Up-regulation of OsBIHD1, a rice gene encoding BELL homeodomain transcriptional factor, in disease resistance responses.

In the present study, we cloned and identified a full-length cDNA of a rice gene, OsBIHD1, encoding a homeodomain type transcriptional factor. OsBIHD1 is predicted to encode a 642 amino acid protein and the deduced protein sequence of OsBIHD1 contains all conserved domains, a homeodomain, a BELL domain, a SKY box, and a VSLTLGL box, which are characteristics of the BELL type homedomain proteins. The recombinant OsBIHD1 protein expressed in Escherichia coli bound to the TGTCA motif that is the characteristic cis-element DNA sequence of the homeodomain transcriptional factors. Subcellular localization analysis revealed that the OsBIHD1 protein localized in the nucleus of the plant cells. The OsBIHD1 gene was mapped to chromosome 3 of the rice genome and is a single-copy gene with four exons and three introns. Northern blot analysis showed that expression of OsBIHD1 was activated upon treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance. Expression of OsBIHD1 was also up-regulated rapidly during the first 6 h after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during the incompatible interaction between M. grisea and a resistant genotype. These results suggest that OsBIHD1 is a BELL type of homeodomain transcription factor present in the nucleus, whose induction is associated with resistance response in rice.

Effect of Organic Amendments on Soilborne and Foliar Diseases in Field-Grown Snap Bean and Cucumber.

Several paper mills in Wisconsin have programs for spreading paper mill residuals (PMR) on land. A growing number of vegetable farmers recognize the agronomic benefits of PMR applications, but there have been no investigations on the use of PMR for control of vegetable crop diseases. Our objective was to determine the effect of PMR amendments on soilborne and foliar diseases of cucumber and snap bean grown on a sandy soil. Raw PMR, PMR composted without bulking agent (PMRC), or PMR composted with bark (PMRBC) were applied annually in a 3-year rotation of potato, snap bean, and pickling cucumber. Several naturally occurring diseases were evaluated in the field, along with in situ field bioassays. All amendments suppressed cucumber damping-off and Pythium blight and foliar brown spot of snap bean. Both composts reduced the incidence of angular leaf spot in cucumber. In a separate field experiment planted with snap bean for two consecutive years, all amendments reduced common root rot severity in the second year. In a greenhouse experiment, the high rate of PMRBC suppressed anthracnose of snap bean. These results suggest that the application of raw and composted PMR to sandy soils has the potential to control several soilborne and foliar diseases.

The formation of a complex community program for diabetes control: lessons learned from a case study of Project DIRECT.

A case study was conducted of the formation of a diabetes initiative in a largely African American urban community. The study focused on how confluent the original project model was with actual formation, what benefits were produced, what areas of needed improvement surfaced, and how different stakeholder groups characterized one another's involvement. The project produced several benefits but also experienced needed improvements in its formation, which suffered from a lack of communication, cooperation, and coordination; unclear goals and personnel roles; and early delays. Lessons include treating project formation as an important developmental stage and reducing bureaucratic management approaches not suited for community partnerships.

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes.

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434-438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

Influence of tomato genotype on growth of inoculated and indigenous bacteria in the spermosphere.

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent, Bacillus cereus UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786-4790, 1999). Here we report results of studies examining the host effect on the support of growth of Bacillus and Pseudomonas strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of B. cereus UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24 B. cereus strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of B. cereus UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains Pseudomonas fluorescens 2-79 or Pseudomonas aureofaciens AB254. Thus, while the host effect on growth extended to all strains of B. cereus examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of B. cereus UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous Bacillus and fluorescent Pseudomonas bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous Bacillus soil bacteria, while the line that supported growth of inoculated B. cereus UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of B. cereus UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains-both Bacillus and Pseudomonas spp.-did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.

Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays.

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).

Activity of nitric oxide is dependent on, but is partially required for function of, salicylic acid in the signaling pathway in tobacco systemic acquired resistance.

When tobacco plants were treated by injection with nitric oxide (NO)-releasing compounds, the sizes of lesions caused by Tobacco mosaic virus (TMV) on the treated leaves and on upper nontreated leaves were significantly reduced. The reduction in TMV lesion size was caused by NO released from the NO-releasing compounds; the byproduct formed after release of NO from the NO-releasing compound NOC-18, diethylenetriamine, did not itself alter lesion size. Treatment of tobacco plants with inhibitors of nitric oxide synthase or an NO scavenger attenuated but did not abolish the systemic acquired resistance (SAR) induced by salicylic acid (SA). In NahG transgenic tobacco plants, NO had no effect on lesion size following TMV infection. These results are consistent with the hypothesis that NO plays an important role in SAR induction in tobacco and that NO is required for the full function of SA as an SAR inducer. The activity of NO is fully dependent on the function of SA in the SAR signaling pathway in tobacco.

Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

Crenarchaeota colonize terrestrial plant roots.

Microorganisms that colonize plant roots are recruited from, and in turn contribute substantially to, the vast and virtually uncharacterized phylogenetic diversity of soil microbiota. The diverse, but poorly understood, microorganisms that colonize plant roots mediate mineral transformations and nutrient cycles that are central to biosphere functioning. Here, we report the results of epifluorescence microscopy and culture-independent recovery of small subunit (SSU) ribosomal RNA (rRNA) gene sequences showing that members of a previously reported clade of soil Crenarchaeota colonize both young and senescent plant roots at an unexpectedly high frequency, and are particularly abundant on the latter. Our results indicate that non-thermophilic members of the Archaea inhabit an important terrestrial niche on earth and direct attention to the need for studies that will determine their possible roles in mediating root biology.

Regression Analyses for Evaluating the Influence of Bacillus cereus on Alfalfa Yield Under Variable Disease Intensity.

ABSTRACT We developed and tested regression methods to exploit the variability in disease inherent in field experiments, and applied the methods to evaluate strains of Bacillus cereus for biocontrol efficacy. Four B. cereus strains were tested for their effect on alfalfa (Medicago sativa) performance in 16 field trials planted during 1993 to 1996 at multiple sites in Wisconsin. To evaluate performance of the strains, we used the ratio of (metalaxyl response)/(untreated control response) as a measure of disease intensity within the experiments. The ratio of (Bacillus response)/(untreated control response) was then regressed as a function of disease intensity. The slope of the resulting line provides a statistical test to compare performance of the Bacillus strain with that of the untreated seed (H(o): slope = 0) and metalaxyl controls (H(o): slope = 1). Under conditions in which disease occurred, forage yield of plots planted with seed treated with B. cereus strain AS4-12 exceeded yield from the untreated control plots (P = 0.002) and was similar to yield of plots planted with metalaxyl-treated seed (P = 0.14). Yield gain associated with AS4-12 and metalaxyl seed treatment averaged 6.1 +/- 2.8% (+/-standard error) and 3.0 +/- 2.8%, respectively. In contrast to the regression approach, means analysis by analysis of variance did not detect differences among treatments. Three other B. cereus strains either did not increase alfalfa yield or increased yield less than did AS4-12. Metalaxyl and three of the Bacillus strains increased seedling emergence, but the improved stands were not predictive of increased forage yield. In six additional studies conducted for one season in 1997, AS4-12 enhanced yield of two cultivars at diverse locations in Wisconsin, but there was an apparent cultivar-location interaction. A strong correlation between response to AS4-12 and metalaxyl treatment suggests that these treatments controlled similar pathogens, most likely the oomycete pathogens Phytophthora medicaginis and Pythium spp.

The Earth's bounty: assessing and accessing soil microbial diversity.

The study of microbial diversity represents a major opportunity for advances in biology and biotechnology. Recent progress in molecular microbial ecology shows that the extent of microbial diversity in nature is far greater than previously thought. Here, we discuss methods to analyse microorganisms from natural environments without culturing them and new approaches for gaining access to the genetic and chemical resources of these microorganisms.

Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus.

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

Genetic basis in plants for interactions with disease-suppressive bacteria.

Plant health depends, in part, on associations with disease-suppressive microflora, but little is known about the role of plant genes in establishing such associations. Identifying such genes will contribute to understanding the basis for plant health in natural communities and to new strategies to reduce dependence on pesticides in agriculture. To assess the role of the plant host in disease suppression, we used a genetic mapping population of tomato to evaluate the efficacy of the biocontrol agent Bacillus cereus against the seed pathogen Pythium torulosum. We detected significant phenotypic variation among recombinant inbred lines that comprise the mapping population for resistance to P. torulosum, disease suppression by B. cereus, and growth of B. cereus on the seed. Genetic analysis revealed that three quantitative trait loci (QTL) associated with disease suppression by B. cereus explained 38% of the phenotypic variation among the recombinant inbred lines. In two cases, QTL for disease suppression by B. cereus map to the same locations as QTL for other traits, suggesting that the host effect on biocontrol is mediated by different mechanisms. The discovery of a genetic basis in the host for interactions with a biocontrol agent suggests new opportunities to exploit natural genetic variation in host species to enhance our understanding of beneficial plant-microbe interactions and develop ecologically sound strategies for disease control in agriculture.

Behavior of pythium Torulosum zoospores during their interaction with tobacco roots and Bacillus cereus.

Bacillus cereus UW85 suppresses seedling damping-off diseases caused by Oomycetes and produces antibiotics that inhibit development of Oomycetes in culture. The goal of this study was to determine how UW85 and its antibiotics affected the behavior of an Oomycete, Pythium torulosum, in its interaction with plant roots. We studied tobacco seedlings inoculated with zoospores of P. torulosum and UW85 culture, culture filtrate, washed cells, antibiotics (zwittermicin A or kanosamine), purified from cultures of UW85, and UW030, a mutant of UW85 that does not suppress disease and does not produce the antibiotics. Microscopic observation revealed that all of the treatments inhibited zoospore activity around roots and encystment on roots. Treatment with UW85 culture, culture filtrate, zwittermicin A, or kanosamine delayed cyst germination and the elongation rate of germ tubes, whereas treatment with UW030 or washed UW85 cells did not. In an in vitro seedling bioassay of disease suppression, the antibiotics, zwittermicin A and kanosamine, suppressed disease singly or together, although UW85 culture suppressed disease more effectively than did the antibiotics. The results show that B. cereus cultures affect zoospore behavior in the presence of roots, and B. cereus-produced antibiotics, zwittermicin A and kanosamine, contribute to disease suppression and inhibition of germ tube elongation in the presence of the plant root.

First Report of Root Rot of Soybeans Caused by Corynespora cassiicola in Wisconsin.

Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei was isolated from diseased soybean plants (Glycine max) collected in two fields near Racine and Arlington, WI. Plants sampled at seedling emergence (VC), late vegetative (V5), and mid-reproductive (R5) stages exhibited reddish to dark brown longitudinal lesions on the exterior of the tap root extending vertically on the hypocotyl to the soil line, and extensive necrosis of lateral roots. Sample size at each growth stage was 144 plants per site. Roots were surface sterilized in 0.5% sodium hypochlorite for 2 min and sections of symptomatic tissue placed on water agar (12 g/liter) containing 100 µg of streptomycin per ml. Sporulation occurred on lesions and on mycelium that had grown out from the plant tissue onto the water agar following a 2-week incubation at 24°C under fluorescent light (280 µmol s-1 m-2). Incidence of isolation of C. cassiicola at both sites was 40% of plants sampled at growth stage VC, 67% at V5, and 78% at R5. Conidia characteristic of C. cassiicola were particularly abundant on the surface of necrotic lateral root tissue. Elongated conidia produced on water agar were 151 ± 5 µm × 15 ± 0.5 µm with an average of 13 ± 0.4 cells separated by hyaline pseudosepta (1). To confirm pathogenicity, a 1-cm lateral slice into each of four 5-day-old soybean seedling roots was made and a plug of agar taken from the margin of a colony of C. cassiicola grown on potato dextrose agar was placed in each wound and incubated for 14 days at 24°C in a growth chamber. Symptoms similar to those of diseased field plants were observed and C. cassiicola was reisolated from all plants inoculated with C. cassiicola; all controls treated with agar alone had no symptoms and C. cassiicola was recovered from none of the noninoculated controls. This is the first report of root rot caused by C. cassiicola on soybean in Wisconsin. Reference: (1) W. L. Seaman and R. A. Shoemaker. Can. J. Bot. 43:1461, 1965.

Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products.

Cultured soil microorganisms have provided a rich source of natural-product chemistry. Because only a tiny fraction of soil microbes from soil are readily cultured, soil might be the greatest untapped resource for novel chemistry. The concept of cloning the metagenome to access the collective genomes and the biosynthetic machinery of soil microflora is explored here.

Ultrasound prenatal diagnosis of the Nail-Patella syndrome.

The Nail-Patella syndrome (NPS) is an autosomal dominant connective-tissue disorder characterized by the absence or hypoplasia of the nails and patella, posterior illiac horns, elbow deformities, congenital nephropathy cervical ribs and eye problems. The presence of the posterior iliac horns is pathognomonic and have been observed in more than 80 per cent of cases. In this report, we present the first case of prenatal diagnosis of NPS by ultrasound. The possible kidney involvement, combined with other, milder, complications make the prenatal diagnosis of this syndrome worthwhile.

Identifying and defining the dimensions of community capacity to provide a basis for measurement.

Although community capacity is a central concern of community development experts, the concept requires clarification. Because of the potential importance of community capacity to health promotion, the Division of Chronic Disease Control and Community Intervention, Centers for Disease Control and Prevention (CDC), convened a symposium in December 1995 with the hope that a consensus might emerge regarding the dimensions that are integral to community capacity. This article describes the dimensions that the symposium participants suggested as central to the construct, including participation and leadership, skills, resources, social and interorganizational networks, sense of community, understanding of community history, community power, community values, and critical reflection. The dimensions are not exhaustive but may serve as a point of departure to extend and refine the construct and to operationalize ways to assess capacity in communities.