Field Pea (Pisum sativum) Germplasm Screening for Seedling Ascochyta Blight Resistance and Genome-Wide Association Studies Reveal Loci Associated with Resistance to Peyronellaea pinodes and Ascochyta koolunga.

Ascochyta blight is a damaging disease that affects the stems, leaves, and pods of field pea (Pisum sativum) and impacts yield and grain quality. In Australia, field pea Ascochyta blight is primarily caused by the necrotrophic fungal species Peyronellaea pinodes and Ascochyta koolunga. In this study, we screened 1,276 Pisum spp. germplasm accessions in seedling disease assays with a mix of three isolates of P. pinodes and 641 accessions with three mixed isolates of A. koolunga (513 accessions were screened with both species). A selection of three P. sativum accessions with low disease scores for either pathogen, or in some cases both, were crossed with Australian field pea varieties PBA Gunyah and PBA Oura, and recombinant inbred line populations were made. Populations at the F3:4 and F4:5 generation were phenotyped for their disease response to P. pinodes and A. koolunga, and genotypes were determined using the diversity arrays technology genotyping method. Marker-trait associations were identified using a genome-wide association study approach. Trait-associated loci were mapped to the published P. sativum genome assembly, and candidate resistance gene analogues were identified in the corresponding genomic regions. One locus on chromosome 2 (LG1) was associated with resistance to P. pinodes, and the 8 Mb genomic region contains 156 genes, two of which are serine/threonine protein kinases, putatively contributing to the resistance trait. A second locus on chromosome 5 (LG3) was associated with resistance to A. koolunga, and the 35 Mb region contains 488 genes, of which five are potential candidate resistance genes, including protein kinases, a mitogen-activated protein kinase, and an ethylene-responsive protein kinase homolog.

Virulence Profiles and Genome-Wide Association Study for Ascochyta lentis Isolates Collected from Australian Lentil-Growing Regions.

Ascochyta lentis, the causal organism of Ascochyta blight (AB) of lentil (Lens culinaris), has been shown to produce an avirulence effector protein that mediates AB resistance in certain lentil cultivars. The two known forms of the effector protein were identified from a biparental mapping population between isolates that have reciprocal virulence on 'PBA Hurricane XT' and 'Nipper'. The effector AlAvr1-1 was described for the PBA Hurricane XT-avirulent isolate P94-24 and AlAvr1-2 characterized in the PBA Hurricane XT-virulent isolate AlKewell. Here, we performed a genome-wide association study to identify other loci associated with AB for a differential set of lentil cultivars from a diverse panel of isolates collected in the Australian lentil-growing regions from 2013 to 2020. The chromosome 3 AlAvr1 locus was strongly associated with the PBA Hurricane XT, 'Indianhead', and Nipper disease responses, but one other genomic region on chromosome 11 was also associated with the Nipper disease trait. Our results corroborate earlier work that identified the AlAvr1 locus for field-collected isolates that span the period before release and after widespread adoption of PBA Hurricane XT. A multiplex PCR assay was developed to differentiate the genes AlAvr1-1 and AlAvr1-2 to predict PBA Hurricane XT avirulence and pathotype designation in the diversity panel. Increasing numbers of the PBA Hurricane XT-virulent pathotype 2 isolates across that time indicate strong selection for isolates with the AlAvr1-2 allele. Furthermore, one other region of the A. lentis genome may contribute to the pathogen-host interaction for lentil AB.

Identification of Novel Sources of Resistance to Ascochyta Blight in a Collection of Wild Cicer Accessions.

Chickpea production is constrained worldwide by the necrotrophic fungal pathogen Ascochyta rabiei, the causal agent of Ascochyta blight (AB). To reduce the impact of this disease, novel sources of resistance are required in chickpea cultivars. Here, we screened a new collection of wild Cicer accessions for AB resistance and identified accessions resistant to multiple, highly pathogenic isolates. In addition to this, analyses demonstrated that some collection sites of C. echinospermum harbor predominantly resistant accessions, knowledge that can inform future collection missions. Furthermore, a genome-wide association study identified regions of the C. reticulatum genome associated with AB resistance and investigation of these regions identified candidate resistance genes. Taken together, these results can be utilized to enhance the resistance of chickpea cultivars to this globally yield-limiting disease.

Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts.

KEY MESSAGE: Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

Near-infrared multispectral endoscopic imaging of deep artificial interproximal lesions in extracted teeth.

BACKGROUND AND OBJECTIVE: A safer alternative method to radiographic imaging is needed. We present a multispectral near-infrared scanning fiber endoscope (nirSFE) for dental imaging which is designed to be the smallest imaging probe with near-infrared (NIR) imaging (1200-2000 nm). MATERIALS AND METHODS: The prototype nirSFE is designed for wide-field forward viewing of scanned laser illumination at 1310, 1460, or 1550 nm. Artificial lesions with varying sizes and locations were prepared on proximal surfaces of extracted human teeth to examine capability and limitation of this new dental imaging modality. Nineteen artificial interproximal lesions and several natural occlusal lesions on extracted teeth were imaged with nirSFE, OCT, and microCT. RESULTS: Our nirSFE system has a flexible shaft as well as a probe tip with diameter of 1.6 mm and a rigid length of 9 mm. The small form factor and multispectral NIR imaging capability enables multiple viewing angles and reliable detection of lesions that can extend into the dentin. Among nineteen artificial interproximal lesions, the nirSFE reflectance imaging operating at 1460-nm and OCT operating at 1310-nm scanned illumination exhibited high sensitivity for interproximal lesions that were closer to occlusal surface. Diagnosis from a non-blinded trained user by looking at real-time occlusal-side nirSFE videos indicate true positive rate of 78.9%. There were no false positives. CONCLUSIONS: This study demonstrates that nirSFE may be used for detecting occlusal lesions and interproximal lesions located less than 4 mm under the occlusal surface. Major advantages of this imaging system include multiple viewing angles due to flexibility and small form factor, as well as the ability to capture real-time video. The multispectral nirSFE has the potential to be employed as a low-cost dental camera for detecting dental lesions without exposure to ionizing radiation. Lasers Surg. Med. 51:459-465, 2019. © 2019 Wiley Periodicals, Inc.

Near-Infrared Imaging of Artificial Enamel Caries Lesions with a Scanning Fiber Endoscope.

Several studies have shown that near-infrared imaging has great potential for the detection of dental caries lesions. A miniature scanning fiber endoscope (SFE) operating at near-infrared (NIR) wavelengths was developed and used in this study to test whether the device could be used to discriminate demineralized enamel from sound enamel. Varying depths of artificial enamel caries lesions were prepared on 20 bovine blocks with smooth enamel surfaces. Samples were imaged with a SFE operating in the reflectance mode at 1310-nm and 1460-nm in both wet and dry conditions. The measurements acquired by the SFE operating at 1460-nm show significant difference between the sound and the demineralized enamel. There was a moderate positive correlation between the SFE measurements and micro-CT measurements, and the NIR SFE was able to detect the presence of demineralization with high sensitivity (0.96) and specificity (0.85). This study demonstrates that the NIR SFE can be used to detect early demineralization from sound enamel. In addition, the NIR SFE can differentiate varying severities of demineralization. With its very small form factor and maneuverability, the NIR SFE should allow clinicians to easily image teeth from multiple viewing angles in real-time.

Influence of multi-wavelength laser irradiation of enamel and dentin surfaces at 0.355, 2.94, and 9.4 µm on surface morphology, permeability, and acid resistance.

OBJECTIVE: Ultraviolet (UV) and infrared (IR) lasers can be used to specifically target protein, water, and mineral, respectively, in dental hard tissues to produce varying changes in surface morphology, permeability, reflectivity, and acid resistance. The purpose of this study was to explore the influence of laser irradiation and topical fluoride application on the surface morphology, permeability, reflectivity, and acid resistance of enamel and dentin to shed light on the mechanism of interaction and develop more effective treatments. METHODS: Twelve bovine enamel surfaces and twelve bovine dentin surfaces were irradiated with various combinations of lasers operating at 0.355 (Freq.-tripled Nd:YAG (UV) laser), 2.94 (Er:YAG laser), and 9.4 µm (CO2 laser), and surfaces were exposed to an acidulated phosphate fluoride gel and an acid challenge. Changes in the surface morphology, acid resistance, and permeability were measured using digital microscopy, polarized light microscopy, near-IR reflectance, fluorescence, polarization sensitive-optical coherence tomography (PS-OCT), and surface dehydration rate measurements. RESULTS: Different laser treatments dramatically influenced the surface morphology and permeability of both enamel and dentin. CO2 laser irradiation melted tooth surfaces. Er:YAG and UV lasers, while not melting tooth surfaces, showed markedly different surface roughness. Er:YAG irradiation led to significantly rougher enamel and dentin surfaces and led to higher permeability. There were significant differences in acid resistance among the various treatment groups. CONCLUSION: Surface dehydration measurements showed significant changes in permeability after laser treatments, application of fluoride and after exposure to demineralization. CO2 laser irradiation was most effective in inhibiting demineralization on enamel while topical fluoride was most effective for dentin surfaces. Lasers Surg. Med. 49:913-927, 2017. © 2017 Wiley Periodicals, Inc.

Near-IR and CP-OCT imaging of suspected occlusal caries lesions.

INTRODUCTION: Radiographic methods have poor sensitivity for occlusal lesions and by the time the lesions are radiolucent they have typically progressed deep into the dentin. New more sensitive imaging methods are needed to detect occlusal lesions. In this study, cross-polarization optical coherence tomography (CP-OCT) and near-IR imaging were used to image questionable occlusal lesions (QOC's) that were not visible on radiographs but had been scheduled for restoration on 30 test subjects. METHODS: Near-IR reflectance and transillumination probes incorporating a high definition InGaAs camera and near-IR broadband light sources were used to acquire images of the lesions before restoration. The reflectance probe utilized cross-polarization and operated at wavelengths from 1,500 to 1,700 nm where there is an increase in water absorption for higher contrast. The transillumination probe was operated at 1,300 nm where the transparency of enamel is highest. Tomographic images (6 × 6 × 7 mm3 ) of the lesions were acquired using a high-speed swept-source CP-OCT system operating at 1,300 nm before and after removal of the suspected lesion. RESULTS: Near-IR reflectance imaging at 1,500-1,700 nm yielded significantly higher contrast (P < 0.05) of the demineralization in the occlusal grooves compared with visible reflectance imaging. Stains in the occlusal grooves greatly reduced the lesion contrast in the visible range yielding negative values. Only half of the 26 lesions analyzed showed the characteristic surface demineralization and increased reflectivity below the dentinal-enamel junction (DEJ) in 3D OCT images indicative of penetration of the lesion into the dentin. CONCLUSION: This study demonstrates that near-IR imaging methods have great potential for improving the early diagnosis of occlusal lesions. Lasers Surg. Med. 49:215-224, 2017. © 2017 Wiley Periodicals, Inc.

Infrared Methods for Assessment of the Activity of Natural Enamel Caries Lesions.

New diagnostic methods are needed for the accurate assessment of caries lesion activity to establish the need for surgical treatment. Detection of the highly mineralized surface layer that forms near the surface of the lesions as a result of remineralization is important for diagnosis of the lesion activity. Previous studies have demonstrated that novel imaging methods can be used to detect remineralization of artificial enamel caries lesions. In this paper, the activity of natural enamel caries lesions was assessed in-vitro via detection of the surface layer with PS-OCT and dehydration rate measurements with NIR reflectance and thermal imaging modalities. An automated approach for detecting the surface layer with PS-OCT yielded high sensitivity (= 0.79) and high specificity (= 0.93) with moderate correlation (R2 = 0.5920) with histology. Significant differences in dehydration rate measurements were found between the active and the arrested lesions using both the NIR reflectance and thermal imaging modalities. These results demonstrate that these novel imaging methods are ideally suited for nondestructive, noninvasive and quantitative measurement of lesion activity during a single clinical examination in real-time.

Near-IR transillumination and reflectance imaging at 1,300 nm and 1,500-1,700 nm for in vivo caries detection.

INTRODUCTION: Several studies suggest that near-IR imaging methods at wavelengths longer than 1,300 nm have great potential for caries detection. In this study, the diagnostic performance of both near-IR transillumination and near-IR reflectance was assessed on teeth scheduled for extraction due to orthodontic treatment (n = 109 teeth on 40 test subjects). METHODS: Three intra-oral near-IR imaging probes were fabricated for the acquisition of in vivo images using a high definition InGaAs camera and near-IR broadband light sources. Two transillumination probes provided occlusal and approximal images using 1,300 nm light which manifests the highest transparency in enamel. A third reflectance probe utilized cross-polarization and operated at wavelengths greater than 1,500 nm where water absorption is higher which reduces the reflectivity of sound tissues, significantly increasing lesion contrast. Teeth were collected after extraction and sectioned and examined with polarized light microscopy and microradiography which served as the gold standard. In addition, radiographs were taken of the teeth and the diagnostic performance of near-IR imaging was compared with radiography. RESULTS: Near-IR imaging was significantly more sensitive (P < 0.05) than radiography for the detection of lesions on both occlusal and proximal surfaces. CONCLUSION: Near-IR imaging methods are ideally suited for screening all tooth surfaces for carious lesions. Lasers Surg. Med. 48:828-836, 2016. © 2016 Wiley Periodicals, Inc.

Clinical monitoring of smooth surface enamel lesions using CP-OCT during nonsurgical intervention.

INTRODUCTION: Studies have shown that cross-polarization optical coherence tomography (CP-OCT) can be used to image the internal structure of carious lesions in vivo. The objective of this study was to show that CP-OCT can be used to monitor changes in the internal structure of early active carious lesions on smooth surfaces during non-surgical intervention with fluoride. METHODS: Lesions on the smooth surfaces of teeth were imaged using CP-OCT on 17 test subjects. Lesion structural changes were monitored during fluoride varnish application at 6-week intervals for 30 weeks. The lesion depth (Ld ), integrated reflectivity (ΔR), and surface zone thickness (Sz ) were monitored. RESULTS: A distinct transparent surface zone that may be indicative of lesion arrestment was visible in CP-OCT images on 62/63 lesions before application of fluoride varnish. The lesion depth and internal structure were resolved for all the lesions. The overall change in the mean values for Ld , ΔR, and Sz for all the lesions was minimal and was not significant during the study (P > 0.05). Only 5/63 lesions manifested a significant increase in Sz during intervention. CONCLUSION: Even though it appears that most of the lesions manifested little change with fluoride varnish application in the 30 weeks of the study, CP-OCT was able to measure the depth and internal structure of all the lesions including the thickness of the important transparent surface zone located at the surface of the lesions, indicating that CP-OCT is ideally suited for monitoring lesion severity in vivo. Lasers Surg. Med. 48:915-923, 2016. © 2016 Wiley Periodicals, Inc.

Sustained antigen delivery improves germinal center reaction and increases antibody responses in neonatal mice.

Neonates and young infants are known to have limited responses to pediatric vaccines due to reduced germinal center formation. Extended vaccine antigen dosing was previously shown to expand germinal center formation and improve humoral responses in adult mice. We report that sustained antigen delivery through sequential dosing overcomes neonatal limitations to form germinal center reactions and improves humoral immunity. Thus, vaccine strategies that extend the release of vaccine antigens may reduce the number of doses, and time needed, to achieve protective immunity in neonates and young infants.

IL6 suppresses vaccine responses in neonates by enhancing IL2 activity on T follicular helper cells.

The inability of neonates to develop CD4+FoxP3-CXCR5hiPD-1hi T follicular helper (TFH) cells contributes to their weak vaccine responses. In previous studies, we measured diminished IgG responses when IL-6 was co-injected with a pneumococcal conjugate vaccine (PCV) in neonatal mice. This is in sharp contrast to adults, where IL-6 improves vaccine responses by downregulating the expression of IL-2Rß on TFH cells and protecting them from the inhibitory effect of IL-2. In this study, we found that splenic IL-6 levels rapidly increased in both adult and neonatal mice following immunization, but the increase in neonatal mice was significantly more than that of adult mice. Moreover, immunized neonatal TFH cells expressed significantly more IL-2 as well as its receptors, IL-2Rα and IL-2Rß, than the adult cells. Remarkably, IL-6 co-injection with PCV vaccine further increased the production of IL-2 and the expression of its receptors by neonatal TFH cells, whereas excess IL-6 had totally opposite effect in immunized adult mice. Underscoring the role of IL-6 in activating the IL-2 mediated suppression of vaccine responses, immunization of IL-6 knock-out neonates led to improved antibody responses accompanied by expanded TFH cells as well as lower levels of IL-2 and IL-2 receptors on TFH cells. Moreover, CpG containing PCV improved TFH response in neonates by suppressing the expression of IL-2 receptors on TFH cells and inhibiting IL-2 activity. These findings unveil age-specific differences in IL-6 mediated vaccine responses and highlight the need to consider age-related immunobiological attributes in designing vaccines.

De Novo Long-Read Whole-Genome Assemblies and the Comparative Pan-Genome Analysis of Ascochyta Blight Pathogens Affecting Field Pea.

Ascochyta Blight (AB) is a major disease of many cool-season legumes globally. In field pea, three fungal pathogens have been identified to be responsible for this disease in Australia, namely Peyronellaea pinodes, Peyronellaea pinodella and Phoma koolunga. Limited genomic resources for these pathogens have been generated, which has hampered the implementation of effective management strategies and breeding for resistant cultivars. Using Oxford Nanopore long-read sequencing, we report the first high-quality, fully annotated, near-chromosome-level nuclear and mitochondrial genome assemblies for 18 isolates from the Australian AB complex. Comparative genome analysis was performed to elucidate the differences and similarities between species and isolates using phylogenetic relationships and functional diversity. Our data indicated that P. pinodella and P. koolunga are heterothallic, while P. pinodes is homothallic. More homology and orthologous gene clusters are shared between P. pinodes and P. pinodella compared to P. koolunga. The analysis of the repetitive DNA content showed differences in the transposable repeat composition in the genomes and their expression in the transcriptomes. Significant repeat expansion in P. koolunga's genome was seen, with strong repeat-induced point mutation (RIP) activity being evident. Phylogenetic analysis revealed that genetic diversity can be exploited for species marker development. This study provided the much-needed genetic resources and characterization of the AB species to further drive research in key areas such as disease epidemiology and host-pathogen interactions.

The novel avirulence effector AlAvr1 from Ascochyta lentis mediates host cultivar specificity of ascochyta blight in lentil.

Ascochyta lentis is a fungal pathogen that causes ascochyta blight in the important grain legume species lentil, but little is known about the molecular mechanism of disease or host specificity. We employed a map-based cloning approach using a biparental A. lentis population to clone the gene AlAvr1-1 that encodes avirulence towards the lentil cultivar PBA Hurricane XT. The mapping population was produced by mating A. lentis isolate P94-24, which is pathogenic on the cultivar Nipper and avirulent towards Hurricane, and the isolate AlKewell, which is pathogenic towards Hurricane but not Nipper. Using agroinfiltration, we found that AlAvr1-1 from the isolate P94-24 causes necrosis in Hurricane but not in Nipper. The homologous corresponding gene in AlKewell, AlAvr1-2, encodes a protein with amino acid variation at 23 sites and four of these sites have been positively selected in the P94-24 branch of the phylogeny. Loss of AlAvr1-1 in a gene knockout experiment produced a P94-24 mutant strain that is virulent on Hurricane. Deletion of AlAvr1-2 in AlKewell led to reduced pathogenicity on Hurricane, suggesting that the gene may contribute to disease in Hurricane. Deletion of AlAvr1-2 did not affect virulence for Nipper and AlAvr1-2 is therefore not an avirulence gene for Nipper. We conclude that the hemibiotrophic pathogen A. lentis has an avirulence effector, AlAvr1-1, that triggers a hypersensitive resistance response in Hurricane. This is the first avirulence gene to be characterized in a legume pathogen from the Pleosporales and may help progress research on other damaging Ascochyta pathogens.

Phenotypic and Genotypic Diversity of Ascochyta fabae Populations in Southern Australia.

Ascochyta fabae Speg. is a serious foliar fungal disease of faba bean and a constraint to production worldwide. This study investigated the phenotypic and genotypic diversity of the A. fabae pathogen population in southern Australia and the pathogenic variability of the population was examined on a differential set of faba bean cultivars. The host set was inoculated with 154 A. fabae isolates collected from 2015 to 2018 and a range of disease reactions from high to low aggressiveness was observed. Eighty percent of isolates collected from 2015 to 2018 were categorized as pathogenicity group (PG) PG-2 (pathogenic on Farah) and were detected in every region in each year of collection. Four percent of isolates were non-pathogenic on Farah and designated as PG-1. A small group of isolates (16%) were pathogenic on the most resistant differential cultivars, PBA Samira or Nura, and these isolates were designated PG-3. Mating types of 311 isolates collected between 1991 and 2018 were determined and showed an equal ratio of MAT1-1 and MAT1-2 in the southern Australian population. The genetic diversity and population structure of 305 isolates were examined using DArTseq genotyping, and results suggest no association of genotype with any of the population descriptors viz.: collection year, region, host cultivar, mating type, or PG. A Genome-Wide Association Study (GWAS) was performed to assess genetic association with pathogenicity traits and a significant trait-associated genomic locus for disease in Farah AR and PBA Zahra, and PG was revealed. The high frequency of mating of A. fabae indicated by the wide distribution of the two mating types means changes to virulence genes would be quickly distributed to other genotypes. Continued monitoring of the A. fabae pathogen population through pathogenicity testing will be important to identify any increases in aggressiveness or emergence of novel PGs. GWAS and future genetic studies using biparental mating populations could be useful for identifying virulence genes responsible for the observed changes in pathogenicity.

Non-destructive clinical assessment of occlusal caries lesions using near-IR imaging methods.

OBJECTIVE: Enamel is highly transparent in the near-IR (NIR) at wavelengths near 1,300 nm, and stains are not visible. The purpose of this study was to use NIR transillumination and optical coherence tomography (OCT) to estimate the severity of caries lesions on occlusal surfaces both in vivo and on extracted teeth. METHODS: Extracted molars with suspected occlusal lesions were examined with OCT and polarization sensitive OCT (PS-OCT), and subsequently sectioned and examined with polarized light microscopy (PLM) and transverse microradiography (TMR). Teeth in test subjects with occlusal caries lesions that were not cavitated or visible on radiographs were examined using NIR transillumination at 1,310 nm using a custom built probe attached to an indium gallium arsenide (InGaAs) camera and a linear OCT scanner. After imaging, cavities were prepared using dye staining to guide caries removal and physical impressions of the cavities were taken. RESULTS: The lesion severity determined from OCT and PS-OCT scans in vitro correlated with the depth determined using PLM and TMR. Occlusal caries lesions appeared in NIR images with high contrast in vivo. OCT scans showed that most of the lesions penetrated to dentin and spread laterally below the sound enamel. CONCLUSION: This study demonstrates that both NIR transillumination and OCT are promising new methods for the clinical diagnosis of occlusal caries.

Current population structure and pathogenicity patterns of Ascochyta rabiei in Australia.

Ascochyta blight disease, caused by the necrotrophic fungus Ascochyta rabiei, is a major biotic constraint to chickpea production in Australia and worldwide. Detailed knowledge of the structure of the pathogen population and its potential to adapt to our farming practices is key to informing optimal management of the disease. This includes understanding the molecular diversity among isolates and the frequency and distribution of the isolates that have adapted to overcome host resistance across agroecologically distinct regions. Thanks to continuous monitoring efforts over the past 6 years, a comprehensive collection of A. rabiei isolates was collated from the major Australian chickpea production regions. To determine the molecular structure of the entire population, representative isolates from each collection year and growing region have been genetically characterized using a DArTseq genotyping-by-sequencing approach. The genotyped isolates were further phenotyped to determine their pathogenicity levels against a differential set of chickpea cultivars and genotype-phenotype associations were inferred. Overall, the Australian A. rabiei population displayed a far lower genetic diversity (average Nei's gene diversity of 0.047) than detected in other populations worldwide. This may be explained by the presence of a single mating-type in Australia, MAT1-2, limiting its reproduction to a clonal mode. Despite the low detected molecular diversity, clonal selection appears to have given rise to a subset of adapted isolates that are highly pathogenic on commonly employed resistance sources, and that are occurring at an increasing frequency. Among these, a cluster of genetically similar isolates was identified, with a higher proportion of highly aggressive isolates than in the general population. The discovery of distinct genetic clusters associated with high and low isolate pathogenicity forms the foundation for the development of a molecular pathotyping tool for the Australian A. rabiei population. Application of such a tool, along with continuous monitoring of the genetic structure of the population will provide crucial information for the screening of breeding material and integrated disease management packages.

Reference genome assembly for Australian Ascochyta lentis isolate Al4.

Ascochyta lentis causes ascochyta blight in lentil (Lens culinaris Medik.) and yield loss can be as high as 50%. With careful agronomic management practices, fungicide use, and advances in breeding resistant lentil varieties, disease severity and impact to farmers have been largely controlled. However, evidence from major lentil producing countries, Canada and Australia, suggests that A. lentis isolates can change their virulence profile and level of aggressiveness over time and under different selection pressures. In this paper, we describe the first genome assembly for A. lentis for the Australian isolate Al4, through the integration of data from Illumina and PacBio SMRT sequencing. The Al4 reference genome assembly is almost 42 Mb in size and encodes 11,638 predicted genes. The Al4 genome comprises 21 full-length and gapless chromosomal contigs and two partial chromosome contigs each with one telomere. We predicted 31 secondary metabolite clusters, and 38 putative protein effectors, many of which were classified as having an unknown function. Comparison of A. lentis genome features with the recently published reference assembly for closely related A. rabiei show that genome synteny between these species is highly conserved. However, there are several translocations and inversions of genome sequence. The location of secondary metabolite clusters near transposable element and repeat-rich genomic regions was common for A. lentis as has been reported for other fungal plant pathogens.

A systematic literature review of vitamin D and ovarian cancer.

OBJECTIVE: We assessed the evidence supporting a reduction in risk for ovarian cancer occurrence or mortality with greater vitamin D exposures. STUDY DESIGN: This review followed standard guidelines for systematic literature reviews. The diverse study designs precluded a quantitative metaanalysis. Therefore studies are summarized via tables and abstracted information. RESULTS: Approximately half of the ecologic and case-control studies reported reductions in incidence or mortality with increasing geographic latitude, solar radiation levels, or dietary/supplement consumption of vitamin D, whereas the other half reported null associations. The cohort studies reported no overall risk reduction with increasing dietary/supplement consumption of vitamin D or with plasma levels of vitamin D prior to diagnosis, although vitamin D intakes were relatively low in all studies. CONCLUSION: There is no consistent or strong evidence to support the claim made in numerous review articles that vitamin D exposures reduce the risk for ovarian cancer occurrence or mortality.