The occurrence of vegetative compatibility groups (VCGs) in Beauveria has been examined by analyzing compatible and incompatible reactions between complementary nitrate non-utilizing mutants. Complementary mutants (nia, and cnx) generated from 25 isolates from different host and insect origins were paired in all combinations revealing 14 different VCGs. Strains isolated from European corn borer in different European countries formed a distinct subspecific group whereas Sitona weevil isolates collected in the same country were found in different VCGs. A high level of polymorphism was detected by fingerprinting through Southern blot hybridization with a telomeric probe. Examination of DNA variations among isolates belonging to different VCGs suggested that variations in telomeric patterns was correlated with VCG groupings; these results underlined the existence of genetic limits to gene flow that would result in genetically diverse subpopulations.

The entomopathogenic hyphomycete Paecilomyces fumosoroseus is a promising candidate for biocontrol of economically important agricultural pests. Assessment of genetic relatedness of this species appears to be essential to gain insight into the monitoring of such biocontrol products. Intraspecific variation within the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) was studied in 48 isolates of P. fumosoroseus. These strains were isolated from different geographical and biological origins, more particularly from the silverleaf whitefly, Bemisia tabaci-argentifolii (Homoptera: Aleyrodidae), a major insect pest in field and greenhouse crops. PCR amplification of the ITS1-5.8S-ITS2 rDNA region followed by restriction analysis of the PCR products underlined the overall highly variable nature of this region. Digestion with the six endonucleases AluI, HaeIII, Hin6I, HpaII, NdeII, and SmaI allowed the separation of the isolates into three distinct groups. The group 1, representing 25 isolates, is composed of strains isolated only from the host B. tabaci-argentifolii. By contrast, the group 3 is more diffuse as it included strains from various insect host and geographical origins. Phylogenetic analysis of rDNA-ITS sequence data strongly supported the conclusions of the PCR-RFLP analysis and recognized three monophyletic groups within the P. fumosoroseus complex. The high level of polymorphism within the P. fumosoroseus species is discussed in the light of their biological origin.

ABSTRACT Protoplast fusion of diauxotrophic mutants of a Beauveria bassiana entomopathogenic strain (Bb28) and a Beauveria sulfurescens toxinogenic strain (Bs2) produced hybrids which were significantly different from the parents in pathogenicity. Some of the hybrids were hypervirulent and killed insects more quickly than the Bb28 strain, probably because these hybrids had acquired the toxic activity of the Bs2 strain. By using six nuclear genes and a telomeric fingerprint probe, the molecular structures of the hybrids were studied. The results demonstrated the occurrence of parasexual events. Hybrids appeared to be diploid or aneuploid, with portions of the genome being heterozygous. A mitochondrial molecular marker indicated homoplasmy of the hybrids and inheritance of mitochondria from strain Bs2 or Bb28. The pathogenicities and the ploidies of the hybrids remained stable after passage through the host insect, showing that somatic hybridization provides an attractive method for the genetic improvement of biocontrol efficiency in the genus Beauveria.

Thirty-eight strains of the entomopathogenic Beauveria bassiana, isolated from diverse species of Lepidoptera (Pyralidae) or Coleoptera (Curculionidae, Chrysomelidae, and Scolytidae) from various geographical sites, were examined by RFLP and RAPD analyses. Similar groupings were recovered from both approaches and these showed clear relationships between the population structure of B. bassiana and some defined host species. Strains isolated from members of the Pyralidae were recovered as two main groups, one group consisted of all strains isolated from Ostrinia irrespective of their origin. The second group consisted primarily of strains isolated from Diatraea in Cuba. All strains isolated from the curculionid genus Sitoma clustered as a single distinct group. separated from strains from other curculionids. In contrast, strains isolated from the pyralid genus Maliarpha, and the coleopteran Chrysomelidae, gave heterogenous patterns and were not recovered as distinct groups. Groups from duster analysis and nonhierarchic ordination methods were compared and the relative merits of the different grouping strategies are discussed.

Erynia neoaphidis is the most widespread aphid pathogen in temperate regions. A few authors have identified this species as infecting some non-aphid hosts. It exhibits high variability with respect to morphology and cultural characteristics that underlines the need for assessment of the genetic variability within this species. We used size of PCR-amplified ribosomal DNA internal transcribed spacer (ITS) coupled with RAPD-PCR pattern in order to investigate genetic variability within a sample of 30 isolates identified as E. neoaphidis and one isolate of E. kondoiensis from diverse countries and hosts. Isolates fell into four discrete ITS size groups: 575 base pairs (bp) in isolates from non-aphid hosts, 1000 (including the E. kondoiensis isolate), 1100, or 1450 bp in those from Aphididae. The 1100-type seems to be the one of most widespread, since two-thirds of the isolates fr om diverse locations belong to this type. ITS types can be correlated with morphological and physiological characteristics. A great deal of genetic variability exists within ITS types, as shown by RAPD-PCR. RAPD groupings could be related with geographical origin in some cases. There was no apparent relationship between host and ITS type or RAPD pattern among isolates from Aphididae. PCR-based technologies provide evidence that could be used to clarify the taxonomy of E. neoaphidis and closely related species, and will help identify markers related to epidemiological characteristics.

Impact of 3 control methods on larval European corn borer, Ostrinia nubilalis Hubner, dynamics on com, Zea mays (L.), was evaluated under field conditions at Versailles, France. The control methods studied were a chemical insecticide, Beauveria bassiana Vuillemin (Deuteromycotina: Hyphomycete), and a transgenic corn hybrid. The experimental study showed that B. bassiana control was similar to chemical control. The transgenic hybrid control was always very high throughout the corn cycle studied. A substantial decrease of B. bassiana and chemical control efficacy was observed with an increase in the delay between treatment and infestation. The complementary studies of B. bassiana persistence, control impact, and pathogen contact showed control-larval behavior interactions, which could explain this decrease in efficacy. To take into account the main factors that condition control efficacy, a modular and mechanistic model was proposed to describe larval dynamic and control impact. The proposed control model made it possible to integrate O. nubilalis dynamics, and thus to describe the time response of control.

Viaud, M., Couteaudier, Y., Levis, C., and Riba, G. 1996. Genome organization inBeauveria bassiana:Electrophoretic karyotype, gene mapping, and telomeric fingerprint.Fungal Genetics and Biology20,175–183. Using telomeric fingerprint and pulsed-field gel electrophoresis, we have analyzed the chromosomal organization among nine entomogenousBeauveria bassianaisolates. A heterologous telomeric probe established that the minimum number of chromosomes was between seven and eight among isolates. Chromosomal DNA was electrophoresed by using contour-clamped homogenous electric field (CHEF) gel electrophoresis. Five to eight chromosomal bands were separated among isolates. Substantial variation in band intensity suggested the presence of some chromosome doublets. The deduced karyotypes were in agreement with the minimum chromosome number reported from telomeric fingerprints. The minimum total genome size was estimated to range from 34.3 to 44.1 Mb. Significant chromosome length polymorphism was observed in karyotypes in the speciesB. bassianaexcept within isolates from one insect,Ostrinia nubilalis.Specific hybridization to chromosomes on CHEF gels was obtained by using homologous genes for proteinase 1, 28S rDNA, β-tubulin, histone 4, nitrate reductase, and the mitochondrial small rDNA. The mitochondrial DNA had a slow mobility in comparison to its circular size, probably because of the presence of very long linear molecules. Comparison of karyotype profiles following Southern analysis demonstrated a very flexible chromosomal organization among isolates from different origins and has confirmed the similarities withinO. nubilalisisolates.

Genetic improvement of two different strains of the entomopathogenic fungus Beauveria bassiana for more effective control of Ostrinia nubilalis and Leptinotarsa decemlineata was obtained by crosses with the insecticidal toxin-producing strain Beauveria sulfurescens. Protoplast fusion between diauxotrophic mutants resulted in the recovery of some stable prototrophic fusion products. The low levels of virulence of the wild type strain B. bassiana 28 isolated originally from L. decemlineata were enhanced both on L. decemlineata and O. nubilalis for one of the hybrids obtained (FP 8) from the cross B. bassiana 28xB. sulfurescens 2. Fusion product 25 obtained from the cross between B. sulfurescens and the highly pathogenic strain B. bassiana 147 showed a three-day reduction in the LT50 towards O. nubilalis. Southern blot hybridization with nine probe-enzyme combinations were conducted on genomic DNAs from the original wild strains, parental mutant strains, and fusion products. Additive banding patterns or unique banding pattern of either parental strain was observed in five hybrids, indicating their status as recombinant and/or partially diploid. Combination of RFLP markers indicative of both parental genomes was never observed with fusion product FP 25. The stability of the virulence following passage through insect-host and stability of molecular structure for the fusion products FP 8 and FP 25 suggest that asexual genetic recombination by protoplast fusion may provide an attractive method for the genetic improvement of biocontrol efficiency in entomopathogenic fungi.

The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the beta-D-glucuronidase gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as the selectable marker. The frequency of cotransformation, as determined by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS+), was very high (up to 75%). Southern hybridization analyses of GUS+ transformants revealed that single or multiple copies of the gusA gene were integrated into the genomes. High levels of GUS activity are expressed in some transformants, but activity in F. oxysporum does not appear to be correlated with the copy number of the gusA gene. Since the highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of F. oxysporum integrated upstream of the gene can act as a promoter or enhancer. Expression of the gusA gene was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a sensitive and easy reporter gene assay in F. oxysporum.

as determined byGUS expression on plates containing mediumsupplemented with5-bromo-4-chloro-3-indolyl glucuronide (GUS'), was very high(upto 75%).Southern hybridization analyses ofGUS'transformants revealed thatsingle or multiple copies ofthe gusAgenewereintegrated into thegenomes. Highlevels ofGUSactivity areexpressed insome transformants, butactivity inF.oxysporum doesnotappear tobecorrelated withthecopy numberofthe gusA gene.Since the highest activity was foundina transformant witha single copy,itcan beassumedthat sequence elements of F.oxysporum integrated upstreamofthegene can actas a promoter orenhancer. Expression ofthegusA gene was alsodetected during growthofthefungus inplants, indicating thattheGUS systemcan beusedas a sensitive andeasyreporter gene assayinF.oxysporum. TheGUS gene fusion system(Escherichia coliP-D-glUCU- ronidase gene) was described byJefferson (14,15)as a powerful toolfortheassessmentofgene activity intrans- genicplants andforthedevelopment ofmolecular genetic analysis. Theutility ofthissystemhasbeenshownfor bacteria, animals, andplants (15)and,more recently, for yeastsandfilamentous fungi (4,20,21,23,25).Thefirst report on theconstruction andtesting ofaplasmid express- ingGUS infilamentous fungi (23) suggested thatthesystem couldbedeveloped forstudies on disease spread andfungal biomass quantification. Ourobjective was toapplythistracking concepttothe important plantpathogen Fusariumoxysporum.Indeed, difficulties areencountered inmonitoring populations ofaF. oxysporum strain inassociation withrootsbecauseofthe presenceoftheF.oxysporumstrain along withmany other fungal species andotherstrains ofthesame species. Isola- tionon selective mediaofa markedstrain by using a pigmentation marker orresistance tobenomyl hasbeenused forthis purpose(2,24), butthistechnique doesnotenable visualization ofhyphae attherootsurface andquantification ofrootcolonization bypathogenic ornonpathogenic strains. Inthis paperwe describe thesuccessful transformation of strains ofF.oxysporum withthegusAgeneincombination withthenitrate reductase structural gene ofAspergillus nidulans (niaD) astheselectable marker(18). Theusefulness oftheGUS genefusion systemforstudies on rootcoloniza- tionbyF.oxysporum was thentested inpreliminary exper- iments byassaying GUS activity invitro andinvivoinorder tolocalize andquantify theactive biomassofthefungus in roots.Moreover, our dataon themolecular structure of

Different mechanisms have been identified or proposed to explain biological control of Fusarium wilt by nonpathogenic strains of Fusarium oxysporum (Alabouvette, 1990; Mandeel and Baker, 1991). One of them is related to competition for nutrients in soil and rhizosphere. This hypothesis was proposed as the result of studies of suppressive soils. In order to explain the suppressiveness of the soil from Châteaurenard, Rouxel et al. (1979) first suggested the existance of intrageneric and/or intraspecific competition between pathogenic and nonpathogenic fusaria; thereafter, it was proposed that suppression was due to nutrient competition between pathogenic and nonpathogenic Fusarium oxysporum (Alabouvette et al., 1984). Recently, Mandeel and Baker (1991) examined the possibility of the association of this mechanism with biological control of Fusarium wilt of cucumber by two nonpathogenic strains of Fusarium oxysporum.