Diversity and functional traits of indigenous soil microbial flora associated with salinity and heavy metal concentrations in agricultural fields within the Indus Basin region, Pakistan.

Soil salinization and heavy metal (HM) contamination are major challenges facing agricultural systems worldwide. Determining how soil microbial communities respond to these stress factors and identifying individual phylotypes with potential to tolerate these conditions while promoting plant growth could help prevent negative impacts on crop productivity. This study used amplicon sequencing and several bioinformatic programs to characterize differences in the composition and potential functional capabilities of soil bacterial, fungal, and archaeal communities in five agricultural fields that varied in salinity and HM concentrations within the Indus basin region of Pakistan. The composition of bacteria with the potential to fix atmospheric nitrogen (N) and produce the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase were also determined. Microbial communities were dominated by: Euryarchaeota (archaea), Actinobacteria, Proteobacteria, Planctomycetota, Firimicutes, Patescibacteria and Acidobacteria (bacteria), and Ascomycota (fungi), and all soils contained phylotypes capable of N-fixation and ACC-deaminase production. Salinity influenced bacterial, but not archaeal or fungal communities. Both salinity and HM altered the relative abundance of many phylotypes that could potentially promote or harm plant growth. These stress factors also appeared to influence the potential functional capabilities of the microbial communities, especially in their capacity to cycle phosphorous, produce siderophores, and act as symbiotrophs or pathotrophs. Results of this study confirm that farms in this region are at risk due to salinization and excessive levels of some toxic heavy metals, which could negatively impact crop and human health. Changes in soil microbial communities and their potential functional capabilities are also likely to affect several critical agroecosystem services related to nutrient cycling, pathogen suppression, and plant stress tolerance. Many potentially beneficial phylotypes were identified that appear to be salt and HM tolerant and could possibly be exploited to promote these services within this agroecosystem. Future efforts to isolate these phylotypes and determine whether they can indeed promote plant growth and/or carry out other important soil processes are recommended. At the same time, identifying ways to promote the abundance of these unique phylotypes either through modifying soil and crop management practices, or developing and applying them as inoculants, would be helpful for improving crop productivity in this region.

Leveraging high-throughput hyperspectral imaging technology to detect cadmium stress in two leafy green crops and accelerate soil remediation efforts.

Cadmium (Cd) is a toxic metal that can accumulate in soils and negatively impact crop as well as human health. Amendments like biochar have potential to address these challenges by reducing Cd bioavailability in soil, though reliance on post-harvest wet chemical methods to quantify Cd uptake have slowed efforts to identify the most effective amendments. Hyperspectral imaging (HSI) is a novel technology that could overcome this limitation by quantifying symptoms of Cd stress while plants are still growing. The goals of this study were to: 1) determine whether HSI can detect Cd stress in two distinct leafy green crops, 2) quantify whether a locally sourced biochar derived from hardwoods can reduce Cd stress and uptake in these crops, and 3) identify vegetative indices (VIs) that best quantify changes in plant stress responses. Experiments were conducted in a tightly controlled automated phenotyping facility that allowed all environmental factors to be kept constant except Cd concentration (0, 5 10 and 15 mg kg-1). Symptoms of Cd stress were stronger in basil (Ocimum basilicum) than kale (Brassica oleracea), and were easier to detect using HSI. Several VIs detected Cd stress in basil, but only the anthocyanin reflectance index (ARI) detected all levels of Cd stress in both crop species. The biochar amendment did reduce Cd uptake, especially at low Cd concentrations in kale which took up more Cd than basil. Again, the ARI index was the most effective in quantifying changes in plant stress mediated by the biochar. These results indicate that the biochar evaluated in this study has potential to reduce Cd bioavailability in soil, and HSI could be further developed to identify rates that can best achieve this benefit. The technology also may be helping in elucidating mechanisms mediating how biochar can influence plant growth and stress responses.

Classifying cadmium contaminated leafy vegetables using hyperspectral imaging and machine learning.

Cadmium (Cd) is a toxic element that can accumulate in edible plant tissues and negatively impact human health. Traditional Cd quantification methods are time-consuming, expensive, and generate a lot of toxic waste, slowing development of methods to reduce uptake. The objective of this study was to determine whether hyperspectral imaging (HSI) and machine learning (ML) can be used to predict Cd concentrations in plants using kale (Brassica oleracea) and basil (Ocimum basilicum) as model crops. The experiments were conducted in an automated phenotyping facility where all environmental conditions except soil Cd concentration were kept constant. Cd concentrations were determined at harvest using traditional methods and used to train the ML models with data collected from the imaging sensor. Visible/near infrared (VNIR) images were also collected at harvest and processed to calculate reflectance at 473 bands between 400 to 998 nm. All reflectance spectra were subject to the feature selection algorithm ReliefF and Principal Component Analysis (PCA) to generate data and provide input to evaluate three ML classification models: artificial neural network (ANN), ensemble learning (EL), and support vector machine (SVM). Plants were categorized according to Cd concentrations higher or lower than the safety threshold of 0.2 mg kg-1 Cd. Wavelengths with the highest ranks for Cd detection were between 519 and 574, and 692 and 732 nm, indicating that Cd content likely altered the plants' chlorophyll content and altered leaf internal structure. All models were able to sort the plants into groups, though the model with the best F1 score was the ANN for the validation subset that utilized reflectance from all wavelengths. This study demonstrates that HSI and ML are promising technologies for the fast and precise diagnosis of Cd in leafy green plants, though additional studies are needed to adapt this approach for more complex field environments.

Changes in the core endophytic mycobiome of carrot taproots in response to crop management and genotype.

Fungal endophytes can influence production and post-harvest challenges in carrot, though the identity of these microbes as well as factors affecting their composition have not yet been determined, which prevents growers from managing these organisms to improve crop performance. Consequently, we characterized the endophytic mycobiome in the taproots of three carrot genotypes that vary in resistance to two pathogens grown in a trial comparing organic and conventional crop management using Illumina sequencing of the internal transcribed spacer (ITS) gene. A total of 1,480 individual operational taxonomic units (OTUs) were identified. Most were consistent across samples, indicating that they are part of a core mycobiome, though crop management influenced richness and diversity, likely in response to differences in soil properties. There were also differences in individual OTUs among genotypes and the nematode resistant genotype was most responsive to management system indicating that it has greater control over its endophytic mycobiome, which could potentially play a role in resistance. Members of the Ascomycota were most dominant, though the exact function of most taxa remains unclear. Future studies aimed at overcoming difficulties associated with isolating fungal endophytes are needed to identify these microbes at the species level and elucidate their specific functional roles.

Crop management system and carrot genotype affect endophyte composition and Alternaria dauci suppression.

Managing pests in carrot production is challenging. Endophytic microbes have been demonstrated to improve the health and productivity of many crops, but factors affecting endophyte dynamics in carrot is still not well understood. The goal of this study was to determine how crop management system and carrot genotype interact to affect the composition and potential of endophytes to mitigate disease caused by Alternaria dauci, an important carrot pathogen. Twenty-eight unique isolates were collected from the taproots of nine diverse genotypes of carrot grown in a long-term trial comparing organic and conventional management. Antagonistic activity was quantified using an in vitro assay, and potential for individual isolates to mitigate disease was evaluated in greenhouse trials using two carrot cultivars. Results confirm that carrot taproots are colonized by an abundant and diverse assortment of bacteria and fungi representing at least distinct 13 genera. Soils in the organic system had greater total organic matter, microbial biomass and activity than the conventional system and endophyte composition in taproots grown in this system were more abundant and diverse, and had greater antagonistic activity. Carrot genotype also affected endophyte abundance as well as potential for individual isolates to affect seed germination, seedling growth and tolerance to A. dauci. The benefits of endophytes on carrot growth were greatest when plants were subject to A. dauci stress, highlighting the importance of environmental conditions in the functional role of endophytes. Results of this study provide evidence that endophytes can play an important role in improving carrot performance and mediating resistance to A. dauci, and it may someday be possible to select for these beneficial plant-microbial relationships in carrot breeding programs. Implementing soil-building practices commonly used in organic farming systems has potential to promote these beneficial relationships and improve the health and productivity of carrot crops.

Tomato Domestication Attenuated Responsiveness to a Beneficial Soil Microbe for Plant Growth Promotion and Induction of Systemic Resistance to Foliar Pathogens.

Crop domestication events followed by targeted breeding practices have been pivotal for improvement of desirable traits and to adapt cultivars to local environments. Domestication also resulted in a strong reduction in genetic diversity among modern cultivars compared to their wild relatives, though the effect this could have on tripartite relationships between plants, belowground beneficial microbes and aboveground pathogens remains undetermined. We quantified plant growth performance, basal resistance and induced systemic resistance (ISR) by Trichoderma harzianum, a beneficial soil microbe against Botrytis cinerea, a necrotrophic fungus and Phytophthora infestans, a hemi-biotrophic oomycete, in 25 diverse tomato genotypes. Wild tomato related species, tomato landraces and modern commercial cultivars that were conventionally or organically bred, together, representing a domestication gradient were evaluated. Relationships between basal and ISR, plant physiological status and phenolic compounds were quantified to identify potential mechanisms. Trichoderma enhanced shoot and root biomass and ISR to both pathogens in a genotype specific manner. Moreover, improvements in plant performance in response to Trichoderma gradually decreased along the domestication gradient. Wild relatives and landraces were more responsive to Trichoderma, resulting in greater suppression of foliar pathogens than modern cultivars. Photosynthetic rate and stomatal conductance of some tomato genotypes were improved by Trichoderma treatment whereas leaf nitrogen status of the majority of tomato genotypes were not altered. There was a negative relationship between basal resistance and induced resistance for both diseases, and a positive correlation between Trichoderma-ISR to B. cinerea and enhanced total flavonoid contents. These findings suggest that domestication and breeding practices have altered plant responsiveness to beneficial soil microbes. Further studies are needed to decipher the molecular mechanisms underlying the differential promotion of plant growth and resistance among genotypes, and identify molecular markers to integrate selection for responsiveness into future breeding programs.

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach.

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.

Accelerated Sample Preparation for Fast Salmonella Detection in Poultry Products.

Salmonella is the most burdensome foodborne pathogen in the USA and a major causal agent of foodborne outbreaks. Detection of a pathogen such as Salmonella can be achieved within a few hours using commercially available rapid methods, but the sample preparation is time consuming and may require multiple days. We have developed and successfully tested an accelerated sample preparation method based on microfiltration, in some cases preceded by a short enrichment step, for the rapid detection of selected pathogens. The time-frame of the overall process, from sample preparation (i.e., food rinse or homogenate preparation, microbial enrichment, and filtration steps) to detection is 8 h or less. While microfiltration has been practiced for 70 years, the complex interactions between food substances and filter membrane surfaces have shown that food pretreatment methods need to be developed on a case by case basis for the recovery of bacteria from food homogenates and/or rinses. We have also demonstrated that addition of protease to treat homogenates of different poultry products prior to microfiltration avoids the rapid decrease in flux that otherwise occurs during microfiltration. This protease treatment minimizes filter clogging, so that the microbial concentration, recovery and detection of 1 to 10 CFU/g of Salmonella in poultry products is possible in less than 8 h.

Human pathogens in plant biofilms: Formation, physiology, and detection.

Fresh produce, viewed as an essential part of a healthy life style is usually consumed in the form of raw or minimally processed fruits and vegetables, and is a potentially important source of food-borne human pathogenic bacteria and viruses. These are passed on to the consumer since the bacteria can form biofilms or otherwise populate plant tissues, thereby using plants as vectors to infect animal hosts. The life cycle of the bacteria in plants differs from those in animals or humans and results in altered physiochemical and biological properties (e.g., physiology, immunity, native microflora, physical barriers, mobility, and temperature). Mechanisms by which healthy plants may become contaminated by microorganisms, develop biofilms, and then pass on their pathogenic burden to people are explored in the context of hollow fiber microfiltration by which plant-derived microorganisms may be recovered and rapidly concentrated to facilitate study of their properties. Enzymes, when added to macerated plant tissues, hydrolyze or alter macromolecules that would otherwise foul hollow-fiber microfiltration membranes. Hence, microfiltration may be used to quickly increase the concentration of microorganisms to detectable levels. This review discusses microbial colonization of vegetables, formation and properties of biofilms, and how hollow fiber microfiltration may be used to concentrate microbial targets to detectable levels. The use of added enzymes helps to disintegrate biofilms and minimize hollow fiber membrane fouling, thereby providing a new tool for more time effectively elucidating mechanisms by which biofilms develop and plant tissue becomes contaminated with human pathogens. Biotechnol. Bioeng. 2017;114: 1403-1418. © 2017 Wiley Periodicals, Inc.

Biostimulant Action of Protein Hydrolysates: Unraveling Their Effects on Plant Physiology and Microbiome.

Plant-derived protein hydrolysates (PHs) have gained prominence as plant biostimulants because of their potential to increase the germination, productivity and quality of a wide range of horticultural and agronomic crops. Application of PHs can also alleviate the negative effects of abiotic plant stress due to salinity, drought and heavy metals. Recent studies aimed at uncovering the mechanisms regulating these beneficial effects indicate that PHs could be directly affecting plants by stimulating carbon and nitrogen metabolism, and interfering with hormonal activity. Indirect effects could also play a role as PHs could enhance nutrient availability in plant growth substrates, and increase nutrient uptake and nutrient-use efficiency in plants. Moreover, the beneficial effects of PHs also could be due to the stimulation of plant microbiomes. Plants are colonized by an abundant and diverse assortment of microbial taxa that can help plants acquire nutrients and water and withstand biotic and abiotic stress. The substrates provided by PHs, such as amino acids, could provide an ideal food source for these plant-associated microbes. Indeed, recent studies have provided evidence that plant microbiomes are modified by the application of PHs, supporting the hypothesis that PHs might be acting, at least in part, via changes in the composition and activity of these microbial communities. Application of PHs has great potential to meet the twin challenges of a feeding a growing population while minimizing agriculture's impact on human health and the environment. However, to fully realize the potential of PHs, further studies are required to shed light on the mechanisms conferring the beneficial effects of these products, as well as identify product formulations and application methods that optimize benefits under a range of agro-ecological conditions.