Mary V. Duke

LOCALIZATION OF ARTEMISININ AND ARTEMISITENE IN FOLIAR TISSUES OF GLANDED AND GLANDLESS BIOTYPES OF ARTEMISIA ANNUA L.

The tissue localization of the antimalarial sesquiterpenoid compound artemisinin in annual wormwood (Artemisia annua L.) was determined by differential extraction of a glanded biotype and through the use of a glandless biotype. A 5-s dip in chloroform extracted 97% of the artemisinin from glanded A. annua leaf tissue. In addition, all of the detectable artemisitene, an artemisinin analog, was extracted. This extraction method caused collapse of the subcuticular space of the capitate glands on the leaf surface, whereas no other damage to the leaf surface was observed with SEM. Light microscopy and TEM revealed that this extraction method, despite causing some organelle structural changes, did not disrupt cell membranes. An A. annua biotype without glands contained neither artemisinin nor artemisitene. These results indicate that artemisinin and artemisitene present in foliar tissue are localized entirely in the subcuticular space of capitate glands of A. annua.

Metagenomic analysis of the turkey gut RNA virus community

Viral enteric disease is an ongoing economic burden to poultry producers worldwide, and despite considerable research, no single virus has emerged as a likely causative agent and target for prevention and control efforts. Historically, electron microscopy has been used to identify suspect viruses, with many small, round viruses eluding classification based solely on morphology. National and regional surveys using molecular diagnostics have revealed that suspect viruses continuously circulate in United States poultry, with many viruses appearing concomitantly and in healthy birds. High-throughput nucleic acid pyrosequencing is a powerful diagnostic technology capable of determining the full genomic repertoire present in a complex environmental sample. We utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease. This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys. Our analysis of this turkey gut RNA metagenome focuses in particular on the turkey-origin members of the Picornavirales, the Caliciviridae, and the turkey Picobirnaviruses.

Quantitative structure-activity relationships of protoporphyrinogen oxidase-inhibiting diphenyl ether herbicides

Abstract Diphenyl ether (DPE) herbicides induce protoporphyrin IX (Proto IX) accumulation in plants by inhibiting the enzyme protoporphyrinogen oxidase (Protox). Others have shown DPE herbicides to inhibit Protox competitively with respect to its substrate, protoporphyrinogen IX (Protogen). We compared the geometrical, bulk, and electronic parameters of Protogen and the DPE acifluorfen. Our analysis suggests that a competitive inhibitor of Protox must be bicyclic, representing roughly one half fraction of Protogen, for fulfilling the steric complementarity. We also examined the relationships between molecular properties of 24 DPE herbicides and their effects on enzyme-inhibitory and herbicidal activities in barley (Hordeum vulgare L.) and cucumber (Cucumis sativus L.). All compounds inhibited Protox although their I50 values ranged from 0.008 to 420 μM. The QSAR involving 24 DPEs showed that (a) the electronic (partial charge, SLUMO, and dipole moment) and lipophilicity properties accounted for the variation in the Protox-inhibiting activity and (b) the molecular bulk and overall electrostatic potentials were responsible for the observed variation in the herbicidal effects. In general, the analogues with substitutions on the m-carbon of the p-nitrophenyl ring were highly active as both enzyme-inhibitors and herbicides. Neither the enzyme-inhibitory activity nor the initial herbicidal activity were changed by substituting a p-chloro for a p-nitro group. In the acifluorfen-methyl structure, the herbicidal activity was completely lost when the CF3 group was moved from the para to the meta position. Compounds with sulfide, sulfoxide, or sulfone bridges between the phenyl rings were herbicidally inactive.

Pyrazole phenyl ether herbicides inhibit protoporphyrinogen oxidase

Abstract Two isomeric pairs of pyrazole phenyl ether herbicides [AH 2.429, 4-chloro-1-methyl-5-(4-nitrophenoxy)-3-(trifluoromethyl)-1 H -pyrazole; AH 2.430, 4-chloro-1-methyl-3-(4-nitrophenoxy)-5-(trifluoromethyl)-1 H -pyrazole; AH 2.431, 5-((4-chloro-1-methyl-5-(trifluoromethyl)-1 H -pyrazol-3-yl)oxy)-2-nitrobenzoic acid; and AH 2.432, 5-((4-chloro-1-methyl-3-(trifluoromethyl)-1 H -pyrazol-5-yl)oxy)-2-nitrobenzoic acid were evaluated for herbicidal activity in both intact plants and in tissue sections. Their capacity to induce accumulation of porphyrins in tissue sections and to inhibit protoporphyrinogen oxidase (Protox) in vitro were determined. In whole plant tests, the order of herbicidal activity was AH 2.430 ⪢ AH 2.431 > AH 2.429 > AH 2.432. AH 2.430 consistently caused light-dependent membrane leakage in both green and far-red light grown cucumber cotyledon and barley primary leaf tissue sections after incubation for 20 hr in darkness in 0.1 m M solutions. The same treatment caused marked increases in protoporphyrin IX (PPIX) content during the 20-hr dark incubation. AH 2.429 and 2.431 were less effective and not effective in all tissues in causing herbicidal damage and PPIX accumulation. AH 2.432 was ineffective in tissue section assays. Mg-PPIX levels were not significantly affected by any of the compounds. Protochlorophyllide levels were decreased by AH 2.430 and 2.431 in barley and increased by AH 2.429, 2.431, and 2.432 in cucumber. A positive relationship was found between herbicidal activity and the amount of PPIX that was caused to accumulate by each compound. All of the compounds inhibited Protox activity. Positive correlations were found between herbicidal activity in planta over a 300-fold range and in vitro Protox inhibition and the amount of PPIX caused to accumulate in vivo . These data support the view that the pyrazole phenyl ethers exert their herbicidal activity entirely through inhibition of Protox.

Evaluation of Ferulic Acid for Controlling the Musty-Odor Cyanobacterium, Oscillatoria perornata, in Aquaculture Ponds

Abstract The cyanobacterium Oscillatoria perornata f. attenuata, a common inhabitant of channel catfish, Ictalurus punctatus, aquaculture ponds, produces the musty compound 2-methylisoborneol that causes fish to become off-flavor and unmarketable. Previous laboratory studies suggest that the natural compound trans-ferulic acid (4-hy-droxy-3-methoxycinnamic acid) is selectively toxic against O. perorna-ta. The present study was conducted to determine the effectiveness of the compound for controlling the growth of O. perornata under field conditions. The study was conducted in 12 0.1-ha earthern ponds in northwest Mississippi. Ponds contained 20,000 catfish/ha and were managed according to commercial culture practices. Half of the ponds were treated six times over a 2-month period with 5μM (0.97 mg/L) trans-ferulic acid, and the other half were untreated controls. Water samples obtained from all ponds preceding and following treatment were analyzed for phytoplankton community structure and concentrations of chlorophyll a, ferulate, and 2-methylisoborneol. Abundance of cyanobacteria, including O. perornata was not consistently affected by applications of ferulic acid. Only one of the six ferulic acid applications resulted in a decrease in abundance of O. perornata in treated ponds relative to untreated ponds (P <0.1). The ineffectiveness of trans-ferulic acid as a cyanobacterial algicide in catfish ponds appears to be caused by rapid dissipation of ferulic acid from pond waters. Use of trans-ferulic acid was neither an effective nor an economical approach to preventing musty off-flavor in pond-cultured channel catfish.

Complete Genome Sequence of Channel Catfish Gastrointestinal Septicemia Isolate Edwardsiella tarda C07-087.

ABSTRACT Edwardsiella tarda is a Gram-negative facultative anaerobe causing disease in animals and humans. Here, we announce the complete genome sequence of the channel catfish isolate E. tarda strain C07-87, which was isolated from an outbreak of gastrointestinal septicemia on a commercial catfish farm.

Microbial phytotoxins as potential herbicides

Abstract Microbes are sources of a diverse array of phytotoxic compounds. These compounds are generally structurally different from commercial herbicides, targeting different molecular sites of action within the plant. These novel structures and sites can be excellent leads for the discovery and development of safer synthetic herbicides. Microbial phytotoxins are often more environmentally benign than synthetic herbicides. Examples of phytotoxins from fungi (AAL‐toxin, cornexistin, cyperin, and tentoxin) with novel structures and sites of action are discussed. AAL‐toxin is toxic to a wide variety of weeds at very low dose rates. AAL‐toxin and many of its analogues kill plants by inhibiting a ceramide synthase‐like enzyme, causing rapid accumulation of free sphingoid bases that disrupt membranes. Cornexistin appears to be metabolically cnverted to an inhibitor of certain aspartate amino transferase isoenzymes. Its activity can be reversed by feeding aspartate and glutamate or with tricarboxylic acid cycle ...

Development of a Large Set of Microsatellite Markers in Zapote Mamey (Pouteria sapota (Jacq.) H.E. Moore & Stearn) and Their Potential Use in the Study of the Species.

Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations (seven wild, three cultivated) from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool (HE = 0.41 and HE = 0.35, respectively), although modified Garza–Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop.

HPLC and in vivo Spectrophotometric Detection of Porphyrins in Plant Tissues Treated with Porphyrinogenic Herbicides

Abstract Protoporphyrinogen oxidase (Protox) inhibitors and other compounds which block or stimulate the porphyrin pathway can cause sufficient levels of porphyrins to accumulate in plant tissues for severe photo dynamic damage to occur. The gross symptomology for all of these porphyrinogenic herbicides is similar. Porphyrin accumulation induced by three porphyrinogenic herbicides acifluorfen (AF), δ-aminolevulinic acid (ALA), and 2,2′-dipyridyl (DY) was determined by in vivo spectrophotometry and HPLC methods. The averaged in vivo difference spectra between untreated and AF-treated (30 μᴍ for 20 h in darkness) yellow cucumber cotyledon discs approximated the absorption spectra of protoporphyrin IX (Proto IX). There was also an enhanced peak near 503 nm. Treatment of cotyledon discs with ALA alone generated a difference spectrum of protochlorophyllide (PChlide) in combination with Mg -Proto IX or Mg-Proto IX monomethyl ester (Mg-Proto IX ME). With ALA and AF in combination , the PChlide and Mg-Proto IX portions of the difference spectrum were reduced and the Proto IX peak and peak near 503 nm were increased. DY treatment yielded a difference spectrum with peaks approximating those of Proto IX and Mg-Proto IX ME , along with a peak near 503 nm . The presence of all porphyrins detected by in vivo spectrophotometry except for the 503 nm peak was confirmed with HPLC . Proto IX monomethyl ester was found by HPLC to be especially elevated in treatments with AF. The in vivo 503 nm peak and in vitro studies with Protox-containing barley etioplast preparations suggest that p rototetrahydroporphyrin IX (an oxidation state intermediate between protoporphyrinogen IX and Proto IX) may accumulate under some conditions. These data demonstrate that rapid in vivo spectrophotometric studies can provide much of the qualitative results of HPLC studies and can confirm that in vitro results correspond with the in vivo situation.