Increasing concerns on environmental and economic issues linked to fossil fuel use has driven great interest in cyanobacteria as third generation biofuel agents. In this study, the biodiesel potential of a model photosynthetic cyanobacterium, Fremyella diplosiphon, was identified by fatty acid methyl esters (FAME) via direct transesterification. Total lipids in wild type (Fd33) and halotolerant (HSF33-1 and HSF33-2) strains determined by gravimetric analysis yielded 19% cellular dry weight (CDW) for HSF33-1 and 20% CDW for HSF33-2, which were comparable to Fd33 (18% CDW). Gas chromatography-mass spectrometry detected a high ratio of saturated to unsaturated FAMEs (2.48-2.61) in transesterified lipids, with methyl palmitate being the most abundant (C16:0). While theoretical biodiesel properties revealed high cetane number and oxidative stability, high cloud and pour point values indicated that fuel blending could be a viable approach. Significantly high FAME abundance in total transesterified lipids of HSF33-1 (40.2%) and HSF33-2 (69.9%) relative to Fd33 (25.4%) was identified using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry, indicating that robust salt stress response corresponds to higher levels of extractable FAME. Alkanes, a key component in conventional fuels, were present in F. diplosiphon transesterified lipids across all strains confirming that natural synthesis of these hydrocarbons is not inhibited during biodiesel production. While analysis of photosynthetic pigments and phycobiliproteins did not reveal significant differences, FAME abundance varied significantly in wild type and halotolerant strains indicating that photosynthetic pathways are not the sole factors that determine fatty acid production. We characterize the potential of F. diplosiphon for biofuel production with FAME yields in halotolerant strains higher than the wild type with no loss in photosynthetic pigmentation.
Apple scab caused by Venturia inaequalis is the most destructive disease of apple worldwide. In this study, genetic diversity of 101 V. inaequalis isolates from cultivated apples and ornamental crabapples in Pennsylvania (PA, USA) was characterized using 14 microsatellite markers. A total of 157 alleles ranging from 5 (Vitg9/99) to 26 (Vica10/154) per locus were detected. Regardless of the host of origin, isolates were grouped into five clusters, which were largely supported by STRUCTURE and principal coordinate analysis. Cluster analyses based on genetic distances and population structure analysis suggest very small differentiation (PhiPT ranging from 0.016 to 0.103, depending on the population comparison) between apple and crabapple isolates of V. inaequalis. Pairwise comparisons among populations from different locations showed very low differentiation, and POPGENE analysis indicated frequent migration of alleles (Nm = 1.47). In pathogenicity tests using a detached leaf assay, isolates of V. inaequalis from crabapple caused characteristic scab symptoms on apple and were highly virulent. Results of the study indicate that scab lesions in crabapple trees in close vicinity to apple orchards could serve as reservoirs for spread of the pathogen.Movement of inoculum among locations and between hosts may be responsible for the limited population structure observed. Understanding the population structure of V. inaequalis isolates is significant for apple scab management as crabapples are often used as pollinizers and rootstock in apple orchards, and as ornamental trees.
The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents. IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.
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