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Funding Acknowledgements Type of funding sources: None. Background/Introduction Exercise is an important factor in the primary and secondary prevention of cardiovascular disease (CVD). However, few data are available on predictors of adopting exercise (AE) in older adults, especially the impact of a new and remote heart disease diagnosis (NHDD and RHDD, respectively) on AE. Purpose The purpose of this study was to examine predictors of AE in older adults using the Health and Retirement Survey (HRS), a prospective, nationally representative household survey examining retirement and health among older persons in the United States that is repeated every two years. Methods Data from respondents who were age 55 or older in 1998 were examined using three waves of the HRS (1998, 2000, and 2002) which provided three distinct person-wave observations for each respondent from which exercise wave changes and AE in older adults with and without CVD was determined resulting in a total of 8,975 respondents for subsequent analyses. The mean age±SD of the respondents was 69±9 years with 56% being female. Logistic regression models were developed to examine predictors of AE while controlling for a variety of demographic and health related variables. Results AE in respondents with and without CVD was relatively similar after the baseline time period, but a significantly (p0.05, respectively). Conclusions A smaller percentage of older adults with CVD were found AE compared to older adults without CVD. The finding that marriage was the strongest significant positive predictor of AE is important and highlights the role of spousal support. The finding that neither a NHDD or RHDD was a significant predictor of AE highlights the important role of cardiac rehabilitation in older adults. Long-term programming for AE and maintenance of exercise in older persons with a NHDD and RHDD is particularly important and requires further investigation. Examination of spousal or similar support while attending cardiac rehabilitation and modifying negative predictors of AE appears warranted and may facilitate older adults AE.

Hydrogenasen sind H2-umsetzende Metalloenzyme und enthalten katalytische Kofaktoren, deren Eisenionen durch biologisch ungewöhnliche Kohlenmonoxid- (CO) und Cyanid- (CN−) Liganden koordiniert sind. Externes CO und CN−hemmt Hydrogenasen jedoch. Der molekulare Mechanismus der Bindung von CN− an [FeFe]-Hydrogenasen ist unbekannt. In dieser Studie präsentieren wir Kristallstrukturen der mit CN− behandelten [FeFe]-Hydrogenase CpI aus Clostridium pasteurianum. Auf Grund der hohen Auflösung von 1.39 Å können wir die intrinsischen CO- und CN−-Liganden voneinander unterscheiden. Wir zeigen, dass externes CN− die offene Bindestelle des Kofaktors besetzt, an die auch externes CO bindet. Im Gegensatz zu anderen Inhibitoren zeigen die CN−-behandelten Kristalle Konformationsänderungen konservierter Reste des Protonentransferpfads, die einen direkten Austausch von Protonen zwischen den Aminosäuren E279 und S319 ermöglichen. Diese Konformation wurde als notwendig für einen effizienten Protonentransfer vorgeschlagen, doch wurde sie bisher nicht strukturell nachgewiesen.

How carbohydrate reserves change in conifers during drought and bark beetle attacks are poorly understood. We investigated changes in carbohydrate reserves and carbon-dependent terpene defenses in ponderosa pine trees experimentally subjected to two levels of drought stress (via root trenching) and two types of biotic challenge treatments (pheromone-induced bark beetle attacks or inoculations with crushed beetles that include beetle-associated fungi) for two consecutive years. Our results showed that trenching did not influence carbohydrates whereas both biotic challenges reduced amounts of starch and sugars of trees. However, only the trenched-beetle attacked trees depleted carbohydrates and died within the first year of bark beetle attacks. While live trees contained higher carbohydrates than dying trees, amounts of constitutive and induced terpenes produced did not vary between live and beetle-attacked dying trees, respectively. Based on these results we propose that reallocation of carbohydrates to terpenes during the early stages of beetle attacks is limited in drought-stricken trees, and that the combination of biotic and abiotic stress leads to tree death. The process tree death is subsequently aggravated by beetle girdling of phloem, occlusion of vascular tissue by bark beetle-vectored fungi, and potential exploitation of host carbohydrates by beetle symbionts as nutrients.

Funding Acknowledgements Type of funding sources: None. Background/Introduction Exercise is an important factor in the primary and secondary prevention of heart disease. However, few data are available on predictors of exercise in older adults, especially the impact of a new and remote diagnosis of heart disease (NHDD and RHDD, respectively) on exercise behavior. It is possible that the motivational impact of a NHDD may be more powerful than a RHDD in promoting exercise in older persons. Purpose The purpose of this study was to examine predictors of exercise in older adults using the Health and Retirement Survey (HRS), a prospective, nationally representative household survey examining retirement and health among older persons in the United States that is repeated every two years. We hypothesized that older persons with a NHDD and RHDD will be more likely to exercise. Methods Data from respondents who were age 55 or older in 1998 were grouped using three waves of the HRS (1998, 2000, and 2002) which provided three distinct person-wave observations for each respondent, yielding 38,634 respondent person-wave observations (9,629 with RHDD and 1,689 with NHDD) with a mean age of 70±9 years (55% female). Logistic regression models were developed to examine the predictors of exercise while controlling for a variety of demographic and health related variables. Results A NHDD was the strongest positive predictor (odds ratio=1.195, p Conclusions A NHDD was a strong positive predictor of exercise in older adults. A RHDD was not a significant predictor of exercise, suggesting that the motivation impact of a heart disease diagnosis lessens over time and secondary prevention efforts such as cardiac rehabilitation continue to be of importance. Long-term programming for maintenance of exercise in older persons with a RHDD appears to be particularly important and requires further investigation.

Interactions between water stress and induced defenses and their role in tree mortality due to bark beetles are poorly understood. We performed a factorial experiment on 48 mature ponderosa pines (Pinus ponderosa) in northern Arizona over three years that manipulated a) tree water stress by cutting roots and removing snow; b) bark beetle attacks by using pheromone lures; and c) phloem exposure to biota vectored by bark beetles by inoculating with dead beetles. Tree responses included resin flow from stem wounds, phloem composition of mono- and sesqui-terpenes, xylem water potential, leaf gas exchange, and survival. Phloem contained 21 mono- and sesqui-terpenes, which were dominated by (+)-α-pinene, (-)-limonene, and δ-3-carene. Bark beetle attacks (mostly Dendroctonus brevicomis) and biota carried by beetles induced a general increase in concentration of phloem mono- and sesqui-terpenes, whereas water stress did not. Bark beetle attacks induced an increase in resin flow for unstressed trees but not water-stressed trees. Mortality was highest for beetle-attacked water-stressed trees. Death of beetle-attacked trees was preceded by low resin flow, symptoms of water stress (low xylem water potential, leaf gas exchange), and an ephemeral increase in concentrations of mono- and sesqui-terpenes compared to surviving trees. These results show a) that ponderosa pine can undergo induction of both resin flow and phloem terpenes in response to bark beetle attack, and that the former is more constrained by water stress; b) experimental evidence that water stress predisposes ponderosa pines to mortality from bark beetles.

Dravet's syndrome (DS) or severe myoclonic epilepsy of infancy is a rare, genetic, and infantile-onset epileptic encephalopathy. DS presents with recurrent febrile seizures and/or febrile status epilepticus in developmentally normal infants, and subsequently evolves into a drug-resistant mixed-seizure disorder with developmental arrest or regression. As many defining clinical features of DS do not become evident until 3 to 4 years of age, diagnosis is often delayed. Early seizure control, particularly the prevention of status epilepticus in infancy, has been shown to correlate with better long-term outcomes. Thus, early diagnosis and seizure control is crucial. Several treatment algorithms have been published in recent years to guide antiepileptic drug selection and escalation. Last year, two agents, stiripentol and cannabidiol, were approved by the U.S. Food and Drug Administration specifically for use in DS, and a third has been submitted (fenfluramine). Additional therapies, including serotonin modulators lorcaserin and trazodone, verapamil, and several first-in-class medications, are currently in various phases of investigation.

Crown gall (CG) is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of CG is Agrobacterium or Allorhizobium strains that harbor a tumor-inducing plasmid (pTi). The microbial community within the CG tumor has not been widely elucidated and it is not known if certain members of this microbial community promote or inhibit CG. This study investigated the microbiotas of grapevine CG tumor tissues from seven infected vineyards located in Hungary, Japan, Tunisia, and the United States. Heavy co-amplification of grapevine chloroplast and mitochondrial ribosomal RNA genes was observed with the widely used Illumina V3–V4 16S rRNA gene primers, requiring the design of a new reverse primer to enrich for bacterial 16S rRNA from CG tumors. The operational taxonomic unit (OTU) clustering approach is not suitable for CG microbiota analysis as it collapsed several ecologically distinct Agrobacterium species into a single OTU due to low interspecies genetic divergence. The CG microbial community assemblages were significantly different across sampling sites (ANOSIM global R = 0.63, p-value = 0.001) with evidence of site-specific differentially abundant ASVs. The presence of Allorhizobium vitis in the CG microbiota is almost always accompanied by Xanthomonas and Novosphingobium, the latter may promote the spread of pTi plasmid by way of acyl-homoserine lactone signal production, whereas the former may take advantage of the presence of substrates associated with plant cell wall growth and repair. The technical and biological insights gained from this study will contribute to the understanding of complex interaction between the grapevine and its microbial community and may facilitate better management of CG disease in the future.

Within the North American boreal forest, a widespread outbreak of the epidermal leaf miner Phyllocnistis populiella Cham. has damaged quaking aspen (Populus tremuloides Michx.) for nearly 20 years. In a series of experiments, we tested the effects of feeding damage by P. populiella on leaf water relations and gas exchange. Relative to insecticide-treated trees, the leaves of naturally mined trees had lower photosynthesis, stomatal conductance to water vapor, transpiration, water-use efficiency, predawn water potential and water content, as well as more enriched foliar δ13C. The magnitude of the difference between naturally mined and insecticide-treated trees did not change significantly throughout the growing season, suggesting that the effect is not caused by accumulation of incidental damage to mined portions of the epidermis over time. The contributions of mining-related stomatal malfunction and cuticular transpiration to these overall effects were investigated by restricting mining damage to stomatous abaxial and astomatous adaxial leaf surfaces. Mining of the abaxial epidermis decreased photosynthesis and enriched leaf δ13C, while increasing leaf water potential and water content relative to unmined leaves, effects consistent with stomatal closure due to disfunction of mined guard cells. Mining of the adaxial epidermis also reduced photosynthesis but had different effects on water relations, reducing midday leaf water potential and water content relative to unmined leaves, and did not affect δ13C. In the laboratory, extent of mining damage to the adaxial surface was positively related to the rate of water loss by leaves treated to prevent water loss through stomata. We conclude that overall, despite water savings due to closure of mined stomata, natural levels of damage by P. populiella negatively impact water relations due to increased cuticular permeability to water vapor across the mined portions of the epidermis. Leaf mining by P. populiella could exacerbate the negative effects of climate warming and water deficit in interior Alaska.

Russet on apple fruits was caused by inoculation with isolates of Aureobasidium pullulans and Rhodotorula glutinis but not with Metschnikowia pulcherrima or two other unidentified yeasts, YT2 and YT17. Another unidentified yeast, YT5, caused russet in 1 of 2 years. Epiphytic survival of isolates on fruit was measured 3 months after inoculation. All isolates that caused russet survived epiphytically on fruit at harvest. With the exception of M. pulcherrima, nonrusseting isolates were not detected on the fruit at harvest. Conidia, hyphae, and chlamydospores characteristic of A. pullulans were observed in naturally russeted tissues of 14 different apple cultivars and Bartlett and Bosc pear collected from 13 orchards in New York state. A. pullulans was the most commonly isolated microorganism, and all A. pullulans isolates caused apple russet but varied in the severity of russet they caused. Isolates were identified using standard identification schemes. In addition, A. pullulans isolates were compared by generating fingerprints of restriction fragments of a PCR-amplified region of ribosomal DNA consisting of ITS-1, the 5.8S gene, and ITS-2.

Agrobacterium vitis was isolated from roots of 41 of 66 feral Vitis riparia vines collected in three different regions of New York State. Two of the regions were more than 150 km from commercial vineyards. The strains were highly diverse as determined by DNA fingerprinting of the chromosomal region lying between the 16S and 23S rRNA genes. Of 24 strains examined, 15 different fingerprints were generated, and none was identical to fingerprints generated by previously identified groups of tumorigenic A. vitis strains. Results of physiological tests that were done to characterize strains from V. riparia conformed closely to those expected for A. vitis, except that 23 of 26 strains did not utilize tartrate. All strains were nontumorigenic, did not hybridize with a probe consisting of T-DNA genes, did not utilize octopine or nopaline, and carried zero to three plasmids. Of 26 strains, 7 inhibited A. vitis strain K306 from causing galls at wound sites on grape as well as or better than a previously studied nontumorigenic A. vitis strain, F2/5, that is known to have biological control activity.

Streptomycin is currently the only antibiotic registered for the control of fire blight, a devastating disease of apple (Malus), pear (Pyrus), and other rosaceous plants caused by the bacterium Erwinia amylovora. Resistance of E. amylovora to streptomycin was first identified in California pear orchards in 1971 and is currently endemic in many parts of the United States. The Northeast remains the only major U.S. apple-growing region without streptomycin-resistant isolates of E. amylovora. In 2002, during a routine survey for streptomycin resistance, isolates from two neighboring orchards in Wayne County, NY were found to be highly resistant to streptomycin at a concentration of 100 μg/ml. This constitutes the first authenticated report of streptomycin resistance in New York State. Infected trees were shipped at the same time from a single nursery in Michigan. Resistance was caused by the acquisition of the strA-strB gene pair, inserted into the ubiquitous nontransmissible E. amylovora plasmid pEA29. Previously, streptomycin-resistant E. amylovora populations from Michigan were described with a similar mechanism of resistance, although the strA-strB genes are not unique to Michigan. These findings illustrate how unintentional movement of nursery material could undermine efforts to prevent the spread of antibiotic-resistant E. amylovora.

Agrobacterium vitis, the primary cause of grape crown gall disease, is known to survive internally in grapevines and to spread in propagation material. In this study, we showed that the bacterium can be detected in dormant grape buds and on surfaces of leaves collected from commercial vineyards. Using a highly selective and sensitive method based on magnetic capture hybridization (MCH) together with real-time PCR, we detected A. vitis in as much as 90% of dormant bud samples and in up to 40% of leaf samples from individual vineyards. The highest percentages of detection occurred in samples collected from vineyards with high incidences of crown gall. A. vitis was also detected in 22% of wild grapevines (Vitis riparia) collected in New York and in 25% of feral grapevines that included V. californica in California. Several of these vines were growing more than 2 km from commercial vineyards, demonstrating that wild grapevines can serve as a significant inoculum reservoir. The specificity of the MCH and real-time PCR assay used to detect tumorigenic A. vitis in the environment was further demonstrated by the finding that 69 nontumorigenic strains from regions across the United States did not amplify a virD2 PCR product.

Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl-coenzyme A (acyl-CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H2 O2 and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild-type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1-only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease.

Agrobacterium vitis, the cause of crown gall disease on grapevine, survives internally in vines and can be spread in cuttings for propagation. The possibility of generating pathogen-free vines through tissue culture makes it essential to understand the distribution of the pathogen in grapevines. A highly sensitive magnetic capture hybridization procedure along with real-time polymerase chain reaction were used to measure the distribution of tumorigenic A. vitis in dormant canes and green shoots of grapevines. Tumorigenic A. vitis was distributed from the basal to apical nodal and internodal tissues of canes as well as in nonlignified green shoots. In experiments conducted in 2013, A. vitis was detected in up to 17% of shoot tips and 52% of meristems of greenhouse-grown plants initiated from known A. vitis-contaminated cuttings. A lower frequency of detection was observed from surface-disinfected shoot tips (7%) as compared with nondisinfected tips (37%), suggesting epiphytic survival on green tissues. In 2014, vines propagated from cuttings collected from crown gall-infected vines from a different vineyard yielded lower incidences of A. vitis from shoot tips, and the bacterium was not detected in meristems. Tumorigenic A. vitis was also detected in cuttings of wild grapevines (Vitis riparia) that were collected both adjacent to and far removed from commercial vineyards.

The effects of tumorigenic and nontumorigenic strains of Agrobacterium vitis on graft strength and growth of grapevines was studied. A procedure was developed for inoculating graft interface surfaces with A. vitis and for measuring the force required to break grafts at different time points. Cuttings were soaked in an aqueous suspension of bacteria, about 106 CFU/ml, and bacteria were spread onto the graft interface during the grafting procedure. Tumorigenic strain CG49 caused reduced bud germination and increased callus (crown gall) at the graft union and at the base of cuttings at 30 days postinoculation (dpi) and significantly reduced shoot growth by 60 dpi whereas, at the same time points, nontumorigenic strain F2/5 inhibited callus formation but did not affect bud germination or shoot growth. Graft strength was enhanced at 30 dpi with CG49, presumably because the crown gall callus served to secure the union; graft strength was weakened by F2/5 over the same period. Between 30 and 60 dpi, the greatest increase in graft strength was observed in the water control. Following graft union inoculations, the A. vitis population increased more than 1,000-fold within 5 days.

Agrobacterium vitis is the primary causal agent of grapevine crown gall worldwide. Symptoms of grapevine crown gall disease include tumor formation on the aerial plant parts, whereas both tumorigenic and nontumorigenic strains of A. vitis cause root necrosis. Genetic and genomic analyses indicated that A. vitis is distinguishable from the members of the Agrobacterium genus and its transfer to the genus Allorhizobium was suggested. A. vitis is genetically diverse, with respect to both chromosomal and plasmid DNA. Its pathogenicity is mainly determined by a large conjugal tumor-inducing (Ti) plasmid characterized by a mosaic structure with conserved and variable regions. Traditionally, A. vitis Ti plasmids and host strains were differentiated into octopine/cucumopine, nopaline, and vitopine groups, based on opine markers. However, tumorigenic and nontumorigenic strains of A. vitis may carry other ecologically important plasmids, such as tartrate- and opine-catabolic plasmids. A. vitis colonizes vines endophytically. It is also able to survive epiphytically on grapevine plants and is detected in soil exclusively in association with grapevine plants. Because A. vitis persists systemically in symptomless grapevine plants, it can be efficiently disseminated to distant geographical areas via international trade of propagation material. The use of healthy planting material in areas with no history of the crown gall represents the crucial measure of disease management. Moreover, biological control and production of resistant grape varieties are encouraging as future control measures.

Although sexual behaviors have been extensively studied among youth in general, they have been relatively understudied among military-dependent youth (MDY). Furthermore, the impact of unique military stressors, such as parental deployment and multiple relocations, on the sexual behaviors of MDY has not been assessed. In this pilot study, we estimated the prevalence of sexual behaviors among MDY, and examined the association between these behaviors and parental deployment and multiple relocations. Between June and September 2011, we recruited youth (N = 208; aged 15-19 years) who attended a military treatment facility in the southern United States, to complete a short, paper-based survey. We computed prevalence estimates and conducted Chi-square analyses, as well as logistic regression analyses, while adjusting for age, gender, and race/ethnicity. More than half (53.7 %) of the youth reported being sexually experienced, and many of these youth reported engaging in risky sexual behaviors. Parental deployment and multiple relocations were significantly associated only with having had sex in the past 3 months. Although with most sexual behaviors there was no significant association between parental deployment and multiple relocations, many MDY are sexually experienced and engage in risky sexual behaviors. MDY should thus be exposed to evidence-based strategies for sexually transmitted infection and pregnancy prevention, as well as provided with teen-friendly health care services and comprehensive sexual/reproductive health counseling.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire) (Coleoptera: Buprestidae), an invasive phloem-feeding buprestid, has killed hundreds of millions of ash (Fraxinus spp.) trees in the United States and two Canadian provinces. We evaluated EAB persistence in post-invasion sites and compared EAB adult captures and larval densities in 24 forested sites across an east-west gradient in southern Michigan representing the Core (post-invasion), Crest (high EAB populations), and Cusp (recently infested areas) of the EAB invasion wave. Condition of green ash (Fraxinus pennsylvanica Marsh) trees were recorded in fixed radius plots and linear transects in each site. Ash mortality was highest in Core sites in the southeast, moderate in Crest sites in central southern Michigan, and low in Cusp sites in the southwest. Traps and trap trees in Crest sites accounted for 75 and 60% of all EAB beetles captured in 2010 and 2011, respectively. Populations of EAB were present in all Core sites and traps in these sites captured 13% of all beetles each year. Beetle captures and larval densities at Cusp sites roughly doubled between 2010 and 2011, reflecting the increasing EAB populations. Sticky bands on girdled trees captured the highest density of EAB beetles per m2 of area, while baited double-decker traps had the highest detection rates and captured the most beetles. Larval densities were higher on girdled ash than on similar ungirdled trees and small planted trees. Woodpecker predation and a native larval parasitoid were present in all three invasion regions but had minor effects on ash survival and EAB densities.

Agrobacterium vitis is the primary causal agent of grapevine crown gall worldwide. Symptoms of grapevine crown gall disease include tumor formation on the aerial plant parts, whereas both tumorigenic and nontumorigenic strains of A. vitis cause root necrosis. Genetic and genomic analyses indicated that A. vitis is distinguishable from the members of the Agrobacterium genus and its transfer to the genus Allorhizobium was suggested. A. vitis is genetically diverse, with respect to both chromosomal and plasmid DNA. Its pathogenicity is mainly determined by a large conjugal tumor-inducing (Ti) plasmid characterized by a mosaic structure with conserved and variable regions. Traditionally, A. vitis Ti plasmids and host strains were differentiated into octopine/cucumopine, nopaline, and vitopine groups, based on opine markers. However, tumorigenic and nontumorigenic strains of A. vitis may carry other ecologically important plasmids, such as tartrate- and opine-catabolic plasmids. A. vitis colonizes vines endophytically. It is also able to survive epiphytically on grapevine plants and is detected in soil exclusively in association with grapevine plants. Because A. vitis persists systemically in symptomless grapevine plants, it can be efficiently disseminated to distant geographical areas via international trade of propagation material. The use of healthy planting material in areas with no history of the crown gall represents the crucial measure of disease management. Moreover, biological control and production of resistant grape varieties are encouraging as future control measures.

Despite the sizable population of military-dependent youth (MDY) in the United States and the military stressors they experience (e.g., relocations, parental deployment), little is known about MDY's sexual behavior, especially about the perceived role that military stressors play in their sexual decisions, such as the decision to initiate sex. We conducted 25 semistructured, in-depth interviews with sexually experienced female MDY aged 15 to 19 years to (a) describe MDY's general perceptions of military life and (b) identify intrapersonal and interpersonal characteristics related to MDY's sexual initiation, including the perceived impact of military stressors. We analyzed life history grids and transcripts to identify common and unique themes across participants' experiences. Most participants reported having positive experiences related to military life, and most did not believe that military stressors influenced their decision to initiate sex. Common intrapersonal and interpersonal characteristics related to sexual initiation were having an older first sexual partner, being in a dating relationship, receiving sexual health education prior to their first sexual experience, and discussing sex with a parent prior to their first sexual experience. These intrapersonal and interpersonal characteristics should be considered when developing sexual health programs for MDY, which should focus on building supportive peer and parental relationships.

The biological characteristics of Xylella fastidiosa (Xf) were investigated in vitro by examining the effect of pure xylem fluid from Pierce?s disease (PD)-resistant Vitis smalliana grapevines on Xf cell multiplication, aggregation, biofilm formation, and motility in microfluidic flow chambers. The aggregations of large clumps were formed in artificial medium, whereas small clumps were observed in V. smalliana xylem fluid. Xylem fluid from V. smalliana negatively affected twitching motility and repressed biofilm formation of Xf, whereas artificial medium enhanced twitching motility and supported robust biofilm development. The results indicate that chemical components of V. smalliana xylem fluid may affect the function of type IV pili of Xf, leading to reduced twitching motility and aggregation as compared to artificial medium.

Assessing emerald ash borer (Agrilus planipennis Fairmaire) impacts in North American forests is essential for projecting future species composition of stands invaded by this phloem-feeding pest. We surveyed all species of overstory trees and regeneration in 2010 and 2011 in 24 forested sites with a major component of green ash (Fraxinus pennsylvanica), representing the Core, Crest, and Cusp of the A. planipennis invasion wave across southern Michigan. By 2011, an average of 78.6% ± 0.10%, 44.8% ± 0.11%, and 19.8% ± 0.07% of overstory ash trees representing 87%, 57%, and 14% of the total ash basal area had been killed in Core, Crest, and Cusp sites, respectively. Green ash seedlings, saplings, and recruits were abundant in all sites, but newly germinated ash seedlings were absent in Core sites and scarce in Crest sites. Canopy gaps resulting from current ash decline and mortality increased available photosynthetically active radiation (PAR) and sapling growth in Crest sites, but PAR was low in Core and Cusp sites. Lateral ingrowth of non-ash overstory trees has largely filled canopy gaps in Core sites, and there was little evidence of green ash recruitment into the overstory. Green ash appears unlikely to persist as a dominant species in forests invaded by A. planipennis.

Concern about calcium supplements, and mainly minor side effects (e.g. constipation) impacting on compliance, means that assessing dietary calcium intake is important. There is no suitable biomarker. Compared to food diaries, a short questionnaire was an efficient way of confirming that patients had adequate calcium intakes (>700 or >1,000 mg) Calcium is usually given alongside treatments for osteoporosis, but recent concerns about potential side effects have led to questioning whether supplements are always necessary. It is difficult to assess calcium intake in a clinical setting and be certain that the patient is getting enough calcium. The aim of this study was to determine whether a short questionnaire for estimating dietary calcium intakes in a clinical setting was fit for purpose. We assessed dietary calcium intakes using a short questionnaire (CaQ) in patients attending an osteoporosis clinic (n = 117) and compared them with calcium intakes obtained from a 7-day food diary (n = 72) and a food frequency questionnaire (FFQ) (n = 33). Mean (SD) daily calcium intakes from the CaQ were 836 (348) mg; from the diaries, 949 (384) mg; and from the FFQ, 1,141 (387) mg. The positive predictive value (PPV) was >80 % for calcium cut-offs > 700 mg and 70 % for cut-offs > 1,000 mg. The calcium intakes for the false positives results were not far below the cut-off. For 1,200 mg, the PPV was 67 % or less. The CaQ is an adequate tool for assessing whether a patient has daily calcium intakes above 700 or 1,000 mg; if below these cut-offs, it is possible that the patient still has enough calcium in the diet, which could be clarified by questioning the patient further. As there were few patients with calcium intakes above 1,200 mg a day, the CaQ cannot be recommended as a tool for confirming higher dietary calcium intakes.

An Sfp-type phosphopantetheinyl transferase (PPTase) encoding gene F-avi5813 in Agrobacterium vitis F2/5 was found to be required for the induction of a tobacco hypersensitive response (HR) and grape necrosis. Sfp-type PPTases are post-translation modification enzymes that activate acyl-carry protein (ACP) domains in polyketide synthases (PKS) and peptidyl-carrier protein (PCP) domains of nonribosomal peptide synthases (NRPS). Mutagenesis of PKS and NRPS genes in A. vitis led to the identification of a PKS gene (F-avi4330) and NRPS gene (F-avi3342) that are both required for HR and necrosis. The gene immediately downstream of F-avi4330 (F-avi4329) encoding a predicted aminotransferase was also found to be required for HR and necrosis. Regulation of F-avi4330 and F-avi3342 by quorum-sensing genes avhR, aviR, and avsR and by a lysR-type regulator, lhnR, was investigated. It was determined that F-avi4330 expression is positively regulated by avhR, aviR, and lhnR and negatively regulated by avsR. F-avi3342 was found to be positively regulated by avhR, aviR, and avsR and negatively regulated by lhnR. Our results suggest that a putative hybrid peptide-polyketide metabolite synthesized by F-avi4330 and F-avi3342 is associated with induction of tobacco HR and grape necrosis. This is the first report that demonstrates that NRPS and PKS play essential roles in conferring the unique ability of A. vitis to elicit a non-host-specific HR and host-specific necrosis.

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 101 CFU/ml compared with 105 CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 104 CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

Nontumorigenic Agrobacterium vitis strain F2/5 is able to prevent crown gall caused by tumorigenic A. vitis on grape but not on other plant species such as tobacco. Mutations in a quorum-sensing transcription factor, aviR, and in caseinolytic protease (clp) component genes clpA and clpP1 resulted in reduced or loss of biological control. All mutants were complemented; however, restoration of biological control by complemented clpA and clpP1 mutants was dependent on the copy number of vector that was used as well as timing of application of the complemented mutants to grape wounds in relation to inoculation with pathogen. Mutations in other quorum-sensing and clp genes and in a gene associated with polyketide synthesis did not affect biological control. It was determined that, although F2/5 inhibits transformation by tumorigenic A. vitis strains on grape, it does not affect growth of the pathogen in wounded grape tissue over time.

Proteorhodopsin (PR) is a wide-spread protein found in many marine prokaryotes. PR allows for the potential conversion of solar energy to ATP, possibly assisting in cellular growth and survival during periods of high environmental stress. PR utilises either blue or green light through a single amino acid substitution. We incubated the PR-bearing bacterium Psychroflexus torquis 50 cm deep within Antarctic sea ice for 13 days, exposing cultures to diurnal fluctuations in light and temperature. Enhanced growth occurred most prominently in cultures incubated under irradiance levels of ∼50 μmol photons m−2 s−1, suggesting PR provides a strong selective advantage. In addition, cultures grown under blue light yielded over 5.5 times more live cells per photon compared to green-light incubations. Because P. torquis expresses an apparently ‘green-shifted’ PR gene variant, this finding infers that the spectral tuning of PR is more complex than previously thought. This study supports the theory that PR provides additional energy to bacteria under sub-optimal conditions, and raises several points of interest to be addressed by future research.

SUMMARY The characterization of Tn5 transposon insertional mutants of Agrobacterium vitis strain F2/5 revealed a gene encoding a predicted LysR-type transcriptional regulator, lhnR (for ‘LysR-type regulator associated with HR and necrosis’), and an immediate upstream operon consisting of three open reading frames (lhnABC) required for swarming motility, surfactant production and the induction of a hypersensitive response (HR) on tobacco and necrosis on grape. The operon lhnABC is unique to A. vitis among the sequenced members in Rhizobiaceae. Mutagenesis of lhnR and lhnABC by gene disruption and complementation of ΔlhnR and ΔlhnABC confirmed their roles in the expression of these phenotypes. Mutation of lhnR resulted in complete loss of HR, swarming motility, surfactant production and reduced necrosis, whereas mutation of lhnABC resulted in loss of swarming motility, delayed and reduced HR development and reduced surfactant production and necrosis. The data from promoter–green fluorescent protein (gfp) fusions showed that lhnR suppresses the expression of lhnABC and negatively autoregulates its own expression. It was also shown that lhnABC negatively affects its own expression and positively affects the transcription of lhnR. lhnR and lhnABC constitute a regulatory circuit that coordinates the transcription level of lhnR, resulting in the expression of swarming, surfactant, HR and necrosis phenotypes.

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

This Part II paper describes source apportionment (SA) for 10 source categories at a 12 km horizontal grid resolution over the eastern U.S. for January and July of 2002 using the Comprehensive Air Quality Model with extensions/Particulate Source Apportionment Technology (CAMx/PSAT). SA results from CAMx/PSAT are contrasted with those from CMAQ/BFM in Part I. The top three sources domainwide in January are identified to be coal combustion (with a monthly–mean contribution of 14.0%, or 1.1 µg m –3 ), biomass burning (11.3%, 0.9 µg m –3 ) and other mobile sources (6.8%, 0.6 µg m –3 ) by CAMx/PSAT but biomass burning (13.7%, 1.1 µg m –3 ), miscellaneous area sources (11.8%, 0.9 µg m –3 ), and coal combustion (10.8%, 0.9 µg m –3 ) by CMAQ/BFM. Both agree that coal combustion, industrial processes, and miscellaneous area sources are the top three sources in July, though they differ in the magnitude of contributions. Both give similar contributions for primary PM, but they differ substantially in SA results for secondary PM, due primarily to their treatments for oxidant–limiting and indirect effects. While CMAQ/BFM inherently accounts for these effects and can provide useful information for primary and secondary PM species, CAMx/PSAT neglects them by linking each PM species only with its direct primary emission precursor. These effects are enhanced in January due to the increased importance of NO 3 – and lower concentrations of oxidants relative to July. CAMx/PSAT is thus accurate for SA of the primary PM species but incorrect in its SA for secondary PM species. For a highly non–linear system studied here, the true SA cannot be obtained with current SA methods. Policy–makers must be mindful of relative strengths and weaknesses of each method, as well as the limitation of current SA methods in the SA of secondary PM species, when using such information in support of state implementation plan and epidemiological studies.

Bacterial biosensor strains have greatly facilitated the rapid discovery, isolation, and study of quorum-sensing systems. In this study, we determined the relative sensitivity of a LasR-based E. coli bacterial bioluminescence biosensor JM109 (pSB1075) for 13 diverse long-chain N-acyl-homoserine lactones (AHLs) including oxygen-substituted and -unsubstituted AHLs containing 14, 16, and 18 carbons and with and without double bonds. Furthermore, we show by bioassay, HPLC, and GC/MS that four long-chain AHLs of the C16-HSL family are encoded by the avsI gene of Agrobacterium vitis strain F2/5, a non-tumorigenic strain that inhibits pathogenic strains of A. vitis from causing crown gall on grape. The four C16-HSLs include: C16-HSL, N-hexadecanoyl homoserine lactone; 3-oxo-C16-HSL, N-(3-oxohexadecanoyl)homoserine lactone; C16:1-HSL, N-(cis-9-octadecenoyl)homoserine lactone; and 3-oxo-C16:1-HSL, N-(3-oxo-cis-11-hexadecenoyl)homoserine lactone. Thus, the LasR-based bioluminescent biosensor tested in this study should serve as a useful tool for the detection of various long-chain AHLs with and without double bonds as well as those oxylated at the third carbon from uninvestigated species.

Following the 2006 annual meeting of The American Phytopathological Society (APS), President Jan Leach appointed an ad hoc committee with the following charge: Based on an assessment of where plant pathology now stands as a profession, develop a vision of where we will be in the future (10-20 yrs) and how we should position ourselves to achieve this vision. Some key questions and background related to this topic include: (1) Disciplinary Balance (2) Institutional Erosion (3) Research Funding and (4) Age Demographics of the Profession. The year following the 100th birthday of APS seems an especially opportune time to take stock of where we stand as a discipline, to identify near-term and long-term challenges, and to discuss how we might meet those challenges in the future. Indeed, the present state and future prospects of our discipline have been recurrent themes of reviews, letters, and editorials from generations of plant pathologists (4,10–17). Most have reflected the experiences and the scholarly but nonetheless personal viewpoint of a single author. What was often lacking were the hard data that would support the stated positions when applied to the discipline as a whole. This report summarizes the efforts of the APS Ad Hoc Committee on the Present Status and Future of the Profession of Plant Pathology to address the above charge. We have attempted to rely upon a dispassionate methodology and go where the data lead us. Our approach has been to study key events and trends of the last several decades at the national level, assemble a base of information from which to properly understand the issues, and attempt to identify and project future trends and challenges facing the profession. Only then would we be in a position to develop recommendations for the profession to meet such challenges. A complete census of plant pathology at U.S. universities, conducted by the Ad Hoc Committee in 2007, formed the foundation for these analyses. Census of plant pathology at U.S. universities. Although both APS and the National Science Foundation (NSF) collect various statistics on departments and sample survey data on graduate degrees in plant pathology, we are not aware of any recent census of plant pathology at U.S. universities. A census differs from a survey in that the aim is to measure an entire population rather than rely upon a statistical sample. A complete and accurate assessment of the number of active plant pathology faculty at U.S. universities, the size of the graduate student population, and membership of both groups in APS was an essential precursor to any demographic analysis. We collected the following responses: (i) the total number of faculty in a department, (ii) the number of plant pathology faculty in the department in the case of multidisciplinary departments (such as plant science or entomology and plant pathology), (iii) the number of faculty who belonged to APS, (iv) the number of faculty who held doctoral degrees in plant pathology, (v) the total number of M.S. and Ph.D. students in the department (some of whom may be pursuing degrees in areas other than plant pathology), and (vi) the number of plant pathology graduate students (both M.S. and Ph.D.). The data were collected by repeatedly sending a blank spreadsheet to department chairs, former students, postdocs, and colleagues across the United States. Missing data were obtained from department websites and the APS member database. All 1862 and 1890 Land Grant institutions were included in the census. Additional plant pathology faculty and students outside the aforementioned systems were located by searching the APS membership database for faculty and student affiliations. Plant pathology faculty, as well as plant patholCorresponding author: David M. Gadoury, Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva, NY; E-mail: dmg4@nysaes.cornell.edu

Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross-linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically. To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Cross-linking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases (assayed on o-dianisidine) were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylan-cross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.

Primary cell walls of grasses and cereals contain arabinoxylans with esterified ferulate side chains, which are proposed to cross-link the polysaccharides during maturation by undergoing oxidative coupling. However, the mechanisms and control of arabinoxylan cross-linking in vivo are unclear. Non-lignifying maize (Zea mays L.) cell cultures were incubated with l-[1-(3)H]arabinose or (E)-[U-(14)C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl-[(3)H]arabinoxylans. The cross-linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL-2B, peaked suddenly and transiently, typically at approximately 9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H(2)O(2) and/or inhibitors. De-esterified arabinoxylans failed to cross-link, supporting a role for the feruloyl ester groups. The cross-links were stable in vivo. Some of them also withstood mild alkaline conditions, indicating that they were not (only) based on ester bonds; however, most were cleaved by 6 m NaOH, which is a property of p-hydroxybenzyl-sugar ether bonds. Cross-linking of [(14)C]feruloyl-arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H(2)O(2). During cross-linking, the feruloyl groups were oxidized, as shown by ultraviolet spectra and thin-layer chromatography. Esterified diferulates were minor oxidation products; major products were: (i) esterified oligoferulates, released by treatment with mild alkali; and (ii) phenolic components attached to polysaccharides via relatively alkali-stable (ether-like) bonds. Thus, feruloyl esters participate in polysaccharide cross-linking, but mainly by oligomerization rather than by dimerization. We propose that, after the oxidative coupling, strong p-hydroxybenzyl-polysaccharide ether bonds are formed via quinone-methide intermediates.

Xylella fastidiosa , a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.

Xylella fastidiosa, an important phytopathogenic bacterium, causes serious plant diseases including Pierce's disease of grapevine. It is reported here that type I and type IV pili of X. fastidiosa play different roles in twitching motility, biofilm formation and cell-cell aggregation. Type I pili are particularly important for biofilm formation and aggregation, whereas type IV pili are essential for motility, and also function in biofilm formation. Thirty twitching-defective mutants were generated with an EZ : : TN transposome system, and several type-IV-pilus-associated genes were identified, including fimT, pilX, pilY1, pilO and pilR. Mutations in fimT, pilX, pilO or pilR resulted in a twitch-minus phenotype, whereas the pilY1 mutant was twitching reduced. A mutation in fimA resulted in a biofilm-defective and twitching-enhanced phenotype. A fimA/pilO double mutant was twitch minus, and produced almost no visible biofilm. Transmission electron microscopy revealed that the pili, when present, were localized to one pole of the cell. Both type I and type IV pili were present in the wild-type isolate and the pilY1 mutant, whereas only type I pili were present in the twitch-minus mutants. The fimA mutant produced no type I pili. The fimA/pilO double mutant produced neither type I nor type IV pili.

Fourteen Xylella fastidiosa isolates from grapevines exhibiting Pierce's disease symptoms in California, Texas, and South Carolina were examined for type IV pilus-mediated twitching motility, a phenotype previously observed in a Temecula isolate from California. All isolates except one from South Carolina (SC 19A97) exhibited colonies with a peripheral fringe on PW agar, a feature indicative of twitching motility; however, when individual cells of SC 19A97 were examined at higher magnifications twitching motility was observed. The presence and width of colony peripheral fringes were related to the amount of bovine serum albumin (BSA) present in the medium; no or low levels of BSA (0-1.8 g L(-1)) permitted development of the widest fringe, whereas higher levels (3.5-6.0 g L(-1)) severely limited, and in many instances prevented, peripheral fringe development. The growth rate of the wild-type Temecula isolate in PW broth with different concentrations of BSA was similar for all tested concentrations of BSA; however, growth was significantly reduced in medium without BSA.

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730+) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730+ indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

Clavibactermichiganensissubsp. michiganensis(Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The goal of the project was to unravel the molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato. The genome of Cmm contains numerous genes encoding for extracellular serine proteases and cell wall degrading enzymes. The first objective was to elucidate the role of secreted serine proteases in Cmm virulence. Mutants of nine genes encoding serine proteases of 3 different families were tested for their ability to induce wilting, when tomato stems were puncture-inoculated, as compared to blisters formation on leaves, when plants were spray-inoculated. All the mutants showed reduction in wilting and blister formation as compared to the wild type. The chpCmutant displayed the highest reduction, implicating its major role in symptom development. Five mutants of cell wall degrading enzymes and additional genes (i.e. perforin and sortase) caused wilting but were impaired in their ability to form blisters on leaves. These results suggest that Cmm differentially expressed virulence genes according to the site of penetration. Furthermore, we isolated and characterized two Cmmtranscriptional activators, Vatr1 and Vatr2 that regulate the expression of virulence factors, membrane and secreted proteins. The second objective was to determine the effect of bacterial virulence genes on movement of Cmm in tomato plants and identify the routes by which the pathogen contaminates seeds. Using a GFP-labeledCmm we could demonstrate that Cmm extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem and formed biofilm-like structures composed of large bacterial aggregates. Our findings suggest that virulence factors located on the chp/tomAPAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates. We constructed a transposon plasmid that can be stably integrated into Cmm chromosome and express GFP, in order to follow movement to the seeds. Field strains from New York that were stably transformed with this construct, could not only access seeds systemically through the xylem, but also externally through tomato fruit lesions, which harbored high intra-and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruit began to ripen. These results highlight the ability of Cmm to invade tomato fruit and seed through multiple entry routes. The third objective was to assess correlation between disease severity and expression levels of Cmm virulence genes and tomato defense genes. The effect of plant age on expression of tomato defense related proteins during Cmm infection was analyzed by qRT-PCR. Five genes out of eleven showed high induction at early stages of infection of plants with 19/20 leaves compared to young plants bearing 7/8 leaves. Previous results showed that Cmm virulence genes were expressed at early stages of infection in young plants compared to older plants. Results of this study suggest that Cmm virulence genes may suppress expression of tomato defense-related genes in young plants allowing effective disease development. The possibility that chpCis involved in suppression of tomato defense genes is currently under investigation by measuring the transcript level of several PR proteins, detected previously in our proteomics study. The fourth objective was to define genome location and stability of virulence genes in Cmm strains. New York isolates were compared to Israeli, Serbian, and NCPPB382 strains. The plasmid profiles of New York isolates were diverse and differed from both Israeli and Serbian strains. PCR analysis indicated that the presence of putative pathogenicity genes varied between isolates and highlighted the ephemeral nature of pathogenicity genes in field populations of Cmm. Results of this project significantly contributed to the understanding of Cmm virulence, its movement within tomato xylem or externally into the seeds, the role of serine proteases in disease development and initiated research on global regulation of Cmm virulence. These results form a basis for developing new strategies to combat wilt and canker disease of tomato.

Xanthomonas axonopodis pv. glycines is the causal agent of bacterial pustule disease of soybeans. A transposon insertional mutant (KU-P-M670) of X. axonopodis pv. glycines derived from wild-type strain KU-P-34017 lost the ability to induce the hypersensitive response (HR) on tobacco and pepper but retained its HR induction capacity on cucumber, sesame, and tomato. The mutation also resulted in loss of ability to cause a potato soft rot and express pectolytic activity at pH 6.5. An approximate 1.4-kb DNA fragment carrying the transposon insertion contained a single open reading frame that showed high homology with PSTRU-3, a pectate lyase gene in X. axonopodis pv. malvacearum. Complemented KU-P-M670 regained HR induction on tobacco and also pectolytic activity. Treatment of plants with inhibitors of eukaryotic metabolism blocked HR induction by wild-type strains and by complemented KU-P-M670. The presence of the pectate lyase homolog, which we designated xagP, in 26 X. axonopodis pv. glycines strains was highly correlated with their ability to induce an HR on tobacco. To our knowledge, this is the first study indicating a role for a functional pectate lyase in induction of a plant HR.

Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI , were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both genes are essential for hypersensitive-like response (HR) and necrosis. Two hypothetical proteins (ORF1 and ORF2) that are positioned downstream of avsR-avsI are also essential for the phenotypes. Profiles of N -acyl-homoserine lactones (AHLs) isolated from the wild type and mutants revealed that disruption of avsI , ORF1, or ORF2 abolished the production of long-chain AHLs. Disruption of avsR reduces long-chain AHLs. Expression of a cloned avsI gene in A. tumefaciens strain NT1 resulted in synthesis of long-chain AHLs. The necrosis and HR phenotypes of the avsI and avsR mutants were fully complemented with cloned avsI . The addition of synthetic AHLs (C 16:1 and 3-O-C 16:1 ) complemented grape necrosis in the avsR , avsI , ORF1, and ORF2 mutants. It was determined by reverse transcriptase PCR that the expression level of avsI is regulated by avsR but not by aviR or avhR , two other luxR homologs which were previously shown to be associated with induction of a tobacco hypersensitive response and grape necrosis. We further verified that avsR regulates avsI by measuring the expression of an avsI :: lacZ fusion construct.

Agrobacterium vitis causes crown gall disease on grapevines. It also induces a specific necrosis on grape roots and a hypersensitive response (HR) on tobacco that are regulated by a complex quorum-sensing regulatory system. Strain F2/5 produces at least six N-acyl-homoserine lactones (AHLs) that function as signal molecules in quorum-sensing. The AHLs differ in acyl side chain length (8–16 carbons) as determined by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry. Mutant derivatives of F2/5 differ in ability to cause necrosis and the HR and show variable AHL profiles as determined by a thin-layer chromatography/biosensor assay. All wildtype A. vitis strains revealed the presence of long-chain AHLs regardless of tumorigenicity or ability to cause the HR. Whereas genes encoding long-chain AHLs are predicted to reside on the F2/5 chromosome, the determinants for short-chain AHLs were shown to be located on conjugal plasmids.