Interactive transcriptome analyses of Northern Wild Rice (Zizania palustris L.) and Bipolaris oryzae show convoluted communications during the early stages of fungal brown spot development.

Fungal diseases, caused mainly by Bipolaris spp., are past and current threats to Northern Wild Rice (NWR) grain production and germplasm preservation in both natural and cultivated settings. Genetic resistance against the pathogen is scarce. Toward expanding our understanding of the global gene communications of NWR and Bipolaris oryzae interaction, we designed an RNA sequencing study encompassing the first 12 h and 48 h of their encounter. NWR activated numerous plant recognition receptors after pathogen infection, followed by active transcriptional reprogramming of signaling mechanisms driven by Ca2+ and its sensors, mitogen-activated protein kinase cascades, activation of an oxidative burst, and phytohormone signaling-bound mechanisms. Several transcription factors associated with plant defense were found to be expressed. Importantly, evidence of diterpenoid phytoalexins, especially phytocassane biosynthesis, among expression of other defense genes was found. In B. oryzae, predicted genes associated with pathogenicity including secreted effectors that could target plant defense mechanisms were expressed. This study uncovered the early molecular communication between the NWR-B. oryzae pathosystem, which could guide selection for allele-specific genes to boost NWR defenses, and overall aid in the development of more efficient selection methods in NWR breeding through the use of the most virulent fungal isolates.

Diversity of Strains in the Pseudomonas syringae Complex Causing Bacterial Stem Blight of Alfalfa (Medicago sativa) in the United States.

Alfalfa growers in the Intermountain West of the United States have recently seen an increased incidence in bacterial stem blight (BSB), which can result in significant herbage yield losses from the first harvest. BSB has been attributed to Pseudomonas syringae pv. syringae and P. viridiflava; however, little is known about the genetic diversity and pathogenicity of these bacteria or their interaction with alfalfa plants. Here, we present a comprehensive phylogenetic and phenotypic analysis of P. syringae and P. viridiflava strains causing BSB on alfalfa. A multilocus sequence analysis found that they grouped exclusively with P. syringae PG2b and P. viridiflava PG7a. Alfalfa symptoms caused by both bacterial groups were indistinguishable, although there was a large range in mean disease scores for individual strains. Overall, PG2b strains incited significantly greater disease scores than those caused by PG7a strains. Inoculated plants showed browning in the xylem and collapse of epidermal and pith parenchyma cells. Inoculation with a mixture of PG2b and PG7a strains did not result in synergistic activity. The populations of PG2b and PG7a strains were genetically diverse within their clades and did not group by location or haplotype. The PG2b strains had genes for production of the phytotoxin coronatine, which is unusual in PG2b strains. The results indicate that both pathogens are well established on alfalfa across a wide geographic range and that a recent introduction or evolution of more aggressive strains as the basis for emergence of the disease is unlikely.

Surfactin and Spo0A-Dependent Antagonism by Bacillus subtilis Strain UD1022 against Medicago sativa Phytopathogens.

Plant growth-promoting rhizobacteria (PGPR) such as the root colonizers Bacillus spp. may be ideal alternatives to chemical crop treatments. This work sought to extend the application of the broadly active PGPR UD1022 to Medicago sativa (alfalfa). Alfalfa is susceptible to many phytopathogens resulting in losses of crop yield and nutrient value. UD1022 was cocultured with four alfalfa pathogen strains to test antagonism. We found UD1022 to be directly antagonistic toward Collectotrichum trifolii, Ascochyta medicaginicola (formerly Phoma medicaginis), and Phytophthora medicaginis, and not toward Fusarium oxysporum f. sp. medicaginis. Using mutant UD1022 strains lacking genes in the nonribosomal peptide (NRP) and biofilm pathways, we tested antagonism against A. medicaginicola StC 306-5 and P. medicaginis A2A1. The NRP surfactin may have a role in the antagonism toward the ascomycete StC 306-5. Antagonism toward A2A1 may be influenced by B. subtilis biofilm pathway components. The B. subtilis central regulator of both surfactin and biofilm pathways Spo0A was required for the antagonism of both phytopathogens. The results of this study indicate that the PGPR UD1022 would be a good candidate for further investigations into its antagonistic activities against C. trifolii, A. medicaginicola, and P. medicaginis in plant and field studies.

Genetic Mapping of Tolerance to Bacterial Stem Blight Caused by Pseudomonas syringae pv. syringae in Alfalfa (Medicago sativa L.).

The bacterial stem blight of alfalfa (Medicago sativa L.), first reported in the United States in 1904, has emerged recently as a serious disease problem in the western states. The causal agent, Pseudomonas syringae pv. syringae, promotes frost damage and disease that can reduce first harvest yields by 50%. Resistant cultivars and an understanding of host-pathogen interactions are lacking in this pathosystem. With the goal of identifying DNA markers associated with disease resistance, we developed biparental F1 mapping populations using plants from the cultivar ZG9830. Leaflets of plants in the mapping populations were inoculated with a bacterial suspension using a needleless syringe and scored for disease symptoms. Bacterial populations were measured by culture plating and using a quantitative PCR assay. Surprisingly, leaflets with few to no symptoms had bacterial loads similar to leaflets with severe disease symptoms, indicating that plants without symptoms were tolerant to the bacterium. Genotyping-by-sequencing identified 11 significant SNP markers associated with the tolerance phenotype. This is the first study to identify DNA markers associated with tolerance to P. syringae. These results provide insight into host responses and provide markers that can be used in alfalfa breeding programs to develop improved cultivars to manage the bacterial stem blight of alfalfa.

Objective Phenotyping of Root System Architecture Using Image Augmentation and Machine Learning in Alfalfa (Medicago sativa L.).

Active breeding programs specifically for root system architecture (RSA) phenotypes remain rare; however, breeding for branch and taproot types in the perennial crop alfalfa is ongoing. Phenotyping in this and other crops for active RSA breeding has mostly used visual scoring of specific traits or subjective classification into different root types. While image-based methods have been developed, translation to applied breeding is limited. This research is aimed at developing and comparing image-based RSA phenotyping methods using machine and deep learning algorithms for objective classification of 617 root images from mature alfalfa plants collected from the field to support the ongoing breeding efforts. Our results show that unsupervised machine learning tends to incorrectly classify roots into a normal distribution with most lines predicted as the intermediate root type. Encouragingly, random forest and TensorFlow-based neural networks can classify the root types into branch-type, taproot-type, and an intermediate taproot-branch type with 86% accuracy. With image augmentation, the prediction accuracy was improved to 97%. Coupling the predicted root type with its prediction probability will give breeders a confidence level for better decisions to advance the best and exclude the worst lines from their breeding program. This machine and deep learning approach enables accurate classification of the RSA phenotypes for genomic breeding of climate-resilient alfalfa.

Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate.

In this article, we describe a set of novel alfalfa (Medicago sativa L.) plants that hyper-accumulate Phosphate ion (Pi) at levels 3- to 6-fold higher than wild-type. This alfalfa germplasm will have practical applications reclaiming Pi from contaminated or enriched soil or be used in conservation buffer strips to protect waterways from Pi run-off. Hyper-accumulating alfalfa plants were generated by targeted mutagenesis of PHOSPHATE2 (PHO2) using newly created CRISPR/Cas9 reagents and an improved mutant screening strategy. PHO2 encodes a ubiquitin conjugating E2 enzyme (UBC24) previously characterized in Arabidopsis thaliana, Medicago truncatula, and Oryza sativa. Mutations of PHO2 disrupt Pi homeostasis resulting in Pi hyper-accumulation. Successful CRISPR/Cas9 editing of PHO2 demonstrates that this is an efficient mutagenesis tool in alfalfa despite its complex autotetraploid genome structure. Arabidopsis and M. truncatula ortholog genes were used to identify PHO2 haplotypes in outcrossing tetraploid M. sativa with the aim of generating heritable mutations in both PHO2-like genes (PHO2-B and PHO2-C). After delivery of the reagent and regeneration from transformed leaf explants, plants with mutations in all haplotypes of PHO2-B and PHO2-C were identified. These plants were evaluated for morphology, Pi accumulation, heritable transmission of targeted mutations, segregation of mutant haplotypes and removal of T-DNA(s). The Agrobacterium-mediated transformation assay and gene editing reagents reported here were also evaluated for further optimization for future alfalfa functional genomic studies.

Pseudomonas viridiflava: An internal outsider of the Pseudomonas syringae species complex.

Pseudomonas viridiflava is a gram-negative pseudomonad that is phylogenetically placed within the Pseudomonas syringae species complex. P. viridiflava has a wide host range and causes a variety of symptoms in different plant parts, including stems, leaves, and blossoms. Outside of its role as a pathogen, P. viridiflava also exists as an endophyte, epiphyte, and saprophyte. Increased reports of P. viridiflava causing disease on new hosts in recent years coincide with increased research on its genetic variability, virulence, phylogenetics, and phenotypes. There is high variation in its core genome, virulence factors, and phenotypic characteristics. The main virulence factors of this pathogen include the enzyme pectate lyase and virulence genes encoded within one or two pathogenicity islands. The delineation of P. viridiflava in the P. syringae complex has been investigated using several molecular approaches. P. viridiflava comprises its own species, within the complex. While seemingly an outsider to the complex as a whole due to differences in the core genome and virulence genes, low average nucleotide identity to other of P. syringae complex members, and some phenotypic traits, it remains as part of the complex. Defining phylogenetic, phenotypic, and genomic characteristics of P. viridiflava in comparison to other P. syringae members is important to understanding this pathogen and for the development of disease resistance and management practices. TAXONOMY: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; Species Pseudomonas syringae species complex, Genomospecies 6, Phylogroup 7 and 8. MICROBIOLOGICAL PROPERTIES: Gram-negative, fluorescent, aerobic, motile, rod-shaped, oxidase negative, arginine dihydrolase negative, levan production negative (or positive), potato rot positive (or negative), tobacco hypersensitivity positive. GENOME: There are two complete genomes, five chromosome-level genomes, and 1,540 genomes composed of multiple scaffolds of P. viridiflava available in the National Center for Biotechnology Information Genome database. The median total length of these assemblies is 5,975,050 bp, the median number of protein coding genes is 5,208, and the median G + C content is 59.3%. DISEASE SYMPTOMS: P. viridiflava causes a variety of disease symptoms, including spots, streaks, necrosis, rots, and more in above- and below-ground plant parts on at least 50 hosts. EPIDEMIOLOGY: There have been several significant disease outbreaks on field and horticultural crops caused by P. viridiflava since the turn of the century. P. viridiflava has been reported as a pathogen, epiphyte, endophyte, and saprophyte. This species has been isolated from a variety of environmental sources, including asymptomatic wild plants, snow, epilithic biofilms, and icepacks.

Phenotyping seedlings for selection of root system architecture in alfalfa (Medicago sativa L.).

BACKGROUND: The root system architecture (RSA) of alfalfa (Medicago sativa L.) affects biomass production by influencing water and nutrient uptake, including nitrogen fixation. Further, roots are important for storing carbohydrates that are needed for regrowth in spring and after each harvest. Previous selection for a greater number of branched and fibrous roots significantly increased alfalfa biomass yield. However, phenotyping root systems of mature alfalfa plant is labor-intensive, time-consuming, and subject to environmental variability and human error. High-throughput and detailed phenotyping methods are needed to accelerate the development of alfalfa germplasm with distinct RSAs adapted to specific environmental conditions and for enhancing productivity in elite germplasm. In this study methods were developed for phenotyping 14-day-old alfalfa seedlings to identify measurable root traits that are highly heritable and can differentiate plants with either a branched or a tap rooted phenotype. Plants were grown in a soil-free mixture under controlled conditions, then the root systems were imaged with a flatbed scanner and measured using WinRhizo software. RESULTS: The branched root plants had a significantly greater number of tertiary roots and significantly longer tertiary roots relative to the tap rooted plants. Additionally, the branch rooted population had significantly more secondary roots > 2.5 cm relative to the tap rooted population. These two parameters distinguishing phenotypes were confirmed using two machine learning algorithms, Random Forest and Gradient Boosting Machines. Plants selected as seedlings for the branch rooted or tap rooted phenotypes were used in crossing blocks that resulted in a genetic gain of 10%, consistent with the previous selection strategy that utilized manual root scoring to phenotype 22-week-old-plants. Heritability analysis of various root architecture parameters from selected seedlings showed tertiary root length and number are highly heritable with values of 0.74 and 0.79, respectively. CONCLUSIONS: The results show that seedling root phenotyping is a reliable tool that can be used for alfalfa germplasm selection and breeding. Phenotypic selection of RSA in seedlings reduced time for selection by 20 weeks, significantly accelerating the breeding cycle.

Pan-transcriptome identifying master genes and regulation network in response to drought and salt stresses in Alfalfa (Medicago sativa L.).

Alfalfa is an important legume forage grown worldwide and its productivity is affected by environmental stresses such as drought and high salinity. In this work, three alfalfa germplasms with contrasting tolerances to drought and high salinity were used for unraveling the transcriptomic responses to drought and salt stresses. Twenty-one different RNA samples from different germplasm, stress conditions or tissue sources (leaf, stem and root) were extracted and sequenced using the PacBio (Iso-Seq) and the Illumina platforms to obtain full-length transcriptomic profiles. A total of 1,124,275 and 91,378 unique isoforms and genes were obtained, respectively. Comparative analysis of transcriptomes identified differentially expressed genes and isoforms as well as transcriptional and post-transcriptional modifications such as alternative splicing events, fusion genes and nonsense-mediated mRNA decay events and non-coding RNA such as circRNA and lncRNA. This is the first time to identify the diversity of circRNA and lncRNA in response to drought and high salinity in alfalfa. The analysis of weighted gene co-expression network allowed to identify master genes and isoforms that may play important roles on drought and salt stress tolerance in alfalfa. This work provides insight for understanding the mechanisms by which drought and salt stresses affect alfalfa growth at the whole genome level.

First Report of Mycoleptodiscus terrestris Causing Crown and Root Rot of Alfalfa (Medicago sativa) in Minnesota.

Mycoleptodiscus terrestris (Gerd.) Ostaz. has been studied extensively as a potential mycoherbicide against aquatic weeds since the early 1970s (Mathur and Gehlot 2018; Shearer 1998). However, it is also a pathogen on many legumes including alfalfa (Gerdemann 1953; Smith et al. 1998). In August 2019, alfalfa plants with stunted yellow foliage and rotted stems at the base of the crown were observed in a variety trial planted in spring 2018 at the University of Minnesota, St. Paul, Minnesota. Affected plants were in patches across the field with approximately 20% of plants showing symptoms. Plants from patches had few lateral and fibrous roots and dark lesions appeared in the crown and root tissue. Tissue samples (5 mm2) from 10 plants with symptomatic crown and root tissues were surface disinfested with 70% ethanol for 5 min, followed by 30 s in 10% NaOCl, and then rinsed three times in sterile distilled water. Samples were air-dried and placed on water agar (WA) amended with 25 µg/ml rifampicin. Plates were incubated at room temperature for 3 days and fungal hyphal tips from tissue samples were transferred to potato dextrose agar (PDA). After 7 days, isolates were tentatively identified by morphological characteristics as M. terrestris (Ostazeski 1967). The hyaline mycelia turned from olive-gray to dark gray with age. Abundant dark microsclerotia formed 5 days after incubation that varied in size and shape, measuring 300-860 x 270-600 µm. Molecular identification of two representative isolates (DAZ5 and DAZ9) was done by sequencing the internal transcribed spacer (ITS) regions of the rDNA, the translation elongation factor 1α (TEF1), and the second largest subunit of RNA polymerase II (RPB2) genes. For both isolates the ITS, TEF1, and RPB2 genes were amplified and sequenced with primers ITS1/4 (White et al. 1990), 983F/1567R (Rehner and Buckley 2005), and fRPB2-7cR/RPB-5F2 (Liu et al. 1999; Sung et al. 2007), respectively. ITS amplification conditions followed White et al. (1990), the TEF and the partial RPB2 gene was obtained by using a touchdown PCR protocol as described in Rehner and Buckley (2005) and Woudenberg et al. (2017), respectively. Based on a BLAST search of the NCBI nucleotide database, the ITS sequences (GenBank MN851265.1 and MN851266.1) had 100% identity with M. terrestris strain CBS 231.53 (MK487754.1). The TEF1 (MN873019, MN873020) and RPB2 sequences (MN873021, MN873022) had 100% identity with M. terrestris strain IMI 159038 (MK495977.1, MK492735.1). A pathogenicity test was performed by inoculating five 3-week-old alfalfa plants per cultivar (cv. DKA44-16RR, Vernal, 53V52, and Agate) with isolates DAZ5 and DAZ9. Plants were inoculated around the exposed stem base with three 5 mm diameter PDA plugs from a culture of M. terrestris covered with microsclerotia. Control plants were inoculated with sterile PDA plugs. After inoculation plants were incubated at 25 0C with a 16-h photoperiod in a growth chamber. The first symptoms appeared two months after inoculation on all cultivars as a dark lesion at the stem base followed by yellowing and rotting of stems. Inoculated plants had more fibrous roots than the controls. The control plants were symptomless with healthy root development. The pathogen was re-isolated from all infected alfalfa cultivars and had the same morphology as isolates DAZ5 and DAZ9, fulfilling Koch's postulates. To our knowledge, this is the first report of M. terrestris causing crown and root rot disease of alfalfa in Minnesota.

Plant defensin antibacterial mode of action against Pseudomonas species.

BACKGROUND: Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. RESULTS: Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. CONCLUSIONS: MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.

Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes.

Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.

A local score approach improves GWAS resolution and detects minor QTL: application to Medicago truncatula quantitative disease resistance to multiple Aphanomyces euteiches isolates.

Quantitative trait loci (QTL) with small effects, which are pervasive in quantitative phenotypic variation, are difficult to detect in genome-wide association studies (GWAS). To improve their detection, we propose to use a local score approach that accounts for the surrounding signal due to linkage disequilibrium, by accumulating association signals from contiguous single markers. Simulations revealed that, in a GWAS context with high marker density, the local score approach outperforms single SNP p-value-based tests for detecting minor QTL (heritability of 5-10%) and is competitive with regard to alternative methods, which also aggregate p-values. Using more than five million SNPs, this approach was applied to identify loci involved in Quantitative Disease Resistance (QDR) to different isolates of the plant root rot pathogen Aphanomyces euteiches, from a GWAS performed on a collection of 174 accessions of the model legume Medicago truncatula. We refined the position of a previously reported major locus, underlying MYB/NB-ARC/tyrosine kinase candidate genes conferring resistance to two closely related A. euteiches isolates belonging to pea pathotype I. We also discovered a diversity of minor resistance QTL, not detected using p-value-based tests, some of which being putatively shared in response to pea (pathotype I and III) and/or alfalfa (race 1 and 2) isolates. Candidate genes underlying these QTL suggest pathogen effector recognition and plant proteasome as key functions associated with M. truncatula resistance to A. euteiches. GWAS on any organism can benefit from the local score approach to uncover many weak-effect QTL.

Sensitivity of Bipolaris oryzae Isolates Pathogenic on Cultivated Wild Rice to the Quinone Outside Inhibitor Azoxystrobin.

The occurrence of fungal brown spot, caused by Bipolaris oryzae, has increased in cultivated wild rice (Zizania palustris) paddies in spite of the use of azoxystrobin-based fungicides. The active ingredient blocks electron transfer at the quinone outside inhibitor (QoI) site in the mitochondrial cytochrome b within the bc1 complex, thus obstructing respiration. The in vitro averaged EC50 of baseline isolates collected in 2007 before widespread fungicide use was estimated to be 0.394 µg/ml with PROBIT and 0.427 µg/ml with linear regression analyses. Isolates collected during 2008, 2015, and 2016 had a range of sensitivity as measured by relative spore germination (RG) at a discriminatory dose of 0.4 µg/ml azoxystrobin. Isolates with a higher (≥80%) and lower RG (≤40%) had the wild type nucleotides at amino acid positions F129, G137, and G143 of cytochrome b, sites known to be associated with QoI fungicide resistance. Two Group I introns were found in the QoI target area. The splicing site for the second intron was found immediately after the codon for G143. A mutation for fungicide resistance at this location would hinder splicing and severely reduce fitness. B. oryzae expresses an alternative oxidase in vitro, which allows the fungus to survive inhibition of respiration by azoxystrobin. This research indicates that B. oryzae has not developed resistance to QoI fungicides, although monitoring for changes in sensitivity should be continued. Judicious use of QoI fungicides within an integrated disease management system will promote an effective and environmentally sound control of the pathogen in wild rice paddies.

Impacts of Sampling Design on Estimates of Microbial Community Diversity and Composition in Agricultural Soils.

Soil microbiota play important and diverse roles in agricultural crop nutrition and productivity. Yet, despite increasing efforts to characterize soil bacterial and fungal assemblages, it is challenging to disentangle the influences of sampling design on assessments of communities. Here, we sought to determine whether composite samples-often analyzed as a low cost and effort alternative to replicated individual samples-provide representative summary estimates of microbial communities. At three Minnesota agricultural research sites planted with an oat cover crop, we conducted amplicon sequencing for soil bacterial and fungal communities (16SV4 and ITS2) of replicated individual or homogenized composite soil samples. We compared soil microbiota from within and among plots and then among agricultural sites using both sampling strategies. Results indicated that single or multiple replicated individual samples, or a composite sample from each plot, were sufficient for distinguishing broad site-level macroecological differences among bacterial and fungal communities. Analysis of a single sample per plot captured only a small fraction of the distinct OTUs, diversity, and compositional variability detected in the analysis of multiple individual samples or a single composite sample. Likewise, composite samples captured only a fraction of the diversity represented by the six individual samples from which they were formed, and, on average, analysis of two or three individual samples offered greater compositional coverage (i.e., greater number of OTUs) than a single composite sample. We conclude that sampling design significantly impacts estimates of bacterial and fungal communities even in homogeneously managed agricultural soils, and our findings indicate that while either strategy may be sufficient for broad macroecological investigations, composites may be a poor substitute for replicated samples at finer spatial scales.

Plant Defensin Peptides have Antifungal and Antibacterial Activity Against Human and Plant Pathogens.

Plant defensins are small, cysteine-rich antimicrobial peptides. These peptides have previously been shown to primarily inhibit the growth of fungal plant pathogens. Plant defensins have a γ-core motif, defined as GXCX3-9C, which is required for their antifungal activity. To evaluate plant defensins as a potential control for a problematic agricultural disease (alfalfa crown rot), short, chemically synthesized peptides containing γ-core motif sequences were screened for activity against numerous crown rot pathogens. These peptides showed both antifungal and, surprisingly, antibacterial activity. Core motif peptides from Medicago truncatula defensins (MtDef4 and MtDef5) displayed high activity against both plant and human bacterial pathogens in vitro. Full-length defensins had higher antimicrobial activity compared with the peptides containing their predictive γ-core motifs. These results show the future promise for controlling a wide array of economically important fungal and bacterial plant pathogens through the transgenic expression of a plant defensin. They also suggest that plant defensins may be an untapped reservoir for development of therapeutic compounds for combating human and animal pathogens.

Antibacterial Activity of Plant Defensins.

Plant defensins are antimicrobial host defense peptides expressed in all higher plants. Performing a significant role in plant innate immunity, plant defensins display potent activity against a wide range of pathogens. Vertebrate and invertebrate defensins have well-characterized antibacterial activity, but plant defensins are commonly considered to display antimicrobial activity against only fungi. In this review, we highlight the often-overlooked antibacterial activity of plant defensins. Also, we illustrate methods to evaluate defensins for antibacterial activity and describe the current advances in uncovering their antibacterial modes of action.

The major nectar protein of Brassica rapa is a non-specific lipid transfer protein, BrLTP2.1, with strong antifungal activity.

Nectar is one of the key rewards mediating plant-mutualist interactions. In addition to sugars, nectars often contain many other compounds with important biological functions, including proteins. This study was undertaken to assess the proteinaceous content of Brassica rapa nectar. SDS-PAGE analysis of raw B. rapa nectar revealed the presence of ~10 proteins, with a major band at ~10 kDa. This major band was found to contain a non-specific lipid transfer protein encoded by B. rapa locus Bra028980 and subsequently termed BrLTP2.1. Sequence analysis of BrLTP2.1 predicted the presence of a signal peptide required for secretion from the cell, eight cysteines, and a mature molecular mass of 7.3 kDa. Constitutively expressed BrLTP2.1-GFP in Arabidopsis displayed accumulation patterns consistent with secretion from nectary cells. BrLTP2.1 was also found to have relatively high sequence similarity to non-specific lipid-transfer proteins with known functions in plant defense, including Arabidopsis DIR1. Heterologously expressed and purified BrLTP2.1 was extremely heat stable and bound strongly to saturated free fatty acids, but not methyl jasmonate. Recombinant BrLTP2.1 also had direct antimicrobial activity against an extensive range of plant pathogens, being particularly effective against necrotrophic fungi. Taken together, these results suggest that BrLTP2.1 may function to prevent microbial growth in nectars.

Comparative Genomic Analyses of Clavibacter michiganensis subsp. insidiosus and Pathogenicity on Medicago truncatula.

Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen, with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,917 genes, with 379 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.