Anne Legrève

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.

Systemic resistance induced by Bacillus lipopeptides in Beta vulgaris reduces infection by the rhizomania disease vector Polymyxa betae.

The control of rhizomania, one of the most important diseases of sugar beet caused by the Beet necrotic yellow vein virus, remains limited to varietal resistance. In this study, we investigated the putative action of Bacillus amylolequifaciens lipopeptides in achieving rhizomania biocontrol through the control of the virus vector Polymyxa betae. Some lipopeptides that are produced by bacteria, especially by plant growth-promoting rhizobacteria, have been found to induce systemic resistance in plants. We tested the impact of the elicitation of systemic resistance in sugar beet through lipopeptides on infection by P. betae. Lipopeptides were shown to effectively induce systemic resistance in both the roots and leaves of sugar beet, resulting in a significant reduction in P. betae infection. This article provides the first evidence that induced systemic resistance can reduce infection of sugar beet by P. betae.

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.

Identification and Quantification of Polymyxa graminis f.sp. temperata and P. graminis f.sp. tepida on Barley and Wheat

Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida are distinguished on the basis of their specific ribosomal DNA sequences. In order to evaluate whether or not host specialization is associated with the special form, the occurrence of infection of both forms on barley and wheat was studied. P. graminis inocula were obtained from soils collected in Belgium and France. Their ribotypes were characterized using molecular tools specific to P. graminis f. sp. temperata or P. graminis f. sp. tepida such as restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified rDNA, nested and multiplex PCR. Both special forms were found in each country and coexisted in some soils. The host specificity of P. graminis special forms for barley and wheat was studied from two soils collected at Gembloux (Belgium) and Chambon-sur-Cisse (France), each infested by bymo- and furoviruses. P. graminis f. sp. temperata is more frequent on barley and P. graminis f. sp. tepida on wheat. Furthermore, the quantification of each form on barley and wheat by two separated real-time quantitative PCR assays confirms the observations on the vector specialization. These results suggest a certain but not exclusive host specificity of P. graminis special forms.

Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Mycosphaerella graminicola in Belgium

Two kinds of propagules play a role in Mycosphaerella graminicola dissemination: splash-dispersed pycnidiospores and airborne sexual ascospores. A method based on real-time polymerase chain reaction (PCR) assay and using Burkard spore traps was developed to quantify M. graminicola airborne inoculum. The method was tested for its reliability and applied in a spore trap network over a 2-year period in order to investigate the spatio-temporal distribution of airborne inoculum in Belgium. At four experimental sites, airborne inoculum was detected in both years. A seasonal distribution was observed, with the highest mean daily quantities (up to 351.0 cDNA) trapped in July and with clusters detected from September to April. The first year of trapping, a mean daily quantity of 15.7 cDNA of M. graminicola airborne inoculum was also detected in the air above a building in a city where the spatio-temporal distribution showed a similar pattern to that in the field. Mean daily quantities of up to 60.7 cDNA of airborne inoculum were measured during the cereal stem elongation and flowering stages, suggesting that it contributes to the infection of upper leaves later in the season. Most detection, however, tended to occur between flowering and harvest, suggesting significant production of pseudothecia during that period. Variations in mean daily quantities from 1.0 to 48.2 cDNA were observed between sites and between years in the patterns of airborne inoculum. After stem elongation, the quantities detected at a site were positively correlated with the disease pressure in the field. Quantities trapped at beginning of the growing season were also well correlated with the disease level the previous year. Multiple regressions revealed that some factors partly explain the daily variations of airborne inoculum.

Genetic Diversity of the Mating Type and Toxin Production Genes in Pyrenophora tritici-repentis

Pyrenophora tritici-repentis, the causal agent of tan spot on wheat, is a homothallic loculoascomycete with a complex race structure. The objectives of this study were to confirm the homothallic nature of the pathogen, characterize mating type diversity and toxin production genes in a global collection of strains, and analyze how these traits are associated between each other and with existing races. The pseudothecia production capacity, race identification, mating type locus (MAT), internal transcribed spacer, and glyceraldehyde-3-phosphate dehydrogenase regions were analyzed in a selection of 88 strains originating from Europe, North and South America, North Africa, and Central and South Asia. Some (60%) strains produced pseudothecia containing ascospores, independent of their origin. Race identification obtained using the multiplex polymerase chain reaction targeting host-selective toxin (HST) genes was consistent, overall, with the results based on the inoculation of a set of differential wheat cultivars and confirmed the predominance of race 1/2 strains ( approximately 83%). However, discrepancies in race identification, differences from the reference tester strains, and atypical ToxA profiles suggest the presence of new races and HSTs. The MAT1-1 and MAT1-2 coding regions are consecutively arranged in a single individual, suggesting putative heterothallic origin of P. tritici-repentis. Upstream from the MAT is an open reading frame of unknown function (ORF1) containing a MAT-specific degenerate carboxy-terminus. The phylogenetic analysis of the MAT locus reveals two distinct groups, unlinked to geographical origin or ToxA profile. Group I, the best-represented group, is associated with typical tan spot lesions caused by races 1, 2, 3, and 5 on wheat. It is more homogenous than group II encompassing race 4 strains, as well as isolates associated primarily with small spot lesions on wheat leaves or other hosts. Group II could contain several distinct taxa.

Silicon Reducs Black Sigatoka development in Banana

The effect of silicon (Si) uptake on the susceptibility of Musa acuminata to Mycosphaerella fijiensis was investigated in three experiments conducted under controlled conditions. Plants were grown in the presence of Si or not, in pots adapted for a hydroponic culture system or in pots filled with compost. The banana leaves were inoculated after 4 or 6 months of plant growth by spraying conidial suspensions or by brushing mycelia fragments. The disease progress over time was assessed using quantitative and qualitative scales. At the end of each experiment, disease severity was also analyzed using the image analysis software ASSESS. The Si concentration in the leaves of plants supplied with Si reached 10 to 28 g/kg of dry matter. The first symptoms appeared 18 days after inoculation. The disease developed more rapidly and more severely on banana plants grown without Si than on plants supplied with Si. The areas under the disease progress curve (AUDPCs) calculated for plants grown with Si were significantly lower than the AUDPCs for plants not supplied with Si, regardless of inoculation method. Thus, Si supply could be a valuable tool in integrated pest management against M. fijiensis by reducing the disease pressure on banana.

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.