA contamination in the isolated RNA solution. The ratios A260/A280, A260/A230, A260/A210 were 1.76–1.82, 2.79–3.21 and 0.81–0.86, respectively. A high yield with 532–881 ng total RNA per gram fresh conidia and mycelium of S. tritici was obtained. The RNA was of sufficient purity for RT-PCR to detect the expression of β-tubulin gene in S. tritici. This protocol may also be suitable for RNA extraction from spores and mycelium of other fungi.