Frozenaire Plant

The refrigerator factory that would soon become the Glenridge Campus.

Does Gamma-aminobutyric acid function as a plant resistance mechanism against phytophagous insect activity? /

Gamma-aminobutyric acid (GAB A) is a ubiquitous non-protein amino acid synthesized via the decarboxylation of L-glutamate in a reaction catalyzed by the cytosolic enzyme L-glutamate decarboxylase (GAD). In animals it functions as an inhibitory neurotransmitter. In plants it accumulates rapidly in response to various stresses, but its function remains unclear. The hypothesis that GABA accumulation in leaf tissue may function as a plant resistance mechanism against phytophagous insect activity was investigated. GABA accumulation in response to mechanical stimulation, mechanical damage and insect activity was demonstrated. In wt tobacco (Nicotiana tabacum cv Samsun), mechanical stimulation or damage caused GABA to accumulate within 2 min from mean levels of 14 to 37 and 1~9 nmol g-l fresh weight (FW), respectively. In the transgenic tobacco strain CaMVGAD27c overexpressing Petunia GAD, the same treatments caused GABA to accumulate from 12 to 59 and 279 nmol g-l FW, respectively. In the transgenic tobacco strain CaMVGADilC 11 overexpressing Petunia GAD lacking an autoinhibitory domain, mechanical stimulation or damage caused GABA to accumulate from 180 to 309 and 630 nmol g-l FW, respectively. Ambulatory activity by tobacco budworm (TBW) larvae (Heliothis virescens) on leaves of CaMVGAD27c tobacco caused GABA to accumulate from 28 to 80 nmol g-l FW within 5 min. Ambulatory and leaf-rolling activity by oblique banded leaf roller (OBLR) larvae (Choristoneura rosaceana cv Harris) on wt soybean leaves (Glycine max cv Harovinton) caused GABA to accumulate from 60 to 1123 nmol g-l FW within 20 min. Increased GABA levels in leaf tissue were shown to affect phytophagous preference in TBW larvae presented with wt and transgenic tobacco leaves. When presented with leaves of Samsun wt and CaMVGAD27c plants, TBW larvae consumed more wt leaf tissue (640 ± 501 S.D. mm2 ) than transgenic leaf tissue (278 ± 338 S.D. mm2 ) nine times out of ten. When presented with leaves of Samsun wt and CaMVGAD~C11 plants, TBW larvae consumed more transgenic leaf tissue (1219 ± 1009 S.D. mm2 ) than wt leaf tissue (28 ± 31 S.D. mm2 ) ten times out of ten. These results indicate that: (1) ambulatory activity of insect larvae on leaves results in increased GABA levels, (2) transgenic tobacco leaves with increased capacity for GABA synthesis deter feeding, and (3) transgenic tobacco leaves with constitutively higher GABA levels stimulate feeding.

Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

Starvation Induces Expression of the Plant-Adhesin Gene, Mad2, of the Entomopathogenic Fungus Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is additionally plant rhizosphere competent. Two adhesin-encoding gens, Mad1 and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. This study examined differential expression of the Mad genes for M robertsii grown on a variety of insectand plant-related substrates. Mad1 was up regulated in response to insect cuticles and up regulation of Mad2 resulted from root exudates, tomato stems and non-preferred carbohydrates. A time course analysis that compared water, minimal media, and nutrient rich broth revealed Mad2 gene expression increased as nutrient availability decreased. The regulation of Mad2 compared to known stress-related genes (Hsp30, Hsp70 and ssgA) under various stresses (nutrient, pH, osmotic, oxidative, temperature) revealed Mad2 to be generally up regulated by nutrient starvation only. Examination of the Mad2 promoter region revealed two copies of a stress-response element (S TRE) known to be regulated under the general stress response pathway.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

Plant rhizosphere specificity and variability in the insect and plant adhesins, Madl and Mad2, within the genus Metarhizium suggest plant adaptation as an evolutionary force

Metarhizium is a soil-inhabiting fungus currently used as a biological control agent against various insect species, and research efforts are typically focused on its ability to kill insects. In section 1, we tested the hypothesis that species of Metarhizium are not randomly distributed in soils but show plant rhizosphere-specific associations. Results indicated an association of three Metarhizium species (Metarhizium robertsii, M. brunneum and M. guizhouense) with the rhizosphere of certain types of plant species. M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense, which was supported by in vitro experiments with grass root exudate. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types. In section 2, we explored the variation in the insect adhesin, Madl, and the plant adhesin, Mad2, in fourteen isolates of Metarhizium representing seven different species. Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions. Phylogenetic analysis of 5' EF-Ia, which is used for species identification, as well as Madl and Mad2 sequences demonstrated that the Mad2 phylogeny is more congruent with 5' EF-1a than Madl. This suggests Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation. While other abiotic and biotic factors cannot be excluded in contributing to divergence, it appears that plant associations have been the driving factor causing divergence among Metarhizium species.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

Feeding and oviposition preferences of the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae) on six Brassicaceae host plant species

Plutella xylostella (diamondback moth, DBM) is a globally distributed Lepidopteran that feeds and oviposits almost exclusively on plants in the Brassicaceae family. DBM disperses from the southern United States and Mexico into Canada in the spring and summer. Establishment of DBM in Ontario is partially dependent upon the quantity and quality of host plants available and the preference of DBM for different hosts. Host plants include many crops such as broccoli, canola and cabbage, as well as landscape ornamentals and wild plants. It has previously been established that DBM are attracted to host plants by chemicals, specifically glucosinolates. I examined the preference of DBM among crop, wild and ornamental host plant species and how preference varies with insect life stage (3rd and 4th instar larvae and adults). Experiments included exposing DBM larvae from five populations coming from different locations in Canada to six Brassicaceae species and evaluating the preferences and weight gain over one hour. Then adult females were exposed to these same plant species and their oviposition preferences were examined. Populations from Alberta, Saskatchewan and Ontario were compared to assess differences in preference associated with geographic region or species of host plant. The ultimate goal of my study was to understand the potential of various Brassicaceae species to act as reservoirs to sustain and promote population growth of DBM, as well as sinks that may decrease DBM abundance. Results showed that garden cress (Lepidium sativum) was highly preferred over other species (wintercress, black mustard, aubretia, broccoli and ornamental kale) for both food and oviposition sources. Previous studies report that garden cress contains saponins, chemicals shown to be toxic to developing DBM larvae, however no studies have yet shown a preference for garden cress. These results provide information on a novel host plant with the potential to control DBM population growth. No difference in preferences was found among populations of DBM from various sources in Canada.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.

Transferring Results of Behavioral Research to Industry to Improve Animal Welfare on the Farm, Ranch and the Slaughter Plant

The article focuses on three results of the study: "(1)Communicate your results outside the research. Write articles in popular and industry magazines. Speak at producer meetings and develop websites that can be used to transfer research results into practice. (2) Choose places (e.g. farms or plants) that have managers who believe in your research, and be prepared to spend a lot of time with the first place that uses your findings. (3) to fail. (4) Do not allow your technology to get tied up in patent disputes."

Designing Meat Packing Plant Handling Facilities for Cattle and Hogs

The focus is on design, more specifically, "animal handling facilities which are labor saving and reduce bruise losses". The article studies: Unloading Chutes, Stockyard Design, Hog Plant Stockyard, General Purpose Small Stockyard, Beef Stockyard, Cattle Crowding Pens, Hog Crowding Pens, Slopes in Chutes and Crowding Pens, Single File Chutes General Recommendations, Single File Chutes for Cattle, Slaughter Restrainers,

The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid

Systemic Acquired Resistance (SAR) is a type of plant systemic resistance occurring against a broad spectrum of pathogens. It can be activated in response to pathogen infection in the model plant Arabidopsis thaliana and many agriculturally important crops. Upon SAR activation, the infected plant undergoes transcriptional reprogramming, marked by the induction of a battery of defense genes, including Pathogenesis-related (PR) genes. Activation of the PR-1 gene serves as a molecular marker for the deployment of SAR. The accumulation of a defense hormone, salicylic acid (SA) is crucial for the infected plant to mount SAR. Increased cellular levels of SA lead to the downstream activation of the PR-1 gene, triggered by the combined action of the Non-expressor of Pathogenesis-related Gene 1 (NPR1) protein and the TGA II-clade transcription factor (namely TGA2). Despite the importance of SA, its receptor has remained elusive for decades. In this study, we demonstrated that in Arabidopsis the NPR1 protein is a receptor for SA. SA physically binds to the C-terminal transactivation domain of NPR1. The two cysteines (Cys521 and Cys529), which are important for NPR1’s coactivator function, within this transactivation domain are critical for the binding of SA to NPR1. The interaction between SA and NPR1 requires a transition metal, copper, as a cofactor. Our results also suggested a conformational change in NPR1 upon SA binding, releasing the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. These results advance our understanding of the plant immune function, specifically related to the molecular mechanisms underlying SAR. The discovery of NPR1 as a SA receptor enables future chemical screening for small molecules that activate plant immune responses through their interaction with NPR1 or NPR1-like proteins in commercially important plants. This will help in identifying the next generation of non-biocidal pesticides.