A serological comparison of some species of microsporida.

Double immunodiffusion techniques were used to investigate the taxonomic relationships between six different microsporidian isolates. Microsporidia used included Nosema bombycis, N. algerae, N. plodiae, and three organisms morphologically similar to N. necatrix. Antigens were extracted from spores after disruption in an MSK Braun cell homogenizer. Cross-reactions were seen between N. plodiae and two of the N. necatrix isolates, while the third N. necatrix, N. bombycis, and N. algerae were antigenically unrelated. One of the N. necatrix isolates revealed temperature-related antigenic differences, but no antigenic differences resulted from aging spores for 5 months before disruption.     

Sequence analysis of the Staphylococcus aureus srrAB loci reveals that truncation of srrA affects growth and virulence factor expression

The SrrAB system regulates metabolism and virulence factors in Staphylococcus aureus. We sequenced the srrAB loci of 21 isolates and performed a phylogenetic analysis. Vaginal and bovine isolates clustered together, while skin isolates were genetically diverse. Few nucleotide polymorphisms were observed, and most were synonymous. Two strains (N2 and N19) with N-terminal truncations in SrrA displayed defects in growth and abnormally upregulated virulence factor expression under low-oxygen conditions.     

Application of Multiple-Locus Variable Number Tandem Repeat Analysis to Identify Outbreak-Associated Neisseria meningitides Serogroup C Sequence Type 4821 in China

Neisseria meningitidis (N. meningitidis) serogroup C sequence type (ST)-4821 caused an outbreak in 2010 in Shandong province of China. Twenty-one non-outbreak-associated strains were isolated, along with twenty-eight N. meningitides serogroup C ST-4821 isolates. Therefore, it’s essential to identify and clarify characterization of the real outbreak-associated strains with a rapid method during an outbreak investigation. In this study, multiple-locus variable number tandem repeat analysis (MLVA) was applied to analyze 84 N. meningitidis strains, among which 58 were recovered from two outbreaks and 26 were sporadic isolates. Three MLVA schemes with different combination of VNTR loci were tested, and two of them were suitable for isolates from China: scheme 2 with six loci was found to separate ST into finer resolution, and scheme 3 with five loci can be used to identify outbreak-associated isolates from the same outbreak that caused by N. meningitidis serogroup C ST-4821.     

Isolation of Pseudomonas stutzeri in wastewater of catfish fish-ponds in the Mekong Delta and its application for wastewater treatment

The aim of this study was to explore the potential for reducing soluble N load in fishpond wastewater using naturally occurring denitrifying bacteria. Twenty-seven isolates were selected from in wastewater (liquid/solid) of catfish-ponds located along the Tien river, in the Mekong Delta, Vietnam in SW-LB medium (artificial seawater Luria-Britani medium) supplemented with 10 mM NH₄ and NO₃ and twenty-five isolates were identified as Pseudomonas stutzeri based on similarity of PCR-16S rRNA using universal primers and specific primers. Four isolates were effective in lowering soluble N (NH₄, NO₂ and NO₃) levels in fishpond water from 10 mg/L to negligible amounts after four days. Further experiments are underway to determine the fate of N lost from solution and the relative activity of ammonia oxidation, and nitrite and nitrate reduction by P. stutzeri isolates.     

The distribution of genetic diversity in the Neofusicoccum parvum/N. ribis complex suggests structure correlated with level of disturbance

Plants and animals adapted to colonize disturbed sites might also be better invaders, but this phenomenon has not been widely considered in fungi. We investigated genetic diversity and structure amongst isolates of Neofusicoccum parvum, N. cordaticola, N. kwambonambiense and N. umdonicola that coexist sympatrically on a native tree, Syzygium cordatum, across its distribution in South Africa. Species composition varied among stands, with dominance of N. parvum in disturbed stands, and absence in undisturbed stands, where the other species dominated. N. parvum populations from trees planted in urban environments were more genetically diverse than populations from human disturbed stands of S. cordatum. Bayesian analysis clustered N. parvum isolates in three sub-populations, suggesting three sources of origin. These results support the hypothesis that as a generalist N. parvum will dominate human disturbed sites and trees in urban areas, indicating strong potential for invasion, and its spreading from non-native hosts to native S. cordatum, rather than vice versa.     

Genetic Variation in Nacobbus aberrans: An Approach toward Taxonomic Resolution

Biochemical and molecular analyses of genetic variation were evaluated to address the taxonomic status of Nacobbus aberrans. Isolates from Mexico, Peru, and Argentina, cultured on tomato in the greenhouse, were analyzed with respect to isozyme and DNA marker variation. Although acid phosphatase and malate dehydrogenase revealed distinct profiles for each isolate, non-specific esterases revealed possible affinities between the Peruvian isolates and between the isolates from Mexico and Peru. Two of l 0 RAPD primers revealed affinities suggested by esterase profiles. RFLP analysis of the rDNA repeating unit with six restriction enzymes revealed identical cleavage patterns between the Peru isolates and a distinct profile shared by isolates from Mexico and Argentina. Nucleotide sequence analysis of the 5.8S rRNA coding region revealed differences among the four isolates at eight of 157 positions; sequences of the Peruvian isolates differed from each other at only one position, whereas the Mexican and Argentine isolates were identical and could be distinguished from the Peruvian isolates. A distance matrix from unweighted pairwise comparisons of the 5.8S rDNA revealed apparent elevated intraspecific divergence in N. aberrans comparable to intergeneric divergence between Heterodera and Globodera. Analysis of additional N. aberrans isolates from throughout the distribution range should help determine the full extent of intraspecific genetic variation that underlies the phenotypic and morphologic diversity of the genus.     

Identification of Fungi in the Botryosphaeriaceae Family Associated with Stem Blight of Vaccinium spp. in the Southeastern United States

Stem blight is a major disease of blueberry caused by Botryosphaeriaceae fungi. Chemical and cultural management options are limited, putting emphasis on breeding efforts to identify sources of resistance. The efficacy and durability of host resistance could be impacted by the species composition of the pathogen population in a region and by the isolates employed in the screenings used to identify the resistance. Samples (365) were collected from southern highbush (SHB) and rabbiteye blueberry (REB) cultivars from 28 sites in the southeastern US (AL, FL, GA, NC, and SC). Colony morphology identified 86% of the isolates as Botryosphaeriaceae. Conidia morphology and Maximum Likelihood analysis of the Internal Transcribed Spacer rDNA regions (ITS), translation elongation factor one alpha (tef1-α), and β-tubulin were used to identify isolates at genera or species level. A PCR-restriction fragment length polymorphism (PCR-RFLP) test was used to identify isolates to genus. Neofusicoccum and Lasiodiplodia were the predominant genera. N. kwambonambiense, N. ribis, L. theobromae and L. pseudotheobromae were the most common species isolated. Phylogenies conducted with a limited number of isolates indicated non-clonal and potentially diverse populations occur on blueberry that warrant additional study. Botryosphaeria corticis, B. dothidea, and Diplodia seriata were isolated infrequently.     

Natural populations of the entomopathogenic fungus Beauveria bassiana in Chinese forest ecosystems are diverse and reveal equal frequencies of mating types within phylogenetic species

Beauveria bassiana is an important entomopathogenic fungus with widespread application in the biological control of harmful insect pests. This species is widely distributed as an anamorph while only two teleomorph specimens have been found in eastern China. However, little is known about the ecological conditions for sexual reproduction in natural populations of B. bassiana. Here, we collected 488 isolates of Chinese B. bassiana sensu stricto from five sites, in which teleomorph or anamorph occurred, and used molecular phylogenetic and haplotype information to determine phylogenetic diversity, mating types, and sexual reproductive potential in these populations. Molecular identification based on denaturing gradient gel electrophoresis (DGGE) and combined data of the nuclear intergenic region Bloc and translation elongation factor-1a (TEF) assemblage resolved five B. bassiana s.s phylogenetic species labeled according to their geographic origin: Europe/N. Africa 1, Asia 3, Asia 4, AFNEO_1, and N. America 2. In Guniujiang and Manshuihe collection sites, teleomorph isolates RCEF 0771 and RCEF 0382 were both identified as Europe/N. Africa 1 phylogenetic species. In addition, more than half of the isolates in five representative sites belonged to Europe/N. Africa 1. However, the teleomorph of B. bassiana s.s. was not detected in Kuankuoshui while isolates within Europe/N. Africa 1 were present at this site, and isolates belonging to Europe/N. Africa 1 were not found in either Jingyuetan or Dinghushan collection sites. Distribution of MAT1 and MAT2 mating type idiomorphs in Europe/N. Africa 1 were 51:69, 37:24, and 15:15 in Guniujiang, Manshuihe, and Kuankuoshui, respectively. The presence of teleomorph and roughly equal frequencies of opposite mating types indicate regular sexual reproduction in B. bassiana natural populations. The data offer a better understanding of the ecological conditions of sexual reproduction in natural populations of B. bassiana. These results also yield insights into the potential for sexual reproduction in other supposedly ‘asexual’ fungi.     

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA)

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689 bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.     

Susceptibility of the cabbage looper, Trichoplusia ni, and the velvetbean caterpillar, Anticarsia gemmatalis, to several isolates of the entomopathogenic fungus Nomuraea rileyi

Cabbage looper larvae, Trichoplusia ni, were equally susceptible to isolates of the entomogenous fungus Nomuraea rileyi from Missouri, Mississippi, and Brazil; an isolate from Florida was 7–17 times less active than the other isolates. Velvetbean caterpillars, Anticarsia gemmatalis, were either only slightly susceptible or not susceptible to the isolates of N. rileyi from Missouri, Florida, and Mississippi. In contrast, larvae of A. gemmatalis cultured from three locations (Missouri, Florida, and Brazil) all were equally susceptible to a Brazilian isolate of N. rileyi.     

Host range and symptom differences between isolates of turnip mosaic virus obtained fromSisymbrium loeselii

Twenty-four isolates of turnip mosaic virus (TuMV) from spontaneously infectedSisymbriutn loeselii plants were collected in Bohemian localities. Single lesions on leaves ofNicotiana tabacum cv. Samsun served for inoculating petunia plants used as infection sources for twelve host species. In no case were two identical isolates obtained. 15 isolates could be transmitted toVicia faba, a new TuMV host. Almost all isolates infectedPhaseolus vulgaris cv. Prince locally andTrifolium incarnatum systemically. Seven isolates were transmissible toPisum sativum. No substantial differences between isolates were observed with infectedAn aranthus caudatus, Chenopodium quinoa, Datura innoxia andSinapis alba plants. Several isolates could not infectNicotiana glutinosa at all, other isolates caused in it latent systemic infection and some isolates only local infection contrary to normal local and systemic infections ofN. glutinosa. Attempts to transmit one isolate to cereals failed.     

Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand

Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species.     

Genetic Diversity of Neisseria meningitidis Serogroup C ST-4821 in China Based on Multiple-Locus Variable Number Tandem Repeat Analysis

Neisseria meningitidis sequence type (ST)-4821 was first reported in China in 2003, and a new hyper-virulent lineage has been designated as the ST-4821 complex. A large number of N. meningitidis ST-4821 strains have been identified in China since 2003; however, the microevolution characteristics of this complex are unclear. Different combinations of variable number of tandem repeats (VNTR) loci were used in multiple-locus VNTR analysis (MLVA) to analyze 118 N. meningitidis serogroup C ST-4821 strains isolated from seventeen provinces between 2003 and 2012. Additionally, MLVA with five VNTR loci was performed due to its high discriminatory power. One hundred and eighteen isolates were found to comprise 112 subtypes based on MLVA, and 16 outbreak-associated strains were clustered into one group. These data indicate a high level of diversity for N. meningitidis ST-4821 due to microevolution in the last decade. In addition, the results revealed high similarity between isolates from the same geographic origins, which is helpful when monitoring the spread of N. meningitidis serogroup C ST-4821 and will provide valuable information for the control and prevention of bacterial meningitis in China.     

Mos1-Mediated Transgenesis to Probe Consequences of Single Gene Mutations in Variation-Rich Isolates of Caenorhabditis elegans

Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs.     

Regulation of hyphal growth rate ofHypoxylon mammatum by amino acids: Stimulation by proline

Variation among isolates ofHypoxylon mammatum caused by amino acids added as sole nitrogen sources demonstrated genetic heterogeneity among the population in the mechanisms regulating hyphal growth rates. Isolates were obtained by a random sample consisting of 22 single ascospore isolates from a local population of hypoxylon cankers onP. tremuloides. Hyphal growth rates were determined from colony diameter measurements on defined media containing either alanine, asparagine, glycine, leucine, lysine, or proline. All isolates but two grew faster on proline than the other amino acids tested. The mean growth rate of the population sample was 3.9 mm day⁻¹ on proline compared to 1.96 mm day⁻¹ on asparagine, the second fastest mean growth rate. Growth rates of the 22 isolates on these six amino acids were largely uncorrelated, indicating independent mechanisms of regulation. Only asparagine gave significant correlations with more than two other amino acids. A more detailed examination of selected isolates representing a range of growth rates on proline showed that the rapid growth rates were also dependent on other materials present in Difco Bacto-Agar. Growth of the proline stimulated isolates was considerably slower on media gelled with Noble agar. Other nitrogen sources, especially glutamate, added with proline stimulated the growth of these isolates. Isolate dependent variation in the stimulatory effect of the N source added with proline was demonstrated. Stimulation of growth rate by proline may be related to drought stress-enhanced canker elongationin vivo, since drought stress caused proline accumulation in the host,Populus tremuloides.     

Symbiosis between the root-nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions

Two hundred forty-three isolates of alfalfa root-nodule bacteria (Sinorhizobium meliloti) were obtained from nodules and soils sampled in the northern Aral region, experiencing secondary salinization. Isolates obtained from nodules (N isolates) were significantly more salt-tolerant than those from soils (T isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild species of alfalfa, melilot, and trigonella preferably formed symbioses with salt-tolerant root-nodule bacteria in both salinized and nonsalinized soils. Only two alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses in soils contrasting in salinity. The formation of efficient symbiosis with alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and T) differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed efficient symbioses in the presence of salt. The increase in the dry weight of the plants was 25–68% higher than in the control group. The efficiency of symbiotic interaction under salinization conditions depended on the symbiotic efficiency of the isolates under standard conditions but did not correlate with the source of root-nodule bacteria (soil or nodule) or their salt tolerance. The results indicate that the strains of root-nodule bacteria forming efficient symbioses under salinization conditions can be found.     

Diversity of Trichoderma spp. causing Pleurotus green mould diseases in Central Europe

The present study includes the molecular characteristics of Trichoderma pleurotum and Trichoderma pleuroticola isolates collected from green moulded cereal straw substrates at 47 oyster mushroom farms in Poland. The screening of the 80 Trichoderma isolates was performed by morphological observation and by using the multiplex PCR assay. This approach enabled specific detection of 47 strains of T. pleurotum and 2 strains of T. pleuroticola. Initial identifications were confirmed by sequencing the fragment of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the rRNA gene cluster and the fragment including the fourth and fifth introns and the last long exon of the translation–elongation factor 1-alpha (tef1) gene. ITS and tef1 sequence information was also used to establish the intra- and interspecies relationship of T. pleurotum and T. pleuroticola originating from the oyster mushroom farms in Poland and from other countries. Comparative analysis of the ITS sequences showed that all T. pleurotum isolates from Poland represent one haplotype, identical to that of T. pleurotum strains from Hungary and Romania. Sequence analysis of the tef1 locus revealed two haplotypes (“T” and “N”) of Polish T. pleurotum isolates. The “T” type isolates of T. pleurotum were identical to those of strains from Hungary and Romania. The “N” type isolates possessed a unique tef1 allele. Detailed analysis of the ITS and tef1 sequences of two T. pleuroticola isolates showed their identicalness to Italian strain C.P.K. 1540.     

Analysis of the abilities of endophytic bacteria associated with banana tree roots to promote plant growth

A total of 40 endophytic bacterial isolates obtained from banana tree roots were characterized for their biotechnological potential for promoting banana tree growth. All isolates had at least one positive feature. Twenty isolates were likely diazotrophs and formed pellicles in nitrogen-free culture medium, and 67% of these isolates belonged to the genus Bacillus sp. The isolates EB-04, EB-169, EB-64, and EB-144 had N fixation abilities as measured by the Kjeldahl method and by an acetylene reduction activity assay. Among the 40 isolates, 37.5% were capable of solubilizing inorganic phosphate and the isolates EB-47 and EB-64 showed the highest solubilization capacity. The isolate EB-53 (Lysinibacillus sp.) had a high solubilization index, whereas 73% of the isolates had low solubilization indices. The synthesis of indole-3-acetic acid (IAA) in the presence of L-tryptophan was detected in 40% of the isolates. The isolate EB-40 (Bacillus sp.) produced the highest amount of IAA (47.88 μg/ml) in medium supplemented with L-tryptophan and was able to synthesize IAA in the absence of L-tryptophan. The isolates EB-126 (Bacillus subtilis) and EB-47 (Bacillus sp.) were able to simultaneously fix nitrogen, solubilize phosphate and produce IAA in vitro. The results of this study demonstrated that the isolates analyzed here had diverse abilities and all have the potential to be used as growth-promoting microbial inoculants for banana trees.     

Genotypic characterization of methicillin-resistant Staphylococcus aureus strains isolated from the anterior nares and catheter of ambulatory hemodialysis patients in Mexico

Methicillin-resistant Staphylococcus aureus (MRSA) is the causal agent of multiple nosocomial infections worldwide, including catheter-associated bacteremia in hemodialysis patients. The purposes of this work were to genetically characterize a group of MRSA isolates from catheter-related infections of ambulatory Mexican hemodialysis patients and to determine whether the strains are the same as those carried by the patients in their anterior nares. Sixteen pairs of MRSA isolates from the catheter (cat) and anterior nares (N) of hemodialysis patients were compared using pulsed-field gel electrophoresis (PFGE), PCR detection of adhesion genes and other virulence markers, and an antibiogram. Three pairs of N/cat MRSA isolates (18.7 %) with identical resistograms also showed the same combination of PCR-detected markers and PFGE pattern; one additional pair showed only an identical electrophoretic PFGE pattern. Of the MRSA isolates, 75 % (n?=?24) were resistant to ≥7 antibiotics, 4 isolates were resistant to 11 antibiotics, and 7 isolates were resistant to the 12 antibiotics tested. The most frequent virulence marker combination found was spa, clfA, clfB, cna, bbp, ebps, map/eap, sdrC, sdrD, sdrE, ica, agr (65.6 %, n?=?21). The SCCmec alleles of the 32 MRSA isolates were IV (n?=?20), I (n?=?7), II (n?=?4), and V (n?=?1), and no SCCmec type III MRSA was found. The genotypic characterization of the MRSA isolates studied in this work will contribute to a better understanding of the virulence gene makeup of catheter-colonizing S. aureus strains and will help to lower the infection risk in these patients.