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.

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.

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.

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.

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.

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.

An evaluation of kits for measuring thyrotropin in newborns.

Several commercial radioimmunoassay kits are now marketed specifically for determination of thyrotropin (TSH) from whole-blood specimens collected on filter paper in neonatal screening programs. We have compared five kits in use in such screening programs in the United States. The reagent kits from Becton Dickinson, Neometrics, and Nuclear Medical Systems gave similar satisfactory results. That from Pharmacia was somewhat more difficult to use and gave greater coefficients of variation. Diagnostic Products' kit appeared to perform satisfactorily, but the analytical values obtained were significantly low, which may suggest erroneous calibrator values within the kit.

Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain.

Pseudomonas aureofaciens Q2-87 produces the antibiotic 2,4-diacetophloroglucinol (Phl), which inhibits Gaeumannomyces graminis var. tritici and other fungi in vitro. Strain Q2-87 also provides biological control of take-all, a root disease of wheat caused by this fungus. To assess the role of Phl in the antifungal activity of strain Q2-87, a genetic analysis of antibiotic production was conducted. Two mutants of Q2-87 with altered antifungal activity were isolated by site-directed mutagenesis with Tn5. One mutant, Q2-87::Tn5-1, did not inhibit G. graminis var. tritici in vitro and did not produce Phl. Two cosmids were isolated from a genomic library of the wild-type strain by probing with the mutant genomic fragment. Antifungal activity and Phl production were coordinately restored in Q2-87::Tn5-1 by complementation with either cosmid. Mobilization of one of these cosmids into two heterologous Pseudomonas strains conferred the ability to synthesize Phl and increased their activity against G. graminis var. tritici, Pythium ultimum, and Rhizoctonia solani in vitro. Subcloning and deletion analysis of these cosmids identified a 4.8-kb region which was necessary for Phl synthesis and antifungal activity.