Trichoderma atroviride as a promising biocontrol agent in seed coating for reducing Fusarium damping-off on maize.

AIMS: The objective of this work was to identify a fungal strain showing potential biocontrol abilities against two Fusarium damping-off agents and to test it as a Biological Control Agent (BCA) in maize seed coating under field conditions. METHODS AND RESULTS: A collection of native fungal strains associated with maize in Belgium was screened for antagonistic potential against Fusarium avenaceum and Fusarium culmorum. The strain with highest biocontrol potential was identified as an endophytic Trichoderma atroviride BC0584. In greenhouse, it significantly improves the emergence of seedlings infected by F. avenaceum or F. culmorum pathogens. In most field trials carried out during the season 2017, it significantly increased the emergence rate of infected seedlings compared to untreated seeds. One slurriable powder formulation allows BCA conidia to survive over a 6-month storage period at 4°C. CONCLUSIONS: The fungal BC0584 strain is a promising BCA that could be an alternative to synthetic fungicides. It is adapted to local environmental conditions, is easily and cheaply produced and can be stored in a low-cost formulation. SIGNIFICANCE AND IMPACT OF THE STUDY: In Belgium, this is the first study to use a T. atroviride native strain against Fusarium damping-off on maize crop. Modes of action and required conditions for ensuring high biocontrol activity in the field have still to be investigated.

Sexual reproduction of Zymoseptoria tritici on durum wheat in Tunisia revealed by presence of airborne inoculum, fruiting bodies and high levels of genetic diversity.

Septoria tritici blotch (STB) caused by the heterothallic ascomycete Zymoseptoria tritici is currently one of the most devastating diseases of wheat worldwide. The extent of sexual reproduction of this pathogen is well documented on bread wheat, but not on durum wheat. The objective of the present study was to quantify the occurrence of Z. tritici sexual reproduction on durum wheat in the Tunisian environment. The assessment was undertaken using a triple approach combining fruiting body assessment, ascospore trapping and population genetic analyses. The results highlighted the formation of pseudothecia on leaves and stubble from the autumn until the end of the growing season. Likewise, qPCR monitoring highlighted a constant release of Z. tritici airborne inoculum during the wheat-growing season, with a peak of production at the end of the season. Genetic investigations using microsatellites revealed high levels of gene and genotypic diversities, an equal distribution of mating types, and a lack of genetic clustering within and between growing seasons. Taken together, these findings indicate that Z. tritici undergoes sexual reproduction on durum wheat in Tunisia at least to the same extent than on bread wheat in Western Europe, and that the dry and warm climate does not affect the mating process of the fungus. Frequent occurrence of sexual reproduction is a valuable knowledge to take into account in STB control strategies on durum wheat.

NEW BNYVV P25 VARIANTS IN BELGIUM.

Rhizomania is a widespread viral plant disease of major importance in sugar beet cropping and breeding. It is caused by the Beet necrotic yellow vein virus (BNYVV), a Benyvirus transmitted by the soil inhabiting plasmodiophorid Polymyxa betae. This vector also transmits other sugar beet virus such as Beet virus Q (BVQ) and Beet soil-borne virus (BSBV). Despite identification of resistance genes, BNYVV remains a major constraint because of resistance-breaking events as well as its ability to survive for long periods in soils in resting spores of P. betae. During the 2014 growing season, severe rhizomania symptoms were detected in Rz1 resistant beet genotypes in ten Belgian fields suggesting resistance-breaking events. Plants from these fields were sampled and total RNA was extracted from root hairs. The presence of BNYVV, BSBV, BVQ and P. betae was assessed by multiplex RT-PCR. Samples were then tested for the presence of BNYVV RNA5 and RNA3 by RT-PCR respectively targeting P26 and P25 genes. PCR products from P25 gene were then purified and sequenced. The results confirmed the presence of P. betae, BSBV and BVQ in all samples. BNYVV was detected in nine fields. Sequencing of P25 partial cDNA sequences revealed the presence of BNYVV types A and B. Two isolates possessed the amino acids motifs AYPR in the so-called tetrad region aa67-70. This motif was previously associated with resistance-breaking events. The Belgian situation will be discussed in the light of the current situation in neighbouring countries.

FUSARIUM HEAD BLIGHT SYMPTOM DISCRIMINATION: A USEFUL TOOL IN THE FIELD EVALUATION OF FUNGICIDE TREATMENTS.

Fusarium head blight (FHB) is one of the major diseases affecting wheat. It is caused by a complex of fungal species, resulting in yield losses and health problems due to mycotoxin production. The presence of multiple fungal species on wheat ears, with varying responses to active fungicide ingredients used in the field, makes the disease difficult to manage. In order to evaluate the efficacy of the timing of applications (at GS 39, GS 61 and GS 39+61) of a prothioconazole + fluoxastrobin-based fungicide, a 2-year field trial was conducted in Belgium. In both years, applications at GS 61 and GS 39+61 resulted in a significant reduction in symptom severity on ears and in deoxynivalenol (DON) content compared with the untreated control in 2013. In 2012, when Microdochium spp. were the most prevalent species, the treatment at GS 39 significantly reduced ear symptoms. Fusarium graminearum was predominant in the second year (2013) and caused significant DON accumulation in the grain after a single foliar spraying. The two genera were characterized by distinct types of symptoms: grouped bleached spikelet's for F. graminearum and isolated bleached spikelet's for Microdochium spp. This difference enabled the significant effect of the double treatments on symptoms caused by Microdochium spp. to be determined in the second year. This effect, which was also visible on leaf symptoms, suggests that Microdochium spp. epidemics in wheat might be polycyclic. Discrimination between symptoms caused by F. graminearum and Microdochium spp. could be a useful tool to study FHB management using fungicide treatments.

Spore traps network: a new tool for predicting epidemics of wheat yellow rust.

A network of Burkard 7-day spore-recording traps was set up in the Walloon Region in Belgium to monitor the airborne inoculum of wheat pathogens. The relationship between the airborne inoculum of Puccinia striiformis f.sp. tritici, the causal agent of stripe rust, and the disease incidence on plants in untreated plots located near each spore traps was studied during the 2008-2009 season. The presence of airborne inoculum was tested in four locations on tapes collected from the Burkard spore traps from 1 April to 14 June 2009. Total DNA from each fragment of spore trap tape corresponding to 1 day sampling was extracted. P. striiformis f.sp. tritici was quantified by real-time polymerase chain reaction (PCR) assay using specific primers and SYBRGreen. The airborne inoculum of P. striiformis was first detected between 7 and 13 April 2009, depending on the location in the Walloon Region. The first symptoms of stripe rust were observed in the fields between 15 May and 2 June 2009. The onset of the disease symptoms was always preceded by a higher peak of airborne inoculum about 15 days earlier. When P. striiformis f.sp. tritici was detected, the daily quantities of spores, collected from a volume of air of 14.4 m3, fluctuated between 0.23 and 154.66. This study shows that spore traps coupled with real-time PCR could be used to assess the airborne inoculum of P. striiformis in order to understand and predict stripe rust outbreaks.

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.

Arabidopsis thaliana, a new tool to investigate Polymyxa betae-host interactions.

Little is known about the genome of Polymyxa betae and its interactions with sugar beet, due partly to the obligate nature of the protist and the patents on Beta vulgaris sequences. The identification of an ecotype of Arabidopsis thaliana compatible with the protist would help to improve this knowledge. The infection and development of P. betae in 14 worldwide ecotypes of A. thaliana were studied. The detection of plasmodia and resting spores and the production of zoospores in the roots of A. thaliana were obtained in three bioassays, using automatic immersion systems and individual glass tubes. Detection was done using molecular detection and microscopy. Compatible interactions were established between 13 A. thaliana ecotypes of the 14 that were tested and the monosporosoric Belgian strain of P. betae, A26-41. The ecotype Cvi-0 (N1096), from the Cape Verde Islands, was the most compatible with the protist. This ecotype is also susceptible to Plasmodiophora brassicae, another plasmodiophorid. Polymyxa betae infection in A. thaliana was relatively very low compared with B. vulgaris, but every stage of the life cycle of the protist was present. The spore-forming phase was promoted at the expense of the sporangial phase, probably caused by the stress of this new environment. In addition, the protist revealed a new phenotype. This new model study will allow molecular tools available for A. thaliana to be used in order to gain a better understanding of the P. betae-plant interaction during the spore-forming phase.

Distribution of airborne Mycosphaerella graminicola inoculum at the field scale.

A network of 10 Burkard 7-day spore-recording traps was set up in the Walloon region in Belgium to monitor the airborne inoculum of wheat pathogens. Three spore traps were used to analyse the distribution of Mycosphaerella graminicola inoculum at the field scale, at 1 m above ground level. Two traps were set up in a wheat field 100 m apart. The third trap was placed 70 m away in a sugar beet field adjacent to the wheat field. Total DNA from each fragment of spore trap tape corresponding to 1 day sampling was extracted and the quantity of M. graminicola was assessed using real-time polymerase chain reaction (PCR) assay. The experiment was conducted from July to October 2009. Positive detections were obtained for between 33 and 36 days, depending on the spore traps. When detected, the daily quantities of cDNA, collected from a volume of 14.4 m3, fluctuated between 4.84E+00 and 6.10E+03. Correlation coefficients higher than 0,82 and no significant differences were observed between the quantities of M. graminicola collected by the three spore traps, indicating that, at 1 m above ground level, the distribution of inoculum can be considered as homogenous at the tested field scale. This study confirms that spore traps coupled with real-time PCR could be used to assess the airborne inoculum of M. graminicola and to understand the development of the disease at this scale.

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.

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.

Peanut Clump virus transmission by Polymyxa graminis under controlled conditions.

More than fifteen soil-borne viruses belonging to the Beny-, Bymo-, Furo- or Pecluvirus, causing diseases on cereals and groundnut, are transmitted by the soil-borne protist root endoparasite Polymyxo graminis. Five special forms are distinguished within this species on the basis of their specific ecological and molecular characteristics, but the specificity of the transmission of the viruses by these forms has been Little investigated. In order to analyse the virus-vector interaction, the transmission assay of the Peanut clump virus by P. graminis f.sp. tropicalis has been conducted under controlled conditions. The major difficulty to be overcome was to combine high levels of PCV and vector infection in the same living plant. This was achieved by using cuttings of PCV-infected sugarcane (variety CP-89327) originating from Burkina Faso showing the typical red leaf mottle symptom as a source for the virus. A culture of P. graminis f.sp. tropicalis isolated from a PCV-infested soil from Niger was used as the vector. Systemic PCV infection in sugarcane at 25-30 degrees C allows plants with new young roots infected by the virus to be produced by placing cuttings of PCV-infected sugarcane in Hoagland nutrient solution for 21 days. These plants were inoculated with aviruliferous zoospores of P. graminis f.sp. tropicalis produced on infected pearl millet roots and maintained in an automatic immersion system. After 21 days' incubation at 25-30 degrees C with a 12 hour photoperiod, zoosporangia were observed in the roots of sugar cane. The zoospores released from these roots were used to infect healthy young pearl millet plants. Twenty days postinoculation, PCV was detected by RT-PCR in roots of inoculated plants. The vector was clearly identified in the roots by microscopy. This result reveals that a PCV isolate from red leaf mottle sugar cane is transmissible by a P. graminis f.sp. tropicalis isolate from a peanut clump infested field. This method should be helpful in the analysis of the specificity of pecluvirus transmission by P. graminis f.sp. tropicalis and P. graminis f.sp. subtropicalis.

Barley yellow mosaic virus is overcoming RYM4 resistance in Belgium.

Barley yellow mosaic virus (BaYMV) is the causal agent of a soil-borne systemic mosaic disease on barley. It has been reported in Belgium since the 1980s. The control of this disease is managed almost exclusively through the use of resistant varieties. The resistance of most commercial barley cultivars grown in Europe is conferred mainly by a single recessive gene, rym4. This monogenic resistance provides immunity against BaYMV pathotype 1 and has been mapped on barley chromosome 3HL and shown to be caused by mutations in the translation initiation factor eIF4E. Another pathotype, BaYMV pathotype 2, which appeared in the late 1980s (in Belgium, in the early 1990s), is able to overcome the rym4-controlled resistance. Until recently, this pathotype remained confined to specific locations. During a systematic survey in 2003, mosaic symptoms were observed only on susceptible barley cultivars collected in Belgian fields. BaYMV was detected by ELISA and RT-PCR on the susceptible cultivars and only by RT-PCR on the resistant cultivars. In 2004, mosaic symptoms were observed on susceptible and resistant cultivars. BaYMV was detected by ELISA and RT-PCR on both cultivars. In addition to developing RT-PCR methods for detecting and identifying BaYMV and Barley mild mosaic virus (BaMMV), an RT-PCR targeting the VPg/NIa viral protein part of the genome, known to discriminate the two BaYMV pathotypes, was set up to accurately identify the pathotype(s) now present in Belgium. The sequences from the generated amplicons revealed the single nucleotide substitution resulting in an amino acid change from lysine to asparagine specific to BaYMV pathotype 2. The possible reasons for the change in the BaYMV pathotype situation in Belgium, such as climatic change or a progressive build-up of soil inoculum potential, will be discussed, as well as the use of eIF4E-based resistance.

A certain but non-exclusive association between Polymyxa graminis special forms and cereals.

Polymyxo graminis, a ubiquitous plasmodiophorid obligate root endoparasite, is recognized as the vector of about 15 viruses on cereals and groundnut in temperate and tropical areas. Within the species, five special forms have been distinguished on the basis of specific ribotypes. Three of them occur in tropical areas: P. graminis f.sp. colombiana on rice, P. graminis f.sp. subtropicalis on cereals cropped in the tropics such as maize, pearl millet and sorghum but also on barley and/or wheat, and P. graminis f.sp. tropicalis mainly on maize, pearl millet and sorghum. Their particular host ranges distinguish them significantly from P. graminis f.sp. temperata and P. graminis f.sp. tepida found in temperate areas on barley and wheat. In order to assess whether these special forms commonly infect these cereals, barley and wheat plants were grown under controlled conditions on two soils from Belgium and France and both infested by P. graminis f.sp. temperata and P. graminis f.sp. tepida. The infection of each cereal species by each form was quantified by real-time quantitative PCR with specific primers and Taqman probes. The infection of P. graminis f.sp. temperata was significantly higher on barley than on wheat, whereas the quantities of P. graminis f.sp. tepida on wheat were higher than on barley. These results show that the distinction between these special forms, based on the ribotype, reflects differences in ecological features.

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.

[Etiology of the sugar beet rhizomania]. / Étiologie de la rhizomanie de la betterave sucrière.

Beet necrotic yellow vein virus is responsible for sugar beet rhizomania. Root proliferation is characteristic of the viral infection and lead to sugar losses. Pathogenicity is particularly linked to the expression of RNA-3-encoded p25. The extensive use of viral tolerant crops allows maintenance of sugar yields but also permits viruliferous vector to be maintained and therefore the appearance of resistance breaking isolates. The resistance breaking isolates present some amino acid variations within the p25 protein sequence, a key determinant in BNYVV pathogenicity. Here, we will review the molecular biology of BNYVV, of its vector and the antiviral strategies that may be used against rhizomania.

Widespread Occurrence of Wheat spindle streak mosaic virus in Belgium.

In order to assess the occurrence of Wheat spindle streak mosaic virus (WSSMV) in Belgium, a reverse-transcription polymerase chain reaction (RT-PCR) was developed, targeting WSSMV isolates from Canada, France, Germany, Italy, and the United States. The primers also were designed for virus quantification by real-time RT-PCR with SYBR-Green. No cross-reaction with soilborne cereal viruses such as Barley mild mosaic virus, Barley yellow mosaic virus, Soilborne cereal mosaic virus, and Soil-borne wheat mosaic virus was observed. The RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection of WSSMV than enzymelinked immunosorbent assay. The incidence of WSSMV in Belgium was evaluated using a bioassay with wheat cvs. Cezanne and Savannah and rye cv. Halo, grown in 104 Belgian soils. The presence of WSSMV was detected from plants grown in 32% of the soils. The RT-PCR methods developed here, combined with large sampling, allowed WSSMV to be detected for the first time in Belgium. The real-time quantitative RT-PCR was developed as a tool for evaluating the resistance to WSSMV by quantifying the virus concentration in wheat cultivars.

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.

[Evaluation of methods of sperm preparation for human in vitro fertilization]. / Evaluation des méthodes de préparation du sperme pour la fécondation in vitro humaine.

OBJECTIVES: Compare results of sperm prepared with centrifiguation-migration, percoll discontinuous gradient (50, 70, 90%) and migration-sedimentation. METHODS: An aliquot of sperm collected from 125 men referred for infertility was prepared according to the three methods. The spermogram after preparation and extration rates were compared using the Student's t test for paired variables and for three subpopulations identified by increasing spermatozoid concentrations. RESULTS: Sperm count were significantly higher after percoll. Mobility and morphology were improved after centrifugation-migration excepting for the samples with the lowest counts. The extration rate was better after percoll and rose for the low-count samples. CONCLUSION: Percoll gradient is the most effective method, particularly for altered sperm. The number of mobile and normal forms can be improved with the centrifugation-migration technique.

Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization.

This study compared swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Sixty trials of in-vitro fertilization (IVF), 38 with normal semen and 22 with abnormal semen, comprising 734 oocytes were included in the study. Each semen sample was prepared by both a swim-up technique and a simplified discontinuous (50%, 70%, 90%) Percoll gradient. The oocytes for each trial were distributed at random between the two sperm preparations and incubated with the same number of motile spermatozoa. Percoll gradient preparation produced a significantly higher final concentration of spermatozoa than swim-up preparation (mean +/- SEM: 6.6 +/- 1.5 x 10(6)/ml versus 1.9 +/- 0.2 x 10(6)/ml; P less than 0.01) but a significantly lower sperm motility (69 +/- 2% versus 94 +/- 1%; P less than 0.001) and a lower number of normal forms (55 +/- 2% versus 64 +/- 2%; P less than 0.01). The ability of the Percoll gradient method to extract motile spermatozoa was higher than that of the swim-up technique (20 +/- 15.6% versus 0.8 +/- 13.6%). Nevertheless, the rates of fertilization (61%), fertilization failure (18%) and polyspermia (9%), embryo quality evaluated by mean embryo scores (3.8 +/- 0.3) and the mean number of spare embryos frozen per trial (1.4 +/- 0.3) were strictly identical in both groups. The 24 pregnancies (including three from frozen--thawed embryos) obtained in these 60 trials (40% per oocyte retrieval) could not be separated according to the sperm preparation method, as embryos from both groups were replaced together.(ABSTRACT TRUNCATED AT 250 WORDS)