Potential of Nano-Formulated Zinc Oxide for Control of Citrus Canker on Grapefruit Trees.

Antimicrobial activity of experimental formulations of two structurally different nano-zinc oxide materials, plate-like Zinkicide SG4 and particulate Zinkicide SG6, was evaluated against Xanthomonas citri subsp. citri, the cause of citrus canker. In vitro assay demonstrated Zinkicide SG4 had a twofold lower minimum inhibitory concentration (MIC) against Escherichia coli and X. alfalfae subsp. citrumelonis (62.5 to 250 µg/ml) compared with copper sulfate (250 µg/ml), copper hydroxide (250 to 500 µg/ml), or cuprous oxide/zinc oxide (125 to 250 µg/ml). Zinkicide SG6 had a sevenfold to eightfold lower MIC against Escherichia coli and X. alfalfae subsp. citrumelonis (31 to 250 µg/ml). Leaves of sweet orange (Citrus sinensis) and fruit of 'Ruby Red' grapefruit (C. paradisi) were evaluated for citrus canker disease control. A greenhouse assay with foliage demonstrated that spray treatment with Zinkicide reduced citrus canker lesion development after injection-infiltration of X. citri subsp. citri into the leaf intercellular space. In field trials conducted in Southeast Florida in 2014 and 2015, Zinkicide SG4 and SG6 reduction of grapefruit canker incidence exceeded that of cuprous oxide and cuprous oxide/zinc oxide bactericides. Zinkicide formulations were also effective against the fungal diseases, citrus scab (Elsinoe fawcetti) and melanose (Diaporthe citri), on grapefruit. No sign of phytotoxicity to the fruit rind was observed during either season. Antimicrobial activity of Zinkicide for protection of leaves and fruit against X. citri subsp. citri was comparable or exceeded that for commercial copper and zinc oxide formulations which may be attributed to translaminar movement of Zinkicide.

Presymptomatic Fibrous Root Decline in Citrus Trees Caused by Huanglongbing and Potential Interaction with Phytophthora spp.

Huanglongbing (HLB), associated with 'Candidatus Liberibacter asiaticus', was first detected in Florida in late 2005 and is now widely distributed throughout the commercial citrus-growing regions. In recent seasons, concurrent with freeze and drought episodes, symptomatic HLB-infected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms exhibited by the stressed trees were excessive leaf loss and premature fruit drop even when HLB-infected trees were managed with good nutritional and irrigation practices recommended to support health of HLB-affected trees. This stress intolerance may be due to a loss of fibrous roots. To assess root status of HLB-infected trees on 'Swingle' citrumelo rootstock (Citrus paradisi × Poncirus trifoliata), blocks of 2,307 3-year-old 'Hamlin' orange trees and 2,693 4-year-old 'Valencia' orange trees were surveyed visually and with a real-time polymerase chain reaction (PCR) assay to determine 'Ca. L. asiaticus' infection status. The incidence of 'Ca. L. asiaticus'-infected trees (presymptomatic: 'Ca. L. asiaticus'+, visually negative; and symptomatic: 'Ca. L. asiaticus'+, visually positive) trees was 89% for the Hamlin block and 88% for the Valencia block. 'Ca. L. asiaticus'+ trees had 30 and 37% lower fibrous root mass density for presymptomatic and symptomatic trees, respectively, compared with 'Ca. L. asiaticus'- trees. In a second survey, 10- to 25-year-old Valencia trees on Swingle citrumelo or 'Carrizo' citrange (C. sinensis (L.) × P. trifoliata) rootstock were sampled within 3 to 6 months after identification of visual HLB status as symptomatic ('Ca. L. asiaticus'+, visually positive) or nonsymptomatic ('Ca. L. asiaticus'-, visually negative) in orchards located in the central ridge, south-central, and southwest flatwoods. Pairs of HLB symptomatic and nonsymptomatic trees were evaluated for PCR status, fibrous root mass density, and Phytophthora nicotianae propagules in the rhizosphere soil. 'Ca. L. asiaticus'+ trees had 27 to 40% lower fibrous root mass density and, in one location, higher P. nicotianae per root but Phytophthora populations per cubic centimeter of soil were high on both 'Ca. L. asiaticus'+ and 'Ca. L. asiaticus'- trees. Fibrous root loss from HLB damage interacted with P. nicotianae depending on orchard location and time of year.

Wind Speed Effects on the Quantity of Xanthomonas citri subsp. citri Dispersed Downwind from Canopies of Grapefruit Trees Infected with Citrus Canker.

The epidemic of citrus canker (Xanthomonas citri subsp. citri) in Florida continues to expand since termination of the eradication program in 2006. Storms are known to be associated with disease spread, but little information exists on the interaction of fundamental physical and biological processes involved in dispersal of this bacterium. To investigate the role of wind speed in dispersal, wind/rain events were simulated using a fan to generate wind up to 19 m·s-1 and spray nozzles to simulate rain. Funnels at ground level and panels at 1.3 m height and distances up to 5 m downwind collected wind-driven splash. Greater wind speeds consistently dispersed more bacteria, measured by concentration (colony forming units [CFU] ml-1) or number sampled (bacteria flux density [BFD] = bacteria cm-2 min-1), from the canopy in the splash. The CFU ml-1 of X. citri subsp. citri collected by panels 1 m downwind at the highest wind speed was up to 41-fold greater than that collected at the lowest wind speed. BFD at the highest wind speed was up to 884-fold higher than that collected at the lowest wind speed. Both panels at distances >1 m and funnels at distances >0 m collected many-fold more X. citri subsp. citri at higher wind speeds compared to no wind (up to 1.4 × 103-fold greater CFU ml-1 and 1.8 × 105-fold the BFD). The resulting relationship between wind speed up to 19 m·s-1 and the mean CFU ml-1 collected by panel collectors downwind was linear and highly significant. Likewise, the mean CFU ml-1 collected from the funnel collectors had a linear relationship with wind speed. The relationship between wind speed and BFD collected by panels was generally similar to that described for CFU ml-1 of X. citri subsp. citri collected. However, BFD collected by funnels was too inconsistent to determine a meaningful relationship with increasing wind speed. The quantity of bacteria collected by panels declined with distance, and the relationship was described by an inverse power model (R2 = 0.94 to 1.00). At higher wind speeds, more bacteria were dispersed to all distances. Windborne inoculum in splash in subtropical wet environments is likely to be epidemiologically significant, as both rain intensity and high wind speed can interact to provide conditions conducive for dispersing large quantities of bacteria from canker-infected citrus trees. Disease and crop management aimed at reducing sources of inoculum and wind speeds in a grove should help minimize disease spread by windborne inoculum.

Plant-pathogenic bacteria as biological weapons - real threats?

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae.

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.

Food web responses to augmenting the entomopathogenic nematodes in bare and animal manure-mulched soil.

Factorial treatments of entomopathogenic nematodes (EPN) and composted, manure mulches were evaluated for two years in a central Florida citrus orchard to study the post-application biology of EPN used to manage the root weevil, Diaprepes abbreviatus. Mulch treatments were applied once each year to study the effects of altering the community of EPN competitors (free-living bactivorous nematodes) and antagonists (nematophagous fungi (NF), predaceous nematodes and some microarthro-pods). EPN were augmented once with Steinernema riobrave in 2004 and twice in 2005. Adding EPN to soil affected the prevalence of organisms at several trophic levels, but the effects were often ephemeral and sometimes inconsistent. EPN augmentation always increased the mortality of sentinel weevil larvae, the prevalence of free-living nematodes in sentinel cadavers and the prevalence of trapping NF. Subsequent to the insecticidal effects of EPN augmentation in 2004, but not 2005, EPN became temporarily less prevalent, and fewer sentinel weevil larvae died in EPN-augmented compared to non-augmented plots. Manure mulch had variable effects on endoparasitic NF, but consistently decreased the prevalence of trapping NF and increased the prevalence of EPN and the sentinel mortality. Both temporal and spatial abundance of NF were inversely related to the prevalence of Steinernema diaprepesi, whereas Heterorhabditis zealandica prevalence was positively correlated with NF over time. The number of weevil larvae killed by EPN was likely greatest in 2005, due in part to non-target effects of augmentation on the endemic EPN community in 2004 that occurred during a period of peak weevil recruitment into the soil.

Cyber security risk assessment for SCADA and DCS networks.

The growing dependence of critical infrastructures and industrial automation on interconnected physical and cyber-based control systems has resulted in a growing and previously unforeseen cyber security threat to supervisory control and data acquisition (SCADA) and distributed control systems (DCSs). It is critical that engineers and managers understand these issues and know how to locate the information they need. This paper provides a broad overview of cyber security and risk assessment for SCADA and DCS, introduces the main industry organizations and government groups working in this area, and gives a comprehensive review of the literature to date. Major concepts related to the risk assessment methods are introduced with references cited for more detail. Included are risk assessment methods such as HHM, IIM, and RFRM which have been applied successfully to SCADA systems with many interdependencies and have highlighted the need for quantifiable metrics. Presented in broad terms is probability risk analysis (PRA) which includes methods such as FTA, ETA, and FEMA. The paper concludes with a general discussion of two recent methods (one based on compromise graphs and one on augmented vulnerability trees) that quantitatively determine the probability of an attack, the impact of the attack, and the reduction in risk associated with a particular countermeasure.

Growth Depression in Mycorrhizal Citrus at High-Phosphorus Supply (Analysis of Carbon Costs).

Mycorrhizal-induced growth depression of plants in high-P soil has been reported in many species. The carbon costs of factors contributing to this growth depression were analyzed in Volkamer lemon (Citrus volkameriana Tan. & Pasq.) colonized by the mycorrhizal (M) fungus Glomus intraradices Schenck and Smith. M and nonmycorrhizal (NM) plants were each grown at two P-supply rates. Carbon budgets of M and NM plants were determined by measuring whole-plant carbon assimilation and respiration rates using gas-exchange techniques. Biomass, M colonization, tissue-P concentration, and total fatty acid concentration in the fibrous roots were determined. Construction costs of the fibrous roots were estimated from heat of combustion, N, and ash content. Root-growth respiration was derived from daily root growth and root-construction cost. M and NM plants grown in high-P soil were similar in P concentration, daily shoot carbon assimilation, and daily shoot dark respiration. At 52 d after transplanting (DAT), however, combined daily root plus soil respiration was 37% higher for M than for NM plants, resulting in a 20% higher daily specific carbon gain (mmol CO2 [mmol carbon]-1 d-1) in NM than M plants. Estimates of specific carbon gain from specific growth rates indicated about a 10% difference between M and NM plants. Absolute values of specific carbon gain estimated by whole-plant gas exchange and by growth analysis were in general agreement. At 52 DAT, M and NM plants at high P had nearly identical whole-plant growth rates, but M plants had 19% higher root dry weight with 10% higher daily rates of root growth. These allocation differences at high P accounted for about 51% of the differences in root/soil respiration between M and NM plants. Significantly higher fatty acid concentrations in M than NM fibrous roots were correlated with differences in construction costs of the fibrous roots. Of the 37% difference in daily total root/soil respiration observed between high-P M and NM plants at 52 DAT, estimated daily growth respiration accounted for only about 16%, two-thirds of which was associated with construction of lipid-rich roots, and the remaining one-third with greater M root growth rates. Thus, of the 37% more root/soil respiration associated with M colonization of high-P plants, 10% was directly attributable to building lipid-rich roots, 51% to greater M root biomass allocation, and the remaining 39% could have been used for maintenance of the fungal tissue in the root and growth and maintenance of the extramatrical hyphae.

Quantitative real-time polymerase chain reaction for bacterial enumeration and allelic discrimination to differentiate xanthomonas strains on citrus.

ABSTRACT Quantitative real-time polymerase chain reaction (QRT-PCR) was developed for identification and enumeration of bacteria in citrus plant samples infected with Xanthomonas axonopodis pvs. citri and citrumelo, the cause of citrus bacterial canker (CBC) and citrus bacterial spot (CBS), respectively. Three sets of primers based on the pathogenicity gene (pth) in X. axonopodis pv. citri, a ribosomal gene in X. axonopodis pv. citrumelo, and the leucine-responsive regulatory protein (lrp) in both pathovars were combined with TaqMan probes and applied for specific strain detection and quantification. Calibration curves for bacterial abundance in plant samples obtained with the three primer-probe combinations were congruent with colony counts on plates of semiselective medium in most of the cases. However, apparent overestimation of bacterial cells by QRT-PCR indicated the presence of nonculturable or nonviable cells in some samples. In addition to quantification, the lrp primers and probes permitted differentiation by allelic discrimination of Xanthomonas strains infecting citrus tissues. This technique is based on the utilization of two probes that detect a single nucleotide difference in the target sequence between different strains and was validated with a collection of cultured Xanthomonas strains as well as tissue with CBC and CBS lesions. Allelic discrimination is demonstrated to be a more specific and sensitive protocol than previously developed PCR-based methods for strain identification and quantification.

Plastic embedding of cutaneous specimens for light microscopy using araldite epoxy resin.

A method is described using araldite epoxy resin for embedding intact 3-mm cutaneous punch biopsies for light microscopy. Sections are cut at 0.5-2.0 micron and stained with hematoxylin-eosin-phloxine. This procedure allows for preservation of cellular detail unobtainable with conventional formalin-fixed, paraffin embedded tissue.

Deep penetrating nevus.

We report a clinical and histologic study of 70 patients, each with a single melanocytic lesion termed "deep penetrating nevus" (DPN). The lesions are most commonly found on the face, upper trunk, or proximal extremities of patients between the ages of 10 and 30 years. Typically they are darkly pigmented. Histologically they are characterized by loosely organized nests of pleomorphic pigmented cells that penetrate deep into the reticular dermis and often to the subcutaneous fat. Follow-up was obtained from 48 patients. It ranged from 1 to 23 years (mean, 7 years). Despite an initial histologic diagnosis of malignant melanoma in 29% of the cases, there were no local recurrences and no distant metastases. It is important to differentiate DPN from malignant melanoma. The characteristic histologic features of DPN also allow its differentiation from spindle cell and epithelioid cell nevi and blue nevi.

Spindle cell and epithelioid cell nevi with atypia and metastasis (malignant Spitz nevus).

We report on the clinical and pathologic features of 32 lesions diagnosed as malignant spindle cell and epithelioid cell nevus (S&E nevus). Because of the clinical or initial histopathologic diagnosis of malignant melanoma, six patients had lymph node dissection. Three of these patients also had an enlarged lymph node. In all six cases, metastatic spindle or epithelioid cells were found in at least one of the resected lymph nodes. Of the 30 patients with follow-up information, including all six patients with lymph node metastases, all are alive and well. No recurrences or further metastases have been found. On histopathologic reevaluation, all the lesions had features of S&E nevi. Study of these cases suggests that although some lesions with features of S&E nevi may involve local lymph nodes, widespread metastases do not result.

WATER RELATIONS OF MYCORRHIZAL AND PHOSPHORUS-FERTILIZED NON-MYCORRHIZAL CITRUS UNDER DROUGHT STRESS.

Rootstock seedlings of Carrizo citrange (Poncirus trifoliata (L.) Raf. x C. Sinensis (L.) Osbeck) and sour orange (Citrus aurontium L.) were grown in a sandy soil low in phosphorus (P) and either inoculated with Glomus intraradices Schenck & Smith (vesicular-arbuscular mycorrhizal; VAM) or fertilized with soluble P (non-mycorrhizal; NM). Five-month-old VAM and NM seedlings of each rootstock were comparable in size, P sufficiency, and relative growth rate whether they were kept well-watered or subjected to two drought-stress cycles of short duration. Under well-watered conditions, whole plant transpiration, leaf water status, and root hydraulic conductivity were similar for VAM and NM plants of each rootstock. During drought-stress and recovery periods, VAM plants also had very comparable whole-plant transpiration rates and leaf water potentials to NM plants, but mycorrhiza reduced root hydraulic conductivity of Carrizo citrange and sour orange 66 and 49%, respectively. These data do not support the hypothesis that mycorrhiza significantly enhance water relations of citrus under the drought-stress conditions studied.

Epithelioma cuniculatum pedis, a variant of squamous cell carcinoma.

The rare lesion of epithelioma cuniculatum pedis may be recognized by the gross appearance of a slow-growing, bulky, "squashy," non-healing, exophytic mass of long duration on the sole, exuding foul-smelling purulent keratin debris from numerous sinuses, and biopsies showing extensive infiltrating plaques of keratinizing, well-formed, squamous epithelium forming walls for numerous sinuses filled with purulent material. The rarity of the lesion, the slow growth, and biopsies showing relatively benign-appearing well-differentiated squamous epithelium caused considerable diagnostic difficulty to clinicians and pathologists. The tumor failed to heal, extensively penetrated the soft tissues, ultimately destroyed the metatarsal bones, and invaded the skin of the dorsum of the fott. The lesion should be treated as a slow-growing, well-differentiated squamous cell carcinoma by wide local excision or by amputation when extensive growth causes deformity of the foot or penetration of the metatarsal bones.

Cocaine-like discriminative stimulus effects of procaine, dimethocaine and lidocaine in rats.

The discriminative stimulus effects of several local anesthetics and (+)-amphetamine were assessed in a drug discrimination based on the psychomotor stimulant cocaine. Two groups of adult male Sprague-Dawley rats were trained in two-lever operant chambers in a cocaine versus saline discrimination, or a cocaine versus procaine or saline discrimination, using a fixed ratio 20 schedule of food reinforcement. Cocaine, (+)-amphetamine and dimethocaine all dose-dependently substituted for the training dose of cocaine (10 mg/kg) in both procedures. While procaine and lidocaine showed partial substitution in the cocaine versus saline procedure, much less substitution occurred in the cocaine versus procaine or saline discrimination. These data demonstrate that it is possible to train rats to discriminate between cocaine and another local anesthetic procaine, resulting in an increased pharmacological specificity of cocaine discrimination. The fact that dimethocaine fully substituted for cocaine in both procedures indicates that this local anesthetic has more cocaine-like effects than others so far tested, which is consistent with results from other behavioral and neurochemical studies with this compound.

Cytomegalovirus infection of the colon associated with ulcerative colitis.

We report a 12th case in which infection of the colon with cytomegalovirus was demonstrated in association with ulcerative collitis. Rectal biopsy has proven to be reliable in identifying cells with cytomegaloviral inclusion bodies if careful histologic evaluation is performed. Thus far, prognosis has been serious when cytomegalovirus of the colon has been documented in ulcerative colitis. Nine of 12 patients have required a colectomy, and only seven of 12 have survived.

Granular cell basal cell carcinoma. A distinct histopathologic entity.

In two cases of basal cell carcinoma with prominent granular cell features, light microscopic examination showed a tumor with the general configuration of a nodular basal cell carcinoma. Focally, there were masses of cells with eosinophilic, granular cytoplasm and large cytoplasmic inclusions, strongly suggestive of granular cell myoblastoma. Ultrastructural observations in one case showed numerous lysosome-like organelles that were similar to those described for granular cell myoblastoma, but were identical to those described for the granular cell variant of ameloblastoma, a tumor that frequently resembles basal cell carcinoma. Additional features included tonofilaments and desmosomes, both of which support an epithelial origin for the granular cells in this rare variant of basal cell carcinoma.

Intravascular papillary endothelial hyperplasia. A benign lesion mimicking angiosarcoma.

Observations were made of three cases of intravascular papillary endothelial hyperplasia (IPEH). Lesions of IPEH usually occur on the head and neck area as a subcutaneous red or blue nodule. This papillary proliferation of endothelial and stromal tissue occurs de novo in organizing venous thrombi or in preexisting hemangiomas or phlebectasias. An adequate understanding of this benign vascular disorder is important, since the lesion may be clinically and histologically mistaken for low grade angiosarcoma.

Macular amyloidosis with localized amyloidosis of upper air passages.

A patient with primary amyloidosis of the upper air passages was found to have cutaneous macular amyloidosis. A review of the literature indicates that this may represent the first report of these two uncommon forms of amyloidosis occurring in the same individual. Biopsy material from the nasopharynx, tonsillar areas, larynx, trachea, and skin showed amyloid in the tissue sections. Special stains were used and polariscopic studies were performed, and the hyalin material met the established criteria for amyloid. Studies of tissues from other parts of the body of this patient failed to demonstrate amyloid.

Potent substituted-3 beta-phenyltropane analogs of cocaine have cocaine-like discriminative stimulus effects.

Two novel phenyltropane analogs of cocaine, 3 beta-(4-chlorophenyl) tropane-2 beta-carboxylic acid methyl ester (RTI-COC-31) and 3 beta-(4-methylphenyl) tropane-2 beta-carboxylic acid methyl ester (RTI-COC-32), were evaluated for cocaine-like discriminative stimulus effects in rats. Two groups of rats were trained to discriminate 10 mg/kg cocaine from saline using a standard 2-lever discrimination procedure with correct-lever responding reinforced under a fixed-ratio 20 schedule of food reinforcement. One group of rats was used to evaluate RTI-COC-31 and the unsubstituted-3 beta-phenyltropane reference compound WIN 35,065-2 in substitution tests. Another group of rats was used to evaluate RTI-COC-32. For purposes of comparison, a cocaine dose-effect curve was also determined in each group. Both RTI-COC-31 and RTI-COC-32, as well as WIN 35,065-2, substituted completely for cocaine. RTI-COC-31 was 26.8-fold and RTI-COC-32 was 6-fold more potent than cocaine for producing cocaine-lever responding. The potent cocaine-like effects of the RTI analogs in vivo correspond with their high affinities for a cocaine recognition site on the dopamine transporter, providing further evidence that this site may mediate behavioral effects of cocaine relevant to its abuse.