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.

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.

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.

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.

Feature and knowledge based analysis for reduction of false positives in the computerized detection of masses in screening mammography.

Previously we presented a morphologic concentric layered (MCL) algorithm for the detection of masses in screening mammograms. The algorithm achieved high sensitivity (92%) but it also generated 3.26 false positives (FPs) per image. In the present study we propose a false positive reduction strategy based on using an artificial neural network that merges feature and knowledge-based analysis of suspicious mammographic locations. The ANN integrates two types of information regarding the suspicious candidates: (i) directional and fractal neighborhood analysis features, and (ii) knowledge-based analysis using an information-theoretic similarity metric. The study hypothesis is that the synergistic application of feature and knowledge-based analysis will be an effective strategy to reduce false positives while still maintaining sufficiently the detection rate for true masses. The study was performed using mammograms from the Digital Database of Screening Mammography. Using the fusion ANN decision strategy 56% of the FPs were reduced while maintaining 95% of the true masses.

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.

Screening Triploid Hybrids of 'Lakeland' Limequat for Resistance to Citrus Canker.

Resistance of citrus genotypes to Xanthomonas axonopodis pv. citri, the cause of Asiatic citrus canker (ACC), was evaluated by injection infiltration of 103 and 104 CFU/ml through stomates on the abaxial surface of immature leaves. Citrus genotypes for screening comprised two autotetraploids and nine triploid hybrids of 'Lakeland' limequat (Citrus aurantifolia × Fortunella japonica) and their progenitors ('Lakeland' limequat, the autotetraploids 'Femminello' lemon (Citrus limon) and 'Giant Key' lime (C. aurantifolia), and the somatic hybrids 'Key' [also known as 'Mexican'] lime + 'Valencia' orange and 'Hamlin' orange + 'Femminello' lemon). 'Meiwa' kumquat (Fortunella crassifolia) and 'Pineapple' sweet orange (C. sinensis) were used as known resistant and susceptible standards, respectively. Lesion number per inoculation site and bacterial population per lesion were recorded 15 to 19 days after inoculation. The assay was performed four times during a spring-summer-fall period under greenhouse conditions. Canker lesions were consistently produced by stomatal inoculation with 104 but not 103 CFU/ml. Susceptible and resistant genotypes were separated based on lesion number per inoculation site and bacterial population per lesion. Spearman's rank correlation analysis for lesion numbers on 15 genotypes common to all four assays showed significant correlations among the genotype rankings. Genotype rankings were also significantly correlated between the two bacterial population assays. Lesion number per inoculation site is sufficient for assessment of resistance of citrus genotypes to ACC without the necessity of conducting bacterial population assays. 'Lakeland' limequat is a promising seed parent for breeding acid citrus fruit that is resistant to ACC.

Lack of Control of Citrus Canker by Induced Systemic Resistance Compounds.

Induced systemic resistance compounds (ISRs), acibenzolar-S-methyl (Actigard), and harpin protein (Messenger) were assayed in the greenhouse against Xanthomonas axonopodis pv. citrumelo, the cause of citrus bacterial spot (CBS), and X. axonopodis pv. citri, the cause of Asiatic citrus canker. Actigard and Messenger applied as foliar sprays 3 to 7 days before inoculation reduced numbers of lesions when either bacterium at 103 or 104 CFU/ml was injection-infiltrated into Swingle citrumelo leaves. Based on this activity, the ISRs were evaluated in southern Brazil in orchards of sweet oranges with low to moderate canker disease incidence in spray programs with and without copper oxychloride (COC) and copper hydroxide (CuOH). Actigard and Messenger were applied full season or in the first two or three sprays of a six-spray program in an attempt to reduce early canker disease on foliage and thereby reduce subsequent fruit infection and premature drop. Sprays of COC and CuOH were moderately to highly effective in reducing canker disease incidence and preventing premature fruit drop. Actigard or Messenger in combination with COC and CuOH, respectively, did not significantly reduce citrus canker incidence on foliage or fruit drop compared with Cu alone. The lack of additional control with ISRs means they cannot be recommended at this time to augment Cu programs for management of citrus canker.

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere.

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P

Incidence of Endemic Entomopathogenic Nematodes Following Application of Steinernema riobrave for Control of Diaprepes abbreviatus.

Control of Diaprepes abbreviatus by endemic and exotic entomopathogenic nematodes (EPN) was monitored during 2000-2001 in two citrus orchards in central Florida (Bartow and Poinciana). Caged sentinel insect larvae were buried beneath citrus trees for 7 days at 1 to 2-month intervals from April to October each year. At Bartow, the survey occurred in experimental plots that were (i) not treated with commercial EPN, (ii) treated twice annually since 1998 with commercially formulated Steinernema riobrave, or (iii) treated twice annually with S. riobrave and liquid fertilization (15 times/year) occurred in place of dry fertilizer (3 times/year) used in the other treatments. Four endemic EPN species, in addition to S. riobrave, were recovered from the sandy soil at Bartow: S. diaprepesi, Heterorhabditis zealandica, H. indica, and H. bacteriophora. Mean insect mortality in control plots was 39.4% (range = 13% to 74%), with seasonal maxima in May to July each year. Endemic EPN were recovered from 55% (range = 22% to 81%) of the cadavers each month. Total numbers of endemic EPN recovered in all plots during 2 years were directly related to the numbers of adult weevils (D. abbreviatus and Pachnaeus litus) captured in modified Tedder's traps and inversely related to recovery of S. riobrave. Insect mortality was higher and cadavers containing endemic EPN were more numerous in untreated control plots than in S. riobrave-treated plots, except during months in which S. riobrave was applied. In treated plots, endemic EPN were recovered from cadavers at twice the rate of S. riobrave. Suppression of endemic EPN in plots treated with S. riobrave, combined with inferior persistence by the introduced species, may have attenuated the net efficacy of S. riobrave against D. abbreviatus. In contrast, H. indica was the only endemic nematode recovered from the sandy clay loam soil at Poinciana, where the average mortality of D. abbreviatus was 12% (range 3% to 20%) and incidence of H. indica did not exceed 8%. Results of these surveys suggest that the regional patterns in the abundance and damage to citrus caused by D. abbreviatus in Florida are regulated by endemic EPN and other soilborne enemies of the weevil.

Phytophthora-Diaprepes Weevil Complex: Phytophthora spp. Relationship with Citrus Rootstocks.

Diaprepes abbreviatus (Coleoptera: Curculionidae) is a root weevil, introduced from the Caribbean Basin into Florida in 1964. The larval stage feeds on fibrous and structural roots of citrus, predisposing the injured root system to infection and girdling by Phytophthora spp. In citrus orchards, the rootstocks trifoliate orange (Poncirus trifoliata) and hybrid 'Swingle' citrumelo (Citrus paradisi × P. trifoliata) are resistant to the complex of P. nicotianae with D. abbreviatus, while 'Cleopatra' mandarin (C. reticulata) is susceptible to this complex. When Phytophthora palmivora is coincident with P. nicotianae in fine-textured, poorly drained soils, Swingle citrumelo is more vulnerable to attack by the complex with P. palmivora than is Cleopatra mandarin. Infestation of 9-month-old seedlings with zero, two, or five neonate larvae resulted in a wide range of fibrous root and taproot damage of trifoliate orange and Cleopatra mandarin. Leakage of reducing sugars increased sharply as injury to the roots exceeded 75%. The relationship between feeding damage and root leakage was similar for the two rootstocks. Thus, reduced root damage was not based on host resistance to larval feeding. Root infection and rhizosphere populations of P. nicotianae were higher on Cleopatra mandarin than on trifoliate orange. Root rot by P. nicotianae did not increase with severity of feeding injury on either rootstock. Root infection and root rot by P. palmivora was more severe on trifoliate orange than on Cleopatra mandarin and increased with severity of larval damage. P. palmivora infected and rotted the taproot of both rootstocks if predisposed by larval feeding, but P. nicotianae did not. Commercial rootstocks are severely damaged by larvae of D. abbreviatus; therefore, tolerance of the Phytophthora-Diaprepes weevil complex should be based on resistance of rootstocks to each Phytophthora sp.

Proteome analysis of highly immunoreactive proteins of Helicobacter pylori.

BACKGROUND: Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori. METHOD: The total complement of protein from seven strains of H. pylori was resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori-infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme-antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease beta-subunit UreB; elongation factor EF-Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains. CONCLUSION: These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF-Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.

Genetic relationship among worldwide strains of Xanthomonas causing canker in citrus species and design of new primers for their identification by PCR.

Partial sequence analysis of the ribosomal operon in Xanthomonas axonopodis allowed discrimination among strains causing the A, B, and C types of citrus bacterial canker (CBC) and quantification of the relationship of these organisms with other species and pathovars in the same genus. Sets of primers based on sequence differences in the internally transcribed spacer and on a sequence from the plasmid gene pthA involved in virulence were designed for specific identification of xanthomonads causing CBC diseases. The two sets were validated with a collection of Xanthomonas strains associated with citrus species. The primer set based on ribosomal sequences had a high level of specificity for X. axonopodis pv. citri, whereas the set based on the pthA gene was universal for all types of CBC organisms. Moreover, the relationships among worldwide Xanthomonas strains causing CBC were analyzed by amplification of repetitive sequences (enterobacterial repetitive intergenic consensus and BOX elements). Under specific conditions, pathotypes of these Xanthomonas strains could be discerned, and subgroups of the pathotypes were identified. Subgroups of strains were associated with certain geographic areas of the world, and on this basis the origin of type A strains introduced into Florida could be inferred.

Eggs of Tylenchulus semipenetrans Inhibit Growth of Phytophthora nicotianae and Fusarium solani in vitro.

In previous greenhouse and laboratory studies, citrus seedlings infested with the citrus nematode Tylenchulus semipenetrans and later inoculated with the fungus Phylophthora nicotianae grew larger and contained less fungal protein in root tissues than plants infected by only the fungus, demonstrating antagonism of the nematode to the fungus. In this study, we determined whether eggs of the citrus nematode T. semipenetrans and root-knot nematode Meloidogyne arenaria affected mycelial growth of P. nicotianae and Fusarium solani in vitro. Approximately 35,000 live or heat-killed (60 degrees C, 10 minutes) eggs of each nematode species were surface-sterilized with cupric sulfate, mercuric chloride, and streptomycin sulfate and placed in 5-pl drops onto the center of nutrient agar plates. Nutrient agar plugs from actively growing colonies of P. nicotianae or F. solani were placed on top of the eggs for 48 hours after which fungal colony growth was determined. Live citrus nematode eggs suppressed mycelial growth of P. nicotianae and F. solani (P

Infection of Citrus Roots by Tylenchulus semipenetrans Reduces Root Infection by Phytophthora nicotianae.

Bioassays and whole-plant experiments were conducted to investigate the interaction between Tylenchulus semipenetrans and Phytophthora nicotianae. Both organisms are parasites of the citrus fibrous root cortex. Nematode-infected and non-infected root segments were excised from naturally infected field roots and placed on water agar in close proximity to agar plugs of P. nicotianae and then transferred to a Phytophthora-selective medium. At 10 and 12 days, 50% fewer nematode-infected segments were infected by P. nicotianae than non-infected segments. In whole-plant experiments in glass test tubes, sour orange seedlings were inoculated with two densities (8,000 or 80,000 eggs and second-stage juveniles) of T. semipenetrans, and after establishment of infection were inoculated with two densities (9,000 and 90,000 zoospores) of P. nicotianae. In the first experiment, fungal protein was 53% to 65% lower in the roots infected by both organisms than in roots infected by the fungus only. Compared to plants infected only by P. nicotianae, shoot weights were 33% to 50% greater (P

Quantitative PCR method for diagnosis of citrus bacterial canker.

For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions.