Project description:Sensitive and specific detection of the boxwood blight pathogen Calonectria pseudonaviculata with a metagenomic sequencing approach
Project description:Boxwood blight is an emerging disease of ornamental and native boxwood plants in the family Buxaceae. First documented in the 1990s at a single location in England, the disease is now reported throughout Europe, Asia, New Zealand, and North America. To address the growing concern over boxwood blight, ongoing research focuses on multiple biological and genetic aspects of the causal pathogens and susceptible host plants. Characterization of genetic variation among the Calonectria fungi that cause boxwood blight shows that two unique sister species with different geographic distributions incite the disease. Studies of the pathogen life cycle show the formation of long-lived survival structures and that host infection is dependent on inoculum density, temperature, and humidity. Host range investigations detail high levels of susceptibility among boxwood as well as the potential for asymptomatic boxwood infection and for other plants in the family Buxaceae to serve as additional hosts. Multiple DNA-based diagnostic assays are available, ranging from probe-based quantitative PCR assays to the use of comparative genomics to develop robust diagnostic markers or provide whole genome-scale identifications. Though many questions remain, the research that continues to address boxwood blight demonstrates the importance of applying a multidisciplinary approach to understand and control emerging plant diseases.
Project description:Rhizosphere microbial communities play a substantial role in plant productivity. We studied the rhizosphere bacteria and fungi of 51 distinct potato cultivars grown under similar greenhouse conditions using a metabarcoding approach. As expected, individual cultivars were the most important determining factor of the rhizosphere microbial composition; however, differences were also obtained when grouping cultivars according to their growth characteristics. We showed that plant growth characteristics were related to deterministic and stochastic assembly processes of bacterial and fungal communities, respectively. The bacterial genera Arthrobacter and Massilia (known to produce indole acetic acid and siderophores) exhibited greater relative abundance in high- and medium-performing cultivars. Bacterial co-occurrence networks were larger in the rhizosphere of these cultivars and were characterized by a distinctive combination of plant beneficial Proteobacteria and Actinobacteria along with a module of diazotrophs namely Azospira, Azoarcus, and Azohydromonas. Conversely, the network within low-performing cultivars revealed the lowest nodes, hub taxa, edges density, robustness, and the highest average path length resulting in reduced microbial associations, which may potentially limit their effectiveness in promoting plant growth. Our findings established a clear pattern between plant productivity and the rhizosphere microbiome composition and structure for the investigated potato cultivars, offering insights for future management practices.
Project description:Calonectria pseudonaviculata (Cps) poses an increasing threat to boxwood, a major nursery crop and iconic landscape plant worldwide. Here, we report on a potent biocontrol agent that produces small sage green (SSG) colonies on potato dextrose agar. SSG is a bacterial strain recovered from Justin Brouwers boxwood leaves with unusual response to Cps inoculation. Water-soaked symptoms developed on leaves 2 days after inoculation then disappeared a few days later. This endophyte affected several major steps of the boxwood blight disease cycle. SSG at 107 cfu/mL lysed all conidia in mixed broth culture. SSG at 108 cfu/mL reduced blight incidence by >98% when applied one day before or 3 h after boxwood were inoculated with Cps. Its control efficacy decreased with decreasing bacterial concentration to 103 cfu/mL and increasing lead time up to 20 days. When applied on diseased leaf litter under boxwood plants, SSG reduced Cps sporulation and consequently mitigated blight incidence by 90%. SSG was identified as a new member of the Burkholderia cepacia complex with distinct characters from known clinical strains. With these protective, curative, and sanitizing properties, this Burkholderia endophyte offers great promise for sustainable blight management at production and in the landscape.
Project description:Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.
Project description:BackgroundGrowing evidence suggests that soil microbes can improve plant fitness under drought. However, in potato, the world's most important non-cereal crop, the role of the rhizosphere microbiome under drought has been poorly studied. Using a cultivation independent metabarcoding approach, we examined the rhizosphere microbiome of two potato cultivars with different drought tolerance as a function of water regime (continuous versus reduced watering) and manipulation of soil microbial diversity (i.e., natural (NSM), vs. disturbed (DSM) soil microbiome).ResultsWater regime and soil pre-treatment showed a significant interaction with bacterial community composition of the sensitive (HERBST) but not the resistant cultivar (MONI). Overall, MONI had a moderate response to the treatments and its rhizosphere selected Rhizobiales under reduced watering in NSM soil, whereas Bradyrhizobium, Ammoniphilus, Symbiobacterium and unclassified Hydrogenedensaceae in DSM soil. In contrast, HERBST response to the treatments was more pronounced. Notably, in NSM soil treated with reduced watering, the root endophytic fungus Falciphora and many Actinobacteriota members (Streptomyces, Glycomyces, Marmoricola, Aeromicrobium, Mycobacterium and others) were largely represented. However, DSM soil treatment resulted in no fungal taxa and fewer enrichment of these Actinobacteriota under reduced watering. Moreover, the number of bacterial core amplicon sequence variants (core ASVs) was more consistent in MONI regardless of soil pre-treatment and water regimes as opposed to HERBST, in which a marked reduction of core ASVs was observed in DSM soil.ConclusionsBesides the influence of soil conditions, our results indicate a strong cultivar-dependent relationship between the rhizosphere microbiome of potato cultivars and their capacity to respond to perturbations such as reduced soil moisture. Our study highlights the importance of integrating soil conditions and plant genetic variability as key factors in future breeding programs aiming to develop drought resistance in a major food crop like potato. Elucidating the molecular mechanisms how plants recruit microbes from soil which help to mitigate plant stress and to identify key microbial taxa, which harbour the respective traits might therefore be an important topic for future research.
Project description:Pathogen detection and identification are key elements in outbreak control of human, animal, and plant diseases. Since many fungal plant pathogens cause similar symptoms, are difficult to distinguish morphologically, and grow slowly in culture, culture-independent, sequence-based diagnostic methods are desirable. Whole genome metagenomic sequencing has emerged as a promising technique because it can potentially detect any pathogen without culturing and without the need for pathogen-specific probes. However, efficient DNA extraction protocols, computational tools, and sequence databases are required. Here we applied metagenomic sequencing with the Oxford Nanopore Technologies MinION to the detection of the fungus Calonectria pseudonaviculata, the causal agent of boxwood (Buxus spp.) blight disease. Two DNA extraction protocols, several DNA purification kits, and various computational tools were tested. All DNA extraction methods and purification kits provided sufficient quantity and quality of DNA. Several bioinformatics tools for taxonomic identification were found suitable to assign sequencing reads to the pathogen with an extremely low false positive rate. Over 9% of total reads were identified as C. pseudonaviculata in a severely diseased sample and identification at strain-level resolution was approached as the number of sequencing reads was increased. We discuss how metagenomic sequencing could be implemented in routine plant disease diagnostics.
Project description:The optimal ranges of protein for healthy adult dogs are not known. This study evaluated the impact of long-term consumption of foods containing low, medium, and high levels of protein on serum, urine, and fecal metabolites, and gut microbiome in beagles. Following maintenance on a prefeed food for 14 days, dogs (15 neutered males, 15 spayed females, aged 2-9 years, mean initial weight 11.3 kg) consumed the low (18.99%, dry matter basis), medium (25.34%), or high (45.77%) protein foods, each for 90 days, in a William's Latin Square Design sequence. In serum and/or urine, metabolites associated with inflammation (9,10-dihydroxyoctadecanoic acid (DiHOME)), 12,13-DiHOME) and kidney dysfunction (urea, 5-hydroxyindole sulfate, 7-hydroxyindole sulfate, p-cresol sulfate) increased with higher protein levels in food, while one-carbon pathway metabolites (betaine, dimethylglycine, sarcosine) decreased. Fecal pH increased with protein consumed, and levels of beneficial indoles and short-chain fatty acids decreased while branched-chain fatty acids increased. Beta diversity of the fecal microbiome was significantly different, with increased abundances of proteolytic bacteria with higher protein food. Feeding dogs a high amount of protein leads to a shift to proteolytic gut bacteria, higher fecal pH, and is associated with increased levels of metabolites linked with inflammation and kidney dysfunction.
Project description:Background: The soil environment is responsible for sustaining most terrestrial plant life on earth, yet we know surprisingly little about the important functions carried out by diverse microbial communities in soil. Soil microbes that inhabit the channels of decaying root systems, the detritusphere, are likely to be essential for plant growth and health, as these channels are the preferred locations of new root growth. Understanding the microbial metagenome of the detritusphere and how it responds to agricultural management such as crop rotations and soil tillage will be vital for improving global food production. Methods: The rhizosphere soils of wheat and chickpea growing under + and - decaying root were collected for metagenomics sequencing. A gene catalogue was established by de novo assembling metagenomic sequencing. Genes abundance was compared between bulk soil and rhizosphere soils under different treatments. Conclusions: The study describes the diversity and functional capacity of a high-quality soil microbial metagenome. The results demonstrate the contribution of the microbiome from decaying root in determining the metagenome of developing root systems, which is fundamental to plant growth, since roots preferentially inhabit previous root channels. Modifications in root microbial function through soil management, can ultimately govern plant health, productivity and food security.