Settling and Ovipositional Behavior of Bactericera cockerelli (Hemiptera: Triozidae) on Solanaceous Hosts Under Field and Laboratory Conditions.

Potato psyllid, Bactericera cockerelli (Sulc), is a seasonal insect pest in the Lower Rio Grande Valley of Texas, where it transmits the bacterial pathogen "Candidatus Liberibacter solanacearum" that causes zebra chip disease of potato. Studies were conducted to evaluate host preference of B. cockerelli adults for different plant species, and plant size and density. Settling and oviposition behavior of B. cockerelli was studied on its wild and cultivated solanaceous hosts, including potato, tomato, pepper, eggplant, and silverleaf nightshade, under both field and laboratory conditions. Naturally occurring B. cockerelli were used to evaluate host preference under open field conditions throughout the growing season. Settling and oviposition preference studies in the laboratory were conducted as cage-release experiments using pairs of plants, and observations were recorded over a 72-h period. Results of field trials indicated that naturally occurring B. cockerelli preferred potato and tomato equally for settling and oviposition, but settled on pepper, eggplant, and silverleaf nightshade only in the absence of potato and tomato. Under laboratory conditions, B. cockerelli adults preferred larger host plants, regardless of the species tested. Results also showed that movement of B. cockerelli was minimal after initial landing and settling behavior was influenced by host plant density. Lone plants attracted the most psyllids and can be used as sentinel plants to monitor B. cockerelli activity. Information from both field and laboratory studies demonstrated that not only host plant species determined host selection behavior of B. cockerelli adults, but also plant size and density.

A Predictive Degree Day Model for the Development of Bactericera cockerelli (Hemiptera: Triozidae) Infesting Solanum tuberosum.

Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) is a pest of potato (Solanum tuberosum L.) that vectors the bacterium that putatively causes zebra chip disease in potatoes, 'Candidatus Liberibacter solanacearum.' Zebra chip disease is managed by controlling populations of B. cockerelli in commercial potato fields. Lacking an integrated pest management strategy, growers have resorted to an intensive chemical control program that may be leading to insecticide-resistant B. cockerelli populations in south Texas and Mexico. To initiate the development of an integrated approach of controlling B. cockerelli, we used constant temperature studies, nonlinear and linear modeling, and field sampling data to determine and validate the degree day parameters for development of B. cockerelli infesting potato. Degree day model predictions for three different B. cockerelli life stages were tested against data collected from pesticide-free plots. The model was most accurate at predicting egg-to-egg and nymph-to-nymph peaks, with less accuracy in predicting adult-to-adult peaks. It is impractical to predict first occurrence of B. cockerelli in potato plantings as adults are present as soon cotyledons break through the soil. Therefore, we suggest integrating the degree day model into current B. cockerelli management practices using a two-phase method. Phase 1 occurs from potato planting through to the first peak in a B. cockerelli field population, which is managed using current practices. Phase 2 begins with the first B. cockerelli population peak and the degree day model is initiated to predict the subsequent population peaks, thus providing growers a tool to proactively manage this pest.

Altering the ratio of phenazines in Pseudomonas chlororaphis (aureofaciens) strain 30-84: effects on biofilm formation and pathogen inhibition.

Pseudomonas chlororaphis strain 30-84 is a plant-beneficial bacterium that is able to control take-all disease of wheat caused by the fungal pathogen Gaeumannomyces graminis var. tritici. The production of phenazines (PZs) by strain 30-84 is the primary mechanism of pathogen inhibition and contributes to the persistence of strain 30-84 in the rhizosphere. PZ production is regulated in part by the PhzR/PhzI quorum-sensing (QS) system. Previous flow cell analyses demonstrated that QS and PZs are involved in biofilm formation in P. chlororaphis (V. S. R. K. Maddula, Z. Zhang, E. A. Pierson, and L. S. Pierson III, Microb. Ecol. 52:289-301, 2006). P. chlororaphis produces mainly two PZs, phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). In the present study, we examined the effect of altering the ratio of PZs produced by P. chlororaphis on biofilm formation and pathogen inhibition. As part of this study, we generated derivatives of strain 30-84 that produced only PCA or overproduced 2-OH-PCA. Using flow cell assays, we found that these PZ-altered derivatives of strain 30-84 differed from the wild type in initial attachment, mature biofilm architecture, and dispersal from biofilms. For example, increased 2-OH-PCA production promoted initial attachment and altered the three-dimensional structure of the mature biofilm relative to the wild type. Additionally, both alterations promoted thicker biofilm development and lowered dispersal rates compared to the wild type. The PZ-altered derivatives of strain 30-84 also differed in their ability to inhibit the fungal pathogen G. graminis var. tritici. Loss of 2-OH-PCA resulted in a significant reduction in the inhibition of G. graminis var. tritici. Our findings suggest that alterations in the ratios of antibiotic secondary metabolites synthesized by an organism may have complex and wide-ranging effects on its biology.

Enzymes as feed additive to aid in responses against Eimeria species in coccidia-vaccinated broilers fed corn-soybean meal diets with different protein levels.

This research aimed to evaluate the effects of adding a combination of exogenous enzymes to starter diets varying in protein content and fed to broilers vaccinated at day of hatch with live oocysts and then challenged with mixed Eimeria spp. Five hundred four 1-d-old male Cobb-500 chickens were distributed in 72 cages. The design consisted of 12 treatments. Three anticoccidial control programs [ionophore (IO), coccidian vaccine (COV), and coccidia-vaccine + enzymes (COV + EC)] were evaluated under 3 CP levels (19, 21, and 23%), and 3 unmedicated-uninfected (UU) negative controls were included for each one of the protein levels. All chickens except those in unmedicated-uninfected negative controls were infected at 17 d of age with a mixed oral inoculum of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Live performance, lesion scores, oocyst counts, and samples for gut microflora profiles were evaluated 7 d postinfection. Ileal digestibility of amino acids (IDAA) was determined 8 d postinfection. Microbial communities (MC) were analyzed by G + C%, microbial numbers were counted by flow cytometry, and IgA concentrations were measured by ELISA. The lowest CP diets had poorer (P < or = 0.001) BW gain and feed conversion ratio in the preinfection period. Coccidia-vaccinated broilers had lower performance than the ones fed ionophore diets during pre- and postchallenge periods. Intestinal lesion scores were affected (P < or = 0.05) by anticoccidial control programs, but responses changed according to gut section. Feed additives or vaccination had no effect (P > or = 0.05) on IDAA, and diets with 23% CP had the lowest (P < or = 0.001) IDAA. Coccidial infection had no effect on MC numbers in the ileum but reduced MC numbers in ceca and suppressed ileal IgA production. The COV + EC treatment modulated MC during mixed coccidiosis infection but did not significantly improve chicken performance. Results indicated that feed enzymes may be used to modulate the gut microflora of cocci-vaccinated broiler chickens.

Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis (aureofaciens) strain 30-84.

The biological control bacterium Pseudomonas chlororaphis (aureofaciens) strain 30-84 employs two quorum sensing (QS) systems: PhzR/PhzI regulates the production of the antibiotics phenazine-1-carboxylic acid, 2-hydroxy-phenazine-1-carboxylic acid, and 2-hydroxy-phenazine, whereas CsaR/CsaI regulates currently unknown aspects of the cell surface. Previously characterized derivatives of strain 30-84 with mutations in each QS system and in the phenazine biosynthetic genes were screened for their ability to form surface-attached biofilm populations in vitro, using microtiter plate and flow cell biofilm assays, and on seeds and roots. Results from in vitro, seed, and root studies demonstrated that the PhzR/PhzI and the CsaR/CsaI QS regulatory systems contribute to the establishment of biofilms, with mutations in PhzR/PhzI having a significantly greater effect than mutations in CsaR/CsaI. Interestingly, phenazine antibiotic production was necessary for biofilm formation to the same extent as the PhzR/PhzI QS system, suggesting the loss of phenazines was responsible for the majority of the biofilm defect in these mutants. In vitro analysis indicated that genetic complementation or AHL addition to the growth medium restored the ability of the AHL synthase phzI mutant to form biofilms. However, only phenazine addition or genetic complementation of the phenazine biosynthetic mutation in trans restored biofilm formation by mutants defective in the transcriptional activator phzR or the phzB structural mutant. QS and phenazine production were also involved in the establishment of surface-attached populations on wheat seeds and plant roots, and, as observed in vitro, the addition of AHL extracts restored the ability of phzI mutants, but not phzR mutants, to form surface attached populations on seeds. Similarly, the presence of the wild type in mixtures with the mutants restored the ability of the mutants to colonize wheat roots, demonstrating that AHL and/or phenazine production by the wild-type population could complement the AHL- and phenazine-deficient mutants in situ. Together, these data demonstrate that both QS systems are involved in the formation of surface-attached populations required for biofilm formation by P. chlororaphis strain 30-84, and indicate a new role for phenazine antibiotics in rhizosphere community development beyond inhibition of other plant-associated microorganisms.

Evolving enzyme technology: impact on commercial poultry nutrition.

The use of exogenous enzymes to improve the nutritional value of poultry diets is a relatively new concept. The technology is rapidly evolving, with new enzymes, enzyme combinations, and novel applications being developed as rapidly as regulatory restrictions will allow. Most researchers in the field of poultry nutrition would consider phytase to be the last significant leap forward in terms of enzyme use in the animal feed industry. However, there is a great deal of ongoing research into the next generation of enzymes with a focus on ingredient quality, predictability of response via least-square models, improvements in food safety, effect of bird age, effect of various side activities and enzyme dose, maximisation of net income and reduction in environmental pollution. It is the purpose of the present review article to summarise the current research in the area of feed enzymes for poultry and to speculate on future applications of enzymes and new enzyme technologies that may be of value to the industry in the coming years.

Negative cross-communication among wheat rhizosphere bacteria: effect on antibiotic production by the biological control bacterium Pseudomonas aureofaciens 30-84.

Phenazine antibiotic production in the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part via the PhzR/PhzI N-acyl homoserine lactone (AHL) system. Previous work showed that a subpopulation of the wheat rhizosphere community positively affected phenazine gene expression in strain 30-84 via AHL signals (E. A. Pierson, D. W. Wood, J. A. Cannon, F. M. Blachere, and L. S. Pierson III, Mol. Plant-Microbe Interact. 11:1078-1084, 1998). In the present work, a second subpopulation, one that negatively affected phenazine gene expression, was identified from this rhizosphere community. Strain 30-84 grown in conditioned medium (CM) from several strains produced lower levels of phenazines (1.5- to 9.3-fold) than control when grown in CM from the strain 30-84I(1)/I(2). Growth of the phzB::lacZ reporter strain 30-84Z in this CM resulted in decreased lacZ expression (4.3- to 9.2-fold) compared to growth of the control strain in CM, indicating that inhibition of phzB occurred at the level of gene expression. Preliminary chemical and biological characterizations suggested that these signals, unlike other identified negative signals, were not extractable in ethyl acetate. Introduction of extra copies of phzR and phzI, but not phzI alone, in trans into strain 30-84Z reduced the negative effect on phzB::lacZ expression. The presence of negative-signal-producing strains in a mixture with strain 30-84 reduced strain 30-84's ability to inhibit the take-all disease pathogen in vitro. Together, the results from the previous work on the positive-signal subpopulation and the present work on the negative-signal subpopulation suggest that cross-communication among members of the rhizosphere community and strain 30-84 may control secondary metabolite production and pathogen inhibition.

KINKY POLLEN encodes a SABRE-like protein required for tip growth in Arabidopsis and conserved among eukaryotes.

In higher plants, pollen tubes and root hairs share an ancient growth process named tip growth. We have isolated three allelic Arabidopsis mutant lines showing kinky-shaped pollen tubes and, when homozygous, showing shorter and thicker root hairs. The ultrastructure of pollen tubes in these kinky pollen (kip) mutants is similar to that of the wild type; however, time-lapse studies suggest that aberrant pollen tube shape is caused by periodic growth arrests alternated with phases of tube axis reorientation. The KIP gene encodes a protein of 2587 amino acids that is predicted to be targeted to the secretory pathway. KIP mRNA was detected in all organs investigated but was most abundant in pollen and roots. KIP has putative homologues in many eukaryotes, including mammals and yeast, and is similar to the Arabidopsis SABRE gene, whose mutation causes a dwarf phenotype. The phenotype of the kip/sab double mutant suggests related functions for both genes, however, the KIP protein is mostly required for tip-growth.

Effects of genetic selection for high or low antibody response on resistance to a variety of disease challenges and the relationship of resource allocation.

Lines of white leghorn chickens were selectively bred for either a high (H) or low (L) antibody response to sheep erythrocytes. The parental lines, HH and LL, and reciprocal crosses, HL (sire line cited first and dam line second) and LH, were compared for their responses to various diseases. High antibody titers were associated with reduced body weight. Lines and their crosses were challenged with infectious diseases. The LL line was most resistant to Mycobacterium avium, whereas the HH line was most susceptible. The HH line was most resistant to Mycoplasma gallisepticum, whereas the LL line was most susceptible. These findings indicate that defense against infectious diseases are resource expensive. In order to save resources, it is possible that different parts of a population might genetically devote high levels of resources against different types of diseases so that the entire population is not susceptible to a single infection.

Antimicrobial susceptibilities of Escherichia coli strains from a turkey operation.

OBJECTIVE: To measure minimum inhibitory concentrations (MIC) of 17 antimicrobials for Escherichia coli isolates from a turkey operation and assess whether small samples provide precise estimates of geometric mean MIC. DESIGN: Prospective study. SAMPLE POPULATION: 105 clinical isolates from birds and 1,104 fecal isolates from 20 flocks (poults and finisher hens). PROCEDURE: A Mueller-Hinton broth dilution panel was used to measure MIC, and MIC of fecal and clinical isolates were compared. We drew random samples of 5,10, 15, 20, 25, 30, 35, 40, and 45 isolates from each finisher flock and between 100 and 105 isolates from 5, 7, 10, and 20 flocks. Antimicrobial usage was determined for enrolled flocks. RESULTS: Six of 12 poult and 18 of 20 finisher flocks had been treated with antimicrobials, often for respiratory illnesses consistent with colibacillosis. All birds received gentamicin at the hatchery. More fecal than clinical isolates were resistant to ampicillin; however, more clinical isolates were resistant to ciprofloxacin, gentamicin, and sulfamethoxazole. Precise estimates of geometric mean MIC for flocks were obtained when > or = 15 fecal isolates were obtained per flock and, for the operation, when 105 isolates were obtained from > or = 7 flocks. CONCLUSION AND CLINICAL RELEVANCE: Antimicrobial usage was common and may have contributed to the resistance patterns of isolates. With a modest allocation of laboratory resources, producers can monitor antimicrobial susceptibilities of clinical and fecal E coli to manage risks of antimicrobial usage and resistance.

Rational analyses of organelle trajectories in tobacco pollen tubes reveal characteristics of the actomyosin cytoskeleton.

To gain insight into the characteristics of organelle movement and the underlying actomyosin motility system in tobacco pollen tubes, we collected data points representing sequential organelle positions in control and cytochalasin-treated cells, and in a sample of extruded cytoplasm. These data were utilized to reconstruct approximately 900 tracks, representing individual organelle movements, and to produce a quantitative analysis of the movement properties, supported by statistical tests. Each reconstructed track appeared to be unique and to show irregularities in velocity and direction of movement. The regularity quotient was near 2 at the tip and above 3 elsewhere in the cell, indicating that movement is more vectorial in the tube area. Similarly, the progressiveness ratio showed that there were relatively more straight trajectories in the tube region than at the tip. Consistent with these data, arithmetical dissection revealed a high degree of randomlike movement in the apex, lanes with tip-directed movement along the flanks, and grain-directed movement in the center of the tube. Intercalated lanes with bidirectional movement had lower organelle velocity, suggesting that steric hindrance plays a role. The results from the movement analysis indicate that the axial arrangement of the actin filaments and performance of the actomyosin system increases from tip to base, and that the opposite polarity of the actin filaments in the peripheral (+-ends of acting filaments toward the tip) versus the central cytoplasm (+-ends of actin filaments toward to the grain) is installed within a few minutes in these tip-growing cells.

Interactive computer-assisted position acquisition procedure designed for the analysis of organelle movement in pollen tubes.

An interactive computer-assisted video microscopy method has been developed for the acquisition of extensive data on the sequential positions of pollen tube organelles, which cannot be automatically tracked using geometric or motion patterns. The method consists of video microscopy, analog and digital contrast enhancement, digital time-lapsing of the images, and interactive selection of positions in a coordinate system corresponding to the cell shape and real size. Data on 15,000 positions acquired with this method have been used to make quantitative analyses of the movement patterns of the organelles. From these analyses and the reconstruction of 900 trajectories, it appears that movements are random in the tip of the pollen tube but become more directed in distal regions of the cell, indicating an increase in axial arrangement of the actin filaments. (All custom-made software is available from the authors on request.)

Homoserine lactone-mediated gene regulation in plant-associated bacteria.

Many plant-associated bacteria produce and utilize diffusible N-acyl-homoserine lactones (AHLs) to regulate the expression of specific bacterial genes and operons. AHL-mediated regulation utilizes two genes that encode proteins similar to the LuxI/LuxR system originally studied in the marine symbiont Vibrio fischeri. The LuxI-type proteins are AHL synthases that assemble the diffusible AHL signal. The LuxR-type proteins are AHL-responsive transcriptional regulatory proteins. LuxR proteins control the transcription of specific bacterial genes in response to the levels of AHL signal. To date, AHL-mediated gene regulation has been identified in a broad range of gram-negative bacteria, most of which are host-associated. However, it seems unlikely that such a widely conserved regulatory mechanism would be limited only to host-microbe interactions. These signals probably play central roles in ecological interactions among organisms in microbial communities by affecting communication among bacterial populations as well as between bacterial populations and their eukaryotic hosts.

Classification of organelle trajectories using region-based curve analysis.

A method based on analysis of the region of movement and the functioning of the acto-myosin cytoskeleton has been elaborated to quantify and classify patterns of organelle movement in tobacco pollen tubes. The trajectory was dilated to the region of movement, which was then reduced to give a one-pixel-wide skeleton, represented by a graph structure. The longest line in this skeleton was hypothesized to represent the basic track of the organelle along a single actin filament. Quantitative features were derived from the graph structure, direction of movement on the longest skeletal line, and distance between skeletal line and particle. These features corresponded to biological events like the amount of linear movement or the probability of attachment of an organelle to the actin filament. From 81 analyzed organelle trajectories, 17 had completely linear, 17 had completely non-linear, and 47 had alternating linear and non-linear movement. Selected features were employed for classification and ranking of the movement patterns of a representative sample of the population of organelles moving in the cell tip. The presented methods can be applied to any field where analysis and classification of particle motion are intended.

Green-fluorescent protein fusions for efficient characterization of nuclear targeting.

The green-fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co-factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue-specific expression of other popular reporter molecules, such as the beta-glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.

Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection.

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells.

Tip-localized calcium entry fluctuates during pollen tube growth.

Studies have been conducted on the dynamics of Ca2+ entry in pollen tubes using ratiometric ion imaging to measure the intracellular gradient and an ion selective vibrating electrode to detect the extracellular influx. A steep tip-focused gradient occurs in all species examined, including Lilium longiflorum, Nicotiana sylvestris, and Tradescantia virginiana. Anlaysis of Lilium pollen tubes loaded with dextran conjugated fura-2 reveals that the gradient derives from Ca2+ entry that is restricted to a small area of plasma membrane at the extreme apex of the tube dome. Since the apical membrane is continually swept to the flanks during tube elongation, either Ca2+ channels are specifically retained at the extreme apex or, as seems more likely, the Ca2+ channels which were active at the tip rapidly inactivate, as new ones are inserted during vesicle fusion. Ratiometric imaging further indicates that the high point of the gradient fluctuates in magnitude from 0.75 to above 3 microM, during measuring intervals of 60 sec, with the elevated points being correlated with an increased rate of tube growth. Independent analysis of the growth at 2- to 3-sec intervals reveals that the rates can fluctuate more than threefold; tubes longer than 700 mu m exhibit oscillations with a period of 23 sec, while tubes shorter than 700 mu m display erratic fluctuations. Inhibition of pollen tube growth caused by mild temperature shock or caffeine (1.5 to 3.0 mM) is correlated with the dissipation of the tip-focused gradient and the Ca2+ influx. Recovery from both treatments is denoted by a global swelling of the pollen tube tip, concomitant with a high transient entry of Ca2+ in the tip. The location of the highest Ca2+ domain within the tip region defines the point from which normal cylindrical elongation will proceed.