bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimized Quantification of Unculturable Candidatus Liberibacter Spp. in Host Plants Using Real-Time PCR
Authors: Li Wenbin , Li Dayan , Twieg Elizabeth , Hartung John S. , Levy Laurene
Published: May 2008

Citrus huanglongbing (HLB) is caused by the phloem-limited and psyllid-vectored Candidatus Liberibacter spp. and is a destructive disease of citrus that is rapidly increasing in importance. The disease was reported recently in the principle citrus-producing areas of São Paulo, Brazil in 2004 and in Florida in 2005. A variety of laboratory methods have been developed to confirm a symptom-based disease diagnosis or for the detection or identification of the pathogen; however, no quantitative information has been available on the pathogen titer in either host or vector interactions because the pathogen remains unculturable in artificial media. We previously developed a quantitative polymerase chain reaction (PCR)-based assay for detection of Ca. Liberibacter spp. and, in this study, we evaluated the effects of sample composition on quantification of the pathogen in citrus plants by TaqMan real-time PCR. Standard curves were established using cloned plasmids containing target DNA from the pathogen and with total DNA samples from field-grown HLB-infected citrus plants. Regression analysis showed that a standard curve established with DNA extracted from naturally infected field-grown plants was more accurate than the standard curve constructed from plasmids containing the amplification targets as cloned inserts. Nontarget DNA and putative PCR inhibitors from citrus plants decreased the sensitivity and the amplification efficiency of real-time PCR when plasmids provided the template target in “spiked” healthy citrus DNA extracts. This effect varied among plant tissue types, citrus species, and geographic locations. Based on these sample effects, a universal standard curve has been established for quantification of the pathogen in various citrus tissues of different citrus species planted in different geographic locations. Sample storage at 4°C for 2 months prior to PCR assay did not affect subsequent quantification of the pathogen. The validated quantitative real-time PCR method and the universal standard curve will be very useful for studies of host–pathogen interactions and epidemiology, and in the development of control strategies for the disease.
Development of a SCAR Marker to Track Canola Resistance Against Blackleg Caused by Leptosphaeria maculans Pathogenicity Group 3
Authors: Dusabenyagasani M. , Fernando W. G. D.
Published: May 2008

Blackleg of rapeseed and canola (Brassica napus) is caused by various pathogenicity groups (PG) of Leptosphaeria maculans. The disease occurring in the Canadian prairies for the last two decades was caused by PG2 and was controlled by host resistance. PG3 and PG4 isolates have been found recently in Canada, but there is no resistance available against these pathogenicity groups in commercial Canadian varieties. This study sought to identify canola cultivars that could be used as sources of resistance to PG3 and to develop molecular markers for marker-assisted selection. Resistance to PG3 specifically was found in B. napus ‘Dunkeld’ and ‘Quinta’, while B. juncea ‘Cutlass’ and ‘Domo’ proved to be resistant to PG2, PG3, and PG4. A set of F2 progeny of ‘Westar’ (susceptible) × ‘Dunkeld’ was used to identify genetic markers linked to PG3 resistance. These markers were physically located on a BAC clone from B. rapa subsp. pekinensis containing a homolog to a serine threonine 20 (ste20)-like kinase in Arabidopsis thaliana. Thus, we have developed a sequence characterized amplified region (SCAR) marker available for marker-assisted selection in breeding canola for resistance against blackleg caused by L. maculans PG3. This work has received a provisional patent (serial # 60/977,933 – Oct. 5, 2007).
Stem and Crown Rot of Chickpea in California Caused by Sclerotinia trifoliorum
Authors: Njambere Evans N. , Chen Weidong , Frate Carol , Wu Bo-Ming , Temple Steve R. , Muehlbauer Fred J.
Published: May 2008

The identities of Sclerotinia isolates obtained from chickpea plants showing stem and crown rot were determined using morphological characteristics, variations in group I introns, and internal transcribed spacer (ITS) sequences. Isolates could be separated into two groups based on growth rates at 22°C, fast growing (about 40 mm per day) versus slow growing (about 20 mm per day). All fast-growing isolates induced stronger color change of a pH-indicating medium than did slow-growing isolates at 22°C. The slow-growing isolates contained at least one group I intron in the nuclear small subunit rDNA, whereas all fast-growing isolates lacked group I introns in the same DNA region. ITS sequences of the slow-growing isolates were identical to sequences of Sclerotinia trifoliorum. Those of the fast-growing isolates were identical to sequences of S. sclerotiorum. Finally, the slow-growing isolates showed ascospore dimorphism, a definitive character of S. trifoliorum, whereas the fast-growing isolates showed no ascospore dimorphism. Isolates of both species were pathogenic on chickpea and caused symptoms similar to those observed in the field. This study not only associated the differences between S. sclerotiorum and S. trifoliorum in growth rates, group I introns, ITS sequences, and ascospore morphology, but also represented the first report that S. trifoliorum causes stem and crown rot of chickpea in North America.
Distribution and Incidence of Sphaeropsis Rot in Apple in Washington State
Authors: Kim Y. K. , Xiao C. L.
Published: May 2008

Sphaeropsis rot, caused by Sphaeropsis pyriputrescens, is a recently recognized postharvest disease of apple in Washington State. To determine the distribution and incidence of this disease as well as other postharvest diseases, decayed fruit were sampled during packing or pre-sizing operations in commercial fruit packinghouses from 26, 72, and 81 grower lots in 2003, 2004, and 2005, respectively. Fungi associated with decayed fruit were isolated and identified. The most common postharvest diseases of apple in the region were blue mold caused by Penicillium spp., primarily P. expansum, gray mold caused by Botrytis cinerea, and Sphaeropsis rot, accounting for 32, 28, and 17% of the decayed fruit, respectively. Percentages of these diseases in the total decayed fruit varied from lot to lot. Bull's eye rot caused by Neofabraea spp. was responsible for 13.4% of the total decay and was most prevalent on Golden Delicious. Other minor diseases included speck rot caused by Phacidiopycnis washingtonensis, Alternaria rot caused by Alternaria spp., Mucor rot caused by Mucor piriformis, and core rot caused by a group of fungi, primarily Alternaria spp. Sphaeropsis stem-end rot was more common than calyx-end rot on Golden Delicious, whereas Sphaeropsis calyx-end rot was more common than stem-end rot on Fuji. On Red Delicious, both stem-end rot and calyx-end rot were common. Sphaeropsis rot resulting from infections through the fruit peel was more commonly seen on Golden Delicious and Fuji than on Red Delicious. The percentage of gray mold was higher on nondrenched fruit than on fruit drenched with thiabendazole (TBZ), whereas blue mold was more prevalent on TBZ-drenched fruit. Our results indicate that Sphaeropsis rot is an important component of storage rots of apples in Washington State.
A Macroarray System for the Detection of Fungal and Oomycete Pathogens of Solanaceous Crops
Authors: Zhang Ning , McCarthy Meaghan L. , Smart Christine D.
Published: May 2008

There are numerous fungal and oomycete pathogens that cause severe damage to solanaceous crops. Rapid and accurate detection and identification of these pathogens is critical for plant disease management. Recently, DNA array technology has been successfully applied for simultaneous detection of multiple microorganisms from various habitats. The goal of this project was to develop a multiplex detection and identification system for major fungal and oomycete pathogens of solanaceous crops. To facilitate this goal, we used a membrane-based macroarray technology that included at least two specific oligonucleotides per pathogen. Based on the internal transcribed spacer sequences of the rRNA genes, 105 oligonucleotides (17 to 27 bases long) specific for 25 pathogens of solanaceous crops were designed and spotted on a nylon membrane. The array was tested against the 25 target pathogen species, 46 infected field samples, and a number of nontarget species. Our results indicate that the oligonucleotide-based macroarray detection system is a reliable and effective method for pathogen detection and identification even when multiple pathogens are present in a field sample.
First Report of Rubus yellow net virus on Rubus idaeus in Alberta, Canada
Authors: Kalischuk M. L. , Kawchuk L. M. , Leggett F.
Published: May 2008

In North America, Rubus yellow net virus (RYNV), a member of the genus Badnavirus, family Caulimoviridae, is vectored in a semipersistent manner by the large raspberry aphid (Amphorophora agathonica Hottes) and is responsible for producing net-like chlorosis of tissue along the leaf veins (2). Red raspberry (Rubus idaeus L.) is commonly grown in Canada and after the dry, hot summer of 2006 in southern Alberta, a group of garden raspberry plants located near Lethbridge, Alberta exhibited interveinal chlorosis resembling viral symptoms. Nonenveloped, 140 × 30 nm, bacilliform particles typical of badnaviruses were observed in infected leaves by transmission electron microscopy. The presence of RYNV was confirmed by immunocapture of virions from extracts of symptomatic leaves using Sugarcane bacilliform virus polycolonal antiserum (Agdia Incorporated, Elkhart, IN) followed by PCR amplification of a 451-bp fragment with RYNV-specific primers based on the highly conserved region of the reverse transcriptase and ribonuclease H genes (5′-ATCCTCAAAGGGTTACGTAGCTGGTT-3′ and 5′-TTCAAGCCACCTTACCCTCGAAGGTTT-3′). Sequence of clones from the PCR product (GenBank Accession No. EU327346) showed 87% identity to a previously sequenced isolate of RYNV (GenBank Accession No. AF468454) (1). To our knowledge, this is the first report of RYNV in Alberta, Canada, and as an important component of raspberry veinbanding disease, it poses a new threat to the Alberta raspberry industry.

References: (1) A. T. Jones et al. Ann. Appl. Biol. 141:1, 2002. (2) R. Stace-Smith. Can. J. Bot. 33:269, 1955.
First Report of Diplodia seriata Causing Shoot Blight and Cankers of Cotoneaster salicifolius in Bulgaria
Authors: Bobev S. G. , Lopes J. , Phillips A. J. L.
Published: May 2008

Shoot and branch blight was observed on shrubs of Cotoneaster salicifolius in the region of Plovdiv, Bulgaria in autumn and winter surveys during 2005 and 2006. Scattered pycnida were found on the withered branches, which bore sunken cankers with marginal bark cracks. Fungal colonies obtained from affected host tissue developed on potato dextrose agar (PDA) at 24 to 25°C in the dark. Colonies grew rapidly, were floccose, initially white, becoming grayish brown, and finally went from gray to black. Dark brown, unilocular pycnida formed after 2 to 3 weeks in culture. Conidia that formed at the tips of cylindrical, percurrently, proliferating, conidiogenous cells were ovoid, straight, pale brown to dark brown, internally verruculose, aseptate or occasionally with a single median septum. The mean conidial dimensions were 20 × 12 μm, the extreme range was 15 to 25 × 11 to 15 μm. These characters correspond to Diplodia seriata De Not., which is the anamorph of “Botryosphaeria” obtusa (2). Identity was confirmed from the nucleotide sequences of the internal transcribed spacer (ITS) from the rRNA repeat as described elsewhere (1). BLAST searches in GenBank showed 100% identity of isolates with reference sequences of D. seriata including that from the ex-epitype culture. The ITS sequence of a representative isolate (CAP337) has been deposited in GenBank (Accession No. EU483658). Pathogenicity was tested by inserting 3-mm-diameter mycelia plugs from 7-day-old PDA cultures into scalpel-wounded mature apple fruits (cv. Granny Smith) and wounded shoots of C. salicifolius. Sterile PDA plugs were placed into similarly made wounds of control specimens. There were three replicates for each treatment and the inoculated wounds were subsequently covered with Parafilm. Small necrotic lesions occurred on the apple fruits 3 to 5 days after inoculation and after 10 to 12 days on the shoots. Within 4 to 5 weeks, all apple fruits were entirely rotten, shrunken, and covered with pycnida, whereas the shoots withered and typical blight symptoms developed above the inoculation sites. The pathogen was reisolated from all inoculated samples but not from any control treatment. To our knowledge, this is the first report of D. seriata on C. salicifolius in Bulgaria. It is likely that this pathogen will be found increasingly more frequently because of the intensive introduction of decorative Rosaceae species into urban districts.

References: (1) A. Alves et al. Mycologia 96:598, 2004. (2) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.
First Report of Bacterial Soft Rot on Vanda Orchids Caused by Dickeya chrysanthemi (Erwinia chrysanthemi) in the United States
Authors: Cating R. A. , Hong J. C. , Palmateer A. J. , Stiles C. M. , Dickstein E. R.
Published: May 2008

Vanda orchids are epiphytes grown for their attractive flowers by commercial producers and hobbyists throughout Florida. In August 2007, five Vanda hybrids, with an economic value of $150 each, were found at a nursery in central Florida with leaves that were macerated, brown, and water soaked. According to the growers, the plants were normal the previous day but symptoms developed rapidly. The plants were immediately removed from the greenhouse to prevent potential disease spread. Bacteria were isolated according to the method of Schaad et al. (1). Isolated bacteria grew at 37°C, were gram negative, degraded pectate, and produced phosphatase. MIDI (Sherlock version TSBA 4.10; Microbial Identification 16 System, Newark, DE) (SIM 0.906) identified the bacteria as Erwinia chrysanthemi (Dickeya chrysanthemi Burkholder et al. 1953) Samson et al. 2005. PCR was performed on the 16S rRNA gene (GenBank Accession No. EU526397) with primers 27f (5′-GAGAGTTTGATCCTG GCTCAG-3′) and 1495r (5′-TACGGCTACCTTGTTACGA-3′) (2). Subsequent DNA sequencing and GenBank search showed the isolated strain is 99% identical to that of Dickeya chrysanthemi. Four leaves each of six Vanda hybrids were inoculated by injecting approximately 150 μl of a bacteria suspension at 1 × 108 CFU/ml into each leaf. One plant was inoculated with water in each of four leaves. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 29°C day and 17°C night temperatures. Within 24 h, soft rot symptoms appeared on inoculated leaves. The water control appeared normal. D. chrysanthemi was reisolated and identified with the above method, thus Koch's postulates were fulfilled. To our knowledge, this is the first report of a soft rot caused by D. chrysanthemi on Vanda hybrids. Because of the popularity and high value of Vanda orchids, proper identification of this rapidly progressing bacterial disease is of great importance for the commercial producer and homeowner alike.

References: (1) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society. St. Paul, MN, 2001. (2) W. G. Weisburg. J. Bacteriol. 173:697, 1991.
Blight Caused by Sclerotium rolfsii on Potted Ornamental Citrus in Sicily
Authors: Pane A. , Lio M. Magnano di San , Raudino F. , Cacciola S. O.
Published: May 2008

Approximately 140,000 container-grown ornamental citrus plants are produced each year in the province of Catania (eastern Sicily). In the spring of 2006, a severe blight was observed in a commercial nursery in Catania on 2-month-old rooted cuttings of lemon (Citrus limon (L.) Burm.) and calamondin (× Citrofortunella mitis (Blanco) J. W. Ingram & H. E. Moore). Approximately 80% of the nursery stock of 2,000 cuttings was affected. Cuttings were grown in 7.5-cm2 pots made with compressed peat and wood pulp at 28 to 30°C with 95 to 100% relative humidity on benches in a greenhouse, The pot mix was composed of peat, perlite, and soil (2:1:2). Cuttings showed a dark brown necrotic lesion at the base of the stem that extended upward, resulting in chlorosis and wilting of the leaves. An invasive, white, cottony mycelium with a fan-like pattern and numerous, small, brown spherical sclerotia (0.5 to 4.0 mm in diameter) developed on infected tissues, in the potting mix as well as on the pot wall. Herbaceous cuttings collapsed within 2 weeks while woody cuttings gradually died. Symptomatic basal stem sections were disinfected for 1 min in 1% NaOCl, rinsed in sterile water, and plated on acidified (pH 4.5) potato dextrose agar (PDA). Isolations consistently yielded a fungus whose morphological characters corresponded to Sclerotium rolfsii Sacc. On PDA, it produced a septate mycelium with clamp connections and numerous olive brown-to-clove brown sclerotia (1 to 3 mm in diameter). Pathogenicity of two S. rolfsii isolates (IMI 396204 and IMI 396205) from citrus was confirmed on 3-month-old lemon cuttings grown in 10-cm-diameter plastic pots filled with a sterilized mix of peat moss and vermiculite (3:1) (10 cuttings for each isolate). Each pot was inoculated with 15 sclerotia harvested from 6-week-old cultures on PDA and placed on the soil surface around the base of the cutting. Ten noninoculated cuttings served as the control. Cuttings were kept in a growth chamber at 28°C and relative humidity at >95%. All inoculated cuttings showed wilting, blight, and stem rot within 3 weeks after inoculation. White mycelium and sclerotia were produced on the stem base and soil surface. Noninoculated controls remained symptomless. S. rolfsii was reisolated from infected cuttings. The pathogenicity test was repeated once with calamondin cuttings and the results were similar. Blight caused by S. rolfsii is widespread in nurseries of ornamentals in Italy (1). However, to our knowledge, this is the first report of this disease on potted ornamental citrus. Probably high temperature and moisture during rooting were conducive to the disease.

References: (1) A. Garibaldi et al. Malattie Delle Piante Ornamentali. Calderini Edagricole, Bologna, Italy, 2000.
Pratylenchus neglectus, P. thornei, and Paratylenchus hamatus Nematodes Causing Yield Reduction to Dryland Peas and Lentils in Idaho
Authors: Riga E. , Porter L. D. , Mojtahedi H. , Erickson D.
Published: May 2008

In June 2006, stunted and chlorotic plants were observed in large patches in two 40.5-ha fields of dryland peas (Pisum sativum) in Latah County, Idaho, which resulted in 90 and 75% crop loss. In the same region, a 121.4-ha field of dryland lentils (Lens culinaris) also had plants showing poor growth, wilting, and yellowing in large patches, which resulted in 40% crop loss. Two species of lesion nematodes (Pratylenchus neglectus and P. thornei) and one species of pin nematode (Paratylenchus hamatus) were extracted from rhizosphere soil and the roots of symptomatic plants from these fields. In a subsequent survey of seven dryland pea fields, under cv. Columbian, in Latah and Nez Perce counties and one dryland pea field, under cv. Small Sieve, in Latah County, plant samples had means of 551 and 2,178 mixed species of lesion nematodes per gram of dry root, respectively. Plant samples from 12 lentil fields in Latah County, six planted with cv. Red Chief and six with cv. Pardina, had means of 279 and 987 mixed species of lesion nematodes per gram of dry root, respectively. Soil samples from the same fields had a mean of 628 and 671 pin nematodes, Paratylenchus hamatus, per 250 cm3 soil for Red Chief and Pardina, respectively. Lentils cv. Pardina and peas cv. Columbian were planted separately in six pots, five seeds per pot containing 250 g of infested soil brought from the field to the greenhouse. Fumigated sandy loam soil was used as control. These assays were repeated three times. In addition, peas and lentils were planted to pots infested singly with each of the three nematode species. For this assay, nematodes were extracted from field soil, surface sterilized, and used to infest 250 g of fumigated sandy loam soil at two nematodes per gram of soil. Six plants per nematode species and an uninoculated control were used in the greenhouse assays, which were repeated three times. Nematodes in all of the assays reduced plant growth in comparison with controls; an average of 50 to 70% reduction in plant height was noted. The lesion nematode populations increased in all pots. The greenhouse assays verified the negative impact of these nematodes on growth of dryland peas cvs. Columbian and Small Sieve and lentils cvs. Red Chief and Pardina. P. neglectus, P. thornei, and Paratylenchus spp. previously have been reported from the semi-arid Pacific Northwest (1). However, to our knowledge, this is the first report attributing plant growth and yield reduction of certain cultivars of lentils and peas to these two species of lesion nematodes and pin nematodes, identified to species level as Paratylenchus hamatus.

Reference: (1) R. W. Smiley et al. J. Nematol. 36:54, 2004.
A Culture Filtrate of Phytophthora infestans Primes Defense Reaction in Potato Cell Suspensions
Authors: Val F. , Desender S. , Bernard K. , Potin P. , Hamelin G. , Andrivon D.
Published: May 2008

Priming of defense reactions by an elicitor results in an enhanced ability of the plant to respond to subsequent pathogen challenges. We previously showed that application of lipopolysaccharides (LPS) to potato cell suspensions causes apoplastic acidification, but does not stimulate lipoxygenase (LOX) activity. Here, we tested the ability of various elicitors to prime and elicit defense reactions in potato cell suspensions. Adding 20 μg ml–1 LPS, laminarin, harpin N, or a concentrated culture filtrate (CCF) of Phytophthora infestans to cell cultures 18 h before a second elicitation with LPS did not alter the intensity of apoplastic acidification compared with a single LPS application. Conversely, high concentrations (200 or 400 μg ml–1) of LPS, laminarin, and harpin N activated LOX in cells pretreated with 1 μg ml–1 CCF, but not in cells pretreated with LPS, laminarin, or harpin N. LOX response was maximal in pretreated cells of potato cv. Bintje when the second elicitation occurred 18 to 24 h after CCF application. These results showed that LOX activation is primed in potato cells by CCF, but not by LPS, harpin N, or laminarin. Finally, bioassays showed a slightly greater reduction of rot weight in half tubers treated with CCF followed by LPS before inoculation with Pectobacterium atrosepticum than in half tubers treated with either preparation alone, indicating a priming effect of CCF on both LOX induction and disease suppression.
Protection Against Pathogen and Salt Stress by Four Plant Growth-Promoting Rhizobacteria Isolated from Pinus sp. on Arabidopsis thaliana
Authors: Barriuso J. , Solano B. Ramos , Gutiérrez Mañero F. J.
Published: May 2008

The ability of four plant growth-promoting rhizobacteria, isolated in a previous study, to induce systemic resistance on Arabidopsis thaliana Col 0 against biotic and abiotic stress was evaluated. All the bacteria enhanced protection against the foliar pathogen Pseudomonas syringae DC3000 and increased plant tolerance to salt stress (NaCl 60 mM). Bacillus sp. strain L81 and Arthrobacter oxidans strain BB1 performed best with a decrease in the disease index of 61.2 and 52.3%, respectively, and a reduction in the mortality due to salt stress of 72.4 and 57.8%, respectively. Additionally, significant differences were found in growth and photosynthesis, again, L81 and BB1 performed best either in normal or under stress conditions. In order to elucidate the pathway elicited by these two strains to induce systemic resistance, experiments with the transgenic line of Arabidopsis thaliana NahG (defective in salicylic acid [SA]) and with the jar1 mutant (defective in jasmonic acid) were carried out. Results showed that the SA-dependent pathway was involved in the defense response induced by strains L81 and BB1. Results from quantitative reverse transcription-polymerase chain reaction analysis of the PR1 gene, related to the SA-dependent pathway and the PDF1.2 gene related to the SA-independent pathway, showed an increased expression of PR1 in BB1-treated plants, confirming involvement of the SA-dependent pathway in the defensive response.
Survival and Spread of Phytophthora capsici in Coastal Peru
Authors: Hurtado-Gonzáles O. , Aragon-Caballero L. , Apaza-Tapia W. , Donahoo R. , Lamour K.
Published: May 2008

Phytophthora capsici is a soilborne pathogen that causes significant losses to pepper production in Peru. Our objective was to investigate the mechanisms by which P. capsici is able to survive and spread. During 2005 to 2007, 227 isolates of P. capsici were collected from four species of pepper (Capsicum annum, C. baccatum, C. chinense, and C. pubescens) and tomato (Solanum lycopersicum) at 33 field sites in 13 provinces across coastal Peru. All 227 isolates were of the A2 mating type and amplified fragment length polymorphism (AFLP) analysis indicates that 221 of the isolates had the same genotype. Analyses of six polymorphic single nucleotide polymorphism (SNP) loci showed fixed heterozygosity suggesting a single clonal lineage is widely dispersed. Members of the same clonal lineage were recovered during 2005 to 2007 from geographically separate locations from each of the host types sampled. Our results indicate that clonal reproduction drives the population structure of P. capsici in Peru. The impact of continuous cropping and irrigation from common river sources on the population structure in Barranca Valley are discussed.
Effect of Temperature, Vector Life Stage, and Plant Access Period on Transmission of Banana bunchy top virus to Banana
Authors: Anhalt M. D. , Almeida R. P. P.
Published: May 2008

The study of the transmission biology of insect-borne plant viruses is important to develop disease control practices. We characterized the transmission of a nanovirus, Banana bunchy top virus (BBTV), by its aphid vector Pentalonia nigronervosa Coquerel (Hemiptera, Aphididae) with respect to temperature, vector life stage, and plant access time. Adult aphids transmitted BBTV more efficiently than third instar nymphs at all temperatures tested. Adult aphids transmitted the virus more efficiently at 25 and 30°C than at 20°C, but temperature had no impact on transmission efficiency by nymphs. By decoupling the relationship between temperature and aphid BBTV acquisition or inoculation, we determined that temperature affected inoculation events more strongly than acquisition. Longer plant access periods increased viral acquisition and inoculation efficiencies in a range of 60 min to 24 h. Both BBTV acquisition and inoculation efficiencies peaked after 18 h of plant access period. We also show that BBTV transmission by P. nigronervosa requires a latent period. Our results demonstrate that vector transmission of BBTV is affected by temperature, vector life stage, and plant access period.
Genetic polymorphisms associated with cigarette smoking and the risk of Graves’ disease
Authors: Bufalo Natássia E. , Santos Roberto B. , Cury Adriano N. , Andrade Renato A. , Morari Joseane , Morari Elaine C. , Leite Janaína L. , Monte Osmar , Romaldini João H. , Ward Laura S.
Published: May 2008

Objective  Cigarette smoking is a well‐recognized risk factor of Graves’ disease and, particularly, Graves’ ophthalmopathy. Hence, germline polymorphisms of detoxification genes and genes belonging to the major DNA repair–apoptosis pathways might have an important role in disease susceptibility. In addition, as some of these genes are regulated by thyroid hormones, they may affect the patients’ outcomes. We aimed to assess the influence of the GST, CYP and TP53 gene polymorphisms in the risk of Graves’ disease and its outcome.

Design  Prospective case‐control study.

Patients  A PCR‐based strategy was used for GSTT1, GSTM1, GSTP1, CYP1A1 and TP53 codon 72 genotypes in a group of 400 Graves’ disease patients, and to compare them to 574 control individuals with similar environmental exposure features.

Results  GSTM1 and GSTT1 genotypes were equally distributed in cases and controls, repectively. However, GSTP1 (P < 0·0001), CYP1A1 (P < 0·0033) and Pro/ProTP53 (P < 0·0035) variants appeared more frequently in Graves’ disease patients than in controls. A multivariate analysis indicated that cigarette smoking and inheritance of GSTP1, CYP1A1 and Pro/ProTP53 variants were important risk factors for Graves’ disease, but only smoking appeared as an independent risk factor for Graves’ ophthalmopathy. There was no association between clinical features, including ophthalmopathy or treatment outcome, and the studied genotypes.

Conclusion  We concluded that GSTP1, CYP1A1 and TP53, but not GSTT1 and GSTM1 germline polymorphisms, may be associated with smoking‐related Graves’ disease susceptibility and configure a risk profile for the disease. However, these polymorphisms do not influence the patients’ response to treatment.
Immunohistochemical staining for thyroid peroxidase (TPO) of needle core biopsies in the diagnosis of scintigraphically cold thyroid nodules
Authors: Yousaf U. , Christensen L. H. , Rasmussen Å. K. , Jensen F. , Mollerup C. L. , Kirkegaard J. , Lausen I. , Rank F. , Feldt‐Rasmussen U.
Published: May 2008

Background  Cold thyroid nodules are common, in particular in iodine‐deficient areas, but only a minority of them are malignant requiring surgery. Thyroid peroxidase (TPO) immunostaining of fine‐needle aspiration cytology (FNAC) material has proven helpful in diagnosing cells from malignant lesions, but the procedure has its limitations in a routine setting.

Purpose  To improve diagnosis and reduce surgery rate, the FNAC procedure was replaced by needle core biopsy (NCB), which was routinely stained for TPO by the monoclonal antibody mAb 47.

Materials and methods  During a 5‐year period 427 consecutive patients with a cold thyroid nodule were evaluated by ultrasound‐guided NCB, which had been routinely stained for TPO in an automated immunostainer. Sensitivity and specificity and predictive values of the TPO immunostaining were estimated, based on the final diagnosis obtained from surgical resection.

Results  The majority of nodules with benign NCB diagnosis were not surgically removed, and thus a subgroup of 140 operated nodules formed the basis for the calculations. Sensitivity and specificity for benign and malignant lesions were 100% if the oxyphilic variant of adenomas and minimally invasive follicular carcinomas were excluded. By inclusion of these, the values fell to 89% and 97%, respectively. The predictive value of a positive test was 96% and the predictive value of a negative test was 97%.

Conclusion  TPO immunostaining was found to be a valuable adjunct to morphology in the diagnosis of cold thyroid nodules of the nonoxyphilic type.
Pegvisomant‐primed GH stimulation test
Authors: Radetti Giorgio , Wu Zida , Elsedfy Heba H. , El Kholy Mohamed , Bozzola Mauro , Strasburger Christian J.
Published: May 2008

Objective  Provocative stimulation tests for GH assessment have poor reproducibility and can often elicit false positive results in normal children. The aim of our study was to evaluate the capability of pegvisomant, as an enhancer of GH secretion, in unmasking false‐positive results in short children undergoing GH testing.

Design  A prospective study was conducted between March 2005 and April 2006.

Patients  Twenty‐one short children (8 males and 13 females), aged 1·0–14·5 years, with a height of < 2 SD scores below the mean were included in the study.

Methods  All subjects underwent an l‐DOPA stimulation test with evaluation of GH. At the end of the test, 1 mg/kg of pegvisomant was given subcutaneously and 3 days later an l‐DOPA stimulation test was repeated.

Results  There was a significant decrease of IGF‐I SDS following pegvisomant (–1·75 ± 0·24 vs.–2·65 ± 0·23; P < 0·0001) and a significant increase of the GH‐peak (6·2 ± 0·91 vs. 15·3 ± 2·30 µg/l; P < 0·0001). Among the 21 patients examined, 18 (85·7%) had an insufficient response (< 10 µg/l) at the first stimulation. Ten of them (55·5%) showed normal secretion after priming with pegvisomant, while insufficient secretory reserve was confirmed in the remaining eight.

Conclusions  Pegvisomant priming before GH stimulation tests can be used to improve the reliability of the diagnostic work up in GH deficiency. Further studies are required, however, to clarify whether this procedure should be recommended in the routine evaluation of GH status.
Total, free and bound leptin and thyroid function in elderly women with different body weights
Authors: Ruscica Massimiliano , Dozio Elena , Gandini Sara , Gnocchi Pierluigi , Devalle Guya Giuseppina , Motta Marcella , Roti Elio , Magni Paolo
Published: May 2008

Objective  The present study was aimed at evaluating the relationship of total leptin, and its free leptin (FL) and bound leptin (BL) fractions with adipose mass in very old euthyroid women, in relationship to thyroid function.

Subjects and methods  Twenty‐five older women (age: 73–95 years) were studied. Subjects representing underweight, normal weight and overweight/obese conditions were included. Plasma leptin, TSH, free T4 (FT4) and free T3, (FT3) total and HDL cholesterol were measured. FL and BL were evaluated by Fast Protein Liquid Chromatography (FPLC) analysis.

Results  Plasma leptin concentration was positively correlated with body mass index (BMI) (r = 0·64, P = 0·0005) and tricipital skin‐fold thickness (TF) (r = 0·46, P = 0·0187). Leptin was positively correlated with TSH (r = 0·50, P = 0·0116) and inversely with FT3 (r = –0·40, P = 0·0477). TSH correlated with the adiposity indexes BMI (r = 0·40, P = 0·05) and TF (r = 0·42, P = 0·0336). Plasma FT3 was positively correlated with FT4 (r = 0·49, P = 0·012). FL and BL were evaluated in 8 out of 25 subjects. FL positively correlated with BMI (r = 0·81, P = 0·0218) and leptin (r = 0·83, P = 0·0004), whereas BL did not correlate with these parameters.

Conclusions  The present results indicate that in very old women, plasma leptin concentrations reflect the extent of adipose mass and suggest that a complex regulatory interaction exists between leptin and thyroid function, possibly taking place at central (hypothalamus–pituitary) and peripheral (deiodinase activity) levels.
Short‐ and long‐term somatostatin analogue treatment in patients with hypoglycaemia related to endogenous hyperinsulinism
Authors: Vezzosi D. , Bennet A. , Courbon F. , Caron P.
Published: May 2008

Background  The long‐term efficacy of somatostatin analogues on insulinomas has not been studied.

Design  A prospective study to evaluate the response of octreotide in 21 patients with hypoglycaemia related to endogenous hyperinsulinism who were not treated by surgery.

Results  Reasons for not undergoing surgery were: refusal (n = 3), old age with multiple diseases (n = 5), unlocalized insulinomas (n = 2), malignant unresectable insulinomas (n = 5), multiple insulinomas (n = 3) and diffuse β‐cell disease (n = 3). Hypoglycaemia was responsive to octreotide in 14 of the 21 patients. A short 100‐µg octreotide test correctly predicted the efficacy of treatment in six patients with benign insulinomas. Octreoscan scintigraphy was positive in 6 of the 16 patients of whom three were responsive and three unresponsive to octreotide. Octreoscan scintigraphy was negative in 10 of the 16 patients, eight of whom were responsive to octreotide. Subcutaneous octreotide treatment was prolonged for > 6 months (7–144 months, 67 ± 47 months) in 11 responsive patients. No tachyphylaxis was observed. However, the octreotide dose had to be increased in two patients after 3 and 18 months, respectively. Only one patient suffered from symptomatic biliary lithiasis after 3 years of treatment.

Conclusion  Long‐term octreotide treatment can be used to control hypoglycaemia in patients with endogenous hyperinsulinism who are not eligible for surgery; octreotide efficacy on hypoglycaemia could be predicted by a short 100 µg‐octreotide test in patients with benign insulinomas, but was not correctly predicted by Octreoscan scintigraphy.
Surgical debulking of pituitary macroadenomas causing acromegaly improves control by lanreotide
Authors: Karavitaki N. , Turner H. E. , Adams C. B. T. , Cudlip S. , Byrne J. V. , Fazal‐Sanderson V. , Rowlers S. , Trainer P. J. , Wass J. A. H.
Published: May 2008

Background  Macroadenomas causing acromegaly are cured surgically in only around 50% of patients. Primary medical treatment with somatostatin analogues has been suggested to be a means of treating patients with a potentially poor surgical outcome. Previous retrospective studies have also suggested that surgical debulking of pituitary tumours causing acromegaly improves control by somatostatin analogues. No prospective study using lanreotide has been carried out thus far to assess whether this is the case.

Objective  We carried out a prospective study to assess whether surgical debulking of pituitary macroadenomas causing acromegaly improved the subsequent control of acromegaly by the somatostatin analogue lanreotide.

Patients and methods  We treated 26 consecutive patients [10 males and 16 females – median age 53·5 years (range 22–70)] with macroadenoma causing acromegaly unselected for somatostatin response for 16 weeks with lanreotide, maximizing GH and IGF‐I suppression, if necessary, by incremental dosing. Surgical resection was carried out and the patients were re‐assessed off medical treatment at 16 weeks following surgery. Those with nadir GH > 2 mU/l in the oral glucose tolerance test (OGTT) and a mean GH in the GH day curve (GHDC) > 5 mU/l were subsequently restarted on lanreotide and the responses were assessed at the same time points as during the preoperative lanreotide treatment.

Results  GH values fell on lanreotide treatment and prior to surgery they were considered ‘safe’ (mean GH in GHDC < 5 mU/l) in eight patients (30·7%). After surgery, they were ‘safe’ in 18 patients (69·2%). The figures for normal IGF‐I were 11 (42·3%) before surgery and 23 (88·5%) after surgery. After surgery, six patients had nadir GH > 2 mU/l in the OGTT and ‘unsafe’ GH levels (mean GH in GHDC > 5 mU/l); on re‐exposure to lanreotide, GH levels fell in all patients and at the end of 16 weeks postsurgery, they were ‘safe’ in three of them (50%) (P < 0·05). Pituitary tumour volume was also assessed prospectively, preoperatively on lanreotide and showed a mean fall of 33·1%. Eighty‐three percent of patients had > 20% shrinkage.

Conclusions  In this first prospective study using lanreotide, surgical debulking of pituitary tumours causing acromegaly improved subsequent postoperative control by the somatostatin analogue lanreotide. Surgery should, therefore, be considered in patients with macroadenoma causing acromegaly, even if there is little prospect of surgical cure. Lanreotide causes significant pituitary tumour shrinkage in the majority of patients.
GH replacement reduces increased lipid peroxidation in GH‐deficient adults
Authors: Karbownik‐Lewinska Malgorzata , Kokoszko Agnieszka , Lewandowski Krzysztof C. , Shalet Stephen M. , Lewinski Andrzej
Published: May 2008

Background  GH replacement improves numerous metabolic abnormalities in GH‐deficient patients; increased lipid peroxidation (LPO) has been observed in GH‐deficient patients; however, it is unknown if LPO is influenced by GH replacement.

Aim and methods  To evaluate the extent to which GH replacement might reverse the increased LPO in GH‐deficient adults and to analyse if this phenomenon might be involved in the improvement of metabolic disturbances due to GH treatment. Serum concentrations of malondialdehyde + 4‐hydroxyalkenals (MDA + 4‐HDA), as an index of LPO, were measured at baseline, and after 12 and 24 months of GH replacement in 40 adult patients with severe GH deficiency (both in adult‐ and childhood‐onset) and in 40 healthy volunteers, matched for sex, age and body mass index (BMI). Correlations were evaluated between LPO and lipids, IGF‐I, metalloproteinase‐2 and ‐9 (MMP‐2, ‐9), vascular endothelial growth factor (VEGF), BMI and GH dose.

Results  LPO values in GH‐deficient patients were several‐fold higher than in controls [55·36 ± 2·27 vs. 4·19 ± 0·42 nmol/mg protein (mean ± SEM), P < 0·0001] and decreased significantly over time with GH replacement to 38·61 ± 2·15 nmol/mg protein (i.e. by approximately 30%), though still remaining markedly elevated compared with controls (P < 0·0001). The proatherogenic lipid profile parameters correlated positively with LPO in the childhood‐onset subgroup before GH replacement. GH replacement restored the positive correlation between LPO and age in male patients (r = 0·57, P = 0·013; r = 0·8, P < 0·001, at 12 and 24 months of GH replacement, respectively).

Conclusions  GH replacement partially reverses the grossly abnormal LPO in GH‐deficient adults. It is highly probable, therefore, that oxidative mechanisms are involved in the overall improvement of metabolic changes due to GH replacement.
Reduced attenuation of bone resorption after oral glucose in type 2 diabetes
Authors: Chailurkit La‐or , Chanprasertyothin Suwannee , Rajatanavin Rajata , Ongphiphadhanakul Boonsong
Published: May 2008

Objective  To investigate the effect of oral glucose on bone resorption and osteoprotegerin (OPG) in subjects with varying degrees of glucose tolerance.

Design and Patients  In a cross‐sectional study, 163 postmenopausal women aged 50–88 years without previous history of diabetes, impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) were recruited. All subjects underwent a 75‐g oral glucose tolerance test (OGTT) and were then classified as having normal glucose tolerance (NGT), IFG, IGT or diabetes according to American Diabetes Association (ADA) criteria.

Measurements  Plasma glucose, serum insulin, C‐terminal telopeptide of type I collagen (CTX‐I) and OPG were measured.

Results  Fasting insulin levels increased progressively from subjects with NGT, IFG/IGT to diabetes. After adjusted for age and body mass index (BMI), there was no significant difference in fasting CTX‐I and OPG levels across the various degrees of glucose tolerance. After oral glucose, there was a significant decrease in serum CTX‐I and OPG (P < 0·001) except for serum OPG in diabetic subjects. In addition, the percentages of change from baseline for both serum CTX‐I and OPG were significantly less in diabetic subjects when compared to those in NGT subjects (–40·9% and 0·6% for diabetes and –50·2% and –10·6% for NGT, respectively).

Conclusions  Oral glucose intake causes suppression of serum CTX‐I and OPG in postmenopausal women. The effect is attenuated in women with type 2 diabetes.
Continuous glucose monitoring in patients with insulinoma
Authors: Munir Alia , Choudhary Pratik , Harrison Barney , Heller Simon , Newell‐Price John
Published: May 2008

Background  Insulinomas are rare neuroendocrine tumours that are usually small and may take time to localize. They cause recurrent life‐threatening spontaneous hypoglycaemia. Recurrent hypoglycaemia causes loss of hypoglycaemia awareness, putting the patient at further risk, but this has rarely been described in insulinoma. We describe the utility of continuous glucose monitoring (CGM) in patients with insulinoma.

Patients and methods  Three patients, aged 72 years (patient 1), 37 years (patient 2) and 24 years (patient 3), with suspected insulinoma attended our investigation unit, in a university teaching hospital. Biochemical diagnosis was confirmed by elevated plasma insulin and C‐peptide during biochemical hypoglycaemia [plasma glucose < 2·2 mM (40 mg/dl)]. Surgery confirmed histology in all. CGM was used to monitor frequency and time of hypoglycaemia during diagnosis and medical treatment, and after definitive surgical treatment.

Results  All patients had evidence of hypoglycaemia unawareness. At diagnosis in patients 1–3, CGM revealed 6·1%, 21·9% and 71·0% of time spent in moderate hypoglycaemia (plasma glucose 2·2–3·0 mM), and 1·4%, 11·4% and 48·1% of time in severe hypoglycaemia (plasma glucose < 2·2 mM), respectively. On diazoxide this reduced to 0·6%, 5·4% and 5·7% time in moderate hypoglycaemia, and no severe hypoglycaemia in patients 1 and 3, and 0·5% in patient 2. Octreotide therapy in patients 2 and 3 resulted in 5·8% and 0% of time in moderate hypoglycaemia, respectively, and no severe hypoglycaemia. After surgical excision CGM confirmed cure in all.

Conclusions  CGM in insulinoma is useful in detecting hypoglycaemia, and hypoglycaemia unawareness, monitoring response to medical therapy and for confirming cure postoperatively, and is useful in the management of this uncommon but dangerous condition.
Host‐plant quality alters grass/forb consumption by a mixed‐feeding insect herbivore, Melanoplus bivittatus (Orthoptera: Acrididae)
Authors: JONAS JAYNE L. , JOERN ANTHONY
Published: May 2008

Abstract 1. Factors affecting the nutritional ecology of mixed‐feeding, polyphagous herbivores are poorly understood. Mixed‐feeding herbivores do better when they consume both forb and grass species although they typically feed primarily on forbs, which are of relatively higher protein content than grasses.

2. In a field experiment, we examined the effects of nitrogen and phosphorus fertilization and associated changes in host‐plant C:N:P on proportional grass consumption by a mixed‐feeding insect herbivore, Melanoplus bivittatus, using natural abundance stable carbon isotope (12C/13C) methods. We also examined a grass‐feeding (Phoetaliotes nebrascensis) and forb‐feeding (Hesperotettix viridis) species.

3. The C isotope signatures of M. bivittatus collected from plots fertilized with nitrogen (+N), phosphorus (+P), nitrogen and phosphorus (+N+P) and no fertilizer were compared with the C isotope signatures of plants in those plots to determine the proportion of assimilated C derived from C4 grasses and C3 forbs in each plot. We also examined the relationship between M. bivittatus diets and plant C:N:P stoichiometry.

4. The proportion of grass assimilated approximately doubled in N‐fertilized treatments (39.1 ± 0.1%) compared with non‐fertilized treatments (19 ± <0.1%), an increase associated with decreased C:N and increased N:P of grasses.

5. These results indicate that mixed‐feeding M. bivittatus can selectively feed to balance C:N:P intake even when choosing between two structurally and chemically different groups of plants.

6. The strong relationship between diet selection and grass stoichiometry also suggests that plant nutrient composition may be more important than defensive chemistry in food choice.