Cover Crop and Crop Rotation Effects on Tissue and Soil Population Dynamics of Macrophomina phaseolina and Yield Under No-Till System.

The effects of crop rotation and winter cover crops on soybean yield and colony-forming (CFU) units of Macrophomina phaseolina, the causal agent of charcoal rot (CR), are poorly understood. A field trial was conducted from 2011 to 2015 to evaluate (i) the impact of crop rotation consisting of soybean (Glycine max [L.] Merr.) following cotton (Gossypium hirsutum L.), soybean following corn (Zea mays L.), and soybean following soybean over a 2-year rotation and its interaction with cover crop and (ii) the impact of different cover crops on a continuous soybean crop over a 5-year period. This trial was conducted in a field with 10 subsequent years of cover crop and rotation treatments. Cover crops consisted of winter wheat (Triticum aestivum L.) and Austrian winter pea (Pisum sativum L. subsp. sativum var. arvense), hairy vetch (Vicia villosa Roth), and a fallow treatment was evaluated with and without poultry litter application (bio-cover). Tissue CFU of M. phaseolina varied significantly between crop rotation treatments: plots where soybean was grown following cotton had significantly greater tissue CFU than plots following soybean. Poultry litter and hairy vetch cover cropping caused increased tissue CFU, though this effect differed by year and crop rotation treatment. Soil CFU in 2015 was substantially lower compared with 2011. However, under some crop rotation sequences, plots in the fallow treatment had significantly greater soil CFU than plots where hairy vetch and wheat was grown as a cover crop. Yield was greater in 2015 compared with 2011. There was a significant interaction of the previous crop in the rotation with year, and greater yield was observed in plots planted following cotton in the rotation in 2015 but not in 2011. The result from the continuous soybean planted over 5 years showed that there were no significant overall effects of any of the cover crop treatments nor was there interaction between cover crop treatment and year on yield. The lack of significant interaction between crop rotation and cover crop and the absence of significant differences between cover crop treatments in continuous soybean planting suggest that cover crop recommendations for midsouthern soybean growers may need to be independent of crop rotation and be based on long-term crop needs.

Screening Tall Fescue for Resistance to Rhizoctonia solani and Rhizoctonia zeae Using Digital Image Analysis.

Brown patch, caused by Rhizoctonia solani, is a destructive disease on tall fescue. Compared with R. solani, Rhizoctonia zeae causes indistinguishable symptoms in the field but varies in geographic distribution. This may contribute to geographic variability observed in the resistance response of improved brown patch-resistant cultivars. This study examined R. solani and R. zeae susceptibility of four cultivars, selected based on brown patch performance in the National Turfgrass Evaluation Program (NTEP), and nine plant introductions (PIs). Twenty genotypes per PI/cultivar were evaluated by using four clonal replicates in a randomized complete block design. Plants were inoculated under controlled conditions with two repetitions per pathogen. Disease severity was assessed digitally in APS Assess, and analysis of variance and correlations were performed in SAS 9.3. Mean disease severity was higher for R. solani (65%) than for R. zeae (49%) (P = 0.0137). Interaction effects with pathogen were not significant for PI (P = 0.0562) but were for genotype (P < 0.001). Moderately to highly resistant NTEP cultivars compared with remaining PIs exhibited lower susceptibility to R. zeae (P < 0.0001) but did not differ in susceptibility to R. solani (P = 0.7458). Correlations between R. solani and R. zeae disease severity were not significant for either PI (R = 0.06, P = 0.8436) or genotype (R = 0.11, P = 0.09). Breeding for resistance to both pathogens could contribute to a more geographically stable resistance response. Genotypes were identified with improved resistance to R. solani (40), R. zeae (122), and both pathogens (26).

Scarless fetal mouse wound healing may initiate apoptosis through caspase 7 and cleavage of PARP.

INTRODUCTION: Apoptotic mechanisms are thought to be important in wound healing for the removal of inflammatory cells and evolution of granulation tissue. However, little is understood about the signal, propagation, and mechanisms responsible for triggering cell death in tissue injury, particularly during fetal wound repair. Understanding these signals may lead to insights regarding scarless wound healing. We hypothesized that differences in apoptosis would exist in mid- (E15) compared with late-gestational (E18) mice subjected to cutaneous wounds. We examined early apoptotic signals that may be initiated following tissue injury. METHODS: Pregnant, time-dated mice underwent laparotomy and hysterotomy on embryonic day 15 (E15) and 18 (E18). Full-thickness, excisional cutaneous wounds were made on the dorsum of the fetuses and dorsal skin harvested 15 and 45 min after wounding. Unwounded dorsal skin from additional fetuses collected at the same time points served as controls. The skin was processed to obtain protein, then levels of caspase 3, caspase 7, and poly ADP-ribose polymerase (PARP) were measured by Western blot. Cyclophilin levels were measured to ensure equal loading of protein. Histone-associated DNA complex formation was examined to provide further evidence of cellular apoptosis. RESULTS: There were no differences in total caspase 3 levels between E15 and E18 fetal tissue with or without wounding, nor was any cleavage of caspase 3 noted in any group. However, cleaved caspase 7 was present in the E15 skin with a >2-fold increase following wounding at both 15 and 45 min, yet absent in the E18 groups. Furthermore, levels of cleaved PARP were also increased by >2-fold at both 15 and 45 min in E15 wound groups, whereas a relatively small amount was only seen in the E18 wound groups at 45 min. DNA-histone fragmentation ELISA assay showed a 5-fold increase in the enrichment of histone-associated DNA fragments in the E15 wounded tissue compared with the time-matched controls at 45 min. This was not seen with the E18 tissue. CONCLUSIONS: Previously, we demonstrated that cutaneous wounds in E15 fetal mice heal in a scarless manner, while similar wounds in E18 mice heal with scar formation. Results from our current work demonstrate differences in apoptosis in mid- compared with late-gestational mouse skin as well as shortly after wounding. Our results suggest that in mid-gestational wounds, activation of apoptotic pathways may be mediated through effector caspase 7 signals with inactivation of PARP. This initiation of apoptotic signals following tissue injury may play a role in scarless wound repair.

Differential expression of procollagen genes between mid- and late-gestational fetal fibroblasts.

BACKGROUND: Previously, we have shown that cutaneous wounds in mid-gestational (E15) mice heal in a scarless manner with decreased procollagen 1 and increased procollagen 3 production compared with wounds in late-gestational (E18) mice, which heal with scars. The aim of the current work was to determine whether E15 and E18 fibroblasts respond to stimulation in culture with differential procollagen expression, suggesting they may preserve their phenotype in vitro. Further, we wanted to determine if fetal fibroblast gene expression patterns persisted in tissue culture. We measured expression of procollagen types 1alpha1 and 3 in response to TGF-beta1 stimulation. We theorized that E15 fibroblasts would respond with a pattern of procollagen that would contribute to a more easily remodeled collagen. METHODS: Mid- and late-gestational fetal fibroblasts were obtained from dorsal skin harvested from fetuses of time-dated CD-1 mice. Cells were grown to confluence in culture plates overnight. Cell monolayers were treated with 0.01% bovine serum albumin (BSA) plus 10 ng/well of TGF-beta1. Cells were harvested at 6 and 24 h following treatment. Additional groups were treated with BSA alone (vehicle controls) and collected at 6 and 24 h. Another group without treatment was harvested after reaching confluence (0 time point). In a separate experiment to determine if gene expression patterns persisted, cells were treated with 0.01% BSA plus 10 ng/well of TGF- beta1 for 24 h, then harvested. A second group of cells were treated again at 24 h and harvested at 48 h. Additional cells were treated with BSA alone for 24 and 48 h, and another group without treatment was harvested after reaching confluence (0 time point). Cells were processed to obtain mRNA, cDNA was made, and then samples analyzed by QPCR. Results were analyzed by ANOVA and Holm-Sidak method. RESULTS: Procollagen 1alpha1 gene expression was decreased in E15 cells at 6 and 24 h following TGF-beta1 treatment, P<0.05. In contrast, procollagen 1alpha1 was increased in E18 cells, P<0.05. Procollagen 3 gene expression was decreased in E18 cells at 6 and 24 h following treatment with TGF-beta1, P<0.05, whereas levels in E15 cells were unchanged at 6 h, and only trended lower at 24 h. We evaluated whether this differential expression of procollagen 3 persisted at 24 and 48 h. At 24 and 48 h, E15 control groups had increased procollagen 3 expression compared with E18 groups, P<0.05. E15 and E18 cells in TGF-beta1-treated groups had decreased procollagen 3 at 48h compared with their respective BSA control groups, P<0.05. However, the degree of difference appeared to be greater in the E15 group than the E18 group. CONCLUSIONS: Our results from this in vitro work demonstrate a differential pattern of gene expression for procollagen 1alpha1 and 3 in E15 and E18 fibroblasts in response to TGF-beta1. E15 cells showed decreased expression of procollagen 1alpha1, while E18 cells showed increased procollagen 1alpha1 and decreased procollagen 3 expression. These patterns of expression in E15 cells are suggestive of increased type 3 to 1 collagen ratio seen in scarless fetal wounds. Interestingly, treatment of either E15 or E18 cells with TGF-beta1 significantly decreased procollagen 3 expression by 48 h, yet this was more profound in E15 groups. This suggests that after 24 h, E15 cells may transition towards an E18 phenotype and corresponding signaling.

Homeobox genes Hoxd3 and Hoxd8 are differentially expressed in fetal mouse excisional wounds.

BACKGROUND: Cell signaling pathways underlying wound repair are under extensive investigation; however, there is still a poor understanding of the mechanisms orchestrating these processes. Hox genes, which are a subgroup of homeobox genes, encode for a family of transcription factors that play a critical role in tissue migration and cell differentiation during embryogenesis and may also serve as master regulatory genes of postnatal wound repair. We have developed a fetal excisional wound healing model whereby mid-gestational wounds heal in a regenerative manner while late-gestational wounds display scar formation. We theorize that Hoxd3 and Hoxd8 will be differentially expressed in mid- and late-gestational wounds compared with normal skin. MATERIALS AND METHODS: Pregnant FVB mice underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional wounds were made on the dorsum of each fetus. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control (c) skin samples were also obtained from unwounded adjacent fetuses. Samples were harvested at 3 and 6 h and real-time polymerase chain reaction was performed for Hoxd3 and Hoxd8 and normalized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by analysis of variance with statistical significance of P < 0.05. RESULTS: Hoxd3 levels were increased in all of the mid-gestational groups, with a significant increase at 3 h compared with late-gestational control groups. In the 3-h time group, Hoxd8 is increased in mid-gestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group, where Hoxd8 is increased in mid-gestational wounds compared with all groups. Also, Hoxd8 in the mid-gestational near wound controls is significantly greater than that in the late-gestational near wound control and control groups. CONCLUSIONS: These data suggest that Hoxd3 is constitutively expressed in the skin of mid-gestational mice. However, Hoxd8 expression is increased in the mid-gestational wounds compared with normal control groups and late gestational wounds, suggesting that it may play a role in scarless wound repair.

Differential use of Erk1/2 and transforming growth factor beta pathways by mid- and late-gestational murine fibroblasts.

BACKGROUND: Previously, we demonstrated the rapid closure of mid-gestational excisional murine wounds at 32 hours. In this study, we theorized that mid-gestational wounds would be completely regenerated, whereas late-gestational wounds would heal with scar formation at 48 hours. Furthermore, we theorized that mid- and late-gestational fibroblasts differentially use the transforming growth factor beta and mitogen-activated protein kinase pathways. METHODS: Three-millimeter excisional cutaneous wounds were made on murine mid- (embryonic day 15 [E15]) and late-gestational (E18) fetuses and harvested at 48 hours for histology. Percent wound closure was calculated. E15 and E18 fibroblasts were cultured overnight for in vitro scratch wound assay in the presence of the activin receptor-like kinase 4-5-7, Erk1/2, and p38 inhibitors. RESULTS: E15 wounds healed in a regenerative manner, whereas E18 wounds exhibited scar formation. In vitro scratch closure was similar in the E15 and E18 groups at 8 hours; yet, it increased in E15 compared with E18 groups with activin receptor-like kinase 4-5-7 and Erk1/2 inhibitors. p38 inhibition resulted in reduced scratch closure in both groups. CONCLUSION: The scarless mid-gestational excisional wounds compared with the scar-forming late-gestational wounds provides a model to study scar formation. This study also suggests that variable transforming growth factor beta and Erk1/2 signaling may influence differences in wound closure between mid- and late-gestational wounds.

Altered procollagen gene expression in mid-gestational mouse excisional wounds.

INTRODUCTION: Many pathologic conditions are characterized by excessive tissue contraction and scar formation. Previously, we developed a murine model of excisional wound healing in which mid-gestational wounds heal scarlessly compared with late-gestational wounds. We theorized that variations in procollagen gene expression may contribute to the scarless and rapid closure. METHODS: Time-dated pregnant FVB strain mice underwent laparotomy and hysterotomy on embryonic days 15 (E15) and 18 (E18). Full-thickness, excisional wounds (3 mm) were made on each of 4 fetuses per doe and then harvested at 32, 48, or 72 h. Control tissue consisted of age-matched normal fetal skin. Procollagen types 1alpha1, 1alpha2, and 3 gene expressions were measured by real-time polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase. Trichrome staining was also performed. RESULTS: Procollagen 1alpha1 expression was decreased in E15 wounds at 32 h compared with their normal skin groups. Procollagen types 1alpha2 and 3 expressions were both increased in the E15 groups compared with the E18 groups at 48 h. At 72 h, the E15 wounds had a collagen density similar to the surrounding normal skin while E18 wounds exhibited increased collagen deposition in a disorganized pattern. CONCLUSIONS: This study demonstrates that the pattern of gene expression for types 1 and 3 collagen varies between mid- and late-gestational mouse excisional wounds. These alterations in procollagen expression may contribute to a pattern of collagen deposition in the mid-gestational fetuses that is more favorable for scarless healing with less type 1 and more type 3 collagen.

Rapid closure of midgestational excisional wounds in a fetal mouse model is associated with altered transforming growth factor-beta isoform and receptor expression.

BACKGROUND: Many pediatric diseases are characterized by excessive tissue contraction. Because of a poor understanding of contraction, few therapies exist. We developed a murine fetal excisional wound model of contraction and theorize that wound closure is associated with changes in transforming growth factor-beta (TGF-beta) expression. METHODS: Pregnant FVB mice underwent hysterotomy at midgestational (E15) or late-gestational (E18) ages. Three-millimeter excisional wounds were made in fetuses and harvested at 32 hours. Real-time polymerase chain reaction was performed for TGF-beta1, TGF-beta2, TGF-beta3, TbetaR-1, and TbetaR-2 in wounds and normal skin and normalized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by paired t test (P < .05). H&E staining of wounds was performed. RESULTS: E15 wounds (80.5% +/- 4.4%) were smaller than E18 wounds (10.4% +/- 10.5%; P < .001) at 32 hours. E15 wounds expressed higher levels of TGF-beta1 compared with normal skin (P = .001). TbetaR-2 levels were elevated in E15 and E18 wounds compared with their respective normal skin (P = .02, P = .01) and in E18 normal skin compared with E15 normal skin (P = .002). CONCLUSION: This study demonstrates that rapid midgestational wound closure in a murine model is associated with increased TGF-beta1 and TbetaR-2 expression. Elucidating the role of the TGF-beta pathways may lead to an improved understanding of wound contraction.

Association of a single nucleotide polymorphism in the matrix metalloproteinase-1 promoter with glioblastoma.

A key feature in the malignant behavior of glioblastoma is the tendency to invade host brain tissue surrounding the primary tumor site. Several members of the matrix metalloproteinase family are thought to contribute to this invasive capacity. A single nucleotide polymorphism has been described in the matrix metalloproteinase-1 (MMP-1) promoter that consists of either the presence or absence of a guanine nucleotide at position -1607. The presence of the guanine base creates a functional binding site for members of the ETS family of transcription factors and has been shown to increase MMP-1 transcription. The purpose of our study was to characterize this polymorphism in human glioblastoma. Promoter genotyping was performed on brain tumor tissue obtained from 81 patients and compared to 57 healthy individuals. The 2G/2G genotype is more prevalent in glioblastoma tissue compared to healthy individuals (p = 0.01). mRNA and protein expression were measured in a subset of brain tumor and normal brain tissue samples. MMP-1 protein levels are significantly higher in glioblastoma tissue compared to normal brain (p = 0.001). Electromobility shift assays and promoter assays were performed to assess binding capability and transcriptional activity, respectively. Proteins present in glioma cell lines can specifically bind the 2G promoter probe. MMP-1 transcription is significantly higher in cells transfected with the 2G promoter when compared to cells transfected with the 1G promoter (p<0.02). This polymorphism may provide a mechanism for increased expression of MMP-1 in malignant gliomas via elevation of MMP-1 mRNA transcription and may underlie the invasive phenotype.