Development of a Quantitative Polymerase Chain Reaction Detection Protocol for Cercospora kikuchii in Soybean Leaves and Its Use for Documenting Latent Infection as Affected by Fungicide Applications.

Cercospora leaf blight (CLB) of soybean, caused by Cercospora kikuchii, is a serious disease in the southern United States. A sensitive TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify C. kikuchii in naturally infected soybean plants. The sensitivity was 1 pg of genomic DNA, which was equivalent to about 34 copies of genome of C. kikuchii. Using this qPCR assay, we documented a very long latent infection period for C. kikuchii in soybean leaves beginning at the V3 growth stage (as early as 22 days after planting). The levels of biomass of C. kikuchii remained low until R1, and a rapid increase was detected from the R2/R3 to R4/R5 growth stages shortly before the appearance of symptoms at R6. The efficacy of various fungicide regimens under field conditions also was evaluated over a 3-year period using this qPCR method. Our results showed that multiple fungicide applications beginning at R1 until late reproductive stages suppressed the development of C. kikuchii in leaves and delayed symptom expression. Different fungicide chemistries also had differential effects on the amount of latent infection and symptom expression during late reproductive growth stages.

First Report of Impatiens Downy Mildew Caused by Plasmopara obducens in Kentucky.

Impatiens downy mildew (Plasmopara obducens (J. Schröt.) J. Schröt. (syn Peronospora obducens) was first reported in the United States in 2004, but widespread outbreaks were observed throughout North America in 2011 (5). In June 2012, symptoms, including severe defoliation while plants retained upright stems, were observed on approximately 100 landscape impatiens (Impatiens walleriana Hook.f.) in Franklin County in central Kentucky. All plants in the landscape were affected. Plants were primarily defoliated and remaining leaves were stunted, mottled, and chlorotic with edges curled downward; no flowers were present. Under examination with a dissecting microscope, white downy fungal growth was observed. Closer examination confirmed that the growth consisted of colorless sporangiophores that were mainly unbranched, straight, and rigid (1,3). Sporangiophores consisted of apical branches attached at right angles to main axes, ranging from 67.2 to 89.9 µm long (1). Sporangia were ovoid and hyaline, measuring 11.2 to 13.3 µm × 8.2 to 10.7 µm (3). No oospores were observed. Pathogenicity tests were performed by inoculating 20 to 40 leaves on three plants each of the cvs. Dazzler and Super Elfin with suspensions of 1 × 105 sporangiophores per ml in sterile distilled water. Sporangia were obtained by washing infected leaves with sterile distilled water, and inoculations were completed by spraying leaves until runoff. Plants sprayed with sterile water served as controls. Plants were covered with black plastic bags for 48 h and then maintained under fluorescent lights for 10 days at room temperature (22 to 25°C). Sporangiophores were recovered from inoculated plants after 10 days, and morphology matched original inoculum; symptoms included chlorotic, downward curling leaves with sporulation on the undersides. Non-inoculated plants did not develop symptoms after 21 days. Molecular identification of the pathogen was conducted using three leaves from one plant from each cultivar. PCR was conducted by amplifying the large ribosomal subunit DNA using primers NL-1 and NL-4 (2). Amplicons of 762 to 691 bp were produced from diseased plant tissue that contained visible sporangiophores, and the bands were extracted from the gel and purified. Sequence results confirmed 100% similarity to accessions from Florida (GenBank Accession No. JX217746.1) and Ohio (JX142134.1) and 99% similarity to amplicons reported from Serbia (HQ246451.1) and UK (AY587558.1). This is believed to be the first report of downy mildew infecting impatiens in Kentucky. References: (1) O. Constantinescu. Mycologia 83:473, 1991. (2) W. Maier et al. Can. J. Bot 81:12, 2003. (3) P. A. Saccardo. Syllogue Fungorum 7:242, 1888. (4) S. N. Wegulo et al. Plant Dis. 88:909, 2004.

Effects of Simplicillium lanosoniveum on Phakopsora pachyrhizi, the soybean rust pathogen, and its use as a biological control agent.

The fungus Simplicillium lanosoniveum was isolated from soybean leaves infected with Phakopsora pachyrhizi, the soybean rust pathogen, in Louisiana and Florida. The fungus did not grow or become established on leaf surfaces until uredinia erupted, but when soybean rust signs and symptoms were evident, S. lanosoniveum colonized leaves within 3 days and sporulated within 4 days. Development of new uredinia was suppressed by about fourfold when S. lanosoniveum colonized uredinia. In the presence of S. lanosoniveum, uredinia became increasingly red-brown, and urediniospores turned brown and germinated at very low rates. Assays using quantitative real time polymerase chain reaction revealed that the fungus colonized leaf surfaces when plants were infected with P. pachyrhizi, either in a latent stage of infection or when symptoms were present. However, when plants were inoculated before infection, there was no increase of DNA of S. lanosoniveum, suggesting that the pathogen must be present in order for the antagonist to become established on soybean leaf surfaces. We documented significantly lower amounts of DNA of P. pachyrhizi and lower disease severity when soybean leaves were colonized with S. lanosoniveum. These studies documented the mycophilic and disease-suppressive nature of S. lanosoniveum.

Paralytic poliomyelitis: seasoned strategies, disappearing disease.

With more than 2 years having elapsed since the last case of paralytic poliomyelitis occurred in the Western Hemisphere, significant progress has been made towards the global eradication of wild polioviruses. Poliomyelitis is disappearing from Europe, North Africa, Southern Africa, the Middle East, China, and the Pacific. Reported poliomyelitis cases declined to 15,587 cases in 1992. Current eradication strategies recommended by the World Health Organization include national mass campaigns administering oral poliovaccine to all children under 5 years of age, enhanced surveillance to detect cases of acute flaccid paralysis, creating a network of laboratories for viral diagnosis, and targeted immunisation to areas and populations where poliovirus transmission is likely to persist. The major obstacles to eradication include inadequate political support for eradication and insufficient funding, especially for the purchase of vaccine. With additional support for the international eradication effort, epidemics of poliomyelitis will cease in developing countries, and industrialised countries will be able to save the large sums spent each year on poliovaccine and rehabilitation.

The WHO-EPI initiative for the global eradication of poliomyelitis.

Since the development of attenuated oral polio vaccine, Dr Albert Sabin consistently maintained that the global eradication of wild poliovirus was possible, but that to achieve polio eradication in developing countries would require the mass administration of the oral vaccine. Experience in Cuba and Czechoslovakia proved the effectiveness of this technique, but it was only with its deployment in Brazil in 1980 that its role in eradicating the virus from a broad geographical area started to be realized. With the declaration in 1985 of a target of regional polio eradication, extension of this policy, allied with the development of effective surveillance of acute flaccid paralysis in children, with laboratory confirmation of diagnosis rapidly led to apparent interruption of wild poliovirus transmission throughout the Americas. The World Health Assembly in 1988 committed WHO to the global eradication of poliomyelitis. Based on experience in the Americas and building on the solid foundation established by the Expanded Programme on Immunization, WHO has defined the strategies through which the global target could be achieved. Progress is encouraging and where the advocated strategies have been fully implemented, the incidence of poliomyelitis has declined dramatically. Significant geographical areas in Western Europe, the Maghreb, the Arabian peninsula, the Pacific basin and Southern Africa, each incorporating several countries, are now thought to be free of the disease caused by wild poliovirus. The target of a world free of polio by the year 2000 can be achieved.

Fundamental studies in reversed-phase liquid-solid extraction of basic drugs; I: Ionic interactions.

Seven basic solutes with known and controlled pKa (7.93-9.5) and log P (0.23-6.63) values have been used as test probes to study the mechanism involved in liquid-solid extraction with C2 and C18 bonded silica phases. A limited comparison has also been made with underivatized silica and CN phases. In addition to the reversed-phase mechanism, cation-exchange was shown to play a very significant role in the retention process. Various cations both organic and inorganic were assessed for their elution strength, and the ordering was similar to that for classical ion-exchange chromatography. Control of selectivity in the elution process can be achieved by varying the concentration of cation or methanol in the eluent. The C2 cartridge in combination with an aqueous ammonium acetate-methanol eluent proved to be the most versatile in that all compounds, irrespective of pKa or log P could be recovered in high yield. The optimal eluent in terms of selectivity with respect to related compounds could be predicted from the solute log P. Blocking of silanols by pre-conditioning the cartridges with cations prior to sample applications was also studied. The order of cation strengths although somewhat variable was similar to that established at the elution stage. To achieve quantitative elution with methanol or aqueous methanol solutions however, high concentrations of inorganic cations, equivalent to 1 ml of a 1 M solution were required to pre-condition a 100 mg cartridge.

Progress towards the global eradication of poliomyelitis.

Poliomyelitis has been virtually eliminated from the industrialized countries by mass campaigns conducted with oral polio vaccine (OPV). In 1988, the World Health Assembly set the goal of global eradication of poliomyelitis by the year 2000. The current WHO strategy for eradication uses three primary activities beyond routine immunization with OPV. They are: (i) improved disease surveillance, (ii) building a global network of laboratories, and (iii) supplemental immunization strategies which include mass immunization campaigns with OPV at the national level, and targeted campaigns at the local level. Eradication of polio from the Region of the Americas is close and may have already been achieved. In other regions, the number of reported polio cases has declined, largely as a result of high immunization coverage. As more countries implement polio eradication strategies, the number of polio cases will continue to fall until eradication is achieved.

PIP: The current status of poliomyelitis in the world is outlined. In May 1988, the World Health Assembly committed the World Health Organization (WHO) to the global eradication of poliomyelitis by the year 2000. Polio eradication cannot be achieved through routine immunization alone using the primary strategy of expanded program of immunization (EPI). Additional strategies recommended by WHO are: 1) Improving the surveillance systems to detect all cases of paralysis which might be polio. 2) Building a global network of laboratories to reliably identify polio virus from specimens. 3) Conducting national vaccination days, outbreak response immunization, targeted mass vaccination campaigns in high-risk areas. An increasing number of countries are adopting these strategies. In 1990, 16,435 cases of polio were reported globally, a 39% decrease from 1989 when 26,916 cases were reported. For 1990, 171 (88%) of 184 countries had reported polio case counts to WHO on October 1 of the following year, compared to 166 (85%) for 1989. Improvements in both timeliness and completeness of reporting at the national level have occurred as more countries improve surveillance for polio as well as for other EPI target diseases. There has been a dramatic decrease in the number of reported polio cases and the eradication of wild polio viruses is imminent in the Americas. WHO estimates that in 1990, approximately 150,000 cases of paralytic poliomyelitis occurred in the world. Although improved immunization coverage with 3 doses of oral polio vaccine given by routine administration can reduce the number of polio cases, polio eradication will require supplemental immunization activities.

Practicalities of a global poliomyelitis control program.

Much progress has been achieved in making the world free of poliomyelitis. The countries that have achieved control should be giving practical help to those where the disease is still endemic, while developing their own competence in surveillance, laboratory support, and effective immunization policies. The polio vaccines have been proved effective. The need is now to implement programs by solving their practical difficulties in order to make use of the presently available vaccines.

Smallpox surveillance in Bangladesh: I - Development of surveillance containment strategy.

A major epidemic of Variola major with an estimated 90 000 cases and 18 000 deaths occurred in Bangladesh in 1972. During a 3-year period methods of active surveillance (market search, outbreak investigation, and house-to-house search) were developed to supplement the passive reporting system. The percentage of smallpox cases detected increased from 11.8% in 1972 to 83% in 1975. Together with the implementation of improved methods of outbreak control, smallpox transmission was interrupted in October 1975.

Treatment of variola major with adenine arabinoside.

A double-blind study of the efficacy of adenine arabinoside in the treatment of patients with variola major was conducted. Adenine arabinoside (20 mg/kg of body weight) was given to patients intravenously every 24 hr for seven days in a 8-hr infusion. Five of the nine patients receiving adinine arabinoside died, and four of 11 patients receiving placebo died. Mortality was related to the severity of illness for both groups of patients. No difference was found between the drug and control groups in number of febrile days after initiation of therapy or in the period during which it was possible to isolate virus from skin lisions, throat swabs, and sources of clotted blood. Formation of scabs on skin lesions was complete 8.3 days after the initiation of therapy for the drug group and after 11.3 days for the control group. The findings suggest that adenine arabinoside is not effective in the chemotherapy of smallpox.