Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Celebrating Professor Rajeev K. Varshney's transformative research odyssey from genomics to the field on his induction as Fellow of the Royal Society.

Professor Rajeev K. Varshney's transformative impact on crop genomics, genetics, and agriculture is the result of his passion, dedication, and unyielding commitment to harnessing the potential of genomics to address the most pressing challenges faced by the global agricultural community. Starting from a small town in India and reaching the global stage, Professor Varshney's academic and professional trajectory has inspired many scientists active in research today. His ground-breaking work, especially his effort to list orphan tropical crops to genomic resource-rich entities, has been transformative. Beyond his scientific achievements, Professor Varshney is recognized by his colleagues as an exemplary mentor, fostering the growth of future researchers, building institutional capacity, and strengthening scientific capability. His focus on translational genomics and strengthening seed system in developing countries for the improvement of agriculture has made a tangible impact on farmers' lives. His skills have been best utilized in roles at leading research centres where he has applied his expertise to deliver a new vision for crop improvement. These efforts have now been recognized by the Royal Society with the award of the Fellowship (FRS). As we mark this significant milestone in his career, we not only celebrate Professor Varshney's accomplishments but also his wider contributions that continue to transform the agricultural landscape.

Linkage Mapping and Genome-Wide Association Study Identified Two Peanut Late Leaf Spot Resistance Loci, PLLSR-1 and PLLSR-2, Using Nested Association Mapping.

Identification of candidate genes and molecular markers for late leaf spot (LLS) disease resistance in peanut (Arachis hypogaea) has been a focus of molecular breeding for the U.S. industry-funded peanut genome project. Efforts have been hindered by limited mapping resolution due to low levels of genetic recombination and marker density available in traditional biparental mapping populations. To address this, a multi-parental nested association mapping population has been genotyped with the peanut 58K single-nucleotide polymorphism (SNP) array and phenotyped for LLS severity in the field for 3 years. Joint linkage-based quantitative trait locus (QTL) mapping identified nine QTLs for LLS resistance with significant phenotypic variance explained up to 47.7%. A genome-wide association study identified 13 SNPs consistently associated with LLS resistance. Two genomic regions harboring the consistent QTLs and SNPs were identified from 1,336 to 1,520 kb (184 kb) on chromosome B02 and from 1,026.9 to 1,793.2 kb (767 kb) on chromosome B03, designated as peanut LLS resistance loci, PLLSR-1 and PLLSR-2, respectively. PLLSR-1 contains 10 nucleotide-binding site leucine-rich repeat disease resistance genes. A nucleotide-binding site leucine-rich repeat disease resistance gene, Arahy.VKVT6A, was also identified on homoeologous chromosome A02. PLLSR-2 contains five significant SNPs associated with five different genes encoding callose synthase, pollen defective in guidance protein, pentatricopeptide repeat, acyl-activating enzyme, and C2 GRAM domains-containing protein. This study highlights the power of multi-parent populations such as nested association mapping for genetic mapping and marker-trait association studies in peanuts. Validation of these two LLS resistance loci will be needed for marker-assisted breeding.

In vitro evaluation of a hybrid negative pressure system for wound therapy.

OBJECTIVE: The objective of this study was to assess whether negative pressure could be maintained accurately and repeatably with a wall-suction-based hybrid negative pressure wound therapy (h-NPWT) system by comparing it with a commercial negative pressure wound therapy (NPWT) device. STUDY DESIGN: In vitro experimental study. METHODS: A commercial NPWT device (control) and three h-NPWT devices, with 0, 3, and 6 meters of additional tubing using the hospital-wall suction (groups 1, 2, and 3 respectively), were applied sequentially to a commercial NPWT dressing on a silicone skin substrate and set to run at a continuous pressure of -125 mmHg. The pressure within the wound space was monitored at 10 second intervals for 24 h. The process was repeated five times for each group. RESULTS: The commercial NPWT device produced an average pressure variance of 3.02 mmHg, and the h-NPWT produced average variances of 4.38, 4.24 and 4.20 mmHg for groups 1, 2 and 3, respectively. All groups produced an average pressure within 0.15 mmHg of -125 mmHg over the 24-hour period, and the h-NPWT systems produced the smallest range with all values remaining within a ±5% variation from -125 mmHg. CONCLUSION: The h-NPWT system achieved negative pressures that were comparable to those of a commercial control NPWT device. The addition of tubing between the skin substrate and the canister did not affect the pressure applied at the wound site. CLINICAL SIGNIFICANCE: The h-NPWT device tested in this study can be considered as an alternative for negative wound therapy when a commercial device cannot be used.

Why Do Plant-Pathogenic Fungi Produce Mycotoxins? Potential Roles for Mycotoxins in the Plant Ecosystem.

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host-pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).

Nested-association mapping (NAM)-based genetic dissection uncovers candidate genes for seed and pod weights in peanut (Arachis hypogaea).

Multiparental genetic mapping populations such as nested-association mapping (NAM) have great potential for investigating quantitative traits and associated genomic regions leading to rapid discovery of candidate genes and markers. To demonstrate the utility and power of this approach, two NAM populations, NAM_Tifrunner and NAM_Florida-07, were used for dissecting genetic control of 100-pod weight (PW) and 100-seed weight (SW) in peanut. Two high-density SNP-based genetic maps were constructed with 3341 loci and 2668 loci for NAM_Tifrunner and NAM_Florida-07, respectively. The quantitative trait locus (QTL) analysis identified 12 and 8 major effect QTLs for PW and SW, respectively, in NAM_Tifrunner, and 13 and 11 major effect QTLs for PW and SW, respectively, in NAM_Florida-07. Most of the QTLs associated with PW and SW were mapped on the chromosomes A05, A06, B05 and B06. A genomewide association study (GWAS) analysis identified 19 and 28 highly significant SNP-trait associations (STAs) in NAM_Tifrunner and 11 and 17 STAs in NAM_Florida-07 for PW and SW, respectively. These significant STAs were co-localized, suggesting that PW and SW are co-regulated by several candidate genes identified on chromosomes A05, A06, B05, and B06. This study demonstrates the utility of NAM population for genetic dissection of complex traits and performing high-resolution trait mapping in peanut.

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time.

BACKGROUND: The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. RESULTS: Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15 mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which encompassed 9 biological super-pathways and 47 sub-pathways. These metabolites were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. CONCLUSIONS: The moderately high aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts also suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.

Deciphering drought-induced metabolic responses and regulation in developing maize kernels.

Drought stress conditions decrease maize growth and yield, and aggravate preharvest aflatoxin contamination. While several studies have been performed on mature kernels responding to drought stress, the metabolic profiles of developing kernels are not as well characterized, particularly in germplasm with contrasting resistance to both drought and mycotoxin contamination. Here, following screening for drought tolerance, a drought-sensitive line, B73, and a drought-tolerant line, Lo964, were selected and stressed beginning at 14 days after pollination. Developing kernels were sampled 7 and 14 days after drought induction (DAI) from both stressed and irrigated plants. Comparative biochemical and metabolomic analyses profiled 409 differentially accumulated metabolites. Multivariate statistics and pathway analyses showed that drought stress induced an accumulation of simple sugars and polyunsaturated fatty acids and a decrease in amines, polyamines and dipeptides in B73. Conversely, sphingolipid, sterol, phenylpropanoid and dipeptide metabolites accumulated in Lo964 under drought stress. Drought stress also resulted in the greater accumulation of reactive oxygen species (ROS) and aflatoxin in kernels of B73 in comparison with Lo964 implying a correlation in their production. Overall, field drought treatments disordered a cascade of normal metabolic programming during development of maize kernels and subsequently caused oxidative stress. The glutathione and urea cycles along with the metabolism of carbohydrates and lipids for osmoprotection, membrane maintenance and antioxidant protection were central among the drought stress responses observed in developing kernels. These results also provide novel targets to enhance host drought tolerance and disease resistance through the use of biotechnologies such as transgenics and genome editing.

High-density genetic map using whole-genome resequencing for fine mapping and candidate gene discovery for disease resistance in peanut.

Whole-genome resequencing (WGRS) of mapping populations has facilitated development of high-density genetic maps essential for fine mapping and candidate gene discovery for traits of interest in crop species. Leaf spots, including early leaf spot (ELS) and late leaf spot (LLS), and Tomato spotted wilt virus (TSWV) are devastating diseases in peanut causing significant yield loss. We generated WGRS data on a recombinant inbred line population, developed a SNP-based high-density genetic map, and conducted fine mapping, candidate gene discovery and marker validation for ELS, LLS and TSWV. The first sequence-based high-density map was constructed with 8869 SNPs assigned to 20 linkage groups, representing 20 chromosomes, for the 'T' population (Tifrunner × GT-C20) with a map length of 3120 cM and an average distance of 1.45 cM. The quantitative trait locus (QTL) analysis using high-density genetic map and multiple season phenotyping data identified 35 main-effect QTLs with phenotypic variation explained (PVE) from 6.32% to 47.63%. Among major-effect QTLs mapped, there were two QTLs for ELS on B05 with 47.42% PVE and B03 with 47.38% PVE, two QTLs for LLS on A05 with 47.63% and B03 with 34.03% PVE and one QTL for TSWV on B09 with 40.71% PVE. The epistasis and environment interaction analyses identified significant environmental effects on these traits. The identified QTL regions had disease resistance genes including R-genes and transcription factors. KASP markers were developed for major QTLs and validated in the population and are ready for further deployment in genomics-assisted breeding in peanut.

Stress Sensitivity Is Associated with Differential Accumulation of Reactive Oxygen and Nitrogen Species in Maize Genotypes with Contrasting Levels of Drought Tolerance.

Drought stress decreases crop growth, yield, and can further exacerbate pre-harvest aflatoxin contamination. Tolerance and adaptation to drought stress is an important trait of agricultural crops like maize. However, maize genotypes with contrasting drought tolerances have been shown to possess both common and genotype-specific adaptations to cope with drought stress. In this research, the physiological and metabolic response patterns in the leaves of maize seedlings subjected to drought stress were investigated using six maize genotypes including: A638, B73, Grace-E5, Lo964, Lo1016, and Va35. During drought treatments, drought-sensitive maize seedlings displayed more severe symptoms such as chlorosis and wilting, exhibited significant decreases in photosynthetic parameters, and accumulated significantly more reactive oxygen species (ROS) and reactive nitrogen species (RNS) than tolerant genotypes. Sensitive genotypes also showed rapid increases in enzyme activities involved in ROS and RNS metabolism. However, the measured antioxidant enzyme activities were higher in the tolerant genotypes than in the sensitive genotypes in which increased rapidly following drought stress. The results suggest that drought stress causes differential responses to oxidative and nitrosative stress in maize genotypes with tolerant genotypes with slower reaction and less ROS and RNS production than sensitive ones. These differential patterns may be utilized as potential biological markers for use in marker assisted breeding.

Protein profiles reveal diverse responsive signaling pathways in kernels of two maize inbred lines with contrasting drought sensitivity.

Drought stress is a major factor that contributes to disease susceptibility and yield loss in agricultural crops. To identify drought responsive proteins and explore metabolic pathways involved in maize tolerance to drought stress, two maize lines (B73 and Lo964) with contrasting drought sensitivity were examined. The treatments of drought and well water were applied at 14 days after pollination (DAP), and protein profiles were investigated in developing kernels (35 DAP) using iTRAQ (isobaric tags for relative and absolute quantitation). Proteomic analysis showed that 70 and 36 proteins were significantly altered in their expression under drought treatments in B73 and Lo964, respectively. The numbers and levels of differentially expressed proteins were generally higher in the sensitive genotype, B73, implying an increased sensitivity to drought given the function of the observed differentially expressed proteins, such as redox homeostasis, cell rescue/defense, hormone regulation and protein biosynthesis and degradation. Lo964 possessed a more stable status with fewer differentially expressed proteins. However, B73 seems to rapidly initiate signaling pathways in response to drought through adjusting diverse defense pathways. These changes in protein expression allow for the production of a drought stress-responsive network in maize kernels.

Effects of Different Shelling Methods on Data Variability during Field Screening for Reduced Aflatoxin Contamination in Maize.

Non-genetic variation limits the identification of novel maize germplasm with genetic markers for reduced Aspergillus flavus infection and aflatoxin contamination. Aflatoxin measurements can vary substantially within fields containing the same germplasm following inoculation with A. flavus. While some variation is expected due to microenvironmental differences, components of field screening methodologies may also contribute to variability in collected data. Therefore, the objective of this study is to test the effects of three different shelling methods (whole ear (WE), ear end removal (EER), and inoculation site-surrounding (ISS)) to obtain bulk samples from maize on aflatoxin measurements. Five ears per row of three inbred lines and two hybrids were inoculated with A. flavus, then shelled using the three different methods, and aflatoxin was quantified. Overall, EER and ISS resulted in reduced coefficients of variance (CVs) in comparison to WE for both inbred and hybrid maize lines, with two exceptions. Susceptible B73 showed increased CVs with both EER and ISS compared to WE, and resistant Mp719's EER CVs marginally increased compared to WE. While WE is the standard practice for most breeding programs due to its technical simplicity, EER and ISS may allow for finely phenotyping parental lines for further breeding applications.

Nondestructive and Rapid Screening of Aflatoxin-Contaminated Single Peanut Kernels Using Field-Portable Spectroscopy Instruments (FT-IR and Raman).

A nondestructive and rapid classification approach was developed for identifying aflatoxin-contaminated single peanut kernels using field-portable vibrational spectroscopy instruments (FT-IR and Raman). Single peanut kernels were either spiked with an aflatoxin solution (30 ppb-400 ppb) or hexane (control), and their spectra were collected via Raman and FT-IR. An uHPLC-MS/MS approach was used to verify the spiking accuracy via determining actual aflatoxin content on the surface of randomly selected peanut samples. Supervised classification using soft independent modeling of class analogies (SIMCA) showed better discrimination between aflatoxin-contaminated (30 ppb-400 ppb) and control peanuts with FT-IR compared with Raman, predicting the external validation samples with 100% accuracy. The accuracy, sensitivity, and specificity of SIMCA models generated with the portable FT-IR device outperformed the methods in other destructive studies reported in the literature, using a variety of vibrational spectroscopy benchtop systems. The discriminating power analysis showed that the bands corresponded to the C=C stretching vibrations of the ring structures of aflatoxins were most significant in explaining the variance in the model, which were also reported for Aspergillus-infected brown rice samples. Field-deployable vibrational spectroscopy devices can enable in situ identification of aflatoxin-contaminated peanuts to assure regulatory compliance as well as cost savings in the production of peanut products.

Macro-micro exploration on dynamic interaction between aflatoxigenic Aspergillus flavus and maize kernels using Vis/NIR hyperspectral imaging and SEM technology.

Aspergillus flavus and its toxic metabolites-aflatoxins infect and contaminate maize kernels, posing a threat to grain safety and human health. Due to the complexity of microbial growth and metabolic processes, dynamic mechanisms among fungal growth, nutrient depletion of maize kernels and aflatoxin production is still unclear. In this study, visible/near infrared (Vis/NIR) hyperspectral imaging (HSI) combined with the scanning electron microscope (SEM) was used to elucidate the critical organismal interaction at kernel (macro-) and microscopic levels. As kernel damage is the main entrance for fungal invasion, maize kernels with gradually aggravated damages from intact to pierced to halved kernels with A. flavus were cultured for 0-120 h. The spectral fingerprints of the A. flavus-maize kernel complex over time were analyzed with principal components analysis (PCA) of hyperspectral images, where the pseudo-color score maps and the loading plots of the first three PCs were used to investigate the dynamic process of fungal infection and to capture the subtle changes in the complex with different hardness of the maize matrix. The dynamic growth process of A. flavus and the interactions of fungus-maize complexes were explained on a microscopic level using SEM. Specifically, fungus morphology, e.g., hyphae, conidia, and conidiophore (stipe) was accurately captured on the microscopic level, and the interaction process between A. flavus and nutrient loss from the maize kernel tissues (i.e., embryo, and endosperm) was described. Furthermore, the growth stage discrimination models based on PLSDA with the results of CCRC = 100 %, CCRV = 97 %, CCRIV = 93 %, and the prediction models of AFB1 based on PLSR with satisfactory performance (R2C = 0.96, R2V = 0.95, R2IV = 0.93 and RPD = 3.58) were both achieved. In conclusion, the results from both macro-level (Vis/NIR-HSI) and micro-level (SEM) assessments revealed the dynamic organismal interactions in A. flavus-maize kernel complex, and the detailed data could be used for modeling, and quantitative prediction of aflatoxin, which would establish a theoretical foundation for the early detection of fungal or toxin contaminated grains to ensure food security.

The right strategy for you: Using the preferences of beef farmers to guide biosecurity recommendations for on-farm management of endemic disease.

Effective on-farm biosecurity measures are crucial to the post-border protection of emerging agricultural diseases and are the foundation of endemic disease control. Implementation of on-farm biosecurity measures are contingent on the priorities of individual producers, which can often be neglected for other aspects of the farming enterprise. The on-farm approach to prevention of endemic diseases, like bovine viral diarrhoea virus (BVDV), is inconsistent between farms and it is not realistic to assume that farmers take an entirely normative approach to on-farm decision making. Multi-criteria decision analysis (MCDA) has been used for disease prioritisation and national disease control in human and animal health; however, it is yet to be used as a decision tool for disease control at the farm level. This study used MCDA to determine the most appropriate biosecurity combinations for management of BVDV, based on the preferences of Australian beef producers. Beef producer preferences were obtained from an online survey using indirect collection methods. Point of truth calibration was used to aggregate producer preferences and the performance scores of 23 biosecurity combinations for control of BVDV based on four main criteria: the probability of BVDV introduction, the on-farm impact of BVDV, the off-farm impact of BVDV and the annual input cost of the practice. The MCDA found that biosecurity combinations that included "double-fencing farm boundaries" used in conjunction with "vaccination against BVDV" were most appropriate for management of BVDV in an initially naïve, self-replacing seasonal single-calving beef herd over a 15-year period. Beef producers prioritised practices that preserved the on-farm health of their cattle more than any other criteria, a finding that was persistent regardless of demographic or farming type. Consequently, combinations with "vaccination against BVDV" were consistently ranked higher than those that included "strategic exposure of a persistently infected cow," which is sometimes used by Australian beef producers instead of vaccination. Findings of this study indicate that the benefits of "double-fencing farm boundaries" and "vaccination against BVDV" outweigh the relatively high cost associated with these practices based on the priorities of the Australian beef producer and may be used to demonstrate the benefits of on-farm biosecurity during discussions between livestock veterinarians and beef farmers.

Understanding biosecurity behaviors of Australian beef cattle farmers using the ten basic human values framework.

Introduction: On-farm biosecurity is an essential component of successful disease management in the beef cattle industry on an individual, regional, and national level. Participation in mandatory or voluntary assurance schemes, knowledge and trusted relationships have all been demonstrated to contribute to the development of behaviors that promote biosecurity. However, compliance with rules, socio-psychological relationships and knowledge-seeking behavior are all contingent upon the motivations and beliefs of the individual. It is widely accepted that the motivations and beliefs of all cultures can be defined by ten basic values (Self-direction, Stimulation, Hedonism, Achievement, Power, Security, Conformity, Tradition, Benevolence and Universalism). In this study, we use the ten basic values to characterize the on-farm biosecurity behaviors of Australian beef farmers to facilitate the identification of interventions that are most likely to align with producer motivations and therefore, more likely to result in wider adoption of effective on-farm biosecurity. Methods: Semi-structured interviews were conducted with 11 Australian beef farmers to discuss the reasons behind decisions to alter or implement biosecurity practices in response to endemic diseases. Thematic analysis was used to identify the motivations, opportunities, and capability of biosecurity behaviors. The ten basic human values were used to characterize these behaviors and inform enablers and barriers to biosecurity adoption. Results and discussion: Benevolence and Self-direction, relating to self-transcendence and an openness to change, were the principal values associated with good biosecurity behaviors. This suggests that farmers will be receptive to education strategies that communicate the actual risk of disease in their area, the impact of disease on animal welfare, and the ability for on-farm biosecurity to mitigate these impacts. Farmers also expressed values of Security which entrenched behaviors as common practice; however, in some cases the Security of trusted relationships was identified as a potential barrier to behavior change. Overall, values associated with biosecurity behaviors were found to align with values that are most important for social cohesion, suggesting that collaborative disease efforts between industry stakeholders and farmers are likely to succeed if designed with these values in mind.

One size does not fit all: Exploring the economic and non-economic outcomes of on-farm biosecurity for bovine viral diarrhoea virus in Australian beef production.

Bovine viral diarrhoea virus (BVDV) is a disease of global importance, affecting the production and welfare of cattle enterprises through poor reproductive performance and calf mortality. In Australia, the prevention of BVDV introduction and spread is primarily achieved with on-farm biosecurity; however, the use of these practices can vary amongst producers. Economic utility is commonly identified as a contributor to the uptake of on-farm biosecurity, but other factors such as animal welfare, producer priorities and introduction risk also influence farmer behaviour. This study uses an individual-based, stochastic simulation model to examine the economic and non-economic value of 23 on-farm biosecurity combinations for the control of BVDV in Australian beef farms without (N0) and with (N1) a neighbouring population of persistently infected (PI) cattle. Combinations of quarantine of purchased bulls (Q), hygiene during herd health events (H), double-fencing adjacent boundaries with neighbouring farms (F) and vaccination against BVDV (V) were tested. This study is the first to simulate the use of strategic PI exposure (PI) as an alternative to V, a contentious practice performed by some Australian beef farmers. Introduction of BVDV into a naïve 300-breeder self-replacing beef herd was achieved through the purchase of PI bulls (N0 and N1 herds) and over-the-fence contact with neighbouring PI animals (N1 herds only). The predicted median cumulative loss due to BVDV over a 15-year period was AUD$172/breeder and AUD$453/breeder for an N0 and N1 herd, respectively. Early establishment of BVDV in the simulation period was found to be the primary factor contributing to economic loss. Consequently, the Q and QF combinations resulted in the highest predicted average annual cost-benefit for BVDV-free N0 and N1 herds. In the five years following establishment of BVDV, use of QP (N0 herds) and V (N1 herds) combinations were most cost-effective. Combinations that involved V and P (in conjunction with F in N1 herds) also resulted in the lowest number of PI animals sold to other farms or feedlots over the simulation period. However, in both N0 and N1 herds, P resulted in the highest number of infected cattle, which has implications for poor animal welfare and increased antimicrobial use on Australian beef farms. The outcomes reported in this study can guide decisions to prevent BVDV introduction and spread on extensive beef farms using on-farm biosecurity, based on the risk of BVDV exposure and the priorities of the individual farmer.

Spatio-temporal patterns of Aspergillus flavus infection and aflatoxin B1 biosynthesis on maize kernels probed by SWIR hyperspectral imaging and synchrotron FTIR microspectroscopy.

The dynamics mechanisms regulating the growth and AFB1 production of Aspergillus flavus during its interactions with maize kernels remain unclear. In this study, shortwave infrared hyperspectral imaging (SWIR-HSI) and synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy were combined to investigate chemical and spatial-temporal changes in incremental damaged maize kernels induced by A. flavus infection at macroscopic and microscopic levels. SWIR-HSI was employed to extract spectral information of A. flavus growth and quantitatively detect AFB1 levels. Satisfactory full-spectrum models and simplified multispectral models were obtained respectively by partial least squares regression (PLSR) for three types of samples. Furthermore, SR-FTIR microspectroscopy coupled with two-dimensional correlation spectroscopy (2DCOS) was utilized to reveal the possible sequence of dynamic changes of nutrient loss and trace AFB1 in maize kernels. It exhibited new insights on how to quantify the spatio-temporal patterns of fungal infection and AFB1 accumulation on maize and provided theoretical basis for online sorting.

Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9.

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.

Modeling the Effect of Bovine Viral Diarrhea Virus in Australian Beef Herds.

Bovine viral diarrhea virus (BVDV) is an economically important disease in Australian beef farming. The disease typically results in low-level production losses that can be difficult to detect for several years. Simulation modeling can be used to support the decision to control BVDV; however, current BVDV simulation models do not adequately reflect the extensive farming environment of Australian beef production. Therefore, the objective of this study was to develop a disease simulation model to explore the impact of BVDV on beef cattle production in south-east Australia. A dynamic, individual-based, stochastic, discrete-time simulation model was created to simulate within-herd transmission of BVDV in a seasonal, self-replacing beef herd. We used the model to simulate the effect of herd size and BVDV introduction time on disease transmission and assessed the short- and long-term impact of BVDV on production outputs that influence the economic performance of beef farms. We found that BVDV can become established in a herd after a single PI introduction in 60% of cases, most frequently associated with the breeding period. The initial impact of BVDV will be more severe in smaller herds, although self-elimination is more likely in small herds than in larger herds, in which there is a 23% chance that the virus can persist for >15 years following a single incursion in a herd with 800 breeders. The number and weight of steers sold was reduced in the presence of BVDV and the results demonstrated that repeat incursions exacerbate long-term production losses, even when annual losses appear marginal. This model reflects the short- and long-term production losses attributed to BVDV in beef herds in southeast Australia and provides a foundation from which the influence and economic utility of BVDV prevention in Australian beef herds can be assessed.