ABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Medicago sativa/genetics , Phosphates , Plants/genetics , Plants, Genetically Modified/genetics , Ubiquitin-Conjugating Enzymes/geneticsABSTRACT
Globally, 15 Pythium species have been found to cause damping-off and seed rot of alfalfa, although surveys of species causing disease on alfalfa in the midwestern United States are lacking. Pathogens were isolated by a seedling baiting technique from soil samples of five alfalfa fields in Minnesota with high levels of damping-off. Of the 149 organisms isolated, 93 (62%) were identified as Pythium spp. and 43 (29%) were identified as Fusarium species. Pythium sylvaticum, P. irregulare, and P. ultimum var. ultimum were aggressive pathogens on germinating alfalfa seedlings. Strains of seven Pythium spp. pathogenic on soybean and corn were also pathogenic on alfalfa. The majority of the Fusarium isolates were identified as F. solani and F. oxysporum with a low number of F. redolens and F. incarnatum-equiseti. The F. oxysporum and F. incarnatum-equiseti strains were the most aggressive in causing seed and root rot. Pythium strains were sensitive to Apron XL (mefenoxam) and pyraclostrobin in vitro but efficacy varied when the fungicides were applied as a seed treatment. Seed treatments with Apron XL were more effective than treatments with Stamina against Pythium. The presence of aggressive, broad-host-range pathogens causing seed rot and damping-off suggests that new strategies are needed for managing this disease in alfalfa production systems.
Subject(s)
Fusarium , Medicago sativa , Plant Diseases , Pythium , Fungicides, Industrial/pharmacology , Fusarium/drug effects , Medicago sativa/microbiology , Medicago sativa/parasitology , Minnesota , Plant Diseases/microbiology , Plant Diseases/parasitology , Pythium/drug effects , Seeds/microbiology , Seeds/parasitologyABSTRACT
We report here the annotated draft genome sequence of Pseudomonas syringae pv. syringae strain ALF3, isolated in Wyoming. A comparison of this genome sequence with those of closely related strains of P. syringae adapted to other hosts will facilitate research into interactions between this pathogen and alfalfa.
