Increase in prevalence of nosocomial non-Candida albicans candidaemia and the association of Candida krusei with fluconazole use

Candida is an important nosocomial pathogen. This study was undertaken to provide information on the rate of candidaemia, to define the risks for candidaemia and to describe and account for the epidemiology of candidaemia at our institution between 1992 and 1999. The overall rate was 0.052 per 1000 patient days and 0.27 per 1000 discharges. The major risks for candidaemia were colonization at a non-sterile site (OR 3.85, 95%CI 1.80-9.09), total parenteral nutrition (TPN) in the absence of neutropenia (OR 11.8, 95%CI 4.5-35.4, P

Breeding for resistance to lentil Ascochyta blight

Ascochyta blight, caused by Ascochyta lentis , is one of the most globally important diseases of lentil. Breeding for host resistance has been suggested as an efficient means to control this disease. This paper summarizes existing studies of the characteristics and control of Ascochyta blight in lentil, genetics of resistance to Ascochyta blight and genetic variations among pathogen populations (isolates). Breeding methods for control of the disease are discussed. Six pathotypes of A. lentis have been reported. Many resistant cultivars/lines have been identified in both cultivated and wild lentil. Resistance to Ascochyta blight in lentil is mainly under the control of major genes, but minor genes also play a role. Current breeding programmes are based on crossing resistant and high-yielding cultivars and multilocation testing. Gene pyramiding, exploring slow blighting and partial resistance, and using genes present in wild relatives will be the methods used in the future. Identification of more sources of resistance genes, good characterization of the host-pathogen system, and identification of molecular markers tightly linked to resistance genes are suggested as the key areas for future study.

Analysis of genetic diversity within Australian lucerne cultivars and implications for future genetic improvement

Lucerne (Medicago sativa L.) is autotetraploid, and predominantly allogamous. This complex breeding structure maximises the genetic diversity within lucerne populations making it difficult to genetically discriminate between populations. The objective of this study was to evaluate the level of random genetic diversity within and between a selection of Australian-grown lucerne cultivars, with tetraploid M. falcata included as a possible divergent control source. This diversity was evaluated using random amplified polymorphic DNA (RAPDs). Nineteen plants from each of 10 cultivars were analysed. Using 11 RAPD primers, 96 polymorphic bands were scored as present or absent across the 190 individuals. Genetic similarity estimates (GSEs) of all pair-wise comparisons were calculated from these data. Mean GSEs within cultivars ranged from 0.43 to 0.51. Cultivar Venus (0.43) had the highest level of intra-population genetic diversity and cultivar Sequel HR (0.51) had the lowest level of intra-population genetic diversity. Mean GSEs between cultivars ranged from 0.31 to 0.49, which overlapped with values obtained for within-cultivar GSE, thus not allowing separation of the cultivars. The high level of intra- and inter-population diversity that was detected is most likely due to the breeding of synthetic cultivars using parents derived from a number of diverse sources. Cultivar-specific polymorphisms were only identified in the M. falcata source, which like M. sativa, is outcrossing and autotetraploid. From a cluster analysis and a principal components analysis, it was clear that M. falcata was distinct from the other cultivars. The results indicate that the M. falcata accession tested has not been widely used in Australian lucerne breeding programs, and offers a means of introducing new genetic diversity into the lucerne gene pool. This provides a means of maximising heterozygosity, which is essential to maximising productivity in lucerne.

Inheritance and mapping of 11 avirulence genes in Phytophthora sojae

Two new crosses involving four races (races 7, 16, 17, and 25) of the soybean root and stem rot pathogen Phytophthora sojae were established (7/16 cross; 17/25 cross). An F-2 Population derived from each cross was used to determine the genetic basis of avirulence towards 11 different resistance genes in soybean. Avirulence was found to be dominant and determined by a single locus for Avr1b, 1d, 1k, 3b, 4, and 6, as expected for a simple gene-for-gene model. We also observed several cases of segregation, inconsistent with a single dominant gene being solely responsible for avirulence, which suggests that the genetic background of the different crosses can affect avirulence. Avr4 and 6 cosegregated in both the 7/16 and 17/25 crosses and, in the 7/16 cross, Avr1b and 1k were closely linked. Information from segregating RAPD, RFLP, and AFLP markers screened on F-2 progeny from the two new crosses and two crosses described previously (a total of 212 F-2 individuals, 53 from each cross) were used to construct an integrated genetic linkage map of P. sojae. This revised genetic linkage map consists of 386 markers comprising 35 RFLP, 236 RAPD, and 105 AFLP markers, as well as 10 avirulence genes. The map is composed of 21 major linkage groups and seven minor linkage groups covering a total map distance of 1640.4 cM. (C) 2002 Elsevier Science (USA). All rights reserved.

Isolation and characterization of microsatellite loci in Bemisia tabaci

Bemisia tabaci (Hemiptera: Aleyrodidae) is a haplo-diploid species with a global distribution demonstrating strong geographical structure with eight recognizable genetic groups. Fifteen microsatellite loci (335 alleles, 6-44 alleles per locus) were derived from four of the eight groups and were then screened across 33 populations. These loci clearly differentiate the populations. The microsatellites amplified best in individuals from genetic groups representing the Mediterranean, Middle East, Asia (three groups) and Australasia/Oceania and amplified less well with populations from sub-Saharan Africa and the New World. This differential amplification pattern is a direct result of the relatedness to the microsatellite source material.

Comparative genetic study confirms exceptionally low genetic variation in the ancient and endangered relictual conifer, Wollemia nobilis (Araucariaceae)

The Wollemi pine, Wollemia nobilis (Araucariaceae), was discovered in 1994 as the only extant member of the genus, previously known only from the fossil record. With fewer than 100 trees known from an inaccessible canyon in southeastern Australia, it is one of the most endangered tree species in the world. We conducted a comparative population genetic survey at allozyme, amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) loci in W. nobilis, Araucaria cunninghatnii and Agathis robusta - representatives of the two sister genera. No polymorphism was detected at 13 allozyme loci, more than 800 AFLP loci or the 20 SSR loci screened in W. nobilis. In Ag. robusta only one of 12 allozyme loci, five of 800 AFLP loci and none of the 15 SSR loci were variable. For A. cunninghamii, 10 of > 800 AFLP loci and five of 20 SSR loci were variable. Thus low genetic diversity characterizes all three species. While not ruling out the existence of genetic variation, we conclude that genetic diversity is exceptionally low in the Wollemi pine. To our knowledge this is the most extreme case known in plants. We conclude that the combination of small population effects, clonality and below-average genetic variation in the family are probable contributing factors to the low diversity. The exceptionally low genetic diversity of the Wollemi pine, combined with its known susceptibility to exotic fungal pathogens, reinforces current management policies of strict control of access to the pines and secrecy of the pine locations.

Pathogenic specialisation within Colletotrichum trifolii in Australia, and lucerne cultivar reactions to all known Australian pathotypes

Anthracnose and crown rot, caused by Colletotrichum trifolii, are serious diseases of lucerne (Medicago saliva L.) in humid regions of the world. A race survey was conducted by inoculating individual lucerne clones (genotypes) with C. trifolii isolates collected from a range of Medicago hosts, locations, and years in south-eastern Queensland. This survey revealed for the first time in Australia the presence of race 2 (virulence on anthracnose resistance gene An I) and the first world report of race 4 (virulence on An(2)). A collection of North American race I and race 2 C. trifolii isolates, when inoculated onto the Australian differential clones, gave responses that were in agreement with their North American reactions. A RAPD analysis was conducted on 9 Australian C. trifolii isolates including races 1, 2, and 4; two C. destructivum and one C. gloeosporioides isolate were included as known outliers. For the C. trifolii isolates, 94.6% similarity was found regardless of host origin or race, compared with 2.2% similarity between this group and the C. gloeosporioides and C. destructivum isolates, confirming that the new races belong to C. trifolii. Currently, it is hypothesised that only plants carrying genes An, and An2 are resistant to the 3 races. Of 22 cultivars screened against the 3 races, only UQL-1, Hallmark, and Pioneer 54Q53 had >30% of plants resistant to the 3 races in separate screenings. The research highlights the need to find new sources of resistance to C. trifolii in lucerne.

Interspecific hybrids between the homothallic Phytophthora sojae and Phytophthora vignae

An interspecific cross was attempted between two homothallic species of Phytophthora, P. sojae and P. vignae. From 1640 single-oospore cultures isolated, DNA was extracted from 800, and two interspecific F-1 hybrids (F(1)1121 and F(1)1426) were putatively identified using RAPD markers. The true hybrid nature of these F-1 hybrids was confirmed using additional AFLP analysis. Single- zoospore cultures were generated for each F-1 hybrid and one single-zoospore culture of each was used in pathogenicity and virulence tests. Both F-1 hybrids were pathogenic to soybean and cowpea, causing symptoms including lesions, wilting and death of susceptible soybean and cowpea cultivars. However, the aggressiveness of the F-1 hybrids was reduced and was substantially more variable when compared with that of the parental isolates on their respective hosts. The F-1 hybrids were reisolated from infected seedlings and their hybrid nature confirmed using RAPD and AFLP analysis. These results provide a basis for further research aimed at obtaining an increased understanding of the genetics of host specificity in the Oomycetes.

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA

A cross between two different races (race 7 x race 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Anur4 and Anur6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F-2 population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0 cM distant from the Anur4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Anur4/6 locus. The chromosome walk spanned a physical distance of 67 kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3 kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Anur4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Anur4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Anur4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24 kb and 4.3 cM that appears to include the Anur4/6 locus. (C) 2003 Elsevier Inc. All rights reserved.

Phylogeny of the Sporobolus indicus complex, based on internal transcribed spacer (ITS) sequences

The entire internal transcribed spacer ( ITS) region, including the 5.8S subunit of the nuclear ribosomal DNA ( rDNA), was sequenced by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified fragments. The study included 40 Sporobolus ( Family Poaceae, subfamily Chloridoideae) seed collections from 14 putative species ( all 11 species from the S. indicus complex and three Australian native species). These sequences, along with those from two out-group species [ Pennisetum alopecuroides ( L.) Spreng. and Heteropogon contortus ( L.) P. Beauv. ex Roemer & Schultes, Poaceae, subfamily Panicoideae], were analysed by the parsimony method (PAUP; version 4.0b4a) to infer phylogenetic relationships among these species. The length of the ITS1, 5.8S subunit and ITS2 region were 222, 164 and 218 base pairs ( bp), respectively, in all species of the S. indicus complex, except for the ITS2 region of S. diandrus P. Beauv. individuals, which was 217 bp long. Of the 624 characters included in the analysis, 245 ( 39.3%) of the 330 variable sites contained potential phylogenetic information. Differences in sequences among the members of the S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur & Schinz and S. jacquemontii Kunth. collections were 0%, while differences ranged from 0 to 2% between these and other species of the complex. Similarly, differences in sequences among collections of S. laxus B. K. Simon, S. sessilis B. K. Simon, S. elongatus R. Br. and S. creber De Nardi were 0%, compared with differences of 1-2% between these four species and the rest of the complex. When comparing S. fertilis ( Steud.) Clayton and S. africanus (Poir.) Robyns & Tourney, differences between collections ranged from 0 to 1%. Parsimony analysis grouped all 11 species of the S. indicus complex together, indicating a monophyletic origin. For the entire data set, pair-wise distances among members of the S. indicus complex varied from 0.00 to 1.58%, compared with a range of 20.08-21.44% among species in the complex and the Australian native species studied. A strict consensus phylogenetic tree separated 11 species of the S. indicus complex into five major clades. The phylogeny, based on ITS sequences, was found to be congruent with an earlier study on the taxonomic relationship of the weedy Sporobolus grasses revealed from random amplified polymorphic DNA ( RAPD). However, this cladistic analysis of the complex was not in agreement with that created on past morphological analyses and therefore gives a new insight into the phylogeny of the S. indicus complex.

A genetic map of macadamia based on randomly amplified DNA fingerprinting (RAF) markers

The first genetic linkage map of macadamia (Macadamia integrifolia and M. tetraphylla) is presented. The map is based on 56 F-1 progeny of cultivars 'Keauhou' and 'A16'. Eighty-four percent of the 382 markers analysed segregated as Mendelian loci. The two-way pseudo-testcross mapping strategy allowed construction of separate parental cultivar maps. Ninety bridging loci enabled merging of these maps to produce a detailed genetic map of macadamia, 1100 cM in length and spanning 70-80% of the genome. The combined map comprised 24 linkage groups with 265 framework markers: 259 markers from randomly amplified DNA fingerprinting (RAF), five random amplified polymorphic DNA (RAPD), and one sequence-tagged microsatellite site (STMS). The RAF marker system unexpectedly revealed 16 codominant markers, one of them a putative microsatellite locus and exhibiting four distinct alleles in the cross. This molecular study is the most comprehensive examination to date of genetic loci of macadamia, and is a major step towards developing marker-assisted selection for this crop.

Genetic divergence in cocoa progenies for backcrossing program to witches' broom disease resistance

Backcrossing has been little used in cacao breeding, particularly due to the long time required to transfer genes and recover the genetic background of the recurrent parent. The objective of this study was to select individuals, resulting from the backcross CEPEC-42 x SIC-19, genetically related to the recurrent parent SIC-19 by using RAPD molecular markers, among those with resistance to witches' broom. Of the 31 plants that clustered with SIC-19, 18 from the replanted material remained free of the disease in the field, with good vegetative aspect and, therefore can be used for backcross to reach the desired objective.

Variabilidade genética de Eugenia uniflora L. em remanescentes florestais em diferentes estádios sucessionais

A compreensão da diversidade genética fornece elementos básicos sobre a dinâmica e funcionamento de populações, auxiliando na conservação e uso sustentável das espécies. Supõe-se que populações sucessionais precoces poderiam ser geneticamente mais diferenciadas do que populações sucessionais mais tardias. Visando testar esta hipótese, o presente trabalho teve como objetivo analisar a variabilidade genética de populações de Eugenia uniflora L. em manchas florestais em diferentes estádios sucessionais. Foram selecionadas duas áreas em diferentes estádios de sucessão, sendo a primeira em estádio inicial e a segunda em estádio avançado. A área de estudo apresenta um remanescente florestal em transição de Floresta Ombrófila Mista e Floresta Estacional Semidecídua. Por meio da técnica de RAPD (Random Amplified Polymorphic DNA) e análise multivariada, a diversidade gênica esperada e a porcentagem de loci polimórficos foram estimadas, além da similaridade genética entre as populações de cada mancha florestal e a diversidade de cada área por meio do índice de diversidade de Simpson. Os resultados indicaram 79% de loci polimórficos para a área em estádio avançado e 70% para a área em estádio inicial de sucessão. A similaridade genética entre pares de indivíduos variou entre 0,55 e 0,86 na área em estádio inicial de sucessão e entre 0,45 e 0,78 para a área em estádio avançado. Não houve diferenças significativas entre a diversidade das duas áreas (P = 89). Um escalonamento multidimensional não-métrico indicou menor distância genética entre os indivíduos da área em estádio inicial. Da mesma forma, uma análise de similaridade - ANOSIM indicou separação entre os indivíduos das duas áreas.

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

Genetic variation between susceptible and non-susceptible snails to Schistosoma infection using random amplified polymorphic DNA analysis (RAPDs)

Susceptibility of snails to infection by certain trematodes and their suitability as hosts for continued development has been a bewildering problem in host-parasite relationships. The present work emphasizes our interest in snail genetics to determine what genes or gene products are specifically responsible for susceptibility of snails to infection. High molecular weight DNA was extracted from both susceptible and non-susceptible snails within the same species Biomphalaria tenagophila. RAPD was undertaken to distinguish between the two types of snails. Random primers (10 mers) were used to amplify the extracted DNA by the polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and silver staining. The results suggest that RAPD represents an efficient means of genome comparison, since many molecular markers were detected as genetic variations between susceptible and non-susceptible snails.