Systemic acquired resistance (SAR) is a state of heightened defense which is induced throughout a plant by an initial infection; it provides long-lasting, broad-spectrum resistance to subsequent pathogen challenge. Recendy we identified a phloem-mobile signal for SAR which has been elusive for almost 30 years. It is methyl salicylate (MeSA), an inactive derivative of the defense hormone, salicylic acid (SA). This discovery resulted from extensive characterization of SA-binding protein 2 (SABP2), a protein whose high affinity for SA and extremely low abundance suggested that it might be the SA receptor. Instead we discovered that SABP2 is a MeSA esterase whose function is to convert biologically inactive MeSA in the systemic tissue to active SA. The accumulated SA then activates or primes defenses leading to SAR. SABP2's esterase activity is inhibited in the initially/primary infected tissue by SA binding in its active site; this facilitates accumulation of MeSA, which is then translocated through the phloem to systemic tissue for perception and processing by SABP2 to SA. Thus, while SABP2 is not the SA receptor, it can be considered the receptor for the SAR signal. This study of SABPs not only illustrates the unexpected nature of scientific discoveries, but also underscores the need to use biochemical approaches in addition to genetics to address complex biological processes, such as disease resistance.

Detection of organic molecules present on the surface of dust particles is important in homeland security, agriculture, and several other applications. The research presented reports the ability of the aerosol mass spectrometer (AMS) to detect molecules on the surface of dust particles without detecting the particle core. Experiments were carried out to detect semi-volatile organic compounds adsorbed onto the surface of particulates without interference from the dust particle core. Methyl salicylate, oleic acid, and organophosphorus pesticides such as Malathion were detected on the surface of particles representative of dust-type materials. Zeolite powders were used as aerosol support, representative of a typical silica mineral aerosol present in the atmosphere. Mass spectral fingerprint information was gained by first directly detecting atomized species to record their clean electron impact mass spectrum. This facilitated detection during later experiments of organic molecules coated on an inorganic support. Spectra obtained give mass spectrometric signatures of molecules coated on inorganic particles without detection of the particle core. An important feature of the AMS is the ability to equate an ion rate detected in the mass spectrometer to a mass concentration of a given chemical species in a sample using its ionization efficiency. Based on an average inlet flow rate of 1.2 cm 3sec -1 the ionization efficiencies obtained were 5.89x10-5, 1.15x10-6, and 1.62x10-5 for Malathion, methyl salicylate, and oleic acid, respectively. These experiments and the results obtained show that detection and characterization of organic species adsorbed onto inorganic dust particles are possible at µg m-3 concentrations using the AMS.

One great issue of reforestation is the infestation of pest insects. The pine weevil (Hylobius abietis L.) is one of the economically most important pest insects in Europe and Asia. When insects feed on the trees the plants emit compounds that repel the insects. Methyl salicylate (MeSA) is one substance known to be emitted by conifer seedlings after herbivore feeding. Similar compounds are emitted by fungus isolated from pine weevil frass. Female pine weevils add frass to the egg laying site to protect the eggs from other pine weevils. The goal of the project was to cultivate the fungi Ophiostoma pluriannulatum and analyze the volatile substances. The fungus was cultivated using natural pine weevil frass (NF) as main substrate. Later new batches were prepared using lignin and potato dextrose broth (PDB), as feeding media to compare the results. Two sampling methods were used, SPME and adsorption with Porapak™ polymer column packing material. These samples were analyzed using GC-MS. Methyl salicylate was found in NF fungi cultures, peaking after ten days. Another, unexpected, product of the fungi was a sesquiterpene hydrocarbon. The lignin and the PDB samples did not produce MeSA. However, the sesquiterpene could be found in the lignin samples.

Leptinotarsa decemlineata (Say) is the most important defoliating pest of potato Solanum tuberosum L., in North America and Europe.  Management of this pest relies heavily on chemical control and insecticide resistance is a persistent problem.  This phenomenon has increased the need for developing novel insecticides, resistance evaluation, and the development of alternative control strategies regarding this insect pest.  From 2010 to 2013, field and lab experiments were conducted to evaluate the efficacy of a novel insecticide tolfenpyrad on L. decemlineata.  In leaf-dip assays, tolfenpyrad was highly toxic to L. decemlineata with LC50 values of 0.013 and 0.164 g ai/L for larvae and adults, respectively.  Tolfenpyrad was also toxic to eggs with 0% hatching after being dipped in a field rate concentration.  In field efficacy trials, potato plots treated with tolfenpyrad at rates as low as 153 g ai/ha effectively controlled L. decemlineata. In 2012, populations of L. decemlineata were collected from the Eastern Shore of VA and subjected to toxicity assays to determine current susceptibility to permethrin and oxamyl.  The toxicity assays indicated an increase in toxicity to permethrin in L. decemlineata larvae (LC50 = 3.931 g ai/L) and an increase in toxicity to oxamyl in adult beetles (LC50 = 9.695 g ai/L) compared with LC50 values previously reported in 1990.  In 2012, populations of L. decemlineata from Cheriton, VA, New Church, VA, Painter, VA, and Plymouth, NC were also evaluated for enzyme activity after exposure to sub-lethal concentrations of permethrin, oxamyl, and tolfenpyrad.  Adult beetles were subjected to enzyme assays to measure the activity of cytochrome P450 mono-oxygenase (P450), glutathione-S-transferase (GST), general esterases, and protein content.  Results from the enzyme assays indicated significantly greater esterase activity in beetles from Painter, VA exposed to permethrin [±-naphthol (F= 11.66, df= 4, 20, P<0.0001) and "-naphthol (F= 11.86, df= 4, 20, P<0.0001)], oxamyl [±- naphthol (F= 10.64, df= 4, 20, P<0.0001) and "-naphthol (F= 6.94, df= 4, 20, P=0.0011)], tolfenpyrad [±- naphthol (F= 407.62, df= 1, 8, P<0.0001) and "- naphthol (F= 28.15, df= 1, 8, P= 0.0007)], and the untreated control [±- naphthol (F= 28.14, df= 3, 16, P<0.0001) and "- naphthol (F= 28.86; df= 3, 16, P<0.0001)] compared to most of the other populations tested.  GST activity was significantly greater in tolfenpyrad exposed beetles compared to the non-treated beetles from Painter VA (F= 17.66, df= 5, 24, P< 0.0001). Through laboratory assays and field experiments in potato, the efficacy of a new bio-pesticide derived from the bacterium Chromobacterium subtsugae was evaluated for the control of L. decemlineata.  Results from the laboratory assays showed L. decemlineata feeding was inhibited by the bio-pesticide derived from C. subtsugae.  However, field efficacy trials in 2010, 2011, and 2012, indicated no control of L. decemlineata. Methyl salicylate is an organic compound produced by potato and other plants in response to insect herbivory.  Abundance of predatory arthropods and L. decemlineata life stages were measured in plots treated with and without 5 g slow-release packets of methyl salicylate (95% methyl salicylate (Predalure")).  Methyl salicylate treatment had no impact on predator recruitment or cumulative mortality of L. decemlineata in potatoes. This research has provided us with a new tool for L. decemlineata management, as well as more information about resistance trends and alternative control strategies from which we can build on to reduce resistance development in L. decemlineata and ultimately formulate a stronger integrated pest management strategy for this insect pest. / Ph. D.

<p>QCR 20161026</p>

This thesis is based on three inter-related studies: the first part deals with the microbial consortium, the identification of microbes and their volatiles, the second part deals with the study of bio-chemical control methods of two conifer pests; the pine weevil Hylobius abietis (L.) and the root rot fungi Heterobasidion spp., and the third part describes the production of styrene by a fungus using forest waste.The large pine weevil (Hylobius abietis L.) is an economically important pest insect of conifers in reforestation areas of Europe and Asia. The female weevils protect their eggs from feeding conspecifics by adding frass (mixture of weevil feces and chewed bark) along with the eggs. In order to understand the mechanism behind frass deposition at the egg laying site and to find repellents/antifeedants for pine weevils, microbes were isolated from the aseptically collected pine weevil frass. Microbial produced volatile organic compounds (VOCs) were collected by solid phase micro extraction and analyzed by GC-MS after cultivating them on weevil frass broth. The major VOCs were tested against pine weevils using a multi-choice olfactometer. Ewingella sp., Mucor racemosus, Penicillium solitum, P. expansum, Ophiostoma piceae, O. pluriannulatum, Debaryomyces hansenii and Candida sequanensis were identified as abundant microbes. Styrene, 6-protoilludene, 1-octene-3-ol, 3-methylanisole, methyl salicylate, 2-methoxyphenol and 2-methoxy-4-vinylphenol were the VOCs of persistently isolated microbes. In behavioral bioassay, methyl salicylate, 3-methylanisole and styrene significantly reduced the attraction of pine weevils to their host plant volatiles. Heterobasidion spp. are severe pathogenic fungi of conifers that cause root and butt rot in plants. Bacterial isolates were tested for the antagonistic activity against fungi on potato dextrose agar. Bacillus subtilis strains significantly inhibited the growth of H. annosum and H. parviporum. Styrene is an industrial chemical used for making polymeric products, currently produced from fossil fuel. A strain of Penicillium expansum isolated from pine weevil frass was investigated for the production of styrene using forest waste. Grated pine stem bark and mature oak bark supplemented with yeast extract produced greater amounts of styrene compared to potato dextrose broth. / <p>QC 20130507</p>

No description available.

Salicylic acid-binding protein 2 (SABP2) is essential for the establishment of systemic acquired resistance (SAR) in tobacco; SABP2's methyl salicylate (MeSA) esterase activity is required in healthy systemic tissues of infected plants to release the active defense phytohormone SA from MeSA, which serves as a long-distance signal for SAR. In the current study, we characterize a new gene family from Arabidopsis thaliana encoding 18 potentially active α/β fold hydrolases that share 32-57% identity with SABP2. Of 14 recombinant AtMES (MES for methyl esterase) proteins tested, five showed preference for MeSA as a substrate and displayed SA inhibition of MeSA esterase activity in vitro (AtMES1, -2, -4, -7, and -9). The two genes encoding MeSA esterases with the greatest activity, AtMES1 and -9, as well as AtMES7 were transcriptionally upregulated during infection of Arabidopsis with avirulent Pseudomonas syringae. In addition, conditional expression of AtMES1, -7, or -9 complemented SAR deficiency in SABP2-silenced tobacco, suggesting that these three members of the AtMES family are SABP2 functional homologs (orthologs). Underexpression by knockout mutation and/or RNAi-mediated silencing of multiple AtMES genes, including AtMES1, -2, -7, and -9, compromised SAR in Arabidopsis and correlated with enhanced accumulation of MeSA in the systemic tissue of SAR-induced plants. Together, the data show that several members of the AtMES gene family are functionally homologous to SABP2 and redundant for MeSA hydrolysis and probably SAR. These data suggest that MeSA is a conserved SAR signal in Arabidopsis and tobacco.

Aphrodisiac and anti-aphrodisiac pheromone production and composition in the green-veined white butterfly Pieris napi L. were investigated. Aphrodisiac pheromone biosynthesis had different time constraints in butterflies from the diapausing and directly developing generations. Effects of stable isotope incorporation in adult butterfly pheromone, in the nectar and flower volatiles of  host plants from labeled substrates were measured by solid phase microextraction and gas chromatography–mass spectrometry. A method to fertilize plants with stable isotopes was developed and found to be an effective method to investigate the transfer of pheromone building blocks from flowering plants to butterflies. The anti-aphrodisiac methyl salicylate was not biosynthesized from phenylalanine in flowers of Alliaria petiolata. Both aphrodisiac and anti-aphrodisiac pheromones in P.napi are produced not only from resources acquired in the larval stage, but also from nutritional resources consumed intheadult stage. Males of P. napi produce the anti-aphrodisiac pheromone from both the essential amino acid L-phenylalanine and from common flower fragrance constituents. / <p>QC 20140311</p>

Soybean [Glycine max (L.) Merr.] is an important crop worldwide. It has been widely consumed for protein, oil and other soy products. To develop soybean cultivars with greater resistance against pests and improved meal quality, it is important to elucidate the molecular bases of these traits. This dissertation aims to investigate the biochemical and biological functions of soybean genes from four gene families, which are hypothesized to be associated with soybean defense against pests and soybean meal quality. There are three specific objectives in this dissertation. The first one is to determine the function of components in the salicylic acid (SA) signaling pathway in soybean resistance against soybean cyst nematode (Heterodera glycines, SCN). The second one is to determine whether insect herbivory induce the emission of volatiles from soybean, and if so, how these volatiles are biosynthesized. The third objective is to identify and characterize soybean mannanase genes that can be used for the improvement of soybean meal quality. The soybean genome has been fully sequenced, which provides opportunities for cross-species comparison of gene families of interest and identification of candidate genes in soybean. The cloned cDNAs of putative genes were expressed in Escherichia coli to produce recombinant enzymes. Through biochemical assays, these proteins were proved to be soybean salicylic acid methyltransferase (GmSAMT1), methyl salicylate esterase (GmSABP2-1), α[alpha]-farnesene synthase (GmTPS1) and E-β[beta]-caryophyllene synthase (GmTPS2), and endo-β[beta]-mannanase (GmMAN1). Through a transgenic hairy root system harboring overexpression of GmSAMT1 and GmSABP2-1, both of these two genes were evaluated for their biological function related to resistance against SCN. The results showed that the over-expression of GmSAMT1 and GmSABP2-1 in the susceptible soybean background lead to enhanced resistance against SCN. Among four putative soybean mannanase genes, one gene was cloned and characterized. GmMAN1 showed the endo-β[beta]-mannanase hydrolyse activity and can hydrolyze cell walls isolated from soybean seeds. In summary, using comparative and functional genomics, a number of genes involved in soybean defense and meal quality were isolated and characterized. This study provides novel knowledge and molecular tools for the genetic improvement of soybean for enhanced resistance and improved meal quality.