Disease Severity Estimates-Effects of Rater Accuracy and Assessment Methods for Comparing Treatments.

Assessment of disease severity is required for several purposes in plant pathology; most often, the estimates are made visually. It is established that visual estimates can be inaccurate and unreliable. The ramifications of biased or imprecise estimates by raters have not been fully explored using empirical data, partly because of the logistical difficulties involved in different raters assessing the same leaves for which actual disease has been measured in a replicated experiment with multiple treatments. In this study, nearest percent estimates (NPEs) of Septoria leaf blotch (SLB) on leaves of winter wheat from nontreated and fungicide-treated plots were assessed in both 2006 and 2007 by four raters and compared with assumed actual values measured using image analysis. Lin's concordance correlation (LCC, ρc) was used to assess agreement between the two approaches. NPEs were converted to Horsfall-Barratt (HB) midpoints and were compared with actual values. The estimates of SLB severity from fungicide-treated and nontreated plots were analyzed using generalized linear mixed modeling to ascertain effects of rater using both the NPE and HB values. Rater 1 showed good accuracy (ρc = 0.986 to 0.999), while raters 3 and 4 were less accurate (ρc = 0.205 to 0.936). Conversion to the HB scale had little effect on bias but reduced numerically both precision and accuracy for most raters on most assessment dates (precision, r = -0.001 to -0.132; and accuracy, ρc = -0.003 to -0.468). Interrater reliability was also reduced slightly by conversion of estimates to HB midpoint values. Estimates of mean SLB severity were significantly different between image analysis and raters 2, 3, and 4, and there were frequently significant differences among raters (F = 151 to 1,260, P = 0.001 to P < 0.0001). Only on 26 June 2007 did conversion to the HB scale change the means separation ranking of rater estimates. Nonetheless, image analysis and all raters were able to differentiate control and treated-plot treatments (F = 116 to 1,952, P = 0.002 to P < 0.0001, depending on date and rater). Conversion of NPEs to the HB scale tended to reduce F values slightly (2006: NPEs, F = 116 to 276, P = 0.002 to 0.0005; and, for the HB-converted values, F = 101 to 270, P = 0.002 to 0.0005; 2007: NPEs, F = 164 to 1,952, P = 0.001 to P < 0.0001; and, for HB-converted values, F = 126 to 1,633, P = 0.002 to P < 0.0001). The results reaffirm the need for accurate and reliable disease assessment to minimize over- or underestimates compared with actual disease, and the data we present support the view that, where multiple raters are deployed, they should be assigned in a manner to reduce any potential effect of rater differences on the analysis.

Cervical spine fractures in the elderly: morbidity and mortality after operative treatment.

PURPOSE: Although there are currently many different strategies and recommendations in the therapy of cervical spine fractures in elderly patients, there are still no generally accepted treatment algorithms. The aim of the present study was to analyze the morbidity, mortality, and outcome of operated cervical spine injuries in the elderly. METHODS: This study presents a retrospective review of 69 patients aged 65 years or older admitted to our level I trauma center with cervical spine injury, who had undergone surgical treatment. The data were acquired by analysis of the hospital inpatient enquiry system and radiological review. RESULTS: The ratio between male and female patients was 37:32. The average age of the patients was 76 years (ranging from 65 to 96 years) for males and 80 years (ranging from 66 to 93 years) for females. Injury to the cervical spine was caused by low-energy trauma in 71 % and high-energy trauma in 29 %, respectively. 55.1 % sustained isolated cervical spine injuries, 39.1 % injuries to two adjacent vertebrae, 2.9 % injuries to three adjacent vertebrae, and 2.9 % an odontoid fracture combined with associated fracture(s) in non-contiguous vertebra(e). Isolated spine injury level was dominated by C2 (47.8 %). The most common site for injuries to two adjacent vertebrae was observed at C6/C7 (14.5 %). The morbidity included cerebral complications, respiratory complications, Clostridium difficile-associated disease, heart failure, and acute renal failure. Operative complications included dislocation/malposition, neurovascular lesions, wound infection, and transient swallowing difficulty. The mortality rate at 3 months was 26.1 %, with an in-hospital mortality of 21.7 %. Age was associated with mortality at 3 months. A cervical fracture-induced neurological deficit was documented in 26.1 %, resulting in a mortality of 44.4 % (8/18). Twenty-seven of 33 patients living at home/nursing home at the time of injury returned to their home/nursing home after their hospitalization. The overall outcome was predominantly related to age and the severity of neurological deficit. CONCLUSIONS: In elderly patients with cervical spine fractures, the hospital course is complicated by medical issues and early mortality rates are significant. Therefore, treatment strategies should be carefully individualized to the patients and their comorbidities.

Targeting highly conserved 3'-untranslated region of pecluviruses for sensitive broad-spectrum detection and quantitation by RT-PCR and assessment of phylogenetic relationships.

The 3'-end region of many virus isolates has been shown to possess conserved sequences in addition to the presence of numerous genomic and subgenomic RNAs. Utilizing these sequences, a broad-spectrum reverse transcription-polymerase chain reaction protocol has been developed to detect all the known Indian peanut clump virus and Peanut clump virus isolates, that cause peanut clump diseases in West Africa and India. The primers were targeted at the highly conserved 3'-untranslated regions of the PCV RNA-1 and RNA-2. The conservation was confirmed by sequencing these untranslated regions of RNA-1 for six isolates and RNA-2 for one isolate. The conserved structure of the RNA-1 and RNA-2 was observed and the importance of this region for the virus survival was confirmed. The primers were also designed for virus quantitation using a Taqman(®)-based real-time RT-PCR. The use of RT-PCR and real-time quantitative RT-PCR improved the sensitivity of PCV detection compared to ELISA. RT-PCR also led to the detection of IPCV and PCV on two new natural hosts: Oldenlandia aspera and Vigna subterranea. Real-time RT-PCR is considered to be an ideal tool for identifying resistant sources to both IPCV and PCV.

Seed, soil and vegetative transmission contribute to the spread of pecluviruses in Western Africa and the Indian sub-continent.

Peanut clump and sugarcane red leaf mottle diseases are caused by viruses of the genus Pecluvirus. Indian peanut clump virus occurs in the Indian sub-continent and Peanut clump virus in West Africa. A feature of these viruses is that they are both seed and soil transmitted. Both modes of transmission contribute to long-term persistence and field spread. Data on seed transmission in pearl millet, virus movement within the plant and virus diversity based on RNA-1 partial sequences are presented. This study emphasizes that pecluviruses are also viruses of cereals infecting sorghum and pearl millet, and highlights a correlation between the countries cultivating these two crops and the virus distribution. Ways of controlling pecluviruses and their vector, Polymyxa graminis, taking into account the virus dissemination routes, are proposed.

Assessing the Accuracy of Simulation Model for Septoria Leaf Blotch Disease Progress on Winter Wheat.

A mechanistic model, PROCULTURE, for assessing the development of each of the last five leaf layers and the progress of Septoria leaf blotch, caused by Septoria tritici (teleomorph Mycosphaerella graminicola), has been applied on susceptible and weakly susceptible winter wheat (Triticum aestivum) cultivars in two locations (Everlange and Reuland) in Luxembourg over a 3-year period (2000 to 2002). A double performance assessment of PROCULTURE was conducted in this study. First, the capability of PROCULTURE to correctly simulate S. tritici incidence was checked. Second, the model's ability to accurately estimate disease severity was assessed on the basis of the difference between simulated and observed levels of disease development at each leaf layer. The model accurately predicted disease occurrence in the 2000 and 2002 seasons, on susceptible and semi-susceptible cultivars, with a probability of detection (POD) exceeding 0.90. However, in 2001, even though the POD never fell below 0.90, the false alarm ratio (FAR) was too high to consider the simulations satisfactory. Concerning the evaluation of disease severity modeling, statistical tests revealed accurate simulations performed by PROCULTURE for susceptible cultivars in 2000 and 2002. By contrast, for weakly susceptible cultivars, the model overestimated disease severity, especially for the upper leaves, for the same period.

First Report of Wheat Leaf Rust in the Grand Duchy of Luxembourg and the Progress of its Appearance over the 2003-2008 Period.

Wheat leaf rust caused by Puccinia triticina Eriks. was identified for the first time in 2000 in the Grand Duchy of Luxembourg on the basis of orange-to-brown, round-to-ovoid, erumpent uredinia (1 to 1.5 mm in diameter) scattered on the upper and lower leaf surfaces and producing orange-brown urediniospores that are subgloboid, approximately 20 µm in diameter, and with up to eight germ pore scattered in thick, echinulate walls. In a second phase, wheat was monitored weekly (starting from Zadoks growth stage 30, pseudo stem erection) during the 2003-2008 cropping seasons for wheat leaf rust. Disease severity (percentage of leaf area with symptoms) was recorded in four, replicated field experiments located in three villages (Diekirch District: Reuler; and Grevenmacher District: Burmerange and Christnach), which are representative of the different agroclimatological zones of Luxembourg. A significant difference in severity was observed between the sites (P < 0.01) and the years (P < 0.05). Over the 6-year period, Burmerange and Reuler consistently showed the highest and lowest disease severity, respectively. In 2003 and 2007, Burmerange (a southern site with the highest average spring temperatures of 13.6 and 14.0°C, respectively) showed the highest disease severity with 66 and 57%, respectively, whereas the lowest severity (<1% for both years) was observed in the north at Reuler (site with the lowest average spring temperatures of 12.0 and 12.4°C, respectively). Christnach, located midway between Reuler and Burmerange, showed an intermediate disease severity with 7% (2003) and 22% (2007). The disease appeared at growth stages 77 (late milk) and 87 (hard dough) in the period 2003-2005, but at an earlier stage (45, boots swollen) for 2006-2008 (P < 0.001). In 2005, low severity was recorded due to a severe drought during May, June, and July. A reason for this earlier appearance of leaf rust occurrences in the two districts may be related to an increase in the average spring temperature (average March to May temperature for Luxembourg was 8.3°C for the 1971-2000 period, 9.5°C for the 2003-2005 period, 9.9°C for the 2006-2008 period, 2007 was exceptional with 11.9°C, P < 0.01). In the past, cereal disease management strategies were oriented toward the control of predominant and yield-reducing diseases such as that caused by Septoria tritici Desm. Because the succession of mild winters and warm springs during the last 5 years allowed the early occurrence and the fast development of wheat leaf rust in the Grand Duchy of Luxembourg, it is advisable to take this disease into account in fungicide application schemes.

First Report of Plasmodiophora brassicae on Rapeseed in the Grand Duchy of Luxembourg.

Rapeseed (Brassica napus L.) is the third most important crop after wheat and barley in the Grand Duchy of Luxembourg. Since 2005, clubroot symptoms in this crop have been reported by farmers in the Gutland Region. In February 2009, plants of the hybrid rapeseed cv. Exocet, with stunted growth, yellow leaves, and club-shaped roots, were sampled from a field in Oberkorn village near Differdange. Microscopic observations of the rapeseed root fragments revealed the presence of the three life stages characteristic of Plasmodiophora brassicae Woronin. Plasmodia and zoosporangia were observed in the root hairs and resting spores were present in root galls. Individual spores were 2 to 3 µm in diameter. Total DNA was extracted from the root galls with a FAST DNA Kit (MP Biomedicals, Irvine, CA). The internal transcribed spacer region (ITS) and 5.8S gene of the rDNA region were amplified with ITS5 and ITS4 primers as described by White et al. (2) and part of this region was sequenced. A BLASTn search in GenBank revealed that the sequence closely resembled (98% identity) sequences of P. brassicae (Genbank Accession No. EF195335) from an isolate of the pathogen from Switzerland. To confirm the presence of the pathogen, seeds of the susceptible ecotype cvi-0 of Arabidopsis thaliana were grown in a soil sample (1 liter) collected near the infected rapeseed plants. After 55 days of growth in a glasshouse at 15 to 20°C, the roots of 11 plants were analyzed. Two showed clear clubroot symptoms and four others exhibited small swellings. The remaining five plants were symptomless, but plasmodia and zoosporangia were found in root hair cells. Clubroot caused by P. brassicae has previously been described on B. napus and other crucifers (1). To our knowledge, this is the first report of clubroot disease caused by P. brassicae in Luxembourg. Because its presence has since been observed in new fields in the Gutland Region and because of the ability of the pathogen to survive for a long period in the soil, this disease could represent a severe threat for cropping of Brassicaceae in Luxembourg and neighboring countries. References: (1) I. R. Crute et al. Plant Breed. Abstr. 50:91, 1980. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic press: San Diego, 1990.

First Report of Fusarium Head Blight on Winter Wheat in the Grand Duchy of Luxembourg.

Following a comparatively mild winter (1.9°C above average [2000-2007]), Fusarium head blight (FHB) on winter wheat was observed during the 2007 season in 17 sites representing all three districts of Diekirch, Grevenmacher, and Luxembourg. The cultivars encountered were diverse and included Achat, Akteur, Aron, Bussard, Cubus, Enorm, Exclusiv, Flair, Rosario, Tommi, and Urban. The preceding crops were maize (six sites), rapeseed (three sites), and one site each of pea, triticale, winter barley, and winter wheat. Rainfalls recorded during the flowering period (June 1-23, mean June 12 for GS 65) ranged from 13 to 62 (mean 38) mm. An overall prevalence of FHB (percentage of infected spikes) of 8.9 ± 15.5% (mean ± SD) and a severity (percentage of infected grains per spike) of 21.0 ± 17.8% were recorded. A significant difference in FHB severity was observed between the cantons north and south of Luxembourg City, 13.4 ± 13.1% (range 0.01 to 46.4) and 35.1 ± 18.1% (range 6.2 to 61.9), respectively (Man-Whitney, P = 0.027), indicating the importance to take regional specificities such as topoclimatological aspects into account. Maize as a preceding crop resulted in significant higher prevalence of FHB as opposed to the other crops (5.9 ± 1.6% versus 3.3 ± 2.2%, Man-Whitney, P = 0.022).

Supplementation with groundnut haulms for sheep fattening in the West African Sahel.

Groundnut haulms along with cowpea hay are major crop residues used for animal fattening in the West African Sahel. In traditional sheep fattening, feeds are always provided ad-hoc and in an unregulated fashion, which is rather wasteful. As a preliminary study to establish the optimal feeding levels of groundnut haulms for profitable sheep fattening, a feeding trial was conducted for 70 days with four levels of groundnut haulms (0, 150, 300 and 450 g/day) and a basal diet of bush hay. The effects of supplementation with groundnut haulms on feed intake, water consumption, live weight changes and economic return were determined. Twenty-four Peuhl Oudah rams with average initial weight of 28.6 kg (SD = 1.4) were randomly allocated to four treatments defined by the four levels of groundnut haulms in the diet. Faeces and urine were collected in weeks 5 and 9 of the trial. Digestible organic matter intake (g/(kg LW)0.75) and nitrogen intake (g/day) increased linearly with the level of groundnut haulms offered. Sheep that were fed only bush hay lost 18.4 g/day, while those that were offered 150, 300 and 450 g of groundnut haulms gained 1.4, 19.3 and 40.2 g/day, respectively. The gross return ranged from 1883 to 4946 FCFA per ram. Net benefit, after removing the feed and veterinary costs from the gross return, ranged from 368 to 1400 FCFA per ram.

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA.

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.

Serological Methods for Detection of Polymyxa graminis, an Obligate Root Parasite and Vector of Plant Viruses.

A purification procedure was developed to separate Polymyxa graminisresting spores from sorghum root materials. The spores were used as im-munogen to produce a polyclonal antiserum. In a direct antigen coating enzyme-linked immunosorbent assay (DAC ELISA), the antiserum could detect one sporosorus per well of the ELISA plate. In spiked root samples, the procedure detected one sporosorus per mg of dried sorghum roots. The majority of isolates of P. graminis from Europe, North America, and India reacted strongly with the antiserum. Interestingly, P. graminis isolates from the state of Rajasthan (northern India), from Pakistan, and an isolate from Senegal (West Africa) reacted weakly with the antiserum. The cross-reactivity of the serum with P. betae isolates from Belgium and Turkey was about 40% of that observed for the homologous isolate. There was no reaction with common fungi infecting roots or with the obligate parasite Olpidium brassicae. However, two isolates of Spongospora sub-terranea gave an absorbance similar to that observed with the homologous antigen. The DAC ELISA procedure was successfully used to detect various stages in the life cycle of P. graminis and to detect infection that occurred under natural and controlled environments. A simple procedure to conjugate antibodies to fluorescein 5-isothiocyanate (FITC) is described. Resting spores could be detected in root sections by using FITC-labeled antibodies. The potential for application of the two serological techniques for studying the epidemiology of peanut clump disease and for the characterization of Polymyxa isolates from various geographical origins is discussed.

Production and characterization of monoclonal antibodies for aflatoxin B1.

Hybridomas that secreted antibodies for aflatoxin B1 were selected using two immunization protocols referred to as A and B. Protocol A is a standard immunization method and resulted in the selection of only two clones that produced monoclonal antibodies against aflatoxin B1. In protocol B a unique immunization schedule which resulted in the generation of 10 hybridomas is described. Of the 10, one antibody was highly specific to B1, four antibodies reacted equally strongly with B1, G1 and weakly with B2. Another four reacted strongly with B1 and weakly with B2 and G1. One clone reacted equally strongly with B1, G1 and B2. Interestingly all the 10 antibodies showed little or no cross-reaction with G2.

Seed Transmission of Indian Peanut Clump Virus (IPCV) in Peanut and Millets.

An enzyme-linked immunosorbent assay (ELISA) procedure was developed to test peanut seed for Indian peanut clump virus (IPCV). A double antibody sandwich form of ELISA detected the Hyderabad isolate (IPCV-H) in seed of peanut. Correlation was established between the results from ELISA performed on cotyledons of peanut seed and grow-out tests. Seed transmission in the field-infected peanut plants ranged from 3.5 to 17%, depending on the genotype. The transmission frequency was 48 to 55% in seed collected from plants infected through seed. Because testae of all seed contained viral antigen, their removal was essential for the determination of frequency of seed transmission. Apparently the virus present only in cotyledons and embryo contributed to the seed transmission. For the first time, IPCV-H was shown to be seed transmitted in finger millet (Eleusine coracana), foxtail millet (Setaria italica), and pearl millet (Pennisetum glaucum) at frequencies of 5.2, 9.7, and 0.9%, respectively. Seed transmission was not observed in sorghum (Sorghum bicolor). Significance of seed transmission in millet crops is discussed.

Structure and characterization of isopyoverdin from Pseudomonas putida BTP1 and its relation to the biogenetic pathway leading to pyoverdins.

Pyoverdin type siderophores produced by six fluorescent Pseudomonas strains isolated from different rhizospheres were purified and characterized. The purified ferri-pyoverdins were tested for their ability to promote the growth of other strains grown under iron deficiency conditions. Only the one obtained from Pseudomonas putida BTP1 did not act as a growth promoter. The structure of the BTP1 siderophore was elucidated by spectroscopic methods and degradation studies. It turned out that it contains a chromophore which differs from the one typical for pyoverdins insofar as it carries the carboxyl group in 3- rather than in 1-position ((3S)-5-amino-1,2-dihydro-8,9-dihydroxy-3H-pyrimido[1,2a]quinoline-3- carboxylic acid). The amino group of the chromophore is substituted with the 5-carboxyl group of L-glutamic acid and its carboxyl group with the N-terminus of the peptide L-Asp-L-Ala-L-Asp-D-N5-Ac-N5-OH-Orn-L-Ser-L-c-N5-OH-Orn. This isopyoverdin fits into the biogenetic scheme which postulates ferribactins as the precursors of pyoverdins.

Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads.

Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.