Screening of Diverse Lupinus spp. Highlights New Resistances to Sclerotinia sclerotiorum.

Stem rot caused by Sclerotinia sclerotiorum is a serious, and sometimes devastating, disease of lupin (Lupinus spp.). Two hundred and thirty-six lupin accessions from across 12 Lupinus species were screened against the prevalent S. sclerotiorum isolate MBRS-1 (pathotype 76). L. angustifolius accession 21655 and L. albus var. albus accession 20589 showed immune and 'near-immune' responses, respectively. Thirteen accessions of L. angustifolius, three accessions each of L. albus and L. albus var. albus, and a single accession each of L. albus var. graecus, L. mutabilis, L. palaestinus and L. pilosus (totalling ~4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, two accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totalling ~10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, one L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, WW-3 (pathotype 10). Against this isolate, five L. angustifolius accessions showed a HR response and four showed a R response, and the L. digitatus accession showed a moderate resistance (MR) response. Overall, isolate WW-3 caused significantly (P<0.05) smaller lesions than MBRS-1 across tested accessions in common. In addition, 328 plants in a 'wild' naturalized field population of L. cosentini were screened in situ in the field against isolate MBRS-1. Five (~1.5%) of the 328 plants of wild lupin showed an immune response, 63 (~19%) showed a HR response, and 146 (~45%) showed a R response. We believe this is the first examination of diverse Lupinus spp. germplasm responses to a prevalent pathotype of S. sclerotiorum. Lupin genotypes exhibiting high level resistance to Sclerotinia stem rot identified in this study can now be used as parental lines for crosses in lupin breeding programs and/or directly as improved cultivars to reduce the adverse impact of this disease on lupin crops.

Genetic and molecular analysis of stem rot (Sclerotinia sclerotiorum) resistance in Brassica napus (canola type).

Identifying the molecular and genetic basis of resistance to Sclerotinia stem rot (Sclerotinia sclerotiorum) is critical for developing long-term and cost-effective management of this disease in rapeseed/canola (Brassica napus). Current cultural or chemical management options provide, at best, only partial and/or sporadic control. Towards this, a B. napus breeding population (Mystic x Rainbow), including the parents, F1, F2, BC1P1 and BC1P2, was utilized in a field study to determine the inheritance pattern of Sclerotinia stem rot resistance (based on stem lesion length, SLL). Broad sense heritability was 0.58 for SLL and 0.44 for days to flowering (DTF). There was a significant negative correlation between SLL and stem diameter (SD) (r = -0.39) and between SLL and DTF (r = -0.28), suggesting co-selection of SD and DTF traits, along with SLL, should assist in improving overall resistance. Non-additive genetic variance was evident for SLL, DTF, and SD. In a genome wide association study (GWAS), a significant quantitative trait locus (QTL) was identified for SLL. Several putative candidate marker trait associations (MTA) were located within this QTL region. Overall, this study has provided valuable new understanding of inheritance of resistance to S. sclerotiorum, and has identified QTL, MTAs and transgressive segregants with high-level resistances. Together, these will foster more rapid selection for multiple traits associated with Sclerotinia stem rot resistance, by enabling breeders to make critical choices towards selecting/developing cultivars with enhanced resistance to this devastating pathogen.

Hsa_circ_0001162 Inhibition Alleviates High Glucose-Induced Human Podocytes Injury by the miR-149-5p/MMP9 Signaling Pathway.

Emerging evidences suggested that circular RNAs (circRNAs) are involved in diabetic nephropathy (DN). Accumulating evidence had suggested that the degree of podocyte is a major prognostic determinant of DN progression. However, the function and in-depth mechanisms of hsa_circ_0001162 in podocyte injury of DN remain unclear. Hsa_circ_0001162 expression was detected by real-time quantitative PCR (RT-qPCR) in peripheral blood of DN patients and high glucose-induced podocytes injury model. The cell counting kit 8, 5-ethynyl-2'-deoxyuridine, flow cytometry with Annexin V-FITC/PI staining, caspase-3 activity assay Kit, enzyme linked immunosorbent assay (ELISA), RT-qPCR and western blotting were used to evaluate the effect of hsa_circ_0001162 / miR-149-5p / MMP9 axis on high glucose-induced podocyte injury. Mechanistically, dual luciferase reporter was used to confirm the relationship of miR-149-5p and hsa_circ_0001162 or MMP9. Furthermore, RNA-pull down and immunoprecipitation assay were implemented to verify the potential regulatory effects of EIF4A3 on biogenesis of hsa_circ_0001162. Our results showed that hsa_circ_0001162 was highly expressed in peripheral blood of DN patients and high glucose-induced podocytes injury model, and the knockdown of hsa_circ_0001162 increased the proliferation, inhibited the apoptosis, and suppressed inflammatory response in high glucose-induced podocytes injury. Mechanism studies demonstrated that EIF4A3 bound with flanking sequences of hsa_circ_0001162 to promote hsa_circ_0001162 expression, upregulated hsa_circ_0001162 increased the MMP9 expression via sponging miR-149-5p, thus aggravating the high glucose-induced podocytes injury. Overall, our data demonstrated that knockdown of hsa_circ_0001162 inhibited high glucose-induced podocytes injury by regulating miR-149-5p/MMP9 axis, and intervention of hsa_circ_0001162/miR-149-5p/MMP9 axis may be a potentially promising therapeutic strategy for podocyte injury in DN patients.

Novel Resistances Provide New Avenues to Manage Alternaria Leaf Spot (Alternaria brassicae) in Canola (Brassica napus), Mustard (B. juncea), and Other Brassicaceae Crops.

Alternaria leaf spot (Alternaria brassicae) can be a devastating disease in canola (Brassica napus) and mustard (B. juncea), but there are no highly effective host resistances available. Screening of 150 diverse Brassicaceae varieties under glasshouse conditions highlighted important novel resistances. In particular, Camelina sativa '4076' and Diplotaxis erucoides 'Wasabi Rocket' had complete resistance across disease assessment parameters (leaf incidence [%LDI]; severity [%LAD]; consequent defoliation [%LCI]). The next most resistant varieties were C. sativa 'CSA' (%LDI 0.6; %LAD 0.4), '4144' (%LDI 1.2; %LAD 0.5), '405' (%LDI 1.7; %LAD 0.7), C. sativa '3274' (%LDI 2.5; %LAD 0.8), Carrichtera annua 'CAN3' (%LDI 7.7; %LAD 4.0), and Sisymbrium irio 'London Rocket' (%LDI 2.1; %LAD 0.8), all with %LCI values of 0. Other genotypes showing high-level resistance included S. erysimoides 'SER 4' (%LDI 11.8; %LAD 5.6; %LCI 0) and D. cardaminoides 'Wild Rocket' (%LDI 15.5; %LAD 7.2; %LCI 0), and those showing moderate resistance were Brassica carinata 'ML-EM-1' (Rungwe), B. insularis 'Moris', B. napus 'ZY006', B. oxyrrhina 'BOX1', B. oleracea var. capitata 'Sugarloaf', B. tournefortii 'CN01-104-2', and Sinapis alba 'Concerta' with %LDI 21.6 to 29.8, %LAD 12.8 to 21.0, and %LCI 0 to 5.7. In particular, B. napus 'ZY006' for canola and B. oleracea var. capitata 'Sugarloaf' can now be directly utilized (i.e., without crossing impairment) for Brassica species and vegetable breeding programs, respectively. While all B. juncea genotypes were susceptible, there were some less susceptible varieties from India in comparison with genotypes from Australia or China. The most susceptible test genotype was Rapistrum sativus (%LDI 89.4; %LAD 83.9; %LCI 71.0), highlighting the value of the resistances identified. These findings not only highlight a range of novel resistances against A. brassicae for canola, mustard, and other diverse Brassicaceae breeding programs to develop resistant commercial varieties, but also emphasize highly susceptible varieties to avoid in both breeding programs and commercial situations conducive to Alternaria leaf spot.

Novel machine learning models to predict pneumonia events in supratentorial intracerebral hemorrhage populations: An analysis of the Risa-MIS-ICH study.

Background: Stroke-associated pneumonia (SAP) contributes to high mortality rates in spontaneous intracerebral hemorrhage (sICH) populations. Accurate prediction and early intervention of SAP are associated with prognosis. None of the previously developed predictive scoring systems are widely accepted. We aimed to derive and validate novel supervised machine learning (ML) models to predict SAP events in supratentorial sICH populations. Methods: The data of eligible supratentorial sICH individuals were extracted from the Risa-MIS-ICH database and split into training, internal validation, and external validation datasets. The primary outcome was SAP during hospitalization. Univariate and multivariate analyses were used for variable filtering, and logistic regression (LR), Gaussian naïve Bayes (GNB), random forest (RF), K-nearest neighbor (KNN), support vector machine (SVM), extreme gradient boosting (XGB), and ensemble soft voting model (ESVM) were adopted for ML model derivations. The accuracy, sensitivity, specificity, and area under the curve (AUC) were adopted to evaluate the predictive value of each model with internal/cross-/external validations. Results: A total of 468 individuals with sICH were included in this work. Six independent variables [nasogastric feeding, airway support, unconscious onset, surgery for external ventricular drainage (EVD), larger sICH volume, and intensive care unit (ICU) stay] for SAP were identified and selected for ML prediction model derivations and validations. The internal and cross-validations revealed the superior and robust performance of the GNB model with the highest AUC value (0.861, 95% CI: 0.793-0.930), while the LR model had the highest AUC value (0.867, 95% CI: 0.812-0.923) in external validation. The ESVM method combining the other six methods had moderate but robust abilities in both cross-validation and external validation and achieved an AUC of 0.843 (95% CI: 0.784-0.902) in external validation. Conclusion: The ML models could effectively predict SAP in sICH populations, and our novel ensemble model demonstrated reliable robust performance outcomes despite the populational and algorithmic differences. This attempt indicated that ML application may benefit in the early identification of SAP.

Faba Bean Gall Pathogen Physoderma viciae: New Primers Reveal Its Puzzling Association with the Field Pea Ascochyta Complex.

Recent morphological and molecular studies confirmed Physoderma viciae, and not Olpidium viciae, to be the causative agent of the devastating Faba Bean Gall (FBG) disease on faba bean (Vicia faba) in Ethiopia and also highlighted its ability to cross-infect with other host genera such as Pisum and Trifolium. In this study, the first pair of specific primer 'Physo 1' and primer pair 'Physo D' are reported from molecular sequences of this pathogen from the conserved LSU (S28) gene. Whereas 'Physo 1' readily detects P. viciae, 'Physo D', clearly separates its identity from the common and confounding presence of Didymella/Phoma spp. The study also reports the presence of the Ascochyta blight pathogen complex, symptomless but almost universal on field pea (Pisum sativum), within faba bean infested by P. viciae. We emphasize historical evidence confirming such unique association in other legumes, such as the subterranean clover (Trifolium subterraneum). This new finding has significant implications for rotations involving different legume crop and/or forage legume genera and possibly provides the first explanation for the widespread occurrence of the field pea Ascochyta blight pathogen complex even in the absence of field pea cropping for many years.

Quantitative Inheritance of Sclerotinia Stem Rot Resistance in Brassica napus and Relationship to Cotyledon and Leaf Resistances.

Sclerotinia sclerotiorum is a necrotrophic fungus causing devastating stem rot and associated yield losses of canola/rapeseed (Brassica napus) worldwide, including in Australia. Developing host resistance against Sclerotinia stem rot is critical if this disease in canola/rapeseed is to be successfully managed, as cultural or chemical control options provide only partial or sporadic control. Three B. napus breeding populations, C2, C5 and C6, including the parents, F1, F2, BC1P1, and BC2P2, were used in a field study with an objective of exploring the inheritance pattern of disease resistance (based on stem lesion length [SLL]) and the genetic relationships of disease with stem diameter (SD) or days to first flowering (DTF), and to compare these new adult plant stem resistances against S. sclerotiorum with those of seedling (cotyledon and leaf) resistances in earlier studies. Heritability (broad sense) of SLL was 0.57 and 0.73 for population C2 at 3 and 5 weeks postinoculation and 0.21 for population C5 at 5 weeks postinoculation. Additive genetic variance was evident within all 3 populations for DTF but not for SD. Narrow-sense heritability for DTF was 0.48 (C2), 0.42 (C5), and 0.32 (C6). SD, DTF, and SLL were all inherited independently, with no significant genetic covariance between traits in bivariate analysis. Genetic variance for SLL in populations C2 and C5 was entirely nonadditive, and there was significant nonadditive genetic covariance of SLL at 3 and 5 weeks postinoculation. Generation means analysis in population C2 supported the conclusion that complex epistatic interactions controlled SLL. Several C2 and C5 progeny showed high adult plant stem resistance, which may be critical in developing enhanced stem resistance in canola/rapeseed. Although population C6 showed no genetic variation for SLL resistance in this study, it showed significant nonadditive genetic variance at the cotyledon and leaf stages in earlier studies. We conclude that host resistance varies across different plant growth stages, and breeding must be targeted for resistance at each growth stage. In populations C2, C5, and C6, resistance to S. sclerotiorum in stem, leaf, and cotyledon was always controlled by nonadditive effects such as complex epistasis or dominance. Overall, our findings in relation to the quantitative inheritance of Sclerotinia stem rot resistance, together with the new high-level resistances identified, will enable breeders to select/develop genotypes with enhanced resistances to S. sclerotiorum.

Decellularized extracellular matrix mediates tissue construction and regeneration / 医学前沿

Contributing to organ formation and tissue regeneration, extracellular matrix (ECM) constituents provide tissue with three-dimensional (3D) structural integrity and cellular-function regulation. Containing the crucial traits of the cellular microenvironment, ECM substitutes mediate cell-matrix interactions to prompt stem-cell proliferation and differentiation for 3D organoid construction in vitro or tissue regeneration in vivo. However, these ECMs are often applied generically and have yet to be extensively developed for specific cell types in 3D cultures. Cultured cells also produce rich ECM, particularly stromal cells. Cellular ECM improves 3D culture development in vitro and tissue remodeling during wound healing after implantation into the host as well. Gaining better insight into ECM derived from either tissue or cells that regulate 3D tissue reconstruction or organ regeneration helps us to select, produce, and implant the most suitable ECM and thus promote 3D organoid culture and tissue remodeling for in vivo regeneration. Overall, the decellularization methodologies and tissue/cell-derived ECM as scaffolds or cellular-growth supplements used in cell propagation and differentiation for 3D tissue culture in vitro are discussed. Moreover, current preclinical applications by which ECM components modulate the wound-healing process are reviewed.

Influence of metabolic syndrome on activities of daily living in middle-aged and elderly population in China: a prospective cohort study / 中华流行病学杂志

Objective: To examine the associations between metabolic syndrome (MS) and the risks for impaired activities of daily living (ADL) in the middle-aged and elderly population in China and provide basis for improving healthy life expectancy. Methods: Prospective cohort study design was used in this study. Based the data of China Health and Retirement Longitudinal Study (CHARLS) from 2011 to 2018 and the baseline data in 2011, the follow up for ADL outcomes was conducted in 2013, 2015 and 2018 respectively, and the participants recruited in 2013 and 2015 were new baseline populations. The participants with impaired ADL in three baseline populations were excluded. Cox proportional hazard model was used to estimate the effect of different components and number of MS components on the risk for ADL impairment in the middle-aged and elderly population in China. Results: In 59 795 person-years of follow-up, a total of 1 011 cases of ADL impairments were recorded. The incidence density of ADL impairment was 16.91 per 1 000 person-year; The findings of Cox regression analysis showed that compared with the middle aged and elderly people without MS, the risk for ADL impairments was 1.29 times higher (95%CI: 1.12-1.50) for those with one component of MS, 1.32 times higher (95%CI:1.07-1.64) for those with hyperlipemia complicated with diabetes, 1.78 times higher (95%CI: 1.22-2.59) for those with obesity and one component of MS, 2.48 times higher (95%CI:1.59-3.85) for those with hypertension complicated with hyperlipemia, 3.51 times higher (95%CI:1.66-7.43) for those with hypertension complicated with diabetes, 1.80 times higher (95%CI: 1.40-2.32) for those with ≥3 MS, respectively. Compared with the middle-aged and elderly population without MS, the risk for impaired ADL increased by 30% (HR=1.30, 95%CI: 1.13-1.51), 54% (HR=1.54, 95%CI: 1.28-1.85) and 87% (HR=1.87, 95%CI:1.45-2.41), respectively, in the middle-aged and elderly with one, two, and more than three components of MS, with a significant dose-response relationship that ADL impaired risks increased as the number of MS components increased (P<0.001). Conclusions: Our findings suggested that MS is a risk factor for ADL impairment in middle-aged and elderly population in China. Prevention and early intervention of obesity, hypertension, hyperlipemia, diabetes and metabolic syndrome could help to reduce or delay the incidence of ADL impairment.

Saikosaponin D Inhibits Peritoneal Fibrosis in Rats With Renal Failure by Regulation of TGFß1/ BMP7 / Gremlin1/ Smad Pathway.

Peritoneal dialysis (PD) can improve the quality of life of patients with kidney disease and prolong survival. However, peritoneal fibrosis can often occur and lead to PD withdrawal. Therefore, it is imperative to better understand how to inhibit and slow down progression of peritoneal fibrosis. This study aimed to investigate the regulatory effect of Saikosaponin d (SSD), a monomer extracted from the plant Bupleurum, on peritoneal fibrosis and the contribution of TGFß1/BMP7/Gremlin1 pathway cross-talk in this process. To this aim, we used a model 5/6 nephrectomy and peritoneal fibrosis in rats. Rats were divided into four groups, namely a control group (saline administration); a model group (dialysate administration; group M); a SSD group (dialysate and SSD administration); and a positive drug group (dialysate and Benazepril Hydrochloride administration; group M + A). Histological analysis indicated that peritoneal fibrosis occurred in all groups. WB, ELISA, and PCR essays suggested that TGFß1 and Gremlin1 levels in group M were significantly higher than those in group C, whereas BMP7 expression was significantly lower. TGFß1, Gremlin1 and BMP7 levels were significantly lower in the group where SSD was administered than in the other groups. The expression of BMP7 in SSD group was significantly increased. In addition, levels of Smad1/5/8 as assessed by PCR, and levels of p-Smad1/5/8 expression as assessed by WB were also significantly higher in the SSD group than in the M group. Expression of vimentin and α-SMA, two important markers of fibrosis, was also significantly decreased. Our study suggests a role for the TGFß1/BMP7/Gremlin1/Smad pathway in peritoneal fibrosis with potential therapeutic implications. Finally, our results also suggest that the monomer SSD may be able to reverse peritoneal fibrosis via regulation of the TGFß1/BMP7/Gremlin1/Smad pathway.

Silicon Nanowire Field-Effect Transistor as Label-Free Detection of Hepatitis B Virus Proteins with Opposite Net Charges.

The prevalence of hepatitis B virus (HBV) is a global healthcare threat, particularly chronic hepatitis B (CHB) that might lead to hepatocellular carcinoma (HCC) should not be neglected. Although many types of HBV diagnosis detection methods are available, some technical challenges, such as the high cost or lack of practical feasibility, need to be overcome. In this study, the polycrystalline silicon nanowire field-effect transistors (pSiNWFETs) were fabricated through commercial process technology and then chemically functionalized for sensing hepatitis B virus surface antigen (HBsAg) and hepatitis B virus X protein (HBx) at the femto-molar level. These two proteins have been suggested to be related to the HCC development, while the former is also the hallmark for HBV diagnosis, and the latter is an RNA-binding protein. Interestingly, these two proteins carried opposite net charges, which could serve as complementary candidates for evaluating the charge-based sensing mechanism in the pSiNWFET. The measurements on the threshold voltage shifts of pSiNWFETs showed a consistent correspondence to the polarity of the charges on the proteins studied. We believe that this report can pave the way towards developing an approachable tool for biomedical applications.

Pathogen Biocontrol Using Plant Growth-Promoting Bacteria (PGPR): Role of Bacterial Diversity.

A vast microbial community inhabits in the rhizosphere, among which, specialized bacteria known as Plant Growth-Promoting Rhizobacteria (PGPR) confer benefits to host plants including growth promotion and disease suppression. PGPR taxa vary in the ways whereby they curtail the negative effects of invading plant pathogens. However, a cumulative or synergistic effect does not always ensue when a bacterial consortium is used. In this review, we reassess the disease-suppressive mechanisms of PGPR and present explanations and illustrations for functional diversity and/or stability among PGPR taxa regarding these mechanisms. We also provide evidence of benefits when PGPR mixtures, rather than individuals, are used for protecting crops from various diseases, and underscore the critical determinant factors for successful use of PGPR mixtures. Then, we evaluate the challenges of and limitations to achieving the desired outcomes from strain/species-rich bacterial assemblages, particularly in relation to their role for plant disease management. In addition, towards locating additive or synergistic outcomes, we highlight why and how the benefits conferred need to be categorized and quantified when different strains/species of PGPR are used in combinations. Finally, we highlight the critical approaches needed for developing PGPR mixtures with improved efficacy and stability as biocontrols for utilization in agricultural fields.

Comparative analysis of draft genome assemblies developed from whole genome sequences of two Hyaloperonospora brassicae isolate samples differing in field virulence on Brassica napus.

Hyaloperonospora brassicae causes downy mildew, a major disease of Brassicaceae species. We sequenced the genomes of two H. brassicae isolates of high (Sample B) and low (Sample C) virulence. Sequencing reads were first assembled de novo with software's SOAPdenovo2, ABySS V2.1 and Velvet V1.1 and later combined to create meta-assemblies with genome sizes of 72.762 and 76.950Mb and predicted gene densities of 1628 and 1644 /Mb, respectively. We could annotate 12.255 and 13,030 genes with high proportions (91-92%) of complete BUSCOs for Sample B and C, respectively. Comparative analysis revealed conserved and varied molecular machinery underlying the physiological specialisation and infection capabilities. BLAST analysis against PHI gene database suggested a relatively higher loss of genes for virulence and pathogenicity in Sample C compared to Sample B, reflecting pathogen evolution through differential rates of mutation and selection. These studies will enable identification and monitoring of H. brassicae virulence factors prevailing in-field.

Dimorphism in Neopseudocercosporella capsellae, an Emerging Pathogen Causing White Leaf Spot Disease of Brassicas.

White leaf spot pathogen: Neopseudocercosporella capsellae causes significant damage to many economically important Brassicaceae crops, including oilseed rape through foliar, stem, and pod lesions under cool and wet conditions. A lack of information on critical aspects of the pathogen's life cycle limits the development of effective control measures. The presence of single-celled spores along with multi-celled conidia on cotyledons inoculated with multi-celled conidia suggested that the multi-celled conidia were able to form single-celled spores on the host surface. This study was designed to demonstrate N. capsellae morphological plasticity, which allows the shift between a yeast-like single-celled phase and the multi-celled hyphal phase. Separate experiments were designed to illustrate the pathogen's morphological transformation to single-celled yeast phase from multi-celled hyphae or multi-celled macroconidia in-vitro and in-planta. Results confirmed the ability of N. capsellae to switch between two morphologies (septate hyphae and single-celled yeast phase) on a range of artificial culture media (in-vitro) or in-planta on the host surface before infection occurs. The hyphae-to-yeast transformation occurred through the production of two morphologically distinguishable blastospore (blastoconidia) types (meso-blastospores and micro-blastospores), and arthrospores (arthroconidia).

Phoma medicaginis Isolate Differences Determine Disease Severity and Phytoestrogen Production in Annual Medicago spp.

Phoma black stem and leaf spot disease of annual Medicago spp., caused by Phoma medicaginis, not only can devastate forage and seed yield but can reduce herbage quality by inducing production of phytoestrogens (particularly coumestrol and 4'-O-methylcoumestrol), which can also reduce the ovulation rates of animals grazing infected forage. We determined the consequent phytoestrogen levels on three different annual Medicago species/cultivars (Medicago truncatula cultivar Cyprus, Medicago polymorpha var. brevispina cultivar Serena, and Medicago murex cultivar Zodiac) after inoculation with 35 isolates of P. medicaginis. Across the isolate × cultivar combinations, leaf disease incidence, petiole/stem disease incidence, leaf disease severity, petiole disease severity, and leaf yellowing severity ranged up to 100, 89.4, 100, 58.1, and 61.2%, respectively. Cultivars Cyprus and Serena were the most susceptible and cultivar Zodiac was the most resistant to P. medicaginis. Isolates WAC3653, WAC3658, and WAC4252 produced the most severe disease. Levels of phytoestrogens in stems ranged from 25 to 1,995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4'-O-methylcoumestrol. There was a significant positive relationship of disease incidence and severity parameters with both coumestrol and 4'-O-methylcoumestrol contents, as noted across individual cultivars and across the three cultivars overall, where r = 0.39 and 0.37 for coumestrol and 4'-O-methylcoumestrol, respectively (P < 0.05). Although cultivar Serena was most susceptible to P. medicaginis and produced the highest levels of phytoestrogens in the presence of P. medicaginis, cultivar Zodiac contained the highest levels of phytoestrogens in comparison with other cultivars in the absence of P. medicaginis. There was a 15-fold increase in coumestrol in cultivar Serena but only a 7-fold increase in cultivar Zodiac from infection of P. medicaginis. The study highlights that the intrinsic ability of a particular cultivar to produce phytoestrogens in the absence of the pathogen, and its comparative ability to produce phytoestrogens in the presence of the P. medicaginis, are both important and highly relevant to developing new annual Medicago spp. cultivars that offer improved disease resistance and better animal reproductive outcomes.

Single-Molecule Ex Situ Atomic Force Microscopy Allows Detection of Individual Antibody-Antigen Interactions on a Semiconductor Chip Surface.

Although in situ atomic force microscopy (AFM) allows single-molecule detection of antibody-antigen binding, the practical applications of in situ AFM for disease diagnosis are greatly limited, due to its operational complexity and long operational times, including the execution time for the surface chemical/biological treatments in the equipped glass liquid cell. Herein, a method of graphically superimposed alignment that enables ex situ AFM analysis of an immobilized antibody at the same location on a semiconductor chip surface before and after incubation with its antigen is presented. All of the required chemical/biological treatments are executed feasibly using standard laboratory containers, allowing single-molecule ex situ AFM detection to be conducted with great practicality, flexibility, and versatility. As an example, the analysis of hepatitis B virus X protein (HBx) and its IgG antibody is described. Using ex situ AFM, individual information on the topographical characteristics of the immobilized single and aggregated IgG antibodies on the chip surface is extracted and the data are analyzed statistically. Furthermore, in a statistical manner, the changes in AFM-measured heights of the individual and aggregated IgG antibodies that occur as a result of changes in conformation upon formation of IgG-HBx complexes are investigated.

Challenges With Managing Disease Complexes During Application of Different Measures Against Foliar Diseases of Field Pea.

Studies were undertaken across five field locations in Western Australia to determine the relative changes in disease severity and subsequent field pea yield from up to four foliar pathogens associated with a field pea foliar disease complex (viz. genera Didymella, Phoma, Peronospora, and Septoria) across four different pea varieties sown at three different times and at three different densities. Delaying sowing of field pea significantly (P < 0.05) reduced the severity of Ascochyta blight (all five locations) and Septoria blight (one location), increased the severity of downy mildew (four locations), but had no effect on seed yield. In relation to Ascochyta blight severity at 80 days after sowing, at all locations the early time of sowing had significantly (P < 0.05) more severe Ascochyta blight than the mid and late times of sowing. Increasing actual plant density from 20 to 25 plants m-2 to 58 to 78 plants m-2 significantly (P < 0.05) increased the severity of the Ascochyta blight (four locations) and downy mildew (one location), and it increased seed yield at four locations irrespective of sowing date and three locations irrespective of variety. Compared with varieties Dundale, Wirrega, and Pennant, variety Alma showed significantly (P < 0.05) less severe Ascochyta blight, downy mildew, and Septoria blight (one location each). Grain yield was highest for the early time of sowing at three locations. Varieties Alma, Dundale, and Wirrega significantly (P < 0.05) outyielded Pennant at four locations. The percentage of isolations of individual Ascochyta blight pathogens at 80 days after the first time of sowing varied greatly, with genus Didymella ranging from 25 to 93% and genus Phoma ranging from 6 to 23% across the five field locations. This fluctuating nature of individual pathogen types and proportions within the Ascochyta blight complex, along with variation in the occurrence of pathogens Peronospora and Septoria, highlights the challenges to understand and manage the complexities of co-occurring different foliar pathogens of field pea. While the search for more effective host resistance continues, there is a need for and opportunities from further exploring and exploiting cultural management approaches focusing on crop sequence diversification, intercropping, manipulating time of sowing and stand density, and application of improved seed sanitation and residue/inoculum management practices. We discuss the constraints and opportunities toward overcoming the challenges associated with managing foliar disease complexes in field pea.

Evidence for Niche Differentiation in the Environmental Responses of Co-occurring Mucoromycotinian Fine Root Endophytes and Glomeromycotinian Arbuscular Mycorrhizal Fungi.

Fine root endophytes (FRE) were traditionally considered a morphotype of arbuscular mycorrhizal fungi (AMF), but recent genetic studies demonstrate that FRE belong within the subphylum Mucoromycotina, rather than in the subphylum Glomeromycotina with the AMF. These findings prompt enquiry into the fundamental ecology of FRE and AMF. We sampled FRE and AMF in roots of Trifolium subterraneum from 58 sites across temperate southern Australia. We investigated the environmental drivers of composition, richness, and root colonization of FRE and AMF by using structural equation modelling and canonical correspondence analyses. Root colonization by FRE increased with increasing temperature and rainfall but decreased with increasing phosphorus (P). Root colonization by AMF increased with increasing soil organic carbon but decreased with increasing P. Richness of FRE decreased with increasing temperature and soil pH. Richness of AMF increased with increasing temperature and rainfall but decreased with increasing soil aluminium (Al) and pH. Aluminium, soil pH, and rainfall were, in decreasing order, the strongest drivers of community composition of FRE; they were also important drivers of community composition of AMF, along with temperature, in decreasing order: rainfall, Al, temperature, and soil pH. Thus, FRE and AMF showed the same responses to some (e.g. soil P, soil pH) and different responses to other (e.g. temperature) key environmental factors. Overall, our data are evidence for niche differentiation among these co-occurring mycorrhizal associates.

Canola Growth Stage at Time of Infection Determines Magnitude of White Leaf Spot (Neopseudocercosporella capsellae) Impact.

White leaf spot (Neopseudocercosporella capsellae) is a persistent and increasingly important foliar disease for canola (Brassica napus) across southern Australia. To define the role of plant growth stage in the development of disease epidemics, we first investigated the response of different canola cultivars (Scoop and Charlton) at five Sylvester-Bradley growth stages against N. capsellae. White leaf spot disease incidence and severity was dependent on plant growth stage and cultivar (both P < 0.001), with plants being most susceptible at plant growth stage 1.00 (cotyledon stage) followed by plant growth stage 1.04 (fourth leaf stage). Then, to quantify the impact of this disease on canola yield, we investigated the in-field relationship of white leaf spot disease incidence and severity with seed yield loss following artificial inoculation commencing at growth stage 1.04 (fourth leaf stage). White leaf spot significantly (P < 0.001) reduced seed yield by 24% in N. capsellae inoculated field plots compared with noninoculated field plots. To our knowledge, this is the first time that serious seed yield losses from this disease have been quantified in the field. The current study demonstrates that N. capsellae disease incidence and severity on canola is determined by host growth stage at which pathogen infestation occurs. Emerging seedling cotyledons were highly susceptible, followed by less susceptibility in first true leaves to emerge, but then increasing susceptibility as plants subsequently aged toward the fourth leaf stage. This explains field observances where white leaf spot readily establishes on emerging seedlings and subsequently becomes more prevalent and severe as plants age.

Novel Disease Host Resistances in the World Core Collection of Trifolium subterraneum.

Glasshouse and field investigations of the phenotypic expressions of resistance of a 97-member World Core Collection of subterranean clover (Trifolium subterraneum) collected from its native Mediterranean habitat and representing approximately 80% of the total genetic diversity within the known 10,000 accessions of the species against the most important damping-off and root rot (Phytophthora clandestina, and Pythium irregulare) and foliar (Kabatiella caulivora, Uromyces trifolii-repentis, and Erysiphe trifoliorum) pathogens were performed. An additional 28 diverse cultivars were also included. Associations of these genotypes among 18 disease parameters and 17 morphological traits, and among these disease parameters and 24 climatic and eco-geographic variables from their collection sites, were examined. Many genotypes showed strong phenotypic expression of novel host disease resistance against one or more pathogens, enabling their potential deployment as disease-resistant parents in subterranean clover breeding programs. These new sources of resistance enable future "pyramiding" of different resistance genes to improve resistance against these pathogens. Of particular value were genotypes with multiple disease-resistance across soilborne and/or foliar diseases, because many of these pathogens co-occur. All diseases had some parameters significantly correlated with one or more morphological traits and with one or more sites of origin variables. In particular, there were significant negative correlations between damping-off (i.e., germination) and 8 of the 17 morphological characters. The outcomes of these studies provide crucial information to subterranean clover breeding programs, enabling them to simultaneously select genotypes with multiple resistance to co-occurring soilborne and foliar diseases and desirable traits to offer renewed hope for re-establishing a more productive subterranean clover livestock feedbase despite multiple diseases prevailing widely.