Hanan R. Shehata

Bacterial endophytes from wild and ancient maize are able to suppress the fungal pathogen Sclerotinia homoeocarpa

The aim of this study was to determine if endophytes from wild and ancient Zea plants (corn family) have anti‐fungal activities, specifically against the most important fungal pathogen (Sclerotinia homoeocarpa) of creeping bentgrass, a relative of Zea, used here as a model grass.

Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed

Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R2) of 0.997–0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05–3.0% (wt/wt) for the bovine and turkey targets, and 0.01–1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species.

Genes Required for the Anti-fungal Activity of a Bacterial Endophyte Isolated from a Corn Landrace Grown Continuously by Subsistence Farmers Since 1000 BC

Endophytes are microbes that inhabit internal plant tissues without causing disease. Some endophytes are known to combat pathogens. The corn (maize) landrace Chapalote has been grown continuously by subsistence farmers in the Americas since 1000 BC, without the use of fungicides, and the crop remains highly valued by farmers, in part for its natural tolerance to pests. We hypothesized that the pathogen tolerance of Chapalote may, in part, be due to assistance from its endophytes. We previously identified a bacterial endophyte from Chapalote seeds, Burkholderia gladioli strain 3A12, for its ability to combat a diversity of crop pathogens, including Sclerotinia homoeocarpa, the most important fungal disease of creeping bentgrass, a relative of maize used here as a model system. Strain 3A12 represents a unique opportunity to understand the anti-fungal activities of an endophyte associated with a crop variety grown by subsistence farmers since ancient times. Here, microscopy combined with Tn5-mutagenesis demonstrates that the anti-fungal mode of action of 3A12 involves flagella-dependent swarming toward its pathogen target, attachment and biofilm-mediated microcolony formation. The mutant screen revealed that YajQ, a receptor for the secondary messenger c-di-GMP, is a critical signaling system that mediates this endophytic mobility-based defense for its host. Microbes from the traditional seeds of farmers may represent a new frontier in elucidating host–microbe mutualistic interactions.

An endophytic microbe from an unusual volcanic swamp corn seeks and inhabits root hair cells to extract rock phosphate

In the animal microbiome, localization of microbes to specific cell types is well established, but there are few such examples within the plant microbiome which includes endophytes. Endophytes are non-pathogenic microbes that inhabit plants. Root hairs are single cells, equivalent to the nutrient-absorbing intestinal microvilli of animals, used by plants to increase the root surface area for nutrient extraction from soil including phosphorus (P). There has been significant interest in the microbiome of intestinal microvilli but less is known about the root hair microbiome. Here we describe a bacterial endophyte (3F11) from Zea nicaraguensis, a wild corn discovered in a Nicaraguan swamp above rock-P lava flowing from the San Cristobal volcano. Rock-P is insoluble and a major challenge for plants. Following seed coating and germination on insoluble-P, the endophyte colonized epidermal surfaces, ultimately colonizing root hairs intracellularly. The endophyte promoted root hair growth and secreted acids to solubilize rock-P for uptake by a larger root hair surface. The most interesting observation was that a seed-coated endophyte targeted and colonized a critical cell type, root hair cells, consistent with earlier studies. The endophyte maintained its targeting ability in two evolutionary divergent hosts, suggesting that the host recognition machinery is conserved.

Draft Genome Sequence of Burkholderia gladioli Strain UCD-UG_CHAPALOTE (Phylum Proteobacteria).

ABSTRACT Here, we present the draft genome of Burkholderia gladioli strain UCD-UG_CHAPALOTE. This strain is an endophyte isolated from surface sterilized seeds of an ancient Mexican landrace of corn, Chapalote. The genome contains 8,527,129 bp in 109 scaffolds.

Turfgrasses as model assay systems for high-throughput in planta screening of beneficial endophytes isolated from cereal crops

Cereal crops including maize (Zea mays L.) are inhabited by non-disease causing microbes known as endophytes that can promote plant growth, aid in host nutrient acquisition and promote host pathogen resistance. Screening endophytes for beneficial traits in planta using large, slow-growing cereals is challenging, thus a rapid but relevant in planta system is needed. Here, we propose that turfgrasses can be used as high-throughput assay systems for screening cereal microbes for beneficial nutrient traits. Turfgrasses are genetic relatives of cereals, but small with fast growth rates; they can be grown in test tubes under sterile conditions on defined media. Five turfgrass genotypes were evaluated for traits ideal for assaying endophytes with nutrient acquisition traits. Based on these criteria, annual ryegrass (Lolium multiflorum) was selected as a high-throughput assay system. Annual ryegrass was then used to test a collection of maize endophytes for their ability to promote plant biomass in the absence of nitrogen. Out of 75 bacterial endophytes tested, one strain (an Enterobacter sp) consistently promoted root and shoot biomass. We discuss the potential of annual ryegrass as a model assay system to test cereal endophytes for acquisition of various nutrients, changes in root/shoot architecture as well as anti-pathogen traits.

A Burkholderia endophyte of the ancient maize landrace Chapalote utilizes c-di-GMP-dependent and independent signaling to suppress diverse plant fungal pathogen targets

Chapalote is a maize (corn) landrace grown continuously by subsistence farmers in the Americas since 1000 BC, valued in part for its broad-spectrum pathogen resistance. Previously, we showed that Chapalote possesses a bacterial endophyte, Burkholderia gladioli strain 3A12, which suppresses growth of Sclerotinia homoeocarpa, a fungal pathogen of a maize relative, used as a model system. Ten mutants that lost the anti-pathogen activities were identified, corresponding to five genes. However, S. homoeocarpa is not a known maize pathogen; hence, the relevance of these anti-fungal mechanisms to its ancient host has not been clear. Here, the strain 3A12 mutants were tested against a known pathogen of maize and many crops, Rhizoctonia solani. Microscopy established that wild-type 3A12 swarms towards, and attaches onto, the pathogen, forming microcolonies, resulting in hyphal cleavage. Analysis of the mutants revealed that 3A12 uses common downstream gene products (e.g. fungicides) to suppress the growth of both S. homoeocarpa and R. solani, but apparently different upstream regulatory machinery, with the former, but not latter pathogen, requiring YajQ, a receptor for the secondary messenger c-di-GMP. We conclude that B. gladioli strain 3A12, an endophyte of an ancient maize, employs both c-di-GMP-dependent and independent signaling to target diverse fungal pathogens.