Importance of Fungicide Seed Treatment and Environment on Seedling Diseases of Cotton.

The importance of fungicide seed treatments on cotton was examined using a series of standardized fungicide trials from 1993 to 2004. Fungicide seed treatments increased stands over those from seed not treated with fungicides in 119 of 211 trials. Metalaxyl increased stands compared to nontreated seed in 40 of 119 trials having significant fungicide responses, demonstrating the importance of Pythium spp. on stand establishment. Similarly, PCNB seed treatment increased stands compared to nontreated seed for 44 of 119 trials with a significant response, indicating the importance of Rhizoctonia solani in stand losses. Benefits from the use of newer seed treatment chemistries, azoxystrobin and triazoles, were demonstrated by comparison with a historic standard seed treatment, carboxin + PCNB + metalaxyl. Little to no stand improvement was found when minimal soil temperatures averaged 25°C the first 3 days after planting. Stand losses due to seedling pathogens increased dramatically as minimal soil temperatures decreased to 12°C and rainfall increased. The importance of Pythium increased dramatically as minimal soil temperature decreased and rainfall increased, while the importance of R. solani was not affected greatly by planting environment. These multi-year data support the widespread use of seed treatment fungicides for the control of the seedling disease complex on cotton.

Sclerotinia blight resistance in Virginia-type peanut transformed with a barley oxalate oxidase gene.

Transgenic peanut lines expressing oxalate oxidase, a novel enzyme to peanut, were evaluated for resistance to Sclerotinia blight in naturally infested fields over a 5-year period. Area under the disease progress curve (AUDPC) for transgenic lines in single rows planted with seed from single-plant selections averaged 78, 83, and 90% lower than nontransgenic parents in 2004, 2005, and 2006, respectively. In addition, AUDPC in 14 transgenic lines planted with bulked seed in two-row plots averaged 81% lower compared with nontransgenic parents in 2005 and 86% lower in 16 transgenic lines in 2006. Six transgenic lines yielded 488 to 1,260 kg/ha greater than nontransgenic parents in 2005, and 10 lines yielded 537 to 2,490 kg/ha greater in 2006. Fluazinam (0.58 kg a.i./ha) fungicide sprays in 2008 and 2009 reduced AUDPC in transgenic and nontransgenic lines but AUDPC was lowest in transgenic lines. Without fluazinam, yields of transgenic lines averaged 1,133 to 1,578 kg/ha greater than nontransgenic lines in 2008 and 1,670 to 2,755 kg/ha greater in 2009. These results demonstrated that the insertion of barley oxalate oxidase in peanut conveyed a high level of resistance to Sclerotinia blight, and negated the need for costly fungicide sprays.

The feasibility of developing a risk assessment for the impact of climate change on the emergence of Crimean-Congo haemorrhagic fever in livestock in Europe: a review.

Crimean-Congo haemorrhagic fever virus (CCHFV) is one of the most widespread of all medically important arboviruses with ticks of the Hyalomma spp. serving as the main vectors. Infection of livestock by CCHFV serves as a route of exposure to humans, as a reservoir of disease and as a route of importation. This study discusses the pathways and data requirements for a qualitative risk assessment for the emergence of CCHFV in livestock in Europe. A risk map approach is proposed based on layers that include the potential routes of release (e.g. by migrating birds carrying infected ticks) together with the main components for exposure, namely the distributions of the tick vectors, the small vertebrate host reservoirs and the livestock. A layer on landscape fragmentation serves as a surrogate for proximity of livestock to the tick cycle. Although the impact of climate change on the emergence of CCHF is not clear, comparing the distribution of risk factors in each layer currently with those predicted in the 2080s with climate change can be used to speculate how potential high-risk areas may shift. According to the risk pathway, transstadial and/or transovarial transmission in the tick vector are crucial for CCHFV spread. Vector competence and tick vector switching, however, remain critical factors for CCHFV colonization of new regions in Europe. The species of migratory bird is also an important consideration in the release assessment with greater abundance and biodiversity of ground-dwelling birds in southern Europe than in northern Europe.

Expression and induction of cathepsins B and D in K562 cells.

Three distinct lysosomal protease activities have been identified in the human leukemia cell line, K562. These include cathepsin D, the classic protease of the mature red blood cell, as well as two proteases, cathepsins B and H, which have been associated with development and differentiation in a variety of tissues. Each of these three lysosomal proteases was expressed in a specific fashion during hemoglobin induction in K562 cells. Both cathepsin B and cathepsin D activities could be induced by growth of K562 cells in medium containing either hemin or heat-treated serum or by increasing the concentrations of untreated serum in the medium. Cathepsin H activity in the same cells remained unchanged. This is the first report of inducible protease activities in K562 cells. Our identification of specific well-characterized protease activities that change differentially during K562 induction provides a framework for additional studies on the role of proteases in hematopoietic differentiation.

Droplets produced by medical nebulizers. Some factors affecting their size and solute concentration.

The effect of nebulizer solution temperature and dilution air humidity on the size and solute concentration of aqueous aerosol droplets were studied. Four combinations of jet-nebulizers with air compressors or oxygen sources and one ultrasonic nebulizer were tested. The temperature to which the nebulizer solution of each system fell during generation was measured. The nebulizers were then kept at set temperatures, generated aerosols collected and either droplet size or solute concentration measured. The droplet solute concentration was found to increase. The droplet size decreased along with the droplet solute concentration increase. The ultrasonic nebulizer also was tested: its high output made the concentration of the solution in the droplets much more stable. However, the proportion of droplets depositing in the tubing and valves changed markedly with aerosol flow rate. The potential for large changes in droplet solute concentration, droplet size and output during nebulization should be considered in therapeutic and diagnostic applications of nebulized aerosols.

Sensitivity and specificity of enzyme immunofiltration and DNA hybridization for the detection of HCMV-infected cells.

The sensitivity and specificity of enzyme immunofiltration and DNA hybridization were compared in human cytomegalovirus (HCMV) (AD 169)-infected MRC-5 cells. The enzyme immunofiltration was carried out on glass fiber filters in microplates, using an HCMV (AD 169) monoclonal antibody and a peroxidase conjugate. The DNA hybridization was carried out with a microfiltration apparatus, using a 32P-labelled HCMV (AD 169) Eco R1 D fragment probe. The sensitivities of enzyme immunofiltration and DNA hybridization were 1.82 X 10(3) and 1.13 X 10(3) infected cells, respectively. Both methods were highly specific, but enzyme immunofiltration was faster and simpler.

Rapid detection of influenza virus infections in human fetal lung diploid cell cultures.

Haemadsorbing foci were found in human fetal lung (HFL) diploid cell cultures 12 h after inoculation with influenza viruses A and B. The size and number of the foci were maximal after 48 h of incubation, being limited by production of an unidentified inhibitor. By contrast, inoculation with parainfluenza virus type 3 led to haemadsorption which increased during 10 days of incubation. For the detection of influenza viruses A and B maximum sensitivity was achieved by changing the medium, the day before use to one that was serum free. The number of foci at 15.5 h post-infection and infectivity for primary African green monkey kidney (AGMK) cultures were similar. Virus infectivity and production of haemagglutinin in HFL cells were entirely cell-associated; they were not affected by treatment with trypsin. Nevertheless, influenza viruses A and B antigens were identified in the infected cells by means of immunofluorescence at 15.5 h and virus was recovered by passage of frozen and thawed cells in AGMK cultures. For rapid routine diagnosis of viral infections, the early haemadsorption test was shown to have the same sensitivity as immunofluorescence tests on specimens and virus detection by the shell-vial technique but was cheaper and simpler to perform.

Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis.

The production of monoclonal antibodies to the bloodstages of the haemoprotozoan parasites Babesia caballi and Babesia equi and the characterization of their corresponding antigens are described. Species specific and immunogenic proteins of both parasites were identified using SDS-PAGE, Western blotting and ELISA. These proteins were then electroeluted from SDS-PAGE gels and used to immunize BALB/c mice for hybridoma production. One monoclonal antibody (Mab), designated BC5.37.70.27 (BC5), recognized a 70 kDa protein of B. caballi as demonstrated by Western blotting under reducing conditions. Another Mab, BE1.24/2.95 (BEI), recognized a 34 kDa protein of B. equi. Both Mabs reacted specifically in indirect ELISA when isolated whole merozoites were used as antigen. Preliminary studies using the two Mabs in a competitive ELISA (cELISA) suggest that the cELISA for the detection of B. caballi infection is more sensitive than the commonly used complement fixation test but that refinement is necessary for the B. equi system.

Evaporation of aqueous aerosols produced by jet nebulizers: effects on particle size and concentration of solution in the droplets.

Aqueous droplets produced by jet nebulizers can lose water by evaporation prior to entry into the patient's mouth, or a sizing device. Evaporation causes increase in the concentration of the solution in the droplet and reduction in size. These changes can complicate interpretation of results from experiments such as in vitro particle sizing or human respiratory tract deposition. We present experimental data obtained by nebulization of isotonic saline using a variety of aerosol delivery systems employing jet nebulizers. The impact of the evaporation phenomena is particularly great when a large volume of dry dilution air is mixed with the aerosol stream--a situation that is quite common in aerosol experiments. The experimental results approach the values calculated from the theoretical mass balance models in the limit of equilibrium between the droplets and the surrounding atmosphere.

Prenatal glucose screening.

Of 3,563 consecutive obstetric patients undergoing glucose screening, 517 (14.5%) were found to have plasma values of greater than or equal to 140 mg%, and 74 (14.3%) of 517 were found to be diabetic on standard oral glucose tolerance testing, for an overall incidence of 2.1%. There was no absolute value on the glucose screen that predicted an abnormal oral glucose tolerance test. Twenty-eight of the 74 diabetics demonstrated none of the classic risk factors for glucose intolerance during pregnancy. Only when combining those patients over 30 years of age and with a mean body weight greater than 120% of the ideal body weight was statistical significance reached when that group was compared to the overall group. Thus, our data support the recommendation for universal prenatal glucose screening.

Collar Rot of Peanut Caused by Lasiodiplodia theobromae.

In August and September of 1993, a collar rot disease of peanut was observed in several fields in Virginia and North Carolina. Only a few scattered plants exhibited symptoms and signs of the disease in Southampton County and Suffolk, Virginia, and Northampton County, North Carolina. The disease was severe at two farm sites in Dinwiddie County, Virginia where the affected areas exceeded 0.4 ha in size. Numerous plants were either chlorotic, wilted, or dead. Symptomatic plants exhibited blackened stem cankers and pods. Diseased stems and tap roots were easily shredded to reveal slate-gray to black internal tissues. Black, erumpent pycnidia were observed on stem lesions at the soil surface. Immature conidia were single-celled and hyaline. Mature conidia were two-celled and dark brown. Morphological features of the fungus on diseased plants and potato dextrose agar were consistent with descriptions of Lasiodiplodia theobromae (Diplodia gossypina). The fungus was isolated from discolored seed and asymptomatic seed from fields having plants which exhibited severe symptoms. Seed treatment with captan 1.125 g + pentachloronitrobenzene 0.375 g + carboxin 0.25 g a.i./kg reduced recovery of the fungus from seed, but did not eradicate the pathogen. This treatment on naturally infested seed provided significant early-season disease suppression and improved yield significantly in 1994. Season-long disease suppression and a significant yield increase were obtained in plots planted to fungicide-treated, commercial seed from non-infested fields. At-plant and mid-season applications of fungicides in 1994 and 1995 did not improve disease suppression over that of fungicide-treated, commercial seed. Overall, Virginia-type cultivars of peanut were more susceptible to collar rot than runner-type cultivars. Among the Virginia-type cultivars, NC-V 11 exhibited moderate susceptibility and the 79-X breeding line from Florida exhibited resistance. Georgia Browne and Southern Runner were the most resistant of the runner-type cultivars.

First Report of the Reniform Nematode Rotylenchulus reniformis on Cotton in Virginia.

In Virginia during September 2002, the reniform nematode, Rotylenchulus reniformis Linford and Oliveira (1), was found for the first time following a grower's concern about poor growth and yield of cotton (Gossypium hirsutum L.) cv. Fiber Max 989BR. The infested field was planted with cotton each year for the last eight growing seasons. The field was located on Hall Road in Southampton County, Virginia at coordinates 77°16'28.8926″W, 36°37'10.6428″N near the town of Branchville. The soil was loamy sand, which is typical of sandy textured soils in the region. Rainfall from May to September at a nearby weather station was nearly 50% below normal, which may have contributed to the suppression of plant growth. The vermiform nematodes were extracted with a North Carolina State University model semiautomatic elutriator and centrifugation/sugar flotation. Populations were 30 to 150 per 500 cm3 of soil in areas with noticeable stunting. Cultures were established on cotton cv. Delta Pine 64 and tomato (Lycopersicon esculentum Mill. cv. Rutgers) and were maintained in a greenhouse. Reproduction was moderate on cotton and high on tomato. Identifications were based on morphology and measurements of vermiform females and males: immature female length (L) = 407 ± 22 (376 to 418) µm, stylet L = 18.5+1.7 (17.0 to 21.3) µm; and male L = 351 ± 17 (339 to 367) µm. Voucher specimens were placed and are maintained in the Virginia Tech Nematode Collection. Reference: (1) M. B. Linford and J. M. Oliveira. Proc. Helminthol. Soc. Wash. 7:35, 1940.

An Algorithm for Predicting Outbreaks of Sclerotinia Blight of Peanut and Improving the Timing of Fungicide Sprays.

Algorithms were evaluated for computing disease risk and improving the timing of fungicide applications for control of Sclerotinia blight of peanut. Disease risk was calculated by multiplying indices of moisture, soil temperature, vine growth, and canopy density each day, and summing values for the previous 5 days to obtain a 5-day risk index (FDI). After fungicide application, the FDI was reset to zero for 3 weeks. Fluazinam at 0.58 kg a.i./ha applied at FDI 24 or 32 in 1994 and 1995 suppressed disease and increased yield as much as or more than programs of weekly scouting and applying fungicide at the initial onset of disease with additional sprays at 3- to 4-week intervals. The FDI algorithm was also more efficient than calendar sprays at 60, 90, and 120 days after planting (DAP). Environmental and host parameters were expanded in 1996 and 1997 by adding new temperature and new vine growth indices. These parameters along with DAP-dependent thresholds consistently improved the timing of fungicide sprays and disease management when using the FDI algorithm in comparison to weekly scouting or calendar sprays at 60, 90, and 120 DAP.

Incidence and Survival of Cylindrocladium parasiticum in Peanut Seed.

Sixty-three commercial seed lots of peanut produced in Virginia were examined for the presence of seed with speckled testae. Speckled seed were present in seed lots from the 1998, 1999, and 2000 growing seasons at average rates of 3, 1.2, and 0.6%, respectively. Speckled and normal seed from 19 seed lots were assayed on a medium selective for C. parasiticum. The fungus was isolated from speckled seed at rates ranging from 40 to 96%. C. parasiticum was isolated only from a single normal seed from one seed lot. The pathogen was recovered at high rates from speckled seed immediately after pods had been dried in commercial drying trailers at temperatures up to 35°C. Ambient temperatures during winter seed storage that fluctuated from -10 to 28°C in 1999 and -8 to 33°C in 2000 greatly reduced pathogen recovery in speckled seed stored for 16 or 24 weeks. In field plots with naturally infested soil, the number of speckled seed harvested was directly correlated to the number of symptomatic plants in plots on 29 September. Based on this finding, the harvest of seed peanuts in areas of a field with high incidence of Cylindrocladium black rot (CBR) should be avoided. Adoption of this policy is expected to lower the number of speckled seed entering commercial seed lots and reduce the risk for spread of CBR.

Stem Canker on Cotton Caused by Phoma exigua in North Carolina and Virginia.

Numerous reports about a disease of unknown etiology on cotton, Gossypium hirsutum L., in northeastern North Carolina and southeastern Virginia were received on 18 June 1999 following several days of cool weather with persistent mist and fog during the week of 14-19 June. Several fields were visited by consultants and county extension staff the following week. In some instances, the cotton stem was girdled, causing the upper portion of the plant to wilt and die. Cotton plants exhibiting various symptoms, including death, wilting, streaking of the vascular system, black sunken lesions on stems, and terminal necrosis were collected for examination and isolation. Pycnidia and spores of the fungus Phoma exigua were abundant in stem and terminal tissues. The fungus was isolated from infected stem tissue and cultured on PDA. A suspension containing 2.5 × 108 spores of P. exigua was sprayed on cotton leaves or injected into the stems to confirm pathogenicity. Controls were sprayed or injected with distilled water. Plants were placed in 100% humidity for 72 h and maintained in the greenhouse thereafter. The experiment was replicated five times and repeated once. Typically, streaking of the vascular system extended 1 to 5 cm from the point of stem inoculation. Inoculated cotton leaves had lesions resembling those attributed to Ascochyta gossypii. Reisolation of the fungus P. exigua from inoculated tissue on potato-dextrose agar (PDA) was successful in all treatments. Crossan (2) considered many isolates of Ascochyta taken from various hosts in North Carolina, including A. gossypii, to be synonymous with Ascochyta phaseolorum. A. phaselorum was subsequently synonomyzed with P. exigua (1). Ascochyta blight (also called ashen spot, or wet weather blight [4]) is usually a minor leaf spot caused by P. exigua (syn. Ascochyta gossypi) and is common in North Carolina. Stem canker caused by P. exigua has not been reported previously in North Carolina (3) or Virginia. The sunken canker at a node is the best diagnostic symptom for cotton stem canker. Dark streaks in vascular tissue extend below and above the canker but do not usually extend to the root system, as with wilt diseases. The disease was widespread and found in most fields north of I-40 in North Carolina into Virginia and east of I-95. Crop consultants and county extension staff estimated disease incidence in individual fields from less than 1 to over 90% in North Carolina and 6 to 25% in Virginia. Disease incidence did not appear to be affected by cotton cultivar, tillage, or crop rotation. This pathogen was also responsible for brittle cotton stems late in the season, resulting in boll loss. Proper identification of the causal organism is essential in formulating management strategies, since P. exigua has an extensive host range and rotation is unlikely to aid in management of this disease in the future. References: (1) G. H. Boerema. Ascochyta phaseolorum synonymous with Phoma exigua. Neth. J. Plant Pathol. 78:113-115, 1972. (2) D. F. Crossan. The relationships of seven species of Ascochyta occurring in North Carolina. Phytopathology 48:248-255, 1958. (3) L. F. Grand, ed. North Carolina Plant Disease Index. Tech. Bul. 240, 1985. (4) G. M. Watkins. Leaf spots. Pages 28-30 in: Compendium of Cotton Diseases, 1st ed. American Phytopathological Society, St. Paul, MN, 1981.

Genetic markers in human bone: I. Deoxyribonucleic acid (DNA) analysis.

Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a "per milligram of starting tissue" basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different single-locus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQ alpha phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3' to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.

Effects of Continuous Cropping of Resistant and Susceptible Cultivars on Reproduction Potentials of Heterodera glycines and Globodera tabacum solanacearum.

The reproductive potentials of Heterodera glycines (mixture of races 3 and 4 and unidentified races) and a tobacco cyst nematode Globodera tabacum solanacearum were studied in the field. The experiments involved four cultivars of soybean Glycine max and four cultivars of Nicotiana tabacum. The reproductive potential of the H. glycines population was high on Essex and Lee 74 soybean but low on Forrest and Bedford over the 3 years (1982-84) of continuous cropping. The reproductive potential of H. glycines was 12% on Forrest and 6% on Bedford in 1982 but increased to 37 and 35% in 1983 and to 71 and 41% in 1984, respectively, on these two cultivars. The reproductive potential of G. tabacum solanacearum was high on McNair 944 and Coker 319 tobacco cultivars and low on VA 81 and PD 4 over the 3 years of cropping. The reproductive potential of G. tabacum solanacearum on VA 81 and PD 4 was 18 and 17% in 1982, 7 and 16% in 1983, and 5 and 5% in 1984, respectively. The changes in reproductive potentials of H. glycines and G. tabacum solanacearum may be related to inherent genetic variability in the systems that control reproduction of the two cyst nematodes and nature of resistance incorporated in the soybean and tobacco cultivars.