Metarhizium spp. isolates from madagascar: Morphology and effect of high temperature on growth and infectivity to the migratory locust,Locusta migratoria

Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams &; Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.     

Isolation of entomopathogenic fungi from Northern Thailand and their production in cereal grains

Spore productivity in six entomopathogenic fungal strains isolated from insect cadavers at four locations in Chiang Mai province was evaluated in five cereal grains: white-rice, wheat, rye, corn and sorghum. According to sequence analysis of the internal transcribed spacer regions of these isolates, they were closely related to Beauveria bassiana (2 isolates), Metarhizium flavoviride (1 isolate), Metarhizium anisopliae (1 isolate), Paecilomyces lilacinus (1 isolate) and Isaria tenuipes (1 isolate). Among all fungal isolates, the maximum amount of spores (530.0?×?10⁹ conidia/g) was yielded P. lilacinus CMUCDMT02 on sorghum grain followed by white-rice (399.3?×?10⁹ conidia/g). Moreover, the highest number of spore in M. flavoviride was 102.8?×?10⁹ conidia/g sorghum whereas white-rice yielded the greatest amount of spore for B. bassiana CMUCDMF03 (141.0?×?10⁹ conidia/g) after 60?days incubation. The fungal growth rate was found highest in corn for all strains and rye showed the lowest with the exception of P. lilacinus CMUCDMT02 among the tested grains. Spore viability was over 80?% for all isolates that had been inoculated for 60?days. Fungal conidia suspension of P. lilacinus obtained highest virulence against Bactrocera spp. at a concentration of 1?×?10⁶ spore/ml. The strains isolated, exhibited good production of conidia suggesting a promising strategy for the mass production of inoculum as biocontrol agents with low production cost.     

Cytotype regulation by telomeric P elements in Drosophila melanogaster: interactions with P elements from M' strains

P strains of Drosophila are distinguished from M strains by having P elements in their genomes and also by having the P cytotype, a maternally inherited condition that strongly represses P-element-induced hybrid dysgenesis. The P cytotype is associated with P elements inserted near the left telomere of the X chromosome. Repression by the telomeric P elements TP5 and TP6 is significantly enhanced when these elements are crossed into M′ strains, which, like P strains, carry P elements, but have little or no ability to repress dysgenesis. The telomeric and M′ P elements must coexist in females for this enhanced repression ability to develop. However, once established, it is transmitted maternally to the immediate offspring independently of the telomeric P elements themselves. Females that carry a telomeric P element but that do not carry M′ P elements may also transmit an ability to repress dysgenesis to their offspring independently of the telomeric P element. Cytotype regulation therefore involves a maternally transmissible product of telomeric P elements that can interact synergistically with products from paternally inherited M′ P elements. This synergism between TP and M′ P elements also appears to persist for at least one generation after the TP has been removed from the genotype.     

Diversity of Telomere Palindromic Sequences and Replication Genes among Streptomyces Linear Plasmids

Streptomyces sp. linear plasmids and linear chromosomes usually contain conserved terminal palindromic sequences bound by the conserved telomeric proteins Tap and Tp, encoded by the tap and tpg genes, respectively, as well as plasmid loci required for DNA replication in circular mode when the telomeres are deleted. These consist of iterons and an adjacent rep gene. By using PCR, we found that 8 of 17 newly detected linear plasmids in Streptomyces strains lack typical telomeric tap and tpg sequences. Instead, two novel telomeres in plasmids pRL1 and pRL2 from the eight strains and one conserved telomere in pFRL1 from the other strains were identified, while multiple short palindromes were also found in the plasmids. The complete nucleotide sequence of pRL2 revealed a gene encoding a protein containing two domains, resembling Tap of Streptomyces and a helicase of Thiobacillus, and an adjacent gene encoding a protein similar to Tpg of Streptomyces and a portion of the telomere terminal protein pTP of adenoviruses. No typical iterons-rep loci were found in the three plasmids. These results indicate an unexpected diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.     

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32°C; several isolates grew at 8 and 37 °C. None of the isolates grew at 40 °C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 °C with several isolates exhibiting optimal growth at temperatures as high as 32 °C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 °C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 °C. However, the two most cold tolerant isolates at 8 °C were M. flavoviride and the three most heat tolerant at 35 °C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans

Terminal restriction fragment analysis is the only method currently available for measuring telomere length in Caenorhabditis elegans. Its limitations include low sensitivity and interference by the presence of interstitial telomeric sequences in the C.elegans genome. Here we report the adaptation of single telomere length analysis (STELA) to measure the length of telomeric repeats on the left arm of chromosome V in C.elegans. This highly sensitive PCR-based method allows telomere length measurement from as few as a single worm. The application of STELA to eight wild-type C.elegans strains revealed considerable strain-specific differences in telomere length. Within individual strains, short outlying telomeres were observed that were clearly distinct from the bulk telomere length distributions, suggesting that processes other than end-replication losses and telomerase-mediated lengthening may generate telomere length heterogeneity in C.elegans. The utility of this method was further demonstrated by the characterization of telomere shortening in mrt-2 mutants. We conclude that STELA appears to be a valuable tool for studying telomere biology in C.elegans.     

Karyotype Rearrangements in a Wine Yeast Strain by rad52-Dependent and rad52-Independent Mechanisms

Yeast strains isolated from the wild may undergo karyotype changes during vegetative growth, a characteristic that compromises their utility in genetic improvement projects for industrial purposes. Karyotype instability is a dominant trait, segregating among meiotic derivatives as if it depended upon only a few genetic elements. We show that disrupting the RAD52 gene in a hypervariable strain partially stabilizes its karyotype. Specifically, RAD52 disruption eliminated recombination at telomeric and subtelomeric sequences, had no influence on ribosomal DNA rearrangement rates, and reduced to 30% the rate of changes in chromosomal size. Thus, there are at least three mechanisms related to karyotype instability in wild yeast strains, two of them not requiring RAD52-mediated homologous recombination. When utilized for a standard sparkling-wine second fermentation, Δrad52 strains retained the enological properties of the parental strain, specifically its vigorous fermentation capability. These data increase our understanding of the mechanisms of karyotype instability in yeast strains isolated from the wild and illustrate the feasibility and limitations of genetic remediation to increase the suitability of natural strains for industrial processes.     

Factors influencing the recombinational expansion and spread of telomeric tandem arrays in Kluyveromyces lactis

We have previously shown that DNA circles containing telomeric repeats and a marker gene can promote the recombinational elongation of telomeres in Kluyveromyces lactis by a mechanism proposed to involve rolling-circle DNA synthesis. Wild-type cells acquire a long tandem array at a single telomere, while telomerase deletion (ter1-Δ) cells, acquire an array and also spread it to multiple telomeres. In this study, we further examine the factors that affect the formation and spread of telomeric tandem arrays. We show that a telomerase⁺ strain with short telomeres and high levels of subtelomeric gene conversion can efficiently form and spread arrays, while a telomere fusion mutant is not efficient at either process. This indicates that an elevated level of gene conversion near telomeres is required for spreading but that growth senescence and a tendency to elongate telomeres in the absence of exogenously added circles are not. Surprisingly, telomeric repeats are frequently deleted from a transforming URA3-telomere circle at or prior to the time of array formation by a mechanism dependent upon the presence of subtelomeric DNA in the circle. We further show that in a ter1-Δ strain, long tandem arrays can arise from telomeres initially containing a single-copy insert of the URA3-telomere sequence. However, the reduced rate of array formation in such strains suggests that single-copy inserts are not typical intermediates in arrays formed from URA3-telomere circles. Using heteroduplex circles, we have demonstrated that either strand of a URA3-telomere circle can be utilized to form telomeric tandem arrays. Consistent with this, we demonstrate that 100-nucleotide single-stranded telomeric circles of either strand can promote recombinational telomere elongation.     

Development of a Model to Predict the Effect of Temperature and Moisture on Fungal Spore Longevity

A model was developed to quantify the effect of temperatureand moisture content on the longevity of conidia of the entomopathogenicfungusMetarhizium flavoviride . This model incorporated a negativesemi-logarithmic relation between longevity and temperatureand a negative logarithmic relation between longevity and moisturecontent. Replacing the latter with a negative semi-logarithmicrelation between longevity and the equilibrium relative humidityof the conidia was also effective. The latter model was appliedsuccessfully to observations on the survival of conidia of afurther four entomopathogenic fungi (Metarhizium anisopliae,Beauveria bassiana ,Beauveria brongniartii, andPaecilomycesfarinosus ) and ascospores, conidia or uredospores of four phytopathogenicfungi (Alternaria porri ,Helminthosporium oryzae ,Uromyces appendiculatus, andSclerotinia sclerotiorum ) across a wide range of differenttemperatures and relative humidities. The sensitivity of sporelongevity to both temperature and equilibrium relative humidity,and the upper and lower relative humidity limits to the applicationof the model, varied considerably between entomopathogenic andphytopathogenic fungi, among species within each group, andamong different strains within certain species. Metarhizium flavoviride W. Gams & J. Roszypal; entomopathogenic fungi; phytopathogenic fungi; spore survival; storage environment; conidia; uredospore; ascospore     

Telomeric repeats act as nucleosome-disfavouring sequences in vivo

Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG_1-3, which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.     

Telomere Variation in Xenopus laevis

Eukaryotic telomeres are variable at several levels, from the length of the simple sequence telomeric repeat tract in different cell types to the presence or number of telomere-adjacent DNA sequence elements in different strains or individuals. We have investigated the sequence organization of Xenopus laevis telomeres by use of the vertebrate telomeric repeat (TTAGGG)_n and blot hybridization analysis. The (TTAGGG)_n-hybridizing fragments, which ranged from less than 10 to over 50 kb with frequently cutting enzymes, defined a pattern that was polymorphic between individuals. BAL 31 exonuclease treatment confirmed that these fragments were telomeric. The polymorphic fragments analyzed did not hybridize to 5S RNA sequences, which are telomeric according to in situ hybridization. When telomeric fragments from offspring (whole embryos) were compared to those from the spleens of the parents, the inheritance pattern of some bands was found to be unusual. Furthermore, in one cross, the telomeres of the embryo were shorter than the telomeres of the parents’ spleen, and in another, the male’s testis telomeres were shorter than those of the male’s spleen. Our data are consistent with a model for chromosome behavior that involves a significant amount of DNA rearrangement at telomeres and suggest that length regulation of Xenopus telomeres is different from that observed for Mus spretus and human telomeres.     

<Emphasis Type="Italic">Metarhizium bibionidarum</Emphasis> and <Emphasis Type="Italic">M. purpureogenum</Emphasis>: new species from Japan

Two new species of Metarhizium, M. bibionidarum and M. purpureogenum are described from Japan. Metarhizium bibionidarum is the phylogenetic sister species of M. pemphigi and a member of the M. flavoviride species complex. It is distinguished morphologically from M. pemphigi by its larger conidia. The species is based on a collection of an infected March fly larva (Diptera: Bibionidae) but is also known to occur on fruit beetle (Coleoptera: Scarabaeidae) encountered in France. Metarhizium purpureogenum was isolated from soil by plating and insect baiting methods and represents a unique phylogenetic lineage placed outside the M. anisopliae and M. flavoviride species complexes. Three isolates of M. purpureogenum excreted a distinctive red-purple pigment into agar medium when co-cultured with M. robertsii or Aspergillus oryzae.     

Nucleotide Sequence Variation Does Not Relate to Differences in Kinetic Properties of Neutral Trehalase from the Insect Pathogenic Fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Genetic variability in a putative virulence factor, the neutral trehalase (Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., K _m , V _max, and K _cat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower K _cat/K _m . However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.     

Identification of a telomeric DNA-binding protein in Eimeria tenella

Coccidiosis is considered to be a major problem for the poultry industry, and coccidiosis control is yet urgent. Due to the roles in telomere length regulation and end protection, telomere-binding proteins have been considered as a good target for drug design. In this work, a putative Gbp1p that is similar to telomeric DNA-binding protein Gbp (G-strand binding protein) of Cryptosporidium parvum, was searched in the database of Eimeria tenella. Sequence analysis indicated E.tenella Gbp1p (EtGbp1p) has significant sequence similarity to other eukaryotic Gbps in their RNA recognition motif (RRM) domains. Electrophoretic mobility shift assays (EMSAs) demonstrated recombinant EtGbp1p bound G-rich telomeric DNA, but not C-rich or double-stranded telomeric DNA sequences. Competition and antibody supershift assays confirmed the interaction of DNA–protein complex. Chromatin immunoprecipitation assays confirmed that EtGbp1p interacted with telomeric DNA in vivo. Collectively, these evidences suggest that EtGbp1p represents a G-rich single-stranded telomeric DNA-binding protein in E.tenella.     

A Family of Telomere-Associated Autonomously Replicating Sequences and Their Functions in Targeted Recombination in Hansenula polymorpha DL-1

A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of H. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5′-GGGTGGCG-3′ which resemble the telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease Bal31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.     

A locus on chromosome 9 is associated with differential response of 129S1/SvImJ and FVB/NJ strains of mice to systemic LPS

Although polymorphisms in TLR receptors and downstream signaling molecules affect the innate immune response, these variants account for only a portion of the ability of the host to respond to microorganisms. To identify novel genes that regulate the host response to systemic lipopolysaccharide (LPS), we created an F2 intercross between susceptible (FVB/NJ) and resistant (129S1/SvImJ) strains, challenged F2 progeny with LPS via intraperitoneal injection, and phenotyped 605 animals for survival and another 500 mice for serum concentrations of IL-1?? and IL-6. Genome-wide scans were performed on pools of susceptible and resistant mice for survival, IL-1??, and IL-6. This approach identified a locus on the telomeric end of the q arm of chromosome 9 (0?C40?Mb) that was associated with the differences in morbidity and serum concentrations of IL-1?? and IL-6 following systemic LPS in FVB/NJ and 129S1/SvImJ strains of mice. Fine mapping narrowed the locus to 3.7?Mb containing 11 known genes, among which are three inflammatory caspases. We studied expression of genes within the locus by quantitative RT-PCR and showed that Casp1 and Casp12 levels are unaffected by LPS in both strains, whereas Casp4 is highly induced by LPS in FVB/NJ but not in 129S1/SvImJ mice. In conclusion, our mapping results indicate that a 3.7-Mb region on chromosome 9 contains a gene that regulates differential response to LPS in 129S1/SvImJ and FVB/NJ strains of mice. Differences in the induction of Casp4 expression by LPS in the two strains suggest that Casp4 is the most likely candidate gene in this region.     

Segregating YKU80 and TLC1 Alleles Underlying Natural Variation in Telomere Properties in Wild Yeast

In yeast, as in humans, telomere length varies among individuals and is controlled by multiple loci. In a quest to define the extent of variation in telomere length, we screened 112 wild-type Saccharomyces sensu stricto isolates. We found extensive telomere length variation in S. paradoxus isolates. This phenotype correlated with their geographic origin: European strains were observed to have extremely short telomeres (<150 bp), whereas American isolates had telomeres approximately three times as long (>400 bp). Insertions of a URA3 gene near telomeres allowed accurate analysis of individual telomere lengths and telomere position effect (TPE). Crossing the American and European strains resulted in F1 spores with a continuum of telomere lengths consistent with what would be predicted if many quantitative trait loci (QTLs) were involved in length maintenance. Variation in TPE is similarly quantitative but only weakly correlated with telomere length. Genotyping F1 segregants indicated several QTLs associated with telomere length and silencing variation. These QTLs include likely candidate genes but also map to regions where there are no known genes involved in telomeric properties. We detected transgressive segregation for both phenotypes. We validated by reciprocal hemizygosity that YKU80 and TLC1 are telomere-length QTLs in the two S. paradoxus subpopulations. Furthermore, we propose that sequence divergence within the Ku heterodimer generates negative epistasis within one of the allelic combinations (American-YKU70 and European-YKU80) resulting in very short telomeres.     

Atlantic moonfishes: independent pathways of karyotypic and morphological differentiation

Fish of the genus Selene, known as lookdowns or moonfish, are one of the most morphologically derived groups of the family Carangidae, whose phylogenetic relationships are still largely unknown. In this study, we discuss karyoevolutionary aspects of three representatives of this genus from the Western Atlantic: Selene brownii (2n = 48; FN = 48), Selene setapinnis (2n = 46; FN = 48), and Selene vomer (2n = 48; FN = 50). Their body patterns were also investigated and compared to one another and in relation to two other species of different genera. Two mechanisms of karyotypic evolution seem to have acted in the diversification of this genus, namely pericentric inversions and centric fusions. Mapping of rDNA sequences showed that chromosome pairs bearing 5S rDNA sites are similar, whereas those bearing 18 rDNA sites are morphologically distinct while apparently also exhibiting interspecies synteny. Although the nucleolar organizer-bearing chromosomes are extremely efficient cytotaxonomic markers among Selene species, others cytogenetic patterns of these species are relatively conserved. Hybridization with telomeric probes (TTAGGG)_n did not exhibit interstitial telomeric sites (ITS), especially in S. setapinnis, where, along with a reduction in diploid number, a large metacentric pair derived from centric fusion is present. Data obtained by geometric morphometrics enable a clear morphological distinction among the three species, as well as in relation to two other species of the genus Caranx and Oligoplites. Data obtained suggest that morphologic evolution in Selene species was primarily dissociated from visible changes that occurred at the chromosomal level.