Philippe Delfosse

Phylogenetic analysis of Polymyxa species based on nuclear 5.8S and internal transcribed spacers ribosomal DNA sequences

A region of the nuclear ribosomal DNA containing the internal transcribed spacer 1 (ITS1), the 5.8S DNA and the internal transcribed spacer 2 (ITS2) was sequenced in 12 Polymyxa graminis and P. betae isolates, with particular emphasis on P. graminis from peanut clump-infested areas of the Indian subcontinent and West-Africa. Four different sequences were obtained from the seven isolates on sorghum or pearl millet, which differed from four sequences previously published for Polymyxa species and obtained for P. graminis isolates on barley, oat and wheat originating from temperate areas in Europe and America (two distinct sequences), for isolates on rice from Colombia and for P. betae isolates on sugar beet from several origins. The sequence variations concerned mainly the composition and length of ITS1 and ITS2 regions. Phylogenetic trees built with the distinct sequences currently known for Polymyxa spp. using parsimony, maximum likeihood and neighbour-joining methods separated P. betae from P. graminis, Within P. graminis, the hierarchy of the clustering partially matched the host range and geographical origin of the isolates. These results confirm the great diversity within P. graminis that has already been observed in host range and temperature requirements studies, and provide new arguments for considering several taxa within the species. On the basis of the ecological requirements and rDNA sequences of distinct P. graminis isolates, five special forms are proposed: P. graminis f. sp. temperata, P. graminis f. sp. tepida, P. graminis f. sp. subtropicalis, P. graminis f. sp. tropicalis and P. graminis f. sp. colombiana.

Differences in temperature requirements between Polymyxa sp. of Indian origin and Polymyxa graminis and Polymyxa betae from temperate areas

The temperature requirements of three single cystosorus strains of Polymyxa sp. from India were studied at 15–18, 19–22, 23–26 and 27–30 °C (night-day temperature), and compared with the temperature requirements of three strains of P. graminis from Belgium, Canada and France and two strains of P. betae from Belgium and Turkey. Sorghum was used as the host-plant for the Indian strains; the strains of P. graminis and P. betae from temperate areas were cultivated on barley and sugar beet, respectively. The cystosori germination and the development of plasmodia, zoosporangia and cystosori of Polymyxa sp. from India were optimal at 27–30 °C. Infection progression was slower at 23–26 °C than at 27–30 °C. At 19–22 °C, infection was insignificant. No infection occurred below 19 °C. In contrast, the infection of barley with P. graminis strains from temperate areas was optimal at 15–18 °C, but at 19–22 °C the progression appeared inconsistent and infection stayed low. Above 22 °C, infection was insignificant. P. betae strains showed consistent infection in the range of 15–18 °C to 27–30 °C. Plasmodia formation and cystosori detection of the Belgian strain were slightly advanced at 23–26 °C compared to 19–22 °C but clearly restrained at 27–30 °C. Fungus development of the P. betae strain from Turkey was almost as high at 27–30 °C as at the lower temperatures. These results strengthen the case for distinguishing between Polymyxa sp. from India and P. graminis or P. betae from temperate areas.

Assessment of energy crops alternative to maize for biogas production in the Greater Region

The biomethane yield of various energy crops, selected among potential alternatives to maize in the Greater Region, was assessed. The biomass yield, the volatile solids (VS) content and the biochemical methane potential (BMP) were measured to calculate the biomethane yield per hectare of all plant species. For all species, the dry matter biomass yield and the VS content were the main factors that influence, respectively, the biomethane yield and the BMP. Both values were predicted with good accuracy by linear regressions using the biomass yield and the VS as independent variable. The perennial crop miscanthus appeared to be the most promising alternative to maize when harvested as green matter in autumn and ensiled. Miscanthus reached a biomethane yield of 5.5 ± 1 × 10(3)m(3)ha(-1) during the second year after the establishment, as compared to 5.3 ± 1 × 10(3)m(3)ha(-1) for maize under similar crop conditions.

Fusarium head blight and associated mycotoxin occurrence on winter wheat in Luxembourg in 2007/2008.

Fusarium head blight (FHB) is among the major causes of reduced quality in winter wheat and its products. In addition, the causal fungi produce a variety of toxins. A relatively high FHB infection rate in winter wheat was observed in 2007 and 2008 in Luxembourg. A fusariotoxin survey was carried out in 17 different geographical locations. Three groups of Fusarium mycotoxins (trichothecenes A and B and zearalenone) were analysed by a multi-detection HPLC–MS/MS method. Fusarium strains were also investigated by morphological and molecular methods. In addition, questionnaires relating to cultural practices were sent to the farmers managing the 17 fields investigated. FHB prevalence ranged from 0.3 to 65.8% (mean: 8.5%) in 2007 and from 0 to 24.5% (mean: 8.3%) in 2008. Results of morphological and molecular identification showed that the most common species isolated from diseased wheat spikes was F. graminearum (33.1%), followed by F. avenaceum (20.3%) and F. poae (17.8%). The chemical analysis revealed that 75% of the investigated fields were contaminated by deoxynivalenol (DON, range 0–8111 µg/kg). The preceding crop was highly and significantly correlated to the number of grains infected and had a significant impact on disease prevalence (p = 0.025 and 0.017, respectively, Fishers F-test). A trend was found for maize as the preceding crop (p = 0.084, Tukeys test) to predict the amount of DON in the fields. This is the first report on the occurrence of DON and ZON in naturally infected wheat grains sampled from Luxembourg.

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 models 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.

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.

Host range of tropical and Sub-tropical isolates of polymyxa graminis

The host range of Polymyxa graminis isolates originating from peanut clump-infested areas in India (Andhra Pradesh and Rajasthan), Pakistan and Senegal was studied on monocotyledonous and dicotyledonous cultivated species, using known quantities of sporosori as inoculum. Profuse multiplication occurred only on some graminaceous species, but the various isolates showed different host specificity. All the isolates produced high infection on sorghum and pearl millet, and all but one isolate from Rajasthan infected maize. Wheat, rye and barley were susceptible to some of the tested isolates. The isolates from Rajasthan and Pakistan produced moderate to severe infection on at least one of these species. On rice, groundnut and sugar beet, only traces of infection by some isolates were detected, whereas no infection was observed on mustard and sunflower. Differences of susceptibility in Pennisetum spp. and Sorghum spp. were demonstrated. The variations in host specificity among isolates from peanut clump-infested areas may result from an adaptation of P. graminis populations to various biotopes. The implications of these results for the management of peanut clump disease are discussed. A comparison of the host ranges of isolates of P. graminis and P. betae from temperate areas demonstrated that distinct types of Polymyxa might be identified based on their relative ability to multiply on susceptible species. Nevertheless, overlapping in the host ranges among the different Polymyxa types, characterised by distinct ecological and genomic features, raises doubts about the host range as a classification criterion for the Polymyxa genus.

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.

Start-up phase of an anaerobic full-scale farm reactor - Appearance of mesophilic anaerobic conditions and establishment of the methanogenic microbial community.

The goal of this study was to investigate how the microbial community structure establishes during the start-up phase of a full-scale farm anaerobic reactor inoculated with stale and cold cattle slurry. The 16S/18S high-throughput amplicon sequencing results showed an increase of the bacterial, archaeal and eukaryotic diversity, evenness and richness during the settlement of the mesophilic anaerobic conditions. When a steady performing digestion process was reached, the microbial diversity, evenness and richness decreased, indicating the establishment of a few dominant microbial populations, best adapted to biogas production. Interestingly, among the environmental parameters, the temperature, alkalinity, free-NH3, total solids and O2 content were found to be the main drivers of microbial dynamics. Interactions between eukaryotes, characterized by a high number of unknown organisms, and the bacterial and archaeal communities were also evidenced, suggesting that eukaryotes might play important roles in the anaerobic digestion process.

Assessment of factors influencing the biomethane yield of maize silages

A large set of maize silage samples was produced to assess the major traits influencing the biomethane production of this crop. The biomass yield, the volatile solids contents and the biochemical methane potential (BMP) were measured to calculate the biomethane yield per hectare (average=7266m(3)ha(-1)). The most influential factor controlling the biomethane yield was the cropping environment. The biomass yield had more impact than the anaerobic digestibility. Nevertheless, the anaerobic digestibility of maize silages was negatively affected by high VS content in mature maize. Late maturing maize varieties produced high biomass yield with high digestibility resulting in high biomethane yield per hectare. The BMP was predicted with good accuracy using solely the VS content.